Determination of R(+)- and S(–)-Lansoprazole Using Chiral Stationary-Phase Liquid Chromatography and Their Enantioselective Pharmacokinetics in Humans

Purpose. Stereoselective and sensitive methods employing chiral stationary phase columns for HPLC determination of enantiomers of lansoprazole in the human serum were developed and pharmacokinetic behaviors of the enantiomers were evaluated in seven subjects. Methods. Five chiral stationary phase columns: Chiralcel OD (cellulose tris(3,5-dimethyl-phenylcarbamate)), OF (cellulose tris(4-chloro-phenylcarbamate)), OG (cellulose tris(4-methylphenylcarbamate)) and OJ (cellulose tris(4-methylbenzoate)), and Chiralpak AS (amylose tris ((S)-1 -phenylethylcarbamate)) were investigated. Results. Chiralcel OD and Chiralpak AS columns gave a good resolution of R(+)- and S(–)-enantiomers from racemic lansoprazole, but Chiralcel OF, OG, and OJ did not. The mean C_max and the AUC values of R(+)-enantiomer were 3–5 times greater than those of S(–)-enantiomer following oral administration of 30 mg of racemic lansoprazole. The CL_tot values of R(+)-enantiomer were significantly smaller than those of S(–)-enantiomer. Binding of R(+)-enantiomer to human serum proteins was significantly greater than that of S(–)-enantiomer. The mean metabolic ratio (metabolites/parent compound) in human liver microsomes of S(–)-enantiomer was significantly greater than that of R(+)-enantiomer. Conclusions. The stereoselective pharmacokinetics of lansoprazole enantiomers is likely due to its Stereoselective protein binding and/ or metabolism.     

Stereoselective Systemic Disposition of Ibuprofen Enantiomers in the Dog

The pharmacokinetics of ibuprofen are complicated by the unidirectional metabolic inversion of the (–)-R- to ( + )-S-enantiomer. Chiral inversion is of therapeutic significance since the drugs pharmacologic activity has been shown to depend upon the ( + )-S-isomer. As a result, the present study was undertaken to determine if chiral inversion occurs systemically and to elucidate further the kinetics of the inversion process. Experiments were performed in the beagle dog after intravenous bolus injections of ibuprofen enantiomers separately [100 mg (–)-R, n = 4; 100 mg ( + )-S, n = 4] and as admixtures of varying proportions [100 mg (–)-R + 100 mg ( + )-S, n = 4; 100 mg (–)-R + 200 mg ( + )-S, n = 2]. Plasma samples of (–)-R-and ( + )-S-enantiomers were measured by a stereospecific HPLC assay after all drug administrations. Based on the area under the plasma concentration–time curves for ( + )-S after administration of each enantiomer alone, chiral inversion was 70 to 75%. A progressive reduction in total plasma clearance of (–)-R-ibuprofen is also observed as increasing amounts of ( + )-S-enantiomer are added to the system. The results demonstrate that chiral inversion occurs to a significant extent in the systemic circulation in dog and that R-to-S inversion of ibuprofen may be inhibited by its ( ( + )-S-enantiomer.     

Diketopiperazine Formation,Hydrolysis, and Epimerization of the New Dipeptide Angiotensin-Converting Enzyme Inhibitor RS-10085

The degradation kinetics, products, and mechanisms of RS-10085(1), 2-[2-(l-ethoxycarbonyl)-3-phenylpropyl]amino-l-oxopropyl]-6,7-dimethoxy-l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(S,S,S), in aqueous solution were investigated at 40, 60, and 80°C from pH 1 to pH 13. Pseudo-first-order kinetics were observed throughout the pH range studied and the log(rate)–pH profiles reflected four kinetic processes (k _o, k_o, k_o, and k _OH) as well as the two pKa's of 1. Excellent (>98%) mass balance was obtained through products 2–5. At pH 4 or below, intramolecular cyclization leading to diketopiperazine 5 accounted for greater than 93% of the observed neutral- or water-catalyzed processes (k _o and k_o). At pH levels greater than 5, hydrolysis giving 2 predominated and was responsible for the observed neutral- or water-catalyzed (k_o) and specific base-catalyzed (k _OH) kinetic processes. Some epimerization leading to the S,S,R drug isomer (4) was also observed at pH levels greater than 7. The relative acidity of the protons at the three chiral centers of 1 was qualitatively compared and was used to explain the observed specificity in epimerization.     

