Validated analytical methodology for the simultaneous determination of a wide range of pesticides in human blood using GC–MS/MS and LC–ESI/MS/MS and its application in two poisoning cases

doi:10.1016/j.scijus.2015.04.007

Science & Justice

Volume 55, Issue 5, September 2015, Pages 307-315

https://doi.org/10.1016/j.scijus.2015.04.007 Get rights and content

Highlights

•
Development of a methodology for the simultaneous quantification of 109 pesticides
•
Validation of the analytical parameters for human whole blood
•
Application to the identification of the pesticide involved in two real cases
•
Semi quantitative application to matrixes other than blood

Abstract

Pesticides are frequently responsible for human poisoning and often the information on the involved substance is lacking. The great variety of pesticides that could be responsible for intoxication makes necessary the development of powerful and versatile analytical methodologies, which allows the identification of the unknown toxic substance. Here we developed a methodology for simultaneous identification and quantification in human blood of 109 highly toxic pesticides. The application of this analytical scheme would help in minimizing the cost of this type of chemical identification, maximizing the chances of identifying the pesticide involved. In the methodology that we present here, we use a liquid–liquid extraction, followed by one single purification step, and quantitation of analytes by a combination of liquid and gas chromatography, both coupled to triple quadrupole mass spectrometry, which is operated in the mode of multiple reaction monitoring. The methodology has been fully validated, and its applicability has been demonstrated in two recent cases involving one self-poisoning fatality and one non-fatal homicidal attempt.

Introduction

The large group of pesticides, which are widely used throughout the world primarily to control pests affecting crops, is often implicated in human poisoning [1]. Morbidity and mortality attributable to these substances vary from country to country, depending on many variables such as the level of socioeconomic development, accessibility to these chemicals, and the importance of the agricultural sector. Fatalities involving pesticides are a consequence of accidents, self-injury or more rarely homicides, and range from less than 1% of deaths from poisoning in EU countries [1], [2] to up to 71% of all violent deaths in the Western Pacific and Southeast Asia [3], [4], [5]. In all these cases chemical analysis to investigate the poison involved is mandatory, and the forensic laboratory is facing a challenge because in most cases there is no information on what the substance involved was. The main difficulties of this type of toxicological identifications are the wide variety of biological matrices that are sent to the laboratory, often in advanced state of decomposition, along with the wide range of pesticides to which the poisoning could be attributed.

In recent years the use of chromatographic techniques (HPLC or GC) combined with detection of analytes by mass spectrometry (MS/MS) has been considered as a very useful tool in forensic toxicology laboratories, since it enables high selectivity along with a very low detection limits. Triple quadrupole mass spectrometers (QqQ) allow operating in the mode of selective multiple reaction monitoring (SRM). This allows the monitoring of parent ions fragmenting into product ions. This mode of operation improves selectivity and sensitivity of the determination, in comparison with one-stage mass spectrometry. With this technique, the virtual elimination of isobaric interferences is allowed, as well as a significant decrease in chemical noise from the matrix [6]. The use of any of these analytical techniques is currently seen as a practical way to overcome the difficulties posed by complex biological matrices, which may contain an excessive amount of potentially interfering substances, such as fat, protein, sugars, and chemicals [7]. In addition, high acquisition speed in the MRM mode allows the development of methods for the simultaneous analysis of tens or even hundreds of compounds belonging to different chemical classes [8], [9], [10], [11], [12].

It needs to be emphasized that when the information of the pesticide involved is lacking it is generally difficult to conduct thorough analytical investigations in complex biological matrices such as ante- or post-mortem blood, and usually several complementary analyses are needed. That is why all the techniques of high sensitivity and specificity that allow the simultaneous analysis of a wide series of chemicals of high toxicity may contribute to reducing the costs associated with this type of analytical, as well as to increase the chances of identifying the “unknown toxic substance”. In this work we have developed an analytical method for the detection and quantification of 109 pesticides in human blood. The pesticides have been selected on the basis of both, their high toxicity to humans [13], and the frequency with which they are involved in cases of poisoning [14], [15]. This methodology is based on a liquid–liquid extraction, clean-up, chromatographic separation, and detection by QqQ operated in the MRM mode, and has been successfully applied to the detection of the toxicant involved in 2 recent cases of poisoning by an unknown pesticide that were submitted to our laboratory. Besides, this methodology has been also applied in our laboratory to the identification of the pesticides in matrices other than blood [16].

Section snippets

Chemicals and reagents

Acetone, acetonitrile, cyclohexane, dichloromethane, ethyl acetate, and methanol (> 99.9%) were purchased from Fisher Scientific (Leicestershire, United Kingdom). Ultrapure (UP) water was obtained from a Milli-Q Gradient A10 (Millipore, Molsheim, France). Blank blood was purchased from Medichem (Medidrug® Basis Line, Medichem, Germany). All the pesticide standards (purity from 97% to 99.5%), as well as the internal standards (ISs, aldicarb-D3, carbofuran-D3, chlorfenvinphos-D10, chloropropham,

Optimization of the instrumental method

LC–MS/MS and GC–MS/MS provided very low detection limits and could be applied to the identification and confirmation of the peak identities. Two transitions were selected for each analyte included in this study (Table 2). The combination of the transitions and their retention times allowed the pesticide identity to be confirmed.

In this work, we simultaneously investigated 90 pesticides suitable for GC (Table 2) to obtain the most efficient quantitative results with maximum separation.

Conclusions

We have shown in this paper the applicability of a methodology based on a liquid–liquid extraction followed by a combination of two chromatographic methods (LC and GC) with mass spectrometry detection for the identification and quantification of 109 toxic pesticides in blood samples from human pesticide poisoning episodes. The validation parameters were satisfactory. For all the pesticides we found good linearity (0.5–500 μg/mL, with r² > 0.98) and low detectability. The recoveries (68 to 105%)

Conflict of interest

There are no financial or other relations that could lead to a conflict of interest.

Acknowledgments

The authors would like to thank to Mrs. María de los Reyes Suárez Hanna and to Dr. Esmeralda Romero-Covo for their technical assistance.

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Technical noteValidated analytical methodology for the simultaneous determination of a wide range of pesticides in human blood using GC–MS/MS and LC–ESI/MS/MS and its application in two poisoning cases

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Optimization of the instrumental method

Conclusions

Conflict of interest

Acknowledgments

J. Forensic Legal Med.

Lancet

J. Chromatogr. A

Sci. Total Environ.

Sci. Total Environ.

Ecotoxicol. Environ. Saf.

J. Chromatogr. A

J. Chromatogr. A

Chemosphere

Homicide by organophosphorus compound poisoning: a case report

Med. Sci. Law

Fatal intoxications in a forensic autopsy material from Epirus, Greece, during the period 1998–2010

J. Forensic Sci.

Technical note
Validated analytical methodology for the simultaneous determination of a wide range of pesticides in human blood using GC–MS/MS and LC–ESI/MS/MS and its application in two poisoning cases