Enantioselective LC-ESI-MS/MS determination of dropropizine enantiomers in rat plasma and application to a pharmacokinetic study.

We developed and validated a simple, sensitive, selective and reliable LC-ESI-MS/MS method for direct quantitation of dropropizine enantiomers namely levodropropizine (LDP) and dextrodropropizine (DDP) in rat plasma without the need for derivatization as per regulatory guidelines. Dropropizine enantiomers and carbamazepine (internal standard) were extracted from 50 µL rat plasma using ethyl acetate. LDP and DDP resolved with good baseline separation (Rs = 4.45) on a Chiralpak IG-3 column. The mobile phase consisted of methanol with 0.05% diethylamine pumped at a flow rate of 0.5 mL/min. Detection and quantitation were done in multiple reaction monitoring mode following the transitions m/z 237 → 160 and 237 → 194 for dropropizine enantiomers and the internal standard, respectively, in the positive ionization mode. The proposed method provided accurate and reproducible results over the linearity range of 3.23-2022 ng/mL for each enantiomer. The intra- and inter-day precisions were in the ranges of 3.38-13.6 and 5.11-13.8 for LDP and 4.19-11.8 and 8.89-10.1 for DDP. Both LDP and DDP were found to be stable under different stability conditions. The method was successfully used in a stereoselective pharmacokinetic study of dropropizine enantiomers in rats following oral administration of racemate dropropizine at 100 mg/kg. The pharmacokinetic results indicate that the disposition of dropropizine enantiomers is not stereoselective and chiral inversion does not occur in rats.

Evaluation of the effects of food on levodropropizine controlled-release tablet and its pharmacokinetic profile in comparison to that of immediate-release tablet.

BACKGROUND: Levodropropizine is a non-opioid antitussive agent that inhibits cough reflex by reducing the release of sensory peptide in the peripheral region. To improve patients' compliance, a controlled-release (CR) tablet is under development. The aim of this study was to compare the pharmacokinetic (PK) profiles of the CR and immediate-release (IR) tablets of levodropropizine. In addition, the effect of food on the PK properties of levodropropizine CR tablet in healthy subjects was evaluated. SUBJECTS AND METHODS: A randomized, open-label, multiple-dose, three-treatment, three-period, six-sequence, crossover study was conducted on 47 healthy subjects. All subjects were randomly assigned to one of the six sequences, which involve combinations of the following three treatments: levodropropizine IR 60 mg three times in the fasted state (R), levodropropizine CR 90 mg two times in the fasted state (T), and levodropropizine CR 90 mg two times in the fed state (TF). Serial blood samples were collected up to 24 h after the first dose. Tolerability was assessed based on the vital signs, adverse events (AEs), and clinical laboratory tests. RESULTS: Levodropropizine CR showed lower maximum drug concentration (Cmax) and similar total exposure compared to levodropropizine IR. The geometric mean ratios (GMRs) (90% confidence intervals [CIs]) of T to R for the Cmax and area under the concentration-time curve from the 0 to 24 h time points (AUC0-24h) were 0.80 (0.75-0.85) and 0.89 (0.86-0.93), respectively. In the fed group, levodropropizine CR showed exposure similar to that in the fasted group. The GMRs (90% CIs) of TF to T for the Cmax and AUC0-24h were 0.90 (0.85-0.97) and 1.10 (1.05-1.14), respectively. No serious AEs occurred with both levodropropizine CR and IR tablets. CONCLUSION: Total systemic exposure for levodropropizine was similar in subjects receiving the CR and IR formulations in terms of the AUC. Although food delayed the absorption of levodropropizine CR, systemic exposure was not affected.

1H nuclear magnetic resonance (NMR)-based serum metabolomics of human gallbladder inflammation.

