Direct high-performance liquid chromatography determination of diastereomeric oxprenolol glucuronides

The β-blocking agent oxprenolol is used therapeutically as the racemate. In humans and animals it is metabolized i.a. to ether glucuronide diastereomers. A stereoselective HPLC assay was developed to determine directly, without hydrolysis to their parent enantiomers, the oxprenolol glucuronides in biological samples. The glucuronide standards for this direct assay are prepared by incubation of rabbit liver microsomes with RS-oxprenolol. The glucuronides obtained are purified and concentrated with solid-phase extraction, and their concentration is measured by an indirect method, i.e. HPLC assay of the oxprenolol enantiomers after enzymatic hydrolysis with β-glucuronidase. The direct assay involves separation by HPLC using a C₁₈-reversed-phase column, with UV detection at 274 nm; nalorphine is used as internal standard. On injection onto the column, without previous hydrolysis, the limit of detection is 20 ng for both glucuronides. The assay is sensitive, accurate and reproducible. The method is suitable for the assay of glucuronides in liver microsomal incubates and plasma.     

Rapid, quantitative high-performance liquid column chromatography of pseudouridine

A rapid, precise, and accurate chromatographic method for the determination of pseudo-uridine (ψ) in urine by high-performance liquid chromatography (HPLC) has been developed. The ribonucleosides were first isolated with an affinity gel containing immobilized phenylboronic acid. The response for ψ was linear well above and below the range necessary to determine urinary ψ. Good precision was obtained for both matrix-dependent and matrix-independent samples. Supporting experimental data are presented on precision, recovery, chromatographic methods, sample cleanup and application to the analysis of urine samples from normal males and females, and patients with advanced colon cancer. In a comparison of 40 normals with 10 colon cancer patients, 9 of the 10 patients had a ψ: creatinine (Cr) ratio greater than + 2σ for the normal population. This HPLC method is now being used extensively in our laboratory as a routine method for determination of ψ in urine from patients with various types of cancer and in chemotherapy response studies. Data are presented on the dynamics of ψ excretion by normal males and females. When the excretion of ψ was normalized with the excretion of creatinine, it was noted that samples collected at random have the same ψ: Cr ratio value as for the 24-h total collection, thus, allowing the use of random samples. The constancy of the ψ: Cr ratio implies that RNA turnover is constant and ψ excretion is independent of diet. Base values are presented for the ψ: Cr     

Direct determination of UDP-glucuronic acid in cell extracts by high-performance liquid chromatography

A simple and sensitive method for the direct determination of UDP-glucuronic acid by high-performance liquid chromatography with simultaneous measurement of UDP-glucose was developed. Optimal resolution and separation of UDP-glucuronic acid was attained under isocratic conditions with the ion-pairing agent n-octylamine. Quantitation was sensitive down to 5 pmol for standards and for liver cell extracts. Because this method directly measures UDP-glucuronic acid, it can be used for quantitation in the presence of drugs that interfere with enzymatic methods.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Standardization of acid hydrolysis procedure for urinary 3-methylhistidine determination by high-performance liquid chromatography

The N-acetylated form of N-methylhistidine (3-methylhistidine, 3-meH), a non-invasive marker of proteolysis, accounts for 80–90% of total 3-meH excretion (acetylated+non-acetylated 3-meH) in the rat. To determine total 3-meH excretion, samples require acid hydrolysis prior to determination by high-performance liquid chromatography. This study evaluated the stability of 3-meH at various times and temperatures of hydrolysis and determined the optimal conditions for hydrolysis of samples. Increasing temperature (120°C) results in significant degradation of 3-meH with no appreciable change in concentration being noted at 80°C. Hydrolysis at 100°C for 1.5 to 4 h or 80°C for 8 to 12 h is recommended for determining total 3-meH concentrations in rat urine.     

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.     

Direct determination by high-performance liquid chromatography of sn-2 monopalmitin after enzymatic lipase hydrolysis

An alternative method to determine the sn-2 monopalmitin in infant formulas was developed and validated. This method offers many advantages over the traditional methods. It follows the official method in the first steps, purification of the fat or oil through an alumina column, and subsequently the triglycerides are incubated with pancreatic lipase in order to obtain the sn-2 monoglycerides. In traditional methods the sn-2 monoglycerides are separated by preparative thin-layer chromatography and then, the 2-monoglycerides are converted into the corresponding fatty acid methyl esters and analysed by gas chromatography. In our method, separation, quantification and identification of the sn-2 monoglycerides were achieved by high-performance liquid chromatography with evaporative light-scattering detection. The detection limit (0.19 μg), quantification limit (0.38 μg), linearity range (r=0.999, range 1–200 μg) and precision (SD=1.10) show the suitability of the proposed method. This method is faster, cheaper and simple and does not consume large quantities of reagents and materials.     

Determination of pseudouridine and other nucleosides in human blood serum by high-performance liquid chromatography

A specific, sensitive, and rapid method to measure pseudouridine in human blood serum is described. The method is based on the following steps: (i) deproteinization of serum samples by filtration on membrane cones or by acetonitrile; (ii) purification of nucleosides and concentration of the sample by affinity chromatography on phenylboronate gel followed by lyophilization; and (iii) separation of nucleosides and their quantitation by reserse-phase high-performance liquid chromatography.The pseudouridine mean value in 30 normal subjects was 2.52 ± 0.28 nmol/ml. The procedure also allows the identification of inosine, uridine, guanosine, and adenosine. Nevertheless, the presence in human blood serum of enzymatic activities which convert adenosine to inosine and cytidine to uridine prevents the precise quantitation of these nucleosides. All the compounds were identified by comparing their retention times and absorbance ratios ($\text{A}{}_{280}\text{A}{}_{254}$) with those of pure compounds, as well as by cochromatography.     

