(T1230-06-36) Development and Validation of an LC-MS/MS Method for the Quantification of Hydrogen Sulfide in Isolated Tissues from Porcine Eyes

Purpose: Hydrogen sulfide (H₂S) is a gasotransmitter that is produced in several tissues including the eye¹. Most of the current methods available to measure H₂S in biological samples such as amperometry and the gold standard methylene blue assay are associated with errors due to the lack of sensitivity and precision². Hence, in this study, we developed and validated a sensitive and robust pre-column derivatization LC-MS/MS method to measure changes in H₂S levels in tissues from isolated porcine eyes.

Methods: 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 was derivatized with monobromobimane using a previously described methodology^3,4. For the LC-MS/MS analysis, a reverse phase Waters Acquity BEH C18 column (1.7 μm, 2.1 x 100 mm) and an injection volume of 2 μL was used. 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). Accuracy and precision were evaluated using intra- and inter-day coefficients of variation (CV). Six porcine ocular samples for four QC levels were analyzed on a single day for intra-day, and six samples were measured on three consecutive days for inter-day CV. The extraction recovery of Sdb was performed by comparing the responses obtained by spiking ocular samples before protein precipitation to responses obtained by spiking after protein precipitation. Internal standard recovery was determined by comparing the responses of the internal standard obtained before protein precipitation to responses obtained after protein precipitation. The matrix factor was analyzed by comparing responses from extracted blank ocular samples spiked with analyte post-extraction with the absolute response of neat solvent spiked with the analyte. Finally, the stability of Sdb was tested at room temperature for 4 h, at 15 ⁰C in the autosampler for 24 h, freeze-thaw, and at -80 ⁰C in the freezer for 14 days. This method was applied 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. Statistical analyses of data were done with ANOVA using the GraphPad Prism software.

Results: The method displayed linearity over a range of concentrations for sulfide dibimane (5 – 2500 ng/ml) in porcine ocular tissue samples. This corresponds to the equivalent H₂S concentration range of 0.41 ng/ml – 205.31 ng/ml. Intraday and inter-day CV ranged from 2.04% to 5.94% and 5.30% to 6.86%, respectively. Extraction recoveries for Sdb were between 92.9% and 113%. The matrix effect was between 5.6% and 10.4%. Sulfide dibimane was stable for up to 4h in porcine ocular samples at room temperature with mean recoveries of 93.3%, 98.3%, and 98.6% at low, medium, and high QC levels, respectively. Auto-sampler stability demonstrated that Sdb is stable in processed ocular samples for up to 24h at 15 ⁰C with mean recoveries of 94.4%, 93.9%, and 95.4% at low, medium, and high QC levels, respectively. The mean recovery of Sdb from porcine ocular samples following three freeze–thaw cycles was 92.6%, 88.3%, and 94.3% at low, medium, and high QC levels, respectively. Also, long-term stability samples at low, medium, and high QC concentrations were within 10% of the nominal concentration. These stability results show that Sdb was stable in ocular samples under expected storage and handling conditions including repeated freeze–thawing and even storage for up to two weeks. There was a significant (p < 0.001) decrease in basal H₂S level in the presence of 100 ng/ml LPS (0.27 ng/mg tissue ± 0.30 ng/mg tissue) compared to blank (0.45 ng/mg tissue ± 0.20 ng/mg tissue) (n=10). The method was able to measure changes in tissue H₂S concentration in the presence of varying concentrations of LPS.

Conclusion: To our knowledge, this is the first study conducted to quantify H₂S in ocular samples by LC-MS/MS. We have developed and validated a specific, sensitive, and reproducible LC-MS/MS method to quantify H₂S in ocular samples. Advantages of this method include small tissue sample size, higher sensitivity than traditional spectrometry methods, as well as the measurement of changes in H₂S levels in the presence of lipopolysaccharide.

References: 1. Kulkarni et al. (2011). Neurochem. Res. 36: 1540-5.
2. Wintner et al. (2010). Br. J. Pharmacol. 160(4): 941-957.
3. Sugiyama et al. (2022). Journal of Pharmaceutical and Biomedical Analysis, 222, 115088.
4. Montoya et al. (2015). Chem Sci. Jan 1;6(1):294-300.

Acknowledgements: This study was funded in part by NIH Grant Numbers: IRI5EY032274 and G12MD007605; CPRIT grant number RP 180748.

Extraction Recovery, Matrix Effect, and Accuracy and Precision data for Sulfide Dibimane

Chromatogram of Sulfide Dibimane with Internal Standard

Stability of Sulfide Dibimane in Porcine Ocular Samples