Development Of Liquid Chromatography-mass Spectrometry Method Research Articles

Ranolazine Quantification in Human Plasma: A QbD-Guided LC-MS Method Development and Validation

The present research outlines a comprehensive and systematic approach for the development and validation of a bioanalytical Liquid chromatography–mass spectrometry (LC-MS) method to quantify ranolazine in human plasma. The study employs principles of quality by design (QbD) to enhance method robustness, precision, and accuracy. The primary goals of this research were to create a reliable LC-MS method for the accurate quantification of ranolazine in human plasma and to validate this approach in accordance with established standards set by regulatory bodies. A secondary objective was to explore the application of QbD principles to optimize method performance. The study resulted in the successful development of an LC-MS method that exhibits exceptional specificity, sensitivity, accuracy, and precision in the quantification of ranolazine in human plasma. Specificity tests revealed no interference from endogenous plasma components. The method demonstrated good sensitivity with limit of quantitation (LoQ) of 5 ng/mL and limit of detection (LoD) of 2 ng/mL. Accuracy, as assessed through recovery studies, showed mean recoveries of 98, 99, and 101% for three different spiked concentrations. Precision, expressed as RSD, was below 5% for both intra-day and inter-day analyses. Linearity studies resulted in a calibration curve with an R² value of 0.9998. By employing QbD principles, the method demonstrated improved robustness, thus minimizing the impact of variability during sample analysis. Robustness experiments revealed minimal effects on precision and accuracy when varying critical parameters. This research not only presents a reliable LC-MS method for ranolazine quantification but also underscores the importance of incorporating QbD principles in bioanalytical method development. The validated method holds significant implications for clinical research, pharmacokinetic studies, and therapeutic drug monitoring. By enhancing the accuracy and robustness of ranolazine quantification, this method contributes to the advancement of pharmaceutical and clinical research, ultimately benefiting patient care and treatment outcomes.

A novel LC-MS method development and validation for the determination of phenyl vinyl sulfone in eletriptan hydrobromide

BackgroundThe aim of the present method is to develop and validate a specific, sensitive, precise, and accurate liquid chromatography-mass spectrometry (LC-MS) method for the estimation of the phenyl vinyl sulfone in the eletriptan hydrobromide. The effective separation of the phenyl vinyl sulfone was achieved by the Symmetry C18 (50 × 4.6 mm, 3.5 μm) column and a mobile phase composition of 0.1%v/v ammonia buffer to methanol (5:95 v/v), using 0.45 ml/min flow rate and 20 μl of injection volume, with methanol used as diluent. The phenyl vinyl sulfone was monitored on atomic pressure chemical ionization mode mass spectrometer with positive polarity mode.ResultsThe retention time of phenyl vinyl sulfone was found at 2.13 min. The limit of detection (LOD) and limit of quantification (LOQ) were observed at 1.43 ppm and 4.77 ppm concentration respectively; the linear range was found in the concentration ranges from 4.77 to 27.00 ppm with regression coefficient of 0.9990 and accuracy in the range of 97.50–102.10%. The percentage relative standard deviation (% RSD) for six replicates said to be injections were less than 10%.ConclusionThe proposed method was validated successfully as per ICH guidelines. Hence, this is employed for the determination of phenyl vinyl sulfone in the eletriptan hydrobromide.

Liquid Chromatography-Mass Spectrometry Method Development and Its Validation for the Estimation of Favipiravir and Remdesivir in the Rat Plasma

Liquid Chromatography-Mass Spectrometry Method Development and Its Validation for the Estimation of Favipiravir and Remdesivir in the Rat Plasma

LC-MS Method Development for Simultaneous Determination of Trans-3′-hydroxycotinine and Three Mercapturic Acids in Urine

The negative impact of tobacco smoke on the human body is due to a wide range of harmful substances including volatile organic compounds (VOCs). Some VOCs of tobacco smoke metabolize in human organism into mercapturic acids (MAs). The determination of the amount of MAs in readily available biological fluids, for example in urine, allows to assess the level of exposure of these VOCs in a particular person. It is useful to assess the impact of individual VOCs on the body together with the assessment of the intake of nicotine. The intake of nicotine can be determined by the content of its metabolites in the urine, in particular by the content of trans-3′-hydroxycotinine (tH-Cot). A joint assessment of the concentrations of trans-3′-hydroxycotinine and MAs in urine allows obtaining selective information about effects of different VOCs and nicotine on the smoker’s body. We have developed a liquid chromatography–mass spectrometry (LC-MS) method for simultaneous quantifying of tH-Cot and three MAs: N-Acetyl-S-(3-hydroxypropyl)cysteine (HPMA), N-Acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (HMPMA), N-Acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA). We used this method to quantify the levels of MAs and tH-Cot in the urine of a group of 15 smokers just before and 5 days after smoking cessation. For all studied compounds, we have found statistically significant changes in concentration on the fifth day of smoking cessation. The method developed can be used to jointly assess the levels of exposure to nicotine and VOCs in the study of various tobacco products.

