#METABOLOMICS WORKBENCH ssah9_20220411_070428 DATATRACK_ID:3191 STUDY_ID:ST002136 ANALYSIS_ID:AN003497 PROJECT_ID:PR001353 VERSION 1 CREATED_ON April 15, 2022, 8:15 am #PROJECT PR:PROJECT_TITLE Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry PR:PROJECT_TITLE Metabolomics to Monitor Ovarian Cancer Progression. PR:PROJECT_SUMMARY The lack of effective screening strategies for high-grade serous carcinoma PR:PROJECT_SUMMARY (HGSC), a subtype of ovarian cancer (OC) responsible for 80% of OC related PR:PROJECT_SUMMARY deaths, emphasizes the need for new diagnostic markers and a better PR:PROJECT_SUMMARY understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled PR:PROJECT_SUMMARY with high-resolution mass spectrometry (HRMS) offers high selectivity and PR:PROJECT_SUMMARY sensitivity, thereby increasing metabolite coverage and consequently enhancing PR:PROJECT_SUMMARY biomarker discovery. Recent advances in CE-MS include small, chip-based CE PR:PROJECT_SUMMARY systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, PR:PROJECT_SUMMARY high-resolution analysis of biological specimens. Here, we describe the PR:PROJECT_SUMMARY development of a targeted microchip (µ) CE-HRMS method to analyze 40 target PR:PROJECT_SUMMARY metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC, PR:PROJECT_SUMMARY with an acquisition time of only 3 min. Extracted ion electropherograms showed PR:PROJECT_SUMMARY sharp, highly resolved peak shapes, even for structural isomers such as leucine PR:PROJECT_SUMMARY and isoleucine. All analytes maintained good linearity with an average R2 of PR:PROJECT_SUMMARY 0.994, while detection limits were in the nM range. Thirty metabolites were PR:PROJECT_SUMMARY detected in mice serum, with recoveries ranging from 78 to 120 %, indicating PR:PROJECT_SUMMARY minimal ionization suppression and good accuracy. We applied the µCE-HRMS PR:PROJECT_SUMMARY method to sequentially-collected serum samples from TKO and TKO-control mice. PR:PROJECT_SUMMARY Time-resolved analysis revealed characteristic temporal trends for amino acids, PR:PROJECT_SUMMARY nucleosides, and amino acids derivatives associated with HGSC progression. PR:PROJECT_SUMMARY Comparison of the µCE-HRMS dataset with non-targeted ultra-high performance PR:PROJECT_SUMMARY liquid chromatography (UHPLC) – MS results revealed identical temporal trends PR:PROJECT_SUMMARY for the 5 metabolites detected on both platforms, indicating the µCE-HRMS PR:PROJECT_SUMMARY method performed satisfactorily in terms of capturing metabolic reprogramming PR:PROJECT_SUMMARY due to HGSC progression, while reducing the total analysis time 3-fold. PR:INSTITUTE Georgia Institute of Technology PR:LAST_NAME Sah PR:FIRST_NAME Samyukta PR:ADDRESS 901 Atlantic Dr NW, Atlanta, GA, 30332, USA PR:EMAIL ssah9@gatech.edu PR:PHONE 5746780124 #STUDY ST:STUDY_TITLE Targeted Microchip Capillary Electrophoresis-Orbitrap Mass Spectrometry ST:STUDY_TITLE Metabolomics to Monitor Ovarian Cancer Progression (calibration standards) ST:STUDY_SUMMARY The lack of effective screening strategies for high-grade serous carcinoma ST:STUDY_SUMMARY (HGSC), a subtype of ovarian cancer (OC) responsible for 80% of OC related ST:STUDY_SUMMARY deaths, emphasizes the need for new diagnostic markers and a better ST:STUDY_SUMMARY understanding of disease pathogenesis. Capillary electrophoresis (CE) coupled ST:STUDY_SUMMARY with high-resolution mass spectrometry (HRMS) offers high selectivity and