Current Pharmaceutical Analysis - Volume 17, Issue 10, 2021

Background: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method had been developed for the quantification of acotiamide in rat plasma and been applied to pharmacokinetic studies. However, there was no LC-MS/MS method been developed for the determination of acotiamide in human plasma and its pharmacokinetic study. Objective: A simple and fast LC-MS/MS method was established and validated for the quantification of acotiamide in human Received: plasma and was applied to a pharmacokinetic study. Methods: Sample preparation was accomplished Revised: Accepted: through protein precipitation, and chromatographic separation was achieved on a Welch, Ultimate XB-C18 column (2.1×50 mm, 3 μm) with a security guard cartridge C18 using a binary gradient with DOI: mobile phase A (Methanol) and B (the solution of 10 mM Ammonium acetate with 0.1% Formic acid) at a flow rate of 400 μL/min. Results: The retention time of acotiamide and its internal standard, acotiamide-d6 was 1.78 min and 1.79 min, respectively. The total run time was 4.0 min. The method was developed and validated over the concentration range of 0.500-100 ng/mL for acotiamide, with correlation coefficient greater than 0.9987. The extraction recovery was more than 108.43% and the matrix effect was not significant. The inter- and intra-day precisions were below 5.80% and accuracies ranged from 92.7 to 103.0%. Acotiamide was demonstrated to be stable in human plasma under the tested conditions. Conclusion: The validated LC-MS/MS method was successfully applied to study the pharmacokinetic profiles of acotiamide in human plasma after oral administration and has achieved satisfactory results.

Background: Establishing R, S-enantiomer (S-KT and R-KT) chiral resolution and determination methods for KT are of great significance. Objective: This study aimed to establish a high-performance liquid chromatography (HPLC) method for the resolution and determination of ketorolac tromethamine (KT) enantiomers. Methods: A CHIRALPAK AGP column (0.4 × 10 cm, 5 μm) was used with 10 mmol/L ammonium acetate (pH 5.5) and isopropanol (97:3) as the mobile phase. The detection wavelength was 324 nm, the flow rate was 1.0 mL/min, the column temperature was 25°C, and the injection volume was 5 μL. Results: The resolution between S-KT and R-KT was 2.8. S-KT and R-KT demonstrated a good linear relationship in the range of 3-60 μg/mL (r > 0.999). The average recoveries of S-KT and RKT were 99.2% and 99.8%, with relative standard deviations of 2.0% and 2.4%, respectively. Conclusion: The established method can be used for the resolution and determination of S-KT and R-KT.

Aim: The aim is to develop and validate a single quantitative method for all DPP-IV inhibitors (Alogliptin, Vildagliptin, Sitagliptin, Saxagliptin, and Linagliptin).

Background: Metformin is the most used antihyperglycemic drug in diabetes, therefore it is preferred to be combined with other classes of antihyperglycemic drugs, specially gliptins. Formulating these two drug classes in one pharmaceutical dosage form would be more efficacious to control hyperglycemia than having either drug alone.

Objective: This study was designed and performed to develop a new, simple, and reliable method of analysis for assaying simultaneously five types of gliptins (Alogliptin, Linagliptin, Saxagliptin, Sitagliptin, and Vildagliptin) and Metformin, which has not been documented before.

Methods: The present method was carried out using a C18 column (250x4.6 mm), 5μm particle size, mobile phase consisting of water to acetonitrile ratio 85:15% (v/v) and the pH was set by orthophosphoric acid at 3, 10 μl injection volume, 0.5 ml flow rate, 25oC temperature, and the eluent was monitored at 232 nm.

Results: The assay's selectivity showed no interference with inactive ingredients in the formulation, and the %recovery from each drug dosage forms at three different concentrations were within the acceptable limits of the ICH guidelines. Although the method showed robustness towards the flow rate, it showed significant change at 230 or 234 nm.

Conclusion: The present analytical method is comprehensive and universal for measuring the five drugs and Metformin. Such an analytical method can be applied to the present available combined drug dosage forms of Metformin and one type of gliptins and the possible future of triple combinations of two gliptins and Metformin.

Background: Breast cancer is a heterogenic disease that comprises of various morphologies with intrinsic subtypes and is principally responsible for casualties among female cancer patients. Triple-negative breast cancer (TNBC) is the most aggressive subtype with a high probability of relapsing, hence successful treatment can be quite challenging. The pathogenesis of TNBC remains ambiguous and the identification of dependable biomarkers for its early diagnosis is crucial to design a strategy for therapeutic armamentarium. Objective: To clarify the folate-dependent mechanism of action causing cell death and to unravel the potential biomarkers of TNBC to defeat this consequential public health burden. Methods: The MTT assay and the morphological examination via microscopy were carried out to examine the viability of the cells upon the administration of the blank folate-conjugated cyclodextrin nanoparticles. An untargeted metabolomic approach using Q-TOF LC/MS was performed. Multivariate analysis of the metabolomic profile was applied to the MDA-MB-231 cell line with the aim of comparing the untreated cells with the folate-conjugated cyclodextrin nanoparticles applied cells to detect possible biomarkers. Results: The spectrophotometric and microscopic analyses revealed that MDA-MB-231 cells underwent early apoptosis following the incubation with the folate-conjugated nanoparticles for 24 h of administration. Moreover, metabolomics profiling pointed out that hexose metabolism was significantly altered. Data mining procedures showed that glycolysis, mannose, fructose, and galactose were the most affected pathways in TNBC upon blank folate-conjugated cyclodextrin nanoparticle administration and this effect was determined to be cell-specific. A perturbed hexose pathway may be the explanation of selective cell death and decelerated cell growth seen in TNBC cells. Conclusion: Our study offers a new understanding of the underlying mechanisms of TNBC since we hereby provide evidence that hexose is one of the main driving forces for the metabolic mechanism over TNBC cells. This alternative mechanistic approach may markedly increase the effect of chemotherapy on TNBC.

