Metabolites In Human Urine Research Articles

A High-throughput HPLC-MS/MS Assay for the Detection, Quantification and Simultaneous Structural Confirmation of 136 Drugs and Metabolites in Human Urine.

Because of its superior sensitivity and specificity, multianalyte high-performance liquid chromatography coupled with tandem mass spectrometry has become the gold standard in clinical toxicology. Although several qualitative and quantitative liquid chromatography coupled with tandem mass spectrometry assays on various mass spectrometry platforms have been described in the literature, most methods either analyze only a limited number of compounds and/or require tedious and time-consuming sample preparation. A major challenge in urine toxicology screening is the minimization of false-negative and false-positive results. This was addressed by screening for a comprehensive panel of 136 compounds of importance for pain and drug addiction clinics, using high-end, high-sensitivity, fast-scanning mass spectrometry in combination with simultaneous structural confirmation based on ion ratios. The assay was validated and successfully participated in proficiency challenges. The assay met all predefined acceptance criteria. The lower limit of quantifications ranged from 10 to 100 ng/mL. Interday trueness and imprecisions ranged from 73.8% to 116.2% and 2.4%-20.0%, respectively. The total assay run time was 10 minutes. We developed and successfully validated a robust, sensitive, and specific liquid chromatography coupled with tandem mass spectrometry-based urine toxicology screening platform that allows for the addition of drugs to quickly adjust to new clinical needs. To date, more than 3000 clinical samples have successfully been analyzed. Our results also indicated that such a quantitative multianalyte assay is pushing against the limits of current fast-scanning MS/MS instrumentation and that the number of analytes and their internal standards must be balanced with acceptable sensitivity, reproducibility, structural confirmation, and the ability to reliably quantify, all of which was achieved.

Magnetic dispersive solid-phase extraction based on modified magnetic nanoparticles for the detection of cocaine and cocaine metabolites in human urine by high-performance liquid chromatography-mass spectrometry.

An easy-to-handle magnetic dispersive solid-phase extraction procedure was developed for preconcentration and extraction of cocaine and cocaine metabolites in human urine. Divinyl benzene and vinyl pyrrolidone functionalized silanized Fe3 O4 nanoparticles were synthesized and used as adsorbents in this procedure. Scanning electron microscopy, vibrating sample magnetometry, and infrared spectroscopy were employed to characterize the modified adsorbents. A high-performance liquid chromatography with mass spectrometrymethod for determination of cocaine and its metabolites in human urine sample has been developed with pretreatment of the samples by magnetic dispersive solid-phase extraction. The obtained results demonstrated the higher extraction capacity of the prepared nanoparticles with recoveries between 75.1 to 105.7% and correlation coefficients higher than 0.9971. The limits of detection for the cocaine and cocaine metabolites were 0.09-1.10ng/mL. The proposed magnetic dispersive solid-phase extraction method provided a rapid, environmentally friendly and magnetic stuff recyclable approach and it was confirmed that the prepared adsorbents material was a kind of highly effective extraction materials for the trace cocaine and cocaine metabolites analyses in human urine.

Electrospun nanofibers-based online micro-solid phase extraction for the determination of monohydroxy polycyclic aromatic hydrocarbons in human urine

In this article, silver nanoparticles (AgNPs) modified nanofibers that represent a novel kind of packing materials for micro-solid phase extraction (μ-SPE) is reported for the first time. The composite material was fabricated through in-situ formation of AgNPs on the polydopamine (PDA) coated polystyrene electrospun fibers (PS@PDA-Ag). The nanofibers displayed desirable hydrophilicity, large surface area, high porosity, good extraction ability and certain capacity to resist matrix interference. A μ-SPE combined with liquid chromatography-mass spectrometry (LC–MS) method was developed for the online determination of three monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) metabolites in human urine. Under the optimal conditions, good linearity (0.02–5ngmL−1) was acquired with the correlation coefficients (R2) being larger than 0.9962. Low limits of detection (0.007–0.032ngmL−1) expressed the satisfactory sensitivity of the method. Moreover, the intraday relative standard deviation values lower than 9.7% and the recoveries among the range of 71–116% were obtained. In general, the online μ-SPE-LC–MS method had the features of simplicity, rapidity, sensitivity and automation, and it was expected to become a promising approach for the online analysis of trace OH-PAHs in complex biological samples.

