TY - JOUR
AU - Barroso,, M
AB - Abstract A microextraction by packed sorbent (MEPS) procedure for rapid concentration of methadone and its primary metabolite (EDDP) in hair samples was developed. The miniaturized approach coupled to gas chromatography with tandem mass spectrometry (GC–MS-MS) was successfully validated. Hair samples (50 mg) were incubated with 1 mL of 1 M sodium hydroxide for 45 min at 50°C, time after which the extract was neutralized by adding 100 μL of 20% formic acid. Subsequently, MEPS was applied using a M1 sorbent (4 mg; 80% C8 and 20% strong cation-exchange (SCX)), first conditioned with three 250-μL cycles of methanol and three 250-μL cycles of 2% formic acid. The extract load occurred with nine 150-μL cycles followed by a washing step involving three 50-μL cycles with 3.36% formic acid. For the elution of the analytes, six 100-μL cycles of 2.36% ammonium hydroxide in methanol were applied. The method was linear from 0.01 to 5 ng/mg, for both compounds, presenting determination coefficients greater than 0.99. Precision and accuracy were in accordance with the statements of international guidelines for method validation. This new miniaturized approach allowed obtaining recoveries ranging from 73 to 109% for methadone and 84 to 110% for EDDP, proving to be an excellent alternative to classic approaches, as well as other miniaturized procedures. Introduction Methadone, (+) -6-dimethylamino-4,4-diphenylheptan- 3-one, is a synthetic narcotic analgesic commonly used for the treatment of heroin and morphine addiction (1). The basic prerequisite for admittance in methadone programs is both detoxification and long-term maintenance therapy (2). Nevertheless, a risk of overdose exists, possibly leading to fatal outcomes, and for that reason, monitoring is necessary in order to detect substance misuse and prevent illicit diversion of prescribed opiates (2, 3). Although urine is usually analyzed from patients undergoing these programs, hair analysis may be an useful alternative to verify drug history and compliance (2, 3). In fact, hair samples present limited possibility of tampering with, becoming more difficult to hide drug intake when compared with urinalysis. Additionally, the hair sampling method is non-invasive, while urine sample collection, which should be performed with strict supervision, may be considered embarrassing for both the individual being tested and supervisor (4). Regarding hair testing in patients under methadone-maintenance programs, methadone and its primary metabolite 2-ethylidine-1,5-dimethyl-3,3-diphenyl-1-pyrrolidine (EDDP) are usually detected (5). However, this specimen is a very complex matrix, since the drugs are strongly bound to inner-hair constituents, and as such the analysis involves, at a first stage, an initial sample pre-treatment step, commonly named incubation or extraction, that will allow the release and solubilization of drugs (6, 7). The resultant extract can be either directly analyzed (3, 8–16) or may require subsequent clean-up, for which liquid–liquid extraction (LLE) (17–22) and solid-phase extraction (SPE) (4, 18, 23–32) are the most commonly used approaches. The pre-treatment of complex matrices by using miniaturized sample preparation methods remains of great interest in the research field (33–35). Indeed, they feature advantages when compared with classical techniques, such as the higher speed of analysis with the higher associated efficiency, low cost of operation due to lower solvents consumption, environmental friendly and highly selective analysis (36). Microextraction techniques, such as liquid-phase microextraction (LPME) (37) and solid-phase microextraction (SPME) (5, 38–42), present these advantages and have been successfully applied as clean-up procedures for the determination of methadone and EDDP in hair. Less explored, concerning drug analysis in hair, is the microextraction by packed sorbent (MEPS) approach, which is a miniaturization of the conventional SPE packed bed cartridges, allowing reducing bed volumes from the millilitre to the microlitre ranges (43–47). MEPS emerged in 2004, developed by Abdel-Rehim (48) and has been accepted as an attractive miniaturized option and powerful sample-preparation technique, because it is fast, simple and requires very small volumes of samples and solvents, resulting in results comparable to those of SPE (43–47). Additionally, MEPS allows full automation, including the sample processing, extraction and injection steps as an online sampling device using the same syringe (43–47). Up until now, and concerning hair testing, this technique has only been applied in the determination of methamphetamine and amphetamine (49) and a number of selected opiate compounds (50). The aim of this work was the development and validation of an analytical method using MEPS to determine methadone and its main metabolite EDDP in hair samples. Materials and Methods Reagents and standards Methadone and EDDP analytical standards, as well as the internal standards (ISs), methadone-d3 and EDDP-d3, were obtained from Sigma-Aldrich (Lisbon, Portugal). The working solutions of both methadone and EDDP were prepared by proper dilution of stock solutions with methanol to the final concentrations of 5 and 0.25 μg/mL, while a working solution of the two ISs was also prepared in methanol at a concentration of 1 μg/mL. All stock and working solutions were stored at −20°C. Formic acid (Panreac Química SA, Barcelona, Spain), ammonium hydroxide (J.T. Baker, Deventer, Holland), methanol (Merck Co, Darmstadt, Germany), isopropanol (Fischer chemical, Loughborough, UK) and acetonitrile (Prolabo, Lisbon, Portugal) were all pro-analysis grade. Deionized (DI) water was obtained from a Milli-Q System (Millipore, Billerica, MA, USA). The MEPS syringe (250 μL) and M1 cartridges (4 