In vivo and in vitro metabolism and pharmacokinetics of cholinesterase inhibitor deoxyvasicine from aerial parts of Peganum harmala Linn in rats via UPLC-ESI-QTOF-MS and UPLC-ESI-MS/MS.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Peganum harmala Linn are a Uighur traditional medicinal herb in China used to treat amnesia, bronchial asthma, and cough. Deoxyvasicine (DVAS), a potent cholinesterase inhibitor exhibiting anti-senile dementia activity, is one of the chief active ingredients in aerial parts of P. harmala and plays a key role in mediating the pharmacological effects of P. harmala. However, the metabolic profiling and in vivo pharmacokinetic characteristics of DVAS still remain unknown. AIM OF THE STUDY: The aim of this present study was to investigate the metabolism and pharmacokinetic properties of DVAS in rats by using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method. MATERIALS AND METHODS: The metabolic profiling of DVAS was evaluated in vitro and in vivo by rat liver microsomes (RLMs) incubation and by rat bio-specimens, such as urine, feces, plasma, and bile, after the oral administration of 45 mg/kg DVAS. An efficient and sensitive UPLC-ESI-MS/MS method was developed and validated to simultaneously determine DVAS and its major four metabolites, namely, vasicine, deoxyvasicinone, vasicinone, and 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-ß-D-glucuronide in rat plasma. For pharmacokinetic studies, 32 Sprague-Dawley rats were randomly divided into four groups, namely, intravenous dosage group (2 mg/kg DVAS) and three oral dosage groups (5, 15, and 45 mg/kg DVAS). In addition, the activity of the components in plasma after intravenous administration of DVAS was evaluated by in vitro anti-butyrylcholinesterase (BChE) assays. RESULTS: A total of 23 metabolites were found in RLMs, plasma, urine, feces, and bile by UPLC-ESI-QTOF-MS. The metabolic pathway of DVAS in vivo and in vitro mainly involved hydroxylation, dehydrogenation, acetylation, methylation, glucuronidation, and O-sulphate conjugation, and the C-3 and C-9 sites were the main metabolic soft spots. All 23 metabolites were detected in the urine sample, and 13, 8, 22, and 6 metabolites were identified from rat feces, plasma, bile, and RLMs, respectively. The standard curves of DVAS and four metabolites in rat plasma showed good linearity in the concentration range of 0.82-524.00 ng/mL with acceptable selectivity, precision, accuracy, recovery, and stability. DVAS exhibited linear dose-proportional pharmacokinetics at doses of 5, 15, and 45 mg/kg after oral administration, and the average oral absolute bioavailability of DVAS was 47.46%. The in vitro anti-BChE assays implied that the inhibitive activities were mainly due to the different concentrations of prototype DVAS. CONCLUSIONS: DVAS can be rapidly absorbed and excreted by blood, and it is also extensively metabolized in vivo, and the anti-BChE activity in blood is mainly attributed to DVAS. These findings can lay a foundation for new drug development for DVAS.

Alterations of Gefitinib Pharmacokinetics by Co-administration of Herbal Medications in Rats.

OBJECTIVE: To evaluate the potential pharmacokinetic interactions of the anticancer agent gefitinib (Iressa®) and the oriental medications Guipi Decoction (, GPD, Guibi-tang in Korean) and Bawu Decoction (, BWD, Palmul-tang in Korean). METHODS: Methylcellulose (MC, control), GPD (1,200 mg/kg), or BWD (6,000 mg/kg) was orally administered to rats either as a single dose or multiple doses prior to gefitinib administration. To examine the effects of a single dose of the herbal medicines, gefitinib (10 mg/kg) was orally administered after 5 min or 1 h of MC or the herbal medicine pretreatments. To examine the effects of the multiple doses of the herbal medicines, gefitinib (10 mg/kg) was orally administered following 7 consecutive days of the administration of MC or each herbal medicine. The plasma concentrations of gefitinib were determined with liquid chromatography-tandem mass spectrometry assay. The plasma concentration-time profiles of gefitinib were analyzed with a noncompartmental analysis. RESULTS: Gefitinib was rapidly absorbed and showed a monoexponential decline with an elimination half-life of 3.7-4.1 h. The pharmacokinetics of gefitinib was not affected by GPD pretreatment. However, a significantly lower maximum plasma concentration (Cmax, P<0.05) and area under the curve (P<0.05), and a delayed time to reach Cmax (Tmax, P<0.01) were observed in both single- and multipledose BWD-pretreated rats compared with the control rats. CONCLUSIONS: BWD and not GPD might delay and interfere with gefitinib absorption. Further evaluations of the clinical significance of these findings are needed.

