Science and Justice 55 (2015) 472–480
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Development and validation of a LC/MS method for the determination of Δ9-tetrahydrocannabinol and 11-carboxy-Δ9-tetrahydrocannabinol in the larvae of the blowfly Lucilia sericata: Forensic applications Sevasti Karampela a,b, Constantinos Pistos a,⁎, Konstantinos Moraitis a, Vasilios Stoukas a, Ioannis Papoutsis a, Eleni Zorba a, Michalis Koupparis b, Chara Spiliopoulou a, Sotiris Athanaselis a a b
Department of Forensic Medicine and Toxicology, School of Medicine, University of Athens 11527, Greece Division of Analytical Chemistry, Department of Chemistry, University of Athens 15771, Greece
a r t i c l e
i n f o
Article history: Received 3 February 2015 Received in revised form 27 May 2015 Accepted 2 June 2015 Keywords: Δ9- tetrahydrocannabinol 11-carboxy-delta-9-tetrahydrocannabinol LC MS fly larva forensic toxicology
a b s t r a c t In a number of forensic toxicological cases, Δ9-tetrahydrocannabinol (THC) and its metabolite 11-carboxy-delta9-tetrahydrocannabinol (THCA) are frequently considered as contributor factors to the event. To that, a liquid chromatographic mass spectrometric method is described for the identification and quantitation of THC and its metabolite THCA in the forensically important larvae of L. sericata. Larvae of Lucilia sericata were fortified with varying concentrations of THC and THCA covering the calibration range between 10 and 500 pg/mg. For the isolation of the analytes from larvae, several extraction techniques were evaluated and finally liquid-liquid extraction under acidic pH was selected using hexane-ethyl acetate (50:50, v/v) as extraction solvent. For the chromatographic separation, a Waters Symmetry® C18 analytical column was used while the mobile phase was acetonitrile-ammonium acetate (2 mM) (30:70, v/v). The detection was performed using electrospray ionization source in negative mode (ESI-) and the selected ions monitored were m/z 313 for THC and m/z 343 for THCA. The proposed method which is simple and sufficiently sensitive for the detection of THC and THCA even in a single larva sampling, assisted the investigation of a forensic case. © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Toxicological analysis of postmortem specimens can often pose special difficulties in comparison with clinically derived samples. Entomotoxicology investigates carrion-feeding insects as alternative toxicological specimens, taking advantage of their bio-accumulation of drugs and toxins [1–3]. This is especially useful in decomposing or skeletonized remains where traditional toxicological samples, such as soft tissue, blood, or urine, are no longer available [4,5] and can offer several technical advantages over the use of putrefied tissue [6,7]. Also in badly decomposed human remains, toxicological analyses can be facilitated using insects as less interferences due to matrix decomposition are observed in the analytical run [8]. In addition, drug concentrations seem to be more stable in insects, while this is not always the case for several post-mortem tissues [4]. From a pure practical point of view, insects are of interest as they are present in high quantities and
⁎ Corresponding author. Tel.: +30 210 746 2433; fax: + 30 210 7716098. E-mail addresses:
[email protected] (S. Karampela),
[email protected] (C. Pistos),
[email protected] (K. Moraitis),
[email protected] (V. Stoukas),
[email protected] (I. Papoutsis),
[email protected] (E. Zorba),
[email protected] (M. Koupparis),
[email protected] (C. Spiliopoulou),
[email protected] (S. Athanaselis).
their remains are present for a long time, even when toxicological samples are no longer available [9]. In cases also where religious and ethical beliefs result in problems collecting samples for toxicological analysis, insects could be also a solution [10]. Phenobarbital was the first drug identified in fly larvae found on a skeletonized corpse in 1980 [11]. Since then, entomotoxicology has become an established avenue for the determination of methadone [12], morphine [13], benzodiazepines [14] amphetamines [15] and significant progress has been made [16–18] as different forensic practitioners were able to detect controlled substances and drugs in fly larvae, pupae, and empty puparia [19]. Several techniques have been used for the toxicological analysis of fly larvae including immunoassay [20–23], gas chromatography (GC) [24–27], and liquid chromatography (LC) [28–31]. The potential for using carrion insects as alternative toxicological specimens is of great interest in countries where high temperatures are observed throughout the year. These climatic conditions lead to a high number of cases of decomposing or skeletonized remains, as decomposition progresses more rapidly. Thus, there is a critical need for research of the commonly encountered drugs and toxins in blowfly larvae. The recreational use of cannabis products like marijuana, hashish, and hashish oil, has been increased since the 1960s. It is estimated that
http://dx.doi.org/10.1016/j.scijus.2015.06.003 1355-0306/© 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.
