Metabolites In Human Plasma Samples Research Articles

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

(Deep eutectic solvent-ionic liquid)-based ferrofluid a new class of magnetic colloids for determination of tamoxifen and its metabolites in human plasma samples

In this research, a novel category of magnetic colloid was created by immobilizing an ionic liquid on the surface of silica-coated magnetic particles. This was followed by utilizing a natural hydrophobic deep eutectic solvent as a carrier. This magnetic colloid served as an adsorbent in dispersive solid-phase microextraction, combined with liquid chromatography-UV detection, for analyzing tamoxifen and its three active metabolites in plasma samples. After optimizing the effective variables in the analysis, the method underwent validation. Linearity was observed in the calibration model between 0.2 and 5.0 µg/L. The range of the detection limit was found to be 0.2–200.0 µg/L. Assessing the method's precision involved evaluating the intra- and inter-day precision at three analyte concentrations, which determined less than 7.5 % and 11.2 %, respectively. Moreover, the accuracy fell between 94.3 % and 105.1 %. The method's feasibility was evaluated using human plasma samples that ranged in concentrations of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen, respectively, from 28.3 to 39.7, 25.9–42.7, ≈1.3, and 3.4–7.2 μg L-1. Among the many benefits of this approach is the removal of the requirement for additional electronic accessories and dispersive solvents. It also minimizes the sample size, making it easily automatable. The method's applicability was demonstrated using human plasma samples, which provided quantification ranges for various tamoxifen metabolites. This technique has a lot of potential for studying the pharmacodynamics, therapeutic drug monitoring, andpharmaceutical kineticsof tamoxifen and its metabolites in bodily fluids, which will help with clinical research and drug analysis.

Quality Control in Targeted GC-MS for Amino Acid-OMICS.

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d0Me) using 2 M HCl in methanol (CH3OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD3OD) generates deuterated methyl esters (d3Me) of AA, which can be used as internal standards in GC-MS. d0Me-AA and d3Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d0Me-AA-PFP and d3Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d0Me-AA-PFP and d3Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d3Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (tR)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d0Me-AA-PFP derivatives (tR(H)) and the d3Me-AA-PFP derivatives (tR(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d3Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS.

Quantitative Analytical and Computational Workflow for Large-Scale Targeted Plasma Metabolomics.

Quantifying metabolites from various biological samples is necessary for the clinical and biomedical translation of metabolomics research. One of the ongoing challenges in biomedical metabolomics studies is the large-scale quantification of targeted metabolites, mainly due to the complexity of biological sample matrices. Furthermore, in LC-MS analysis, the response of compounds is influenced by their physicochemical properties, chromatographic conditions, eluent composition, sample preparation, type of MS ionization source, and analyzer used. To facilitate large-scale metabolite quantification, we evaluated the relative response factor (RRF) approach combined with an integrated analytical and computational workflow. This approach considers a compound's individual response in LC-MS analysis relative to that of a non-endogenous reference compound to correct matrix effects. We created a quantitative LC-MS library using the Skyline/Panorama web platform for data processing and public sharing of data. In this study, we developed and validated a metabolomics method for over 280 standard metabolites and quantified over 90 metabolites. The RRF quantification was validated and compared with conventional external calibration approaches as well as literature reports. The Skyline software environment was adapted for processing such metabolomics data, and the results are shared as a "quantitative chromatogram library" with the Panorama web application. This new workflow was found to be suitable for large-scale quantification of metabolites in human plasma samples. In conclusion, we report a novel quantitative chromatogram library with a targeted data analysis workflow for biomedical metabolomic applications.

