High performance liquid chromatography–tandem mass spectrometry quantification of tryptophan metabolites in human serum and stool – Application to clinical cohorts in Inflammatory

Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis. Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD),

Journal of Chromatography A 1685 (2022) 463602 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma High performance liquid chromatography–tandem mass spectrometry quantification of tryptophan metabolites in human serum and stool – Application to clinical cohorts in Inflammatory Bowel Diseases ✩,✩✩,★ Aurore Desmons a,∗ , Lydie Humbert a , Thibaut Eguether a , Pranvera Krasniqi a , Dominique Rainteau a , Tarek Mahdi b , Nathalie Kapel b , Antonin Lamazière a a Clinical metabolomic department, Sorbonne Université, INSERM, Centre de Recherche Saint-Antoine (CRSA), Saint Antoine Hospital, Assistance Publique des Hôpitaux de Paris (AP-HP.Sorbonne Université), Paris, France Laboratoire de Coprologie Fonctionnelle, Hôpitaux Universitaires Pitié-Salpêtrière - Charles Foix, Assistance Publique des Hôpitaux de Paris (AP-HP.Sorbonne Université), Paris, France b a r t i c l e i n f o Article history: Received 12 July 2022 Revised 12 October 2022 Accepted 23 October 2022 Available online November 2022 Keywords: tryptophan metabolites profile LC-MS/MS Inflammatory bowel diseases a b s t r a c t Tryptophan, an essential amino acid, and its metabolites are involved in many physiological processes including neuronal functions, immune system, and gut homeostasis Alterations to tryptophan metabolism are associated with various pathologies such as neurologic, psychiatric disorders, inflammatory bowel diseases (IBD), metabolic disorders, and cancer It is consequently critical to develop a reliable, quantitative method for the analysis of tryptophan and its downstream metabolites from the kynurenine, serotonin, and indoles pathways An LC-MS/MS method was designed for the analysis of tryptophan and 20 of its metabolites, without derivatization and performed in a single run This method was validated for both serum and stool The comparisons between serum and plasma, collected with several differing anticoagulants, showed significant differences only for serotonin References values were established in sera and stools from healthy donors For stool samples, as a proof of concept, the developed method was applied to a healthy control group and an IBD patient group Results showed significant differences in the concentrations of tryptophan, xanthurenic acid, kynurenic acid, indole-3-lactic acid, and picolinic acid This method allowed an extensive analysis of the three tryptophan metabolic pathways in two compartments Beyond the application to IBD patients, the clinical use of this method is wide-ranging and may be applied to other pathological conditions involving tryptophan metabolism, such as neurological, psychiatric, or auto-inflammatory pathologies © 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Tryptophan (Trp) is an essential aromatic amino acid involved in protein synthesis and is the precursor of many bioactive compounds Trp and its metabolites are implicated in many physiological processes such as neuronal functions, immunoregulation, and inflammation Trp is also identified as a key marker in gut homeostasis and its metabolism is closely linked to the intestinal microbiome Around 90% of Trp is metabolized through the kynurenine ✩ Disclosure statement: no All authors declare no competing financial interests and consent for publication ★ Data and material are available ∗ Corresponding author at: Hôpital Saint-Antoine, AP-HP Sorbonne Université, 27, rue Chaligny, 75012 Paris, France E-mail address: aurore.desmons@aphp.fr (A Desmons) ✩✩ pathway, also called the indoleamine-2,3-dioxygenase (IDO) pathway [1–3] This pathway leads to kynurenine production as well as other neurologically active compounds such as kynurenic acid (KA), quinolinic acid (QA) and 3-hydroxykynurenine (3-HK) QA and 3-HK have neurotoxic properties while KA has neuroprotective effects [4] The second pathway utilizing Trp, which is quantitatively less important and contributes less than 5% of Trp degradation, is the serotonin pathway This pathway leads to the production of 80% of total serotonin by intestinal compartment and plays an important role in neurotransmission and neurological functions [3,5] The last pathway, the aryl hydrocarbon receptor (AhR) pathway utilizes Trp in the synthesis of indole and indoles derivatives by intestinal bacteria Many of these derivatives, such as indole3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3- https://doi.org/10.1016/j.chroma.2022.463602 0021-9673/© 2022 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Fig Tryptophan metabolism via kynurenine (blue), serotonin (green) and indoles (orange) pathways Chemicals structures of tryptophan and metabolites quantified by the LC-MS/MS method developed Figure adapted from Agus et al [3] carboxaldehyde, can activate the AhR expressed by some immune and intestinal cells [6,7] Alterations to Trp metabolism are associated with many pathological states such as neurodevelopmental, neurologic and psychiatric disorders, metabolic disorders, and cancer [8,9] Many publications have highlighted the modifications of tryptophan metabolism in gastrointestinal disorders including inflammatory bowel diseases (IBD) and irritable bowel syndrome (IBS) [3,10] In IBD, these alterations of Trp metabolism may be involved in the pathogenesis of the disease [3,11] Indoles metabolites are highly of interest in several disorders, a recent study reported that metabolic disorders are associated with a decrease of AhR agonists produced from trp [12] Depletion of trp metabolites including AhR agonists may be affect the severity of the disease [13] It is consequently critical to develop a reliable, quantitative method for the analysis of Trp and its metabolites for research and clinical purposes To explore Trp metabolism, several quantitative methods have been developed The first methods consisted of liquid chromatography (LC) separation associated with detection based on UV absorbance, fluorescence, or electrochemistry [14] Recent methods have been developed using LC coupled to tandem mass spectrometry (LC-MS/MS) focusing on the major metabolites of kynurenine and serotonin pathways, and quantification performed mainly on serum or plasma matrices [15,16] Global analysis of Trp metabolites was developed in different biological samples but quantifications were dedicated to non-human matrices [17,18] The purpose of our study was to explore the metabolism of Trp in the two compartments (i.e matrices) relevant in IBD and IBS contexts [12,13] Beyond theses pathologies, exploration of trp metabolism is highly of interest in neurological diseases, such as multiple sclerosis and Huntington’s disease [19,20] This panel may be useful to follow new therapies targeting the gut microbial metabolism [13] Published methodology studies have mainly been performed either in serum or stool, but not in both compartments of patients Here, we developed a LC-MS/MS method for the quantification of tryptophan and 20 of its metabolites in three different human biological matrices: feces, serum, and plasma (Fig 1) This method allows an extensive analysis of the three stated metabolic pathways in a single test, without derivatization Quantification was es- tablished and validated in human serum, plasma, and stool using both internal standards and external calibration, which allowed the establishment of references values in the two compartments As a proof of concept, the developed method was applied to a control healthy group and a patients group diagnosed with IBD, including ulcerative colitis (UC) and Crohn’s disease Material and methods 2.1 Chemicals and reagents Tryptophan (Trp) (≥ 99 %), picolinic acid (Pico) (99 %), quinolinic acid (QA) (99 %), 3-hydroxy-kynurenine (3HK) (≥ 98 %), serotonin (5HT) (≥ 98 %), 5-hydroxy-tryptophan (5HTP)(≥ 98 %), 3-hydroxy-anthranilic acid (3-HAA) (97 %), kynurenine (KYN) (≥ 98 %), xanthurenic acid (XA) (96 %), tryptamine (TA) (≥ 97 %), kynurenic acid (KA) (≥ 98 %), 5-hydroxyindole acetic acid (5HIAA) (≥ 98 %), N-acetylserotonin (NAS) (≥ 99 %), indole-3acetamide (IAM) (98 %), indole-3- lactic acid (ILA) (99 %), indole3-carboxaldehyde (I-3CA) (97 %), melatonin (MELA) (≥ 98 %), tryptophol (TOL) (97 %), indole-3-acetic acid (IAA) (≥ 98 %), indole3-propionic acid (3-IPA) (99 %), indoxyl-sulfate (3-IS) (≥ 97 %) and methanol (hypergrade for LC-MS LiChrosolv®) were purchased from Sigma-Aldrich (St Louis, Missouri, USA) Chemicals purity were provided in brackets for each standards molecules Internal standards (ISs), L-Tryptophan-d8 (Trp-d8) (98 %) and anthranilic acid 15N (A15N) (98 %) were obtained from Eurisotop (Saint-Aubin, France) and from Cambridge Isotope Laboratories, Inc (Andover,USA), respectively Formic acid (FA) was acquired from Honeywell-Fluka Fisher scientific (IllKirch France), HiPerSolv CHROMANORM acetonitrile (ACN) for HPLC LC-MS grade from VWR (Radnor, USA) 2.2 Analytical protocol 2.2.1 Calibration standards The stocks solutions of unlabeled standards (1mg/mL for each molecule) were prepared in water/methanol (50/50) (v/v) for KYN and I3CA TA, TOL, IAM, IAA, 3-IPA, 5-HIAA, ILA, MELA, A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 3-IS, Pico and NAS were dissolved in methanol (1mg/mL for each molecule) XA, KA were dissolved in dimethylsulfoxide (DMSO) at 1mg/ml QA, 3-HAA were dissolved in dimethylsulfoxide (DMSO)/methanol 10/90 (v/v) at 1mg/ml 