Absolute bioavailability, dose proportionality, and tissue distribution of rotundic acid in rats based on validated LC-QqQ-MS/MS method.

Rotundic acid (RA), an ursane-type pentacyclic triterpene acid isolated from the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), possesses diverse bioactivities. To further study its pharmacokinetics, a simple and sensitive liquid chromatography with triple quadrupole mass spectrometry (LC-QqQ-MS/MS) method was developed and validated to quantify RA concentration in rat plasma and tissue using etofesalamide as an internal standard (IS). Plasma and tissue samples were subjected to one-step protein precipitation. Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 column (4.6 mm × 50 mm, 5 µm) under gradient conditions with eluents of methanol:acetonitrile (1:1, V/V) and 5 mM ammonium formate:methanol (9:1, V/V) at 0.5 mL/min. Multiple reaction monitoring transitions were performed at m/z 487.30 → 437.30 for RA and m/z 256.10 → 227.10 for IS in the negative mode. The developed LC-QqQ-MS/MS method exhibited good linearity (2-500 ng/mL) and was fully validated in accordance with U.S. Food and Drug Administration bioanalytical guidelines. Dose proportionality and bioavailability in rats were determined by comparing pharmacokinetic data after single oral (10, 20, and 40 mg/kg) and intravenous (10 mg/kg) administration of RA. Tissue distribution was studied following oral administration at 20 mg/kg. The results showed that the absolute bioavailability of RA after administration at different doses ranged from 16.1% to 19.4%. RA showed good dose proportionality over a dose range of 10-40 mg/kg. RA was rapidly absorbed in a dose-dependent manner and highly distributed in the liver. In conclusion, this study is the first to systematically elucidate the absorption and distribution characteristics of RA in rats, which can provide additional information for further development and evaluation of RA in drug metabolism and pharmacokinetic studies.

Arylboronic Acid Deborylation Deuteration via Synergistic Thiol, Lewis Base, and Photoredox Catalysis.

A mild method for the deborylation deuteration of arylboronic acids with D2O, mediated by the synergistic combination of a thiol, a Lewis base, and photoredox catalysis, is reported. This reaction showed a broad substrate scope, excellent deuterium incorporation, and functional group tolerance. Therefore, this method is practical for the site-selective D-labeling of bioactive molecules and drug molecules.

Intestinal absorption mechanism of rotundic acid: Involvement of P-gp and OATP2B1.

ETHNOPHARMACOLOGICAL RELEVANCE: Ilicis Rotundae Cortex (IRC), the dried barks of Ilex rotunda Thunb. (Aquifoliaceae), has been used for the prevention or treatment of colds, tonsillitis, dysentery, and gastrointestinal diseases in folk medicine due to its antibacterial and anti-inflammatory effects. However, there is no report about the intestinal absorption of major compounds that support traditional usage. AIM OF STUDY: Considering the potential of rotundic acid (RA) - major biologically active pentacyclic triterpenes found in the IRC, this study was purposed to uncover the oral absorption mechanism of RA using in situ single-pass intestinal perfusion (SPIP) model, in vitro cell models (Caco-2, MDCKII-WT, MDCKII-MDR1, MDCKII-BCRP, and HEK293-OATP2B1 cells) and in vivo pharmacokinetics studies in rats. MATERIALS AND METHODS: The molecular properties (solubility, lipophilicity, and chemical stability) and the effects of principal parameters (time, compound concentrations, pH, paracellular pathway, and the different intestinal segments) were analyzed by liquid chromatography-tandem mass spectrometry. The susceptibility of RA to various inhibitors, such as P-gp inhibitor verapamil, BCRP inhibitor Ko143, OATP 2B1 inhibitor rifampicin, and absorption enhancer EGTA were assessed. RESULTS: RA was a compound with low water solubility (12.89 µg/mL) and strong lipophilicity (LogP = 4.1). RA was considered stable in all media during the SPIP and transport studies. The SPIP and cell experiments showed RA was moderate absorbed in the intestines and exhibited time, concentration, pH, and segment-dependent permeability. In addition, results from the cell model, in situ SPIP model as well as the in vivo pharmacokinetics studies consistently showed that verapamil, rifampicin, and EGTA might have significant effect on the intestinal absorption of RA. CONCLUSION: The mechanisms of intestinal absorption of RA might involve multiple transport pathways, including passive diffusion, the participation of efflux (i.e., P-gp) and influx (i.e., OATP2B1) transporters, and paracellular pathways.

