The impurities produced during the synthesis of methyl 2-cyano-4-pyridinecarboxylate mainly include methyl isonicotinate and methylisonicotinic acid N-oxide.
Methyl 2-cyano-4-pyridinecarboxylate and main impurity methyl isonicotinate are similar in polarity, CN201610560075.4 provides a kind of isocratic elution to realize methyl 2-cyano-4-pyridinecarboxylate and phase The adjacent impurity peaks can be completely separated, and the method with good peak shape can realize the accurate analysis of the content of methyl 2-cyano-4-pyridinecarboxylate. A high-performance liquid chromatography purity detection method for complete separation of methyl 2-cyano-4-pyridine carboxylate and its main impurities involved in the present invention comprises the following steps:
a. Preparation of the test solution: take methyl 2-cyano-4-pyridinecarboxylate, dissolve it with mobile phase or methanol, and prepare a solution with a concentration of 0.5mg/ml to 1.2mg/ml as the test solution .
b. Preparation of impurity reference solution: take methyl isonicotinate and methylisonicotinic acid N-oxide, dissolve with mobile phase or methanol, and prepare a solution with a concentration of 0.5mg/ml ~ 1.2mg/ml as impurities reference solution.
c. Use high performance liquid chromatography to detect the test solution and the impurity reference solution respectively. The detection conditions are: the chromatographic column is a C18 column; the mobile phase is methanol-buffer salt, and the volume ratio of methanol to the buffer solution is 10%: 90%～90%: 10%; the injection volume is 20ul, the flow rate is 0.5ml/min～1.5ml/min; the column temperature is 10℃～40℃; the detection wavelength is 200nm～300mn.
d. Calculate the peak areas of methyl isonicotinate and methylisonicotinic acid N-oxide according to the peak area normalization method.