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© 2000 Waters Corp

Carbamates - Analysis byAPI LC/MS

Eric Block, John Van Antwerp

LC/MS

Waters Corporation

Introduction to carbamate analysis by Atmospheric Pressure Ionization (API)LC/MS.

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Appropriate ChromatographicConditions

HPLC: Waters Alliance™ SystemColumn: Waters Symmetry™C18, 1.0 x150 mmTemperature: 35°CInjection Volume: 10µLMobile Phase:

A: 10% Methanol/10 mM NH4AcB: 90% Methanol/10 mM NH4Ac

Flow Rate: 75 µL/min

Gradient: Time %A %B Curve Initial 90 1010 10 90 612 10 90 6

Note that the chromatographic conditions are different from the HPLC post-columnderivatization method .

A Symmetry C18 with a 1.0 mm ID (inner diameter) was used.

An ammonium acetate buffer was added to the mobile phase in order to createbetter ionization for the mass spectrometer.

Optimum flow rate for the column dimension was 75 µL/min.

The gradient program for the HPLC post-column derivatization method is fairlyintricate, because fluorescence and UV detection are 2-dimensional. For goodquantitation, each peak must be baseline resolved from one another. Mass Spec, onthe other hand, adds the additional dimension of mass, allowing the massspectrometer to “resolve” co-eluting compounds on the basis of their molecularweight. For this reason, the simpler and faster gradient, shown here as used.

Note. Later work with spiked matrix used a 2.1 mm x 150 mm column. By simply scalingup the flow rate, the same chromatography could be obtained without the excessive delaytimes incurred with the microbore column.

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Mass Spectrometer ConditionsInstrument: Waters ZMD ZSpray™ Mass Detector

Interface: Positive Electrospray (ESI+)

Scan Function: Multiple Selected-Ion Recording (SIR):

Time Cone Dwell Group mins. Compound Mass Voltage Time

1 0-9 Aldicarb Sulfoxide 207.1 18V 0.5 secsAldicarb Sulfone 223.2 25V 0.5 secsOxamyl 237.2 10V 0.5 secsMethomyl 163.2 15V 0.5 secs

2 9-11 3-OH Carbofuran 238.2 15V 1.5 secs3 10.5-12.5 Aldicarb 208.2 8V 1.5 secs4 11.5-14 Propoxur 210.2 18V 0.4 secs

Carbofuran 222.2 22V 0.4 secsCarbaryl 202.2 18V 0.4 secs

5 14-20 Methiocarb 226.2 19V 0.6 sec

MassLynx ™ Software

The ZMD mass detector can easily switch between positive and negative ionmonitoring. However, all of the analytes in this case worked best using theelectrospray interface in the positive ion mode. The ZMD operates in full scan orselected ion recording (SIR) acquisitions modes.

The best sensitivity for carbamates is obtained running in SIR mode. TheMassLynx software group function tells the mass spectrometer which ions tomonitor. This slide shows the acquisition program used for quantitative analysis. Aspecific ion is monitored for each analyte over its retention time window. As eachion is monitored, the mass spectrometer uses the optimum cone voltage setting forthat analyte, increasing sensitivity.

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00T im e0

100

%

12.51

9.95

6.67

6.05 7.3911.44

15.17

12.96

0.00 2.50 5.00 7.50 10.00T ime0

100

%

1

100

%

4

100

%

1

100

%

B : A ldicarb Su lfon eC : O xamylD : MethomylE : 3-OH Carbofura nF : A ldicarbG : P ropoxurH: C arbofuranI : C arbarylJ : Meth iocarb

A : A ldicarb S ulfoxideCarbamates Full-Scan TIC

AB,C

D

E

F

G,H

IJ

Oxamyl [M+NH4]+1 = 237.2

Methomyl [M+H]+1 = 163.2

Aldicarb [M+H]+1 = 223.2Sulfone

Aldicarb [M+H]+1 = 207.1Sulfoxide

Selected Ion Recording

20 ng Each on-column

Minutes

This is a good illustration of how the mass spectrometer can resolve co-elutingpeaks. The large plot is the total ion chromatogram, which plots the sum of all ionsignal in each scan versus time. It is roughly equivalent to a UV or fluorescencedetector output. As can be seen, the early-eluting compounds are poorly resolvedand co-elute. However, since each mass acts as an independent data channel, byplotting masses specific to each compound (inset), the mass spectrometer is able toextract four discrete peaks. Since the masses are distinct for each compound, theycan be quantitated even though they co-elute.

