Capillary electrophoresis-laser induced fluorescence-electrospray ionization-mass spectrometry: a case study

The simultaneous hyphenation of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and electrospray ionization-mass spectrometry (ESI-MS) as a novel combined detection system for CE is presented. beta-Carbolines were chosen as model analytes with a forensic background. Nonaqueous CE as well as conventional CE with an aqueous buffer system are compared concerning efficiency and obtainable detection limits. The distance between the optical detection window and the sprayer tip was minimized by placing the optical cell directly in front of the electrospray interface. Similar separation efficiencies for both detection modes could thus be obtained. No significant peak-broadening induced by the MS interface was observed. The high fluorescence quantum yield and the high proton affinity of the model analytes investigated resulted in limits of detection in the fg (nmol/L) range for both detection methods. The analysis of confiscated ayahuasca samples and ethanolic plant extracts revealed complementary selectivities for LIF and MS detection. Thus, it is possible to improve peak identification of the solutes investigated by the use of these two detection principles.     

Laser-induced fluorescence detection at 266 nm in capillary electrophoresis. Polycyclic aromatic hydrocarbon metabolites in biota

The separation of five phenolic polycyclic aromatic hydrocarbon metabolites (hydroxy-PAHs) has been performed by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using a 30 mM borate buffer (pH 9.0) containing 60 mM sodium dodecyl sulfate and varying concentrations of gamma-cyclodextrin (gamma-CD). A concentration of 12.5 mM gamma-CD was found to provide a baseline separation of the five hydroxy-PAHs. We applied conventional fluorescence and laser-induced fluorescence (LIF) detection, using a new, small-size, quadrupled Nd-YAG laser emitting at 266 nm. The best limits of detection, in the low ng/ml range, were achieved using LIF detection. For all analytes, linearity was observed up to ca. 100 ng/ml. As an application, conjugated pyrene metabolites in hepatopancreas samples from the terrestrial isopods Oniscus asellus and Porcellio scaber were separated and detected. Finally, flatfish bile samples from individuals exposed to polluted sediment or crude oil, which were part of an interlaboratory study, were analyzed by CD-MEKC with conventional fluorescence and LIF detection to determine the 1-hydroxypyrene concentrations.     

In-capillary derivatization and analysis of amino acids,amino phosphonic acid-herbicides and biogenic amines by capillary electrophoresis with laser-induced fluorescence detection

This paper describes a general approach for the in-capillary derivatization of amino compounds and the subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) or capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection. Amino acids, biogenic amines and amino phosphonic acid-herbicides were chosen as model analytes to evaluate the analytical potential of this approach. Fulfilment of the in-capillary reaction of the analytes using LIF detection hinged on the excellent labeling chemistry of 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) and the good resolution achieved in the separation of derivatized analytes. Careful optimization of the electrophoretic conditions in the mixing step of this protocol allowed the determination of amino acids, biogenic amines and phosphorus-containing amino acid-herbicides with concentration limits of detection at the nug/L level and relative standard deviations from 3.5 to 5.8%. The whole analysis is carried out within 20 min, resulting in a very simple, fast and practical approach for the fully automated analysis of amino acids and related compounds in low-volume and low-concentration samples.     

Method development for the determination of coumarin compounds by capillary electrophoresis with indirect laser-induced fluorescence detection

A capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence detection (ILIFD) method is described for the simultaneous determination of esculin, esculetin, isofraxidin, genistein, naringin and sophoricoside. The baseline separation was achieved within 5 min with running buffer (pH 9.4) composed of 5mM borate, 20% methanol (v/v) as organic modifier, 10(-7)M fluorescein sodium as background fluorophore and 20 kV of applied voltage at 30 degrees C of cartridge temperature. Good linearity relationships (correlation coefficients >0.9900) between the second-order derivative peak-heights (RFU) and concentrations of the analytes (mol L(-1)) were obtained. The detection limits for all analytes in second-order derivative electrophoregrams were in the range of 3.8-15 microM. The RSD data of intra-day for migration times and second-order derivative peak-height were less than 0.95 and 5.02%, respectively. This developed method was applied to the analysis of the courmin compounds in herb plants with recoveries in the range of 94.7-102.1%. In this work, although the detection sensitivity was lower than that of direct LIF, yet the method would extend the application range of LIF detection.     

