Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.     

Stereoselective determination of hydroxychloro-quine and its major metabolites in human urine by solid-phase microextraction and HPLC

The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid-phase microextraction. The chromatographic separation was performed on a Chiralcel OD-H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25 degrees C and the desorption time was 3 min using methanol (100%). PDMS-DVB 60 microm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50-1000 ng/mL for HCQ enantiomers and over the range of 42-416 ng/mL for DCQ and DHCQ enantiomers. Within-day and between-day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)-(S)-enantiomer excreted was higher than its antipode.     

Simultaneous determination of ketoprofen enantiomers and probenecid in plasma and urine by high-performance liquid chromatography.

A rapid, sensitive, stereospecific reversed-phase high-performance liquid chromatographic method was developed for simultaneous quantitation of ketoprofen enantiomers, probenecid and their conjugates in biological fluids. Following addition of the internal standard, indoprofen, the constituents were extracted into isooctane-isopropanol (95:5), water-washed, extracted with chloroform, then evaporated and the residue sequentially derivatized with ethyl chloroformate and L-leucinamide hydrochloride. The formed diastereomers were chromatographed on a reversed-phase column with a mobile phase of 0.06 M KH2PO4-acetonitrile-triethylamine (65:35:0.1) at a flow-rate of 1 ml/min and a detection wavelength of 275 nm. The minimum quantifiable concentration was 0.5 micrograms/ml in 100 microliters of rat plasma and urine samples. The intra- and inter-day coefficients of variation for this method are less than 10%. The assay is successfully applied to a pharmacokinetic study. The simultaneous analysis of probenecid with several other non-steroidal anti-inflammatory drugs was also successful.     

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed-phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Determination of the S(+)- and R(-)-enantiomers of baclofen in plasma and urine by gas chromatography using a chiral fused-silica capillary column and an electron-capture detector

A sensitive and enantiospecific gas chromatographic method for the determination of the S(+)- and R(-)-enantiomers of baclofen (I and II) in plasma and urine has been developed and validated. The method is based on the complete resolution of the derivatized enantiomers on a chiral fused-silica capillary column. The hydrochloride salt of a (-)-fluoro analogue of baclofen (III.HCl) was used as the internal standard in plasma, the hydrochloride salt of a (+)-fluoro analogue of baclofen (IV.HCl) as the internal standard in urine. Rapid and convenient isolation of the compounds was achieved using reversed-phase Bond-Elut C18 columns. After elution, the compounds were converted into isobutyl esters and purified by base-specific solvent extraction. The isobutyl esters were then N-acylated with heptafluorobutyric anhydride. The derivatives were quantitated after separation on the chiral column using electron-capture detection. The analysis of spiked plasma and urine samples demonstrated the good accuracy and precision of the method, with limits of quantitation of 25 nmol/l for I and II in plasma and of 2 mumol/l for I and II in urine. The method appears to be suitable for use in pharmacokinetic studies of the enantiomers in plasma and urine from animals and man after administration of the racemic baclofen.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Enantioselective high-performance liquid chromatography with fluorescence detection of methamphetamine and its metabolites in human urine.

A simple, rapid and highly sensitive high-performance liquid chromatographic method with fluorescence detection for determining the enantiomers of methamphetamine and its major metabolites, amphetamine and p-hydroxymethamphetamine, in urine samples was developed. Using a newly developed reagent for amines, namely, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, six enantiomers were derivatized under mild conditions (i.e., 10 min at room temperature, pH 9.0) and separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column following a pre-separation on an ODS column within 42 min, and the effluent was monitored at 440 nm (lambda ex 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05-100 mumol dm-3 with correlation coefficients > or = 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8-8.8 fmol per 5 microliters injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were < or = 7.4%. The method was successfully applied to discriminate between (S)-(+)-methamphetamine and its corresponding metabolites found in abusers' urine and their antipodes in a sample taken from a Parkinsonian patient on selegiline (Deprenyl) therapy.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection

A column-switching HPLC method was described for the direct analysis of clarithromycin in human plasma using electrochemical detector without sample pre-purification step. Plasma samples were diluted with washing solvent, i.e. acetonirile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (5:2:93, v/v) and then, injected to the precolumn. After plasma proteins had flowed out from the precolumn, clarithromycin and internal standard (roxithromycin) were eluted to a Luna 2 C(18) column and separated with acetonitrile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (41:6:53, v/v). Electrochemical oxidation of clarithromycin occurred at 0.87 V vs. Ag/AgCl reference electrode with glassy carbon electrode. The calibration curve was linear in the concentration range 0.1-4 mug ml(-1) with correlation coefficient of 0.998. This method showed excellent precision (RSD 3.8% at 0.1 mug ml(-1)) and accuracy (+/-2%) with the total analysis time per sample of 30 min. The present method was successfully applied to the pharmacokinetic study of clarithromycin in volunteers receiving a single oral administration of clarithromycin.     

Simultaneous determination of cefamandole and cefamandole nafate in human plasma and urine by high-performance liquid chromatography with column switching

A high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of cefamandole and cefamandole nafate in plasma and urine. The plasma and urine samples were injected onto a precolumn packed with Corasil RP C18 (37-50 microns) after simple dilution with an internal standard solution in 0.05 M phosphoric acid. Polar plasma and urine components were washed out using 0.05 M phosphoric acid. After valve switching, the concentrated drugs were desorbed in back-flush mode and separated by a reversed-phase C8 column with methanol-5 mM tetrabutylammonium bromide (45:55, v/v) as the mobile phase. The method showed excellent precision with good sensitivity and speed, and a detection limit of 0.5 microgram/ml. The total analysis time per sample was less than 30 min, and the mean coefficients of variation for intra- and inter-assay were both less than 4.9%. The method has been successfully applied to plasma and urine samples for human volunteers after intravenous injection of cefamandole nafate.     

Stereospecific high-performance liquid chromatographic determination of tocainide

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

Determination of remoxipride in human plasma and urine by reversed-phase ion-pair high-performance liquid chromatography.

A sensitive method is described for the measurement of remoxipride in human plasma and urine. Remoxipride and its internal standard are extracted from plasma or urine at pH 12 with a mixture of hexane and methyl tert.-butyl ether. After washing the organic phase with base, the compounds are extracted into acid and analyzed on a C18 column with ultraviolet detection at 214 nm. The mobile phase is composed of acetonitrile and aqueous buffer (sodium perchlorate and phosphoric acid, pH 1.7). The limits of reliable quantitation for remoxipride are 12.5 and 50 ng/ml for plasma and urine, respectively. The run times are 6 min for plasma and 3 min for urine. The method has been successfully used to assay remoxipride clinical study samples. This mobile phase has also been successfully applied to the analysis of other basic drugs such as cimetidine, codeine, diltiazem and quinidine with minor modifications.     

Enantioselective CE method for pharmacokinetic studies on ibuprofen and its chiral metabolites with reference to genetic polymorphism

A stereospecific CE method was elaborated for the quantification of ibuprofen enantiomers and their major phase I metabolites: 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen in plasma and urine. Optimal temperature and pH of BGE were established to obtain complete separation of eight ibuprofen chiral compounds and (+)-S indobufen, applied as an internal standard, during one analytical run. After isolation from biological matrices using SPE on an octadecyl stationary phase, the analytes were separated and resolved up to 10 min in a silica capillary filled with BGE, consisting of heptakis 2,3,6-tri-O-methyl-beta-CD in triethanolamine-phosphate buffer, pH 5.0. Complete enantioseparation of the all analytes confirmed specificity of the method. The calibration curves were linear in the range of 0.1-25.0 mg/L for IBP enantiomers and their chiral metabolites in 0.5 mL of plasma and 1.0-200.0 mg/L in 0.05 mL of urine. Following SPE procedure, recovery of the chiral analytes from the two media was in the ranges of 82-87%, 90-95% and 70-76% for ibuprofen, 2'-hydroxy-ibuprofen and 2'-carboxy-ibuprofen enantiomers, respectively. The validated method was successfully applied in pharmacokinetic investigations of IBP enantiomers as well as free chiral metabolites in reference to the genetic polymorphism of CYP450 2C isoenzymes.     