Note: The poor peak shape of the first compound is a result of the fact that thesample was injected from a 100% organic solution. Subsequent work usedstandards/samples in more aqueous solutions, which improved peak shape of theearly-eluting components.

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00Time0

100%

0

100%

0

100%

0

100%

0

100%

9.68

6.94

6.27

6.07

5.28

2

100%

4

100%

-18

100%

1

100%

4

100%

15.02

12.83

12.36

12.31

11.23

Ald. Sulfoxide

Ald. Sulfone

Oxamyl

Methomyl

3-OH Carbofuran

Aldicarb

Propoxur

Carbofuran

Carbaryl

Methiocarb

Composite SIR Chromatograms of 10 CarbamatesSensitivity: 50 pg On-Column

s/n=6:1

s/n=80:1

This slide illustrates the sensitivity of the method. A mixture of all compounds (5pg/µL) was injected and analyzed. Displayed are all the chromatograms of all themonitored ions. This represents 50 pg (<500fmol) of each compound on-column.Signal to noise varied from 6:1 for methomyl, to approximately 80:1 for propoxur

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m/z

[M+H]+

120 140 160 180 200 220 240 260 280 300 320 340 360 380 4000

100

%

163

185 [M+Na]+

Background-subtracted Full-scan ESI+ Spectrumof Methomyl

[M+K]+ [2M+H]+

[2M+Na]+

This slide shows the mass spectrum of methomyl acquired by the ZMD using theelectrospray interface in positive ion mode. The most intense ion is the [M+H] + ionat 161. However, several other ions are seen, including the sodium and potassiumadduct ions as well as the dimer and its adduct ion.

This is a good illustration of how instrument response is compound-dependent.

Note: These slides are from infusion of individual carbamate standards atconcentrations of approximately 20 ppm. Dimerization is not uncommon wheninfusing relatively high concentrations of sample.

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120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

m/z

0

100

%

237

223

242

278

[M+NH4]+

[M+Na]+

[M+H]+

Background-subtracted Full-scan ESI+ Spectrumof Oxamyl

For Oxamyl, the expected protonated molecular ion at m/z 200 is minisculecompared to the adduct ions. Clearly this compound is very ionophoric, and willscavenge any cations in solution. For this reason the mobile phase contained 10mM ammonium acetate in order to provide a constant level of ammonium cationsand the ammonium adduct at m/z 237 was monitored in subsequent quantitativeanalyses.

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120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z

0

100

%

202

145

219

[M+H]+

[M+NH4]+

Background-subtracted Full-scan ESI+ Spectrumof Carbaryl

This slide shows the resulting ESI spectrum of Carbaryl. This compound gave asimpler spectrum where the protonated molecular ion at m/z 202 displayed thegreatest intensity. This ion would be monitored for quantitative studies.

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Quantitation of Carbamates via LC/MS

• Once the retention times and mass spectral behavior ofcarbamates is known under appropriate chromatographicconditions, we are ready to quantitate.

• Must still achieve acceptable limits of detection andquantitation to insure reliable pesticide screening.

• Must still achieve the same limits of detection andquantitation with actual samples in matrix.

In order to quantitate, we must first evaluate the sensitivity, linearity, accuracy, andprecision of the method using standards. Then we can run actual samples.

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0.0 200.0 400.0 600.0 800.0 1000.0ng/mL

0

Compound 4 name: methomyl (m/z 163)Coefficient of Determination: 0.995876Response type: External Std, AreaCurve type: Linear, Weighting: 1/x 5.96e6

Response

Methomyl Calibration Curve; 5-1000 ng/mL

Here is a representative calibration curve for methomyl.

