#METABOLOMICS WORKBENCH alvaroferochoa_20250902_024126 DATATRACK_ID:6363 STUDY_ID:ST004172 ANALYSIS_ID:AN006926 PROJECT_ID:PR002632
VERSION             	1
CREATED_ON             	September 9, 2025, 12:45 pm
#PROJECT
PR:PROJECT_TITLE                 	Untargeted metabolomic approach to assess the acute metabolism and urinary
PR:PROJECT_TITLE                 	excretion of olive leaf bioactive compounds in humans
PR:PROJECT_SUMMARY               	Olive (Olea europaea) leaves are a rich source of bioactive phytochemicals with
PR:PROJECT_SUMMARY               	reported health benefits, which may be mediated by their in vivo metabolites
PR:PROJECT_SUMMARY               	rather than the native compounds. However, their human metabolism remains
PR:PROJECT_SUMMARY               	insufficiently characterized. In this study, we aimed to identify olive
PR:PROJECT_SUMMARY               	leaf-derived metabolites in human urine. A randomized, double-blind,
PR:PROJECT_SUMMARY               	placebo-controlled, acute dietary intervention was conducted in 17 healthy
PR:PROJECT_SUMMARY               	volunteers supplemented with an olive leaf extract. Urine samples were collected
PR:PROJECT_SUMMARY               	before and after intake and analyzed by untargeted metabolomics using
PR:PROJECT_SUMMARY               	HPLC-ESI-QTOF-MS. Data processing yielded >18,500 molecular features, from which
PR:PROJECT_SUMMARY               	513 were significantly associated with the intervention, and 176 were
PR:PROJECT_SUMMARY               	successfully annotated. Identified metabolites included oleuropein conjugates,
PR:PROJECT_SUMMARY               	hydroxytyrosol- and elenolic acid-derived compounds, as well as flavonoids and
PR:PROJECT_SUMMARY               	phenolic acids, mainly excreted 6–12 h post-intake. Results highlight the
PR:PROJECT_SUMMARY               	extensive phase I and II biotransformation of olive leaf phytochemicals in
PR:PROJECT_SUMMARY               	humans and provide a comprehensive urinary metabolic profile supporting the use
PR:PROJECT_SUMMARY               	of untargeted metabolomics to uncover novel bioactive compounds.
PR:INSTITUTE                     	University of Granada
PR:DEPARTMENT                    	Department of Analytical Chemistry
PR:LABORATORY                    	Bioactive Ingredients AGR274
PR:LAST_NAME                     	Fernández-Ochoa
PR:FIRST_NAME                    	Álvaro
PR:ADDRESS                       	Avda Fuentenueva S/N
PR:EMAIL                         	alvaroferochoa@ugr.es
PR:PHONE                         	+34958240794
PR:FUNDING_SOURCE                	Ministerio de Economía y Competitividad (Spain)
#STUDY
ST:STUDY_TITLE                   	Untargeted metabolomic approach to assess the acute metabolism and urinary
ST:STUDY_TITLE                   	excretion of olive leaf bioactive compounds in humans
ST:STUDY_SUMMARY                 	Olive (Olea europaea) leaves are a rich source of bioactive phytochemicals with
ST:STUDY_SUMMARY                 	reported health benefits, which may be mediated by their in vivo metabolites
ST:STUDY_SUMMARY                 	rather than the native compounds. However, their human metabolism remains
ST:STUDY_SUMMARY                 	insufficiently characterized. In this study, we aimed to identify olive
ST:STUDY_SUMMARY                 	leaf-derived metabolites in human urine. A randomized, double-blind,
ST:STUDY_SUMMARY                 	placebo-controlled, acute dietary intervention was conducted in 17 healthy
ST:STUDY_SUMMARY                 	volunteers supplemented with an olive leaf extract. Urine samples were collected
ST:STUDY_SUMMARY                 	before and after intake and analyzed by untargeted metabolomics using
ST:STUDY_SUMMARY                 	HPLC-ESI-QTOF-MS. Data processing yielded >18,500 molecular features, from which
ST:STUDY_SUMMARY                 	513 were significantly associated with the intervention, and 176 were
ST:STUDY_SUMMARY                 	successfully annotated. Identified metabolites included oleuropein conjugates,
ST:STUDY_SUMMARY                 	hydroxytyrosol- and elenolic acid-derived compounds, as well as flavonoids and
ST:STUDY_SUMMARY                 	phenolic acids, mainly excreted 6–12 h post-intake. Results highlight the
ST:STUDY_SUMMARY                 	extensive phase I and II biotransformation of olive leaf phytochemicals in
ST:STUDY_SUMMARY                 	humans and provide a comprehensive urinary metabolic profile supporting the use
ST:STUDY_SUMMARY                 	of untargeted metabolomics to uncover novel bioactive compounds.
