Simplified processing and rapid quantification of buprenorphine, norbuprenorphine, and their conjugated metabolites in human plasma using UPLC-MS/MS: Assessment of buprenorphine exposure during opioid use disorder treatment.

Opioid use disorder (OUD) is a chronic neurobehavioral ailment and is prevalent in pregnancy. OUD is commonly treated with methadone or buprenorphine (BUP). Pregnancy is known to alter the pharmacokinetics of drugs and may lead to changes in drug exposure and response. A simple, specific, and sensitive analytical method for measuring the parent drug and its metabolites is valuable for assessing the impact of pregnancy on drug exposure. A new liquid chromatography-tandem mass spectrometric method that utilized a simple protein precipitation procedure for sample preparation and four deuterated internal standards for quantification was developed and validated for BUP and its major metabolites (norbuprenorphine [NBUP], buprenorphine-glucuronide [BUP-G], and norbuprenorphine-glucuronide [NBUP-G]) in human plasma. The standard curve was linear over the concentration range of 0.05-100 ng/mL for BUP and NBUP, and 0.1-200 ng/mL for BUP-G and NBUP-G. Intra- and inter-day bias and precision were within ±15% of nominal values for all the analytes. Quality controls assessed at four levels showed high recovery consistently for all the analytes with minimal matrix effect. Adequate analyte stability was observed at various laboratory conditions tested. Overall, the developed method is simple, sensitive, accurate and reproducible, and was successfully applied for the quantification of BUP and its metabolites in plasma samples collected from pregnant women in a clinical study assessing BUP exposure during OUD treatment.

Monitoring medication and illicit drug consumption in a prison by wastewater-based epidemiology: Impact of COVID-19 restrictions.

Drug consumption in prisons is a concern for the safety of incarcerated people and staff. Typically, drug use prevalence in prisons is estimated through urinalysis and intelligence operations, which can be intrusive and stressful. An alternative approach, wastewater-based epidemiology (WBE), was used in this study to estimate the consumption of licit and illicit drugs for the entire population of a prison in Australia. Wastewater samples were collected from March to December 2020, covering periods of no restrictions and periods when prison access was restricted to prevent the transmission of COVID-19. Target biomarkers were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The average consumption of common illicit drugs (MDMA, methamphetamine and cocaine) over the sampling period in the prison (0.5 - 4.5 mg/1000 people/day) was two to three orders of magnitude lower than in the community population (254 - 1000 mg/1000 people/day). Comparison of WBE estimates against pharmacy dispensing data suggested potential illicit buprenorphine consumption at the prison. Methamphetamine and buprenorphine use decreased when no visitors were allowed (18% - 72% decrease for methamphetamine; about half decrease for buprenorphine) and increased once these restrictions were eased (22% - 39% increase for methamphetamine; 44% - 67% increase for buprenorphine). The changes in drug use may be attributed in part to a reduction of drug trafficking into the prison from visitors or non-essential staffs and in part to the reduced contribution of urine from staff who used toilets within the prison. This study provided useful information on the scale of illicit drug use and extra-medical use of licit drugs in prison, and its changes under different security conditions.

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.

Determination of buprenorphine, norbuprenorphine, naloxone, and their glucuronides in urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), naloxone (NAL), and their glucuronide conjugates BUP-G, NBUP-G, and NAL-G in urine samples was developed. The method, omitting a hydrolysis step, involved non-polar solid-phase extraction, liquid chromatography on a C18 column, electrospray positive ionization, and mass analysis by multiple reaction monitoring. Quantification was based on the corresponding deuterium-labelled internal standards for each of the six analytes. The limit of quantification was 0.5 µg/L for BUP and NAL, 1 µg/L for NAL-G, and 3 µg/L for NBUP, BUP-G, and NBUP-G. Using the developed method, 72 urine samples from buprenorphine-dependent patients were analysed to cover the concentration ranges encountered in a clinical setting. The median (maximum) concentration was 4.2 µg/L (102 µg/L) for BUP, 74.7 µg/L (580 µg/L) for NBUP, 0.9 µg/L (85.5 µg/L) for NAL, 159.5 µg/L (1370 µg/L) for BUP-G, 307.5 µg/L (1970 µg/L) for NBUP-G, and 79.6 µg/L (2310 µg/L) for NAL-G.

