Isocratic Method Research Articles

Development and Validation of an RP-HPLC-PDA Method for Determination of Paracetamol, Caffeine and Tramadol Hydrochloride in Pharmaceutical Formulations.

Paracetamol (acetaminophen) (PAR), caffeine (CAF) and tramadol hydrochloride (TRA) are important drugs widely used for many clinical purposes. Determination of their contents is of the paramount interest. In this respect, a quick, simple and sensitive isocratic RP-HPLC method with photodiode array detection was developed for the determination of paracetamol, caffeine and tramadol in pharmaceutical formulations. An improved sensitive procedure was also evolved for tramadol using a fluorescence detector system. A C18 column and a mobile phase constituted by methanol/phosphate were used. LODs were found to be 0.2 μg/mL, 0.1 μg/mL and 0.3 μg/mL for paracetamol, caffeine and tramadol hydrochloride, respectively, using photodiode-array detection. Alternatively, LOD for tramadol decreased to 0.1 μg/mL with the fluorescence detector. Other notable analytical figures of merit include the linear concentration ranges, 0.8–270 μg/mL, 0.4–250 μg/mL and 1.0–300 (0.2–40) μg/mL, for the same ordered analytes (including the fluorescence detector). The proposed method was successfully applied for the quantitative determination of the three drugs in tablet dosage forms.

Applying eco‐friendly micellar liquid chromatography for the simultaneous determination of two ternary mixtures utilized for cold treatment using monolithic column

AbstractRapid, isocratic, and sensitive micellar liquid chromatographic methods were developed successfully for the analysis of two ternary mixtures: phenylephrine hydrochloride (PHE), ibuprofen (IBU) and chlorpheniramine maleate (CP) (mixture I), and pseudoephedrine hydrochloride (PSE), IBU and CP (mixture II) in their synthetic mixtures and pharmaceutical dosage forms. Analytical separation was achieved in 7 min using monolithic column adopting UV detector at 233 and 218 nm for mixture I and II, respectively. For both mixtures, a micellar mobile phase solution composed of 0.15 M sodium dodecyl sulphate, 15% n‐propanol, and 0.3% triethylamine adjusted to pH 4.0 by 0.02 M phosphoric acid was utilized applying a flow rate of 1.2 ml/min. The methods were fully validated in accordance with the International Conference on Harmonization guidelines. The proposed methods were linear in the range of 0.5–20.0 μg/ml for PHE and 10.0–400.0 μg/ml for IBU, and CP 0.25–8.0 μg/ml (mixture I) and in the range of 3.0–60.0 μg/ml for PSE and 20.0–400.0 μg/ml for IBU, and CP 0.2–4.0 μg/ml (mixture II) and methanol was utilized as diluting solvent. Furthermore, the proposed methods exhibited good accuracy and repeatability (R.S.D. < 2.0%). The limits of detection were 0.07 (PHE), 1.85 (IBU), 0.04 (CP) for mixture I and were 0.41 (PSE), 2.37 (IBU), 0.02 (CP) for mixture II. The limits of quantitation were 0.22 (PHE), 5.61 (IBU), 0.12 (CP) for mixture I and were 1.24 (PSE), 7.17 (IBU), 0.07 (CP) for mixture II, respectively. The new chromatographic method could successfully substitute traditional hazardous liquid chromatographic methods for the separation of the provided polar compounds consuming lower percent of environmentally toxic organic solvents.