ST:INSTITUTE                     	University of Granada
ST:LAST_NAME                     	Fernández Ochoa
ST:FIRST_NAME                    	Álvaro
ST:ADDRESS                       	Avda Fuentenueva S/N
ST:EMAIL                         	alvaroferochoa@ugr.es
ST:PHONE                         	+34958240794
ST:TOTAL_SUBJECTS                	17
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	blank	B1blk1-r001	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:1	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221215_Neg_blk1-r001.mzML; Sex=blank; Age=blank; Batch=1
SUBJECT_SAMPLE_FACTORS           	blank	B1blk1-r002v2	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:2	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221215_Neg_blk1-r002.mzML; Sex=blank; Age=blank; Batch=1
SUBJECT_SAMPLE_FACTORS           	blank	B1blk2-r001	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:61	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221215_Neg_blk2-r001.mzML; Sex=blank; Age=blank; Batch=1
SUBJECT_SAMPLE_FACTORS           	blank	B1blk2-r002	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:62	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221215_Neg_blk2-r002.mzML; Sex=blank; Age=blank; Batch=1
SUBJECT_SAMPLE_FACTORS           	blank	B2blk1-r001	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:63	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221219_Neg_blk1-r001.mzML; Sex=blank; Age=blank; Batch=2
SUBJECT_SAMPLE_FACTORS           	blank	B2blk1-r002v2	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:64	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221219_Neg_blk1-r002v2.mzML; Sex=blank; Age=blank; Batch=2
SUBJECT_SAMPLE_FACTORS           	blank	B2blk2-r001	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:123	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221219_Neg_blk2-r001.mzML; Sex=blank; Age=blank; Batch=2
SUBJECT_SAMPLE_FACTORS           	blank	B2blk2-r002	Sample source:Urine | Treatment:blank | Time Interval:blank | Injection order:125	Collection Time (s)=blank; RAW_FILE_NAME(Raw_File Name)=20221219_Neg_blk2-r002.mzML; Sex=blank; Age=blank; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE1	OE1-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:42	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_001-00.mzML; Sex=Male; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE1	OE1-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:46	Collection Time (s)=39; RAW_FILE_NAME(Raw_File Name)=20221215_001-01.mzML; Sex=Male; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE1	OE1-02	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:44	Collection Time (s)=224; RAW_FILE_NAME(Raw_File Name)=20221215_001-02.mzML; Sex=Male; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE1	OE1-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:43	Collection Time (s)=400; RAW_FILE_NAME(Raw_File Name)=20221215_001-03.mzML; Sex=Male; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE1	OE1-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:45	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_001-04.mzML; Sex=Male; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE2	OE2-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:24	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_002-01.mzML; Sex=Male; Age=49; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE2	OE2-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:21	Collection Time (s)=126; RAW_FILE_NAME(Raw_File Name)=20221215_002-02.mzML; Sex=Male; Age=49; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE2	OE2-02	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:25	Collection Time (s)=254; RAW_FILE_NAME(Raw_File Name)=20221215_002-03.mzML; Sex=Male; Age=49; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE2	OE2-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:22	Collection Time (s)=422; RAW_FILE_NAME(Raw_File Name)=20221215_002-04.mzML; Sex=Male; Age=49; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE2	OE2-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:23	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_002-05.mzML; Sex=Male; Age=49; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE3	OE3-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:56	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_003-00.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE3	OE3-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:57	Collection Time (s)=174; RAW_FILE_NAME(Raw_File Name)=20221215_003-01.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE3	OE3-02	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:55	Collection Time (s)=354; RAW_FILE_NAME(Raw_File Name)=20221215_003-02.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE3	OE3-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:58	Collection Time (s)=548; RAW_FILE_NAME(Raw_File Name)=20221215_003-03.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE3	OE3-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:54	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_003-04.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE4	OE4-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:7	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_OO4_00.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE4	OE4-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:6	Collection Time (s)=171; RAW_FILE_NAME(Raw_File Name)=20221215_OO4_01.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE4	OE4-02	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:9	Collection Time (s)=279; RAW_FILE_NAME(Raw_File Name)=20221215_OO4_02.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE4	OE4-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:5	Collection Time (s)=419; RAW_FILE_NAME(Raw_File Name)=20221215_OO4_03.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE4	OE4-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:8	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_OO4_04.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	OE5	OE5-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:75	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_005-00.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE5	OE5-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:74	Collection Time (s)=146; RAW_FILE_NAME(Raw_File Name)=20221219_005-01.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE5	OE5-02	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:77	Collection Time (s)=321; RAW_FILE_NAME(Raw_File Name)=20221219_005-02.