Design of strategies to study the metabolic profile of highly polar compounds in plasma by reversed-phase liquid chromatography-high resolution mass spectrometry

Amino acids and related compounds are paramount analytes which are involved in numerous metabolic pathways. Most of these compounds are unable to be retained on Liquid Chromatography with Reversed Phase stationary phases due to their high hydrophilic character. An interesting strategy is to reduce their polarity through their derivatization with a labelling reagent, such as the commercially available 9-fluorenylmethyloxycarbonyl (FMOC) which forms stable complexes with primary and secondary amine moieties rapidly. Although some derivatization reagents have been employed in the study of metabolic profiles, as far as we know, FMOC has never been employed for this purpose. In this work, it is demonstrated that the use of RP-LCMS(TOF) using a C18 column and FMOC as labelling agent enables the determination of a larger number of hydrophilic compounds (proteinogenic amino acids, non-proteinogenic amino acids, and biogenic amines) when compared to the use of a fully -wettable pentafluorophenyl column in fully-aqueous conditions (gradient starting in 0% of organic solvent) and HILIC column, both without using compound derivatization. Different strategies for plasma protein elimination were also carefully evaluated. Results revealed that ultrafiltration (UF) offered a lower variability from sample to sample when compared to the protein precipitation (PP) method (from 2 to 12 times lower variability found in UF). Additionally, UF preserved a larger number of possible compounds when compared to the PP approach: 4631 unique molecular features with UF, 666 unique molecular features with PP. (C) 2017 Elsevier B.V. All rights reserved

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils.

Accepted, revised and published in "Journal of Cromatography A", 2001, 1218 (30), pp. 4944-4951. DOI: 10.1016/j.chroma.2011.01.045A new analytical methodology based on capillary electrophoresis–mass spectrometry (CE–MS2) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS2 detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD 0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS2 experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication.Acknowledgements Authors thank the Spanish Ministry of Science and Innovation (project CTQ2009-09022) and the Comunidad Autónoma of Madrid (Spain) and European funding from FEDER programme (project S2009/AGR-1464, ANALISYC-II). They also thank the University of Alcalá and the Comunidad Autónoma of Madrid for project CCG10-UAH/AGR-5950. Laura Sánchez-Hernández thanks the Comunidad Autónoma of Madrid for her research contrac

Comparison of charged cyclodextrin derivatives for the chiral separation of atropisomeric polychlorinated biphenyls by capillary electrophoresis

Charged cyclodextrin (CD) derivatives were used as chiral selectors in electrokinetic chromatography (EKC) for the chiral separation of highlyhydrophobic neutral racemates such as atropisomeric polychlorinated biphenyls (PCBs). -CD-phosphated, -CD sulfated, succinylated--CD (Succ--CD) and succinylated- -CD (Succ- -CD) were used as anionic CDs. As cationic CD, 6-monodeoxy-6-monoamino- -CD ( -CD-NH2) was tested for the first time in order to separate PCBs. From the different CD derivatives employed, the best separations were obtained with the cationic CD derivative. Thus, the use of -CD-NH2 in phosphate buffer at pH 2.0 containing urea allowed the chiral recognition of eleven PCBs (45, 84, 88, 91, 95, 131, 136, 144, 149, 176, and 197). In this case, the addition of 2 M urea to the buffer solution was crucial to achieve the chiral separation of PCBs. The addition of acetonitrile to 10 mM phosphate buffer (pH 2.0) with 30 mM -CD-NH2 and 2 M urea improved considerably the chiral resolution obtained for PCBs 91, 95, 136, 144, 149, and 197 although an increase in the analysis time was also observed. All the results obtained were compared with those previously obtained with the dual CD system carboxymethyl--CD/ -CD.