OBJECTIVE AND DESIGN: We present in this article 1H nuclear magnetic resonance (NMR)-based metabolic approach to screen the serum metabolic alterations in human gallbladder inflammation with chronic cholecystitis (CC). MATERIAL/METHODS: Total of 71 human serum samples was divided into two groups, (n = 41, CC) and (n = 30 control). 1H NMR metabolic profiling was carried out for investigation of metabolic alterations. Multivariate statistical analysis was applied for pattern recognition and identification of metabolites playing crucial role in gallbladder inflammation. Receiver operating curve (ROC) and pathway analysis on NMR data were also carried out to validate the findings. RESULTS: Serum metabolites such as glutamine, low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), alanine, branch chained amino acids (BCAA), histidine and tyrosine were found to be depleted whereas formate, lactate, 1,2-propanediol were found to be elevated in CC. Metabolic pathways associated with metabolite alteration have also been reported. CONCLUSIONS: NMR has been established for disease diagnosis along with identification of metabolic pattern recognition in biofluids. Gallstones cause inflammation of the gallbladder in the form of CC. Inflammation plays a major role in causation of gall bladder cancer and leads the way to malignancy. Metabolic analysis of CC may lead to early diagnosis of disease and its progression to gallbladder cancer.

Analysis of eight glycols in serum using LC-ESI-MS-MS.

A liquid chromatography coupled with electrospray tandem mass spectrometry method was developed for the analysis of ethylene glycol, diethylene glycol, triethylene glycol, 1,4-butanediol, 1,2-butanediol, 2,3-butanediol, 1,2-propanediol and 1,3-propanediol, in serum after a Schotten-Baumann derivatization by benzoyl chloride. Usual validation parameters were tested: linearity, repeatability and intermediate precision, limits of detection and quantification, carry over and ion suppression. Limits of detection were between 0.18 and 1.1 mg/L, and limits of quantification were between 0.4 and 2.3 mg/L. Separation of isomers was possible either chromatographically or by selecting specific multiple reaction monitoring transitions. This method could be a useful tool in case of suspected intoxication with antifreeze agents, solvents, dietary supplements or some medical drug compounds.

Subchronic exposure to ethyl tertiary butyl ether resulting in genetic damage in Aldh2 knockout mice.

Ethyl tertiary butyl ether (ETBE) is biofuel additive recently used in Japan and some other countries. Limited evidence shows that ETBE has low toxicity. Acetaldehyde (AA), however, as one primary metabolite of ETBE, is clearly genotoxic and has been considered to be a potential carcinogen. The aim of this study was to evaluate the effects of ALDH2 gene on ETBE-induced genotoxicity and metabolism of its metabolites after inhalation exposure to ETBE. A group of wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice were exposed to 500ppm ETBE for 1-6h, and the blood concentrations of ETBE metabolites, including AA, tert-butyl alcohol and 2-methyl-1,2-propanediol, were measured. Another group of mice of WT and KO were exposed to 0, 500, 1750, or 5000ppm ETBE for 6h/day with 5 days per weeks for 13 weeks. Genotoxic effects of ETBE in these mice were measured by the alkaline comet assay, 8-hydroxyguanine DNA-glycosylase modified comet assay and micronucleus test. With short-term exposure to ETBE, the blood concentrations of all the three metabolites in KO mice were significantly higher than the corresponding concentrations of those in WT mice of both sexes. After subchronic exposure to ETBE, there was significant increase in DNA damage in a dose-dependent manner in KO male mice, while only 5000ppm exposure significantly increased DNA damage in male WT mice. Overall, there was a significant sex difference in genetic damage in both genetic types of mice. These results showed that ALDH2 is involved in the detoxification of ETBE and lack of enzyme activity may greatly increase the sensitivity to the genotoxic effects of ETBE, and male mice were more sensitive than females.

Relative bioavailability of levodropropizine 60 mg capsule and syrup formulations in healthy male Korean volunteers: a singledose, randomized-sequence, open-label, two-way crossover study.