Direct determination of tramadol glucuronides in human urine by high-performance liquid chromatography with fluorescence detection

A sensitive and selective reversed-phase high-performance liquid chromatography method has been developed for the direct determination of three glucuronides of the centrally acting analgesic tramadol (1). Separation of these glucuronides into their diastereomers was achieved by HPLC using ion pair chromatography with nonanesulfonic acid sodium salt and LiChrospher 100 RP 18 as stationary phase. Quantification of O-demethyltramadol glucuronide and N,O-didemethyltramadol glucuronide in human urine was performed by fluorescence detection. The urine samples were purified by a two-step solid-phase extraction. The glucuronides were found to be highly enriched in the 1S,2S-diastereomers. The results of a study with three healthy volunteers are presented.     

Analysis of purines in urinary calculi by high-performance liquid chromatography

A high-pressure liquid chromatography method has been developed for the analysis in urinary calculi of six purines: uric acid, 2, 8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol, and oxypurinol. Separation was conducted isocratically on a reversed-phase column, using 50 mM phosphate buffer (pH 5.5) / methanol (97/3, v/v) as mobile phase. Limits of detection, depending on compound, ranged from 7 to 28 microg/g stone weight. Hitherto, no reports have appeared on other purines present with uric acid in stones, due to lack of a sensitive and specific analytical method. We have now found that all calculi with more than 4% uric acid also contained 1-methyluric and 7-methyluric acids and trace amounts of hypoxanthine, xanthine, and 2,8-dihydroxyadenine. Accurate identification and quantitation of purines in urinary calculi are important for the diagnosis of rare metabolic diseases leading to urolithiasis (xanthinuria, dihydroxyadeninuria), as well as for prevention of iatrogenic complications during treatment with allopurinol of uric acid urolithiasis. The method may be used for reference purposes in clinical laboratories and for research on the pathogenesis of urolithiasis in disorders of purine metabolism.     

Tissue porphyrin pattern determination by high-speed high-performance liquid chromatography

A double-label two-dimensional electrophoresis procedure has been developed which is specifically designed for the comparison of serum or plasma proteins in two different samples. Proteins are labeled by reductive methylation with [14C]- or [3H] formaldehyde. The procedure is economical because small quantities of relatively inexpensive isotopes are used and it is at least as sensitive as silver staining in detecting proteins. A fourfold increase in the sensitivity of autoradiography over existing methods was obtained by performing autoradiography before processing the gel for fluorography. A spot in the electrophoretic gel that contains 17-28 ng of labeled protein is detectable. This corresponds to proteins present in serum at a concentration of 5-10 micrograms/ml. Even greater sensitivity can be achieved, at greater expense, by increasing the quantities of the radioisotopes in the labeling reaction. The particular value of the double label approach is that complex mixtures from two different sources are resolved together thus eliminating the possibility of differences arising from the resolving procedure itself. The procedure was applied to a mixture of serum and plasma from a single subject and a number of qualitative and quantitative differences were observed.     

Free malonaldehyde determination in tissues by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of free malonaldehyde (MA) in tissues. HPLC separation was performed using a TSK G1000 PW column (7.5-mm i.d. X 30 cm) with a mobile phase of 0.1 M Na3PO4 buffer, pH 8.0, at a flow rate of 0.6 ml/min. The eluant was monitored at 267 nm. Free MA in the tissue sample was separated and quantified in approximately 50 min. The lowest amount of MA that can be determined by this HPLC technique is approximately 1 ng per injection. This method was successfully applied to rat liver and beef, pork, and chicken muscle and was compared to the thiobarbituric acid (TBA) test. It was found to be more sensitive, accurate, and specific for the determination of free MA than the TBA method.     

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.     

Direct assay for creatine kinase by reversed-phase high-performance liquid chromatography

A direct assay for creatine kinase (CK) activity was developed based on the separation and quantitation of adenosine triphosphate (ATP) by high-performance liquid chromatography. The total incubation time is 13 min and the elution time for ATP is 16 min. Using lyophilized CK as the sample, a sensitivity in the range of 8 U/l (units/liter) was obtained. The method presented also has clinical significance in that CK levels in serum can easily be determined with minimal sample preparation. Using serum samples from a healthy patient and a heart attack victim, activities of 26.6 U/l and 609.0 U/l, respectively, were obtained. Because of the direct measurement of ATP, this method eliminates the coupled reactions encountered in the common spectrophotometric and colorimetric methods of analysis resulting in a simpler and inexpensive assay.     

Quantitative determination of epinastine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the quantitative determination of epinastine, a non-sedating histamine H₁ receptor antagonist, in rat plasma, was developed. A 100-μl volume of plasma sample was spiked with a solution of internal standard (diphenidol) and extracted with dichloromethane under alkaline conditions. The extract was applied onto the HPLC system and detected by ultraviolet absorption at a wavelength of 220 nm. The linearity of the calibration curve was preserved over the concentration range of 20--1000 ng/ml. Both intra-assay variation and relative error were less than 5% for the plasma sample containing 50 ng/ml or 1000 ng/ml of epinastine hydrochloride. The analytical method presented here should be useful for the investigation of the pharmacokinetic properties of epinastine, which is of clinical significance.