Rapid Assessment of Opioid Exposure and Treatment in Cities Through Robotic Collection and Chemical Analysis of Wastewater.

INTRODUCTION: Accurate data regarding opioid use, overdose, and treatment is important in guiding community efforts at combating the opioid epidemic. Wastewater-based epidemiology (WBE) is a potential method to quantify community-level trends of opioid exposure beyond overdose data, which is the basis of most existing response efforts. However, most WBE efforts collect parent opioid compounds (e.g., morphine) at wastewater treatment facilities, measuring opioid concentrations across large catchment zones which typically represent an entire municipality. We sought to deploy a robotic sampling device at targeted manholes within a city to semi-quantitatively detect opioid metabolites (e.g., morphine glucuronide) at a sub-city community resolution. METHODS: We deployed a robotic wastewater sampling platform at ten residential manholes in an urban municipality in North Carolina, accounting for 44.5% of the total municipal population. Sampling devices comprised a robotic sampling arm with in situ solid phase extraction, and collected hourly samples over 24-hour periods. We used targeted mass spectrometry to detect the presence of a custom panel of opioids, naloxone, and buprenorphine. RESULTS: Ten sampling sites were selected to be a representative survey of the entire municipality by integrating sewer network and demographic GIS data. All eleven metabolites targeted were detected during the program. The average morphine milligram equivalent (MME) across the nine illicit and prescription opioids, as excreted and detected in wastewater, was 49.1 (standard deviation of 31.9) MME/day/1000-people. Codeine was detected most frequently (detection rate of 100%), and buprenorphine was detected least frequently (12%). The presence of naloxone correlated with city data of known overdoses reversed by emergency medical services in the prehospital setting. CONCLUSION: Wastewater-based epidemiology with smart sewer selection and robotic wastewater collection is feasible to detect the presence of specific opioids, naloxone, methadone, and buprenorphine within a city. These results suggest that wastewater epidemiology could be used to detect patterns of opioid exposure and may ultimately provide information for opioid use disorder (OUD) treatment and harm reduction programs.

Simultaneous quantification of buprenorphine, naloxone and phase I and II metabolites in plasma and breastmilk by liquid chromatography-tandem mass spectrometry.

Opioid abuse during pregnancy is associated with fetal growth restriction, placental abruption, preterm labor, fetal death, and Neonatal Abstinence Syndrome. Current guidelines for medication-assisted opioid addiction treatment during pregnancy are methadone or buprenorphine monotherapy. Buprenorphine/naloxone combination therapy (Suboxone(®)) has not been thoroughly evaluated during pregnancy and insufficient naloxone safety data exist. While methadone- and buprenorphine-treated mothers are encouraged to breastfeed, no studies to date investigated naloxone concentrations during breastfeeding following Suboxone administration. For this reason, we developed and fully validated a liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine, buprenorphine-glucuronide, norbuprenorphine, norbuprenorphine-glucuronide, naloxone, naloxone-glucuronide and naloxone-N-oxide in 100µL human plasma and breastmilk in a single injection following protein precipitation and solid-phase extraction. Lowest limits of quantification were 0.1-2µg/L with 20-100µg/L upper limits of linearity. Bias and imprecision were <±16%. Matrix effects ranged from -57.9 to 11.2 and -84.6 to 29.3% in plasma and breastmilk, respectively. All analytes were stable (within ±20% change from baseline) under all tested conditions (24h room temperature, 72h at 4°C, 3 freeze/thaw cycles at -20°C, and in the autosampler for 72h at 4°C). For proof of concept, buprenorphine and its metabolites were successfully quantified in authentic positive maternal and infant plasma and paired breastmilk specimens. This comprehensive, highly sensitive and specific method detects multiple buprenorphine markers in a small specimen volume.

Chemometric approach to open validation protocols: Prediction of validation parameters in multi-residue ultra-high performance liquid chromatography-tandem mass spectrometry methods.

