Determination of zidovudine/lamivudine/nevirapine in human plasma using ion-pair HPLC

A new high-performance liquid chromatography (HPLC) assay was developed for the simultaneous determination of zidovudine (AZT)/lamivudine (3TC)/nevirapine in human plasma. Plasma samples were treated using a solid-phase extraction procedure. The compounds were separated using a mobile phase of 20 mM sodium phosphate buffer (containing 8 mM 1-octanesulfonic acid sodium salt)-acetonitrile (86:14, v/v) with pH adjusted to 3.2 with phosphoric acid on an octylsilane column (150x3.9 mm i.d.) with UV detection at 265 nm. Aprobarbital was chosen as the internal standard (IS). The method was validated over the range of 57.6-2880 ng/ml for AZT, 59.0-17650 ng/ml for 3TC and 53.2-13300 ng/ml for nevirapine. The method was shown to be accurate, with intra-day and inter-day accuracy from 0.1 to 11% and precise, with intra-day and inter-day precision from 0.4 to 14%. Extraction recoveries of the analytes and IS from plasma were higher than 92%. The assay should be suitable for use in pharmacokinetic studies and routine plasma monitoring of this triple-drug therapy in AIDS patients.     

Development and validation of an HPLC method for the determination of gatifloxacin stability in human plasma

A simple reversed-phase high performance liquid chromatography (HPLC) method for the determination of gatifloxacin stability in human plasma was developed and validated. Using ciprofloxacin as an internal standard (IS), separation was achieved on X Terra MS C18 (3 mm x 50 mm, 5 microm) column. The mobile phase, 0.025 M disodium hydrogen phosphate (pH 3.0) and acetonitrile (80:20 v/v), were delivered at a flow rate of 1.0 ml/min. The eluent was monitored using spectrophotometeric detection at 293 nm. Plasma samples were deproteinized using Amicon Centrifree system. No interference in blank plasma or of commonly used drugs was observed. The relationship between gatifloxacin concentration and peak height ratio of gatifloxacin to the IS was linear over the range of 0.10-6.0 microg/ml. The intra-day and inter-day coefficients of variation were < or = 2.77 and < or = 4.59%, respectively. The extraction recovery of gatifloxacin and the IS from plasma samples was > or = 85%. Gatifloxacin was found to be stable for at least 5 h at RT, 7 weeks at -20 degrees C, and after 3 freeze-thaw cycles in plasma; 16 h at RT and 48 h at -20 degrees C in deproteinized plasma; and 24 h at RT and 7 weeks at -20 degrees C in phosphate buffer.     

Liquid chromatography tandem mass spectrometry assay to determine the pharmacokinetics of aildenafil in human plasma

A simple, sensitive and specific liquid chromatography/tandem mass spectrometry method for the quantitation of aildenafil, a new phosphodiesterase V inhibitor, in human plasma is presented. The analyte and internal standard, sildenafil, were extracted by a one-step liquid-liquid extraction in alkaline conditions and separated on a C(18) column using ammonia:10mM ammonium acetate buffer:methanol (0.1:15:85, v/v/v) as the mobile phase. The detection by an API 4000 triple quadrupole mass spectrometer in multiple-reaction monitoring mode was completed within 2.5 min. The calibration curve exhibited a linear dynamic range of 0.05-100 ng/ml with a 10 pg/ml limit of detection. The intra- and inter-day precisions measured as relative standard deviation were within 8.04% and 5.72%, respectively. This method has been used in a pharmacokinetic study of aildenafil in healthy male volunteers each given an oral administration of one of the three dosages.     

Stability-indicating HPLC assays for the determination of prilocaine and procaine drug combinations

