Current Pharmaceutical Analysis - Volume 17, Issue 2, 2021

This paper includes different analytical methods available for the estimation of the Perindopril Erbumine. Perindopril Erbumine is an Angiotensin-Converting Enzyme (ACE) inhibitor used to treat hypertension and heart failure. The drug is officially listed in the monograph of USP, BP & EP. This drug is available in various marketed formulations. There are so many methods reported for the estimation of Perindopril Erbumine and its related substances. Various methods including UVspectroscopic method, stability-indicating liquid chromatography method, Thermo-Radio, and Photostability study, Stability indicating UHPLC-DAD method, LC-MS/MS electrospray ionization validated method, and LC-MS/MS bioanalytical method are discussed in this paper.

Introduction: Scutellaria Baicalensis Georgi (SBG), a Traditional Chinese Medicine (TCM) used for the treatment of antiviral therapy, contains many flavonoids. Materials and Methods: A simple, fast and accurate ¹H quantitative nuclear magnetic resonance (¹HqNMR) method was established for simultaneously determination of four flavonoids (Baicalin, Baicalein, Wogonin and Wogonoside) in SBG extract by using 3,4,5-trichloropyridine as an internal standard (IS). All the NMR determination work was performed at 308 K on an NMR sample tube inserted with a co-axial tube. The NMR sample tube was added with the sample (SBG extract and IS), the co-axial insert tube was added with D2O. Quantification of four flavonoids was carried out by calculating the relative peak area ratio of the selected proton signals from the target compounds and the IS. Results: The validated ¹H-qNMR method was successfully used to determine the four flavonoids in 9 batches of SBG from different origins and the results were in good accordance with those obtained from HPLC. Conclusion: The established ¹H-qNMR method could be used as a powerful tool for the quality control of SBG from different sources and had potential in the quantification of main components from other TCMs.

Background: Formaldehyde is a key intermediate/reagent in the synthesis of many significant pharmaceutical compounds. It is genotoxic as it interacts with the DNA, RNA and hence there is a pressing need to develop sensitive analytical methods for its trace level determination. Objective: The present study aims to develop a simple and robust Ultra-High-Performance Liquid Chromatographic (UHPLC) method for the trace level quantification of a carcinogen-formaldehyde, in pharmaceutical drug substance. Methods: This method was developed on a conventional pre-column derivatization technique with brady’s reagent followed by fast analysis on fused core C18 Ascentis Express (150 x 4.6 mm, 2.7 μm) column using ultraviolet (UV) detection. Optimization of the derivatization reaction time was conducted in different pH conditions. The optimized analytical method was fully validated in accordance with the current International Conference on Harmonization (ICH) Q2 guidelines, which demonstrated the developed method to be fast, specific, linear, sensitive, repeatable, accurate and convenient for routine quality control. Results: The developed method was linear, accurate and precise in the concentration of 12.8 ng/mL to 510.7 ng/mL. The LOD and LOQ were found to be 3.8 ng/mL and 12.8 ng/mL, respectively. Conclusion: The developed UHPLC can be used effectively for trace level quantification of formaldehyde in drug substances or drug products.

Background: ZT55 is a novel natural product isolated from Radix isatidis. It is a highlyselective tyrosine kinase inhibitor against myeloproliferative neoplasms. Although earlier research has described the pharmacodynamic properties of ZT55 in vivo and in vitro, the quantitative determination and pharmacokinetic profile in vivo have not been thoroughly studied. Methods: A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of ZT55 in rat plasma. A Waters symmetry C18 column was used for chromatographic separation; 0.1% formic acid in acetonitrile and 0.1% formic aqueous solution was used as the mobile phase. Detection was performed by Multiple Reaction Monitoring (MRM) mode using electrospray ionization in the positive ion mode. UPLC-QTOF-MS was used for the identification of metabolites. Results: The method was linear (R²=0.9988) over the concentration range of 1-2500 ng/mL. The lower limit of quantification was 1 ng/mL. The intra-day and inter-day precision of ZT55 showed a relative standard deviation within 8.47%, whereas the accuracy (RE) ranged from -4.84% to 4.45%. The recoveries ranged from 92.89% to 97.21%. ZT55 reached the highest plasma concentration at 0.5h. The peak concentrations with three dosages were 103.59±10.11, 185.23±29.56, and 355.98±28.86 ng/mL. The AUC0-24 of three dosages were 874.70±72.33, 433.80±49.33, and 231.65±19.41 ng•h/ml respectively. Five metabolites of ZT55 from plasma were confirmed. The main pathways of ZT55 in vivo were hydrolysis, N-dealkylation, glycosylation, and sulfonation. Conclusion: LC-MS/MS method was successfully applied to the pharmacokinetic study of ZT55 after oral administration and intravenous. ZT55 exhibited rapid oral absorption, high elimination, and low absolute bioavailability. This study provides important pharmacokinetic and metabolism information for further pharmacological and toxicological research on ZT55.

