Verification of Exposure to Organophosphates: Detection of an Unknown Cholinesterase Inhibitor

Verification of exposure to nerve agents is relevant because the health risks of even low level exposures have not yet been fully explored. Within this context it is important to verify non-exposure in order to reassure worried military personnel or citizens. Phosphylated butyrylcholinesterase is one of the most important biomarker to verify an exposure to nerve agents. Nerve agent adducts can be analyzed with LCMS/MS by detection of a phosphylated nonapeptide that is derived after digestion of butyrylcholinesterase with pepsin. For a sensitive analysis (low degree of BuChE inhibition) the identity of the cholinesterase inhibitor has to be known in order to operate the LC-MS/MS instrument in the most sensitive SRM mode. In practice the identity of the cholinesterase inhibitor will not be known beforehand and the number of possible organophosphates is larger than thousand. However, the number of possible molecular masses of relevant organophosphates is approximately 170. By smart selection of these masses the number of MRM transitions that have to be acquired can even be reduced to 34 transitions, when all nerve agents from the OPCW schedule 1 list need to be screened. Furthermore, a generic method will be presented that is based on the conversion of the phosphyl moiety conjugated to BuChE. After digestion of BuChE the phosphyl group is eliminated from the peptide under mild alkaline conditions. Subsequently, the formed dehydroalanine residue reacts with a specific nucleophile which results in the formation of a generic adduct that can be measured with LC-MS/MS using the most sensitive SRM mode. With the presented methodology a larger spectrum of BuChE inhibitors can be detected without paying too much compromise to the sensitivity. 1.0 INTRODUCTION Recently we have developed several methods for the verification of exposure to nerve agents [1-3]. Besides the hydrolysis products of nerve agents, the most important biomarker is the adduct of the nerve agent to butyrylcholinesterase. One method utilizes the fluoride reactivation technique in which reactivatible phosphyl groups are released from the enzyme upon incubation with fluoride ions [4]. The other method focuses on the enzyme itself: after isolation from plasma and digestion with pepsin a phosphylated nonapeptide is analyzed with LC-MS/MS [5]. The mass of the phosphylated peptide is a reference to the identity of the nerve agent. In view of the low concentrations of the biomarkers, analyses are often acquired in the SIM (Single Ion Monitoring) or SRM (Single Reaction Monitoring) mode to adjust the mass spectrometer in the most sensitive mode. It means that the identity of the nerve agent must be known in advance in order to adjust the mass spectrometer for the adequate acquisition mass. In practice, the identity of the ChE inhibitor will not be known always, which means that the mass spectrometer must be operated in a scan mode to screen for possible ChE inhibitors. Three common van der Schans, M.J.; Fidder, A.; Hulst, A.; Noort, D. (2007) Verification of Exposure to Organophosphates: Detection of an Unknown Cholinesterase Inhibitor. In Defence against the Effects of Chemical Hazards: Toxicology, Diagnosis and Medical Countermeasures (pp. 9-1 – 9-10). Meeting Proceedings RTO-MP-HFM-149, Paper 9. Neuilly-sur-Seine, France: RTO. Available from: http://www.rto.nato.int. Verification of Exposure to Organophosphates: Detection of an Unknown Cholinesterase Inhibitor 9 2 RTO-MP-HFM-149 daughter ions of the nonapeptide are 602, 673 and 778, which can serve as fixed product ions to perform a parent ion scan. It will be studied whether the identity of the ChE inhibitor can be noticed after a fixed product ion scan. Secondly, the consequences on the sensitivity of the assay will be studied as well. An alternative approach is to convert the nerve agent adduct into a common adduct which can be measured with LC-MS/MS in the SRM mode. The identity of the nerve agent can not be deduced anymore, but the LC-MS analysis can be performed in the most sensitive (SRM) mode. The method is truly generic because all phosphylation groups that bind to the active site of BuChE will be detected. The conversion will be performed according to a method that is inspired from the detection of phosphylation sites as used in proteomic studies [6-8]. The phosphyl group will be eliminated by mild alkaline hydrolysis, which results in the formation of a dehydroalanine group. Next, the double bond reacts with a specific nucleophile that is added to the reaction mixture. The adduct can later be analyzed as a specific mass tag to the nonapeptide. The fixed product ion scan and conversion of phosphylated BuChE will be discussed in this report. 2.0 EXPERIMENTAL 2.1 Materials Purified human butyrylcholinesterase (HuBuChE, E.C. 3.1.1.8) was obtained from Dr. B.P. Doctor of the Walter Reed Hospital, Washington DC. Centrifugal ultrafilters (Centricon YM-3, 3 kD or Amicon Ultra15, 100 kD), were purchased from Millipore (Bedford, MA, USA). Pepsin (EC 3.4.23.1) was purchased from Roche Diagnostics (Almere, The Netherlands), Sarin, soman and VX were used from stocks within our laboratory. Human plasma was purchased from a blood bank (Sanquin, Leiden, The Netherlands). Bariumhydroxide was obtained from Riedel-de Haën. 2-(3-aminopropyl-amino)ethanolwas purchased from Fluka (Buchs, Switzerland). 