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.     

A degradation study of cefepime hydrochloride in solutions under various stress conditions by TLC–densitometry

A rapid, accurate and sensitive thin‐layer chromatography (TLC) method with densitometric detection has been developed and validated for the determination of cefepime in pharmaceuticals. Chromatographic separation was achieved on a silica gel TLC F₂₅₄ plates with a mobile phase consisting of ethanol–2‐propanol–glacial acetic acid 99.5%–water (4:4:1:3, v/v). Densitometric detection was carried out at wavelength of 266 nm in reflectance/absorbance mode. The validation of the method was found to be satisfactory with high accuracy (from 99.24 to 101.37%) and precision (RSD from 0.06 to 0.36%). Additionally, the stability of cefepime in solution was investigated, including the effect of pH, temperature and incubation time. Favorable retention parameters (R_f, R_s, α) were obtained under the developed conditions, which guaranteed good separation of the studied components. The degradation process of cefepime hydrochloride was described by kinetic and thermodynamic parameters (k, t_0.1, t_0.5 and E_a). Moreover, the chemical properties of degradation products were characterized by the R_f values, absorption spectra, HPLC‐MS/MS and TLC‐densitometry analysis. As the method could effectively separate the active substance from its main degradation product (1‐methylpyrrolidine), it can be employed as a method to indicate the stability of this drug. Copyright © 2014 John Wiley & Sons, Ltd.     

Use of a database of plots of pesticide retention (R F) against mobile-phase composition.Part I. Correlation of pesticide retention data in normal- and reversed-phase systems and their use to separate a mixture of ten pesticides by 2D TLC

Summary Ten pesticides have been completely separated by two-dimensional (2D) development on TLC plates coated with coupled layers of octadecyl silica (reversed-phase, RP) and plain silica (normal-phase, NP). The binary mobile phases, aqueous-organic for RP chromatography and nonaqueous for NP chromatography, were chosen from plots ofR _F against mobile-phase composition and graphicalR _F(RP)-R _F(NP) correlations. The different selectivity of the RP and NP systems enabled dispersion of spots over the plate area and good separation.     

The use of selected ion flow tube mass spectrometry to detect and quantify polyamines in headspace gas and oral air

Polyamines are a class of aliphatic compounds which include putrescine, cadaverine, spermine and spermidine. They are involved in a variety of cellular processes and have been implicated in a number of different pathophysiological mechanisms. Polyamines are volatile compounds having a distinctive odour normally perceived as being unpleasant. The measurement of their abundance has, however, been restricted to compounds present in the aqueous phase. Using selected ion flow tube mass spectrometry (SIFT‐MS) we have shown that the polyamines react with the ions H₃O⁺, NO⁺ and O to form distinctive product ions allowing their levels to be quantified in the vapour phase. The low volatility of spermine did not allow extensive analysis of this compound by SIFT‐MS while the adherent properties of cadaverine and putrescine required the use of PTFE transfer lines and couplers. Our data suggested the presence of cadaverine and putrescine in both oral air and the headspace of putrefying bovine muscle, while product ions corresponding to putrescine and spermidine were found in the headspace of human semen. SIFT‐MS therefore appears to be a practical means of measuring vapour‐phase polyamine levels, having applications in biology, medicine and dentistry, and food science. Copyright © 2009 John Wiley & Sons, Ltd.     

Heparin-sepharose as a tool in the subcellular fractionation of a polyamine-rich organ (rat ventral prostate)

Heparin-sepharose forms complexes with putrescine, spermidine, and spermine and indirect measurements of its affinity for polyamines gives values similar to those obtained with free heparin. A direct measurement of the binding of heparin-sepharose to spermine gives an apparent dissociation constant (K _d) of 1.5×10⁻⁶ M spermine. Unlike free heparin, heparin-sepharose does not cause either disruption of the nuclei or more sutble modifications able to modify their sedimentation behavior. The heparin-sepharose polyamine complex formed by the addition of heparin-sepharose to the homogenate can easily be removed and the homogenate can be processed according to normal schedules. Heparin-sepharose is able to sequester 85% of the exchangeable spermine present in the homogenate of rat ventral prostate. The distribution of the marker enzyme galactosyltransferase (Golgi apparatus) on a sucrose density gradient was followed to assess the usefulness of heparin-sepharose in minimizing the aggregation of cellular organelles brought about by polyamines.     

