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"PROJECT":{"PROJECT_TITLE":"Methods for improved identification and quantification in GC-MS-based metabolomic profiling of human plasma","PROJECT_TYPE":"GC-MS non targeted qualitative analysis","PROJECT_SUMMARY":"The field of metabolomics as applied to human disease and health is rapidly expanding. However, studies reporting experiences with quality-control and method validation are lacking. In this study, we sought to identify and modify steps in GC-MS-based metabolomic profiling of human plasma that could influence metabolite identification and quantification. Our experimental design included two studies: 1) the limiting-dilution study, which investigated the effects of dilution on analyte identification and quantification, and 2) the concentration-specific study, which compared the optimal plasma extract volume established in the first study with the volume used in the current institutional protocol. We confirmed that contaminants, concentration, intra- and inter-experiment variability are major factors influencing metabolite identification and quantification. In addition, we established methods for improved metabolite identification and quantification, which were summarized to provide recommendations for experimental design of GC-MS-based profiling of human plasma.","INSTITUTE":"Duke University","DEPARTMENT":"Duke Molecular Physiology Institute","LAST_NAME":"Wang","FIRST_NAME":"Hanghang","ADDRESS":"300 North Duke Street, Durham, NC, 27701, USA","EMAIL":"hanghang.wang@duke.edu","PHONE":"+1 919 884 0025","FUNDING_SOURCE":"U.S. Department of Health & Human Services, National Institutes of Health (NIH) - T32HL007101; Thoracic Surgery Foundation for Research and Education (TSFRE) - Braunwald Fellowship"},

"STUDY":{"STUDY_TITLE":"Effects of dilution on analyte identification and quantification","STUDY_TYPE":"GC-MS non-targeted metabolomic profiling","STUDY_SUMMARY":"The limiting-dilution study evaluated the effects of sample dilution on the ability to identify and quantify analytes in plasma. The study was divided into 10 batches with identical experimental design spanning over a 16-day period. Each batch consisted of 33 aliquots with 11 different plasma extract volumes (0 – 700 µL) corresponding to 11 plasma concentrations repeated three times.","INSTITUTE":"Duke University","DEPARTMENT":"Duke Molecular Physiology Institute","LAST_NAME":"Wang","FIRST_NAME":"Hanghang","ADDRESS":"300 North Duke Street, Durham, NC, 27701, USA","EMAIL":"hanghang.wang@duke.edu","PHONE":"+1 919 884 0025","NUM_GROUPS":"10"},

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{
"Subject ID":"-",
"Sample ID":"5-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-200B",
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},
{
"Subject ID":"-",
"Sample ID":"5-200C",
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},
{
"Subject ID":"-",
"Sample ID":"6-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-200C",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-200C",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-200C",
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},
{
"Subject ID":"-",
"Sample ID":"9-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-200C",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-200A",
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},
{
"Subject ID":"-",
"Sample ID":"10-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-200C",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-200B",
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},
{
"Subject ID":"-",
"Sample ID":"11-200C",
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},
{
"Subject ID":"-",
"Sample ID":"12-200A",
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},
{
"Subject ID":"-",
"Sample ID":"12-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-200C",
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},
{
"Subject ID":"-",
"Sample ID":"13-200A",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-200B",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-200C",
"Factors":{"Plasma Volume Extracted":"200 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-300C",
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},
{
"Subject ID":"-",
"Sample ID":"5-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-300A",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-300B",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-300C",
"Factors":{"Plasma Volume Extracted":"300 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-400C",
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},
{
"Subject ID":"-",
"Sample ID":"10-400A",
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},
{
"Subject ID":"-",
"Sample ID":"10-400B",
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},
{
"Subject ID":"-",
"Sample ID":"10-400C",
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},
{
"Subject ID":"-",
"Sample ID":"11-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-400A",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-400A",
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},
{
"Subject ID":"-",
"Sample ID":"13-400B",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-400C",
"Factors":{"Plasma Volume Extracted":"400 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-600C",
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},
{
"Subject ID":"-",
"Sample ID":"7-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-600B",
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},
{
"Subject ID":"-",
"Sample ID":"9-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-600A",
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},
{
"Subject ID":"-",
"Sample ID":"10-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-600A",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-600B",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-600C",
"Factors":{"Plasma Volume Extracted":"600 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"4-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"5-700B",
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},
{
"Subject ID":"-",
"Sample ID":"5-700C",
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},
{
"Subject ID":"-",
"Sample ID":"6-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"6-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"7-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"8-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"9-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"10-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"11-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"12-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-700A",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-700B",
"Factors":{"Plasma Volume Extracted":"700 uL"}
},
{
"Subject ID":"-",
"Sample ID":"13-700C",
"Factors":{"Plasma Volume Extracted":"700 uL"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Plasma from blood drawn intravenously","SAMPLE_TYPE":"Blood","COLLECTION_METHOD":"IV","ADDITIVES":"EDTA","BLOOD_SERUM_OR_PLASMA":"Plasma"},

"TREATMENT":{"TREATMENT_SUMMARY":"no treatment"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Protein precipitation and drying followed by derivatization via methoxyamine and MSTFA","EXTRACTION_METHOD":"Protein precipitation","SAMPLE_DERIVATIZATION":"18 mg/mL methoxyamine hydrochloride in pyridine for 30 min at 50°C, followed by trimethylsilylation with N-methyl-N- (trimethylsilyl)trifluoroacetamide (MSTFA) for 30 min at 50°C","SAMPLE_SPIKING":"retention-time-lock internal standard of perdeuterated myristic acid"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"GC/MS methods follow previous studies using a 6890 N GC connected to a 5975B Inert single quadrupole MS (Agilent Technologies, Santa Clara, CA) (Bonikos et al. 1975; Fiehn 2008; Kind et al. 2009). The two wall-coated, open-tubular GC columns connected in series are both from J&W/Agilent (part 122–5512), DB5-MS, 15 meters in length, 0.25 mm in diameter, with an 0.25-l m luminal film. Positive ions generated with conventional electron-ionization at 70 eV are scanned broadly from 600 to 50 m/z in the detector throughout the 45 min cycle time.","CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 6890N","COLUMN_NAME":"Agilent DB5-MS (15m × 0.25mm, 0.25um)","FLOW_RATE":"2.0 mL/min","TIME_PROGRAM":"initial GC oven temperature of 60°C increased at 10°C/min to a final temperature of 325°C"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 5975B","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE"},

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}

}