The recent technological advancements of liquid chromatography-tandem mass spectrometry allow the simultaneous determination of tens, or even hundreds, of target analytes. In such cases, the traditional approach to quantitative method validation presents three major drawbacks: (i) it is extremely laborious, repetitive and rigid; (ii) it does not allow to introduce new target analytes without starting the validation from its very beginning and (iii) it is performed on spiked blank matrices, whose very nature is significantly modified by the addition of a large number of spiking substances, especially at high concentration. In the present study, several predictive chemometric models were developed from closed sets of analytes in order to estimate validation parameters on molecules of the same class, but not included in the original training set. Retention time, matrix effect, recovery, detection and quantification limits were predicted with partial least squares regression method. In particular, iterative stepwise elimination, iterative predictors weighting and genetic algorithms approaches were utilized and compared to achieve effective variables selection. These procedures were applied to data reported in our previously validated ultra-high performance liquid chromatography-tandem mass spectrometry multi-residue method for the determination of pharmaceutical and illicit drugs in oral fluid samples in accordance with national and international guidelines. Then, the partial least squares model was successfully tested on naloxone and lormetazepam, in order to introduce these new compounds in the oral fluid validated method, which adopts reverse-phase chromatography. Retention time, matrix effect, recovery, limit of detection and limit of quantification parameters for naloxone and lormetazepam were predicted by the model and then positively compared with their corresponding experimental values. The whole study represents a proof-of-concept of chemometrics potential to reduce the routine workload during multi-residue methods validation and suggests a rational alternative to ever-expanding procedures progressively drifting apart from real sample analysis.

Ex vivo studies for the passive transdermal delivery of low-dose naltrexone from a cream; detection of naltrexone and its active metabolite, 6ß-naltrexol, using a novel LC Q-ToF MS assay.

Naltrexone (NTX) is a long-acting opiate antagonist. Low-dose naltrexone (LDN) therapy has shown promising results in the treatment of several autoimmune disorders. Our aim was to formulate NTX into a cream for the delivery of LDN and develop an analytical technique for the quantification of NTX and its active metabolite 6-ß-naltrexol (NTXol) during transdermal diffusion cell permeation studies. A 1% w/w NTX cream was formulated and drug permeation was examined over 24 h using static Franz diffusion cells mounted with pig skin. A Liquid Chromatography Quadrupole-Time of Flight Mass Spectrometry (LC-MS Q-ToF) method was developed for the detection of NTX and NTXol in the receptor solution, skin membrane and residual cream on the donor chamber after completion of the diffusion studies. The cream formulation exhibited steady state release of NTX over 24 h after an initial lag time of 2.74 h. The bioconversion of NTX to NTXol in the skin membrane was 1.1%. It was concluded that the cream may be an effective formulation for the sustained transdermal delivery of LDN. The novel LC Q-ToF MS method allowed the accurate measurement of NTX and NTXol levels across the diffusion cell assemblies and the quantification of NTX metabolism in the skin.

Hair analysis for long-term monitoring of buprenorphine intake in opiate withdrawal.

BACKGROUND: Buprenorphine (BUP) is a psychoactive pharmaceutical drug largely used to treat opiate addiction. Short-term therapeutic monitoring is supported by toxicological analysis of blood and urine samples, whereas long-term monitoring by means of hair analysis is rarely used. Aim of this work was to develop and validate a highly sensitive ultrahigh-performance liquid chromatography tandem mass spectrometry method to detect BUP and norbuprenorphine (NBUP) in head hair. METHODS: Interindividual correlation between oral dosage of BUP and head hair concentration was investigated. Furthermore, an intra-individual study by means of segmental analysis was performed on subjects with variable maintenance dosage. Hair samples from a population of 79 patients in treatment for opiate addiction were analyzed. RESULTS: The validated ultrahigh-performance liquid chromatography tandem mass spectrometry protocol allowed to obtain limits of detection and quantification at 0.6 and 2.2 pg/mg for BUP and 5.0 and 17 pg/mg for NBUP, respectively. Validation criteria were satisfied, assuring selective analyte identification, high detection capability, and precise and accurate quantification. Significant positive correlation was found between constant oral BUP dosage (1-32 mg/d) and the summed up head hair concentrations of BUP and NBUP. Nevertheless, substantial interindividual variability limits the chance to predict the oral dosage taken by each subject from the measured concentrations in head hair. In contrast, strong correlation was observed in the results of intra-individual segmental analysis, which proved reliable to detect oral dosage variations during therapy. CONCLUSIONS: Remarkably, all hair samples yielded BUP concentrations higher than 10 pg/mg, even when the lowest dosage was administered. Thus, these results support the selection of 10 pg/mg as a cutoff value.

