Analytical Approaches for the Determination of Buprenorphine, Methadone and Their Metabolites in Biological Matrices.

The abuse of buprenorphine and methadone has grown into a rising worldwide issue. After their consumption, buprenorphine, methadone and their metabolites can be found in the human organism. Due to the difficulty in the assessment of these compounds by routine drug screening, the importance of developing highly sensitive analytical approaches is undeniable. Liquid chromatography tandem mass spectrometry is the preferable technique for the determination of buprenorphine, methadone and their metabolites in biological matrices including urine, plasma, nails or oral fluids. This research aims to review a critical discussion of the latest trends for the monitoring of buprenorphine, methadone and their metabolites in various biological specimens.

Ultra-High Performance Liquid Chromatography-High Resolution Mass Spectrometry and High-Sensitivity Gas Chromatography-Mass Spectrometry Screening of Classic Drugs and New Psychoactive Substances and Metabolites in Urine of Consumers.

The use of the new psychoactive substances is continuously growing and the implementation of accurate and sensible analysis in biological matrices of users is relevant and fundamental for clinical and forensic purposes. Two different analytical technologies, high-sensitivity gas chromatography-mass spectrometry (GC-MS) and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) were used for a screening analysis of classic drugs and new psychoactive substances and their metabolites in urine of formed heroin addicts under methadone maintenance therapy. Sample preparation involved a liquid-liquid extraction. The UHPLC-HRMS method included Accucore™ phenyl Hexyl (100 × 2.1 mm, 2.6 µm, Thermo, USA) column with a gradient mobile phase consisting of mobile phase A (ammonium formate 2 mM in water, 0.1% formic acid) and mobile phase B (ammonium formate 2 mM in methanol/acetonitrile 50:50 (v/v), 0.1% formic acid) and a full-scan data-dependent MS2 (ddMS2) mode for substances identification (mass range 100-1000 m/z). The GC-MS method employed an ultra-Inert Intuvo GC column (HP-5MS UI, 30 m, 250 µm i.d, film thickness 0.25 µm; Agilent Technologies, Santa Clara, CA, USA) and electron-impact (EI) mass spectra were recorded in total ion monitoring mode (scan range 40-550 m/z). Urine samples from 296 patients with a history of opioid use disorder were examined. Around 80 different psychoactive substances and/or metabolites were identified, being methadone and metabolites the most prevalent ones. The possibility to screen for a huge number of psychotropic substances can be useful in suspected drug related fatalities or acute intoxication/exposure occurring in emergency departments and drug addiction services.

Review of LC techniques for determination of methadone and its metabolite in the biological samples.

Methadone (MTD) is a synthetic analgesic drug used for treating opioid dependence and effectively used clinically for patients with severe pain. The abuse of MTD may lead to poisoning, disorder in the central nervous system and even death. The regular monitoring of MTD in biological matrices including serum, plasma and urine samples is an effective way to control abuse of MTD. In this manner, the selection of analytical monitoring of MTD in biological matrices is of paramount importance. This study was conducted to review high-performance liquid chromatography (HPLC) techniques carried out on MTD and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the biological samples during 2015-June 2021.

Screening for Opioid and Stimulant Exposure In Utero Through Targeted and Untargeted Metabolomics Analysis of Umbilical Cords.

BACKGROUND: Neonatal abstinence syndrome is an array of signs and symptoms experienced by a newborn due to abrupt discontinuation of intrauterine exposure to certain drugs, primarily opioids. In the United States, the incidence of neonatal abstinence syndrome has tripled over the past decade. The current standard of care for drug testing includes the analysis of infant urine and meconium. Sample collection is associated with several limitations, including diaper media interferences, limited sample amount, sample heterogeneity, and the need for professional staff for collection. Umbilical cord tissue has emerged as a convenient sample matrix for testing owing to its universal availability. The purpose of this study was to examine umbilical cords using an untargeted metabolomics approach to determine the detected drugs and validate an analytical method to confirm and quantify the identified drugs. METHODS: A metabolomics analysis was performed with 21 umbilical cords to screen for drugs and drug metabolites by liquid chromatography-mass spectrometry. Drugs were identified using the National Institute of Standards and Technology database, and an analytical method was developed and validated using secondary liquid chromatography-mass spectrometry instrument for positive confirmation and quantitative analysis. RESULTS: Twenty-one random umbilical cords from women were tested: 4 were positive for cocaine and the primary and secondary metabolites; one was positive for methadone, the primary metabolite; 3 were positive for cotinine, the metabolite of nicotine; and 5 were positive for acetyl norfentanyl. CONCLUSIONS: Our research is a prospective method development study using untargeted and targeted approaches to characterize steady-state drug metabolite levels in the umbilical cord matrix at the time of delivery. By characterizing drug type and concentration, this methodology can be used to develop a reliable complementary testing method for meconium toxicology screens.

