Uncoated Fused-silica Capillary Research Articles

Method Development for Determination of Doripenem in Human Plasma via Capillary Electrophoresis Coupled with Field-Enhanced Sample Stacking and Sweeping.

In this study, we established a novel capillary electrophoresis method for monitoring the concentration of doripenem in human plasma. As a time-dependent antibiotic, doripenem maximizes its antibacterial effects and minimizes the potential for antibiotic resistance through careful therapeutic drug monitoring. Two online preconcentration techniques, field-enhanced sample stacking (FESS) and sweeping, were coupled to enhance the detection sensitivity. Briefly, an uncoated fused silica capillary (40 cm × 50 μm i.d) was rinsed with a high conductivity buffer (HCB) composed of 150 mM phosphate buffer (NaH2PO4, pH 2.5) and 20% methanol. A large sample plug prepared in a low-conductivity phosphate buffer (50 mM NaH2PO4, pH 2.5) was then hydrodynamically injected (5 psi, 80 s) into the capillary. Under an applied voltage of -30 kV, the analyte was accumulated at the FESS boundary and swept by the negatively charged micelles toward the UV detector. Plasma samples were pretreated by solid-phase extraction (SPE) to eliminate endogenous interferences. The validation results demonstrated a high coefficient of determination (r2 > 0.9995) for the regression curve with impressive precision and accuracy: relative standard deviation (RSD) <5.86% and relative error <4.63%. The limit of detection (LOD, S/N = 3) for doripenem was determined to be 0.4 μg/mL. Compared to the conventional micellar electrokinetic chromatography method, our developed method achieved a sensitivity enhancement of up to 488-fold for doripenem. Furthermore, the newly developed method successfully quantified doripenem concentrations in plasma samples obtained from patients accepting doripenem regimens, proving its application potential in the clinical realm.

On-line preconcentration of fluorescein derivatives of essential amino acids using sweeping in a nonionic micellar system

A rapid and effective method of nonionic micellar electrokinetic chromatography with laser-induced fluorescence detection and sweeping as an online preconcentration method was developed for the simultaneous determination of free essential amino acids in supplement formulations. The analytes were derivatized using fluorescein isothiocyanate isomer I dissolved in dimethyl sulfoxide. Optimal separation conditions were achieved using an uncoated fused silica capillary (40.0 cm effective length, 50.2 cm total length, 50 µm internal diameter), 20 mM Na2B4O7, (pH 9.3), 50 mM Brij-35, and 15% (v v-1) methanol as running buffer. Complete separation of all analytes was achieved within 15 min. The optimized method exhibited good linearity (r2 > 0.997), acceptable precision (1.3 – 12.4%), and accuracy (88 – 112%) in concentration range 20 – 1000 ng mL−1. The limit of detection was 6 ng mL−1. The method successfully determined the quantity of free essential amino acids in a dietary supplement containing spirulina.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30–1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120–210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1–13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Simultaneous determination of triclosan，triclocarban and p-chloro-m-xylenol in disinfectant，personal care products and oiltment by nonaqueous capillary electrophoresis

