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"PROJECT":{"PROJECT_TITLE":"Antibiotics in Cystic Fibrosis Sputum","PROJECT_SUMMARY":"Antibiotics have enormous impact on microbial communities, yet many studies focused on microbiome and health are confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antibacterial properties, investigating the type, concentration and location would help us understand what microbes encounter at an infection site. Here, we developed Liquid Chromatography Mass Spectrometry (LC-MS) methods to detect 18 antibiotics in clinical samples using cystic fibrosis sputum as an application. Three liquid extraction methods were compared with spike-in controls of antibiotics on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was then used to detect antibiotics in sputum samples from 11 people with cystic fibrosis in comparison to self-reported antibiotic use. For the sputum samples, the Quattro Premier XE method detected nanomolar or micromolar concentrations of 16 antibiotics. In 71 of the 158 sputum samples, at least one antibiotic that was not reported by the subject was detected by the LC-MS method, a discordance largely explained by alternating usage and the long half-life of azithromycin. For ~37% of samples, antibiotics were marked by the subject but were not detected, which has implications for in vivo antibiotic penetrance and efficacy. Our study takes the first step in detecting antibiotics at the infection site, with future goals of incorporating accurate antibiotic data into microbiome studies.","INSTITUTE":"University of California, Irvine","DEPARTMENT":"Molecular Biology & Biochemistry","LABORATORY":"Katrine Whiteson","LAST_NAME":"Gallagher","FIRST_NAME":"Tara","ADDRESS":"3315 McGaugh Hall, UC IRVINE, Irvine, CA, 92697, USA","EMAIL":"tgallagh@uci.edu","PHONE":"9498243509"},

"STUDY":{"STUDY_TITLE":"Antibiotics in Cystic Fibrosis Sputum","STUDY_TYPE":"Clinical","STUDY_SUMMARY":"Detection of 18 antibiotics in 171 cystic fibrosis sputum samples from the Phase 1 Clinical Study.","INSTITUTE":"University of California, Irvine","LAST_NAME":"Gallagher","FIRST_NAME":"Tara","ADDRESS":"3315 McGaugh Hall, UC IRVINE, Irvine, CA, 92697, USA","EMAIL":"tgallagh@uci.edu","PHONE":"9498243509"},

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],
"COLLECTION":{"COLLECTION_SUMMARY":"Sputum samples from 11 subjects with cystic fibrosis were selected from a larger airway microbiome study that was approved by the University of Michigan Medical School Institutional Review Board. Subjects were 6 males and 5 females, age 21 to 56 years (median 37). Sputum samples were collected by subjects at home and stored at 4˚C for up to 23 days and then transferred to -80˚C.","SAMPLE_TYPE":"Sputum","STORAGE_CONDITIONS":"Described in summary"},

"TREATMENT":{"TREATMENT_SUMMARY":"The goal of this study was to determine which antibiotics are present in cystic fibrosis sputum using a LC-MS method. There were no experimental conditions. The types of samples included in the LC-MS run were: cystic fibrosis sputum, external standard curve, quality control pools of external standards run every ~50 samples, and washes (blanks)."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The extraction solvent was chilled 1% DTT and spiked with 1.33 µM of both internal standards, linezolid-d3 and levofloxacin-d8. Solvent (150 µL) was added to 50 µL of sputum. The samples were vortexed for 30 seconds, shaken at 4˚C on a shaking platform with moderate agitation for 15 minutes, and centrifuged at 13,200 RCF for 10 minutes at 4˚C. The supernatant was pipetted into amber glass vials and injected directly into the LC-MS.","PROCESSING_STORAGE_CONDITIONS":"On ice","EXTRACT_STORAGE":"On ice"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity I-Class","COLUMN_NAME":"Waters Acquity BEH C18 (50 x 2.1mm, 1.7 um)","FLOW_RATE":"0.3 ml/min","COLUMN_TEMPERATURE":"50","METHODS_FILENAME":"qopt_A2B2_3g_c9_5min.wvhp","SOLVENT_A":"0.1% v/v formic acid and 2 mM ammonium acetate in water","SOLVENT_B":"d 0.1% v/v formic acid and 2 mM ammonium acetate in methanol","INJECTION_TEMPERATURE":"10"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"CF_antibiotics_quant.mdb","ACQUISITION_PARAMETERS_FILE":"CF_antibiotics.exp"},

"MS":{"INSTRUMENT_NAME":"Waters Quattro Premier XE","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The retention times (RT) and MS/MS parameters for reach antibiotic were determined using the QuanOpt function in the Waters Masslynx software. Peaks were automatically picked and filtered in QuanLynx. Downstream filtering was conducted in R. Data were imported into R and the following criteria for filtering were applied: minimum peak area under the curve of 20 and signal to noise ratio of 10. Because several of the antibiotics had high carry-over rates (including ciprofloxacin, levofloxacin, and trimethoprim), sample peaks were also filtered out when the AUC was lower than the wash ran directly before the sample. Scripts can be found at the following github link: https://github.com/tgallagh/LCMS_Antibiotics.","CAPILLARY_VOLTAGE":"3.3 kV","COLLISION_GAS":"Nitrogen","SOURCE_TEMPERATURE":"125","DESOLVATION_GAS_FLOW":"800 L/h","DESOLVATION_TEMPERATURE":"400"},

"MS_METABOLITE_DATA":{
"Units":"micromolar",

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