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.     

Dialkyl phosphates determination by gas chromatography: Evaluation of a microwave‐assisted derivatization†

Dialkyl phosphates are organophosphate insecticide metabolites and their urinary analysis is useful for assessing human exposure to these compounds. This study presents a sample preparation method with microwave‐assisted derivatization for two dialkyl phosphates to make the process faster before gas chromatographic analysis. The optimized conditions for derivatization procedure were: 250 μL of 2,3,4,5,6‐pentafluorobenzyl bromide 3% in acetonitrile for derivative; microwave for 5 min with intensity of 160 W. The electron ionization mass spectrometric analysis was performed using a gas chromatography with mass spectrometry QP‐2010 from the Shimadzu^® equipped with RTx^®‐5MS capillary column. Ions were monitored at selected‐ion monitoring mode at m/z 350 for diethyl thiophosphate and m/z 366 for diethyl dithiophosphate. The developed method was linear for both metabolites. The intra‐assay precision was the values ranged between 1.1 and 9.1%, for diethyl thiophosphate, and between 4.06 and 6.9%, for diethyl dithiophosphate. The interassay precision showed relative standard deviation between 10.3 and 15.1%, for diethyl thiophosphate and between 4.9 and 11.9%, for diethyl dithiophosphate. The results obtained suggests that derivatization assisted by microwave, before gas chromatography with mass spectrometry analysis, can be applied to monitoring of exposure to organophosphates once is fast, sensible, and precise method to determinate dialkyl phosphates.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.     

Microalgae amino acid extraction and analysis at nanomolar level using electroporation and capillary electrophoresis with laser‐induced fluorescence detection

Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser‐induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave‐assisted derivatization at 80°C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and β‐cyclodextrin (10 mM), and the limits of quantification were 3–30 nM. The developed capillary electrophoresis with laser‐induced fluorescence method was used to analyze amino acids in Dunaliella salina green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home‐made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated‐solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in Dunaliella salina cells.     

Enantioselective analysis of bambuterol in human plasma using microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry

In this study, an enantioselective analytical method based on microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry was developed for the determination of bambuterol enantiomers in human plasma. The chiral derivatization reaction was greatly accelerated by microwave irradiation. Under the optimized conditions, both the derivatization time and separation time on column was only 3 min, and the lower limit of quantification was 2.5 pg/mL. The recoveries were in the range of 90.1–93.0% without significant matrix effect. Compared with the conventional heating chiral derivatization, microwave‐assisted chiral derivatization obtained higher chiral derivatization yields with much shorter time due to the effect of microwave irradiation. Furthermore, the racemization during the derivatization reaction was systematically investigated. The results showed the concentration of acetic acid and the reaction time had significant effects on the racemization, which could be well controlled during microwave‐assisted chiral derivatization for the short reaction time. Finally, this novel approach was demonstrated by determining bambuterol in human plasma of a clinical pharmacokinetic study in eight healthy volunteers. On the basis of the results, microwave‐assisted chiral derivatization coupled with ultra high performance liquid chromatography and tandem mass spectrometry as a simple and effective enantioselective analysis technique for the determination of chiral drugs in complex biological samples showed great promise.     

Analysis of low‐molecular mass aldehydes in drinking waters through capillary electrophoresis with laser‐induced fluorescence detection

The potential of CZE with LIF detection in the separation and determination of low‐molecular mass aldehydes involving precolumn derivatization with fluorescein 5‐thiosemicarbazide was investigated. Different variables that affect derivatization (pH, fluorescein 5‐thiosemicarbazide concentration, time and temperature) and separation (pH and concentration of the BGE, kind and concentration of surfactants at levels higher and lower than CMC, and applied voltage) were studied. The separation was conducted within 16 min by using borate buffer (60 mM; pH 10) with 10 μM polyethylene glycol tert‐octylphenyl ether as modifier. Good linearity relationships (correlation coefficients ranged from 0.9978 to 0.9994 for aldehydes) were obtained between the peak areas and concentration of the analytes (0.5–100 μg/L). The LODs for aldehydes were achieved at submicrogram‐per‐liter level (0.15–0.35 μg/L), which indicated that the proposed method surpassed other electrophoretric alternatives in terms of LOD, in many cases even at ca. 1000‐fold. The inter‐day precision (RSD, %) of the aldehydes ranged from 5.2 to 8.3%. Finally, the method was successfully applied to bottled drinking‐water samples, and the aldehydes were readily detected at 0.6–4.4 μg/L levels with average recoveries ranging from 99.1 to 103.5%.     