柱前衍生化RP-HPLC法分离苯乙醇胺类化合物对映体

目的以2,3,4,6-四-O-乙酰基-β-D-葡萄糖异硫氰酸酯(GITC)为手性衍生化试剂,建立苯乙醇胺类化合物对映体RP-HPLC分离分析方法。方法采用RP-HPLC法。考察了衍生化反应中碱化试剂和衍生化试剂的浓度等反应条件对衍生化产率的影响,并考察流动相的组成和pH值等因素对生成的非对映异构体分离的影响。讨论了化合物的分子结构对手性衍生化及衍生后的非对映异构体色谱分离的影响。结果在三乙胺和GITC的浓度分别为10和5 mmol.L-1的乙腈溶液中,室温下反应20 min后,有5个苯乙醇胺化合物转化成相应的非对映异构体的硫脲衍生物。在色谱条件为:Diamonsil C18色谱柱(150 mm×4.6 mm,5μm),30 mmol.L-1醋酸铵(pH6.0)-乙腈(体积比为50∶50)为流动相,检测波长254 nm,流速1.0 mL.min-1,室温下,5个苯乙醇胺化合物对映体衍生化后非对映异构体的分离度达到4以上。结论该方法可作为苯乙醇胺类化合物对映体分离的方法之一。     

The Measurement of Warfarin Enantiomers in Serum Using Coupled Achiral/Chiral,High-Performance Liquid Chromatography (HPLC)

An assay for the serum concentration of the enantiomers of warfarin, R-warfarin and S-warfarin, has been developed using a bovine serum albumin chiral stationary phase (BSA-CSP) coupled to a Pinkerton internal-surface reverse-phase (ISRP) achiral column. The ISRP column is used to separate R,S-warfarin from the serum components and warfarin metabolites and to quantitate the total R,S-warfarin concentration. The eluent containing R,S-warfarin is then selectively transferred to the BSA-CSP, where the enantiomers are stereochemically resolved ( = 1.19) and the enantiomeric composition is determined. This system is sensitive and accurate, does not require extensive precolumn manipulations, and can be automated for use in large-scale clinical studies.     

Effects of Hyperlipidemia on the Pharmacokinetics of Nifedipine in the Rat

Purpose. The effect of hyperlipidemia on nifedipine pharmacokinetics was studied. The mechanisms by which hyperlipidemia affects pharmacokinetics of drugs are mainly undetermined. Hyperlipidemia may decrease the fraction of unbound drug in plasma and/or decrease intrinsic ability of the cytochrome P-450 systems due to excess membrane cholesterol. Hyperlipidemia is a primary risk factor for coronary artery disease leading to hypertension and ischemic heart disease, for which nifedipine, a calcium channel blocker, is used. Methods. Poloxamer 407 (P407)-induced hyperlipidemic rat model was used to study the effects of hyperlipidemia on the pharmacokinetics of nifedipine (6 mg kg^–1 given iv, ip and po). Total plasma cholesterol levels increased from 0.82–2.02 to 5.27–11.05 mmol L^–1 48 h post P407 administration (Ig kg^–1, ip). Protein binding studies were conducted by an ultrafiltration method. Results. Hyperlipidemia significantly decreased CL_TB by 38% and CL_TB/F by 45 and 42% following po and ip doses, respectively, thereby increasing AUC_0–, C_max and half-life. Absolute bioavailability and Vd_ss remained unchanged. AUC_0– was affected to the same extent in each route of administration, therefore, the effect was mainly systemic rather than presystemic. Hyperlipidemia significantly lowered the fraction unbound in plasma by approximately 31%. Conclusions. The altered pharmacokinetics of nifedipine by P407-induced HYPERLIPIDEMIA may be, at least in part, due to the decrease in fraction unbound in plasma. A decrease in intrinsic clearance, however, cannot be ruled out.     

Measurement of Underivatized Propranolol Enantiomers in Serum Using a Cellulose –Tris(3,5 -Dimethylphenylcarbamate) High-Performance Liquid Chromatographic (HPLC) Chiral Stationary Phase

A commercially available high-performance liquid chromatographic (HPLC) chiral stationary phase (HPLC-CSP) has been used to measure serum levels of d- and l-propranolol. The HPLC-CSP is based upon cellulose–tris(3,5-dimethylcarbamate) and is able to stereochemically resolve d- and l-propranolol without precolumn derivatization using a mobile phase composed of hexane:2-propranol:N,N-dimethyloctylamine (92:8:0.01, v/v/v). Under these conditions the observed stereochemical resolution () of the two enantiomers was = 2.2. A subject's concentration–time curve of the two isomers was determined following the ingestion of 160 mg racemic propranolol.     

