Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography–electrospray mass spectrometry (LC–ES-MS) was developed and evaluated for the simultaneous quantitative determination of caffeine metabolites 1U, 1X and AAMU in human urine. This method involved a simple dilution of urine samples. The chromatographic separation was achieved on a C₁₈ reversed-phase column using a gradient of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was performed in the negative ion, selected ion monitoring mode. This method yielded acceptable accuracy and precision within the range 0.25–50 μg/ml. This analytical method was applied to investigate the N-acetylator phenotype of HIV-infected patients and compared with high-performance liquid chromatography with UV detection. Its specificity was better, which appeared to be absolutely necessary to prevent errors in metabolic ratios and phenotype interpretation.     

Identification of substance P metabolites using a combination of reversed-phase high-performance liquid chromatography and capillary electrophoresis

Gradient elution reversed-phase high-performance liquid chromatographic and capillary electrophoretic separations were optimised to separate substance P (SP) and twelve of its fragments. The methods were applied to a study of the in vivo metabolism of substance P in the rat after intrastriatal injection of the peptide (10 nmol). SP and significant amounts of its N-terminal fragments, SP(1-7) and SP(1-4), were detected but no major C-terminal fragments could be identified. At the concentration studied, the metabolism of SP was shown to follow zero order elimination kinetics with a rate of decay of 0.2 nmol/min. As we have shown that SP(1–4) and SP(1–7) can be produced in vivo in the striatum in relatively large amounts, it is conceivable that these fragments contribute to the overall pharmacological pattern of activity of the parent peptide.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Determination of N-acetyl-L-glutamate using high-performance liquid chromatography

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.     

Isolation of alpha-latrotoxin using high-performance liquid chromatography methods

alpha-Latrotoxin, a main toxic component of the Latrodectus mactans tredecimguttatus venom is a large polypeptide with molecular weight of 130 KDa. A rapid method is suggested for isolating this protein using high-effective liquid chromatography on chromatograph FPLC ("Pharmacia", Sweden). The isolated protein does not differ from the previously described alpha-latrotoxin in the main physicochemical parameters as well as in physiological properties.     

Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis

Chemically modified phosphorothioate oligodeoxynucleotides (ODNs) have become critical tools for research in the fields of gene expression and experimental therapeutics. Bioanalytical assays were developed that utilized fast anion-exchange high-performance liquid chromatography (HPLC) and capillary gel electrophoresis (CGE) for the determination of 20-mer ODNs in biological fluids (plasma and urine) and tissues. A 20 mer ODN in the antisense orientation directed against DNA methyltransferase (denoted as MT-AS) was studied as the model ODN. The anion-exchange HPLC method employed a short column packed with non-porous polymer support and a ternary gradient elution with 2 M lithium bromide containing 30% formamide. Analysis of the MT-AS is accomplished within 5 min with a detection limit of approximately 3 ng on-column at 267 nm. For plasma and urine, samples were diluted with Nonidet P-40 in 0.9% NaCl and directly injected onto the column, resulting in 100% recovery. For tissue homogenates, a protein kinase K digestion and phenol–chloroform extraction were used, with an average recovery of about 50%. Since the HPLC assay cannot provide one-base separation, biological samples were also processed by an anion-exchange solid-phase extraction and a CGE method to characterize MT-AS and its catabolites of 15–20-mer, species most relevant to biological activity. One base separation, under an electric field of 400 V/cm at room temperature, was achieved for a mixture of 15–20-mer with about 50 pg injected. Assay validation studies revealed that the combined HPLC–CGE methods are accurate, reproducible and specific for the determination of MT-AS and its catabolites in biological fluids and tissue homogenates, and can be used for the pharmacokinetic characterization of MT-AS.     

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.     

Comparison of high-performance liquid chromatography and capillary zone electrophoresis in penciclovir biodegradation kinetic studies

High-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) were used in biodegradation kinetic studies. This paper describes a rapid penciclovir separation using CZE with detection limits comparable to HPLC. The ionic-strength mediated stacking technique was employed while good resolution was maintained. With a shorter analysis time, comparable detection limits and no organic solvent consumption, CZE is a better method for penciclovir biodegradation studies than conventional reversed-phase HPLC (RP-HPLC).     

Analysis of ellagic acid in pomegranate rinds by capillary electrophoresis and high-performance liquid chromatography

Two novel methods for the analysis of ellagic acid in pomegranate (Punica granatum) rinds are proposed. Capillary electrophoresis (CE) was performed in a bare fused-silica capillary using a buffer solution of tri(hydroxymethyl)aminomethane:potassium dihydrogen phosphate (pH 8.4) with an applied voltage of 20 kV and UV detection at 254 nm. HPLC analysis was performed with a Zobax SB C(18) column and a mobile phase consisting of methanol:ethyl acetate:potassium dihydrogen phosphate: phosphoric acid at a flow rate of 1.0 mL/min. Under optimised conditions, the HPLC retention and the CE migration times for ellagic acid were 10.32 and 12.23 min, respectively. Calibration curves of peak area vs. concentration gave correlation coefficients of 0.9999 for HPLC and 0.9990 for CE. The detection limits for HPLC and CE were 2.8 and 2.2 microg/mL, respectively. Average recoveries were 98.32 +/- 1.2% for HPLC and 96.52 +/- 2.8% for CE. Both methods were shown to be suitable for the determination of ellagic acid in pomegranate rinds extraction; however, the CE method required less solvent and gave better column efficiency, whilst the HPLC provided superior precision.     