Increase in drugs-of-abuse findings in post-mortem toxicology due to COVID-19 restrictions-First observations in Finland.

A lot has been published on the anticipated effects of the current COVID-19 pandemic on users of illegal drugs. In this study, we present evidence-based data on such effects, namely, the increased number of drug findings in post-mortem investigations. All post-mortem toxicology cases positive for at least one of the following: buprenorphine, amphetamine or cannabis, were investigated in the first 8 months of the year 2020, and the monthly numbers were compared to those in the previous 5 years from 2015 to 2019. These substances served as indicator analytes that could reveal changes in the drug using population. Right after the government restrictions came into force in March 2020, the numbers of buprenorphine, amphetamine and cannabis findings increased. The increase was most noticeable for amphetamine and was evident in all age groups. Our findings indicate that the assumptions on the increased risk of drug-related harm (including death) have become reality. Reduced access to harm-reduction services seems to have increased the mortality among individuals that use buprenorphine, amphetamine or cannabis. Significant and prompt actions need to be taken in order to find new ways in helping this vulnerable group of people.

High Prevalence of Buprenorphine in Prenatal Drug Screens in an Appalachian City.

OBJECTIVES: To define the magnitude of buprenorphine presence in the urine drug screens of pregnant women and to assess the presence of illicit buprenorphine use versus the presence of prescribed buprenorphine use. METHODS: Initial prenatal drug screen results for all pregnant patients in our practice for a 1-year period were analyzed and tabulated. RESULTS: Buprenorphine was found in the urine drug screens of 16% of pregnant patients. The presence of buprenorphine was by far the highest for any substance associated with neonatal abstinence syndrome (NAS). We estimate that the exposure to buprenorphine of approximately one-third of individuals in our population is associated with illicit buprenorphine use. CONCLUSIONS: The high rate of NAS in our region is primarily associated with both illicit and prescribed buprenorphine rather than other substances. Buprenorphine usage at the time that prenatal care is initiated, rather than opiate use at the onset of prenatal care, is the underlying factor that must be addressed if our region is to successfully combat our high rates of NAS.

Highly selective extraction and voltammetric determination of the opioid drug buprenorphine via a carbon paste electrode impregnated with nano-sized molecularly imprinted polymer.

An electrochemical sensor for the opioid drug buprenorphine (BUP) is described. Molecularly imprinted polymer nanoparticles (nanoMIP) were prepared and used to modify a carbon paste electrode (CPE). The BUP-imprinted polymer was synthesized using precipitation polymerization. The resulting polymer along with multiwalled carbon nanotubes (MWCNT) was used to fabricate the modified CPE which exhibited an anodic peak at about +0.73 V (vs. Ag/AgCl) for BUP. The MIP on the CPE functions as selective recognition element with an imprinting factor of 5.6. The assay consists of two-steps, viz. analyte extraction at the electrode surface and differential pulse voltammetric determination of BUP. The effects of various parameters on the electrochemical signal were optimized, and the selectivity of the modified CPE over cross reactants was studied. At optimum experimental conditions, the response is linear in the 1 nM to 50 µM BUP concentration range, and the detection limit is 0.6 nM (at S/N = 3). This method was applied to the determination of BUP in spiked urine with acceptable relative standard deviations (3.2-4.4%). Graphical abstract Schematic representation of buprenorphine (BUP) recognition and voltammetric determination at the surface of carbon paste electrode modified with imprinted polymer and carbon nanotubes.

In situ synthesis of rhodium nanoparticles - Mesoporous carbon hybrid via a novel and facile nanocasting method for simultaneous determination of morphine and buprenorphine.

