Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ _ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure. In‑capillary derivatization and separation of amino acids with different concentrations. From the top to bottom the concentrations are 1.11×10⁻⁵ M, 5.55×10⁻⁶ M, 2.78×10⁻⁶ M, 6.95×10⁻⁷ M. for valine; 1.26×10⁻⁵ M, 6.30×10⁻⁶ M, 3.15×10⁻⁶ M, 7.88×10⁻⁷ M for alanine; 3.78×10⁻⁵ M, 1.89×10⁻⁵ M, 9.45×10⁻⁶ M, 2.36×10⁻⁶ M for glutamic acid;, 4.27×10⁻⁵ M, 2.14×10⁻⁵ M, 1.07×10⁻⁵ M, 2.68×10⁻⁶ M for aspartic acid. Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10⁻² M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.     

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.     

Micellar electrokinetic capillary chromatographic separation and fluorescent detection of amino acids derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

Another method has been developed for the separation of amino acids (1 min derivatization plus 22 min separation) by micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence detection. Interestingly enough, such work has never been performed on essential amino acids derivatized by 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Fifteen L-amino acid standards were labelled with NBD-F at 60 degrees C for 1 min, and separated in a buffer system containing 20 mM borate, 25 mM sodium cholate, 10 mM Brij 35 and 2.5% methanol. Methanol was employed to expand the MECC migration time window; whereas Brij 35 was used to improve the fluorescence intensity of amino acid derivatives. This method also indicates that bile salt is effective for MECC separation of ionic analytes. Surprising though, improvements in resolution, sensitivity and speed for amino acids analysis are obtained in this work, which are not initially apparent in just employing another derivatizing reagent. Under optimal conditions, 15 amino acids were separated in a short 22 min analysis time, the shortest ever reported, and detection limits of nanomolar concentration and attomole mass were obtained. Furthermore, RSDs of migration time and peak height are better than 1% and 1.8%, respectively, again the smallest ever reported in the literature.     

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).     

Determination of fluvoxamine in rat plasma by HPLC with pre-column derivatization and fluorescence detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

A sensitive, simple and reliable method using high-performance liquid chromatographic (HPLC) assay of fluvoxamine (FLU), a selective serotonin reuptake inhibitor (SSRI), in rat plasma after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed in this study. Extracted plasma samples were mixed with NBD-F at 60 degrees C for 5 min and injected into HPLC. Retention times of FLU and an internal standard (propafenone) derivative were 15.5 and 13.5 min, respectively. The calibration curve was linear over the range 0.015-1.5 microg/mL (r2 = 0.9985) and the lower limits of detection and quantification of FLU were 0.008 and 0.015 microg/mL, respectively, in 100 microL of plasma. The derivative sample was stable at 4 degrees C for 1 day. The coefficients of variation for intra-day and inter-day assay of FLU were less than 8.3 and 9.6%, respectively. Other SSRIs and centrally acting drugs did not interfere with the peak of the FLU derivative. The method was applied for analysis of the plasma samples from rats treated with FLU. These results indicate that the method presented is useful to determine the FLU levels in rat plasma of volumes as small as 100 microL and can be applied to pharmacokinetic studies.     

Amino acid composition analysis of minute amounts of cysteine-containing proteins using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-fluoro-7-nitro-2,1,3-benzoxadiazole in combination with HPLC

The simultaneous determination of amino acid composition including cysteine of egg albumin, a model protein containing a/s cysteine residue, is reported. All the thiol groups of the cysteine residue(s) of egg albumin were labelled with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole, a fluorogenic reagent for thiol groups. The labeled egg albumin was hydrolyzed in 6N HCl at 110 degrees C for 24 h. The hydrolysate was lyophilized, derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, a fluorogenic reagent for amines, and subjected to HPLC. 18 derivatized amino acids including double labelled cysteine were separated within 90 min on a Nucleosil ODS column (150 mm X 4.6 mm i.d.; 5 microns), and detected at 530 nm (ex. 470 nm) in a range from 90 fmol (aspartic acid) to 1.3 pmol (cysteine) (S/N = 3). Composition ratios of amino acids of egg albumin were similar to theoretical values except for methionine, which would be destroyed under the present acid hydrolysis condition. Analytical methods for cysteine residues are reviewed, and the availability of fluorogenic reagents having the benzofurazan structure is also discussed.     

Reversible labeling of tyrosine residue in peptide using 4-fluoro-7-nitro-2,1,3-benzoxadiazole and N-acetyl-L-cysteine.

