High performance liquid chromatography of a new 1,4-dihydropyridine: applications to pharmacokinetic study in dogs

A new nitrendipine derivative [ethyl-2-(1-piperidino)ethyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)- 3,5-pyridine dicarboxylate hydrochloride;1] was assayed in plasma by high-performance liquid chromatography. After deproteinization and extraction, 1 and nitrendipine, as the external standard, were separated by ion-pair chromatography and measured by UV detection (254 nm). The method is rapid and specific: the limit of sensitivity of the assay was 5 ng/mL and the concentration range was linear between 5 and 2000 ng/mL. In vitro studies showed that, in contrast to nifedipine and nicardipine, 1 and nitrendipine were stable when exposed to light for at least 4 h. Pharmacokinetic parameters obtained in three beagle dogs after oral and intravenous administration are reported. Comparison with a nicardipine pharmacokinetic study showed similar results for the distribution and elimination characteristics of these two drugs.     

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.     

Development and validation of an HPLC method for vancomycin and its application to a pharmacokinetic study

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of vancomycin in artificial perfusion fluid and lung tissue samples has been developed and validated. Chromatographic separation was carried out in a Nucleosil 120 C(18) 5 microm column (length, 15 cm; inner diameter, 0.4 cm) using a mixture of 0.05 M NH(4)H(2)PO(4) (pH 4)-acetonitrile (92:8, v/v) as the mobile phase at a flow rate of 1 mL/min, with UV detection at 220 nm. The method used for the vancomycin quantification showed linearity for concentration ranges of 0.1-2, 2-15 and 15-250 microg/mL, with r(2)=0.9985, 0.9996 and 0.9985, respectively. The limit of quantification of the method was 0.1 microg/mL and the coefficients of variation of the between- and within-day precision showed values between 0.6% and 7.0%. The retention time of vancomycin was 8.5 min. The method was used successfully to study the pharmacokinetics of vancomycin in isolated rat lung after its administration through the systemic and inhalatory routes.     

HPLC enantiomer separation of a chiral 1,4-dihydropyridine monocarboxylic acid

An enantioselective anion exchanger based on tert-butylcarbamoylquinine as chiral selector and thiol-modified silica as chromatographic support was applied for the enantiomer separation of the shortacting calcium antagonist clevidipine after its hydrolysis to methyl 4-(2',3'-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate. This hydrolyzed derivative of clevidipine is the primary metabolite and precursor of the parent drug. Method development included the steps of optimization of the composition of the mobile phase (pH, type and content of organic modifier, polar organic mode), screening of various structural analogs of above mentioned CSP, and evaluation of the effect of the flow rate. These detailed studies gave insight into the operational mode of the CSP, which is basically an enantioselective anion exchange mechanism. The polar organic mode turned out to be advantageous with regard to enantioselectivity and resolution. The optimized method makes use of an eluent composed of 0.125% acetic acid in acetonitrile with flow rate of 1 ml/min at a constant temperature of 25 degrees C, and allows the separation of the both enantiomers with enantioselectivity alpha of 1.25 and a resolution RS of 3.0 within 10 min. On the above mentioned quinine carbamate phase the (R)-enantiomer is stronger retained, while a change to the corresponding quinidine carbamate CSP allows the reversal of the elution order.     

液相色谱法测定羟基喜树碱含量及药动学研究

目的:建立一个简单、快速、灵敏的柱切换高效液相色谱法测定人血清中10-羟基喜树碱(HCPT)含量。方法:生物样品首先在装有限进介质(RAM)填料的预柱中净化和富集,然后转移到C18分析柱分析待测物,用荧光法检测。结果:整个分析时间8 min。在1~1000 ng/mL浓度范围内,HCPT显良好的线性关系,日内、日间变异小于5%,最低检测限为0.1 ng/mL。结论:本法已应用于临床病人注射HCPT后的药代动力学研究,结果符合一房室模型并计算出它的药动学参数。     

HPLC method for the determination of rosiglitazone in human plasma and its application in a clinical pharmacokinetic study

Rosiglitazone (CAS 155141-29-0, Avandia) is a novel insulin sensitizer used in the treatment of type 2 diabetes. A sensitive high performance liquid chromatography (HPLC) method for its determination in human plasma using fluorescence detection (excitation: 247 nm, emission: 367 nm) with a suitable internal standard (I. S.) is described. Ethyl acetate was used as extraction solvent. A mobile phase consisting of phosphate buffer, acetonitrile and methanol was used at a flow rate of 1.0 ml/min on a C18 column. The absolute recovery was > 90% and the lower limit of quantitation was 5 ng/ml. The intra- and inter-day relative standard deviations ranged from 0.58-6.69% and 0.82-6.63%, respectively. The method described is simple, economical, precise and accurate and has been successfully applied in a pharmacokinetic study conducted in healthy human volunteers.     

