Determination of the Enantiomers of Mianserin and its Metabolites in Plasma by Capillary Electrophoresis After Liquid-Liquid Extraction and On-Column Sample Preconcentration

Capillary electrophoresis has drawn considerable attention in the past few years, particularly in the field of chiral separations because of its high separation efficiency. However, its routine use in therapeutic drug monitoring is hampered by its low sensitivity due to a short optical path. We have developed a capillary zone electrophoresis (CZE) method using 2mM of hydroxypropyl-β-cyclodextrin as a chiral selector, which allows base-to-base separation of the enantiomers of mianserin (MIA), desmethylmianserin (DMIA), and 8-hydroxymianserin (OHMIA). Through the use of an on-column sample concentration step after liquid-liquid extraction from plasma and through the presence of an internal standard, the quantitation limits were found to be 5 ng/mL for each enantiomer of MIA and DMIA and 15 ng/mL for each enantiomer of OHMIA. To our knowledge, this is the first published CE method that allows its use for therapeutic monitoring of antidepressants due to its sensitivity down to the low nanogram range. The variability of the assays, as assessed by the coefficients of variation (CV) measured at two concentrations for each substance, ranged from 2 to 14% for the intraday (eight replicates) and from 5 to 14% for the interday (eight replicates) experiments. The deviations from the theoretical concentrations, which represent the accuracy of the method, were all within 12.5%. A linear response was obtained for all compounds within the range of concentrations used for the calibration curves (10-150 ng/mL for each enantiomer of MIA and DMIA and 20-300 ng/mL for each enantiomer of OHMIA). Good correlations were calculated between [(R) + (S)]-MIA and DMIA concentrations measured in plasma samples of 20 patients by a nonchiral gas chromatography method and CZE, and between the (R)- and (S)-concentrations of MIA and DMIA measured in plasma samples of 37 patients by a previously described chiral high-performance liquid chromatography method and CZE. Finally, no interference was noted from more than 20 other psychotropic drugs. Thus, this method, which is both sensitive and selective, can be routinely used for therapeutic monitoring of the enantiomers of MIA and its metabolites. It could be very useful due to the demonstrated interindividual variability of the stereoselective metabolism of MIA.

Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.

Differential protein labeling with 2-DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared-based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide-conjugated infrared dyes DY-680 and DY-780 followed by 1- and 2-DE. The procedure allows amounts of less than 5 microg of cysteine-labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2-DE step with an LOD of individual proteins in the femtogram range; however, co-migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol-labeled proteins, such as tubulin, were identified by MS, with cysteine-containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared-labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low-abundant cysteine-containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.

Biochemical identification of three sympatric Apodemus species by protein electrophoresis of blood samples

The allelic pattern of seralbumine and general protein 1 of the three sympatric Apodemus species Apodemus sylvaticus, A. flavicollis, and A. alpicola, were studied using electrophoretic analysis of blood samples, This method appears to be a sensitive tool for distinguishing the three Apodemus species in the Alps, Their identification on the basis of external characteristics in the field is sometimes extremely difficult, even more so for juvenile specimens. Compared to previously described methods the electrophoretic analysis does not require killing animals and can be used on juveniles.

Analysis of Markers for Recent Alcohol Consumption by Capillary Zone Electrophoresis and an Immunochemical Method

