Development and validation of a MEKC method for the direct determination of cefozopran in human serum

A method for determining the concentration of cefozopran, a cephem anti-microbial agent which has a broad spectrum, in human serum using micellar electrokinetic capillary chromatography (MEKC) by serum direct injection is developed and the validation of the assays of this method is performed. A borate buffer (25mM; pH 10.0) containing sodium dodecyl sulfate (SDS) (50mM) is used as a run buffer. The electrophoresis of serum samples is carried out at 25kV and the detection of cefozopran at 244nm as its absorption maximum at the cathodic side of the capillary. The migration time of cefozopran is 6.5min. Linearity (0-200mg/l) is good and limit of quantification is 0.5mg/l at a signal-to-noise ratio of 3. Coefficient of variation (CV) of intra-day precision and that of inter-day precision are 2.4-4.0% (7.3-92.0mg/l) and 2.9-7.7% (22.5-71.4mg/l), respectively, and the recovery rate is 92-109%. The detection results of 12 other cephem anti-microbial agents under the analytical conditions of this method show that the migration time of cefmetazole is identical with that of cefozopran, making it impossible to separate these two anti-microbial agents. This method is characterized by the fact that simple and economic determination can be achieved by directly injecting the serum samples of micro-quantities into the capillary.     

Method development for determining the antibacterial linezolid in human serum by micellar electrokinetic capillary chromatography

A precise method for determining linezolid concentration in human serum by micellar electrokinetic capillary chromatography has been developed and validated. Serum was deproteinized with acetonitrile and etofylline was used as an internal standard. A borate buffer (pH 10.0; 25 mM) containing sodium dodecyl sulfate (80 mM) was used as a running buffer. Detection was performed at UV253 nm by applying 25 kV voltage to a fused-silica capillary tube. Migration time of linezolid was approximately 14 min. Good linearity (0-100 mg/l) was obtained and the limit of detection was 0.5 mg/l (S/N=3). This technique covered the clinical concentration (4 mg/l) measurement of this drug enough. The intra- and inter-day reproducibility was good. Serum recovery was 95-102%. No interference from other anti-microbial agents was observed. Linezolid after serum deproteinization showed high stability. This method was easy to operate as well as economical as a method for determining linezolid in serum.     

Development, evaluation and application of an isocratic high-performance liquid chromatography (HPLC) assay for the simultaneous determination of aciclovir and its metabolite 9-carboxymethoxymethylguanine in human serum and cerebrospinal fluid

9-Carboxymethoxymethylguanine (CMMG), the main metabolite of aciclovir (ACV), is a putative neurotoxin. Measurement of CMMG in body fluids may aid patient management. We describe the development, validation and application of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of ACV and CMMG in human serum and cerebrospinal fluid (CSF). Recovery was between 94% and 100% at all concentrations both from serum (range 0-20 mg/L) and CSF (0-5 mg/L). The intra-assay precision (coefficient of variation (CV)) was <2% and the inter-assay precision (CV) was <5%. The limits of detection and quantification were 0.1 and 0.25 mg/L, respectively, in both body fluids. Significant interference from endogenous material or from drugs in clinical samples was not seen. CMMG was detected in most of the 55 clinical samples containing ACV, but little correlation was found between the levels of the drug and its metabolite.     

Chiral CE separation of warfarin in albumin containing samples

A capillary zone electrophoresis method with ultraviolet (UV) absorbance detection for the chiral separation of warfarin enantiomers using highly sulfated beta-cyclodextrin (beta-CD) was developed and optimized. Enantiomeric separation of warfarin was characterized by high resolution and efficiency. The optimized electrophoretic conditions were subsequently applied to the analysis of warfarin extracted from spiked human serum albumin samples. This assay showed acceptable precision, with linearity in the warfarin enantiomer concentration range of 0.1-25 mg/l. The limits of detection (LOD) and quantitation (LOQ) evaluated as warfarin enantiomer concentrations in the serum samples were 0.05 and 0.15 mg/l, respectively, for each warfarin enantiomer.     

Further method development for measurement of linezolid in human serum by MEKC

A method for determining linezolid concentration in human serum using micellar electrokinetic capillary chromatography by direct injection of serum is described. A borate buffer (pH 8.0) containing sodium dodecyl sulfate was used as a run buffer and detection of linezolid was performed at 250 nm (its absorption maximum). The migration time of linezolid was 5.5 min and the detection limit was 0.5 mg/l (S/N = 3). The precision and accuracy of this method was good with no interference with the detection from bilirubin, hemoglobin and chyle of high concentrations. This provides a simple and easy method where samples of micro-quantity are used.     

