Sensitive determination of THC and main metabolites in human plasma by means of microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

Sensitive determination of THC and main metabolites in human plasma by means of microextraction in packed sorbent and gas chromatography–tandem mass spectrometry

G Model ARTICLE IN PRESS CHROMB-20244; No. of Pages 11 Journal of Chromatography B, xxx (2016) xxx–xxx Contents lists available at ScienceDirect ...

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ARTICLE IN PRESS

CHROMB-20244; No. of Pages 11

Journal of Chromatography B, xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Sensitive determination of THC and main metabolites in human plasma by means of microextraction in packed sorbent and gas chromatography–tandem mass spectrometry T. Rosado a,b,1 , L. Fernandes a,1 , M. Barroso c , E. Gallardo a,b,∗ a

Centro de Investigac¸ão em Ciências da Saúde, Faculdade de Ciências da Saúde da Universidade da Beira Interior (CICS-UBI), Covilhã, Portugal Laboratório de Fármaco-Toxicologia—UBIMedical, Universidade da Beira Interior, Covilhã, Portugal c Servic¸o de Química e Toxicologia Forenses, Instituto de Medicina Legal e Ciências Forenses-Delegac¸ão do Sul, Rua Manuel Bento de Sousa, Lisboa, Portugal b

a r t i c l e

i n f o

Article history: Received 31 May 2016 Received in revised form 1 September 2016 Accepted 5 September 2016 Available online xxx Keywords: Cannabis Plasma Microextraction by packed sorbent Gas chromatography–tandem mass spectrometry

a b s t r a c t Cannabis is one of the most available and consumed illicit drug in the world and its identification and quantification in biological specimens can be a challenge given its low concentrations in body fluids. The present work describes a fast and fully validated procedure for the simultaneous detection and quantification of 9 -tetrahydrocannabinol (9 THC) and its two main metabolites 11-hydroxy 9 tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-9 - tetrahydrocannbinol (THC-COOH) in plasma samples using microextraction by packed sorbent (MEPS) and gas chromatography–tandem mass spectrometry (GC–MS/MS). A small plasma volume (0.25 mL) pre-diluted (1:20), was extracted with MEPS M1 sorbent as follows: conditioning (4 cycles of 250 ␮L methanol and 4 cycles of 250 ␮L 0.1% formic acid in water); sample load (26 cycles of 250 ␮L); wash (100 ␮L of 3% acetic acid in water followed by 100 ␮L 5% methanol in water); and elution (6 cycles of 100 ␮L of 10% ammonium hydroxide in methanol). The procedure allowed the quantification of all analytes in the range of 0.1–30 ng/mL. Recoveries ranged from 53 to 78% (THC), 57 to 66% (11-OH-THC) and 62 to 65% (THC-COOH), allowing the limits of detection and quantification to be set at 0.1 ng/mL for all compounds. Intra-day precision and accuracy revealed coefficients of variation (CVs) lower than 10% at the studied concentrations, with a mean relative error within ±9%, while inter-day precision and accuracy showed CVs lower than 15% for all analytes at the tested concentrations, with an inaccuracy within ±8%. © 2016 Published by Elsevier B.V.

1. Introduction Cannabis is one of the oldest and most widely abused substances whose legal status is, at the present date, of world-wide concern. If, in the one hand, cannabis potential therapeutic benefits may lead to an approval for medical use, in the other hand, there is also evidence that it may adversely affect the brain’s neural connections, leading to dependence and behavioral disturbances, and the impairment it may cause on driving ability gains special attention in forensic contexts [1,2]. Among the listed compounds of cannabis, 9 - tetrahydrocannabinol (9 THC) is considered the most psychoactive component. Microsomal hydroxylation of 9 THC generates the

∗ Corresponding author at: Centro de Investigac¸ão em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6201-556 Covilhã, Portugal. E-mail address: [email protected] (E. Gallardo). 1 These authors contributed equally to this paper.

psychoactive compound 11-hydroxy 9 tetrahydrocannabinol (11OH-THC) and further oxidation generates the inactive, but of huge interest for diagnostic purposes, 11-nor-9-carboxy-9 tetrahydrocannbinol (THC-COOH) [2–5]. The analysis of cannabinoids may present some difficult challenges because THC and 11-OH-THC are highly lipophilic, and consequently are present in body fluids at low concentrations. In addition, whole-blood cannabinoid concentrations are approximately one-half the concentrations found in plasma specimens, due to the low partition coefficient of drug into erythrocytes, and THC and THC-COOH are predominantly found in the plasma fraction of blood, where 95−99% are bound to lipoproteins [6]. A suitable sample preparation is an important prerequisite for GC–MS analysis, with great impact on the analyte isolation from complex matrices such as plasma. Solid phase extraction (SPE) is the most commonly used sample preparation technique to extract THC and its main metabolites from biologic specimens [7–13]. The partition of solutes, guiding

http://dx.doi.org/10.1016/j.jchromb.2016.09.007 1570-0232/© 2016 Published by Elsevier B.V.

