supplemental information for · supplemental information for by steven j. lehotay, lijun han, and...

Supplemental information for

by Steven J. Lehotay, Lijun Han, and Yelena Sapozhnikova

“Automated mini-column solid-phase extraction cleanup for rapid analysis of chemical contaminants in foods by low-pressure gas chromatography –

tandem mass spectrometry”

USDA Agricultural Research ServiceEastern Regional Research Center

Wyndmoor, Pennsylvania; USA

Contact: Steven.Lehotay@ars.usda.gov

Automated Mini-SPE of QuEChERS Reagent Blank ExtractsGC-MS Full Scan m/z 100-500, 1 µL injection including APs

Rgt Blk (no cleanup)

MeCN

200 µL

400 µL

600 µL600 µL

400 µL

200 µL

45 mg MgSO4+PSA+C18+CarbonX 30 mg Z-Sep+C18+CarbonX

Peaks are from Analyte Protectantsand Reagent Co-Extractives,

Not SPE Contaminants!

Water in extracts when MgSO4 not included in SPE yields worse cleanup

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Kale ExtractsGC-MS Full Scan m/z 100-500, 1 µL injection including APs

No Cleanup

MeCN

200 µL

300 µL

500 µL

600 µL

400 µL

Conclusion: Not much cleanup, but 200-300 µL is ok

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Salmon ExtractsGC-MS Full Scan m/z 100-500, 1 µL injection including APs

No Cleanup

MeCN

200 µL

300 µL

500 µL

600 µL

400 µL

Conclusion: Not much cleanup

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Pork ExtractsGC-MS Full Scan m/z 100-500, 1 µL injection including APs

No Cleanup

MeCN

300 µL

500 µL

600 µL

400 µL

Conclusion: Not much cleanup

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Avocado ExtractsGC-MS Full Scan m/z 100-500, 1 µL injection including APs

MeCN

No Cleanup

200 µL

500 µL

600 µL

400 µL

Conclusion: Not much cleanup

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Kale ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

600 µL 500 µL 400 µL 200 µL None300 µL

Chlorophyllsand Xanthophylls

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Salmon ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

600 µL 500 µL 400 µL 200 µL None300 µL

45 mg MgSO4+C18+PSA+CarbonX Mini-SPE of Salmon ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

600 µL 500 µL 400 µL 200 µL None300 µL

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Pork ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

600 µL 500 µL 400 µL 200 µL None300 µL

45 mg MgSO4+C18+PSA+CarbonX Mini-SPE of QuEChERS Pork ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

600 µL 500 µL 400 µL 200 µL None300 µL

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Avocado ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

Chlorophylls

600 µL 500 µL 400 µL 200 µL None300 µL

45 mg MgSO4+C18+PSA+CarbonX Mini-SPE of Avocado ExtractsUV/Vis Absorbance Results

Spectrum of Extract before Cleanup

Ab

sorb

ance

Ab

sorb

ance

Spectrum of 300 µL Cleaned Up Extract

Wavelength (nm)

Chlorophylls

600 µL 500 µL 400 µL 200 µL None300 µL

Comparison of Mini-Cartridges to Remove Chlorophyll

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

100 200 300 400 500 600

%R

emo

ved

bas

ed

on

Ab

sorb

ance

at

68

0 n

m

Added Extract Volume (uL)

ITSP Cleanup of Chlorophyll

Kale 30 mg 3-sorbentsKale 45 mg 4-sorbentsAvocado 30 mg 3-sorbentsAvocado 45 mg 4-sorbents

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS Kale ExtractsChlorophyll Removal and HCB Results vs. Extract Vol. Added

Conclusion: 200 µL extract needed for 80% HCB elution

50%

60%

70%

80%

90%

100%

50%

60%

70%

80%

90%

100%

150 200 250 300 350 400 450 500 550 600 650

%R

em

ove

d b

ased

on

Ab

sorb

ance

at

68

0 n

m

HC

B P

eak

Are

a vs

. Max

Added Extract Volume (µL)

% vs. Max HCB

Chlorophyll removal

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS ExtractsRecovery vs. Extract Vol. Added

50%

60%

70%

80%

90%

100%

110%

120%

150 200 250 300 350 400 450 500 550 600 650

Pea

k ar

ea v

s. t

hat

fo

r 6

00

µL

Added Extract Volume (µL)

Rel. Responses of bifenthrin in ITSP

Kale Salmon

Pork Avocado

Conclusion: most analytes were not retained by the sorbents

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS ExtractsRecovery vs. Extract Vol. Added

