Residue study of doxycycline and 4-epidoxycycline in pigs medicated via-drinking water.

A study was performed to determine the residues in edible tissues of healthy pigs after continuous administration of doxycycline with drinking water for five consecutive days at a dose rate of 10.5 mg doxycycline kg-1 body weight (BW) per day. Quantitation was performed using a validated HPLC method with fluorescence detection. The method was able to separate doxycycline and its 4-epimer, 4-epidoxycycline. This epimer was found in kidney, liver, skin, fat and muscle tissue. The method was validated at the maximum residue limit (MRL), at half the MRL and at double the MRL for both doxycycline and 4-epidoxycycline. Linear calibration curves were obtained in spiked tissues (r > 0.99). The accuracy of the calibrators of the calibration curves was within -20% to +10%. The accuracy and precision (expressed as the within-run repeatability) were found to be within the required ranges for the specific concentration. The limits of detection and limits of quantification were below one-half of the MRL. The quantification limits were 50 micrograms kg-1 for doxycycline and 100 micrograms kg-1 for 4-epidoxycycline in kidney and liver, 20 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in skin and fat and 10 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in muscle tissue. The withdrawal time was calculated according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95) and was set at 3 days. The plasma concentration of doxycycline and the stability of doxycycline in drinking water were also determined during the treatment period. The mean plasma concentration of doxycycline during the treatment period ranged from 0.83 to 0.96 microgram ml-1. Thirty-six hours after the withdrawal from medicated drinking water, no plasma levels could be detected. Samples of medicated water were taken at time zero and at 24 h after addition of doxycycline to the drinking water. No statistically significant difference in the mean drinking water concentration was seen at time zero and at time 24 h (Student's t-test, alpha = 0.05).     

The assay of a novel histamine H2-receptor antagonist, SK&F 93479, in human plasma by normal-phase high-performance liquid chromatography

A selective assay of a new histamine H2-receptor antagonist, SK&F 93479, in human plasma has been developed. The method uses liquid-liquid extraction from the biological sample and analysis of the resulting extract by normal-phase high-performance liquid chromatography with UV detection for quantitation of the drug and an added standard. The assay is sufficiently accurate and precise to determine the compound at concentrations as low as 0.025 mg 1(-1). The coefficient of variation of the assay averages 5.7% at concentrations between 0.1 and 2.0 mg 1(-1), but increases to 21.8% at 0.02 mg 1(-1). SK&F 93479 can be determined in spiked plasma samples, at concentrations between 0.05 and 0.80 mg 1(-1) with a bias of between -7.5 and +3.6%, but at 0.02 mg 1(-1) concentrations were underestimated by 15% on average. The assay has been used for pharmacokinetic and bioavailability studies: after a single 0.5 mg kg-1 oral dose in man, plasma concentrations can be monitored for up to 70 h after dosing.     

Determination of dextromoramide in plasma and whole blood using high-performance liquid chromatography with ultraviolet absorbance detection.

Dextromoramide was determined in plasma and whole blood after solid-phase isolation by high-performance liquid chromatography using dextropropoxyphene as internal standard and ultraviolet detection at 215 nm. Owing to its good selectivity, sensitivity and reproducibility, the technique is available for forensic toxicology purposes as well as for clinical pharmacology. The concentrations of dextromoramide were determined in three cancer patients receiving intravenous treatment with one to three 5-mg daily doses. On the fourth day the plasma level was 13.85 +/- 3.27 ng ml-1 just before the first daily dose and 84.28 +/- 12.60 ng ml-1 30 min after dosing. The whole blood concentration, determined in one of the patients, was undetectable just before the dose and was 76 ng ml-1 30 min after dosing.     

Pharmacokinetics of Tetra (m-hydroxyphenyl)chlorin in Human Plasma and Individualized Light Dosimetry in Photodynamic Therapy

The pharmacokinetics of the photosensitizer 5,10,15,20-tetra( m -hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re-action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug-light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light-induced fluorescence spectroscopy using an optical fiber-based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.     

Pharmacokinetics of brain natriuretic peptide in rats.

The pharmacokinetics of rat brain natriuretic peptide (rBNP) was compared with that of alpha-rat atrial natriuretic peptide (alpha-rANP) in rats. After intravenous infusion in rats (600 pmol min-1kg-1 for 2 min), the disappearance of plasma rBNP was 4-fold slower than that of alpha-rANP. The estimated mean plasma clearance rates for rBNP and alpha-rANP were 45.9 ml min-1kg-1 and 74.4 ml min-1kg-1, respectively. The affinity of rBNP for the clearance receptor or degradation enzyme was considered to be lower than that of alpha-rANP.     

