Current Pharmaceutical Analysis - Volume 18, Issue 10, 2022

Background: The pharmaceutical industry is in constant development, aiming to increase its portfolio, optimizing time, product quality and efficacy along with patient safety. The main goal of developing and validating an analytical method is to achieve a balance between costs and risks within the wide array of technical possibilities in order to assure that the method is capable of meeting its expectations, ensuring effective quality control. Objective: The objective of this mini-review is to discuss the analytical aspects of development and validation for analysis of pharmaceutical products, focusing on the complete evaluation package in a systematic way to demonstrate the optimal performance of the method. Methods and Results: Validation results are obtained following strict protocols, typically starting with the assessment of selectivity/specificity parameters, followed by parameters such as linearity and precision. Moreover, accuracy, detection limit, quantification limit and method robustness are also evaluated. Conclusion: This paper may serve as a guide for the pharmaceutical-chemical laboratory, conceptualizing quality and current analytical needs, according to Green Analytical Chemistry, for the development and validation of reliable methods, ensuring clarity to the analyst and assisting in decision making.

Background: Perampanel (PER) is a third-generation antiepileptic drug (AED). Several methods have been developed for the quantification of perampanel in plasma. The pharmacokinetic characteristics of perampanel in healthy Chinese ubjects have not been comprehensively reported. Objective: A simple, fast and sensitive LC-MS/MS method was established and validated for the quantification of perampanel in human plasma and its application to a bioequivalence study. Methods: Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-Phenyl column (4.6 mm × 75 mm, 3.5 μm) using a binary gradient with mobile phase (A) (water containing 5 mmol/L ammonium acetate and 0.1% formic acid and (B) acetonitrile-water (95:5, v/v) at a flow rate of 0.9 mL/min and sample preparation was by one-step protein precipitation via acetonitrile. Results: The total run time in this study was 4.5 min and the retention time of perampanel and perampanel-d5 (internal standard) were 2.30 min and 2.32 min, respectively. The method was developed and validated over the concentration range of 2.00-500 ng/mL for perampanel, with a correlation coefficient greater than 0.9992. The inter-day precision was 3.1%-3.8% and accuracy was 98.9%-103.5%. The intra-day precision was 2.4%-6.8% and the accuracy was 97.6%- 104.9%. The extraction recovery ranged from 99.23%-103.84% and the matrix effect was not significant. Perampanel was proved to be stable in solution and human plasma under different tested conditions. The validated method was successfully applied to a randomized, open-label, 2- period, crossover bioequivalence study in healthy Chinese subjects, and the results indicated that bioequivalence was achieved for 2 formulations of the 4-mg perampanel tablet under both fasting and fed conditions, and both treatments were safe and well-tolerated by all study subjects. Conclusion: The validated method was successfully applied to a bioequivalence study of perampanel in human plasma and has achieved satisfactory results.

Background: Polymyxin E (PME), a complex of cationic cyclic lipodecapeptides, is used to treat multidrug-resistant gram-negative bacterial infections. Besides the main components PME1 and PME2, polymyxin containing unsaturated fatty acyl (FA) group with lower contents can hardly determine the structure without chromatographic preparations and NMR. Introduction: The peptide sequences of PME components have been carried out based on highperformance liquid chromatography-quadrupole / time-of-flight mass spectrometry (HPLCQ/ TOF-MS). However, the components with double bonds on the FA, such as 2’, 3’-dehydro PME1, were difficult to be determined or easily misjudged by MS/MS. The transformation of such unsaturated components to be epoxidized or di-hydroxylated components can promote the acquisition of more fragment ions in the MS/MS to assist in judging the position of double bonds on FA. Methods: In this paper, the PME mixtures were dissolved in an equal proportion of 20% ACN aqueous solution and 2-acetylpyridine. The above PME solution was transferred to a quartz cuvette and irradiated with the ultraviolet lamp at 254 nm for 8h. The dehydro PME components were converted to epoxy PMEs and dihydroxy PMEs. A fragmentation pathway of epoxidized or di-hydroxylated components based on Q/TOF-MS/MS was proposed for the first time. Results: According to the characteristic ions of epoxidized components and di-hydroxylated components, 2’, 3’-epoxy PME1/E2 and 2’, 3’-dihydroxy PME1/E2 were confirmed. It can be inferred that the double bond is located at the 2’, 3’-position of FA. Conclusion: The structure of unsaturated PME components with double bonds on the FA is elucidated by HPLC-Q/TOF-MS combined with photochemical reaction. This strategy applies to other lipopeptides containing unsaturated FA chains.

