Photosensitizing properties of 6-methoxy-2-naphthylacetic acid, the major metabolite of the phototoxic non-steroidal anti-inflammatory and analgesic drug nabumetone

The photobiological properties of 6-methoxy-2-naphthylacetic acid (6-MNAA) were studied using a variety of in vitro phototoxicity assays: photohemolysis, photoperoxidation of linoleic acid, photosensitized degradation of histidine and thymine and the Candida phototoxicity test. 6-MNAA was phototoxic in vitro. 6-MNAA reduced nitro blue tetrazolium (NBT) when irradiated with lambda > or = 300 nm in deoxygenated aqueous buffer solution (pH 7.4). NBT can be reduced by reaction with the excited state of 6-MNAA subject to interference with molecular oxygen. The photohemolysis rate was inhibited by the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO), sodium azide (NaN3) and reduced glutathione (GSH). Photoperoxidation of linoleic acid and photosensitized degradation of histidine and thymine were significantly inhibited by sodium azide and reduced glutathione. 6-MNAA was phototoxic to C. albicans, C. lipolytica and C. tropicalis. A mechanism involving singlet oxygen, radicals, and electron transfer reactions is suggested for the observed phototoxicity.     

Simultaneous determination of piroxicam and its main metabolite in plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid Chromatographic procedure is described for the simultaneous determination of piroxicam and its main metabolite, such as 5′-hydroxypiroxicam, in plasma and urine. Acidified plasma (pH 3.0) was extracted with ethyl ether and indomethacin was used as an internal standard. The organic extract was reduced to dryness, the resultant residue redissolved in the mobile phase, and aliquots of this solution chromatographed on a Lichrosorb RP-18 (7 μm) column using a mobile phase consisting of an acetonitrile-water-acetic acid (58:38:4) mixture. The flow rate was 1.2 ml/min and the effluent was monitored at 365 nm with 0.02 aufs. The sensitivities of this method were 0.05 μg/ml levels of piroxicam and 5′-hydroxypiroxicam in the plasma and urine samples.     

Simultaneous determination of cefepime and grepafloxacin in human urine by high-performance liquid chromatography

A liquid chromatographic method with UV detection for simultaneous determination of cefepime and grepafloxacin has been developed. The method uses a C18 column, equipped with a pre-column of the same material, and acetonitrile-0.1 M phosphoric acid/sodium hydroxide buffer (pH 3.0)-0.01 M n-octylamine (pH 3.0) as mobile phase in gradient mode. Mobile flow rate and sample volume injected were 1.3 mL min(-1) and 20 microL, respectively. Detection wavelengths were 259 nm for cefepime and 278 nm for grepafloxacin. The retention times were 4.03 min for cefepime and 8.85 min for grepafloxacin, with detection limits of 1.0 and 1.1 microg mL(-1), respectively. The method was applied to the determination of both antibiotics in spiked samples of human urine.     

Simultaneous quantitation of buspirone and its major metabolite 1-(2-pyrimidinyl)piperazine in human plasma by high-performance liquid chromatography with coulometric detection

Buspirone is a member of the azapirone group of anxiolytic drugs and has one major metabolite, 1-(2-pyrimidinyl)piperazine (1-PP). The analyte, its metabolite and the internal standard were extracted from plasma utilizing solid-phase extraction columns. Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The calibration graph was linear over the range 0–50 ng ml⁻¹ of plasma. The lower limits of quantitation for buspirone and 1-PP were 0.5 and 2 ng ml⁻¹, respectively, when 1 ml of plasma was extracted. The intra-assay relative standard deviations (RSD) over the range of the calibration graph varied from 4 to 12.5% for buspirone and 1-PP. The inter-assay RSD was 6.9% for 1-PP and 9.6% for buspirone. The recovery averaged 96% for buspirone and 66% for 1-PP. Plasma profiles of buspirone and 1-PP following oral dosing are presented.     

