CZE-UV Method Research Articles

Front Cover: Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE‐UV

DOI: 10.1002/elps.202200028 The cover picture illustrates the first work screening prolyl hydroxylase 2 (PHD2) inhibitory activity from traditional Chinese medicines (TCMs). PHD2 is a key oxygen receptor participating in the regulation of oxygen homeostasis in the human body and thus, it is one of the crucial targets for drug discovery and development for hypoxia-based diseases. The present work utilizes the advantage of the high separation efficiency of the CZE-UV method to avoid the interference of the complex matrix on the results of inhibitor screening. Astragali Radix and Codonopsis pilosula were found to have good PHD2 inhibitory activities for the first time in PHD2 screening, suggesting their potential anti-hypoxic activities in oxygen-regulated pathways in vivo. The present work provides a novel approach for screening the PHD2 inhibitory activity of TCMs extracts and the discovery of anti-hypoxia bioactive compounds.

Rapid and simple CZE-UV method for quality control of B1 and B6 vitamins in drugs and dietary supplements

Abstract The application of hydrodynamically closed capillary zone electrophoresis combined with convenient ultraviolet (UV) detection allows fast, simple, environmentally friendly and cost-effective analysis of ions or ionisable molecules. This technique has been used to determine two selected B vitamins (thiamine, pyridoxine) in various drug formulations. The developed method was characterised by excellent validation parameters, such as linearity, precision, accuracy, limit of detection and limit of quantification. The total time of analysis was lower than 13.5 min. The results indicate that the method is suitable for implementation in routine quality control of selected B vitamins in pharmaceutical and food samples.

Assessment of enzymatic hydrolysis of lactose in lactose-free milk production - A comparative study using capillary zone electrophoresis and cryoscopy

A comparison of the capillary zone electrophoresis under indirect detection at ultraviolet region (CZE-UV) method with the traditional cryoscopic method for lactose hydrolysis process monitoring is proposed. Two different lactose enzymatic hydrolysis processes (in batch and sterile) were assessed. The hydrolysis levels obtained by the CZE-UV method differed statistically from those obtained by cryoscopy, at 95% confidence, showing variations up to 20% at the same time of processing. During sterile hydrolysis, 90% of lactose reduction was detected after 1 h by CZE-UV and after 3 h by cryoscopy. For in batch hydrolysis, the CZE-UV method indicated 50% of lactose hydrolysis after 3 h of processing, while the cryoscopic method did not identify such lactose reduction during the process simulation. Our results demonstrated that CZE-UV method is precise and reliable analytical method, acting as a good alternative for lactose hydrolysis process monitoring.

A CZE-UV Method for Saturated and Unsaturated Fatty Acids Determination in Hops

Fatty acids (FA) are minor compounds in beer and its raw materials, but can develop important roles in brewing and require monitoring. The aim of this work was to show the potential of capillary electrophoresis for fast hop analysis and to propose a simple method for the determination of saturated and unsaturated FA in different hop varieties. Sample preparation was investigated and evaluated and validation procedures were carried out to determine palmitic, stearic, oleic and linolenic FA in hops. Low values of relative standard deviation for areas (< 5.32%) and good recoveries (85-110%) were achieved. Laborious extraction and derivatization reactions were not necessary and only a saponification reaction was employed in sample preparation. Also, specific columns were not required and analysis time was only 10 min. The proposed methodology appears promising for faster monitoring of FA in terms of hop quality control in the brewing industry.

Establishment and development of a CZE-UV method for rapid measurement of aprotinin potency.

A new method for the measurement of aprotinin potency by CZE-UV detector was established for the first time. The on-line mixing of substrate, trypsin and aprotinin using at-inlet technology was realized by the established method. Enzymatic reaction, separation, and detection of substrate and product can be performed simultaneously online. The aprotinin potency can be measured within 4min. The response surface methodology was used to optimize the incubation conditions of trypsin and substrate, and the optimized conditions were obtained under 17.39mM phosphate buffer at pH 7.6, 1.40min of incubation time. The repeatability of proposed method was evaluated in three different systems of capillary zone electrophoresis: (i) only substrate; (ii) trypsin and substrate; (iii) aprotinin, trypsin and substrate, and the RSDs of migration times and peak areas of substrate were less than 2.7 and 3.1%, respectively. The RSDs of migration times and peak areas of product were less than 2.1 and 3.0%, respectively. A formula was also developed to calculate the aprotinin potency in this method. In a word, the established CZE-UV method was convenient, fast, and environmentally friendly for the measurement of aprotinin potency.