Triclosan （TCS）， triclocarban （TCC）， and p-chloro-m-xylenol （PCMX） are some of the most widely used antibiotics because of their broad-spectrum and highly-efficient bactericidal effects. In the context of disinfection， the National Standard GB 38598-2020 stipulates that the contents of the effective ingredients present in a disinfectant must be specified， wherein their range must fall within 90%-110% of the specified central value. To ensure a suitable product quality， analysis by high performance liquid chromatography （HPLC） is recommended by both the GB/T 27947-2020 and GB/T 34856-2017. However， the results analyzed according to the National Standard method often exceed the declared contents， thereby indicating the necessity to establish a new method based on a completely different principle （e. g.， capillary electrophoresis）， especially since it was not possible to analyze TCS， TCC， and PCMX in a single injection using the National Standard method. Moreover， using this method， large amounts of methanol were consumed， which could be potentially harmful to both operators and the environment. In terms of their water solubilities， this decreases in the order of PCMX>TCS>TCC， wherein TCC is insoluble in water. As such， the use of nonaqueous capillary electrophoresis （NACE） based on running buffer solutions prepared in pure organic solvents （e. g.， methanol or acetonitrile） is necessary. In this paper， a new NACE approach combined with an ultraviolet detection method was developed for the simultaneous analysis of TCS， TCC， and PCMX in disinfectants， personal care products， and ointments. For this purpose， an uncoated fused silica capillary （20 cm×50 μm， total length=30.2 cm） was used as the separation column with a separation buffer composed of 14 mmol/L sodium borate， 2 g/L polyethylene glycol （PEG） 20000， and 0.5 mmol/L dodecyltrimethylammonium bromide （DTAB） in methanol. Following optimization of the separation parameters， the complete and simultaneous separation of TCS， TCC， and PCMX was achieved when the sample solution was prepared using 5 g/L PEG 20000 in methanol-acetonitrile （50∶50， v/v）. It was possible to directly inject the sample into the analysis system after a simple dilution with the sample medium， and no interference was observed in any of the sample electropherograms when a separation voltage and detection wavelength of -12 kV and 214 nm were employed， respectively. Furthermore， TCS， TCC， and PCMX showed good linear relationships with their corrected peak areas within a mass concentration range of 1-100 mg/L， and the correlation coefficients （r） were all greater than 0.99. Moreover， the limits of detection （LODs， S/N=3） and limits of quantification （LOQs， S/N=10） were determined to be 0.2 and 1 mg/L， respectively. The spiked recoveries ranged from 94.5% to 104.4% with relative standard deviations of ≤4.8% in all cases. Subsequently， the established method was used to analyze 31 commercial samples， including hand sanitizer， disinfectant， baby powder， and antibacterial cream. A comparative analysis of HPLC， the developed NACE method， and our previously reported micellar electrokinetic chromatographic （MEKC） method was also carried out for the quantitative determination of TCS， TCC， and PCMX. Although no statistically significant differences were observed among the three methods， the results determined for 16 out of the 31 samples did not match the claimed contents. These results therefore indicate the necessity to further control the compositions of disinfectant products. Our results indicate that the newly established NACE method can be an important alternative to HPLC for routine laboratory analyses， especially considering that it minimizes waste generation， requires only a simple sample pretreatment process， and exhibits a good selectivity to the target compounds. It is therefore hoped that the NACE method will be incorporated into the National Standard method in the near future.

A Capillary Electrophoresis-Based Method for the Measurement of Hydroxychloroquine and Its Active Metabolite Desethyl Hydroxychloroquine in Whole Blood in Patients with Rheumatoid Arthritis.

A capillary electrophoresis method was developed to detect and measure hydroxychloroquine (HCQ) and its active metabolite desethyl hydroxychloroquine (DHCQ) in whole blood in patients with rheumatoid arthritis. The best separation in terms of peak area reproducibility, migration time, peak shape, and resolution of adjacent peaks was obtained in a 60 cm, 75 µm i.d. uncoated fused-silica capillary using a background electrolyte mixture of an aqueous 55 mmol/L TRIS solution brought to pH 2.6 with phosphoric acid and methanol (85:15) and a voltage and a temperature of separation of 20 kV and 30 °C, respectively. Analytes were separated in less than 12 min, with excellent linearity (R2 ≥ 0.999) in the concentration range of 0.5–8 µmol/L. The recovery of analytes spiked in whole blood was 99–101% for HCQ and 98–99% for DHCQ. Analysis of five samples from patients with rheumatoid arthritis receiving HCQ 400 mg daily yielded mean steady-state concentrations of 2.27 ± 1.61 and 1.54 ± 0.55 μmol/L for HCQ and DHCQ, respectively, with a HCQ to DHCQ ratio of 1.40 ± 0.77.

