No abstract

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

A cylindrical quadrupole ion trap (r(0) = 2.5 mm, z(0) = 2.88 mm, ∼(1)/(64) of the volume of commercial hyperbolic ion traps) has been constructed, its geometry optimized, and its performance examined in the mass-selective instability scan mode. Spectra of ionized perfluorotributylamine and o-dichlorobenzene show a resolution (m/Δm, 50% valley definition) of ∼100. The instrument has been coupled to a membrane introduction system to test its applicability for on-line reaction monitoring and to determine detection limits. Simulations using the ion trap simulation program are used to explore the effects of geometry on performance and to validate the experimental results.

Organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BChE) can be used to confirm exposure in humans. A highly accurate method to detect G-series and V-series OPNA adducts to BChE in 75 μL of filtered blood, serum, or plasma has been developed using immunomagnetic separation (IMS) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS). The reported IMS method captures > 88% of the BChE in a specimen and corrects for matrix effects on peptide calibrators. The optimized method has been used to quantify baseline BChE levels (unadducted and OPNA-adducted) in a matched set of serum, plasma and whole blood (later processed in-house for plasma content) from 192 unexposed individuals to determine the interchangeability of the tested matrices. The results of these measurements demonstrate the ability to accurately measure BChE regardless of the format of the blood specimen received. Criteria for accepting or denying specimens were established through a series of sample stability and processing experiments. The results of these efforts are an optimized and rugged method that is transferrable to other laboratories and an increased understanding of the BChE biomarker in matrix.

Ricin is a toxalbumin derived from the castor bean plant, Ricinus communis. Ricinine is an alkaloid (3-cyano-4-methoxy-N-methyl-2-pyridone) that shares a common plant source with ricin, and its presence in urine infers ricin exposure. A new quantification method for ricinine was developed that uses solid-phase extraction to prepare 1-mL urine samples (81% recovery) for a 5-min, isocratic high-performance liquid chromatography method, followed by electrospray ionization tandem mass spectrometry. Protonated molecular ions were selected in the multiple reaction monitoring mode and quantified by isotope dilution with (13)C(6)-labelled ricinine as the internal reference. Urine pools enriched with ricinine at two concentrations were characterized as quality control materials and then used to validate the method. The method limit of quantification was 0.083 ng/mL, even with a confirmation ion of low relative abundance. Ricinine was stable in human urine when heated at 90 degrees C for 1 h, and during storage at 25 degrees C and 5 degrees C for 3 weeks. The method was applied to an animal exposure study, a crude ricin preparation scheme, and a forensic analysis. These studies show that ricinine can be measured in rat urine at least 48 h after exposure. Ricinine is present in crude preparations of ricin, and it can be found in human urine after a lethal exposure to ricin.

US Centers for Disease Control and Prevention.

Hydrolysis of G- and V-series organophosphorus nerve agents containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of “aging” and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has occurred, common therapeutics like oximes cannot reactivate the cholinesterase enzyme and relieve cholinergic inhibition. Until now, a direct, quantitative method for determination of the MeP adduct to BChE was unavailable. Aged adducts in serum samples were processed by immunomagnetic separation of BChE by antibody conjugated bead, isotope-dilution, pepsin digestion, followed by UHPLC separation and detection by conventional electrospray ionization tandem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 → 778.3 was used for quantitation. The analytical response ratio was linearly proportional to the serum concentration of MeP adducted peptide (MeP-P) over the nominal concentration range of 2.0–250 ng/mL, with a coefficient of determination R2 ≥0.997. Intrarun accuracy expressed as %Relative Error (%RE) was ≤13.5, 16.3 and 3.20% at 2.0, 16 and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was ≤11.9, 6.15 and 3.39%. Interday %RSD was ≤7.13, 5.69 and 1.91%. Recovery of MeP-P from serum was ≥68% across the validated concentration range, and contributions from matrix effects were minimal. The method provides a direct, quantitative measurement of MeP-P found in clinical samples suspected of nerve agent exposure and subjected to such post-sampling stresses as elevated temperature and extended shipping.

Methylenecyclopropylglycine (MCPG) and hypoglycin A (HGA) are naturally-occurring amino acids found in some soapberry fruits. Fatalities have been reported worldwide as a result of HGA ingestion, and exposure to MCPG has been implicated recently in the Asian outbreaks of hypoglycemic encephalopathy. In response to an outbreak linked to soapberry ingestion, the authors developed the first method to simultaneously quantify MCPG and HGA in soapberry fruits from 1 to 10,000 ppm of both toxins in dried fruit aril. Further, this is the first report of HGA in litchi and longan arils. This method is presented to specifically address the laboratory needs of public health investigators in the hypoglycemic encephalitis outbreaks linked to soapberry fruit ingestion.