High-performance liquid chromatographic separation of kyotorphin, a basic TyrArg dipetide, in rat brain tissue and quantification using fluorimetric detection

Two HPLC procedures based on sample derivatization at the N-terminal Tyr moiety with agents yielding fluorescent derivatives were applied to the selective and sensitive detection as well as quantification of the basic kyotorphin, TyrArg dipeptide, in rat brain tissue. The first one is a post-column fluorescence derivatization method, whereby the peptides extracted from the brain tissue are separated on an octadecylsilyl-silica gel column, followed by on-line fluorescence derivatization for detection. The other one is a pre-column derivatization method, where the extracted peptides are first reacted with fluorogenic agents at the N-terminal Tyr moiety to their corresponding fluorescent derivatives, subsequently separated on an octadecyl-poly(vinyl alcohol) copolymer gel column, and signal responses are measured fluorimetrically. Both methods permitted the quantification of the synthetic kyotorphin added to the rat brain tissues. The concentration range of kyotorphin-like biogenic peptide was 60–100 pmol/g in the cortex, striatum and hypothalamus tissues.     

High-performance liquid chromatographic determination of 1,2,3,4-tetrahydroisoquinoline in rat brain with fluorescence detection

A high-performance liquid chromatographic method for the fluorometric determination of 1,2,3,4-tetrahydroisoquinoline in rat brain is described. 1,2,3,4-Tetrahydroisoquinoline and 4-phenylpiperidine (internal standard) are isolated by liquid-liquid extraction, and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 60 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of 1,2,3,4-tetrahydroisoquinoline is 1.0 pmol/g in rat brain (S/N = 3).     

High-performance liquid chromatographic determination of beta-phenylethylamine in human plasma with fluorescence detection

A simple and highly sensitive method for the determination of beta-phenylethylamine in human plasma is investigated. The method employs high-performance liquid chromatography with fluorescence detection. beta-Phenylethylamine and p-methylbenzylamine (internal standard) in human plasma are isolated by cation-exchange chromatography on a Toyopak SP cartridge and then converted into the corresponding fluorescent derivatives with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbonyl chloride, a fluorescence derivatization reagent for amines. The derivatives are separated within 30 min on a reversed-phase column, TSK gel ODS-120T, with isocratic elution, and detected fluorometrically. The detection limit of beta-phenylethylamine is 0.3 pmol/ml in plasma (S/N = 3).     

Combined use of micro-preparative gel electrophoresis and reversed-phase high-performance liquid chromatography for purification of amyloid beta peptides deposited in brains of Alzheimer's disease patients

A new micro-technique is developed for purification of amyloid beta peptides (A beta) extracted from brain tissues of patients with Alzheimer's disease (AD). It includes SDS-polyacrylamide gel electrophoresis of the extracted brain tissue material, electroblotting onto supporting membranes, and reversed-phase HPLC of the proteins eluted from membranes. By this technique, the extracted A beta are first separated electrophoretically from the higher and lower molecular mass tissue components, and then purified by reversed-phase HPLC from the contaminants having similar molecular masses, but different retention times on the column. In contrast to the common large-scale isolation procedures employing density gradient centrifugation, enzymatic digestions and size-exclusion chromatography, the developed micro-technique might be applied for biochemical analysis of A beta contained in small AD brain tissue specimens.     

A simple liquid chromatographic method based on intramolecular excimer-forming derivatization and fluorescence detection for the determination of tyrosine and tyramine in urine

A liquid chromatographic (LC) method for sensitive and selective fluorometric determination of p-hydroxyphenylethylamino group containing compounds is described. This method is based on an intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butanoyl chloride, followed by reversed-phase LC. The analytes, containing an amino moiety and a phenolic hydroxyl moiety in a molecule, were converted to the corresponding dipyrene-labeled derivatives by one-step derivatization. The dipyrene-labeled derivatives afforded intramolecular excimer fluorescence (440-540 nm), which can clearly be discriminated from the normal fluorescence (360-420 nm) emitted from reagent blanks. The derivatives of tyrosine and tyramine could be separated by reversed-phase LC on ODS column under conditions of isocratic elution. The detection limits (signal-to-noise ratio = 3) for tyrosine and tyramine were 4.5 and 2.6 fmol per 20 microL injection, which corresponded to analyte concentrations of 0.9 and 0.5 nM, respectively.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Determination of γ-aminobutyric acid by liquid chromatography with electrochemical detection

γ-Aminobutyric acid (GABA) has been determined in rat brain by derivatization with 2,4,6-trinitrobenzenesulfonic acid. The derivative and an internal standard, 2,4,6-trinitrophenyl-δ-aminovaleric acid, are extracted into toluene and separated by reversed-phase chromatography. Electrochemical reduction of these derivatives permits picomole measurements of GABA in microgram amounts of brain tissue.     