mg; 80% C8 and 20% SCX), both from SGE Analytical Science, were acquired from VWR international (Alfragide, Portugal). Statistical analyses used for optimization were carried out with Minitab Statistical Software version 17 and SPSS version 25. Hair samples Blank hair samples for methadone and EDDP were provided by laboratory staff (CICS, Covilhã, Portugal) and were used for MEPS optimization and method validation. Authentic hair samples were obtained from individuals undergoing methadone treatment program at Centro de Atendimento ao Toxicodependente—Casas de Santiago (Belmonte, Portugal) and, subsequently, sent to the health sciences research center (CICS, Covilhã, Portugal). Both blank and authentic samples were stored in paper envelopes in a dry, dark environment at room temperature, away from direct sunlight. Gas chromatographic and mass spectrometric conditions An HP 7890A gas chromatography (GC) system coupled to a 7000B triple quadrupole mass spectrometer (MS), both from Agilent Technologies (Waldbronn, Germany), was used for analysis. Automated injections were performed with an MPS2 auto-sampler and a PTV-injector from Gerstel (Mülheim an der Ruhr, Germany). Methadone and EDDP chromatographic separation was possible using a capillary column (30 m × 0.25-mm I.D., 0.25-μm film thickness) with 5% phenylmethylsiloxane (HP-5 MS), supplied by J & W Scientific (Folsom, CA, USA). The oven gradient of temperatures started at 150°C for 2 min, after which it was raised to 300°C at 20°C/min and was held at that temperature for 3 min, originating a total run time of 12.5 min. The temperatures of the injection port and transfer line were 220 and 280°C, respectively. The sample extract (2 μL) was injected into the GC in the splitless mode, and helium was used as a carrier gas at a constant flow of 0.8 mL/min. The MS operated with a filament current of 35 μA and an electron energy of 70 eV in positive electron ionization mode. Nitrogen was used as a collision gas with a flow rate set of 2.5 mL/min. All data were acquired in the multiple reaction monitoring mode with the help of the MassHunter WorkStation Acquisition software rev. B.02.01 (Agilent Technologies). The injection of methanolic standard solutions of methadone and EDDP at different collision energies and dwell times contributed for the optimization of the final MS conditions. The transitions were chosen based on selectivity and abundance in order to maximize the signal-to-noise ratio in hair extracts (Table I). Table I Retention times and selected transitions for the identification of analytes Analyte . Retention time (min) . Quantifying transition (m/z) . Qualifying transition (m/z) . Collision energy (eV) . Dwell time (μs) . EDDP 8.17 275.4–232.3 275.4–247.2 20 (15)a 50 EDDP-d3 8.16 237.2–220.2 – 10 50 MTD 8.68 222.1–105.1 222.1–117.0 20 (20)a 50 MTD-d3 8.67 297.0–297.0 – 5 50 Analyte . Retention time (min) . Quantifying transition (m/z) . Qualifying transition (m/z) . Collision energy (eV) . Dwell time (μs) . EDDP 8.17 275.4–232.3 275.4–247.2 20 (15)a 50 EDDP-d3 8.16 237.2–220.2 – 10 50 MTD 8.68 222.1–105.1 222.1–117.0 20 (20)a 50 MTD-d3 8.67 297.0–297.0 – 5 50 a Collision energy used for the qualifying transition. Open in new tab Table I Retention times and selected transitions for the identification of analytes Analyte . Retention time (min) . Quantifying transition (m/z) . Qualifying transition (m/z) . Collision energy (eV) . Dwell time (μs) . EDDP 8.17 275.4–232.3 275.4–247.2 20 (15)a 50 EDDP-d3 8.16 237.2–220.2 – 10 50 MTD 8.68 222.1–105.1 222.1–117.0 20 (20)a 50 MTD-d3 8.67 297.0–297.0 – 5 50 Analyte . Retention time (min) . Quantifying transition (m/z) . Qualifying transition (m/z) . Collision energy (eV) . Dwell time (μs) . EDDP 8.17 275.4–232.3 275.4–247.2 20 (15)a 50 EDDP-d3 8.16 237.2–220.2 – 10 50 MTD 8.68 222.1–105.1 222.1–117.0 20 (20)a 50 MTD-d3 8.67 297.0–297.0 – 5 50 a Collision energy used for the qualifying transition. Open in new tab Sample preparation Hair decontamination and extraction The washing procedure adopted for all hair samples prior to analysis involved a sequential soaking in dichloromethane, DI water and methanol, 15 min each, at room temperature with agitation. This procedure allowed the removal of hair care products, sweat, sebum or surface material that could interfere with the chromatographic analysis and/or reduce extraction recovery. This step also becomes important to remove potential external drug contamination. For that reason, the last wash was stored for further analysis, in order to check for the presence of the target compounds. After the decontamination procedure, hair samples were left to dry at room temperature. After completely dried, each hair sample was cut into fragments of less than 1 mm, and 50 mg was weighed into glass tubes. Then, 1 mL of 1 M sodium hydroxide was added and the tubes were tightly closed. The tubes were vortex-mixed and incubated for 45 min at 50°C. After digestion of the hair samples, these were neutralized by adding 100 μL of 20% formic acid in water, vortex-mixed and centrifuged at 3500 rpm for 15 min. The extracts were transferred into new glass tubes ,and 25 μL of ISs working solution was added. Microextraction by packed sorbent The MEPS procedure used for sample clean-up was optimized, resulting in the following final conditions. The MEPS cartridge was previously conditioned with three 250-μL cycles of methanol and three 250-μL cycles of 2% formic acid in water. Sample load was performed with nine withdraw–dispense cycles of 150 μL. A subsequent washing step was performed by three cycles with 50 μL of 3.36% formic acid in water, time after which the retained analytes were eluted from the sorbent with six 100-μL cycles of 2.36% ammonium hydroxide in methanol. The eluted solution was, then, evaporated to dryness under a