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

The free solution electrophoretic behavior of DNA‐protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA‐protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA‐methyl‐CpG‐binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA‐protein complexes. This method is based on the effective separation of free DNA and DNA‐protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide‐coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA‐MBD2b complexes, we found that the values of (μ₀/μ)‐1 were directly proportional to the charge‐to‐mass ratios of formed complexes, where the μ₀ and μ are the mobility of free DNA probe and DNA‐protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge‐to‐mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA‐binding properties of the purified MBD2b protein and help to analyze other DNA‐protein complexes. Additionally, this study enhances the understanding of the influence of the charge‐to‐mass ratios of formed DNA‐protein complexes on their separation and electrophoretic behaviors.     

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.     

毛细管电泳-激光诱导荧光法测定草甘膦、草胺膦及氨甲基膦酸

建立了一种快速、有效的毛细管电泳分离-激光诱导荧光检测有机磷除草剂草甘膦、草胺膦和草甘膦的代谢物氨甲基膦酸的方法。将荧光衍生试剂5-(4, 6-二氯三嗪基)氨基荧光素(DTAF)成功用于衍生上述3种化合物。最佳衍生条件: DTAF的浓度为1.0 μmol/L,以50 mmol/L硼酸(pH 9.5)作为缓冲溶液,在30 ℃下反应40 min。以pH 9.5的30 mmol/L硼酸缓冲溶液(含15 mmol/L Brij-35)作为电泳背景电解质,3种衍生物得到基线分离。在优化的条件下,草甘膦、草胺膦、氨甲基膦酸的检出限分别为3.21、6.14和1.99 ng/kg。将该方法应用于环境水样和土壤中除草剂及代谢物的测定,回收率为91.3%～106.0%。该方法准确、灵敏,可满足环境样品中有机磷农药及其代谢物残留的检测要求。     

Analysis of red inks by micellar electrokinetic capillary chromatography with laser‐induced fluorescence detection

A combination of MEKC with a—highly sensitive but not commonly used—LIF detector was tested regarding the possibility of differentiation of red inks. The separation process was conducted in a fused silica capillary (id 50 μm, 60/50 cm total/effective length) in BGE consisting of 40 mM sodium borate, 20 mM SDS and 10% v/v ACN with 30 kV applied. The optimized temperature of storage and capillary was 10 and 25°C, respectively. The samples were prepared using 20 dots (ø 0.5 mm), extracted in 35 μL BGE and diluted with 30 μL of water. The proposed method showed excellent repeatability and reproducibility (RSD (t_m) < 0.8 and < 2.5%, respectively). It was applied to group identification and differentiation of different brands, models, and batches of red printing, stamp, and pen inks. It was demonstrated that differentiation can be performed effectively on the basis of migration times and ratios of peak areas. The high efficiency of the developed method was indicated by discriminating power ranging from 87.3 to 98.8%, for stamp and pen inks, respectively. The results showed that the proposed procedure can be valuable for an objective examination of the red parts of questioned documents.     

Novel solvent‐free microwave‐assisted extraction coupled with low‐density solvent‐based in‐tube ultrasound‐assisted emulsification microextraction for the fast analysis of organophosphorus pesticides in soils

A novel and rapid solventless microwave‐assisted extraction coupled with low‐density solvent‐based in‐tube ultrasound‐assisted emulsification microextraction has been developed for the efficient determination of nine organophosphorus pesticides in soils by GC analysis with microelectron capture detection. A specially designed, homemade glass tube inbuilt with a scaled capillary tube was used as an extraction device to collect and measure the separated extractant phase easily. Parameters affecting the efficiencies of the developed method were thoroughly investigated. From experimental results, the following conditions were selected for the extraction of organophosphorus pesticides from 1.0 g of soil sample to 5 mL of aqueous solution under 226 W of microwave irradiation for 2.5 min followed by ultrasound‐assisted emulsification microextraction with 20 μL toluene for 30 s and then centrifugation at 3200 rpm for 3 min. Detections were linear in the range of 0.25–10 ng/g with detection limits between 0.04 and 0.13 ng/g for all target analytes. The applicability of the method to real samples was assessed on agricultural contaminated soils and the recoveries ranged between 91.4 and 101.3%. Compared to other methods, the present method was shown to be highly competitive in terms of sensitivity, cost, eco‐friendly nature, and analysis speed.     