Ultra performance liquid chromatography coupled with electrospray and atmospheric pressure chemical ionization (ESCi)-quadrupole time-of-flight mass spectrometry with novel mass spectrometry(Elevated Energy) (MS(E)) data collection technique: determination and pharmacokinetics, tissue distribution and biliary excretion study of ergone in rat

Ergosta-4,6,8(14),22-tetraen-3-one (ergone) has been proved to have novel antitumor effects on HepG2 cells. The aim of this study was to investigate the pharmacokinetics, tissue distribution, and biliary excretion of ergone in rats following a single oral administration (5, 10, and 20 mg/kg). The levels of ergone in plasma, tissues, and bile were measured by ultra performance liquid chromatography coupled with electrospray and atmospheric pressure chemical ionization (ESCi)-quadrupole time-of-flight mass spectrometry with novel mass spectrometry(Elevated Energy) (MS(E)) data collection technique method. The results show ergone was distributed and eliminated from rat plasma and in non-linear pharmacokinetics from a dose range of 5-20 mg/kg. The ergone was found to distribute widely in the internal organs, with tissue concentrations in order of lungs, spleen, liver, intestine, kidneys, heart, stomach, parorchis, teasticles, and brain. At 12 h after dosing, the tissue concentrations in the organs were markedly decreased. The lungs, spleen, and liver were the dominant organs with high tissue concentrations that might be the primary sites for metabolism and elimination of ergone. Total recoveries of ergone within 24 h in bile were 34.14%.     

Development,Validation, and Comparison of Two Mass Spectrometry Methods (LC-MS/HRMS and LC-MS/MS) for the Quantification of Rituximab in Human Plasma

Rituximab is a chimeric immunoglobulin G1-kappa (IgG1κ) antibody targeting the CD20 antigen on B-lymphocytes. Its applications are various, such as for the treatment of chronic lymphoid leukemia or non-Hodgkin’s lymphoma in oncology, and it can also be used in the treatment of certain autoimmune diseases. Several studies support the interest in therapeutic drug monitoring to optimize dosing regimens of rituximab. Thus, two different laboratories have developed accurate and reproductive methods to quantify rituximab in human plasma: one using liquid chromatography quadripolar tandem mass spectrometer (LC-MS/MS) and the other, liquid chromatography orbitrap tandem mass spectrometer (LC-MS/HRMS). For both assays, quantification was based on albumin depletion or IgG-immunocapture, surrogate peptide analysis, and full-length stable isotope-labeled rituximab. With LC-MS/MS, the concentration range was from 5 to 500 µg/mL, the within- and between-run precisions were <8.5%, and the limit of quantitation was 5 µg/mL. With LC-MS/HRMS, the concentration range was from 10 to 200 µg/mL, the within- and between-run accuracy were <11.5%, and the limit of quantitation was 2 µg/mL. Rituximab plasma concentrations from 63 patients treated for vasculitis were compared. Bland–Altman analysis and Passing–Bablok regression showed the interchangeability between these two methods. Overall, these methods were robust and reliable and could be applied to routine clinical samples.     

Pharmacokinetic Study of ZS-1, a Targeted Peptide to NCI-H1299, in Rats Following Intravenous Administration

To investigate the pharmacokinetics of ZS-1 following intravenous injection in rats, ZS-1 was administered at doses of 20, 30 and 45 mg kg^?1, respectively. Blood samples were collected at 0.5, 3, 8, 12, 15, 20, 30, 40 and 45 min. ZS-1 in rat plasma was measured by LC. The limit of detection (LOD) was 0.02 μg mL^?1. The relative standard deviation (RSD) of intra- and inter-day precisions were <10%, and the accuracy of intra- and inter-day were >94%. The mean extraction recovery of ZS-1 was 86.1%. After intravenous injection at doses of 20, 30 and 45 mg kg^?1, the concentration–time curves of ZS-1 fitted well to one compartment model. Area under the concentration–time curves (AUC) increased with dose. Clearance rates (CL) and elimination half-lives (T _1/2) had no significant difference between different dose groups (P > 0.05). ZS-1 was stable in plasma after at 25 °C for 2, 4, 6 h, after three freeze–thaw cycles, after ?20 °C for a month, and after ?80 °C for 3 months. The accuracy of ZS-1 was between 96.8 and 106.9%. The results indicated there was no significant degradation. These data indicated that the method for analysis of ZS-1 was reliable and the pharmacokinetic data could guide dosing regimens to be tested in future clinical pharmacokinetic study.     

