High-performance liquid chromatographic determination of glyoxylate in rat liver

A high-performance liquid chromatographic method was developed for the determination of glyoxylate in the liver. Alpha-keto acids in charcoal-treated acid-extract of the liver were converted to the corresponding 2,4-dinitrophenylhydrazones and purified as the derivatives by successive extractions with ethyl acetate and sodium bicarbonate solution. The dinitrophenylhydrazones were then quantitatively converted to the corresponding substituted 2-hydroxyquinoxalines by reaction with o-phenylenediamine, followed by analysis by high-performance liquid chromatography with fluorescence detection. As a control to correct the recovery of tissue glyoxylate, an acid-extract of the liver prepared with the addition of standard glyoxylate (25-50 nmol/g wet weight of tissue) was simultaneously subjected to the analytical procedure. The maximum sensitivity of the glyoxylate measurement as 2-hydroxyquinoxaline (the quinoxaline derivative corresponding to glyoxylate) was defined as the peak area reading five times as high as the blank value obtained without sample and was approximately 10 pmol per injection. Glyoxylate in the addition compound with tris(hydroxymethyl)aminomethane was quantitatively recovered as 2-hydroxyquinoxaline. The addition compounds of glyoxylate with bisulfite and cysteine did not react with 2,4-dinitrophenylhydrazine under the conditions employed and were not detectable as glyoxylate by this method, while the adduct-forming substances added to the acid-extract of the liver did not interfere with the glyoxylate determination. No glyoxylate was detected when the liver extract had been incubated at neutral pH with a large excess of cysteine, indicating that little artificial production of glyoxylate occurred during the analytical procedure. Among 64 compounds tested for possible artificial production of glyoxylate or possible interference with the chromatographic determination of 2-hydroxyquinoxaline, p-hydroxyphenylpyruvate was the only compound which was converted to glyoxylate during the procedure. However, p-hydroxyphenylpyruvate was easily removed from the acid-extract of the tissue by charcoal treatment. The amount of glyoxylate in the liver of fasted rat was measured by the present method to be approximately 5 nmol per g of wet weight.     

High-performance liquid chromatographic analysis of dipyridamole in plasma and whole blood

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of dipyridamole in plasma and whole blood. The method involves a single extraction of an alkalinized sample with diethyl ether followed by evaporation of the organic solvent and ion-pair chromatography using fluorescence detection. The lower limit of sensitivity for dipyridamole is 1 ng/ml. Concentrations of dipyridamole between 1 and 500 ng per sample are measured with an average coefficient of variation of 4.5% in plasma and 7.4% in whole blood.     

High-performance liquid chromatographic assay for amiodarone N-deethylation in microsomes of rat liver

A reversed-phase high-performance liquid chromatographic assay using ultraviolet detection is described for determining the production of the major N-dealkylated metabolite of amiodarone in rat liver microsomes. The principal advantages of this method are its simple sample preparation (protein precipitation by acetonitrile), low detection limit for N-desethylamiodarone (0.05 μmol/l) and relatively short analysis time (16 min). Its analytical applicability is demonstrated by the comparison of the kinetic parameters (maximum velocity and Michaelis—Menten constant) between Sprague-Dawley and Dark-Agouti rats.     

New high-performance liquid chromatographic method for amphotericin B analysis using an internal standard

A simple and reproducible HPLC method for the analysis of amphotericin B (AmB) in serum, lung and liver using natamycin as the internal standard was developed. AmB and natamycin were extracted from serum, lung and liver and were separated using an isocratic elution from a C₁₈ reversed-phase column. The mobile phase consisted of acetonitrile-10 mM acetate buffer pH 4.0 (37:63, v/v). The HPLC system had two detectors in series. One was set at 303 nm and the other at 383 nm for the detection of natamycin and AmB, respectively. The retention times of AmB and natamycin were 15 and 6 min, respectively. The recovery efficiency was 96-70%. The limit of quantification was 0.1 μg/ml. The assay was reproducible, the within-day coefficient of variation (n=6) was <8% for serum, lungs and liver. The between-day variability (n=6) was <7.7% for serum, liver and lungs at 1 μg/ml or 1 μg/g tissue concentration. The assay was linear within the range 1–40 μg/ml (r²=0.999).     

