a s u pple m e the n t o l c g application o r t h a...

SUPPLEMENT TO

THE

APPLICATION NOTEBOOK

June 2014

www.chromatographyonline.com

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ES443973_LCGCSUPP0614_CV1.pgs 05.22.2014 23:40 ADV blackyellowmagentacyan

Now for my next trick: Essential Macromolecular Characterization™ without SEC-MALS

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ES445920_LCGCSUPP0614_CV2_FP.pgs 05.28.2014 01:27 ADV blackyellowmagentacyan

THE APPLICATION

NOTEBOOK

Medical/Biological

8 Utilization of CESI Technology for

Comprehensive Characterization of Biologics

Rajeswari Lakshmanan, PhD, AB Sciex

10 High Resolution LC–MS Analysis of Reduced IgG1

Monoclonal Antibody Fragments Using HALO Protein C4

Stephanie A. Schuster and Barry E. Boyes , Advanced Materials Technology, Inc.

11 Intrada Amino Acid Separates

Intact Amino Acids in Human Serum

Piotr Macech, Robert Puryear, and Itaru Yazawa, Imtakt USA

12 Separation of Nucleobases Using

TSKgel® SuperSW mAb HTP Column in HILIC Mode

Justin Steve and Atis Chakrabarti, PhD, Tosoh Bioscience LLC

14 Characterization of Tobacco Extracts by

Gas Chromatography High Resolution

Time of Flight Mass Spectrometry (GC-HRT)

David E. Alonso, Jonathan Byer, Jeff Patrick, and Joe Binkley, Life Science

and Chemical Analysis Centre, LECO Corporation

Chiral

15 Isolation of Structurally Related Components in Natural

Product Extracts Using the RegisPack and Whelk-O1 Chiral

Selectors

Andrew Geyer and Ted Szczerba, Regis Technologies, Inc.

THE APPLICATION NOTEBOOK – JUNE 2014 3

TABLE OF CONTENTS

ES445790_LCGCSUPP0614_003.pgs 05.28.2014 00:06 ADV blackyellowmagentacyan

Environmental

16 Keeping Water Safe: Detecting Pharmaceutical

and Personal Care Products in Water Using Liquid

Chromatography–Mass Spectrometry

Joe Anacleto, Zicheng Yang, Helen (Qingyu) Sun, and Kefei Wang, Bruker

Daltonics

18 Solid Phase Extraction of Organophosphorus

Pesticides and Triazine Herbicides in Water

Using a New Polymeric Sorbent

Xiaoyan Wang, UCT, LLC

Food and Beverage

19 Determination of Phenolic Compounds in Virgin Olive Oil

Using Comprehensive 2D-LC

Sonja Krieger and Sonja Schneider, Agilent Technologies Inc.

20 The Analysis of Chlorinated Dioxins, Difurans, and

Polychlorinated Biphenyls in Edible Oils

FMS, Inc.

21 Structural Differences in Modified Starches

Malvern Instruments Ltd.

22 Detection of Low-Level Sulfur Compounds in

Spearmint Oil Using the Pulsed Flame Photometric

Detector (PFPD)

Gary Engelhart and Cynthia Elmore, OI Analytical

23 HILIC with Increased Sensitivity for the Analysis of

Sugars

Melissa Turcotte and Satoko Sakai, Showa Denko America and Showa Denko

K.K.

24 Veterinary Drug Residue Analysis Using the

AutoMate-Q40: An Automated Solution to QuEChERS

Tyler Trent, Teledyne Tekmar

4 THE APPLICATION NOTEBOOK – JUNE 2014

TABLE OF CONTENTS


Pharmaceutical/Drug Discovery

25 Fast Screening Methods for Analgesics and Non-Steroidal

Anti-Inflammatory (NSAIDS) Drugs by HPLC with Agilent

Poroshell 120 Columns

William Long, Agilent Technologies, Inc.

28 Separation of Five Steroids on a C18-Functionalized

Polymeric Reversed-Phase HPLC Column (PRP™-C18)

Derek Jensen and Mark Carrier, Hamilton Company

29 Measuring Antibody Molecular Weight by SEC-MALS

Malvern Instruments Ltd.

30 Accurate Pain Management Analysis in

Under 5 Min on Raptor™ Biphenyl

Superficially Porous Particle LC Columns

Sharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation

32 Analysis of Alprostadil by HPLC

with Post-Column Derivatization

Pickering Laboratories, Inc.

33 Antibody Drug Conjugate (ADC) Analysis

Wyatt Technology

34 Characterization of PLGA Using SEC–MALS-VIS

Wyatt Technology

General

35 Separation of a Mix of Acidic, Basic, and Neutral

Compounds at High pH Conditions

Diamond Analytics

36 Concentrating Phenolic Compounds Using

the XcelVap® Concentration System

David Gallagher, Horizon Technology, Inc.

37 Recent Improvements in Time of Flight

Mass Spectrometer Detection Technologies

PHOTONIS USA

THE APPLICATION NOTEBOOK Ð JUNE 2014 5

TABLE OF CONTENTS


Articles

38 The 2014 LCGC Awards

Meg L’Heureux

43 Recent Progress in Chiral Stationary Phase Development

and Current Chiral Applications

Timothy J. Ward and Karen D. Ward

47 The Fundamental Shift to Tandem Mass Spectrometry

St. John Skilton, Eric Johansen, and Xu Guo

Departments

51 Call for Application Notes

Cover Photography: Getty Images


TABLE OF CONTENTS



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MEDICAL/BIOLOGICAL

Utilization of CESI Technology for Comprehensive Characterization of BiologicsRajeswari Lakshmanan, PhD, AB Sciex

Monoclonal antibodies (mAbs) form a major class of biologics and

recently biosimilars and biobetters are being added to the grow-

ing inventory of therapeutics. In-depth characterization of mAbs at

various stages of development and manufacturing is essential to

maintain product safety and eff cacy. However, analysis of mAbs is

challenging due to their high molecular weight, the microheteroge-

neity presented by the glycans, and degradative modif cations that

occur during production. Any analytical technique that provides

greater depth of information without a time penalty is an advantage.

A recent advancement to meet this need was the introduction of

CESI–MS. CESI is the integration of capillary electrophoresis (CE)

and electrospray ionization (ESI) in a dynamic process, within the

same device. In this technology, the analytes are separated inside

an open nontapered capillary based on their electrophoretic mobil-

ity, and electrosprayed directly into the MS (2). At operating f ow

rates less than 30 nL/min, very eff cient desolvation and, thus, ion-

ization is achieved.

Though high speed CESI separations reduce analysis time, it also

necessitates the use of high speed MS to preserve the separation

eff ciency. The TripleTOF® 5600+ system (AB Sciex) offers the

necessary high acquisition speed, high resolution, and high mass

accuracy, in both MS and MS-MS modes. CESI performance was

evaluated by analyzing a tryptic digest of trastuzumab using the

SCIEX CESI 8000 - TripleTOF® 5600+ MS platform.

Figure 1: (a) Extracted ion electropherogram of N-terminal peptide with Glu and pyroGlu separated by CESI and (b) MS-MS identif cation of N-terminal peptide with pyroGlu.

4.0e6

3.5e6

3.0e6

2.5e6

2.0e6

1.5e6

1.0e6

5.0e5

0.0e0

6500

6000

5500

5000

4500

4000

3500

3000

2500

2000

1500

1000

500

0

Inte

nsit

yIn

tens

ity

29.5

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_1to5WithChargeSel_2.wiff (sample 1) - Her10, Experiment 10, +TOF MS^2 (100 - 2500) from 35.322 minPrecursor: 932.5 Da

150 200

b2

250 300

*424.2592

*339.1698

*452.2540

b5

*551.3233

b6

b7

*680.3647

b10

b11

b9

*938.4616

350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z

30.0 31.0 32.0 33.0Time (min)

34.0 35.0 36.0 37.036.535.534.533.532.531.530.5

N-terminal Glu

N-terminal pyroGlu

(a)

(b)

b4

*586.3339

y6

*714.3923

*813.4611

y7

y8

*983.5663

y10 *1040.5852

y11

*1097.6123

y12

*1184.6458

y13

*1313.6853

*1314.6921

y9

b3

y14

*1412.7647

y15

Experimental Conditions

Trastuzumab was reduced, alkylated, and digested with trypsin.

After drying, it was resuspended in the leading electrolyte (100

mM ammonium acetate at pH 4) and 50 nL (100 fmol) was

injected into the separation capillary. The background elec-

trolyte used was 10% acetic acid and a separation voltage of

20 kV (normal polarity) was applied for 60 min. Information

dependent acquisition (IDA) was utilized to trigger MS-MS. IDA

parameters were optimized so that the duty cycle of the MS

was matched to the high speed CE separation. Data analysis

was performed using BioPharmaView™ software (AB Sciex,

Massachusetts).

Results and Discussion

Primary Sequence Coverage: 100% primary sequence coverage of

both the light and heavy chains of the antibody were obtained. Pep-

tides ranging from 4 to 63 amino acids in length without any missed

cleavages were detected. Electrophoretic separation is based on the

charge-to-mass ratio of the peptides and is not dependent on rela-

tive hydrophobicity. Thus, small hydrophilic peptides often lost in

the LC void volume, and large hydrophobic peptides, which tend to

be retained on the column, can be identif ed by CESI–MS, resulting

in the high sequence coverage observed.

PTM Characterization: Data from CESI-MS analysis showed the

presence of several PTM hotspots such as N-terminal pyroGlu

formation, methionine oxidation, and asparagine deamidation.

Pyroglutamination leads to loss of a positive charge which re-

sults in the electrophoretic mobility of the modif ed peptide being

lower than the unmodif ed one. This is advantageous since the

modif ed and unmodif ed forms can be separated by CESI–MS

and the MS-MS spectra conf rmed the presence of the pyroGlu-

tamate residue (Figure 1). Oxidative degrada-

tions at Met255 and Met431 and deamidations at

Asn55 and Asn387 in the heavy chain and at Asn30

in the light chain were also identif ed. A typical

identif cation from the CESI–MS data is shown

in Figure 2.

Glycosylation Heterogeneity: Trastuzumab pos-

sesses one N-glycosylation site at Asn300 in

the HC where different glycoforms such as a-

fucosylated or fucosylated glycans can be pres-

ent (3). By using CESI-MS, the G0F, G1F, and

G2F forms of the peptide TKPREEQYNSTYR

were separated well as shown in Figure 3. Fur-

thermore, the identification of G0F, G1F, and

G2F forms of peptide EEQYNSTYR without the

missed cleavage (at the arginine residue in the

peptide TKPREEQYNSTYR) also confirmed the

presence of these glycoforms. In addition, the

a-fucosylated forms of this peptide, such as G0

and G1, were identified, but it has to be further



MEDICAL/BIOLOGICAL

confirmed that the a-fucosylated forms were not generated due

to source fragmentation of fucosylated counterparts.

Conclusions

We have presented CESI–MS, a robust ultra-low f ow and highly

eff cient separation technology in combination with TripleTOF MS,

a high resolution accurate mass measurement system for qualita-

tive analysis of biopharmaceuticals. CESI–MS is attractive for simul-

taneous analysis of primary sequence coverage and glycopeptide

prof ling, without carry-over concerns. The combination of high

Figure 2: MS-MS identif cation of asparagine deamidation with (a) showing unmodif ed peptide and (b) deamidation at Asn55 in the heavy chain of trastuzumab.

Figure 3: Extracted ion electropherograms of peptide TKPREEQYNSTYR with G0F, G1F, and G2F modif cations.

AB Sciex

500 Old Connecticut Path, Framingham, MA 01701

tel. (877) 740-2129, fax (800)343-1346

Website: www.sciex.com/cesi

separation eff ciency and high sensitivity allows the

analysis of all peptides including modif ed and low

abundant species, in addition to conf rming the

amino acid sequence of the antibody.

References

(1) J.M. Busnel, B. Schoenmaker, R. Ramautar, A.

Carrasco-Pancorbo, C. Ratnayake, J. Feitelson, J. Chap-

man, A. Deelder, and O. Mayboroda, Anal. Chem. 82,

9476–9483 (2010).

(2) A. Beck, S. Sanglier-Cianferani, and A. Van Dorsselaer,

Anal. Chem. 84, 4637–4646 (2012).


4.5e4

4.0e4

3.5e4

3.0e4

2.5e4

2.0e4

1.5e4

1.0e4

5.0e3

0.0e0

Inte

nsit

y

3.0e4

2.5e4

2.0e4

1.5e4

1.0e4

5.0e3

0.0e0

Inte

nsit

y

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_NOChargeSel_2.wiff (sample 1) - Her8, Experiment 19, +TOF MS^2 (100 - 2500) from 27.027 min

Precursor: 542.8 Da

Spectrum from 6Dec13Her2ugPerul_100mMLE_250TOF501DA30ions5sec500cps_NOChargeSel_2.wiff (sample 1) - Her8, Experiment 9, +TOF MS^2 (100 - 2500) from 28.180 min

Precursor: 543.3 Da

Unmodifed peptide

Deamidated at Asn55

*136.0803

*171.1181*199.1135

*249.1643

*486.2354*496.2534

*611.2995

*610.2927

*711.3387 (1)

712.3390 (1)

*790.3809 (1)

809.3908 (1)

971.4573

*808.3885 (1)

*277.1595 (1)

*404.7045 (2)

405.2075 (2)

278.1583 (1)

b2

y1

*136.0805

*249.1646

*171.1177

*277.1594

*405.1974

*405.7015

*486.7298

*496.2536

*611.2788 (1)

612.2888 (1)

*712.3253

*722.3113

*810.3828

*809.3766

b2

y1

y4

y5

y6

y7

y4

y5

y6

y8

971.4457y

8

y7

N T P Y

N* T P Y

150 200 250 300 400 500 600 700 800 900 950850750650550450350

m/z

150 200 250 300 400 500 600 700 800 900 950850750650550450350

m/z

(a)

(b)

5.2e5

5.0e5

4.8e5

4.6e5

4.4e5

4.2e5

4.0e5

3.8e5

3.6e5

3.4e5

3.2e5

3.0e5

2.8e5

2.6e5

2.4e5

2.2e5

2.0e5

1.8e5

1.6e5

1.4e5

1.2e5

1.0e5

8.0e4

6.0e4

4.0e4

2.0e4

0.0e0

Inte

nsit

y

24.8 24.9 25.0 25.1

25.10

25.42G0F

25.7

25.95

G1F

G2F

25.2 25.3 25.4 25.5 25.6 25.7Time (min)

25.8 25.9 26.0 26.1 26.2 26.3 26.4 26.5

XIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1039.800+/-0.025DaXIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1093.780+/-0.025DaXIC from 09292013_Tras1mgmL_100mMLE_R7.wiff(sample1)-09292013_Tras1mgmL_100mMLE_R7, Experiment1, +TOF MS (100-2000): 1147.8200+/-0.025Da


MEDICAL/BIOLOGICAL

A new fused-core particle designed specif cally for the sepa-

ration of proteins and monoclonal antibodies with 3.4 µm

particle size and a thin 0.2 µm porous shell demonstrates high

resolution of the heavy and light chain fragments of IgG1.

The focus of many pharmaceutical companies has shifted to larger

biotherapeutic molecules as potential treatments for disease. These

biomolecules present a new set of challenges compared to their

small molecule predecessors when complete characterization is

considered. Variants can be found through different glycans, de-

amidations, enzymatic clipped polypeptides, and so on, not to men-

tion the potential for aggregate formation. Rapid reversed-phase LC

analysis is convenient for its ability to be interfaced to online mass

spectrometry. The HALO Protein C4 bonded phase is ideal for this

application with its optimized design for high recovery and stability

at the high temperatures and low pH required by the analysis.


Column: 100 mm × 2.1 mm Halo Protein C4; gradient: 29–32%

B in 20 min; mobile phase A: 0.5% (v/v) formic acid with 20 mM

ammonium formate; mobile phase B: 45% acetonitrile, 45% iso-

propanol/0.5% (v/v) formic acid with 20 mM ammonium formate;

temperature: 80 °C; f ow rate: 0.4 mL/min; instrument: Shimadzu

LCMS 2020; detection: 280 nm; MS-2020 single quadrupole MS in-

strument using ESI at +4.5 kV capillary voltage, 2 pps scan rate from

500 to 2000 amu m/z. Component masses for these measurements

were determined by deconvolution using MagTran software, v. 1.02,

based on the ZScore algorithm developed by Zhang and Marshall (1).

Results

The addition of a small amount of ammonium formate to formic acid

in the mobile phase improves the peak shapes. An optimized, shallow

gradient allows resolution of the variants of both the heavy and light

chain fragments of IgG1 as shown in Figure 1. The MS spectra can then

be deconvoluted to determine the masses associated with each peak.

Conclusions

HALO Protein C4 demonstrates the low pH and high temperature

stability that is required to analyze reduced and alkylated IgG1 us-

ing mass-spec friendly mobile phase. The use of 80 °C enables

improved peak shape while the high resolution allows complete

analysis of the IgG1 fragments that are present.

References

(1) Z. Zhang and A.G. Marshall, J. Am. Soc. Mass Spectrom. 9, 225 (1998).

High Resolution LC–MS Analysis of Reduced IgG1 Monoclonal Antibody Fragments Using HALO Protein C4Stephanie A. Schuster and Barry E. Boyes, Advanced Materials Technology, Inc.

Figure 1: LC–MS separation of reduced IgG1. Column: 2.1 mm i.d ×100 mm HALO Protein C4; Flow rate: 0.4 mL/min. Mobile phase A: 0.5 % formic acid with 20 mM ammonium formate; mobile phase B: 45% AcN/45% IPA/ 0.5 % formic acid with 20 mM ammonium formate; gradi-ent: 29–32% B in 20 min.; temperature: 80 °C; detection: 280 nm; MS conditions: Shimadzu LCMS-2020, ESI +4.5 kV, 2 pps, 500–2000 m/z.

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MAC-MOD Analytical, Inc. 103 Commons Court, Chadds Ford, PA 19317

tel. (800) 441-7508; fax (610) 358-5993

Website: www.mac-mod.com



MEDICAL/BIOLOGICAL

We have previously shown the ability of a novel stationary phase to sepa-

rate 55 amino acids without derivatization (LCGC 32(3), 211 [2014]).

One of the recurring comments we received has been that our samples

were standards which could potentially behave differently compared to

samples derived from more complex matrices. Here, we show that the

Intrada Amino Acid column separates amino acids without derivatiza-

tion in complex samples equally effective as it does for standard sam-

ples, with very little change in retention time.

This new technology will undoubtedly prove to be a valuable ad-

dition to the industries currently analyzing amino acids, such as

nutritional supplements, clinical testing laboratories and cell culture

monitoring. In this experiment we sought to prove that our previous

results with samples derived from standard amino acids are similar

to those derived from complex sources, further implicating the value

of this novel stationary phase to these industries.

Experimental

First, 0.5 mL of human serum (commercial grade reagent, Cosmo

Bio Co. Ltd) was subject to protein crash with 0.5 mL of 0.4 N HClO4

and centrifuged (6000 rpms, 10 min). Supernatant was f ltered (0.2

µm, Amicon Ultrafree-MC). Next, 5 µL of supernatant was injected

onto Intrada Amino Acid, 50 × 3 mm (length × i.d.). Eluent was ion-

ized using ESI (SIM, positive), and analyzed by LC–MS-2020 (Single

Quad, Shimadzu Corp.). Other conditions are shown in Figure 1.

Intrada Amino Acid Separates Intact Amino Acids in Human SerumPiotr Macech, Robert Puryear, and Itaru Yazawa, Imtakt USA

Imtakt USA1104 NW Overton St., Portland, OR 97209

tel. (888) 456-HPLC, (215) 775-8902, fax (501) 646-3497

Website: www.imtaktusa.com

Figure 2: Comparison of intact amino acids separation in human se-rum (black lines, bottom) and standards (red lines, top) using Intrada Amino Acid column. Separation conditions as in Figure 1.

Figure 1: LC–MS: Separation of intact amino acids in human serum using Intrada Amino Acid.

