Current Pharmaceutical Analysis - Volume 16, Issue 1, 2020

The role of the analytical methods and their validations has been important in quantification of drugs from their dosage forms or biological samples in recent years. Development of analytical methodscoupled with each other, is useful for the investigation of behavior of drugs or metabolites or impurities, and is also a useful tool for sensitive detections. The recent roles of spectroscopy, chromatography, titrimetry, electrochemistry and capillary electrophoresis have been explained here.

Introduction: Malaria, an infectious disease caused by protozoa of the genus Plasmodium, is highly prevalent in the Brazilian Amazon. Chloroquine is the first-choice drug for the treatment of malaria caused by P. vivax and P. malariae. The humid and hot climate characteristic of the Brazilian endemic region favors drug degradation and modification of its biopharmaceutical properties, which may result in subtherapeutic dosage, formation of degradation products that can be toxic to humans and appearance of parasitic resistance. Thus, it is necessary to monitor the quality of chloroquine tablets. Materials and Methods: An analytical method was developed and validated to determine chloroquine content in tablets by ultraviolet spectrophotometry. The diluent consisted of 0.06 M monosodium phosphate buffer pH 6.8 and detection was performed at 343 nm. Results and Conclusion: The method proved to be linear in the range of 7.2 to 19.2 μg.mL-1, precise, accurate, selective, robust, and statistically equivalent to a liquid chromatographic method by the United States Pharmacopeia. The developed method was applied to determine chloroquine content in six batches of the drug. The evaluated batches were considered adequate for identification, assay, dissolution, disintegration and uniformity of dosage units, and were found to be inadequate in terms of friability.

Objective: A simple, accurate and economical reverse phase- high performance liquid chromatographic method has been developed for the simultaneous quantitative estimation of two anti-retro viral drugs, Etravirine and Elvitegravir in nanoparticulate formulations for the first time. This method is a novel analytical technique for the detection of the both the drugs concurrently, as there is no method available for their simultaneous quantification, to the best of our knowledge. Methods: Optimization and validation of the chromatographic conditions were completed according to the standard ICH guidelines. The separation was done on a C18 column (250 mm x 4.6mm, 5μm) using methanol and phosphate buffer of pH (5.6) as the mobile phase in the ratio 78:22 v/v at a flow rate of 1ml/min for a short run time of 13 min. The detection wavelength was 285nm and the column temperature was maintained at 32oC. Results: The developed method was linear over 10 to 160 μg/ml with a regression coefficient of 0.999 for each. The LOD values were 4.83 and 9.25 μg/ml while LOQ values were 14.63 and 28.01 μg/ml for etravirine and elvitegravir respectively. The recovery values obtained by etravirine and elvitegravir were between 97.6% and 100.8%. Conclusion: The method was specific, precise, fast and accurate with good inter and intra day precision. The method was also effectively employed for the characterization and simultaneous quantification of both drugs in nanoparticulate formulation.

Background: Glycyrrhizic Acid (GRA), a potent antioxidant triterpene saponin glycoside and neuroprotective properties exhibits an important role in the treatment of neurological disorders i.e. cerebral ischemia. GRA is water soluble, therefore it’s have low bioavailability in the brain. Objective: To enhance brain bioavailability for intranasally administered Glycyrrhizic Acidencapsulated- chitosan-coated-PCL-Nanoparticles (CS-GRA-PCL-NPs). Methods: Chitosan-coated-PCL-Nanoparticles (CS-PCL-NPs) were developed through double emulsification- solvent evaporation technique and further characterized for particle size, zeta potential, size distribution, encapsulation efficiency as well as in vitro drug release. UPLC triple quadrupole Qtrap MS/MS method was developed to evaluate brain-drug uptake for optimized CS-GRA-PCL-NPs and to determine its pharmacokinetic in rat’s brain as well as plasma. Results: Mean particles size (231.47±7.82), polydispersity index (PDI) i.e. (0.216±0.030) and entrapment efficiency (65.69±5.68) was determined for developed NPs. UPLC triple quadrupole Qtrap MS/MS method study showed a significantly high mucoadhesive potential of CS-GRA-PCL-NPs and least for conventional and homogenized nanoformulation; elution time for GRA and internal standard (IS) Hydrocortisone as 0.37 and 1.94 min at m/z 821.49/113.41 and 363.45/121.40 were observed, respectively. Furthermore, intra and inter-assay (%CV) of 0.49-5.48, %accuracy (90.00-99.09%) as well as a linear dynamic range (10.00 ng/mL -2000.0 ng/mL), was observed. Pharmacokinetic studies in Wistar rat brain exhibited a high AUC0-24 alongwith an amplified Cmax (p** < 0.01) as compared to i.v. treated group. Conclusion: Intranasal administration of developed CS-coated-GRA-loaded-PCL-NPs enhanced the drug bioavailability in rat brain along with successfully UPLC-MS/MS method and thus preparation of GRA-NPs may help treat cerebral ischemia effectively. The toxicity studies performed at the end revealed safe nature of optimized nanoformulation.

