Effervescent tablet‐assisted demulsified dispersive liquid–liquid microextraction based on solidification of floating organic droplet for determination of methadone in water and biological samples prior to GC‐flame ionization and GC‐MS

A novel effervescent tablet‐assisted demulsified dispersive liquid–liquid microextraction based on the solidification of floating organic droplet was developed to determine methadone prior to gas chromatography with flame ionization detection and gas chromatography with mass spectrometry. In this method, a tablet composed of citric acid, sodium carbonate, and 1‐undecanol was utilized. The resulting effervescent tablet generated carbon dioxide in situ to disperse 1‐undecanol in the sample. Thus, the dispersive and extraction processes were performed in one synchronous step. An aliquot of acetonitrile as the demulsifier solvent was used for the separation of two phases instead of centrifugation. Under optimal conditions, the developed method was linear up to 50 000 µg/L with correlation coefficients higher than 0.99. Moreover, limits of detection and limits of the quantification were in the range of 3‐10  and 7‐30 µg/L in water and biological samples, respectively. Intra‐ and interday precisions (n = 6) of the spiked methadone at a concentration level of 50 µg/L were over ranges of 5.1‐6.8% and 5.7‐7.1%, respectively. The preconcentration factors and recovery values were obtained in the range of 140‐145 and 98.1 to 101.6% in real samples, respectively.     

GC-MS Analysis of Methadone and EDDP in Addicted Patients under Methadone Substitution Treatment: Comparison of Urine and Plasma as Biological Samples

(1) Background: Methadone, along with buprenorphine, is the most commonly used drug for the treatment of opioid dependence. This study aimed to analyze methadone and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenyl pyrrolidine (EDDP), in the urine and plasma of opiate addicts. The study group consisted of drug users voluntarily admitted to the detoxification center C.E.T.T.T. “St. Stelian” of Bucharest. Secondly, the study aimed to identify whether urine or plasma provides better results for the proposed method. (2) Methods: A GC-MS method, using an internal standard (diphenylamine) in the FULL-SCAN and SIM modes of operation and using the m/z = 72 ion for methadone and the m/z = 277 ion for EDDP, combined with a liquid–liquid extraction procedure was performed. (3) Results: The applied procedure allows the detection and quantification of methadone in both urine and plasma samples. EDDP was identified in patients with higher levels of methadone. Higher levels of methadone were detected in urine than in plasma samples. (4) Conclusions: This procedure can be used in clinical laboratories for the rapid determination of methadone levels in urine rather than in plasma. The procedure can be applied for the monitoring of methadone substitution treatment.     

Suppressor capacity of iron nanoparticles biosynthesized using Salvia chloroleuca leaf aqueous extract on methadone-induced cell death in PC12: Formulation a new drug from relationship between the nanobiotechnology and neurology sciences

Metallic nanoparticles have gained significant attention in the area of biomedical technology. Because of its high surface area, metallic nanoparticles are being widely used in various fields including the medical and engineering sciences. One of the valuable applications of metallic nanoparticles especially copper, zinc, and iron nanoparticles is increasing the physiological function of central nervous system. Besides, Iranian people are using the Salvia chloroleuca for neuroprotective properties. In the present research, iron nanoparticles were biosynthesized by S. chloroleuca leaf aqueous extract as reducing and stabilizing agents. Also, we revealed the protective effect of FeNPs in methadone-treated PC12 cells. FeNPs were characterized and analyzed using common nanotechnology techniques including FT-IR, UV–Vis. spectroscopy; EDS, TEM, and FE-SEM. TEM and FE-SEM images revealed a uniform spherical morphology for FeNPs. In the biological part of the current study, the both treatments of FeNPs significantly (p ≤ 0.01) reduced the cell cytotoxicity and cell death index as well as increased the cell viability and cell proliferation in methadone-treated PC12 cells. In these treatments, mitochondrial membrane potential significantly (p ≤ 0.01) increased compared to methadone-induced PC12 cells. DPPH free radical scavenging test was did to evaluate the antioxidant potentials of FeCl₃, S. chloroleuca, and FeNPs. DPPH test indicated similar antioxidant activities for S. chloroleuca, FeNPs, and butylated hydroxytoluene. In current experiment, we concluded that iron nanoparticles biosynthesized by S. chloroleuca leaf aqueous extract suppressed methadone-induced cell death in a dose-dependent manner in PC12 cells.     

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane

An enantioselective assay for the determination of methadone and its main metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ‐cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ‐cyclodextrin. The developed assay is precise (intra‐ and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.     

Mixed-mode solid-phase extraction for sample cleanup in hair analysis for methadone and its main metabolite

A simple and rapid method for the determination of methadone and its main metabolite EDDP in hair has been developed and validated. The analytes were completely extracted from the matrix after a short alkaline incubation, and the extracts were further cleaned up by solid‐phase extraction using mixed‐mode cartridges. Linearity was obtained from 0.1 (lower limit of quantitation, LLOQ) to 30 ng/mg for both compounds, with correlation coefficients higher than 0.99. Intra‐ and interday precision and accuracy were in conformity with internationally accepted guidelines for bioanalytical method validation, and the cleanup procedure presented mean extraction efficiencies higher than 90% for both analytes. This high efficiency greatly contributed to the low limits of quantitation achieved, and therefore this method can be successfully applied in the determination of methadone and EDDP in hair samples in clinical and forensic scenarios where these compounds are involved. Copyright © 2010 John Wiley & Sons, Ltd.     

Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine

The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL⁻¹ for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10–1000 ng mL⁻¹ and 21–1000 ng mL⁻¹, respectively.     

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.     

Physicochemical and Microbiological Stability of Two Oral Solutions of Methadone Hydrochloride 10 mg/mL

In this article, we studied physicochemical and microbiological stability and determined the beyond-use date of two oral solutions of methadone in three storage conditions. For this, two oral solutions of methadone (10 mg/mL) were prepared, with and without parabens, as preservatives. They were packed in amber glass vials kept unopened until the day of the test, and in a multi-dose umber glass bottle opened daily. They were stored at 5 ± 3 °C, 25 ± 2 °C and 40 ± 2 °C. pH, clarity, and organoleptic characteristics were obtained. A stability-indicating high-performance liquid chromatography method was used to determine methadone. Microbiological quality was studied and antimicrobial effectiveness testing was also determined following European Pharmacopoeia guidelines. Samples were analyzed at days 0, 7, 14, 21, 28, 42, 56, 70, and 91 in triplicate. After 91 days of storage, pH remained stable at about 6.5–7 in the two solutions, ensuring no risk of methadone precipitation. The organoleptic characteristics remained stable (colorless, odorless, and bitter taste). The absence of particles was confirmed. No differences were found with the use of preservatives. Methadone concentration remained within 95–105% in all samples. No microbial growth was observed. Hence, the two oral methadone solutions were physically and microbiologically stable at 5 ± 3 °C, 25 ± 2 °C, and 40 ± 2 °C for 91 days in closed and opened amber glass bottles.     

UHPLC‐MS/MS quantification of buprenorphine,norbuprenorphine, methadone,and glucuronide conjugates in umbilical cord plasma

Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC‐MS/MS method using solid‐phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were <15% for each validated concentration. Our data establishes a 2 week maximum freezer storage window in order to achieve the most accurate cord plasma concentrations of these analytes. Additionally, we found that the umbilical cord tissue analysis was less sensitive compared with analysis with umbilical cord blood plasma, indicating that this may be a more appropriate matrix for determination of buprenorphine and metabolite concentrations. This method was successfully applied to the analysis of cord blood from women with known buprenorphine or methadone use during pregnancy. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.