Spectrofluorimetric method for determination of bupropion in pharmaceutical dosage forms.

OBJECTIVE: Bupropion is a noradrenaline and dopamine reuptake inhibitor which is used as an antidepressant drug. Few HPLC and spectrophotometric methods have been reported before for the determination of bupropion. Most of the previous methods reported for determination of bupropion in pharmaceutical dosage forms are somehow dangerous to health and environment because of using organic solvents. METHOD: In the present method bupropion was determined in pharmaceutical dosage forms by spectrofluorimetry after ion-pair complex formation with eosin Y. The ion-pair complex formation was optimized for reagent amount, buffer pH and time. RESULT: The developed method was linear over the range of 3-120µgmL-1 with an acceptable precision (CV<1.5%) and accuracy (Error<1%). CONCLUSION: The present method is applicable for determination of bupropion in pharmaceutical dosage forms for routine quality control analysis.

Degradation of Bupropion: Implications for Interpretation of Postmortem Case Data.

The interpretation of postmortem bupropion is often a challenge to the forensic toxicology community because of the instability of the parent compound. At the North Carolina Office of the Chief Medical Examiner (NC OCME) toxicology laboratory, one of the active metabolites, threobupropion, is used as a complementary indicator for the extent of exposure to the parent compound. Metabolite data will address postmortem normal concentrations as well as when bupropion was attributed to the cause of death. For 55 natural cases where bupropion was unattributed to the cause of death, the blood and liver mean threobupropion concentrations were 1.8 mg/L and 12.1 mg/kg, respectively, with median concentrations of 1.5 mg/L and 10 mg/kg, respectively. For the 51 suicidal ingestion cases when bupropion was attributed to the cause of death, the blood and liver mean threobupropion concentrations were 15.8 mg/L and 131.5 mg/kg, respectively, with median concentrations of 13.5 mg/L and 110 mg/kg, respectively. The laboratory completed a stability study over the course of 50 days to evaluate how bupropion and threobupropion degrade in postmortem blood, liver and liver homogenate. The samples were subjected to forensically relevant conditions by storing them at room temperature (RT, 20°C), refrigerated (4°C) and frozen (-20°C). While the concentration of bupropion decreased in all specimens, the rate of degradation of the RT samples was the most dramatic. The threobupropion metabolite appeared to be relatively stable. The postmortem case data along with the evaluation of potential degradation products should provide an overall picture to assist the toxicological community with the interpretation of bupropion found in routine casework.

Bupropion Overdose Resulted in a Pharmacobezoar in a Fatal Bupropion (Wellbutrin® ) Sustained-release Overdose: Postmortem Distribution of Bupropion and its Major Metabolites.

Bupropion (BUP) overdose commonly causes generalized seizures and central nervous system depression. The case of a 28-year-old woman who died from a massive lethal overdose with sustained-release bupropion (Wellbutrin® 300 mg) is herein presented. The autopsy revealed the presence of a pharmacobezoar consisting of at least 40 tablets in the stomach. Determination of bupropion and its active metabolites (hydroxybupropion, threobupropion, erythrobupropion) was achieved by a liquid chromatographic mass spectrometry (LC-MS/MS) method. Postmortem concentrations for bupropion, hydroxybupropion, threobupropion, and erythrobupropion were obtained in intracranial blood, urine, bile, liver, kidney, and vitreous humor. In this case, intracranial blood level of the parent drug was 1.9 mg/L. Threobupropion was the most abundant metabolite in both blood and urine, 59.3 and 890.6 mg/L. Tissue distribution showed the highest concentration in the liver, 12.3 mg/kg. The 0.8 bupropion concentration ratio vitreous/blood suggested that vitreous could be a valuable specimen for toxicological analysis should postmortem blood be unavailable.

In vivo effect of borneol on rat hepatic CYP2B expression and activity.