Determination of plasma theophylline by straight-phase high-performance liquid chromatography: Elimination of interfering caffeine metabolites

Several authors have recently reported interference in theophylline analysis by paraxanthine (1,7-dimethylxanthine), an important metabolite of caffeine. A method for the determination of theophylline in plasma is described, eliminating caffeine and related compounds by means of straight-phase high-performance liquid chromatography. The resulting procedure is sufficiently rapid, accurate and sensitive to be applied in routine monitoring of therapeutic levels in patients as well as for pharmacokinetic purposes. Although only 0.1 ml of sample is required, concentrations as low as 0.2 mg/l can be measured with acceptable precision. A brief comparative evaluation of this procedure with a radioimmunoassay is made.     

Determination of chlorhexidine in saliva using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of chlorhexidine in saliva is reported. The method developed includes a simple and short sample preparation with a one-step extraction procedure and a short total chromatographic run time of 5 min. In a preliminary pharmacokinetic study with a healthy volunteer the chlorhexidine concentration found in saliva after 12 h was 0.8 μg/ml.     

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.     

Effect of temperature on the separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis: a comparative study

This study investigates the effect of experimental temperature on the separation of DNA fragments, 21–587 bp, by both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The results show that the temperature plays an important role in the HPLC separation of DNA fragments. The optimum temperature was found to be between 40 and 50°C for HPLC, while 25°C was the optimum temperature for the CE separation. Also, although CE migration times became shorter, efficiency and resolution decreased with an increase in temperature from 25 to 50°C, but the separation was not significantly affected. Also, the optimum HPLC temperature might be different depending on the fragment sizes to be resolved.     

Investigation of the stereoselective in vitro biotransformation of glutethimide by high-performance liquid chromatography and capillary electrophoresis

Due to our interest in drugs with a glutarimide structure, we reinvestigated the stereoselectivity of the in vitro biotransformation of the chiral hypnotic-sedative drug glutethimide. Glutethimide enantiomers were separated on a preparative scale by HPLC on cellulose tris(4-methylbenzoate) as chiral stationary phase. The enantiometric purity was higher than 99%. A reversed-phase HPLC method was developed to determine the metabolites of glutethimide. After incubations with rat liver microsomes both enantiomers formed 5-hydroxyglutethimide as the main metabolite, as well as additional metabolites, of which some were formed stereoselectivity. Mass spectrometry of the unknown metabolites indicated a hydroxylation in the ethyl side chain for two of the metabolites. A third metabolite was tentatively identified as desethylgutethimide.     

High-throughput determination of urinary hexosamines for diagnosis of mucopolysaccharidoses by capillary electrophoresis and high-performance liquid chromatography

Mucopolysaccharidoses (MPS) diagnosis is often delayed and irreversible organ damage can occur, making possible therapies less effective. This highlights the importance of early and accurate diagnosis. A high-throughput procedure for the simultaneous determination of glucosamine and galactosamine produced from urinary galactosaminoglycans and glucosaminoglycans by capillary electrophoresis (CE) and HPLC has been performed and validated in subjects affected by various MPS including their mild and severe forms, Hurler and Hurler-Scheie, Hunter, Sanfilippo, Morquio, and Maroteaux-Lamy. Contrary to other analytical approaches, the present single analytical procedure, which is able to measure total abnormal amounts of urinary GAGs, high molecular mass, and related fragments, as well as specific hexosamines belonging to a group of GAGs, would be useful for possible application in their early diagnosis. After a rapid urine pretreatment, free hexosamines are generated by acidic hydrolysis, derivatized with 2-aminobenzoic acid and separated by CE/UV in ∼10 min and reverse-phase (RP)-HPLC in fluorescence in ∼21 min. The total content of hexosamines was found to be indicative of abnormal urinary excretion of GAGs in patients compared to the controls, and the galactosamine/glucosamine ratio was observed to be related to specific MPS syndromes in regard to both their mild and severe forms. As a consequence, important correlations between analytical response and clinical diagnosis and the severity of the disorders were observed. Furthermore, we can assume that the severity of the syndrome may be ascribed to the quantity of total GAGs, as high-molecular-mass polymers and fragments, accumulated in cells and directly excreted in the urine. Finally, due to the high-throughput nature of this approach and to the equipment commonly available in laboratories, this method is suitable for newborn screening in preventive public health programs for early detection of MPS disorders, diagnosis, and their treatment.     

Comparative determination of purine compounds in carotid plaque by capillary zone electrophoresis and high-performance liquid chromatography

Allantoin, uric acid (UA), hypoxanthine (Hx) and xanthine (X) were determined on carotid plaque by capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC). Comparison of the results showed that capillary zone electrophoresis may have similar or even superior analytical performance to HPLC, especially for the determination of allantoin in biological samples.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Recent developments of enantioseparation techniques for adrenergic drugs using liquid chromatography and capillary electrophoresis: a review

This review provides the achievements of enantioseparation of adrenergic drugs and application of these methods in clinical and pharmaceutical analysis. The adrenergic agonist and antagonist drugs are analyzed in the direct and indirect modes by liquid chromatography (LC) and capillary electrophoresis (CE). Other chromatographic enantioseparation methods including super- and sub-critical fluid chromatography (SFC), and capillary electrochromatography (CEC) are presented likewise to analyse the cited compounds. The different separation processes for these drugs are briefly discussed and some applications are presented.