Development and Validation of a Liquid Chromatographic Method for Determination of Efavirenz in Human Plasma

A simple, rapid, and sensitive high-performance liquid chromatographic method for estimation of efavirenz in human plasma has been developed and validated. Chromatography was performed with C₁₈ analytical column and 50:50 acetonitrile–phosphate buffer (pH 3.5) as mobile phase. Compounds were monitored by UV detection at 247 nm. The retention time for efavirenz was 6.45 min and that for the internal standard, nelfinavir, was 2.042 min. Response was a linear over the concentration range of 0.1 μg–10 μg mL⁻¹ in human plasma. The method was simple, specific, precise and accurate and was useful for bioequivalence and pharmacokinetic studies of efavirenz.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

Development and Validation of a High Performance Liquid Chromatographic Method for the Determination of Voriconazole Content in Tablets

This paper describes the validation of an isocratic HPLC method for the assay of voriconazole in tablets. The method employs a Merck LiChrospher^? 100 RP-8 (125 × 4.6 mm I.D., 5 μm particle size) column, with a mobile phase of methanol : triethylamine solutions 0.6 %, pH 6.0 (50:50, v/v) and UV detection at 255 nm. A linear response (r > 0.9999) was observed in the range of 20.0–100.0 μg mL⁻¹. The method showed good recoveries (average 100.4%) and the relative standard deviation intra and inter-day were ≤ 1.0 %. Validation parameters as specificity and robustness were also determined. The method can be used for both quality control assay of voriconazole in tablets and for stability studies as the method separates voriconazole from its degradation products and tablet excipients.     

Development and Validation of a Micellar Liquid Chromatographic Method with UV Detection for Determination of Azithromycin in Tablets and Capsules

A rapid and simple micellar liquid chromatographic method that does not require use of specific chromatographic columns has been developed and validated for azithromycin determination. The method uses a Hypersil C₁₈ column at 60 °C, 1-butanol–pH 6.86 phosphate buffer solution–water, 15:25:60 (v/v), containing 0.10 M sodium dodecyl sulfate, as mobile phase, and UV-detection at 215 nm. Different characteristics of the method were validated satisfactorily. The specificity, accuracy, linearity, precision (repeatability), and robustness of the method were demonstrated. The method proved suitable for determination of the azithromycin content of capsules and uncoated tablets.Revised: 5 February and 11 March 2004     

整体柱高效液相色谱法测定尿中核苷的方法研究

尿中修饰核苷已被研究用作可能的肿瘤标记物,目前最常用的分析方法为反相高效液相色谱法,其不足之处是分析周期太长。为此,建立了应用整体柱的高效液相色谱方法对尿中修饰核苷进行分析,所用缓冲液为25 mmol/L磷酸二氢钾溶液(pH 4.55)和60%(体积分数）的甲醇水溶液,线性梯度洗脱,检测波长为260 nm。12种目标核苷可被完全分离。所建立的方法在分离度、线性、重现性、灵敏度及回收率等指标上与普通反相液相色谱法相近,但分析周期大大缩短,仅为23 min,适合于大量临床样品的分析。     

Simple Liquid Chromatographic Determination of Minocycline in Brain and Plasma

A new high-performance liquid chromatography assay was developed for the determination of minocycline in plasma and brain. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC system. The system requires a mobile phase consisting of acetonitrile:water:perchloric acid (26:74:0.25, v/v/v) adjusted to pH 2.5 with 5 M sodium hydroxide for elution through a RP8 column (250 × 3.0 mm, i.d.) with UV detection set at 350 nm. The method proved to be accurate, precise (RSD < 20%) and linear between 0.15–20 μg mL⁻¹ in plasma and 1–20 μg mg⁻¹ in brain. The method was successfully applied to a blood-brain barrier minocycline transport study.     

