TY - JOUR
AU - Carmo, Helena
AB - Abstract The quantification of drugs of abuse in keratinized matrices is becoming of special relevance for monitoring consumption and for post-mortem investigations. We aimed to implement an analytical method for the simultaneous detection of morphine (MORF), 6-monoacetylmorphine (6-MAM), methadone (MET), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrrolidine (EMDP) in nails. After decontamination, the nail samples (30 mg) were submitted to an alkaline digestion followed by a two-step liquid–liquid and SPE extraction using mixed-mode cation exchange cartridges. The analytes were eluted with 5% NH4OH/methanol. After derivatization with N-methyl-N-(trimethylsilyl) trifluoroacetamide, the analytes were quantified by gas chromatography-mass spectrometry. The method was optimized and fully validated only for MET, EDDP and EMDP, since for MOR and 6-MAM it was not possible to obtain adequate recovery rates after extraction, although detection of MOR was still possible. The method was selective, accurate and precise. Regression analysis demonstrated linearity over a concentration range of 20.8–333.3 ng/mg for MET and 10.4–166.7 ng/mg for EDDP and EMDP. Limits of detection and quantification values ranged from 3.3 to 6.0 ng/mg and 10.4 to 20.8 ng/mg, respectively, and recovery rates ranged from 82% to 98%. The applicability of the method was demonstrated by analyzing nail and urine samples obtained from heroin consumers under substitution therapy with MET. Introduction Besides forensic purposes, the analytical monitoring of drugs of abuse and/or prescribed medications with abuse potential is determinant for the control and prevention of drug abuse. The drug stability in biological matrices, the toxicokinetic profile of the substances, and even post-mortem redistribution phenomena, are fundamental parameters that must be considered for these toxicological analyses and data interpretation. The final purpose of the laboratorial analysis will determine the choice of the analytical method, as well as the most suitable biological matrix to be tested. Opioids are among the most frequently monitored drugs and many analytical methods were already developed for their quantification in biological matrices, mainly for forensic, but also for clinical purposes. Blood and urine are the conventional matrices for such analyses but both have the major drawback of enabling detection for only a limited period of time. Keratinized matrices, including hair and nails, have emerged as alternatives or as a complement to the use of the conventional matrices since they can stably accumulate some drugs, enabling monitoring for extended periods (1). For this reason, these matrices are now regarded as having exceptional forensic and clinical interest. There are several methods already reported for the quantification of opioids in hair, but considerably fewer in nails. In the case of methadone (one of the opiates most frequently administered for the therapy of heroin abuse), there are currently no methods for the simultaneous determination of the parent drug and both metabolites in nails. Such a method could be useful to widen the time and probability of detection of the drug and of compliance with the drug treatment. We have therefore developed and validated an analytical method for the simultaneous quantification of methadone (MET) and the metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrrolidine (EMDP) (2) in nails. A brief review addressing the most relevant issues for the detection of drugs of abuse, and of opioids in particular, in nails, is also presented herein. Opioid Abuse Drug overdose is the primary contributor to the global number of drug-related deaths, and opioids (including heroin and the non-medical use of prescription opioids) are the main drug type implicated in those deaths, with 43,000 deaths attributed to opioid abuse in 2010 (3). The 2016 and 2017 World drug reports flag the alarming rise in heroin use in some regions of the world. In 2014, an estimated 914,000 people aged 12 years or older had used heroin in the past year, worldwide, representing a 145% increase since 2007, while mortality related to heroin use has increased 5-fold since 2000 (4, 5). Europe has experienced different waves of heroin addiction, the first affecting many western countries from the mid-1970s and a second wave affecting other countries especially those in central and Eastern Europe in the mid to late 1990s. From 2010/11 new recruitment into heroin use seems to be declining (6). Heroin itself or its metabolites are present in the majority of fatal overdoses reported in Europe, often in combination with other substances (6). Also, opiates continue to be the drugs most commonly associated with the more harmful forms of use, including injecting drug use (6). The estimated number of high-risk opioid users in Europe in 2014 was 1.3 million. In 2015, opioids were detected in the majority of drug-related deaths in Portugal, with heroin mentioned in 18 out of a total number of 40 cases registered by the National Institute of Forensic Medicine (6, 7). In our country, 48% of treatment entrants in specialized drug treatments were attributed to heroin abuse. (6). Due to its extensive oral bioavailability and long elimination time, methadone has been long used to treat heroin abuse. The use of this drug has greatly contributed to reduce mortality while maintaining abstinence or at least a significant reduction of heroin consumption. This in turn also results in decreased criminal activities and risk of acquiring drug-related infectious diseases (8). Use of Nails in the Analytical Monitoring of Drugs of Forensic Interest In several countries, the inclusion in detoxification treatments implies a mandatory urine analysis to detect drug abuse withdrawal and treatment compliance. At detoxification treatment centers, health technicians frequently use point-of-care immunoassays methodologies such as the enzyme multiplied immunoassay technique (EMIT) to analyze drug levels in urine (9). However, poor limits of detection and cross-reactivity due to other drugs and/or endogenous substances are major drawbacks and therefore, gas (GC) or liquid (LC) chromatographic separation followed by mass spectrometry (MS) detection are much preferred (10, 11). These confirmation analysis are usually not available at the detoxification treatment centers, and urine samples are easily adulterated enabling a limited and short retrospective analysis period. In these analytical settings keratinized matrices, including hair and nails can be considered highly advantageous (11). Over the past years, quantitative analysis of drugs of abuse in keratinized matrices including hair and nails has emerged as a complement to the analysis of conventional matrices (blood and urine) (12). These matrices have the great advantages of: (i) enabling a much wider time-window for detection, (ii) the non-invasive collection and (iii) easiness of storage and transport conditions at room temperature (1). External contamination, low incorporation of test drugs and/or metabolites, and limited amount of sample are however, major drawbacks (13, 14). These matrices are becoming specially relevant for post-mortem investigations and particularly useful for drug analysis in corpses at an advanced state of decomposition. Another analytical scenario where these type of samples can be extremely helpful is for the screening of intrauterine exposure to drugs of abuse in newborns, to help diagnose withdrawal syndromes and predict possible deleterious effects on the physical and mental development of children (14, 15). Nails incorporate drugs via the nail matrix, a highly proliferative epidermal tissue, or the nail bed, a noncornified tissue below the keratinized nail plate (14). However, exposure to environmental contamination and biological fluids including sweat, sebum, saliva and urine are also considered possible ways for drug incorporation in nails (14). The extent of nail incorporation is also largely determined by the characteristics of the drug, namely the molecular weight, lipophilia, dissociation constant, bioavailability and route of administration (14, 16). Unlike hair, nails grow continuously without resting phases, which can be an advantage in terms of exposure monitoring (13). In addition to the forensic interest, the use of nails in quantifying various compounds extends to the determination of clinically used drugs including, antifungal drugs (14) and metals in cases of environmental/occupational exposure (17). The quantification of trace elements on nails by ICP-MS was also recognized to be clinically useful for the diagnosis and prevention of chronic diseases, including cancer, diabetes and cardiovascular diseases, since the excess or the deficiency of these elements may be associated with risk of developing such diseases (18). The nail quantification of fetal steroids in infants has been proposed as a promising non-invasive and retrospective biomarker of intrauterine exposure to maternal stress (19). The possibility to detect drugs of abuse in nails was first reported by Suzuki and co-workers, in 1984, for the GC-MS quantification of methamphetamine in frequent users of the drug (20). Since then, several methods have been proposed for the analysis of different drugs in nails. Among the most frequently used analytical methods for drug and/or medications with abuse potential in nails are GC and LC coupled with MS. Such methods have been successfully applied to: (i) amphetamines and ketamine (20–26); (ii) opiates, cocaine and major metabolites (12, 15, 27–40); (iii) cannabinoids (22, 41, 42); (iv) sedatives and antipsychotic drugs (13, 43–46); (v) steroids (19, 47–49); (vi) ethylglucuronide (50–54); among others (17, 18, 55–65) (Table S1). For the monitoring of intrauterine exposure to drugs of abuse, GC-MS methods that allowed the non-invasive quantification of cocaine, benzoylecgonine, morphine, methadone, caffeine, nicotine and cotinine in the nails of newborns have been developed (15). Use of Nails in the Analytical Monitoring of Opioids Compared to the number of analytical methods proposed for the detection of drugs of abuse and/or major metabolites in blood, urine, tissue and hair, there are only a few methods developed for their quantification in nails. Therefore, there is great interest in developing new methods that can foster the application of nail analysis, namely opioids such as heroin and methadone. A summary of the methods reporting the quantification of opioids in nails is presented in Table I. In humans, opiates undergo extensive metabolism (Figure 1). Heroin rapidly undergoes deacetylation yielding 6-monoacetylmorphine (6-MAM). The 6-MAM metabolite undergoes a slower deacetylation into morphine (66). Morphine is further metabolized into the active metabolite morphine-6-glucuronide (M6G) and to the inactive morphine-3-glucuronide (M3G) and to a minor extent, by N-demethylation, to normorphine (66). Methadone is mainly metabolized in the liver (Figure 2) and the main reaction involved is a N-demethylation which produces the inactive metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) which is subsequently demethylated into 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP) (8). Due to the extended half-life of the metabolites, their determination is particularly useful for forensic purposes but also to help monitor the compliance with detoxification treatments and to ascertain abstinence from drug abuse. We therefore aimed at validating an analytical method that could help monitor individuals undergoing substitution therapy with methadone by simultaneously quantifying the parent drug and its two major metabolites EDDP and EMDP. Table I. Methods reporting the quantification of opioids in nails Analyte  Sample  Decontamination  Hydrolysis  Extraction  Derivatization  Analytical method  LOD (ng/g)  LOQ (ng/g)  Reference  6-MAM  fingernails toenails  2 × MeOH  MeOH, 4 h, sonication    PFPA  GC-CI-MS  100    (38)  MORF  100  Codeine  200  MORF  toenails  3 × MeOH  0.1 M PB (pH = 5); 1 h, sonication + 72 h, RT  SPE  MSTFA  GC-EI-MS    0.3 ng  (30)  6-MAM  Codeine  Hydrocodone  MORF  fingernails  1 × 0,1% SDS 3 × H2O 3 × MeOH  NaOH (1 M); 60°C, 1–2 h  L/L    RIA HPLC  50    (34)  MET  fingernails  3 × 0,1% SDS 3 × H2O 3 × MeOH  NaOH (1 M); 90°C, 30–40 min  SPE    EIA GC-EI-MS  10    (33)  5      Codeine  fingernails  1 × iPrOH, 3 × PB (pH = 6)  Enzyme digestion 40°C, overnight  SPE  BSTFA/1% TMCS  GC-EI-MS    100  (12)  MORF  fingernails toenails  3 × MeOH  0.1 M PB (pH = 5); 1 h sonication + 72 h RT  L/L  MSTFA  GC-EI-MS    0.1 ng  (29)  6-MAM  Codeine  Hydromorphone  Oxycodone  Hydrocodone  MORF  toenails  2 × DCM  HCl (37%); 100°C, 30 min + 12hRT  L/L  Propionic anhydride  GC-EI-MS    100  (28)  6-MAM  MORF  fingernails toenails  1 MeOH  HCl (0,1 M); overnight  SPE  BSTFA/1%  GC-EI-MS  25  25  (15)  MET  TMCS  50  6-MAM  fingernails  2 × H2O 2 × Acetone  BB (pH = 9.2), 30 min, RT; sonication  L/L    LC-MS-MS  10  50  (36)  MORF  20  50  Codeine  30  50  Acetylcodeine  10  50  Heroin  10  50  25 opiates  fingernails  Acetone  HCl (0,1 M); 53°C, overnight  SPE    LC-MS-MS    40  (37)  Buprenorphine  fingernails  2 × H2O 2 × CHCl3 2 × MeOH  NaOH (1 M); 55°C, 3 h; sonication  L/L    LC-ESI-MS  2.3  8.9  (39)  Norbuprenorphine  5.5  20.7  Naloxone  8.1  27.0  MET  fingernails toenails  1 × H2O 2 × Acetone    S/L    UHPLC-MS  3.8  6.6  (32)  EDDP  4.5  5.3  (76 drugs including several opiates)      6-MAM  fingernails  1 × H2O 1 × Acetone    S/L    LC-MS-MS    50  (27)  MORF  toenails  50  Codeine              50  Methadone  50  EDDP  50  Analyte  Sample  Decontamination  Hydrolysis  Extraction  Derivatization  Analytical method  LOD (ng/g)  LOQ (ng/g)  Reference  6-MAM  fingernails toenails  2 × MeOH  MeOH, 4 h, sonication    PFPA  GC-CI-MS  100    (38)  MORF  100  Codeine  200  MORF  toenails  3 × MeOH  0.1 M PB (pH = 5); 1 h, sonication + 72 h, RT  SPE  MSTFA  GC-EI-MS    0.3 ng  (30)  6-MAM  Codeine  Hydrocodone  MORF  