Quantification and validation of HPLC‐UV and LC‐MS assays for therapeutic drug monitoring of ertapenem in human plasma

Rapid and simple HPLC‐UV and LC‐MS methods were developed and validated for the quantification of ertapenem (Invanz?) in human plasma. Ertapenem is a unique drug in that current dosing recommendations call for a 1 g dose for normal renal function patients, despite body weight. These assays, which involve a protein precipitation followed by liquid–liquid extraction, allow for fast therapeutic drug monitoring of ertapenem in patients, which is especially useful in special populations. Both methods were sufficient to baseline resolve meropenem (internal standard) and ertapenem, and were validated over 3 days using a six‐point calibration curve (0.5–50 µg/mL). Validation was collected using four different points on the calibrations curve yielding acceptable precision (<15% inter‐day and intra‐day; <20% for lower limit of quantitation, LLOQ) as well as accuracy (<15% inter‐day and intra‐day; <20% for LLOQ). The lower limit of detection (LOD) was determined to be 0.1 and 0.05 µg/mL for the HPLC‐UV and LC‐MS methods, respectively. The developed HPLC‐UV and LC‐MS methods for ertapenem quantification are fast, accurate and reproducible over the calibration range and can be used to determine ertapenem plasma concentrations for monitoring clinical efficacy. Copyright © 2012 John Wiley & Sons, Ltd.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

反相离子对高效液相色谱法测定动物血浆中的恩诺沙星及其代谢物

建立了用反相离子对ＨＰＬＣ测定动物血浆中恩诺沙星（ＥＲＦＸ）及其代谢产物环丙沙星（ＣＰＦＸ）浓度的方法。血浆中药物用二氯甲烷萃取,选用ＯＤＳ柱,甲醇－四丁基氢氧化铵溶液为流动相,吡哌酸（ＰＰＡ）作内标,检测波长２７２ｎｍ。方法简便、快速、灵敏、准确,适用于ＥＲＦＸ及其代谢物ＣＰＦＸ血药浓度的测定和药代动力学研究。并首次测定了黄牛血浆中的ＥＲＦＸ和ＣＰＦＸ。     

Amantadine hydrochloride monitoring by dried plasma spot technique: High‐performance liquid chromatography–tandem mass spectrometry based clinical assay

Amantadine plasma concentrations correlate well with desired therapeutic effects and adverse outcomes; information on amantadine exposure could be useful when multiple amantadine clearance pathways are impaired or non‐compliance is suspected. Micro‐sampling strategies, like dried plasma spot, would be particularly useful because ambulatory patients that do not attend a clinic can easily sample a few drops of blood by themselves at the required time of the dosing interval. We developed and validated a dried‐plasma‐spot‐based high performance liquid chromatography–tandem mass spectrometry assay to quantify amantadine. This assay met relevant validation requirements within a hematocrit range of 20–50% and was linear from 100 to 2000 ng/mL. Amantadine was stable in dried plasma spots for up to 21 days at room temperature, regardless of whether the dried plasma spot was protected from light or not. The correlation between paired dried and wet plasma concentrations was assessed in 52 patients. Deming regression coefficients between wet plasma and simultaneously pipetted dried plasma spots were used to predict plasma concentrations. Bland–Altman plots revealed a strong agreement between dried and wet plasma concentrations, supporting the clinical usefulness of dried plasma spots for amantadine monitoring with a self‐sampling strategy at a convenient time and place for the patient.     

Pharmacokinetics of a new sustained-release formulation of theophylline sodium glycerinate in healthy subjects with a new asymmetric dosage regimen

Pharmacokinetics of a new sustained-release formulation of theophylline sodium glycerinate in healthy subjects was studied. In this study, a new asymmetric dosage regimen was presented to achieve a better accordance with the chronotherapy of asthma. Each of 10 subjects was administered one tablet (equivalent to 0.1 g anhydrous theophylline) in the morning and four tablets in the evening for a consecutive 6 days and blood samples were collected at the predetermined time and analyzed by a validated HPLC method. This new regimen produced a steady and effective level of theophylline in plasma for the whole day, especially in the evening. A lower dose in the morning could reach the effective level (C(min)4.97 +/- 1.60 microg/mL and C(max)10.68 +/- 1.80 micro g/mL over the a.m. dosing interval) and a higher dose in the evening did not result in toxic levels but led to a reasonable concentration range (C(max)9.72 +/- 1.56 microg/mL over p.m. dosing interval), which could maintain a higher plasma theophylline concentration without the risk of serious adverse events and control asthmatic symptoms probably occurring during the night or early in the morning. The results suggested that the proposed asymmetric regimen was necessary, practicable and safe for twice daily sustained-release tablets of theophylline sodium glycerinate and also provides the basis for the clinical dosage regimen of other theophylline formulated products.     