The reversible labeling of tyrosine (Tyr)-containing peptide, which involves detection and recovery, is described in this paper. The phenolic-OH in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under a mild condition at room temperature in alkaline medium. The resulting derivative absorbed at around 280 nm and 380 nm. However, the fluorescence was very weak. The NBD moiety in the derivative was efficiently removed by the treatment of N-acetyl-L-cysteine (N-AcCys), and the original peptide before the labeling was completely recovered. The proposed procedure was successfully applied to the reversible labeling of N-terminal amine-blocked peptides, i.e., N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Although the proposed method could not recover the N-terminal amine-free peptides without blocking, the selective detection and the recovery of Tyr-containing peptide fragments were possible by the combination with enzyme digestion. The reversible labeling of Tyr-containing peptide was demonstrated with [Tyr8]-bradykinin as a model for high-molecular-mass peptides and proteins. The peptide fragments containing NBD-O-Tyr moiety, obtained after the digestion, were easily discriminated from various peptides with the monitoring of UV and FL, because the target peptide did not fluoresce, but absorbed at both 280 nm and 380 nm. The peptide fragment containing Tyr was finally recovered from the de-labeling reaction with N-AcCys. The proposed method hence provides a novel technique for the reversible labeling of Tyr-containing peptides, which will enable the selective detection and the recovery of the original peptide.     

Catecholamines derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole: characterization of chemical structure and fluorescence properties

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was evaluated as a fluorogenic derivatization reagent for the analysis of the catecholamines, dopamine, epinephrine, norepinephrine, and their naturally occurring metabolites, metanephrine and normetanephrine, homovanillic acid, 3,4-dihydroxyphenyl acetic acid. These compounds reacted rapidly with NBD-F under mild conditions to form stable derivatives. The optimal reaction conditions were found to be 12.5 mM borate buffer pH 8.0 in water:acetonitrile (1:1) at 50 °C for 5 min. New NBD derivatives of all the catecholamines and metabolites were prepared and purified and were shown by electrospray mass spectrometry to be fully reacted at all available catechol and amine sites, resulting in di- or tri-substituted derivatives. Homovanillic acid and 3,4-dihydroxyphenyl acetic acid reacted with NBD-F but gave non-fluorescent derivatives. The fluorescence excitation wavelength maximum demonstrated a red shift for the derivatives with increasing polarity of the solvent and the fluorescence intensity increased linearly with increasing organic ratio in the solvent-aqueous buffer complex. The presence of electrolyte in the solvent and the electrolyte concentration in the solvent-electrolyte complex had little effect on the fluorescent intensity. The fluorescence quantum yields in acetonitrile were also obtained. The separation behavior of the NBD-catecholamines was determined by high-performance liquid chromatography (HPLC). The studies demonstrated good potential for the application of NBD-F derivatization to the quantitative analysis of catecholamines and related compounds in biological matrices.     

Chiral separation by capillary electrophoresis with oligosaccharides.

Maltodextrins, i.e., mixtures of linear alpha-(1-4)-linked D-glucose polymers, were found to be effective as chiral electrolyte modifiers to perform direct, rapid separations by capillary electrophoresis of racemic mixtures of 2-arylpropionic acid non-steroidal anti-inflammatory compounds and coumarinic anticoagulant drugs, and also diastereomeric cephalosporin antibiotics. Enantioselectivity seemed to be dependent on an as yet unidentified combination of variables.     

Simultaneous liquid chromatographic assay of amantadine and its four related compounds in phosphate-buffered saline using 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorescent derivatization reagent

Simultaneous HPLC assay of 1-adamantanamine hydrochloride (amantadine) and its four related compounds [2-adamantanamine hydrochloride (2-ADA), 1-adamantanmethylamine (ADAMA), 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) and 3,5-dimethyl-1-adamantanamine hydrochloride (memantine)] in phosphate-buffered saline (pH 7.4) after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed. Phosphate-buffered saline samples were mixed with borate buffer and NBD-F solution in acetonitrile at 60 degrees C for 5 min and injected into HPLC. Five derivatives were well separated from each other. The lower limits of detection of amantadine, 2-ADA, ADAMA, rimantadine and memantine were 0.008, 0.001, 0.0008, 0.0015 and 0.01 microg/mL, respectively. The coefficients of variation for intra- and inter-day assay were less than 6.4 and 8.2%, respectively. The method presented was applied to a binding study of these compounds to human alpha(1)-acid glycoprotein. While affinity constants and capacities for ADAMA, rimantadine and memantine were calculated by means of Scatchard plots, those for the others were not determined. ADAMA, rimantadine and memantine were bound with different affinities and capacities. These results indicate that NBD-F is a good candidate as a fluorescent reagent to simultaneously determine amantadine and its four related compounds by HPLC after pre-column derivatization. Our method can be applied to binding studies for protein.     

