Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near‐infrared laser‐induced fluorescence detection using a new BODIPY‐based probe as labeling reagent

A CZE with near‐infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ‐glutamylcysteine, cysteinylglycine, and N‐acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(4'‐iodoacetamido)difluoroboradiaza‐s‐indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N‐acetylcysteine to 0.31 nmol/L for γ‐glutamylcysteine, which are better than or comparable to those reported with other derivatization‐based CE‐LIF methods. As the first trial of NIR CE‐LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5–104.3%.     

Design, characterization, and utilization of a fast fluorescence derivatization reaction utilizing o-phthaldialdehyde coupled with fluorescent thiols

We have developed a chemical derivatization scheme for primary amines that couples the fast kinetic properties of o-phthaldialdehyde (OPA) with the photophysical properties of visible, high quantum yield, fluorescent dyes. In this reaction, OPA is used as a cross-linking reagent in the labeling reaction of primary amines in the presence of a fluorescent thiol, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA fluorescein), thereby incorporating fluorescein (epsilon = 78 000 M(-1), quantum yield of 0.98) into the isoindole product. Detection is based on excitation and emission of the incorporated fluorescein using the 488 nm laser line of an Ar(+) laser rather than the UV-excited isoindole, thereby eliminating the UV light sources for detection. Using this method, we have quantitatively labeled biologically important primary amines in less than 10 s. Detection limits for analysis of glutamate, glycine, GABA, and taurine were less than 2 nM. We present the characterization of OPA/SAMSA-F reaction and the potential utility of the derivatization reaction for dynamic chemical monitoring of biologically relevant analytes using CE.     

Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells

This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N‐acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7‐tetramethyl‐8‐phenyl‐(2‐maleimide)‐difluoroboradiaza‐s‐indacene (TMPAB‐o‐M), as the labeling reagent. TMPAB‐o‐M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2–3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE‐based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

Capillary electrophoresis‐laser‐induced fluorescence detection of rat brain catecholamines with microwave‐assisted derivatization

In this study, a rapid and sensitive method is described for the catecholamines detection in rat brain. CE with LIF detection for the determination of FITC derivatized catecholamines (dopamine, epinephrine, and norepinephrine) was demonstrated. Conventional water bath and microwave‐assisted derivatization methods were employed and a significant reduction in the derivatization time from 2 h for the conventional water bath at room temperature (ca. 25°C) to 2 min for the microwave‐assisted derivatization was achieved. Online sample concentration of field‐amplified sample stacking (FASS) method was employed to achieve higher sensitivities (the detection limits obtained in the normal injection mode ranged from 2.6 to 4.5 ng L^?1 and in the FASS mode ranged from 22 to 34 pg L^?1). Furthermore, this microwave‐assisted derivatization CE–LIF method successfully determined catecholamines in rat brain with as low as 100 ng L^?1 (FASS mode) to 10 μg L^?1 (normal injection mode). This CE–LIF method provided better detection ability when compared to the best reports on catecholamines analyses.     

Boron‐chelating fluorescent probe (BOPB) in the red region combined with CE‐LIF for the detection of NO in mice liver

Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8‐(3’,4’‐Diamino phenyl)‐3,5‐(2‐hydroxyphenyl)‐dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE‐LIF detection. Therefore, BOPB was used in CE‐LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB‐T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H₃BO₃–NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10^?9–5.0 × 10^?7 M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization‐based CE‐LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.     

Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser‐induced fluorescence detection

The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2‐(diethylamino)ethanol and triethanolamine. A buffer solution containing 2‐(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene‐2,3‐dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4–28) of proteins, compared with conventional absorbance detection.     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Analysis of alpha-1-acid glycoprotein isoforms using CE-LIF with fluorescent thiol derivatization

The analysis of glycoprotein isoforms is of high interest in the biomedical field and clinical chemistry. Many studies have demonstrated that some glycoprotein isoforms could serve as biomarkers for several major diseases, such as cancers and vascular diseases, among others. Capillary zone electrophoresis (CZE) is a well-established technique to separate glycoprotein isoforms, however, it suffers from limited sensitivity when UV-Vis detection is used. On the other hand, with laser-induced fluorescence (LIF) detection, derivatization reaction to render the proteins fluorescent can destroy the resolution of the isoforms. In this work, a derivatization procedure through the thiol groups of glycoproteins using either 5-(iodoacetamide) fluorescein (5-IAF) or BODIPY iodoacetamide is presented with the model protein of alpha-1-acid glycoprotein (AGP). The derivatization process presented enabled high-resolution analysis of AGP isoforms by CZE-LIF. The derivatization procedure was successfully applied to label AGP from samples of serum and secretome of artery tissue, enabling the separation of the AGP isoforms by CE-LIF in natural samples at different concentration levels.     

