Determination of Chlorpyrifos, Chlorpyrifos Oxon, and 3,5,6-Trichloro-2-Pyridinol in Rat and Human Blood

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Abstract
Analytical methods to quantitate chlorpyrifos and two potential metabolites, chlorpyrifos oxon (oxon) and 3,5,6-trichloro-2-pyridinol (TCP), in human and rat blood are described. Chlorpyrifos and the oxon were extracted simultaneously with a methanol/hexane mixture from 0.5 mL blood that was deactivated with an acidic salt solution. The extract was then concentrated and analyzed by negative-ion chemical ionization gas chromatography-mass spectrometry (NCI-GC-MS). TCP was extracted from a separate 0.1-mL aliquot of blood, also deactivated by the addition of acid. The t-butyldimethylsilyl derivative of TCP was formed using MTBSTFA, and the analysis was performed by NCI-GC-MS. Stable isotope analogues of chlorpyrifos (−13C2−15N), oxon (−13C2−15N), and TCP (−13C2) were used as internal standards. Oxon was observed to partially degrade to TCP during the sample analysis. Accurate oxon and TCP measurements were obtained with the use of oxon−13C2−15N, TCP−13C2, and TCP−13C2−15N internal standards, which compensated for both the degradation of oxon and the formation of artifactual TCP during analysis. The limits of quantitation were 1 ng/mL blood for both chlorpyrifos and oxon and 10 ng/mL for TCP. Calibration curves were linear over the concentration range of 2.5–2500 ng/mL solvent for chlorpyrifos and oxon and between 5 and 1060 ng/mL solvent for TCP. Taking concentration factors and extraction efficiencies into account, these linear ranges represent blood concentrations of approximately 0.3–300 ng/mL blood for chlorpyrifos and the oxon and 6–1300 ng/mL blood for TCP. The lowest spike level for chlorpyrifos and the oxon was 1 ng/mL blood, and the lowest spike level for TCP was 10 ng/ml. blood. Recoveries from rat blood were as follows: 106–119% for chlorpyrifos, 94–104% for oxon, and 85–102% for TCP. In addition, chlorpyrifos and oxon were incubated with rat and human blood for various time intervals before deactivation to determine precautions that needed to he taken when collecting and handling specimens. No change in chlorpyrifos concentration was observed in rat blood up to 180 min at 37°C. In contrast, the oxon was rapidly hydrolyzed to TCP in both rat (t½ ≈ 10 s) and human (t½ ≈ 55 s) blood held at 37°C. The hydrolysis rate for the oxon was independent of whether a rat had been administered chlorpyrifos previously, the initial oxon concentration, the presence of chlorpyrifos, and the age or gender of the human volunteers. These results suggest rapid sample preparation is critical for accurate determinations of the oxon metabolite of chlorpyrifos. These methods provide excellent tools for use in chlorpyrifos pharmacokinetic modeling studies.

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