Improvement in precision of the liquid chromatographic–electrospray ionization tandem mass spectrometric analysis of 3′-C-ethynylcytidine in rat plasma

During the development of the liquid chromatography with electrospray ionization–tandem mass spectrometry for the quantitative determination of 3′-C-ethynylcytidine (I) in rat plasma, ion suppression caused by the matrix components was observed for I and its structural analogue, 3′-C-ethylcytidine (II) as the internal standard. In the method initially designed, I/II peak area ratios varied according to the degree of matrix effect, which led to the poor precision of the assay. From the examination of the ion suppression behavior for I and II, it was assumed that this phenomenon is attributed to the difference in the retention time between I and II. Based on this assumption, therefore, the methanol content in the mobile phase was changed from 5 to 25% so as to make I and II the same retention time. As a result of this modification of the initial method, the precision expressed as relative standard deviation was improved from 5.2–16.2 to 2.7–4.2% in intra-assay and from 6.8–14.9 to 3.5–7.2% in inter-assay validations.