On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

Simultaneous determination of p-hydroxybenzoic acid and parabens by capillary electrophoresis with improved sensitivity in nonaqueous media

New methods based on nonaqueous capillary electrophoresis (NACE) were developed as promising alternatives for the simultaneous separation and determination of p-hydroxybenzoic acid (PHBA) and a group of parabens (methyl, ethyl, propyl, butyl and benzyl p-hydroxybenzoates), with good resolution and excellent sensitivity. As an effective on-line preconcentration technique, large-volume sample stacking (LVSS) was successfully combined with NACE allowing significant sensitivity enhancement. Identification and quantification of the analytes were performed by diode array detection (DAD). The influence of different parameters, such as buffer apparent pH, concentration of electrolyte, temperature, applied voltage and sample volume, on the efficiency, resolution and sensitivity of the electrophoretic separation was studied. The analytical performance was evaluated, and both NACE-DAD and LVSS-NACE-DAD methods showed good linearity, precision and instrumental LODs at low ng/mL levels. These LODs were compared with those described in the literature, and it was found that NACE-DAD method was comparable to GC-MS, while LVSS-NACE-DAD procedure achieved sensitivity similar to LC-MS, LC-MS/MS and GC-MS/MS, even using conventional ultraviolet-visible absorption detection. To test their suitability, proposed methods were evaluated for the analysis of PHBA and parabens at low and sub-ng/mL levels in environmental water samples.     

Field-amplified online sample stacking capillary electrophoresis UV detection for plasma malondialdehyde measurement

Malondialdehyde (MDA) determination is the most widely used method for monitoring lipid peroxidation. Here, we describe an easy field-amplified sample injection (FASI) CE method with UV detection for the detection of free plasma MDA. MDA was detected within 8 min by using 200 mmol/L Tris phosphate pH 5.0 as running buffer. Plasma samples treated with ACN for protein elimination were directly injected on capillary without complex cleanup and/or sample derivatization procedures. Using electrokinetic injection, the detection limit in real sample was 3 nmol/L, thus improving of about 100-fold the LOD of the previous described methods based on CE. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.11%) and for areas (CV = 2.05%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 2.55% and CV = 5.14%, respectively). Suitability of the method was tested by measuring MDA levels in 44 healthy volunteers.     

Double sample preconcentration by in‐line coupled large volume single drop microextraction and sweeping in capillary electrophoresis

Single drop microextraction (SDME) is a convenient and powerful preconcentration method for CE before injection. By simple combination of sample‐handling sequences without modification of the CE apparatus, a drop of an aqueous acceptor phase covered with a thin organic layer was formed at the tip of a capillary; 10 min SDME of fluorescein and 6‐carboxyfluorescein from a donor phase of pH 1 to an acceptor phase of pH 9 provided 110‐fold enrichments without stirring the donor phase. To improve the concentration effect further, SDME was coupled with an on‐line (after injection) sample preconcentration method, sweeping, in which analytes in a long sample zone are accumulated at the boundary of a pseudostationary phase penetrating into the sample zone. It is thus necessary to inject a sample of much larger volume than that of a drop in typical SDME. A Teflon sleeve over the capillary inlet allowed a large volume drop to be held stably during extraction. By in‐line coupling 10 min SDME and sweeping of a 30 nL sample using a cationic surfactant dodecyltrimethylammonium, enrichment factors of the double preconcentration were increased up to 32 000.     

