ms/ms spectral library with embedded linear … · ms/ms spectral library with embedded linear...

MS/MS SPECTRAL LIBRARY WITH EMBEDDED LINEAR RETENTION INDICES: UNIQUE

SUPPORT FOR A RELIABLE NON-TARGETED PESTICIDE IDENTIFICATION

E. Trovato 1, M. Utczás 1,2, F. Alibrando 1, F. Vento 1, Luigi Mondello 1,3

1 Chromaleont s.r.l., c/o University of Messina, Polo Annunziata I-98168, Messina, Italy2 Center of Sports Nutrition Science, University of Physical Education, H-1123,

Budapest, Hungar3 Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali,

University of Messina, Polo Annunziata, I-98168, Messina, Italy

[email protected]

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40th International Conference on

Environmental & Food Monitoring, Santiago

de Compostela (Spain) 19-22 June 2018

mailto:[email protected]

Identification in GC-MS

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✓Commercial libraries

✓Chromaleont GC-MS libraries✓Commercial libraries

✓Chromaleont GC-MS libraries

MS spectrum

50.0 100.0 150.0 200.0 250.00

5

10

15

20

25%

125 204

89

4113863 102

113 23977

5.0 15.0 25.0 35.0 45.0 55.0 65.0 75.0 85.00

0.2

0.4

0.6

0.8

1.0

1.2

tR(z+1)

tRi

tRz

5.0 10.0 15.0 20.0 25.0 30.0 35.0

0.05

0.25

0.45

0.65

0.85

1.05

C8

C9

C10

C11

C12

C13

C14

C15

C7

Linear retention index (LRI)

Retention behavior as identification parameter

At the 1st analysis

Intraday (48 h) repeatability of retention time

At the 35th analysis

At the 15th analysis

0.1 min (6 s)

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The absolute retention time

varies not only with all

chromatographic variables

(temperature program, gas

linear velocity, phase ratio

and column length) but also

between normal runs on the

same column

26.00 26.05 26.10 26.15 26.20 26.25 26.30 26.35 26.40

2.5

5.0

7.5

(x100,000)

5.0 15.0 25.0 35.0 45.0 55.0 65.0 75.0

0.05

0.25

0.45

0.65

0.85

1.05(x1,000,000)

GC-MS chromatogram n-alkanes (C7-C30)

Linear retention index (LRI) approach

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Linear retention index for a specific analyte (LRI) [1,2]:

LRI = 100n[(tRi-tRz)/(tR(z+1)-tRz)]+100z

z: carbon atom number

n: the difference in carbon atom number of the two standards

(n-alkanes: n=1, even or odd FAMEs and FAEEs: n=2)

[1] H. Van den Dool and P.D. Kratz, J. Chromatogr. 11 (1963) 463

[2] M.B. Evans and J.F. Smith, J. Chromatogr. 8 (1962) 303

tR(z+1)

tRztRi

10 20 30 40 50 60 70 80

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(x1,000,000)

C7

C8

C9

C10

C11

C12

C13

C14

C15

C16

C17

C18

C19 C20

C21

C22 C

23 C

24 C25

C26

C27

C28

C29

C30

C10

C11

LRI: 1031

LRI: 1000LRI: 1100

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Linear retention index for a specific analyte (LRI)

LRI = 100n[(tRi-tRz)/(tR(z+1)-tRz)]+100z

z: carbon atom number

n: the difference in carbon atom number of the two

standards

(n-alkanes: n=1, even or odd FAMEs and FAEEs: n=2)

tR(z+1)

tRi

tRz


Compound type LRI range

Average peak width

at half height

(LRI)

Average peak width

at half height

(s)

Super volatile <800 2 2.16

High volatile 800 1200 2 3.00

Volatile 1200 1600 2 3.96

Medium volatile 1600 2000 2 3.72

Low volatile >2000 3 4.02

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LR

I (u

nit

)0

500

1000

1500

2000

2500

3000

0 20 40 60 80

tr (min)


Mass spectral databases on the market

Library Name DateEI MS

spectraMS/MS spectra

GC and retention

index records

NIST MS '17 (recommended base library) 2017-06 NEW 306622 652475 404045

NIST GC '17 (included with NIST MS) 2017-06 404045

NIST MSMS '17 (included with NIST MS) 2017-06 652475

Wiley Registry 11th 2016-03 775550

Wiley 10th+NIST14 combined 2014-11 918000 234284 346757

Wiley METLIN Mass Spectral Database 2017 NEW >72000 (+ NISTMSMS)