Background: Spinosin is one of the major bioactive constituents among the total flavonoids in semen ziziphi spinosae, which has sedation and hypnosis actions. Methods: A simple and rapid high-resolution ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was developed and validated for predicting the structures of its spinosin metabolic products. This paper presents the first research focused on the metabolites of spinosin in human liver microsomes. Results: Based on the analytical strategy, 8 spinosin metabolites were detected in human liver microsome incubation samples, and the metabolic pathways required to generate these metabolites were proposed. However, no phase II metabolites were found. The cytochrome P450 enzyme is the main metabolic enzyme involved in drug metabolism, accounting for approximately 75% of the total number of different metabolic reactions. Conclusion: The in vitro metabolism of spinosin was proposed. These results allow us to learn about spinosin metabolism, leading to a better understanding of drug biotransformation and providing a basis for clinical applications. Moreover, this study laid the foundation for developing new pharmaceutical drugs.

Background: Doxofylline (DXE) is a novel methylxanthine derivative used in the treatment of asthma and Chronic Obstructive Pulmonary Diseases (COPD). Therapeutic Drug Monitoring (TDM) has been proposed in adults, while the adapted analytical method and TDM data are still missing in children. Methods: A highly sensitive and stability indicating High-Performance Liquid Chromatography (HPLC) method of DXE with caffeine as the internal standard, was developed and validated by separating its metabolites, β-Hydroxyethyltheophylline (HPE) and Theophylline (TPE). HPLC separation is achieved on C18 column connected to an ultraviolet detector (276 nm), using acetonitrile and ultra-pure water in a gradient mode of elution at a flow rate of 0.9 mL/min at 25°C. A liquid-liquid extraction method using ethyl acetate was developed with a small sample volume of plasma of 50 μL. Trough concentration was monitored in children receiving DXE therapy. Results: The method was linear over the concentration ranges from 0.4-20 μg/mL for DXE, HPE and TPE, respectively, in plasma. The limits of quantification were 0.4 μg/mL. Intra- and interday coefficients of variation did not exceed 6.5%, and the accuracy ranged from 94.9% to 112.5%. A total of 39 children (mean age of 1.8 years, range: 0.3-5.7 years) were included. The pediatric patients had detectable DXE concentrations with a mean value of 1.78 μg/mL (range from 0.49 to 6.36 μg/mL), and HPE measurable concentrations with a mean value of 0.52 μg/mL (range from 0.40 to 0.82 μg/mL), while the TPE could not be measured in any patient. Conclusion: A sensitive, reliable, and adapted HPLC method has been developed for the simultaneous analysis of DXE and its metabolites in children. The DXE and its metabolites trough concentrations showed large inter-individual variability.

