反相高效液相色谱法测定小鼠血浆及组织中的阿昔洛韦浓度

 建立了测定小鼠血浆、肝、肾、脾、肺等组织中阿昔洛韦 (ACV)浓度的高效液相色谱法。色谱柱为HypersilODS ,流动相为甲醇 水 冰醋酸 (体积比为 1∶99∶0 5 )混合溶液 ,流速为 1 5mL/min ,检测波长为 2 5 2nm。ACV血浆最低检测浓度为 2 0 μg/L ,各组织最低检测浓度为 5 0ng/g。血浆及组织匀浆中的ACV浓度在 0 1mg/L～ 4mg/L及 0 1μg/g～ 4μg/ g时线性关系良好 (r >0 99)。血浆及肝匀浆中的ACV回收率分别为 97 5 %～ 10 0 0 %和 10 0 0 %～ 10 6 0 % (n =5 )。该法精密度高 ,方便 ,快捷 。     

Simultaneous determination of four lignan compounds in Herpetospermum caudigerum by normal-phase high-performance liquid chromatography

A normal-phase high-performance liquid chromatographic method with diode array UV detection is developed for the simultaneous quantitation of four lignan compounds in Herpetospermum caudigerum. This analysis provides a good resolution and reproducibility. Chromatography is carried out with a mobile phase of N-hexane-dichlormethane-methanol (42.5:42.5:5, v/v) at a flow rate of 1.0 mL/min. UV detection is performed at 280 nm. The calibration curve for lignans concentration is linear over the range of 2.10 to 42.0 microg/mL, 15.26 to 305.2 microg/mL, 6.15 to 123.0 microg/mL, and 6.24 to 124.8 microg/mL, respectively. The limit of quantitation and detection for compounds 1, 2, 3, and 4 is 1.31, 2.74, 2.63, and 2.17 microg/mL and 0.28, 0.25, 0.27, and 0.31 microg/mL, respectively. The validation data show that the assay is sensitive, specific, accurate, and reproducible for the simultaneous quantitation of four compounds. This rapid method is therefore appropriate to quantitate these lignans in Herpetospermum caudigerum.     

Analysis of acyclovir by high performance capillary electrophoresis with on-column amperometric detection

The separation of acyclovir (ACV) by high performance capillary electrophoresis (HPCE) with on-column amperometric detection using alpha-amino-5-mercapto-3,4-dithiazole (AMD) as internal standard is described. The calibration line was linear in the range of 0.5-20 mg/L of ACV. The detection limit was 0.15 mg/L of ACV. Its recovery ranged from 98 to 101% with relative standard deviations (RSDs) from 1.9 to 3.2% (n = 5). This method was successfully used for determining ACV in some pharmaceuticals and human urine. Comparable results with HPCE with ultraviolet (UV) detection and amperometric detection were obtained.     

Determination of ranolazine in human plasma by liquid chromatographic-tandem mass spectrometric assay

A highly sensitive liquid chromatographic-tandem mass spectrometric method (LC-MS-MS) is developed to quantitate ranolazine in human plasma. The analyte and internal standard tramadol are extracted from plasma by liquid-liquid extraction using diethyl ether-dichloromethane (60:40 v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (60:40 v/v, pH 4.0) at a flow of 1.0 mL/min. Detection is carried out by multiple reaction monitoring on a QtrapTM LC-MS-MS system with an electrospray ionization interface. The assay is linear over the range 10-5000 ng/mL with a limit of quantitation of 10 ng/mL and a lower limit of detection (S/N > 3) of 1 ng/mL. Intra- and inter-day precision are < 3.1% and < 2.8%, respectively, and the accuracy is in the range 96.7-101.6%. The validated method is successfully used to analyze the drug in samples of human plasma for pharmacokinetic studies.     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

Development and validation of a novel RP-HPLC method for the analysis of reduced glutathione

The objective of this study was the development, optimization, and validation of a novel reverse-phase high-pressure liquid chromatography (RP-HPLC) method for the quantification of reduced glutathione in pharmaceutical formulations utilizing simple UV detection. The separation utilized a C18 column at room temperature and UV absorption was measured at 215 nm. The mobile phase was an isocratic flow of a 50/50 (v/v) mixture of water (pH 7.0) and acetonitrile flowing at 1.0 mL/min. Validation of the method assessed the methods ability in seven categories: linearity, range, limit of detection, limit of quantification, accuracy, precision, and selectivity. Analysis of the system suitability showed acceptable levels of suitability in all categories. Likewise, the method displayed an acceptable degree of linearity (r(2) = 0.9994) over a concentration range of 2.5-60 μg/mL. The detection limit and quantification limit were 0.6 and 1.8 μg/mL respectively. The percent recovery of the method was 98.80-100.79%. Following validation the method was employed in the determination of glutathione in pharmaceutical formulations in the form of a conjugate and a nanoparticle. The proposed method offers a simple, accurate, and inexpensive way to quantify reduced glutathione.     

