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 Table of Contents  
Year : 2017  |  Volume : 3  |  Issue : 1  |  Page : 22-25

Determination of Chlorpyrifos in Human Blood by Gas Chromatography-Mass Spectrometry

Department of Forensic Toxicological Analysis, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, China

Date of Web Publication31-Mar-2017

Correspondence Address:
Prof. Linchuan Liao
8th Floor, Fa Yi Building, No. 16, Section 3, Renmin Nan Road, Chengdu, Sichuan 610041
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jfsm.jfsm_2_17

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Gas chromatography-mass spectrometry method was developed for the qualitative and quantitative analyses of chlorpyrifos in human blood samples. The chlorpyrifos and parathion (internal standard) in human blood were extracted with a mixed solvent of hexane and acetonitrile. Chlorpyrifos was well separated from the internal standard. The linear range of chlorpyrifos was 0.01–2 μg/ml in blood. The limit of detection and limit of quantification were estimated at 0.002 and 0.01 μg/ml, respectively. The inter- and intra-day precisions, accuracy, and recovery were assessed to verify this method. The results showed that the developed method is rapid, sensitive, and reliable. It is suitable for the determination of chlorpyrifos in forensic toxicological analysis and clinical diagnosis.

Keywords: Blood, chlorpyrifos, forensic toxicological analysis, gas chromatography-mass spectrometry

How to cite this article:
Dai X, Fan F, Ye Y, Chen F, Wu Z, Lu X, Wei Q, Chen J, Yan Y, Liao L. Determination of Chlorpyrifos in Human Blood by Gas Chromatography-Mass Spectrometry. J Forensic Sci Med 2017;3:22-5

How to cite this URL:
Dai X, Fan F, Ye Y, Chen F, Wu Z, Lu X, Wei Q, Chen J, Yan Y, Liao L. Determination of Chlorpyrifos in Human Blood by Gas Chromatography-Mass Spectrometry. J Forensic Sci Med [serial online] 2017 [cited 2023 Jan 28];3:22-5. Available from: https://www.jfsmonline.com/text.asp?2017/3/1/22/203549

  Introduction Top

Chlorpyrifos [O, O-diethyl-O-(3, 5, 6-trichloro-2-pyridinyl) phosphorothioate], also known as clopidogrel, is a widely used broad-spectrum organophosphorus pesticide.[1] It is used as an alternative to highly toxic organophosphorus pesticides such as methamidophos owing to its broad spectrum, high efficiency, and low toxicity. With the prohibition of highly toxic organophosphate insecticides and subsequent widespread use of chlorpyrifos, cases of chlorpyrifos poisoning have increased significantly in the recent past.[2],[3] As with the mechanism of organophosphorus pesticides, chlorpyrifos can inhibit the activity of acetylcholinesterase, which could affect the nervous system, causing typical symptoms of acute organophosphate poisoning such as nausea, dizziness, or obnubilation. Excessive exposure to chlorpyrifos can even cause respiratory paralysis and death.[4]

In the recent years, analytical methods for chlorpyrifos such as gas chromatography (GC),[5] high-performance liquid chromatography (HPLC),[6] GC–mass spectrometry (GC-MS),[7] and liquid chromatography–MS [8] have been reported. However, these studies paid more attention to the issues of environmental pollution or food residues and less to biological samples. To develop an effective approach to qualify and quantify chlorpyrifos in blood, a method based on GC-MS was established. With this method, it is possible to suggest treatments for clinical chlorpyrifos poisoning and forensic toxicological analysis.

  Subjects and Methods Top

Apparatus and reagents

An Agilent 7890A-5975CGC-MS (CA, USA) chromatograph fitted with an autoinjection system was utilized. The data system contains the software required to collect GC-MS chromatograms and spectra and contains the National Institute of Standards and Technology library. Separation was achieved on a DB-5MS capillary column (Agilent, CA, USA) (30 m × 0.25 mm i.d., 0.25 μm).

A chlorpyrifos standard solution (100 μg/ml in methanol [GBW(E) 081170]) and internal standard parathion (100 μg/ml in acetone (GSB05-2284-2008)) were purchased from the Argo-environmental Protection Institute, Ministry of Agriculture (Beijing, China). Methanol and acetonitrile (both HPLC grade) were obtained from Fisher Scientific (Waltham, MA, USA); hexane, ethyl acetate, diethyl ether, and dichloromethane (HPLC grade) were purchased from Kelong Chemical Company (Chengdu, China).

