US FDA Guidelines Research Articles

Greenness and whiteness appraisal for bioanalysis of quetiapine, levodopa and carbidopa in spiked human plasma by high performance thin layer chromatography.

A sustainable HPTLC-densitometric method was developed for quantitative determination of Quetiapine (QUET), Levodopa (LD) and Carbidopa (CD) in presence of Dopamine (DOP) as an internal standard. This applicable technique was achieved by spiking human plasma and extraction was performed using the protein precipitation approach. The mobile phase used was acetone, dichloromethane, n-butanol, glacial acetic acid and water (3: 2.5: 2: 2: 1.75, by volume). Method validation was done according to US-FDA guidelines and was able to quantify Quetiapine, Levodopa and Carbidopa in the ranges of 100-4000, 200-8000 and 30-1300 ng/mL, respectively. Bioanalytical method validation parameters were assessed for the studied drugs. Finally, the analytical suggested methodology was evaluated using various green and white analytical chemistry metrics and other tools, such as the green solvent selection tool, analytical eco-scale, green analytical procedure index, analytical greenness metric approach and the red-green-blue algorithm tool. The results revealed that the applied analytical method had a minor impact on the environment and is a relatively greener option than other previously reported chromatographic methods.

Enantioselective Separation and Pharmacokinetics of a Chiral 1,4-Dihydropyrimidine Derivative in Rats: A Combined Chromatography and Docking Approach.

Chirality in 1,4-Dihydropyrimidines influences their pharmacological properties and synthetic strategies. Enantiomers of chiral drugs often exhibit different pharmacokinetic profiles. Therefore, separating and studying individual enantiomers is crucial to optimize drug efficacy and safety. Enantiomeric separation of ±4-(4-chlorophenyl)-6-methyl-2-oxo-N-(O-toyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (DP-1), which is a 1,4-Dihydropyrimidine derivative is achieved on CHIRALCEL® OD-H column (particle size: 5 μm, inner diameter: 4.6 mm, length:150 mm), following by investigating the kinetic properties of (R) and (S) enantiomers. The separation was achieved with a mobile phase composed of 70% (v/v) isopropyl alcohol and 30% (v/v) n-hexane. For the bioanalytical study, acetonitrile was used to precipitate the rat plasma samples and validated the method according to USFDA guidelines. The validated bioanalytical method was then successfully applied to determine the pharmacokinetic parameters of the drug in biological samples. Molecular modeling techniques, specifically docking simulations, were employed to predict the elution order of DP-1 enantiomers. The docking results revealed moderate binding interactions between the enantiomers and the chiral stationary phase (CSP), which aligns with the theoretical expectation that stronger interactions lead to longer retention times on the column.

Novel Quantitative Estimation of Rucaparib and Bevacizumab in Rat Plasma Using LC-MS/MS & Study of Pharmacokinetics.

Rucaparib and Bevacizumab‘s bioanalytical evaluation was performed using an easy-to-use, effective, and repeatable LC-MS/ MS technique, with D6-Rucaparib and D6-Bevacizumab serving as internal standards. This work provides a summary of current developments in bioanalytical LC-MS/MS techniques using an organic mobile phase consisting of 50:50 formic acid and acetonitrile IN 0.1% as well as a Waters Symmetry C18 column. The results for stability, matrix effect, accuracy, precision, and recovery were all within allowable bounds. To test the targeted analytes in body fluids using pharmacokinetic research, a simple and efficient approach was created. This application included all accuracy, system appropriateness, linearity, and specificity characteristics that were used successfully for pharmacokinetic investigations in rats and that complied well with USFDA guidelines.

