Development and validation of spectrophotometric methods for simultaneous estimation of citicoline and piracetam in tablet dosage form.

CONTEXT: Citicoline (CN) and piracetam (PM) combination in tablet formulation is newly introduced in market. It is necessary to develop suitable quality control methods for rapid and accurate determination of these drugs. AIM: The study aimed to develop the methods for simultaneous determination of CN and PM in combined dosage form.
MATERIALS AND METHODS: The first method was developed by formation and solving simultaneous equations using 280.3 and 264.1 nm as two analytical wavelengths. Second method was absorbance ratio in which wavelengths selected were 256.6 nm as its absorptive point and 280.3 nm as λmax of CN. According to International Conference on Harmonization (ICH) norm, the parameters - linearity, precision, and accuracy were studied. The methods were validated statistically and by recovery studies.
RESULTS: Both the drugs obeyed Beer-Lambert's law at the selected wavelengths in concentration range of 5-13 μg/ml for CN and 10-22 μg/ml for PM. The percentage of CN and PM in marketed tablet formulation was found to be 99.006 ± 0.173 and 99.257 ± 0.613, respectively; by simultaneous equation method. For Q-Absorption ratio method the percentage of CN and PM was found to be 99.078 ± 0.158 and 99.708 ± 0.838, respectively.
CONCLUSIONS: The proposed methods were simple, reproducible, precise and robust. The methods can be successfully applied for routine analysis of tablets.

Pharmaceutical products formulated with more than one drug, typically referred to as combination products, are intended to meet previously unmet patients’ need by combining the therapeutic effects of two or more drugs in one product.[1] These combination products can present daunting challenges to the analytical chemist responsible for development and validation of analytical methods. Piracetam (PM), 2-(2-Oxopyrrolidin-1-yl) acetamide, is often used in the treatment of Alzheimer's, senile dementia, clotting, coagulation, vasospastic disorders, stroke, and ischemia; and for symptoms of dyspraxia, dysgraphia, and schizophrenia. It is a positive allosteric modulator of the AMPA receptor, improves the function of the neurotransmitter acetylcholine via muscarinic cholinergic (ACh) receptors which are implicated in memory processes [Figure 1].[23]

Figure 1

Structure of piracetam

Citicoline (CN) is cytidine 5‘(trihydrogen diphosphate) p’- 2 (trimethyl aminoethyl) ester inner salt, is used in treatment of pharmacotherapy of brain insufficiency and other related neurological disorders viz., as stroke, brain trauma, and Parkinson's disease.[4] It repairs the damaged cholinergic neurons via potentiation of acetylcholine production and reduces free fatty acid build up at the site of stroke-induced nerve damage [Figure 2].[56]

Figure 2

Structure of citicoline

Second order derivative method and absorbance correction method are reported for simultaneous ultraviolet (UV) spectrophotometric estimation of PM and CN in tablet formulation.[7]

The main disadvantage of this technique is its dependence on instrumental parameters like speed of scan and the slit width. No official procedure is given in pharmacopoeias for simultaneous spectrophotometric estimation of PM and CN. A liquid chromatography method for the determination of CT in injection, oral drops, and tablet dosage form;[891011] and spectrophotometric methods like colorimetry by complexation and UV-visible spectrophotometric methods using standard absorptivity value for determination of CT in pharmaceutical dosage form were also reported.[1213141516] Literature survey revealed high performance liquid chromatography (HPLC) methods for the estimation of PM in biological fluids.[17] Capillary electrophoresis, thin layer densitometric determination, and micellar electrokinetic chromatography methods were also developed for the estimation of PM in biological fluids.[18] In this present study, a successful attempt has been made to determine PM and CN in tablet dosage form by two methods based on simultaneous equation and Q-absorption ratio method. Both the methods were validated as per International Conference on Harmonization (ICH) norms.

Materials and Methods

Chemicals and reagents

Pure drug samples of PM and CN were obtained as gift sample from Akums Drugs and Pharmaceuticals, Mumbai. The combined dose PM (800 mg) + CN (500 mg) tablets (Strocit Plus) manufactured by Akums Drugs and Pharmaceuticals was procured from local medical shop. 0.1N methanolic HCl was used as solvent.