Simultaneous determination of mycophenolic acid and its acyl and phenol glucuronide metabolites in human serum by capillary zone electrophoresis

A simple capillary electrophoretic method was developed for simultaneous determination of mycophenolic acid (MPA) and its metabolites, acyl glucuronide (AcMPAG) and phenol glucuronide (MPAG), in human serum. The method utilized only running buffer in the separation, prior acidification of serum, and extraction with ethyl acetate. Separation was performed by capillary zone electrophoresis using 20 mM sodium acetate-acetic acid (pH 4.9) as running buffer, applied voltage of 15 kV, and UV detection at 217 nm. Each electrophoretic run was completed within 14 min. The optimized method demonstrated good performance concerning specificity, linearity (r>0.998), sensitivity (limit of detection: for MPA, 0.10 microg/ml; for AcMPAG, 0.10 microg/ml; MPAG, 0.42 microg/ml), accuracy (87-108%) and precision (<9.3%). This method was successfully applied to measurements of MPA, AcMPAG, and MPAG in renal transplant patient samples and could be a useful alternative to HPLC-based methods.     

Assay of terpene alcohols in pharmacopoeial essential oils by micellar electrokinetic capillary chromatography (MEKC)

Micellar electrokinetic capillary chromatography (MEKC) was used to separate and determine terpene alcohols of wide occurrence in herbal extracts and essential oils, namely eugenol, linalool, geraniol, citronellol and thymol. In the present paper sodium dodecyl sulfate (SDS) has been used as a micelle-forming additive to the CZE background electrolytes. Effects of SDS concentration, buffer type, its pH and concentration, addition of organic solvents on the migration times and separation efficiency were investigated. The optimal electrolyte system consisted of 20 mM TAPSO and 30 mM SDS in aqueous 10% (v/v) acetonitrile of pH 7.5 (adjusted by the addition of TRIS). The separation capillary was a fused silica tube (50 microm I. D., total length 75 cm, 42 cm effective length) maintained at 25 degrees C. The separations were performed at the applied voltage of 20 kV. Samples were injected hydrodynamically at a pressure of 50 mbar for 6 s. Detection was carried out at 200 nm. The calibration curves were rectilinear for 50-200 mg l(-1) (for eugenol, thymol and geraniol) and 100-400 mg l(-1) (for linalool and citronellol). The limits of detection varied between 5 mg l(-1) (for thymol) and 16 mg l(-1) (for linalool). The devised MEKC method was employed for the determination of the cited terpene alcohols as major quality-affecting constituents in commercial pharmacopoeial essential oils such as Geranii etheroleum, Caryophylli floris etheroleum, Lavandulae etheroleum and Thymi etheroleum. The results agreed well with those of a reference gas chromatographic method.     

Liquid chromatographic determination of piroxicam in serum

A sensitive method for monitoring serum piroxicam (Feldene) is described. The assay requires 1.0 ml of specimen and involves chloroform extraction from an acidified mixture followed by concentration and injection into a liquid chromatograph. Column effluent is monitored at 330 nm. Retention times of piroxicam and the internal standard (naproxen) are 6.6 and 11.0 min, respectively. The lower limit of detection in serum is 0.5 mg/L. Within-day precision (coefficient of variation, CV) of piroxicam in serum (5-13 mg/L range) varied from 3.6 to 6.3%; between-day CV at concentrations of 5 to 20 mg/L varied from 6.5 to 9.8%. Analytical recovery of piroxicam at 20 mg/L was 88%. Several other drugs were analyzed but none interfered with the assay. Serum concentrations found in 28 patients on a 20-30 mg/day dose ranged from 1.5 to 15.2 mg/L.     

Determination of ketoprofen enantiomers in human serum by capillary zone electrophoresis: man pharmacokinetic studies after administration of rac-ketoprofen tablets

A rapid and stereospecific capillary zone electrophoresis (CZE) method to quantify ketoprofen (KTP) enantiomers was developed. The KTP enantiomers and (+)-S-naproxen [(+)-S-NPX] as an internal standard (IS) were extracted with methylene chloride from serum acidified. Recovery of both enantiomers was in the range of 85-91%. The enantiomers were determined using a background electrolyte (BGE), consisting of 0.05 M heptakis 2,3,6-tri-O-methyl-beta-cyclodextrin (TMbetaCD) in a phosphate-triethanolamine buffer, which filled a fused silica capillary of 75 micrometer i.d. The linear range of calibration curves was between 0.25 and 50 mg l(-1), with detection limit of 0.1 mg l(-1) (signal-to-noise baseline ratio (S/N) >4). Intra- and interday precision and accuracy of the calibration curves, expressed by the coefficient of variation (CV), did not exceed 15.0%. The validated method has been successfully applied for pharmacokinetic studies of KTP enantiomers from tablets with rac-KTP in man.     

