Development of LC-MS/MS method for quantification of Lurasidone using volumetric absorptive microsampling (VAMS); a comparative study between dried blood and plasma samples.

The ecological impact of biological, chemical, and analytical research practices, including toxic reagents and biohazardous waste, has led to the development of alternative sampling and extraction techniques for bioanalysis. Microsampling (sample volume < 50 µL) aligns with the 3Rs principle, allowing multiple sampling points from the same animal at different time points and improving animal welfare. A bioanalytical method was developed to investigate factors related to bioanalytical challenges and the implementation of microsampling techniques. An LC-MS/MS method for Volumetric Absorptive Microsampling (VAMS), 20 µL, was developed for quantifying Lurasidone using a liquid-liquid extraction technique. The method uses a C18, Phenomenex column for chromatographic separation and a mobile phase composition of Methanol, Acetonitrile, and Water with 0.1 % HFBA. The method was validated over a concentration range of 5.0 to 1200.0 ng/mL and achieved acceptable precision and accuracy. The recovery for analyte from VAMS was approximately 40% at four different concentrations and is consistent (%CV < 15), with no significant differences among HCT levels. The matrix factor ranged between 85.00 and 115.00 %, showing no substantial issues with reduced or enhanced signal. The stability data showed no significant degradation of LUR in VAMS samples when stored at room temperature for 15 days. The newly established method for Lurasidone confirmed the use of VAMS sampling method and its analysis on LC-MS/MS. Further, the data obtained from microsampling techniques was compared with conventional (plasma) technique, as proof-of-concept, and it confirms the agreement between the two methods. The study supports the advantages of microsampling in protecting the environment and animals while maintaining scientific judgement.

Biodistribution of lipid nanoparticle, eGFP mRNA and translated protein following subcutaneous administration in mouse.

Aim: Increased knowledge of biodistribution and pharmacokinetics of lipid nanoparticle (LNP)-encapsulated mRNA drug components may aid efficacy and safety evaluation. Methods: Mice were subcutaneously administrated LNP encapsulated enhanced green fluorescent protein mRNA and sampled up to 72 h after dosing. LNP, mRNA and translated protein were quantified by LC-MS, branched DNA and ELISA. Results: Highest levels of LNP and mRNA were detected in skin, followed by spleen, but also rapidly distributed to circulation. Translated protein showed high concentration in skin and spleen, but also in liver and kidney across 24 h where the LNP was cleared at 4 h. Conclusion: Subcutaneously dosing LNP encapsulated mRNA in mice resulted in a nonlinear relationship of LNP, mRNA and protein concentration across multiple tissues.

[Box: see text].

Preclinical Pharmacokinetic Study and Lung Penetration of a Coumarin Extracted from Zanthoxylum tingoassuiba.

The compound 6-methoxyseselin, derived from Zanthoxylum tingoassuiba, demonstrates various therapeutic properties, including vasorelaxation, antinociceptive, anti-inflammatory, and immunomodulatory effects, along with recently discovered antiasthmatic properties. This study aimed to evaluate its preclinical pharmacokinetics and pulmonary delivery in Balb/c mice. The method involved administering the compound via inhalation and intravenous routes, followed by blood sample collection for analysis using high-performance liquid chromatography with diode array detection (HPLC-DAD). The results indicated good linearity, precision, accuracy, and stability of the compound in the biological samples. Pharmacokinetic parameters such as the rate of elimination, half-life, clearance, volume of distribution, area under the curve, and mean residence time were determined for both administration routes, showing similar profiles. The lung concentrations were notably higher than the plasma concentrations, indicating significant lung penetration. These findings suggest 6-methoxyseselin as a promising candidate for new anti-asthmatic drugs, supported by its favorable pharmacokinetic profiles and high lung penetration factors. This study represents the first exploration of the pharmacokinetics and pulmonary delivery of 6-methoxyseselin in mice, highlighting its potential for further drug development.