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principle of liquid-liquid extraction (LLE), has also been described to be efficient for these target analytes isolation [14,15]; however, nowadays, there is the growing need to search for sample preparation procedures that are simple, inexpensive, allow good analyte recovery and adequate selectivity, reducing simultaneously the use of organic solvents and sample volumes, and, whenever possible, automated [16]. Microextraction by packed sorbent (MEPS) is usually described as a miniaturization of the conventional SPE technique, in which the sample and solvents volumes are greatly reduced. The sorbent is packed into a syringe which can be used several times, and this originates an easy-to-use and rapid procedure that can be automated [17–19]. In fact, this miniaturized procedure has been successfully applied in oral fluid samples to identify and quantify THC and metabolites by liquid chromatography tandem mass spectrometry (LC–MS/MS) [20]. The authors presented a simple extraction technique, applying 5 sample strokes and obtaining recoveries from 50 to 100% when 50 mM NH4 OH in methanol was used as elution solvent [20]. Although oral fluid can be considered one of the most interesting alternative biological sample due to its noninvasive and simple collection procedure, plasma is still one of the most common samples to be used when drug monitoring is the main goal, hence MEPS applicability to plasma has become emergent. Nevertheless, this miniaturized extraction technique has also been successfully applied to the most diverse fields of analytical development and quantification such as environmental analysis [21,22], food analysis [23], biomarkers of diseases [24–26], therapeutic drugs [27–29], drugs of abuse and other toxic substances [30–33]. The application of MEPS to determine cannabinoids in human plasma has not been yet reported, and this sample may present added value in order to obtain a dose-response evaluation, which is not possible using oral fluid, due to oral cavity contamination after smoking [34]. The main goal of the present work is the development and optimization of the first application of MEPS coupled to gas chromatography–tandem mass spectrometry (GC–MS/MS) to identify and quantify THC and metabolites (11-OH-THC and THC-COOH) in plasma samples, which are the most commonly analyzed specimens in a forensic toxicology laboratory. Its application will result in a simple, fast, highly sensitive and less expensive procedure to determine the target analytes with the possibility to be automated and applied on a routine basis.

2. Materials and methods 2.1. Reagents and standards The analytical standards of 9 -tetrahydrocannabinol (9 THC), 11-hydroxy 9 tetrahydrocannabinol (11-OH-THC), 11-nor-9carboxy-9 -tetrahydrocannbinol (THCCOOH) and the internal standards (ISs): 9 -tetrahydrocannabinol-d3 (9 THC-d3); 11hydroxy 9 tetrahydrocannabinol-d3 (11-OH-THC-d3); 11-nor-9carboxy-9 - tetrahydrocannbinol-d3 (THC-COOH-d3) were purchased from LGC Promochem, (Barcelona, Spain). Methanol (Merck Co, Darmstadt, Germany), Acetic acid (Sigma Aldrich, Lisbon, Portugal), acetonitrile (Prolabo, Lisbon, Portugal) and Ethyl Acetate (Fischer chemical, Loughborough, UK) were all of analytical grade. Deionised (DI) water was obtained from a Milli-Q System (Millipore, Billerica, MA, USA) and ammonium hydroxide was purchased from J.T. Baker (Deventer, Holland). N-methylN-(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane (TMCS) were acquired from Macherey-Nagel (Düren, Germany). A MEPS syringe (250 ␮L) and M1 cartridges (4 mg; 80% C8 and 20% SCX), both SGE Analytical Science, Australia, were used. Stock solutions of each analyte were prepared at 1 mg/mL in methanol.

Working solutions were prepared by proper dilution of stock solutions with methanol to the final concentrations of 50 and 1 ng/mL for 9 THC, 11-OH-THC and THCCOOH, and a working solution of all ISs at 10 ng/mL was prepared also in methanol. All those solutions were stored in the absence of light at 4 ◦ C.

2.2. Biological specimens Drug-free plasma samples used in all experiments were provided by the Portuguese blood institute (Coimbra, Portugal). Authentic plasma samples used for analysis were provided by the emergency services of Hospital of Cova da Beira, Covilhã, Portugal. These samples were stored refrigerated at −21 ◦ C until analysis.

