RP-HPLC method for simultaneous estimation of lamivudine, tenofovir disoproxil fumarate and efavirenz in tablet formulation

A simple, rapid, and precise reversed-phase high-performance liquid chromatographic method for the simultaneous determination of lamivudine, tenofovir disoproxil fumarate and efavirenz in bulk and tablet dosage form has been developed and validated. Chromatography was performed on a 150 mm × 4.6 mm i.d., 5-μm particle, Phenomenex Luna C₁₈ column with 30: 45: 25 (v/v/v) acetonitrile: methanol: water as mobile phase at a flow rate of 0.5 mL/min. UV detection was done at 258 nm; lamivudine, tenofovir disoproxil fumarate and efavirenz were eluted with retention times of 3.27, 4.58 and 10.90 min, respectively. The method was validated in accordance with ICH guidelines. Validation revealed the method is specific, rapid, accurate, precise, reliable and reproducible. Calibration plots were linear over the concentration ranges 1–6 μg/mL for lamivudine and tenofovir disoproxil fumarate and 2–12 μg/mL for efavirenz. Limits of detection were 0.05, 0.09 and 0.11 μg/mL and limits of quantification were 0.15, 0.28 and 0.34 μg/mL for lamivudine, tenofovir disoproxil fumarate and efavirenz, respectively. The high recovery and low coefficients of variation confirm the suitability of the method for the simultaneous determination of these three drugs in bulk and tablets.     

Development and validation of RP-HPLC,HPTLC and UV-visible spectrophotometric methods for simultaneous estimation of alprazolam and propranolol hydrochloride in their combined dosage form

Three accurate, sensitive and reproducible methods are described for the quantitative determination of alprazolam (ALP) and propranolol hydrochloride (PNL) in their combined dosage form. The first method involves an RP-HPLC separation on the C₁₈ column using acetonitrile-25 mM ammonium acetate buffer and 0.2% triethylamine (pH of buffer adjusted to 4 with glacial acetic acid) in the ratio of 35: 65 (v/v) as mobile phase. Symmetrical peaks with good separation, ALP at 9.3 min and PNL at 3.5 min, were achieved. Quantification was done with photo diode array detection at 255 nm over the concentration ranges of 0.5–50 and 10–250 μg/mL for ALP and PNL, respectively. The second method is based on the separation of drugs by HPTLC using chloroform-methanol-ammonia 7: 0.8: 0.1 (v/v/v) as mobile phase. Quantification was achieved using UV detection at 248 nm over the concentration range of 100–600 ng/spot and 5–30 μg/spot for ALP and PNL, respectively. The third method involves dual wavelength UV-visible spectrophotometric method. It is based on the determination of PNL at 319.4 nm using its absorptivity value and ALP at 258.2 nm after deduction of absorbance due to PNL. Quantification was achieved over the concentration range of 1–40 and 80–200 μg/mL for ALP and PNL, respectively. All methods were validated according to ICH guidelines and successively applied to marketed pharmaceutical formulation, and the results of all three methods were compared statistically as well. No interference from the tablet excipients was found.     

A Stability Indicating High Performance Liquid Chromatographic Assay of Isradipine in Pharmaceutical Preparations

Abstract

A high performance liquid chromatographic procedure has been developed for the assay of isradipine in bulk form and tablet and capsule pharmaceutical preparations. The separation is achieved within 20 min on an octadecylsilane column at ambient temperature with a mobile phase of 60:40 v/v methanol - water, a flow rate of 1 mL/min, and detection at 325 nm. Degradation studies showed no peak interference between isradipine and degradation products. It was also determined that the excipients in the commercial tablet and capsule preparations did not interfere with the assay. The method was linear in the range 10–60 μg/mL with accuracy and precision in the 0.40 - 1.53% range.     

