CE-UV Method Research Articles

CE-UV method for the determination of catecholamine metabolites from baby pee-covered diapers

A method has been developed for the analysis of vanillylmandelic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from baby urine as biomarkers of neuroblastoma in infants. Disposable diapers were employed as sampling devices in order to guarantee a low invasiveness during this step. The proposed method consists on a simple extraction step with water from the used diaper followed by the measurement using capillary electrophoresis with UV detection. The Box-Behnken design (BBD) was utilized to optimize the process of extracting catecholamine metabolites from the examined samples. The variables of the sample preparation step were optimized and the method was validated obtaining limits of quantification of 1.65 μg mL−1, good intraday and inter-day precision with RSDs under 15 %. Finally the method was applied to real samples collected from the Department of Neonatology, University Clinical Centre (Gdańsk, Poland). The greenness of the proposed method was also evaluated with different tools (i.e., AGREEPrep and GAPI) with satisfactory results, which allow to state that the method can be considered green. Moreover, its practicality was evaluated by application of BAGI tool, proving to be a practical and economical method to be applied in routine laboratories for determination of catecholamine metabolites in urine-type samples.

Separation, identification and quantification of associated impurities in cobratide using sheathless CE-MS and CE-UV.

Cobratide is a peptide drug extracted from the venom of Chinese cobra, and has been widely used in the clinical treatment of chronic, intractable and persistent pain. In a recent study, it was reported that it has the potential to treat COVID-19. In order to control the quality of commercial cobratide drugs, a protocol was established for the separation, identification and quantification of cobratide and its associated impurities, in which sheathless capillary electrophoresis-mass spectrometry (CE-MS) was used for identification and a rapid capillary electrophoresis-ultraviolet-visible detector (CE-UV) method was developed for accurate quantification. Separation conditions that affect the resolution and MS intensities of cobratide and its associated impurities were investigated, including pH value, concentration of background electrolyte (BGE), ratio of organic additive and sample solution. The optimized CE conditions (BGE: 50 mM NH4Ac, pH 4.0; sample solution: deionized water) were used for both sheathless CE-MS and CE-UV methods. Three associated impurities were separated and identified for the first time by sheathless CE-MS. Then, a rapid CE-UV method was validated and used for accurate quantification of cobratide and its associated impurities. The CE-UV method showed good linearity between concentration and corrected peak area of cobratide in the concentration range of 5.36-536.30 μg mL-1. The limit of quantification of the CE-UV method was 4.16 μg mL-1. The relative standard deviations of migration time were less than 1% for both intra-day and inter-day experiments, and those of corrected peak area were less than 5%. Finally, different cobratide drugs were analyzed to evaluate the batch-to-batch consistency. This established protocol combining sheathless CE-MS and CE-UV methods would provide useful information for both quality control and process analysis of peptide drugs.

Comparison of capillary electrophoresis and zwitterionic-hydrophilic interaction capillary liquid chromatography with ultraviolet and mass spectrometry detection for the analysis of microRNA biomarkers

This study evaluates zwitterionic-hydrophilic interaction capillary liquid chromatography (capZIC-HILIC) and capillary electrophoresis (CE) with ultraviolet (UV) and mass spectrometry (MS) detection for the direct, label-free and multiplex analysis of microribonucleic acids (miRNAs). CapZIC-HILIC-UV and CE-UV methods were first optimized, resulting in similar separations for a mixture of three miRNAs (hsa-iso-miR-16-5p, hsa-let-7g-5p, and hsa-miR-21-5p) but with reversal of elution/migration orders and small differences in repeatability, linearity, limit of detection (LOD) and separation efficiency. The established UV methods were transferred and validated in these terms with mass spectrometry (MS) detection, which allowed identifying the miRNAs and characterizing their post-transcriptional modifications. LOD by capZIC-HILIC-MS was 1 μM of miRNA, around 5 times lower than by CE-MS due to the analyte dilution with the sheathflow CE-MS interface and to the slightly increased abundance of alkali metals adducts in the CE-MS mass spectra. In addition, the suction effect promoted by the nebulizer gas in CE-MS negatively affected the already compromised separations. In contrast, CE-MS showed superior repeatabilities with spiked serum samples, as well as reduced costs, extended capillary column durabilities and shorter conditioning times. The comparison of the different methods allows disclosing the current advantages and disadvantages of capZIC-HILIC and CE for the analysis of miRNA biomarkers.

