Abstract
A new, simple, precise, accurate, reproducible, and efficient Vierordt’s method or simultaneous equation method was developed and validated for simultaneous estimation of paracetamol and flupirtine maleate in pure and pharmaceutical dosage form. The method was based on the measurement of absorbance at two wavelengths 245 nm and 344.5 nm, of paracetamol and flupiritine maleate in 0.1 N HCl correspondingly. Calibration curves of paracetamol and flupiritine maleate were found to be linear in the concentration ranges of 5–15 μg/mL and 1.53–4.61 μg/mL, respectively, with their correlation coefficient values (R2) 0.999. LOD and LOQ were 185.90 ng/mL and 563.38 ng/mL for paracetamol and 78.89 ng/mL and 239.06 ng/mL for flupiritine maleate. In the precision study, the % RSD value was found within limits (%). The percentage recovery at various concentration levels varied from 99.18 to 100.02% for paracetamol and 98.47 to 100.09% for flupiritine maleate confirming that the projected method is accurate. It could be concluded from the results obtained in the present investigation that this method for simultaneous estimation of paracetamol and flupirtine maleate in pure and tablet dosage form is simple, accurate, precise, and economical. The proposed method can be applied successfully for the simultaneous estimation of paracetamol and flupiritine maleate in pure and pharmaceutical dosage form.
1. Introduction
Paracetamol (PAR) is chemically N-(4-hydroxyphenyl)acetamide (Figure 1). It is centrally and peripherally acting nonopioid analgesic and antipyretic [1]. Flupirtine maleate (FLU) is chemically ethyl 2-amino-6-[(4-fluorobenzyl) amino] pyridin-3-yl carbamate maleate (Figure 2). FLU is a unique centrally acting nonopioid analgesic with muscle relaxants and neuroprotective properties. It is an aminopyridine derivative [2].
Literature survey publicized that certain UV [2–9], HPLC [1, 10–16], HPTLC [17], and LC-MS [18] methods were reported for the estimation of these drugs either individually or combined with other drugs. On the other hand simultaneous equation (SE) or Vierordt’s method was not reported for this new combination. Simultaneous equation (SE) or Vierordt’s method is typically applied to estimate drug combinations that contain two drugs or more than two drugs in combined dosage form. Technical hitches involved in this method is very less when compared to other UV methods. Hence an attempt has been made to develop a simple and a reproducible SE method to ensure the safety and efficacy of this selected combination. This developed method was fully validated and applied successfully for the simultaneous estimation of PAR and FLU in pure and pharmaceutical dosage form.
2. Materials and Methods
2.1. Apparatus
Shimadzu 1650 UV-VIS double beam spectrophotometer with UV probe software was used. Absorbance measurements were recorded with a pair of 1 cm matched quartz cells.
2.2. Chemicals and Reagents
Paracetamol and flupirtine maleate were kindly supplied by Safetab pharma (Pondicherry, India) and analytical grade Hydrochloric acid (S.D fine chemical Ltd., Mumbai, India) was used. The marketed tablet (LUPIRTINE) used contains 325 mg paracetamol and 100 mg flupiritine maleate and was manufactured by Lupin Pvt Ltd. (Mumbai, India).
2.3. Preparations of Standard Stock Solutions
Standard stock solution of paracetamol (125 μg/mL) was prepared by dissolving 12.50 mg of paracetamol in 60 mL of 0.1 N HCL. Resulting solution was sonicated for 10 minutes and the final volume was adjusted to 100 mL with 0.1 N HCL. From this standard stock solution, 2 mL was withdrawn and diluted to 25 mL using same solvent to get working standard solution of 10 μg/mL. Similarly standard stock solution of flupirtine maleate (38.46 μg/mL) was prepared by dissolving 3.846 mg of flupirtine maleate in 60 mL of 0.1 N HCL. Resulting solution was sonicated for 10 minutes and the final volume was adjusted to 100 mL with 0.1 N HCL. This solution was further diluted to get working standard solution of 3.07 μg/mL.
2.4. Simultaneous Equation Method Development
Working solutions of both drugs were scanned in the UV range 200–400 nm. The overlay spectra of both drugs were recorded (Figure 3). From overlain spectra, wavelengths 245 nm ( of PAR) and 344.5 nm ( of FLU) were selected for analysis of both drugs using simultaneous equation method (-245 nm for PAR and -344.5 nm for FLU). Consequently, it may be possible to determine both drugs by the technique of from method or simultaneous equation method[19–22].
