Simultaneous extraction of acidic and basic drugs via on-chip electromembrane extraction using a single-compartment microfluidic device.

In this study, a new chip was designed for simultaneous extraction of acidic and basic drugs by a single chamber on-chip electromembrane extraction (CEME) followed by high performance liquid chromatography. Diclofenac (DIC) and nalmefene (NAL) were selected as acidic and basic model analytes, respectively. In this device, simultaneous extraction of the analytes was carried out using a single compartment. The chip was composed of three PMMA (polymethyl methacrylate) parts with sandwiched structures and carved spiral microfluidic channels in each part. The middle part was cut and an "M" pattern provided interfaces for contact between the sample solution flow and two porous polypropylene sheets on both sides. Two other parts had the same spiral channels dedicated to the corresponding acceptor phases of the acidic and basic analytes and were located at both sides. Each polypropylene sheet was impregnated with the appropriate organic solvent for the acidic and basic analytes. Two platinum electrodes connected to a power supply were mounted at the bottom of the acceptor channels. These electrodes provided the electrical fields across SLMs to extract the analytes from a single sample flow. When the extraction was completed, the acceptor solutions were collected, mixed, and then injected into the chromatographic system. The effective parameters on the extraction efficiency were investigated and optimized. Under the optimal conditions, the calibration curves were linear in the range of 9.0-500 µg L-1 for NAL and 11.0-500 µg L-1 for DIC with the coefficient of determination (R2) higher than 0.9913. The relative standard deviations (RSD%) based on five replicate measurements were less than 6.3%. LOD values were 4.0 and 3.0 µg L-1 for DIC and NAL, respectively. Finally, the method was successfully applied to determine DIC and NAL in the human urine samples and satisfactory results were obtained (recovery ≥90).

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.

Procedure for the Selection and Validation of a Calibration Model I-Description and Application.

Calibration model selection is required for all quantitative methods in toxicology and more broadly in bioanalysis. This typically involves selecting the equation order (quadratic or linear) and weighting factor correctly modelizing the data. A mis-selection of the calibration model will generate lower quality control (QC) accuracy, with an error up to 154%. Unfortunately, simple tools to perform this selection and tests to validate the resulting model are lacking. We present a stepwise, analyst-independent scheme for selection and validation of calibration models. The success rate of this scheme is on average 40% higher than a traditional "fit and check the QCs accuracy" method of selecting the calibration model. Moreover, the process was completely automated through a script (available in Supplemental Data 3) running in RStudio (free, open-source software). The need for weighting was assessed through an F-test using the variances of the upper limit of quantification and lower limit of quantification replicate measurements. When weighting was required, the choice between 1/x and 1/x2 was determined by calculating which option generated the smallest spread of weighted normalized variances. Finally, model order was selected through a partial F-test. The chosen calibration model was validated through Cramer-von Mises or Kolmogorov-Smirnov normality testing of the standardized residuals. Performance of the different tests was assessed using 50 simulated data sets per possible calibration model (e.g., linear-no weight, quadratic-no weight, linear-1/x, etc.). This first of two papers describes the tests, procedures and outcomes of the developed procedure using real LC-MS-MS results for the quantification of cocaine and naltrexone.

A stability-indicating HPLC method for simultaneous determination of morphine and naltrexone.

This study developed a stability-indicating reversed-phase HPLC method for the simultaneous determination of morphine sulfate and naltrexone hydrochloride content in bulk, Solid dosage forms and in-vitro dissolution samples to support product development and quality control efforts. Chromatographic separation of the pharmaceutical compound was achieved on a perfectSil™ MZ C18 column (250×4.6mm, 5µm) with an isocratic mobile phase composed of a mixture of acetate buffer (10mM, pH 4, containing 0.1% of 1-heptanesulfonic acid sodium salt) and acetonitrile with 80/20 at a flow rate of 1.5mlmin(-1). Both analytes were quantified using a photodiode array detector set at a wavelength of 280nm and column temperature was set to 30°C. naltrexone, morphine and a mixture of the two were subjected to thermal, peroxide, acid, base and photolytic degradation and their peak homogeneity was obtained using a photodiode array detector, demonstrating the specificity of method. These pharmaceuticals were spiked in biological fluid to examine method selectivity. The method was validated for system suitability, linearity, accuracy, precision, detection and quantification limits and robustness and was found it is acceptable in range of 2-250µgml(-1) for morphine and 4-100µgml(-1) for naltrexone.

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.

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.

Voltammetry of naltrexone in commercial formulation and human body fluids: Quantification and pharmacokinetic studies.

