A Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method

A spectrouorimetric approach has been developed and validated for determination of sulfur-containing drug; ixabepilone in raw powder, vials and human plasma. This approach studies the quenching effect of IXA on the uorescence intensity of acetoxymercuric uorescein (AMF) reagent at λ em of 530 nm and λ ex of 500 nm. All the parameters that can affect the reaction as pH, AMF solution concentration, temperature, time and solvents were studied and optimized. The linearity range of the studied approach was 20-100 ng mL -1 with correlation coecient of (r = 0.9998). The proposed approach was validated and approved regarding to ICH guidelines in terms of accuracy, precision, linearity, LOD and LOQ, with mean percentage recovery of 99.79 and RSE% of 1.64. The previously obtained resultes were already statistically compared with that of established reported methods indicating no signicant differences in accuracy and precision. Finally, the proposed approach is easy, sensitive, and inexpensive so it is suitable for routine determination of IXA in raw powder, vials and human plasma with no need for any prior separation or sample extraction.

) is an orally bioavailable semisynthetic analogue of epothilone B with antineoplastic activity, a natural chemical compound produced by Sorangium cellulosum [2]. Epothilone B itself might not be created as a pharmaceutical drug since of low metabolic stability and pharmacokinetics [3]. The epothilones parallel taxanes in that they connect to β-tubulin and trigger microtubule nucleation at numerous spots farther from the centriole. This chaotic microtubule stabilization triggers cell-cycle capture at the G2-M interface and apoptosis. Epothilones connect to a location de nite from that of taxanes. In colon cancer cell lines, p53 and Bax trigger apoptosis in ixabepilone-treated cells. In vitro application, advise that ixabepilone is less inclined to P-glycoprotein-mediated multidrug resistance when compared to taxanes. Other instrument involved in epothilone resistance incorporate mutation of the β-tubulin active site of binding and upregulation of isoforms of βtubulin [4]. Ixabepilone was constructed through medicinal chemistry advanced upon these properconnects [3]. It is very potent, able of harming cancer cells in exceptionally low concentrations, and holds action in cases where tumor cells are heartless to taxanes brand drugs [5]. As with the taxanes and other agents that target tubulin, the epothilones, counting ixabepilone, connect to the btubulin subunits of microtubules to initiate microtubule polymerization and stabilization, which lead to capture of cells within the G2-M stage of the cell cycle and the initiation of apoptosis ( Figure 2). A lack of chemical methods deduced for determining of IXA, rather than LC [6][7][8], appeared in the literature as enlisted in this review.
Non-uorescent compounds holding sulphide or sulphydryl moieties, were determined quantitatively with acetoxymercuric uorescein (AMF), a mercuric acetate substituted uorescein; which consider a widely used uorescent agent, depending on the reaction of Hg 2+ incorporated in (AMF) with the sulfur containing groups in the analyzed compounds ( Figure 3) [9], this reaction decreases the intensity of the (AMF) uorescence that measured quantitatively with the tested compounds [10][11][12]. Many compounds successfully determined quantitively using this method such as mesna, acetylcysteine, timonacic corrosive [13], penicillamine [14] and mirabegron [15]. In this study, the reaction of IXB with its sul de group with AMF and the quenching effect on the uorescence were measured spectro uorimetricaly at ( em 530 nm) [9]. It is worth to mention that there is no publication conducted for the IXA assay spectro uorimetricaly either in bulk, dosages forms or human biological uids.
This work aimed to construct a spectro uorimetric method privileged with validity, sensitivity, simplicity and reliability along with the advantages of being costly effective and rapid when compared with other widely used techniques, for the purpose of quantitative determination of IXA in bulk, pharmaceutical dosages forms or human biological uids.
In spite of the non-existence of a procedure conducted for the assay of IXA spectro uorimetricaly until now, more improvements needed eagerly to attain more suitable conditions and better analytical performance.

Instrumentation
All the measurements were carried out on Agilent Cary Overshadow Fluorescence Spectro uorimeter (USA); prepared with a 150 W xenon streak light and 1 cm quartz cell were utilized. The excitation and emanation opening width was 10 nm, worked with Cary overshadow check application program adaptation 1.2. pH estimations were made with HANNA pH 211 Chip pH Meter with two-fold intersection glass anode. Digital pH meter 3310 Jenway.

