Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction

An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.6 mm I.D. Kromasil C8 5 microm column was performed using a mobile phase of water-acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 microg/mL with a 50-microL sample size. The method was successfully applied to in vitro pharmacokinetic-pharmacodynamic studies.     

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.     

Determination and pharmacokinetics of orientin in rabbit plasma by liquid chromatography after intravenous administration of orientin and Trollius chinensis Bunge extract

A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet (UV) absorbance detection. Orientin is the active constituent of purified herbal extract (TRO PE) from the flower of Trollius chinensis Bunge. Protein precipitation was used as the sample preparation technique. A Diamonsil C18 column (150 mmx4.6 mm, 5 microm) was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile (80/5/15, v/v/v). The calibration curve of orientin in rabbit plasma was linear in the concentration range of 0.530-53.0 microg/mL. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin and TRO PE at three different doses.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Separation and determination of astaxanthin from microalgal and yeast samples by molecularly imprinted microspheres

In this work, molecularly imprinted microspheres (MIMs) were synthesized by aqueous microsuspension polymerization using astaxanthin (3,3'-dihydroxy-beta,beta'-carotene-4,4'-dione) as imprinting molecule. The MIMs obtained were subsequently packed into the stainless steel column and the chromatographic characterization of the column was investigated. The effects of pH and composition of the mobile phase on the retention factor (k') were investigated in detail. The mixture of methanol and dichloromethane (DCM) (8:2, v/v) was used as mobile phase A while the mixture of methanol and water (5:5, v/v) as mobile phase B. The separation of astaxanthin and zeaxanthin (3,3'-dihydroxyl-beta-carotene) was obtained when the concentration of mobile phase B was higher than 30% (v/v) due to their strong lipophilicity. The method developed was successfully applied to separate astaxanthin in the saponified samples of the microalga Haematococcus pluvialis and the yeast Phaffia rhodozyma. The recovery of adding 40 mg astaxanthin to 1.0 g microalgal sample was 95.5% with an R.S.D. (n =5) of 5.3%. The results of determination of astaxanthin in the microalga and the yeast were 3.7% (R.S.D (n = 1.5%, n = 9) and 0.041% (R.S.D n= 7.3%, n = 9), respectively.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Determination of mitiglinide in rat plasma by high-performance liquid chromatography with UV detection

A selective and sensitive high-performance liquid chromatography method has been developed and validated for determination of mitiglinide (MGN) in rat plasma using 2-(4-biphenylyl) propionic acid (BPA) as internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved on a C(18) column using acetonitrile and 0.02 mol/l KH(2)PO(4) buffer (pH 4.0) (45:55, v/v) as mobile phase delivered at 1.0 ml/min. The UV detector was set at 210 nm. The assay was linear over the range 0.1-20 microg/ml for MGN. The average extraction recoveries of MGN and BPA from rat plasma were 98.6 and 97.4%, respectively. The developed method has been applied to the pharmacokinetic study of MGN in rats.     

Simultaneous determination of the antipsychotic drugs levomepromazine and clozapine and their main metabolites in human plasma by a HPLC-UV method with solid-phase extraction

A HPLC method with UV detection has been developed for the simultaneous determination of levomepromazine, clozapine and their main metabolites: N-desmethyl-levomepromazine, levomepromazine sulphoxide, O-desmethyl-levomepromazine, N-desmethylclozapine and clozapine N-oxide. The analytes were separated on a C8 reversed-phase column using a mobile phase composed of acetonitrile and a pH 2.0, 34 mM phosphate buffer containing 0.3% triethylamine (29:71, v/v). Loxapine was used as the internal standard. A reliable biological sample pre-treatment procedure by means of solid-phase extraction on C1 cartridges was implemented, which allows to obtain good extraction yields (>91%) for all analytes and appropriate sample purification from endogenous interference. The method was validated in terms of extraction yield, precision and accuracy. These assays gave RSD% values for precision always lower than 4.9% and mean accuracy values higher than 92%. The method is suitable for the therapeutic drug monitoring (TDM) of patients undergoing polypharmacy with levomepromazine and clozapine.     

Fully automated high-performance liquid chromatography of ciprofloxacin with direct injection of plasma and Mueller–Hinton broth for pharmacokinetic/pharmacodynamic studies

An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C₈ 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.     

Automated liquid-liquid extraction based on 96-well plate format in conjunction with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the quantitation of methoxsalen in human plasma

A sensitive, specific and high throughput bioanalytical method using automated sample processing via 96-well plate liquid-liquid extraction and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has been developed for the determination of methoxsalen in human plasma. Plasma samples with ketoconazole as internal standard (IS) were prepared by employing 0.2mL human plasma in ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on a Waters Acquity UPLC BEH C18 column using isocratic mobile phase, consisting of 10mM ammonium formate and acetonitrile (60:40, v/v), at a flow rate of 0.5mL/min. The linear dynamic range was established over the concentration range 1.1-213.1ng/mL for methoxsalen. The method was rugged and rapid with a total run time of 1.5min. It was successfully applied to a pivotal bioequivalence study in 12 healthy human subjects after oral administration of 10mg extended release methoxsalen formulation under fasting condition.     