Determination of main taxoids in Taxus species by microwave‐assisted extraction combined with LC‐MS/MS analysis

A method based on microwave‐assisted extraction (MAE) has been developed for the determination of paclitaxel and five related taxoids, namely 10‐deacetylbaccatin III (10‐DAB III), cephalomannine, 10‐deacetylpaclitaxel (10‐DAT), 7‐xyl‐10‐ deacetylpaclitaxel (7‐xyl‐10‐DAT), and 7‐epi‐10‐deacetylpaclitaxel (7‐epi‐10‐DAT) in Taxus species in this study. The influential parameters of the MAE procedure were optimized, and the optimal conditions were as follows: extraction solvent 80% ethanol solution, solid/liquid ratio 1:10 (g/mL), temperature 50°C, and three extraction cycles, each cycle 10 min. The method validation for LC‐MS/MS analysis was performed. The LOD and LOQ were 3.16–9.20 and 12.20–30.45 ng/mL, respectively. Repeatability and reproducibility for the six taxiods with RSD ranged from 2.78 to 3.85% and from 5.26 to 6.60%. The recoveries of the method for the six taxoids were 92.6–105.6%. The developed MAE‐LC‐MS/MS method was also successfully applied to determine the contents of six taxoids in different Taxus species.     

Capillary electrophoresis coupled with in‐column fiber‐optic laser‐induced fluorescence detection for the rapid separation of neodymium

In this study, in‐column fiber‐optic (ICFO) laser‐induced fluorescence (LIF) detection technique is coupled with capillary electrophoresis (CE) for the rapid separation of neodymium for the first time. The effects of buffer concentration, buffer pH, and separation voltage on the CE behaviors, including electrophoretic efficiency and detection sensitivity, are investigated in detail. Under the optimal condition determined in this study (15 mM borate buffer, pH 10.50, separation voltage 24 kV), neodymium could be separated effectively from the neighboring lanthanides (praseodymium and samarium) within several minutes, and the limit of detection for neodymium is estimated to be at the ppt level. The ICFO‐LIF‐CE system assembled in this study exhibits unique performance characteristics such as low cost and flexibility. Meanwhile, the separation efficiency and detection sensitivity of the assembled CE system are comparable to or somewhat better than those obtained in the previous traditional CE systems, indicating the potential of the assembled CE system for practical applications in the fields of spent nuclear fuel analysis, nuclear waste disposal/treatment, and nuclear forensics.     

Study on the PEG‐based microwave‐assisted extraction of flavonoid compounds from persimmon leaves

A method for PEG‐based microwave‐assisted extraction (MAE) of flavonoid compounds from persimmon leaves has been successfully developed. The extraction efficiency of total flavonoid content was evaluated by the chromatographic peak areas of quercetin and kaempferol, which are two bioactive components typically found in persimmon leaves. The best combination of extraction parameters was obtained with response surface methodology. A microwave power of 525 W, liquid to solid ratio of 17:1 mL/g, and PEG aqueous solution concentration of 60% w/w were identified as the optimum parameters. Extraction dynamics analysis indicated that the quercetin, kaempferol, and total flavonoid contents were rising with increasing extraction time up to 20–25 min, from which point onwards they all decreased. Under the optimum conditions, quercetin, kaempferol, and total flavonoid contents obtained from the sample were 1.20 ± 0.05, 0.64 ± 0.11, and 16.90 ± 0.06 mg/g, respectively. Compared with ethanol‐based MAE, and ethanol‐based and PEG‐based ultrasonic‐assisted extractions, PEG‐based MAE had higher efficiency for the extraction of flavonoid compounds from persimmon leaves. Overall, PEG‐based MAE represents an efficient choice for the extraction of bioactive substances from traditional Chinese medicines.     

Electrochemical derivatization‐capillary electrophoresis‐contactless conductivity detection: A versatile strategy for simultaneous determination of cationic,anionic, and neutral analytes

The simultaneous determination of cationic, anionic, and neutral analytes in a real sample was demonstrated by coupling electrochemical (EC) derivatization with counter‐EOF CE‐C⁴D. An EC flow cell was used to oxidize alcohols from an antiseptic mouthwash sample into carboxylic acids at a platinum electrode in acid medium. The carboxylates formed in the derivatization process and other sample ingredients, such as benzoate, saccharinate, and sodium ions, were separated in counter‐flow mode and detected in one run in Tris‐HCl buffer, pH 8.6. Fewer than 5 min were needed to complete each analysis with the automated flow system comprising solenoid pumps for the management of solutions. Insights into the electrochemistry of benzoic acid, present in the sample matrix, were also gained by EC‐CE‐C⁴D; more specifically, by applying potentials higher than 1.47 V to the platinum electrode, some formiate and minute amounts of salicylate were detected.     

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser‐induced fluorescence detection

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK_a determination of small molecule analytes is demonstrated with the multilane microchip.     

