Evaluation of the enantioselective binding of imazalil to human serum albumin by capillary electrophoresis

In this work, a methodology for the evaluation of enantioselective binding of imazalil (IMA) enantiomers to human serum albumin (HSA) that does not require the separation of free and bound to HSA fractions is developed. This methodology comprises the incubation of IMA–HSA designed mixtures for 30 min directly in the capillary electrophoresis system and the subsequent direct injection and chiral separation of IMA employing highly sulfated β‐cyclodextrin as chiral selector and the complete filling technique. Two mathematical approaches were used to estimate apparent affinity constants (K₁), protein binding and enantioselectivity (ES) for both enantiomers of IMA. Moderate enantioselective binding of IMA enantiomers to HSA (ES = 2.0) was shown by the 1:1 stoichiometry and log K₁ values of 3.4 ± 0.4 and 3.1 ± 0.3 for the first and second eluted enantiomers, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S‐omeprazole, S‐OME) and its R‐enantiomer (R‐omeprazole, R‐OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10³ M⁻¹ and 5.36 × 10³ M⁻¹, respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs.     

Characterization of Interactions Between Fluoroquinolones and Human Serum Albumin by CE–Frontal Analysis

The binding of fluoroquinolones to the transport protein, human serum albumin (HSA), under simulated physiological conditions has been studied by capillary electrophoresis–frontal analysis (CE–FA). The binding of these drugs to human plasma was evaluated by using ultrafiltration and capillary electrophoresis. The free drug concentration [D]_f at each HSA concentration was determined by the plateau height in the electropherograms and the calibration lines. The binding constants of fluoroquinolones and HSA were estimated using nonlinear regression with origin 7.5 software. The fluoroquinolones were found to show low affinity toward HSA, with binding constants ranging from 1.73 × 10² to 5.40 × 10² M⁻¹. The percentages of protein binding (PB) for fluoroquinolones to HSA were between 8.6 and 22.2%, while the PB percentages for fluoroquinolones to human plasma were between 10.2 and 33.1%. It can be found that the PB percentages for fluoroquinolones to HSA are mostly lower than those for fluoroquinolones to human plasma. It suggests that HSA is the primary protein responsible for the binding of fluoroquinolones in human plasma. The thermodynamic parameters were obtained by CE–FA. The positive ∆H and ∆S values obtained by CE–FA showed that the binding reaction was an endothermic process, and the entropy drive the binding and hydrophobic interaction played major roles in the binding of fluoroquinolones to HSA.

     

罗布麻活性成分与人血清白蛋白结合的光谱学研究

应用荧光和紫外光谱研究了人血清白蛋白与罗布麻活性成分槲皮素(QUE)、芸香苷(RUT)和儿茶素(CAT)的结合机理. 在QUE与蛋白质浓度比小于3.5时, 其荧光猝灭机理主要是静态猝灭, 在药物浓度较高时动态猝灭所占的比例增加; RUT在整个实验浓度范围内对蛋白质的荧光猝灭机理为静态猝灭; CAT与蛋白质之间不能形成复合物, 其荧光猝灭主要由动态猝灭产生. QUE和RUT分别与蛋白质形成1∶1的复合物, 结合常数分别为(1.51±0.13)×10⁵和(0.81±0.08)×10⁵ L•mol^－1. 由于激发态质子转移, 与蛋白质的相互作用引起QUE和RUT内源荧光发射峰强度的明显增加, 进一步证实了它们与蛋白质的结合. 与蛋白质的结合也引起了QUE紫外吸收带的明显红移, 说明药物分子中的酚羟基发生了解离, 以离子形式与蛋白质发生作用. RUT的紫外吸收谱带没有明显移动, 说明它主要以中性状态与蛋白质结合. 应用与蛋白质作用后药物分子紫外吸收光谱的二阶导数谱, 对药物与蛋白质的结合模式进行了深入探讨.     

Characterization of interaction between doxycycline and human serum albumin by capillary electrophoresis‐frontal analysis

The binding of doxycycline to HSA under simulated physiological conditions (pH 7.4, 67 mM phosphate, I=0.17, drug concentration 100 μM, HSA concentration up to 475 μM, 36.5°C) was studied by CE‐frontal analysis. The number of primary binding sites, binding constant and physiological protein‐binding percentage were 1.9, 1.51×10³ M^?1 and 59.80%, respectively. In addition, the thermodynamic parameters including enthalpy change (ΔH), entropy change (ΔS) and free energy change (ΔG) of the reaction were obtained in order to characterize the acting forces between doxycycline and HSA. Furthermore, to better understand the nature of doxycycline–HSA binding and to get information about potential interaction with other drugs, displacement experiments were performed. The results showed that doxycycline binds at site II of HSA.     

