Metabolites In Clinical Samples Research Articles

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

A Circular Network of Coregulated L-Threonine and L-Tryptophan Metabolism Dictates Acute Lower Limb Ischemic Injury.

Lower limb ischemia is characterized by reduced arterial perfusion in the lower limbs, leading to tissue ischemia and cell death. It is primarily caused by thrombosis and the rupture of arterial plaques, resulting in damage to ischemic muscle tissues. Metabolic processes are crucial in its development. Herein we combined single-cell data with metabolomics data to explore the pathways and mechanisms influencing lower limb ischemia. We analyzed single-cell and metabolomics data. In single-cell analysis, we identified different cell subpopulations and key regulatory genes, and biological enrichment analysis was performed to understand their functions and relationships. For metabolomics, mass spectrometry and chromatography techniques were employed to analyze metabolites in clinical samples. We performed differential analysis, correlation analysis, and Mendelian randomization to determine the relationships between key metabolites and genes. Nebl, Dapl1, Igfbp4, Lef1, Klrd1, Ciita, Il17f, Cd8b1, Il17a, Cd180, Il17re, Trim7, and Slc6a19 were identified to play a crucial role in lower limb ischemia. Important metabolites included L-threonine and L-tryptophan. The metabolism of L-threonine and L-tryptophan is linked to lower limb ischemia and thrombosis. B0AT1, encoded by SLC6A19, is closely related to these metabolites and appears to play a key role in lower limb ischemia development. Our analysis revealed the roles of key genes and metabolites in lower limb ischemia. These findings enhance our understanding of the pathogenesis of lower limb ischemia and provide new insights into its prevention and treatment.

Quantitative determination of lurbinectedin, its unbound fraction and its metabolites in human plasma utilizing ultra-performance LC-MS/MS.

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods to quantify total lurbinectedin, its plasma protein binding to derive the unbound fraction and its main metabolites 1',3'-dihydroxy-lurbinectedin (M4) and N-desmethyl-lurbinectedin (M6) in human plasma, were developed and validated. For lurbinectedin, sample extraction was performed using supported liquid extraction. For metabolites, liquid-liquid extraction with stable isotope-labeled analogue internal standards was used. Plasma protein binding was evaluated using rapid equilibrium dialysis. In vitro investigations at different plasma protein concentrations were carried out to estimate dissociation rate constants to albumin and alpha-1-acid glycoprotein (AAG). Calibration curves displayed good linearity over 0.1 to 50 ng/mL for lurbinectedin and 0.5 to 20 ng/mL for the metabolites. Methods were validated in accordance with established guidance. The inter-day precision and accuracy ranged from 5.1% to 10.7%, and from -5% to 6% (lurbinectedin in plasma); from 3.1% to 6.6%, and from 4% to 6% (lurbinectedin in plasma:PBS); from 4.5% to 12.9%, and from 4% to 9% (M4); and from 7.5% to 10.5%, and from 6% to 12% (M6). All methods displayed good linearity (r2 >0.99). Recovery was evaluated for lurbinectedin in plasma:PBS (66.4% to 86.6%), M4 (7.82% to 13.4%) and M6 (22.2% to 34.3%). The method for lurbinectedin in plasma has been applied in most clinical studies, while the plasma:PBS and metabolites methods were used to evaluate the impact of special conditions on lurbinectedin PK. Lurbinectedin plasma protein binding was 99.6% and highly affected by AAG concentration. These UPLC-MS/MS methods enable the rapid and sensitive quantification of lurbinectedin and its main metabolites in clinical samples.

Label-free and de-conjugation-free workflow to simultaneously quantify trace amount of free/conjugated and protein-bound estrogen metabolites in human serum