Examining naltrexone and alcohol effects in a minority population: results from an initial human laboratory study.

Prior clinical findings have indicated a potential lack of naltrexone efficacy among African Americans with alcohol dependence. However, no definitive conclusions have been drawn due to the relatively small numbers of African Americans in most alcohol treatment trials. The purpose of this study was to examine alcohol and naltrexone effects on healthy African-American individuals in a laboratory environment. Nonalcohol-dependent social drinking adults of African descent (n = 43) were recruited for participation. After consenting and completing the baseline assessment, they participated in four separate alcohol challenge sessions each separated by at least 10 days. During each of the sessions, subjects were administered alcohol or sham drinks, after pretreatment with either naltrexone (50 mg/day) or placebo in a double-blind fashion. The order of the four sessions was randomly assigned. During each session, breath alcohol levels and subjective responses were measured. Results indicate an alcohol effect among these subjects for subjective responses, but no naltrexone effect. Similar to the apparent lack of clinical efficacy findings, naltrexone does not appear to impact alcohol effects in African-American social drinkers. Future studies should investigate African-American populations with heavy drinking as well as alcohol-dependent subjects in order to strengthen the parallels to clinical findings.

Effect of surfactants and pH on naltrexone (NTX) permeation across buccal mucosa.

The objective of this pre-formulation study was to systematically investigate the effects of two surfactants (Brij 58(®) and Tween 80(®)) and change in solution pH on in vitro permeation of naltrexone HCl (NTX-HCl) across tissue engineered human buccal mucosa. For the study, 10mg/ml solutions of Tween 80(®) (0.1 and 1%, w/v) and Brij 58(®) (1%, w/v) were prepared in standard artificial saliva buffer solution (pH 6.8). For studying pH effects, solution pH was adjusted to either 7.5 or 8.2. As controls, three concentrations of NTX-HCl (2.5, 10 and 25mg/ml) were prepared. Using NTX standard solution (10mg/ml; pH 6.8), the permeation was observed between in vitro human and ex vivo porcine mucosa. It was observed that Brij 58(®) increased the permeation rates of NTX significantly. The flux of 10mg/ml solution (pH 6.8) increased from 1.9 ± 0.6 (× 10(2)) to 13.9 ± 2.2 (× 10(2))µg/(cm(2)h) (approximately 6-fold) in presence of 1% Brij 58(®). Increasing pH of NTX-HCl solution was found to increase the drug flux from 1.9 ± 0.6 (× 10(2)) (pH 6.8) to 3.0 ± 0.6 (× 10(2)) (pH 7.4) and 8.0 ± 3.5 (× 10(2)) (pH 8.2)µg/(cm(2)h), respectively. Histological analyses exhibited no tissue damage due to exposure of buccal tissue to Brij 58(®). The mean permeability coefficients (K(p)) for 2.5, 10 and 25mg/ml solutions of NTX-HCl (pH 6.8) were 5.0 (× 10(-2)), 1.8 (× 10(-2)) and 3.2 (× 10(-2))cm/h, respectively, consistent with data from published literature sources. Increase of NTX flux observed with 1% Brij 58(®) solution may be due to the effects of ATP. Increase in flux and the shortening of lag time observed by increasing in solution pH confirmed earlier finding that distribution coefficient (logD) of NTX is significantly affected by small increments in pH value and therefore plays an important role in NTX permeation by allowing faster diffusion across tissue engineered human buccal tissue.

Buprenorphine and norbuprenorphine findings in hair during constant maintenance dosage.

