A stability-indicating HPLC assay for metronidazole benzoate

A simple and rapid stability-indicating HPLC assay procedure has been developed and validated for metronidazole benzoate. The HPLC conditions were as follows, column: Waters Symmetry C8, 5 microm packing, 4.6 mm x 250 mm; detection: UV at 271 nm; injection volume: 20 microl; mobile phase: acetonitrile-0.1% glacial acetic acid in monobasic potassium phosphate (0.01 M) (40:60, v/v); isocratic elution under ambient temperature at 2.0 ml min(-1). The procedure separated metronidazole benzoate and its potential degradation products, metronidazole and benzoic acid, in an overall analysis time of about 6 min with metronidazole benzoate eluting at about 5 min. The injection repeatability was 0.03%, and the intraday and interday repeatability were 0.4 and 0.7%, respectively. The procedure provided a linear response over the concentration range 0.2-800 microg ml(-1) (r=1.0000) with the limits of detection and quantitation 0.03 and 0.2 microg ml(-1), respectively. The solubilities of metronidazole benzoate in water, 0.01 M hydrochloric acid and 0.05 M phosphate buffer, pH 6.8, determined each in triplicate using the procedure, were 0.2 mg ml(-1) (R.S.D. 7%), 0.4 mg ml(-1) (R.S.D. 2%) and 0.2 mg ml(-1) (R.S.D. 8%), respectively. The results show no detectable hydrolysis of metronidazole benzoate in 0.01 M hydrochloric acid at 37 degrees C or in the mobile phase at ambient temperature in 10 h.     

Determination of cetylpyridinium chloride and tetracaine hydrochloride in buccal tablets by RP-HPLC

The HPLC method for simultaneous determination of cetylpyridinium chloride (CPC), tetracaine hydrochloride (TTC) in Xipiluan buccal tablets was developed and validated. The HPLC method was performed on a CN column (150 x 4.6 mm i.d., 5 microm particle size); the mobile phase was methanol-tetramethylammonium hydroxide (20 mM)-potassium dihydrogen phosphate (3 mM) (90:10:3, v/v/v) (pH* 5.0), pumped at a flow rate 1.5 ml min(-1). The UV detector was set at 230 nm. The retention time for CPC and TTC was 3.52 and 3.10 min, respectively. Calibration curves were linear (r=0.9999, n=6) in the range of 5-2000 microg ml(-1) for CPC and 1-500 microg ml(-1) for TTC. Limit of detection and quantitation for CPC was 0.033 and 0.11 microg ml(-1), for TTC were 0.0056 and 0.019 microg ml(-1). The R.S.D. of repeatability and intermediate precision for CPC and TTC were less than 2.0%.     

Simultaneous determination of benazepril hydrochloride and hydrochlorothiazide by micro-bore liquid chromatography

A micro-bore liquid chromatographic method was developed for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in pharmaceutical dosage forms. The use of a BDS C-18 micro-bore analytical column, results in substantial reduction in solvent consumption and increased sensitivity. The mobile phase consisted of a mixture of 0.025 M sodium dihydrogen phosphate (pH 4.8) and acetonitrile (55:45, v/v), pumped at a flow rate of 0.40 ml min(-1). Detection was set at 250 nm using an ultraviolet detector. Calibration graphs are linear (r better than 0.9991, n = 5), in concentration range 5.0-20.0 microg ml(-1) for benazepril hydrochloride and 6.2-25.0 microg ml(-1) for hydrochlorothiazide. The intra- and interday R.S.D. values were <1.25% (n = 5), while the relative percentage error (Er) was <0.9% (n = 5). The detection limits attained according to IUPAC definition were 0.88 and 0.58 microg ml(-1) for benazepril hydrochloride and hydrochlorothiazide, respectively. The method was applied in the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

HPLC determination of sertraline in bulk drug, tablets and capsules using hydroxypropyl-beta-cyclodextrin as mobile phase additive

