Pesticide Protocols Pesticide Protocols

Pesticides in Human Serum/Plasma 37
several methods involving blood, serum, or plasma measurements of a variety of contemporary
pesticides have been published (10–35). The pesticides measured using
these methods include primarily organophosphate and carbamate insecticides. The
majority of these methods were developed for forensic applications or for diagnosis of
acute pesticide intoxication and have LOD ranges in the high nanograms per milliliter
to micrograms per milliliter. In all cases, these methods lack the sensitivity or the
selectivity to measure pesticides in blood or blood products resulting from low-level
exposures.
We have developed a sensitive and accurate method for quantifying 29 contemporary
pesticides in human serum or plasma (Table 1). Our method uses a simple solidphase
extraction (SPE) followed by a highly selective analysis using isotope dilution
gas chromatography–high-resolution mass spectrometry (GC–HRMS).
2. Materials
1. Standards of the pesticides and labeled pesticides (Table 2) with purity higher than 99%.
2. Ammonium sulfate.
3. Anhydrous sodium sulfate.
4. Oasis ® HLB SPE columns (Waters, Milford, MA).
5. Disposable empty sorbent cartridges.
6. SPE extraction manifold.
7. Pesticide quality solvents: dichloromethane, methanol, and toluene.
8. Bioanalytical grade I water.
9. SuporCap-100 filtration capsule.
10. Volumetric flasks.
11. Automatic pipets with disposable tips.
12. 15-mL glass centrifuge tubes.
13. 1-mL conical autosampler vials (silanized) with crimp tops.
14. Qorpak glass bottles with Teflon caps (30 mL).
15. TurboVap LV.
16. MAT900 magnetic sector mass spectrometer (ThermoFinnigan, Bremen, Germany).
17. GC (e.g., Agilent 6980, Palo Alto, CA) equipped with autosampler (e.g., CTC A200S)
and operated using XCalibur ® software
18. DB5 MS column ([14% cyanopropylphenyl]-methyl polysiloxane, 30 m, 0.25-mm id, 25-
µm film) (e.g., J & W Folsom, CA).
19. Helium (>99.999%).
3. Methods (36)
3.1. Standards
3.1.1. Native Standards
1. Prepare individual stock solutions by dissolving 3 mg of each standard into 15 mL toluene
and mixing well to obtain a concentration of 200 ng/µL.
2. Divide into 1-mL aliquots, flame seal in ampules, and store at −20°C until used.
3.1.2. Internal Standards
1. Prepare stock internal standard solutions by dissolving 5 mg of each stable isotope labeled
standard into 50 mL toluene and mixing well (see Note 1).
2. An internal standard spiking solution is prepared by diluting 1 mL of each stock solution