An in situ and facile nanocasting procedure has been developed to embed Rhodium nanoparticles (RhNPs) in mesoporous carbon (MC) matrix via carbonizing ß-Cyclodextrin capped RhNPs (ß-CDs@RhNPs) as a source of both carbon and metal, in the presence of SBA-15 as hard template. Firstly, ß-CDs was used to coat and stabilize the RhNPs, and then coated RhNPs was applied as carbon source during the carbonization step. In this way, biocompatible materials are used as much as possible. The transmission electron microscopy, field emission scanning electron microscopy, Fourier transform infrared, BET surface area and X-ray diffraction devices were used to characterize the nanomaterials. The nanocomposite (RhNPs-MC) was casted on the glassy carbon electrode (GCE) to fabricate an electrochemical sensor (RhNPs-MC/GCE). This sensor shows high efficiency toward simultaneous determination of Morphine (Mp) and Buprenorphine (Bp), electrochemically, what other modified electrodes cannot do. For Mp, the linear range and limit of detection were obtained 0.1-20 µM and 40 nM, respectively, and these data were obtained about 0.1-14 µM and 45 nM for Bp determination. Less steps of synthesis, biocompatibility and facility are the major advantages of the process, and some benefits of the sensor are its separate signals, fast response time, sensitivity, and simple use without need for pretreatment.

Opioid Deaths in Milwaukee County, Wisconsin 2013-2017: The Primacy of Heroin and Fentanyl.

Heroin and fentanyl are the overwhelming and increasing cause of opioid deaths in Milwaukee County, Wisconsin. We reviewed all drug and opioid deaths from 2013 to 2017 to delineate the specific opioid drugs involved and changes in their incidence. From 2013 to 2017, 980 deaths were due to opioids, rising from 184 in 2013 to 337 in 2017. In 2017, opioid deaths exceeded combined non-natural deaths from homicide and suicide. Illicit heroin and fentanyl/analogs caused 84% of opioid deaths and 80% of drug deaths, with no increase in deaths due to oral prescription drugs such as oxycodone and hydrocodone. Any approach to decreasing this dramatic increase in opioid deaths should first focus on interdicting the supply and cheap availability of these illicit opioids. Fentanyl and its analogs represent the most deadly opioids and the greatest threat to human life in our population.

Quantitative Analysis of Novel Synthetic Opioids, Morphine and Buprenorphine in Oral Fluid by LC-MS-MS.

The opioid epidemic has become a national health emergency in the USA. While heroin and prescription opioid abuse is not uncommon, synthetic opioid use has risen dramatically, creating a public safety concern. Like traditional opioids, novel synthetic opioids are abused due to their analgesic and euphoric effects. Some adverse side effects include respiratory distress, nausea and decreased consciousness. Synthetic opioids have emerged into the illicit and online drug market, including AH-7921, MT-45, U-series and W-series. Though originally developed by pharmaceutical companies, these substances are not well studied in humans and comprehensive analytical methods for detecting and quantifying these opioids are limited. Oral fluid is a useful biological matrix for determining recent drug use, does not require a trained medical professional, and can be collected under direct observation, deterring adulteration. The purpose of this research was to develop and validate a comprehensive analytical method for the detection and quantification of morphine, 6-acetylmorphine, buprenorphine, U-47700, U-49900, U-50488, AH-7921, MT-45, W-18 and W-15 in oral fluid collected via Quantisal. This was achieved by solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. The limits of detection and quantitation were 5 ng/mL and 10 ng/mL, respectively. Linearity was observed between 10 and 500 ng/mL (R2 ≥ 0.9959). Bias and imprecision were <±11.1%. Matrix effects ranged from -21.1 to 13.7%. No carryover was detected following injection of the highest calibrator. All analytes were stable (within ±15% change from baseline) under all tested conditions (24 h at room temperature, 72 h at 4°C, and in the autosampler for 60 h at 4°C).

Suicide by Medication Overdose in Prison: A Study of Three Cases.

Suicide is one of the principal causes of mortality in a prison environment. Although suicide by medication overdose is less frequent than suicide by hanging, self-strangulation, or vein cutting, it raises questions as to how the medications are obtained, particularly in view of the specific organization of the medication circuit in prisons. We present three cases of suicide by medication overdose involving different therapeutic classes with different distribution circuits and review the regulatory requirements and the measures that could be taken to prevent such suicides.

Interpreting quantitative urine buprenorphine and norbuprenorphine levels in office-based clinical practice.