Trp (m/z 205.0) Glu (m/z 148.0) Arg (m/z 175.1)

5.0 7.5 10.0 (min)2.50.0

Human Serum (commercial reagent)

Intrada Amino Acid, 50 x 3 mmA: ACN/THF/25mM HCOONH4/HCOOH = 9 / 75 / 16 / 0.3B: ACN/100mM HCOONH4 = 20 / 800 %B (0-25 min), 0-17 %B (2.5-6.5 min), 100 %B (6.5-10 min)0.6 mL/.min (6MPa), 35 deg.C, 5 uLESI (SIM, positive)

0.4N HCIO4 (1:1, vol)

0.0 2.5 5.0 7.5

(min)

10.0

%

mixing, centrifugation

Supernatant

0.2µm filtration

Filtrate (Injection Sample)

Arg (m/z 175.1)

Lys (m/z 147.0)

His (m/z 156.0)

(Cys)2 (m/z 241.0)

Asn (m/z 133.0)

Gly (m/z 76.0)

Gln (m/z 147.0)

Ser (m/z 106.1)

Ala (m/z 90.0)

Asp (m/z 134.0)

Thr (m/z 120.1)

Pro (m/z 116.0)

Glu (m/z 148.0)

Val (m/z 118.2)

Leu, lle (m/z 132.0)

Met (m/z 150.0)

Tyr (m/z 182.0)

Phe (m/z 166.0)

Trp (m/z 205.0)


Figure 1 shows separation of intact amino acids from human serum

using Intrada Amino Acid column with a method optimized for LC–MS.

In addition to the separation of these amino acids by LC–MS, critical

isobaric compounds (that is, Leu and Ile) are chromatographically re-

solved. These results are shown here in samples prepared from stan-

dard compounds as well as those found in human serum (Figure 2).

As expected, the results in Figure 1 are comparable with previously

published data for analysis using standards (LCGC 32(3), 211 [2014]).

While we were not able to include this previously published data here, we

show comparisons of retention times for three common amino acids (Fig-

ure 2). These data show that there is very little shift in the retention times

whether the sample is from a standard mixture or from human serum.

We did notice a trend that, while the elution order remains the same,

less polar amino acids which elute early on this column do show a slight

difference in retention times, while strongly retained amino acids remain

virtually unaffected. Specif cally, we found that these less-retained com-

pounds are retained slightly better when coming from human serum. We

are not sure why this is the case, but it is likely that this is an effect of the

complex matrix of the human serum sample. It could be an interruption

of the ionic interaction to a certain extent but this was not examined

further in this work. This could be a potential area for study in the future.

Alternatively, it is also possible that the effect is not signif cant such that it

falls within the expected range of experimental variability.

In the work presented here, we show that there is no change in elu-

tion order or for the most part, retention times of amino acids whether

from a standard mixture or from human serum. Therefore, regardless of

the source of the sample, amino acids are reliably analyzed by LC–MS

with the use of this new novel stationary phase. In summary, the Intrada

Amino Acid column provides excellent and fast separation of amino

acids without the need of pre- or post-derivatization for both real life

samples with complex matrices as well as standards.



MEDICAL/BIOLOGICAL

Separation of Nucleobases Using TSKgel® SuperSW mAb HTP Column in HILIC ModeJustin Steve and Atis Chakrabarti, PhD, Tosoh Bioscience LLC

Hydrophilic interaction liquid chromatography (HILIC) is one of the

fastest growing modes of separation, in which any polar chromato-

graphic surface can be used. Chemically bonded diol coated phas-

es, as found in TSKgel SW size exclusion chromatography (SEC)

columns, demonstrate high polarity and hydrogen bonding proper-

ties and do not contain ionizable groups other than the unreacted

residual silanols, making them appropriate for HILIC mode.

For many years, SEC columns have been used to separate vari-

ous nucleic acid species such as DNA, RNA, and tRNA as well as

their constituent bases, adenine, guanine, thymine, cytosine, and

uracil. In medicine, several primary nucleobases are the basis for

the nucleoside analogues and other synthetic analogs which are

used as anticancer and antiviral agents. Nucleobase modif cations

are the basis of oligonucleotide-based therapeutics, making their

purif cation very important.

The TSKgel SuperSW mAb HTP column is a newly introduced

SEC column designed for the high throughput separation of mono-

clonal antibodies from their high and low molecular mass variants.

TSKgel SuperSW mAb HTP has a diol coating to minimize second-

ary interactions which may occur in SEC separations. This note

demonstrates the benef ts of using a TSKgel SuperSW mAb HTP

column in HILIC mode for the superior resolution of four nucleo-

bases, as opposed to using the column in SEC mode or using a

HILIC column.

Materials and Methods

Instrumentation: Agilent 1100 HPLC system run by Chemstation

(ver B.04.02)

Columns: TSKgel SuperSW mAb HTP, 4 µm,

4.6 mm i.d. × 15 cm

TSKgel Amide-80, 5 µm, 2.0 mm i.d. × 10 cm

Mobile phase: A: acetonitrile (HILIC mode)

B: 15 mmol/L ammonium bicarbonate, pH 7.4

(HILIC mode)

Mobile phase: 100 mmol/L phosphate/100 mmol/L sodium

sulfate, pH 6.7 + 0.05% NaN3 (SEC mode)

Gradient: Isocratic

Flow rate: 0.4 mL/min

Detection: UV @ 280 nm

Injection vol.: 1 μL

Temperature: ambient

Samples: uracil (1.5 mg/mL), adenine (1.5 mg/mL),

cytosine (1.5 mg/mL), cytidine (1.5 mg/mL)

from Sigma Aldrich


Figure 1 illustrates the separation of four nucleobases using the

TSKgel SuperSW mAb HTP column in HILIC mode with 15 mmol/L

ammonium bicarbonate, pH 7.4 as mobile phase B. It is important

to note that the order of elution of the analytes does not correlate

with their molecular mass (as in SEC separations), but instead is

based on their relative hydrophilicity.

Figure 2 illustrates the separation of the four nucleobases on the

TSKgel SuperSW mAb HTP column using conventional SEC con-

ditions. As expected, due to the similarities in molecular masses

between the four compounds, signif cant interference is observed

amongst the peaks of interest, particularly the three pyrimidine de-

rivatives, when separated on the TSKgel SuperSW mAb HTP col-

umn under SEC conditions. The late elution of adenine (relative to

Figure 1: Separation of four nucleobases using TSKgel SuperSW mAb HTP column in HILIC mode at pH 7.4.

Figure 2: Separation of nucleobases using the TSKgel SuperSW mAb HTP column under conventional SEC conditions.



MEDICAL/BIOLOGICAL

the other three compounds) may be attributed to possible interac-

tions between the stationary phase and the derivatized purine com-

pound, leading to a shift towards longer retention time.

In an effort to explore the novelty of the separation of nucleobas-

es using the TSKgel SuperSW mAb HTP column in HILIC mode, the

same separation was carried out using a TSKgel Amide-80 HILIC

column. The use of the TSKgel Amide-80 column yields very poor

separation of the four nucleobases with virtually no retention of any

of the components (Figure 3).

Conclusions

This work illustrates the novelty and utility of the TSKgel SuperSW

mAb HTP column as a diol-functionalized HILIC column for the

high resolution separation of nucleobases on the basis of their rela-

tive hydrophilicity, rather than differences in their relative molecu-

lar mass. As shown, markedly different separation prof les are ob-

served with the use of the TSKgel Amide-80 HILIC column under

identical chromatographic conditions. Additionally, nucleobase sep-

aration using the TSKgel SuperSW mAb HTP under conventional

SEC conditions yielded poor resolution of all components, making

it an ineffective mode of separation for this application. The TSKgel

SuperSW mAb HTP column, while designed for SEC separation of

monoclonal antibodies, is an extremely effective tool in HILIC mode

that should be considered for the fast separation of nucleobases.

Tosoh Bioscience and TSKgel are registered trademarks of Tosoh Corporation.

Tosoh Bioscience LLC

3604 Horizon Drive, Suite 100, King of Prussia, PA 19406

tel. (484) 805-1219, fax (610) 272-3028

Website: www.tosohbioscience.com

Figure 3: Separation of nucleobases using the TSKgel Amide-80 HILIC column.



MEDICAL/BIOLOGICAL

High resolution time-of-f ight mass spectrometry was used

for comprehensive prof ling of tobacco plant extracts.

The f avor and health risks of tobacco products are a direct result of

their chemical composition. Alkaloids, organic acids, amino acids, sac-

charides, terpenes, and volatile aromatics are important contributors to

tobacco leaf quality. It is well known that tobacco quality can vary signif -

cantly depending on where it is harvested. Comprehensive prof ling is

critical for understanding the chemical complexity responsible for aroma

and f avor differentiation of tobacco leaves grown under different envi-

ronmental conditions.

Experimental

Green leaf and cured tobacco extracts were analyzed using the

Pegasus GC-HRT. The analysis workf ow included EI-HRT and CI-

HRT data acquisition to maximize the number of conf dently identi-

f ed metabolites. Instrument parameters are listed below:

GC: Agilent 7890

Column: Rxi-5 Sil MS, 30 m × 0.25 mm × 0.25 µm

Injection: Splitless at 250 °C

Carrier: Helium at 1.0 mL/min, constant f ow

Oven: 50 °C (1 min), 10 °C/min to 300 °C (10 min)

MS: LECO Pegasus GC-HRT

Mass range: 29–510 m/z (CI 45–800)

Acquisition rate: 6 spectra/s

Source temperature: 250 ºC (CI 200 ºC)

Transfer Line: 300 °C

Reagent Gas: Methane


Some of the major components in green leaf tobacco are terpenes, ter-

penoids, and sterols. An expanded analytical ion chromatogram (AIC)

shows some of the plant sterols in the sample including campesterol,

stigmasterol, sitosterol, and α-Amyrin. The extracted ion chromato-

gram (XIC) illustrates the deconvolution power of LECO’s ChromaTOF

HRT software. The tocopherol and cholesterol MS spectral data is easily

separated to provide high quality spectral data as shown by the excellent

library match (LM = 880/1000) and mass accuracy value (-0.98 ppm)

for cholesterol.

Tabular data for a representative set of additional compounds in the

extract are listed in Table I. The quality of spectral data obtained us-

ing the GC-HRT is clearly evident from good library similarity matches

(758–895/1000) and excellent mass accuracy values (Avg. = 0.95

ppm) which aided in library match conf rmation for these analytes.

Conclusion

The Pegasus GC-HRT is an ideal tool for metabolomic prof ling of plant

extracts. Excellent mass accuracy values and high quality spectral data

facilitated characterization of numerous classes of compounds in to-

bacco. For brevity, the CI-HRT data was not shown in this note, but will

be included in a full application note at LECO.com.

Characterization of Tobacco Extracts by Gas Chromatography High Resolution Time of Flight Mass Spectrometry (GC-HRT) David E. Alonso, Jonathan Byer, Jeff Patrick, and Joe Binkley, Life Science and Chemical Analysis Centre, LECO Corporation

LOGO GOES HERE

LECO Corporation3000 Lakeview Avenue, St. Joseph, MI 49085

tel. (269) 985-5496, fax (269) 982-8977

Website: www.leco.com

Figure 1: AIC (B) and XIC (A) of a green leaf tobacco extract. Peak true mass spectrum of cholesterol (C).

Table I: Representative compounds in green leaf tobacco extract

Name Formula SimilarityObserved

Ion m/z

Mass

Accuracy

(ppm)

Benzene, ethyl~ C8H

10895 106.07780 0.95

Benzene, 1-ethyl-2,3-

dimethyl-C

lOH

14 579 134.10893 -0.52

Ethylmethylmaleimide C7H

9NO

2758 139.06272 -0.41

Nicotine (CAS) C10

H14

N2

889 162.11507 -0.48

1,1,5-Trimethyl-1,2-

dihydronaphthaleneC

13H

16889 172.12451 -0.85

Neophytadiene C20

H38

876 278.29627 -1.93

Cembrene C20

H32

803 272.24940 -1.67

Pregnenolone C21

H32

02

826 316.23944 -0.78

Avg.= 0.95 ppm

HOHO

HO

HO

HO

HO

OH

dl-

a-T

oco

ph

ero

l

Ch

ole

stero

l (C

AS)

Are

a (

Ab

un

dan

ce)

XIC (165.0909)

O

1650

1.2e7

1.0e7

8.0e6

6.0e6

4.0e6

2.0e6

0.0e0Time(s)

16601650 1700

m/zLibrary Hit - Library: Wiley9 - Cholesterol (CAS)

50

1000

800

600

400

200

0

Are

a (

Ab

un

dan

ce) 1.2e3

1.0e3

8.0e2

6.0e2

4.0e2

2.0e2

0.0e0

100 150 200 250 300 350 400 450 500

m/z 50 100 150 200 250 300 350 400 450 500

1750 1850AIC

1800

A B

C Peak True

LM = 88/1000

Campesterol

Stigmasterol

Sitosterol

α-Amyrin

C27H46O-0.98 ppm

18

36

43

55

67

81

91 9

5

105

121

133

145

159

173

213 247

255

275

353

368

386

121,1

0111

39,0

2315 43,0

5449

55,0

5439

67,0

5431

71,0

8556

275,2

7292

255,2

1050

247,2

4172

213,1

6363

199,1

4803

173,1

3243

163,1

4806

159,1

1679

133,1

0111

129,0

6981

121,1

0111

121,1

0111

95,0

8561

301,2

8867

353,3

1997

368,3

4333

105,06991

145,1011581,06991



CHIRAL

Chiral separations of multi-component natural product mixtures

comprised of enantiomers, diastereomers, and unrelated mol-

ecules are often diff cult to resolve in a single isocratic method.

Utilizing different chiral stationary phases can often provide the

selectivity needed for a multiple pass isolation approach.

A proprietary compound mixture recently screened consisted of mul-

tiple isomers and structurally similar components. Even though this was

not specif cally an enantiomeric separation, we desired to develop chiral

analytical and preparative methods under SFC mobile phase. Upon ini-

tial screening, distinct differences between RegisPack® and Whelk-O1®

were noted. Major differences in retention times between key peaks of

interest indicated a multi-pass method was needed.

Under analytical SFC conditions the multi-component mixture

resolved somewhat but not suff cient for a preparative separation

(Figure 1).

Due to interfering peaks, isolation of the desired peaks at tr = 5.9

min and tr = 8.8 min would require a multiple chiral stationary phase

Isolation of Structurally Related Components in Natural Product Extracts Using the RegisPack and Whelk-O1 Chiral SelectorsAndrew Geyer and Ted Szczerba, Regis Technologies, Inc.

Regis Technologies, Inc.8210 Austin Ave., Morton Grove, IL 60053

tel. (847) 583-7661, fax (847) 967-1214

Website: www.registech.com

Figure 1: Sample: proprietary. Column: (R,R) Whelk-O1, 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.

Figure 2: Sample: proprietary. Column: RegisPack 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Methanol 80:20. Flow rate: 4.0 mL/min.

approach. Upon rescreening with the amylose based RegisPack chiral

stationary phase we found enhanced resolution and a much higher re-

tention of the minor desired component now at tr = 14 min (Figure 2).

Using a multiple pass approach, the minor desired peak was iso-

lated in high eff ciency using RegisPack with its purity conf rmed

on Whelk-O1 (Figure 3). Reprocessing of the balance of the multi-

component mixture using (R,R) Whelk-O1 allowed for the clean

isolation of the remaining desired major component at tr = 8.8 min.

Conclusion

Although SFC systems are optimally designed for collecting rela-

tively pure binary mixtures, creative applications of multiple chiral

stationary phases can often allow one to isolate complex mixture

components that would otherwise be isolated in poor yield and pu-

rity. This application to a crude botanical extract is a typical example

of how a comprehensive chiral screening layout can help design an

optimal preparative separation process.

Figure 3: Sample: proprietary. Column: (R,R) Whelk-O1, 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.

Figure 4: Sample: proprietary. Column: (R,R) Whelk-O1 25 cm × 4.6 mm, 5 µm. Mobile phase: CO2/Ethanol 80:20. Flow rate: 4.0 mL/min.



ENVIRONMENTAL

The Problem with PPCPs

Pharmaceutical and personal care products (PPCPs) are products

used for personal health or cosmetic reasons. This category includes

a broad group of chemical substances such as human and veterinary

medicines and cosmetics. The presence of PPCPs in environmental

and potable water is a widespread concern due to the potentially harm-

ful environmental effects. Evidence suggests PPCPs are linked to some

ecological damage such as the delayed development in f sh (1).

To ensure the safety of water, PPCP concentrations are stringently

monitored by environmental regulatory bodies, including the United

States Environmental Protection Agency (US EPA) (2). Detection

of PPCPs is traditionally a complicated process due to the range of

substances potentially present. Here we explore a simple, more con-

venient method than traditional solid phase extraction (SPE) based

methods for highly sensitive PPCP detection, using triple quadrupole

liquid chromatography–mass spectrometry (LC–MS-MS).

Detecting PPCPs

Conventional methods of PPCP detection in clean water have

followed the defined EPA 1694 "template" for analysis which

requires the pre-concentration of large volume water samples

and tedious solid phase extraction cleanup, followed by liq-

uid chromatography–mass spectrometry analysis in order to

achieve the low ng/L (ppt) level detection necessary to comply

with regulations (3).

Keeping Water Safe: Detecting Pharmaceutical and Personal Care Products in Water Using Liquid Chromatography–Mass SpectrometryJoe Anacleto, Zicheng Yang, Helen (Qingyu) Sun, and Kefei Wang, Bruker Daltonics

Figure 1: PPCPs in environmental water and nearby soil is a widespread concern.

Figure 2: Selected MRM chromatograms for PPCPs at 2 ppt.

Trimethoprim; (+) 291.1 > 230.0Hydroxy Atrazine; (+) 198.0 > 156.0

Thiabendazole; (+) 202.0 > 175.0Caffeine; (+) 195.1 > 138.0

Sildenafil; (+) 355.0 > 143.9Sulfamethoxazole; (+) 254.1 > 156.0

Cyanazine; (+) 241.0 > 213.9Hexazinone; (+) 253.0 > 171.0Dapoxetine; (+) 306.0 > 156.9

Bentazone; (-) 239.0 > 132.1Carbamazepine; (+) 237.1 > 194.1

Atrazine; (+) 216.0 > 173.9Alpazolam; (+) 309.0 > 280.8Prometryn; (+) 242.0 > 157.9

MCPA; (-) 199.2 > 141.1Metolachlor; (+) 284.0 > 176.0

1

0

10

20

30

40

2 3 4 5 6 7 8

Minutes

kCps

Tables Ia & Ib: Instrumentation set up for analysis of PPCPs

in clean water

Mass Spectrometer Parameters (EVOQ Elite)

HV 4000 V

Cone gas f ow 15 units

Cone gas temperature 300 °C

Heated probe gas f ow 40 units

Heated probe temperature 450 °C

Nebulizer gas f ow 50 units

Exhaust gas On

Q2 pressure 1.5 mTorr (Argon)

Chromatography Parameters (Advance UHPLC)

Trap columnYMC-Pack ODS-AQ, 3 µm, 35 mm

× 2.0 mm I.D.

Column temperature 40 °C

Injection volume 400 µL

Flow rate 400 µL/min

Solvent A2 mM ammonium formate, 0.1%

FA in water

Solvent B2 mM ammonium formate, 0.1%

FA in MeOH

Solvent C2 mM ammonium formate, 0.1%

FA in water

Gradient conditions 0.0 min, 10% B

0.2 min, 10% B

0.8 min, 25% B

8.0 min, 95% B

9.0 min, 95% B

9.1 min, 10% B

12.0 min, 10% B



ENVIRONMENTAL

Bruker has explored how LC–MS-MS can be employed specif -

cally for the analysis of PPCPs in clean water. PPCPs were detected

at 1–2 ppt with a linear response up to 200 or 500 ppt. Excellent

system robustness was obtained throughout the extended method

development and sample analysis period.

Case Study: Using LC–MS-MS

to Analyze PPCPs in Clean Water

The study was carried out using ultrahigh-performance liquid chro-

matography (UHPLC) with an integrated on-line extraction (OLE)

option coupled to a triple quadrupole mass spectrometer. The OLE

module enables convenient method-driven on-line sample cleanup

or sample pre-concentration.

Several water samples were analyzed for a range of PPCP spe-

cies, including tap water samples along with bottles and creek water.

Samples were analyzed targeting a wide range of PPCP species

representing compounds displaying varied properties and concen-

trations. Tables Ia and Ib illustrate the Advance UHPLC and EVOQ

instrumentation set up respectively.

All of the PPCPs were detected at 2 ppt or better with the injec-

tion of 0.4 mL samples with a linear response range up to 200 or

500 ppt. The fast polarity switch can analyze positive and nega-

tive PPCPs in the same analytical segment with excellent linear

response for both polarities (Figures 2 and 3). The results for the

analysis of tap, creek, and bottled waters are shown in Table II.

