Development and application of LC-MS/MS method for the quantification of hydrogen sulfide in the eye

• Published in: Analytical biochemistry 2024-04, Vol.687, p.115448, Article 115448
• Main Authors: Okolie, Anthonia, Nigro, Maria Rincon, Polk, Sharhazad, Stubbs, Keyona, Chelliah, Selvam, Ohia, Sunny E, Liang, Dong, Mbye, Ya Fatou Njie
• Format: Article
• Language: English
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2023.115448

There are limited studies that report the physiological levels of H S 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 H S levels in tissues from isolated porcine eyes. H S 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). H S 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 H S and was then successfully adapted to measure H S 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. H S in the supernatant was then derivatized with monobromobimane and quantified.