Fast methods for simultaneous determination of arginine, ascorbic acid and aspartic acid by capillary electrophoresis

Arginine (ARG), ascorbic acid (ASC) and aspartic acid (ASP) are very popular and widely consumed active ingredients used for fatigue treatment or improvement of physical performance. For this reason, these compounds are usually available in the same pharmaceutical formulation. In the current paper, we describe for the first-time methods for simultaneous determination of ARG, ASC and ASP using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D) and with UV spectrophotometric detection (CE-UV). The proposed methods are simple, rapid (78 and 23 injections h−1, respectively) and have low environmental impact (minimal waste generation). The separation by CE-C4D was achieved with a background electrolyte (BGE) composed by 20 mmol L−1 N-tris(hydroxymethyl)- methyl]-3-aminopropanesulfonic acid (TAPS) and 10 mmol L−1 of NaOH (pH 8.7). The limits of detection (LOD) were 0.01, 0.02 and 0.04 mmol L−1 for ARG, ASC and ASP, respectively. The proposed CE-UV method was optimized with a BGE composed by 10 mmol L−1 sodium tetraborate (pH 9.4). The limits of detection (LOD) were 0.03, 0.02 and 0.04 mmol L−1 for ARG, ASC and ASP, respectively. No statistically significant differences were observed (95% confidence level) between the results obtained by the developed CE methods and reference procedures (HPLC for ARG, iodometry for ASC and, acid-base titration for ASP).

Development and validation of a capillary electrophoresis method applied to the analysis of l-citrulline in an oral formulation for pediatric use.

A simple and highly sensitive CE-UV method was applied in the determination of l-ctrulline, which was developed from an oral formulation for pediatric use. The novel method was based on the analysis of l-citrulline for direct ultraviolet detection at 198nm. The BGE consisted of 10 mM sodium tetraborate and 50 mM SDS at pH 9, and the electrophoretic parameters were optimized. The method was validated in terms of specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The LOD and LOQ obtained were 1.36 and 4.54 μg/mL, respectively. In addition, the method offers higher sensitivity and specificity compared with the results obtained from HPLC method using UV-detectors, in which l-citrulline needs to be derivatizated. Furthermore, low cost and simplicity of the system allowed the rapid and simple quantitation of l-citrulline in the oral formulation for quality control and stability indicated method.

A Capillary Electrophoresis UV Detection-Based Method for Global Genomic DNA Methylation Assessment in Human Whole Blood.

Quantitative analysis of DNA methylation patterns is of special importance in several developmental and pathological situations. The development of simple and robust methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly reproducible CE-UV method for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolyzed samples were dried and successively dissolved with water and then injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH3.25 and 60mmol/L sodium acetate buffer at pH3.60 (4:1, v/v) allowed a baseline analytes separation within 12min. Precision tests indicated an elevated reproducibility with an inter-assay CV of 1.98%.

Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the “charge-tagging” Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure.

Highly sensitive detection of copper(i) and copper(ii) in cell specimens by CE-UV with large-volume sample stacking

A CE-UV method has been developed for the simultaneous separation and sensitive determination of copper(i) and copper(ii) in cell specimens.

Higher detectability method for the analysis of nucleosides, putative tumor biomarkers, in blood serum samples by CE-UV with reversed EOF.