Wiley Lipids Mass Spectral Database 2017 NEW 430

Wiley Pesticides Mass Spectral Library with LRI, 2nd Edition 2017 NEW

Wiley Designer Drugs 2016-06 23477

(include

~12700

Wiley Flavors/Fragrances (FFNSC), 3rd Edition 2014-12 3462 Yes

Wiley Drugs/Poisons/Pesticides/Pollutants & Metabolites(Maurer/Pfleger/Weber) 2017 NEW 10430 Yes

Wiley LC-MSn Library of Drugs, Poisons and Their Metabolites(Maurer/Wissenbach/Weber) 2014 >10000

Wiley FAMEs Fatty Acid Methyl Esters: Mass Spectral Database 2011-11 240 YES

Wiley Physiologically Active Substances (drugs, steroid hormones, and endocrine disruptors) 2011-05 4182

Wiley Pesticides (Rolf Kühnle) 2009 1238

Wiley Androgens/Estrogens/Steroids 2008 3722

Wiley Organic Compounds 2004 37055

Wiley Geochemicals/Petrochemicals/Biomarkers 2003 1100

Wiley Tandem MSMS (MSforID) 2012 Mar 12048

NIST17 supersedes NIST14.

Chart lists number of spectra or data records for each library.

Many libraries contain Linear Retention Indices (LRI) calculated by using different

columns (e.g. non polar, polar, medium polarity)

But these values represent only an average of the experimental data

obtained by various authors

NO ACCURATE searching!

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• Retention index types in the databases: Normal alkane RI, Linear RI, Kovats RI

• Average experimental RI values and estimated values based purely on

structure

• Average retention index: expressed as the median of experimental RI on similar

stationary phases with standard deviation

median ± standard deviation (n: number of values used for the calculation)

• Retention index prediction: from the cited values of the given compound or

structurally related compounds in the database on non-polar stationary phase (n-

alkane scale)

estimated value

confidence interval (diverse functional groups) at 50% and 95% confidence level

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Propanil

Estimated non-polar retention index (n-alkane scale): Value: 1752

Confidence interval (Diverse functional groups): 89(50%) 382(95%) iu

Column type median RI SD n experimental LRI

Standard non-polar 1669 24 4 1611

Semi-standard non-polar 1746 8 30 1748

Bromoxynil



ΔLRI: +207




Polar 3197 0 1 -

ΔLRI: +63ΔLRI: +21

ΔLRI: +58

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Metamitron






Ethofumesate



ΔLRI: +103




ΔLRI: +207ΔLRI: +45

ΔLRI: +20

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A comprehensive database was realized containing the MS spectra of about 1300

pure compounds. Each component is associated to multiple LRIs calculated by using

different columns and n-alkanes homologous series.

Experimental LRI conceptOur concept of MS spectral database is different from the other

The concept of stability of LRIs was emphasized

To obtain a reliable identification a filter window of ±5 LRI units should be used

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HIT CAS Common name Molecular

weight

Molecular

formulaLRIlib LRIexp1 LRIexp2 LRIexp3 LRIaverage

Repeatibility

intralaboratoryΔLRI

562 1194 - 65 - 6 Dichlobenil 171 C7 H3 Cl2 N 1332 1334 1336 1335 1335 1.0 3

596 759 - 94 - 4 EPTC 189 C9 H19 N O S 1352 1356 1352 1356 1355 0.9 3

526 1918 - 16 - 7 Propachlor 211 C11 H14 Cl N O 1601 1605 1604 1606 1605 1.2 4

639 81777 - 89 - 1 Clomazone 239 C12 H14 Cl N O2 1746 1750 1748 1747 1748 0.6 2

527 709 - 98 - 8 Propanil 217 C9 H9 Cl2 N O 1872 1874 1873 1876 1874 0.7 2

643 61213 - 25 - 0 Flurochloridon 311 C12 H10 Cl2 F3 N O 2012 2009 2010 2011 2010 0.5 -2

536 101 - 27 - 9 Barban 257 C11 H9 Cl2 N O2 2186 2184 2187 2189 2187 0.4 2

551 74070 - 46 - 5 Aclonifen 264 C12 H9 Cl N2 O3 2269 2272 2275 2271 2273 1.1 4

525 137641 - 05 - 5 Picolinafen 376 C19 H12 F4 N2 O2 2465 2470 2467 2469 2469 0.9 3

578 66441 - 23 - 4 Fenoxaprop ethyl ester 361 C18 H16 Cl N O5 2656 2661 2660 2658 2660 0.9 3

588 76578 - 14 - 8 Quizalofop-ethyl 372 C19 H17 Cl N2 O4 2834 2835 2840 2838 2838 1.3 5

