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.     

Validation of a high-performance liquid chromatographic method for the determination of ibuprofen enantiomers in plasma of broiler chickens

To characterise the pharmacokinetic properties of each enantiomer of ibuprofen in broiler chickens, a stereospecific HPLC method based on a α₁-acid glycoprotein bonded chiral stationary phase has been validated. S-(+)-naproxen was used as internal standard. Enantiomers of ibuprofen and S-(+)-naproxen were baseline separated using a mobile phase consisting of 0.1 M phosphate buffer pH=7 and 0.4% 2-propanol. The method is precise, specific, accurate and reproducible. Recoveries were higher than 80% and the limits of quantification for R-(−)- and S-(+)-ibuprofen were 1.16 and 1.37 μg ml⁻¹, respectively. The method seemed suitable for the pharmacokinetic studies of ibuprofen in chickens.     

A new high-performance liquid chromatographic method for determination of warfarin enantiomers

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.     

Improved high-performance liquid chromatographic assay for the determination of ethionamide in serum

A solid-phase extraction (SPE) method was developed to simplify the preparation of human serum prior to high-performance liquid chromatography of ethionamide (ETA). Octadecyl SPE columns were used. Serum constituents were removed from the column with water, and ETA was eluted with methanol. Samples were evaporated to dryness, reconstituted in mobile phase, and assayed. The method is reproducible, with a recovery of ETA of 64%, comparable to the more tedious liquid-liquid extraction method for ETA.     

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.     

Stereospecific high-performance liquid chromatographic assay of ibuprofen: improved sensitivity and sample processing efficiency

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of the non-steroidal anti-inflammatory drug ibuprofen (IB) in plasma was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), samples are acidified and extracted with a mixture of isooctane—isopropanol (95:5, v/v). After evaporation of the organic layer, the drug and I.S. are derivatized with S-(−)-1(1-naphthyl)ethylamine (S-NEA) after addition of ethyl chloroformate as the coupling reagent. Ethanolamine is added 3 min after the addition of S-NEA to react with the excessive ethyl chloroformate. The resultant diastereomers corresponding to IB and I.S. were chromatographed at ambient temperature on a 100 mm × 4.6 mm I.D. C₁₈ reversed-phase column using acetonitrile—water—acetic acid—triethylamine (60:40:0.1:0.02) as the mobile phase pumped at a flow-rate of 1.2 ml/min. Detection of the fluorescent chromophore was at 280 and 320 nm for excitation and emission, respectively. The suitability of the assay for clinical pharmacokinetic studies of IB was determined by the analysis of plasma samples obtained from a healthy volunteer, following administration of a single 400-mg oral dose of racemic IB.     

A high-performance liquid chromatographic method for the assay of cholesterol 7 alpha-hydroxylase activity

A high-performance liquid chromatographic method has been developed for the measurement of cholesterol 7 alpha-hydroxylase activity in liver microsomes. 7 alpha-Hydroxycholesterol generated from endogenous cholesterol was derivatized with anthroyl 1-carbonitrile, chromatographed on a reverse-phase column, and detected fluorometrically. The detection limit of 7 alpha-hydroxycholesterol was 1 ng/tube. The cholesterol 7 alpha-hydroxylase activity in rat liver microsomes was assayed by this method, and the effects of some detergents and of the addition of exogenous cholesterol together with detergents on the enzyme activity were investigated. The endogenous 7 alpha- and 7 beta-hydroxycholesterol could be also measured by this method.     

Novel sensitive high-performance liquid chromatographic method for assay of coumarin 7-hydroxylation

In this paper, a novel HPLC-based method with fluorometric detection of coumarin 7-hydroxylase is presented. The described method provides a time-effective, more sensitive and specific alternative to the previously used spectrofluorometric assay. Using the developed method, metabolism of coumarin in 11 samples of human liver microsomes was evaluated and 1790±690 pmol/min/nmol cytochrome P450 (CYP) activity was found. Kinetic parameters and linearity of coumarin 7-hydroxylation were studied in a reconstituted system consisting of recombinant CYP2A6 expressed in Escherichia coli, rat NADPH-CYP reductase and usual components. It was found that a 3.5 to 30 min time of incubation is suitable for estimation of coumarin 7-hydroxylase activity. Observed K_m and V_max values in the CYP2A6 reconstituted system were 1.48±0.37 μM and 3360±180 pmol product/min/nmol CYP, respectively.     

Improved high-performance liquid chromatographic method in the analysis of adenovirus particles

We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.     

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.     

