Intoxications involving methoxyacetylfentanyl and U-47700: a study of 3 polydrug fatalities.

Novel synthetic opioids (NSOs) represent an emerging group of novel psychoactive substances, acting as agonists at the opioid receptors. NSOs include fentanyl-related compounds, e.g. methoxyacetylfentanyl (MeACF), and non-fentanyl analogs, e.g. "U compounds" including U-47700. Here we present three cases of death involving MeACF and U-47700, with particular reference to preliminary data on pharmacokinetics and tissue distribution.After a complete post-mortem examination, general unknown screenings and analysis of drugs of abuse were performed on postmortem samples by immunoassays, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. To quantify the analytes of interest in post-mortem blood and tissues, the standard addition method was used. A toxicological significance score (TSS), weighing the role of the NSO in each death case, was assigned.Case 1 died at the hospital after consumption of U-47700, methadone (serum levels: 2,600 ng/ml and 37 ng/ml), tilidine and benzodiazepines. In case 2, U-47700 (204 ng/ml) together with methadone (290 ng/ml), flubromazepam (480 ng/ml) and diazepam (300 ng/ml) were detected in peripheral blood. In case 3, methoxyacetylfentanyl (266 ng/ml), furanylfentanyl (4.3 ng/ml) 4-ANPP (15 ng/ml) and alprazolam (69 ng/ml) were quantified in femoral blood. In all cases, the NSO likely contributed to the death (TSS = 3).NSOs appear to be often consumed in the setting of polydrug intoxications, especially in combination with other opioids and benzodiazepines, which often exert synergistic effects. The standard addition method remains the most reliable in post-mortem analysis and toxicological results should always be evaluated together with circumstantial and autopsy data.

Validation and application of an automated multitarget LC-MS/MS method for drugs of abuse testing using exhaled breath as specimen.

Aerosol microparticles in exhaled breath carry non-volatile compounds from the deeper parts of the lung. When captured and analyzed, these aerosol microparticles constitute a non-invasive and readily available specimen for drugs of abuse testing. The present study aimed to evaluate a simple breath collection device in a clinical setting. The device divides a breath sample into three parallel "collectors" that can be individually analyzed. Urine was used as the reference specimen, and parallel specimens were collected from 99 patients undergoing methadone maintenance treatment. Methadone was used as the primary validation parameter. A sensitive multi-analyte method using tandem liquid chromatography - mass spectrometry was developed and validated as part of the project. The method was successfully validated for 36 analytes with a limit of detection of 1 pg/collector for most compounds. Based on the validation results tetrahydrocannabinol THC), cannabidiol (CBD), and lysergic acid diethylamide (LSD) are suitable for qualitative analysis, but all other analytes can be quantitively assessed by the method. Methadone was positive in urine in 97 cases and detected in exhaled breath in 98 cases. Median methadone concentration was 64 pg/collector. The methadone metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was detected in 90 % of the cases but below 10 pg/collector in most. Amphetamine was also present in the urine in 17 cases and in exhaled breath in 16 cases. Several other substances were detected in the exhaled breath and urine samples, but at a lower frequency. This study concluded that the device provides a specimen from exhaled breath, that is useful for drugs of abuse testing. The results show that high analytical sensitivity is needed to achieve good detectability and detection time after intake.

Use of psychoactive substances by goods carriage drivers associated with Kerala, India.

