Method validation of gamma-Hydroxybutyric acid detection upon Herpes Simplex Virus-Type 1 infection using LC-MRM-MS...

Volatile organic compounds (VOCs) release triggered by infection of DNA virus has not been studied extensively. Previously, we reported that gamma-butyrolactone (GBL), a VOC, was released upon Herpes Simplex Virus Type-1 (HSV-1) acute infection. Based on the metabolic pathway and chemical conversion of GBL, we hypothesized that infected cells produce gamma-Hydroxybutyric acid (GHB) as a key pathway intermediate for the subsequent production of GBL. An analytical technique for the rapid detection of GHB is crucial for further understanding its role in the cellular response to HSV-1 infection. To address this, we developed a sensitive, reliable, and specific method for the detection and quantification of GHB in mammalian cell culture using a pre-column derivatization approach. Our data showed that the carboxylic acid functional group of GHB could be derivatized with 3-nitrophenylhydrazine hydrochloride (3-NPH) to produce its hydrazineyl derivative. Unlike GHB, the derivative could be detected seamlessly in HPLC-MS. We also demonstrate quantitive conversion of GHB into the derivative with over 95% yield at a range of 1 μg/mL- 6 μg/mL GHB concentration. This method offers a rapid quantification of GHB in aqueous mixtures, especially in cultured extracts.