Highly sensitive determination and validation of gabapentin in pharmaceutical preparations by HPLC with 4-fluoro-7-nitrobenzofurazan derivatization and fluorescence detection

A sensitive HPLC method with pre-column fluorescence derivatization using 4-Fluoro-7-Nitrobenzofurazan (NBD-F) has been developed for the determination of gabapentin in pharmaceutical preparations. The method is based on the derivatization of gabapentin with (NBD-F) in borate buffer of pH 9.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Inertsil C(18) column (250 mm × 4.6 mm) using a mobile phase of methanol water (80:20, v/v) solvent system at 1.2 mL/min flow rate. Mexiletine was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 521 nm (emission). The assay was linear over the concentration range of 5 50 ng/mL. The method was validated for specificity, linearity, limit of detection, limit of quantification, precision, accuracy, robustness. Moreover, the method was found to be sensitive with a low limit of detection (0.85 ng/mL) and limit of quantitation (2.55 ng/mL). The results of the developed procedure for gabapentin content in capsules were compared with those by the official method (USP 32). Statistical analysis by t- and F-tests, showed no significant difference at 95 confidence level between the two proposed methods.     

Development and validation of the HPLC method for the analysis of trimetazidine hydrochloride in bulk drug and pharmaceutical dosage forms

A simple, economic, selective, precise, and accurate high-performance liquid chromatographic (HPLC) method for the analysis of trimetazidine hydrochloride in both bulk drug and pharmaceutical formulations was developed and validated in the present study. The mobile phase consisted of water: methanol: triethylamine (75: 25: 0.1 v/v/v), and pH 3.3 was adjusted with orthophosphoric acid. This system was found to give a sharp peak of trimetazidine hydrochloride at a retention time of 3.375 ± 0.04 min. HPLC analysis of trimetazidine hydrochloride was carried out at a wavelength of 232 nm with a flow rate of 1.0 mL/min. The linear regression analysis data for the calibration curve showed a good linear relationship with a regression coefficient of 0.997 in the concentration range of 5–90 μg/mL. The linear regression equation was y = 35362x − 8964.2. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.6 and 10.9 μg/mL, respectively. The developed method was employed with a high degree of precision and accuracy for the analysis of trimetazidine hydrochloride. The developed method was validated for accuracy, precision, robustness, detection, and quantification limits as per the ICH guidelines. The wide linearity range, accuracy, sensitivity, short retention time, and composition of the mobile phase indicated that this method is better for the quantification of trimetazidine hydrochloride. The text was submitted by the authors in English.     

A sensitive and rapid determination of ranitidine in human plasma by HPLC with fluorescence detection and its application for a pharmacokinetic study

A novel precolumn derivatization reversed-phase high-performance liquid chromatography method with fluorescence detection is described for the determination of ranitidine in human plasma. The method was based on the reaction of ranitidine with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole forming yellow colored fluorescent product. The separation was achieved on a C(18) column using methanol-water (60:40, v/v) mobile phase. Fluorescence detection was used at the excitation and emission of 458 and 521 nm, respectively. Lisinopril was utilized as an internal standard. The flow rate was 1.2 mL/min. Ranitidine and lisinopril appeared at 3.24 and 2.25 min, respectively. The method was validated for system suitability, precision, accuracy, linearity, limit of detection, limit of quantification, recovery and robustness. Intra- and inter-day precisions of the assays were in the range of 0.01-0.44%. The assay was linear over the concentration range of 50-2000 ng/mL. The mean recovery was determined to be 96.40 ± 0.02%. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of ranitidine.     

An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study

A new high-performance liquid chromatographic method is described for the determination of bisoprolol in human plasma. The proposed method was based on the derivatization of bisoprolol with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluorescent product. Chromatographic separation of bisoprolol was achieved by using isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed-phase column (Inertsil, 4 μm, 150 4.6 mm) at 40°C. The mobile phase used for the analysis was methanol-water (70:30, % v/v). Fluorescence detector was used at the excitation and emission wavelengths of 458 and 525 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery and system suitability. The assay was linear over the concentration range of 10-2000 ng/mL. This method was applied in pharmacokinetic studies of bisoprolol preparations in healthy volunteers.     

