Diego García-Gómez

Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease

Abberant lipid metabolism is implicated in Alzheimers disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases. In a subsequent validation study (n = 141), PC variation in a bigger sample set was investigated, and the same 3 PCs were found to be significantly lower in AD patients: PC 16:0/20:5 (p < 0.001), 16:0/22:6 (p < 0.05), and 18:0/22:6 (p < 0.01). A receiver operating characteristic (ROC) analysis of the PCs, combined with apolipoprotein E (ApoE) data, produced an area under the curve predictive value of 0.828. Confirmatory investigations into the background biochemistry indiciated no significant change in plasma levels of 3 additional PCs of similar structure, total choline containing compounds or total plasma omega fatty acids, adding to the evidence that specific PCs play a role in AD pathology.

Development and validation of a hydrophilic interaction chromatography-tandem mass spectrometry method with on-line polar extraction for the analysis of urinary nucleosides. Potential application in clinical diagnosis.

The present paper describes the development, validation and application of a quantitative method for the determination of endogenous nucleosides and nucleobases in urine based on the on-line coupling of a solid-phase extraction step with hydrophilic interaction chromatography-tandem mass spectrometry. The method combines the use of a highly polar restricted-access material (RAM), based on an N-vinylacetamide copolymer, for efficient analyte extraction and matrix removal, with separation by zwitterionic hydrophilic interaction chromatography (ZIC-HILIC), that revealed a satisfactory retention of the polar analytes studied. Detection using a triple quadrupole analyser allowed reliable identification and high-sensitivity quantitation of the target compounds. The on-line configuration developed, RAM-ZIC-HILIC-MS/MS, provides a convenient approach to automate the application to urine analysis, with minimum sample manipulation. The whole method was validated according to European Legislation for bioanalytical methods. The validation steps included the verification of matrix effects, calibration curve, precision, accuracy, selectivity, stability and carry-over in real samples. The results of the validation process revealed that the proposed method is suitable for the reliable determination of nucleosides and nucleobases in human urine, showing limits of detection from 0.1 to 1.3 ng mL(-1). The application to clinical samples was also checked; the results obtained in analyses of urine samples from healthy volunteers and cancer patients using Principal Component Analysis, Hierarchical Cluster Analysis and Soft Independent Modeling of Class Analogy are also shown.

Capillary electrophoresis coupled to mass spectrometry for the determination of anthelmintic benzimidazoles in eggs using a QuEChERS with preconcentration as sample treatment

Benzimidazoles (BZDs) are anthelmintic agents widely used in veterinary medicine. Their use in food-producing animals increases the possibility of residues appearing in animal tissues and products. Most analytical procedures reported for the determination of BZDs have been developed based on liquid chromatography (LC) because of their polar nature - zwitterionic - and thermal lability. To our knowledge, the determination of these compounds by capillary electrophoresis coupled to mass spectrometry (CE-MS) has not yet been described. In this work CE-MS is proposed for the identification and simultaneous quantification of several benzimidazoles in egg samples. The target compounds were 2-aminobenzimidazole, carbendazim, albendazole-2-aminosulphone, 5-hydroxy-thiabendazole, oxibendazole, albendazole, fenbendazole, oxfendazole, albendazole-sulphone, fenbendazole-sulphone. Optimization of the composition and nature - organic/aqueous - of both the electrophoretic separation buffer and the injection medium was carried out with a view to obtaining the best sensitivity and separation efficiency for the CE-MS coupling. A comparative study was carried out on different sample treatments for analyte extraction from egg samples. Two of them comprised a solvent extraction step followed by clean-up using a new commercial polymeric sorbent (Evolute ABN(©)), and the third was a particularization of the general extractive method so called Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS). Different modifications of the QuEChERS method were assayed, which included a later preconcentration step based on either SPE with MCX(©) sorbents or evaporation. The whole optimized method (QuEChERS with preconcentration prior to CE-MS) was validated according to the 2002/657/EC decision obtaining a CE-MS method sufficiently reliable and robust to determine residues of these compounds in egg samples of different origins with limits of detection between 3 and 51 μgL(-1) (S/N=3) and recoveries in the 74-112% range.