OBJECTIVE: This study aimed to measure the prevalence of drugs of abuse (DOA) among the goods carriage drivers associated with the southern State of India, Kerala. METHODS: Point-of-collection testing (POCT) of oral fluid collected from the participants (n = 249) was done using the Evidence MultiSTAT DOA Oral Fluid II Assay kits and the Evidence MultiSTAT analyzer. RESULTS: Out of the total samples, 53 (21.29%) were positive for one or more DOA. A high prevalence of tetrahydrocannabinol (THC) (10.04%) and synthetic cannabinoids were detected in the samples. The use of ketamine, alpha-PVP, LSD, methamphetamine, opiate, 6-MAM, benzodiazepines I, methadone, PCP, tramadol, and amphetamine was also detected and their frequency of use ranged between 4.02 and 0.80%. An association between drug abuse and distance of travel was found in drivers in this study, χ2 (5, N = 249) = 123.5, p < 0.001. Confirmatory analysis using ultra-high performance liquid chromatography-tandem mass spectrometry showed excellent agreement with the results of the screening test. CONCLUSIONS: This was the first study conducted among drivers in India for the detection of DOA. Tetrahydrocannabinol (THC) was used more by the goods carriage drivers associated with Kerala State, India. The use of psychoactive substances significantly increased with the distance of travel. Point-of-collection testing (POCT) by the biochip array technology is an efficient method for the detection of these substances.

Comparing presumptive with direct-to-definitive drug testing in oral fluid vs. urine for a U.S. national sample of individuals misusing drugs.

BACKGROUND: The aims are to compare the results of presumptive drug testing with confirmation of positives vs. direct-to-definitive drug testing, combined with investigation of urine vs. oral fluid as test matrices. METHODS: Paired oral fluid and urine specimens were collected voluntarily and anonymously from 1098 individuals applying for methadone treatment in 11 clinics across 7 U.S. states. All specimens were analyzed by immunoassay (IA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). RESULTS: Confirmed IA prevalences for urine were significantly higher than for oral fluid for 7 out of 10 drug classes - benzodiazepines, cannabis, cocaine, methadone, opiates, oxycodone and tramadol. Drug prevalences by direct-to-definitive LC-MS-MS were either the same or higher than prevalences by confirmed IA. Drug prevalences by LC-MS-MS were higher in urine for two drug classes (cocaine, methadone) and higher in oral fluid for two drug classes (buprenorphine, tramadol), but were equivalent in urine and oral fluid when averaged over all 10 drug classes. Certain drugs of special concern such as heroin and buprenorphine were more frequently detected in oral fluid than urine. CONCLUSIONS: Urine analysis showed some technical advantage over oral fluid in sensitivity to several drug classes within a confirmed IA testing protocol, but this may be outweighed if there is reason to believe that tampering with urine specimens is a significant problem. Overall drug detection by direct-to-definitive testing was similar for oral fluid and urine, but one matrix may be preferable if there is a particular drug of clinical or epidemiological interest.

Detection of Three Opioids (Morphine, Codeine and Methadone) and Their Metabolites (6-Monoacetylmorphine and 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) in Larvae of Lucilia sericata Species by UHPLC-TF-MS and Validation.

Insects on corpses could be a useful tool for the detection of exogenous substances such as drugs of abuse. The identification of exogenous substances in carrion insects is critical for proper estimation of the postmortem interval. It also provides information about the deceased person that may prove useful for forensic purposes. High-performance liquid chromatography coupled with Fourier transform mass spectrometry is a highly sensitive analytical technique that can identify substances even at very low concentrations, such as in the case of searching for exogenous substances in larvae. In this paper, a method is proposed for the identification of morphine, codeine, methadone, 6-monoacetylmorphine (6-MAM) and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the larvae of Lucilia sericata, a common carrion fly widely distributed in temperate areas of the world. The larvae, which were reared on a pig meat substrate, were killed once they reached their third stage by immersion in hot water at 80 °C and aliquoted into 400 mg samples. The samples were fortified with 5 ng of morphine, methadone and codeine. After solid-phase extraction, the samples were processed with a liquid chromatograph coupled to a Fourier transform mass spectrometer. This qualitative method has been validated and tested on larvae from a real case. The results lead to the correct identification of morphine, codeine, methadone and their metabolites. This method could prove useful in cases where toxicological analysis must be conducted on highly decomposed human remains, where biological matrices are very limited. Furthermore, it could help the forensic pathologist to better estimate the time of death, as the growth cycle of carrion insects can undergo changes if exogenous substances are taken.

