Current Pharmaceutical Analysis - Volume 15, Issue 7, 2019

There has been ever increasing interest in impurities present in Active Pharmaceutical Ingredient’s (API’s). Nowadays, not only purity profile but also impurity profile has become mandatory according to the various regulatory authorities. In the pharmaceutical world, an impurity is considered as an inorganic or organic material, or residual solvents other than the drug substances, or ingredients, arising out of synthesis or unwanted chemicals that remains with APIs. Impurity profiling includes identification, structure elucidation and quantitative determination of impurities and degradation products in bulk drug materials and pharmaceutical formulation. The control of impurities in Formulated products and API’s were regulated by various regulatory authorities like ICH, USFDA, Canadian Drug, and Health Agency. Impurity profiling is very important in the modern pharmaceutical analysis due to the fact that unidentified, potentially toxic impurities are hazardous to health and in order to increase the safety of drug therapy, impurities should be identified and determined by the selective method. Nowadays, it is a mandatory requirement in various pharmacopeias to know the impurities present in APIs and finished drug products. Thus, impurity profiling can act as a Quality Control tool. It can provide crucial data regarding the toxicity, safety, various limits of detection and limits of quantitation of several organic and inorganic impurities, usually accompany with APIs and finished products. There is a strong requirement to have unique specifications/standards with regard to impurities.

The frequency of Type 2 Diabetes Mellitus worldwide is rising rapidly and is estimated to reach 4.4% of the world’s population or about 366 million people by 2030. In the view of National Diabetes Statistics report 2017, 30.3 million people have diabetes in the United States (9.4%) of total population, out of which, 23.1 million are diagnosed and 7.2 million (23.8%) people with Diabetes are undiagnosed. One of the recently introduced therapeutic classes is Sodium Glucose co- Transporter 2 (SGLT2) Inhibitors. Since 2010 USFDA has approved 6 single dose and 7 fixed dose combinations for treatment of Type 2 Diabetes Mellitus. SGLT2 Inhibitors such as Canagliflozin, Dapagliflozin and Empagliflozin are in combinations with most of the dosage forms. This article reviews published analytical methods reported so far in the literature to find SGLT2 inhibitors. They include various techniques like spectrophotometry, high-performance liquid chromatography, liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance thin layer chromatography for their routine, impurity profiling and stability indicating determination in different pharmaceutical matrices.

Introduction: (COPD) Chronic Obstructive Pulmonary Disease is a partially reversible and treatable lung disease, characterized by progressive limitation of airflow. It is one of the main causes of mortality and morbidity worldwide. Methods: An easy, precise and selective reversed-phase liquid chromatographic method, with stabilityindicating assay was established and validated for the determination of indacaterol maleate and glycopyrronium bromide in the mixture. In addition, a forced degradation study was performed for indacaterol maleate, comprised of hydrolysis by acid and base, degradation by oxidation and heat, and photo-degradation. Separation and forced degradation were done by isocratic elution using a reversed phase phenyl column and (methanol: phosphate buffer) at ratio (65:35, v/v) with 3.5 pH buffer as an eluent at 1 mL min-1 as a flow rate. Quantitation was accomplished using a UV detector at 210 nm. Results: The method showed good separation of glycopyrronium bromide, indacaterol maleate and its degradation products. Accuracy, linearity, and precision were acceptable over 10-160 μg mL-1 and 10- 80 μg mL-1 concentration range for indacaterol maleate and glycopyrronium bromide, respectively. Conclusion: The proposed method does not require any previously done separation steps, making it applicable for the analysis of the drugs under investigation in their pharmaceutically marketed preparations.

Background: Metoclopramide is a potent dopaminergic antagonist utilized for treating gastrointestinal disorders. The aim of this study was to examine the pharmacokinetics of oral formulation of metoclopramide in the Pakistani volunteers. Methods: This open label, single dose, pharmacokinetic study was executed in non-smoking, 12 Pakistani healthy males, treated with a single 20 mg oral tablet dose of metoclopramide on test day. Maximum plasma concentration (Cmax), time to achieve Cmax (Tmax), and area under curve (AUC0-t) of metoclopramide were determined analyzing the serial blood samples using a validated HPLC-UV method. Results: The determined values of Cmax, Tmax, and AUC0-t of metoclopramide were 34.51 ± 4.91 ng/ml, 1.56 ± 0.18 and 273.88 ± 114.49 ng.h/ml, respectively. Conclusion: The results reveal that the findings of Pakistani individuals are slightly different when compared with the published data of other ethnic origins.

