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.

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.

On-chip pulsed electromembrane extraction as a new concept for analysis of biological fluids in a small device.

In the present work, an on-chip pulsed electromembrane extraction technique followed by HPLC-UV was developed for the analysis of codeine, naloxone and naltrexone as model analytes in biological fluids. The chip consisted of two channels for the introduction of the donor and acceptor phases. The channels were carved in two poly (methyl methacrylate) plates and a porous polypropylene membrane, which is impregnated by an organic solvent separating the two channels. Two platinum electrodes were mounted on the bottom of these channels and a pulsed electrical voltage was applied as an electrical driving force for the migration of ionized analytes from the sample solution through the porous sheet membrane into the acceptor phase. Using the pulsed voltage provided effective and reproducible extractions and could successfully overcome the disadvantages of applying constant voltages. Effective parameters of on-chip pulsed electromembrane extraction such as chemical composition of the organic solvent, applied voltage, pH of the donor and acceptor phases, flow rate and pulse duration were optimized using one-variable-at-a-time method. Under the optimized conditions, the model analytes were effectively extracted from different matrices and good linearity in the range of 10.0-500.0µgL-1 was achieved for calibration curves with coefficients of determinations (R2) higher than 0.997. Extraction recoveries and%RSDs were obtained in the ranges of 28.6-32.9% and 2.15-3.8, respectively. Also, limits of detection were obtained in the ranges of 5-10µgL-1 and 2-5µgL-1 in plasma and urine samples, respectively.

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.

Determination of residual levels of naloxone, yohimbine, thiophanate, and altrenogest in porcine muscle using QuEChERS with liquid chromatography and triple quadrupole mass spectrometry.

A quick, easy, cheap, effective, rugged, and safe QuEChERS (method) was used for the simultaneous detection of four veterinary drug residues, namely naloxone, yohimbine, thiophanate, and altrenogest, in porcine muscle, using liquid chromatography with electrospray ionization triple quadrupole tandem mass spectrometry. Because of the unavailability of a suitable internal standard, matrix-matched calibrations were used for quantification, with determination coefficients ≥ 0.9542. The accuracy (expressed as recovery %) ranged from 60.53 to 83.25%, and the intra- and interday precisions (expressed as relative standard deviations) were <12%. The limits of quantification were 5, 0.5, 2, and 5 ng/g for naloxone, yohimbine, thiophanate, and altrenogest, respectively. Samples purchased from local markets in Seoul, Republic of Korea, revealed no traces of the target analytes. The developed method described herein is sensitive and reliable and can be applied to quantify the tested veterinary drugs in animal tissues.

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.

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.

Inhibition of morphine-induced cAMP overshoot: a cell-based assay model in a high-throughput format.

Opiates are not only potent analgesics but also drugs of abuse mainly because they produce euphoria. Chronic use of opiates results in the development of tolerance and dependence. Dr Marshall Nirenberg's group at the National Institutes of Health (NIH) was the first to use a cellular model system of Neuroblastoma × Glioma hybrid cells (NG108-15) to study morphine addiction. They showed that opiates affect adenylyl cyclase (AC) by two opposing mechanisms mediated by the opiate receptor. Although the cellular mechanisms that cause addiction are not yet completely understood, the most observed correlative biochemical adaptation is the upregulation of AC. This model also provides the opportunity to look for compounds which could dissociate the acute effect of opiates from the delayed response, upregulation of AC, and thus lead to the discovery of non-addictive drugs. To identify small molecule compounds that can inhibit morphine-induced cAMP overshoot, we have validated and optimized a cell-based assay in a high throughput format that measures cellular cAMP production after morphine withdrawal. The assay performed well in the 1536-well plate format. The LOPAC library of 1,280 compounds was screened in this assay on a quantitative high-throughput screening (qHTS) platform. A group of compounds that can inhibit morphine-induced cAMP overshoot were identified. The most potent compounds are eight naloxone-related compounds, including levallorphan tartrate, naloxonazine dihydrochloride, naloxone hydrochloride, naltrexone hydrochloride, and naltriben methanesulfonate. The qHTS approach we used in this study will be useful in identifying novel inhibitors of morphine induced addiction from a larger scale screening.

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.

Development and validation of a HPLC method for the determination of buprenorphine hydrochloride, naloxone hydrochloride and noroxymorphone in a tablet formulation.