Improving sensitivity by large-volume sample stacking combined with sweeping without polarity switching by capillary electrophoresis coupled to photodiode array ultraviolet detection

An easy, simple, and highly efficient on-line preconcentration method for polyphenolic compounds in CE was developed. It combined two on-line concentration techniques, large-volume sample stacking (LVSS) and sweeping. The analytes preconcentration technique was carried out by pressure injection of large-volume sample followed by the EOF as a pump pushing the bulk of low-conductivity sample matrix out of the outlet of the capillary without the electrode polarity switching technique using five polyphenols as the model analytes. Identification and quantification of the analytes were performed by photodiode array UV (PDA) detection. The optimal BGE used for separation and preconcentration was a solution composed of 10 mM borate-90 mM sodium cholate (SC)-40% v/v ethylene glycol, without pH adjustment, the applied voltage was 27.5 kV. Under optimal preconcentration conditions (sample injection 99 s at 0.5 psi), the enhancement in the detection sensitivities of the peak height and peak area of the analytes using the on-line concentration technique was in the range of 18-26- and 23-44-fold comparing with the conventional injection mode (3 s). The detection limits for (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG), and (-)-epicatechin gallate (ECG) were 4.3, 2.4, 2.2, 2.0, and 1.6 ng/mL, respectively. The five analytes were baseline-separated under the optimum conditions and the experimental results showed that preconcentration was well achieved.     

Field-amplified on-line sample stacking for determination of carnosine-related peptides by capillary electrophoresis

An on-line sample stacking method, namely field-amplified sample injection, has been developed for the separation and determination of carnosine, anserine, and homocarnosine by capillary electrophoresis. Using electrokinetic injection, about 130- to 160-fold improvement of sensitivity was achieved without loss of separation efficiency when compared to conventional sample injection. For conventional injection, the samples were dissolved in running buffer and then hydrodynamically injected for 10 s (3.45 kPa). Various parameters affecting separation and sample stacking were optimized. Under optimum conditions, linear responses were obtained over two orders of magnitude and the detection limits (defined as S/N = 3) of carnosine, anserine, and homocarnosine were 1.5 x 10(-8) to 1.6 x 10(-8) mol/L.     

Acupuncture injection for field amplified sample stacking and glass microchip‐based capillary gel electrophoresis

Acupuncture sample injection is a simple method to deliver well‐defined nanoliter‐scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost‐effective acupuncture sample injection method can be used for PDMS microchip‐based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA‐HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.     

On-line two-step stacking in capillary zone electrophoresis for the preconcentration of strychnine and brucine

An on-line sample preconcentration method by two-step stacking i.e., sweeping and micelle to solvent stacking, in capillary zone electrophoresis (CZE) has been developed for the determination of strychnine and brucine in traditional Chinese herbal medicines. After experimental optimizations, the best separation was achieved by using 75 mM phosphate buffer (pH 2.5) with 30% methanol (v/v). Compared with normal CZE injection, 51- and 38-fold improvement in concentration sensitivity was achieved for strychnine and brucine, respectively. The calibration curve was linear in the range of 0.1–5.0 μg mL⁻¹ for both strychnine and brucine, with the correlation coefficients of 0.9998 and 0.9997, respectively. The limits of detection (S/N = 3) for both alkaloids were 0.01 μg mL⁻¹. The inter-day (n = 8) and intra-day (n = 5) reproducibilities expressed as the relative standard deviations for corrected peak area were less than 9.5%. The method was applied to determine strychnine and brucine in two Chinese herbal medicines, with recoveries ranging from 94.2% to 105.4%. The results indicated that the method is simple, rapid, reliable, and can be applied to determine strychnos alkaloids in traditional Chinese herbal medicines.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

Sweeping and new on-line sample preconcentration techniques in capillary electrophoresis

Sweeping is a powerful on-line sample preconcentration technique that improves the concentration sensitivity of capillary electrophoresis (CE). This approach is designed to focus the analyte into narrow bands within the capillary, thereby increasing the sample volume that can be injected, without any loss of CE efficiency. It utilizes the interactions between an additive [i.e., a pseudostationary phase (PS) or complexing agent] in the separation buffer and the sample in a matrix that is devoid of the additive used. The accumulation occurs due to chromatographic partitioning, complexation or any interaction between analytes and the additive through electrophoresis. The extent of the preconcentration is dependent on the strength of interaction involved. Both charged and neutral analytes can be preconcentrated. Remarkable improvements—up to several thousandfold—in detection sensitivity have been achieved. This suggests that sweeping is a superior and general approach to on-line sample preconcentration in CE. The focusing mechanism of sweeping under different experimental conditions and its combination with other on-line preconcentration techniques are discussed in this review. The recently introduced techniques of transient trapping (tr-trapping) and analyte focusing by micelle collapse (AFMC) as well as other novel approaches to on-line sample preconcentration are also described.