It is still a matter of debate whether a positive correlation between the dose and the amount of drug in the hair exists. Drugs such as buprenorphine (BUP) used under controlled conditions present an opportunity to prove a possible relationship. Due to discrepant findings of BUP/norbuprenorphine (NBUP) ratios in hair, in vitro degradation of both analytes in diluted acid was also investigated. The levels of BUP and NBUP in proximal hair sections from 18 subjects participating in a maintenance program were determined by liquid chromatography/tandem mass spectrometry following incubation with methanol and subsequent liquid/liquid extraction. BUP and NBUP were incubated in diluted hydrochloric acid at 60°C for up to 24 h. The alleged rearrangement products were simultaneously monitored. All hair samples tested positive for BUP (lower limit of detection-0.238 ng/mg hair) and NBUP (0.043-0.961 ng/mg hair). The concentration of NBUP in hair was consistently higher than that of BUP except for a single specimen. Degradation of BUP and NBUP was dependent on time; hydrolysis of NBUP occurred faster than that of BUP. The concentration of BUP and NBUP will be underestimated if analytes are recovered by acidic procedures. NBUP should be monitored in hair samples besides BUP for the sum of both BUP and NBUP may provide an estimate of BUP exposure following long-term administration of the drug.

Determination of naloxone and nornaloxone (noroxymorphone) by high-performance liquid chromatography-electrospray ionization- tandem mass spectrometry.

A highly sensitive method was developed to measure naloxone and its metabolite nornaloxone in human plasma, urine, and human liver microsomes (HLM). Naltrexone-d(3) and oxymorphone-d(3) were used as respective internal standards. Solid-phase extraction, using mixed mode extraction columns and 0.1 M phosphate buffer (pH 5.9), was combined with high-performance liquid chromatography interfaced by electrospray ionization to tandem mass spectrometry. The calibration range in plasma was 0.025 to 2 ng/mL for naloxone and 0.5 to 20 ng/mL for nornaloxone. It was 10 to 2000 ng/mL in urine and 0.5 to 20 ng/mL in HLM for both. Enzymatic hydrolysis of urine was optimized for 4 h at 40 degrees C. Intra- and interrun accuracy was within 15% of target; precision within 13.4% for all matrices. The mean recoveries were 69.2% for naloxone and 32.0% for nornaloxone. Analytes were stable in plasma and urine for up to 24 h at room temperature and in plasma after three freeze-thaw cycles. In human subjects receiving 16 mg buprenorphine and 4 mg naloxone, naloxone was detected for up to 2 h in all three subjects and up to 4 h in one subject. Mean AUC(0-24) was 0.303 +/- 0.145 ng/mL.h; mean C(max) was 0.139 +/- 0.062 ng/mL; and T(max) was 0.5 h. In 24-h urine samples, about 55% of the daily dose was excreted in either conjugated or unconjugated forms of naloxone and nornaloxone in urine. When cDNA-expressed P450s were incubated with 20 ng of naloxone, nornaloxone formation was detected for P450s 2C18, 2C19, and 3A4. Naloxone utilization exceeded nornaloxone formation for 2C19 and 3A4, indicating they may produce products other than nornaloxone. These results demonstrate a new method suitable for both in vivo and in vitro metabolism and pharmacokinetic studies of naloxone.

Brain opioid receptor binding in early abstinence from alcohol dependence and relationship to craving: an [11C]diprenorphine PET study.

The importance of the opioid receptor system in substance dependence is increasingly recognised. We used PET with the non-selective tracer [11C]diprenorphine to examine opioid receptor binding in early abstinence from alcohol dependence and the relationship to craving. We recruited 11 alcohol dependent patients and 13 controls. Subjects underwent one [11C]diprenorphine PET scan in early abstinence from dependent alcohol use (approximately 2 weeks) and 2 months later if continuously abstinent. Global and regional [11C]diprenorphine volumes of distribution (VD) were increased in alcohol dependent patients compared with controls but did not reach significance. We demonstrated a correlation between global and regional [11C]diprenorphine VD and craving in alcohol dependent patients which persisted in the anterior cingulate cortex into extended abstinence. This confirms previous work showing increased opioid receptor availability in early abstinence from substances of abuse and correlation with craving suggesting that the opioid system plays a fundamental role in this phase of addiction.

Simultaneous quantification of buprenorphine, norbuprenorphine, buprenorphine-glucuronide and norbuprenorphine-glucuronide in human umbilical cord by liquid chromatography tandem mass spectrometry.

