Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described. Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde. The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media. The limit of detection is 20-30 pmol, and the response of the fluorescence detection is linear up to 4 nmol. The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation. The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine [N-(2-aminooxyethyl)-1,4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane] in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.     

Reversed-phase liquid chromatographic separation and simultaneous fluorimetric detection of polyamines and their monoacetyl derivatives in human and animal urine, serum and tissue samples: an improved, rapid and sensitive method for routine application

A highly sensitive and precise method for the determination of the polyamines putrescine, cadaverine, spermidine and spermine and all their monoacetyl derivatives in a single analysis in human and animal urine, serum and tissue samples is described. For polyamine separation, an ion-pairing reversed-phase high-performance liquid chromatographic (HPLC) method is used, followed by post-column derivatization with o-phthalaldehyde and consecutive fluorescence detection. Urine and serum samples are purified with a Bond Elut silica cartridge. The detection limit for polyamines is 0.5-1.0 pmol and excellent linearity is achieved in the range from 3 pmol up to more than 10 nmol. The influence of some modifications of different analytical steps such as the temperature of the HPLC column and the derivatization reaction coil and the o-phthalaldehyde flow-rate is described. Quality control data and measurements of the reproducibility of the method are presented. In order to establish a rapid analytical method for easy routine use, all steps for preparation and quantitative analysis are minimized. This method was applied to the determination of total polyamines in human urine and serum hydrolysate and of free and acetylated polyamines in human urine and pancreatic tissue of the rat. Values for normal polyamine concentrations in the urine and serum of fifteen male and fifteen female healthy volunteers and in the pancreas of ten normal rats are presented.     

Simultaneous detection of S-adenosylmethionine and S-adenosylhomocysteine in mouse and rat tissues by capillary electrophoresis.

A capillary electrophoresis method for the determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in rat liver and kidney and mouse liver is described. The method can also be used to determine SAM in whole blood. The method provides rapid (approximately 16 min sample to sample) resolution of both compounds in perchloric extracts of tissues. Separation was performed by using an uncoated 50 microm ID capillary with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV and the separation running buffer was 200 mM glycine pH 1.8 (with HCl). The method compares favorably to HPLC methods (r (2) = 0.994 for SAM, r (2) = 0.998 for SAH) and has a mass detection limit of about 10 fmol for both SAM and SAH at a signal-to-noise ratio of 3. The method is linear over ranges of 1-100 microM SAM and 1-250 microM SAH. This method can be used to determine tissue concentrations of SAM and SAH, two metabolites that can provide insight into many biological processes.     

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.     

HPLC determination of polyamines in the femtomole range

A new method for the chromatographic determination of polyamines is presented. The procedure consists of the reaction of 9-fluorenylmethyl chloroformate (FMOC-Cl) with polyamines at 50 +/- 1 degrees C for 10 min, followed by stepwise elution chromatography. The reaction is simple and the derivatives are stable. All the polyamine-FMOC derivatives can be separated within 13 min. The linearity of this method is satisfactory in the range of 0.5-100 pmol. The detection limits for putrescine, cadaverine, spermidine and spermine are 83.3 fmol, 109 fmol, 25.5 fmol and 22.7 fmol respectively.     

A new technique for the determination of polyamines: Overpressured ion-exchange thin-layer chromatography

Summary An overpressured, thin-layer ion-exchange, chromatographic method was developed for the separation of basic amino acids and natural polyamines. This technique, combined with video-densitometric evaluation, appeared to be suitable for the quantitative determination of polyamines in tissue extracts prepared without any previous purification.     

Facile Chemoenzymatic Strategies for the Synthesis and Utilization of S‐Adenosyl‐L‐Methionine Analogues

A chemoenzymatic platform for the synthesis of S‐adenosyl‐L ‐methionine (SAM) analogues compatible with downstream SAM‐utilizing enzymes is reported. Forty‐four non‐native S/Se‐alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non‐native SAM analogues. As a proof of concept for the feasibility of natural product “alkylrandomization”, a small set of differentially‐alkylated indolocarbazole analogues was generated by using a coupled hMAT2–RebM system (RebM is the sugar C4′‐O‐methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.     

Spectrophotometric assay for total polyamines by immobilized amine oxidases

Immobilized bovine plasma and pig kidney amine oxidases coupled with the hydrogen peroxide-peroxidase system are proposed for the detection of di- and polyamines in biological samples. The measurements were carried out spectrophotometrically with a flow reactor filled with the enzymes immobilized on Sepharose. The simplicity of the procedure and the short analysis time are the main advantages. The kinetic characteristics of the immobilized system, amine oxidases and peroxidase, are also reported. This method, which appears useful for routine analyses, permits the determination of polyamines in tissue homogenates and isolated cells.     

