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PubMedDate not supplied

High performance liquid chromatography coupled with radioactivity detection: a powerful tool for determining drug metabolite profiles in biological fluids.

High performance liquid chromatography coupled with continuous radioactivity detection represents an advancement in drug metabolism research. Using radioactive substances labelled in biologically stable positions, all metabolites can be specifically detected by radioactivity measurement. Thus no clean-up of biological fluids is required prior to HPLC. This can prevent artefact formation from unstable metabolites, reduces recovery problems and facilitates quantitation. Separation of highly polar and unpolar metabolites is possible in a single chromatographic run using gradient elution and reversed phase materials. This technique is also well-suited for preparative isolation and purification of metabolites for subsequent structure elucidation. Various metabolite profiles of drugs labelled with carbon-14 or tritium are shown. Metabolites of the following drugs are presented: norfenefrine, etozolin, thymoxamine, naloxone, and levobunolol. We review the general methodology and report our experience with this technique. In principle, this technique may be useful for all biological systems in which tracer techniques are applied.

2-Hydroxyphenethylamine↗

PubMed1984

Leukotriene D4 and E4 formation by plasma membrane bound enzymes.

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C4, D4 and E4. The enzymes responsible for the formation of LTA4 and LTB4 are in the soluble fraction while the enzymes for LTC4, LTD4 and LTE4 are particulate (10,000 X g pellet). Centrifugation of the 10,000 X g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet). The fractions were incubated with LTC4, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes gamma-glutamyl transpeptidase and amino peptidase which convert LTC4 to LTD4 and LTD4 to LTE4, respectively. When incubated with LTC4, the membrane fraction showed a dose dependent formation of LTD4 and a time course which reached a plateau at 30 to 45 minutes. Addition of serine.borate blocked the formation of LTD4, and cysteine blocked LTE4 production. The sucrose pellet showed little conversion of LTC4 to LTD4. We conclude that the gamma-glutamyl transpeptidase and the amino peptidase which produce LTD4 and LTE4 respectively are plasma membrane bound.

Aminopeptidases↗

PubMedDate not supplied

Preparation of asialoorosomucoid-polylysine conjugates.

Asialoorosomucoid-polylysine (ASOR-PL) conjugates have been recently developed as carriers of electrostatically bound DNA for targeted delivery to the hepatic asialoglycoprotein receptor (ASGPr) for gene therapy. Using acid-urea gel electrophoresis we have found that previously reported procedures for the fractionation of ASOR-PL conjugates do not efficiently remove noncovalently bound polylysine (PL) from ASOR-PL. DNA complexes prepared with these conjugates have low solubilities, which limits their usefulness for subsequent experimentation, particularly in vivo. For ASOR-PL made by carbodiimide-mediated crosslinking with 5-kDa PL, dialysis against 1 M guanidine hydrochloride is effective to remove the low molecular weight unbound PL. Dialysis is not feasible when using higher molecular weight PLs, but preparative elution acid-urea gel electrophoresis was used to isolate crude ASOR-PL fractions free of unbound PL. ASOR-PL freed of PL by dialysis or electrophoresis was further fractionated by cation-exchange HPLC on carboxymethyl-functionalized columns eluted with a mixed pH-salt gradient. Early-eluting ASOR-PL fractions isolated by a combination of preparative elution acid-urea gel electrophoresis and cation-exchange HPLC were found to be preferred for the formation of soluble DNA complexes.

Animals↗

PubMed1983

In vitro synthesis of thymosin beta 4 encoded by rat spleen mRNA.

Thymosin beta 4, containing 43 amino acids and acetylated at the NH2 terminus, is synthesized in vitro in a rabbit reticulocyte lysate or in a yeast protein-synthesis system in the presence of mRNA from rat spleen. The product formed was identified as beta 4 by immunoprecipitation by a specific anti-beta 4 antiserum, comigration with authentic beta 4 in NaDodSO4/polyacrylamide gel electrophoresis and in HPLC, and identity of peptide fragments. The immunoprecipitable product generated in the wheat germ protein-synthesizing system emerged slightly ahead of beta 4 in HPLC and appeared to lack the NH2-terminal acetyl group. There was no evidence for formation of a larger polypeptide precursor of beta 4 in any of the three systems used. In sucrose density gradient centrifugation, the mRNA coding for beta 4 was recovered in the 7-8S mRNA fraction.