S. Karampela et al. / Science and Justice 55 (2015) 472–480
in 2009, between 125 and 203 million people of the world population aged 15–64 (corresponding to between 2.8% and 4.5%) had consumed cannabis at least once in the past year [32]. Furthermore, in a number of forensic toxicological cases, Δ9-tetrahydrocannabinol (THC) and its metabolite 11-carboxy-delta-9-tetrahydrocannabinol (THCA) are frequently considered as contributor factors to the event. To that, the purpose of the present study was to develop a sensitive LC/MS method for the simultaneous quantitation of THC and THCA in larvae in order to investigate the potential of Lucilia sericata (Diptera, Calliphoridae) as an alternative toxicological specimen for their detection. This species is of forensic importance since it is among the first insects to detect and colonize human remains. Hypothesis of this study is that THC and THCA are bio-accumulated in larvae reared on a putrefied body. To the authors' knowledge, this is the first report in the literature describing the development and validation of an assay for the determination of THC and THCA in blowfly larvae. In addition, extended study was performed investigating the effect of different approaches on extraction of the analytes from larvae samples. The effects of these different extraction procedures on recovery and ion suppression are also reported. The developed method which provides simple clean-up followed by LC/MS detection was assessed on authentic forensic samples of blowfly larvae and the results are reported. In the literature, only Tracqui et al. [33] reported two cases positive for THCA in arthropod larvae. The cases were reported at the Medico-Legal Institute of Strasburg between the period 1988–2002 and the concentrations were determined to be 16 and 39 pg/mg THCA. 2. Materials and methods 2.1. Reagents and materials Acetonitrile and absolute methanol of HPLC grade, n-Hexane, heptane, diethylether and methyltert-butylether of analytical grade were obtained from Merck (Darmstadt, Germany). Ethyl acetate of HPLC grade was purchased from SDS (Solvent Documentations Synthèse, Vitry, France) and ammonium acetate was purchased from Ferak (Berlin, West-Germany). Water was deionized and further purified by means of a Direct-Q water purification system from Millipore SA (Molsheim, France). THC, THCA, THC-D3 and THCA-D3 were purchased from LGC Promochem (Molsheim, France). Symmetry C18 and Xˊ Terra MS C8 analytical columns were purchased from Waters Corporation (Milford, MA, USA) while solid phase extraction cartridges (Bond Elute C18 and Bond Elute Certify II) were purchased from Varian Inc. (Lake Forest, CA, USA). First instar larvae of L. sericata were obtained from BioMonde GmbH (Barsbüttel, Germany). The larvae were shipped to the Department of Forensic Medicine and Toxicology at the University of Athens Medical School in sterile flasks containing an appropriate substrate to maintain their viability. For method development and validation purposes, a pool of 100 drug-free larvae were sacrificed after their receipt by placing them in distilled water at 80 °C for 5 seconds and then transferring them in 30 mL of distilled water at room temperature. Following homogenization, the pool larvae matrix was stored at − 20 °C until use. The homogenate (blank pooled larvae matrix) was then fortified by spiking it with appropriate amounts of THC and THCA. 2.2. Apparatus Chromatography was performed using an LC/MS 2010EV system (Shimadzu, Kyoto, Japan) consisting of LC 20AB pump equipped with a DGU 20A5 degasser, SIL 20 AC autosampler and a LCMS 2010EV single quadrupole mass spectrometer with an electrospray ionization (ESI) source operation. Chromatographic separations were achieved on a Symmetry C18, analytical column (150 mm x 2.1 mm i.d., 5 μm) using an isocratic program and ammonium acetate (2 mM)-acetonitrile (70:30, v/v) as mobile phase. The flow rate was 0.25 mL/min, the column temperature was set at 35 °C and the injection volume was
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10 μL. The data were acquired and processed using LCMS solution software (version 3, Shimadzu). 2.3. Methods The method was optimized and validated according to European Union guidelines [34] and thus, parameters such as selectivity, specificity, linearity, precision, accuracy, recovery, stability, robustness and matrix effect were evaluated. Retention time, m/z and S/N ratios were set as acceptance criteria in order to assure the proper identification of the compounds. The retention time of the analytes with specific m/z should correspond to that of the calibrator at a tolerance of ± 2.5%. In addition, the identification and quantification of the analytes were evaluated based on S/N ratios (N3 and N10, respectively). All these acceptance criteria should be met after an additional assay of the samples on a second column with a different polarity in order to gather the appropriate identification points. In the proposed method two identification points are obtained from each m/z and other two identification points are obtained from each retention time originated from the two different analytical columns. For this purpose a second analysis using a different column (X'Terra MS C8, 250 mm × 2.1 mm i.d., 5 mm; Waters Corporation, Milford, MA, USA) was performed. 