Development of a simple and sensitive high-performance liquid chromatography method for determination of glucosamine in pharmaceutical formulations

A column high-performance liquid chromatography (HPLC) method was developed for the determination of glucosamine in dosage forms. Glucosamine was derivatized by addition of a solution containing orthophthaldialdehyde. The HPLC separation was achieved on a Spherimage 80 ODS2 column (250 x 4 mm id, 5 microm particle size) using an isocratic mobile phase containing phosphate buffer-methanol (90 + 10, v/v, pH 6.50) and methanol-tetrahydrofuran (97 + 3, v/v) in proportions of 85 + 15 at a flow rate of 1 mL/min, followed by fluorescence detection. The method was validated for specificity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The detector response for glucosamine HCI was linear over the concentration range of 0.1-20 microg/mL with a correlation coefficient of 0.9980. The accuracy was between 99.4 and 100.8%. The LOD and the LOQ were 0.009 and 0.027 microg/mL, respectively. The method was applied to determination of glucosamine in solid dosage forms.     

磁性壳聚糖微球的制备及对Cr(Ⅵ)的吸附性能研究

用壳聚糖包埋磁流体,用戊二醛交联制成磁性壳聚糖微球,并用红外光谱表征其结构。用制备的磁性壳聚糖微球吸附Cr(Ⅵ)离子,考察了其对Cr(Ⅵ)离子的吸附性能;探讨了吸附时间、溶液pH值、吸附剂用量、温度、Cr(Ⅵ)起始浓度以及其他离子存在对Cr(Ⅵ)离子去除率的影响。实验结果表明,磁性壳聚糖微球吸附Cr(Ⅵ)离子的最佳条件为:吸附平衡时间40 min,最佳吸附pH值6左右,磁性壳聚糖微球用量10 mg,温度升高有利于提高磁性壳聚糖微球的吸附效率,Cr(Ⅵ)离子起始质量浓度为12μg/mL,无机盐的存在引起磁性壳聚糖微球的吸附性能降低。并且考察了吸附剂的再生性能,实验结果表明磁性壳聚糖微球具有良好的重复使用性。     

Determination of vitamin E acid succinate in biodegradable microspheres by reversed-phase high-performance liquid chromatography

A simple, rapid, and reproducible reversed-phase high-performance liquid chromatographic (HPLC) method is applied to the routine assay of vitamin E acid succinate in biodegradable microspheres. Vitamin E acid-succinate-containing poly-(D,L-lactic-co-glycolic acid) microspheres are prepared by the solvent evaporation method. The starting drug-polymer ratio is 1:10 (w/w) and the total amount of drug and polymer processed is always 440 mg. The content of vitamin E acid succinate in the microspheres is evaluated by HPLC. Chromatography is carried out isocratically at 25 degrees C +/- 0.5 degrees C on an Extrasil ODS-2 column with a mobile phase composed of methanol-water (97:3, v/v) (pH 5.6) at a flow rate of 2 mL/min and UV detection at 284 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, specificity, and ruggedness are studied as reported in the International Conference on Harmonization guidelines. The stability of vitamin E acid succinate is also studied with satisfactory results after 48 h at 25 degrees C. The method is selective and linear for drug concentrations in the range 15-210 micro g/mL. The LOQ and LOD are 15 and 3 micro g/mL, respectively. The results for accuracy studies are good. Values for coefficient of variation for intra- and interassay are 2.08% and 2.32%, respectively. The mean percentage of vitamin E acid succinate in the recovery studies is 99.52% +/- 0.81%. The mean loading efficiency for microspheres is 96.53% +/- 1.31%.     

用硼酸作为显色剂光度法测定阿昔洛韦

在pH12.00的缓冲溶液中,阿昔洛韦(ACV)与H3BO3形成组成比为1∶1的反应产物,其最大吸收波长λmax=289 nm,ACV的质量浓度在0.48～57.6 mg/L范围内与吸光度成良好关系,线性回归方程A=-0.01796+0.01624ρ,相关系数r=0.9990,表观摩尔吸光系数ε=3.7×103L.mol-1.cm-1,回收率为99.9%～101.6%。据此建立了测定ACV的新方法,能够直接用于药物样品中ACV的测定。     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Development and validation of column high-performance liquid chromatographic and ultraviolet spectrophotometric methods for citalopram in tablets

Column high-performance liquid chromatographic (LC) and UV spectrophotometric methods for the quantitative determination of citalopram, a potent and selective serotonin reuptake inhibitor, in tablets were developed. The parameters linearity, precision, accuracy, specificity, robustness, limit of detection, and limit of quantitation were studied according to International Conference on Harmonization guidelines. Chromatography was carried out by the reversed-phase technique on an ACE C18 column with a mobile phase composed of 0.30% triethylamine solution-acetonitrile (55 + 45, v/v) adjusted to pH 6.6 with 10% ortho-phosphoric acid at a flow rate of 1.0 mL/min and 25 degrees C. The UV spectrophotometric method was performed at 239 nm. The linearity of the LC method was in the range of 10.00-70.00 microg/mL, and 2.50-17.50 microg/mL for the UV spectrophotometric method. The interday and intraday assay precision was < 1.5% (relative standard deviation) for the LC and UV spectrophotometric methods. The recoveries were in the range 100.70-101.35% for the LC method and 98.48-98.65% for the UV spectrophotometric method. Statistical analysis by Student's t-test showed no significant difference between the results obtained by the 2 methods. The proposed methods are highly sensitive, precise, and accurate and can be used for the reliable quantitation of citalopram in tablets.     

