Current Pharmaceutical Analysis - Volume 18, Issue 5, 2022

Background: Lamotrigine (LTG) is a broad-spectrum and first-line anti-epileptic drug. To monitor the serum levels of LTG in epileptic seizures patients, high-performance liquid chromatography with diode-array detection (HPLC-DAD) and ultra-performance liquid chromatography-- tandem mass spectrometry (UPLC-MS/MS) methods were established and compared. Methods: Imatinib was used as the internal standard (IS) for both methods. LTG and IS were detected at 246 nm by HPLC-DAD. In UPLC-MS/MS, LTG and IS positive ion were detected by multiple reaction monitoring (MRM), with m/z of 256/210.9 and 494/394.02, respectively. A total of 37 blood samples from epileptic patients were determined and studied by these two methods. Results: There was an acceptable linearity for the two methods. The concentration range of LTG was 0.59 ∼ 22.20 mg/L by HPLC, and 0.28 ∼ 23.97 mg/L by UPLC-MS/MS. The Pearson regression coefficient of Deming regression was 0.9653 (95% CI: 0.9332 to 0.9821). Bland–Altman method demonstrated that the concentration of LTG determined by UPLC-MS/MS was 8.3% higher than that determined by HPLC (limits of agreement, -32.0% to +48.6%). Conclusion: There was a significant correlation between the two methods. Both HPLC and UPLC- MS/MS can be used for routine clinical monitoring of LTG.

Background: Platinum derivatives, including cisplatin and its later generations carboplatin, and oxaliplatin remain the most largely used drugs in the therapy of malignant diseases. They exert notable anticancer activity towards numerous types of solid tumors such as gastric, colorectal, bladder, ovary, and several others. The chemotherapeutic activity of these compounds, however, is associated with many unwanted side effects and drug resistance problems, limiting their application and effectiveness. Proper dosage is still an inherent problem, as these drugs are usually prescribed in small doses. Objective: Several analytical methods have been reported for the accurate quantification of cisplatin, carboplatin, and oxaliplatin and their metabolites either alone or in combination with other chemotherapeutic drugs, in different matrices, such as pharmaceutical formulations, biological fluids, cancer cells, and environmental samples. The main goal of this review is to systematically study the analytical methods already used for the analysis of cisplatin, carboplatin, and oxaliplatin in various matrices during the last two decades. Results and Conclusion: In the literature, reviews showed that numerous analytical methods, such as electroanalytical, UV-visible spectrophotometry, chromatographic, fluorescence, atomic absorption spectrophotometry, and other spectroscopic methods combined with mass spectrometry were used for the determination of these compounds in various matrices.

Background: Rabdosia rubescens drop pills have the effects of clearing away heat and toxin, detumescence, relieving pain. Objective: A simple and sensitive method for simultaneous determination of oridonin, ponicidin, and rosmarinic acid in rat plasma was developed based on ultra performance liquid chromatography- tandem mass spectrometry (UPLC-MS/MS). Methods: Chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with a mobile phase consisting of water containing 0.2% formic acid (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.3 mL/min over a total run time of 3.8 min. All analytes were measured with optimized multiple reaction monitoring (MRM) in positive and negative ion ESI mode. Results: The transitions of oridonin, ponicidin, rosmarinic acid, diphenhydramine, and chloramphenicol were 365.3→347.3, 363.3→345.2, 359.0→160.9, 256.0→167.2, and 321.1→151.9, respectively. The linear ranges were 1-256 ng/mL for ponicidin and rosmarinic acid and 2-512 ng/mL for oridonin. The validated method was stable and reliable. There was no significant difference in the half-life (t1/2) of the three analytes at three doses. The area under the curve (AUC0-t) and peak concentration (Cmax) of the three analytes decreased linearly in each dose range, and the linear correlation R² of each analyte under the three doses was greater than 0.95. Conclusion: This method was successfully applied to pharmacokinetic studies of oridonin, ponicidin, and rosmarinic acid in rat plasma after intragastric administration of Rabdosia rubescens drop pills.

