A rapid, specific and high-throughput stable isotope-dilution LC-MS/MS method was developed and validated with high sensitivity for the quantification of R-phencynonate (a eutomer of phencynonate racemate) in rat and dog plasma. Plasma samples were deproteinized using acetonitrile and then separated on a C8 column with an isocratic mobile phase containing acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) at a flow rate of 0.2 mL/min. Each sample had a total run time of 3 min. Quantification was performed using triple quadrupole mass spectrometry in selected reaction monitoring mode with positive electrospray ionization. The method was shown to be highly linear (r2  > 0.99) and to have a wide dynamic range (0.1-100 ng/mL) with favourable accuracy and precision. No matrix effects were observed. The detailed pharmacokinetic profiles of R-phencynonate at therapeutic doses in rats and dogs were characterized by rapid oral absorption, quick clearance, high volume of distribution and poor absolute bioavailability. R-Phencynonate lacked dose proportionality over the oral dose range, based on the power model. However, the area under concentration-time curve and the maximum plasma concentration increased linearly in a dose-dependent manner in both animal models. The absolute bioavailability of R-phencynonate was 16.6 ± 2.75 and 4.78 ± 1.26% in dogs and rats, respectively.

The herb‐derived compounds silymarin, glycyrrhizin, and oxymatrine are widely used to treat chronic hepatitis C virus infections in China. They are often prescribed in combination with ribavirin, which has a narrow therapeutic index. We investigated the influence of these compounds on ribavirin pharmacokinetics following concurrent administration at the human dose in rats. Pharmacokinetic parameters were determined in rats following oral (PO) administration of ribavirin (30 mg/kg) with or without silymarin (40 mg/kg, PO), glycyrrhizin (15 mg/kg, intraperitoneal [IP]), or oxymatrine (60 mg/kg, PO). Compared with the animals in ribavirin group, silymarin significantly decreased the area under the plasma concentration‐time curve (AUC0–t) and the peak plasma concentration (Cmax) of ribavirin and ribavirin base by 31.2–44.5% and 48.9–50.0%, respectively. Glycyrrhizin significantly decreased the Cmax and AUC0–t of both ribavirin and its metabolite by 35.3–37.6% and 38.6–39.8%, respectively. However, silymarin or glycyrrhizin did not change the ribavirin metabolite/parent ratios of the AUC and Cmax. Oxymatrine did not induce significant changes in ribavirin concentration, but it significantly decreased the Cmax (26.6%) and AUC (21.8%) of the metabolite. This study indicates that the therapeutic efficacy of ribavirin may be compromised by the concurrent administration of herbal medicines/dietary supplements containing silymarin, glycyrrhizin, or oxymatrine. Copyright © 2016 John Wiley & Sons, Ltd.

Objective To establish a sensitive,simple and accurate HPLC-MS/MS method to quantify glycyrrhetic acid(glyc?yrrhetinic acid)in mice blood,and to further study pharmacokinetic profiles of glycyrrhetic acid after oral administration of glycyrrhi?zin and Bu-Zhong-Yi-Qi-Wan(BY). Methods Rats were intragastric administered of glycyrrhizin(glycyrrhizic acid,61.5 mg/kg) and BY extract(3 g/kg,with the same mole of glycyrrhizin moiety),respectively. Plasma samples were collected after administration and extracted with liquid-liquid extraction,then by separated by liquid chromatography on a C8 reversion phase chromatographic col?umn with gradient elution. Concentration of glycyrrhetic acid was detected by the validated HPLC-MS/MS. Non-compartmental pharma?cokinetic profiles were constructed using the software of Das 2.0 software(Shanghai,China),and the pharmacokinetic parameters were compared using unpaired Student′s t-test. Results This bioanalytical method was fully validated and showed good linearity(r>0.99),wide dynamic range(5-1000 ng/ml),and favorable accuracy and precision. Compared with the glycyrrhizin pure form group, BY significantly reduced the Cmax and AUC0-t of glycyrrhetic acid by 56%and 76%,respectively. Whereas no significant differences in Tmax,T1/2 and MRT were observed between the two groups. Conclusion The constituents in the BY prescription have significantly reduced the oral bioavailability of glycyrrhetic acid in rats than those in the glycyrrhizin pure form and the results indicate that some components in the BY have an inhibition effect on the absorption process of glycyrrhizin in the gut.

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.