Objective: The aim of the present work is to achieve a novel highly sensitive chromatographic method for the simultaneous determination of hepatitis C agents, sofosbuvir and velpatasvir from human plasma using ritonavir as an internal standard. Methods: Chromatographic separation was achieved using Hypersil C18 column (50mm x 4.6mm, 3μm) with an isocratic elution mode using the mobile phase composition 10 mM ammonium formate buffer (pH 5.0): acetonitrile (20:80 v/v) pumped at a flow rate of 0.5 ml/min. The detection was carried out by tandem mass spectrometry using Multiple Reaction Monitoring (MRM) positive Electrospray Ionization (ESI) with proton adducts at m/z 530.10 > 243.10, 883.40 > 114.0 and 721.25 > 197.0. Results: The method validated as per USFDA guidelines with respect to linearity, accuracy, and precision was found to be acceptable over the concentration range of 0.2–2000 ng/ml and 5-2000 ng/ml for sofosbuvir and velpatasvir respectively and the method was found to be highly sensitive and selective. Conclusion: The developed tandem mass spectrometric method is robust and can be applied for the monitoring of plasma levels of the analyzed drug in preclinical and clinical pharmacokinetic studies.

Background: Gliclazide Impurity A (GI-A) is one of the gliclazide impurities, as described in the European Pharmacopoeia. Objective: The objective of this study was to develop and validate simple, robust and accurate Reverse- Phase High-Performance Liquid Chromatography (RP-HPLC) method for estimation of gliclazide along with GI-A in bulk by optimising chromatographic parameters using Box Behnken design in response surface methodology. Methods & Results: Box Behnken design was employed for optimizing flow rate, injection volume and strength of the buffer in order to minimize retention time of both gliclazide and GI-A. The optimized strength of orthophosphoric acid buffer in a mixture of Acetonitrile (50:50 v/v), flow rate and injection volume were found to be 25mM, 1mL/min, 20 μL respectively. Linearity was observed in concentration range of 25-150 μg/mL (r2=0.999). The retention time of gliclazide and GI-A was found to be 5.799 minutes and 3.819 minutes, respectively. The limit of detection for Gliclazide and GI-A was found to be 0.0066, and 0.0075 μg/mL and the limit of quantification limit was found to be 0.0202, 0.0228 μg/mL, respectively. The developed method was validated as per the ICH guidelines. Conclusion: The proposed method is useful for best analysis of Gliclazide and GI-A in pharmaceutical dosage forms. QbD approach was found to be an effective tool for optimising chromatographic conditions of the proposed method.

Background: Four major degradation products (1-4) of pralatrexate injection were formed under hydrolytic and light stress conditions. The impurities 1 and 2 were the potential photo degradation products and the impurities 3 and 4 were the potential hydrolytic degradation products. Objective: To prepare and characterize the novel degradation impurities 1, 2, 3 and 4 of pralatrexate injection using NMR, HR MS and IR techniques; and to develop and validate stability indicating analytical reverse phase HPLC-UV method for quantitative simultaneous determination of potential degradation impurities, related substances of pralatrexate and pralatrexate active in pralatrexate liquid formulation. Methods: Gradient HPLC-UV method was developed for the quantification of degradation impurities, related substances and pralatrexate in pralatrexate injection. The separation was achieved on C18 column (250 mm X 4.6 mm, 5μm) using a mobile phase composed of sodium dihydrogen phosphate monohydrate in water (pH 3.0; 0.01M) and methanol. The components were monitored by the UVvisible detector at 242 nm with a flow rate of 1.0 mL/min. Results: The method validation parameters such as accuracy, selectivity, linearity, LOD, LOQ, precision, ruggedness and robustness were demonstrated successfully for pralatrexate and its degradation impurities. The stability-indicating capability of the developed HPLC method was demonstrated by adequate separation of all potential pralatrexate related substances from pralatrexate stressed drug product samples. Conclusion: The developed stability indicating HPLC method was found to be suitable for the simultaneous quantitative determination of potential degradation impurities and related substances of pralatrexate and pralatrexate active in pralatrexate liquid formulation.

