Rapid determination of tamsulosin in human plasma by high-performance liquid chromatography using extraction with butyl acetate

A high-performance liquid chromatographic method with fluorescence detection for the determination of tamsulosin in human plasma is reported. The sample preparation involved liquid-liquid extraction of tamsulosin from alkalised plasma with butyl acetate and back-extraction of the drug to the phosphate buffer (pH 2). Butyl acetate is preferable to more commonly used ethyl acetate because of its much lower solubility in water. Liquid chromatography was performed on an octadecylsilica column (55 mm x 4 mm, 3 microm particles), the mobile phase consisted of acetonitrile-30 mM dihydrogenpotassium phosphate (25:75 v/v). The run time was 3.5 min. The fluorimetric detector was operated at 228/326 nm (excitation/emission wavelength). An analogue of tamsulosin, (R)-5-[2-[(3-(2-ethoxyphenoxy)propyl)amino]-2-methylethyl]-2-methoxybenzensulfonamide was used as the internal standard. The limit of quantitation was 0.4 ng/ml using 1 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 5%. The assay was applied to the analysis of samples from several pharmacokinetic studies.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Rapid determination of omeprazole in human plasma by protein precipitation and liquid chromatography-tandem mass spectrometry

A rapid, sensitive and reliable method was developed to quantitate omeprazole in human plasma using liquid chromatography-tandem mass spectrometry. The assay is based on protein precipitation with acetonitrile and reversed-phase liquid chromatography performed on an octadecylsilica column (55 mm x 2mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (60:40, v/v). Omeprazole and flunitrazepam, the internal standard, elute at 0.80+/-0.10 min with a total run time 1.35 min. Quantification was through positive ion mode and selected reaction monitoring mode at m/z 346.1-->197.9 for omeprazole and m/z 314.0-->268.0 for flunitrazepam, respectively. The lower limit of quantitation was 1.2 ng/ml using 0.25 ml of plasma and linearity was observed from 1.2 to 1200 ng/ml. Within-day and between-day precision expressed by relative standard deviation was less than 5% and inaccuracy did not exceed 12%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Liquid chromatographic method for the determination of ganciclovir and/or acyclovir in human plasma using pulsed amperometric detection

A rapid high-performance liquid chromatographic method for the quantitation of pseudoephedrine in human plasma is presented. The sample preparation involved liquid-liquid extraction of pseudoephedrine from alkalised plasma with hexane-isoamylalcohol (9:1, v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on an octadecylsilica column (50 x 4 mm, 5 microm particles); the mobile phase consisted of acetonitrile-phosphate buffer containing 0.1% of triethylamine, pH 2.4 (5:95, v/v). The run time was 4 min. The spectrophotometric detector was operated at 195 nm. Codeine was used as the internal standard. The limit of quantitation was 5.8 ng/ml using 0.5 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 8%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of mirtazapine in human plasma by liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of mirtazapine in human plasma is presented. The method is based on a liquid-liquid extraction and reversed-phase chromatography with fluorimetric detection. The separation was performed on a Luna microm C(18)(2) 50 x 4.6 mm I.D. column using an isocratic elution. Zolpidem hemitartrate was used as the internal standard. The between-day precision expressed by relative standard deviation was less than 5% and inaccuracy does not exceed 6%. A low limit of quantitation (1.5 ng/ml) and a short time of analysis (4 min) makes this assay suitable for pharmacokinetic studies.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Determination of free and total (free plus protein-bound) melatonin in plasma and cerebrospinal fluid by high-performance liquid chromatography with fluorescence detection

A simple, sensitive and accurate method for the estimation of free and total (free plus protein-bound) melatonin (MLT) in human plasma and cerebrospinal fluid (CSF) is described. Via Chem-Elut cartridges, free and total MLT (the latter obtained after a deproteinization step) were quantified in dichloromethane-extracted samples and analyzed in one chromatographic run by high-performance liquid chromatography (HPLC) with fluorimetric detection. The column used was an Extrasil ODS-2 (3 microm, 150 x 4.6 mm I.D.), while the mobile phase consisted of 75 mM sodium acetate-acetonitrile (72:28, v/v) (pH 5.0). Repeatability and reproducibility of the method were 3.24 and 9.4%, respectively. The recovery of melatonin from plasma and CSF was 99.9+/-4.0% for non-deproteinized samples and 93.2+/-4.8% for deproteinized samples. The detection limit of the assay was 0.5 pg/ml. In human plasma, the mean+/-SD concentrations in the darkness period were 23.18+/-7.44 pg/ml for free melatonin and 82.5+/-36.48 pg/ml for total melatonin, while the lowest concentrations detected during daytime were 2.23+/-2.22 and 7.40+/-5.68 pg/ml, respectively. Detection of MLT in CSF was 5.01+/-2.31 and 28.55+/-6.95 pg/ml for the free and total fraction, respectively.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of alendronate in human urine as 9-fluorenylmethyl derivative by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of alendronate as the 9-fluorenylmethyl derivative (FMOC) in human urine is presented. The sample preparation involved coprecipitation with calcium phosphate, separation on diethylamine (DEA) solid-phase extraction (SPE) cartridge and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer pH 11.9. Liquid chromatography was performed on an octadecylsilica column (150 x 4.6 mm, 3 microm particles); a gradient method with starting mobile phase acetonitrile-methanol-citrate/pyrophosphate buffer (20:15:65 v/v) was employed. The total run time was 21 min. The fluorimetric detector was operated at the following wavelengths: 260 nm (excitation) and 310 nm (emission). Pamdronate was used as the internal standard. The limit of quantitation was 3.5 ng/ml using 5 ml of urine. Within-day and between-day precision expressed by relative standard deviation was less than 8% and inaccuracy did not exceed 9%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of 25-OH-PPD in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive and specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the investigation of the pharmacokinetics of 20(R)-dammarane-3beta,12beta,20,25-tetrol (25-OH-PPD) in rat. Ginsenoside Rh(2) was employed as an internal standard. The plasma samples were pretreated by liquid-liquid extraction and analyzed using LC/MS/MS with an electrospray ionization interface. The mobile phase consisted of methanol-acetonitrile-10 mmol/l aqueous ammonium acetate (42.5:42.5:15, v:v:v), which was pumped at 0.4 ml/min. The analytical column (50 mm x 2.1 mm i.d.) was packed with Venusil XBP C8 material (3.5 microm). The standard curve was linear from 10 to 3000 ng/ml. The assay was specific, accurate (accuracy between -1.19 and 2.57% for all quality control samples), precise and reproducible (within- and between-day precisions measured as relative standard deviation were <5% and <7%, respectively). 25-OH-PPD in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 6h. The method had a lower limit of quantitation of 10 ng/ml, which offered a satisfactory sensitivity for the determination of (25-OH-PPD) in plasma. This quantitation method was successfully applied to pharmacokinetic studies of 25-OH-PPD after both an oral and an intravenous administration to rats and the absolute bioavailability is 64.8+/-14.3%.     

