Fluorometric analysis of polyamines, histamine, and 1-methylhistamine.

This paper describes an automated cation-exchange chromatographic method for the simultaneous analysis of di- and polyamines, histamine, and its metabolite 1-methylhistamine. Recommendations for the selection of an effective system are made after evaluating sodium- and potassium-containing buffers, pH, temperature, column packings, and detection methods. The method can be used to analyze di- and polyamines in biological fluids, such as urine and tissue extracts, and may be useful for monitoring the effectiveness of cancer chemotherapy.     

Rapid chromatographic method to determine polyamines in urine and whole blood

A procedure is described for the rapid determination of putrescine, spermine and spermidine in urine and whole blood. The samples are hydrolyzed with barium hydroxide and are neutralized with sulfuric acid. The polyamines are concentrated and separated from amino acids on a small bed of ion-exchange resin that then serves to load the samples on a two-channel, automated ion-exchange chromatography apparatus. As many as 100 samples can be analyzed in a 24-h period. The method has been shown to be applicable to the analysis of urine and whole blood samples, but further development is needed for application to serum samples.     

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues.The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method.Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.     

An Improved Isolation Procedure for the Gas Chromatographic Analysis of Urinary Polyamines

The isolation of polyamines from urinary hydrolysates in a sufficiently pure state for subsequent analysis by gas chromatography has proved to be difficult. However, by using columns of Porapak-Q and ion-exchange resins, urinary hydrolysates are readily purified and formation of trifluoroacetyl derivatives of polyamines proceeds in high yield without carryover of artifacts in the gas chromatographic elution profile. Good yields from the trifluoroacetylation reaction are not achieved if large quantities of salts or urinary pigments are present. By obtaining the polyamine carbonates in the final stages of the method described, the trifluoroacetylation reaction yields excellent derivatives of nanogram or microgram amounts, particularly after standing over-night at room temperature. The procedure described in detail should permit routine urinary polyamine analysis where rapidity, ease of handling many samples, freedom from complications and artifacts are a consideration. The recent reports by Russell^{1, 2} that the urinary excretion of polyamines are greatly elevated in cancer patients has stimulated interest in these compounds as possible biological “markers” for the diagnosis of cancer. The polyamines usually considered are: putrescine, 1, 4-diaminobutane; cadaverine, 1, 5-diaminopentane; spermidine, and spermine. An extensive literature has developed over the last 50 years concerning the isolation and determination of polyamines including many excellent reviews. ^3–5 However, the isolation and determination of small quantities of polyamines from biological sources has proven to be difficult. This has led to conflicting conclusions among investigators as to which polyamine is the major excretion product in the urine of cancer patients. ^{2, 6, 7, 8} The following report presents in detail a new procedure of isolation of urinary polyamines in high yield and pure state that facilitates quantitation of these amines by gas chromatography.     

Liquid chromatographic analysis of penem antibiotic FCE22101 in biological fluids with a liquid-liquid extraction procedure

A sensitive high-performance liquid chromatographic method has been developed for the determination of penem antibiotic FCE22101 in plasma and urine. FCE22101 was extracted from plasma and urine with ethyl acetate. After evaporating, the sample solutions were analyzed by a reversed-phase high-performance liquid chromatographic system using a two-sided bracketing injection technique. The determination limit of FCE22101 was 5 ng/ml in plasma. Analysis of the spiked plasma samples demonstrated the good accuracy and precision of the method. The proposed method improved from five- to ten-fold on the analytical sensitivity in comparison with the most commonly ultrafiltration method.     

Biological and biomedical applications of isoelectric focusing : edited by N. Catsimpoolas and J. Drysdale, Plenum Press, New York, XV + 351 pp., Price US$ 39.00, ISBN 0-306-34603-6

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5–9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

Determination of polyamines in human prostate by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination of polyamines in human prostate has been developed. This method is based on pre-column derivatization with dansyl chloride (Dns-Cl). The derivatives were separated on a μBondapak C₁₈ column (250×4.6 mm I.D.; 10 μm), and eluted with methanol and distilled water using a one-step linear gradient. The column eluate was monitored by fluorescence detection (excitation, 370 nm; emission, 506 nm). The within-assay precision of the study (C.V.) was as follows: putrescine (PUT) 2.88%, spermidine (SPD) 2.94% and spermine (SP) 1.17%. The between-assay precision (C.V.) was: PUT 2.66%, SPD 3.06%, SP 2.79%. The recovery was greater than 97%. The detection limit for PUT, SPD and SP were 0.05, 0.08 and 0.06 nmol/ml, respectively. In contrast to other studies, sample or polyamine derivatives did not require extraction with an organic solvent such as ethanol, evaporation under vacuum or other condensation procedures. This is a simple, rapid and sensitive method that can be applied to the determination of polyamines in nearly all biological tissues and body fluids, such as urine and serum.     

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE II EX robotic liquid handling system.     

An improved and easy technique for polyamine determination in biological samples : Application to cell-free system from hypertrophied rat heart

An accurate, improved cation-exchange chromatographic method using o-phthalaldehyde and ultraviolet detection at 280 nm for the determination of free polyamines (putrescine, spermidine, spermine) has been developed. Different samples, such as the 105,000 g supernatant of reticulocyte or heart muscle, and KCl ribosomal wash containing initiation factors, can be analysed. The minor modification of reagents results in a good precision and sensitivity, which is demonstrated by a relative standard deviation of 5–9% and recoveries of 98%. This technique is of particular interest because it allows polyamine determination in biological samples with high concentrations of salt.     

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.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.     

