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

A Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method Hesham Salem, Amany abdelaziz, Aliaa Gamal, Ramy El Sabaa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-931324/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 1 You are reading this latest preprint version Abstract A spectrofluorimetric 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 fluorescence intensity of acetoxymercuric fluorescein (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 coefficient 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 significant 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. Clinical Pharmacology Spectrofluorimetric approach Ixabepilone (IXA) Acetoxymercuric fluorescein reagent (AMF) quenching effect Vials Human serum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Ixabepilone (IXA) is (1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy 8, 8, 10, 12, 16-pentamethyl-3-[(E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-17-oxa-4-azabicyclo [14.1.0] heptadecane-5,9-dione [1] (Figure 1) 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 definite 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 β-tubulin [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 b-tubulin 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 [6-8], appeared in the literature as enlisted in this review. Non-fluorescent compounds holding sulphide or sulphydryl moieties, were determined quantitatively with acetoxymercuric fluorescein (AMF), a mercuric acetate substituted fluorescein; which consider a widely used fluorescent 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)fluorescence that measured quantitatively with the tested compounds [10-12]. 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 sulfide group with AMF and the quenching effect on the fluorescence were measured spectrofluorimetricaly at (ƛ em 530 nm) [9]. It is worth to mention that there is no publication conducted for the IXA assay spectrofluorimetricaly either in bulk, dosages forms or human biological fluids. This work aimed to construct a spectrofluorimetric 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 fluids. In spite of the non-existence of a procedure conducted for the assay of IXA spectrofluorimetricaly until now, more improvements needed eagerly to attain more suitable conditions and better analytical performance. Experimental 2.1 Instrumentation All the measurements were carried out on Agilent Cary Overshadow Fluorescence Spectrofluorimeter (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. 2.2 Materials and reagents Ixabepilone (IXA) was gifted from Bristol-Myers Squibb (USA, Akhenaton office (Egypt)). Acetoxymercuric fluorescein (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 refined 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 flask 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 office (Egypt). Plasma was achieved from Minia University Hospital, blood bank, Minia, Egypt and were kept solidified until utilize after delicate defrosting. 2.3 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. 2.4 Spectrofluorimetric 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 flasks 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 flask was weakened quantitatively with methanol. The fluorescence intensity was detected at λ em of 530 nm after excitation at λ ex 500 nm. At that point the fluorescence change was determined by subtracting the fluorescence 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 fluorescence 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 flask 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. 2.5.2 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 flasks 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 flask, the required volumes of buffer and AMF were taken and the volume was completed to the line with methanol. The relative fluorescence intensities were measured utilizing the previous cited fluorescence 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 fluorescein (AMF) (a reagent with green fluorescence) [17], 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 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 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) [19]. 3.1 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 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 [17] 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 λ em 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. 3.2.2. 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 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 [21]. 3.2.3. pH 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 [22]. 3.3 Validation of the proposed spectrofluorimetric method The established method has been validated according to ICH guidelines [23]. 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. 3.3.4. 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) 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 [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 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. Conclusion In this work an easy, reproducible, and fast spectrofluorimetric 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 fluorescence quenching of AMF due to the presence of the sulfide 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 specific as they gave satisfactory recoveries and the correlation coefficient (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 spectrofluorimetric approach can be suggested to consider the pharmacokinetics of the drug in numerous preparations and combinations and human plasma. Declarations Author Declarations - Ethics Approval and consent to participate Collection of samples was authorized by Ethics Committee of Minia Hospital. - Consent to Participate Accept - Availability of data and materials Available upon request - Competing interests No conflict of interests - Funding No funding was received for this work from any. - Author Contributions All authors contributed to the study conception and design. Data collection and analysis were performed by all authors. The first draft of the manuscript was written by Hesham Salem and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. - Acknowledgements Not applicable - Authors information - Compliance with Ethical Standards - Disclosure of potential conflicts of interest The authors declare that they have no conflict of interest. - Research involving Human Participants and / or Animals Not applicable - Informed consent Not applicable References https://pubchem.ncbi.nlm.nih.gov/compound/Ixabepilone . S., G., "Novel cytotoxic agents: epothilones". Am J Health Syst Pharm, (May 2008). 