Le but de ce travail doctoral était le développement de méthodes analytiques pour la détermination dethyl glucuronide et dethyl sulfate. Ces deux substances sont des métabolites directs de lethanol qui peuvent être détectées pendant des heures jusqu'à des jours dans des fluides corporels, après que léthanol ait été complètement éliminé du corps humain. Ce sont donc des marqueurs de consommation récente d'alcool.La majorité des expériences ont été effectuées en utilisant l'électrophorèse capillaire. Il était envisagé de fournir des méthodes utilisables dans des laboratoires de routine. Des méthodes électrophorétiques ont été développées et optimisées pour la détermination dethyl sulfate dans le sérum et l'urine ainsi que pour lethyl glucuronide dans le sérum. Lethyl glucuronide urinaire a pu être déterminé par un immunoassay commerciale qui a en plus été adapté avec succès pour des échantillons de sérum. Avec toutes ces méthodes d'analyse il était possible d'observer les deux marqueurs de consommation d'alcool récente, même une consommation aussi basse qu'un verre de boissons alcooliques.Finalement, une étude englobant plus de 100 échantillons aété effectuée avec l'ambition de déterminer les valeurs de référence pour lethyl glucuronide dans le sérum et l'urine. De plus, la nécessité de normaliser les échantillons d'urine par rapport à la dilution a été investiguée. Grâce à cette étude des valeurs de cut-off et une base statistique pour l'interprétation probabiliste ont pu être proposées.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

Information transfer between large and small two-dimensional polyacrylamide gel electrophoresis.

To determine the feasibility of data transfer, an interlaboratory comparison was conducted on colon carcinoma cell line (DLD-1) proteins resolved by two-dimensional polyacrylamide gel electrophoresis either on small (6 x 7 cm) or large (16x18 cm) gels. The gels were silver-stained and scanned by laser densitometry, and the image obtained was analyzed using Melanie software. The number of spots detected was 1337+/-161 vs. 2382+/-176 for small vs. large format gels, respectively. After gel calibration using landmarks determined using pl and Mr markers, large- and small-format gels were matched and 712+/-36 proteins were found on both types of gels. Having performed accurate gel matching it was possible to acquire additional information after accessing a 2-D PAGE reference database (http://www.expasy.ch/ cgibin/map2/def?DLD1_HUMAN). Thus, the difference in gel size is not an obstacle for data transfer. This will facilitate exchanges between laboratories or consultation concerning existing databases.

Simultaneous analysis of saturated and unsaturated fatty acids present in pequi fruits by capillary electrophoresis

In the current study, an alternative method has been proposed for simultaneous analysis of palmitic, stearic, oleic, linoleic, and linolenic acids by capillary zone electrophoresis (CZE) using indirect detection. The background electrolyte (BGE) used for the analysis of these fatty acids (FAs) consisted of 15.0 mmol L−1 NaH2PO4/Na2HPO4 at pH 6.86, 4.0 mmol L−1 SDBS, 8.3 mmol L−1 Brij 35, 45% v/v acetonitrile (can), and 2.1% n-octanol. The FAs quantification of FAs was performed using a response factor approach, which provided a high analytical throughput for the real sample. The CZE method, which was applied successfully for the analysis of pequi pulp, has advantages such as short analysis time, absence of lipid fraction extraction and derivatization steps, and no significant difference in the 95% confidence intervals for FA quantification results, compared to the gas chromatography official method (AOCS Ce 1h-05).

Allelopathic potential and systematic evaluation of secondary compounds in extracts from roots of Canavalia ensiformis by capillary electrophoresis

Organic extracts were obtained from roots of Canavalia ensiformis and evaluated for allelopathic potential on the germination of the weed seeds: Mimosa pudica, Cassia tora and Cassia occidentalis showing a strong allelopathic potential. After that, a systematic study of these crude extracts was made using specific protocols developed in capillary electrophoresis (CE) in order to determine some classes of secondary metabolites. Capillary electrophoresis protocols were highly specific, which makes it possible to identify 5 classes of compounds using the same crude extract samples and analyze them fartly. Some of the compounds identified show activity in the inhibition of seeds germination.

Comparison of Potassium and Sodium Content in Diet and Non-Diet Soft Drinks by Using Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) was used for determination of sodium and potassium concentrations in diet and non-diet soft drinks. Higher sodium concentrations were found in the diet samples due to the utilization of sodium salts of cyclamate and saccharine as sweeteners. The CE-C4D method can be used by food industries and health regulatory agencies for monitoring sodium and potassium content, not only in soft drink but in many others food products.