Methadone-induced encephalopathy: a case series and literature review.

BACKGROUND: Accidental ingestion or consumption of supra-therapeutic doses of methadone can result in neurological sequelae in humans. We aimed to determine the neurological deficits of methadone-poisoned patients admitted to a referral poisoning hospital using brain magnetic resonance (MR) and diffusion weighted (DW) imaging. METHODS: In this retrospective study, brain MRIs of the patients admitted to our referral center due to methadone intoxication were reviewed. Methadone intoxication was confirmed based on history, congruent clinical presentation, and confirmatory urine analysis. Each patient had an MRI with Echo planar T1, T2, FLAIR, and DWI and apparent deficient coefficient (ADC) sequences without contrast media. Abnormalities were recorded and categorized based on their anatomic location and sequence. RESULTS: Ten patients with abnormal MRI findings were identified. Eight had acute- and two had delayed-onset encephalopathy. Imaging findings included bilateral confluent or patchy T2 and FLAIR high signal intensity in cerebral white matter, cerebellar involvement, and bilateral occipito-parietal cortex diffusion restriction in DWI. Internal capsule involvement was identified in two patients while abnormality in globus pallidus and head of caudate nuclei were reported in another. Bilateral cerebral symmetrical confluent white matter signal abnormality with sparing of subcortical U-fibers on T2 and FLAIR sequences were observed in both patients with delayed-onset encephalopathy. CONCLUSIONS: Acute- and delayed-onset encephalopathies are two rare adverse events detected in methadone-intoxicated patients. Brain MRI findings can be helpful in detection of methadone-induced encephalopathy.

Simultaneous chiral separation of tramadol and methadone in tablets, human urine, and plasma by capillary electrophoresis using maltodextrin as the chiral selector.

The stereoselective analysis and separation of racemic drugs play an important role in pharmaceutical industry to eliminate the unwanted isomer and find the right therapeutic control for the patient. Present study suggests a maltodextrin-modified capillary electrophoresis method for a single-run chiral separation of two closely similar opiate pain relief drugs: tramadol (TRA) and methadone (MET). The best separation method possible for the both enantiomers was achieved on an uncoated fused-silica capillary at 25°C using 100 mM phosphate buffer (pH 8.0) containing 20% (w v-1 ) maltodextrin with dextrose equivalent of 4-7 and an applied voltage of 16 kV. Under optimal conditions, the baseline resolution of TRA and MET enantiomers was obtained in less than 12 minutes. The relative standard deviations (n = 3) of 20 µg mL-1 TRA and MET were 2.28% and 3.77%, respectively. The detection limits were found to be 2 µg mL-1 for TRA and 1.5 µg mL-1 for MET. This method was successfully applied to the measurement of drugs concentration in their tablets, urine, and plasma samples.

Characteristics and Outcome of Male and Female Methadone Maintenance Patients: MMT in Tel Aviv and Las Vegas.