The use of an RP-HPLC-UV method for the analysis of oxcarbazepine in the presence of its preservatives; stability studies and application to human plasma samples

AbstractA simple, sensitive, selective, accurate and precise method was developed and fully validated for determination of oxcarbazepine (OXC) in presence of their preservatives and determination of oxcarbazepine (OXC) in human plasma. A reversed phase liquid chromatography (RP-HPLC) with UV detection techniques were applied for separation and quantification of studied drug OXC. Successful separation of the drug from methyl paraben (M.P.), propyl paraben (P.P.) and potassium sorbate (P.ST.) was achieved on a Kromasil C18 column (5 μm particle size, pore size 300 Å, l × I.D. 250 × 4.6 mm). The mobile phase that contain aqueous 0.05M potassium dihydrogen phosphate buffer (pH 7): acetonitrile, (50: 50, %v/v). The method was linear over concentration ranges 5.0–50 μg mL−1 for OXC. Bioanalytical validation of the developed method was carried out according to US-FDA guidelines and revealed a good linear relations over a range of (5.0–50), (0.5–10), (0.05–0.15), and (1.0–10) μg mL−1 for OXC, M.P, P.P, and P.ST, respectively, with a correlation coefficient (R2) of more than 0.999. Limit of detection (LOD) were 1.15, 0.03, 0.01 and 0.04 μg mL−1 for OXC, M.P, P.P, and P.ST, respectively, Intra and inter-day precisions, calculated as percentage relative standard deviation (% RSD), were lower than 2.0%. The developed method can be applied for routine drug analysis, therapeutic drug monitoring and bioequivalence studies through the analysis of plasma samples taken from blood bank.

Simultaneous estimation of paclitaxel and bortezomib via LC-MS/MS: pharmaceutical and pharmacokinetic applications.

Aim & Objective: This study evaluates the potential of combining paclitaxel (PTX) and bortezomib (BTZ) for breast cancer therapy.Materials & Methods: The nanoformulation was optimized via Box-Behnken Design (BBD), with method validation adhering to US-FDA guidelines.Results: Multiple reaction monitoring transitions for PTX, BTZ and internal standard were m/z 855.80→286.60, 366.80→226.00 and 179.80→110.00, respectively. Elution done on C18 Luna column with 0.1% FA in MeOH:10mM ammonium acetate. The size of nanoformulation was 133.9±1.97nm, PDI 0.19±0.01 and zeta potential -19.20±1.36mV. Pharmacokinetics showed higher Cmax for PTX-BTZ-NE (313.75±10.71ng/ml PTX, 11.92±0.53ng/ml BTZ) versus free PTX-BTZ (104±13.06ng/ml PTX, 1.9±0.08ng/ml BTZ).Conclusion: Future findings will contribute to the treatment of breast cancer using PTX and BTZ.

Evaluation of pharmacokinetic herb-drug interaction of diabecon and losartan by UHPLC-MS/MS

The diabecon is an ayurvedic herbal formulation that contains a mixture of herbs traditionally used as antidiabetic which is reported in the ayurvedic pharmacopeia of India and Indian Materia medica. The diabetic population has a common co-morbidity of hypertension for which losartan drug is commonly used for the treatment of hypertension. However, there is a lack of research on the pharmacokinetics interaction between diabecon and losartan. This research aims to investigate the influence of diabecon on the pharmacokinetics of losartan drugs in rats by establishing a highly sensitive ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) method. The method was validated according to the USFDA guidelines and was applied for the pharmacokinetic study. The lowest concentration of losartan detection in rat plasma was found to be 1 ng/mL and the accuracy and precision were within the linear range (1–1500 ng/mL). The results revealed that after 28 days of dosing diabecon, it has altered the pharmacokinetic parameters like area under the curve (AUC0-t), drug clearance (Cl/F), and rate of elimination (Ke) of losartan, which may happen due to enzyme induction. Although there was a change in the pharmacokinetic parameters of losartan when administered in the presence of diabecon, it was found to be nonsignificant in rats (p > 0.05). According to the best of our knowledge, this is the first article that discusses the pharmacokinetic herb-drug interaction between diabecon and losartan.