65 (10 Suppl 3)(doi: 10.2146/ajhp080089 . PMID 18463327.). Lee FY, Borzilleri R., Fairchild CR, Kamath A., Smykla R., Kramer R., Vite G, "Preclinical discovery of ixabepilone, a highly active antineoplastic agent". Cancer Chemother. Pharmacol., (December 2008). 63(1)(doi: 10.1007/s00280-008-0724-8 . PMID 18347795.): p. 157–66. Bareaton, L.L., R. Hilal-Dandan, and B.C. Knollmann, Goodman & Gilman's the pharmacological basis of therapeutics. 2018: McGraw-Hill Education New York. M. 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Q2 (R1), Validation of analytical procedures: Text and methodology, international conference on harmonization, November 2005, Geneva, http://www.ich.org/fileadmin/public_web_site/ICH_products/guidlines/quality/Q2_R1/step4/Q2_R1_guidlines.pdf . Tables Table 1 Detailed data of the optimization of the reaction parameters. AMF Con. (µg mL -1 ) Fluorescence Difference pH Fluorescence Difference Time (min.) Fluorescence Difference Temp. ( o C) Fluorescence Difference Diluting solvent Fluorescence Difference 0.1 65.689 5 18.6 0 207.987 0.5 215.564 6 47.387 5 352.123 Water 220.699 1 240.30 6.4 366.211 10 357.254 25 270.689 Methanol 260.898 1.5 115.958 7 116.057 15 352.68 40 235.865 Chloroform 15.785 2 70.032 8 22.288 20 356.68 60 237.868 DMF 17.789 9 27.998 25 355.964 80 188.547 30 351.871 100 59.98 35 355.329 40 351.985 45 354.259 Mean of (n = 3) experiments for each parameter Table 2 Assay parameters and conditions for determination of IXA by the proposed spectrofluorimetric method. Parameters Proposed method AMF concentration 1x10 -4 M AMF volume 1.0 mL Temperature 25 o C Time 10 min Diluting solvent Methanol pH 6.4 Table 3 Regression parameters and test results for the determination of IXA by the proposed spectrofluorimetric procedure. Parameters Spectral data λ ex & λ em (nm) 500 & 530 Linearity range (ng mL -1 ) 20-100 LOD (ng mL -1 ) 5.145 LOQ (ng mL -1 ) 16.987 Slope ± Sb 10.14± 0.99 Intercept ± Sb -105.81± 2.67 %RSD of Sb 1.39 Regression equation Intensity 530 = 10.14 – 105.81 Significance F 5.84 x 10 -6 Correlation coefficient (r) 0.9998 Table 4 Intra-day and inter-day accuracy and precision for the determination of IXA by the proposed spectrofluorimetric method. IXA (ng mL -1 ) Intra-day Inter-day Found ±SD (ng mL -1 ) Accuracy (%) Precision (%RSD) Found ±SD (ng mL -1 ) Accuracy (%) Precision (%RSD) 40 40.58±1.06 101.45 1.045 40.82±0.75 102.05 0.735 60 59.66±0.99 99.43 0.996 59.38±0.88 98.96 0.889 80 79.23±0.89 99.04 0.899 80.63±0.69 100.79 0.685 Table 5 Robustness of the proposed method for the determination of IXA Parameters %RSD AMF Volume (±0.2 mL) 1.04 Reaction time (± 2 min) 0.84 Mean of % RSD Table 6 Recovery of IXA by applying standard addition technique Ixempra ® vials (ng mL -1 ) Drug added (ng mL -1 ) Drug found (ng mL -1 ) % Recovery 80 40 40.74 101.84 80 60 60.34 100.56 80 80 81.74 102.18 80 100 98.49 98.49 Mean 100.77 RSD % 1.85 Table 7 Statistical analysis of the results obtained by the proposed and reported procedures for the determination of IXA in Ixempra ® vials Parameters Ixempra ® vials Proposed method Reported method [6] N a 5 5 Recovery % 101.62 100.94 SD 0.739 0.939 RSD% 0.716 0.937 t b (2.262) 1.27 F value b (5.05) 1.615 a Number of experiments. b The values in parenthesis are tabulated of t and F at (P = 0.05) Table 8 Statistical analysis of the results obtained by the proposed and reported procedures for the determination of IXA in human plasma Parameters IXA Proposed method Reported method [6] N a 5 5 Recovery % 99.79 100.94 SD 1.597 0.939 RSD% 1.643 0.937 t b (2.262) 1.676 F value b (5.05) 2.898 a Number of experiments. b The values in parenthesis are tabulated of t and F at (P = 0.05) Supplementary Files Scheme1.png Scheme 1. The proposed mechanism of the reaction between IXA and AMF. Cite Share Download PDF Status: Under Review Version 1 posted First submitted to journal 22 Sep, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-931324","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":54936147,"identity":"eb7e8053-aabd-4e60-aefa-61aa95038d6a","order_by":0,"name":"Hesham Salem","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACAxDxAMxkPsDA2ECslgQwky2BZC08BsRpMWdgf/ghoWIbAz//mm8SP3fYyDGwHz66AZ8WywYeY4mEM7cZJGe83SbZeybNmIEnLe0GXocd4GGQSGy7zWBw4+w2Cd62w4kNEjxmBLSwP/6R+O82g/2NM88k/xKnhcFMIrEBaAt/D5s0kbbwmFkkHLvNIHGDzdhati3NmI2wX9gf3/hQc5uBv//ww5tv22zk+NkPH8OrhUH+AZiqb5BIYJEAsdjwKkcB/AeYPxCvehSMglEwCkYSAAD+H0zptyi8pQAAAABJRU5ErkJggg==","orcid":"","institution":"Deraya University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hesham","middleName":"","lastName":"Salem","suffix":""},{"id":54936148,"identity":"dd01a721-f8a2-4e18-9823-db1c6f127b81","order_by":1,"name":"Amany abdelaziz","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amany","middleName":"","lastName":"abdelaziz","suffix":""},{"id":54936149,"identity":"71f8df51-8875-46f7-a391-e452519c65bc","order_by":2,"name":"Aliaa Gamal","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aliaa","middleName":"","lastName":"Gamal","suffix":""},{"id":54936150,"identity":"44cc404c-32d6-4d59-87da-59e8980404df","order_by":3,"name":"Ramy El Sabaa","email":"","orcid":"","institution":"Deraya University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ramy","middleName":"El","lastName":"Sabaa","suffix":""}],"badges":[],"createdAt":"2021-09-23 18:08:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-931324/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-931324/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14206369,"identity":"26b334b2-3e45-4b9d-b12c-ee837967b4cc","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33011,"visible":true,"origin":"","legend":"Chemical structure of ixabepilone","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/4b985c53ef2b697aa8c5c0fc.png"},{"id":14206379,"identity":"751a953c-874b-41a6-8eed-16da0c57a62e","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":605635,"visible":true,"origin":"","legend":"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. ","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/23c301f3cc89179de3631692.png"},{"id":14206457,"identity":"c0cf0a4d-bd64-4710-8a45-d27780354a4f","added_by":"auto","created_at":"2021-10-01 21:42:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55067,"visible":true,"origin":"","legend":"Chemical structure of Acetoxymercuric fluorescein reagent. ","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/826a5e08a9508d9d5a6bad5c.png"},{"id":14206460,"identity":"226d7307-46bd-47b4-88c3-a06066505f29","added_by":"auto","created_at":"2021-10-01 21:42:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":21382,"visible":true,"origin":"","legend":"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.