Analysis of cerebrospinal fluid γ‐aminobutyric acid by capillary electrophoresis with laser‐induced fluorescence detection

The measurement of γ‐aminobutyric acid (GABA) is suitable for investigating various neurological disorders. In this study, a sensitive and selective method for free GABA quantification in cerebrospinal fluid (CSF) has been standardised. This method is based on CE with LIF detection using 4‐fluoro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐F) as a derivatisating agent. The reaction conditions (NBD‐F concentration, pH, temperature and reaction time) and the electrophoretic parameters (run buffer composition and pH and separation voltage) were optimised to obtain the maximum derivatisation efficiency and electrophoretic resolution. The best resolution was obtained using 200 mM sodium borate, 10 mM SDS, 8.5 mM β‐CD, pH 10 and 20 kV voltage. The method was linear in the concentration range of 2.5–1000 nM with good inter‐ and intra‐assay precision values. The effects of CSF handling on free GABA concentrations were also evaluated. Our results show that the time delay between CSF collection and freezing strongly increases the CSF GABA values. Age‐related reference values were established in 55 paediatric controls. The influence of antiepileptic therapy on free CSF GABA was studied in 38 neuropaediatric patients. Significantly, higher GABA values were obtained in patients taking valproic acid or vigabatrin therapy, which are antiepileptic drugs that modulate GABA metabolism.     

Determination of plant growth regulators in pears by microwave‐assisted extraction and liquid chromatography with electrospray ionization mass spectrometry

A new method for the determination of six plant growth regulators, 3‐indolylacetic acid, 3‐indolepropionic acid, 2‐naphthoxyacetic acid, 2,4‐dicholrophenoxyacetic acid, 1‐naphthlcetic acid, and methyl naphthalene‐1‐acetate, in pears was established by liquid chromatography with electrospray ionization mass spectrometry. In this study, a microwave‐assisted extraction technique was first applied for the determination of plant growth regulators in fruit and three cleanup techniques were, respectively, investigated for the purification of pear samples. The chromatographic separation was performed on a Diamonsil C₁₈ column by using 0.01 mol/L formic acid/ammonium formate buffer solution (pH 3.5)/methanol (35:65, v/v) as the mobile phase with a flow rate of 0.7 mL/min in 1:1 split mode. The LODs ranged from 0.3 to 1.9 μg/kg. Under optimized conditions, the average recoveries (five replicates) for six plant growth regulators (spiked at 0.01, 0.05, and 0.5 mg/kg) ranged from 78.9 to 118.0%, and the RSDs were 1.4–10.3%.     

Separation and purification of steroidal saponins from Paris polyphylla by microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection

A method of microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection was successfully developed for the separation and purification of steroidal saponins from Paris polyphylla. The main extraction conditions including microwave power, liquid/solid ratio, irradiation time, and extraction temperature were optimized using an orthogonal array design method. A suitable two‐phase solvent system consisting of n‐heptane/n‐butanol/acetonitrile/water (10:19:6:20, v/v/v/v) was employed in the separation and purification of the extracts of P. polyphylla. A total of 7.1 mg polyphyllin VII, 4.3 mg gracillin, 9.2 mg dioscin, and 10.2 mg polyphyllin I were obtained from 1.5 g P. polyphylla in less than 300 min, the purities of which determined by HPLC were 96.7, 97.3, 98.7, and 98.6%, respectively. The identification and characterization of these compounds were performed by LC–ESI‐MS and ¹H NMR spectroscopy. The results demonstrated that the proposed method is feasible, economical and efficient for the extraction, separation and purification of effective compounds from natural products.     

Analysis of volatile oil composition of Citrus aurantium L. by microwave‐assisted extraction coupled to headspace solid‐phase microextraction with nanoporous based fibers

Nanoporous silica was prepared and functionalized with amino propyl‐triethoxysilane to be used as a highly porous fiber‐coating material for solid‐phase microextraction (SPME). The prepared nanomaterials were immobilized onto a stainless steel wire for fabrication of the SPME fiber. The proposed fiber was evaluated for the extraction of volatile component of Citrus aurantium L. leaves. A homemade microwave‐assisted extraction followed by headspace (HS) solid‐phase apparatus was used for the extraction of volatile components. For optimization of factors affecting the extraction efficiency of the volatile compounds, a simplex optimization method was used. The repeatability for one fiber (n = 4), expressed as RSD, was between 3.1 and 8.6% and the reproducibility for five prepared fibers was between 10.1 and 14.9% for the test compounds. Using microwave‐assisted distillation HS‐SPME followed by GC‐MS, 53 compounds were separated and identified in C. aurantium L., which mainly included limonene (62.0%), linalool (7.47%), trans‐β‐Ocimene (3.47%), and caryophyllene (2.05%). In comparison to a hydrodistillation method, the proposed technique could equally monitor almost all the components of the sample, in an easier way, which was rapid and required a much lower amount of sample.