Multi-class pesticide analysis in human hair by gas chromatography tandem (triple quadrupole) mass spectrometry with solid phase microextraction and liquid injection
Graphical abstract
Highlights
► SPME and GC–MS/MS were combined for the detection of 22 pesticides in human hair. ► High sensitivity was obtained despite of dealing with 7 different chemical classes. ► All the field samples analyzed here contained target pesticides. ► Some pesticides never analyzed in hair so far were detected here at high levels. ► This work will help studying pesticide exposure and associated biological effects.
Introduction
First thoroughly investigated in forensic contexts for the detection of drugs of abuse, hair has been increasingly considered a relevant matrix for the biomonitoring of human exposure to environmental pollutants. Indeed, the possibility to reach extended windows of detection and to obtain information representative of the average level of xenobiotics concentration/entrance into the body from a single specimen is particularly relevant in the context of chronic exposure biomonitoring. For this purpose, hair has been tested for several environmental pollutants such as metals, PCBs, PAHs, and pesticides such as organochlorines, organophosphates or pyrethroids [1], [2], [3], [4]. The easy sampling of hair, which does not require medical staff and generally facilitates patient compliance, is a further advantage in the case of epidemiological studies focusing on population exposure. On the other hand, the small amounts generally collected (limited to a few tens to a few hundreds milligrams) and the low levels of concentration of xenobiotics in hair, particularly in the case of environmental exposure, require the use of highly sensitive analytical methods. Analytical sensitivity is also directly affected by the specificity of the method, in that sensitivity generally decreases with increasing number of compounds from different chemical families (with different physicochemical properties) that are simultaneously analyzed in one run. Nevertheless, priority lists defined by authorities are based on risks associated with chemicals and generally include compounds from different chemical families without taking into account technical feasibility [5], [6]. Moreover, the importance of cumulative exposure to chemical mixtures, even at low levels, is increasingly pointed out [7], [8]. These considerations highlight the need to develop multi-class methods with high sensitivity for the biomonitoring of human exposure to pesticides based on hair analysis.
Solid phase microextraction (SPME), developed by Pawliszyn and co-workers in 1989 [9], [10], consists in a coated metal- or glass-fiber on which analytes are adsorbed directly from an aqueous extract (direct immersion: DI-SPME) or from the associated headspace (HS-SPME) that initially contains the analytes to be analyzed, before their desorption into the chromatographic system. Presenting the ability to perform at the same time analyte extraction from sample, concentration and purification, as well as being a versatile injection tool, SPME represents a significant advance in analytical chemistry for the handling of environmental and biological matrices containing low level of target analytes or/and high concentration of impurities. Associated with gas chromatography and microelectron-capture, mass spectrometry or high-resolution time-of-flight mass spectrometry detection, SPME has already been proven to be suitable for multi-class pesticide analysis in environmental matrices (e.g. rain, water, atmosphere) and food (e.g. bovine milk) [11], [12], [13], [14], [15]. SPME has also been tested for the determination of pesticides in human biological fluids (blood, urine, human milk), but the studies were limited to the detection of specific compounds or families of compounds and did not aim at performing multi-class analysis [16]. Until now and despite its obvious suitability for pesticide analysis, SPME has never been used for the detection of pesticides in hair and the use of this technique in hair analysis was limited to the detection of medical drugs and drugs of abuse [16], [17]. This probably lies in the fact that hair, as a solid matrix, requires pretreatment such as hydrolysis (generally acidic or alkaline) or extraction with aqueous buffer, as it is performed for the detection of drugs before exposing the SPME fiber to the aqueous extract. For several pesticides, acidic or alkaline hydrolysis is not recommended for protecting analytes from degradation. Extraction with aqueous buffer is neither adapted as it is not compatible with the hydrophobic properties of most pesticides. Until now, in most of the studies that investigated pesticide detection in hair, extraction was performed with organic solvent (directly from pulverized hair or from acidic hydrolyzate) and after possible clean-up on solid phase extraction and concentration by evaporation to dryness and reconstitution in a smaller volume of solvent, the liquid extract was injected into the gas chromatography system coupled with mass spectrometry or electron capture detection [4], [18], [19], [20]. In the published literature, neither SPME nor gas chromatography coupled with tandem (triple quadrupole) mass spectrometry (GC–MS/MS) has been tested for the detection of pesticides in human hair.
The aim of the present study has been to develop a highly sensitive method for the analysis of multi-class pesticides in human hair based on SPME coupled with GC–MS/MS. Among other parameters, the development of the full methodology included the setting up of an adapted extraction method of analytes from hair, the comparison of DI-SPME vs HS-SPME, the optimization of the time and temperature of fiber exposure and the validation of the method. The list of analytes investigated comprised 22 pesticides from different chemical classes including organochlorines, organophosphates, dinitroanilin, nicotianilin, phenol, azole and pyrethroids. An optimized protocol was designed on the basis of the validation parameters and was finally applied to the analysis of field samples that were tested for the presence of the analytes investigated.
Section snippets
Chemicals, reagents and standard solutions
All solvents (HPLC grade methanol, HPLC grade acetonitrile, methylene chloride, ethyl acetate) were purchased from Labscan Analytical Sciences (Dublin, Ireland), acetic anhydride, sodium dihydrogen phosphate and disodium hydrogen phosphate were supplied by Merck (Darmstadt, Germany). Ultrapure water was produced by an AFS-8 from Millipore (Brussels, Belgium). Pesticides standards were purchased from Dr. Ehrenstorfer (Augsburg, Germany) (the complete list of standards and internal standards is
Extraction of analytes from hair
Solid phase microextraction is based on the affinity of target compounds for the SPME-fiber compared with the aqueous extract (DI-SPME) or the associated head-space (HS-SPME) that initially contains the analytes to be analyzed. In liquid matrices, SPME can be performed directly without pre-analytical treatment when the matrix does not contain too many impurities, such as rain water or river water [14]. In the case of biological fluids (e.g. blood, urine, milk), pre-analytical treatment such as
Conclusions
Solid phase microextraction coupled with gas chromatography-tandem (triple quadrupole) mass spectrometry is particularly useful to the analysis of multi-class pesticides in human hair. For each analyte, the sensitivity of the method was proven to significantly depend on the injection mode. Besides, the combination of two successive steps of direct immersion-SPME, performed at low (30 °C) and high (90 °C) temperature on the same extract along with liquid injection was finally necessary to cope
Acknowledgment
This work was supported in part by the French Agency for Food, Environmental and Occupational Health Safety, in the framework of the Pesticide Residue Observatory action.
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