A highly sensitive fluorimetric protocol based on isoindole formation for determination of gabapentin

A new, simple, highly sensitive and selective spectrofluorimetric method was developed for determination of gabapentin through its derivatization with o-phthalaldehyde in the presence of 2-mercaptoethanol. The resulting product was highly fluorescent and its emission intensity was measured at 431 nm after excitation at 335 nm. The effect of different experimental parameters on the formation and stability of the fluorescent product was carefully studied and optimized. The fluorescence–concentration plot was rectilinear over the range of 25–125 ng mL−1. The lower detection and quantification limits were 3.4 mL−1 and 11.2 ng mL−1, respectively. The procedure was fully validated according to the guidelines of the International Conference on Harmonization. The proposed method was successfully applied for the determination of the investigated drug in its pharmaceutical capsules and the results were in agreement with those of the reported method, in terms of the accuracy and precision. The low cost of analysis and high sensitivity make the proposed method ideally suited for analysis of the investigated drug in quality control laboratories.


Introduction
Gabapentin (Fig. 1), chemically known as 1-(amino methyl) cyclohexane acetic acid, is a structural analogue of the inhibitory neurotransmitter gamma-amino butyric acid (GABA) and its action is attributed to the irreversible inhibition of the enzyme GABA-transaminase, thus preventing the physiological degradation of GABA in the brain. It is an anticonvulsant drug used in the treatment of epilepsy and neuropathic pain, as an adjunct therapy for partial seizures in adults and children. 1 An analytical literature survey revealed that several methods have been reported for determination of gabapentin in pure forms and pharmaceutical dosage forms including spectrophotometry, 2-4 spectrouorimetry, 5-8 high-performance liquid chromatography (HPLC), [9][10][11] gas chromatography-mass spectrometry (GC-MS), 12 capillary electrophoresis 13 and electrochemical techniques. 14 The reported spectrouorimetric methods of analysis are either suffer from low sensitivity [5][6][7] or require heating and tedious extraction procedures. 8 In addition, the chromatographic, electrophoretic and electrochemical methods require highly sophisticated instruments and/or require expensive detector which is not available in most laboratories.
The aim of the present work is to develop a simple and highly sensitive spectrouorimetric method for determination of gabapentin. The amino group of the investigated drug was condensed with o-phthalaldehyde to form a highly uorescent product. The proposed method was applied for the determination of gabapentin in its capsules without any interference from the excipients. The advantage of spectrouorimetric method over other methods of analysis is that it does not require highly sophisticated instruments and more sensitive than the reported spectrophotometric methods of analysis.

Apparatus
PerkinElmer UK model LS 45 Luminescence Spectrometer was used for performing all the spectrouorimetric measurements. The instrument is equipped with a 150 W xenon arc lamp and a 1 cm quartz cell, connected to an IBM PC computer loaded with the FL WINLAB™ soware. Grating excitation and emission monochromators slit width for both monochromators were set at 10 nm.
Spectronic™ Genesys™ 2PC Ultraviolet/Visible Spectrophotometer (Milton Roy Co, USA) with a matched 1 cm quartz cell, connected to IBM computer loaded with Winspec™ application soware. Milwaukee SM 101 pH meter, Portugal and Digital Analytical Balance (AG 29, Mettler Toledo, Glattbrugg, Switzerland) were also used.

Chemicals and reagents
Gabapentin was kindly provided by Delta Pharm Company (10 th of Ramadan city, El Sharkeya, Egypt). It was used without further purication. o-Phthalaldehyde (Loba Chemie, Pvt. Ltd. 107, Woodhouse road, Mumbai, India) was freshly prepared by dissolving 10 mg in 3 mL of methanol and complete to 100 mL with distilled water. Working solution of the reagent (1 mg mL À1 ) was prepared by further dilution with distilled water. 2-Mercaptoethanol (Alpha Chemika, Mumbai, India) was freshly prepared as 0.5% (v/v) in water. Sodium hydroxide (El-Nasr Chemical Co., Cairo, Egypt) (0.1 M) was prepared by dissolving 400 mg in 100 mL of distilled water. Methanol was obtained from Merck, Darmstadt, Germany. All the reagents used were of analytical grade and the solutions were prepared with double distilled water.

Pharmaceutical formulations
The following available commercial preparation was analyzed; Gaptin® capsules (Delta Pharma S.A.E, 10 th of Ramadan City, El Sharkeya, Egypt) labeled to contain 100 mg gabapentin per capsule.

Preparation of standard solution
A stock solution of gabapentin was prepared by dissolving 10.0 mg of the investigate drug in 100 mL methanol. This solution was further diluted with the same solvent as appropriate to obtain the required working concentrations. The standard solutions were stable for at least 7 days when kept in the refrigerator.