Quantification of vincristine and its major metabolite in human plasma by high-performance liquid chromatography/tandem mass spectrometry

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 x 150 mm) with a 5-mum particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3--> 271.7), vincristine (m/z 413.2--> 362.2), and M1 (m/z 397.3 --> 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 +/- 9.6% for intra-day, n = 5 each concentration; 90.9 +/- 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.     

Enhanced skin permeation of 6-methoxy-2-naphthylacetic acid by salt formation

Abstract

The aim of this work was to prepare salts of 6-methoxy-2-naphthylacetic acid (6-MNA) to improve its physicochemical properties for percutaneous application. 6-MNA, an active metabolite of non-steroidal anti-inflammatory drug nabumetone has long half life and has the tendency to penetrate well into synovial fluid. The physicochemical properties of 6-MNA salts were investigated by solubility measurements, differential scanning calorimetry (DSC) and infrared (IR). The DSC thermograms and Fourier transform infrared (FT-IR) spectra indicated that 6-MNA formed salts with organic and alkali metal bases. Among the series, salts formed with amine bases (ethanolamine, diethanolamine, triethanolamine and diethylamine) had lower melting points while alkali metal salt (sodium) had higher melting point than 6-MNA. The salts had higher solubilities than 6-MNA as determined in phosphate buffer at pH 5.0 and 7.4. There is no significant difference in partition coefficient (log P) values between salts and 6-MNA at pH 5.0 but, at pH 7.4, the log P values for the salts increased by 4–10 times as compared to 6-MNA. In vitro permeation studies showed that all the salts increased the flux in comparision to 6-MNA, and the ethanolamine salt (1b) was found to be having 7.7 and 9.4 times higher permeability as compared to 6-MNA at pH 5.0 and 7.4, respectively.     

Determination of theophylline and its metabolites in human urine by high-performance liquid chromatography

High-performance liquid chromatographic method with UV detection was developed for the determination of theophylline and its metabolites, in human urine using β-hydroxyethyl theophylline (β-HET) as an internal standard. For extraction of urine sample, quality control sample and xanthine-free blank urine were mixed with decylamine (ion-paring reagent) and β-HET. After saturation with ammonium sulfate, the mixture was then extracted with organic solvent at pH values of 4.0∼4.5. All separations were performed with ion-pair chromatography using decylamine as an ion-pairing reagent and 3 mM sodium acetate buffered mobile phase (pH 4.0) containing 1% (v/v) acetonitrile and 0.75 mM decylamine. The detection limits of theophylline, 1,3-DMU, 1-MU, 3-MX and 1-MX in human urine were 0.17, 0.17, 0.39, 0.19 and 0.19 μg/ml, based on a signal-to-noise ratios of 3.0. The mean intraday coefficients of variation (C.V.s) of each compound on nine replicates were lower than 2.0%, while mean interday C.V.s on three days were lower than 1.6%. All separations were finished within 40 minutes.     

Simultaneous determination of warfarin enantiomers and its metabolite in human plasma by column-switching high-performance liquid chromatography with chiral separation

A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of warfarin enantiomers and their metabolites, 7-hydroxywarfarin enantiomers, in human plasma is described. Warfarin enantiomers, 7-hydroxywarfarin enantiomers, and an internal standard, diclofenac sodium, were extracted from 1 mL of a plasma sample using diethyl ether-chloroform (80:20, v/v). The extract was injected onto column I (TSK precolumn BSA-C8, 5 microm, 10 mm x 4.6 mm inside diameter) for cleanup and column II (Chiralcel OD-RH analytical column, 150 mm x 4.6 mm inside diameter) coupled with a guard column (Chiralcel OD-RH guard column, 10 mm x4.6 mm inside diameter) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (84:16 v/v, pH 2.0) for clean-up and phosphate buffer-acetonitrile (45:55 v/v, pH 2.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 312 nm, and total time for chromatographic separation was approximately 25 minutes. The validated concentration ranges of this method were 3 to 1000 ng/mL for (R)- and (S)-warfarin and 3 to 200 ng/mL for (R)- and (S)-7-hydroxywarfarin. Intra- and interday coefficients of variation were less than 4.4% and 4.9% for (R)-warfarin and 4.8% and 4.0% for (S)-warfarin, and 5.1% and 4.2% for (R)-7-hydroxywarfarin and 5.8% and 5.0% for (S)-7-hydroxywarfarin at the different concentrations. The limit of quantification was 3 ng/mL for both warfarin and 7-hydroxywarfarin enantiomers. This method was suitable for therapeutic drug monitoring of warfarin enantiomers and was applied in a pharmacokinetic study requiring the simultaneous determination of warfarin enantiomers and its metabolite, 7-hydroxywarfarin enantiomers, in human volunteers.     

