Determination of methylparaben,propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC

A novel reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of active component triamcinolone acetonide, its degradation product triamcinolone (occurring in formulation after long-term stability tests) and two preservatives presented in the cream-methylparaben and propylparaben, using hydrocortisone as an internal standard.The chromatographic separation was performed on a Supelco Discovery C18 column; the mobile phase for separation of all compounds consists of a mixture of acetonitrile and water (40:60 v/v). The analysis time was less than 9 min, at a flow rate of 0.6 mL min(-1) and detection at 240 nm. The method was found to be applicable for routine analysis (stability tests, homogeneity) in the pharmaceutical product topical cream Triamcinolon cream 0.1%.     

Enantio- and chemo-selective HPLC separations by chiral-achiral tandem-columns approach: the combination of CHIROBIOTIC TAG and SCX columns for the analysis of propionyl carnitine and related impurities

We describe a new tandem-columns chiral-achiral HPLC arrangement by using a chiral column (CHIROBIOTIC TAG) connected in series with an achiral column (Spherisorb S5 SCX), based on a strong cationic exchange mechanism; this approach is very useful for the analysis of chiral molecules, containing cationic groups in their structures. We used this special combination to develop an easy and convenient procedure for the enantio- and chemo-selective dosage of propionyl L-carnitine (1) and relative impurities (2-6), which allowed for the simultaneous separation and quantitation within 30 min. Under the best chromatographic conditions (acetonitrile-10 mM sodium dihydrogen phosphate 65:35, v/v (pHa 6.80) as the mobile phase and UV detection at 205 nm], all the individual peaks were well separated. The applicability of the method, fully validated, was demonstrated by the analysis of a pharmaceutical batch of propionyl L-carnitine, where we found the following contents: 98.5% for 1 (drug substance); 0.15% for 3; 0.1% for 5 and 0.2% for 6. The enantiomeric excess (e.e.%) measured for the drug substance was 98.9%. Finally, a single mixed-bed column, packed with a binary mixture of the chiral and achiral phases, in a 1:1 ratio, gave similar chromatographic results as the tandem-columns approach, and thus, offered an easy alternative solution to the separation of the considered mixture.     

高效液相色谱法直接拆分泮托拉唑钠对映体

首次使用Kromasil CHI-TBB手性柱在正相条件下直接拆分了泮托拉唑钠对映异构体,考察了流动相组成、酸碱调节剂用量以及流速对对映体分离的影响。优化后的分析条件为:流动相组成为正己烷-异丙醇-乙酸(体积比为95∶5∶0.1)混合溶液,流速2.0 mL/min,柱温25 ℃。所建立的方法具有柱效高、简便快捷及重复性好等优点。     

纤维素键合手性固定相拆分6种萘满酮衍生物对映体

使用Chiralpak IC（纤维素-三（3,5-二氯苯基氨基甲酸酯）共价键合硅胶）手性柱,建立了采用手性固定相高效液相色谱拆分6种 α -芳基萘满酮类衍生物对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温和流速对对映体分离的影响。结果显示6种化合物在异丙醇为改性剂的条件下均可获得较高的对映体分离度。热力学研究表明6种化合物对映体的手性拆分过程均受焓驱动影响,且低温有利于对映体分离。最终推荐分离化合物Ⅰ对映体的流动相是正己烷-异丙醇（90：10,v/v）;分离化合物Ⅱ、Ⅲ、Ⅳ对映体的流动相是正己烷-异丙醇（99：1,v/v）;分离化合物Ⅴ对映体的流动相是正己烷-异丙醇（85：15,v/v）;分离化合物Ⅵ对映体的流动相是正己烷-异丙醇（80：20,v/v）。柱温为25℃,流速为1.0 mL/min。6种化合物对映体均可在Chiralpak IC手性固定相上得到完全分离,证明该色谱柱对6种化合物具有较高的对映体选择性。     

Quantitative determination of valienamine and validamine by thin-layer chromatography

A simple and valid thin-layer chromatographic method for the separation and quantitative determination of valienamine and validamine is described. The two compounds are separated using a Silica gel G plate as the stationary phase and a mixture of 1-PrOH-AcOH-H2O (4:1:1, v/v/v) as the mobile phase. The plate is developed for 1 h at 25 degrees C and dried by a hairdrier, then immersed in 0.1% ninhydrin aqueous solution and heated for 5 min at 121 degrees C. The reacted spots are scanned with a single wavelength at 420 nm in the measurement mode of absorption. The limits of detection of the two compounds are both 0.4 microg. The responses of the densitometry are highly correlated with the amounts of valienamine and validamine in the range of 0.4-2.8 pg. Moreover, the method shows good accuracy and high precision.     