stream of nitrogen. Since the same extraction cartridge is reused for the clean-up of several samples, the sorbent was sequentially washed with 1% ammonium hydroxide in acetonitrile:methanol (1:1) and 1% formic acid in isopropanol:water (10:90) (four cycles of 250 μL each) before each new sample extraction cycle. The dry extracts were reconstituted with 50 μL of methanol, and a 2-μL aliquot of the resulting solution was injected onto the chromatographic system. Validation procedure The full validation of the developed analytical method followed the guiding principles of the Food and Drug Administration (51), the International Conference on Harmonization (52) and the Scientific Working Group for Forensic Toxicology (53). A 5-day validation protocol was adopted, and the evaluated parameters were selectivity, linearity and limits, intra- and inter-day precision and accuracy, recovery and auto-sampler stability. Results and Discussion Optimization of the MEPS procedure Although simplicity is a feature usually associated to MEPS, this clean-up procedure should follow a range of optimization steps in order to obtain a fine tuning of extraction efficiency (46, 54). For instance, the appropriate selection of the sorbent is of extreme importance to obtain satisfactory clean-up and analyte recovery (46, 54). As previously mentioned, MEPS is a miniaturization of SPE, and the sorbent selection was based on the available literature that applied the classic procedure to pre-concentrate EDDP and methadone from hair extracts. Moreover, Agilent Bond Elut Certify sorbent that consists of a nonpolar C8 sorbent and an SCX has been the most described sorbent (4, 24, 29, 30). Also, Oasis® MCX cartridges, with a mixed-mode polymeric sorbent, have been reported because they present high selectivity and sensitivity to extract basic compounds with cation-exchange groups (25, 27). In addition, Phenomenex® Strata X polymeric sorbent, ideal for clean-up of neutral, acidic or basic small molecule compounds, was successfully applied for methadone, amongst other drugs (18, 26). However, Phenomenex® Strata X was only used after an initial LLE of the hair extract. ISOLUTE® HCX, a mixed-mode sorbent, was efficiently used for methadone and EDDP determination in hair from human subjects following a maintenance program (31), and SPE Cationic Exchange from StepBio was also described for a multimethod that included both target analytes (28). To the best of our knowledge, only one work has reported octadecyl-modified silica phase sorbent to pre-concentrate these compounds from hair extracts (23), and for this reason, the microextraction procedure was carried out using mixed mode sorbent containing a mixture of 80% C8 and 20% SCX, labeled as M1 on the MEPS BIN. Depending on the compounds to extract, some steps can be simplified or skipped (46, 54). Nevertheless, the number of sample extraction cycles, also known as strokes, the solvents used in the washing and elution steps, as well as their volumes, can be optimized for each application, leading to greater recoveries (46, 54). Considering a previous successful MEPS application from the same work group to determine selected opiates in hair samples, 3.36% formic acid in water and 2.36% ammonium hydroxide in methanol were selected as washing and elution solvents, respectively (50). Three cycles with 250 μL of methanol and 250 μL 2% formic acid were maintained for the sorbent conditioning step. A two-level full factorial design with three factors (23) was developed in order to study the effect they had on the methadone and EDDP recoveries. The studied factors were the number of sample load strokes (3–9 × 150 μL), the number of washes (1–3 × 50 μL) and the number of elution cycles (2–6 × 100 μL). This study was performed using the Design of Experiments statistical tool, which rapidly evaluates, in a multivariate fashion, the critical factors that may have a significant impact on compounds’ recoveries. A central point (n = 3) was added to the design matrix for precision evaluation. The evaluation was performed with blank hair samples spiked at 1 ng/mg. The ISs were added only after extraction. According to the pareto charts obtained from the experimental design (Figure 1), the only factor that revealed a significant influence on both methadone and EDDP recoveries was the number of sample load strokes. Through the main effect plots (Figure 1), it is possible to observe a greater response when a higher number of strokes, in this case nine cycles, were adopted. The other two factors under study resulted in a very low response when compared with the sample strokes number, and therefore, those conditions that originated an apparent better response (considering the main effects plots) were chosen: number of washes (3 × 50 μL) and number of elution cycles (6 × 100 μL). The monitoring of the experimental design through the central point resulted in relative standard deviations (RSDs) of 5.9 and 3.2% for EDDP and methadone, respectively. Figure 1 Open in new tabDownload slide Pareto charts and main effects plots obtained for EDDP and methadone after experimental design. Figure 1 Open in new tabDownload slide Pareto charts and main effects plots obtained for EDDP and methadone after experimental design. Once the number of withdraw–dispense cycles appeared as a significant factor, it seemed pertinent to evaluate if the increment of these cycles number above nine would result in the improvement of the recoveries. A subsequent study was made, in which the number of strokes was increased up to 18, while the other conditions were kept unchanged. Figure 2 shows the graphical representation of the obtained results, when 9, 12, 15 and 18 strokes were applied on the sample load step (n = 3). One observes that, although recoveries increased with the number of strokes, the