The development and validation of methodologies for the analysis of biological samples is of outcome importance in order to obtain trustworthy results. This work reports a novel CE-UV method for the assessment of nucleosides, putative tumor biomarkers, in blood serum. The separation of seven nucleosides within c.a. 20 min has been achieved with: BGE 30 mmol/L borate at pH 9.90, 50 mmol/L CTAB, and 10% methanol; V = -10 kV; T = 20°C; and capillary dimensions of 56 cm × 50 μm. The sample plug was concentrated by a modified large volume sample stacking strategy that provided better detectability. Validation showed that the method is suitable for bioanalytical purposes and initial applications in serum samples from healthy subjects are also presented. Finally, statistical methods were applied to verify the effect of characteristics such as age, smoking habits, and alcohol consumption on nucleoside concentrations in blood serum. Univariate statistical analysis tests emphasized the need for age matching, which was confirmed by PCA-DA and PLS-DA. Cancer history in the nearby family may also interfere in nucleoside levels in blood serum, since adenosine concentrations were statistically higher for volunteers who declared having diseased relatives.

Determination of sulfate and chloride ions in highly saline oilfield water by capillary electrophoresis using bilayer-coated capillaries and indirect absorption detection

Analysis of highly saline oilfield waters for anions presents challenges. Traditional analytical techniques used for such analysis tend to suffer from both poor sensitivity and selectivity due to the high concentrations of salt present in the samples. A capillary electrophoresis method was developed for the simultaneous determination of chloride and sulfate anions which is relevant to the oilfield analysis industry and of economic value. Due to the extremely high concentrations of chloride in highly saline oilfield waters, it is difficult to achieve baseline electrophoretic separation necessary for accurate quantitation. By using a capillary with a noncovalently bound bilayer coating using Polybrene, a cationic polymer and sodium dodecyl sulfate (SDS), an anionic surfactant and a buffer consisting of 50 mM TRIS, 30 mM SDS, 5% methanol and 26 mM chromium trioxide (CrO3) at pH 6.7, baseline separation (Rs > 1.5) of chloride and sulfate was achieved. To mimic possible oilfield water samples, model water solutions of 5%, 10%, 15% and 20% chloride containing low ppm sulfate were prepared and successfully analysed using the method developed. In addition, the method was applied to determine chloride and sulfate anions in highly saline oilfield water samples. The accuracy of the method developed was verified by analysing NIST certified standards of chloride and sulfate. The results obtained for chloride and sulfate with the indirect CE-UV method were in close agreement (94–100% accuracy; <2.5% relative standard deviations) with those of the certified standard analysed by ion chromatography.

Simultaneous separation and sensitive detection of four β2-agonists in biological specimens by CE-UV using a field-enhanced sample injection method

A rapid, sensitive and cost-effective CE-UV method has been developed for the simultaneous separation and sensitive determination of four beta2-agonists.

Analysis of ecstasy tablets using capillary electrophoresis with capacitively coupled contactless conductivity detection.

A method for the identification of 3,4-methylenedioxymethamphetamine (MDMA) and meta-chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4) D). Sample extraction, separation, and detection of "Ecstasy" tablets were performed in <10min without sample derivatization. The separation electrolyte was 20mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE-C(4) D and compared against routine gas chromatography-mass spectrometry (GC-MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90sec. The advantages of using C(4) D instead of traditional CE-UV methods for in-field analysis are also discussed.

Simultaneous determination of erlotinib and metabolites in human urine using capillary electrophoresis

The purpose of this study was to develop a simple and sensitive CE-UV method to quantify erlotinib and metabolites in urine. Following liquid-liquid extraction, erlotinib, and metabolites were separated with a BGE whose composition was phosphate buffer (pH 2.5, 65 mM) with 0.5% Tween 20. The applied voltage was 22 kV, capillary temperature 25°C and the sample injection was performed in the hydrodynamic mode. All the analyses were carried out in a fused silica capillary with an internal diameter of 75 μm and a total length of 37 cm. The detection of target compounds was performed at 240 nm. The calibration was linear in the range 0.15-20 mg/L for erlotinib and metabolites. Inter-and intraday imprecision were less than 4%. This simple, sensitive, accurate, and cost-effective method can be used in routine clinical practice to monitor erlotinib concentrations in urine from nonsmall cell lung cancer patients.

