Original Research

European Journal of Forensic Sciences www.ejfs.co.uk DOI: 10.5455/ejfs.203527

Method validation for simultaneous determination of pesticide residues in post-mortem samples by highperformance liquid chromatographyultraviolet method Deepti S Deshpande, Ashwini K Srivastava ABSTRACT Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai 400098, Maharashtra, India Address for correspondence: A. K. Srivastava, Department of Chemistry, Lokmanya Tilak Bhavan, University of Mumbai, Vidyanagari, Santacruz (East), Mumbai - 400 098, Maharashtra, India. E-mail: aksrivastava@chem. mu.ac.in. Received: October 02, 2015 Accepted: February 08, 2016 Published: 26 February, 2016

Introduction: In forensic or clinical toxicology, a correct interpretation of toxicological findings needs reliable analytical data. The analytical methods that are to be used for routine analysis need careful method development and thorough validation. Blood, body fluids, and certain organs are mostly used in examinations of deaths due to intoxication. However, validated methods for the analysis of post-mortem samples, especially tissues are very few. The aim of this work was to validate a simple reversed-phase high-performance liquid chromatographyultraviolet method for the simultaneous determination of carbamate (propoxur), organophosphorus (malathion, quinalfos, profenofos, and chlorpyrifos), and organochlorine (endosulfan) pesticides in post-mortem tissue and blood samples. Materials and Methods: The pesticides incorporated in the method were those commonly encountered in suicidal or homicidal poisonings in India. Before analysis, QuEChERS isolation technique was conveniently used to clean the tissue and blood samples. Validation proved the applicability of matrix-matched calibration for routine screening of the pesticides in post-mortem samples. Results: The working range of all the pesticides showed good linearity with correlation coefficients (r) ranging from 0.9996 to 0.9999. The limits of detection for all pesticides range from 0.37 to 0.70 μg/g in tissue while it varies from 0.14 to 0.35 μg/ml in blood. The limit of quantitation ranges from 1.16 to 2.50 μg/g in tissue and from 0.44 to 1.50 μg/ml in blood. Conclusion: The developed method can be effectively used for routine screening and quantitation of the pesticides belonging to diverse groups which otherwise require different time consuming or costly analytical techniques for confirmation and quantitation " KEY WORDS: Forensic sciences, forensic toxicology, liquid chromatography, method validation, pesticides

INTRODUCTION The process of method validation has a direct impact on the quality of residue analytical data. Forensic laboratories are specifically in need of effective methods for the correct interpretation of toxicological findings. Unreliable data can lead to unjustified legal consequences or to wrong treatment of the patient. The importance of method validation is emphasized by Peters et al. [1] not only to develop a method carefully for the correct interpretation of the toxicological findings in daily routine work but also to demonstrate the inherent quality of any analytical method to prove its applicability for a certain purpose, especially in the context of quality management and accreditation. As mentioned by them as a general agreement for quantitative bio-analytical procedures, the essential validation parameters to be evaluated are selectivity, calibration model (linearity), stability, accuracy (bias), precision (repeatability, intermediate precision), and Eur J Forensic Sci

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the lower limit of quantification. At the same time, they also mention that though no general validation guideline is available for a qualitative procedure, there is an agreement that at least selectivity and limits of detection (LOD) are significant evaluation parameters with additional parameters such as precision, recovery, and robustness. Although there are no set maximum limits for the presence of pesticides in postmortem tissues in clinical and forensic toxicology, a precise analytical method is favored for procuring authentic results to prove the intake of a substance. Cases involving fatalities due to consumption of pesticides are common in India. A study of suicidal behavior in India indicates that poisoning (93%) is the most common mode of deliberate self-harm irrespective of gender [2]. It also reveals that pesticides are the most common lethal poisons in the household as they are easily available and due to the ignorance and carelessness about their self-storage [3]. 1