A LCMS method was developed and validated for the simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine-glucuronide (BUP-Gluc) and norbuprenorphine-glucuronide (NBUP-Gluc) in human umbilical cord. Quantification was achieved by selected ion monitoring of precursor ions m/z 468.4 for BUP; 414.3 for NBUP; 644.4 for BUP-Gluc and 590 for NBUP-Gluc. BUP and NBUP were identified by MS(2), with m/z 396, 414 and 426 for BUP, and m/z 340, 364 and 382 for NBUP. Glucuronide conjugates were identified by MS(3) with m/z 396 and 414 for BUP-Gluc and m/z 340 and 382 for NBUP-Gluc. The assay was linear 1-50 ng/g. Intra-day, inter-day and total assay imprecision (%RSD) were <14.5%, and analytical recovery ranged from 94.1% to 112.3% for all analytes. Extraction efficiencies were >66.3%, and process efficiency >73.4%. Matrix effect ranged, in absolute value, from 3.7% to 7.4% (CV<21.8%, n=8). The method was selective with no endogenous or exogenous interferences from 41 compounds evaluated. Sensitivity was high with limits of detection of 0.8 ng/g. In order to prove method applicability, an authentic umbilical cord obtained from an opioid-dependent pregnant woman receiving BUP pharmacotherapy was analyzed. Interestingly, BUP was not detected but concentrations of the other metabolites were NBUP-Gluc 13.4 ng/g, BUP-Gluc 3.5 ng/g and NBUP 1.2 ng/g.

Prevalence and disposition of drugs of abuse and opioid treatment drugs in oral fluid.

Testing oral fluid for drugs of abuse has been studied under many conditions but rarely has been evaluated in large population databases. We evaluated oral fluid tests in a database from a commercial laboratory in the United Kingdom composed of 8679 confirmed positive results. The results originated from 635,000 specimens collected over the period of May 2004 through September 2006. Oral fluid specimens were collected with the Intercept oral fluid collection device, screened by enzyme immunoassay, and confirmed by GC-MS or GC-MS-MS. The database was organized by collection settings (legal/treatment, N = 8198 specimens; and workplace, N = 481 specimens) and by drug groups (without consideration of collection setting). The drug groups were as follows (number of confirmed positives): amphetamines (468); benzodiazepines (892); buprenorphine (276); cannabinoids (725); cocaine (1443); methadone (998); and opiates (5739). The goal of the study was to provide drug/metabolite prevalence data, concentrations, and drugs/metabolite patterns encountered in oral fluid. Comparison of results by collection setting indicated differences in relative frequency, primarily for opiates and cannabinoids. Opiate positives were most frequently observed for specimens collected in legal/treatment settings, whereas cannabinoids were most frequently reported in the workplace. An array of information on drug and metabolite occurrences and concentration arose from evaluation of the data by drug groups. Amphetamine was the predominant drug reported for the Amphetamines Group; approximately 10% were also positive for MDA and/or MDMA; and methamphetamine was rarely reported. Multiple combinations of diazepam, nordiazepam, oxazepam, temazepam, chlordiazepoxide, and lorazepam were reported for the Benzodiazepine Group. Buprenorphine, an opioid treatment drug, was the predominant analyte reported, but low concentrations of norbuprenorphine were frequently reported. THC was the predominant analyte reported in the Cannabinoids Group and was frequently reported in combination with cannabidiol and cannabinol. THCCOOH was reported in only 10.8% of these specimens and was never reported in the absence of THC. HO-THC was reported in 5.7% of the specimens. In the Cocaine Group, cocaine was present, often in combination with BZE, but also as the sole analyte in 17.3% of the specimens. AEME and cocaethylene were reported in 10.4% and 5.5% of the specimens. Methadone, another opioid treatment drug, was reported in all specimens for the Methadone Group; EDDP was reported in 30.1% of the specimens. In the Opiates Group, morphine, codeine and 6-acetylmorhine were most frequently reported, often in combination. The frequency of detection of 6-acetylmorphine when morphine was present (N = 4575 specimens) was 77.5%. Surprisingly, heroin (19.0%; N = 1091 specimens) and 6-acetylcodeine (24.9%; N = 1431 specimens) were frequently reported. The results from analysis of this large oral fluid database offer a rich mixture of new information on detection frequency, drug and metabolite patterns, and concentration data on drugs of abuse.