Background: Econazole nitrate and triamcinolone acetonide are one of the topical antifungal corticosteroid fixed combinations. Although this combination has been literately investigated, yet their ternary mixture with benzoic acid as preservative lack of a simple method for its determination. Objective: The objective of this work is to present simple, accurate, precise and low cost reversed-phase liquid chromatography (RP-LC) method for the separation and determination of econazole nitrate, triamcinolone acetonide and benzoic acid in a ternary mixture. Methods: Separation was conducted on Mediterranea C18 (150mm x 4.6mm, 5μm) column using methanol: 50 mM potassium dihydrogen phosphate buffer (pH 2.60±1), (70:30, v/v) as a mobile phase at ambient temperature. Isocratic flow rate at 1 ml/min was used and UV detection was carried out at 230 nm. The Linearity, accuracy and precision of the developed method investigated as per ICH guidelines. Results: Good separation of two drugs, benzoic acid as preservative and excipients of dosage form was achieved with acceptable retention times (< 8 min). Validation parameters tested including linearity, accuracy and precision were found to be satisfactory over the concentration range (10-200 μg/ml) for econazole nitrate, (1-20 μg/ml) for triamcinolone acetonide and (2-40 μg/ml) for benzoic acid. Conclusion: The proposed method showed good separation and demonstrated adequate specificity, robustness and accuracy for the quantification of the tested drugs in laboratory prepared mixtures and in pharmaceutical preparations.

Background: Retinol and vitamin E are fat-soluble vitamins crucial for human health, yet their isomers’ distributions in the human body are still known roughly. In order to figure out the physical condition and evaluate the nutritional status of an individual, it is imperative to analyze retinol and VE isomers in human serum. Objective: This work aims to establish a rapid and simple high-performance liquid chromatography with fluorescence detection for simultaneous determination of retinol and vitamin E isomers in human serum. Methods: Separation was accomplished on a common C18 column thermostated at 25 oC, using a simple isocratic elution program of methanol/acetonitrile (85:15, v/v) at a flow rate of 1.0 mL/min. Fluorescence detection was operated using excitation/emission wavelengths of 329 nm/472 nm for retinol and 294 nm/338 nm for VE isomers, respectively. Results: Rapid separation was achieved within 13 min. Linear ranges of the method were 0.020-50.0 μg/mL, with correlation coefficients greater than 0.999. Detection limits and the quantification limits were 0.001-0.004 μg/mL and 0.003- 0.013 μg/mL, respectively. Mean recoveries were 84.1%- 98.2%, with intra-day and inter-day relative standard deviations less than 12.3% and 13.6%, respectively. This method has been applied to the simultaneous determination of retinol and 8 VE isomers in human serum samples with satisfactory results. Conclusion: A rapid, simple and robust method was developed for routine analysis of retinol and eight vitamin E isomers in human serum, providing a useful tool for clinical diagnosis and nutritional evaluation.

Introduction: A suitable LC-MS method for the quantitative determination of genotoxic impurities such as alkyl p-toluene sulfonates in Cabazitaxel was developed. Alkyl p-toluene sulfonates were estimated by LC-MS method using Waters Symmetry C18 (75x4.6 mm), 3.5 μ column. Materials and Methods: Column temperature was maintained 40 °C. Injection volume was 10 μL and flow rate was set as 0.8 mL/min. Sampler temperature was maintained to 25 °C and run time was set as 25 minutes. The mobile phase was a mixture of ammonium acetate buffer and acetonitrile in 70:30(v/v) was used. Results: The method validation has been carried as per ICH guidelines. LOQ was found to be 2.66 μg/mL, 2.75 μg/mL and 2.55 μg/mL for MPTS, EPTS and IPPTS Alkyl p-Toluene Sulfonates (APTS) respectively. Conclusion: The proposed Liquid chromatography-Mass spectroscopy method that can quantify genotoxic APTS in Cabazitaxel at low-level concentration has been developed and validated as per ICH guidelines. Hence, the proposed method was recommended for the assay of genotoxic impurities of cabazitaxel in dosage forms in busy pharmaceutical laboratories.