LC Determination of Thiols Derivatized with 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole after SPE

Solid-phase extraction (SPE) coupled with high-performance liquid chromatography?Cfluorescence detection (LC?CFL) was developed for the determination of three thiol compounds including glutathione, cysteine and acetylcysteine. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was used for derivatization of thiols. Factors affecting derivatization and extraction efficiency were optimized. Sample solution (2?mL) was extracted on a SPE column for 2?min and then eluted with 400???L methanol. The analytes were injected onto the LC system for separation on a C₁₈ column, and eluted with methanol?Cacetate buffer. The analytes were detected by fluorescence at an emission wavelength of 515?nm with excitation at 385?nm. The linearity of the method was in the range of 0.1?C60???M, with correlation coefficients ranging from 0.9979 to 0.9990. The detection limits of the method were in the range of 5?C20?nM. The proposed method was applied to the analysis of human plasma samples with recoveries of 86?C112.9%.     

Chiral separation of cetirizine by capillary electrophoresis

Chiral separation of cetirizine, a second-generation H(1)-antagonist, was studied by CD-mediated CE. Several parameters, including pH, CD type, buffer concentration, type of co-ion, applied voltage and temperature, were investigated. The best conditions for chiral separation were obtained using a 75 mM triethanolamine-phosphate buffer (pH 2.5) containing 0.4 mg/mL heptakis(2,3-diacetyl-6-sulfato)-beta-CD and 10% ACN. Online UV detection was performed at 214 nm, a voltage of 20 kV was applied and the capillary was temperature controlled at 25 degrees C by liquid cooling. Hydrodynamic injection was performed for 1 s. The method was validated for the quantification of levocetirizine in tablets and for enantiomeric purity testing of the drug substance. Selectivity, linearity, LOD and LOQ, precision and accuracy were evaluated for both methods. The amount of levocetirizine dihydrochloride in the commercially available tablets was quantified and was found to be within the specification limits of the claimed amount (5 mg). The amount of distomer in levocetirizine drug substance was found to be 0.87 +/- 0.09% w/w, which is in agreement with the certificate of analysis supplied by the company.     

Chiral separation of diastereomeric flavanone-7-O-glycosides in citrus by capillary electrophoresis

The 2S- and 2R-diastereomers of major flavanone-7-O-glycosides found in sweet orange (Citrus sinensis), mandarine (Citrus deliciosa), grapefruit (Citrus paradisi), lemon (Citrus limon), and sour or bitter orange juice (Citrus aurantium) were separated for the first time by chiral capillary electrophoresis (CE) employing various buffers with combined chiral selectors. Native cyclodextrins (CDs), neutral and charged CD derivatives were examined as chiral additives to the background electrolyte (BGE). Separation efficiency has not proved satisfactory with one single CD as chiral selector in the buffer, a full and simultaneous separation could often be achieved only by using combined buffer with two different CDs. Chiral separation of major flavanones in sweet orange, mandarine and grapefruit juices raised more difficulties than in lemon and sour orange juices as narirutin will not readily build complexes with most CDs. Diastereomeric flavanones of mature and immature grapefruits were compared and some differences were found: naringin showed different diastereomeric ratio and 2S-prunin appeared only in immature grapefruit. Marmalade was also examined by chiral CE. Its major flavanones corresponded to flavanone pattern of mixed sour and sweet oranges.     

Fluorogenic reagent for thiols: 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole

4-(N,N-Dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was synthesised for use as a more reactive, thiol-specific fluorogenic reagent than 4-(aminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The former had negligible fluorescence whereas its thiol derivatives fluoresced intensely at about 510 nm (excitation occurred at about 380 nm). The DBD-F reacted quantitatively with thiols after 10 min at 50 degrees C and pH 8.0 and the reaction rates were several times higher than those with ABD-F; it is suggested that the electron withdrawing effect of the dimethylsulphonamide group (SO2NMe2) is larger than that of the sulphonamide group (SO2NH2). No reaction occurred with alanine, proline, cystine or cysteic acid under the same conditions. The fluorescence intensities of the derivatives were found to be higher in neutral and acidic media than in alkaline solutions. The thiol derivatives of DBD-F were separated by high-performance liquid chromatography and detected fluorimetrically, the detection limits being 0.92, 0.16, 0.13, 0.16 and 0.32 pmol for cysteine, glutathione, homocysteine, N-acetylcysteine and alpha-mercaptopropionylglycine, respectively. The method was applied to the determination of thiols in rat tissues.     

