Development of a validated UPLC-MS/MS method for PK/PD analysis of SYL930 and its two major metabolites in dogs.

A sensitive and specific UPLC-MS/MS method was developed and validated for the simultaneous determination of 2-amino-2-(2-(4'-(2-propyloxazol-4-yl)-[1,1'-biphenyl]-4-yl)ethyl)propane-1,3-diol (SYL930), phosphorylated metabolite (SYL930-P) and hydroxylated metabolite (SYL930-M) in dog blood using SYL927 and SYL927-P, analogues of SYL930, as the internal standards. Analytes were extracted with protein precipitation followed by chromatographic separation on a ZorbaxSB-C18 column (3.5 µm, 2.1 × 100 mm) with a gradient elution of methanol-water containing 0.1% formic acid (v/v). A triple quadrupole tandem mass spectrometer operating in the positive electrospray ionization mode was used to detect SYL930, SYL930-P, SYL930-M and IS transitions of 381.2 → 364.2, 461.2 → 334.2, 397.3 → 380.3, 367.1 → 350.4 and 447.5 → 320.2, respectively. The linear calibration curves for SYL930, SYL930-P and SYL930-M were 0.5-500, 0.2-100 and 0.5-100 ng/mL, respectively (r2 > 0.99). The intra-day and inter-day precisions (RSD, %) of analytes did not exceed 9.16% except for low QCs (≤16.22%), and the accuracy (RE, %) ranged from -14 to 11.4%. The mean recoveries for SYL930, SYL930-P and SYL930-M in dog blood were 85.13-107.94, 73.84-80.08 and 85.64-95.44%, respectively. The validated method was successfully applied to pharmacokinetic and PK/PD studies of SYL930 and its two major metabolites in dogs after an oral administration of SYL930.

[Role of breast cancer resistance protein in drug disposition].

Human breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) efflux transporter, is mainly responsible for the transport of many endogenous and xenobiotics. BCRP is expressed in different tissues, such as placental, intestinal epithelium, endothelial cells of brain microvessels, and renal proximal tubular cells. BCRP is considered as one of the key factor for the drug-drug interaction and individual difference in drug therapy. The review will provide an overview of the current knowledge on the discovery of BCRP and its physical function, transport mechanism, substrate and inhibitors, as well as its effect on the drug pharmacokinetics.

Stereoselective formation and metabolism of 20(S)-protopanaxadiol ocotillol type epimers in vivo and in vitro.

(20S,24S)-epoxy-dammarane-3,12,25-triol (24S-epimer) and (20S,24R)-epoxy- dammarane-3,12,25-triol (24R-epimer), a pair of ocotillol type epimers, were identified as the main metabolites of 20(S)-protopanaxadiol (PPD). The aim of this study was to systematically investigate the formation and metabolism of this pair of epimers in vivo and in vitro and to elucidate the isoforms of cytochrome P450 enzymes responsible for the stereoselective metabolism of both epimers. The result showed that 24S-epimer was a more predominant ingredient in rat plasma after oral administration of PPD with higher area under the curve (AUC) values. Both the enzyme kinetic evaluations of the formation and elimination of 24S-epimer and 24R-epimer in rat liver microsomes (RLM) and human liver microsomes (HLM) indicated that 24S-epimer had a higher formation rate and a lower oxygenation metabolism rate than 24R-epimer, and the stereoselective differences were more obvious in HLM than in RLM. The chemical inhibition and recombinant human P450 isoforms assay showed that CYP3A4 was the predominant isoform responsible for the further metabolism of 24R-epimer in HLM. The biliary excretion ratio of the 24S-epimer glucuronide was more than 28-fold higher than that of 24R-epimer glucuronide after intravenous administration to rats, which also indicated 24S-epimer was more preferential to be metabolized as the glucuronide conjugate than 24R-epimer.

[Study on excretion of 20 (S) -protopanaxadiolocotillol type epimers in rats].

Gindenosides are the active ingredients of Panax ginseng. 20 (S) -protopanaxadiolocotillol type epimers are the main metabolites of 20 (S) -protopanaxadiol. The previous studies showed that there are stereoselectivity difference in pharmacodynamics and pharmacokinetics between 24R-epimer and 24S-epimer. The purpose of this study was to explore the excretion of the epimers in bile, feces and urine of rat. Liquid chromatography tandem mass spectrometry method has been performed for determination of 24R-epimer and 24S-epimer in bile, feces and urine. 24R-epimer or 24S-epimer was intragastric administered to rats at a single dose of 10 mg x kg(-1). Results showed that after administration the recovery of 24R-epimer and 24S-epimer in feces was 17.69% and 17.09%, respectively, while both of the two epimers were hardly detected in urine. The 48 h cumulative biliary excretion rate of 24R-epimer was 8.01% after administration, while only 1.47% for 24S-epimer. It indicated that there are stereoselectivity in biliary excretion of the epimers with intragastric administration.

