Micellar enhanced synchronous spectrofluorimetric method for determination of dasatinib in tablets, human plasma and urine: application to in vitro drug release and content uniformity test

Abstract

A highly sensitive and simple spectrofluorimetric method has been developed and validated for the determination of dasatinib (DSB) in its pharmaceutical formulations, spiked human plasma and urine. The suggested method depended on studying the fluorescence spectral behavior of DSB in Cremophor EL (Cr EL) micellar system using synchronous scan technique (Δλ = 50 nm). In aqueous solution, the fluorescence intensity of DSB was markedly enhanced in the presence of Cr EL. The fluorescence–concentration plot was rectilinear over the range 25–500 ng mL⁻¹, with lower detection limit of 2.70 ng mL⁻¹. The proposed method was successfully applied to the assay of commercial tablets, spiked human plasma and urine samples as well as content uniformity test. The application of the proposed method was extended to test the in vitro drug release of DSB tablets, according to United States Pharmacopeia (USP) guidelines.

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1. Introduction

Dasatinib (DSB; N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide monohydrate) (Fig. 1) is an oral tyrosine kinase inhibitor (TKI) approved for the treatment of chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia. DSB is more potent than imatinib by several times and also inhibits imatinib-resistant BCR–ABL mutants.^1,2 The BCR–ABL oncogene encodes a chimeric BCR–ABL protein that has constitutively activated ABL tyrosine kinase activity. This protein is the principal reason of CML.³ DSB is also evaluated for treatment of numerous other cancers, including prostate cancer.⁴

Fig. 1 Chemical structure of dasatinib (DSB).

The effective and safe therapy with DSB is basically depending on the quality of its pharmaceutical preparations (tablets), and assessing its concentrations in tablets for the purposes of quality control. As a consequence, there is an increasing demand for a proper analytical method for determination of DSB in its bulk drug and finished pharmaceutical formulations. In vitro and in vivo studies were performed to characterize the pharmacokinetics, oral bioavailability, and metabolism of DSB. Therefore, it is crucial to develop simple and sensitive method for determination of DSB in both plasma and urine. A detailed understanding of correlations of drug levels with drug action is an important aspect of the routine use of drugs. The accurate quantification of agents in biological matrices such as blood, serum, urine, and tissue samples is the cornerstone of therapeutic drug monitoring. Clinical decisions and further treatment options for patients can only be based on accurate validated analytical methods. Likewise, when determining the efficacy, dose limiting toxicity or pharmacokinetics of new agents and/or new combinations of agents and accurate and precise quantification methods are required. Therefore, detailed specific, reproducible and accurate method for the quantitation of DSB in biological fluids is necessary. Additionally, there is a study published by Kamath et al.⁵ that indicate that less 15% of DSB was excreted unchanged in urine, bile, and the gastrointestinal tract, suggesting that DSB is primarily cleared via metabolism, these results reflect the significance of DSB determination in urine as any increase in its concentration may affect clinical decision on different patients especially with renal or hepatic impairment.

The literature survey revealed that there are few analytical techniques were reported for the determination of DSB in pharmaceutical preparations or human plasma including colorimetry,⁶ spectrophotometry,⁷ HPTLC,⁸ HPLC with UV detection,^8–10 HPLC with fluorescence detection,¹¹ LC-MS^4,12–18 and isotope dilution-MS.¹⁹ There is just one reported method for determination of DSB in urine using HPLC with UV detection.⁵ Even though colorimetry and spectrophotometry characterized by their simplicity, they suffered from lacking selectivity and sensitivity. Chromatographic techniques are selective and sensitive but suffered from being laborious, tedious, time consuming or need highly skilled technical expertise and expensive instruments that minimize their usage in quality control laboratories in developing countries as a result of their very high cost.