BACKGROUND: Quantitative urine buprenorphine testing is used to monitor patients receiving buprenorphine for the treatment of opioid use disorder (OUD), however the interpretation of urine buprenorphine testing is complex. Currently, interpretation of quantitative buprenorphine testing is guided by data from drug assay development studies and forensic labs rather than clinical treatment cohorts. METHODS: In this retrospective study, we describe the patterns of urine buprenorphine and norbuprenorphine levels in patients prescribed sublingual buprenorphine for OUD in an office-based addiction treatment clinic. Urine buprenorphine and norbuprenorphine levels were analyzed in patients who reported having adulterated their urine, patients clinically suspected of adulterating their urine, and patients without concern for urine adulteration. Finally, we tested the accuracy of urine buprenorphine, norbuprenorphine, and norbuprenorphine: buprenorphine ratio (Norbup:Bup) to identify adulterated urine samples. RESULTS: Patients without suspicion for urine adulteration rarely provided specimens with buprenorphine >=1000ng/ml (4.4%), while the proportion provided by those who endorsed or were suspected of urine adulteration was higher (42.9%, 40.6%, respectively). Compared to patients without reported urine adulteration, specimens from patients who reported or were suspected of urine adulteration had significantly higher buprenorphine (p=0.0001) and lower norbuprenorphine (<0.0001) levels, and significantly lower Norbup:Bup ratios (p=0.04). Buprenorphine >=700ng/ml offered the best accuracy for discriminating between adulterated and non-adulterated specimens. CONCLUSION: This study describes the patterns of urine buprenorphine and norbuprenorphine levels from patients with OUD receiving buprenorphine treatment in an office-based addiction treatment clinic. Parameters for identifying urine adulterated by submerging buprenorphine medication in the urine specimen are discussed.

[The peculiarities of the comprehensive study of buprenorphine]. / Osobennosti kompleksnogo issledovaniia buprenorfina.

The authors describe the methods of pharmaceutical and criminalistics analysis that are finding the increasingly wider application for the drug expertise (identification) and elucidation of the circumstances conducive to the commission of an offence. The special emphasis is laid on the buprenorphine studies with the use of the colour chemical reactions, thin-layer chromatography, gas chromatographic analysis, high-performance liquid chromatography, IR spectrometry, and other modern techniques. The methods based on the recent achievements in pharmaceutical and criminalistics sciences can be employed in the activities intended to control the illegal drug circulation. Moreover, they may be of importance for obtaining valuable information about the actions of the persons involved in the trafficking or synthesis (production) of the prohibited substances after they are brought to criminal responsibility and/or appear before the court.

Do all screening immunoassay positive buprenorphine samples need to be confirmed?

Background Interference from opiates in the Microgenics CEDIA® Buprenorphine assay is known to produce false-positive buprenorphine screening immunoassay results necessitating confirmatory buprenorphine testing by chromatography/mass spectrometry methods. Method We reviewed data on falsely positive buprenorphine immunoassay screen (cut-off ≥ 5 µg/L) but negative for buprenorphine by gas chromatography mass spectrometry (cut-off ≥ 5 µg/L) and had a positive opiate immunoassay result (cut-off ≥ 300 µg/L). The results were collected over three months, and the data were evaluated to determine whether there is an opiate immunoassay screen concentration below which a false-positive buprenorphine result will not occur. Results We found that cross-reactivity in the CEDIA® buprenorphine immunoassay by opiates at concentrations <2000 µg/L will not cause a false-positive buprenorphine result. After changing our practice to not proceed with confirmatory buprenorphine gas chromatography mass spectrometry assay when the opiate screening concentration is below an even more conservative cut-off of <1500 µg/L, we estimate a potential cost-saving of AU$ 17,810 per year without compromising clinical care. Conclusion Samples with CEDIA® opiate immunoassay result <2000 µg/L and a positive CEDIA® buprenorphine immunoassay screen do not require confirmatory testing for buprenorphine.

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.

Challenges and opportunities in using wastewater analysis to measure drug use in a small prison facility.