Instrumental conditions

Gas chromatography conditions

A GC-MS analysis was performed using the autoinjection system with a DB-5MS capillary column. Ultrapure helium was used as a carrier gas with a flow rate of 1 ml/min. The injection port and auxiliary heater temperatures were 250° and 230°, respectively. The GC oven temperature was set at 60° for 2 min, programed from 60° to 180° at 20°/min, and then increased at a rate of 10°/min–280°. The temperature was maintained at 280° for 10 min. The total running time was 28 min, and splitless injection of 1 μl was carried out.

Mass spectrometry conditions

MS was carried out in the electron impact ionization mode using energy of 70 eV. The ion source and quadrupole temperatures were maintained at 230° and 150°, respectively. A mass spectral analysis was performed in the full scan mode using the mass range of 40–400 amu and in the selected ion monitoring mode. The characteristic ion fragmentation for quantitative analysis was m/z = 197 and 314 for chlorpyrifos and m/z = 109 and 291 for parathion.

Working solution preparation

A chlorpyrifos standard working solution was prepared in methanol by diluting the stock standard solution (100 μg/ml) to 10 and 1 μg/ml. The same operation was used to create a working solution of 5 μg/ml for internal standard parathion. All solutions were stored at 4° until analyzed.

Extraction of chlorpyrifos from blood samples

Samples were processed according to the following liquid–liquid extraction procedure: a 0.5 ml aliquot of each sample was transferred to a 10 ml glass vial containing internal standard parathion at 1 μg/ml. After vortexing for 1 min, the samples were extracted with 0.5 ml of acetonitrile and 4.0 ml of hexane and then mixed and centrifuged at 3500 rpm for 5 min. The hexane layer was collected in another glass vial and dried in a gentle stream of N2 gas. The residue was reconstituted in 100 μl of methanol for GC-MS analysis.

  Results and Discussion Top

Selection of the internal standard

The internal standard should have chemical and physical properties that are as similar to the analyte as possible.[9] The structure of internal standard parathion was similar to that of chlorpyrifos, and they also had similar physical and chemical properties, which effectively reduced the bias caused by the extraction of the analytical method. Moreover, chlorpyrifos was well separated from parathion and had a similar retention time as parathion. Therefore, we selected parathion as the internal standard for chlorpyrifos.

Selection of the extraction method

To obtain better extraction efficiency, different types of extraction and dispersive solvents were analyzed in this study. Each extraction method was repeated three times within 1 day.

For the selection of the extraction solvent, ethyl acetate, diethyl ether, hexane, and dichloromethane were investigated to extract chlorpyrifos from blood. By comparing the four extraction solvents, we found that diethyl ether and hexane showed higher extraction efficiency than ethyl acetate and dichloromethane. Chlorpyrifos is a weakly polar organophosphorus compound; thus, when a nonpolar solvent such as hexane was used as the extraction solvent, higher extraction efficiency for chlorpyrifos in blood was obtained. However, through a matrix blank test, ethyl acetate and diethyl ether also showed great extraction efficiency for the impurities in blood samples. When hexane and dichloromethane were used as extraction solvents, there was less matrix interference in the GC-MS chromatogram. Therefore, hexane was chosen as the extraction solvent.

However, hexane can result in serious emulsification during extraction, which may affect the extraction efficiency. Studies have shown that methanol and acetonitrile, which have usually served as dispersive solvents, have been introduced as chemical demulsifiers to eliminate emulsification, considering their low surface tension and high surface activity. Therefore, the selection of an appropriate dispersive solvent was a great way to break out emulsions. In our study, a suitable dispersive solvent should be miscible with both aqueous and organic phases to ensure the formation of the cloudy state, which increases the contact between the two phases, thus facilitating extraction. In previous studies, Brzak et al.[10] chose 0.25 ml of methanol as the dispersive solvent combined with 1.5 ml of hexane to extract chlorpyrifos and its metabolites from blood. Sinha et al.[11] selected 0.75 ml of methanol and 4.5 ml of hexane to extract chlorpyrifos from human blood. In reference to previous studies, we compared four extraction methods in this study, which simultaneously added 4 ml of hexane as the extraction solvent and different types or different volumes of dispersive solvents to determine an appropriate dispersive solvent and its volume: method 1: 4 ml of hexane and 1 ml of methanol, method 2: 4 ml of hexane and 0.5 ml of methanol, method 3: 4 ml of hexane and 1 ml of acetonitrile, and method 4: 4 ml of hexane and 0.5 ml of acetonitrile. The results showed that the extraction recovery of method 4, which reached 90%, was higher than that of the other methods, and there was also less matrix interference in chromatogram for method 4. Therefore, 4 ml of hexane and 0.5 ml of acetonitrile were selected as the extraction and dispersive solvents, respectively, for extracting chlorpyrifos from blood.