2.3.1. Sample extraction optimization-matrix effect For the extraction of the analytes liquid-liquid extraction (LLE), back-extraction, centrifugation speed, ultracentrifugation, filtering of the samples, sampling larvae amount and solid phase extraction were investigated in order to obtain the optimum recovery values for both analytes with the less matrix effect. The extraction efficiency of five different solvents (hexane, heptane, methyltert-butylether, ethyl acetate, diethylether) and their solvent mixtures in different percentages and mixtures, and two solid phase cartridges (Bond Elute C18, Bond Elute Certify II), were investigated. For the investigation of ion suppression effect caused by co-eluting matrix substances, six different blank pooled larvae samples fortified with THC and THCA at 500 pg/mg were analyzed. Ion suppression is quantitative expressed by comparing the response of the analytes and internal standards in aqueous solution to the response of the analytes and internal standards spiked into a blank matrix sample that has been carried through the sample preparation process [35]. % Ion suppression ¼ Analyteðor ISÞpeak area in matrix 100=Analyteðor ISÞpeak area in standard solution
2.3.2. Selectivity–specificity Selectivity of the method was examined by assaying six drug-free pooled larvae samples. Specificity was evaluated by analyzing six larvae samples fortified with 250 pg/mg of twenty five other drugs (11hydrohy-THC, cannabinol, cannabidiol, Δ8-THC, acetaminophen, aspirin, cetirizine, pseudoephedrine, methamphetamine, amphetamine, ephedrine, morphine, codeine, 6-monoacetylmorphine, methadone, ketamine, alprazolam, diazepam, phenobarbital, 7-amineflunitrazepam, cocaine, benzoylecgonine, ecgonine methylester, nordiazepam and γhydroxybutyric acid) than THC and THCA. All compounds were assayed using the same chromatographic conditions as the proposed method. Interference was determined by evaluating the signal to noise ratio (b3) at the retention time of THC and THCA. Carry-over effect was also assayed by injecting six fortified larvae samples at the upper limit of the calibration curve, and evaluating any potential transfer or interferences at the next samples. 2.3.3. Calibration curves For quantification, the calibration samples were prepared by spiking blank pooled larvae matrix with appropriate amounts of THC and THCA. A mixed working standard solution of THC and THCA at 10 μg/mL was
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prepared from ampoules of 1 mg/mL and 100 μg/mL, respectively. This mixture was then used for spiking to provide calibration curves. A mixed solution (1 μg/mL) of deuterated internal standards was prepared from ampoules of 1 mg/mL THC-D3 and THCA-D3. For the preparation of larvae matrix a modification of Roeterdink et al. [36] method was adopted. To that, a pool of 100 drug-free larvae (BioMonde GmbH) were sacrificed by placing them in distilled water at 80 °C for 5 seconds and then transferring them in 30 mL of distilled water at room temperature. The average weight of one larva was calculated at 75 (± 0.2) mg. Following homogenization, pool larvae matrix was stored at − 20 °C until use. THC and THCA working solutions were prepared by dilution of 10 μg/mL mixed standard solution with water in order to achieve fortified larvae samples in the seven point's calibration curve (10, 20, 40, 100, 200, 300 and 500 pg/mg). The range was selected according to the limited published cases including quantitative data for THCA in fly larvae [30]. Fifty μL of the deuterated internal standards mixture was then added in each sample corresponding to 250 pg/mg. The concentration of the internal standard was selected in order to correspond to the half of the upper limit of the calibration curve [37]. Calibration curves were assayed in five different runs and were plotted between the analyte concentrations and the peak area ratios of the analytes to the relative internal standards. Calibration points were evaluated according to acceptance criteria as they are set by the FDA guidelines [38] (±15% for all calibration levels, ±20% for LOQ level).
2.3.4. Precision, accuracy and recovery Quality control samples were prepared by appropriate dilutions using ampoules of different batch to obtain final larvae samples containing THC and THCA at 30, 250 and 400 pg/mg. Within-run (n = 6) and between-run (n = 30) precision, accuracy and recovery were determined by extracting the fortified samples. Recovery was calculated by comparing the responses obtained when the internal standards were added after the extraction step with those obtained from unextracted standard solution at the same concentrations.