Amentoflavone (AMF) is a biflavone found in many herbal dietary supplements. To investigate the pharmacokinetic profile of AMF in rats, a sensitive, simple, and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and used to monitor AMF and its conjugated metabolites in plasma. AMF was administered to rats by oral gavage (po), or by intravenous (iv) or intraperitoneal (ip) injection. Plasma samples (with apiolin as an internal standard) were liquid/liquid extracted after hydrolysis with β-glucuronidase/sulfatase in vitro. Following chromatographic separation on a C18 column with a methanol:water:formic acid (70:30:0.1, v/v/v) mobile phase, AMF and internal standard were determined by electrospray ionization in negative ion mode and their precursor-product ion pairs (m/z 537.1 → 374.9 and m/z 269.2 → 224.9, respectively) were used for measurement. This bioanalytical method was fully validated and showed good linearity (r(2) > 0.99), wide dynamic range (0.93-930 nmol/L), and favorable accuracy and precision. After iv or ip AMF (10 mg/kg) injection, 73.2% ± 6.29% and 70.2% ± 5.18% of the total AMF detected in plasma was present as conjugated metabolites. Furthermore, AMF and AMF conjugates showed similar time courses with no significant differences in the time to reach the maximum plasma concentration (tmax) and terminal half-life (t1/2) (p > 0.05). Following po AMF administration (300 mg/kg), 90.7% ± 8.3% of the total AMF was circulating as conjugated metabolites. When compared with iv administration (with dose correction), the bioavailability of po AMF was very low (0.04% ± 0.01% for free AMF; 0.16% ± 0.04% for conjugated AMF). Collectively, these data provided a preliminary pharmacokinetic profile for AMF that should inform further evaluations of its biological efficacy and preclinical development.

Chronic hepatitis B virus (HBV) infection may lead to liver cirrhosis and hepatocellular carcinoma, but few drugs are available for its treatment. Acyclic nucleoside phosphonates (ANPs) have remarkable antivirus activities but are not easily absorbed from the gastrointestinal tract and accumulate in the kidneys, resulting in nephrotoxicity. Therefore, there is a need to find effective liver site-specific prodrugs. The dipivaloyloxymethyl ester of 9-(2-phosphonylmethoxyethyl)adenine (PMEA)—adefovir dipivoxil (ADV)—is a first-line therapy drug for chronic hepatitis B with a low therapeutic index because of renal toxicity and low hepatic uptake. In this study, a series of PMEA derivatives were synthesized to enhance plasma stability and liver release. The metabolic stability of ADV (Chemical I) and its two analogues (Chemicals II and III) was evaluated in rat plasma and liver homogenate in vitro. An ion-pair reverse-phase HPLC–UV method and a hybrid ion trap and high-resolution time-of-flight mass spectrometry (LC-IT-TOF-MS) were used to evaluate the degradation rate of the analogues and to identify their intermediate metabolites, respectively. Chemicals I and II were hydrolyzed by cleavage of the C–O bond to give monoesters. Sufficient enzymatic activation in the liver homogenate through a relatively simple metabolic pathway, in addition to a favorable stability profile in rat plasma, made Chemical II an optimal candidate. Next, six analogues based on the structure of Chemical II were synthesized and evaluated in plasma and liver homogenate. Compared to Chemical II, these compounds generated less active PMEA levels in rat liver homogenate. Therefore, chemical modification of Chemical II may lead to new promising PMEA derivatives with enhanced plasma stability and liver activation.

Objective To study the bioavailability and the metabolic conversion in vitro of MCC-478,a purine nucleotide analogue prodrug which can anti-HBV,in Beagle dogs.Methods Pharmacokinetic parameters were determined for 602076 following intravenous and oral administration of 602076 and MCC-478,respectively,MCC-478 or 602076 was incubated with dog plasma,small intestinal and liver homogenates of male Beagle dogs.Plasma and tissue homogenates concentrations of MCC-478 and 602076 were quantitated with a validated LC-MS/MS assay.Results The overall absolute oral bioavailability of 602076 from MCC-478 was determined to be 2.83% in male Beagle dogs.The metabolism of MCC-478 in plasma,liver and small intestinal homogenates were similar,all hydrolysised,their t1/2 were 10.76,11.20,59.23 min,respectively;however,the active product 602076 was only observed in plasma fraction,which exhibited low transformation ratio(5.90%).On the contrast,602076 was nearly steady both in liver and intestine homogenates,t1/2 were 533.08,693.00 min,respectively.Conclusion Given the transformation ratio,the limited in vitro conversion of MCC-478 to 602076 in plasma is probably consistent with the low absolute bioavailability of 602076 from MCC-478.

Objective To develop a HPLC-MS/MS method for the determination of aconitine and study the in vitro metabolic stability of aconitine in dog tissue homogenates.Methods The chromatographic separation was performed on a C 18 column.The mobile phase consisted of acetonitrile and water with 0.2% formic acid and 5 mmol/L ammonium acetate.A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface source was used for the quantitative determination in the positive selective reaction monitor mode.Aconitine was incubated with dog tissue homogenates and samples were withdrawn at different time points and precipitated by acetonitrile with internal standards citalopram.Results Aconitine showed good linear relationship over the range from 5 to 500 ng/ml.The recoveries of aconitine were between 85.73% and 92.12% at three QC concentration levels.The intra-and inter-day precisions were 5.32%-8.95% and 5.45%-8.86%,respectively.After incubation,about 20% of aconitine were cleared in the liver and small intestine,and t1/2 were 460.6 and 521.3 min,respectively.But none was metabolized in the stomach and kidney.Conclusion These results demonstrated that aconitine was mainly metabolized in the liver and small intestine at a slow rate.