高效毛细管电泳分析血清中头孢曲松钠

建立了一种简单、灵敏的高效毛细管电泳分析犬血清中头孢曲松钠的方法。用乙腈为蛋白沉淀剂,硼酸盐为缓冲体系,以苯甲酸为内标物。电泳条件：检测波长２５４ｎｍ ,非涂渍石英毛细管３７ｃｍ ×７５μｍ（ｉ．ｄ．）,电压１０ｋＶ,自动压力进样５ｓ,电泳时间４ｍ ｉｎ。结果：头孢曲松钠测定的线性范围为１～１０－ ４ｍ ｇ／ｍｌ,最低检测限为５×１０－ ６ｍ ｇ／ｍ ｌ;本法的ＲＳＤ日内和ＲＳＤ日间分别为１．１％ 和１．２％ ;头孢曲松代谢产物及血清中的其他成分对分析无干扰。     

Method development and validation for the simultaneous determination of meloxicam and pridinol mesylate using RP-HPLC and its application in drug formulations

A simple and reliable reversed-phase high-perfomance liquid chromatographic method has been developed and validated for the simultaneous determination of meloxicam and pridinol mesylate in their synthetic mixtures and combined tablet formulations. Both drugs were separated on a 250 mm x 4.6mm C18 column packed with 5 microm particles. The mobile phase, optimized through an experimental design, was a 51:9:40 (v/v/v) mixture of methanol, isopropanol and 50mM potassium phosphate buffer (pH 5.9), pumped at a flow rate of 1.0 ml min(-1). UV detection was performed at 225 nm. The method was validated in the sample concentration ranges of 33.7-61.8 mg l(-1) for meloxicam and 8.8-16.8 mg l(-1) for pridinol mesylate, where it demonstrated good linearity with r=0.9989 and 0.9987 (n=15), respectively. The assay was shown to be repeatable at concentration levels of 70%, 100% and 130%, with relative standard deviation values of 1.09% and 0.82% for meloxicam and pridinol, respectively. For independent 100% level samples, the intra-day precision was 0.4% and 1.0% while the intermediate precision was 0.7% and 1.0% for the drugs. The method demonstrated to be robust, resisting to small deliberate changes in pH, flow rate and composition (organic:aqueous ratio) of the mobile phase. The LOD values were 0.22 and 0.20 mg l(-1), while the LOQ were 1.7 and 1.1 mg l(-1), for meloxicam and pridinol, respectively. The applicability of the method was demonstrated by determining the drug content of two commercial pharmaceutical formulations, where it exhibited good performance.     

Determination of ibuprofen and flurbiprofen in pharmaceuticals by capillary zone electrophoresis

Capillary zone electrophoresis with spectrophotometric detection was used for the determination of ibuprofen (IB) and flurbiprofen (FL) in pharmaceuticals. The separation was carried out in a fused silica capillary (60 cm x 100 microm i.d. effective length 45 cm) at 30 kV with UV detection at 232 nm. The optimized background electrolyte was 20mM N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) with 20mM imidazole and 10mM alpha-cyclodextrin of pH 7.3. 2-Naphthoxyacetic acid was used as internal standard. A single analysis took less than 5 min. Rectilinear calibration ranges were 2-500 mg l(-1) for IB and 1-60 mg l(-1) for FL. The relative standard deviations (R.S.D.) values (n=6) were 1.53% for IB and 1.29% for FL (for 200 mg l(-1) IB and 10 mg l(-1) FL). This validated method has been successfully applied for the routine analysis of 10 commercially available pharmaceutical preparations (syrup, tablets, cream and gel).     