5HT, 5HTP, were dissolved in water/methanol/acetic acid 90/9.9/0.1 (v/v/v) at 1mg/ml 3HK was dissolved in water/methanol/NaOH0.2M 90/9.9/0.1 (v/v/v) at 1mg/ml Trp solution (2mg/ml) was prepared in water/sodium hydroxide 0,2 M (75/25) Stock solutions of ISs Trp-d8 (1mg/ml) and A15N (1mg/ml) were dissolved in methanol For serum, a stock calibration standards solution was prepared by mixing equal volumes of the twenty tryptophan metabolites Twelve calibration standards levels were prepared from the stock solution (ranging from 23.8 μg/ ml to ng/ml) by serial dilutions in methanol Seven levels of calibration standards were prepared separately for tryptophan (ranging from 125 μg/ml to 60 ng/ml) by serial dilutions in methanol All stocks solutions were stored at -80°C until analysis 2.3 HPLC-ESI-MS/MS analysis Samples were analyzed using a LC-20ADXR (Shimadzu, Kyoto, Japan) chromatographic system in tandem with a linear ion trap quadrupole MS/MS spectrometer QTRAP 5500 system (SCIEX, Ontario, Canada) Chromatographic separation was performed with a kinetex biphenyl column (100×2.1 mm; particle size 2.6μm) (Phenomenex, Torrance, USA) with a 2.1 mm C8 SecurityGuardTM ULTRA Cartridges UHPLC guard column (Phenomenex, Torrance, USA) The mobile phases were composed of 0.4 % formic acid (FA) in water (v/v) (mobile Phase A) and 0.4% FA in acetonitrile (mobile phase B) The column temperature was set at 17°C, the gradient elution was performed at 0.3 mL/min, starting at 3% of phase B; then increasing to 20 % of phase B from to 0.1 min; then increasing to 65 % B from 0.1 to min; finally increasing to 95 % of phase B from to 7.5 min, and completed to 2.5 at 95% of phase B for wash The column reequilibration consisted in a plateau of 3% B for 2.5 at 0.3 mL/min The injection volume was μL (for plasma, serum, standards and stool) MS detection was performed using electrospray ionisation (ESI) in positive and negative modes, using the Multiple Reaction Monitorig (MRM) function of the analyser For each analyte, the MS conditions were determined via direct infusion of individual standard solutions.” Compressed air was used as the desolvation gas and nitrogen was used as the collision gas The instrument parameters were set as follows: nebuliser gas and turbo gas: 40 psi, curtain gas: 20 psi, ion spray voltage: +/- 4500 V for positive or negative ionization respectively, source temperature: 450°C Declustering potentials (DPs) were set at 60 V, except for QA and Pico, at 33 V A dwell-time of 10 ms was set for all transitions at positive ionization and 20 ms for negative ionization 2.2.2 Serum calibration standards preparation 20 μL of calibration standards were transferred to 1.5 mL Eppendorf tube, 50 ng of each internal were added from stock solution (1mg/mL) for Trp d-8 and A15N, 50 μL of “lyophilized blank serum” (Chromsystems, Gräfelfing, Germany) and 430 μL of methanol were added Samples were mixed for 15 s and placed for 30 at 4°C After centrifugation (18 0 g for 15 at +4°C), the supernatant was transferred to a glass vial We injected μl of the mix into the HPLC-MS/MS system for analysis 2.2.3 Serum and plasma samples preparation 50 μL of plasma or serum sample were transferred to 1.5 mL Eppendorf tube, 50 ng of each internal were added from stock solution (1mg/mL) for Trp d-8 and A15N, and 450 μL of cold methanol was added The samples were vortex-mixed for 15 s and placed for 30 at 4°C The samples were centrifuged (18 0 g for 15 at +4°C) The total content of the supernatant was transferred to a vial from which μl were injected into the HPLCMS/MS system for analysis 2.4 Data processing Spectral data acquisitions were processed using Analyst (v1.6.3) software and quantifications were performed with Multiquant (v3.0.2) software (SCIEX, Ontario, Canada) GraphPad Prism (v6.01) (San Diego, CA, USA) and SIMCA (v16.0.2.10561) (Göttingen, Germany) softwares were used for statistical analysis 2.2.4 Stool calibration standards preparation 20 μL of calibration standards were transferred to 1.5 mL Eppendorf tube, 50 ng of each internal were added from stock solution (1mg/mL) for Trp d-8 and A15N, and 480 μL of methanol were added Samples were vortex-mixed again for 15 s and transferred into glass vials before injection of μL into the HPLC-MS/MS system for analysis 2.5 Method validation The method validation was based on the recommendations of NF EN ISO 15189 criteria and international guidelines dedicated to LC-MS/MS methods [21–23] 2.5.1 Linear range Suitable calibration ranges were determined based upon analysis of pooled sample of serum and adjusted accordingly For each analyte using an internal standard, peak area response ratios were calculated and plotted against the nominal concentration A linear fit was employed, and a 1/x weighting factor was applied 2.2.5 Stool