Alterations in Pharmacokinetics of Gemcitabine and Erlotinib by Concurrent Administration of Hyangsayukgunja-Tang, a Gastroprotective Herbal Medicine.

Gemcitabine and erlotinib are the chemotherapeutic agents used in the treatment of various cancers and their combination is being accepted as a first-line treatment of advanced pancreatic cancer. Hyangsayukgunja-tang (HYT) is a traditional oriental medicine used in various digestive disorders and potentially helpful to treat gastrointestinal adverse effects related to chemotherapy. The present study was aimed to evaluate the effect of HYT on the pharmacokinetics of gemcitabine and erlotinib given simultaneously in rats. Rats were pretreated with HYT at an oral dose of 1200 mg/kg/day once daily for a single day or 14 consecutive days. Immediately after pretreatment with HYT, gemcitabine and erlotinib were administered by intravenous injection (10 mg/kg) and oral administration (20 mg/kg), respectively. The effects of HYT on pharmacokinetics of the two drugs were estimated by non-compartmental analysis and pharmacokinetic modeling. The pharmacokinetics of gemcitabine and erlotinib were not altered by single dose HYT pretreatment. However, the plasma levels of OSI-420 and OSI-413, active metabolites of erlotinib, were significantly decreased in the multiple dose HYT pretreatment group. The pharmacokinetic model estimated increased systemic clearances of OSI-420 and OSI-413 by multiple doses of HYT. These data suggest that HYT may affect the elimination of OSI-420 and OSI-413.

UPLC-ESI-MS/MS study of the effect of green tea extract on the oral bioavailability of erlotinib and lapatinib in rats: Potential risk of pharmacokinetic interaction.

Green tea (GT) is one of the most consumed beverages worldwide. Tyrosine kinase inhibitors (TKIs) belong to the oral targeted therapy that gained much interest in oncology practice, among which are erlotinib (ERL) and lapatinib (LAP). Since green tea polyphenols (GTP) are known to be inhibitors of receptor tyrosine kinases, GTE could likely potentiate the anticancer effect of TKIs, but with a possibility of pharmacokinetic (PK) interaction with co-administered TKIs. In this study, the effect of GTE on the PK of ERL/LAP in rats was studied. UPLC-ESI-MS/MS method has been developed and validated for the quantification of ERL and LAP in rat plasma, using gefitinib (GEF) as the internal standard. Plasma samples were treated extensively by protein precipitation (PPT) followed by solid phase extraction (SPE) using octadecyl C 18/14% cartridges. Chromatographic analysis was carried out on Acquity UPLC BEH™ C18 column with a mobile phase consisting of water: acetonitrile (20: 80, v/v), each with 0.15% formic acid. Quantification was performed in the positive electrospray ionization (ESI+) mode with multiple reaction monitoring (MRM) of the transitions m/z 394.29→278.19 (ERL), m/z 581.07→365.13 (LAP), and m/z 447.08→128.21 (GEF). The method was fully validated as per the FDA guidelines showing linearity over the range of 0.4-1000 (ERL) and 0.6-1000 (LAP) ng/mL with very low lower limit of quantification (LLOQ) of 0.4 and 0.6ng/mL for ERL and LAP, respectively. The applicability of the method was extended to perform a comparative study of the PK of ERL/LAP following short-term and long-term administration of GTE, compared with their single oral administration. The results revealed that a significant reduction in the oral bioavailability was recorded with both ERL and LAP following the ingestion of GTE particularly for short-term administration. A reduction in Cmax (AUC) by 67.60% (69.50%) and 70.20% (73.96%), was recorded with short-term administration of GTE, compared with only 16.03% (21.09%) and 13.53% (22.12%) reduction for ERL and LAP, respectively, with long-term administration. Thus patients taking TKIs should preferably avoid drinking GT or ingesting GTE capsules during the period of treatment with TKIs.

Detection and characterization of the metabolites of rutaecarpine in rats based on ultra-high-performance liquid chromatography with linear ion trap-Orbitrap mass spectrometer.