Stability-indicating, reversed phase high-performance liquid chromatographic (HPLC) methods have been developed for the determination of several procaine hydrochloride and prilocaine hydrochloride combinations. The separation and quantitation of epinephrine-prilocaine and epinephrine-procaine drug combinations were achieved on a phenyl column using a mobile phase of 80:20% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid sodium salt-acetonitrile at a flow rate of 1 ml x min(-1) and UV detection at 254 nm. The method showed linearity for the epinephrine and prilocaine hydrochloride mixture in the 0.25-2.5 and 8-200 micro g ml(-1) ranges, respectively. The intra- and inter-day relative standard deviations (RSDs) ranged from 0.26 to 2.05% and 0.04 to 0.61% for epinephrine and prilocaine hydrochloride, respectively. The epinephrine and procaine hydrochloride mixture yielded linear ranges of 0.25-2.0 and 5-100 micro g ml(-1) and intra- and inter-day RSDs ranged from 0.23 to 1.88% and 0.07 to 0.26% for epinephrine and procaine hydrochloride, respectively. The assays were shown to be suitable for measuring epinephrine-prilocaine and epinephrine-procaine combinations in their respective injection dosage forms. Stability-indicating HPLC assays were also developed for several other procaine drug combinations since their monographs are present in the USP 24; however, quantitation was not investigated since these combinations are not commercially available. A mobile phase consisting of 80:20% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid-acetonitrile was utilized for the levonordefrin-tetracaine-procaine drug combination, while a mobile phase consisting of 70:30% v/v 25 mM phosphate buffer (pH 3.0) containing 50 mM heptanesulfonic acid sodium salt-acetonitrile was utilized for the separation of levonordefrin-procaine-propoxycaine and norepinephrine-procaine-propoxycaine. All separations were achieved on a phenyl column at a flow rate of 1 ml x min(-1) and UV detection at 254 nm.     

Determination of Huperzine A in rat plasma by high-performance liquid chromatography with a fluorescence detector

A simple reversed-phase high-performance liquid chromatography (HPLC)-fluorescence method for the determination of Huperzine A in rat plasma was developed and validated. Separation was achieved on Kromasil C(8) column (5 microm, 150 mm x 4.6mm i.d.). The mobile phase, methanol-water-triethanol amine (45:55:0.05, v/v/v), was delivered at a flow rate of 1.0 ml/min. The eluent was monitored by a fluorescence detector with excitation wavelength at 310 nm and emission wavelength at 370 nm. No interfering peaks were observed in rat blank plasma. The relationship between Huperzine A concentration and peak-area ratio of Huperzine A to the IS was linear over the range of 2.5-250 ng/ml. The intra- and inter-day coefficients of variation were 相似文献   

Quantification of trimetazidine in human plasma by liquid chromatography-electrospray ionization mass spectrometry and its application to a bioequivalence study

A rapid, sensitive and specific liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method has been developed for the quantification of trimetazidine in human plasma. The analyte and the internal standard (pseudoephedrine) were extracted from plasma samples with n-hexane-dichloromethane (1:1, v/v) and analyzed on a C18 column. The chromatographic separation was achieved within 3.5 min using the mobile phase consisting of methanol/0.05% formic acid (80:20, v/v) and the flow rate was 1.0 ml/min. Ion signals m/z 181.0 and 148.0 were measured in the positive mode for trimetazidine and pseudoephedrine, respectively. The calibration curves were linear within the range of 0.4 to approximately 120 ng/ml. The lower limit of quantification (LLOQ) was 0.4 ng/ml with 0.5 ml plasma sample. The intra- and inter-day precisions were lower than 12% in terms of relative standard deviation (RSD). The inter-day relative error (RE) as determined from quality control samples (QCs), ranged from -1.4% to 3.3%. This validated method was successfully applied to the bioequivalent evaluation of two brands of trimetazidine tablets in 20 healthy volunteers.     

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma: method and clinical applications

A sensitive LC-ESI-MS method for the determination of indapamide in human plasma using glibenclamide as the internal standard (IS) was established. Following acidification with 1 M hydrochloric acid solution, plasma samples were extracted with ethyl acetate and separated on a C(18) column with a mobile phase of 10 mM ammonium acetate-methanol (22:78, v/v). Indapamide was determined using electrospray ionization in a single quadrupole mass spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 364.3 for indapamide and m/z 492.4 for the IS. Calibration curves were linear over the ranges of 0.1-100 ng/ml for indapamide. The lower limit of quantification was 0.1 ng/ml. The intra- and inter-assay precisions were less than 9.5% and 10.6%, respectively. The mean plasma extraction recovery of indapamide was 90.5-93.9%. The method has been successfully applied to study the pharmacokinetics of indapamide in healthy male Chinese volunteers.     