Ambient mass spectrometry has significantly decreased the complexity associated with LC-MS/MS for the analysis of drug molecules from body-fluids. However, there is no such report of analysis of DSB using ambient mass spectrometry.^20,21

Spectrofluorimetric analysis constitutes a widespread, effective technique to improve analysis selectivity and sensitivity. Relying on its advantages, spectrofluorimetry provide adequately sensitive, simple and accurate tool for the determination of several drugs either in bulk form or in different matrices including biological fluids such as human plasma and urine. Micelle-enhanced spectrofluorimetric method has been reported for the quantification of numerous drugs^22–25 because of the ability of formed micelle to intensify the fluorescence intensity of the weakly fluorescent compounds. Furthermore, these methods presented sensitive and eco-friendly methodology, since no organic solvents were used.

The surfactants usually used on micellar enhanced analytical methods are sodium dodecyl sulphate (SDS),^22,25 tween^23,26 and cyclodextrin.^27,28 Yet, the application of the non-ionic surfactant “Cremophor EL” in enhanced micellar drug determination has never been described. Cremophor EL (Cr EL) is manufactured by reacting 35 mol of ethylene oxide with castor oil. It contains mainly the tri-ricinoleate ester of ethoxylated glycerol, with fewer amount of polyethylene glycol ricinoleate and the corresponding free glycols.²⁹ Therefore, the aim of the current study is to develop and validate a simple and rapid micelle enhanced spectrofluorimetric method for the determination of DSB in spiked human plasma and urine in addition to pharmaceutical tablets. This method does not need derivatization of DSB due to the native fluorescent property of the drug. Additionally, the application of the proposed method was extended to test the content uniformity and in vitro drug release of DSB tablets, according to USP guidelines.

2. Experimental

2.1. Apparatus

- Fluorescence measurements were carried out on a Jasco FP-8200 Fluorescence Spectrometer (Jasco Corporation, Japan) equipped with a 150 W xenon lamp and 1 cm quartz cells. The slit widths for both the excitation and emission monochromators were set at 5.0 nm. The calibration and linearity of the instrument were frequently checked with standard quinine sulphate (0.01 μg mL⁻¹). Wavelength calibration was performed by measuring λ_ex at 275 nm and λ_em at 430 nm; no variation in the wavelength was observed. All recorded spectra converted to ASCII format by SpectraManager® software.

- A Hanna pH-Meter (Romania) was used for pH adjustments.

- Automatic dissolution tester (8 cup system), Abbota Corporation, 178 Franklin Road, New Jersey 07869, United States.

2.2. Reagents and materials

All the chemicals used were of analytical reagents grade, and the solvents were of HPLC grade.

- Dasatinib reference standard (purity ∼ 99.6%) was purchased from LC Laboratories (New Boston, USA). Sprycel® tablets (Bristol-Myers Squibb Pharmaceutical Limited, New Jersey U.S.A.) labeled to contain 50 mg (as monohydrate) per tablet was procured from the local market.

- Polyoxyl 40 hydrogenated castor oil (Cremophor RH 40), polyoxyl 35 hydrogenated castor oil (Cremophor EL) were purchased from BASF (Ludwigshafen, Germany) and used as 1% w/v aqueous solution for both Cremophor RH 40 and Cremophor EL.

- Sodium dodecyl sulphate (SDS; 95%) was purchased from Winlab (UK) and used as 1% w/v aqueous solution.

- β-Cyclodextrin (β-CD) and carboxymethylcellulose (CMC) were obtained from Merck (Germany) and used as 1% w/v aqueous solution.

- Tween 20, tween 80 and tween 85 (Techno Pharmchem Haryana Company, India), used as 1% v/v aqueous solution.

- Methanol, ethanol (Prolabo, France) and acetonitrile (Sigma-Aldrich Chemie GmbH, Germany).

- Boric acid, sodium hydroxide, phosphoric acid, potassium chloride, potassium dihydrogen phosphate and disodium hydrogen phosphate were all of spectroscopic grade.

- Phosphate buffer (0.1 M, pH 2–7), and borate buffer (0.1 M, pH 8–10) solutions were freshly prepared.

- Ultrapure water of 18 μΩ was obtained from a Millipore Milli-Q® UF Plus purification system (Millipore, Bedford, MA, USA) was used throughout the study.

- Human plasma was kindly provided by King Khalid University Hospital (King Saud University, Riyadh, KSA). After informed consent was obtained, fasting blood samples were taken and plasma was separated and stored at −70 °C.