L-羟脯氨酸寡肽混合物的高效液相色谱分离与质谱分析

研究了三氯氧磷辅助下L-羟脯氨酸的成肽反应,建立了采用反相高效液相色谱-质谱/质谱联用技术分离鉴定羟脯氨酸寡肽混合物的方法,优化了L-羟脯氨酸寡肽混合物的色谱分离条件。实验以YWG C8柱(10 μm,250 mm×10 mm)为分离柱,以乙腈-0.06%三氟乙酸水溶液(体积比为2∶98)为流动相进行等度洗脱,在正离子模式下对洗脱物进行了电喷雾电离串联质谱鉴定。结果显示,分离出的各组分分别为L-羟脯氨酸二肽、L-羟脯氨酸环二肽和L-羟脯氨酸三肽。     

反相离子对高效液相色谱法测定铁强化酱油中的乙二胺四乙酸铁钠

建立了用反相离子对高效液相色谱法分离和测定铁强化酱油中的铁营养强化剂乙二胺四乙酸铁钠(NaFeEDTA)的方法。样品经甲醇沉淀后,以Zorbax C8 色谱柱(150 mm×4.6 mm i.d.,5 μm)进行分离,以含12.5%甲醇、0.13%四丁基氢氧化铵(TBAOH)和0.052%甲酸的水溶液（pH 3.5）作为流动相,流速为1.00 mL/min,检测波长为254 nm,整个分离过程在30 min内完成。考察了NaFeEDTA在不同品牌酱油中的回收率,其中NaFeEDTA的添加量为0.50~4.00 g/L时,其回收率为94.15%~101.5%。对NaFeEDTA添加量为2.00 g/L的铁强化酱油样品重复测定6次,其峰面积的相对标准偏差为0.89%。NaFeEDTA标准溶液的最低检测限为0.03 mg/L。本方法简单、快速、重现性好,可用于铁强化酱油中NaFeEDTA含量的检测。     

漆酚酯键合硅胶液相色谱固定相的制备与应用

以甲基丙烯酸漆酚酯为色谱配体,制备了一种新型色谱固定相。首先以漆酚和甲基丙烯酰氯为原料制备得到甲基丙烯酸漆酚酯,并通过物理吸附涂覆到由3-（甲基丙烯酰氧）丙基三甲氧基硅烷化学修饰的硅胶上,再通过自由基引发与硅烷化硅胶的双键聚合制得漆酚酯键合硅胶固定相（USP）。对固定相进行傅里叶红外光谱（FT-IR）、热重分析（TGA）、扫描电子显微镜（SEM）和元素分析（EA）表征,结果表明通过共聚反应成功地将漆酚酯固定在硅烷化硅胶上,且制备出的固定相具有良好的单分散性。采用匀浆法装柱,以乙腈-0.05%磷酸溶液（3：97,v/v）为流动相,流速为0.4 mL/min,检测波长为220 nm,考察固定相对天麻浸膏的分离性能。以乙腈-水（50：50,v/v）为流动相,流速为0.5 mL/min,检测波长为290 nm,考察固定相对吴茱萸浸膏的分离性能。结果表明该固定相对天麻浸膏和吴茱萸浸膏均具有良好的分离性能,从天麻浸膏中分离出5个色谱峰,从吴茱萸浸膏中分离出2个色谱峰。与商品化C₁₈ 柱相比,USP柱可以从天麻浸膏中分离出更多的有效组分并实现基线分离,分离吴茱萸浸膏的色谱条件更为环保和安全。采用低流速对天麻浸膏和吴茱萸浸膏进行分离,减少了流动相的使用量,分离结果令人满意。以天然产物漆酚制备色谱固定相,既为分离纯化天麻素和吴茱萸碱提供了一种新的方法,又为液相色谱固定相制备提供了新的思路,还拓展了生漆在色谱分离材料方面的应用。     