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for determination of sertraline in bulk drug, tablets and capsules was developed. Chromatography resolution of the sertraline enantiomeric forms and trans diastereoisomers was performed on Alltima C18 (250 mm x 4.6mm i.d., 5 microm) column with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as mobile phase additive. The composition of the mobile phase was 68:32 (v/v) aqueous 170 mM phosphate buffer, pH 3.0 (adjusted with 85% phosphoric acid) containing 18 mM HP-beta-CD/acetonitrile at a flow rate of 1.0 ml x min(-1). The UV detector was set at 225 nm. Calibration curves were linear (r=0.9999, n=9) in the range of 1-120 microgml (-1) for sertraline. Limit of detection and quantitation for sertraline was 0.029 and 0.097 microg x ml (-1). The values of R.S.D. of repeatability and intermediate precision for bulk drug, tablets and capsules of sertraline hydrochloride were less than 1.0%.     

基于一测多评法鉴别蓝芩制剂中的黄柏与关黄柏

目的 建立蓝芩制剂中盐酸黄柏碱、盐酸巴马汀及盐酸小檗碱的HPLC一测多评法,并基于含量测定结果建立蓝芩制剂中黄柏与关黄柏的鉴别方法。方法 采用Agilent 1260色谱仪,Waters Atlantis T3(250 mm×4.6 mm,5 μm)色谱柱,以乙腈-0.2%磷酸溶液(每100 mL加十二烷基磺酸钠0.2 g)梯度洗脱,流速为1 mL·min^-1,检测波长为280 nm,柱温30℃,以盐酸小檗碱为参照物,计算盐酸黄柏碱及盐酸巴马汀的相对校正因子,所得结果与外标法结果进行比较,判断该方法的可靠性。利用聚类分析含量测定结果,讨论黄柏与关黄柏对3种生物碱含量的影响,建立鉴别方法,对不同厂家的蓝芩制剂进行测定。结果 盐酸黄柏碱、盐酸巴马汀及盐酸小檗碱分别在1.087~54.35 μg·mL^-1,0.100 9~20.18 μg·mL^-1,0.536 5~26.82 μg·mL^-1内线性关系良好;平均加样回收率分别为91.57%(RSD=3.0%),97.96%(RSD=2.1%),100.6%(RSD=1.1%);相对校正因子分别为0.368,1.239。所得结果与外标法所测结果的RSD<2%,2种方法无显著差异。聚类分析显示使用黄柏与使用关黄柏的蓝芩制剂3种生物碱含量有明显差异;以盐酸黄柏碱与盐酸巴马汀的含量比值为鉴别标准,建议蓝芩制剂中\     

An enantiospecific HPLC method for the determination of (S)-enantiomer impurities in (R)-tolterodine tartarate

A high-performance liquid chromatographic method was developed for the separation of the enantiomers of tolterodine tartarate. The proposed method was applied to the determination of (S)-isomer in (R)-tolterodine tartarate, and satisfactory results were obtained. The enantiomers of tolterodine tartarate were separated on a Chiralpak AD-H (250 mm x 4.6 mm) column containing amylose tris-(3,5-dimethylphenyl-carbamate) at room temperature. The mobile phase consisted of n-hexane and isopropyl alcohol in the ratio of 85:15 (v/v) with 0.075% triethylamine (TEA) and 0.05% trifluoroacetic acid (TFA) as the additive. The flow rate was kept at 0.5 ml/min, and UV detection wavelength was set at 283 nm. The calibration curves of (S)-enantiomer in the concentration range from 0.05 microg/ml to 1 microg/ml range were linear. The relative standard deviations of within-day and between-day were less than 2% (n = 3). The limit of detection (LOD) was 0.75 ng (S/N = 3) and the limit of quantification (LOQ) was 0.05 microg/ml (RSD < 4.1%, n = 3). The determination recoveries of the (S)-enantiomer were in the range of 98.2-104.8%. The results demonstrated that the developed HPLC method was a reliable, simple technique and was applicable to the purity determination of (R)- tolterodine tartarate.     