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/6b17ec5c843fb1b1a0cde652.png"},{"id":14206372,"identity":"3ae78d50-f13a-4e17-8e12-c34fe70d4e23","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15146,"visible":true,"origin":"","legend":"Stoichiometry of the reaction of IXA (1x10-4 M) and 1x10-4 M) AMF by continuous variation (Job,s) method.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/824d9b70b9b2a55ef1cd71ea.png"},{"id":14206370,"identity":"04aae483-5e18-42f0-894c-ef40b64fb486","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15797,"visible":true,"origin":"","legend":"Effect of AMF volume on the fluorescence difference, after the reaction with 60 ng mL-1 IXA at 530 nm.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/d4b0197b5621a292426d95bc.png"},{"id":14206681,"identity":"dc8e100f-e52d-41c2-8be9-a95e91d178f4","added_by":"auto","created_at":"2021-10-01 21:45:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14033,"visible":true,"origin":"","legend":"Effect of temperature on the fluorescence quenching 1 mL AMF after the reaction with 60 ng mL-1 IXA at 530 nm.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/de3c1d3531fccfc66e3efde3.png"},{"id":14206377,"identity":"505241af-15b2-4253-b5a0-6dfd2653b207","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":17131,"visible":true,"origin":"","legend":"Effect of medium pH on the fluorescence quenching of 1 mL AMF after reaction with 60 nm mL-1 IXA at 530 nm.","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/2859e74de528ba0b7cfcd9aa.png"},{"id":14206375,"identity":"a206caed-dd7d-473c-8682-5402a1b64e34","added_by":"auto","created_at":"2021-10-01 21:39:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":11959,"visible":true,"origin":"","legend":"Effect of reaction time on the fluorescence quenching of 1 mL AMF after the reaction with 60 ng mL-1 IXA at 530 nm.","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/5d84d58c1de40677282628c4.png"},{"id":14206454,"identity":"259b096f-5ac4-43f9-a89a-6ff2f27661dc","added_by":"auto","created_at":"2021-10-01 21:42:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":17764,"visible":true,"origin":"","legend":"Calibration graph of IXA with 1x10-4 M AMF at λem 530 nm.","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/1dfad57e11268d3312853429.png"},{"id":14206736,"identity":"0493b433-e104-42b6-9788-70f59c8055a6","added_by":"auto","created_at":"2021-10-01 21:48:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1209068,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/b125da3c-f019-4bef-a68b-961b5ccb2f03.pdf"},{"id":14206732,"identity":"b91be178-ee5c-4e1a-af3a-0fb8ac1dbab9","added_by":"auto","created_at":"2021-10-01 21:48:48","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":113395,"visible":true,"origin":"","legend":"Scheme 1. The proposed mechanism of the reaction between IXA and AMF.","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-931324/v1/7dd2d152160e071bbba97f90.png"}],"financialInterests":"","formattedTitle":"\u003cp\u003eA Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIxabepilone (IXA) is (1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy 8, 8, 10, 12, 16-pentamethyl-3-[(E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-17-oxa-4-azabicyclo [14.1.0] heptadecane-5,9-dione [1] (Figure 1) is an orally bioavailable semisynthetic analogue of epothilone B with antineoplastic activity, a natural chemical compound produced by \u003cem\u003eSorangium cellulosum\u003c/em\u003e [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 \u0026beta;-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 definite 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 \u0026beta;-tubulin active site of binding and upregulation of isoforms of \u0026beta;-tubulin [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 b-tubulin 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 [6-8], appeared in the literature as enlisted in this review.\u003c/p\u003e\n\u003cp\u003eNon-fluorescent compounds holding sulphide or sulphydryl moieties, were determined quantitatively with acetoxymercuric fluorescein (AMF), a mercuric acetate substituted fluorescein; which consider a widely used fluorescent agent, depending on the reaction of Hg\u003csup\u003e2+\u003c/sup\u003e incorporated in (AMF) with the sulfur containing groups in the analyzed compounds (Figure 3) [9], this reaction decreases the intensity of the (AMF)fluorescence that measured quantitatively with the tested compounds [10-12]. 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 sulfide group with AMF and the quenching effect on the fluorescence were measured spectrofluorimetricaly at (ƛ \u003csub\u003eem\u003c/sub\u003e 530 nm) [9]. It is worth to mention that there is no publication conducted for the IXA assay spectrofluorimetricaly either in bulk, dosages forms or human biological fluids.\u003c/p\u003e\n\u003cp\u003eThis work aimed to construct a spectrofluorimetric 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 fluids.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In spite of the non-existence of a procedure conducted for the assay of IXA spectrofluorimetricaly until now, more improvements needed eagerly to attain more suitable conditions and better analytical performance.