Liquid extraction surface analysis in-line coupled with capillary electrophoresis for direct analysis of a solid surface sample

A surface-sampling technique of liquid extraction surface analysis (LESA) was in-line coupled with capillary electrophoresis (CE) to expand the specimen types for CE to solid surfaces. The new direct surface analysis method of LESA–CE was applied to the determination of organophosphorus pesticides, including glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate on the external surface of a fruit such as apple. Without any sample pretreatment, the analytes sprayed on the surface of a half apple were directly extracted into a liquid microjunction formed by dispensing the extractant from the inlet tip of a separation capillary. After extraction, the analytes were derivatized in-capillary with a fluorophore 4-fluoro-7-nitro-2,1,3-benzoxadiazole and analyzed with CE-laser induced fluorescence (LIF). The limits of detection for glufosinate-ammonium, aminomethylphosphonic acid, and glyphosate were 2.5, 1, and 10 ppb, respectively, which are at least 20 times lower than the tolerance limits established by the U.S. Environmental Protection Agency. Thus, we demonstrated that LESA–CE is a quite sensitive and convenient method to determine analytes on a solid surface avoiding the dilution from sample pretreatment procedures including homogenization of a bulk sample.     

The Combination of Laser-Induced Fluorescence and Intensified Linear Diode Array Detection in Column Liquid Chromatography

Abstract

The frequency-doubled 514-nm argon-ion laser line providing excitation at 257 nm is combined with an intensified linear diode array (ILDA) detector to enable on-the-fly identification by laser-induced fluorescence (LIF) in conventional-size column liquid chromatography (LC). The potential of this detection system is demonstrated for the analysis of a standard mixture of polynuclear aromatic hydrocarbons (PAHs). Detection limits are at the 1.5 μg 1^?1 level (15 pg injected); the identitication limits are about one order of magnitude higher.     

A high‐sensitivity LIF detector with silver mirror coating detection window and small‐angle optical deflection from collinear system for CE

An LIF detector was integrated into a CE system based on silver mirror coating detection window and small‐angle optical deflection from collinear configuration. For this detection scheme, the incident light beam was focused on capillary through the edge of a lens, resulting in a small deflection angle that deviated 18° from the collinear configuration. Meanwhile, the excitation light and emitted fluorescence were effectively reflected by silver mirror coating at the detection window. The fluorescence was collected through the center of the same lens and delivered to a PMT in the vertical direction. In contrast to conventional collinear LIF detection systems, the fluorescence intensity was greatly enhanced and the background level was significantly eliminated. FITC and FITC‐labeled amino acids were used as model analytes to evaluate the performance with respect to design factors of this system. The limit LOD was estimated to be 0.5 pM for FITC (S/N = 3), which is comparable to that of optimized confocal LIF systems. All the results indicate that the proposed detection scheme will be promising for development of sensitive and low‐cost CE system.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

HPLC determination of γ‐aminobutyric acid and its analogs in human serum using precolumn fluorescence labeling with 4‐(carbazole‐9‐yl)‐benzyl chloroformate

In this study, a simple analytical method for the determination of γ‐aminobutyric acid, gabapentin, and baclofen by using high‐performance liquid chromatography with fluorescence detection was developed. An amidogen‐reactive fluorescence labeling reagent, 4‐(carbazole‐9‐yl)‐benzyl chloroformate was first used to sensitively label these analytes. The completed labeling of these analytes can be finished rapidly only within 5 min at the room temperature (25°C) to form 4‐(carbazole‐9‐yl)‐benzyl chloroformate labeled fluorescence derivatives. These labeled derivatives expressed strong fluorescence property with the maximum excitation and emission wavelengths of 280 and 380 nm, respectively. The labeled derivatives were analyzed using a reversed‐phase Eclipse SB‐C18 column within 10 min with satisfactory shapes. Excellent linearity (R² > 0.995) for all analytes was achieved with the limits of detection and the limits of quantitation in the range of 0.25?0.35 and 0.70?1.10 μg/L, respectively. The proposed method was used for the simultaneous determination of γ‐aminobutyric acid and its analogs in human serum with satisfactory recoveries in the range of 94.5–97.5%.     

Performance and selectivity of polymeric pseudostationary phases for the electrokinetic separation of amino acid derivatives and peptides

Two polymeric pseudostationary phases, one an acrylamide polymer and the second a siloxane polymer, have been investigated for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized amino acids and small peptides. The dervatized amino acids were detected by UV absorbance and laser-induced fluorescence (LIF) detection. The polymers provided very high efficiency and good selectivity for the separation of the amino acids. The separation selectivity using the polymers was significantly different from that of SDS micelles, and there were subtle differences in selectivities between the polymers. Although very good detection limits were obtained with LIF detection, a significant background signal was observed when the polymers were not washed to remove fluorescent impurities. The polymers did not separate the peptides very well. It is postulated that the fixed covalent structure of the polymers prevents them from interacting strongly or efficiently with the peptides, which are large in relation to the analytes typically separated by electrokinetic chromatography using polymers.     