Chiral separation of a basic drug with two chiral centers by electrokinetic chromatography for its pharmaceutical development

A chiral method using capillary electrophoresis was developed for the separation of the four stereoisomers of a new chiral substance currently undergoing drug development as single enantiomer. After the selection of highly sulfated beta-CD as chiral selector, an exhaustive study on the influence of several experimental variables on the resolution was performed, being the substitution degree of the CD a very decisive factor. Run time and resolutions were about 20 min and higher than 2.0, respectively. The method was validated in terms of selectivity, linearity, accuracy, precision, and limits of detection and quantitation according to the requirements of the International Conference on Harmonisation for the determination of the chiral purity of a drug substance. The usefulness of the method was demonstrated in the control of stereoisomeric impurities in raw material as well as in the determination of the chiral stability of the drug in the solid state and in dosage forms used in safety assessment. Finally, the chiral method was used to investigate the possible in vivo inversion in biological samples. (C) 2016 Elsevier B.V. All rights reserved

Development of chiral methodologies by Capillary Electrophoresis with ultraviolet and Mass Spectrometry detection for duloxetine analysis in pharmaceutical formulations

Two chiral methodologies were developed by capillary electrophoresis (CE) with UV and mass spectrometry (MS) detection to ensure the quality control of the drug duloxetine, commercialized as a pure enantiomer. Both methods were optimized to achieve a high baseline enantioresolution (Rs > 2) and an acceptable precision (RSD values 0.05). (C) 2014 Elsevier B.V. All rights reserved

Determination of betaines in vegetable oils by capillary electrophoresis tandem mass spectrometry. Application to the detection of olive oil adulteration with seed oils.

The final publication is available at Springer via http://dx.doi.org/10.1002/elps.201100005A capillary electrophoresis-tandem mass spectrometry methodology enabling the simultaneous determination of betaines (glycine betaine, trigonelline, proline betaine and total content of carnitines) in vegetable oils was developed. Betaines were derivatized with butanol previous to their baseline separation in 10 min using a 0.1 M formic acid buffer at pH 2.0. Ion trap conditions were optimized in order to maximize selectivity and sensitivity. Analytical characteristics of the proposed method were established by evaluating its selectivity, linearity, precision (RSDs ranged from 4.8% to 10.7% for corrected peak areas), accuracy by means of recovery studies (from 80% to 99%) and LODs and LOQs at 0.1 ppb level. The method was applied to the determination of the selected betaines in seed oils and extra virgin olive oils. MS2 experiments provided the fingerprint fragmentation for all betaines studied in seed oils. In extra virgin olive oils, carnitines were not detected being possible to propose them as a feasible novel marker for the detection of adulterations of olive oils. Application of the developed method to the analysis of different mixtures of extra virgin olive oil with seed oil (between 2-10 %) enabled the detection and quantitation of the total content of carnitines. The results obtained show the high potential of the developed method for the authentication and quality control of olive oils.Authors thank the Spanish Ministry of Science and Innovation (project CTQ2009- 09022) and the Comunidad Autónoma of Madrid (Spain) and european funding from FEDER programme (project S2009/AGR-1464, ANALISYC-II). They also thank the University of Alcalá and the Comunidad Autónoma of Madrid for project CCG10- UAH/AGR-5950. Laura Sánchez-Hernández thanks the Comunidad Autónoma of Madrid for her research contract

Recent contributions of Capillary Electrophoresis to neuroscience

Contributions to neuroscience are necessary to understand the behavior of the brain. Powerful analytical techniques are needed to monitor neuroactive molecules and their concentrations in biological samples (fluids, cells, and brain tissues). Capillary electrophoresis (CE) is well known for its high resolution power, short analysis times, and low consumption of reagents and samples. It presents analytical advantages for the determination of neuroactive molecules not easily determined by other analytical techniques. CE also offers the possibility of controlling more than one neuroactive molecule at a time, making it interesting to detect changes as a result of a stimulus. CE is well established to accomplish enantioseparations, contributing a better understanding of the properties of a neuroactive chiral molecule. This review focuses on the most relevant articles published from January 2008 to July 2014, based on the determination in biological samples of potentially interesting molecules in neuroscience using CE and microchip-CE. (C) 2014 Elsevier B.V. All rights reserved