General analytical procedure
Aliquots of gabapentin standard solutions covering the working concentration ranges (25-125 ng mL À1 as nal concentrations) were quantitatively transferred into a series of 10 mL volumetric asks. To each ask 0.6 mL of 2-mercaptoethanol solution (0.5% v/v) was added, followed by 0.8 mL of 0.1 M sodium hydroxide and mixed well. The solutions were allowed to stand for 10 min. Then, 1.7 mL of o-phthalaldehyde was added. The reaction mixture was allowed to stand for 25 min. The volume was completed to the mark with methanol and the uorescence intensity of the reaction product was measured at 431 nm aer excitation at 335 nm. The relative uorescence intensity was plotted against the nal concentration of the drug (ng mL À1 ) to get the calibration graph.

Analysis of pharmaceutical formulations
Twenty capsules containing the investigated drug were accurately weighed and mixed thoroughly. An amount of the powder equivalent to 10 mg of the drug was dissolved in 100 mL methanol. The solution was further diluted with the same solvent and a portion of the resulting solution was subjected for drug analysis using the previously described general analytical procedure. The nominal content of the capsules were calculated using the corresponding regression equation.

Result and discussion
o-Phthalaldehyde in combination with a thiol compound, such as 2-mercaptoethanol, is widely utilized as a uorescence derivatizing agent for amino compounds. This approach has been applied for the determination of several pharmaceutical compounds. [15][16][17] A condensation reaction occurs between the aldehydic groups of o-phthalaldehyde and primary amine group of gabapentin lead to formation of a high uorescent reaction product. Addition of 2-mercaptoethanol to the solution is essential to stabilize the formed reaction product. A proposal for the reaction pathway is presented in Fig. 1. The formed product exhibited high uorescence at 431 nm aer excitation at 335 nm. The uorescence spectra of the reaction product obtained by using 75 ng mL À1 of gabapentin against the blank treated in the same manner are shown in Fig. 2.
The developed spectrouorimetric method has several advantages over the previous reported methods. The current spectrouorimetric method is characterized by high sensitivity (in ng mL À1 ) compared with Hassan et al. 5 and Prasad et al. 7 methods. Also, the method is cheap as it does not depend on using expensive reagent compared with Belal et al. method. 6 In addition, the proposed spectrouorimetric method is highly simple as there is no need for heating, using buffer or extraction with ethyl acetate as in Ulu et al. method. 8 As a result, the current spectrouorimetric method is a good alternative to the previous reported methods and could be ideally suited for quality control laboratory.

Optimization of the reaction condition
The uorescence properties of the reaction product, as well as the different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. Each factor was changed individually while others were kept constant. The examined factors included; volumes of 2-mercaptoethanol, sodium hydroxide and ophthalaldehyde, reaction time and diluting solvents.
3.1.1 Effect of volume of 2-mercaptoethanol. The addition of 2-mercaptoethanol is necessary to stabilize the reaction product of the studied drug with o-phthalaldehyde. Different volumes of 0.5% 2-mercaptoethanol solution (0.2-1.2 mL) were used in performing the general analytical procedure. It was found that, increasing the volume of the reagent produces a proportional increase in the uorescence intensity of the reaction product up to 0.4 mL. The uorescence intensity remains unchanged up to 0.8 mL (Fig. 3). Therefore, 0.6 mL of 0.5% of 2-mercaptoethanol solution was chosen as the optimal volume of the reagent.
3.1.2 Effect of sodium hydroxide volume. The uorescence intensity of the reaction products of the investigated drug was examined using different volumes (0.2-1.5 mL) of 0.1 M sodium hydroxide solution. Increasing the reagent volume resulted in increasing the uorescence intensity. Maximum values were obtained at 0.5 mL of sodium hydroxide and remain constant up to 1.0 mL. However, higher volumes of the reagent gradually decreased the uorescence intensity of the reaction products. Therefore, 0.8 mL of sodium hydroxide was selected as optimum volume throughout the study (Fig. 3).
3.1.3 Effect of volume of o-phthalaldehyde. The inuence of changing o-phthalaldehyde volume was studied using different volumes (0.5-2.5 mL) of the reagent (Fig. 4). It was observed that, increasing the volume of the reagent produces a proportional increase in the uorescence intensity of the  reaction product up to 1.6 mL. The uorescence intensities remained unchanged until l.8 mL aer which, a gradual decrease in the uorescence intensity were observed. Consequently, 1.7 mL of o-phthalaldehyde solution was chosen as the optimal.
3.1.4 Effect of reaction time. The reaction was allowed to proceed at different time intervals were tested (Fig. 4) and it was found that maximum relative uorescence intensity was obtained aer 20 min and remains stable up to 30 min. Hence, allowing the reaction mixture to stand for 25 min was adequate for maximum uorescence intensity.
As, the uorescence intensity is directly proportional to the uorescent product concentration. By increasing the reaction time, volume of 2-mercaptoethanol, sodium hydroxide or ophthalaldehyde, the concentration of the uorescent product (uorophore) increase and subsequently the uorescent intensity increased up to saturation. Further increasing in the uorophore concentration, the uorophore absorbs the exciting and possibly emitted light (the inner lter effect), reducing the amount of light detected and hence reduces the uorescence intensity.
3.1.5 Effect of diluting solvent. The formed uorophore was diluted with different solvents. The examined solvents included; water, methanol, ethanol, isopropyl alcohol, dimethyl formamide and acetone (Fig. 5). Of all the studied solvents, the highest uorescence intensity was obtained upon using methanol. The variable effect of different types of solvents on the uorescence intensity of the formed product could be attributed to that the changes in the solvent polarity and the surrounding environment have a great effect on the formed uorophore.