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE5	OE5-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:73	Collection Time (s)=482; RAW_FILE_NAME(Raw_File Name)=20221219_005-03.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE5	OE5-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:76	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_005-04.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE6	OE6-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:109	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_OO6-00.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE6	OE6-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:108	Collection Time (s)=261; RAW_FILE_NAME(Raw_File Name)=20221219_OO6-01.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE6	OE6-02	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:110	Collection Time (s)=467; RAW_FILE_NAME(Raw_File Name)=20221219_OO6-02.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE6	OE6-03	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:107	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_OO6-03.mzML; Sex=Female; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE7	OE7-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:90	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_OO7-00.mzML; Sex=Male; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE7	OE7-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:88	Collection Time (s)=347; RAW_FILE_NAME(Raw_File Name)=20221219_OO7-01.mzML; Sex=Male; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE7	OE7-02	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:92	Collection Time (s)=408; RAW_FILE_NAME(Raw_File Name)=20221219_OO7-02.mzML; Sex=Male; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE7	OE7-03	Sample source:Urine | Treatment:Olive | Time Interval:2 | Injection order:89	Collection Time (s)=487; RAW_FILE_NAME(Raw_File Name)=20221219_OO7-03.mzML; Sex=Male; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE7	OE7-04	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:91	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_OO7-04.mzML; Sex=Male; Age=22; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE8	OE8-00	Sample source:Urine | Treatment:Olive | Time Interval:0 | Injection order:129	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_OO8_00.mzML; Sex=Female; Age=26; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE8	OE8-01	Sample source:Urine | Treatment:Olive | Time Interval:1 | Injection order:130	Collection Time (s)=205; RAW_FILE_NAME(Raw_File Name)=20221219_OO8_01.mzML; Sex=Female; Age=26; Batch=2
SUBJECT_SAMPLE_FACTORS           	OE8	OE8-02	Sample source:Urine | Treatment:Olive | Time Interval:3 | Injection order:131	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_OO8_02.mzML; Sex=Female; Age=26; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB1	PB1-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:81	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB1_00.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB1	PB1-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:83	Collection Time (s)=177; RAW_FILE_NAME(Raw_File Name)=20221219_PB1_01.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB1	PB1-02	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:80	Collection Time (s)=353; RAW_FILE_NAME(Raw_File Name)=20221219_PB1_02.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB1	PB1-03	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:79	Collection Time (s)=519; RAW_FILE_NAME(Raw_File Name)=20221219_PB1_03.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB1	PB1-04	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:84	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB1_04.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB2	PB2-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:95	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB2-00.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB2	PB2-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:96	Collection Time (s)=218; RAW_FILE_NAME(Raw_File Name)=20221219_PB2-01.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB2	PB2-02	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:93	Collection Time (s)=342; RAW_FILE_NAME(Raw_File Name)=20221219_PB2-02.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB2	PB2-03	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:97	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB2-03.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB3	PB3-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:51	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_PB3-00.mzML; Sex=Female; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB3	PB3-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:48	Collection Time (s)=197; RAW_FILE_NAME(Raw_File Name)=20221215_PB3-01.mzML; Sex=Female; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB3	PB3-02	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:49	Collection Time (s)=424; RAW_FILE_NAME(Raw_File Name)=20221215_PB3-02.mzML; Sex=Female; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB3	PB3-03	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:50	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_PB3-03.mzML; Sex=Female; Age=19; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB4	PB4-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:115	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB4-00.mzML; Sex=Female; Age=30; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB4	PB4-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:112	Collection Time (s)=237; RAW_FILE_NAME(Raw_File Name)=20221219_PB4-01.mzML; Sex=Female; Age=30; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB4	PB4-02	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:114	Collection Time (s)=574; RAW_FILE_NAME(Raw_File Name)=20221219_PB4-02.mzML; Sex=Female; Age=30; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB4	PB4-03	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:113	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB4-03.mzML; Sex=Female; Age=30; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB5	PB5-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:102	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB5-00.mzML; Sex=Female; Age=49; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB5	PB5-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:104	Collection Time (s)=182; RAW_FILE_NAME(Raw_File Name)=20221219_PB5-01.mzML; Sex=Female; Age=49; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB5	PB5-02	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:101	Collection Time (s)=381; RAW_FILE_NAME(Raw_File Name)=20221219_PB5-02.mzML; Sex=Female; Age=49; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB5	PB5-03	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:105	Collection Time (s)=576; RAW_FILE_NAME(Raw_File Name)=20221219_PB5-03.mzML; Sex=Female; Age=49; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB5	PB5-04	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:103	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB5-04.mzML; Sex=Female; Age=49; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:17	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-00.