Simultaneous determination of docetaxel and ketoconazole in rat plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for simultaneous quantification of docetaxel and ketoconazole in rat plasma with paclitaxel as internal standard (IS). The analytes were extracted from rat plasma by using a liquid-liquid extraction technique with ethyl acetate and the LC separation was performed on a Cosmosil-C(18) analytical column (150 mm x 2.0 mm i.d., Nacalai Tesque Inc., Japan). The extracted samples were analyzed with LC/MS/MS, operating in selected reaction monitoring (SRM) mode. The SRM transitions of precursor ions to product ions were 830.3-->549.1 (m/z) for docetaxel, 531.2-->489.3 (m/z) for ketoconazole, and 876.7-->307.9 (m/z) for the IS. The calibration curves were linear over the range of 2-500 ng/mL for docetaxel and 50-20 000 ng/mL for ketoconazole, with coefficients of correlation above 0.999. The limits of quantification for docetaxel and ketoconazole were both 2 ng/mL. The limit of detection for each analyte was 1 ng/mL. The intra- and inter-day precision (CV) of analysis were within 7%, and the accuracy ranged from 95 to 110%. The overall recoveries for docetaxel and ketoconazole were about 89.0% and 91.1%, respectively. The total analysis time was only 9.0 min. This quantitation method was successfully applied to the simultaneous determination of docetaxel and ketoconazole in rat plasma and some potential interaction was found in the current coadministration pharmacokinetic study. This established method was also utilized in the in vitro and in vivo drug-drug interaction study of docetaxel and ketoconazole (to be published).     

Analysis of aromatic and terpenic constituents of pepper extracts by capillary electrochromatography

An original method based on CEC has been developed for the determination of aromatic and terpenic compounds in extracts of spices obtained from Piper nigrum. The method is based on the use of a fused silica capillary (effective length: 23.5 cm, internal diameter: 100 microm) packed with a C18 sorbent (packing length: 23 cm, particle size: 5 microm). The mobile phase is a 50 mM, pH 6.0 ammonium acetate/ACN (10:90 v/v) mixture. Applying a 30 kV voltage, the following 11 compounds were separated and analysed: terpinen-4-ol, caryophyllene oxide, limonene, alpha-pinene, 3-carene, beta-pinene, alpha-humulene, beta-caryophyllene, alpha-phellandrene, eugenol and piperine. Compound determination is carried out using a diode-array detector set at 265 and 338 nm for alpha-phellandrene and piperine, respectively, and at 210 nm (reference subtraction at 282 nm) for all the other analytes. The optimised method has been validated with good results in terms of linearity, limits of quantitation, detection and precision. The CEC method was successfully applied to the analysis of essential oils and methanolic extracts of 'black', 'white' and 'green' pepper.     

Analysis of ethambutol and methoxyphenamine by capillary electrophoresis with electrochemiluminescence detection

A capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection method for the analysis of ethambutol (EB) and methoxyphenamine (MP) has been investigated. Complete separation of EB and MP was achieved in 8 min using a background electrolyte of 20 mM sodium phosphate at pH 10.0 and a separation voltage of 9 kV. ECL detection was performed with an indium/tin oxide (ITO) working electrode biased at 1.4 V (versus a Pt wire reference) in a 200 mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)3(2+) (where bpy = 2,2'-bipyridyl). Linear correlation (r > or = 0.993) between ECL intensity and drug concentration was obtained in the range 2-50 ng/ml. The limits of detection (LODs) for EB and MP in water were 1.0 and 0.9 ng/ml, respectively. The relative standard deviation values on peak size (10 ng/ml level) and migration time for the two drugs were in the ranges 5-8 and 0.2-0.7% (n = 7), respectively. Applicability of the CE-ECL method to the analysis of human plasma spiked with EB and MP was examined. The LODs for EB and MP in plasma were 0.4 and 0.3 microg/ml, respectively.     

