Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.     

Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase II or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-beta-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6sbeta(1-->3)gal, accounting for approximately equals 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase II) from Bacillus sp. generates two major products, the monosulfated disaccharide galbeta(1-->4)glcNAc6s ( approximately equals 50% nmol product) and the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s ( approximately equals 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximately equals 108 pmol, 50 ng, to 2,160 pmol, 1,000 ng, for the disaccharide galbeta(1-->4)glcNAc6s, and from 92 pmol, 50 ng, to 1,840 pmol, 1,000 ng, for the disaccharide gal6sbeta(1-->4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80+/-0.34 microg/ml/10(6) cells and composed of approximately equals 71% nmol of disaccharide galbeta(1-->4)glcNAc6s and 18% nmol of the disulfated disaccharide gal6sbeta(1-->4)glcNAc6s having approximately equals 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( approximately 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.     

Fluorometric determination of guanidino compounds by new postcolumn derivatization system using reversed-phase ion-pair high-performance liquid chromatography

A new postcolumn derivatization system using 1,2-naphthoquinone-4-sulfonate as fluorogenic reagent for the fluorometric determination of guanidino compounds is described. The guanidino compounds were separated by reversed-phase ion-pair high-performance liquid chromatography with an isocratic mobile phase containing the fluorogenic reagent and octane-sulfonate as the counterion. Fluorophors were derived from a condensation of guanidino compounds with the fluorogenic reagent in an alkaline solution. The chromatographic system using the mobile phase containing the fluorogenic reagent was simplified because only two pumps were required to deliver the mobile phase and the alkaline solution. Separation of guanidino compounds was completed within 25 min using a Nucleosil C8 column (5 microns, 15 cm X 4.6 mm i.d.). This method was applied to serum obtained from patients on hemodialysis therapy.     

Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.     

Fluorometric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization

A reliable and semi-automated high-performance liquid chromatographic (HPLC) method is described for the determination of total vitamin C in whole blood. After deproteinization of whole blood and enzymatic oxidation of l-ascorbic acid to dehydro-l-ascorbic acid, the latter is condensed with o-phenylenediamine to its quinoxaline derivative. This derivative is separated on a reversed-phase HPLC column and detected fluorometrically. Total vitamin C in whole blood can be determined in concentrations as low as 0.2 μmol/l.Special attention was paid to the stability of vitamin C in whole blood and of its quinoxaline derivative in the extract. Results of our investigations showed that total vitamin C in whole blood is stable for eight days at −20°C, provided ethyleneglycol-bis-(β-amino-ethyl ether)-N,N,N′,N′-tetraacetic acid and glutathione are immediately added to the blood sample. The quinoxaline derivative of vitamin C in the blood extract is stable for at least 24 h if stored in the dark at 4°C.Routine vitamin C determinations can be carried out in a series of 100 samples within 48 h. The within-assay and between-assay coefficients of variation were 3.7% and 4.6%, respectively. The between-assay analytical recovery of l-ascorbic acid added to whole blood samples was 97.0 ± 7.0% (mean ± S.D.). Reference values of vitamin C in whole blood of normal healthy Dutch adults were found in the range 20–80 μmol/l.     

Analysis of tamoxifen-DNA adducts by high-performance liquid chromatography using postcolumn online photochemical activation

Tamoxifen, a widely used nonsteroidal antiestrogen in the treatment of breast cancer, forms several metabolites. 4-Hydroxytamoxifen (4-OHTam), a metabolite found in the bloodstream, has much higher affinity for the estrogen receptor than tamoxifen itself. Oxidative activation of 4-OHTam induces DNA damage. DNA isolated from HL-60 cells exposed to 10 microM 4-OHTam in the presence of 1 microM hydrogen peroxide was digested enzymatically to release both normal and modified nucleosides. The modified nucleosides were enriched by butanol extraction. Using UV detection, HPLC analysis of the butanol extract from 200 microg DNA digest detected approximately 4 4-OHTam-dG adducts per 10(7) nucleotides (n = 3). Online postcolumn UV irradiation in HPLC and fluorescence detection improved the detection sensitivity by 3 x 10(2) times. Using 4-OHTam as an example, this report demonstrated for the first time the power of the technique to assay tamoxifen-DNA adducts directly in the DNA digest without relying on postlabeling.     