Improving wastewater-based epidemiology to estimate cannabis use: focus on the initial aspects of the analytical procedure

Wastewater-based epidemiology is a promising and complementary tool for estimating drug use by the general population, based on the quantitative analysis of specific human metabolites of illicit drugs in urban wastewater. Cannabis is the most commonly used illicit drug and of high interest for epidemiologists. However, the inclusion of its main human urinary metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in wastewater-based epidemiology has presented several challenges and concentrations seem to depend heavily on environmental factors, sample preparation and analyses, commonly resulting in an underestimation. The aim of the present study is to investigate, identify and diminish the source of bias when analysing THC-COOH in wastewater. Several experiments were performed to individually assess different aspects of THC-COOH determination in wastewater, such as the number of freeze-thaw cycles, filtration, sorption to different container materials and in-sample stability, and the most suitable order of preparatory steps. Results highlighted the filtration step and adjustment of the sample pH as the most critical parameters to take into account when analysing THC-COOH in wastewater. Furthermore, the order of these initial steps of the analytical procedure is crucial. Findings were translated into a recommended best-practice protocol and an inter-laboratory study was organized with eight laboratories that tested the performance of the proposed procedure. Results were found satisfactory with z-scores ≤ 2.

Quantification of 7-aminoflunitrazepam in human urine by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry

Using a simple liquid-liquid extraction (LLE) procedure for sample pretreatment, 7-Aminoflunitrazepam (7-aminoFM2), a major metabolite of flunitrazepam (FM2), was determined in urine samples by polymeric monolith-based capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The linearity was found in the range of 0.1–50ngmL−1 with a method detection limit (signal-to-noise ratio of 3) estimated at 0.05ngmL−1. Using the proposed method, good precision and recovery were also found in spiked urine samples at the levels of 0.5, 5.0, and 50ngmL−1 (intra-day/inter-day precision: 0.6–1.8% / 0.1–0.8%; post-spiked/pre-spiked recovery: 95.4–102.9% / 96.3–102.5%). In addition, acceptable relative differences (−24.2 – 0.8%) were observed by analyzing clinical urine samples using this monolith-based capillary LC-MS/MS method compared with the results obtained by the routine GC-MC method. Using the monolithic column, no noticeable deterioration of separation efficiency or carry-over was observed for more than 200 injections of urine samples. The applicability of the developed monolith-based capillary LC-MS/MS method was demonstrated by quantifying 7-aminoFM2 in various clinical urine samples. Based on these experimental results, the proposed LLE-monolith-based capillary LC-MS/MS method shows the potential for routine determination of drug metabolites in human urine for clinical and forensic applications.

Estimation of caffeine intake from analysis of caffeine metabolites in wastewater

Caffeine metabolites in wastewater were investigated as potential biomarkers for assessing caffeine intake in a population. The main human urinary metabolites of caffeine were measured in the urban wastewater of ten European cities and the metabolic profiles in wastewater were compared with the human urinary excretion profile. A good match was found for 1,7-dimethyluric acid, an exclusive caffeine metabolite, suggesting that might be a suitable biomarker in wastewater for assessing population-level caffeine consumption. A correction factor was developed considering the percentage of excretion of this metabolite in humans, according to published pharmacokinetic studies. Daily caffeine intake estimated from wastewater analysis was compared with the average daily intake calculated from the average amount of coffee consumed by country per capita. Good agreement was found in some cities but further information is needed to standardize this approach. Wastewater analysis proved useful to providing additional local information on caffeine use.

Identification of (2-aminopropyl)benzofuran and its metabolites in human urine

An HPLC−HRMS method for the detection of the benzofuran analog of amphetamine and its metabolites in human urine using both library search and target search of the most probable metabolites based on their calculated accurate weights and fragmentation regularities is proposed. The proposed method is confirmatory. Gas chromatography–electron impact mass spectrometry with library search is proposed as the main screening method.

Synthetic and analytical strategies for the quantification of phenyl-γ-valerolactone conjugated metabolites in human urine.

