Quantitative Analysis of Alendronate by Capillary Electrophoresis with 4-Methoxybenzenesulfonyl Fluoride

A low-cost, highly sensitive pre–column capillary electrophoresis (CE) method with diode-array detector (DAD) for determination of alendronate in pharmaceutical dosage forms was developed. The analyte was labeled with 4–methoxybenzenesulfonyl fluoride (MOBS–F) at 40 °C. Solid pharmaceutical preparations tablets of alendronate sodium were analyzed as real samples by proposed method with satisfied results.

In this work, we aimed at development of a low-cost, high sensitive and quantitative method for determination of alendronate by CE and using a new derivatization reagent with facile conditions and simple operation procedure.4methoxybenzenesulfonyl fluoride (MOBS-F) is a compound with chromophore and strong ultraviolet absorption.There were no literatures about applications of MOBS-F in CE detection.MOBS-F is much cheaper than DMS-Cl so as to reduce the detection cost.

Apparatus
All CE experiments were performed on the Beckman MDQ P/ACE system (Beckman Fullerton, CA, USA) equipped with an on-column DAD detector used at 235 nm.The Beckman 32 Karat software Version 4.01 (1999-2000 Beckman-Coulter) allowed instrument control and data analysis.An uncoated fused silica capillary tube (375μm O.D., 75μm I.D.) from Yongnian Optic Fiber (Hebei, China) was used for all CE separations.The total length of the capillary was 60 cm, with an effective length of 50 cm.The capillary was conditioned with 0.1 M NaOH (20 min at 20 psi), and then with water (10 min at 20 psi).Additionally, the capillary was washed with 0.1 M NaOH (2 min at 20 psi), with water (2 min at 20 psi), and with the running buffer (5 min at 20 psi) before each run.The temperature of the capillary was maintained at 25 •C.UV-visible spectra were recorded in 1-cm cells with UV-2550 spectrophotometer (SHIMADZU, Japan).The TDL80-2B tabletop high-speed centrifuge was employed.
Reagent Co. (Shanghai, China).MOBS-F was provided by the Institute of Nanjing Zhongyi Chemistry.Ultrapure water was prepared with a Milli-Q system (Millipore, Milford, MA, USA).Buffers were daily prepared in high purity water and filtered through 0.45 μm membrane filter.The separation buffer was a borate buffer (pH 9.3, 20 mM).The reagent MOBS-F solution (100 mM) was prepared in ACN.Alendronate stock solutions, at 5.0 mg/ml were prepared in ultra-purewater; the solution was prepared each month and stored at −20 •C.

Derivatization reaction
To 50 μL of standard solution, 1.50 mL of sodium borate buffer (20 mM, pH 9.3) and 200 μL of MOBS-F solution (100 mM in ACN) were added and then the mixture was heated for 10 min at 30 •C.After filtration with a 0.45 μm membrane, the mixture was injected into the CE.

Spectra of MOBS-F and derivation reaction
Alendronate sodium was labeled using MOBS-F under alkaline conditions, in order to obtain highly ultraviolet derivative.Fig. 2   UV-vis spectra of MOBS-F were investigated in acetonitrile solution.The absorption wavelengths of MOBS-F were obtained with the scanning range of 200 -400 nm.Maximum ultraviolet absorption responses were observed at the wavelengths of 268 nm.The molar absorption coefficients (ε) are 1.15×10 4 L mol −1 cm −1 .UV-vis spectra of MOBS-OH (λmax=230 nm) and derivative of alendronate sodium ((λmax=232 nm)) were investigated in running buffer (pH 9.30, 20 mM BBS).Therefore, the UV-vis detector was set at the wavelength of 232 nm in this method.
The yield of the derivatization reaction depends on pH, reagent concentration, reaction mixture composition and reaction time.The results show the derivative yield is strongly pH dependent, gradually increasing to pH 11.5 and rapidly falling down above pH 12.The derivatization is fast, prolonged reaction times had no effect on the reaction yield.

Stabilities of the reagent (MOBS-F) and its
derivative After an anhydrous acetonitrile solution of MOBS-F had been stored at room temperature (25 °C) for three months, the alendronate sodium derivatization yields obtained with the MOBS-F barely changed.The corresponding solution of derivative was stored at room temperature within 48 h, during which time they were analyzed ten times.No by-products peaks were found within the period of time.The results showed that MOBS-F and the alendronate sodium derivative were very stable.

CE Separation
A number of experiments optimized the resolution and efficiency in the separation by CE.The type of running buffer, the concentration and pH of the running buffer were optimized.Several types of running buffers were evaluated for the separation, including phosphate buffers, borate buffers, nitric acid, citric buffer and Tris buffer.In the nitric acid and Tris buffer, there were no alendronate sodium peaks.The results showed that borate buffers were the best choice.
The concentration (5-50 mM) and pH value (8.0-10.0) of borate buffer had to be varied.With a further high buffer, a large amount of Joule heating will affect separation efficiency.20 mM was the best.In the series of experiments of pH, the concentration of borate buffer 20 mM) was unchanged.The results showed that pH 9.30 was the best choice.

Linearity, precision, accuracy
A linear relationship between the peak area (monitored at 232 nm) and the concentration of alendronate was established over the examined concentration range (8.3-370 μg/ml).The average regression equation (n = 5) was calculated by the method of linear regression and found to be Y = 4347.2+ 194.5 * X.The method was found to be linear with a good correlation coefficient (0.992) over the examined concentration range.
The precision of the assay procedure as applied to alendronate raw material was determined at low (8.0 μg/ml), intermediate (54.0 μg/ml) and high (370 μg/ml) concentrations within the same day and over a period of 5 days.Results are summarized in Table 1.The recovery of the proposed CE method was estimated by analyzing laboratory mixtures of standard alendronate and excipients at concentrations that are expected to be found in tablets.The obtained average recovery (n = 5) for the mixture was 96.3 % (R.S.D. =1.2) which indicated good recovery.

Application to real samples
Commercially available tablets of alendronate sodium (Fosamax® 70 mg) were subjected to analysis by the proposed method (CE-DAD).The accuracy of the method can be inferred from the demonstrated lack of interference from commonly used tablet-additives (selectivity) as well as from the demonstrated precision of the methods.However, the precision of the proposed methods (experiments were repeated five times) appeared to be very well.

Copyright
This paper submitted to ICIAE 2018 was original and unpublished research.It has not been submitted to other conferences or journals.

Conclusions
Alendronate sodium can be determined in pharmaceutical tablets based on reaction with MOBS-F.One of the most attractive features of this method is the simplicity of the preparation of derivatives.The proposed spectrophotometric method is based on the fact that only the derivatized drug has absorbance at the measurement wavelength of 232 nm.It is a simple and nonexpensive method which is suitable for routine quality control of

Fig. 2 .
Fig. 2. Putative reaction scheme of the derivatization of Alendronate sodium with MOBS-F as a derivative agent.
demonstrates the chromatogram of the Alendronate sodium standard derivatized with MOBS-F.