1,2-Diarylethylenediamines as fluorogenic reagents for catecholamines

Twenty-eight 1,2-diarylethylenediamines were investigated in a search for highly sensitive fluorogenic reagents for catecholamines (norepinephrine, epinephrine and dopamine). They all react with epinephrine used as a model catecholamine under mild conditions (pH 6.5–6.8, 37–50°C) in the presence of potassium hexacyanoferrate (III) to produce fluorescence. Of the compounds tested, 1,2-bis(3,4-dimethoxyphenyl)ethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis (4-ethoxyphenyl)ethylenediamine, all in the meso-form, are the most sensitive for all the catecholamines. 1,2-Diphenylethylenediamine, 1,2-bis(4-methoxyphenyl)ethylenediamine and 1,2-bis (4-methylphenyl) ethylenediamine in the dl-form are more sensitive to dopamine than the corresponding compounds in the meso-form. Catecholamines can be determined at concentrations as low as 10–15 pmol ml^?1 by using those compounds as reagents. The reactions of other catechol compounds are reported. Other types of biologically important compounds do not interfere.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10-10,000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17 degrees C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

High performance liquid chromatography determination of cyanide in urine by pre-column fluorescence derivatization

A method for the determination of cyanide in human urine has been developed. The method is based on the reaction of cyanide with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product for reversed-phase HPLC separation and fluorometric detection. After centrifugation followed by dilution of urine samples, the specimens could be analysed directly by this method. The recovery of cyanide added to urine at concentration levels of 50-1000 pmol/mL was 85-96%. The detection limit of cyanide was 30 pmol/mL in urine. The method was successfully applied to the analysis of urine from smokers and nonsmokers. The mean concentrations of cyanide were found to be 215 pmol/mL for the former and 84 pmol/mL for the latter.     

Improved determination of the bisphosphonate pamidronate disodium in plasma and urine by pre-column derivatization with fluorescamine, high-performance liquid chromatography and fluorescence detection.

An improved method for the determination of 1-hydroxy-3-aminopropylidene-1,1-bisphosphonate (pamidronate) in human urine and plasma is described. The procedure is based on a co-precipitation of the bisphosphonates (pamidronate and 6-amino-1-hydroxypentilidene-bisphosphonate, used as internal standard) with calcium phosphate. After centrifugation the precipitate is redissolved in hydrochloric acid, followed by a second precipitation. Then the bisphosphonates are dissolved in ethylenediaminetetraacetic acid, derivatized with fluorescamine, and separated by high-performance liquid chromatography. Using fluorescence detection, the limit of quantitation for pamidronate was 0.8 mumol/l in plasma and 0.7 mumol/l in urine.     

High-performance liquid chromatographic determination of forphenicine in mouse serum and muscle by pre-column fluorescence derivatization using 1,2-diamino-4,5-ethylenedioxybenzene as fluorogenic reagent

A simple and sensitive high-performance liquid chromatographic method has been developed for the determination of forphenicine in biological samples. Forphenicine in the deproteinized sample is converted by reaction with 1,2-diamino-4,5-ethylenedioxybenzene into a fluorescent derivative. The derivative is separated on a reversed-phase column (TSK gel ODS-120T) by isocratic elution with acetonitrile-30 mM phosphate buffer (pH 6.5) (5:1, v/v) and monitored fluorimetrically. The method allows the quantification of forphenicine in serum (100 microliters) and muscle (0.1 g) of mice dosed with forphenicine or forphenicinol. The limits of detection (signal-to-noise ratio of 3) are 7.35 pmol/ml in serum and 5.36 pmol/g in muscle. The distribution of forphenicine and forphenicinol in the mouse serum and muscle after oral administration of these compounds is also described.     