Different chemical forms of sex hormones including free/conjugated metabolites as well as their protein/DNA adducts in human serum are a panel of important indicators of health conditions. It is, however, hard to quantify all species simultaneously due to the lack of general extraction, derivatization, and de-conjugation methods. Here we developed a label-free and de-conjugation-free workflow to quantify 11 free/conjugated estrogen metabolites including depurinating DNA and protein adduct forms of 4-hydroxyestradiol (4OHE2) in human serum. Acetonitrile acts as an excellent solvent to purify adducted and non-adducted human serum albumin (HSA) by precipitation as well as to extract free/conjugated metabolites and depurinating DNA adducts from the supernatant by salting-out effect. The adduction level of 4OHE2 on HSA was determined by proteomics; free/conjugated metabolites were quantified by a newly developed microflow liquid chromatography (microflow LC)-nanoelectrospray ionization (nanoESI)-multiple reaction monitoring (MRM) method with high reproducibility (7–22% RSD, n > 3) and sub-picogram levels (0.6–20 pg/mL) of quantification limits (S/N = 8) by using non-pulled capillary as nano-ESI emitter. This workflow was demonstrated to reveal endogenous adduction level of 4OHE2 on HSA as well as circulation levels of free/conjugated metabolites in clinical samples. 4OHE2 in human serum were solely detected as protein-bound form, indicating the merit of such integrated platform covering unstable or active metabolites. Compared to traditional methods using labeling or de-conjugation reaction, this workflow is much simplier, more sensitive, and more specific. Moreover, it can be widely applied in omics to concurrently access various bio-transformed known and un-known markers or drugs.

Quantitation of mycophenolic acid and metabolites by UPLC-MS/MS in renal transplant patients.

Mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), specific inhibitors of lymphocyte proliferation, are commonly used as adjuvant therapy with calcineurin inhibitor agents after kidney transplantation. After administration, MMF and EC-MPS are hydrolyzed to mycophenolic acid (MPA), the active form of the drug, which must be monitored due to its narrow therapeutic window, drug-drug interactions, and large intra-and inter-individual pharmacokinetic variability despite a fixed dose. Monitoring plasma MPA level is recommended to maintain the drug within the therapeutic window, optimize its efficacy, and minimize side effects. This study aims to develop a method for quantifying MPA and its major metabolites (mycophenolic acid glucuronide [MPAG]) using on-line solid phase extraction (SPE) coupled with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in kidney transplant patients. The linearity of MPA and MPAG were 0.3-13.6 μg/mL and 2.6-232.9 μg/mL, respectively (r2 > 0.999). The relative error of accuracy was <15%. The within-run and between-run imprecision was <5.8%. No carryover, ion suppression, or ion enhancement were observed. This method was used to analysis of 351 plasma samples from renal transplant patients after MMF or EC-MPS using this method showed large pharmacokinetic variability between patients. Analysis of the same samples by immunoassay showed a large positive bias compared with our validated UPLC-MS/MS method, averaging 15.1%. These results suggest that this UPLC-MS/MS method is more effective than immunoassay for quantitation of MPA and its metabolites in clinical samples.

Molecular tools for the diagnosis of periodontitis

Periodontitis is a multifactorial chronic inflammatory disease associated with dysbiotic biofilms and aberrant host inflammatory response. It is characterized by destruction of the tissues that support the teeth. Periodontitis is the major cause of tooth loss in adults significantly affecting the quality of life and is associated with many chronic non communicable diseases by contributing to systemic inflammatory burden. Early and accurate diagnosis is the key to the successful management of periodontitis as the entire treatment plan, prognosis, and maintenance directly depend on the quality of periodontal diagnosis. Traditionally the diagnosis of Periodontitis is based on recording medical and dental history, periodontal examination and radiographic findings. The current periodontal diagnostic process reveals only historical tissue destruction and does not provide any information regarding current disease activity, future progression or for monitoring response to therapy. For these reasons, new molecular diagnostic aids are being developed that allow an early detection of disease, determine the presence of current disease activity, predict sites at risk for future breakdown and monitor the response to periodontal therapy. Advanced molecular diagnostic techniques are a class of diagnostic tests that are used to detect and measure nucleic acids, proteins or metabolites in clinical samples to identify risk factors, screen asymptomatic patients, provide more accurate diagnosis and guide the process of development of an ideal therapeutic intervention. This paper provides a review of the molecular diagnostic tools that have the potential to be utilized for diagnosis and management of periodontitis.

An Ultrafast Ultrahigh-Performance Liquid Chromatography Coupled With Tandem Mass Spectrometry Method for Cannabidiol Monitoring in Pediatric Refractory Epilepsy.