METHOD DEVELOPMENT AND VALIDATION OF IXAZOMIB DRUG BY RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORM

A novel approach was used for develop and validate a rapid, accurate, and an isocratic RP-HPLC method with PDA detector for the estimation of ixazomib drug in pharmaceutical dosage forms. Ixazomib was seperated using Agilent 4.6*150 mm, 5μm analytical column, a Waters HPLC system (USA) and a mobile phase consisting of water and acetonitrile in the ratio of 40:60 V/V. The flow rate was set to 0.7 mL/min with 10µL injection volume. The column was maintained at ambient temperature, detector was set at wavelength of 274 nm. The retention time of ixazomib was found to be 2.17 min. The system suitability parameters for ixazomib such as theoretical plates and tailing factor were found to be 4146. Linearity was established for ixazomib such as theoretical plates and telling factor were found to be 4146. Linearilty was established for ixazomib in the range of 50-250 µg/ml concentration levels with correlation coefficients (r2) of 0.999. The intra-and inter-day precision % RSD values were found to be 0.47 and 0.31, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.03 and 6.17 µg/mL respectively. The method was validated for all of the above parameters according to the International Conference on Harmonization (ICH) guidelines. This method can be used for estimation and analysis of ixazomib drug in active pharmaceutical ingredients and pharmaceuticals.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION METHOD FOR TRANSDERMAL DIFFUSION STUDY OF GRANISETRON

Objective: The study is aimed to develop a simple, isocratic and sensitive reversed-phase high-performance liquid chromatography method for the analysis of granisetron during transdermal permeation studies through porcine ear epithelium.
 Methods: A reversed-phase (C18) column was used with ultraviolet detection at 210 nm. Selected mobile phase contained 25% (v/v) of acetonitrile and 75% (v/v) of 0.25 mM potassium dihydrogen orthophosphate solution pH adjusted to 3.0 using 1% orthophosphoric acid solution.
 Results: Calibration curve showed good linearity over 0.1–30 μg/mL concentration range of porcine ear permeates in 80% ethanol and 20% water (blank permeates). The applicability of the method was demonstrated by the analysis of diffusion study samples of granisetron through porcine ear epithelium and the steady-state permeability flux (J) from ethanolic solution was found to be 4.707 µg/square cm/h.
 Conclusion: The developed method can be used in the analysis of diffusion study samples of granisetron transdermal formulations through the porcine ear epithelium.

Analysis of Organic Impurities of Besifloxacin Hydrochloride by High-Performance Liquid Chromatography with Isocratic and Gradient Elution

Introduction: Impurity analysis is an important step in the quality control of pharmaceutical ingredients and the final product. Impurities can arise from drug synthesis or excipients and even at small concentrations may affect product efficacy and safety. In this work, two methods using highperformance liquid chromatography (HPLC) were developed and validated for the evaluation of besifloxacin and its impurity synthesis, with isocratic elution and another with gradient elution. Method: The analysis by HPLC in isocratic elution mode was performed using a cyano column maintained at 25 °C. The mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 mL/min with detection at 330 nm. The gradient elution method was carried out with the same column and mobile phase components only modifying the rate between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, observing results within acceptable limits. Results: The methods presented were found to be linear in the 140 to 260 μg/ mL range for besifloxacin and 0.3 to 2.3 μg/ mL for an impurity named A. The limits of detection and quantification were, respectively, 0.07 and 0.3 μg/ mL for impurity A, with a 20 μL injection volume. The precision achieved for all analyses performed provided RSD inter-day equal to 6.47 and 6.36% for impurity A with isocratic elution and gradient, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method, an analysis time of 25 min and 15 min was obtained for the gradient. For impurity A, the number of theoretical plates in the isocratic mode was about 5000 while in the gradient mode it was about 45000, hence, it made the column more efficient by changing the mobile phase composition during elution. In besifloxacin raw material and in the pharmaceutical product used in this study, other related impurities were present but impurity A was searched for and not detected. Conclusion: The proposed methods can be applied for the quantitative determination of impurities in the analysis of the besifloxacin raw material, as well as in ophthalmic suspension of the drug, considering the quantitation limit.

