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

3 Results and discussions3.

1 Optimizing the UPLC-MS/MS conditionsTooptimize the LC condition, different mobile phases including MeOH, ACN, ammoniaacetate, 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 thesignal response in MS, methanol (containing 0.1% formic acid)/water (containing0.

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1% formic acid) and HSS T3 column were selected as the mobile and thestationary phases. A gradientelution was established based on the shape of the MC-A peak to increase thethrough-put of the method. In addition, both positive and negative scan moodwere tested. The results showed that positive scan was moresensitive. Compound dependent parameter and instrument dependent parameterswere optimized by infusing the compound solution into the MS directly using asyringe pump. MRM scan type was used to improve the specificity. The MS/MS fragmentationpatterns of MC-A and the I.S.

are shown in Fig. 2A and 2B.3.2. Specificity, linearity, LLODThe 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 theanalyte and I.

S. (S/N>3, Fig. 3), indicating the specificity of this methodis acceptable. The standard curves were linear in theconcentration range of 2,000.00-0.49 ng/mL in the plasma. The LLOD was 0.24ng/mL.

3.5. Recovery and matrix effectThe extraction recoveries weredetermined using three replicates of QC samples at four concentrations asdescribed above. The recovery was > 78.1% (Table 2), suggesting that theextraction procedure is suitable for MC-A.

The matrix effect at fourconcentrations were <15%, indicating that matrix effect of this extractionis in the acceptable range. 3.4. Accuracy andPrecisionThequantification accuracy and inter/intra-day precisions of this method wasdetermined using the QC samples at four different concentrations. All theresults of the tested samples were within the acceptable criteria (RSD% <15%, Table 3) according to the FDA guidance, suggesting that this method isaccurate and precise. 3.

6. Stability in theplasmaThe 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 atthese different conditions (Table 4), indicating this method was suitable forbioanalysis of MC-A.  3.7 PK studies using SDratsThevalidated method was used to quantify MC-A in the plasma in PK studies. The mean plasma concentration-timeprofiles of MC-A are shown in Fig. 4 afteroral and i.v.

administration. The main PK parameters are listed in Table 5. In the i.

v. injection, thehalf-life (t1/2) of MC-A was 57.73 ± 2.43 min, suggesting theclearance 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 datashowed that it is a challenge to develop MC-A as an drug administratedthrough oral route. Since there is an acetyl in the structure (Fig. 1), hydrolysis could be one of thepossible metabolism causing rapid clearance and low oral bioavailability. Further studies are needed to verifythe 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 toquantify MC-A in rat plasma. The method was successfully used to quantify MC-Ain PK studies using SD rats. The main PK parameters and the oralbioavailability of MC-A were calculated. Since the oral bioavailability of MC-Ais extremely low, efforts on absorption/metabolism are needed in order to developthis compound as a drug administered through oral route.

Other ent-kaurane-typediterpenes may also suffer from the same challenge.