High-performance liquid chromatographic determination of citalopram and four of its metabolites in plasma and urine samples from psychiatric patients

Determination of atrazine and its metabolites in mouse urine and plasma by LC–MS analysis

Determination of ivabradine and its N-demethylated metabolite in human plasma and urine, and in rat and dog plasma by a validated high-performance liquid chromatographic method with fluorescence detection

Changes of Human Gut Microbiome Correlated with Metabolomics After Cranberry Juice Consumption in a Double-Blinded, Placebo Controlled, Crossover Study

Free radicals are thought to play an important role in many types of tissue injury. Recently, we reported that a series of prostaglandin F2-like compounds (F2-isoprostanes) capable of exerting potent biological activity are produced in vivo by free radical-induced lipid peroxidation. Their formation is independent of the cyclooxygenase enzyme and has been shown to increase profoundly in animal models of free radical injury and lipid peroxidation. We now report the identification of F-ring isoprostane metabolites in human urine and plasma utilizing a gas chromatographic/mass spectrometric assay for the major urinary metabolite of prostaglandin D2 (9 alpha,11 beta-dihydroxy-15-oxo-2,3, 18,19-tetranorprost-5-ene-1,20-dioic acid). Evidence confirming these metabolites as tetranor, dicarboxylic acid compounds containing one double bond, cis-cyclopentane ring hydroxyls, and one keto group similar in structure to the major urinary metabolite of prostaglandin D2 was obtained by analysis of human urine by electron ionization mass spectrometry. Levels of these metabolites in normal human urine were determined and found to be unaffected by cyclooxygenase inhibitors. Evidence that these metabolites arise from F2-isoprostanes was obtained by demonstrating that (a) marked increases in plasma levels and urinary excretion of these metabolites, which were unaffected by coadministration of indomethacin, occurred in rats administered CCl4 to induce F2-isoprostane formation and (b) marked increases in levels of these metabolites in plasma and urine resulted from the intravenous infusion of F2-isoprostanes into a rat. Quantification of these isoprostane metabolites in urine and plasma may provide a reliable index of endogenous isoprostane production which could prove to be an important advance in our ability to assess oxidant stress in vivo in humans.

A method has been developed for the determination of trace levels of alpha-endosulfan, beta-endosulfan, endosulfan sulfate, and endosulfan diol in rat plasma and tissue samples. Endosulfan and its metabolites in the plasma samples were extracted with solid-phase extraction Chromabond-end-capped C18 cartridges and analyzed by a Shimadzu QP-5050A gas chromatograph-mass spectrometer (GCMS) with quadrupole detector in selected-ion-monitoring mode. The analysis of endosulfan and its metabolites in liver and kidney samples involved solvent extraction, Florisil solid-phase-extraction cleanup, and quantitation by GCMS. Recovery experiments for the plasma and tissue samples were conducted over concentration ranges of 10-100 ng mL(-1) and 100-1000 ng mL(-1), respectively. The method was applied to the analysis of trace levels of endosulfan and its metabolites in plasma and tissue samples collected from an animal study. Trace levels of alpha-endosulfan and beta-endosulfan in the ranges of undetectable to 3.11 microg g(-1) and undetectable to 1.19 microg g(-1), respectively, were detected in the kidney samples, whereas trace levels of endosulfan sulfate in the range of 0.02-0.22 microg g(-1) were detected in the liver samples of rats. Neither endosulfan nor its metabolites was detected in any of the plasma samples.

Background The purpose of this study was to identify plasma and urinary metabolites that can be used to better identify the effects of cadmium exposure than N-acetyl-β-D-glucosaminidase (NAG) using capillary electrophoresis - mass spectrometry (CE-MS). Methods Urinary cadmium (U-Cd) was measured as an indicator of cadmium exposure. Fasting plasma and urine samples were collected from 1,412 men and 2,022 women in Tsuruoka Metabolomics Cohort Study. Charged 94 plasma and 123 urinary metabolites were detected and determined. Regression analysis was performed for urinary NAG, plasma, and urinary metabolites as dependent variables and U-Cd in quartiles as an independent variable. Multivariate regression model included age, SBP, smoke, rice intake, BMI, HbA1c, LDLc, alcohol consumption, physical activity, educational history, dietary energy intake, urinary Na/K ratio, and uric acid. Results The mean U-Cd of the population was 2.65 μg/g creatinine (SD: 1.63). NAG was positively associated with U-Cd, but the association was not significant with lower U-Cd quartiles. In the plasma metabolites, 10 metabolites had significantly negative association with U-Cd in all models and Cd quartiles. Among urinary metabolites, 27 metabolites had significantly positive association with U-Cd. Alanine was negatively associated with U-Cd in urinary metabolites. The trend test also showed significant dose-response trends for 9 plasma and all 28 urinary metabolites (p < 0.05). Conclusions We found that the levels of Cd exposure, which did not cause an increase in NAG, caused changes in plasma and urinary metabolites. Key messages This study indicated that metabolomics might be promising and useful as markers of Cd exposure.