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e\u003cstrong\u003e2.1 Instrumentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the measurements were carried out on Agilent Cary Overshadow Fluorescence Spectrofluorimeter (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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Materials and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIxabepilone (IXA) was gifted from Bristol-Myers Squibb (USA, Akhenaton office (Egypt)). Acetoxymercuric fluorescein (AMF), 1x10\u003csup\u003e-4\u003c/sup\u003e 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 refined 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 flask 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\u0026reg; vials 45 mg per vials (Batch no. 69019) is a brand of Bristol-Myers Squibb (USA, Akhenaton office (Egypt). Plasma was achieved from Minia University Hospital, blood bank, Minia, Egypt and were kept solidified until utilize after delicate defrosting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Preparation of standard stock solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStandard stock solution of IXA (0.25 mg mL\u003csup\u003e-1\u003c/sup\u003e) was prepared by dissolving 0.025 of powder in 100 mL methanol and kept in fridge.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Spectrofluorimetric procedure and construction of the calibrated curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 flasks to achieve a IXA concentration area of 20-100 ng mL\u003csup\u003e-1\u003c/sup\u003e 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 flask was weakened quantitatively with methanol. The fluorescence intensity was detected at \u0026lambda;\u003csub\u003eem\u003c/sub\u003e of 530 nm after excitation at \u0026lambda;\u003csub\u003eex\u003c/sub\u003e 500 nm. At that point the fluorescence change was determined by subtracting the fluorescence 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 fluorescence contrasts at \u0026lambda;\u003csub\u003eem\u003c/sub\u003e 530 nm to the comparing drug concentrations in ng mL\u003csup\u003e-1\u003c/sup\u003e was developed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Application procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.1 Procedure for pharmaceutical preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIxempra \u0026reg; vials: (45 mg per vial). An aliquot of 1 mL from the blended substance of Ixempra \u0026reg; vials was precisely relocated to to a 100 mL volumetric flask 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5.2 Procedures for spiked human plasma\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne-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 flasks 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 flask, the required volumes of buffer and AMF were taken and the volume was completed to the line with methanol. The relative fluorescence intensities were measured utilizing the previous cited fluorescence approach and subtracted from the comparing resultes of an essentially treated blank.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eA 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 fluorescein (AMF) (a reagent with green fluorescence) [17], 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 Hg\u003csup\u003e2+\u003c/sup\u003e complexes would replace the acetoxy moiety in AMF to make a solid chelate with Hg\u003csup\u003e2+\u003c/sup\u003e cation [17]. 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) [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1 The stoichiometry of the reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Continuous Variation Method (Job\u0026apos;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\u003csup\u003e\u0026minus;4\u003c/sup\u003e M) solutions were arranged. Precisely measured various volumes from (1 x 10\u003csup\u003e\u0026minus;4\u003c/sup\u003e 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\u0026apos;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), \u0026nbsp;so it can be clarified by the trade of two acetoxy moieties in AMF by two moles of IXA [17] Scheme 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Optimization of the reaction parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVarious 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 \u0026amp; 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.1. AMF concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of AMF solution concentration was considered utilizing various volumes (0.1\u0026ndash;2 mL) of 1 x 10\u003csup\u003e\u0026minus;4\u003c/sup\u003e M AMF to respond with a certain concentration of IXA in a solution of 10-mL volumetric flasks. The flasks\u0026apos; substance was blended and completed to the line with methanol and waiting for 10 min at room temperature. The fluorescence contrast was observed, at \u0026lambda;\u003csub\u003eem\u003c/sub\u003e 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 \u0026plusmn; 0.2 mL of 1 x10\u003csup\u003e\u0026minus;4\u003c/sup\u003e M AMF was appropriate for the proposed approach.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.2. Temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ideal temperature for total quenching was considered by warming the reaction blend at various temperatures (40\u0026ndash;100 \u0026deg;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 \u0026deg;C, while diminished at temperatures over 60 \u0026deg;C and up to 100 \u0026deg;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 [21].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.3. pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026ndash;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\u0026apos;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.4. The reaction time\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 \u0026plusmn; 2 min, past which there were nearly slight changes within the measured fluorescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2.5. Weakening solvent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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 [22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Validation of the proposed spectrofluorimetric method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe established method has been validated according to ICH guidelines [23]. All validation parameters are shown in Tables 3\u0026ndash;5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1. Linearity range\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u003csup\u003e\u0026minus;1\u003c/sup\u003e which was found to be direct within the area of (20\u0026ndash;100 ng mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e). 