related-samples Friedman’s two-way analysis of variance by ranks gave no statistical difference for either EDDP (P = 0.068) or methadone (P = 0.060). Figure 2 Open in new tabDownload slide Graphical representation of number of strokes influence on EDDP and methadone recoveries (n = 3). Figure 2 Open in new tabDownload slide Graphical representation of number of strokes influence on EDDP and methadone recoveries (n = 3). After the MEPS steps optimization, and according to the reported results, the final procedure to clean-up hair extracts in order to determine EDDP and methadone was obtained. Method Validation Selectivity Selectivity of the method was evaluated by the analysis of blank hair samples from 10 different origins (laboratory staff). These samples were analyzed and checked for possible interferences at the retention times and selected transitions of the analytes. Identification criteria for positivity included the use of ion ratios, retention times and signal-to-noise evaluation. The maximal accepted deviations for the studied parameters were those specified in the World Anti-Doping Agency’s document (55). By using these criteria, no analyte could be identified in any of the analyzed samples. Figure 3 represents a chromatogram of a blank hair sample (on the left) and a chromatogram of a sample spiked at 0.01 ng/mg—the lower limit of quantification (LLOQ)—(on the right). By means of these criteria, the method was considered selective, since no compound could be identified in the blank hair specimens. Figure 3 Open in new tabDownload slide Chromatogram of a blank hair specimen and a hair specimen spiked at 0.01 ng/mg. Figure 3 Open in new tabDownload slide Chromatogram of a blank hair specimen and a hair specimen spiked at 0.01 ng/mg. Calibration curves and limits Calibration curves were constructed by plotting the ratio between each target compound and the respective IS peak areas against compound concentration. As acceptance criteria, a determination coefficient (R2) of at least 0.99 and the calibrators’ accuracy within a ±15% interval from the nominal value (± 20%, for the LLOQ) were adopted. Method linearity was, then, obtained in the range of 0.010–5 ng/mg for EDDP and methadone (n = 5). However, variational approach was adopted relying on a weighted least squares criterion (1/x) to compensate for heterocedasticity. The LLOQ was considered the lowest concentration that could be measured with an RSD equal or lower than 20% and a relative error (RE, %) within ±20% of the nominal concentration. Considering the above, 0.010 ng/mg was obtained as the LLOQ for both EDDP and methadone with the present analytical method. Table II resumes calibration data. Table II Linearity data (n = 5) Analyte . Weight . Linear range (ng/mg) . Linearity . R 2 * . LLOQ (ng/mg) . slope* . Intercept* . EDDP 1/x 0.010–5 0.159 ± 0.0109 0.017 ± 0.7882 0.9965 ± 0.0025 0.010 Methadone 1/x 0.010–5 0.001 ± 0.0006 0.001 ± 0.0145 0.9970 ± 0.0012 0.010 Analyte . Weight . Linear range (ng/mg) . Linearity . R 2 * . LLOQ (ng/mg) . slope* . Intercept* . EDDP 1/x 0.010–5 0.159 ± 0.0109 0.017 ± 0.7882 0.9965 ± 0.0025 0.010 Methadone 1/x 0.010–5 0.001 ± 0.0006 0.001 ± 0.0145 0.9970 ± 0.0012 0.010 a Mean values ± standard deviation. Open in new tab Table II Linearity data (n = 5) Analyte . Weight . Linear range (ng/mg) . Linearity . R 2 * . LLOQ (ng/mg) . slope* . Intercept* . EDDP 1/x 0.010–5 0.159 ± 0.0109 0.017 ± 0.7882 0.9965 ± 0.0025 0.010 Methadone 1/x 0.010–5 0.001 ± 0.0006 0.001 ± 0.0145 0.9970 ± 0.0012 0.010 Analyte . Weight . Linear range (ng/mg) . Linearity . R 2 * . LLOQ (ng/mg) . slope* . Intercept* . EDDP 1/x 0.010–5 0.159 ± 0.0109 0.017 ± 0.7882 0.9965 ± 0.0025 0.010 Methadone 1/x 0.010–5 0.001 ± 0.0006 0.001 ± 0.0145 0.9970 ± 0.0012 0.010 a Mean values ± standard deviation. Open in new tab The LLOQs reached with the present method can be considered good, comparing with the available literature that had the same goals. For instance, Concheiro et al. (26) used the same amount of hair, 50 mg, to determine cocaine, heroin and methadone to evaluate in utero drug exposure and reported an LLOQ of 0.020 ng/mg for methadone. The authors used both classic clean-up procedures, first LLE and then SPE, and analysis was carried out with liquid chromatography coupled to tandem mass spectrometry (LC–MS-MS). The same working group had already developed a multimethod for target screening and confirmation of 35 drugs and metabolites in 50 mg of hair by LC–MS-MS, again using LLE followed by SPE, and reported identical LLOQ for methadone (18). Additionally, De la Torre et al. (28) developed a high throughput analysis of drugs of abuse in 50 mg of hair, using SPE as clean-up technique and GC–MS, obtaining an LLOQ of 0.1 ng/mg for both EDDP and methadone. Skender et al. (30) performed a quantitative determination of amphetamines, cocaine and opiates in 50 mg of hair using SPE followed by GC–MS and achieved a limit of detection (LOD) of 0.3 ng/mg. Girod and Staub (31) determined methadone and EDDP in 50 mg of hair from human subjects following a maintenance program. The authors used SPE for sample clean-up with analysis by GC–MS and reported LLOQs of 0.05 ng/mg for methadone and 0.2 ng/mg for EDDP. Bermejo et al. (21) had the goal to simultaneously determine methadone, heroin and metabolites in 50 mg of hair using LLE followed by GC–MS and obtained an LLOQ of 0.13 ng/mg for methadone. All these authors used a classical clean-up procedure after hair samples incubation, but several other researchers excluded this additional step, still achieving higher LLOQs than those obtained in the herein described method. Di Corcia et al. (11) published the simultaneous determination of multiclass drugs of abuse, also in 50 mg of hair, by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS-MS). These