Background: Cefoperazone Sulbactam injection is a cephalosporin antibiotic with a β- lactamase inhibitor used in the treatment for intra abdominal infections, Urinary track infections, surgical infections, etc. The combination is not official in any of the pharmacopeia for their content and impurities determination. Introduction: The present study involves the development of a simple, rapid, accurate, sensitive and stability indicating RP-HPLC method for the quantitative estimation of Cefoperazone Sulbactam mixture and its impurities in bulk and pharmaceutical dosage forms. Methods: 0.005 M Tetrabutyl ammonium hydroxide buffer solution pH adjusted to 6.80 and Acetonitrile combination has been used in a gradient programme with a flow rate of 1.0 ml/min. The retention time of Cefoperazone and Sulbactam were observed at around 8.5 and 19.5 minutes respectively. The UV detection was carried out at a wavelength of 230 nm. The chromatographic separation was achieved using Waters xbridge C18-150*4.6 mm, 3.5 μm HPLC column. The method has been validated according to the current International Council for Harmonization (ICH) guidelines for the method validation parameters such as Specificity, linearity, range, accuracy, precision, robustness and sensitivity. Results: The validation results indicate that the method is specific, as the known impurities and other impurities formed during the forced degradation studies were not co-eluting with the main components. Moreover, all these impurities were found to be spectrally pure, proving the stability indicating power of the method. The linearity and range of the method is in the range of 0.01-150%, highly accurate (100.2%), precise (<1%) and robust. Conclusion: The proposed method was accurate and specific for the quantitative analysis of Cefoperazone and Sulbactam and their related impurities in the sterile mixture. Hence the proposed method can be used for the quantification of impurities in routine as well as stability analysis in the development as well as quality control laboratories.

Background: As first-line treatments for diabetes, sitagliptin and metformin have been widely prescribed as a combination to enhance the therapeutic effect. Objective: To establish a methodology to simultaneously monitor the two drugs in vivo by a reversedphase Liquid Chromatography-Tandem Mass Spectrometric (LC-MS/MS) method. Methods: The two drugs were extracted from 50 μl human plasma by ion-pair solid phase extraction. The separation of the plasma samples was implemented on an Agilent Zorbax SB-CN column (150×4.6 mm, 5.0 μm). The mobile phase was the mixture (80:20, v/v) of methanol and 5.0 mM ammonium formate in water (pH 4.5). An ion trap spectrometer equipped with an electrospray ionization source was utilized to detect the elution in positive mode. Quantification of the analytes was achieved by Multiple Reaction Monitoring (MRM) using the transitions of m/z 408.3→235.1 for sitagliptin and m/z 130.1→ 60.2 for metformin. Results: Sitagliptin and metformin demonstrated good linearity among the range of 1.00-1000 ng/mL and 5.00-4000 ng/mL. The intra-day and inter-day investigations displayed precisions of ≤ 3.6% and an accuracy range of -7.5% to 6.0% for the two drugs. The mean recovery of the two drugs was 96.0% and 98.5%. Under mandatory storage conditions, both the drugs gave an acceptable stability. The throughput of the assay was found to be more than 100 plasma samples per day ascribed to the run time of 3.0 min for each sample. Conclusion: The developed method was successfully applied to a pharmacokinetic study for a fixeddose tablet formulation containing 50 mg sitagliptin and 500 mg metformin in 12 healthy volunteers.