High performance liquid chromatographic determination of N-nitrosoamines by pre-column fluorescence derivatization with acridone-N-acetyl chloride

A sensitive high performance liquid chromatographic method for the determination of N-nitrosoamines with pre-column fluorescence derivatization has been developed. N-nitrosoamines are first changed into secondary amines using denitrosation reagent, then react with acridone-N-acetyl chloride (ARC-Cl) to produce corresponding secondary amine derivatives, which exhibit a strong fluorescence. Maximum emission for ARC derivatives is 430 nm (lambda(ex) 404 nm). The labelled derivatives are very stable, less than 4% decomposition occurs after heating at 40 degrees C for 24 h. Fluorescence intensities of derivatives are higher in neutral and alkaline than in acidic solutions. This method, in conjunction with a multi-gradient program, offers a baseline resolution of the ARC derivatives from a linear acetonitrile gradient. Separation is carried out on a reverse phase C(18) column. Derivatization and chromatographic conditions are optimized. The relative standard deviation (n=6) at an analytical concentration of 10 pmol of each N-nitroamine is less than 4.5%. The detection limits at the fmol level. The method described is also suitable for analysis of other amino compounds in different biological samples.     

High-performance liquid chromatographic determination of valproic acid in plasma using a micelle-mediated pre-column derivatization

A method for the determination of valproic acid (2-propylpentanoic acid) in plasma by high-performance liquid chromatography (HPLC) after pre-column derivatization is described. The derivatization of valproic acid with a fluorophore and UV label, 4-bromomethyl-7-methoxycoumarin, is performed in plasma diluted with an aqueous micellar system. No extraction or solvent evaporation steps are required. The mechanism of the derivatization of the carboxylic acid is based on phase-transfer catalysis. The sample preparation, including the derivatization step, is rapid and very simple. The proposed HPLC-method was evaluated and compared with a standard immunological assay used for the determination of valproic acid in plasma.     

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.     

Determination of fluoxetine enantiomers in rat plasma by pre-column fluorescence derivatization and column-switching high-performance liquid chromatography

A column-switching HPLC method employing both octadecylsilica (ODS) and chiral columns with fluorescence detection was developed for the determination of enantiomer of fluoxetine (FLX), an antidepressant drug, in rat plasma. Racemic FLX was derivatized with a fluorescent reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl) or 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and the enantiomeric separation of the resultant derivatives was examined on an amylose-based chiral column (CHIRALPAK AD-RH) in the reversed-phase mode. The derivative with NBD-COCl (NBD-FLX) showed a sufficient separation factor (a) and resolution (Rs) compared with that with DBD-COCl. Thus, FLX was derivatized with NBD-COCl and the resultant NBD-FLX was first quantified on the ODS column and then introduced to the CHIRALPAK AD-RH column via a six-port switching valve to examine the enantiomeric ratio. The intra- and inter-day accuracy (97.6-112.7%) and precision (1.47-10.60%) were satisfactory in the range 10-1000 nM FLX and the limit of quantification was approximately 10 nM. The absolute recoveries of FLX with hexane from rat plasma were in the range 87.5-92.2% (n = 3). The method was applied to determine FLX enantiomers in the plasma of rats administered FLX orally, and it was shown that the R-isomer was eliminated faster than the S-isomer.     

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.     

Automated solid-phase catalyzed pre-column derivatization of fatty acids for reversed-phase high-performance liquid chromatographic analysis with fluorescence detection

A method is described to automate the solid phase catalyzed reaction of a carbonyl group with labels containing a bromomethyl group. The acids to be determined must be soluble in an aprotic, non-aqueous reaction medium, such as acetonitrile. Special emphasis is placed on the solubility of the base, which serves as a catalyst. A suspension of potassium carbonate was prepared in acetonitrile. In this way, the effective surface area of the base is drastically enlarged resulting in faster reaction kinetics. The procedure is illustrated with the detection of fatty acids in serum and rat brain tissue with bromomethylmethoxycoumarin as label. The generality of our approach for HPLC determination of carbonyl compounds is discussed.     

High-performance liquid chromatographic determination of alpha-keto acids in human serum and urine using 1,2-diamino-4,5-methylenedioxybenzene as a precolumn fluorescence derivatization reagent

A simple and highly sensitive high-performance liquid chromatographic (HPLC) method for the determination of alpha-keto acids in human serum and urine is described. In an acidic solution, twelve species of alpha-keto acids examined were converted by reaction with 1,2-diamino-4,5-methylenedioxy-benzene into highly fluorescent derivatives. The derivatives were separated isocratically by reversed-phase HPLC on a TSK gel ODS-80TM column and detected fluorimetrically. Eight alpha-keto acids in human serum and eleven alpha-keto acids in human urine can be determined simultaneously. The detection limits (signal-to-noise ratio = 5) are 6-44 fmol in an injection volume of 5 microliters. The intra-assay relative standard deviations for both serum and urine sample analyses are usually ca. 5%.     

