Lazeeza Sattar Omer1

Lazeeza Sattar Omer1, Rasul Jameel Ali2 1 Department of Pharmaceutical Chemistry, College of Pharmacy, Hawler Medical University, Erbil, Iraq 2 Department of Clinical Biochemistry, College of Health Sciences, Hawler Medical University, Erbil, Iraq Abstract Extraction-spectrophtometric technique has been applied and approved for the estimation of orphenadrine citrate in the presence of paracetamol in binary synthetic mixture and in combined drugs. The process is built on the formation of a soluble red colour orphenadrine citrate eriochrom black T ion pair complex at pH 1.40, while the paractamol not paired. The produced red colour ion-pair complex was extracted by chloroform, and showed maximum absorption at 509 nm. For quantitative evaluation Beers law applied to plot the absorbance against concentration , the relation was liner in the concentration range of 0.10-6.00 mg L -1 with the molar absorptivity 4.4025 x104 L mol-1 cm-1.The limit of detection and limit quantification were 0.024 mg L -1, and 0.100 mg L -1 respectively. The intra-assay precision evaluated in terms of relative standard deviation (RSD) ( 2). and accuracy were validated with recovery (98.8-102.5).The results showed that orphenadrin citrate could be determined successfully in the combined tablet without interference by paracetamol and other common co-formulated substance. Orphenadrine citrate is diphenhydramine-mono methylated derivative of class tertiary amino ethers (ethanol amines or amino alkyl ether) figure-1, with potent antihistamines muscle-relaxant and analgesic properties, and it has some usefulness in the treatment of Parkinsonism including that induced by drugs such as the phenothiazines.2, 3 Figure1-1 Structural formulae of (A) diphenhydramine (B) orphenadrine citrate . Orphenadrine citrate is an N-methyl-D-aspartate receptor antagonist, which is possible of analgesic effects. Associated side effects with orphenadrine citrate use are partially related to its anticholinergic action and include dry mouth, urinary retention, confusion, blurred vision, agitation, and restlessness. 4 .Orphenadrine citrate is available in a combined form with paracetamol, aspirin, and nonsteroidal anti-inflammatory drugs .The Combination between Orphenadrine citrate and paracetamol has appeared predominant pain relieving impacts, compared to each fixing when utilized alone. 5, 6 In the present study the chromogenic reagent is the acidic dye Eriochrome black T (EBT) Sodium 1-(1-hydroxy-2-naphthylazo)-6-nitro-2-naphthol-4-sulphonate (NaH2In) figure-2 ,its of class azo (-NN-) compound ,and contains SO3Na group allowing it to be soluble in water .At low pH (e.g. 1.4) the basic drug protonated (positively charged),and EBT is a completely ionized forming anion (H2In- ).The electrostatic attraction between cationic drugs and the anionic dyes produce an ion-pair complex, which definitely extracted using organic solvents. 19. EMBED ChemDraw.Document.6.0 Figure 2- The Chemical Structure of EBT 2- Experimental 2.1 Instruments A double beam UV-visible spectrophotometer -Perkin Elmer Lambda 25 was used to carry out spectral runs. JENWAY 6305 UV-visible spectrophotometer with glass cells (L1cm) was used for all other absorbance measurements. A pH-meter HANNA was used for fast and dependable pH measurements. 2.2 Material Paracetamol and orphenadrine citrate were kindly provided from Awa medica pharmaceutical company., Hawler, Iraq.All other chemicals were pure analytical reagent grade. The pharmaceutical products Myogesic (Naur- Jordan , Dar Al Dawa ), KANAGESIC (Syria ,KANAWATI),and Muscadol (Ras Al Khaimah,U.A.E,Gulf pharmaceutical industries/Julphar) have been obtained from the local drugstores. 2.3 Reagents 2.3.1 Paracetamol and Orphenadrine Citrate Standard Solutions. (100 mg/L) 0.01gm each paracetamol or orphenadrine citrate was dissolved in distilled water and diluted to 100ml. Appropriate dilute working solutions were prepared from the stock solution. The standard solution stayed unchanged for 1 month when kept in dark bottle and refrigerated. 