#METABOLOMICS WORKBENCH Ahsan1629_20241104_134429 DATATRACK_ID:5343 STUDY_ID:ST003576 ANALYSIS_ID:AN005871 PROJECT_ID:PR002206 VERSION 1 CREATED_ON November 14, 2024, 5:49 pm #PROJECT PR:PROJECT_TITLE Comparison of the capillary and venous blood lipidomes- validation of the Tasso PR:PROJECT_TITLE SST capillary blood collection device for circulating lipid biomarker analysis PR:PROJECT_SUMMARY Cumulative evidence identifies many circulating lipids can act as biomarkers of PR:PROJECT_SUMMARY human health and status. To date, lipid biomarkers have largely been correlated PR:PROJECT_SUMMARY to dietary patterns to reveal the beneficial effects of specific nutritional PR:PROJECT_SUMMARY regimens and at the same time, for screening, early diagnosis, and therapeutic PR:PROJECT_SUMMARY decision making in cardio-metabolic diseases, and in certain types of cancers. PR:PROJECT_SUMMARY These studies use biofluids or tissues as a source of material for lipidomics. PR:PROJECT_SUMMARY Blood is a readily accessible and most commonly analyzed biological fluid not PR:PROJECT_SUMMARY only for plasma/serum based lipidomics studies but also for laboratory based PR:PROJECT_SUMMARY multi-analyte panels (enzyme, metabolite, electrolyte, lipid, hormone, or PR:PROJECT_SUMMARY vitamin levels) and biomarkers for diseases. Therefore, collection of PR:PROJECT_SUMMARY high-quality volumetric blood for these purposes is important as composition of PR:PROJECT_SUMMARY blood provides a wealth of reliable information concerning a patient’s health. PR:PROJECT_SUMMARY Venipuncture is the clinical gold standard for blood collection, however, PR:PROJECT_SUMMARY recently a single-use, clinical-grade, automatic at-home capillary blood PR:PROJECT_SUMMARY collecting device called Tasso+ (formely called TassoOne™ hemolink) PR:PROJECT_SUMMARY (https://www.tassoinc.com) has been introduced in both educational and PR:PROJECT_SUMMARY healthcare institutions. A few studies have already reported the use of Tasso+ PR:PROJECT_SUMMARY in clinical and non-clinical settings and have also been assessed for clinical PR:PROJECT_SUMMARY chemistry hematology, and serology with overall good analytical performance and PR:PROJECT_SUMMARY participant acceptance, there are still no studies in literatures evaluating the PR:PROJECT_SUMMARY suitability of Tasso-based collected blood for lipidomics. Therefore, the aim of PR:PROJECT_SUMMARY this pilot study was to establish and validate the suitability of Tasso+-drawn PR:PROJECT_SUMMARY blood plasma as an alternative to venous blood plasma for clinical lipidomics. PR:PROJECT_SUMMARY For this purpose, blood was collected from healthy (non-fasting) consenting PR:PROJECT_SUMMARY adults (n =10, age = >18 years old) under an approved IRB protocol at Wake PR:PROJECT_SUMMARY Forest University School of Medicine. Subjects provided written consent to PR:PROJECT_SUMMARY participate in the study and provided matched blood using traditional PR:PROJECT_SUMMARY venipuncture and TASSO+ device. Comprehensive plasma lipidomics analysis of PR:PROJECT_SUMMARY blood samples collected through venipuncture and Tasso+ devices was carried out PR:PROJECT_SUMMARY to investigate and compare the performance and suitability of Tasso+ devices for PR:PROJECT_SUMMARY lipidomics. Plasma was extracted from the respective venous and capillary blood PR:PROJECT_SUMMARY samples and prepared for the global lipidomics on LC-MS coupled online via an PR:PROJECT_SUMMARY ESI with a Q Exactive™ MS instrument. The lipidomics data was processed using PR:PROJECT_SUMMARY MS-DIAL v. 4.9.221218 software and only lipids with MS2 level identification was PR:PROJECT_SUMMARY used for statistical analyses. comprehensive statistical analysis with cross PR:PROJECT_SUMMARY validation and multiple testing adjustments show that the lipid composition of PR:PROJECT_SUMMARY paired Tasso+ and venous blood samples do not significantly differ (adjusted PR:PROJECT_SUMMARY p-value > 0.05). A linear regression model with Spearman correlation analysis PR:PROJECT_SUMMARY also showed a significant-to-perfect level (r = 0.95-0.99) of concordance PR:PROJECT_SUMMARY between the matched samples. With the exception of monoacylglycerols (MG) and PR:PROJECT_SUMMARY cardiolipins (CL), every class of lipid biomarkers also demonstrated PR:PROJECT_SUMMARY substantially high degree of correlation (0.9-0.99) between paired venous and PR:PROJECT_SUMMARY capillary blood. These findings demonstrate that capillary and venous blood PR:PROJECT_SUMMARY plasma lipidomes are similar; indicating that self-administered capillary PR:PROJECT_SUMMARY sampling could be used interchangeably with standardized venipuncture in