Quantitative TLC Analysis of Sterol (24β-Ethylcholesta-5,22E,25-triene-3β-ol) in Agnimantha (Clerodendrum phlomidis Linn)

A quantitative method using silica gel 60F₂₅₄ high performance thin layer chromatography plates, automated bandwise sample application, and automated visible mode densitometric method has been developed for the determination of 24β-ethylcholesta-5,22E,25-triene-3β-ol (ECTO) in the aerial part of Clerodendrum phlomidis. ECTO was used as a chemical marker for the standardization of C. phlomidis plant extracts. The separation was performed on silica gel 60F₂₅₄ TLC plates using chloroform-methanol (98.5: 1.5, v/v) as mobile phase. The quantitation of ECTO was carried out using the densitometric reflection/absorption mode at 650 nm after post chromatographic derivatization with anisaldehyde reagent. A precise and accurate quantification can be performed in the linear working concentration range of 150–400 ng band⁻¹ with good correlation (r ² = 0.996). The method was validated for peak purities, precision, robustness, limit of detection (LOD) and quantitation (LOQ), etc. as per ICH guidelines.     

Determination of polyamines in urine via electrospun nanofibers–based solid-phase extraction coupled with GC-MS and application to gastric cancer patients

The simultaneous determination of polyamines and their metabolites in urine samples was achieved by gas chromatography–mass spectrometry in the selected ion monitoring mode. After conjugating with the ion-pair reagent bis-2-ethylhexylphosphate in the aqueous phase, the polyamines in the samples were extracted with polystyrene nanofiber-based packed-fiber solid-phase extraction followed by a derivatization step using pentafluoropropionyl anhydride. With optimal conditions, all analytes were separated well. For analytes of putrescine, cadaverine, N-acetylputrescine, and N-acetylcadaverine, the linearity was good in the range of 0.05–500 μmol/L (R² ≥ 0.993). While for spermidine, spermine, acetylspermidine, N⁸-acetylspermidine, and N-acetylspermine, the linearity was good in the range of 0.5–500 μmol/L (R² ≥ 0.990). The recoveries of three spiked concentrations (0.5, 5, 300 μmol/L) were 85.6%–108.4%, and relative standard deviations for intra- and interday were in the range of 2.9%–13.4% and 4.5%–15.1%, respectively. The method was successfully applied to the analysis of urine samples of gastric cancer patients. The results showed that the levels of most polyamines and N-acetylated polyamines from the patient group were significantly higher than those from the control group. The altered concentrations of the above-mentioned metabolites suggest their role in the pathogenesis of gastric cancer, and they should be further evaluated as potential markers of gastric cancer.     

Biphenylene units possessing flammable and nonflammable characteristics in fluoroalkyl end-capped vinyltrimethoxysilane oligomeric silica gel matrices after calcination at 800 °C

Two kinds of fluoroalkyl end-capped vinyltrimethoxysilane oligomer [R_F-(VM) _n -R_F] silica nanocomposites containing biphenylene units were prepared by the sol-gel reactions of the corresponding oligomer with biphenylene-bridged ethoxysilanes or 4,4′-biphenol under alkaline conditions, respectively. One is the fluorinated oligomer/silica nanocomposites containing biphenylene units [R_F-(VM-SiO₂) _n –R_F/Ar-SiO ₂ ], of whose biphenylene units were incorporated into nanocomposite cores through the siloxane bondings, and the other is the fluorinated oligomer/silica nanocomposites containing biphenylene units [R_F-(VM-SiO₂) _n –R_F/Biphenol], of whose biphenylene units were directly encapsulated into nanocomposite cores through the sol–gel process. Interestingly, the shape of R_F-(VM-SiO₂) _n –R_F/Ar-SiO ₂ nanocomposites is morphologically controlled cubic particles; although the shape of R_F-(VM-SiO₂) _n –R_F/Biphenol nanocomposites is spherically fine particles. Thermogravimetric analyses ²H magic-angle spinning nuclear magnetic resonance, Ultraviolet visible, and fluorescent spectra showed that biphenylene units in R_F-(VM-SiO₂) _n –R_F/Ar-SiO ₂ nanocomposites have a flammable characteristic after calcinations at 800 °C; in contrast, biphenylene units in R_F-(VM-SiO₂) _n –R_F/Biphenol nanocomposites have a nonflammable characteristic even after calcination at 800 °C. X-ray photoelectron spectroscopy analyses of these two kinds of fluorinated nanocomposites showed that nonflammable characteristic toward biphenylene units in the silica gel matrices is due to the formation of ammonium hexafluorosilicate during the sol–gel process.     