Development and validation of an enantioselective LC‐MS/MS method to quantify enantiomers of (±)‐TAK‐700 in rat plasma: lack of in vivo inversion of (+)‐TAK‐700 (Orteronel) to its antipode

A highly sensitive, specific and enantioselective assay has been developed and validated for the estimation of TAK‐700 enantiomers [(+)‐TAK‐700 and (?)‐TAK‐700] in rat plasma on LC‐MS/MS‐ESI in the positive‐ion mode. Liquid–liquid extraction was used to extract (±)‐TAK‐700 enantiomers and IS (phenacetin) from rat plasma. TAK‐700 enantiomers were separated using methanol and 5 mm ammonium acetate (80:20, v/v) at a flow rate of 0.7 mL/min on a Chiralcel OJ‐RH column. The total run time was 7.0 min and the elution of (+)‐TAK‐700, (?)‐TAK‐700 and IS occurred at 3.71, 4.45 and 4.33 min, respectively. The MS/MS ion transitions monitored were m/z 308.2 → 95.0 for TAK‐700 and m/z 180.2 → 110.1 for IS. The standard curves for TAK‐700 enantiomers were linear (r² > 0.998) in the concentration range 2.01–2015 ng/mL for each enantiomer. The inter‐ and intra‐day precisions were in the ranges 3.74–7.61 and 2.06–8.71% and 3.59–9.00 and 2.32–11.0% for (+)‐TAK‐700 and (?)‐TAK‐700, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method was applied to the study of stereoselective oral pharmacokinetics of (+)‐TAK‐700 and it was unequivocally demonstrated that (+)‐TAK‐700 does not undergo chiral inversion to its antipode in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

抗肿瘤药物5-氟尿嘧啶与人血清白蛋白相互作用的热力学研究

在298.15 K下,根据本结合过程的假设和Langmuir结合理论, 用等温滴定微量热和圆二色谱分析法研究了抗肿瘤药物5-氟尿嘧啶(5-FU)与人血清白蛋白(HSA)的相互作用. 研究结果表明, 蛋白质(HSA)与药物配体5-氟尿嘧啶的相互作用存在两类结合位点. 第一类结合, 结合位点数N＝71±0.1, 结合常数 K＝(1.46±0.016)×10⁵ L•mol^－1, 结合焓ΔH＝(39.61±0.220) kJ•mol^－1, 结合熵ΔS＝(231.68±0.025) J•mol^－1•K^－1, 结合自由能ΔG＝(－29.48±0.030) kJ•mol^－1. 结合过程为熵驱动过程, 疏水相互作用是过程的主要推动力;第二类结合, 结合位点数N＝140±0.2, 结合常数 K＝(1.49±0.032)×10⁵ L•mol^－1, 结合焓ΔH＝(－19.31±0.103) kJ•mol^－1, 结合熵ΔS＝(34.30±0.055) J•mol^－1•K^－1, 结合自由能ΔG＝(－29.53±0.041) kJ•mol^－1, 结合过程为焓-熵协同驱动过程, 氢键和静电相互作用是过程的主要推动力. 圆二色谱分析结果表明, 在两类结合过程中, 药物5-氟尿嘧啶(5-FU)的作用致使蛋白质(HSA)二级结构单元的相对含量发生了变化.     

