Rapid screening and determination of nerve agent metabolites in human urine by LC-MS/MS

ISSN 1061�9348, Journal of Analytical Chemistry, 2014, Vol. 69, No. 9, pp. 909–916. © Pleiades Publishing, Ltd., 2014.

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1 In cases of allegations of use of chemical warfare(CW) agents, environmental [1] or biomedical sam�ples [2] may be collected for forensic analysis.Unequivocal identification of agents, degradationproducts, and metabolites provides key supportingevidence of non�compliance with the ChemicalWeapons Convention. The perceived increasing threatof terrorist use of CW agents has further stimulated therequirement for forensic methods of analysis. Theanalysis of biomedical samples was extensivelyreported following two releases of the nerve agent sarinby terrorists in Japan in 1994 and 1995 [3–8], and fol�lowing the use of VX for an assassination [9, 10].

In general, the primary metabolites of nerve agentsare their simple hydrolysis products. Exposure to nerveagents resulted in the parent compounds hydrolyzingto their corresponding phosphonic acids and covalent�ly binding to enzymes and tissue proteins [11–13].Nerve agents readily degrade by rapid hydrolysis to thecorresponding alkyl methylphosphonic acids in ourbodies (Fig. 1): Sarin (GB) to isopropyl methylphos�phonic acid (IMPA), Soman (GD) to pinacolyl meth�ylphosphonic acid (PMPA), VX to ethyl methylphos�phonic acid (EMPA), Russian VX to isobutyl meth�ylphosphonic acid (iBuMPA), GF to cyclohexylmethylphosphonic acid (CMPA). These alkyl meth�ylphosphonic acids are finally slowly hydrolyzed to

1 The article is published in the original.

methylphosphonic acid (MPA). Urinary excretion ofthe phosphonic acid metabolite is the primary elimi�nation route for nerve agents following exposure [14].In the body the primary leaving group is fluorine forGB, GD and GF. For VX the leaving group is the thiomoiety. As the degradation products are more stablethan their parent compounds, they have been used asagent specific markers for quantitating each nerveagents following exposure [15].

Owing to its sensitivity and selectivity, gas chroma�tography (GC) [16], especially gas chromatographymass spectrometry (GC–MS) [17–22], has been usedmost frequently for the separation and identificationof the chemical warfare agents and related com�pounds. However, all GC�based methods suffer fromsome obvious limitations in the quantitation of thenerve agent degradation products. Since aqueous sam�ples or extracts, which are common in laboratory, can�not be analyzed directly by GC. The analytes must betransferred from the water phase to an organic solventby extracting or concentrating to dryness, and then po�lar degradation products of nerve agents need to be de�rivatized into volatile, nonpolar compounds before be�ing injected into a GC column. All these procedures aretime�consuming and may result in unstable recovery.

LC–MS/MS seems to be an attractive alternativeto GC–MS for the determination of chemical warfareagent degradation products, which are frequentlynonvolatile, highly polar and very water soluble, sinceit allows the direct analysis of aqueous samples or

Rapid Screening and Determination of Nerve Agent Metabolitesin Human Urine by LC⎯MS/MS1

Jin Young Lee* and Yong Han LeeAgency for Defense Development (ADD)

PO BOX 35�5, Yuseong�gu, Daejeon, 305�600 Republic of Korea*e�mail: [email protected]

Received June 4, 2012; in final form, March 27, 2013

Abstract—Qualitative screening procedures have been developed for the rapid detection and identificationof the metabolites of nerve agents in the urine samples and extracts using liquid chromatography–tandemmass spectrometry (LC–MS/MS). The combination of negative electrospray ionization (ESI) using a C18column and water–methanol mobile phase modified with ammonium formate provides a rapid screeningprocedure for nerve agent degradation products with limit of detection of 1 ng/mL in the precursor�ion anal�ysis. Also, determination of the alkyl methylphosphonic acids was carried out by the SRM scan mode withthe limit of detection of 0.1 ng/mL. These procedures will be applicable to the trace analysis of metabolites ofnerve agents in human urine matrices in the Organisation for the Prohibition of Chemical Weapons (OPCW) pro�ficiency test.

Keywords: screening, determination, nerve agent, OPCW, LC⎯MS/MS

DOI: 10.1134/S1061934814090081

ARTICLES

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JOURNAL OF ANALYTICAL CHEMISTRY Vol. 69 No. 9 2014

JIN YOUNG LEE, YONG HAN LEE

aqueous extracts with little or no sample preparation.In recent years, with the development of new interfac�es and ionization methods, Black and Read [23–25]developed a qualitative screen procedure for the detec�tion of the hydrolysis and related products of nerveagents, sulfur and nitrogen mustard, and 3�quinuclid�inyl benzylate using liquid chromatography–atom�spheric pressure chemical ionization mass spectrome�try and liquid chromatography electrospray ionizationmass spectrometry (LC–ESI–MS).