2.3.5. Stability All samples were stored in glass vials in order to avoid any undesired loss of the analytes [39]. The stability of THC and THCA were investigated in pooled larvae samples fortified at three different concentrations (QC levels). All samples were assayed at room temperature (20 °C), 6 °C, − 20 °C and 3 cycles freeze-thaw test. These samples were compared with the freshly prepared samples (n = 6). Stability was also investigated in standard solution (15 ng/mL) which was placed at 80 °C for 2 min (n = 6). This temperature was selected in order to assure that in authentic samples, the analytes do not degrade during the sacrificing procedure. These samples were compared with freshly prepared samples. During this procedure the effect of temperature on matrix was also evaluated.
2.3.6. Robustness Robustness of the method was evaluated by slightly altering (±10%) experimental parameters on extraction procedures and chromatographic separation. To that, column temperature, CDL temperature of mass spectrometer, vortex time and percentage of solvents during liquid-liquid extraction, were evaluated.
2.4. Statistical analysis Validation results are expressed as mean ± standard deviation or % relative standard deviation. Statistical analysis was carried out using SPSS software package (version 10.0 for Windows).
3. Results 3.1. Optimization of chromatographic conditions During the preliminary experiments isocratic and gradient elution were investigated on a Symmetry C18 analytical column. Although both analytes are highly lipophilic compounds, the presence of an additional carboxylic group in THCA provides increased polarity to the molecule and its retention on a non-polar analytical column is very shorter than THC. Thus, a gradient elution program was initially investigated with the aim to increase THCA retention time avoiding thus the matrix effect. While the optimum total run time was 13 min, the effect of vertical and linear increase of organic modifier on the mobile phase composition after the elution of THCA, eluted simultaneously THC and the highly lipophilic extract of larvae, increasing thus the matrix effect on THC. Linear increase of organic modifier led to extensive retention of THC (N25 min) without any decrease of matrix effect leading us to proceed with isocratic elution. In isocratic program several percentages of mobile phase composition and pH values were investigated, in order to obtain the optimum separation and response of THC and THCA. To that, the acetonitrile percentage (10–80%), the pH of mobile phase by the addition of triethylamine (0.1%), and the ammonium acetate buffer concentrations (2, 5, 7.5, 10, 15 and 20 mM) were examined. By decreasing acetonitrile composition, no increase on the THCA retention time was observed. Thus, seventy percent of acetonitrile was chosen as the compromised percentage that led to sufficient retention time for the analytes and ion suppression. Two mM ammonium acetate buffer concentration in the mobile phase was found to give the best response. The pH of mobile phase was also examined using an Xʹ Terra analytical column that enables the use of mobile phases with pH values ranging between 1 and 12. The pH affected slightly the retention time of the analytes, while their response did not change significantly. The best separation and response for THC and THCA on the Symmetry C18 column was achieved using a mobile phase of acetonitrile-ammonium acetate (2 mM) (70:30, v/v) (pH = 6.79) without pH adjustment, in order to obtain retention times of 16.7 min for THC and 2.5 min for THCA. Although THCA was eluted at the retention time of matrix, no interference was observed allowing the proper identification and quantification of this metabolite. The analytes were detected using an electrospray ionization (ESI) source operation in negative mode. Positive mode was also applied; however, the ions abundance was much lower than negative mode. The instrument parameters were optimized by optimizing the signal of [M-H]− ions (m/z 313 for THC, m/z 343 for THCA) by direct infusion of standard solutions of the analytes and also by flow injection analysis. The Nebulizer gas flow was set to 1.5 L/min. CDL and Heat Block temperatures were optimized and kept at 230 °C. Detector voltage and interface voltage were optimized at 1.8 and 4.5 kv, respectively, while the appropriate Q-array DC and Q-array RF voltages were adjusted at 10 and 150 V, respectively. 3.2. Sample extraction-matrix effect Preliminary results showed that solvents, such as hexane and heptane, which are characterized by low polarity comparing to other solvents (log P hexane: 3.9; log P heptane: 4.4) show good distribution for both analytes. Tetramethylbutylether, having a higher polarity (log P 0.9) was shown to have reduced distribution while, ethyl acetate seems to favor the distribution of analytes in the organic layer to a greater degree than the diethylether. The combination of hexane with ethyl acetate provided the optimum recovery values of 98% for THC and 95% for THCA from larvae samples (Fig. 1). The effect of hexane/ethyl acetate mixture composition was further investigated and was found not to affect significantly the distribution of the two analytes. Hexane/ethyl acetate 50/50 (v/v) solvent mixture was finally selected as the optimum mixture composition providing recovery 98% for THC and 97% for THCA. A major problem after liquid-liquid extraction was the increased
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Fig. 1. Plots of the different Liquid-Liquid (L-L) extraction solvents versus the recovery of THC (RTHC) and THCA (RTHCA) (n-Hex: n-Hexane; n-Hep: n-Heptane; TMBE: Tetramethylbutylether; EA: Ethyl Acetate; DE: Diethylether).
