Micellar enhanced Synchronous Fluorescence for the assay of Sumatriptan succinate in Pharmaceutical and Biological Samples

Safwan M. Fraihat
Department of Chemistry, Faculty of Science
The University of Jordan 11942, Amman-Jordan
ABSTRACT: A Sensitive, simple and rapid spectrofluorimetric method for the assay of Sumatriptan succinate (SUM) in both pharmaceutical and Biological samples has been developed. The method is based on measuring the synchronous fluorescence of SUM using ?? of (120nm) and at a wavelength of excitation and emission of 230 and 325 nm respectively in methanol at pH of (5.5). The calibration curves were obtained with a range of (50-150) ng/ml with a limit of detection of 16.3 (ng/ml), then the developed method has been validated statistically for precision and accuracy as per ICH guidelines and the results compared favorably with those obtained from the reference method, the developed method was also applied successfully for the determination of SUMS in drug formulations and biological samples with good accuracy and precision.
Keywords: Sumatriptan; Spectrofluorimetry; Pharmaceutical preparations; Biological fluids.
INTRODUCTION

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Triptans are used for the treatment of prophyllaxis and acute migraine headaches in human, the most commonly used drug among triptans is sumatriptan succinate Fig.1. it is chemically 3-2-(diaminoethyl)-N-methyl-indole-5-methane sulfonamide succinate. Sumatriptan is a specific and selective 5-hydroxyl tryptan receptor (5HT1D) agonist with no effect on other 5HT receptor (5HT2-5HT7) subtypes.
It is official in European pharmacopeia 1 and united states pharmacopeia 2.
Several methods were suggested for the determination of sumatriptan in different types of samples, including, UV 3, spectrophotometric 4-6, Other methods were based on high-performance liquid chromatography with various detecting systems such as UV detection 7, fluorescence detection 8 and MS detection 9,10. Other methods were based on and voltammetry 11. Few methods were based on the fabrication of nanomaterial-based sensing methods 12-14. According to the privous methods, it can be seen that most of them are based on using expensive and sophistigated techniques, this encourage us to develop a simple method for the assay of sumatriptan in pharmaceutical and biological fluids. The developed method is based on the enhancement of the intrinsic Fluorescence of SUM, then measuring Synchronous Fluorescence intensities in different media. All the factors affecting the fluorescence intensities were studied, including the type of solvent, pH, temperature and micellar additives. The developed methods were validated and applied to real pharmaceutical samples and biological fluids and were compared with official methods. Synchronous fluorescence technique has many priorities compared to conventional fluorescence spectroscopy, including: simple spectra, higher selectivity, sensitivity and lower interferences which are due to sharper and narrower fluorescence peaks 15-17. It is will known that the presence of surfactants may increase the rigidity of the molecules being surrounded by the surfactant which restricts the movement of the fluorophore and so decrease the energy transfer to the surrounding environment, As a result, increases the quantum yield and enhance the fluorescence intensity of the guest molecules 18. The anionic surfactant Sodium Lauryl Sulfate (SLS) has recently found many applications as a fluorescence enhancer for the determination of some pharmaceutical compounds 19,20.
The developed method is considered as a low cost, time-saving and sensitive method that has the advantages of not using derivatization reactions using toxic chemicals. Moreover, another method of the conventional spectrofluorimetric technique was applied using the Synchronous scanning fluorescence at (?? = 120 nm), which allowed simpler spectrum with less overlapping 20.
The developed method was validated and applied successfully for quantitative determination of (SUM) in pharmaceutical samples and biological fluids.