Development and validation of a stability-indicating RP-UPLC method for the quantitative analysis of nabumetone in tablet dosage form

High efficiency and less run time are the basic requirements of high-speed chromatographic separations. To fulfill these requirements, a new separation technique, ultra-performance liquid chromatography (UPLC), has shown promising developments. A rapid, specific, sensitive, and precise reverse-phase UPLC method is developed for the determination of nabumetone in tablet dosage form. In this work, a new isocratic chromatographic method is developed. The newly developed method is applicable for assay determination of the active pharmaceutical ingredient. The chromatographic separation is achieved on a Waters Acquity BEH column (100 mm, i.d., 2.1 mm, 1.7 μm) within a short runtime of 2 min using a mobile phase of 5 mM ammonium acetate-acetonitrile (25:75, v/v), at a flow rate of 0.3 mL/min at an ambient temperature. Quantification is achieved with photodiode array detection at 230 nm, over the concentration range of 0.05-26 μg/mL. Forced degradation studies are also performed for nabumetone bulk drug samples to demonstrate the stability-indicating power of the UPLC method. Comparison of system performance with conventional high-performance liquid chromatography is made with respect to analysis time, efficiency, and sensitivity. The method is validated according to the ICH guidelines and is applied successfully for the determination of nabumetone in tablets.     

Simultaneous determination of cefuroxime axetil and ornidazole in tablet dosage form using reversed-phase high performance liquid chromatography

 A simple, accurate and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of cefuroxime axetil and ornidazole in combined tablet dosage form has been developed. The method was performed with a HiQ-SiL C18 column (250 mm×4.6 mm) and photodiode array (PDA) detector, using 0.01 mol/L potassium dihydrogen orthophosphate-methanol (56∶44, v/v) as the mobile phase and tinidazole as the internal standard. Beer’s law obeys in the concentration ranges of 5-25 μg/mL and 10-50 μg/mL for cefuroxime axetil and ornidazole, respectively. The method has been successfully validated statistically and applied for the analysis of the drugs in pharmaceutical formulation.     

Simultaneous determination of four hormonal compounds in oral contraceptive tablet formulations by high performance liquid chromatography

A rapid, accurate, and precise HPLC method has been developed for simultaneous determination of four contraceptive hormonal compounds namely ethinylestradiol (EE), drospirenone (DR), gestodene (GS), and levonorgestrel (LV) in oral contraceptive tablet dosage form. The chromatographic separation was achieved on a C18 (150 × 4.6 mm, 5μ) column; the mobile phase consists of acetonitrile: water (50:50, v/v) pumped at a flow rate of 1.0 mL/min; and UV detection was set at 200 nm. The limit of detection was 0.0086 µg/mL for (EE), 0.0397 µg/mL for (GS), 2.80 µg/mL for (DR), and 0.229 µg/mL for (LV), whereas the limit of quantitation (LOQ) was 0.028 µg/mL for (EE), 0.132 µg/mL for (GS), 9.500 µg/mL for (DR), and 0763 µg/mL for (LV), respectively. The correlation coefficient (r) values of the four compounds ranged from 0.99995 to 0.99999. The method was validated as per ICH guidelines and USP 34 for estimation of (EE), (DR), (GS), and (LV) in commercially available tablet dosage form. The validation results were found satisfactory. The proposed method can be useful in quality control of bulk manufacturing and pharmaceutical dosage forms.     

Stability-indicating RP-HPLC method development and validation for determination of nine impurities in apixaban tablet dosage forms. Robustness study by quality by design approach

A quality by design (QbD) based high-resolution HPLC method is described for determination of impurities in apixaban (APX) in the tablet dosage form. Employing a simple and stability-indicating HPLC method, nine known impurities were quantified with good peak resolution. Mobile phase A (MP-A) was prepared with buffer and acetonitrile 90:10 v/v, while mobile phase B (MP-B) contained water and acetonitrile 10:90 v/v. The gradient program was 0 min, MP-A 75%, B 25%; 20 min, MP-A 65%, B 35%; 30 min, MP-A 40%, B 60%; 40min, MP-A 40%, B 60%; 42 min, MP-A 75%, B 25%; and 50 min, MP-A 75%, B 25%. The chromatographic separation was achieved using a Zorbax RX C₁₈ 250 × 4.6 mm column, 5 μm (1.0 ml min⁻¹, 280 nm, 50 μl) and a column temperature of 40°C. Several separation studies were carried out using design of experiments to optimize the method. Validation results confirm the applicability of the developed method for quality analysis and stability studies of the regular product on the manufacturing stream.     

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.     