BACKGROUND: Levodropropizine is an oral non-opioid anti-tussive drug used in treatment of cough. A new generic 60 mg capsule formulation of levodropropizine has recently been developed. OBJECTIVES: The aim of this study was to assess the pharmacokinetics and bioequivalence of the test (capsule) formulation and reference (syrup) formulation of levodropropizine (60 mg) in healthy, fasted, male Korean volunteers. METHODS: This was a single-dose, randomized sequence, open-label, 2-period crossover study conducted in healthy male Korean volunteers in the fasted state at Kyung Hee University Medical Center (Seoul, Republic of Korea). A single oral dose of the test or reference formulation was followed by a 1-week washout period, after which subjects received the alternative formulation. Blood samples were collected at 0 (predose), 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 hours after study drug administration. Plasma concentration of levodropropizine was determined using a validated liquid chromatography tandem mass spectrometry (LCMS/ MS) method. The formulations were considered bioequivalent if the 90% CIs for C(max), AUC(0-12h) and AUC(0-∞) were within the predetermined bioequivalence range (80 - 125%, according to the guidelines of the Korea Food and Drug Administration (Korea FDA)). Tolerability was evaluated throughout the study based on vital sign measurements, laboratory analysis (blood biochemistry, hematology, hepatic function and urinalysis) and subject interviews concerning adverse events (AEs). RESULTS: A total of 36 male Korean subjects (mean (SD) age, 23.9 (2.4) years (range 19 - 30 years); height, 176.2 (6.1) cm (range 161 - 190 cm); weight, 69.8 (9.1) kg (range 54.0 - 92.2 kg); body mass index, 22.4 (2.1) kg/m2 (range 19.1 - 28.3 kg/m2)) was enrolled and completed the study. The mean values for C(max), t(max), AUC(0-12h), and AUC(0-∞) with the test formulation of levodropropizine were 331.51 ng/ml, 0.60 hours, 784.32 ng×h/ml, and 825.82 ng×h/ml, respectively; for the reference formulation, the values were 332.81 ng/ml, 0.44 hours, 726.46 ng×h/ml, and 769.46 ng×h/ ml, respectively. The 90% CIs for the logtransformed ratios of C(max) (92.74 - 111.24), AUC(0-12h) (104.31 - 113.67) and AUC(0-∞) (103.87 - 113.57) were within the predetermined range for the assumption of bioequivalence. No serious adverse events were reported. CONCLUSIONS: This single-dose (60 mg) study found that the test (capsule) and reference (syrup) formulations of levodropropizine met the regulatory criterion for assuming bioequivalence in these healthy, fasted, male Korean subjects. Both formulations were well tolerated in the population studied. Korea FDA registration number: BED-1784.

LC-MS/MS determination of FTY720 and FTY720-phosphate in murine intracellular compartments and human plasma.

A new analytical method for the quantitation of the orally active immunomodulatory drug FTY720 and its phosphate derivative in human plasma and murine subcellular compartments has been developed. The samples undergo a liquid-liquid extraction process before they are injected into a liquid chromatographic system coupled to a tandem mass spectrometer operating in positive ion mode. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the analytical process is around 80% for all analytes. Intra- and interday precision and accuracy, as relative standard deviation and relative error, respectively, are lower than 12.5% in all cases. No important matrix effects were observed. The lower limits of quantitation for the analysed substances were 0.875 ng/mL and 2 ng/mL for FTY720 and FTY720-phosphate, respectively. Since no deuterated derivatives of the analytes were commercially available, the developed method was applied for quantifying the studied compounds using C17-sphingosine and C-17-sphingosine-1-phosphate as internal standards, in subcellular compartments of murine splenocytes. This method could be applied in the future for monitoring purposes in multiple sclerosis patients, since FTY720 has been approved by the American Food and Drug Administration and the European Medicines Agency for the pharmacological treatment of this disease.

Pharmacodynamic effects of steady-state fingolimod on antibody response in healthy volunteers: a 4-week, randomized, placebo-controlled, parallel-group, multiple-dose study.