Influence of verapamil on the pharmacokinetics of rotundic acid in rats and its potential mechanism.

CONTEXT: Rotundic acid (RA), a plant-derived pentacyclic triterpene acid, has been reported to possess extensive pharmacological activities. The poor bioavailability limits its further development and potential clinic application. OBJECTIVE: To clarify the potential mechanism for poor oral bioavailability. MATERIALS AND METHODS: The single-dose pharmacokinetics of orally administered RA (10 mg/kg) in Sprague-Dawley rats without or with verapamil (25 or 50 mg/kg) were investigated. Additionally, MDCKII-MDR1 and Caco-2 cell monolayers, five recombinant human cytochrome P450 (rhCYP) enzymes (1A2, 2C8, 2C9, 2D6 and 3A4), and rat liver microsomes were also conducted to investigate its potential mechanism. RESULTS: Verapamil could significantly affect the plasma concentration of RA. Co-administered verapamil at 25 and 50 mg/kg, the AUC0-∞ increased from 432 ± 64.2 to 539 ± 53.6 and 836 ± 116 ng × h/mL, respectively, and the oral clearance decreased from 23.6 ± 3.50 to 18.7 ± 1.85 and 12.2 ± 1.85 L/h/kg, respectively. The MDCKII-MDR1 cell assay showed that RA might be a P-gp substrate. The rhCYPs experiments indicated that RA was mainly metabolized by CYP3A4. Additionally, verapamil could increase the absorption of RA by inhibiting the activity of P-gp, and slow down the intrinsic clearance of RA from 48.5 ± 3.18 to 12.0 ± 1.06 µL/min/mg protein. DISCUSSION AND CONCLUSIONS: These findings indicated that verapamil could significantly affect the pharmacokinetic profiles of RA in rats. It was demonstrated that P-gp and CYP3A were involved in the transport and metabolism of RA, which might contribute to the low oral bioavailability of RA.

In vitro metabolism and in vivo pharmacokinetics of bentysrepinine (Y101), an investigational new drug for anti-HBV-infected hepatitis: focus on interspecies comparison.

The objective of this study was to clarify the species differences of pharmacokinetics of Y101 (N-[N-benzoyl-O-(2-dimethylaminoethyl)-l-tyrosyl]-l-phenylalaninol hydrochloride), a derivative of herbal ingredient with anti-HBV hepatitis activity, in rats, dogs, monkeys and humans.The metabolic stability and metabolite identification studies using liver microsomes in vitro, plasma protein binding using a rapid equilibrium dialysis in vitro, pharmacokinetic studies in vivo were carried out to evaluate the interspecies differences. The toxicokinetic study in monkeys was also investigated.The metabolic profiles were similar in monkeys and humans, which were significant different from rats and dogs in vitro. In vitro plasma protein binding showed no major differences between species with medium to high protein binding rates. After single oral dose to rats, dogs, and monkeys, the absolute oral bioavailability of Y101 was 44.9%, 43.1%, and 19.2%, respectively. There was no accumulation for Y101 toxicokinetics in monkeys after oral administration for 90 d.The metabolic profiles indicated monkey was the very animal model for preclinical safety evaluation of Y101. Our results have demonstrated the favorable pharmacokinetics profile of Y101, which supports the clinical trials in humans.

Direct quantification of intact FIM in monkey plasma using a selective chromatography-tandem mass spectrometry method: Application in a pharmacokinetic study.

FIM protein, which consists of 155 amino acids, was developed as a novel GLP-1 analog to reduce blood glucose, and pharmacodynamic results showed that it had a certain effect when used in treating Alzheimer's disease. The molecular weight of FIM is 16,304 Da. In theory, the concentration of FIM in biological samples should be determined by the ligand binding assay method or indirectly quantified using LC-MS/MS instrumentation. However, the above methods are complex and time-consuming. In this study, we successfully developed a simpler LC-MS/MS method for directly quantifying the intact FIM protein in monkey plasma for the first time. The chromatographic separation of FIM was achieved using an InertSustain Bio C18 column with a mobile phase of acetonitrile containing 0.1% formic acid (A)-water containing 0.1% formic acid (B) at a flow rate of 0.3 ml/min. Good linearity was observed in the concentration range of 5-500 ng/ml (r2 > 0.99). The intra- and inter-day precisions (expressed as relative standard deviation, RSD) of FIM were 2.30-12.8 and 7.30-13.2%, respectively. The intra- and inter-day accuracies (expressed as a relative error, RE) were -12.7-6.55 and - 10.1-0.892%, respectively. This method was successfully applied for a pharmacokinetic study of the FIM protein in four monkeys after subcutaneous administration.

