Bioanalytical applications of capillary electrophoresis with laser-induced native fluorescence detection.

In this article we describe recent developments and applications of capillary electrophoresis (CE) coupled with laser-induced native fluorescence (LINF) detection in the analysis of biological, pharmaceutical and environmental samples. Compared with traditional UV absorbance detection used in CE, the LINF technique can greatly improve the concentration sensitivity of CE without the need for derivatization; the only requirement being that the analyte must have native fluorescence. Instrumentation and laser sources used in CE-LINF are summarized and specific applications of CE-LINF to small-biomolecule analysis, profiling of human biofluids, detection of native fluorescent peptides and proteins, single-cell analysis and the use of online sample preconcentration methods are also reviewed in detail.

A mutation in amino acid permease AAP6 reduces the amino acid content of the Arabidopsis sieve elements but leaves aphid herbivores unaffected.

The aim of this study was to investigate the role of the amino acid permease gene AAP6 in regulating phloem amino acid composition and then to determine the effects of this altered diet on aphid performance. A genotype of Arabidopsis thaliana (L.) was produced in which the function of the amino acid permease gene AAP6 (At5g49630) was abolished. Plants homozygous for the insertionally inactivated AAP6 gene had a significantly larger mean rosette width than the wild type and a greater number of cauline leaves. Seeds from the aap6 mutant were also significantly larger than those from the wild-type plants. Sieve element (SE) sap was collected by aphid stylectomy and the amino acids derivatized, separated, and quantified using Capillary Electrophoresis with Laser Induced Fluorescence (CE-LIF). In spite of the large variation across samples, the total amino acid concentration of SE sap of the aap6 mutant plants was significantly lower than that of the wild-type plants. The concentrations of lysine, phenylalanine, leucine, and aspartic acid were all significantly lower in concentration in the aap6 mutant plants compared with wild-type plants. This is the first direct demonstration of a physiological role for an amino acid transporter in regulating SE composition in vivo. The amino acid availability in sieve element sap is thought to be the major limiting factor for aphid growth and reproduction. Despite the changes in their diet, the aphid Myzus persicae (Sulzer) displayed only small changes in feeding behaviour on mutant plants when measured using the Electronic Penetration Graph (EPG) technique. Salivation by the aphid into the SE (E1 phase) was increased on mutant plants but there was no significant effect on other feeding EPG behaviours, or in the rate of honeydew production. Consistent with the small effect on aphid feeding behaviour, there was only a small effect of reduced sieve element amino acid concentration on aphid reproduction. The data are discussed in relation to the regulation of phloem composition and the role of phloem amino acids in regulating aphid performance.

Analysis of mono-, di- and oligosaccharides by CE using a two-stage derivatization method and LIF detection.

A sensitive CE with LIF method has been developed for quantitative analysis of small carbohydrates. In this work, 17 carbohydrates including mono-, di- and oligosaccharides were simultaneously derivatized with 4-fluoro-7-nitrobenzofurazane (NBD-F) via a two-step reaction involving reductive amination with ammonia followed by condensation with NBD-F. Under the optimized derivatization conditions all carbohydrates were successfully derivatized within 2.5 h and separated within 15 min using borate buffer (90 mmol/L, pH 9.2). For sugar standards LODs were in the range of 49.7 to 243.6 nmol/L. Migration time and peak area reproducibility were better than RSD 0.1 and 3%, respectively. The method was applied to measure sugars in nanoliter volume samples of phloem sap obtained by stylectomy from wheat and to honeydew samples obtained from aphids feeding from wheat and willow.

Micellar electrokinetic biofluid analysis of biogenic amines using on-line sample concentration and UV laser-induced native fluorescence detection.

A simple and sensitive sweeping micellar electrokinetic chromatography method coupled with UV laser-induced native fluorescence detection has been developed for quantitative analysis of biogenic amines in biofluids. The background electrolyte comprised 30 mmol L(-1) phosphoric acid and 20 mmol L(-1) sodium dodecyl sulfate. The concentration limits of detection of analytes using sweeping-micellar electrokinetic chromatography (sweeping-MEKC) were in the range 7-100 nmol L(-1), which were 250-3600-fold improvement for dopamine, DOPA and epinephrine compared with conventional capillary zone electrophoresis. An improvement of approximately 20-fold was observed for all analytes compared with typical micellar electrokinetic chromatography conditions. Baseline separation was achieved for the all analytes within 12 min and migration-time and peak-area repeatability were better than RSD 0.35% and 5.68%, respectively. The developed method was applied to measure the biogenic amines in biofluids extracted from wheat phloem sap, human plasma and human urine.

