A High-Throughput Diagnostic Method for Measuring Human Exposure to Organophosphorus Nerve Agents

Abstract

An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL.

Direct Quantitation of Methyl Phosphonate Adducts to Human Serum Butyrylcholinesterase by Immunomagnetic-UHPLC-MS/MS

“…The validated concentration range is consistent with earlier measurements for OPNA adducts to BChE in clinical samples and the general BChE abundance of 40–80 nM expected in plasma. 7,11,16 The Taylor 17 calculation for the lower limit of detection was 0.96 ng/mL and was only slightly higher than that reported for the detection of the free acid in human urine. 18 The reportable range was from the lowest calibrator 2 ng/mL to the highest calibrator 250 ng/mL.…”

“…10 The need for a method to measure the aged adduct is apparent due to the extensive detection capabilities of the MeP adduct and the absence of a quantitative, direct measurement. Therefore, we to present a quantitative analytical method that possesses the specificity and sensitivity of immunomagnetic separation-isotope dilution-UHPLC-MS/MS 11,14 for the direct measurement of MeP adduct to BChE in human serum.…”

A high‐throughput UHPLC–MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents

“…(Carter et al, 2013, Fidder et al, 2002, Knaack et al, 2012, Sporty et al, 2010) When adducted BChE peptides (FGESAGAAS) are fragmented using collision-induced dissociation, β-elimination of the OPNA adduct is observed, converting the adducted serine into a dehydroalanine ( m/z 778.3) (Figure 4). For the MeP-BChE, EtP-BChE, PrP-BChE, and P-BChE peptides, quantitation was based on the transition of each respective [M+H] + precursor ion to the product ion m/z 778.3.…”

“…(Johnson et al, 2015, Knaack et al, 2012, Pantazides et al, 2014) Briefly, a DynaMag-15 (Invitrogen, Carlsbad, CA) was applied to remove the solvent from 2 mL of Dynabeads Protein G magnetic beads. The beads were washed twice with 4 mL aliquots of PBST, and then 8 mL of PBST was added to the beads followed by 400 μg of anti-BChE monoclonal antibody.…”

“…OPNA-BChE adducts have relatively long half-lives (8–12 days), are abundant in serum and plasma, and can easily be extracted from blood since BChE is not membrane-bound. (Knaack et al, 2012, Pantazides et al, 2014, Sporty et al, 2010)…”