authors used no clean-up procedure after hair incubation and achieved an LLOQ 0.03 ng/mg for methadone. Musshoff et al. (14), with no clean-up added after incubation of 50 mg of hair, had the goal to determine opioid analgesics using LC–MS-MS with application to patients under palliative care and achieved an LLOQ of 0.03 ng/mg for methadone. Kelly et al. (15) used a greater amount of hair, 75 mg, to perform a chiral analysis of methadone and major metabolites by LC–MS-MS. The authors also described a sample preparation that consisted only of incubation and no additional clean-up procedure, obtaining LLOQs of 0.05 and 0.03 ng/mg for methadone and EDDP, respectively. It is consensual that the amount of hair sample used may influence the limits of determination of an analytical method. Although the amount of 50 mg is widely applied, nowadays, there is a tendency for the reduction of the sample weight. Tournel et al. (17) used 20 mg of hair to determine methadone exposure in pediatric deaths with LLE and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS-MS), reporting a LOD of 0.1 ng/mg for both compounds. Also, Fernández et al. (25) used 20 mg of hair for the simultaneous analysis of 33 basic drugs in hair. These authors used SPE as clean-up procedure and UHPLC–MS-MS for analysis reaching an LLOQ of 0.03 ng/mg. These limits are also higher than those presented in this work. However, a method development for methadone and other illegal drugs in 20 mg of hair from children with parents under maintenance treatment in a German community was performed by Pragst et al. (56) and should be highlighted. These authors used a liquid chromatography–hybrid quadrupole time-of-flight mass spectrometry and obtained LLOQs of 0.001–0.003 ng/mg for the target compounds, values much lower than the ones achieved with the present work. The greater sensitivity was achieved with the use of a high-resolution detector. Nevertheless, the amount of 10 mg of hair sample is also extensively applied for the accurate measure of these analytes (9, 12, 20, 27, 57) with LLOQs reported in the range of 0.01–0.1 ng/mg, values equal or greater than those herein described. To the best of our knowledge, the fewer amounts of hair samples used to determine methadone are described by three authors. Leung et al. (8) performed a surveillance of drug abuse in Hong Kong by the analysis of 5 mg of hair using LC–MS-MS, obtaining an LLOQ of 0.04 ng/mg for methadone. Sheibani et al. (58) determined methadone in 2 mg of human hair by headspace extraction and ion mobility spectrometry with an LLOQ of 0.03 ng/mg. Lastly, and of great interest, is the work by Zhu et al. (10) that presents a microfluidic chip based on nano liquid chromatography coupled to tandem mass spectrometry (nano-HPLC–Chip-MS-MS) for the determination of abused drugs and metabolites in 2 mg of human hair, reporting an LLOQ of 0.0005 ng/mg. Regarding miniaturized procedures, only SPME and LPME have been described in order to determine EDDP and methadone in hair specimens, all of them resulting in greater LLOQs than ours. SPME differs from MEPS on the diffusion of the analytes mediated by stirring, in the first, or flow-through, in the second (54). Aleksa et al. (38) proposed the simultaneous detection of 17 drugs of abuse and metabolites in 10 mg of hair using headspace SPME (HS-SPME) and GC–MS achieving an LLOQ of 0.6 ng/mg for methadone. The authors used the most commonly applied 100-μm polydimethylsiloxane (PDMS) fiber, a non-polar fiber adding yet another level of selectivity and a reduction in background noise. Merola et al. (39) proposed the same miniaturized technique and fiber to determine different recreational drugs in 10 mg of hair with GC–MS obtaining an LLOQ of 0.16 ng/mg. Also, Musshoff et al. (5) with the same technique and fiber studied the dose–concentration relationships of EDDP and methadone in 10 mg of hair belonging to patients on a maintenance program. The authors reported LLOQs of 0.05 and 0.3 ng/mg for EDDP and methadone, respectively, using a GC–MS. Gentili et al. (40) aimed at a rapid screening procedure based on HS-SPME (100-μm PDMS) and GC–MS for the detection of many recreational drugs in 20 mg of hair, achieving an LLOQ of 1.05 ng/mg for methadone. A different fiber was, however, used by Sporkert and Pragst (42) for the determination of methadone and EDDP in 10 mg of human hair by HS-SPME and GC–MS. The authors described a clean-up procedure using a 65-μm PDMS/divinylbenzene fiber, obtaining an LLOQ of 0.16 and 0.1 ng/mg for EDDP and methadone, respectively. Lucas et al. (41) used another variant of SPME with direct immersion (DI-SPME) for the determination of methadone and EDDP in 50 mg of human hair by GC–MS. The fiber used was, again, a 100-μm PDMS, achieving LLOQs of 0.36 ng/mg for EDDP and 3.46 ng/mg for methadone. When compared with MEPS, the SPME approach has often been mentioned as hardly suitable for high-throughput applications, mainly because of the long time required to establish equilibrium and the resultant low absolute recoveries obtained. The lower recoveries might justify the greater LLOQs obtained in the works described (54, 59). Other adopted miniaturized technique was the surfactant-enhanced (SE) LPME, described by Yazdi and Es’haghi (37) to help determine basic drugs of abuse in 50 mg of hair. With this technique, the analytes were concentrated through an aqueous solution (donor phase) into an organic liquid immobilized within the pores of 2.0-cm length of polypropylene hollow fiber before they were trapped with the aqueous acceptor phase, contained within the lumen of the porous hollow fiber. The authors reported an LLOQ of 16 ng/mL for methadone when 2 mL of methanol was added to 50 mg of hair in incubation. Intra-day, inter-day and intermediate precision and accuracy The evaluation