Hair analysis by LC-MS as evidence of nalbuphine abuse by a nurse.

Individuals in any profession can succumb to chemical abuse. Among the healthcare profession, nurses represent a specific group because of their ease of access to drugs, particularly narcotics. Opioids, potentially highly addictive agents, are usually their drug of choice. Nalbuphine, a synthetic opioid analgesic, is prescribed for moderate-to-severe acute pain, for chronic pain syndromes, and in obstetrics to decrease the adverse respiratory effect of opioid epidural administration. The case of a nurse who was suspected of drug misuse after the disappearance of two nalbuphine ampules in an obstetrics service is described. Because of discrepancies in the results of her blood and urine samples, a sample of head hair was subsequently collected from the nurse. A hair analysis of nalbuphine by liquid chromatography-mass spectrometry has not been previously described. Following decontamination and grinding, hair was mixed with a Söerensen buffer, then subjected to ultrasonic treatment (1 h), and extracted with ethyl acetate. A quantitative analysis was performed with two channels (30 and 45 V), and it is based on a m/z 358 for nalbuphine and a m/z 330 for methylclonazepam as an internal standard. The method was linear from 0.020 to 12 ng/mg of hair (R(2) = 0.972), and the limit of detection and limit of quantitation are 0.020 ng/mg. Accuracy (CV), assessed at 0.4 and 1.6 ng/mg of hair, was 6.18% and 5.77%, respectively, for intraday assays and 4.5% and 10.9% for interday assays. Recovery efficiency at 1.6 ng/mg and 8 ng/mg of hair was 100% and 97.4%, respectively. The hair specimen from the nurse (6 cm) was cut into three equal lengths. Nalbuphine, venlafaxine, and nordiazepam were detected. The concentration of nalbuphine was similar in the three hair locks: 5.07, 7.06, and 5.70 ng/mg of hair. A hair analysis revealed the repeated intake of nalbuphine by the nurse. This person was treated for depression for several months with Effexor (venlafaxine) and Nordaz (nordiazepam) prior to the investigation. Hair appears to be a unique matrix to provide evidence for chronic drug exposure by establishing a historic record that is not possible by blood or urine analysis.

Nano-level detection of naltrexone hydrochloride in its pharmaceutical preparation at Au microelectrode in flowing solutions by fast fourier transforms continuous cyclic voltammetry as a novel detector.

An easy and fast Fourier transform continuous cyclic voltammetric technique for monitoring of ultra trace amounts of naltrexone in a flow-injection system has been introduced in this work. The potential waveform, consisting of the potential steps for cleaning, stripping and potential ramp, was continuously applied on an Au disk microelectrode (with a 12.5 microm in radius). The proposed detection method has some of advantages, the greatest of which are as follows: first, it is no more necessary to remove oxygen from the analyte solution and second, this is a very fast and appropriate technique for determination of the drug compound in a wide variety of chromatographic analysis methods. The method was linear over the concentration range of 0.34-34000 pg/mL (r = 0.9985) with a limit of detection 8.0 x 10(-4) nM. The method has the requisite accuracy, sensitivity, precision, and selectivity to assay naltrexone in tablets. The influences of pH of eluent, accumulation potential, sweep rate, and accumulation time on the determination of the naltrexone were considered.

Diffusion of naltrexone across reconstituted human oral epithelium and histomorphological features.

In transbuccal absorption a major limitation could be the low permeability of the mucosa which implies low drug bioavailability. The ability of naltrexone hydrochloride (NLX) to penetrate a resembling histologically human buccal mucosa was assessed and the occurrence of any histomorphological changes observed. We used reconstituted human oral (RHO) non-keratinised epithelium as mucosal section and a Transwell diffusion cells system as bicompartmental model. Buccal permeation was expressed in terms of drug flux (J(s)) and permeability coefficients (K(p)). Data were collected using both artificial and natural human saliva. The main finding was that RHO does not restrain NLX permeation. Drug transport across the epithelium was observed also in presence of various concentrations of penetration enhancers, without any significant differences. On the contrary, the flux throughout the mucosa was extensively affected by iontophoresis. Histologically, no sign of flogosis was observed in any specimen under experiment without iontophoresis, whereas cytoarchitectural changes, up to nuclear pycnosis or cellular swelling, were determined as a consequence of the application of electric fields.

