Return to study ST002283 main page

MB Sample ID: SA218536

Local Sample ID:D3_TS_A_Hilic_Neg
Subject ID:SU002369
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:61-91
Gender:Female

Select appropriate tab below to view additional metadata details:


Subject:

Subject ID:SU002369
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:61-91
Gender:Female

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
D3_TS_A_Hilic_NegSA218536FL02649661Age
D3_TS_A_Hilic_NegSA218536FL026496834PMI
D3_TS_A_Hilic_NegSA218536FL026496BurialDeposition
D3_TS_A_Hilic_NegSA218536FL026496SkeletonisedState

Collection:

Collection ID:CO002362
Collection Summary:Bone samples (ca. 1 cm3) of the anterior midshaft tibia (left) were collected prior to placement of the body outdoors, and again upon retrieval of the completely skeletonized remains (right). Each body was in “fresh” stage of decomposition when pre-placement samples were taken, and in “skeletonization” stage when post-placement samples were collected, based on scoring of the gross morphological changes37. The duration of each placement and the deposition context are reported in Table 1. The soft tissue was incised with a disposable scalpel, and a 12 V Dremel cordless lithium-ion drill with a diamond wheel drill bit was used at max. 5000 revolutions to collect ~1 cm3 of bone. Sampling instruments were cleaned with bleach and deionised water between each individual sample collection. A total of eight samples were collected in Ziploc bags, transferred immediately to a -80 °C freezer, and subsequently shipped overnight on dry ice to the Forensic Science Unit at Northumbria University, U.K. The samples were then transferred to a lockable freezer at -20 °C as per UK Human Tissue Act regulations (licence number 12495). The bone samples were defrosted, and fine powder was obtained with a Dremel drill equipped with diamond-tipped drill bits operated at speed 5000 rpms, to avoid heat damage caused by the friction with the bone. The collected powder was homogenised and stored in 2 mL protein LoBind tubes (Eppendorf UK Limited, Stevenage, UK) at -80 °C until extraction and testing. The powder sample was later divided into 25 mg aliquots. The research and bone sample analyses were reviewed and approved by the Ethics committee at Northumbria University (ref. 11623).
Sample Type:Bone
Storage Conditions:-20℃

Treatment:

Treatment ID:TR002381
Treatment Summary:Anterior midshaft tibial bone was collected from four female body donors in a fresh stage of decomposition before placement of the bodies to decompose outdoors at the human taphonomy facility managed by the Forensic Anthropological Center at Texas State (FACTS). Bone samples were again collected at selected PMIs (219, 790, 834 and 872 days)

Sample Preparation:

Sampleprep ID:SP002375
Sampleprep Summary:Chloroform (Chl), AnalaR NORMAPUR® ACS was purchased from VWR Chemicals (Lutterworth, UK). Water Optima™ LC/MS Grade, Methanol (MeOH) Optima™ LC/MS Grade, Pierce™ Acetonitrile (ACN), LC-MS Grade and Isopropanol (IPA), Optima™LC/MS Grade were purchased from Thermo Scientific (Hemel Hempstead, United Kingdom). In total two replicates for each of the six specimens were extracted according to a modified Folch et al. [17] as follow: 25 mg of bone powder was placed in tube A and 750μL of 2:1 (v/v) Chl:MeOH were added, vortexed for 30s and sonicated in ice for additional 20 min. 300μL of LC-MS grade water was added to induce phase separation and sonicate for another 15 mins. The sample were then centrifuged at 10°C for 5 mins at 2000 RPM. The respective upper and lower fractions were collected and transferred to fresh Eppendorf tubes and the samples were re-extracted with a second time using 750μL of 2:1 (v/v) Chl:MeOH. The two respective fractions were combined and concentrated. The organic lipid fraction was preconcentrated using a vacuum concentrator at 55oC for 2.5 hours or until all organic solvents has been removed. The aqueous metabolite fractions were flash frozen in liquid nitrogen and preconcentrated using a lyophilizer cold trap -65oC over night to remove all water content. The respective dry fractions were then stored at -80 until analysis. The metabolite fraction was resuspended in 100μL in 95:5 ACN/water (v/v) and sonicated for 15 mins and centrifuged for 15 min at 15K RPM at 4oC and supernatant was then transferred to 1.5mL autosampler vials with 200μL microinsert and caped. 20μL of each sample were collected and pooled to create the pooled QC. The lipid extracts were resuspended in 100μL of 1:1:2 (v/v) water:ACN:IPA and sonicated for sonicated for 15 min and centrifuged for 15 min at 15K RPM at 10oC and supernatant was then transferred to 1.5mL autosampler vials with 200μL microinsert and caped. 20μL of each sample were collected and pooled to create the pooled QC. The sample set was then submitted for analysis
Processing Storage Conditions:On ice
Extraction Method:Chloroform methanol biphasic extraction
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003728 AN003729 AN003730 AN003731
Analysis type MS MS MS MS
Chromatography type HILIC HILIC Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish Thermo Vanquish Thermo Vanquish
Column Waters Acquity BEH Amide (150 x 2.1mm,1.7um) Waters Acquity BEH Amide (150 x 2.1mm,1.7um) Waters Acquity BEH Amide (150 x 2.1mm,1.7um) Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Obitrap ID-X Tribrid Obitrap ID-X Tribrid Obitrap ID-X Tribrid Obitrap ID-X Tribrid
Ion Mode POSITIVE NEGATIVE POSITIVE NEGATIVE
Units Area integration Area integration Area integration Area integration

