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.     

Determination of pK a Values of Cefdinir and Cefixime by LC and Spectrophotometric Methods and Their Analysis in Pharmaceutical Dosage Forms

The pK _a values of cefdinir and cefixime, which are used in the treatment of bacterial infections, have been determined precisely in water and methanol?Cwater binary mixtures (20% v/v) using spectrophotometric titration and LC, respectively. A simple, fast and precise isocratic high-performance liquid chromatographic (LC) procedure has been developed for the determination of cefdinir and cefixime in drug formulations. This method was validated successfully for specificity, precision, linearity, range, accuracy, limit of detection, and limit of quantitation as per the ICH guidelines. The proposed method can be used for routine analysis of studied cephalosporin compounds and as an alternative tool for drug quality control laboratories.     

Development and validation of a reversed-phase column liquid chromatographic method for the determination of five cephalosporins in pharmaceutical preparations

A new, simple, rapid, and precise RP-HPLC method has been developed and validated for the determination of five cephalosporins, namely, cefalexin, cefoperazone, ceftriaxone, ceftazidime, and cefepime. The method has been applied successfully for simultaneous determination of cefalexin in a binary mixture with sodium benzoate in a suspension, and cefoperazone in a binary mixture with sulbactam in vials. Chromatographic separation was achieved on a Waters microBondapak C18 column (250 x 4.6 mm id, 10 pm particle size) using the mobile phase monobasic potassium phosphate (50 mM, pH 4.6)-acetonitrile (80 + 20, v/v) with UV detection. A flow rate of 1 mL/min was applied. Linearity, accuracy, and precision were found to be acceptable over the concentration range of 30-300, 3-30, and 15-120 microg/mL for the studied cephalosporins, sodium benzoate, and sulbactam, respectively. The optimized method proved to be specific, robust, and accurate for QC of the cited drugs in their pharmaceutical preparations.     

反相高效液相色谱法同时测定片剂中的加替沙星与盐酸氨溴索

 A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm×4.6 mm, 5 μm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70∶30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10-60 μg/mL and that for AM was found to be 5-30 μg/mL. The correlation coefficients were 0.9996 and 0.9993 for GA and AM respectively. The results of the tablet analysis (n=5) were found to be 99.94% with ±0.25% standard deviation (SD) and 99.98% with±0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92%-100.02% and that of AM was 99.86%-100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.     

Determination of aflatoxin in processed dried cassava root: validation of a new analytical method for cassava flour

A new method that uses HPLC with a photochemical reactor for enhanced detection was developed and validated for the determination of aflatoxins in cassava flour. Samples were spiked with a mixture of four aflatoxins at 5, 10, and 20 microg/kg mixed with either 1 or 5 g NaCI and extracted with methanol-water (80 + 20, v/v) by shaking for 10 or 30 min. An immunoaffinity column was used for cleanup. HPLC with postcolumn derivatization, for enhancement of aflatoxin fluorescence, and fluorescence determination were used for quantitation of the toxin concentration. The method was validated for recovery, linearity, and precision at the three concentrations tested. Recovery ranges were 52-70, 69-85, and 80-89% for the spiking levels of 5.0, 10.0, and 20.0 microg/kg, respectively. It appears that the amount of salt (NaCl) and the shaking time are critical factors in this method; optimal performance was obtained when 1 g salt was used and the shaking time was 10 min. The good linearity and precision of the method allowed baseline separation from interferences, e.g., coumarins.     

Simultaneous determination of chlordiazepoxide and selected antidepressants using CZE

A simple CE method was developed and validated for the simultaneous determination of chlordiazepoxide (CHL), amitriptyline, and nortriptyline (mixture I) or the determination of CHL and imipramine (mixture II) using the same BGE. Sertraline and amitriptyline were used as internal standards for the first and second mixtures, respectively. The method allows amitriptyline to be completely separated from its impurity and main metabolite nortriptyline, which can be quantified from 0.2 μg/mL. The separation was achieved using 20 mM potassium phosphate buffer pH 5 containing 12 mM β‐cyclodextrin and 1 mM carboxymethyl‐β‐cyclodextrin. UV detection was performed at 200 nm and a voltage of 15 kV was applied on an uncoated fused‐silica capillary at 25°C. These experimental conditions allowed separation of the compounds to be obtained in 7 min. Calibration graphs proved the linearity up to 40 μg/mL for CHL, up to 100 μg/mL for amitriptyline and imipramine, and up to 5 μg/mL for nortriptyline. The accuracy and precision of the method have been determined by analyzing synthetic mixtures and pharmaceutical formulations. The analytical results were quite good in all cases indicating that the method was linear, sensitive, precise, accurate, and selective for both mixtures.     