2.3. Gas chromatographic and mass spectrometric conditions An HP 7890A gas chromatography system (Agilent Technologies, Waldbronn, Germany), equipped with a model 7000B triple quadrupole mass selective detector (Agilent Technologies, Waldbronn, Germany), a MPS2 autosampler and a PTV-injector from Gerstel (Mülheim an der Ruhr, Germany) was used for chromatographic analysis. The separation of the analytes was achieved using a capillary column (30 m × 0.25-mm I.D., 0.25-␮m film thickness) with 5% phenylmethylsiloxane (HP-5 MS), supplied by J & W Scientific (Folsom, CA, USA). The oven temperature started at 120 ◦ C for 1.85 min, followed by an increase at 20 ◦ C/min during 9 min until 300 ◦ C and held for 9.10 minutes. The volume of injection was 2 ␮L on splitless mode, and the inlet and ion source temperatures were set at 250 ◦ C and 280 ◦ C, respectively. Carrier gas (helium) was set at a constant flow rate of 0.8 mL/min and data were acquired on the multiple reaction monitoring (MRM) mode. The mass spectrometer was operated with a filament current of 35 ␮A and electron energy 70 eV in the positive electron ionization mode. The transitions were chosen based on selectivity and abundance in order to maximize the signal- to-noise ratio in matrix extracts (Table 1).

2.4. Sample preparation Frozen plasma samples were allowed to thaw at room temperature. As most of the target analytes are highly bound to proteins, a previous protein precipitation step with frozen acetonitrile (3:1) was performed, and the mixture was centrifuged at 4500 rpm for 15 min. The supernatant was transferred into a glass tube and evaporated under a gentle stream of nitrogen at room temperature. Pre-treated plasma samples (0.25 mL) were diluted with 5 mL of 0.1 mM potassium phosphate buffer (pH = 6) and fortified with 50 ␮L of the IS working solution at 10 ng/mL. The mixture was homogenized by rotation/inversion movements during 15 min. The MEPS procedure was optimized previously (Section 3.1), and the final conditions were as follows. M1 MEPS cartridge was previously conditioned with 4 cycles of 250 ␮L of methanol and 4 cycles of 250 ␮L of 0.1% formic acid in water. Sample loading was performed with 26 cycles of 250 ␮L of the diluted plasma sample. Endogenous interferences were removed from the sorbent with 100 ␮L of 3% acetic acid in water followed by 5% aqueous methanol. The retained analytes were eluted from the sorbent with 6 cycles of 100 ␮L of 10%ammonium hydroxide in methanol, which was evaporated to dryness under a gentle stream of nitrogen. The dry extracts were derivatized using 65 ␮L of MSTFA with 5% TMCS, on a thermo block at 85 ◦ C for 40 min, and 2 ␮L aliquot of the resulting solution was injected into the GC–MS system.

Please cite this article in press as: T. Rosado, et al., Sensitive determination of THC and main metabolites in human plasma by means of microextraction in packed sorbent and gas chromatography–tandem mass spectrometry, J. Chromatogr. B (2016), http://dx.doi.org/10.1016/j.jchromb.2016.09.007

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Table 1 Retention time and selected transitions for the identification of analytes (quantitation ions underlined). Analyte

Retention time (min)

Precursor ion (m/Z)

Fragment ions (m/Z)

Collision Energy (eV)

