Green Spectrofluorimetric Technique for the Determination of Vildagliptin Through Quenching Lanthanide Luminescence in Pure, Pharmaceutical Dosage Form and Spiked Plasma

Objectives: The objective of the proposed method is to develop a validated sensitive, selective, rapid, and low-cost spectrofluorimetric technique for the estimation of Vildagliptin depending on its quenching effect on the fluorescence intensity of Terbium (Tb). Methods: The proposed method involves quantitative fluorescence quenching of terbium measured at 477 nm after excitation at 239 nm. Various experimental conditions were investigated as pH, time of reaction, order of mixing of reagents, concentrations of Tb and buffer. Results: Under optimum conditions, good linearity was obtained within the range 20.0-3000.0 ng/mL with correlation coefficients of 0.9997. LOD and LOQ were 1.8 ng/mL and 19.2 ng/mL, respectively. Conclusion: The proposed method accomplished greener parameters; accordingly, it is a green choice for the determination of Vildagliptin in bulk, pharmaceutical dosage form, and spiked plasma.

Lanthanide ions (terbium) form stable chelates with organic ligands. Luminescence of lanthanide chelate was identified by large stock shift, narrow emission bands, and long fluorescence lifetimes. Therefore, they improve fluorometric detection of certain organic compounds 13 as fluoroquinolones 14 , fexofenadine 15 , and DNA 16 . Different organic compounds can quench the background luminescence of lanthanide ions especially when they are in the form of chloride salts, because the probability of collisions leading to energy transfer is larger for the chloride salts 17 . The proposed method provides a new simple method for quantitative spectrofluorimetric measurement of VIL through quenching the fluorescence of Terbium-Tris complex.
Recently analytical techniques have directed toward substituting existing techniques with greener ones 18 . Green techniques are usually rapid and less consuming in solvents and reagents; accordingly, researchers have established spectroscopic techniques especially fluorometric ones depending on their greenness and high selectivity of the methods 19 .
Many greenness assessment tools have been developed. Recently the analytical eco-scale was used to calculate a greenness score for each method by subtracting penalty points for each factor from total of 100 points. A total eco-scale score represents ˃ 75 (=excellent greenness), ˃ 50 (=acceptable greenness), and ˂ 50 (=inadequate greenness) 20 . Another method, the green analytical procedure index (GAPI) was used to assess the greenness of the technique by giving an accurate picture about the effect of each step of the proposed technique 21 , and a new assessment called the 'green certificate' classifies the methods by colors and letters from A (the greenest) to G 22 .
VIL has no intrinsic fluorescence. Thus, the proposed method depends on developing a valid, green, sensitive and selective spectrofluorimetric technique for the estimation of the drug in bulk powder, pharmaceutical dosage form, and spiked plasma.

Market samples
Icandra® tablet (Mash premiere, Egypt) 50 mg of VIL per tablet (batch no. M1133320) was purchased from the local market.

Reagents
All solvents used were of HPLC grade and chemicals were of analytical reagent grade. Terbium (ΙΙΙ) Chloride hexahydrate 99.9 ٪(Aldrich, Germany) 1×10 -4 M methanolic solution of terbium was prepared. Tris (hydroxymethyl) aminomethane buffer (Tris) (El-Nasr pharmaceutical company, Cairo, Egypt) 0.1 M methanolic solution of tris buffer was prepared at pH 7 with 1 N HCl. Methanol, acetonitrile, and isopropanol (Fisher chemical, United States), Fresh frozen human plasma samples were obtained from VACSIRA (Cairo, Egypt) and were kept frozen until use after gentle thawing in room temperature.

Standard Solutions
Stock standard solution of 1 mg/mL VIL was prepared by dissolving 10 mg of VIL in methanol in 10 mL volumetric flask.
Working standard solution of 40 μg/mL VIL was prepared from the stock solution. The standard solutions were kept in a refrigerator at approximately 4°C and remained stable for at least 1 month.

Application to pharmaceutical preparation
10 tablets of Icandra ® 50 mg were ground and an accurate weight equivalent to 50 mg was added to 50 mL volumetric flask containing 30 mL of methanol, sonicated for 20 min, then the volume was completed with methanol, filtered using 0.45 μm filter and proceeded as under construction of calibration curve.

Application to spiked plasma
A 0.5 mL aliquot of human plasma was transferred into a series of 5 mL centrifuge tubes and spiked with different amounts of VIL to give a final concentration range of 0.2-10.0 µg/mL, then 4.0 mL acetonitrile were added, vortexed for 30 s, then centrifuged at 6000 rpm for 6 min and the supernatant was separated carefully. 1 mL of the supernatant was taken separately from each centrifugation tube and was treated as under construction of calibration curve. Apparatus All the fluorescence spectra were recorded using a JASCO FP-6200 Spectrofluorometer, equipped with 150 W Xenon lamp, grating excitation and emission monochromators, and a recorder. Slit widths for both monochromators were set at 10 nm. A 1.0 cm quartz cell was used. Spectra were evaluated using Spectra Manager FP-6200 Control Driver software, Version 1.54.03, JASCO Corporation.