Pharmacokinetic Evaluation of Phytochemicals From Selected Herbs and Spices in Human Plasma Samples

ObjectivesHerbs and spices (H/S) are rich sources of bioactive compounds with a limited understanding of their absorption and metabolism in humans. This study aimed to characterize H/S metabolites in human plasma samples over 24 h after H/S intake. MethodsPlasma samples from a randomized, single-blinded, 4-arm, 24-h, crossover clinical trial (Clincaltrials.gov NCT03926442) were used for this research. Subjects (n = 24, aged 37 ± 3 years, BMI = 28.4 ± 0.6 kg/m2) consumed a high-fat/high-carbohydrate meal with salt and pepper only (control) or with three different H/S mixtures: Italian herbs (rosemary, basil, thyme, oregano, and parsley), cinnamon, and pumpkin pie spice (cinnamon, ginger, nutmeg, and allspice), and blood samples were collected at 0, 0.5, 1, 2, 4, 5.5, 7 and 24 h. Meals contained 1 g H/S per 135 kcal and delivered 35% of each subject’s energy needed to maintain weight. Quantitative analysis of H/S metabolites was conducted using UHPLC-QQQ based on reference standards and reported multiple reaction monitoring transitions. ResultsWe have tentatively identified 58 metabolites in plasma samples, including 32 phenolic acids, 11 terpenoids, 6 flavonoids, and 9 other polyphenol metabolites. Preliminary analysis (n = 13) showed that coumarin glucuronide increased after cinnamon and pumpkin pie meals and peaked at 2 h (2656.8 ± 481.3 nmol/L and 1542 ± 275.7 nmol/L respectively), and returned to baseline concentration at 24 h. After consuming Italian herbs meal, carnosol appeared in plasma early peaking at 1 h (48.2 ± 7.0 nmol/L); carnosic acid and 12-methoxy carnosic acid peaked at 2 h (366.4 ± 142.2 nmol/L and 588.6 ± 66.3nmol/L, respectively), and these metabolites circulated in the body for up to 24 h; apigenin-7-O-glucuronide peaked at 7 h (6.0 ± 1.7 nmol/L); carnosic acid glucuronide peaked at 24 h (510.9 ± 86 nmol/L). 6- and 10-gingerol glucuronide peaked at 1 h and 2 h after consuming pumpkin pie meal (1.4 ± 0.6 nmol/L and 6.7 ± 1.7 nmol/L), and returned to baseline concentration at 5.5 and 7 h, respectively. All data are expressed as mean ± SEM. ConclusionsH/S bioactive compounds are absorbed and metabolized in the human body to early and late phase metabolites, peaking at various time-points across 24 h in response to different H/S meals. Funding SourcesThis project was supported by a gift from McCormick Science Institute and various donor funds.

Simultaneous quantitative detection of afatinib, erlotinib, gefitinib, icotinib, osimertinib and their metabolites in plasma samples of patients with non-small cell lung cancer using liquid chromatography-tandem mass spectrometry

Background and aimsAs numerous studies have reported the concentration-exposure relationships of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), therapeutic drug monitoring is a promising approach in lung cancer treatment, aiming to avoid treatment failure or toxicity. A new method for the simultaneous analysis of five EGFR-TKIs (afatinib, erlotinib, gefitinib, icotinib and osimertinib) and their metabolites in human plasma samples was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Materials and methodsAfatinib-d6, erlotinib-d6, OSI-420-d4, gefitinib-d6 and osimertinib-C13,d3 were used as internal standards (ISs). The samples were prepared by liquid-liquid extraction using tert-butyl methyl ether. Chromatographic separation was undertaken on an XBridge C18 column using a linear gradient elution. LC-MS/MS was conducted in positive ionization mode with multiple reaction monitoring. ResultsThe proposed method showed satisfactory results in terms of linearity, sensitivity, specificity, precision (intra- and inter-day coefficients of variation ranged from 1.1 to 13.9%), and accuracy (from 93.3 to 111.1%). The IS-normalized matrix factors were below 15%. The sensitivity and linearity were highly appropriate for the expected concentrations according to the analysis of samples from non-small cell lung caner (NSCLC) patients who received EGFR-TKIs. ConclusionsThe proposed method showed an acceptable reproducibility, high sensitivity and selectivity, and low matrix effects. This method could be significant for monitoring plasma concentrations of the mentioned EGFR-TKIs in NSCLC patients, aiming to improve the efficacy and safety of targeted therapies.