A Validated high Performance Liquid Chromatographic Method for the Quantification of Favipiravir by PDA Detector

Favipiravir is an antiviral agent showing activity for the treatment of various life threatening viruses such as Ebola virus, Lassa virus and also recent virus for COVID-19. It is a pyrazine carboxamide derivative with activity against RNA viruses which targets RNA-dependent RNA polymerase enzymes which are necessary for the transcription and replication of viral genomes. The lack of research work and no compendial methods available for the estimation of this drug influenced the current research investigation to give a simple, sensitive, rapid, precise, accurate and robust isocratic high performance liquid chromatographic and UV Spectroscopic method for the determination and quantification of Favipiravir. The elution was done by using SHIMADZU Prominence-i, LC-2030 C system equipped with Shim-Pack GIST C18 (250X 4.6 mm, 5μm) column with a mobile phase mixture of 10 mM potassium dihydrogen ortho phosphate buffer (pH 4.0) and acetonitrile in the ratio of 90:10 v/v at a flow rate of 1.0 ml/min. The ultraviolet detection was done at the wavelength of 315 nm by maintaining column temperature at 30°. The total run time was 8.0 min. Calibration plot showed best regression over the concentration range of 10-60 μg/ml of Favipiravir standard solutions. The LOD and LOQ was found to be 0.18 μg/ml and 0.53 μg/ml, respectively. The accuracy of the proposed method was determined by performing recovery studies and was found to be between 99.47-100.80%. The repeatability testing for both sample and standard solutions was found as %RSD<2.0% which is within the acceptable limits showing that the method is precise as well. The proposed method was successfully applied for the marketed formulations of Favipiravir tablets. In addition the main features of the proposed method are economic and eco-friendly with less retention time around 4.622 min.

Simultaneous Achiral/Chiral HPLC Separation of Ketoprofen, Ibuprofen, Flurbiprofen, and Naproxen

An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, Ibuprofen, and Flurbiprofen) and three of the four pairs of enantiomers in one run is reported. The NSAID samples were prepared with 1 mg/mL of Ketoprofen, Naproxen, Ibuprofen and Flurbiprofen, and 0.5 mg/mL thiourea as a t0 marker in a sample solvent consisting of 1: 1 acetonitrile and 0.2% formic acid. The separation of the enantiomers of the NSAIDs was investigated using a chiral column in two different lengths (5-cm, 10-cm) packed with Amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel and using a tandem column that consists of a biphenyl column connected by a zero dead volume connector to a 5-cm chiral column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel. Both isocratic and step-gradient methods were investigated, and the results of the different methods were compared to determine the benefits and drawbacks of each approach, with an emphasis on the resolution and analysis time.

Development and Validation of RP-UPLC Method for 2,6-Dimethylaniline, Its Isomers, and Related Compounds Using Design of Experiments

2,6-Dimethlyaniline (2,6-DMA) is a key starting material which is used in the synthesis of many classes of drugs, such as anesthetics drugs Lidocaine (Xylocaine®), Bupivacaine, Mepivacaine, Etidocaine, Ropivacaine, Pyrrocaine, Xylazine and anti-anginal drug like Ranolazine and anti-diarrheal drug like Lidamidine. 2,6-DMA together with its five positional isomers and related compounds were separated using a simple isocratic and reverse-phase ultra-performance liquid chromatographic (UPLC) method within a shorter runtime. The developed UPLC method was capable to detect and quantify the impurities at lower levels, i.e., Limit of Detection (LOD) 0.007 µg mL−1 and Limit of Quantification (LOQ) 0.02 µg mL−1. The chromatographic separation of the impurities was successfully accomplished on Acquity UPLC CSH Phenyl hexyl (100 mm × 2.1 mm × 1.7 µm) column at a flow rate of 0.3 mL min−1 and signal detection of 210 nm at a sampling rate of 10 points second−1. The column oven temperature was maintained at 40 °C. A mixture of sodium phosphate buffer (10 mM, pH 3.5)—acetonitrile (86:14, v/v) was used as mobile phase with an isocratic mode of elution. Optimization of the chromatographic conditions was carried out by conducting column scouting experiments, studying effect of PH, and organic ratio on individual peak separation. The current method was developed with a systematic approach. Robustness of the developed method was assessed by varying critical factors, such as PH of the buffer, organic ratio, and column temperature, at two levels [low (−), high (+)]. Final method conditions were selected using global desirability with numerical optimization. The resolution (Rs) between each isomer was > 1.5, and column efficiency (N) > 1900 in all the varied conditions. The developed method was fully validated according to ICH Q2 guidelines and USP general chapter requirements.

