(T1330-09-52) Comparative Evaluation of Colorimetric, Chromatographic, and Electrochemical Techniques for Hydrogen Sulfide Quantification

Purpose: Hydrogen sulfide (H₂S), once known as a toxic gas, is now known to have endogenous roles in mammalian pathophysiology. H₂S is reported to play a role in learning and memory, inflammation, regulation of blood pressure, modulation of synaptic activities in central nervous system, neuroprotection, etc.¹ Owing to the critical role of H₂S in several physiological and pharmacological processes, several studies have been conducted to investigate its potential application in Alzheimer’s and Parkinson’s disease, glaucoma, cataract, ischemia, myocardial infarction, etc.^2-4 One of the major challenges of investigating   H₂S therapeutic potential and, developing a safe and efficient delivery system is its quantification in physiological solutions which is not optimal for the stability of chemicals capable of generating H₂S. Being a gas at standard temperature and pressure, it rapidly escapes from solutions and gets lost to the environment. Moreover, it is oxidized by dissolved oxygen in water to form sulfur dioxide. Several techniques have been developed to accurately determine, with each technique having its own advantages and limitations. In this study, we have compared and contrasted three analytical techniques (a colorimetric, a chromatographic, and two electrochemical techniques), differing in their working principles, widely used in H₂S detection and quantification in aqueous solutions for their specific limitations and usefulness.

Methods: H₂S donors sodium hydrogen sulfide (NaSH) and sodium sulfide (Na₂S) were used as sources of H₂S to prepare standard sulfide solutions. The colorimetric technique involved preparation of acidic solutions of N, N-diethyl-p-phenylenediamine (DEPD) and ferric chloride. Standard hydrogen sulfide donor solution prepared in water was added to the acidic DEPD-ferric chloride solution and kept at room temperature for 30-45 minutes for the development of ethylene blue complex. The intensity was determined by measuring the absorbance of the ethylene blue complex spectrophotometrically using UV/Vis detector at 671nm. The intensity of similarly developed ethylene blue complex was determined by a reverse-phase HPLC method using similar UV/Vis detector at 671nm. Lastly, two commercially available H₂S specific electrodes (one from Lazar Research Laboratories, hereafter referred as electrode A and other from World Precision Instruments, hereafter referred to as electrode B) were evaluated for detection and quantification of H₂S following their respective vendor provided instructions.

Results: The colorimetric method assesses the extent of ethylene blue complex formation and provides the equivalent quantity of H₂S. Using the colorimetric method, H₂S was detected in the range of 1.5-1,000 µmole/mL. On the other hand, the HPLC method using the same ethylene blue complex was validated for the USP specifications for specificity (well resolved peak at 670 nm with retention time of 3.3 minutes), linearity (R²=0.9998), precision (95-105%), accuracy (±10%) and the results were found to be in conformity with the official limits in the range of 0.3-9 µmole/mL. Both these methods needed a preparation time of 30-45 minutes. On the other hand, the two electrodes offered a quick and easy detection method with real-time analysis. The electrochemical method detected sulfide ions, which were in equilibrium with hydrogen sulfide in water. Electrode A used an antioxidant solution to prevent oxidation of H₂S and detected H₂S in the range of 1-100 nmole/mL while electrode B used EDTA solution purged with argon and yielded a range of 2.5-10,000 pmole/mL.

Conclusion: The colorimetric, chromatographic and electrochemical methods offer high specificity. All the 4 methods described above can be used for simple solutions as well as biological samples. Both spectrophotometric and HPLC methods were capable of quantifying H₂S in micromole range. However, the HPLC method needed significantly less amount of sample than the spectrophotometric method. Whereas the electrochemical methods were able to quantify H₂S in nanomole range, electrode B further detected the gas in picomole range. Additionally, electrochemical methods were not dependent on the development of any colored complex, rendering them less time-consuming and minimizing error due to any loss/degradation of H₂S in the aqueous samples.

References: 1. Mhatre, Susmit, Catherine A. Opere, and Somnath Singh. "Unmet needs in glaucoma therapy: The potential role of hydrogen sulfide and its delivery strategies." Journal of Controlled Release 347 (2022): 256-269.
2. Bos, Eelke M., et al. "Hydrogen sulfide: physiological properties and therapeutic potential in ischaemia." British journal of pharmacology 172.6 (2015): 1479-1493.
3. Szabó, Csaba. "Hydrogen sulphide and its therapeutic potential." Nature reviews Drug discovery 6.11 (2007): 917-935.
4. Shen, Yaqi, et al. "The cardioprotective effects of hydrogen sulfide in heart diseases: from molecular mechanisms to therapeutic potential." Oxidative Medicine and Cellular Longevity 2015 (2015).

Acknowledgments: Financial support from School of Pharmacy and Health Professions, Creighton to the graduate students involved in this study. We acknowledge the contributions of our past students (Hatim, Akash, Richa, and Neetu Singh) for incrementally increasing our understanding of this gasotransmitter.

Funding: This study was funded by Wareham Research Fund Award and NIH NEI 1R15EY032275-01A1
Conflict of Interest Statements: We confirm that there are no conflicts of interest associated with this work and there has been no significant financial support for this work that could have influenced its outcome.