2.3.2 Mixture Solutions Paracetamol plus orphenadrine citrate lab-made mixtures have been prepared by mixing different volume from their particular stock standard solutions (100 mg/L) equivalent to (35,40,and 45 mg/L) for orphenadrine citrate , and (400.0,450.0 and 500.0 mg/L), surrounding the dosage form ratio. Phosphate buffer (pH 4) was prepared by dissolving 3.58 g of disodium hydrogen phosphate dodecahydrate in water and diluted to 500 mL with the same solvent. The pH value was adjusted with phosphoric acid 2.3.3 EBT Solution (0.01) A fresh prepared EBT in small quantities was prepared by dissolving the correct weight in distilled water. Phosphate buffer (pH 4) was prepared by dissolving 3.58 g of disodium hydrogen phosphate dodecahydrate in water and diluted to 500 mL with the same solvent. The pH value was adjusted with phosphoric acid 2.3.4 Buffer Solution Different buffer solutions were freshly prepared using standard methods. The involved buffer systems are HCl-KCl (pH 1.5-pH 4.2), HCl-CH3COONa(pH 1.99- pH 4.92), HCl- KHpthalate (pH 2.0-pH 6.0), and CH3COOH-CH3COONa(pH 2.8- pH 6.0). 2.4 Recommended Procedure In order to found the maximum wavelength of detection .3 mL of 25 mg/mL working solution of each Paracetamol , orphenadrine citrate , and laboratory prepared mixture of both drugs was mixed with 0.2 mL of 0.01 EBT solution , 3 mL buffer solution pH 1.4 and the volume filled up to 25 mL by distilled water .The solution mixture transferred to a separating funnel, the funnel was shaken twice by 5ml chloroform for 2 mints .Once a clear separation of the two phases were obtained the chloroform layers were collected and decanted to a spectrophotometric cell. These solutions were scanned in the visible region between 400-800 nm the. The blank was prepared in a similar way without drug solution. 3- Results and Discussion 3.1 Absorption Spectra The current method depend on the formation of red colour ion pair complex by means of the reaction of orphenadrine citrate with EBT in the buffer pH 1.4, extracted ion pair complex was showed maximum absorbance at 509 nm figure-3a, orphenadrine citrate in the presence of paracetamol in laboratory prepared mixture solutions figure-3b. While paracetamol alone not react with EBT in the buffer pH 1.4, therefore the paracetamol aqueous layer figure-3c, and paracetamol organic chloroform layer figure-3d appear no absorbance. Figure 3- Absorption spectrum of the a- orphenadrine citrate -EBT ion pair complex b- mixture of paracetamol and orphenadrine citrate -EBT ion pair complex c- paracetamol aqueous layer ,and d- paracetamol organic chloroform layer. 3.2 Reaction Mechanism Orphenadrine citrate is amino ether the amine group is of a class tertiary amine(R3N) ,that contain unshared electron pair on nitrogen, At low pH, nonbonding pair of electrons on nitrogen is able to accept acidic proton forming protonated orphenadrine citrate charged positively . The anion of acidic dye (that results from the removal of the proton from the acidic dye) attracts protonated orphenadrine citrate forming ion- pair neutral species. The orphenadrine- ion- pair neutral species was extracted into chloroform phase though the chloroform phase changes colour from clear to red .Whereas paracetamol is phenolic drugs (pKa 9.5) are 50 ionized when the pH equals their pKa, and it will only ionize to approximately 1 at the pH of blood (7.4)20, thus at low pH paracetamol remain unionized and not paired. The colour of the chloroform layer remains unchanged In the blank experiment. The mechanism of the reaction and ion- pair formation described in scheme-1 EMBED ChemDraw.Document.6.0 Scheme1 – Reaction scheme of Orphenadrine-EBT ion pair formation. 3.4 Optimization of Variables Parameters affecting on Orphenadrine EBT ion-pair formation were optimized through a number of elementary tests. 3.4.1 Effect of type, pH, and volume of Buffer Solution Buffers of a type HCl-KCl (pH 1.5-pH 4.2), HCl-CH3COONa (pH 1.99- pH 4.92), HCl- KHpthalate (pH 2.0-pH 6.0), and CH3COOH-CH3COONa(pH 2.8- pH 6.0) at different pH ranges were examined figure-4.The maximum colour intensity and highest absorbance values were recorded in potassium chloride- hydrochloric acid (pH1.4). Effect of buffer volume was also tested by addition of different volumes of HCl-KCl buffer solution (pH1.4) in the range of 1-4 mL .It was found that 2 mL gave highest absorbance value figure-4. 