Fluorescence spectral characteristics
Terbium chloride (Tb 3+ ) does not show characteristic fluorescence while by adding Tris buffer intense fluorescence was observed. When excited at λ 239 nm, two emissions bands at λ 477 and λ 545 nm were obtained as shown in Figure 2. By adding VIL to Tb 3+ /Tris complex, a decrease in the fluorescence intensity was observed based on the quenching effect of the studied drug (Figure 3), the quenching effect was proportional to the concentration of VIL. Quantitative determinations were made by excitation at λ 239 nm and measuring the fluorescence at λ 477 nm. The emission peak at λ 477 nm was selected for the fluorescence quenching measurements as it has higher fluorescence intensity than the emission peak at λ 545 nm leading to greater sensitivity while measuring the concentration of the studied drug, so our study will be at λex/λem = 239/477 nm.

Optimization of the experimental conditions Effect of concentration of Tris buffer
The effect of the concentration of Tris buffer was studied and as shown in Figure 4, the quenching in fluorescence intensity reached a maximum at concentration 0.01M Tris buffer. Thus, 1 ml of 0.1 M Tris-HCl in 10 ml volumetric flask was the optimum buffer volume for analysis.

Effect of concentration of terbium
The effect of the concentration of Tb 3+ was studied and as shown in Figure 5, the quenching in fluorescence intensity reached a maximum at concentration of 0.1 × 10 -4 M Tb 3+ . Thus, 1 mL of 1×10 -4 M Tb +3 in 10 mL volumetric flask was selected for further analysis.

Effect of pH
A series of 10 mL volumetric flask containing Tb +3 / Tris complex and 800ng/mL of VIL at different pH values with their corresponding blank solutions were prepared and measured at λex/λem = 239/477 nm. As shown in Figure 6, the quenching in fluorescence intensity of the system reached a maximum when pH was 7.0. At higher pH (pH >7.0) the intensity decreased due to the precipitation of terbium hydroxide. Thus, pH 7 was selected for the following measurements.

Effect of reaction time
The time of the reaction was studied at room temperature, and it was found that the complex was formed after 10 min and remained stable up to 50 min. In this method, 15 min was set as the standard time for the reaction.

Effect of different solvent
Different solvents were investigated as acetonitrile, water, isopropanol, and methanol, high fluorescence intensity of Tb 3+/ Tris complex was only observed when methanol was used as the diluting solvent.

Effect of order of addition of reagents
Series of solution with the same concentrations of reagent but differ in the order of mixing and their corresponding blank solution was prepared and measured at λex/λem = 239/477. The results showed no significant difference can impact fluorescence quenching (F°/F).

Method validation Linearity
Under the above condition, the calibration curve was constructed by plotting F°/F against VIL concentration (Figure 7). A linear relationship was obtained over concentration range 20-3000 ng/mL. The limit of quantitation (LOQ) and the limit of detection (LOD) were calculated according to ICH guidelines 23 . The results were shown in Table 1. = 10σ/ S, LOD = 3.3 / , Where σ = the standard deviation of response and S = slope of the calibration curve.
A comparison of the analytical performances of the previously reported methods and the proposed method for the determination of VIL is summarized in Table  2.
Compared with the previous spectroflourimetric methods, the proposed method was relatively simple with higher sensitivity and lower detection limit that can detect minor amount of VIL in spiked plasma.

Accuracy
The accuracy of the proposed method was determined by the standard addition method after spiking with three-level concentrations of 50,100,150 ng/mL of VIL solution. The percentage of recoveries based on the average of triplicate determinations are shown in table 3. The results prove the accuracy of the proposed method for the determination of VIL as indicated by high values of recovery percentages.

Precision
The intra-day and inter-day precision were investigated through triplicate analysis of three concentration on the same day and on 3 successive days, respectively. The results are shown in Table 4.

Application on analysis of pharmaceutical dosage form
The proposed method was applied successfully for the determination of VIL in bulk and dosage form Table 5. The results of our method were compared with those of the reported method. No significant difference was observed between the two methods regarding accuracy as indicated by student's t test and precision as indicated by F test.

Application in spiked plasma
The proposed method was efficiently applied for the determination of VIL in spiked plasma ( Table 6). The results prove the efficiency of the proposed method for the determination of VIL in spiked plasma by high values of recovery percentages.

Proposed mechanism of the reaction
The intense lanthanide-sensitized luminescence originates from an intramolecular energy transfer through the excited triplet state of the ligand to the emitting resonance level of the ion followed by radiative emission from the cation. The efficiency of the energy transfer depends on the matching between the triplet level of the organic compound and the resonance level of the ion. The energy of the triplet level should be close to but higher than, that of the resonance level of the ion. In some instances, when the organic compound has a triplet state level below the excited state-level of the lanthanide ion the organic compound can quench the background luminescence of the ion 17 . The proposed mechanism between terbium and VIL is that VIL is coordinated to the metal ion via the amino group and Carbonyl-O groups (Figure 8).

Quenching mechanism
The quenching mechanism was studied using stern Volmer equation 26 F0/F=1+Q KSV Where F0/F is the emission intensity in the absence and the presence of the quencher, Q is quencher concentration and KSV is stern volmer constant and calculated From the slope by plotting F0/F vs concentration of quencher. Ksv was found to decrease with increasing the temperature (298,308 K) which is a property for static quenching (Figure 10, Table 7).

Assessment of method greenness
Assessment of method greenness was determined through the quantitative analytical eco-scale, GAPI and green certificate methods. As shown in Table  8 and Figure 10, the proposed method has excellent greenness.

CONCLUSION
A validated, rapid, and low-cost spectrofluorimetric method was developed for the quantitative determination of VIL in pure form and pharmaceutical dosage form. The suggested method is economical compared to the reported HPLC method as there is no need to consume a large amount of solvents. The proposed method could be successfully used for routine analysis of the cited drug.

Funding Acknowledgment
No external funding was received.