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of cinnarizine and nicergoline in pharmaceutical preparations

A rapid, simple, and highly sensitive second-derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixtures of cinnarizine (CN) and nicergoline (NIC). The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 80 nm in aqueous methanol (50%, v/v). The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.025-1.5 and 0.25-5.5 microg/mL for CN and NIC, respectively, with lower detection limits of 0.58 and 0.82 ng/mL and quantitation limits of 1.93 and 2.73 ng/mL for CN and NIC, respectively. The proposed method was successfully applied for the determination of the studied compounds in synthetic mixtures and in commercial tablets. The results obtained were in good agreement with those obtained with reference methods. The high sensitivity attained by the proposed method allowed the determination of CN in real and spiked human plasma. The mean recovery in the case of spiked human plasma [number of trials (n) = 3] was 102.82 +/- 2.17%, while that in real human plasma (n = 3) was 105.25 +/- 2.05.     

Determination of remoxipride in human plasma and urine by reversed-phase ion-pair high-performance liquid chromatography.

A sensitive method is described for the measurement of remoxipride in human plasma and urine. Remoxipride and its internal standard are extracted from plasma or urine at pH 12 with a mixture of hexane and methyl tert.-butyl ether. After washing the organic phase with base, the compounds are extracted into acid and analyzed on a C18 column with ultraviolet detection at 214 nm. The mobile phase is composed of acetonitrile and aqueous buffer (sodium perchlorate and phosphoric acid, pH 1.7). The limits of reliable quantitation for remoxipride are 12.5 and 50 ng/ml for plasma and urine, respectively. The run times are 6 min for plasma and 3 min for urine. The method has been successfully used to assay remoxipride clinical study samples. This mobile phase has also been successfully applied to the analysis of other basic drugs such as cimetidine, codeine, diltiazem and quinidine with minor modifications.     

Capillary electrophoresis of baclofen with argon-ion laser-induced fluorescence detection

A capillary electrophoretic method with laser-induced fluorescence detection for baclofen (4-amino-3-p-chlorophenylbutyric acid) has been developed. 6-Carboxyfluorescein succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the lowest derivatizable concentration limit for baclofen in aqueous solution was 10 nM (2 ng baclofen/ml). Coupled with a simple clean up procedure, the method can be applied to the analysis of baclofen in human plasma at micromolar level. Recovery of spiked baclofen in plasma was 95%. The relative standard deviation values on peak size (0.5 microM level) and migration time were 8.2 and 1.0% (n=7), respectively. The limit of detection of baclofen in plasma was 0.1 microM (21 ng/ml).     

Sensitive micromethod for column liquid chromatographic determination of fluoxetine and norfluoxetine in human plasma.

A rapid, selective and sensitive micromethod has been developed for the determination of fluoxetine (FLU) and its demethylated metabolite norfluoxetine (N-FLU) using a 250-microliters plasma sample and column liquid chromatography with ultraviolet detection at 226 nm. The limit of detection is 2.0 ng/ml for both FLU and N-FLU. Peak-height ratios are linear over a concentration range of 10-800 and 10-1000 ng/ml for FLU and N-FLU, respectively. Acceptable coefficients of variation are demonstrated for both within-run and day-to-day assays. Selected drugs were checked for interference. The method, which requires a very small volume of plasma, is sensitive enough for pharmacokinetic studies in animals, clinical pharmacology studies and drug monitoring in children or adult patients.     

Spectrophotometric determination of acyclovir and ribavirin in their dosage forms