larvae matrix remained in the final reconstituted sample, resulting in drastic suppression of the signal; ion suppression was found to be 48.3% for THC and 42.6% for THCA. Thus, the main concern was to further optimize the clean-up of the sample without any significant decrease of recovery. Various techniques were examined such as: a) the effect of the sampling larvae amount, b) back-extraction, c) centrifugation speed, d) ultracentrifugation, e) filtering of the samples with nylon filters, and f) solid phase extraction. In each sample the degree of ion suppression was calculated and the results are presented in Fig. 2. 3.2.1. Effect of pooled larvae amount Different amounts of the pooled larvae homogenate were examined. It was observed that recovery was not significantly affected by the concentration of homogenate used in contrast with the suppression of the signal which was improved as the amount of larvae was decreased. Two hundred microliters (200 μL) pooled larvae homogenate was selected since it provided the lowest signal suppression while allows the detection of the analytes even in one larva. 3.2.2. Back-extraction technique Data from back-extraction technique using HCl and NaOH as pH adjustment solvents showed that it provides samples with less matrix interferences and ion suppression (11.2% for THC, 12.6% for THCA); however recovery results were lower than in single step liquid-liquid
extraction and this difference was statistically significant only for THCA (13.2% for THC and 22.1% for THCA). 3.2.3. Effect of centrifuge speed Samples were centrifuged at different speed and time values in order to increase their purity without affecting the recovery of the analytes. For THC and THCA 4500 rpm were selected as the speed with less ion suppression and minimum effect on recovery (Table 1). 3.2.4. Effect of ultracentrifugation Ultracentrifugation was further investigated at 12000 rpm for 10 min and it was observed that the recovery was significantly reduced (70.3% for THC and 73.6% for THCA), while the purity of the sample was not improved. The suppression of the signal remained high (49.7% for THC and 46.2% for THCA). 3.2.5. Filtration of samples through filters Solvent mixture was filtered through GHP Acrodisk (0.45 microns, 13 mm) filters prior to evaporation. Recovery was found not to be significantly affected (94.9% for THC and 93.4% for THCA), however, the purity of the sample was not improved. The suppression of the signal remained high (53.2% for THC and 49.6% for THCA). 3.2.6. Solid phase extraction Further sample cleaning was further attempted through solid phase extraction (SPE) cartridges after a liquid-liquid extraction step. Thus Bond Elut C18 and Bond Elut Cerify II SPE cartridges were examined. Bond Elut C18 cartridges are characterized by non-polar interactions while Bond Elut Cerify II by combined non-polar/strong anionic interactions (silanol groups associated with eight carbon atoms and quaternary amines, C8/SAX). Table 1 Effect of centrifuge speed in THC and THCA recovery and ion suppression.
Fig. 2. Plots of the different larvae amount versus the recovery and ion suppression of THC (RTHC; IsTHC) and THCA (RTHCA; IsTHCA).
THC
THCA
rpm
% Ra
% Isb
%R
% Is
2500 3000 3500 4000 4500
93.4 96.2 101.8 98.5 97.4
30.4 30.5 31.9 28.7 27.3
94.2 104.6 92.3 96.2 98.7
33.1 31.7 31.3 29.8 28.4
a b
Recovery. Ion suppression.
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Fig. 3. Plots of the different extraction procedures versus the recovery and ion suppression of THC (RTHC; IsTHC) and THCA (RTHCA; IsTHCA) (LA: Larvae Amount; BE: Back Extraction; CE: Centrifuge; UCE: Ultracentrifuge; Af: Acrodisc filters; C18; C18 Solid Phase Extraction cartridges; BEC: BondElut Certify Solid Phase Cartridges; L-L: Liquid-Liquid extraction).