The contribution of the gut microbiota to the metabolism of catechins and proanthocyanidins remains unclear. Phenyl-γ-valerolactones have been identified as the most representative metabolites of these dietary flavan-3-ols, but their accurate quantification has posed problems because of a lack of appropriate bioanalytical standards. This work aimed at synthesizing a novel set of sulphate- and glucuronide-conjugated phenyl-γ-valerolactones and at developing an analytical platform using UHPLC-ESI-MS/MS for their quantification in urine. Eight glucuronide and sulphate conjugates of hydroxyphenyl-γ-valerolactones were synthesized and used as analytical standards, together with five phenyl-γ-valerolactone aglycones, for the development of a high-throughput, validated analytical method. Chromatographic and MS conditions were optimized. The method validation showed acceptable linearity, intra-day and inter-day repeatability, and accuracy, with the analytical range, limit of detection (LOD), and lower limit of quantification (LLOQ) varying notably among compounds. The method was used to calculate the excretion of phenyl-γ-valerolactones in healthy subject consuming green tea, providing novel information on the real concentrations of phenyl-γ-valerolactones in urine. This work opens the door to better studying the bioavailability of flavan-3-ols and the real exposition to flavan-3-ol sources, as well as to define the bioactivity of these colonic metabolites in cell assays.

Development of a solid-phase extraction method with simple MEKC-UV analysis for simultaneous detection of indole metabolites in human urine after administration of indole dietary supplement

This work presents the development of a solid phase extraction method with simple MEKC-UV analysis for the simultaneous determination of indole-3-carbinol (I3C) and its metabolites (3, 3′-diindolylmethane (DIM), indole-3-carboxaldehyde (I3CAL), indole-3-acetonitrile (I3A)) in human urine after oral administration of an indole dietary supplement. Solid phase extraction (SPE) method was applied for the first time for simultaneous analysis of these indole metabolites. The MEKC separation method was developed in a previous work. Three commercial SPE cartridges, each with different sorbent materials, were investigated: Sep-Pak® C18, Oasis® HLB and Oasis® WCX. The Sep-Pak® C18 material provided the highest extraction recovery of 88–113% (n = 9), for the four target indole metabolites (I3C, DIM, I3CAL and I3A). The optimal washing and elution solutions were 40% methanol/water (v/v) and 100% methanol, respectively, and optimal elution volume was 2.0mL. The specificity of the proposed SPE method was evaluated with negative control urine samples (n = 10) from healthy volunteers who had not taken the dietary supplement or vegetables known to contain indole compounds. Linear calibration curves were in the range of 0.2–25μgmL−1 (r2 > 0.998) using diphenylamine (DPA) as the internal standard. Intra-day and inter-day precisions were 3.5–12.3%RSD and 2.7–14.1%RSD, respectively. Limits of detection and quantification were 0.05–0.10μgmL−1 and 0.10–0.50μgmL−1, respectively. The four target indole compounds were separated within only 5min by MEKC-UV analysis. Urine from 5 subjects who had taken a dietary supplement containing I3C and DIM were found to contain only the DIM metabolite at concentrations ranging from 0.10 to 0.35µgmL−1. Accuracy of the proposed method based on the percentage recovery of spiked urine samples were 70–108%, 82–116%, 82–132% and 80–100% for I3C, I3CAL, I3A and DIM, respectively. The Sep-Pak®C18 cartridge was highly effective in extraction and sample cleanup for the downstream simultaneous detection of urinary indole metabolites by MEKC-UV method.

Screening with Quantification for 64 Drugs and Metabolites in Human Urine using UPLC-MS-MS Analysis and a Threshold Accurate Calibration.

Drug and metabolite (analytes) identification together with quantification is an important analytical tool in forensic and clinical toxicology. We report the development and validation of a definitive detection and quantification method (UPLC-MS-MS) for initial screening of 64 analytes in urine. The principle of the method is a quantitative extension of a recently reported threshold accurate calibration (TAC) technique which employs a rapid dual-specimen analysis i.e., with and without addition of a reference-analyte standard for normalization of matrix effects. Analytes include pharmaceutical and illicit agents from opiate and opioid agonist, opiate-antagonist, benzodiazepine, amphetamine, designer amphetamine, cathinone, cocaine, hallucinogen, gabapentinoid and sedative drug classes. Using a 96-well plate format, the protocol employs glucuronidase hydrolysis, 27-fold urine dilution and a 3 min UPLC-MS-MS acquisition. Subsequent data management includes calculation of a normalized TAC ratio response and weighted least squares calibration. The method utilizes analyte-specific calibration ranges from 2.5 to 1,500 ng/mL with quality control (QC) monitoring of transition-ion ratio, calibrator re-analysis, injection precision and multi-level QC analysis. Method precision, bias, calibration linearity, detection limit, carryover, crossover studies and external proficiency performance were evaluated based on pre-established criteria. The validated method provides an alternative to stable-isotope internal standardization methods of quantification and is applicable to screening with quantification in routine toxicology practice.