Background: To explore the pharmacokinetic data of Astragali Radix, a simple, one-step deproteinization procedure was followed to prepare plasma samples, and separation was achieved on an InfinityLab Poroshell 120 column (3.0 mm × 50 mm, 1.8 μm) with a gradient mobile phase consisting of solution A (water containing 0.1% formic acid) and solution B (methanol) at a flow rate of 0.3 mL/min. Aims: In this study, a rapid, sensitive and selective Triple Quadrupole LC/MS method was developed to determine Calycosin-7-glucoside, Formononetin, Calycosin, Ononin, Cycloastragenoland Astragaloside IV from the extractive of Astragali Radixin in rat plasma, and it was validated for rat plasma as the matrix and applied for a pharmacokinetic study in rat plasma, while the internal standard was Sulfamethoxazole. Objective: Multiple reaction monitoring (MRM) was used with an electrospray ionization source with the Agilent Jet Steam System (AJS-ESI) in the positive mode. Methods: A rapid, sensitive and selective Triple Quadrupole LC/MS method was developed to determine Calycosin-7-glucoside, Formononetin, Calycosin, Ononin, Cycloastragenoland Astragaloside IV from the extractive of Astragali Radixin rat plasma, and it was validated for plasma as the matrix and applied for a pharmacokinetic study in rats, while the internal standard was Sulfamethoxazole. Results: There were different pharmacokinetic characteristics after orally administering AR, including absorption and elimination rate, exposure degree, plasma distribution and retention rate. Conclusion: Apart from that, a good linear response was observed within all analytes. The pharmacokinetic study on the six analytes in rats after oral administration of Astragali Radix was successfully completed by adopting this method, thus filling a blank in the pharmacokinetic studies of Astragali Radix.

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide for their analgesic, anti-inflammatory, and antipyretic properties. Objective: The article highlights the development of zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) methods to quantify three NSAIDs as active ingredients in pharmaceutical formulations simultaneously. Methods: For NSAIDs analysis, two homemade ZIC-HILIC stationary phases were carried out by adding sulfobetaine monomers to polystyrene-divinylbenzene particles. The methods were developed by examining the effect of HILIC mode parameters like the kind of organic modifier (methanol or acetonitrile), acetonitrile content, pH and concentration of the acetate buffer in the eluent. Results: The experimental data exhibit the predominant mechanism of NSAIDs with two ZICHILIC stationary phases. The different chain lengths in the two ZIC-HILIC stationary phases are employed to investigate NSAIDs retention behaviour. The strategies were evaluated for their specificity, linearity, accuracy, and precision. The linear ranges were 0.01-5.0, 0.05-17.5 and 0.12-13.5 μg ml^-1, LOD 0.005. 0.015 and 0.046 μg ml^-1 and LOQ 0.015, 0.045 and 0.139 μg ml^-1 for nimesulide, tenoxicam and diclofenac, respectively. Conclusion: They represent interactions with three NSAIDs, ZIC-HILIC columns, and mobile phase in a mixed-mode of hydrophilic and hydrophobic interactions. In contrast to the ZICHILIC- 1 stationary phase with a shorter chain length, the ZIC-HILIC-4 stationary phase with a longer chain length exhibited a longer retention time, lower detection and limit of quantitation for three NSAIDs.The verification findings demonstrate the suitability of the proposed ZIC-HILIC methods for quantifying NSAIDs in pharmaceutical formulations.