of the inter-day precision and accuracy was performed within a 5-day period at eight concentration levels, the same levels applied to build the calibration curve. The obtained coefficients of variation (CVs) were typically lower than 8% for methadone at the tested concentration levels with an accuracy within a ±12% interval, except for the LLOQ, for which an accuracy within a ±20% interval was obtained. Regarding EDDP, the CVs observed were typically lower than 11%, with exception of LLOQ (lower than 16%). The accuracy was within a ±5% interval for all tested concentration levels. The intra-day precision and accuracy were evaluated by the analysis, on the same day, of six replicates of blank hair samples spiked at four concentration levels, LLOQ included. The observed CVs were lower than 9 and 13% for EDDP and methadone, respectively, at all studied concentrations, both analytes measurements resulting in a mean RE within ±9%. Lastly, intermediate (combined intra- and inter-day) precision and accuracy were also evaluated with the help of quality control (QC) samples at three concentration levels (0.035, 0.75 and 3.5 ng/mg). This study involved the preparation of the QC (n = 3) samples and their simultaneous analysis with the calibration curves on the 5-day period (n = 15). The measurement of EDDP QCs resulted in CVs typically lower than 10% and accuracy within ±10% interval. Regarding methadone, the obtained CVs were usually equal or lower than 9% with an accuracy within ±11% interval. All data are shown in Table III. Table III Inter- and intra-day and intermediate precision and accuracy Analyte . Spiked . Inter-day precision and accuracy (n = 5) . Intra-day precision and accuracy (n = 6) . Intermediate precision and accuracy (n = 15) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . EDDP 0.01 0.01 ± 0.002 16.00 4.08 0.01 ± 0.004 4.72 −8.84 0.025 0.025 ± 0.003 10.27 0.61 0.026 ± 0.002 6.91 5.91 0.035 0.035 ± 0.003 9.54 0.72 0.05 0.05 ± 0.002 3.85 −4.25 0.1 0.10 ± 0.003 2.63 −3.49 0.5 0.51 ± 0.015 3.01 1.25 0.48 ± 0.035 7.33 −4.49 0.75 0.75 ± 0.064 8.45 0.54 1 1.03 ± 0.008 0.77 3.40 2.5 2.46 ± 0.262 10.67 −1.78 3.5 3.83 ± 0.193 5.05 9.24 5 5.01 ± 0.259 5.18 0.19 5.13 ± 0.424 8.27 2.58 Methadone 0.01 0.01 ± 0.0001 0.58 19.55 0.01 ± 0.001 12.09 2.24 0.025 0.026 ± 0.002 7.38 5.99 0.026 ± 0.002 9.32 2.95 0.035 0.034 ± 0.003 9.00 −4.01 0.05 0.05 ± 0.002 4.72 −8.98 0.1 0.09 ± 0.004 4.97 −11.28 0.5 0.51 ± 0.009 1.74 2.56 0.54 ± 0.039 7.25 8.63 0.75 0.67 ± 0.023 3.42 −10.57 1 0.92 ± 0.034 3.65 −7.77 2.5 2.42 ± 0.055 2.29 −3.24 5 5.16 ± 0.035 0.69 3.16 5.37 ± 0.260 4.83 7.49 3.58 ± 0.203 5.66 2.34 Analyte . Spiked . Inter-day precision and accuracy (n = 5) . Intra-day precision and accuracy (n = 6) . Intermediate precision and accuracy (n = 15) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . EDDP 0.01 0.01 ± 0.002 16.00 4.08 0.01 ± 0.004 4.72 −8.84 0.025 0.025 ± 0.003 10.27 0.61 0.026 ± 0.002 6.91 5.91 0.035 0.035 ± 0.003 9.54 0.72 0.05 0.05 ± 0.002 3.85 −4.25 0.1 0.10 ± 0.003 2.63 −3.49 0.5 0.51 ± 0.015 3.01 1.25 0.48 ± 0.035 7.33 −4.49 0.75 0.75 ± 0.064 8.45 0.54 1 1.03 ± 0.008 0.77 3.40 2.5 2.46 ± 0.262 10.67 −1.78 3.5 3.83 ± 0.193 5.05 9.24 5 5.01 ± 0.259 5.18 0.19 5.13 ± 0.424 8.27 2.58 Methadone 0.01 0.01 ± 0.0001 0.58 19.55 0.01 ± 0.001 12.09 2.24 0.025 0.026 ± 0.002 7.38 5.99 0.026 ± 0.002 9.32 2.95 0.035 0.034 ± 0.003 9.00 −4.01 0.05 0.05 ± 0.002 4.72 −8.98 0.1 0.09 ± 0.004 4.97 −11.28 0.5 0.51 ± 0.009 1.74 2.56 0.54 ± 0.039 7.25 8.63 0.75 0.67 ± 0.023 3.42 −10.57 1 0.92 ± 0.034 3.65 −7.77 2.5 2.42 ± 0.055 2.29 −3.24 5 5.16 ± 0.035 0.69 3.16 5.37 ± 0.260 4.83 7.49 3.58 ± 0.203 5.66 2.34 All concentrations in ng/mg; CV—Coefficient of variation; RE—Relative error [(measured concentration-spiked concentration/spiked concentration)] x 100; Mean values ± standard deviation. Open in new tab Table III Inter- and intra-day and intermediate precision and accuracy Analyte . Spiked . Inter-day precision and accuracy (n = 5) . Intra-day precision and accuracy (n = 6) . Intermediate precision and accuracy (n = 15) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . EDDP 0.01 0.01 ± 0.002 16.00 4.08 0.01 ± 0.004 4.72 −8.84 0.025 0.025 ± 0.003 10.27 0.61 0.026 ± 0.002 6.91 5.91 0.035 0.035 ± 0.003 9.54 0.72 0.05 0.05 ± 0.002 3.85 −4.25 0.1 0.10 ± 0.003 2.63 −3.49 0.5 0.51 ± 0.015 3.01 1.25 0.48 ± 0.035 7.33 −4.49 0.75 0.75 ± 0.064 8.45 0.54 1 1.03 ± 0.008 0.77 3.40 2.5 2.46 ± 0.262 10.67 −1.78 3.5 3.83 ± 0.193 5.05 9.24 5 5.01 ± 0.259 5.18 0.19 5.13 ± 0.424 8.27 2.58 Methadone 0.01 0.01 ± 0.0001 0.58 19.55 0.01 ± 0.001 12.09 2.24 0.025 0.026 ± 0.002 7.38 5.99 0.026 ± 0.002 9.32 2.95 0.035 0.034 ± 0.003 9.00 −4.01 0.05 0.05 ± 0.002 4.72 −8.98 0.1 0.09 ± 0.004 4.97 −11.28 0.5 0.51 ± 0.009 1.74 2.56 0.54 ± 0.039 7.25 8.63 0.75 0.67 ± 0.023 3.42 −10.57 1 0.92 ± 0.034 3.65 −7.77 2.5 2.42 ± 0.055 2.29 −3.24 5 5.16 ± 0.035 0.69 3.16 5.37 ± 0.260 4.83 7.49 3.58 ± 0.203 5.66 2.34 Analyte . Spiked . Inter-day precision and accuracy (n = 5) . Intra-day precision and accuracy (n = 6) . Intermediate precision and accuracy (n = 15) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . Measured . CV (%) . RE (%) . EDDP 0.01 0.01 ± 0.002 16.00 4.08 0.01 ± 0.004 4.72 −8.84 0.025 0.025 ± 0.003 10.27 0.61 0.026 ± 0.002 6.91 5.91 0.035 0.035 ± 0.003 9.54 0.72 0.05 0.05 ± 0.002 3.85 −4.25 0.1 0.10 ± 0.003 2.63 −3.49 0.5 0.51 ± 0.015 3.01 1.25 0.48 ± 0.035 7.33 −4.49 0.75 0.75 ± 0.064 8.45 0.54 1 1.03 ± 0.008 0.77 3.40 2.5 2.46 ± 0.262 10.67 −1.78 3.5 3.83 ± 0.193 5.05 9.24 5 5.01 ± 0.259 5.18 0.19 5.13 ± 0.424 8.27 2.58 Methadone 0.01 0.01 ± 0.0001 0.58 19.55 0.01 ± 0.001 12.09 2.24 0.025 0.026 ± 0.002 7.38 5.99 0.026 ± 0.002 9.32 2.95 0.035 0.034 ± 0.003 9.00 −4.01 0.05 0.05 ± 0.002 4.72 −8.98 0.1 0.09 ± 0.004 4.97 −11.28 0.5 0.51 ± 0.009 1.74 2.56 0.54 ± 0.039 7.25 8.63 0.75 