Rapid Quantitation of desmosine content in tissue hydrolysates by high-performance liquid chromatography

The lysine-derived crosslinks in elastin, desmosine, and isodesmosine, are quantitated in tissue hydrolysates by monitoring high-performance liquid chromatography eluents at 275 nm. The results from this method compare favorably with results from the amino acid analyzer. However, this more sensitive method (1) eliminates ninhydrin-positive artifacts which elute with the desmosines from some tissue hydrolysates on the amino acid analyzer, and (2) makes possible elastin quantitation in a tissue with the minimum amount of manipulations.     

Analysis of histidine and urocanic acid isomers by reversed-phase high-performance liquid chromatography

The qualitative separation performance of a C₁₈, C₈ and C₄ reversed-phase column was investigated for the separation of histidine and its metabolites histamine, 1-methyihistamine and trans- and cis-urocanic acid. Trans- and cis-urocanic acid were baseline separated from their precursor histidine on all three columns using isocratic elution with a mobile phase composed of 0.01 M aqueous TEAP pH 3.0 and acetonitrile at a ratio of 98:2 (v/v). However, histidine was not separated from histamine and 1-methyihistamine. Selecting the C₈ column and introducing 0.005 M of the ion pairing reagent 1-octanesulfonic acid sodium salt into the aqueous solution and acetonitrile at a ratio of 90:10 (v/v), significantly improved the separation. The separation was also followed by a change in the retention times and the order of elution. The sequence of elution was histidine, cis-urocanic acid, trans-urocanic acid, histamine and 1-methylhistamine with retention times of 5.58±0.07, 7.03±0.15, 7.92±0.18, 18.77±0.24 and 20.79±0.21 min (mean±SD; n=5). The separation on the C₈ column in the presence of the ion-pairing reagent was further improved with gradient elution that resulted in a reduction in the retention times and elution volumes of histamine and 1-methylhistamine. The detection limits of histidine and trans-urocanic acid at a wavelength of 210 nm and an injection volume of 0.05 ml were 5×10⁻⁸ mol l⁻¹ (n=3). The kinetic of the in-vitro conversion of trans- into the cis-isomer after UV irradiation was depending on the time of exposure and the energy of the light source. UVB light induced a significantly faster conversion than UVA light. TUCA and cUCA samples kept at −25°C were stable for up to 50 weeks. Samples, eluted from human skin showed various concentrations of histidine and trans- and cis-urocanic acid with an average of 1.69±0.33×10⁻⁵ mol l⁻¹, 1.17±0.43×10⁻⁵ mol l⁻¹ and 1.67±0.33×10⁻⁵ mol l⁻¹, respectively (n=8).     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography

An HPLC method for quantifying the 3-hydroxypyridinium crosslinks of collagen is described. It can be applied to crude hydrolysates of all types of connective tissue. Mineralized tissues can be hydrolyzed directly and analyzed without interference from the mineral ions. The hydroxylysyl (HP) and lysyl (LP) forms of hydroxypyridinium residue were resolved on a reverse-phase C18 column using a gradient of acetonitrile in water and 0.01 M n-heptafluorobutyric acid as an ion-pairing agent. The crosslinking amino acids were accurately quantified down to 2 PM (1 ng) injected, by detecting their natural fluorescence with a spectrofluorometer. Tissues in which hydroxypyridinium crosslinks were plentiful included all forms of cartilage, bone, dentin, ligament, tendon, fascia, intervertebral disc, lung, gut, cervix, aorta, and vitreous humor. Among normal tissues, LP, the minor form of the crosslink, was present in significant amounts relative to HP only in bone and dentin. Both crosslinks were essentially absent from skin, cornea, rat tail tendon, and basement membranes.     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Separation of porphyrin isomers by high-performance liquid chromatography.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.     

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.     

Quantitation of harringtonine and homoharringtonine in serum by high-performance liquid chromatography

Harringtonine and homoharringtonine are naturally occurring alkaloids with demonstrated antineoplastic activity against certain types of leukemias in cell cultures, experimental animals, and initial clinical trials. Sample preparation consists of addition of the internal standard (one compound used as the internal standard for the other), solvent extraction with methylene chloride, washing with ammonium formate, and evaporation to dryness. The residue is dissolved in the mobile phase (40% methanol—60% 0.1M ammonium formate) and an aliquot is chromatographed on μC₁₈ reversed-phase column (flow-rate 1.5 ml/min). Peaks are detected with a spectrophotofluorimeter by monitoring the emission at 320 nm with excitation wavelength of 280 nm. Limit of detection is 10 ng/ml (20 nM) for both compounds; reproducible quantitation can be made to 30 ng/ml (60 nM).     