PR:PROJECT_SUMMARY clinical research settings. PR:INSTITUTE University of Arkansas for Medical Sciences PR:DEPARTMENT Arkansas Children Nutrition Center, University of Arkansas for Medical Sciences PR:LABORATORY MCAC PR:LAST_NAME Hameed PR:FIRST_NAME Ahsan PR:ADDRESS 15 Childern's way, Little Rock, AR, 72202, USA PR:EMAIL ahameed@uams.edu PR:PHONE 5019441248 PR:FUNDING_SOURCE This work was supported by grants from the NIH and Department of Veterans PR:FUNDING_SOURCE Affairs to AJM (U54NS121688 and BX006469-01). Drs. Morris and Rahbar are members PR:FUNDING_SOURCE of the Blood Biomarkers Core Group of the U54 NS121688 supported Care4Kids PR:FUNDING_SOURCE Consortium. #STUDY ST:STUDY_TITLE Comparison of the capillary and venous blood lipidomes- validation of the Tasso ST:STUDY_TITLE SST capillary blood collection device for circulating lipid biomarker analysis ST:STUDY_SUMMARY Venipuncture of the upper extremities continues to be the standard method of ST:STUDY_SUMMARY whole blood collection used for lipidomics, but self-administered Tasso+TM ST:STUDY_SUMMARY capillary sampling is now an accepted clinical method. Wide adoption of the ST:STUDY_SUMMARY capillary blood test method for lipid biomarker measurements would require ST:STUDY_SUMMARY distinguishing between the universal global lipid levels variations in venous ST:STUDY_SUMMARY versus capillary blood. As lipids are biomarkers for a number of disorders, and ST:STUDY_SUMMARY because they may provide valuable information on nutritional and toxicological ST:STUDY_SUMMARY status, we view this study as an example of broader biomarker assay consistency. ST:STUDY_SUMMARY Because exchange of blood components including lipids occurs in capillaries, the ST:STUDY_SUMMARY capillary and venous blood lipidomes might be different. Here we compared the ST:STUDY_SUMMARY lipidomes of Tasso+ drawn capillary blood plasma to venous blood plasma by ST:STUDY_SUMMARY high-resolution mass spectrometry based lipidomics, in 10 human subjects. While ST:STUDY_SUMMARY we found substantial inter individual variability when comparing lipid profiles, ST:STUDY_SUMMARY comprehensive statistical analysis with cross validation and multiple testing ST:STUDY_SUMMARY adjustments show that the lipid composition of paired Tasso+ and venous blood ST:STUDY_SUMMARY samples do not significantly differ (adjusted p-value > 0.05). A linear ST:STUDY_SUMMARY regression model with Spearman correlation analysis also showed a ST:STUDY_SUMMARY significant-to-perfect level (r = 0.95-0.99) of concordance between the matched ST:STUDY_SUMMARY samples. With the exception of monoacylglycerols (MG) and cardiolipins (CL), ST:STUDY_SUMMARY every class of lipid biomarkers also demonstrated substantially high degree of ST:STUDY_SUMMARY correlation (0.9-0.99) between paired venous and capillary blood. These findings ST:STUDY_SUMMARY demonstrate that capillary and venous blood plasma lipidomes are similar; ST:STUDY_SUMMARY indicating that self-administered capillary sampling could be used ST:STUDY_SUMMARY interchangeably with standardized venipuncture in clinical research settings. ST:INSTITUTE University of Arkansas for Medical Sciences ST:DEPARTMENT Arkansas Children Nutrition Center, University of Arkansas for Medical Sciences ST:LABORATORY MCAC ST:LAST_NAME Hameed ST:FIRST_NAME Ahsan ST:ADDRESS 15 Childern's way, Little Rock, AR, 72202, USA ST:EMAIL ahameed@uams.edu ST:PHONE 5019441248 ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 10 ST:NUM_MALES 5 ST:NUM_FEMALES 5 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:AGE_OR_AGE_RANGE 30.8 ± 10.4 SU:WEIGHT_OR_WEIGHT_RANGE >100 lbs SU:HEIGHT_OR_HEIGHT_RANGE not recorded SU:GENDER Male and female SU:HUMAN_RACE 90% white, 10% asian SU:HUMAN_ETHNICITY 0% hispanic SU:HUMAN_EXCLUSION_CRITERIA Exclusion criteria were known pregnancy, any known inflammatory disease, a SU:HUMAN_EXCLUSION_CRITERIA positive COVID-19 test or case within the past 2 weeks, and any aspirin or NSAID SU:HUMAN_EXCLUSION_CRITERIA use within the past 24-48 hrs. #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 Sample_001_Veni Sample_001_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_01_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_01_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_003_Veni Sample_003_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_02_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_02_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_004_Veni Sample_004_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_03_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_03_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_005_Veni