Deshpande and Srivastava: Pesticide residues in post-mortem samples

Among the pesticides, the organophosphorus group (OP) is responsible for the largest number of deaths followed by the organochlorines (OCs) and carbamates (Carbs). The OPs and Carbs are cholinesterase inhibitors while OCs impair nervous system function by depolarization of the membrane. When post-mortem tissue or blood in such poisoning cases is referred to clinical/forensic toxicology, the correct interpretation of toxicological findings is an important objective of the toxicologist for which a reliable analytical method becomes an important prerequisite. Literature survey shows documentation of various reviews on analytical methods for monitoring pesticides and their metabolites in different matrices such as rice, fatty vegetables and food, fruits, and vegetables [4-14]. An increased trend of gas chromatography and liquid chromatography with MS in the analysis of pesticides [15-19] is evident from a large number of reviews. High-performance liquid chromatographyultraviolet (HPLC-UV)-visible detection is common for analysis of pesticides belonging to one particular class such as the OPs, OCs, or Carbs [20-27], wherein the pesticides are determined individually or with other pesticides of the same class. Blood, urine, or plasma is the most common matrix of determination in most of the reported analytical methods [28-36] while very few methods document their analysis in bone marrow [37], hair [38], or tissue materials [39,40]. A recent work reports HPLC with photodiode array detection for pesticide analysis in bovine tissue [41]. Similarly, QuEChERS method of sample preparation since its inception by Anastassiades et al. [42], has not only found its application for matrices such as foods [43], fruits, and vegetables [44] but also for matrices such as sea-food, beef, and human whole blood [45]. However, it is obvious from one of the latest reviews that there is a lack of method developments in postmortem tissues and blood though there is an array of analytical methods to determine pesticides in several matrices. Method validation is problematic in post-mortem analysis because not only the acquisition of representative validation data virtually impossible owing to varying composition of samples but also the concentration of the analyte is no longer what it was at the time of death. The composition of partly decomposed or putrefied samples may vary considerably from case to case, and hence, validation studies carried out using matrix samples from one or several post-mortem cases may become questionable. However, if the tissues are properly preserved and are not decomposed before analysis, then the validation data can ensure sufficient quality of the results. Validation studies are certainly justified if an analytical method is to be used routinely. Since in India deaths due to pesticide consumption is a common phenomenon the analysis of multiple - class pesticides in post-mortem tissues and blood is a routine. Keeping in view the above considerations, the simple reversed-phase HPLC-UV method is validated, for the simultaneous determination of pesticides commonly found in poisoning, viz., propoxur (Carbs), malathion, quinalfos, profenofos, chlorpyrifos (OPs), and endosulfan (OC). 2

MATERIALS AND METHODS Chemicals and Reagents All reagents were of HPLC grade (unless otherwise specified) and used as supplied by the manufacturers. High purity water, acetic acid, and acetonitrile (ACN) were procured from Merck Specialties Pvt. Ltd., India. The pesticide standards (Malathion, Quinalfos, Profenofos, and Chlorpyrifos) were from Sigma-Aldrich, India. Propoxur and endosulfan were from Accustandard, India with a certified purity of ≥97%. The Agilent SampliQ QuEChERS kits procured from Agilent Technologies, India were made up of an extraction kit and a dispersive SPE kit. SampliQ QuEChERS extraction kit: The kit consists of 50 ml extraction tube and pack of 6 g magnesium sulfate and 1.5 g sodium acetate. SampliQ QuEChERS dispersive solid phase extraction (SPE) kit: The kit consists of 15 ml and 2 ml SPE tubes containing primary secondary amine (PSA), C18 and magnesium sulfate and was used for clean-up of extracts obtained after extraction with ACN.

Materials The post-mortem tissue sample and blood were obtained from the Forensic Science Laboratory, Mumbai. The samples used for development and validation of the method were those which were confirmed to be negative for any toxicological findings after routine analysis while the samples used to demonstrate the applicability of the developed method were those which had poisoning history and were confirmed positive after the routine analysis. The samples were stored at −4°C until analysis.

Preparation of Different Solutions Stock/working solutions: A stock solution of each analyte at 1 mg/ml was prepared in ACN. Working solutions were prepared in ACN in different concentration levels every alternate day by appropriately diluting the stock standard solutions and analyzed. The stock solutions, as well as working standard solutions, were stored under refrigeration.

Sample Extraction QuEChERS for tissue: 10 g (± 0.01) of tissue sample cut into fine pieces was weighed in the 50 ml centrifuge tube and spiked with known spiking solution followed by addition of 10 ml of acidified ACN (1% acetic acid/ACN). If the tissue contains sufficient water quantity to achieve partition after addition of extraction salts, then the addition of water was not necessary during this step. However, if the tissue is dry, then 2 ml of water was added to achieve the desired consistency. The tightly capped tube was sonicated for 5 min, and then Agilent QuEChERS SampliQ extraction salts were added to Eur J Forensic Sci