Two simple, accurate, and reliable spectrophotometric methods have been developed for the determination of 2 antiviral drugs, acyclovir (ACV) and ribavirin (RBV), in their pharmaceutical formulations. These methods are based on oxidation of the 2 drugs with either cerium (IV) ammonium sulfate (Method A) or potassium persulfate (Method B). The products of oxidation in both methods are coupled with 3-methylbenzothiazolin 2-one hydrazone, producing a deep blue color with a maximum absorption wavelength at 630 nm. In Method A, the absorbance-concentration plots were linear over the ranges of 5-50 and 10-60 microg/mL with detection limits of 0.18 microg/mL (8 x 10(-7) M) and 0.63 microg/mL (2.58 x 10(-6) M) for ACV and RBV, respectively. In Method B, the ranges were 5-45 and 20-50 microg/mL with detection limits of 0.11 microg/mL (4.88 x 10(-7) M) and 1.40 microg/mL (5.73 x 10(-6) M) for the 2 drugs, respectively. The molar absorptivities were 4.1 x 10(3) and 3.65 x 10(3) L/mol/cm in Method A and 5.03 x 10(3) and 3.97 x 10(3) L/mol/cm in Method B for the 2 drugs, respectively. The proposed methods were applied successfully for the determination of the 2 drugs in their pharmaceutical formulations. The percentage recoveries +/- standard deviation were 99.57 +/- 0.86 and 100.82 +/- 0.46 for ACV; 99.41 +/- 1.08 and 100.35 +/- 1.03 for RBV. The results obtained were compared statistically with those given by official methods and showed no significant differences regarding accuracy and precision.     

Determination of catechin isomers in human plasma subsequent to green tea ingestion using chiral capillary electrophoresis with a high-sensitivity cell

A simple and reliable analytical electrophoretic method using chiral capillary electrophoresis (CCE) with a high-sensitivity cell of special design has been established for simultaneous determination of (+)-catechin (C) and (-)-epicatechin (EC) in aqueous and human plasma media. The application of a capillary with high-sensitivity cell has led to an improvement of 10-fold and 5-fold time-corrected peak area over a standard cell and a capillary with bubble cell, respectively. Analysis has involved the electrophoretic separation of C and EC in less than 4.0 min at 210 nm. The running buffer consist of 50.0 mmol L(-1) borate buffer with 1.0 mmol L(-1) beta-cyclodextrin at pH 8.5. CCE system has been proved for its intended use by applying procedure starting from calibration of CE instrument into validation of all experimental parameters. The resolution between catechin isomers under optimal conditions has been found to be more than 3.0. The detection limits of C and EC have been calculated to be 3.2 and 1.0 ng mL(-1), respectively. Good linearity has been obtained with correlation coefficient (r(2)) ranging between 0.995 and 0.996 at 99% confidence level (CL). Application of the proposed method to human plasma after ingestion of green tea has successfully been achieved and has statistically been proved. The unchanged amounts of C and EC in plasma were about 17.4 and 1.8% of the administered dose after 2 h of starting tea ingestion. The detection limits of C and EC in human plasma at 210 nm were 4.1 and 1.5 ng mL(-1), respectively.     

高效液相色谱法测定环境水中痕量4,4′-二氨基联苯、4-硝基酚和苯酚

采用ＰｈｅｎｏｍｅｎｅｘＳｐｈｅｒｅｘＣ１８色谱柱,以含５０ｍｍｏｌ／Ｌ乙酸－乙酸钠缓冲溶液（ｐＨ６．０）的Ｖ（乙腈）∶Ｖ（乙醚）∶Ｖ（水）＝１２∶１０∶７８混合溶液为流动相,用分光光度法检测,检测波长为２７５ｎｍ,在１６ｍｉｎ内实现了４,４′－二氨基联苯、４－硝基酚和苯酚的同时分离测定。检测限分别为０．１４,０．１９和０．０８ｎｇ。标准回收率分别为９８．８５％,９８．１５％和９８．１０％。方法灵敏度高,用于环境水样分析时结果令人满意。     

Analysis of baclofen by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. Naphthalene-2,3-dicarboxaldehyde was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) and a He-Cd laser (excitation at 442 nm, emission at 500 nm). Linearity (r > or = 0.99) over three orders of magnitude was generally obtained and the concentration limit of detection was in the nanomolar level. Coupled with a simple cleanup procedure, the method was successfully applied to the analysis of baclofen in human plasma. Recovery of spiked baclofen in plasma was 98%. The relative standard deviation values on peak size and migration time were 7.9% and 0.4%, respectively. The limit of detection of baclofen in plasma was 10 ng/ml.     