fingernails  1 × 0,1% SDS 3 × H2O 3 × MeOH  NaOH (1 M); 60°C, 1–2 h  L/L    RIA HPLC  50    (34)  MET  fingernails  3 × 0,1% SDS 3 × H2O 3 × MeOH  NaOH (1 M); 90°C, 30–40 min  SPE    EIA GC-EI-MS  10    (33)  5      Codeine  fingernails  1 × iPrOH, 3 × PB (pH = 6)  Enzyme digestion 40°C, overnight  SPE  BSTFA/1% TMCS  GC-EI-MS    100  (12)  MORF  fingernails toenails  3 × MeOH  0.1 M PB (pH = 5); 1 h sonication + 72 h RT  L/L  MSTFA  GC-EI-MS    0.1 ng  (29)  6-MAM  Codeine  Hydromorphone  Oxycodone  Hydrocodone  MORF  toenails  2 × DCM  HCl (37%); 100°C, 30 min + 12hRT  L/L  Propionic anhydride  GC-EI-MS    100  (28)  6-MAM  MORF  fingernails toenails  1 MeOH  HCl (0,1 M); overnight  SPE  BSTFA/1%  GC-EI-MS  25  25  (15)  MET  TMCS  50  6-MAM  fingernails  2 × H2O 2 × Acetone  BB (pH = 9.2), 30 min, RT; sonication  L/L    LC-MS-MS  10  50  (36)  MORF  20  50  Codeine  30  50  Acetylcodeine  10  50  Heroin  10  50  25 opiates  fingernails  Acetone  HCl (0,1 M); 53°C, overnight  SPE    LC-MS-MS    40  (37)  Buprenorphine  fingernails  2 × H2O 2 × CHCl3 2 × MeOH  NaOH (1 M); 55°C, 3 h; sonication  L/L    LC-ESI-MS  2.3  8.9  (39)  Norbuprenorphine  5.5  20.7  Naloxone  8.1  27.0  MET  fingernails toenails  1 × H2O 2 × Acetone    S/L    UHPLC-MS  3.8  6.6  (32)  EDDP  4.5  5.3  (76 drugs including several opiates)      6-MAM  fingernails  1 × H2O 1 × Acetone    S/L    LC-MS-MS    50  (27)  MORF  toenails  50  Codeine              50  Methadone  50  EDDP  50  6-MAM, 6-monoacetylmorphine; BSTFA, N,O-Bis-(Trimethylsilyl)-trifluoroacetamide; CI, chemical ionization; EDDP, 1,5-dimethyl-3,3-diphenylpyrrolidine; EI, electron impact ionization; EIA, enzyme immunoassay; ESI, electron spray ionization; GC, Gas chromatography; iPrOH, 2-propanol; L/L, liquid/liquid extraction; LOD, Limit of detection; LOQ, Limit of quantification; LC, Liquid chromatography; MeOH, Methanol; MS, mass spectrometry; MET, methadone; MORF, morphine; MS(-MS), tandem mass spectrometry; MSTFA, N-Methyl-N-(trimethylsilyl)trifluoroacetamide; PB, Phosphate buffer; PFPA, Pentafluoropropionic anhydride; RIA, Radioimmunoassay; RT, Room temperature; SDS, sodium dodecyl phosphate; S/L, solid/liquid extraction; SPE, Solid phase extraction; TMCS, Trimethylchlorosilane; UHPLC, Ultra-high performance liquid chromatography. Figure 1. View largeDownload slide Main metabolic pathways for heroin. Figure 1. View largeDownload slide Main metabolic pathways for heroin. Figure 2. View largeDownload slide Main metabolic pathways for methadone. Figure 2. View largeDownload slide Main metabolic pathways for methadone. Materials and Methods Chemicals and reagents All chemicals and reagents were of analytical grade or of the highest grade commercially available. Methadone (MET), 2-ethylidene-1,5-dimethyl-3,3-difenilpirrolidine (EDDP), 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), morphine (MOR), 6-monoacetylmorphine (6-MAM) and ethylmorphine (internal standard, IS) were obtained from Lipomed (Arlesheim, Switzerland). Methanol (HPLC grade), n-hexane, N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and Type HP-2 β-glucuronidase from Helix pomatia were obtained from Sigma-Aldrich (St Louis, MO, USA). Ammonium hydroxide (NH4OH) (25%), methyl tert-butyl ether (MTBE) (99.5%), hydrochloric acid (HCl) (32%), sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS), ethyl acetate, sodium acetate and potassium dihydrogen phosphate were acquired from Merck (Darmstadt, Germany). The solid phase extraction (SPE) was carried out with OASIS® (MCX) columns, 10 mg, with 1 mL capacity, obtained from Waters (Milford, MA, USA) and using the device Supleco TM Visiprep SPE vacuum manifold obtained from Sigma-Aldrich. Helium (99.99%) was obtained from Gasin, Portugal. Collection of nail and urine samples Control nail and urine samples used for the optimization and validation of the analytical method were obtained from adult volunteers, recruited at our research center, of both genders, who had never consumed drugs of abuse. At the time of sample collection, these volunteers were also not taking any prescribed drug. Control fingernails and urine samples obtained from volunteers of both genders and from both left and right hands were pooled before storage until analysis. The fingernails from both left and right hands and urine test samples were obtained from volunteers of the Addicts Treatment Centre (CAT) of Cedofeita—Porto, Portugal, with documented prior use of heroin and undergoing substitution therapy with MET. All individuals participating in the study provided written consent after a brief explanation of the stages of the study and its main objectives. Each participant also filled a very simple and concise questionnaire aimed at obtaining demographic data (age, gender, ethnics) and drug consumption patterns (heroin abuse pattern, date/time from last abuse, alcohol and tobacco habits, other drugs of abuse intake, date from first MET treatment and dosage regimen). The confidentiality of the data was assured, as samples and questionnaires were always identified by number and never by name. For the collection of nail samples, volunteers previously washed their hands with soap and water and then with the help of a commercially available cosmetic nail clipper, proceeded to the removal of small nail fragments in the presence of a member of the research team. For this study, fingernails were preferred due to the easier collection of the sample. Differences between finger and toenail drug concentrations are known and while in some cases concentrations in fingernails are higher, in other cases the opposite occurs (14). After collection, the nail fragments were conditioned in a plastic bag that was hermetically sealed until laboratory analysis at room temperature. Urine samples were obtained through collection into sterile recipients. In the case of the test samples, all urine collections occurred within minutes after the daily intake of the MET dose. The urine samples were stored at −20°C until analysis. The study was conducted in strict accordance to the ethical principles of the Declaration of Helsinki. The protocol was reviewed and approved by the Ethical Committee of the Faculty of Pharmacy of the University of Porto. All volunteers participating in the study provided written informed consent. The study protocol was also reviewed and approved by the Coordinator of Treatment at the Addicts Treatment Centre (CAT) of Cedofeita—Porto, Portugal that allowed the sample collection at their facilities. Preparation of stock and working standard solutions All stock solutions were prepared in methanol at a concentration of 1 mg/mL and stored at −20°C until use. Intermediate concentrations were also prepared by successively diluting the respective concentrated solutions in methanol and stored at −20°C until use. For the simulation of the matrix, nail hydrolysates were obtained as described below and spiked with the appropriate amounts of standard solutions to obtain the range of the test concentrations (0; 312.5; 625; 1,250; 2,500; 