Current Pharmaceutical Analysis - Volume 19, Issue 7, 2023

Background: The Bai ethnologic herb Radix Dactylicapnotis, the root and tuber of Dactylicapnos scandens (Papaveraceae), is used for clearing heat, relieving pain, and achieving hemostasis and antihypertensive effects. Objective: The study aimed to develop a quantitative method for determining the protopine content in Radix Dactylicapnotis by using proton nuclear magnetic resonance (¹H NMR) spectroscopy. Methods: The deuterium solvent, internal standard, and NMR parameters were optimized. The quantitative method was validated by linearity, precision, accuracy, repeatability, and stability, as well as limit-of-detection (LOD) and limit-of-quantitation (LOQ) assays. Results: A mixture solution consisting of 500 μL of DMSO-d6 and 20 μL of D2O enabled satisfactory separation of the signals to be integrated into the ¹H NMR spectrum. Trimethyl benzene-1,3,5- tricarboxylate (TMBT) was selected as an internal standard. The integration of δ 6.05-6.08 corresponding to OCH2O was selected to quantify protopine. The developed quantitative method was found to be precise and accurate and to exhibit excellent linearity and range. The protopine content in Radix Dactylicapnotis could be quantified accurately using the featured signal. Conclusion: This is the first study to report quantitative ¹H NMR determination of protopine in Radix Dactylicapnotis. The study results indicate that quantitative ¹H NMR represents a feasible alternative to HPLC-based methods for the quantitation of protopine in Radix Dactylicapnotis, and is suitable for the quality control of Radix Dactylicapnotis.

Introduction: Drug abuse is a real threat that threatens the Indonesian nation in the form of non-military threats. The second most abused illicit substance in the world is methamphetamine (MA). Most of the methamphetamine users in the world live in East and Southeast Asia. Methamphetamine is a powerful stimulant of the central nervous system (CNS) that can make someone feel an increase in sympathomimetic. Methods: First, the preliminary test was conducted using a BioCare multi-drug rapid test and a specific methamphetamine rapid test that works based on the Lateral Flow Assay (LFA) principle. Second, the sample preparation used Liquid-Liquid Extraction (LLE). Third, GC-MS confirmed the test using GCMS Shimadzu-QP2020NX-GC-2030 (HP-5MS column, Length 30 m, diameter 0.25 m, film 0.25). Results and Discussion: The Preliminary test shows that the urine is positive and contains methamphetamine and amphetamine. Based on the sample chromatogram, There are 2 peaks, which are methamphetamine and amphetamine compounds it can be seen by the retention time is 5.560 minutes (methamphetamine) a similarity index of 98% to methamphetamine compounds, and 4.885 minutes (amphetamine) with a similarity index of 96% to amphetamine compounds. Conclusion: The urine samples with code 210DGB from methamphetamine users were detected due to the presence of methamphetamine and amphetamine, which is the metabolite of methamphetamine.

Introduction: Lipid raft is found on the cell membrane and is considered a microstructure rich in cholesterol, phospholipids and target proteins that are insoluble in nonionic detergents at low temperatures. Methods: In this study, detergent and non-detergent methods were used to extract lipid rafts from different cells. With β-cyclodextrin as the negative control group, we analyzed and compared the effects of different extraction methods on the composition of lipid rafts in Caco-2 and U251 cells using three kinds of lysate, namely detergent method 1, detergent method 2 and non-detergent method, which could be extracted and collected via sucrose density gradient centrifugation. Western blotting and immunofluorescence were utilized to determine the location of lipid rafts via the proteins Caveolin-1 and Flotillin-1, which are the characteristic proteins P-gp and TrkA in cells. The total protein in the lipid raft was quantitatively determined through the BCA (detergent compatible) kit method. Results: The results showed that the total amount of lipid raft proteins extracted via the detergent method was more than that of the non-detergent method, while the content of β-cyclodextrin control histone that caused disruption of lipid rafts structure was the lowest. Conclusion: The detergent method extracted more abundant lipid rafts than the non-detergent method. Detergent method 2 did not only extract more fat raft layers, but also the extracted highest total protein content, wherein it demonstrated better extraction effect with more lipid raft layers and higher expression of target protein P-gp.

Background: Schisandra chinensis has been widely used. It has many pharmacological activities. Lignans, including schizandrol A, schizandrin A, schisandrin B, schisanhenol, gomisin E, gomisin H, gomisin J, gomisin N, etc., are the major active ingredients of Schisandra chinensis. Objective: In the present study, the liquid chromatography-tandem mass spectrometric (LCMS/ MS) method was developed for the simultaneous quantification of Schisandra lignans in normal rats. Methods: Nifedipine was used as an internal standard, and chromatographic separation was achieved on Agela Venusil C18 Plus (4.6*100 mm, 3μm). Aqueous solution containing 0.1% (v/v) formic acid was used as the mobile phase A, and methanol solution containing 0.1% (v/v) formic acid was used as the mobile phase B for gradient elution. The flow rate was 0.8 mL/min. Multiple reaction monitoring (MRM) mode with positive electrospray ionization was used to detect the analytes. Results: The calibration curves provided reliable responses at concentrations of 0.5-200 ng/ml for schizandrin A, schisandrin B, schisanhenol, gomisin E, gomisin H, gomisin N, concentrations of 10-200 ng/ml for schizandrol A, and concentrations of 5-200 ng/ml for gomisin J. The inter- and intra-day coefficients of variations (CVs) for the precision ranged from 6.70% (3.44%) to 11.66% (10.38%). The inter- and intra-day accuracies of eight lignans ranged from 95.70% (93.89%) to 104.59% (106.13%). No significant variation of any of the lignans occurred in the stability tests. Conclusion: The established method can be successfully applied to the pharmacokinetic study of the Schisandra lignan extract in normal rats.