ST:STUDY_SUMMARY sensitivity, thereby increasing metabolite coverage and consequently enhancing ST:STUDY_SUMMARY biomarker discovery. Recent advances in CE-MS include small, chip-based CE ST:STUDY_SUMMARY systems coupled with nanoelectrospray ionization (nanoESI) to provide rapid, ST:STUDY_SUMMARY high-resolution analysis of biological specimens. Here, we describe the ST:STUDY_SUMMARY development of a targeted microchip (µ) CE-HRMS method to analyze 40 target ST:STUDY_SUMMARY metabolites in serum samples from a triple-mutant (TKO) mouse model of HGSC, ST:STUDY_SUMMARY with an acquisition time of only 3 min. Extracted ion electropherograms showed ST:STUDY_SUMMARY sharp, highly resolved peak shapes, even for structural isomers such as leucine ST:STUDY_SUMMARY and isoleucine. All analytes maintained good linearity with an average R2 of ST:STUDY_SUMMARY 0.994, while detection limits were in the nM range. Thirty metabolites were ST:STUDY_SUMMARY detected in mice serum, with recoveries ranging from 78 to 120 %, indicating ST:STUDY_SUMMARY minimal ionization suppression and good accuracy. We applied the µCE-HRMS ST:STUDY_SUMMARY method to sequentially-collected serum samples from TKO and TKO-control mice. ST:STUDY_SUMMARY Time-resolved analysis revealed characteristic temporal trends for amino acids, ST:STUDY_SUMMARY nucleosides, and amino acids derivatives associated with HGSC progression. ST:STUDY_SUMMARY Comparison of the µCE-HRMS dataset with non-targeted ultra-high performance ST:STUDY_SUMMARY liquid chromatography (UHPLC) – MS results revealed identical temporal trends ST:STUDY_SUMMARY for the 5 metabolites detected on both platforms, indicating the µCE-HRMS ST:STUDY_SUMMARY method performed satisfactorily in terms of capturing metabolic reprogramming ST:STUDY_SUMMARY due to HGSC progression, while reducing the total analysis time 3-fold. ST:INSTITUTE Georgia Institute of Technology ST:LAST_NAME Sah ST:FIRST_NAME Samyukta ST:ADDRESS 901 Atlantic Dr NW, Atlanta, GA, 30332, USA ST:EMAIL ssah9@gatech.edu ST:PHONE 5746780124 #SUBJECT SU:SUBJECT_TYPE Synthetic sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - sample1-2 factor :standard mix RAW_FILE_NAME=sample1-2 SUBJECT_SAMPLE_FACTORS - sample1-3 factor :standard mix RAW_FILE_NAME=sample1-3 SUBJECT_SAMPLE_FACTORS - sample2-1 factor :standard mix RAW_FILE_NAME=sample2-1 SUBJECT_SAMPLE_FACTORS - sample2-2 factor :standard mix RAW_FILE_NAME=sample2-2 SUBJECT_SAMPLE_FACTORS - sample3-1 factor :standard mix RAW_FILE_NAME=sample3-1 SUBJECT_SAMPLE_FACTORS - sample3-2 factor :standard mix RAW_FILE_NAME=sample3-2 SUBJECT_SAMPLE_FACTORS - sample4-1 factor :standard mix RAW_FILE_NAME=sample4-1 SUBJECT_SAMPLE_FACTORS - sample4-2 factor :standard mix RAW_FILE_NAME=sample4-2 SUBJECT_SAMPLE_FACTORS - sample5-1 factor :standard mix RAW_FILE_NAME=sample5-1 SUBJECT_SAMPLE_FACTORS - sample5-2 factor :standard mix RAW_FILE_NAME=sample5-2 SUBJECT_SAMPLE_FACTORS - sample6-1 factor :standard mix RAW_FILE_NAME=sample6-1 SUBJECT_SAMPLE_FACTORS - sample6-2 factor :standard mix RAW_FILE_NAME=sample6-2 SUBJECT_SAMPLE_FACTORS - 0.1uM_run1 factor :standard mix RAW_FILE_NAME=0.1uM_run1 SUBJECT_SAMPLE_FACTORS - 0.1uM_run2 factor :standard mix RAW_FILE_NAME=0.1uM_run2 SUBJECT_SAMPLE_FACTORS - 0.05uM_run1 factor :standard mix RAW_FILE_NAME=0.05uM_run1 SUBJECT_SAMPLE_FACTORS - 0.05uM_run2 factor :standard mix RAW_FILE_NAME=0.05uM_run2 SUBJECT_SAMPLE_FACTORS - 0.025uM_run1 factor :standard mix