Cholesterol-modified sphingomyelin chimeric lipid bilayer for improved therapeutic delivery.

Cholesterol (Chol) fortifies packing and reduces fluidity and permeability of the lipid bilayer in vesicles (liposomes)-mediated drug delivery. However, under the physiological environment, Chol is rapidly extracted from the lipid bilayer by biomembranes, which jeopardizes membrane stability and results in premature leakage for delivered payloads, yielding suboptimal clinic efficacy. Herein, we report a Chol-modified sphingomyelin (SM) lipid bilayer via covalently conjugating Chol to SM (SM-Chol), which retains membrane condensing ability of Chol. Systemic structure activity relationship screening demonstrates that SM-Chol with a disulfide bond and longer linker outperforms other counterparts and conventional phospholipids/Chol mixture systems on blocking Chol transfer and payload leakage, increases maximum tolerated dose of vincristine while reducing systemic toxicities, improves pharmacokinetics and tumor delivery efficiency, and enhances antitumor efficacy in SU-DHL-4 diffuse large B-cell lymphoma xenograft model in female mice. Furthermore, SM-Chol improves therapeutic delivery of structurally diversified therapeutic agents (irinotecan, doxorubicin, dexamethasone) or siRNA targeting multi-drug resistant gene (p-glycoprotein) in late-stage metastatic orthotopic KPC-Luc pancreas cancer, 4T1-Luc2 triple negative breast cancer, lung inflammation, and CT26 colorectal cancer animal models in female mice compared to respective FDA-approved nanotherapeutics or lipid compositions. Thus, SM-Chol represents a promising platform for universal and improved drug delivery.

Impact of aging and ergothioneine pre-treatment on naphthalene toxicity in lung.

Aging increases susceptibility to lung disease, but the topic is understudied, especially in relation to environmental exposures with the bulk of rodent studies using young adults. This study aims to define the pulmonary toxicity of naphthalene (NA) and the impacts of a dietary antioxidant, ergothioneine (ET), in the liver and lungs of middle-aged mice. NA causes a well-characterized pattern of conducting airway epithelial injury in the lung in young adult mice, but NA's toxicity has not been characterized in middle-aged mice, aged 1-1.5 years. ET is a dietary antioxidant that is synthesized by bacteria and fungi. The ET transporter (ETT), SLC22A4, is upregulated in tissues that experience high levels of oxidative stress. In this study, middle-aged male and female C57BL/6 J mice, maintained on an ET-free synthetic diet from conception, were gavaged with 70 mg/kg of ET for five consecutive days. On day 8, the mice were exposed to a single intraperitoneal NA dose of 50, 100, 150, or 200 mg/kg. At 24 hours post NA injection samples were collected and analyzed for ET concentration and reduced (GSH) and oxidized glutathione (GSSG) concentrations. Histopathology, morphometry, and gene expression were examined. Histopathology of mice exposed to 100 mg/kg of NA suggests reduction in toxicity in the terminal airways of both male (p ≤ 0.001) and female (p ≤ 0.05) middle-aged mice by the ET pretreatment. Our findings in this study are the first to document the toxicity of NA in middle-aged mice and show some efficacy of ET in reducing NA toxicity.

A universal fluorescence polarization high throughput screening assay to target the SAM-binding sites of SARS-CoV-2 and other viral methyltransferases.

SARS-CoV-2 has caused a global pandemic with significant humanity and economic loss since 2020. Currently, only limited options are available to treat SARS-CoV-2 infections for vulnerable populations. In this study, we report a universal fluorescence polarization (FP)-based high throughput screening (HTS) assay for SAM-dependent viral methyltransferases (MTases), using a fluorescent SAM-analogue, FL-NAH. We performed the assay against a reference MTase, NSP14, an essential enzyme for SARS-CoV-2 to methylate the N7 position of viral 5'-RNA guanine cap. The assay is universal and suitable for any SAM-dependent viral MTases such as the SARS-CoV-2 NSP16/NSP10 MTase complex and the NS5 MTase of Zika virus (ZIKV). Pilot screening demonstrated that the HTS assay was very robust and identified two candidate inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the NSP14 MTase with low micromolar IC50. We used three functional MTase assays to unambiguously verified the inhibitory potency of these molecules for the NSP14 N7-MTase function. Binding studies indicated that these molecules are bound directly to the NSP14 MTase with similar low micromolar affinity. Moreover, we further demonstrated that these molecules significantly inhibited the SARS-CoV-2 replication in cell-based assays at concentrations not causing cytotoxicity. Furthermore, NSC111552 significantly synergized with known SARS-CoV-2 drugs including nirmatrelvir and remdesivir. Finally, docking suggested that these molecules bind specifically to the SAM-binding site on the NSP14 MTase. Overall, these molecules represent novel and promising candidates to further develop broad-spectrum inhibitors for the management of viral infections.

Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing.

Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as Cne and Cga. Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 µg/mL against the wild-type Cne-H99 (Cne-WT or Cne). In contrast, CMN inhibited the intein-less mutant strain (Cne-Mut) with a 16-fold higher MIC. Interestingly, Aspergillus fumigatus and a few Candida species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in Cne. CMN also inhibited Cne intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC50 of CMN was 4.6 µg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating Cne cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat Cne and possibly Cga infections.

Differential Effects of Arsenic in Drinking Water on Mouse Hepatic and Intestinal Heme Oxygenase-1 Expression.

Arsenic exposure has been associated with the risks of various diseases, including cancers and metabolic diseases. The aim of this study was to examine the effects of arsenic exposure via drinking water on the expression of heme oxygenase-1 (HO-1), a major responsive gene to arsenic-induced oxidative stress, in mouse intestinal epithelial cells which is the first site of exposure for ingested arsenic, and the liver, a known target of arsenic toxicity. The expression of HO-1 was determined at mRNA, protein, or enzymic activity levels in mice exposed to sodium arsenite through drinking water, at various doses (0, 2.5, 10, 25, 100 ppm), and for various time periods (1, 3, 7, or 28 days). HO-1 was significantly induced in the intestine, but not liver, at arsenic doses of 25 ppm or lower. The intestinal HO-1 induction was seen in both males and females, plateaued within 1-3 days of exposure, and was accompanied by increases in microsomal HO activity. In mice exposed to 25-ppm of arsenite for 7 days, total arsenic and As(III) levels in intestinal epithelial cells were significantly higher than in the liver. These findings identify intestinal epithelial cells as likely preferential targets for arsenic toxicity and support further studies on the functional consequences of intestinal HO-1 induction.

In vitro and in vivo characterization of erythrosin B and derivatives against Zika virus.

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

Rational design of a deuterium-containing M2-S31N channel blocker UAWJ280 with in vivo antiviral efficacy against both oseltamivir sensitive and -resistant influenza A viruses.

Seasonal influenza A virus (IAV) infections are among the most important global health problems. FDA-approved antiviral therapies against IAV include neuraminidase inhibitors, M2 inhibitors, and polymerase inhibitor baloxavir. Resistance against adamantanes (amantadine and rimantadine) is widespread as virtually all IAV strains currently circulating in the human population are resistant to adamantanes through the acquisition of the S31N mutation. The neuraminidase inhibitor-resistant strains also contain the M2-S31N mutant, suggesting M2-S31N is a high-profile antiviral drug target. Here we report the development of a novel deuterium-containing M2-S31N inhibitor UAWJ280. UAWJ280 had broad-spectrum antiviral activity against both oseltamivir sensitive and -resistant influenza A strains and had a synergistic antiviral effect in combination with oseltamivir in cell culture. In vivo pharmacokinetic (PK) studies demonstrated that UAWJ280 had favourable PK properties. The in vivo mouse model study showed that UAWJ280 was effective alone or in combination with oseltamivir in improving clinical signs and survival after lethal challenge with an oseltamivir sensitive IAV H1N1 strain. Furthermore, UAWJ280 was also able to ameliorate clinical signs and increase survival when mice were challenged with an oseltamivir-resistant IAV H1N1 strain. In conclusion, we show for the first time that the M2-S31N channel blocker UAWJ280 has in vivo antiviral efficacy in mice that are infected with either oseltamivir sensitive or -resistant IAVs, and it has a synergistic antiviral effect with oseltamivir.

RNA-Seq Reveals Sub-Zones in Mouse Adrenal Zona Fasciculata and the Sexually Dimorphic Responses to Thyroid Hormone.

