3 Results and discussions3.1 Optimizing the LC-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 thepeak 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 themobile and the stationary 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 and instrument dependent parameters were optimized byinfusing the compound solution into the MS directly using a syringe pump. MRMscan type was used to improve the specificity.
The MS/MS of MC-A and the I.S. are shown inFig. 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 wereevaluated using QC samples (n=3) at 0.80, 40.00, 500.
00, and 1,500.00 ng/mL. The recovery was > 78.
1% (Table 2), suggesting thatthis protein precipitation method couldextract MC-A from the plasma. The matrix effect at 0.80, 40.00, 500.00, and1,500.00 ng/mL were <15%, indicating that matrix effectof this extraction is in the acceptable range.
3.4. Accuracy andPrecisionThequantification accuracy and inter/intra-day precisions of this method wasdetermined using the QC samples at 0.80, 40.
00, 500.00, and 1,500.00 ng/mL.
All the results of the tested samples werewithin the acceptable criteria (RSD% < 15%, Table 3) according to the FDAguidance, suggesting that this method is accurate 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 57.74 ± 2.43 min, suggesting theclearance was rapid. The AUC(0-t) of MC-A in the i.
v. administration(43857.52 ± 3806.46 µg/L*min) is ~ 10-fold higher than that (4558.10 ± 979.56 µg/L*min) of the p.
o. administration.The absolute oral bioavailability is only 2.9 %. These data showed that it is achallenge to develop MC-A as an drug administrated through oral route. Sincethere is an acetyl in the structure (Fig.
1),hydrolysis could be one of the possible metabolism causing rapid clearance andlow oral bioavailability.Further studies are needed to verify the mechanism that lead to low oralbioavailability. 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 developMC-A as a drug administered through oral route. Other ent-kaurane-type diterpenesmay also have the same challenge.