Rapid and simple methods for quantitative analysis of some antidepressant in pharmaceutical formulations by using first derivative spectrophotometry and HPLC

Two rapid, simple and accurate first derivative spectrophotometry and HPLC method for the determination of nefazodone hydrochloride and sertraline hydrochloride in pharmaceutical formulations are discussed. The first one is a derivative spectrophotometric procedure and the second one is based on a HPLC method with a UV detector. In the first method, first derivative spectrophotometry, nefazodone hydrochloride or sertraline hydrochloride by measurement of their first derivative signals at 241.8-256.7 nm (peak-to-peak amplitude), or 271.6-275.5 nm (peak-to-peak amplitude), respectively. Calibration graphs were established for 10.0-42.0 microg ml(-1) nefazodone hydrochloride, or 8.0-46.0 microg ml(-1) sertraline hydrochloride. In the other method, HPLC, the UV detection was carried out at 265.0 nm (nefazodone hydrochloride) and 270.0 nm (sertraline hydrochloride). The samples were chromatographed on a Supercosil RP-18 column. The mobile phases were methanol:acetonitrile:phosphate buffer at pH 5.5 (10:50:40 v/v/v) (nefazodone hydrochloride) and methanol:phosphate buffer at pH 4.5 (20:80 v/v) (sertraline hydrochloride). The results obtained from first derivative spectrophotometric method were comparable with those obtained by using HPLC. It was concluded that both the developed methods are equally accurate, sensitive, and precision could be applied directly and easily to the pharmaceutical formulations of nefazodone hydrochloride and sertraline hydrochloride, respectively.     

HPLC法同时测定芩连片中盐酸巴马汀和盐酸小檗碱的含量

目的建立同时测定芩连片中盐酸巴马汀和盐酸小檗碱含量的方法,用于芩连片的质量控制。方法采用HPLC法。色谱柱为Diamonsil ODS柱(4.6 mm×200 mm,5μm),流动相为50 mmol.L-1磷酸二氢钠(pH 4.6)-乙腈(体积比72∶28),流速为1.0 mL.min-1,检测波长为343 nm,柱温为35℃。结果芩连片中盐酸巴马汀、盐酸小檗碱与其他成分分离良好;盐酸巴马汀的质量浓度在5.2~52.0 mg.L-1(r=0.9998)内、盐酸小檗碱的质量浓度在30.4~304.0 mg.L-1(r=0.9997)内与峰面积呈良好的线性关系;盐酸巴马汀、盐酸小檗碱的回收率分别为100.1%、99.9%,RSD分别为1.1%、0.80%。结论所建立的同时测定芩连片中盐酸巴马汀和盐酸小檗碱含量的HPLC法准确、重现性好,可用于芩连片的质量控制。     

Determination of amphotericin B in human plasma using solid-phase extraction and high-performance liquid chromatography

A rapid and selective HPLC method is described and validated for measuring amphotericin B (AB) in plasma. The procedure involves the solid phase extraction of AB from plasma by incorporating 1-amino-4-nitronaphthalene as an internal standard during the last elution step in extraction followed by HPLC analysis with UV detection at 407 nm. The chromatographic separation is achieved in less than 10 min on a reversed-phase C-18 column using acetonitrile-disodium edetate (20 mM) (45:55, v/v) at pH 5.0 as eluent. A linear response over the concentration range of 0.0100--2.00 microg ml(-1) is obtained having a detection limit of 0.00500 microg ml(-1) for AB. The mean extraction recovery is found to be 98.1+/-1.1% (n=15). The within-day and day-to-day R.S.D. were less than 2% (n=15) and 6.54% (n=45) respectively. This method is applied for quantifying AB trough levels in the plasma of cancer patients who have been on antifungal therapy with AmBisome. It can further be applied either for AB therapeutic monitoring or single/multiple pharmacokinetic analysis of AB in plasma.     