Determination of American ginseng saponins and their metabolites in human plasma, urine and feces samples by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Determination of midazolam and its unconjugated 1-hydroxy metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive reversed‐phase high performance liquid chromatography (HPLC) method was developed for the determination of the novel antithrombotic agent acetylsalicylic acid maltol ester (aspalatone, CAS: 147249‐33‐0) and its four metabolites, salicylic acid maltol ester (SME), salicylic acid (SA), salicyluric acid (SUA), and gentisic acid (GA), in rat plasma and urine. After a treatment of plasma or urine sample by liquid‐liquid extraction, the compounds were analyzed on an HPLC system with ultraviolet detection at 229 nm for aspalatone and SME, and at 313 nm for SA, SUA, and GA. HPLC analysis was carried out using reversed‐phase isocratic elution with a C18 column (4.6 mm×250 mm, 5 µm), a mobile phase of a mixture of butanol, acetic acid, doubly deionized water, and sodium sulfate (2∶5∶83∶10, v/v/v/w %) at a flow rate of 1.0 mL/min. The chromatograms showed good resolution and sensitivity and no interference of plasma and urine. The calibration curves for all substances in both plasma and urine samples were linear over the concentration range of 0.05–200 µg/mL for both plasma and urine. The intra‐ and inter‐day assay accuracies of this method were within 100±11% of nominal values and the precision did not exceed 13% of relative standard deviation. The lower limits of quantitation were 50 ng/mL for aspalatone and its metabolites in plasma and urine, which were sensitive enough for pharmacokinetic studies.

Determination of amifloxacin and two of its principal metabolites in plasma and urine by high-performance liquid chromatography using automated column switching

The need for appropriate methodology for the analysis of drugs and their metabolites in complex biological matrices led to a growing interest in developing new techniques for sample preparation, particularly microextraction techniques because they are highly selective and require a minimum consumption of organic solvents. Allied to these developments, the employment of modern and efficient analytical technologies, such as capillary electrophoresis (CE) and high-performance liquid chromatography coupled to mass spectrometry (LC-MS-MS), has resulted in a considerable improvement in quality in the analytical methodologies available for bioanalysis. In this context, it is worth to mention the use of such techniques to develop enantioselective methodologies, allowing the quantification of the enantiomers of drugs administered as racemates. Therefore, we proposed the development and validation of enantioselective methodologies for the analysis of the enantiomers of mirtazapine (MRT) and of its main metabolites in plasma and urine, using the CE and LC-MS-MS. Solid phase microextraction (SPME) and liquid phase microextraction (LPME) were used for sample preparation. In the first method, LPME was used to extract the analytes from plasma samples (1 ml), previously diluted, alkalinized with 3.0 mL 0.5 mol L -1 pH 8 phosphate buffer solution and supplemented with 15% (w/v) sodium chloride. N-hexyl ether and 0.01 mol L -1 acetic acid solution were used as solvent extractor and acceptor phase, respectively. The analyses were carried out on a CHIRALPAK AD-RH column and acetonitrile: methanol: ethanol (98:1:1, v / v / v) plus 0.2% of diethylamine was used as mobile phase, at a flow rate of 1 mL min -1 . The detection was performed by LC-MS-MS equipped with a triple-quadrupole analyzer and ionization by eletrospray positive. Under these conditions, recoveries were from 18.3 to 45.5%; linear response over the 1,25-125 ng ml -1 concentration range and limit of quantification (LOQ) of 1.25 ng ml -1 for all enantiomers evaluated were obtained. CE and LPME were also used for the analysis of MRT and its main metabolites in urine. Before the extraction, urine samples (1 mL) were submitted to enzymatic hydrolysis at 37 C for 16 hours, the enzyme was precipitated with trichloroacetic acid, the pH was adjusted to 8 with 0.5 mol L -1 phosphate buffer solution (pH 11) and 10% (w/v) sodium chloride was further added. Then, the LPME extraction was performed according to the procedure previously developed. The electrophoretic analyses were carried out in 50 mmol L -1 phosphate buffer solution (pH 2.5) containing 0.55% (w/v) carboxymethyl--cyclodextrin (CM--CD). The method was linear over the concentration range of 62.5-2500 ng mL-1 for each MRT and 8-OHM enantiomer and 62.5-1250 ng mL -1 for each DMR enantiomer. The quantification limit (LOQ) was 62.5 ng mL -1 for all the enantiomers. A SPME method was also developed for the simultaneous enantioselective determination of MRT and its metabolites in urine using CE and LC-MS-MS. The target analytes were transferred from the hydrolyzed aqueous solution to the polydimetylsiloxane-divinylbenzene (PMDS-DVB) fiber coating and then desorbed in methanol. The means recoveries were 12 % for the enantiomers of MRT, 3.8 % for DMR and 0.72 % for 8-OHM. The method was linear over the concentration range of 62.5-2500 ng mL -1 with suitable LOQ (62.5 ng mL -1 ) for all the enantiomers. The precision and accuracy were lower than 15% for all developed methods. Moreover, the methods were successfully employed for the determination of MRT, 8-OHM and DMR enantiomers in plasma and urine samples obtained after oral administration of a single dose of rac-MRT to healthy volunteers.