Determination of thiamin and its phosphate esters in cultured neurons and astrocytes using an ion-pair reversed-phase high-performance liquid chromatographic method.

A sensitive method, based on fluorescence detection, for the determination of thiamin derivatives after precolumn derivatization is described. The separation is achieved on a PRP-1 column using ion-pair reversed-phase HPLC. This method is especially well adapted to the detection of thiamin triphosphate in complex mixtures such as tissue extracts. The detection limit for TTP is 50 fmol. The contents of thiamin derivatives were determined in primary cultures of rat cerebellar granule neurons and cerebral astrocytes. The amount of TTP is about five times higher in neurons than in astrocytes. Thus in rat brain TTP seems to be essentially associated with neurons and the intracellular concentration is estimated to be about 0.2 microM. Our results suggest the existence, in nerve cells, of specific regulatory mechanisms not related to the blood-brain barrier and responsible for the maintenance of thiamin homeostasis in brain.     

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO₂)LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells. The quantitation of 3-NT content in samples was achieved through either fluorescence spectrometry or boronate affinity chromatography with detection by specific fluorescence (excitation and emission wavelengths of 360 and 510 nm, respectively); the respective limits of detection were 95 and 68 nM (19 and 13 pmol total amount) of 3-NT. Importantly, the derivatized peptides show a strong retention on a synthetic boronate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to separate the derivatized peptides from large amounts (milligrams) of nonderivatized peptides and to enrich them for fluorescent detection and mass spectrometry (MS) identification. Tandem MS analysis identified chemical structures of peptide 3-NT fluorescent derivatives and revealed that the fluorescent derivatives undergo efficient backbone fragmentations, permitting sequence-specific identification of protein nitration at low concentrations of 3-NT in complex protein mixtures.     

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching high-performance liquid chromatography with fluorescence detection

An HPLC method for the direct and simultaneous determination of estriol 3- and 16-glucuronides in pregnancy urine is described. The method is based on direct derivatization of the glucuronic acid moiety in estriol glucuronides in urine with 6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or urine sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide at 37°C. The resulting fluorescent derivatives were separated by column-switching chromatography using a first column (YMC-Pack C₄) for clean-up of the derivatives and a second column (YMC Pack Ph) for the complete separation of the derivatives. The derivatives were detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise ratio=3) for estriol 3- and 16-glucuronides were 150 and 180 fmol in a 5 μl of urine (14 and 17 ng ml⁻¹ urine), respectively. The present method is highly sensitive and simple without any clean-up such as conventional solid-phase extraction.     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.     

An improved method for the fluorometric determination of tissue catecholamines

Catecholamines extracted from tissues are readily measured by fluorescence following trihydroxyindole derivatization. A complicating factor however, has been the variably high background fluorescence levels obtained which adversely affect the precision of the assay. Employing EDTA is shown to reduce the high background fluorescence to a consistently low level, and to improve the accuracy of the trihydroxyindole method. It is suggested that high background fluorescence readings obtained by previous workers were due to metal ions extracted from tissue and carried over into the derivatization procedure.Measurement of tissue catecholamines by fluorescent methods is improved by the addition of EDTA which gives a consistently low background with a concommitant increase in accuracy.     

Fluorescence derivatizing procedure for 5-hydroxytryptamine and 5-hydroxyindoleacetic acid using 1,2-diphenylethylenediamine reagent and their sensitive liquid chromatographic determination

A pre-column derivatization method using a fluorogenic reagent, 1,2-diphenylethylenediamine (DPE) was studied for the sensitive HPLC determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), which are biosubstances used in the diagnosis of several diseases. For the quantitative determination, the biogenic indole compounds were converted to their corresponding fluorescent derivatives with DPE in the presence of potassium hexacyanoferrate (III) at room temperature, and then the derivatives were separated by reversed-phase liquid chromatography with fluorescence detection. The chromatographic detection limits of the fluorescent peaks at a signal-to-noise ratio of 3 were 0.3 fmol for 5-HT and 0.2 fmol for 5-HIAA. The proposed method permits the simultaneous quantification of 5-HT and 5-HIAA at concentrations higher than 2.4 nM in human urine without a clean-up procedure.     

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.     