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Deshpande and Srivastava: Pesticide residues in post-mortem samples

the tube. Again, after shaking vigorously for another two min, the tube was centrifuged for 3 min at 4000 rpm to get a clear upper layer [Figure 1]. 6 ml of the supernatant was transferred to the Agilent SampliQ dispersive SPE 15 ml tube and shaken vigorously for 2-3 min and centrifuged for another 3 min to get a clear supernatant for analysis. QuEChERS for blood: 0.5 ml of blood was diluted three-fold with distilled water in a tube containing 0.5 g pre-packed extraction preparation (Agilent SampliQ) and a stainless steel bead. After spiking with a known spiking solution and adding 2 ml acidified ACN (1% acetic acid/ACN), the tube was shaken vigorously for a minute and then centrifuged at 4000 rpm for 5 min. The supernatant (1 ml) obtained [Figure 2] after centrifugation was then transferred to a 2 ml centrifuge tube containing SPE sorbent (SampliQ), shaken vigorously and centrifuged at 4000 rpm for 1 min to get a clear supernatant for analysis.

Specificity: The specificity of the method as demonstrated by analyzing 15 blank extracts from tissue and blood samples did not show any interfering peaks [Figure 3b] at the retention time (Rt) of the analytes. The other system suitability parameters evaluated by the optimized method by adding pesticides at the upper limit of calibration range to extracts obtained from pesticide free tissue and blood established the absence of any endogenous interference. Linearity: The calibration plots obtained by plotting the peak area versus analyte concentration for all the pesticides

Equipment Liquid chromatography was performed with a binary-solvent delivery module liquid chromatograph model LC-10ATvp, (Shimadzu Corporation, Kyoto, Japan) equipped with UV-visible detector model SPD-10Avp, and column oven model CTO-20A Prominence. Mobile phase for isocratic elution consisted of ACN: water 60:40 (v/v) mixed together with a binary-solvent delivery module, model LC-10ATvp. Chromatographic separation was performed on a Phenomenex C18 endcapped column (250 × 4.6 mm i.d., particle size 5 μm), (Phenomenex, USA). Manual sample injector model 7725i, (Rheodyne, Co., Berkeley, CA, USA) having a 20 μL sample loop was used. Winchrom-Ex software module was used for data acquisition and processing.

Figure 1: Flow chart of QuEChERS extraction method for tissue

RESULTS The optimized method with a flow of 1.5 ml/min for sample 20 μL efficiently resolves the six pesticides within 20 min. A liquid chromatogram in Figure 3a clearly indicates that the peaks are distinguished from the baseline as narrow, sharp, well resolved, and non-tailed. The other optimization parameters considered were peak area, separation efficiency and peak asymmetry as presented in Table 1.

Figure 2: Flow chart of QuEChERS extraction method for blood

Table 1: System suitability parameters and separation characteristics Pesticide

Propoxur Malathion Quinalfos Profenofos Endosulfan β Endosulfan α Chlorpyrifos a

Retention time±SDa tR (min)

No. of theoretical plates/ 25 cm, (N)

Retention factor (k)

Asymmetry ratio (T)

a

3.36±0.01 7.47±0.02 10.20±0.02 16.14±0.02 20.16±0.04 26.21±0.04 27.96±0.04

8211 10709 11615 12124 15292 13263 12829

2.35 6.43 9.15 15.06 19.05 24.13 26.88

1.0 0.84 0.79 0.77 0.82 0.81 0.84

b

Represents the standard deviation for the retention time (n=10)

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Figure 3: (a) Typical liquid chromatogram of the pesticides. The elution order and peak numbering is as follows: 1. Propoxur, 2. Malathion, 3. Quinalfos, 4. Profenofos, 5. Endosulfan β, 6 Endosulfan α, 7. Chlorpyrifos, (b) A representative of blank tissue/blood extract 3

Deshpande and Srivastava: Pesticide residues in post-mortem samples

show good linearity with correlation coefficients (r) ranging from 0.9996 to 0.9999. The concentration range used for tissue varied from 0.5 to 50 μg/g while that for blood varied from 0.5 to 25 μg/ml. The experimental LOD and limits of quantitation (LOQ) are based on the standard deviation of the response and slope. As summarized in Tables 2 and 3, the LOD ranges from 0.37 to 0.70 μg/g in tissue and 0.14 to 0.35 μg/ml in blood. The limit of quantitation in tissue and blood ranges from 1.16 to 2.50 μg/g and 0.44 to 1.50 μg/ml, respectively. The precision determined as relative standard deviation (RSD) in terms of repeatability (intra-day) and reproducibility (interday) at three fortification levels for tissues [Table 2] and blood [Table 3] were satisfactory with the intra- and inter-day RSD values ≤ 15% and 10%, respectively. The recoveries determined at two different concentrations were ≥85% for tissue and ≥90% for blood, which proves the accuracy of the method [Tables 2 and 3].