Fingolimod, a first-in-class oral sphingosine 1-phosphate receptor (S1PR) modulator, is approved in many countries for relapsing-remitting multiple sclerosis, at a once-daily 0.5-mg dose. A reduction in peripheral lymphocyte count is an expected consequence of the fingolimod mechanism of S1PR modulation. The authors investigated if this pharmacodynamic effect impacts humoral and cellular immunogenicity. In this double-blind, parallel-group, 4-week study, 72 healthy volunteers were randomized to steady state, fingolimod 0.5 mg, 1.25 mg, or to placebo. The authors compared T-cell dependent and independent responses to the neoantigens, keyhole limpet hemocyanin (KLH), and pneumococcal polysaccharides vaccine (PPV-23), respectively, and additionally recall antigen response (tetanus toxoid [TT]) and delayed-type hypersensitivity (DTH) to KLH, TT, and Candida albicans. Fingolimod caused mild to moderate decreases in anti-KLH and anti-PPV-23 IgG and IgM levels versus placebo. Responder rates were identical between placebo and 0.5-mg groups for anti-KLH IgG (both > 90%) and comparable for anti-PPV-23 IgG (55% and 41%, respectively). Fingolimod did not affect anti-TT immunogenicity, and DTH response did not differ between placebo and fingolimod 0.5-mg groups. Expectedly, lymphocyte count reduced substantially in the fingolimod groups versus placebo but reversed by study end. Fingolimod was well tolerated, and the observed safety profile was consistent with previous reports.

Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with liquid chromatography-tandem mass spectrometry.

Fingolimod (Gilenya; FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In the present study, we have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify FTY720 and FTY720-P in human blood. The sample preparation involves the sample dilution with a solution made of dimethylhexylamine (DMHA), ortho-phosphoric acid and methanol prior to the on-line solid phase extraction (SPE) on a C(18) cartridge. The samples were then eluted on a C(18) column with a gradient elution of DMHA solution and acetonitrile and analyzed by LC-MS/MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.5 min. Standard curves were linear over the ranges of 0.0800 ng/mL (LLOQ) to 16.0 ng/mL for FTY720 and 0.100 ng/mL (LLOQ) to 20.0 ng/mL for FTY720-P with correlation coefficient (r(2)) greater than 0.997. The method selectivities for FTY720 and FTY720-P were demonstrated in six different batches of human blood. Intra-run and inter-run precision and accuracy within ± 20% (at the LLOQ) and ± 15% (other levels) were achieved during a 3-run validation for quality control samples (QCs). In addition, stability data obtained during freeze-thaw (3 cycles), at room temperature (24h), and in an auto-sampler were determined and reported. The method robustness was demonstrated by the consistent data obtained by reanalyzing human blood samples for several clinical studies. In addition comparative data for FTY720 and FTY720-P were obtained between our current method and those of two available separate LC-MS/MS assays. The results of the present work demonstrated that our bioanalytical LC-MS/MS method is rapid, sensitive, specific and reliable for the simultaneous quantitative analysis of FTY720 and FTY720-P in human blood.

Absorption and disposition of the sphingosine 1-phosphate receptor modulator fingolimod (FTY720) in healthy volunteers: a case of xenobiotic biotransformation following endogenous metabolic pathways.

Fingolimod [(FTY720), Gilenya; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol], a new drug for the treatment of relapsing multiple sclerosis, acts through its phosphate metabolite, which modulates sphingosine 1-phosphate receptors. This represents a novel mechanism of action. In the present work, the absorption and disposition of (14)C-labeled fingolimod were investigated in healthy male volunteers after a single oral dose of 4.5 mg. Total radioactivity was determined in blood, urine, and feces. Fingolimod was quantified in blood. Metabolite profiles were determined in blood and excreta, and metabolite structures were elucidated by mass spectrometry, wet-chemical methods, and comparison with reference compounds. Fingolimod was absorbed slowly but almost completely. The biotransformation of fingolimod involved three main pathways: 1) reversible phosphorylation to fingolimod phosphate [(S)-enantiomer, active principle]; 2) ω-hydroxylation at the octyl chain, catalyzed predominantly by CYP4F enzymes, followed by further oxidation to a carboxylic acid and subsequent ß-oxidation; and 3) formation of ceramide analogs by conjugation with endogenous fatty acids. This metabolism is quite unusual because it follows metabolic pathways of structurally related endogenous compounds rather than biotransformations typical for xenobiotics. The elimination of fingolimod was slow and occurred predominantly by oxidative metabolism whereas fingolimod phosphate was eliminated mainly by dephosphorylation back to fingolimod. Drug-related material was excreted mostly in the urine in the form of oxidation products.

