Stereoselective pharmacokinetics of flobufen in rats

Stereoselectivity of the pharmacokinetics of the nonsteroidal anti-inflammatory drug flobufen, 4-(2', 4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in male Wistar rats after intravenous administration. Pharmacokinetic parameters and chiral inversion of flobufen enantiomers were studied after a bolus injection of the racemate and individual enantiomers (5 mg/kg). Determinations of the enantiomers in rat plasma were performed using chiral HPLC (terguride column). After i.v. administration of flobufen racemate, plasma levels of R-enantiomer decreased more rapidly. The S-/R-enantiomer ratio of AUCs after rac-flobufen was 13.3. The total plasma clearance value of S-flobufen was more than 10-fold lower than R-flobufen. The other pharmacokinetic parameters of the enantiomers were also significantly different. While only traces of R-enantiomer (less than 1%) were detected in rat plasma after S-flobufen administration, considerable conversion to the S-enantiomer was found after injection of R-flobufen (R-enantiomer AUC/S-enantiomer AUC = 0.52). The results indicate substantial stereoselectivity in the disposition of flobufen enantiomers in the rat, which is, at least in part, attributed to chiral bioconversion.     

Stereospecificity and stereoselectivity of flobufen metabolic profile in male rats in vitro and in vivo: phase I of biotransformation.

Flobufen (F) is the original nonsteroidal antiinflammatory drug (NSAID) containing two enantiomers. The aim of this investigation was to elucidate the biotransformation pathway of F at chiral level in phase I of biotransformation. Stereoselectivity and stereospecificity of the respective enzymes were studied in male rats in vitro (microsomal and cytosolic fractions, hepatocytes suspension) and in vivo. The rac-F, (+)-R-F and (-)-S-F were used as substrates. Amounts of F enantiomers, 4-dihydroflobufen diastereoisomers (DHF) and other metabolites (M-17203, UM) were determined with a chiral HPLC method in two chromatographic runs on R,R-ULMO and allyl-terguride bonded columns. Stereoselective biotransformation of the two enantiomers of F was observed at all tested levels and significant bidirectional chiral inversion of enantiomers of F was observed in hepatocytes. Mean enantiomeric ratios of F concentrations (S-/R-), after rac-F incubations, ranging from 1.09 in cytosolic fraction to 18.23 in hepatocytes. Stereospecificity of the respective F reductases was also observed. (2R;4S)-DHF and (2S;4S)-DHF are the principal metabolites of F in microsomes and hepatocytes. Neither DHF diastereoisomers nor M-17203 were found in cytosolic fraction. Only the nonchiral metabolite, M-17203, was found in all urine and feces samples after oral administration of F.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

Stereospecificity of flobufen metabolism in guinea pigs in vitro and in vivo: phase I of biotransformation

Flobufen (F) is an original nonsteroidal antiinflammatory drug that exists in two enantiomeric forms. Its biotransformation was investigated in male guinea pigs because of the similarities shown in a preliminary F metabolic study between guinea pig and man. Stereospecificity of the respective enzymes was studied in vitro, using microsomes and cytosol, and in vivo, in urine and feces. Rac-F, R-F, and S-F served as substrates. The amount of 4-dihydroflobufen stereoisomers (DHF) and other metabolites (M-17203 and UM-2) was determined by chiral HPLC using an R,R-ULMO column. It was observed that F reductases were distributed differently in microsomes and cytosol. The microsomal fraction showed higher activity and different stereospecificity in rac-F, R-F, and S-F reduction compared to cytosol. (2R;4S)-DHF was the principle metabolite in microsomes and (2S;4S)-DHF was the principle metabolite in cytosol. In vivo experiments revealed the excretion of a main metabolite UM-2 in addition to other metabolites M-17203 and DHF stereoisomers. UM-2 was predominantly excreted after S-F administration. Stereoselectivity of DHF stereoisomers excretion was different in urine and in feces. The absence of UM-2 and M-17203 in microsomes and cytosol and their presence in urine and feces showed that both could arise in some other extrahepatic tissue or cell compartment or that their formation depends on liver cell integrity.     

