"Productivity and Simplicity" - Streamlining Cumbersome Sample Preparation in the Lab

“Productivity and Simplicity” Streamlining Cumbersome Sample Preparation

Workflows in the Analytical Lab

Oscar G. Cabrices Ph.D.GERSTEL, Inc.Linthicum, MD

Industrial MS Symposium – New Jersey June 9th 2015

OutlineGERSTEL’s goal is to deliver customer focused solutions with intelligent automation for GC/MS & LC/MS in the Industrial research laboratory.

This presentation will exemplify how can tedious and routine sample preparation workflows can be simplified and improved for analysis:

Dilutions and Liquid Liquid Extractions Calibration Curve Generation and IS Spiking Enzymatic Reactions and Derivatizations Post QuEChERS or QuPPE sample cleanup and enrichment Centrifugation and Solid Phase Extraction Filtration and Large Volume Injections

MultiPurposeSampler MPSfor LC/MS

MPS Workstation

Our Technology…

MultiPurposeSampler MPSfor GC/MS

MultiPurposeSampler MPSfor GC/MS

Disposable PipetteExtraction DPX

Solid PhaseExtraction SPE

Twister

MAESTROSoftware

ThermalDesorptionSystem TDS

ThermalDesorptionUnit TDU

Automated TDULiner Exchange ATEX

AutomatedLiner EXchange ALEX

MultiFiber EXchangeMFX

DynamicHeadspaceDHS

Cooled InjectionSystem CIS

PreparativeFractionCollector PFC

Olfactory DetectionPort OPD

MAESTROPrepAhead

easy LinerExchange eLEX

MultiPurposeSampler MPSfor LC/MS

MPS Workstation

µFlowManagerTDU PYRO

Selectable1D/2DGC/MS

LC/MSEffluentOptimizer LEO

MultiPositionEvaporationStation mVAP

A “Snack” of our Portfolio of Techniques

Puzzled?

Historically, liquid-liquid extractions are a basic technique in chemical laboratories and are used to extract analytes from a variety of different matrices.

A partitioning method is used to separate compounds based on their relative solubility in two different immiscible liquids (e.g., water and an organic solvent).

However, as a general rule, liquid-liquid extractions are manually intensive.

Example # 1: Automating LLE

LLE of Drugs from SerumHere is a typical manual LLE workflow:

1mL of serum sample is placed into a 10mL vial and capped with a magnetically transportable cap and placed onto the autosampler.Add 100mL of 5000ng/mL working internal standard to the sample.Add 1mL of 10mM potassium hydroxide to the sample.Add 5mL of (7:3) methyl-t-butyl ether: dichloromethane to the sample.Vortex for 5minutes at 2000rpm.Centrifuge samples for 5minutes at 3000rpm.Transfer 4mL of the organic layer to a clean, 10mL vial that had been previously capped with a magnetically transportable cap.Evaporate to dryness at 40oC for 20minutes.Reconstitute residues using 0.500mL of (90:10) 0.05% formic acid in water: methanol.Filter resulting sample using a 0.45mm nylon filter into a clean 2mL autosampler vial and Inject into LC/MS/MS

Can it be automated and optimized?

After sample is initially received, load an aliquot on the GERSTEL MPS for automated LLE processing:

Load vials containing biological sample and extraction solvents on MPS

MPS mixes sample Buffer, Organic solvents and Internal Standard

Evaporate organic layer and Reconstitute in Mobile Phase

Filter extract with 4 mm filter

Inject 2 µL sample to LC/MS/MS

LLE of Drugs from Serum

Four organic solvents Polarity Index• (99:1) Hexane:IPA 0• MTBE 2.5• Methylene Chloride 3.1• Ethyl Acetate 4.4Three (3) pH levels• 10mM HCl acid• 10mM Ammonium Acetate neutral• 10mM Potassium Hydroxide base

Test Matrices and Compounds:•Bovine Serum for Ketamine•Bovine Lyophilized Plasma:• Buprenorphine and Norbuprenorphine

with deuterated Internal StandardsThree extractions with each organic solvent (90%rule)

Automated LLE Method Development

Automated BenchPrep for LLE

Automated LLE-LC/MS/MS workflow

Just

released!