2.3. Standard solutions

Preparation of DSB stock solution (1 mg mL⁻¹) was done by dissolving 25 mg of DSB reference standard powder into 25 mL acetonitrile in a 25 mL volumetric flask and completing the volume properly. This stock solution was further diluted with acetonitrile to produce a working standard solution of 1 μg mL⁻¹. The standard solutions were stable for at least 14 days when kept in the refrigerator at −4 °C.

2.4. Construction of the calibration graph

Different volumes of DSB standard solution were transferred into a series of 5 mL volumetric flasks to provide final concentrations of 25–500 ng mL⁻¹. 700 μL of Cremophor EL solution (Cr EL) (1% w/v) and 1 mL of phosphate buffer of pH 7 were added and the volume was completed with distilled water. The flasks' contents were mixed well and then measured by synchronous scanning fluorescence in the range of 300–450 nm employing a Δλ of 50 nm. The intense band observed at 370 nm was used for quantitative purposes. Relative fluorescence intensity (RFI) at 370 nm was plotted vs. the final drug concentration in ng mL⁻¹ to obtain the calibration graph. The regression equation for the data was then calculated.

2.5. Assay of tablet samples

Ten commercial tablets (Sprycel® 50 mg tablets; batch no. 3E6016B) were weighed and finely powdered. An accurately weighed quantity of the powder equivalent to 50 mg of the active ingredient (DSB) was transferred into a 100 mL calibrated flask, and dissolved in about 50 mL of acetonitrile. The contents of the flask were swirled, sonicated for 30 min, and then completed to volume with acetonitrile. This solution (0.5 mg mL⁻¹) was diluted quantitatively with acetonitrile to obtain suitable concentrations for the analysis.

2.6. Assay of human plasma samples

The extraction procedure was based on liquid–liquid extraction. Plasma samples were kept at −20 °C and thaw before processing at room temperature. A volume of 20 μL of standard drug solutions (at different concentrations of DSB) was spiked into 1 mL of human plasma and mixed properly for 1 min to achieve spiked plasma concentrations of 60 ng mL⁻¹, 70 ng mL⁻¹, 80 ng mL⁻¹ and 100 ng mL⁻¹ respectively. One mL portion of 100 mM NaOH/glycine pH 11 buffer was added then the tube was then mixed for 10 seconds on a vortex mixer. Five mL of diethyl ether was added and the solution was allowed to vortex for 30 seconds and the samples were then centrifuged at 10 000 rpm for 15 minutes to assure phase separation. Afterward 4 mL of the upper organic layer was transferred into glass vials and dried using gentle stream of nitrogen. Ultimately, residue reconstitution took place in acetonitrile and the procedures depicted under “construction of the calibration graph” were then followed. A blank plasma sample was treated in a similar way. RFI was measured at 370 nm using Δλ of 50 nm and the concentration of the drug was determined from the corresponding regression equation of DSB in micellar medium.

2.7. Assay of human urine samples

Human drug free urine (1 mL) was spiked with volume of 20 μL of standard drug solutions (at different concentrations of DSB) and mixed for 60 seconds. One mL portion of 100 mM NaOH/glycine pH 11 buffer was added then the tube was vortexed for 10 seconds. Five mL of diethyl ether was added and the solution was vortexed for 30 seconds and the samples were then centrifuged at 10 000 rpm for 15 minutes to assure phase separation. Then 4 mL of the upper organic layer was transferred into glass vials and dried under a gentle stream of nitrogen. Finally, reconstitution of the residue occurred in acetonitrile, then suitable dilutions were made to give final concentrations of 80 ng mL⁻¹, 120 ng mL⁻¹, 160 ng mL⁻¹ and 200 ng mL⁻¹ respectively. Ultimately, the procedures described under “construction of the calibration graph” were performed. A blank urine sample was treated similarly. Fluorescence intensity was measured at 370 nm using Δλ of 50 nm and the concentration of the drug was determined from its corresponding regression equation in micellar medium.

2.8. Procedure for content uniformity testing for DSB

Ten different tablets were analyzed using the same procedure applied for the analysis of the studied compound in tablets. The uniformity of their contents was tested by applying the official USP guidelines³⁰ (Chapter 905: Uniformity of Dosage Units).