THC THC-d3 11-OH-THC 11-OH-THC −d3 THC-COOH THC-COOH −d3

10.68 10.68 11.73 11.73 12.43 12.43

313.9 374.3 371.3 374.3 370.8 374.3

219.1 374.3 371.3 374.3 289.2 374.2

15 10 10 10 15 10

2.5. MEPS procedure optimization In order to minimize the number of interferences and get better efficiency in the procedure, the extraction technique was optimized. A total of 7 different approaches were tested, in order to evaluate washing and elution solvents, which had been selected according to the existing literature on the matter [35] and to the physical-chemical properties of the studied compounds. After selecting the technique, it was further optimized using the Design of Experiments (DOE) approach. The factors (independent variables) with significant relevance on the recovery of the target analytes, as well as their main effects, were screened by means of a two- level-four factor full factorial design (24 ) [36]. These variables were selected according to literature [17–19,35], and were found to be the number of strokes, acetic acid amount in the washing solvent, amount of methanol in the washing solvent and ammonium hydroxide amount in the elution solvent. To cover all possible combinations of factors’ levels, a total of 16 experiments (treatment combinations) were needed [36]. The independent variables were studied at 2 levels (low and high) as follows. Number of strokes: 6 and 18; Acetic acid (%): 1 and 5; Methanol (%): 0 and 10; Ammonium hydroxide (%): 5 and 15; These experiments were carried out in a random order with a central point in triplicate, to avoid the influence of noise factors, minimizing systematic errors [37]. An additional study was performed on the effect of different number of extraction cycles, as this factor revealed to be significant, ranging from 16 to 26. The whole optimization study was performed using samples spiked at 20 ng/mL. 2.6. Validation procedure The described method was fully validated according to the guiding principles of the Food and Drug Administration (FDA) [38] and International Conference on Harmonization (ICH) [39]. The 5 day validation protocol and the studied parameters included selectivity, linearity and limits, intra- and inter-day precision and accuracy, process efficiency and stability. The method’s selectivity was evaluated with the analysis of 6 pools of blank plasma samples, each pool containing 3 different origins, resulting in the search for eventual interferences at the retention times and selected ions of the studied compounds. Identification criteria for positivity and the guarantee of a suitable confidence in identification followed WADA statements [40], and the method would be considered selective if no analyte could be identified in the blank samples by means of those criteria. Linearity of the method was established on spiked samples prepared and analyzed using the extraction procedure, in the range of 0.1–30 ng/mL for all target analytes in plasma samples (five replicates). Calibration curves were obtained by plotting the peak area ratio between each analyte and the IS against analyte concentration. The acceptance criteria included a determination coefficient (R2 ) value of at least 0.99 and the calibrators’ accuracy within a ±15% (except at the lower limit of quantification (LLOQ), where ±20% was considered acceptable). Together with each calibration curve, a zero sample (blank sample with IS) and three QC

273.2 289.2 305.2

10 15 15

samples at 0.3, 5, and 25 ng/mL (n = 3) were also analyzed. The LLOQ was defined as the lowest concentration which could be measured with adequate precision and accuracy, i.e. with a coefficient of variation (CV, %) of less than 20% and a relative error (RE, %) within ±20% of the nominal concentration. The limits of detection (LOD) were determined by analyzing ten replicates of spiked samples, and were considered as the lowest concentrations yielding a discrete peak clearly distinguishable from the blank with a signal-to-noise ratio of at least 3. Intra-day precision was evaluated by analyzing in the same day 6 replicates of blank plasma samples spiked with the studied analytes at 5 concentration levels. Inter-day precision was evaluated at a minimum of six concentrations within a 5-day period. The method’s accuracy was characterized in terms of the mean RE between the concentrations measured using the calibration equation and the spiked concentrations; the accepted limit was 15% for all concentrations, except at the LLOQ, where 20% was accepted. For the analysis of extraction efficiency, two sets of samples (n = 3) were prepared at 3 concentration levels (0.5, 10, and 30 ng/mL). Set 1 represented post-extraction spikes (representing 100% efficiency), while set 2 consisted of pre-extraction spikes. The IS was added to the two sets of sample after extraction. The efficiency results were obtained by comparison of peak areas ratio of sample set 2 to those of the corresponding peaks in sample set 1. The stability of the analytes was studied at 3 concentration levels (0.3, 5, and 25 ng/mL) (n = 3) under specific conditions and time intervals (processed samples, short-term and freeze/thaw stability). To study the stability in processed samples, the extracts that were previously analyzed were re-analyzed after being stored at room temperature in the autosampler for 24 h, and their concentrations were determined on the basis of the original calibration curve. To evaluate short- term stability, blank samples were spiked and were left at room temperature for 24 h and for the freeze and thaw stability, plasma samples were spiked and were stored at −20 ◦ C for 24 h. After this period the frozen samples were thawed unassisted at room temperature, and then refrozen for 12–24 h under the same conditions. This freeze/thaw cycle was repeated twice more, and the samples were analyzed after the third cycle. During the entire stability procedure the analyzed samples were compared with samples prepared and analyzed freshly in the same day. For each stability study, the analyte was considered stable if the CV between the two sets of samples was below 15%. 3. Results and discussion 3.1. Optimization of the extraction procedure 3.1.1. Extraction procedure selection The first step for this study was the selection of the most suitable extraction technique. In total, 7 extraction procedures were tested according to both the analytes’ properties and literature [35]. Maintaining the conditioning solvents and volumes, as well as the diluted sample number of extraction cycles (12 cycles), different washing and elution steps were tested, according to Table 2. Each extraction technique was performed in triplicate, and the internal standard was added to the extracts before the evaporation step. This