CONTEXT: Rutaecarpine is an active indoloquinazoline alkaloid ingredient originating from Evodia rutaecarpa (Wu-zhu-yu in Chinese), which possesses a variety of effects. However, its metabolism has not been investigated thoroughly yet. OBJECTIVE: This study develops a highly sensitive and effective method for detection and characterization of the metabolites of rutaecarpine in Sprague-Dawley (SD) rats. MATERIALS AND METHODS: In this study, an efficient method was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UHPLC-LTQ-Orbitrap MS) to detect the metabolism profile of rutaecarpine in rat plasma. First, a blood sample (1 mL) was withdrawn 2 h after oral administration of rutaecarpine in SD rats (50 mg/kg). Second, the blood was centrifuged at 4000 rpm for 10 min and pretreated by solid-phase extraction method. Third, 2 µL of the plasma was injected into UHPLC-LTQ-Orbitrap MS for analysis. Finally, the metabolites of rutaecarpine were tentatively identified based on accurate mass measurements, fragmentation patterns and chromatographic retention times. RESULTS: A total of 16 metabolites (four new metabolites, viz., dihydroxylation and sulphate conjugation products of rutaecarpine (M8-M11)) as well as parent drug itself, including three phase I and 12 phase II metabolites were detected and identified in rat plasma. Hydroxylation, sulphate conjugation and glucuronidation were confirmed as the primary metabolic pathways for rutaecarpine in rat plasma. DISCUSSION AND CONCLUSION: These results provide an insight into the metabolism of rutaecarpine and also can give strong indications on the effective forms of rutaecarpine in vivo.

Preclinical assessment of the interactions between the antiretroviral drugs, ritonavir and efavirenz, and the tyrosine kinase inhibitor erlotinib.

PURPOSE: Prevalence of non-AIDS-defining cancers (NADCs) has increased in the era of potent antiretroviral treatments. Incidence rates of NADCs now exceed AIDS-defining cancers in HIV-positive patients. Treatment of NADCs may be complicated by interactions between antiretrovirals and chemotherapy mostly via inhibition or induction of CYP3A4. Erlotinib is used to treat non-small cell lung and pancreatic cancer and is primarily metabolized by CYP3A4 into multiple products including the active metabolite (OSI-420). Preclinical in vivo assessment was performed to gain a better understanding of CYP3A4-mediated interactions between antiretrovirals and erlotinib. METHODS: Erlotinib (50 mg/kg p.o.) was administered to male FVB mice in the presence and absence of dexamethasone (10 mg/kg p.o. QDx4), efavirenz (25 mg/kg p.o. QDx4), ketoconazole (50 mg/kg p.o.), or ritonavir (12.5 mg/kg p.o.). Blood samples were collected to characterize exposure (AUC). RESULTS: Administration of erlotinib with CYP3A4 inducers (dexamethasone) and inhibitors (ketoconazole and ritonavir) resulted in significant alterations in erlotinib exposure. Ketoconazole and ritonavir resulted in a 1.7- and 3.0-fold increase in erlotinib AUC, respectively, while dexamethasone results in a 0.6-fold decrease in erlotinib AUC. The CYP3A4 inducer efavirenz did not have a significant effect on erlotinib exposure. CONCLUSION: CYP3A4 inducers and inhibitors altered the exposure of erlotinib. Until a definitive clinical trial is performed, erlotinib should be used with caution in patients on a ritonavir-containing antiretroviral regimen, while standard doses may be appropriate for patients on an efavirenz-containing antiretroviral regimen.

[Pharmacokinetics behavior of raltitrexed in rats after repeatedly injected with Huangqi injection].