An Insight Into Risk Assessment and Reformulation of Drug Products Manufactured Using Benzene Grade Carbomer: A Regulatory Perspective.

Cancer has been an enormous pain point for patients and regulatory bodies across the globe. In Dec. 2023, the US FDA released guidance on benzene-grade carbomer formulations, which triggered pharmaceutical manufacturers to assess risk, test finished products, and reformulate drug products with benzene-grade carbomer. The immediate implementation of the stoppage of finished products with benzene-grade carbomers has threatened pharmaceutical excipients and finished product manufacturers. The gravity of this situation prompted the US Pharmacopeia to extend the deadline for discontinuation from August 1, 2025, to August 1, 2026, allowing manufacturers ample time for reformulation and regulatory compliance.There is an immediate need to understand the guidance and to learn how manufacturers should do the risk assessment and approach reformulation. This review provides an in-depth analysis of the risk assessment and reformulation processes involved in various dosage forms utilizing benzene-grade carbomer, supported by specific case studies.This review offers insights into navigating the USFDA guidelines to ensure formulation safety and compliance, thus enabling pharmaceutical practitioners to uphold the highest standards of patient care and tackle life cycle management challenges.The decision of the USFDA to restrict the usage of high benzene content of carbomer in the formulation is a welcome move. This article has shown a way for researchers to see opportunities in the path and provide best-in-class medicines to patients with a better formulation safety profile.

Application of newly developed and validated LC-MS/MS method for pharmacokinetic study of adagrasib and pembrolizumab simultaneously in rat plasma

Non-small cell lung cancer (NSCLC) is a significant subtype of lung cancer, and poses a dangerous global threat. One of the current approaches of NSCLC treatment is a combination therapy of adagrasib and pembrolizumab. Accurate monitoring of these drug concentrations in biological fluids is critical for treatment efficacy. Since no method was reported for simultaneous estimation of these drugs, this study focuses on the development of a validated LC-MS/MS bioanalytical method for simultaneous quantification of Adagrasib and Pembrolizumab in rat plasma. The analytes were extracted from the biological matrix through liquid–liquid extraction techniques using acetonitrile as extraction solvent. The analytes were separated on a Waters X-bridge phenyl C18 column, with a mixture of acetonitrile: 0.1 % TFA in water (50: 50 v/v) as mobile phase at an isocratic flow rate of 1.0 mL/min with a runtime of about 5 min. Adagrasib (m/z 605.12 → 201.62), Pembrolizumab (m/z 146.32 → 85.15), and Sotorasib (m/z 561.59 → 218.92) were determined by recording the mass spectra through multiple reaction monitoring in positive mode. The method was validated according to USFDA guidelines. The results demonstrate satisfactory linearity with an r2 value of 0.9998 in the ranges of 40–800 and 10–200 ng/mL, accuracy with mean percentage recovery of 95.22–98.59 % and 96.98–98.57 %, precision indicated with %RSD ranged between 0.39–1.91 % and 0.85–9.03 % for Adagrasib and Pembrolizumab respectively, and other key parameters. The developed method can determine the pharmacokinetic parameters to indicate the efficacy and safety of the drugs, and also can quantify selected drugs simultaneously in biological samples.

Integrated approach of white analytical chemistry and design of experiments to microwave-assisted sensitive and eco-friendly spectrofluorimetric estimation of mirabegron using 4-chloro-7-nitrobezofuran as biosensing fluorescent probe