High-performance liquid chromatographic determination of benzodiazepines in human plasma

A simple and sensitive method for determination of benzodiazepines in plasma has been developed using high-performance liquid chromatography in a reverse-phase mode. The method is illustrated by application to plasma samples containing diazepam and N-desmethyldiazepam at concentrations which would be encountered during therapy, with limits of detection of 10 ng/ml and 2 ng/ml for diazepam and N-desmethyldiazepam, respectively.     

Rapid simultaneous determination of four anthracene derivatives using a single non-linear variable-angle synchronous fluorescence spectrum

A rapid, simple and inexpensive spectrofluorimetric method has been developed for the simultaneous identification and quantification of anthracene (ANT), 9,10-dimethylanthracene (DIM), 2-aminoanthracene (AMI) and dibenz[ah]anthracene (DIB). A well-resolved spectrum for the mixture of these four compounds is obtained based on a single non-linear variable-angle synchronous scanning. The linear concentration ranges are 10-1,000, 5-500, 50-1,000 and 10-200 ng mL(-1) for ANT, DIM, AMI and DIB, respectively, at lambdaex/lambdaem = 358/380, 399/408, 414/465 and 298/394 nm, respectively. The analyses are performed in cyclohexane. Recoveries of 90.0-111.0% in synthetic mixtures are obtained. The detection limits are 2.0 ng mL(-1) for DIM, 2.7 ng mL(-1) for ANT, 15.8 ng mL(-1) for AMI and 4.2 ng mL(-1) for DIB. The method has also been applied to several real water samples with satisfactory results.     

Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

Development and validation of an HPLC method for the determination of seven penicillin antibiotics in veterinary drugs and bovine blood plasma

Herein a quantitative method for the determination of seven penicillins in bovine plasma and veterinary drugs has been developed. Amoxicillin (AMO), ampicillin (AMP), penicillin G (PENG), penicillin V (PENV), oxacillin (OXA), cloxacillin (CLO) and dicloxacillin (DICLO) were separated on a Perfectsil ODS‐2 (250×4 mm, 5 μm) column, using gradient elution, with a mobile phase of 0.1% v/v TFA and ACN–methanol (90:10 v/v). PDA detection was used at 240 nm. Penicillins were isolated from bovine plasma by SPE on Lichrolut RP‐18 cartridges with mean recoveries from 85.7 to 113.5%. Colchicine (3 ng/μL) was used as an internal standard. The developed method was validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity. Repeatability (n = 5) and between‐day precision (n = 5) revealed RSD < 12%. The detection limits in the bovine plasma were estimated as 18 ng for AMO and AMP, 25 for PENG, PENV and OXA, 3 ng for CLO and 12 ng for DICLO. Spiked plasma samples were stable for 1 wk, except for AMP and CLO, which were stable for 3 wk and OXA for 4 wk. AMO, PENG and PENV were stable for two freeze–thaw cycles, OXA, CLO and DICLO for four, while AMP only for one.     

Determination of risperidone and 9-hydroxyrisperidone in plasma, urine and animal tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the simultaneous determination of the new antipsychotic risperidone and its major metabolite 9-hydroxyrisperidone in plasma, urine and animal tissues. The alkalinized plasma samples were extracted with ethyl acetate and further purified prior to reversed-phase chromatography with ultraviolet detection at 280 nm. The method could also be applied to urine samples and animal tissue homogenates. Quantification limits were 2 ng/ml for plasma and urine and 10 ng/g for animal tissue. The method was applied to pharmacokinetic studies in experimental animals, human volunteers and patients.     

High-performance liquid chromatographic determination of fluvoxamine and fluvoxamino acid in human plasma.