The 2nd OPCW confidence building exercise onbiomedical sample analysis coordinated by the Prepa�ratory Commission for the Organisation for the Prohi�bition of Chemical Weapons/Provisional TechnicalSecretariat took place in February 2012. The partici�pating laboratories were asked to identify all analytesdeemed relevant to the Chemical Weapons Conven�tion: beta�lyase metabolites of sulfur mustard, thiodig�lycol and metabolites of nerve agents in urine samples.Our primary interest is the development of rapidscreening and identification techniques for a wide

range of CW agents and their analogues as included inthe lists of scheduled chemicals in the Annex to theChemical Weapons Convention. These schedules in�clude literally hundreds of possible analogues of nerveagents and their precursors, which produce a similarnumber of possible degradation products.

In the present paper, we report the use of precursor�ion analysis and selected reaction monitoring (SRM)tandem mass spectrometry to rapidly screen and quan�titate the metabolites of nerve agents in urine samplesand extracts. These techniques are demonstrated in theidentification and characterization of alkyl meth�ylphosphonic acids in unknown urine samples.

EXPERIMENTAL

Materials. MPA, EMPA, and PMPA were pur�chased from Aldrich (Seoul, Korea). IMPA, iBuMPAand CMPA were synthesized in our laboratory andwere >95% pure by nuclear magnetic resonance andLC–MS. The silica bonded strong anion�exchange Ac�

H3C P

O

F

O H3C P

O

OH

O

H3C P

O

F

O H3C P

O

OH

O

H3C P

O

F

O H3C P

O

OH

O

H3C P

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OEt

SN H3C P

O

OH

OEt

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O

H3C P

O

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OH

H2O

fast

H2O

fast

H2O

fast

H2O

fast

H2O

fast

H2O

slow

GB

GD

GF

VX

RVX

iBuMPA

EMPA

CMPA

PMPA

IMPA

MPA

Fig. 1. The major metabolites of nerve agents GB, GD, GF, VX and Russian VX from hydrolysis.


RAPID SCREENING AND DETERMINATION OF NERVE AGENT METABOLITES 911

cuBond II (SAX) cartridges (200 mg, 3 mL) and strongcation�exchange AccuBond II (SCX) cartridges(200 mg, 3 mL) were purchased from Agilent Technolo�gies (Milwaukee, WI, USA). HPLC�grade solventswere used; water was obtained from a Milli�Q system(Millipore).

Liquid chromatography conditions. A Thermo�Scien�tific Accela LC system was used consisting of a Thermo�Scientific Accela autosampler plus Accela 600 pump.The system was fitted with a 150 mm × 2.1 mm AlltechC18 column (Thermo Electron Co., USA), with 5 mmparticle size and 10 nm pore size. The mobile phaseconsisted of water (solvent A) and methanol(solvent B), each modified with 20 mM ammoniumformate. Chromatographic conditions were initiallydeveloped to separate a mixture of alkyl methylphos�phonic acids consisting of MPA, EMPA, IMPA,iBuMPA, CMPA, and PMPA. The gradient was as fol�lows: 5% of B (from 0 to 2 min), linear increase up to90% B at 8 min and 100% B at 10 min at a flow�rate of0.3 µL/min. The injection volume for LC experimentwas 10 µL using the autosampler.

Mass spectrometer conditions. The column efflu�ent was introduced into a Thermo�Scientific TSQVantage triple stage quadrupole mass spectrometer(Thermo Finnigan Surveyor, San Jose, CA, USA) viaan atmospheric pressure ionization source/interfaceoperated in ESI mode. Ionization was performed innegative mode for alkyl methylphosphonic acids. Thespray voltage was 3.5 kV, capillary temperature 350°C,vaporizer temperature 300°C, sheath gas 40 arbitraryunits, and auxiliary gas 10 arbitrary units. Both Q1 andQ3 were operated at the peak width of 0.7 amu. Thecollision gas was argon at 173.32 Pa. Detection of an�alytes was conducted in precursor�ion analysis andSRM mode. The Xcalibur software was used for in�strument control, data acquisition and data handling.

RESULTS AND DISCUSSION

Rapid screening of analytes using LC–ESI–MS/MSprecursor�ion analysis. First, six kinds of alkyl meth�ylphosphonic acids were spiked with the aqueous solu�tion at the 100 ng/mL levels.

As shown in the Fig. 2, all kinds of alkyl meth�ylphosphonic acids have the same fragment ion at m/z95 by LC–ESI–MS/MS in the negative ion mode.Thus, we selected the ion at m/z 95 as product ion and

screened the aqueous sample with spiking standards byLC–ESI–MS/MS under precursor�ion analysis.