Figure1. Chemical Structure of Sumatriptan succinate
Experimental
Apparatus
Fluorescence intensities were measured using Agilent Technology, Cary eclipse, G9800AA model Luminescence spectrometer (Australia) equipped with a xenon arc lamp, The slit width for excitation and emission measurements were set at 5.0 nm and measured simultaneously with a constant ?? scan (?emi-?exi) of 120 nm. A 1.0 cm quartz cell was used at 25.0 °C. A pH meter (HANNA model: HI 2211) was used for all pH measurements.
Materials and Reagents
Reagents used were of analytical grade and deionized water was used throughout. Pure grade Sumatriptan succinate were kindly supplied from Egyptian company. The pharmaceutical preparations were purchased from local drug stores Oratab®100mg tablet Tabouk company-Saudi Arabia. Sodium lauryl sulfate SLS 96 %, Tween 80, ?-cyclodextrin, glacial Acetic acid were from BDH laboratory supplies, Sodium acetate, Sulphuric acid. Analytical grade solvents: Acetonitrile 99.5%, Methanol 99.9% Dioxan 99% were obtained from Sharlau-Spain.
Standard solution
Stock solution of SUM was prepared by dissolving 0.01 g in 100 ml deionized water, then further dilutions were prepared using the given diluting solvents as appropriate and kept in the refrigerator.
General Procedures
Calibration graphs. Aliquots of (SUM) standard solution were transferred into a series of 10-ml calibrated volumetric flasks, then 1 ml of 0.2 M acetic acid/acetate buffer of pH (5.5) was added, followed by 0.5 ml 2% SLS, mixed and then diluted to the mark using appropriate diluting solvent to give a final concentration of (50-150 ng/ml). Then stored in the refrigerator at 10 °C for 15 minutes, after that the Fluorescence intensity was measured at appropriate excitation and emission wavelengths corresponding to the selected diluting solvent. The calibration curves were constructed by plotting the fluorescence intensity versus (SUM) concentration (ng/ml) and the regression equations were obtained.
Analysis of Tablet samples. The contents of ten tablets were crushed and powdered, then a mass equivalent to 10.0 mg was weighed and transferred into a 100 ml volumetric flask, about 75 ml deionized water was added, the mixture was sonicated for 10 minutes, then the volume is completed to 100 ml with water, mixed and filtered through a 0.45 µm membrane filter. Serial dilutions covering the working concentration range of (50-150 ngml-1) were transferred into a series of 10.0 ml volumetric flasks. Then the procedure mentioned in the preparation of calibration graph was followed.
Assay of human urine and plasma samples
The proposed method was applied to the determination of SUM in the spiked urine and plasma samples provided from several healthy volunteers. A 1.0 ml Spiked urine sample was 50-fold diluted with deionized water. Aliquots of human plasma (1.0 ml) were transferred into a series of centrifuge tubes. The plasma samples were spiked with aliquots of different concentration of standard SUM so that the final concentration was in the range of (50-150 ) ngml-1. The solutions were mixed well and then completed to 5 ml with acetonitrile after fixation with methanol volume in all tubes. After vortex mixing for 5 minutes, the mixtures were centrifuged at 4000 rpm for 15 minutes. The supernatant in each tube was aspirated. Then 0.1 ml aliquots of the supernatant were quantitatively transferred into a series of 10.0 ml volumetric flasks and diluted to the volume with acetonitrile. A blank sample was analyzed, then the previous procedure mentioned in the preparation of calibration curve was followed and the fluorescence values were measured at the same conditions.
RESULTS AND DISCUSSION
To our knowledge there is no analytical method based on native fluorescence of SUM, in this study, it was found that SUM exhibits an emission fluorescence spectrum at about 350 nm after excitation at 225 nm in aqueous medium Figure 2. So there is a need to investigate the effect of different media on such behavior, because of its relatively low relative fluorescence intensity (RFI). It is important to develop simple and sensitive spectrofluorimetric method for the determination of SUM in a different type of samples. In this study, various experimental factors that influence the RFI of SUM were studied in order to decrease the detection limit. So different factors were studied as follow:

Figure 2: Fluorescence spectra of SUM (100 ngml-1) in water (A) and Methanol (B)
Effect of diluting solvent
In order to study the effect of different media on the enhancement of fluorescence intensity, different organic solvents were used as diluting solvents including Acetonitrile, Methanol, Ethanol, and Dioxan. It was found that the fluorescence intensity is enhanced with the order of Methanol, Acetonitrile, Dioxan and then ethanol as can be seen in Figure 3. This behavior is probably due to the dynamic stabilization of the studied drug. The emission wavelengths of SUM in Dioxan were 357and 350 for Acetonitrile and 294 nm for Methanol. The excitation wavelength were 298, 262 nm and 253nm of the three solvents respectively.

Figure 3. Effect of diluting solvent on RFI of SUM (100 ngml-1)
Effect of organized media
For the better enhancement of the analytical characteristics of the fluorescence spectra of SUM, a study of the effect of different types of surfactants (cationic, Anionic and nonionic) was performed using the different effect of equal volume each surfactant used were summarized in Figure (4) it can be shown that SLS gave the best enhancement of the RFI which is due to dynamic properties of limited movement of the micellar medium of SUM 21.

Figure 4. Effect of the addition of organized media on RFI of SUM (100 ngml-1), after the addition of 1ml of 0.5% of each medium of the RFI of SUM.
Effect of pH
The effect of pH value on the fluorescence intensity was studied by varying the values of pH using different buffer solutions, it was found that the maximum value of fluorescence intensity was obtained at pH around 5 using acetate buffer as shown in Figure 5, which is probably due to the formation of a nonionizable form of the studied drug at this pH value.

Figure 5. Effect of pH on RFI of SUM (100 ngml-1) using acetate buffer
In order to minimize the broadening of the emission fluorescence peaks and thus possible overlapping and minimize scattering. An alternative method for the conventional fluorescence spectrum is the use of Synchronous scanning fluorescence. 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 difference between ?em. and ?ex. (??) is a very important factor in the synchronous spectrum for determining the position of the bands and their intensities. In the present work the optimization of (??) was performed by selecting different values of (??) then interpretation of the fluorescence spectrum, as a result, ??=120 nm was selected because it gave sharper peak with maximum intensity, least peak overlap and increase in (RFI) as a function of concentration of SUM as shown in Figure 6 below.