Naltrexone HCl (NAL.HCl) has been reduced at the mercury electrode in Britton-Robinson universal buffer of pH values 2-11 with a mechanism involving the quasi-reversible uptake of the first transferring electron followed by a rate-determining protonation step of its C=O double bond at position C-6. Simple, sensitive, selective and reliable linear-sweep and square-wave adsorptive cathodic stripping voltammetry methods have been described for trace quantitation of NAL.HCl in bulk form, commercial formulation and human body fluids without the necessity for sample pretreatment and/or time-consuming extraction steps prior to the analysis. Limits of quantitation of 6.0×10(-9)M and 8.0×10(-10)M NAL.HCl in bulk form or commercial formulation and of 9.0×10(-9) and 1.0×10(-9)M NAL.HCl in spiked human serum samples were achieved by the described linear and square-wave stripping voltammetry methods, respectively. Furthermore, pharmacokinetic parameters of the drug in human plasma samples of healthy volunteers following the administration of an oral single dose of 50mg NAL.HCl (one Revia(®) tablet) were estimated by means of the described square-wave stripping voltammetry method without interferences from the drug's metabolites and/or endogenous human plasma constituents. The estimated pharmacokinetic parameters were favorably compared with those reported in literature.

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.

Ex vivo delta opioid receptor autoradiography: CNS receptor occupancy of two novel compounds over their antihyperalgesic dose range.

Discovered as part of an effort to identify delta opioid (DOPr or DOR) agonist analgesics, JNJ-20788560 and JNJ-39204880 exhibited high DOR affinity, with K(i) values of 1.7 and 2.0nM, respectively, and were selective for DOR over the mu opioid receptor (MOPr or MOR), with 596- and 122-fold selectivity, respectively. Both compounds stimulated DOR but not MOR induced GTPgammaS binding and were effective antihyperalgesic agents in the complete Freund's adjuvant model of thermal hyperalgesia in the rat, with oral ED(50) values of 13.5 and 35mg/kg, corresponding to plasma levels of 1 and 9microM, respectively. Autoradiographic analysis of DOR and MOR occupancy in sections of brain (striatum) and lumbar spinal cord (L4-L6) was determined ex vivo, using radiolabeled naltrindole or DAMGO. Quantitative image analysis resulted in striatal DOR ED(50) values of 6.9 and 10.7mg/kg, for JNJ-20788560 and JNJ-39204880 respectively, and spinal cord values of 6.4 and 3.2mg/kg, respectively. Neither compound dose-dependently occupied MOR within the dose range studied. Thus, this study confirmed the DOR selectively over MOR of both compounds following their oral administration, and further demonstrated dose-dependent DOR occupancy by each compound across its antihyperalgesic dose range. Importantly, these in vitro, in vivo, and ex vivo data revealed that the greater in vitro potency of JNJ-20788560 was paralleled by its greater in vivo potency, although JNJ-39204880 achieved higher plasma levels following its oral administration. The receptor occupancy levels observed at the pharmacologic ED(50) doses of these compounds suggest the need for greater target engagement by JNJ-39204880 than by JNJ-20788560 to elicit a similar therapeutic response.

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.

Structural determination of the novel fragmentation routes of morphine opiate receptor antagonists using electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Electrospray ionization quadrupole time-of-flight (ESI-QqToF) mass spectra of naltrindole hydrochloride 1, naltriben mesylate 2, and naltrexone hydrochloride 3, a common series of morphine opiate receptor antagonists, were recorded using different declustering potentials. Low-energy collision-induced dissociation (CID) MS/MS experiments established the fragmentation routes of these compounds. In addition, re-confirmation of the various established fragmentation routes was effected by conducting a series of ESI-CID-QqTof-MS/MS experiments using non-conventional quasi MS(n) (up to MS8) product ion scans, which were initiated by CID in the atmospheric pressure/vacuum interface using a higher declustering potential. Precursor ion scan analyses were also performed with a conventional quadrupole-hexapole-quadrupole tandem mass spectrometer and allowed the confirmation of the genesis of some diagnostic ions.

Preparation of naltrexone hydrochloride loaded poly (DL-lactide-co-glycolide) microspheres and the effect of polyvinyl alcohol (PVA) as surfactant on the characteristics of the microspheres.