Validation of the proposed method
According to ICH Q2 recommendations, 18 the analytical validity of the method was tested regarding; linearity, sensitivity, accuracy, and precision. Linearity and range: the calibration curve for studied drug was constructed by plotting the relative uorescence intensities (RFI) against the nal investigated drug concentrations (ng mL À1 ). Linear regression analysis of the data was performed and the calculated analytical parameters including slope, intercept, standard deviation of slope and intercept and correlation coefficient are summarized in Table 1. From the calibration curves, we could observe that the RFI values and the drug concentrations was linear dependent within the range of 25-125 ng mL À1 of gabapentin. The excellent linearity of the proposed method was indicated by the high correlation coefficients. In addition, the small values of standard deviation of the intercept (S a ), and standard deviation of the slope (S b ) indicate low scattering of the points around the calibration curves.
Accuracy: in general, the accuracy could be dened as the closeness or agreement of the obtained analytical value to the true value and measured by calculating the percentage (%) recoveries. For the proposed method, the accuracy was checked by preparation of three standard solutions containing different concentrations of the studied drug within the specied linear range. The concentration of each standard solution was measured by our proposed method in triplicate manner. As shown in Table 2, the obtained % recoveries are close to 100% which indicates the good accuracy of the proposed method.
Precision: the precision is closeness or agreement of obtained analytical values to each other and is measured by calculating the relative standard deviation (RSD). The intra-and inter-day precisions were evaluated by applying the general analytical procedure for the analysis of standard drug solutions having three different concentrations within the specied linear range. The analysis was repeated three times within the same day for the intra-day precision and at three successive days for inter-day precision. The results were expressed in the form of % recovery and % relative standard deviation ( Table 3). The calculated relative standard deviations were found to be very small and below 2%, indicating good repeatability and reliability of the proposed method.
Sensitivity: the sensitivity of the proposed method was evaluated by calculating the limit of detection (LOD) and limit of quantitation (LOQ). Based on the standard deviation of the intercept (s) and the slope of calibration curve (S), the Limit of detection (LOD) and limit of quantitation (LOQ) were calculated using the following formula (LOD ¼ 3s/S and LOQ ¼ 10s/S). The obtained detection and quantitation limits were 3.4 and 11.2 ng mL À1 , respectively (Table 1). These values indicate the high sensitivity of the proposed method.
Robustness of the proposed method was evaluated by introducing minor changes in the experimental parameters such as volumes of o-phthalaldehyde, 2-mercaptoethanol, and sodium hydroxide and the reaction time (Table 5). These minor changes have no effect on the method performance as the obtained uorescence intensities were nearly the same. As a result, the proposed method could be considered as robust.

Application to pharmaceutical capsules
The general analytical procedure was applied for the quantitative analysis of commercial capsules containing gabapentin. The obtained results were statistically compared with those of

Conclusion
A highly sensitive procedure for the selective spectro-uorimetric determination of gabapentin through derivatization with o-phthalaldehyde was developed and validated. The proposed method was successfully applied for determination of investigated drug in their commercial pharmaceutical capsules with good accuracy and precision without any interference from common excipients. The involved methodology does not require elaborate treatment for the sample or tedious extraction steps. The high sensitivity together with the recognized advantage of the relatively low cost of spectrouorometric instrumentation make the application of the proposed method is feasible in routine analysis. Therefore, the proposed method is ideally suited for determination of gabapentin in quality control laboratories.

Conflicts of interest
There are no conicts to declare.  a Tabulated values at 95% condence limit; t ¼ 2.306 and F ¼ 6.338.