Determination of the enantiomers of salmeterol xinafoate in salmeterol fluticasone powder inhalant by chiral nonaqueous capillary electrophoresis

昔萘酸沙美特罗是目前治疗哮喘夜间发作和哮喘维持治疗的理想药物之一,它在临床上以外消旋体形式给药。昔萘酸沙美特罗的两个对映体在药理活性和毒理作用等方面差异较大,建立昔萘酸沙美特罗对映体的手性分离分析方法对提高手性药物质量、保证临床用药安全有效具有重要意义。该文以L(+)-酒石酸-硼酸络合酸为手性选择剂,建立了测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体含量的非水毛细管电泳法。实验考察了L(+)-酒石酸浓度、硼酸浓度和表观pH(apparent pH, pH* )对手性分离效果的影响。优化的缓冲溶液为:含120.0 mmol/L L(+)-酒石酸和120.0 mmol/L硼酸的甲醇溶液,pH* 为0.93;其他实验条件为:未涂层弹性熔融石英毛细管(内径50.0 μm,总长度64.5 cm,有效长度55.5 cm),重力进样17.5 cm×10.0 s,检测波长225 nm,室温,工作电压20.0 kV。在优化的实验条件下,昔萘酸沙美特罗的两个对映体在18.0 min内获得了2.18的分离度;在27.5~800.0 mg/L质量浓度范围内,与峰面积呈现良好的线性关系,相关系数(r)大于0.9990;检出限和定量限分别为7.5和25.0 mg/L;加标回收率为98.1%~101.9%,相对标准偏差为1.2%~1.9%。随机购买市面上出售的沙美特罗替卡松粉吸入剂,对其昔萘酸沙美特罗对映体的含量进行了分析检测。结果显示,昔萘酸沙美特罗对映体1和对映体2的标示量百分含量均为98.7%, RSD分别为2.5%和2.7%。该方法操作简便易行,结果准确可靠,消耗低,可用于市售沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体的含量测定。

Cyclodextrins as modulators for separation of charged variants of mAbs by capillary zone electrophoresis

Charge heterogeneity is a critical quality attribute for mAb-based therapeutics. Its characterization involves the identification of charge variant species in the final formulation. Capillary zone electrophoresis (CZE) is a valuable tool to assess charge heterogeneity, but typically offers incomplete separation of the different charged isoforms. Cyclodextrin-mediated chiral CE is a well-established electromigration method. In this paper, we propose the use of cyclodextrins as modulators for CZE based separation of mAb charge variants. Uncoated fused silica capillaries have been used for examining charge heterogeneity in biosimilars of Rituximab and Trastuzumab in presence of different cyclodextrins. Both the negatively charged CMβCD and uncharged HPβCD have been explored. The effect of cyclodextrin concentration has been found to impact the extent of modulation of resolution of charge variants. Addition of 10 mM CMβCD not only improved resolution but also resulted in separation of two additional basic and one additional acidic species for Rituximab. Similarly, besides improvement in the resolution of separation, one additional basic variant was observed with 2.5 mM CMβCD with another sample, Trastuzumab. The paper demonstrates that the effect of CMβCD is mAb specific. No improvement in resolution was, however, observed with HPβCD, suggesting the selective nature of cyclodextrins for mAb variants.

Development of a Capillary Electrophoretic Method for the Determination of Huperzine A Concentration in Vietnamese Huperzia serrata

Huperzine A, isolated from Huperzia serrata, is a potent, specific, and reversible inhibitor of acetylcholinesterase with high efficiency and low toxicity. To evaluate the presence of huperzine A in Vietnamese H serrata, a reliable capillary zone electrophoresis method was developed. The analytical conditions were established using 80 mM ammonium acetate buffer, pH 6.0, hydrodynamic injection at 50 mbar for 5 s, applied voltage of 20 kV, temperature at 25 °C, uncoated fused-silica capillary, 56 cm (50 cm effective length) × 70 µm inner diameter, and ultraviolet detection at 310 nm. The recovery rates ranged from 98.05% to 100.64%, with a relative standard deviation &lt;2%. Good linear regression was observed in the concentration range of 1 to 500 µg/mL, with a correlation coefficient of 0.9994. The limit of detection and limit of quantification were 0.33 and 1.0 µg/mL, respectively. These results demonstrate that this method is simple, selective, and suitable for performing quality control for huperzine A derived from Vietnamese H serrata.