A diurnal component to the variation in sieve tube amino acid content in wheat.

We have used high-sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection method to quantify 16 amino acids in wheat (Triticum aestivum) sieve tube (ST) samples as small as 2 nL collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual STs. Some of the total concentrations of amino acids recorded are higher than those reported previously. The results obtained show variation in the concentrations of phenylalanine (Phe), histidine/valine (His/Val), leucine/isoleucine (Leu/Ile), arginine, asparagine, glutamine, tyrosine (Tyr), and lysine (Lys) across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (Tyr, Lys, Phe, His/Val, and Leu/Ile) that showed striking covariation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of Tyr, Lys, Phe, Leu/Ile, His/Val, asparagine, arginine, and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analyzing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed: samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.

Profiling of amine metabolites in human biofluids by micellar electrokinetic chromatography with laser-induced fluorescence detection.

A rapid and sensitive capillary electrophoresis (CE) method has been developed for profiling organic metabolites containing amine functional groups in mammalian biofluids. Metabolites containing an amine group were derivatized with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection. The optimized MEKC background electrolyte conditions were: 50 mmol L-1 sodium cholate, 5 mmol L-1 beta-cyclodextrin, and 20 mmol L-1 Brij 35 in 20 mmol L-1 aqueous borate buffer, pH 9.3, containing 7% methanol. Under these conditions all the amine compounds in mammalian biofluids, for example plasma, saliva, and urine, were derivatized directly, without extraction, in a minimum volume of 100 nL and the derivatives could be separated within 16 min. Up to 90% of the amine-containing metabolites in plasma and saliva could be identified by reference to standard compounds. For twenty amine standards linearity of calibration was better than R2=0.99. Migration-time and peak-area reproducibility were better than RSD 1.5% and 15% respectively. In replicate analysis of human plasma bioanalytical precision ranged between 0.7 and 3.8 RSD% for a 5.0-microL volume and between 1.7 and 5.5 RSD% for 100-nL volume. The concentrations measured were found to be in agreement with literature values.

Sensitivity improvement on detection of Coptidis alkaloids by sweeping in capillary electrophoresis.

A simple and rapid sweeping method for the online improvement of detection sensitivity of the main alkaloids of Coptidis Rhizoma has been developed in this work. Optimum separation conditions were found as follows: electrophoretic running solution comprising 100 mM phosphoric acid, 15 mM sodium dodecyl sulfate (SDS) and 10% (v/v) tetrahydrofurane with pH 1.82; running voltage of -25 kV; sample matrix composed of 50 mM phosphoric acid and sample injection at 1000 mbar for 60 s (sample injection volume ca. 2.75 microl). With this sweeping method, the concentration limits of detection of berberine, coptisine and palmatine were found to be 2.5 ppb (ng/ml), which was about 500 times lower than those from conventional sample injections. Baseline separation was achieved for the main alkaloids within 15 min. After validation, the developed method was applied to determine the quantity of berberine, coptisine and palmatine in a Coptidis Rhizoma sample. The method should be able to be used in identification and quantitative evaluation of the crude drugs requiring only a minor amount of sample.

Improved detection of Coptidis alkaloids by field-amplified sample stacking in capillary electrophoresis.

To improve the on-line detection of Coptidis alkaloids in capillary electrophoresis the field-amplified sample stacking was studied for them. In this work the peak height enhancements of stacking with hydrodynamic and electrokinetic injections were compared with respect to the conventional sample injection. It was found that the stacking efficiency of electrokinetic injection was more than ten times greater than that of hydrodynamic injection. No peak height enhancement was observed with the pre-injection of a short water plug before sample injection with electrokinetic injection. The concentration limits of detection of berberine, coptisine and palmatine obtained with electrokinetic injection were about 5 ng/ml (ppb), which was approximately 240 times lower than those from conventional sample injections. Baseline separation was also achieved for the main alkaloids. After validation the developed method was applied to determine the quantity of berberine, coptisine and palmatine in a Coptidis Rhizoma sample. The method is simple, rapid and should be able to be used in identification and quantitative evaluation of the crude drugs.