3.2.7. Bond Elut C18 After liquid-liquid extraction, the sample was reconstituted with 5 mL 0.1 M acetic acid and stirred for 30 s before SPE. Cartridges were conditioned with 2 mL methanol following with 2 mL of acetic acid 0.1 M prior to loading the sample. Wash step included 1 mL of acetic acid 0.1 M, 1 mL of acetonitrile/water (40:60, v/v) and the cartridge was allowed to dry for 5 min. Finally, the analytes were eluted with 4 mL of acetonitrile. The elute was evaporated to dryness using nitrogen gas, reconstituted with 100 μL mobile phase and 10 μL were injected into
the chromatographic system. Ion suppression was improved when compared to liquid-liquid extraction (THC: 15.4%, THCA: 20.9%), while recovery was drastically decreased (THC: 21%, THCA: 44.5%). 3.2.8. Bond Elut Cerify II After liquid-liquid extraction, the sample was reconstituted with sodium acetate buffer (pH 7)/acetonitrile (95/5, v/v) and stirred for 30 s before SPE. Cartridges were conditioned with 2 mL methanol following with 2 mL of sodium acetate buffer (pH 7) prior to loading
Fig. 4. Representative MS chromatogram of blank larvae sample. m/z 313 (THC), m/z 343 (THCΑ), m/z 316 (THC-D3), m/z 346 (THCΑ-D3).
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the sample. Wash step included 2 mL of sodium acetate buffer (pH 7)/ methanol (95/5, v/v) and the cartridge was allowed to dry for 5 min. THC was eluted with 4 mL of hexane/ethyl acetate (95/5, v/v), while for THCA the cartridge was again washed with 5 mL of 50% methanol and the sample was allowed to dry for 10 min. THCA was finally eluted with 4 mL of hexane/ethyl acetate (95:5 v/v) acidified with 1% acetic acid. Both elutes were evaporated to dryness using nitrogen gas, reconstituted with 100 μL mobile phase and 10 μL were injected into the chromatographic system. Although Bond Elut Cerify II cartridges provided higher recovery for THC compared to Bond Elut C18 cartridges, however it was again lower than liquid-liquid extraction. (THC: 81.2%, THCA 40.5%). In addition, ion suppression of the signal did not improve (THC: 20.4%, THCA: 19.8%) comparing to Bond Elut certify II cartridges. Conclusively, SPE provided poor recovery and increased variability on results although the improvement of ion suppression. To that, liquidliquid was selected as the optimum extraction procedure using a prior centrifugation step of the extraction solvent. In all the above experiments, the extent of the ion suppression did not depend on the concentration of the analytes. Finally, whatever effect of temperature (80 °C) used for larvae sacrification, in larvae matrix was found to be negligible when compared with untreated larvae samples.
vortex at speed 4 and further centrifuging for 10 min at 4500 rpm. The upper (organic) layer was then transferred into a 10 mL conical glass tube and evaporated to dryness at room temperature under a gentle stream of nitrogen. The residue was reconstituted in 100 μL of mobile phase and an aliquot of 10 μL was injected into the LC/MS system. Representative MS chromatograms of a blank larvae sample and a fortified sample at LOQ are presented at Figs. 4 and 5, respectively. In all cases, the proper identification and quantification of the analytes were confirmed if all acceptance criteria were met as they are described in Section 2.3.
3.2.9. Optimized extraction procedure Liquid-Liquid extraction using n-hexane-ethyl acetate (50:50, v/v) as extraction solvent compromised the optimum recovery with acceptable ion suppression (Fig. 3) while the extraction includes only a single step with short preparation time. Two hundred μL of pooled larvae homogenate was added in a 10 mL conical glass tube. Seven hundred μL of sodium acetate (pH 7.0; 0.1 M) and 0.3 mL of HCL (1 M) were then added and mixed for 10 seconds on a vortex. Each sample was extracted with 5.0 mL of n-hexane-ethyl acetate (50:50, v/v) after 5 min
The peak area ratios of the analytes to their deuterated analogues were linearly related to concentrations of THC and THCA for L. sericata. The response of the deuterated internal standards appeared to be lower than the respective analytes, which might be due to a matrix effect. The correlation coefficient of the calibration curves in five consecutive days was higher than 0.998 for both THC and THCA using 1/x2 as weighting factor (Table 2). Percentage R.S.D. of the slopes between the different calibration curves was better than 3.67 and 4.37 for THC and THCA, respectively. Back calculation of the calibrators provided higher
3.3. Selectivity–specificity Blank samples provided lower interference than S/N N 3 for THCA and no interference for THC. In addition, all twenty-five drugs did not yield any signal at the elution times of THC and THCA. Furthermore, carry over did not provide any interferences since in all cases, peak areas of the analytes in the sample that follows the high concentration sample, were less than 10% of the peak areas of the LOD concentration. 3.4. Linearity
Fig. 5. Representative MS chromatogram of spiked larvae sample with the two analytes at LOQ level (10 pg/mg) and their internal standards. Retention time of THC and THCΑ are 16.7 min and 2.5 min, respectively. m/z 313 (THC), m/z 343 (THCΑ), m/z 316 (THC-D3), m/z 346 (THCΑ-D3).