Chromatography:

Chromatography ID:CH002761
Chromatography Summary:Hilic ESI+
Methods Filename:AB_Human_Bone_Chromatography.pdf
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:95% B at T0 hold for 1.5 min, linear decrease to 50% B at 11 min, hold for 4 mins
Flow Rate:200 μL/min
Injection Temperature:300°C
Sample Injection:3μL
Solvent A:100% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:100% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Preconditioning:4.5 mins B
Chromatography Type:HILIC
  
Chromatography ID:CH002762
Chromatography Summary:Hilic ESI-
Methods Filename:AB_Human_Bone_Chromatography.pdf
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:95% B at T0 hold for 1.5 min, linear decrease to 50% B at 11 min, hold for 4 mins
Flow Rate:200 μL/min
Injection Temperature:300°C
Sample Injection:5μL
Solvent A:100% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:100% acetonitrile; 10 mM ammonium formate; 0.1% formic acid
Preconditioning:4.5 mins B
Chromatography Type:HILIC
  
Chromatography ID:CH002763
Chromatography Summary:RP ESI+
Methods Filename:AB_Human_Bone_Chromatography.pdf
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:60% B at T0 hold for 1.5 min, linear increase to 85% B at 7 min, increase to 95% B at 12.5 min and hold for 4.5 min
Flow Rate:200 μL/min
Injection Temperature:300°C
Sample Injection:3μL
Solvent A:40% water/60 % acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 0.1% formic acid; 10mM ammonium formate
Preconditioning:4.5 mins B
Chromatography Type:Reversed phase
  
Chromatography ID:CH002764
Chromatography Summary:RP ESI-
Methods Filename:AB_Human_Bone_Chromatography.pdf
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:45°C
Flow Gradient:60% B at T0 hold for 1.5 min, linear increase to 85% B at 7 min, increase to 95% B at 12.5 min and hold for 4.5 min
Flow Rate:200 μL/min
Injection Temperature:300°C
Sample Injection:5μL
Solvent A:40% water/60 % acetonitrile; 0.1% ammonia; 10 mM ammonium formate
Solvent B:90% isopropanol/10% acetonitrile; 0.1% ammonia solution; 10mM ammonium formate
Preconditioning:4.5 mins B
Chromatography Type:Reversed phase

MS:

MS ID:MS003476
Analysis ID:AN003728
Instrument Name:Obitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list.
Ion Mode:POSITIVE
  
MS ID:MS003477
Analysis ID:AN003729
Instrument Name:Obitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list.
Ion Mode:NEGATIVE
  
MS ID:MS003478
Analysis ID:AN003730
Instrument Name:Obitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list.
Ion Mode:POSITIVE
  
MS ID:MS003479
Analysis ID:AN003731
Instrument Name:Obitrap ID-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Mass spectrometry (MS) data were acquired using the AcquieX acquisition workflow (data dependent analysis). The MS operating parameters were as follows: MS1 mass resolution 60K, for MS2 30K, stepped energy (HCD) 20, 25, 50, scan range 100-1000, RF len (%) 35, AGC gain, intensity threshold 2e4, 25% custom injection mode with an injection time of 54 ms. An extraction blank was used to create a background exclusion list and a pooled QC was used to create the inclusion list.
Ion Mode:NEGATIVE
  logo