S-Adenosyl methionine/S-adenosyl-L-homocysteine ratio determination by capillary electrophoresis employed as a monitoring tool for the antiviral effectiveness of adenosine analogs

S-Adenosyl-L-homocysteine hydrolase (SAHh) inhibitors have long been used as broad-range antivirals and have been recently evaluated as an experimental therapy of filovirus infections. In response to the need for a rapid laboratory testing method that could assess antiviral potency in vivo, our group developed a capillary electrophoresis (CE) method for the determination of the S-adenosyl-L-homocysteine (SAH) to S-adenosyl-L-methionine (SAM) ratio. After chloroacetaldehyde derivatization, SAH and SAM were detected using laser-induced fluorescence detection with a HeCd laser. Separation and quantitation of both SAH and SAM in human plasma were achieved in less than 1 min. The proposed method is rapid and reliable, and could be easily applied to routine monitoring of clinical and preclinical trials subjects.     

Molecularly imprinted polymer prepared with bonded beta-cyclodextrin and acrylamide on functionalized silica gel for selective recognition of tryptophan in aqueous media

The rapid, sensitive and simultaneous determination of six polyamines, i.e., ornithine (ORN), 1,3-diaminopropane (DAP), putrescine (PUT), cadaverine (CAD), spermidine (SPD) and spermine (SPM), in human hairs was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS). The primary (-NH(2)) and secondary (-NH) amines in the polyamine structures were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30min in the mixture of 0.1M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting derivatives were perfectly separated using an ACQUITY UPLCtrade mark BEH C(18) column (1.7mum, 100mmx2.1mm i.d.) by a gradient elution with a mixture of water-acetonitrile containing 0.1% formic acid (HCOOH). The separated polyamine derivatives were sensitively detected with both FL and TOF-MS. The detection limits in FL and TOF-MS were 11-86 and 2-5fmol, respectively. However, the determination of several polyamines by FL detection was interfered with by endogenous substances in the hair. Therefore, the simultaneous determination in hair was carried out by the combination of UPLC separation and the ESI-TOF-MS detection. The structures of the polyamines were identified from the protonated-molecular ions [M+H](+) obtained from the TOF-MS measurement. A good linearity was achieved from the calibration curves, that was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 1,6-diaminohexane (DAH), against the injected amounts of each polyamine (0.05-50pmol, r(2)>0.999). The proposed method was applied to the determination in the hairs of healthy volunteers. The mean concentrations of ORN, DAP, PUT, CAD, SPD and SPM in 1mg of human hairs (n=20) were 1.46, 0.18, 1.18, 0.11, 1.97 and 0.98pmol, respectively. Because the proposed method provides a good mass accuracy and the trace detection of the polyamines in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair.     

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.     

Fully automated standard addition method for the quantification of 29 polar pesticide metabolites in different water bodies using LC-MS/MS

A reliable quantification by LC-ESI-MS/MS as the most suitable analytical method for polar substances in the aquatic environment is usually hampered by matrix effects from co-eluting compounds, which are unavoidably present in environmental samples. The standard addition method (SAM) is the most appropriate method to compensate matrix effects. However, when performed manually, this method is too labour- and time-intensive for routine analysis. In the present work, a fully automated SAM using a multi-purpose sample manager “Open Architecture UPLC®-MS/MS” (ultra-performance liquid chromatography tandem mass spectrometry) was developed for the sensitive and reliable determination of 29 polar pesticide metabolites in environmental samples. A four-point SAM was conducted parallel to direct-injection UPLC-ESI-MS/MS determination that was followed by a work flow to calculate the analyte concentrations including monitoring of required quality criteria. Several parameters regarding the SAM, chromatography and mass spectrometry conditions were optimised in order to obtain a fast as well as reliable analytical method. The matrix effects were examined by comparison of the SAM with an external calibration method. The accuracy of the SAM was investigated by recovery tests in samples of different catchment areas. The method detection limit was estimated to be between 1 and 10 ng/L for all metabolites by direct injection of a 10-μL sample. The relative standard deviation values were between 2 and 10 % at the end of calibration range (30 ng/L). About 200 samples from different water bodies were examined with this method in the Rhine and Ruhr region of North Rhine-Westphalia (Germany). Approximately 94 % of the analysed samples contained measurable amounts of metabolites. For most metabolites, low concentrations ≤0.10 μg/L were determined. Only for three metabolites were the concentrations in ground water significantly higher (up to 20 μg/L). In none of the examined drinking water samples were the health-related indication values (between 1 and 3 μg/L) for non-relevant metabolites exceeded.     