2.9. Procedure for in vitro drug release test (dissolution test) for DSB

Dissolution test was performed on Sprycel® 50 mg tablets. The dissolution USP apparatus II³⁰ stirred at 60 rpm for 60 min using 1000 mL of citrate buffer (pH 3.4) at 37 ± 0.50 °C. These conditions were applied as it is published in the literature.³¹ Five mL sample was withdrawn through a 0.45 μm syringe filter and replaced with another 5 mL of a suitable fresh dissolution medium at preselected intervals up to 60 min. The procedure described under ‘construction of the calibration graph’ was applied on the filtered samples to calculate the percentage of drug release.

3. Results and discussion

Upon severe literature survey, it was clear that few reported methods were reported for the determination of DSB in its pharmaceutical preparations or human plasma and urine. For this reason, it is crucial to develop simple, sensitive and unified method for DSB quantitative determination in different matrices such as tablets, human plasma and urine. Spectrofluorimetry by virtue of its advantages such as sensitivity, selectivity, simplicity, and wide availability in quality control laboratories was selected in this study. Various experimental factors that influence the RFI of DSB were studied and optimized carefully; changing one factor at a time while others kept constant.

3.1. Fluorescence spectra and characteristics of DSB

DSB exhibits excitation and emission wavelengths of 320 and 370 nm respectively. The fluorescence spectra of DSB in both aqueous and Cr EL micellar systems were carefully studied. The RFI of DSB in Cr EL medium was enhanced significantly in comparison with its native fluorescence in aqueous medium as shown in Fig. 2A. This enhancement in micellar medium may be due to restrictions imposed on the free rotational movements that may decrease the luminescent emission.²⁴ But on examining the fluorescence spectrum of the Cr EL (Fig. 2B), it was found that there is a clear overlap between DSB emission spectrum and that of Cr EL due to the presence of a scattering peak at 370 nm (peak no. 4 in Fig. 2B). Thus, a good alternative for the conventional fluorescence spectrum is the use of synchronous scanning fluorescence to minimize this spectral overlap. In this technique, the excitation and emission monochromator are scanned simultaneously and the emission intensity is recorded as a function of the excitation wavelength. The Δλ (difference between λ_em and λ_ex) plays an important role and is responsible for the intensity and the position of the bands in the synchronized spectrum. In the present work, the optimum Δλ was already determined by the interpretation of the fluorescence spectrum. We selected Δλ = 50 nm because it gave a considerable reduction of the spectral overlap and the RFI increased linearly with the corresponding increase in DSB concentrations as shown in Fig. 3A. A linear calibration curve of DSB is anticipated in Fig. 3B.

Fig. 2 Excitation (1) and emission (2) spectra of DSB (300 ng mL⁻¹) in Cr EL (1%, w/v); (A) excitation (3) and emission (4) spectra of DSB (300 ng mL⁻¹) in water; (B) excitation (3) and emission (4) spectra of Cr EL (1%, w/v) in water.

Fig. 3 (A) Synchronous fluorescence spectra of different concentrations of DSB (25–500 ng mL⁻¹) in (1%, w/v) Cr EL (solid lines), Cr EL (1%, w/v) in water (dotted line) (Δλ = 50 nm); (B) calibration curve of DSB under the optimum conditions.

3.2. Optimization of the experimental conditions

3.2.1. Effect of organized media. The influence of various organized media on the RFI of DSB was studied by adding 0.5 mL, 1% w/v solution of each surfactant to DSB solution. The final concentrations of the tested surfactants and the drug were 0.1% w/v and 400 ng mL⁻¹ respectively. The final surfactants' concentration are above their reported critical micelle concentration, cmc. Anionic surfactants such as tween 20, tween 80, tween 85, Cr El and Cr RH 40, anionic surfactant sodium such as dodecyl sulfate (SDS) and carboxymethylcellulose (CMC) and macromolecules such as β-cyclodextrin were tested. The highest RFI was obtained using nonionic surfactants in the following order; Cr EL > tween 80 > tween 85 > Cr RH 40 > tween 20 (as shown in Fig. 4). Generally nonionic surfactants have better solubilizing characteristics than ionic surfactants for hydrophobic drugs, due to their, comparatively, lower cmc values.³²

Fig. 4 Effect of the type of organized media (0.5 mL, 1% w/v solution of each) on RFI of DSB (400 ng mL⁻¹).

