Summary of Study ST001365

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000933. The data can be accessed directly via it's Project DOI: 10.21228/M8398Z This work is supported by NIH grant, U2C- DK119886.

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Study IDST001365
Study TitleAntibiotics in Cystic Fibrosis Sputum
Study TypeClinical
Study SummaryDetection of 18 antibiotics in 171 cystic fibrosis sputum samples from the Phase 1 Clinical Study.
Institute
University of California, Irvine
Last NameGallagher
First NameTara
Address3315 McGaugh Hall, UC IRVINE, Irvine, CA, 92697, USA
Emailtgallagh@uci.edu
Phone9498243509
Submit Date2020-04-23
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2020-06-01
Release Version1
Tara Gallagher Tara Gallagher
https://dx.doi.org/10.21228/M8398Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000933
Project DOI:doi: 10.21228/M8398Z
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

Subject:

Subject ID:SU001439
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:21-56
Gender:Male and female
Human Inclusion Criteria:Cystic Fibrosis, adults

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Factor
SA099129WaterBlank34Blank
SA099130WaterBlank35Blank
SA099131WaterBlank14Blank
SA099132WaterBlank36Blank
SA099133WaterBlank15Blank
SA099134WaterBlank32Blank
SA099135WaterBlank33Blank
SA099136WaterBlank11Blank
SA099137WaterBlank37Blank
SA099138WaterBlank39Blank
SA099139WaterBlank40Blank
SA099140WaterBlank7Blank
SA099141WaterBlank8Blank
SA099142WaterBlank9Blank
SA099143WaterBlank16Blank
SA099144WaterBlank38Blank
SA099145WaterBlank10Blank
SA099146WaterBlank17Blank
SA099147WaterBlank23Blank
SA099148WaterBlank22Blank
SA099149WaterBlank1Blank
SA099150WaterBlank24Blank
SA099151WaterBlank25Blank
SA099152WaterBlank26Blank
SA099153WaterBlank27Blank
SA099154WaterBlank21Blank
SA099155WaterBlank20Blank
SA099156WaterBlank30Blank
SA099157WaterBlank31Blank
SA099158WaterBlank18Blank
SA099159WaterBlank19Blank
SA099160WaterBlank28Blank
SA099161WaterBlank29Blank
SA099162WaterBlank6Blank
SA099163WaterBlank12Blank
SA099164WaterBlank1ABlank
SA099165WaterBlank3bBlank
SA099166WaterBlank2ABlank
SA099167WaterBlank42Blank
SA099168WaterBlank4Blank
SA099169WaterBlank41Blank
SA099170WaterBlank2bBlank
SA099171WaterBlank1bBlank
SA099172WaterBlank1aBlank
SA099173WaterBlank2Blank
SA099174WaterBlank2aBlank
SA099175WaterBlank44Blank
SA099176WaterBlank43Blank
SA099177WaterBlank5Blank
SA099178WaterBlank3ABlank
SA099179Sample_13283CF_sample
SA099180Sample_8331CF_sample
SA099181Sample_1038CF_sample
SA099182Sample_6496CF_sample
SA099183Sample_5572CF_sample
SA099184Sample_11186CF_sample
SA099185Sample_4158CF_sample
SA099186Sample_5547CF_sample
SA099187Sample_8330CF_sample
SA099188Sample_5551CF_sample
SA099189Sample_9286CF_sample
SA099190Sample_12267CF_sample
SA099191Sample_9282CF_sample
SA099192Sample_1161CF_sample
SA099193Sample_12295CF_sample
SA099194Sample_1137CF_sample
SA099195Sample_11158CF_sample
SA099196Sample_13356CF_sample
SA099197Sample_5549CF_sample
SA099198Sample_10141CF_sample
SA099199Sample_9085CF_sample
SA099200Sample_13288CF_sample
SA099201Sample_1034CF_sample
SA099202Sample_9284CF_sample
SA099203Sample_15158CF_sample
SA099204Sample_11185CF_sample
SA099205Sample_8304CF_sample
SA099206Sample_5111CF_sample
SA099207Sample_5575CF_sample
SA099208Sample_8303CF_sample
SA099209Sample_1036CF_sample
SA099210Sample_13309CF_sample
SA099211Sample_1160CF_sample
SA099212Sample_1362CF_sample
SA099213Sample_5336CF_sample
SA099214Sample_1159CF_sample
SA099215Sample_6492CF_sample
SA099216Sample_4139CF_sample
SA099217Sample_4154CF_sample
SA099218Sample_6490CF_sample
SA099219Sample_1054CF_sample
SA099220Sample_15134CF_sample
SA099221Sample_13287CF_sample
SA099222Sample_9285CF_sample
SA099223Sample_9287CF_sample
SA099224Sample_13355CF_sample
SA099225Sample_4138CF_sample
SA099226Sample_9250CF_sample
SA099227Sample_12294CF_sample
SA099228Sample_6515CF_sample
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Collection:

Collection ID:CO001434
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 ID:TR001454
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).

Sample Preparation:

Sampleprep ID:SP001447
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

Combined analysis:

Analysis ID AN002272
Analysis type MS
Chromatography type Reversed phase
Chromatography system Waters Acquity I-Class
Column Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Triple quadrupole
MS instrument name Waters Quattro Premier XE
Ion Mode POSITIVE
Units micromolar

Chromatography:

Chromatography ID:CH001671
Methods Filename:qopt_A2B2_3g_c9_5min.wvhp
Instrument Name:Waters Acquity I-Class
Column Name:Waters Acquity BEH C18 (50 x 2.1mm,1.7um)
Column Temperature:50
Flow Rate:0.3 ml/min
Injection Temperature:10
Solvent A:100% water; 0.1% acetic acid; 10 mM ammonium acetate
Solvent B:100% methanol; 0.1% formic acid; 2 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS002116
Analysis ID:AN002272
Instrument Name:Waters Quattro Premier XE
Instrument Type:Triple quadrupole
MS Type:ESI
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.
Ion Mode:POSITIVE
Capillary Voltage:3.3 kV
Collision Gas:Nitrogen
Source Temperature:125
Desolvation Gas Flow:800 L/h
Desolvation Temperature:400
Acquisition Parameters File:CF_antibiotics.exp
Analysis Protocol File:CF_antibiotics_quant.mdb
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