samples preparation Stool samples were frozen at -80°C then, all samples were feeeze-dried (Freeze dryer Buchi L-200 with scroll pump qm/h) at -55.9°C, 0.300 mbar, for 24h 10 mg of freeze-dried stool were transferred to 1.5 mL Eppendorf tube and 500 μL of milliQ water were added The samples were vortex-mixed for 15 s 500 μL of methanol and 50 ng of each internal were added from stock solution (1mg/mL) for Trp d-8 and A15N and samples vortex-mixed again for 15 s After centrifugation at 18 0 g for 15 at +4°C, the supernatant is collected A second extraction was made by adding ml of methanol / water (90/10) (v/v) to the pellet Samples were vortex-mixed for 15 s After centrifugation (18 0 g for 15 at +4°C) the two supernatants were pooled Evaporation was performed under a nitrogen flow Five hundred μl of methanol were added to the residue Five μl were injected onto the HPLCMS/MS system for analysis 2.5.2 Lower limit of detection (LOD) and lower limit of quantification (LLOQ) To determine LOD and LLOQ, analysis of spiked samples with decreasing concentrations of analytes were performed The LOD was accepted for a signal-to-noise ratio (S/N) ≥ and the LLOQ for a signal-to-noise ratio (S/N) ≥ 10 2.5.3 Intra- and interday accuracy and precision Accuracy and precision were estimated from the analysis of levels of quality control (QC) samples prepared for each analyte For intraday analysis, 20 samples were prepared and assayed in A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 the same day for serum and stool For interday analysis, samples and samples were analyzed in separate days for serum and stool, respectively Acceptance criteria for accuracy was determined as a bias within ± 15% of the nominal value and within ± 20% of the LLOQ Acceptance criteria for precision were defined as within 15% of relative standard deviation (R.S.D) and 20% of R.S.D of the LLOQ Results and discussion from these common losses For molecules with very close precursor ions (m/z 206.1 for XA and 206.2 for ILA), product ions used for quantification were different (m/z 160.1 and 130.1 for XA and ILA, respectively) and the chromatographic retentions times were also different (RT: XA 2.2 and ILA 3.3 min) For KA and 3IPA, with the same precursor ion (m/z 190.1), the assigned product ions were different (m/z 115.9 KA and 130.1 IPA), and peaks were chromatographically resolved (RT: 2.4 for KA and 4.6 for 3IPA) The same ion products were shared for quantification of some indoles derivatives (m/z 130.1): for IAM, ILA, 3IPA but the peaks were chromatographically resolved (RT: 3.0 (IAM); 3.3 (ILA); 4.6 (3-IPA)) All metabolites, except for indoxyl sulfate, were analyzed using a positive ESI and were all metabolites were tuned for maximum sensitivity across the linear range (Table 1) A challenge for the development of this method was the presence of metabolites in serum, plasma, and stool at very different levels This heterogeneity in metabolite concentrations required an instrument with a great dynamic range and the appropriate preparation of standard solutions at linear ranges Thus, two solutions were prepared, one containing 20 metabolites, ranging from ng/ml to 23.8 μg/ml, and another for Trp, ranging from 60 ng/ml to 125 μg/ml Both solutions were obtained by serial dilutions in methanol During preparation of stock solution, poor solubility and stability of some metabolites in methanol or in aqueous solutions were observed This phenomenon of instability was previously described for some metabolites from the kynurenines pathway in aqueous solutions (16) For these metabolites, a water/methanol mix, with the addition of sodium hydroxide, acetic acid, or DMSO, was required, as detailed in the materials and methods section For chromatographic separation, a C18 column ˚ 100ì2.1 mm) C18 was tested rst (Kinetexđ m, C18, 100 A, columns, which use octaldecylsilane, were widely used in other quantitative methods dedicated to Trp and its metabolites because of the use of non polar solvents for these methods (e.g water, methanol and acetonitrile)[14–16,18] We also tested a biphenyl column, a new phase, that have short alkyl biphenyl ligands covalently bound to the silica surface, stable under 100% aqueous conditions and exhibited good reverse phase retention and aromatic selectivity Eventually, separation was performed on this column because we obtained an enhanced retention for the aromatic derivatives Different column temperatures were used, from 15°C to 55°C [18,24,25], and was maintained at 17°C following assays, which corresponded to the lowest value available for our device and environment This temperature value, in comparison with higher values, allowed increased retention times and better separation for the compounds eluted first Different percentages of FA were tested in