3.2.2. Effect of the volumes of nonionic surfactants. The influence of the nonionic surfactants on the RFI of DSB was studied using different volumes of 1% w/v solutions. On increasing their volumes, Cr EL showed the highest results (Fig. 5). The RFI of DSB using Cr EL increased significantly up to 600 μL (1% w/v), where after this volume no more significant increase in RFI was observed. This is may be due to that, any further increase in the surfactant concentrations will not correspond to an increment in micelle formation. Therefore, 700 μL 1% w/v Cr EL solution was chosen for DSB analysis (Fig. 5).

Fig. 5 Effect of Cr EL, Cr-RH 40, tween 85, tween 80 and tween 20 volumes (1% w/v) on RFI of DSB (500 ng mL⁻¹).

3.2.3. Effect of pH. The effect of pH on the RFI of the DSB, DSB–Cr EL and Cr EL was studied using different kinds of buffers that cover the pH range 2–10. For pH range 2–7, 0.1 M phosphate buffer was used, while 0.1 M borate buffer was used for pH range 8–10. Fig. 6 showed that the RFI of DSB alone was almost the same and decrease significantly after pH 7. This may be due to changing the ionization of DSB. For Cr EL alone, its RFI was stable over the whole pH range, which offer an advantage of using Cr EL in this study. Finally, the RFI of DSB in presence of Cr EL increased initially as the pH increased and maximum RFI was attained at pH 7.0 ± 0.2, then RFI decreased with pH increase, till reaching minimum at pH 10. From all these findings, it was thought that at pH 7, DSB is in the nonionized form and thus favorably interact with the nonionic Cr EL surfactant. This thought was supported by the calculations performed by Chemicalize which indicates that the charge on DSB was minimal at pH 7–8 that reflects the domination of nonionized form of DSB at this pH range.³³

Fig. 6 Effect of pH on the RFI of 500 ng mL⁻¹ DSB in 700 μL 1%, w/v Cr EL solution in water (black column DSB with surfactant, white column DSB without surfactant and grey column surfactant only).

3.2.4. Effect of diluting solvent. The influence of different diluting solvents (water, methanol, ethanol or acetonitrile) on the RFI of 400 ng mL⁻¹ DSB in 1% w/v Cr EL was also investigated (Fig. 7). The results revealed that water was the best solvent for dilution in presence of CR EL, as it gave the highest RFI and the lowest blank reading, while distinct and sharp decrease in the RFI was observed in the Cr EL system using other solvents. The denaturating effect of methanol and ethanol on the formed micelle could be the primary reason for this effect as these alcohols solubilized in water and consequently, changing the solvent properties which may decrease micelle formation. Moreover, addition of these alcohols also results in a reduction of the size of the micelles but with a progressive breakdown of the surfactant aggregate at very high concentration.³⁴

Fig. 7 Effect of the diluting solvent on RFI of DSB (400 ng mL⁻¹).

3.2.5. Effect of time. In order to determine the effect of time on the stability of the RFI of DSB in micellar system, the RFI was monitored for 60 min and it was found that the RFI developed instantaneously and persisted stable through the whole time (60 min).

3.3. Validation of the method

The proposed methods were validated in accordance with the ICH-guidelines for validation of the analytical procedures³⁵ regarding linearity, sensitivity, accuracy, specificity, repeatability and reproducibility.

3.3.1. Linearity and range. The calibration graph for the determination of DSB by the proposed method was constructed by plotting the RFI against the concentration. The graph was found to be rectilinear over the concentration range 25–500 ng mL⁻¹ (Table 1).