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:11	Collection Time (s)=86; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-01.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-02	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:15	Collection Time (s)=197; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-02.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-03	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:14	Collection Time (s)=336; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-03.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-04	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:16	Collection Time (s)=483; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-04.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-05	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:18	Collection Time (s)=591; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-05.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB6	PB6-06	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:13	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_PB6-06.mzML; Sex=Female; Age=34; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:32	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-00.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:30	Collection Time (s)=171; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-01.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-02	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:34	Collection Time (s)=288; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-02.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-03	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:27	Collection Time (s)=341; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-03.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-04	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:36	Collection Time (s)=470; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-04.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-05	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:29	Collection Time (s)=512; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-05.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-06	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:35	Collection Time (s)=545; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-06.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-07	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:31	Collection Time (s)=602; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-07.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB7	PB7-08	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:28	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221215_PB7-08.mzML; Sex=Female; Age=22; Batch=1
SUBJECT_SAMPLE_FACTORS           	PB8	PB8-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:70	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB8_00.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB8	PB8-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:67	Collection Time (s)=285; RAW_FILE_NAME(Raw_File Name)=20221219_PB8_01.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB8	PB8-02	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:69	Collection Time (s)=543; RAW_FILE_NAME(Raw_File Name)=20221219_PB8_02.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB8	PB8-03	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:68	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB8_03.mzML; Sex=Male; Age=23; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-00	Sample source:Urine | Treatment:Control | Time Interval:0 | Injection order:121	Collection Time (s)=0; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-00.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-01	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:126	Collection Time (s)=158; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-01.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-02	Sample source:Urine | Treatment:Control | Time Interval:1 | Injection order:124	Collection Time (s)=216; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-02.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-03	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:117	Collection Time (s)=426; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-03.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-04	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:120	Collection Time (s)=471; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-04.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-05	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:127	Collection Time (s)=517; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-05.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-06	Sample source:Urine | Treatment:Control | Time Interval:2 | Injection order:122	Collection Time (s)=594; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-06.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	PB9	PB9-07	Sample source:Urine | Treatment:Control | Time Interval:3 | Injection order:118	Collection Time (s)=1440; RAW_FILE_NAME(Raw_File Name)=20221219_PB9-07.mzML; Sex=Female; Age=21; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B1QC1	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:3	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC1.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC2	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:4	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC2.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC3	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:10	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC3.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC4	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:12	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC4.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC5	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:19	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC5.