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Linearity and SensitivityLinearity was assessed from triplicate analysis of a series of calibration standards(5-1000 ng/mL). Instrumental LOD and LOQ were defined as 3X and 10X thestandard deviation of the calculated concentrations, respectively. LOD and LOQwere determined from 5 replicate injections of a standard mixture (50 pg eachanalyte on-column).

Coefficient of determination LOD* LOQ*

Aldicarb Sulfoxide 0.9969 8 26Aldicarb Sulfone 0.9982 18 61

Oxamyl 0.9990 7 22

Methomyl 0.9959 16 55

3-OH Carbofuran 0.9970 4 14

Aldicarb 0.9963 2 5

Propoxur 0.9967 17 55

Carbofuran 0.9981 9 30

Carbaryl 0.9994 3 11

Methiocarb 0.9995 4 14

* pg on-column

Linearity was determined over a 200-fold concentration range with very goodresults.

Note: Unlike UV detection, the mass spectrometer is inherently non-linear. Thedegree f non-linearity is compound dependent and also varies with analyteconcentration range and matrix effects. This as an inherent behavior of APIionization, and is not instrument specific. For this reason, weighting is often used.This is NOT CHEATING! Also note that, because linear regression used aweighting factor, the familiar term “R2” is replaced with statistically equivalent“coefficient of determination.”

The lowest standard was injected in replicate (n=5) in order to determine theinstrumental limits or detection (LOD) and quantitation (LOQ). This slide listsinstrumental sensitivity limits calculated for the amount of material injected on-column.

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Precision and AccuracyFive replicate injections of a 5 ng/mL standard solution were made. Precision isdefined as the percent coefficient of variation of the calculated concentrations.Accuracy is defined as the percent difference from theoretical of the meancalculated concentration.

Mean(ng/mL)

S.D. % C.V. % Diff.

Aldicarb Sulfoxide 7.48 0.259 3.5 49.6Aldicarb Sulfone 6.32 0.606 9.6 26.4Oxamyl 6.68 0.217 3.2 33.6Methomyl* 5.50 0.548 10.0 10.03-OH Carbofuran 6.80 0.141 2.1 36.0Aldicarb 7.36 0.055 0.7 47.2Propoxur 4.90 0.552 11.3 -2.0Carbofuran* 3.98 0.299 7.5 -20.5Carbaryl 6.26 0.114 1.8 25.2Methiocarb 5.70 0.141 2.5 14.0

*n=4

Linearity is nice so is sensitivity, but the bottom line is accuracy and precision.This slide presents the accuracy and precision results from replicate injections ofthe lowest standard. Precision (defined as % C.V.) and accuracy (defined as the %difference from theoretical of the mean) are quite good.

Note: These values are probably higher than what people are used to seeing withLC/UV methods, but it should be borne in mind that we are talking about very lowamounts (<500 fmol) on-column; levels that are unobtainable by UV detection.Obviously, precision and accuracy values are much better for higherconcentrations. This slide shows “worst case” data. Also, remember that unlike aUV detector, LC/MS is a very dynamic process. When working at the ragged edgeof concentration, precision and accuracy often fall off. Still, by LC/MC standards,these results are very respectable.

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0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

Time

1

100

%

S/N:PtP=22.84

Overlaid SIR Chromatograms of Aldicarb(50 pg on-column) and a Blank Injection

Blank

This Selected -Ion Recording chromatogram of Aldicarb represents 50 pg(<500fmol) on column. Signal to noise is 22.84 to 1.

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But What About Real Samples?

Based on standards, we have demonstrated that our method provides adequatesensitivity, linearity, precision, and accuracy. Unfortunately, analytical chemistsare not paid to analyze standards. We have to look at real samples, which have atendency to be messy. A better test of this method would be to apply it to theanalysis of real samples. Let’s take a look at the results of some actual samples.