Table 1 Analytical performance data for the spectrofluorimetric determination of DSB

Parameter	DSB
a Percentage relative standard deviation for three replicate samples. b Percentage relative error for three replicate samples. c Limit of detection. d Limit of quantitation.
Synchronous scanning range (nm)	300–450
Δλ (nm)	50
Linearity range (ng mL^−1)	25–500
Intercept (a)	1.679
Slope (b)	−3.370
Correlation coefficient (r)	0.9998
S.D. of residuals (Sy/x)	5.942
S.D. of intercept (Sa)	0.005
S.D. of slope (Sb)	1.372
% RSD^a	1.66
% Error^b	1.04
LOD^c (ng mL^−1)	2.70
LOQ^d (ng mL^−1)	8.17

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Statistical analysis³⁶ of the data gave high values of the correlation coefficients (r) of the regression equations, small values of the standard deviation of residuals (S_y/x), of intercept (S_a), and of slope (S_b), and small value of the percentage relative standard deviation and the percentage relative error (Table 1). These data proved the linearity of the calibration graph for the studied drug.

3.3.2. Limit of quantitation (LOQ) and limit of detection (LOD). Limit of quantitation (LOQ) and limit of detection (LOD) were calculated in accordance with the ICH Q2 (R1) recommendation.³⁵ LOQ was determined by establishing the lowest concentration that can be measured below which the calibration graph is nonlinear. LOD was determined by evaluating the lowest concentration of the drug that can be detected. The results are also presented in Table 1. The values of LOQ and LOD were calculated according to the following equation:
where, σ was the standard deviation of the intercept of regression line and S was the slope of regression line of the calibration curve. The results are given in Table 1.

The reported data in the literature⁴ support that our LOQ (8.17 ng mL⁻¹) is much lower than maximum plasma concentration (C_max) and hence can easily quantify DSB in plasma (reported C_max of DSB around 150 ng mL⁻¹).

3.3.3. Accuracy and precisions. Accuracy, intra-day and inter-day precisions of the proposed method were determined (anticipated in Tables 2 and 3). Three replicate samples in the same day, as well as on three consecutive days were analyzed for intra-day and inter-day precision at different concentrations. Accuracy was calculated as % bias using the following equation,

Table 2 Accuracy of the spectrofluorimetric method for determination of DSB^a

Days	Actual conc. (ng mL^−1)	Mean conc. (ng mL^−1)	±SD	% Nominal	% RSD	% Bias	SEM
a SEM standard error of the mean; 1, 2, 3 represents measurements obtained on day 1, day 2 and day 3 respectively (n = 3 for each day).
1	50	48.79	0.41	97.59	0.83	2.44	0.17
2	50	49.67	0.71	99.33	1.44	0.67	0.41
3	50	51.47	0.78	102.93	1.52	−2.88	0.39
1	100	100.57	1.91	100.57	1.9	−0.55	0.78
2	100	104.51	1.65	104.51	1.58	−4.41	0.95
3	100	101.33	1.88	101.33	1.85	−1.31	0.94
1	200	201.54	4.05	100.77	2.01	−0.75	1.66
2	200	199.42	1.02	99.71	0.51	0.29	0.59
3	200	205.62	3.45	102.81	1.68	−2.76	1.72
1	400	400.76	6.53	100.19	1.63	−0.18	2.67
2	400	397.02	3.39	99.26	0.85	0.75	1.96
3	400	393.68	5.45	98.42	1.38	1.60	2.72

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Table 3 Intra-assay and inter-assay precision and accuracy for determination of DSB by the proposed spectrofluorimetric method

Nominal conc. (ng mL^−1)	Intra-assay		Inter-assay
	Measured conc. (ng mL^−1)	Recovery^a (% ± RSD)	Measured conc. (ng mL^−1)	Recovery^a (% ± RSD)
a Mean of three determinations.
50	49.67	99.33 ± 1.44	51.47	102.93 ± 1.52
100	100.74	104.51 ± 1.58	101.13	101.13 ± 1.67
200	203.63	99.71 ± 0.51	205.54	102.77 ± 1.46
400	394.64	99.26 ± 0.85	393.68	99.55 ± 1.38