Cannabidiol (CBD) is a nonpsychoactive natural product that has been increasingly used as a promising new drug for the management of neurological conditions such as refractory epilepsy. Development of rapid and sensitive methods to quantitate CBD is essential to evaluate its pharmacokinetics in humans, particularly in children. The objective of this work was to develop and validate an ultrafast ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) method for CBD quantitation that is capable of detecting major CBD and tetrahydrocannabinol (THC) metabolites in the plasma of pediatric refractory epilepsy patients. Eight-point CBD calibration curves were prepared using 60 µL of plasma from healthy volunteers. Samples were analyzed in a Shimadzu Nexera X2 UHPLC system, which was coupled to a Sciex QTRAP 6500 mass spectrometer. Chromatography was optimized in acetonitrile (ACN)/water with a 70%-90% gradient of ACN in 2 minutes. Multiple reaction monitoring transitions of major CBD and THC metabolites were optimized in patient plasma. The optimized UHPLC-MS/MS method was validated for the linear range (1-300 ng/mL) of CBD (r2 = 0.996). The limit of quantification and limit of detection were 0.26 and 0.86 ng/mL, respectively. Accuracy and precision met the acceptable validation limits. CBD recovery and matrix effects were 83.9 ± 13.9% and 117.4 ± 4.5%, respectively. The method was successfully applied to quantify CBD and detect the major CBD and THC metabolites in clinical samples. 7-COOH-CBD was the most intensely detected metabolite followed by glucuronide conjugates. A simple and sensitive method for rapidly monitoring CBD and identifying relevant metabolites was developed. Its applicability in samples from children treated for epilepsy was demonstrated, making it an excellent alternative for performing pharmacokinetic studies.

Voltammetric Methods Used in the Determination of Nucleoside Reverse Transcriptase Inhibitors

Background: The Human Immunodeficiency Virus (HIV) has now been established as the causative agent of the Acquired Immunodeficiency Syndrome (AIDS) and exactly 25 antiretroviral drugs have been formally approved for clinical use in the treatment of AIDS. The life quality and duration of HIV-positive patients have increased with the usage of antiretroviral drugs in the treatment of AIDS. Nucleoside Reverse Transcriptase Inhibitors (NRTIs) are one of the subgroups of antiretroviral. Objective: The quantification of drugs is important, as they make positive contributions to dose adjustments in practice. Voltammetric methods are very powerful analytical methods used in the pharmaceutical industry because of the determination of therapeutic agents and/or their metabolites in clinical samples at extremely low concentrations (10-50 ng/ml). Methods: This review mainly includes the pharmacological properties and recent determination studies by voltammetric methods from pharmaceutical dosage forms and biological samples of eight NRTIs group antiretroviral drugs (zidovudine, abacavir, adefovir, entecavir, zalcitabine, didanosine, emtricitabine, lamivudine) that are used in the clinic and show electroactive properties, were performed. Conclusion: Due to the variety of working electrodes in voltammetric methods, it is possible to choose the electrode that best responds. In this way, the analysis of NRTIs was possible at lower concentrations in pharmaceuticals and biological samples with voltammetric methods in these studies without the necessity for the sample pre-treatment or time-consuming extraction steps. The voltammetric methods provide good stability, repeatability, reproducibility and high recovery for the analysis of the analyte. They could be used for the pharmacokinetic studies as well as for quality control laboratory studies.

Dilute-and-shoot-based direct nano-electrospray ionization tandem mass spectrometry as screening methodology for multivitamins in dietary supplement and human urine

IntroductionIn recent years, analytical screening methods for simultaneous detection of multivitamins have gained substantial attention to ensure quality and public confidence in dietary supplements. Even so, few analytical methods have been proposed for simultaneous analysis of multivitamin constituents due to the large divergence in chemical characteristics. ObjectivesIn the present study, the objective was to develop a simple and rapid direct nano-electrospray ionization-tandem mass spectrometry (DI-nano-ESI-MS/MS) method for targeted detection of water soluble vitamins, fat soluble vitamins, amino acids, royal jelly, ginkgo biloba, and ginseng in a dietary supplement. The applicability of dilute-and-shoot-based DI-nano-ESI-MS/MS to analyze the same tested compounds and their related metabolites in clinical samples was also examined. MethodsIntact urine mixed with the ionization solvent was loaded (4-μL aliquot) into a nanospray (NS) capillary of 1-μm tip diameter. The NS capillary was then fitted into an off-line ion source at a distance of 5 mm from MS aperture. The sample was directly injected by applying a voltage of 1.1 kV, producing a numerous of m/z peaks for analysis in mere minutes. ResultsThe DI-nano-ESI-MS/MS method successfully identified almost all dietary supplement components, as well as a plethora of component-related metabolites in clinical samples. In addition, a new merit of the proposed method for the detection of index marker and chemical contaminants as well as subspecies identification was investigated for further quality evaluation of the dietary supplement. ConclusionsThe previous findings illustrated that DI-nano-ESI-MS/MS approach can emerge as a powerful, high throughput, and promising analytical tool for screening and accurate detection of various pharmaceuticals and ingredient in dietary supplements as well as biological fluids.