Rapid enantiomeric separation of polychlorinated biphenyls by electrokinetic chromatography using mixtures of neutral and charged cyclodextrin derivatives

Electrokinetic chromatography with cyclodextrin derivatives (CD-EKC) was used to achieve the rapid enantiomeric\ud separation of chiral polychlorinated biphenyls (PCBs). Thirteen of the 19 chiral PCBs stable at room temperature were individually separated into their two enantiomers by using 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.5) containing carboxymethylated g-cyclodextrin (CM-g-CD) as pseudostationary phase mixed with b-cyclodextrin (b-CD) or permethylated b-cyclodextrin (PM-b-CD). Urea was also added to increase the solubility of PCBs and cyclodextrins in the aqueous separation buffer. Several experimental parameters such as the nature, concentration, and pH of the buffer, nature and concentration of the cyclodextrin derivatives used, and the addition of different additives were studied in order to improve the enantiomeric separation. In addition, the effect of some instrumental parameters such as separation temperature and applied voltage was also investigated. PCBs were enantiomerically separated in less than 12 min by using a 50 mM MES buffer (pH 6.5) containing 20 mM CM-g-CD, 10 mM b-CD or 20 mM PM-b-CD, and 2 M urea at a temperature of\ud 458C and an applied voltage of 20 kV

Determination of non-protein amino acids and betaines in vegetable oils by flow injection triple-quadrupole tandem mass spectrometry: a screening method for the detection of adulterations of olive oils.

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review.A novel screening method using an automated flow injection electrospray ionization tandem mass spectrometry system is proposed for the simultaneous determination of five nonprotein amino acids (β-alanine, alloisoleucine, ornithine, citrulline, pyroglutamic acid) and three betaines (glycine betaine, trigonelline, proline betaine) after derivatization with butanolic HCl. MS/MS experiments were carried out in a triple-quadrupole instrument using multiple reaction monitoring mode in <2 min. The proposed method provided high fingerprinting power to identify the presence of five of the studied compounds in different types of vegetable oils (soybean, sunflower, corn, olive) with LODs at parts per billion levels. The method was validated, and different mixtures of extra virgin olive oil with seed oils were analyzed, achieving the typification for the detection of adulterations in extra virgin olive oils up to 2% w/w. The nonprotein amino acid ornithine was confirmed as a marker for adulteration in the olive oils analyzedThe projects involved in this work have been: project CTQ2009-09022 from the Spanish Ministry of Science and Innovation (Spain), project S2009/AGR-1464 from the Comunidad Autónoma of Madrid (Spain) and European funding from FEDER programme (ANALISYC-II), and project CCG10-UAH/AGR-5950 from the University of Alcalá and the Comunidad Autónoma of Madrid (Spai

Detection of saffron adulteration with gardenia extracts through the determination of geniposide by liquid chromatography-mass spectrometry

A new and sophisticated saffron adulteration method with gardenia was recently discovered in the European saffron market. In this work, an analytical methodology using liquid chromatography (quadrupole-time of flight)-mass spectrometry has been developed for the detection of the adulteration of saffron samples with gardenia through the determination of geniposide as an adulteration marker. A fused-core C18 column was employed using an isocratic elution with water:acetonitrile (85:15 v/v) containing 0.1% formic acid. After optimization of the mass spectrometry conditions, the analytical characteristics related to the determination of geniposide in negative electrospray ionization mode were evaluated. Then, it was possible to detect up to 10 ng/mL geniposide after a dilution step of 50-fold of the saffron extract (LOD of 41.7 mu g of geniposide per gram of sample analysed (i.e up to 0.004%)). The developed LC-MS methodology was applied to the analysis of different authentic and suspicious saffron samples. (C) 2016 Elsevier Inc. All rights reserved