High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: application to a pharmacokinetic study in humans

A high-performance liquid chromatographic (HPLC) method was developed using diethylamine (DEA) solid-phase extraction (SPE), 9-fluorenylmethyl derivative (FMOC) and fluorescence detection for quantifying alendronate in human plasma. Sample preparation involved a manual protein precipitation with trichloroacetic acid, a manual coprecipitation of the bisphosphonate with calcium phosphate and derivatization with 9-fluorenylmethyl chloroformate in citrate buffer at pH 11.9. Liquid chromatography was performed on a Capcell Pak C(18) column (4.6 mm x 150 mm, 5 microm particles), using a gradient method starting with mobile phase acetonitrile/methanol-citrate/pyrophosphate buffer (32:68, v/v). The total run time was 25 min. The fluorometric detector was operated at 260 nm (excitation) and 310 nm (emission). Pamidronate was used as the internal standard. The limit of quantification was 1 ng/ml using 3 ml of plasma. The intra- and inter-day precision expressed as the relative standard deviation was less than 15%. The assay was applied to the analysis of samples from a pharmacokinetic study. Following the oral administration of 70 mg of alendronate sodium to volunteers, the maximum plasma concentration (C(max)) and elimination half-life were 40.94 +/- 19.60 ng/ml and 1.67 +/- 0.50 h, respectively. The method was demonstrated to be highly feasible and reproducible for pharmacokinetic studies including bioequivalence test of alendronate sodium in humans.     

Liquid chromatographic assay of moxidectin in human plasma for application to pharmacokinetic studies

A sensitive and selective high-performance liquid chromatography (HPLC) method is presented for the analysis of moxidectin in human plasma. Solid phase extraction using Oasis HLB cartridges is used for sample preparation. The fluorescent derivative is obtained by a dehydrative reaction with trifluoroacetic anhydride and N-methylimidazole. Separation is achieved on a Bondapak C(18) reversed-phase column with a mobile phase composed of tetrahydrafuran-acetonitrile-water (40:40:20, v/v/v). Detection is by fluorescence, with excitation at 365 nm and emission at 475 nm. The retention times of moxidectin and internal standard, ivermectin are approximately 10.7 and 18.6 min, respectively. The assay is linear over the concentration range 0.2-1000 ng/ml for moxidectin in human plasma (r=0.9999, weighted by 1/concentration). Recoveries at concentrations 0.2, 400, 1000 ng/ml are 94, 75, and 71%, respectively. The analysis of quality control (QC) samples for moxidectin (0.2, 400, and 1000 ng/ml) demonstrates excellent precision with relative standard deviations of 11.9, 5.7, and 2.7%, respectively (n=6). The method is accurate with all intra- (n=6) and inter-day (n=18) mean concentrations within (5.0%) from nominal at all QC sample concentrations. Moxidectin was found to be stable after three free-thaw cycles, and with storage at -20 and -80 degrees C for 12 weeks. The method is suitable for pharmacokinetic studies of moxidectin after oral administration to humans.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

A simple and sensitive HPLC-UV method for the determination of swainsonine in rat plasma and its application in a pharmacokinetic study

A simple, rapid, selective and sensitive HPLC-UV method was developed and validated for the determination of swainsonine (SWSN) in rat plasma. The analyte was extracted from rat plasma with methanol as the extraction solvent. The LC separation was performed on a Diamonsil? C18 (250×4.6 mm, 5 μm) analytical column with a mobile phase consisting of acetonitrile-potassium dihydrogen phosphate (25 mmol/l, pH=7.5) at a flow rate of 1.0 ml/min. There was a good linearity over the range of 10-500 ng/ml (r=0.9995) with a weighted (1/C2) least square method. The lower limit of quantification was proved to be 10 ng/ml. The accuracy was within 4.8% in terms of relative error and the intra- and inter-day precisions were less than 9.0% in terms of relative standard deviation. After validation, the method was successfully applied to characterize the pharmacokinetics of SWSN in rats.     

Validated high performance liquid chromatographic method for simultaneous determination of rosiglitazone, cilostazol, and 3,4-dehydro-cilostazol in rat plasma and its application to pharmacokinetics

A high performance liquid chromatographic (HPLC) method for simultaneous determination of rosiglitazone, CAS 122320-73-4, RSG), cilostazol (CAS 73963-72-1, CLZ) and its active metabolite 3, 4-dehydro-cilostazol (DCLZ), using pioglitazone (PIO) as internal standard (IS), in rat plasma is described. The plasma was extracted with methyl t-butyl ether, the dry extract was reconstituted in mobile phase and the aliquot was injected. The eluent drugs were detected by UV at dual wavelength of 226 nm (RSG and DCLZ) and 257 nm (CLZ). The mobile phase consisting of acetonitrile:potassium di-hydrogen phosphate buffer (35:65 v/v) was used at the flow rate of 1.2 ml/min on a reverse phase C18 column. The absolute recovery was above 90% of all analytes over the concentration range of 25-2500 ng/ml for RSG and CLZ and 20-2000 ng/ ml for DCLZ. The relative standard deviation (RSD) of the inter-day and intra-day precision ranged from 2.8 to 8.4% and 0.9 to 5.9%, respectively. The method is simple, rapid, accurate and sensitive and was applied to pharmacokinetic studies.     

