secrets to successful gc-ms/ms pesticide method development · secrets to successful gc-ms/ms...

NACRW, Agilent Users Meeting, July 25, 2013

Secrets to Successful

GC-MS/MS Pesticide

Method Development

Katerina Mastovska

Nutritional Chemistry and Food Safety

Covance Laboratories

NACRW, Agilent Users Meeting, July 25, 2013 2

QuEChERS vs. Traditional Methods

“Quick, Easy, Cheap, Effective, Rugged, and Safe”

• Lower cost of chemicals and consumables

• Minimum waste disposal (no chlorinated solvents)

• Basically no glassware washing and storage

• Faster turn-around-times

• Less training

• Larger scope of analytes (including modern, polar pesticides)

• Directly compatible with GC-MS(/MS) and LC-MS(/MS)

• Suitable for analysis of other contaminants

Many labs have implemented QuEChERS

BUT some are still hesitant or are having problems


Problems with QuEChERS Implementation Major problem:

Compatibility of QuEChERS extracts with old GC methods

Why?

• Acetonitrile as the GC injection solvent

• Matrix co-extractives

Solutions:

• Optimize GC injection conditions, ideally use PTV in solvent vent

mode to eliminate acetonitrile

• Use GC-MS or GC-MS/MS for improved detection selectivity

• Transfer more polar (troublemaker) pesticides to LC-MS(/MS)

• Use column backflushing to remove less volatile matrix

components from the GC system

• Use analyte protectants to deactivate GC system in every injection


GC-MS/MS Reference Guide


Acetonitrile as the GC Injection Solvent

Solvent Mr

(g/mol)

(g/mL)

b.p.

(°C) P’

pv

(kPa)

Vvapor

(L)

Vinj max (L)

10 psi 20 psi 40 psi 60 psi

Acetonitrile 41 0.78 82 6.2 9.6 486 1.2 1.7 2.7 3.7

Acetone 58 0.79 56 5.4 24.6 348 1.7 2.4 3.8 5.2

Ethyl acetate 88 0.90 77 4.3 9.7 261 2.3 3.2 5.1 6.9

Hexane 86 0.66 69 0.0 16.3 196 3.1 4.3 6.8 9.3

Toluene 92 0.87 111 2.3 2.9 242 2.5 3.5 5.5 7.5

Isooctane 114 0.69 99 -0.4 5.1 155 3.9 5.5 8.6 11.7

Mr ............ molecular mass

................. solvent (liquid) density (at 20°C, patm)

b.p. .............. boiling point (at patm)

P’ ................ polarity index

pv ................ vapor pressure (at 20°C)

Vvapor ........... vapor volume generated by 1 L injection (Vinj = 1 L) of the given solvent at 10 psi (a pressure close to a head pressure in

typical GC-MS pesticide analysis without a pressure pulse) and injection temperature t inj = 250°C; calculated from the

following equation:

Vvapor = 22.4103 (/Mr) [(tinj + 273)/273] [patm/(pi + pa)] Vinj

where patm = 14.7 psi (101 kPa) and pa is ambient pressure, usually taken as patm

Vinj max ...... maximum safe injection volume for the 800 L liner used at different column head pressures (10, 20, 40, and 60 psi) and t inj =

250°C, i.e. injection volume that generates 600 L of vapors (75% of the liner volume)

K. Mastovska, S.J. Lehotay, J. Chromatogr. A 1040 (2004) 259-272.


Acetonitrile as the GC Injection Solvent

• Large expansion volume

• Not compatible with relatively non-polar GC stationary phases

• Forms droplets rather than a continuous film upon re-

condensation in the GC column, causing peak distortions for

early eluting analytes

• Splitless injection: the initial oven temperature has to be at or

above its boiling point (e.g. at 90°C) to prevent acetonitrile

condensation in the GC column

• PTV (programmable temperature vaporizer) with solvent vent

– ideal to eliminate acetonitrile from the inlet prior reaching the

GC column

• Alternative approach: solvent exchange into toluene (or

adding toluene to the acetonitrile extract)


PTV Solvent Vent Injection

• Injection at a lower temperature → less discrimination, better results for thermally-labile analytes, reduced matrix effects

• Solvent elimination → large volume injection (LVI), better peak shapes of early eluting peaks in acetonitrile:

• Better column protection → better long-term performance, reduced matrix effects

Dichlorvos HCH isomers

K. Mastovska, P.L. Wylie, J. Chromatogr. A 1265 (2012) 155-164.


8

Important inlet parameters:

• Solvent vent period:

• vent temperature

• vent time

• vent flow

• vent pressure

• Analyte transfer period:

• inlet temperature program

• splitless time

• Post-transfer period:

• purge flow

• gas saver flow and time Dimpled liner, 2 mm i.d.

