Direct determination of urinary pseudouridine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed for the determination of pseudouridine in urine. This method does not need pretreatment by boronate affinity gel. Therefore, it can be used in screening patients with malignant disease and for monitoring clinical response to chemotherapy with other tumor markers.     

Rapid separation of urinary acids by high-performance liquid chromatography

Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.     

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.     

Analysis of inositol by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method has been developed for identification and quantification of inositol isomers and monosaccharides in inositol-containing glycans. The method, which can determine 10 pmol of inositol, utilizes an Aminex HPX-87C column packed with an 8% crosslinked cation-exchange resin in the calcium form eluted with deionized water at 50 degrees C. NaOH solution is added to the column effluent through a postcolumn tee to increase the pH (pH greater than 11.6) before entering a pulsed amperometric detector which is highly sensitive for polyhydroxylated compounds. Samples in which inositol is linked to sugar through a glycosidic bond are hydrolyzed with 5.5 N trifluoroacetic acid, 100 degrees C, 4 h, and then reduced with NaBH4. Samples in which inositol is linked via a phosphate ester are hydrolyzed with 6 N HCl, 110 degrees C, 24 h. This method has been applied to the analysis of inositol in the hamster prion proteins (PrP) PrP27-30, and PrPSc.     

Analysis of urinary 3-methoxy-4-hydroxyphenylglycol by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection for the quantitation of total 3-methoxy-4-hydroxyphenylglycol (MHPG) in human urine is described. Existing methods for deconjugation and extraction have been optimized. The present method is simpler than existing methods with a high precision. Urinary MHPG is deconjugated enzymatically and subsequently extracted with ethyl acetate. The organic layer is extracted with acetic acid and a sample of the aqueous layer is injected into a reversed-phase column. In one run 90 samples can be processed. The critical parameters of deconjugation, extraction and chromatography are described. Data for reproducibility and selectivity are presented.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A™ column with mobile phase consisting of 0.05 M citric acid–0.1 M dipotassium hydrogen phosphate (pH 4.0)–methanol (97+3). The detection limits were 100–1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2–22.2 pmol/ml), ADP (5.5–22.2 pmol/ml), AMP (0.8–3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.     

Analysis of prednisolone in plasma by high-performance liquid chromatography

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet detector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentrations in plasma following administration of a 10-mg single oral dose to a human subject is described.     

Analysis of barbiturates in blood by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for the identification and quantification of barbiturates in blood at therapeutic levels has been developed. An ODS-silica column is used with an eluent of 40% methanol at pH 8.5. The barbiturates are detected at 240 nm. The sample preparation procedure involves extraction of unfractionated blood (100 μl) with hexane—diethyl ether (50:50, v/v) and is very rapid. Talbutal is used as an internal standard.The method has been applied to the determination of five barbiturates (amylobarbitone, butobarbitone, cyclobarbitone, pentobarbitone and quinalbarbitone) in blood after therapeutic doses of the drugs. An application of the HPLC assay to forensic casework is demonstrated.     

Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography

An isocratic high-performance liquid chromatography procedure was developed for the analysis of five urinary metabolites of caffeine; caffeine or 1,3,7-trimethylxanthine (137X), paraxanthine or 1,7-dimethylxanthine (17X), 1,7-dimethylurate (17U), 1-methylxanthine (1X), 1-methylurate (1U) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU). A standardized procedure was used for oral intake of caffeine and for urine collection. Conditions for sample storage and preparation were optimized, resulting in no detectable loss of caffeine metabolites after storage of the urine samples for four months. Urine samples were extracted with chloroform–2-propanol (4:1, v/v) and separated on a reversed-phase column with acetic acid (33%)–tetrahydrofuran–acetonitrile–water (1:2.5:44:925.5, v/v) as the eluent. Peaks were monitored at 280 nm. Peak heights were measured and the five metabolites were quantified using calibration curves. Cytochrome P4501A2 (CYP1A2) activity was calculated from the molar ratio (AFMU+1X+1U)/17U, N-acetyltransferase (NAT) from the ratio AFMU/1X, XO from the ratio 1U/1X+1U and cytochrome P4502A6 (CYP2A6) from the ratio 17U/(17U+17X+1U+1X+AFMU). The inter-assay coefficients of variation ranged from 1.7% for 17U to 5.7% for 1X. The intra-individual variation in metabolite ratios determined in two people, with intervals of a few days to several weeks between measurements, ranged from 2.1% for XO to 11.0% for CYP2A6. Using this procedure, metabolic ratios were determined for four groups of subjects; healthy, non-smoking females using oral contraceptives (OC users, n=5) and non-users (n=5), healthy non-smoking males (n=9) and children (n=7). Results found in this study were comparable to results reported in the literature for subjects with similar characteristics. A significantly higher CYP1A2 ratio was found for males (4.87±0.47) compared to females (3.62±0.91; p=0.005, Mann-Whitney). For the other enzyme activities, no significant differences were found between the groups of subjects in this study.     

Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with β-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C₁₈ reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 μM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 μM 5-HTOL and a standard solution of 2.0 μM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r² = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic—mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 μM, but after an acute dose of alcohol they increased to 0.5–15 μM.     

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 mm x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges.     

Analysis of trimethylselenonium ion in urine by high-performance liquid chromatography

A rapid and simple method for the separation of trimethylselenonium ion and other cationic forms of selenium in urine by HPLC on a strong cation exchanger is described. Most of the inorganic salts in urine are removed prior to chromatography by means of ethanolic precipitation, thus minimizing interferences. Following sample loading and elution with 0.003 M ammonium phosphate (pH 4), a linear gradient to 0.33 M ammonium phosphate (pH 4) is employed. Complete separation of the trimethylselenonium ion from four other selenonium compounds was achieved, and good recovery of the compounds was obtained for the desalting and chromatographic procedures. The procedure was successfully employed to demonstrate that dimethylselenocysteineselenonium iodide and Se-methylselenomethionineselenonium iodide are extensively metabolized when administered to rats, and that trimethylselenonium ion is a major urinary metabolite of both compounds.     

Analysis of phospho- and phosphonosphingolipids by high-performance liquid chromatography

A simple and efficient method for the separation of phosphosphingolipids including phosphonosphingolipids by high-performance liquid chromatography is described. A mixture of authentic lipids consisting of sphingomyelin, ceramide phosphorylethanolamine, ceramide 2-aminoethylphosphonate, and ceramide N-methylaminoethylphosphonate was completely separated using a silica gel (Zorbax SIL) column with acetonitrile-methanol-water 72:40:10 (v/v) as eluting solvent. The elution of these sphingolipids was monitored directly with an ultraviolet spectromonitor at 207 nm. The practical limit of detection of each sphingolipid was about 0.2 microgram or 0.3 nmol. Using this method, we found that from one to four different phosphono- and/or phosphosphingolipids in fresh-water shellfish can be routinely identified and reproducibly quantified.