There are limited studies that report the physiological levels of H2S in the eye. The currently available UV/Vis methods lack the required sensitivity and precision. Hence, the purpose of this study was to develop and validate a sensitive and robust pre-column derivatization LC-MS/MS method to measure changes in H2S levels in tissues from isolated porcine eyes. H2S was derivatized and an LC-MS/MS method was developed to monitor the derivatized product, Sulfide-dibimane (Sdb) using a reverse phase Waters Acquity BEH C18 column (1.7 μm, 2.1 × 100 mm). H2S quantification was performed using multiple-ion reaction monitoring (MRM) in positive mode, with the transitions of m/z 415.0 → m/z 223.0 for Sdb and m/z 353.0 → m/z 285.0 for internal standard (griseofulvin). This method provided a suitable way to quantify H2S and was then successfully adapted to measure H2S levels in isolated porcine iris-ciliary body tissues previously treated in the presence or absence of varying concentrations of lipopolysaccharide (LPS, 5-100 ng/ml), a pro-inflammatory agent. Isolated iris-ciliary bodies (ICB) from porcine eyes were cut into quadrants of approximately 50 mg and homogenized using a 1:3 volume of homogenizing buffer. H2S in the supernatant was then derivatized with monobromobimane and quantified.
Keywords: Derivatization; Hydrogen sulfide; Inflammation; LC-MS/MS; Monobromobimane; Ocular study.
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