CYP2B subfamily accounts for 2-10% of total hepatic CYP450 enzymes and participate in the metabolism of around 8% of clinical drugs. Borneol has been widely used in traditional Chinese medicine for thousands of years. There are many studies about borneol-induced promoting penetration role for a number of drugs through various physiologic barriers, whereas there is no report involved the effect of borneol on hepatic CYP2B. The present work studied the in vivo effect of borneol on the expression and activity of rat hepatic CYP2B. The results indicated that the oral administration of borneol (33, 100 and 300 mg/kg/d) to rats for consecutive 7 days increased the hepatic CYP2B1/2 activity by 1.4-, 1.7- and 2.8-fold, hepatic CYP2B1 mRNA expression by 6.3-, 8.7- and 18.1-fold, and hepatic CYP2B1/2 protein expression by 1.2-, 1.9- and 2.6-fold, respectively compared to the control. Additionally, in the borneol pre-dosing (300 mg/kg/d for consecutive 7 days) rats, the increased Clint and decreased AUC0-24 of bupropion were observed as compared to the control. Moreover, there were no obvious effects on CAR protein level in rat liver microsome and nucleus following the borneol treatment. Taken together, our observations indicate that borneol is an in vivo inducer of rat hepatic CYP2B with different regulatory mechanism from phenobarbital-like inducers which caused CYP2B induction with CAR activation.

Bupropion therapy during pregnancy: the drug and its major metabolites in umbilical cord plasma and amniotic fluid.

BACKGROUND: Bupropion is used for treatment of depression during pregnancy. However, its use as a smoking cessation aid for pregnant women is currently under evaluation. OBJECTIVE: The aim of this opportunistic study was to investigate the transfer of bupropion and its major pharmacologically active metabolites, hydroxybupropion and threohydrobupropion, across the placenta in vivo. In addition, the concentrations of the drug and its metabolites were determined in the amniotic fluid. STUDY DESIGN: The following samples were collected at deliveries from 22 women taking bupropion: maternal blood (n = 22), umbilical cord venous blood (n = 22), and amniotic fluid (n = 9). The concentrations of the drug and its metabolites in blood plasma and amniotic fluid were determined by means of liquid chromatography-mass spectrometry. Placental passage was calculated as a ratio of umbilical cord venous plasma to maternal plasma concentrations. RESULTS: The levels of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were invariably lower than their corresponding concentrations in maternal plasma. The concentrations of bupropion in umbilical cord plasma were lower than in maternal plasma in the majority of the maternal-cord blood pairs. The median values of the umbilical cord venous plasma to maternal plasma ratios were: bupropion, 0.53 (interquartile range 0.35, n = 18), hydroxybupropion, 0.21 (interquartile range 0.12, n = 18), and threohydrobupropion, 0.61 (interquartile range 0.11, n = 21). In umbilical cord venous plasma, the median concentration of bupropion was 5.3 ng/mL; hydroxybupropion, 103.6 ng/mL; and threohydrobupropion, 59.6 ng/mL. Bupropion and its metabolites were detectable in the amniotic fluid but the concentrations of threohydrobupropion were higher than those in the corresponding umbilical cord venous plasma. CONCLUSION: Bupropion and its active metabolites cross the placenta to the fetal circulation. The concentrations of hydroxybupropion and threohydrobupropion in umbilical cord venous plasma were higher than bupropion concentrations suggesting a higher fetal exposure to the metabolites than the parent drug. The higher levels of threohydrobupropion in the amniotic fluid than those in umbilical cord venous plasma suggest that enzymes involved in the metabolism of bupropion to threohydrobupropion are most likely active in the fetus. The biological consequences of fetal exposure to maternally administered bupropion and/or its active metabolites via placental transfer and recirculation of the amniotic fluid are yet to be determined.

Resolution of enantiomers of bupropion and its metabolites by liquid chromatography.

Bupropion, a tricyclic aminoketone, is used primarily in the treatment of depression, the management of patients with bipolar and schizo-affective disorder, and the treatment of Parkinson's disease. Bupropion is marketed as a racemate, but the racemic mixture is known to have several disadvantages while the two isomers of bupropion and its metabolite differ significantly in their pharmacological activities. Therefore, the stereoselective determination of the drug enantiomers in pharamaceutical dosages, plasma or urine is of potential clinical and analytical importance. Different chromatographic methods have been employed for the separation of the two enantiomers. This is the first attempt to review the methods of enantiosepartion of bupropion using both direct and indirect approaches in both HPLC and TLC. The review presents a detailed discussion on the use of chiral stationary phase (based on polysaccharide, α1 acid glycoprotein and ovomucoid column) and chiral derivatizing reagents (based on isothiocyanate and cyanuric chloride) along with TLC separation of bupropion enantiomers using ligand exchange and impregnation methods. The focus is also on the separation mechanism for enantioresolution using the various methods described herein.

