Development and validation of a high performance liquid chromatographic method for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets

A simple, sensitive and rapid high performance liquid chromatographic method was developed and validated for the simultaneous determination of potassium clavulanate and cefadroxil in synthetically prepared tablets. Chromatographic separation and detection was carried out on a C-18 column using 0.05 M potassium dihydrogen phosphate buffer (pH 5.0) and acetonitrile in the ratio of 94: 06 (v/v) as mobile phase at wavelength of 225 nm. The method was linear in the concentration range of 3.75–22.5 μg/mL for potassium clavulanate and 15–90 μg/mL for cefadroxil. The flow rate was 1.0 mL/min and the total analysis time was less than 10 min. The mean recoveries was found to be greater than 99% with RSD less than 1.0%. The proposed method was validated by performing linearity, recovery, specificity, robustness, LOD/LOQ and within-day and between-day precision. The chromatographic results obtained from the synthetically prepared tablets show that the method is highly precise and accurate for the simultaneous quantitation of clavulanate potassium and cefadroxil.     

A comparative study of first-derivative spectrophotometry and column high-performance liquid chromatography applied to the determination of repaglinide in tablets and for dissolution testing

A fast and reliable method for the determination of repaglinide is highly desirable to support formulation screening and quality control. A first-derivative UV spectroscopic method was developed for the determination of repaglinide in tablet dosage form and for dissolution testing. First-derivative UV absorbance was measured at 253 nm. The developed method was validated for linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) in comparison to the U.S. Pharmacopeia (USP) column high-performance liquid chromatographic (HPLC) method. The first-derivative UV spectrophotometric method showed excellent linearity [correlation coefficient (r) = 0.9999] in the concentration range of 1-35 microg/mL and precision (relative standard deviation < 1.5%). The LOD and LOQ were 0.23 and 0.72 microg/mL, respectively, and good recoveries were achieved (98-101.8%). Statistical comparison of results of the first-derivative UV spectrophotometric and the USP HPLC methods using the t-test showed that there was no significant difference between the 2 methods. Additionally, the method was successfully used for the dissolution test of repaglinide and was found to be reliable, simple, fast, and inexpensive.     

Separation,determination of six impurities in methotrexate drug substance using ultra-performance liquid chromatography

Methotrexate(MTX) is an antineoplastic therapeutic medicine as antimetabolite of folic acid. In this paper, a sensitive and rapid ultra-performance liquid chromatographic(UPLC) method was developed and validated for the separation and determination of impurities in MTX drug substances. The UPLC method was accomplished on an Agilent Zorbax Extend C-18(50 mm 4.6 mm, 1.8 mm) with a gradient elution system composed of sodium dihydrogen phosphate in water(20 mmol/L, pH 3.0) and acetonitrile. The flow rate was 2.2 mL/min. The method was validated. The calibration curves displayed good linearity(r 0.999) within the tested concentration ranges. The limit of detection(LOD) and limit of quantification(LOQ) of the six analytes were all less than 0.774 mg/mL and 1.03 mg/mL. The relative standard deviation(RSD) for intra- and inter-day precision of the six analytes was less than 9.8%, including at the LOQ. The average recovery ranged from 95.2% to 103% except at the LOQ, where recovery ranged from 82.7% to 117%. The validated method was successfully used to determine the relative abundance of six impurities in the MTX drug substances.     

Development and validation of stability-indicating HPLC method for the simultaneous determination of paracetamol, tizanidine, and diclofenac in pharmaceuticals and human serum

A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.     

Applying green analytical chemistry for development and validation of RP-HPLC stability indicating assay method for estimation of fenoverine in bulk and dosage form using quality by design approach

A green and robust reverse-phase liquid chromatographic method has been developed for the determination of fenoverine (FEN), by applying combined principles of green analytical chemistry and quality by design approaches on a Spherisorb C18 column (150?×?4.6?mm, 3?µm) with UV detection at 262?nm. A two level fractional factorial design (2^^7-3) Res IV was used for screening of influential chromatographic factors. The critical method parameters actively affecting critical quality attributes (CQAs) were identified and further optimized using Box–Behnken design. The predicted optimum assay conditions comprised of methanol and ammonium acetate buffer 20?mM, in an extent of 81:19% v/v individually having a flow rate of 1.0?mL/min with a column oven temperature of 33°C. The drug was stressed in hydrolytic, oxidative, reductive, thermal, and photolytic conditions. The developed method was validated successfully. The detector response was linear in the concentration of 0.5–160?µg/mL with a limit of detection (LOD) and limit of quantitation (LOQ) as 0.1 and 0.3?µg/mL, respectively. The % recovery was found to be 99.7%. The analytical method volume intensity value for developed method was 45?mL and the environment assessment tool (EAT) score was 41.07. The method is simple, environmentally benign, rapid, and robust for the determination of FEN in bulk and in its dosage form.     