5,000 ng/mL for MET and 0; 156; 312.5; 625; 1,250; 2,500 ng/mL for EDDP and EMDP, which corresponded to 0; 20.8; 41.7; 83.3; 166.7; 333.3 ng/mg nail for MET and 0; 10.4; 20.8; 41.7; 83.3; 166.7 ng/mg nail for EDDP and EMDP). Additionally, nail hydrolysates spiked with MORF and 6-MAM were also prepared to obtain the final test concentrations of 0; 30; 60; 120; 280; 480 ng/mL. To the spiked nail hydrolysates, 15 μL of ethylmorphine (IS; 100 μg/mL) were added to obtain a final IS concentration of 1.5 μg/mL. Sample Preparation for GC-MS Analysis Nail sample decontamination External contamination of keratinized samples is one of the major limitations to the use of these alternative matrices. However, by using adequate decontamination steps this limitation can be easily overcome. The nail samples were decontaminated in the ultrasonic bath by successively washing with 5 mL of an aqueous detergent solution (0.1% SDS; 2 × 15 min, followed by 1 × 30 min), 5 mL deionized water (2 × 15 min, followed by 1 × 30 min) and finally with 5 mL of methanol (2 × 15 min) (Figure 3A). For each decontamination step, the washing solution was always discarded and replaced by fresh solution after each cycle in the ultrasounds bath. This decontamination protocol was adapted from previous protocols that reported an efficient removal of external contaminants (33, 67). Figure 3. View largeDownload slide Analytical procedure adopted in this study for samples of nails. (A) Decontamination of nail samples. (B) hydrolysis (alkaline). (C) Liquid–Liquid Extraction. (D) Solid Phase Extraction. (E) Derivatization. SDS, sodium dodecyl sulfate; NaOH, sodium hydroxide; MTBE, methyl tert-butyl ether; MCX, mixed-mode cation exchange; HCl, hydrochloric acid; NH4OH, ammonium hydroxide; MSTFA, N-methyl-N-(trimethylsilyl) trifluoroacetamide. Figure 3. View largeDownload slide Analytical procedure adopted in this study for samples of nails. (A) Decontamination of nail samples. (B) hydrolysis (alkaline). (C) Liquid–Liquid Extraction. (D) Solid Phase Extraction. (E) Derivatization. SDS, sodium dodecyl sulfate; NaOH, sodium hydroxide; MTBE, methyl tert-butyl ether; MCX, mixed-mode cation exchange; HCl, hydrochloric acid; NH4OH, ammonium hydroxide; MSTFA, N-methyl-N-(trimethylsilyl) trifluoroacetamide. Alkaline hydrolysis and dissolution of the nail fragments The hydrolyzed matrix can be obtained with alkaline, acidic or methanolic digestion protocols. During the development of this method several possibilities were tested including an acidic digestion by incubating the nail fragments with 1 mL 0.1 M HCl during 16 h at 40°C in a ultrasonic bath, followed by neutralization with 1 mL 1 M NaOH and 0.1 M phosphate buffer (pH = 7). This procedure was efficient for all analytes tested except for the heroin metabolite 6-MAM that was degraded under such conditions. The alkaline hydrolysis was therefore preferred. After the decontamination of the samples, the nail fragments were dried at room temperature, cut into small fragments and incubated (40–60 min) with 1 mL of 1 M NaOH at 90°C until they were fully dissolved. The alkaline hydrolysates obtained from 30 mg control nail sample constituted the blank analysis matrices that were spiked for the optimization and validation of the analytical method (Figure 3B). Extraction Procedure of the Analytes Liquid–liquid extraction The nail hydrolysate obtained is a complex matrix, with many potentially interfering substances and with high viscosity. If applied directly onto a SPE column for drug extraction the obtained eluates present a turbidity that will after the derivatization step produce turbid derivatizates that cannot be injected into the GC-MS. Therefore, previously to the SPE extraction of the nail hydrolysates a liquid/liquid extraction at acidic pH was performed to obtain cleaner extracts. For this purpose, several extraction solvents were tested including n-hexane/ethyl acetate (9 : 1, 8 : 2 and 7 : 3) and chloroform:2-propanol that were mixed with the previously acidified hydrolysates followed by centrifugation at 4°C for phase separation. After at least three extractions the organic phases were discarded and the hydrolysates were then further submitted to the extraction with the SPE columns. However, the turbidity of the obtained eluates persisted. An alternative liquid/liquid extraction was then tested which involved the prior extraction of the test analytes into an organic phase. For this purpose 1 mL of MTBE was added to a volume of 500 μL of alkaline hydrolysate diluted with 500 μL of 0.1 M phosphate buffer (pH 6; 13.6 g/L KH2PO4). After vigorous vortex mixing and centrifugation (4,000 rpm, 5 min, 4°C) the organic phase was collected. This procedure was repeated three times sequentially. Finally, the organic phases were dried under a stream of nitrogen and the extract was dissolved in 3 mL of phosphate buffer (pH = 6) before the SPE extraction (Figure 3C). With this procedure the extracts were no longer turbid nor were the subsequent derivatizates. However, the extraction efficiencies for MOR and 6-MAM were very low (below 20%). Therefore, although detection was still possible, the method could not be further validated for MOR and 6-MAM. Solid phase extraction For further clean-up of the extracts, SPE was performed using the Supelco Visiprep SPE vacuum manifold apparatus and OASIS® MCX 1cc columns. The columns were conditioned with 1 mL methanol and 1 mL of deionized water. Then, the samples (the reconstituted extracts obtained from working standards, blank and test samples) were transferred into the columns followed by two sequential washes with 2 × 1 mL 0.1 M HCl and 2 × 1 mL methanol. Finally, the analytes of interest were eluted into a glass tube using 2 × 1 mL of a 5% NH4OH/methanol solution. The obtained eluates were evaporated to dryness on a hot plate set at 30°C under a stream of nitrogen. To eliminate residual water all tubes were dried under reduced pressure over P2O5 and KOH and left open in the desiccator overnight (Figure 3D). During the method development, additional washing steps with deionized water and with trimethylamine were tested but resulted in a significant loss of sensitivity. Also other eluents were tested, including the elution with dicloromethane:2-propanol:NH4OH (78:20:2), which is reported for opioids for other analytical matrices (11), but these also performed worse than the 5% NH4OH/methanol eluent that was used in the validated procedure. To assure that the 2 mL of eluent were sufficient to elute all analytes from the column a third elution with 1 mL of the eluent was additionally tested but no analytes could be found in this third fraction. Derivatization procedure Prior to the chromatographic analysis the derivatization of the analytes may be necessary to increase volatility and chromatographic performance. Given the chemical nature of methadone and main metabolites, the most common derivatization reactions involve either perfluoroacylation and/or trimethylsilylation. For opioids, the most frequently used derivatization