[Preparation of 1 µm non-porous C18 silica gel stationary phase for chiral-pressurized capillary electrochromatography].

Non-porous C18 silica gel stationary phase (1 µm) was prepared and applied to chiral separation in pressurized capillary electrochromatography (pCEC) for the enantioseparation of various basic compounds. The non-porous silica particles (1 µm) were synthesized using modified St6ber method. C18 stationary phase (1 µm) was prepared by immobilization of chloro-dimethyl-octadecylsilane. Using carboxymethyl-ß-cyclodextrin (CM-ß-CD) as the chiral additive, the pCEC conditions including the content of acetonitrile (ACN), concentration of buffer, pH, the concentration of chiral additive and flow rate as well as applied voltage were investigated to obtain the optimal pCEC conditions for the separation of four basic chiral compounds. The column provided an efficiency of up to 190,000 plates/m. Bupropion hydrochloride, clenbuterol hydrochloride, metoprolol tartrate, and esmolol hydrochloride were baseline separated under the conditions of 5 mmol/L ammonium acetate buffer at pH 4. 0 with 20% (v/ v) acetonitrile, and 15 mmol/L CM-ß-CD as the chiral additive. The applied voltage was 2 kV and flow rate was 0.03 mL/min with splitting ratio of 300:1. The resolution were 1.55, 2.82, 1. 69, 1. 70 for bupropion hydrochloride, clenbuterol hydrochloride, metoprolol tartrate, esmolol hydrochloride, respectively. The C18 coverage was improved by repeating silylation method. The synthesized 1 µm C18 packings have better mechanical strength and longer service life because of the special, non-porous structure. The column used in pCEC mode showed better separation of the racemates and a higher rate compared with those used in the capillary liquid chromatography (cLC) mode. This study provided an alternative way for the method of pCEC enantioseparation with chiral additives in the mobile phase and demonstrated the feasibility of micron particle stationary phase in chiral separation.

A novel dosage form for buccal administration of bupropion

Bupropion is an antidepressant used in the treatment of smoking. The purpose of this study was to prepare controlled-release hydrogel films for buccal administration of bupropion and investigate its physicochemical and cytotoxic properties. The films were prepared from ultrapure sodium carboxymethylcellulose, hydroxypropylmethylcellulose K4M, and medium-viscosity chitosan. Evaluation of film physicochemical characteristics was based on scanning electron microscopy, bupropion content, mechanical strength (burst strength, relaxation, resilience, and traction), and cytotoxicity. Bupropion content in bilayer films was 121 mg per 9 cm2. The presence of bupropion modified film mechanical strength, but did not compromise the use of this pharmaceutical form. As shown by the cytotoxicity results, films containing bupropion did not cause cellular damage. Bupropion administration in the form of hydrogel films is a potentially useful alternative in the treatment of smoking.

A bupropiona é um antidepressivo utilizado no tratamento do tabagismo. O objetivo deste trabalho foi a preparação de filmes hidrogelatinosos de liberação controlada para administração bucal de bupropiona. Os filmes foram preparados utilizando carboximetilcelulose sódica ultrapurificada, hidroxipropilmetilcelulose K4M e quitosana de média viscosidade. As características físico-químicas dos filmes foram avaliadas por microscopia eletrônica de varredura, teor de bupropiona, resistência mecânica (perfuração, relaxação, resiliência e tração) e citotoxicidade. Os resultados mostraram que os filmes em bicamada apresentaram teor de bupropiona de 121 mg por 9 cm2 de filme e que a bupropiona modifica a resistência mecânica dos filmes, sem, no entanto, inviabilizar o uso desta forma farmacêutica. Os estudos de citotoxicidade mostraram que as formulações dos filmes contendo bupropiona não causam dano celular. Este estudo mostrou que a bupropiona veiculada na forma de filme hidrogelatinoso pode ser uma alternativa útil no tratamento do tabagismo.

