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Micellar enhanced Synchronous Fluorescence Determination 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:
Sensitive, simple and rapid spectrofluorimetric method for the determination 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 wavelength of excitation and emission of 220 and 330 nm respectively in methanol at pH of (5.5), the fluorescence intensity was enhanced using micellar medium. The calibration curves were obtained with a range of (50-150) ngml -1 with a limit of detection of 30 (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.
Key words: Sumatriptan, Spectrofluorimetry, Pharmaceutical preparations, Biological fluids.
Introduction:

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) sub types.
It is official in European pharmacopeia 1 and united states pharmacopeia 2.
Several methods were suggested for the determination of sumatriptan in pharmaceutical and biological samples, including liquid chromatography methods with different detecting systems such as UV 3, Fluorescence 4 and MS detection 5,6. Other methods were based on spectrophotometric 7,8 and voltammetry 9. Few methods were based on the fabrication of nanomaterial based sensing methods 10,11. The objective of this work is to develop and validate simple, sensitive and selective spectrofluorimetric methods for the determination of sumatriptan in pharmaceutical and biological fluids in an easy way without the need to use high sophisticated and expensive methods. The method is based on measuring Synchronous Fluorescence intensities of Sumatriptan succinate in different solvents. All the factors affecting the fluorescence intensities were studied, including solvent compositions, 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 spectroscopy has advantages over conventional fluorescence spectroscopy, which includes simple spectra, higher selectivity, sensitivity and lower interferences which is due to sharper and narrower fluorescence peaks 12,13. Micellar media provide a rigid micro environment capable of restricting the freedom of fluorophore and so diminish the probabilities of non-radiative processes and provide relatively high viscous environment that can inhibit quenching by molecular oxygen. As a result, these factors may increase the quantum yield and enhance the fluorescence signal of the guest molecules 14.
The anionic surfactant Sodium Lauryl sulfate (SLS) has recently found many applications as a fluorescence enhancer for the determination of some pharmaceutical compounds 15,16
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 16.
The developed method was validated and applied successfully for quantitative determination of (SUM) in pharmaceutical samples and biological fluids.

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Fig. 1. 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 sulphate SLS 96% , Tween 80, ?-cyclodextrin, glacial Acetic acid were from BDH laboratory supplies, Sodium acetate, Sulphuric acid. Analytical grade solvents: Acetonitril 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 volummetric 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 (µg/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 were 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 volummetric 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 ng/ml. 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 quantitavely 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 Discussions:
The Literature survey revealed that 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 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 spectrum of SUM in water and Methanol.
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 if 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
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 17.

Figure 4: Effect of the addition of organized media on 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
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 synchronous spectrum for determining the position of the bands and their intensities. In the present work the optimization of (??) was performed by selection 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 showed in Fig. 6 below.
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 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 guidline. Validation on Analytical procedures: text and methodology, 18 Q2(R1), 2005:http://www.ich.org/LOB/MEDIA417.pdf 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
Synchonous scanning range (nm)
?? (nm)
Linearity range (ng/ml)
Correlation coefficient 0.994
Slope
Intercept
SD of the intercept (Sa)
SD of the slope (Sb)
% RSDa
% errorb
LODc (ng/ml)
LOQd (ng/ml)
a Percent relative standard deviation of three replicate samples, b Percent relative error of three replicates samples, c Limit of detection, d 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 19 HPLC….. , statistical analysis of the results using student’s t-test and variance ratio F-test 20 were 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 tablet form 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 5.0 4.910
10.0 9.876 10.0 10.074
20,0 20.143 20,0 19.948
50.0 49.654 50.0 50.204
X? ± SD
t-value
F-value

Oratab®
100mg tablet 20.0
40.0
60.0
X? ± SD
t-value
F-value

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

Table 3 Intra-assay and inter assay precision and accuracy for the determination of SUM by 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
100
150
a Mean of three determinations.
X? ± SD
t-value
F-value

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

Pharmaceutical applications
The proposed method was successfully applied for the assay of SUM in tablet 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 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 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 5.0 4.910
10.0 9.876 10.0 10.074
20,0 20.143 20,0 19.948
50.0 49.654 50.0 50.204
X?
SD
Each result is the mean value of three determinations
Number in the parentheses is the critical tabulated values of t and F at (P=0.05)

Figure 6: Calibration Synch-Sum-MeOH (10-200)

Calibration Synch-Sum-MeOH (10-200)

Effect of Temperature and time

Table 2 Assay results of the proposed methods in pure form and pharmaceutical forms

Proposed Method
Pure Form Taken Found
(µg/ml) (µg/ml) (µg/ml)
Parameter
X? ± SD
5.0
% Recovery ±SD
R.S.D (%)
t-value
F-value

Method
Tablet form Taken Found
(100 mg) (µg/ml) (µg/ml)

5.0
% Recovery ±SD
RSD (%)
t-value
F-value

*Theoretical values of (t) and (F) at p=0.05 Official Method
Taken Found
(µg/ml) (µg/ml)

Official Method
Taken Found
(µg/ml) (µg/ml)

Table 3: Accuracy and precision data for the determination of SUM using the proposed methods

Parameter Amount taken Amount found % Recovery

Method
Intrday precision
X? ± SD
% RSD
%Error
Interday precision

First day
Second day
Third day
X? ± SD
% RSD
%Error

Table 4 Assay results of the studied drug using the proposed method in spiked biological samples

Proposed Method
Urine samples Amount added Amount Found
(µg/ml) (µg/ml) (µg/ml)
Parameter
X? ± SD
5.0
% Recovery ±SD
R.S.D (%)
t-value
F-value

Serum samples
Amount added Amount Found
(µg/ml) (µg/ml)

5.0
% Recovery ±SD
RSD (%)
t-value
F-value

*Theoretical values of (t) and (F) at p=0.05
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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