Conclusion: 300 µL extract needed for >70% elution of PBDEs

40%

50%

60%

70%

80%

90%

100%

110%

120%

150 200 250 300 350 400 450 500 550 600 650

Pea

k ar

ea v

s. t

hat

fo

r 6

00

µL

Added Extract Volume (µL)

Rel. Responses of BDE 153 in ITSP

Kale Salmon

Pork Avocado

45 mg MgSO4+C18+PSA+CarbonX ITSP of QuEChERS ExtractsRecovery vs. Extract Vol. Added

50%

60%

70%

80%

90%

100%

110%

120%

150 200 250 300 350 400 450 500 550 600 650

Pea

k ar

ea v

s. t

hat

fo

r 6

00

µL

Added Extract Volume (µL)

Rel. Responses of BDE 153 in ITSP

Kale Salmon

Pork Avocado

Conclusion: 300 µL extract needed for >70% elution of PBDEs

30 mg C18+Z-Sep+CarbonX Mini-SPE of QuEChERS ExtractsRecovery vs. Extract Vol. Added

Conclusion: ≥600 µL extract needed for full elution of PAHs

40%

50%

60%

70%

80%

90%

100%

150 200 250 300 350 400 450 500 550 600 650

Pea

k ar

ea v

s. t

hat

fo

r 6

00

µL

Added Extract Volume (µL)

Rel. Responses of 2 PAHs in ITSP

Kale Salmon

Pork Avocado

Rel. responses of benz(a)anthracene+chrysene

45 mg MgSO4+C18+PSA+CarbonX Mini-SPE of QuEChERS ExtractsRecovery vs. Extract Vol. Added

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

150 200 250 300 350 400 450 500 550 600 650

Pea

k ar

ea v

s. t

hat

fo

r 6

00

µL

Added Extract Volume (µL)

Rel. Responses of 2 PAHs in ITSP

Kale Salmon

Pork Avocado

Conclusion: ≥600 µL extract needed for full elution of PAHs

Rel. responses of benz(a)anthracene+chrysene

Relative Recovery Differences for the Analytes in Mini-SPEfor the 300 µL Extract Volumes vs. Avg. 600 µL Result with45 mg mini-cartridge results – 30 mg mini-cartridge results

(n = 12 for 4 matrices)

Conclusion: Similar results using either mini-cartridge

-60%

-40%

-20%

0%

20%

40%

60%

Re

lati

ve R

eco

very

Dif

fere

nce

(4

5 m

g -

30

mg)

Analyte No. from Table 1

Validation ExperimentsDetermine performance results in the use of automated mini-SPE

cleanup in the LPGC-MS/MS analysis of pesticides and other contaminants in QuEChERS extracts of 10 different matrices.

Used mini-cartridgesshowing removal of chlorophyll and othermatrix components

Robotic liquid handler:3 min cleanup step at 2 µL/s+ 5 min for addition of APs and switching/washing syringes

Final extract volumes = 278 ± 5 µL (n = 255) after 50 µL addition of APs (and/MeCN) solution

Injection liner and septa after the 5 day experimentincluding 230 matrix extracts of diverse commodities

A little “dirt” here and there, but the analyte protectantsdid their job and results still looked great from start to finish.

Coumaphos in salmon after 230 injections of QuEChERS matrix extracts using mini-SPENo internal standard needed to still yield R2 = 0.999 calibration curve

Quant. Ion

Qual. Ion

25 ng/mL std in salmon extract

green boxes = atrazine-d5 (IS)black circles = stds in solventblue triangles = matrix-matched stds

10 ng/mL std in kale

quant. ion qual. ion

Methamidophos Results (vs. atrazine-d5 IS) – all 325 analyses combined!

green boxes = atrazine-d5 (IS)black circles = stds in solventblue triangles = matrix-matched stds

25 ng/mL std in orange

quant.ion

qual. ion

Acephate Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in carrot

quant.ion

qual.ion

Omethoate Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in pork

quant.ion

qual.ion

Dicrotophos Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in kiwi

quant.ion

qual.ion

Dimethoate Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

10 ng/mL std in orange

quant.ion

qual.ion

Carbofuran Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

10 ng/mL std in wheat

quant.ion

qual.ion

Diazinon Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in basil

quant.ion

qual.ion

Chlorothalonil Results (vs. atrazine-d5 IS) – all 325 analyses combined!

quant. ion

qual. ion

Chlorothalonil 5 ng/mL calibration standards over the course of 5 days (inj. #)RO = reagent-only; ion ratios within horizontal lines < |±10%| of ref. ratio

Day1

Day2

Day3

Day4

Day5

5) RO-a 12) apple 36) kiwi 59) RO-b

70) RO-a 77) carrot 101) kale 124)RO-b

135)RO-a

142)orange

166)olive 189)

RO-b

200)RO-a

207)wheat 231)

basil254)RO-b

265)RO-a

272)pork

296)salmon

319)RO-b

Matrix contaminationof system from canned black olive extracts slowlydissipated duringsequence, but did not cause instrument or analytical performance problems.

