Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy specimens by high-performance liquid chromatography on a standard reversed-phase column

The concentrations of putrescine, spermine and spermidine were measured in human serum, children's duodenal biopsy specimens and mouse brain homogenates by high-performance liquid chromatography. The chromatographic analysis was performed on dansyl derivatives of the polyamines using a reverse-phase system with an ion-pairing retention mechanism (heptane sulphonate). Capacity factors were determined at different concentrations of acetonitrile. Simple linear gradients were set up for fast (15 min) or routine (25 min) analysis. Three fluorescence detectors were compared for these determinations and their detection limits determined. The minimum detectable amount of polyamines was 25 fmol compared to 500 fmol with standard detectors. While samples prepared from tissues did not require a high sensitivity, a detector of better performance was needed to assay the polyamines in human serum.     

Fluorimetric high-performance liquid chromatography of prostaglandins and its application to their determination in human seminal fluid

A highly sensitive and simple high-performance liquid chromatographic method with fluorescence detection for the determination of prostaglandins is described. Prostaglandins are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 34 min (the total run time per injection, 56 min) on a reversed-phase column (YMC Pack C8) by a stepwise elution with mixtures of acetonitrile, methanol and water and detected fluorimetrically. The detection limits are 10-15 fmol at a signal-to-noise ratio of 5 in a 10-microliter injection volume. Prostaglandins E1, E2, F1 alpha and F2 alpha in human seminal fluids are measured by this method.     

Sensitive fluorometric detection of prostaglandins by high performance liquid chromatography after precolumn labelling with 4-(N,N-dimethylaminosulphonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiazole (DBD-PZ).

A highly sensitive and simple method for the determination of prostaglandins (PGs) by HPLC with fluorescence detection is described. PGs are converted to the corresponding fluorescence derivatives by the reaction with 4-(N,N-dimethylaminosulphonyl)-7-(1-piperazinyl)-2,1,3-benzoxadiaz ole (DBD-PZ) in the presence of 2,2'-dipyridyl disulphide and triphenyl phosphine in acetonitrile. The reaction is completed at room temperature after 30 min. The DBD derivatives of nine PGs are separated within a single 45 min chromatographic run on a reversed phase ODS column with a linear gradient elution using water and acetonitrile. The detection limits (signal-to-noise ratio of 3) calculated from the standard mixture of PGs (6-keto-F1 alpha, F1 alpha, F2 alpha, E1, E2, D2, limaprost, A1 and B1) are in the range 1.7-5.0 fmol. The applicability of the proposed procedure is evaluated to the detection of PGs added to rat plasma.     

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.     

4,5-Diaminophthalhy drazide as a highly sensitive chemiluminescence reagent for α-keto acids in liquid chromatography

4,5-Diaminophthalhydrazide dihydrochloride is studied as a highly sensitive and selective chemiluminescence derivatization reagent for α-keto acids in liquid chromatography (LC). The reagent reacts selectively with α-keto acids in dilute hydrochloric acid to give derivatives which produce chemiluminescence by reaction with hydrogen peroxide and potassium hexacyanoferrate(III). The derivatives in the reaction mixture of eight biologically important α-keto acids are separated within 50 min by reversed-phase LC with isocratic elution, followed by chemiluminescence detection. The detection limits for the acids are in the range 4–50 fmol for a 20-μl injection.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Development of hydrophilic fluorogenic derivatization reagents for thiols: 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

Sensitive, reactive, and hydrophilic fluorogenic reagents for thiols with the benzofurazan skeleton, 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (AcABD-F) and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) have been developed. These reagents reacted with thiols within 10 min at 60 degrees C. AcABD-F and TCAcABD-F themselves do not fluoresce but are strongly fluorescent after the reaction with thiol compounds. The generated derivatives were highly water-soluble, since they dissociated a proton and ionized in the neutral pH region. The derivatives with four biologically important thiol compounds were separated on a reversed-phase HPLC column and detected fluorometrically at 504 nm with excitation at 388 nm. The detection limit attained for homocysteine with AcABD-F was 25 fmol on column (11 nM) (signal-to-noise ratio = 3), and that for glutathione with TCAcABD-F was 45 fmol on column (20 nM).     