Background: A normal phase- High Performance Liquid Chromatographic (HPLC) method was developed for the enantioseparation of Oxomemazine. Methods: Separation of enantiomers was attained on Amylose Tris (5-chloro-2-methylphenylcarbamate) using n-hexane: Iso-propyl Alcohol (IPA): Diethylamine (DEA) (60: 40: 0.1) as the mobile phase and the peaks were observed at 227nm using PDA detector. The run time of the analysis was set to 30 min. Results: Linearity was found in the range 10-50 μgmL-1. The enantiomers were separated at retention times 16.87 min and 21.37 min. Conclusion: The developed method was validated as per the ICH guidelines, thus proving the method to be selective, precise and showing linear response of Oxomemazine peak areas. Additionally, the method of chiral separation was being understood by docking simulation study. The method was appropriate for analysis of Oxomemazine in the pure form and its formulation.

Background: Linagliptin (LNG) is an oral hypoglycemic agent that acts by inhibiting the enzyme dipeptidyl peptidase - 4 (DPP-4) and reduces blood sugar levels in type-II diabetic patients. To date, the literature presents few analytical methods for the determination of LNG. However, no reversed phase-high performance liquid chromatography (RP-HPLC) method has been reported for the determination of LNG in nanotransfersomes and in vitro skin permeation samples. Objective: The present study involves the development and validation of RP-HPLC method to quantify LNG in both nanotransfersomes and in vitro skin permeation and deposition samples. Methods: The chromatographic analysis was performed on Luna C18 (2) column (250 x 4.6 mm, 5μm particle size) with a mobile phase consisting of a mixture of methanol: 0.2% orthophosphoric acid (50:50, v/v) at a flow rate of 1.0 mL/min, detection wavelength of 227 nm, and column temperature of 40 ^°C. Results: The method was found to be specific, linear (r² ≥ 0.999; 2-12 μg/mL), precise at both intra and inter-day levels (percentage relative standard deviation; % RSD < 2.00), accurate (percentage recovery 100.21-103.83%), and robust. The detection and quantification limits were 0.27 and 0.82 μg/mL, respectively. The mean % entrapment efficiency and the cumulative amount of LNG permeated across the rat skin from different transfersomal formulations ranged between 40.78 ± 2.54 % to 52.26 ± 2.15 % and 79.54 ± 16.67 to 200.74 ± 35.13 μg/cm² respectively. Conclusion: The method was successfully applied to determine the entrapment efficiency, in vitro skin permeation and deposition behavior of LNG-nanotransfersomes.