Isotopic labeling of multiple functional groups for improving the quantification of urine metebolome

A high proportion of metabolites in human urine possess hydroxy and amino groups. Dangly chloride has been widely used in chemical isotope labeling to detect the compounds with phenol and amines by liquid chromatography‐mass spectrometry (LC‐MS), but not for alcohols. Therefore, we carried out the chemical isotope labeling with two kinds of catalysts (DMAP and DIPEA) for labeling both amino group and hydroxy group simultaneously using 12C or 13C‐dansyl chloride. After labeling, the products were extracted with water for three times and collected the organic layer. We dried the extracted sample, separated as two aliquots which were labeled separated with 12C / 13C‐dansyl chloride. The mixtures of 12C / 13C‐labeled metabolites were analyzed by LC‐MS for metabolite identification and quantification. Using penal and cholesterol as standards, this new dansylation method is proved be able to label the compounds with phenol and hydroxyl groups. To optimize the whole work flow, we optimized the variables including reaction solvent ratio, reaction time, reaction volume and compared this technique with other dansylation methods in the peak pairs, retention time and the functional group of labeled metabolites. In summary, this technique improves the detection of many metabolites in LC‐MS analysis, particularly hydroxy group and amino group.

Development of microextraction techniques in combination with GC-MS/MS for the determination of mycotoxins and metabolites in human urine.

Simple and highly efficient sample preparation procedures, namely, dispersive liquid-liquid microextraction and salting-out liquid-liquid extraction for the analysis of ten Fusarium mycotoxins and metabolites in human urine were compared. Various parameters affecting extraction efficiency were carefully evaluated. Under optimal extraction conditions, salting-out liquid-liquid extraction showed a better accuracy (84-96%) and precision (<14%) than dispersive liquid-liquid microextraction. Hence, a multibiomarker method based on salting-out liquid-liquid extraction followed by gas chromatography with tandem mass spectrometry was proposed. Satisfactory results in terms of validation were achieved. The method resulted in low limits of detection and quantitation within the range of 0.12-4 and 0.25-8μg/L, respectively. The method accuracy and precision were evaluated at three spiking levels (8, 25 and 100μg/L) and the recoveries were in a range from 70 to 120% with relative standard deviations lower than 15%. Matrix effect was evaluated and matrix-matched calibrations were used for quantitation purpose. The developed method was applied in 12 human urine samples as a pilot study before and after sample treatment with β-glucuronidase before the analysis to quantify the mycotoxin conjugates. Total deoxynivalenol (free + conjugated) was found in 83% of samples at an average concentration in positive samples of 31.6μg/L.

Simultaneous quantification of antofloxacin and its major metabolite in human urine by HPLC-MS/MS, and its application to a pharmacokinetic study.

This study presents a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of antofloxacinin and its main metabolite - N-demethylated metabolite (N-DM) - in human urine. Ornidazole was used as the internal standard. This was a clinical urine recovery study, in which 10 healthy Chinese volunteers were intravenously administered a single 200 mg dose of antofloxacin hydrochloride. Compounds were extracted by albumen precipitation, after which samples were isocratically eluted using a Poroshell 120 SB-C18 column, and were analysed using HPLC-MS/MS under electronic spray ionization positive ion mode. The method was successfully applied in a urine pharmacokinetic study of antofloxacinin, with a detection range of 0.02/0.01 to 200/100 μg/mL (for antofioxacin/N-DM).The average percentages of antofioxacin/N-DM measured in urinary excretion frp, 10 volunteers were 54.9 ± 5.7/8.2 ± 2.5% in 120 h duration.

Simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites in human urine by high performance liquid chromatography-tandem mass spectrometry.

A rapid, simple and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their main metabolites, N-(2,4-dimethylphenyl)-N-methyl-formamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA), 4-chloro-2-methylaniline and 3-hydroxyacetanilide in human urine. The urine samples were mixed with buffer solutions (pH 8) and subsequently cleaned up by solid supported liquid/liquid extraction (SLE). The target analytes were efficiently separated with a Waters Atlantis T3 column (150mm×4.6mm, 5μm), ionized with electrospray ion source in positive mode, and quantitatively determined by tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. In order to minimize matrix effects, the matrix-matched calibration curves of eight analytes were adopted with correlation coefficients (R2) above 0.99. The method were further validated by determining the limits of detection (LODs, 0.3-0.6ng/mL), the limits of quantitation (LOQs, 1.0-2.0ng/mL) and recoveries (89.1%-108.4%) with intra-day and inter-day relative standard deviation (RSD, <11%). The established method was applied and demonstrated in a real case by assaying a urine sample from a female poisoned by formetanate. The achieved results proved this method to be rapid, sensitive and accurate for simultaneous quantitation of eight analytes in human urine for intended forensic cases of human poisoning.