Liquid-liquid extraction and ultrafiltration based sample preparation technique for Q-TOF LC/MS analysis of non-polar metabolites in human plasma samples

Liquid-liquid extraction and ultrafiltration based sample preparation technique for Q-TOF LC/MS analysis of non-polar metabolites in human plasma samples

Simultaneous LC–MS/MS analysis of simvastatin, atorvastatin, rosuvastatin and their active metabolites for plasma samples of obese patients underwent gastric bypass surgery

Statins, HMG-CoA reductase inhibitors, are considered the first line treatment of hyperlipidemia to reduce the risk of atherosclerotic cardiovascular diseases. The prevalence of hyperlipidemia and the risk of atherosclerotic cardiovascular diseases are higher in obese patients. Published methods for the quantification of statins and their active metabolites did not test for matrix effect of or validate the method in hyperlipidemic plasma. A sensitive, specific, accurate, and reliable LC–MS/MS method for the simultaneous quantification of simvastatin (SMV), active metabolite of simvastatin acid (SMV-A), atorvastatin (ATV), active metabolites of 2-hydroxy atorvastatin (2-OH-ATV), 4-hydroxy atorvastatin (4-OH-ATV), and rosuvastatin (RSV) was developed and validated in plasma with low (52–103 mg/dl, <300 mg/dl) and high (352–403 mg/dl, >300 mg/dl) levels of triglyceride. The column used in this method was ACQUITY UPLC BEH C18 column (2.1 × 100 mm I.D., 1.7 μm). A gradient elution of mobile phase A (10 mM ammonium formate and 0.04% formic acid in water) and mobile phase B (acetonitrile) was used with a flow rate of 0.4 ml/min and run time of 5 min. The transitions of m/z 436.3 → 285.2 for SMV, m/z 437.2 → 303.2 for SMV-A, m/z 559.2 → 440.3 for ATV, m/z 575.4 → 440.3 for 2-OH-ATV and 4-OH-ATV, m/z 482.3 → 258.1 for RSV, and m/z 412.3 → 224.2 for fluvastatin (internal standard, IS) were determined by Selected Reaction Monitoring (SRM) method to detect transitions ions in the positive ion mode. The assay has a linear range of 0.25 (LLOQ) −100 ng/ml for all six analytes. Accuracy (87–114%), precision (3–13%), matrix effect (92–110%), and extraction recovery (88–100%) of the assay were within the 15% acceptable limit of FDA Guidelines in variations for plasma with both low and high triglyceride levels. The method was used successfully for the quantification of SMV, ATV, RSV, and their active metabolites in human plasma samples collected for an ongoing clinical pharmacokinetic and pharmacodynamic study on patients prior to and post gastric bypass surgery (GBS).

Quantitation of escitalopram and its metabolites by liquid chromatography-tandem mass spectrometry in psychiatric patients: New metabolic ratio establishment.

Therapeutic drug monitoring (TDM) is used to determine the concentration of drug in plasma/serum to adjust the dose of the therapeutic drug. Selective and sensitive analytical methods are used to determine drug and metabolite levels for the successful application of TDM. The aim of the study was to develop and validate using LC-MS/MS to analyse quantitative assay of escitalopram (S-CT) and metabolites in human plasma samples. In order to provide a convenient and safe treatment dose, it was aimed to determine the levels of S-CT and its metabolites in the patients' plasma. A new method with short sample preparation and analysis time was developed and validated using LC-MS/MS to analyse quantitative assay of S-CT and its metabolites in plasma. Also, plasma samples of 30 patients using 20mg S-CT between the ages of 18 and 65years were analysed by the validated method. The mean values of S-CT, demethyl escitalopram and didemethyl escitalopram in plasma of patients were 27.59, 85.52 and 44.30ng/mL, respectively. At the end of the analysis, the metabolic ratio of S-CT and metabolites was calculated. It is considered that the method for the quantitative analysis of S-CT and its metabolites in human plasma samples may contribute to the literature on account of its sensitive and easy application. Additionally, the use of our data by physicians will contribute to the effective drug treatment for their patients who take S-CT.