Rapid and sensitive LC-MS/MS method for determination of felbamate in mouse plasma and tissues and human plasma.

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a second generation antiepileptic drug used to treat seizures refractory to other antiepileptic drugs. With approximately 3500 new patients exposed annually, several important pharmacologic interaction questions remain unanswered necessitating the need for rapid and accurate methods of felbamate analysis in biological matrices. To this end, a rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the measurement of felbamate in mouse plasma and tissues and human plasma. Plasma (100 µL) and tissues homogenates (100 µL of 100 mg/mL) were spiked with internal standard (carisoprodol) prior to protein precipitation with acetonitrile. Samples were chromatographed on a XBridge Phenyl, 2.5 µm, 4.6 mm×50 mm column with quantitation by internal standard reference monitoring of the ion transitions m/z 239→117 for felbamate and m/z 261→176 for carisoprodol. Calibration curves were linear from 2.5 to 500 ng/mL in mouse or human plasma and 25-5000 pg/mg in tissue homogenates. Recoveries were greater than 97% for plasma and homogenates with accuracies >92% in any of the mouse matrices and >88% in human plasma. Comparable accuracies and precision were found with and without the use of the internal standard in preparation of the calibration curves and suggest that the internal standard may not be required.

Simultaneous HPLC-UV analysis of rufinamide, zonisamide, lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy.

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 microL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 microm Hydro-RP, 250 mm x 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2-40 microg/mL for RFN and 2-80 microg/mL for ZNS. The limit of quantitation was 2 microg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).

Drug monitoring and toxicology: a simple procedure for the monitoring of felbamate by HPLC-UV detection.

This article describes a simple isocratic high-performance liquid chromatographic (HPLC) method with UV detection for the determination of felbamate in the serum of patients with epilepsy. Sample preparation requires only protein precipitation with a single-step methanol extraction. After centrifugation, the resulting supernatant was injected directly onto the HPLC system. Separation was achieved by reversed-phase HPLC, using a 5-microm Microsorb-MV C(18) column (250 x 4.6 mm) connected to a Silica C(18) guard column (20 x 4.6 mm) and a mobile phase consisting of a mixture of phosphate buffer (pH = 6.9, 0.05 M), methanol, and acetonitrile (64:18:18, v/v/v). The flow rate was at 1.0 mL/min and column temperature was set at 35 degrees C. Quantitation was performed by measurement of the UV absorbance at a wavelength of 210 nm. Calibration curves were linear over a range of 2-200 mg/L, which covered the proposed range of 50-150 mg/L for reference. Both within-run and between-run precision were lower than 5%. Recoveries ranged between 97% and 105% for spiked and pooled samples. No interferences with other common antiepileptic drugs (except zonisamide) were observed. The method requires only 100 microL of serum or less. It is simple and fast (sample preparation and analysis time) and suitable for routine clinical use.

The immunomodulator FTY720 is phosphorylated and released from platelets.

The novel immunomodulator FTY720 causes lymphocytes from peripheral blood to accumulate in lymphoid tissues. In vivo, FTY720 is phosphorylated to FTY720-P, which binds to the sphingosine 1-phosphate receptor S1P(1). So far, it has been unclear where FTY720-P is produced. We demonstrate that platelets efficiently convert FTY720 to FTY720-P and release it into the extracellular space. This release is mostly independent of platelet activation, but is slightly increased upon thrombin stimulation. These results suggest that platelets are a major source of plasma FTY720-P, and that FTY720-P release is mediated by two different transporters.

