An improved liquid chromatographic method for the analysis of tylosin and its impurities

A selective reversed-phase (RP) liquid chromatographic (LC) method coupled with UV for the determination of tylosin and its related substances is described. The gradient method uses a Capcell pak C18 ACR column (25 cm×4.6 mm id, 5 μm) maintained at a temperature of 60°C. The mobile phases consist of acetonitrile, phosphate buffer pH 5.5 and water: (A; 27.5:10:62.5 v/v/v) and (B; 50:10:40 v/v/v). The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. It allows the separation of all known and 22 other unknown related substances (≥0.02%) from the main compound and from one another. The method shows good precision, sensitivity, linearity (between 0.2 μg/mL and 1.25 mg/mL) and robustness. The limit of quantification is 0.2 μg/mL, corresponding to 0.020%. Seven bulk tylosin samples containing a large number of impurities were examined using this method.     

反相高效液相色谱法测定肾复康胶囊中的野黄芩苷和芦丁

建立了肾复康胶囊中野黄芩苷和芦丁的反相高效液相色谱测定方法。以甲醇-水为提取溶剂,采用超声提取法对样品进行前处理。以0.02 mol/L磷酸二氢钾缓冲溶液(含1.0％（体积分数）冰醋酸)-甲醇（体积比为63∶37）为流动相,于330 nm波长下检测,肾复康提取液中野黄芩苷和芦丁可达基线分离,分析时间在20 min内。野黄芩苷和芦丁在10 ~300 mg/L内,其峰面积与浓度之间线性关系良好,目标物的加标回收率大于98％。该方法适用于肾复康胶囊及相关药材中野黄芩苷和芦丁黄酮类化合物的测定和质量控制。     

Column and thin-layer chromatographic methods for the simultaneous determination of acediasulfone in the presence of cinchocaine, and cefuroxime in the presence of its hydrolytic degradation products

Column liquid chromatography (LC) and thin-layer chromatography (TLC)-densitometry methods are described for simultaneous determination of acediasulfone (Ace) and cinchocaine (Cinco). In the LC method, the separation and quantitation of the 2 drugs was achieved on a Zorbax C8 column (5 microm, 150 x 4.6 mm id) using a mobile phase composed of methanol-phosphate buffer, pH 2.5 (66 + 34, v/v), at a flow rate of 1 mL/min and ultraviolet detection at 300 and 327 nm for Ace and Cinco, respectively. The method showed linearity over concentration ranges of 20-200 and 45-685 microg/mL, respectively. In the TLC-densitometry method, a mobile phase composed of methanol-tetrahydrofuran-acetic acid (45 + 5 + 0.5, v/v/v) was used for the separation of the 2 drugs. The linearity range was 0.5-4 and 2-9 microg/spot, respectively. In addition, stability indicating TLC-densitometry method has been developed for determination of cefuroxime sodium in the presence of 5-70% of its known hydrolytic degradation products. The mobile phase butanol-methanol-tetrahydrofuran-concentrated ammonium hydroxide (50 + 50 + 50' + 5, v/v/v/v) was used. The concentration range was 2-10 microg/spot. The optimized methods proved to be specific and accurate for the analysis of the cited drugs in laboratory-prepared mixtures and dosage forms. The obtained results agreed statistically with those obtained by the reference methods.     

Sensitive LC-ESI/MS/MS assay for the quantification and pharmacokinetic study of roxithromycin in human serum

A simple and sensitive LC-ESI/MS/MS method is developed and evaluated to determine the concentrations of roxithromycin in human serum. Serum proteins are precipitated with methanol with clarithromycin as the internal standard. In order to reduce the pollution of sample, after vortex mixing and centrifugation, the supernatants are diluted with mobile phase before analysis on a Phenomenex Luna CN column (100 mm × 2.0mm i.d., 3 μm). The mobile phase composes of methanol, acetonitrile and 0.1% formic acid and 0.1% ammonium acetate in water (3: 3: 4, v/v/v) at a flow rate of 0.2 mL/min. The linearity ranges from 10 to 20480 ng/mL. The extraction recoveries of roxithromycin range from 97 to 101%. The method is successfully used to pharmacokinetic study of roxithromycin after an oral administration dose of 300 mg roxithromycin tablets to 20 healthy volunteers.     