Background: Current guidelines for the treatment of hypertension recommend combination therapy, which intends to control blood pressure and enhance cardiovascular protection. Materials and Methods: A sensitive, reliable and selective tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous quantification of amlodipine (AML), valsartan (VAL) and hydrochlorothiazide (HCTZ) in human plasma. The chromatographic system was equipped with ACE 5 C8 (50 X 2.1 mm) column and utilized a mobile phase composition of 0.5 mM Ammonium Chloride & 0.04% FA-Methanol (45:55% v/v). The method used three internal standards; AML-D4, HCTZ-D2 C13 and VAL-D3 with 10% intra- and inter-day precision, and 6% bias for all the analytes. Results: The assay was found to be linear with R^-2 > 0.998, and the limits of quantification for AML, VAL and HCTZ were 0.2, 50.0 and 2.0 ng/mL, respectively. The analytes were found to be stable in plasma samples over short and long term storage. The developed method is rapid with a run time of 3.5 min and cost-effective since the simple sample preparation method is adopted. This method was successfully applied for the bioequivalence study of AML, VAL, and HCTZ in human plasma after administration of the fixed-dose combination tablet of (10/160/25 mg). Pharmacokinetic parameters (Cmax and AUC0-72) for AML and (Cmax, AUC0-t, AUC0-∞) for VAL and HCTZ were used for bioequivalence assessment. These were determined by noncompartmental analysis of concentration data. Conclusion: The result showed 90% confidence intervals (obtained by ANOVA) which were within the predefined ranges. As a consequence, this method can be successfully applied for measuring and quantifying a large number of samples.

Background: Meloxicam is a non-steroidal anti-inflammatory drug, known to inhibit cyclooxygenase, a key enzyme of prostaglandin biosynthesis at the site of inflammation. Objectives: Two sensitive and fast spectrophotometric methods (direct and indirect) using flOW INJection Analysis (FIA) have been described for the assay of meloxicam in pure and in pharmaceutical formulations. Methods: The direct method (method A) depended on the coupling of meloxicam with diazotized procaine benzylpenicillin in alkaline medium. In contrast, the indirect method (method B) involved a charge transfer reaction between the alkaline hydrolytic product of meloxicam as n-donor with metol (p-methylaminophenol sulfate) as a π- acceptor using sodium periodate as an oxidant. Results: The colored products exhibited absorption maxima at 492 and 656 nm for method A and B respectively. The methods obeyed Beer’s low over concentration ranges of 5-80 and 15-225 μg/mL, respectively. The detection limits were 2.73 and 5.26 μg/mL for direct and indirect methods, respectively. The chemical and physical parameters associated with FIA manifolds were optimized to construct the most sensitive and reproducible results. Conclusion: Methods described herein are cheap, environmentally friendly and showed no significant difference with those of the official method indicated no significant difference in accuracy and precision.

Background: There is no worldwide recognized reference internal quality control method for Therapeutic Drug Monitoring (TDM) of voriconazole (VCZ) by Liquid Chromatography (LC). In this study, we aimed to develop an internal quality control method for TDM of VCZ, evaluate it by the Westgard multi-rule theory, and guarantee the analytical quality of the assays. Methods: The plasma concentration of VCZ was detected by two-dimensional liquid chromatography with ultraviolet detection (2D-LC-UV) method. The internal quality control results accompanying with TDM of VCZ in our laboratory from July 2019 to January 2020 were collected and retrospectively studied. The Levey-Jennings quality chart and Z-score quality chart were drawn and Westgard multirules of 12s/13s/22s/R4s/41s/10x were applied to assess the suitable quality control method for TDM of VCZ. Results: The 2D-LC-UV method was well suited to monitor the plasma concentration of VCZ and increase the real-time capability of TDM for VCZ. Combined with Westgard multi-rules, the quality control charts of Levery-Jennings and Z-score both can timely discover and judge the systematic errors and random errors for the internal quality control results. 86 batches of quality control products were assessed and 7 times warnings and 6 times out of control were detected. Conclusion: The Westgard multi-rules, with high efficacy in determining detection errors, has important application value in the internal quality control for TDM of VCZ. The developed quality control method can improve the accuracy and reliability for VCZ measurement by the 2D-LC-UV method and further promote the clinical rational use of the drug.