Characterization of capillary inner surface conditions with streaming potential.

Streaming potential is created when an electrolyte solution is forced to flow pass a charged surface. For an uncoated fused silica capillary, the streaming potential is measured between the inlet and outlet vials while applying a pressure across the capillary. The changes in streaming potential can be used to characterize the properties of the capillary inner surface. In this work, HCl, NaCl, and NaOH solutions ranging from 0.4 to 6mM were used as the background electrolyte (BGE) at temperatures of 15 to 35 °C for the mesurements. The streaming potential decreases with the increase in BGE concentration, and the trend is amplified at higher temperatures. When buffer solutions in the pH range of 1.5 to 12.7 were used as the BGE, streaming potential was shown to be sensitive to changes in pH but reaches a maximum at around 9.5. At pH <3.3, no streaming potentials were observed. The pH of zero surface charge (streaming potential equals 0) changes with temperature, and is measured to be 3.3 to 3.1 when the temperature is changed from 15 to 35°C. Zeta potentials can be calculated from the measured streaming potential, conductivity, and the solution viscosity. Surface charge densities were calculated in this work using the zeta potentials obtained. We demonstrated that capillary surface conditions can significantly change the streaming potential, and with three different solutions, we showed that analyte-dependent adsorption can be monitored and mitigated to improve the peak symmetry and migration times reproducibility.

A rapid electrokinetic chromatography method using short-end injection for the enantioselective separation of tryptophan

A rapid enantioselective methodology for the analysis of tryptophan was developed in this work by electrokinetic chromatography using short-end injection and an anionic cyclodextrin as chiral selector. No previous derivatization of tryptophan was necessary. The influence of different experimental variables on the enantiomeric separation was investigated. The use of a 100 mM formate buffer (pH 2.2) containing 1.25% sulfated-γ-cyclodextrin with an uncoated fused-silica capillary of 50 µm inner diameter with a total length of 48.5 cm (effective length of 8.5 cm), and an injection by applying a pressure of −50 mbar (short-end injection) for 4 s, enabled the enantiomeric separation of tryptophan within 2.5 min with a resolution of 7.4. As desirable, the enantiomeric impurity, D-tryptophan, was the first-migrating enantiomer. The analytical characteristics of the developed methodology were evaluated in terms of linearity, precision, accuracy, and limits of detection and quantification, showing its good performance to be applied to the analysis of tryptophan-based dietary supplements. A relative limit of detection of 0.1% was obtained for the enantiomeric impurity, D-tryptophan, in the presence of the L-enantiomer. Results showed that the developed methodology is an interesting alternative for the enantioselective analysis of tryptophan enabling the rapid quality control of dietary supplements.

Simultaneous determination of four phenolic acids in traditional Chinese medicine by capillary electrophoresis-chemiluminescence.

Chlorogenic, ferulic, vanillic, and caffeic acids are phenolic acids found in natural drugs. They possess the biological activities of scavenging free radicals and inhibiting thrombus formation. Phenolic acids can inhibit the oxidation of low-density lipoprotein, as well as have anti-inflammatory effects. This paper reports for the first time a capillary electrophoresis–chemiluminescence (CE–CL) method for the simultaneous determination of the four phenolic acids found in traditional and proprietary Chinese medicine, including Lycium chinense Miller, Shuanghuanglian oral liquid, and Taraxacum mongolicum granules. Capillary electrophoretic separation was performed on a self-assembled CE–CL device with an uncoated fused-silica capillary (66 cm effective length, 50 μm i.d.), and the background electrolyte was composed of 3.0 × 10−5 M Ag(iii) (pH = 12.01), 3.0 mM luminol (pH = 9.20), and 10 mM sodium tetraborate solution. The injection time was 12 s (under gravity) and the separation voltage was 22 kV. The combination of solid-phase extraction (SPE) and CE–CL improves the sensitivity. Under optimal conditions, calibration graphs displayed a linear range between 0.625 and 20.0, 1.000 and 30.0, 0.150 and 1.50, and 0.045 and 1.00 μg mL−1 for chlorogenic, ferulic, vanillic, and caffeic acid, respectively. The detection limit ranged from 0.014 to 0.300 μg mL−1. The practicality of using the proposed method to determine the four target analytes in traditional Chinese medicine was also validated, in which recoveries ranged from 90.9% to 119.8%. Taken together, these results indicate that the developed method is sensitive and reliable. Furthermore, the method was successfully applied to real traditional Chinese medicine samples.