BIO ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULANEOUS ESTIMATION OF NITAZOXANIDE AND OFLOXACIN IN HUMAN PLASMA BY RP-HPLC

An isocratic Reversed-Phase High Performance Liquid Chromatography method has been developed for rapid and simultaneous separation and estimation of two antibiotics, namely, nitazoxanide and ofloxacin, in human plasma. Separation was carried out on Altima C8 (150 x 4.6 mm, 5µ) column using a mobile phase of 0.1% ortho phosphoric acid: acetonitrile (50:50, V/V) at 260 nm. The retention time of nitazoxanide and ofloxacin was noted to be 4.850 and 7.949 min, respectively. The average % recovery for nitazoxanide and ofloxacin were 98.012 % and 94.176 %, respectively and reproducibility was found to be satisfactory. The linearity was investigated in the concentration range of 0.02-2 µg/ml (r2=0.9996) for nitazoxanide and 0.008-0.8 µg/ml (r2=0.9998) for ofloxacin. The lower limits of quantification were 0.0196 µg/ml and 0.0079 µg/ml for nitazoxanide and ofloxacin, respectively, which reach the level of both drugs possibly found in human plasma. The proposed method can be applied for etermination of nitazoxanide and ofloxacin from dosage forms during pharmacokinetic study.

Analytical Study of the Antifungal Posaconazole in Raw Material: Quantitative Bioassay, Decomposition Chemical Kinetics, and Degradation Impurities by LC-QTOF-MS.

Posaconazole is a triazole antifungal drug that was approved by the U.S. Food and Drug Administration in 2006. No bioassay of it is available in the literature nor official codes for potency determination in bulk. To conduct an analytical study focused on posaconazole in bulk. An alternative microbiological assay was validated for drug quantitation, applying agar diffusion technics (3 × 3 design), using Saccharomyces cerevisiae ATCC MYA 1942 as a test microorganism (2% inoculum). An isocratic HPLC-DAD method, with C8 Shim-pack column (250 × 4.6 mm, 5 μm) and methanol-water (75:25 v/v) mobile phase was used for stress stability by photolysis and oxidation, indicating the formation of degradation products, which were investigated by ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry. The established conditions for the bioassay were satisfactory. It was linear in the range evaluated (2.5-10.0 µg/mL), as well as precise, accurate, and robust. Stress tests showed drug susceptibility to the factors evaluated (60% of degradation after 120 min). Kinetics curves for photolytic decomposition followed first-order kinetics. From a photolytic and oxidative degraded matrix, three major degradation products were identified as being derivatives with modifications in the piperazine central ring and in the triazole and triazolone side chains, whose mass spectra results were m/z 683 (DP1), m/z 411 (DP2), and m/z 465 (DP3). The microbiological method was adequately validated and demonstrated to be equivalent to physico-chemical ones. The impurities found are described for the first time in studies with posaconazole raw material. A microbiological bioassay was developed for posaconazole, first-order kinetics was determined for photolytic degradation, and structures for new degradation products were suggested.

A Simple Isocratic HPLC Method for the Quantitation of 17 Cannabinoids

Although cannabis has been used for several thousand years, the exact composition of the cannabinoids patients are administered for different symptoms has remained largely unknown. While this absence of catalogued information may be accepted in some cultures, the use of cannabis as a human product in the registered medicines setting requires knowing its composition so that doses can be standardised between patients. This is particularly so in clinical trials that are currently under way to determine the efficacy of a product. Although the major cannabinoids of interest to prescribers are well known – tetrahydrocannabinol and cannabidiol and the corresponding acids tetrahydrocannabinolic acid and cannabidiolic acid, the cannabis plant contains many more phytocannabinoids. We have developed and validated a robust and fast (11 min) isocratic HPLC method for the analysis of 17 phytocannabinoids. The method had an analytical range of 1–150 μg mL−1 for tetrahydrocannabinolic acid and cannabidiolic acid, 0.5–75 μg mL−1 for tetrahydrocannabinol and cannabidiol, and 0.5–20 μg mL−1 for the remaining 13 cannabinoids. The method had excellent repeatability with a relative standard deviation of between 5 and 14 % and a bias of between –8.6 and 6 % for the 17 cannabinoids. The method was applied to the analysis of medicinal cannabis products, including both flos and oils with results matching the supplier’s certificate of analysis. This simple fast isocratic method with basic HPLC equipment can be easily transferred to any analytical laboratory interested in the identification and quantitation of cannabinoids.