Impact of introducing a new biological matrix into a validated bioanalytical method: Focus on matrix protein content.

International guidance on bioanalytical method validation recommends the practice of partial validation when introducing a new matrix from the same species into a previously fully validated assay. Planning the partial validation protocol should include an evaluation of analyte chemistry, consideration of sample container materials, and a comparison of properties between the relevant biological matrices. Transition of a serum/plasma-validated bioanalytical method to analysis from a low-protein matrix, such as urine, cerebral spinal fluid, or oral fluid can result in inconsistent analyte recovery. The low recovery can potentially be mistaken for signal suppression or lack of drug stability and may be more pronounced in low-concentration or low-volume samples. In addition, adsorption and absorption interactions with containers may be exacerbated in low-protein matrices. Several possibilities exist for mitigating the impact of non-specific binding and low-protein matrices, including surfactants, bovine serum albumin, and ß-cyclodextrin. Finally, higher matrix protein can facilitate analyte stability. Given all this, matrix protein content should not be overlooked when anticipating a partial bioanalytical method validation.

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 1-600 ng/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 RAL 13.53 ± 2.72, L-ORM nanoemulsion 65.07 ± 14.0 and RAL-nanoemulsion 59.27 ± 17.44 ng/ml. Conclusion: Future findings will contribute to the treatment of postmenopausal osteoporosis and breast cancer using L-ORM and RAL.

[Box: see text].

LC-MS/MS method for simultaneous estimation of raloxifene, cladrin in rat plasma: application in pharmacokinetic studies.

Aim: A newer LC-MS/MS method was developed and validated for the simultaneous quantification of raloxifene (RL) and cladrin (CL). Methodology: Both drugs were resolved in RP-18 (4.6 × 50 mm, 5 µ) Xbridge Shield column using acetonitrile and 0.1% aqueous solution of formic acid (FA) (70:30% v/v) as mobile phase by using biological matrices in female Sprague-Dawley rats using-MS/MS. Results: The developed method was found to be linear over the concentration ranges of 1-600 ng/ml, and lower limit of quantification was 1 ng/ml for RL and CL, respectively. Pharmacokinetic results of RL+CL showed Cmax = 4.23 ± 0.61, 26.97 ± 1.14 ng/ml, at Tmax(h) 5.5 ± 1.00 and 3.5 ± 1.00, respectively. Conclusion: Pharmacokinetic study results will be useful in the future for the combined delivery of RL and CL for osteoporosis treatment.

Matrix effect in bioanalytical assay development using supercritical fluid chromatography-mass spectrometry.

Matrix effect (ME) is commonly caused by coelution of compounds with target analytes, resulting in either suppression or enhancement of analyte ionization. Thus, to achieve the desired accuracy, precision, and sensitivity, ME needs to be evaluated and controlled during bioanalytical method development. As the application of supercritical fluid chromatography-mass spectrometry (SFC-MS) for analysis of biological samples has increased, ME using SFC-MS has also been investigated with a focus on the difference in ME in SFC-MS compared to other chromatographic techniques used for achiral separation in biological samples. Here, we provide a summary of the status of ME evaluation and mitigation in SFC-MS methods. This review presents an overview of the phenomenon of ME and methods for evaluating ME in bioanalysis. Next, the factors that can impact ME in SFC-MS-based bioanalytical methods are discussed in detail with an emphasis on SFC. A literature review of the evaluation of ME in targeted bioanalytical methods using SFC-MS is included at the end. Robust instrumentation, effective sample preparation, and superb separation selectivity are the foundations of reliable analytical methods as well as the ability to mitigate detrimental ME in SFC-MS methods.

Lessons learned from regulatory submissions involving endogenous therapeutic analyte bioanalysis.

