TITLE: Metabolite Extraction and Derivatization of Plasma/ Serum Samples for High Resolution GC-MS- based Metabolomics
AUTHORS: Biswapriya Misra, Michael Olivier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This protocol provides details on preparation of human and non-human primate blood plasma/ serum extraction, and derivatization for GC-MS based metabolomics data acquisition. </span></div></div>

[STEPS]
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
Freshly thawed aliquots of serum or plasma  (30 µL) samples from non-human primate (i.e., baboons, vervets) are subjected to sequential solvent  extraction once each with 1 mL of acetonitrile: isopropanol: water  (3:3:2, v/v ratio) stord in -20 C  and 500 µL of acetonitrile: water (1:1, v/v) ratio  mixtures stored at 4 °C.22
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
Adonitol  (2 µL from 10 mg/mL stock in methanol, stored in -20 C) is added to each aliquots as  internal standard to the sample tube prior to the start of  extraction.
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
The pooled extracts (~ 1500 µL) from the two steps are  dried under vacuum at 4 °C prior to chemical derivatization.
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
Dummy  extractions are also performed on blank microcentrifuge tubes that serve as extraction blanks to  account for background (extraction solvent, derivatization reagents)  noise and other sources of contamination (septa, liner, column, vials,  handling etc.).
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
Blanks are only used to see that no carry overs  occurred during randomized run orders and to manually filter out  contaminating chemicals from the combined list of features.
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
The steps involve the addition of 10 μL of MeOX (20 mg/mL stock in pyridine, freshly prepared and stable for a week at maximum) in pyridine  incubated under shaking at 55 °C for 60 min followed by  trimethylsilylation at 60 °C for 60 min after adding 90 μL MSTFA.
?. [PROTOCOL OVERVIEW BASED ON Lisec et al., 2006, Fiehn , 2016]
Dried samples are then sequentially derivatized with  methoxyamine hydrochloride (MeOX) and 1% TMCS in N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) as described elsewhere.23, 24
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Prepare methoxyamine (MeOX, Sigma/ Supelco): Prepare a 20 mg/ml MeOX solution in Pyridine; vortex and then Thermomix at 60 °C for 15 min to dissolve; 1 ml is good for a week only if stored in desiccator at room temperature.
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Prepare Adonitol ( synonym, ribitol)  (Sigma) stock: 2 mg/ ml (Prepare 10 ml, use 20 mg in methanol, i.e., 20 mg/ ml): Store in −20°C for months! Used to (i) monitor extraction efficiency, and (ii) as an internal standard to normalize peak area.
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Prepare Extraction Solvent I: 3:3:2 (v/v/v) acetonitrile/isopropanol/water: Make 100 ml (or, as needed): Store in −20°C
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Prepare Extraction Solvent II: 1:1 (v/v) acetonitrile/water : Make 50 ml (or, as needed) : Store in -20 °
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Retrieve plasma/ serum samples form -80 C freezer on to ice/ dry ice. Can take 30 min to 1 hr for thawing.
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Add 2.5µl adonitol (from stock) to each ‘thawed’ sample (i.e., 30 µL plasma/ serum) kept on ice. Hence forth, called "sample".
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Add 1 ml cooled extraction solvent to one 1.5 mL microfuge tube containing an aliquot of the sample (30 µl) and ‘dummy’ control (B1, empty tube).
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The rest of the tubes should be kept ice chilled (dry ice) at  4°C on ice.
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Vigorously vortex for 10 sec and on Thermomixer shake for 5 min at 4°C (1000 rpm shaking needed)
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Centrifuge for 2 min at 14,000 × g, room temperature.
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Aliquot two (x2) 450 μl portions of the supernatant to separate tubes: one for analysis and one for a backup in -80 C.
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Evaporate both aliquots to complete dryness using a speed vacuum concentrator. Preferably operated at 4 C. Store the backup aliquot at −20°C for up to 4 weeks.
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Resuspend the other dried aliquot in 450 μL nitrogen-degassed 50:50 (v/v) acetonitrile/water at room temperature.
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Centrifuge for 2 min at 14,000 × g, room temperature.
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Transfer supernatant (400 μL) to a fresh 1.5-ml polypropylene microcentrifuge tube and evaporate to dryness in a Speedvac evaporator.
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Add 10 µl MeOX solution to samples [Stock 20 mg/ ml in Pyridine]. Slow vortex for 10 sec.
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Use Thermomixer to heat for 1 hr at 55°C. (no shaking needed)
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Add 90 μl MSTFA[ Sigma, Original bottle] to each sample and QC. Slow vortex for 10 sec.
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Use Thermomixer to heat for 1 hr at 60°C. (no shaking needed)
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Collect the tubes, vortex slowly for for 10 seconds, centrifuge for 30 seconds and collect the supernatant into autosampler vials (with 250 uL inserts, screw capped, Amber color); and submit to the GC-MS autosampler.