PTV Solvent Vent Injection


Matrix Co-Extractives in GC

(1) Non-volatile

(2) Volatile

Backflush less volatile matrix

Counts vs. Acquisition Time (min)

3 x10

0

1

+EI MRM [email protected] (284 -> 249) 409_spike_2_A.D

2 x10

0

5

+EI MRM [email protected] (274 -> 88) 409_spike_2_A.D

3 x10

0

2

+EI MRM (272 -> 237) 409_spike_2_A.D

7 7.4 7.8 8.2 8.6 9 9.4 9.8

Improve selectivity

by using GC-MS/MS

- Keep them in the liner!

Use analyte

protectants

to deactivate


Column Backflushing

Backflushing can eliminate less volatile matrix components from

the GC column by reversing the column flow at a pressure

junction point:

Inlet

Flow

MS

Flow

Inlet

Flow

MS

Flow


Column Backflushing

• Post-Run Backflushing

- begins after the last analyte has been detected

- the entire column is backflushed

- typically uses a short restriction capillary installed at the column outlet

• Concurrent Backflushing

- begins after the last analyte has eluted from the first “column”

- different options:

▪ retention gap = a short uncoated capillary

▪ mid-column set-up (e.g. two 15-m columns)

▪ short column = a short coated capillary

Column 1: HP 5-MS UI, 5 m x 0.25 mm x 0.25 µm

Column 2: HP 5-MS UI, 15 m x 0.25 mm x 0.25 µm



Column Flow Program

MS/MS

EPC

Inlet

A) Elution of the analytes from the first column

Time:

0 – 15.8 min 1.1 mL/min 1.2 mL/min

Column 1

5 m

Column 2

15 m

B) Backflushing of the first column to remove less volatile matrix components

MS/MS

EPC

Inlet

Time:

15.8 – 18.6 min

18.6 – 20.0 min

2.5 mL/min

5.0 mL/min

1.2 mL/min

2.4 mL/min

Column 1

5 m

Column 2

15 m

MS/MS

EPC

Inlet

A) Elution of the analytes from the first column

Time:

0 – 15.8 min 1.1 mL/min 1.2 mL/min

Column 1

5 m

Column 2

15 m

B) Backflushing of the first column to remove less volatile matrix components

MS/MS

EPC

Inlet

Time:

15.8 – 18.6 min

18.6 – 20.0 min

2.5 mL/min

5.0 mL/min

1.2 mL/min

2.4 mL/min

Column 1

5 m

Column 2

15 m



Elimination of Less Volatile Matrix Components

Dandelion root powder full scan (m/z 45-650) analysis with backflushing

9x10

0.5

1

1.5

2

2.5

3

9x10

0.5

1

1.5

2

2.5

3


4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Acetonitrile blank analysis (no backflushing, additional 15 min at 290°C)

after the dandelion root powder analysis with backflushing

No matrix peaks from the previous injection observed!

Run time = 20 min

Backflushing starts(after deltamethrin safely elutes from the first column)

Last analyte RT = 18.45 minDeltamethrin (m/z 253>172)

2x10

0

1

2

3

4

5

6


18.2 18.3 18.4 18.5 18.6

9x10

0.5

1

1.5

2

2.5

3

9x10

0.5

1

1.5

2

2.5

3


4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Acetonitrile blank analysis (no backflushing, additional 15 min at 290°C)

after the dandelion root powder analysis with backflushing

No matrix peaks from the previous injection observed!

Run time = 20 min

Backflushing starts(after deltamethrin safely elutes from the first column)

Last analyte RT = 18.45 minDeltamethrin (m/z 253>172)

2x10

0

1

2

3

4

5

6


18.2 18.3 18.4 18.5 18.6



Elimination of Less Volatile Matrix Components

Dandelion root powder full scan (m/z 45-650) analysis without backflushing

9x10

1

2

3

9x10

1

2

3

9x10

1

2

3


4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Acetonitrile blank analysis after the dandelion root powder analysis without backflushing

and run time of 20 min

Run time = 20 min

Run time = 35 min (additional 15 min at 290°C)

Matrix peaks observed in two

subsequent blank injections!

Additional 10 min at 290°C needed to elute the less

volatile matrix components (e.g. sterols).



