The determination of (-)-(S)- and (+)-(R)-ifosfamide in plasma using enantioselective gas chromatography: a validated assay for pharmacokinetic and clinical studies

An enantioselective gas chromatographic method has been developed and validated for the determination of the plasma concentration of the enantiomers of the anticancer drug ifosfamide (IFF). In this approach, the IFF enantiomers are separated from the plasma matrix by solid phase extraction, chromatographically resolved by gas chromatography on a chiral stationary phase, and detected by mass selective detection using selective ion monitoring. The assay has been validated for routine clinical and pharmacokinetic use and has a limit of detection in plasma of 250 ng/ml of each isomer.     

Preliminary clinical studies with L-prolyl-L-leucyl-glycine amide in Parkinson's disease

The present study, carried out in 16 parkinsonian patients, indicates that L-prolyl-L-leucyl-glycine amide possesses some antiparkinsonian activity and that, if used with levodopa, it can reduce some of the drug-induced dyskinesias. These preliminary observations should be submitted to a controlled trial in a large number of patients before therapeutic applications are claimed.     

Liquid chromatographic assay of abouthiouzine in plasma and its application to pharmacokinetic studies

Abouthiouzine is a newly synthesized antithyroid agent with a proposed less adverse effects profile than other currently used drugs. A simple and rapid reversed phase high performance liquid chromatography assay was developed to determine the concentration of abouthiouzine in human plasma. The procedure involved extraction of the drug and propranolol (internal standard) from the plasma using ethylacetate. The extract was evaporated under nitrogen and the residue was constituted with the mobile phase and injected onto micro-Bondapack phenyl column (10 microm, 3.9 mm x 150 mm). The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer, acetonitrile, and methanol in the ratio of 60:25:15 (v/v/v, pH=3.0), which was delivered at a rate of 1.5 ml/min. Abouthiouzine and the internal standard were monitored using UV detection at 240 nm; the run time was less than 5 min. The detection limit of abouthiouzine is 0.5 microg/ml. The within- and between-day coefficients of variation were less than 7%. Our method has been successfully used to measure abouthiouzine plasma concentrations in a rabbit model following an intravenous administration of the drug.     

Emotional reactions to odours in Parkinson's disease A clinical application of ethological methods

The behavioral reactions of 24 Parkinson patients to six kinds of odours were analyzed in relation to the subjective assessments of the odours. The reactions of the patients were normal in character, but reduced in intensity for two of the six odours. This was attributed primarily not to motor retardation but to impaired subjective perception.     

Stereoselective features of (R)- and (S)-atenolol: Clinical pharmacological,pharmacokinetic, and radioligand binding studies

In a randomized, double-blind, cross-over study in 12 healthy volunteers, the effects of single oral doses of 100 mg rac-atenolol were compared during exercise to those of equal amounts of the optically pure enantiomers, i.e., 50 mg (R)- and 50 mg (S)-atenolol. The mean rate pressure product decreased with rac-atenolol (?37%; P < 0.01) and half-dosed (S)-atenolol (?35%; P < 0.01) to the same extent, whereas (R)-atenolol caused no effect. Radioligand binding studies in beta-adrenergic receptors of the guinea pig heart yielded a eudismic ratio of 46 for (S)- to (R)-atenolol. The mean AUCs, maximal plasma concentrations, and plasma half-lives of the enantiomers were similar regardless of whether they were administered as optically pure enantiomers or as racemic mixture. On the other hand, the AUC of (R)-atenolol was 1.08-fold greater (P < 0.01) than that of the (S)-enantiomer. The reason for this finding remains unclear. We conclude that only (S)-atenolol, but not (R)-atenolol, contributes to the beta-blocking effect of currently used rac-atenolol since the same effect can be elicited with the (S)-enantiomer alone. © 1993 Wiley-Liss, Inc.     

Improved method for the rapid determination of isosorbide dinitrate in human plasma and its application in pharmacokinetic studies

A rapid, accurate and highly sensitive method was developed for the determination of isosorbide dinitrate in human plasma. Concentrations in the lower nanogram and subnanogram range are determined by a one-step extraction of 2 ml plasma, containing 4 ng/ml nitroglycerine as internal standard, with 5.5 ml n-pentane. The extract is subjected to gas—liquid chromatography—electron capture detection analysis. The lower limit of quantitation is 200 pg/ml, but concentrations as low as 50 pg/ml are still detectable. The method allows the quantitative determination of isosorbide dinitrate plasma levels in man following a 5 mg sublingual administration up to four hours after application.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

Sensitive, high-throughput gas chromatographic–mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies

A sensitive, robust gas chromatographic–mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean±SD): C_max, 32.73±9.21 ng/ml; AUC_0–∞, 1627±1372 ng/ml h; T_max, 3.08 h (median); k_e, 0.022±0.007 h⁻¹; elimination half-life, 37.69±21.70 h.     

