3.8.2. Derivatization of 2-methyloctanoic Acid with 2-phenylglycine Methyl Ester

To generate a 1:1 standard mixture of both possible diastereomers of 2-methyloctanoic acid, 6.0 mg (37.9 μmol) of racemic 2-methyloctanoic acid was combined with 1-[bis(dimethylamino)methylene]- 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (14.4 mg, 37.9 μmol), (*S*)-(+)-2-phenylglycine methyl ester hydrochloride (7.6 mg, 37.9 μmol) and *N*,*N*-diisopropylethylamine (DIPEA) (30 μL) in dimethylformamide (DMF) (300 μL). This was stirred overnight at room temperature and ambient atmosphere. The reaction mixture was then diluted with 1.0 mL of EtOAc, washed with saturated aqueous NH4Cl (3 × 1.0 mL), concentrated under vacuum, and prepared for LC-MS analysis. To generate a chiral standard, 1.8 mg (11.4 μmol) of (*S*)-2-methyloctanoic acid was combined with HATU (4.3 mg, 11.4 μmol), (*S*)-(+)-2-phenylglycine methyl ester hydrochloride (2.3 mg, 11.4 μmol) and DIPEA (30 μL) in DMF (300 μL), and stirred overnight at room temperature and ambient atmosphere. The reaction mixture was then diluted with 1.0 mL of EtOAc, washed with saturated aqueous NH4Cl (3 × 1.0 mL), concentrated under vacuum, and prepared for LC-MS analysis. The diastereomeric ratio of the chiral standard was 3:1 by area-under-curve analysis.

### 3.8.3. Derivatization of Lysine with Marfey's Reagent (FDAA)

To generate a racemic standard, 0.8 mg (4 μmol) of racemic lysine hydrochloride and 0.1 M NaHCO3 (200 μL) were added to a solution of l-FDAA (4.4 mg, 16 μmol) in acetone (600 μL). The reaction mixture was sealed in a vial with ambient atmosphere, stirred at 90 ◦C for 5 min, neutralized with 6 M HCl, concentrated under vacuum, and prepared for LC-MS analysis. To generate a chiral standard, 1.0 mg (6 μmol) of l-lysine monohydrate and 0.1 M NaHCO3 (200 μL) were added to a solution of l-FDAA (6.5 mg, 24 μmol) in acetone (600 μL). The reaction mixture was sealed in a vial with ambient atmosphere, stirred at 90 ◦C for 5 min, neutralized with 6 M HCl, concentrated under vacuum, and prepared for LC-MS analysis.