Novel isocratic RP-HPLC method for simultaneous estimation of Berberine and Aloe-emodin

The present paper describes a novel, isocratic, simple, precise, accurate, and robust reversed-phase high-performance liquid chromatographic (RP-HPLC) method development and validation for simultaneous quantitative estimation of berberine and aloe-emodin in polyherbal formulation. The proposed chromatographic estimation was carried out isocratically using Cosmosil C18 (250 × 4.6mm) SH 5.0μm column, the mobile phase was a mixture of 0.05 M potassium dihydrogen phosphate buffer (pH adjusted to 2.5 using ortho-phosphoric acid): acetonitrile in a ratio of 60: 40 v/v with a flow rate of 1 mL/min and column oven adjusted to 30°Cwith injection volume 10μL. The ultraviolet (UV) detection was carried out at 225 nm. The retention time of berberine and aloe-emodin was found to 4.78±0.2 min and 15.49±0.2 min respectively. Calibration curves were linear over the tested concentration range of 4 to 20μg/mL. The present study reveals that this novel isocratic HPLC method is well validated, reliable and can be used for routine analysis of berberine and aloe-emodin in polyherbal formulations containing these phytoconstituents as one of the ingredients.

Development and validation of a short runtime method for separation of trace amounts of 4-aminophenol, phenol, 3-nitrosalicylic acid and mesalamine by using HPLC system

In a short runtime process (about 6 minutes), with isocratic method without need of reconditioning before the next run, a mixture of some molecules containing 4-aminophenol (4APh), phenol, 3-nitrosalicylic acid (3NSA), and mesalamine (5-aminosalicylic acid) (MLZ), were separated and detected by means of HPLC/UV-Vis system. The resolutions of the separated and sharp peaks referring to the mentioned compounds were at least more than 2, and the tailing factor (TF) was about 1, even in 1 µg/l (ppm) concentration. In order to optimize the method, the effects of the pH, eluent percentage, flow rate, and buffer concentration, and wavelength on the chromatogram were investigated. Also, the results of the validation processes showed that the method trustable to be used in laboratories.

Determination of candesartan or olmesartan in hypertensive patient plasma using UPLC-MS/MS.

Candesartan and olmesartan are angiotensin II receptor blockers (ARBs) used for the treatment of hypertension and heart failure. Quantitation methods for candesartan and olmesartan were developed using ultra-high performance liquid chromatography-tandem mass spectrometry following protein precipitation. Candesartan was separated using 5 mM ammonium formate (A) and 100% acetonitrile (B) and olmesartan was separated using 2 mM ammonium formate with 0.1% formic acid (A) and 100% acetonitrile (B). Separation was performed using an isocratic method with a Thermo hypersil GOLD C18 column. Electrospray ionization was used for analyte ionization and detection of candesartan, olmesartan, and the internal standards by multiple reaction monitoring. Developed method showed excellent linearity (r > 0.99) in the concentration range of 2–500 ng/mL for candesartan and 5–2,500 ng/mL for olmesartan. Accuracies were 86.70–108.8% for candesartan and 87.87–112.6% for olmesartan. These methods were able to successfully measure plasma candesartan or olmesartan concentrations in hypertensive patients. This study can be used for pharmacokinetic studies of candesartan or olmesartan in humans.