Background: Ambrisentan is a drug used to treat the pulmonary arterial hypertension symptoms, commercialized as coated tablets. Drug quality control is an essential part for the development and release of drugs for consumption; however, there are few studies related to the proposition of analytical methods and stability study for ambrisentan. Objective: The development of an UPLC assay of ambrisentan in tablets with degradation product`s elucidation was proposed. Methods: Tests with different solvents and chromatographic columns were carried out, achieving an optimal condition using mobile phase in gradient mode, Waters® BEH C18 column and detection at 260 nm. Results: Satisfactory system suitability was obtained (theoretical plates, sensitivity and resolution among peaks), with a reduced analysis time (6 minutes). The method was validated in accordance with the international guidelines and it demonstrated adequate specificity, either for the drug assay as for the identification and quantification of degradation product. It showed linearity (r= 0.999), accuracy (degradation products recovery: 98.47 - 102.44; assay recovery: 99.98 - 104.32%) and precision (RSD: 0.69), with limits of quantification and detection in suitable magnitude in order to evaluate possible drug degradation. Conclusion: UPLC method demonstrated to be fast with satisfactory robustness. The main ambrisentan degradation product formed under thermal stress conditions was elucidated by UPLC-MS/MS and its structure was suggested.

Background: Hydrochlorothiazide (HCTZ) is potent diuretic that is used alone or in combination with other drugs such as labetalol (Lab) (mixtures ) or nebivolol (Neb) (mixtures ) to control moderate to sever hypertension. Introduction: This paper demonstrates the establishment of different validated spectrophotometric and chemometric methods for simultaneous estimation of these mixtures in pure form and pharmaceutical formulations in the presence of HCTZ related impurities in quality control laboratories. Methods: (A) Derivative method (D3) of Lab and HCTZ and its related impurities at 245.3nm and 278.5nm respectively, (D1) of Neb and HCTZ at 294.2nm and 282.2nm, respectively. (B) First derivative of ratio spectra method (DD 1) of Lab at 244.3nm, HCTZ at 261.2nm and 275.4nm, while at 294nm for Neb and 269.4nm for HCTZ. (C) Ratio difference method which depends on measuring the distinction between the amplitudes of ratio spectra at 240nm and 288.3nm for Lab and at 270.1nm and 277.4nm for HCTZ for mixture while at 290.4nm and 299.2nm for Neb and at 232.2nm and 254nm for HCTZ for mixture . (D) Mean centering of ratio spectra (MC) and (E) partial least squares regression (PLS) and principal component regression (PCR). Results: These methods were applied over concentration ranges of 10-100 μg/ml, 10-75 μg/ml and 2.5- 25 μg/ml of Lab, Neb and HCTZ, respectively. Methods were validated according to ICH guidelines and statistical comparison of results of reported and proposed methods revealed no difference. Conclusion: The methods were successfully used for the frequent analysis of selected mixtures in quality control laboratories.

Background: Ginsenoside Rg1 (Rg1) is the main active compound of ginseng herbs. Objective: The aim of this study is to develop a rapid, selective and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the levels of Rg1 in rat plasma and investigate the pharmacokinetics and bioavailability of Rg1 in rats. Methods: Chromatographic separation was achieved on an UHPLC-MS/MS system with an UPLC BEH C18 column using an elution gradient of a mixture of acetonitrile and water (with 0.1% formic acid). The analytes were quantitatively determined by negative-mode electrospray tandem MS. Results: The linearity of the calibration curve was from 2 to 1,000 ng/mL (r ≥ 0.9956), and the lower limit of quantification was 2 ng/mL. The inter-day and intra-day precision were both lower than 12.0%, and the accuracy ranged from 90.6 to 109.7%. The recovery of the targets was higher than 87.0%, and the matrix effect at three different analyte concentrations were from 89.0 to 97.2%. The bioavailability of Rg1 was only 6.1% due to a poor oral absorption. Conclusion: This new quantitative method was found to be sensitive, rapid and selective, and was successfully used to study the pharmacokinetics of Rg1 after intravenous and oral administration in rats.