Stability of Prostacyclin Analogues: An Unusual Lack of Reactivity in Acid-Catalyzed Alkene Hydration

Prostacyclin analogue 5 undergoes specific acid-catalyzed hydration (k _H+ = 1.9 × 10^–7 M ^–l sec^–1 at 25°C) and a pH-independent oxidation reaction (k ₀ = 1.2 × 10^–10 sec^–1 at 25°C) above pH5. The hydration reaction for 5 is much slower than for other structurally similar exocyclic alkenes, even though the rate-determining step is proton transfer. This slowness of reaction and an analysis of the pH–rate profile show that 5 does not exhibit significant intramolecular general acid catalysis, as does prostacyclin.     

Apparent Lack of Mrp1-Mediated Efflux at the Luminal Side of Mouse Blood-Brain Barrier Endothelial Cells

Purpose. The purpose of this work was to determine mrp1-mediated efflux across the luminal membrane of endothelial cells at the blood-brain barrier (BBB) in mice. Methods. The transport of radiolabeled etoposide, 17-estradiol-D-17-glucuronide (E₂17G), vincristine, and doxorubicin across the BBB of mrp1(–/–) and wild-type mice was evaluated by in situ brain perfusion. Etoposide transport was also determined in P-glycoprotein-deficient mdr1a(–/–) mice perfused with both etoposide and mrp1 inhibitors like probenecid or MK571. Cerebral vascular volume was determined by co-perfusion with labeled sucrose. Results. Sucrose perfusion indicated that the vascular space was close to normal in all the studies, indicating that the BBB remained intact. The transport of etoposide, E₂17G, vincristine, and doxorubicin into the brain was not affected by the lack of mrp1. Trans-efflux studies in mrp1-deficient mice with etoposide and E₂17G confirmed that mrp1 was not involved in the efflux of these substrates across the BBB. There was also a significant P-gp-mediated efflux of etoposide in studies with P-glycoprotein-deficient mdr1a(–/–) mice. Perfusion of mdr1a(–/–) mice etoposide plus probenecid or MK571 did not affect the brain transport of etoposide. Conclusion. Efflux mediated by mrp1 does not seem to occur across the luminal membrane of the endothelial cells forming the mouse BBB.     

Chiral Amines Derived from 2-Arylpropionic Acids: Novel Reagents for the Liquid Chromatographic (LC) Fluorescence Assay of Optically Active Carboxylic Acid Xenobiotics

For the enantiospecific analysis of optically active carboxylic acids, the availability of readily detectable coupling components is desirable, but highly fluorescent chiral amines are rare. From activated enantiomers of fluorescent 2-arylpropionic acids fluorescent chiral amines were synthesized via Curtius degradation, i.e., under formation of the acyl azide, the isocyanate, and finally, the amine. The formation of isocyanates and of amine hydrochlorides led to an inversion of the direction of rotation of polarized light. Amines derived from R- and S-flunoxaprofen, R- and S-naproxen, and R/S-benoxaprofen were characterized. The amines were found to be applicable for the chiral separation of carboxylic acids (such as 2-arylpropionic acids) as diastereomeric derivatives via high-performance liquid-chromatographic (normal and reversed-phase) and thin-layer chromatographic techniques.     

Reversed-phase liquid chromatographic separation of enantiomeric and diastereomeric bases related to chloramphenicol and thiamphenicol

The important antimicrobial agents chloramphenicol and thiamphenicol are N-acylated amines whose chemical structures include two chiral centers. Each drug is the single enantiomer of R,R configuration. The N-deacylated bases of the drugs are important intermediates in their synthesis and optical resolution. In this report, reversed-phase HPLC methods are described for the separation of enantiomeric and diastereomeric bases of the two drugs and of two closely related bases used in some syntheses of the drugs. The stereoisomeric bases were derivatized with a homochiral isothiocyanate and the resulting diastereomeric thioureas were separated on C18 columns with methanol:water mixtures as mobile phases and detection at 254 nm. The four stereoisomeric bases of chloramphenicol and those of its unnitrated analogue were thus separable after derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate. This reagent also allowed the separation of the D-threo isomer of the p-mercaptomethyl analogue of thiamphenicol base from its stereoisomers. The stereoisomers of thiamphenicol base were similarly separated with (R)-alpha-methylbenzyl isothiocyanate as the derivatizing agent. The diastereomers of chloramphenicol base and of thiamphenicol base were chromatographically separable after derivatization with the nonchiral reagent benzyl isothiocyanate. The procedures developed may be useful in the determination of the stereoisomeric composition of the drugs in research and in quality control, and may be applicable to other similar agents whose chemistry and pharmacology are receiving considerable attention.     