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.     

Critical evaluation of experimental conditions influencing the surface-enhanced Raman spectroscopic (SERS) detection of substances separated by layer liquid chromatographic techniques

Summary Surface-enhanced Raman spectra (SERS) ofp-dimethylaminobenzylidenerhodanine have been recorded on silica gel 60 F₂₅₄ and Si60 F₂₅₄ Raman TLC plates. Spectra were enhanced by use of a silver sol prepared according to the modified Lee-Meisel procedure. The standard deviations of the intensities and the band ratios for the seven most intense peaks were calculated for 30 parallel measurements. Although the Raman plate gives more reproducible results, several experimental difficulties are encountered in the development of chromatograms. SERS detection of ascorbigen and 1′-methylascorbigen was performed after chromatography on silica gel 60 F₂₅₄ TLC and HPTLC plates and on Si60 F₂₅₄ Raman TLC plates. Traditional development was used for the silica gel 60 F₂₅₄ TLC plates and Si60 F₂₅₄ Raman plates, and the personal OPLC technique for the silica gel 60 F₂₅₄ HPTLC plates. It was found that the SERS spectrum gave information about the indole ring only. Because bonding of the analyte to the stationary phase results in a change in molecular conformation-in contrast with the behaviour of rhodanine-the type of the plateused and the development procedure employed can significantly influence the quality of the SERS spectrum. Presented at Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary September 1–3, 1999     

Determination of polyamines in human plasma by high‐performance liquid chromatography coupled with Q‐TOF mass spectrometry

A high‐performance liquid chromatography coupled with Q‐time of flight mass spectrometry (HPLC/Q‐TOF MS) method was developed and validated for the determination of 1, 3‐diaminopropane, putrescine, cadaverine, spermidine and spermine in human plasma. The plasma samples were first pretreated by 10% HClO₄ and then derived by benzoyl chloride with 1, 6‐diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column using a gradient program. The detection was performed on a Q‐TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentration range of 0.4 ~ 200.0 ng ? ml^?1, with limit of detection of 0.02 ~ 0.1 ng ? ml^?1. The intra‐ and inter‐day RSD for all polyamines were 2.5–14.0% and 2.9 ~ 13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancer patients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higher than that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicators in clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.     

Monitoring of biologically active amines in cereals and cereal based food products by HPLC

Summary Biologically active amines (putreanine sulphate, N-acetyl putrescine, putrescine, cadaverine, histamine, agmatine, N-acetyl spermidine, spermidine, spermine) were separated and quantified in cereal flour and cereal products by a liquid chromatographic method. The method consists of the separation of ion pairs formed between biologically active amines and octanesulphonic acid on a reversed-phase column, postcolumn derivatization with o-phtalaldehyde-2-mercapthoethanol and spectrofluorometric detection. Results of the reliability study were satisfactory. The method was linear for each amine at 1–10 mg L⁻¹. Putrescine and spermidine were the only amines always detected in cereal flour and cereal products, ranging from 2.45 to 47.83 mg kg⁻¹ for putrescine and 3.27 to 37.14 mg kg⁻¹ for spermidine. The most important differences among types of samples were found in polyamine derivatives. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Automated Determination of Urinary Polyamines

Abstract

A system for the determination of urinary polyamines by ion exchange chromatography with fluorescence detection is presented. It provides a sensitive and specific assay of putrescine, cadaverine, spermidine and spermine in urine hydrolyzates. An internal standard is used, which makes the automated determination of series of samples easy.     