0.67 ± 0.023 3.42 −10.57 1 0.92 ± 0.034 3.65 −7.77 2.5 2.42 ± 0.055 2.29 −3.24 5 5.16 ± 0.035 0.69 3.16 5.37 ± 0.260 4.83 7.49 3.58 ± 0.203 5.66 2.34 All concentrations in ng/mg; CV—Coefficient of variation; RE—Relative error [(measured concentration-spiked concentration/spiked concentration)] x 100; Mean values ± standard deviation. Open in new tab Recovery Recoveries of the clean-up step for EDDP and methadone were studied by comparing two sets of samples. The first set involved blank hair extracts spiked with the target compounds, prior to MEPS procedure, while in the second set of samples, the spike only occurred after the MEPS procedure. This study was performed at three concentration levels: 0.035, 0.75 and 4 ng/mg (n = 3). The ISs were added to both sets only after MEPS procedure. The ratio of the relative peak areas obtained in the first set with those obtained for the second set allowed the calculation of the mean recoveries. Overall, the recoveries obtained with the MEPS procedure were very good, ranging from 84 to 110% for EDDP and 73 to 109% for methadone (Table IV). Table IV Recovery (%) of EDDP and methadone under the optimized MEPS procedure (n = 3) Analyte . Concentration (ng/mg) . 0.035a . 0.75a . 4a . EDDP 84.22 ± 7.74 99.33 ± 6.44 110.69 ± 10.63 MTD 84.16 ± 8.42 73.17 ± 7.31 109.21 ± 9.43 Analyte . Concentration (ng/mg) . 0.035a . 0.75a . 4a . EDDP 84.22 ± 7.74 99.33 ± 6.44 110.69 ± 10.63 MTD 84.16 ± 8.42 73.17 ± 7.31 109.21 ± 9.43 a Mean values ± standard deviation. Open in new tab Table IV Recovery (%) of EDDP and methadone under the optimized MEPS procedure (n = 3) Analyte . Concentration (ng/mg) . 0.035a . 0.75a . 4a . EDDP 84.22 ± 7.74 99.33 ± 6.44 110.69 ± 10.63 MTD 84.16 ± 8.42 73.17 ± 7.31 109.21 ± 9.43 Analyte . Concentration (ng/mg) . 0.035a . 0.75a . 4a . EDDP 84.22 ± 7.74 99.33 ± 6.44 110.69 ± 10.63 MTD 84.16 ± 8.42 73.17 ± 7.31 109.21 ± 9.43 a Mean values ± standard deviation. Open in new tab The recovery values are comparable to those reported by Concheiro et al. (26) who used LLE followed by SPE with Strata X cartridges and obtained recoveries greater than 85% for methadone. The same extraction techniques were adopted by Lendoiro et al. (18) resulting in recoveries ranging from 102 to 106% for methadone. Girod et al. (31) used the classic technique SPE with Isolute HCX cartridges for hair analysis of human subjects following methadone maintenance program, reporting recoveries between 80 and 86% for both methadone and EDDP. Also, Barroso et al. (27) presented recoveries from 90 to 99% for both compounds, using SPE Oasis® MCX cartridges. Slightly lower recoveries, even still comparable, were those presented by Moeller et al. (23) with SPE Chromabond C18 cartridges, 70–80% for EDDP and methadone. Regarding LLE extractions, similar results to the herein presented were obtained by Bermejo et al. (21), who used ToxiTubes A® for the simultaneous determination of methadone, heroin and metabolites in hair, obtaining recoveries of 85% for methadone. The same technique was used by Wilkins et al. (22) for a quantitative analysis of methadone and two major metabolites in hair, resulting, however, in lower recoveries, 70% for both compounds. Considering the above, it is fair to assume that the proposed MEPS procedure is a quite efficient technique to be applied on routine hair samples clean-up. It can undoubtedly assure great recoveries for methadone and EDDP, being in accordance with green chemistry standards, such as the environmentally friendly procedure due to lower organic solvents consumption. If compared with other miniaturized techniques described for the same goal, the present work results in similar recoveries to those shown by Gentili et al. (40) who used HS-SPME with PDMS 100-μm fiber and obtain recoveries of 98% for methadone. Additionally, Lucas et al. (41) adopted DI-SPME for the determination of methadone and EDDP in human hair and achieved recoveries between 102 and 107% for EDDP and methadone, respectively. Also, Yazdi and Es’haghi (37) proposed a clean-up method for basic drugs of abuse in hair with SE-LPME and showed recoveries from 89 to 93% for methadone. However, as previously mentioned, SPME has also been known for its resultant low absolute recoveries. This is observed in the work of Merola et al. (39) that applied HS-SPME to determine different recreational drugs in hair reporting a recovery of 9.5% for methadone. Sporkert and Pragst (42) also used HS-SPME, although with a different fiber, aiming at the determination methadone and its metabolites in human hair, and achieve recoveries of 10.5–14.5% for both EDDP and methadone. Lastly, Lachenmeier et al. (57) described a method with headspace solid-phase dynamic extraction for the determination of drugs of abuse in hair samples and obtained recoveries of 16 and 23.5% for EDDP and methadone, respectively. When comparing the proposed MEPS method in this work with other miniaturized techniques, specially SPME, the present work can be associated with good recoveries, high sensitivity, low carry over and low cost. On the other hand, SPME is commonly known for its low recovery, low sensitivity, high carry over and great costs associated (54). Stability Hair samples differ from other human specimens, such as blood or urine, used for toxicological analysis, due to its solid and durable nature, strong tissue, being less affected by adulterants (6, 60). Once drugs are incorporated into the hair, they remain fixed as hair grows, and for this reason, the sample can be collected and stored at room temperature (61). Nevertheless, after hair samples extraction through incubation and after the adopted clean-up MEPS procedure, the extracts should reveal enough stability over the anticipated run time for batch size. In this sense, it is important to evaluate the so-called