2.2 Instrumentation Fixed product ion scan analyses were performed on a TSQ Quantum Ultra triple quad mass spectrometry instrument from Thermo Scientific (Breda, The Netherlands). HPLC system was an Acquity system from Waters (Milford, MA, USA). Derivatized BuChE was analyzed on a Q-TOF mass spectrometer from Micromass (Altrincham, UK) equipped with a standard Z-spray electrospray interface. The LC system consisted of an Alliance 2690 HPLC gradient system (Waters, Milford, MA, USA). 2.3 Procedures 2.3.1 Inhibition of plasma samples with organophosphates Human plasma was inhibited with sarin, soman, VX or dichlorvos. The concentration of the OP in plasma was 3.7 – 7 μM, which is a 75-140 fold excess compared to the approximate concentration of HuBuChE in plasma (50 nM). Inhibition of the sample was allowed for 2 hrs at room temperature. As a blank, noninhibited plasma (0.5 ml) was used. The plasma samples were further processed as described below. 2.3.2 Isolation of HuBuChE from human plasma A disposable 10 mL mini-extraction column (tube ABIMED AMS 422 peptide synthesizer, Gilson, Villiers le Bel, France) was filled with 2 mL procainamide-gel, which was washed with 20 mL of phosphate buffer (15 mM KH2PO4 and 5 mM Na2HPO4, pH 6.9) Then, 1 mL of plasma sample was gently Verification of Exposure to Organophosphates: Detection of an Unknown Cholinesterase Inhibitor RTO-MP-HFM-149 9 3 mixed with the procainamide-gel. After 30 min at room temperature, the gel was washed with 5 mL phosphate buffer and 7 mL 350 mM sodium chloride (350 mM NaCl in phosphate buffer). Finally, HuBuChE was eluted with 10 ml 1000 mM NaCl in phosphate buffer. 2.3.3 Digestion of HuBuChE with pepsin The HuBuChE solution obtained after procainamide affinity extraction was concentrated using a 100 kD cut-off filter. The retentate was washed with 5% formic acid (2x 2 mL). The retentate (approximately 200 μL) was transferred to a 4 ml glass vial; the filter was rinsed with 250 μl 5% formic acid. The rinse fluid was combined with the retentate. Pepsin solution (50 μl of a 0.2% (i.e., 2 mg/ml) solution in 5% formic acid) was added. After incubation for 2 h at 37 °C, the incubation mixture was filtrated through a prewashed (0.5 ml water) 3 kD cut-off filter. The filter was washed with 150 μl 5% formic acid solution and the fluid was filtrated and pooled with the first filtrate. This solution was used for LC-tandem MS experiments. 2.3.4 LC-tandem MS of pepsin digests Stationary phase was a PepMap C18 column (15 cm x 1 mm, 3 μm particles) from LC-Packings (Amsterdam, The Netherlands). The mobile phase consisted of a gradient of A: 0.2% formic acid in water and B: 0.2% formic acid in acetonitrile. Gradient program was 0’-5’: 100%A, flow 0.1 0.6 mL/min; 5’60’: 100% A->70% B, flow 0.6 mL/min. The pump flow (0.6 mL/min) was reduced to a column flow of approx. 50 μL/min by a splitter (LC-packings). Injection volume was 10-20 μl. QTOF: Electrospray MSMS spectra of the protonated molecular ion were recorded using a cone voltage of approximately 35 V and a collision energy of approximately 30 eV. Subsequently, ion chromatograms of m/z 778.4, the most selective fragment originating from the loss of the phosphyl moiety from the protonated molecular ion, were generated. Triple quad: MRM transitions of daughter ion 778 were recorded. Source CID was 12 V and collision energy was 31 V. 2.3.5 Conversion of pepsin digests of HuBuChE with Ba(OH)2 and 2-(3aminopropylamino)ethanol and subsequent LC-tandem MS analysis Isolation of HuBuChE from plasma and subsequent pepsin digestion was carried out as described above. The filtrate was concentrated, coevaporated with 50 mM NH4HCO3 (2x 0.5 ml), and dissolved in an aqueous solution of Ba(OH)2 and 2-(3-aminopropylamino)ethanol (100 and 50 mM, respectively; 0.2 mL). After incubation for 1 h at 37 oC, the reaction was quenched by the addition of acetic acid (10 microliter). The resulting solution was analyzed with LC/MS/MS (QTOF configuration) as described above, but with a collision energy of 18 eV. Verification of Exposure to Organophosphates: Detection of an Unknown Cholinesterase Inhibitor 9 4 RTO-MP-HFM-149 3.0 RESULTS 3.1 Generic assay: Fixed product ion scan Human plasma was incubated with the n-propyl analogue of VX to inhibit BuChE completely. BuChE was isolated using the procainamide affinity extraction, digested with pepsin and analyzed with LCMS/MS. It was supposed that the identity of the inhibitor was not known beforehand, which means that a specific acquisition mass could not be established. The nonapetides have three daughter ions in common (602, 673 and 778) which can serve as fixed product ion in a parent ion scan. Normally the ionchromatogram of a transition to daughter ion 778 is the cleanest chromatogram, therefore a parent ion scan of daughter ion 778 was acquired and Figure 1 shows the result. R T : 0 . 0 0 4 0 . 0 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 T i m e ( m i n ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 7 0 7 5 8 0 8 5 9 0 9 5 1 0 0 R el at iv e A bu nd an ce 2 7 . 2 7 1 7 . 4 2