Conclusion

The Bruker Advance UHPLC with OLE coupled to EVOQ LC–MS-

MS detected PPCP samples at 2 ppt or better within 0.4 mL sam-

ples. Excellent linearity, sensitivity, and robustness were achieved

throughout. The technique presents a more convenient and simpler

approach to PPCP analysis than traditional SPE-based methods.

References

(1) K. Hirsch, "Pharmaceuticals and Personal Care Products," (2013). Available

at: http://serc.carleton.edu/NAGTWorkshops/health/case_studies/pharma-

ceutical.html.

(2) US EPA, "PPCPs Basic Information," (2010). Available at: http://www.epa.

gov/ppcp/basic2.html.

(3) US EPA, "EPA Method 1694: Pharmaceuticals and Personal Care Products

in Water, Soil, Sediment, and Bio-

solids by HPLC/MS/MS," (2007).

Bruker Daltonics Inc.40 Manning Road, Billerica, MA 01821

tel. (978)663-3660, fax (978) 667-5993

Website: www.bruker.com

Figure 3: Selected calibration curves.

100 200Amount (xxx)

400300100

5

0 0

1

2

3

4

5

10

15

20

11 13 11113 1 1 1Replicates Replicates1 1

11 13 11112 1 1 1Replicates Replicates1 1

M M

200Amount (xxx)

Peak S

ize

Peak S

ize

400300

100 200Amount (xxx)

40030050

5.0

0.0

2.5

0

5

10

15

20

7.5

10.0

12.5M M

100Amount (xxx)

Peak S

ize

Peak S

ize

150

BentazoneCurve Fit Linear, 1/0X2Resp. Fact. RSD: 10.46%. CoeII. Det. (r2):0.998783y = +6.7794e+4x + 1.7995e+4

DepoxetineCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 11.45%. CoeII. Det. (r2):0.998810y = +4.9513e+4x + -1.9343e+4

BentazoneCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 7.925%. CoeII. Det. (r2):0.996309y = +4.2244e+4x -940.3596

DepoxetineCurve Fit Linear, Ignore, 1/nX2Resp. Fact. RSD: 6.052%. CoeII. Det. (r2):0.999951y = +1.0610e+4x + -2882.1665

Table II: Test results for selected PPCPs in real water samples

Compound NameTap Water

1

Tap Water

2

Creek

Water

Bottle

Water

Trimethoprim <2 <2 5 <2

Hydroxyatrazine 4 <2 7 <2

Thiabendazole ND <2 <2 <2

Ciproxacin ND ND ND ND

Caffeine ND <2 <2 10

Sildenaf l ND ND ND <2

Sulfamethoxazole <2 <2 ND <2

Cyanazine ND ND ND <2

Simazine 3 <2 5 ND

Metribuzin ND ND ND ND

Hexazinone 17 3 3 ND

Dapoxetine ND ND ND ND

Bentazone ND ND ND ND

Ametryn ND ND <2 ND

Carboxine ND ND ND ND

Carbamazepine <2 <2 <2 ND

Atrazine <2 ND ND ND

Alpazolam ND ND ND ND

Diuron 9 <2 6.2 ND

Prometryn ND ND ND <2

2,4-D 9 <2 13 <2

MCPA <2 <2 <2 ND

Mecoprop <2 <2 11 2

Metolachlor 22 <2 <2 <2

Pyriproxifen ND <2 ND <2



ENVIRONMENTAL

This application describes a solid phase extraction (SPE) method

for EPA method 8141B analytes, including organophosphorus

compounds, and triazine herbicides in water. Target analytes are

extracted onto Styre Screen® HL DVB polymeric sorbent from water

samples at neutral pH. Proper sample pH is critical for good ana-

lyte recoveries as organophosphorus esters hydrolyze under acidic

or basic conditions. The retained analytes are eluted with acetone

and dichloromethane (DCM), the eluate is then dried, concentrated,

and exchanged into n-hexane or 1:1 acetone:n-hexane if a loss of

polar analytes occurs.

SPE Procedure

a) Adjust 1 L (or less) water sample to pH 7.

b) Connect sample transfer tubes to the top of the SPE cartridges

(ECHLD156) and attach the cartridges to a SPE manifold.

c) Wash the SPE cartridges with 3 × 5 mL DCM, condition with

2 × 5 mL methanol, and equilibrate with 2 × 5 mL DI water.

d) Insert the stainless steel ends of the transfer tubes into the

sample containers, and draw the entire sample through the SPE

cartridge at a fast drop-wise fashion (about 15 mL/min).

e) Remove the transfer tubes from the SPE cartridges, and dry the

cartridges under full vacuum for 10 min.

f) Insert collection vials into the manifold for eluate collection.

g) Rinse the sample bottles with 5 mL acetone and, using the

transfer tubes, slowly draw the rinsates through the SPE car-

tridges. Repeat with 10 mL DCM.

h) Remove the transfer tubes from the SPE cartridges. Continue

the elution with 5 mL DCM.

i) Dry the eluates with 10–15 g anhydrous sodium sulfate

(ECSS25K) held in 15 mL reservoirs (RFV1F15P) or glass funnels

with glass wool.

j) Rinse the collection vials with 2 × 5 mL DCM, pass the rinses

through the sodium sulfate and collect.

k) Concentrate the dried eluates to about 0.5 mL under a gentle

stream of nitrogen at 40 ºC.

Solid Phase Extraction of Organophosphorus Pesticidesand Triazine Herbicides in Water Using a New Polymeric Sorbent Xiaoyan Wang, UCT, LLC

UCT, LLC 2731 Bartram Road, Bristol, PA 19007

tel. (800) 385-3153; Email: [email protected]

Website: www.unitedchem.com

Table I: SPE materials

ECHLD156Styre Screen® HL DVB

500 mg / 6 mL cartridge

ECSS25K25 kg sodium sulfate, anhydrous,

ACS grade, granular 60 mesh

RFV1F15P 15 mL reservoirs with 1 frit

l) Rinse the surface of the eluate containers with 3 mL n-hexane;

continue concentrating to 2 mL.

m) Adjust the f nal volume to 2 mL with n-hexane. Add internal-

standards. The samples are ready for GC analysis.

Instrumental

GC–MS: Agilent 6890N GC with 5975C MSD Injector: 250 ºC

Injection Vol.: 2 μL

GC column: Restek Rxi®-5sil MS 30 m × 0.25 mm, 0.25 µm,

integrated with 10-m guard column

Carrier gas: Helium at a constant f ow of 1.2 mL/min

Oven: Initial temperature at 60 ºC, hold for 1 min; ramp at 10 ºC/min

to 300 ºC, hold for 2 min

Tune: dftpp.u

Full Scan: 45–450 amu

Results

Conclusion

Excellent recoveries ranging from 85.5% to 120% and relative stan-

dard deviations of less than 7.3% were obtained using UCT’s new

Styre Screen® HL DVB polymeric sorbent (ECHLD156) for the ex-

traction of EPA 8141B analytes in water.

Table II: Accuracy and precision data

Analyte Recovery % RSD % (n+5)

Thionazin 100.9 1.8

Sulfotep 96.2 0.9

Phorate 93.0 1.2

Dimethoate 108.7 7.3

Simazine 104.2 2.1

Atrazine 101.5 1.2

Disulfoton 85.5 1.7

Methyl parathion 112.4 1.8

Malathion 110.7 1.3

Parathion 106.8 1.3

Ethion 107.8 0.7

Famphur 120.0 1.8



FOOD & BEVERAGE

Determination of Phenolic Compounds in Virgin Olive Oil Using Comprehensive 2D-LCSonja Krieger and Sonja Schneider, Agilent Technologies Inc.

This application note demonstrates how comprehensive 2D-

LC can be used to resolve the complex mixture of hydrophilic

phenols found in virgin olive oil and investigates differences

in the phenol composition of several olive oils.

Virgin olive oil is associated with the health and nutritional benef ts

of the Mediterranean diet. In this respect, the presence of antioxi-

dants, which are represented by hydrophilic phenols among others,

plays an important role. Hydrophilic phenols contained in virgin ol-

ive oil include phenolic acids and alcohols, f avonoids, secoiridoids,

and lignans (1,2).

One-dimensional liquid chromatography is not able to resolve

completely the complex mixture of hydrophilic phenols present in

virgin olive oil (3). Due to its high separation capability, comprehen-

sive 2D-LC can be deployed to improve the separation.


Comprehensive 2D-LC analysis was achieved with the Agilent 1290

Inf nity 2D-LC solution. The f rst dimension separation used an

Agilent ZORBAX RRHD Eclipse Plus Phenyl-Hexyl column (2.1 ×

150 mm, 1.8 µm) with a gradient of water and methanol, each with

0.1% formic acid, at a f ow rate of 0.05 mL/min. In the second di-

mension, an Agilent ZORBAX RRHD Eclipse Plus C18 column (3.0

× 50 mm, 1.8 µm) was used with shifted gradients of water and

acetonitrile, each with 0.1% formic acid, at a f ow rate of 3.0 mL/min.

Modulation was realized using the Agilent 2-position/4-port duo-valve,

equipped with two 60 µL loops and with a modulation time of 30 s.

Detection was performed at 260 nm and by ESI-TOF-MS in negative

ionization mode. Preparation of olive oil samples was carried out ac-

cording to the protocol from the International Olive Council (4).

Results

Four different olive oil samples with high phenol content (3) purchased

from Italian olive oil farms were analyzed by comprehensive 2D-LC. Fig-

ure 1 (top) exemplarily shows the 2D-LC chromatogram of one olive oil.

MS detection showed that the main hydrophilic phenols present

in the analyzed olive oils are aglycons of oleuropein, ligstroside, and

their derivatives. Further, elenolic acid, luteolin, apigenin, hydroxy-

tyrosol, and hydroxytyrosol acetate were identif ed in all olive oils

analyzed. Compared to the olive oils A–C (from the same farm),

olive oil D (from another farm) showed higher percent responses of

the f avonoids apigenin and luteolin (Figure 1, bottom).

Conclusions

The Agilent 1290 Inf nity 2D-LC solution can be used to improve sig-

nif cantly the separation of hydrophilic phenols contained in virgin

olive oil. This enables the investigation of differences between the

compositions of hydrophilic phenols in olive oils.

References

(1) M. El Riachy et al., Eur. J. Lipid Sci. Technol. 113, 678–691 (2011).

(2) Y. Ouni et al., Food Chemistry, 127, 1263–1267 (2011).

(3) S. Schneider, “Quality Analysis of Virgin Olive Oils – Part 6,” Agilent

Application Note, publication number 5991-3801EN, (2014).

(4) “Determination of biophenols in olive oils by HPLC,” International Olive

Council: COI/T.20/DOC. 29, (2009).

Agilent Technologies Inc.5301 Stevens Creek Blvd., Santa Clara, CA 95051

Website: www.agilent.com

Figure 1: Two-dimensional-LC chromatogram of an olive oil at 260 nm (top) and visualization of differences between the olive oils analyzed (bot-tom); blue circles indicate higher percent responses and white circles lower percent responses of substances detected, areas indicate differences.

2.8

02.6

024.0

22.0

20.0

18.0

16.0

14.0

12.0

10.0

22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0 42.0 44.0 46.0 48.0 50.0 52.0 54.0 56.0 58.0 60.0 62.0 64.0 66.0 68.0 70.0 72.0 74.0

Rete

nti

on

Tim

e II (S

ec)

Retention Time I (min)

Apigenin

Luteolin

ApigeninApigenin

Apigenin

Luteolin

Luteolin

Luteolin

25.00

20.00

15.00

10.00

5.00

0.00

RT

II

(se

c)R

T I

I (s

ec)

RT

II

(se

c)R

T I

I (s

ec)

20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Olive oil A

25.00

20.00

15.00

10.00

5.00

0.0020.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Olive oil B

25.00

20.00

15.00

10.00

5.00

0.0020.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Olive oil C

25.00

20.00

15.00

10.00

5.00

0.0020.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00

Olive oil D

RT I (min)

RT I (min)

RT I (min)

RT I (min)



FOOD & BEVERAGE

FMS, Inc.

580 Pleasant Street, Watertown, MA 02472

tel. (617) 393-2396, fax (617) 393-0194

Website: www.fmsenvironmental.com

The dioxin family consists of 210 compounds, of which 17 contain

the 2,3,7,8 pattern of chlorination. These 2,3,7,8 containing com-

pounds are of extreme human health concern due to their high level

of toxicity. Similarly, 12 of the 209 polychlorinated biphenyls have

also been identif ed as human toxins. For this reason, the U.S. FDA

and EU have established strict regulations for the monitoring of food

products for human consumption, in particular edible oils.

Manual extractions of oils can be a time consuming procedure of-

ten delaying lab turnaround times. By automating the process with

the PowerPrep®, food oil samples can be reliably processed with

routine 24 h turnaround times.

Instrumentation

• FMS, Inc. PowerPrep®

• FMS, Inc. SuperVap® Concentrator

• Thermo Trace Ultra GC with DFS HRMS

• Restek Dioxin 2, 60 m GC column

Procedure

Sample Prep

• Various oil matrices obtained (lard, olive oil, corn oil, cod oil, red

palm oil, unreå ned pumpkin oil, unreå ned vegetable oil)

• Aliquots of 5 g samples are spiked with 13C labeled surrogate stan-

dards

PowerPrep

• Automated Multi Column Sample Cleanup

SuperVap

• Automatic Direct-to-Vial Concentration

Conclusions

Analysis of the six matrices processed yielded acceptable recoveries for

all analytes with standard deviations below 20%. Analysis of an n-hex-

ane blank sample resulted in no detectable target analytes measured

within the calibration range of each respective compound.

With a total processing time of less than 2.5 h, the FMS PowerPrep®

and SuperVap® Concentrator delivers an eff cient, totally automated

sample prep process for edible oils.

The Analysis of Chlorinated Dioxins, Difurans, and Polychlorinated Biphenyls in Edible Oils FMS, Inc.

Table I: Mean recoveries and deviations for labeled compounds; concentration of blank

Analyte Mean Dev Blk Conc.

2,3,7,8-TCDD 78 8.6 <.1 pg/g

1,2,3,7,8-PeCDD 81 11.6 <.5 pg/g

1,2,3,4,7,8-HxCDD 81 11.3 <.5 pg/g

1,2,3,6,7,8-HxCDD 77 9.4 <.5 pg/g

1,2,3,7,8,9-HxCDD NA NA <.5 pg/g

1,2,3,4,6,7,8-HpCDD 75 7.1 <.5 pg/g

OCDD 70 3.6 <1 pg/g

2,3,7,8-TCDF 70 8.5 <.1 pg/g

1,2,3,7,8-PeCDF 83 13.5 <.5 pg/g

2,3,4,7,8-PeCDF 81 10.7 <.5 pg/g

1,2,3,4,7,8-HxCDF 70 7.1 <.5 pg/g

1,2,3,6,7,8-HxCDF 62 3.6 <.5 pg/g

1,2,3,7,8,9-HxCDF 66 6.9 <.5 pg/g

2,3,4,6,7,8-HxCDF 71 10.0 <.5 pg/g

1,2,3,4,6,7,8-HpCDF 73 5.0 <.5 pg/g

1,2,3,4,7,8,9-HpCDF 85.3 9.0 <.5 pg/g

OCDF NA NA <1 pg/g

PCB-77 73 14.9 <.5 pg/g

PCB-81 64 11.0 <.5 pg/g

PCB-105 75 15.2 <.5 pg/g

PCB-114 73 11.4 <.5 pg/g

PCB-118 73 8.5 <.5 pg/g

PCB-123 72 8.0 <.5 pg/g

PCB-126 88 19.7 <.5 pg/g

PCB-156 63 7.4 <.5 pg/g

PCB-157 53 8.7 <.5 pg/g

PCB-167 63 6.1< <.5 pg/g

PCB-169 75 10.4 <.5 pg/g

PCB-170 79 9.4 <.5 pg/g

PCB-180 77 14.2 <.5 pg/g

PCB-189 80 9.8 <.5 pg/g



FOOD & BEVERAGE

Modif ed starches are important materials used in many applications

including foodstuffs. The starches are modif ed by a number of

methods — both physical and chemical — to tailor the properties to

the required application. Most commonly the starches are modif ed

to give a particular texture to a f nished foodstuff; for example, to give

extra thickening in puddings.

In this application note we show how two modified starch samples

with essentially the same molecular size in solution can be easily

differentiated and characterized by triple detection size-exclusion

chromatography (TD-SEC).

Triple Detection SEC

In the advanced technique of TD-SEC, the sample, after separation on

the chromatography column, is passed though a series of detectors

to provide a complete analysis of the molecules: The low angle light

scattering detector (LALS) provides a direct measure of the molecular

weight; the refractive index (RI) detector measures the concentration;

and the differential viscometer measures the intrinsic viscosity (IV).

From the measured IV and molecular weight (MW) values a Mark-

Houwink (M-H) plot showing structural changes can be made.

Instrumentation and Conditions

SEC system comprising the Viscotek GPCmax (degasser, pump,

autosampler) with the Viscotek TDA detector equipped with the

following detectors: Low angle light scattering; differential viscometer;

RI. The data were all calculated using OmniSEC software.

Discussion

The triple chromatogram of one of the modif ed starch samples

is shown in Figure 1. The signal-to-noise on all three detectors is

excellent, which ensures the quality of the calculated data. The data

are calculated directly from the chromatograms by the OmniSEC

software and the results for both samples are shown in Table I. Note

that the hydrodynamic radius (RH) of both samples is within 0.2 nm.

This means that by traditional GPC/SEC techniques the molecular

weights based on retention volumes would be the same. However,

TD-SEC clearly shows the weight average molecular weight of sample

A is only 60% of sample B. We can also see that the viscosity of A,

despite the lower molecular weight, is higher than B.

By looking at the structure plot (M-H plot, Figure 2) of both modified

samples (with a dextran T70 sample as reference), it is clear that the two

modified starches have very different molecular structures. Sample B

has a much more compact structure than sample A; shown by the fact it

appears lower on the M-H plot. This means that despite higher molecular

weight the molecules in sample B are denser — because of the different

modification — resulting in a lower intrinsic viscosity. The dextran T70

material is shown for reference. It indicates, as expected, that modified

starches have a much more compact structure than dextran.

Conclusions

The Viscotek triple detection system provides a convenient and rapid

way to characterize starches and modif ed starches. The instrument

allows determination of molecular weight and molecular size in a single

run using normal conditions and sample concentrations. The IV and

size data allow differentiation between molecules of differing structures.

The technique is equally applicable to other polysaccharides and all

other synthetic or natural polymers such as proteins and DNA.

Structural Differences in Modif ed StarchesMalvern Instruments Ltd.

Malvern Instruments Ltd.Enigma Business Park, Grovewood Road, Malvern, UK

tel. +44 (0) 1684 892456

E-mail: [email protected]

Website: www.malvern.com

1200

Dete

cto

r si

gn

al (m

V) 1000

800

600

400

200

-2002 6 10 14 18 22

Retention volume (mL)

RIViscometerLALS

0

-0.2

-0.5

-0.7

-0.9

-1.1

-1.3

-1.54.5 5.0 5.5 6.0 6.5

Log molecular weightLo

g in

trin

sic

vis

cosi

ty

DextranModifed starch AModifed starch B

Figure 1: Triple chromatogram of a modif ed starch sample.

Figure 2: Mark-Houwink (structure) plot.

Table I: Weight average molecular weight, number average

molecular weight, intrinsic viscosity, and hydrodynamic

radius data

Sample Mw(D) Mn(D) IV(dL/g) RH(nm)

Modif ed starch A 241.780 123.780 0.117 7.2

Modif ed starch B 399.020 169.620 0.081 7.4



FOOD & BEVERAGE

Two species of spearmint oil (mentha spicata and mentha gracilis)

are cultivated in the United States. In 2008, 1.09 million kilograms

of spearmint oil were produced in the US (1). Approximatley, 45%

of mint oil produced in the US is used to f avor chewing gum. One

55-gallon drum of mint oil can ý avor 5,200,000 sticks of gum or

400,000 tubes of toothpaste (2).

Spearmint oil is stored in glands on the underside of leaves. Mature

mint plants are cut and left to dry before being chopped and transferred

to a distillery. Pressurized steam vaporizes the mint oil, which passes

through a condenser to be collected as a liquid. A separator takes mint

oil from the liquid and transfers it into drums that are placed in a tem-

perature-controlled warehouse. Essential oil companies test samples at

this point to decide whether to purchase the oil. Gas chromatography

is commonly used to assess oil quality. Oils containing impurities may

undergo rectif cation, a re-distillation step used to purify the oil.