Direct comparison of LC-MS/MS and RIA methods for the pharmacokinetics assessment of human insulin in preclinical development.

Insulin is an effective therapeutic for diabetes, and the level of insulin in vivo is directly related to the health of diabetic patients. Traditionally, the concentrations of insulin in vivo are determined by the radioimmunoassay (RIA) method. In this study, we developed an LC-MS/MS method for the quantification of human insulin in dog plasma and directly compared the RIA and LC-MS/MS methods. Our LC-MS/MS method exhibited superior accuracy, efficiency and cost-effective for the pharmacokinetic (PK) assessment of human insulin. The LC-MS/MS method can quantitate human insulin and canine insulin simultaneously without cross-reactivity, making the analysis more efficient. The LLOQ of our LC-MS/MS method was 38.5 pg/mL, which was necessary to fully describe the PK profiles of endogenous and exogenous insulin in vivo. The direct comparison of PK data obtained from the two methods demonstrated that LC-MS/MS could be an alternative to the RIA method and should be widely used for the quantification of insulin drugs, especially in preclinical studies.

Determination of liraglutide in rat plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetics study.

A simple, sensitive and selective LC-MS/MS method was developed for the quantitative analysis of liraglutide and validated in rat plasma. Human insulin was used as the internal standard. After a simple protein precipitation step, liraglutide was chromatographically separated using an InertSustain Bio C18 column with mobile phases comprising acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B). Detection was achieved using positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the concentration range 0.5-250 ng/mL (r2 > 0.99). The intra- and inter-day precision values (expressed as relative standard deviation, RSD) of liraglutide ranged from 1.97-7.63% and 5.25-11.9, respectively. The accuracy (expressed as relative error, RE) ranged from -8.79-11.4%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied for the pharmacokinetics study of liraglutide in rats after subcutaneous administration.

Determination of human insulin in dog plasma by a selective liquid chromatography-tandem mass spectrometry method: Application to a pharmacokinetic study.

A simple, sensitive and selective LC-MS/MS method for quantitative analysis of human insulin was developed and validated in dog plasma. Insulin glargine was used as the internal standard. After a simple step of solid-phase extraction, the chromatographic separation of human insulin was achieved by using InertSustain Bio C18 column with a mobile phase of acetonitrile containing 1% formic acid (A)-water containing 1% formic acid (B). The detection was performed by positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the concentration range of 1-1000 µIU/mL (r2 > 0.99), and the lower limit of quantification was 1 µIU/mL (equal to 38.46 pg/mL). The intra- and inter-day precision (expressed as relative standard deviation, RSD) of human insulin were ≤12.1% and ≤13.0%, respectively, and the accuracy (expressed as relative error, RE) was in the range of -7.23-11.9%. The recovery and matrix effect were both within acceptable limits. This method was successfully applied for the pharmacokinetic study of human insulin in dogs after subcutaneous administration.

[Simultaneous determination of salidroside and tyrosol in Beagle dog plasma using UHPLC-MS/MS after pre-column dansyl chloride derivatization].

A pre-column derivatization method combined with UHPLC-MS/MS was developed for the simultaneous determination of salidroside and tyrosol in Beagle dog plasma. After protein precipitation by acetonitrile, the liquid supernatant was treated with dansyl chloride under dark conditions at 60 ℃ for 30 min, and then, the sample solution was extracted using methyl tertiary butyl ether. The multiple reaction monitoring in positive ion mode was used for MS detection of the tested analytes with the specific ion transitions of m/z 534.2→372.0 for salidroside derivative, m/z 372.0→171.0 for tyrosol derivative and m/z 506.0→171.0 for arbutin derivative. The chromatograph separation was achieved on an ACQUITY UPLC® BEH C18 column (100 mm × 2.1 mm, 1.7 µm) with a gradient mobile phase consisting of acetonitrile (0.1% formic acid)-water (10% acetonitrile, 0.1% formic acid) for 9 min. The assay showed a good linearity over the range of 0.02/0.1 − 20/10 µmol·L−1 with a lower limit of quantitation of 0.02 and 0.1 µmol·L−1 for salidroside and tyrosol in dog plasma, respectively. The intra- and inter-day precisions were all less than 8.68%, and the accuracy was within ±11.4%. The established method with a high sensitivity, good specificity and reliability was appropriate for simultaneous determination of salidroside and tyrosol in dog plasma and successfully applied to a pharmacokinetic study after intragastric administration of salidroside to Beagle dogs.

