Isoabsorptive Point Method for Simultaneous Determination of Paracetamol and Orphenadrine Citrate in Their Combined Pharmaceutical Dosage Forms

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Introduction
Paracetamol (PAR); N-(4-Hydroxyphenyl)acetamide ( Figure 1) is related to a non-steroidal anti-inflammatory drugs (NSAID) which acts centrally and peripherally for treatment of non-inflammatory conditions in patients with gastric symptoms [1]. Orphenadrine citrate (ORP); (±)-N,N-Dimethyl-2-[(o-methyl-a-phenylbenzyl) oxy] ethylamine citrate ( Figure 1) is a skeletal muscle relaxant which acts centrally by depressing a specific neurons in the nervous system so that impulses of the somatic nerves can't be generated [1]. The combination of nonsteroidal anti-inflammatory drug and a skeletal muscle relaxant is better than single agents alone [2]. ORP can be used in combination with PAR as it prolongs and increases its antinociceptive effect [1]. The literature revealed that several methods have been carried out for the analysis of PAR and ORP in their mixture form or in their combination with other drugs. PAR & ORP were determined by spectrophotometric methods, HPLC methods, TLC and microemulsion HPLC method and square wave voltammetric method [1, [3][4][5][6][7][8][9][10][11][12][13]. To the best of our knowledge, there is no reported method for the determination of this drug mixture using Isoabsorptive point technique. As such, the aim of work is to develop a spectrophotometric method which is accurate, fast and non-complicated for determination of PAR & ORP combination without the interference of their additives or their excipients in pharmaceutical formulations.

I Apparatus
JASCO dual beam UV-visible spectrophotometer model V-630 (Japan), connected to an ACER compatible computer with spectra manager II software was used. The spectral slit width was 2 nm and it could scan at speed up to 8000 nm/min. All the measurements were carried out in 1

v Laboratory Prepared Mixtures
Solutions of different ratios of PAR & ORP were prepared by transferring accurate aliquots from their standard solutions to 10 mL volumetric flasks and then diluting with 90% methanol.

For Isoabsorptive Point Method
A calibration curve was constructed relating absorbance of the zeroorder spectra of PAR at = 280 nm (Plateau region of ORP in which its absorbance is zero) and the zero-order spectra of PAR and ORP at = 211 nm (Isoabsorptive point) to their corresponding concentrations in g/mL (Figure 2, 3) then the regression equation was computed. For PAR quantitation, Plateau region ( = 280 nm) was applied for estimation of the concentration of PAR. For ORP quantitation, isoabsorptive method was applied for estimation of total concentration of PAR and ORP. Absorption spectra of 20 g/mL of PAR, of 20 g/mL of ORP, and of a mixture containing 10 g/mL of each of PAR and ORP displayed isoabsorptive point at 211 nm point in the absorption spectrum, the total concentration of PAR and ORP in the mixture could be determined and consequently ORP concentration was calculated by subtraction of PAR concentrations. The total concentration of PAR and ORP could be calculated using the following equation: where represents the concentration of total concentration of PAR and ORP in g/mL, represents the absorbance of PAR or ORP at 211 nm, and represents the correlation coefficient. The major limitation of this method is that it needs a plateau region or a complementary analytical method for determination of one of the analytes in the mixture.

ii Analysis of Laboratory Prepared Mixtures
After preparation of different ratios of laboratory prepared mixtures, the spectra of these mixtures were measured and treated in the same way as described under the proposed methods. iii Application to Pharmaceutical Formulation 10 Tablets of Orphenadrine plus ® were weighed and crushed then an amount equivalent to 50 mg PAR and 5.55 mg ORP in each tablet was transferred into a 50 mL volumetric flask and diluted with 90% methanol as follows: First, 30 mL of 90% methanol were added and sonicated then dilution was carried out to the mark and filtered. Second, 10 mL of the dilution was transferred into a 100 mL volumetric flask to give a concentration equivalent to 100 µg/mL PAR and 11.11 µg/mL ORP. Third, any further dilutions were done in 10 mL volumetric flasks and treated in the same way as described under the proposed methods.

I Method Optimization
Two major problems were found during the analysis of PAR & ORP binary mixture; first, the overlapped spectra between the absorptivities of the drugs, and second, PAR, the major constituent in the dosage forms, had unfortunately high absorbance, while ORP the minor component in the dosage forms, had low absorbance values. As such, sample enrichment technique was used in which the concentration of the minor component ORP in its binary mixture was increased to facilitate its determination [14]. This was done by the addition of fixed amount of standard ORP to each experiment when combined with PAR, then subtracting its concentration before calculating the claimed concentration of the drug. Sample enrichment technique was used to solve the same problem for analyzing other drug mixtures of different drug ratios [15,16] .  (Table 1). Isoabsorptive point is very easy and simple as it depends on zero absorption spectra without the need of extra processing. On the other hand, it has two limitations; one is the need for some specific calculations to determine the values of Isoabsorptive point and the other is requiring more time for performing the standard addition on each mixture.

II Method validation
All methods were validated according to ICH guidelines [17]. The linear regression data for the calibration curve showed good linear relationship ( Table 1). The accuracy was calculated by analyzing the standard addition where satisfactory results were obtained as shown in (Table 1). The specificity of the method was calculated by assaying the laboratory prepared mixtures of PAR & ORP within the linearity range and good results were obtained (Table 1). The intra-and inter-day precisions were calculated by the analysis of 3 different concentrations of the drugs 3 times on the same day and on 3 successive days (Table 1).

III Application to Pharmaceutical Formulation
The proposed method was successfully applied for determination of PAR and ORP in their pharmaceutical formulation (Orphenadrine plus ® tablets). The results were acceptable and with sufficient agreement with the labelled amounts. The standard addition technique was applied and showed that no interference of the excipients was observed ( Table 2)..

IV Statistical Analysis
Statistical comparison of the proposed method was performed through One-way ANOVA method by using PASW statistics 18® software program in which there was no significant difference between the proposed method and the reference method [4] as shown in (Table 3). Table 3: Statistical comparison of the results obtained by the proposed method and the reference method using One-way ANOVA.

Conclusion
Isoabsorptive point method was successfully applied for the determination of paracetamol and orphenadrine citrate in their binary mixtures and in their dosage form. The proposed method is simple, sensitive and accurate and could be used for routine analysis by using simple technology or instruments. By comparison with the previous reported methods, it was concluded that Isoabsorptive point method does not require extra processing but it may need a plateau region or a complementary technique for determination of one of the analytes. Statistical comparison revealed that there is no observed significant difference between the proposed method and the reference one.