Volatile sulfur compounds impart undesirable odors to essential

oils and have extremely low olfactory thresholds. Essential oil com-

panies require a rapid screening technique to detect and quantify

volatile sulfur compounds.


Instrumentation used in this study was an Agilent 7890A GC equipped

with an OI Analytical 5380 Pulsed Flame Photometric Detector. Two

different samples of neat spearmint oil were tested. The identity of

sulfur compounds in the sample was unknown, only suspected.

Results

The PFPD provides two independent channels of data. One chan-

nel provides a carbon chromatogram and the second channel a

sulfur chromatogram. The PFPD carbon channel chromatograms

contained 65 peaks which were compared to the client’s current

FID chromatograms. The percent area report obtained for carbon

from the PFPD closely matched the expected carbon percentages

from the FID detector for hydrocarbon peaks.

The PFPD sulfur chromatograms contained nine peaks. Five

peaks were conå rmed as sulfur peaks in the spearmint oil using the

integration time gate function of the PFPD and WinPulse software.

The percent total sulfur of the smallest peak that was detected and

conf rmed was .00206%.

Conclusions

The study demonstrated that low-level sulfur compounds can be

detected and isolated in spearmint oil using an OI Analytical 5380

PFPD detector, dual integration time gates, and comparative carbon

peak matching with FID chromatograms.

References

(1) V.D. Zheljakov, C.L. Cantrell, T. Astatkie, and M.W. Ebelhar, “Productivity,

Oil Content, and Composition of Two Spearmint Species in Mississippi,”

Agronomy Journal, Vol. 102, Issue 1 (2010).

(2) Mint Industry Research Council, www.usmintindustry.org.

Detection of Low-Level Sulfur Compounds in Spearmint Oil Using the Pulsed Flame Photometric Detector (PFPD) Gary Engelhart and Cynthia Elmore, OI Analytical

OI AnalyticalP.O. Box 9010, College Station, TX 77842

tel. (800) 653-1711 or (979) 690-1711, fax (979) 690-0440

Website: www.oico.com

Figure 1: PFPD carbon channel chromatogram of spearmint oil show-ing 65 peaks labeled with retention times.

Figure 2: PFPD sulfur channel chromatogram of spearmint oil show-ing nine peaks labeled with retention times.



FOOD & BEVERAGE

HILIC with Increased Sensitivity for the Analysis of SugarsMelissa Turcotte* and Satoko Sakai†, *Showa Denko America and †Showa Denko K.K.

Hydrophilic interaction chromatography (HILIC) provides an al-

ternative approach for the separation of highly polar substances.

Using a high polarity packing material, separation is based on the

hydrophilic interactions of the analyte on the stationary phase sur-

face. Shodex Asahipak NH2P series is packed with a durable poly-

mer based packing material modiå ed with chemically stable amino

functional groups. Shodex Asahipak NH2P-40 3E column is suit-

able for saccharide analysis, has demonstrated the separation of

sugars at low concentration, and has achieved 2–3 times improved

detection sensitivity even with RI detection.


The separation was carried out by Shodex Asahipak NH2P-40 3E

(3.0 mm i.d. × 250 mm, 4 μm), a polymer-based amino HILIC

column. The separation is compared to the same analysis using

Shodex Asahipak NH2P-50 4E (4.6 mm i.d. × 250 mm, 5 μm).

Column temperature was set at 30 °C and f ow rate was 0.35 mL/

min for the NH2P-40 3E analysis and 1.0 mL/min for the NH2P-50

4E analysis. Eluent conditions are 75% acetonitrile in water. Injec-

tion volume of 10 μL of 0.2% each sugar used for each experiment.

HPLC system was coupled with RI detector.

Results

The saccharides, arabinose, mannose, glucose, sucrose, and lac-

tose, were analyzed successfully by HPLC with RI detection with

NH2P-40 3E and NH2P-50 4E (Figure 1). Shodex NH2P-40 3E col-

umn has smaller dimensions of 3.0 mm i.d. × 250 mm length with

a smaller particle size of 4 µm. The decrease in column dimensions

requires a decrease f ow rate of 0.35 mL/min. The decrease in par-

ticle size allows for an increase in the number of theoretical plates

allowing for increased sensitivity of analytes. Of note, the NH2P-40

3E analysis uses one third the amount of eluent and provides up to

three times improved detection sensitivity.

Conclusions

Shodex Asahipak NH2P-40 3E, a polymer-based hydrophilic interac-

tion (HILIC) chromatography column suitable for saccharide analysis,

has demonstrated the separation of these sugars at low concentration

and achieved 2–3 times improved detection sensitivity even with RI

detection. The presented method using

NH2P-40 3E provides an eff cient analy-

sis of saccharides that can be detected

in small quantities while saving the

amount of eluent used per analysis.

Shodex™/Showa Denko America, Inc.

420 Lexington Avenue Suite 2335A, New York, NY, 10170

tel. (212) 370-0033 x109 , fax (212) 370-4566

Website: www.shodex.net

TM

Figure 1: Increased sensitivity using NH2P-40 3E for the analysis of sugars. Column: Shodex NH2P-40 3E (left) and NH2P-50 4E (right); column

temperature: 30 °C; injection volume: 10 μL; eluent: CH3CN/H2O = 75/25; f ow rate: 0.35 mL/min and 1.0 mL/min, respectively; detector: Shodex RI.



FOOD & BEVERAGE

QuEChERS is a quick-easy-cheap-effective-rugged-safe extraction

method developed in 2003 for the extraction of pesticide residues

in agricultural commodities (1,2). Modif cations to the method have

expanded the scope to include many additional matrices and target

analytes such as veterinary drug residues.

With the amounts of samples being required for residue analysis

continually increasing, Teledyne Tekmar has developed the AutoMate-

Q40. This revolutionary system is designed to automate the QuEChERS

extraction worký ow, allowing laboratories to be more efå cient and timely

in meeting their customer requirements for fast and reliable results.

The intent of this application is to evaluate the performance of the

AutoMate-Q40 by monitoring veterinary drug residues in bovine liver.

The target compounds (sulfonamides) will be analyzed using LC–MS-MS.

Extraction and Clean-Up

Figure 1 shows the f ow chart for the QuEChERS extraction proce-

dure for bovine liver using the AutoMate-Q40.


A precision and accuracy study was performed on the bovine liver

samples using the AutoMate-Q40. A 2.0 µg/mL stock solution was

used to fortify the liver samples. The AutoMate-Q40 spiked the sam-

ples with 50.0, 100.0, and 250.0 µL of the standard to produce 50.0,

100.0, and 250.0 ng/g check samples. These QC samples were

quantitated against a corresponding matrix matched calibration.

Table I shows that when using the AutoMate-Q40 to extract veteri-

nary drug residues from bovine liver samples, it exhibits recoveries

ranging from 74.2% to 103.6%. By using the AutoMate-Q40 for the

QuEChERS extraction, it showed great precision ranging from 1.2%

to 9.4% RSD for the spiked QC samples.

Conclusion

This study demonstrates the feasibility of automating the QuEChERS

extraction method using the AutoMate-Q40. The extraction process

is faster, more reliable, and easier. This enables time and labor sav-

ings, while improving consistency and repeatability of the extraction. As

shown above in Table I combined veterinary residues spikes recovered

at 89.5% with an average RSD of 4.5%. These numbers indicate su-

perb precision and accuracy thus validating the performance of the

AutoMate-Q40 and its use as an excellent analytical tool.

References

(1) European Committee for Standardization/Technical Committee CEN/TC275,

“Foods of plant origin: Determination of pesticide residues using GC–MS

and/or LC–MS-MS following acetonitrile extraction/partitioning and cleanup

by dispersive SPE QuEChERS-method” (2008).

(2) AOAC Official Method 2007.07 Pesticide Residues in Food by Acetonitrile

Extraction and Partitioning with Magnesium Sulfate. Gas Chromatography–

Mass Spectrometry and Liquid Chromatography–Tandem Mass Spectrom-

etry, First Action (2007).

Veterinary Drug Residue Analysis Using the AutoMate-Q40:An Automated Solution to QuEChERS Tyler Trent, Teledyne Tekmar

Analyze by LC-MS/MS

Dilute sample extract 10x into Mobile Phase A

Transfer 4.0 ml to fnal extract vial

Shake for 1 minute, centrifuge for 5 minutes

Transfer 8.0 mL to dSPE 15mL centrifuge tube

Centrifuge for 5 minutes

Cap and shake vigorously for 1 minutes

Add 100µL of is solution and QC spike solution if necessary

Add 15.0ml of 1.0% ACN

Add 8.0ml of ACN

Weight out 2.0 g +/-0.1 of Bovine Liver into 50.0 mLCentrifuge tubePlace Sample into AutoMate-Q40

ADD 7.5g of AOAC QuEChERSextraction salt

Figure 1: QuEChERS extraction for bovine liver.

Table I: Data table for veterinary drug residues

50.0 ng/g Spike100.0 ng/g

Spike

250.0 ng/g

Spike

Compounds R2 %

Recovery

%

RSD

%

Recovery

%

RSD

%

Recovery

%

RSD

Sulfapyridine (IS) 8.5 8.5 8.5

Sulfamethoxazole 0.9992 99.7 7.1 85.0 4.9 74.2 1.7

Sulfamethazine 0.9995 103.5 5.6 87.4 9.4 84.8 6.5

Sulfadimethoxine 0.9992 103.6 2.2 86.9 2.7 77.6 2.1

Sulfadizine 0.9995 102.2 5.6 89.9 3.6 78.7 2.2

Teledyne Tekmar4736 Socialville Foster Rd., Mason, OH 45040

tel. (513) 229-7000

Website: www.teledynetekmar.com



PHARMA/DRUG DISCOVERY

Using Selectivity to Enhance Separation of Analgesics

Selectivity is the most powerful tool to optimize separations in HPLC.

This parameter is changed by using different bonded phases, in-

cluding C18, polar embedded, phenyl bonded phases and perf o-

rophenyl, or by changing the mobile phase. In this work, 4.6 × 50

mm Poroshell 120 columns are used to quickly evaluate method

development choices for the analysis of non-steroidal anti-inf am-

matory drugs (NSAIDS). The short column length and high eff cien-

cy provide short analysis times and rapid equilibration, leading to

fast investigations of selectivity.


Instrument: Agilent 1260 Inf nity Binary LC System

Columns: Noted below

Flow rate: 2 mL/min

Mobile Phase: A: 20 mM NH4HCO

2 pH 3.0 B: Acetonitrile

Temperature: 40 °C

Detection: UV, 254 nm

Gradient:

Time % Organic

0 8

6 100

7 100

8 8

The Agilent 1260 Inf nity Binary LC System was conf gured as follows:

• G1312B Binary Pump SL, capable of delivering up to 600 bar

• G1316C Thermostatted Column Compartment (TCC)

• G1376D High Performance Autosampler SL Plus

• G4212A Diode Array Detector equipped with a G4212-60008 10

mm path length, 1 µL volume f ow cell

The following columns were used in this study.

• Agilent Poroshell 120 PFP, 4.6 × 50 mm, 2.7 µm (p/n 699975-408)

Fast Screening Methods for Analgesics and Non-Steroidal Anti-Inf ammatory (NSAIDS) Drugs by HPLC with Agilent Poroshell 120 ColumnsWilliam Long, Agilent Technologies, Inc.

Figure 1: Structures of selected analgesics.

Figure 2: Separation of analgesics using Agilent Poroshell 120 columns using methanol.




• Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7 µm (p/n 699975-902)

• Agilent Poroshell 120 Bonus-RP, 4.6 × 50 mm, 2.7 µm (p/n

699968-901)

• Agilent Poroshell 120 Phenyl-Hexyl, 4.6 × 50 mm, 2.7 µm

(p/n 699975-912)

A generic gradient separation was used to evaluate these columns

consisting of ammonium formate (20 mM NH4HCO

3 pH 3.0) using

either methanol or acetonitrile.

The analgesic materials all possess a wide variety of functional groups

including f uorine (sulindac and dif unisal) and chlorine (diclofenac).

The structures of the compounds examined are shown in Figure 1 and

Table I. All samples were prepared at 10 mg/mL in acetonitrile and were

diluted in water to a f nal concentration of 0.1 mg/mL.

Column Choice to Enhance Selectivity

The columns were chosen to improve selectivity in the separation.

They included a highly end capped C18 column recommended as a

å rst choice in method development (Poroshell120 EC- C18).

Figure 3: Separation of analgesics using Agilent Poroshell 120 columns with acetonitrile.

Table I: Retention time, Log P, and pKa data for selected analgesics

Compound log P pKatr PFP

MeCN

trPFP

MeOH

tr C18

MeCN

trC18

MeOH

trBrp

MeCN

trBrP

MeOH

trPH

MeCN

trPH

MeOH

Acetominophen 0.46 9.38 0.863 1.252 0.803 1.123 0.99 1.235 0.781 1.039

Phenacetin 1.58 2.2 1.966 2.912 2.147 2.943 2.176 2.774 20.59 2.959

Piroxicam 3.06 6.3 2.536 3.876 2.849 3.688 2.744 3.415 2.732 4.027

Tolemetin 2.79 3.5 2.868 4.137 2.928 4.265 3.173 4.073 2.893 4.395

Ketoprofen 3.12 4.45 3.008 4.258 3.109 4.308 3.342 4.146 3.137 4.468

Naproxen 3.18 4.15 3.112 4.505 3.249 4.436 3.342 4.218 3.167 4.468

Sulindac 3.42 4.7 2.934 4.656 3.249 4.308 3.173 4.288 2.995 4.594

Diclofenac 4.51 4.15 3.53 4.795 3.9 5.046 4.043 4.87 3.711 5.106

Dif unisal 4.41 2.69 3.659 5.094 3.249 4.567 3.867 4.919 3.091 4.559

Poroshell 120 Bonus-RP can be used for many of the same sepa-

rations as a C18 column while avoiding some of the disadvantages

of C18, such as poor wettability in high aqueous mobile phases. In

addition, it is much more retentive for those molecules that can

interact by hydrophobic interactions and also by H-bonding with

the amide group. Compared to alkyl only phases, Bonus-RP has

enhanced retention and selectivity for phenols, organic acids, and

other polar solutes due to strong H-bonding between polar group

(H-bond acceptor) and H-bond donors, like phenols and acids.

Bonus-RP gives retention slightly less than a C18 allows, for easy

column comparison without the need to change mobile phase con-

ditions. The Bonus-RP phase gives different selectivity than C18 for

polar compounds. It is also compatible with 100% water.

Poroshell 120 Phenyl-Hexyl columns deliver unique selectivity for

compounds with aromatic groups, providing superior resolution for

these samples. Poroshell 120 Phenyl-Hexyl can also provide optimum

separations of moderately polar compounds where typical alkyl

phases (C18 and C8) do not provide adequate resolution. Acetoni-




Agilent Technologies2850 Centerville Road, Wilmington, DE 19808

Website: www.agilent.com/discoverporoshell

Figure 4: Poroshell 120 EC-C18 retention time vs. log P values.

Figure 5: Poroshell 120 EC-C18 retention time vs. Poroshell 120 PFP retention time.

trile tends to decrease the π–π interactions between aromatic and

polarizable analytes and the phenyl-hexyl stationary phases, but

methanol enhances those same interactions, giving both increased

retention and changes in selectivity. This does not mean that acetoni-

trile should not be used with a phenyl bonded phase or that it might

not provide an acceptable separation, but methanol is more likely to

deliver the different selectivity that is desired from a phenyl phase.

Poroshell 120 PFP columns possess a pentaf uorophenyl li-

gand. This can provide an orthogonal separation mechanism to

traditional reverse phase columns. By specif cally targeting many

polar retention mechanisms, PFP phases can separate analytes

based on small differences in structure, substitution, and steric

access to polar moieties. The resulting selectivity for positional iso-

mers, halogenated compounds, and polar analytes is particularly

useful in the analysis of complex mixtures, and small molecule

pharmaceuticals


The separation of all nine compounds was attempted on all columns

surveyed. The Poroshell 120 PFP and Poroshell 120 Bonus RP col-

umns both fully resolved all compounds in the same order, although the

spacing of the peaks is more even on PFP. The Poroshell 120 Phenyl

Hexyl column does not yield the same elution order as the Poroshell

120 PFP column. This means that the PFP column is not just a stronger

phenyl column, other interactions beside π-π and hydrophobic interac-

tions are in play. All four columns elute acetominophen (APAP) and

phenacetin å rst. The Poroshell 120 EC-C18 column did not fully sepa-

rate three compounds (Tolmetin, Ketoprofen, and Sulindac).

Figure 3 shows the separation on all four columns using acetoni-

trile. In this case, only Poroshell 120 PFP resolves all compounds,

and Poroshell 120 EC-C18 and Poroshell Phenyl Hexyl columns

elute all compounds in the same order. Typically π-π interactions

with Phenyl Hexyl columns are overwhelmed in acetonitrile. Again,

the PFP and Bonus RP Columns have very similar elution orders

(with the exception of the last two peaks).

Since the Poroshell 120 PFP phase almost separates all nine

compounds when using methanol or acetonitrile, it provides the

best method development option for further development.

Table I lists the retention time of all nine analytes on the four col-

umns using both methanol and acetonitrile. Log P and pKa data are

also listed. The Log P refers to the equilibrium distribution of a single

substance between two solvent phases separated by a boundary.

It was discovered that the narcotic action of many simple organic

solutes was ref ected rather closely by their oil-water partition coef-

f cients. The oil was later replaced by octanol (5). In Figure 4, Log

P values correspond to retention time on the Poroshell 120 EC-C18

column using methanol or acetonitrile. Figure 5 shows the correla-

tion of Poroshell EC-C18 and PFP retention time data in methanol

and acetonitrile for the last seven analgesics. The f rst two analgesics,

acetominophen and phenacetin, remain in the same elution order in

all solvent column combinations. With these compounds removed,

the correlation between EC-C18 and PFP retention in methanol (5a)

and acetonitrile (5b) is poor and highly indicative of orthogonality.

Conclusions

Analysis problems can be quickly resolved by including survey meth-

ods with generic gradients as part of the method development scheme.

This work demonstrates how different chemistries and organic modif ers

such as acetonitrile and methanol can develop different selectivity and

can be used to optimize the separation. In this case, using an alternative

selectivity column such as Poroshell 120 PFP yielded better results than

a more commonly used C18 chemistry. Fluorinated stationary phases

are useful because of their enhanced interaction with halogens, and

conjugated compounds.

References

(1) A. Streitweiser Jr. and C.H. Heathcock, Introduction to Organic Chemistry,

(MacMillan, New York, 1981).

(2) http://en.wikipedia.org/wiki/Non-steroidal_anti-inflammatory_drug.

(3) J. Sangster, “Logkow: A Database of Evaluated Octanol/Water Partition

Coefficents,” (Log P), http://logkow.cisti.nrc.ca/logkow/index.jsp.




Steroids represent a chemically distinct class of hormones with

wide-ranging biological functions. Synthetic derivatives of endog-

enous steroid prototypes are used medically in birth control and in

the treatment of asthma, arthritis, inf ammation, and osteoporosis.

Steroids share a characteristic, polycyclic structure and have

varying degrees of lipophilicity (log P). In this study, a reversed-

phase HPLC method was developed for separation of f ve steroid

hormones with partition coefå cients ranging from 1.47 (cortisone)

to 4.5 (pregnenolone) on a Hamilton PRP-C18 HPLC column.

© 2014 Hamilton Company. All rights reserved. All trademarks are owned and/or registered by

Hamilton Company in the U.S. and/or other countries. 04/2014 Lit. No. L80086 Rev. B

Separation of Five Steroids on a C18-Functionalized Polymeric Reversed-Phase HPLC Column (PRP™-C18)Derek Jensen and Mark Carrier, Hamilton Company

Hamilton Company4970 Energy Way, Reno, NV 89502

tel. (775) 858-3000, (800) 648-5950

Website: www.hamiltoncompany.com

Table I: Experimental conditions

Column: PRP-C18, 5 µm, 4.6 × 150 mm

Part Number: 79676

Mobile Phase A: 20/80 acetonitrile/water

Mobile Phase B: 90/10 acetonitrile/water

Flow Rate: 1.0 mL/min

Gradient: 20 to 95% B in 20 min

Temperature: Ambient

Injection Volume: 1.0 µL

Sample Concentration: 0.1 mg/mL

Detection: UV at 210 nm

Table II: Compounds

Compounds Lipophilicity

1. Estriol (log P = 2.5)

2. Cortisone (log P = 1.5)

3. Testosterone (log P = 3.4)

4. Estrone (log P = 3.1)

5. Pregnenolone (log P = 4.5)

300

250

200

150

100

50

0

-5 0 5

Time (min)m

AU

210 n

m10

1

4

5

3

2

15 20

Figure 1: Separation of f ve steroids on a 4.6 × 150 mm PRP-C18.




