High-performance liquid chromatographic determination of proteins by post-column fluorescence derivatization with thiamine reagent.

A post-column derivatization method for the high-performance liquid chromatography of peptides and proteins giving a fluorescence intensity proportional to the number of peptide bonds is described. Peptide bonds were chlorinated with hypochlorite and the N-chlorite formed was allowed to react with thiamine to give fluorescent thiochrome. This method was applied the determination of membrane-forming proteins of microorganisms.     

High-performance liquid chromatographic method for determining trichothecene mycotoxins by post-column fluorescence derivatization

The method described here is based on a separation of deoxynivalenol, nivalenol and fusarenon-X on a C18 column using aqueous acetonitrile, and successive post-column fluorescence derivatization involving an alkaline decomposition to form formaldehyde and modified Hantzsch reaction with methyl acetoacetate and ammonium acetate (lambda ex = 370 nm and lambda em = 460 nm). By this method, 5-10 ng of the standard trichothecenes could be determined. By employing a clean-up procedure with a florisil column and a Sep-Pak CN cartridge, 61.4-96.9% recoveries were obtained for deoxynivalenol and nivalenol added to corn, wheat and barley at concentration levels of 0.05-1 ppm.     

High-performance liquid chromatographic determination of clenbuterol and cimaterol using post-column derivatization.

A general high-performance liquid chromatographic method for the simultaneous and rapid determination of cimaterol and clenbuterol is described. Solid samples, such as animal tissues, faeces and feeding-stuffs, are extracted with dilute acid saturated with ethyl acetate. The resulting extracts or liquid samples, such as urine, plasma, blood and bile, are purified via Chem Elut columns. Separation is achieved by ion-pair chromatography on a Nova-Pak C18 column, and highly specific detection is obtained with an adapted version of the post-column derivatization described previously for the determination of clenbuterol in urine and animal tissues. Detection limits for liquids and solids are 0.1 ng/ml and 0.2 ng/g, respectively. The results are in complete agreement with analysis by high-performance thin-layer chromatography and gas chromatography-mass spectrometry, applied for confirmation after the same sample pretreatment. With this simple method, complete analysis of a liquid sample needs about 30 min and, even without an automatic sampler, 40 samples can be completely analysed in one day. This method has been used on a routine scale for nearly two years.     

High-performance liquid chromatographic determination of selected amino acids in rat brain by precolumn derivatization with phenylisothiocyanate

We describe here a simple, sensitive, selective and reproducible assay method for quantitative determination of aspartate, glutamate, serine, glutamine, glycine and gamma-aminobutyric acid in rat brain using reversed-phase high-performance liquid chromatography. The method is based upon formation of phenylthiocarbamyl derivatives of the amino acids. Good resolution of the six amino acids and the internal standard norvaline is achieved within 40 min. Other amino acids which have been reported to be present in rat brain do not interfere with the analysis. Standard curves for each of the amino acids exhibited good linearity (r greater than 0.9993) over the range 0.5-20 nmol. The coefficient of variation for the intra-day and inter-day determinations ranged from 0.4% at the highest to 11% at the lowest concentration limit. Storage of whole brains at -0 degrees C for up to 8 weeks did not affect mean concentrations of the six amino acids.     

High-performance liquid chromatographic method for monitoring leupeptin in mouse serum and muscle by pre-column fluorescence derivatization with benzoin

A high-performance liquid chromatographic method is described for the determination of leupeptin, a possible therapeutic drug for muscular dystrophy, in mouse serum and muscle. Leupeptin is reduced with sodium borohydride to leupeptinol, and then converted to a fluorescent derivative with benzoin. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) with isocratic elution and determined with fluorescence detection. The detection limits of leupeptin in serum and muscle are 250 pmol/ml (107 ng/ml) and 500 pmol/g (214 ng/g), respectively, corresponding to approximately 150 fmol each in a 100-microliters injection volume. This method is simple and sensitive enough to permit the quantification of leupeptin in biological samples from mice dosed with leupeptin.     

High-performance liquid chromatographic determination of taurine in biological fluids by post-column fluorescence reaction with thiamine.

A high-performance liquid chromatographic method is described for the selective determination of taurine in biological fluids by post-column fluorescence reaction. Taurine was separated on an adsorption-distribution type Shodex Ionpac KC-811 column. Then it was converted with hypochlorite into the corresponding N-chloramine, which was allowed to react with thiamine to give fluorescent thiochrome. As little as 6 ng per injection of taurine could be determined. The average recoveries of spiked taurine in serum and urine were 99.5 +/- 2.7 and 101.8 +/- 2.9%, respectively. The method could be applied to the assay of taurine in human serum and urine with simple pretreatment.     

Quantitative liquid chromatographic determination of cefatrizine in serum and urine by fluorescence detection after post-column derivatization

A fast, specific and sensitive high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. The drug is determined by the internal standard method, using cephradine as the internal standard. The separation is carried out on a reversed-phase column, filled with octadecylsilane chemically bonded microparticles. The eluent is a mixture of acetonitrile with 0.025 M sodium phosphate buffer (pH 7). Quantitation is effected by fluorescence detection of the fluorophores formed after post-column derivatization with fluorescamine in a packed-bed reactor. The chromatographic conditions and the conditions for the post-column derivatization are discussed. The method has been applied to serum and urine samples, which were analysed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by the determination of the cefatrizine content in spiked serum and urine samples.     

