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

Abstract

Background:

Hydrogen sulfide (H₂S), an endogenous gasotransmitter, has potential applications in several conditions. However, its quantification in simulated physiological solutions is a major challenge due to its gaseous nature and other physicochemical properties.

Aim:

This study was designed to compare four commonly used H₂S detection and quantification methods in aqueous solutions.

Methods:

The four techniques compared were one colorimetric, one chromatographic and two electrochemical methods.

Results:

Colorimetric and chromatographic methods quantified H₂S in millimolar and micromole ranges, respectively. The electrochemical methods quantified H₂S in the nanomole and picomole ranges and were less time-consuming.

Conclusion:

The H₂S quantification method should be selected based on the specific requirements of a research project in terms of sensitivity, response time and cost-effectiveness.

METHOD SUMMARY

This work assessed four different methods (colorimetric, chromatographic, voltametric and amperometric) to determine the concentration of hydrogen sulfide (H₂S) in aqueous medium. The colorimetric method is relatively simple and inexpensive but requires a greater amount of sample and time for the completion of the process. The chromatographic method is built on the colorimetric method that requires significantly less sample (e.g., 25 μl vs 1 ml for colorimetric) but exhibits much greater sensitivity (e.g., nM vs μM) without involving a time-consuming sample preparation step. However, they are relatively more expensive than other methods.

Tweetable abstract

Four validated analytical techniques for quantifying and detecting hydrogen sulfide in micromolar to nanomolar range in physiological simulated solutions were compared and contrasted.

Hydrogen sulfide (H₂S) is reported to elicit several physiological and pharmacological functions in mammalian cells [1–5]. In addition to modulating neuronal messages in the brain [6,7], H₂S also mediates critical processes such as neurotransmission [6,8], cytoprotection [9–13], neuroprotection [14,15], smooth muscle relaxation [16,17], vasorelaxation and regulation of blood pressure [7,17–22], anti-inflammation [10,23–28] and cellular respiration [29–33].

There are numerous reports on the potential application of H₂S in conditions like Alzheimer's disease [34], Parkinson's disease [35], intracellular hemorrhage [36], traumatic brain injury [37], myocardial ischemia/reperfusion injury [38], myocardial fibrosis [39], atherosclerosis [40], hepatic disorders [41], breast cancer [42], glaucoma [43], retinopathy [44], macular degeneration [45] and cataract [46]. The most investigated H₂S donor, GYY 4137, exhibited relaxation of arteries, precontracted using phenylephrine in the concentration range of 100 nM to 100 mM with an IC₅₀ value of 13.4 ± 1.9 mM [47]. GYY4137 at concentrations ranging from 50 to 500 μM has also shown antitumor effects in a wide range of cancerous cell types, including breast, lung and colon cancer cells [48]. At these concentrations, not only did GYY4137 inhibit cell proliferation but it also induced apoptosis in tumor cells. The fast-releasing H₂S donor sodium hydrogen sulfide (NaSH) in concentrations ranging from 40 to 500 μM has also shown suppression of tumor cells in prostate cancer [49,50]. This understanding of the role of H₂S in mammalian pathophysiology has generated much interest in utilizing this molecule as a therapeutic agent.

Despite the reported therapeutic potential of this gas, its investigation as a drug candidate in preclinical as well as clinical settings is limited due to several challenges [43,51,52]. Its gaseous nature at physiologically relevant temperatures and pressure, inherent toxicity, narrow therapeutic activity at low concentrations, aqueous instability and need for an efficient, sustained-release delivery system are some of the major challenges. However, the most critical challenge is the development of an accurate and sensitive assay method capable of quantifying this gas in therapeutically relevant concentrations (micronanomolar range) in release media that simulates physiological conditions. The cumulative amount of H₂S released from any sustained-release delivery systems must be less than its solubility in the releasing media, which is typically low, for example, 3.9 g/l (120 mM) at 20°C [53], to minimize measurement error due to its gaseous nature and volatility.

Accurate and reliable measurement of H₂S in physiological conditions is needed for the translation of this gasotransmitter into clinical applications. This can provide critical information about H₂S concentrations found with various normal as well as abnormal physiological and biochemical processes. Therefore, in this study, the efficiency of four techniques based on different principles for quantification of H₂S were compared in aqueous media (e.g., simulated tear fluids and phosphate-buffered saline [PBS] at pH 7.4) frequently used during the development of sustained-release drug delivery systems.

