Development and Validation of a Liquid Chromatographic Method Useful for the Determination of Amino Acids in New and Commercial Alimentary Supplements

In this study, a new liquid chromatographic method with pre-column derivatization was developed and validated for the simultaneous quantification of acetylcarnitine taurinate, asparagine, potassium aspartate, asparagine and carnosine in new and commercial alimentary supplements. The effect of complex matrices was evaluated by the study of the amino acid derivatization reaction with 2,5-dimethyl-1H-pyrrole-3,4-dicarbaldehyde in placebo solutions (DPD). Experimental parameters affecting the derivatization and chromatographic separation were investigated. The reaction was carried out at room temperature for 10 min. The adducts were separated on a Synergi Hydro-RP 80 Å column using a mobile phase consisting of 11 mM aqueous tetrapropylammonium bromide (pH 3.5)/methanol by gradient elution conditions at a flow rate of 0.8 mL/min. UV absorbance detection was set at λ = 320 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, specificity and ruggedness were highly satisfactory. Linear responses were obtained by placebo solutions (determination coefficient ≤ 0.9994). Intra-day precision (RSD) was ≤1.06 % for corrected peak area and ≤1.14 % for retention times (t _R) without significant differences between intra- and inter-day data. Recovery studies showed good results for all the examined compounds (from 97.72 to 101.5 %) with RSD ≤1.30 %.

     

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 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.     

Environmentally Sustainable Achiral and Chiral Chromatographic Analysis of Amino Acids in Food Supplements

Two LC methods were developed for the achiral and chiral reversed-phase (RP) analysis of an amino acid (AA) pool in a food supplement, in compliance with the main paradigms of Green Chromatography. A direct achiral ion-pairing RP-HPLC method was optimized under gradient conditions with a water-ethanol (EtOH) eluent containing heptafluorobutyric acid (0.1%, v/v), to quantify the eight essential AAs (Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val) contained in the food supplement. Thus, the usually employed acetonitrile was profitably substituted with the less toxic and more benign EtOH. The method was validated for Leu and Phe. The chiral LC method performed with a teicoplanin chiral stationary phase was developed with a water-EtOH (60:40, v/v) eluent with 0.1%, v/v acetic acid. The enantioselective analysis was carried out without any prior derivatization step. Both developed methods performed highly for all eight AAs and revealed that: (i) the content of six out of eight AAs was consistent with the manufacturer declaration; (ii) only L-AAs were present. Furthermore, it was demonstrated that a two-dimensional achiral–chiral configuration is possible in practice, making it even more environmentally sustainable. A molecular modelling investigation revealed interesting insights into the enantiorecognition mechanism of Lys.     

氨基酸柱前衍生化的3种新荧光试剂的光谱特性及高效液相色谱研究

合成了３种新的荧光标记试剂：吖啶－Ｎ－乙酰氯，咔唑－９－乙酰氯和咔唑－９－丙酰氯。它们的最大发射降激发波长分别为４３０ｎｍ，３６８ｎｍ，和３６５ｎｍ。３种衍生化试剂与氨基酸形成的衍生物在ｐＨ６．５的条件下结合梯度洗脱程序在Ｃ１８反相柱上对色谱条件进行了优化。     

High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Four Flavonols in Food Supplements and Pharmaceutical Formulations

A method using high-performance liquid chromatography with diode array detection (HPLC-DAD) as a powerful separation technique has been developed for the simultaneous determination of the four flavonols rutin, quercetin, kaempferol and isorhamnetin in food supplements and pharmaceutical formulations. The chromatographic separation was achieved in 36?min using a Symmetry C₁₈ column (250?×?3?mm; 5?µm) as the stationary phase and a mixture of methanol, acetonitrile, and pH 2.5 aqueous acetic acid as the mobile phase in gradient elution mode. The analytical wavelengths were 256?nm for rutin, quercetin and isorhamnetin, and 368?nm for kaempferol. An ultrasound-assisted extraction protocol was performed using methanol as solvent. The detection and quantification limits were lower than 0.03?µg mL^?1 and 0.08?µg mL^?1, respectively. The inter-day and intra-day precisions were less than 4.8 and 5.1%, respectively, and the average recoveries were in the range from 96 to 107%. The method was applied for the determination of the studied flavonols in food supplements and pharmaceutical preparations. The satisfactory recovery values demonstrate the potential of the developed method for the determination of the analytes in these samples. In addition, the method is suitable for routine quality control due its ease of operation.     

