1. Instruments and reagents Shimadzu LC-10A high performance liquid chromatography; SPD-10A tunable wavelength detector; C-R7Aeplus data processor.
Nabumetone reference substance (content of 99.85%), raw materials, Southwest Synthetic Pharmaceutical Factory; theophylline, China Pharmaceutical and Biological Products Inspection; nabumetone tablets, Jiangxi Dawn Pharmaceutical Factory. Methanol, sodium acetate, glacial acetic acid were of analytical grade; water was distilled water.
2. Chromatographic conditions Column: Shimadzu Shim-pack CLC-ODS column, 150 mm × 4.6 mm; mobile phase: methanol -0.05 mol.L-1 acetate buffer (0.05 mol·L -1 sodium acetate aqueous solution with glacial acetic acid Adjusted to pH 3.5) (65:35); flow rate: 1.0mL.min-1; detection wavelength: 254nm; injection volume: 10μL.
Nabumetone and internal standard chromatograms in the chromatographic conditions shown in Figure 1.
3. Linear relationship Precision weighed nabumetone reference substance and the internal standard amount of theophylline, respectively, formulated with methanol 1.08mg.mL-1 and 1.05mg.mL-1 solution. The precise amount of reference substance solution 2.0,3.0,4.0,5.0,6.0,7.0,8.0mL were set 25mL volumetric flask, the precise addition of internal standard solution 5mL, diluted with methanol to the mark, shake, injection determination. The results showed that there was a good linear relationship between the peak area ratio of nabumetone in the range of 86.4 ~ 345.6μg.mL-1 and the concentration of nabumetone. The regression equation (n = 7) was Y = 3.43 × 10-3 5.64 × 10 -3Xr = 0.99981. Theophylline (1.88 min) 2. Nabumetone (3.75 min)
4. Sample Determination 4.1 Determination of the raw material Precision Weigh the sample about 100mg, set 100mL flask, add methanol and dilute to the mark. Precision draw 5mL set 25mL volumetric flask, add precise internal standard solution 5mL, diluted with methanol to the mark, shake, injection determination. Use internal standard method to calculate the content. 3 batches of raw materials measured results in Table 1.
Sample mark% RSD /% 199.470.5299.680.7399.450.7 Table 1 Determination of raw material (n = 3)
4.2 Tablet Determination Take tablets 20, accurately weighed, fine study, accurately weighed amount (equivalent to about 500mg nabumetone), set 500mL volumetric flask, dissolved in methanol and diluted to the mark, shake , Filtered. Precision drawing continued filtrate 5mL set 25mL volumetric flask, add precise internal standard solution 5mL, add methanol to the mark, shake, the sample was measured.
Three batches of samples measured results in Table 2.
Sample Marked% RSD /% 195.890.7297.660.43100.00.65 Discussion 5.1 The author according to the ratio of nalidiximene tablet prescription adjuvant and nabumetone reference substance, the recovery test study, 5 times the average recovery of the test results 99.46%, RSD 0.6%. Experiments show that this method using theophylline as an internal standard, can improve the reproducibility of the analysis results, to avoid system errors due to injection and equipment factors, but also to avoid impurities, accessories, etc. to participate in the UV-induced errors .
5.2 The amount of methanol in the mobile phase and the pH of the mobile phase have a significant effect on the internal standard, especially the peak time and peak shape of the sample. The greater the amount of methanol, the lower the pH, can speed up the peak time, improve the peak shape. After several experiments, the ratio of methanol-0.05mol.L-1 acetate buffer (65:35, pH3.5) was better, the peak was fast, the peak shape, the resolution was good, the result was satisfactory .
5.3 The product solution set colorless and transparent volumetric flask, placed for 1 week, the ratio of sample and internal standard peak area is essentially the same, the solution is stable.