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SPE Recovery; 500 ppt in Tap Water

0 50 100

Ald.Sulfoxide

Ald. Sulfone

Oxamyl

Methomyl

3-OH Carbofuran

Aldicarb

Propoxur

Carbofuran

Carbaryl

MethiocarbC

om

po

un

d

% Recovery

LC/MS

LC/PCFD

The first matrix we looked at was water. Milford drinking water was spiked with acarbamate mixture at 500 ppt, and extracted and concentrated by SPE cleanup usingOasis HLB cartridges. The sample was analyzed by both traditional post-columnderivatization fluorescence detection (red) as well as by LC/MS (green), and therecoveries were compared.

As can be seen, there is good agreement between the two methods. The differencesfor aldicarb sulfoxide are believed to be due to sample degradation in the timeinterval between the two analyses.

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Carbamates LC/MSA comparison of results obtained from 500 ng/L spiked drinking water samples.Each extract was analyzed by LC/MS and also by LC with post-columnderivatization/fluorescence detection (PCFD). [% recovery (% RSD)]

Compound LC/MS LC/PCFDaldicarb sulfoxide 74.8 (19) 54.7 (0.5)aldicarb sulfone 88.7 (16) 98.7 (4.0)oxamyl 83.2 (18) 90.8 (7.0)methomyl 92.3 (8.0) 99.9 (6.4)3-hydroxycarbofuran 101 (8.6) 98.7 (2.3)aldicarb 79.4 (9.3) 90.7 (9.3)propoxur 103 (13) 97.5 (5.6)carbofuran 95.6 (7.5) 97.2 (4.7)carbaryl 97.7 (14) 89.6 (2.2)

methiocarb 81.2 (14) 91.6 (2.2)

Here is a comparison of the actual results.

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LC/PCFD Analysis of HarvestedBell Pepper Extract # 00329

mV

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.0055.00

Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

1- Methomyl 46.0 ppb

2- Propoxur 293.8 ppb *

* spiked in sample

Bell peppers are more complex than water. We were provided with several extractsfrom peppers with incurred (and therefore unknown) levels of carbamates exceptfor propoxur, which was spiked into all the samples.

The samples were analyzed by the traditional post-column derivatizationfluorescence detection method. Methomyl was detected in this sample.

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LC/PCFD Analysis of HarvestedBell Pepper Extract # 00336

Minutes

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

3

1- Oxamyl- 32.4 ppb2- Propoxur- 280.6 ppb *3- Carbaryl- 14.2 ppb

* spiked in sample

mV

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.0055.00

Here is a chromatogram of a different sample where oxamyl and carbaryl weredetected.

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LC/PCFD Analysis of HarvestedBell Pepper Extract # 00340

mV

-5.000.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.0055.00

Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

1

2

3

1- Oxamyl- 54.1 ppb

2- Propoxur- 290.5 ppb *

3- Carbaryl- 136.8 ppb

* spiked in sample

Detection of carbamates in another sample of bell pepper.

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0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

Time

1

100

%

1

100

%

1

100

%

5.23

5.26

2.97

1.33 1.90

Low Standard 200 pg on-column

Positive Pepper Sample 00329 approx. 47 pg on-column

Negative Pepper Sample 00340

LC/MS Analysis of Harvested BellPeppers: Methomyl, 2µL Injections

The same samples were also analyzed by LC/MS. Quantitation was done on thebasis of a 3-point standard. This slide compares the methomyl chromatogram fromthe low standard (top) with chromatograms from positive (middle) and negative(bottom) pepper extract. As may be seen from the extracts, the selectivity providedby LC/MS results in extremely clean baselines.

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0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50Time

4

100

%

4

100

%

4

100

%

4.14

4.11

Low Standard 240 pg on-column

Positive Pepper Sample 00340approx. 150 pg on-column

Negative Pepper Sample 00329

LC/MS Analysis of Harvested BellPeppers : Oxamyl, 2 µL Injections

These chromatograms compare a standard, positive, and negative sample foroxamyl.