The analysis of clobazam and its metabolite desmethylclobazam by high-performance liquid chromatography.

The analysis of clobazam and its metabolite desmethylclobazam by high-performance liquid chromatography is described. After adding an internal standard 500 microliters of plasma is extracted under basic conditions into dichloroethane. The organic solvent is then evaporated to dryness and the residue reconstituted in 100 microliters of mobile phase prior to injecting an aliquot (30 microliters) onto a Hypersil 5 MOS column, which is eluted with acetonitrile/acetate buffer (pH 5.4) 40:60 vol/vol. The components are separated in approximately 12 min. Using this method, 15 micrograms L-1 of clobazam and 30 micrograms L-1 of desmethylclobazam can be detected. The method is suitable for the therapeutic monitoring of these two drugs in patient samples.     

Determination of oxprenolol and its glucuronide metabolite in plasma and urine using high-performance liquid chromatography

A HPLC assay is presented for the determination of oxprenolol (1) and its glucuronic acid conjugate (2) in human plasma and urine. The procedure employs a selective re-extraction using alprenolol (3) as the internal standard, followed by reversed-phase chromatography and UV-detection. The minimal detectable concentration is 10 ng/ml in plasma and 50 ng/ml in urine, using 1.0 and 0.5 ml of plasma and urine, respectively. Within-run and day-to-day variations are below 10% at all concentrations examined. Plasma and urine samples of either healthy volunteers or patients with renal failure are free of interferences from endogenous compounds and drugs frequently used in these patients. The glucuronic acid conjugate of oxprenolol is determined as the parent compound after hydrolytic cleavage with beta-glucuronidase/arylsulfatase. The specificity and selectivity of this cleavage are also demonstrated.     

Simultaneous determination of thiopental and its metabolite, pentobarbital, in blood by high-performance liquid chromatography and post-column photochemical reaction

A novel approach for the simultaneous high-performance liquid chromatographic (HPLC) analysis of thiopental and its major metabolite, pentobarbital, in blood plasma is described. On-line irradiation of the column eluate with UV-Iight leads to a significant bathochromic shift in the absorbance spectrum of pentobarbital, allowing now sensitive UV-detection of both barbiturates at 270 nm. At this longer wavelength, plasma matrix constituents are less interfering in the analysis, thus less sample preparation is necessary and blood plasma can be directly injected into the HPLC system only after protein precipitation with acetonitrile. Because of the minimal sample handling, the described HPLC method has good accuracy and reproducibility and thiopental and pentobarbital can be determined in small plasma volumes down to 0.2 μg ml⁻¹.     

HPLC法同时测定人尿液中霉酚酸及其代谢物葡糖苷酸结合物的浓度

目的用HPLC测定人尿液中霉酚酸(MPA)及其代谢物葡糖苷酸结合物(MPAG)的浓度。方法分析柱为Zorbax Eclipse XDB-C8(150 mm×4．6mm,5μm);流动相为甲醇-0．1％三氟乙酸溶液(55:45,V／V);尿稀释5倍后直接进样。MPAG采用紫外检测,检测波长为250nm;MPA采用柱后添加0．2 mol·L-1 NaOH溶液进行荧光检测,荧光激发波长325 nm,发射波长435 nm。结果MPA和MPAG的线性范围分别为0．1-50 mg·L-1和10-500 mg·L-1,MPA、MPAG日内和日间RSD均<10％。结论此法能简便、灵敏、准确地测定人体尿液中MPA与MPAG的浓度,可用于临床药动学研究和治疗药物浓度监测。     

Determination of amygdalin and its major metabolite prunasin in plasma and urine by high pressure liquid chromatography

A method is described for the determination of amygdalin and prunasin in plasma ultrafiltrate and urine. Both compounds are separated by high pressure liquid chromatography on a reversed phase column and subsequently detected at 215 nm. The identity of an amygdalin metabolite with prunasin was confirmed by mass spectrometry.     