Optimization of chromatographic systems for the high-performance thin-layer chromatography of flavonoids

Summary To characterize the retention and selectivity of separations of 23 flavonoids (aglycones and glycosides) relationships betweenR _F and modifier concentration were determined for silica and diol adsorbents (with mixtures of ethyl acetate and methanol as mobile phases), for cyanopropyl silica (with mixtures of ethyl acetate and dichloromethane as mobile phases), for aminopropyl silica (with mixtures of ethyl acetate, methanol and water as mobile phases) and for octadecyl silica (with mixtures of methanol and water as mobile phases). Owing to large polarity differences between aglycones and glycosides, these groups of compounds cannot be separated other than by use of reversed-phase systems, for which the selectivity is lower. It follows from correlation plots ofR _F1 againstR _F2 that for some pairs of adsorbents (e. g. silica and diol) selectivity differences are small; for others the points in the plot are widely dispersed, indicating selectivity differences. The chemometric database obtained can be used to choose optimum chromatographic systems for the separation of given sets of flavonoids and for planning gradient elution programs for separation of flavonoid aglycones and glycosides in a single TLC experiment.     

Determination of protonation constants of some fluorinated polyamines by means of <Superscript>13</Superscript>C NMR data processed by the new computer program HypNMR2000. Protonation sequence in polyamines

The pK_a values of 6-fluoro-4,8-diazadodecane-1,12-diamine (6-fluorospermine) (1), 6,6-difluoro-4,8-diazadodecane-1,12-diamine (6,6-difluorospermine) (2), 6-fluoro-4-azaoctane-1,8-diamine (6-fluorospermidine) (3) and 6,6-difluoro-4-azaoctane-1,8-diamine (6,6-difluorospermidine) (4) in D₂O solution have been determined at 40 °C from ¹³C NMR chemical shifts data using the new computer program HypNMR2000. The enthalpies of protonation of compounds 1–4 and the parent amines spermine (5) and spermidine (6) have been determined from microcalorimetric titration data. The values of H° were used to derive basicity constants relative to 25 °C. The NMR data have been analysed by two different methods to obtain information on the protonation sequence in the polyamines 1–5. The protonation sequence for spermine is related to its biological activity.Abbreviations PKC Protein kinase C - PS Phosphatidylserine - VB Microsoft Visual BasicPresented at the Spanish-Italian Congress on the Thermodynamics of Metal Complexes, Santiago de Compostela, Spain, June 2–6, 2002     

Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry

A simple and sensitive HPLC/Q-TOF MS method for simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine and acetyl-spermine in human urine was developed in electrospray-ionization source by positive ion mode. The samples were firstly pretreated by 10% HClO₄ and then derivatized by benzoyl chloride with 1,6-diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column by a gradient elution with methanol-water, and then sensitively detected with Q-TOF MS. The limits of detection for polyamines ranged from 0.02 to 1.0 ng ml⁻¹ with excellent linearity within the range from 1 to 1000 ng ml⁻¹ except acetyl-spermine from 5 to 1000 ng ml⁻¹. The intra- and inter-day R.S.D. for all polyamines were 2.0-14.7% and 3.9-12.9%, respectively. The method was applied to determine the polyamines in human urine from 10 cancer patients and 15 healthy volunteers. Results showed that the mean levels of polyamines in urine of patients were all higher than those in healthy volunteers. The cluster analysis was used to establish the distinction mode between cancer sufferers and healthy individuals.     

Optimization of experimental variables in the dabsyl chloride derivatization of biogenic amines for their determination by RP-HPLC

Summary In the last few years special attention has been paid to the pre-column derivatization of biogenic amines with dabsyl chloride because proper experimental conditions for this reaction are very important. In this study, an experimental design (Doehlert design) was used to optimize the variables involved in the dabsylation of the following amines: histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermidine, and spermine. The optimum experimental conditions for forming the dabsyl derivatives are: reagent concentration, 1.75.10⁻³ M; pH, 8.2; temperature, 70°C; heating time (t _h ), 21 min. Under these conditions good chromatographic repeatability is obtained.     

