Methyl chloroformate

Thawing was conducted in an ice bath to ensure the quality of serum samples. A 150 µl aliquot of serum was then mixed with 50 µl 4 M NaOH, 4 µl internal standard (10 mM 2,3,3,3-D4-alanine, Sigma, USA), and 200 µl methanol. After a 15-minute incubation at room temperature, the mixture underwent centrifugation at 12,000 rpm for 15 min. Following centrifugation, 300 µl of the supernatant was carefully transferred to a new glass test tube, where 34 µl pyridine (Sigma, USA) was added and thoroughly mixed. Next, 20 µl methyl chloroformate (Sigma, USA) was added twice, with a vortex mix for 30 s after each addition. Subsequently, 400 µl of chloroform and 400 µl of NaHCO₃ solution were sequentially added, with a vortex mix for 10 s after each addition. To facilitate phase separation, the blended liquid was centrifuged at 2000 rpm for 10 min, and the upper water and intermediate protein layers were carefully discarded. Following this step, sodium sulfate, similar to mung beans, was added to the lowest chloroform layer for water absorption. Finally, 200 µl of the resulting liquid phase, containing derivatized metabolites, was transferred into the GS automatic sampler sample bottle for subsequent analysis.

LTG
(>98%) and 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (98.5%) were purchased
from TCI (Zwijndrecht, Belgium). LTG-N₂-oxide (>95%),
LTG-¹³C3, and 5-desamino 5-oxo-2,5-dihydro LTG (OXO-LTG;
98%),
2-methyllamotrigine methanesulfonate (LTG-N₂-methyl) were
purchased from TRC. All solvents (acetone, acetonitrile, methanol,
and water) were LC-MS grade (Biolab Co., Jerusalem, Israel). NaOH
was purchased from Honeywall Fluka (Tokyo, Japan). The following reagents
were purchased from Sigma-Aldrich (St. Louis, MO): methyl chloroformate
(99%), potassium carbonate, allyl bromide (99%), propionyl chloride
(98%), 2-(2-bromoethyl)-1,3-dioxolane (96%), iodomethane (99%), formamide
(99%), and sodium methoxide (99%).

All chemicals used in this study were of analytical grade. Methanol, chloroform, sodium bicarbonate, and sodium hydroxide were obtained from MERK (Damstadt, Germany). The internal standard 2,3,3,3-d₄-alanine, the derivatisation reagent methyl chloroformate (MCF), and pyridine were purchased from Sigma-Aldrich (St. Louis, USA). Anhydrous sodium sulphate and farnesol were obtained from Fluka (Steinheim, Germany). U-¹³C5 l-proline and U-¹³C6 arginine were acquired from Cambridge Isotope Laboratory Inc. (Tewksbury, USA). Two types of DNA polymerases were utilised in this study: KOD hot start DNA polymerase from NovgenR (Darmstadt, Germany), used to construct the SAT1 flipper disruption cassette, and TaKaRa Ex TaqTM from Takara Bio Inc. (Shiga, Japan), used for colony PCR amplifications. The antibiotic nourseothricin was purchased from WERNER BioAgents (Jena, Germany). The QIAquick Gel Extraction kit was supplied by Qiagen (Valencia, CA, USA) and the Alkali-cation yeast transformation kit was obtained from Qbiogene (Heidelberg, Germany).