高效液相色谱法同时测定发酵液中赤藓糖醇和L-赤藓酮糖的含量

建立了采用高效液相色谱(HPLC)同时测定发酵液中底物赤藓糖醇和产物L-赤藓酮糖含量的方法。采用Lichrospher 5-NH2色谱柱(250 mm×4.6 mm),柱温30 ℃,以乙腈-水(体积比为9:1)为流动相,流速1.0 mL/min。用示差折光检测器检测赤藓糖醇,检测器温度为35 ℃。用紫外检测器在室温下检测L-赤藓酮糖,检测波长为277 nm。所得赤藓糖醇的线性范围为1.00～100.00 g/L,相关系数为0.9985,检出限为0.10 g/L,定量限为0.45 g/L;所得L-赤藓酮糖的线性范围为1.00～100.00 g/L,相关系数为0.9958,检出限为0.50 g/L,定量限为0.87 g/L;赤藓糖醇的日内和日间相对标准偏差(RSD)分别小于3.28%和5.30%, L-赤藓酮糖的日内和日间RSD分别小于2.16%和2.25%;回收率均大于99%。取不同时间的发酵液样品分别用上述方法测定,结果表明所建立的HPLC法不受发酵液中其他组分的影响,可同时测定底物赤藓糖醇和产物L-赤藓酮糖的含量。     

Monitoring of biogenic amines and drugs of various therapeutic groups in urine samples with use of HPLC

A high‐performance liquid chromatography method for simultaneous separation and determination of biogenic amines [dopamine, epinephrine, serotonin and its six metabolites (normetanephrine, metanephrine, 3,4‐dihydroxyphenylacetic acid, 4‐hydroxy‐3‐methoxyphenylglycol, homovanilic acid and 5‐hydroxyindoloacetic acid)] with drugs from different therapeutically groups [analgesics (paracetamol, metamizol), diuretics (furosemide) and antibiotics (cefazolin, fluconazole)] was developed. A chromatographic column with pre‐column with octadecylsilane phase (C_18e) and two detectors – diode array serial connected and fluorescence – was used. Gradient elution of mixture of acetate buffer (pH 4.66) and methanol as a mobile phase was applied. The limit of detection (LOD) of 8–10 ng/mL and limit of quantitation (LOQ) of 24–30 ng/mL for biogenic amines, as well as the LOD of 50–100 ng/mL and the LOQ of 150–300 ng/mL for drugs, were determined. The applied sample preparation method allowed recoveries of 93% for the biogenic amines and 92% for the drugs to be achieved. The developed procedure has been applied to simultaneous determination of the examined compounds in urine samples and could be used in clinical analysis. Copyright © 2015 John Wiley & Sons, Ltd.     

A Novel Ultra-High Performance Liquid Chromatography Method for the Determination of Erdosteine,Related Impurities and Degradation Products in New Effervescent Tablets

A novel and automated, stability-indicating, reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantitative determination of erdosteine, its known impurities and two novel degradation products in a new pharmaceutical dosage form (effervescent tablets). The chromatographic separations were performed on a Waters Acquity UPLC HSS T3, 1.8 µm (2.1 mm?×?150 mm, I.D.) stainless steel column. The mobile phase consisted of 0.1% TFA in water and methanol under gradient elution conditions, at a flow rate of 0.29 mL/min, for the assay and impurities analysis. UV detection was set at a wavelength of 238 nm. Erdosteine raw material, placebo and effervescent tablets were subjected to forced degradation. The new degradation products (labeled OX1 and OX2) were found after oxidative treatment and characterized by ultra performance liquid chromatography mass spectrometry. The validation parameters such as linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision, specificity and robustness were highly satisfactory for all analyzed compounds. LOD (0.020 and 0.011–0.385 µg/mL for erdosteine and impurities, respectively) and LOQ values show the high sensibility of the method. Specificity of the method was confirmed by testing the matrix components. The validated method demonstrated to be suitable for routine quality control purposes and for routine stability studies of erdosteine in effervescent formulations.     

