A reaction named the Wronski reaction  which describe the complexometric reaction occurred between a mercuriated compounds and the sulfur containing compounds is happened here between a mercuriated derivative of fluorescein (AMF) (a reagent with green fluorescence) , and mercury complexing agents such as sulfides, arising in quenching of its fluorescence. Upon the reaction is occurred, AMF is changed over to weak fluorescent ones. This is due to the alteration within the chromophore structure of the reagent particle. For encourage clarification of the reaction mechanisms, it was presumed that anions which can shape stable Hg2+ complexes would replace the acetoxy moiety in AMF to make a solid chelate with Hg2+ cation . The proposed pathway is shown in scheme 1. Figure 4 appears the fluorescence quenching of the reagent within the nearness of IXA. The quenching pathway was examined by developing Stern-Volmer plot. It is a plot that appears a connection between (Io/I) and the quencher concentration. A linear curve was achieved upon plotting (Io/I) against concentration of the drug which demonstrates either inactive or energetic quenching happens in an inactive mechanism, as the quencher got to be a portion of the complex shaped amid the chemical reaction agreeing to (Eq. (1)) which speaks to a ground-state quenching model [19, 20]. This association constant Ka was determined and it is 0.1079. Io/I = 1 + Ka[Q] (1) Io is the fluorescence intensity of AMF in nonattendance of quencher whereas I is its fluorescence intensity in nearness of the quencher. Ka is the association constant and [Q] is concentration of the quencher (drug) .
3.1 The stoichiometry of the reaction
The Continuous Variation Method (Job's Approach)  has been generally utilized with isomolar solutions to examine the complexation cases in these solutions and to decide the transcendent complexes of the reaction. It was accepted in this work to examine the reaction stoichiometry between IXA and AMF. Iso-molar concentrations of IXA and AMF (1 x 10−4 M) solutions were arranged. Precisely measured various volumes from (1 x 10−4 M) stocks of each IXA and AMF were included together into a set of test tubes in numerous proportions to get a volume of 5 mL. A connection between the achieved fluorescence difference and the proportion between the drug and the reagent was outlined in Job's plot (Figure. 5). It showed that 2.0 mol of IXA were required to full the quenching reaction of 1.0 mol of AMF, so the stoichiometric ratio between (drug: AMF) was (2:1), so it can be clarified by the trade of two acetoxy moieties in AMF by two moles of IXA  Scheme 1.
3.2 Optimization of the reaction parameters
Various parameters influencing the reaction were optimized to have the most sensitivity, counting concentration of AMF reagent solution, temperature, ideal pH, time and weakening solvents. The resultes of optimization of the reaction parameters are appeared in Tables 1 & 2.
3.2.1. AMF concentration
The impact of AMF solution concentration was considered utilizing various volumes (0.1–2 mL) of 1 x 10−4 M AMF to respond with a certain concentration of IXA in a solution of 10-mL volumetric flasks. The flasks' substance was blended and completed to the line with methanol and waiting for 10 min at room temperature. The fluorescence contrast was observed, at λem 530 nm, for each test solution against a fresh prepared blank solution for each estimation. The connection between AMF volume and the fluorescence contrast of the reaction blend was shown to in (Figure 6). It uncovered that; 1.0 ± 0.2 mL of 1 x10−4 M AMF was appropriate for the proposed approach.
The ideal temperature for total quenching was considered by warming the reaction blend at various temperatures (40–100 °C), and its impact on the fluorescence quenching is shown in (Figure 7). This appeared that, the greatest fluorescence quenching was achieved at room temperature, whereas it remained nearly consistent when the temperature was raised up to 60 °C, while diminished at temperatures over 60 °C and up to 100 °C. The diminish in fluorescence quenching at great temperature may be due to the separation of the shaped weak complexes that are greatly important for quenching the fluorescence .
The pH plays a vital part within the sensitivity of this reaction. The impact of pH on quenching the fluorescence was examined in the pH area (5–9) utilizing the universal Britton Robinson buffer. The connection between various pH and comparing fluorescence contrast in (Figure 8) appeared that the most extreme sensitivity was achieved within the solution's pH 6.4. This data is due to the reality that at pH ranges from 6 to 7, AMF appeared exceptionally solid fluorescence. This could be due to the nearness of AMF as a doubly charged anion. It was moreover found that upon diminishing the pH underneath 6.0 or increasing it past 7.0, a drop within the fluorescence intensity of AMF happened leading to diminish within the predictable quenching by the addition of IXA.