High-performance liquid chromatographic analysis of corticosteroids

This review presents recent developments in high-performance liquid chromatographic (HPLC) analysis of corticosteroids for the determination of clinically important steroids in biological specimens. Various sample preparation techniques are described.     

High-performance liquid chromatographic analysis of polyhydroxyflavones using solid-phase borate-complex extraction

A high-performance liquid chromatographic method using a solid-phase borate-complex extraction pretreatment was studied for the selective quantitation of polyhydroxyflavones in vegetables, red wine and human blood plasma. Homogenate, extract and intact samples were applied to phenylboric acid cartridges to retain polyhydroxyflavones on the solid-phase by forming the borate-complex, followed by elution of the retained analytes with an acidic solvent. Reversed-phase chromatography with diode array detection allowed the simultaneous separation of rutin, myricetin, fisetin, morin, quercetin and kaempferol without significant interference from other components, indicating high selectivity of the solid-phase borate-complex extraction. The absolute recoveries of quercetin, fisetin and rutin were superior to those of kaempferol, myricetin and morin, suggesting a difference in the complex formation efficiency between 1,2- and 1,3-diol structures. When using fisetin as an internal standard, polyhydroxyflavones were quantified in the concentration range 0.10–30.0 μg/ml. In replicate spiking experiments with standards, the mean relative recoveries ranged between 75.7 and 104.6%, and the intra- and inter-assay C.V.s ranged between 0.8 and 10.2% for onion, wine and plasma samples. The proposed method will be applicable to nutritional and pharmacokinetic experiments of polyhydroxyflavones.     

High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes

Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4′-amidinophenoxy)-3-pentanol and 1,5-di-(4′-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.     

High-performance liquid chromatographic analysis of angiotensin II receptor antagonist valsartan using a liquid extraction method

PURPOSE: To develop an easy assay for the quantitation of the angiotensin II receptor antagonist valsartan in human plasma using a liquid extraction procedure. METHOD: The method involves acid extraction from 1 ml human plasma with methyl-tert.-butyl ether followed by back-extraction into a basic medium. An isocratic HPLC equipped with reverse phase column and a fluorescence detector was used at room temperature. RESULTS: The response to 10-2000 ng/ml valsartan was linear. In plasma of three human subjects given 160 mg valsartan orally, concentrations of 25-1540 ng/ml were observed. CONCLUSION: This convenient method is suitable for pharmacokinetic studies of valsartan.     

High-performance liquid chromatographic determination of papaverine in whole blood

The development and application of an assay method for papaverine in whole blood is reported. A single, simple extraction procedure at pH 10.0 using chloroform—n-hexane (2:3) as the solvent, results in pure extracts which can be chromatographed without further purification. Chromatography is performed on a nitrile-bonded phase, using n-hexane—dichloromethane—acetonitrile—propylamine (50:25:25:0.1) as mobile phase. This method is characterized by a between-day precision of 4% at the 200 ng/ml level and a detection limit of 5 ng/ml, and was successfully applied in a pharmacokinetic study.     

An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components

A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4 degrees C prior to chromatography on a 5-microns octadecylsilyl column. AA concentrations (mean +/- SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 +/- 0.05, 15.2 +/- 6.28, and 2.43 +/- 1.63 micrograms/10(8) cells, respectively; the mean plasma AA concentration was 0.97 +/- 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 X SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.     

High-performance liquid chromatographic analysis of glycoamines in serum

This report describes the development of an HPLC-UV method for studies of glycoamines and glycoamine-like compounds in normal human serum and osteosarcoma patient serum as potential biological markers of cancer. The glycoamines, a newly recognized class of endogenous, low-molecular-mass biopolymers, are conjugates of amino acids and sugar units, containing 5 to 29 amino acid and 1 to 17 sugar units. After ultrafiltration of serum samples, reversed-phase HPLC separation with diode-array detection was used to obtain standard profiles of serum ultrafiltrates below M_r 10 000 in healthy subjects. These highly reproducible profiles utilized two-dimensional peak identification and were used to develop a statistical profile of the major glycoamine peaks in normal serum. This newly developed analytical method was subsequently used to address a key question: whether or not there is a single tumor-specific glycoamine or a family of tumor-specific glycoamines in cancer patient serum. Preliminary results suggest that this method can separate and detect glycoamines and glycoamine-like compounds in various types of cancer patient serum with a high degree of reproducibility on the basis of comparative two-dimensional identification of natural compounds and a panel of synthetic glycoamine analogs. Moreover, the method is useful for following the relative changes in the amount of a given glycoamine over an extended clinical time course. Initial results suggest that a glycoamine or glycoamine-like compound, GA-4.63, may have clinical utility in human osteosarcoma studies.     

