Development of primary standards for mass spectrometry to increase accuracy in quantifying environmental contaminants.

Internal standards are essential in electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) to correct for systematic error associated with ionization suppression and/or enhancement. A wide array of instrument setups and interfaces has created difficulty in comparing the quantitation of absolute analyte response across laboratories. This communication demonstrates the use of primary standards as operational qualification standards for LC-MS instruments and their comparison with commonly accepted internal standards. In monitoring the performance of internal standards for perfluorinated compounds, potassium hydrogen phthalate (KHP) presented lower inter-day variability in instrument response than a commonly accepted deuterated perfluorinated internal standard (d3-PFOS), with percent relative standard deviations less than or equal to 6%. The inter-day precision of KHP was greater than d3-PFOS over a 28-day monitoring of perfluorooctanesulfonic acid (PFOS), across concentrations ranging from 0 to 100µg/L. The primary standard trometamol (Trizma) performed as well as known internal standards simeton and tris (2-chloroisopropyl) phosphate (TCPP), with intra-day precision of Trizma response as low as 7% RSD on day 28. The inter-day precision of Trizma response was found to be greater than simeton and TCPP, across concentrations of neonicotinoids ranging from 1 to 100µg/L. This study explores the potential of primary standards to be incorporated into LC-MS/MS methodology to improve the quantitative accuracy in environmental contaminant analysis.

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS).

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 µL samples were prepared using a (13)C(4)-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60°C, and reconstituted in 50 µL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 µm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92-13.4%) and accuracy (96.4-111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments-none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.

Rapid analysis of Lewisite metabolites in urine by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry.

A high-throughput method has been developed for determining Lewisite [dichloro(2-chlorovinyl)arsine] exposure by measuring the urine metabolite 2-chlorovinylarsonous acid (CVAA) and the oxidized metabolite 2-chlorovinylarsonic acid (CVAOA). The rapid sample preparation included a simple dilution of 400 microL of urine with 40 microL of water and 1 mL of buffer containing an internal standard and brief centrifugation prior to analysis by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (ICP-MS). CVAOA and CVAA were eluted isocratically with retention factors of approximately 3.0 and 4.2, respectively, from a reversed-phase polar embedded column with a cycle time of 5 min per sample. The dynamic reaction cell, typically used to remove polyatomic isobaric interferences, was not required for ICP-MS analysis because of the resolution of chloride from arsenical peaks of interest. This method was used to detect CVAA and CVAOA in the urine of a rat administered Lewisite up to 24 h after exposure. The method demonstrated linearity over at least three orders of magnitude and had a method detection limit of 1.3 microg/L as CVAA (1.4 microg/L CVAOA). The relative standard deviations for quality control samples ranged from 3 to 6%. The method was sensitive and selective with no false positives in 100 different urine samples collected from individuals with no known exposure to Lewisite. Ninety-six samples could be analyzed in an 8-h day.

Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry.

The rodenticide monofluoroacetate (MFA) and monochloroacetate (MCA), a chemical intermediate from several chemical syntheses, have been identified as potential agents of chemical terrorism due to their high toxicity. In preparation for response to poisonings and mass exposures, we have developed a quantification method using isotopic dilution to determine MFA and MCA in urine from 50 to 5000 ng/mL. Both analytes were extracted from urine using solid-phase extraction; extraction recoveries were 62% (MFA) and 76% (MCA). The extracts were then separated with isocratic high-performance liquid chromatography and identified using electrospray ionization tandem mass spectrometry, with detection limits of 0.9 and 7.0 ng/mL for MFA and MCA, respectively. Selectivity was established for both analytes with unique chromatographic retention times which were correlated with isotopically labeled internal standards and the use of two mass spectral transitions for each compound. The intra-day variability was less than 5% for both analytes and the inter-day variability was 7% for MFA and 6% for MCA.

Enhancing the response of alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry by post-column addition of organic solvents.

A method to enhance the signal intensity and signal-to-noise of several alkyl methylphosphonic acids in negative electrospray ionization liquid chromatography tandem mass spectrometry (ESI LC-MS/MS) is presented. This class of compound represents the initial metabolites and environmental degradants of the nerve agents: VX, rVX (Russian VX), GB (Sarin), GF (Cyclosarin), and GD (Soman). Compared with the post-column addition of the mobile phase, the post-column addition of aprotic solvents and longer chain alcohols enhance the signal intensity and signal-to-noise ratio (S/N) of the chromatographic peaks by factors of up to 60 and 19, respectively. The post-column addition of water, methanol, and ethanol resulted in little or no relative signal enhancement. It is proposed that the post-column addition of these solvents do not result in the same enhancements due to stabilization of analyte solvation through hydrogen bonding.

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry.

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention (k'=3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (-0.5 to +3.4%) and precise (coefficients of variation of 2.1-3.6%) quantitative results over the clinically relevant urine concentration range of 1-200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.

Quantification of 31 volatile organic compounds in whole blood using solid-phase microextraction and gas chromatography-mass spectrometry.

The prevalence of exposure to volatile organic compounds (VOCs) has raised concern about possible health effects resulting from chronic human exposure. To support studies exploring the relation between VOC exposure and health effects, we developed an automated analytical method using solid-phase microextraction (SPME), capillary gas chromatography (GC), and quadrupole mass spectrometry (MS). This method quantifies trace levels (low parts per trillion) of 14 halogenated alkanes, 5 halogenated alkenes, 10 aromatic compounds, and 2 other VOCs in human blood. Detection limits for the SPME-GC-MS method range from 0.005 to 0.12 microg/L, with linear calibration curves spanning three orders of magnitude. The improved throughput of this method will enable us to expand biomonitoring efforts to assess nonoccupational VOC exposure in large epidemiological studies.