分析单克隆抗体电荷异质性的成像毛细管等电聚焦电泳技术平台方法的建立

目的  采用成像毛细管等电聚焦电泳技术（imaged capillary isoelectric focusing, iCIEF）建立分析单克隆抗体（单抗）电荷异质性的平台方法,并用不同亚型单抗（IgG1、IgG2、IgG4）确认该平台方法的适用性。方法  优化该平台方法的部分参数,包括两性电解质和阴极稳定剂体积、聚焦时间和尿素浓度。采用3种亚型单抗（IgG1、IgG2、IgG4）对该平台方法的专属性、精密度、线性、准确度和耐用性进行验证。结果   对样品（单抗）的处理条件为：3 mol/L尿素-0.5%甲基纤维素溶液70 μl、两性电解质（pH3~10）4 μl、阴极稳定剂（500 mmol/L 精氨酸）2 μl、等电点 6.14和9.99 Marker 各2 μl,最终完成0.2 mg/ml单抗（样品）的制备。检测参数：预聚焦1 500 V、1 min,聚焦3 000 V、8 min。该平台方法的专属性良好,制剂缓冲液对检测无干扰。重复检测6份平行样品以及不同分析员于不同时间检测12份样品各成分含量的相对标准偏差均符合规定的要求。单抗（样品）终浓度为0.1～0.3 mg/ml时,主要和酸性成分的线性决定系数（R2）≥0.99,碱性成分的线性R2≥0.98。该平台方法检测样品各成分的准确度为92～105%。耐用性实验设计结果表明,两性电解质（pH3~10）体积和毛细管批次对该平台方法有显著影响。结论  建立的iCIEF平台方法分离度较高,精密度、准确度和耐用性良好,为单抗制品的电荷异质性表征和质量控制提供了更有效的工具。     

气相色谱-质谱联用法测定Beagle犬血清中托吡酯浓度及药动学研究

目的:建立测定犬血清中托吡酯浓度的方法,并用于药动学研究。方法:采用气相色谱-质谱联用法。以盐酸阿米替林为内标,色谱柱为DM-5弹性石英毛细管柱,150～280℃程序升温,选择m/z为202和324的离子碎片峰分别对盐酸阿米替林和托吡酯进行检测。将4只Beagle犬灌胃给予托吡酯(20mg·kg-1),于不同时间点(0.25、0.5、0.75、1、1.5、2、4、6、10、14、20、26、36h)采集血样,计算主要的药动学参数。结果:托吡酯检测浓度线性范围为0.18～35.93mg·L-1(r=0.9994),日内RSD≤8.53%,日间RSD≤14.19%,方法回收率为101.4%,萃取回收率为64.09%;t1/2α为(1.113±0.307)h,t1/2β为(10.209±8.89)h,cmax为(10.96±2.45)mg·L-1,AUC0～∞为(60.317±17.828)mg·h·L-1。结论:所建立的方法灵敏、准确,可用于血清托吡酯浓度的测定及药动学研究。     

Development and validation of a capillary electrophoresis method for the enantiomeric purity determination of RS86017 using experimental design

A selective capillary electrophoresis method for determination of enantiomeric purity of RS86017, a new antiarrhythmic agent with two chiral centers, was developed and validated using sulfobutyl ether-β-cyclodextrin as chiral selector. The concentration of the chiral selector and organic modifier, pH of background electrolyte (BGE), capillary temperature, and applied voltage were systematically optimized by using orthogonal design and concentration of chiral selector was further optimized. The optimal conditions included 25mM phosphate buffer at pH 8.0, containing 28mg/mL sulfobutyl ether-β-cyclodextrin and 20% acetonitrile as running buffer, an applied voltage of 22kV, and a temperature of 20°C. The detection wavelength was 206nm. The obtained method was capable of separating RS86017 from its potential chiral impurities, the S,R-enantiomer, the R,R-diastereomer and the S,S-diastereomer with a short analysis time of 10min. The separation was validated with respect to its selectivity, repeatability, linearity, precision, accuracy, limits of detection (LOD), limits of quantitation (LOQ) and robustness testing. The LODs and LOQs were 0.8μg/mL and 2.5μg/mL for all isomers of RS86017, respectively. Finally, the method was used to investigate the chiral purity of RS86017 in bulk samples.     

Separation and determination of clotrimazole, methylparaben and propylparaben in pharmaceutical preparation by micellar electrokinetic chromatography

In this study, micellar electrokinetic chromatography (MEKC) method was developed for the determination of clotrimazole (CLO), methylparaben (MP) and propylparaben (PP) in a pharmaceutical preparation. Separation was carried out in a fused silica capillary (60 cm x 75 microm i.d.) at 25 kV with UV detection at 212 nm. Optimized background electrolyte (BGE) was 15 mM phosphate buffer (pH 7.2) containing 30 mM sodium dodecyl sulfate (SDS) as a surfactant. Rectilinear calibration ranges were 50-500 mg l(-1) for CLO, 10-100 mg l(-1) for MP and 2.5-25 mg l(-1) for PP. The total analysis time was < 12 min.     