海南地不容药材定性定量方法研究

目的:建立海南地不容药材的定性定量方法。方法:采用薄层色谱法对海南地不容药材进行定性鉴别;采用高效液相色谱法测定巴马汀、延胡索乙素、防己诺林碱、粉防己碱的含量,色谱柱为Hypersil-C18(4.6 mm×250 mm,5μm),流动相为甲醇-0.1%三乙胺体系,梯度洗脱,流速1.0 mL.min-1,柱温25℃,检测波长281 nm。参照中国药典2010年版一部所述方法测定水分及总灰分。结果:在薄层色谱鉴别中,延胡索乙素和巴马汀斑点分离效果良好,具专属性,可用于海南地不容药材的定性鉴别;高效液相色谱测定中,巴马汀、延胡索乙素、防己诺林碱、粉防己碱进样量分别在0.288～2.88,0.0978～0.815,1.44～9.60,0.116～0.776μg范围内线性关系良好(r=0.9997～0.9999);平均加样回收率(n=6)分别为97.4%,98.3%,100.8%,97.9%。结论:所建立的方法可准确地对海南地不容进行定性、定量分析,用于其质量控制。     

HPLC determination of aminophylline,methoxyphenamine hydrochloride,noscapine and chlorphenamine maleate in compound dosage forms with an aqueous-organic mobile phase

A high-performance liquid chromatography procedure for the simultaneous determination of aminophylline, methoxyphenamine hydrochloride, noscapine and chlorphenamine maleate in commercially available compound capsule dosage forms has been developed and validated. The separation and quantification were achieved on an Ultrasphere C18 column using a mobile phase of dichloromethane-methanol-0.25% (v/v) diethylamine aqueous solution (20:60:20, v/v/v) at a flow rate of 1 ml min(-1) with detection of all analytes at 264 nm. The separation was achieved within 6 min for each drug mixture. The method showed good linearity for the aminophylline, noscapine, chlorphenamine maleate and methoxyphenamine hydrochloride mixture in the 125-750, 35-210, 10-60 and 62.5-375 microg ml(-1) ranges, respectively. The intra- and inter-day R.S.D.s ranged from 0.4 to 0.5%, 0.4-0.6%, 0.5-0.7% and 0.4-0.6% for aminophylline, noscapine, chlorphenamine maleate and methoxyphenamine hydrochloride, respectively. The recoveries (mean+/-S.D.) of low, middle and high concentrations were 99.9+/-0.9, 100.4+/-1.3 and 99.7+/-0.7% for aminophylline; 99.9+/-1.1, 100.4+/-0.7 and 100.1+/-0.8% for noscapine; 99.8+/-1.1, 99.7+/-1.0 and 100.7+/-0.8% for chlorphenamine maleate; and 99.8+/-0.9, 100.4+/-1.6 and 99.9+/-0.9% for methoxyphenamine hydrochloride, respectively.     

Quantification of huperzine A in Huperzia serrata by HPLC-UV and identification of the major constituents in its alkaloid extracts by HPLC-DAD-MS-MS

A rapid, simple and reliable high performance liquid chromatography (HPLC) method has been established for the analysis of the major alkaloids in Huperzia serrata, a traditional Chinese medicine (TCM) herb. After chromatographic separation on a reversed-phase C18 column with methanol-ammonium acetate (pH 6.0; 80 mM, 30/70, v/v) as the mobile phase, nine alkaloid compounds in the alkaloid extracts of H. serrata were identified by online diode array detection-MS and by comparing with data from literature and standard samples. One compound in the extract, huperzine A, is a drug for treating Alzheimer's disease. Its content was quantified by HPLC coupled with UV-vis. The method was the validated. The recovery rates were 96.8-97.7% with R.S.D <2.44%. The intra- and inter-day precisions, expressed as R.S.D., ranged from 0.53% to 1.51%. Good linear regression was observed in the concentration range of 5-100 microg/ml (r = 0.9997). The results demonstrate that this method is simple, selective, and suitable for the quality control of this TCM herb.     