The sex-specific prevalence of adrenal diseases has been known for a long time. However, the reason for the high prevalence of these diseases in females is not completely understood. Mouse studies have shown that the adult adrenal gland is sexually dimorphic at different levels such as transcriptome, histology, and cell renewal. Here we used RNA-seq to show that in prepubertal mice, male and female adrenal glands were not only sexually dimorphic but also responded differently to the same external stimulus. We previously reported that thyroid hormone receptor ß1 (TRß1) in the adrenal gland is mainly expressed in the inner cortex and the fate of this TRß1-expressing cell population can be changed by thyroid hormone (triiodothyronine; T3) treatment. In the present study, we found that adrenal glands in prepubertal mice were sexually dimorphic at the level of the transcriptome. Under T3 treatment, prepubertal females had 1162 genes differentially expressed between the saline and T3 groups, whereas in males of the same age, only 512 genes were T3-responsive. Immunostaining demonstrated that several top sexually dimorphic T3-responsive genes, including Cyp2f2 and Dhcr24, were specifically expressed in the adrenal inner cortex, precisely in an area partially overlapping with the X-zone. Under T3 treatment, a unique cortical layer that surrounds the adrenal X-zone expanded significantly, forming a distinct layer peculiar to females. Our findings identified novel marker genes for the inner adrenal cortex, indicating there are different sub-zones in the zona fasciculata. The results also highlight the sex-specific response to thyroid hormone in the mouse adrenal gland.

Determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain tissue by liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of IMM-H004, a novel neuroprotective agent, in rat plasma and brain was developed. Plasma and brain tissue homogenate samples containing IMM-H004 and propranolol (internal standard, IS) were prepared by using a direct protein precipitation of acetonitrile. Separation was carried out in Zorbax SB-C18 column at a flow rate of 0.3mL/min utilizing acetonitrile/water as mobile phases which contain 0.5% formic acid (v/v). Triple quadrupole mass spectrometer was used for detection with selective reaction monitoring. The mass transition ion-pairs were 305→248 for IMM-H004 and 260→183 for IS in positive ion mode. The linear ranges of IMM-H004 were 5-1000ng/mL in plasma and 1-200ng/mL in brain tissue homogenate. The intra- and inter-day precisions were within ±14.9% for analyte in both matrices (±17.0% at the lowest limit of quantification level), while the deviation of assay accuracy was within ±12.9%. No obvious matrix effect was observed. The recovery of the analyte was higher than 85.3%. IMM-H004 was stable during the whole analytic process. The method was applied successfully to the plasma and brain pharmacokinetic study of IMM-H004 in rats after a single intravenous administration.

Metabolism of F18, a Derivative of Calanolide A, in Human Liver Microsomes and Cytosol.

10-Chloromethyl-11-demethyl-12-oxo-calanolide (F18), an analog of calanolide A, is a novel potent nonnucleoside reverse transcriptase inhibitor against HIV-1. Here, we report the metabolic profile and the results of associated biochemical studies of F18 in vitro and in vivo. The metabolites of F18 were identified based on liquid chromatography-electrospray ionization mass spectrometry and/or nuclear magnetic resonance. Twenty-three metabolites of F18 were observed in liver microsomes in vitro. The metabolism of F18 involved 4-propyl chain oxidation, 10-chloromethyl oxidative dechlorination and 12-carbonyl reduction. Three metabolites (M1, M3-1, and M3-2) were also found in rat blood after oral administration of F18 and the reduction metabolites M3-1 and M3-2 were found to exhibit high potency for the inhibition of HIV-1 in vitro. The oxidative metabolism of F18 was mainly catalyzed by cytochrome P450 3A4 in human microsomes, whereas flavin-containing monooxygenases and 11ß-hydroxysteroid dehydrogenase were found to be involved in its carbonyl reduction. In human cytosol, multiple carbonyl reductases, including aldo-keto reductase 1C, short-chain dehydrogenases/reductases and quinone oxidoreductase 1, were demonstrated to be responsible for F18 carbonyl reduction. In conclusion, the in vitro metabolism of F18 involves multiple drug metabolizing enzymes, and several metabolites exhibited anti-HIV-1 activities. Notably, the described results provide the first demonstration of the capability of FMOs for carbonyl reduction.

Simultaneous quantification of 2',3',5'-tri-O-acetyl-N6-(3-hydroxylaniline)adenosine and its principal metabolites in hamster blood by LC-MS/MS and its application in pharmacokinetics study.

