Current Pharmaceutical Analysis - Volume 18, Issue 9, 2022

Epilepsy is one of the chronic diseases seen in 1.0% of the world’s population. For the study of antiepileptic medications, a variety of approaches have been used extensively. These methods provide reliable, accurate, and reproducible results. The available analytical methods for the determination of AEDs in API, biological fluids, and pharmaceutical formulations are reviewed in this context. The drugs categorized under AEDs were studied for different analytical procedures and methods, and were systematically classified based on their applications. The analytical instruments used for the determination of AEDs include various chromatographic techniques such as High-performance liquid chromatography (HPLC), Ultra-performance liquid chromatography (UPLC), Gas chromatography (GC), etc., and other techniques such as hyphenated, spectrophotometric, electrochemical analysis including UV, MS/MS, ECD, CLND, Fluorescence, colometric electrochemical detector (CED) are covered in this review. Supporting electrolyte, pH, panel, mobile phase, measuring or detection potential, sensitivity, and selectivity are all recorded as part of the analytical method formulation and validation process. This review is beneficial for various researchers for further study and advancement in research related to antiepileptic drugs.

Background: Digoxin, a cardiac glycoside, is one of the most significant drugs of choice for treating congestive heart failure. As digoxin is a BCS class IV drug, dissolution is a critical quality attribute during its solid dosage formulation. Methods: This study aims to quantify the drug release during dissolution for low dosage digoxin of 0.0625 mg tablets with a targeted drug release of more than 80% at 60 minutes. We used a highly sensitive, fast, and versatile UPLC-MS technique for this work. UPLC-MS method was operated by positive ionization mode with ACQUITY UPLC C18 (2.1 mm x 100 mm, 1.8 μm) column and at a flow rate of 0.3 ml/minute. Subsequently, this method was developed and validated for parameters like linearity, precision, accuracy, ruggedness, the limit of detection (LOD), and the limit of quantification (LOQ) as per the ICH guidelines. Results: LOD was found to be 2.1 ng/ml. Collision energy for digoxin was observed as 35 eV for QDa mass detector along with 803.5 m/z precursor ion and 651 m/z daughter ion. An optimal custom experimental design was employed to optimize the final dissolution conditions. The critical dissolution factors for optimization were pH of dissolution media, dissolution media volume, and rpm (rotations per minute). The % drug release (DR) was selected as a critical quality attribute with the desired response of drug release > 80% at 60 minutes. Outcomes of the design were further evaluated by statistical tools, including ANOVA. The final optimized dissolution method consisted of 500 mL of pH 7.4 buffer with a USP apparatus of I (Basket) rotating at 120 rpm. Conclusion: We optimized the dissolution conditions using QbD and developed a sensitive UPLC-MS method for quantifying digoxin that can be efficiently used in routine quality control purposes in dissolution testing and quantifying low-dose digoxin tablets.

Background: Early antidepressant Active Pharmaceutical Ingredients (API), like imipramine hydrochloride and clomipramine hydrochloride, have not been assessed for Genotoxic Impurities (GTI) as per ICHM7. Objective: The main aim of the study was to develop a new simple and sensitive method for the determination of genotoxic impurities in imipramine hydrochloride and clomipramine hydrochloride active pharmaceutical ingredients. Methods: A simple, selective, and sensitive Gas Chromatography (GC) liquid injector method was developed for the quantitative determination of N, N-Dimethyl Aminopropyl Chloride (DAPC) in APIs. This method provided an excellent sensitivity and a typical target analyte level of 4.0 ppm. The DPAC content in the sample was analyzed on ZB-624, 30m x 0.53mm x 3.0μm column interfaced with a Flame Ionization Detector (FID). The developed method was validated as per ICH guidelines. Results: The validated method showed good linearity over the concentration range of LOQ to 120% (1.18 ppm to 4.8 ppm), with a correlation coefficient of 0.9986. Limit Of Detection (LOD) and Limit Of Quantification (LOQ) were found to be 0.36 ppm and 1.18 ppm, respectively. This method showed high sample recovery (>90.0%). Conclusion: The developed method was found to be a simple and sensitive gas chromatography liquid injector method. This method was validated as per ICH guidelines and could be very useful for the determination of a potential genotoxic impurity (DAPC) in imipramine hydrochloride and clomipramine hydrochloride active drug substances, thus ensuring the safety of the patients.