Background: High-performance liquid chromatography is one of the most used analytical techniques in quality control in the pharmaceutical industry. Since it is a complex technique, it needs good practices that can contribute to compliance with regulatory requirements. Objective: This study aims to establish a diagnostic tool for Good Chromatographic Practices (GCP) for the self-assessment of a Quality Control Laboratory (QCL). Methods: The research was carried out on scientific bases, pharmaceutical legislation, as well as guides published by manufacturers. Results: Seven axes of action were identified: implementation, management, and continuous improvement of GCP in the laboratory; GCP in the installation, operationalization, qualification, and validation processes of the equipment and software; GCP in processes related to data management, including guidelines regarding access, generation, integrity, and traceability; GCP related to the management and use of consumables; GCP related to handling, maintenance, analytical and operational troubleshooting; GCP in the processes of preparation, use, and storage of analytical solutions and reagent solutions; and GCP related to the acquisition and processing of standards, samples, and results. These axes resulted in a diagnostic tool with 124 questions. Conclusion: The application of the GCP diagnostic tool provides the mapping of the routine and procedures related to the execution of the HPLC technique for quality control in the pharmaceutical industry, contributing to meeting regulatory requirements.

Introduction: The coronavirus disease-2019 (COVID-19) outbreak all over the world has led researchers to strive to develop treatment and preventive measures to control its progression. Methods: Molnupiravir, a prodrug of the synthetic nucleoside derivative N-4-hydroxycytidine was found to be a promising candidate against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Results: It could significantly reduce the risk of hospitalization and mortality among patients with positive SARS-CoV-2 reports. In this study, an RP-HPLC method with UV detection was developed to determine its dissolution and release in the capsule dosage form. The developed method was validated as per International Council for Harmonization (ICH) guidelines. Conclusion: The method was evaluated and validated for its applicability using various parameters. It was found to be a simple, rapid, selective, sensitive, accurate, precise, robust and rugged method.

Background: The Siddha-based polyherbal formulation known as “Kabusura Kudineer (Marketed)" and developed as “HYDALJSS08” hydroalcoholic polyherbal formulation contains some fifteen plant materials in a dried raw form. Due to its immuno-booster properties, the Ministry of Ayush, Govt of India, highly recommended the use of "Kabusura Kudineer" during the pandemic of COVID-19. Objective: The present study intends to expand and validate the analytical profile for Andrographolides (AP), and isolated Andrographolides (AP) from the Andrographis paniculata whole plant and in the Polyherbal Formulations (Marketed-Kabusura Kudineer, & Developed “HYDALJSS08”). Methods: One of the active components of “Kabusura Kudineer” marketed and developed as “HYDALJSS08” Hydroalcoholic Polyherbal formulation is kalmegh, also known as the king of bitter (Andrographis paniculata-Acanthaceae). Kalmegh composes active principal components of Andrographolides (AP), which are proven for their Anti-viral and immunomodulatory activity. The preliminary identification of AP and the sample was carried out by TLC and FT-IR. The liquid chromatography was performed on a Zorbaz SB C8 (250*4.6 mm & 5 μm). The mobile phase incorporated pH 2.8 phosphate buffer with Acetonitrile: Methanol (60:30:10). The flow rate of the mobile phase was 1 ml/min, and effluents were kept an eye on at 223 nm in a UV detector. The run time on the chromatogram was 10 min, and retention time was also observed. Results: The Rf value of Andrographolides (AP) was found to be 0.62. ICH guidelines were followed to carry out the Validation parameter. The retention time of AP was 2.5 min, and the Valid parameters of AP and system precision were as follows: SD (1831.11), % RSD (0.2), regression equations y = 41978 + x−10763, and correlation coefficient (R2) 0.9994. The adequate Linearity concentration was found to be 5 to 50 μg/ml, the value of LODs was 0.61μg /ml, LOQs was 2.01 μg/ml, method precision % RSD was 0.2, SD was 1597.1, and recovery was 99.9% and 101%. AP content found in a formulation (“Kabusura Kudineer” 1.48 μg/mL, developed “HYDALJSS08” Hydroalcoholic Polyherbal formulation- 0.48 μg/ml) and isolated Andrographolides from Andrographis paniculata was 112.4 μg/ml. Conclusion: The developed HPLC methods enabled simple, novel, rapid, easy, accurate, reproducible, and linear analysis of isolated andrographolides, and Siddha-based Polyherbal formulations.