Endogenous therapeutic analytes include hormones, neurotransmitters, vitamins, fatty acids and inorganic elements that are naturally present in the body because either the body produces them or they are present in the normal diet. The accurate measurement of endogenous therapeutic analytes poses a challenge when the administered exogenous therapeutic analyte and its endogenous counterpart cannot be distinguished. In this article, real case examples with endogenous therapeutic analyte bioanalysis during drug development in support of regulatory submissions are collected and presented. The article highlights common challenges encountered and lessons learned related to bioanalysis of endogenous therapeutic analytes and provides practical tips and strategies to consider from a regulatory perspective.

Simple HPLC-UV Method for Therapeutic Drug Monitoring of 12 Antiepileptic Drugs and Their Main Metabolites in Human Plasma.

The objective of the present report was to develop and validate a simple, selective, and reproducible high-performance liquid chromatography method with UV detection suitable for routine therapeutic drug monitoring of the most commonly used antiepileptic drugs and some of their metabolites. Simple precipitation of plasma proteins with acetonitrile was used for sample preparation. 10,11-dihydrocarbamazepine was used as an internal standard. Chromatographic separation of the analytes was achieved by gradient elution on a Phenyl-Hexyl column at 40 °C, using methanol and potassium phosphate buffer (25 mM; pH 5.1) as a mobile phase. The method was validated according to the FDA guidelines for bioanalytical method validation. It showed to be selective, accurate, precise, and linear over the concentration ranges of 1-50 mg/L for phenobarbital, phenytoin, levetiracetam, rufinamide, zonisamide, and lacosamide; 0.5-50 mg/L for lamotrigine, primidone, carbamazepine and 10-monohydroxycarbazepine; 0.2-10 mg/L for carbamazepine metabolites: 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine and carbamazepine-10,11-epoxide; 0.1-10 mg/L for oxcarbazepine; 2-100 mg/L for felbamate and 3-150 mg/L for ethosuximide. The suitability of the validated method for routine therapeutic drug monitoring was confirmed by quantification of the analytes in plasma samples from patients with epilepsy on combination antiepileptic therapy.

Novel aspects of taxifolin pharmacokinetics: Dose proportionality, cumulative effect, metabolism, microemulsion dosage forms.

Taxifolin (TFL) is a small drug molecule with a broad therapeutic potential limited by its poor aqueous solubility and excessive metabolism. Despite comprehensive research, some aspects of the TFL pharmacokinetics, e.g., dose proportionality and possible cumulative effect, remain unexplored. In the current study, we have tried to fill this gap. Our results revealed that the TFL pharmacokinetics in rats had nonlinear character in the dose range of 10-50 mg/kg after its single oral administration (AUC). For Cmax, the data are ambiguous: linearity was confirmed via the equivalence criterion and was disproved using the power model approach. Also, the cumulative drug effect was observed on the 4th day after its multiple-dose oral administration (25 mg/kg; compared to the 1st day). Interestingly, biologically active TFL metabolites such as aromadendrin and luteolin were putatively found in plasma samples, although they were previously detected only in feces. In addition, oil-in-water and water-in-oil microemulsions were fabricated to design novel drug delivery systems. These carrier dosage forms did not improve the TFL bioavailability but significantly affected its metabolism. To support pharmacokinetic studies, the bioanalytical liquid chromatography-tandem mass spectrometry method was developed and validated in the concentration range of 1-1000 ng/mL using candesartan as an internal standard. Liquid-liquid extraction with methyl tert-butyl ether was used to isolate the analytes from plasma followed by evaporation and reconstitution of the residues in acetonitrile. Thus, the present findings broaden our understanding of the TFL behavior in vivo and provide novel ideas and reference directions for its continued use in medical practice.

How does the order of sample analysis influence the matrix effect during LC-MS bioanalysis?