GERSTEL CF-200 Centrifuge

System prompts user for offline Centrifugation when needed

Automated LLE-LC/MS/MS workflow

Ketamine Automated Liquid-Liquid Extraction Results

Sample Name Response ISTD Resp Resp Ratio1000ng/mL neat Ketamine std 1 11878597 16748195 0.70921000ng/mL neat Ketamine std 2 12544625 17637186 0.71131000ng/mL neat Ketamine std 3 12259205 17294488 0.7089

mean 12227475 17226623 0.7098SD 334146 448364 0.00129

%CV 2.73 2.60 0.182

LiqLiq Extr rep 1 9794014 15573317 0.6289LiqLiq Extr rep 2 9891262 15714821 0.6294LiqLiq Extr rep 3 9484299 15152067 0.6259LiqLiq Extr rep 4 9761104 15417946 0.6331

mean 9732670 15464538 0.6293SD 174558 241028 0.00294

%CV 1.79 1.56 0.467%Recovery 79.6 89.8 88.7

Results from Automated LLE-LC/MS/MS

Results from Automated LLE-LC/MS/MS

Hex:IP

A_ acid

ic

Hex:IP

A_ bas

ic

EtOAc_

neutr

al

MTBE_ acid

ic

MTBE_ bas

ic

MeCl2_

neutr

al90.0

94.0

98.0

102.0

106.0

Norbuprenorphine %Recovery by Ratio

Hex:IP

A_ acid

ic

Hex:IP

A_ bas

ic

EtOAc_

neutr

al

MTBE_ acid

ic

MTBE_ bas

ic

MeCl2_

neutr

al90

94

98

102

Buprenorphine %Recovery by Ratio

Main Objective:To develop a completely automated urine sample handling and analysis workflow using LC/MS/MS for comprehensive screening of multiple drug classes.

Clinical/forensic chemists perform a lengthy variety of sample handling steps prior to analyses in order to determine the final concentrations of analytes in urine samples:

1. Asess the sample2. Hydrolyze (15 min for Acid/Base or

2hrs enzymatic)3. Cleanup and Derivatization4. Analysis by GC/MS or LC/MS

Enzymatic Hydrolysis Reaction (Source: Sigma-Aldrich)

Example # 2: Enzymatic Reactions

After urine sample is initially received, centrifuge to remove any particulates or proteins present. Then load an alliquot on the GERSTEL MPS

Load plates and reagents on MPS

MPS mixes urine sample Buffer, Enzyme and Internal Standard

Incubate sample for 15 minutes

MPS dilutes hydrolyzed sample with Mobile Phase A

Inject 2 µL sample to LC/MS/MS

Automated Prep and Shoot hydrolysis

GERSTEL MPS 2 multi-purpose sampler configured with an AB SCIEX QTRAP® 4500 LC/MS/MS system.

Automated Prep and Shoot hydrolysis

MT-96 Back Plate containing Urine Sample

MT-96 Front Plate containing Mobile Phase

A

Buffer/Enzyme/ISsolutions

DW-96 Empty Plate

Well plate incubating

station

LC/MS/MS Injection valve

The efficiency of the automated enzymatic hydrolysis was examined by spiking blank urine with different glucuronide conjugates.

Morphine-3-glucuronideOxymorphone-glucuronide

Codeine-6-glucuronideTapentadol-glucuronide

Buprenorphine-glucuronideOxazepam-glucuronideLorazepam-glucuronideTHC-COOH-glucuronide

Automated Hydrolysis performance

XIC of +MRM (30 pairs): Exp 1, 222.000/107.000 amu Expected RT: 3.0 ID: Tapentadol 1 from Sample 6 (100) of UrineHydrolysis_Cal... Max. 7.3e4 cps.

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

Inte

ns

ity, c

ps

3.15

XIC of +MRM (30 pairs): Exp 1, 398.200/222.300 amu Expected RT: 2.8 ID: tapentadol gluc 1 from Sample 6 (100) of UrineHydrolysis... Max. 1.3e4 cps.

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.3e4

Inte

ns

ity, c

ps

2.83

XIC of -MRM (6 pairs): Exp 2, 343.200/299.300 amu Expected RT: 4.9 ID: THC-COOH 1 from Sample 6 (100) of UrineHydrolysis_Calc... Max. 1.1e4 cps.

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0Time, min

0.00

500.00

1000.00

1500.00

2000.00

2500.00

3000.00

3500.00

4000.00

4500.00

5000.00

5500.00

6000.00

6500.00

7000.00

7500.00

8000.00

8500.00

9000.00

9500.00

1.00e4

1.05e4

1.10e4

Inte

ns

ity, c

ps

4.92

XIC of -MRM (6 pairs): Exp 2, 494.900/318.900 amu Expected RT: 4.1 ID: lorazepam gluc 1 from Sample 6 (100) of UrineHydrolysis_C... Max. 370.0 cps.