Background: Meloxicam is as a non-steroidal anti-inflammatory drug that indicates a strong anti-inflammatory, analgesic and antipyretic activity. It is used in the treatment of osteoarthritis arthritis, osteoarthritis and rheumatoid arthritis in the form of various pharmaceutical preparations. Objective: The aim of the work was an elaboration of chromatographic conditions enabling the complete separation of impurities A and B from meloxicam and also its quantitative determination in tablets with use of TLC combined with densitometry as well as the comparison of the method proposed with that described in the literature by Starek and Krzek. Methods: The mixture of ethyl acetate: toluene: n-butylamine (2:2:1, v/v/v) was used as a mobile phase. Determination of meloxicam was performed on silica gel and aluminium oxide plates. Chromatographic conditions presented in this work are better than those described by Starek and Krzek. Results: Linearity of the method for both types of plates was in the range from 1.0 to 5.0 μg/spot. Limit of quantification for silica gel plates was 0.18 μg/spot, while for aluminium oxide plates it was 0.26 μg/spot. Limit of detection has been also specified, 0.06 μg/spot for silica gel plates and 0.08 μg/spot for aluminium oxide plates. The average amount of meloxicam in tablets obtained on silica gel plates was 100.4%, and on the aluminium oxide plates it was 100.3%. Conclusion: The developed method of determination of meloxicam using thin layer chromatography combined with densitometry turned out to be accurate, precise and specific. It can be successfully applied in quality control of meloxicam.

Background: CoQ10 is a very important compound which is found in every tissue of our organism. It participates in the processes of cellular respiration and ATP production. Also, it acts as a strong antioxidant. In an organism, it is represented in two forms: oxidized (ubiquinone) and reduced (ubiquinol). Its low blood level may be a signal for a list of diseases. Materials and Methods: This study developed and compared two methods of CoQ10 determination in order to find the fastest and the most convenient one. The first one involved HPLC-UV with the wavelength of ubiquinone determination equivalent to 290 nm and 275 nm for ubiquinol, respectively. The second one was carried out on an HPLC/MS/MS system utilizing Electrospray Ionization (ESI) and triple quadrupole mass analyzer for quantification in MRM positive mode. Results: Two methods of ubiquinol and ubiquinone determination were developed and validated. HPLC-UV included sample preparation based on liquid-liquid extraction. The LLOQ was 0.50 μg/ml. HPLC-MS/MS method sample preparation was based on protein precipitation. The LLOQ was 0.10 μg/ml. Conclusion: During the investigation, a conclusion was drawn that the HPLC-UV method is too insensitive for simultaneous determination of ubiquinol and ubiquinone. Furthermore, ubiquinol is very unstable and during exogenous factors’ exposure, it rapidly turns into ubiquinone. While, the HPLCMS/ MS method turned out to be sensitive, selective, rapid as it provides an accurate determination of both forms of CoQ10 in spiked human plasma.

Background: Hedyotis diffusa is one of the most extensively used Chinese herbs for the treatment of varieties of cancers in the folk of China. The ingredients of the preparation are still not very clear and the quality control method of the herb medicine requires further improvement. Introduction: In this paper, the quantitative methods for the quality control of H. diffusa were originally developed by using UPLC-UV. Methods: Using the five irdoid glycosides and three flavonoid glycosides previously obtained from H. diffusa as index components, referring to 10-Acetyl Scandoside Methyl Ester (ASME), Scandoside (SD), Deacetyl Aspemloside (DA), Asperulosidic Acid (AA), Asperuloside (AD), Qurecetin-3-OSophoroside (QS), Quercetin-3-O-[2-(6-O-E-Sinapoyl-β-D-Glucopyranosyl]-β-D-Glucopyranosyl (QESGG), and Quercetin-3-O-[2-O-(6-O-E-Feruloyl)-β-D-Glucopyranosyl]-β-Glucopyanoside (QEFGG), the extraction conditions, including solvents, durations, and cycles were optimized firstly. Subsequently, a new UPLC-UV method was originally established to simultaneously determine the eight ingredients in the Chinese herbal medicine. Results: The validated method exhibited good linearity (R2 > 0.999), repeatability (RSD < 3.01%), intra- and inter-day precision (RSD <6.19%) and recoveries (96.31% to 104.22%) for the simultaneous determination of the eight ingredients studied and had been used for the quality analysis of 6 batches of H. diffusa sameples. Conclusion: The established UPLC-UV method could serve as a rapid and effective tool for quality evaluation of the herbal medicine, H. diffusa.