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LC/MS Analysis of Harvested BellPeppers : Carbaryl, 2µL Injections

10.60 10.80 11.00 11.20 11.40 11.60 11.80 12.00 12.20 12.40Time

6

100

%

6

100

%

6

100

%

Negative pepper Sample 0329

Positive pepper Sample 0336approx. 27 pg on-column

Low Standard120 pg on-column

One last slide showing carbaryl response.

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Harvested Bell Pepper Samples

Sample HPLC LC/MS HPLC LC/MS HPLC LC/MS HPLC LC/MSID (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb) (ppb)

329 46 46.5 - - - - 293.8 276.5

330 411.5 342.5 - - - - 319.7 323.5

332 32.5 40.5 - - - - 298.6 241.0

336 - - 32.4 48.0 14.2 13.5 280.6 263.5

340 - - 54.1 76.0 136.7 154.5 290.5 341.5

Methomyl Oxamyl Carbaryl Propoxur

These samples had incurred carbamate levels except for propoxur, which wasspiked into all the samples, and so the actual concentration was unknown, outside ofthe LC/MS determination. As an additional check, the samples were analyzed viathe traditional post-column derivatization fluorescence method, and the meandetermination of both methods were compared. As may be seen, there is goodagreement between the two methodologies.

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Confirmation/Screening

• “Three-ion rule” used to eliminate false positives

• In this example, “In-Source CID” was used togenerate fragment ions from the analyte(Methomyl)

• Relative abundance ofpseudomolecular/fragment ion ratios werecompared for standard and spiked spinachextracts (n=10)

The “3-ion rule” is a convention which arose from the early days of GC/MS. Inorder to provide confirmation and avoid false positives, three structurally significantions are monitored by SIR for each compound. Identification is consideredconfirmed if all three ions have the same retention time/chromatographic profileand their relative intensities match those of an authentic standard. Unfortunately,atmospheric-pressure ionization spectra typically do not yield abundant fragmentions as is the case for electron-impact

ionization used in GC/MS. However, fragmentation can be induced by increasingthe voltage on the sampling cone in a process known as “in-source CollisionallyInduced Dissociation.”

Note: CID fragmentation is extremely compound and tune-dependent; CID spectraare not nearly as reproducible as electron-impact spectra. Also, bear in mind thatas sampling cone voltage is increased to induce fragmentation, very often absolutesignal will decrease, meaning that the analyst will have to strike a balance betweenoverall sensitivity and confirmation.

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50 60 70 80 90 100 110 120 130 140 150 160 170 180m/z

0

100

%

106

88

163

122

OH3 CS

CH3

N NHCH3

O882H

106

CID Fragmentation of Methomyl

[M+H]+

This is an abbreviated spectrum of methomyl under CID conditions. As may beseen, several structurally significant fragment ions are observed. The insert showsthe proposed identity of these fragments.

Note: The ion at m/z 122 cannot be explained by a simple bond cleavage andprobably arises from rearrangement during the CID process. Because of theunknown identity of this ion, it was not considered in the following slides.

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Methomyl Confirmation in Spinach

Ion m/z 88 Da 106 Da 163 Da 88 Da 106 Da 163 Da

% 100 51 34 100 55 34Relative 100 56 36 100 62 36Abundance 100 54 34 100 55 30

100 51 32 100 65 35100 55 31 100 63 36100 52 30 100 65 37100 54 30 100 69 38100 54 30 100 71 37100 54 30 100 72 38100 55 30 100 51 31

Mean 100 53.6 31.7 100 62.8 35.2S.D. - 1.71 2.21 0 7.16 2.78% C.V. - 3.2 7.0 0 11.4 7.9

Standard Spinach

In a separate experiment, both spiked spinach extract and a solvent standard wereinjected 10 times, and mass spectra were obtained under CID conditions.

This table compares the methomyl ion ratios for the two samples. Ion ratios arenormalized relative to the most intense ion at m/z 88.