Trace analysis of D-tyrosine in biological samples by microchip electrophoresis with laser induced fluorescence detection

A rapid and sensitive microchip electrophoresis (MCE) method with laser induced fluorescence (LIF) detection has been developed for the quantification of D-tyrosine (Tyr) in biological samples. The assay was performed using a MCE-LIF system with glass/poly(dimethylsiloxane) (PDMS) hybrid microchip after pre-column derivatization of amino acids with fluorescein isothiocyanate (FITC). Chiral separation of the derivatives was achieved by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using γ-CD as chiral selector in the running buffer. D/L-Tyr enantiomer was well separated in less than 140s. The limit of detection (S/N=3) was 3.3 × 10(-8) M. Using the present method, D-Tyr level in human plasma was found to vary significantly from normal humans to patients suffering from renal failure.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Quantification of hydrolyzed peptides and proteins by amino acid fluorescence

Reliable quantification of peptides and proteins is essential for drug discovery. We report the successful development and validation of an accurate and broadly applicable high performance liquid chromatography hyphenated to fluorescence detector procedure for the quantitative determination of the aromatic amino acids tyrosine, phenylalanine, and tryptophan, without relying on derivatization chemistry. Using ion‐pair chromatography, fluorescent amino acids were clearly separated within 10 minutes. The hydrolysis of peptides was performed under acidic and heated conditions to yield the monomeric building blocks. Various protecting agents were tested to ensure tryptophan stability. The presented analytical method accurately (>95%) quantifies all fluorescent residues. The power of the method was confirmed by correct quantification of protein reference standard to 98.6% over all fluorescence traces. The method allowed us to identify pre‐analytical differences between the nominal and actual concentrations of 12 peptide solutions. Salt formation, weighing errors, and other pre‐analytical pitfalls resulted in noteworthy differences of up to 85% between the indicated and actual concentration of peptide solutions, subsequently leading to false positive or negative interpretation of activity data. Finally, only one solution is needed to perform quantification as well as UV‐purity tests and can further be used as stock solution for activity testing.     

Determination of N-acetyl-L-glutamate using high-performance liquid chromatography

Acetylglutamate in HClO4 tissue extracts is first separated from glutamate by ion exchange. It is then deacylated with aminoacylase, and the resulting glutamate, after adsorption to and elution from an AG 50 column, is quantitated by a fast-HPLC method using o-phthaldialdehyde precolumn derivatization, separation in a C18 reverse-phase column, and fluorescence detection. A linear response is obtained up to 2 nmol, the detection limit is 5 pmol, and the method is suitable for assay in 1 mg liver tissue and thus for needle biopsies. When samples were analyzed by this procedure and by earlier procedures based upon detection of glutamate with glutamate dehydrogenase or upon activation of carbamoyl phosphate synthetase the results were similar. The method, which is highly specific, compares favorably in sensitivity, precision, and accuracy with all other published procedures. Using this assay, no acetylglutamate has been found in chicken liver and rat kidney.     

High performance liquid chromatographic determination of topiramate in human serum using UV detection

Topiramate has no ultraviolet, visible or fluorescence absorption. Analysis of the drug in human serum has been reported by high performance liquid chromatography (HPLC) with either mass detector or fluorescence detection after precolumn derivatization using 9-fluorenylmethyl chloroformate as fluorescent labeling agent. This study was aimed to validate derivatization and analysis of topiramate in human serum with HPLC using UV detection. The drug was extracted from human serum by liquid-liquid extraction and subjected to derivatization with 9-fluorenylmethyl chloroformate. Analysis was performed on a phenyl column using of spectrophotometer detection operated at wavelength of 264 nm. A mixture of phosphate buffer (0.05M) containing triethylamine (1 ml/l, v/v; pH 2.3) and methanol (28:72, v/v) at a flow rate of 2.5 ml/min was used as mobile phase. No interference was found with endogenous substances. Validity of the method was studied and the method was precise and accurate with a linearity range from 40 ng/ml to 40 microg/ml. The limit of quantification was 40 ng/ml of serum. The correlation coefficient between HPLC methods using fluorescence and UV detections was studied and found to be 0.992.     

Determination of bioactive eicosanoids in brain tissue by a sensitive reversed-phase liquid chromatographic method with fluorescence detection

Arachidonic acid (AA) is metabolized to prostaglandins (PGs) via cyclooxygenases (COX) catalysis, and to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DiHETrEs), and hydroxyeicosatetraenoic acids (HETEs) via cytochrome P450 (CYP450) enzymes. A reliable and robust fluorescence based HPLC method for these eicosanoids was developed. A new selective reverse-phase solid phase extraction (SPE) procedure was developed for PG, DiHETrEs, HETE, and EETs of interest from rat cortical brain tissue. The eicosanoids were derivatized with 2-(2,3-naphthalimino)ethyl-trifluoromethanesulphonate (NE-OTf), followed by separation and quantification at high sensitivity using reverse-phase HPLC with fluorescent detection, and further identified via LC/MS. The derivatization was studied and optimized to obtain reproducible reactions. Various PGs, DiHETrEs, HETEs, EETs, and AA were sensitively detected and baseline resolved simultaneously. LC/MS under positive electrospray ionization selected ion monitoring (SIM) mode was developed to further identify the peaks of these eicosanoids in cortical brain tissue. The method was applied in the traumatic brain injured rat brain.