Occurrence and sources of residues of drugs of abuse in an urban aquifer: Chemical analysis and solute transport modelling.

This study investigated the occurrence and potential sources of residues of drugs of abuse in an urban aquifer beneath the City of Ljubljana using water analysis and a solute transport model designed to predict nitrogen distribution. Samples were collected from three sources: 28 wastewater samples (24-h composites), 4 aquifer-recharging river samples (grab), and 22 groundwater samples. The samples were analysed for residues of commonly (ab)used licit drugs (nicotine and alcohol), medications of abuse (morphine, methadone, codeine, and ketamine), and illicit drugs (tetrahydrocannabinol - THC, cocaine, amphetamines, and heroin) using liquid-liquid (alcohol residue) and solid-phase extraction, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, we used solute transport modelling to predict the spatial distribution of drug residues in the aquifer and their potential sources. Nicotine (up to 45,7 ng/L), cotinine (up to 5.86 ng/L), trans-3'-hydroxycotinine (up to 0.528 ng/L) and benzoylecgonine (up to 0.572 ng/L) were the most commonly detected drug residues in groundwater, followed by cocaine (

Validation of an HPLC-HR-MS Method for the Determination and Quantification of Six Drugs (Morphine, Codeine, Methadone, Alprazolam, Clonazepam and Quetiapine) in Nails.

Keratinized matrices, including nails, are among the most resistant matrices that can be analyzed in cases where remains are deeply decomposed and relatively non-invasive for living people. In order to exploit these new matrices in the search for exogenous substances, it is necessary to develop analytical technologies capable of achieving high levels of sensitivity. In this technical note, an easy method is presented for the simultaneous extraction and quantification of three narcotic substances (morphine, codeine and methadone), two benzodiazepines (BDZs) (clonazepam and alprazolam) and an antipsychotic (quetiapine) from nail matrix by analysis in ultra-high-performance liquid chromatography at high-resolution mass spectrometry. The method has been validated following the Standard Practices for Method Validation in Forensic Toxicology of the Scientific Working Group for Forensic Toxicology. Nail specimens from eight authentic postmortem (PM) cases and 13 living donor samples were extracted and analyzed. Of the eight PM samples, five resulted positive for at least one of the three substances searched. Ten of the 13 living donor specimens were positive for at least one of the targeted BDZs or quetiapine.

[Estimation of the consumption level of four drugs in Beijing using wastewater-based epidemiology].

Objective: To estimate the consumption level of four drugs in Beijing using wastewater-based epidemiology (WBE). Methods: The primary sludge from one large wastewater treatment plants (WWTPs) was collected in Beijing from July 2020 to February 2021. The concentrations of codeine, methadone, ketamine and morphine in the sludge were detected through solid-phase extraction-liquid chromatography-tandem mass spectrometry. The consumption, prevalence and number of users of four drugs were estimated by using the WBE approach. Results: Among 416 sludge samples, codeine had the highest detection rate (82.93%, n=345) with a concentration [M (Q1, Q3)] of 0.40 (0.22-0.8) ng·g-1, and morphine had the lowest detection rate (28.37%,n=118) with a concentration [M (Q1, Q3)] of 0.13 (0.09, 0.17) ng·g-1. There was no significant difference in the consumption of the four drugs on working days and weekends (all P values>0.05). Drug consumption was significantly higher in winter than that in summer and autumn (all P values <0.05). The consumption [M (Q1, Q3)] of codeine, methadone, ketamine and morphine in winter was 24.9 (15.58, 38.6), 9.39 (4.57, 26.72), 9.84 (5.18, 19.45) and 5.67 (3.57, 13.77) µg·inhabitant-1·day-1, respectively. For these drugs, there was an upward trend in the average drug consumption during summer, autumn and winter (the Z values of the trend test were 3.23, 3.16, 2.19, and 3.32, respectively and all P values<0.05). The prevalence [M (Q1, Q3)] of codeine, methadone, ketamine and morphine were 0.0056% (0.003 4%, 0.009 2%), 0.0148% (0.009 6%, 0.026 7%),0.0333% (0.0210%, 0.0710%) and 0.0072% (0.003 8%, 0.011 7%), respectively. The estimated number of drug users [M (Q1, Q3)] was 918 (549, 1 511), 2 429 (1 578, 4 383), 5 451 (3 444, 11 642) and 1 173 (626, 1 925),respectively. Conclusion: Codeine, methadone, ketamine and morphine have been detected in the sludge of WWTPs in Beijing, and the consumption level of these drugs varies in different seasons.

