1. IntroductionMaoecrystal A (MC-A, Fig.
1)is an ent-kaurane-type diterpene isolated from Rabdosia eriocalyx (Dunn)Hara, a well-known herbalmaterial used in a few China Traditional Medicines. 1-3 The leaves and roots of this plant areused by local folk for the treatment of enteritis, jaundice, hepatitis,laryngopharyngitis, lepromatous leprosy, and ascariasis 4. Due to longhistory of usage and promising efficacy, chemists have spent lot of efforts toidentify ent-kaurane-type diterpenes as the active components in this plant 5-7.In addition, total synthesis and structure modifications of maoecrystals havebeen conducted by medicinal chemists trying to develop this class of compoundsas drugs for the treatment of different types of diseases. 8, 9Pharmacologicalstudies showed that ent-kaurane diterpenes possess different activities such asanticancer, anti-diabetes, antibacterial, anti-inflammatory, and bacteriostaticfunctions 10, 11. For example, Li et al reported that an ent-kauraneisolated from isodon Phyllostacys washighly active against a human leukemia cancer cell line K562 and its activitywas even higher than that of cisplatin. 12 Mechanism studies showed thatent-kaurane diterpenes could selectively inhibit 11?-HSD1 11, activate Aktsignaling 13, or induce apoptosis through the mitochondrial pathway 14, 15.
Although the bioactivities of ent-kauranediterpenes are promising, their pharmaceutical properties have not beenwell-studied. The reason could be because of lack of bioanalytical method asthis class of compounds is not sensitive in UV detector. In this paper, wedeveloped a sensitive UPLC-MS/MS method to quantify MC-A in biological samplesand apply the method in in vivosample analysis and determine the PK properties and oral bioavailability ofMC-A. 2. Experimental 2.1 Chemicals and reagents.
Thestandard compound MC-A (purity >98%) was purchasedfrom Chengdu Must Bio-Technology Co., Ltd (Chengdu China). The standard oridonin(purity > 98%), which was used as the internal standard, was obtained from theNational Institutes for Food and Drug Control (Beijing, China). LC-MS grade acetonitrileand methanol were from Merck and formic acid was from Sigma. DD water wasprepared in our lab using a GWA-UN ultra-pure water apparatus (PurkinjeGeneral, China). 2.2 Instrument and conditions.
The separation was performed using a Waters HSS T3 column(50 mm× 2.1 mm, 1.8 µm) in an AcquityTM ultra performance liquidchromatography (UPLC) system. Methanol and water containing 0.1% formic acid wasused as mobile phase A (A) and mobile phase B (B), respectively. Elutiongradient was: 0-0.5 min (95-95% B), 0.
5-3.0 min (95-5% B), 3.0-3.
5 min (5-5%B), 3.5-5.0 min (5-95% B) and 5.0-5.
5 min (95-95% B). The flow rate was 0.3mL/min. The temperature for the auto-sampler and the column was 18°C and 40°C,respectively.The analyte wasquantified using a waters XEVO TQ triple quadrupole mass spectrometer equippedwith an electro-spray ionization (ESI) source. Multiple reactions monitoring(MRM) scan type was used to increase the specificity of the analysis.
The MSparameters were listed in Table 1. Masslynx 4.1 was used to control theinstrument and data anlaysis. 2.3 Preparation of stock solution, workingsolution, calibration curve in rat plasma, quality control (QC) samples, and PKsamples. The stock solutions of MC-Aand oridonin (1,000 µg/mL) were prepared in 50% methanol (containing 0.1%formic acid) by accurately weighing appropriate amounts of MC-A or oridonin anddissolved the powder into the solvent in a volumetric bottle. The workingsolution were prepared by diluting the stock solution of MC-A into 50% methanolat final concentrations of 4,000.
00, 2000.00, 1000.00, 500.00, 250.
00, 125.00,62.50, 31.25, 15.63, 7.81, 3.91, 1.
95, 0.98, 0.49, and 0.24 ng/mL. The standard curve samples in rat plasma were prepared byspiking the each of the above working solution (20 µL) and I.
S. solution (20 µLin methanol, 200 ng/mL) into blank rat plasma (100 µL) and extract with 1.0 mLof ethyl acetate.
After centrifugation at 14,000 rpm for 15 min at 4 ?C, the supernatantwas transferred into a clean microcentrifuge tube and the solvent was evaporatedunder N2 flow. The residue was re-constituted in 50% methanol (100µL) for injection after centrifugation (14,000 rpm, 15 min, 4 ?C). The QCsamples were prepared at 0.
80, 40.00, 500.00, and 1,500.
00 ng/mL following thesame procedure. The PK plasma samples were prepared by spiking the blanksolvent (50% methanol, 20 µL) and I.S. (20 µL in methanol, 200 ng/mL) into the plasmasamples (100 µL) and were extracted using ethyl acetate as described above. 2.4 Method validation.
The method was validated according to the guidance fromthe FDA by evaluating the specificity, linearity, lower limit of detection (LLOD), recovery, matrix effect, accuracy, precision,and stability.2.4.1 Specificity, linear range, and LLOD. The specificity of themethod was determined by injecting the plasma samples prepared from blankplasma (pooled from 6 rats), blank plasma spiked with the I.
S. or MC-A (at theLLOQ level), and post-dosing plasma samples. The linear range of the standard curve was determined by injectingthe standard curve samples prepared in the plasma according to the methoddescribed above. The LLOD was determined by checking the signal to noise (S/N) ratioin the chromatogram.
2.4.2 Recovery and matrix effect. The extraction recovery wasdetermined by comparing the peak areas obtained from samples prepared from blankplasma spiked with the QC samples at 0.80, 40.00, 500.
00, and 1,500.00 ng/mL with those from samples preparedfrom water spiked with the same concentrations. Matrix effect was determined bycomparing the peak areas of samples prepared from residues of pooled blankplasma spiked with QC samples at 0.80, 40.00, 500.
00, and 1,500.00 ng/mL with those from residues of mobilephase A spiked with the same volume of QC samples. 2.
4.3 Accuracy andprecision.The accuracy was calculated using QC samples at 0.80, 40.00, 500.00, and 1,500.
00ng/mL. The intra-day and inter-day precisions were determined by injecting theQC samples at these four concentrations on the same day or on three continuousdays. 2.5 Pharmacokinetic study using SD rats. Male Sprague-Dawley rats (250 – 300 g) were supplied bythe Animal Center at Hubei University of Medicine. Animals were maintained inan environment control animal facility (22 ± 2°C and 55 ± 5% relative humidityon a 12 h light/12 h dark cycle) for 5 days before the experiment.
Before the PK studies, the rats were fasted overnight withfree access of water. Blood samples (~ 0.2 mL) were collected into heparinized microcentrifugetubes from the fossa orbitalis vein at 10, 30, 60, 90, 105, 120, 150, 180, 210,240, 300 and 480 min after a single oral administration of MC-A (10 mg/kg inoral suspending vehicle). Similarly, after I.V. administration (2 mg/kg in saline),blood samples were collected at 0, 5, 15, 30, 60, 120, 240, 360, 480, and 1440min. The samples were centrifuged at 14,000 rpm for 10 min immediately aftercollection to afford the plasma.
All the samples were stored at -80°C untilanalysis. The pharmacokinetic parameters were calculated with DAS (Drug andStatistics) version 2.1.
1 software (edited by the Chinese MathematicalPharmacology Society) using non-compartment model.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. 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 applied to quantifyMC-A in PK studies using SD rats. The plasma concentrations of MC-A werequantified and the main PK parameters including the absolute oralbioavailability of MC-A were afforded. 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.