Determination of LongR3-IGF-I, R3-IGF-I, Des1-3 IGF-I and their metabolites in human plasma samples by means of LC-MS

According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR3-IGF-I, R3-IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection. Noteworthy, the usage of a specific antibody for LongR3-IGF-I enables the determination in low ng/mL levels despite the presence of an enormous excess of endogenous human IGF-I. In addition, different metabolism studies (in-vitro and in-vivo) were performed using sophisticated strategies such as incubation with skin tissue microsomes, degradation in biological fluids (for all analogues), and administration to rats (for LongR3-IGF-I). Herewith, several C-and N-terminally truncated metabolites were identified and their relevancy was additionally confirmed by in-vivo experiments with rodents. Especially for LongR3-IGF-I, a metabolite ((Des1-11)-LongR3-IGF-I) was identified that prolonged the detectability in-vivo by a factor of approximately 2. The method was validated for qualitative interpretation considering the parameters specificity, identification capability, recovery (26–60%), limit of detection (0.5ng/mL), imprecision (<25%), linearity, stability, and matrix effects. A stable isotope labelled (15N)-IGF-I was used as internal standard to control all sample preparation steps.

Development and validation of a quantitative assay for the determination of cinacalcet and its main metabolites in human plasma using RP-HPLC method

A novel and sensitive methodology for the quantitative determination of cinacalcet (CIN), and its main metabolites (M2a-Glu, M2b-Glu, M5, and M6) in plasma sample is suggested. The analytical assay is based on reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a fluorescence detector. The method has been fully validated at range covering the plasma concentrations in patients receiving therapeutic dosages of CIN. In this investigation, 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (NI-ETFMS) was used as a pre-column derivation agent according to interaction of amine and carboxylate groups of CIN and its metabolites with NI-ETFMS. The sample pretreatment consists of protein precipitation with acetonitrile using only 100μL of plasma, and chromatographic separation was achieved in isocratic mode on Kinetex C18 100Å column with mobile phase of acetonitrile, methanol, and 50mM phosphate buffer (40:20:40v/v). The analytical method was fully validated in terms of selectivity, linear range (0.2 to 5.5ngmL−1), linearity (r2>0.997), sensitivity (LOD=0.05 to 0.75ngmL−1 and LOQ=0.36 to 2.52ngmL−1), intra and inter-days accuracy (−4.6 to 8.1%), precision (<7.4%), and robustness (<3.5%). As results, the proposed method can be useful in the routine analysis for the determination of CIN and its main metabolites in human plasma samples.

Determination of tamoxifen and its metabolites in human plasma by nonaqueous capillary electrophoresis with contactless conductivity detection.

A new approach for the quantification of tamoxifen and its metabolites 4-hydroxytamoxifen, N-desmethyltamoxifen, and 4-hydroxy-N-desmethyltamoxifen (endoxifen) in human plasma samples using NACE coupled with contactless conductivity detection (C4 D) is presented. The buffer system employed consisted of 7.5 mM deoxycholic acid sodium salt, 15 mM acetic acid, and 1 mM 18-crown-6 in 100% methanol. The complete separation of all targeted compounds (including endoxifen racemate) could be achieved within 6 min under optimized conditions. The proposed method was validated and showed good linearity in the range from 100 to 5000 ng/mL with correlation coefficients between 0.9922 and 0.9973, LODs in the range of 25-40 ng/mL, and acceptable reproducibility of the peak area (intraday RSD 2.2-3.1%, n = 4; interday (3 days) RSD 6.0-8.8%, n = 4). The developed method was successfully demonstrated for the quantification of tamoxifen and its metabolites in human plasma samples collected from breast cancer patients undertaking tamoxifen treatment.

Liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of simvastatin, lovastatin, atorvastatin, and their major metabolites in human plasma.