reagents are N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS), N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and pentafluoropropionic anhydride (PFPA) with pentafluoropropanol (PFPOH) (11, 68, 69). We used the following derivatization procedure: 60 μL of MSTFA were added to the dried residue obtained after the extraction, the tubes were then vortexed vigorously and heated for 30 min at 80°C. After cooling to room temperature, the derivatized samples were again vortex-mixed and injected into the GC-MS equipment (Figure 3E). Urine samples For the pre-treatment of urine samples the protocol previously described for the nail alkaline hydrolysates was adopted. However, for urine samples, before proceeding to the liquid–liquid extraction, an enzymatic hydrolysis was performed by adding 500 μL of 0.1 M phosphate buffer (pH 6) to 500 μL of urine sample spiked with 15 μL IS, 1,000 μL of 0.2 M sodium acetate buffer (pH = 5.2) and 50 μL of β-glucuronidase. The samples were then incubated overnight at 37°C before extraction. Analytical instrument settings The derivatized samples were analyzed by gas chromatography (GC Trace™ 2000 Series ThermoQuest) with detection by mass spectrometry with an ion-trap type detector (ThermoQuest Finnigan GCQ Plus). The chromatographic separation was performed on a capillary column Agilent® VF-5 ms (30 m × 0.25 mm × 0.25 mM) using helium as carrier gas with C-60 high purity at a constant flow rate of 1 mL/min. The temperatures of the quadrupole and ion source were 150°C and 230°C, respectively. One μL of each sample was injected into the injector in splitless mode at 280°C. The oven temperature program for the chromatographic separation was: 80°C, held for 1 min, increasing to 300°C at a rate of 10°C/min. The total run time was 28 min. All acquisitions were in full scan mode, allowing full detection of ions in the range of m/z 50–650. For quantification, the selective ion monitoring mode (SIM) was used, in which m/z fragments were pre-selected according to their specificity and relative abundance, thereby affording increased sensitivity. The identification of each analyte in the samples was achieved by comparing their retention times and mass spectra with those of standards injected under the same chromatographic conditions. Fragments (m/z) selected for quantification are presented in Table II. Table II. GC-MS parameters for the detection of the analytes Analyte  Retention time (min)  Ions (m/z)a  MORF  21.87  324/414/429  6-MAM  22.42  204/287/340/399  MET  18.78  73/223/294  EDDP  17.79  220/262/277  EMDP  16.86  115/130/165/193/208  Ethylmorphine (IS)  21.74  146/192/385  Analyte  Retention time (min)  Ions (m/z)a  MORF  21.87  324/414/429  6-MAM  22.42  204/287/340/399  MET  18.78  73/223/294  EDDP  17.79  220/262/277  EMDP  16.86  115/130/165/193/208  Ethylmorphine (IS)  21.74  146/192/385  aIons used for quantification in the SIM mode. Method validation and acceptance criteria The validation of the analytical method was performed according to the international standards recommended by the European Medicines Agency (70). The parameters studied to ensure that the method produces reproducible and reliable results were linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy and recovery. All working calibrators were prepared by spiking blank nail hydrolysates with the appropriate amounts of methanolic standard solutions of each analyte that were subsequently submitted to the pre-treatment procedure summarized in Figure 3. Method linearity The linearity was obtained by calculating the linear regression (ratio of analyte peak area and IS peak area versus analyte concentration) and the respective correlation coefficient (r2) of five independent calibration curves (y = mx + b) obtained from six different working calibrators for MET (0; 312.5; 625; 1,250; 2,500; 5,000 ng/mL; 20.8–333.3 ng/mg) and EDDP and EMDP (0; 156; 312.5; 625; 1,250; 2,500 ng/mL; 10.4–166.7 ng/mg). The mean slopes were obtained to calculate the concentrations of the test samples. Linearity was accepted at r2 > 0.99. Limits of detection and quantification The limit of detection (LOD) and the limit of quantification (LOQ) were determined by analysing successive dilutions of the least concentrated working calibrator of each analyte. For well-defined and symmetrical chromatographic peaks, a signal-to-noise ratio of 3 was considered acceptable for estimating the LOD and a signal-to-noise ratio of 10 was deemed acceptable for the LOQ. Peaks that were excessively broad, showed tailing or shoulders, did not resolve to within 10% baseline or if the relative % of the calibration ions were not maintained were not considered. The lowest concentrations of the analytes found to fit these criteria were injected five times onto the GC-IT/MS instrument. For the LOQ imprecision ≤20% was accepted. Precision and accuracy Precision was assessed by calculating the mean, standard deviation and coefficient of variation (% CV) of the obtained results. For all precision determinations three different concentrations of spiked control samples, spanning the linear dynamic range of the method of each analyte were always tested: 312.5, 1,250, 5,000 ng/mL for MET, and 156, 625, 2,500 ng/mL for EDDP and EMDP. The intra-day precision of the GC-MS apparatus was determined by analysing three times, on the same day, the three different concentrations of MET, EDDP and EMDP spiked control samples. The intra-day precision of the extraction method was calculated by extracting five times, on the same day, three different concentrations of MET, EDDP and EMDP spiked control samples. The inter-day precision of the extraction method was determined on five different days, by extracting three different spiked control samples at three different concentrations of MET, EDDP and EMDP on each day. A CV value ≤15% was considered acceptable. The accuracy of the method (A%) was investigated by the standards addition of low, medium and high concentration spiked control samples (312.5, 1,250, 5,000 ng/mL for MET, and 156, 625, 2,500 ng/mL for EDDP and EMDP). The percent deviation between the calculated value obtained from the calibration curve and the nominal value was determined [accuracy (%) = (mean calculated concentration/nominal concentration)×100]. A CV value ≤15% was considered acceptable. Extraction recovery Recovery of each analyte was assessed at three different concentrations (312.5; 1,250 and 5,000 ng /mL for MET and 156; 625 and 2,500 ng/mL EDDP and EMDP) by adding the analytes to one set of low, medium and high concentration control samples before extraction and to a second set after extraction but before evaporation to dryness. The procedure was repeated five times and the recovery was calculated by comparing the peak area ratios of analyte to IS for extracted and non-extracted samples. Analytical recovery between 80% and 120% was considered acceptable. The recovery of the IS was independently tested at the 1.5 μg/mL concentration. The calculated recovery percentage for