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 \u0026gt; 0.9998), % RSD of the slope (Sb% \u0026lt; 2%) and the small value of significance F that shown a small grade of empirical points diffusing around the regression line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.2. Limit of detection (LOD) and limit of quantitation (LOQ)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLOD 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.3. Accuracy and precision\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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\u0026ndash;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.4. Robustness\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe already detailed approach was performed beneath little varieconnects within the optimized parameters such as volume of AMF solution (\u0026plusmn; 0.2 mL) and the reaction time (\u0026plusmn; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Analytical applications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.1. Pharmaceutical preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proposed approach was practiced for the assurance of IXA in Ixempra\u0026reg; 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\u0026reg; 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\u0026apos;s t-test and the variance ratio F-test (Table 7). The calculated values didn\u0026apos;t pass the hypothetical ones showing no significant difference between the proposed approach and the reported one with respect to precision and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4.2. In plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u0026ndash;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.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work an easy, reproducible, and fast spectrofluorimetric approach was created for the determination of IXA in bulk as well as in Ixempra\u0026reg; vials and human plasma. The approach depends on the measured fluorescence quenching of AMF due to the presence of the sulfide 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 specific as they gave satisfactory recoveries and the correlation coefficient (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 spectrofluorimetric approach can be suggested to consider the pharmacokinetics of the drug in numerous preparations and combinations and human plasma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Ethics Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollection of samples was authorized by Ethics Committee of Minia Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Consent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccept\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Availability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAvailable upon request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo conflict of interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Funding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo funding was received for this work from any.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Author Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Data collection and analysis were performed by all authors. The first draft of the manuscript was written by Hesham Salem and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Authors information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e-\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Disclosure of potential conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Research involving Human Participants and / or Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e- Informed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/compound/Ixabepilone\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eS., G., \u0026quot;Novel cytotoxic agents: epothilones\u0026quot;. Am J Health Syst Pharm, (May 2008). 65 (10 Suppl 3)(doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2146/ajhp080089\u003c/span\u003e\u003c/span\u003e. PMID 18463327.).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLee FY, Borzilleri R., Fairchild CR, Kamath A., Smykla R., Kramer R., Vite G, \u0026quot;Preclinical discovery of ixabepilone, a highly active antineoplastic agent\u0026quot;. Cancer Chemother. Pharmacol., (December 2008). 63(1)(doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00280-008-0724-8\u003c/span\u003e\u003c/span\u003e. PMID 18347795.): p. 157\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBareaton, L.L., R. Hilal-Dandan, and B.C. Knollmann, Goodman \u0026amp; Gilman\u0026apos;s the pharmacological basis of therapeutics. 2018: McGraw-Hill Education New York.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eM. Vulfovich; Rocha-Lima, C.e.a., \u0026quot;Novel advances in pancreatic cancer treatment\u0026quot;. Expert Rev Anticancer Ther., (2008). 8 (6)(doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1586/14737140.8.6.993\u003c/span\u003e\u003c/span\u003e. PMID 18533808. S2CID 20049942): p. 993\u0026ndash;1002.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZeng J.,Mylott W., Arnold M., Waltrip J., Lacono L., Mariannino T., Stouffer B., Liquid chromatography and tandem mass spectrometry for the quantitative determination of ixabepilone (BMS-247550, Ixempra\u0026trade;) in human plasma: Method validation, overcoming curve splitting issues and eliminating chromatographic interferences from degradants. Journal of Chromatography B, 2010. 878(5-6): p. 525-537.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBeumer, JH., Garner RC., Cohen MB., Galbraith S., Duncan GF., Griffin T., Beijnen JH., Schellens JHM., Human mass balance study of the novel anticancer agent ixabepilone using accelerator mass spectrometry. Investigational new drugs, 2007. 25(4): p. 327-334.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e\u0026Ccedil;\u0026ouml;mezoğlu, S.N., LY Van T., Zhang D., Humphreys WG., Bonacorsi SJ., Everett DW., Cohen MB., Gan J., Beumer JH., Beijnen JH., Biotransformation profiling of [14C] ixabepilone in human plasma, urine and feces samples using accelerator mass spectrometry (AMS). Drug metabolism and pharmacokinetics, 2009. 24(6): p. 511-522.