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Table 2 Different extraction procedures applied to spiked plasma samples (n = 3). Extraction Procedure

Washing step (1 stroke of 100 ␮L)

Elution step (6 strokes of 100 ␮L)

1

Ammonium acetate 25 mM 5% Methanol (Water) Water: Acetonitrile:Ammonium (84:15:1) 5% Ammonium hydroxide (water) 1% Acetic acid (water) 10% Methanol (water) 8% Isopropanol in 2% formic acid (water) 5% Isopropanol in 1% Ammonium hydroxide (water) 10 mM Formic acid (water)

Formic acid in methanol (5:95)

2 3 4 5 6 7

Hexane: Ethyl acetate: Acetic acid (75:20:5) Dichloromethane: Isopropanol (75:25) 5% Ammonium hydroxide (methanol) Methanol: Acetonitrile (70:30) 3% Formic acid in Acetonitrile:Methanol (60:40) 50 mM Ammonium hydroxide (methanol)

Fig. 1. Graphical representation of the different extraction conditions affecting THC, 11-OH-THC and THC-COOH.

approach allows comparing the different techniques using relative peak areas, and minimizes the influence of small variations in the injection of extracts in the chromatographic system. The selection of the extraction procedure should reflect the greater recovery of the target analytes, which may result in different techniques for each of the compounds we intend to identify and quantify. This selection should have a critical interpretation depending on the necessity of a greater recovery of a specific analyte when compared to the others present in the analytical method. For instance, Fig. 1 reveals the extraction procedure 3 to be the one resulting in better recoveries for THC and 11-OH-THC, while for THC-COOH greater recoveries are obtained when extraction procedure 4 is applied. One can also observe in the same figure that technique 4 reveals to be the second best extraction procedure to be applied on plasma samples in order to obtain good recoveries for THC and 11-OH-THC. A complementary statistical study was needed to observe the differences between these two procedures leading to the selection of a unique technique that would then be optimized. ANOVA and T-student tests revealed no significant differences between the extraction procedures for THC (F(1,4) = 3.76, p < 0.05) and 11-OH-THC (F(1,4) = 4.93, p < 0.05). However, procedure 4 appears to have a significant better influence on THC-COOH, resulting in greater recoveries for this analyte (F(1,4) = 39.55, p < 0.05). Therefore, this approach was selected for further optimization.

3.1.2. Optimization of extraction procedure with DOE DOE is the statistical tool to be applied when a decision has to be made, evaluating in a multivariate fashion the critical factors that have a significant impact on the extraction procedure leading, to greater recoveries of the target analytes. The studied factors were: number of strokes; acetic acid (%); methanol (%); and ammonium hydroxide (%), in order to have a global approach of the load, washing and elution steps of MEPS. One can observe in Fig. 2 the results of the multivariate study of the factors affecting the recovery of THC, 11-OH-THC and THCCOOH. All analytes have a similar Pareto chart, indicating the number of strokes as the only variable with significant influence on the extraction procedure. The same influence is also observed on the main effects plot, showing a greater recovery of all analytes when a larger number of strokes is used. Regarding the other variables studied with DOE, although no significant influence was observed, it’s possible to see an apparently better response when an intermediate percentage of solvents (centre point) was used. The main effects plot shows greater recoveries for 11-OH-THC and THC-COOH when 3% acetic acid, 5% methanol and 10% ammonium hydroxide are applied as solvents in the MEPS steps. 3.1.3. Optimization of the number of strokes The final optimization step was performed in a univariate matter, since the number of extraction cycles (strokes) was the only

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Fig. 2. Graphical representation of DOE optimization for THC, 11-OH-THC and THC-COOH.

Fig. 3. Graphical representation of the different number of sample strokes influence on THC, 11-OH-THC and THC-COOH.

parameter with a significant influence on the recovery of the 3 analytes. To complete this step, all MEPS conditions were maintained in the apparent optimal conditions, whereas the number of

strokes varied from 16 to 26 (16, 18, 20, 22, 24 and 26). The results are shown on Fig. 3. A number of strokes higher than 26 was not evaluated for practical reasons.

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6 Table 3 Linearity data (n = 5). Compound

Weight

Linear Range (ng/mL)

a

THC 11-OH-THC THC-COOH a

1/x 1/x2 1/x2

0.1–30

R2 a

Linearity a

Slope

Intercept

0.030 ± 0.01 0.889 ± 0.065 0.200 ± 0.025

0.128 ± 0.039 0.036 ± 0.017 0.013 ± 0.009

0.996 ± 0.003 0.996 ± 0.003 0.996 ± 0.005

Mean values ± standard deviation.

