Evaluation of amino acids and other related metabolites levels in end-stage renal disease (ESRD) patients on hemodialysis by LC/MS/MS and GC/MS.

End-stage renal disease (ESRD) is a rapidly increasing health problem, and every year, about 2 million ESRD cases are reported worldwide. Hemodialysis (HD) is the vital renal reinstatement therapy for ESRD, and HD patterns play a crucial role in patients' health. Plasma metabolomics is the potential approach to understanding the HD process, effectiveness, and patterns. The lack of protein vitality is a primary problem for HD patients, and the quantities of amino acids intracellularly and in the blood are considered to be a symbolic index of protein metabolism and nutrition conditions. In the current study, LC/MS/MS and GC/MS methods were developed for 29 targeted plasma metabolites and validated as per ICH bioanalytical method validation M10 guidelines. The 29 metabolites included 20 proteinogenic amino acids and nine other related metabolites. The methods were employed to measure the absolute quantities (µM) of the targeted metabolites in HD patients (n=60) before and after dialysis (PRE-HD and POST-HD), and compared with the healthy control (HC) group (n=60). Phenylacetylglutamine was found to be higher in both PRE-HD (72.88±14.5 µM) and POST-HD (26.62±7.9 µM), when compared to HC (1.61±0.6 µM). On the other hand, glutamic acid was lower in PRE-HD (14.90±6.5 µM), and POST-HD (13.6±6.1 µM) than that of HC (245.4±37.8 µM). The dialytic loss was found to be 52-45% for arginine, lysine, and histidine, while it was 38-26% for glycine, cysteine, proline, alanine, threonine, glutamine, valine, and methionine. The dialytic loss was low (≤12%) for aspartic acid, glutamic acid, asparagine, leucine, tyrosine, tryptophan, and isoleucine. Graphical abstract adapted from mass spectrometry templates by Biorender.com retrieved from https://app.biorender.com/biorender-templates .

Assessment of uremic toxins in advanced chronic kidney disease patients on maintenance hemodialysis by LC-ESI-MS/MS.

INTRODUCTION: In the advanced stage of chronic kidney disease (CKD), electrolytes, fluids, and metabolic wastes including various uremic toxins, accumulate at high concentrations in the patients' blood. Hemodialysis (HD) is the conventional procedure used worldwide to remove metabolic wastes. The creatinine and urea levels have been routinely monitored to estimate kidney function and effectiveness of the HD process. This study, first from in Indian perspective, aimed at the identification and quantification of major uremic toxins in CKD patients on maintenance HD (PRE-HD), and compared with the healthy controls (HC) as well as after HD (POST-HD). OBJECTIVES: The study mainly focused on the identification of major uremic toxins in Indian perspective and the quantitative analysis of indoxyl sulfate and p-cresol sulfate (routinely targeted uremic toxins), and phenyl sulfate, catechol sulfate, and guaiacol sulfate (targeted for the first time), apart from creatinine and urea in PRE-HD, POST-HD, and HC groups. METHODS: Blood samples were collected from 90 HD patients (both PRE-HD and POST-HD), and 74 HCs. The plasma samples were subjected to direct ESI-HRMS and LC/HRMS for untargeted metabolomics and LC-MS/MS for quantitative analysis. RESULTS: Various known uremic toxins, and a few new and unknown peaks were detected in PRE-HD patients. The p-cresol sulfate and indoxyl sulfate were dominant in PRE-HD, the concentrations of phenyl sulfate, catechol sulfate, and guaiacol sulfate were about 50% of that of indoxyl sulfate. Statistical evaluation on the levels of targeted uremic toxins in PRE-HD, POST-HD, and HC groups showed a significant difference among the three groups. The dialytic clearance of indoxyl sulfate and p-cresol sulfate was found to be < 35%, while that of the other three sulfates was 50-58%. CONCLUSION: LC-MS/MS method was developed and validated to evaluate five major uremic toxins in CKD patients on HD. The levels of the targeted uremic toxins could be used to assess kidney function and the effectiveness of HD.

Lutein and ß-carotene biosynthesis in Scenedesmus sp. SVMIICT1 through differential light intensities.

The study evaluated the biosynthesis of lutein and ß-carotene by Scenedesmus sp. SVMIICT1 under five different light intensities (50, 250, 500, 750 and 1000 µE/m2/s). Liquid chromatography/mass spectrometry (LC/MS) was used to determine relative quantities of lutein and ß-carotene. Relatively, high lutein content of 1.43 ± 0.04 and 0.70 ± 0.02 mg/g was found with 50 and 500 µE/m2/s conditions respectively. ß-Carotene content was quantified as 0.15 ± 0.01, 0.1 ± 0.01 and 0.12 ± 0.02 mg/g with 50, 250 and 500 µE/m2/s conditions respectively. The light intensities altered photosystem II and photosystem I. At 50 µE intensity, high chlorophyll content and high photosystem II quantum efficiency (FV/FM) was observed. Low FV/FM ratio of around 0.3 was detected in high light intensities (750 µE and 1000 µE) due to photoinhibition. Lipid fraction increased with increasing light intensity and the fatty acid profiles were similar in all five conditions.

ESI-MS/MS analysis of protonated N-methyl amino acids and their immonium ions.

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry-based metabolomics workstation. As it is possible to encounter all the N-methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N-methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI-MS/MS data of all methylated proteinogenic amino acids, except that of mono-N-methyl amino acids. In this study, the N-methyl amino acids of all the amino acids (1-21; including one isomeric pair) were synthesized and characterized by ESI-MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N-methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]+ ions of most N-methyl amino acids showed respective immonium ions by the loss of (H2 O, CO). The other most common product ions detected were [MH-(NH2 CH3 ]+ , [MH-(RH)]+ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N-methyl group. The isomeric/isobaric N-methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α-nitrogen of the N-methyl amino acids.

Characterization of degradation products of macitentan under various stress conditions using liquid chromatography/mass spectrometry.

RATIONALE: Stress testing of a drug candidate is an important step in the drug discovery and development process. The presence of degradation products in a drug affects the quality as well as the safety and efficacy of drug formulation. Hence, it is essential to develop an efficient analytical method which could be useful for the separation, identification and characterization of all possible degradation products (DPs) of a drug. Macitentan (MT) is an endothelin receptor antagonist (ERA) drug used to treat high blood pressure in the lungs. Comprehensive stress testing of MT was carried out as per ICH guidelines to understand the degradation profile of the drug. METHODS: MT was subjected to various stress conditions such as acidic, basic, neutral hydrolysis, oxidation, photolysis and thermal conditions; and the resulting degradation products were investigated using liquid chromatography/diode-array detection/electrospray ionization high-resolution mass spectrometry (LC/DAD/ESI-HRMS) and tandem mass spectrometry (MS/MS) techniques. An efficient and simple ultra-high-performance liquid chromatography (UHPLC) method has been developed using an Accucore C18 column (4.6 × 150 mm, 2.6 µm) using a gradient elution of 5 mM ammonium formate and acetonitrile as mobile phases. RESULTS: MT was found to degrade under acid and base hydrolysis stress conditions; whereas it was stable under oxidation, neutral hydrolysis, thermal and photolytic conditions. MT formed nine DPs (DP1 to DP9) and one DP (DP10) under acidic and basic hydrolytic conditions, respectively. All the degradation products (DP1 to DP10) were identified and characterized by LC/MS/MS in positive ion mode with accurate mass measurements. CONCLUSIONS: MT was found to be labile under hydrolytic conditions. The structures of the DPs were characterized by appropriate mechanisms. The proposed method can be effectively used for the characterization of MT and its DPs.