Development of a high-performance liquid chromatography-based assay for carotenoids in human red blood cells: application to clinical studies

Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of many diseases; for example, there is an abnormal increase of phospholipid hydroperoxides in red blood cells (RBCs) of dementia patients. Dietary carotenoids have gained attention as potent inhibitors of RBC phospholipid hydroperoxidation, thereby making them plausible candidates for preventing disease. However, the occurrence of carotenoids in human RBCs is still unclear. This is in contradistinction to plasma carotenoids, which have been investigated thoroughly for analytical methods as well as biological significance. In this study, we developed a method to analyze RBC carotenoids using high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) diode array detection (DAD) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS). Under optimized conditions that included extraction, separation, and detection procedures, six carotenoids (lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene) were separated, detected by DAD, and concurrently identified based on APCI/MS and UV spectra profiles when an extract from human RBCs was subjected to HPLC-DAD-APCI/MS. The amounts of carotenoids varied markedly (1.3-70.2 nmol/L packed cells), and polar oxygenated carotenoids (xanthophylls) were predominant in RBCs. The HPLC-DAD-APCI/MS method would be a useful tool for clinical studies for evaluating the bioavailability of RBC carotenoids.     

A simple bioanalytical assay for determination of montelukast in human plasma: application to a pharmacokinetic study

An analytical method based on high-performance liquid chromatographic (HPLC) was developed for the determination of montelukast in human plasma using mefenamic acid as an internal standard. After precipitation of plasma proteins with acetonitrile, chromatographic separation was carried out using a Zorbax Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) with mobile phase consisted of methanol-acetonitrile-0.04M disodium hydrogen orthophosphate (22:22:56, v/v, pH 4.9). The wavelengths of fluorescence detection were set at 350 nm for excitation and 450 nm for emission. The linearity was confirmed in the concentration range of 5-1000 ng/ml in human plasma. Intra- and inter-day accuracy determined from quality control samples were 101.50 and 107.24%, and 97.15 and 100.37%, respectively. Intra- and inter-day precision measured as coefficient of variation were < or =4.72 and < or =9.00%, respectively. Extraction recoveries of drug from plasma were >48.14%. The protocol herein described was employed in a pharmacokinetic study of tablet formulation of montelukast in healthy Thai male volunteers.     

A validated enantioselective assay for the simultaneous quantitation of (R)-, (S)-fluoxetine and (R)-, (S)-norfluoxetine in ovine plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS)

A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantitation of (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine in ovine plasma. The analytes were extracted from ovine plasma at a basic pH using a single-step liquid-liquid extraction with methyl-tert-butyl ether. Chromatographic separation of all enantiomers was achieved using an AGP-chiral column with a run time of 10 min. (R)-, (S)-fluoxetine, and (R)-, (S)-norfluoxetine were quantitated at the total ion current (TIC) of multiple reaction monitoring (MRM) transitions of m/z 310.2→44.1, m/z 310.2→147.7 for (R)-, (S)-fluoxetine, and m/z 296.2→30.3, m/z 296.2→133.9 for (R)-, (S)-norfluoxetine. This method was validated for accuracy, precision, linearity, range, limit of quantitation (LOQ), selectivity, recovery, dilution integrity, matrix effect, and evaluation of carry-over. Observed accuracy ranges were as follows: (R)-fluoxetine -8.82 to 3.75%; (S)-fluoxetine -10.8 to 1.46%; (R)-norfluoxetine -7.50 to 0.37% and (S)-norfluoxetine -8.77% to -1.33%. Observed precision ranges were as follows: (R)-fluoxetine 5.29-11.5%; (S)-fluoxetine 3.91-11.1%; (R)-norfluoxetine 4.32-7.67% and (S)-norfluoxetine -8.77% to -1.33%. The calibration curves were weighted (1/X(2), n=4) and observed to be linear for all analytes with the following r(2) values: (R)-fluoxetine ≥ 0.997; (S)-fluoxetine ≥ 0.996; (R)-norfluoxetine ≥ 0.989 and (S)-norfluoxetine ≥ 0.994. The analytical range of the method was 1-500 ng/ml with an LOQ of 1 ng/ml for all analytes, using a sample volume of 300 μL.     

High performance liquid chromatography assay with ultraviolet detection for moxifloxacin: validation and application to a pharmacokinetic study in Chinese volunteers

A specific, sensitive and widely applicable high performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of moxifloxacin in human plasma was developed and validated in this study. The method involved a single step of liquid-liquid extraction with dichloromethane and the extraction yields more than 80% were achieved. The separation was performed on a common Kromasil C(8) column with an isocratic mobile phase. The total time was within 10 min per run. The calibration curve for moxifloxacin was linear in the concentration range of 0.05-5.0 μg/ml with correlation coefficient of 0.9997. The developed method was validated with excellent specificity, sensitivity, accuracy, precision and stability. Using this developed method, the pharmacokinetics of moxifloxacin in healthy Chinese volunteers was studied.     

