Determination of polyamines in urine of normal human and cancer patients by capillary gas chromatography

A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic-anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 +/- 0.041 (micrograms/mg creatinine, mean +/- SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).     

Determination of polyamines by flow injection analysis with a chemiluminescence detector based on their complexation with copper(II).

Through the flow injection analysis experiments, we discovered that an unsaturated complex of Cu(II) and polyamines (spermine, spermidine, putrescine) had a strongly catalytic effect on luminol-H(2)O(2) chemiluminescence (CL) reaction, and that the CL intensity is proportional to the concentrations of polyamines. Based on the automatic formation of an unsaturated complex of polyamines and Cu(II) when the solution containing polyamines passed through a column packed with solid Cu(OH)(2), a new flow injection chemiluminescence analysis method was proposed for the determination of polyamines. The effects of pH, buffer concentration, the concentration of chemiluminescence reagent, and the influence of mixing coil length were examined. Under optimal conditions, the linear range was from 1.0 x 10(-7) mol L(-1) to 1.0 x 10(-5) mol L(-1), and the detection limits were 0.17, 0.38, 0.44 pmol for spermine, spermidine, and putrescine, respectively. Compared with other methods, the advantages of this method include convenience, time-saving and low cost.     

Determination of polyamines in hydrolysates of uremic plasma by high-performance cation-exchange column chromatography

The levels of putrescine, cadaverine, spermidine and spermine in uremic plasma were determined with an automatic polyamine analyzer with a 7.5 X 0.2 cm I.D. cation-exchange column using a stepwise sodium chloride gradient. All four polyamines were higher in ten patients with chronic renal failure than in eight normal subjects. The total polyamine content was also measured in the patients' plasma before and after maintenance dialysis; putrescine and spermidine levels were significantly lowered by the procedure.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.     

HPLC analysis of polyamines and their acetylated derivatives in the picomole range using benzoyl chloride and 3,5-dinitrobenzoyl chloride as derivatizing agent

Analytical methods are described for the quantitative determination of putrescine, cadaverine, spermidine, spermine and the acetylated derivatives of spermidine and spermine in biological fluids using pre-column derivatization with either benzoyl chloride or 3,5-dinitrobenzoyl chloride, which were added to each sample as solutions in diethyl ether. Putrescine, spermidine and spermine can be analysed in seminal plasma at nanogram levels when benzoyl chloride is used as derivatizing agent. In the analysis of putrescine, cadaverine, spermidine and acetyl derivatives of spermidine and spermine, higher sensitivity is obtained with 3,5-dinitrobenzoyl-chloride. This method can readily be used in the determination of acetylated polyamines in urine samples.     

Liquid chromatographic determination of polyamines in human urine based on intramolecular excimer-forming fluorescence derivatization using 4-(1-pyrene)butanoyl chloride

A liquid chromatographic method for highly sensitive and selective fluorometric determination of polyamines (putrescine, cadaverine, spermidine and spermine) in human urine is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride (PBC), followed by reversed-phase liquid chromatography. The method offers higher sensitivity for determination of spermidine and spermine than previously reported method utilizing 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester as a derivatization reagent. Samples containing free polyamines in diluted human urine were directly derivatized with PBC and separated on an octyl column. The derivatives were detected at excitation 345 and emission 475 nm wavelengths. For determination of total polyamine content, the conjugated polyamines were first hydrolyzed in 4 M HCl. The detection limits (signal-to-noise ratio = 3) for polyamines in urine were 1.1-3.4 pmol/mL. At optimized derivatization and chromatographic conditions, interferences such as biogenic monoamines gave no peaks or the peaks did not interfere with the peaks of polyamine derivatives. In conclusion, the present derivatization method allows direct determination of polyamines in human urine samples without the need for sample clean-up procedures.     

Determination of 5-dimethylaminonaphthalene-1-sulfonyl derivatives of urinary polyamines by ion-pair high-performance liquid chromatography

A sensitive and specific method for the determination of diamines and polyamines by ion-pair high-performance liquid chromatography is described. The 5-dimethylaminonaphthalene-1-sulfonyl derivatives of putrescine, 1,6-diaminohexane, spermidine and spermine are separated on a muBondapak C18 reversed-phase column with 1-heptanesulfonic acid and acetonitrile as the mobile phase. All compounds are eluted within 30 min using a programmed solvent gradient system. The method has a lower detection limit of 1 pmole on column. Because of the simplicity of the method, its application provides a better means for closely monitoring patients undergoing treatment for various types of genito-urinary neoplastic diseases.     