Do the lockdown-imposed changes in a wastewater treatment plant catchment's socio-demographics impact longitudinal temporal trends in psychoactive pharmaceutical use?

Wastewater-based epidemiology (WBE) includes the analysis of human metabolic biomarkers of xenobiotics in influent wastewater. WBE complements existing drug utilization approaches and provides objective, spatio-temporal information on the consumption of pharmaceuticals in the general population. This approach was applied to 24-h composite influent wastewater samples from Leuven, Belgium. Daily samples were analysed from September 2019 to December 2019 (n = 76), and on three days of the week (Monday, Wednesday, Saturday) from January 2020 to April 2022 (n = 367). Sample analysis consisted of 96-well solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry. Measured concentrations of 21 biomarkers for antidepressant and opioid use were converted to population-normalized mass loads (PNML) by considering the flow rate and catchment population. To capture population movements, mobile phone data was used. Amitriptyline, hydroxy-bupropion, norcitalopram, citalopram, normirtazapine, trazodone, O-desmethylvenlafaxine, codeine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), methadone, morphine, O-desmethyltramadol, and tramadol were included in the temporal assessment since concentrations were above the lower limit of quantification. The PNML of most biomarkers increased (with 3-119 %) throughout the sampling period. The population disruption during the COVID-19 pandemic led to a major change in the socio-demographics of the catchment area, resulting in temporal differences in the PNML of the different biomarkers. As such, higher PNML were observed during the different lockdown phases, which were characterized by the outflow of university students and a decreasing commuting in and out the catchment area. The effects of the fluctuating socio-demographics of the catchment population were further evidenced by the different week-weekend pattern of PNMLs over the course of the sampling campaign. Mean parent/metabolite ratios (i.e., citalopram/norcitalopram, tramadol/O-desmethyltramadol, venlafaxine/O-desmethylvenlafaxine, and methadone/EDDP) remained relatively stable throughout the entire sampling campaign (RSD% below 25 % for all ratios, except for methadone/EDDP) and therefore were not affected by this population change.

Dental Plaque Concentrations of Methadone, Morphine and Their Metabolites in Opioid Replacement Therapy and in Postmortem Cases.

Non-mineralized dental biofilm (plaque) has potential as a novel alternative matrix in forensic toxicology to prove drug use. The incorporation of illicit and medicinal drugs in dental plaque could take place through direct contact after oral or nasal intake, which can lead to high drug levels in the oral cavity, or indirectly via the secretion of drug-containing saliva, e.g., after intravenous application. Therefore, plaque samples from patients in opioid replacement therapy (ORT) and postmortem plaque samples were analyzed and the drug concentrations were compared. The study comprised 26 plaque samples from ORT patients with different daily doses, which were analyzed for methadone, morphine and their respective metabolites. Plaque samples were taken directly before the oral administration of the regular daily dose. Seventeen postmortem plaque samples were analyzed, either from cases of lethal drug intoxications or after pain therapy with morphine. Plaque analysis was performed using liquid chromatography--tandem mass spectrometry after liquid extraction with acetonitrile. Plaque concentrations in ORT for methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) ranged from 42 to approximately 49,000 pg/mg (median 1,300 pg/mg) and from below 10 to 610 pg/mg (median 31 pg/mg), respectively. Morphine plaque concentrations in ORT ranged from 120 to 480 pg/mg (median 400 pg/mg). In lethal intoxication cases, plaque concentrations were generally at least one order of magnitude higher than those in the study groups with therapeutic substance use. These data will help to interpret drug findings in plaque. Furthermore, the EDDP/methadone concentration ratio in plaque was lower after oral intake with contamination of the oral cavity (e.g., syrup) compared to cases with suspected intravenous application of methadone. Therefore, the EDDP/methadone concentration ratio could therefore indicate the drug administration route.