RAW_FILE_NAME=0.025uM_run1 SUBJECT_SAMPLE_FACTORS - 0.025uM_run2 factor :standard mix RAW_FILE_NAME=0.025uM_run2 SUBJECT_SAMPLE_FACTORS - 1uM_run1 factor :standard mix RAW_FILE_NAME=1uM_run1 SUBJECT_SAMPLE_FACTORS - 1uM_run2 factor :standard mix RAW_FILE_NAME=1uM_run2 SUBJECT_SAMPLE_FACTORS - 2.5uM_run1 factor :standard mix RAW_FILE_NAME=2.5uM_run1 SUBJECT_SAMPLE_FACTORS - 2.5uM_run2 factor :standard mix RAW_FILE_NAME=2.5uM_run2 SUBJECT_SAMPLE_FACTORS - 5uM_run1 factor :standard mix RAW_FILE_NAME=5uM_run1 SUBJECT_SAMPLE_FACTORS - 5uM_run2 factor :standard mix RAW_FILE_NAME=5uM_run2 SUBJECT_SAMPLE_FACTORS - 10uM_run1 factor :standard mix RAW_FILE_NAME=10uM_run1 SUBJECT_SAMPLE_FACTORS - 10uM_run2 factor :standard mix RAW_FILE_NAME=10uM_run2 SUBJECT_SAMPLE_FACTORS - 25uM_run2 factor :standard mix RAW_FILE_NAME=25uM_run2 SUBJECT_SAMPLE_FACTORS - 25uM_run3 factor :standard mix RAW_FILE_NAME=25uM_run3 SUBJECT_SAMPLE_FACTORS - 0.1uMstdmix_run1 factor :standard mix RAW_FILE_NAME=0.1uMstdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 0.1uMstdmix_run2 factor :standard mix RAW_FILE_NAME=0.1uMstdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - 0.05uM_stdmix_run1 factor :standard mix RAW_FILE_NAME=0.05uM_stdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 0.05uM_stdmix_run2 factor :standard mix RAW_FILE_NAME=0.05uM_stdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - 1uM_stdmix_run1 factor :standard mix RAW_FILE_NAME=1uM_stdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 1uM_stdmix_run2 factor :standard mix RAW_FILE_NAME=1uM_stdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - 2.5uM_stdmix_run1 factor :standard mix RAW_FILE_NAME=2.5uM_stdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 2.5uM_stdmix_run2 factor :standard mix RAW_FILE_NAME=2.5uM_stdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - 25uM_stdmix_run1 factor :standard mix RAW_FILE_NAME=25uM_stdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 25uM_stdmix_run2 factor :standard mix RAW_FILE_NAME=25uM_stdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - 10uM_stdmix_run1 factor :standard mix RAW_FILE_NAME=10uM_stdmix_run1chip2955 SUBJECT_SAMPLE_FACTORS - 10uM_stdmix_run2 factor :standard mix RAW_FILE_NAME=10uM_stdmix_run2chip2955 SUBJECT_SAMPLE_FACTORS - sample2-2-run2 factor :standard mix RAW_FILE_NAME=sample2-2-run2 SUBJECT_SAMPLE_FACTORS - sample2-2-run3 factor :standard mix RAW_FILE_NAME=sample2-2-run3 #COLLECTION CO:COLLECTION_SUMMARY To improve CE peak shape and support electrophoretic focusing, a sample diluent CO:COLLECTION_SUMMARY consisting of 133 mM ammonium acetate, and 0.1% formic acid was prepared.13 This CO:COLLECTION_SUMMARY sample diluent was spiked with 1 µM 13C phenylalanine, 3 µM 13C6 arginine and CO:COLLECTION_SUMMARY 0.8 µM 13C methionine D3 as internal standards. Calibration mixtures were CO:COLLECTION_SUMMARY prepared from serial dilution of stock standard solutions using the spiked CO:COLLECTION_SUMMARY sample diluent in a 1:4 ratio. Each calibration standard was analyzed twice to CO:COLLECTION_SUMMARY yield calibration curves, calculate figures of merit, and perform metabolite CO:COLLECTION_SUMMARY quantification. CO:SAMPLE_TYPE Synthetic Mixture #TREATMENT TR:TREATMENT_SUMMARY To improve CE peak shape and support electrophoretic focusing, a sample diluent TR:TREATMENT_SUMMARY consisting of 133 mM ammonium acetate, and 0.1% formic acid was prepared.13 This TR:TREATMENT_SUMMARY sample diluent was spiked with 1 µM 13C phenylalanine, 3 µM 13C6 arginine and TR:TREATMENT_SUMMARY 0.8 µM 13C methionine D3 as internal standards. Calibration mixtures were TR:TREATMENT_SUMMARY prepared from serial dilution of stock standard solutions using the spiked TR:TREATMENT_SUMMARY sample diluent in a 1:4 ratio. Each calibration standard was analyzed twice to TR:TREATMENT_SUMMARY yield calibration curves, calculate figures of merit, and perform metabolite TR:TREATMENT_SUMMARY quantification. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To improve CE peak shape and support electrophoretic focusing, a sample diluent SP:SAMPLEPREP_SUMMARY consisting of 133 mM ammonium acetate, and 0.1% formic acid was prepared.13 This SP:SAMPLEPREP_SUMMARY sample diluent was spiked with 1 µM 13C phenylalanine, 3 µM 13C6 arginine and SP:SAMPLEPREP_SUMMARY 0.8 µM 13C methionine D3 as internal standards. Calibration mixtures were SP:SAMPLEPREP_SUMMARY prepared from serial dilution of stock standard solutions using the spiked SP:SAMPLEPREP_SUMMARY sample diluent in a 1:4 ratio. Each calibration standard was analyzed twice to SP:SAMPLEPREP_SUMMARY yield calibration curves, calculate figures of merit, and perform metabolite SP:SAMPLEPREP_SUMMARY quantification. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME ZipChip (908 Devices) CH:COLUMN_NAME HS chip #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS All experiments were performed in positive ionization mode in the 50-500 m/z MS:MS_COMMENTS range at a mass resolution setting of 17,500. The capillary temperature was set MS:MS_COMMENTS to 200 °C and the sheath gas flow rate was 2 psi. The automatic gain control MS:MS_COMMENTS (AGC) target value was set to 3E6 and the maximum injection time was 20 ms. Data MS:MS_COMMENTS were acquired using Xcalibur 3.0 (Thermo Scientific) and were imported to MS:MS_COMMENTS Skyline software14 for peak picking and integration. The peak picking procedure MS:MS_COMMENTS used the analyte accurate m/z and migration time. Peak areas obtained from MS:MS_COMMENTS Skyline were exported as spreadsheets for further analysis. Quantitation was MS:MS_COMMENTS performed with the analyte peak areas relative to the peak area of one of the MS:MS_COMMENTS three isotopically labeled internal standards (13C6 arginine, 13C methionine D3 MS:MS_COMMENTS and 13C phenylalanine) chosen based on migration time similarities. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Micromolars MS_METABOLITE_DATA_START Samples sample6-1 sample6-2 sample5-1 sample5-2 sample4-1 sample4-2 sample3-1 sample3-2 sample2-1 sample2-2 sample1-2 sample1-3 sample2-2-run2 sample2-2-run3 0.1uM_run1 0.1uM_run2 0.05uM_run1 0.05uM_run2 0.025uM_run1 0.025uM_run2 1uM_run1 1uM_run2 2.5uM_run1 2.5uM_run2 5uM_run1 5uM_run2 10uM_run1 10uM_run2 25uM_run2 25uM_run3 0.1uMstdmix_run1 0.1uMstdmix_run2 0.05uM_stdmix_run1 0.05uM_stdmix_run2 1uM_stdmix_run1 1uM_stdmix_run2 2.5uM_stdmix_run1 2.5uM_stdmix_run2 25uM_stdmix_run1 25uM_stdmix_run2 10uM_stdmix_run1 10uM_stdmix_run2 Factors factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix factor :standard mix 5'-Hydroxy-L-tryptophan 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Acetylcholine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Alanine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 2-Aminoisobutyric acid 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Arginine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Asparagine 