ethylmorphine, after five independent determinations, was of 98.38% ± 1.18% (CV% = 1.2). Evaluation of interferences and specificity To evaluate interference and method specificity, several different pooled blank nail hydrolysates (no analyte or IS added) were evaluated for co-eluting peaks that might interfere with the detection of analytes of interest or IS. Three independent analysis were performed with at least three replicates of pooled blank nail hydrolysates. Proof of applicability Nail samples obtained from ten patients with prior documented use of heroin, that were currently under methadone-substitution treatment were analyzed with the implemented method. Of these, six individuals also provided urine samples at the time of the collection of the nail samples. These urines were analyzed using the method as described for the nail alkaline hydrolysates after enzymatic hydrolysis. Although a validation of the method was not performed for the urine matrix, adequate linearity was assured within the detected concentration range. Results and Discussion Sample preparation Although the analytical procedures should be kept as simple as possible to increase efficiency and reduce cost and time consumption, due to the complexity of the analytical matrix, several steps that included the decontamination, hydrolysis, and two sequential extraction protocols of the analytes from the nail samples had to be implemented during the development of the method. In spite of the multiple steps involved in the sample preparation prior to the GC-IT/MS injection the good results obtained with the reproducibility studies assured the final accurate results as can be seen below. Although the entire procedure of sample preparation and chromatographic run can be considered long and time-consuming, using a less complex protocol would significantly compromise the data quality due to the turbidity of the extracts and of the corresponding derivatives. Gas chromatographic separation The adopted GC conditions resulted in well resolved peaks eluting in less than 30 min. The acquisition in Full Scan mode guaranteed the identification of the target peaks in each chromatogram. This mode also allows the detection of other chromatographic peaks of potential interest and later identified in the test samples. On the other hand the use of specific ions for the integration allowed more precise peak integration which is specially important for small peaks. The use of splitless mode also increased sensitivity without compromising the column overloading and peak resolution. The first analyte of interest to elute is EMDP at 16.86 min, followed by EDDP at 17.79. Parent drug methadone elutes at 18.78 min followed by the IS at 21.74 min as can be depicted in the representative chromatogram shown in Figure 4A. Figure 5 shows the chromatogram of a working calibrator with MET, EMDP and EDDP metabolites, and also MORF and 6-MAM. It is therefore possible to detect MOR that elutes at 21.87 min. However, with the validated extraction protocol 6-MAM that elutes at 22.42 min, could not be easily detected in the full scan chromatograms. Using SIM mode this analyte can be detected but with this extraction the recovery is extremely limited (<20%). Figure 4. View largeDownload slide Reconstructed GC-EI/MS selective ion monitoring mode chromatogram for EMDP, EDDP and MET (2.5 μg/mL) and IS (1.5 μg/mL) (A) and of a blank sample fortified with only IS (B). Figure 4. View largeDownload slide Reconstructed GC-EI/MS selective ion monitoring mode chromatogram for EMDP, EDDP and MET (2.5 μg/mL) and IS (1.5 μg/mL) (A) and of a blank sample fortified with only IS (B). Figure 5. View largeDownload slide Reconstructed GC-EI/MS selective ion monitoring mode chromatogram for EMDP, EDDP, MET, MOR and 6-MAM (0.96 μg/mL) and IS (1.5 μg/mL). Figure 5. View largeDownload slide Reconstructed GC-EI/MS selective ion monitoring mode chromatogram for EMDP, EDDP, MET, MOR and 6-MAM (0.96 μg/mL) and IS (1.5 μg/mL). Quantification procedure and choice of suitable IS Several chromatographic methods have been developed for opiates in different analytical matrices but methods for analysis in nails are fewer. Among these methods, GC-MS techniques are frequently adopted since they seem to provide high sensitivity for the target analytes (Table I). The selection of an adequate IS is mandatory to limit systematic errors that will hamper the precision and reproducibility of the method. The deuterated analogs are often preferred (33) but are usually expensive. Given the similarity of ethylmorphine with our target analytes and since it is frequently used in opioid quantification methods (11) we tested this IS that performed well in our extraction methods (mean recovery 98.38% ± 1.18%; CV% = 1.2) as well as in the derivatization step and presented a suitable retention time. Its unlikely presence in the analytical matrix makes this choice useful for clinical and forensic purposes. Method Validation Selectivity Several blank hydrolysates were analyzed to evaluate chromatographic interference (Figure 4B). No interference peaks were detected, neither at the retention time of the analytes nor at the IS retention time. All working calibrators were prepared with blank hydrolysates to mimic real analytical conditions and matrix complexity. Linearity Regression analysis of calibration data achieved satisfactory linearity over a wide concentration range (0–333.3 ng/mg for MET and 0–166.7 ng/mg for EDDP and EMDP). Square determination coefficients (r2) were always higher than 0.99, indicating a linear relationship from six-point calibration curves. The linearity data are shown in Table III. These data show that the linearity achieved for methadone in this method is higher than that observed with the methods by Lemos et al. (33) and Mari et al. (15) that reported r2 values of 0.945 and 0.9799 for a concentration range of 1–25 ng/mg and 0.05–0.4 ng/mg, respectively. A recent LC-MS/MS method reported a linearity range of 0.05–20 ng/mg for both methadone and EDDP (27). To the best of our knowledge there are no methods reporting validation data for the quantification of the metabolite EMDP. Table III. Linear regression analysis from five independent calibration curves   Linearity  Equation (y = mx + b)  Concentration range  r2  ng/mL  ng/mg    MET  y = 0.0005x – 0.0215  312.5–5,000  20.8–333.3  0.9995  EDDP  y = 0.0006x – 0.064  156–2,500  10.4–166.7  0.994  EMDP  y = 0.0004x + 0.0031  156–2,500  10.4–166.7  0.9968    Linearity  Equation (y = mx + b)  Concentration range  r2  ng/mL  ng/mg    MET  y = 0.0005x – 0.0215  312.5–5,000  20.8–333.3  0.9995  EDDP  y = 0.0006x – 0.064  156–2,500  10.4–166.7  0.994  EMDP  y = 0.0004x + 0.0031  156–2,500  10.4–166.7  0.9968  Sensitivity LOQ and LOD were determined using the SIM mode (Table IV). The LOQ and LOD values range from 3.3 to 6.0 ng/mg, and 10.4 to 20.8 ng/mg, respectively. The methods reported by Mari et al. and by Lemos et al. have significantly lower LOD and LOQ values in the range of 0.005–0.05 ng/mg for MET (Table