A new HPLC/UV method for the determination of clindamycin in dog blood serum

A new HPLC method for the quantitative determination of clindamycin in dog blood serum at levels down to 80 ng/ml has been developed. Samples were deproteinised with acetonitrile and clindamycin was extracted with dichloromethane. Chromatographic analysis was carried out on a C(18) reversed-phase analytical column in the presence of tetra-n-butylammonium hydrogen sulfate (TBA), as an ion-pairing agent. UV detector wavelength was set at 195 nm. The assay was validated for a concentration range from 80 to 6000 ng/ml serum. Good linearity was observed in the entire concentration range. The limit of quantification (LOQ) was 80 ng/ml and the limit of detection (LOD) was 60 ng/ml. Regression of accuracy data yielded an overall mean recovery value (+/-S.E.M.) of 93.98+/-0.42%, while precision data revealed coefficient of variation (CV (%)) values lower than 4.41%. The method was successfully applied to determine drug concentrations in serum samples from dogs that had been orally administered clindamycin hydrochloride.     

Determination of gomisin A (TJN-101) and its metabolite in rat serum by gas chromatography-mass spectrometry]

Gomisin A (TJN-101) is one of the lignan components isolated from Schisandra Fruits. A high sensitive and precise method for the determination of TJN-101 and its major metabolite (Met. B) in the rat serum was developed by selected ion monitoring (SIM) with gas chromatography-mass spectrometry (GC/MS) using a fused silica capillary column (SPB-1, Supelco). A 100 microliter serum sample was used for the solid phase extraction. The calibration curves of TJN-101 and Met.B both showed a good linearity between 2.0 and 2000.0 ng/ml. The analytical precision (intra-assay, C.V. less than 4.7%), recoveries (98.4 +/- 10.1%), and detection limit (2 ng/ml) of TJN-101 indicated that this system was suited for the determination of TJN-101 in biological fluid. In case of Met.B, the same results as TJN-101, were obtained. After oral administration of TJN-101 at a dose of 10 mg/kg to male rats, the average values of the maximal serum concentration of TJN-101 and Met.B were 1446.1 +/- 131.8 and 317.4 +/- 18.5 ng/ml, respectively. The serum concentrations of these substances could be monitored sufficiently for 8 h after dosing.     

Optimisation, validation and application of a capillary electrophoretic method for the determination of zafirlukast in pharmaceutical formulations

Zafirlukast is a selective and competitive orally administered inhibitor of the cysteinyl leukotrienes and currently indicated for the prophylaxis and treatment chronic asthma. A simple, rapid, reliable capillary zone electrophoresis method for the determination of ZAF in pharmaceutical formulations was developed and validated. The influence of buffer concentration, buffer pH, organic modifier, capillary temperature, applied voltage and injection time was systemically investigated in a fused silica capillary (i.d. 50 microm, total length 80.5 cm and effective length 72.0 cm). Optimum results were obtained with 50mM borate buffer at pH 8.50, capillary temperature 25 degrees C and applied voltage 30 kV. The samples were injected hydrodynamically for 3s at 50 mbar. Detection wavelength was set at 240 nm. Meloxicam was used as internal standard. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision, selectivity, robustness and ruggedness. The linear calibration range was 2.00-80.00 microg mL(-1) and the limits of detection and quantification were 0.75 and 2.00 microg mL(-1) with R.S.D. of 3.88 and 2.75%, respectively. The proposed method was applied for the determination of ZAF in its pharmaceutical formulations. The results obtained from developed method were compared with a HPLC method reported in the literature and no significant difference was found statistically.     

Indirect determination of neomycin trisulphate as sulphate by column coupling capillary isotachophoresis

Anionic capillary isotachophoresis (cITP) with conductometric detection was used for indirect determination of neomycin trisulphate (NMS) as sulphate anion. The following electroylets were tested: leading (LE)--8 mM HCl adjusted with beta-alanine to pH = 3.55, 3 mM bis-tris-propane and 0.2% hydroxyethylcellulose and terminating (TE): 5 mM citric acid; 5 mM formic acid and 5 mM caproic acid. The calibration graphs of sulphates concentration were linear in the range 3.55 to 25.90 mg/dm3 with recoveries between 94.50-99.70%. The method was tested on pharmaceutical preparation Enterogast.