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.

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 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.

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.

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.

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).

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.

The effect of biogenic amine modifiers on morphine analgesia and its antagonism by naloxone.

The stimulation of dopaminergic receptors, inhibition of serotonin synthesis or blockade of muscarinic receptors by various modifiers led to inhibition of morphine analgesia in mice. Blockade of dopaminergic receptors or the increase in serotonergic or cholinergic activity resulted in the enhancement of morphine analgesia. Serotonergic and cholinergic systems are proposed as positive and the dopaminergic system as negative modulators of morphine analgesia. The modulation of naloxone antagonism was much more complicated than that of morphine analgesia and often the effect of biogenic amine modifiers on antagonism differed from that on analgesia. The fact than biogenic amine modifiers do not affect morphine analgesia and naloxone antagonism by a similar pattern suggests that interaction of narcotics and narcotic antagonists with analgesic receptors may not be exactly the same.

Pharmacokinetics of morphine and its surrogates. VIII: Naloxone and naloxone conjugate pharmacokinetics in dogs as a function of dose and as affected by simultaneously administered morphine.

Reversed-phase HPLC assays with electrochemical detection, developed to quantify naloxone, 6 beta-naloxol, and their hydrolyzed conjugates in biological fluids provided assay sensitivities of 10 to 20 ng/mL in plasma, urine, and bile. These fluids were monitored in dogs after iv bolus administrations of 0.47 and 4.7 mg/kg of naloxone. Plasma concentration-time data were well fitted by the sums of two exponentials with two sequential half-lives of 11 +/- 1 (SEM) and 56 +/- 3 min. Pharmacokinetics were dose-independent; total and renal clearances were 1334 +/- 133 mL/min and 42 +/- 9 mL/min, respectively, with a renal clearance of 65 +/- 5 mL/min for the conjugate. The percentage of the dose excreted in the urine as naloxone was 4.4 +/- 1.0%. There was a possible dose-dependent excretion of conjugate with 46 +/- 1 and 22 +/- 5% of the dose renally excreted at the high and low doses, respectively. In incomplete biliary cannulation, 13 and 18% were collected as conjugate in the bile of two bile-cannulated dogs. There was negligible biliary secretion of unchanged naloxone. Neither 6 beta-naloxol nor its conjugates were metabolites of naloxone in dogs. The simultaneous administration of naloxone does not reverse the dose-dependent pharmacokinetic perturbations of morphine. Morphine significantly lessened its own body, renal, and biliary clearances, as well as those of naloxone, and also lowered their apparent overall volumes of distribution. Plasma levels of naloxone and its conjugate were elevated with simultaneous morphine administration. Urinary flow rates were also greatly lessened and initial renal shut-down was implied at the higher morphine dose. Thus, the established competitive antagonistic action of naloxone on morphine does not extend to the reversal of the biological feedback effects of morphine on the metabolism and excretion of itself and simultaneously administered naloxone.

Systemic morphine pharmacokinetics after ocular administration.

Pharmacokinetics of a single dose of morphine ocularly applied is reported in rabbits before and after lacrimal canaliculi ligature. Our investigations are based on a sensitive reversed-phase ion-pair chromatographic determination of morphine. This study describes the various resorption sites of morphine when administered through the conjunctiva. After ocular administration, morphine rapidly reaches high blood levels compatible with pharmacological activity. Ocular bioavailability of morphine is higher than after non-parenteral routes. Canaliculi ligature modifies the morphine pharmacokinetic profile without significant modification of drug bioavailability. Our results suggest a great capacity of drug resorption for the conjunctiva, and indicate the major role of nasal mucosa in physiological conditions.

Improved method for morphine extraction from biological samples.

Methadone morphine, or naloxone extraction from brain homogenates, plasma, and urine is described. An aqueous sample was loaded on a surgical gauze support, which was washed with extracting solvents. Aqueous samples remained on the support, and nonpolar drugs partitioned into the lipophilic extracting solvent. The procedure recovered 80-100% of nanogram levels of methadone, morphine, or naloxone from biological samples. In addition, an approximate 10-fold timesaving capacity was demonstrated compared to standard liquid-liquid extraction techniques.

Biosynthesis, isolation, and identification of 6-beta-hydroxynaltrexone, a major human metabolite of naltrexone.

Chemical reduction of naltrexone is described in an attempt to synthesize 6-beta-hydroxynaltrexone. Only the epimer, 6-alpha-hydroxynaltrexone, was produced. Pilot metabolic studies on naltrexone in the dog, rat, and guinea pig were made to determine which animal produced the greatest amount of 6-beta-hydroxynaltrexone. The guinea pig was selected and used to produce the metabolite. Isolation and purification methods are described, and spectral data are presented for structural confirmation of the metabolite.

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.

Quantification of naloxone binding sites in brains from suckled beef cows during postpartum anestrus and resumption of estrous cycles.

Thirty beef cows, approximately 3 yr of age, were randomly assigned to be slaughtered on d 7, 14, 28, 42 or 56 postpartum. Each cow suckled one calf until slaughter. Data from cows slaughtered on d 42 and 56 were pooled and further classified as anestrous or cyclic based on the presence of a corpus luteum and elevated serum concentrations of progesterone at slaughter. Specific binding of [3H]naloxone (3H-NAL) to homogenates of tissue from hypothalamus (HYP), preoptic area (POA) and basal forebrain (BF) was assessed using multiple-point Scatchard analyses. Nonspecific binding was estimated in the presence of 10(-6) M naloxone. Separation of bound from free 3H-NAL was achieved by centrifugation at 20,000 X g. Concentration (fmol/mg original tissue wet wt) of 3H-NAL binding sites in POA tissue was higher (P less than .05) on d 28 postpartum in anestrous cows than in cyclic cows on d 42 + 56 postpartum (2.58 +/- .32 vs 1.58 +/- .10). When all anestrous cows were compared with cyclic cows, concentrations of 3H-NAL binding sites in POA tissues and in BF tissue were higher (P less than .05) in anestrous cows (anestrous POA, 2.12 +/- .17, cyclic POA, 1.58 +/- .10; anestrous BF, 2.94 +/- .41, cyclic BF, 2.19 +/- .16). Compared across brain regions for all cows, the concentration of specific binding sites for 3H-NAL was greater (P less than .01) in BF (2.5 +/- .2) than in POA (1.9 +/- .1) and greater (P less than .01) in POA than in HYP (1.5 +/- .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of high pressure liquid chromatographic retention indices of narcotic analgetics and related drugs.

A method for the prediction of the retention index of drugs on C18 reversed-phase columns was developed and then applied to a series of drugs that were structurally related to morphine and to a series related to fentanyl. The HPLC retention index (/) of the test compounds was estimated using the equation: / = 200 pi + /ref. where pi was the sum of the Hansch substituent constants for the compound, and /ref. was the index experimentally observed for the reference compound of the series. It was also found that the observed index could be used to assign the stereochemistry of the fentanyl analogues.