Fig. 4. Ion chromatogram of a blank sample.

It is possible to observe the increase on the analytes’ recovery with the increase on the number of sample extraction cycles. THC-COOH was the most affected compound when the number of strokes raised up to 26 strokes, this number showing a significant statistical difference when compared to 24 cycles. This result complies, somehow, with the ones reported on Fig. 2, as it is possible to observe THC-COOH as the most affected analyte in what concerns the number of strokes. The final extraction conditions were then the following. Conditioning (4 cycles of 250 ␮L methanol and 4 cycles 250 ␮L of 0.1% formic acid in water); sample load (26 cycles of 250 ␮L); wash (100 ␮L of 3% acetic acid in water followed by 100 ␮L 5% aqueous methanol); and elution (6 cycles of 100 ␮L of 10% ammonium hydroxide in methanol).

3.2. Method validation parameters The present method was fully validated according to the guiding principles of the Food and Drug Administration (FDA) [38] and International Conference on Harmonization (ICH) [39]. The parameters included selectivity, extraction efficiency, linearity and calibration model, limits, intra- and interday precision and trueness, and stability. 3.2.1. Selectivity The method’s selectivity was evaluated by analyzing 6 pools of blank plasma samples (each pool containing 3 samples) collected from non consumers, and it was checked for interferences of endogenous compounds at the retention times and selected transi-

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Fig. 5. Ion chromatogram of a spiked sample at the LOD (0.1 ng/mL).

Table 4 Intra-day precision and accuracy (n = 6).

and the other compounds were observed when our method’s conditions were applied (Figs. 4 and 5).

Compound

Spiked

Measured

CV (%)

RE (%)

THC

0.1 0.5 1 10 30

0.11 ± 0,006 0.49 ± 0.047 1.06 ± 0,074 9.19 ± 0.771 28.46 ± 1.131

5.41 9.50 7.02 8.34 4.01

8.01 −1.01 5.56 −8.07 −5.13

11-OH-THC

0.1 0.5 1 10 30

0.10 ± 0.006 0.53 ± 0.028 1.00 ± 0.061 9.45 ± 0.272 29.14 ± 0.491

6.55 5.18 5.98 2.83 1.69

−4.83 6.99 0.36 −5.54 −2.86

THC-COOH

0.1 0.5 1 10 30

0.09 ± 0.007 0.51 ± 0.037 0.94 ± 0.056 9.82 ± 0.472 27.51 ± 1.681

8.12 7.31 5.99 4.74 6.11

−8.87 2.25 −5.93 −1.76 −8.29

All concentrations in ng/mL; CV – Coefficient of variation; RE – Relative error [(measured concentration-spiked concentration/spiked concentration)] × 100; Mean values ± standard deviation.

tions of the studied analytes. In addition, the ability to identify the compounds of interest several other abused drugs, alkaloids, and therapeutic drugs [including amphetamines, opiates, cocaine and metabolites, antidepressant, antipsychotics, nicotine and caffeine] were also tested. No interferences from endogenous substances,

3.2.2. Calibration curves and limits The method was considered linear from 0.1 to 30 ng/mL for all analytes. Still, weighted least squares regressions had to be adopted in order to compensate for heterocedasticity. Six weighting fac√ √ tors were evaluated for each compound (1/ x, 1/x, 1/x2, 1/ y, 1/y, 1/y2), and the one revealing the best results was selected, given the data obtained during the assessment of the inter-day precision and accuracy [38,39]. In order to select the most appropriate factor, the mean relative errors (RE) for each factor were summed. The factor that revealed the lowest sum of errors and simultaneously presented a mean R2 value of at least 0.99 was chosen (Table 3). The present analytical method obtained linear relationships, by means of these weighted least squares regressions, with calibrators’ accuracy (mean relative error (bias) between the measured and spiked concentrations) within a ±15% interval for all concentrations. Table 3 shows calibration data. The obtained LLOQ was 0.1 ng/mL. The limits of detection (LOD) were not systematically evaluated, and were considered to be the same as the LLOQ, since positive results below this value are not reported. These limits can also be considered quite satisfactory, especially when compared to analytical methods published by other authors for the same analytes. The present method results in better LLOQs comparing to the literature, namely for: THC

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Fig. 6. Ion chromatogram of an authentic sample (THC 4.01 ng/mL, 11-OH-THC 0.83 ng/mL and THC-COOH 10.46 ng/mL).