Identification and characterization of infectious bursal disease virus subviral particles by capillary zone electrophoresis: potential application for vaccine production and quality control

The infectious bursal disease (IBD) causes immunosuppression in chicken of all ages and high mortality in young chicken, posing serious threat to poultry industry worldwide. One promising strategy for preventing this highly contagious disease is using recombinant subunit vaccine, employing VP2 subviral particles (SVP) as epitomic antigen. Analytical techniques of viral-like particles such as SDS-PAGE, western blot, or high-performance size-exclusion chromatography have been widely applied, but mostly unsatisfactory. In the present study, a simple, fast and cost-effective capillary zone electrophoresis (CZE) method with UV-detection was developed to analyze purified IBDV-SVP (expressed by Escherichia coli system) using commercial monoclonal antibody (mAb) against VP2. To find satisfying CZE conditions, injection mode, separation voltage, and separation buffer were explored. Through the modified CZE, mAb and SVP could be well separated and shown distinct peaks in the electropherogram. Furthermore, to determine the stoichiometry, the area of the mAb peak versus SVP/mAb binding ratio was plotted and indicated that 2 or 3 receptor molecules were bound per SVP. The purity and integrity of SVP and the interactions between SVP and mAb could be analyzed by the developed simple CZE-UV method in less than half hour. This CZE-UV method proved to be a valuable and useful tool in detection, characterization, and quantification of IBDV-SVP and the mAb, offering potential applications of in-process quality control of vaccine production, surveillance of serum antibody produced against IBDV infection, or vaccine immunization.

First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms.To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100–1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000–2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.

Lactulose determination in UHT milk by CZE-UV with indirect detection

Lactulose is not found naturally in milk, but it has been detected in small quantities in heated dairy products. It can therefore be used as an indicator of the severity of heat treatment for dairy products. This study proposes an alternative method for lactulose quantification in milk using capillary zone electrophoresis with ultraviolet detection (CZE-UV). The investigation used several Brazilian UHT milk samples, and various sample pretreatment methods and injection conditions were considered. Optimal separation results were achieved using Carrez clarification solutions. Low detection and quantification limits were obtained, and good precision and high recovery rates indicated the applicability of the method for analysis of milk. Due to matrix effects, single-point standard addition was used for lactulose quantification. High lactulose concentrations (>1000 mg L−1) suggested that recirculation may have occurred during milk processing. The findings demonstrate that the CZE-UV method is a viable alternative for quality process control.

Development of a capillary zone electrophoresis method to quantify E. colil-asparaginase and its acidic variants

A capillary zone electrophoresis (CZE) method with UV detection was developed for the quantification of the E.colil-asparaginase (l-ASNase) and its acidic variants. During the initial method development, a variety of experimental conditions were screened. Subsequently, a Design of Experiments (DoE) was used to optimize the pH and concentration of the selected background electrolyte (BGE) containing both TRIS and boric acid. Optimization was performed taking into account both the separation efficiency of l-ASNase and its acidic variants as well as overall method robustness. A repeatable separation between E.colil-ASNase and its acidic variants was achieved on a bare fused silica capillary in combination with a BGE consisting of both 400 mM TRIS and boric acid. The method was validated for linearity, accuracy, precision, LOD, LOQ and robustness. The recovery for l-ASNase was 97.9–104.4% with a precision RSD of 1.5–3.2%, while the recovery of impurities was 92.1–109.8% with a RSD of 1.7–4.6%. The quantification limit was 1.9% (m/m). Moreover, the CZE-UV method was applied to determine the degradation rate in the presence of ammonium bicarbonate, confirming the suitability of the method. The degraded, partially charged l-ASNase was evaluated for its in-vitro enzymatic activity showing an insignificant different enzyme activity compared to the unmodified sample.

Online sample concentration and analysis of drugs of abuse in human urine by micelle to solvent stacking in capillary zone electrophoresis.

A sensitive and rapid CZE-UV method was developed to determine drugs and their metabolites' presence in human urine. Ten drugs of abuse were analyzed including four amphetamines, cocaine, cocaethylene, heroin, morphine, 6-monoacetylmorphine, and 4-methylmethcathinone. An MSS (micelle to solvent stacking) approach was evaluated to enhance method sensitivity. This method considers composition of the micellar sample solution matrix and the injection time. Several analytical conditions influencing the resolution of the drugs mixture as pH and buffer concentration, organic solvent content, were also investigated. The base-line separation of all studied analytes in the same run was achieved within 18 min in an uncoated fused silica capillary (50 μm id × 60 cm) using a background solution containing 50 mM phosphate buffer pH 2.5 and 30% ACN v/v. Other experimental parameters such as applied voltage and capillary temperature were set up at 20 kV and 20°C, respectively. LOD values ranging between 15 and 75 ng/mL for all studied compounds were obtained. From a comparison with conventional CZE, the proposed method provides an increase of sensitivity (39- to 55-fold enhancement factor). Under optimal MSS-CZE conditions, good linearity was achieved (R2 ≤ 0.9998). The method was finally applied to the analysis of urine samples spiked with a standard mixture after a sample pretreatment, reaching satisfactory recovery values.

Quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets by CZE-UV and identification of related degradation products by heart-cut CZE-CZE-MS.

Capillary electrophoresis is commonly applied for the analysis of pharmaceutical products due to its high separation efficiency and selectivity. For this purpose, electrospray-ionization-(ESI)-interfering additives or electrolytes are often required, which complicates the identification of impurities and degradation products by mass spectrometry (MS). Here, a capillary zone electrophoresis (CZE) method with ultraviolet (UV) absorption detection for the simultaneous determination and quantification of ascorbic acid and acetylsalicylic acid in effervescent tablets was developed. Related degradation products were identified via CZE-CZE-MS. Systematic optimization yielded 100 mM tricine (pH = 8.8) as appropriate background electrolyte, resulting in baseline separation of ascorbic acid, acetylsalicylic acid, and related anionic UV-active degradation products. The CZE-UV method was successfully validated regarding the guidelines of the Food and Drug Administration. The validated method was applied to trace the degradation rate of the active pharmaceutical ingredients at defined ambient conditions. A heart-cut CZE-CZE-MS approach, including a 4-port-nL-valve, was performed for the identification of the observed degradation products. This 2D setup enables a precise cutting of accurate sample volumes (20nL) and the independent operation of two physically separated CZE dimensions, which is especially beneficial regarding MS detection. Hence, the ESI-interfering tricine electrolyte components were separated from the analytes in a second electrophoretic dimension prior to ESI-MS detection. The degradation products were identified as salicylic acid and mono- and diacetylated ascorbic acid. This setup is expected to be generally applicable for the mass spectrometric characterization of CZE separated analytes in highly ESI-interfering electrolyte systems. Graphical Abstract A CZE-UV method for the quantification of effervescent tablet ingredients and degradation products was developed and validated. In order to identify unknown degradation products separated in the CZE-UV, a 2D heart-cut approach was performed applying a mechanical 4-port-valve. The unknown substances were transferred from the 1st to the 2nd dimension followed by the separation of ESI-interfering tricine from the analytes prior to mass spectrometric detection.

New multilayer coating using quaternary ammonium chitosan and κ-carrageenan in capillary electrophoresis: application in fast analysis of betaine and methionine.

The aim of this study was to develop a new multilayer coating with crosslinked quaternary ammonium chitosan (hydroxypropyltrimethyl ammonium chloride chitosan; HACC) and κ-carrageenan for use in capillary electrophoresis. A new semi-permanent multilayer coating was formed using the procedure developed and the method does not require the presence of polymers in the background electrolyte (BGE). The new capillary multilayer coating showed a cathodic electroosmotic flow (EOF) of around 30×10(-9) m(2) V(-1) s(-1) which is pH-independent in the range of pH 2 to 10. The enhanced EOF at low pH obtained contributed significantly to the development of a fast method of separation. The multilayer coating was then applied in the development of a fast separation method to determine betaine and methionine in pharmaceutical formulations by capillary zone electrophoresis (CZE). The BGE used to determine the betaine and methionine concentrations was composed of 10 mmol L(-1) tris(hydroxymethyl) aminomethane, 40 mmol L(-1) phosphoric acid and 10% (v/v) ethanol, at pH 2.1. A fused-silica capillary of 32 cm (50 µm ID×375 µm OD) was used in the experiments and samples and standards were analyzed employing the short-end injection procedure (8.5 cm effective length). The instrumental analysis time of the optimized method was 1.53 min (approx. 39 runs per hour). The validation of the proposed method for the determination of betaine and methionine showed good linearity (R(2)>0.999), adequate limit of detection (LOD <8 mg L(-1)) for the concentration in the samples and inter-day precision values lower than 3.5% (peak area and time migration). The results for the quantification of the amino acids in the samples determined by the CZE-UV method developed were statistically equal to those obtained with the comparative LC-MS/MS method according to the paired t-test with a confidence level of 95%.