A Reliable and Cost-Effective Method for Determination of Endocrine-Disrupting Compounds in Coastal Waters, Suspended Particulate Matter, and Sediments by Ultrafast Liquid Chromatography Coupled to Photodiode Array and Fluorescence Detectors

Analytical methods for determining 14 endocrine-disrupting compounds (EDCs) in coastal waters, suspended particles, and sediment samples were successfully performed by ultrafast liquid chromatography with photodiode array and fluorescence detections (UFLC-PDA-FLD). Solid‑phase extraction (SPE) and ultrasound-assisted extraction (USE) were used for sample preparation. Two chromatographic methods have been developed. An isocratic separation method was used to separate bisphenol A (BPA) and steroids and another gradient elution method to separate phthalates and alkylphenols. The detection by fluorescence was used for alkylphenols, BPA, and steroids and photodiode array (PDA) for phthalates. Limits of detection (LOD) ranged from 0.41 (4-tert-octylphenol (4tOP)) to 63 ng L-1 (dibutylphthalate (DBP)), 0.41 (4tOP) to 63.2 ng g-1 dried weight (dw) coastal waters, and solid samples (suspended particulate matter (SPM) and sediment samples), respectively. Recoveries ranged from 52 (diethylphthalate (DEP)) to 116% (DBP) for water, from 54 (DEP) to 108% (estrone (E1)) for SPM, and from 62 (4-n-nonylphenol (4nNP)) to 117% (4-n-octylphenol (4nOP)) for sediment samples. Finally, with the minimization of reagents and energy, the proposed methods were applied to samples collected from Todos os Santos Bay (BTS), Bahia, Northeastern Brazil.

Isocratic RP-UHPLC method development and validation of stability-indicating for simultaneous determination of teneligliptin and metformin in fixed-dose combination

The pharmaceutical combination of Teneligliptin Hydrobromide hydrate (TEN) and Metformin Hydrochloride (MET) drugs is used in the treatment of type 2 diabetes mellitus. A new analytical method: QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) has been developed for the quantification of Teneligliptin (TEN) and Metformin (MET) in bulk and tablet dosage forms. The analysis was performed on Agilent symmetry analytical column Eclipse plus C18 (150 mm × 4.6 mm, 5 μm) ultra- performance liquid chromatography-Diode Array Detectors (UHPLC-DAD), while the detection was performed on 233 nm using Diode Array Detectors. Buffer and acetonitrile (65:35 v/v) were the mobile phase, run at a flow rate of 0.7 mL min−1 for isocratic elution. The buffer used in the mobile phase contained 50 mM potassium di-hydrogen phosphate, pH adjusted to 3.5±0.02 with orthophosphoric acid. The mean values of recovery were found to be 100.50% and 99.81%. The proposed method could be ideal for quantitative evaluation in pharmaceutical preparations of these drugs and also for their quality control in bulk manufacturing. Stress test covers: acid, base, peroxide, thermal and photolytic degradation; were conducted to show the specificity of the method and degradation.

LC-UV and UPLC-MS/MS Methods for Analytical Study on Degradation of Three Antihistaminic Drugs, Ketotifen, Epinastine and Emedastine: Percentage Degradation, Degradation Kinetics and Degradation Pathways at Different pH

Evaluation of pH-dependent reactivity of drugs is an essential component in the pharmaceutical industry. Thus, the stability of three antihistaminic drugs, i.e., ketotifen, epinastine and emedastine, was tested, in solutions of five pH values, i.e., 1.0, 3.0, 7.0, 10.0 and 13.0, at high temperature (70 °C). LC-UV isocratic methods were developed to estimate percentage degradation as well as the kinetics of degradation. Generally, epinastine was shown to be the most stable compound with degradation below 14%. Emedastine was labile in all pH conditions, with degradation in the range 29.26–51.88%. Ketotifen was moderately stable at pH 1–7 (degradation ≤ 14.04%). However, at pH ≥ 10, its degradation exceeded 30%. The kinetics of degradation of ketotifen, epinastine and emedastine was shown as a pseudo-first-order reaction with the rate constants in the range 10−4–10−3 min−1 Finally, the UPLC-MS/MS method was applied to identify the main degradants and suggest degradation pathways. Degradation of ketotifen proceeded with oxidation and demethylation in the piperidine ring of the molecule. As far as epinastine was concerned, opening of the imidazole ring with formation of the amide group was observed. Unfortunately, no degradation products for emedastine were detected. The present results complete the literary data and may be important for both manufacturing of these drugs and their administration to patients.

Analysis of Aspirin, Prasugrel and Clopidogrel in Counterfeit Pharmaceutical and Herbal Products: Plackett-Burman Screening and Box-Behnken Optimization.

An isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of aspirin, prasugrel HCl and clopidogrel bisulfate in the presence of clopidogrel-related compound (impurity-A) in focus on counterfeit. This method was used to determine counterfeited antiplatelet drugs in two substandard Indian pharmaceutical products sold on the market in Yemen and two traditional herbal medicines sold on the market in China. Thin layer chromatography and mass spectrometry of counterfeit herbal medicines have additionally been carried out to verify the identification of adulterants. Chromatographic separation was performed on Inertsil ® ODS-3 C18 (4.6 × 250mm, 5μm) with isocratic mobile phase elution containing a mixture of acetonitrile: (25mM) potassium dihydrogen phosphate buffer, pH2.7 adjusted with 0.1M o-phosphoric acid (79: 21, v/v), at a flow rate of 1mL/min and UV detection at 220nm. Designs of experiment methodology, Plackett-Burman and Box-Behnken designs were used for the screening and optimization of the mobile phase composition. The method validation was also performed in accordance with the International Council on Harmonization (ICH) guidelines. The method developed for routine analysis was found to be sensitive, simple, accurate and highly robust. The results were statistically compared to reference methods using Student's t-test and variance ratio F-test at P<0.05.

New liquid chromatography assays for simultaneous quantification of antihypertensives atenolol and valsartan in their dosage forms.

Nowadays, various single-pill combinations are used as the best choice in hypertension management. However, these pills made a high challenge to analysts in terms of quality control assays. We have developed three sensitive, selective, fast, simple, green, accurate, precise, and robust isocratic high-performance liquid chromatography methods for simultaneous determination of valsartan and atenolol in dosage forms. To find the appropriate high-performance liquid chromatography conditions for the separation of the examined drugs, various columns, isocratic mobile phase systems were tried, and successful attempts were performed. The used columns proved to be indispensably applicable and gave a shorter analysis time and peak symmetries. This reduction in total run time leads to low solvent consumption and makes all methods more economical. The linearity, accuracy, and precision remained within the acceptable limits. Therefore, all developed methods are suitable for the routine quality control analysis of any pharmaceutical preparation containing the two tested drugs with the proposed chromatographic methods advantages for checking quality during stability studies of their pharmaceutical preparations.

Estimation of four drugs: Ambroxol hydrochloride, Levocetirizine hydrochloride, Phenylephrine hydrochloride and Paracetamol by RP-HPLC in tablet dosage form

A simple, specific, precise, accurate and economic isocratic RP-HPLC method was developed for the simultaneous determination of Ambroxol hydrochloride, Levocetirizine hydrochloride, Phenylephrine hydrochloride and Paracetamol in bulk and tablet dosage form. A reverse phase Nucleosil C18 column (250 mm x 4.6 mm x 5 µm) with mobile phase consisting of Methanol: Sodium Phosphate dibasic anhydrous Buffer (65:35 V/V) having (pH of buffer 7.0 ± 0.02 adjusted with ortho phosphoric acid) was used. The flow rate was 1.0 mL/min and the effluents were monitored at 230 nm. The retention times of Paracetamol, Levocetirizine hydrochloride, Phenylephrine hydrochloride and Ambroxol hydrochloride were found to be 3.117, 4.925, 6.217 and 12.308 min. respectively. The method was validated in terms of linearity, range, specificity, accuracy, precision and robustness. The proposed method was successfully applied for the estimation of paracetamol, levocetirizine hydrochloride, phenylephrine hydrochloride and ambroxol hydrochloride in combined tablet dosage form. Keywords: Paracetamol (PCM), Levocetirizine hydrochloride (LEVO), Phenylephrine hydrochloride (PHEN), Ambroxol hydrochloride (AMB), Isocratic; RP-HPLC.