Liquid chromatographic high-throughput analysis of ketamine and its metabolites in human plasma using a monolithic silica column and solid phase extraction

A rapid and sensitive liquid chromatographic (LC) assay was developed for the simultaneous determination of ketamine (KE) and its two main metabolites, namely, norketamine (NK) and dehydronorketamine (DHNK) in human plasma. Each compound together with an internal standard (Labetalol) was extracted from the plasma matrix using solid phase extraction (SPE). The applicability of monolithic LC phases in the field of quantitative bioanalysis has been evaluated. The existing method with UV detection set at 220 nm was successfully transferred from a conventional reversed phase column to a 10 cm × 4.6 mm i.d. monolithic silica column. By simply increasing the mobile phase flow-rate, run times were about six-fold reduced and consumption of mobile phase were about two-fold decreased, while the chromatographic resolution of the analytes remain unaffected. The method was validated over the range 25-2000 ng/mL for KE, 25-1500 ng/mL for NK, and 15-750 ng/mL for DHNK. The method proved to be precise (within-run precision ranges from 2.2 to 7.2% and between-run precision ranges from 3.7 to 8.2%) and accurate (within-run accuracies ranged from 1.3 to 7.2% and between-run accuracies ranged from 1.5 to 8.7%). The mean absolute recoveries were 95.3, 96.9, and 103.9% for KE, NK and DHNK, respectively. The limit of quantitation (LOQ) and limit of detection (LOD) for KE and NK in human plasma were 25 and 12.5 ng/mL, respectively, and for DHNK were 15 and 7.5 ng/mL (S/N = 3). The assay should be suitable for use in routine determination of KE and its metabolites in human plasma.     

Development and validation of gas chromatography-mass spectroscopy method for determination of prilocaine HCl in human plasma using internal standard methodology

The accurate determination of prilocaine HCl levels in plasma is important in both clinical and pharmacological/toxicological studies. Prilocaine HCl is quickly hydrolyzed to o-toluidine, causing methemoglobinemia. For this, the present work describes the methodology and validation of a GC-MS assay for determination of prilocaine HCl with lidocaine HCl as internal standard in plasma. The validation parameters of linearity, precision, accuracy, recovery, specificity, limit of detection and limit of quantification were studied. The range of quantification for the GC-MS was 20-250 ng/mL in plasma. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 6.0%, and accuracy (relative error) was better than 9.0% (n = 6). The analytical recovery of prilocaine HCl and IS from plasma has averaged 94.79 and 96.8%, respectively. LOQ and LOD values for plasma were found to be 20 and 10 ng/mL, respectively. The GC-MS method can be used for determination from plasma of prilocaine HCl in routine measurement as well as in pharmacokinetic studies for clinical use.     

Determination of levodopa and biogenic amines in urine samples using high-performance liquid chromatography

A chromatographic system is developed for the separation and determination of levodopa, biogenic amines, and their metabolites from the catecholamines group: dopamine (DA), epinephrine (E), normetanephrine (NMN), metanephrine (MN), 3,4-dihydroxyphenylacetic acid (DOMA), 3-metoxy-4-hydroxyphenyl-glycol (MHPG), and homovanillic acid (HVA); and indoloamines group: serotonin (5HT) and 5-hydroxyindole-3-acetic acid (5HIAA) in urine. The limit of detection (LOD) and limit of quantitation (LOQ) are determined for all compounds with signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD 10 (ng/mL) and LOQ 30 (ng/mL) are determined for L-DOPA, DOMA, E, NMN, DA, MN, and MHPG, as well as LOD 8 (ng/mL) and LOQ 24 (ng/mL) for HVA, 5HT, and 5HIAA. A fluorescence detector is used. Gradient elution with acetate buffer (pH=4.66) with methanol is applied. In urine samples from patients treated with levodopa, the following concentrations (microg/mL) of analytes are determined: L-DOPA 3.73-46.80, DOMA 1.43-28.43, E 0.83-13.57, NMN 2.58-8.81, DA 24.07-62.11, MN 0.89-66.20, MHPG 6.72-63.64, 5HT 22.96-95.27, 5HIAA 1.45-14.77, and HVA 0.21-15.07.     