Study of retention behaviour and mass spectrometry compatibility in zwitterionic hydrophilic interaction chromatography for the separation of modified nucleosides and nucleobases

A study has been made of the chromatographic behaviour of modified nucleosides and nucleobases using different stationary phases with functional groups of polar nature, all of them compatible with aquoorganic mobile phases. The stationary phases assayed were a pentafluorophenylpropyl (PFP) column for reverse phase separation, and another two for hydrophilic interaction chromatography (HILIC) separation. Six modified nucleosides and nucleobases (hydroxylated and methylated derivatives) were chosen as the target analytes. In the study, chromatographic resolution as well as the sensitivity in detection by mass spectrometry were taken into account. The results obtained showed that the zwitterionic (ZIC-HILIC) column was the most suitable one for the separation of these analytes. From the study of the different parameters affecting separation it may be concluded that in the ZIC-HILIC column separation is based on a mechanism of partition and interaction through weak electrostatic forces.

Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates.

A fast and efficient method for the simultaneous separation of highly polar compounds, in this case nucleosides and nucleotide mono-, di- and triphosphates, using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) is proposed. This new separation method revealed the possibilities of the formation of hydrophilic ion-pairing compounds. Three stationary phases (HILIC XBridge-Amide, HILIC-CoreShell and ZIC-HILIC) were assayed for the separation of 20 target analytes, and a detailed study of the composition of the mobile phase was made using different salts at different concentrations in a organic-rich mobile phase. We report that in order to prevent the adsorption of nucleotides on the LC-MS setup and to enhance their retention on the HILIC stationary phase, a mobile phase containing hexafluoro-2-propanol and different cations should be used. Four cations were evaluated: ammonium, diethylammonium, triethylammonium and tetrabutylammonium. The results revealed the formation of an ionic-association compound between the phosphorylated analytes and the cationic ion-pairing reagents, whose retention increased with the polarity of the cationic ion-pairing reagent. HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100mM hexafluoro-2-propanol and 50mM diethylamine (w(w)pH 9-w(s)pH 10). In a total analysis time of 8min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out.

Analysis of free nucleotide monophosphates in human milk and effect of pasteurisation or high-pressure processing on their contents by capillary electrophoresis coupled to mass spectrometry

A simple, efficient and green analytical method for the determination of free nucleotide monophosphates in human milk is proposed. It involves centrifugal ultrafiltration (CUF) as sample treatment and capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) for separation and simultaneous quantification. The optimised method, applied to the analysis of human milk samples, included their dilution (1:5) with water followed by CUF treatment. No matrix effects were found. The method provided limits of detection between 0.08 and 0.13 μg mL(-1) and limits of quantification between 0.26 and 0.43 μg mL(-1). The intralaboratory repeatability and reproducibility afforded relative standard deviation values lower than 10%. The method was applied to the study of the effects of Holder pasteurisation and high-pressure processing on the nucleotide contents in samples from a human milk bank. The results showed concentration values between 0.5 and 10 μg mL(-1), with higher concentrations for the samples treated by pasteurisation. The effect of freezing time on the content of nucleotides was also assessed.

Capillary electrophoresis–mass spectrometry for direct determination of urinary modified nucleosides. Evaluation of synthetic urine as a surrogate matrix for quantitative analysis

This work describes the development of a fast and reliable method based on capillary zone electrophoresis coupled with electrospray ionization-mass spectrometry (CZE-ESI-MS) for the determination of modified nucleosides in untreated human urine. The target compounds were guanine, 1-methyl-guanine, 7-methyl-guanine, 9-methyl-guanine, adenosine, 1-methyl-adenosine, cytidine, guanosine, 7-methyl-guanosine. As internal standards, ribose-2-(13)C-adenosine and 8-(13)C-guanine were used. The CZE separation was carried out in acidic medium (pH 2.5). MS detection with a single quadrupole, with ESI operating in positive-ion mode, was optimized. For the analysis of urine samples, owing to the endogenous character of these analytes different quantification strategies were explored. The standard additions method, matrix-matched calibration in synthetic urine and calibration in pure aqueous medium were compared in order to evaluate the endogenous levels of these compounds in human urine. The results obtained showed that calibration in synthetic urine as a surrogate matrix was an appropriate alternative to the method of standard additions for the accurate quantitation of compounds such as guanine, 1-methyl-guanine, 7-methyl-guanine, adenosine, 1-methyl-adenosine and cytidine by CE-ESI-MS directly in the urine matrix; values in the range 0.1μg/mL for cytidine and 6.4μg/mL for 7mGua, as the lowest and the highest level, were found in untreated urine from healthy volunteers. These results were confirmed by LC-MS/MS detection. It can be concluded that the electrophoretic CZE-ESI-MS methodology offers a valid and reliable alternative for the determination of urinary nucleosides at naturally occurring levels in healthy individuals.