High-performance liquid chromatographic determination of vertilmicin in rat plasma using sensitive fluorometric derivatization

A sensitive and reliable high-performance liquid chromatographic method was developed for the determination of vertilmicin in rat plasma. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C(18) reversed-phase chromatography allowed the fluorimetric detection of vertilmicin. Optimal conditions for the derivatization of vertilmicin are described. The limit of quantification was 0.02 mg/L. The pharmacokinetics of vertilmicin was studied in 24 rats following intramuscular injection (i.m.) of different doses (4, 8, 16, 32 mg/kg of body weight). The pharmacokinetic parameter values were estimated by use of 3P97 program. In this study, we assessed the dose proportionality of vertilmicin after single intramuscular injection doses and obtained new information on the pharmacokinetics of the compound.     

High-performance liquid chromatographic determination of midazolam in rat brain

A high-performance liquid chromatography method for the determination of midazolam in rat brain is described. Midazolam and the internal standard halazepam were extracted with toluene and analyzed isocratically on a reversed-phase column with a mobile phase consisting of methanol, acetonitrile and potassium phosphate buffer. Detection was monitored by ultraviolet absorption at 240 nm. The standard curves were linear over the range of 25--350 ng midazolam per 50 mg brain tissue. The day-to-day coefficient of variation ranged from 1.7 to 6.9%. The limit of quantification was 80 ng/g brain tissue. The method is rapid, simple and reproducible for brain analysis.     

High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver.

The folate coenzyme 5,10-methylenetetrahydrofolate is an important folate metabolite which cannot be determined directly by HPLC near neutral pH because it dissociates to formaldehyde and tetrahydrofolate. A method for the determination of 5,10-methylenetetrahydrofolate in liver is described. This method involves (1) determination of liver 5-methyltetrahydrofolate; (2) chemical reduction of liver 5,10-methylenetetrahydrofolate (stabilized at pH 10) to 5-methyltetrahydrofolate; and (3) determination of total liver 5-methyltetrahydrofolate. Subtraction of (1) from (3) gives the concentration of 5,10-methylenetetrahydrofolate in liver.     

High-performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood

Zinc protoporphyrin and protoporphyrin free acid in blood were determined by high-performance liquid chromatography on a C₁₈ column. Results for 63 human blood samples obtained through a lead poisoning detection program compared favorably with the widely-used ethyl acetate—acetic acid extraction determination. Blood from 16 rats which had been maintained on water heavily spiked with chloroform or bromodichloromethane and blood from a lead-poisoned cow were examined by this procedure.     

High-performance liquid chromatographic analysis of nitroxoline in plasma and urine

A high-performance liquid chromatographic method has been developed for the determination of nitroxoline in 50-μl plasma and urine samples.A structural analogue of nitroxoline, 8-hydroxyquinoline, was added to the eluent in order to suppress peak asymmetry. Several parameters of the eluent were studied for the optimisation of the chromatographic system.Plasma concentration—time curves were constructed for three volunteers after they had received an oral dose of 100 mg of nitroxoline. Plasma half-life was about 1 h. Within 12 h, about 1% of the dose was excreted in the urine as free nitroxoline and about 30% as conjugated metabolite of the parent compound.     

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition

A significantly improved method for base composition analysis of synthetic oligodeoxyribonucleotides is presented. This highly accurate and sensitive method used enzymatic digestion followed by high-resolution HPLC of the nucleosides to determine the empirical base composition of the parent compound. The enzymatic digestion reaction is quantitative and is not blocked by modified bases, thus allowing the degree of base deprotection and chemical modification to be assessed. Digestion data are presented for oligodeoxyribonucleotides which range from 18 to 150 bases in length with excellent agreement of experimental and theoretical composition. The method is also applicable to high-molecular-weight genomic DNA.