Quantitation of ortho‐cresyl phosphate adducts to butyrylcholinesterase in human serum by immunomagnetic‐UHPLC‐MS/MS

Abstract

Tri-ortho-cresyl phosphate (ToCP) is an anti-wear, flame retardant additive used in industrial lubricants, hydraulic fluids, and gasoline. The neurotoxic effects of ToCP arise from the liver-activated metabolite 2-(o-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (cresyl saligenin phosphate or CBDP), which inhibits esterase enzymes including butyrylcholinesterase (BChE). Following BChE adduction, CBDP undergoes hydrolysis to form the aged adduct ortho-cresyl phosphoserine (oCP-BChE), thus providing a biomarker of CBDP exposure. Previous studies have identified ToCP in aircraft cabin and cockpit air, but assessing human exposure has been hampered by the lack of a laboratory assay to confirm exposure. This work presents the development of an immunomagnetic-UHPLC-MS/MS method for the quantitation of unadducted BChE and the long-term CBDP biomarker, oCP-BChE, in human serum. The method has a reportable range from 2.0 ng/mL to 150 ng/mL, which is consistent with the sensitivity of methods used to detect organophosphorus nerve agent protein adducts. The assay demonstrated high intraday and interday accuracy (≥ 85%) and precision (RSD ≤ 15%) across the calibration range. The method was developed for future analyses of potential human exposure to CBDP. Analysis of human serum inhibited in vitro with CBDP demonstrated that the oCP-BChE adduct was stable for at least 72 hours at 4, 22 and 37 °C. Compared to a previously reported assay, this method requires 75% less sample volume, reduces analysis time by a factor of 20, and demonstrates a 3-fold improvement in sensitivity.

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

“…(Schopfer et al, 2010) Previously reported methods were used to confirm that the CBDP-inhibited serum was not fully aged. (Johnson et al, 2015) Therefore, the level of P-BChE in this material was expected to increase with time resulting in a greater %RSD. The imprecision observed in this QC is a reflection of adduct stability in the material and not the assay.…”

“…This is different from other methods developed in this laboratory which have used GB-inhibited serum or GA-inhibited serum as a matrix blank. (Carter et al, 2013, Johnson et al, 2015, Pantazides et al, 2014) These inhibited matrices could not be selected for this method since GB inhibited BChE ages to MeP-BChE and GA inhibited BChE can age to form P-BChE or ExP-BChE. All three of these aged products are measured in this assay so use of GA or GB inhibited serum would interfere with the detection of aged biomarkers as the materials aged.…”

“…(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.…”

“…Samples were prepared as previously described with additional optimization (Johnson et al, 2015;Knaack et al, 2012;Pantazides et al, 2014). Briefly, a DynaMag-15 (Invitrogen, Carlsbad, CA, USA) was applied to remove the solvent from 2 mL of Dynabeads Protein G magnetic beads.…”

High-Confidence Qualitative Identification of Organophosphorus Nerve Agent Adducts to Human Butyrylcholinesterase

“…Therefore, the same products ions are always observed regardless of which OPNA is bound. 24,25,26 These conserved product ions are signature b-ions (containing the nonapeptide amino terminus after loss of the OPNA adduct) 27 that can be broadly used by qualitative screening assays to determine OPNA exposure regardless of the initial precursor ion mass. For example, a blood sample taken from an exposed individual days after an exposure event may contain several distinct biomarkers of OPNA exposure, which requires complicated targeted analysis.…”