[8–10,13–15,20,41–46]; 11-OH-THC [8–10,13–15,20,41,44,45] and THC-COOH [7–15,41,43–45,47–50]. The mentioned papers report higher LLOQs than ours when analysis were carried out using a GC–MS system [41–44,46], GC–MS/MS [14,49,51], and also with higher selectivity systems, such as LC–MS/MS [7,8,10,45–48,50,52]. The present work describes the first application of MEPS to plasma samples with the purpose to quantify THC and its main metabolites, this extraction procedure being known for its advantages such as: reduced time to prepare and inject samples (around 3 min each); reduced solvent volumes from milliliters to microliters; and the reduced sample volume required, with an extreme importance regarding multiple analysis and/or re-analysis when the available sample for analysis is limited in volume. This extraction procedure allows reaching LLOQs which are significantly lower than previously published literature, yet using SPE with greater volumes, and usually not allowing re- utilization of the cartridges, all combined resulting in a much more expensive sample pre-treatment [8,9]. To the best of our knowledge, the results obtained with the present method are only surpassed in the papers by Jamey et al. [11], who used 1 mL of whole blood and SPE coupled to UPLC-MS/MS, obtaining lower LLOQS for THC, but not for the metabolites; and Sergi [20], the only application of MEPS to cannabinoids, using 125 ␮L of oral fluid and LC–MS/MS, reaching lower LLOQs for THC and THC-COOH, but not for 11-OH-THC. The present method is the first applying MEPS and GC–MS/MS to plasma samples to identify and quantify cannabinoids in a fast way, requiring only 0.25 mL of specimen and

reaching LLOQs that can be considered adequate according with the literature, making it an excellent option to apply routinely in forensic toxicology laboratories. 3.2.3. Intra- and inter-day precision and accuracy Intra-day precision and accuracy were evaluated by analyzing, on the same day, 6 replicates of blank plasma spiked with THC and metabolites at 5 concentration levels. The obtained coefficients of variation (CVs) were lower than 10% at all studied concentrations, with a mean relative error within ± 9% (Table 4). The evaluation of inter-day precision and accuracy was made within a 5-day period at 8 concentration levels. The obtained CVs were lower than 15% for all analytes at the tested concentrations, with an inaccuracy within ±8% (Table 5). 3.2.4. Extraction efficiency To study the extraction efficiency, two sets of samples (n = 3) were prepared by spiking blank plasma with the target analytes at three concentration levels: 0.5; 10; and 30 ng/mL. Set 1 represented post-extraction spikes (representing 100% recovery), while set 2 consisted of pre-extraction spikes. The efficiency results were obtained by comparison of peak area ratios of sample set 2 with those of the corresponding peaks in sample set 1. Table 6 shows extraction efficiencies of the target analytes. When compared to the only published paper applying MEPS for screening of THC and metabolites [20] our extraction technique

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T. Rosado et al. / J. Chromatogr. B xxx (2016) xxx–xxx Table 5 Inter-day precision and accuracy (n = 5). Compound

Spiked

Measured

CV (%)

RE (%)

THC

0.1 0.5 1 5 10 15 25 30

0.11 ± 0.014 0.50 ± 0.045 1.00 ± 0.080 4.79 ± 0.681 10.19 ± 0.552 15.10 ± 0.800 24.47 ± 0.761 30.45± 2.101

13.19 8.96 7.97 14.25 5.43 5.32 3.12 6.90

7.20 0.76 −0.11 −4.13 1.87 0.68 −2.12 1.49

11-OH-THC

0.1 0.5 1 5 10 15 25 30

0.10 ± 0.001 0.54 ± 0.032 1.06 ± 0.060 5.03 ± 0.281 10.16 ± 0.571 14.94 ± 0.400 23.75 ± 0.941 28.37 ± 0.920

1.02 6.01 5.99 5.63 5.66 2.66 3.97 3.26

−1.87 7.37 6.12 0.64 1.60 −0.43 −4.99 −5.44

THC-COOH

0.1 0.5 1 5 10 15 25 30

0.10 ± 0.002 0.48 ± 0.040 0.97 ± 0.049 4.88 ± 0.161 10.55 ± 0.270 15.52 ± 0.431 25.37 ± 1.600 29.54 ± 0.942

1.99 8.34 5.03 3.19 2.55 2.77 6.30 3.17

1.27 −4.23 −3.39 −2.39 5.51 3.49 1.49 −1.55

All concentrations in ng/mL; CV – Coefficient of variation; RE – Relative error [(measured concentration-spiked concentration/spiked concentration)] × 100; Mean values ± standard deviation. Table 6 Extraction efficiency (%) of the target analytes under the optimized extraction conditions (n = 3). Analyte