Animals↗

PubMed1993

Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography.

We developed an assay which permits measurement of aspartate carbamoyltransferase (ACTase) activity. Cytosol from human peripheral blood mononuclear cells was used as the enzyme source. Using [14C]carbamoyl phosphate as the radiolabeled substrate, the formation of [14C]carbamoyl aspartate was quantitated by high performance liquid chromatography (HPLC) using an anion-exchange column with UV detection at 200-280 nm and an on-line liquid scintillation detector. A gradient method from an initially low concentration of ammonium phosphate, 1 mM (pH 3.0), to a higher concentration, 38 mM (pH 4.5), was used. The apparent Km values of carbamoyl phosphate and aspartate were 58 microM and 1.9 mM, respectively. ACTase inhibition by N-(phosphonacetyl)-l-aspartate (PALA) was consistent with a competitive model with respect to carbamoyl phosphate. The assay conditions were optimized to permit measurement of ACTase activity prior to and following therapy with PALA; ACTase was inhibited in a dose-dependent manner. This HPLC method permits direct quantitation of both the product of the reaction and the initial integrity of the substrate, [14C]carbamoyl phosphate, which is unstable in aqueous solutions.

Antimetabolites, Antineoplastic↗

PubMed1997

Bile acid analysis in biological fluids: a novel approach.

In contrast to current methods of bile acid analysis that require the separation of bile acids into different groups prior to their analysis, the HPLC method using a reverse phase column and gradient elution that we developed permits the separation and detection of nonconjugated, glycine-conjugated, and esterified bile acids as their fluorescent dimethoxycoumarin esters. The mild conditions for ester formation make possible the identification of allylic bile acids characteristic of metabolic errors in bile acid synthesis. Quantification is obtained using 7 alpha,12 alpha-dihydroxy-5 beta-cholanoic acid as an internal standard. In addition to identification based on retention time, peak-shift strategy is used by treatment of aliquots with cholyglycine hydrolase and/or solvolysis. Loss of the parent peak and appearance of the derivative provide further assurance of the identity of each bile acid in biologic fluids that contain other organic acids.

Adult↗

PubMed2000

A method for the determination of the hepatic enzyme activity catalyzing bile acid acyl glucuronide formation by high-performance liquid chromatography with pulsed amperometric detection.

A method for the determination of the activity of hepatic glucuronyltransferase catalyzing formation of bile acid 24-glucuronides using high-performance liquid chromatography (HPLC) with pulsed amperometric detection (PAD) has been developed. Bile acid 24-glucuronides were simultaneously separated on a semimicrobore column, Capcell Pak C18UG120, using 20 mM ammonium phosphate (pH 6.0)-acetonitrile (27:10 and 16:10) as the mobile phase in the stepwise gradient elution mode. A 1 M potassium hydroxide solution for the hydrolysis of the 24-glucuronides, which liberates the corresponding bile acids and glucuronic acid, was mixed with the mobile phase in a post-column mode, and the resulting eluant was heated at 90 degrees C, the 24-glucuronides being monitored using a pulsed amperometric detector; the limit of detection was 10 ng. The proposed method was applied to the determination of the hepatic enzyme activity catalyzing bile acid 24-glucuronide formation and the result exhibited the efficient 24-glucuronide formation of the monohydroxylated bile acid, lithocholic acid.

Acetonitriles↗

PubMed2006

Monitoring lipase-catalyzed methanolysis of sunflower oil by reversed-phase high-performance liquid chromatography: elucidation of the mechanisms of lipases.