Reduction of flobufen in pig hepatocytes: effect of pig breed (domestic,wild) and castration

Knowledge of the biotransformation processes of veterinary drugs and food supplements in food-producing animals is increasingly important. Residual levels of parent compounds or their metabolites in food of animal origin may differ with the breed, breeding conditions, and gender of animals. The nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (racemic or its individual enantiomers) was used as a model to evaluate differences in activity, stereoselectivity, and stereospecificity of reductases in primary cultures of hepatocytes from intact male or castrated male domestic pigs (Sus scrofa domestica) or male wild pig (Sus scrofa scrofa). Time-dependent consumption of flobufen enantiomers and formation of dihydroflobufen (DHF) diastereoisomers as their principal metabolites in hepatocytes were measured using chiral HPLC. Flobufen reduction in hepatocytes from all three experimental groups of animals was stereoselective ((+)-R-flobufen was predominantly metabolized) and stereospecific (2R;4S-DHF and 2S;4S-DHF diastereoisomers were preferentially formed). Flobufen reductases activity in male domestic pigs was 30 times higher compared to castrated pigs. Flobufen reductases activity was similar in domestic and wild pigs. The stereospecificity and stereoselectivity of DHF production did not significantly differ with breed or castration of animal. Chiral inversion of flobufen enantiomers was also studied and differences between castrated and intact male pigs were seen.     

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.     

Direct resolution of optically active isomers on chiral packings containing ergoline skeleton. 6. Enantioseparation of profens

The enantioselective behaviour of some underivatized 2-arylpropionic acids (profens) and flobufen by HPLC using a terguride-based chiral stationary phase was tested. X-ray analysis of crystals of the chiral selector and its complexes with naproxen allowed a deeper insight into the enantiodiscriminative process. The column stability and reproducibility, and the potential of the packing for semipreparative scale separations were also determined. A method for determining flobufen enantiomers and metabolites in plasma samples is described.     

Stereoselective pharmacokinetics of ornidazole after intravenous administration of individual enantiomers and the racemate

The pharmacokinetics of ornidazole (ONZ) were investigated following i.v. administration of racemic mixture and individual enantiomers in beagle dogs. Plasma concentrations of ONZ enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OB-H column with quantification by UV at 310 nm. Notably, the mean plasma levels of (-)-ONZ were higher in the elimination phase than those of (+)-ONZ. (-)-ONZ also exhibited greater t1/2, MRT, AUC(0-t) and smaller CL, than those of its antipode. The area under the plasma concentration-time curve (AUC(0-t)) of (-)-ONZ was about 1.2 times as high as that of (+)-ONZ. (+)-ONZ total body clearance (CL) was 1.4 times than its optical antipode. When given separately, there were significant differences in the values of AUC(0-infinity) and CL between ONZ enantiomers (P < 0.05), indicating that elimination of (+)-ONZ was more rapid than that of (-)-ONZ. No significant differences were found between the estimates of the pharmacokinetic parameters of (+)-ONZ or (-)-ONZ, obtained following administration as the individual and as a racemic mixture. This study demonstrates that the elimination of ONZ enantiomers is stereoselective and chiral inversion and enantiomer/enantiomer interaction do not occur when the enantiomers are given separately and as racemic mixture.     

Pharmacokinetics of ibutilide and its enantiomers in dogs

Ibutilide fumarate, a new drug for the treatment of cardiac arrhythmias, contains a stereogenic center bearing a secondary alcohol group. Several single dose and multiple dose studies of racemic ibutilide or its enantiomers were performed by the oral and intravenous routes in dogs. A chiral assay was used to examine racemization and the individual enantiomer pharmacokinetics. Following low oral or intravenous doses (approximately 0.3 mg/kg), the pharmacokinetics of the enantiomers were nearly identical, with no substantial chiral conversion. Both enantiomers exhibited high clearance rates, large volumes of distribution, and low oral bioavailability. As the dose increased, pharmacokinetic differences between the enantiomers were observed. The greatest differences (3-fold) were seen after oral administration at 4 mg/kg, indicating that first-pass metabolism of ibutilide was highly enantioselective at high doses. The clearances of the enantiomers differed by up to 34% at 5 mg/kg followed intravenous administration of the racemate. At high doses, other non-linear pharmacokinetic behavior was also apparent. The intravenous clearance of ibutilide declined from 5.3 L/h/kg at 0.3 mg/kg to 3.7 L/h/kg at a dose of 5 mg/kg. The absolute oral bioavailability of the racemate increased from 2% at 0.3 mg/kg to as much as 84% at 5 mg/kg. © 1996 Wiley-Liss, Inc.     

Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.     

Stereoselective metabolism of fenoxaprop‐ethyl and its chiral metabolite fenoxaprop in rabbits

The stereoselective metabolism of the enantiomers of fenoxaprop‐ethyl (FE) and its primary chiral metabolite fenoxaprop (FA) in rabbits in vivo and in vitro was studied based on a validated chiral high‐performance liquid chromatography method. The information of in vivo metabolism was obtained by intravenous administration of racemic FE, racemic FA, and optically pure (−)‐(S)‐FE and (+)‐(R)‐FE separately. The results showed that FE degraded very fast to the metabolite FA, which was then metabolized in a stereoselective way in vivo: (−)‐(S)‐FA degraded faster in plasma, heart, lung, liver, kidney, and bile than its antipode. Moreover, a conversion of (−)‐(S)‐FA to (+)‐(R)‐FA in plasma was found after injection of optically pure (−)‐(S)‐ and (+)‐(R)‐FE separately. Either enantiomers were not detected in brain, spleen, muscle, and fat. Plasma concentration–time curves were best described by an open three‐compartment model, and the toxicokinetic parameters of the two enantiomers were significantly different. Different metabolism behaviors were observed in the degradations of FE and FA in the plasma and liver microsomes in vitro, which were helpful for understanding the stereoselective mechanism. This work suggested the stereoselective behaviors of chiral pollutants, and their chiral metabolites in environment should be taken into account for an accurate risk assessment. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Determination of esmolol and metabolite enantiomers within human plasma using chiral column chromatography

A high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous determination of each of esmolol's enantiomers at the 25–1000 ng/ml concentrations observed in human plasma upon intravenous administration of this rapidly metabolized beta-adrenergic receptor blocking agent. Alternatively, a high performance liquid chromatography (HPLC) UV detection method has been developed for the simultaneous determination of each of the enantiomers for esmolol's metabolite which, in turn, achieve 2.5–50 μg/ml concentrations in human plasma. Utilizing chiral columns, these methods do not require a precolumn asymmetric derivatization step. Linearity in all cases was >0.99. Precision and accuracy at all but the lowest concentrations were within ±6% for the esmolol enantiomers and within ±2.5% for the esmolol metabolite enantiomers. These values should be suitable for performing thorough pharmacokinetic studies for all of the stereoisomers of this prototypical soft drug and its corresponding metabolite.     

Stereoselective disposition of S- and R-licarbazepine in mice

The stereoselective disposition of S-licarbazepine (S-Lic) and R-licarbazepine (R-Lic) was investigated in plasma, brain, liver, and kidney tissues after their individual administration (350 mg/kg) to mice by oral gavage. Plasma, brain, liver, and kidney concentrations of licarbazepine enantiomers and their metabolites were determined over the time by a validated chiral HPLC-UV method. The mean concentration data, attained at each time point, were analyzed using a non-compartmental model. S-Lic and R-Lic were rapidly absorbed from gastrointestinal tract of mouse and immediately distributed to tissues supplied with high blood flow rates. Both licarbazepine enantiomers were metabolized to a small extent, each parent compound being mainly responsible for the systemic and tissue drug exposure. The stereoselectivity in the metabolism and distribution of S- and R-Lic was easily identified. An additional metabolite was detected following R-Lic administration and S-Lic showed a particular predisposition for hepatic and renal accumulation. Stereoselective processes were also identified at the blood-brain barrier, with the brain exposure to S-Lic almost twice that of R-Lic. Another finding, reported here for the first time, was the ability of the mouse to perform the chiral inversion of S- and R-Lic, albeit to a small extent.     