Determination of endocrine disruptors in honey by CZE-MS using restricted access materials for matrix cleanup.

An analytical method based on CZE coupled to ESI‐MS is proposed for the identification and simultaneous quantification of several endocrine‐disrupting chemicals in honey. The target compounds were the chlorophenols: 2,4‐dichlorophenol, 2,4,5‐trichlorophenol and pentachlorophenol, and bisphenol‐A, 4‐tert‐butylphenol, and 4‐tert‐butylbenzoic acid. A two‐step optimization of the ESI‐MS detection was carried out. First, the organic solvent present in the sheath liquid was selected and its effect on the analytical signal was studied. The best results in terms of the intensity of the MS signals were obtained with methanol. Thus, an experimental design technique (Doehlert type) was used for the optimization of the other parameters: the NH3 concentration in the sheath liquid, the flow of the sheath liquid, the nebulizer pressure in ESI, and the drying gas temperature and flow. Here, we developed a new sample treatment based on the combined use of a restricted access material and a polymeric sorbent for SPE. The LOD achieved were in the range of 5–31 ng/g. The intraday precision of the proposed method was determined from replicate analyses (n=4) at a concentration level of 50 ng/g, with RSD values in the range of 15–23%. The results revealed that the proposed method is suitable for the reliable quantification of endocrine‐disrupting chemicals in honey at nanograms per gram levels.

A validated method for the determination of nucleotides in infant formulas by capillary electrophoresis coupled to mass spectrometry.

In this work CE‐ESI‐MS is proposed for the identification and simultaneous quantification of several ribonucleotide 5′‐monophosphates in infant formula (IF) samples. The target compounds were adenosine 5′‐monophosphate, cytidine 5′‐monophosphate, guanosine 5′‐monophosphate, uridine 5′‐monophosphate, and inosine 5′‐monophosphate. To our knowledge, the application of CE for the determination of these bioactive compounds in IFs has not yet been described. Optimization of the composition of the electrophoretic separation buffer and ‐mainly‐ the injection medium was carried out with a view to obtaining the best sensitivity and separation efficiency for the CE‐MS coupling. Different sample treatments were assayed and one based on centrifugal ultrafiltration proved to be the simplest and most compatible with CE separation of the analytes and their ionization by the electrospray source. The whole optimized method (centrifugal ultrafiltration treatment prior to CE‐MS) was validated according to the 2002/657/EC decision, obtaining a reliable and robust CE‐MS method to determine these compounds in IF samples, with LODs between 0.8 and 1.8 μg/g (S/N = 3) and recoveries in the 90–106% range.

Development and validation of a method for the detection and confirmation of biomarkers of exposure in human urine by means of restricted access material-liquid chromatography-tandem mass spectrometry

The present article describes the development and validation of a LC-MS/MS method for the determination and confirmation of biomarkers of exposure to different types of xenobiotics in human urine. The method combines the use of a restricted access material (RAM) coupled on-line to a LC-IT-MS system; in this way, a rapid and efficient matrix cleanup was achieved, reducing manual sample preparation to freezing and sample filtration. The ion trap (IT) mass spectrometry detector provided the selectivity, sensitivity and ruggedness needed for confirmatory purposes. The on-line RAM-LC-MS/MS method developed here has been validated as a quantitative confirmatory method according to the European Union (EU) Decision 2002/657/EC. The validation steps included the verification of linearity, repeatability, specificity, trueness/recovery, reproducibility, stability and ruggedness in fortified urine samples. Repeatability and within-laboratory reproducibility, measured as intraday and interday precisions, were evaluated at two concentration levels, being 12.7% or below at the concentration corresponding to the quantification limits. Matrix effects and non-targeted qualitative analyses were also evaluated in fortified urine samples. Decision limits (CC(alpha)) and detection capabilities (CC(beta)) were in the range of 3.6-16.5 and 6.0-28.1ngmL(-1) respectively. The results of the validation process revealed that the proposed method is suitable for reliable quantification and confirmation of biomarkers of exposure to xenobiotics in human urine at low ngmL(-1) levels. In addition, working in Data-Dependent Scan mode the proposed method can be used for the screening of these compounds in urine samples.