To compare in methadone maintenance treatment (MMT) gender characteristics and outcomes, all patients ever admitted to Tel Aviv (TA) MMT clinic (N = 837) (June 1993-December 2014) and Las Vegas (LV) MMT clinic (N = 1256) (February 2000-June 2015) were prospectively followed-up (until June 2016). Drugs in urine on admission and after one year and long-term retention up to 23 and 16 years, respectively, were analyzed. Females in both clinics admitted younger than males and following shorter duration of opioid usage (TA: n = 215, 25.7%, age 35.0 ± 7.9 vs. 40.6 ± 9.8 years, p < .0005, duration 12.4 ± 7.0 vs. 18.1 ± 10 years, p < .0005; LV, n = 494, 39.3%, age 38.0 ± 12.6 vs. 39.2 ± 12.8, p = .08 duration 12.9 ± 11.0 vs. 14.8 ± 12.7 years, p = .008). On admission, higher proportion of female than male had positive urine for cocaine in TA (30.4% vs. 21.8%, p = .02) and for benzodiazepine in LVs (33.9% vs. 26.6%, p = .006). After 1 year, both genders had similar retention rate (TA: 76.1% LV: 49.8%) and opioid abstinence (TA: 67.6%, LV: 74.9%), and cumulative retention (TA: 8.2 years, 95% Confidence-Interval 7.6-8.8; LV 2.2 years, 95% confidence interval 2.0-2.4). CONCLUSIONS: Clinics differed in their characteristics and outcome, however in both clinic similar outcome between genders despite the difference in characteristics on admission was observed, as did the known women "telescoping effect."

Dispersion of magnetic graphene oxide nanoparticles coated with a deep eutectic solvent using ultrasound assistance for preconcentration of methadone in biological and water samples followed by GC-FID and GC-MS.

Magnetic graphene nanoparticles coated with a new deep eutectic solvent (Fe3O4@GO-DES) were developed for efficient preconcentration of methadone. The extracted methadone was then analyzed by gas chromatography-flame ionization detection (GC-FID) or gas chromatography-mass spectrometry (GC-MS). Fe3O4@GO-DES were characterized by Fourier transform IR and X-ray diffraction techniques. Ultrasound was used to enhance the dispersion of the sorbent, with a high extraction recovery. Some parameters affecting the extraction recovery, such as pH, type of deep eutectic solvent, sample volume, amount of sorbent, extraction time, and type of eluent, were investigated. Under optimum conditions, the method developed was linear in the concentration range from 3 to 45,000 µg L-1 for GC-FID and from 0.1 to 500 µg L-1 for GC-MS, with a detection limit of 0.8 µg L-1 for GC-FID and 0.03 µg L-1 for GC-MS. The relative standard deviations (n = 6) as the intraday and interday precisions of the methadone spike at a concentration of 100 µg L-1 were 5.8% and 8.4% respectively for GC-FID. The preconcentration factor was 250. Relative recoveries from spiked plasma, urine, and water samples ranged from 95.1% to 101.5%.

Solid-phase microextraction of methadone in urine samples by electrochemically co-deposited sol-gel/Cu nanocomposite fiber.

Electrochemically co-deposited sol-gel/Cu nanocomposites have been introduced as a novel, simple and single-step technique for preparation of solid-phase microextraction (SPME) coating to extract methadone (MDN) (a synthetic opioid) in urine samples. The porous surface structure of the sol-gel/Cu nanocomposite coating was revealed by scanning electron microscopy. Direct immersion SPME followed by HPLC-UV determination was employed. The factors influencing the SPME procedure, such as the salt content, desorption solvent type, pH and equilibration time, were optimized. The best conditions were obtained with no salt content, acetonitrile as desorption solvent type, pH 9 and 10 min equilibration time. The calibration graphs for urine samples showed good linearity. The detection limit was about 0.2 ng mL-1 . Also, the novel method for preparation of nanocomposite fiber was compared with previously reported techniques for MDN determination. The results show that the novel nanocomposite fiber has relatively high extraction efficiency.

Body pushing, prescription drugs and hospital admission.

A 39-year-old man died of multi-organ failure complicating mixed drug toxicity that included methadone, oxazepam, oxycodone and nitrazepam. His past medical history involved alcohol and poly-substance abuse with chronic self-harm and suicidal ideation. There had been multiple hospital admissions for drug overdoses. At autopsy the most unusual finding was of two packages of 10 tablets each, wrapped in thin plastic film within the rectum. The insertion of drugs into body orifices and cavities has been termed body pushing to distinguish it from body packing where illicit drugs are wrapped and swallowed for transport and smuggling, and body stuffing where small amounts of loosely wrapped or unwrapped drugs are swallowed to conceal evidence from police. This case demonstrates that body pushing may not always involve illicit drugs or attempted concealment from police or customs officials. It appears that the drugs had been hidden to ensure an additional supply during the time of residence in hospital. The extent to which body pushing is currently being used by patients to smuggle drugs into secure medical facilities is yet to be determined.