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of fluvoxamine and its major metabolite fluvoxamino acid in plasma. Fluvoxamine and fluvoxamino acid in plasma were extracted using a C18 bonded-solid phase cartridge, followed by C4 reversed-phase HPLC separation.Fluvoxamine, fluvoxamino acid and moperone as an internal standard were detected by ultraviolet absorbance at 254 nm. It was possible to determine both fluvoxamine and fluvoxamino acid in the concentration range of 25.0-200.0 ng/mL, respectively. The detection limits of both fluvoxamine and fluvoxamino acid were 10.0 ng/mL, respectively. The mean recoveries of fluvoxamine and fluvoxamino acid added to plasma were more than 94.0% and 96.5%, with a coefficient of variation of less than 7.6% and 8.2%, respectively. This method has been used for the simultaneous determination of steady-state plasma concentration (Css) of fluvoxamine and fluvoxamino acid in depressive patients treated with 200 mg of oral fluvoxamine dosed as 100 mg twice-daily. The Css values of fluvoxamine and fluvoxamino acid in twelve Japanese patients were showed individual variations, which were in the range of 48.3-532.9 ng/ml and 35.6-307.1 ng/ml, respectively.     

Gas chromatographic determination of ifosfamide in microvolumes of urine and plasma.

In oncology, particularly in pediatric malignancies, high doses (5-10 g/m2) of the oxazaphosphorine ifosfamide play an important role in the treatment of sarcomas. Pharmacokinetic data of ifosfamide and its metabolites in these cases are scanty. Considering the special demands of the determination of ifosfamide in plasma of young children, a very sensitive capillary gas chromatographic method, requiring only 50 microliters of plasma, has been developed. This bioanalysis of ifosfamide shows good linearity and accuracy in the concentration range 10 ng to 100 micrograms per ml of plasma and 25 ng to 1 mg per ml of urine. The absolute limits of detection in plasma and urine are 2 ng/ml and 5 ng/ml, respectively. The stability of various solutions of ifosfamide and trofosfamide was tested and proved to be satisfactory, except for ifosfamide in plasma and urine kept in the refrigerator. The validity of the method for pharmacokinetic purposes is shown in the case of one patient.     

High-performance liquid chromatographic assay of cefotaxime, desacetylcefotaxime and ceftriaxone in rat plasma

A simple and selective high-performance liquid chromatographic method is described for the analysis of the cephalosporins cefotaxime (CXM), desacetylcefotaxime (DACXM) and ceftriaxone (CFX) in rat plasma. Plasma was deproteinized with methanol, and the supernatant was directly injected into the chromatograph and monitored at 254 nm. For determination of the unbound drugs, a centrifugal ultrafiltration method was employed. The calibration curves were linear (r = 0.999) from 2.5 to 500 micrograms/ml; the detection limits were 100 ng/ml for DACXM and 250 ng/ml for CXM and CFX. The method was not interfered with by other plasma components, nor by barbital sodium or caffeine, and has been applied to study the pharmacokinetics of the cephalosporins in rats.     

Determination of narciclasine in serum by reversed-phase high-performance liquid chromatography: comparison of amperometric, ultraviolet photometric and fluorescence detection

Narciclasine was determined in the blood of mice by reversed-phase high-performance liquid chromatography, using a C18 stationary phase and a mobile phase of methanol-0.025 M potassium dihydrogen phosphate (50:50, v/v) of pH 5.5. Amperometric detection at a carbon fibre array working electrode held at +1.8 V (Ag/AgCl) permitted determination down to concentrations of 10 and 15.4 ng ml-1 (at a signal-to-noise ratio of 2) in aqueous solution and in serum, respectively. Fluorescence detection (excitation and emission wavelengths of 360 and 480 nm, respectively) exhibited somewhat poorer sensitivities for aqueous and serum samples: the respective limits of detection were 25 and 32 ng ml-1 at a signal-to-noise ratio of 2. Both the amperometric and the fluorescence detection were free from interference from blood components, but the fluorescence measurement required a post-column pH adjustment. UV photometric detection at 254 nm exhibited detection limits of 15 and 65 ng ml-1 in aqueous samples and in serum, respectively, and suffered from interferences from blood components that strongly absorbed in the ultraviolet region. All three detection techniques exhibited good linearity and precision.