Isolation of three antibacterial naphthoquinones from Plumbago indica roots and development of a validated quantitative HPLC analytical method

Three naphthoquinones, plumbagin (1), 3,3'-biplumbagin (2) and elliptinone (3), isolated from Plumbago indica roots by antibacterial bioassay-guided isolation, were used as standard markers for quantitative determination. A reversed-phase HPLC method was established for the simultaneous determination of the naphthoquinones in P. indica root extracts. The method utilised a Phenomenex? ODS column (4.6?×?150?mm, 5?μm) at 25°C with a mixture of methanol and 5% aqueous acetic acid (80?:?20 v/v) as the mobile phase at a flow rate of 0.85?mL/min, and UV detection at 260?nm. The parameters of linearity, precision, accuracy specificity and sensitivity of the method were evaluated. The recovery of the method was 98.6-100.6% with good linearity (r (2?)≥?0.9997) for all three naphthoquinones. A high degree of sensitivity, specificity as well as repeatability and reproducibility (R.S.D. values less than 5%) were also achieved.     

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.     

离子色谱法测定葡萄酒中的高氯酸盐

建立了离子色谱测定红酒中高氯酸盐的分析方法。以4种葡萄酒为典型样品,测定了其中的高氯酸盐含量。使用Metrosep A Supp5阴离子分析柱（150 mm×4.0 mm）进行分离,柱温为40℃,流动相为1.0 mmol/L碳酸钠水溶液-丙酮（85：15,v/v）,流速为0.8 mL/min。结果表明,高氯酸盐在0.1~10 mg/L内具有良好的线性关系,相关系数为0.9998,方法回收率大于86.0%,相对标准偏差小于2.6%。该方法前处理方便快捷、检测灵敏度高,可满足红酒中高氯酸盐含量的测定。     

Simultaneous determination of methotrexate and metoclopramide in physiological fluids using RP-HPLC with ultra-violet detection; application in evaluation of polymeric nanoparticles

Methotrexate (MTX) is an anticancer drug while metoclopramide (MCP) is an antiemetic agent. Both the drugs are commonly coprescribed to avoid the emesis caused by anticancer drug. In this study, a novel, rapid, sensitive, and cost-effective reverse-phase high-performance liquid chromatography method was developed and validated for simultaneous determination of the methotrexate and metoclopramide in biological and pharmaceutical samples using sparfloxacin as internal standard. The analytes were separated on a Kromasil 100-5C18 RP (250?×?4.6?mm, 5?µm) column, methanol, and 0.05% trifloroacetic acid (36:64?v/v) as mobile phase with a flow rate of 1?mL/min, detection wavelength of 290?nm, and column oven temperature at 40°C. Both the analytes were extracted from physiological fluids (bovine aqueous humor, vitreous humor, and human plasma) using mixture of methanol and 10% perchloric acid (50:50 v/v). The method was linear over the concentration range of 0.025–1.0?µg/mL for methotrexate and 0.030–1.0?µg/mL for metoclopramide. The % recovery from human plasma was 98.57 and 96.74% for MTX and MCP, respectively, while from aqueous humor and vitreous humor was 95.84 and 98.51% for MTX.

The developed method was applied for in vitro release of MTX from polymeric nanoparticles and can be applied for analysis of pharmaceutical and biological samples containing both the drugs.     

Development and validation of an RP-HPLC method to quantitate acyclovir in cross-linked chitosan microspheres produced by spray drying

An accurate, simple, reproducible, and sensitive liquid chromatographic method is developed and validated to quantitate acyclovir (ACV) in cross-linked chitosan microspheres produced by spray drying. The analysis is carried out using a reversed-phase C18 column with UV-vis detection at 254 nm. The mobile phase is diluted with pure water and acetonitrile (95:5 v/v) at a flow-rate of 0.8 mL/min. The parameters used in the validation process are: linearity, range, quantitation limit, detection limit, accuracy, specificity precision, and ruggedness. The retention time of acyclovir is approximately 3.5 min with symmetrical peaks. The linearity in the range of 1-10 microg/mL presents a correlation coefficient of 0.9999. The chitosan and the tripolyphosphate in the formulation do not interfere with the analysis, and the recovery is quantitative. Results are satisfactory, and the method proves to be suitable to quantitate ACV in cross-linked chitosan microspheres.     

