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MB Sample ID: SA198209

Local Sample ID:D0D1_A-63_2018-03-28
Subject ID:SU002166
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:47-89 years
Gender:Male and female
Human Inclusion Criteria:Patients with lung cancer

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:SU002166
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:47-89 years
Gender:Male and female
Human Inclusion Criteria:Patients with lung cancer

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
D0D1_A-63_2018-03-28SA198209FL0240921Time before death (weeks)

Collection:

Collection ID:CO002159
Collection Summary:The study was conducted at six sites (hospitals and hospices) in the North West of England (UK) from June 2016 to September 2018. Patients with lung cancer were recruited prospectively and multiple urine samples were collected up to three times a week while an inpatient. Ethical approval was provided by North Wales (West) Research Ethics Committee (REC reference 15/WA/0464). Research team members collected 20 mL of urine from participants in a universal container. For those participants with a urinary catheter, the urine was collected using a needle and syringe from the catheter port. The samples were stored on site in a locked freezer at −20°C. An anonymised record of the medication administered was collected. Collection protocol described previously by Coyle et al. (BMJ Open. 2016; 6(11) e011763. doi: 10.1136/bmjopen-2016-011763).
Sample Type:Urine
Volumeoramount Collected:20 mL
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002178
Treatment Summary:Clinical observational study. Serial urine samples collected from patients with lung cancer in a palliative care setting at varying time points up until death; >12 weeks - 1 week before death (see 'study design' information). Although multiple samples were collected from patients, only the final sample was included in the analysis.

Sample Preparation:

Sampleprep ID:SP002172
Sampleprep Summary:Individual patient samples were thawed at room temperature, vortexed and separated into four replicate aliquots in individual 96-well plates (Waters, UK) which were stored at -80 °C until analysis by one of four different methods; two different chromatography conditions in negative and positive ionisation polarity. Pooled quality control samples were created following the protocol described by Norman et al. (Clin Chem. 2019;65(4):530-39. doi: 10.1373/clinchem.2018.295345). For each sample group (time before death), a separate representative pool was created by pooling an equal volume of each individual urine sample for quality control purposes. An overall pool was also created by pooling equal proportions of the above group pools. Analysis of individual and pooled samples was performed following dilution of 1:3 urine:deionised water (DIRECT-Q 3UV Millipore water purification system) as previously described by Norman et al. (2019).

Combined analysis:

Analysis ID AN003396 AN003397 AN003398 AN003399
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase HILIC HILIC
Chromatography system Agilent 6550 Agilent 6550 Agilent 6550 Agilent 6550
Column Waters Atlantis dC18 (100 x 3mm,3um) Waters Atlantis dC18 (100 x 3mm,3um) Waters BEH Amide (150 x 3.0mm,1.7um) Waters BEH Amide (150 x 3.0mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type QTOF QTOF QTOF QTOF
MS instrument name Agilent 6550 QTOF Agilent 6550 QTOF Agilent 6550 QTOF Agilent 6550 QTOF
Ion Mode NEGATIVE POSITIVE NEGATIVE POSITIVE
Units Values are raw peak area raw peak area raw peak area Values are raw peak area

Chromatography:

Chromatography ID:CH002511
Chromatography Summary:LC method 1: employed an Atlantis dC18 column (3x100 mm, 3 µm, Waters, UK) maintained at 60 °C with flow rate at 0.4 mL/min. Mobile phases were (A) water and (B) methanol both containing 5 mmol/L ammonium formate and 0.1 % formic acid. The elution gradient started at 5 % B at 0 to 1 min increasing linearly to 100 % by 12 min, held at 100 % B until 14 min, returning to 95 % A for 5 min.
Instrument Name:Agilent 6550
Column Name:Waters Atlantis dC18 (100 x 3mm,3um)
Column Temperature:60
Flow Gradient:The elution gradient started at 5 % B at 0 to 1 min increasing linearly to 100 % by 12 min, held at 100 % B until 14 min, returning to 95 % A for 5 min
Flow Rate:0.4 mL/min
Solvent A:100% water; 0.1 % formic acid; 5 mM ammonium formate
Solvent B:100% methanol; 0.1 % formic acid; 5 mM ammonium formate
Chromatography Type:Reversed phase
  
