3 including MeOH, ACN, ammonia acetate, and formic acid,

3 Results and discussions

3.1 Optimizing the UPLC-MS/MS conditions

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To
optimize the LC condition, different mobile phases including MeOH, ACN, ammonia
acetate, and formic acid, and different types of columns including C18,
HSS T3, BEH Amide columns, were tested. Based on the shape of the peaks and the
signal response in MS, methanol (containing 0.1% formic acid)/water (containing
0.1% formic acid) and HSS T3 column were selected as the mobile and the
stationary phases. A gradient
elution was established based on the shape of the MC-A peak to increase the
through-put of the method. In addition, both positive and negative scan mood
were tested. The results showed that positive scan was more
sensitive. Compound dependent parameter and instrument dependent parameters
were optimized by infusing the compound solution into the MS directly using a
syringe pump. MRM scan type was used to improve the specificity. The MS/MS fragmentation
patterns of MC-A and the I.S. are shown in Fig. 2A and 2B.

3.2. Specificity, linearity, LLOD

The specificity of the method was determined by injecting blank plasma,
blank plasma spiked with MC-A and I.S., and plasma samples from the PK study.
The results revealed that there is no interference at the retention times of the
analyte and I.S. (S/N>3, Fig. 3), indicating the specificity of this method
is acceptable. The standard curves were linear in the
concentration range of 2,000.00-0.49 ng/mL in the plasma. The LLOD was 0.24
ng/mL.

3.5. Recovery and matrix effect

The extraction recoveries were
determined using three replicates of QC samples at four concentrations as
described above. The recovery was > 78.1% (Table 2), suggesting that the
extraction procedure is suitable for MC-A. The matrix effect at four
concentrations were