Simple determination of betamethasone and chloramphenicol in a pharmaceutical preparation using a short monolithic column coupled to a sequential injection system

This contribution describes the use of a new separation method based on a reversed-phase sequential injection chromatography (SIC) technique for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops. A short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25 x 4.6 mm column with a 5 mm precolumn (Merck, Germany) and a FIA1ab 3000 system (USA) with a 6-port selection valve and 5 mL syringe were used for sequential injection chromatographic separations in this study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 mL/min; UV detection was at two wavelengths, i.e., 241 and 278 nm (absorption maxima of betamethasone and chloramphenicol, respectively). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) >0.999; repeatability of determination (RSD) in the range 0.8-1.7% at two different concentration levels, and detection limits in the range 0.5-1.0 mg/mL. The chromatographic resolution between compound peaks was greater than 2.1 and the analysis time was less than 8 min under optimal conditions. The developed sequential injection chromatography method was compared with the HPLC method and was found to be applicable for routine analysis of active compounds in pharmaceutical preparations.     

Development and validation of an HPLC method for the quantitation of 1,3-dihydroxy-2-methylxanthone in biodegradable nanoparticles

A rapid and simple high-performance liquid chromatographic method for the analysis of 1,3-dihydroxy-2-methylxanthone (DHMXAN) in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanosphere and nanocapsule formulations is developed and validated. The method does not require any complex sample extraction procedure. Chromatographic separation is made with a reversed-phase C18 column, using methanol-water (90:10, v/v) containing 1% (v/v) acetic acid as a mobile phase at a flow rate of 1 mL/min. Identification is made by UV detection at 237 nm. The isocratic system operates at ambient temperature and requires 7.5 min of chromatographic time. The developed method is statistically validated according to ICH guidelines and USP 29 for its specificity, linearity, accuracy, and precision. The assay method proposed in this study is specific for DHMXAN in the presence of nanosphere and nanocapsule excipients. Diode-array analyses confirm the purity of DHMXAN peak in stress conditions (> 99.0%). The method is shown to be linear (r > or = 0.999) over the concentration range of 0.25-3.0 microg/mL. Recovery ranges from 99.0% to 102.7% (RSD: 1.49%) and from 98.3% to 101.6% (RSD: 1.07%) for nanospheres and nanocapsules, respectively. Repeatability (intra-assay precision) and intermediate precision is acceptable with RSD values ranging from 0.6% to 1.9% and from 0.3% to 2.0%, respectively. The method is shown to be suitable for the evaluation of DHMXAN content entrapped in PLGA nanoparticles.     

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).     

Development and validation of a liquid chromatography-mass spectrometry method for the determination of 4,5-diazafluoren-9-one

A new liquid chromatography-mass spectrometry (LC-MS) method is developed and validated for the identification and determination of novel 4,5-diazafluoren-9-one compound. The method employs a Waters XTerra RP-18 column (150 mmx4.6 mm, i.d. 5 microm) with a mobile phase comprised of a (50:50, v/v) mixture of deionized water containing 0.2% acetic acid (solvent A) and methanol (solvent B) at a flow rate of 1 mL/min, at 35 degrees C. The detection is performed with photodiode-array (PDA) set at 210-400 nm and single quadropole mass spectrometer with electrospray ionization (ESI) positive ion mode. The chromatographic separation is achieved in less than 3 min. The linearity is established over the concentration range of 0.1-0.5 mg/mL (r2=1.000). The mean RSD values for intra- and inter-day precision studies are <2%. The recovery of 4,5-diazafluoren-9-one ranged between 99.84 and 99.97%. The limits of detection and quantitation are determined to be 0.58 and 0.1 mg/mL, respectively.     

Simultaneous determination of diclofenac potassium and drotaverine hydrochloride in human plasma using reversed-phase high-performance liquid chromatography

A simple high-performance liquid chromatographic method with ultraviolet detection is proposed for the estimation of diclofenac potassium and drotaverine hydrochloride in human plasma. Liquid-liquid extraction was carried out with a mixture of dichloromethane-isopropyl alcohol (80:20, v/v). Chromatographic separation of the analytes and internal standard was achieved on an analytical 250 × 4.6 mm i.d. reversed-phase Thermo BDS Hypersil C8 (5 μm particle size) column using a mobile phase of acetonitrile-0.02M ammonium acetate buffer (53:47, v/v) at pH 3.5. The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration ranges of 25-1500 ng/mL and 32-960 ng/mL was obtained for diclofenac potassium and drotaverine hydrochloride, respectively. The limit of quantification was 25 and 32 ng/mL for diclofenac potassium and drotaverine hydrochloride, respectively. Recoveries of diclofenac potassium and drotaverine hydrochloride from plasma were 97.45% and 98.27%, respectively.     