DISCUSSION Since the post-mortem tissues are preserved in saturated salt solution, the sample extraction was based on the AOAC 2007.01 version of the quick, easy, cheap, effective, rugged, and safe (QuECHERS) method as it does not incorporate sodium chloride in the initial extraction step, excess of which could prohibit partitioning and separation of the ACN layer. As described in the experimental section, the method involves two steps: Extraction and dispersive SPE (d-SPE). In the first step, ACN is added to the sample, followed by salting out of

water from the sample by addition of the salts. ACN facilitates protein precipitation while liquid-liquid partitioning gets induced by salting out with sodium acetate and magnesium sulfate thereby making the process non-clumpy and nonsticky. Pesticides are known to accumulate in fatty tissues of living organisms including humans. In the second step, d-SPE minimizes the matrix effects with a combination of PSA, C 18 (octadecylsilane), and anhydrous MgSO4. The amine removes fatty acids, sugars, and some ionic lipids, whereas C18 effectively removes the fats and lipids while MgSO4 remove the remaining water in the extracts. To optimize the operating conditions, systematic investigation of the influence of various parameters, viz., nature of bonded stationary phase, composition, and flow rate of the mobile phase was studied. Initial scanning of UV spectra for individual pesticides facilitated selection of detector wavelength at 230 nm. RP C18 columns are commonly available in routine analytical laboratories and hence chosen for the present study. The mobile phase was degassed in an ultrasonic bath and filtered through 0.45 μm membrane (Millipore Corporation, USA) before use. The method was kept simple by choosing isocratic elution with varying proportion of ACN and water. To study the effect of ACN on the retention behavior for all the pesticides curves corresponding to the Rt versus ACN in the mobile phase were constructed. The curves clearly indicated that the retention factor of all the pesticides decrease with increase in ACN in the mobile phase. Chlorpyrifos and endosulfan-α, overlap at 67% ACN as evident from Figure 4, but further reduction of

Table 2: Performance characteristics of the HPLC-UV method for determination of pesticides in tissue samples Pesticide

LOD at 3.3 σ/S (μg/g)

LOQ at 10 σ/S (μg/g)

Linearity (μg/g)

Correlation coefficient (r)

Precision at different concentration levels (RSD) (μg/g) Intra-day Rp

Propoxur Malathion Quinalfos Profenofos Endosulfan β Endosulfan α Chlorpyrifos

0.45 0.51 0.37 0.38 0.70 0.50 0.70

1.37 1.50 1.10 1.16 2.10 1.60 2.00

1.37-50 1.50-50 1.10-50 1.16-50 2.10-50 1.60-50 2.00-50

0.9996 0.9997 0.9997 0.9998 0.9996 0.9996 0.9998

Inter-day Rc

% Recovery at different spiking levels (μg/g)±SD (n=5)

2

10

25

2

10

25

2

25

13.6 11.4 11.8 10.1 10.3 12.5 8.5

8.4 6.4 9.2 9.8 11.5 7.6 3.8

9.7 8.3 10.1 7.5 10.2 6.3 3.3

14.1 10.9 13.4 12.6 9.3 14.8 12.3

12.1 12.4 8.5 6.2 7.2 5.4 7.1

11.5 8.3 8.9 10.1 10.5 4.3 5.3

82±8 84±4 86±5 85±4 83±2 87±2 82±3

84±4 87±5 93±3 98±3 90±1 91±3 98±1

Rp: Repeatability (n=5), Rc: Reproducibility (n=5), HPLC-UV: High-performance liquid chromatography-ultraviolet, LOQ: Limits of quantitation, LOD: Limits of detection, SD: Standard deviation, RSD: Relative standard deviation

Table 3: Performance characteristics of the HPLC-UV method for determination of pesticides in blood Pesticide

LOD at 3.3 σ/S (μg/ml)

LOQ at 10 σ/S (μg/ml)

Linearity (μg/ml)

Correlation coefficient (r)

Precision at different concentration levels (RSD) (μg/ml) Intra-day Rp

Propoxur Malathion Quinalfos Profenofos Endosulfan β Endosulfan α Chlorpyrifos

0.27 0.19 0.16 0.14 0.52 0.48 0.70

0.84 0.50 0.47 0.44 1.50 1.01 2.00

0.84-25 0.50-25 0.47-25 0.44-25 1.50-25 1.01-25 2.00-50

0.9997 0.9998 0.9997 0.9998 0.9996 0.9996 0.9998

Inter-day Rc

% Recovery at different spiking levels (μg/ml)±SD (n=5)