Determination of sugars and organic acids in pulp samples by capillary electrophoresis with UV detection

This MSc work was done in the project of BIOMECON financed by Tekes. The prime target of the research was, to develop methods for separation and determination of carbohydrates (sugars), sugar acids and alcohols, and some other organic acids in hydrolyzed pulp samples by capillary electrophoresis (CE) using UV detection. Aspen, spruce, and birch pulps are commonly used for production of papers in Finland. Feedstock components in pulp predominantly consist of carbohydrates, organic acids, lignin, extractives, and proteins. Here in this study, pulps have been hydrolyzed in analytical chemistry laboratories of UPM Company and Lappeenranta University in order to convert them into sugars, acids, alcohols, and organic acids. Foremost objective of this study was to quantify and identify the main and by-products in the pulp samples. For the method development and optimization, increased precision in capillary electrophoresis was accomplished by calculating calibration data of 16 analytes such as D-(-)-fructose, D(+)-xylose, D(+)-mannose, D(+)-cellobiose, D-(+)-glucose, D-(+)-raffinose, D(-)-mannitol, sorbitol, rhamnose, sucrose, xylitol, galactose, maltose, arabinose, ribose, and, α-lactose monohydratesugars and 16 organic acids such as D-glucuronic, oxalic, acetic, propionic, formic, glycolic, malonic, maleic, citric, L-glutamic, tartaric, succinic, adipic, ascorbic, galacturonic, and glyoxylic acid. In carbohydrate and polyalcohol analyses, the experiments with CE coupled to direct UV detection and positive separation polarity was performed in 36 mM disodium hydrogen phosphate electrolyte solution. For acid analyses, CE coupled indirect UV detection, using negative polarity, and electrolyte solution made of 2,3 pyridinedicarboxylic acid, Ca2+ salt, Mg2+ salts, and myristyltrimethylammonium hydroxide in water was used. Under optimized conditions, limits of detection, relative standard deviations and correlation coefficients of each compound were measured. The optimized conditions were used for the identification and quantification of carbohydrates and acids produced by hydrolyses of pulp. The concentrations of the analytes varied between 1 mg – 0.138 g in liter hydrolysate.

Determination of decomposition products of flotation collector chemicals using capillary electrophoresis

Vaahdotusta käytetään yleisesti erottamaan eri mineraaleja malmista. Tässä menetelmässä käytetään erityisiä pinta-aktiivisia aineita, joita kutsutaan kokoojakemikaaleiksi, muuntamaan halutut mineraalit hydrofobisiksi ja erottamaan ne hydrofiilisistä partikkeleista ilmakuplien avulla. Eräs tärkeimmistä kokoojakemikaalien ryhmistä on ksantaatit. Ksantaateilla on havaittu taipumusta hajota useiksi erilaisiksi hajoamistuotteiksi vaahdotusprosessin aikana. Näillä hajoamistuotteilla voi olla monia haitallisia vaikutuksia vaahdotuksen tuloksiin. Näiden tuotteiden tunnistaminen ja määrittäminen on tärkeää vaahdotusprosessin paremman ymmärtämisen kannalta. Työn kirjallisuusosassa vaahdotusprosessi, ksantaatit ja niiden yleisimmät hajoamistuotteet on esitelty, kuten myös käytetty analyysimenetelmä, kapillaarielektroforeesi. Työn kokeellisessa osassa etsittiin sopivaa erotusmenetelmää etyyliksantaatin, etyylitiokarbonaatin, etyyliperksantaatin ja etyyliksantyylitiosulfaatin erottamiseksi kapillaarilelektroforeesilla. Pääasiassa keskityttiin kahteen eri erotusmenetelmään. Ensimmäinen menetelmä kykeni erottamaan kaikki tutkitut tuotteet puhdasvesinäytteissä, ja toinen menetelmä oli sopiva näiden tuotteiden erottamiseen prosessivesinäytteissä. Jälkimmäistä menetelmää kokeiltiin käytännössä rikastamolla, jossa sillä kyettiin erottamaan isobutyyliksantaatti, isobutyylitiokarbonaatti, ja suurella todennäköisyydellä myös isobutyyliperksantaatti.