Biocompatible Solid-Phase Microextraction Nanoelectrospray Ionization: An Unexploited Tool in Bioanalysis.

In recent years, different geometrical configurations of solid-phase microextraction (SPME) have been directly coupled to mass spectrometry, resulting in benefits such as diminishing matrix effects, improvement of detection limits, and considerable enhancement of analysis throughput. Although SPME fibers have been used for years, their potential for quantitative analysis when directly combined with mass spectrometry has not been explored to its full extent. In this study, we present the direct coupling of biocompatible SPME (Bio-SPME) fibers to mass spectrometry via nanoelectrospray ionization (nano-ESI) emitters as a powerful tool for fast quantitative analysis of target analytes in biofluids. Total sample preparation time does not exceed 2 min, and by selecting an appropriate fiber length and sample vessel, sample volumes ranging between 10 and 1500 µL can be used. Despite the short extraction time of the technique, limits of detection in the subnanogram per milliliter with good accuracy (≥90%) and linearity (R(2) > 0.999) were attained for all the studied probes in phosphate-buffered saline (PBS), urine, and whole blood. Given that Bio-SPME-nano-ESI efficiently integrates sampling with analyte extraction/enrichment, sample cleanup (including elimination of matrix effects in the form of particles), and ionization, our results demonstrated that it is an advantageous configuration for bioanalytical applications such as therapeutic drug monitoring, doping in sports, and pharmacological studies in various matrixes.

Development and validation of a magnetic solid-phase extraction with high-performance liquid chromatography method for the simultaneous determination of amphetamine and methadone in urine.

The simultaneous determination of amphetamine and methadone was carried out by magnetic graphene oxide nanoparticles, a magnetic solid-phase extraction adsorbent, as a new sample treatment technique. The main factors (the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, the ionic strength of extraction medium, and agitation rate) influencing the extraction efficiency were investigated and optimized. Under the optimized conditions, good linearity was observed in the range of 100-1500 ng/mL for amphetamine and 100-1000 ng/mL for methadone. The method was evaluated for determination of AM and methadone in positive urine samples, satisfactory results were obtained, therefore magnetic solid-phase extraction can be applied as a novel method for the determination of drugs of abuse in forensic laboratories.

Risk factors for weight gain during methadone maintenance treatment.

BACKGROUND: Weight gain was reported during methadone maintenance treatment (MMT). However, its relation to eating habits and specific risk factors, including methadone dose or serum level, was limited. The aims of this study were to characterize risk factors for weight gain and to study current eating habits, food preferences, and nutrition knowledge. METHODS: Patients with available measures of weight and height (body mass index [BMI]) at admission to MMT and at follow-up, when methadone serum levels were determined (after 1 year or when stabilized) (N = 114), were studied (using the Addiction Severity Index [ASI], drugs in urine, methadone doses, and serum levels). In addition, 109 current patients with available earlier (5.8 ± 2.6 years earlier) BMI completed eating behavior rating and nutrition knowledge questionnaires, and their current and earlier BMI were compared. RESULTS: The BMI of 114 newly admitted patients increased from 22.5 ± 3.8 to 24.4 ± 4.3 (P < .0005). Once stabilized on methadone, BMI increased further (24.3 ± 4.5 to 25.6 ± 5.0; P < .0005; n = 74), with no change in methadone doses (125.6 ± 32.5 to 128.0 ± 34.1; F = 1.4, P = .2) or serum levels (495.6 ± 263.7 to 539.8 ± 306.2; F = 1.3, P = .2). Repeated-measures analyses revealed that BMI elevation was higher among 45 hepatitis C virus seronegative and 46 non-benzodiazepine-abusing on-admission patients. Those who scored lower on knowledge about healthy diet and showed a higher sweet-foods preference had a higher BMI. CONCLUSION: BMI increased over time, but independent of methadone dosage and blood levels. As expected, worse diet habits and a desire for sweet foods are related to higher BMI. Paradoxically, healthier status (i.e., hepatitis C seronegative, no benzodiazepine abuse) at admission is predictive of greater weight gain during MMT. Education about nutrition habits is recommended.