The USFDA recently approved mirabegron, a novel once-daily β-3 adrenoceptor agonist for oral administration, as a transformative treatment for overactive bladder. Despite the existence of numerous analytical methods for the assay and bioanalysis of mirabegron, it’s perplexing that none have explored the domain of microwave-assisted sensitive spectrofluorimetric method for mirabegron estimation, even after extensive literature review. Adding to the enigma is the insistence of current analytical methods on using expensive and harmful organic solvents, posing a threat to marine life and the broader environment. Recently, the white analytical chemistry approach has been introduced to develop analytical methods that are cost-effective, environmentally friendly, and user-friendly. Consequently, a white analytical chemistry-based, sensitive, and eco-friendly spectrofluorimetric estimation of mirabegron has been initiated, using 4-Chloro-7-nitrobenzofurazan as a fluorescent biosensing probe. The development of this robust method involved a series of experiments designed to minimize solvent and time wastage. Through a combination of fractional factorial and Box-Behnken designs, researchers identified the critical variables and optimized the method to perfection. This method was validated according to the stringent ICH Q2 (R2) and USFDA guidelines, ensuring its reliability and accuracy. Once approved, this sensitive spectrofluorimetric method was tested, accurately estimating mirabegron levels in commercial formulations and rat plasma samples. To further enrich the study, a comprehensive evaluation of existing analytical methods was conducted alongside the proposed spectrofluorimetric method, using advanced tools like the AGREE calculator, GAPI software, and RGB model to assess their eco-friendliness and effectiveness in mirabegron estimation.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Enhanced oral bioavailability of levormeloxifene and raloxifene by nanoemulsion: simultaneous bioanalysis using liquid chromatography-tandem mass spectrometry.

Aim & objective: Levormeloxifene (L-ORM) and raloxifene (RAL) are selective estrogen receptor modulators used in the treatment of postmenopausal osteoporosis and breast cancer. Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous estimation of both drugs. Materials & methods: A quality-by-design (QbD) approach was used for the optimization of the nanoemulsion, and US FDA guidelines were followed for method validation. Results: Multiple reaction monitoring transitions were used for L-ORM (459.05→98.50), RAL (475.00→112.02) and internal standard (180.10→110.2). Analytes were resolved in a C18 column with 80:20 v/v% acetonitrile (ACN), 0.1% formic acid in triple-distilled water as a mobile phase. The developed method was linear over a concentration range of 1-600ng/ml. Pharmacokinetic results of free L-ORM-RAL and the L-ORM-RAL nanoemulsion showed Cmax of free L-ORM- 70.65±16.64, free RAL13.53±2.72, L-ORM nanoemulsion 65.07±14.0 and RAL-nanoemulsion 59.27±17.44ng/ml. Conclusion: Future findings will contribute to the treatment of postmenopausal osteoporosis and breast cancer using L-ORM and RAL.

Validation and Suitability Assessment of Multiplex Mesoscale Discovery Immunogenicity Assay for Establishing Serological Signatures Using Vaccinated, Non-Vaccinated and Breakthrough SARS-CoV-2 Infected Cases.

Antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are multi-targeted and variable over time. Multiplex quantitative serological assays are needed to provide accurate and robust seropositivity data for the establishment of serological signatures during vaccination and or infection. We describe here the validation and evaluation of an electro-chemiluminescence (ECL)-based Mesoscale Discovery assay (MSD) for estimation of total and functional IgG relative to SARS-CoV-2 spike, nucleocapsid and receptor binding (RBD) proteins in human serum samples to establish serological signatures of SARS-CoV-2 natural infection and breakthrough cases. The 9-PLEX assay was validated as per ICH, EMA, and US FDA guidelines using a panel of sera samples, including the NIBSC/WHO reference panel (20/268). The assay demonstrated high specificity and selectivity in inhibition assays, wherein the homologous inhibition was more than 85% and heterologous inhibition was below 10%. The assay also met predetermined acceptance criteria for precision (CV < 20%), accuracy (70-130%) and dilutional linearity. The method's applicability to serological signatures was demonstrated using sera samples (n = 45) representing vaccinated, infected and breakthrough cases. The method was able to establish distinct serological signatures and thus provide a potential tool for seroprevalence of SARS-CoV-2 during vaccination or infection.