Simultaneous determination of gliquidone, pioglitazone hydrochloride, and verapamil in formulation and human serum by RP-HPLC

In the present study, a reverse-phase high performance liquid chromatography method was developed, validated and applied for the simultaneous determination of gliquidone, pioglitazone hydrochloride and verapamil in tablets and human serum. Chromatographic separation was achieved on a C18 column (5 μm, 25 × 0.46 cm) with a mobile phase consisting of methanol-water-acetonitrile (80:10:10 v/v/v) with a flow rate of 0.7 mL/min and pH adjusted to 3.50 with phosphoric acid at 230 nm. Glibenclamide was used as internal standard. The experimentally derived limit of detection and limit of quantitation were determined to be 0.24, 0.93, 0.40, and 0.80, 3.11, 1.36 μg/mL for gliquidone, pioglitazone, and verapamil, respectively. There were no interfering peaks due to the excipients present in the pharmaceutical tablets. Thus, the proposed method is simple and suitable for the simultaneous analysis of active ingredients in dosage forms and human serum.     

Rapid and economic chiral-HPLC method of nebivolol enantiomers resolution in dosage formulation

A fast, economic, reproducible, accurate, effective, rugged and selective chiral-HPLC method was developed and validated for the enantiomeric resolution of nebivolol enantiomers [(+)-RRRS and (-)-SSSR)] in dosage formulation. The method was rapid as chiral separation occurred within only 12 min. The mobile phase used was n-heptane-ethanol-DEA (85:15:0.1, v/v) at 3.0 mL/min flow-rate with 225 nm detection. The column used was an amylase-based 3-AmyCoat (150 × 46 mm) [tris-(3,5-dimethylphenyl carbamate)]. The capacity factors of (+)-RRRS and (-)-SSSR enantiomers were 7.85 and 10.90 while the separation and resolution factors were 1.39 and 1.83, respectively. The limits of detection and quantitation for (+)-RRRS enantiomer were 4.5 and 10.00 μg/mL, while these values for (-)-SSSR enantiomer were 4.1 and 8.2 μg/mL, respectively. The linearity was observed in the concentrations range of 0.10-1.0 mg/mL for both enantiomers. The π-π interactions, hydrogen bonds, dipole-dipole interactions and steric effects control the chiral resolution of nebivolol enantiomers on the reported chiral column. The reported method can be used for the quality control of nebivolol in pharmaceutical preparations with good economy. In addition, this method can also be used for the analysis of (+)-RRRS and (-)-SSSR) enantiomers in biological and environmental samples.     

An improved high-performance liquid chromatographic method for simultaneous determination of tocopherols, tocotrienols and γ-oryzanol in rice

An improved normal phase high performance liquid chromatographic (NP-HPLC) method was developed for simultaneous quantification of eight vitamin E isomers (α-, β-, γ- and δ-tocopherols and α-, β-, γ- and δ-tocotrienols) and γ-oryzanol in rice. A complete separation of all compounds was achieved within 25 min using an Inertsil CN-3, SIL-100A 5 μM (4.6 mm × 250 mm) column and an isocratic elution system of hexane/isopropanol/ethylacetate/acetic acid (97.6:0.8:0.8:0.8, v/v/v/v) at a flow rate varying from 0.7 to 1.5 mL min(-1). A linear correlation coefficient (r(2)>0.99) and high reproducibility were obtained at concentrations ranging 0.05-10 μg mL(-1) for vitamin E isomers and 0.5-500 μg mL(-1) for γ-oryzanol. This method proved to be rapid, accurate and reproducible.     

Determination of aliskiren in human serum quantities by HPLC–tandem mass spectrometry appropriate for pediatric trials

The orally active direct renin inhibitor aliskiren is approved for the treatment of essential hypertension in adults. Analytical methods utilized in clinical studies on efficacy and safety have not been fully described in the literature but need a large sample volume ranging from 200 to 700 μL, rendering them unsuitable particularly for pediatric applications. In the assay presented only 100 μL of serum is needed for mixed‐mode solid‐phase extraction. The chromatographic separation was performed on Xselect^TM C₁₈ CSH columns with mobile phase consisting of methanol–water–formic acid (75:25:0.005, v/v/v) and a flow rate of 0.4 mL/min. Running in positive electrospray ionization and multiple reaction monitoring the mass spectrometer was set to analyze precursor ion 552.2 m/z [M + H]⁺ to product ion 436.2 m/z during a total run time of 5 min. The method covers a linear calibration range of 0.146–1200 ng/mL. Intra‐run and inter‐run precisions were 0.4–7.2 and 0.6–12.9%. Mean recovery was at least 89%. Selectivity, accuracy and stability results comply with current European Medicines Agency and Food and Drug Administration guidelines. This successfully validated LC‐MS/MS method with a wide linear calibration range requiring small serum amounts is suitable for pharmacokinetic investigations of aliskiren in pediatrics, adults and the elderly. Copyright © 2012 John Wiley & Sons, Ltd.     