Validation of the optimized method

The optimized conditions were used to validate the method for the qualitative and quantitative analyses of chlorpyrifos.

For the qualitative analysis of chlorpyrifos, the selectivity of the method and the characteristic ion fragmentation were investigated in this study. The selectivity of the method was assessed for the endogenous interference by analyzing three different blank blood samples and three blank blood samples spiked with chlorpyrifos and internal standard parathion. The total ion chromatogram of blank blood and spiked blood [Figure 1] reflected that no significant peaks were found at the retention time of chlorpyrifos or the internal standard, which were, respectively, detected at 12.607 and 12.759 min. It was shown that there was no endogenous interference during the determination of chlorpyrifos from human blood, and the separation efficiency of the target compound and internal standard were great. The mass spectrograms of chlorpyrifos and internal standard parathion are shown in [Figure 2].
Figure 1: Total ion current chromatograms of (a) blank blood and (b) spiked blood. Blood sample spiked with the 1 μg/ml internal standard and 2 μg/ml chlorpyrifos. The retention times of chlorpyrifos and the internal standard were 12.607 and 12.759 min, respectively. No endogenous interference was observed during the determination of chlorpyrifos

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Figure 2: Mass spectrograms of (a) chlorpyrifos and (b) parathion. The full scan mode was utilized for the mass range of 40–400 amu. The characteristic ion fragmentation for the quantitative analysis was as follows: m/z = 197 and 314 for chlorpyrifos and m/z = 109 and 291 for parathion

Click here to view

To determine the linearity of the method, six different concentrations of spiked samples ranging from 0.01 to 2 μg/ml were submitted to the entire analytical procedure. The spiked samples used a 0.5 ml aliquot of blood spiked with the chlorpyrifos working solution to obtain the different concentrations of 0.01, 0.02, 0.1, 0.2, 1, and 2 μg/ml. The calibration curve was measured using the ratio of the peak areas of the target ion chosen for chlorpyrifos and those of the internal standard. A correlation coefficient of r2 = 0.9998 with an equation of Y = 1.2928X − 0.005 (n = 6) was obtained. The obtained results showed that the linearity was excellent in the concentration range of 0.01–2 μg/ml. Zheng et al.[12] reported four cases of chlorpyrifos poisoning, in which the concentrations of chlorpyrifos in blood were 0.941, 0.096, 0.013, and 0.369 μg/ml, which were all within the range of our calibration curve. Therefore, the linearity and sensitivity could meet the needs of real cases.

The limit of detection (LOD) of the developed method was determined by the analytes of the chromatographic extracts of the aqueous solution spiked with decreasing amounts of the analytes until a signal-to-noise (S/N) ratio of 3:1 was reached.[13] The limit of quantification (LOQ) was estimated from the analysis as the concentration of an analyte giving an S/N ratio of 10:1. The LOD and LOQ were estimated to be 0.002 and 0.01 μg/ml, respectively.

The precision and accuracy of the method were expressed as the relative standard deviation (RSD) by treating three quality control samples at low, medium, and high concentrations (0.02, 0.2, and 1 μg/ml, respectively) of six replicates, and they were calculated using the calibration curves. All replicated samples were tested under repeatable conditions (same analyst, same concentration, same preprocessing, same instrument, and same materials). The intraday precision was determined by analyzing the six same-day replicates of blood samples spiked at three levels. The interday precision was processed by an analysis of the samples on three consecutive days of six replicates. The accuracy was determined by the ratio of the measured concentration to the target concentration and the RSD at three levels of six replicates. The method showed good intra- and inter-day precision with RSD values between 4.0%–14.8% and 4.3%–18.4%, respectively, and the accuracy was 91.8%–114.8% [Table 1]. An acceptable precision with RSD values of 20% at the lowest calibration point and 15% for higher concentrations is usually acceptable.[14] The results were consistent with this finding.
Table 1: Accuracy, precision, and recovery of the method (%)

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The recovery was determined by comparing unspiked samples to spiked blood samples for three concentration levels (0.02, 0.2, and 1 μg/ml) with each level performed six times. The recoveries of this analytical method ranged from 89.3% to 93.8%, as shown in [Table 1]. It can be observed that the extraction efficiency of the method was excellent, which can meet the requirements of forensic toxicology analysis.