Metabolite identification of bentysrepinine (Y101), a novel anti-HBV agent in rats using a five-step strategy based on a combined workflow with two different platforms of liquid chromatography-tandem mass spectrometry.

Bentysrepinine (Y101), a derivative of repensine (a compound isolated from Dichondrarepens Forst), is a novel phenyalanine dipeptide inhibiting DNA-HBV and cccDNA activities and is currently under development for the treatment of hepatitis B virus (HBV)-infected hepatitis. Our previous study implied that there might be an existence of extensive metabolism of Y101 in rats. Therefore, it is necessary to perform metabolic profiling study to further evaluate its safety and drug-like properties. In this study, the metabolism of Y101 in rats was investigated by a convincible five-step strategy to characterize metabolites in plasma and that excreted into urine, bile and feces. The five-step strategy was realized by using an combined workflow on two different MS platforms, including various scan modes of liquid chromatography with hybrid quadruple-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and various post-acquiring data mining tools of liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). QTOF MS/MS was employed as a powerful complementary tool to enable high confidence of metabolites identification using its functions of accurate MS and MS/MS fragmentation. As a result, a total of 30 metabolites were detected, including 25 phase I and 5 phase II metabolites. Among them, four primary metabolites (M6-M9) were further identified by comparing with the authentic standards chemically synthesized. The possible metabolic pathways of Y101 in rats were proposed to be amide hydrolysis, monohydroxylation, dihydroxylation, N-oxidation, demethylation, methylation, glucosidation and glucuronidation. This is the first study of the metabolism of Y101 in rats. The five-step strategy was successfully used to systematically characterize metabolites of Y101 in rats, and it would be generally applied for metabolite identification of new drug candidate.

Effect of age on the pharmacokinetics of polymorphic nimodipine in rats after oral administration.

The previous investigation has proved that their existed pharmacokinetic difference between the different crystal forms of the polymorphic drugs after oral administration. However, no systemic investigations have been made on the change of this pharmacokinetic difference, resulted either from the physiological or from the pathological factors. In this paper, we used polymorphic nimodipine (Nim) as a model drug and investigated the effect of age difference (2- and 9-month old) on the pharmacokinetics after oral delivery in rats. As the results shown, for L-form of Nim (L-Nim), the AUC0-24 h in 2-month-old rats was 343.68±47.15 ng·h/mL, which is 23.36% higher than that in 9-month-old rats. For H-form of Nim (H-Nim), the AUC0-24 h in 2-month-old rats was 140.91±19.47 ng·h/mL, which is 54.64% higher than that in 9-month-old rats. The AUC0-24 h ratio between H-Nim and L-Nim was 2.44 in 2-month-old rats and 3.06 in 9-month-old rats. Since age difference could result in unparallelled change of the absorption and bioavailability of the polymorphic drugs, the results in this experiment are of value for further investigation of crystal form selection in clinical trials and rational clinical application of the polymorphic drugs.

[The in vitro transport mechanism of bentysrepinine: a novel anti-hepatitis B virus drug candidate].

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (P(app)) and efflux ratio (RE); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-h OATP1B1, HEK293-h OATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1, OATP2B1 and PEPT1. The results suggest that Y101 has a weak inhibitory effect on OATP1B1 and OATP2B1, and Y101 may not be substrates of OATP1B1, OATP2B1 or PEPT1. The above results can be used to explain the in vivo absorption and distribution characteristics, and to provide a scientific basis for the further development of Y101.

[The in vitro inhibition and induction of cytochrome P450 activities by bentysrepinine: a novel candidate of anti-hepatitis B virus drug].