mobile phases, ranging from to 0.4%, to check for molecule specific ionization; the percentage of 0.4 % was set The presence of formic acid (FA) at the highest percentage (i.e 0.4 %) in mobile phases allowed a better ionization of molecules, improved the shapes of the peak, and targeted analytes exhibited MS better responses.The elution gradient was tested with H2 O/Methanol and H2 O/ACN respectively for mobile phases A/B; better shapes of peaks were obtained when ACN was used 3.1 LC-MS/MS method development 3.2 Method validation For a specific detection, MRM transitions were selected for each metabolite and internal standard, as shown in Table A representative chromatogram of all targeted metabolites was shown in Fig Ion products resulting from the losses of 18 or 44 m/z, corresponding to the loss of stable neutral molecules H2 O and CO2 respectively, were excluded where possible This allowed us to obtain more specific transitions, and to avoid cross contamination The method validation was performed based on different international guidelines including Food and Drug Administration (FDA) and European Medicines Agency (EMA) recommendations [21–23] The results for intraday and interday precision and accuracy are reported in Table and for serum and stool All metabolites demonstrated a CV < 20% for the lowest values and a CV < 15% for intraday and interday precisions For the intraday assay, analytical 2.5.4 Stability Stability was evaluated using serum calibration standards obtained as described in part 2.2.2 Calibration standards were prepared and quantified immediately, which served as a reference Other aliquots from the same solution were stored at -80°C These samples were subjected to three freeze-thaw cycles, 10 days, one month, and months after initial freezing Stability was acceptable if the mean concentration was between 85% and 115% of the reference mean concentration 2.5.5 Type of collection tube The impact of the type of collection tube was determined by analyzing serum and plasmas collected in citrate, heparin, and EDTA anticoagulants from healthy donors at fed state 2.5.6 Matrix effects Peaks areas were determined in different sets of samples: a blank matrix sample of stool (set 1), a blank matrix sample of stool spiked with standard solutions at different levels after extraction (set 2), and one prepared from blank matrix from the same stool but spiked just before extraction (set 3) The matrix effect was determined as follows and expressed as a percentage: (set 2-set 1) / (spiked concentration in methanol) An extraction yield was evaluated as follows and expressed as a percentage: (set 3)/(set 2)[21] 2.6 Biological sample testing 2.6.1 Application of the assay to clinical samples The assay protocol was applied to clinical samples from a control group and a group of patients diagnosed with IBD (including UC and Crohn’s disease) 2.6.2 Sample collection information Plasma and serum samples were obtained from the French blood establishment (EFS, Saint Antoine Hospital) Stool samples were received from the routine workload of the laboratory for the follow-up of patients The study using stool residues was approved by the French public health organization (CSP-article L11213, amended by the law n°2011-2012, December 29, 2011-article 5) Blood, from non-fasting or fasting subjects, was collected in tubes containing coagulant, a clot activator, or anticoagulant (heparin, sodium citrate, EDTA) After collection, tubes were centrifuged (3,0 0 g for 10 at 8°C) and serum or plasma aliquoted in Eppendorf tubes, ensuring that no red blood cells or clots were carried over, and stored at -80°C before analysis Stool samples were collected and immediately stored at -80°C; samples were freezedried before analysis A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Table MS conditions and retention times for metabolites and labeled internal standards (ordered by retention time) ∗ quantifier ion Component Names Positive Mode Piconilic acid Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Internal Standards L Tryptophan D8 Anthranilic acid 15N Negative Mode Indoxyl sulfate Retention Time (min) Precursor ion (m/z) Products ions (m/z) Collision energy (V) Internal Standard 1.2 1.3 1.4 2.0 2.0 2.1 2.1 2.2 2.3 2.4 2.4 2.6 2.6 3.0 3.3 3.7 3.7 3.8 3.9 4.6 [M+H] 124.2 168.1 225.1 177.2 221.1 154.1∗ ; 136.1 209.2 206.1 205.2 161.2 190.1 192.1 219.1 175.1 206.2 146.1 233.2 162.1 176.1 190.1 [M+H] 77.9 ∗ ; 96.0 78.0∗ ; 124.0 162.1∗ ; 208.1 115.1∗ ; 132.2 204.2; 162.2 136.1∗ ; 108.2 146.1∗ ; 94.1 160.1∗ ; 132.0 118.1∗ ; 146.1 117.1∗ ; 144.2 115.9∗ ; 144.1 146.1∗ ; 118.0 160.0∗ 130.1∗ ; 103.1 130.1∗ ; 118.1 91.1∗ ; 118.1 174.2∗ 115.1∗ ; 144.1 103.1∗ ; 130.1 130.1∗ 30∗ ; 30∗ ; 25∗ ; 30∗ ; 25∗ ; 25∗ ; 25∗ ; 25∗ ; 55∗ ; 30∗ ; 40∗ ; 30∗ ; 25∗ 20∗ ; 30∗ ; 25∗ ; 22∗ 