Anti-fibrosis attributes: UHPLC-MS/MS-based pharmacokinetics profiling of a novel autotaxin inhibitor with excellent in vivo efficacy in rat

3,4-Difluorobenzyl(1-ethyl-5-(4-((4-hydroxypiperidin-1-yl)-methyl)thiazol-2-yl)-1H-indol-3-yl)carbamate (NAI59), a small molecule with outstanding therapeutic effectiveness to anti-pulmonary fibrosis, was developed as an autotaxin inhibitor candidate compound. To evaluate the pharmacokinetics and plasma protein binding of NAI59, a UPLC–MS/MS method was developed to quantify NAI59 in plasma and phosphate-buffered saline. The calibration curve linearity ranged from 9.95 to 1990.00 ng/mL in plasma. The accuracy was −6.8 to 5.9%, and the intra- and inter-day precision was within 15%. The matrix effect and recovery, as well as dilution integrity, were within the criteria. The chromatographic and mass spectrometric conditions were also feasible to determine phosphate-buffered saline samples, and it has been proved that this method exhibits good precision and accuracy in the range of 9.95–497.50 ng/mL in phosphate-buffered saline. This study is the first to determine the pharmacokinetics, absolute bioavailability, and plasma protein binding of NAI59 in rats using this established method. Therefore, the pharmacokinetic profiles of NAI59 showed a dose-dependent relationship after oral administration, and the absolute bioavailability in rats was 6.3%. In addition, the results of protein binding showed that the combining capacity of NAI59 with plasma protein attained 90% and increased with the increase in drug concentration.     

The effect of β-carotene supplementation on the pharmacokinetics of nelfinavir and its active metabolite M8 in HIV-1-infected patients

β-Carotene supplements are often taken by individuals living with HIV-1. Contradictory results from in vitro studies suggest that β-carotene may inhibit or induce cytochrome P450 enzymes and transporters. The study objective was to investigate the effect of β-carotene on the steady-state pharmacokinetics of nelfinavir and its active metabolite M8 in HIV-1 infected individuals. Twelve hour nelfinavir pharmacokinetic analysis was conducted at baseline and after 28 days of β-carotene supplementation (25,000 IU twice daily). Nelfinavir and M8 concentrations were measured with validated assays. Non-compartmental methods were used to calculate the pharmacokinetic parameters. Geometric mean ratios comparing day 28 to day 1 area under the plasma concentration-time curve (AUC(0-12 h)), maximum (C(max)) and minimum (C(min)) concentrations of nelfinavir and M8 are presented with 90% confidence intervals. Eleven subjects completed the study and were included in the analysis. There were no significant differences in nelfinavir AUC(0-12 h) and C(min) (-10%, +4%) after β-carotene supplementation. The M8 C(min) was increased by 31% while the M8 AUC(0-12 h) and C(max) were unchanged. During the 28 day period, mean CD4+ % and CD4+:CD8+ ratio increased significantly (p < 0.01). β-carotene supplementation increased serum carotene levels but did not cause any clinically significant difference in the nelfinavir and M8 exposure.     

Pharmacokinetics of clenbuterol in the ostrich.

The aim of this study was to investigate the pharmacokinetics of clenbuterol in the ostrich as no such data is available. Clenbuterol (2 mg) was given as a single oral dose to nine ostriches. Blood samples were collected over a period of 96 h after administration and urine for a period of 5 d. Plasma and urine samples were frozen at -20 degrees C pending analysis. Clenbuterol was quantified using a gas chromatograph-mass selective detector. The method for quantification of clenbuterol in plasma was validated by analysing spiked quality control samples at different concentrations. The limit of quantification was determined to be 0.75 ng ml-1 with an absolute recovery of more than 80%. The geometric mean maximum plasma clenbuterol concentration was 4.40 ng ml-1 with 3.0 h as the median time for maximum concentration. The plasma elimination half-life was 19.7 h. The clenbuterol concentration was above 0.75 ng ml-1 in plasma for 48 h and above 1.0 ng ml-1 in urine for 5 d. These data can be useful in residue analysis for clenbuterol in ostriches.     