Nateglinide quantification in rabbit plasma by HPLC: optimization and application to pharmacokinetic study

A rapid, simple, and sensitive HPLC method with UV detection was developed and validated for the determination of nateglinide (NTG) from rabbit plasma. The retention behavior of NTG and gliclazide (GLZ, internal standard-IS) as a function of mobile phase pH, composition and flow rate was investigated. Separation was developed on a reverse-phase C(18) column (250 mm x 4.6mm i.d., 5 microm particle size), using a mixture of acetonitrile (ACN):10mM phosphate buffer (PBS, pH 3.0) in the ratio of 70:30(%v/v) at a flow rate of 1.0 ml/min with UV detection at 203 nm within 8 min, and quantified based on drug/IS peak area ratios. The plasma samples were prepared by a simple deproteinization with a mixture of methanol and acetonitrile, yielding more than 97.86% extraction efficiencies. The calibration curve was linear (correlation coefficient of 0.9984) in the concentration range of 10-2500 ng/ml. The limit of detection (LoD) and limit of quantitation (LoQ) were found to be 2.91 and 9.70 ng/ml, respectively. Both the intra-day and inter-day precisions at four tested concentrations were below 1.32% R.S.D. The present method was selective enough to analyze NTG in rabbit plasma without any tedious sample clean-up procedure and was successfully applied for estimating the pharmacokinetic parameters of NTG following oral administration of a single 15 mg NTG to white albino rabbits.     

HPLC法测定人体血浆中盐酸二甲双胍的浓度

目的建立测定人体血浆中盐酸二甲双胍的HPLC法。方法取血浆样品0.4ml,乙腈沉淀蛋白0.6ml。流动相为甲醇：乙腈：0.01mol/L磷酸盐缓冲液（pH7.0,65：20：15）,流速1.0ml/min,检测波长233nm。结果在0.25～5.0μg/ml范围内,盐酸二甲双胍的峰高与浓度呈良好的线性关系（r=0.9993,n=7）,最低检测限为50ng/ml,低、中、高3种浓度的日内和日间RSD分别为4.93%、2.73%、1.50%（n=5）和5.57%、3.02%、1.72%（n=5）,相对回收率分别为95.53%,101.0%,100.6%。结论该法操作简便,灵敏度高,适用于盐酸二甲双胍的药动学研究及治疗药物浓度监测。     

Validated liquid chromatography-tandem mass spectrometric method for the quantitative determination of daidzein and its main metabolite daidzein glucuronide in rat plasma

A highly selective and sensitive liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed and validated to determine daidzein and its main metabolite daidzein glucuronide in rat plasma. The analytes and internal standard genistein were extracted from plasma samples by n-hexane-diethyl ether (1:4, v/v), and separated on a C18 column. The mobile phase consisted of acetonitrile-water-formic acid (80 : 20: 1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24-1000 ng/ml. The intra- and inter-day precisions were lower than 13.2% in terms of % RSD. The accuracy ranged from -0.5% to 2.4% in terms of % RE (relative error). This method was successfully applied to the determination of plasma concentration of daidzein and its main metabolite daidzein glucuronide in rats after an oral administration of 20 mg/kg daidzein.     

Quantitative determination of nitrendipine in human plasma using high-performance liquid chromatography-mass spectrometry

A sensitive and highly selective liquid chromatography-mass spectrometry (LCMS) method was developed to determine nitrendipine (4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3, 5-pyridinedicarboxylic acid ethyl methyl ester, CAS 39562-70-4) in human plasma. The analyte and the internal standard nimodipine (CAS 66085-59-4) were extracted from plasma samples by n-hexane-isopropanol (95:5, v/v), and analyzed on a commercially available column Interfaced with a mass spectrometer. Positive atmospheric pressure chemical ionization (APCI) was empolyed as the ionization source. The samples were detected by the use of selected ion monitoring (SIM) mode. The mobile phase consisted of methanol-water (75:25, v/v). The method has a limit of detection (LOD) of 0.1 ng/ml. The linear calibration curves were obtained in the concentra tions range of 0.3-40 ng/ml (r2 > or = .99). The intra- and inter-day batch precisions were lower than 10% in terms of relative standard deviation (R. S. D.), and the accuracy ranged from 85 to 110% in terms of percent accuracy. The overall extraction recoveries were determined to be about 75% on average. This validated method was successfully applied to the evaluation of the pharmacokinetic profiles of nitrendipine tablets administered to 8 Chinese healthy volunteers.     