THC 11-OH-THC THC-COOH a

9

errors within a ±13% interval. The obtained results confirm the possibility of performing a re-analysis after 24 h in the autosampler with no significant change in the target analytes’ concentration. Short-term stability evaluation was carried out for blank plasma samples spiked at the same concentration levels (3 concentration levels related with the linearity range), also analyzed in triplicate. For this study, the plasma samples were spiked and left at room temperature for 24 h. The analysis of these samples was then compared with freshly prepared samples. The CVs obtained were lower than 14% for all of target analytes, while mean relative error was within a ±15% interval. Freeze/thaw stability was also studied in plasma samples at same 3 concentration levels (n = 3). This evaluation was made by freezing the spiked samples at −20 ◦ C for 24 h, after which they were thawed unassisted at room temperature. When completely thawed, the samples were re-frozen for 24 h. In total, three cycles of freeze/thaw were performed, after which samples were reanalyzed and subsequently compared to samples prepared and analyzed on the same day. All compounds were considered stable in plasma after 3 freeze/thaw cycles. 3.2.6. Method applicability The present method was successfully applied in routine analysis for THC, 11-OH-THC and THC-COOH in plasma samples belonging to individuals for whom cannabis consumption was suspected (provided by the emergency services of Hospital Cova da Beira, Covilhã, Portugal). A chromatogram of an authentic sample is shown in Fig. 6, and analyte concentrations were 4.01, 0.83 and 10.46 ng/mL for THC, 11-OH-THC and THC-COOH, respectively. 4. Conclusions

Recovery (%)a 0.5

10

30

78 ± 1.8 66 ± 2.0 63 ± 5.3

76 ± 8.9 57 ± 2.6 62 ± 1.4

53 ± 1.3 58 ± 1.0 65 ± 2.4

Mean values ± standard deviation.

allows greater efficiencies for THC, but lower ones for the metabolites in the present method. Still, the recovery of the analytes can be considered adequate as our results are similar, taking into account the different biological specimen and chromatographic equipment. Overall, the extraction efficiency can also be considered suitable considering other authors’ results, using molecularly imprinted solid phase extraction (MIPS) [41] polymer monolith microextraction (PMME) [42], disposable pipette extraction (DPX) [43] and the classical LLE [53]. According to our results, MEPS can be considered a powerful technique, revealing a fast and efficient extraction of the target analytes with a lower sample and solvent consumption. 3.2.5. Stability In order to simulate the normal conditions of handling and storage of real samples, the stability of the studied analytes was studied in plasma stored under specific conditions and time intervals. In this study, short-term, freeze/thaw and processed samples stability was conducted in order to evaluate the behavior of the analytes under different storage situations and the present method’s applicability after sample storage. The study of processed samples stability was carried out at the same concentrations than quality control samples (n = 3), in which previously analyzed samples were re-analyzed after 24 h in the autosampler. THC and its metabolites, as well as each internal standard, were assessed over the anticipated run time for batch size, and their concentrations were determined on the basis of the original calibration curve, obtaining CVs lower than 7% and mean relative

An easy and fast execution, sensitive, selective, accurate and fully validated procedure is herein described for the simultaneous detection and quantification of THC and its two main metabolites (11-OH-THC and THC-COOH) in plasma samples using MEPSGC–MS/MS. The design of experiments approach proved to be an excellent tool in order to find the critical factors on MEPS procedure and allowing a full optimization of the process in order to maximize the analytes’ recovery and consequently reach lower limits of detection and quantification. The present analytical method has shown to be linear within the adopted range for all target analytes with a LLOQ of 0.1 ng/mL, using a small volume of plasma (0.25 mL). Being successfully applied to authentic samples, and given the fast extraction, small specimen and solvent consumption, and over 200 reutilizations of the mixed mode cartridge, its usefulness for routine drug monitoring has been shown. This is the first time that microextraction by packed sorbent combined with GC–MS/MS is used to quantify THC and metabolites in plasma samples. Acknowledgments This work is supported by FEDER funds through the POCI – COMPETE 2020 – Operational Programme Competitiveness and Internationalisation in Axis I – Strengthening Research, Technological Development and Innovation (Project POCI-010145-FEDER-007491) and National Funds by FCT – Foundation for Science and Technology (Project UID/Multi /00709/2013). References [1] M.A. Huestis, Deterring driving under the influence of cannabis, Addiction 110 (2015) 1697–1698.

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