A simple isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) was developed for the simultaneous determination of buprenorphine hydrochloride, naloxone hydrochloride dihydrate and its major impurity, noroxymorphone, in pharmaceutical tablets. The chromatographic separation was achieved with 10 mmol L(-1) potassium phosphate buffer adjusted to pH 6.0 with orthophosphoric acid and acetonitrile (17:83, v/v) as mobile phase, a C-18 column, Perfectsil Target ODS3 (150 mm x 4.6mm i.d., 5 microm) kept at 35 degrees C and UV detection at 210 nm. The compounds were eluted isocratically at a flow rate of 1.0 mL min(-1). The average retention times for naloxone, noroxymorphone and buprenorphine were 2.4, 3.8 and 8.1 min, respectively. The method was validated according to the ICH guidelines. The validation characteristics included accuracy, precision, linearity, range, specificity, limit of quantitation and robustness. The calibration curves were linear (r>0.996) over the concentration range 0.22-220 microg mL(-1) for buprenorphine hydrochloride and 0.1-100 microg mL(-1) for naloxone hydrochloride dihydrate and noroxymorphone. The recoveries for all three compounds were above 96%. No spectral or chromatographic interferences from the tablet excipients were found. This method is rapid and simple, does not require any sample preparation and is suitable for routine quality control analyses.

Simultaneous stopped-flow determination of morphine and naloxone by time-resolved chemiluminescence.

The first application of the flow analysis coupled with chemiluminescence detection and based on stopped-flow chemistry to the simultaneous determination of two components, using a two equation system, is described. The proposed method to determine simultaneously morphine and naloxone is based on the chemiluminescence oxidation of these compounds by their reaction with potassium permanganate in an acidic medium. The main feature of the system used is that the recording of the whole chemiluminescence intensity-versus-time profiles can be obtained, using the stopped-flow technique in a continuous-flow system. Then, the chemiluminescent signals obtained at two times of these profiles can be used to determine the concentration of both opiate narcotics. The effect of common emission enhancers on the chemiluminescence emission of these compounds in different acidic media, using the above-mentioned technique, was studied, in order to achieve the best conditions in which, the CL profiles of both compounds should be additive. The parameters selected were sulphuric acid 1.0 mol L(-1), permanganate 0.2 mmolL(-1) and formaldehyde 0.8 mol L(-1). Taken in account the different profiles of the transient CL signal obtained with each compounds, using the selected chemical conditions, two measurement times (1.4 and 4.8s) of these responses curves were considered with the purpose to establish a simple 2 x 2 matrix calculation. Using the chemiluminiscent signals obtained at these times, a linear calibration graph was obtained for each one of the compounds between 0.01 and 1.00 mg L(-1) for morphine and 0.10-1.50 mg L(-1) for naloxone. The present chemiluminescence procedure was applied to the determination of both compounds in mixtures and was found to be satisfactory.

Multivariate calibration applied to the time-resolved chemiluminescence for the simultaneous determination of morphine and its antagonist naloxone.

A novel alternative for the simultaneous determination of compounds with similar structure is described, using the whole chemiluminescence-time profiles, acquired by the stopped-flow technique, in combination with mathematical treatments of multivariate calibration. The proposed method is based on the chemiluminescent oxidation of morphine and naloxone by their reaction with potassium permanganate in an acidic medium, using formaldehyde as co-factor. The whole chemiluminescence-time profiles, acquired using the stopped-flow technique in a continuous-flow system, allowed the use of the time-resolved chemiluminescence (CL) data in combination with multivariate calibration techniques, as partial least squares (PLS), for the quantitative determination of both opiate narcotics in binary mixtures. In order to achieve overcoat the additivity of the CL profiles and beside to obtain CL profiles for each drug the most separated as possible in the time, the optimum chemical conditions for the CL emission were investigated. The effect of common emission enhancers on the CL emission obtained in the oxidation reaction of these compounds in different acidic media was studied. The parameters selected were sulphuric acid 1.0 mol L(-1), permanganate 0.2 mmol L(-1) and formaldehyde 0.8 mol L(-1). A calibration set of standard samples was designed by combination of a factorial design, with three levels for each factor and a central composite design. Finally, with the aim of validating the chemometric proposed method, a prediction set of binary samples was prepared. Using the multivariate calibration method proposed, the analytes were determined in synthetic samples, obtaining recoveries of 97-109%.