Determination of fenthion and oxidation products in personal protection equipment by gas chromatography

A method for the co-extraction and simultaneous chromatographic determination of fenthion and its five oxidation products (or metabolites) fenoxon, fenoxon-sulfoxide, fenoxon-sulfone, fenthion-sulfoxide, and fenthion-sulfone in personal protection equipment (PPE) of pesticide applicators was developed and validated. Capillary gas chromatography-nitrogen-phosphorus detection was used for the analytical determination of all the aforementioned compounds over the concentration range of 0.1-0.5 microg/mL. All necessary validation criteria of the method were met. The method was found to be highly selective, accurate, and precise, gave satisfactory recovery (>70%) and RSD values (<20%) for fenthion and its metabolites in all tested specimens of personal protection equipment and in air samplers. The limit of quantification (LOQ) and linearity were determined for the most relevant PPE (i.e. inner coverall) for the parent compound and for the metabolites. The LOQ values ranged from 0.05 to 0.1 ppb while the linearity in the tested range of 0.1-0.5 ppm had r(2)>0.994 for all analytes.     

Simultaneous densitometric determination of anthelmintic drug albendazole and its metabolite albendazole sulfoxide by HPTLC in human plasma and pharmaceutical formulations

A new, simple, accurate and precise high‐performance thin‐layer chromatographic method has been developed and validated for simultaneous determination of an anthelmintic drug, albendazole, and its active metabolite albendazole, sulfoxide. Planar chromatographic separation was performed on aluminum‐backed layer of silica gel 60G F₂₅₄ using a mixture of toluene–acetonitrile–glacial acetic acid (7.0:2.9:0.1, v /v/v) as the mobile phase. For quantitation, the separated spots were scanned densitometrically at 225 nm. The retention factors (R _f) obtained under the established conditions were 0.76 ± 0.01 and 0.50 ± 0.01 and the regression plots were linear (r ² ≥ 0.9997) in the concentration ranges 50–350 and 100–700 ng/band for albendazole and albendazole sulfoxide, respectively. The method was validated for linearity, specificity, accuracy (recovery) and precision, repeatability, stability and robustness. The limit of detection and limit of quantitation found were 9.84 and 29.81 ng/band for albendazole and 21.60 and 65.45 ng/band for albendazole sulfoxide, respectively. For plasma samples, solid‐phase extraction of analytes yielded mean extraction recoveries of 87.59 and 87.13% for albendazole and albendazole sulfoxide, respectively. The method was successfully applied for the analysis of albendazole in pharmaceutical formulations with accuracy ≥99.32%.     

Development and validation of a high-performance liquid chromatographic method for determination of eprosartan in bulk drug and tablets

A simple, precise, and accurate isocratic RP-HPLC method was developed and validated for determination of eprosartan in bulk drug and tablets. Isocratic RP-HPLC separation was achieved on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using the mobile phase 0.5% formic acid-methanol-acetonitrile (80 + 25 + 20, v/v/v, pH 2.80) at a flow rate of 1.0 mL/min. The retention time of eprosartan was 7.64 +/- 0.05 min. The detection was performed at 232 nm. The method was validated for linearity, precision, accuracy, robustness, solution stability, and specificity. The method was linear in the concentration range of 10-400 microg/mL with a correlation coefficient of 0.9999. The repeatability for six samples was 0.253% RSD; the intraday and interday precision were 0.21-0.57 and 0.33-0.71% RSD, respectively. The accuracy (recovery) was found to be in the range of 99.86-100.92%. The drug was subjected to the stress conditions hydrolysis, oxidation, photolysis, and heat. Degradation products produced as a result of the stress conditions did not interfere with detection of eprosartan; therefore, the proposed method can be considered stability-indicating.     

Single‐run capillary electrophoresis method for the fast simultaneous determination of amoxicillin,clavulanate, and potassium

We report a new fast method for the simultaneous determination of amoxicillin, clavulanate, and potassium by capillary electrophoresis with capacitively coupled contactless conductivity detection. Samples containing potassium as the cation, and both amoxicillin and clavulanate as anions were determined simultaneously in a single run (in less than 45 s) using 10 mmol/L of both 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol and 3‐{[2‐hydroxy‐1,1‐bis(hydroxymethyl)ethyl]amino}‐1‐propanesulfonic acid (pH 8.4) as the background electrolyte. Limits of detection were 25.0, 5.0, and 4.0 μmol/L for amoxicillin, clavulanate, and potassium, respectively. The proposed method is inexpensive, simple, fast (75 injections h⁻¹), environment friendly (minimal waste generation), and accurate (recovery values between 98 and 103%). The results obtained with the proposed method were statistically similar (95% confidence level) to those obtained by using high‐performance liquid chromatography (amoxicillin and clavulanate) and flame photometry (potassium).     