A HPLC method for determination of nicousamide in dog plasma and its application to pharmacokinetic studies

A sensitive and reproducible high performance liquid chromatography (HPLC)-UV method for determination of nicousamide, an inhibitor of rennin and transforming growth factor-beta1 (TGF-beta1) type II receptors, has been developed and validated. Following acetonitrile deproteiniation, samples were separated by isocratic reversed-phase HPLC on an Aichrom Bond-AQ C(18) column and quantified using UV detection at 320 nm. The mobile phase was acetonitrile/water (ratio 62:38 containing 0.1% H(3)PO(4)), with a flow-rate of 1.0 ml/min. A linear curve over the concentration range 5-200 ng/ml (r(2)=0.9978) was obtained. The coefficients of the variation for the intra- and inter-day precisions ranged from 1.4-10.7% and 1.8-7.1%, respectively. The percentage of relative recovery was 91.56-105.45%. The method was used to determine the plasma concentration-time profiles for nicousamide after oral doses of 30, 100 and 300 mg/kg in dogs. A nonlinear pharmacokinetics was found in dogs at doses from 30 to 300 mg/kg. Following 30 mg/kg oral dose, the C(max) and AUC in females were lower than that in male. There is a potential for accumulation in dogs following multiple doses.     

Validated LC-MS-MS method for determination of m-nisoldipine polymorphs in rat plasma and its application to pharmacokinetic studies

A sensitive and specific liquid chromatography-tandem mass spectrometric (LC-MS-MS) method has been developed to determine m-nisoldipine in rat plasma. Sample was pretreated by a single-step protein precipitation with acetonitrile, in contrast to the liquid-liquid procedure frequently used for the extraction of 1,4-dihydropyridines from biologic samples. Separation of analyte and internal standard (I.S.) was performed on a Symmetry RP-C(18) analytic column (50 mm x 4.6 mm, 3.5 microm) with a mobile phase consisting of acetonitrile-water (80:20, v/v) at a flow rate of 0.5 ml/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) scan mode using TurboIonSpray ionization (ESI) source. The method was sensitive with a lower limit of quantification (LLOQ) of 0.2 ng/mL, with good linearity (r>or=0.9982) over the linear range of 0.2-20 ng/mL. All the validation data, such as accuracy, precision, and inter-day repeatability, were within the required limits. The method was successfully applied to pharmacokinetic and relative bioavailability studies of m-nisoldipine polymorphs in rats.     

Quantitative analysis of procarbazine, procarbazine metabolites and chemical degradation products with application to pharmacokinetic studies

Quantitative analytical methods are described for the analysis of the anticancer drug procarbazine and eight known metabolites including those known to have cytotoxic activity. A direct sample insertion mass spectrometric assay for procarbazine and the urinary excretion product, N-isopropyl-terephthalamic acid, has been developed. This method employs stable isotope labeled variants in a procedure that minimizes analytical errors that may be encountered in the quantitation of the chemically unstable parent drug. A liquid chromatographic method is described for the analysis of seven known procarbazine metabolites. Use of these methods is demonstrated by the analysis of procarbazine metabolism during incubation in a 9000-g rat liver homogenate preparation. Procarbazine disappearance and metabolite appearance are also monitored in rat plasma following intraperitoneal administration of a 150 mg/kg bolus dose. Applications to patient pharmacokinetics is demonstrated using the liquid chromatographic assay to follow the appearance of active procarbazine metabolites on the first and fourteenth day of an oral 250 mg/kg/day course of therapy of a patient being treated for cancer.     

Simple and rapid determination of carboplatin in plasma by high-performance liquid chromatography. Error pattern and application to clinical pharmacokinetic studies

Carboplatin is an antitumor agent widely employed in cancer chemotherapy. A specific and selective method for the determination of carboplatin in human plasma and its applications to pharmacokinetic investigations is described. One ultrafiltration step, through a Centrifree micropartition system (Amicon) at 2000 g for 10 min, is the only requirement as sample treatment. The resulting solution is injected into an Inertsil ODS-2 (5 μm, 25 cm×4.6 mm I.D.) analytical column. The mobile phase consisted of 0.1 M potassium dihydrogenphosphate with 1 mM dipotassium edetate adjusted to a pH between 3 and 3.5. The limit of quantitation was 1 mg/l. The method showed good recovery (100.68±5.49%) and precision: the within-day relative standard deviation (RSD) for carboplatin (3–350 mg/l) was 2.07% and the between-day RSD for carboplatin, in the previously described range, was 1.31%. We determined the assay error pattern for proper weighting of serum level data in pharmacokinetic models. The selectivity (discrimination between the parent drug and platinum-containing species such as carboplatin metabolites), simplicity and speed of this assay for free carboplatin quantitation should facilitate pharmacokinetic investigations and therapeutic drug monitoring.     

A useful route to (R)- and (S)-tert-leucine

Resolution of 1-(2-furyl)-2,2-dimethylpropylamine, an intermediate on a synthetic route to tert-leucine, followed by oxidation of the respective enantiomers, constitutes an interesting and useful strategy to (R)- and (S)-tert-leucine.