2

10

25

2

10

25

2

25

6.6 4.4 8.4 7.1 8.7 9.4 5.1

5.1 5.4 6.3 3.6 5.6 9.5 3.8

5.7 2.7 2.5 1.1 7.3 6.2 3.3

8.7 2.3 4.6 5.2 6.2 4.8 4.9

4.2 1.1 5.3 4.1 2.3 5.5 4.1

6.7 1.2 3.4 1.1 1.7 3.3 2.3

92±5 91±2 94±2 91±5 92±3 91±6 90±4

94±3 96±1 93±1 92±2 95±1 92±4 98±1

Rp: Repeatability (n=5), Rc: Reproducibility (n=5), LOQ: Limits of quantitation, LOD: Limits of detection, SD: Standard deviation, RSD: Relative standard deviation, HPLC-UV: High-performance liquid chromatography-ultraviolet 4

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Deshpande and Srivastava: Pesticide residues in post-mortem samples

coefficients (r) of the linear regression curves obtained for each analyte established the linearity. The method is applicable only for the quantification of these pesticides after screening and reliable identification by thin layer chromatographic technique using target specific chromogenic spray reagents or any other hyphenated technique.

CONCLUSIONS

Figure 4: Effect of acetonitrile on retention factor of pesticides

ACN to 60% in the mobile phase separates them efficiently (resolution > 1.0). The identity of all analyte peaks in samples was confirmed by comparing their Rts with the Rts of reference standards under similar HPLC conditions. The method was evaluated in terms of selectivity, limit of detection (LOD), linear range of calibration, precision, and accuracy as per the important considerations mentioned by Peters et al. (2007) in validation of new methods to be used for forensic or clinical toxicology. The specificity of the method which establishes the fact that there is no interfering signal with that of the analytes was achieved by analyzing 15 different blank tissue/blood samples and standards spiked in them. The other system suitability parameters in terms of various retention and separation characteristics, such as Rt, column efficiency (N), retention factor (k), peak tailing or asymmetry ratio (T), and resolution (Rs), were also evaluated to show the absence of any endogenous interference. The lowest concentration of the analyte that can be detected is the LOD and the LOQ that determine the sensitivity of the method are based on the standard deviation (σ) of the response and the slope (S), where LOD is 3.3 σ/S and LOQ is 10 σ/S. Precision was determined in terms of RSD for the repeatability and reproducibility of the method. Repeatability was assessed by running five tissue/blood extracts spiked at three concentration levels (2, 10, and 25 ug/g) while reproducibility was evaluated by running five replicate samples that were spiked at same concentrations (2 and 25 ug/g) and analyzed on five different days. The recovery efficiencies of the spiked samples at the lower and higher concentrations by the optimized method evaluated the accuracy of the method. To achieve this, two sets of samples were prepared: Set 1 was prepared in a blank matrix extract and spiked after extraction, whereas Set 2 was prepared by spiking before extraction. Recovery was calculated by relating the areas of the extracts before and after extraction, as follows: Recovery % = set 2area/set 1area × 100 The matrix-matched calibration method demonstrates an effective method of calibration, wherein a series of standard solutions including one at LOQ level were prepared in blank extracts of tissue and blood samples and the correlation

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The analyst can face many problems during the performance of any method. In forensic toxicology accurate quantification, especially in post-mortem tissues is always a subject of debate. Conventionally, the method of standard addition is preferred in forensic analyses of post-mortem samples in which the calibration and quantification are performed in the sample matrix of the case in question. This can be tedious and time consuming for routine analysis if the number of analyzes is sufficiently high. The present validation study has confirmed that if the samples are properly preserved, matrix-matched calibration using matrix samples from several post-mortem samples can be applied for routine analysis of these pesticides in both blood and tissues with a good precision and accuracy. QuEChERS sample preparation reduces potential crosscontamination of samples thereby facilitating rapid and efficient analyzes of a large number of samples with an ordinary chromatographic instrumentation. However, the method cannot be applied to tissues that are decomposed or putrefied since the composition of such specimens varies considerably.

ACKNOWLEDGMENTS The authors are thankful to Dr.M.K.Malve, Director and Shri.S.M.Bakre, Assistant Director, Directorate of Forensic Science Laboratories, Mumbai, for their support in providing tissue and blood samples.

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Eur J Forensic Sci

● Jul-Sep 2016 ● Vol 3 ● Issue 3