1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0Time, min

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

360370

Inte

ns

ity, c

ps

4.09

3.97

ESI+ ESI-

ESI+ ESI-

XIC of +MRM (105 pairs): 414.000/187.000 amu Expected RT: 2.6 ID: Norbuprenorphine 1 from Sample 2 (Test Hydrolysis Sample) of TestH... Max. 3.3e4 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

3.4e4

3.6e4

3.8e4

4.0e4

4.2e4

4.4e4

4.6e4

4.8e4

5.0e4

5.2e4

5.4e45.5e4

Inte

nsity, c

ps

2.58

3.072.38 2.77

XIC of +MRM (105 pairs): 590.000/414.000 amu Expected RT: 2.2 ID: Norbuprenorphine-gluc 1 from Sample 2 (Test Hydrolysis Sample) of ... Max. 1333.0 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

900

950

1000

1050

1100

1150

1200

1250

13001333

Inte

ns

ity, c

ps

2.69

2.53

1.97

2.32 2.452.171.75

Norbuprenorphine

Norbuprenorphine-G

Over 95% hydrolysis was achieved for most compounds

Analyte % HydrolyzedMorphine-3G 99.70%

Oxymorphone-G 99.80%Codeine-6G 80.50%

Tapentadol-G 97.90%Buprenorphine-G 99.60%

Oxazepam-G 95.80%Lorazepam-G 99.30%THC-COOH-G 94.40%

Norbup-G 99.80%


Ten (10) incurred urine samples with semi quantitative results were automatically hydrolyzed and injected into the LC/MS/MS system. Each sample was hydrolyzed, diluted and injected a total of 96 times.

XIC of +MRM (105 pairs): 287.000/241.000 amu Expected RT: 3.1 ID: Oxazepam 1 from Sample 48 (13188) of Plate2_053114.wiff (Tur... Max. 3.1e6 cps.

1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0Time, min

0.0

2.0e5

4.0e5

6.0e5

8.0e5

1.0e6

1.2e6

1.4e6

1.6e6

1.8e6

2.0e6

2.2e6

2.4e6

2.6e6

2.8e6

3.0e6

3.2e6

3.3e6

Inte

ns

ity, c

ps

3.11Sample ID #13188 O

XA

OXA-G

AnalyteSample ID

#13188

Automated Hydrolysis Avg. Concentration

n=96 (ng/mL) %RSDNorfentanyl 857.9 9.00%

Fentanyl 412.6 6.80%Oxazepam 2444.1 8.00%

Temazepam 1843.2 9.80%Nordiazepam 559.1 10.40%


The combined automation of urine hydrolysis, injection and analysis was optimized using the PrepAhead intelligent algorithms in the GERSTEL MAESTRO software to allow the processing of more than 200 samples in a 24 hr span.

Incubation

Combination of Urine , IS ,

Buffer and Enzyme

LC/MS/MS

Automated Hydrolysis optimization

Glyphosate and glufosinate are non-selective, post emergence herbicides used for the control of a broad spectrum of grasses and broad-leaf weed species in agricultural and industrial fields.

Aminomethyl-phosphonic acid (AMPA) is the major metabolite of glyphosate

.

According to recent reports, there has been a dramatic increase in the usage of these herbicides which are of risk to both human health and the environment.

Example # 3: Derivatization and sample cleanup

Main Objective: To develop a fully automated sample preparation workflow for the detection and quantitation of Glyphosate and other polar pesticides in water and food samples that meet the established MRLs.

Load Derivatize Extract and Analyze

Automated analysis of polar pesticides

Glufosinate (MW = 181 g/moL)

Glyphosate (MW = 169 g/moL)

AMPA (MW = 111 g/moL)

Due to their high polarity – difficult to retain on common reversed phase LC columns

Derivatization with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) used to improve extraction and separation of glyphosate and related compounds.

NH POH

OH

OO

OH

O

ClO

O

NO

OOH

P OH

O

OH

+

NH POH

OH

OO

OH

O

ClO

O

NO

OOH

P OH

O

OH

+

Glyphosate

FMOC-Cl

Gly-FMOCMW = 391 g/moL


Automation workflow part 1: Derivatization and Injection with the MPSAdd 100 μL of borate buffer (pH=9) to 1 mL

of sample.