Liquid chromatographic determination of S-adenosyl-L-methionine in dietary supplement tablets

A simple and reliable liquid chromatographic method was developed for the determination of S-adenosyl-L-methionine (SAM) in dietary supplement tablets. SAM in products was extracted with a phosphate buffer and separated from the mixture on a reversed-phase C8 column by ion-pair chromatography. A gradient mobile phase containing phosphate buffer, sodium octanesulfonate as the ion-pair reagent, and acetonitrile at a flow rate of 1.2 mL/min was used in the analysis. The UV detection wavelength was set at 257 nm. The calibration curve was linear over a range of 75-375 microg/mL for the SAM active ion with R2 = 0.9999. Replicate tests indicated good reproducibility of the method with a relative standard deviation of 0.9% (n = 8). The multiple extractions and recoveries from fortified products showed the high accuracy of the analysis. The use of the acidic buffer for SAM extraction and elution and the use of a fresh standard for each calibration to counteract the instability of the SAM compound significantly improved the accuracy of the method.     

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.     

HPLC separation of tetracycline analogues: comparison study of laser-based polarimetric detection with UV detection

A sensitive and specific high-performance liquid chromatography (HPLC) method based upon laser-based polarimetric detection is developed for the determination of six tetracycline analogues. By interfacing the laser-based polarimeter online with an HPLC system, the specific rotation of each analogue is obtained as compounds elute from the separation system. The six structurally similar tetracycline analogues exhibit significant differences in specific rotations. The experiments suggest that specific rotation can be useful in identifying closely related tetracycline analogues. Linear relationships are found to be in the range of 0.342-0.0043 mg for the tetracycline analogues. Five of the six analogues exhibit excellent linearity (R(2) value >/= 0.99). The polarimetric results are compared with UV detection. The HPLC-laser-based polarimetric detection instrument is able to quantitate the studied tetracycline analogues with high precision, accuracy, and sensitivity, which make it useful for the development of a standard method for the determination of tetracyclines in biological specimens. The performance of the HPLC-polarimetric system for the analysis of tetracyclines in a biological matrix is evaluated. The selectivity of polarimetric detection provides a distinct advantage in the analysis of tetracycline analogues in milk. The HPLC-polarimetric system provides a rapid and sensitive technique that involves minimal sample cleanup and pretreatment for the analysis of tetracyclines in milk.     

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.     

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.     

Validation of an ultra high pressure liquid chromatographic method for the determination of biologically active amines in food

Biologically active amines include the so called biogenic amines, such as histamine, tyramine and cadaverine, and polyamines such as spermidine and spermine. Ultra high pressure liquid chromatography (UHPLC) is a new generation of separation techniques that takes full advantage of chromatographic principles to increase speed flow which drastically reduce analysis time. The aim of the present work was to validate a rapid method of UHPLC to detect the presence of biogenic amines and polyamines in food. Different food matrixes (wine, fish, cheese, and dry fermented sausage) were used in order to test the versatility of the method. The UHPLC method described in this article has been demonstrated as a reliable procedure to determine 12 biogenic amines and polyamines in less than 7 min of chromatographic elution. The method provides a satisfactory linearity and chromatographic sensitivity with a detection limit lower than 0.2 mg/L and a determination limit falling below 0.3 mg/L for all amines. The precision, in terms of relative standard deviation, was lower than 5% and the accuracy, as mean recovery, was between 93% and 98%, depending on the food matrix.     

Improved method for the determination of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine in tissue using high-performance liquid chromatography with electrochemical detection.

A liquid chromatographic method with electrochemical detection is described for the determination of the 5-hydroxytryptamine (5-HT) neurotoxins 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT) in rat brain tissue. This method has also been used for the determination of 5-hydroxyindoleacetic acid, homovanillic acid and 5-HT in other tissue samples. The method is based on extraction of the indoles from brain samples with perchloric acid followed by reversed-phase liquid chromatography with electrochemical detection. The detection limit is 1 ng per 100 mg of tissue. This paper describes a quick and reliable method of assaying the 5-HT neurotoxins 5,6-DHT and 5,7-DHT in brain tissue, which is improved compared to currently available assays.     

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.