Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection

The capillary electrophoresis (CE) system with optical fiber light-emitting diode (optical fiber LED) induced fluorescence detector was developed for the analysis of the excitatory amino acids (EAAs) tagged with naphthalene-2,3-dicarboxaldehyde (NDA). The separation of EAAs was carried out in an uncoated fused-silica capillary (50 cm x 75 microm i.d.) with a buffer of 10 mM borate at pH 9.3 and an applied voltage of 20 kV. High sensitivity was obtained by the use of optical fiber LED induced fluorescence detector with a violet LED as the excitation light source. The limits of detection (S/N = 3) for glutamic acid (Glu) and aspartic acid (Asp) were 2.1 x 10(-8) and 2.3 x 10(-8) M, respectively. The detection approach was successfully applied to the analysis of Glu and Asp in biological fluids including human serum, rabbit serum and human cerebrospinal fluid (CSF) with satisfactory results.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization with fluorescein isothiocyanate (FITC) and the conditions for derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids.

The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate determination of amino acids in biological samples and in metal-assisted peptide hydrolysis reactions. It is especially critical for the relative fluorescence intensities (RFI) of equal amounts of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as organic solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although determination of tryptophan is problematic, this difficulty is overcome by the addition of beta-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids.     

Assessment of in situ cellular glutathione labeling with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography

We have presently studied a dialdehydic reagent, i.e. naphthalene-2,3-dicarboxaldehyde (NDA), as a fluorogenic probe for the labeling of intracellular reduced glutathione (GSH), using a yeast strain Candida albicans as a cell model. Chemical reactivity of NDA with both amino and sulfhydryl groups of the GSH molecule leads to a highly selective detection. Moreover, fluorescence properties of the resulting adduct fit well with most of modern instruments adapted for in situ measurements, and equipped with an argon laser. After incubation of cells with 100 microM of NDA for 20 min, cells were harvested and corresponding lysates obtained after a freezing cycle, were suspended in 0.2M borate buffer pH 9.2 and analysed with HPLC (column: Spherisorb ODS-2 (125 mm x 4.6 mm i.d.) 5 microm; mobile phase: methanol-0.01 M phosphate buffer pH 6.5 (20:80, v/v) at a flow rate of 0.8 mL min(-1); spectrofluorimetric detection: lambda(exc)=430 nm and lambda(em)=530 nm). The GSH-NDA adduct was identified in the yeast strain extracts using the reported HPLC technique and quantified versus a calibration curve of NDA derivatized with an excess of GSH (linearity range: 9-230 nM). The cell loading step of the free probe NDA and the extraction efficiency of the resulting NDA-GSH adduct were optimized.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

Simultaneous determination of histamine and polyamines by capillary zone electrophoresis with 4-fluor-7-nitro-2,1,3-benzoxadiazole derivatization and fluorescence detection

Capillary zone electrophoresis (CZE) with fluorescence detection was applied to the simultaneous determination of histamine and polyamines including spermine, spermidine, diaminopropane, putrescine, cadaverine, diaminohexane with 4-fluor-7-nitro-2,1,3-benzoxadiazole (NBD-F) as the fluorescent derivatization reagent. The seven NBD-F labeled amines was separated within 200 s using 85 mM phosphate running buffer at pH 3.0. The concentration limits of these amines ranged from 5.1 x 10(-8) M for spermine to 2.1 x 10(-8) M for histamine. The relative standard deviations for migration time and peak height were less than 1.5% and 6.0%, respectively. The method was successfully applied to the analysis of biogenic amines in the lysate of tobacco mesophyll protoplasts, and spermidine and putrescine were detected in the lysate with satisfying recovery.     

Detection of d-Serine in rat brain by capillary electrophoresis with laser induced fluorescence detection

A capillary zone electrophoresis method with laser induced fluorescence detection for the chiral separation of highly fluorescent enantiomeric derivatives of d/l-Serine from 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-d/l-Serine) was developed and optimized. Enantiomeric separation of NBD-d/l-Serine was accomplished by using 40 mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD) contained in 100 mM borate buffer, pH 10.0. A 70 cm (effective length of 50 cm) uncoated fused-silica capillary at a voltage of 15 kV was used for the separation. The optimized electrophoretic conditions were subsequently applied to the analysis of d-Serine in rat brain, and satisfactory analytical results with respect to accuracy were obtained. This assay showed acceptable precision, with linearity in the d-Serine concentration range of 0.2-20.0 microM. The limit of detection for d-Serine was 3.0 x 10(-7)M.     