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.

Evidence for the transfer of methadone and EDDP by sweat to children's hair.

In cases where there is a question as to whether children have come into contact with drugs, examinations of their scalp hair are frequently carried out. Positive test results are often discussed in the forensic community due to the various possible modes via which drugs and their metabolites can be incorporated into the hair. These include drug uptake by the child (e.g. oral ingestion or inhalation), but also contamination of hair via contact with the sweat from drug users. In this study, the possibility of methadone and its metabolite EDDP being incorporated into children's hair by contact with sweat from persons undergoing opiate maintenance therapy (methadone) was examined. The transfer of methadone and EDDP via sweat from methadone patients (n = 15) to children's hair was simulated by close skin contact of drug-free children's hair, encased in mesh-pouches, for 5 days. Sweat-collecting patches (hereafter referred to as 'sweat patches') were applied to the test persons' skin. One strand of hair and one sweat patch were collected daily from each patient. Analyses were performed using GC-MS/MS (hair) and LC-MS/MS (serum, sweat patches). After 4 days of skin contact, methadone was detectable in the formerly drug-free hair strands in all 15 study participants. EDDP was detectable in 34 of 75 hair strands, with the maximum number of positive results (11 EDDP-positive hair strands) being detected after 5 days. These results show that transfer of methadone and EDDP to drug-free hair is possible through close skin contact with individuals taking part in methadone substitution programmes. A correlation between serum concentration, sweat concentration and substance concentration in hair strands could not be demonstrated, but a tendency towards higher concentrations due to longer contact time is clearly evident.

Solid-phase microextraction of methadone by using a chitosan nanocomposite incorporated with Polyoxomolibdate nanocluster/Graphene oxide.

In the present study, we report on the simple sol-gel preparation of a nanocomposite composed of chitosan/ polyoxometalate /graphene oxide, and its application in the headspace solid-phase microextraction combined with the ion mobility spectrometry for the analysis of methadone in biological matrices. The developed nanocomposite was characterized through the infrared spectroscopy and thermogravimetric analyses. The ternary nanocomposite coating offers good mechanical and thermal stability and high extraction efficiency thanks to its large specific surface. A central composite statistical design was used to study the main variables affecting the extraction efficiency. Afterward, to study the relationship between different input and output variables as well as to identify the optimal operating conditions, response surface methodology was used, whereby a second-order polynomial equation was fit to the experimental data. The optimized extraction conditions were as follows: temperature, 70°C; extraction time, 15 min; and concentration of NaCl, 5%w/v. The detection limit of 0.12 ng/mL was obtained at the optimized extraction conditions, and the calibration plot was linear in the concentration range of 0.30-200 ng/mL. With relatively low limit of detection and good precisions, the proposed method has the potential for the extraction and determination of methadone in biological samples.

Quantification of methadone and its metabolites: EDDP and EMDP determined in autopsy cases using LC-MS/MS.