Stereoselective disposition of carvedilol in man after intravenous and oral administration of the racemic compound

The racemic compound carvedilol is a multiple-action oral antihypertensive drug that exhibits both vasodilator and non-selective beta-adrenergic blocking activities. The effects of the levorotatoryS-enantiomer [S( – )-CARV] are vasodilatation and beta-blockade. TheR (+)-enantiomer [R (+)-CARV] is a pure vasodilating agent. Quantitative determination of the enantiomers in human plasma by HPLC was carried out after formation of diastereoisomers with the chiral reagent 2,3,4,6-tetraO-acetyl--d-glucopyranosyl isothiocyanate (GITC). The pharmacokinetics of the enantiomers were studied following i. v. (12.5 mg in 1 h) and p. o. (50 mg) administration of racemic carvedilol in ten healthy male subjects according to a randomized crossover design. The AUCs ofS (–)-CARV were significantly lower than those ofR (+)-CARV after both i. v. and p. o. administration. The systemic clearance of the two enantiomers was significantly different, whereas half-lives and apparent distribution volumes were comparable. Following p. o. administration, the absolute bioavailability (31.1% and 15.1%, respectively) and maximal plasma concentrations ofR (+ )-CARV were twice those ofS (–)-CARV A similar difference was found in the half-lives. A close correlation existed between enantiomeric ratios after i.v. and after p. o. administration, demonstrating slight intraindividual variability. The preferential systemic clearance of theS ( – )-enantiomer suggests stereoselective hepatic metabolism of carvedilol, becoming especially apparent after p. o. administration. The small intrasubject variability in enantiomer ratios indicates a relatively constant relation of beta-blockade to vasodilation during chronic treatment.     

Biotransformation of Estradiol in the Human Keratinocyte Cell Line HaCaT: Metabolism Kinetics and the Inhibitory Effect of Ethanol

Purpose. The aim of our study was to investigate the kinetics of -estradiol (E₂) metabolism in the human keratinocyte cell line HaCaT and to estimate the effect of the potential inhibitor ethanol on the biotransformation reaction. Methods. The formation rates of estrone (E₁) in dependence on substrate concentrations were determined in HaCaT cells using tritium labelled E₂. Experiments were conducted with and without addition of dehydroepiandrosterone (DHEA) and ethanol. Possible toxic effects on the cells due to ethanol were investigated by cytotoxicity tests. Results. The metabolism of E₂ in HaCaT cells exhibited Michaelis-Menten kinetics with K_m and V_max values of 3.5 M and 216 pmol × mg^–1 protein × h^–1, respectively. The reaction was inhibited by DHEA and ethanol. The alcohol showed a reversible competitive inhibition mechanism for concentrations of 4 to 8% (v/v). Lower ethanol concentrations had no effect, whereas levels 10% significantly decreased cell viability leading to a different inhibition mechanism. Conclusions. The HaCaT cell line seems to be a suitable model for studying enzyme kinetics equivalent to the human skin. The concentration dependent inhibitory effect of ethanol observed in this cell line may be relevant for the transdermal E₂ application in patients.     

Snake F(ab′)2 Antivenom from Hyperimmunized Horse: Pharmacokinetics Following Intravenous and Intramuscular Administrations in Rabbits

Purpose. The pharmacokinetics of a currently available horse F(ab)₂ antivenoms to Vipera aspis, V. ammodytes, and V. berus (Ipser Europe) and a new more purified and pasteurized preparation (SAV) was investigated in the rabbit. Methods. An immunoradiometric assay using an affinity-purified goat IgG horse F(ab)₂ specific and the same IgG labelled with iodine 125 as a tracer was developed. The limit of quantification in plasma was 0.032 µg/ml. Specificity study showed that mouse F(ab)₂ and Fab did not cross-react. Results. Pharmacokinetic analysis showed that the plasma F(ab)₂ concentration followed a biexponential decline after intravenous bolus administration with distribution and elimination half-lives of 2.66 ± 0.18 hrs and 49.69 ± 4.13 hrs, respectively. The total volume of distribution (Vdss or Vd) was between 209 and 265 ml.kg^–1 and was similar to the volume of the extracellular fluid in the rabbit (300 ml.kg^–1). Total body clearance ranged from 3.33 to 3.96 ml. h^–1 · kg^–1. After intramuscular administration which was only investigated with SAV, Tmax was 48 hrs and the absolute bioavailability was 42%. Conclusions. No difference in pharmacokinetics was observed between the two antivenom preparations following the intravenous administration. In contrast, a reduced rate and extent of absorption was shown following intramuscular administration.     