Quality-by-design optimized RP-HPLC approach for the therapeutic drug monitoring of glibenclamide and fluoxetine in human plasma

Glibenclamide (GLB) and fluoxetine (FLX) were reported to have a potential drug-drug interaction when clinically co-administered. FLX is an inhibitor of CYP2C9 (the enzyme responsible for the metabolism of GLB), thus resulting in increased serum levels of GLB followed by severe hypoglycemia. Therefore, a sensitive, simple, and selective HPLC method is urgently needed for their simultaneous determination in plasma to be utilized for monitoring their therapeutic blood concentration levels. The current study introduces the first RP-HPLC approach for the simultaneous estimation of this binary mixture in less than 7 min. Adopting a quality-by-design (QbD) approach allowed for the designed approach to be optimized. Using a two-level full factorial design (25), the effects of various variables on the chromatographic responses were investigated and optimized. Hypersil BDS C18 column was used for the achievement of the optimum chromatographic separation at 19.9 °C using acetonitrile: sodium dihydrogen phosphate buffer (7.5 mM; pH 4, 60:40 % v/v) as a mobile phase, pumped isocratically at a flow rate of 1.036 mL/min. An injection volume of 30.0 μL was used for the binary mixture with UV detection at 230 nm. FLX and GLB calibration curves revealed an excellent linearity over the ranges of 0.025–10.00 and 0.02–10.00 µg/mL with limits of quantitation of 0.018 and 0.02 µg/mL, respectively. Due to the high sensitivity and selectivity, the method was successfully applied to the assay of the two drugs in spiked human plasma samples with reasonable % recoveries (92.70–108.50 %), indicating good bioanalytical applicability. Therefore, this study could be applied for the therapeutic drug monitoring of GLB and FLX in human plasma to control their potential drug-drug interaction. The suggested approach was also applied to estimate the investigated drugs in their dosage forms with reasonable % recoveries (96.80–102.80 %) and small % RSD values (less than 2 %). The method's excellent greenness was evaluated using three metric tools, including Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness (AGREE). Validation of the proposed method was performed according to ICH and US FDA guidelines.

Development and Validation of Bioanalytical Method for Estimation of Rivaroxaban using RP-HPLC with Liquid liquid extraction in Human Blood Plasma and its application in Bioequivalence Study

Rivaroxaban is andirect acting oralanticoagulant and factor Xa inhibitor. A simple, selective, precise and rapid RP-HPLC method for estimation of Rivaroxaban (RIVA) in human blood plasma was developed and validated. The sample spike in plasma was extracted using liquid liquid extraction were extracted with the organic solvent ethyl acetate as organic solvent. Apixaban as an internal standard. The compounds were analysed by Agilent HPLC was used with control panel software using UV detector on a Inertsil ODS (250mm x 4.6mm ID;5μ) column with an Flow rate of 1.2mL/min, an isocratic mobile phase consisting of 0.02M Ammonium acetate buffer: Acetonitrile (70:30%v/v). Different sample pre-treatment techniques were evaluated, but Liquid Liquid extraction was found to be satisfactory, with good recovery values of 93.70% for RIVA. The developed method is validated by ICHM10 and USFDA guidelines over the concentration range of 5.00 to 200.00 ng/ml in human blood plasma with R² =0.9993. Within-day precisions and accuracy for RIVA were found in 0.36% to 4.73% and 92.58% to101.82% respectively. The validated RP-HPLC method has been used successfully for both preliminary pharmacokinetic studies and therapeutic drug monitoring

Co-stimulatory pathway competitive assay development using Liquid chromatography–tandem mass spectrometry (LC-MS/MS)