Instrumentation

Spectrophotometric analysis was performed on a double-beam Shimadzu 1800 UV-Visible spectrophotometer with data processing system, spectral bandwidth of 2 nm, wavelength accuracy ± 0.5 nm, and a pair of 10-cm matched quartz cells was used.

Preparation of stock and standard solutions for linearity

Stock solutions were prepared by dissolving PM and CN in 0.1 N methanolic HCl separately to obtain a concentration of 100 μg/ml of each compound. The standard solutions were prepared by dilution of the stock solutions in 0.1N methanolic HCl to reach concentration ranges of 10 μg/ml and 20 μg/ml for CN and PM, respectively.

Simultaneous equations method (method 1)

This method of analysis was based on the absorption of drugs CN and PM at the wavelength maxima of each other. The stock solutions of CN and PM were scanned separately in range of 200-350 nm to determine the wavelength of maximum absorption. From the overlain spectra [Figure 3], the wavelengths 280.3 and 264.1 nm was selected for CN and PM, respectively. For constructing a calibration curve, solutions of three series of different concentrations in range of 5-13 μg/ml for CN and 10-22 μg/ml for PM were prepared by appropriate dilution of stock solution with 0.1N methanolic HCl. Absorbance of these solutions were measured at 280.3 and 264.1 nm and calibration curves were plotted. The absorptivities (A1 %, 1 cm) of both the drugs at these two wavelengths were determined. These calculated values were the mean of six independent determinations. The concentration of the two drugs in mixture was calculated by using following equations:[19]

Figure 3

Overlain spectra of citicoline and piracetam

Where, Cx and Cy are the concentrations of CN and PM, respectively in mixture and in sample solutions. A1 and A2 are the absorbances of sample at 280.3 and 264.1 nm, respectively; ax1 and ax2 are the absorptivities of CN at 280.3 and 264.1 nm, respectively; and ay1 and ay2 are the absorptivities of PM at 280.3 and 264.1 nm, respectively. The validity of the formed equations was checked by carrying out six different determinations.

Q-absorption ratio method (method 2)

This method is applicable to the drugs that obey Beer's law at all wavelengths and the ratio of absorbance at any two wavelengths is a constant value, independent of concentration and path length. From the overlain spectra of CN and PM, two wavelengths 256.6 nm as isoabsorptive point and 280.3 nm (λmax of CN) were selected for formation of Q-absorption ratio equation. The calibration curves were determined in the concentration range 5-13 μg/ml for CN and 10-22 μg/ml for PM. The absorptivities (A1 %, 1 cm) of both the drugs at both the wavelengths were determined. These calculated values were the mean of six independent determinations. The concentration of the individual components can be calculated by using the following equations. [20]

Where, Cx and Cy are the concentrations of CN and PM, respectively in mixture and in sample solutions. A1 and A2 are the absorbances of sample at 280.3 and 256.6 nm, respectively. Qm is the ratio of absorbance of sample at 256.6-280.3 nm, QX is the ratio of absorptivity of CN at 256.6-280.3 nm, QY is the ratio of absorptivity of PM at 256.6-280.3 nm, ax1 and ay1 are the absorptivities of CN and PM at 280.3 nm.

Estimation of CN and PM in tablets

Twenty tablets of brand Strocit Plus (Akums drugs and pharmaceuticals, Mumbai, label claim CN 500 mg and PM 800 mg) were weighed and finely powdered. The powder equivalent to about 500 mg PM was weighed accurately, transferred to a 100 ml volumetric flask and suspended in 50 ml 0.1N HCl. The mixture was sonicated for 15 min and the volume was made to the mark with 0.1N HCl. The mixture was filtered through Whatmann No. 41 filter paper. Aliquot portion of the filtrate was further diluted with 0.1N HCl to achieve the final concentration of 9.37 μg/ml of CN and 15 μg/ml of PM. The absorbances were noted at respective wavelengths. The concentration of CN and PM was determined by the above methods.

Validation of methods

Linearity/range

Five aliquots of each drug solutions were taken from standard stock solution and transferred to 10 ml volumetric flask to get a final concentration of 5-13 μg/ml of CN and 10-22 μg/ml of PM using 0.1 N methanolic HCl. For simultaneous equation method, the absorbance of all standard solutions were measured at 280.3 and 256.6 nm, the calibration curves of absorbance vs concentration were plotted and correlation coefficient and regression line equations for both CN and PM were determined. For Q-Absorption ratio method, the wavelengths selected were 265.6 (isoabsorptive point) and 280.3 nm (λmax of CN). The absorbances of all standard solutions of both CN and PM were measured at these two wavelengths.

Accuracy

Accuracy of the developed method was confirmed by doing recovery study as per ICH norms at three different concentration levels 80, 100, and 120%. The absorbances were noted at respective wavelengths. The concentration of CN and PM were determined by the above methods in triplicate.

Precision

In intraday study, concentration of two drugs was calculated on the same day at an interval of 1 h. In interday study, concentration of drug contents were calculated on three different days. In both intra- and interday precision study, percentage relative standard deviation (% RSD) was calculated.

Ruggedness

Ruggedness of the two proposed methods was determined by carrying out analysis by three different analysts on different days.

Results and Discussion

In overlain spectra, CN and PM show prominent peaks at 280.3 and 264.1 nm. Hence, absorbances were measured at these two wavelengths for their determinations by simultaneous equation method. The linearity of CN and PM were found to be in the concentration range of 5-13 μg/ml and 10-22 μg/ml. For Q-Absorption ratio method, two wavelengths 256.6 nm as isoabsorptive point and 280.3 nm (λmax of CN) were selected from the overlain spectra and the linearity was found to be 5-13 μg/ml and 10-22 μg/ml, respectively. Calibration curves were plotted and regression analysis was carried out [Table 1]. The calibration curves indicated a good correlation between concentration and absorbance within the concentration range tested. The calibration curves are shown in Figures 4-9.

Table 1

Regression analysis of calibration curve

Figure 4

Calibration curve of citicoline at 280.3 nm

Figure 9

Calibration curve of piracetam at 256.6 nm

Calibration curve of citicoline at 264.1 nm

Calibration curve of citicoline at 256.6 nm

Calibration curve of piracetam at 280.3 nm

Calibration curve of piracetam at 264.1 nm

The percentage of CN and PM in marketed tablet formulation containing CN 500 mg and PM 800 mg were found to be 99.001 and 99.257%, respectively by simultaneous equation method. For Q-Absorption ratio method, the percentage of CN and PM were found to be 99.078 and 99.708%, respectively. The results obtained were comparable with the corresponding labeled amounts [Table 2].

Table 2

Results of analysis of piracetam and citicoline in tablets

The accuracy of the methods was determined at 80, 100, and 120% level. The percent recovery by simultaneous equation method ranges from 99.55-99.933% and 99.167-100.112% for CN and PM, respectively. By Q-Absorption ratio method the ranges were found to be 98.73-99.797% and 99.283-99.981% for CN and PM, respectively.

The precision was confirmed by intermediate precision. The analysis of formulation was carried out for three times in the same day and on three successive days. The % RSD values for inter- and intraday analysis of formulation was found to be less than 2%.

The ruggedness was confirmed by different analysts. The % RSD values for different analysts were found to be less than 2%. Hence, the intermediate precision and ruggedness were confirmed. The results for intermediate precision and ruggedness are shown in Table 3.

Table 3

Summary of validation parameters

Conclusion

Based on the results obtained, it can be concluded that the proposed UV-spectrophotometric methods (simultaneous equation method and Q-absorption ratio method) for simultaneous determination of CN and PM is rapid, economical, accurate, precise, and reproducible. The utility of the developed methods have been demonstrated by analysis of combined dose tablet formulation. Hence, the proposed method can be employed for quantitative determination of CN and PM in combined dose tablet formulation. Simultaneous equation method can be used to carry out dissolution study in combination tablet formulation.