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC6	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:20	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC6.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC7	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:26	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC7.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC8	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:33	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC8.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC9	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:37	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC9.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC10	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:38	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC10.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC11	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:39	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC11.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC12	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:40	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC12.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC13	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:41	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC13.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC14	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:47	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC14.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC15	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:52	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC15.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC16	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:53	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC16.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC17	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:59	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC17.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B1QC18	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:60	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221215_QC18.mzML; Sex=QC; Age=QC; Batch=1
SUBJECT_SAMPLE_FACTORS           	QC	B2QC1	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:65	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC1.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC2	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:66	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC2.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC3	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:71	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC3.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC4	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:72	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC4.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC5	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:78	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC5.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC6	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:82	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC6.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC7	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:85	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC7.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC8	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:86	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC8.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC9	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:87	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC9.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC10	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:94	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC10.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC11	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:98	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC11.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC12	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:99	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC12.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC13	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:100	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC13.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC14	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:106	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC14.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC15	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:111	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC15.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC16	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:116	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC16.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC17	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:119	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC17.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC18	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:128	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC18.mzML; Sex=QC; Age=QC; Batch=2
SUBJECT_SAMPLE_FACTORS           	QC	B2QC19	Sample source:Urine | Treatment:QC | Time Interval:- | Injection order:132	Collection Time (s)=QC; RAW_FILE_NAME(Raw_File Name)=20221219_QC19.mzML; Sex=QC; Age=QC; Batch=2
#COLLECTION
CO:COLLECTION_SUMMARY            	Urine samples were collected in plastic containers at baseline (prior to capsule
CO:COLLECTION_SUMMARY            	ingestion, T0) and at different time points during the acute intervention,
CO:COLLECTION_SUMMARY            	depending on each volunteer, until the end of the study. Additionally, a final
CO:COLLECTION_SUMMARY            	urine sample was collected from all volunteers at 24 h post-intake. For
CO:COLLECTION_SUMMARY            	statistical comparison between samples, the collected urines were grouped into
CO:COLLECTION_SUMMARY            	the following intervals: time 0, interval 1 (0–6 h), interval 2 (6–12 h),
CO:COLLECTION_SUMMARY            	and interval 3 (12–24 h). A pooled quality control (QC) sample was prepared by
CO:COLLECTION_SUMMARY            	mixing equal aliquots of each experimental sample.Urine samples were aliquoted
CO:COLLECTION_SUMMARY            	in Wilmut tubes and stored at -80 ºC until analysis.
CO:SAMPLE_TYPE                   	Urine
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	The Olea europaea (OE) leaf bioactive extract was provided by NATAC Biotech S.L.