Millions of individuals are treated with a variety of statins that are metabolized to a variety of active metabolites. A single assay capable of simultaneously quantifying commonly used statins and their major metabolites has not been previously reported. Herein we describe the development and validation of a novel and robust liquid chromatography-tandem mass spectrometry assay for simultaneously quantifying simvastatin, lovastatin, atorvastatin, and their metabolites, simvastatin acid, lovastatin acid, para-hydroxy atorvastatin, and ortho-hydroxy atorvastatin in human plasma. Plasma samples were processed with a simple protein precipitation technique using acetonitrile, followed by chromatographic separation using an Agilent Zorbax Extend C18 column. A 12.0min linear gradient elution was used at a flow rate of 400μL/min with a mobile phase of water and methanol, both modified with 2mM ammonium formate and 0.2% formic acid. The analytes and internal standard, hesperetin, were detected using the selected reaction monitoring mode on a TSQ Quantum Discovery mass spectrometer with positive electrospray ionization. The assay exhibited a linear range of 1-1000nM for simvastatin acid and lovastatin acid, and a linear range of 0.1-100nM for the other analytes in human plasma. The accuracy and the within- and between-day precisions of the assay were within acceptable ranges, and the method was successfully utilized to quantify the statins and their metabolites in human plasma samples collected from an ongoing pharmacokinetic study.

Derivation of uncertainty functions from validation studies in biological fluids: Application to the analysis of caffeine and its major metabolites in human plasma samples

Procedures for estimating the measurement uncertainty (MU) of the concentration of a given analyte in a sample are of major concern for analytical chemists. Unfortunately, it is still unclear how and why MU should be assessed. While several possibilities exist, an appropriate approach consists in using method validation data for the evaluation of MU. This was demonstrated by a validation study achieved in the framework of a clinical study related to caffeine in sports medicine, where the results were used for the evaluation of MU. After validation of the method developed using ultra-high pressure liquid chromatography–mass spectrometry for caffeine and its three main metabolites, accuracy profiles were built for each analyte. The first important conclusion is that the developed method was valid for all compounds and met the given specifications for the application (fit for purpose). Relevant estimates of combined standard uncertainty were computed to obtain uncertainty functions, which allow obtaining values of MU as a function of the concentration of the analyte. The great advantage of both uncertainty function and uncertainty profile is the development of a continuous model that enables easy calculation of the standard, expanded and relative expanded uncertainty at any concentration within the validation domain. In fact, the expanded uncertainty interval is assumed to contain 95% of all possible measurements, regardless of the concentration. Finally, the uncertainty function enables the determination of the lowest limit of quantification by selecting adequate acceptance limits, with the limit of quantification being defined as the point where the relative uncertainty equals the acceptance limit threshold. It has to be noted that further discussions remain mandatory to establish which criteria should be applied to define an adequate decision threshold, and the proposal afforded in this work may open new avenues in this direction.

In matrix derivatization of trichloroethylene metabolites in human plasma with methyl chloroformate and their determination by solid-phase microextraction–gas chromatography-electron capture detector

Trichloroethylene (TCE) is a common industrial chemical that has been widely used as metal degreaser and for many industrial purposes. In humans, TCE is metabolized into dichloroacetic acid (DCA), trichloroacetic acid (TCA) and trichloroethanol (TCOH). A simple and rapid method has been developed for the quantitative determination of TCE metabolites. The procedure involves the in situ derivatization of TCE metabolites with methyl chloroformate (MCF) directly in diluted plasma samples followed by extraction and analysis with solid-phase microextraction (SPME) coupled to gas chromatography-electron capture detector (GC-ECD). Factors which can influence the efficiency of derivatization such as amount of MCF and pyridine (PYR), ratio of water/methanol were optimized. The factors which can affect the extraction efficiencies of SPME were screened using 2(7-4) Placket-Burman Design (PBD). A central composite design (CCD) was then applied to further optimize the most significant factors for optimum SPME extraction. The optimum factors for the SPME extraction were found to be 562.5mg of NaCl, pH at 1 and an extraction time of 22 min. Recoveries and detection limits of all three analytes in plasma were found to be in the range of 92.69-97.55% and 0.036-0.068 μg mL(-1) of plasma, respectively. The correlation coefficients were found to be in the range of 0.990-0.995. The intra- and inter-day precisions for TCE metabolites were found to be in the range of 2.37-4.81% and 5.13-7.61%, respectively. The major advantage of this method is that MCF derivatization allows conversion of TCE metabolites into their methyl esters in very short time (≤30 s) at room temperature directly in the plasma samples, thus makes it a solventless analysis. The method developed was successfully applied to the plasma samples of humans exposed to TCE.