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Betaine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 Carnitine 0.02 0.02 0.03 0.03 0.06 0.06 0.2 0.2 0.3 0.3 0.6 0.6 Choline 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 N,N-Dimethylglycine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Glucosamine 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Glutamine 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Glycine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Histidine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Trans-4-Hydroxy-L-proline 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Methionine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Normetanephrine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 Phenylalanine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 Proline 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Serine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Threonine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Trimethylamine-N-oxide 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Tryptophan 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Tyrosine 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Valine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Nicotinamide 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Aspartic acid 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Nicotinic acid 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 γ-Aminobutyric acid 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Aminoadipic acid 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Cytidine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Citrulline 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Kynurenine 0.167 0.167 0.25 0.25 0.5 0.5 1.67 1.67 2.5 2.5 5 5 Isoleucine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 10 10 Leucine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 1 1 2.5 2.5 25 25 10 10 Ornithine 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 0.1 0.1 0.05 0.05 0.025 0.025 1 1 2.5 2.5 5 5 10 10 25 25 Lysine 0.033 0.033 0.05 0.05 0.1 0.1 0.333 0.333 0.5 0.5 1 1 Glutamic acid 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Adenosine 0.066 0.066 0.1 0.1 0.2 0.2 0.666 0.666 1 1 2 2 Adenine 0.02 0.02 0.03 0.03 0.06 0.06 0.2 0.2 0.3 0.3 0.6 0.6 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Migration time (min) 5'-Hydroxy-L-tryptophan 221.09206 1.8 Acetylcholine 146.117 0.9 Alanine 90.05495 1.2 2-Aminoisobutyric acid 104.0706 1.3 Arginine 175.1189 0.9 Asparagine 133.06076 1.5 Betaine 118.08625 1.9 Carnitine 162.1124 1.1 Choline 104.107 0.8 N,N-Dimethylglycine 104.07059 1.7 Glucosamine 180.08664 1.3 Glutamine 147.07641 1.6 Glycine 76.0393 1.1 Histidine 156.07675 1 Trans-4-Hydroxy-L-proline 132.06551 2 Methionine 150.05832 1.5 Normetanephrine 166.086 1.3 Phenylalanine 166.08625 1.6 Proline 116.0706 1.6 Serine 106.04986 1.4 Threonine 120.06551 1.5 Trimethylamine-N-oxide 76.07569 0.8 Tryptophan 205.09715 1.6 Tyrosine 182.08116 1.7 Valine 118.08625 1.3 Nicotinamide 123.05528 1.3 Aspartic acid 134.04478 1.8 Nicotinic acid 124.0393 1.9 γ-Aminobutyric acid 104.0706 1 Aminoadipic acid 162.07608 1.6 Cytidine 244.09279 1.4 Citrulline 176.1029 1.7 Kynurenine 209.09206 1.6 Isoleucine 132.1019 1.3 Leucine 132.1019 1.4 Ornithine 133.09715 0.9 Lysine 147.1128 0.9 Glutamic acid 148.0604 1.6 Adenosine 268.104 1.7 Adenine 167.0896 1 METABOLITES_END #END