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 210 nm was used to monitor the formation of the major compounds during the lipase-catalyzed transesterification reaction of sunflower oil with methanol. Individual triacylglycerols, diacylglycerols, monoacylglycerols as well as fatty acids and their corresponding methyl esters were separated using acetonitrile/acetone as a mobile phase and a combined linear gradient-isocratic-step gradient-isocratic elution procedure. Another relatively short method consisting of a linear gradient elution followed by an isocratic elution gave similar results, yet with lower resolution. HPLC/mass spectrometry with an ion trap analyzer and atmospheric pressure chemical ionization source was used for the identification of the individual compounds. Individual calibration curves obtained with UV detection at 210 nm were found to be of use for quantitative analyses of double-bond containing methyl esters and acylglycerols. The use of the RP-HPLC methods in the elucidation of the mechanisms of three immobilized lipases, namely Lipozyme TL IM, Lipozyme RM IM and Novozym 435, in biodiesel production was described.

Chromatography, High Pressure Liquid↗

PubMed2001

Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis.

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.

Base Sequence↗

PubMed1990

[Behavior of the enzymes of the salvage pathway of purine bases in leukemia lymphocytes].

The Authors present a procedure for the determination of adenine phosphoribosyltransferase (APRT) and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes which exhibits high sensitivity and requires low quantities of lymphocytes. 5 normal subjects and 4 patients affected by chronic lymphocytic leukemia (CLL) were considered. Human lymphocytes were prepared and treated as previously reported. To the incubation mixtures buffered with 50 mM TRIS-HCl pH 7.4 either 14C-adenine or 14C-hypoxanthine was added: after deproteinization and neutralization we followed the formation of either 14C-adenylic acid (AMP) or 14C-inosinic acid (IMP) by HPLC. A Supelcosil C18 5 microns (250 X 4.5 mm) column was used: IMP was eluted with 20 mM KH2PO4 pH 5.5 while AMP with a linear gradient to 40% B in 20 min., where A was 20 mM KH2PO4 pH 5.5 and B methanol/water 60:40. Evaluation of AMP and IMP formed was carried out by determination of the radioactivity of the collected peaks. The values of APRT in leukemic patients were enhanced when referred to the proteins and those of HGPRT decreased: the Authors propose to complete the study evaluating the intracellular content of adenine and hypoxanthine.

Adenine Phosphoribosyltransferase↗

PubMed1998

Formation of carbonyls during attack on insulin by submolar amounts of hypochlorite.

Bovine insulin was reacted at pH 4.0 with submolar amounts of hypochlorite. At least one molecule of insulin was modified per two molecules of hypochlorite added, as estimated by HPLC of native and modified insulin. About 5% of the hypochlorite-modified insulin reacted with dinitrophenylhydrazine (DNPH), a reagent which specifically labels carbonyl groups. The major DNPH-labeled product was isolated from the native insulin on reverse-phase HPLC, using trifluoroacetic acid/water/acetonitrile gradients. The UV spectrum of the major peak on the HPLC diode-array detector was representative of DNPH adducts, with lambda max = 365 nm. Several methods, including total amino acid analysis, tryptic digestion, and collision-induced dissociation-electrospray MS, indicate that the major carbonyl in the DNPH-labeled product was on the amino-terminal phenylalanine of the insulin B-chain. Amino acid analysis indicated that tyrosine was also degraded by hypochlorite, but we could not detect a carbonyl group formed at tyrosine. These findings suggest that the terminal amino groups of proteins are highly vulnerable to carbonyl formation during hypochlorite attack. The use of relatively low amounts of active oxygen species (such as hypochlorite), followed by chromatographic isolation of the protein labeled with a carbonyl-specific reagent, can be a useful approach to the study of reactive sites on proteins.

Amino Acid Sequence↗

PubMed1994

Determination of purine bases and nucleosides by conventional and, microbore high-performance liquid chromatography and gas chromatography with an ion-trap detector.