Background: Schisandra chinensis Turcz. (Baill.) is a perennial deciduous woody vine plant, which is beneficial to all systems of the body. Objective: The goals of the present study were to compare the pharmacokinetics of schisandrol B in rats after the oral administration of schisandrol B monomer (10 mg/kg) and S. chinensis extract (equivalent to 10 mg/kg schisandrol B) and to explore interactions among the components in S. chinensis extract. Methods: Twelve Sprague-Dawley rats of SPF grade were randomly divided into the monomer and S.chinensis extract groups. Plasma samples were extracted with methyl tert-butyl ether, and chromatographic separation was performed on an Agilent ZORBAX Eclipse XDB-C18 (4.6 x 150 mm, 5 μm) column with the mobile phase consisting of methanol (containing 0.1% formic acid)-water (containing 0.1% formic acid and 5 mmol ammonium acetate). This analysis was achieved by multiple reaction monitoring modes in an electrospray interface. Results: The seven lignans had a good linear relationship within the determination range (r>0.9950); the intra- and inter-day precision was < 12.08% and accuracy was 88.64%-111.61%. The pharmacokinetic parameters (T1/2, Tmax, MRT0-∞, CL, AUC0-t, and AUC0-∞) of schisandrol B showed significant differences between the two groups (P <0.05). Conclusion: The validated method has been successfully applied to the pharmacokinetics of schisandrin, schisandrol B, schisandrin A, schisandrin B, schisandrin C, schisanhenol, and schisantherin A. The pharmacokinetic differences indicate that other components in the extract may increase the absorption of schisandrol B, decrease the rate of elimination, and improve the bioavailability of schisandrol B.

Background: Linifanib (LFB) is a tyrosine kinase inhibitor with antineoplastic activity. The existing methods for the analysis of LFB in bulk and dosage forms do not meet the requirements of quality control (QC) analysis. Objective: The present study was devoted to the development of two methods with high throughputs for determination of LFB. These methods are 96-microwell plate assay with microplate fluorescence reader (MWP-FR) and high-performance liquid chromatography with fluorescence detection (HPLC-FD). Methods: The MWP-FR assay was carried out in white opaque 96-well assay plates and the native fluorescence signals of LFB were measured at 360 nm for excitation and 500 nm for emission. In the HPLC-FD, the chromatographic separation of LFB and quinine sulphate (QS) as internal standard (IS) was performed on μ-Bondapack CN HPLC column using a mobile phase consisting of acetonitrile:water (60:40, v/v) pumped at a flow rate of 1 ml/min in an isocratic mode. The fluorescence detector was set at 350 nm for excitation and 454 nm for emission. Results: The linear ranges of the MWP-FR and HPLC-FD were 1-12 μg/well and 10-500 ng/ml, respectively. The limits of detection were 0.85 μg/well and 8.24 ng/ml for MWP-FR and HPLC-FD, respectively. Both MWP-FR and HPLC-FL methods were successfully applied for the determination of LFB in both bulk and tablets. Conclusion: Both methods have high analytical throughputs, they are suitable for use in QC laboratories for analysis of large numbers of LFB samples, and are environmentally friendly as they consume low volumes of chemicals and solvents.

Background: The main goal of process-related substances investigations is to understand how impurities should be controlled during the synthetic or purification process to produce a high-quality drug substance. The simultaneous determination of related substances is a very important factor in the quality control of API and pharmaceutical dosage forms. Methods: A new HPLC method was developed for the simultaneous analysis of Ipratropium bromide and its seven related substances. The separation was performed on a waters HPLC ZORBAX Eclipse XDB- C8 column (150 x 4.6 mm, 3.5 μm). The gradient method used mobile phases containing methanol, acetonitrile, trifluoroacetic acid (TFA), water: A: (methanol, acetonitrile, TFA, 500:500:0.3 v/v/v) and B (0.3% TFA in water v/v). The gradient elution conditions were as follows: 0-32.0 min, linear from 10% A to 35% A; 32.0-36.0 min isocratic 35% A; 36.0-42.0 min linear from 35% to 10%A. The wavelength by UV detector was 220 nm and the flow rate was 1.2 mL/min. The injection volume of sample was 20 μL, and the column temperature was 30 ^ºC. The proposed RP-HPLC method was applied for the determination of seven related substances. Results: All standard curves obtained exhibited good linear regression (r > 0.9939) within the tested range. The average recovery rates were in the range of 98.2-102.0% and RSD was less than 2.9% (n = 3). The process-related impurities were not observed in different batches (<0.025% purity). Conclusion: The method was applied to determine 7 process-related substances in ipratropium bromide and proved to be robust, linear, repeatable, sensitive, selective and easy to perform. The results are valuable for quality control in the manufacturing of ipratropium bromide.