A purif ed polyclonal antibody (IgG) is separated and fully

characterized using the Viscotek SEC-MALS 20, allowing

calculation of molecular weight and radius of gyration (Rg).

Therapeutic recombinant antibodies represent a growing proportion

of biopharmaceuticals and are primarily classed as Immunoglobulin

G (IgG). However, proteins have a tendency to aggregate over

time and one challenge for biologic drugs is that the presence

of aggregates will stimulate an immune response. Size-exclusion

chromatography (SEC) is a powerful tool that is commonly used to

look at the aggregation of proteins. While most SEC systems use a

single concentration detector such as ultraviolet (UV), the addition

of light scattering allows the molecular weight of the protein to be

measured independent of its retention volume. The new SEC-MALS

20 detector, which uses multi-angle light scattering (MALS), is

ideal for this application. In addition, the MALS detector makes it

possible to measure the radius of gyration (Rg) of molecules that

scatter light anisotropically.

In this application note, a purified polyclonal antibody (IgG)

is separated using SEC and characterized using the Viscotek

SEC-MALS 20.


Samples were analyzed using a Viscotek TDAmax system connected

to Viscotek SEC-MALS 20. The mobile phase was phosphate

buffered saline, which was also used to prepare the IgG for analysis.

Results

The SEC-MALS results are presented in Table I. The monomer

(15.80 mL) and dimer (14.00 mL) peaks are clearly identif ed by

the measured molecular weights and low polydispersity (Mw/Mn).

No size (Rg) can be measured for these peaks as they are below

the isotropic scattering threshold of 10–15 nm. Studying Figure 1,

it is just possible to see that the SEC-MALS 20 show the same

response for the monomer peaks at all angles. The aggregate peak

(13.23 mL) is clearly different. The molecular weight is higher and

more polydisperse, which shows that there is a variable composition

of molecules within the aggregate peak. Because it is large, the light

scattering response varies with angle and can be used to measure

the Rg.

Measuring Antibody Molecular Weight by SEC-MALSMalvern Instruments Ltd.

Malvern Instruments Ltd.Enigma Business Park, Grovewood Road, Malvern, UK

tel. +44 (0) 1684 892456

E-mail: [email protected]

Website: www.malvern.com

Table I: Measured molecular weights of the different peaks

of the IgG sample

Aggregates Dimer Monomer

Peak RV - (mL) 13.23 14.00 15.80

Mn - (kDa) 674.12 308.6 147.2

Mw - (kDa) 7661.00 309.2 147.4

Mw / Mn 11.364 1.002 1.001

Rg (w) - (nm) 26.6 N/C N/C

Wt. Fr. (Peak) 0.014 0.065 0.921

Weight Fraction % (Peak) 1.4 6.5 92

-0.6

02

6.1

5m

V

8.15 Ret Vol20.04

Figure 1: Overlay of MALS detector responses for IgG.




Pain management LC analyses can be diff cult to optimize due

to the limited selectivity of C18 and phenyl-hexyl phases. In

contrast, the selectivity of Raptor™ Biphenyl superf cially po-

rous particle (SPP) LC columns provides complete resolution

of isobaric pain medications with a total cycle time of 5 min.

Accurate, reliable analysis of pain medications is a key component

in monitoring appropriate medical use and preventing drug diversion

and abuse. As the demand for fast, multicomponent methods grows,

LC–MS-MS methods are increasingly desired for pain management

and therapeutic drug monitoring due to the low detection limits that

can be achieved with this highly sensitive and selective technique.

However, despite the selectivity offered by mass spectrometry, hy-

drophilic matrix components can still interfere with early-eluting drug

compounds resulting in ion suppression. In addition, isobaric pairs

must be chromatographically separated for positive identif cation.

The need for highly selective and accurate methods makes LC col-

umn selection critical.

While C18 and phenyl-hexyl phases are frequently used for bio-

analytical LC–MS-MS applications, Restek’s Biphenyl phase offers

better aromatic retention and selectivity for pharmaceutical and drug-

like compounds, giving it a signif cant advantage over other phases

for the analysis of pain management medications or other drugs

of abuse. The Biphenyl phase, originally developed a decade ago

by Restek, has recently been combined with Raptor™ SPP (“core-

shell”) silica particles to allow for faster separations without the need

for expensive UHPLC instrumentation. Here, we demonstrate the

fast, selective separation of commonly tested pain drugs that can be

achieved using the new Raptor™ SPP Biphenyl LC column.


A standard containing multiple pain management drugs was pre-

pared in blank human urine and diluted with mobile phase as fol-

lows, urine:mobile phase A:mobile phase B (17:76:7). The å nal

concentration for all analytes was 10 ng/mL except for lorazepam,

which was 100 ng/mL. Samples were then analyzed by LC–MS-

MS using an AB SCIEX API 4000™ MS-MS in ESI+ mode. Chro-

matographic conditions, retention times, and mass transitions are

presented here and in Tables I and II:

Column: Raptor™ Biphenyl, 50 mm × 3.0 mm i.d. × 2.7 µm

Sample: Fortif ed urine

Inj. vol.: 10 μL

Inj. temp.: 30 °C

Mobile phase A: Water + 0.1% formic acid

Mobile phase B: Methanol + 0.1% formic acid

Results

As shown in Figure 1, 18 commonly tested pain management

drugs were analyzed with the last compound eluting in less than

3.5 min, giving a total cycle time of 5 min on Restek’s Raptor™

SPP Biphenyl LC column. Analyte retention times are presented in

Table II. Important isobaric pairs (morphine/hydromorphone and

codeine/hydrocodone) were completely resolved and eluted as

symmetrical peaks, allowing accurate identif cation and integra-

tion. In addition, early-eluting compounds such as morphine, oxy-

morphone, and hydromorphone are separated from hydrophilic

matrix interferences, resulting in decreased ion-suppression and

increased sensitivity. Similar analyses on C18 and phenyl-hexyl

columns often exhibit poor peak shape and resolution (for exam-

ple, peak tailing between closely eluting isobars), which makes

identif cation and accurate quantif cation more diff cult.

Accurate Pain Management Analysis in Under 5 Min on Raptor™ Biphenyl Superf cially Porous Particle LC Columns Sharon Lupo, Ty Kahler, and Paul Connolly, Restek Corporation

Figure 1: Baseline resolution of isobaric pain management drugs in sub-5-min runs on the Raptor™ Biphenyl column.

Table I: Mobile phase gradient

Time (min) Flow (mL/min) %A %B

0.00 0.6 90 10

1.50 0.6 55 45

2.50 0.6 0 100

3.70 0.6 0 100

3.71 0.6 90 10

5.00 0.6 90 10




Conclusions

Complete separation of critical pain management drug analytes

from hydrophilic matrix components and isobaric interferences was

achieved using the new Raptor™ SPP Biphenyl LC column in less

than 5 min. The fast, complete separations produced in this method

allow accurate quantif cation of pain management drugs and sup-

port increased sample throughput and improved lab productivity.

To learn more, visit www.restek.com/raptor

Restek Corporation110 Benner Circle, Bellefonte, PA 16823

tel. (800) 356-1688, fax (814) 353-1309

Website: www.restek.com/raptor

Table II: Analyte retention times and transitions

Peaks tR (min) Precursor Ion Product Ion 1 Product Ion 2

Morphine* 1.34 286.2 152.3 165.3

Oxymorphone 1.40 302.1 227.3 198.2

Hydromorphone* 1.52 286.1 185.3 128.2

Amphetamine 1.62 136.0 91.3 119.2

Methamphetamine 1.84 150.0 91.2 119.3

Codeine* 1.91 300.2 165.4 153.2

Oxycodone 2.02 316.1 241.3 256.4

Hydrocodone* 2.06 300.1 199.3 128.3

Norbuprenorphine 2.59 414.1 83.4 101.0

Meprobamate 2.61 219.0 158.4 97.2

Fentanyl 2.70 337.2 188.4 105.2

Buprenorphine 2.70 468.3 396.4 414.5

Flurazepam 2.73 388.2 315.2 288.3

Sufentanil 2.77 387.2 238.5 111.3

Methadone 2.86 310.2 265.3 105.3

Carisoprodol 2.87 261.2 176.3 158.1

Lorazepam 3.03 321.0 275.4 303.1

Diazepam 3.31 285.1 193.2 153.9

*An extracted ion chromatogram (XIC) of these isobars is presented in the inset of Figure 1.




Alprostadil (Prostaglandin E1) is a drug that has vasodilation proper-

ties and is used to treat erectile dysfunction and other medical condi-

tions. This medication is available in injectable form and in suppository

form. Since Prostaglandin E1 has low UV absorbance, the analysis

of the formulations could be diff cult. The addition of post-column

derivatization increases the sensitivity and specif city of the analysis.

Analytical Conditions

Analytical column: Reversed-phase C18 column (150 × 4.6 mm)

Temperature: 37 ºC

Flow rate: 1 mL/min

Mobile phase: 30% Acetonitrile – 70% of 0.02 M Potassium

Phosphate monobasic (adjusted to pH 3)

Or Alternative Conditions

Mobile phase: 25% Acetonitrile – 75% of 0.0067 M Potassium

Phosphate buffer pH 6.3

Injection volume: 20 µL

Post-Column Conditions

Post-column system: Pinnacle PCX

Reactor volume: 2 mL

Reactor temperature: 60 ºC

Reagent: 1 mol/L Potassium Hydroxide

Reagent ý ow rate: 1 mL/min

Detection: UV 278 nm

Calibration

Alprostadil: 0.1 µg/mL – 10 µg/mL

β-Naphthol (Internal Standard) 0.25 µg/mL – 10 µg/mL

Make stock solutions of Alprostadil and β-Naphthol in anhydrous

Ethanol. Prepare working standards by appropriate dilution of stock

solution with mobile phase.

Analysis of Alprostadil by HPLC with Post-Column Derivatization Pickering Laboratories, Inc.

Pickering Laboratories, Inc.1280 Space Park Way, Mountain View, CA 94043

tel. (800) 654-3330, (650) 694-6700

Website: www.pickeringlabs.com

Figure 3: Chromatogram of Alprostadil and β-Naphthol (internal standard).

Figure 1: Calibration curve for Alprostadil.

Figure 2: Calibration curve for β-Naphthol.

min

b-Naphthol

Alprostadil

0 5 10 15 20

R² = 1

0

1

2

3

4

5

6

0 2 4 6 8 10 12

are

a

ppm

R² = 0.9999

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 6 8 10 12

are

a

ppm




Antibody Drug Conjugate (ADC) Analysis Wyatt Technology

A novel method for drug antibody ratio determinations

based on SEC-MALS in conjunction with UV absorption and

differential refractive index detection.

There has been a signif cant resurgence in the development of anti-

body-drug conjugates (ADC) as target-directed therapeutic agents for

cancer treatment. Among the factors critical to effective ADC design is

the drug antibody ratio (DAR). The DAR describes the degree of drug

addition which directly impacts both potency and potential toxicity of

the therapeutic, and can have signif cant effects on properties such as

stability and aggregation. Determination of DAR is, therefore, of critical

importance in the development of novel ADC therapeutics.

DAR is typically assessed by mass spectrometry (MALDI-TOF or ESI-

MS) or UV spectroscopy. Calculations based on UV absorption are of-

ten complicated by similarities in extinction coefå cients of the antibody

and small molecule. Mass spectrometry, though a powerful tool for Mw

determination, depends on uniform ionization and recovery between

compounds — which is not always the case for ADCs.

We present here a method for DAR determination based on SEC-

MALS in conjunction with UV absorption and differential refractive

index detection. Figure 1 shows UV traces for two model ADCs; mo-

lecular weights of the entire ADC complexes are determined directly

from light scattering data.

Component analysis is automated within the ASTRA 6 software

package by using the differential refractive index increments (dn/

dc) and extinction coefå cients, which are empirically determined for

each specie or mined from the literature, to calculate the molar mass

of the entire complex as well as for each component of the complex.

In this example an antibody has been alkylated with a compound

having a nominal molecular weight of 1250 Da (Figure 2). Molar

masses of the antibody fractions are similar, which indicates that

the overall differences between the two formulations ref ect distinct

average DARs which are consistent with values obtained by orthog-

onal techniques. Note that the molar mass traces for the conjugated

moiety represent the total amount of attached pendant groups; the

horizontal trends indicate that modif cation is uniform throughout

the population eluting in that peak.

Wyatt Technology6300 Hollister Avenue, Santa Barbara, CA 93117

tel. +1 (805) 681-9009, fax +1 (805) 681-0123

Website: www.wyatt.com

Antibody-Drug Conjugate Analysis

(■) Mw of complex

(+) Mw of antibody

(x) Mw of conjugated drug

1.0x105

1.0x104

9.0 9.5 10.0 10.5 11.0 11.5 12.0time (min)

Complex Antibody Drug

DAR

ADC1

ADC2

167.8 (±1.2%)

163.7 (±1.2%)

155.2 (±1.8%)

155.6 (±1.2%)

12.6

8.1 6.5

10.1

Mw (kDa)

Mo

lar

Ma

ss (

g/m

ol)

ADC1

ADC2

2.0x105

Molar mass vs. time

167.8 kDa

ADC1

ADC2

163.7 kDa1.8x105

1.6x105

1.4x105

1.2x105

Mo

lar

Ma

ss (

g/m

ol)

1.0x105

8.0x104

9.0 9.5 10.0 10.5Time (mn)

11.0 11.5 12.0

Figure 2: Molar masses for the antibody and total appended drug are calculated in the ASTRA software package based on prior knowledge of each component’s extinction coeff cent and dn/dc, allowing deter-mination of DAR based on a nominal Mw of 1250 Da for an individual drug.

Figure 1: Molar masses for two distinct ADC formulations are determined using SEC-MALS analysis.




Poly(lactic-co-glycolic acid) (PLGA) is a copolymer based on glycol-

ic acid and lactic acid. The two monomer units are linked together

by ester linkages and form linear polyester chains. The obtained

product is biodegradable and biocompatible, and it is approved by

the Food and Drug Administration (FDA) for production of various

therapeutic devices as well as for drug delivery applications. The

properties of PLGA can be tuned by the ratio of the two monomers

and by its molar mass distribution.

The characterization of PLGA by means of conventional size-ex-

clusion chromatography (SEC) is problematic because of the lack

of suitable calibration standards. In addition, the linear polyester

structure can be modif ed by the addition of small amounts of poly-

functional monomer to obtain branched chains of differing degrees

of branching. The degree of branching becomes an additional pa-

rameter that can be used to adjust PLGA properties — all of which

renders conventional column calibration an inadequate analytical

technique.

In this application note, two commercially available samples

were analyzed by SEC coupled to a multi-angle light scattering

(MALS) detector (HELEOS), a refractive index detector (Optilab

rEX), and a viscosity (VIS) detector (ViscoStar). The ViscoStar was

used in order to discover additional information about the molecu-

lar structure of the analyzed polymers. In addition to molar mass

distributions, the SEC–MALS-VIS system yields the relationship

between intrinsic viscosity and molar mass (Mark-Houwink plot)

that can provide deep insight into the molecular structure of the

polymers being analyzed.

In Figure 1, the molar mass distributions are given as differential

distribution plots. As seen from the plots, the two samples span

markedly different molar mass ranges. The Mark-Houwink plots of

the two samples are shown in Figure 2 together with the plot of

linear polystyrene that is shown simply for the sake of compari-

son. The slope of the Mark-Houwink plot of the linear polystyrene is

0.71, a typical value for linear random coils in thermodynamically

good solvents. The slope of the red sample roughly corresponds

to a linear structure as well. However, there is a slight indication of

deviation from linearity at the region of high molar masses that may

indicate the presence of branched molecules. The Mark-Houwink

plot of the blue sample is curved. Curvature of the Mark-Houwink

plot generally reveals branching. In addition, the slope of the higher

molar mass portion of the Mark-Houwink plot of 0.48 suggests sig-

nif cant branching.

SEC-MALS-VIS is an excellent method for the characterization

of PLGA polyesters as it has the ability to determine not only the

molar mass distribution, but to reveal subtle differences in PLGAs

molecular structure.

Characterization of PLGA Using SEC–MALS-VISWyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, CA 93117

tel. (805) 681-9009, fax (805) 681-0123

Website: www.wyatt.com

Figure 1: Differential molar mass distribution curves of two PLGA samples.

Figure 2: Mark-Houwink plots of two samples of PLGA (red and blue) and linear polystyrene (magenta). The lines are linear extrap-olations of the data.



GENERAL

Separation of a Mix of Acidic, Basic,and Neutral Compounds at High pH Conditions Diamond Analytics

The unique surface chemistry of the Flare diamond core-

shell column combines ionic and hydrophobic separation

mechanisms to effectively retain a variety of chemical

species in a single run.

HPLC Conditions

Column Name: Flare C18 Mixed-Mode ColumnDimensions: 4.6 × 33 mm, 3.6 μm, 180 ÅHPLC System: Waters® 1525 Binary PumpInjection Volume: 5 μLDetection: UV at 254 nmFlow Rate: 1.0 mL/minSeparation Mode: IsocraticMobile Phase: A: 10 mM phosphate buffer, pH 12; B: acetonitrile A/B (70:30)Temperature: 35 °C Analytes:

0 1

1

2

3

4

5

6

2 3

Retention Time (minutes)

4 5

1st injection

50th injection

6

Diamond Analytics11260 South 1600 West, Orem, UT 84058

tel. (801) 235-9001, fax (801) 235-9141

Website: diamond- analytics.com

Conclusions

1) Fast separation at high pH2) Separation possible with 100% aqueous mobile phase3) Straightforward method transfer to any system

10

9

8

7

6

5k

4

3

2

1

00 5 10 15 20 25

No. of injections

30 35 40 45 50

6

5

4

3

2

1

1) 2, 4-D 2) Propazine

3) Ethylbenzene

5) Imipramine 6) Amitriptyline

4) Nortriptyline



GENERAL

Concentrating Phenolic Compounds Using the XcelVap®

Concentration SystemDavid Gallagher, Horizon Technology, Inc.

Natural and synthetic phenolic compounds have been used in a

wide range of applications, from antiseptics and fungicides to food

additives. With the adoption of the Clean Water Act (CWA), many

phenolic compounds were placed on a list of “Priority Pollutants.”

This list encompasses highly manufactured compounds which were

found in water with a frequency of 2.5% or more (1). In addition,

the entire class of compounds or specif c compounds may be writ-

ten into National Pollution Discharge Elimination (NPDES) permits

to control release into public waterways (2).

Although the general analysis process contains extraction, extract

drying, and evaporation steps prior to chromatography, this note

will describe a procedure for the evaporation and concentration

of organic solvent extracts containing phenolic compounds. It will

make use of an automated pressure ramp during the concentra-

tion procedure to ensure that a maximum throughput of samples

is achieved.

Experimental

The XcelVap benchtop, heated water bath, nitrogen blow-down

system (Horizon Technology) was used with 200-mL nipple tipped

concentrator tubes. The following procedure was followed:

1. Set the XcelVap conditions using the parameters given in Table I

and allow the bath to equilibrate.

2. Place 200 mL of dichloromethane (DCM) into each of the six

200-mL XcelVap concentrator tubes.

3. Using a spiking mix of phenolic compounds and surrogates pre-

pared at 100 µg/mL, add 0.5 mL of the mix to each of the six

concentrator tubes.

4. Start the XcelVap.

5. When complete, remove the rack from the XcelVap unit to pre-

vent excess heat transfer to the extracts.

6. Using a pipette, transfer the concentrated extract to an auto-

sampler vial, being sure to rinse the concentrator tube with DCM

and bring the f nal volume to 1 mL.

Results

Table II shows the results of 12 replicate extracts concentrated

using the procedure as given above. Each was concentrated

using a single, automated

pressure ramp and resulted

in 0.5–1 mL of solvent. The

extract was then brought

up to a final volume of 1 mL

prior to being injected onto

the GC–MS.

