Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes.

Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC-MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum.

Combining data acquisition modes in liquid-chromatography-tandem mass spectrometry for comprehensive determination of acylcarnitines in human serum.

Acylcarnitines (ACs) are metabolites involved in fatty acid ß-oxidation and organic acid metabolism. Metabolic disorders associated to these two processes can be evaluated by determining the complete profile of ACs. In this research, we present an overall strategy for identification, confirmation, and quantitative determination of acylcarnitines in human serum. By this strategy we identified the presence of 47 ACs from C2 to C24 with detection of the unsaturation degree by application of a data-independent acquisition (DIA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Complementary, quantitative determination of ACs is based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in data-dependent acquisition (DDA) to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-day variability were below 15-20%, respectively; and the accuracy, expressed as bias, was always within ± 25%. The proposed method was tested with 40 human volunteers to determine the relative concentration of ACs in serum and identify predominant forms. Significant differences were detected by comparing the ACs profile of obese versus non-obese individuals.

Fully automated method for quantitative determination of steroids in serum: An approach to evaluate steroidogenesis.

Steroidogenesis is a set of metabolic reactions where the enzymes play a key role to control the physiological levels of steroids. A deficiency in steroidogenesis induces an accumulation and/or insufficiency of steroids in human blood and can lead to different pathologies. This issue added to the low levels of steroids (pg mL-1 to ng mL-1) in this biofluid make of their determination an analytical challenge. In this research, we present a high-throughtput and fully automated method based on solid-phase extraction on-line coupled to liquid chromatography with tandem mass spectrometry detection (SPE-LC-MS/MS) to quantify estrogens (estrone and estradiol), androgens (testosterone, androstenedione, dihydrotestosterone and dehydroepiandrosterone), progestogens (progesterone, pregnenolone, 17-hydroxyprogesterone and 17-hydroxypregnenolone), glucocorticoids (21-hydroxyprogesterone, 11-deoxycortisol, cortisone, corticosterone and cortisol) and one mineralocorticoid (aldosterone) in human serum. The performance of the SPE step and the multiple reaction monitoring (MRM) mode allowed reaching a high sensitivity and selectivity levels without any derivatization reaction. The fragmentation mechanisms of the steroids were complementary studied by LC-MS/MS in high-resolution mode to confirm the MRM transitions. The method was characterized with two SPE sorbents with similar physico-chemical properties. Thus, limits of quantification were at pg mL-1 levels, the variability was below 25% (except for pregnenolone and cortisone), and the accuracy, expressed as bias, was always within ±25%. The proposed method was tested in human serum from ten volunteers, who reported levels for the sixteen target steroids that were satisfactorily in agreement with the physiological ranges reported in the literature.

Development of a quantitative method for determination of steroids in human plasma by gas chromatography-negative chemical ionization-tandem mass spectrometry.

Sex steroids are involved in biological functions that encompass from the complete sexual development of individuals up to the deregulation of metabolic pathways leading to some pathologies. Steroids are present in blood at low concentration levels from pg mL-1 to ng mL-1. For this reason, a high sensitive and selective method based on gas chromatography-negative chemical ionization-tandem mass spectrometry (GC-NCI-MS/MS) is here proposed to quantify either androgens (androstenedione, dehydroepiandrosterone, dihydrotestosterone and testosterone), estrogens (estrone and estradiol) and a progestogen (progesterone) in human plasma. The sample preparation steps, protein precipitation and solid phase extraction, were optimized to ensure the sample matrix removal and to extract steroids with high efficiency. The NCI-MS/MS detection approach was compared with that based on electron impact to evaluate the incidence of the ionization source in the determination of steroids. The quantification limits for determination of these analytes were in a range from 10 pg mL-1 to 5 ng mL-1, with a high sensitivity for estrogens, typically found at low concentrations. The proposed method was tested for the determination of steroids in male blood samples, in which 6 out of 7 steroids were detected and quantified to report concentration values in agreement with those described in the literature.

Study of sample preparation for determination of endocannabinoids and analogous compounds in human serum by LC-MS/MS in MRM mode.

Endocannabinoids are lipids with a key role in physiological processes such as the immune response or the metabolism. This involvement explains their association to pathologies such as cancer, obesity or multiple sclerosis. The determination of endocannabinoids constitutes a challenge for clinical laboratories due to the variety of biological matrices and the wide range of concentrations at which they can be found. This research deals with the comparison of three sample preparation strategies (viz., on-line SPE, off-line SPE for interferents removal, and protein precipitation) for subsequent LC-MS/MS analysis of 14 endocannabinoids and analogous compounds in serum. As a result, the on-line coupling between SPE and LC-MS/MS is proposed as the best approach for this determination. The proposed method allows full automation of the overall process, shortening of the analysis time, and avoidance of errors associated with sample preparation steps. The improvement in sensitivity and selectivity thus achieved allows obtaining quantification limits at the pg mL-1 level, which makes possible the application of the method for clinical studies.