Optimization and validation of a new approach based on CE-HRMS for the screening analysis of novel psychoactive substances (cathinones, phenethylamines, and tryptamines) in urine.

The continuous introduction in the market of new psychoactive drugs (NPS) represents a well-known international emergency. Indeed, the European Monitoring Centre for Drugs and Drug Addiction and the United Nations Office on Drugs and Crime are paying great attention to the spread of NPS. In addition to the traditional analytical approaches based on GC-MS and HPLC-MS, also CE coupled with MS has proved to be a precious tool for the toxicological screening of biosamples. On these grounds, the aim of the present work was to test the application of CE-HRMS as a new screening tool for the rapid detection of these novel drugs in urine. Separations were performed in an uncoated fused-silica capillary with id of 75μm with a total length of 100cm, by applying a constant voltage of 15kV. The QTOF-MS was implemented with an electrospray ion source operating in positive ionization full scan mode in the range of 100-1000 m/z. Under these conditions, different NPS has been tested, including eight cathinones, five phenethylamine, and seven tryptamines. The method was validated after optimization of the following analytical parameters: BGE composition and pH, separation voltage, sheath liquid composition, and flow rate and ESI source settings. The applicability of the method was successfully tested by analyzing a series of real urine samples obtained from drug users.

A capillary electrophoresis method for the determination of the linagliptin enantiomeric impurity

Linagliptin is a highly specific, long-acting inhibitor that is used as an orally administrable agent for type-2 diabetes treatment. Because only the R-enantiomer is of clinical use, we developed a capillary electrophoresis method for the determination of the enantiomeric impurity of this compound. Carboxymethyl-β-cyclodextrin was selected as the chiral selector for the separation of linagliptin enantiomers. Design of experiments and desirability functions were used for the analytical optimization, which was focused on understanding and improving the electrophoretic process. The effects of significant parameters (background electrolyte concentration and pH, cyclodextrin concentration, temperature, and voltage) were thoroughly investigated. The complete separation of linagliptin and its enantiomeric impurity with baseline resolution was achieved within 10min on an uncoated fused-silica capillary (50μm inner diameter, 365μm outer diameter, 64.5/56cm in total/ effective length) maintained at 25°C, under an applied voltage of 28.0kV. The background electrolyte contained 70mM sodium acetate and 4.7mM carboxymethyl-β-cyclodextrin, and the pH was adjusted to 6.10. The method was validated, and a limit of quantitation of 0.05% for the impurity was estimated.

Application of micellar electrokinetic capillary chromatography for identification, quantitative detection and unfolding analysis of interleukin-12

To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process. An uncoated fused-silica capillary (inner diameter 50 μm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states. A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak. This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.

Development, validation, and application of capillary zone electrophoresis method for determination of pyrimidine glucosides in seeds of Vicia faba L. var. minor.

