improved lc methods in food qc and routine food analysis€¦ · · 2016-09-04improved lc methods...

Improved LC Methods in Food QC and Routine Food Analysis

Dayin Lin, Ph.D.

Credits: Mr. Adam Horkey, Schwabe NA

(USA) Dr. Udo Huber & Dr. Sonja

Schneider (Agilent DE)

Agenda

• Agilent UHPLC for Food QC

• Faster QC with 1290 Infinity LC

• Agilent 1200 Infinity HDR DAD and cost-saving solution

• 1220 Mobile LC for Beer QC

Agilent Food Seminar

2

Agilent Liquid Phase Separations Portfolio Overview

1290 Bin

1260

Bin

1220

Grad

1220

Iso

SFC 1260 SFC and SFC/MS

1260 Hybrid SFC/UHPLC-MS

Bio-inert LC 1260 Bio-inert LC

1260 Bio-SEC

1290 Quat

1260

Quat

1260

Iso

Dissolution 708/709, 400, BIO-DIS,

8000, MQ 850

High-end UHPLC

Core UHPLC

HPLC-Chip/MS UHC-NCE, Phospho Peptide

mAB-Glyco, HiP Protein ID

Lowflow LC 1290 Micro, 1260 Cap

1260 Nano

GPC-SEC 1260 GPC, 1260 MDS,

GPC 50, GPC 220

Cap. Electrophoresis 7100 CE and CE/MS

Workflow Automation Solutions Multi-method/Walk-up, Method Dev., ISET, 2D-LC, Online SPE, HT LC/MS, ACR,

Online Enrichment and Clean-up, HDR-DAD, Mobile LC, Online Sampling, etc

Preparative LC 1260 uS, 1260 AS, 1260 PS

218, SD1

HDR


Analysis of Curcumins with UHPLC

1290 Infinity

Binary LC

Abstract: Improving traditional USP method in analysis of curcuminoids

with Agilent 1290 Infinity LC and STM column

Food Testing

Pub. number: 5991-3340EN

Agilent 1290 Infinity LC Agilent’s leading UHPLC

Requirement/benefit:

Significantly improvement over

current USP analysis method for

curcumins: much faster and huge

savings in labor and solvent use.


4

World of Turmeric Spice

• Natural extract from the Turmeric spice

• Various known benefits

• Arthritis, digestive problems and urinary issues

• Low energy

• Variety of skin and wound conditions

• Alzheimer’s disease, diabetes, allergies

• USP method for Analysis


5

Current USP Method

Curcuminoids in turmeric extract using USP method with an Agilent

ZORBAX Eclipse Plus C18, 4.6x250 mm, 5µm column

USP Curcuminoids RS

Lot F0H161 (Cat. 1151866)

USP Monograph: Curcuminoids (PF 33 (6))

Test: Content of curcuminoids

Solution: Standard Solution 1


6

Agilent 1290 Infinity LC Method with STM column

Curcuminoids in turmeric extract using an Agilent 1290 Infinity LC with

an Agilent ZORBAX Eclipse Plus C18, 2.1x50 mm, 1.8 µm column


7

Method Comparison


8

Summary of Improvements - 1290 LC method vs. USP method

• 30x faster with Agilent 1290 Infinity LC method

• Similar peak resolution, much higher sensitivity

• 95% reduction in solvent consumption

• Significant cost saving in labor


9

QA/QC of Vitamin Tablets

1290 Infinity

Binary LC

Preliminary: Analysis of high-dosed and low-dosed vitamins in one run

using the Agilent Infinity High Dynamic Range DAD

Food Testing & Agriculture


Agilent 1200 Infinity Series High Dynamic Range (HDR) DAD

solution 30x Wider Linear Range


Quantitation of high abundant

(e.g. vit. C) and low abundant

vitamins (e.g. vit. B12) for the

QA/QC of the tablets in a single

run. Saves time, solvent, waste

and therefore money for the

customer.