Background: Telmisartan and rosuvastatin calcium fixed-dose combination double-layer tablet is used for hypertension and hyperlipidemia treatment. Owing to the critical problem in establishing the specificity, precision, and accuracy of the USP analysis method and simultaneous analysis method for both components, an analysis method that could be applied to researchers in the field is urgently needed. Objective: To develop and validate a reversed phase-high-performance liquid chromatography method for the quantitative analysis of dissolution samples of telmisartan and rosuvastatin calcium fixed-dose combination double-layer tablets, as a hypertension and hyperlipidemia treatment. Methods: The developed analysis method was validated according to USP Category I requirements. The validity of the quantitative assay of the dissolution test solution was determined based on the system suitability, specificity, linearity, accuracy, precision, and solution stability of the assay for the components. Results: The developed method was selective and precise. The retention times of telmisartan and rosuvastatin calcium was 8.1 min and 2.8 min, respectively, and system suitability was 0.217 and 0.17, respectively. Both components showed linearity at 10–120% concentration. The accuracy and precision were also within acceptable limits. The drugs in the dissolution samples were stable for a long time, no interaction occurred between the excipients and active pharmaceutical ingredients, and no interference was observed in the analysis. Conclusion: The quantitative analysis of the double-layered formulation of telmisartan and rosuvastatin calcium was accurate, selective, and precise. Therefore, this method can be recommended for use in the industrial quality control of telmisartan and rosuvastatin calcium formulations.

Background: Vildagliptin is a drug for the treatment of diabetes. DPP-IV inhibitor represents a new class of oral antihyperglycemic agents to treat patients with type 2 diabetes. Several RP-HPLC methods have been reported to determine Vildagliptin alone. However, it has been noted that there are no available stability-indicating methods in pharmacopeias (USP/BP/EP/JP) nor in the available literature to quantify known and unknown impurity patterns for vildagliptin in vildagliptin tablets. Objective: The aim of this study is to develop a new single, sensitive, robust and specific gradient RP-HPLC method to quantify known and unknown impurities and degradants of Vildagliptin in Vildagliptin tablets. Methods: Chromatographic separation has been accomplished on the Hypersil ODS column (250 x 4.6) mm, 5 μm with a mobile phase consisting of a mixture of Perchloric acid Buffer, methanol, acetonitrile and Triethylamine delivered at a flow rate of 1.0 mL minute^-1 and the detection wavelength 210 nm. The developed method was validated as per ICH guidelines. Results: Vildagliptin was found degraded significantly under oxidative and alkaline stress conditions. The degradation products were well resolved from Vildagliptin and its impurities. An analytical method was found linear, accurate and precise from LOQ (Limit of Quantification) level to 150% of impurity specification limit (0.5%). Conclusion: The method found sensitive, rapid and accurate quantification of known, unknown impurities and degradants. The peak purity results confirmed that the Vildagliptin peak was homogeneous and pure in all stress samples, thus proving the stability-indicating nature of the method.

Background: Impurities in pharmaceutical compounds can influence their clinical effects and represent a potential health risk. To ensure the safety and effectiveness of a drug, it is necessary to investigate potential impurities. Methods: In this paper, a new impurity was separated and characterized by two-dimensional high performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (2D HPLC-Q/TOF-MS) in negative electrospray ionization mode. The peak containing the new impurity, eluted from the first dimension chromatographic system, was selectively trapped by a switching valve based on its retention time and transferred to the second dimension chromatographic system, which was connected to the mass spectrometer. We obtained MET-TA by chemical synthesis, and its structure was characterized by MS/MS and further confirmed by nuclear magnetic resonance (NMR). Results: The impurity was found to be (2S, 3S)-2,3.-dihydroxy-4-((1R,2S)-1-hydroxy-1-(3-hydroxyphenyl) propan-2-yl)amino)-4-oxobutanoic acid, labelled as MET-TA. In this study, we investigated the mechanism of formation of MET-TA, and found that it was the amidation product of metaraminol and tartaric acid. Conclusion: The identification, structural elucidation, synthesis and most probable mechanism of formation of MET-TA are discussed in detail in this paper.