3.2.4. The reaction time
The impact of time on the quenching of the fluorescence of AMF by IXA was considered by calculating the reactions each 5 min for 45 min, and it was shown in (Figure 9). The results shown that the overall reaction and consequently the greatest sensitivity was achieved after 10 ± 2 min, past which there were nearly slight changes within the measured fluorescence.
3.2.5. Weakening solvent
The impact of various weakening solvents was followed after the same approach. Various solvents of different polarities were attempted counting: chloroform, isopropanol, methanol, dimethylformamide (DMF) and refined water. It was found that the chief solvents to be utilized for achieving highest sensitivity at 530 nm was methanol. Typically due to the low energy gap among methanol vibrational energy levels related to water, so sensitivity in case of methanol is greater .
3.3 Validation of the proposed spectrofluorimetric method
The established method has been validated according to ICH guidelines . All validation parameters are shown in Tables 3–5.
3.3.1. Linearity range
The linearity of the proposed approach was built up beneath the already optimized conditions employing a set of solutions of various concentrations. A calibration curve (Figure 10) was built to show the relationship of the fluorescence contrast between the signals of blank solutions of AMF and those achieved after reaction of IXA to the comparing drug concentrations in ng mL−1 which was found to be direct within the area of (20–100 ng mL−1). Regression analysis was achieved by least squares analysis of the calibration results to determine the relation coefficient (r), slope (b), intercept (a), standard deviation of slope (Sb) and standard deviation of intercept (Sa) (Table 3), which confirmed acceptable linearity of the proposed approach as shown by the high relationship coefficient (r > 0.9998), % RSD of the slope (Sb% < 2%) and the small value of significance F that shown a small grade of empirical points diffusing around the regression line.
3.3.2. Limit of detection (LOD) and limit of quantitation (LOQ)
LOD is considered as the concentration which can be spoken to by 3 S/m and LOQ by 10 S/m, where, S is the standard deviation and m is the slope of the calibration line. The values of LOD and LOQ displayed in (Table 3) affirmed the sensible sensitivity of the proposed approach in qualitative and quantitative analysis of IXA.
3.3.3. Accuracy and precision
To evaluate the reliability and repeatability of the proposed approach, the precision and accuracy of estimations have been assessed as beneath the main method. Three readings at each concentration level were done (Table 4). Recovery % and RSD % were determined for each level. The resultes were inside the satisfactory limits of 98–103% and 2% for recoveries and RSD% separately. The intra-day and inter-day precision were evaluated utilizing concentrations inside the linearity area, on the same day and on three distinctive days individually. The little RSD % shown the great precision of the proposed approach (Table 4) and affirmed the reliability of the approach for quality control tests of IXA.
The already detailed approach was performed beneath little varieconnects within the optimized parameters such as volume of AMF solution (± 0.2 mL) and the reaction time (± 2 min). Low RSD% values appeared in (Table 5) affirmed that little varieties within the previously detailed had no critical impact on the analysis of IXA by the recommended approach.
3.4. Analytical applications
3.4.1. Pharmaceutical preparation
The proposed approach was practiced for the assurance of IXA in Ixempra® vials. The resultes achieved are appeared in (Table 6). Recovery was achieved by applying the standard addition technique where various concentrations of standard IXA solution (40-80 ng) were included to already analyzed Ixempra® vials. There was no obstructions from co-formulated excipients. Statistical analysis of the resultes achieved by the proposed approach and those achieved by the reported approach  was done utilizing the student's t-test and the variance ratio F-test (Table 7). The calculated values didn't pass the hypothetical ones showing no significant difference between the proposed approach and the reported one with respect to precision and accuracy.
3.4.2. In plasma
The sensitivity of the proposed spectrofluorimetric approach permitted the analysis of IXA drug in spiked human plasma. To defeat lattice interferences, tests were subjected to a clean-up method. In this regard, acetonitrile was utilized for protein precipitation. Three concentrations were spiked for the drug and spiked concentration was reproduced three times to affirm the accuracy and precision of the proposed approach. The recoveries were calculated and they were between 95–97% (Table 8). Appropriately, this work about spiked plasma tests propose that the proposed approach is performed for the in vivo test of the drug in real biological samples.