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1 Instruments and reagents HPLC: Agilent110 (Agilent Technology Development Co., Ltd.); acetonitrile is chromatographically pure, water is purified water, and triethylamine and phosphoric acid are analytically pure. Tomoxetine hydrochloride reference substance (lot number: 040726, content: 99.6%), raw material medicine (lot number: 040625, 040706, 040715), synthesized by Yangzijiang Pharmaceutical Group Co., Ltd.
2 Chromatographic conditions using Inertsil CN-3 column, 4.6mm Ã— 250mm (5Î¼m), with acetonitrile-1% triethylamine (phosphoric acid adjusted pH = 5.8 (40:60) as mobile phase; flow rate 1.0ml / min; detection wavelength At 217nm, the injection volume is 10Î¼l, and the number of theoretical plates should be no less than 2000 based on the tomoxetine peak. Under chromatographic conditions, tomoxetine hydrochloride and each impurity peak are well separated.
3 Method specificity inspection 3.1 The resolution of tomoxetine hydrochloride and each impurity According to the production process and the molecular characteristics of tomoxetine hydrochloride, in the determination of related substances by high performance liquid chromatography, acetonitrile-1% triethylamine is selected (Phosphoric acid adjusted to pH = 5.8) (40:60) As the mobile phase, tomoxetine hydrochloride can be completely separated from the four synthetic intermediates and by-products, and the resolution is greater than 1.5.
3.2 Destructive test Weigh 5 samples, each about 10mg, respectively, acid, alkali, oxidation, high temperature, light damage. The test results showed that the related substances of tomoxetine hydrochloride increased slightly under acid, oxidation and light conditions, and the resulting impurity peak and main peak could be separated well.
4 Limit of detection and limit of quantification Precisely weigh the appropriate amount of tomoxetine hydrochloride reference substance, add mobile phase to make the solution diluted quantitatively. Based on the triple signal-to-noise ratio, the detection limit of tomoxetine hydrochloride is 1.2ng, and with the ten-fold signal-to-noise ratio, the limit of quantification of tomoxetine hydrochloride is 4.2ng.
5 Determination of the relevant substances of the sample and the results Take an appropriate amount of this product, add the mobile phase to dissolve and make a solution containing 0.25mg of tomoxetine hydrochloride per 1ml, as the test solution; take 1ml of precise measurement in a 100ml measuring bottle Dilute the mobile phase to the mark, shake well, and use it as the control solution; precisely measure 10Î¼l of the control solution into the liquid chromatograph, adjust the sensitivity of the instrument, so that the peak height of the main component is about 10% to 25% of the full scale; 10Î¼l each of the control solution and the test solution were injected into the liquid chromatograph and the chromatogram was recorded to 3 times the retention time of the main component peak. If there is an impurity peak in the chromatogram of the test solution, the area formed by the baseline of each impurity Called the peak area. A = Ã— Ïƒ Ã— h = 2.507Ïƒh = 1.064 Wh / 2h> The sum of the peak areas should not be greater than the main peak area of â€‹â€‹the control solution. The test results of related substances of tomoxetine hydrochloride reference substance and 3 batches of raw materials are shown in Table 1.
Table 1 Related substance test results of tomoxetine hydrochloride reference substance and 3 batches of samples (omitted)
6 Discussion The chromatogram of the test solution for this test was recorded for 90 minutes. From the chromatogram, the purity of the tomoxetine hydrochloride reference and the three batches of raw materials are higher. There is basically no impurity peak after the main peak. At standard time, record the chromatogram of the test solution to three times the retention time of the main component peak.
When choosing the chromatographic conditions, I have tried methanol-1% triethylamine (30:70), methanol-tetrahydrofuran-1% triethylamine (phosphoric acid adjusted pH = 5.8) (10:30:60), acetonitrile-1 % Triethylamine (50:50) and other mobile phase systems, but the resolution between the main peak and each impurity peak does not meet the requirements, the final determination of acetonitrile-1% triethylamine (phosphoric acid adjusted pH = 5.8) (40: 60) As the mobile phase, the system can get better separation. When choosing the chromatographic column, the Zorbax Extend-C18 column (4.6mm Ã— 150mm, 5Î¼m) and the Inertsil CN-3 column (4.6mm Ã— 250mm, 5Î¼m) were tested separately. The test results showed that the Inertsil CN-3 column (4.6mm Ã— 250mm, 5Î¼m) The main peak has good symmetry, the main peak and the intermediate are well separated, and the column efficiency is high.
This article comes from the determination of related substances of tomoxetine hydrochloride by high performance liquid chromatography | Scientific Instrument Online Original Link: http: //
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