Sensitive determination of clarithromycin in human plasma by high-performance liquid chromatography with spectrophotometric detection

A rapid, selective and sensitive high-performance liquid chromatographic method with spectrophotometric detection was developed for the determination of clarithromycin in human plasma. Liquid-liquid extraction of clarithromycin and norverapamil (as internal standard) from plasma samples was performed with n-hexane/1-butanol (98:2, v/v) in alkaline condition followed by back-extraction into diluted acetic acid. Chromatography was carried out using a CN column (250 mm x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-50 mM aqueous sodium dihydrogen phosphate (32:68, v/v), pH 4.5. Detection was made at 205 nm and analyses were run at a flow-rate of 1.0 ml/min at 40 degrees C. The analysis time was less than 11 min. The method was specific and sensitive with a quantification limit of 31.25 ng/ml and a detection limit of 10 ng/ml in plasma. The mean absolute recovery of clarithromycin from plasma was 95.9%, while the intra- and inter-day coefficient of variation and percent error values of the assay method were all less than 9.5%. Linearity was assessed in the range of 31.25-2000 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method was used to analyze several hundred human plasma samples for bioavailability studies.     

Liquid chromatographic assay of abouthiouzine in plasma and its application to pharmacokinetic studies

Abouthiouzine is a newly synthesized antithyroid agent with a proposed less adverse effects profile than other currently used drugs. A simple and rapid reversed phase high performance liquid chromatography assay was developed to determine the concentration of abouthiouzine in human plasma. The procedure involved extraction of the drug and propranolol (internal standard) from the plasma using ethylacetate. The extract was evaporated under nitrogen and the residue was constituted with the mobile phase and injected onto micro-Bondapack phenyl column (10 microm, 3.9 mm x 150 mm). The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer, acetonitrile, and methanol in the ratio of 60:25:15 (v/v/v, pH=3.0), which was delivered at a rate of 1.5 ml/min. Abouthiouzine and the internal standard were monitored using UV detection at 240 nm; the run time was less than 5 min. The detection limit of abouthiouzine is 0.5 microg/ml. The within- and between-day coefficients of variation were less than 7%. Our method has been successfully used to measure abouthiouzine plasma concentrations in a rabbit model following an intravenous administration of the drug.     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).     

Development and validation of a sensitive assay of valproic acid in human plasma by high-performance liquid chromatography without prior derivatization

Sensitive and selective determination of valproic acid in plasma by high-performance liquid chromatography (HPLC) is usually achieved with pre-column derivatization. In the present work, the derivatization is omitted due to using a simple but highly selective plasma extraction procedure and an optimized chromatographic condition. Valproic acid and the internal standard octanoic acid were extracted from plasma samples with n-hexane under acidic condition followed by back-extraction into diluted triethylamine. Chromatography was performed on a CN column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-40 mM aqueous sodium dihydrogen phosphate (30:70, v/v), pH 3.5. Detection was made at 210 nm and analyses were run at a flow-rate of 1 ml/min. The method was specific and sensitive with a quantification limit of 1.25 microg/ml and a detection limit of 0.1 microg/ml in plasma. The mean absolute recovery for valproic acid using the present plasma extraction procedure was 75.8%. The intra- and inter-day coefficient of variation and percent error values of the assay method were all in acceptable range. Calibration curves were linear (r>0.999) from 1.25 to 320 microg/ml in plasma.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of phenylephrine in human plasma and its application to a pharmacokinetic study

A sensitive and reliable method was developed to quantitate phenylephrine in human plasma using liquid chromatography-electrospray tandem mass spectrometry. The assay was based on solid-phase extraction with C18 cartridges and hydrophilic interaction chromatography performed on a pentafluorophenylpropylsilica column (50 mm x 4 mm, 3 microm particles), the mobile phase consisted of methanol-10 mM ammonium acetate (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 168.1-->135.0 for phenylephrine and m/z 182.1-->135.0 for internal standard etilefrin, respectively. The lower limit of quantitation was 51 pg/ml using 0.25 ml of plasma and linearity was observed from 51 to 5500 pg/ml. Within-day and between-day precision expressed by relative standard deviation was less than 12% and inaccuracy did not exceed 8% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid-liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-(S)- and (-)-(R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm x 4.6 mm, 10 microm particle size) protected with a CN guard column, using hexane-ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-(S)- and (-)-(R)-mirtazapine were in the 88-111% range with a linear response over the 6.25-625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.