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eG. Colovos, M. Haro, H. Frewer. Reactions of 2-, 7\u0026mdash;Bis (Acetoxymercuri)-fluorescein with certain complexing anions. Talanta, 17 (1970), 273-278.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eH.D. Axelrod, H.J. Cary, J.E. Bonelli and J.P. Lodge. Fluorescence determinationof sub-parts per billion hydrogen sulfide in the atmosphere, Anal. Chem. (1969), 41(13), 1856-1858.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eS. Jayaraman, R. Walia and N. Alagirisamy. Fluorescein mercuric acetate \u0026ndash; a novel sensor for oral malodour detection. Sens. Actuators, B (2010), 148 (1), 54-58.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eK. Toda, S.I. Ohira and M. Ikeda. Micro-gas analysis system gas comprising a microchannel scrubber and a micro fluorescence detector for measurement of hydrogen sulfide. Anal. Chim Acta. (2004), 511(1), 3-10.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eR.S. Haggag, D.A. Gawad, S.F. Belal, H.M. Elbardisy. Spectrophotometric and spectrofluorimetric determination of mesna, acetylcysteine and timonacetic acid through the reaction with acetoxymercuri fluorescein. Anal. Methods, 8(11) (2016) 2479-2493, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c5ay02279g\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eR. Shaalan. Improved spectrofluorimetric methods for determination of penicillamine in capsules, Open Chemistry 8(4) (2010), \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2478/s11532-010-0049-4\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eS. Morshedy, G. Omran, O.A. Abduuatef, M. Omar W. Talaat. Validated spectrofluorimetric method for determination of mirabegron by utilizing its quenching effect on acetoxymercuric fluorescein reagent. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 239 (2020) 118509.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eC. Mongay, V. Cerda. A Britton Robinson buffer of known ionic strength. Ann. Chim. 64 (1974) 409-412.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eG. Colovs, M. Haro, H. Frewer. Reactions os 2-, 7\u0026mdash;Bis(Acetoxymercuri)-fluorescein with certain complexing anions, Talanta (1970) 17, 273-278.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eA. Gomez-Hens and M. Valchrcel. Spectrofluorimetric determination anions: A review, Analyst (1982), 107 (1274) 465-494.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eR. William, B. Paul, Fluorescence quenching studies: analysis of nonlinear Stern-Volmer data, Methods Enzymol. 210 (1992) 448\u0026ndash;463, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0076-6879(92)10023-7\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eW.R. Carmody, Demonstrating Job\u0026rsquo;s method with colorimeter or spectrophotometer, J. Chem. Educ. 41 (11) (1964) 615.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eM. Wieslaw, L. Tadeusz, The effect of temperature on the fluorescence quenching of perylene by tetrachloromethane in mixtures with cyclohexane and benzene, Z. Naturforsch. 47a (1992) 533\u0026ndash;535\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eP.W. Atkins, J.D. Paula, J. Keeler, Atkins Physical Chemistry, Oxford University Press, Oxford, 2018.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eICH. Q2 (R1), Validation of analytical procedures: Text and methodology, international conference on harmonization, November 2005, Geneva, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ich.org/fileadmin/public_web_site/ICH_products/guidlines/quality/Q2_R1/step4/Q2_R1_guidlines.pdf\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eDetailed data of the optimization of the reaction parameters.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003eAMF Con. (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003cp\u003eDifference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003cp\u003eDifference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003eTime (min.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003cp\u003eDifference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003eTemp. (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003cp\u003eDifference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003eDiluting solvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003eFluorescence\u003c/p\u003e\n \u003cp\u003eDifference\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e65.689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e18.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e207.987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e215.564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e47.387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e352.123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003eWater\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e220.699\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e240.30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e366.211\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e\u003cstrong\u003e10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e357.254\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u003cstrong\u003e25\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e270.689\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethanol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u003cstrong\u003e260.898\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e115.958\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e116.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e352.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e235.865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003eChloroform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e15.785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e70.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e22.288\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e356.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e237.868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003eDMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e17.789\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e27.998\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e355.964\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e188.547\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e351.871\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e59.