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in black olive

quant.ion

qual.ion

Chlorpyrifos Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in black olive

quant.ion

qual.ion

Cyprodinil Results (vs. atrazine-d5 IS) – all 325 analyses combined!

10 ng/mL in wheat grain

TBECH Results (vs. atrazine-d5) – all 325 analyses combined!

quant.ion qual. ion

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL in salmon

Thiabendazole Results (vs. atrazine-d5) – all 325 analyses combined!

quant.ion

qual. ion

black circles = stds in solventblue triangles = matrix-matched stds

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in kale

quant.ion

qual.ion

Flutriafol Results (vs. atrazine-d5 IS) – all 325 analyses combined!

Imazalil Results (vs. atrazine-d5) – all 325 analyses combined!

quant.ion

qual. ion

5 ng/mL std in apple

Note: imazalil was incurredin orange at 1243 ng/mL

black circles = stds in solventblue triangles = matrix-matched stds

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in apple

quant.ion

qual.ion

Myclobutanil Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in apple

quant.ion

qual.ion

Endosulfan II Results (vs. atrazine-d5 IS) – all 325 analyses combined!

black circles = stds in solventblue triangles = matrix-matched stds

10 ng/mL std in orange

quant.ion

qual.ion

Endosulfan Sulfate Results (vs. atrazine-d5 IS) – all 325 analyses combined!

Endosulfan sulfate 5 ng/mL reagent-only and matrix-matched calibration standards

LOQ ≈2 ng/mL in all matrices; even after 325 injections, including 230 food extracts

black circles = stds in solventblue triangles = matrix-matched stds

5 ng/mL std in basil

quant.ion

qual.ion

Bifenthrin Results (vs. atrazine-d5 IS) – all 325 analyses combined!

Benzo(b+k)fluoranthene (vs. benzo(a)pyrene-d12 IS) – all 325 analyses combined!

quant.ion

qual.ion

5 ng/mL std in wheat grain

IS

black circles = stds in solventblue triangles = matrix-matched stds

10 ng/mL std in orange

quant.ion

qual.ion

atrazine-d5(IS)

a ghost peak occurs in orange 1 injection later

(Es)fenvalerate Results (vs. atrazine-d5 IS) – all 325 analyses combined!

Matrix effects occur at later tR

Dechlorane Plus Results (vs. FBDE 126 IS) – all 325 analyses combined!

quant.ion

qual.ion

5 ng/mL std in wheat grain

IS

black circles = stds in solventblue triangles = matrix-matched stds

0

20

40

60

80

100

120

140

apple kiwi carrot kale orange olive wheat basil pork salmon

Ave

rage

Re

cove

rie

s (%

)

Flame Retardants vs IS wo IS

0

20

40

60

80

100

120

140

apple kiwi carrot kale orange olive wheat basil pork salmon

Ave

rage

Re

cove

rie

s (%

)

Pesticides vs IS wo IS

0

20

40

60

80

100

120

apple kiwi carrot kale orange olive wheat basil pork salmon

Ave

rage

Re

cove

rie

s (%

)

PCBs vs IS wo IS

0

20

40

60

80

100

120

140

apple kiwi carrot kale orange olive wheat basil pork salmon

Ave

rage

Re

cove

rie

s (%

)

PAHs vs IS wo IS

-100

-80

-60

-40

-20

0

20

40

60

80

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15M

E %

(vs

IS)

Flame Retardants Apple

Kiwi

Carrot

Kale

Orange

Olive

Wheat

Basil

Pork

Salmon

-60

-40

-20

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

ME

% (

vs IS

)

PAHs Apple

Kiwi

Carrot

Kale

Orange

Olive

Wheat

Basil

Pork

Salmon

-40

-30

-20

-10

0

10

20

30

40

0 1 2 3 4 5 6 7 8 9 10 11 12 13

ME

% (

vs IS

)

PCBs AppleKiwiCarrotKaleOrangeOliveWheatBasilPorkSalmon

Numbers correspond to

analytes listed in Table 1

PBTTBNPA

naphthalene was ubiquitous