Benefits of Column Backflushing

• Elimination of long “baked out” at a high temperature to remove less volatile, late eluting matrix components

• Reduced analysis time

• Increased column life time

• Prevention of the MS source contamination

• Less frequent MS source maintenance

• Improved ruggedness


Deltamethrin

m/z 253→174

Dichlorvos

m/z 185→93

Malathion

m/z 173→99

Ethion

m/z 231→129

Phosalone

m/z 367→182

Overlays of GC-MS/MS chromatograms obtained within a 2.5-day sequence of 125 dietary supplement injections:


Sample Preparation and Analysis

• Samples: 5 dietary supplement matrices representing root powders (ginseng and dandelion), fruit (freeze-dried) powders (saw palmetto berry and mangosteen) and full-plant powdered extracts (scutellaria)

• Spiking level: 50 ng/g

= lower limit for the majority of pesticides listed in the European Pharmacopoeia monograph 2.08.13

- corresponding matrix-matched standards (MMstd) and standards in acetonitrile (solvent std) were prepared at 5 ng/mL (equivalent to 50 ng/g)


Sample Preparation and Analysis

• Sample extraction and clean-up: - based on the QuEChERS method

• Long-term GC-MS/MS system evaluation: 150 injections (incl. 125 matrix injections) consisting of:

5 repeated sets of 30 runs, which included the following

6 injections for each of the 5 matrices:

(1) solvent std (2) MMstd (3) spike 1 (4) spike 2 (5) spike 3 (6) MMstd

2.5-day uninterrupted sequence (no maintenance)

to mimic an over-the-weekend run


Long-term System Performance

Overlays of GC-MS/MS chromatograms for selected analytes in spiked samples

obtained within the sequence of 125 matrix injections (sets: 1, 2, 3, 4, 5):