Simultaneous determination of isoline and its two major metabolites using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatographic (HPLC) assay was developed for a simultaneous determination of isoline, a potent hepatotoxic pyrrolizidine alkaloid, and its two major metabolites, namely M1 (bisline) and M2 (bisline lactone, a new pyrrolizidine alkaloid). The latter two metabolites were produced during in vitro metabolism of isoline by rat and mouse microsomal enzyme systems. The analysis was conducted by a direct injection of aliquots of supernatant of the microsomal reaction mixture treated with the equal volume of ice-cold methanol onto a conventional reversed-phase analytical column (150 x 4.6 mm). The analytes were separated by a gradient elution with mobile phases A (0.01 M dihydro-potassium phosphate, pH 4.8) and B (acetonitrile). The assay has shown excellent precision and accuracy with less than 10% of overall intra- and interday variations and higher than 94% of overall accuracy. The developed HPLC method was successfully applied for the determination of the intact isoline and its two pH- and thermally labile metabolites produced in rat and mouse liver microsomal incubations.     

LC-MS/MS法同时测定人血浆中氯吡格雷及其羧酸代谢物的浓度

目的建立快速测定人血浆中氯吡格雷及其羧酸代谢物SR26334含量的LC-MS/MS法。方法乙醚-正己烷(4:1,V:V)2次液-液提取法(中性和酸化条件下),采用Teknokroma C_(18)色谱柱,以那格列奈和吡格列酮为内标,同时测定血浆中氯吡格雷和SR26334的浓度。流动相:甲醇-0.1%甲酸(80:20,V:V);流速:0.2mL·min~(-1);以多反应离子监测方式检测:氯吡格雷[M+H]~+,m/z 322.1→212.1;那格列奈[M+H]~+,m/z 318.3→166.2;氯吡格雷羧酸代谢物SR26334[M+H]~+,m/z 308.1→q98.1;吡格列酮[M+H]~+,m/z 357.2→134.2。结果氯吡格雷和内标那格列奈的保留时间分别在4.4和3.7min,SR26334和内标吡格列酮的保留时间分别在1.3和1.7min,氯吡格雷的线性范围为5～5000ng·L~(-1);SR26334的线性范围为20～2500μg·L~(-1)。提取回收率大于75%,方法回收率大于90%,日内、日间RSD小于10%(n=5)。结论本方法简便快速,适用于氧吡格雷制剂的新药临床研究和临床长期治疗病人血药浓度的常规监测。     

Determination of naproxen in human urine by solid-phase microextraction coupled to liquid chromatography

An SPME-LC-UV method for the determination of the non-steroidal anti-inflammatory drug (NSAID) naproxen and, after hydrolysis, its glucuronide in human urine samples was developed for the first time using a carbowax/templated resin (CW/TPR-100)-coated fibre. The procedure required a very simple sample pre-treatment, an isocratic elution, and provides a highly selective extraction. All the aspects influencing adsorption (extraction time, temperature, pH and salt addition) and desorption (desorption and injection time and desorption solvent mixture composition) of the analyte on the fibre have been investigated. The linear range investigated in urine was 0.2-20 microg/ml (that covers the typical naproxen urinary concentration) and almost quantitative recoveries were obtained. Within-day and between-days R.S.D.% in urine were 4.5 and 6.0, respectively. The LOD and LOQ in spiked urine were 0.03 and 0.20 microg/ml, well below the usual naproxen urinary level.     