Mass spectrometry coupled with liquid chromatography is a valuable tool for drug development and personalised drug therapy. The matrix effect is caused by enhancing or suppressing the analyte signal intensity by the interfering compounds of biological fluids. The matrix effect may influence the reliability of the quantitative results. Thus, its evaluation is a critical part of bioanalytical method validation. Identified factors affecting the matrix effect are the physicochemical properties of the analyte, type of biological material, analytical conditions, the ion source construction and calculation method. The order of analysis of test samples (pure solutions and post-extraction spiked samples) is another factor possibly affecting quantifying the matrix effect variability between sources. Our primary goal was to find which experimental design - interleaved or set of blocks - is more sensitive to detect matrix effect variability. Additionally, to better understand the reason of variability, we evaluated the influence of chromatographic elution and the type of plasma (normal, lipemic or hemolyzed), co-elution, and carry-over of phospholipids. We used chemometric methods: Principal Component Analysis and Partial Least-Squares Discriminant Analysis. Although a comparable (but statistically different) matrix effect (%RSDMF) is observed using the interleaved and block schemes, for some compounds, the order of the samples strongly influences the results. The interleaved scheme was generally more sensitive in detecting the matrix effect than the block scheme. Thus, reporting the order of samples is needed to ensure the repeatability of experiments. Chemometrics suggests that lipemic samples analyzed in isocratic conditions are most prone to the matrix effect. Different compositions of matrix lots of the same type - especially lipemic - may influence method reliability. Thus, evaluating more than one source of lipemic and hemolyzed plasma is recommended.

A Validated Chiral LC-MS/MS Method for the Enantioselective Determination of (S)-(+)- and (R)-(-)-Ibuprofen in Dog Plasma: Its Application to a Pharmacokinetic Study.

The purpose of this study was to develop a method for simultaneously separating ibuprofen enantiomers using electrospray ionization (ESI) liquid chromatography with tandem mass spectrometry (LC-MS/MS). LC-MS/MS was operated with negative ionization and multiple reaction monitoring modes; transitions were monitored at m/z of 205.1 > 160.9 for ibuprofen enantiomers, 208.1 > 163.9 for (S)-(+)-ibuprofen-d3 [internal standard 1 (IS1)], and 253.1 > 208.9 for (S)-(+)-ketoprofen (IS2), respectively. In a one-step liquid-liquid extraction, 10 µL plasma was extracted with ethyl acetate:methyl tertiary-butyl ether of 7:3. Enantiomer chromatographic separation was carried out with an isocratic mobile phase consisting of 0.008% formic acid in water-methanol (v/v) at a flow rate of 0.4 mL/min on a CHIRALCEL® OJ-3R column (150 × 4.6 mm, 3 µm). This method was fully validated for each enantiomer and results were in compliance with the regulatory guidelines of the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. The validated assay was executed for nonclinical pharmacokinetic studies after oral and intravenous administration of racemic ibuprofen and dexibuprofen in beagle dogs.

A validated LC-MS/MS method for analysis of Cabergoline in human plasma with its implementation in a bioequivalent study: investigation of method greenness.

Cabergoline (CAB) is effective prolactin lowering drug. Evaluation of the bioequivalence for the new test product (0.5 mg CAB film-coated tablets) in Egypt is strongly needed for approval of the drug by the official health authority. Therefore, a highly sensitive and rapid (LC-MS/MS) method was validated for CAB analysis in human plasma. CAB was extracted from plasma via diethyl ether using Quetiapine (QUE) as an internal standard. Multiple reaction monitoring (MRM) in positive ion mode was used, m/z 452.3 → 381.2 for CAB and 384.2 → 253.1 for QUE. Separation was accomplished on a reversed-phase C18. FDA procedures for the bio-analytical method were followed. The method was used in the bioequivalence study to compare the test product (0.5 mg CAB) versus Dostinex tablets, on 24 healthy Egyptian volunteers. The total analysis time was 5.5 min for each sample which permits analysis of various samples per day. The linearity range was from 2.00 to 200.00 pg/mL for CAB. LOD and LOQ were found to be 0.5 and 1.6 pg/mL, respectively. The final greenness numerical value was 0.63 using AGREE tool. The results of pharmacokinetic parameter Tmax were 2.17, and 2.33 h; for test and reference products, respectively. The generic formulation of test product is considered bioequivalent to the reference product Dostinex 0.5 mg tablets and satisfies the requirements of the Egyptian market. The merits of the method over the previous published methods are low cost; availability of cheap internal standard; rapidness; use of acetonitrile-free solvents mobile phase.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

A versatile high-performance LC-MS/MS assay for the quantification of voriconazole and its N-oxide metabolite in small sample volumes of multiple human matrices for biomedical applications.

