Download - Evaluation of simultaneous MS and MS2 workflows of LC/Q … · 2016. 6. 6. · 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

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<2 ppm 2-3 ppm 3-5 ppm

ddMS2

vDIA (5 segments/35K) AIF

Evaluation of simultaneous MS and MS2 workflows of LC/Q-Orbitrap for analysis of

pesticides in fruits and vegetables Łukasz Rajski, María del Mar Gómez Ramos, Víctor Cutillas, Amadeo R. Fernández-Alba

European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables. University of Almeria, Agrifood Campus of International Excellence (ceiA3). Spain. e-mail: [email protected]

INTRODUCTION

Acknowledgements: The authors acknowledge funding support from the European Commission, DG SANTE (Specific Agreement No. 7 to Framework Partnership Agreement No. SANCO/2005/FOOD

SAFETY/0025-Pesticides in fruit and vegetables).

EXPERIMENTAL

RESULTS

During last decade high resolution accurate mass spectrometers have improved qualitative (resolution, mass accuracy) as well as quantitative (sensitivity, linear range) aspects. HARMS instruments are well known for their high selectivity in full MS

mode. Nevertheless even spectrometers which have very high resolution may produce false positive results. Isotopic pattern and alternative adducts not always can correctly discard false positives. A solution to reduce number of false positive results is

application of simultaneous full MS and MS2.

The objective of this work was to compare three workflows of simultaneous MS and MS2: All Ion Fragmentation (no precursor ion selection, ions from entire mass range fragmented at the same time), variable Data Independent Acquisition (no precursor

ion selection, mass range divided into smaller segments before fragmentation) and data dependent MS2 (selection of precursor ion). Acatonitrile extracts (blanks and spiked with 166 pesticides) of 11 fruits and vegetables were used for the evaluation. Blank

extracts were used to evaluate potential false positives (considering retention time window of 0.2 min) whereas spiked extracts (at 0.01 mg/kg and 0.1 mg/kg) to evaluate the false negatives. Samples were analysed with Q Exactive Focus working with

resolution of 70,000 (at m/z 200) in full scan MS and 17,500 or 35,000 in MS2 mode.

Chromatography Mobil phase:

- A: 98% H2O 2% MeOH 5mM HCOONH4 0.1% HCOOH

- B: 98% MeOH 2% H2O 5mM HCOONH4 0.1% HCOOH

Flow: 0.35 mL/min

Gradient time: 14 min + 3 min reequilibration

Column: Accucore™ aQ C18,

100 mm x 2.1 mm x 2.6 µm

Column temperature: 30°C

Ijnection volume: 10 µL

Acquisition mode: full scan MS -resolution 70,000

-AGC target 1e6

-max IT auto

-scan range 120-1000 Da Acquisition mode: MS2 -dd MS2 resolution 17,500

-vDIA resolution 17,500 or 35,000,

3 or 5 mass segments - AIF resolution 70,000

CONCLUSIONS

Matrices

RT: 11.00 - 11.70

11.00 11.05 11.10 11.15 11.20 11.25 11.30 11.35 11.40 11.45 11.50 11.55 11.60 11.65

Time (min)

0

50

1000

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1000

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1000

50

100

Re

lative

Ab

un

da

nce

0

50

1000

50

100 NL: 1.01E8

Base Peak m/z= 307.1790-307.1820

F: FTMS + p ESI Full ms

[120.00-1000.00] MS

Tomate_100ppb_dil5_dd_150pest_R

2

NL: 8.99E7

Base Peak m/z= 307.1790-307.1820


[120.00-1000.00] MS

Tomate_100ppb_dil5_AIF_R2

NL: 9.66E7

Base Peak m/z= 307.1790-307.1820


[120.00-1000.00] MS

Tomate_100ppb_dil5_vDIA_17500_3

segm_R2

NL: 9.34E7

Base Peak m/z= 307.1790-307.1820


[120.00-1000.00] MS

Tomate_100ppb_dil5_vDIA_17500_5

segm_R2

NL: 9.48E7

Base Peak m/z= 307.1790-307.1820


[120.00-1000.00] MS

Tomate_100ppb_dil5_vDIA_3segm_

R2

NL: 9.28E7

Base Peak m/z= 307.1790-307.1820


[120.00-1000.00] MS

Tomate_100ppb_dil5_vDIA_5segm_

R2

ddMS2

cycle time 0.27s

AIF

cycle time 0.62s

vDIA (3segm x 17,500)

cycle time 0.61s


cycle time 0.79 s


cycle time 0.83 s


cycle time 1.28 s

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RSD 0-5% RSD 0-10% RSD 0-20%