Stability-indicating HPLC method for determination of naftazone in tablets. Application to degradation kinetics and content uniformity testing

A simple, sensitive, stability-indicating HPLC method was developed and validated for the quantitative determination of the vasoprotective drug, naftazone in presence of its degradation products. The analysis was carried out on a Nucleosil 100-5 phenyl column (250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of methanol-0.02 M sodium dihydrogen phosphate mixture (60:40, v/v) of pH 6.0. The analyses were performed at ambient temperature with a flow rate of 1.0 mL/min and UV detection at 270 nm. The method showed good linearity over the concentration range of 0.1-10.0 μg/mL with a lower detection limit of 0.032 and quantification limit of 0.096 μg/mL. The suggested method was successfully applied for the analysis of naftazone in its commercial tablets. Moreover, it was utilized to investigate the kinetics of alkaline, acidic and oxidative degradation of the drug. The apparent first-order rate constants, half-life times, and activation energies of the degradation process were calculated. The pH-rate profile curve was derived. Furthermore, the proposed method was successfully applied to the content uniformity testing of naftazone tablets.     

Assay and stability‐indicating micellar electrokinetic chromatography method for the simultaneous determination of valacyclovir,acyclovir and their major impurity guanine in pharmaceutical formulations

A micellar electrokinetic chromatography (MEKC) method for the simultaneous determination of the antiviral drugs acyclovir and valacyclovir and their major impurity, guanine, was developed. The influences of several factors (surfactant and buffer concentration, pH, applied voltage, capillary temperature and injection time) were studied. Using tyramine hydrochloride as internal standard, the analytes were all separated in about 4 min. The separation was carried out in reversed polarity mode at 28°C, 25 kV and using hydrodynamic injection (15 s). The separation was effected in a fused‐silica capillary 100 μm × 56 cm and a background electrolyte of 20 mM citric acid–1 M Tris solution (pH 2.75), containing 125 mM sodium dodecyl sulphate and detection at 254 nm. The method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 0.1–1 μg/mL (guanine) and from 0.1 to 120 μg/mL for both valacyclovir and acyclovir. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 5.0%. The proposed method was successfully applied to the determination of the analytes in tablets and creams. From the previous study it is concluded that the stability‐indicating method developed for acyclovir and valacyclovir can be used for analysis of the drug in various stability samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a validated liquid chromatographic method for the determination of related substances and assay of tenofovir disoproxil fumarate

The present study describes the development and validation of a selective liquid chromatographic (LC) method for the analysis of tenofovir disoproxil fumarate (TDF) and its related substances. The gradient method uses a base deactivated C18 column (Hypersil BDS column; 25 cm×4.6 mm I.D.) maintained at a temperature of 30°C. The mobile phases consist of acetonitrile, tetrabutylammonium/phosphate buffer pH 6.0 and water: (A; 2:20:78 v/v/v) and (B; 65:20:15 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 260 nm. Good separation of TDF and 21 impurities was achieved. A system suitability test (SST) to check the quality of separation is also specified. The developed method was further validated with respect to robustness, precision, sensitivity and linearity. The method is proved to be robust, precise, sensitive and linear between 0.1 μg/mL and 0.15 mg/mL. The limit of detection and limit of quantification are 0.03 and 0.1 μg/mL, respectively. The method was successfully applied to the quantification of related substances and assay of commercial TDF samples (bulk substances and tablets).     

高效液相色谱法分析十溴二苯乙烷产品

采用Zorbax C18色谱柱(5 μm,150 mm×4.6 mm)于40 ℃下分离十溴二苯乙烷产品,以甲醇-四氢呋喃(体积比为70∶30)为流动相,在230 nm波长下检测。实验结果显示,在十溴二苯乙烷的质量浓度为0.001～0.100 g/L时,其浓度与峰面积有较好的线性关系。该方法对十溴二苯乙烷的回收率大于96%,相对标准偏差为4.0%,可替代热分析法分析十溴二苯乙烷,且能满足工业生产检测的要求。