Introduction 1 1 Fig. 1 Structural formula of oxamniquine  2 3 5 6 7 8 13 11 14 15 16 17 8 13 18 24 17 Experimental Apparatus The spectrofluorimetric measurements were recorded using ARF-1501 Shimadzu Spectrofluorometer, equipped with Xenon arc lamp. Materials and reagents Oxamniquine pure sample was kindly provided by Pfizer (Sandwich, UK). Capsules containing 250 mg of Oxamniquine each (Vansil capsules) were obtained from commercial sources in the local market. 1-Dimethyl aminonaphthalene-5-sulphonyl chloride (dansyl chloride), purchased from Sigma (St. Louis, USA). A stock solution containing 0.1% of dansyl chloride was freshly prepared in acetone and was further diluted with the same solvent to obtain 0.001% solution. Sodium carbonate (BDH, UK) 0.5 M aqueous solution (pH 10). Sodium hydroxide (BDH, UK) 1 M aqueous solution. Isobutylmethyl ketone (IBMK) (Merck, Darmstadt, Germany). Plasma was kindly provided by Mansoura University Hospital, and kept frozen until assay after gentle thawing. Methanol and diethyl ether (Merck, Darmstadt, Germany). Standard solutions A stock solution was prepared by dissolving 20.0 mg of OXM in 20 ml of acetone and 80 ml of 0.5 M sodium carbonate solution. This solution was further diluted with the same solvent mixture as appropriate. The standard solutions were stable for seven days when kept in the refrigerator. General procedure −1 Applications Procedure for commercial capsules 1 Table 1 Performance data of the proposed method Parameter Proposed method −1 0.02–0.2 −1 0.007 −1 0.02 Correlation coefficient ( r). 0.9999 Slope 3,473.114 Intercept 0.574 S y x 1.273 S a 7.733 S b 8.965 % Error 0.28 S y x S a S b % Error = RSD%/√ n Procedure for spiked human plasma −1 5 Results and discussion 18 20 21 24 2 Fig. 2 A B A B −1  Optimization of experimental parameters The spectrofluorimetric properties of the colored product as well as the different experimental parameters affecting the development of the reaction product and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. The factors include pH, concentration of the reagent, type of buffer, temperature, reaction time and dilution time. Effect of pH 25 25 Effect of concentration of dansyl chloride 3 Fig. 3 −1  Effect of temperature Increasing the reaction temperature higher than the room temperature would result in a subsequent decrease in the fluorescence intensity of the reaction product. Effect of reaction time 4 Fig. 4 −1  Effect of diluting solvent Different solvents were tried to dilute the reaction mixture through out the study. It was observed that isobutyl methyl ketone gave the highest fluorescence intensity. Dilution with 0.5 M sodium carbonate solution, water, acetone–water produced almost very week fluorescence and did not reduce the blank fluorescence intensity. While upon using isobutyl methyl ketone, the fluorescence intensity attained its highest value, this was attributed to the low fluorescence value of the reagent. Effect of dilution time 5 Fig. 5 −1  Analytical performance Validation of the proposed methods 26 Linearity 1 \documentclass[12pt]{minimal}
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\begin{document}$$ F = 0.574 + 3,473.114C\quad {\left( {r = 0.9999} \right)} $$\end{document} F C −1 r 26 1 1 26 \documentclass[12pt]{minimal}
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\begin{document}$$ {\text{LOQ}} = 10\sigma  \mathord{\left/  {\vphantom {\sigma  S}} \right.  \kern-\nulldelimiterspace} S $$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$$ {\text{LOD}} = 3.3\sigma  \mathord{\left/  {\vphantom {\sigma  S}} \right.  \kern-\nulldelimiterspace} S $$\end{document} σ S 1 2 Table 2 Application of the proposed and official methods to the determination of Oxamniquine in pure form Parameters Spectrofluorimetric method 2 No. of experiments 6 3 Mean found (%) 100.13 100.09  ± SD 0.68 0.87  % RSD 0.68 0.87 Variance 0.462 0.76 t 0.081 (2.31)  F 1.65 (5.41)  t F p 27 Accuracy t F 27 2 S y x S a S b 1 Precision Repeatability 3 Intermediate precision 3 3 Table 3 Validation of the proposed method for the determination of oxamniquine in pure form Sample concentration % Recovery (repeatability) % Recovery intermediate precision −1 99.64 101.08 100.72 99.28 101.44 100.89 X 100.60 100.42  ± SD 0.91 0.99  %RSD 0.91 0.99  % Error 0.53 0.57 −1 99.74 101.05 100.44 100.53 101.29 98.95 X 100.49 100.18  ± SD 0.78 1.09  %RSD 0.78 1.09  % Error 0.45 0.63 Robustness of the method The robustness of the method adopted is demonstrated by the constancy of the fluorescence intensity with the deliberated minor changes in the experimental parameters such as change in the volume of dansyl chloride (0.001%), 0.7 ± 0.1 ml, the change in reaction time 35 ± 5 min and the change in dilution time 15 ± 5 min. These minor changes that may take place during the experimental operation didn’t affect the fluorescence intensity of the reaction product. Pharmaceutical applications The proposed methods were then applied to the determination of OXM in its capsules. The methods were tested for linearity, specificity, accuracy, repeatability and precision according to ICH Q2B recommendations. Specificity The specificity of the method was investigated by observing any interference encountered from the common capsule excepients, such as talc, lactose, starch, avisil, gelatine, and magnesium stearate. These excepients did not interfere with the proposed method. Accuracy 2 27 t F 4 Table 4 Application of the proposed methods to the determination of oxamniquine in capsules Preparations % Recovery Proposed method 2 Vansil capsules (250 mg oxamniquine/capsule) 98.25  101.00  99.38  99.80  99.30   Mean found (%)± SD. 99.55 ± 0.99 100.56 ± 0.77 t 1.67  F 1.66  t F p 27 Analysis of biological fluid 28 −1 5 Table 5 Application of the proposed methods to the determination of oxamniquine in spiked human plasma Sample −1 −1 % Recovery 1-a-Plasma (inter-day precision) 0.02 0.01957 97.85 0.04 0.03856 96.40 0.10 0.09754 97.54 0.20 0.19856 99.28  Mean   97.77  ± SD   1.19 1-b-Plasma (inter-day precision) 0.20 0.1914 95.70 0.20 0.1969 98.45 0.20 0.1948 97.40 0.20 0.1955 97.75 0.20 0.1979 98.95 0.20 0.1945 97.25  Mean   97.58  ± SD   1.13 Each result is the average of three separate determinations 7 Precision 5 −1 5 Mechanism of the reaction 29 6 1 Fig. 6 a b  Scheme 1 Proposed reaction pathway between dansyl chloride and oxamniquine  Conclusion The proposed method has the advantage of being simple, sensitive and suitable for routine analysis in quality control laboratory. Also, it is suitable for the determination of oxamniquine in spiked human plasma with minimum detection limit lower than the reported value. In addition, it could be applied to the determination of OXM in its pharmaceutical preparation.