Simultaneous eicosanoid profiling and identification by liquid chromatography and hybrid triple quadrupole-linear ion trap mass spectrometry for metabolomic studies in human plasma / Multiparametrische Bestimmung und Identifizierung von Eikosanoiden mit Flüssigchromatographie in Kombination mit Hybrid-Quadrupol/lineare Ionenfallen Massenspektrometrie für Metabolom-Studien in Humanplasma

Abstract Eicosanoids play a key role in many physiological and pathological processes and might therefore serve as interesting diagnostic targets. Methods for the analysis of arachidonic acid metabolites in cells and body fluids require high sensitivity and specificity because of the very low concentrations, similar chemical structures and short half-lives of these metabolites. We established a mass spectrometric method for the simultaneous identification and quantification of arachidonic acid metabolites in human plasma samples using solid phase extraction followed by liquid chromatography (LC) and hybrid triple quadrupole-linear ion trap (QqLIT) mass spectrometry. Quantitative analysis was performed using the 4000 QTrap tandem mass spectrometer in multiple reaction monitoring (MRM) mode. As part of an independent data acquisition experiment MRMs were used as survey scans, which dependently triggered enhanced product ion (EPI) scans. Compound identification was carried out by library search using a library based on EPI spectra of standard components (prostaglandins, thromboxanes, leukotrienes and isoprostanes). The newly developed compound library enables the verification of known and structural elucidation of unknown eicosanoid metabolites in human plasma. In conclusion, our mass spectrometric method allows the simultaneous identification and quantification of arachidonic acid metabolites in one single LC-MS/MS run.

Algorithm for Thorough Background Subtraction of High-Resolution LC/MS Data: Application to Obtain Clean Product Ion Spectra from Nonselective Collision-Induced Dissociation Experiments

Nonselective collision-induced dissociation (CID) is a technique for producing fragmentation products for all ions generated in an ion source. It is typical of liquid chromatography/mass spectrometry (LC/MS) analysis of complex samples that matrix-related components may contribute to the resulting product ion spectra and confound the usefulness of this technique for structure interpretation. In this proof-of-principle study, a high-resolution LC/MS-based background subtraction algorithm was used to process the nonselective CID data to obtain clean product ion spectra for metabolites in human plasma samples. With buspirone and clozapine metabolites in human plasma as examples, this approach allowed for not only facile detection of metabolites of interest but also generation of their respective product ion spectra that were clean and free of matrix-related interferences. This was demonstrated with both an MS(E) technique (where E represents collision energy) with a quadrupole time-of-flight (QTOF) instrument and an in-source fragmentation technique with an LTQ Orbitrap instrument. The combined nonselective CID and background subtraction approach should allow for detection and structural interpretation of other types of sample analyses where control samples are obtained.

Determination of amitriptyline and its main metabolites in human plasma samples using HPLC-DAD: application to the determination of metabolic ratios after single oral dose of amitriptyline

A simple and sensitive HPLC-DAD method for the simultaneous determination of amitriptyline, nortriptyline, E-10-hydroxyamitriptyline, Z-10-hydroxyamitriptyline, E-10-hydroxynortriptyline, Z-10-hydroxynortriptyline and desmethylnortriptyline in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation with isocratic elution. Precision assays showed R.S.D % lower than 12.1% and accuracy was in the range of 93.1 to 102.5%. Lowest Limit of detection was 5 ng mL-1 for all analytes. Metabolic ratios of amitriptyline demethylation were evaluated in individuals genotyped for CYP2C19, with clear differences between volunteers with zero or two active alleles. The method is suitable for therapeutic drug monitoring of patients under amitriptyline treatment, also allowing the indication of CYP2C19 activity.

