Development of high throughput LC/MS/MS method for analysis of perfluorooctanoic acid from serum, suitable for large-scale human biomonitoring.

A simple method for determination of perfluorooctanoic acid (PFOA) from human serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and utilizing high-throughput sample preparation was developed by New York State Public Health Emergency Preparedness Laboratory and used for biomonitoring studies. 50µl of serum was mixed with a 0.4ml of acetonitrile containing isotopically labeled internal standard, followed by phospholipid removal/protein precipitation. The extract was partially dried and analyzed by LC/MS/MS. The linear range of PFOA analysis was 0.5-100ng/ml. LLOQ was selected as 0.5ng/ml. The method was validated following APHL guidelines for LRN-C laboratories and about 6000 specimens were successfully prepared and analyzed using this simple LC/MS method. Due to sample preparation simplicity, the presented method can be used in large-scale clinical testing, such as public health surveillance studies.

Cyanide adducts with human plasma proteins: albumin as a potential exposure surrogate.

Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.

A sensitive method for quantitation of beta-lyase metabolites of sulfur mustard as 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) in human urine by isotope dilution liquid chromatography-positive ion-electrospray-tandem mass spectrometry.

A method for measurement of an important biological marker, 1,1'-sulfonylbis[2-(methylthio)ethane] (SBMTE) of sulfur mustard agent HD [bis-(2-chloroethyl)sulfide] in human urine, to quantify HD exposure, is presented. It employs TiCl3 reduction of beta-lyase metabolites to SBMTE, and automated solid-phase extraction sample preparation, followed by isotope dilution liquid chromatography-positive ion-electrospray ionization-tandem mass spectrometry with 7.5 min/sample cycle time, to achieve SBMTE quantitation of up to 200 samples/24h a day. Percent relative standard deviations over the calibration range varied from 12.0% at 0.1 ng/mL to 0.9% at 100 ng/mL, and the limit of detection from a 0.5 mL sample was below the lowest level calibration standard of 0.1 ng/mL.