Voriconazole (VRC) pharmacokinetics, in particular its complex metabolism, is still not fully understood which challenges its optimal therapeutic use. To increase knowledge on the pharmacokinetics of this antifungal drug, it is essential to broaden the perspective and expand in vitro and clinical in vivo investigations in particular to aspects such as unbound plasma, target-site and metabolite concentrations. Innovative sampling approaches such as microdialysis, a minimally-invasive technique for the analysis of compound concentrations in target-site human tissue fluids, are associated with bioanalytical challenges, i.e. small sample volumes and low concentrations. Thus, a bioanalytical LC-MS/MS assay for the simultaneous quantification of VRC and its main N-oxide (NO) metabolite in human plasma, ultrafiltrate and microdialysate was developed and validated according to the European Medicines Agency guideline. Quantification was rapid, simple and feasible for clinically relevant concentrations from 5 to 5000 ng/mL in plasma and ultrafiltrate as well as from 4 to 4000 ng/mL in microdialysate. Due to the high sensitivity of the assay, only 20 µL of plasma or ultrafiltrate and 5 µL of microdialysate were required. For VRC and NO in all matrices, between-run accuracy was high with a maximum mean deviation of 7.0% from the nominal value and between-run precision was demonstrated by ≤ 11.8% coefficient of variation. Both compounds proved stable under various conditions. The assay suitability was demonstrated by the application to a clinical study quantifying simultaneously VRC and NO concentrations in plasma, ultrafiltrate and microdialysate. Additionally, the assay was successfully adapted for pharmacokinetic analyses in human tissue-derived in vitro experiments. Overall, by reducing the required sample volume, the bioanalytical method allows for an increased number of plasma samples in vulnerable populations, e.g. infants, and enables the generation of concentration-time profiles with a higher temporal resolution in microdialysis studies. Consequently, the developed assay is apt to elucidate the complex pharmacokinetics of VRC in clinical settings as prerequisite for therapy optimisation.

Replicates Number for Drug Stability Testing during Bioanalytical Method Validation-An Experimental and Retrospective Approach.

BACKGROUND: The stability of a drug or metabolites in biological matrices is an essential part of bioanalytical method validation, but the justification of its sample size (replicates number) is insufficient. The international guidelines differ in recommended sample size to study stability from no recommendation to at least three quality control samples. Testing of three samples may lead to results biased by a single outlier. We aimed to evaluate the optimal sample size for stability testing based on 90% confidence intervals. METHODS: We conducted the experimental, retrospective (264 confidence intervals for the stability of nine drugs during regulatory bioanalytical method validation), and theoretical (mathematical) studies. We generated experimental stability data (40 confidence intervals) for two analytes-tramadol and its major metabolite (O-desmethyl-tramadol)-in two concentrations, two storage conditions, and in five sample sizes (n = 3, 4, 5, 6, or 8). RESULTS: The 90% confidence intervals were wider for low than for high concentrations in 18 out of 20 cases. For n = 5 each stability test passed, and the width of the confidence intervals was below 20%. The results of the retrospective study and the theoretical analysis supported the experimental observations that five or six repetitions ensure that confidence intervals fall within 85-115% acceptance criteria. CONCLUSIONS: Five repetitions are optimal for the assessment of analyte stability. We hope to initiate discussion and stimulate further research on the sample size for stability testing.

Fosfomycin therapeutic drug monitoring in real-life: development and validation of a LC-MS/MS method on plasma samples.