Liquid chromatographic method for the simultaneous determination of eprosartan and hydrochlorothiazide in tablets and human plasma

A new, specific, and sensitive RP-HPLC method was developed for the simultaneous determination of eprosartan (EPR) and hydrochlorothiazide (HCT). Good chromatographic separation was achieved using a 250 x 4.6 mm id, 5 microm particle size Symmetry C18 column. The mobile phase acetonitrile-0.1 M phosphate buffer (35+65, v/v), pH 4.5, was pumped at a flow rate of 1 mL/min, with UV detection at 275 nm. The method showed good linearity in the ranges of 0.5-50 and 0.1-10 microg/mL, with LOD of 0.06 and 0.02 microg/mL and LOQ of 0.20 and 0.08 microg/mL for EPR and HCT, respectively. The proposed method was successfully applied for the analysis of the studied drugs in their synthetic mixture and co-formulated tablets. The method was further extended to the in vitro and in vivo determination of the two drugs in spiked and real human plasma. Interference likely to be encountered from the co-administered drugs was studied.     

A modified HPLC method for the determination of ochratoxin A by fluorescence detection

A high-performance liquid chromatographic method (HPLC) with fluorescent detector is described for the determination of ochratoxin A (OTA). A mobile phase consisting of acetonitrile:water:acetic acid (99:99:2, v/v/v) was used for the resolution of the compound on a C(18) Hypersil column. The retention time for OTA and diflunisal which was used as an internal standard (IS) were 11.7 and 12.8 min, respectively. The method is selective, reliable, reproducable with relative standard deviation (RSD) of 1.70 and linear in the range of 2.5 x 10(-9)-1.5 x 10(-8) M OTA. The limit of detection (LOD) and limit of quantification (LOQ) were 2.5 x 10(-10) M corresponding to 0.1 ng mL(-1) and 8.2 x 10(-10) corresponding to 3.3 ng mL(-1), respectively. Recovery studies were 81.2 +/- 1.9 (SD). The method was applied for analysis of OTA in wheat, corn, red pepper, cheese and wine. The proposed method can be used for the routine analysis of OTA in food and animal feed.     

Quantitative determination of indapamide in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection for the determination of the diuretic indapamide using a muBondapak C18 column is developed. The mobile phase consists of an acetonitrile-water mixture (45:55, 5 mM) in KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1200 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is performed prior to chromatographic analysis to avoid the interferences found in urine matrix. Percentages of recovery are 88.3 +/- 5.6 and 82.9 +/- 7.8 for liquid-liquid and solid-liquid extraction, respectively. The developed method has a linear concentration range from 25 to 315 ng/mL with a reproducibility in terms of relative standard deviation of 4% for a concentration level of 0.5 microgram/mL and a quantitation limit of 1 ng/mL. The method is applied to the determination of indapamide in tablets and urine obtained from hypertensive patients after the ingestion of Tertensif (indapamide 2.5 mg).     

Determination of folic acid in tablets by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of folic acid, a water-soluble vitamin, in a commercial tablet formulation. The analysis was performed using a microemulsion containing 0.5% (w/w) ethyl acetate, 1.2% (w/w) butan-1-ol, 0.6% (w/w) sodium dodecyl sulfate, 15% (v/v) 2-propanol and 82.7% (w/w) 10 mmol L(-1) sodium tetraborate aqueous buffer at pH 9.2. Direct UV detection at 214nm led to an adequate sensitivity without interference from sample excipients. For quantitative purposes, niacin was used as internal standard. Acceptable precision (<1.2% relative standard deviation (R.S.D.)), linearity (r = 0.9992; range from 160.0 to 240.0 microg/mL), sensitivity (limit of detection (LOD) = 2.98 microg/mL; limit of quantification (LOQ) = 9.05 microg/mL) and recovery (99.8 +/- 1.8% at three concentration levels) were obtained. Based on the performance characteristics, the proposed methodology was found suitable for the determination of folic acid in tablet formulations.     