Rapid and sensitive high-performance liquid chromatographic method for busulfan assay in plasma

A reversed-phase liquid chromatographic method with ultraviolet detection has been developed to determine busulfan concentrations in plasma of children undergoing bone marrow transplantation. Plasma samples (200 μl) containing busulfan and 1,6-bis(methanesulfonyloxy)hexane as an internal standard were prepared by a simple derivatization method with diethyldithiocarbamate followed by extraction with ethyl acetate and solid-phase purification on C₈ columns conditioned with methanol and water and eluted with acetonitrile (recovery 99%). Chromatography was accomplished using a Hypersil octadecylsilyl column (10 cm×4.6 mm I.D.) and a mobile phase of acetonitrile, tetrahydrofuran and distilled water (65:5:30, v/v). The limit of detection was 25 ng/ml (signal-to-noise ratio of 5). Calibration curves were linear up to 25 000 ng/ml. Intra-day and inter-day coefficients of variation of the assay were ≤5%. This method was used to analyse busulfan plasma concentrations after oral administration within the framework of therapeutic drug monitoring and pharmacokinetic studies in children.     

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.     

Validation of a high-performance liquid chromatographic assay method for pharmacokinetic evaluation of busulfan

The development and validation of a high-performance liquid chromatographic (HPLC) assay for determination of busulfan concentrations in human plasma for pharmacokinetic studies is described. Plasma samples containing busulfan and 1,6-bis(methanesulfonyloxy)hexane, and internal standard, were prepared by derivatization with sodium diethyldithio-carbamate (DDTC) followed by addition of methanol and extraction with ethyl acetate. The extract was dried under nitrogen and the samples reconstituted with 100 μl of methanol prior to HPLC determination. Chromatography was accomplished using a Waters NovaPak octadecylsilyl (ODS) (150×3.9 mm I.D.) analytical column, NovaPak ODS guard column, and mobile phase of methanol-water (80:20, v/v) at a flow-rate of 0.8 ml/min with UV detection at 251 nm. The limit of detection was 0.0200 μg/ml (signal-to-noise ratio of 6) with a limit of quantitation (LOQ) of 0.0600 μg/ml for busulfan in plasma. Calibration curves were linear from 0.0600 to 3.00 μg/ml in plasma (500 μl) using a weighting scheme. Precision of the assay, as represented by C.V. of the observed peak area ration values, ranged from 4.41 to 13.5% (13.5% at LOQ). No day-to-day variability was observed in predicted concentration values and the bias was low for all concentrations evaluated (bias: 0 to 4.76%; LOQ: 2.91%). The mean derivatization and extraction yield observed for busulfan in plasma at 0.200, 1.20 and 2.00 μg/ml was 98.5% (range 93.4 to 107%). Plasma samples containing potential busulfan metabolites and co-administered drugs, which may be present in clinical samples, provided no response indicating this assay procedure is selective for busulfan. This method was used to analyze plasma concentrations following administration of a 1 mg/kg oral busulfan dose.     

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.     

Tissue extraction and high-performance liquid chromatographic determination of ketoprofen enantiomers

Local transcutaneous delivery of non-steroidal anti-inflammatory drugs avoids gastrointestinal side effects and concentrates drugs in the intended tissues. An extraction and HPLC method was developed for ketoprofen in skin, fascia and muscle. Tissue samples were homogenized in NaHCO₃. After methylene chloride removal of lipids, the aqueous layer was acidified with HCl and back extracted into isooctane/isopropanol. Ketoprofen was derivatized with ethylchloroformate/S-(−)-α-phenylethylamine in triethylamine, then detected by HPLC. Ketoprofen recovery was linear (1–33 μg/g) and was detected in these tissues following in vivo cathodic iontophoresis (160 mA*min). This represents the first non-radioactive method for determination of ketoprofen in tissues following transcutaneous iontophoresis.     

Rapid reversed-phase high-performance liquid chromatographic method with double derivatization for the assay of urinary hydroxyproline

An HPLC method with two derivatizations, the first with o-phthaldehyde in order to eliminate interferences due to some primary amino acids eluting with retention times similar to those of hydroxyproline and the second with dabsyl chloride, was developed and evaluated. Calibration graph linearity, influence of agitation and temperature on the preparation of the first derivative and the influence of the detection wavelength were assessed. The analysis time is shorter in comparison with other available methods, and therefore this method is suitable for laboratories that analyse both small and large series of samples.     

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.     

Simple high-performance liquid chromatographic method to verify the direct barbituric acid assay for urinary cotinine

An isocratic HPLC method is described to determine urinary concentrations of nicotine and cotinine after derivatization with cyanogen chloride and barbituric acid. This method has been used to assess the reliability of the direct barbituric acid assay to determine smoking status. It is concluded that the direct barbituric acid assay is a very reliable indicator of smoking status, provided that urine blank samples are prepared to correct for background absorbance. If the direct barbituric acid assay is in disagreement with self-reported smoking status, this HPLC procedure is a useful method to resolve the discrepancy.     

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.