Chiral separation of basic drugs by capillary electrophoresis with carboxymethylcyclodextrins

Capillary electrophoresis (CE) with carboxymethylated beta- or gamma-cyclodextrins was used to achieve the rapid enantiomeric separation of a set of basic drugs. The enantiomers of 12 chiral amino-containing pharmaceutical compounds belonging to various therapeutic categories were analyzed by CE using an uncoated 60 cm x 75 microm I.D. silica capillary. Several experimental parameters such as the nature, concentration and pH of the buffer, nature and concentration of the anionic cyclodextrin and temperature were studied in order to optimize the enantiomeric separation. The variation of the solute partition coefficient for the chiral selector, the enantioselectivity and resolution factors are used to assess the quality of the chiral separation. It is shown that the solute affinity for the chiral selector is not related to its enantioresolution factor. None of the two cyclodextrin selectors used was able to separate the whole set of basic drugs.     

Determination of thiols and disulfides in normal rat tissues and hamster pancreas treated with N-nitrosobis(2-oxopropyl)amine using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate

Biological thiols and disulfides in rat and hamster tissues were simultaneously determined by HPLC-fluorescence detection using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). The coefficients of variation (CV) of the method for reduced glutathione (GSH) and oxidized glutathione (GSSG) in liver and for cysteine (CySH) and cystine (CySSCy) in kidney were less than 3.1%. In 11 tissues of Wistar rats (liver, spleen, heart, lung, stomach, bladder, ovary, uterus, adrenal, kidney and pancreas), only CySH, CySSCy, GSH and/or GSSG were detected. Other thiols and disulfides were at extremely low levels in all samples. Both concentrations of CySH and CySSCy in the livers of old rats (111 weeks old, F344) were significantly higher than those of young rats (8 weeks old) (CySH, 0.246 +/- 0.099 vs 0.130 +/- 0.020 mumol/g; CySSCy, 0.051 +/- 0.027 vs 0.013 +/- 0.002 mumol/g). Administration of N-nitrosobis(2-oxopropyl)amine (BOP), a selective carcinogen of hamster pancreatic cancer, to Syrian golden hamsters (38 weeks old) resulted in the increase in the pancreas of GSH to a level 19 times as high and of GSSG to a level 14 times as high as those in untreated hamsters (GSH, 1.173 +/- 0.272 vs 0.062 +/- 0.017 mumol/g; GSSG, 0.155 +/- 0.063 vs 0.011 +/- 0.001 mumol/g).     

Chiral separation of local anaesthetics with capillary electrophoresis

Summary A chiral capillary electrophoresis system for the highresolution separation of the enantiomers of the local anaesthetics mepivacaine, ropivacaine, bupivacaine and prilocaine is described. Triethanolamine was added to the background electrolyte to obtain a negative electroosmotic flow and hence higher resolutions. The interactions of the local anaesthetics and their chemical analogues with the chiral selector, dimethyl--cyclodextrin, were studied. From a model describing chiral capillary electrophoresis, the association equilibrium constants were determined by curve-fitting. The separation of mepivacaine, ropivacaine and bupivacaine was due to the different mobilities of the free analytes in solution, whereas the separation of a pair of enantiomers of a single analyte was due to differences between the association equilibrium constantsK ₁ andK ₂. Branching of the alkyl chain, which was situated close to the cavity in the inclusion complex, had strong effects on the chiral separation of the enantiomers.     

Sensitive determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, in a rat biological sample by HPLC with fluorescence detection after pre-column derivatization using 4-fluoro-7-nitro-2,1,3-benzoxadiazole

4-(4-Chlorophenyl)-4-hydroxypiperidine (CPHP), one of the metabolites of haloperidol, is considered to exhibit brain toxicity. CPHP concentrations in plasma and tissue homogenates (each 200 microL) from rats were analyzed by HPLC fluorescence detection after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). After basic extraction of the samples with benzene, the derivatization with NBD-F was conducted in borate buffer (pH 8.0) at 60 degrees C for 3 min. Mexiletine was carried through the procedure as an internal standard. The regression equation for CPHP showed a good linearity in the range of 0.03-1 microg/mL with a detection limit of 0.008 microg/mL. The coefficient of variation was less than 11.6%. Plasma concentration-time courses of CPHP after intraperitoneal or per oral administration of CPHP, haloperidol or reduced haloperidol were examined, and the pharmacokinetic parameters were estimated. Additionally, CPHP levels in various tissues at 8 h after intraperitoneal administration of these compounds were compared. The method was simple and sensitive, useful for determination of CPHP in rat biological samples using as little as 200 microL of sample volume and could be applied for pharmacokinetic study.