stability of processed samples, also known as auto-sampler stability. This study was performed at the QC concentration levels (n = 3) by the re-analysis of these samples after a period of 24 h unassisted in the auto-sampler. Their concentrations were determined on the basis of a newly prepared calibration curve on the day of re-analysis. Both EDDP and methadone presented a good stability in the extracts over the period of 24 h. The results obtained on the stability assay are shown on Table V. The CVs obtained were typically lower than 10% with RE within a ±7% interval for EDDP, while methadone is measured with CVs commonly lower than 14% and with an RE within ±8% interval. The latter assures the possibility of a re-analysis after 24 h in the auto-sampler with no significant change in the concentration determination of the two target compounds. Table V Analyte stability in processed samples (n = 3) Analyte . Spiked . Measured . CV (%) . RE (%) . EDDP 0.035 0.037 ± 0.003 9.56 5.70 0.75 0.700 ± 0.069 9.92 −6.61 3.5 3.693 ± 0.286 7.76 5.51 Methadone 0.035 0.037 ± 0.003 6.94 5.98 0.75 0.692 ± 0.059 8.50 −7.73 3.5 3.505 ± 0.464 13.25 0.14 Analyte . Spiked . Measured . CV (%) . RE (%) . EDDP 0.035 0.037 ± 0.003 9.56 5.70 0.75 0.700 ± 0.069 9.92 −6.61 3.5 3.693 ± 0.286 7.76 5.51 Methadone 0.035 0.037 ± 0.003 6.94 5.98 0.75 0.692 ± 0.059 8.50 −7.73 3.5 3.505 ± 0.464 13.25 0.14 All concentrations in ng/mg; CV—Coefficient of variation; RE—Relative error [(measured concentration-spiked concentration/spiked concentration)] x 100; Mean values ± standard deviation. Open in new tab Table V Analyte stability in processed samples (n = 3) Analyte . Spiked . Measured . CV (%) . RE (%) . EDDP 0.035 0.037 ± 0.003 9.56 5.70 0.75 0.700 ± 0.069 9.92 −6.61 3.5 3.693 ± 0.286 7.76 5.51 Methadone 0.035 0.037 ± 0.003 6.94 5.98 0.75 0.692 ± 0.059 8.50 −7.73 3.5 3.505 ± 0.464 13.25 0.14 Analyte . Spiked . Measured . CV (%) . RE (%) . EDDP 0.035 0.037 ± 0.003 9.56 5.70 0.75 0.700 ± 0.069 9.92 −6.61 3.5 3.693 ± 0.286 7.76 5.51 Methadone 0.035 0.037 ± 0.003 6.94 5.98 0.75 0.692 ± 0.059 8.50 −7.73 3.5 3.505 ± 0.464 13.25 0.14 All concentrations in ng/mg; CV—Coefficient of variation; RE—Relative error [(measured concentration-spiked concentration/spiked concentration)] x 100; Mean values ± standard deviation. Open in new tab Method applicability Method applicability was verified by the analysis of authentic hair samples obtained from two individuals undergoing methadone treatment program at Centro de Atendimento ao Toxicodependente—Casas de Santiago (Belmonte, Portugal). As example, Figure 4 represents the chromatogram obtained from the analysis of one of those samples, positive for EDDP and methadone with measured concentrations of 0.11 and 0.37 ng/mg, respectively. Figure 4 Open in new tabDownload slide Chromatogram of authentic hair sample positive for EDDP and methadone. Figure 4 Open in new tabDownload slide Chromatogram of authentic hair sample positive for EDDP and methadone. The same two specimens were additionally assessed as described by Barroso et al. (27) who used a mixed-mode SPE for hair samples clean-up to determine EDDP and methadone. The results obtained by the reproduction of Barroso et al. method were, then, compared with those obtained in the present work, and the obtained concentrations were similar (the resulting CVs were lower than 5% for EDDP and 9% for methadone). Thus, the proposed MEPS procedure may be considered as a great alternative to SPE, due to lower solvent volumes consumption and the possibility of sorbent reusage (approximately 100 samples clean-up). Conclusions This work describes the application of MEPS for the determination of methadone and EDDP in hair by GC–MS-MS. The MEPS procedure was optimized, and the analytical method fully validated. Overall, the procedure has proven to be simple with an ease and fast operation, selective, precise and accurate. Additionally, MEPS resulted in great recoveries (73–110%), high sensitivity, low carry over and low cost. The method was linear between 0.010 and 5 ng/mg for both compounds, with the LLOQ assured at 0.010 ng/mg, and was successfully applied to the analysis of hair samples from patients undergoing a methadone maintenance program. The developed method results in the first work to couple MEPS to GC–MS-MS for the determination of methadone and EDDP in hair samples, resulting in a great alternative to the classic clean-up techniques, such as LLE and SPE. Acknowledgments The authors acknowledge the European Regional Development Fund (FEDER) through the POCI-COMPETE 2020—Operational Programme Competitiveness and Internationalization in Axis I—Strengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491) and National Funds by Fundação para a Ciência e a Tecnologia (UID/Multi/00709/2019). T. Rosado acknowledges the Centro de Competências em Cloud Computing in the form of a fellowship C4_WP2.6_M1—Bioinformatics; Operação UBIMEDICAL—CENTRO-01-0145-FEDER-000019—C4—Centro de Competências em Cloud Computing, supported by Fundo Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional Regional Centro (Centro 2020). References 1. Marsh , A. , Evans , M.B. ( 1994 ) Radioimmunoassay of drugs of abuse in hair. Part 1: methadone in human hair, method adaptation and the evaluation of decontamination procedures . Journal of Pharmaceutical and Biomedical Analysis , 12 , 1123 – 1130 . 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TI - Microextraction by Packed Sorbent as a Novel Strategy for Sample Clean-Up in the Determination of Methadone and EDDP in Hair
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkaa040
DA - 2020-12-12
UR - https://www.deepdyve.com/lp/oxford-university-press/microextraction-by-packed-sorbent-as-a-novel-strategy-for-sample-clean-sNrXUUXQL0
SP - 840
EP - 850
VL - 44
IS - 8
DP - DeepDyve
ER -