Deltamethrinm/z 253>174

Ginseng

Root

Powder

Saw

Palmetto

Berry

Powder

Scutellaria

Powdered

Extract

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


18.1 18.2 18.3 18.4 18.5 18.6

3x10

0

0.5

1

1.5

2

2.5

3

3.5


4 4.05 4.1 4.15 4.2

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4


4 4.05 4.1 4.15 4.2

Dichlorvosm/z 185>93

3x10

0

1

2

3

4

5


4 4.05 4.1 4.15 4.2

Malathionm/z 173>99

Ethionm/z 231>129

3x10

0

0.5

1

1.5

2

2.5


9.9 10 10.1 10.2 10.3 10.4

3x10

0

0.5

1

1.5

2

2.5

3


9.9 10 10.1 10.2 10.3 10.4

3x10

0.5

1

1.5

2

2.5


9.9 10 10.1 10.2 10.3 10.4

2x10

0

1

2

3

4

5

6

7


15.3 15.4 15.5 15.6 15.7

Phosalonem/z 367>182

3x10

0

0.5

1

1.5

2

2.5

3

3.5


13.2 13.3 13.4 13.5 13.6

3x10

0.5

1

1.5

2

2.5

3

3.5

4


13.2 13.3 13.4 13.5 13.6

3x10

0.5

1

1.5

2

2.5

3


13.2 13.3 13.4 13.5 13.6

2x10

0

1

2

3

4

5

6

7


15.3 15.4 15.5 15.6 15.7

2x10

0

1

2

3

4

5

6

7

8


15.3 15.4 15.5 15.6 15.7

2x10

0

0.5

1

1.5

2

2.5

3

3.5


18.1 18.2 18.3 18.4 18.5 18.6

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4


18.1 18.2 18.3 18.4 18.5 18.6

Deltamethrinm/z 253>174

Ginseng

Root

Powder

Saw

Palmetto

Berry

Powder

Scutellaria

Powdered

Extract

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5


18.1 18.2 18.3 18.4 18.5 18.6

3x10

0

0.5

1

1.5

2

2.5

3

3.5


4 4.05 4.1 4.15 4.2

3x10

0

0.5

1

1.5

2

2.5

3

3.5

4


4 4.05 4.1 4.15 4.2

Dichlorvosm/z 185>93

3x10

0

1

2

3

4

5


4 4.05 4.1 4.15 4.2

Malathionm/z 173>99

Ethionm/z 231>129

3x10

0

0.5

1

1.5

2

2.5


9.9 10 10.1 10.2 10.3 10.4

3x10

0

0.5

1

1.5

2

2.5

3


9.9 10 10.1 10.2 10.3 10.4

3x10

0.5

1

1.5

2

2.5


9.9 10 10.1 10.2 10.3 10.4

2x10

0

1

2

3

4

5

6

7


15.3 15.4 15.5 15.6 15.7

Phosalonem/z 367>182

3x10

0

0.5

1

1.5

2

2.5

3

3.5


13.2 13.3 13.4 13.5 13.6

3x10

0.5

1

1.5

2

2.5

3

3.5

4


13.2 13.3 13.4 13.5 13.6

3x10

0.5

1

1.5

2

2.5

3


13.2 13.3 13.4 13.5 13.6

2x10

0

1

2

3

4

5

6

7


15.3 15.4 15.5 15.6 15.7

2x10

0

1

2

3

4

5

6

7

8


15.3 15.4 15.5 15.6 15.7

2x10

0

0.5

1

1.5

2

2.5

3

3.5


18.1 18.2 18.3 18.4 18.5 18.6

2x10

0

0.5

1

1.5

2

2.5

3

3.5

4


18.1 18.2 18.3 18.4 18.5 18.6


Analyte Protectants = compounds that strongly interact with the active sites

in the GC system, thus protecting susceptible analytes

against adsorption and/or degradation

M. Anastassiades, K. Mastovska, S.J. Lehotay, J. Chromatogr. A 1015 (2003) 163-184

solvent

matrix

solvent + analyte protectants

matrix + analyte protectants


Analyte Protectants Combination of analyte protectants for GC pesticide analysis

K. Mastovska, S.J. Lehotay, M. Anastassiades, Anal. Chem. 77 (2005) 8129-8137

retention time

O

OH OH

OOH

OH

gulonolactone (1 g)

OH O

OH ethylglycerol (10 g)

OH

OH

OH

OHOH

sorbitol (1 g)

moderate

strong

Signal enhancement:


Calibration without AP

21

R 2 = 0.9994

R 2 = 0.9966

R 2 = 0.9894

0.0

0.5

1.0

1.5

2.0

2.5

0 100 200 300 400 500 600

Concentration (ng/mL)

Norm

aliz

ed p

ea

k a

rea

Fruit

Vegetable

MeCN

o-phenylphenol



Calibration with AP

22

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 100 200 300 400 500 600

Concentration (ng/mL)

Norm

aliz

ed p

ea

k a

rea

o-phenylphenol

R 2 = 0.9995

Fruit

R 2 = 0.9998

Vegetable

R 2 = 0.9997

MeCN



23

lindane phosalone o-phenylphenol

1.4 higher response

6.5 higher response

response diminishment

AP = Deactivation in Every Injection!



ISTD

p,p’-DDT p,p’-Methoxychlor

Accuracy (%) RSD (%) Accuracy (%) RSD (%)

None 95.5 14 94.3 13

TPP 100 7.8 98.0 6.9

13C12- p,p’-DDT 100 1.5 98.3 2.0

K. Mastovska, Agilent Application Note 5991-1054EN

Use of Internal Standards


Why Triple Quadrupole GC-MS/MS?

• Selectivity

• Sensitivity

• Ruggedness

0.5 ng/mL chlorpyrifos

in ginseng root extract

(5 ng/g in the sample)

314>258 314>286

MS/MS is TARGETED ANALYSIS

• Analyte-specific conditions required

• We don’t see what we don’t look for!


GC-MS/MS Selectivity: Selection of Ions

(mainlib) Fonofos

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260

0

50

100

14

27

29

32

3945 51

63

69

81

83 90 95

109

121

137

141 157174

185 202 217

246

P

S

O

S

Fonofos

137>109

246>109 246>109

246>137 137>109

246>109

3-fold higher

signal


27

Atrazine EI-MS spectrum (NIST)

(mainlib) Atrazine

20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 2300

50

100

27

43

55

58

62

68

96 104 122 132145

158

173

187

200

215HN

N N

HN N Cl



28

Analysis of atrazine (RT = 7.9 min) in flour (at 5 ng/g) using MS/MS transitions m/z 215 > 200 and m/z 215 > 58

m/z 215 > 200 m/z 215 > 58



(mainlib) Malathion

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340

0

50

100

15

18

29

47 55

59

63

73

79

87

93

99

111

125

143

158

173

184 193211

227 238256

271285

330

SP

O

O

OO

S

O O

Malathion

173>117

173>99 173>99

173>117

173>99

158>47




MS/MS Optimization - Example

(mainlib) 1,2,4-Thiadiazole, 5-ethoxy-3-(trichloromethyl)-

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 2600

50

100

15

27

29

32 38

46

49 5461 73

79

8493

101

108

114

140

157 167

177

183

202

211

231

248

Cl Cl

Cl

N N

S

O

Etridiazole: Full scan EI MS spectrum

Etridiazole

183

185

211

213

246 248



Etridiazole: Overlay of product ion spectra of m/z 211 for CE = 0 - 60 V



Etridiazole: Overlay of m/z 211 > 183 chromatograms at CE = 0 - 60 V

Optimum CE = 10 V


MS/MS Optimization - Example Etridiazole: Pesticides and Environmental Pollutants MRM Database (G9250AA)


34

“Secrets” to Rugged GC-MS/MS Method

• Well optimized injection conditions (PTV solvent vent for direct analysis of QuEChERS extracts)

• Suitable internal standard(s)

• Column backflushing

• Analyte protectants

• Well optimized MS/MS transitions (evaluated in target or worst-case matrices)

NACRW, Agilent Users Meeting, July 25, 2013

Thank you for your attention!

[email protected]

Your questions?

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secrets to successful gc-ms/ms pesticide method development · secrets to successful gc-ms/ms...

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