3 segments/ R=35000

5 segments/ R=35000

3 segments/ R=17500

5 segments/ R=17500

AIF

dd MS2

Tomato 0.01 mg/kg

Peak area repeatability

In workflows with shorter cycle times more

points per chromatographic peak is obtained,

by that peak area repeatability is better.

Are

a

Linearity

0,0E+00

2,0E+08

4,0E+08

6,0E+08

8,0E+08

1,0E+09

1,2E+09

1,4E+09

1,6E+09

1,8E+09

2,0E+09

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

Concentration [mg/kg]

0,0E+00

2,0E+08

4,0E+08

6,0E+08

8,0E+08

1,0E+09

1,2E+09

1,4E+09

1,6E+09

1,8E+09

2,0E+09

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6

Concentration [mg/kg]

Pirimiphos-methyl

Ethoprophos

Flusilazole

dd-MS2 vDIA (5 semgents/35,000)

Independently of the workflow selected the detector response is characterised

by very good linear range and no saturation is observed.

Percentage of identified pesticides

80%

85%

90%

95%

100%

garlic

onion

spinach

green bean

lettuce

apple orange

cucumber

parsley

leek

tomato

0.1 mg/kg

0.01 mg/kg

dd-MS2

80%

85%

90%

95%

100%

garlic

onion

spinach

green bean

lettuce

apple orange

cucumber

parsley

leek

tomato

0.1 mg/kg

0.01 mg/kg

0.1 mg/kg without ion ratio


vDIA (5 semgents/35,000)

80%

85%

90%

95%

100%

garlic

onion

spinach

green bean

lettuce

apple orange

cucumber

parsley

leek

tomato

0.1 mg/kg

0.01 mg/kg



AIF

0.01 mg/kg of Propargite in leek

Full scan MS

368.1890 ± 5ppm

Full scan MS

368.1890 ± 5ppm

MS2 (120 – 1000 m/z)

231.1745 ± 5ppm

All Ion Fragmentation vDIA

MS2 (295 – 405 m/z)

231.1745 ± 5ppm

Resolving power

70,000

Resolving power

35,000

Mass errors in full scan MS

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Tomato

Orange % o

f co

mp

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ddMS2

vDIA (5 segments/35K) AIF

Tomato

% o

f co

mp

ou

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s Orange

% o

f co

mp

ou

nd

s

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Tomato Apple Orange Onion

-50%<ME

-21%<ME<-50%

-20%<ME<20%

21%<ME<50%

50%<ME

Selectivity in AIF and vDIA

MS2

151.0326 ± 5 ppm

0.01 mg/kg 0.02 mg/kg 0.05 mg/kg

Full Scan MS

343.0529 ± 5 ppm

Thiophanate methyl in onion (vDIA, 5 segments, 35K).

vDIA provides more selectivity than AIF

Quantitation in MS2 (AIF, vDIA)

If full scan MS does not have enough

selectivity, quantitation is possible in MS2

Matrix effects

Mass errors in MS2

LC–Q Exactive Focus MS operated in full scan at the resolution of 70,000 detected close to 100% of the selected pesticides in all matrices with mass errors below 2 ppm and

mass errors < 5 ppm in MS2. From the point of view of peak area repeatability the most robust workflow was dd MS2. Matrix effects were lower than 20% in the majority of the

cases facilitating quantification. AIF and vDIA offer the possibility to quantify with accurate MS2, this can help with "difficult" matrices. Dd MS2 had the highest identification rate

(96-100%, depending on the matrix). In vDIA it was 86-100% and in AIF 81-100%.