Validated HPLC method for determination of chlorzoxazone in human serum and its application in a clinical pharmacokinetic study

A high performance liquid chromatographic (HPLC) method for the determination of chloroxazone in human serum using phenacetin as internal standard (IS) is described. Protein precipitation is used for preparation of the sample. A mobile phase consisting of acetonitrile and 0.5% acetic acid in water mixture (40:60 v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/VIS detector set at 287 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 96% over a concentration range of 1 to 100 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 1, 10, 50, and 100 micrograms/ml ranged from 0.9 to 5.1%. The inter-day RSD ranged from 0.6 to 3.0%. The method is simple, sensitive and has been successfully used in pharmacokinetic study conducted in healthy human volunteers.     

HPLC determination of telmisartan in human plasma and its application to a pharmacokinetic study

A sensitive, simple, and accurate HPLC method was developed for the assay of telmisartan in human plasma. Using naproxen as internal standard, the assay involved liquid-liquid extraction of the compound from acidified plasma into organic solvent and reversed-phase chromatography with fluorescence detection. The assay was shown to be linear from 0.5 to 1000 ng/mL. In 24 healthy volunteers, the plasma concentrations of the drug were determined after a single oral dose of 160 mg.     

高效液相色谱-紫外法检测家兔血浆美托洛尔的浓度及药动学

目的:建立高效液相色谱-紫外(HPLC-UV)方法测定家兔血浆中美托洛尔浓度并进行酒石酸美托洛尔片剂家兔体内药动学研究。方法:以Phenomenex C18柱(250 mm×4.6 mm,5μm)为分析柱,甲醇-水相(60∶40)为流动相(其中水相由385mL水、1.6 mL三乙胺和0.5 mL磷酸组成),紫外检测波长为223 nm,以苯妥英钠为内标,建立HPLC-UV法检测家兔血浆内美托洛尔的浓度。采用DAS3.0.1药动学软件计算药动学参数。结果:美托洛尔的线性范围为0.078～7.81μg·mL-1,最低定量限为0.078μg·mL-1,日内和日间精密度均小于10%,低、中、高3个质控浓度的提取回收率为87.5%～92.3%,准确度的范围在95.4%～108.6%。家兔口服酒石酸美托洛尔片后的主要药动学参数AUC0-t、tmax和Cmax分别为(338.8±30.9)mg.L-1.min、(45.0±0.0)min、(2.2±0.3)μg·mL-1。结论:该方法准确、快速、方便,为美托洛尔的药动学研究提供了简单易行的分析方法。     

A new and simple HPLC method for determination of etamsylate in human plasma and its application to pharmacokinetic study in healthy adult male volunteers

A new and simple HPLC assay method was developed and validated for the determination of etamsylate in human plasma. After protein precipitation with 6% perchloric acid, satisfactory separation was achieved on a HyPURITY C₁₈ column (250 mm × 4.6 mm, 5 μm) using a mobile phase comprising 20 mM sodium dihydrogen phosphate-2 hydrate (pH was adjusted to 3.5 by phosphoric acid) and acetonitrile at a ratio of 95:5 v/v. The elution was isocratic at ambient temperature with a flow rate of 0.75 ml/min. Allopurinol was used as internal standard. The calibration curve was linear over the range from 0.25 to 20 μg/ml (r² = 0.999). The limit of quantification for etamsylate in plasma was 0.25 μg/ml. The within day coefficient of variance (%CV) ranged from 3.9% to 10.2%, whereas the between-day %CV ranged from 3.1% to 8.7%. The assay method has been successfully used to estimate the pharmacokinetics of etamsylate after oral administration of a 500 mg tablet under fasting conditions to 24 healthy Egyptian human male volunteers. Various pharmacokinetic parameters including AUC_0–t, AUC_0–∞, C_max, T_max, t_1/2, MRT, Cl/F, and V_d/F were determined from plasma concentration–time profile of etamsylate.     