Sensitive determination of methadone in human serum and urine by dispersive liquid-liquid microextraction based on the solidification of a floating organic droplet followed by HPLC-UV.

Dispersive liquid-liquid microextraction based on solidification of floating organic droplet was developed for the extraction of methadone and determination by high-performance liquid chromatography with UV detection. In this method, no microsyringe or fiber is required to support the organic microdrop due to the usage of an organic solvent with a low density and appropriate melting point. Furthermore, the extractant droplet can be collected easily by solidifying it at low temperature. 1-Undecanol and methanol were chosen as extraction and disperser solvents, respectively. Parameters that influence extraction efficiency, i.e. volumes of extracting and dispersing solvents, pH, and salt effect, were optimized by using response surface methodology. Under optimal conditions, enrichment factor for methadone was 134 and 160 in serum and urine samples, respectively. The limit of detection was 3.34 ng/mmL in serum and 1.67 ng/mL in urine samples. Compared with the traditional dispersive liquid-liquid microextraction, the proposed method obtained lower limit of detection. Moreover, the solidification of floating organic solvent facilitated the phase transfer. And most importantly, it avoided using high-density and toxic solvents of traditional dispersive liquid-liquid microextraction method. The proposed method was successfully applied to the determination of methadone in serum and urine samples of an addicted individual under methadone therapy.

Application of drug testing using exhaled breath for compliance monitoring of drug addicts in treatment.

AIM: Exhaled breath has recently been identified as a possible matrix for drug testing. This study explored the potential of this new method for compliance monitoring of patients being treated for dependence disorders. METHODS: Outpatients in treatment programs were recruited for this study. Urine was collected as part of clinical routine and a breath sample was collected in parallel together with a questionnaire about their views of the testing procedure. Urine was analyzed for amphetamines, benzodiazepines, cannabis, cocaine, buprenorphine, methadone and opiates using CEDIA immunochemical screening and mass spectrometry confirmation. The exhaled breath was collected using the SensAbues device and analyzed by mass spectrometry for amphetamine, methamphetamine, diazepam, oxazepam, tetrahydrocannabinol, cocaine, benzoylecgonine, buprenorphine, methadone, morphine, codeine and 6-acetylmorphine. RESULTS: A total of 122 cases with parallel urine and breath samples were collected; 34 of these were negative both in urine and breath. Out of 88 cases with positive urine samples 51 (58%) were also positive in breath. Among the patients on methadone treatment, all were positive for methadone in urine and 83% were positive in breath. Among patients in treatment with buprenorphine, 92% were positive in urine and among those 80% were also positive in breath. The questionnaire response documented that in general, patients accepted drug testing well and that the breath sampling procedure was preferred. CONCLUSION: Compliance testing for the intake of prescribed and unprescribed drugs among patients in treatment for dependence disorders using the exhaled breath sampling technique is a viable method and deserves future attention.

SELF-REPORTS OF ILLEGAL ACTIVITY, SCL-90-R PERSONALITY SCALES, AND URINE TESTS IN METHADONE PATIENTS.

In routine work, medical staff usually has to rely on the patient's self-reports of criminal activity and of recent involvement in fights. This study examines how these self-reports of crime correlate with the patients' routine urine tests and personality measures. Pearson correlations of these self-reports by 55 methadone patients (M age = 34.1 yr., SD = 9.1; 35 men, 20 women) were calculated to their urine screening tests (those for opiates, benzodiazepines, and cocaine) and to personality scores on the Symptom Checklist 90-Revised (SCL-90-R). Patients who reported being involved in recent illegal activities to obtain drugs had significantly higher scores on the SCL-90-R scale assessing obsessive-compulsive symptoms (r = .28) and had more frequent positive urine tests for cocaine (r = .35). Those who reported having engaged in fights within the last 12 mo. had higher scores on SCL-90-R measures of somatic complaints (r = .32), anxiety (r = .31), and depression (r = .29), and of overall psychopathology (r = .29), and they also had more often positive urine tests for cocaine (r = .28) than other patients. Studies on larger samples are needed to help clinicians to predict criminal or hostile behavior during methadone treatment.