Capillary Electrophoresis with UV Detection to Determine Cocaine on Circulated Banknotes

A new methodology employing capillary electrophoresis with UV detection (CE-UV) was developed, validated, and applied to determine the presence of cocaine on Brazilian banknotes. Some of the banknotes analyzed were obtained directly from general circulation (well used) while others were collected from Automated Teller Machines (ATMs) (relatively new). The background electrolyte optimized using Peakmaster 5.1 software was composed of 60 mmol L−1 TRIS(hydroxymethyl)aminomethane and 20 mmol L−1 2-hydroxyisobutyric acid, at pH 8.4. The separation time achieved for cocaine was only 2.5 minutes. The figures of merit obtained in the evaluation of the proposed method were good linearity (r&gt;0.999) in the concentration range of 0.8–8.0 mg L−1 and acceptable limits of detection and quantification (0.2 mg L−1 and 0.8 mg L−1, resp.). The relative standard deviations of the instrumental precision, repeatability (intraday), and intermediate precision (interday) were less than 4.5% (peak area). The accuracy evaluated through comparing the cocaine results for the banknotes determined by the proposed CE-UV method and using an LC-MS/MS method showed no significant difference between the methods (95% confidence level). In the analysis of the samples cocaine was detected on 93% of the circulating banknotes in amounts ranging from 11.5 μg to 2761.9 μg per note.

Determination of fluoroquinolones in bovine milk samples using a pipette-tip SPE step based on multiwalled carbon nanotubes prior to CE separation

A simple CE-UV method was developed for the simultaneous determination of ciprofloxacin, norfloxacin, and ofloxacin in milk samples. The optimum separation was obtained using a 20 mM ammonium dihydrogenphosphate solution with 2 mM cetyltrimethylammonium bromide at pH 3.0 as the BGE. Satisfactory resolution for structurally very similar analytes, like norfloxacin and ciprofloxacin, was achieved without including any organic solvent. Milk samples were prepared using a simple/extraction procedure based on acidic protein precipitation followed by an SPE step using only 5 mg of multiwalled carbon nanotubes as the sorbent material. The LODs for the three compounds were between 7.5 and 11.6 μg/L and the RSDs for the peak areas were between 2.6 and 4.9%. The complete method was applied to spiked real milk samples with satisfactory recoveries for all analytes (84-106%).

Merging a sensitive capillary electrophoresis–ultraviolet detection method with chemometric exploratory data analysis for the determination of phenolic acids and subsequent characterization of avocado fruit

Herein we present the development of a powerful CE–UV method able to detect and quantify an important number of phenolic acids in 13 varieties of avocado fruits at 2 ripening stages. All the variables involved in CE separation were exhaustively optimized and the best results were obtained with a capillary of 50μm i.d.×50cm effective length, sodium tetraborate 40mM at a pH of 9.4, 30kV, 25°C, 10s of hydrodynamic injection (0.5psi) and UV detection at 254nm. This optimal methodology was fully validated and then applied to different avocado samples. The number of phenolic acids determined varied from 8 to 14 compounds; in general, they were in concentrations ranging from 0.13ppm to 3.82ppm, except p-coumaric, benzoic and protocatechuic acids, which were found at higher concentrations. Principal component analysis (PCA) was applied to highlight the differences between varieties and ripening degrees, looking for the most influential analytes.

A fast and sensitive method for the determination of nitrite in human plasma by capillary electrophoresis with fluorescence detection

Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE–UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (−30kV) were used to shorten the analysis time. The good separation was achieved with 20mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4min. The deproteinized plasma sample was injected hydrodynamically for 5s at −50mbar into a 60cm×75μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240–400nm), and the emitted light was measured at 418nm by the use of a cutoff filter. A good linearity (R2=0.9975) was obtained in the range from 2 to 500nM. The detection limit of nitrite was 0.6nM in original plasma samples, which is 750 times lower than our previous CE–UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.