LC-MS/MS法测定血浆中山奈酚-3-O-芸香糖苷浓度及其在大鼠药动学研究中的应用

目的:建立能够灵敏、特异、准确、可靠地测定血浆中山奈酚-3-O-芸香糖苷浓度的分析方法。方法:优化山奈酚-3-O-芸香糖苷(kaempferol-3-O-ruti-noside,NFR)的离子化及其断裂方式,确定相关液相色谱条件,选择能有效地从血浆样品中提取NFR的样品前处理方法;开展方法学考察,以验证新建分析方法的准确性、精密度等,在此基础上将该方法用于分析大鼠静脉注射NFR后所得的实际血浆样品,以验证新建分析方法的适用性。结果:ESI(+)为NFR提供最佳的离子化条件,采用SRM工作模式,用m/z595→287来检测NFR。同时,以左旋千金藤啶碱(l-Stepholidine)为内标物化合物(IS),其SRM检测采用m/z328→178。NFR及IS的色谱保留时间(tR)分别为2.3和2.2min。用乙酸乙酯(EtOAc)提取经HCl酸化的大鼠血浆样品,NFR和IS的回收率分别为58.5%~70.1%和72.5%。NFR和IS在整个分析过程中稳定。在0.192~600ng·ml-1的浓度范围内,对NFR与IS的峰面积比值和NFR的血药浓度进行线性回归,其回归曲线线性良好(r=0.9999,n=6×5)。批内准确性为92%~107%,其精密度为1.0%~5.7%;批间准确性为94%~99%,其精密度为1.5%~8.4%。本方法的LLOQ为0.192ng·ml-1。大鼠静脉给药单剂量NFR(30mg·kg-1)后,NFR的消除半衰期(T1/2)为1.27h、体内平均滞留时间(MRT)为0.32h,NFR在大鼠体内的清除率(CL)为2.73L·h·kg-1、稳态分布体积(VSS)为0.92L·kg-1。结论:应用LC-MS/MS技术建立的测定血浆中山奈酚-3-O-芸香糖苷浓度的新方法灵敏可靠,这项研究工作为全面开展NFR注射液的临床前药代动力学研究打下了重要的分析方法学基础。     

Validated HPLC method for the determination of celecoxib in human serum and its application in a clinical pharmacokinetic study

A simple high performance liquid chromatographic method using UV detection for the determination of celecoxib, a specific COX 2 inhibitor, in serum was developed. Serum samples containing the internal standard, tolbutamide, are eluted through a C18, Wakosil column. After extracting with dichloromethane, the eluent is monitored at 250 nm. The mobile phase comprised of 10 mM potassium dihydrogen ortho phosphate (pH 3.2) and acetonitrile (50:50 v/v) with a flow rate of 1 ml/min. Retention times of celecoxib and tolbutamide were 9.6 and 3.5 min, respectively. The mean absolute recovery value was about 70-80%, while the intra day and inter day coefficient of variation and percent error values of the assay method were less than 10%. The calibration curve was linear over a concentration range of 10-1000 ng/ml.     

Validated HPLC method for the determination of tinidazole in human serum and its application in a clinical pharmacokinetic study

A high performance liquid chromatographic (HPLC) method for the determination of tinidazole in human serum using metronidazole as internal standard (IS) is described. Protein precipitation is used for the preparation of sample. Mobile phase consisting of 0.002 M phosphate buffer, methanol and acetonitrile mixture (85:7.5:7.5/v/v/v) was used at a flow rate of 1 ml/min on a C18 column. The eluate was monitored using an UV/Vis detector set at 320 nm. Ratio of peak area of analyte to IS was used for quantification of serum samples. The absolute recovery was greater than 95% over a concentration range of 0.5 to 30 micrograms/ml and the limit of quantitation was 0.05 microgram/ml. The intra-day relative standard deviation (RSD) measured at 0.5, 5, 15 and 30 micrograms/ml ranged from 0.36 to 6.14%. The inter-day RSD ranged from 1.14 to 4.21%. The method is simple, sensitive and has been successfully used in a pharmacokinetic study conducted in healthy human volunteers.     