On-line sample stacking and short-end injection CE for the determination of fluoxetine and norfluoxetine in plasma: Method development and validation using experimental designs

A short-end injection CE method combining field-amplified sample stacking (FASS) is presented for the analysis of fluoxetine (FL) and norfluoxetine in plasma. In this study, FASS enhanced the sensitivity about 1100-fold, while short-end injection reduced the analysis time to less than 4 min. Parameters involved in the separations were investigated using a central composite design (CCD) and response surface methodology to optimize the separation conditions in a total of only 32 runs. Samples injected into the capillary for 99.9 s at a voltage of -5 kV were stacked in a water plug (0.5 psi, 9 s). Baseline resolution of FL and its major metabolite was achieved using a BGE formulation consisting of phosphate-triethanolamine at low pH, and a separation voltage of -10 kV. Five percent methanol was added as organic modifier to enhance selectivity and resolution. The linear range was between 10 and 500 ng/mL (r >0.9946), covering the expected plasma therapeutic ranges. The LOD in plasma were 4 ng/mL (S/N = 3), a value comparable to that obtained using LC-MS, showing the success of the on-line stacking technique. Our method was also successfully validated in quantification and pharmacokinetic studies with three volunteer plasma samples and could be applied to pharmacogenetic studies.     

Comparative study for the analysis of parabens by micellar electrokinetic capillary chromatography with and without large-volume sample stacking technique

The separation and determination of four parabens (methyl, ethyl, propyl, and butyl p-hydroxybenzoate) which are commonly used as preservatives in cosmetic products, by micellar electrokinetic capillary chromatography (MEKC) with and without large-volume sample stacking (LVSS) technique were compared. As an effective on-line concentration technique, LVSS was successfully combined with MEKC to determine neutral parabens in an acidic media. The effects of some typical parameters such as sample volume, buffer pH, temperature, and concentration of surfactant were examined. The detection limits for this LVSS-MEKC method were found to be 3.0 × 10⁻⁷ M for each of the parabens based on the signal-to-noise ratio of 3, which were around 300 times lower than normal MEKC technique. The curves of peak response versus concentration were linear from 1.0 × 10⁻⁶ to 5.0 × 10⁻⁵ M with regression coefficients of 0.9987, 0.9960, 0.9925 and 0.9864, respectively. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available cosmetic samples. It was found that with current sample preparation process and instrumentation system, 0.5 g of sample is enough for the analysis of parabens preservatives in cosmetic product with satisfactory results.     

Simultaneous enrichment and separation of neutral and anionic analytes through combining large volume sample stacking with sweeping in CE

In this work, we overcame the deficiencies of large volume sample stacking (LVSS) in separating low‐mobility and neutral analytes through combining LVSS with sweeping in CE, and employed this new approach to enrich and separate neutral and anionic analytes simultaneously. This technique was carried out with pressure injection of large‐volume sample followed by EOF as a pump pushing the bulk of low‐conductivity sample matrix out of the outlet of the capillary while analytes were swept by micelles and separated via MEKC without the electrode polarity switching. Careful optimization of the enrichment and separation conditions allowed the enrichment factors (EFs) of peak height and peak area of the analytes to be in the range of 9–33 and 21–35 comparing with the conventional injection mode, respectively. The five analytes were baseline separated in 15 min and the detection limits ranged from 26.5 to 55.8 ng/mL (S/N = 3). The developed method was successfully applied to determine adenine, caffeine, theophylline, reduced L‐glutathione (GSH) and oxidized L‐glutathione (GSSG) in two different teas with recoveries that ranged from 84.4 to 105.2%.     