Direct determination of S-(-)- and R-(+)-propranolol glucuronide in rat hepatic microsomes by RP-HPLC

Propranolol, available commercially as a racemic mixture, is a non-selective beta-adrenergic blocking agent used in the treatment of hypertension, angina pectoris and cardiac arrhythmias. We have developed and validated an RP-HPLC assay method for direct determination of R-(+)- and S-(-)-propranolol glucuronide in rat hepatic microsomes to investigate the enantioselectivity of propranolol glucuronidation metabolism. A baseline separation of propranolol glucuronide enantiomers was achieved on a 5 microm reversed-phase ODS column, with a mixture of phosphate buffer (pH 3.5, 0.067 mol/L) and methanol (55:45, v/v) as mobile phase. Ultraviolet detection was set at 220 nm, and p-nitrobenzoic acid was used as internal standard. The standard curve of assay for R-(+)- and S-(-)-propranolol glucuronide in spiked microsomal incubate showed good linearity throughout the concentration range from 0.50 to 20.0 micromol/L. The analytical method affords average recovery of 99.8 and 100.1% for R-(+)- and S-(-)-propranolol glucuronide, respectively. The method provides a high sensitivity and good precision for R-(+)- and S-(-)-propranolol glucuronide (RSD < 10%). The LOD was 0.15 micromol/L and the LOQ was 0.5 micromol/L (RSD < 8%, n = 5) for both R-(+)- and S-(-)-propranolol glucuronide. The method is simple, precise and accurate, and is suitable for quantifying the propranolol glucuronides enantiomers in rat hepatic microsomes.     

A validated enantioselective HPLC assay of dexibuprofen in dexibuprofen tablet formulations

A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed and validated for the quantitation of dexibuprofen in dexibuprofen tablets using ovomucoid chiral stationary phase (Ultron ES-OVM). The mobile phasewas composed of 0.025 M potassium phosphate dibasic (pH 4.5)-methanol-ethanol (85:10:5 v/v/v). The method was validated for specificity, linearity, range, accuracy, precision and robustness. The method was enantiomerspecific for the determination of dexibuprofen [S-(+)-isomer ibuprofen] in the presence of R-(-)-isomer ibuprofen in bulk drug, pharmaceutical dosage form and under stress degradation. The method was linear over the range 15-35 mg/mL with r2 = 0.9995; accuracy and precision were acceptable with %RSD < 2.0%. The method was found to be specific, precise, accurate, robust and stability-indicating, and can be successfully applied for the routine analysis of dexibuprofen in bulk drug and pharmaceutical dosage form.     

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.     

Spectrophotometric, difference spectroscopic, and high-performance liquid chromatographic methods for the determination of cefixime in pharmaceutical formulations

Three simple and sensitive spectrophotometric, difference spectroscopic, and liquid chromatographic (LC) methods are described for the determination of cefixime. The first method is based on the oxidative coupling reaction of cefixime with 3-methyl-2-benzothiazolinon hydrazone HCI in presence of ferric chloride. The absorbance of reaction product was measured at the maximum absorbance wavelength (wavelength(max)), 630 nm. The difference spectroscopic method is based on the measurement of absorbance of cefixime at the absorbance maximum, 268 nm, and minimum, 237 nm. The measured value was the amplitude of maxima and minima between 2 equimolar solutions of the analyte in different chemical forms, which exhibited different spectral characteristics. The conditions were optimized, and Beer's law was obeyed for cefixime at 1 to 16 microg/mL and 10 to 50 microg/mL, respectively. The third method, high-performance LC, was developed for the determination of cefixime using 50 mM potassium dihydrogen phosphate (pH 3.0)-methanol (78 + 22, v/v) as the mobile phase and measuring the response at wavelength(max) 286 nm. The analysis was performed on a Lichrospher RPC18 column. The calibration curve was obtained for cefixime at 5 to 250 microg/mL, and the mean recovery was 99.71 +/- 0.01%. The methods were validated according to the guidelines of the U.S. Pharmacopoeia and also assessed by applying the standard addition technique. The results obtained in the analysis of dosage forms agreed well with the contents stated on the labels.     

高效液相色谱法同时检测乳制品中3种β-内酰胺酶抑制剂的残留量

建立了乳制品中3种β-内酰胺酶抑制剂克拉维酸钾、他唑巴坦和舒巴坦的提取和固相萃取净化方法。样品经水溶后,用丙酮沉淀蛋白质。上清液在弱酸性条件下用PWAX固相萃取小柱(60 mg/3 mL)富集、净化,0.05%(如无特殊说明均为体积分数)氨水-甲醇溶液洗脱。然后采用高效液相色谱-二极管阵列检测器(HPLC-PAD)分离检测。分析柱为资生堂CAPCELL PAK MG-C18(150 mm×4.6 mm,5μm);流动相为0.03%磷酸-乙腈。该方法对克拉维酸钾、他唑巴坦和舒巴坦的最低检测质量浓度均为0.03 mg/L;纯牛奶、酸奶和奶粉中的回收率在84.6%~101.7%之间,RSD在2.2%~7.4%之间(n=6);在0.05~5 mg/L范围内均呈良好的线性关系,线性回归系数r>0.999。该方法的前处理净化效果好、检测灵敏度高、回收率和重现性良好,适用于乳制品中β-内酰胺酶抑制剂的测定。     

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.     