High-Performance Liquid Chromatographic Assay of Zonisamide (1,2-benzisoxazole-3-methanesulfonamide) in Human Plasma using a Solid-Phase Extraction Technique

A selective and sensitive liquid chromatographic method was developed for the determination of zonisamide in small volumes of plasma. Zonisamide and the internal standard methyl 4-hydroxybenzoate were extracted from 0.2 mL of plasma with solid-phase extraction columns and eluted with methanol. Analysis of the extracts was performed on a Symmetry C₁₈ column with ultra-violet spectrophotometric detection. The calibration curve was linear over the concentration range of 0.05–5 g mL^–1 in plasma. Recoveries were reasonable for routine analyses; the limit of quantification was 0.05 g mL^–1 with a signal-to-noise ratio of 5. This method could be useful for the pharmacokinetic study of zonisamide in a limited volume of human plasma and for therapeutic drug monitoring.     

Development of an Ion-Pairing Liquid Chromatography Method for the Determination of Phenoxyacetic Herbicides and Their Main Metabolites: Application to the Analysis of Soil Samples

A modified ion-pairing liquid chromatography method is developed for the analysis of 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA) and their phenolic metabolites in water and soil samples. The separation and determination of all compounds of interest can be performed with a mobile phase containing 30% acetonitrile and 10 mM tetrabutylammonium hydroxide as the ion-pairing reagent. Separation of the two phenoxyacids is strongly affected by the pH of the mobile phase, which has to be buffered to pH 7.2 with phosphoric acid. The increase in the hydrophobicity of the sample solvent results in wider peaks for the two phenoxyacids, which reduces the resolution in their separation. The evaluation of different methanol:water solvent mixtures shows that no baseline separation for the two phenoxyacids is obtained when the methanol content in the mixture is higher than 60%.     

High-Performance Liquid Chromatographic Analysis of Amino Acids in Edible Seaweeds after Derivatization with Phenyl Isothiocyanate

Summary A reversed-phase high-performance liquid-chromatographic method has been used for analysis of the amino acids in edible seaweed. Sample proteins were hydrolysed with hydrochloric acid and the amino acids produced were derivatized with phenyl isothiocyanate. The resulting phenylthiocarbamyl amino acids were chromatographed on an ODS2 column with UV detection at 254 nm. The mobile phase was a mixture of 0.14 M ammonium acetate buffer, pH 6.4, containing 0.05% triethylamine (A) and 60:40 (v/v) acetonitrile–water (B), at a flow rate of 1.1 mL min^–1; the elution gradient (min:A%) was: 0:90, 8:90, 10:70, 12:70, 18:52, 20:0, 25:0, 28:90, 35:90. Method precision for the different amino acids was between 1.33 and 3.88% (relative standard deviation); detection limits were between 6.9 and 14.3 ng mL^–1. The amino acid content of the algae analysed ranged from 22.4 ± 1.9 to 138.0 ± 5.6 mg g^–1 d.w. The amino acids present at highest concentrations were glutamic acid, alanine, and phenylalanine.     

Development and Validation of a LC Method for the Determination of Pramipexole Using an Experimental Design

A rapid and sensitive RP-HPLC method with UV detection for routine control of pramipexole in tablets was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile/phosphate buffer (60/40; v/v) with a flow rate of 0.8 mL min⁻¹. Quantitation was accomplished with the internal standard method; the procedure was validated by linearity (correlation coefficient = 0.99892), accuracy, robustness and intermediate precision. Limit of quantitation and limit of detection were found to be 4.5 μg and 1.4 μg respectively, which indicates the method is highly sensitive. Experimental design was used during validation to calculate method robustness and intermediate precision, for robustness test three factors were considered; percentage v/v of acetonitrile, flow rate and pH; an increase in the flow rate results in a decrease of concentration found of the drug, while the percentage of organic modifier and temperature have no important effect on the response. For intermediate precision measure the considered variables were: analyst, equipment, days and obtained RSD value (0.56%, n=24) which indicated a good precision of the analytical method. The method was found to be applicable for determination of the drug in tablet formulations and the results of the developed method were compared with those of the UV spectrophotometric method to access the active pramipexole content. Revised: 13 March and 25 April 2006     

Development of an HPLC Assay Method for Lenalidomide

Chromatographic separation of lenalidomide and its impurities was achieved on an Inertsil ODS-3 V column using a mobile phase consisting of a mixture of buffer, acetonitrile and methanol in the ratio 80:8:12 v/v. Degradation studies were performed on bulk samples of lenalidomide subjected to 0.5 N hydrochloric acid, 0.5 N sodium hydroxide, 10% v/v hydrogen peroxide, heating to 60 °C and UV light at 254 nm. Degradation was observed only under base hydrolysis conditions. The developed LC method gave a mass balance close to 99.5%, proving it to be suitable for stability studies and was validated with respect to linearity, accuracy, precision and robustness.     