10

1.4 1.8 2.0 1.3

>10

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

1040 > 1090 1090 > 1050 1050 > 1100 1100 > 1200 1200 >1290

History of DAD Sensitivity Gain The last 30 years

Detector Model (Intro Year)

Se

nsitiv

ity G

ain

(1988) (1986) (1995) (2006) (2010)

1260 / 1290 Infinity

Diode Array Detector


11

History of DAD Linearity Gain The last 30 years

Detector Model (Intro Year)

1.4 1.7 2.0 1.4 2.9

> 30

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

1040 to 1090 1090 to 1050 1050 to 1100 1110 to 1200 1200 to 1290/60 1200 to HDR

Lin

ea

rity

Ga

in

(1988) (1986) (1995) (2006) (2010) (2011)

1200 Infinity HDR-DAD

With HDR

>30X Linearity


12

Deuterium

Lamp

Max-Light Cartridge Cell

10 mm or 60 mm path length

Programmable

or fixed slit

1024 element

diode-array

Grating Mirror

Optofluidic Waveguides: Max-Light Flow Cells Total-internal reflection in a non-coated fused silica fiber

1260 / 1290 Infinity DAD


13

Effects of path length increase

Peak Dispersion

60 mm pathlength

large geom. cell volume (78 µL)

lower light transmission

- Loss of resolution

- Loss of signal height

10 mm pathlength

small geom. cell volume (13 µL)

high light transmission

Conventional flow cells:

60 mm pathlength

4 µL σV dispersion volume

Max-Light High Sensitivity cell:

Optofluidic waveguides

(total internal reflection)


14

10x Higher Sensitivity 1290/1260 Infinity DAD compare to 1200 Series DAD SL

1260/1290 DAD

60 mm

1200 DAD SL

10mm

Height [mAU] 28.876 4.938

Noise [mAU] 0.0098 0.0190

Signal/ noise 2944 259

Increase 11.4 x

min 0 1 2 3 4 5

mAU

0

5

10

15

20

25

30

Columns: 150 x 4.6mm Zorbax SB C18, 5µm

Sample: Anthracene: 835 pg/µL

Mobile phase: A: Water, B: Acetonitrile

Elution: isocratic 80 % B

Injection volume: 5 µL

Flow: 1.5 mL/min

DAD: 251/4nm, Ref= 450/80nm, 2.5Hz, slit width 4nm

1200 Series

DAD / DAD SL

1260/1290

Infinity DAD


15

Linearity, linear range and dynamic range 1200 Series Diode Array Detector, 10 mm flow cell

Conc [µg/mL]

Detector Signal

[AU]

LOD

(3x S/N) Conc (upper limit)

linear range

Detector

noise

7*10-6

Conc (upper limit)

dynamic range

Linear range

Dynamic range

2.0

Slope = Response Factor

(Sensitivity)

HDR- DAD

HDR- DAD

Upper limit

5

10_3

__

NoiseDetector

LimitUpperrangeLinear

10 mm


16

30x Wider Linear Range with HDR-DAD 3.7 mm and 60 mm and Max-Light flow cell

60 mm path-length

3.7 mm path-length

Computing the signals from 60 mm path-length for the low concentration

3.7 mm path-length for the high concentration

Detector Signal

[AU/cm]

Conc

[µg/mL]


17

30x Wider Linear Range with HDR-DAD 3.7 mm and 60 mm and Max-Light flow cell

Conc

[µg/mL]

Detector Signal

[AU/cm]

10 x higher sensitivity ~3x higher upper limit

30x lower Limit of Detection (LOD)

30x lower Limit of Detection (LOD) for impurity analysis

- for more reliable automated peak integration

- higher area precision

60 mm

3.7 mm


18

1290/1260 High Dynamic Range – HDR Increasing Linear UV Range by >30x

Technology Cluster of two DADs

DAD 1: 60 mm Cell – for low

concentrations

DAD 2: 3.7 mm Cell – for high

concentrations

Output: One, combined signal,

normalized to 10 mm path length (HDR

range: 0.6 x 10-6 to 6.7 AU/cm) vs for

1200 Series: 7 x 10-6 to 2 AU/cm)

Control / Usability Like standard 1290/1260 DAD: Control,

data analysis, reporting

Investment Protection Existing 1290/1260 DAD can be upgraded

(by 2nd DAD)

Existing 1100/1200 Systems can be

upgraded

(by two 1290 or 1260 DAD) 3.7mm

60mm

Clustered DAD

DAD 1:

DAD 2:


19

QA/QC of Vitamin Tablets Scope

• DAD analysis of main and trace components might require two separate

analyses to be able to quantify all components.

• This can be solved by using the 1200 Infinity HDR DAD solution by the

enhanced dynamic range.

• In vitamin tablets some vitamins are present at high concentrations (e.g. vit. C),

whereas for example vitamin B 12 is present in very low concentrations.