Characterization of multi-cationic aminopyrene-based tag for oligosaccharide labeling by capillary electrophoresis with laser-induced fluorescence detection

In this work, we characterize a previously synthesized multi-cationic aminopyrene-based labeling tag for oligosaccharide analysis by capillary electrophoresis with laser-induced fluorescence detection (CE/LIF). The fluorescent tag, 4,4',4''-(8-aminopyrene-1,3,6-trisulfonyl)tris(1-methylpiperazine) (APTMP), was characterized by reaction with standard maltooligosaccharides and the labeling parameters such as fluorescent tag concentration, labeling temperature, and time as well as influence of a reducing agent and its solvent were investigated in terms of labeling efficiency. The nanomolar limit of detection of CE/LIF analysis of APTMP labeled maltopentaose was determined. However, significant amount of the oligosaccharides was reduced to alditols, which negatively affects the yield and rate of the labeling reaction. Under optimized conditions, a highly reproducible labeling by multi-cationic APTMP was obtained; however, the most commonly used labeling by multi-anionic 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) is superior compared to APTMP labeling. Lower reactivity of APTMP compared to APTS can be explained by the loss of nucleophilicity induced by substitution of the sulfonate groups with more electron-withdrawing aminosulfonyl ones. On contrary, APTMP is still a promising tag for oligosaccharide labeling followed by CE-MS in a positive ion mode, which is considered to be more sensitive than MS detection of APTS in a negative ion mode.     

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.     

Comparison of oligosaccharide labeling employing reductive amination and hydrazone formation chemistries

In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.     

Microemulsion electrokinetic chromatography: an application for the simultaneous determination of suspected fragrance allergens in rinse-off products

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.     

Laser‐Induced Fluorometry for Capillary Electrophoresis

Laser‐induced fluorometry (LIF) has achieved the detection of single molecules, which ranks it among the most sensitive of detection techniques, whereas capillary electrophoresis (CE) is known as a powerful separation method with resolution that is beyond the reach of many other types of chromatography. Therefore, a coupling of LIF with CE has established an unrivaled analytical technique in terms of sensitivity and resolution. CE‐LIF has demonstrated excellent performance in bioanalytical chemistry for the high‐resolution separation and highly sensitive detection of DNAs, proteins, and small bioactive molecules. This review describes the CE‐LIF methods developed by the author's group that include indirect and direct detection using diode lasers, post‐column derivatization, and Hadamard transformation, as well as applications to the binding assays of specific DNA immunoassays of proteins and to the determination of anticancer drugs.     

Improved sample preparation method for glycan analysis of glycoproteins by CE‐LIF and CE‐MS

CE is a high‐resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ≥100 fold excess of fluorophore reagent, 8‐aminopyrene‐1,3,6‐trisulfonic‐acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:≥100 ratio) maintained the >95% derivatization yield at 55°C with only 50 min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8‐aminopyrene‐1,3,6‐trisulfonic‐acid simplified the removal of excess reagent, important in both CE‐LIF (electrokinetic injection bias) and CE‐MS (ion suppression). Coupling CE‐ ESI‐MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values.     

Nonaqueous capillary electrophoresis coupled with laser-induced native fluorescence detection for the analysis of berberine, palmatine, and jatrorrhizine in Chinese herbal medicines

LIF detection is one of the most sensitive detection methods for CE. However, its application is limited because the analyte is usually required to be derivatized with a fluorescent label. As a result, LIF is seldom used to analyze active ingredients in plants. In this work, we introduce a rapid, simple, and sensitive method of nonaqueous CE (NACE) coupled with laser-induced native fluorescence detection for the simultaneous analysis of berberine, palmatine, and jatrorrhizine. This method skillfully utilizes the native fluorescence of these alkaloids and requires no troublesome fluorescent derivatization. As these alkaloids can fluoresce to some degree, they were simply detected by a commercially available 488 nm Ar+ laser. The native fluorescence of the analytes was greatly enhanced by nonaqueous media. Compared with the reported UV detection method, much lower LOD was achieved (6.0 ng/mL for berberine, 7.5 ng/mL for palmatine, and 380 ng/mL for jatrorrhizine). This method was successfully applied to analyze berberine, palmatine, and jatrorrhizine in two Chinese herbal medicines, Rhizoma coptidis and Caulis mahoniae.     