Recent advances in affinity capillary electrophoresis

Use of the specificity of (bio)interactions can effectively overcome the selectivity limitation faced in capillary electrophoresis (CE), and the resulting technique usually is referred to as affinity capillary electrophoresis (ACE). Despite the high selectivity of ACE, several important problems still need to be addressed. A major issue in all CE separations, including ACE, is the concentration detection limit. Using UV detection, this is usually in the order of 10(-6) M whereas laser-induced fluorescence (LIF) detection can provide detection limits down to the sub-10(-10) M range. However, a marked disadvantage of LIF is that labeling of the analytes is usually required, which might change the interaction behavior of the solutes under investigation. Additionally, labeling reactions at sub-10(-10) M concentration levels are certainly not trivial and often difficult to perform quantitatively. Alternative and universal detection approaches, particularly mass spectrometric (MS) detection, look very promising but (A) CE-MS techniques are still far from routine application. Important future progress in sensitive detection strategies is likely to increase the use of ACE in the future.     

Rapid quantitative determination of ephedra alkaloids in tablet formulations and human urine by microchip electrophoresis

Microchip electrophoresis with fluorescence detection has been applied for fast separation and determination of ephedra alkaloids in pharmaceutical formulations and body fluids. A custom epifluorescence microscope setup was employed and the compounds were separated within 40?s, allowing the detection of less than 200?ng/L for both analytes. Quantitation of the two stimulants was performed via a derivatization step using FITC without any extraction or preconcentration steps. The effects of different microchip types and excitation light sources were investigated and the method was successfully applied for the analysis of these compounds in tablet formulations, yielding recovery rates from 100.2 to 101.1% and relative standard deviations from 1.5 to 3.4%. Analysis of ephedrines was also carried out with human urine samples at detection limits of 500-1000?ng/L and relative standard deviations from 2.2 to 3.3% using argon ion LIF detection.     

Analysis of organic pollutants in water at trace levels using fully automated solid-phase extraction coupled to high-performance liquid chromatography

Summary A method has been developed for the determination of trace levels of 32 pesticides, 19 explosives and 16 polycyclic aromatic hydrocarbons (PAH) in water in three individual steps. Solid-phase enrichment (SPE) is coupled to high-performance liquid chromatography (HPLC) with a fully automated system. The organic pollutants are enriched on reusable cartridges packed with adsorbent materials: pesticides and explosives on a mixed bed of divinylbenzene-ethylvinylbenzene copolymers (LiChrolut EN^?) and perfluorinated polyethylene (PolyF^?), and polycyclic aromatic hydrocarbons on C₁₈-modified silica (Zorbax^? ODS1). Thermally assisted desorption (TAD) has been shown to increase the recovery of analytes significantly. As all enriched analytes are transferred to the detector, only fifty millilitres of sample is needed for each single on-line analysis, compared with at least a litre for conventional methods. The separation of the enriched organic analytes is performed on specialized HPLC columns based on reversed-phase materials. The limits of detection of the system employed were found to be below 100 ng L⁻¹. Use of fluorescence detection for the polycyclic aromatic hydrocarbons resulted in limits of detection in the upper pg L⁻¹ range. Thek values, number of theoretical plates, the recovery rates and the limits of detection of this method for fast screening of organic pollutants from three fifty-millilitre aqueous samples are described. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Quantification of glutathione and glutathione disulfide in human plasma and tobacco leaves by capillary electrophoresis with laser-induced fluorescence detection

A new capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection was developed for the rapid separation and sensitive detection of glutathione (GSH) and glutathione disulfide (GSSH) after derivatization by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl). The derivatization and separation conditions were investigated in detail and the optimums were obtained. Under the optimum experiment conditions, linear relationships between the peak height and concentrations of the analytes in normal and second-derivative electrophoregrams were obtained (0.22-45.00 μM). The detection limits for glutathione and glutathione disulfide in normal and second-derivative electrophoregrams were 0.046 and 0.012 μM and 0.046 and 0.014 μM, respectively. The method was applied to the analysis of glutathione and glutathione disulfide in human plasma and tobacco leaves with satisfactory results.     