Five standard solutions having concentrations of 5, 7.5, 10, 12.5, and 15 μg/mL for PAR and 1.53, 2.30, 3.07, 3.84, and 4.61 μg/mL for FLU were prepared in 0.1 N HCL and their corresponding absorbance was measured at 245 nm and 344.5 nm. The concentration of drugs x (PAR) and y (FLU) in sample solutions were determined by the SE method using the following formula: where and are the concentration of PAR and FLU, and are the absorbance of sample solution at 245 nm and 344.5 nm, respectively, and are absorptivity of PAR at 245 nm and 344.5 nm, and and are absorptivity of FLU at 245 nm and 344.5 nm, respectively.
2.5. Determination of Absorptivity Value
The absorptivity value of PAR and FLU from each solution was calculated using following formula [23] and the results were presented in Tables 1 and 2: Developed method was validated as per ICH [24] guidelines.
3. Results and Discussion
3.1. Specificity
Specificity of the method was dogged by measuring the absorbance of PAR and FLU individually at 245 nm and 344.5 nm against the blank and synthetic excipients and their absorbance was compared with the blank and synthetic excipients. No interference was observed at 245 nm and 344.5 nm indicating that the method is specific.
3.2. Linearity
The calibration curves (Figures 4 and 5) were constructed by plotting the absorbance versus the concentration ranges from 5, 7.5, 10, 12.5, to 15 μg/mL and 1.53, 2.30, 3.07, 3.84, to 4.61 μg/mL for PAR and FLU. It was found that, the calibration curves were linear in these concentration ranges with their correlation coefficient values 0.999 for PAR and FLU. Results revealed that good correlation exists between the concentration of the sample and their absorbance.
3.3. Accuracy
Accuracy of the method was determined by applying this described method to synthetic excipients containing known amount of each drug corresponding to 50%, 75%, 100%, 125%, and 150% of the label claim of PAR and FLU. The amount of PAR and FLU recovered in each level was calculated and results were presented in Table 3.
The percentage recovery varied from 99.18 to 100.02 for PAR and 98.47 to 100.09 for FLU, indicating good accuracy of the method.
3.4. Precision
Precision study was established by measuring the absorbance of sample solution without changing the assay procedure and the results were presented in Table 4.
3.5. Repeatability
This study was performed with a minimum of three replicate measurements of absorbance of sample solution at 0 hrs, 8 hrs, and 16 hrs in the same day.
3.6. Intermediate Precision
Intermediate precision was performed by measuring the absorbance of sample solution in three different days by different analysts and in different instruments.
3.7. Reproducibility
Reproducibility of the method was checked in two laboratories and the results were compared.
The low % RSD (<2%) for PAR and FLU indicated that the method is precise.
3.8. Robustness
Robustness of the method was determined by changing the wavelength ±1 nm from 245 nm to 344.5 nm and the results were offered in Table 5.
The % RSD value calculated from the robustness study was found to be less than 2% for PAR and FLU, indicating that the method is robust.
3.9. Limit of Detection (LOD) and Limit of Quantification (LOQ)
LOD and LOQ were calculated based on the standard deviation of the response (50% concentration solution) and the slope of calibration graph. LOD and LOQ for PAR and FLU were found to be 185.91 ng/mL and 563.38 ng/mL for PAR and 78.89 ng/mL and 239.06 ng/mL for FLU.
3.10. Stability
Stability of the standard and sample solutions were checked for three days at room temperature and the absorbance was measured on each day. Amount of drug present in the sample solution was calculated and the results confirmed that the sample solution is stable for three days without any degradation at room temperature.
3.11. Application of Developed Method to Marketed Dosage Forms
20 tablets were weighed and flattened into powder. Powder weight equivalent to 12.500 mg of PAR and 3.846 mg of FLU were transferred into a 100 mL volumetric flask. 50 mL of solvent was added and sonicated for 20 minutes. Then the final volume was diluted up to the mark with the solvent (0.1 N HCl) and filtered. 2 mL of the above filtrate was transferred into a 25 mL volumetric flask, and the final volume was adjusted up to mark with the same solvent to get sample solution with the concentration of 10 μg/mL of PAR and 3.07 μg/mL of FLU. The absorbance of the resulting solution was measured at 245 and 344.5 nm and the amount of PAR and FLU present in each tablet was found to be 325 mg and 100 mg, respectively. The assay results were described in Table 6.
Finally the developed new simple simultaneous equation method was applied successfully to the marketed tablet dosage form and the assay results were indicating that this method can be effectively used for the estimation of both drugs in combined dosage form.
4. Conclusion
The developed simultaneous equation method is simple, precise, specific, and accurate. Statistical analysis proved that the method was repeatable and selective for the simultaneous estimation of PAR and FLU in pure and pharmaceutical dosage forms without any interference from the excipients. This new simple method can be used routinely for the estimation of these drugs.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.