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.
administration, 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(44.87 ± 3.81 mg/L*min) is ~ 10-fold higher than that (4.
56 ± 0.98 mg/L*min) of the p.o. administration. The absoluteoral bioavailability is only 2.
9 %. These data showed that it is a challenge todevelop MC-A as an drug administrated through oral route. Since there isan 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. Inconclusion, an accurate, precise, sensitive, and rapidUPLC-MS/MS method was developed and validated to quantify MC-A in rat plasma.The method was successfully applied to quantify MC-A in PK studies using SDrats. The plasma concentrations of MC-A were quantified and the main PKparameters including the absolute oral bioavailability of MC-A were afforded. Sincethe oral bioavailability of MC-A is extremely low, efforts onabsorption/metabolism are needed in order to develop MC-A as a drugadministered through oral route.
Other ent-kaurane-type diterpenes may also havethe same challenge.