T-cells play a significant role in the development of autoimmune diseases. The CD28-B7 costimulatory pathway is crucial for activating T-cells, and blocking this pathway is essential for treating autoimmune diseases. Therapeutic antibodies and fusion proteins that target costimulatory molecules like CD80, CD86, CTLA-4, and CD28 have been developed to explore the costimulation process and as targeted treatments. To advance our understanding of costimulation in autoimmunity and the inhibition of the costimulatory pathway, it is crucial to have an accurate, precise, and direct method for detecting and quantifying the soluble form of these molecules in body fluids and various biological systems. Herein, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying the four costimulatory proteins depending on the signature peptides derived from the soluble isoform of these proteins in multiple reaction monitoring (MRM) mode. The method was validated using the US FDA guidelines. The LOQ was determined as ∼0.5 nM for the four analytes, with quantification extended to 20 nM with a correlation coefficient of R2>0.998. The developed MRM method was used to analyze on-bead digested protein mixtures to establish a competitive assay for the CD28-B7 costimulatory pathway using CTLA4-Ig (Abatacept ™) as an FDA-approved drug for rheumatoid arthritis. The IC50 was determined to be 2.99 and 159.8 nM for sCD80 and sCD86, respectively. A straightforward MRM-based competitive assay will advance the knowledge about the costimulatory role in autoimmunity and the autoimmune therapeutic drug discovery, with the need for broad application on different in vitro and in vivo models to discover new targeted inhibitors.

Ocular bioanalysis of moxifloxacin and ketorolac tromethamine in rabbit lacrimal matrix using liquid chromatography-tandem mass spectrometry.

Aim: The fixed-dose combination of moxifloxacin (MOXI) and ketorolac tromethamine (KTR) is widely used for the treatment of bacterial keratitis. Thus, a new LC-MS/MS method was developed to determine MOXI and KTR in lacrimal fluid. Methods: Bioanalysis was performed using aShimadzu 8050 LC-MS/MS in electrospray ionization-positive mode and the method was validated per US FDA guidelines. Isocratic separation wasperformed with aWaters Symmetry C18 column using methanol and 0.1% formic acid containing deionized water (85:15, v/v). Results & conclusion: An easy, quickand selective method was established and applied to assess the ocular pharmacokinetic profile of a commercially available formulation containing MOXI and KTR. Based on thepharmacokinetic data, this work describes pharmacokinetics-based dosage regimen calculations and their clinical significance.

MARALIXIBAT IN RAT PLASMA AND ITS PHARMACOLOGICAL STUDIES (BIO-ANALYTICAL METHOD DEVELOPMENT AND VALIDATION BY LC-MS)

The bio-analytical method development of Maralixibat using Elobixibat as an internal standard is a convenient, fast, accurate, and consistent new LC-MS technique and was validated. The present work explains the development of the LC-MS/MS bio-analytical method by RP-18(150x4.6 mm, 3.5µ) column and a binary mixture(0.1% formic acid &amp; methanol) of organic mobile phase in the ratio 60:40. By using liquid-liquid extraction process, these drugs are removed from rat plasma. The linearity in the standard curve was observed under the experimental concentration range. 10%-200% (6-12ng/ml) of Maralixibat. The calibration plots were linear with a regression coefficient of R2&gt; 0.999. Precision, Accuracy, Stability results, and Matrix effect were observed within the acceptable limit. The method is more accessible and effective for analyzing the sample in the body fluids. The work represents that Specificity, Suitability, Accuracy, and linearity parameters ideally agree with the USFDA guidelines and are efficiently practiced in rat plasma for pharmacokinetic studies

Concurrent determination of anti-microbial and anti-inflammatory drugs in lachrymal fluid and tissue by LC-MS/MS: A potential treatment for microbial keratitis and its PK-PD evaluation