Consumption of fava beans seeds can be harmful to both humans and animals due to the presence of pyrimidine glucosides: vicine and convicine. Therefore, seeds, feed supplements, and protein extracts derived from fava beans should be monitored for the contents of pyrimidine derivatives. The main objective of this work was the development and validation of an inexpensive, uncomplicated, quick, and robust method for the quantitation of vicine and convicine in seeds of fava beans. Plant material was extracted using a dual-phase extraction system to remove lipophilic contaminants and deactivate residual enzymes. Pyrimidine glucosides together with the internal standard: 5-bromouridine were separated using capillary zone electrophoresis (CZE) on an uncoated fused-silica capillary with alkaline, tetraborate-based electrolyte. Linearity, limits of detection and quantitation, precision, accuracy, recovery, and robustness of the method were investigated. The method was applied for quantitation of pyrimidine glucosides in seeds. Validation results suggest that the method is suitable for quantitation of pyrimidine glucosides, although it may not be sensitive enough for low-concentrated samples. Application of the method showed that seeds of the investigated variety of fava beans contain both vicine at 10-15 mg/g of dry weight and convicine at 3-5 mg/g of dry weight. Developed and validated CZE-ultraviolet (UV) method is suitable for analysis of pyrimidine glucosides in seeds of Vicia faba L. var. minor. It can be used as an inexpensive, greener alternative to more advanced methods while providing equally reliable and accurate results.

Simple and Rapid Micellar Electrokinetic Chromatography Method for Simultaneous Determination of Febuxostat and its Related Impurities

A micellar electrokinetic capillary chromatography (MEKC) method has been developed and validated for simultaneous analysis of febuxostat and its related substances in active pharmaceutical ingredients and pharmaceutical formulations. Analysis was performed in an uncoated fused silica capillary (34 cm effective length, 50 µm diameter). Method sensitivity was enhanced using an extended light path capillary, with an optical path 150 µm and the choice of 320 nm detector operating wavelength due to lower background noise and higher selectivity. BGE consisted of 20 mM borate buffer pH 9.3 and 50 mM SDS and 2% acetonitrile. Analysis was performed at 30 kV separation voltage and 32 °C capillary temperature. The method was linear over the range 1.0–10.0 µg mL−1 for each impurity corresponding to reporting limits regarding the ICH Q3A guidelines. The method has proven to be selective, precise, accurate and robust. Proposed MEKC method has been successfully applied for the quantitative determination of febuxostat and purity assay of active pharmaceutical ingredient and tablet dosage forms.

Simultaneous determination of twelve biogenic amines in human urine as potential biomarkers of inflammatory bowel diseases by capillary electrophoresis – tandem mass spectrometry

Biogenic amines (BA) are a broad group of biologically active substances, the presence of which in the human body can provide important diagnostic information for many various pathologies, including chronic inflammation. In this work, a capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of twelve BA (histamine, serotonin, dopamine, norepinephrine, epinephrine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine, phenylethylamine) in human urine as potential biomarkers of inflammatory bowel diseases (IBD). The electrophoretic separations were carried out in an uncoated fused silica capillary (I.D. 50 μm) using 50 mM formic acid (pH 2.0) as a background electrolyte. A reliable identification of the analytes was based on the combination of time resolution in CE and mass resolution in triple quadrupole MS/MS. The total analysis time of the proposed CEMS/MS method was less than 10 min with the limits of detection in the range of 4.47–144 ng/mL. The intra- and inter-day accuracy ranged in the intervals 89.75–109.4% and 89.99–110.2%, respectively, with the RSD values for the intra- and inter-day precision lower than 14 and 13 %, respectively. The recovery values for the samples spiked at three concentration levels ranged from 81.73–105.6% with a precision not exceeding 9.9 %. The favorable performance parameters of the CEMS/MS method highlighted its usefulness for routine clinical applications. In this work, the CEMS/MS method was applied, for the first time, to the analytical profiling of the BA in clinical human samples. The obtained results showed a statistically significant decrease of serotonin and norepinephrine, and an increase of histamine and spermidine, in the studied group of IBD patients when compared with the control group. These findings could be utilized in studying and clarifying the mechanisms of IBD or relevant therapy.