Stability indicating HPLC method for the simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations

ABSTRACT: BACKGROUND: A simple, specific, and fast stability indicating reverse phase liquid chromatographic method was established for instantaneous determination of moxifloxacin and prednisolone in bulk drugs and pharmaceutical formulations. RESULTS: Optimum chromatographic separations among the moxifloxacin, prednisolone and stressinduced degradation products were achieved within 10 minutes by use of BDS Hypersil C8 column (250 X 4.6 mm, 5 mum) as stationary phase with mobile phase consisted of a mixture of phosphate buffer (18 mM) containing 0.1% (v/v) triethylamine, at pH 2.8 (adjusted with dilute phosphoric acid) and methanol (38:62 v/v) at a flow rate of 1.5 mL min-1. Detection was performed at 254 nm using diode array detector. The method was validated in accordance with ICH guidelines. Response was a linear function of concentrations over the range of 20-80 mug mL-1 for moxifloxacin (r2 [greater than or equal to] 0.998) and 40-160 mug mL-1 for prednisolone (r2 [greater than or equal to] 0.998). The method was resulted in good separation of both the analytes and degradation products with acceptable tailing and resolution. The peak purity index for both the analytes after all types of stress conditions was [greater than or equal to] 0.9999 indicated a complete separation of both the analyte peaks from degradation products. The method can therefore, be regarded as stabilityindicating. CONCLUSIONS: The developed method can be applied successfully for simultaneous determination of moxifloxacin and prednisolone in pharmaceutical formulations and their stability studies.     

HPLC assay for determination of amphotericin B in biological samples

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1-10.0 microg/mL was obtained. Intra-day and inter-day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided.     

Chromatographic methods for the separation of biocompatible iron chelators from their synthetic precursors and iron chelates

Chromatographic methods have been developed for the separation of the three novel biocompatible iron chelators pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), and pyridoxal 2-chlorobenzoyl hydrazone (o-108) from their synthetic precursors and iron chelates. The chromatographic analyses were achieved using analytical columns packed with 5 microm Nucleosil 120-5 C18. For the evaluation of all chelators in the presence of the synthetic precursors, EDTA was added to the mobile phase at a concentration of 2 mM. The best separation of PIH and its synthetic precursors was achieved using a mixture of phosphate buffer (0.01 M NaH2PO4, 5 mM 1-heptanesulfonic acid sodium salt; pH 3.0) and methanol (55:45, v/v). For separation of SIH and its synthetic precursors, the mobile phase was composed of 0.01 M phosphate buffer (pH 6.0) and methanol (60:40, v/v). o-108 was analyzed employing a mixture of 0.01 M phosphate buffer (pH 7.0), methanol, and acetonitrile (60:20:20, v/v/v). These mobile phases were slightly modified to separate each chelator from its iron chelate. Furthermore, a RP-TLC method has also been developed for fast separation of all compounds. The chromatographic methods described herein could be applied in the evaluation of purity and stability of these drug candidates.     

Sensitive and rapid analytical method for the quantification of glucosamine in human plasma by ultra high performance liquid chromatography with tandem mass spectrometry

A highly sensitive and rapid ultra high performance liquid chromatography with tandem mass spectrometry method has been developed and validated for the determination of glucosamine in human plasma using miglitol as the internal standard. Special attention was paid to achieve the high throughput and sensitivity of the established method, and the absence of a matrix effect on the analytes. The sample preparation procedure involved a simple deproteinization step. The chromatographic separation was achieved on a Waters ACQUITY HSS Cyano column using a mixture of acetonitrile/2 mM ammonium acetate solution containing 0.03% formic acid (80:20, v/v) as the mobile phase with a very short run time of 1.5 min. This method was validated over the concentration range of 10–3000 ng/mL for glucosamine. The intra‐ and inter‐batch precision was <13.9% for the low, medium, and high quality control samples. The established method is highly sensitive with a lower limit of quantification of 10 ng/mL, low enough to determine the circadian rhythm on endogenous glucosamine level in human plasma, which has not been reported in detail until now. The method was successfully applied to characterize the pharmacokinetic profile of glucosamine in healthy volunteers following a single oral administration of 750 or 1500 mg glucosamine hydrochloride.     

Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate

A liquid chromatographic method for the determination of aniline in cyclamate sweeteners based on a pre-column derivatization with 1,2-naphthoquinone-4-sulfonate (NQS) is proposed. Aniline traces were extracted from the cyclamate samples using dichloromethane. After solvent evaporation, the dry residue was derivatized with NQS at pH 9.5 and 85 degrees C for 1 min. The aniline derivative, which was extracted from the reacting mixture, was redissolved in the eluent solution and injected into the chromatographic system. The separation of aniline derivative from other amine impurities was carried out in a C18 column using a 2% acetic acid-methanol (40:60, v/v) mobile phase. Results from the analysis of aniline in the sweetener samples with the proposed method were compared with those from the standard method. A good concordance between the two methods was observed.     

Determination of phenyltoloxamine salicylamide, caffeine, paracetamol, codeine and phenacetin by HPLC

A reversed-phase high-performance liquid chromatographic method for the determination of six common analgesics (phenyltoloxamine dihydrogen citrate, salicylamide, caffeine, paracetamol, codeine phosphate and phenacetin) is presented. The method is specific for detection and determination of each of these compounds in a complex mixture, without pretreatment. A 10-mum C(18) silica gel stationary phase is used with a methanol-acetonitrile-water-tetrahydrofuran mixture (20:20:55:5 v/v) and spectrophotometric detection at 254 nm. All six components are eluted within 7 min. The method has given good results for three commercial products containing two, three and five active ingredients respectively. Phenacetin, a common analgesic which might be found in other formulations, is used as an internal standard.     

A LC Separation and Quantification of o‐Toluene Sulphonamide and its Related Positional Isomers Using a Cellulose tris(3,5‐dimethyl phenylcarbamate)

A simple, rapid, selective and sensitive high performance liquid chromatographic method was developed for the separation and quantification of o‐Toluene Sulphonamide and its positional isomers which is an intermediate of Zafirlukast drug substance using a chiral column. Elution time was below 10 min in normal phase mode and ultra violet detection was carried out at 220 nm. Efficient separation was achieved on a Chiralcel OD‐H column using of n‐Hexane and 2‐Propanol mixture (50:50 v/v) as isocratic at 1.0 mL min⁻¹ flow rate. Resolutions between m‐Toluene Sulphonamide and p‐Toluene Sulphonamide isomers of o‐Toluene Sulphonamide were found to be >; 2.5. The calibration graphs for m‐Toluene Sulphonamide and p‐Toluene Sulphonamide isomers of o‐Toluene Sulphonamide were linear (R² > 0.999) when ranging from the limit of quantitation to 0.5%. The method is found to be selective, precise, linear, accurate and also robust. It was used for not only Quality assurance but also monitoring the synthetic reactions involved in the process development of Zafirlukast. The liquid chromatographic method is found to be specific and reliable for the determination of unreacted levels of positional isomers in reaction mass. This method was successfully validated according to the International Conference Harmonization (ICH) guidelines (Validation of Analytical Procedures: Test and Methodology Q2).     

A validated LC method for determination of 2,3-dichlorobenzoic acid and its associated regio isomers

A simple, selective and sensitive gradient reversed-phase liquid chromatography method has been developed for the separation and determination of 2,3-dichlorobenzoic acid, which is an intermediate of the lamotrizine drug substance, and its regio isomers. The separation was achieved on a reversed-phase United States Pharmacopeia L1 (C-18) column using 0.01 M ammonium acetate buffer at pH 2.5 and methanol (50:50 v/v) mixture as mobile phase A and a methanol and water mixture (80:20 v/v) as mobile phase B in a gradient elution at flow rate 1.2 mL/min with ultraviolet detection at 210 nm. The method is found to be selective, precise, linear, accurate and robust. It was used for quality assurance and monitoring the synthetic reactions involved in the process development of lamotrizine. The method is found to be simple, rapid, specific and reliable for the determination of unreacted levels of raw materials and isomers in reaction mixtures and finished product lamotrizine. The method was fully validated as per International Conference of Harmonization guidelines and results from validation confirm that the method is highly suitable for its intended purpose.     