Conclusions

The XcelVap Concentration System yields excellent recoveries for

phenolic compounds when used in a fully automated capacity. With

its large volume concentration tubes, the XcelVap System can be

used with sample extracts from a wide range of extraction proce-

dures including solid phase extraction (Method 3535), liquid-liq-

uid extraction (Method 3510), continuous liquid-liquid extraction

(Method 3520), TCLP extracts (Method 1311), pressurized f uid

extraction (Method 3545), and ultrasonic extraction (Method 3550).

References

(1) http://water.epa.gov/scitech/methods/cwa/pollutants-background.cfm.

(2) http://www.ecfr.gov/cgi-bin/retrieveECFR?gp=&SID=37bb5003c18921554

522c0c1e206c8e3&r=PART&n=40y30.0.1.1.19.

Horizon Technology, Inc. 16 Northwestern Drive, Salem, NH 03079

tel. (603) 893-3663, fax (603) 893-4994

Website: www.horizontechinc.com

Table I: XcelVap conditions

Parameter Setting

Source nitrogen 90 psi

Nitrogen ramp 6–24 psi

Bath temperature 40 °C

Timer 57 min

Table II: Average recovery and precision of 12 replicate

evaporations

Average Recovery (%) RSD (%)

2-Fluorophenol 74.52 8.58

Phenol-d5 87.00 9.14

Phenol 77.80 9.29

2-Chlorophenol 71.71 9.37

2-Methyl phenol 81.78 9.13

3+4 Methyl phenol 84.88 9.04

2-Nitrophenol 67.78 10.58

2,4-Dimethylphenol 77.60 9.47

2,4-Dichlorophenol 74.29 9.68

2,6-Dichlorophenol 76.71 9.60

4-Chloro-3-methylphenol 90.35 9.55

2,4,6-Trichlorophenol 78.38 8.56

2,4,5-Trichlorophenol 88.40 7.84

2,4-Dinitrophenol 90.95 8.52

4-Nitrophenol 112.04 6.82

2,3,4,6-Tetrachlorophenol 91.26 6.24

4,6-Dinitro-2-methylphenol 97.82 6.09

2,4,6-Tribromophenol 103.22 4.82

Pentachlorophenol 96.19 4.15

Dinoseb 100.00 4.58



GENERAL

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The 2014 LCGC Awards

Meg L’Heureux, Managing Editor, LCGC North America and Spectroscopy

The seventh annual LCGC Awards continue the time-honored tradition of celebrating the careers of out-standing chromatographers. We are proud to announce

that the 2014 Lifetime Achievement in Chromatography Award is granted to Fred E. Regnier and the 2014 Emerging Leader in Chromatography Award is presented to André de Villiers. Table I shows the complete listing of our prestigious award winners over the past seven years.

Fred E. RegnierOne of the greatest aspects of the LCGC Lifetime Achievement in Chromatography Awards is that it presents us with the opportunity to appreciate the legacy and scientific impact of our winners. The passion and insight these scientists offer the community cannot easily be expressed, but everyone we inter-view gives resounding comments

in that regard. For Fred E. Regnier, the passion, legacy, and scientific impact are just a few of his many achievements in a life-long scientific journey.

Regnier’s path to chromatography did not start in the usual fashion. He says that he came to science “through the back door.” “Getting into science didn’t occur to me until I was out of college,” he adds. In fact, he claims he only started college as a way to get out of working outside in the cold Nebraska winter. Despite his professed lack of interest in sci-ence early on, as an undergraduate at Nebraska State College Regnier had a triple major in physics, chemistry, and math — quite an undertaking! It was during those undergraduate years that Regnier first came across separation science — while shelving books at the library on chromatography and electrophoresis. Those subjects clearly captured his interest; Regnier’s undergraduate thesis was on paper electrophoresis of amino acids. After graduating from Nebraska State Col-lege in 1960, Regnier went on to get his PhD from Oklahoma State University in 1965. The focus of his doctoral disserta-tion was the biosynthesis of terpenes and pheromones.

Regnier went on to do postdoctoral research at Oklahoma State University in 1965, the University of Chicago from 1966 to 1967, and Harvard University in 1968. That work at Harvard, under the direction of Ed Wilson and John Law, focused on the identification of insect hormones and phero-mones. He built a high performance liquid chromatography (HPLC) system to purify trail pheromones from 100-lb bags of fire ants and also did work with gas chromatography (GC) and mass spectrometry (MS).

Following his postdoctoral research, Regnier took an as-sistant professor position in the biochemistry department at

Purdue University, where he began a long-lasting professorship that would span more than four decades. He was promoted to associate professor of biochemistry in 1971. In 1976 he was promoted to associate director of the agricultural experiment station and then to full professor of biochemistry. In 1990 his title changed to a professor of chemistry, and in 2004 he be-came the John H. Law distinguished professor of chemistry.

Major Scientific Contributions

Regnier is known as an innovator who has often bridged the gap between biochemistry and analytical science. He cites Wilson and Law as big influences in that regard. “They told me I should focus on method development, their rationale being that developing new, innovative, original methods al-ways keeps one at the cutting edge,” said Regnier. “‘You will always be relevant,’ they said.”

Regnier took that advice to heart: Among his many ac-complishments, he has received 45 patents related to the separation and detection of proteins and peptides. His work includes developing the first high-performance chromatog-raphy columns for size-exclusion chromatography (SEC), an-ion-exchange and cation-exchange chromatography, hydro-phobic interaction chromatography (HIC), and macroporous reversed-phase chromatography separations of proteins; de-scribing “footprint” retention models for ion-exchange chro-matography (IEC), HIC, and reversed-phase chromatography of proteins; fabricating a variety of capillary and collocated monolith support structure capillary electrochromatography systems; and synthesizing multiple polymeric bonded phases for SEC, IEC, HIC, and affinity chromatography that enabled silica, titania, zirconia, alumina, and poly(styrene–divinyl-benzene) supports to be used for protein separations; and the synthesis of gigaporous chromatographic media with en-hanced mass transfer and pore diameters exceeding 400 nm.

When we asked Regnier’s colleagues, past students, and friends about his biggest contributions to the field, the re-sponses were as varied as his work. Barry Karger, the James L. Waters Chair in Analytical Chemistry and the director of the Barnett Institute at Northeastern University, feels Reg-nier’s biggest contribution was the development of perfusion particles for preparative-scale separations of biopolymers. “These packings, developed over 25 years ago, are still widely used today for purification,” he said.

Tim Schlabach, a marketing manager at Agilent Technolo-gies and a former graduate student of Regnier’s, agrees with Karger. “Critical advances in the fast separation of biomol-ecules including size-exclusion, ion-exchange, and perfu-sion chromatography were Regnier’s biggest contributions,” Schlabach said.

Joseph J. Kirkland, vice-president of R&D at Advanced Materials Technology Inc., points to Regnier’s work char-

Fred E. Regnier



acterizing biomolecules using HPLC. “Regnier was an early leader in rec-ognizing the potential of HPLC for characterizing biomolecules and estab-lishing effective research programs to demonstrate this as a premier method,” said Kirkland. “His contributions in this area have been enormously im-portant in describing new science that has had a serious impact on improving health issues.”

Wolfgang Lindner, a professor emeritus at the Institute of Analytical Chemistry at the University of Vienna, believes that Regnier’s knowledge of biochemistry, proteins, and biological systems led to his biggest contribu-tions. “Regnier was always aware of the complexity of biological samples and systems and the great demand to have tools available to deconvolute the samples’ complexity,” said Lindner. He went on to explain that this awareness allowed Regnier to advance concepts of how LC can separate biomolecules, including peptides and large proteins. “This knowledge makes Regnier a very unique scientist indeed,” he said. “There are not many colleagues in the world who have this spectrum of in-sights in the biochemical and separa-tion science fields.”

Karger echoed this sentiment. “Reg-nier combined his biochemical back-ground with separation principles in very creative ways,” he said. “He has made significant contributions to the advancement of separation science over this career.”

Tim Wehr, a staff scientist at Bio-

Rad Laboratories, agreed that Regnier did groundbreaking work with biomol-ecules. “Fred is a pioneer in the field of HPLC of biomolecules, and the first large-pore chromatography columns designed specifically for protein sepa-rations were created in his lab,” Wehr said.

Michael Dong, a senior scientist at Genentech, thinks that Regnier’s greatest contributions were not just his work advancing the fundamentals of protein separations and microfluidics, but also “the idea of using analytical chemistry to solve the most impor-tant questions for mankind, which are mostly in biology.”

Xiang Zhang, a professor of chem-istry at the University of Louisville, agrees with Dong. “Regnier’s contribu-tion to separation science is not only his development of chromatography systems for protein and small molecule separations, but also the application of these technologies to solve real-life problems,” Zhang said.

Ira Krull, a professor emeritus of Northeastern University, points to the specifics of many of Regnier’s contribu-tions, such as the development of new stationary phases or instrumentation for HPLC separation of biopolymers, immobilized enzyme reactors, chip-based HPLC separations, and biophar-maceutical characterization. “Regnier has made countless contributions in separations, mostly in new and novel stationary phases, and instrumenta-tion such as the Bio-Cad systems from PerSeptive Biosystems and other small firms that Regnier started.” Krull is referring to one of the five companies related to separations and analytical chemistry that Regnier cofounded:

Bioseparations, PerSeptive Biosystems, BG Medicine, Quadraspec, and Perfin-ity Biosciences.

Andrew Alpert, the president of PolyLC, Inc., and a former graduate student of Regnier’s, also feels strongly about Regnier’s work on the commer-cial side of analytical science. “Regnier ensured that his inventions became commercial products so that scientists everywhere would have ready access to them, and sometimes he started the companies needed to implement this,” said Alpert. “That example particularly inspired me.”

Alpert also points to Regnier’s work adapting every mode of chromatogra-phy used for life-science separations from low-pressure media to HPLC media, adding that Regnier system-atically worked out the requirements for a successful or optimal material in such applications. “In some cases, there was no precedent for this,” Alpert said. “This set the agenda for everyone who has followed in that field.”

Academic Contributions

Regnier was not only interested in de-veloping products, however. He also focused on the development of his stu-dents. As an educator for more than 40 years, Regnier mentored more than 80 graduate students and more than 30 postdoctoral researchers. Edward Pfannkoch, the director of technol-ogy development in North America for Gerstel Inc., and a former graduate student of Regnier’s, emphasizes the importance of this aspect of Regnier’s career. “It is easy to cite Regnier’s many contributions such as his fundamental work on coating wide-pore silica for HPLC packings for biological separa-tions,” he said. “However, I feel one of his biggest contributions is the train-ing, guidance, and example he set for a whole generation of students that are now working in areas related to separa-tion science.”

Deena Krestel-Rickert, the owner of Pettec, LLC, and a previous gradu-ate student of Regnier’s, agrees with Pfannkoch. She explained that Reg-nier is an innovative thinker, is a great speaker, is very well-liked and respected, and is extremely willing to

Figure 1: Regnier with his wife Linda and his f rst and last PhD students. From left to right: Nishi Rochelle (PhD 2011), Fred Regnier, Linda Regnier, and Peter Dunn (PhD 1973).

Figure 2: Regnier’s retirement party. Note the chromatogram on the cake.



help people both in their career and personal lives. “I was extremely fortu-nate to have been his student,” Krestel-Rickert said. Schlabach also noted Reg-nier’s willingness to help his students during both their academic and profes-sional careers. “Regnier helped me on more than one occasion secure a posi-tion with a company, “ he said.

Work in Regnier’s laboratory was both challenging and exciting for many of his students. Alpert came up with a great analogy: “Working with Regnier was like panning for gold. His numer-ous ideas and concepts were like a rush-ing river. It was the job of his graduate students and postdocs to reach in and grab the gold nuggets as the f low car-ried them by — that is, to figure out which ideas were both practical and worthwhile to implement, then work out a way to do so.”

Zhang added that Regnier had a broad vision of science and was always

excited and full of passion about his research. “Everyone who worked with Regnier knew that he was always the right person to talk with about research and the source to get encouragement,” Zhang said.

Jim Pearson, a partner at Bioscience Advisors and a graduate student of Regnier’s, says that Regnier never told him what experiment to do; instead he let Pearson explore and discussed the results afterwards. “This instilled a sense of scientific curiosity in me, because I could test my own ideas and bounce the results off Regnier,” he said. Pearson’s favorite example of this was in 1980 when he coated various porous silicas with an experimental reversed-phase coating containing an adaman-tyl “box-like” group at the end of the n-alkyl ligand. The goal was to see if peptide or protein selectivity could be enhanced, and the silicas had various pore diameters, ranging from 55 to 1000 Å. “To my surprise, the 330-Å diameter porous silica worked the best by far, and I was excited about my ada-mantyl ‘box-like’ ligand find,” he said. “Regnier just smiled when he saw the data because he knew I stumbled upon the understanding that the pore diam-eter effect is key for resolution.” Regnier told Pearson to make an HPLC column with that 330-Å porous silica using a C8 ligand instead, which resulted in even better selectivity. “C8-coated 330-Å porous silica reversed-phase columns soon became the universal standard for peptide and protein separations for the next 25 years,” said Pearson.

Pfannkoch had a similar experi-ence of being given independence in Regnier’s laboratory, with his first real exposure to HPLC instrumentation. Pfannkoch explained that Regnier had an HPLC instrument that was also used to pack HPLC columns, and someone had precipitated buffer in the lines. “Regnier handed me some wrenches and a manual and said, ‘Fix it,’” said Pfannkoch. “After that day I was never intimidated by analytical instrumenta-tion.”

Krestel-Rickert also said that Regnier taught her a lot about HPLC and GC and entrusted her with maintaining the HPLC system. What was much bigger

than that, however, was that Regnier took her into his laboratory under a Na-tional Science Foundation grant on dog olfaction after she lost her major pro-fessor at Florida State and still had two years left to complete her PhD. “Regnier made a lot of things possible for me and always had my and all of his student’s best interests and futures in mind,” said Krestel-Rickert.

Pfannkoch summarized Regnier’s role as an educator nicely. “Regnier’s leadership, enthusiasm, example, and humility serve as a tremendous role model for all the best in science,” he said.

The affection that Regnier’s stu-dents have for him was demonstrated recently when many of them gathered together for a surprise party in honor of his retirement from Purdue Uni-versity in May 2013. Jianming Lei, an analytical chemist at the office of In-diana State Chemist at Purdue Univer-sity and a former graduate student of Regnier’s, helped to organize the party and reached out to all of Regnier’s for-mer students and colleagues to create a book of congratulation letters, stories, and photos of Regnier throughout the years, titled Fred Regnier’s Indy 500 of Chromatography and Other Stories (1). A few of those photos are included here. In the book, Regnier’s wife, Linda said that for Regnier, his students were “his life, his passion, and his pride and joy.”

Contributions to the Field

Beyond his work in academia and commercial start-ups, Regnier has also contributed to the scientific commu-nity as a whole through his involve-ment in various boards, organizing committees, and journals. He has been on the editorial boards of 13 journals, including LCGC North America since its inception in 1983 as LC Magazine. Regnier has also published more than 300 journal articles, two books, more than 30 book chapters, and more than 70 review papers.

Regnier explained that researchers in academia have a contract with society in which society provides money to ex-amine scientific problems and, in turn, academics have an obligation to share what they find with society. “I view our

Figure 3: 2013 ACS medal symposium. Back row, left to right: Ashraf G. Madian, Samir Julka, and Hamid Mirzaei. Front row, left to right: Mary Wirth, Fred Reg-nier, and Andy Alpert.

Figure 4: The chairmen at the 2nd ISPPP conference in December 1982, in Balti-more, Maryland. From left to right: Jan-Christer Janson, Tim Wehr, Klaus Unger, Milton Hearn, and Fred Regnier.



relationship with the American tax-payer as a collaboration for which I am enormously grateful,” he said. Regnier also described his role as peer-reviewer of manuscripts as “trying to help people improve the presentation of their work to society in the form of a publication.”

Regnier was also one of the first or-ganizers of the International Sympo-sium on HPLC of Peptides, Proteins, and Polynucleotides (ISPPP) along with Wehr, Milton Hearn, Klaus Unger, and Jan-Christer Janson. The first meeting they organized was held in Washing-ton, D.C., in 1980. “The conference still takes place 34 years later, although none of the original committee members are involved with it,” said Wehr. A photo of the 1982 organizing committee appears on this page.

Scientific Accolades

As one might expect, someone with this rich of a history in scientific achieve-ments is sure to have received a num-ber of awards from the community. Indeed, Regnier has received many accolades throughout his career. Some of the most notable awards he has re-ceived are the David B. Hime Award for Achievement in Chromatography (1982); the Stephen Dal Nogare Award for Achievements in Chromatography (1987); the ACS Award in Chromatog-raphy (1989); the Martin Gold Medal (1993); the Eastern Analytical Sym-posium Award for Achievements in Separation Science (1996); the CASSS Scientific Achievement Award (2000); the Golay Award (2001); and the Out-standing Commercialization Award, presented by Purdue University and the Central Indiana Corporate Partnership (2006) — just to name a few.

Personal Accounts

Regnier has made many friends during the course of his career. Many of these have fond memories and personal sto-ries of their experiences with Regnier that they wanted to share.

Krestel-Rickert told us how she used to go running at the track with Reg-nier and would jokingly refer to him as “Mr. Popularity” because he could never make it around the track with-out someone wanting to talk to him. “If

the person wasn’t running, Regnier was so gracious that he would stop running and walk or stop to talk with them,” she said. “So we kidded with him about whether he was going to the gym to run or to socialize.”

Dong remembers when he invited Regnier to give an informal talk at a dinner event for the Chinese American Chromatography Association in 2012. There, Regnier said there should be an LC–MS instrument in every doctor’s office so that doctors could make im-mediate diagnoses of diseases or treat-ment progression. Dong realized this could be possible if the front-end of a sample extraction could be automated so that the right biomarkers (often a protein isoform) could be isolated and determined. “Listening to him, it dawned on me that this could be the greatest contribution that an analyti-cal chemist could make — to solve the great biology problems of today, such as those in disease diagnostics,” said Dong.

Karger has been friends and col-leagues with Regnier for more than 35 years and taught short courses with him in the 1980s on HPLC. He shared a light-hearted memory from the 1992 HPLC conference in Baltimore, Mary-land, where Regnier convinced well-known chromatographers (including Karger) to dress the way people dressed 500 years ago, in honor of the 500th anniversary of Columbus’ discovery of America. “We stunned the attendees as we walked in to the conference to open the meeting,” said Karger.

André de

Villiers

LCGC ’s 2014 E m e r g i n g L e a d e r i n Chromatog-raphy award w i n n e r , André de Vil-liers, received his Bachelor

of Science degree in chemistry and bio-chemistry (1997), his Honors Bachelor of Science degree in chemistry (cum laude, 1998), his Masters of Science degree in analytical chemistry (cum laude, 2000),

and his doctoral degree in analytical chemistry (2004) from Stellenbosch University in South Africa. De Villiers says his interest in analytical science began with the start of his postgradu-ate studies. In 1999 he was very unsure of his future plans, but decided to meet with two professors, Henk Lauer and Pat Sandra. At that meeting, de Villiers decided to pursue analytical chemistry. “Looking back now, it seems a highly fortuitous conglomeration of circum-stances that made this possible,” said de Villiers. “Essentially, my career path was determined by a 30-minute discussion with Pat Sandra and Henk Lauer.”

From the point of view of Lauer, who is currently the managing director of HLCE and was one of de Villiers’s su-pervisors of his masters and PhD the-ses, the timing of de Villiers’s decision was perfect, given the changes that were going on at Stellenbosch Univer-sity at the time. Ben Burger, a professor and the director of the Laboratory for Ecological Chemistry (LECUS), had decided that Stellenbosch University needed a chemistry department with a focus on separation science, so he enlisted Pat Sandra, who was then at the University of Ghent and also a di-rector of his own Research Institute of Chromatography (RIC), to set up such a department. According to Lauer, Sandra made sure that a strong pro-gram was established, and he imported a lot of instrumentation from Europe and helped secure funding for the new department. “De Villiers brought his talent at the right time and the right place,” said Lauer. “He showed his tal-ent with excellent study results, a great understanding of the analytical prob-lems, and use of the instrumentation available then.”