2',3',5'-Tri-O-acetyl-N6-(3-hydroxylaniline)adenosine (IMM-H007, once called WS070117) is being developed as a novel anti-hyperlipidemia agent for its high efficacy and low toxicity. In this study, a sensitive and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was established for the simultaneous quantification of IMM-H007 and its two major metabolites (3S,4R,5R)-2-(hydroxymethyl)-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahdrofuran-3,4-diol (M1) and ((2R,3S,4R,5R)-3,4-dihydroxy-5-(6-((3-hydroxyphenyl)amino)-9H-purin-9-yl)tetrahydrofuran-2-yl)methyl dihydrogen phosphate (MP) in hamster blood. An analogue of IMM-H007, WS070119 was used as the internal standard. Blood samples were prepared by a simple protein precipitation with acetonitrile. The chromatographic separation was performed on a ReproSil-Pur 120C18 column (3µm, 2mm×100mm) with a gradient mobile phase of methanol/water containing 0.1% formic acid (v/v) in a flow rate of 0.2mL/min. Detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) in positive ion selective reaction monitoring (SRM) mode. The monitored transitions were 486.2→228.1 for IMM-H007, 360.0→228.0 for M1, 440.0→228.0 for MP and 374.1→242.0 for the internal standard, respectively. Satisfactory linearity was obtained for the analytes over the range of 1-500ng/mL for IMM-H007, 2-1000ng/mL for M1 and 10-5000ng/mL for MP. The lower limits of the quantification (LLOQs) were 1ng/mL for IMM-H007, 2ng/mL for M1 and 10ng/mL for MP. The intra-day and inter-day precisions (RSD, %) of the analytes were within 14.2%, and the accuracy (RE, %) ranged from -9.4% to 10.7%. The average recoveries of the analytes were more than 80.0%. The analytes were proved to be stable during given storage, preparation, and analytic procedures. The method was successfully applied to the pharmacokinetic study in hamsters after oral administration of IMM-H007.

Stereoselective property of 20(S)-protopanaxadiol ocotillol type epimers affects its absorption and also the inhibition of P-glycoprotein.

Stereoselectivity has been proved to be tightly related to drug action including pharmacodynamics and pharmacokinetics. (20S,24R)-epoxy-dammarane-3,12,25-triol (24R-epimer) and (20S,24S)-epoxy-dammarane-3,12,25-triol (24S-epimer), a pair of 20(S)-protopanaxadiol (PPD) ocotillol type epimers, were the main metabolites of PPD. Previous studies have shown that 24R-epimer and 24S-epimer had stereoselectivity in pharmacological action and pharmacokinetics. In the present study, the aim was to further study the pharmacokinetic characteristics of both epimers, investigate their absorption mechanism and analyze the selectivity effects of ocotillol type side chain and C24 stereo-configuration on P-glycoprotein (P-gp) in vivo and in vitro. Results showed that the absolute bioavailability of 24R-epimer was about 14-fold higher than that of 24S-epimer, and a linear kinetic characteristic was acquired in doses of 5-20 mg/kg for both epimers after oral administration. Furthermore, the apparent permeability coefficients of 24R-epimer were 5-7 folds higher than that of 24S-epimer having lower efflux ratios in Caco-2 cell models. Moreover, both 24R-epimer and 24S-epimer had similar inhibitory effects on P-gp by increasing cellular retention of rhodamine 123 in Caco-2 cells and decreasing efflux of digoxin across Caco-2 cell monolayers. In situ in vivo experiments showed that the inhibition of 24R-epimer on P-gp was stronger than that of 24S-epimer by single-pass intestinal perfusion of rhodamine 123 in rats. Western blot analyses demonstrated that both epimers had no action on P-gp expression in Caco-2 cells. In conclusion, with respect to the stereoselectivity, C24 S-configuration of the ocotillol type epimers processed a poor transmembrane permeability and could be distinguished by P-gp. Sharing a dammarane skeleton, both 24R-epimer and 24S-epimer were potent inhibitors of P-gp. This study provides a new case of stereoselective pharmacokinetics of chiral compounds which contributes to know the chiral characteristics of P-gp and structure-action relationship of PPD type and ocotillol type ginsenosides as a P-gp inhibitor.

Determination of 20(S)-protopanaxadiol ocotillol type epimers in rat plasma by liquid chromatography tandem mass spectrometry.

To study stereoselectivity in the pharmacokinetics of each epimer, a rapid, specific and reliable liquid chromatography tandem mass spectrometry method has been established for simultaneous quantitation of both epimers in rat plasma. Plasma samples were pretreated by liquid-liquid extraction. Chromatographic separations were performed on a Shim-pack XR-ODS C18 column (50 mm × 2.1mm, i.d., 2.2 µm) with an isocratic elution. Both epimers and the internal standard tanshinone II A were ionized with an ESI source operated in positive ion mode and measured by selective reactions monitoring mode. Calibration curve was linear over the concentration range of 1-1000 ng/mL with the lower limit of quantitation of 1 ng/mL for both epimers. Intra and inter-day precisions were less than 6.7% and 9.5%, and the accuracy was within ±5.8% for both epimers. The validated method has been successfully applied to a pharmacokinetic study of the two epimers in rats after oral administration.