TR:TREATMENT_SUMMARY             	(Cáceres, Spain). This extract was obtained through a solid–liquid extraction
TR:TREATMENT_SUMMARY             	process using a mixture of ethanol and water in a ratio of 80:20 (v:v) for a
TR:TREATMENT_SUMMARY             	duration of 2 h, maintaining a solvent-to-plant ratio of 20:1. The extraction
TR:TREATMENT_SUMMARY             	temperatures was set to 45 °C. After extraction, the extract was vacuum-dried,
TR:TREATMENT_SUMMARY             	stored at room temperature, and protected from light until the encapsulation
TR:TREATMENT_SUMMARY             	process. Previous reports have detailed the phytochemical characterization, and
TR:TREATMENT_SUMMARY             	bioactivity of the extract (Villegas-Aguilar, Sánchez-Marzo, et al., 2024). The
TR:TREATMENT_SUMMARY             	acute, randomized, controlled, double-blind clinical trial was conducted at the
TR:TREATMENT_SUMMARY             	premises of the University of Miguel Hernández. Following an overnight fast, 8
TR:TREATMENT_SUMMARY             	participants consumed an OE capsule containing 500 mg of extract, while 9 took a
TR:TREATMENT_SUMMARY             	placebo capsule. The placebo capsule was based on microcrystalline cellulose. A
TR:TREATMENT_SUMMARY             	polyphenol-free breakfast, which included toast with butter, was provided to the
TR:TREATMENT_SUMMARY             	volunteers 30 minutes after consuming the supplement. Furthermore,
TR:TREATMENT_SUMMARY             	polyphenol-free snacks and lunch were offered to participants at 2, 4 and 9
TR:TREATMENT_SUMMARY             	hours after breakfast intake. All participants consumed identical quantities of
TR:TREATMENT_SUMMARY             	these meals to ensure dietary consistency and comparability across the study
TR:TREATMENT_SUMMARY             	population. Water was provided ad libitum.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	All urine samples were normalized to a standardized osmolality value before
SP:SAMPLEPREP_SUMMARY            	HPLC-ESI-QTOF-MS analysis to compensate for variations in hydration levels among
SP:SAMPLEPREP_SUMMARY            	individuals. Urine samples were thawed on ice, and osmolality was measured in
SP:SAMPLEPREP_SUMMARY            	duplicate using two 50 μL aliquots per sample with an OSMOMAT3000 osmometer
SP:SAMPLEPREP_SUMMARY            	(Gonotec®, Berlin, Germany). Based on these osmolality measurements, a 100 μL
SP:SAMPLEPREP_SUMMARY            	aliquot of each sample was diluted with LC-MS grade water to achieve the
SP:SAMPLEPREP_SUMMARY            	normalized osmolality value of 100 mOsm/kg. All samples were diluted, except for
SP:SAMPLEPREP_SUMMARY            	two specific samples that required pre-concentration because their measured
SP:SAMPLEPREP_SUMMARY            	osmolality was below 100 mOsm/kg. These two samples were dried using an
SP:SAMPLEPREP_SUMMARY            	Eppendorf Concentrator Plus (Eppendorf™, Hamburg, Germany), and then
SP:SAMPLEPREP_SUMMARY            	reconstituted with LC-MS grade water to reach the target osmolality value. Once
SP:SAMPLEPREP_SUMMARY            	all samples were normalized to 100 mOsm/kg, they were vortexed (ZX3 Vortex
SP:SAMPLEPREP_SUMMARY            	Mixer, Fisherbrand™, Waltham, Massachusetts, USA) for 10 seconds, and
SP:SAMPLEPREP_SUMMARY            	centrifuged (Centrifuge 5430/5430 R, Eppendorf™, Hamburg, Germany) at 14,000
SP:SAMPLEPREP_SUMMARY            	rpm and 4 °C for 10 minutes to remove undesired solid particles. Then, 60 μL
SP:SAMPLEPREP_SUMMARY            	of supernatant from each sample was transferred to HPLC vials. Furthermore, a
SP:SAMPLEPREP_SUMMARY            	pooled quality control (QC) sample was prepared by mixing equal aliquots of each
SP:SAMPLEPREP_SUMMARY            	experimental sample. All urine aliquots were stored at -20 °C until the day of
SP:SAMPLEPREP_SUMMARY            	analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1260
CH:COLUMN_NAME                   	WATERS ACQUITY UPLC BEH SHIELD RP18 (150 x 2.1mm, 1.7 um)
CH:SOLVENT_A                     	100% Water; 0.1% Formic Acid
CH:SOLVENT_B                     	100% Acetonitrile
CH:FLOW_GRADIENT                 	time in minutes; %B: (0.00; 5), (5.00; 10), (18.00; 85), (24.00; 100), (25.50;
CH:FLOW_GRADIENT                 	100), (26.50; 5) and (32.50; 5).