• In the literature for vitamin B12 different sample preparation procedures and

different chromatographic methods are used to be able to detect and quantify

vitamin B 12 in vitamin preparations.1,2

• Until now it was not possible by applying one sample preparation and one

chromatographic method to detect and quantify all water soluble vitamins

present in in a vitamin tablet.

1 U.Kirchner, K.Degenhardt, G.Raffler, Journal of AOAC International Vol.95, No4, 2012 2 Rada Amidzic et al, J.Serb.Chem.Soc.70 (10) 1229-1235 (2005)


20

QA/QC of Vitamin Tablets Chromatogram

No: Name:

1 Vitamin C (B)

2 Niacinamid (B)

3 Pantothenic acid (A)

4 Vitamin B6 (A)

5 Vitamin B1 (B)

6 Vitamin B12 (C)

7 Vitamin B2 (C)

Column ZORBAX RRHT Eclipse plus C18, 4.6 x 150 mm, 1.8 µm

Mobile phase A A=Water + 4 g/L KH2HPO4 + 0.5 g/L hexanesulfonate, pH 3 with phosphoric acid

Mobile phase B Methanol

Gradient at 0 min 3%B, at 1 min 3%B, at 10 min 34%B, at 11 min 50%B

Flow rate 1.2 mL/min

Col.temp. 40 °C

Inj. volume 2 µL

Data acq. (DAD) A: 220/10 nm, B: 265/10 nm, C: 360/10 nm (ref. off)

min 2 4 6 8 10 12

mAU

0

500

1000

1500

2000

1

2

3

4 5

6

7


21

QA/QC of Vitamin Tablets 1260 Infinity DAD with 10 mm path length cell

min 2 4 6 8 10 12

mAU

0

500

1000

1500

2000

>2000 mAU linearity limit

Red: 220 nm

Blue: 265 nm

Green: 360 nm

min 10 11 12

mAU

0

0.1

0.2

0.3

0.4

0.5

0.6

Peak height = 0.145998 mAU

PtoP noise = 0.03282 mAU

S/N = 4.4 Vit.

B1

2


22

QA/QC of Vitamin Tablets 1260 Infinity DAD with 60 mm path length cell

min 2 4 6 8 10 12

mAU

0

500

1000

1500

2000

2500

3000

3500

4000


min 10 10.2 10.4 10.6 10.8 11

mAU

0

0.2

0.4

0.6

0.8

Peak height = 0.768691 mAU

PtoP noise = 0.04776 mAU

S/N = 16.1

Vit.

B1

2


23

QA/QC of Vitamin Tablets 1260 Infinity HDR DAD

min 0 2 4 6 8 10 12

mAU/cm

0

250

500

750

1000

1250

1500

1750

2000


Typically 6700 mAU

min 9.5 10 10.5 11

mAU/cm

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16 Peak height = 0.126319 mAU/cm

PtoP noise = 0.007922 mAU/cm

S/N = 16

Vit.

B1

2


24

QA/QC of Vitamin Tablets Summary of results

Compound

Quantitation with

1260 Infinity DAD

with 10 mm cell

Quantitation with

1260 Infinity DAD

with 60 mm cell

Quantitation with

1260 Infinity DAD

HDR

Vitamin C

Niacinamid

Pantothenic acid

Vitamin B6

Vitamin B1

Vitamin B12

Vitamin B2


25

QA/QC of Vitamin Tablets Summary of results

• The analysis of vitamin B 12 and other water soluble

vitamins present in vitamin tablets is possible in a single

run using the Agilent 1200 Infinity HDR DAD

• 200 pg (injected amount) of vitamin B12 can be quantified

• 6000 ng (injected amount) of vitamin C can be quantified

simultaneously

• Tedious sample preparation procedures for vitamin B 12

can be avoided


26

Onsite QC of Beers

1220 Infinity

Mobile LC

Quantification of Isohumulones and Reduced Isohumulones Using the

Agilent 1220 Infinity Mobile LC Solution


Agilent 1220 Infinity Mobile LC Now the LC is near your sample!


With the mobile LC the analysis can

be easily done at the site that

produces beers.