In this study, the variation of pharmacokinetics behavior of raltitrexed (RTX) in rats after repeatedly injected with Huangqi injection was investigated. Twelve SD rats were divided into two groups: the multidose group and the RTX group. Rats in multidose group were iv. injected with Huangqi injection (dose of 1.575 mL x kg(-1)) everyday at 8 am for a week, and had free accesses for food and water. The rats were fasted for food but not water since 8 h before the eighth day. At the eighth morning, firstly, rats were injected with Huangqi injection (dose of 1.575 mL x kg(-1)), and 5 min later, were injected with RTX (dose of 0.467 mg x kg(-1)); rats in RTX group were not disposed in the previous seven days, also had free accesses for food and water, and were iv. injected with raltitrexed at the same time as Multidose group at the eighth day morning. Rat plasma was collected at different time and processed with methanol to precipitate the protein before HPLC assays. The pharmacokinetics parameters for two groups were calculated by software 3P97. Through the observation of drug concentration in plasma and time curve, we found that at almost every time point the concentration of RTX in plasma in multidose group was lower than the RTX group. When comparing the pharmacokinetics parameters between the multidose group and the RTX group, the average of AUC(0-t) and half-life(t1/2) of multidose group were decreased from 56 080 microg x min x L(-1) and 15.07 min to 35 834 microg x min x L(-1) and 8.95 min, respectively, while the clearance (CL) was increased from 0.51 to 0.83 mL x h(-1). Therefore, it could be deduced that repeatedly injected with AR injection may influence the renal excretion and glycometabolism of RTX, thus change pharmacokinetics behavior of raltitrexed in rats plasma. This result may give us a hint to prudantly manage the drug combination of RTX and Huangqi injection.

Simultaneous determination and pharmacokinetic analysis of seven alkaloids and two flavonoids from rat plasma by HPLC-DAD after oral administration of Wuzhuyu decoction.

A simple and sensitive high-performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic analysis of seven alkaloids dehydroevodiamine (DHED), 10-hydroxyrutaecarpine (HDR), evodiamine (EDM), rutaecarpine (RCP), 1-methyl-2-n-nonyl-4(1H)quinolone (MNQ), evocarpine (ECP), and dihydroevocarpine (DHE), and two flavonoids isorhamnetin-7-O-rutinoside (RIM) and diosmetin-7-O-ß-d-glucopyranoside (GRD) in rat plasma after oral administration of Wuzhuyu decoction. The flow rate was kept at 1.0 ml/min and the detection wavelength was set at 300 nm. The calibration curves were linear in the range of 0.5013-30.076 µg/ml for DHED, 0.2161-21.608 µg/ml for RIM, 0.161-12.876 µg/ml for HDR, 0.2146-21.457 µg/ml for GRD, 2.0464-40.928 µg/ml for EDM, 1.0398-31.194 µg/ml for RCP, 0.5970-35.818 µg/ml for MNQ, 0.8371-20.928 µg/ml for ECP, and 0.5167-31.003 µg/ml for DHE. The precision (relative standard deviation (RSD), %) for all was less than 10% and the accuracy (relative error (RE), %) was within ± 10%. The results demonstrated that the assay had remarkable reproducibility with acceptable accuracy and precision. The lower limit of quantifications for the compounds in plasma ranged from 0.12 to 0.23 µg/ml and the lower limit of detections ranged from 0.024 to 0.076 µg/ml. This validated method has been successfully applied in the pharmacokinetics study of seven alkaloids and two flavonoids after orally administrating the Wuzhuyu decoction to rats.

Pharmacokinetic comparisons of rutaecarpine and evodiamine after oral administration of Wu-Chu-Yu extracts with different purities to rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Wu-Chu-Yu is a well-known herbal drug used for hypertension. Rutaecarpine and evodiamine are main bioactive components of the medicine. MATERIALS AND METHODS: A sensitive and specific HPLC method was developed to analyze rutaecarpine (Rut) and evodiamine (Evo) in rat whole blood. The pharmacokinetics of Rut and Evo after oral administration of Wu-Chu-Yu extracts with different purities to rats was compared to evaluate the effect of purity of Wu-Chu-Yu extracts on the absorption of Rut and Evo. Male Sprague-Dawley rats were given Wu-Chu-Yu extracts with different purities (high, medium and low) approximately the same doses of equivalent to Rut (40 mg/kg) and Evo (31 mg/kg). The contents of Rut and Evo were 45 and 35%, 28 and 21%, 9 and 7% in high, medium and low purity extracts, respectively. At different time points (0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3 and 4h) after administration, the concentrations of Rut and Evo in rat whole blood were determined by HPLC, and main pharmacokinetic parameters were calculated. RESULTS: The results indicated that the absorption of Rut and Evo in Wu-Chu-Yu extracts was improved when compared with the pure Rut and Evo and there were significant differences among different groups. CONCLUSIONS: The bioavailability of Rut and Evo was increased along with the increasing of purity (16%-80%) in Wu-Chu-Yu extracts.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

[Determination of evodiamine and rutaecarpine of compound Wuzhuyu cataplasm in plasma by SPE-HPLC: application to its pharmacokinetics].