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection

A method for the residue analysis of the veterinary antimicrobial agent enrofloxacin and its active desethyl metabolite ciprofloxacin in chicken muscle tissue has been developed and validated. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd laser (lambda(ex) = 325 nm) providing an enhancement in sensitivity and selectivity compared to conventional UV detection. The assay has been validated with satisfying results. The limits of quantification for enrofloxacin and ciprofloxacin were 5 microg/kg and 20 microg/kg, respectively, with a fivefold preconcentration yielded by a sample clean-up with a simple liquid-liquid extraction procedure. Calibration graphs were linear from 5 to 1000 microg/kg for enrofloxacin and from 20 to 1000 microg/kg for ciprofloxacin. The assay allows the detection of contaminated muscle samples at the required maximum residue limit of the European Union, which is 100 microg/kg for the sum of enrofloxacin and ciprofloxacin.     

LC Determination of Nosiheptide in Swine Kidney and Liver

A liquid chromatographic method was developed for determination of nosiheptide in swine kidney and liver. The tissue samples were extracted with acetonitrile and defatted with hexane. The analytes were determined at a fluorescence excitation/emission wavelength of 357/515 nm and with gradient elution program. The limits of detection and limits of quantification were 5 and 20 ng g^?1, respectively, for swine kidney and liver. The mean recoveries for nosiheptide in swine kidney and liver ranged from 70.3 to 97.4% with coefficients of variation below 11.3% at 20–100 ng g^?1 fortification levels.     

Rapid analysis of anthracycline antibiotics doxorubicin and daunorubicin by microchip capillary electrophoresis

A simple and rapid method has been developed for the analysis of anthracycline antibiotics doxorubicin (DOX) and daunorubicin (DAU) in human serum using mirochip-based capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. In this study, method development included studies of the effect of buffer pH, buffer concentration, organic solvents and separation voltage on sensitivity and separation efficiencies for the CE separation of DOX and DAU. Acetonitrile was found to have significantly improved the sensitivity and separation efficiency. The method was validated with regard to reproducibilities, linearity and limit of detection (LOD). The optimum electrophoretic separation conditions were 10 mM sodium tetraborate buffer at pH 9.5 with 40% acetonitrile (V/V) and a separation voltage of 2.1 kV. DOX and DAU were separated in 60 s under the optimum separation conditions. Linear relationships were obtained between the concentration and peak area (or peak height) in the 1–75 µg mL^{− 1} range and with the detection limits of 0.3 and 0.2 μg mL^{− 1} for DOX and DAU, respectively. The stability of both migration time and peak height of the analytes showed relative standard deviations of less than 5% (n = 9). The potential of this method was verified by spiking a human serum sample with the two drugs and analyzing the recovery ratios.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

Polymer-Enhanced Capillary Transient Isotachophoresis with Boronic Acid-Functionalized Squarylium Dyes for the Fluorescent Determination of Digoxin and Digoxigenin

Here is reported a new application of polymer-enhanced capillary transient isotachophoresis for the separation and quantification of the drug digoxin and its primary metabolite digoxigenin coupled with laser-induced fluorescence (LIF) detection facilitated by labeling with two boronic acid-functionalized squarylium dyes of different alkyl side chain lengths, SQ-BA1 and SQ-BA2. The conditions for drug–dye complex formation were optimized, as determined by absorbance and fluorescence spectra, according to solution pH and buffer composition. As digoxin has a digitoxose sugar moiety in its structure, it was shown to exhibit better enhancement in the fluorescence intensity of both dyes than digoxigenin, which lacks this moiety, presumably through the formation of a cyclic boronate ester complex. A comparison of analyte labeling in pre-column and on-column modes was conducted in subsequent capillary electrophoresis-LIF studies, with the latter labeling mode yielding superior sensitivity. However, to achieve the complete resolution of labeled digoxin and digoxigenin analytes, it was necessary to use the modified isotachophoresis method, with added borate ions that may differentially interact with the drug and its metabolite, hence affecting their mobilities. Limits of quantification of the method for the determination of digoxin with SQ-BA1 and SQ-BA2 were 2.61?×?10^?3 and 2.82?×?10^?3?M and limits of detection were 7.83?×?10^?4 and 8.47?×?10^?4?M while sensitivities were as great as 5.06?×?10⁹ and 2.89?×?10⁹?M^?1, respectively, indicating that the method is suitable for practical analysis.     

Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids

Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids.