Summary of Study ST002025

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 PR001287. The data can be accessed directly via it's Project DOI: 10.21228/M8BX22 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002025
Study Title	Metabolomics Analysis of Blood Plasma and Stool from Six Week Flaxseed Dietary Intervention in Postmenopausal Women (Serum/GC)
Study Summary	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.
Institute	University of California, Davis
Last Name	folz
First Name	jake
Address	451 Health Science Drive
Email	jfolz@ucdavis.edu
Phone	7155636311
Submit Date	2021-12-21
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2023-01-02
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001287
Project DOI:	doi: 10.21228/M8BX22
Project Title:	Metabolomics Analysis of Blood Plasma and Stool from Six Week Flaxseed Dietary Intervention in Postmenopausal Women
Project Summary:	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.
Institute:	University of California, Davis
Last Name:	Folz
First Name:	jake
Address:	451 Health Science Drive
Email:	jfolz@ucdavis.edu
Phone:	7155636311

Subject:

Subject ID:	SU002107
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

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

mb_sample_id	local_sample_id	Treatment (pre-post)
SA189741	RS-02152009	Pre-intervention
SA189742	RS-02146133	Pre-intervention
SA189743	RS-02142009	Pre-intervention
SA189744	RS-02152490	Pre-intervention
SA189745	RS-02145607	Pre-intervention
SA189746	RS-02155235	Pre-intervention
SA189747	RS-02162522	Pre-intervention
SA189748	RS-02159492	Pre-intervention
SA189749	RS-02156423	Pre-intervention
SA189750	RS-02139721	Pre-intervention
SA189751	RS-02153108	Pre-intervention
SA189752	RS-02138650	Pre-intervention
SA189753	RS-02128352	Pre-intervention
SA189754	RS-02127671	Pre-intervention
SA189755	RS-02126796	Pre-intervention
SA189756	RS-02124349	Pre-intervention
SA189757	RS-02129862	Pre-intervention
SA189758	RS-02135566	Pre-intervention
SA189759	RS-02137274	Pre-intervention
SA189760	RS-02136527	Pre-intervention
SA189761	RS-02136451	Pre-intervention
SA189762	RS-02163042	Pre-intervention
SA189763	RS-02165754	Pre-intervention
SA189764	RS-02184819	Pre-intervention
SA189765	RS-02180401	Pre-intervention
SA189766	RS-02179400	Pre-intervention
SA189767	RS-02177759	Pre-intervention
SA189768	RS-02185095	Pre-intervention
SA189769	RS-02186113	Pre-intervention
SA189770	RS-02193435	Pre-intervention
SA189771	RS-02189574	Pre-intervention
SA189772	RS-02186657	Pre-intervention
SA189773	RS-02176958	Pre-intervention
SA189774	RS-02174862	Pre-intervention
SA189775	RS-02169817	Pre-intervention
SA189776	RS-02168982	Pre-intervention
SA189777	RS-02167382	Pre-intervention
SA189778	RS-02166761	Pre-intervention
SA189779	RS-02171890	Pre-intervention
SA189780	RS-02173280	Pre-intervention
SA189781	RS-02174609	Pre-intervention
SA189782	RS-02173913	Pre-intervention
SA189783	RS-02173508	Pre-intervention
SA189784	RS-02119140	Pre-intervention
SA189785	RS-02118840	Pre-intervention
SA189786	RS-02046599	Pre-intervention
SA189787	RS-02045370	Pre-intervention
SA189788	RS-02041308	Pre-intervention
SA189789	RS-02040155	Pre-intervention
SA189790	RS-02047137	Pre-intervention
SA189791	RS-02049971	Pre-intervention
SA189792	RS-02055157	Pre-intervention
SA189793	RS-02052504	Pre-intervention
SA189794	RS-02051836	Pre-intervention
SA189795	RS-02037571	Pre-intervention
SA189796	RS-02037282	Pre-intervention
SA189797	RS-02020293	Pre-intervention
SA189798	RS-02019510	Pre-intervention
SA189799	RS-02017643	Pre-intervention
SA189800	RS-01975659	Pre-intervention
SA189801	RS-02022737	Pre-intervention
SA189802	RS-02023824	Pre-intervention
SA189803	RS-02029507	Pre-intervention
SA189804	RS-02027065	Pre-intervention
SA189805	RS-02024202	Pre-intervention
SA189806	RS-02055215	Pre-intervention
SA189807	RS-02057574	Pre-intervention
SA189808	RS-02099922	Pre-intervention
SA189809	RS-02099604	Pre-intervention
SA189810	RS-02096868	Pre-intervention
SA189811	RS-02096722	Pre-intervention
SA189812	RS-02101117	Pre-intervention
SA189813	RS-02107455	Pre-intervention
SA189814	RS-02116664	Pre-intervention
SA189815	RS-02108921	Pre-intervention
SA189816	RS-02108617	Pre-intervention
SA189817	RS-02095379	Pre-intervention
SA189818	RS-02091843	Pre-intervention
SA189819	RS-02062802	Pre-intervention
SA189820	RS-02061579	Pre-intervention
SA189821	RS-02061501	Pre-intervention
SA189822	RS-02060161	Pre-intervention
SA189823	RS-02080484	Pre-intervention
SA189824	RS-02080521	Pre-intervention
SA189825	RS-02089132	Pre-intervention
SA189826	RS-02088574	Pre-intervention
SA189827	RS-02082111	Pre-intervention
SA189828	RS-02198195	Pre-intervention
SA189829	RS-02203857	Pre-intervention
SA189830	RS-02353557	Pre-intervention
SA189831	RS-02349825	Pre-intervention
SA189832	RS-02349202	Pre-intervention
SA189833	RS-02348475	Pre-intervention
SA189834	RS-02353973	Pre-intervention
SA189835	RS-02505087	Pre-intervention
SA189836	RS-02621406	Pre-intervention
SA189837	RS-02620835	Pre-intervention
SA189838	RS-02613837	Pre-intervention
SA189839	RS-02583622	Pre-intervention
SA189840	RS-02348427	Pre-intervention

Showing page 1 of 4 Results: 1 2 3 4 Next Showing results 1 to 100 of 356

Collection:

Collection ID:	CO002100
Collection Summary:	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR002119
Treatment Summary:	Samples were taken before and after 6 weeks of flaxseed dietary intervention. Serum and fecal metabolomics were performed to characterize the metabolomic profiles of serum and stool metabolites pre- and post-intervention.

Sample Preparation:

Sampleprep ID:	SP002113
Sampleprep Summary:	Sample preparation of blood plasma or serum samples for CSH, HILIC and GC analysis Purpose: This SOP describes sample extraction and preparation of blood plasma or serum for lipid profiling on the CSH, and HILIC platform by liquid chromatography/ mass spectrometry (LC-MS) as well as primary metabolomics platform on GC/MS. This method is to be used when there is low sample volume for separate extractions, and when more than one platform is to be used in a project. References: Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A and Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lip Res 2008, 49: 1137-1146 Starting material: Plasma/serum: 20 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 20-200µL and 100-1000µL Multi-Tube Vortexer (VWR VX-2500) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Chemicals: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Eppendorf tubes 2 mL, uncolored Eppendorf 022363352 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 MTBE, HPLC Grade Acros Organics 389050010 Methanol, LC/MS Grade Fisher A456-4 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Acetonitrile, HPLC Grade Fisher Optima A955-4 Iso-Propanol, HPLC Grade Fisher A461-4 Sample Preparation: Preparation of extraction solvent Combine 120 mL of chilled MeOH/QC mix with 400 mL of chilled MTBE/Cholesterol Ester 22:1 in a clean 500 mL stock bottle. Mix thoroughly by swirling or stir plate and store at -20°C until use. *See SOP “QC mix for LC-MS lipid analysis” for preparation of MeOH/QC mix and MTBE/Cholesterol Ester 22:1. Preparation of Clean Up solvent For 1 L of extraction solvent, combine 375 mL of acetonitrile, 375 mL of isopropanol, and 250 mL water in a 1 L bottle conditioned with the aforementioned chemicals. If a different total volume of extraction solvent is needed, simply mix acetonitrile, isopropanol, and water in volumes in proportion 3:3:2. Purge the extraction solution mix for 5 min with nitrogen with small bubbles. Make sure that the nitrogen line is flushed out of air before using it for degassing the extraction solvent solution. Store at -20°C until use. Note: if solvent freezes, sonicate until thawed and mix before use. Extraction Thaw raw samples/controls at room temperature (or in the refrigerator at 4˚C) and either invert the tube or vortex 10 sec at low speed to homogenize. Aliquot 20 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 975 µL ice-cold 3:10 (v/v) MeOH/MTBE + QC mix/CE 22:1 extraction solvent mixture to each aliquot, keeping the extraction solvent on ice during the procedure. Vortex samples for 10 seconds, then shake for 5 minutes at 4°C on the orbital mixer. Add 188 µL room temperature LC/MS grade water to each tube. Vortex tubes for 20 seconds and then centrifuge for 2 min at 14,000 rcf. Transfer the upper organic phase to two separate tubes (350 µL/each tube) for lipidomics analysis. Transfer 75 µL of the remaining organic phase to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Transfer the bottom aqueous phase to two separate tubes (110 µL/each tube) for HILIC/GC-TOF analysis. Dry down one tube from each phase by centrivap, keeping the undried tubes as backups. Store all tubes at -20˚C until ready for analysis. Clean up step for GC only (and pooling) Resuspend the dried aliquot with 500 μL 3:3:2 (v/v/v) ACN:IPA:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 450 uL supernatant to a clean 1.5 mL eppendorf tube. Tranfering remainder to a 2, 15, 50 mL Tube, dependent on number of samples. Aliquot out 1.9 mL uL of supernatant to new 2ml eppendorf tubes. Centrifuge for 2 min at 14000 rcf Aliquot out 4x450 uL of supernatant into clean 1.5 mL Eppendorf tubes. Evaporate to comeplete dryness in the Labconco Centruvap cold trap concentrator. Submit to derivatization . Pooling (CSH platform only) Transfer multiple 350 µL aliquots of pooled samples to 1.5 mL Eppendorf tubes, one aliquot for every 10 samples in the study. If there is still pool remaining, prepare additional aliquots for backup. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Store all tubes at -20°C until ready for analysis. Quality assurance For every 10 samples, extract a method blank (20 µL of H2O) and a sample control (20 µL human Bioreclamation or analogous species plasma) in addition to samples. For large studies (>100 samples), for every 100 samples a NIST plasma extract should be prepared in the same manner as positive controls. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Collect residual plasma/serum samples in specifically designed red ‘biohazard’ waste bags. Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Sampleprep ID:

SP002113

Sampleprep Summary:

Sample preparation of blood plasma or serum samples for CSH, HILIC and GC analysis Purpose: This SOP describes sample extraction and preparation of blood plasma or serum for lipid profiling on the CSH, and HILIC platform by liquid chromatography/ mass spectrometry (LC-MS) as well as primary metabolomics platform on GC/MS. This method is to be used when there is low sample volume for separate extractions, and when more than one platform is to be used in a project. References: Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A and Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. J Lip Res 2008, 49: 1137-1146 Starting material: Plasma/serum: 20 µL sample volume or aliquot Equipment: Centrifuge Eppendorf 5415 D Calibrated pipettes 20-200µL and 100-1000µL Multi-Tube Vortexer (VWR VX-2500) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Chemicals: Product Manufacturer & Part Number Eppendorf tubes 1.5 mL, uncolored Eppendorf 022363204 Eppendorf tubes 2 mL, uncolored Eppendorf 022363352 Crushed ice UC Davis Water, LC/MS Grade Fisher Optima W6-4 MTBE, HPLC Grade Acros Organics 389050010 Methanol, LC/MS Grade Fisher A456-4 Bioreclamation human plasma (disodium EDTA) Bioreclamation HMPLEDTA Acetonitrile, HPLC Grade Fisher Optima A955-4 Iso-Propanol, HPLC Grade Fisher A461-4 Sample Preparation: Preparation of extraction solvent Combine 120 mL of chilled MeOH/QC mix with 400 mL of chilled MTBE/Cholesterol Ester 22:1 in a clean 500 mL stock bottle. Mix thoroughly by swirling or stir plate and store at -20°C until use. *See SOP “QC mix for LC-MS lipid analysis” for preparation of MeOH/QC mix and MTBE/Cholesterol Ester 22:1. Preparation of Clean Up solvent For 1 L of extraction solvent, combine 375 mL of acetonitrile, 375 mL of isopropanol, and 250 mL water in a 1 L bottle conditioned with the aforementioned chemicals. If a different total volume of extraction solvent is needed, simply mix acetonitrile, isopropanol, and water in volumes in proportion 3:3:2. Purge the extraction solution mix for 5 min with nitrogen with small bubbles. Make sure that the nitrogen line is flushed out of air before using it for degassing the extraction solvent solution. Store at -20°C until use. Note: if solvent freezes, sonicate until thawed and mix before use. Extraction Thaw raw samples/controls at room temperature (or in the refrigerator at 4˚C) and either invert the tube or vortex 10 sec at low speed to homogenize. Aliquot 20 μL of plasma sample into a 1.5 mL Eppendorf tube. Keep all samples on ice. Add 975 µL ice-cold 3:10 (v/v) MeOH/MTBE + QC mix/CE 22:1 extraction solvent mixture to each aliquot, keeping the extraction solvent on ice during the procedure. Vortex samples for 10 seconds, then shake for 5 minutes at 4°C on the orbital mixer. Add 188 µL room temperature LC/MS grade water to each tube. Vortex tubes for 20 seconds and then centrifuge for 2 min at 14,000 rcf. Transfer the upper organic phase to two separate tubes (350 µL/each tube) for lipidomics analysis. Transfer 75 µL of the remaining organic phase to a 2, 15, or 50 mL tube for pools, depending on number of samples in the study. Transfer the bottom aqueous phase to two separate tubes (110 µL/each tube) for HILIC/GC-TOF analysis. Dry down one tube from each phase by centrivap, keeping the undried tubes as backups. Store all tubes at -20˚C until ready for analysis. Clean up step for GC only (and pooling) Resuspend the dried aliquot with 500 μL 3:3:2 (v/v/v) ACN:IPA:H2O (degassed as given above) and vortex for about 10 sec. Centrifuge for 2 min at 14000 rcf. Remove 450 uL supernatant to a clean 1.5 mL eppendorf tube. Tranfering remainder to a 2, 15, 50 mL Tube, dependent on number of samples. Aliquot out 1.9 mL uL of supernatant to new 2ml eppendorf tubes. Centrifuge for 2 min at 14000 rcf Aliquot out 4x450 uL of supernatant into clean 1.5 mL Eppendorf tubes. Evaporate to comeplete dryness in the Labconco Centruvap cold trap concentrator. Submit to derivatization . Pooling (CSH platform only) Transfer multiple 350 µL aliquots of pooled samples to 1.5 mL Eppendorf tubes, one aliquot for every 10 samples in the study. If there is still pool remaining, prepare additional aliquots for backup. Evaporate to complete dryness in the Labconco Centrivap cold trap concentrator. Store all tubes at -20°C until ready for analysis. Quality assurance For every 10 samples, extract a method blank (20 µL of H2O) and a sample control (20 µL human Bioreclamation or analogous species plasma) in addition to samples. For large studies (>100 samples), for every 100 samples a NIST plasma extract should be prepared in the same manner as positive controls. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules. Collect residual plasma/serum samples in specifically designed red ‘biohazard’ waste bags. Extraction of Mammalian Tissue Samples: Liver 1. References: Fiehn O, Kind T (2006) Metabolite profiling in blood plasma. In: Metabolomics: Methods and Protocols. Weckwerth W (ed.), Humana Press, Totowa NJ (in press) 2.Starting material: Liver sample: weigh 4mg per sample into 2mL Eppendorf tubes. 3. Equipment: Centrifuge (Eppendorf 5415 D) Calibrated pipettes 1-200μl and 100-1000μl Eppendorf tubes 2mL, clear (Cat. No. 022363204) Centrifuge tubes 50mL, polypropylene Eppendorff Tabletop Centrifuge (Proteomics core Lab.) ThermoElectron Neslab RTE 740 cooling bath at –20°C MiniVortexer (VWR) Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) Speed vacuum concentration system (Labconco Centrivap cold trap) Turex mini homogenizer 4. Chemicals Acetonitrile, LCMS grade (JT Baker; Cat. No.9829-02) Isopropanol, HPLC grade (JT Baker; Cat. No. 9095-02) Methanol Acetone Crushed ice 18 MΩ pure water (Millipore) Nitrogen line with pipette tip pH paper 5-10 (EMD Chem. Inc.) 5. Procedure Preparation of extraction mix and material before experiment: Switch on bath to pre-cool at –20°C (±2°C validity temperature range) Check pH of acetonitrile and isopropanol (pH7) using wetted pH paper Make the extraction solution by mixing acetonitrile, isopropanol and water in proportions 3 : 3 : 2 De-gas the extraction solution for 5 min with nitrogen. Make sure that the nitrogen line was flushed out of air before using it for degassing the extraction solvent solution Sample Preparation Weigh 4mg tissue sample in to a 2mL Eppendorf tube. Add 1mL extraction solvent to the tissue sample and homogenize for 45 seconds ensuring that sample resembles a powder. In between samples, clean the homogenizer in solutions of methanol, acetone, water, and the extraction solvent in the order listed. Vortex samples for 10 seconds, then 5 minutes on 4°C shaker. Centrifuge the samples for 2 minutes at 14,000 rcf. Aliquot 500µL supernatant for analysis, and 500µL for a backup. Store backup aliquots in the -20°C freezer. Evaporate one 500µl analysis aliquot in the Labconco Centrivap cold trap concentrator to complete dryness (typically overnight). The dried aliquot is then re-suspended with 500μl 50% acetonitrile (degassed as given) Centrifuge for 2 minutes at 14,000 rcf using the centrifuge Eppendorf 5415. Remove supernatant to a new Eppendorf tube. Evaporate the supernatant to dryness in the the Labconco Centrivap cold trap concentrator. Submit to derivatization. The residue should contain membrane lipids because these are supposedly not soluble enough to be found in the 50% acetonitrile solution. Therefore, this ‘membrane residue’ is now taken for membrane lipidomic fingerprinting using the nanomate LTQ ion trap mass spectrometer. Likely, a good solvent to redissolve the membrane lipids is e.g. 75% isopropanol (degassed as given above). If the ‘analysis’ aliquot is to be used for semi lipophilic compounds such as tyrosine pathway intermediates (incl. dopamine, serotonine etc, i.e. polar aromatic compounds), then these are supposedly to be found together with the ‘GCTOF’ aliquot. We can assume that this mixture is still too complex for Agilent chipLCMS. Therefore, in order to develop and validate target analysis for such aromatic compounds, we should use some sort of Solid Phase purification. We re-suspend the dried ‘GCTOF’ aliquot in 300 l water (degassed as before) to take out sugars, aliphatic amino acids, hydroxyl acids and similar logP compounds. The residue should contain our target aromatics .We could also try to adjust pH by using low concentration acetate or phosphate buffer. The residue could then be taken up in 50% acetonitrile and used for GCTOF and Agilent chipMS experiments. The other aliquot should be checked how much of our target compounds would actually be found in the ‘sugar’ fraction. 6. Problems To prevent contamination disposable material is used. Control pH from extraction mix. 7. Quality assurance For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample. 8. Disposal of waste Collect all chemicals in appropriate bottles and follow the disposal rules.