Investigation of urinary steroid metabolites in calf urine after oral and intramuscular administration of DHEA

DHEA (3β-hydroxy-androst-5-en-17-one) is a natural steroid prohormone. Despite a lack of information on the effect, DHEA and other prohormones are frequently used as a food supplement by body-builders. DHEA is suspected for growth promoting abuse in cattle as well. Considering the latter, urine samples from a previous exposure study in which calves were exposed to 1 g DHEA per day for 7 days, were used. The calves were divided in three groups: one orally treated, one intramuscularly injected, and a control group. The effect of this treatment on the urinary profile of several precursors and metabolites of DHEA was investigated. Urine samples were collected several days before and during the 7 days of administration and were submitted to a clean-up procedure consisting of a separation of the different conjugates (free, glucuronidated, and sulfated forms) of each compound on a SAX column (Varian). An LC-MS/MS method was developed for the detection and quantification of several metabolites of the pathway of DHEA including 17α- and 17β-testosterone, 4-androstenedione, 5-androstenediol, pregnenolone, and hydroxypregnenolone. Elevated levels of DHEA, 5-androstenediol, and 17α-testosterone were observed in the free and sulfated fraction of the urine of the treated calves, thus indicating that the administered DHEA is metabolized mainly by the ∆⁵-pathway with 5-androstenediol as the intermediate. Sulfoconjugates of DHEA and its metabolites were found to constitute the largest proportion of the urinary metabolites. The free form was also present, but in a lesser extent than the sulfated form, while glucuronides were negligible.     

Antitumor activity and pharmacokinetics of podophyllotoxin incorporated into solid lipid nanoparticles

To evaluate the antitumor activity and pharmacokinetics of podophyllotoxin (PPT) incorporated into solid lipid nanoparticles (SLN), Kunming mice inoculated with flesh tumor were used as animal model. The mice received a single daily intraperitoneal injection of PPT in 20% ethanol (5 mg/kg) and PPT-SLN (5 mg/kg in PPT) for 3 weeks. Gross tumor volumes, body weight and clinical observations were recorded daily. The mice were sacrificed for 24 h after the last administration, and the tumor inhibition rate was calculated with the tumor weight. For the pharmacokinetics research, the mice were treated with intraperitoneal injection of PPT (10 mg/kg) and PPT-SLN (10 mg/kg in PPT). Blood samples were collected at different time to determine the PPT concentration in plasma by HPLC. Blood drug level-time curve was made and pharmacokinetic parameters were calculated. As a result of drug administration, the tumor volume and weight of the mice injected with PPT-SLN were significantly restrained compared with mice treated with PPT or negative control. The tumor inhibition rate of 58.13% showed a significant antitumor activity of PPT-SLN. At the same time, the increased weight gain of the mice injected with PPT-SLN suggested a reduced toxicity of PPT in SLN. Pharmacokinetics study displayed a higher blood concentration, a prolonged circulation time, and an increased bioavailability of PPT-SLN compared with those of PPT. Our results demonstrated that PPT-SLN could optimize pharmacokinetics, enhance antitumor activity and attenuate toxicity, so it has a promising prospect for the application in anti-tumor treatment. Supported by the National Natural Science Foundation of China (Grant No. 50673078), the Innovation Program of Shanghai Municipal Education Commission (Grant No. 08ZZ21) and the Shanghai Key Fundamental Project (Grant No. 07DZ19603)     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.     

A quantitative LC/MSMS method for determination of edoxaban,a Xa inhibitor and its pharmacokinetic application in patients after total knee arthroplasty

Edoxaban was extracted from human plasma by simple protein precipitation with acetonitrile, followed by quantitative determination using a liquid chromatography–mass spectrometry method. The recoveries of edoxaban and the internal standard (ticlopidine) from human plasma were >85%, and the within‐ and between‐day coefficients of variation were within 15%. The limit of quantification in human plasma was 1 ng/mL. The concentration of edoxaban in blood decreased at room temperature, but remained unchanged for 1 week at 4°C. On the other hand, the concentration in plasma at both −20 and −80°C remained unchanged for 5 months. These results indicated that blood samples should be centrifuged immediately or stored at 4°C, and that plasma samples should be stored below −20°C until analysis. This method was applied to human plasma obtained from four patients after total knee arthroplasty. Analysis of edoxaban pharmacokinetics demonstrated an absorption time lag of 4h, a maximum concentration of 110 ± 26 ng/mL and an oral clearance of 37 ± 16 L/h. The analytical methods established in this study will be suitable for determining the concentrations of edoxaban in human plasma.