Agitate for 20 min at 50°C.

Cool to bring to ambient temperature.

Add 130 μL 2% H3PO4.

Inject 1mL to SPE-LC/MS/MS

Add 200 μL of 10mM FMOC-Cl solution.


Automation workflow part 2: SPE Cleanup and Analysis

Inject 1mL

Sample

SPE Cartridge Transport and Exchange

Condition GERSTEL SPEXOS C8EC-SE (18.5 mg)

cartridge with methanol and water + 100 mM

formic acid

Load 1 mL of derivatized sample onto SPE cartridge

Wash with water + 100 mM formic acid

Elute with LC pump gradient

Elute clean

sample


Analytical Column: Phenomenex Gemini, 3 μm (150 x 2.0 mm) Mobile Phase: 50mM Amm Acetate (pH=9)/ ACN (Gradient, time= 25 min) Flow Rate: 250 μL/min Column Temperature: 40°C

XIC of -MRM (9 pairs): 402.000/179.600 Da ID: glufosinate 1 from Sample 2 (0.1 ug/L) of Glyphosate-WaterReproducibility_062613.wiff (Turbo Spr... Max. 1.2e5 cps.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e5

2.2e5

Inte

ns

ity, c

ps

6.47

Standard water chromatogram at a spiked concentration of 10 ng/mL

Glyphosate at 4.9 minGlufosinate at 6.5 minAMPA at 6.9 min

Compound Q1 Q3 CE (V)Glyphosate 390 168, 150 -18, -34

Glufosinate 402 179.6, 205.4 -16, -20

AMPA 322 110, 136 -12, -22

MS/MS Detection Optimized for the QTRAP®4500 LC/MS/MS system equipped with a Turbo V™ source with ESI probe .Negative polarity


Triplicate analysis of polar pesticides in a spiked water sample at0.1 μg/L (injection volume of 10 μL)

Compound Concentration (μg/L) %CV of MRM 1 %CV of MRM 2

Glyphosate0.1 4.0 3.9

10 7.7 8.9

Glufosinate0.1 2.3 4.5

10 4.6 5.4

AMPA0.1 1.4 5.3

10 5.1 5.4


Corn and Soy samples were pre-treated using the QuPPe “Quick, Polar Pesticides Method” (www.qppe.com)

Weigh 5g of food subsample

Add 10 mL of Water and soak for 10 min

Add 10 mL of acidified methanol (1% formic acid).

Shake vigorously and Centrifuge

Filter extract (top) layer

After filtration, transfer to vial to MPS forautomated derivatization, extraction and

analysis

WorkflowAutomation for LC/MS/MS Polar

Pesticide Screening


10 and 100 μg/kg of glyphosate spiked into corn and soy and analyzed using automatic derivatization, dilution, and online SPE

Corn 10 Soy 100Soy100Corn 10


10 and 100 μg/kg of AMPA spiked into corn and soy and analyzed using automatic derivatization, dilution, and online SPE

Corn 10 Soy 100Soy100Corn 10


Compound Concentration (μg/kg) %CV of MRM 1 %CV of MRM 2 Ion ratio

(%RSD)Glyphosate 100 (in corn) 3.6 6.0 0.36 (1.9%)

100 (in soy) 5.1 5.9 0.31 (1.9%)

Glufosinate 100 (in corn) 1.6 12.5 0.71 (8.9%)

100 (in soy) 5.2 7.7 0.67 (3.9)%

AMPA 100 (in corn) 5.7 4.8 0.43 (0.9%)

100 (in soy) 5.3 6.2 0.38 (2.2%)

Triplicate analysis of polar pesticides spiked into corn and soy samples.

The slightly higher %RSD of the ion ratio of glufosinate in corn can be explained by interfering matrix signals.

Stable LC separation was essential for confident identification and accurate quantitation of glufosinate.


Mycotoxins are secondary metabolites produced by different types of filamentous fungi genus:- Aspergillus (Aflatoxins) - Penicillum (Ochratoxin A)- Fusarium (trichothecenes, fumonisins,

deoxynivalenol and zearalenone)- Patulin

More than 25% of all agricultural commodities are contaminated with mycotoxins.