Materials and reagents
Ixabepilone (IXA) was gifted from Bristol-Myers Squibb (USA, Akhenaton o ce (Egypt)). Acetoxymercuric uorescein (AMF), 1x10 -4 M solution was made by dissolving 82.3 mg of AMF raw material in 20 mL of 0.1 N NaOH, weakened with 100 mL of 0.1 M boric acid solution and the volume was completed to 1.0 L utilizing re ned water [9], and the solution is suggested to be kept secured from light in fridge. Britton Robinson buffer utilized in optimization trials was made by infusing match volumes of boric acid (0.1 M), phosphoric acid (0.1 M) and acetic acid (0.1 M) in a 100 mL volumetric ask at that point the pH was adapted within the wanted area (5-9) by including acceptable volumes of sodium hydroxide (0.1 N) [16]. Methanol, ethanol, isopropanol, chloroform and dimethylformamide (DMF) solvents were acquired from El-Nasr Co. Egypt. All reagents and solvents utilized were of analytical grade.
A fresh arranged bi-distilled water was utilized through all tests. Ixempra® vials 45 mg per vials (Batch no. 69019) is a brand of Bristol-Myers Squibb (USA, Akhenaton o ce (Egypt). Plasma was achieved from Minia University Hospital, blood bank, Minia, Egypt and were kept solidi ed until utilize after delicate defrosting.

Preparation of standard stock solution
Standard stock solution of IXA (0.25 mg mL -1 ) was prepared by dissolving 0.025 of powder in 100 mL methanol and kept in fridge.

Spectro uorimetric procedure and construction of the calibrated curve
The proposed approach was practiced beneath the optimized conditions that will be examined afterward. Precisely measured volumes of the stock solution were relocated into a set of 10-mL volumetric asks to achieve a IXA concentration area of 20-100 ng mL -1 followed by the inclusion of 1.0 mL of 1 AMF. The solutions were blended well applying a vortex and left to stand at room temperature for 10 min. Each ask was weakened quantitatively with methanol. The uorescence intensity was detected at λ em of 530 nm after excitation at λ ex 500 nm. At that point the uorescence change was determined by subtracting the uorescence intensity of the reaction admixtures from the comparing values of so also treated blank (a solution contains 1.0 mL of AMF reagent and weakened with methanol). A calibration curve detailing the uorescence contrasts at λ em 530 nm to the comparing drug concentrations in ng mL -1 was developed.
2.5 Application procedures 2.5.1 Procedure for pharmaceutical preparation Ixempra ® vials: (45 mg per vial). An aliquot of 1 mL from the blended substance of Ixempra ® vials was precisely relocated to to a 100 mL volumetric ask and broken down in methanol, then the volume was completed to the line with methanol. 0.5 mL of this solution was weakened with methanol to earn an eventual IXA working solution concentration, then the approach was completed as already explained.

Procedures for spiked human plasma
One-milliliter aliquots of plasma were delocated into two solution of centrifuge tubes. The plasma were spiked with 0.1, 0.2 and 0.3 mL from 12.5 mg% stock solution of IXA. The tubes were blended well by employing a vortex blender. The solutions were deproteinized twice with acetonitrile. The centrifugation was done for 15 min at 8000 rpm. The centrifugates were delocated to new and clean centrifuge tubes then vaporized. The residues were transformed in to methanol and delocated to 5 mL volumetric asks and the volumes were adapted to the line with the same solvent. Aliquots of 2 mL from each solution were delocated to a 25 mL volumetric ask, the required volumes of buffer and AMF were taken and the volume was completed to the line with methanol. The relative uorescence intensities were measured utilizing the previous cited uorescence approach and subtracted from the comparing resultes of an essentially treated blank.

Results And Discussion
A reaction named the Wronski reaction [18] which describe the complexometric reaction occurred between a mercuriated compounds and the sulfur containing compounds is happened here between a mercuriated derivative of uorescein (AMF) (a reagent with green uorescence) [17], and mercury complexing agents such as sul des, arising in quenching of its uorescence. Upon the reaction is occurred, AMF is changed over to weak uorescent ones. This is due to the alteration within the chromophore structure of the reagent particle. For encourage clari cation of the reaction mechanisms, it was presumed that anions which can shape stable Hg 2+ complexes would replace the acetoxy moiety in AMF to make a solid chelate with Hg 2+ cation [17]. The proposed pathway is shown in scheme 1. Figure 4 appears the uorescence 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 uorescence intensity of AMF in nonattendance of quencher whereas I is its uorescence intensity in nearness of the quencher. Ka is the association constant and [Q] is concentration of the quencher (drug) [19].