Background: Bispecific antibody (BsAb) therapeutics have emerged as the nextgeneration immuno-oncology therapy. The architecture of bsabs is inherently more complex than that of mAb therapeutics. As a result, prior knowledge of critical quality attributes (CQAs) assessment of mAbs is no longer inclusive for bsabs. Objective: The main objective of this work is to develop a fully automated one-step capillary isoelectric focusing - mass spectrometry (cIEF-MS) workflow for the charge variant analysis of a bispecific antibody molecule. Methods: A number of critical factors for the method development are investigated: the performance of two commonly used ampholytes compared; the impact of protein concentration for the cIEF-MS assay is examined; as for sample preparation, off-line and on-line desalting are compared; various combinations of Pharmalyte® 3-10 and 8-10.5 are considered. Results: In this fully automated workflow, the charge variants of this BsAb molecule are clearly separated and accurately identified. Based on six repeat injections, RSDs of the migration time of the identified charge variants are between 3 and 6%. The identified masses of each charge variant show a variation between 0.48 and 1.40 Da. The delta masses of the basic and acidic variants are from the most basic to the most acidic, -58.59, 162.26, 453.44, -907.47, 1,563.60, and 1,566.98 Da, respectively. Conclusion: Overall, the separation resolution, system sensitivity, robustness, and reproducibility of this fully automated cIEF-MS workflow, as demonstrated using this BsAb example, proves it a powerful assay for the quality assessment of recombinant protein therapeutics.

Guanfacine, an α2-adrenergic (α2A) agonist long indicated to treat hypertension, is now being used to treat attention deficit hyperactivity disorder (ADHD) in adolescents. A simple, rapid high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method to detect and quantify guanfacine provides a basis for studying its bioequivalence and pharmacokinetics in human plasma. This assay involves quantitation of guanfacine using its stable isotope-labeled internal standard (IS) guanfacine-¹³C-¹⁵N3 without the impact of ion suppression in the plasma matrix. Electrospray Ionization (ESI) in positive mode and Multiple Reaction Monitoring (MRM) was used for guanfacine and guanfacine-¹³C-¹⁵N3 at the transitions m/z 246.1→60.1 and m/z 250.0→159.1 respectively. This method's sample preparation is optimized with an accurate and simple protein precipitation method employing methanol. Linearity was demonstrated within the range of 0.0500-10.0 ng/mL for guanfacine in plasma with correlation coefficients greater than 0.99. The method showed excellent reproducibility. The Aaccuracy (Relative Error) ranged from -2. to 8.9%, even at the lower limit of quantification (LLOQ), and total precision, expressed as the coefficient of variation was between 1.6% and 10.5%. The average recoveries of guanfacine at three spiked levels of 0.150, 1.00 and 7.50 ng/mL were 103.93, 97.91 and 100.22%, respectively. The validated method was applied successfully to a bioequivalence study of a fixed-dose of extended-release guanfacine hydrochloride (GXR) tablet (Test formulation) and Intuniv® (Reference formulation) in healthy Chinese subjects, 42 subjects under fasting conditions and 30 subjects under feeding condition. Pharmacokinetic parameters were calculated using DAS 3.2.8, and 90% confidence intervals (CIs) of AUC0-t, AUC0-∞ and Cmax for guanfacine were all within 80.00- 125.00%, suggesting that the two formulations were bioequivalent in terms of rate and extent of absorption.