Novel monofunctional and bifunctional boronic acid‐functionalized squarylium dyes as precolumn and on‐column labels for protein analysis by capillary electrophoresis with laser‐induced fluorescence

This article presents a continuous capillary electrophoresis with laser‐induced fluorescence (CE‐LIF) following spectral studies of the noncovalent interactions between novel Squarylium Boronic Acid 4 (SQ‐BA4) & Squarylium Diboronic Acid 2 (SQ‐DBA2) squarylium dyes and human serum albumin (HSA). Two protocols were used wherein the on‐column‐labeling protocol was found to be more sensitive than the precolumn one by showing a better enhancement in the peak area of the HSA–dye complex besides lower limits of detection (LODs) for HSA. Also, stability studies were conducted with or without HSA using precolumn‐labeling mode over one week exhibiting the superiority of SQ‐BA4 to SQ‐DBA2. Then, a mixture containing three model proteins, HSA, β‐lactoglobulin B, and transferrin, was labeled on‐column with both dyes and completely resolved by CE‐LIF after optimization of several parameters. Both dyes provided lower LODs for HSA than those of β‐lactoglobulin B and transferrin with higher sensitivities. In addition, the SQ‐BA4 dye showed again greater sensitivities with all the three proteins than SQ‐DBA2.     

Simultaneous LIF and retro-reflected beam interference detection for CE

This Short Communication describes a novel optical detection method for CE, based on the combination of LIF detection and retro-reflected beam interference detection. By the use of a side-illuminated laser beam, an on-column multifunctional detection for CE has been developed, and some key elements in the scheme have been optimized. In addition, two miscellaneous samples including fluorescent dyes, carbohydrates and amino acids have been determined to evaluate its performance. Without any additional pretreatment, sufficient LOD and linear ranges have been achieved for most analytes. Its performance in retro-reflected beam interference detection is better than those mentioned in a former report, and its fluorescent sensitivity is comparable with the achievable sensitivity by conventional LIF systems, all of which together combine high sensitivity and universal analysis to a certain extent.     

Optimized sample preparation strategy for the analysis of low molecular mass adducts of a fluorescent cisplatin analogue in cancer cell lines by CE‐dual‐LIF

Pt‐based anticancer drugs, such as cisplatin, are known to undergo several (bio‐)chemical transformation steps after administration. Hydrolysis and adduct formation with small nucleophiles and larger proteins are their most relevant reactions on the way to the final reaction site (DNA), but there are still many open questions regarding the identity and pharmacological relevance of various proposed adducts and intermediates. Furthermore, the role of buffer components or additives, which are inevitably added to samples during any type of analytical measurement, has been frequently neglected in previous studies. Here, we report on adduct formation reactions of the fluorescent cisplatin analogue carboxyfluorescein diacetate platinum (CFDA‐Pt) in commonly used buffers and cell culture medium. Our results indicate that chelation reactions with noninnocent buffers (e.g., Tris) and components of the cell culture/cell lysis medium must be taken into account when interpreting results. Adduct formation kinetics was followed up to 60 h at nanomolar concentrations of CFDA‐Pt by using CE‐LIF. CE‐MS enabled the online identification of such unexpected adducts down to the nanomolar concentration range. By using an optimized sample preparation strategy, unwanted adducts can be avoided and several fluorescent adducts of CFDA‐Pt are detectable in sensitive and cisplatin‐resistant cancer cell lines. By processing samples rapidly after incubation, we could even identify the initial, but transient, Pt species in the cells as deacetylated CFDA‐Pt with unaltered complexing environment at Pt. Overall, the proposed procedure enables a very sensitive and accurate analysis of low molecular mass Pt species in cancer cells, involving a fast CE‐LIF detection within 5 min.