Enantioseparation of N‐acetyl‐glutamine enantiomers by LC–MS/MS and its application to a plasma protein binding study

A novel chiral method was developed and validated to determine N‐acetyl‐glutamine (NAG) enantiomers by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Enantioseparation was achieved on a Chiralpak QD‐AX column (150 × 4.6 mm i.d., 5 μm) using methanol–water (50 mm ammonium formate, pH 4.3; 70:30, v/v) at a flow rate of 500 μL/min. The detection was operated with an electrospray ionization source interface in positive mode. The ion transition for NAG enantiomers was m/z 189.0 → 130.0. The retention time of N‐acetyl‐l ‐glutamine and N‐acetyl‐d ‐glutamine were 15.2 and 17.0 min, respectively. Calibration curves were linear over the range of 0.02–20 μg/mL with r > 0.99. The deviation of accuracy and the coefficient of variation of within‐run and between‐run precision were within 10% for both enantiomers, except for the lower limit of quantification (20 ng/mL), where they deviated <15%. The recovery was >88% and no obvious matrix effect was observed. This method was successfully applied to investigate the plasma protein binding of NAG enantiomers in rats. The results showed that the plasma protein binding of NAG enantiomers was stereoselective. The assay method also exhibited good application prospects for the clinical monitoring of free drugs in plasma.     

Validation of an enantioselective LC–MS/MS method to quantify enantiomers of (±)‐OTX015 in mice plasma: Lack of in vivo inversion of (−)‐OTX015 to its antipode

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)‐OTX015 and (−)‐OTX015] in mice plasma on LC–MS/MS‐electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)‐OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(−)‐OTX015] and inactive [(+)‐OTX015] enantiomers were resolved on a Chiralpak‐IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v /v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)‐OTX015, (−)‐OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r ² > 0.998) in the concentration range 1.03–1030 ng/mL. The inter‐ and intraday precisions were in the range 2.20–13.3 and 8.03–12.1% and 3.80–14.4 and 8.97–13.6% for (+)‐OTX015 and (−)‐OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)‐OTX015 and unequivocally demonstrated that (−)‐OTX015 does not undergo chiral inversion to its antipode in vivo in mice.     

Total Synthesis and Absolute Configuration Assignment of MRSA Active Garcinol and Isogarcinol

A short total synthesis of (±)‐garcinol and (±)‐isogarcinol, two endo‐type B PPAPs with reported activity against methiciline resistant Staphylococcus aureus (MRSA), is presented. The separation of framework‐constructing from framework‐decorating steps and the application of two highly regio‐ and stereoselective Pd‐catalysed allylations, that is, the Pd‐catalysed decarboxylative Tsuji–Trost allylation and the diastereoselective Pd‐catalysed allyl–allyl cross‐coupling, are key elements that allowed the total synthesis to be accomplished within 13 steps starting from acetylacetone. After separation of the enantiomers the absolute configurations of the four natural products (i.e., (?)‐garcinol, (+)‐guttiferone E (i.e., ent‐garcinol), (?)‐isogarcinol, and (+)‐isoxanthochymol (i.e., ent‐isogarcinol)) were assigned based on ECD spectroscopy.     

Analysis of primaquine and its metabolite carboxyprimaquine in biological samples: enantiomeric separation, method validation and quantification

The clinical formulation of primaquine (PQ) is a mixture of (−)‐(R)‐ and (+)‐(S)‐ primaquine enantiomers which may show different pharmacokinetic and pharmacodynamic properties. To assess the efficacy and toxicity of primaquine enantiomers, a method using LC‐MSD‐TOF has been developed. The enantiomers were well separated using a Chiralcel OD column (250 × 4.6 mm, 10 µm) with a linear gradient of mobile phase consisting of acetonitrile (0.1% formic acid) and aqueous ammonium formate (20 mm ; 0.1% formic acid) adjusted to pH 5.9 at a flow rate of 0.7 mL/min. The method was validated for linearity, precision, accuracy and limits of detection and quantification. The calibration curves were linear with all correlation coefficients being >0.999. The average recoveries of (−)‐(R)‐ and (+)‐(S)‐primaquine and (−)‐(R)‐ and (+)‐(S)‐carboxyprimaquine were 88 and 92%, respectively, in spiked human plasma and 89 and 93% respectively in spiked mouse plasma samples. The RSD of (−)‐(R)‐ and (+)‐(S)‐primaquine and (−)‐(R)‐ and (+)‐(S)‐carboxyprimaquine were 2.15, 1.74, 1.73 and 2.31, respectively, in spiked human plasma and 2.21, 1.09, 1.95 and 1.17% in spiked mouse plasma, respectively. The intra‐day and inter‐day precisions expressed as RSD were lower than 10% in all analyzed quality control levels. The method as reported is suitable for study of the pharmacokinetic and pharmacodynamic properties of the enantiomers of primaquine. The method was successfully applied to study plasma pharmacokinetic profile of enantiomers of primaquine and carboxyprimaquine in mice administered with primaquine in racemic form. The analytical method was found to be linear, accurate, precise and specific. Copyright © 2010 John Wiley & Sons, Ltd.     