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e355.329\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e351.985\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"4.483695652173913%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"6.657608695652174%\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e354.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"7.065217391304348%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.869565217391305%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.771739130434783%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Mean of (n = 3) experiments for each parameter\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eAssay parameters and conditions for determination of IXA by the proposed spectrofluorimetric method.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eProposed method\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eAMF concentration\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e1x10\u003csup\u003e-4\u003c/sup\u003e M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eAMF volume\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e1.0 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eTemperature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e25 \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eTime\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e10 min\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eDiluting solvent\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eRegression parameters and test results for the determination of IXA by the proposed spectrofluorimetric procedure.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eSpectral data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e\u0026lambda;\u003csub\u003eex\u003c/sub\u003e \u0026amp; \u0026lambda;\u003csub\u003eem\u0026nbsp;\u003c/sub\u003e(nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e500 \u0026amp; 530\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eLinearity range (ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e20-100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eLOD (ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e5.145\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eLOQ (ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e16.987\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eSlope \u0026plusmn; Sb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e10.14\u0026plusmn; 0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eIntercept \u0026plusmn; Sb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e-105.81\u0026plusmn; 2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e%RSD of Sb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eRegression equation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eIntensity 530 = 10.14 \u0026ndash; 105.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eSignificance F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e5.84 x 10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eCorrelation coefficient (r)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e0.9998\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eIntra-day and inter-day accuracy and precision for the determination of IXA by the proposed spectrofluorimetric method.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.426229508196721%\"\u003e\n \u003cp\u003eIXA\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003eIntra-day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003eInter-day\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.426229508196721%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003eFound \u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003eAccuracy (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003ePrecision (%RSD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003eFound \u0026plusmn;SD\u003c/p\u003e\n \u003cp\u003e(ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003eAccuracy (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003ePrecision (%RSD)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.426229508196721%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e40.58\u0026plusmn;1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e101.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e1.045\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e40.82\u0026plusmn;0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e102.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e0.735\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.426229508196721%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e59.66\u0026plusmn;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e99.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e0.996\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e59.38\u0026plusmn;0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e98.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e0.889\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"14.426229508196721%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e79.23\u0026plusmn;0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e99.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e0.899\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.573770491803279%\"\u003e\n \u003cp\u003e80.63\u0026plusmn;0.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e100.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.60655737704918%\"\u003e\n \u003cp\u003e0.685\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u0026nbsp;\u003c/strong\u003eRobustness of the proposed method for the determination of IXA\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e%RSD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eAMF Volume (\u0026plusmn;0.2 mL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e1.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003eReaction time (\u0026plusmn; 2 min)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMean of % RSD\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u0026nbsp;\u003c/strong\u003eRecovery of IXA by applying standard addition technique\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003eIxempra\u003csup\u003e\u0026reg;\u003c/sup\u003e vials\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(ng mL\u003csup\u003e-1\u003c/sup\u003e) \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003eDrug added\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003eDrug found\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;(ng mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e% Recovery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e40.