INTRODUCTION AND AIMS: Wastewater analysis (WWA) is intended to be a direct and objective method of measuring substance use in large urban populations. It has also been used to measure prison substance use in two previous studies. The application of WWA in this context has raised questions as to how best it might be used to measure illicit drug use in prisons, and whether it can also be used to measure prescription misuse. We applied WWA to a small regional prison to measure the use of 12 licit and illicit substances. We attempted to measure the non-medical use of methadone and buprenorphine and to compare our findings with the results of the prison's mandatory drug testing (MDT). DESIGN AND METHODS: Representative daily composite samples were collected for two periods of 12 consecutive days in May to July 2013 and analysed for 18 drug metabolites. Prescription data and MDT results were obtained from the prison and compared with the substance use estimates calculated from WWA data. RESULTS: Daily use of methamphetamine, methadone, buprenorphine and codeine was detected, while sporadic detection of ketamine and methylone was also observed. Overall buprenorphine misuse appeared to be greater than methadone misuse. DISCUSSION AND CONCLUSIONS: Compared with MDT, WWA provides a more comprehensive picture of prison substance use. WWA also has the potential to measure the misuse of medically prescribed substances. However, a great deal of care must be exercised in quantifying the usage of any substance in small populations, such as in prisons.

Quantitation of Total Buprenorphine and Norbuprenorphine in Meconium by LC-MS/MS.

Buprenorphine (Suboxone, Zubsolv, Buprenex, Butrans, etc.) is an opioid drug that has been used to treat opioid dependence on an outpatient basis, and is also prescribed for managing moderate to severe pain. Pregnant women may be prescribed buprenorphine as part of a treatment plan for opioid addiction. This chapter quantitates buprenorphine and norbuprenorphine in meconium by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Determination of buprenorphine, norbuprenorphine and naloxone in fingernail clippings and urine of patients under opioid substitution therapy.

The aim of this study was to develop and validate a method for the determination of buprenorphine (BUP), norbuprenorphine (NBUP) and naloxone (NAL) in fingernails and urine samples collected from former heroin users under suboxone substitution therapy. The analytes were extracted by solid-liquid or solid-phase extraction and were analyzed by liquid chromatography-mass spectrometry. The validation of the analytical methods developed included linearity, recovery, accuracy, precision, ion suppression, sensitivity of interfaces and limits of determination and quantification. The validated methods were applied to samples from 46 individuals. The majority of the urine samples were positive for all analytes (93.5% for BUP, 95.7% for NBUP and 84.8% for NAL). In nails, a higher detection rate was observed for NBUP and BUP (89.1%), compared with NAL (10.9%). The median values of the NBUP/BUP and the NAL/BUP ratio were 2.5 and 0.3 in urine and 0.8 and 0.3 in nails, respectively. A statistically significant correlation was found between the BUP, NBUP and total BUP (BUP and NBUP) concentrations in urine and those in nails. A weak correlation was observed between the daily dose (mg/day) and total BUP (P = 0.069), or NBUP (P = 0.072) concentrations in urine. In contrast, a strong correlation was found between the total amount of BUP administered during the last 12 months and total BUP (P = 0.038), or NBUP (P = 0.023) concentrations in urine. Moreover urine BUP, NBUP and total BUP concentrations correlated significantly. Our study demonstrated successfully the application of the developed method for the determination of the three analytes in urine and nails.

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.

An unusual case of death probably triggered by the association of buprenorphine at therapeutic dose with ethanol and benzodiazepines and with very low norbuprenorphine level.

Buprenorphine is largely prescribed for maintenance treatment in opioid dependence due to its safety profile. Nevertheless, fatalities at therapeutic dose have been described when associated with other central nervous system depressants, such as ethanol or benzodiazepines. Here, we report a case of death due to association of buprenorphine at therapeutic dose with benzodiazepines and ethanol. Although toxicity has been often attributed to its metabolite norbuprenorphine rather than to buprenorphine itself, in our case, norbuprenorphine was not detected in urine and bile and only in traces in blood. Moreover, the presence in blood of free buprenorphine but not of glucuronide metabolites argues for an unusual early death, at the beginning of buprenorphine metabolism. We propose that in the context of prior toxic impregnation, buprenorphine directly (and not via its metabolite norbuprenorphine) acted as a triggering factor by blocking the ventilatory response, rapidly leading to fatal respiratory depression.