Chromatography ID:CH002512
Chromatography Summary:LC method 2: used a BEH amide column (3x150 mm, 1.7 µm, Waters, UK) maintained at 40 °C with flow rate at 0.6 mL/min. Mobile phases were (A) water and (B) acetonitrile both containing 0.1 % formic acid. The elution gradient started at 99 % B, decreasing linearly to 30 % from 1 to 12 min, held at 30 % B until 12.6 min, returning to 99 % B for 3.4 min. Sample injection volume was 1 µL for both LC methods.
Instrument Name:Agilent 6550
Column Name:Waters BEH Amide (150 x 3.0mm,1.7um)
Column Temperature:40
Flow Gradient:The elution gradient started at 99% B, decreasing linearly to 30% from 1 to 12 min, held at 30% B until 12.6 min, returning to 99% B for 3.4 min.
Flow Rate:0.6 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B: 100% acetonitrile; 0.1% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS003163
Analysis ID:AN003396
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry conditions: The mass spectrometer was tuned and calibrated according to protocols recommended by the manufacturer. Acquisition was performed in 2 GHz mode and mass range 50-1700. The capillary voltage was 4000 V and fragmentor voltage 380 V. The desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min. The nebulizer pressure was 40 psig and nozzle voltage 1000 V (± for positive and negative ionisation modes). The acquisition rate was 3 spectra/second. The reference mass solution was continually infused at a flow rate of 0.5 mL/min by a separate isocratic pump for constant mass correction. Repeat injections of each pooled sample were interspersed throughout the analytical sequence, as per the quality control procedure described by Norman et al. (Clin Chem. 2019;65(4):530-539. doi: 10.1373/clinchem.2018.295345). The individual sample analysis order was randomised computationally. Data Pre-processing and Quality Control: All data were acquired using the MassHunter suite (Agilent build 6.0) with quality checks being performed by Qualitative Analysis (build 07.00). Mass accuracy was checked using extracted ion chromatograms of reference masses: the resulting accuracy was ±5 ppm during the run. Additionally, chromatographic reproducibility was checked by overlaying binary pump pressure curves across each analytical sequence. Data was filtered based upon the pooled QC samples, with compounds being retained if observed in 100 % of replicate injections for at least 1 pool and with peak area coefficient of variation (CV) <25 % across all replicate injections for each pool. A comprehensive semi-targeted approach was employed to assign the identity of urinary metabolites using an in-house compound library that included a broad range of metabolites involved in intermediary metabolism. Targeted feature extraction was performed on each dataset based on matching of metabolite chemical features against an accurate mass and retention time (AMRT) database previously generated from analysis of the IROA Technology MS metabolite library of tandards by each LC method described above, combined with the same QTOF analytical parameters used in this study (databases publicly available: https://doi.org/10.6084/m9.figshare.c.4378235.v2). In addition to accurate mass and retention time, MS/MS (data-dependent, employed for 'hit' metabolites) was also used in the confirmation of metabolite identity (i.e. level 1 identification) as per Sumner et al. Feature extraction was performed in MassHunter Profinder (build 10.0); accurate mass window of 10 ppm and retention time window of 0.3 min against the respective AMRT database. Data were exported in the format of a csv file for statistical analysis. Unidentified compounds are 'known unknowns' of interest from previous experiments; detection by matched AMRT and named in the format 'RT_neutral-mass'. 'BDRM'; unidentified bone-derived metabolite.
Ion Mode:NEGATIVE
  
MS ID:MS003164
Analysis ID:AN003397
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry conditions: The mass spectrometer was tuned and calibrated according to protocols recommended by the manufacturer. Acquisition was performed in 2 GHz mode and mass range 50-1700. The capillary voltage was 4000 V and fragmentor voltage 380 V. The desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min. The nebulizer pressure was 40 psig and nozzle voltage 1000 V (± for positive and negative ionisation modes). The acquisition rate was 3 spectra/second. The reference mass solution was continually infused at a flow rate of 0.5 mL/min by a separate isocratic pump for constant mass correction. Repeat injections of each pooled sample were interspersed throughout the analytical sequence, as per the quality control procedure described by Norman et al. (Clin Chem. 2019;65(4):530-539. doi: 10.1373/clinchem.2018.295345). The individual sample analysis order was randomised computationally. Data Pre-processing and Quality Control: All data were acquired using the MassHunter suite (Agilent build 6.0) with quality checks being performed by Qualitative Analysis (build 07.00). Mass accuracy was checked using extracted ion chromatograms of reference masses: the resulting accuracy was ±5 ppm during the run. Additionally, chromatographic reproducibility was checked by overlaying binary pump pressure curves across each analytical sequence. Data was filtered based upon the pooled QC samples, with compounds being retained if observed in 100 % of replicate injections for at least 1 pool and with peak area coefficient of variation (CV) <25 % across all replicate injections for each pool. A comprehensive semi-targeted approach was employed to assign the identity of urinary metabolites using an in-house compound library that included a broad range of metabolites involved in intermediary metabolism. Targeted feature extraction was performed on each dataset based on matching of metabolite chemical features against an accurate mass and retention time (AMRT) database previously generated from analysis of the IROA Technology MS metabolite library of tandards by each LC method described above, combined with the same QTOF analytical parameters used in this study (databases publicly available: https://doi.org/10.6084/m9.figshare.c.4378235.v2). In addition to accurate mass and retention time, MS/MS (data-dependent, employed for 'hit' metabolites) was also used in the confirmation of metabolite identity (i.e. level 1 identification) as per Sumner et al. Feature extraction was performed in MassHunter Profinder (build 10.0); accurate mass window of 10 ppm and retention time window of 0.3 min against the respective AMRT database. Data were exported in the format of a csv file for statistical analysis. Unidentified compounds are 'known unknowns' of interest from previous experiments; detection by matched AMRT and named in the format 'RT_neutral-mass'. 'BDRM'; unidentified bone-derived metabolite.
Ion Mode:POSITIVE
  