High-performance liquid chromatographic enantioseparation of (RS)-bupropion using isothiocyanate-based chiral derivatizing reagents.

A high-performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate-based chiral derivatizing reagents, (S)-1-(1-naphthyl) ethyl isothiocyanate, (S)-NEIT, and (R)-α-methyl benzyl isothiocyanate, (R)-MBIT. The diastereomers synthesized with (S)-NEIT were enantioseparated by reversed-phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)-MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040-0.043 µg/mL for each of the diastereomers prepared with (S)-NEIT.

Simultaneous quantitative determination of bupropion and its three major metabolites in human umbilical cord plasma and placental tissue using high-performance liquid chromatography-tandem mass spectrometry.

A liquid chromatography in tandem with electro-spray ionization mass spectrometry method has been developed and validated for the quantitative determination of bupropion and its major metabolites (hydroxybupropion, threo- and erythrohydrobupropion) in human umbilical cord plasma and placental tissue. The samples were acidified with trichloroacetic acid, and protein precipitated by adding acetonitrile. Chromatographic separation of drug and metabolites was achieved by using a Waters Symmetry C(18) column, with an isocratic elution of 31% methanol and 69% formic acid (0.04%, v/v) aqueous solution at a flow rate of 1.0 mL/min. Detection was carried out by mass spectrometry using positive electro-spray ionization mode, and the compounds were monitored using multiple reactions monitoring method. Deuterium-labeled isotopes of the compounds were used as internal standards. Calibration curves were linear (r(2)>0.99) in the tested ranges. The lower limit of quantification of analytes in umbilical cord plasma samples is <0.72 ng/mL and 0.92 ng/g in placental tissue samples. The relative deviation of this method was <15% for intra- and inter-day assays, and the accuracy ranged between 88% and 105%. The extraction recovery of the four analytes ranged between 89% and 96% in umbilical cord plasma, and 64% and 80% in placental tissue. No significant matrix effect was observed in the presented method.

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine.

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 µm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.

Application of ion-pair complexation reaction for the spectrophotometric determination of bupropion hydrochloride in pharmaceuticals.

This is the first report on the use of visible spectrophotometry for the determination of bupropion hydrochloride (BUPH), a second-generation antidepressant, in pharmaceuticals. Two sensitive, selective, and cost-effective spectrophotometric methods are described. The first method (method A) is based on the formation of yellow-coloured ion-pair complex between the BUPH and methyl orange (MO) at pH 3.80 ± 0.10 which was extracted into dichloromethane and the absorbance measured at 425 nm. The second method (method B) is based on the breaking of the yellow BUPH-MO ion-pair complex in acid medium followed by the measurement of the red-pink colour at 520 nm. Beer's Law is obeyed over the concentration ranges of 1.00-12.0 and 0.48-7.20 µg ml(-1) BUPH for method A and method B, respectively. The molar absorptivities are calculated to be 2.18 × 10(4) and 3.79 × 10(4) l mol(-1) cm(-1) for method A and method B, respectively, and the corresponding Sandell sensitivity values are 0.0127 and 0.0073 µg cm(-2) . The limits of detection and quantification have also been reported. The proposed methods were applied successfully to the determination of BUPH in pure drug and commercial tablets. The accuracy and reliability of the proposed methods were further ascertained by recovery studies via standard addition technique.

A simple and sensitive LC-ESI-MS (ion trap) method for the determination of bupropion and its major metabolite, hydroxybupropion in rat plasma and brain microdialysates.