Materials & methods

Materials

Supplementary Table 1 lists some of the important chemicals used in this study. All other chemicals used were of analytical reagent quality.

Colorimetric technique

Preparation of mixed diamine reagent

A total of 33 μl of N, N-diethyl-p-phenylenediamine was added to 10 ml of 7.2 M hydrochloric acid to a prepared diamine solution. A second solution was made by adding 48 mg of FeCl₃ to 10 ml of 1.2 M hydrochloric acid. The two solutions were then mixed, resulting in the formation of the mixed N, N-diethyl-p-phenylenediamine solution, which was stored in a refrigerator.

Preparation of standard NaSH solutions

Simulated tear fluid (STF) was prepared following the methods reported in the literature [54,55]. The final composition of the STF solution was as follows: sodium chloride (6.80 g), sodium bicarbonate (2.2 g), calcium chloride 2H₂O (0.08 g) and potassium chloride (1.40 g) in ultrapure deionized water q.s. 1 l. The pH of the resulting solution was adjusted to 7.4 by 0.1 M HCl. Next, 200 ml of stock solution of NaSH was prepared by adding 2.5 mg of NaSH in 100 ml of STF. Toluene (0.045 g) was added to 100 ml of the stock solution while the remaining volume served as a control.

Quantification of H₂S

A total of 20 μl of mixed diamine reagent was added to 1 ml of standard NaSH solution, which was subjected to vortexing and left undisturbed for 10 min at room temperature to allow the development of the colored complex. Next 200 μl of the sample was transferred to a 96-well plate and the intensity of the resultant colored solution was determined by measurement of absorbance at 671 nm using a microplate reader (Synergy H1 Hybrid Reader, Biotek, Agilent Technologies, CA, USA). According to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, the method was validated for specificity, linearity, precision and accuracy. To create a standard curve, the diamine reagent solution was mixed with different concentrations of NaSH and the resulting absorbances were recorded.

Chromatographic technique

Preparation of standard NaSH solutions

A total of 28 mg of NaSH was weighed and transferred to a 1-l volumetric flask and the volume was brought to 1 l using STF. Standard solutions in the range of 0.04–5.60 μg/ml of NaSH that would release 0.03–3.40 μg/ml of H₂S were prepared by diluting the stock solution using STF.

Quantification of H₂S by HPLC

A total of 100 μl mixed diamine reagent (prepared as previously described) was added to 5-ml aliquots of standard NaSH solution. The resultant solution was shaken vigorously and set aside for 10 min after which its 20-μl aliquot was injected into HPLC for the determination of H₂S content using an Alltech C-18 (150 mm × 4.6 mm, 5 μm) column (GL Sciences Inc., CA, USA) and isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (15 mM; 70:30 v/v). The flow rate was set at 1.2 ml/min with a total run time of 6 min and the retention time of H₂S was 3.3 min. A PDA UV-visible detector was used at 670 nm for the detection of the analyte. The method for HPLC was validated as per US Pharmacopeia (USP)/ICH guidelines.

Electrochemical techniques

Voltametric technique

Preparation of antioxidant buffer

Antioxidant buffer was prepared by adding 25 g sodium salicylate, 6.5 g ascorbic acid and 8.5 g NaOH to 100 ml q.s. deionized water. The solution was heated (60°C, 1 h) and stirred (magnetic stirring, 800 r.p.m.) to dissolve the insoluble components and form a pale yellow solution. The solution was refrigerated for use as needed within 1 week. During each experiment, it was diluted fourfold to prepare the diluted antioxidant buffer (DAOB) that did not require heating.

Preparation of standard sulfide solutions

A standard solution of NaSH (1000 μM) was made by adding 0.056 g of NaSH to 100 ml DAOB. This stock solution was stored under refrigerated conditions for up to 30 days which was further diluted as needed for the development of a standard curve.

Measurement of H₂S content

A Shelf Scientific Lazar electrode was soaked in the lowest NaSH concentration (e.g., 0.1 μM) for 30 min. Then, the electrode was rinsed with deionized water and baseline measurement was recorded with DAOB. Subsequent solutions of NaSH were added from lowest to highest concentration and a stabilized reading for each concentration was recorded. A final graph was plotted using the signal (mV) of each dilution (μM).