OBJECTIVE: To establish a SPE-HPLC method for the determination and pharmacokinetic study of evodiamine and rutacarpine in rat plasma. METHOD: A Kromasil C18 column (4.6 mm x 250 mm, 5 microm) was used with acetonitrile-water-tetrahydrofuran-acetic acid (51:48:1:0.1) as a mobile phase and at a flow rate of 1 mL x min(-1), and the UV detection was at 225 nm. The column temperature was 35 degrees C. After the analytes were extracted from the plasma of rats by solid phase extraction (SPE), the content of evodiamine and rutaecarpine was measuared by HPLC method using halcinonide as an internal standard solution. RESULT: After transdermal administration to rats, the pharmacokinetic behavior of evodiamine and rutaecarpine belongs to the one-compartment model. The main pharmacokinetic parameters was as follows: K(a) 0.224 h(-1) and 0.220 h(-1), K(e) 0.114 h(-1) and 0.118 h(-1), C(max) 0.211 mg x L(-1) and 0.272 mg x L(-1), T(peak) 6.132 h and 6.102 h, respectively. CONCLUSION: The method in this study is simple, rapid and sensitive. It is proved to be suitable for pharmacokinetic study of evodiamine and rutaecarpine.

Short preoperative treatment with erlotinib inhibits tumor cell proliferation in hormone receptor-positive breast cancers.

PURPOSE: To administer the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib to patients with operable untreated breast cancer during the immediate preoperative period and to measure an antiproliferative and/or a proapoptotic effect in the post-therapy specimen and determine a biomarker profile associated with evidence of erlotinib-mediated cellular activity. PATIENTS AND METHODS: Newly diagnosed patients with stages I to IIIA invasive breast cancer were treated with erlotinib 150 mg/d orally for 6 to 14 days until the day before surgery. Erlotinib plasma levels were measured by tandem mass spectrometry the day of surgery. Drug-induced changes in tumor cell proliferation and apoptosis were assessed by Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling analysis, respectively, in biopsies from the pretherapy and surgical specimens. Biopsies were also evaluated for P-EGFR, P-HER-2, P-MAPK, P-Akt, P-S6, and S118 P-ER alpha. RESULTS: In drug-sensitive PC9 xenografts, 5 days of treatment with erlotinib were enough to induce a maximal inhibition of cell proliferation and induction of apoptosis. Forty-one patients completed preoperative treatment with erlotinib. Grade

Potentiometric sensors enabling fast screening of the benign prostatic hyperplasia drug alfuzosin in pharmaceuticals, urine and serum.

The construction and characterization of potentiometric membrane electrodes are described for the quantification of alfuzosin, a drug used in a mono- and combined therapy of benign prostatic hyperplasia (BPH). The membranes of these electrodes consist of alfuzosin hydrochloride-tetraphenyl borate, (Az-TPB), chlorophenyl borate (Az-ClPB), and phosphotungstate (Az(3)-PT) ion associations as molecular recognition reagent dispersed in PVC matrix with dioctylpthalate as plasticizer. The performance characteristics of these electrodes, which were evaluated according to IUPAC recommendations, revealed a fast, stable and liner response for alfuzosin over the concentration ranges of 8.3 x 10(-6) to 1.0 x 10(-2) M, 3.8 x 10(-6) to 1.0 x 10(-2) M, 7.5 x 10(-7) to 1.0 x 10(-2) M AzCl with cationic slopes of 57.0, 56.0 and 58.5 mV/decade, respectively. The solubility product of the ion-pair and the formation constant of the precipitation reaction leading to the ion-pair formation were determined conductometrically. The electrodes, fully characterized in terms of composition, life span and usable pH range, were applied to the potentiometric determination of alfuzosin hydrochloride ion in different pharmaceutical preparations and biological fluids without any interference from excipients or diluents commonly used in drug formulations. The potentiometric method was also used in the determination of alfuzosin hydrochloride in pharmaceutical preparations in four batches with different expiration dates. Validation of the method showed suitability of the proposed electrodes for use in the quality control assessment of alfuzosin hydrochloride. This potentiometric method offers the advantages of high-throughput determination, simplicity, accuracy, automation feasibility, and applicability to turbid and colored sample solutions.

Determination of evodiamine and rutecarpine in human serum by liquid chromatography-tandem mass spectrometry.