Individualization of fosfomycin dosing based on therapeutic drug monitoring (TDM) of plasma concentrations could reduce drug-related adverse events and improve clinical outcome in complex clinical conditions. Quantification of fosfomycin in plasma samples was performed by a rapid ultraperformance liquid chromatography mass spectrometry method. Sample preparation involved protein precipitation with [13C3]-fosfomycin benzylamine salt as internal standard. The calibration curve ranged from 2 to 800 mg/L. Within- and between-day precision and accuracy, sensitivity, selectivity, dilution integrity, recovery were investigated and the results met the acceptance criteria. In patients, multiple drug dosing (every 6 or 8 hours) or in continuous administration were adopted, resulting in a large interpatient variability in drug concentrations (from 7.4 mg/L and 644.6 mg/L; CV: 91.1%). In critical care patient setting TDM can represent an important tool to identify the best fosfomycin dosing in single patients, taking into consideration clinical characteristics, infection sites and susceptibility of the treated pathogens.

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples.

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

A Pharmacokinetic Study of Ephedrine and Pseudoephedrine after Oral Administration of Ojeok-San by Validated LC-MS/MS Method in Human Plasma.

A sensitive and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was developed and fully validated for the simultaneous determination of ephedrine and pseudoephedrine in human plasma after oral administration of the herbal prescription Ojeok-san (OJS); 2-phenylethylamine was used as the internal standard (IS). Both compounds presented a linear calibration curve (r2 ≥ 0.99) over a concentration range of 0.2-50 ng/mL. The developed method was fully validated in terms of selectivity, lower limit of quantitation, precision, accuracy, recovery, matrix effect, and stability, according to the regulatory guidelines from the U.S. Food and Drug Administration and the Korea Ministry of Food and Drug Safety. This validated method was successfully applied for the pharmacokinetic assessment of ephedrine and pseudoephedrine in 20 healthy Korean volunteers administered OJS.

Development, validation, and application of an LC-MS/MS method for the quantification of the novel antibiotic drug lefamulin (Xenleta®) and its main metabolite 2R-hydroxy lefamulin in human plasma.

Lefamulin (Xenleta®) is a first in-class systemic pleuromutilin antibiotic that inhibits bacterial protein synthesis and selectively binds to a highly conserved region of the peptidyl transferase center of the bacterial 50S ribosomal subunit. A total of twenty-five Phase 1 clinical studies, one Phase 2 study in acute bacterial skin and skin structure infections (ABSSSI), and two pivotal Phase 3 studies in adults with community acquired bacterial pneumonia (CABP) have been completed. Xenleta® (lefamulin) has been approved by the FDA on August 19, 2019, by Health Canada on July 10, 2020, and by the EMA on July 28, 2020 for the oral and IV treatment of CABP in adults. For and during the clinical development, simple, sensitive, precise, and selective LC-MS/MS methods were developed and validated, first for lefamulin alone and later for the simultaneous quantification of lefamulin and its main metabolite 2R-hydroxy lefamulin in human plasma. Chromatographic separation in the current method was achieved on a reverse phase C18 column using gradient elution at a flowrate of 500 µL/min consisting of a mobile phase of water (A) and methanol (B) each containing 0.1 % formic acid (v/v) with a run time of 8.0 min. The detection and quantification of the analytes were performed on AB Sciex Triple Quad 5500 using multiple reaction monitoring operated in positive electrospray ionization mode after a simple plasma protein precipitation cleanup and dilution. The method was linear over the concentration range of 1.00-1 000 ng/mL (r ≥ 0.999) for lefamulin und 1.00-500 ng/mL (r ≥ 0.999) for 2R-hydroxy lefamulin. No significant matrix effects and a good extraction recovery were observed. The within- and between-run precision and accuracy were within the acceptable limits, and both analytes were found to be stable throughout the short term, long term, and freeze thaw stability studies. This current validated method was successfully applied in five Phase 1 and two Phase 3 studies.