Stereospecific high-performance liquid chromatographic assay of metoprolol

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (R_s > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Stereospecific high-performance liquid chromatographic analysis of ibuprofen in rat serum

A simple, rapid and sensitive high-performance liquid chromatographic method was developed for determination of ibuprofen, (+/-)-(R, S)-2-(4-isobutylphenyl)-propionic acid, enantiomers in rat serum. Serum (0.1 ml) was extracted with 2,2,4-trimethylpentane/isopropanol (95:5, v/v) after addition of the internal standard, (S)-naproxen, and acidification with H(2)SO(4). Enantiomeric resolution of ibuprofen was achieved on ChiralPak AD-RH column with ultraviolet (UV) detection at 220 nm without interference from endogenous co-extracted solutes. The calibration curve demonstrated excellent linearity between 0.1 and 50 microg/ml for each enantiomer. The mean extraction efficiency was >92%. Precision of the assay was within 11% (relative standard deviation (R.S.D.)) and bias of the assay was lower than 15% at the limit of quantitation (0.1 microg/ml). The assay was applied successfully to an oral pharmacokinetic study of ibuprofen in rats.     

Stereospecific high-performance liquid chromatographic assay of isosakuranetin in rat urine

A stereospecific method of analysis of racemic isosakuranetin (5,7-dihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study pharmacokinetics. A simple high-performance liquid chromatographic method was developed for the determination of isosakuranetin enantiomers. Separation was achieved on a Chiralpak AD-RH column with ultraviolet (UV)-detection at 286 nm. The standard curves in urine were linear ranging from 0.5 to 100.0 microg/ml for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV) and was within 12% at the limit of quantitation (0.5 microg/ml). Bias of the assay was <15% and was within 6% at the limit of quantitation. The assay was applied successfully to stereospecific disposition of isosakuranetin enantiomers in rat urine.     

Stereospecific high-performance liquid chromatographic assay of ketoprofen in human plasma and urine

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of ketoprofen (KT), a 2-arylpropionic acid (2-APA) non-steroidal antiinflammatory drug (NSAID), in plasma and urine was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), plasma containing the KT enantiomers and I.S. was extracted by liquid-liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in mobile phase and injeted into the HPLC system. The enantiomers were separated at ambient temperature on a commercially available 250 × 4.3 mm amylose carbamate-packed chiral column (Chiralpak AD) column with hexane-isopropyl alcohol-trifluoroacetic acid (80:19.9:0.1, v/v/v) as the mobile phase pumped at 1.0 ml/min. The enantiomers of KT were quantified by ultraviolet detection with the wavelength set at 254 nm. The assay described allows for the direct quantification of KT enantiomers without pre-column derivatization, and is suitable for clinical studies of KT enantiomers in human plasma and urine after administration of therapeutic doses.     

Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

Rapid high-performance liquid chromatographic assay for atovaquone

A rapid high-performance liquid chromatography assay has been developed for the drug atovaquone, which is currently being used to treat Pneumocystis carinii pneumonia and Taxoplasma gondii encephalitis associated with the acquired immunodeficiency syndrome (AIDS). Protein is precipitated from plasma with acetonitrile-aqueous 1% acetic acid (85:15). The supernatant is assayed on a C₆ column using methanol-10 mM triethylamine in aqueous 0.2% trifluoroacetic acid (76:24) with detection at 254 nm. The working assay range was 0.5 to 50 μg/ml. Recovery was 97% and the between-day coefficients of variation were 2.1% at 50 μg/ml and 10.3% at 1 μg/ml. A number of drugs commonly used to treat AIDS and its complications did not interfere with the assay.     

Sensitive high-performance liquid chromatographic determination of nifedipine in cat plasma following improved sample treatment

A simple, easy and accurate reversed-phase high-performance liquid chromatographic method is described for the determination of nifedipine in cat plasma. The procedure involves extraction of nifedipine from plasma using a Sep-Pak C₁₈ cartridge and ultraviolet detection at 350 nm. The present method provides the required reproducibility and sensitivity for the determination of low concentrations of nifedipine without interference from plasma components or photodegradation products. The method was validated over the range 1–50 ng/ml nifedipine. Accuracy and precision were, respectively, 97% or more and 5% or less over the concentration range examined. The minimum quantifiable concentration of nifedipine was found to be 1 ng/ml.     

Automated high-performance liquid chromatographic assay of enzymatic activities

High-performance liquid chromatography (HPLC) is a powerful technique which enables a reliable and quantitative determination of enzyme activities. The purpose of the work reported here was to develop an automatic assay of enzymatic activity. Using an automatic sample processor and injector, a program was developed which allows the complete automation of each step of analysis (calibration, enzymatic reaction, HPLC determination). This program can be adapted to different experimental requirements as each step can be performed independently and each input (time, volume, number of standards) is made by answering questions asked by instrument. Using this approach both kinetic and single-point determinations can be carried out, and in the latter case different samples can be analysed sequentially. This paper reports the automated analysis of trypsin.     

Paired-ion high-performance liquid chromatographic assay for plasma choline

Choline was isolated from deproteinized plasma by cation-exchange chromatography. Isolated choline was directly converted to the 3,5-dinitrobenzoate derivative and was analyzed by paired-ion high-performance liquid chromatography with UV detection at 254 nm. An internal standard, 3-hydroxy-N,N,N-trimethylpropanaminium iodide was used for quantitation of plasma choline.Linearity was achieved from 1–500 nmole/ml with a reproducibility of ± 6%. Plasma choline concentrations below 1 nmole/ml could not be accurately measured while plasma choline concentrations in the μmole/ml range deviated from linearity.     

Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions

This paper reports change to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml⁻¹ 6-MMP were 99.0±6.0% and 96.3±4.0% respectively and the limit of quantification was lowered to 5 ng ml⁻¹. This method, which does not require radioactive S-adenosyl- -methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltranferase activity in red blood cells.     

Ion-pair high-performance liquid chromatographic assay of 4-aminopyridine in serum

An assay for the quantitative estimation of 4-aminopyridine in biological fluids has been developed using 2-aminopyridine as internal standard and ion-pair reversed-phase (C_1$$$) high-performance liquid chromatography with detection at 263 nm. A 7.5% solution of acetonitrile in water containing tetrabutylammonium iodide and sodium heptanesulfonate buffered at pH 3.0 provided excellent separation of the analytes from each other and from an interfering peak that was occasionally observed in the outdated human sera used in these studies. Sensitivity, specificity, precision, accuracy and reproducibility all were judged sufficient for the routine use of this assay for pharmacokinetic and pharmacodynamic studies.     

Automation of high-performance liquid chromatographic sample clean-up for mass fragmentographic assays

A previously described high-performance liquid chromatographic (HPLC) sample clean-up procedure has been automated by attaching a (DuPont) auto-sampler and a time-controlled fraction collector to the HPLC equipment. To obtain the required reliability for unattended operation, the sample intake was controlled by volume rather than by time, and the system was protected against sample loss due to non- or improper operation of the injection valve. The capacity of the system depends on the HPLC run time per sample but varies from 45 to 135 samples per 24 h. The recovery and reproducibility are comparable to the manually operated system, while carry-over to subsequent samples is prevented by intermittent injection of the HPLC solvent system as flush fluid.     

Sensitive high-performance liquid chromatographic assay for norfloxacin utilizing fluorescence detection

A rapid, sensitive and reproducible reversed-phase high-performance liquid chromatographic assay was developed for the determination of norfloxacin. Following protein precipitation with 10% trichloroacetic acid, norfloxacin and the internal standard enoxacin were extracted from plasma with chloroform, dried and reconstituted in the mobile phase. The chromatographic separation of norfloxacin and the internal standard enoxacin was achieved on a C₈ column with fluorescence detection set at 280 and 418 nm for excitation and emission, respectively. The peaks with a resolution factor greater than 1.5 were free from interferences. Excellent linearity (r² 0.998) was observed over the concentration range 0.025–5.0 μg/ml in plasma. The inter-assay variability was 13.6% or less at all concentrations examined. The suitability of the assay for pharmacokinetic studies was determined by measuring norfloxacin concentration in rat plasma after administration of a single intravenous 10 mg/kg dose.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the chiral reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C₈ column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Stability-indicating high-performance liquid chromatographic assay of busulfan in aqueous and plasma samples

A sensitive, specific and stability-indicating high-performance liquid chromatographic (HPLC) assay, involving pre-column derivatization and solid-phase extraction (SPE), was developed and validated for the quantitation of busulfan (BU) in aqueous and plasma samples. The linearity of the assay was in the concentration ranges of 0.15–10 μg/ml and 0.15–3 μg/ml for aqueous and plasma samples, respectively. The within-day and between-day variations were 2.90 and 3.31%, respectively, for the aqueous samples, and 9.24 and 14.56%, respectively, for the plasma samples. The overall recovery, derivatization yield and SPE efficiency of BU from plasma samples were 82.03, 108.01 and 86.69%, respectively. Forced degraded samples, either in highly acidic, neutral or basic medium, produced no interfering peaks in the chromatogram. The reported assay requires only 0.2 ml of plasma for the analysis, and its sensitivity is 150 ng/ml by monitoring samples at a wavelength of 254 nm, sufficient to study the plasma pharmacokinetics of BU in rats after a clinically relevant oral dose. Moreover, the sensitivity of the assay can be significantly increased to 30 ng/ml by monitoring samples at a wavelength of 278 nm. The applications of the assay were demonstrated with BU solubility measurements in two aqueous systems and with plasma samples from a Sprague–Dawley rat for an in vivo pharmacokinetic study. In addition, the assay has been employed in the development of a patented intravenous formulation, and in evaluations of stability, preclinical pharmacokinetics in rats and dogs, and clinical phase I trial of the formulation. The assay is readily adaptable to clinical therapeutic drug monitoring.     

Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay

A new liquid–liquid extraction is described for thiopurine methyl transferase (TPMT, EC 2.1.1.67) activity determination: the use of a pH 9.5 NH₄Cl buffer solution, before adding the solvent mixture, allows more rapid extraction, avoiding a centrifugation step, and reduces the global cost of analysis. After the extraction step, 6-methylmercaptopurine, synthesised during the enzymatic reaction, is determined by a liquid chromatographic assay. Analytical performance of the assay was tested on spiked erythrocyte lysates. The linear concentration range was 5–250 ng ml ⁻¹ (r≥0.997, slope=1.497, intercept=−0.367). The recoveries were 82.8, 89.9 and 82.2% for 75, 125 and 225 ng ml⁻¹, respectively. The coefficients of variation were ≤6.1% for within-day assay (n=6) and ≤9.5% for between-day assay precision (n=6; 14 days). TPMT activity was determined in a French adult Caucasian population (n=70). The results ranged from 7.8 to 27.8 nmol h⁻¹ ml⁻¹ packed red blood cells and the frequency distribution histogram is similar to that previously published.     

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2·10⁸ particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.