Quantitation of hyaluronic acid in tissues by ion-pair reverse-phase high-performance liquid chromatography of oligosaccharide cleavage products

A method for quantifying hyaluronic acid in biological tissues and fluids is described. The assay uses ion-pair HPLC to resolve and quantify the oligosaccharide end products of Streptomyces hyaluronidase digestion. Tissue samples were solubilized by papain, and the nondiffusate after dialysis was exhaustively digested with Streptomyces hyaluronidase. The resulting tetrasaccharide and hexasaccharide cleavage products were resolved by reverse-phase high-performance liquid chromatography in the presence of the ion-pairing agent, tetrabutylammonium phosphate. The saccharides were detected and quantified by their absorbance at 232 nm due to the alpha, beta-unsaturated carboxyl group generated by the eliminase reaction. In control experiments 93 +/- 3% of a hyaluronic acid standard so treated was reproducibly recovered as its tetra- and hexasaccharide cleavage products. As little as 0.5 microgram of the oligosaccharides could be quantified with no interference from a vast excess of chondroitin sulfate or other tissue components. The assay was applied to various types of human, bovine, and rabbit cartilage and to samples of other tissues including nucleus pulposus, annulus fibrosus, skin, aorta, cervix, cockscomb, synovial fluid, and vitreous humor. Results on human articular cartilage showed a linear increase in the content of hyaluronate from 0.1 to 0.5% of tissue dry weight between birth and 80 years of age.     

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.     

Amino acid analysis of collagen hydrolysates by reverse-phase high-performance liquid chromatography of 9-fluorenylmethyl chloroformate derivatives

A recently described procedure for amino acid analyses has been modified and adapted for use in quantitating the unique mixture of products commonly found in hydrolysates of the collagens. The method involves precolumn derivatization of hydrolysates with 9-fluorenylmethyl chloroformate (FMOC-CL), chromatographic separation of the derivatives and excess reagent on a reverse-phase column, and quantitation based on the fluorescent properties of the derivatives. The method takes advantage of the ease with which stable derivatives are formed with the FMOC reagent. Using a ternary gradient system, a complete amino acid analysis with good resolution of all components can be performed within 35 min. The sensitivity of the method is comparable to levels attained by other derivatives and the fluorescence response of each derivative is linear over the total range of 1-800 pmol. Given these parameters, the method allows complete amino acid analyses to be performed on 100 ng of collagen corresponding to a single picomole of a collagen chain (Mr 100,000).     

Quantitation of cocaine and cocaethylene in canine serum by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic procedure for the determination of cocaine and cocaethylene in canine serum has been developed. The compounds were extracted from 1 ml of alkalinized canine serum with hexane. Chromatographic separation was achieved with a cyanopropyl column (250 × 4.6 mm I.D., 5 μm) using a mobile phase of acetonitrile and phosphate buffer, pH 7.40 (38:62, v/v) flowing at 1 ml/min. Eluate was monitored by a variable-wavelength UV detector set to 230 nm. The extraction procedure yields an average recovery of 99 and 96% for cocaine and cocaethylene, respectively. The between-day coefficients of variation, at 2400 ng/ml, for cocaine and cocaethylene were both 8.6% and the within-day coefficients of variation, at 400 ng/ml, for cocaine and cocaethylene were 7.3 and 8.0%, respectively. A concentration-time profile resulting from administration of 3 mg/kg cocaine and cocaethylene to the dog revealed a similar disposition between cocaine and cocaethylene, with a clearance and volume of distribution at steady-state values of 72.8 and 61.0 ml/min/kg and 2.6 and 2.7 1/kg, respectively.     

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4′-bromo-[1,1′-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen-1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO₂), were isolated from the basified biological matrix using a liquid–liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate–hexane–triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.     

Separation of positional and geometrical fatty acid isomers as 2-nitrophenylhydrazide derivatives by high-performance liquid chromatography

Previously, following derivatization to their 2-nitrophenylhydrazide (2-NPH) derivatives, fatty acid (FA) abundances have been evaluated using high-performance liquid chromatography (HPLC). Although the method was sensitive, resolution was insufficient for many of the biologically important FAs. We have developed an enhanced separation of 24 FAs by use of different column temperature, stationary phase, and mobile phase gradient conditions. We applied this method to analysis of mouse skeletal muscle phospholipid and triglyceride. This further development of the chromatographic separation of 2-NPH FAs may lead to greater utility of this HPLC approach.