Selective quantification of doxycycline in human plasma and urine with optimised chromatography

Summary A method for the selective quantification of doxycycline in human plasma and urine has been developed using HPLC with UV-detection at 350 nm. Analyte and internal standard were extracted from plasma by extraction columns filled with octadecylsilica. Urines were only mixed with diluted acid prio to injection. The influence of pH on peak shapes is discussed as well as comparative investigations on selectivity and peak symmetry on commerical octadecylsilica. The method was successfully applied to the samples of a clinical study with an oral single dose of 100 mg. Precision and accuracydata of the assay and calculated pharmacokinetic parameters are presented.     

Determination of Polyvinyl Alcohol Using Gel Filtration Liquid Chromatography

Gel filtration chromatography using a TSKgel G2000 SW column coupled with differential refractive index detection was used to analyse five grades of polyvinyl alcohol. Limits of detection and quantification for the assay were 0.14 mg mL^–1, 0.47 mg mL^–1 respectively. The inter and intra-day co-efficient of variance were both <7%. There was a significant difference (p<0.05, n=5) between the calibration curves across the five grades of PVA due to a refractive index range of 13.0790 –1.3181 (n=3). The assay accuracy was 98.99% ± 8.97% (n=5) and 90.60% ± 7.87% (n=5) of a spiked PVA sample was recovered from a commercial formulation.     

High-Performance Liquid Chromatographic Determination of Cinnarizine in Workplace Air

Summary Cinnarizine is a pharmaceutical drug used in the treatment of cerebral and peripheral vascular diseases. A reversed-phase liquid chromatographic method with fluorescence detection has been developed for determination of the drug in workplace air. Air sampling in the workplace is performed on perchlorovinyl filters (FPP), the filters are extracted with methanol for 40 min, and the extract (50 L) is injected and separated on a 250 mm × 4.6 mm i.d., 5 m particle, C₈ reversed-phase column with 1% ammonium acetate (pH 4.5)–acetonitrile, 1:4 (v/v), as mobile phase at a flow rate of 1 mL min^–1.     

High Performance Liquid Chromatographic Analysis of Pharmaceuticals Using Oil-In-Water Microemulsion Eluent and Monolithic Column

Novel and efficient separations of pharmaceutical substances were achieved using oil-in-water microemulsion eluent and a conventional C18 packing with a flow rate of 1 mL/min⁻¹. Attempts to decrease analysis time was limited due to the high viscosity of the microemulsion which generated relatively high back-pressures. Monolithic columns gave 3-fold lower back-pressures and allowed flow rates of 4 mL/min⁻¹. with the same microemulsion mobile phase which permitted rapid separations to be achieved. Separation of a test-mix of paraben preservatives was achieved in both isocratic and gradient mode in less than 1 min. The monolith-microemulsion combination was applied to rapidly quantitatively analyse two formulated products with excellent linearity, accuracy and repeatability. Quantitative analysis times were under 90 seconds. Successful quantitation of both nicotine lozenges and naprosyn tablets was performed using this approach.     

Analysis of Spectinomycin by HPLC with Evaporative Light-Scattering Detection

A high-performance liquid chromatographic method with evaporative light-scattering detection (ELSD) has been developed for analysis of spectinomycin and related impurities. Separation of spectinomycin from structurally related impurities was achieved on a C₁₈ column. The optimized mobile phase was 25 mmol L⁻¹ ammonium acetate (pH 7.5)-methanol, 90:10 (v/v), at a flow rate of 0.6 mL min⁻¹. The temperature of the drift tube of the ELSD was 95°C and the flow rate of carrier gas was 2.2 L min⁻¹. The accuracy, specificity, precision, linearity, sensitivity, and robustness of the method were validated in accordance with ICH guidelines. In addition to determination of spectinomycin and related impurities, the method is also ideal for determination of the salts spectinomycin hydrochloride and spectinomycin sulfate.     