Background: Brivaracetam is a novel antiepileptic drug clinically approved for the treatment of partial onset seizures in adults and adolescents. It has some abuse potential and assigns to Schedule V category under the Controlled Substance Act by the Drug Enforcement Administration. It is essential to develop a faster, simple, and highly sensitive method for the quantification of Brivaracetam in human plasma by employing simple liquid-liquid extraction. Objective: The objective of this study is to develop and validate a novel UHPLC-MS/MS method for the estimation of brivaracetam in human plasma samples and application to pharmacokinetic study. Methods: An ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated according to current regulatory guidelines for bioanalytical methods. Sample processing (50 μL) involved only a simple liquid-liquid extraction by ethyl acetate as extraction solvent. Brivaracetam-d7 was used as an internal standard. The chromatographic analysis was performed by a Unisol C18 (4.6 X 100 mm, 5μm) column using 0.1% formic acid in water/acetonitrile (20/80 V/V) as an isocratic mobile phase, at a flow rate of 1.0 mL/min with a run time of 2.2 min. Brivaracetam and its internal standard Brivaracetam D7 were detected and quantified in positive ion mode using multiple reaction monitoring transitions at m/z 213.100→168.100 and m/z 220.000→175.100, respectively. The developed method was applied to assess pharmacokinetic parameters like Cmax, Tmax, t1/2 and AUC for Brivaracetam in healthy, male, and adult humans. Results: The method was validated over a concentration range of 20.000 ng/mL to 4000. 000 ng/mL. Both intra- and inter-assay precision and accuracy were <15% for all quality control samples. No matrix effect was observed. Pharmacokinetic results showed that test formulation is bioequivalent with reference formulation. Conclusion: The present assay is faster, highly sensitive and simpler than previously published analytical reports for brivaracetam in human plasma samples and is suitable for pharmacokinetic evaluation of any marketed formulation.

Background: Quality is one of the three main characteristics of medicinal products. The quality assurance process is multi-stage: during the manufacturing, quality control is the commitment of the manufacturer, but after medicinal products become part of the distribution and pharmacy network, analytical quality control is carried out within the program for Market Surveillance. There are different approaches in conducting quality control of medicinal products under the Market Surveillance Program. Aim: The aim of the study is to compare the results obtained under two approaches: individual testing and testing by groups with the same active substance. Methods: In this study, comparative tests for assay and purity were carried out within two groups of medicinal products from the antihypertensive group containing Amlodipine besilate and Valsartan. Analyses were performed in accordance with the available pharmacopoeial monographs, as well as those from literature sources. Results: The results from the assay tests show a significant difference in the same product tested. Analytical methods for the determination of impurities also show different results when analyzing the same medicinal product. Conclusion: Considering the performed analytical tests, the obtained results can be used to make several conclusions and suggestions concerning the optimisation of the Annual Market Surveillance Program.

Objective: The study aims to investigate the pharmacokinetic profile of Praeruptorin A and khellactone and in vitro hydrolysis of praeruptorin A to khellactone in different biological samples. Methods: A LC-MS/MS method was established. Analytes and internal standard (IS) were isolated using the protein precipitation method and then separated on a Thermo BDS Hypersil C18 (2.1 mm×50 mm, 2.4μm) column using a mobile phase consisting of 0.05% formic acid solution and acetonitrile. Samples were analyzed in positive electrospray-ionization (ESI) mode using multiple reaction monitoring (MRM). Results: The calibration plots gave desirable linearity (r²>0.99) in the concentration range from 0.99-990.0 and 2.0-2000.0 ng/mL for Praeruptorin A and khellactone, respectively. In addition, the LOQs of these analytes were sufficient for vivo pharmacokinetic study and vitro hydrolysis study of Praeruptorin A. The intra-batch and inter-batch precision were all within 14.05%, and the accuracy was between 89.39% and 109.50%. The extraction efficiency of PA and khellactone ranged from 76.35 ∼ 89.58%. The matrix effects of analytes and the IS were between 89.67% ∼ 105.26%. Conclusion: The liver CYPs mediated by the metabolism of PA may contribute to the systemic exposure of its active metabolite, khellactone, in rats.

Objective: Linezolid is a significant antibiotic used against severe infections initiated by multi-resistant bacterial pathogens. Aims: The aim of this study is to develop and validate a simple, selective and accurate highperformance liquid chromatographic HPLC method for the analysis of linezolid LZD. Methods: Linezolid extraction from plasma is obtained using methanol. Chromatographic separation is achieved isocratically on a C18 column [Zorbax C18, 5 μm particle size, 150 mm* 4.6 mm] making use of a mobile phase of acetonitrile / 0.05 M phosphate buffer, pH = 4.5 (30: 70 v/v) at a flow rate of 1.2 mL/min with photodiode array detector DAD, at a wavelength of 256 nm. Results: The retention time of linezolid was 2.5 min. The analytical method was linear (r² > 0.998) over the calibration range of 0.30 to 50.0 μg/mL. The extraction recoveries of linezolid range from 71.03 to 91.93 %. The limit of quantification and the limit of detection were 0.112 μg and 0.037 μg, respectively. The RSDs for intraday and interday assays were < 7.77 and 4.32 %, respectively. The intraday and interday accuracies were in the range 80.6-112 % and 77.44- 104.85 %, respectively. Conclusion: The applied method is precise, accurate and appropriate for pharmacokinetic studies and therapeutic drug monitoring of linezolid in routine clinical practice.