CE analysis and molecular characterisation of depurinated DNA samples

A DNA sample was partially degraded by scalar heat-acid treatments to study the extent of apurinic-apyrimidinic (A-P) lesions produced along the molecule. A CE-UV method allowed us to measure the rate of depurination at pH 5.0 and 70°C which was calculated to be 5.41×10(-6) s(-1) for adenine and 6.27×10(-6) s(-1) for guanine. CE identified depurination on treated samples when it occurred with a loss of >4% of the basic moieties. The molecular features of the A-P enriched samples were investigated by using molecular assays (agarose gel electrophoresis, UV spectrophotometry and quantitative PCR) and the consistency of the results of the STR typing were compared with the degree of depurination of the PCR template. The treated DNA samples showed molecular features such as fragmentation, altered OD(260) /OD(280) ratios and decreased ability of the quantitative PCR to synthesise the human target, related to the severity of depurination. A satisfactory correlation between the degree of damage and the amount of residual PCR-sensitive target sequences was also demonstrated (r(2) =0.9717). The conventional and mini-STR typing of the samples showed that the genetic outcome was influenced by a depurination damage that exceeded 4% when locus drop-outs and artefactual PCR results were evident. As the success of STR typing depends on the integrity of the DNA recovered from the samples, the CE-UV, physical and molecular assays described here are proposed as a set of useful methods in the analysis of certain forensic and clinical samples, for a critical evaluation of the outcome of the genetic testing.

Simultaneous discrimination of jasmonic acid stereoisomers by CE‐QTOF‐MS employing the partial filling technique

Jasmonic acid (JA), an essential plant hormone controlling the plant defense signaling system and developmental processes, has stereospecific bioactivities that have not been well understood mainly due to the limitation in separation and detection methodologies. In this work, a fast CE-UV method based on short-end injection technique and a sensitive CE-QTOF-MS method based on partial filling technique were successfully developed for the enantioseparation of racemic JA. The successive coating technique was also involved by modifying the capillary with multiple ionic polymer layers of polybrene-dextran sulfate-polybrene. This was the first report on the direct resolution of both pairs of JA enantiomers, including two naturally occurring JA stereoisomers. Although no pure JA stereoisomers were commercially available, all the separated JA stereoisomers were identified indirectly by comparing the difference between the racemic standard and plant samples based on the presence and the ratio of each stereoisomer. Satisfactory results were obtained in terms of sensitivity (LOD, 24 ng/mL or 0.7 fmol for single JA stereoisomer) using 45 mmol/L ammonium acetate at pH 4.5 containing 70 mmol/L α-CD as the buffer system. This established CE-QTOF-MS method was later successfully applied for the study of the naturally occurring JA stereoisomers in wounded tobacco leaves.

Simple and rapid CE‐UV method for the assessment of trail pheromone compounds of leaf‐cutting ants' venom glands

Leaf-cutting ants cause large losses in several crops around the world. In the ant species Atta sexdens, each colony comprises up to 5 million individuals. In order to keep a close connection among such a large number of individuals, an efficient chemical communication system is necessary. Among other different substances, these animals use alkylpyrazines to mark their trails and to guide ant workers from the nest to their sources of food. In this study, CE-UV was used to apply a method for qualitative analysis of venom gland components of leaf-cutting ants. Mobility of these compounds proved to be a function of the ionization capability of these bases as well as their volumes. Migration order was thoroughly explained in terms of such parameters. The best analysis conditions were achieved with a BGE composed by 0.8% formic acid plus 20% methanol in water, hydrodynamic injection, and application of external pressure. Such analysis conditions may be easily applied in CE-MS analyses as well. CE-UV analyses proved to be as adequate as GC to analyze such compounds due to system detectability (LD ≅ 0.005 mmol/L), separation efficiency (from 5.07×10(4) to 1.23×10(5) theoretical plates), and resolution (minimum of 2.35). In addition, analysis time was ca. 15 min, which shows another advantage of CE analysis when compared with GC. Although the analytes are found in concentrations as low as 50 ng/venom glands, four putative pyrazine ring moiety substances could be detected in real samples, due to sample stacking and use of a capillary with extended detection cell.