Over 100 countries have regulations for the control of mycotoxins in food and feed

Aspergillus fungiSource:

http://www.iaqm.com

Example # 4: SPE and Evaporative Reconstitution

http://www.iaqm.com/

Weigh 25 g food subsample

Add 100 mL 84:16 ACN:H2O, Vortex and Centrifuge

Collect 8 mL supernatant for SPE cleanup

Main Objective: To develop a fully automated sample preparation workflow for the screening of mycotoxins in food products, that meet the established MRLs.

Typical Mycotoxin sample preparation workflow:

Evaporate to dryness and reconstitute in Mob. Phase

Transfer to Vial for LC/MS analysis

Add Internal Std

Automated Extraction of Mycotoxins

Weigh 1 g food subsample

Add 4 mL 84:16 ACN:H2O, Vortex and Centrifuge

Collect 250-500 uL supernatant for SPE cleanup

Original Mycotoxin workflow is microscaled 25x for effective MPS handling.

Evaporate to dryness and reconstitute in Mob. Phase

Transfer to Vial for LC/MS analysis

Add Internal Std

Workstation Automation for

MycotoxinSample

Handling



CentrifugeOption

Tray for Raw

Samples

Trays for DPX cleanup and final

Samples for LC/MS/MS

analysis

mVORX Option for DPX and 2mL vial mixing

mVAP Option for

solvent evaporative

concentration


Weigh 1 g of homogenized

sample to a sealed 10mL vial and place

on MPS

MPS adds 4 mL of 84:16 ACN:H2O to

vial moves it to agitator for mixing

(1 h, 55°C, 500 rpm) followed by

Centrifugation

Vortex and Centrifuge (1900 RPM, 10 min)

Centrifugation of first sample batch occurs

while the following batch is being mixed

Prep Aheadenabled !

Mycotoxins

Weigh 1 g of homogenized

sample to a sealed 10mL vial and place

on MPS

MPS adds 4 mL of 84:16 ACN:H2O to

vial moves it to agitator for mixing

(1 h, 55°C, 500 rpm) followed by

Centrifugation

MPS transfers 500 μL of supernatantto Test Tube for DPX cleanup

Vortex and Centrifuge (1900 RPM, 10 min)

Whole Corn Samples

Mycotoxins

Cleaned Sample is injected for analysis

on the AB SCIEX 4500

QTRAP® LC/MS/MS System

200 μL of cleaned extract is transferred to a sealed 2 mL vial for evaporation (5 min, 55°C) and reconstitution in 500 μL of Mobile Phase A using the mVAP

Mycotoxins

Representative MRM chromatograms from a matrix matched corn sample extract at 10 µg/kg.

TIC: from Sample 52 (10 ng/mL) of Myco... Max. 1.1e5 cps.

8 10 12 14 16 18Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e51.13e5

Intensity, cps

14.17

11.76

10.97

10.60

12.327.03 10.12

XIC of +MRM (25 pairs): Exp 1, 404.000/... Max. 9.3e4 cps.

8 10 12 14 16 18Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

Inte

ns

ity, c

ps

14.16

XIC of -MRM (10 pairs): Exp 2, 317.100/1... Max. 4.6e4 cps.

8 10 12 14 16 18Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4In

ten

sity

, cp

s14.22

3-AcD

ON

DONFUS-X

ZON

AFB1

AFB2AFG1

DAS

FB1

OTA

HT-2

NEO T-2AFG2

ESI positive

ESI negative

Mycotoxins

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480Concentration

0.0e0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

Area

Calibration for AFB1 1: y = 5741.12177 x + 1276.09662 (r = 0.99965) (weighting: 1 / x)

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480Concentration

0.0e0

5.0e6

1.0e7

1.5e7

2.0e7

Area

Calibration for OTA 1: y = 4.22125e4 x + -7984.53762 (r = 0.99937) (weighting: 1 / x)

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480Concentration

0e0

1e5

2e5

3e5

4e5

5e5

6e5

7e5

Area

Calibration for DON 1: y = 1371.26551 x + 95.47439 (r = 0.99891) (weighting: 1 / x)

DON (R2 = 0.9989)

AFB1 (R2 = 0.9997)

OTA (R2 = 0.9994)

Calibration lines of the quantifier and qualifier MRMsMycotoxins

Compound (MRM)

QC sampleConcentration

(ng/g)

% Average Accuracy

(n=6)

% RSD (n=6)

DON (1)250

94.60% 10.38%

DON (2) 94.97% 10.50%

Peak review of wheat middlings containing 250 µg/kg DON Mycotoxins

Peak review of corn containing 12-20 µg/kg OTA

Compound (MRM)

QC sampleConcentration

(ng/g)

% Average Accuracy

(n=6)

% RSD (n=6)

OTA (1)12-20

82.33% 8.94%

OTA (2) 82.79% 7.90%

Mycotoxins

TIC chromatogram of an incurred Corn sample.