Evodiamine and rutecarpine are two kinds of indole alkaloids contained in the fruit of Evodiae fructus, which have been shown to exhibit various bioactivities in humans. A liquid chromatography-tandem mass spectrometric method (LC-MS/MS) was developed for the determination of evodiamine and rutecarpine in human serum. The serum was extracted by solid-phase extraction (SPE) and analyzed using a C18 column and a mobile phase consisting of methanol-water (85:15) solution containing 5 mmol/L ammonium formate at a flow rate of 0.5 mL/min. The mass spectrometer was operated in positive mode, employing the extracted ion chromatogram (EIC) for detection and quantitation of evodiamine (m/z 288) and rutecarpine (m/z 304). Good linear relationships between the peak area and the concentration were obtained in the ranges of 5.2-1040 ng/mL and 10.2-1020 ng/mL, with correlation coefficients (r) of 0.999 and 0.998, for evodiamine and rutecarpine, respectively. The repeatabilities (RSD, n=6) of quantitation for evodiamine and rutecarpine were 2.18-4.00% and 2.99-5.67%, respectively, and the recovery ranged from 90.5% to 98.1%. A comparative study of the different ionization and quantitation modes, including ESI-MS, ESI-MS/MS, APCI-MS and APCI-MS/MS, was also accomplished. The MS/MS fragmentation mechanism of the base peak ([M+H](+), m/z 304) of evodiamine was investigated in order to identify the analytes in more complicated body fluid samples.

Pharmacokinetics of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat and dog.

The pharmacokinetics of gefitinib and its metabolites in rat and dog were investigated in preclinical studies conducted to support the safety evaluation and clinical development of gefitinib, the first EGFR tyrosine kinase inhibitor approved for the treatment of non-small-cell lung cancer. Following intravenous dosing (5 mg kg(-1), gefitinib plasma half-life was 3-6h in rats and dogs, although studies using a more sensitive HPLC-MS assay produced longer estimates of half-life (7-14h). In these studies, plasma clearance was high (male rat: 25 ml min(-1) kg(-1); female rat: 16 ml min(-1) kg(-1); male dog: 16 ml min(-1) kg(-1)), as was the volume of distribution (8.0-10.41 kg(-1) in rat; 6.31 kg(-1) in dog), and exposure in female rats was double that in males. Following administration of [14C]-gefitinib, concentrations of radioactivity in plasma exceeded gefitinib throughout the profile, indicating the presence of circulating metabolites in both rat and dog. An HPLC-MS assay was developed to measure concentrations of gefitinib and five potential metabolites in plasma. All five metabolites were detected in the rat, but at levels much lower than gefitinib. In the dog, exposure to gefitinib and M523595 was similar, with much lower concentrations of M537194 and only trace levels of the other metabolites. This profile of metabolites is similar to that observed in man.

Metabolic disposition of gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, in rat, dog and man.

Following oral administration of [14C]-gefitinib to albino and pigmented rats, radioactivity was widely and rapidly distributed, with the highest levels being found in liver, kidney, lung and gastrointestinal tract, but with only low levels penetrating the brain. Levels of radioactivity persisted in melanin-containing tissues (pigmented eye and skin). Binding to plasma proteins was high (86-94%) across the range of species examined and was 91% in human plasma. Substantial binding occurred to both human serum albumin and alpha-1 acid glycoprotein. Following oral and intravenous administration of [14C]-gefitinib, excretion of radioactivity by rat, dog and human occurred predominantly via the bile into faeces, with < 7% of the dose being eliminated in urine. In all three species, gefitinib was cleared primarily by metabolism. In rat, morpholine ring oxidation was the major route of metabolism, leading to the formation of M537194 and M608236 as the main biliary metabolites. Morpholine ring oxidation, together with production of M523595 by O-demethylation of the quinazoline moiety, were the predominant pathways in dog, with oxidative defluorination also occurring to a lesser degree. Pathways in healthy human volunteers were similar to dog, with O-demethylation and morpholine ring oxidation representing the major routes of metabolism.

High-performance liquid chromatographic determination of evodiamine in rat plasma: application to pharmacokinetic studies.

A previously published simple and sensitive high-performance liquid chromatographic method for determination and identification of rutaecarpine in rat plasma was used for evodiamine determination. However, the ultraviolet detection was not 344 nm, but 227 nm. The method was applied to a pharmacokinetic study of evodiamine in rats after 2 mg/kg intravenous administration. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. Compartmental analysis yielded a two-compartment model.