Ethnic sensitivity study of fingolimod in white and Asian subjects.

OBJECTIVE: The authors compared the pharmacokinetics and pharmacological effects of the immunomodulator fingolimod in healthy white and Asian subjects for potential ethnic differences. METHODS: White and Asian (Japanese) healthy subjects were demographically matched for sex, age and weight. Subjects received single 1.25 mg doses of fingolimod (6 ethnic pairs), 2.5 mg (7 pairs), 5 mg (6 pairs) or 5 mg/day for 7 days (6 pairs). The pharmacokinetics of fingolimod, major metabolites, peripheral blood lymphocyte counts and heart rate were characterized over 1 month after single-dose and 2 months after multiple-dose administration. RESULTS: There were no clinically relevant differences in the fingolimod dose Cmax or dose AUC relationships between Asian subjects (slopes 0.84 and 1.05) versus white subjects (slopes 1.13 and 1.26) after single-dose administration. During multiple-dose administration, there were no clinically relevant interethnic differences in fingolimod accumulation ratios (6.6 +/- 0.4 for whites, 7.0 +/- 0.7 for Asians), area under the concentration-time curve (390 +/- 73 versus 382 +/- 106 ng x h/ml), or elimination half-life (7.4 +/- 0.8 versus 7.9 +/- 2.0 days). The acute decrease in lymphocyte counts after single- and multiple-dose fingolimod were similar in the two ethnic groups. The lymphocyte recovery rate to baseline after a 5 mg single dose and 5 mg/day multiple dose was reduced by 36 and 15% in Asian subjects compared with white subjects. The transient, acute decrease in heart rate after the first dose of fingolimod and the subsequent return to baseline was similar in the two ethnic groups. CONCLUSION: There were no marked differences between healthy white and Asian subjects in fingolimod single-dose and multiple-dose pharmacokinetics, lymphocyte trafficking and heart rate responses.

Oral pharmacokinetic and pharmacodynamic effects of FTY720 in cats.

The aim of the study was to determine pharmacokinetic and pharmacodynamic profiles of FTY720 in cats and identify any toxic side effects. Six adult cats were used for the experimental study. Single oral dosages were tested at 0.05, 0.3 and 1.0 mg/kg. Whole blood drug concentration, total white blood cell and differential counts were monitored. Flow cytometry evaluated the effects on lymphocyte subsets. A toxicity study consisted of cats receiving a dose of 0.15 mg/kg daily for 30 days. Daily observation, physical examination and bloodwork were evaluated to assess for toxicity. All single doses resulted in > or =80% reduction in circulating lymphocytes within 12 h after administration, with the duration of lymphopenia being dose dependent. CD4+ and CD5+ T cells were specifically depleted. Peripheral neutrophils declined by approximately 70% at all dosages tested. No other toxic side effects were observed. Results of this study suggest that FTY720 is effective at inducing a peripheral lymphopenia in cats without any toxic side effects. Currently, cats appear to be the only species in which FTY720 induces a neutropenia. This study provides the foundation for future clinical transplantation trials using FTY720 in cats. By using combination therapy of FTY720 and low dose cyclosporine, the incidence of serious side effects may be reduced while still preventing allograft rejection.

HPLC determination and steady-state bioavailability study of levodropropizine sustained-release tablets in dogs.