3.4.2 Effect of EBT Volume The volume range 0.05-0.4 mL of 0.01 solution of EBT have been selected to mixed with the drug to investigate the colour intensity of Orphenadrine-EBT ion-pair complex. The results figure -4 showed the absorbance of Orphenadrine-EBT ion- pair was directly proportional to the volume EBT up to 0.3 mL, which remained approximately constant by further addition. More addition make the colour opaque, therefore 0.3 mL of the EBT was used for the determination of orphenadrine 3.4.3 Effect of Shaking Time To extract the ion-pair complex from aqueous layer to chloroform layer .Shaking times ranging from 0.5 to 3.5 mints was studied, figure-4. Between 1.0 to 3.5 mints, the absorbance stayed fairly constant, and the optimum time for maximum extraction was 2 mints. Figure 4- Effect of pH , buffer volume, EBT(0.01) volume , and shaking time on absorbance of the Orphenadrine EBT ion-pair. 3.4.4 Effect of Temperature and Stability of The Ion Pair In this method, the red colour ion pair complex of orphenadrine-EBT was created directly after mixing at room temperature .The temperature effect on ion pair was studied at 25,and 30 0C,neglible change in the absorbance values was found and the results were constant at room temperature.After extraction the ion pair complex remained stable for at least 5 hours Figure-5 Figure 5- Effect of the time on the stability of ion pair complex. 3.4.5 Effect of Extracting Solvent To find efficient extraction solvent, the organic solvents such as ethyl acetate ,butanol , propanol, chloroform, carbon tetrachloride, and dichloromethane, were tested for extraction of the ion pair complex of Orphenadrine-EBT .Chloroform was chosen because it gave maximum absorbance of the extracted complex table-3. 1. Table 3.1- Solvent effect on Orphenadrine-EBT ion-pair complex absorbance. SolventEthyl acetateButaneolPropanolChloroform Carbon tetra chloride Dichloro- methaneAbsorbance0.125-0.063-0.0380.8820.2960.116 3.5 Justification of Recommended Method 3.5.1 Analytical Parameters A standard graph of the ion pair complex was constructed by plotting absorbance against concentration ,under the optimum conditions figure-6. Figure-6 calibration graph of the Orphenadrine-EBT ion-pair complex The standard graph indicate that the extracted ion pair was followed Beers Law in the concentration range of 0.1 – 6 mg/L ,with high correlation coefficient value R 0.9996. At high concentrations (greater than 6 mg/L) there is adeviation from Beers Law and the line loses its linearity .The Sandells sensitivity, and molar absorptivity were 0.0122 mg / cm2 , 4.4025 x 10 4 L/mol.cm respectively. The limits of detection and quantification were found to be 0.024 mg/L and 0.100 mg/L respectively, these low values designate to a good sensitivity of the method. The analytical parameter of the standard graph was shown in table 3.2. 3.5.2 Repeatability (Precision) 0.5041 0.732 RSD Average of six determination. 2mg/L, 4mg/L,and 6 mg/L of orphenadrine citrate. 3.5.3 Trueness (Accuracy) The trueness of the method was evaluated by addition of a known amounts of standard orphenadrine citrate solution to pre-quantified orphenadrine citrate solution. The analysis was done in a set of six replicate measurements at three different spiking table -3.3 .Precent recovery calculated using the following formula. Recovery (amount found / total amount) x 100 Table 3.3- Accuracy evaluation of the determination orphenadrine citrate Spiking Actual concentration, mg/LAdded concentration ,mg/LFound concentration ,mg/LRecovery S.D50212.96498.8 0.011100224.101102.50.032150234.97399.40.121 3.5.4 Interference To study the efficiency and selectivity of the suggested method, procedure of the analysis under the optimum conditions was carried out for the effect of excipients type (binders, disintegrate and diluents) as Cellulose-micro crystalline, Mg stearate,Silica-colloidal,Starch, dextrose , mannitol that are present in a tablet dosage. In this study, the solutions containing 3 mg L-1 of orphenadrine citrate and 10-fold of excipients were used following the recommended procedure. Experimental results achieved were nearly the same as those obtained for orphenadrine citrate solutions without excipients (error not more than 1) table- 3.4. 4. Conclusion In the suggested method, orphenadrine citrate in the presence of paracetamol react directly with EBT in acidic medium forming stable ion pair complex. Under the optimized extraction conditions the method was validate. The developed method are selective, accurate, precise ,rapid, and economical, and the drug was successfully estimated in both pure and pharmaceuticals samples for the determination orphenadrine citrate in single- drug or in combine drug in the presences of paracetamol without any interference from the commonly used ecipients. References Martindale. 2007. The complete drug reference .35th Edition, vol 1.published by the pharmaceutical press.UK, p 730. Wilson Gisvolds. Textbook of Organic Medicinal and Pharmaceutical Chemistry11th Edition. Published by Philadelphia. 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