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e101.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e60.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e100.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e81.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e102.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e98.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e98.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e100.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003eRSD %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 7\u0026nbsp;\u003c/strong\u003eStatistical analysis of the results obtained by the proposed and reported procedures for the determination of IXA in Ixempra\u003csup\u003e\u0026reg;\u003c/sup\u003e vials \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eIxempra\u003csup\u003e\u0026reg;\u003c/sup\u003e vials \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eProposed method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eReported method [6]\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eN\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRecovery %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e101.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e100.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.739\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.939\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRSD%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.716\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003et\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(2.262)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e1.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eF value \u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(5.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e1.615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Number of experiments.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e The values in parenthesis are tabulated of t and F at (P = 0.05)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 8\u003c/strong\u003e Statistical analysis of the results obtained by the proposed and reported procedures for the determination of IXA in human plasma \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eIXA \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eProposed method\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eReported method [6]\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eN\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRecovery %\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e99.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e100.94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e1.597\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.939\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eRSD%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e1.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e0.937\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003et\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(2.262)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e1.676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eF value \u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(5.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e2.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Number of experiments.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e The values in parenthesis are tabulated of t and F at (P = 0.05)\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"investigational-new-drugs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"drug","sideBox":"Learn more about [Investigational New Drugs](https://www.springer.com/journal/10637)","snPcode":"10637","submissionUrl":"https://submission.nature.com/new-submission/10637/3","title":"Investigational New Drugs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Spectrofluorimetric approach, Ixabepilone (IXA), Acetoxymercuric fluorescein reagent (AMF), quenching effect, Vials, Human serum","lastPublishedDoi":"10.21203/rs.3.rs-931324/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-931324/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA spectrofluorimetric 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 fluorescence intensity of acetoxymercuric fluorescein (AMF) reagent at λ\u003csub\u003eem\u003c/sub\u003e of 530 nm and λ\u003csub\u003eex\u003c/sub\u003e 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\u003csup\u003e-1\u003c/sup\u003e with correlation coefficient 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 significant 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.\u003c/p\u003e","manuscriptTitle":"A Study of the Interaction of Ixabepilone As Anticancer Drug With Acetoxymercuric Fluorescein Reagent by Fluorescence Quenching Approach: A Validated Method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-01 21:39:46","doi":"10.21203/rs.3.rs-931324/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"submitted","content":"Investigational New Drugs","date":"2021-09-22T05:29:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"investigational-new-drugs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"drug","sideBox":"Learn more about [Investigational New Drugs](https://www.springer.com/journal/10637)","snPcode":"10637","submissionUrl":"https://submission.nature.com/new-submission/10637/3","title":"Investigational New Drugs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"606fe51d-f6c4-46fd-bc9b-4957917fadaf","owner":[],"postedDate":"October 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":7596410,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2021-10-01T21:39:46+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-01 21:39:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-931324","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-931324","identity":"rs-931324","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}