MS ID:MS003165
Analysis ID:AN003398
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry conditions: The mass spectrometer was tuned and calibrated according to protocols recommended by the manufacturer. Acquisition was performed in 2 GHz mode and mass range 50-1700. The capillary voltage was 4000 V and fragmentor voltage 380 V. The desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min. The nebulizer pressure was 40 psig and nozzle voltage 1000 V (± for positive and negative ionisation modes). The acquisition rate was 3 spectra/second. The reference mass solution was continually infused at a flow rate of 0.5 mL/min by a separate isocratic pump for constant mass correction. Repeat injections of each pooled sample were interspersed throughout the analytical sequence, as per the quality control procedure described by Norman et al. (Clin Chem. 2019;65(4):530-539. doi: 10.1373/clinchem.2018.295345). The individual sample analysis order was randomised computationally. Data Pre-processing and Quality Control: All data were acquired using the MassHunter suite (Agilent build 6.0) with quality checks being performed by Qualitative Analysis (build 07.00). Mass accuracy was checked using extracted ion chromatograms of reference masses: the resulting accuracy was ±5 ppm during the run. Additionally, chromatographic reproducibility was checked by overlaying binary pump pressure curves across each analytical sequence. Data was filtered based upon the pooled QC samples, with compounds being retained if observed in 100 % of replicate injections for at least 1 pool and with peak area coefficient of variation (CV) <25 % across all replicate injections for each pool. A comprehensive semi-targeted approach was employed to assign the identity of urinary metabolites using an in-house compound library that included a broad range of metabolites involved in intermediary metabolism. Targeted feature extraction was performed on each dataset based on matching of metabolite chemical features against an accurate mass and retention time (AMRT) database previously generated from analysis of the IROA Technology MS metabolite library of tandards by each LC method described above, combined with the same QTOF analytical parameters used in this study (databases publicly available: https://doi.org/10.6084/m9.figshare.c.4378235.v2). In addition to accurate mass and retention time, MS/MS (data-dependent, employed for 'hit' metabolites) was also used in the confirmation of metabolite identity (i.e. level 1 identification) as per Sumner et al. Feature extraction was performed in MassHunter Profinder (build 10.0); accurate mass window of 10 ppm and retention time window of 0.3 min against the respective AMRT database. Data were exported in the format of a csv file for statistical analysis.
Ion Mode:NEGATIVE
  
MS ID:MS003166
Analysis ID:AN003399
Instrument Name:Agilent 6550 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Mass spectrometry conditions: The mass spectrometer was tuned and calibrated according to protocols recommended by the manufacturer. Acquisition was performed in 2 GHz mode and mass range 50-1700. The capillary voltage was 4000 V and fragmentor voltage 380 V. The desolvation gas temperature was 200 °C with flow rate at 15 L/min. The sheath gas temperature was 300 °C with flow rate at 12 L/min. The nebulizer pressure was 40 psig and nozzle voltage 1000 V (± for positive and negative ionisation modes). The acquisition rate was 3 spectra/second. The reference mass solution was continually infused at a flow rate of 0.5 mL/min by a separate isocratic pump for constant mass correction. Repeat injections of each pooled sample were interspersed throughout the analytical sequence, as per the quality control procedure described by Norman et al. (Clin Chem. 2019;65(4):530-539. doi: 10.1373/clinchem.2018.295345). The individual sample analysis order was randomised computationally. Data Pre-processing and Quality Control: All data were acquired using the MassHunter suite (Agilent build 6.0) with quality checks being performed by Qualitative Analysis (build 07.00). Mass accuracy was checked using extracted ion chromatograms of reference masses: the resulting accuracy was ±5 ppm during the run. Additionally, chromatographic reproducibility was checked by overlaying binary pump pressure curves across each analytical sequence. Data was filtered based upon the pooled QC samples, with compounds being retained if observed in 100 % of replicate injections for at least 1 pool and with peak area coefficient of variation (CV) <25 % across all replicate injections for each pool. A comprehensive semi-targeted approach was employed to assign the identity of urinary metabolites using an in-house compound library that included a broad range of metabolites involved in intermediary metabolism. Targeted feature extraction was performed on each dataset based on matching of metabolite chemical features against an accurate mass and retention time (AMRT) database previously generated from analysis of the IROA Technology MS metabolite library of tandards by each LC method described above, combined with the same QTOF analytical parameters used in this study (databases publicly available: https://doi.org/10.6084/m9.figshare.c.4378235.v2). In addition to accurate mass and retention time, MS/MS (data-dependent, employed for 'hit' metabolites) was also used in the confirmation of metabolite identity (i.e. level 1 identification) as per Sumner et al. Feature extraction was performed in MassHunter Profinder (build 10.0); accurate mass window of 10 ppm and retention time window of 0.3 min against the respective AMRT database. Data were exported in the format of a csv file for statistical analysis.
Ion Mode:POSITIVE
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