Determination of related substances in oxytocin for injection by HPLC-principal component self-control with correction factor
Objective:To determine seven impurities in oxytocin for injection and investigate the limit values.Methods:HPLC and principal component self-control with correction factor were adopted.The determination was performed on a Waters Xbridge C18column(150 mm ×4.6 mm,5 μm).The mobile phase consisted of 0.1 mol·L-1dihydrogen phosphate solution(adjusted to pH 5.4)-acetonitrile(90∶10,phase A),and acetonitrile(phase B)with gradient elution at a flow rate of 1.5 mL·min-1.The column temperature was maintained at 32℃,and the detection wavelength was set at 220 nm.The injection volume was 100 μL.The linear equations of oxytocin,impurities Ac-Oxy,Oxy[Glu4],Oxy[+Gly10],Oxy[-NH2],Oxy[trisulfide],Oxy[cis-dimer]and Oxy[trans-dimer]were drawn.The correction factors of each impurity related to oxytocin were calculated by slope.The contents of impurities in 3 batches of oxytocin for injection were determined and compared with the results of impurity reference method.Results:The limits of quantification for seven impurities were 2.75-5.66 ng,while the detection limits were 1.38-2.83 ng.The linear ranges of seven impurities were 0.03-3.40 μg· mL-1with good linearity(r＞0.999).The correction factors of Ac-Oxy,Oxy[Glu4]and Oxy[-NH2]were 1.1,while the correction factors of Oxy[+Gly10]and Oxy[trisulfide]were 1.2 and 0.9,respectively.The cor-rection factors of Oxy[cis-dimer]and Oxy[trans-dimer]were both 1.3.The seven impurities were determined in 3 batches of samples by principal component self-control with correction factor.The contents of impurity Ac-Oxy were 0.96％,0.93％and 1.01％,respectively.The contents of impurity Oxy[Glu4]were 0.07％,0.06％and 0.08％,respectively.The contents of impurity Oxy[+Gly10]were 0.07％,0.04％and 0.04％,respec-tively.The contents of impurity Oxy[-NH2]were 0.09％,0.05％and 0.07％,respectively.The contents of impurity Oxy[trans-dimer]were 0.27％,0.18％and 0.22％,respectively.The maximum single impurity con-tents were 0.18％-0.19％,while the total impurity contents were 1.88％-2.06％.Compared the results measured by principal component self-control with correct factor method and the impurity reference method,there was no significant difference between two methods(p＞0.05).Conclusion:The method is proved to be simple,repeatable and accurate for the content determination of related substances in oxytocin for injection.
oxytocinrelated substancesHPLCprincipal component self-control with correct factorknown impurities
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