A specific and highly sensitive liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) method for the direct determination of bupropion (BUP) and its main metabolite hydroxybupropion (HBUP) in rat plasma and brain microdialysate has been developed and validated. The analysis was performed on a Bonus RP C18 (100 mm × 2.1mm i.d., 3.5 µm particles) column using gradient elution with the mobile phase consisting of acetonitrile and ammonium formate buffer (10mM, pH 4). Plasma samples were analyzed after a simple, one-step protein precipitation clean-up with trichloroacetic acid (TCA), however clean-up for microdialysis samples was not necessary, enabling direct injection of the samples into the LC-ESI-MS system. Signals of the compounds were monitored under the multiple reaction monitoring (MRM) mode of the LC-ESI-MS (ion trap) for quantification. The precursor to product ion transitions of m/z 240-184 and m/z 256-238 were used to measure BUP and HBUP, respectively. The method was validated in both plasma and microdialysate samples, and the obtained lower limit of quantification (LLOQ) was 1.5 ng mL(-1) for BUP and HBUP in both matrices. The intra- and inter-day assay variability was less than 15% for both analytes. This LC-ESI-MS method provided simple sampling, rapid clean-up and short analysis time (<9 min), applicable to the routine therapeutic monitoring and pharmacokinetic studies of BUP and HBUP.

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis.

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.

A fatal case involving extremely high levels of ethylene glycol without elevation of its metabolites or crystalluria.

A unique case of an intentional overdose of ethylene glycol resulting in a fatality is described. The decedent had a very high concentration of ethylene glycol without elevated concentrations of its metabolites or crystalluria. The ethylene glycol concentrations in blood, urine, and vitreous fluid were 2340, 2261, and 1028 mg/dL, respectively. Osmolality of blood and vitreous fluid was also very high at 1426 and 534 mOsm/kg, respectively. No crystals were found in the urine. Furthermore, on the urine organic acids profile the ethylene glycol metabolites oxalic, glycolic, and glyoxylic acids were within the reference ranges. In addition to ethylene glycol, the decedent had an elevated level of mirtazapine, an antidepressant, and a low level of bupropion. It was estimated that the subject consumed 1034 g of ethylene glycol. To our knowledge, this is the first case of death from severe ethylene glycol poisoning in the absence of ethylene glycol metabolites or crystalluria.

Recomendaciones para el tratamiento farmacológico del tabaquismo. Propuestas de financiación / Recommendations for pharmacological tobacco cessation treatments: proposals for financing

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Stereoselective analysis of hydroxybupropion and application to drug interaction studies.

A sensitive and stereoselective assay has been developed for the quantitation of the enantiomers of hydroxybupropion, an active metabolite of bupropion, in human plasma. The assay used liquid-liquid extraction and a Cyclobond I 2000 HPLC column with a mobile phase containing 3% acetonitrile, 0.5% triethylamine, and 20 mM ammonium acetate (pH 3.8). The technique was linear over the concentration range of 12.5-500 ng/ml for (2R,3R)- and (2S,3S)-hydroxybupropion. The method was reproducible as both interday and intraday variabilities were less than 10% for both hydroxybupropion enantiomers. Overall extraction recovery of hydroxybupropion enantiomers and the internal standard phenacetin from plasma was greater than 80% and reproducible over the concentration range of 12.5-500 ng/ml for each enantiomer. The limit of quantification (LOQ) of hydroxybupropion enantiomers was 12.5 ng/ml. The stereoselective pharmacokinetics of both (2R,3R)- and (2S,3S)-hydroxybupropion in healthy male subjects (n = 16) were investigated after a single dose of (rac)-bupropion either alone or during rifampicin administration. (2R,3R)-Hydroxybupropion was the predominant enantiomer present in plasma. A stereoselective effect of rifampicin on hydroxybupropion concentrations was observed, with rifampicin influencing the pharmacokinetics of each hydroxybupropion enantiomer in a different manner. The ratio of (2R,3R)-hydroxybupropion (AUC(0-24)) to (2S,3S)-hydroxybupropion (AUC(0-24)) increased from 4.9 +/- 1.6 to 8.3 +/- 1.9 during rifampicin administration (P < 0.001). A time-dependent change in the hydroxybupropion enantiomeric ratio was observed after (rac)-bupropion administration both before and during rifampicin coadministration, with an increase in the relative proportion of (2S,3S)-hydroxybupropion over the 24 h postdose period.