Semi-automatic liquid chromatographic analysis of pamidronate in urine after derivatization with 1-naphthylisothiocyanate

An existing sensitive chromatographic assay for pamidronate in urine has considerably been automated. Using the same sample processor, the solid-phase extraction (SPE) was automated separately from the derivatization with 1-naphthylisothiocyanate, the two-fold ion-pair liquid–liquid-extraction and the treatment with hydrogen peroxide for the 2–20 ng/ml concentration range. The automatic procedure was preceded by a triple calcium precipitation and interrupted by evaporation of the SPE eluate under nitrogen. For the 0.5–5 μg/ml concentration range one automatic sequence was used by avoiding evaporation during the sample treatment. In addition to the labour-saving of the semi-automatic procedure, the daily sample-throughput was improved compared to the existing manual assay. Further, the validation showed marginal improvements in the precision, accuracy and lower limit of quantification.     

On-line extraction using an alkyl-diol silica precolumn for racemic citalopram and its metabolites in plasma: Results compared with solid-phase extraction methodology

Sample preparation is usually the most critical and time consuming step when using HPLC for drug analysis in biological matrixes. Sample extracts have to be clean considering both chromatographic interferences and column maintenance. To meet some of these criteria a fully automated on-line extraction (OLE) analysis method was developed for the antidepressant drug citalopram and its two demethylated metabolites, using an RP-C4-ADS extraction column. A comparison between the new OLE method and an off-line solid-phase extraction method showed that the two methodologies were equal in analytical precision but that the OLE method was faster and therefore superior in sample capacity per day.     

Simultaneous determination of free and N-acetylated polyamines in urine by semimicro high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

We have developed a simple and highly sensitive semimicro high-performance liquid chromatographic method for the simultaneous determination of free and N-acetylated polyamines in urine. Polyamines and N-acetylated polyamines were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 50 degrees C for 15 min at pH 9. The fluorescent derivatives were separated on a reversed-phase column with a gradient elution using water-acetonitrile-methanol at 50 degrees C and detected by fluorescence measurement at 318 nm (excitation) and 406 nm (emission). The detection limits (signal-to-noise ratio=3) of the polyamines and N-acetylated polyamines were 0.7-4.5 fmol/injection. The within-day and day-to-day relative standard deviations were 3.2-7.9 and 3.0-7.7%, respectively. Significant differences were found in the urinary excretion of polyamines between cancer patients and normal subjects.     

Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography.

A fully automated method for assaying the collagen crosslinking amino acids, pyridinoline and deoxypyridinoline, in human urine samples or tissue hydrolysates is described. Samples were processed using a Gilson ASPEC system with solid-phase extraction of the crosslinks on columns containing 100 mg of microgranular cellulose. Introduction of an additional solvent step during sample preparation allowed direct analysis by reversed-phase HPLC and elimination of the drying step used previously in a manual method. Use of a synthetic pyridinoline derivative as internal standard enabled accurate quantification of the crosslinks by correcting for recoveries through the whole assay. Samples were analyzed in sequential mode with a total assay time of 30 min. The automated assay showed close correlation with the manual method for both free and total crosslink determinations in human urine (r > 0.97). Reproducibility was improved, as seen from replicate analyses of human urine (CV < 3% for automated pyridinoline measurement compared with 8-12% previously observed for the manual method). Crosslink excretion is the most useful marker of collagen degradation in metabolic bone diseases and arthritic disorders. The automated assay which has been developed is rapid, convenient, and reliable and will greatly facilitate the monitoring of urinary collagen crosslinks and their tissue levels in clinical investigations.     

Simultaneous determination of ampicillin and metampicillin in biological fluids using high-performance liquid chromatography with column switching

A new high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of metampicillin and its metabolite ampicillin in biological fluids. The plasma, urine and bile samples were injected onto a precolumn packed with LiChrosorb RP-8 (25–40 μm) after simple dilution with an internal standard solution in 0.05 M phosphate buffer (pH 7.0). The polar plasma components were washed out using 0.05 M phosphate buffer (pH 7.0). After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Ultracarb 5 ODS-30 column with a gradient system of acetonitrile-0.02 M phosphate buffer (pH 7.0) as the mobile phase. The method showed excellent precision, accuracy and speed with a detection limit of 0.1 μg/ml. The total analysis time per sample was less than 40 min and the coefficients of variation for intra- and inter-assay were less than 5.1%. This method has been successfully applied to plasma, urine and bile samples from rats after intravenous injection of metampicillin.     

Determination of uric acid in biological fluids by high-pressure liquid chromatography

A high-pressure liquid chromatographic assay for uric acid in biological fluids has been developed. Blood uric acid can be analyzed in as little as 20 μl of plasma. The mean and range of plasma uric acid concentrations in healthy adults determined by high-pressure liquid chromatography were similar to these obtained by enzymatic analysis. One of the advantages of the present method is that naturally occurring metabolites in biological fluids or drugs do not interfere with the analysis. Data are presented for blood and urine specimens obtained from mice fed a known uricase inhibitor, potassium oxonate. Comparisons are made between the present method and methods previously employed for uric acid determination.     

Direct determination of 4-hydroxy-3-methoxyphenylacetic (homovanillic) acid in urine by high-performance liquid chromatography with amperometric detection

The improvement of high-performance liquid chromatographic analysis with electrochemical detection for urinary homovanillic acid is described. The method permits the chromatographic resolution of authentic homovanillic acid from coeluting interfering compounds in human and nonhuman primate, and rat urine. The electrochemically derived results are compared with post-column derivatized fluorescence results, and quality-control checks necessary to maintain assay precision in automated analysis are described.     

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.