  Conclusion Top

GC-MS method with a convenient extraction procedure was developed for the qualification and quantification of chlorpyrifos in blood samples. This analytical method is simple, sensitive, and reliable. For all the assayed analyses of interest, the figures of merit obtained from the validation, selectivity, linearity, LOD, LOQ, precision, accuracy, and recovery were quite reasonable. The results showed that the method could be used to meet the requirements of forensic toxicology analysis and provide suggestions for the treatment of clinical chlorpyrifos poisoning cases.

Financial support and sponsorship

This study was financially supported by the Project of the National Natural Sciences Foundation of China (81373239).

Conflicts of interest

There are no conflicts of interest.

  References Top

Hines CJ, Deddens JA. Determinants of chlorpyrifos exposures and urinary 3,5,6-trichloro-2-pyridinol levels among termiticide applicators. Ann Occup Hyg 2001;45:309-21.  Back to cited text no. 1
Sharma VK, Jadhav RK, Rao GJ, Saraf AK, Chandra H. High performance liquid chromatographic method for the analysis of organophosphorus and carbamate pesticides. Forensic Sci Int 1990;48:21-5.  Back to cited text no. 2
Sogorb MA, Vilanova E, Carrera V. Future applications of phosphotriesterases in the prophylaxis and treatment of organophosphorus insecticide and nerve agent poisonings. Toxicol Lett 2004;151:219-33.  Back to cited text no. 3
Eddleston M, Eyer P, Worek F, Sheriff MH, Buckley NA. Predicting outcome using butyrylcholinesterase activity in organophosphorus pesticide self-poisoning. QJM 2008;101:467-74.  Back to cited text no. 4
Chandra S, Mahindrakar AN, Shinde LP. Determination of cypermethrin and chlorpyrifos in vegetables by GC-ECD. Int J Chemtech Res 2010;2:908-11.  Back to cited text no. 5
Ata S, Wattoo FH, Feroz M, Wattoo M, Tirmizi SA, Asad MJ. Analytical investigation of selected pesticide residues from fruits and vegetables by an improved extraction method using reverse phase high performance liquid chromatograph. Ethiop J Environ Stud Manage 2013;6:342-6.  Back to cited text no. 6
Cajka T, Mastovská K, Lehotay SJ, Hajslová J. Use of automated direct sample introduction with analyte protectants in the GC-MS analysis of pesticide residues. J Sep Sci 2005;28:1048-60.  Back to cited text no. 7
Zhang Z, Jiang WW, Jian Q, Song W, Zheng Z, Wang D, et al. Changes of field incurred chlorpyrifos and its toxic metabolite residues in rice during food processing from-RAC-to-consumption. PLoS One 2015;10:e0116467.  Back to cited text no. 8
Jones GR, Peat MA. Forensic toxicology laboratory guidelines. J Anal Toxicol 1999;23:636.  Back to cited text no. 9
Brzak KA, Harms DW, Bartels MJ, Nolan RJ. Determination of chlorpyrifos, chlorpyrifos oxon, and 3,5,6-trichloro-2-pyridinol in rat and human blood. J Anal Toxicol 1998;22:203-10.  Back to cited text no. 10
Sinha SN, Pal R, Dewan A, Mansuri MM, Saiyed HN. Effect of dissociation energy on ion formation and sensitivity of an analytical method for determination of chlorpyrifos in human blood, using gas chromatography-mass spectrometer (GC-MS in MS/MS). Int J Mass Spectrom 2006;253:48-57.  Back to cited text no. 11
Zheng Q, Hui Y, Zhuo XY, Shen BH. Chlorpyrifos determined in human blood by UPLC-MS/MS and its application in poisoning cases. J Forensic Med 2015;31:112-6.  Back to cited text no. 12
Plotka-Wasylka J, Simeonov V, Namiesnik J. An in situ derivatization-Dispersive liquid-liquid microextraction combined with gas-chromatography-Mass spectrometry for determining biogenic amines in home-made fermented alcoholic drinks. J Chromatogr A 2016;1453:10-8.  Back to cited text no. 13
Robertso J. Clarke's Analytical Forensic Toxicology. London: Pharmaceutical Press; 2008.  Back to cited text no. 14


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  [Table 1]

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