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5 and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and stereoselectively metabolized to CIT-PROP.

This report describes the development and validation of an LC-MS/MS method for the quantitative determination of glyburide (GLB), its five metabolites (M1, M2a, M2b, M3 and M4) and metformin (MET) in plasma and urine of pregnant patients under treatment with a combination of the two medications. The extraction recovery of the analytes from plasma samples was 87-99%, and that from urine samples was 85-95%. The differences in retention times among the analytes and the wide range of the concentrations of the medications and their metabolites in plasma and urine patient samples required the development of three LC methods. The lower limit of quantitation (LLOQ) of the analytes in plasma samples was as follows: GLB, 1.02 ng/mL; its five metabolites, 0.100-0.113 ng/mL; and MET, 4.95 ng/mL. The LLOQ in urine samples was 0.0594 ng/mL for GLB, 0.984-1.02 ng/mL for its five metabolites and 30.0 µg/mL for MET. The relative deviation of this method was <14% for intra-day and inter-day assays in plasma and urine samples, and the accuracy was 86-114% in plasma, and 94-105% in urine. The method described in this report was successfully utilized for determining the concentrations of the two medications in patient plasma and urine.

Determination of the new fluoroquinolone fleroxacin and its N-demethyl and N-oxide metabolites in plasma and urine by high-performance liquid chromatography with fluorescence detection

Introduction: Hypertension is the leading preventable cause of premature death worldwide and high sodium (HS) intake is the strongest dietary risk factor for hypertension. Salt sensitivity of blood pressure (SSBP), defined as an increase in blood pressure in parallel to an increase in salt intake, affects ~50% of hypertensive patients but is not routinely examined in clinical settings due to the complexity of current testing procedures. Previously, we sought to identify unique plasma biomarkers for SSBP to differentiate salt-sensitive (SS) versus salt-resistant (SR) participants using metabolomics and lipidomic profiling of plasma samples from the Dietary Approaches to Stop Hypertension-Sodium (DASH-Sodium) Trial. We identified that a HS diet was associated with changes in three plasma metabolites, an increase in citramalic acid and decreases in 2-ketoisocaproic acid and tocopherol alpha compared to low sodium (LS) diet, in SS but not SR participants. Here, we sought to validate these findings using additional plasma and urine samples from DASH-Sodium Trial and to identify novel biomarkers for SSBP by directly comparing plasma and urine metabolomic profiles between SS and SR participants after HS diet. We hypothesized that: 1). SS and SR participants would exhibit different alterations in the plasma and urine metabolomic profiles after LS or HS diets. 2). SS participants would exhibit different metabolomic profiles compared to SR participants after HS diet. METHOD: Theplasma and urine samples from 59 SS and 44 SR participants (total of 103 participants) from the DASH-Sodium Trial were sent to the West Coast Metabolomics Center at University of California at Davis for an untargeted metabolomic screening. Then, metabolomics data were provided to GeneVia Technologies (Tampere, Western Finland, Finland) for analysis. Result: For hypothesis 1, plasma metabolomic profiles showed that HS diet consumption was associated with changes in 11 metabolites in SS participants and 10 metabolites in SR participants compared to LS diet (p&lt;0.05). There was no difference in urine metabolites after LS or HS diets in SS and SR participants (P&gt;0.05). For hypothesis 2, metabolomic profiles showed that SR participants had differences in 11 plasma metabolites and 12 urine metabolites compared to SS participants after HS diet (p&lt;0.05). Discussion: In this study, we observed a decrease in plasma 2-ketoisocaproic acid after a HS diet in SS participants, which validates the finding from our previous study, but we did not see any changes in plasma tocopherol alpha and citramalic acid. We also observed that nine plasma metabolites differed similarly between LS and HS diets in those participants regardless of being SS or SR and changes in some of those metabolites are associated with hypertension such as increases in cholesterone, glutamyl-valine, methionine sulfoxide and a decrease in a glutamine. Importantly, only SR participants showed increased plasma glutamic acid, a BP-lowering compound, and only SS participants showed an increased plasma anthranilic acid, which is observed in hypertensive patients. When directly comparing metabolites from SS and SR participants after a HS diet, SR participants exhibited a higher plasma level of 3-aminoisobutyric acid, which has been shown to attenuate SSBP in rats. Furthermore, 2-picolinic acid levels were higher in plasma and lower in urine of SR compared to SS participants, which may suggest its protective role against the SSBP in SR participants. In summary, these findings provide the preliminary evidence for the use of identified metabolites as novel biomarkers for SSBP to advance the testing procedures of SSBP in clinical settings. This study was funded by the National Institutes of Health (R01 AG075963 &amp; R01 AG062515) and by Hevolution Foundation Grant (HF-GRO-23–1199246-43). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.