Analysis of hydroxyproline in urine by high-performance liquid chromatography after dabsyl-chloride derivatization

Summary In order to analyse hydroxyproline (HYP) in urine, a high-performance liquid chromatographic method was modified. The primary amino groups were blocked with o-phthalaldehyde, and then the secondary amino groups were derivatized with 4-dimethylaminoazobenzene-4-sulphonyl chloride. In addition, the dabsylated samples were treated with ethyl acetate to obtain a simple elution profile in high-performance liquid chromatography. The dabsyl-HYP and -proline were eluted at 4.7 min and 8.0 min, respectively. The chromatographic analysis was completed within 10 min, including the time needed for reequilibration of the column. Using the present method, the concentration of HYP in urine was determined to 260 ± 6µmol/l.     

Multiresidue determination of sulfonamides in edible catfish, shrimp and salmon tissues by high-performance liquid chromatography with postcolumn derivatization and fluorescence detection

A liquid chromatographic (LC) method for determining 14 sulfonamide (SA) (sulfanilamide, sulfadiazine (SDZ), sulfathiazole, sulfapyridine, sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethizole, sulfamethoxypyridazine, sulfachloropyridazine (SCP), sulfamonomethoxine, sulfadoxine, sulfamethoxazole, sulfadimethoxine (SDM), and sulfaquinoxaline (SQX)) residues in edible catfish, shrimp and salmon tissues was developed and validated at 5, 10 or 20 ng g(-1). The method was then used to determine residues in tissues of catfish, shrimp and salmon dosed with six selected sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfadimethoxine and sulfaquinoxaline). All assays were within U.S. Food and Drug Administration guidelines for recovery and intra-assay variability. The method was developed to determine possible sulfonamide residues in aquacultured catfish, shrimp and salmon produced for food.     

Acetylcholine measurement by high-performance liquid chromatography using an enzyme-loaded postcolumn reactor

Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed.     

Analysis of aspartate and glutamate in human cerebrospinal fluid by high-performance liquid chromatography with automated precolumn derivatization

A method is described for the analysis of the neuroexcitatory amino acids, aspartate and glutamate, in human cerebrospinal fluid (CSF) by reverse-phase, high-performance liquid chromatography. Fluorescent isoindole derivatives of the amino acids were prepared by reacting the amino acids with ortho-phthalaldehyde in an automated, precolumn procedure. Chromatographic conditions were developed that resolve the isoindole derivatives of aspartate and glutamate from those of at least 10 unidentified components of CSF. Amino acids were reliably quantified in 5-microliter samples of CSF, and deproteinization of the specimens was not required. Furthermore, it was found that deproteinization by precipitation with strong acid can lead to artifactually high measurements of glutamate. The concentrations of free aspartate and glutamate in lumbar CSF from 15 neurologically normal children were 0.30 +/- 0.11 and 0.48 +/- 0.26 microM (mean +/- SD), respectively. The value for glutamate is considerably lower than has been reported in any previous study of human CSF.     

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C₁₈ column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 ± 3.7% (n = 45) and 100 ± 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 μmol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.     

A reversed-phase ion-pair high-performance liquid chromatography method for bovine testicular hyaluronidase digests using postcolumn derivatization with 2-cyanoacetamide and ultraviolet detection

A reversed-phase ion-pair HPLC method for separating hyaluronic acid oligomers, using a polymeric C18 column at alkaline pH, is described. As the concentration of the ion-pairing agent tetrabutylammonium hydroxide increased, over the range of 0.01 to 0.06M, the capacity factors (k') of tetra- to dodecasaccharide decreased. The change in k', for each increment in pairing agent, increased with oligomer molecular weight. When changing mobile phase pH from 7 to 8, k' dramatically decreased and remained unchanged from pH 8 to 11. The isocratic separation was optimized to resolve tetrato dodecasaccharide at pH 9.0 in under 19 min. The postcolumn derivatizing agent 2-cyanoacetamide reacted with the reducing N-acetylglucosamine end groups of hyaluronic acid oligomers to yield reaction products that were monitored at 27 nm. In a series of control experiments using decasaccharide and N-acetylglucosamine, it was found that maximum product formation took place at pH 9 and was greatly influenced by borate buffer concentration. The optimum concentration for 2-cyanoacetamide was 0.33% and a temperature of 100 degrees C gave the best signal to noise ratio for the postcolumn reaction. The method is linear and reproducible, and has a lower limit of detection for tetrasaccharide of 20 ng (25 pmol). This system is suitable for studying the degradation kinetics of purified hyaluronic acid oligomers by bovine testicular hyaluronidase. Extension of the method to fluorescent and electrochemical detection and its applicability to other glycosaminoglycans is discussed.     