Determination of Norepinephrine in Microdialysis Samples by Microbore Column Liquid Chromatography with Fluorescence Detection Following Derivatization with Benzylamine

A microbore column liquid chromatographic method is described for the determination of norepinephrine (NE) in microdialysis samples from rat brain. The method is based on precolumn derivatization of NE with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in a highly fluorescent and stable benzoxazole derivative. Typically, a 10-microl sample was mixed with 10 microl derivatization reagent containing 0.45 M Caps buffer (pH 12.0), 0.2 M benzylamine, 10 mM potassium hexacyanoferrate(III), and N,N-dimethylformamide (1:1:1:15, v/v). The derivatization was carried out at 50 degrees C for 20 min. Under these conditions only NE and epinephrine produced high fluorescence yields at excitation and emission wavelengths of 345 and 480 nm, respectively, while fluorescence of other catechols and 5-hydroxyindoles was quenched by 10-100 times. The NE derivative was separated on a reversed-phase column (100 x 1.0 mm i.d., packed with C18 silica, 5 microm) within 10 min with no late eluting peaks. The mobile phase consisted of 40 mM Britton-Robinson buffer (pH 7.5) containing 1 mM didodecyldimethylammonium bromide and acetonitrile (34%, v/v), the flow rate was 40 microl/min. The limit of detection (signal-to-noise ratio of 3) for NE was 90 amol in 10 microl sample injected. Microdialysis samples were collected in 5-min intervals from the probes implanted in the hippocampus, frontal cortex, or hypothalamus of awake rats. The basal extracellular NE levels in the respective areas were 4.7 +/- 0.9, 1.8 +/- 0.3, and 0.8 +/- 0.2 fmol/10 microl (mean +/- SE, n = 7). Perfusion with a Ringer solution containing 100 mM K+ increased hippocampal NE levels by 700%, while NE uptake inhibitors maprotiline and amitriptyline administered orally or subcutaneously increased extracellular NE in the frontal cortex by about 300%. On the other hand, reserpine (5 mg/kg) reduced cortical NE levels by 40% 3 h after the administration. This new fluorescence derivatization method provides better selectivity, sensitivity, and speed for NE determination than the electrochemical detection since no late-eluting compounds such as dopamine, serotonin, and their metabolites are detectable in the chromatograms of the microdialysis samples.     

Separation and quantitative analysis of O-linked phosphoamino acids by isocratic high-performance liquid chromatography of the 9-fluorenylmethyl chloroformate derivatives

Phosphoamino acids derivatized with 9-fluorenylmethyl chloroformate were separated on an anion-exchange column (Partisil 10 SAX) at pH 3.90 using an isocratic elution with 10.0 mM potassium phosphate, 1.0% tetrahydrofuran, and 55% methanol. Phosphoamino acids were eluted with baseline resolution in the following order: phosphotyrosine, phosphothreonine, and phosphoserine. Each phosphoamino acid was separated from its parent amino acid, dicarboxylic amino acids, sugaramine phosphates, as well as the other common amino acids. The turn-around time from injection to injection was 35 min. The linearity for all three O-linked phosphoamino acids extended from 0.5-1000 pmol and has been shown to be directly applicable to the analysis of isolated phosphoproteins.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100mM boric acid buffer containing 10mM SDS and 10mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0 x 10(-7) to 9.0 x 10(-7) M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.     

Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM     

Determination of hexafluoroisopropanol,a sevoflurane urinary metabolite,by 9-fluorenylmethyl chloroformate derivatization

A reversed-phase HPLC method with fluorescence detection for the quantification of hexafluoroisopropanol (HFIP) in urine is presented. HFIP, a metabolite of the inhalation anesthetic sevoflurane, is excreted mainly in urine as glucuronic acid conjugate. After enzymatic hydrolysis of the glucuronate, primary amino groups of interferent urinary compounds are blocked by reaction with o-phthalic dicarboxaldehyde and 3-mercaptopropionic acid, followed by labeling of HFIP with 9-fluorenylmethyl chloroformate. The derivatization reaction proceeds in a water-acetonitrile (1:1) solution at room temperature with a borate buffer of pH 12.5 as a catalyst. A stable fluorescent derivative of HFIP is formed within 5 min. The HFIP-FMOC derivative is separated by reversed-phase chromatography with isocratic elution on an octadecyl silyl column (33x4.6 mm, 3 microm) and guard column (20x4.0 mm, 40 microm), at 35 degrees C, and detected by fluorescence detection at an excitation wavelength of 265 nm and an emission wavelength of 311 nm. The method detection limit is 40 pg, per 10-microl injection volume, corresponding to 16 microg/l of HFIP in urine. The among-series relative standard deviation is <6% at 200 microg/l (n=6). As a preliminary application, the method was used to detect HFIP concentration in the urine of two volunteers exposed for 3 h to an airborne concentration of sevoflurane in the order of 2 ppm.     

