Comparison of colorimetric, spectroscopic and electrochemical techniques for quantification of hydrogen sulfide

**Background:** Hydrogen sulfide (H₂S) is an endogenous gasotransmitter with potential therapeutic applications in conditions such as Alzheimer's disease, Parkinson's disease, glaucoma, retinopathy, and cataract. Despite its promise, accurate quantification of H₂S in simulated physiological solutions is challenging due to its gaseous nature, volatility, and instability. This study aimed to compare four commonly used H₂S detection and quantification methods in aqueous solutions to help researchers select the most appropriate technique for their specific needs.

**Methods:** The four techniques compared were: (1) a modified colorimetric method using N,N-diethyl-p-phenylenediamine (ethylene blue complex) with toluene to reduce oxidative loss; (2) a reverse-phase HPLC method paired with the ethylene blue complex; (3) a voltametric method using an ion-selective electrode; and (4) an amperometric method using a WPI ISO-H₂S-100 microelectrode. All methods were validated according to ICH or USP guidelines for specificity, linearity, precision, and accuracy. Standard solutions of sodium hydrogen sulfide (NaSH) were prepared in simulated tear fluid (STF) or phosphate-buffered saline (PBS) at pH 7.4. For the colorimetric method, the mixed diamine reagent was added to NaSH solutions, and absorbance was measured at 671 nm after 10 minutes. For HPLC, 100 μl of mixed diamine reagent was added to 5 ml of NaSH solution, and 20 μl was injected into a C-18 column with isocratic elution (acetonitrile:ammonium formate 70:30 v/v) at 1.2 ml/min, detection at 670 nm. The voltametric method used a Lazar electrode soaked in the lowest NaSH concentration, with measurements taken from lowest to highest concentration. The amperometric method involved polarizing the sensor in 0.1 M PBS for 12 hours, then immersing it in 0.05 M PBS and injecting aliquots of NaSH standard solutions (5, 10, 20, and 40 μl) sequentially.

**Key Results:** The colorimetric method was linear (R² = 0.9987) over 1.5–100 μM, with intraday RSD 1.39–5.84% and interday RSD 6.67–12.23%, accuracy >94%, and detection limit 1000 nM. The chromatographic method was linear (R² = 0.9982) over 0.1–3.5 μM (later stated as 0.5–60 μM in comparison table), with intra- and interday RSD <5% (except at 0.54 μM interday RSD 7.00% and 15.18 μM intraday RSD 9.44%), accuracy within acceptable limits (<10% error), and detection limit 100 nM. The voltametric method was linear (R² = 0.9983) over 0.3–2.17 μM (later stated as 30–1000 μM in comparison table), with intra- and interday RSD <1.1%, accuracy error <7%, and detection limit 400 nM. The amperometric method was linear (R² = 0.9992) over 0–2000 nM, with average intra- and interday RSD <8%, accuracy error <10%, and detection limit 5 nM. Response times were 30 minutes for colorimetric, 10 minutes for chromatographic, and 20–60 seconds for both electrochemical methods. Minimum detection volumes were 2–3.5 ml for colorimetric, 0.5–2 ml for chromatographic, and <1 ml for electrochemical methods.

**Clinical Implications:** The choice of H₂S quantification method depends on the specific requirements of the research project. For applications requiring high sensitivity (nanomolar range), such as measuring H₂S in biological samples or validating controlled-release delivery systems, the amperometric technique is most suitable due to its low detection limit (5 nM) and fast response time (20–60 seconds). The voltametric method offers a balance of sensitivity (micromolar range) and speed, while the chromatographic method provides improved sensitivity over colorimetric but requires more expensive equipment. The colorimetric method, though cost-effective, has the lowest sensitivity and longest response time, making it less ideal for precise quantification in physiological studies. These findings help researchers select the appropriate method for their experimental needs, considering trade-offs between sensitivity, response time, and cost.