40∗ ; 50∗ ; 20∗ L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 L-Tryptophan-d8 Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N Anthranilic acid 15N 213.3 139.2 [M-H] 212.0 151.3∗ ; 195.3 65.1∗ ; 121.1 [M-H] 132.4∗ ; 80.4 2.2 3.0 2.6 20 13 13 25 20 25 25 30 45 30 25 45 55 30 25 30 25 25∗ ; 25 30∗ ; 30 27∗ ; 27 Anthranilic acid 15N Fig Chromatogram of the 21 standards of analysis ordered by retention times (in brackets, transitions of precursor and quantifier ions (m/z)) accuracy was < 15% (recovery comprised between 85 % and 115%), and for the interday assay some recoveries were higher than 15%, particularly for the lowest values in serum Accuracies in serum ranged between 85 and 115% except for compounds for which percentages moderately under 85%: NAS (84%), IAA (84%) and 3IPA(83%) (Table 2) For intraday and interday assays in stool, analytical accuracy and CV were < 15% for all molecules quantified (Table 3) Dynamic ranges were determined according to the physiological and pathological values previously described, and results are reported in Table for LOD and LOQ, and Table for ULOQ, which corresponded to the highest calibration standards quantified To account for matrix effect for serum, calibration standards were prepared using lyophilized blank serum For stool samples, matrix effects were evaluated as described in material and methods section Matrix effects calculated at levels were in accordance with published studies, ranging from 50 % to 125%, but few components exhibited highest matrix effects (34%, 196%, 41 %, 36 %, for picolinic acid, tryptophan, melatonin, N acetyl serotonin, respectively) (Table 5) These resuts were obtained for the lowest level of spike for all of the metabolites, but the levels of spike tested in our method validation were lower than other published methods [18] Recoveries ranging from 62% to 134% (Table 5) Despite high variance, these results were consistent with those previ- A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Table Intraday and interday precision in serum Means, coefficients of variation (CVs) and recoveries were calculated from internal quality controls (number of assays: 20 for intra- and inter-assays precisions, respectively) for two levels Intra-Day Inter-Day Component Names Nominal concentration (nM) CV(%) Recovery (%) CV(%) Recovery (%) Piconilic acid 177.2 5669.1 261.0 4180.0 195.0 779.2 31.0 3966.1 99.0 3173.0 143.0 4562.0 105.0 3356.0 27.0 3405.0 595.0 152364.7 36.0 4362.5 115.0 3693.0 29.0 3655.0 25.0 3202.0 62.0 4012.0 106.0 3405.0 150.0 4813.0 23.0 3008.0 34.0 4335.0 31.0 3989.0 29.0 3693.0 103.0 3309.0 13.7 9.6 15.7 9.3 8.1 8.1 11.1 10.0 16.2 6.4 8.8 6.5 7.2 5.9 9.3 4.0 10.7 3.5 8.5 5.9 7.0 8.2 10.5 6.7 13.9 4.6 6.8 6.7 11.7 7.8 8.1 5.6 11.7 3.3 11.7 6.9 10.9 7.8 13.3 5.5 9.8 5.4 89.7 88.1 88.2 109.1 98.6 107.8 92.1 92.5 99.1 102.0 90.0 107.2 104.8 105.3 92.0 114.4 95.4 88.0 88.2 102.5 104.8 101.8 102.5 89.1 85.7 85.0 108.5 92.3 91.9 95.8 107.5 87.8 93.8 103.3 94.0 94.6 95.1 109.8 93.8 98.5 93.0 88.1 17.3 11.4 14.8 11.5 11.7 15.4 12.0 12.6 17.7 9.7 11.9 6.4 8.7 6.1 10.5 4.3 6.7 2.8 11.1 6.6 8.9 9.7 10.4 6.9 13.0 6.9 6.8 9.0 13.2 7.3 11.5 6.7 12.2 12.4 12.7 8.3 11.3 8.5 13.9 7.5 10.0 11.7 99.9 89.5 91.8 103.4 113.7 102.4 91.5 90.4 90.2 101.7 88.1 99.9 102.3 99.3 97.4 108.2 94.8 85.5 97.6 98.6 104.7 102.6 106.8 94.4 91.6 84.3 95.0 93.8 99.5 92.7 87.9 98.5 96.5 100.6 96.0 92.0 84.0 89.1 83.1 104.9 96.1 112.3 Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate ously described in a similar matrix [18] Matrix effects could be minimized by adding a larger number of stable isotope-labeled analogue to the targeted molecule Some preanalyticals steps based on on phospholipid removal and interferent proteins may be performed to minimize matrix effect In blood, method validation was performed in serum samples, but a comparison was done between blood collected on different anticoagulants (EDTA, heparin, citrate) No major difference was found regarding the presence and type of anticoagulant except for with serotonin, where levels were higher in serum than in plasma (supplemental data, Fig 1) These results for serotonin were already described [15], however, it was necessary to evaluate the percentage of difference when developing our method Residue stabilities are shown in supplemental data, Table After the first thawing, at 10 days, compounds (3-HK, XA, KA, 5-HIAA, I3A, ILA, IS) were under acceptable values and highlights changes in both host metabolism (serum) and microbial metabolism (stool)[26] Following analytical validation, the method was tested on 24 human serum samples and 22 human stool samples from healthy donors (Table 6) Reference values were in agreement with previously published reports [26] The few differences between our data and published results could