Selective fluorometric detection of polyamines using micellar electrokinetic chromatography with laser-induced fluorescence detection

The polyamines putrescine, cadaverine, spermine and spermidine were separated and quantified by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection. The derivatization reagent, 1-pyrenebutanoic acid succinimidyl ester (PSE), allowed for the selective detection of the polyamines at 490 nm. Multiple labeling of the polyamines with PSE allows the formation of intramolecular excimers that emit at longer wavelengths (450-520 nm) than mono-labeled analytes (360-420 nm). Optimal separation of the labeled polyamines was achieved using a separation buffer consisting of 10 mM phosphate pH 7.2, 30 mM cholate, and 30% acetonitrile. Using these conditions, the four polyamines were separated in under 10 min. Limits of detection for putrescine, cadaverine, spermine and spermidine were 6, 5, 15 and 13 nM, respectively. These are superior or comparable to those previously reported in the literature using fluorescence detection.     

对酞内酰胺苯甲酰氯柱前衍生反应HPLC分析痕量多胺

本文提出了一种快速、简便、灵敏检测痕量多胺的新方法。用对酞内酰胺苯甲酰氯同多胺进行柱前衍生反应,以反相高效液相色谱定量测定多胺含量。在4～500 pmol范围内有良好的定量线性关系,腐胺、精脒和精胺的检测限均为0.1 pmol。     

Application of pharmacia automated FPLC System and PepRPC HR 5/5 bonded phase column for determination of dansylated polyamines

Summary The Pharmacia Automatized FPLC System equipped with a PepRPC HR 5/5 bonded-phase column was tested with dansyl polyamines, toluene, dimethyl- and dibutylphthalate, using either isocratic conditions or linear gradient-elution program of methanol in water as the mobile phase. A simple and reproducible method is described for the quantiation of dansylated natural polyamines, such as putrescine, spermidine and spermine originating from P388/S leukemia cells. Comparing the data from analyses performed in parallel by the Automated FPLC/PepRPC HR 5/5, and a Hewlett-Packard Model 1084B/LiChrosorb RP-8 systems a linear correlation has been found, with a regression coefficient of r=0.974.     

Sensitive fluorimetric method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles. The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 +/- 3.1, and spermine 8.4 +/- 2.8 nmoles/ml packed erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml. The polyamine content of the erythrocytes from patients with malignant neoplasms was significantly elevated.     

Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmoles for spermine to 12 pmoles for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the three polyamines. The procedure was applied successfully to urine and serum samples.     

Polyamines and corresponding aminoacids measured together

Summary A two-column switching technique plus gradient elution have been developed to separate polyamines (putrescine, spermine and spermidine) and related aminoacids (aspartic, glutamic, glycine, taurine and gaba) as isoindole derivatives. The system uses two columns (CN and C18) arranged in series, via a valve which allows the effluent to be switched from the primary column to the secondary column or directly to the detector.     

Rapid and Simple Technique for the Quantitation of Polyamines in Biological Samples

Abstract

A rapid and simple technique has been developed to quantify putrescine, spermidine, and spermine in biological tissue. The method, based upon several published procedures, involves protein precipitation with perchloric acid followed by dansylation with 5-dimethylamino-1-naphthalenesulfonyl chloride (dansyl chloride). After extraction on a Waters C₁₈ Sep-Pak cartridge, the samples are analyzed by high pressure liquid chromotography using a step solvent change and a 3μ C₁₈ reverse phase column. The chromotographic conditions allowed complete analysis of the three polyamines within 10 min with a total run time of 13 min (sample injection and re-equilibrium of column). Standard curves were linear up to 1 μg polyamine and the coefficient of variation for the assay ranged from 4% at l μg polyamine per sample to 11% at 50 ng polyamine per sample. The assay is therefore both rapid and simple. Moreover, unlike other available methods, the present technique does not require duel pumps, ion pairing agents, solvent extraction or a gradient control system. The concentrations of putrescine, spermidine and spermine in rat lung, liver and kidney are reported.     

Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer

To evaluate the potential relationship between cancer and polyamine metabolome, a UHPLC–MS/MS method has been developed and validated for simultaneous determination of polyamine precursors, polyamines, polyamine catabolite in human plasma and urine. Polyamine precursors including l-ornithine, lysine, l-arginine and S-adenosyl-l-methionine; polyamines including 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, agmatine, N-acetylputrescine, N-acetylspermine and N-acetylspermidine; polyamine catabolite including γ-aminobutyric acid had been determined. The analytes were extracted from plasma and urine samples by protein precipitation procedure, and then separated on a Shim-pack XR-ODS column with 0.05% heptafluorobutyric acid (HFBA) in methanol and 0.05% HFBA in water. The detection was performed on UHPLC–MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The limits of quantitation for all analytes were within 0.125–31.25 ng mL⁻¹ in plasma and urine. The absolute recoveries of analytes from plasma and urine were all more than 50%. By means of the method developed, the plasma and urine samples from hepatic cancer patients and healthy age-matched volunteers had been successfully determined. Results showed that putrescine and spermidine in hepatic cancerous plasma were significant higher than those in healthy ones, while spermidine, spermine and N-acetylspermidine in hepatic cancerous urine were significant higher than those in healthy ones. The methods demonstrated the changes of polyamine metabolome occurring in plasma and urine from human subjects with hepatic cancer. It could be a powerful manner to indicate and treat hepatic cancer in its earliest indicative stages.     

Determination of polyamines by high-performance liquid chromatography with chemiluminescence detection

A method was developed for the determination of polyamines (PA) by high-performance liquid chromatography with chemiluminescence detection. It is based on the unsaturated complex of PA with Cu(II) which had a strong catalytic effect on the luminol-H₂O₂ chemiluminescence reaction. The separation of PA was carried out on a reveres phase C18 column using methanol/water (25/75, v/v) as a mobile phase. The method was applied to the analysis of putrescine and the total amount of spermine and spermidine in apple leaves and strawberry fruit. The results indicated that the method is practical and useful.     

The polyamines of Xanthium strumarium and their response to photoperiod

Flowering plants of Xanthium strumarium L., grown in 8 h photoperiods, were analysed for polyamines. Putrescine, spermidine and spermine were found throughout the plant in three forms: (a) as free polyamines; (b) conjugates soluble in 5% trichloracetic acid (TCA); and (c) bound to the TCA-insoluble precipitate. On a fresh weight basis, total polyamines are most abundant in young leaves and buds, especially flower buds. Spermidine predominates in the free polyamine fractions, while spermine is dominant in the conjugated fraction. Transfer of vegetative plants from 16 h photoperiods to 1, 2, 3, or 4 inductive cycles (8 h light + 16 h uninterrupted dark) caused rapid and marked changes in the polyamine titer of the leaves and ultimately, floral initiation. The titer of free putrescine per mg protein declined progressively with induction in all leaf sizes, while the titers of free spermidine and spermine rose during days 2 and 3 in small and expanding leaves. Conjugated putrescine, spermidine and spermine rose sharply after only 1 inductive cycle, especially in small and expanding leaves, and maintained the higher level for at least several cycles. In plants given 4 inductive cycles, buds harvested after 4 additional days had sharply elevated levels of conjugated polyamines, especially spermine, on a protein basis.     

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.     

Representative applications of heparin-sepharose in the removal of polyamines from biological materials

Many experimental systems would greatly benefit from the availability of a simple and effective technique to remove polyamines from biological materials. We have examined the possibility of utilizing heparin-sepharose in the removal of polyamines from rat heart mitochondria, DNA-spermine complex, and fetal calf serum. Heparin-sepharose removes 90% of spermine adsorbed to the cytoplasmic surface of rat heart mitochondria. Heparin-sepharose almost totally removes spermine from DNA-spermine complex, leaving less than 0.003 mol spermine/mol DNA phosphorus. Heparin-sepharose is highly effective in removing spermine and spermidine (99.5 and 95% adsorbed, respectively) from fetal calf serum. Under the same experimental conditions only 50% of putrescine is adsorbed. A higher amount of resin corresponding to an increased capacity for putrescine must be used to achieve a satisfactory removal of putrescine.     

Indirect photometric detection of polyamines in biological samples separated by high-performance capillary electrophoresis.

A rapid separation of polyamines and some related amino acids in cultured tumor cells by high-performance capillary zone electrophoresis with indirect photometric detection is demonstrated. 60 cm x 75 microns I.D. fused-silica capillary was used for the separation and quinine sulfate was used as a background electrolyte (BGE). Several polyamines (putrescine, spermidine and spermine), amino acids (lysine, arginine, histidine) and simple cations (K+, Na+) were easily separated in less than 10 min. Using the indirect photometric detection method, femtomole amounts of polyamines extracted from the tumor cells were detected from nanoliter injection volumes, and the signal response was linear over two orders of magnitude.