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Table 2 Analytical parameters of the calibration equations for the determination of THC and THCA in L. sericata (n = 5). Concentration range (pg/mg) THC THCA
10 – 500 10 – 500
Regression equationa Ss = 0.0073 × Cs + 0.0031 Ss = 0.0058 × Cs + 0.0039
Table 4 Accuracy, precision and recovery for THCA in blowfly larvae Lucilia sericata samples. Concentration added (pg/mg)
THCA
rb 0.998 0.998
a
Ratio of the peak area amplitude of the analyte to that of the internal standard, Ss, vs. the corresponding concentration; Cs is the concentration of THC or THCA. b Correlation coefficient.
accuracy than 85% (N 89.37%). The lower limit of detection was 3.2 pg/mg, while the lower limit of quantification was 10 pg/mg for both THC and THCA; as they were calculated by the signal to noise ratio (LOD = 3.3, LOQ = 10). 3.5. Precision, accuracy and recovery Intraday precision and bias for THC were found to be less than 7.2% and − 4.5% in L. sericata, respectively. Interday precision and bias for THC in five different days (n = 30) were found to be less than 7.3% and 4.2%, respectively (Table 3). Intraday precision and bias for THCA were found to be less than 5.3% and 14.3% in L. sericata, respectively. Interday precision and bias for THCA in five different days (n = 30) were found to be less than 5.5% and 12.5%, respectively (Table 4). 3.6. Stability THC and THCA concentrations in L. sericata homogenate were only slightly altered at room temperature, 6 °C −20 °C and 3 cycles freezethaw test (Table 5). These results are in accordance with published data regarding the stability of both compounds in biological fluids. Johnson et al. [40] reported that THC is stable until one month at room temperature, four months at 6 °C and six months at −20 °C. Degradation at room temperature was observed after the 1st month and was increased significantly until the end of the 6th month. The authors reported also that THA did not present any significant change even until the 6th month of storage. McCurdy et al. [41] found that THCA is stable in whole blood for one month of storage. Finally, the analytes were further found to be stable when 15 ng/mL of standard solution was stressed at 80 °C for 2 min, while the composition of the matrix was not found to be affected by the temperature. 3.7. Robustness No effect was found for both analytes by the slight modification (±10%) of the evaluated parameters. 3.8. Forensic case The application of the method was further applied in a forensic case. The body of a 30-year-old woman with a history of psychiatric disorder
%R.S.D.a
%Er.b
Intraday (n = 6) 30 250 400
5.3 4.7 3.1
14.3 13.1 11.4
Interday (n = 30) 30 250 400
5.5 5.2 2.0
12.5 11.5 9.6
was found hanged. The body was in the early stage of decomposition and had extensive population of fly larvae all over the body, that were identified as Lucilica sericata (Diptera, Calliphoridae). After police investigation and complete autopsy, suicide was determined as the manner of death. Initially, a urine sample was screened for drugs of abuse. Second instar larvae samples were further assayed according to the proposed LC/MS method. For the determination of THC and THCA in the respective liver samples, the method from Kudo et al. [42] was modified by replacing the final step of derivatization with a reconstitution step using100 μL of mobile phase. All samples were stored at −20 °C until analysis. The toxicological analysis of the urine sample, showed the presence only of cannabinoids while they were negative for cocaine, opiates and benzodiazepines. Alcohol was determined in the fluid obtained from squeezed liver and was found to be also negative. In larvae samples only THCA was detected at 43 pg/mg, while in liver samples both THC and THCA were detected at 2.1 and 3.4 ng/g, respectively (Table 6). 4. Discussion Although a number of other articles in the literature, report the determination of these analytes in whole blood and urine, this is the first reported method for the determination of both compounds in fly larvae. In addition, this article provides for first time essential information regarding the effect of larvae on experimental parameters such as the recovery, the ion suppression and a number of extraction techniques. The results obtained from larvae samples are similar to the two cases reported by Tracqui et al. [30] On the other hand the reported liver concentrations of our study were lower than the reported THC concentration of 38 μg/g of a man believed to have died of acute oral overdosage with THC [43]. Considering that metabolism of THC is considered to be hepatic blood flow limited; [44] the present results obtained from liver, show low concentrations of THC and higher concentrations of the THCA that are similar to the study of Gronewold and Skopp. [45] These authors studied the distribution of cannabinoids in man in five case reports. In two of the reported cases both THC and THCA were detected while two other cases were found positive only Table 5 Stability study of THC and THCA mean concentrations in Lucilia sericata (n = 6).