Amperometric technique

Preparation of 1.0 mM NaSH standard solution

First, 5 mg of ethylenediaminetetraacetic acid was dissolved in 100 ml of distilled water. The solution was purged vigorously with argon gas for 15 min. NaSH (5.6 g) was dissolved in the argonated ethylenediaminetetraacetic acid solution (100 ml) under an argon atmosphere. The flask was sealed with a rubber stopper, kept away from light and refrigerated (2–8°C) until further use.

Calibration of amperometry-based H₂S electrode

A WPI ISO-100-H₂S sensor was immersed and polarized in 20 ml of 0.1 M PBS for 12 h. Calibration and subsequent measurements were performed maintaining the same temperature and salinity of the solution. The polarized sensor was then immersed in 20 ml 0.05 M PBS and plugged into the free radical analyzer. The range was set to 10 nA and the poise voltage to +150 mV. A magnetic stirrer was placed in the 20-ml vial containing 0.05 M PBS, which was placed on a magnetic stirring plate. Measurements were taken once the background current due to the 0.05 M PBS was stabilized. Into the vial containing 20 ml of PBS solution, four aliquots of the NaSH standard solutions (5, 10, 20 and 40 μl) were injected sequentially. A plot of current output (nA) versus H₂S content (nM) was plotted.

Results

Colorimetric method

The selectivity of a method in analyzing the compound of interest and not eliciting signal for other compounds is termed specificity. Supplementary Figure 1 represents the UV spectrum from 400 nm to 800 nm of ethylene diamine solution containing NaSH. The detection of a peak at 671 nm indicated the presence of ethylene blue with maximum absorption. The absence of any additional peaks or interference in the spectrum suggests that the method used is specific for detecting NaSH.

For linearity assessment, a concentration range was identified where the response of the analyte was directly proportional to its concentration. The standard curve depicted in Supplementary Figure 2 was linear (R² = 0.9987) within the range of 1.5 to 100 μM.

Precision refers to the ability of a method to produce consistent and reproducible results. Interday precision was assessed by analyzing four standard curves on the same day, while intraday precision was determined by analyzing the same over 5 days during a 30-day period, with the solutions kept refrigerated. The relative standard deviation (RSD) values were calculated for both intraday and interday precision and ranged from 1.39 to 5.84% and 6.67 to 12.23%, respectively, for all concentrations in the range of 1.5–100 μM, as shown in Supplementary Table 2. The higher RSD observed for interday samples compared with those of intraday samples can be attributed to using the same reagents at different times of the day whereas fresh reagents were used in intraday experiments.

The accuracy of the method was assessed by measuring three samples of known strengths and was found to be over 94%, as shown in Supplementary Table 3.

An experiment was conducted to investigate the effect of toluene on preventing the oxidative loss of H₂S. The results showed a significantly (p < 0.05) greater amount of H₂S in the sample compared with the control at all time points, with a biphasic pattern observed in the sample. The concentration of H₂S decreased at a slower rate for up to 5 days, after which a faster rate of decrease was observed (as shown in Supplementary Figure 3).

Chromatographic technique

A comparison of the chromatograms obtained from a blank sample and a standard solution sample showed a specific peak without any overlapping adjacent peak(s) at 670 nm with a retention time of 3.3 min (Figure 1).

Figure 1. . Chromatogram depicting specificity for ethylene blue complex.

The graph shown in Figure 2 demonstrates that the chromatographic quantification method was linear in the range of 0.1–3.5 μM concentrations with a high degree of correlation as indicated by an R² value of 0.9982.

Figure 2. . Calibration curve for quantification of hydrogen sulfide using chromatographic technique.

The precision of the quantification method was evaluated by determining intra- and interday variability. The RSDs for both intra- and interday measurements were less than 5% (Table 1).

Table 1. . Mean peak area for each concentration of hydrogen sulfide obtained in chromatographic technique (n = 3).

Concentration (μM)	Interday		Intraday
	Mean peak area (×103)	% RSD	Mean peak area (×103)	% RSD
0.54	14.6 ± 1.0	7.00	15.6 ± 0.5	3.12
0.89	23.8 ± 0.7	2.74	24.1 ± 0.1	4.09
3.75	100.5 ± 3.4	3.41	99.1 ± 0.3	0.31
7.68	216.6 ± 5.7	2.66	223.2 ± 11.4	5.12
15.18	497.5 ± 37.17	7.47	416.1 ± 39.1	9.44
30.36	983.3 ± 32.4	3.30	902.1 ± 38.9	4.31
60.71	1857.3 ± 81.8	4.40	1829.1 ± 75.8	4.15

RSD: Relative standard deviation.

