| Abstract: |
The initial objective of this section was to develop isocratic reversed phase HPLC procedure for the simultaneous determination of drotaverine hydrochloride - nifuroxazide in presence of 4-HBH, mixture”A” and drotaverine hydrochloride - paracetamol, mixture ”B”.To optimize the HPLC assay parameters, the mobile phase composition, polarity and pH- values were studied for HPLC separation and determination(213)The mobile phase acetonitrile - methanol - water - acetic acid (70: 10: 20: 1 % v/v/v) was eluted at flow rate 1ml/min, nifuroxazide - drotaverine hydrochloride were separated at (4.52 and 8.33 min), respectively, but with poor sensitivity and a broadness in drotaverine hydrochloride peak was observed.The mobile phase, acetonitrile - methanol - water - acetic acid (65: 5: 30: 1% v/v/v) was tested at flow rate 1 ml/min, poor resolution and selectivity was observed, with retention time ”Rt” (3.3 and 3.2min) for drotaverine hydrochloride and nifuroxazide, respectively.Different mobile phases with different pH-values containing acetate buffer or phosphate buffer were prepared and tested, the results indicate poor resolution.Mobile phase acetonitrile - water at different composition ratios and various pH-values (3.2 - 4), was studied, a bad separation and poor sensitivity were observed at various wavelengths.Mobile phase, methanol and water at different pH-values (2.5 - 5) were tested and the selected pH was found to be 4.8 ± 0.1Different acids, orthophosphoric acid, glacial acetic acid and 0.1M HCl were used to adjust the pH-value, good resolution was observed on using glacial acetic acid. Different volumes of acetic acid ( 0.5 - 2 ml % v/v) were added to the prepared mobile phase, 1ml ± 0.1 % v/v glacial acetic acid was adequate to give good resolution.To maximize detector sensitivity, the response at various wavelength 302, 285, 250and 230 nm were tested, good quantification was achieved on using UV- detector at 250 nm for the simultaneous determination of the components in mixture ”A & B”.The selectivity of HPLC method was illustrated in fig (47,48) where the elution order in mixture “A” was 4-HBH (Rt = 1.15 min), paracetamol as I.S (Rt = 1.45 min), nifuroxazide (Rt = 3.99 min) and drotaverine hydrochloride (Rt = 6.93). While in mixture “B” the elution order was paracetamol (Rt = 2.05 min), nifuroxazide as I.S. (Rt = 5.53 min) and drotaverine hydrochloride (Rt =9.64 min).Calibration curves were constructed representing the relationship between the ratio of peak area and the corresponding concentration in the range 0.25 - 8 µg/ml for drotaverine hydrochloride, 0.5 - 16 µg/ml of nifurozaxide and paracetamol , respectively. Linear correlation coefficients were obtained as shown in fig (49 - 52). The regression equations(30 - 33) were computed and found to be:Mixture ”A”:Y=0.1769C-0.0037 r2=0.9997 ….. Drotaverine…………...(30)Y=0.2146C-0.0019 r2=1.0000 …... Nifuroxazide………… (31)Mixture ”B”:Y=0.4141C-0.0055 r2=0.9997 .…. Drotaverine…………... (32)Y=1.1866C-0.0176 r2=1.0000 ….. Paracetamol……………(33)Where, y is the ratio of peak area of each component in the two studied mixtures, C is the corresponding concentration in µg/ml and r2 is the regression coefficient.In mixture ”A&B” the selectivity was checked by analyzing the cited drugs in the laboratory prepared mixtures which prepared in different ratios mainly 1:5 as that of Drotazide capsules mixture ”A” and Anaspasm tablets mixture ”B” table (69-71).By the proposed HPLC technique, nifuroxazide in mixture ”A” could be determined in presence of drotaverine hydrochloride and 4-HBH ( nifuroxazide impurity ”A”), in the range (0.01-10 % w/w) ,table (70) without any interference, where the limited specification in the British pharmacopoeia(40)is not more than 0.05 %.For further study of the validity, recovery experiment were carried out b
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