I) (15, 33). The LC-MS/MS method reported by Capelle at al, obtained a LOQ of 0.05 ng/mg for both MET and EDDP (27). Although our method is less sensitive, probably due to the more complex protocol for the sample preparation, we can still detect low amounts of methadone in our test samples that are within the range of the concentrations detected in drug abusers under methadone replacement therapy. This was confirmed in the present study and also conformed with the previous findings of Lemos et al. in which nail levels of methadone were below 3.3 ng/mg for only 9 out of 30 cases (33). We are additionally able to monitor both major metabolites enabling a wider window for detection and therefore minimizing to some extent the loss of sensitivity for the parent drug, again providing evidence of the applicability of the method for clinical and forensic purposes. Table IV. LOD and LOQ values   Sensitivity  (n = 5)  LOD (ng/mg)  LOQ (ng/mg)  MET  3.3 (7.8)  20.8 (8.5)  EDDP  6.0 (8.4)  10.4 (8.7)  EMDP  6.0 (3.7)  10.4 (6.4)    Sensitivity  (n = 5)  LOD (ng/mg)  LOQ (ng/mg)  MET  3.3 (7.8)  20.8 (8.5)  EDDP  6.0 (8.4)  10.4 (8.7)  EMDP  6.0 (3.7)  10.4 (6.4)  (CV%) Precision and Accuracy This GC-IT/MS method also showed satisfactory (CV < 15%) intra- and inter-assay precision. The intra-day precision of the extraction method was estimated based upon five independent manipulations of three different concentrations. The CV% values ranged from 1.3% to 10.9%. (Table V). The intra-day precision of the apparatus was determined after three injections of three different extracts. The CV% values ranged from 3.4% to 8.9% (Table V). Table V. Precision, accuracy and recovery data   Intra-day precision (CV%)  Inter-day precision (CV%)  Accuracy (%)  Recovery (%)  Concentration (ng/mL)  GC/MS apparatus  Extraction method        METH   312.5  4.2  8.5  8.1  97.5 (14.6)  98 (1.0)   1,250  5.6  9.7  10.7  104.0 (14.9)  89 (7.4)   5,000  6.5  10.9  10.8  102.3 (9.6)  91 (2.7)  EDDP             156  5.8  9.9  13.6  105.0 (10.1)  96 (9.2)   625  8.9  7.9  8.8  96.4 (14.7)  82 (4.8)   2,500  4.1  10.1  10.4  105.6 (10.2)  86 (1.6)  EMDP             156  8.5  9.6  5.8  112.8 (11.0)  85 (2.7)   625  3.4  1.3  5.3  93.4 (14.9)  98 (1.6)   2,500  7.4  9.5  12.8  98.5 (8.9)  91 (3.9)    Intra-day precision (CV%)  Inter-day precision (CV%)  Accuracy (%)  Recovery (%)  Concentration (ng/mL)  GC/MS apparatus  Extraction method        METH   312.5  4.2  8.5  8.1  97.5 (14.6)  98 (1.0)   1,250  5.6  9.7  10.7  104.0 (14.9)  89 (7.4)   5,000  6.5  10.9  10.8  102.3 (9.6)  91 (2.7)  EDDP             156  5.8  9.9  13.6  105.0 (10.1)  96 (9.2)   625  8.9  7.9  8.8  96.4 (14.7)  82 (4.8)   2,500  4.1  10.1  10.4  105.6 (10.2)  86 (1.6)  EMDP             156  8.5  9.6  5.8  112.8 (11.0)  85 (2.7)   625  3.4  1.3  5.3  93.4 (14.9)  98 (1.6)   2,500  7.4  9.5  12.8  98.5 (8.9)  91 (3.9)  (CV%) Inter-day precision and accuracy of the method The results for inter-day precision of the method were obtained at three different levels of concentrations within the linear range, with spiked control samples independently extracted and injected in five different days. The obtained CV% values were always lower than 15% (Table V), as recommended by the EMA (2011) and ranged from 5.3% to 13.6%. The accuracy, calculated as the percentage of the added target concentration ranged between 96.4% and 112.8%. The proposed acceptance limits for this parameter were 100 ± 20%, and the obtained accuracy results were within these limits for the three analytes (Table V). Extraction recovery At three different concentration levels, spanning the linear dynamic range of the assay, the recovery ranged from 82% to 98% (Table V). The values obtained are within the acceptance limits proposed for this parameter (100 ± 20%) (EMA, 2011). The CV% values ranged from 1.0% to 9.2%. Proof of applicability Samples of nails and urine were obtained from 10 caucasian individuals (3 females and 7 males, aged 32–52 years old), with documented prior use of heroin and undergoing substitution therapy with MET. Demographic data, pattern of heroin abuse and methadone dose/frequency of administration are presented in Table VI. All test samples were processed and analyzed by the method developed and validated as described above. The obtained data are shown in Table VII. It is clear from these data that the extension of deposition of methadone and main metabolites in nails is not directly related with dose or frequency of administration. It could be expected that individuals with higher dosages and frequency of administration would present higher MET levels but this was not the case (e.g., cases 1 and 11 present similar methadone levels around 13 ng/mg but doses are markedly different: 40 mg and 100 mg, respectively). Instead, a high inter-individual variability was found among our test samples. The low number of samples (n = 10) and the fact that the mechanism of MET deposition in nails is not fully understood can help explain the disparity of the data. High variability is expected due to physiological, biochemical and pharmacokinetic differences. For example, older individuals as well as those with hepatic or renal pathologies may have decreased drug metabolism and elimination. Gender differences are also likely to occur in respect to drug absorption, distribution, metabolism and excretion. Increased treatment time would also be expected to produce higher deposition of MET and/or main metabolites in nails. Again no such correlation could be found in these individuals. With the exception of case 14, all volunteers had been treated with methadone for several years (range between 3 and 10 years). However, it was not clear from the interviews if the patients fully complied with the methadone treatment over the years after the first therapeutic intervention. This is also a probable reason for the noted variability. A similar data dispersion and absence of dose-concentration relationship was reported in a study that involved 29 adult patients attending the methadone-maintenance clinic of the Edinburgh Drug Addiction Study, where a range of 0–362.5 ng/mg was observed for methadone (33). Levels of metabolites in nails were not reported in that study. Table VI. Demographic data and drug use pattern from individuals with a previous history of heroin abuse and currently under methadone-substitution therapy   Heroin    Methadone    Code  Age  Sex  Time since last use (years)  Route  Frequency (per day)  Other drugs  Nail products/drug treatments  Time since first dose (years)  Dose (mg)  Frequency (per day)  Urine  1*  52  M  7  IV Inhaled  3 ×  Hax, Coc  No  7  40  1 ×  Yes  2*  39  M  3  IV  3 ×    No  3  160  1 ×  Yes  4  43  M  1  IV  3 ×  Hax  No  7/8  100  1 ×  No  8  44  F  3      Hax  No  3  10  1 ×  Yes  9**  33  M  12–24 h  Inhaled  2 ×  Hax  No  10  60  1 ×  Yes  10  52  M  10        No  10  80  1 ×  Yes  11**  46  M  24–48 h  IV Inhaled  1 ×  Coc  No  3  100  1 ×  No  12  32  M  3        No  10  20  1 ×  No  13  44  F  20        No  4  45  1 ×  