Silica hydride based phases for small molecule separations using automated liquid chromatography–mass spectrometry method development

Silica hydride, or Type C silica, has been developed as an alternative chromatographic support material for liquid chromatography. There are various bonded phases available with this new support. For four such phases (Cholesterol, Bidentate C18, Diamond Hydride, and Diol), retention and selectivity behavior were investigated using liquid chromatography coupled with triple quadrupole mass spectrometry. A set of small molecules from several chemical classes of interest, and varying in their physicochemical properties, were chromatographed under both reversed-phase and aqueous normal phase modes. To screen the columns, column switching was performed using an automated platform controlled by associated software and an additional valve. A typical scouting gradient was implemented. The separation conditions were not further optimized since the goal was simply to evaluate the variable retention behavior of the phases and selectivity under generic conditions. Further, retention of the analytes were evaluated under isocratic conditions with varying percentages of organic phase to visualize the potential for dual retention modes on the same column for certain analytes. Four analytes (fentanyl, hydrocodone, hydromorphone, and matrine) showed dual mode retention behavior with all four phases. Especially, fentanyl exhibited dramatic “U-shaped” retention profiles on Cholesterol and Bidentate C18 phases. Overall, changes in the retention order between reversed phase and aqueous normal phases emphasized the potential for altered selectivity. Results showed that the Cholesterol phase provided the highest retention for most analytes compared to the other phases. The more polar Diol phase still provided good retention in reversed phase mode. Retention and selectivity were all highly reproducible.

Liquid chromatography-mass spectrometry method development for monitoring stress-related corticosteroids levels in pig saliva.

Biochemical response stressors results in an increase of adrenocortical activity. Before knowing the corticosteroid levels in saliva in a stressful situation, baselines salivary levels should be established. A method for simultaneous determination of five corticosteroids was developed, validated and applied to pig saliva at farms. The method employs solid-phase extraction (SPE) coupled with clean-up extraction step using silica cartridge in the same step followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS), using electrospray ionization (ESI) in positive mode. The overall method quantification limits range from 0.050 to 0.30μg/L for the enrichment of 1.0mL saliva samples and analyte recoveries are between 60 and 90% (RSD<11%). Some factors studied were: pig sex, breeds, and time at farm. The analytical method clearly shows that CRL and CRS levels of, respectively, 3.0 and 4.0μg/L in saliva can be indicative of maxima non-stress levels in different pig breeds at farm.

Orthogonal analytical screening for liquid chromatography–mass spectrometry method development and preparative scale-up

An analytical HPLC–MS screening methodology has been developed to improve preparative RP-HPLC–MS purifications in medicinal chemistry laboratories. Although several approaches have been previously described to optimize analytical separations, none of them met our needs for the optimization of preparative conditions. Our screening protocol is based on searching among several orthogonal conditions to find the optimum preparative separation. Five different buffer conditions, from low to high pH, two organic solvents, acetonitrile and methanol, and five stationary phases of different polarities and characteristics were used. The orthogonality of the system was demonstrated using both, a standard mixture and mixtures from synthesis. To carry out the screening one of the analytical “open access” HPLC–MS systems was modified to perform the analytical screening while maintaining the open-access functionality for synthesis reaction monitoring. A software tool for automated sample programming and data reporting was also developed.

A component tracking algorithm for accelerated and improved liquid chromatography–mass spectrometry method development

A method for tracking of sample components during liquid chromatography–mass spectrometry (LC–MS) method development has been proposed. The method manages to, fully automatically and without user intervention, find the chromatographic peaks in the data sets, discriminate them to sample components and track them when the separation conditions have been changed. The algorithm utilises the resolution obtained from all considered data sets and has the ability to discriminate the non informative parts. The technique has a great sensitivity even in cases where a majority of the tracked components cannot easily be spotted by means of traditional total ion chromatogram (TIC) or base peak chromatogram (BPC) representations. The method was tested on an experimental sample using six different columns and an average of 79% of the suggested sample components could be successfully tracked at a minimum area of 0.05% of the main component in the sample. 66 components with 79–92% of the total suggested component area were able to be tracked between all data sets. The method could be used to rapidly investigate selectivity during different types of separation conditions.

Development of LC-MS Method for Detection of Mutant Uromodulin Protein

Mutations in the uromodulin gene cause the autosomal disorders familial juvenile hyperuricemic nephropathy (FJHN) and medullary cystic kidney disease type 2 (MCKD2). However, methods to detect the mutant form of the uromodulin protein have not been developed. In this study, we developed a liquid chromatography-mass spectrometry (LC-MS) method for detection of the mutated uromodulin peptide (C148W). Our method can distinguish the mutant peptide, GWHWE, from wildtype peptide, GWHC*E. Using MS/MS analysis with a selected reaction monitoring (SRM) mode, peptide-specific fragment ions (m/z 714 → 381, 471, 567, and 679 for GWHWE and m/z 688 → 381, 445, 541, and 653 for GWHC*E) were detected.

Negative ion electrospray high-performance liquid chromatography–mass spectrometry method development for determination of a highly polar phosphonic acid/sulfonic acid compound in plasma: Optimization of ammonium acetate concentration and in-source collision-induced dissociation

A method, based on negative ion electrospray ionization (ESI) single-stage mass spectrometry coupled with HPLC, was developed for the determination of a squalene synthase inhibitor, BMS-187745, in human plasma. BMS-187745, a highly polar compound with both phosphonic acid and sulfonic acid groups, presented difficulties in developing plasma extraction and HPLC procedures. Precipitation of the plasma protein with methanol was finally chosen as the basis for sample preparation since extraction with water-immiscible solvents or with solid-phase extraction columns failed. It was essential to add ammonium acetate to the HPLC mobile phase, not only to enhance the retention of BMS-187745 but also to ensure a well-shaped chromatographic peak. While the use of ammonium acetate had the desired chromatographic effects, it had the undesirable consequence of suppressing the negative ion ESI signal. With the plasma extracts, the [M–H 2O–H] − ion ( m/ z 367) showed significantly lower chemical noise than the [M–H] − ion ( m/ z 385), and was thus chosen as the analytical ion for the selected ion monitoring. The signal of the m/ z 367 ion was significantly enhanced by the optimization of the in-source collision-induced dissociation (CID) of m/ z 385 to m/ z 367.