be explained by the non-fasting state of our donors, which was imposed by the composition of the available patient cohort Subsequently, we tested our method on IBD patient samples to investigate its potential in clinical practice Samples were obtained from two groups: patients diagnosed with IBD (27 patients) and a control group of non-IBD patients (13 patients) (Fig 3A) Fecal calprotectin was used to classify patients into three different groups among IBD patients according to the established cut-offs used in IBD management and diagnosis [27]: 1) Patients with fecal calprotectin < 50 μg/g, 2) patients with fecal calprotectin between 50 and 200 μg/g, which corresponds to an intermediate state, and 3) patients with fecal calprotectin > 200 μg/g which corresponds to an acute phase of the disease Fecal calprotectin was also assayed in patients from the control group, who all had a calprotectin level < 50 μg/g The multivariate analysis identified the most discriminant variables (VIP > 1) to stratify non IBD patients from IBD patients: 3.3 Application of the method: human serum and stool concentrations The main objective of our work was the development of a reliable method for the exploration of Trp metabolism in two relevant compartments, serum and stool, in cases of IBD and IBS This method allows extensive metabolite profiling of Trp metabolism A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Table Intraday and interday precision in stool A Intraday precison in stool B Interday precision in stool Means, coefficients of variation (CVs) and recoveries were calculated from internal quality controls (number of assays: 20 for intra- and inter-assays precisions, respectively) for two levels Sample-1 Sample-2 Sample-3 Component Names Mean (nmol/g) CV (%) Mean (nmol/g) CV (%) Mean (nmol/g) CV (%) Piconilic acid Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate A B 393.7 < LOQ 1.4 16.8 3.8 4.0 < LOQ 0.7 106.0 1.2 6.6 1.1 0.3 < LOQ < LOQ 8.4 < LOQ 0.6 29.2 29.1 2.6 5.8 116.1 3.2 < LOQ 3.2 1.2 7.2 < LOQ 4.6 34.4 2.2 16.6 0.28 0.2 < LOQ < LOQ 9.4 < LOQ 1.3 20.9 11.4 2.7 8.5 8.5 524.4 2.6 < LOQ 4.3 2.6 4.5 1.7 9.3 1492.0 49.6 5.5 < LOQ 0.6 < LOQ 9.1 6.9 < LOQ 1.7 32.6 54.6 1.0 5.6 9.0 7.4 6.7 3.0 6.7 6.5 4.4 4.9 4.4 3.9 4.1 5.6 10.3 5.1 6.2 5.5 Sample-1 2.4 11.0 5.2 6.5 7.9 9.4 5.2 4.72 4.7 3.9 7.5 7.6 5.3 6.9 Sample-2 3.3 11.3 6.2 7.1 3.7 3.1 5.6 5.9 4.9 5.7 6.2 7.6 5.5 5.7 10.7 Sample-3 Component Names Mean (nmol/g) CV (%) Mean (nmol/g) CV (%) Mean (nmol/g) CV (%) Piconilic acid Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate 383.1 < LOQ 1.4 16.2 3.5 3.6 < LOQ 0.7 110.2 1.6 6.1 1.0 0.3 < LOQ < LOQ 8.4 < LOQ 0.6 26.4 27.7 2.2 10.3 106.7 3.1 < LOQ 3.2 1.1 6.7 < LOQ 4.8 34.5 2.7 15.5 0.3 0.2 < LOQ < LOQ 8.8 < LOQ 1.2 19.7 12.0 2.5 4.3 9.6 466.9 2.7 < LOQ 4.2 2.5 4.2 1.6 9.8 1502.0 45.4 5.6 < LOQ 0.6 < LOQ 8.6 6.7 < LOQ 1.7 31.7 55.3 1.0 11.1 9.7 4.6 6.5 5.0 6.6 4.5 10.3 5.5 10.5 2.5 2.0 6.6 7.6 8.4 10.7 6.2 3.7 8.3 10.5 3.5 3.7 2.6 1.8 7.6 8.5 3.5 6.0 5.4 7.3 7.7 6.0 5.8 7.4 7.2 5.2 1.5 4.6 7.8 2.7 10.7 7.7 2.1 9.1 8.7 5.1 Fig Analysis of clinical samples Stool samples from non IBD and IBD patients A Score plot of patients obtained from OPLS-DA B Variable importance in projection (VIP) exhibited significant differences for XA, KA, ILA, calprotectin, trp, Pico XA, KA, ILA, calprotectin and trp are increased (red bar plot) and pico is decreased (blue bar plot) in IBD patients compared to control group A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Table Limit of detection (LOD) and limit of quantification (LOQ) LOD was determined by a signal-to-noise ratio (S/N) of 3:1 and LOQ by a signal-to-noise ratio (S/N) of 10:1 Piconilic acid Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate Lower limit of Detection (nmol/L) Lower limit of Quantification (nmol/L) 1.4 65.3 48.7 31 3.1 35.6 3.3 0.8 28.3 34 3.3 3.6 3.1 3.9 0.8 4.7 0.7 16.9 15.5 0.9 25.8 88.5 130 97.5 124 49.6 71 105 26.6 37.2 136 105 14.3 6.2 62.5 26.5 37.6 5.9 67.5 62.3 14.5 103 Table Matrix effect and yield extraction in stool at levels (low, medium, high) Component Names Spiked Concentration μg/ml Matrix effect % Recovery % Piconilic acid 1.6 12.2 1.2 9.0 0.9 6.7 1.1 8.5 0.9 6.8 1.3 9.8 1.0 7.2 1.0 7.3 1.0 7.3 1.2 9.4 1.1 7.9 1.0 7.8 0.9 6.9 1.1 8.6 1.0 7.3 1.4 10.3 0.9 6.5 1.2 9.3 1.1 8.6 1.1 7.9 0.9 7.0 34 57 125 93 81 82 57 50 71 55 89 94 95 74 107 86 196 68 60 76 108 118 76 84 36 57 48 55 89 74 93 82 41 60 108 93 80 84 106 79 100 77 90 123 80 103 96 80 82 72 75 86 87 84 78 86 94 96 99 101 76 62 94 93 89 80 86 83 95 93 89 85 73 65 91 85 79 82 97 80 93 91 123 134 Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate A Desmons, L Humbert, T Eguether et al Journal of Chromatography A 1685 (2022) 463602 Table Metabolites concentrations in serum and feces References