Simultaneous high-performance liquid chromatographic determination of catecholamine-related compounds by post-column derivatization involving coulometric oxidation followed by fluorescence reaction

A highly selective and sensitive high-performance liquid chromatographic method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) and related compounds (L-DOPA, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid, 3,4-dihydroxyphenylethylene glycol, 4-hydroxy-3-methoxyphenylethylene glycol and 4-hydroxy-3-methoxyphenylethanol) with a post-column technique involving coulometric oxidation followed by fluorescence derivatization is described. These compounds, 3,4-dihydroxybenzylamine and ferulic acid are separated within 35 min by ion-pair reversed-phase chromatography using acidic buffers (pH 3.1) with methanol-acetonitrile (3:2, v/v) gradient elution, and then oxidized by a commercial coulometric detector to the corresponding o-quinones, which are converted into fluorescent derivatives by reaction with 1,2-diphenylethylenediamine. The detection limits (signal-to-noise ratio = 3) on-column are 1.5-4 pmol for the two mandelic acids, 600 fmol for L-DOPA and 20-70 fmol for the others.     

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 analysis of serum short-chain fatty acids by direct derivatization

The application of direct derivatization in conjunction with high-performance liquid chromatography is described for the analysis of short-chain fatty acids in serum. The method is based on the reaction of these acids with acidic 2-nitrophenylhydrazine hydrochloride, without complicated isolation steps, which produces their non-volatile hydrazine derivatives. The hydrazides of fourteen saturated and unsaturated, straight and branched, short-chain fatty acids were separated from other acid hydrazides and interfering components by a simple solvent extraction, and were eluted isocratically on a reversed-phase C8 column within 24 min. UV detection demonstrated that the detection limits for the acids were 200-400 fmol per injection with linearity over the range from 400 fmol to 5 nmol per injection. Analytical recoveries ranged from 96.8% to 103.1% and coefficients of variation ranged from 0.9% to 3.8%. The present method is simple, accurate and adequate for the analysis of short-chain fatty acids in biological fluids and tissues of patients suffering from organic acidemias.     

Measurement of N-acetylneuraminic acid in human serum and urine by high performance liquid chromatography with chemiluminescence detection.

A highly sensitive method for the determination of N-acetylneuraminic acid in human serum and urine is investigated. This method employs high performance liquid chromatography with chemiluminescence detection. N-Acetylneuraminic acid, released by hydrochloric acid hydrolysis of serum and urine, and N-glycolylneuraminic acid (internal standard) are converted into chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for alpha-keto acids. The derivatives are separated within 35 min on a reversed phase column, TSKgel ODS-120T, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(III) in alkaline solution. The detection limit for N-acetylneuraminic acid is 9 fmol (signal-to-noise ratio = 3). This sensitivity permits precise determination of N-acetylneuraminic acid in 10 nL of serum or 50 nL of urine. The method is applied to the determination of the N-acetylneuraminic acid in human sera from normal subjects and cancer patients and in normal urine.     

High-performance liquid chromatographic assay of N(G)-monomethyl-L-arginine, N(G),N(G)-dimethyl-L-arginine, and N(G),N(G)'-dimethyl-L-arginine using 4-fluoro-7-nitro-2, 1,3-benzoxadiazole as a fluorescent reagent

N(G)-Monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (ADMA), and N(G),N(G)'-dimethyl-L-arginine (SDMA) are emerging cardiovascular risk factors. A high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of L-NMMA, ADMA and SDMA is described. The assay employed 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a fluorescent derivatization reagent. After solid phase extraction with cation-exchange column, the methylated arginines were converted to fluorescent derivatives with NBD-F, and the derivatives were separated within 32 min on a reversed-phase column. Nomega-Propyl-L-arginine was Used as an internal standard. Extrapolated detection limits were 12 nM (12 fmol per injection) for L-NMMA and 20 nM (20 fmol per injection) for ADMA and SDMA, respectively, with a signal-to-noise ratio of 3. The calibration curves for L-NMMA, ADMA and SDMA were linear within the range of 50-5000 fmol. The method was applied to the quantitative determination of L-NMMA, ADMA and SDMA in 200 microl of rat plasma. The concentrations of L-NMMA, ADMA and SDMA in rat plasma were 0.16 +/- 0.03, 0.80 +/- 0.25 and 0.40 +/- 0.21 microM, respectively (n = 5).     