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The % bias was ranged from −4.41 to 2.44%, showing the accuracy of the method. The intra-day and inter-day precisions were expressed as recovery (% ± RSD). The average recovery percentages were almost 100% and the low value of relative standard deviations (RSD) showed the inter-day and intra-day precision of the method (Table 3). Additionally, comparing the results of the assay of the drug in pure form with those obtained by reported spectrophotometric method⁷ gave another evidence for the accuracy of the proposed method. It was found that the calculated t- and F-values (0.894 and 1.445 for t- and F-value, respectively) were lower than the tabulated ones (2.228 and 5.050 for t- and F-value, respectively). This indicated that there were no significant differences between the means and variance between the two methods in terms of the accuracy and precision (Table 4). Overall, these results indicate that the proposed spectrofluorimetric method is accurate and precise.

Table 4 Statistical comparison between analysis results of DSB in pure powder form by proposed spectrofluorimetric method and the reported method⁷

Value	Proposed method	Reported method^7
a Tabulated value at p = 0.05.
Mean	99.92	99.33
SD	1.247	1.036
RSD%	1.25	1.04
N	6	6
Variance	1.56	1.07
F value	1.445 (5.050)^a
T test	0.894 (2.228)^a

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3.3.4. Robustness. Robustness was examined by evaluating the influence of small variation in the method variables on its analytical performance. In these experiments, one parameter was changed whereas the others were kept unchanged, and the recovery percentage was calculated each time. It was found that deliberate variations that may occur during the experimental conditions did not significantly affect the RFI of the suggested method. The results are shown in Table 5.

Table 5 Robustness of the proposed spectrofluorimetric method

Experimental parameter variation	Recovery (%) ± SD^a
a Mean of three determinations. b Following the general calibration procedures.
No variation^b	99.68 ± 0.88
Cremophor EL volume (μL)
650	103.60 ± 1.93
750	107.77 ± 2.25
pH
6.8	104.76 ± 0.64
7.2	101.62 ± 1.4
Buffer volume (μL)
0.95	102.37 ± 0.98
1.05	102.59 ± 1.12
Temperature (°C)
20	106.92 ± 1.67
30	96.02 ± 1.27

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3.3.5. Specificity. The specificity of the method was also estimated by monitoring any interference that may occur from the common excipients of the dosage form (Sprycel® tablets). It was found that these compounds did not interfere with determination of DSB by the suggested fluorimetric method (Table 6).

Table 6 Results of the determination of DSB in pure form, Sprycel tablets (batch no. 3E6016B) human plasma and human urine samples

Parameter	Pure form			Sprycel tablets		Plasma samples		Urine samples
	Amount taken (ng mL^−1)	Amount added (ng mL^−1)	% Found	Amount taken (ng mL^−1)	% Found	Amount taken (ng mL^−1)	% Found	Amount added (ng mL^−1)	% Found
	50	99.33	99.33	49.34	98.69	60	91.38	80	96.26
	100	102.62	102.62	99.02	99.02	70	91.2	120	101.85
	200	99.2	99.2	196.96	98.48	80	92.83	160	105.08
	400	99.57	99.57	390.78	97.69	100	92.49	200	98.89
Mean			100.18		98.47		91.98		100.52
S.D.			1.63		0.57		0.81		3.80

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3.4. Applications

3.4.1. Application of procedure to analysis of DSB in tablets. It is evident from the above-mentioned results that the proposed method gave satisfactory results with DSB in bulk powder. Thus, its pharmaceutical dosage form (Sprycel® tablets) was subjected to the analysis of their DSB contents by the proposed method. The percentage found from the label claim was 98.47 ± 0.57% (Table 6). These results indicated the excellent accuracy of the proposed method.

3.4.2. Application of procedure to analysis of DSB in human plasma. It was clear from the previous data that the proposed method is sensitive enough to determine DSB in spiked human plasma. DSB reaches peak plasma concentration at one hour after oral administration.⁴ The maximum plasma concentration (C_max) of DSB is about 150 ng mL⁻¹.⁴ Hence, the drug plasma level is lied in the working dynamic range of our fluorimetric method (Table 1). The results abridged in Table 6 reveal that the % RSD and mean absolute recoveries of DSB in spiked plasma samples are 0.81%, 91.98%, respectively. The recovery values are in good agreement with the findings of Kassem et al.¹¹ concerning HPLC with fluorescence detection for determination of DSB in human plasma. Relatively, high plasma protein binding (96% in humans) could be responsible for low recovery values (about 92%).