Analysis of cytokinin nucleotides by capillary zone electrophoresis with diode array and mass spectrometric detection in a recombinant enzyme in vitro reaction

A capillary zone electrophoresis (CZE) method for separation of adenosine and N6-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20min with high theoretical plate number (up to 400000 for isopentenyladenosine triphosphate) was achieved using a 100mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27μmolL−1). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R2>0.999) was achieved over the concentration range 5–1000μmolL−1. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.

Determination of selected biogenic amines in red wines by automated on-line combination of capillary isotachophoresis–capillary zone electrophoresis

A screening analytical method based on an automated on-line combination of capillary isotachophoresis–capillary zone electrophoresis (cITP–CZE) in hydrodynamically closed separation system, equipped with photometric detection at 280nm, was developed for a routine determination of the selected biogenic amines, namely histamine, 2-phenylethylamine and tyramine, in red wines. The evaluated limits of detection (LODs) were 0.35mgL−1 for histamine, 0.33mgL−1 for 2-phenylethylamine and 0.37mgL−1 for tyramine. The repeatability of the migration time and peak area for histamine were 1.1% and 2.6%, respectively, for 2-phenylethylamine 0.7% and 2.0%, respectively, and for tyramine 0.8% and 2.1%, respectively. The method recoveries were 92.1% for histamine, 96.4% for 2-phenylethylamine and 95.5% for tyramine. The developed automated cITP–CZE–UV method was applied for the determination of histamine, 2-phenylethylamine and tyramine in seven red wine samples originating from Czech Republic.

Electrokinetic supercharging with a system‐induced terminator and an optimized capillary versus electrode configuration for parts‐per‐trillion detection of rare‐earth elements in CZE

A further improvement of electrokinetic supercharging (EKS) methodology has been proposed, with the objective to enhance the sensitivity of the conventional CZE-UV method down to a single-digit part per trillion (ppt) level. The advanced EKS procedure is based on a novel phenomenon displaying the formation of a zone with an increased concentration of the hydrogen ion, capable to perform the function of a terminator, behind the sample zone upon electrokinetic injection. In combination with a visualizing co-ion of BGE, protonated 4-methylbenzylamine, acting as the leading ion, such system-induced terminator a effected the transient ITP state to efficiently concentrate cationic analytes prior to CZE. Furthermore, to amass more analyte ions within the effective electric field at the injection stage, a standard sample vial was replaced with an elongated vial that allowed the sample volume to be increased from 500 to 900 μL. Alongside, this replacement made the upright distance between the electrode and the capillary tips prolonged to 40.0 mm to achieve high-efficiency electrokinetic injection. The computer simulation was used for profiling analyte concentration, pH, and field strength in order to delineate formation of the terminator during sample injection. The proposed preconcentration strategy afforded an enrichment factor of 80,000 and thereby the LODs of rare-earth metal ions at the ppt level, e.g. 0.04 nM (6.7 ng/L) for erbium(III).

Manganese speciation in human cerebrospinal fluid using CZE coupled to inductively coupled plasma MS

The neurotoxic effects of manganese (Mn) at elevated concentrations are well known. This raises the question, which of the Mn species can cross neural barriers and appear in cerebrospinal fluid (CSF). CSF is the last matrix in a living human organism available for analysis before a compound reaches the brain cells and therefore it is assumed to reflect best the internal exposure of brain tissue to Mn species. A previously developed CE method was modified for separation of albumin, histidine, tyrosine, cystine, fumarate, malate, inorganic Mn, oxalacetate, alpha-keto-glutarate, nicotinamide-dinucleotide (NAD), citrate, adenosine, glutathione, and glutamine. These compounds are supposed in the literature to act as potential Mn carriers. In a first attempt, these compounds were analyzed by CZE-UV to check whether they are present in CSF. The CZE-UV method was simpler than the coupled CZE-inductively coupled plasma (ICP)-dynamic reaction cell (DRC)-MS method and it was therefore chosen to obtain a first overview information. In a second step, the coupled method (CZE-ICP-DRC-MS) was used to analyze, in detail, which of the compounds found in CSF by CZE-UV were actually bound to Mn. Finally, 13 Mn species were monitored in CSF samples, most of them being identified: Mn-histidine, Mn-fumarate, Mn-malate, inorganic Mn, Mn-oxalacetate, Mn-alpha-keto glutarate, Mn-carrying NAD, Mn-citrate and Mn-adenosine. By far the most abundant Mn species was Mn-citrate showing a concentration of 0.7 +/- 0.13 microg Mn/L. Interestingly, several other Mn species can be related to the citric acid cycle.