Polar metabolite extraction for LC-MS

Extract polar metabolites from snap-frozen coral larvae, juveniles or adult clippings in cold methanol:acetonitrile:water with formic acid, neutralize, and load LC-MS autosampler vials.

Version 1.0 · last updated 2026-10-05 · Used in 10 notebook entries ↓

At a glance

Input	Snap-frozen larvae (≥ 20 per sample), juveniles on plugs, or an adult clipping (weighed), stored at −80 °C
Output	Duplicate 100 µL LC-MS autosampler vials + the remaining extract, all at −80 °C
Time	~15 min per sample; plan around how many clean dounces you have (estimate)
Tested on	Porites astreoides adults and larvae; Montipora capitata larvae, recruits and juveniles

Materials

Equipment and consumables

• 2 mL glass dounce homogenizers (Sigma D8938), autoclaved between samples
• 100 µm cell strainers and 50 mL tubes
• 11 mm snap-cap autosampler vials and caps
• Refrigerated centrifuge (4 °C), analytical balance (adults), dry ice
• Sterile clippers or razor blades; fume hood

Reagents (LC-MS grade)

• Methanol, acetonitrile, water, formic acid
• Ammonium bicarbonate
• 10% bleach and alkaline lab soap for cleaning

Solutions to prepare

Solution	Recipe	Storage
Extraction buffer: 40:40:20 methanol:acetonitrile:water + 0.5% formic acid	200 mL: 80 mL methanol + 80 mL acetonitrile + 40 mL water + 1 mL formic acid	4 °C, flammables fridge
Saturated ammonium bicarbonate (“100%”, 220 g L⁻¹)	55 g in 250 mL LC-MS water	4 °C
15% ammonium bicarbonate	37.5 mL saturated stock + 212.5 mL LC-MS water	4 °C

Before you start

• Autoclave all glassware (dounces, pestles). Wipe benches and the fume hood with 10% bleach.
• Aliquot the day’s extraction buffer and ammonium bicarbonate into 50 mL tubes and keep them cold.
• Pre-cool the centrifuge to 4 °C. Get dry ice.
• Label per sample: a weighing vial (adults), a temporary 1.5 mL tube, and two autosampler vials (A and B).

Procedure

A. Get the sample into extraction buffer

Larvae (in tubes)

1. Put a dounce on dry ice. Take the sample from −80 °C and remove any residual seawater.
2. Add 500 µL extraction buffer to the tube, pipette-mix, and transfer to the dounce. Rinse the tube with another 500 µL and add it to the dounce.

Juveniles on plugs

3. Add 500 µL extraction buffer to a 1.5 mL tube on dry ice. Scrape the juvenile off the plug with a sterile razor blade straight into the buffer (push stuck tissue down with a pipette tip). Transfer to the dounce and rinse with another 500 µL.

Adult clippings

4. Weigh an empty vial, then the vial + 1 mL extraction buffer. Clip a small piece of tissue (~0.5 cm) into it and weigh again (tissue mass = difference). Transfer to the dounce.

B. Homogenize and clear

5. Incubate 5 min on dry ice (or on ice).
6. Homogenize with the glass pestle for ~1 min, until the liquid is visibly pigmented.
7. If the sample is strongly pigmented and opaque, rinse the pestle with another 500 µL buffer into the dounce. If it is faint (small samples), skip this to keep the extract concentrated, and record the total volume used.
8. Pour through a 100 µm cell strainer into a 50 mL tube, then transfer to a 1.5 mL tube.
9. Vortex and spin 15,000 × g, 10 min, 4 °C.

C. Neutralize and load

10. Move 500 µL of supernatant to a new tube and add 44 µL cold ammonium bicarbonate. Vortex and spin down.
11. Load 100 µL into each of two autosampler vials (A, B) and cap.
12. Store the vials and the remaining extract at −80 °C, in a labeled box (record the box and freezer).

Clean-up

• Soak dounces in alkaline soap, rinse well, and autoclave. With 10 dounces, plan to autoclave after every ~5 samples or between lab days.

Troubleshooting

Problem	Likely cause	Fix
Residue or soap in a dounce	Not rinsed well before autoclaving	Don’t use it; re-wash. Put the sample back at −80 °C if it hasn’t touched buffer (2020-10-06)
Low signal from small samples	Sample too diluted	Use less buffer for small larvae/recruit samples, and record the volume (2021-03-25)
Normalization unclear	Different buffer volumes between samples	Record buffer volume and larva number or tissue mass for every sample

ImportantAmmonium bicarbonate concentration

The 2020 protocol used 15% ammonium bicarbonate; the 2021 Symbiont Integration batches recorded 10%. Use one concentration across a whole project. TODO: confirm which is current.

Notebook entries using this protocol

Changelog

Date	Version	Change
2026-10-05	1.0	Merged the 2020 protocol with the 2021 juvenile and dry-ice modifications
2021-03	–	Juveniles on plugs; dry-ice incubation; skip the rinse step for faint samples (M. capitata)
2020-10	–	Adult P. astreoides extractions (Porites July Bleaching)
2020-02	–	Protocol developed on P. astreoides larvae and adults