High performance liquid chromatography analysis method of o-methylphenylacetic acid and 2-chloromethylphenylacetic acid impurity content in 3-isochromanone
A technology of isochromanone and chloromethylphenylacetic acid, applied in the field of high-performance liquid chromatography analysis, to achieve the effects of ensuring stability, improving symmetry, and reliable detection data
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Embodiment 1
[0036] Selection of HPLC conditions:
[0037] 1.1 Selection of mobile phase
[0038] According to the influence of solvent strength, solubility, polarity and viscosity on HPLC separation, the influence of methanol-water and acetonitrile-water composition flow relative separation was investigated respectively. The results show that the solubility of 3-isochromanone in acetonitrile is very good, its viscosity is lower than that of methanol, and its impact on machine pressure is small, so acetonitrile-water is used as the flow. In addition, the effect of different volume ratios of acetonitrile-water solution on the separation effect was investigated. Experiments have found that when the proportion of acetonitrile in the mobile phase is between 20% and 40%, the impurities in 3-isochromanone can be separated well, so the gradient elution procedure is used to make the impurity peak more effective Separate. In the mobile phase selected above, in order to improve the peak shape, the sep...
Embodiment 2
[0042] Preparation of standard samples
[0043] Accurately weigh 400.00mg o-methylphenylacetic acid and 2-chloromethylphenylacetic acid impurity standard reference substance, place it in a 10ml volumetric flask, dilute to the mark with acetonitrile, and shake it well with ultrasound. A standard with a concentration of 40mg / ml is formulated. Dilute 10ml successively to obtain 5 concentration points, respectively 10mg / ml, 20mg / ml, 30mg / ml, 40mg / ml, 50mg / ml.
Embodiment 3
[0045] Production of standard curve
[0046] Choose the best HPLC conditions, that is, the column is ZORBAX Eclipse XDB-C184.6×150nm, 5μm (Agilent); the mobile phase is pH 3.0 sodium dihydrogen phosphate buffer solution; the gradient elution is within 0-30 minutes ; The mobile phase ratio is 80%-60%; the flow rate: 1ml / min; the column temperature: 40°C; the injection volume is 5μl, and the detection wavelength is 230nm.
[0047] The peak area is plotted against the concentration. When the concentration of o-methylphenylacetic acid is in the range of 0-50mg / ml, the sample concentration is directly proportional to the peak area. The linear regression equation is:
[0048] A=4.128x-0.358, r=0.99994
[0049] The peak area is plotted against the concentration. The concentration of 2-chloromethylphenylacetic acid is in the range of 0-50mg / ml, and the sample concentration is proportional to the peak area. The linear regression equation is:
[0050] A=1.296x+0.421, r=0.99992
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