CH:FLOW_RATE                     	0.4 ml/min
CH:COLUMN_TEMPERATURE            	60
CH:SAMPLE_INJECTION              	5 ul
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6540 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Detection was performed over a range of 50-1200 m/z in negative ion mode. Two
MS:MS_COMMENTS                   	reference masses, TFA anion (m/z 112.98558700) and HP-921 (m/z 1033.98810900),
MS:MS_COMMENTS                   	were used for continuous mass calibration, ensuring accurate mass measurements.
MS:MS_COMMENTS                   	Optimal MS parameters for maximal analyte detection were applied as follows:
MS:MS_COMMENTS                   	drying gas (ultrapure nitrogen) temperature, 200 °C; gas flow, 10 L/min;
MS:MS_COMMENTS                   	nebulizer, 20 psig; sheath gas temperature, 350 °C; sheath gas flow, 12 L/min.
MS:MS_COMMENTS                   	The MS scan acquisition parameters were set in the following manner: capillary
MS:MS_COMMENTS                   	voltage, 4000 V; nozzle voltage, 500 V; fragmentor, 130 V; skimmer 45 V; and
MS:MS_COMMENTS                   	octopole RF Peak, 750 V. All samples were analyzed in full-scan mode (MS1) using
MS:MS_COMMENTS                   	an acquisition rate of 3 spectra/s. Pooled QC samples were also acquired in auto
MS:MS_COMMENTS                   	MS/MS fragmentation mode using nitrogen as the collision gas at collision
MS:MS_COMMENTS                   	energies of 10, 30, and 60 eV. Since some signals of interest were not
MS:MS_COMMENTS                   	fragmented in the autoMS/MS analyses of the QCs, targeted MS/MS analyses were
MS:MS_COMMENTS                   	also carried out after the statistical analysis on the significant signals
MS:MS_COMMENTS                   	detected in the specific biological samples where they were detected. In
MS:MS_COMMENTS                   	addition, targeted analyses of the commercial standards were carried out to
MS:MS_COMMENTS                   	confirm metabolite identification. Due to the large number of samples, they were
MS:MS_COMMENTS                   	split into two different analytical batches. Two analytical blanks (BK) were
MS:MS_COMMENTS                   	analyzed at the beginning and end of both sequences to control potential
MS:MS_COMMENTS                   	contaminants. After the initial blanks, 5 injections of the QC sample were
MS:MS_COMMENTS                   	performed to equilibrate the system. In addition, the pooled QC samples were
MS:MS_COMMENTS                   	injected every 6 biological samples to ensure analytical reproducibility and
MS:MS_COMMENTS                   	correct within- and between-batches effects. Biological samples from the studied
MS:MS_COMMENTS                   	groups (placebo or OE supplements) were randomized within the sequences, but the
MS:MS_COMMENTS                   	samples from each volunteer were injected consecutively. Initially, the raw
MS:MS_COMMENTS                   	HPLC-ESI-QTOF-MS data were converted from the .d vendor format to .mzML format
MS:MS_COMMENTS                   	using the open-source ProteoWizard msConvert software (version 3.0.) (Adusumilli
MS:MS_COMMENTS                   	& Mallick, 2017). Next, MZmine software (version 3.4.27.) was used for data
MS:MS_COMMENTS                   	preprocessing, which involved the following steps: Mass Detection, ADAP
MS:MS_COMMENTS                   	Chromatogram Builder, ADAP Chromatogram Resolver, 13C Isotope Grouper,
MS:MS_COMMENTS                   	Alignment, and Feature List Filtering.
MS:MS_RESULTS_FILE               	ST004172_AN006926_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END