Combined analysis:

Analysis ID	AN003295
Analysis type	MS
Chromatography type	GC
Chromatography system	Leco Pegasus IV GC
Column	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Leco Pegasus IV TOF
Ion Mode	UNSPECIFIED
Units	normalized peak height

Chromatography:

Chromatography ID:	CH002434
Instrument Name:	Leco Pegasus IV GC
Column Name:	Restek Rtx-5Sil (30m x 0.25mm,0.25um)
Chromatography Type:	GC

MS:

MS ID:	MS003065
Analysis ID:	AN003295
Instrument Name:	Leco Pegasus IV TOF
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	Gas Chromatography conditions: A 30 m long, 0.25 mm i.d. Rtx-5Sil MS column (0.25 μm 95% dimethyl 5% diphenyl polysiloxane film) with additional 10 m integrated guard column is used (Restek, Bellefonte PA). 99.9999% pure Helium with built-in purifier (Airgas, Radnor PA) is set at constant flow of 1 ml/min. The oven temperature is held constant at 50°C for 1 min and then ramped at 20°C/min to 330°C at which it is held constant for 5 min. Mass spectrometer settings: A Leco Pegasus IV time of flight mass spectrometer is controlled by the Leco ChromaTOF software vs. 2.32 (St. Joseph, MI). The transfer line temperature between gas chromatograph and mass spectrometer is set to 280°C. Electron impact ionization at 70V is employed with an ion source temperature of 250°C. Acquisition rate is 17 spectra/second, with a scan mass range of 85-500 Da.
Ion Mode:	UNSPECIFIED