Yes  14**  43  F  12 h  Inhaled  1 ×  EtOH  No  12–24 h  130  2 ×  Yes    Heroin    Methadone    Code  Age  Sex  Time since last use (years)  Route  Frequency (per day)  Other drugs  Nail products/drug treatments  Time since first dose (years)  Dose (mg)  Frequency (per day)  Urine  1*  52  M  7  IV Inhaled  3 ×  Hax, Coc  No  7  40  1 ×  Yes  2*  39  M  3  IV  3 ×    No  3  160  1 ×  Yes  4  43  M  1  IV  3 ×  Hax  No  7/8  100  1 ×  No  8  44  F  3      Hax  No  3  10  1 ×  Yes  9**  33  M  12–24 h  Inhaled  2 ×  Hax  No  10  60  1 ×  Yes  10  52  M  10        No  10  80  1 ×  Yes  11**  46  M  24–48 h  IV Inhaled  1 ×  Coc  No  3  100  1 ×  No  12  32  M  3        No  10  20  1 ×  No  13  44  F  20        No  4  45  1 ×  Yes  14**  43  F  12 h  Inhaled  1 ×  EtOH  No  12–24 h  130  2 ×  Yes  Coc-cocaine; EtOH-ethanol; F-female; Hax- hashish; IV-intravenous; M-male. *sample collection immediately after methadone administration; **individuals with recent heroin abuse. Table VII. Concentration of methadone and metabolites found in nail and urine samples from individuals with a previous history of heroin abuse and currently under methadone-substitution therapy   Concentration  Matrix  Sample  MET  EDDP  EMDP  EDDP/MET  EMDP/MET  MORF  Nails (ng/mg)  1  26.23  18.3  6.6a  0.70  0.25    2  78.0  44.7  15.8  0.57  0.20    4  46.1  15.1  ND  0.33      8  7.5a  ND  ND        9  15.3a  14.2  ND  0.93    D  10  14.1a  32.1  ND  2.28      11  26.0  ND  ND      ND  12  16.0a  ND  6.1a    0.38    13  18.8a  ND  11.2    0.60    14  17.6a  ND  23.8    1.36  ND  Urine (μg/mL)  1  17.9  1.4  0.5  0.08  0.03    2  384.9  76.8  3.2  0.2  0.01    8  38.9  7.5  2.2  0.19  0.06    9  45.8  2.4  ND  0.05    D  13  177.9  13.1  ND  0.07      14  61.0  114.5  1.0  1.88  0.02  ND    Concentration  Matrix  Sample  MET  EDDP  EMDP  EDDP/MET  EMDP/MET  MORF  Nails (ng/mg)  1  26.23  18.3  6.6a  0.70  0.25    2  78.0  44.7  15.8  0.57  0.20    4  46.1  15.1  ND  0.33      8  7.5a  ND  ND        9  15.3a  14.2  ND  0.93    D  10  14.1a  32.1  ND  2.28      11  26.0  ND  ND      ND  12  16.0a  ND  6.1a    0.38    13  18.8a  ND  11.2    0.60    14  17.6a  ND  23.8    1.36  ND  Urine (μg/mL)  1  17.9  1.4  0.5  0.08  0.03    2  384.9  76.8  3.2  0.2  0.01    8  38.9  7.5  2.2  0.19  0.06    9  45.8  2.4  ND  0.05    D  13  177.9  13.1  ND  0.07      14  61.0  114.5  1.0  1.88  0.02  ND  D-detected; ND-not detected. aBelow LOQ. External contamination has also been pointed out as a probable explanation for data variability in determinations in keratinized matrices including nail and hair samples. We applied an extensive decontamination protocol that had previously demonstrated to overcome this limitation (33). It is accepted that the successive washes with detergent and methanol solutions are highly efficient in limiting the influence of external contamination in the quality of the data (14). However, this cannot be ruled out, as well as the variability in nail incorporation of drug that is eliminated in sweat (32). Furthermore, although we did not validate the method for urine, the detected urinary levels also showed great inter-individual variability that does not seem to be lower for this matrix when compared to nails, but our urine sample number (n = 6) is even lower than for the tested nails. The parent drug/metabolite ratios also differed significantly among the different individuals. This was noted both in nails and in urine. This could be attributed to the high variability in the nail deposition of the target analytes and is also most certainly due to an inter-individual variability in the extent of drug metabolism and excretion. To the best of our knowledge there is only one study in the literature that reports the ratio of MET/EDDP found in the nails of three post-mortem whole nail samples that were split into segments before analysis (32). The authors also report variability in the parent drug/metabolite ratio among the three cases. The relative metabolic profile of MET varies according to the analytical matrix. While much higher concentrations of the parent drug are expected in blood, saliva, sweat or hair, urinary levels of EDDP metabolite are frequently higher than those of methadone with much lower amounts of EMDP (71). In subjects on MET maintenance, up to 33% of the dose is excreted as unchanged drug and up to 43% as EDDP, while EMDP accounts for 5–10% of the dose. Urinary excretion is largely determined by urinary pH, being increased in acidic urine (72). Our data also show that EDDP is the major urinary metabolite but a wide variation in the parent drug/metabolite ratio can be observed among our participants. Conclusion Our method is clearly less sensitive than other reported in the literature but it should be considered that it enabled the successful and more robust detection of methadone and metabolites in all suspected samples and is therefore suitable for monitoring compliance and abstinence of patients enrolled in methadone-maintenance programs. However, this could be a serious limitation in cases as those reported by Mary and co-workers that determined methadone levels in the nails of newborns exposed during pregnancy. They found extremely low MET levels that ranged from 0.12 to 0.26 ng/mg (15), which could be expected since the nail incorporation of the drug from the fetal blood occurs only after the drug reaches the placenta through the maternal blood. Other analytical methods as is the case of UHPLC-triple quadrupole-mass analyzers offer great sensitivity but are not as easily available, specially at detoxification centers or other point-of-care facilities. The costs and equipment involved are also higher than with the presently proposed method. The review of the literature herein presented also shows that there are few analytical methods available but none addressed the quantification of both methadone metabolites. We therefore believe that the present method may be of value for forensic and clinical applications. Supplementary Data Supplementary data are available at Journal of Analytical Toxicology online. Acknowledgments The authors wish to express their sincere gratitude to the Coordinators Adelaide Gomes and Joana Barroso Coutinho, and to all the staff of the Addicts Treatment Centre (CAT) of Cedofeita—Porto, Portugal, for their support in the study design and for providing all the necessary conditions for sample collection. Funding This work was supported by the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. The study is a result of the project NORTE-01-0145-FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBI-OtecHealth—New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF). 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TI - Quantification of Methadone and Main Metabolites in Nails
JF - Journal of Analytical Toxicology
DO - 10.1093/jat/bkx099
DA - 2018-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/quantification-of-methadone-and-main-metabolites-in-nails-kfAsSZAW6S
SP - 192
EP - 206
VL - 42
IS - 3
DP - DeepDyve
ER -