Bentysrepinine (Y101), a derivative of phenyalanine dipeptide, has a novel mechanism in the treatment of hepatitis B virus (HBV) infection with a good anti-HBV effect. In the present study, a fluorometric-based high throughput method using cytochrome P450 (CYP) screening kit was adopted to evaluate in vitro inhibition potential of Y101 on CYP isoenzymes by calculating remaining enzyme activities and inhibitory potential (IC(50) values) using the determined values of fluorescence intensity. The result showed that Y101 exhibited little activity in the inhibition of CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 (IC(50) > 100 µmol·L(-1)). Y101 was used to treat human primary hepotocytes for 72 h, and the enzyme activities of CYP1A2, CYP2B6 and CYP3A4 were determined with a cocktail of probe substrates for the three CYP isoforms. The metabolites were simultaneously determined using a LC-MS/MS method. Y101 had no activity in the induction of CYP1A2, CYP2B6 and CYP3A4 on the basis of the following results: 1 The ratio of enzyme activities between test and control groups were all below than 1 (varied from 0.662 to 0.928); 2 The induction potential of Y101 were lower than forty percent compared with that of positive groups. The above results suggest that Y101 has little activity in the regulation of metabolic drug-drug interactions based on the CYP isoform changes following co-administration of drugs.

Development of an LC-MS/MS method for the quantitation of deoxyglycychloxazol in rat plasma and its application in pharmacokinetic study.

Deoxyglycychloxazol (TY501) is a glycyrrhetinic acid derivative which exhibits high anti-inflammatory activity and reduced pseudoaldosteronism compared to glycyrrhetinic acid. In this study, a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantitation of TY501 in rat plasma. Plasma samples were treated by precipitating protein with methanol and supernatants were separated by a Symmetry C8 column with the mobile phase consisting of methanol and 10 mM ammonium formate (containing 0.1% of formic acid) (90:10, v/v). The selected reaction monitoring (SRM) transitions were performed at m/z 647.4→191.2 for TY501 and m/z 473.3→143.3 for astragaloside aglycone (IS) in the positive ion mode with atmospheric pressure chemical ionization (APCI) source. Calibration curve was linear over the concentration range of 5-5000 ng/mL. The lower limit of quantification was 5 ng/mL. The mean recovery was over 88%. The intra- and inter-day precisions were lower than 6.0% and 12.8%, respectively, and the accuracy was within ±1.3%. TY501 was stable under usual storage conditions and handling procedure. The validated method has been successfully applied to a pharmacokinetic study after oral administration of TY501 to rats at a dosage of 10 mg/kg.

The Targeted-liposome Delivery System of Antitumor Drugs.

The liposome delivery system has been intensively explored as novel drug delivery system (DDS) for antitumor drugs, due to its safety, selective cytotoxicity, long circulation and slow elimination in blood, which is favorable for cancer therapy. The liposome-based chemotherapeutics are used to treat a variety of cancers to enhance the therapeutic index of antitumor drugs. Here, the author reviewed the important targets for cancer therapy and the pharmacokinetic behavior of liposomal drugs in vivo, as well as the application of the targeting liposomal system in cancer therapy. Considering further application for clinical use, the great challenges of the liposome-based delivery system were also proposed as follows: 1) prepare stealth liposome with steric stabilization and further enhance the therapeutic effects and safety; 2) explore more safe clinical targets and complementary or different types of targeting liposome; 3) thirdly, more investment is needed on the research of pharmacokinetics of the elements such as the ligands (antibody), PEG and lipids of liposome delivery system as well as safety evaluation. Considering the complex process of the liposomal encapsulation drugs in vivo, the author inferred that there are maybe different forms of the encapsulation drug to be internalized by the tumor tissues at the same time and space, although there are little reports on it.

Interactions between Transporters and Herbal Medicines/Drugs: A Focus on Hepatoprotective Compounds.

Many herbal medicines and drugs are available in the clinic as potent hepatoprotective agents for the treatment of commonly occurring liver diseases. Recently, herbal medicines such as silymarin and curcumin have gained more attention and popularity for the treatment of various liver diseases because of their safety and efficacy profiles. Some of them are related to transporters for drug disposition processes, therapeutic efficacy and/or adverse drug reactions. Currently, herbal medicines and diet supplements made from natural products are widely used in patients who are being treated with conventional prescription medicines, which are related to an increasing risk of herbal-drug interactions (HDIs) and/or drug-drug interactions (DDIs). The purpose of present review is to summarize the contemporary knowledge of transporter-mediated HDIs or DDIs for herbal medicines/drugs focusing on hepatoprotective compounds. Several herbal medicines/drugs are discussed in detail in this review.