The analysis of the complete range of hydrolysisproducts in a single procedure required a column ca�pable of separating basic, acidic and neutral com�pounds whilst using the same mobile phase system.The C18 column employed was initially developed tochromatograph basic compounds, without the need touse basic mobile phase modifiers, but it is also suitablefor acidic analytes. This column provided acceptablechromatography for the complete range of nerve agenthydrolysis products using a conventional water–meth�anol–ammonium formate mobile phase with gradientelution. For precursor�ion analysis experiment, theprecursors of all alkyl methylphosphonic acids werescanned simultaneously during analysis of aqueoussamples. Screening the precursors of the ion at m/z 95detected six precursor ions at m/z 95, 123, 137, 151,177, and 179 which corresponded to the molecular an�ions of MPA (tr = 1.06 min), EMPA (tr = 1.55 min),IMPA (tr = 2.44 min), iBuMPA (tr = 5.77 min), CMPA(tr = 6.86 min), and PMPA (tr = 7.43 min), respectively(Fig. 3). The least sensitivity was experienced with themost acidic analyte such as methylphosphonic acid.Peak shapes were generally good with minimal tailing.Injection of 10 µL or less gave the best resolution; larg�er volume injections gave enhanced detection limitsbut at the expense of resolution.

Quantitation of alkyl methylphosphonic acids byLC–ESI–MS/MS SRM mode. The precursor�ionanalysis experiment conducted on a Q1qQ3 analyzerwas particularly useful to rapidly screen the alkyl me�thylphosphonic acids in the unknown samples. How�ever, precursor�ion analysis alone cannot discriminatethe isomers even though these can be separated byHPLC. The alkyl methylphosphonic acids gave acommon fragment ion at m/z 95 in electrospray nega�tive mode spectra. This common product ion can beutilized for the application of LC–ESI–MS/MS tothe trace analysis of alkyl methylphosphonic acids,with Q3 set to detect the common product ion and Q1set for selected quasimolecular ions [M–H]–. SRManalysis experiment was also conducted to furtheridentify the alkyl methylphosphonic acids havingproducts ion at m/z 95 detected during precursor�ionanalysis.

The followed transitions and collision energies aregiven in Table 1. Two transitions were followed for allalkyl methylphosphonic acids. The alkyl methylphos�

HO P

O

OH

O R O P

O

CH3

O R O P

O

CH3

OH–

–

+ alkene

m/z = 95

Fig. 2. Fragmentation scheme for alkyl methylphosphonic acids (negative ion mode).

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phonic acids produced two common product ions atm/z 95 and 79. The major abundant transitions ofquasimolecular ions [M–H]– to m/z 95 were selectedfor quantitative analysis of alkyl methylphosphonicacids. Identical elution sequence (MPA > EMPA >IMPA > iBuMPA > CMPA > PMPA) as shown previ�ously by LC–ESI–MS/MS precursor�ion analysiswas obtained during LC–ESI–MS/MS analysis usingSRM detection.

Application of LC–ESI–MS/MS precursor�ionanalysis and SRM mode in the analysis of unknownurine samples. In the 2nd OPCW confidence buildingexercise on biomedical sample analysis test scenario,the five urine samples (201–205) were collected insupport of an investigation of alleged use. There wasevidence of nerve agent use and the concentration ofthe urine samples was under 50 ppb level. The partici�pating laboratories were asked to identify all alkyl me�thylphosphonic acids deemed relevant to the Chemi�cal Weapons Convention in the five urine samples.

Sample preparation. One milliliter of urinary sam�ple was diluted in 1 mL of deionized water. To removeextraneous materials such as inorganic cations in theurine, the diluted sample (2 mL) was passed throughAg/Ba cation exchange resin cartridge followed bySCX (3 mL, Supelco, USA) cartridge preconditionedwith 1 mL of methanol followed by 1 mL of deionizedwater. The cartridges were rinsed with 2 mL of water.The eluted analyte (4 mL) was loaded onto SAX(3 mL, Supelco, USA) cartridge preconditioned with 1mL of methanol followed by 1 mL of deionized water.The cartridge was then washed with 2 mL of water and2 mL of methanol before elution. The analytes were elut�ed by 2 mL of acidic methanol (0.2 M HCl/MeOH) andconcentrated to dryness at room temperature under agentle stream of nitrogen. The dried residue was dis�solved in 0.15 mL of water and neutralized to pH 7with 1 M KOH solution.