Quantifying endogenous androgens, estrogens, pregnenolone and progesterone metabolites in human urine by gas chromatography tandem mass spectrometry

A method for the quantitation of 22 urinary steroids (androgens, estrogens and the main pregnenolone and progesterone metabolites) by means of gas chromatography tandem mass spectrometry using a triple quadrupole analyzer has been developed. Two different enzymatic hydrolysis protocols were investigated; one capable of releasing steroids present as both sulfates and glucuronides (total fraction), and another with β-glucuronidase activity only.After selecting adequate internal standards and choosing the optimal instrumental parameters, i.e. chromatographic separation and ion transition conditions, the method was fully validated using both hydrolysis protocols. The method was shown to be linear (r >0.99) in the range of endogenous concentrations for all studied steroids with extraction recoveries higher than 80%. The use of labeled internal standards allowed for both a correct quantification and the evaluation of the rate of deconjugation for sulfates and glucuronides in every sample. In general, the sensitivity of the method was suitable for the detection of the endogenous levels, with limits of quantification ranging from 0.1 to 20ng/mL. Accuracies ranging from 80% to 120%, and relative standard deviations below 25% in intra- and inter- assay experiments were found for most of the analytes.The applicability of the validated method was tested by quantifying twenty-two metabolites in 24-h urine samples collected from healthy individuals. The ranges for the excretion of steroids in the total and glucuronide fractions obtained with the new method were compared with those available in the literature. By comparing the figures in both fractions, an estimation of the percentage that the sulfation represents for each steroid was also calculated. The presence of side enzymatic activities and the utility of the method for clinical studies as well as for doping control analysis is discussed.

Photochemical transformation and wastewater fate and occurrence of omeprazole: HRMS for elucidation of transformation products and target and suspect screening analysis in wastewaters

Omeprazole is one of the world-wide most frequently prescribed and administered pharmaceuticals in humans for the relief of gastro-intestinal disorders. Despite its high worldwide consumption, omeprazole is rarely detected in urban wastewaters and environmental waters. On the other hand, its human urinary metabolites and transformation products formed through abiotic processes in the environment have been recently documented. Despite these available data, however, there is still a lack of information on the extent of environmental fate and occurrence, on elimination mechanisms and efficiencies in waste water treatment plants. In this study, the relevance of photodegradation processes on the environmental persistence of omeprazole under simulated and solar irradiation was investigated. Photodegradation experiments were performed in distilled water, lake, river and seawater, and revealed that the different matrix in natural waters significantly affect the photolytic behavior of the investigated compound. Overall, the results highlight that photodegradation process by solar irradiation significantly contributes to omeprazole degradation and elimination from the aquatic environment. TPs formed through the process were identified and elucidated by using liquid chromatography high resolution mass spectrometry. In total seven TPs were identified, among of which four were also detected as hydrolysis TPs. The Microtox bioassay showed that solar photolysis is efficient to detoxify omeprazole and its TPs in aqueous solutions. Finally, a systematic investigation was conducted in order to provide information on removal efficiency and occurrence of omeprazole and its metabolites/TPs in 8 WWTPs in North West Greece by performing target and suspect screening analysis. The findings revealed the presence of both parent compound and metabolites/TPs in wastewaters. Given, however, the scarce detection of omeprazole at fairly low concentration levels, from analytical and environmental point of view, further attention should be given to metabolites/TPs instead of the parent compound.

Simultaneous determination of glipizide and its four hydroxylated metabolites in human urine using LC–MS/MS and its application in urinary phenotype study