To determine the accuracy of the measurement method, three quality control samples of known concentrations of H₂S equal to 1.96, 9.10 and 36.41 μM were analyzed. Accuracy was calculated by comparing the measured concentration to the actual known concentration of the samples and expressed as a percentage as shown in Supplementary Table 4.

Electrochemical techniques

Voltametric technique

A plot of log concentration (μM) versus voltage (mV) over the range of 0.3–2.17 μM indicated a linear relationship with a correlation coefficient of linearity of R² equal to = 0.9983 (Figure 3).

Figure 3. . Calibration curve for hydrogen sulfide for voltametric technique.

The %RSDs of the inter- and intraday variations were less than 1.1% as indicated in Table 2.

Table 2. . Corresponding voltage for each concentration of hydrogen sulfide solution obtained in voltametric technique.

Log concentration (μM)	Intraday		Interday
	Voltage (mV)	% RSD	Voltage (mV)	% RSD
2.17	-412.33	0.28	-406.75	0.86
1.47	-408.33	0.14	-403	1.03
1	-406.33	0.14	-401.5	0.96
0.69	-405.33	0.14	-400.5	0.97
0.30	-404.00	0.25	-398.5	0.97

RSD: Relative standard deviation.

The accuracy of this method was determined by injecting three quality control samples of known H₂S concentrations of 300, 75 and 50 μM. The percentage error was less than 7% as shown in Supplementary Table 5.

Amperometric technique

Linearity was evaluated over the range of 0–2000 nM. The plot of the concentration of H₂S in nM against the corresponding voltage in picoamperes (pA) was linear (R² = 0.9992) over the range of 0–2000 nM (Figure 4).

Figure 4. . Calibration curve for hydrogen sulfide for amperometric technique.

Precision was measured on an intraday and interday basis. The average RSDs for intra- and interday variability were less than 8% (Table 3).

Table 3. . Corresponding current for each concentration of hydrogen sulfide obtained by amperometric technique.

Concentration (nM)	Intraday		Interday
	Current (pA)	% RSD	Current (pA)	% RSD
0	0	0	0	0
249.93	906.5	5.2	1178.0	0.6
499.62	2146.5	1.7	2908.0	4.1
996.26	4159.5	6.8	4663.5	2.4
1992.00	7816.5	7.1	8137.5	1.8

RSD: Relative standard deviation.

To assess the accuracy of the measurement technique, three quality control samples with known concentrations of NaSH (300, 75 and 50 μM) were analyzed. The percentage error was less than 10% (Supplementary Table 5), which falls within acceptable limits for accuracy assessment.

Discussion

The classical colorimetric technique of H₂S quantification using methylene blue assay has been adopted by several researchers owing to its feasibility and cost-effectiveness [56,57]. The working principle of this method involves a reaction between sulfide (S^2-) and an acidified solution of N,N-dimethyl-p-phenylenediamine sulfate (DPD) in the presence of an oxidizing agent ferric chloride (FeCl₃) that produces a colored methylene blue complex [58]. The intensity of the color produced, which can be determined by measuring its absorbance using a spectrophotometer, is used to calculate the quantity of sulfide present in the medium.

However, this method needs a preparation time of 30–60 min for the formation of the colored methylene blue complex, which could lead to erroneous H₂S quantification due to its volatility and limited temperature-dependent aqueous solubility as well as instability. Moreover, the dissolved oxygen present in aqueous medium reacts instantaneously with H₂S, resulting in the production of sulfur dioxide and consequently leading to a considerable reduction in the amount of H₂S detected [59]. We found that strategies such as nitrogen purging, the use of antioxidants and maintaining the colored complex at lower temperatures are inefficient at completely improving detection limits [59]. Therefore, we adopted three subtle modifications in the classic methylene blue method. First, instead of DPD, its diethyl derivative was used. Second, we used toluene in the release medium to prolong the period during which H₂S could be quantified more accurately. The release of H₂S in the presence of toluene was biphasic and we hypothesize that this biphasic pattern was due to changes in toluene concentrations over time, which we confirmed by measuring the absorbances of a toluene solution. A decrease in absorbance was observed between days 1 and 13. Therefore, we repeated the addition of toluene on day 5 to minimize the loss of H₂S. Lastly, the diamine reagent was maintained at an acidic pH (<2) to avoid oxidation of H₂S, which is more susceptible to oxidation at pH over 6 [60].