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.

Mu opiate active substances trefentanil and naloxonazine: tritiation at high specific activity.

Methods are presented to synthesize and characterize [propyl-(3)H] trefentanil 3 and [propenyl-(3)H] naloxonazine 6.

RP-HPLC method and its validation for the determination of naloxone from a novel transdermal formulation.

The aim of the present work was to develop a simple and reliable liquid chromatographic method for the quantitative determination of naloxone (NLX) in a novel transdermal formulation. Chromatography was carried out by reversed-phase technique on a C-18 column with a mobile phase composed of methanol, acetonitrile and 50 mM phosphate buffer (pH 7) in the proportion of 40:20:40 v/v/v, at a flow rate of 1 ml/min. The UV spectrophotometric determination was performed at 220 nm. This method was found to be specific and accurate with the mean recovery of 98.72% in the range of 2-50 microg/ml, and a run time of 15 min (retention time of NLX 11.3 min). Method was applied for stability testing of novel transdermal formulation developed in our laboratory. Assay content of NLX in the formulation was determined in stability samples and compared with the control samples. Statistical analysis by Student's t-test showed no significant difference between the assay content of NLX in control and test samples at 95% confidence interval. Overall, the proposed method is highly sensitive, precise and accurate and can be used for the reliable quantitation of NLX in developed transdermal formulation with the added advantage of simple procedure.

Investigation of 4,5-epoxymorphinan degradation during analysis by HPLC.

Compounds of the 4,5-epoxymorphinan series have been shown to degrade in solution to the corresponding 2,2'-dimers when stored in amber glass HPLC vials. A colorant in the glass has been shown to catalyze the degradation. Although amber glass is routinely used to protect solutions from light degradation, it should not be used without evaluating its effect on sample stability.

Biosensing of opioids using frog melanophores.

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.

Opioid binding profile of morphiceptin, Tyr-MIF-1 and dynorphin-related peptides in rat brain membranes.

Opioid properties of several morphiceptin- (Tyr-Pro-Phe-Pro-NH2), Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and dynorphin-derivatives were characterized in rat brain in vitro receptor binding assay and in electrically stimulated longitudinal muscle strip preparation of guinea pig ileum. In the case of morphiceptin-related peptides, an excellent correlation was found between the [3H]-naloxone binding displacement data and the agonist potencies determined in the bioassay. The "turning point' was the C-terminal amidation in the tri- and tetrapeptide pairs in both series. Tyr-MIF-1 derivatives showed weak affinity in the opioid receptor binding assay and none of them had any remarkable effect in the bioassay either as agonist or antagonist. The dynorphin A(1-10)-peptides modified at positions 5 and 8 retained their affinity with Pro5-, Pro8-, and Ala8-substituents, whereas some loss of affinity was observed in the case of Gly8-Dyn A(1-10).

Adrenergic mediation of the naloxone-induced GnRH release from hypothalami of ovariectomized, steroid-treated immature rats.

The present study examines the effect of naloxone on GnRH release in vitro under different steroid milieus. Naloxone (6.1 mumol/kg) administered 30 min before decapitation was highly effective in evoking GnRH release from superfused hypothalamic tissues derived from ovariectomized, estradiol- and progesterone-treated immature rats, while ineffective in altering GnRH release from intact, ovariectomized and vehicle- or estradiol-treated rats. To further explore the possible involvement of catecholamines in the naloxone-stimulated GnRH release, diethyldithiocarbamic acid (2.9 mmol/kg), an inhibitor of noradrenalin synthesis, was administered ip 30 min before naloxone injection into ovariectomized, estradiol- and progesterone-treated rats. Diethyldithiocarbamic acid markedly reduced the naloxone-evoked GnRH release, although it was ineffective in modifying the spontaneous release of GnRH. A blockade of alpha-adrenergic receptor with phenoxybenzamine significantly suppressed the naloxone-stimulated GnRH release, whereas treatment with propranolol, a beta-adrenergic receptor blocker, failed to alter GnRH release. The present data suggest that the endogenous opioid peptide may participate in the regulation of GnRH release under a particular steroid milieu, and the inhibitory action of endogenous opioid peptide seems to require the mediation of adrenergic neurotransmission, presumably through alpha-adrenergic receptor.