A strategy for high‐throughput analysis of levosimendan and its metabolites in human plasma samples using sequential negative and positive ionization liquid chromatography/tandem mass spectrometric detection

Levosimendan (Simdax) is an approved drug in approximately 40 countries and currently in phase III clinical studies in the USA and Europe. An accurate, high-throughput and rugged assay is critical to support these clinical trials. Due to the mechanism of drug metabolism, the drug and its active metabolites often have significant differences in their chemical properties. In order to achieve high assay throughput and low sample volumes, a single bioanalytical assay for the drug and its metabolites is preferred. However, this need may prevent the optimization of both high-performance liquid chromatography (HPLC) and mass spectrometric ionization conditions. The chemical properties of levosimendan are significantly different from those of its two active metabolites, OR-1855 and OR-1896. Here, we present a novel strategy for high-throughput analysis of levosimendan and its metabolites. A 96-well liquid/liquid extraction procedure was developed for sample preparation. A single liquid chromatography/tandem mass spectrometry (LC/MS/MS) system with two separate mobile phases, shared backwash solvent and conditioning solvent, was developed to perform sequential LC separation for levosimendan and the metabolites. Levosimendan was eluted by 5 mM ammonium acetate in 33.3% acetonitrile and detected using negative ionization mode MS/MS monitoring. The metabolites were eluted by 5 mM ammonium acetate and 0.2% acetic acid in 20% acetonitrile and detected with positive ionization mode MS/MS monitoring. The method has been demonstrated to have excellent precision and accuracy, with high assay ruggedness during method validation and clinical sample analysis. The linear dynamic ranges were approximately 200-50,000 pg/mL for levosimendan and approximately 500-130,000 pg/mL for both metabolites. The coefficient of determination (r2) for all analytes was greater than 0.9985. The intra-assay %CVs for QC samples were from 0.9% to 2.0% for levosimendan, 0.9% to 3.2% for OR-1855, and 0.4% to 4.9% for OR-1896. The inter-assay %CVs for QC samples were from 1.2% to 1.8% for levosimendan, 1.3% to 2.7% for OR-1855, and 1.4% to 3.4% for OR-1896. The mean % biases for QC samples were from 1.5% to 5.5% for levosimendan, -1.4% to 2.6% for OR-1855, and -0.3% to 4.5% for OR-1896. By using a single extraction approach coupled with sequential LC/MS/MS analysis for levosimendan and its metabolites, the assay maintained high throughput and low sample volume usage.

A highly sensitive method for the detection of PKC412 (CGP41251) and its metabolites by high-performance liquid chromatography

Introduction PKC412 has proven activity in the treatment of acute myelogenous leukemia (AML). The drug is extensively metabolized, and recently it was shown that its metabolites have differing efficacies against malignant cells. Therefore, we established a new isocratic HPLC method, which sensitively detects PKC412 and five of its metabolites. Method Quantification was performed using N-phenyl-1-naphtylamine as an internal standard. We could demonstrate linearity in a range from 10 to 10,000 ng/ml PKC412 with an intra-day variability of less than 7.5% and an inter-day-variability of less than 11%. Results The assay was used to monitor plasma samples from patients with AML treated within a clinical trial. Here we could demonstrate the ability of the assay to detect five metabolites of PKC412 and describe the application of the assay for drug monitoring in clinical situations. Conclusion The assay described here will enable a discriminative analysis of PKC412 and its metabolites in human plasma samples. First clinical application of the assay suggests different rates of metabolism of the individual metabolites of PKC412 with e.g. accumulation of CGP52421 epimer 2 even after cessation of therapy. Since it is assumed that different metabolites of PKC412 have a very individual mode of actions, determination of PKC412 and its metabolites within clinical studies of the drug will be important.