High performance liquid chromatographic assay of Amicar, epsilon-aminocaproic acid, in plasma and urine after pre-column derivatization with o-phthalaldehyde for fluorescence detection

A simple, reliable and highly sensitive procedure was devised for measuring the levels of Amicar in blood and urine. 100 microL of serum or urine sample was added to 10 microL of a 10% w/v zinc sulfate solution and 100 microL of methanol, as previously described (Lam et al., 1980) for the removal of proteins by precipitation. 50 microL of the supernatant was then mixed with 300 microL of 1 M borate buffer containing D-valine as the internal standard before derivatization with o-phthalaldehyde. The amino acids were then separated by a stereoselective reversed-phase system using a mobile phase containing 10% of acetonitrile in 2.5 mM Cu(II) complexes of L-proline. The chromatography is highly selective, resolving Amicar from L-valine which in turn is resolved from its unnatural D-antipode, the internal standard. The procedure including sample preparation and separation required a total of 15 min. As little as 50 ng/mL of Amicar in body fluids could be detected as the o-phthalaldehyde derivative by fluorescence.     

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.     

An automated high-performance liquid chromatographic method for the determination of aminoglycosides in serum using pre-column sample clean-up and derivatization

An automated high-performance liquid chromatographic method for the determination of the aminoglycosides amikacin, dibekacin, gentamicin, netilmicin, sisomicin and tobramycin is described. The procedure involves sample clean-up by adsorption of the aminoglycosides on a pre-column, subsequent derivatization with o-phthalaldehyde and on-line separation of derivatives by column switching. A short cation-exchange column serving concurrently as a guard column in combination with a reversed-phase column was used for separation. Except for the determination of netilmicin an internal standard consisting of an aminoglycoside was used in each assay. The signals of the aminoglycosides determined were linear within the range of 1-16 mg/l serum. The inter-assay imprecision (n = 10) calculated as coefficient of variation was less than 6%. The results were obtained within 20 min after injection of the serum sample. Easy performance and flexibility make the procedure feasible for therapeutic drug monitoring.     

Monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine by high-performance liquid chromatography after pre-column derivatization with glyoxal reagents

A new, simple and sensitive pre-column fluorescence derivatization high-performance liquid chromatographic method for the determination of the oxidative DNA stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, was developed. Solid-phase extraction using an Oasis HLB cartridge avoided troublesome sample preparation steps, interference from charged species and frequent and essential electrode maintenance in electrochemical procedures. 8-Oxo-7,8-dihydro-2'-deoxyguanosine and other guanine compounds were selectively derivatized with glyoxal reagents (phenylglyoxal, 3,4-methylenedioxyglyoxal, 2-naphtylglyoxal and 6-methoxynaphthylglyoxal) at 40-60 degrees C. Derivatization with 6-methoxynaphthylglyoxal at 40 degrees C for 30 min gave the strongest fluorescence product. The fluorescence derivatives from reaction with 6-methoxynaphthylglyoxal were separated on a Capcell Pak C18 SG 120A column (4.6 mm i.d. x 150 mm, 5 microm) with acetonitrile-5 mM phosphate buffer (pH 6.0; 3:7, v/v) as mobile phase. The detection wavelength of the fluorescence derivative of 8-oxo-7,8-dihydro-2'-deoxyguanosine was lambda(ex) 400 nm and lambda(em) 510 nm. The detection limit of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 1 ng/mL using 50 mL of urine. The calibration graphs were linear up to 30 microg/mL for 8-oxo-7,8-dihydro-2'-deoxyguanosine. The relative standard deviation of 20 ng/mL of 8-oxo-7,8-dihydro-2'-deoxyguanosine was 7.0%. The proposed method was compared with the enzymatic ELISA 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis method (8-OH-dG Check, JaICA, Shizuoka, Japan). The correlation coefficient was 0.79 (n = 20) and y = 0.85x + 5.34. The proposed method was applied to the monitoring of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine from male heavy smokers.