Determination and validation of duloxetine hydrochloride in capsules by HPLC with pre-column derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of duloxetine hydrochloride in capsules. The method was based on pre-column derivatization with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole using the fluorimetric detection technique. Duloxetine hydrochloride was analyzed by HPLC using an Inertsil C18 column (5 μm, 150 × 4.6 mm) and mobile phase consisted of methanol and water (65:35, v/v). The fluorescence detector was adjusted at excitation and emission wavelengths of 461 and 521 nm, respectively. The linearity of the method was in the range of 10-600 ng/mL. Limits of detection and quantification were 0.51 and 1.53 ng/mL, respectively. The proposed method was successfully applied for determination of duloxetine hydrochloride in its pharmaceutical preparation. The results were in good agreement with those obtained using a reference method.     

反相高效液相色谱法测定普通鹿蹄草中的黄酮苷类成分

建立了普通鹿蹄草中5种黄酮苷(金丝桃苷、异槲皮苷、2″-O-没食子酰基金丝桃苷、槲皮素-3-O-呋喃阿拉伯糖苷和槲皮苷)的高效液相色谱检测方法。采用Zorbax Extend-C18色谱柱(250 mm×4.6 mm,5 μm),以乙腈-水(体积比14∶86)为流动相,在波长为350 nm处检测。在此条件下,样品中5种黄酮苷分离良好且无杂质峰干扰,低、中、高浓度下的回收率为96.3%~104.2%,相对标准偏差（RSD）小于5%。该方法简便快速、结果准确、重现性好,可以作为普通鹿蹄草药材及相关制剂质量控制的一个有效方法。     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

反相高效液相色谱法测定蟾酥中的3种蟾毒内酯

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。。     

Determination of glimepiride in pharmaceutical formulations using HPLC and first-derivative spectrophotometric methods

Two validated analytical methods have been developed to determine glimepiride in pharmaceutical formulations using HPLC and 1st order derivative spectrophotometric techniques. Employing reverse phase HPLC method, the drug was analyzed by pumping a mixture of acetonitrile and 2% formic acid solution, pH 3.5 (80: 20 v/v) through a C₁₈ column (250 × 4.6 mm, 5 μm) and detecting the eluents at 228 nm. The linearity range was found to be 20–140 μg/mL with mean recovery of 100.52 ± 0.33%. The second method was based on the formation of a complex of the drug with 2,3,5-triphenyl-2H-tetrazolium chloride in basic media. 1st order derivative spectrum made it possible to detect the complex at 413.5 nm. The linearity range was found to be 40–160 μg/mL, with mean recovery of 100.33 ± 0.47%. Both the proposed methods can reliably be used for routine analysis of glimepiride in raw material as well as in pharmaceutical formulations. The article is published in the original.     

Simultaneous Determination of Ceftriaxone Sodium and Non Steroidal Anti‐Inflammatory Drugs in Pharmaceutical Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of ceftriaxone in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher Star, C18 (5 μm, 250 × 4.6 mm) column at room temperature using methanol:water:acetonitrile (80:15:5 v/v/v) as a mobile phase, pH adjusted at 2.8 with ortho‐phosphoric acid and at a flow rate of 1.0 mL/minute, while UV detection was performed at 270 nm. The results obtained showed a good agreement with the declared content. The method shows good linearity in the range of 2.5‐25 μg/mL ceftriaxone serum concentrations with a correlation coefficient 0.999 (inter‐ and intra‐day RSD < 2.0%). The limit of detection and quantification for ceftriaxone and NSAID's in pharmaceutical formulation and serum were in the range 0.51‐1.54 μg/mL. Analytical recovery was >98.1%. The proposed method may be used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs i.e. tiaprofenic acid, naproxen sodium, flurbiprofen, diclofenac acid and mefenamic acid alone or in combination with ceftriaxone from raw materials, dosage formulations and in serum. The established HPLC method is rapid, accurate and selective, because of its sensitivity and reproducibility.