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girard_t_derivatization.md

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Overview

This protocol describes how to derivatize samples containing ketones or aldehydes with Girard's T reagent for improved LC-MS analysis.

Equipment

PPE

Safety glasses and nitrile gloves are required for this protocol.

Samples

Samples for this protocol are assumed to be already-extracted, clarified liquids. They might be plant tissue extracts or filtered microbial culture supernatants, or any sample that is suitable for injection on a C18 chromatography column and LC-MS system.

  • Thus, samples cannot contain particulates, precipitates surfactants, or high concentrations of non-volatile salts.

Reagents

  1. Girard's T reagent, e.g. Aldrich catalog # 89397.

  2. Glacial acetic acid, e.g. Fisher catalog #A35-500

  3. LC-MS-grade methanol

  4. Suitable vials and vial closures (lids) for holding samples dissolved in organic and aqueous samples and for LC-MS injection.

Protocol

  1. Prepare stock solution of T reagent.

    1. weight 20 mg of Girard's T reagent into an LC-MS vial or similar container.
    2. Add 1 mL of HPLC-grade methanol.
    3. Mix until completely dissolved.
      • This stock solution is not shelf stable for long periods and is best prepared fresh before use.

  2. Mix 100 μL of each sample with 100 μL of Girard T stock solution in an LC-MS vial.

    • LC-MS 96-well plates are perhaps compatible with this protocol: it may be difficult to seal the plate tightly during the heating step below. Without a tight seal, solvent will evaporate in an uncontrolled way during derivatization. Test your equipment before use on precious samples.

  3. Add 20 μL of glacial acetic acid.

  4. Incubate samples at 70 °C for 1 hr.

  5. Let samples cool to near room temperature. They are now ready to inject.

Chemistry

The Girard's T reagent:

  • is (carboxymethyl)trimethylammonium chloride hydrazide
  • has CAS number of 123-46-6
  • has a PubChem CID of 67156
  • has a SMILES structure of C[N+](C)(C)CC(=O)NN.[Cl-]
  • is detectable (in unreacted form and if present in excess or unreacted with carbonyls) at an $m/z$ of 132.1131 .
    • also shows a prominent ion corresponding to (M+ - H2O - N2)+, i.e. C5H11N+, at $m/z$ of 86.0964 .
  • replaces the carbonyl oxygen with a hydrazone moiety.
  • A SMARTS reaction model for the derivatization might be [#6:3]-[C:1]=[O:2]>>[#6:3]-[C:1]=NNC(=O)C[N+](C)(C)(C)
  • increases the exact mass of derivatized analytes by 114.1026 Da and the charge of analytes by +1.
  • increases the measured m/z of derivatized analytes by 113.0953 Da.
  • was first described in 1936 by Andre Girard & Georges Sandulesco in Helvetica Chimica Acta (in French).

Acknowledgements

Ryan Nett contributed to this protocol.

CF & RN / cf