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): Implications for adenosine receptor subclassification

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N⁶-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.90%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A₁ receptor (IC₅₀ 240 nM) could entirely be attributed to its content of R-PIA (IC₅₀ 7.8 nM). Calculation of a theoretical IC₅₀ of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable. Correspondence to: R. Mathôt at the above address(–)-N⁶-(R-phenylisopropyl)adenosine is referred to in literature as L-PIA, I-PIA, (–)-PIA and R-PIA. The abbreviation R-PIA is commonly used     

Distribution of Gacyclidine Enantiomers in Spinal Cord Extracellular Fluid

Purpose. Determination of the pharmacokinetics of gacyclidineenantiomers, a non-competitive NMDA antagonist, in plasma and spinal cordextracellular fluid (ECF) of rats. Methods. Implantation of microdialysis probes in spinal cord (T9).Serial collection of plasma samples and ECF dialysates over 5 hoursafter IV bolus administration of (±)-gacyclidine (2.5 mg/kg). Plasmaprotein binding determined in vivo by equilibrium dialysis. ChiralGC/MS assay. Results. Plasma concentrations of (+)-gacyclidine were 25% higherthan those of (–)-gacyclidine over the duration of the experiment inall animals. Plasma concentrations decayed in parallel in a biphasicmanner (t_1/2 9 min; t_1/2 90 min) with no significant differencebetween enantiomers. Clearance and volume of distribution of(–)-gacyclidine were approximately 20% higher than those of its opticalantipode (CL: 248 vs 197 ml.kg^–1.min^–1;Vd: 31.6 vs 23.5 l/kg).Protein binding (90%) was not stereoselective. Both gacyclidineenantiomers were quantifiable in spinal cord ECF 10 min after drugadministration and remained stable over the duration of the experimentin spite of changing blood concentrations. Penetration of(–)-gacyclidine was significantly higher (40%) than that of (+)-gacyclidine inall animals. Yet, exposure of spinal cord ECF was similar for bothenantiomers, and not correlated with plasma AUCs. Conclusions. The disposition of gacyclidine enantiomers isstereoselective. Both enantiomers exhibit a high affinity for spinal cord tissueand their distribution may involve a stereoselective and active transportsystem. This hypothesis could also explain the discrepancy betweendrug concentrations in plasma and spinal cord ECF.     

Enantiomer separation of α-hydroxy acids in high-performance immunoaffinity chromatography

In this study, a monoclonal anti-d-hydroxy acid antibody was immobilized onto a synthetic high-flow-through chromatographic support material to produce a chiral stationary phase suitable for enantiomer separation of free α-hydroxy acids. Chiral separation of several aliphatic and aromatic members of this class of compounds was achieved in HPLC under mild isocratic buffer conditions using phosphate buffered saline, pH 7.4, as mobile phase. Due to the high degree of stereoselectivity exhibited by the immobilized antibody, in all cases the l-enantiomer eluted with the void volume, while the d-enantiomer was retained and eluted second. The effect of the mobile phase parameters flow rate, temperature, pH, and ionic strength on the enantiomer separation of the model analyte mandelic acid was investigated. While it was found that variations in the flow rate did not change the retention factor k₂, dramatic effects on the interaction between the immobilized antibody and d-mandelic acid were observed when any of the other mobile phase parameters were modulated.     

The activity of the neuronal and extraneuronal catecholamine-metabolizing enzymes of the perfused rat heart