Assay of amikacin in the skin by high-performance liquid chromatography

Amikacin is used in the systemic treatment of serious infections, but also locally for the treatment of skin infections. The aim of this work was to develop and validate a simple procedure for amikacin determination inside the epidermal tissue: this implies a simple method for an efficient drug extraction from the skin and a clean and easy HPLC analysis. Amikacin was extracted from epidermis samples with 500 microl of a mixture methanol-water-0.05 M NaOH (5:5:2 v/v/v) at 60 degrees C for 1 h. After filtration, the obtained solution was derivatized (1-fluoro-2,4-dinitrobenzene at 90 degrees C for 10 min) and analyzed by HPLC, on a C18 microBondapack 300 mmx4.6 mm column thermostatted at 45 degrees C. The mobile phase was a mixture of acetonitrile-water-acetic acid (47:53:0.1 v/v/v) at a flow rate of 1.5 ml/min and the UV detector was set at 365 nm. The derivatization and HPLC analysis were validated in the concentration interval 1.64-49.21 microg/ml. The linearity resulted very good (R=0.9995); the R.S.D.% varied between 0.20% and 3.89% depending on the concentration and the ER% was included between 5.4 and 0.9. The extraction method used demonstrated to be specific and the recovery resulted about 93%. The extraction, derivatization and HPLC assay has good reproducibility, sensitivity and specificity resulting in a reliable method for biopharmaceutical studies of AK distribution in the epidermis.     

基于含量测定和化学计量分析的消渴灵片质量标准研究

目的:建立13个厂家111批消渴灵片中黄连的6个生物碱、丹皮酚等多指标性成分含量测定方法,并结合主成分分析及聚类分析等化学计量学方法对其进行质量评价研究.方法:采用HPLC-UV法建立6个黄连生物碱类成分(非洲防己碱、表小檗碱、盐酸药根碱、盐酸黄连碱、盐酸巴马汀、盐酸小檗碱)、丹皮酚的含量测定方法及通过Chempatt...     

HPLC determination of rifampicin and related compounds in pharmaceuticals using monolithic column

A rapid, sensitive and reproducible HPLC method using C18 monolithic column was developed and validated for the analysis of rifampicin (RIF) and its four related compounds, including rifampicin quinone (RQ), rifamycin SV (SV), rifampicin N-oxide (RNO) and 3-formylrifamycin SV (3-FR). Chromatographic separation was achieved by using the mixture of methanol-acetonitrile-monopotassium phosphate (0.075 M)-citric acid (1.0M) (28:30:38:4, v/v) as the mobile phase at a flow rate of 2 mL/min and with UV detection at 254 nm. Calibration curves were obtained in the concentration ranges of 1-40 microg/mL for SV, RNO and 3-FR, 1.5-60 microg/mL for RQ and 5-200 microg/mL for RIF. Limit of quantitation (LOQ) was determined to be 1 microg/mL and the limit of detection (LOD) was 0.2 microg/mL for all studied compounds with a 10 microL injection. The intra-day R.S.Ds. and inter-day R.S.Ds. for the above five compounds were all less than 2.5%. The recoveries of rifampicin from placebo tablets were from 99.7% to 100.5%. The total run time was less than 11 min, as opposed to around 60 min by using C18 particle-packed column. In conclusion, by this developed method, RIF and its related compounds can be determined rapidly with good precision and accuracy in pharmaceuticals.     