Asymmetric Synthesis of Both Enantiomers of Protected 5‐Hydroxynorvaline by Hetero‐Diels‐Alder Addition of ethyl 2‐Nitrosoacrylate to (R)‐ and (S)‐1‐Phenylbutyl Vinyl Ether

Both enantiomers of protected 5‐hydroxynorvaline were prepared by hetero‐Diels‐Alder addition of ethyl 2‐nitrosoacrylate to readily available (R)‐ and (S)‐1‐phenylbutyl vinyl ether and a further three‐step manipulation. Attempted synthesis of (±)‐vigabatrin from protected (±)‐5‐hydroxynorvaline was unsuccessful.     

Simultaneous stereoselective analysis of naftopidil and O‐desmethyl naftopidil enantiomers in rat feces using an online column‐switching high‐performance liquid chromatography method

A novel online column‐switching chiral high‐performance liquid chromatography method was developed and validated for the simultaneous determination of naftopidil (NAF) and its O‐desmethyl metabolites (DMN) enantiomers in rat feces. Direct and multiple injections of supernatant from rat feces homogenate were allowed through the column‐switching system. Analyte extraction was performed on the Capcell Pak mixed‐functional column by acetonitrile–phosphate buffer (pH 7.4; 10 mm ; 8:92, v/v) flowing at 1 mL/min. Separation of NAF and DMN enantiomers was achieved on the Chiralpak IA column by methanol–acetonitrile–acetate buffer (pH 5.3; 5 mm ; 45:33:22, v/v/v) flowing at 0.5 mL/min. The analytes were measured with a fluorescence detector at 290 nm (λ_ex) and 340 nm (λ_em). The validated method showed a good linearity [22.5–15,000 ng/mL for (+)‐/(?)‐NAF; 35–25,000 ng/mL for (+)‐/(?)‐DMN] and the lowest limits of quantification for NAF and DMN enantiomers were 22.5 and 35 ng/mL, respectively. Both intra‐ and inter‐day variations were <10%. The assay was successfully applied to the fecal excretion of NAF and DMN enantiomers in rat after single oral administration of (±)‐NAF. Nonstereoselective excretion of (+)‐ and (?)‐NAF was found in feces, while stereoselective excretion of (+)‐ and (?)‐DMN was observed with higher excretion levels of (+)‐DMN, indicating that there may exist stereoselective metabolism for NAF enantiomers. Copyright © 2014 John Wiley & Sons, Ltd.     

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: Application to fluoxetine metabolism by CYP2D6

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in‐capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β‐CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis–Menten kinetic parameters (K_m and V_max) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis–Menten equation. K_m values have been found to be 30 ± 3 μM for S‐FLX and 39 ± 5 μM for R‐FLX. V_max estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min^?1·(pmol CYP)^?1 for S‐ and R‐FLX, respectively. Similar results were obtained using a single enantiomer (R‐FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R‐FLX.     

Optical Resolution and Structure Determination of New Indolizidine Alkaloids from Elaeocarpus sphaericus

Two new indolizidine alkaloids, (±)‐3‐oxoisoelaeocarpine ( 1 ) and (±)‐elaeocarpine N‐oxide ( 2 ), along with three known alkaloids, (±)‐isoelaeocarpine ( 3 ), (±)‐elaeocarpine ( 4 ), and (?)‐isoelaeocarpiline ( 5 ), were isolated from an EtOH extract of the branches and leaves of Elaeocarpus sphaericus. The structures of these compounds were determined by spectroscopic and chemical methods. Furthermore, enantiomers of compounds 1 and 3 were separated on a chiral CD‐Ph column, and their absolute configurations were determined by TD‐DFT (=time‐dependent density‐functional theory) quantum‐chemical calculations of their electronic circular dichroism (ECD) spectra.     