Summary In a comparative study the neuronal and extraneuronal metabolism of several ³H-catecholamines (all of which were tritiated in the C-7 position of the side chain only) was determined in isolated rat hearts perfused at a concentration of the 3H-amines of 50 nmol/1. While the neuronal MAO activity was determined after inhibition of extraneuronal uptake (100 mol/1 OMI) and COMT (10 mol/1 U-0521), the extraneuronal MAO activity was estimated after inhibition of neuronal uptake (30 mol/1 cocaine) and COMT. The extraneuronal COMT activity was determined under conditions of inhibition of both neuronal uptake and MAO (pretreatment with pargyline). Hearts were perfused with the 3H-catecholamines until the rate of appearance of the various ³H-metabolites in the venous effluent has reached a steady state. From these rates (v _st-st) and the steady-state content of the unchanged ³H-catecholamines in the tissue (S _i), the rate constants (V _max/K _m) for the unsaturated intracellular enzymes COMT (_COMT) and MAO (_MAO) were calculated. The _COMTvalues for all four catecholamines, (–)-noradrenaline, dopamine, (–)-adrenaline and (±)-isoprenaline exhibit a range from 0.24 to 0.78 min^–1; the metabolism of the catecholamines by the COMT differs: (-)-noradrenaline = dopamine < (–)-adrenaline < (±)-isoprenaline. The extraneuronal MAO activity was low for all three catecholamines, (–)-adrenaline, (–)-noradrenaline and dopamine (range of _MAOfrom 0.05 to 0.28 min^–1) and declined in the order: (–)-adrenaline < (–)-noradrenaline < dopamine. The neuronal MAO activity for (–)-adrenaline, (–)-noradrenaline and dopamine was slightly higher than that in the extraneuronal cells (range of kMAO from 0.08 to 0.35 min^–1), but the ranking order showed the same pattern: (–)-adrenaline < (–)-noradrenaline = dopamine.Abbreviations MAO monoamine oxidase - COMT catechol-Omethyltransferase - NMN normetanephrine - MN metanephrine - MT 3-methoxytyramine - OMI 3-O-methyl-isoprenaline - DOPEG dihydroxyphenylglycol - DOPET dihydroxyphenylethanol - DOMA dihydroxymandelic acid - DOPAC dihydroxyphenylacetic acid - U-0521 3,4-dihydroxy-2-methyl propiophenone     

Gas Chromatographic–Mass Spectrometric (GC-MS) Determination of MDL 72,222 and Four Metabolites in Monkey Plasma

MDL 72,222, a potent serotonin antagonist, is being developed for use as an antiemetic drug in cancer chemotherapy. An assay method has been developed for the determination of MDL 72,222 and four metabolites: N-desmethyl-MDL 72,222 (1), 3,5-dichlorobenzoic acid (2), glycine conjugate of 2 (3), and MDL 72,222-N-oxide (4). The method involves liquid–liquid extractions, derivatization with trifluoroacetic anhydride for metabolite 1, methylation with diazomethane for metabolites 2 and 3, reduction with titanous chloride for 4, and detection of each analyte by GC-MS. In this method d ₃-MDL 72,222, a 3-methyl-5-chlorobenzoate analogue of 1 (5), and 3,4-dichlorobenzoate analogues of 2–4 (6–8) are used as internal standards for the determination of MDL 72,222 and metabolites 1, 2, 3, and 4, respectively. The method is suitable for quantification of MDL 72,222 and the metabolites 1-4 over a concentration range of 1–150, 0.5–75, 1–150, 0.5–75, and 1–150 ng/ml, respectively. The intraday precision and accuracy values are within 10% RSD and 92–110%, respectively. The interday precision and accuracy values are within 14% RSD and 87.6–116%, respectively. The method is specific and sensitive for the analysis of MDL 72,222 and four metabolites in monkey plasma. The assay method has been utilized in analyzing pharmacokinetic study samples.     

The Stereospecific Determination of Fluoxetine and Norfluoxetine Enantiomers in Human Plasma by High-Pressure Liquid Chromatography (HPLC) with Fluorescence Detection

A quantitative method for the simultaneous HPLC resolution and detection of the enantiomers of (R,S) fluoxetine (F) and their metabolites (R,S) norfluoxetine (N) in human plasma has been developed. F is a serotonin uptake inhibitor used in the treatment of depression and is administered as a racemate. After liquid–liquid extraction and derivatization with (R) napthyl ethyl isocyanate (NEI), the separation and detection of the resultant diasteriomers were achieved using normal phase HPLC and fluorescence. The four NEI diastereomers and the internal standard [(–)-N-methyl--(2-methylphenoxy) benzenepropanamine hydrochloride], representing the enantiomers S-F, R-F, S-N, and R-N were resolved within 15 min. The assay for each analyte was linear using two concentration ranges of 1–10 and 10–500 ng/ml of human plasma. The precision and accuracy are reported as the coefficient of variation (%CV) and relative error (%RE). The sum of the chiral HPLC results from plasma samples were compared to the achiral gas chromatographic/electron capture (GC/EC) results. The correlation between these two methods, for total F and N, resulted in r ² values of 0.98 and 0.89, respectively. The chiral HPLC method is currently being applied to clinical studies for the evaluation of the enantiomeric disposition of F.