Background: Cardiovascular diseases (CVD) have been reported in 8%-16% of patients with 2019 coronavirus disease (COVID-19). Digoxin is one of the main drugs to treat CVD. Objective: The clinician conducted therapeutic drug monitoring (TDM) of digoxin according to the drug usage on patients to monitor the concentration of digoxin, so as to avoid its toxic and side effects, and provide a theoretical reference for clinical usage of digoxin in patients with COVID-19. Methods: A method for quantifying digoxin concentration in plasma with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was developed. After simple protein precipitation of plasma with methanol, digoxin and its internal standard (digoxin-d3) were detected in the positive ion mode using multiple reaction monitoring. Results: Plasma digoxin in the range of 0.2 - 10 ng/mL had good linearity. The UPLC-MS/MS method was validated with inter-run accuracies ranging from 91.3% to 107.4% and precision less than 13%. Nine plasma samples (5 at valley concentration and 4 at follow-up after stopping dosing) from three patients with COVID-19 were tested. The mean plasma digoxin concentration was 0.73 ng/mL (ranged from 0 to 1.31 ng/mL). Digoxin was detected at the concentration of 0.93 ng/mL after stopping drug administration for 14 days. Conclusion: In this study, we established a simple UPLC-MS/MS method using protein-precipitation to perform TDM of digoxin in patients with COVID-19, and found that about 56% of digoxin plasma concentration was within the treatment window (0.8 - 2.0 ng/mL). Digoxin can be remained in the body for nearly 14 days in severe patients with COVID-19 after stopping dosing.

Background: There is no single chiral method for the quantitation of teneligliptin stereoisomers by high performance liquid chromatography (HPLC). Hence, there is a need for the quantification of teneligliptin (TNGP) and its stereoisomers. Objective: The main aim of the research work is to develop a novel simple, selective, precise and accurate HPLC method for the separation of TNGP and its stereoisomers. Methods: Different screening trials were executed by changing the mobile phase compositions to normal phase and polar mode and also by utilizing the different immobilized polysaccharide chiral columns like CHIRALPAK IA, IC, ID, IE, IF and IG. All the stereoisomers were eluted with high resolution, on CHIRALPAK IC-3 (4.6×250 mm), 3 μm chiral stationary phase (CSP) with a flow rate of 0.7 mL/min. The chromatographic system was processed with isocratic mode comprising ethanol: acetonitrile: ethanolamine in the proportion of 90:10:0.1% v/v/v with a column oven temperature of 15°C and detection wavelength of 250 nm. Results: The limit of detection (LOD) and limit of quantification (LOQ) values of TNGP(API), R,S-isomer, S,R-isomer and R,R-isomer were found to be 0.036/0.11, 0.029/0.09, 0.038/0.011 and 0.020/0.06 μg/mL, respectively. The method was found to be precise, accurate and linear (R2 > 0.999). Conclusion: The developed method was successfully applied for the quantification of bulk drug without any interference with the extraneous components. Hence, the method can be utilized successfully in the pharmaceutical organizations for the separation and quantification of TNGP isomers.

Background: Thiazide diuretics are listed as one of the first-line antihypertensive treatment agents. Hydrochlorothiazide is mainly formulated with many antihypertensive agents as combined pill therapy. Objective: In this study, a new, simple, rapid and sensitive LC-MS/MS assay was developed, optimized and validated for the simultaneous quantification of commonly prescribed cardiovascular combined medications that are used for hypertension management, including Hydrochlorothiazide (HCT) in the presence of irbesartan (IRB), candesartan (CAN) and bisoprolol (BISO) in human plasma. Methods: RP-HPLC separation of analytes and internal standard (atorvastatin) was performed on Zorbax Eclipse XDB-C18 (4.6×50 mm 5-Micron) using acetonitrile: (0.05%) formic acid (80:20), as an eluting system with a run time of less than 5.0 min. Results: Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 84.30%, the accuracy was within 86.82–110.17 % and the RSD was below 10% for all the studied compounds. Conclusion: The suggested method is highly appropriate for routine assays of the studied combination therapy in view of the lower LLOQ, simple extraction process, and short run time. The method can be further investigated and used for pharmacokinetics studies of the studied different antihypertensive combinations.

Background: The reaction between N¹-methylnicotinamide iodide (NMNI) and the active methylene group-containing compounds yields a fluorescent product, which can be used in the quantitative determination of certain drugs. Objective: The objective is to develop, adapt, and validate a simple spectrofluorometric method for the quantitative analysis of modafinil 1, budesonide 2, allicin 3, and florfenicol 4. Methods: Spectral analysis (elemental analysis, ¹H, ¹³C NMR, MS), computational methods (geometry optimization, Gibbs free energy and pKa calculations) and spectrofluorometric approaches were conducted. Result: A C4 cyclization pathway was confirmed for the formation of the fluorophore. The spectrofluorometric method showed good linearity (R2= 0.996-0.999) over concentrations of 1-50 ng/mL, 0.5-5 ng/mL, 60-150 pg/mL, and 1-15 ng/mL of the standard solutions of 1, 2, 3, and 4, respectively. Conclusion: The proposed method is simple and suitable for quality control and assurance studies.