values were determined from healthy individuals in serum (n=24) and stool (n=22), results were expressed as mean ± standard deviation (SD) Piconilic acid Quinolinic acid 3-Hydroxy Kynurenine Serotonin 5-Hydroxytryptophan 3-Hydroxyanthranilic acid Kynurenine Xanthurenic acid Tryptophan Tryptamine Kynurenic acid 5-Hydroxyindole acetic acid N-Acetylserotonin Indole-3-acetamide Indole-3- lactic acid Indole-3-carboxaldehyde Melatonin Tryptophol Indole-3- acetic acid Indole-3-propionic acid Indoxyl sulfate Serum (nmol/L) Feces (nmol/g) < LOQ 231 ± 139 12 ± 14 377 ± 177 < LOQ 33 ± 47 1652 ± 272 332 ± 52 51460 ± 9187 < LOQ < LOQ 57 ± 49 < LOQ < LOQ 823 ± 228 < LOQ < LOQ < LOQ 1489 ± 475 1337 ± 1039 3130 ± 1451 69390 ± 8730 190 ± 120 < LOQ 910 ± 790 170 ± 180 1730 ± 1000 70 ± 50 510 ± 550 10320 ± 6400 770 ± 1120 1790 ± 1900 230 ± 240 50 ± 50 < LOQ 450 ± 450 1150 ± 900 < LOQ 340 ± 300 5360 ± 4760 6500 ± 3380 70 ± 320 serum of IBD patients [16,31] but no studies have determined PA levels in feces for these patients Eventually, despite a rather small number of patients in the cohort, the results obtained through our developed method evidenced significative differences of tryptophan derivatives patterns between IBD and non IBD patients Conclusions A HPLC-ESI-MS/MS method has been developed for the analysis of Trp and 20 metabolites for application to human serum, plasma, and stool samples This method allows for an extensive analysis of the three Trp metabolic pathways in two compartments and was applied to a clinical study of IBD patients Increased concentrations of Trp, XA, KA, ILA, and a decrease of PA were observed in IBD patients compared to healthy controls using this approach The clinical use of this method is wide-ranging and may be applied to other pathological conditions involving Trp metabolism such as neurological, psychiatric, or auto-inflammatory pathologies Authors contribution A.D and A.L performed the design of research L.H., A.D and D.R performed experiments and data acquisition A.D., N.K and A.L performed data analysis and interpretation A.D wrote the paper All authors critically revised the article All authors approved the final version to be published calprotectin, Trp, and four Trp metabolites, xanthurenic acid (XA), kynurenic acid (KA), indole-3-lactic acid (ILA) and picolinic acid (Pico) (Fig B) These results were confirmed by univariate statistical analysis as shown in supplemental data, Fig The results obtained showed that Trp derivatives are important markers for study in clinical settings with our method Three patients (numbers 30, 31, and 37) tested very close to the control group in the score plot Patient 37 was suffering from Crohn’s disease and presented consecutive normal levels of calprotectin over months, which was probably due to a deep remission Patient 31 also presented Crohn’s disease in deep remission and did not receive drug treatment Patient 30 was suffering from UC under treatment and presented two consecutive normal levels of calprotectin over months, which was probably due to a steady state of the disease Most published studies of Trp derivatives in IBD patients have been performed in serum or plasma samples Trp metabolites, such as PA and XA, were reported at a lower concentration in serum or plasma than control groups [16,26] The increase we observed for Trp in stool samples supports previous results, which showed an increase in fecal amino acids [26] Trp increase in feces could be explained by inflammation and mucosal damage in IBD due to a compromised gut epithelial barrier [26,28] An extensive quantification of amino-acid profiles in blood and stool may be useful to highlight the impaired absorption of all amino acids in the gut in patients suffering from IBD Regarding the indoles pathway, which corresponds to indoles derivatives produced by commensal bacteria, an increase of ILA in feces was observed Some studies have shown how indoles molecules derived from bacteria are implicated in intestinal inflammation, IPA, an indole molecule, have been reported to be decreased in serum [29] This result is consistent with the increase of ILA found in feces, a downstream product of IPA Other results showed an increase for two downstream products of the kynurenine pathway, KA and XA, and a decrease in PA The increase of KA and XA, which are derived from kynurenine metabolism by indolemanine 2,3 dioxygenase (IDO), could be supported by the previously reported increase of IDO activity in IBD patients [30] A decrease for PA in feces may be explained by an increase of the production of QA, however, in our study, no significance was found for the increase in QA A decrease in PA was previously reported in the Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that 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