Two-step high-performance liquid chromatography method for the determination of myo-inositol and sorbitol

A simple two-step HPLC method for the separation and quantitation of myo-inositol and sorbitol in extracts of glomeruli from rat kidneys is described. The limit of detection is 2 ng. The procedure involves fractionation of the sugar alcohols on a Waters Sugar Pak column, preparation of the p-nitrobenzoate derivatives, and further purification with quantitation by absorbance at 254 nm using a Waters mu Porasil column. The applicability of the procedure to determination of sorbitol and myo-inositol in biological samples was demonstrated by the finding of marked alterations in sorbitol and myo-inositol content of glomeruli isolated from diabetic compared to that from normal rat kidneys.     

Quantitative analysis of myo-inositol in urine, blood and nutritional supplements by high-performance liquid chromatography tandem mass spectrometry

Myo-inositol plays key physiological functions, necessitating development of methodology for quantification in biological matrices. Limitations of current mass spectrometry-based approaches include the need for a derivatisation step and/or sample clean-up. In addition, co-elution of glucose may cause ion suppression of myo-inositol signals, for example in blood or urine samples. We describe an HPLC-MS/MS method using a lead-form resin based column online to a triple quadrupole tandem mass spectrometer, which requires minimum sample preparation and no derivatisation. This method allows separation and selective detection of myo-inositol from other inositol stereoisomers. Importantly, inositol was also separated from hexose monosaccharides of the same molecular weight, including glucose, galactose, mannose and fructose. The inter- and intra-assay variability was determined for standard solutions and urine with inter-assay coefficient of variation (CV) of 1.1% and 3.5% respectively, while intra-assay CV was 2.3% and 3.6%. Urine and blood samples from normal individuals were analysed.     

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 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 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 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.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Analysis of minocycline by high-performance liquid chromatography in tissue and serum

A sensitive and rapid reversed-phase high-performance liquid chromatography assay can be used to accurately determine serum and tissue minocycline concentrations. Minocycline is a broad spectrum tetracycline derivative with many applications. Tissue and serum samples were obtained from guinea pigs that had received either topical or intravenous minocycline. Samples were extracted using a Sep-Pak C₁₈ cartridge and were injected into a μBondapak C₁₈ column with an isocratic methanol mobile phase. Samples were analyzed using UV detection and produced sharp peaks with a retention time of 2.5 min. The lower limit of detection was 100 ng and drug recovery was 61%. This method greatly facilitated the analysis of minocycline while allowing for sensitivity.     

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 polysulfated chondroitin disaccharides by high-performance liquid chromatography

A high-performance liquid chromatography method for analyzing disaccharides derived from chondroitin sulfate glycosaminoglycans has been developed which employs a Whatman Partisil-10 PAC amino-cyano column and an acetonitrile/methanol/ammonium acetate solvent to resolve disulfated, monosulfated, and unsulfated disaccharides in a chromatographic run of less than 20 min. The single known trisulfated chrondroitin disaccharide can be eluted in an alternate solvent system containing the same mobile phase components in different proportions. Disaccharides were prepared for chromatography from glycosaminoglycans and proteoglycans of known compositions by digestion with chondroitinase ABC, with the exception of king crab cartilage glycosaminoglycan which was incubated sequentially with hyaluronidase and chondroitinase ABC. Disaccharides were extracted from the digestion mixtures in 80% ethanol, dried over nitrogen, resuspended in the HPLC solvent, and chromatographed at a flow rate of 1 ml/min. Unsaturated disaccharides in the column eluate were detected by continuous ultraviolet absorbance monitoring at 232 nm; alternatively, fractions were collected and assayed for uronic acid content or radioactivity. By utilizing the HPLC technique in conjunction with chondroitinase ABC and AC digestion and sulfatase hydrolysis, the epimeric structures of chondroitin sulfates E and H were confirmed. With this technique, rapid and reproducible analyses of chondroitin sulfate disaccharides generated from mouse mast cell proteoglycan and from glycosaminoglycans of squid cranial cartilage, shark skin, hagfish skin, and hagfish notocord were in close agreement with compositions obtained by other techniques.     

Analysis of abscisic acid by high-performance liquid chromatography.

Methodology for the ready analysis of abscisic acid (ABA) in plant tissues based upon application of high-performance liquid chromatography (hplc) has been developed. The method involves isolation of the acid fraction, preparation of the methyl esters with diazomethane, and hplc using a combination procedure of two columns: (1) reversed-phase C₁₈, and (2) porous silica in the absorption mode. Only isocratic elution is required so the method is readily adaptable to laboratories having limited hplc capability. Measured recoveries are 70% and the use of an internal standard allows quantification of ABA levels to 1 ng/g of tissue with minimum absolute detectable levels of ABA of 20 ng. The method is illustrated by analysis of ABA concentration in potato tubers at various times postcutting.     

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.     

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.     

Analysis of nucleotides from Tetrahymena by high-performance liquid chromatography

Procedures for the complete extraction and isocratic high-performance liquid chromatography analysis of cellular nucleoside di- and triphosphate are reported. Maximum nucleotide recovery from cultures of the ciliate Tetrahymena pyriformis is achieved if (a) cells are cooled to 4°C prior to harvest; (b) dicarbonic acid diethyl ester, a protein crosslinking reagent, is added prior to neurtralization of the cellular acid extract; and (c) 350 mm tri-n-octylamine in Freon is used as a neutralizing agent. These procedures also extend the useful lifetime of the Waters μBondapak NH₂ column used for high-performance liquid chromatography analysis. Nucleoside triphosphates are resolved on this column in under 25 min using 125 mm ammonium phosphate, pH 3.15. Nucleoside diphosphates are separated by reducing the buffer concentration to 75 mm.