Cytochrome P450 (CYP) 2C9 and CYP2C19 genetic mutant could influence the plasma concentration of glipizide in human subjects, which refers to glipizide safety and adverse effects in clinic practice. A further study to investigate the relationship of the concentrations between glipizide and its metabolites in human with different CYP mutants was valuable. We firstly develop a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for simultaneous quantification of glipizide and its hydroxylated metabolites in human urine. After simple protein precipitation with methanol including 4′-OH-tolbutamide and gliclazide (both are internal standards), the analytes were chromatographed on a reversed-phased column with a mobile phase of 0.1% formic acid in acetonitrile and 0.1% formic acid in water by a gradient elution. The ion transitions of the precursor to the product ion were principally protonated ions [M+H]+ at m/z 446.4→m/z 321.1 for glipizide, m/z 462.2→m/z 321.1 for the four hydroxylated forms of glipizide, m/z 287.2→m/z 188.0 for 4′-OH-tolbutamide, and m/z 324.1→m/z 127.1 for gliclazide. The method was linear over a concentration range of 0.02–20.0ng/mL. The intraday and inter-day variances were less than 9.9%, and accuracy was within ±6.8%. The method was successfully applied to the urinary phenotyping study in volunteers after a single oral administration of 5-mg glipizide tablet, and two new hydroxycyclohexyl metabolites of glipizide (OH-gp), 4-cis-OH-gp and 3-trans-OH-gp, were found in this study.

Flavonoid Metabolites in Human Urine during Blueberry Anthocyanin Intake.

The human health benefits of anthocyanins (Anc) and other flavonoids are widely recognized. However, the flavonoid-based urinary metabolites arising in vivo after Anc intake are not well described. Human (n = 17) urine was collected while blueberry juice (BJ) was consumed daily for 28 days and once after a 7 day washout. MS/MS scanning of 664 urine samples for 18 parent Anc (PAnc) and 42 predicted Anc metabolites (AncM) yielded 371 products (i.e., MS/MS × retention time (RT)). Flavonoid-based AncM, which were likely underestimated, were almost 20 times more abundant than PAnc. Together, PAnc and AncM accounted for about 1% of the daily Anc dose. Aglycone forms were >94% of the total. Cluster analysis of the 371 Anc identified about 55 major Anc that contributed about 80% to the total Anc. The abundance of flavonoid-based Anc-derived products in the gastrointestinal tract could contribute to the health benefits of Anc-rich berries.

Simultaneous Determination of Erlotinib and its Metabolites in Human Urine and Serum Samples by High-Performance Liquid Chromatography

A simple and rapid reverse-phase high-performance liquid chromatographic method for the simultaneous separation and determination of erlotinib, an antineoplastic agent, which is used in the therapy of advanced or metastatic pancreatic or non-small cell lung cancer, and its metabolites has been developed. This method has been applied to urine and serum samples of patients under erlotinib treatment. After a dilution step, erlotinib and its metabolites have been separated on an Ultrabase 120 A C18 column (4.6 mm i.d. × 150 mm, 5 μm) using methanol: 20 mM phosphate buffer pH 5.0 (60:40; v:v) as mobile phase at a flow rate of 1.4 mL min−1 and detected at 248 nm with a diode array detector. The limits of detection were between 2.1 and 5.1 ng mL−1. Within- and between-day accuracy and precision of the validated method were below the acceptable limits of 15% relative standard deviation at all concentrations tested. The quantitation method was successfully applied for the simultaneous determination of erlotinib and its metabolites in urine and serum samples from patients under clinical treatment of non-small cell lung cancer. The concentration ranges of erlotinib and desmethyl erlotinib in serum were 380–6510 and 66–558 ng mL−1, respectively, in patients under treatment. The objective is to propose a fast and simple method for a direct and reliable quantification of erlotinib and its metabolites in human urine and serum without previous sample treatment steps. The direct injection of diluted urine and serum can be highlighted as the greatest advantage that can be achieved by this method.

Identification of phenolic metabolites in human urine after the intake of a functional food made from grape extract by a high resolution LTQ-Orbitrap-MS approach

Grape pomace (GP) is known to be a rich source of polyphenols with biological activity which may be used as functional ingredients for the development of new health-promoting products. Numerous studies have reported that bioactive compounds may act through multiple mechanisms. In order to verify the oral absorption and metabolism of grape polyphenols, we performed a prospective, randomised and cross-over acute study in 12 volunteers with two interventions: 500mL of a functional beverage enriched with 200mL of GP and 500mL of a control beverage without GP. In this work, liquid chromatography coupled with an electrospray ionization hybrid linear ion trap quadrupole-Orbitrap-mass spectrometry (LC/ESI-LTQ-Orbitrap-MS) technique has been used to accurately identify phenolics in GP and human urine. In GP, 41 phenolic compounds were identified mainly procyanidins, phenolic acids and flavonols, and in human urine over 70 metabolites of phenolic compounds including microbiota metabolites, glucuronides and sulfate derivatives were detected. Overall, high resolution mass spectrometry (HR-MS) enhances the identification of a large variety of polyphenols and their metabolites with great mass accuracies for all molecular ions.