Connecting simulated,bioanalytical, and molecular docking data on the stereoselective binding of (±)-catechin to human serum albumin

The stereoselective binding of the frequently ingested nutraceutical (±)-catechin, with demonstrated differential biological activity between enantiomers, to human serum albumin (HSA), with the largest complexation and enantioselectivity potential among the plasmatic proteins, is studied by combining simulations to optimize the experimental design, robust in vitro electrokinetic chromatographic data, and molecular docking–chiral recognition estimates. Methodological and mathematical drawbacks in previous reports on (±)-catechin–HSA are detected and eliminated. Recent and novel direct equations extracted from the classical interaction model allows advantageous univariate mathematical data treatment, providing the first evidence of quantitative (±)-catechin–HSA enantioselectivity. Also, the binding site in HSA of the enantiomers is approached, and both the experimental enantioselectivity and the main binding site information are contrasted with a molecular docking approach.     

Comparison of three methods for analyzing loureirin B and human serum albumin interaction using capillary electrophoresis

Loureirin B (LB), a bioactive drug, is widely used in the treatment of biological diseases. However, due to its poor solution in water, it is important to find the approach which helps LB to specific biological targets. As the most abundant protein in plasma, HSA plays the role of a carrier of numerous drug ligand. Thus, the interaction between LB and HSA was explored by ACE, CE frontal analysis, and pressure‐mediated ACE under simulated physiological conditions (pH 7.4). The binding constants were calculated as 13.14 × 10⁴ L/mol, 7.00 × 10⁴ L/mol, and 2.78 × 10⁴ L/mol for each method, respectively. At the same time, the binding site number (n = 1.429) could be only calculated by the CE frontal analysis method. Furthermore, good experimental repeatability was obtained by pressure‐mediated ACE with RSDs for retention times and peak areas within 2.149 and 1.228, respectively.     

Kinetic and thermodynamic characterization of camptothecin hydrolysis at physiological pH in the absence and presence of human serum albumin

To accurately derive the kinetic and thermodynamic parameters governing the hydrolysis of the lactone ring at physiological pH, a derivative spectrophotometric technique was used for the simultaneous estimation of lactone and carboxylate forms of camptothecin (CPT). The hydrolysis of the CPT‐lactone and the lactonization of CPT‐carboxylate at 310.15 K followed a first‐order decay with apparent rate constants equal to 0.0279 ± 0.0016 min^?1 and 0.0282 ± 0.0024 min^?1, respectively. The activation energy associated with the hydrolysis of the CPT‐lactone and the lactonization of the CPT‐carboxylate as calculated from the Arrhenius equation was 89.18 ± 0.84 and 86.49 ± 2.7 kJ mol^?1, respectively. The enthalpy and entropy of the thermodynamically favored hydrolysis reaction were on average 10.49 kJ mol^?1 and 48.00 J K^?1 mol^?1, respectively. The positive enthalpy and entropy values of the CPT‐lactone hydrolysis indicate that the reaction is endothermic and entropically driven. The stability of CPT‐lactone in the presence of human serum albumin (HSA) was also analyzed. Notwithstanding the much faster hydrolysis of the CPT‐lactone in the presence of HSA at various temperatures, the energy of activation was determined to be similar to the one estimated in the absence of HSA, suggesting that HSA does not catalyze the hydrolysis reaction, but it merely binds, sequesters, and stabilizes the CPT‐carboxylate species. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 704–715, 2009     

Characterization of Porous Graphitic Carbon LC Phase with some Carbohydrate Derivatives

Stereoselective CE method for investigations of pharmacokinetics of ketoprofen enantiomers (KTP) in patients taking also other drugs is proposed, to establish relation between levels of KTP enantiomers in blood and synovial fluid of patients with rheumatoid arthritis. Resolution of the analytes was obtained in silica capillary filled with chiral selector-heptakis 2,3,6-tri-O-methyl–β-cyclodextrin in triethanolamine-phosphate buffer. Calibration curves for enantiomers in plasma and synovial fluid were linear in the range of 0.25–50.0 mg L⁻¹, but 1.0–250.0 mg L⁻¹ in urine. Concentrations of KTP enantiomers in synovial fluid measured at 4 h after the administration of a tablet with racemic KTP were insignificantly greater [(−)-R = 1.07 ± 0.66; (+)-S-KTP = 1.13 ± 0.65 mg L⁻¹] than in plasma [(−)-R = 0.86 ± 0.37; (+)-S-KTP = 0.96 ± 0.42 mg L⁻¹]. The validated method has been successfully applied for the determination of KTP enantiomers in biological fluids of patients with rheumatoid arthritis.