Determination of tobramycin in human serum by capillary electrophoresis with contactless conductivity detection

A study on the determination of the antibiotic tobramycin by CE with capacitively coupled contactless conductivity detection is presented. This method enabled the direct quantification of the non-UV-absorbing species without incurring the disadvantages of the indirect approaches which would be needed for optical detection. The separation of tobramycin from inorganic cations present in serum samples was achieved by optimizing the composition of the acetic acid buffer. Field-amplified sample stacking was employed to enhance the sensitivity of the method and a detection limit of 50 microg/L (S/N = 3) was reached. The RSDs obtained for migration time and peak area using kanamycin B as internal standard were typically 0.12 and 4%, respectively. The newly developed method was validated by measuring the concentration of tobramycin in serum standards containing typical therapeutic concentrations of 2 and 10 mg/L. The recoveries were 96 and 97% for the two concentrations, respectively.     

Large‐volume sample stacking with polarity switching for monitoring of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) reactions by capillary electrophoresis

Nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is a membrane glycoprotein involved in the hydrolysis of extracellular nucleotides. Its main substrate is ATP yielding AMP and pyrophosphate. NPP1 has been proposed as a novel drug target, for diabetes type 2 and the treatment of calcium pyrophosphate dihydrate deposition disease leading to inflammatory arthritis. The monitoring of NPP1 reactions is difficult because its velocity is very slow requiring highly sensitive analytical procedures. In this study, a method of large‐volume sample stacking with polarity switching was developed, and separations were optimized. Large sample volumes were loaded by hydrodynamic injection (5 psi, 13 s) followed by removal of a large plug of sample matrix from the capillary using polarity switching (?10 kV). The stacked analytes were subsequently separated in phosphate buffer (100 mM, pH 9.2) at 20 kV. The validated method was found to be linear (R² = 0.9927) in the concentration range of 0.05–50 μM of AMP, with high accuracy and precision. The determined LOD and LOQ of AMP were 18 nM and 60 nM, respectively. Compared to a previously reported CE procedure using sweeping technique, a fivefold improvement of sensitivity was achieved. Moreover, the new technique was faster, and reproducibility of migration times was improved (RSD value = 1.2%). Importantly, adenine nucleotide analogs and derivatives tested as NPP1 inhibitors could be completely separated from the substrate ATP and the enzymatic product AMP. The method was applied to NPP1 inhibition assays investigating nucleotide‐derived inhibitors in the presence of ATP.     

Capillary electrophoresis determination of biogenic amines by field-amplified sample stacking and in-capillary derivatization

A sensitive CE method for determining biogenic amines in wines based on in-capillary derivatization with 1,2-naphthoquinone-4-sulfonate is presented. In this method, reagent and buffer solutions are introduced hydrodynamically into the capillary whereas the sample is injected electrokinetically, thus, allowing a selective preconcentration of the analytes by field-amplified sample stacking. Amines are labeled inside the capillary using a zone-passing derivatization approach in mixed tandem mode. The most relevant variables influencing on the derivatization and separation as well as significant interactions have been evaluated using experimental design. Multi-criteria decision making is utilized for the simultaneous optimization of interacting variables through overall desirability response surfaces. The validation of the method has proven an excellent separation performance and accuracy for the determination of biogenic amines such as histamine, tryptamine, phenylethylamine, tyramine, agmatine, ethanolamine, serotonin, cadaverine, and putrescine in red wines. Detection limits range from 0.02 mg/L for ethanolamine to 0.91 mg/L for serotonin. The RSDs for migration time and peak area are around 1.2 and 6.2%, respectively. Red wines from different Spanish regions have been analyzed using the proposed method.     