The present study aim to investigate the metabolic stability and degradation of cleavage sites of human parathyroid hormone peptide, hPTH (1-34), in rat tissue homogenate, and to identify the types of proteases involved in hPTH (1-34) processing degradation. The stability of hPTH (1-34) in rat kidney, lung and liver homogenates was evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by MALDI-TOF MS and LC-ESI-MS/MS. The ability of protease inhibitors to inhibit hPTH (1-34) degradation was used to identify the class of proteases involved in the metabolism of hPTH (1-34). hPTH (1-34) peptide was readily degraded in rat kidney, liver, and lung homogenates, with half-lives of 5.7, 32.2, and 18.9 min, respectively. The degradation of hPTH (1-34) in each tissue can be inhibited by inhibitors of serine and metalloproteases. The major degradation products of hPTH (1-34) are similar in each tissue and suggest that hPTH (1-15) and hPTH (16-34) appear as the major degradation products. The degradation patterns of hPTH (1-34) incubated in rat kidney, liver and lung homogenates are largely overlapping, and a majority of the fragments are generated via cleavages at sites of Leu15-Asn16 peptide bond.

Salmon calcitonin (sCT), a 32-amino-acid peptide, is the active component in many pharmaceuticals used for the management of bone diseases. In this study, the stability of sCT in rat kidney and liver homogenates were evaluated by LC-ESI-MS, and the structures of the major degradation products were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results show that the half life of sCT was 13.18 min in rat kidney homogenate (2.5 mg/ml, protein concentration) and 43.07 min in rat liver homogenate (2.5 mg/ml, protein concentration). MALDI-TOF MS results indicated that sCT was initially cleaved at Leu9-Gly10 and Gly10-Lys11 bonds in rat kidney homogenate in vitro, at the same time, the major degradation fragment, Lys11-Pro32-NH2 Was metabolized at the C-terminal amide by deamidation, whereas in rat liver homogenate, the initial cleavage sites were at Val8-Leu9 and His17-Lys18. The results indicated that the metabolism of sCT proceeds by initial endoproteolytic cleavage and subsequent exoproteolytic digestion.

On the basis of comprehensive experience in the analysis of small drug molecules,pharmaceutical research is one of the developing technologies for analysis of a growing number of peptide drug candidates.The pharmaceutical application of several technologies to sample preparation,various aspects of peptide chromatography,important characteristics of ESI-MS,selectivity of MS-detection modes,the large variability of internal standards,and modern instrumentation are discussed in this review.

Reversed phase high performance liquid chromatography (RP-HPLC) method was developed to investigate and compare in vitro metabolisms of luteinizing hormone releasing hormone(LHRH), leuprorelin acetate (TAP-144-SR) and new drug LH antagonist(LXT101) in six different enzymatic systems. The separation of peptide drugs and their digested fragments was achieved by gradient or isocratic elution program at 216 nm (for LHRH and TAP-144-SR) or 225 nm(for LXT101). Liquid chromatography-electrospray ionization mass spectrometry(LC-ESI-MS) was used to identify the metabolites of LHRH, TAP-144-SR and LXT101. The good linear range is 4.0-400 mg/L(r > 0.9990) for LHRH, TAP-144-SR and LXT101. The recoveries of three peptide drugs ranged from 95.0% to 98.7% in chymotrypsin, trypsin, pepsin and pancreatin systems, 60.0% to 71.0% in liver homogenate and plasma. The RSDs of intra-day and inter-day were less than 1.5% and 2.5%, respectively. The order of digested half-life of the three peptide drugs was LXT101 > TAP-144-SR > LHRH. The results obtained by LC-ESI-MS indicated that LHRH was easy to be cleaved at its Trp(3)-Ser(4) and Tyr(5)-Gly(6) bonds, but TAP-144-SR only at its Trp(3)-Ser(4) bond. The selected enzymatic systems can be successfully used to evaluate in vitro metabolisms of peptide drugs.

The Caco-2 cell monolayers drug transport model containing most of the intestinal metabolic enzymes is widely used for screening lead compounds in drug discovery to assess intestinal transport and predict absorption rates. This paper discusses basic characteristics and applications of the Caco-2 cell monolayers model in assessment of drug absorption,metabolism and high throughput screening.

目的:建立一种快速、简单的测定大鼠血浆中阿德福韦酯水解产物阿德福韦单特戊酸甲基酯和阿德福韦的离子对-反相高效液相色谱方法.方法:用Hypersil ODS C18反相柱,乙腈-50 mmol/L醋酸铵缓冲液(2∶ 98和50∶ 50)为流动相,采用梯度洗脱,流速1.0 ml/min,紫外检测波长为262 nm.结果:测定血浆中阿德福韦浓度的线性范围为1.25～60.0 μg/ml(r＝0.9999),最低检测限为0.40 μg/ml(S/N>3),绝对回收率为76.72%～80.61%.结论:本方法准确,灵敏度较高,重复性好,适于对阿德福韦酯结构类似物进行体外血浆稳定性评估筛选,并可为临床前/临床药代研究提供重要的方法学参考.