Background and Objectives: Tissue engineering skin is a three-dimensional skin substitute cultured in the gas-liquid interface using the immortalized keratinocytes (HaCaT cells). In this study, the preliminary metabolism of betamethasone dipropionate by tissue engineering skin was studied and the pharmacokinetics methodology was established using betamethasone dipropionate gel as the target drug. Methods: The betamethasone dipropionate gel was applied on the tissue engineering skin after the skin was cultured. Then the medium (receiving liquid) and skin were taken on 0.25, 0.75, 1.75, 3, 5, 8, 12, 24, 36, 48 h time points. The betamethasone concentration in the medium and skin was determinated by the LC-MS method. Chromatographic analysis was conducted using isocratic elution on a C18 column (150 mm x 2.0 mm, 5 μm) in mobile phase consisting of methanol and water (70 : 30, v/v). The mobile phase was pumped at a flow rate of 0.2 mL/min. Results: This method exhibited linearity within the concentration range of 0. 1 to 50 μg /mL of betamethasone. The LLOQ was 0. 1 μg /mL. The intra- and inter-day precisions of betamethasone in the blank medium were all less than 10.69 % (RSD, %), while in the blank, skin homogenates were all less than 13.96 % (RSD, %). As a result, the betamethasone concentration in the medium and skin could both be detected, which suggested that betamethasone dipropionate could be metabolized to betamethasone through the tissue engineering skin. Conclusion: It was feasible to use tissue engineering skin as a model to study the dermatopharmacokinetics of topical betamethasone dipropionate gel. The research could build a foundation for the dermato-pharmacokinetic study approach.

Background: The purpose of the current study was to develop a selective, precise, fast economical and advanced reverse phase ultra-high-performance liquid chromatography (UHPLC UV) method and validate it for the simultaneous estimation of cholecalciferol and its analogue 25- hydroxycholecalciferol in lipid-based self-nano emulsifying formulation (SNEDDS). Methods: The chromatographic separation was simply performed on a Dionex® UHPLC systems (Ultimate 3000, Thermo scientific) by using HSS C18 (2.1x50 mm, 1.8 μm) analytical column. The elution was carried out isocratically with the mobile phase consisting of acetonitrile and methanol in the ratio of 50:50 %v/v with a flow rate of 0.4 ml/min, followed by the UV detection at 265 nm. The injection volume was 1μl and the column temperature was maintained at 45°C. FDA regulatory guidelines were used to develop and validate the method. Results: The current developed UHPLC-UV method was found to be rapid (run time 2 min), and selective with the high resolution of cholecalciferol and 25-hydroxycholecalciferol (RT=0.530 min & 1.360 min) from different lipid matrices. The method was highly sensitive (Limit of Detection and Lower Limit of Quantification were 0.13 ppm & 0.51ppm, and 0.15 ppm & 0.54 ppm, respectively). The linearity, accuracy and precision were determined as suitable over the concentration range of 0.5-50.0 ppm for both the analytes. Conclusion: The proposed UHPLC-UV method can be used for the determination of cholecalciferol and 25-hydroxycholecalciferol in SNEDDS and marketed Vi-De 3® as pure forms (intact) with no interference of excipients or drug-related substances.

Background: Schiff base is an advanced key starting material of Sertraline hydrochloride. Schiff base is synthesized using two raw materials 1-Naphthol and 1,2-Dichlorobenzene which are potentially genotoxic impurities. Objective: Genotoxic impurities need to be controlled in key starting material to avoid carry forward in the active pharmaceutical ingredient. For trace level quantification of impurities a sensitive, accurate and cost effective method is developed by simultaneous estimation of both impurities. Methods: Reverse phase high performance liquid chromatography (HPLC) method was developed and validated for determination of both impurities in Schiff base. HPLC column Cosmosil MS-II C18, 100 mm X 4.6 mm, 3 μm particle size with ultra-violet detector (UV) was used. Results: The calibration curve of 1-Naphthol and 1,2-Dichlorobenzene showed good linearity over the concentration range of 0.25 μg/g to 7.5 μg/g and 1.5 μg/g to 7.5 μg/g and the regression coefficient was 0.999 and 0.998 respectively. Method had very low limit of detection (LOD) and limit of quantification (LOQ) of both analytes which proves that the method is sensitive and suitable for quantification of compounds at trace level. Conclusion: The proposed method is specific, linear, accurate, rugged and precise. Genotoxic impurities 1-Naphthol and 1,2-Dichlorobenzene are quantified and controlled in the key starting material of Sertraline hydrochloride. The validated method can be used in quality control unit in pharmaceutical industry.