Food Testing


27

Mobile Beer Testing is Now Possible

• Analysis of isohumulones in different beer samples

• Direct injection of beer samples and isocratic elution mode enables the

user to perform easy on-site measurements in a mobile laboratory

• With this setup, nonreduced and reduced isohumulone standards were

analyzed with high precision and linearity

• Isohumulones were determined and quantified in 14 different beer

samples

• International Bitterness Units (IBU) were calculated for the tested beer

samples revealing significant differences from, for example, lager beer to

pils

• Reduced isohumulones could only be detected in one beer sample, an

American lager beer

28


Chromatographic Conditions

29

Solvent

Flow rate

Stoptime

Injection volume

Temperature TCC

DAD

Peak width

ACN/H2O + H3PO4 to pH 2.8 (52:48, v/v) +

1mL EDTA 0.1M per liter solvent

1.8 mL/min

20 min

5 (standards) or 20 µl (beer samples)

injection with needle wash

35°C

270 nm/ 4 nm, Ref.: OFF

>0.025 min (0.5 s response time) (10 Hz)


One Step Separation

30

1

2

3

4

5

6

7

9

8

Nr. Substance

1 Trans-isocohumulone

2 Trans-isohumulone

3 Trans-isoadhumulone.

4 Tetrahydro-trans-isocohumulone

5 Tetrahydro-cis-isocohumulone

6 Tetrahydro-trans-isohumulones

7 Tetrahydro-cis-isohumulones

8 Tetrahydro-itrans-soadhumulone

9 Tetrahydro-cis-isoadhumulone

min 2 4 6 8 10 12 14 16 18

mAU

0

50

100

150

200

250

300

Separation of nine isohumulone standards, nonreduced (Peaks 1–3) and

reduced (Peaks 4–9) with isocratic elution


Linearity and precision in RSD (%) for all nine analyzed iso-α-acids

31

Nr. Iso-α-acids

Correlation

coefiicient

R

RT

RSD

[%]

Area

RSD

[%]

1 Trans-isocohumulone 0.99987 0.097 0.199

2 Trans-isohumulone 0.99987 0.083 0.157

3 Trans-isoadhumulone. 0.99988 0.099 0.158

4 Tetrahydro-trans-isocohumulone 0.99989 0.102 0.093

5 Tetrahydro-cis-isocohumulone 0.99988 0.065 0.148

6 Tetrahydro-trans-isohumulones 0.99989 0.092 0.142

7 Tetrahydro-cis-isohumulones 0.99987 0.062 0.148

8 Tetrahydro-itrans-soadhumulone 0.99957 0.089 0.693

9 Tetrahydro-cis-isoadhumulone 0.99989 0.064 0.145


Comparison of IBUs, literature vs. experimental

32

Yeast type Beer type IBU experimental IBU literature*

top-fermented

(Saccharomyces cerevisiae)

Weizen 11 8-15

Kölsch 23 20-30

Irish Stout 40 30-45

Northern English Brown

Ale 24 20-30

bottom-fermented

(Saccharomyces

carlsbergensis)

Premium Lager 26 18-30

American Premium Lager 19

Lager 14 8-15

Export 27 23-30

Bock 29 23-35

Pils 26

25-45, partly up to

100

Pils 38

Pils 27

Pils 60

Pils, alcohol-free 49

*The literature IBUs were taken from the Beer Judge Certification Program, Inc.


Separation of isohumulones (A) and of reduced ones (B)

33

min 2 4 6 8 10 12 14 16 18

mAU

0

2

4

6

8

1

2

3

4

5 6

1 Trans-tetrahydroisocohumulone

2 Cis-tetrahydroisocohumulone

3 Trans-tetrahydroisohumulone

4 Cis-tetrahydroisohumulone

5 Trans-tetrahydroisoadhumulone.

6 Cis-tetrahydroisoadhumulone.

Be

er

ma

trix

min 2 4 6 8 10 12 14 16 18

mAU

0

5

10

15

20

25

1

2

3

4

5

6

1 Trans-isocohumulone

2 Cis-isocohumulone

3 Trans-isohumulone

4 Cis-isohumulone

5 Trans-isoadhumulone.

6 Cis-isoadhumulone.

Be

er

ma

trix

A

B


Summary of Beer Bitterness Analysis

34

• Easy analysis with direct injection (without SPE) and

isocratic elution

• Analysis of nonreduced and reduced isohumulones was

highly precise and linear with correlation coefficients over

0.999 %.

• The IBUs were calculated, and, as expected, significant

differences were found from weizen beer to pils

• Nonreduced isohumulones were detected in most beers

except for the American premium lager, which contained

only reduced isohumulones.

• The 1220 Infinity Mobile LC Solution is a robust and rugged

system that enables easy on-site measurement of

isohumulones in beer in a simple analytical setup



35

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improved lc methods in food qc and routine food analysis€¦ · · 2016-09-04improved lc methods...

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