High performance liquid chromatographic analysis of dehydroepiandrosterone and its pharmaceutical tablet formulation

A rapid, sensitive and stability indicating high performance liquid chromatographic method was developed and validated for the analysis of dehydroepiandrosterone (DHEA) in pharmaceutical tablet formulation. The analysis was done on a Supelcosil C(18) column (25 cm x 4.6 mm i.d., 5 microm). The mobile phase consisted of methanol:sodium acetate buffer solution (5 g/L):acetic acid (500 mL/L), 57:42:1, v/v/v, adjusted to pH 5 at a flow rate of 1 mL/min. Detection was carried out at a wavelength of 258 nm. The polynomial regression data for the calibration curve showed good linear relationship in the concentration range of 0.2-1 mg/mL with r = 0.9996. The method was validated for precision, accuracy and recovery. The limit of detection was found to be 50 ng/ microL. The method was applied for the analysis of DHEA in its pharmaceutical tablet formulation. The effects of different buffers and alcohols on the retention of DHEA were studied and the role of acetic acid as an organic phase modifier was also investigated.     

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).     

超高效合相色谱法快速测定保健食品中10种脂溶性维生素

脂溶性维生素作为保健食品重要的功效指标,现有的标准方法存在测定组分少、样品前处理过程复杂、对人员操作能力要求较高等问题,因此建立一种快速、简便、准确,且能同时检测多种常见脂溶性维生素的方法具有重要的现实意义。该研究采用超高效合相色谱(UPC²)建立了同时测定保健食品中维生素A棕榈酸酯、维生素A醋酸酯、维生素E醋酸酯、维生素K₁、α-生育酚、β-生育酚、γ-生育酚、δ-生育酚、维生素D₂、维生素D₃等10种常用脂溶性维生素的方法。样品经含75%二甲基亚砜(DMSO)的水溶液在45 ℃水浴超声15 min破乳,再加入正己烷振摇提取90 min, 3000 r/min离心10 min,取上清液过滤后进行分析。使用LC-Simulator软件对色谱条件进行模拟及优化,选用ACQUITY UPC² Viridis HSS C18 SB色谱柱进行分离,CO₂和乙腈-甲醇(85∶15, v/v)为流动相,梯度洗脱,流速1.9 mL/min,柱温30 ℃,选取10种脂溶性维生素各自的最大吸收波长检测,外标法进行定量。结果表明,10种脂溶性维生素在各自范围内线性关系良好,相关系数(r)均大于0.9997,检出限(LOD)和定量限(LOQ)分别为片剂:0.2~30 μg/g和0.8~75 μg/g,胶囊:0.4~60 μg/g和2~150 μg/g,样品平均加标回收率在96.5%~113.9%之间,RSD均小于4%,精密度、稳定性、重复性测定结果的RSD值也均小于2%。经比较,该方法测定结果与现有的国家食品安全标准基本一致,但该方法简单、快速、灵敏、准确,可满足保健食品中10种脂溶性维生素的检测要求,为保健食品中脂溶性维生素的快速同时检测奠定基础。     

基质固相分散-高效液相色谱法测定鲫鱼肌肉中残留的辛硫磷

建立了鲫鱼肌肉中残留的辛硫磷的基质固相分散-高效液相色谱-二极管阵列检测(MSPD-HPLC-DAD)的分析方法。通过优化样品处理条件,确定选取0.50 g鲫鱼肌肉样品与1.5 g弗罗里硅土、0.5 g无水硫酸钠混合研磨,并采用丙酮-正己烷溶液(体积比为40:60)为洗脱剂,洗脱剂用量为25 mL。优选的最佳色谱条件为：ODS色谱柱(250 mm×4.6 mm,5 μm),流动相为甲醇-水(体积比为50:50),流速0.6 mL/min,检测波长270 nm,进样量为20 μL。在上述条件下,辛硫磷质量浓度在0.01～10 mg/L范围内与响应信号呈良好的线性关系(r20.9994),检出限为3.3 μg/kg;相对标准偏差为1.1%～6.3%(n7);3个添加水平(0.05,0.1,1 mg/kg)下得到的回收率为88%～112%。该方法操作简单,耗时少,精密度高,符合农残分析的要求。     