New high-performance liquid chromatographic method for serum analysis of oxazepam: application to bioequivalence and pharmacokinetic study

A rapid and sensitive high-performance liquid chromatographic method was developed and validated for determination of oxazepam in serum. Oxazepam was isolated from biological fluid using a simple liquid-liquid extraction with dichloromethane. Nordazepam was used as the internal standard. The chromatographic separation was accomplished using a 125 x 4-mm (inner diameter) stainless-steel (5 microm) Perfectsil Target ODS-3 reversed phase column with a mobile phase consisting of ammonium dihydrogen phosphate buffer (0.05 mol x L(-1), pH 5.8) and methanol (50:50, v/v), running at a flow rate of 1.5 ml x min(-1). The absorbance of the fluent was monitored at 254 nm. The developed method resulted in totally symmetrical peaks. It has been applied to assess the pharmacokinetics of oxazepam. Also the bioequivalence of two different oxazepam preparations following oral administration in healthy volunteers was assessed by this method.     

用HPLC法测定头孢丙烯的血浆浓度并应用于健康人体药动学研究

目的：建立测定人血浆中头孢丙烯浓度的HPLC法，并应用于健康人体药动学研究。方法：10名男性健康志愿者单剂量口服500mg头孢丙烯片，以HPLC法测定血浆中头孢丙烯浓度，采用非房室模型法计算药动学参数。结果：主要的药动学参数如下：cmax（8．64±1．00）μg／mL，tmax（2．00±0．85）h，t1／2（1．47±0．27）h，MRT（2.88±0．49）h，C1／F（17．964±2．50）L／h，AUC0～10（28．05±4．60）μg·h·mL^-1和AUC0-∞（2842±4．75）μg·h·mL^-1结论：本方法的准确度、灵敏度、专属性及重现性等符合生物样品的分析要求，适用于头孢丙烯片的人体药动学研究。     

大鼠血浆中岩白菜素含量分析方法的建立及其体内药动学

目的建立血浆中岩白菜素的HPLC的测定方法,研究岩白菜素及岩白菜素磷脂复合物在大鼠体内的药动学行为。方法采用Odyssil C18色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇-乙腈-质量分数为0.2%的磷酸水溶液(体积比为4∶16∶80),流速为0.8 m L·min-1,检测波长为220 nm。应用液-液萃取法处理血浆,测定两只大鼠分别口服岩白菜及其磷脂复合物后不同时刻血浆中岩白菜素的质量浓度。结果岩白菜素质量浓度在0.01~5 mg·L-1内线性关系良好,定量下限为0.01 mg·L-1;提取回收率均在78.6%~90%之间;日内、日间精密度均小于13.7%。结论该方法准确度高、灵敏度强、专属性好,可作为测定大鼠血浆岩白菜素质量浓度的方法,为探讨岩白菜素的药动学行为提供方法依据。     

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%.     

HPLC method for the determination of fluvoxamine in human plasma and urine for application to pharmacokinetic studies

A simple, specific and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the assay of fluvoxamine in human plasma and urine. The method was based on reaction of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) forming orange colored product. The fluvoxamine-NQ derivative was separated by isocratic reversed-phase HPLC and detected at 450 nm. The chromatographic conditions were as follows: Phenomenex C(18) (250 mm x 4.6 mm i.d., 5 microm) column, mobile phase consisting of acetonitrile/water (80:20 v/v) at a flow rate of 1 ml/min. Tryptamine was selected as an internal standard. The assay was linear over the concentration range of 5-145 and 2-100 ng/ml for plasma and urine, respectively. The limits of detection (LOD) were 1.4 and 1 ng/ml for plasma and urine estimation at a signal-to-noise (S/N) ratio of 3. The limits of quantification (LOQ) were 5 and 2 ng/ml for plasma and urine, respectively. The extraction recoveries were found to be 96.66+/-0.69 and 96.73+/-2.17% for plasma and urine, respectively. The intra-day and inter-day standard deviations (S.D.) were less than 1. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay was demonstrated to be applicable for clinical pharmacokinetic studies.