Background: Recombinant activated human coagulation factor VIIa (rFVIIa), a vitamin K-dependent serine protease, was originally developed by Novo Nordisk® for the treatment of patients with FVII deficiency as well as patients with hemophilia A and B with inhibitors against FVIII or FIX. The gene for human FVII is cloned and expressed in baby hamster kidney (BHK) cells, and the protein is secreted into the culture media, which is then converted to the active form during chromatographic purification steps. When secreted into the culture media, measuring the concentration levels of FVII is needed in order to monitor, control and optimize the production processes. However, because of the high complexity of such media, reliable analytical techniques are required for this purpose. Objective: This work focuses on the analytical application of VIISelect resin (GE Healthcare) as a highly selective adsorbent for cleanup and preconcentration of rFVII in culture supernatant before analysis by reversed-phase high-performance liquid chromatography (RP-HPLC). Methods: Empty 1 ml SPE cartridges were packed with VIISelect resin. Four types of solutions were used for the sample preparation. The RP-HPLC separation was conducted on a C4 column with UV detection. Results: The method shows recoveries greater than 97% in culture medium with relative standard deviations (RSDs) of less than 2%. The limits of detection and quantification were 0.039 mg/L and 0.12 mg/L, respectively. Conclusion: The method showed better performance in terms of precision and accuracy for rFVIIa determination in cell culture supernatant compared to other techniques, like ELISA and SDSPAGE.

Background: Mixtures of Fluorinated Quinolones and Nitroimidazole antibacterial drugs take a significant place in the treatment of different inflammatory diseases. The necessity to develop analytical techniques for quality control is inextricably related to the introduction of new mixed dose forms into clinical practice. Objective: The objective of our study is to develop HPLC methods for the analysis of Fluorinated Quinolones in combinations with Nitroimidazole antibiotics. Methods: We developed and described an HPLC method for the quantitative determination of model mixtures composed of Metronidazole and Ofloxacin, Tinidazole, and Ciprofloxacin. HPLC method has been developed for the quantitative determination of Metronidazole and Ciprofloxacin in model tablets. The methods have been validated according to the requirements of European Pharmacopoeia 7.0 and the ICH criteria in terms of: selectivity, linearity, repeatability, accuracy, limit of detection, and limit of quantification. Results: The tests are highly efficient liquid chromatographic with and without the use of highly specialized consumables (chiral chromatographic column) and are characterized by excellent reproducibility, accuracy, high sensitivity, and selectivity. Conclusion: The methods would be useful and applicable in routine analytical practice, as well as for regulatory institutions in the control of newly registered generic products.

Background: Fisetin (FIS) is a bioactive flavonoid found in various plants, reported for many pharmacological activities, and presently marketed as a nutraceutical. To overcome less water solubility and bioavailability issues, FIS cubosomal nanoformulation has been prepared and characterized. Objective: To estimate FIS in prepared novel cubosomes, an RP-HPLC analytical method development with the most sensitivity, economical, robust, and wide applicability in marketed FIS formulations and plant extracts also. Methods: An RP-HPLC method was developed and validated as per ICH Q2R1 guidelines by using C-18 Phenomenex Luna 5μ, 100A0 column, LC-20 AD pump, and Shimadzu LC solution 1.25 software. The combination of acetonitrile and formic acid (0.1%v/v) in the ratio of 25:75 v/v was used as a mobile phase for chromatographic separation using a PDA detector at 360 nm and a flow rate of 1 ml/min. Results: The developed method was remarkably linear in the range of 0.1 to 16 μg/ml (R² #131; 0.999). This method was found to be accurate (recovery 98.24 to 100.65 %), precise, robust (% RSD #130; 2), and more sensitive than the earlier reported method with LOD and LOQ values of 17.26 and 52.31 ng/ml, respectively. The FIS estimation was also performed using the developed method in the marketed FIS formulation Doctor’s Best ® Fisetin, and different plant extracts such as strawberry, grapes, black tea, and green tea. The forced degradation study suggests that FIS was unstable in alkaline and oxidative stress conditions. Conclusion: For FIS estimation in cubosomal nanoformulation, a widely applicable, novel, robust, most sensitive, and economical RP-HPLC method was developed and validated and also applied to marketed formulations and plant extracts.