Sensitive Laser induced fluorescence detection of Polyamine-Fluoresceinisothiocyanate-derivatives after capillary zone electrophoretic separation

Capillary zone electrophoresis (CZE) has been applied for the separation and quantification of the polyamines putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), after their derivatization with the fluoresceinisothiocyanate isomer I (FITC).The derivatization conditions (character of the derivatization medium and concentration, organic modifier, pH and temperature) and CZE operation conditions have been optimized with respect to a sensitive Laser induced fluorescence detection (_excitation=488 nm/_detection=520 nm). A complete separation of CAD from PUT was impossible under the conditions chosen. The detection limits and relative standard deviations for SPM, SPD, CAD, PUT were determined to be 2.9 (12%), 3.2 (7.6%), 0.7 (4.6%), 1.2 (7.6%) nmol/L, respectively. The method has been applied to a pine needle extract.List of abbreviations a intercept - b slope - b_norm normalized slope of the calibration plot - CAD cadaverine - CZE capillary zone electrophoresis - DL detection limit - EOF electroosmotic flow - FITC fluoresceinisothiocyanate - FMOC 9-fluorenylmethyl chloroformate - HEC hydroxyethyl-cellulose - LIF Laser induced fluorescence - MW molecular weight - OPA o-phthalaldehyde - P/ACE programmable/automatical capillary electrophoresis - PUT putrescine - q charge - RSD relative standard deviation - SDS sodium dodecylsulfate - SPD spermidine - SPM spermine - t_migr migration time - w basic peak width - _eff effective mobility     

Preparation of a variety of fluoroalkyl end‐capped N‐(1,1‐dimethyl‐3‐oxobutyl)acrylamide oligomer/silica nanocomposites possessing no weight loss characteristic at 800°C

A variety of fluoroalkyl end‐capped N‐(1,1‐dimethyl‐3‐oxobutyl)acrylamide oligomer [R_F‐(DOBAA)_n‐ R_F]/silica nanocomposites, in which the oligomer contents are 18–96%, were prepared by reactions of the corresponding fluorinated oligomer with tetraethoxysilane and silica nanoparticles under alkaline conditions. Each fluorinated oligomer/silica composite thus obtained is nanometer size‐controlled very fine particles (22–68 nm) possessing a good dispersibility and stability in a variety of solvents including water. Interestingly, the weight loss of R_F‐(DOBAA)_n‐R_F/silica nanocomposites, in which the oligomer contents are 18–72%, were not observed at all even at 800°C, as well as the original silica nanoparticles, although the corresponding sub‐micrometer size‐controlled R_F‐ (DOBAA)_n‐R_F/silica composites (particle size: 359 nm) decomposed completely at 800°C to afford the weight loss in proportion to the content of R_F‐(DOBAA)_n‐R_F oligomer in composites. On the other hand, a slight weight loss of R_F‐(DOBAA)_n‐R_F/silica nanocomposites, in which the oligomer contents are 75–94%, was observed at 800°C compared to that of the original silica nanoparticles. Copyright © 2008 John Wiley & Sons, Ltd.     

SPE–TLC Profiling of Impurities in 1-(3,4-Methylenedioxyphenyl)-2-nitropropene, an Intermediate in 3,4-Methylenedioxy- methamphetamine (MDMA) Synthesis

Impurity profiling of 1–(3,4-methylenedioxyphenyl)-2-nitropropene, an intermediate in the synthesis of 3,4-methylenedioxymethamphetamine (MDMA), has been studied by SPE combined with TLC. Extraction of impurities was performed on C₁₈ columns. TLC separation was performed on 0.2 mm silica gel 60 plates, with fluorescent indicator F₂₅₄, in a horizontal developing chamber. To improve the quality of the profile (increase the TLC sensitivity) SPE extracts were concentrated by evaporation in a stream of nitrogen. The usefulness of methanol, ethanol, ethyl acetate, chloroform, acetonitrile, and their mixtures as mobile phases was tested by use of Gibbss triangle. Spots of the separated impurities were observed under UV light (_exc=254 and 366 nm). The proposed characteristics of profile quality (optimization criteria) are based on matrix presentation of the TLC pattern. They take into account, simultaneously, the number of spots revealed, differences between R_F values, and the intensity of fluorescence.