Determination of closantel residues in milk and animal tissues by HPLC with fluorescence detection and SPE with oasis MAX cartridges

A liquid chromatographic method for the determination of closantel residues in milk and tissues is developed and validated. An acetonitrile-acetone solution (80:20, v/v) is used for the extraction of closantel residues from milk and animal tissues, and the extract is purified by solid-phase extraction with Oasis MAX cartridges and a mixture of formic acid-acetonitrile (5:95, v/v) as the elution solution. A C(18) bonded silica column is used for chromatographic separation. The mobile phase consists of acetonitrile-water (85:15, v/v) containing 0.05% triethylamine at pH 2.5, adjusted with phosphoric acid with the flow-rate set at 1.0 mL/min. Using the fluorescence emission of closantel at lambda(ex) = 335 nm and lambda(ex) = 510 nm, the calibration curve is linear, with a correlation coefficient of 0.9999 over the concentration range of 10-5000 microg/kg for the tissue sample and 10-5000 microg/L for the milk sample. The detection limit (s/n = 3) is 3 microg/kg for tissue sample and 3 microg/L for milk sample. The intra- and inter-day repeatabilities are between 3.35-7.66% and 4.04-8.67%, respectively. The proposed method enables the quantitative determination of closantel residues at levels as low as 10 microg/kg in animal tissue samples and 10 microg/L in milk samples.     

配位色谱法从葛根素浸膏中分离纯化葛根素

建立了采用配位色谱柱从葛根素浸膏中分离纯化葛根素的方法。以铜离子为中心离子,制备了中心离子含量为7%的配位色谱柱。样品上样于配位色谱柱后,以氯仿-甲醇（体积比为10∶1）混合溶剂洗脱,得到了较纯的葛根素,较之用传统的硅胶色谱柱纯化,纯度提高了11%,回收率提高了12%,且柱容量提高了两倍。配位色谱改变了葛根素在传统硅胶柱上的洗脱顺序,对目标物质的分辨率比传统硅胶色谱柱高。     

Development of validated stability-indicating chromatographic method for the determination of fexofenadine hydrochloride and its related impurities in pharmaceutical tablets

ABSTRACT: A simple reversed phase high performance liquid chromatographic method with diode array detector (HPLC-DAD) has been developed and subsequently validated for the determination of fexofenadine hydrochloride (FEX) and its related compounds; keto fexofenadine (Impurity A), meta isomer of fexofenadine (Impurity B), methyl ester of fexofenadine (Impurity C) in addition to the methyl ester of ketofexofenadine (Impurity D). The separation was based on the use of a Hypersil BDS C-18 analytical column (250 × 4.6 mm, i.d., 5 μm). The mobile phase consisted of a mixture of phosphate buffer containing 0.1 gm% of 1-octane sulphonic acid sodium salt monohydrate and 1% (v/v) of triethylamine, pH 2.7 and methanol (60:40, v/v). The separation was carried out at ambient temperature with a flow rate of 1.5 ml/min. Quantitation was achieved with UV detection at 215 nm using lisinopril as internal standard, with linear calibration curves at concentration ranges 0.1-50 μg/ml for FEX and its related compounds. The optimized conditions were used to develop a stability-indicating HPLC-DAD method for the quantitative determination of FEX and its related compounds in tablet dosage forms. The drugs were subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. Complete separation was achieved for the parent compounds and all degradation products. The method was validated according to ICH guidelines in terms of accuracy, precision, robustness, limits of detection and quantitation and other aspects of analytical validation.     

直链淀粉手性固定相法分离利阿唑对映体

建立了利阿唑对映体的高效液相色谱拆分方法。采用Chiralpak AD-H手性柱在正相条件下直接拆分利阿唑对映体,考察了流动相中有机极性调节剂的种类和浓度、酸碱的种类和含量、柱温及流速等对利阿唑对映体分离的影响。确定了最佳的拆分条件:流动相为正己烷-乙醇(含0.3%二乙胺和0.1%冰醋酸)(体积比为80∶20),流速0.6 mL/min;检测波长254 nm;柱温20 ℃。在此条件下利阿唑对映体的分离度为3.4。该法简单快速,重现性好。