Unforeseen surfacing of microbial keratitis (MKT) over the years has led to a requisite for promising treatment strategy involving combination of antifungal and antibacterial agents. Subsequently, symptoms associated with MKT including inflammation and watery eyes require treatment with anti-inflammatory agents. Thus, a requirement of functional clinical treatment strategy involving combination of anti-inflammatory corticosteroids (Betamethasone) with antifungal polyene (Amphotericin B, AmB) and antibacterials macrolide (Azithromycin, AZT) and aminoglycoside (Neomycin, NEO). In the ensuing pursuit, a sensitive and fast simultaneous LC-MS/MS method of four drastically different analytes in rabbit tear fluid and cornea was developed and validated as per US-FDA guidelines. The gradient LC set-up was used with C18 column and flow rate of 0.55 mL/min along with short run time of 7 min. The calibration curves showed good linearity over the concentration range of 0.07–300 ng/mL, 1.00–400 ng/mL, 3.00–600 ng/mL and 8.00–900 ng/mL for AZT, AmB, NEO and BEM respectively. The bioanalytical method requires only 10 µL of ocular sample and analytes were extracted with fast protein precipitation with acidic methanol. Finally, the developed method was validated for selectivity, linearity (r2 > 0.99), precision, accuracy, matrix effects, and stability. PK-PD indices and dosing frequency was predicted using Phoenix WinNonlin Software, based on single dose ocular pharmacokinetics and MIC values of AmB, AZT and NEO. According to the PK-PD simulation, S. aureus and E. coli required 6 and 12 instillations of AZT per 24 h, respectively whereas 12 instillation of NEO requires per 24 h for S. aureus. The result suggests that to minimize antimicrobial resistance; drug, dose and dosing schedule depend upon the pathogen as well as the strain.

An eco-friendly ultra-performance liquid chromatography-mass spectrometry method for quantification of rivaroxaban and ticagrelor in rat plasma: grapefruit interactions.

Aim: An eco-friendly ultra-performance liquid chromatography-tandem mass spectrometry method was developed to study the pharmacokinetics of rivaroxaban and ticagrelor in rat plasma, utilizing moxifloxacin as an internal standard. The food-drug interaction between grapefruit juice and these drugs was also investigated. Methods: Liquid-liquid extraction was used. A nonporous stationary phase Agilent® Poroshell 120EC C18 column was used with methanol: 0.1% aqueous formic acid (95:5 v/v) as a mobile phase. The detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method's validation was conducted in accordance with US FDA and European Medicines Agency guidelines. Results & conclusion: Grapefruit juice should be ingested with caution in patients treated with antithrombotic medications as it may increase their plasma concentration, inducing bleeding, and requires close clinical monitoring.

Development and validation of a LC–MS/MS assay for tenofovir and tenofovir alafenamide in human plasma: Application in a bioequivalence study

Tenofovir alafenamide (TAF), a prodrug of tenofovir (TFV), is currently used for treatment of chronic hepatitis B as first line recommendation. The Vietnamese market is currently circulating a tenofovir alafenamide generic. This formulation has not been assessed for in vivo bioequivalence. This study has been performed with the aim of development, validation, and application of LC-MS/MS procedure for quantitation of TAF and TFV in human plasma. Internal standard (IS), and analytical parameters were investigated to find out the suitable IS and conditions. Chromatographic conditions were optimized by considering the column type, mobile phase component, concentration of the buffer solutions and strength, oven temperature, flow rate, and injection volume. Human plasma samples were treated by protein precipitation with acetonitrile. The assay was validated in compliance with US-FDA and EMA guidelines. This assay was applied to evaluate the bioequivalence of the generic and reference products of 25 mg TAF in the healthy Vietnamese subjects under fed conditions. TAF, TFV, IS were ionized using ESI and detected by MRM mode to obtain molecular and fragment ions for quantification. The recovery of all analytes from human plasma was above 70%. The chromatographic conditions contained a C6-Phenyl column and mobile phase including acetonitrile and 0.5% formic acid. The specificity, precision, accuracy, matrix effect of all the analytes were in the acceptable range. Thirty six subjects finished the fed trial. The two products’ geometric mean ratios for AUC0-t, AUC0-∞, and Cmax (80.00% to 125.00%) met the bioequivalence acceptance criteria. A LC-MS/MS procedure for simultaneous quantitation of TAF and TFV in human plasma was developed, validated, applied.