Point-of-care testing and optimization of sample treatment for fluorometric determination of hydrogen sulphide in plasma of cardiovascular patients

• A comparative sample preparation methodology for fluorometric determination of H 2 S in plasma. • Sample treatment protocol is critical to reliable results for determination of gasotransmitter. • Portable H 2 S Analyser was designed, manufactured and verified as POCT for the gas in the ambulance. • Validation in human plasma proved efficiency of H 2 S Analyser in determining H 2 S in MI patients. • H 2 S is elevated in MI patients compared to normal controls up to 10 h from emergence of symptoms. Introduction: Hydrogen sulphide (H 2 S) is one of the gasotransmitters that was reported to have a cardioprotective effect at its physiological levels in blood. Previous determinations of H 2 S levels in cardiovascular disease (CVD) patients suffered from diversity of analytical methods, different targeted chemical forms of the gas, and multitude of matrices assessed. Objectives: In this study, a comparative biological sample preparation study is detailed for optimum selective determination of the unionized form of H 2 S in blood of CVD patients using a new in-house POCT portable spectrofluorometer together with a Reagent-Analyser system. Methods: Dansyl azide was synthesized to react with hydrogen sulfide in biological matrix to produce the fluorescent dansyl amide. Fluorescence was measured at λ ex 340 nm and λ em 517 nm in the new in-house POCT portable spectrofluorometer. The method was validated according to ICH guidelines. Several blood sample treatments and reaction protocols were compared to achieve maximum fluorescence yield. Results: The H 2 S Analyser was verified in comparison to a benchtop spectrofluorometer where linearity was confirmed in the range of 3–300 μM, LOD being 1 μM, at λ ex 340 nm and λ em 517 nm. Sample treatment involving blood centrifugation followed by addition of reagent on plasma produced maximum fluorescence yield. Analysis of blood samples of myocardial infarction (MI) patients and controls showed elevated levels of H 2 S in MI patients (28 μM ± 1.111) vs. controls (23 μM ± 1.036) at p = 0.0015. Conclusion: The study is novel in being a POCT approach for selective determination of H 2 S molecular form in plasma after simple optimized sample treatment. The study confirms that MI is associated with H 2 S elevated levels up to 10 hours from emergence of symptoms.