There is good agreement between the two data sets, indicating the utility of CIDfragmentation for compound confirmation.

Note: More alert people may comment on the fact that the ion ratios expressed inthis table differ from those in the previous slide, in which the ion at m/z 106 was themost intense ion. We believe that this is due to the fact that the data from bothslides was acquired under slightly different conditions and/or on different days.While the table shows good INTR-assay reproducibility, whish is perfectlyacceptable for confirmation of target analytes, some people might be tempted toconclude that CID spectra are well-suited to library matching. This is anassumption that other manufacturers are willing to foster. However, the utility ofCID spectra for library-based identification requires excellent INTER-assayreproducibility, which front-end CID does not offer. This is a very importantdistinction which should be borne in mind when addressing claims of othermanufacturers.

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0

1 0

20

3 0

40

5 0

60

7 0

80

90

1 00

Re

lati

ve A

bu

nd

an

c

S tandard S pinach

m/z 88 m/z 106 m/z 163

Methomyl Confirmation in Spinach

This is a more graphic representation of the results presented in the previous table.It is clear that compound identity can be confirmed even in the presence of acomplex matrix.

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time

0

100

%

7.04e514.69

6.45

2.271.66

10.277.69

15.53

19.29

Carbaryl, monitoringm/z 145, 202

Propoxur,monitoring m/z 210

Methomyl, monitoringm/z 88, 106, 122, 163

Composite of a Three Component StandardSIR of 7 Channels

In a separate experiment, the chromatogram of three carbamates; methomyl,propoxur, and carbaryl, is displayed. They were analyzed by monitoring the sevenfragment ions corresponding to the three compounds.

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60 80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z

0

100

%

0

100

%

0

100

%

SIR of 7 Channels ES+ 1.02e5

145

88

202

163 210

SIR of 7 Channels ES+ 2.73e5

210

88 122106 202

SIR of 7 Channels ES+ 6.05e4

106

88

163122 145 210

Carbaryl response at 15.53 min.

Propoxur response at 14.69 min.

Methomyl response at 6.45 min.

Characteristic Mass Spectral Behavior

Here is the corresponding mass spectra obtained from the three carbamates.

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2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

Time

0

100

%

SIR acquisition, extracted chromatogram at m/z 162.9

10.13

9.78

2.19

0.723.97

Peak at 6.42 min from Methomyl

25 pg on-column

S/N ~ 10:1

Detection of Methomyl in Orange JuiceExtract, 8.5 ng/ml, 3 µµµµl Injection

An orange juice extract was analyzed under the same conditions. Shown here is theextracted chromatogram at m/z 162.9 from the orange juice sample detectingmethomyl at 8.5 ng/ml (25 pg on column) with excellent signal/noise ratio.

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Conclusion

• LC/MS adds an additional dimension to on-linechromatographic analyses.– “Resolution” of co-eluting peaks

– Detection of weak chromophores

• LC/MS enables you to perform qualitative and/orquantitative analyses.– SIR for sensitivity

– CID for structural information

It should be pointed out that all of the preceding data was derived from a variety ofexperiments performed over several months in different laboratories. Theyrepresent ‘real’ results; no effort was made to produce “perfect” data. What isimportant is that, taken together, they all tell the same story, lab-to-lab, operator-to-operator, and day-to-day. Hopefully, the moral is clear:

First, LC/MS adds a third dimension to typical on-line separations. It allows you to“peek” under a chromatographic peak. As we saw, this allows the chemist a lot ofleeway when it comes to methods development. As was shown, the massspectrometer can ‘resolve’ co-eluting peaks, allowing significant reductions in runtime. Response does not require a chromophoric or fluorescent group, so there isnot need for derivatization, again, simplifying the analysis.

Second, LC/MS is unique in that it can provide very sensitive and specificquantitative information (when operated in the SIR mode), as well as qualitative,structural information (full-scan analysis provides molecular weight, CID providesstructurally-significant fragment ions). Other analytical instruments are limited toone or the other.