Determination of collector chemicals from flotation process waters using capillary electrophoresis

Vaahdotusprosessia käytetään yleisesti erottamaan arvokkaita mineraaleja malmeista. Toimiakseen tehokkaasti prosessi tarvitsee kokoojakemikaaleja, joiden tehtävänä on sitoa halutut mineraalit ilmakupliin. Jotta näiden kemikaalien käyttäytymistä prosessissa voitaisiin ymmärtää paremmin ja prosessin ohjausta tehostaa, pitää kokoojia pystyä analysoimaan prosessivesistä. Työn kirjallisuusosassa on koottu ja vertailtu erilaisia kirjallisuudesta löytyneitä analyysimenetelmiä kokoojakemikaaleille. Kokeellisessaosassa on kehitetty kaksi kapillaarielektroforeesimenetelmää näiden kemikaalien tutkimiseen. Menetelmien toteamisrajat tutkituille kemikaaleille olivat seuraavanlaiset: natrium diiosobutylditiofosfaattille (DTP) 2,7 mg/L puhtaassa vedessä ja 6,7 mg/L prosessivedessä; natrium diisobutyldithiofosfinaatille (DTPI) vastaavasti 4,5 mg/L ja 6,7 mg/L; etyyli ksantaatille 0,025 mg/L ja 0,16 mg/L; ja isobutyyli ksantaatille 0,41 mg/L ja 0,62 mg/L. Näitä menetelmiä voidaan tulevaisuudessa kehittää kokoojien hajoamistuotteiden analysointia varten sekä prosessien on-line mittauksiin.

Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

Bioprocess technology is a multidisciplinary industry that combines knowledge of biology and chemistry with process engineering. It is a growing industry because its applications have an important role in the food, pharmaceutical, diagnostics and chemical industries. In addition, the current pressure to decrease our dependence on fossil fuels motivates new, innovative research in the replacement of petrochemical products. Bioprocesses are processes that utilize cells and/or their components in the production of desired products. Bioprocesses are already used to produce fuels and chemicals, especially ethanol and building-block chemicals such as carboxylic acids. In order to enable more efficient, sustainable and economically feasible bioprocesses, the raw materials must be cheap and the bioprocesses must be operated at optimal conditions. It is essential to measure different parameters that provide information about the process conditions and the main critical process parameters including cell density, substrate concentrations and products. In addition to offline analysis methods, online monitoring tools are becoming increasingly important in the optimization of bioprocesses. Capillary electrophoresis (CE) is a versatile analysis technique with no limitations concerning polar solvents, analytes or samples. Its resolution and efficiency are high in optimized methods creating a great potential for rapid detection and quantification. This work demonstrates the potential and possibilities of CE as a versatile bioprocess monitoring tool. As a part of this study a commercial CE device was modified for use as an online analysis tool for automated monitoring. The work describes three offline CE analysis methods for the determination of carboxylic, phenolic and amino acids that are present in bioprocesses, and an online CE analysis method for the monitoring of carboxylic acid production during bioprocesses. The detection methods were indirect and direct UV, and laser-induced frescence. The results of this work can be used for the optimization of bioprocess conditions, for the development of more robust and tolerant microorganisms, and to study the dynamics of bioprocesses.

In-House Validation of Capillary Electrophoresis Method

Capillary electrophoresis method designed originally for the analysis of monosaccharides was validated using reference solutions of polydatin. The validation was conducted by studying and determining the concentration levels of LOD and LOQ and the range of linearity and by determining levels of uncertainty in respect to repeatability and reproducibility. The reliability of the gained results is also discussed. A guide with recommendations considering the validation and overall design of analysis sequences with CE is also produced as a result of this study.