固相萃取结合高效液相色谱测定人血浆中万古霉素的浓度

目的建立固相萃取结合高效液相色谱法测定人血浆中万古霉素的浓度。方法以去甲万古霉素为内标,用Cleanert PEP固相萃取柱预处理样品,水洗涤杂质,60%甲醇溶液洗脱药物;采用SHIMADZUVP-ODS C18（150mm×4.6 mm,5μm）色谱柱,流动相为乙腈-0.05 mol.L-1磷酸二氢钾缓冲液（8.5∶91.5,pH=3.2）,流速1mL.min-1,柱温为35℃,二极管阵列检测器检测波长230 nm。结果人血浆中万古霉素在1.080.0 mg.L-1线性关系良好,平均萃取回收率〉78%,方法回收率为88%109%,日内RSD〈6.9%（n=5）,日间RSD〈7.5%（n=15）。结论本法简便、灵敏、准确,适用于人体内万古霉素的血药浓度监测。     

Liquid chromatographic determination of plasma ropivacaine for assessing pharmacokinetics of the viscous preparation

We developed assay method for determination of plasma ropivacaine by using reversed-phase high performance liquid chromatography (HPLC) equipped with ordinary octadecylsilyl silica-gel (ODS) column. Plasma samples spiked with internal standard (bupivacaine) were treated by ethylacetate to extract ropivacaine and internal standard. The ropivacaine and internal standard separated on ODS column were detected by an ultra violet (UV) detector set at 215 nm. The mobile phase solvent consisted of acetonitrile, methanol and 0.05 M phosphate buffer adjusted to pH 4.0 (10 : 30 : 60, v/v) was pumped at a flow rate of 0.8 ml/min. The calibration curve of ropivacaine was linear at the concentration of 25-1,000 ng/ml (r=0.9998). The recoveries of ropivacaine from plasma were greater than 87.9% with the coefficient of variations (CVs) less than 6.1%. The CVs for intra- and inter-day assay of ropivacaine were 2.0-12.0% and 1.7-14.8%, respectively. This HPLC method was applied to determining plasma ropivacaine in two healthy subjects after receiving 0.5% ropivacaine viscous preparation, which was prepared in our hospital. Our preliminary pharmacokinetic data showed that ropivacaine viscous could be used safely based on the plasma ropivacaine concentrations (C(max): 89-125 ng/ml) for pain relief in oral mucosa.     

Validated HPLC method for determination of amlodipine in human plasma and its application to pharmacokinetic studies

A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for quantification of amlodipine in plasma. The assay enables the measurement of amlodipine for therapeutic drug monitoring with a minimum detectable limit of 0.2 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was about 97%. The separation was performed on an analytical 125 x 4.6 mm i.d. Nucleosil C8 column. The wavelength was set at 239 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (63:37, v/v) adjusted to pH 3.5 at a flow rate of 1.5 ml min(-1). The calibration curve was linear over the concentration range 0.5-16 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 10%.     

Quantitative determination of atorvastatin and ortho-hydroxy atorvastatin in human plasma by liquid chromatography tandem mass spectrometry and pharmacokinetic evaluation

A specific, sensitive and simple method was developed to determine the levels of both atorvastatin and ortho-hydroxy atorvastatin in human plasma. The analytes and internal standard pitavastatin were extracted from plasma by liquid-liquid extraction, separated on a Zorbax SB-C18 column, eluted with a mobile phase of water:acetonitrile (45:55 v/v), both containing 5% methanol and 0.01% formic acid. Detection was performed with an electrospray ionization triple quadrupole mass spectrometer in positive ion mode using multiple reaction monitoring. The standard calibration curves of atorvastatin and ortho-hydroxy atorvastatin were linear in the concentration range of 0.2-20 and 0.1-20 ng/mL, respectively. The intra- and inter-day precisions were < 7.7% and the accuracy was within ± 5.9%. The method has been successfully used for the study of the pharmacokinetics of atorvastatin and ortho-hydroxy atorvastatin in Chinese patients with coronary heart disease after a single oral dose of 20 mg atorvastatin. The mean values for the area under the plasma concentration-time curve for atorvastatin and ortho-hydroxy atorvastatin were 63.1 and 46.9 ng.h/mL, respectively.