Combination of large volume sample stacking with polarity switching and cyclodextrin electrokinetic chromatography (LVSS-PS-CDEKC) for the determination of selected preservatives in pharmaceuticals

In this study, a large volume sample stacking (LVSS) with polarity switching (PS) and cyclodextrin electrokinetic chromatography (CDEKC) method has been developed for the simultaneous separation and determination of 8 preservatives: methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), isobutylparaben (IBP), sorbic acid (SA), benzoic acid (BA), p-hydroxybenzoic acid (PHBA) in pharmaceuticals. The effects of some typical parameters such as sample volume, applied voltage, composition and pH of the running buffer and organic modifier concentration were examined and optimized. Moreover, the impact of type and concentration of cyclodextrin as electrolyte modifiers was also investigated. The detection limits of analytes for the elaborated LVSS-PS-CDEKC method were found to be in 0.8–5 ng mL−1 range, which were around 500 times lower than normal CDEKC without preconcentration technique. All analytes were completely resolved in less than 11 min in an uncoated fused-silica capillary of 75 μm internal diameter (I.D) x 50 cm length. The electrophoretic separation was performed in a 2 mM α-cyclodextrin and 25 mM tetraborate system (pH = 9.3) with an applied voltage of 25 kV. The established method was validated and confirmed to be applicable for the determination of the preservatives in a quality control of pharmaceuticals.

Micellar electrokinetic capillary chromatographic determination of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients receiving treatment for idiopathic pulmonary fibrosis (IPF)

A micellar electrokinetic capillary chromatographic method, a variant of capillary electrophoresis, was developed for the detection and measurement of pirfenidone and 5-carboxy-pirfenidone by direct injection of plasma from patients under treatment for idiopathic pulmonary fibrosis. To achieve the best separation a 35 mmol/L N-lauroylsarcosine sodium salt surfactant added with a 16 mmol/L sodium 1-heptanesulfonate solution was used as a running buffer. Analytes were eluted in a fairly short time, 7.35 and 9.40 min for pirfenidone and 5-carboxy-pirfenidone, respectively, at 15 °C and with an applied voltage of 30 kV in an 80 cm, 75 µm i.d. uncoated fused-silica capillary. In the range of 6.25–200 µmol/L, calibration curves exhibited acceptable linearity with the coefficient of determination higher than 0.999. The recovery of analytes spiked in samples of plasma was found to be 95–109% and 93–106% for pirfenidone and 5-carboxy-pirfenidone, respectively. Intra- and inter-day precisions were around 5%. LOD and LOQ for pirfenidone and 5-carboxy-pirfenidone were, respectively, 1.0 and 4.0 µmol/L. The method, applied to measure analytes in six idiopathic pulmonary fibrosis male patients, provided mean concentrations of 68.91 ± 26.31 and 36.21 ± 26.39 µmol/L for pirfenidone and 5-carboxy-pirfenidone, respectively.

Database of free solution mobilities for 276 metabolites

Although databases are available that provide mass spectra and chromatographic retention information for small-molecule metabolites, no publicly available database provides electrophoretic mobility for common metabolites. As a result, most compounds found in electrophoretic-based metabolic studies are unidentified and simply annotated as “features”. To begin to address this issue, we analyzed 460 metabolites from a commercial library using capillary zone electrophoresis coupled with electrospray mass spectrometry. To speed analysis, a sequential injection method was used wherein six compounds were analyzed per run. An uncoated fused silica capillary was used for the analysis at 20 °C with a 0.5% (v/v) formic acid and 5% (v/v) methanol background electrolyte. A Prince autosampler was used for sample injection and the capillary was coupled to an ion trap mass spectrometer using an electrokinetically-pumped nanospray interface. We generated mobility values for 276 metabolites from the library (60% success rate) with an average standard deviation of 0.01 × 10−8 m2V−1s−1. As expected, cationic and anionic compounds were well resolved from neutral compounds. Neutral compounds co-migrated with electro-osmotic flow. Most of the compounds that were not detected were neutral and presumably suffered from adsorption to the capillary wall or poor ionization efficiency.