The stoichiometry of the reaction
The Continuous Variation Method (Job's Approach) [20] 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 uorescence 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 clari ed by the trade of two acetoxy moieties in AMF by two moles of IXA [17] Scheme 1.

Optimization of the reaction parameters
Various parameters in uencing 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 asks. The asks' substance was blended and completed to the line with methanol and waiting for 10 min at room temperature. The uorescence contrast was observed, at λ em 530 nm, for each test solution against a fresh prepared blank solution for each estimation. The connection between AMF volume and the uorescence 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.

Temperature
The ideal temperature for total quenching was considered by warming the reaction blend at various temperatures (40-100 °C), and its impact on the uorescence quenching is shown in (Figure 7). This appeared that, the greatest uorescence 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 uorescence quenching at great temperature may be due to the separation of the shaped weak complexes that are greatly important for quenching the uorescence [21].

pH
The pH plays a vital part within the sensitivity of this reaction. The impact of pH on quenching the uorescence was examined in the pH area (5-9) utilizing the universal Britton Robinson buffer. The connection between various pH and comparing uorescence 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 uorescence. 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 uorescence intensity of AMF happened leading to diminish within the predictable quenching by the addition of IXA.

The reaction time
The impact of time on the quenching of the uorescence 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 uorescence.

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 re ned 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 [22].

Validation of the proposed spectro uorimetric method
The established method has been validated according to ICH guidelines [23]. All validation parameters are shown in Tables 3-5.

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 uorescence 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 coe cient (r), slope (b), intercept (a), standard deviation of slope (Sb) and standard deviation of intercept (Sa) ( Table 3), which con rmed acceptable linearity of the proposed approach as shown by the high relationship coe cient (r > 0.9998), % RSD of the slope (Sb% < 2%) and the small value of signi cance F that shown a small grade of empirical points diffusing around the regression line.

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) a rmed the sensible sensitivity of the proposed approach in qualitative and quantitative analysis of IXA.

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 a rmed the reliability of the approach for quality control tests of IXA.

Robustness
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) a rmed that little varieties within the previously detailed had no critical impact on the analysis of IXA by the recommended approach.

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 [6] 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 signi cant difference between the proposed approach and the reported one with respect to precision and accuracy.

In plasma
The sensitivity of the proposed spectro uorimetric 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 a rm 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.

Conclusion
In this work an easy, reproducible, and fast spectro uorimetric approach was created for the determination of IXA in bulk as well as in Ixempra® vials and human plasma. The approach depends on the measured uorescence quenching of AMF due to the presence of the sul de moiety in IXA. The approach was statistically validated with regard to precision, accuracy, linearity, LOD, LOQ and robustness. All parameters were established to be inside satisfactory limits. Linearity and area were found to be greatly speci c as they gave satisfactory recoveries and the correlation coe cient (r) was 0.9998. In addition, it is sensibly delicate and reasonable for dependable investigation of low concentrations of the IXA. Both inter-day and intra-day precisions were considered. Resultes of this experiment were found to be inside satisfactory. Subsequently, the proposed spectro uorimetric approach can be suggested to consider the pharmacokinetics of the drug in numerous preparations and combinations and human plasma. Mean of (n = 3) experiments for each parameter   Mechanism of action of ixabepilone. Ixabepilone binds to the b-tubulin subunits of microtubules to induce microtubule polymerization and stabilization, which lead to G2-M arrest and the induction of apoptosis.

Figure 4
Excitation and emission spectra of 1.0 mL (10-4 M AMF solution) in the absence and presence of (120 ng mL-1) IXA at 500 and 530 nm, respectively. a: Excitation blank of AMF, a-: Excitation of AMF+IXA, b: Emission blank of AMF and b-: Emission of AMF+IXA.  Effect of AMF volume on the uorescence difference, after the reaction with 60 ng mL-1 IXA at 530 nm.

Figure 7
Effect of temperature on the uorescence quenching 1 mL AMF after the reaction with 60 ng mL-1 IXA at 530 nm.

Figure 8
Effect of medium pH on the uorescence quenching of 1 mL AMF after reaction with 60 nm mL-1 IXA at 530 nm.

Figure 9
Effect of reaction time on the uorescence quenching of 1 mL AMF after the reaction with 60 ng mL-1 IXA at 530 nm.