?

Mycotoxins

Extracted spectra and automated library search with Purity Score values for the incurred corn sample

Summary of all MTS results

TIC

Overlaid Acquired and Library MS/MS spectra

Library MS/MS hitsStructure of

FB1

Mycotoxins

Main Goal: To develop a comprehensive LC/MS/MS screening method for different classes of environmental contaminants present in water samples with low ppt detection levels.

Compounds of interest:- Pesticices- PCPPs- PFCs- Drugs of Abuse

Dichlorvos Bolstar Carbendazim MCPP (Mecoprop)Mevinphos Tokuthion Cypermethrin DichloropropDemeton-O Azinphos-methyl Pentachlorophenol DalaponDemeton-S-methyl Bromacil DCPA MCPAMalathion Molinate Amoxicilin BentazonEthoprophos Cyanazine Azithromycin TriclosanNaled Prometon Caffeine Merphos OxidePhorate Metribuzin Primidone FensulfothionDiazinon Butachlor Acetaminophen 2,4-DDisulfoton Aldicarb Carbamazepine 2,4,5TPParathion-methyl Aldicarb-sulfoxide Ciprofloxacin 2,4,5TFenthion Aldicarbsulfone Erythromycin 2,4-DBChlorpyrifos Oxamyl Fluoxetine DicambaCoumaphos Methomyl Sulfamethoxazole PicloramRonnel 3-Hydroxycarbofuran Trichloronate PendimethalinParathion Propoxur Stirophos PFOSCarbofuran Carbaryl Methiocarb PFOA

Example # 5: Filtration and Large Volume Injection

1. QTRAP® 6500 sensitivity was sufficient to eliminate the need for sample enrichment. (Fast Polarity Switching)

2. 500 µL sample injection facilitated ≤ ppt levels of detection for many analytes Larger volume injection requires

larger particle size column (i.e., 4.6 um) with higher flow rates.

Suggest filtering the samples prior to analysis to remove any contaminants or particles that can affect column lifetime

If the samples are really dirty, an automated dilution followed by filtration can be done

Automated Water Analysis

Load Vials with original Sample

Picks up a 4mm filter, and filters sample

Injects 500uL of filtered sample for LC/MS/MS analysis

WorkflowAutomation for

LC/MS/MS MultiresidueScreening

In water samples

MPS pipettes a determined volume of sample


High Level SummaryMost compounds observed ≤ 1 ng/L


PPCP MixCaffeine

Amoxicillin Azithromycin

Primidone Triclosan

1 ppt

50 ppt 50 ppt

1 ppt 2 pptAutomated Water Analysis

2,4-D

2 ppt

2,4,5-T Dichlorprop

5 ppt 10 ppt


PFC Mix

PFOA

10 ppt

PFOS

1 ppt


Method performance for Amoxicillin

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950Concentration

0.0e0

5.0e6

1.0e7

1.5e7

Area

Calibration for Amoxicilin-1: y = 1.43309 x̂ 2 + 14961.22889 x + -272.33235 (r = 0.99993) (weighting: 1 / x)

Dynamic Range5 – 1000 pptR2 = 0.9993

10 ppt in River Water (n = 3)


Sample performance for Chlorpyrifos

1 – 1000 pptr = 0.9985

10 ppt in River Water (n = 3)0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

Concentration

0.0e0

5.0e6

1.0e7

1.5e7

2.0e7

Area

Calibration for Chlorpyrifos-1: y = 20319.10260 x + -346.97809 (r = 0.99850) (weighting: 1 / x)


Automation is the best tool for being prepared in case “Hells breaks loose” in the lab.Several workflows were intelligently automated using the GERSTEL MPS 2XL autosampler:Enzymatic ReactionsLiquid Liquid ExtractionsDerivatization and sample enrichmentQuick cleanupsEvaporative ReconstitutionFiltration and large volume injections

.

In Summary

Please visit www.gerstelus.com for more information!

Also..visit our YouTube Channel for more automation examples!

StillPuzzled?

GERSTEL brings it

together!!!

Science

"Productivity and Simplicity" - Streamlining Cumbersome Sample Preparation in the Lab