541 142891 - 20 - 1 Cinidon ethyl 393 C19 H17 Cl2 N O4 3191 3200 3189 3194 3194 0.3 1

Identification in GC-MS

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GCMS Solution SoftwareHighly Reliable

Identification

✓Commercial libraries

✓Chromaleont GC-MS libraries✓Commercial libraries

✓Chromaleont GC-MS libraries

MS spectrum

50.0 100.0 150.0 200.0 250.00

5

10

15

20

25%

125 204

89

4113863 102

113 23977

5.0 15.0 25.0 35.0 45.0 55.0 65.0 75.0 85.00

0.2

0.4

0.6

0.8

1.0

1.2

tR(z+1)

tRi

tRz

5.0 10.0 15.0 20.0 25.0 30.0 35.0

0.05

0.25

0.45

0.65

0.85

1.05

C8

C9

C10

C11

C12

C13

C14

C15

C7

Linear retention index (LRI)

Inject the sample

WORK-FLOW

Inject the standard series (n-

alkanes) using the same

chromatographic method as the

sample

Register the retention time of each

standard compound and assign it the

conventional LRI value (e.g.

C10=1000). Save the data file

Calculate the LRI of your target

molecule according to your

standard mixture

Compare the LRI value with the

value reported in the Library

Load retention time series

from the data file

Use linear retention index filter

simultaneously with MS similarity

search in the Library

Manual GCMSsolution

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PESTICIDES GC-MS Library

The most extensive pesticide database

New version included also metabolites and artifacts

for a total of 1300 compounds

925 compounds

Columns made by Merck-Millipore

(length 30m × inner diameter 0.25mm × film thickness 0.25μm)

ColumnReference

seriesNo.

SLB-5ms ALKANEs 1300

EQUITY-1 ALKANEs 147

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PESTICIDES GC-MS Library Ver. 2

Containing:

✓ Systematic name

✓ Common name

✓ CAS number

✓ Molecular weight

✓ Molecular formula

✓ Molecular class

✓ Standard and column

information

✓ 2 experimental LRI values

Class No.

Adjuvants 12

Acaricides 93

Alkanes 34

Artifacts/Degradation products 63

Avicides 1

Bactericides 29

Fungicides 187

Herbicides 248

Insecticides 236

Molluscicides 7

Nematicides 11

Pesticides 112

Pesticides intermediers 48

Pesticides metabolites 80

Pheromones 56

Plant growth regulators 37

Preservative 2

Repellents 17

Rodenticides 10

Solvents 17

Total 1300

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Identification of HCH isomers

1,000 times more

toxic than other

diastereomers

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with two independent analytical information

50 100 150 200 250 3000.0

5.0

(x1,000)181

219

111 217

51

14777

254121 15896 290258193 234

GCMSsolution MS similarity filter (>90%)

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?

By using only the MS similarity filter the identification is

not reliable!

GCMSsolution MS similarity filter (>90%)

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50 100 150 200 250 3000.0

5.0

(x1,000)181

219

111 217

51

14777254121 15896 290258193 234

50 100 150 200 250 300

0.0

5.0

(x1,000)181

219

111 217

51

14777254121 15896 135 290258193 234

50 100 150 200 250 3000.0

5.0

(x1,000)181

219109

51

85 1458773 216113 156 254133 169 292193 277235



GCMSsolution MS similarity filter (>90%) and ±5 LRI filter

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!



GCMSsolution MS similarity filter (>90%) and ±5 LRI filter

The LRI filter allows to obtain one single match

excluding possible wrong identification

2,4 isomer:

insecticide activity

Other positional isomers:

nematicide activity

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Identification of dimethyl phenol isomers

?


LRI: 1146

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50.0 75.0 100.0 125.00.0

5.0

(x1,000)107 122

779151

65 10355 80 8974 119

50.0 75.0 100.0 125.00.0

5.0

(x1,000)107 122

779151

65 10355 8074 89 11995 125110130

50.0 75.0 100.0 125.00.0

5.0

(x1,000)107 122

779151

65 10355 80 8974 11995

10 20 30 40 50 60 70 80

0.5

1.0

1.5

2.0

2.5

3.0

3.5(x10,000,000)


The LRI filter allows to obtain one single match

excluding possible wrong identification !