Sample_005_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_04_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_04_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_007_Veni Sample_007_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_05_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_05_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_008_Veni Sample_008_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_06_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_06_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_009_Veni Sample_009_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_07_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_07_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_010_Veni Sample_010_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_08_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_08_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_011_Veni Sample_011_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_09_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_09_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_012_Veni Sample_012_Veni Mode of whole blood collection:Venipuncture | Type of blood:Venous | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_010_Veni_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_010_Veni_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_001_Tasso Sample_001_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_01_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_01_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_003_Tasso Sample_003_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_02_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_02_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_004_Tasso Sample_004_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_03_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_03_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_005_Tasso Sample_005_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_04_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_04_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_007_Tasso Sample_007_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_05_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_05_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_008_Tasso Sample_008_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_06_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_06_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_009_Tasso Sample_009_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_07_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_07_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_010_Tasso Sample_010_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_08_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_08_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_011_Tasso Sample_011_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_09_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_09_Tasso_neg.mzML SUBJECT_SAMPLE_FACTORS Sample_012_Tasso Sample_012_Tasso Mode of whole blood collection:Tasso+ drawn | Type of blood:Capillary | Sample source:Plasma RAW_FILE_NAME(Raw file name)=Sample_010_Tasso_pos.mzML; RAW_FILE_NAME(Raw file name)=Sample_010_Tasso_neg.mzML #COLLECTION CO:COLLECTION_SUMMARY Blood was collected from healthy (non-fasting) consenting adults (>18 years old) CO:COLLECTION_SUMMARY under an approved IRB protocol at Wake Forest University School of Medicine. CO:COLLECTION_SUMMARY Subjects provided written consent to participate in the study and provided CO:COLLECTION_SUMMARY matched blood using traditional venipuncture and TASSO+ device. In both methods, CO:COLLECTION_SUMMARY blood was drawn into an EDTA tube; for venipuncture we used BD EDTA vacutainers CO:COLLECTION_SUMMARY (3mL) and for the Tasso+ device we used a BD Microtainer® EDTA (light purple CO:COLLECTION_SUMMARY top) blood collection tube (250-500 ul) that was compatible with the TASSO+ CO:COLLECTION_SUMMARY device. Exclusion criteria were known pregnancy, any known inflammatory disease, CO:COLLECTION_SUMMARY a positive COVID-19 test or case within the past 2 weeks, and any aspirin or CO:COLLECTION_SUMMARY NSAID use within the past 24-48 hrs. In this pilot we recruited 10 healthy CO:COLLECTION_SUMMARY subjects. Once collected, blood was inverted gently a few times to ensure mixing CO:COLLECTION_SUMMARY with EDTA and then was centrifuged at 1500g for 15 minutes at 4C to obtain CO:COLLECTION_SUMMARY plasma. Plasma was aliquoted and stored in cryovials at -80C for future CO:COLLECTION_SUMMARY biomarker testing. CO:SAMPLE_TYPE Blood (plasma) CO:COLLECTION_METHOD venipuncture and TASSO+ CO:COLLECTION_LOCATION arm CO:COLLECTION_FREQUENCY once CO:COLLECTION_DURATION <10 min CO:STORAGE_CONDITIONS -80℃ CO:STORAGE_VIALS EDTA CO:COLLECTION_TUBE_TEMP room temperature #TREATMENT TR:TREATMENT_SUMMARY Not Applicable TR:HUMAN_FASTING none #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipid extraction was performed in 4 mL borosilicate glass tubes with Teflon SP:SAMPLEPREP_SUMMARY lined cap employing the modified Folch method. Briefly, aliquot 50 µL plasma SP:SAMPLEPREP_SUMMARY samples were thawed at room temperature and extracted with 2 : 1 ratio of SP:SAMPLEPREP_SUMMARY CH2Cl2/MeOH containing 450 µL 0.1M HCl. Every sample was spiked with 10 µL SP:SAMPLEPREP_SUMMARY lipid internal standard purchased from Avanti (Splash Lipidomix, Alabaster, SP:SAMPLEPREP_SUMMARY United States) and included phosphatidylcholine 15:0–18:1(d7) PC, SP:SAMPLEPREP_SUMMARY phosphatidylethanolamine 15:0–18:1(d7) PE, phosphatidylserine 15:0–18:1(d7) SP:SAMPLEPREP_SUMMARY PS, phosphatidylglycerol 15:0–18:1(d7) PG, phosphatidylinositol SP:SAMPLEPREP_SUMMARY 15:0–18:1(d7), PI, phosphatidic acid 15:0–18:1(d7) PA, SP:SAMPLEPREP_SUMMARY lysophosphatidylcholine 18:1(d7) LPC, lysophosphatidylethanolamine 18:1(d7) LPE, SP:SAMPLEPREP_SUMMARY cholesteryl ester 18:1(d7) CE, monoacylglycerol 18:1(d7) MG, diacylglycerol SP:SAMPLEPREP_SUMMARY 15:0–18:1(d7) DG, triacylglycerol 15:0–18:1(d7)-15:0 TG, sphingomyelin SP:SAMPLEPREP_SUMMARY 18:1(d9) SM and Cholesterol (d7). The solvent mixture was vortexed for 10 min SP:SAMPLEPREP_SUMMARY after which 1 mL of chloroform and 1.3 mL of 0.1M HCl was added for phase SP:SAMPLEPREP_SUMMARY separation. The mixture was vortexed again for another 10 min followed by SP:SAMPLEPREP_SUMMARY centrifugation at 4000 rcf for 10 min. The lower phase was carefully transferred SP:SAMPLEPREP_SUMMARY to a new 4 mL borosilicate glass vial and then dried under nitrogen gas using SP:SAMPLEPREP_SUMMARY TurboVap evaporator. The dried samples were reconstituted in methanol stored at SP:SAMPLEPREP_SUMMARY -80 ℃ until analysis. Two quality control (QC) samples were prepared by SP:SAMPLEPREP_SUMMARY combining material from each biological sample (n = 10; 10 µL of each SP:SAMPLEPREP_SUMMARY sample) of each group. SP:PROCESSING_STORAGE_CONDITIONS On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC BEH C8 (50 mm x 2.1 mm, 1.7 µm) CH:SOLVENT_A 40% acetonitrile/60% water; 10 mM ammonium formate; 0.1% formic acid CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 10 mM ammonium formate; 0.1% formic acid CH:FLOW_GRADIENT 0–24.00 min, 32% B; 25.0– 28.0 min, 32–97% B; 29.0–29.1 min, 97–32% B; CH:FLOW_GRADIENT 29.2–35.0 min, 32– 32% B CH:FLOW_RATE 0.250 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME MCAC AN:OPERATOR_NAME Ahsan Hameed #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS A Dionex UltiMate 3000 ultra-high performance liquid chromatography (UHPLC) MS:MS_COMMENTS system (Santa Clara, CA, USA) coupled to an electrospray ionization source (ESI) MS:MS_COMMENTS with a Q Exactive™ Plus MS instrument (Thermo Fisher Scientific, USA) was used MS:MS_COMMENTS for the untargeted lipidomic analysis. For data aquisition, 10 µL aliquots of MS:MS_COMMENTS sample solution, maintained at 4 ̊C in an auto-sampler, was injected. Separate MS:MS_COMMENTS datasets were acquired in positive and negative ionization modes using a heated MS:MS_COMMENTS electrospray ionization source. The source and ion transfer parameters applied MS:MS_COMMENTS were as follows: spray voltage 3.5 kV (positive) and 2.8 kV (negative). For both MS:MS_COMMENTS ionization modes, the sheath gas, aux gas, capillary temperature and heater MS:MS_COMMENTS temperature were maintained at 12, 5 (arbitrary units), 320 and 300, MS:MS_COMMENTS respectively. The S-Lens RF level was set at 60. The Orbitrap mass analyzer was MS:MS_COMMENTS operated at a resolving power of 70 000 in full-scan mode (scan range: 80–1200 MS:MS_COMMENTS m/z; automatic gain control (AGC) target: 5 x 106 ) and 15 000 in the Top 5 MS:MS_COMMENTS data-dependent MS2 mode (stepped normalized collision energy: 20; injection MS:MS_COMMENTS time: 250 ms; isolation window: 1.0 m/z; AGC target: 5 x 106 ) with a dynamic MS:MS_COMMENTS exclusion setting of 6.0 s. LC-MS grade methanol was used to minimize intensity MS:MS_COMMENTS of the background signal. Product ion scans in positive and negative ion mode MS:MS_COMMENTS were performed on each sample, to maximize numbers of lipid species identified. MS:MS_COMMENTS Sampling order was randomized prior to analysis. MS:MS_RESULTS_FILE ST003576_AN005871_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END