3.4.3. Application of procedure to analysis of DSB in urine. Less than 1% of DSB usual adult dose (which is 100 mg daily for leukemia) is excreted in the urine unchanged.³⁷ Therefore, the drug level in urine (1 μg mL⁻¹) is above our working range. The results reported in Table 6 reveal that the % RSD and mean absolute recovery of DSB in spiked urine samples are 3.80%, 100.52%, respectively. These excellent results of the urine analysis when compared to that of plasma analysis (around 100% mean absolute recovery in urine while in plasma around 92%) may be attributed to the low amount of the endogenous amino acids that present in urine when compared to plasma.

3.4.4. Content uniformity testing for DSB. Due to the advantages of the proposed method of being highly sensitive and its rapidity and simplicity, the method is preferably suitable for content uniformity testing which is a time consuming process when using conventional assay techniques. The steps of the test were adopted according to the USP³⁰ (Chapter 905: Uniformity of Dosage Units) procedure. The acceptance value (AV) was calculated and it was found to be smaller than the maximum allowed acceptance value (L1). The results demonstrated excellent drug uniformity as anticipated in Table 7.

Table 7 Results of content uniformity testing of DSB tablets using the proposed spectrofluorimetric method

Parameter	Tablet no.	Percentage of the label claim
Data	1	99.87
	2	100.83
	3	101.73
	4	101.24
	5	97.63
	6	99.54
	7	101.62
	8	101.28
	9	102.91
	10	101.94
Mean	100.86
S.D.	1.50
% RSD	1.49
Acceptance value (AV)^30	3.59
Max. allowed AV (L1)^30	15

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3.4.5. In vitro drug release (dissolution test) for DSB. Dissolution test was performed on Sprycel® tablets (50 mg). The amount of drug released was then determined with the help of the calibration curve and the percentage of drug released was calculated. Results showed that about 100% of the labeled amount of DSB is dissolved in 30 min (Fig. 8).

Fig. 8 Dissolution profile for DSB tablets according to USP guidelines.

3.5. Mechanism of the micellar-enhancement effect of Cr EL

To discover if the increase of RFI of DSB is attributed to increment of the quantum yield and/or increment in absorption at λ_ex, the molar absorptivity of DSB in Cr EL micellar system was calculated at 320 nm (λ_ex). The molar absorptivities of DSB in micellar medium and acetonitrile which indicated that the RFI increment is not due to an increase in the absorption of the drug in micellar system at its λ_ex. On the other hand, the quantum yield of DSB in acetonitrile and Cr EL was 0.012 and 0.043 respectively. The increase in the quantum yield of DSB in Cr EL may be due to the protection of the lowest excited singlet state from non-radiative processes by the formed micelle. The quantum yield was calculated using the following equation (eqn (1)):³⁸where Ø_d and Ø_q represent the fluorescence quantum yields of the drug and quinine, respectively; F_d and F_q are the integral fluorescence intensities of the drug and quinine, respectively; A_d and A_q are the absorbance values of the drug and quinine at the excitation wavelength, respectively. The concentration was chosen so that the absorbance was less than 0.05 to minimize the error due to the inner effect.³⁹

4. Conclusions

A simple and sensitive synchronous spectrofluorimetric method was established for the determination of DSB in different matrices. The present study is considered the first spectrofluorimetric method for DSB analysis. The proposed method characterized by its rapidity, saving time and does not require complicated treatment when compared with chromatographic techniques. By virtue of its simplicity and sensitivity, the proposed method could be applied to the analysis of the studied drug in pharmaceutical tablets and biological fluid. The proposed method is very suitable to be applied in content uniformity testing. Additionally, it has been adapted for dissolution testing of DSB tablets as a rapid and simple method. From the economical point of view, the method involved the native fluorescence property of DSB, rather than expensive derivatizing analytical reagents.

Acknowledgements

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding this work through the Research Group Project no. RGP-VPP-322.

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