Measurement of gut microbial metabolites in cardiometabolic health and translational research.

The human gut microbiota is a functioning endocrine organ and stands at the intersection between dietary components and health or disease. There are very many microbial metabolites with numerous structures and functions arising from the gut microbial fermentation of foods and become signals for biological communication in the human body. These small molecules can be absorbed and delivered to distant organs through the circulatory system to build the gut-systemic axis. The gut microbial metabolomes are thus believed to play important roles in regulating cardiometabolic health and provide opportunities in mechanistic research and new drug discovery. Measurement of these novel microbial metabolites in clinical samples may serve as a tool for investigating disease biomarkers. In the past decade, the development of untargeted and targeted metabolomics approaches using NMR, LC/MS, and GC/MS has contributed to the exploration of gut microbial metabolomes in cardiometabolic health and disease. Some important targets are currently being translated into clinical applications. In this review article, we introduce an oral carnitine challenge test developed as an example to demonstrate the potential applications in personalized nutrition based on the function of gut microbiota. It is a method taking the gut microbiota as a bioreactor and provides fermentable materials as inputs and measures the outputs of targeted microbial byproducts in the blood or urine. This challenge test may be extended to measure metabolites from microbial fermentation related to other endocrinological or inflammatory diseases. We review current gut metabolome research approaches and propose a gut microbial functional measurement using a challenge test. We suggest that the maturation in measuring gut microbial metabolites may provide an important piece to complete the puzzle of precision medicine.

Validation of an improved liquid chromatography tandem mass spectrometry method for rapid and simultaneous analysis of plasma catecholamine and their metabolites

Catecholamines [dopamine (DA), epinephrine (E), and norepinephrine (NE)] and their metabolites [metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT)] are functionally important in humans. Their overexpression can indicate the presence of neuroendocrine tumors. Accurate and rapid quantitation of catecholamines and their metabolites may function in differential diagnosis of neuroendocrine tumors. Herein, we diluted 200 μL plasma using isotope labelled internal standards (IS), and extracted using solid phase extraction. The performance of isotope diluted liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) was evaluated and applied to quantify the level of catecholamines and metabolites in clinical samples from 73 apparently healthy adults. The total analysis time of the ID-LC-MS/MS method was 4 min. The improved method was highly sensitive, with a limit of quantification (LOQ) for MN, NMN, 3-MT, and E of 1 pg/mL, a LOQ for DA of 5 pg/mL, and for NE of 10 pg/mL. After correction using IS, no significant matrix effects were observed. Good reproducibility was obtained, with total CVs of 3.2–13.1% (DA), 4.8–10.0% (E), 6.2–6.9% (NE), 3.8–7.9% (MN), 4.1–8.8% (NMN), 3.4–8.9% (3-MT). Recoveries were in the range of 91.1–109.7% for the six analytes. Also, the mean concentration of catecholamines were as follows: MN, 22.9 ± 7.2 pg/mL; NMN, 41.4 ± 17.2 pg/mL; 3-MT, 2.34 ± 2.01 pg/mL; DA, 10.2 ± 4.6 pg/mL; E, 29.3 ± 14.2 pg/mL and NE 427.0 ± 190.6 pg/mL. A reliable ID-LC-MS/MS method for the determination of catecholamines and their metabolites using small volumes of plasma was verified. This method is rapid, simple, and may serve as an essential diagnostic tool for neuroendocrine tumors in clinical practice.

Electroanalytical Methods for Determination of Calcium Channel Blockers

Background:Calcium Channel Blockers (CCBs) are widely used in the treatment of cardiovascular and ischemic heart diseases in recent years. They treat arrhythmias by reducing cardiac cycle contraction and also benefit ischemic heart diseases. Electroanalytical methods are very powerful analytical methods used in the pharmaceutical industry because of the determination of therapeutic agents and/or their metabolites in clinical samples at extremely low concentrations (10-50 ng/ml). The purpose of this review is to gather electroanalytical methods used for the determination of calcium channel blocker drugs in pharmaceutical dosage forms and biological media selected mainly from current articles.Methods:This review mainly includes recent determination studies of calcium channel blockers by electroanalytical methods from pharmaceutical dosage forms and biological samples. The studies of calcium channel blockers electroanalytical determination in the literature were reviewed and interpreted.Results:There are a lot of studies on amlodipine and nifedipine, but the number of studies on benidipine, cilnidipine, felodipine, isradipine, lercanidipine, lacidipine, levamlodipine, manidipine, nicardipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, diltiazem, and verapamil are limited in the literature. In these studies, DPV and SWV are the most used methods. The other methods were used less for the determination of calcium channel blocker drugs.Conclusion:Electroanalytical methods especially voltammetric methods supply reproducible and reliable results for the analysis of the analyte. These methods are simple, more sensitive, rapid and inexpensive compared to the usually used spectroscopic and chromatographic methods.