柱切换技术-高效液相色谱法快速检测大鼠血浆中的普萘洛尔对映体

建立了快速检测大鼠血浆中普萘洛尔对映体浓度的柱切换-高效液相色谱法。将自制限进填料柱作为预处理柱,通过直接进样方式,使普萘洛尔对映体在预处理柱上保留,同时除去血浆中的蛋白质等大分子;再通过柱切换技术,使普萘洛尔对映体在键合型纤维素-三(3,5-二甲基苯基氨基甲酸酯)(Chiralcel OD-RH)分析柱上得到手性拆分。通过条件优化,确定切换前预处理流动相为硼酸盐缓冲液(pH 8.5)-甲醇(95:5, v/v),流速为1.0 mL/min;切换后分析流动相为异丙醇-乙醇-0.2 mmol/L硼酸盐缓冲液(pH 8.5)(30:30:40, v/v/v),流速为0.8 mL/min;切换时间为3 min;柱温为25 ℃;检测波长为293 nm。普萘洛尔两对映体在25～500 mg/L的质量浓度范围内具有良好的线性关系(r=0.9995), 3个加标水平(50、100、250 mg/L)的平均回收率为97.89%～101.56%,日内和日间精密度均小于5%。该方法简便、快速、灵敏、准确,适于血浆样本中手性药物的药代动力学研究。     

High-performance liquid chromatographic assay for 1-aminocyclopropanecarboxylic acid from plasma and brain.

A reversed-phase high-performance liquid chromatographic method for the analysis of 1-aminocyclopropanecarboxylic acid (ACPC) from plasma or brain tissue is described. Samples were deproteinized with perchloric acid, centrifuged, alkalinized with potassium hydroxide and recentrifuged. The supernatants were derivatized with o-phthaldialdehyde and injected onto a C18 3-microns column (100 mm x 4 mm I.D.) pumped with 1 ml/min methanol-acetonitrile-0.1 M sodium phosphate buffer pH 6.0 (28:5:67, v/v). The retention times for ACPC and the internal standard were 15 and 31 min, respectively. The minimum detectable amount of ACPC was 0.08 nmol. The extraction recovery of ACPC (2.7-270 nmol) from spiked plasma or brain tissue ranged from 88 to 109%. The intra- and inter-day coefficients of variation for 27 nmol ACPC were 3.9 and 4.9%, respectively. This method was utilized to obtain preliminary pharmacokinetic parameters following ACPC administration to mice.     

Determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma by high-performance liquid chromatography and fluorescence detection

A high-performance liquid chromatographic method for the simultaneous determination of 7-amino-flunitrazepam (Ro 20-1815) and 7-amino-desmethylflunitrazepam (Ro 5-4650) in plasma is described. After extraction with an organic solvent, the compounds and their internal standard (7-amino-methylclonazepam or Ro 5-3384) are derivatized with fluorescamine and chromatographed on a reversed-phase muBondapak C18 column using pH 8 buffer solution-acetonitrile (3:1) as mobile phase. The detection is performed by a fluorometer at excitation and emission wavelengths of 390 and 470 nm, respectively. The sensitivity limit is about 1 ng/ml of plasma for both 7-amino-flunitrazepam and 7-amino-desmethylflunitrazepam. The method has been applied to the determination of plasma levels of these substances during pharmacokinetic studies of flunitrazepam, desmethylflunitrazepam and 7-amino-flunitrazepam.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.