According to MS similarity search a long list of candidates

can be obtained with almost identical similarity

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Advantages:

• Non-target pesticide identification

• Reduced false-positive and negative identification

• 50% coverage of the EU Pesticides Database and the

Japan Positive List

10 20 30 40 50 60 70 80

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

(x1,000,000)TIC

1,2

-Dic

hlo

robenzene

Eth

iola

te

Dic

hlo

benil

EP

TC

Pebula

teT

ebuth

iuro

nM

ecopro

pm

eth

yleste

r

MC

PA

meth

yleste

r

Pro

pachlo

rA

trazin

e-d

esis

opro

pyl

Sulfalla

teIs

opro

pyl2,4

-D e

ste

rC

lom

azone

Pro

flura

linA

zip

rotr

yn

Monalid

eP

ropanil

Medin

ote

rb a

ceta

teE

thofu

mesate

Flu

rochlo

ridon

isom

er

I

Chlo

rflu

recol-m

eth

yl

Halo

xyfo

p-m

eth

yl

Flu

oro

difen

Barb

an A

ciflu

orf

en

meth

yleste

rA

clo

nifen

TD

CP

PC

hlo

ridazon

Flu

roxypyr

1-m

eth

ylh

epty

l

este

rN

itra

lin

Pic

olin

afe

n

Mefe

nacet

Fenoxapro

peth

yleste

r

Quiz

alo

fop-P

-eth

yl

Flu

ridon

Pyridate

Cin

idon

eth

yl

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Reliable non-target identification

using Pesticides Library

0

• Low limit of identification and quantification

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26.50 26.75 27.00 27.25 27.50 27.75 28.00 28.25 28.50 28.75 29.00 29.25 29.50 29.75 30.00

0.25

0.50

0.75

1.00

1.25

1.50

(x1,000,000)

26.50 26.75 27.00 27.25 27.50 27.75 28.00 28.25 28.50 28.75 29.00 29.25 29.50 29.75 30.00

0.50

0.75

1.00

1.25

1.50

1.75(x100,000)

100 ppm

10 ppm

Identification of a spectrum acquired in Fullscan

mode by Shimadzu GCMS-TQ8040

Dic

hlo

be

nil

Dic

hlo

be

nil

S/N = 5

0

0.25

171

136100

TIC

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1 ppm

• Low limit of identification and quantification

The compound signal at 1 ppm level is similar to the baseline signal. The

use of LRI index makes possible the identification

Identification of a spectrum acquired in Fullscan


Dic

hlo

be

nil

S/N ~ 0 TIC171

136100

91 Similarity

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27.0 27.1 27.2 27.3 27.4 27.5 27.6 27.7 27.8 27.9 28.0 28.1 28.2

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2.25

(x1,000)

At the same concentration, the signal of the compound aquired

in MRM mode is much higher respect to those in full scan mode

Identification of a spectrum acquired in MRM


Dic

hlo

be

nil

170.90>110.00 (7.02)

170.90>136.00 (1.10)

170.90>100.00 (1.00)

TIC

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Good capability to reach lower identification

and quantification limits

y = 12.404,194x + 806,267

R² = 1,000

0

200000

400000

600000

800000

1000000

1200000

1400000

0 20 40 60 80 100

Pea

k a

rea

(-)

Absolute quantity (pg)

Dichlobenil

Identification of a spectrum aquired in MRM mode

(by Shimadzu GCMS-TQ8040)

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By using only the MS similarity filter the identification is not reliable!


(by Shimadzu GCMS-TQ8040) with MRM library

and CromatoPlusSpectra Software

170.90>110.00 (7.02)

170.90>136.00 (1.10)

170.90>100.00 (1.00)

Aquisition time window: 27.00-28.25 min

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✔

170.90>110.00 (7.02)

170.90>136.00 (1.10)

170.90>100.00 (1.00)

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The LRI filter allows to

obtain one single match

excluding possible wrong

identification




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• The use of triple quadrupole (QqQ) MS as a detector offers

advantages due to its high sensitivity and reproducibly

generated spectra

• Possibility to identify unknown compounds at a

concentration level 10 times lower than the legal limit

• On the market only a limited number of MS/MS libraries

can be found, furthermore using only spectral information in

many cases misidentification could occur due to the highly

similar fragmentation pattern even after the collision.

Remarks

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t.it• The simultaneous use of spectral similarity and LRI filter

information in the integrated MS/MS-LRI database makes

automatic the identification procedure, reduces false-positive

and negative identification, and provides an important

support in untargeted pesticide analysis

• The Linear Retention Indices (LRI) approach used in

combination with mass spectral search is a powerful

tool for boosting the reliability of the identification of

challenging molecules and also decrease significantly

the limit of identification (LOI).

Remarks

Thank you for your kind

attention!

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ms/ms spectral library with embedded linear … · ms/ms spectral library with embedded linear...

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