A stability-indicating LC method for the simultaneous determination of ramipril and hydrochlorothiazide in dosage forms

A simple, rapid and sensitive HPLC method has been developed for the simultaneous determination of ramipril and hydrochlorothiazide in their dosage forms. Acetonitrile: sodium perchlorate solution (0.1 M) adjusted to pH 2.5+/-0.2 with phosphoric acid (46:54 v/v), was used as the mobile phase, at a flow rate of 1.5 ml/min. A supelcosil LC-8 column (5 microm), 15 cm x 4.6 mm i.d. was utilized as stationary phase. Detection was affected spectrophotometrically at 210 nm. Clobazam was used as an internal standard. The method was also applied for the determination of ramipril in the presence of its degradation products. Linearity ranges for ramipril and hydrochlorothiazide were 4.5-45 and 0.6-14 microg/ml, respectively. Minimum detection limits (S/N = 2) obtained were 180 and 23 ng/ml for ramipril and hydrochlorothiazide, respectively. The proposed method was further applied to the analysis of tablets containing the two drugs, the percentage recoveries +/- S.D. (n = 5) were 100.45%+/-0.63 and 99.55%+/-0.78 for ramipril and hydrochlorothiazide, respectively.     

宣肺祛风合剂的质量标准研究

目的 制定宣肺祛风合剂的质量标准.方法 采用薄层色谱法(TLC)对处方中的前胡、钩藤进行鉴别;采用高效液相色谱法(HPLC)测定方中麻黄碱类成分含量,Supelco ODS C18柱(250 mm×4.6 mm,5 μm),流动相:乙腈-0.2 %磷酸水溶液(3∶97);检测波长:210 nm.柱温:30 ℃;流速:1.0 mg·L-1.结果 前胡、 钩藤TLC鉴别斑点清晰,阴性无干扰,可作为定性鉴别;HPLC测定盐酸麻黄碱在 0.843 75~54.00 mg·L-1范围内,与峰面积性关系良好, 回归方程为Y=19.48X + 12.554,r2=0.999 9,平均回收率为 98.2%,RSD=2.4% (n=5);盐酸伪麻黄碱在0.781 25~50.00 mg·L-1范围内,与峰面积的积分值呈良好的线性关系,回归方程Y=19.816X+12.468 (r2=0.999 9) ,平回收率均为 97.9%,RSD=1.7% (n=5).结论 TLC鉴别方法操作简单,专属性强;HPLC含量测定方法准确、重现性良好,可作为该制剂的质量控制方法.     

高效液相色谱-二极管阵列检测器同时测定清热祛浊胶囊中6种活性成分的含量

目的：建立高效液相色谱同时测定清热祛浊胶囊中绿原酸、栀子苷、芍药苷、盐酸小檗碱、连翘苷和丹皮酚含量的方法。方法：采用Agilent Eclipse XDB-C₁₈色谱柱,流动相为乙腈（A）和0.1%的磷酸水溶液（B）,梯度洗脱;流速：1.0 mL·min^-1;柱温：30℃;检测波长：绿原酸为327 nm,栀子苷、芍药苷和连翘苷为230 nm,盐酸小檗碱和丹皮酚为274 nm。结果：绿原酸、栀子苷、芍药苷、盐酸小檗碱、连翘苷和丹皮酚的线性范围分别为8.64~216 μg·mL^-1（r=0.999 8）,4.08~102 μg·mL^-1（r=0.999 8）,4.20~105 μg·mL^-1（r=0.999 9）,9.12~228 μg·mL^-1（r=0.999 8）,3.28~82.0 μg·mL^-1（r=0.999 9）和4.84~121 μg·mL^-1（r=0.999 8）,6种成分的平均加样回收率（n=6）分别为99.2%,99.1%,98.6%,98.6%,98.9%和99.3%,RSD分别为1.0%,1.1%,1.2%,1.0%,0.8%和0.9%。结论：本法结果准确,重现性好,回收率高,可用于清热祛浊胶囊的质量控制。     

Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with beta-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 degrees C. The standard curves were linear over the range of 0.0975-7.800 and 0.1744-13.95 microg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze-thaw cycles, storage at room temperature for 4 h, and at -20 degrees C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).     

A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.