Figure 6 Calibration of SUM using the proposed synchronous method using
(??= 120nm) in methanol with SLS added, the concentration range is (50-150 ngml-1)
Validation of the proposed method
The developed method was validated using the following parameters: Linearity, sensitivity, LOD, LOQ, specificity, accuracy and precision.
Linearity and range
A linear relationship was established for SUM by plotting relative fluorescence intensities against different drug concentrations, the calibration curve was linear over the range 50-150 ng/ml with a high value of correlation coefficient (r) Table 1.

Limit of detection (LOD) and limit of Quantitation (LOQ)
LOD and LOQ were calculated according to ICH Q2 (R1) recommendationsICH Expert Working Group. ICH harmonized tripartite guidelines. Validation of Analytical procedures: text and methodology 22, using the following equations and the results are shown in Table 1
LOD = 3.3Sa/b
LOD = 10 Sa/b
Where Sa: standard deviation of the intercept and Sb: standard deviation of the slope of the calibration curve.
Validation of the developed Spectrofluorimetric methods
Table 1 Analytical Performance data of the proposed method
Parameter SUM
Synchronous scanning range (nm) 200-300
?? (nm) 120
Linearity range (ng/ml) 50-150
Correlation coefficient 0.992
Slope (b) 0.939
Intercept (a) 68.4
SD of the intercept (Sa) 4.65
SD of the slope (Sb) 0.0021
% RSDa 1.3
LODb (ng/ml) 16.3
LOQc (ng/ml) 49.5
a Percent relative standard deviation of three replicate samples, b Limit of detection, c Limit of quantitation
Accuracy and precision
To investigate the accuracy and precision of the developed method, the assay results of SUM were compared from standard reference method 23, statistical analysis of the results using student’s t-test and variance ratio F-test 24 showed no significant differences between the performance of the proposed method and reference method regarding accuracy and precision (Table 2)
The intra-day and inter-day precision were evaluated by determining three concentrations of SUM in its pure form on three successive days, the recovery results were obtained

Table 2 Assay results of the proposed method for SUM in pure and tablet forms compared with a reference method
Parameter: Proposed method % Found Reference method % Found
Amount Amount Amount Amount
taken found taken found
(ng/ml) (ng/ml) (ng/ml) (ng/ml)
5.0 4.974 99.48 5.0 4.910 99.20
10.0 9.876 98.60 10.0 10.074 100.27
20,0 20.143 100.7 20.0 19.948 99.74
50.0 49.651 99.30 50.0 50.204 100.41
x? ± SD 99.52 ± 0.87 99.82 ±0.61
t-value 0.32
F-value 0.57

Oratab®
100mg tablet 20.0 19.93 99.6 20.0 20.07 100.4
40.0 39.76 99.4 40.0 39.89 99.7
60.0 58.85 98.3 60.0 59.87 99.8
x? ± SD 99.1 ± 0.7 99.9 ± 0.38
t-value 0.065 (3.18)
F-value 0.450 (9.55)

Each result is the mean value of three determinations
Numbers in the parentheses are the critical tabulated values of t and F at (P=0.05)

Table 3 Intra-assay and inter-assay precision and accuracy of the determination of SUM in tablets using the proposed method
Intra-assay Inter-assay

Nominal conc. Measured conc. Recoverya Nominal conc. Measured conc. Recovery
(ng/ml) (ng/ml) (%±RSD) (ng/ml) (ng/ml) (%±RSD)
50 50.23 100.5 50 49.65 99.3
100 99.68 99.7 100 97.86 97.9
150 149.57 99.7 150 147.83 98.6
a Mean of three determinations.
x? ± SD 99.9 ± 0.46 98.6 ± 0.7

Each result is the mean value of three determinations
Pharmaceutical applications
The proposed method was successfully applied for the assay of SUM in tablets which is the conventional pharmaceutical formulation of SUM. Percent recovery results of different concentrations based on three determinations are shown in Table 2. The results were comparable with those obtained from the reference method.
Application to human urine and plasma
The proposed method was also applied for the assay of SUM in spiked urine and human plasma. The % recovery results shown in Table 4 revealed that the proposed method is applicable for the assay of SUM in urine and plasma samples.
Table 4 Assay results of the proposed method for the assay of SUM in spiked urine and plasma

Parameter: urine % Found Plasma % Found
Amount Amount Amount Amount
added found taken found
(ng/ml) (ng/ml) (ng/ml) (ng/ml)
5.0 4.974 98.8 5.0 4.910 98.2
10.0 9.876 98.8 10.0 9.674 96.7
20.0 20.143 100.7 20.0 19.848 99.2
50.0 48.654 97.3 50.0 49.204 98.4
x? 98.9 98.1
SD 1.4 1.0
Each result is the mean value of three determinations

Conclusion
In this study, a new simple and sensitive was developed for the determination of Sumatriptan succinate based on the enhancement of native fluorescence of the studied drug using sodium lauryl sulfate as a micellar medium. The new method utilized the use of synchronous scanning for the determination of SUM in both pharmaceutical tablets and biological fluids, the method was validated and compared with reference methods.

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