Capillary electrophoresis with laser-induced fluorescence and pre-column derivatization for the analysis of illicit drugs

In the current paper, we report the development of a new capillary electrophoresis method using pre-column derivatization and laser-induced fluorescence detection for the determination of ephedrine and amphetamine drugs. Our new method allows for the identification and quantification of six commonly used illicit drugs namely pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxymethylamphetamine, respectively, as well as propafenone (internal standard). Following derivatization with fluorescein isothiocyanate, a total of six amphetamine drugs and the internal standard could readily be separated using a fused-silica 75 micromID x 60 cm length (effective length: 50.2 cm) capillary column. The mobile phase consisted of buffer containing 20mM borate (pH 12, adjusted with sodium hydroxide). Samples were injected in pressure mode with the capillary being operated at 25kV/25 degrees C, and the detection of the derivatized compounds was sought using a laser-induced fluorescence (LIF) detector (lambda(ex)=488 nm and lambda(em)=520 nm), with a run-time of 20 min. The current method was validated with regard to precision (relative standard deviation, RSD), accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ). In human blood and urine samples, detection limits were 0.2 ngmL(-1), and the linear range of the calibration curves was 0.5-100 ngmL(-1). The intra-day and inter-day precisions were both less than 13.22%.     

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2-9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 microl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 degrees C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.     

Liquid chromatographic method for the quantitative determination of Nepsilon-carboxymethyllysine in human plasma proteins

The modification of the lysine moieties of proteins to Nepsilon-carboxymethyllysine (CML) is supposed to play a major role in the development of long-term complications in patients with diabetes mellitus. This paper presents an analytical method for the quantitative determination of CML in plasma proteins, which could be used for studying the development of diabetic complications. The method is based on isolating proteins from plasma by precipitation with trichloroacetic acid and hydrolysing these under acidic conditions (6M hydrochloric acid at 110 degrees C for 20 h) to the individual amino acids. After hydrolysis, CML is derivatised along with the other amino acids to 9-fluorenylmethoxycarbonyl (FMOC) derivatives, which are subsequently separated by reversed-phase column liquid chromatography using a 150 mm x 4.6 mm C8 column and a mobile phase of 25 mM potassium phosphate buffer (pH 2.0) and acetonitrile (80:20 (v/v)) and detected using fluorescence detection (excitation at 260 nm and emission at 310 nm). Quantification of the protein-bound CML content of a plasma sample is achieved using standard addition. The impact of several aspects of the sample preparation and chromatography on method performance is discussed. Method evaluation results are reported and show that this method is capable of determining CML with good accuracy and precision (below 10%) in the relevant concentration range (1-10 microg/ml), with a limit of detection of 0.2 microg/ml.     

Amino Acid Analysis by Reverse-Phase High-Performance Liquid Chromatography: Improved Derivatization and Detection Conditions with 9-Fluorenylmethyl Chloroformate

An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.     

Sensitive determination of trimetazidine in spiked human plasma by HPLC with fluorescence detection after pre-column derivatization with 9-fluorenylmethyl chloroformate

A high-performance liquid chromatographic method for the determination of trimetazidine dihydrochloride (TMZ) in spiked human plasma is described. The method is based on the pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) using the fluorimetric detection technique. Fluoxetine HCl (FLX) was used as internal standard. Both, TMZ and FLX were completely derivatized after heating at 50 degrees C for 20 min in borate buffer pH 8.0. Samples were analyzed by high performance liquid chromatography (HPLC) using Zorbax-TMS column (250 mm x 4.6 mm, i.d., 5 microm) and mobile phase consist of acetonitrile, methanol and 20 mM sodium acetate pH 4.7 (44:6:50; v/v/v). Fluorescence detector (FLD) was adjusted at excitation and emission wavelengths; 265 and 311 nm, respectively. The linearity of the method was in the range of 4.5-200 ng/ml. Limits of detection (LOD) and quantification (LOQ) were 1.5 and 4.5 ng/ml, respectively. Trimetazidine recovery was 96.5+/-1.3% (n=6; RSD=2.1%).