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 <15%, indicating that matrix effect of this extraction is in the acceptable range. 3.4. Accuracy and Precision The quantification accuracy and inter/intra-day precisions of this method was determined using the QC samples at four different concentrations. All the results of the tested samples were within the acceptable criteria (RSD% < 15%, Table 3) according to the FDA guidance, suggesting that this method is accurate and precise. 3.6. Stability in the plasma The bench, short-term, long-term, and freeze-thaw stabilities of MC-A in rat plasma were evaluated. The results showed that MC-A was stable (variation<15%) in the plasma at these different conditions (Table 4), indicating this method was suitable for bioanalysis of MC-A.   3.7 PK studies using SD rats The validated method was used to quantify MC-A in the plasma in PK studies. The mean plasma concentration-time profiles of MC-A are shown in Fig. 4 after oral and i.v. administration. The main PK parameters are listed in Table 5. In the i.v. injection, the half-life (t1/2) of MC-A was 57.73 ± 2.43 min, suggesting the clearance was rapid. The AUC(0-t) of MC-A in the i.v. administration (44875.52 ± 3806.47 µg/L*min) is ~ 10-fold higher than that (4558.096 ± 979.556 µg/L*min) of the p.o. administration. The absolute oral bioavailability is only 2.9 %. These data showed that it is a challenge to develop MC-A as an drug administrated through oral route. Since there is an acetyl in the structure (Fig. 1), hydrolysis could be one of the possible metabolism causing rapid clearance and low oral bioavailability. Further studies are needed to verify the mechanism that lead to low oral bioavailability. 4. Conclusion. In conclusion, an accurate, precise, sensitive, and rapid UPLC-MS/MS method was developed and validated to quantify MC-A in rat plasma. The method was successfully used to quantify MC-A in PK studies using SD rats. The main PK parameters and the oral bioavailability of MC-A were calculated. Since the oral bioavailability of MC-A is extremely low, efforts on absorption/metabolism are needed in order to develop this compound as a drug administered through oral route. Other ent-kaurane-type diterpenes may also suffer from the same challenge.