High-performance liquid chromatographic determination of peptides in biological fluids by automated pre-column fluorescence derivatization with fluorescamine

Peptides containing a free alpha- or epsilon-amino group react with fluorescamine under mild alkaline conditions to generate a highly fluorescent but unstable reaction product and, consequently, practical high-performance liquid chromatographic (HPLC) approaches to analysis have typically involved the use of postcolumn derivatization. An automated precolumn approach is reported in which peptides are reacted with fluorescamine just prior to HPLC analysis by a commercially available autoinjector with derivatization capabilities. The autoinjector added base and fluorescamine reagent solutions to a sample vial containing peptide analytes, and the derivatization reaction was allowed to proceed for 5 min at room temperature prior to injection into the HPLC system. The derivatized peptides were analyzed by reversed-phase HPLC with fluorescence detection (excitation at 390 nm; emission 470-nm cut-off filter) on an octylsilica column. Optimization of the precolumn reaction conditions and the use of narrower HPLC columns (2 mm I.D.) resulted in a typical on-column detection limit of 30-50 fmol of peptide, which was substantially lower than that in previously reported post-column methods. This approach was applied to the HPLC of several naturally occurring and synthetic peptides containing alpha- and epsilon-amino groups. In combination with solid-phase extraction, prior to automated precolumn fluorescence derivatization and chromatographic analysis, the methodology was used for the determination of a synthetic growth hormone-releasing peptide in plasma samples.     

High-performance liquid chromatographic determination of cyanide in human red blood cells by pre-column fluorescence derivatization.

A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.     

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.     

High-performance liquid chromatographic measurement of guanidino compounds of clinical importance in human urine and serum by pre-column fluorescence derivatization using benzoin

High-performance liquid chromatographic microanalyses for guanidino compounds in human physiological fluids have been accomplished by means of a pre-column fluorescence derivatization method using benzoin. The guanidino compounds in urine or deproteinized serum after ultrafiltration are converted to the fluorescent derivatives with benzoin in an alkaline medium, and the derivatives are separated simultaneously within 25 min on a reversed-phase column (mu Bondapak Phenyl) with a linear gradient elution of methanol in aqueous mobile phase (pH 8.5). The method permits the quantitative determination of guanidinosuccinic acid, methylguanidine, taurocyamine and guanidinobutyric acid at concentrations of as low as 8-78 pmol/ml in human urine and serum.     

Simultaneous determination of 2-deoxy-D-glucose and D-glucose in rat serum by high-performance liquid chromatography with post-column fluorescence derivatization

A simple high-performance liquid chromatographic method for the determination of 2-deoxy-D-glucose and D-glucose in rat serum is described; this method is based on a post-column fluorescence derivatization. The sugars are automatically converted into fluorescent derivatives by reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium after their separation on a strong anion exchanger column (TSK gel Sugar AXG). The detection limits (S/N = 3) for 2-deoxy-D-glucose and D-glucose in rat serum are 0.52 and 0.56 nmol/ml, respectively.     

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.     

Determination of alpha-tocopherolquinone in human serum samples by liquid chromatography with fluorescence detection and on-line post-column derivatization

A HPLC/fluorescence method with on-line post-column derivatization by a photoreactor was developed, where alpha-, beta + gamma- and delta-tocopherolquinone (TQ) are separated on a 250 mm x 4.6 mm RP-18 column. The LOD is about 250 pg for all TQs. In combination with a two-step sample preparation procedure, this method was successfully employed for measurement of alpha-TQ in human serum samples. Recovery for alpha-TQ from spiked serum was excellent (99 +/- 5%) and results of alpha-TQ determinations in 111 serum samples are reported. Additionally, possibilities for determination of other TQs in serum and alternative derivatization with a zinc reduction column are discussed.     

High-performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization

In this study, we investigated a simple, sensitive and reliable liquid chromatography‐fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl). For the first time, FMOC‐Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC‐Cl to form memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions, which can be very compatible for LC‐FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C₁₈ column (DIAMONSIL 150×4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025–5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra‐day and inter‐day assays were 4.46–12.19 and 5.23–11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.     

High-performance liquid chromatographic determination of peptides released by tryptic degradation from opioid peptide precursors in rat brain.

A high-performance liquid chromatographic method involving postcolumn fluorescence derivatization is described for the quantification of five fragment peptides (methionine-enkephalin-Thr-Ser-Glu-Lys, methionine-enkephalin-Lys, methionine-enkephalin-Arg, leucine-enkephalin-Lys and leucine-enkephalin-Arg) released by tryptic digestion from the opioid peptide precursors (proopiomeranocortin and proenkephalins A and B) in rat brain tissues. The tissue proteins containing the precursors are hydrolyzed with trypsin to the fragment peptides. The peptides are separated on an Asahipak ODP-50 column and on-line detected fluorometrically by using hydroxylamine, cobalt(II) and borate buffer reagents. The detection limits (S/N = 3) for the peptides are 0.7-2.8 pmol per 100 microliters injected. The distribution of the precursors in the brain tissues was also discussed on the basis of the determined values of the fragment peptides.     

Determination of alkylamines by high-performance liquid chromatography with post-column fluorescence derivatization

A styrene/divinylbenzene polymer column and an amino column are compared for the non-aqueous separation of primary, secondary and tertiary alkylamines. Post-column derivatization with o-phthalaldehyde/2-mercaptoethanol is selective for primary amines and derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is selective for secondary amines after on-line masking of primary amines. This procedure can tolerate 0.4 M butylamine. The limit of detection is 18.5 mM for dioctylamine (with NBD-Cl) and 0.18 mM for decylamine and tetraethylenepentamine (with o-phthalaldehyde/2-mercaptoethanol).