Background: Lung cancer remains one of the leading cancers with increasing mortality rates in the world. The clinicians in our hospital summarized “Fu Zheng Fang (FZF)” as a Chinese medicine prescription with good therapeutic effect and low adverse reactions to treat lung cancer. Objective: This research aimed to conduct an in-depth study on the essence and internal rules of the effect of FZF. Methods: Serum samples from twenty lung cancer patients who received FZF were subjected to metabolomic profiling using UPLC-Q-Exactive-MS combined with multivariate statistical analysis. Results: A total of 17 significantly differential metabolites were found in NC and FZF groups, which mainly participated in phenylalanine metabolism, apelin signaling pathway, sphingolipid signaling pathway, and others. Seven metabolites were increased in FZF group relative to NC group, while ten metabolites were decreased in FZF group; most of them were proven to be consistent with previous experiments. This indicated that FZF had a definite therapeutic effect on lung cancer by regulating the contents of metabolites through amino acid metabolism, metabolism of cofactors and vitamins, carbohydrate metabolism, and cancer. Conclusion: This study provides a deeper insight into the comprehensive understanding of molecular mechanisms of FZF treatment against lung cancer.

Background: Long-term nitroglycerin (NTG) therapy causes tolerance, which limits its clinical application. Previous studies have reported a novel Danshensu/tetramethylpyrazine derivative ADTM, which displays strong cardioprotective effects. However, the effect of ADTM is not known in the NTGinduced tolerance model. In this study, we aimed to evaluate the potential improvement and underlying mechanism of ADTM for preventing the development of NTG-induced tolerance in in vivo and in vitro experiments. Methods: Firstly, the effect of ADTM was determined on NTG-induced tolerance using isolated thoracic aortic rings obtained from rats (50 μM for 45 min). After intragastric administration of ADTM (30 mg/kg, twice a day) for 7 days, NTG solution (10 mg/kg) was subcutaneously injected into male Sprague-Dawley (SD) rats once a day for 7 consecutive days. The systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) were recorded using the PowerLab system. The iTRAQ-based proteomics analysis was used to clarify the underlying mechanism of ADTM in NTG-induced tolerance. Results: ADTM markedly enhanced relaxation sensitivity and vasodilator responses to NTG tolerance in the isolated rat thoracic aorta, and this effect was independent of the vascular endothelium. ADTM prevented the development of NTG-induced tolerance in rats by improving hemodynamic parameters, such as SAP, DAP, and MAP. The iTRAQ-based proteomic analysis suggests that ADTM prevention of NTGinduced tolerance may be related to the regulation of ribosomal metabolism and tight junctions. Conclusion: These results indicate that ADTM has therapeutic potential for NTG-induced tolerance, and is worthy of further studies.

Background: Angiotensin II receptor blockers (ARBs) are helpful medications for treating hypertension and heart failure and have been proposed as a possible alternative to standard angiotensin-converting enzyme inhibitors. Objective: The present article describes and validates sensitive hydrophilic interaction liquid chromatography methods to simultaneously analyse four angiotensin II receptor blockers: valsartan, telmisartan, losartan, and irbesartan. Methods: Numerous chromatographic parameters were studied and optimized in detail. As a mobile phase, the best separation was obtained on two handmade columns (ZIC-S1 and ZIC-S4) using acetonitrile/sodium acetate buffer (40 mM, pH 4.75). The detection of target pharmaceuticals was obtained at 220 nm. Results: The linear ranges of target drugs (valsartan, telmisartan, losartan, and irbesartan) were 0.02-4, 0.06-7, 0.005-6 and 0.08-5 μg/mL on the ZIC-S1 and ZIC-S4 columns, respectively. The suggested methods demonstrated high precision (RSD ≤ 1.15), linearity (r2 ≥ 0.9964), and accuracy (between 98.88 and 101%) with detection and quantitation limits of (0.0021-0.0550 and 0.0063-0.1666 μg/mL, respectively) for all target pharmaceuticals. Conclusion: The suggested methods have been validated and applied to pharmaceutical formulations, indicating that it is suitable for therapeutic drug monitoring of these medications.