The paper presents a method for the determination of methadone, EDDP, and EMDP in postmortem biological materials using liquid-liquid extraction with ethyl acetate (pH9) and UHPLC-MS/MS technique. Methadone-d9 and EDDP-d3 were used as the internal standards. The method validation results for blood and urine were as follows: linearity: 0.5-1000 ng/ml; R2  > 0.9993 for methadone, EDDP and R2  > 0.9944 for EMDP. Intra- and inter-day precision: 0.1%-7.5% and 0.3%-8.6%, respectively; intra- and inter-day accuracy: -11.8% to 13.9% and -9.3 to 14.8%, respectively; recovery: 91.5%-123.0%; matrix effect: 83.5%-123.9%. This study also describes 18 postmortem cases, where methadone concentrations ranged 2.3-1180 ng/ml in blood (n = 17), from 11.0 to >10,000 ng/ml in urine (n = 13) and 135.2-409.0 in vitreous humor (VH, n = 3). EDDP concentrations ranged from not detectable to 180 ng/mL in blood, from 42.4 to >10,000 ng/ml in urine and 18.3-36.5 in VH. EMDP concentrations were found in four cases in blood from below LLOQ to 1.8 ng/ml and in seven cases in urine, ranged 2.1-243.0 ng/ml. EMDP was not detected in VH samples. The EDDP/methadone ratios and blood/urine ratios for methadone and EDDP in EMDP-positive and negative cases were performed. The paper presents mass spectra of other methadone metabolites, than EDDP and EMDP (ring hydroxylated methadone, ring hydroxylated EDDP, ring hydroxylated EMDP, methadol, and DDP). Simultaneous determination of methadone and its metabolites in order to unequivocally interpret the results of toxicological tests seems to be useful in cases related to prescription/illicit use of methadone.

Brain/blood ratios of methadone and ABCB1 polymorphisms in methadone-related deaths.

Methadone is an opioid that often leads to fatalities. Interpretation of toxicological findings can be challenging if no further information about the case history is available. The aims of this study were (1) to determine whether brain/blood ratios can assist in the interpretation of methadone findings in fatalities; (2) to examine whether polymorphisms in the gene encoding the P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)), which functions as a multispecific efflux pump in the blood-brain barrier, affect brain/blood ratios of methadone. Femoral venous blood and brain tissue (medulla oblongata and cerebellum) from 107 methadone-related deaths were analysed for methadone by gas chromatography-mass spectrometry. In addition, all the samples were genotyped for three common ABCB1 single nucleotide polymorphisms (SNPs rs1045642, rs1128503, and rs2032582) using ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). In nearly all cases, methadone concentrations were higher in the brain than in the blood. Inter-individual brain/blood ratios varied (0.6-11.6); the mean ratio was 2.85 (standard deviation 1.83, median 2.35). Moreover, significant differences in mean brain/blood ratios were detected among the synonymous genotypes of rs1045642 in ABCB1 (p = 0.001). Cases with the T/T genotype had significantly higher brain/blood ratios than cases with the other genotypes (T/T vs. T/C difference (d) = 1.54, 95% CI [1.14, 2.05], p = 0.002; T/T vs. C/C d = 1.60, 95% CI [1.13, 2.29], p = 0.004). Our results suggest that the rs1045642 polymorphisms in ABCB1 may affect methadone concentrations in the brain and its site of action and may be an additional factor influencing methadone toxicity.

Revealing the Kinetic Advantage of a Competitive Small-Molecule Immunoassay by Direct Detection.

Small-molecule detection in an immunoassay format generally employs competition or labeling. A novel direct-detection label-free primary immunoassay utilizing second harmonic generation (SHG) has been developed and the utility of the method has been demonstrated for several small-molecule narcotics. Specifically, the binding of morphine, methadone, and cocaine to antimorphine, antimethadone, and anticocaine antibodies was measured by SHG, allowing binding affinities and rates of dissociation to be obtained. The SHG primary immunoassay has provided the first kinetic measurements of small-molecule hapten interactions with a receptor antibody. The kinetics reveal for the first time that competitive immunoassays achieve their selectivity by taking advantage of the kinetics of association and dissociation of the labeled and unlabeled target and nontarget small-molecule to the capture antibody. In particular, the induced fit of the target small-molecule to their antibody pairs prolongs their residence time, while the nontarget small-molecule dissociate rapidly in comparison.

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.

Microextraction by Packed Sorbent as a Novel Strategy for Sample Clean-Up in the Determination of Methadone and EDDP in Hair.