Analysis of urine sample by LC–ESI–MS/MSprecursor�ion analysis and SRM mode. The developed

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Fig. 3. LC–ESI–MS/MS chromatogram and mass spectra of negative precursor�ion analysis; (a) MPA, (b) EMPA, (c) IMPA(see in the next page), (d) iBuMPA, (e) CMPA, (f) PMPA.



procedures were applied to the identification for allalkyl methylphosphonic acids present in the unknownurine samples. To rapidly screen where alkyl meth�ylphosphonic acids are present in the urine samples,we selected the common fragment ion at m/z 95 asproduct ion and analyzed the each urine sample underprecursor�ion analysis. As shown in Fig. 4, two uni�dentified peaks are eluted at tr = 1.52 and 7.43 min in

the 202 urine sample under LC–ESI–MS/MS pre�cursor�ion analysis. Compared to retention time of thepeaks of standard alkyl methylphosphonic acids, thesetwo peaks were anticipated as EMPA and PMPA dueto the same retention time. To further confirm theseunidentified peaks, SRM analysis experiment wasconducted with Q3 set for detecting the product ion atm/z 95 and Q1 set for selected quasimolecular

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ions [M–H]– at m/z 123 for EMPA and at m/z 179 forPMPA. Based on the result of precursor ion analysisand SRM scan mode, these two peaks in the unknownurine sample were confirmed as EMPA and PMPA.

As the result of the same screening procedure,IMPA was present in the 203 urine sample and IMPA

and PMPA were present in the 204 urine sample at atrace level.

Quantitative analysis and limit of detection (LOD)for unknown sample by LC–ESI–MS/MS SRMmode. To ascertain the reliability of the method, thequantitation of analytes in the unknown sample wascarried out at different concentrations. A calibrationgraph was constructed by plotting the ratio of the peakareas for EMPA and PMPA within the spiking con�centration range of 10–100 ng/mL in the SRM scanmode. Linearity was observed over the specified con�centration range with γ2 = 0.996 and 0.999 for EMPAand PMPA, respectively.

The two compounds which were confirmed as EMPAand PMPA in the 202 urine sample were analyzed bySRM scan mode to calculate the concentration of eachcompound. The intergrated areas of each compoundwere ratioed against those of the external standard forquantitation purposes. The value of intergrated areasfor each analyte is calculated on a calibration curvesobtained from the standard samples. In the case ofEMPA and PMPA, each compound was spiked at the20 ng/mL for EMPA and 25 ng/mL for PMPA in the202 sample (Fig. 5). Limit of detection of each com�pound and quantitative value in the 202, 203, 204urine samples showed in the Table 2.

Table 1. SRM transition and collision energies for the ana�lytes

Chemical Transitions, m/z Collisionenergy, eV

Retention time, min

MPA 95 [M−H] → 95 10 1.0879 18

EMPA 123 [M−H] → 95 15 1.5079 27

IMPA 137 [M−H] → 95 16 2.4579 36

iBuMPA 151 [M−H] → 95 16 5.7379 34

CMPA 177 [M−H] → 95 19 6.8979 46

PMPA 179 [M−H] → 95 20 7.4379 51

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180.01

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Fig. 4. The chromatogram and mass spectra of negative precursor�ion analysis of 202 urine sample; (a) EMPA, (b) PMPA.



* * *

Qualitative screening procedures have been devel�oped for the rapid detection and identification of themetabolites of nerve agents in the urine samples andextracts, using liquid chromatography–tandem massspectrometry. The combination of negative electro�spray ionization using a C18 column and water–meth�anol mobile phase modified with ammonium formateprovides a rapid screening procedure for nerve agentdegradation products with limit of detection of 1 ng/mLin the precursor�ion analysis. Also, quantitative anal�ysis of the alkyl methylphosphonic acids was carriedout by the SRM scan mode with limit of detection of0.1 ng/mL. These procedures will be applicable to thetrace analysis of metabolites of nerve agents in humanurine matrices in the Organisation for the Prohibitionof Chemical Weapons proficiency test.

ACKNOWLEDGEMENTS

We thank the Agency for Defense Development forfinancial support.

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2000

1000

0 7654321Time, min

10

Inte

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ty

RT: 7.43

98

AA: 12300

1000

500

RT: 1.51AA: 5868

0

500RT: 1.51AA: 2028

0

RT: 7.43AA: 4091

Fig. 5. The chromatogram of negative SRM scan mode of urine sample 202.

Table 2. LOD of each compound and quantitative data in each urine sample

Sample Chemical LOD of SIM mode, ng/mL

LOD of SRM mode, ng/mL

Reported concentration, ng/mL

Spiking concentration, ng/mL

202EMPA 5 1 20 25

PMPA 1 0.1 25 25

203 IMPA 5 1 7 5

204IMPA 5 1 7 5

PMPA 1 0.1 8 5

916



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Rapid screening and determination of nerve agent metabolites in human urine by LC-MS/MS