Automated analysis of alditols by anion-exchange chromatography with photometric and fluorimetric postcolumn derivatization

Eight alditols were separated in ca. 80 min as their borate complexes by stepwise elution with three borate buffers on a column packed with Hitachi 2633 resin. The alditols in the eluate were derivatized automatically to colored, fluorescent products by applying sequential reactions of periodate oxidation and Hantzsch condensation, and the products were detected either photometrically or fluorimetrically. This automated method allowed simultaneous determination of 20–500 and 20–200 nmol amounts of alditols by photometric and fluorimetric monitorings, respectively. The lower limits of detection were ca. 2 and 0.5 nmol, respectively. The interference by aldoses was slight. Aldoses may be also determined as alditols by direct injection of aqueous solutions to which excess amounts of sodium borohydride have been added. This method was applied with success to urinary alditol assay and to molecular weight determination by end group analysis.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

Determination of ethambutol in plasma by high-performance liquid chromatography after pre-column derivatization

A new HPLC assay using UV detection (200 nm) was developed to determine ethambutol (EMB) concentrations in plasma. Following extraction (0.1 ml plasma) with chloroform, EMB and octylamine (used as internal standard) were derivatized with phenylethylisocyanate. Quantitation in plasma was achieved at 200 nm. There were no interferences from endogenous compounds. Intra- and inter-day variabilities were lower than 5.2 and 7.6%, respectively. The limit of quantitation of the method was 0.2 μg/ml. In plasma, ethambutol was found to be stable for at least one month when samples were stored at −20°C. This assay was applied to the therapeutic monitoring of EMB concentrations in 19 patients suffering from tuberculosis.     

Femtomole peptide mapping by derivatization, high-performance liquid chromatography, and fluorescence detection.

A highly sensitive peptide mapping method using derivatization and fluorescence detection is described. Bovine cytochrome c was digested using a buffer compatible with the derivatization that followed. The derivatization was performed with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The peptide mapping of the tagged digest was conducted with both HPLC and capillary LC (CLC) systems. A capillary LC-electrospray ionization mass spectrometer (MS) was set up for measuring the molecular weights of the tagged peptides. Optimization was made of the conditions used for digestion, derivatization, and mapping. MS measurements of the tagged peptides suggested that there was only one derivatization product produced from all peptides (except one) and that all the identified peptides were fully tagged. Peptide mapping of the tagged digest reviews a larger number of peptides, covering almost the entire sequence. Peptide mapping of a 20 fmol amount of tagged digest was readily performed with the CLC system. By using derivatization and fluorescence detection, the sensitivity of peptide mapping could be improved 2000 times compared to that observed with uv detection of untagged peptides.     

Electrochemical detection of proteins in high-performance liquid chromatography using on-line, postcolumn photolysis.

Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective.     

Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes

Glutathione (GSH) and glutathione disulfide (GSSG) are important thiols, which provide defence against oxidative stress by scavenging free radicals or causing the reduction of hydrogen peroxide. The ratio GSH/GSSG is often used as a sensitive index of oxidative stress in vivo. In this paper, a direct electrochemical method using an electrode modified with functionalized carbon nanotubes as electrochemical detector (ED) for liquid chromatography (LC) was described. The electrochemical behaviors of GSH and GSSG on this modified electrode were investigated by cyclic voltammetry and it was found that the functionalized carbon nanotubes exhibited efficiently electrocatalysis on the current responses of GSH and GSSG. In LC-ED, both of the analytes showed good and stable current responses. The detection limit of GSH was 0.2 pmol on column and that of GSSG was 1.2 pmol on column, which were low enough for the analysis of real small samples. The method was sensitive enough to detect difference in concentration of GSH and GSSG in hepatocytes from animals with and without introduction of oxidation stress by glucose or hydrogenperoxide.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.