Background: Cytidine Disodium Triphosphate (CTP-2Na) for injection is mainly used for treating nervous system diseases. Currently, there are few studies focused on the separation and identification of polar impurities in CTP-2Na for injection, which is important for ensuring drug safety and efficacy. Objective: The study aimed to establish an HPLC-Q/TOF method for the separation and identification of polar impurities in CTP-2Na for injection. Methods: Chromatographic separation was achieved on a Waters Atlantis T3 column using 5 mM aqueous ammonium acetate solution as the mobile phase in an isocratic elution mode. A postcolumn compensation technology was used to improve the ionization efficiency of impurities in the spray chamber. Results: Three polar impurities (disodium cytidine tetraphosphate, disodium cytidine diphosphate, disodium cytidine monophosphate) were detected in CTP-2Na for injection. The former one is probably the overreaction product during the production of CTP-2Na, the latter two were reported as degradation products. The fragmentation patterns of cytidine phosphate compounds in negative ion mode are summarized. Conclusion: This study provides a good reference for the separation and identification of polar impurities in nucleotide drugs.

Background: Compound Aluminum Hydroxide Tablets (CAHTs) are widely used in the Chinese domestic market, and strict quality control is required to ensure their clinical efficacy. Purpose: In this study, we established a comprehensive strategy of acid-neutralization, in vitro dissolution and an assay of magnesium trisilicate to evaluate the overall quality and monitor the consistency of CAHTs. Methods: The acid-neutralization profiles of 38 batches of CAHTs were generated using the dissolution and release method III (the cup method, the Chinese pharmacopeia) combined with potentiometric titration. To directly reflect the disintegration and release process of the preparation, we optimized the sample pretreatment method by omitting the grinding step to determine the profiles of complete tablets. In addition, in vitro dissolution was conducted in the hydrochloric acid medium at pH 1.0 by using the assay of magnesium trisilicate through a validated approach of Flame Atomic Absorption Spectrophotometry (FAAS) to evaluate the similarity of the dissolution profiles. Results: Acid-neutralization tests showed that the quality of the samples from manufacturers B and F was poor. In vitro dissolution experiments showed that the samples from manufacturer A had the highest similarity with the reference preparation, which indicated their good quality consistency. Besides, the optimized acid-neutralization method had the advantage of simple operation and enabled direct characterization of pharmacodynamics in the quality consistency evaluation of antacids. Conclusion: A successful synthetic evaluation strategy was established to assess the overall quality of CAHTs, which demonstrated that the improvement in the quality of this formulation is imperative.

Background: Vitexin is the main bioactive compound of hawthorn (Crataegus pinnatifida), a famous traditional Chinese medicine, and vitexin for injection is currently in phase I clinical trial in China. Objective: This investigation systematically evaluated the metabolism and toxicokinetics of vitexin in rats and dogs. Methods: Rats and beagle dogs were administrated different doses of vitexin, and then the plasma concentration, tissue distribution, excretion, metabolism, pharmacokinetics and plasma protein binding were investigated. Results: The elimination half-life (t1/2) values in rats after a single intravenous dose of 3, 15 and 75 mg/kg were estimated as 43.53±10.82, 22.86±4.23, and 21.17±8.64 min, and the values of the area under the plasma concentration-time curve (AUC0→∞) were 329.34±144.07, 974.79±177.27, and 5251.49±786.98 mg•min/L, respectively. The plasma protein binding rate in rats was determined as about 65% by equilibrium dialysis after 72 hr. After 24 hr of intravenous administration, 16.30%, 3.47% and 9.72% of the given dose were excreted in urine, feces and bile, respectively. The metabolites of the vitexin were hydrolyzed via deglycosylation. The pharmacokinetics of dogs after intravenous administration revealed t1/2, AUC0-∞ and mean residence time (MRT0-∞) values of 20.43±6.37 min, 227.96±26.68 mg•min/L and 17.12±4.33 min, respectively. The no-observed-adverse- effect level (NOAEL) was 50 mg/kg body weight/day. There was no significant accumulation effect at 8 or 20 mg/kg/day in dogs over 92 days of repeated administration. For the 50 mg/kg/- day dose group, the exposure (AUC, Cmax) decreased significantly with prolonged administration. This trend suggests that repeated administration accelerates vitexin metabolism. Conclusion: The absorption of vitexin following routine oral administration was very low. To improve the bioavailability of vitexin, the development of an injectable formulation would be a suitable alternative choice.