A simple HPLC method using UV detection was developed and validated for the determination of levodropropizine (LDP) in dog plasma. The sample was prepared for injection using a liquid-liquid extraction method with 1-phenypiperazine as the internal standard. The mobile phase was methanol-diethylamine solution (0.05 M) (20:80, v/v, pH adjusted to 3.0 with H3PO4) with a detection wavelength of 240 nm. The limit of quantitation (LOQ) of LDP in a biological matrix was determined to be 25.25 ng/mL. The calibration curve was linear across the concentration range of 25.25 to 2020 ng/mL. The intra-day and inter-day precision values (CV %) were within 7% and accuracy (R.E. %) was within 6% of the nominal values for medium (252.5 ng/mL) and high (2020 ng/mL) LDP concentrations. For the LDP concentration at the LOQ, the intra-day and inter-day precision and accuracy were within 20% and 10%, respectively. The average absolute recovery for LDP was 70.28%. This method was successfully used to analyze plasma samples in a steady-state bioavailability study of a newly developed sustained-release LDP tablets (SR) using immediate-release tablets (IR) as the reference. The relative bioavailability of the SR was determined to be 106.3 +/- 12.8% (n=6). The Cmax of the SR was significantly lower (P<0.05), and the tmax was significantly longer than that of the IR (P<0.05). The results of ANOVA and two one-sided tests indicated that the SR exhibited acceptable sustained release properties and was bioequivalent to the IR.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3-->255.3). The assay was linear from 0.2 to 25 microg/l (r(2)>0.997, n=5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 microg/l) were 95.8-103.2 and <5.5%, respectively. At the lower limit of quantification (0.2 microg/l) the inter- and intra-day analytical recovery was 99.0-102.8% with imprecision of <7.6% (n=5). The assay had a mean relative recovery of 100.5+/-5.8% (n=15). Extracted samples were stable for 16 h. FTY720 quality control samples were stable at room temperature for 16 h, at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans.

Physiologically based pharmacokinetic modeling of FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) in rats after oral and intravenous doses.

FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-1,3-diol hydrochloride) is a new sphingosine-1-phosphate receptor agonist being developed for multiple sclerosis and prevention of solid organ transplant rejection. A physiologically based pharmacokinetic model was developed to predict the concentration of FTY720 in various organs of the body. Single oral and intravenous doses of FTY720 were administered to male Wistar rats, with blood and tissue sampling over 360 h analyzed by liquid chromatography/tandem mass spectrometry. A well stirred model (perfusion rate-limited) described FTY720 kinetics in heart, lungs, spleen, muscle, kidneys, bone, and liver, with a permeability rate-limited model being required for brain, thymus, and lymph nodes. Tissue-to-blood partition coefficients (RT) ranged from 4.69 (muscle) to 41.4 (lungs). In lymph nodes and spleen, major sites for FTY720-induced changes in sequestration of lymphocytes, RT values were 22.9 and 34.7, respectively. Permeability-surface area products for brain, thymus, and lymph nodes were 39.3, 122, and 176 ml/min. Intrinsic hepatic clearance was 23,145 l/h/kg for the free drug in blood (f(ub) 0.000333); systemic clearance was 0.748 l/h/kg and terminal half-life was 23.4 h. The fraction orally absorbed was 71%. The model characterized well FTY720 disposition for this extensive dosing and tissue collection study in the rat. On scaling the model to dogs and humans, good agreement was found between the actual and predicted blood concentration-time profiles. More importantly, brain concentrations in dogs were well predicted from those of the rat. In absolute terms, the predictions were slightly lower than observed values, just under a 1.5-fold deviation, but the model accurately predicted the terminal elimination of FTY720 from the brain.

Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in plasma of patients with epilepsy.

A very simple and fast method has been developed and validated for simultaneous determination of the new generation antiepileptic drugs (AEDs) lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine and felbamate in plasma of patients with epilepsy using high-performance liquid chromatography (HPLC) with spectrophotometric detection. Plasma sample (500 microL) pre-treatment was based on simple deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi 4 microm Hydro-RP, 150 mm x 4 mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm. Calibration curves were linear (mean correlation coefficient >0.999 for all the three analytes) over a range of 1-20 mg/mL for lamotrigine, 2-40 microg/mL for monohydroxycarbamazepine and 10-120 microg/mL for felbamate. Both intra and interassay precision and accuracy were lower than 7.5% for all three analytes. Absolute recoveries ranged between 100 and 104%. The present procedure describes for the first time the simultaneous determination of these three new antiepileptic drugs. The simple sample pre-treatment, combined with the fast chromatographic run permit rapid processing of a large series of patient samples.