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.     

Determination of anti-virus drug, ganciclovir, in human serum by HPLC with precolumn fluorescence derivatization using phenylglyoxal.

A selective and highly sensitive liquid chromatographic method for the determination of ganciclovir (anti-virus drug) in human serum was described. After ganciclovir and acyclovir (internal standard; IS) were extracted with solid-phase extraction cartridge from serum, they were converted into fluorescent derivatives by reaction with phenylglyoxal in a phosphate buffer (pH 5.8) at 20 degrees C for 30 min. The derivatives were separated by reversed-phase column with a mixture of acetonitrile-1 mM phosphate buffer (pH 6.2) (18:82, v/v), and were then detected spectrofluorometrically at 512 nm with excitation at 365 nm. Extraction recoveries were 87.0-91.6% for ganciclovir and 86.8-92.3% for IS. The detection limit for ganciclovir spiked to serum was 5 ng ml-1 serum (306 fmol on column) at a signal-to-noise ratio of three. The accuracy and precision of this method were 7.6% and 5.0% even at low concentration (20 ng ml-1). The within- and between-day variations are lower than 7.6% and 8.1%, respectively.     

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.     

Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68 degrees C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at lambda(em) = 460 nm with lambda(ex) = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10-450 and 35-1400 fmol, respectively.     

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.     

High-performance liquid chromatography of arachidonic acid metabolites and its application to the determination of leukotriene B4 in stimulated leukocytes

A highly sensitive high-performance liquid chromatography with fluorescence detection for the determination of arachidonic acid metabolites is described. The metabolites are converted into corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-methylenedioxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Inertsil ODS) with aqueous acetonitrile and detected fluorimetrically. The detection limits are 5-15 fmol at a signal-to-noise ratio of 3 in a 10-microliter injection volume. The method is applied to the determination of leukotriene B4 produced in stimulated leukocytes.     

First evidence for the use of polyamines as nucleophiles in a regioselective palladium-catalyzed allylic amination reaction

Palladium-catalyzed reaction of unsymmetrical allylic electrophiles with polyamines gives rise to regioisomeric allylic polyamines. An original catalytic procedure providing an efficient method for the regioselective synthesis of new classes of polyamino derivatives of biological interest is reported. Based on experimental considerations, a mechanistic rationale involving a thermodynamically controlled isomerization of the initially formed branched product is proposed to account for the total regioselectivity observed.     

Simultaneous analysis of plasma thiols by high-performance liquid chromatography with fluorescence detection using a new probe, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene

The design, synthesis and properties of a new derivatizing reagent, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene (TMPAB-I), for thiol groups are presented. Using the derivatization of TMPAB-I with thiols, a new high-performance liquid chromatographic method for measuring low-molecular-weight thiol-containing compounds, including coenzyme A (CoA), glutathione, N-acetylcysteine, cysteine, homocysteine (HCys) and 6-mercaptopurine has been developed. The reaction of TMPAB-I with thiols is specific, fast and stable for both TMPAB-I and the derivatives. A baseline separation of all the six derivatives is achieved by isocratic elution on reversed-phase column within 20 min with detection wavelengths of 500 and 510 nm for the excitation and emission, respectively, and the limits of detection (signal-to-noise ratio = 3) are from 1.8 fmol (CoA) to 14.0 fmol (HCys), respectively, per 20 μL injection. The utility of the proposed method has been validated by measuring thiol-containing compounds in human plasma samples from healthy persons and patients with hypertension, with recoveries of 94.2–106.8%.