De Villiers did his postdoctoral stud-ies at the Pfizer Analytical Research Centre (PARC) at Ghent University in Belgium, from 2004 to 2006. From there he decided to return to Stellen-bosch University as a lecturer in chem-istry, a position he held from August 2006 to July 2008. In August 2008, de Villiers was promoted to senior lecturer of chemistry, and he remained in that position until December 2012. In Janu-ary 2013, he was promoted to associate

André de Villiers



professor of chemistry and continues to hold that position today.

Contributions to the Field

De Villiers’s research interests include fundamental studies that push the boundaries of the chemical charac-terization of complex mixtures using state-of-the-art techniques such as mul-tidimensional LC and GC combined with MS, and the application of these methods, primarily to natural product analysis. He has published 50 papers in peer-reviewed journals, and his papers have been cited 925 times — quite an ac-complishment for such a young scientist!

Sandra, who is now an emeritus pro-fessor with the Research Institute for Chromatography and was de Villiers’s professor and thesis supervisor at Stel-lenbosch, feels that de Villiers’s greatest contribution to the field of separation science so far is his work developing new LC methods and techniques, in-cluding comprehensive LC×LC for the analysis of natural products, such as South African wines.

Lauer agrees, citing “his endeavor to understand and nail down the complex-ity of molecules that define the color, taste, and bitterness of South African wines with all the available separation techniques he could lay his hands on.”

Tadeusz Górecki, a professor at the University of Waterloo, said he values de Villiers’s contributions to the area of multidimensional LC “from his early work with Isabelle François to his recent foray into hydrophilic interac-tion reversed-phase LC×LC.” Górecki also mentioned that de Villiers’s more theoretical work, such as his papers on

kinetic optimization of LC separations, are also of high quality.

Emily Hilder, a professor of chemis-try and director of the ARC Training Centre for Portable Analytical Separa-tion Technologies at the University of Tasmania (as well as the 2012 LCGCEmerging Leader in Chromatography award winner), agreed with Górecki’s thoughts on de Villiers’s contributions in multidimensional LC. “His work has demonstrated how 2D LC can be ap-plied to the analysis of very complex samples from natural products (wine, food, and so on),” said Hilder. “Such practical applications of this technol-ogy are what is needed to guide future developments.”

Scientific Accolades

De Villiers has received a number of awards from the separation science com-munity, including the 2009 Csaba Hor-váth Memorial Award from the Interna-tional Symposium on High-Performance Liquid Phase Separations and Related Techniques (HPLC) and the 2012 Chro-matographer of the Year award from the Chromatographic Society of South Africa. He has also been invited to de-liver lectures at prestigious international conferences, such as HPLC and the In-ternational Symposium on Hyphenated Techniques in Chromatography.

De Villiers is currently chairing the Western Cape board of the Chromato-graphic Society of South Africa. He was also responsible for the organization of two successful conferences that took place in Stellenbosch: the 39th National South African Chemical Institute con-vention in 2008 and Analitika 2010.

Future Contributions

Given what de Villiers has already ac-complished in his career, we asked

several of his mentors and peers where they thought his work might take him.

Sandra expects de Villiers to make contributions to the fundamental un-derstanding of chromatographic pro-cesses, because he has a very strong theoretical background. He also thinks de Villiers will play an important role in education. “He will definitely have a great impact in the education of students in Africa on state-of-the-art analytical techniques and, more specif-ically, chromatography and electropho-resis combined with high-resolution mass spectrometry,” said Sandra.

Górecki feels de Villiers will continue the legacy of other great South African separation scientists, like Victor Pre-torius or Ben Burger. “He has already made his mark on separation science, and the trajectory from here can only be up, especially knowing de Villiers’s talent and work ethic,” Górecki said.

Barend (Ben) V. Burger, an emeritus professor from Stellenbosch University, thinks that de Villiers might branch into GC, even though that is not his primary area of research. “I think there is still much scope for the development of more affordable two-dimensional instrumentation,” said Burger.

Frédéric Lynen, an associate professor at Ghent University, said de Villiers’s re-search would continue in natural prod-uct analysis with the “discovery of new, thus far, biologically active compounds via the combination of high-end chro-matography and the elucidation of struc-tures of unknown natural solutes.”

Table I: Winners of the LCGC Awards

Year Lifetime

Achievement

Emerging

Leader

2008 Walt Jennings Gert Desmet

2009 Harold McNair Kevin Schug

2010 Georges Guiochon

Jared Anderson

2011 James W. Jorgenson

Dwight Stoll

2012 Lloyd Snyder Emily Hilder

2013 Peter W. Carr Davy Guillarme

2014 Fred E. Regnier André de Villiers

Figure 5: Pat Sandra with his former co-workers in April 2008 in Stellenbosch, South Africa. From left to right: Pat San-dra, Frédéric Lynen, Andreas Tredoux, André de Villiers, Deirdre Cabooter, and Martina Sandra.

Figure 6: De Villiers at Cape Point, South Africa.

Continued on page 46



Recent Progress in Chiral Stationary Phase Development and Current Chiral ApplicationsChiral separations remain a decided area of interest, particularly in the pharmaceuti-cal and agrochemical fields. Although high performance liquid chromatography (HPLC) remains a strong choice for separations because of its robustness, transferability, and instrument availability, the use of chiral supercritical fluid chromatography (SFC) contin-ues to expand in analytical and preparative techniques. Several chiral stationary phases continue to enjoy wide use because of their broad application in both HPLC and SFC.

Timothy J. Ward and Karen D. Ward, Millsaps College, Jackson, Mississippi

Chiral separations continue to be of great interest because of the prevalence of racemates in markets such as the phar-maceutical and agrochemical (pesticide) industries. In fact,

a review of the importance of pharmaceutical chiral separations in single-enantiomer patent cases was recently published (1), and another review estimates that about 30% of pesticides are chiral with about half of these having multiple chiral features (2). The individual pesticide enantiomers may exhibit different effects on the environment. Although the separation of enantiomers can be challenging because of their identical physical and chemical prop-erties in an achiral environment, chiral stationary phases (CSPs) have greatly facilitated enantioseparations in high performance liquid chromatography (HPLC) and supercritical fluid chroma-tography (SFC). Research on specialized separation techniques using novel CSPs, particularly derivatized polysaccharides and cyclodextrins, continues for the resolution of specific individual enantiomers, and chiral separation on commercially available CSPs remains a mature and widely used technique with some new entries to the market. Polysaccharide and macrocyclic gly-copeptides CSPs continue to be the most widely used commercial chiral phases, with cyclodextrins, cyclofructans, π-complex, and protein-based CSPs also finding use. For non-HPLC separations such as gas chromatography (GC) and capillary electrophoresis (CE), cyclodextrins continue to dominate. Enantioseparations of larger, more-complex molecules with multiple chiral centers have increased as biotech continues to grow, meaning that more com-pounds must be resolved simultaneously, and chiral separations of more-polar molecules are needed, especially for the agrochemi-cal and pharmaceutical fields.

There are plenty of resources and information available for performing chiral separations, both from the commercial sup-pliers of CSPs and in the literature. There are numerous reviews of the widely used CSPs, including recent reviews of cellulose and polysaccharide-based CSPs (3), protein and glycoprotein CSPs (4), macrocyclic antibiotic CSPs (5), cyclodextrin CSPs (6,7), and

chiral ion- and ligand-exchange CSPs (8). Reflecting the burgeon-ing interest in SFC chiral separations of pharmaceuticals, several reviews specific to SFC have recently been published (9–12).

The State of Current CSPsChiral separation continues to be the primary technique of choice, with many companies seeing an increase of about 20% for both analytical and preparative enantioseparations in their laboratories. The market continues to enjoy growth and matura-tion as older technologies are replaced by newer and improved technologies. New CSPs continue to be introduced to the market, including the zwitterionic phases, Chiralpak Zwix(+) and Zwix(-) from Chiral Technologies, a new immobilized crown-ether phase, Chiralpak CR-I also from Chiral Technologies, and an immobi-lized ovomucoid phase, C18-Ovo-5-120 from Separation Meth-ods Technologies. Companies such as YMC America, Inc., Chiral Technologies, Diacel, and Separation Methods Technologies are continuing to expand their offerings of immobilized polysaccha-ride-derived CSPs because these columns offer greater stability, can be used with a wider variety of mobile phases, and are useful in both liquid chromatography (LC) and SFC applications.

One apparent trend in the market is toward the increased use of SFC for chiral separations. The advantages of SFC are the re-duced environmental impact and operating costs with increased throughput. Although SFC has traditionally found more use in preparative-scale chiral separations, where the waste reduction and decreased solvent use is attractive to industry, these immo-bilized CSPs are also seeing increased use in analytical applica-tions as well. SFC is currently receiving a lot attention from the pharmaceutical industry for screening and method development of chiral separations (13–15).

Polysaccharide CSPsAs in previous years, chiral separations achieved by polysaccha-ride CSPs in HPLC account for approximately one-third of all

ES443939_LCGCSUPP0614_043.pgs 05.22.2014 23:29 ADV blackyellowmagenta


HPLC chiral separations in the literature, and most of these separations are carried out on commercially available columns. The most commonly used phase on cel-lulose or amylose continues to be 3,5-di-methylphenyl carbamate, which includes immobilized columns such as Chiralpak IA (Chiral Technologies), Chiralpak IB, Lux Cellulose-1 (Phenomenex), and coated phases such as Chiralcel OD-H (Chiral Technologies), Kromasil Cel-luCoat (Akzo Nobel), and AmyCoat (Akzo Nobel). Because the conformation of the polymer is influenced by how the stationary phase is attached to the pack-ing material, coated and immobilized columns can exhibit different selectiv-

ity. Chloro-substituted polysaccharide CSPs have also found much use, includ-ing tris(3,5-dichlorophenyl carbamate) (Chiralpak IC), tris(3-chloro-4-meth-ylphenyl carbamate) (Lux Cellulose-2), tris(5-chloro-2-methylphenyl carbamate) (Chiralpak AY-H), and tris(3-chloro-4-methylphenyl carbamate) (Chiralcel OZ-H). The selectivity of the Chiralpak IC column toward different compounds is demonstrated in Figure 1.

A reversed-phase study using tris(chloromethylphenyl carbamate) de-rivatives of cellulose and amylose con-cluded that using these CSPs in screen-ing protocols yields higher success rates in achieving baseline separations with

shorter screening times (16). An updated generic separation strategy in normal-phase HPLC was reported using the fol-lowing commercial CSPs: Lux Cellulose-1, Lux Cellulose-2, Lux Amylose-2, Lux Cel-lulose-4 and Chiralpak AD-H, Chiralcel OD-H, and Chiralcel OJ-H (17). Reversed-phase screening strategies for HPLC with Chiralpak IA, Chiralpak IB, and Chiral-pak IC with applications compatible with liquid chromatography–mass spectrom-etry (LC–MS) were also reported (18). Simplified screening protocols for chiral separations in HPLC and SFC on Chi-ralpak IA, IB, IC, and ID in reasonable time frames and high success rates were recently published (14).

Cyclodextrin-Based CSPsAlthough no new cyclodextrin-based CSPS have been brought to market lately, the use of cyclodextrins still accounts for over one-fourth of all publications in chiral HPLC in recent years. The bonded cyclodextrin–based CSPs are popular because of their robustness, wide selec-tivity, and ability to enantioseparate in the reversed and polar organic phases. The mechanical stability of the cyclo-dextrin CSPs lends itself to preparative-scale use as well.

Macrocyclic Glycoprotein CSPsThe macrocyclic glycoprotein CSPs con-tinue to fill a broad and useful niche in chiral HPLC because of their unique versatility and broad selectivity, and are unsurpassed in the enantioseparation of chiral amino acids. Sigma-Aldrich offers a Chirobiotic method development kit containing V2, T, R, and TAG columns for screening in polar ionic, polar organic, reversed-phase, and normal-phase modes.

Figure 1: The separation of three compounds using a 250 mm × 4.6 mm, 5-µm dp Chiralpak IC column (Chiral Technologies). Mobile phase: 70:30:0.1 (v/v/v) n-hexane–ethyl acetate–diethylamine. Adapted from reference 24.

0 2 4 6

1-Benzoyl-2-tert-butyl-3-methyl-4-imadazoline 1-Phenylethyl-3,5-dinitrobenzoate

8

DevinrolCH

3

CH3

CH3

N

NN

O2N

NO2

O

O

O

O

O

OO

10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14

Figure 2: Examples of separations of pharmaceutical compounds on CF6 and CF7 CSPs. Adapted from reference 25.

O

O

OO

Thalidomide

Bendrofumethiazide

20 55Time (min)

N

NH

NH2

HN

O O O O

SS

NH

CF3



A review of macrocyclic glycopeptide-based CSPs in HPLC methods for amino acid enantiomers and related analogues was published in 2010 (19) and recently updated (5).

Cyclofructan CSPsThe recently released cyclofructan-based Larihc CSPs from AZYP, a new supplier of novel chiral and achiral phases for HPLC, hydrophilic-interaction chromatography (HILIC) and SFC, also available from Supelco/Sigma-Aldrich, continue to have great application in chiral HPLC sepa-rations. (Larihc is “chiral” spelled back-wards.) These columns include Larihc CF6-P, considered the “king column” for separation of racemic primary amines, Larihc CF6-RN, which separates nonpri-mary amines, and Larihc CF7-DMP, the only commercialized cyclofructan 7 col-umn, which shows complementary enan-tioselectivity to the CF6-RN CSP.

Larihc CF6-P is the only column that separates primary amines in a nonaque-ous solvent using the polar organic mode. The Larihc CF6-P was reported to be use-ful in the separation of chiral illicit drugs and controlled substances, with the other Larihc CSPs also yielding enantiosepara-

tions (20). A comparison of separations of 46 chiral reagents to determine enan-tiomeric purity using Larihc, Cyclobond (Supelco/Sigma-Aldrich), and Chirobi-otic CSPs was recently reported, with this being the first use of Larihc CSP for this purpose (21). Recent reports indicate that the Larihc CF6-P CSP is broadly ap-plicable to the separation of nonamine containing racemates as well (20–22).

All Larihc CSPs are reported to work well in SFC because of their use in the normal-phase or polar organic phase modes. A comparison of dimethylphenyl carbamate cyclofructan 7 CSP use in SFC and HPLC was recently made (23) and the retention and enantiodiscrimination properties and the effect of different SFC modifiers was reported.

Chiral Separations TodayAlthough the chiral market is matur-ing and stable, perhaps the biggest need in the field remains an increased overall understanding of chiral methodologies and more practical training. Because in-formation is largely supplied by vendors to newcomers to chiral HPLC, novices can sometimes stumble around for a while wading through vendor literature and

advice. In addition to an understandable sales objective, vendors occasionally mis-understand the customers’ end-to-end chiral operation, so promised increases are sometimes not realized at the labora-tory level. Furthermore, increased per-formance is also sometimes not realized because of low-tech reasons that may be related more to laboratory layout and operation, rather than the lack of latest technology and equipment. Newcomers should make use of the short courses on chiral HPLC that give a wealth of unbi-ased information to the participants and are offered at major conferences such as Eastern Analytical Symposium (EAS), The Pittsburgh Conference (Pittcon), and Chirality.

Increased selectivity remains a chal-lenge in chiral separations. With in-creased selectivity, loading can be in-creased in preparative chiral separations, which will provide the greatest cost sav-ings. With significant cost savings in the separation process, companies can shift from using chiral selective synthesis for chiral purification to purification by chro-matography. Although HPLC is used for preparative-scale separations, SFC con-tinues to gain ground as the most cost-effective way to purify enough material for further studies.

The market is maturing, but technol-ogy has not moved forward in any revolu-tionary way. Chiral HPLC still lacks (and may always lack) a CSP that can operate in all modes for enantioseparation of mol-ecules from low to high polarity.

AcknowledgmentsWe gratefully thank Daniel W. Arm-strong, Elena Eksteen, Hafeez Fatunmbi, J.T. Lee, and Gary Yanik for their sugges-tions in the preparation of this manu-script.

References (1) C. Weekes, Drugs Pharm. Sci. 211, 304–311

(2012).

(2) E. Ulrich, C. Morrison, M. Goldsmith, and W.

Foreman, Rev. Environ. Contam. Toxicol. 217,

1–74 (2012).

(3) J. Shen and Y. Okamoto, Compr. Chirality 8,

200–226 (2012).

(4) J. Haginaka, Compr. Chirality 8, 153–176 (2012).

(5) I. Ilisz, Z. Pataj, A. Aranyi, and A. Peter, Sep.

Purif. Rev. 41, 207–249 (2012).

Figure 3: Chromatograms showing chiral separations in normal-phase, polar organic phase, and reversed-phase modes on CF6-RN CSP. Adapted from reference 26.

(a)

(b)

(c)

0 5 10Time (min)

15

NO2

NH2

H2N

NH2

OH

Ph

O2N

O

NH

Ph

R CO2H



(6) Y. Xiao, S. Ng, T. Tan, and Y. Wang, J. Chro-

matogr. A 1269, 52–68 (2012).

(7) X. Zhang, Y. Zhang, and D.W. Armstrong,

Compr. Chirality 8, 177–199 (2012).

(8) B. Natalini and R. Sardella, Compr. Chirality 8,

115–152 (2012).

(9) R. Wang, T. Ong, S. Ng, and W. Tang, Trends

Anal. Chem. 37, 83–100 (2012).

(10) K. De Klerck, D. Mangelings, and Y. Vander

Heyden, J. Pharm. Biomed. Anal. 69, 77–92

(2012).

(11) C. West, Curr. Anal. Chem. 10(1), 99–120

(2014).

(12) K. De Klerck, Y. Vander Heyden, and D. Mange-

lings, J. Chromatogr. A 1328, 85–97 (2014).

(13) L. Kott, Am. Pharm. Rev. 16(1), 5/1–5/8 (2013).

(14) K. De Klerck, C. Tistaert, D. Mangelings, and

Y. Vander Heyden, J. Supercrit. Fluids 80, 59–59

(2013).

(15) W. Schafer, T. Chandrasekaran, Z. Pirzada, C.

Zhang, G. Chaowei, B. Xiaoyi, and R. Mirlinda,

Chirality 25(11), 799–804 (2013).

(16) L. Peng, S. Jayapalan, B. Chankvetadze, and T.

Farkas, J. Chromatogr. A 1217(44), 6942–6955

(2010).

(17) A.A. Younes, D. Mangelings, and Y. Vander

Heyden, J. Pharm. Biomed. Anal. 56(3), 521–537

(2011).

(18) T. Zhang, D. Nguyen, and P. Franco, J. Chro-

matogr. A 1217(7), 1048–1055 (2010).

(19) I. Ilisz, Z. Pataj, and A. Peter, Macrocyclic Chem.

129–157 (2010).

(20) N. Padivitage, E. Dodbiba, Z. Breitbach, and

D.W. Armstrong, “Enantiomeric separations

of illicit drugs and controlled substances using

cyclofructan-based (LARIHC) and cyclobond I

2000 RSP HPLC chiral stationary phases,” Drug

Test. Anal. doi:10.1002/dta.1534 (2013).

(21) H. Qiu, N. Padivitage, L. Frink, and D.W. Arm-

strong, Tetrahedron: Asymmetry, 24(18), 1134–

1141 (2013).

(22) J. Smuts, X. Hao, Z. Han, C. Parpia, M. Krische,

and D.W. Armstrong, Anal. Chem. 86(2), 1282–

1290 (2014).

(23) J. Vozka, K. Kalikova, C. Roussel, D.W. Arm-

strong, and E. Tesarova, J. Sep. Sci. 36(11), 1711–

1719 (2013).

(24) http://immobilizedchiralcolumns.com/csp-

robustness.

(25) http://www.sigmaaldrich.com/etc/medialib/

docs/Supelco/Posters/1/daw-chiral-010711.

Par.0001.File.tmp/daw-chiral-010711.pdf.

(26) http://www.sigmaaldrich.com/etc/medialib/

docs/Supelco/Posters/1/daw-chiral-010711.

Par.0001.File.tmp/daw-chiral-010711.pdf.

Timothy J. Ward is a professor of

chemistry and associate dean of sciences

at Millsaps College (Jackson, Mississippi).

His research interests include chiral sepa-

rations, the development of analytical LC

and CE methods, and their application to

pharmaceutical and archaeological analy-

sis. Karen D. Ward is an instructor at

Millsaps College. She previously worked in

the pharmaceutical industry at the Analytical

Environmental Research Division at Syntex

Pharmaceuticals (Palo Alto, California). Direct

correspondence to: [email protected] ◾

For more information on this topic, please visit our homepage at:


A Testament to De Villiers’s Character

Other scientists describe de Villiers as friendly and down to earth. Hilder recalls first meeting him at an HPLC conference in 2006, and said their friendship has grown since then. “The separation science community is very supportive and there is now a good group of young people all at a similar stage in their careers,” she said. “We catch up at meetings, and this makes for a great, fun support network of scien-tists for bouncing off ideas and sharing advice.” Hilder also said that she shares a love of cricket with de Villiers.