A Simple Liquid Chromatography Method for the Determination of Captopril in Urine

A simple and specific method for the determination of total captopril in human urine was developed. 2-Chloro-1-methylquinolinium tetrafluoroborate was used as a thiol precolumn derivatizing reagent after conversion of a disulfide forms to free captopril with tris(2-carboxyethyl)phosphine hydrochloride. The 2-S-quinolinium derivative of captopril was separated on a Zorbax SB C-18 column using reversed-phase ion-paring chromatography and monitored by spectrophotometric detector at 355 nm. The calibration curve for the derivatized captopril showed linearity in the range 0.1–200 μmol L⁻¹ of urine with a regression coefficient corresponding to 0.9999. The detection and quantitation limits were 0.05 and 0.1 μmol L⁻¹, respectively. The intra-day imprecision was from 0.01 to 10.58%. This method can be used for routine clinical monitoring of the thiol-drug. Omission of the reduction step gives result for concentration of the reduced form of captopril.     

Supported Liquid Membrane Work-Up of Blood Plasma Samples Coupled On-Line to Liquid Chromatographic Determination of Basic Antidepressant Drugs

An automated sample pretreatment of human blood plasma for liquid chromatographic determination of three antidepressant drugs, dibenzepine; a tricyclic antidepressant (TCA), reboxetine; a selective noradrenaline reuptake inhibitor (SNRI) and fluvoxamine; a selective serotonin reuptake inhibitor (SSRI), based on supported liquid membrane (SLM) for unsurpassed sample clean-up and analyte enrichment, has been developed. The chromatograms after enrichment of plasma blank and aqueous blank are virtually indistinguishable. The entire analytical procedure revealed good linearity and low detection limits of 5, 15 and 20 ng mL^–1 for dibenzepine, reboxetine and fluvoxamine, respectively. No carry-over effects were noted. The repeatability of extraction at three concentrations in the range 40–150 ng mL^–1 for the three drugs was between ca. 3% and 7% as relative standard deviation. The reproducibility relative standard deviation during three different days (replacing the membrane each day) was not significantly higher.     

Development and Validation of an HPLC Method for Determination of Ceftiofur Sodium

An isocratic high-performance liquid chromatographic method has been developed for assay of ceftiofur sodium in drug substance and in sterile powder for injection. Chromatography was performed on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column with a 78:22 (v/v) mixture of 0.02 m disodium hydrogen phosphate buffer (pH adjusted to 6.0 with 85% orthophosphoric acid) and acetonitrile as mobile phase, at a flow rate of 1.0 mL min⁻¹. The separation was monitored by UV detection at 292 nm. Validation of the method for linearity and range, intra- and inter-day precision, accuracy, specificity, recovery, robustness, and limits of quantification and detection yielded good results. The calibration plot was linear from 20.0–120.0 μg mL⁻¹ and the correlation coefficient was 0.9999. It was shown that ceftiofur was degraded under acidic, alkaline, oxidative, and photolytic conditions. The method was found to be stability-indicating and could be used for routine analysis of ceftiofur sodium for injection.     

High-Performance Liquid Chromatographic Determination of Isofraxidin in Rat Plasma

For the first time a high-performance liquid chromatographic (HPLC) method, with liquid-liquid extraction and ultraviolet (UV) absorbance detection, has been developed for quantification of isofraxidin in rat plasma. The analysis was performed on a Diamonsil C₁₈ column (200 mm × 4.6 mm i.d., 5 μm particle size) with acetonitrile–0.05% phosphoric acid, 26:74 (v/v), as isocratic mobile phase. The linear range was 0.05–8.0 μg mL⁻¹ and the lower limit of quantification was 0.05 μg mL⁻¹. The intra and inter-day relative standard deviation (RSD) for measurement of 0.25, 2.0, and 6.0 μg mL⁻¹ quality-control (QC) samples ranged from 5.7 to 6.4% and from 6.3 to 7.9%, respectively. Accuracy, as relative error (RE), was from ±5.8% to ±7.3%. The method was validated for specificity, accuracy, and precision and was successfully used in a pharmacokinetic study of isofraxidin in rat plasma after administration of Ciwujia extract.