Table 3 Accuracy, precision and recovery for THC in spiked blowfly larvae Lucilia sericata samples. Concentration added (pg/mg)
THC %R.S.D.a
%Er.b
Intraday (n = 6) 30 250 400
7.2 3.6 4.4
3.7 −4.5 −0.7
Interday (n = 30) 30 250 400
7.3 3.9 5.1
4.2 −2.3 −1.7
(%) degradationa pg/mg
RTb (24 h)
6°C (1 month)
−20°C (1 month)
3 cycles Freeze-thaw
30 THC 250 400
−13.4
−9.9
−14.0
−1.8
−11.7 −12.3
−9.1 −8.6
−12.9 −14.5
−1.6 −2.7
THCA 30 250 400
−8.6 −10.1 −7.9
−8.7 −8.2 −8.6
−2.8 −3.3 −3.1
−0.6 −1.8 −1.5
a b
Percentage of degradation when compared to fresh samples. RT: Room temperature (20 °C).
S. Karampela et al. / Science and Justice 55 (2015) 472–480 Table 6 THC and THCA mean concentrations in real forensic autopsy samples (Lucilia sericata, liver; n = 6). Larvae THCc (pg/mg) THCAc (pg/mg)
43
Liver THCc (ng/g) THCAc (ng/g)
2.1 3.4
THCc: Concentration of THC. THCAc: Concentration of THCA.
for THCA. The verified cannabis use could be a contributing factor to the manner of death. Until today, no correlation studies exist between cannabinoids concentrations in human material and larvae found on human corpses. Although our results originated from a single case study, and quantitative correlation between larvae and liver cannot be obtained due to the limited data, they suggest the accumulation of THCA in maggots. In such a case, we should consider that absence of drugs in the maggots does not mean absence of drugs in the cadaver, and thus positive larvae samples should be considered as advantage. Although there are researchers who are skeptical about the usefulness of entomotoxicology in forensic medicine [30], we believe that all reasonable steps should be taken to perform a comprehensive toxicological analysis, if such is required to clear doubts related to a case. We always have to keep in mind factors affecting toxicological analyses and the limitations of their interpretation. Maggots, even if used only for qualitative analysis, could play an important part in detecting drugs of abuse and can contribute to the determination of the cause, mechanism and manner of death. 5. Conclusion In the absence of other biological samples, the use of blowfly larvae as an alternative sample is a well established procedure for the toxicological investigation of several classes of drugs. For this purpose, a method has been developed and validated for the simultaneous determination of THC and THCA in these insects. The method consists of liquid-liquid extraction followed by LC/MS detection and its applicability was confirmed through the successful analysis of larvae samples collected from a forensic case. Conflict of interest The authors declare no conflict of interest. References [1] L.M.L. Carvahlo, Toxicology and forensic entomology, in: J. Amendt, C.P. Campobasso, M.L. Goff, M. Grassbecrger (Eds.), Current Concepts in Forensic Entomology, Springer Science 2010, pp. 163–178. [2] http://www.forensicmag.com/news/2013/10/when-crime-scene-evidence-crawlsaway?cmpid=related_content (accessed: 21 May 2015). [3] http://www.forensicmag.com/articles/2013/08/entomotoxicology-alternativematrices-forensic-toxicology?cmpid=related_content (accessed: 21 May 2015). [4] M. Gosselin, S.M. Wille, M. Del Mar Ramírez Fernández, V. Di Fazio, N. Samyn, G. De Boeck, et al., Entomotoxicology, experimental set-up and interpretation for forensic toxicologists, Forensic Sci. Int. 208 (2011) 1–9. [5] O.H. Drummer, J. Gerostamoulos, Postmortem drug analysis: analytical and toxicological aspects, Ther. Drug Monit. 24 (2002) 9–199. [6] F. Introna, C.P. Campobasso, M.L. Goff, Entomotoxicology, Forensic Sci. Int. 120 (2001) 42–47. [7] R. Gagliano-Candela, L. Aventaggiato, The detection of toxic substances in entomological specimens, Int. J. Legal Med. 114 (2001) 3–197. [8] P. Kintz, A. Tracqui, B. Ludes, J. Waller, A. Boukhabza, P. Mangin, A.A. Lugnier, A.J. Chaumont, Fly larvae and their relevance in forensic toxicology, Am. J. Forensic Med. Pathol. 11 (1990) 63–65. [9] D.W. Sadler, C. Fuke, F. Court, D.J. Pounder, Drug accumulation and elimination in Calliphora vicina larvae, Forensic Sci. Int. 71 (1995) 191–197.
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