Comprehensive automation of the solid phase extraction gas chromatographic mass spectrometric analysis (SPE-GC/MS) of opioids, cocaine, and metabolites from serum and other matrices.

The analysis of opioids, cocaine, and metabolites from blood serum is a routine task in forensic laboratories. Commonly, the employed methods include many manual or partly automated steps like protein precipitation, dilution, solid phase extraction, evaporation, and derivatization preceding a gas chromatography (GC)/mass spectrometry (MS) or liquid chromatography (LC)/MS analysis. In this study, a comprehensively automated method was developed from a validated, partly automated routine method. This was possible by replicating method parameters on the automated system. Only marginal optimization of parameters was necessary. The automation relying on an x-y-z robot after manual protein precipitation includes the solid phase extraction, evaporation of the eluate, derivatization (silylation with N-methyl-N-trimethylsilyltrifluoroacetamide, MSTFA), and injection into a GC/MS. A quantitative analysis of almost 170 authentic serum samples and more than 50 authentic samples of other matrices like urine, different tissues, and heart blood on cocaine, benzoylecgonine, methadone, morphine, codeine, 6-monoacetylmorphine, dihydrocodeine, and 7-aminoflunitrazepam was conducted with both methods proving that the analytical results are equivalent even near the limits of quantification (low ng/ml range). To our best knowledge, this application is the first one reported in the literature employing this sample preparation system.

Determination of amphetamine and methadone in human urine by microextraction by packed sorbent coupled directly to mass spectrometry: an alternative for rapid clinical and forensic analysis.

Speed and low cost, together with regulatory approval, are the most important requirements of clinical assays. Therefore, a fast and automated on-line sample preparation method is essential for the routine analysis of biological samples. Microextraction by packed sorbent is an option for optimal sample preparation due to its easy automation, minimal requirements for the sample and elution solvent volumes, elimination of evaporation and reconstitution steps, and ability to integrate sample preparation and injection into one step. The use of effective sample preparation steps circumvents the need for chromatographic separation and therefore allows more rapid and less expensive sample analysis in clinical and forensic practice. Two biologically active compounds, amphetamine and methadone, were chosen as representative drugs of abuse for the application of microextraction by packed sorbent coupled directly to mass spectrometry. The developed method was validated, with the results confirming the suitability of the combination of these techniques for the analysis of biological samples. The approach was confirmed to be appropriate for use in clinical and forensic practice with regard to cost and time requirements for analysis.

Laboratory and clinical evaluation of on-site urine drug testing.

AIM: Products for on-site urine drug testing offer the possibility to perform screening for drugs of abuse directly at the point-of-care. This is a well-established routine in emergency and dependency clinics but further evaluation of performance is needed due to inherent limitations with the available products. METHODS: Urine drug testing by an on-site product was compared with routine laboratory methods. First, on-site testing was performed at the laboratory in addition to the routine method. Second, the on-site testing was performed at a dependency clinic and urine samples were subsequently sent to the laboratory for additional analytical investigation. RESULTS: The on-site testing products did not perform with assigned cut-off levels. The subjective reading between the presence of a spot (i.e. negative test result) being present or no spot (positive result) was difficult in 3.2% of the cases, and occurred for all parameters. The tests performed more accurately in drug negative samples (specificity 96%) but less accurately for detecting positives (sensitivity 79%). Of all incorrect results by the on-site test the proportion of false negatives was 42%. The overall agreement between on-site and laboratory testing was 95% in the laboratory study and 98% in the clinical study. CONCLUSION: Although a high degree of agreement was observed between on-site and routine laboratory urine drug testing, the performance of on-site testing was not acceptable due to significant number of false negative results. The limited sensitivity of on-site testing compared to laboratory testing reduces the applicability of these tests.