Pentazocine monitoring in rat hair and plasma by HPLC-fluorescence detection with DIB-Cl as a labelling reagent.

Pentazocine (PZ) in rat hair and plasma was determined by HPLC-fluorescence detection with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a labelling reagent and cyclazocine (CZ) as an internal standard (IS). PZ and IS extracted from hair or plasma sample were derivatized with DIB-Cl and the resulted solution was cleaned up with solid phase extraction. The isocratic separation of DIB-PZ and -CZ within 20 min could be achieved by a Wakopak Handy-ODS column (250 x 4.6 mm i.d.) using a mobile phase composed of 0.1 mol/L acetate buffer (pH 6.2):acetonitrile (25:75, v/v). The detection limits of PZ at a signal-to-noise ratio of 3 for rat hair and plasma were 0.18 ng/mg and 0.57 ng/mL, respectively. Reproducible and precise results could be obtained by an IS method with RSD values less than 6.6% for within- and between-day measurements. The method was successfully applied for the monitoring of PZ levels in Zucker rat hair and plasma samples after a single administration of 25 mg/kg PZ. Moreover, incorporation rates of PZ into black and white hair of Zucker rat were evaluated.

A survey of buprenorphine related deaths in Singapore.

Buprenorphine is available in Singapore as substitution treatment for opioid dependence since 2002. This study surveys buprenorphine related deaths in Singapore between September 2003 and December 2004. The aims are to establish the autopsy prevalence of buprenorphine related deaths and the demographical and toxicological profile of the cases. Toxicological screening was performed for all unnatural deaths, deaths involving known drug addicts, as well as when autopsy revealed no obvious cause of death. Twenty-one cases had buprenorphine detected in post-mortem blood and/or urine samples. Eighteen were sudden deaths. There were two fatal falls from height and one death by hanging. All subjects were male. The age range was 24-48 years. Fourteen subjects were between 30 and 39 years of age. The mean age was 35 years. The majority (62%) were Chinese. Eleven (52%) were known drug abusers. For sudden deaths, two groups were identified. Six cases died from natural causes. Blood buprenorphine levels ranged from undetected (detected in urine) to 3.2 ng/mL (mean 1.4 ng/mL). Twelve cases were attributed directly and indirectly to mixed drug poisoning. Blood buprenorphine levels ranged from undetected (detected in urine) to 17 ng/mL (mean 3.2 ng/mL). Nineteen cases showed concurrent abuse of buprenorphine and benzodiazepine, diazepam being the most frequently detected, followed by nitrazepam and midazolam. The availability of buprenorphine as substitution therapy is associated with an increase in buprenorphine related deaths. The danger of co-abuse of buprenorphine and benzodiazepines is highlighted.

Extraction and determination by liquid chromatography and spectrophotometry of naloxone in microparticles for drug-addiction treatment.

This paper discusses the development and validation of two analytical methods for the assay of naloxone in microparticles, as used in the therapy of opioid drug addiction (weaning). A UV-Vis spectrophotometric method is proposed to study drug loading and drug release, due to its ease and simplicity of performance, while a high performance liquid chromatographic method is developed as a means of stability-indication. Both analytical procedures were validated according to the International Committee for Harmonization (ICH) guidelines. Although the ranges and wavelengths were different for the two analytical methods, they were both found to be specific, linear, precise, and accurate under the determined conditions.

Buprenorphine detection in biological samples.

Buprenorphine is a narcotic analgesic used in the treatment of moderate-to-severe pain. In Italy, buprenorphine is now being used as an alternative in the therapeutic treatment of heroin abusers. Even if confirmation methods are needed for identification and quantitation, a rapid and sensitive test, such as an immunoassay, is essential for screening. We evaluated the Buprenorphine One-step ELISA test (designed for serum specimens) for the qualitative determination of buprenorphine in other matrices, such as urine, saliva and hair.