The aim of this study was to investigate the effect of the surfactant properties of polyvinyl alcohol (PVA) in enhancing the yield of small size microspheres. Naltrexone microspheres were prepared by solvent-solvent extraction evaporation process. PVA of various concentrations were added into the aqueous phase prior to the mixing process. The addition of PVA was expected to influence the shape, size distribution, drug loading and drug release profile. The results indicated that it is desirable to increase the weight fraction of the microspheres with size range below 106 mm for the highest possible yield.

Quantification of naltrexone and 6,beta-naltrexol in plasma and milk using gas chromatography-mass spectrometry. Application to studies in the lactating sheep.

A selective gas chromatography-mass spectrometry method using solid-phase extraction has been developed for the detection and quantification of naltrexone and its metabolite, 6,beta-naltrexol in plasma and milk from humans and sheep at pharmacologically relevant concentrations. Di- or tri-acetyl derivatives were formed and quantified by selected-ion monitoring. Recoveries of naltrexone (30 microg/l) and 6,beta-naltrexol (250 microg/l) from both human plasma and milk were greater than 70%. Intra-assay and inter-day precision ranged from 3 to 21% for naltrexone and 2-18% for 6,beta-naltrexol for all matrices investigated, with an overall mean accuracy of 104% for naltrexone, and 99% for 6,beta-naltrexol. Human samples containing these analytes were stable for at least 3 weeks at -20 degrees C or 6 weeks at -80 degrees C. Analysis of the plasma and milk from the lactating sheep showed mean milk-to-plasma ratios of 55 for naltrexone and 3 for 6,beta-naltrexol.

Chemiluminescence determination of naltrexone based on potassium permanganate oxidation.

A rapid method by chemiluminescence is described for the determination of naltrexone. The method is based on the chemiluminescence reaction with potassium permanganate in sulfuric acid medium. The optimum conditions for the chemiluminescence emission were investigated. With the integrated chemiluminescence intensity for 10 s after KMnO4 injection as a quantitative parameter, naltrexone can be determined over the concentration range 50-1000 ng ml-1 with a detection limit of 2.5 nm ml-1 and with a RSD (n = 10) of 0.9% and 0.5% at levels of 1000 and 100 ng ml-1, respectively. The method was applied to the determination of naltrexone in pharmaceutical preparations.

Naltrexone is not detected in preweaning rats following transplacental exposure: implications for growth modulation.

Extracts of brain and heart from rats at birth and postnatal days 2 and 10 were evaluated for naltrexone following maternal injection of 50 mg/kg opioid antagonist throughout gestation. Samples were prepared by ultrafiltration, lyophilized, reconstituted in mobile phase, and separated by reversed-phase high performance liquid chromatography with ultraviolet detection. Qualitative analysis revealed the presence of naltrexone in tissues from neonates, but not in rats of 2 and 10 days, that were transplacentally exposed to drug. These results confirm earlier reports showing that naltrexone, maternally administered, passes through the placenta and enters the fetus. Moreover, the data suggest that the somatic and neurobiological acceleration observed in offspring exposed to naltrexone during gestation is not due to opioid receptor blockade during the postnatal period.

Flow injection analysis with amperometric detection of naltrexone in pharmaceuticals.

Flow injection analysis (FIA) with amperometric detection using a carbon paste electrode is applied to the determination of naltrexone. The sample solution was injected into the carrier stream of 0.1 M perchloric acid, being determined by oxidation at +1.0 V vs. Ag/AgCl/sat. KCl using a flow rate of 4 ml min-1. A relative standard deviation of 1.5% was calculated for a concentration level of 10(-5) M (n = 17) without carrying out a carbon paste electrode pretreatment. Calibration curves were found to be linear between 2 x 10(-8) and 10(-5) M (almost three orders of magnitude) and the method has a detection limit of 2 x 10(-8) M. A simple and reproducible procedure is proposed for the determination of naltrexone in pharmaceuticals. The results compared favourably with those obtained by an HPLC-UV method.

Stability of Revex, nalmefene hydrochloride injection.

The stability of Revex, nalmefene hydrochloride injection, has been studied at several temperatures for periods up to 36 months. The data were obtained using a HPLC method for the potency determination, and for the level of the sole degradation product (2,2'-bisnalmefene). These methods were found to be characterized by excellent precision, linearity, and accuracy over the analyte concentration ranges established. The stability data were found to be interpretable using first-order kinetics, and essentially comparable rate constants were calculated for both the potency loss and the formation of 2,2'-bisnalmefene. Applying the Arrhenius equation to these data, a rate constant of 0.00441 month-1 was deduced for the reactions taking place at 25 degrees C. This low value is consistent with the excellent stability exhibited by the product, and amply justifies its shelf life.