Methods for isolating, identifying, and quantifying anthocyanin metabolites in clinical samples.

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 ± 0.002) and sensitivity (limits of detection (LODs): urine, 100 ± 375 nM; serum, 104 ± 358 nM; feces 138 ± 344 nM), and the final SPE methods provided recoveries of 88.3 ± 17.8% for urine, 86.5 ± 11.1% for serum, and 80.6 ± 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.

Simultaneous Screening of 177 Drugs of Abuse in Urine Using Ultra-performance Liquid Chromatography with Tandem Mass Spectrometry in Drug-intoxicated Patients

ObjectiveThe demand for rapid and broad clinical toxicology screening methods to identify drugs of abuse and medicinal drugs is increasing steadily. Liquid chromatography-tandem mass spectrometry (LC-TMS) is increasingly used to screen for drugs of abuse and to identify a wide range of drugs and metabolites in clinical samples. We revised a high-throughput and rapid ultra-performance (UP) LC-TMS method for simultaneous screening of 177 of the most prevalent medicinal drugs and drugs of abuse in urine and validated the quality of performance using system suitability mixture (SSM) and quality control (QC) materials.MethodsWe assessed the limits of detection (LOD) using high concentrations of the test substances. The method was applied to 473 urine samples obtained from patients intoxicated with drugs who visited the emergency center.ResultsThe retention time, peak area, and total ion chromatogram of the SSM and QC materials were within the acceptance criteria of the pre-defined acceptance interval. The LODs were <62 ng/ml for 12 commonly encountered drugs. In total, 418 patients (88.4%) tested positive for one or more medicinal drugs or drugs of abuse. Twenty-eight drugs were detected over ten times; the most commonly detected were zolpidem, ephedrine, paracetamol, and chlorpheniramine.ConclusionThe UPLC-TMS method provided excellent performance for simultaneous screening of a large number of the drugs of abuse in urine samples. We conclude that this robust technique is useful for screening for a large number of drugs and for rapid screening of the most commonly encountered substances in emergency cases.

Determination of Ketobemidone and Its Metabolites in Plasma and Urine Using Solid-Phase Extraction and Liquid Chromatography-Mass Spectrometry

The authors developed a sensitive, specific, and rapid liquid chromatography--mass spectrometry (LC-MS) method for determining ketobemidone and its major metabolites in plasma and urine. The method involves a solid-phase extraction, high-performance liquid chromatography (HPLC), and electrospray mass spectrometry. The limit of quantification for ketobemidone and norketobemidone was 3 nmol/L. Recovery rates for ketobemidone and norketobemidone were 84.8% and 81.1%, respectively. Coefficients of variation (CV) ranged from 2.8 % to 9.5%. The method was used to determine ketobemidone and its major metabolites in clinical samples from relevant patient groups.

Rapid monitoring of benzodiazepines in clinical samples by using on-line column switching HPLC

We have determined routinely administered benzodiazepines and some of their metabolites in clinical samples by using on-line column switching for sample clean-up and reversed-phase high-pressure liquid chromatography (HPLC). The plasma sample is directly injected onto a cyanopropyl (CN) pre-column, washed with buffer, and eluted onto an analytical column (C18, octadecylsilane). Analytical recovery from drug-supplemented plasma samples was greater than 85% for all the benzodiazepines tested. The response of the analytical column varied linearly with drug concentrations over the range 0.01-1 mg/L in plasma, with CVs ranging from 0.3% to 1.8% within-day and 5.5% to 15.7% between days. The detection limit (signal-to-noise ratio greater than 3) was 0.01 mg/L (1 ng on column) for all of the benzodiazepines and their metabolites. The advantages of the technique include the extreme cleanliness of the traces, no requirement for off-line sample preparation, and ease of automation.