A microextraction by packed sorbent (MEPS) procedure for rapid concentration of methadone and its primary metabolite (EDDP) in hair samples was developed. The miniaturized approach coupled to gas chromatography with tandem mass spectrometry (GC-MS-MS) was successfully validated. Hair samples (50 mg) were incubated with 1 mL of 1 M sodium hydroxide for 45 min at 50°C, time after which the extract was neutralized by adding 100 µL of 20% formic acid. Subsequently, MEPS was applied using a M1 sorbent (4 mg; 80% C8 and 20% strong cation-exchange (SCX)), first conditioned with three 250-µL cycles of methanol and three 250-µL cycles of 2% formic acid. The extract load occurred with nine 150-µL cycles followed by a washing step involving three 50-µL cycles with 3.36% formic acid. For the elution of the analytes, six 100-µL cycles of 2.36% ammonium hydroxide in methanol were applied. The method was linear from 0.01 to 5 ng/mg, for both compounds, presenting determination coefficients greater than 0.99. Precision and accuracy were in accordance with the statements of international guidelines for method validation. This new miniaturized approach allowed obtaining recoveries ranging from 73 to 109% for methadone and 84 to 110% for EDDP, proving to be an excellent alternative to classic approaches, as well as other miniaturized procedures.

Rapid and sensitive detection of drugs of abuse in sweat by multiplexed capillary based immuno-biosensors.

Rapid and sensitive detection of drugs of abuse plays an important role in monitoring of drug use and treatment compliance. Sweat based drug analysis shows great advantages due to its non-invasive nature. However, most of the related methods developed to date are qualitative, slow, or costly, which significantly hinders their application in field use. Here we report rapid, sensitive, quantitative detection of drugs of abuse in sweat based on capillary arrays combined with competitive enzyme-linked immunosorbent assay. Using four common drugs of abuse, methadone, methamphetamine, amphetamine, and tetrahydrocannabinol, spiked in artificial sweat as a model system, we demonstrate rapid, quantitative, and multiplexed detection of the four drugs in ∼16 minutes with a low sweat volume (∼4 µL per analyte) and a large dynamic range (methadone: 0.0016 ng mL-1-1 ng mL-1; METH: 0.016 ng mL-1-25 ng mL-1; amphetamine: 0.005 ng mL-1-10 ng mL-1; THC: 0.02 ng mL-1-1000 ng mL-1). In addition, we show that the detection range can be tuned for different applications by adjusting the competitors' concentrations. Our work paves a way to develop an autonomous, portable, and cost-effective device for hospital testing, workplace drug-use screening, roadside testing, and patient monitoring in drug rehabilitation centers.

ICH Q3D based elemental impurities study in liquid pharmaceutical dosage form with high daily intake - comparative analysis by ICP-OES and ICP-MS.

Guideline for Elemental Impurities-Q3D of the International Conference on Harmonisation represents a new paradigm in the control of elemental impurities (EIs) in pharmaceuticals. It changes the approach toward control of EIs from the historical 'heavy metals test', to a scientific-based risk assessment and testing by modern analytical instrumentation such as inductively coupled plasma-optical emission spectroscopy (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS). Management of EIs related to all finished drug products must be implemented in strict compliance with the regulatory requirements of pharmaceutical industry due to their quality and safety concerns. Testing for presence of EIs from Class 1 and Class 2a in methadone hydrochloride 1 mg/ml oral solution with recommended daily intake of 150 mg methadone hydrochloride was initially performed on ICP-OES using in-house validated method according to the requirements of pharmacopoeias, in line with Q3D. During the procedure, it became apparent that ICP-OES has its own limitations, especially when it comes to testing arsenic and lead in low concentrations. ICP-MS in-house validated method was developed and employed for determination of trace concentrations of arsenic and lead, providing resourceful information that were compared and correlated to the data obtained by ICP-OES analysis. Sample preparation using microwave digestion technique was applied for the analyses by both techniques. Although the applied ICP-OES in-house method is suitable for determination of Hg, Cd, Co, V, and Ni, more sensitive technique such as ICP-MS is required for accurate determination of As and Pb concerning pharmaceuticals with high daily intakes.