Validation of a Reversed-Phase Liquid Chromatographic Method for the Determination of Metronidazole in Transparent Oil-Water-Gel Formulations

A high performance liquid chromatographic (HPLC) method for the determination of metronidazole in new gel formulations was developed and validated according to the recommendations of the International Harmonization Conference. An adaptation of the method described for metronidazole determination in plasma has been used, and the step of extraction has been developed. The method was demonstrated to be linear over a range of 60–140% of nominal label claim, accurate (100.08±0.63%) and precise (0.67% for intra-assay and 1.68% for inter-assay). Single point calibration was chosen for analysis because of its simplicity and wide use in the pharmaceutical industry for content uniformity analysis.     

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     

Development and Validation of a Liquid Chromatographic Method for the Determination of Leflunomide: Application to in vitro Drug Metal Interactions

Leflunomide is a leading drug for the treatment of rheumatoid arthritis. The principle aim of this study was to develop and validate an RP‐HPLC method for the determination of leflunomide in bulk and pharmaceutical dosage form using diclofenac sodium as an internal standard. For this purpose, chromatography was accomplished on a Purospher Start, C₁₈ (5 (m, 12.5 cm×0.46 cm) column at ambient temperature. Methanol:water (80:20, V/V) solvent system was selected as mobile phase, the pH of which was adjusted to 3.4 by ortho‐phosphoric acid and delivered at a flow rate of 1.2 mL·min⁻¹. Seperation of leflunomide and diclofenac sodium was carried out on a Purospher Start, C₁₈ equipped with a UV‐visible detector at 248 nm. The suitability of the method for the quantitative determination of the drugs is proven by validation in accordance with the requirements laid down by the International Conference on Harmonization (ICH) guidelines. The method was accurate (99.55%–100.03%), specific, linear (R²>0.999) and precise (intra‐day precision 0.023%–0.93% and inter‐day precision 0.26%–0.944%) in the range of 0.5–20 (g·mL⁻¹. The minimum limit of detection and quantification in pharmaceutical formulation were 0.05 and 0.15 (g·mL⁻¹, respectively. Thus the proposed method is simple, accurate, reproducible and suitable for the routine analysis of leflunomide in pharmaceutical formulations and was applied to study in vitro drug‐metal interactions.     

磺胺二甲嘧啶及有关物质HPLC检测方法验证

建立了磺胺二甲嘧啶及其可能存在的5种杂质成分:对氨基苯磺酸、4,6-二甲基-2-羧基嘧啶、磺胺脒、磺胺和2-氨基-4,6-二甲基嘧啶的高效液相色谱分离和定量分析方法.采用C18色谱柱,pH 4.0的乙腈-醋酸(体积比1:9)流动相,分离有关杂质,而水-乙腈-冰醋酸(体积比87:12:1)流动相用于主成分磺胺二甲嘧啶(SM2)含量的测定;二极管阵列检测器,检测波长275 nm.上述5种杂质的检出限分别为0.023、0.023、0.009、0.009、0.030 mg/L,满足测定要求.     

Comparison of Reverse-Phase High-Performance Liquid Chromatographic Methods for Precolumn-Derivatized Amino Acids

Abstract

A comparison was made among five precolumn derivatization techniques for amino acid analysis using reverse-phase high-performance liquid chromatography (HPLC). All chromatographic analyses were conducted using the same instrumentation and a C₁₈ Ultrasphere ODS column (5 μm, 250 × 4.6 mm). The precolumn derivatization methodologies studied included the formation of OPA (o-phthaldialdehyde), DANSYL (dimethylaminonaphthalenesulphonyl), DABSYL (dimethylaminoazobenzenesulphonyl), PTH (phenylthiohydantoin), and PTC (phenylthiocarbamyl) derivatives. The derivatization procedures were evaluated for simplicity, time required, and derivative stability. HPLC analyses of the amino acid derivatives were compared in terms of resolution, sensitivity, reproducibility, and time of analysis.     