In addition to this modified colorimetric technique, we also developed a reverse-phase HPLC (RP-HPLC) method for quantifying H₂S in aqueous solution. There are many methods in the literature employing HPLC for quantification of H₂S in tissues and plasma [61–63]. These methods use monobromobimane reagent, which requires long and tedious preparation steps with long retention times [64]. Hence, we paired our modified ethylene blue complex strategy with the HPLC method. The goal of using HPLC over the colorimetric technique was to achieve faster and more accurate quantification of H₂S. The chromatographic peak depicted in Figure 4 has a significant peak tailing, however, the linearity between peak area as well as the peak height versus concentration was linear at all concentrations studied with R² value >0.99. In addition, there was no interference from the blank, indicating that the method was specific to H₂S. These findings allowed us to proceed with the analysis without a perfect chromatographic peak shape. The method also passed other validation tests. It conforms to the standards set by USP, which state that an analytical procedure must be able to produce test results that are directly proportional to the concentration of the substance being analyzed or can be transformed mathematically to show this relationship, within a specific range. The percentage error was less than 10%, which falls within acceptable limits.

We also evaluated two commercially available electrochemical techniques and compared them with the colorimetric and chromatographic methods developed in our laboratory. Ion-selective electrochemical methods are now being widely used in the quantification of H₂S, owing to their ability for real-time detection/quantification along with faster response, higher sensitivity, accuracy and reciprocity [65,66]. The voltametric technique used in this study utilizes an ion-selective electrode to measure sulfide and H₂S in biological solutions. H₂S in aqueous solutions follows an equilibrium with sulfide and bisulfide [67]. The equilibrium is inclined toward H₂S in acidic solution while alkaline solutions have more sulfide content. Since H₂S is more toxic than sulfide, the solutions in this study were maintained at alkaline pH. Hence, pH is an important parameter and must be kept constant across different measurements. The method met precision criteria on an interday and intraday basis according to the acceptable ICH range. The amperometric technique using a WPI ISO-H₂S-100 microelectrode is an H₂S-specific quantification technique with WPI's proprietary combination electrode technology containing an H₂S sensing material and a reference electrode.

The modified colorimetric technique using ethylene blue complex offered several advantages over the conventional methylene blue method [68]. The formation of the ethylene blue complex is based on 2:1 stoichiometry between DPD and sulfide (Figure 8) [60]). It has advantages such as less toxicity compared with its parent compound, higher molar absorptivity (87,700 mol^-1 l cm^-1) than methylene blue (71,090 mol^-1 l cm^-1) and less deviation from Beer's law due to a lower tendency to form dimers and trimers [69]. The use of toluene in the preparation of STF resulted in the detection of 1.5-times more H₂S. Moreover, the dual addition of toluene on days 1 and 5 was significant (p < 0.05) in maintaining higher sulfide concentrations in STF. Additionally, FTIR and UV studies confirm there is no reaction between toluene and STF or H₂S. Hence, the efficiency of the colorimetric method for the detection and quantification of H₂S can be improved with the combined use of these two strategies. Although this method can offer an accessible way to detect H₂S, it is associated with several drawbacks, such as time involved in the development of the colored complex, detection limit only up to mM range and involvement of multiple steps increasing the exposure of H₂S to the atmosphere leading to low accuracy.

The chromatographic technique is an improvement over the colorimetric method since it offers higher sensitivity (0.5–60 μM vs 1.5–100 μM; Table 4). The sample volume requirement (0.5–2 ml) is also lower than that for the colorimetric method (2–3.5 ml). The sample preparation time for the HPLC method is reduced to 10 min and the specificity of this method is also superior to the colorimetric method. Although many of the limitations of the colorimetric method are overcome by the HPLC method, the latter requires a more expensive setup than the prior method. The preparation time of 10 min could still be detrimental for some experimental setups. Run time may vary from minutes to hours based on the equipment and the nature of the experiment. Although the sample requirement of 0.5–2 ml is better than that for the colorimetric method, it is still higher than is possible for some experiments. Finally, the method does not allow real-time quantification of H₂S. Table 4 provides a brief comparison of the methods compared bin this work.

Table 4. . Comparison of hydrogen sulfide quantification techniques.