Determination of 3'-amino-3'-deoxythymidine, a cytotoxic metabolite of 3'-azido-3'-deoxythymidine, in human plasma by ion-pair high-performance liquid chromatography

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of 3'-amino-3'-deoxy-thymidine (AMT), a cytotoxic metabolite of 3'-azido-3'-deoxy-thymidine (AZT, zidovudine), in human plasma. The sample pretreatment involved solid-phase extraction using cation-exchange extraction columns. Chromatography was carried out on a C₈ column, using a mobile phase of methanol—0.01 M ammonium acetate (pH 5)—0.25 M sodium dioctylsulfosuccinate (60:40:4, v/v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The lower limit of quantitation is 5 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used for the determination of AMT in patients with AIDS who used AZT.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.     

Simple high-performance liquid chromatographic method for the determination of metformin in human plasma

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

Simple liquid chromatographic method for the determination of cefotaxime in human and rat plasma

A high-performance liquid chromatographic method with ultraviolet (UV) detection was developed for measuring cefotaxime in rat and human plasma. The method used direct injection of the plasma supernatant after deproteinization with 70% perchloric acid. Degradation of cefotaxime in acidic medium was retarded by adding phosphate buffer before centrifuging the sample. The mobile phase was 0.05 M aqueous ammonium acetate-acetonitrile-tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5. Analysis was run at a flow-rate of 1.0 ml/min, and a detection wavelength of 254 nm was used. The method has a quantification limit of 0.20 microgram/ml. The within- and between-day coefficients of variation and accuracy values were less than 8% and +/-3%, respectively, while the recovery values were greater than 87% over the concentration range tested (0.20-50 microgram/ml). The speed, sensitivity, specificity and reproducibility of this method make it particularly suitable for the routine determination of cefotaxime in human plasma. Moreover, only a relatively small sample plasma volume (100 microliter) is required, allowing this method to be applied to samples taken from neonates.     

Determination of nifedipine in human plasma by ultra performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study

A fast, sensitive and selective ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method was developed for the determination of nifedipine in human plasma. Nitrendipine was used as the internal standard. The sample preparation employed liquid-liquid extraction with a mixture of n-hexane-diethyl ether (1:3, v/v). Chromatographic separation was performed on an ACQUITY UPLC? BEH C(18) column. The mobile phase was composed of acetonitrile-10 mmol/L ammonium acetate (75:25, v/v) with a flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. A high throughput was achieved with a run time of 1.4 min per sample. The linear calibration curves were obtained in the concentration range of 0.104-52.0 ng/mL (r(2)≥ 0.99) with a lower limit of quantification (LLOQ) of 0.104 ng/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were below 15% and the accuracy (relative error, RE) was -4.0% to 6.2% at three quality control levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of nifedipine sustained-release tablet in healthy male volunteers.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Determination of roxithromycin residues in the flounder muscle with electrospray liquid chromatography–mass spectrometry

A highly sensitive and specific method for the determination of roxithromycin in the flounder muscle by LC–MS was developed and validated. A dichloromethane extract of the sample was separated on C₁₈ reversed-phase column with acetonitrile–50 mM ammonium acetate (80:20, v/v) as the mobile phase and analyzed by LC–MS via atmospheric pressure ionization/electrospray ionization interface. The limit of detection and limit of quantitation were 0.01 and 0.1 ng/g, respectively. Mean recoveries from spiked muscles were 81.1% (ranged from 71.0 to 90.3%) for roxithromycin. The method has been successfully applied to determine roxithromycin in the flounder muscle.     

Rapid determination of solanesol in tobacco by high-performance liquid chromatography with evaporative light scattering detection following microwave-assisted extraction

Solanesol is the starting material for many high-value biochemicals, including coenzyme Q(10) and Vitamin-K analogues. The aim of the current study was to develop a reliable and fast analytical procedure for the determination of solanesol in tobacco using high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique. The HPLC conditions were Agilent C18 column using acetonitrile-isopropanol (60:40, v/v) as mobile phase at a flow rate of 1 ml/min. ELSD conditions were optimized at nebulizer-gas flow rate of 1.5 l/min and drift tube temperature of 65 degrees C. The method was validated to achieve the satisfactory precision and recovery, and the calibration range was 0.1-1.5 mg/ml. The developed analytical procedure was successfully applied to determine solanesol content in tobacco samples from different growing regions in China.