Aspartate-modified doxorubicin on its N-terminal increases drug accumulation in LAT1-overexpressing tumors.

L-type amino acid transporter 1 (LAT1), overexpressed on the membrane of various tumor cells, is a potential target for tumor-targeting therapy. This study aimed to develop a LAT1-mediated chemotherapeutic agent. We screened doxorubicin modified by seven different large neutral amino acids. The aspartate-modified doxorubicin (Asp-DOX) showed the highest affinity (Km = 41.423 µmol/L) to LAT1. Aspartate was attached to the N-terminal of DOX by the amide bond with a free carboxyl and a free amino group on the α-carbon atom of the Asp residue. The product Asp-DOX was characterized by HPLC/MS. In vitro, Asp-DOX exerted stronger inhibition on the cancer cells overexpressing LAT1 and the uptake of Asp-DOX was approximately 3.5-fold higher than that of DOX in HepG2 cells. Pharmacokinetic data also showed that Asp-DOX was expressed over a longer circulation time (t1/2 = 49.14 min) in the blood compared to DOX alone (t1/2 = 15.12 min). In HepG2 and HCT116 tumor-bearing mice, Asp-DOX achieved 3.1-fold and 6.4-fold accumulation of drugs in tumor tissue, respectively, than those of the unmodified DOX. More importantly, treatment of tumor-bearing mice with Asp-DOX showed a significantly stronger inhibition of tumor growth than mice treated with free DOX in HepG2 tumor models. Furthermore, after Asp modification, Asp-DOX avoided MDR mediated by P-glycoprotein. These results suggested that the Asp-DOX modified drug may provide a new treatment strategy for tumors that overexpress LAT1 and MDR1.

[Simultaneous determination of clevidipine butyrate and its metabolite clevidipine acid in dog blood by liquid chromatography-tandem mass spectrometry].

A rapid, sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of clevidipine butyrate and its primary metabolite clevidipine acid in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150 mm x 4.6 mm, 5 µm) with a gradient mobile phase consisting of methanol and 5 mmol · L(-1) ammonium formate. A chromatographic total run time of 13.0 min was achieved. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M-H]- --> m/z 234.1 for clevidipine butyrate, m/z 354.0 [M-H]- --> m/z 208.0 for clevidipine acid and m/z 256.1 [M-H]- --> m/z 227.1 for elofesalamide (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The linear calibration curves for clevidipine butyrate and clevidipine acid were obtained in the concentration ranges of 0.5-100 ng · mL and 1-200 ng · mL(-1), separately. The lower limit of quantification of clevidipine butyrate and clevidipine acid were 0.5 ng · mL(-1) and 1 ng · mL(-1). The intra and inter-assay precisions were all below 12.9%, the accuracies were all in standard ranges. Stability testing indicated that clevidipine butyrate and clevidipine acid in dog blood with the addition of denaturant methanol was stable under various processing and/or handling conditions. The validated method has been successfully applied to a pharmacokinetic study of clevidipine butyrate injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg · h(-1) for 0.5 h.

Quantitation of clevidipine in dog blood by liquid chromatography tandem mass spectrometry: application to a pharmacokinetic study.

Clevidipine, a vascular selective calcium channel antagonist of the dihydropyridine class, is rapidly metabolized by ester hydrolysis because of incorporation of an ester linkage into the drug molecule. To characterize its pharmacokinetic profiles in dogs, a simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of clevidipine in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C18 column (150mm×4.6mm, 5µm) with a gradient mobile phase consisting of methanol and 5mM ammonium formate at a flow rate of 0.5mL/min. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m/z 454.1 [M-H](-)→m/z 234.1 for clevidipine and m/z 256.1 [M-H](-)→m/z 227.1 for elofesalamide (internal standard) in the negative ion mode with electrospray ionization (ESI) source. This validated LC-MS/MS method showed good linearity over the range 0.5-100ng/mL with the lower limit of quantitation (LLOQ) of 0.5ng/mL together with the satisfied intra- and inter-day precision, accuracy, extraction recovery and matrix effect. Stability testing indicated that clevidipine in dog blood with the addition of denaturant methanol was stable on workbench for 1h, at -80°C for up to 30 days, and after three freeze-thaw cycles. Extracted samples were also observed to be stable over 24h in an auto-sampler at 4°C. The validated method has been successfully applied to a pharmacokinetic study of clevidipine injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5mg/h for 0.5h.