Optimization of dispersive liquid–liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC‐UV

A simple, rapid, and sensitive method based on dispersive liquid–liquid microextraction combined with HPLC‐UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 μL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min^?1, neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C₁₈ column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min^?1. The linear response (r² > 0.998) was achieved in the range of 0.005–10 μg mL^?1 with detection limit 0.0005 μg mL^?1. The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3–102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid–liquid microextraction combined with HPLC‐UV is a fast and simple method for the determination of chlordiazepoxide in real samples.     

High Performance Liquid Chromatographic Determination of Pirenzepine Dihydrochloride in Its Pharmaceutical Formulation

Abstract

A high-performance liquid chromatographic procedure for the determination of pirenzepine dihydrochloride as a bulk material and in its tablet dosage form (Gastrozepin^R) is presented. Normal phase liquid chromatography has been performed on a Micro-pack Si-10 column using ammonium hydroxide (28–30% NH₃) in methanol (0.75: 99.25% v/v) as mobile phase at a flow rate of 2 ml/min. Clobazam has been used as internal standard with retention times of 1.9 and 2.8 minutes for clobazam and pirenzepine dihydrochloride, respectively at 254 nm. Analytical calibration yields a linear relationship between 5 and 25 μg/ml, with correlation coefficient of 0.999. Tablets each labelled to contain 25 mg pirenzepine dihydrochloride give mean percentage found of 99.98 ± 0.4. A plot of logarithm of concentration against time for a solution in 6 N hydrochloric acid gives a straight line with a slope of - 0.197 day^?1. The proposed method is, therefore, a stability indicating method.     

Stability indicating method for famotidine in pharmaceuticals using porous graphitic carbon column

A simple, sensitive and rapid HPLC method was developed and validated for the simultaneous determination of famotidine (FMT) and related impurities in pharmaceuticals. Chromatographic separation was accomplished within 10 min on a porous graphitic carbon (PGC) column using 50:50 v/v ACN-water containing 0.5% pentane sulphonic acid (PSA) as the mobile phase. Separation was achieved with a flow rate of 1 mL/min and a detection wavelength of 265 nm. The calibration curves were linear over a concentration range of 1.5-100 microg/mL. The intra- and interday RSDs (n = 5) for the retention times and peak area were all less than 2%. The method was sensitive with an LOD (S/N = 3) of 0.1 microg/mL for FMT, imp. C and 0.05 microg/mL for imp. 2, A and D. All recoveries were greater than 98%. The method was demonstrated to be precise, accurate and specific with no interference from the tablet ingredients and separation of the drug peak from the peaks of the degradation products (oxidative degradation and acid and base degradation). The results indicated that the proposed method could be used for the determination of FMT in commercial dosage forms and as a stability-indicating assay.     

Analytical Quality by Design (AQbD)-oriented RP-UFLC method for quantification of lansoprazole with superior method robustness

A reversed-phase ultrafast liquid chromatography method was developed for quantification of lansoprazole in pharmaceutical dosage form using analytical quality by design approach. Systematic planning and experimentation using design of experiment approach were used for method development and optimization studies. A central composite design was used for optimizing the chromatography, by choosing organic phase proportion and flow rate as the critical method variables for evaluating their effect on critical analytical attributes like resolution, plate number, and tailing factor. The optimal chromatography was accomplished on a C-18 column (250?×?4.6?mm, 5?µm) using methanol:water (70:30, v/v) as mobile phase at a flow rate of 1.0?mL/min. Photo diode array (PDA) detection was performed at 284?nm. Caffeine was used as the internal standard. Method validation studies revealed that the calibration curve was linear over 1.0–300?µg/mL. The method was found accurate with average recovery between 98.99 and 102.87%. The percent relative standard deviation values obtained for precision were as per ICH guideline and within the acceptance limits (<2%). Results of system suitability indicated superior method robustness. In a nutshell, the method was found to be highly suitable for its applicability in the determination of lansoprazole in bulk and tablet dosage form.