Route-dependent stereoselective pharmacokinetics of tramadol and its active O-demethylated metabolite in rats

The effects of route of administration on the stereoselective pharmacokinetics of tramadol (T) and its active metabolite (M1) were studied in rats. A single 20 mg/kg dose of racemic T was administered through intravenous, intraperitoneal, or oral route to different groups of rats, and blood and urine samples were collected. Samples were analyzed using chiral chromatography, and pharmacokinetic parameters (mean +/- SD) were estimated by noncompartmental methods. Following intravenous injection, there was no stereoselectivity in the pharmacokinetics of T. Both enantiomers showed clearance values (62.5 +/- 27.2 and 64.4 +/- 39.0 ml/min/kg for (+)- and (-)-T, respectively) that were equal or higher than the reported liver blood flow in rats. Similar to T, the area under the plasma concentration-time curves (AUCs) of M1 did not exhibit stereoselectivity after intravenous administration of the parent drug. However, the systemic availability of (+)-T was significantly (P < 0.05) higher than that of its antipode following intraperitoneal (0.527 +/- 0.240 vs. 0.373 +/- 0.189) and oral (0.307 +/- 0.136 vs. 0.159 +/- 0.115) administrations. The AUC of the M1 enantiomers, on the other hand, remained mostly nonstereoselective regardless of the route of administration. Pharmacokinetic analysis indicated that the stereoselectivity in the pharmacokinetics of oral T is due to stereoselective first pass metabolism in the liver and, possibly, in the gastrointestinal tract. The direction and extent of stereoselectivity in the pharmacokinetics of T and M1 in rats were in agreement with those previously reported in humans, suggesting that the rat may be a suitable model for enantioselective studies of T pharmacokinetics.     

Species differences in the chirality of the carbonyl reduction of [14C] fenofibrate in laboratory animals and humans

The prochiral carbonyl group of fenofibrate (isopropyl 2-[4-(4-chlorobenzoyl)phenoxy]-2-methyl propionate) is reduced during its metabolism giving rise to a chiral secondary alcohol, "reduced fenofibric acid." Chiral and diastereomeric HPLC methods have been developed for the determination of its enantiomeric composition and these have been applied to the measurement of the "reduced fenofibric acid" enantiomers in urine of rats, guinea pigs, dogs, and human volunteers given [14C]fenofibrate. In the three animal species, the reduction is markedly enantioselective for the (-)-isomer, the enantiomeric ratios (-/+) being 95:5. This was not due to differences in the excretion of the enantiomers, since when racemic "reduced fenofibric acid" was given to rats it was recovered in the urine with the same enantiomeric composition as the dose form. In humans the ratio was 52:48 showing the lack of stereoselectivity of reduction in this species.     

Vancomycin as chiral selector for enantioselective separation of selected profen nonsteroidal anti-inflammatory drugs in capillary liquid chromatography

The chiral selector vancomycin was used either as mobile phase additive or bound as a chiral stationary phase (CSP) for the stereoselective separation of seven racemic nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen, carprofen, flurbiprofen, indoprofen, flobufen, ketoprofen, and suprofen, by capillary liquid chromatography. The effect of the type of stationary phase, the chiral column Chirobiotic V or the achiral stationary phases Nucleosil 100 C8 HD and Nucleosil 100 C18 HD, and the concentration of vancomycin in the mobile phase on separation of the drug enantiomers were evaluated. All the drugs, except flobufen, were successfully enantioseparated on Nucleosil 100 C8 HD with 4 mM vancomycin present in the mobile phase (composed of methanol and buffer) in the reversed phase mode. On the vancomycin-bonded chiral stationary phase, it was difficult to get enantioseparations of the profen NSAIDs. However, flobufen gave better enantioseparation on the vancomycin CSP. The better enantioresolution of the majority of profen derivatives on the achiral columns with vancomycin added to the mobile phase can be attributed in particular to the higher separation efficiency of this capillary chromatographic system. In addition, vancomycin dimers, formed in the mobile phase, seem to offer a better steric arrangement for stereoselective interaction to these analytes than the vancomycin bonded on the CSP. These substantial differences in the CS structure significantly influence the chiral discrimination mechanism.     