Simultaneous determination of Ciprofloxacin and the active metabolite of Prulifloxacin in aqueous human humor by high-performance liquid chromatography

A rapid and simple method for determining two fluoroquinolones (FQNs), namely Ciprofloxacin and Ulifloxacin, this being the last active metabolite of Prulifloxacin, in aqueous human humor (AHH) has been developed and validated. The calibration data resulted linearly correlated in the 4-500 ng/mL concentration range with 8 ng/mL lower limit of quantification (LLOQ) for Ciprofloxacin, and 5-600 ng/mL concentration range with 6 ng/mL LOD for Ulifloxacin. The proposed analytic procedure has been validated by testing quality control sample (QCS) of AHH probed with the two FQNs at 10, 50, 500, and 1000 ng/mL concentration values. Validation of the method has been checked by accuracy and precision data of intra-day and long-term experiments. The two FQN concentrations have been measured by HPLC technique with UV detection at 278-nm wavelength for the AHH of patients to whom were supplied oral doses of FQNs (500 mg) twice in a day, within 1-24h before the surgery intervention of cataract. The average concentration of Ciprofloxacin resulted 186 ng/mL and that of Ulifloxacin 78 ng/mL. The nice quality of the proposed analytic procedure means that it may be suitable for in vivo studies of pharmacokinetics regarding these substances in the AHH medium.     

Quantification of oxcarbazeipine and its active metabolite 10-hydroxycarbazepine in human plasma by high-performance liquid chromatography

A simple, sensitive and selective high-performance liquid chromatography (HPLC) method with ultraviolet detection (230 nm) was developed and validated for the quantification of oxcarbazepine (CAS 28721-07-5), a new antiepileptic drug, and its active metabolite 10-hydroxycarbazepine (CAS 29331-92-8) in human plasma. Following solid-phase extraction, the analytes and internal standard (zaleplon, CAS 151319-34-5) were separated using an isocratic mobile phase on a reverse phase C18 column. The lower limit of quantification was 50 ng/mL for oxcarbazepine and 100 ng/mL for 10-hydroxycarbazepine with a relative standard deviation of less than 10%. A linear dynamic range of 50 to 5000 ng/mL for oxcarbazepine and of 100 to 10000 ng/mL for 10-hydroxycarbazepine was established. This HPLC method was validated with between-batch precision of 0.8 to 8.6% and 3.2 to 7.5% for oxcarbazepine and 10-hydroxycarbazepine respectively. The between-batch accuracy was 94.0 to 102.4% and 95.4 to 105.6%, respectively. Stability of oxcarbazepine and 10-hydroxycarbazepine in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days storage in a freezer. This validated method is sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Determination of fluvoxamine and its metabolite fluvoxamino acid by liquid-liquid extraction and column-switching high-performance liquid chromatography

This study describes a new simultaneous determination of fluvoxamine and fluvoxamino acid by automated column-switching high-performance liquid chromatography. The test compounds were extracted from 1.5 ml of plasma using chloroform-toluene (15:85, v/v), and the extract was injected into a hydrophilic metaacrylate polymer column for clean-up and a C18 analytical column for separation. The mobile phase for separation consisted of phosphate buffer (0.02 M, pH 4.6), acetonitrile and perchloric acid (60%) (62.4:37.5:0.1, v/v/v) and was delivered at a flow rate of 0.6 ml/min. The peak was detected using a UV detector set at 254 nm. The method was validated for the concentration range 0.8-153.6 ng/ml for fluvoxamine and 0.6-115.2 ng/ml for fluvoxamino acid, and their good linearity (r > 0.998) were confirmed. Intra-day coefficient variations (CVs) for fluvoxamine and fluvoxamino acid were less than 6.6 and 6.0%, respectively. Inter-day CVs for corresponding compounds were 6.3 and 6.5%, respectively. Relative errors ranged from -18 to 9% and mean recoveries were 96-100%. The limit of quantification was 1.2 and 0.9 ng/ml for fluvoxamine and fluvoxamino acid, respectively. This method shows successful application for pharmacokinetic studies and therapeutic drug monitoring.