De Villiers is indeed a big sports en-thusiast — not just as a fan, according to Górecki. De Villiers regularly plays pickup football games, and is an avid biker and an aspiring surfer. “He often comes to the university in the summer with a surfboard attached to the roof of his car,” said Górecki. “This earned him the nickname ‘Professor Dude’ among his students.”

Górecki shared a story about de Vil-liers’s participating in a grueling bike race around Cape Peninsula that is more than 100 km long and has numerous climbs and strong winds. Several years

ago de Villiers crashed and injured him-self rather badly, so he could not com-plete the race. It took several hours for an ambulance to reach him. “De Villiers declared that he would never do that race again and stopped biking entirely. After a few months, though, he registered for the next year’s race and started to train again,” said Górecki. “This dedication is what drives his career as well.”

Lynen paraphrased one of de Villiers’s favorite quotes: Something worth doing is worth doing well. “I always found this phrase very characteristic of his person-ality,” said Lynen. He also mentioned a research problem that de Villiers worked on in 2003 to address peak distortion problems when drawing the calibration lines of organic acids in capillary elec-trophoresis. Lynen explained that the problem looked very strange and dif-ficult to solve, but de Villiers was able to correctly deduce that the increasing concentrations of each organic acid cali-brant were effectively lowering the pH of the migrating zone and, as a result, creating an electrodispersion phenom-enon that could be fixed by adjusting the sample pH (2). “The meticulous ap-

proach with which he addressed that problem (also by studying a lot of litera-ture on the topic) was very impressive and demonstrated the eye for detail that is characteristic of a true scientist,” said Lynen.

More About the Winners

In-depth interviews with Fred E. Regnier and André de Villiers, focused on their re-search, challenges, and accomplishments will be published in upcoming editions of our newsletter, E-Separations Solutions.

References

(1) Fred Regnier’s Indy 500 of Chromatography and

Other Stories, available at: http://www.blurb.

com/bookstore/invited/3531763/965fa935d16

7f1c23c7e4c18630aff06249d65b6?ce=blurb_

ew&utm_source=widget (2013).

(2) A. de Villiers, F. Lynen, A. Crouch, and P. San-

dra, Eur. Food Res. Technol. 217(6), 535–540

(2003). ◾



Continued from page 42



The Fundamental Shift to Tandem Mass SpectrometryIn this article, we examine how tandem and tandem hybrid mass spectrometry has opened up new frontiers already. We go further and examine how lesser-known experi-ments are breaking new ground, with alternative fragmentation techniques, as well as the addition of extra levels of orthogonality by parallel separations techniques.

St. John Skilton, Eric Johansen, and Xu Guo

Today, in the biopharmaceutical industry mass spec-trometry (MS) is a critically useful and efficient tool for routine and investigational analysis in therapeu-

tic discovery, development, and production. Almost every analytical department now routinely uses MS at some stage in the process of therapeutic development.

However, there is one intriguing aspect that is some-what surprising given the prevalence of MS; that is, a monolithic view held by some whereby all mass spectrom-eters or all techniques are lumped into a broad category labeled “MS.” This is all the more surprising given that the experiments performed are enormously varied. It is the view of these authors that such convenient shorthand results from a predominance of a small number of MS experiment types adopted by the industry. Although many may know that alternative experiments exist, few have the time to explore them and many may be unaware of the extreme utility of these experiments for greater efficiency and information, with little time penalty or method de-velopment. In this article, we touch on how tandem mass spectrometry (MS-MS) has developed and how alterna-tive uses of it may better inform the industry and speed up therapeutic design and development, with particular reference to the biopharmaceutical area.

A Brief View of MS-MS HistoryThe development of MS-MS has not been seen as obvi-ous, and has relied partly on fortuitous results and typical scientific curiosity about fundamental gas phase reactions (1,2). In the 1970s the use of MS-MS was extremely infor-mative about the behavior of ions in the gas phase and their dissociation, although it remained highly academic (3,4). In experiments that often used enormous magnetic sector instruments, advanced research was still looking intently at what was later termed “fundamentals,” re-f lecting how the field was aiming to understand the very mechanisms of what was occurring (5). In fact, MS-MS research had been a steady thread of activity right from the very start of mass spectrometry, beginning more than a century ago (1). However, the 1970s saw the massive rise of a plethora of instrument types, including some ambi-tious multiple-sector instrumentation. One type was the

tandem quadrupole, which opened up what has argu-ably been the most commercially successful type of mass spectrometer ever invented, and which still dominates the market today (8). In common terminology, this has become known as a triple quadrupole, on the basis that the middle quadrupole segment was the collision cell, al-though this mechanism has long been superseded.

But here too lies one of the continuing puzzles for many people in the field: Why has the variety of experiment types not been used more widely? The most predominant MS-MS experiment remains that used for quantification of analytes: multiple reaction monitoring (MRM), whereby a precursor is selected, and a small subset of the frag-ments are subsequently monitored to determine very pre-cisely how much of the analyte is present — mostly with reference to isotopically labeled standard analog species. However, almost all tandem mass spectrometers have the inherent capability of looking “backward” by using the fragment ion species to reconstruct what the precursor molecule was like. This has been extensively explored in the metabolite identification world — for example, where predictable biotransformations can be mapped by inte-grating the MS and MS-MS information with informat-ics packages (10). Additionally, it is also possible to look backward and mark out the parts of a molecule that are not present because they didn’t ionize or were broken into pieces that are not recognizable. Examples of this are “con-stant neutral loss” experiments, or “parent–precursor ion scans” (13). It is all the more surprising that these types of experiments are not performed more frequently because they can be done almost simultaneously in certain types of mass spectrometers (for example, tandem quadrupoles and quadrupole time-of-f light [QTOF] systems).

Tandem in Space and TimeHere too, the subtle differences in what tandem mass spec-trometers actually are is instructive: tandem itself is a con-f lation of “tandem in space” and “tandem in time.” The examples all mentioned above are of the tandem-in-space type, whereby the instrument components that analyze each of the successive fragments are separated physically. This is not the case for instruments that are “trapping,”



in which ions can be stored for some period of time, potentially forever. This distinction is important, because the differences in tandem types have largely determined which types of in-struments have been sold for specific applications in the pharmaceutical market. Traps that store specific ions and subject them to even more analy-sis are extremely good at homing in on one specific thing, and providing an extremely in-depth view of what that analyte is — even though it may be at the expense of other molecules present simultaneously. On the other hand, tandem-in-space instruments are extremely well suited for looking at a number of things simultaneously, perhaps at the expense of losing de-tail on specific items. So, why not have a combination of both attributes? In many ways, this is partly where the instrumentation industry has headed, with some surprisingly creative results.

Economically Viable AdoptionBy the time the tandem quadrupole was being widely adopted for clini-cal work in the mid 2000s, it was al-ready profiting from rapid advances,

so the invention of what many have described as a new “class” of instru-ment was also well accepted (9). But addit iona l ly, by this stage many researchers were working directly with the nascent biopharmaceutical industry and were extremely ori-ented toward the actual samples that needed to be analyzed. In an example of this, Hopfgartner and colleagues (9) commented:

“. . . the uniqueness of the in-

strument is that the same mass

analyzer Q3 can be run in two

different modes [quantitative and

qualitative] . . . [one mode,] EMC

. . . offers obvious advantages, in

particular for samples containing

very low peptide levels. For many

analytical challenges, selectivity

of ten becomes more important

than sensitivity.”

In that article, Hopfgartner and colleagues also compared the capabil-ities of trapping with “axial” ejection, which coped with the limited storage capacity of three-dimensional (3D) traps. The limited storage capacity

can be a hindrance when a mixture of large, highly charged species are examined together, where the ions that are more predisposed to become highly charged may force the exclu-sion of lower abundance species from the spectrum. In instruments with “ linear” ion traps, it is one of the reasons why low relative abundance is not as problematic (9,13). The ca-pability to sequence peptides of low abundance was noted.

Perhaps two of the answers as to why more MS-MS experiment types are not used are profit reasons and the relative slowness in the rise of truly intuitive software. The profit aspect relates to economic imperatives that determine whether instruments are able to provide value for money — and of course in constrained eco-nomic times this is all the more ap-parent. These authors suggest that as we move forward, greater emphasis will be placed on the biopharmaceu-tical industry obtaining tools that are able to simultaneously provide extensive experiment types — pro-ductivity — as well as allow post-experiment investigation — security.

LC–UV

LC–MS and CE–MS

2014 2015

Va

lue

in

bio

log

ics

an

aly

sis

2016 2017 2018

Figure 1: Graph showing the estimated relative adoption rates of MS-based detection versus optical detection for the bio-pharmaceutical market (2014–2018). Both techniques grow above 5% per annum, but MS-based techniques accelerate as more biotherapeutics reach the market and pipeline. (Data sources: various, including FiercePharma, PhRMA reports, public company reports; collated by the author.)



Figure 2: (a) An example of an automatically assigned peptide map by LC–MS-MS where each of the peaks is labeled by software. The panels at the bottom indicate the orthogonal evidence available to the reviewer in the event of queries. The coverage achieved was 98% over the 60-min run. (b) An example of an automatically assigned peptide map of the molecule trastuzumab by capillary electrophoresis electrospray ionization (CESI) separation with a color-coded assignment of the peptides identif ed (100% coverage).

TIC/XIC

Peptide Results

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Protein Sequence Coverage = 97.7% View Sequence Explain... Update Assay Information

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50%

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T7

5.34 5.647.86

11.5914.95

19.62 21.65 24.8027.40

35.9238.33 40.14 46.48 49.47

48.89

49.83

6 8 10 12 14 15 18 20 22 24 26 28 30 32 34 36 38

Time (min)

40 42 44 46 48 50 52 54 56 58 60 62 64 66 68

% I

nte

nsi

ty%

In

ten

sity

100%

50%

0%

% I

nte

nsi

ty

Peaks

TIC from 20130221_IDA 10ug_10Kppm_01.wiff (sample 1) - 20130221_peps\20130221_IDA 10ug_10Kppm_01, Experiment1: +TOF MS (400-1600)

+TOF MS (400-1600) from 20130221_IDA 10ug_10Kppm_01.wiff@RT:2.26, Mono m/z 503.2819 from 2.05 to 2.78 min +MS/MS (100-1600) from 20130221_IDA [email protected] min, Precursor: 503.2822 Da.

Graph Peaks Graph Fragments

1.2e7

1.1e7

1.0e7

9.0e6

8.0e6

7.0e6

6.0e6

5.0e6

4.0e6

3.0e6

2.0e6

1.0e6

0.0e6

Inte

nsi

ty (

cps)

Time (min)13

Graph

m/z Tolerance: ±

Project

Intact Protein Workfow

Peptide Mapping Workfow

System

20.0 ppm

Automatic

Time Selection – –/ min

6Dec13Her2ugPerul_100mMLE_250TOF50IDA30ions5sec500cps_1to5WithChargeSel_1.wiff

Processing Parameters

Assay Information

Characterize Standard

Create Batch

Review Results

View Queue

Create Report

Characterize Standard

Create Batch

Review Results

RT Range Processing

TIC/XIC

Characterize Standard for Peptide Mapping

Open File...Process

14

XIC from 6Dec 13Her2ugPerul_100mMLE_250TOF50IDA30ions5sec500cps_1to5WithChargeSel_1.wiff(sample 1) - Her9. Experiment 1: +TOF MS (100-2000) 284.1755 ±0.0125 Da

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

(a)

(b)



Now that many experiment types are instantaneous within the constraints of typical experimentation, the abil-ity to go back to find data that may have always been there becomes much more feasible. In fact, many of these advances have been quietly adopted as standard tools, including the abil-ity to automatically assign peptide sequences and look at the sequence simultaneously (14). The use of in-formatics has progressed, but perhaps by virtue of not being as emotionally exciting as advances in expensive capital equipment, it is paradoxically perceived as less exciting. But there has been a crucial development in the industry: the ability to go back and re-examine data with fresh hypoth-eses without new sample injections. So, the combination of instrument capability and informatics has pro-vided some real benefits, not least of which is the automation of many te-dious tasks otherwise performed by humans.

Conclusion

The practical application of MS-MS has long been recognized, and a sub-set of the experiments has long been a default tool in the industry (MRMs). But it is reasonable to argue that the depth of capability has remained

untouched. It can be argued that it is economic imperatives that push organizations to start to use more of the tools already at their disposal to obtain more out of their capital ex-penditures. In some cases, this might mean finding contaminant host cell proteins more rapidly, or identifying previously unknown sequence vari-ants in an effort to improve product quality as quickly as possible (15). Under all circumstances, MS-MS will only increase in value, usage, and, un-derstanding.

References

(1) http://www.asms.org/docs/history-post-

ers/tandem-ms-poster-2012.pdf?sfvrsn=2.

(2) R.G. Cooks and J.H. Beynon, J. Chem.

Educ. 51(7), 437Ð43 (1974).

(3) E. Gustafsson and E. Lindholm, Arkiv foer

Fysik 18, 219Ð39 (1960).

(4) J.H. Beynon et al., Int. J. Mass Spectrom.

Ion Phys. 3(5), 313Ð21 (1969).

(5) F.W. McLafferty, D.J. McAdoo, and J.S.

Smith, J. Am. Chem. Soc. 91(19), 5400Ð1

(1969).

(6) A.L. Yergey, J.R. Coorssen, P.S. Backlund,

P.S. Blank, G.A. Humphrey, J. Zimmer-

berg, J.M. Campbell, and M.L. Vestal, J.

Am. Chem. Soc. 13(7), 784Ð791 (2002).

(7) B.A. Mamyrin, V.I. Karataev, D.V.

Shmikk, and V.A. Zagulin, Zh. Eksp. Teor.

Fiz. 64(1), 82Ð9 (1973).

(8) M.L. Vestal and J.H. Futrell, Chem. Phys.

Lett. 28(4), 559Ð61 (1974).

(9) G. Hopfgartner, E. Varesio, V. TschŠp-

pŠt, C. Grivet, E. Bourgogne, and L.A.

Leuthold, J. Mass Spectrom. 39, 845Ð855

(2004). DOI: 10.1002/jms.659. Available

at: http://medchem.rutgers.edu/AnalMed-

Chem511/pd f_ f i le s /R B _pdf/LIT%20

and%20QQQ.pdf.

(10) G. Hopfgartner, I.V. Chernushevich,

T. Covey, J.B. Plomley, and R. Bonner,

J. Am. Soc. Mass Spectrom. 10, 1305

(1999).

(11) G. Hopfgartner and F. Vilbois, Analusis

28, 906 (2001).

(12) J.W. Hager, Rapid Commun. Mass Spec-

trom. 17, 1389 (2003).

(13) I.V. Chernushevich, A.V. Loboda, and

B.A. Thomson, J. Mass Spectrom. 36,

849Ð865 (2001).

(14) L.C. Gillet, P. Navarro, S. Tate, H. Ršst,

N. Selevsek, L. Reiter, R. Bonner, and R.

Aebersold, Mol. Cell . Proteomics 11(6),

PMID 22261725 (2012).

(15) J. Hogan and S.J. Skilton, ÒEff iciency

Gains with Sequence Variant Analysis by

Mass Spectrometry,Ó Genet. Eng. Biotech-

nol. News webinar (2014). Available at:

http://www.genengnews.com/webinars/

eff iciency-gains-with-sequence-variant-

analysis-by-mass-spectrometry/219/.

(16) http://www.absciex.com/applications/

bioma rker-d i s cover y-a nd-omic s -

research/swath-acquisition.

(17) ht tp://w w w.absciex .com/Documents/

Appl ic a t ion s /HCP_Tech _ Note%20 _

Detailed_FINAL.pdf.

St. John Skilton, PhD, is a

senior global marketing manager for

biologics with AB Sciex in Framingham,

Massachusetts. Eric Johansen,

PhD, is a global technical marketing

manager for biopharmaceutical applica-

tions at AB Sciex. Xu Guo is an appli-

cation scientist in the product applica-

tion lab at AB Sciex.

Direct correspondence to: stjohn.

[email protected] ◾

Peptide A = I

Peptide B = I

Peptide C = I

Figure 3: Graphical illustration of data acquisition using informatics software (Swath, AB Sciex) where precursor and fragment ions are collected through an entire experiment. This technique provides a comprehensive dataset in a peptide map that can be remined at a later date with new hypotheses or to extract infor-mation that was not predicted. For more information see reference 16.




THE APPLICATION NOTEBOOK

Call for Application Notes

LCGC is planning to publish the next issue of

T e Application Notebook special supplement in

September. T e publication will include vendor

application notes that describe techniques and

applications of all forms of chromatography and

capillary electrophoresis that are of immediate in-

terest to users in industry, academia, and govern-

ment. If your company is interested in participat-

ing in these special supplements, contact:

Michael J. Tessalone, Group Publisher,

(732) 346-3016

Edward Fantuzzi, Associate Publisher,

(732) 346-3015

Stephanie Shaf er, East Coast Sales Manager,

(774) 249-1890

Lizzy T omas, Account Executive,

(574) 276-2941

Application Note Preparation

It is important that each company’s mate-

rial f t within the allotted space. T e editors

cannot be responsible for substantial editing

or handling of application notes that deviate

from the following guidelines:

Each application note page should be no more

than 500 words in length and should follow

the following format.

Format

• Title: short, specif c, and clear

• Abstract: brief, one- or two-

sentence abstract

• Introduction

• Experimental Conditions

• Results

• Conclusions

• References

• Two graphic elements: one is the company

logo; the other may be a sample chromato-

gram, f gure, or table

• T e company’s full mailing address,

telephone number, fax number,

and Internet address

All text will be published in accordance with

LCGC ’s style to maintain uniformity through-

out the issue. It also will be checked for gram-

matical accuracy, although the content will not

be edited. Text should be sent in electronic for-

mat, preferably using Microsoft Word.

Figures

Refer to photographs, line drawings, and

graphs in the text using arabic numerals in

consecutive order (Figure 1, etc.). Company

logos, line drawings, graphs, and charts must

be professionally rendered and submitted as

.TIF or .EPS f les with a minimum resolution

of 300 dpi. Lines of chromatograms must be

heavy enough to remain legible after reduc-

tion. Provide peak labels and identif cation.

Provide f gure captions as part of the text,

each identif ed by its proper number and title.

If you wish to submit a f gure or chromato-

gram, please follow the format of the sample

provided below.

Tables

Each table should be typed as part of the main

text document. Refer to tables in the text by

Roman numerals in consecutive order (Table I,

etc.). Every table and each column within the

table must have an appropriate heading. Table

number and title must be placed in a continu-

ous heading above the data presented. If you

wish to submit a table, please follow the format

of the sample provided below.

References

Literature citations must be indicated by arabic

numerals in parentheses. List cited references

at the end in the order of their appearance. Use

the following format for references:

(1) T.L. Einmann and C. Champaign, Science

387, 922–930 (1981).

T e deadline for submitting application notes for the September issue of T e Application Notebook is:

July 30, 2014

T is opportunity is limited to advertisers in LCGC North America. For more information, contact:

Mike Tessalone at (732) 346-3016, Ed Fantuzzi at (732) 346-3015, Stephanie Shaf er at (774) 249-1890, or Lizzy T omas at (574) 276-2941.

Table I: Factor levels used in the designs

Factor Nominal value Lower level (−1) Upper level (+1)

Gradient profile 1 0 2

Column temperature (°C) 40 38 42

Buffer concentration 40 36 44

Mobile-phase buffer pH 5 4.8 5.2

Detection wavelength (nm) 446 441 451

Triethylamine (%) 0.23 0.21 0.25

Dimethylformamide 10 9.5 10.5

Figure 1: Chromatograms obtained using the conditions under which the ion sup-pression problem was originally discov-ered. The ion suppression trace is shown on the bottom. Column: 75 mm × 4.6 mm ODS-3; mobile-phase A: 0.05% heptaf uo-robutyric acid in water; mobile-phase B: 0.05% heptaf uorobutyric acid in aceto-nitrile; gradient: 5–30% B in 4 min. Peaks: 1 = metabolite, 2 = internal standard, 3 = parent drug.



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