Stereoselective chiral inversion of pantoprazole enantiomers after separate doses to rats

(±)-Pantoprazole ((±)-PAN), (±)-5-(difluoromethoxy)-2-[[(3.4-dimethoxy-2-pyridinyl)methyl]sulfinyl]-1H-benzimidazole is a chiral sulfoxide that is used clinically as a racemic mixture. The disposition kinetics of (+)-PAN and (−)-PAN given separately has been studied in rats. Serum levels of (+)- and (−)-PAN and its metabolites, pantoprazole sulfone (PAN-SO₂), pantoprazole sulfide (PAN-S), 4′-O-demethyl pantoprazole sulfone (DMPAN-SO₂), and 4′-O-demethyl pantoprazole sulfide (DMPAN-S) were measured by HPLC. Following single intravenous or oral administration, both enantiomers were rapidly absorbed and metabolized, resulting in similar serum concentrations, suggesting that the two enantiomers have approximately the same disposition kinetics. The major metabolite of both (+)- and (−)-PAN was PAN-SO₂, while DMPAN-SO₂ was also detected as a minor metabolite. Serum levels of PAN-S and DMPAN-S could not be quantified after intravenous or oral administration of either enantiomer. Significant chiral inversion occurred after intravenous and oral administration of (+)-PAN. The AUCs of (−)-PAN after intravenous and oral dosing of (+)-PAN were 36.3 and 28.1%, respectively of those of total [(+) + (−)] PAN. In contrast, the serum levels of (+)-PAN were below quantitation limits after intravenous or oral administration of (−)-PAN. Therefore, chiral inversion was observed only after administration of (+)-PAN, supporting the hypothesis that stereoselective inversion from (+)-PAN to (−)-PAN occurs in rats. Chirality 10:747–753, 1998. © 1998 Wiley-Liss, Inc.     

Separation of the stereoisomers of the main metabolite of a non-steroidal anti-inflammatory drug,flobufen, by chiral high-performance liquid chromatography

The major metabolite of a novel non-steroidal anti-inflammatory drug, dl-4-(2′-4′-difluorobiphenyl-4-yl)-4-oxo-2-methylbutonoic acid (flobufen, I), namely 4-(2′,4′-difluorobiphenyl-4-yl)-2-methyl-γ-butyrolactone (4-dihydroflobufen lactone, III), has four stereoisomers consisting of two racemic pairs of enantiomers. Of three chiral stationary phases tested, Cyclobond I β-RSP (Astec) (β-cylodextrin derivatized with R,S-hydroxypropyl) was best able to separate the (++)(−−) racemate, with a liquid phase containing acetonitrile as modifier and triethylamine acetate as buffer. Using the Box-Wilson Central Composite Design for three factors, an optimum combination of pH and concentrations of the modifier and buffer was eventually obtained. A chromatographic response function based on a combination of the Kaiser peak separation function, P_i, and retention time of the second eluting enantiomer, t_RL, served as a response criterion for the process of optimization. The optimum conditions developed for the (++)(−−) racemate were also found to be suitable for separating the (+−)(−+) racemate, for which earlier studies had shown the separation to be more facile. Separation of the four stereoisomers of III, for which the chiral chromatographic system optimized in this study is proposed as the second stage, is targeted at a biochemical study of the stereoisomeric metabolism of I.     

Enantiospecific determination of DL-methylphenidate and DL-ethylphenidate in plasma by liquid chromatography-tandem mass spectrometry: application to human ethanol interactions

In humans, concomitant DL-methylphenidate (DL-MPH) and ethanol results in the carboxylesterase 1 (hCES1) mediated biotransformation of MPH to the transesterification metabolite DL-ethylphenidate (DL-EPH). The separate enantiomers of MPH and EPH are found at low ng/ml to pg/ml plasma concentrations. Substantial pharmacological differences exist between D- and L-isomers of MPH and EPH, both in terms of pharmacological potencies and receptor selectivity, as well as in pharmacokinetic properties. Accordingly, a sensitive, accurate and precise enantiospecific analytical method is required in order to fully explore pharmacokinetic-pharmacodynamic correlations regarding the MPH-ethanol interaction. The present study describes a novel liquid chromatographic-tandem mass spectrometric method for simultaneous analysis of D- and L-MPH as well as D- and L-EPH concentrations from human plasma. This assay provides baseline resolution of the individual MPH and EPH isomers utilizing a vancomycin-based chiral column. The lower limit of quantification was 0.025 ng/ml for each isomer when extracting 0.5 ml plasma aliquots. Calibration curves were linear over the range from 0.025 ng/ml to 25 ng/ml for all analytes (r(2)>0.995). Assay accuracy and precision were excellent and stability studies and assessment of potential matrix effects contributed to the validation of the method. Application of the method to human plasma samples collected after the administration of dl-MPH with or without ethanol is included, and the implications of this pharmacokinetic drug interaction discussed.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.