A reversed-phase high-performance liquid chromatographic method has been developed for the analysis of purine and pyrimidine bases, uric acid and nucleosides largely relating to the purine synthetic and degradation metabolic pathways, with particular attention to the separation of hypoxanthine, xanthine and guanine. Complete separation and quantitation of the purines has been accomplished in the nanogram-microgram scale on conventional 4.6 mm I.D. columns with a standard gradient HPLC instrumentation as well as on 1 mm I.D. microbore columns with a dedicated isocratic micro-HPLC system using a dioxane-sodium acetate buffer. For the definite identification of components in excreta of ticks a GC-MS method has been described involving formation and GC of the trimethysilyl derivatives on a 25-m DB-5 column directly coupled with an ion trap detector. The methods are demonstrated on the analysis of the purine metabolites having an assembly pheromone effect on argasid ticks.

Animals↗

PubMed2004

[Separation of tannins in Rhubarb and its analysis by high performance liquid chromatography-mass spectrometry].

In order to investigate the pharmaceutical actions of rhubarb, a method for extracting, separating and analyzing the tannin components in rhubarb was studied. At first, a procedure for the group separation of tannins from the water-ethanol extract of rhubarb was established based on the formation of tannins-caffein precipitation. Then, a high performance liquid chromatographic (HPLC) method for the analysis of tannins in rhubarb was developed. This HPLC method is based on a reversed-phase C18 column and polar mobile-phase such as water and methanol with gradient elution, and the tannins can be well separated. Finally, the identification of the tannin components in rhubarb was carried out by high performance liquid chromatography-mass spectrometry (HPLC-MS). The structures of the main tannin components (gallic acid, catechin, the dimer, trimer, tetramer and pentamer of catechin) in rhubarb are suggested. The fragmentation laws of the tannin components are summarized. In comparing with previous methods, it is simple and without the interference of co-existed compounds.

Chromatography, High Pressure Liquid↗

PubMed2003

Separation and quantification of chicken and bovine growth plate cartilage matrix vesicle lipids by high-performance liquid chromatography using evaporative light scattering detection.

Matrix vesicles (MV) are lipid bilayer-enclosed nanoscale structures that initiate extracellular mineral formation in most vertebrate species. Little attention has been given to differences between species in membrane lipid composition or to how new mineral is formed in MV. To explore more precisely the lipids of MV isolated from avian and bovine species, we developed a new high-performance liquid chromatography (HPLC) method used in combination with evaporative light scattering detection (ELSD) to quantify their lipid composition. HPLC analyses were performed on a Lichrosorb silica column using separate binary gradient elution systems for analyzing polar and nonpolar lipids. Standard mixtures of both phospholipids and nonpolar lipids were used to prepare calibration curves for each lipid, which were analyzed mathematically by polynomial regression for accurate quantitation. This new methodology provides high-resolution separations and quantitation of both the polar and the nonpolar lipids typically present in biological membranes, including lyso- (monoacyl-) phospholipids. We have applied this method to quantitate the phospholipid and nonpolar lipid composition of MV isolated from chicken and bovine growth plate cartilage. We also compared the ability of these MV to induce mineral formation. While the ability to induce mineralization and the lipid composition were generally similar, some significant differences between MV from these two disparate species were seen.

Animals↗

PubMed1999

Determination of the oral platelet aggregation inhibitor Sibrafiban in rat, dog, and human plasma utilising HPLC-column switching combined with turbo ion spray single quadrupole mass spectrometry.

