High-performance liquid chromatography of coproporphyrin isomers.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.     

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 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.     

Application of ion-pair high-performance liquid chromatography to detection of the atypical coproporphyrin isomers II and IV in human faeces

A highly selective and sensitive method has been developed for the detection of small amounts of the atypical isomers II and IV of coproporphyrin in human faeces. This method combines liquid—liquid extraction and solid-phase sampling techniques using talc and C₁₈-modified silica gel as the sorbents. Simultaneous separation of the four coproporphyrin isomers I–IV was achieved by isocratic ion-pair high-performance liquid chromatography. Stool samples of healthy subjects (n = 12) contained 1.1 ± 0.4% (mean ± S.D.) isomer II and 2.2 ± 0.9% isomer IV of total coproporphyrins. A somewhat higher content of isomer II (2.7%) and isomer IV (5.4%) was found in faeces of a patient suffering from porphyria variegata.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.     

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 hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.     

Determination of malondialdehyde by ion-pairing high-performance liquid chromatography

A method for the analysis of malondialdehyde (MDA) by ion-pairing HPLC is described. The method is direct, no derivitization is required, and sample preparation is minimal. After removal of particulates, the samples are injected directly onto an octadecylsilane column which is eluted with 14% (v/v) acetonitrile in 50 mM myristyltrimethylammonium bromide. 1 mM phosphate, pH 6.8. Detection is accomplished by monitoring absorbance at 254 nm or for greater sensitivity at 267 nm. The lower limit for reliable quantitation is 5 pmol MDA and the dynamic range extends to at least 4 nmol MDA. The method has been applied to the quantitation of MDA production during microsomal lipid peroxidation and to an assessment of the stability of MDA in microsomal and urine samples.     

Determination of acyclovir by ultrafiltration and high-performance liquid chromatography

A simple, sensitive and rapid high-performance liquid chromatographic (HPLC) procedure to determine total serum acyclovir concentrations is described. The assay involves a heat inactivation step at 56°C to prevent risk of infection, ultrafiltration as a pretreatment step prior to ion-pair reversed-phase liquid chromatography using guanosine as internal standard, and ultraviolet detection at 254 nm. This method has excellent recovery (97–100%), linearity (0.5–100 mg/l) and precision (1.2–8.0% coefficient of variation). The detection limit is 50 μg/l. The assay proved to be suitable for therapeutic drug monitoring of acyclovir.     

Determination of glutamate decarboxylase by high-performance liquid chromatography

An improved method for the determination of glutamate decarboxylase (GAD) activity is described. The enzyme was evaluated by incubation with glutamic acid (l-Glu) in the presence of pyridoxal 5 ′-phosphate (PLP): the γ-aminobutyric acid (GABA) formed was derivatized to PTC-GABA; the latter was subsequently separated and assayed by isocratic HPLC (LiChrospher RP-18 column; isocratic elution with pH 5.8 acetate buffer in acetonitrile-water) with UV absorbance detection at 254 nm. The method described is a sensitive, reproducible and specific assay useful for following variations of GAD activity in vitro; this assay was subsequently used for the evaluation of GAD activity variations after irradiation with low doses of HeNe laser radiation.     

Dermatan sulfate isomers in human articular cartilage characterized by high-performance liquid chromatography

The Constituents of dermatan sulfate isomers in human articular cartilage were studied at the disaccharide level by high-performance liquid chromatography. Appreciable amounts of dermatan sulfate components, i.e., dermatan sulfate, chondroitin sulfate types G and B, could be detected after digestion with chondroitinases-B or -ABC. The oversulfated dermatan sulfate isomers were isolated only after digestion with chondroitinase-ABC but not with the AC-lyase. The dermatan sulfate isomers were found to be markedly increased in weight loading parts of articular cartilage. It is postulated that the dermatan sulfate isomers are formed as a result of the weight loading reaction, which may be responsible for the fibrosis of articular cartilage.     

Analysis of angiotensins I,II, III,and iodinated derivatives by high-performance liquid chromatography

Angiotensins I, II, and III were separated by reversed-phase high-performance liquid chromatography on an octadecylsilyl column. The peptides were isocratically eluted with 50 mm NaH₂PO₄-25% ($\text{v}\text{v}$) acetonitrile, pH 6.0. The retention times were 3.3, 6.0, and 9.6 min for angiotensin II, III, and I, respectively. ¹²⁵I-Angiotensins II, III, and I eluted with retention times of 5.4, 16.8, and 19.9 min, respectively, under the same chromatographic conditions used for the unlabeled angiotensins. The effect of iodination of the tyrosine residue on the retention time was also demonstrated by chromatographic comparison of tyrosine and diiodotyrosine. Saralasin (Sar¹, Ala⁸-angiotensin II), a partial agonist of angiotensin II, and des-Asp¹, Ile⁸-angiotensin II, an inhibitor of angiotensin III, eluted with retention times of 2.5 and 3.9 min, respectively.     

Determination of medrogestone in plasma by high-performance liquid chromatography

Interference with the UV absorbance of medrogestone by endogenous steroids in plasma was prevented by reacting plasma with oxalyl chloride. The reduction of interference was effective when oxalyl chloride was in the range 10–50 μl/ml plasma. Reaction of oxalyl chloride with plasma for 10 min could reduce interference approximately 5.5-fold, and there was no significant reduction after 30 min. The limit of quantitative concentration for medrogestone in HPLC was 1 ng/ml. The standard curves were linear with the correlation coefficient greater than 0.999 in the range of 1–30 ng/ml. The coefficients of variation of both intra- and inter-day mean values were <12% and <10% of the actual values, respectively. The developed method for plasma sample preparation and the evaluated HPLC condition were further applied to an in vivo pharmacokinetic study.     

Determination of metformin in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of metformin, an oral antidiabetic agent, in plasma is described. Plasma samples containing the internal standard, phenformin, are eluted through Amprep extraction columns before injection into the chromatographic column, packed with μBondapak phenyl. The eluent is monitored at 236 nm. At a mobile phase flow-rate of 1.35 ml/min, the retention times of metformin and phenformin are 2.8 and 5.6 min, respectively. The intra-day coefficients of variation are 1.5 and 4.3% at metformin concentrations of 0.05 and 1 mg/l, respectively.