反相高效液相色谱法同时测定丁酸氯维地平中10种相关杂质

建立反相高效液相色谱辅-光电二极管阵列检测器(RP-HPLC)法同时测定丁酸氯维地平原料药中的10种杂质。色谱柱为Symmetry C18柱(250 mm×4.6 mm,5μm),流动相为0.05 mol/L Na H2PO4溶液(pH 2.5)-乙腈/甲醇(3∶2,V/V),梯度洗脱,柱温35℃,流速为1.5 m L/min,检测波长220 nm。丁酸氯维地平及其10个已知杂质能够达到良好的分离,且各组分在各自测定浓度范围内与峰面积的线性关系良好(r≥0.9970);丁酸氯维地平及杂质1~10的检出限(S/N=3)在0.15~0.90 mg/L之间。本方法快速、简便、有效,可用于丁酸氯维地平原料药的质量控制管理。     

Liquid Chromatographic Determination of Quazepam in Commercial Tablets

Abstract

A simple and rapid reverse-phase liquid chromatography method is described for quantification of quazepam in tablets. Quazepam is extracted with methanol. An aliquot (10 ul) of the diluted methanolic extract was injected into the chromatograph. Chromatographic separations were made on a Adsorbosphere C₈ column (10 cm × 4.6 mm I. D.). Chromatograph was operated at ambient temperature with a mobile phase of 0.002 M phophate buffer (pH 4.0)-methanol (40:60) using a flow rate of 1.5 ml/min. Effluents were monitored at 265 nm. Retention times for diazepam (internal standard) and quazepam were 5.41 min and 8.67 min, respectively. Excellent day-to-day reproducibility of the slope of the standard curve and recovery data were obtained.     

Development and Validation of a Fast Reversed-Phase Ion-Pairing Liquid Chromatographic Method for Simultaneous Determination of Eight Cephalosporin Antibiotics in Pharmaceutical Formulations

A fast and reliable single method was developed for rapid screening of cephalosporin oral dosage forms aimed to the detection of counterfeit and substandard drugs that might be illegally commercialised. Nine cephalosporin compounds, ceftibuten, cefatrizine, cefadroxil, cefaclor, cefprozil (Z) and (E)-isomers, cefixime, cephalexin and cefradine were separated in a six minutes chromatographic run by using a Symmetry^® C18 column (50 × 4.6 mm I.D., 3.5 μm particle size) and an UV detector set at 254 nm. The mobile phase consisted of a mixture of acetonitrile-methanol-phosphate buffer (50 mM) containing 1-pentanesulfonic acid sodium salt (7 mM) adjusted to pH=2.1 with phosphoric acid (9:13:78 v/v/v). Validation of the method showed it to be robust, precise, accurate and linear over the concentration range of analysis.     

High Performance Liquid Chromatographic Determination of Amino Acids in Biological Samples by Precolumn Derivatization with O-Phthaldialdehyde

Abstract

A suitable gradient system has been developed for rapid analysis of amino acids in biological samples using O-phthaldialdehyde as a precolumn derivatizing agent and fluorescence detection. Resolution of 21 amino acids has been accomplished with 3 μm Ultrasphere ODS column by using a multi-step gradient system of two solvents (0.1M sodium acetate, pH 7.2/methanol:tetrahydrofuran) in less than 1 hour. Within-assay and between-assay coefficients of variation of retention times and fluorescence yield show good reproducibility. The fluorometric detection response is linear from 25 to 500 pmoles with a minimum detection limit of less than 1 pmol. High resolution, rapid analysis and high sensitivity of this method facilitates amino acid analysis in samples of less than 1 mg of tissue.     

High Performance Liquid Chromatographic Separation of Phenylthiohydantoin Derivatives of Amino Acids

Abstract

Phenylthiohydantoin derivatives of amino acids have been separated by modern liquid chromatography. The amino acid thiohydantoins were divided into three groups according to their relative polarity and three solvent systems were used to separate the groups.     

高效液相色谱Pico－Tag法测定野蕨菜氨基酸成分

高效液相色谱Ｐｉｃｏ－Ｔａｇ法测定野蕨菜氨基酸成分陈柏林，郭敏（四川农业大学动物营养研究所四川雅安６２５０１４）１前言高效液相色谱（ＨＰＬＣ）Ｐｉｃｏ－Ｔａｇ法因采用柱前衍生，反相色谱分离￣［１］，具有分析时间短、灵敏度高、重现性好等优点，广泛地用于...     

PICO－TAG反相色谱法测定鸡蛋中氨基酸含量

采用ＰＩＣＯ－ＴＡＧ反相色谱法测定了氨基酸，方法的相对标准偏差为０．７５％，平均回收率为９７．１２％。对鸡蛋样品进行了测定，发现不同产地不同皮色鸡蛋中氨基酸含量无显著差异，样品测定相对标准偏差为１．６７％。结果表明测定方法快速、灵敏、准确、重现性好。