Parameter	Colorimetric	Chromatographic	Voltametric	Amperometric
Range	1.5–100 mM	0.5–60 μM	30–1000 μM	0.25–5 nM
Response time	30 min	10 min	20–60 s	20–60 s
Detection limit	1000 nM	100 nM	400 nM	5 nM
Linearity	0.9987	0.9982	0.9983	0.9992
Minimum detection volume	2–3.5 ml	0.5–2 ml	<1 ml	<1 ml

The electrochemical techniques are further improvements over the chromatographic method. First, the preparation time for samples was largely reduced, from minutes to seconds. Second, the smaller size of the electrodes allows real-time analysis of sulfide content. Since only the electrode tips require contact with the sample, as low as 100–200 μl of sample volume is sufficient to obtain a measurement. The voltametric technique employed in this study detected sulfide content in the μM range, like the colorimetric and chromatographic methods, although the response time and needed sample volume were significantly lower. The analytical sensitivity was further improved using the amperometric technique, which can detect H₂S at the nM level. Thus, all the limitations of colorimetric and chromatographic techniques identified earlier can be overcome using sophisticated electrochemical and amperometric techniques.

Conclusion

Colorimetric, chromatographic, electrochemical and amperometric methods offer high specificity when used and compared for the detection and quantification of H₂S. All four methods can be used for aqueous solutions as well as biological samples. Although both the colorimetric and HPLC methods were capable of quantifying H₂S in micromole ranges, the HPLC method needed less sample and was sensitive to lower micromolar ranges of sample concentrations. Furthermore, the electrochemical methods were able to quantify H₂S in the micromolar range but at a faster rate using less sample. Finally, the amperometric technique was sensitive to the nanomolar range, thus more suitable for biological samples. Additionally, the electrochemical and amperometric methods were not dependent on the development of any colored complex. Therefore, they were less time-consuming, minimizing error due to any loss/degradation of H₂S in the aqueous samples. The electrochemical/amperometric techniques have higher accuracy than other methods but can be expensive. Therefore, the H₂S quantification method should be selected based on the specific requirements of a research project as the methods differ widely in sensitivity, response time and cost-effectiveness. The findings of this study should help researchers choose an appropriate H₂S quantification method suitable to a specific experimental need.

Future perspective

The low potential therapeutic concentration range (100–100 μM) [47] of H₂S warrants an efficient controlled-release delivery system, which requires an ultrasensitive detection and quantitation method to validate its release profile. However, its volatility and instability pose significant challenges in such endeavors. Moreover, being gaseous, it can be released rapidly and cause handling problems. Thus, an effective method to accurately measure H₂S concentration in each sample is important for this gaseous molecule that can be lost easily to the environment. Literature suggests that approaches such as gas chromatography and ion chromatography [70], fluorescent probes [71], methylene blue assay [72] and monobromobimane assay [73] with HPLC, ion-selective or polarographic electrodes [74] can be used for the detection of H₂S [75]. However, the primary limitation posed by these approaches is their inability to measure the real-time concentration of H₂S while achieving the necessary sensitivity and response time.

Executive summary.

• This work compared four analytical methods for hydrogen sulfide (H₂S) quantification. Colorimetric and chromatographic methods quantified H₂S in the millimolar and micromole ranges, respectively. The electrochemical methods quantified H₂S in the nanomole and picomole ranges and were less time-consuming.

• The classical methylene blue colorimetric technique for H₂S quantification has limitations due to its preparation time, volatility and interference from dissolved oxygen. Strategies such as nitrogen purging and antioxidants have shown limited effectiveness in improving detection limits.

• The authors made three subtle modifications to the method: using N,N-diethyl-p-phenylenediamine (DEPD) instead of N,N-dimethyl-p-phenylenediamine (DMPD), adding toluene to prolong the period for accurate quantification and maintaining the diamine reagent in an acidic pH (<2) to prevent oxidation of H₂S, which improved the accuracy and sensitivity of the H₂S quantification process.

• A reverse-phase HPLC method offered faster and more accurate quantification compared with traditional colorimetric techniques.

• Electrochemical techniques did not rely on the formation of colored complexes and were less time-consuming.

• The voltametric technique utilized an ion-selective electrode to measure sulfide and H₂S in biological solutions, with alkaline pH maintained to prioritize H₂S detection.

• The amperometric technique using a WPI ISO-H2S-100 microelectrode demonstrated H₂S-specific quantification, meeting precision criteria within the acceptable International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use range for interday and intraday measurements.

• Electrochemical/amperometric techniques have higher accuracy, are less time-consuming and require a smaller sample size than other methods but can be expensive.

• The H₂S quantification method should be selected based on the specific requirements of a research project as the methods differ widely in sensitivity, response time and cost-effectiveness.