在线固相萃取净化-高效液相色谱法测定人血浆中的霉酚酸

建立了在线固相萃取净化-高效液相色谱法联用(Online SPE-HPLC)测定人血浆中霉酚酸浓度的分析方法。联用系统以Capcell PAK MF Ph-1为净化柱,Poroshell 120?EC-C18为分析柱,血浆样品经甲醇沉淀蛋白后,直接进样分析。结果表明,霉酚酸在0.20~50.00μg/mL范围内线性关系良好,相关系数(r)为0.9998,检出限和定量下限分别为0.07、0.20μg/mL。在0.39、25.00、50.00μg/mL 3个质量浓度下的平均回收率为96.2%~105%,日内和日间相对标准偏差(RSD)分别为2.6%~3.1%和2.9%~3.3%。该法操作简便、快速,可用于人血浆中霉酚酸浓度的分析。     

Method Development and Validation of a Rapid Determination of Ropinirole in Tablets by LC-UV

A reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed and validated for the determination of ropinirole (ROP) in tablets. The assay utilized UV detection at 250 nm and a Luna CN column (250 × 4.6 mm I.D, 5 μm). The mobile phases were comprised of acetonitrile: 10 mM nitric acid (pH 3.0) (75:25, v/v). Validation experiments were performed to demonstrate linearity, accuracy, precision, limit of quantitation (LOQ), limit of detection (LOD), and robustness. The method was linear over the concentration range of 0.5–10.0 μg mL⁻¹. The method showed good recoveries (99.75–100.20%) and the relative standard deviations of intra and inter-day assays were 0.38–1.69 and 0.45–1.95%, respectively. The method can be used for quality control assay of ropinirole.     

Determination of atazanavir in human plasma by high-performance liquid chromatography with UV detection

A high-performance liquid chromatographic method for the determination of atazanavir (ATV) in human plasma is developed and validated. The method involves a rapid and simple solid-phase extraction (SPE) of ATV using Bond-elut C18 3 mL cartridge. The separation of ATV from internal standard and endogenous components is achieved using an isocratic elution on an octyl column and an UV detector set at 260 nm. The method is linear from 20 to 10,000 ng/mL (mean r2 = 0.9991, n = 10). The observed intra- and inter-day assay precision ranged from 2.2% to 14.7%[at the lower limit of quantitation (LOQ)], whereas accuracy varies between 1.0% and 14% (at LOQ). Mean drug recovery is 80.5% for ATV and 78.4% for IS. The method is found to be precise and accurate, practical enough for therapeutic drug monitoring in routine clinical practice and is applied for the assessment of 24-h ATV plasma concentration-time profiles in HIV-infected pregnant women.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.     

Determination of endocrine-disrupting phenols in water samples by a new manual shaking-enhanced, ultrasound-assisted emulsification microextraction method

Manual shaking-enhanced, ultrasound-assisted emulsification microextraction (MS-USAEME) combined with ultraperformance liquid chromatography (UPLC) with UV detection has been developed for the determination of five endocrine-disrupting phenols (EDPs) in seawater samples and detergent samples: 4-tert-butylphenol (4-t-BP), 4-cumylphenol (4-CP), 4-tert-octylphenol (4-t-OP), 2,4-di-tert-butylphenol (2,4-di-t-BP) and 4-nonylphenol (4-NP). Optimum conditions were found to be: 25 μL 1-bromohexadecane as extraction solvent, 5 mL of aqueous sample and 1 g of NaCl to control the ionic strength; manual shaking for 10 s; ultrasonication for 1 min; centrifugation for 3 min at 5000 rpm (speed). For MS-USAEME, manual shaking for 10 s is essential for effective extraction when the ultrasonic extraction time is as brief as 1 min. The small volume of aqueous sample enhances the effect of manual shaking significantly. For seawater samples, the limit of detection (LOD) was 0.5-2.8 ng mL(-1), the limit of quantification (LOQ) was 1.8-9.3 ng mL(-1) with the relative standard deviation (RSD) in the range 4.2-10.3%. For detergent samples, the LOD was 0.4-2.4 ng mL(-1), LOQ was 1.6-8.2 ng mL(-1) and RSD 4.7-10.0%. The relative recovery was 96-109% for seawater samples and 81-106% for the detergent samples.