A sensitive and selective HPLC-column switching method with single quadrupole mass spectrometric detection was developed for the simultaneous determination of the oral platelet aggregation inhibitor Sibrafiban (double protected prodrug), its prodrug and the active metabolite in rat, dog, and human plasma. The three analytes together with their tri-deuterated internal standards were isolated from plasma by protein precipitation (0.5 M perchloric acid). The de-proteinated samples were injected onto a standard-bore trapping column (4.0 mm i.d., LC-ABZ) of an HPLC-column switching system. Polar plasma components were removed by flushing the trapping column with ammonium formate (pH 3.6; 5 mM). Enriched compounds (including the analytes of interest) were backflushed onto a narrow-bore analytical column (2.1 mm i.d., Inertsil ODS-2) and separated by gradient elution (formic acid/ methanol). The whole effluent (200 microl/min) from the analytical column was passed to the turbo ion spray interface without splitting. Selected ion monitoring (SIM) was used for mass spectrometric detection. The limit of quantification for all three analytes was 1 ng/ml, using a 250-microl specimen of plasma. The mean precision and inaccuracy for the three analytes in all species were < 6 and < 5%, respectively. The practicability of the new analytical method was demonstrated by the analysis of about 500 rat and dog plasma and about 14,000 human plasma samples. The new method represents a successful example for the application of LC single MS with ionspray ionisation to the analysis of small molecule drugs in biological matrices from toxicokinetic studies and large clinical trials.

Administration, Oral↗

PubMed1995

Apparent intramolecular acyl migration and hydrolysis of furosemide glucuronide in aqueous solution.

The stability of furosemide glucuronide (FG) was investigated in buffer solutions ranging from pH 1 through 10. This glucuronic acid conjugate was the major metabolite of furosemide (F) excreted in human urine. FG, obtained by extraction from human urine, was purified by ion-exchange chromatography. The concentration of FG, acyl migration isomers of FG (FG-iso), and F were determined simultaneously with an HPLC method that included fluorescence detection and gradient elution. FG was found to be unstable in highly acidic and in neutral to alkaline solutions. Hydrogen ion and hydroxy ion catalyzed the hydrolysis of FG below pH 2.8 and above pH 5.6, respectively. Above pH 3.7, FG instability led to the formation of eight FG-iso compounds. Though beta-glucuronidase cleaved FG, the FG-iso compounds were resistant to the enzyme. The half-life of FG in a buffer solution at pH 7.4 and 37 degrees C was 4.4 h. The maximum stability of FG (half-life about 62 d) occurred at approximately pH 3.2. Below pH 3.7, acyl migration products of FG were not detected. Instead, the hydrolysis of FG to F and glucuronic acid was followed by the formation of 4-chloro-5-sulfamoylanthranilic acid (CSA), a secondary product in acidic media.

Acylation↗

PubMed1991

Analysis of glutathione S-transferase-catalyzed S-alkylglutathione formation by high-performance liquid chromatography.

Metabolism of alkyl halides and some organophosphorous compounds by glutathione S-transferases (EC 2.5.1.18) leads to formation of an S-alkylglutathione as a common product. We have developed an HPLC assay for formation of S-methylglutathione and S-ethylglutathione that is applicable to measuring enzyme activity toward a variety of xenobiotic substrates. The conjugates are derivatized with 1-fluoro-2,4-dinitrobenzene to form the corresponding N-2,4-dinitrophenyl derivatives, which are then separated by reverse-phase HPLC with gradient elution. The utility of the method is illustrated by the use of partially purified preparations of rat liver glutathione S-transferases and several prototypic substrates including iodomethane, iodoethane, dichlorvos, and methyl parathion. The limit of detection is about 50 pmol of N-(2,4-dinitrophenyl)-S-alkylglutathione. Advantages of the method over other assays of S-alkyl transferase activity are discussed.

2,4-Dinitrophenol↗

PubMed2004

Reversed-phase HPLC methods for purity test and assay of pioglitazone hydrochloride in tablets.

A reversed-phase gradient HPLC method was developed for the evaluation of pioglitazone hydrochloride (PG-HCl) in tablets. Limit of detection for PG-HCl was found to be 42 ng/ml. Analyses were performed on a C18 column (Symmetry C18, 5 microm, 250 x 4.6 mm), mobile phase was a mixture of ammonium formate buffer adjusted with formic acid to pH 4.1 and acetonitrile. Shortened purity method was used as the assay method. Methods were validated.

Chromatography, High Pressure Liquid↗