Identification and determination of inorganic anions in real extracts from pre- and post-blast residues by capillary electrophoresis

Fast, selective, and sensitive analysis of inorganic anions is compulsory for the identification of explosives in post-blast or environmental samples. For the last twenty years, capillary electrophoresis (CE) has become a valuable alternative to ion chromatography (IC) for the analysis of inorganic-based explosives because of its low running costs and its simplicity of use. This article focuses on the development and validation of a CE method for the simultaneous analysis of 10 anions (chloride, nitrite, nitrate, thiosulphate, perchlorate, chlorate, thiocyanate, carbonate, sulphate, and phosphate) which can be found in post-blast residues, plus for the first time azide anion, possibly present in the composition of detonators, and the internal standard (formate) in 20 min total runtime. Intermediate precisions were 2.11% for normalized areas and 0.72% for normalized migration times. Limits of detection close to 0.5 ppm for all anions were obtained with the use of preconcentration techniques, thanks to a fast and simple sample preparation allowing the analysis of a large variety of matrices with the developed generic CE method. The matrix effects were statistically studied for the first time in the explosive field for different matrices, containing interfering anions and cations, sometimes at high levels. In fact, no significant matrix effect occurred (tests with blank matrix extracts of soil, cloth, glass, plastic, paper, cotton, and metal). Finally, analyses of real post-blast residues and real detonator extracts were performed. The CE results were compared with those obtained with the IC method used routinely and showed excellent correlation.     

Determination of mercaptopurine and its four metabolites by large-volume sample stacking with polarity switching in capillary electrophoresis

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.     

Large volume sample stacking of cationic tetracycline antibiotics toward 10 ppb level analysis by capillary electrophoresis with UV detection

This paper aimed to build up a sensitive CE method for the analysis of tetracyclines (TCs) antibiotics (including tetracycline, chlorotetracycline, oxytetracycline, and doxycycline) with conventional UV detection. Here, the large volume sample stacking was applied to achieve in capillary preconcentration of the targets. To achieve large volume sample stacking, the essential step was a large volume of sample (around 83.3% of total capillary length from inlet to detection window) hydrodynamically loaded. Then, the reserved voltage was added in order to push the sample matrix out of the capillary. Due to different pH between sample solution (pH 4.6) and BGE (pH 11.0), the cationic TCs would turn into negatively charged while the sample matrix was removing from the capillary. Finally, the anionic TCs were stacked at the inlet for the subsequent separation. Although the loss of sample existed during their charge transformation, the LODs could be improved around 40 times than that obtained by normal hydrodynamic injection CE method. Here, the LODs were in the range of 8.1–14.5 μg/L, around 10 ppb that close to the level by electrochemiluminescence or laser‐induced fluorescence detection of TCs by CE. The precision was characterized by RSDs of migration times and peak areas, which were in the range of 0.19–0.24% and 0.97–2.54%, respectively. The recoveries of the developed method were in the range of 95–112% by spiking TCs in the tap water. The proposed inline preconcentration CE method could be a simple, speed, and sensitive method for the quantitative analysis of TCs.     

On-line sample enrichment for the determination of proteins by capillary zone electrophoresis with poly(vinyl alcohol)-coated bubble cell capillaries

Field-amplified sample stacking (FASS) is used to separate basic proteins in a poly-(vinyl alcohol)-coated bubble cell capillary. To our knowledge, this is the first paper describing the on-column stacking of proteins (as cations) using FASS in bubble cell capillary. The bubble cell capillary is fabricated using a one-step method. Cetyltrimethylammonium chloride is added into the running buffer to reverse the EOF and, thus, to pump the water plug out during the sample stacking step. The effect of the water plug lengths and sample injection durations were investigated and optimized. The results obtained were compared with those for the normal capillary without bubble cell in terms of resolution and sensitivity enhancement. Under the optimal condition, this method can improve the sensitivity of the peak areas ranging from 5000- to 26 000-fold. The RSDs (n = 5) of the migration time and peak area are satisfactory (less than 0.6 and 12%, respectively). Application of the capillary electrophoresis method with bubble cell, FASS, and UV detection thereby leads to the determination of these proteins at concentrations ranging from 3 to 10 ng/mL, based on a signal-to-noise ratio of 3:1.