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

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 h 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 threefold improvement in sensitivity. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

Exposure to tri-o-cresyl phosphate detected in jet airplane passengers.

The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.

Pharmacokinetics and metabolism of a single subneurotoxic oral dose of tri-o-cresyl phosphate in hens.

Hens were given a single oral dose of 50 mg (4.6 microCi)/kg [14C] tri-o-cresyl phosphate (TOCP). Four groups of three hens each were killed after 0.5, 1, 2, and 5 days. The half-life of 14C in plasma was 2 days. TOCP and its metabolites in the plasma, liver, kidneys, and lungs were analyzed by high-performance liquid chromatography and liquid scintillation counting. TOCP reached its highest concentration in plasma between 0.5 and 1 day after administration. Under these experimental conditions, the disappearance of TOCP from the plasma followed monoexponential kinetics with a half-life of 2.2 days. Appreciable concentrations of saligenin cyclic-o-tolyl phosphate, the active neurotoxic metabolite, were detected in the plasma as well as in the liver, kidneys, and lungs at all time points and had half-lives of 2.06, 1.36, 1.11 and 4.44 days, respectively. The presence of this active metabolite of TOCP might contribute to the sensitivity of the hen to TOCP-induced delayed neurotoxicity. Other hydrolytic and oxidative products of TOCP were also identified in tissues.

Disposition of [14C]tri-o-cresyl phosphate and its metabolites in various tissues of the male cat following a single dermal application.

The disposition and metabolism of a single dermal dose of 50 mg (5.8 muCi)/kg of tri-o-cresyl [phenyl-U-14C]phosphate (TOCP) were investigated in adult male cats. TOCP was applied on a preclipped area on the back of the animals neck. Three treated cats were sacrificed at each time interval 0.5, 1, 2, 5, and 10 days following application. Plasma, brain, spinal cord, sciatic nerve, liver, kidneys, and lungs were extracted and analyzed for TOCP and its various metabolites by high performance liquid chromatography and liquid scintillation counting. TOCP reached its highest concentration in plasma at 12 hr, while its metabolites attained their maximum concentration levels between 24 and 48 hr after dosing. The disappearance of TOCP from the plasma followed a monoexponential kinetics with a half-life of 1.2 days. Di-o-cresyl hydrogen phosphate and o-cresyl dihydrogen phosphate were the major metabolites in the plasma while dihydroxymethyl TOCP was present in trace amounts. Appreciable concentrations of saligenin cyclic-o-tolyl phosphate, which is believed to be the active neurotoxic metabolite, were detected in the plasma at all time points. TOCP was the predominant compound in the brain, spinal cord, and sciatic nerve, while the liver, kidneys, and lungs contained mostly metabolites. The major metabolite identified in the liver, kidneys, and lungs was o-hydroxybenzoic acid (salicylic acid) followed by di-o-cresyl hydrogen phosphate whereas di-o-cresyl hydrogen phosphate and o-cresyl dihydrogen phosphate were the most abundant metabolites in the brain, spinal cord, and sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Three new contaminants detected in postmortem blood samples.

Three contaminants were identified during drug screening of postmortem blood samples which had been stored in glass bottles with a black rubber seal. Two of these contaminants, cyanoethyl dimethyldithiocarbamate and N-phenyl-2-naphthylamine, were found to come from the rubber seal of some bottles. The third contaminant was not a single compound but rather a mixture of aryl phosphates with a composition very similar to technical grade tritolyl phosphate. The origin of these phosphates is at present unknown.

High-performance liquid chromatographic analysis on radial compression column of the neurotoxic tri-o-cresyl phosphate and metabolites.

A method utilizing high-performance liquid chromatography (HPLC) has been developed for the analysis of tri-o-cresyl phosphate (TOCP) and its possible metabolites, o-cresyl dihydrogen phosphate, di-o-cresyl hydrogen phosphate, o-hydroxybenzyl alcohol, o-cresol, saligenin cyclic-o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one], salicylic acid, salicylaldehyde, hydroxymethyl TOCP (di-o-cresyl o-hydroxymethylphenyl phosphate), and dihydroxymethyl TOCP (o-cresyl di-o-hydroxymethylphenyl phosphate). TOCP and its possible metabolites were analyzed on a reverse-phase C18 cartridge fitted into RCM-100 radial-compression separation system. Compounds were separated using a linear gradient of 25 to 80% acetonitrile in 2% aqueous acetic acid at a flow rate of 1.3 ml/min in a period of 22 min. Quantification was achieved by monitoring the ultraviolet absorbance of the column eluates at 254 nm and measuring peak areas. Retention times and peak areas were highly reproducible for all compounds analyzed. The relationship between peak area and amount injected was linear over a 100-fold range for all compounds analyzed. The minimum detectable level was 3 ng for salicylaldehyde, 25 ng for o-hydroxybenzyl alcohol and salicylic acid, and 50 ng for the remaining compounds. A mixture of TOCP and its possible metabolites was added to samples of cat liver, kidney, and plasma and then extracted and analyzed. High recovery and reproducibility for most compounds was observed in tissues analyzed.