Improving the Determination of Nitrovin in Feeds by Reversed-Phase LC with SPE

A simple reversed-phase liquid chromatographic method with ultraviolet detector (378 nm) for the determination of nitrovin in feeds was improved and validated. The mobile phase was a mixture of acetonitrile and 0.1% formic acid solution (v/v) in the ratio of 50:50 (v/v), and the flow rate was set at 1.2 mL min^?1. The extraction solution was a mixture of dimethyl formamide, acetonitrile and methanol (50:25:25, v/v), the sample was cleaned-up with reversed-phase solid phase extraction cartridge. The standard nitrovin was purified with crude nitrovin product by ethylene glycol monoethyl ether and identified by elemental analyzer. The limit of detection was 0.05 mg kg^?1 and the limit of quatification was 0.2 mg kg^?1 in feeds. The assay had satisfactory selectivity, recovery, linearity and precise repeatability and trueness.     

Development and validation of an analytical method for metformin hydrochloride and its related compound (1-cyanoguanidine) in tablet formulations by HPLC-UV

A simple, and stability-indicating liquid chromatographic method was developed and validated for the analysis of metformin hydrochloride and its related compound (1-cyanoguanidine) in tablet formulations. Liquid chromatography with a UV detector at a wavelength of 232 nm using a Nova-Pak silica column was employed in this study. Isocratic elution was employed using a mixture of ammonium dihydrogen phosphate buffer and methanol (21:79, v/v). This new method was validated in accordance with USP requirements for new methods for assay determination, which include accuracy, precision, specificity, linearity and range. The current method demonstrates good linearity over the range of 0.01-0.03 mg mL⁻¹ of metformin hydrochloride. The accuracy of the method is 100.4%. The precision of this method reflected by relative standard deviation of replicates is 0.30%. Validation of the same method for 1-cyanoguanidine determination was also performed according to USP requirements for quantitative determination of impurities which include accuracy, precision, linearity and range, selectivity, and limit of quantification (LOQ). Low LOQ of 1-cyanoguanidine using this method enables the detection and quantification of this impurity at low concentration.     

Simultaneous quantification of brexpiprazole and sertraline HCl in synthetic mixture by thin-layer chromatography method

According to the International Council for Harmonisation (ICH) Q2 (R1) guideline, a sensitive, precise, accurate and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the simultaneous quantification of a newer combination of brexpiprazole (BREX) and sertraline HCl (SERT) in bulk and synthetic mixture. Stationary phase selected was pre-coated silica gel aluminum plate 60 F_254, and n-propanol‒hexane‒toluene‒triethylamine (7:2:1:0.1, V/V) was used as developing mobile phase. An appreciable absorbance shows at 254 nm, therefore the common detection wavelength was selected for the simultaneous quantification of BREX and SERT. The method was validated for different parameters: linearity, precision, accuracy, robustness, limit of detection and limit of quantification as per ICH guideline. The correlation coefficients (r²) for BREX and SERT were found to be 0.9940 and 0.9911, respectively. The mean of percentage recoveries for BREX and SERT were found to be 99.40–102.10% and 99.52–101.05%, respectively. The proposed HPTLC method has potential application for the quantification of BREX and SERT simultaneously in bulk and synthetic mixture both qualitatively and quantitatively.

     

Simultaneous Determination of Losartan Potassium, Atenolol and Hydrochlorothiazide in Pharmaceutical Preparations by Stability-Indicating UPLC

A stability-indicating UPLC method was developed for the simultaneous quantitative determination of losartan potassium, atenolol, and hydrochlorothiazide in pharmaceutical dosage forms in the presence of degradation products. The separation was achieved on a simple isocratic method (water: acetonitrile: triethyl amine: ortho phosphoric acid (60:40:0.1:0.1, v/v) at 0.7 mL min^?1, a detection wavelength of 225 nm). The retention times of losartan potassium, atenolol, and hydrochlorothiazide were 2.3, 0.6 and 0.9 min. The total runtime was 3 min. Losartan potassium, atenolol, and hydrochlorothiazide were subjected to different ICH prescribed stress conditions. The method was validated with respect to linearity, accuracy, precision, robustness and ruggedness.