Untargeted characterization of extracts from Cannabis sativa L. cultivars by gas and liquid chromatography coupled to mass spectrometry in high resolution mode.

Elucidation of Cannabis composition is required to evaluate the potential of this plant for pharmacological uses, but also for implementation in breeding programs with agronomical purposes. The aim of the present study was to develop a method for untargeted analysis of polar and non-polar Cannabis extracts. For this purpose, extracts from 17 cultivars of Cannabis sativa L. were analyzed by gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) and liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF MS/MS) in high resolution mode. One hundred sixty-nine compounds were identified in the extracts by searching MS and MS/MS information. Among identified families, there were mainly cannabinoids, terpenoids, lipids and flavonoids, but also some interesting compounds such as amino and organic acids, among others. Relative contents of terpenoids and cannabinoids in the same cultivars grown in greenhouse and field were compared. Compositional differences in the profile of terpenoids and cannabinoids between both types of grown conditions were found.

The potential of near infrared spectroscopy to estimate the content of cannabinoids in Cannabis sativa L.: A comparative study.

Cannabis has been one of the oldest source of food, textile fiber and psychotropic substances. Cannabinoids are the main biologically active constituents of the Cannabis genus, with a demonstrated medicinal value. Its production is becoming legalized and regulated in many countries, thus increasing the need for a rapid analysis method to assess the content of cannabinoids. Gas chromatography (GC) is the preferred analytical method for the determination of these compounds, although is a slow and costly technique. Near infrared spectroscopy (NIR) has the potential for the quantitative prediction of quality parameters, and also of pharmacologically active compounds, but no references about cannabinoids prediction has been previously reported. The aim of the present research was to develop a fast, economical, robust and environmentally friendly method based on NIR technology that allow the quantification of the main cannabinoids present in Cannabis sativa L. SAMPLES: A total of 189 grinded and dried samples from different genotypes and registered varieties were used. The content of the cannabinoids CBDV, Δ9-THCV, CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN were determined by gas chromatography. Spectra were collected in a dispersive NIR Systems 6500 instrument, and in a Fourier transform near Infrared (FT-NIR) equipment. The sample group was divided into calibration and validation sets, to develop modified partial lest squares (PLS) regression models with WINISI IV software with the dispersive data, and PLS models using OPUS 7.2 with the FT-NIR ones. Excellent coefficient of determination of cross validation (R2CV from 0.91 to 0.99), were obtained for the prediction of CBD, CBC, Δ8-THC, Δ9-THC, CBG and CBN, with standard error of prediction (SEP) values among 1.5-3 times the standard error of laboratory (SEL); and good for CBDV and Δ9-THCV cannabinoids (R2 values of 0.89 and 0.83, respectively) with the dispersive instrument. Similar calibration and validation statistics have been obtained with the FT-NIR instrument with the same sample sets, using its specific OPUS software. In conclusion, a methodology of quantitative determination of cannabinoids in Cannabis raw materials has been developed for the first time using NIR and FT-NIR instruments, with similar good predictive results. This new analytical method would allow a simpler, more robust and precise estimation than the current standard GC.

Quantitative analytical method to evaluate the metabolism of vitamin D.

A method for quantitative analysis of vitamin D (both D2 and D3) and its main metabolites - monohydroxylated vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3) and dihydroxylated metabolites (1,25-dihydroxyvitamin D2, 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3) in human serum is here reported. The method is based on direct analysis of serum by an automated platform involving on-line coupling of a solid-phase extraction workstation to a liquid chromatograph-tandem mass spectrometer. Detection of the seven analytes was carried out by the selected reaction monitoring (SRM) mode, and quantitative analysis was supported on the use of stable isotopic labeled internal standards (SIL-ISs). The detection limits were between 0.3-75pg/mL for the target compounds, while precision (expressed as relative standard deviation) was below 13.0% for between-day variability. The method was externally validated according to the vitamin D External Quality Assurance Scheme (DEQAS) through the analysis of ten serum samples provided by this organism. The analytical features of the method support its applicability in nutritional and clinical studies targeted at elucidating the role of vitamin D metabolism.

Stable isotopic internal standard correction for quantitative analysis of hydroxyeicosatetraenoic acids (HETEs) in serum by on-line SPE-LC-MS/MS in selected reaction monitoring mode.

The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels.

Anthocyanidins, proanthocyanidins, and anthocyanins profiling in wine lees by solid-phase extraction-liquid chromatography coupled to electrospray ionization tandem mass spectrometry with data-dependent methods.

A method has been developed to study the content of anthocyanidins, proanthocyanidicins, and anthocyanins in wine lees, an abundant byproduct from wineries. Detection/quantitation of the target compounds was carried out by a hyphenated system consisting of a solid-phase extraction workstation (Prospekt-2 unit) online coupled to a liquid chromatograph-triple-quadrupole tandem mass spectrometer (LC-MS/MS), where standards were used for identification/quantitation of both anthocyanidins and proanthocyanidins. Owing to the lack of anthocyanins standards, advantages from the use of data-dependent methods were taken for their identification and confirmatory analysis. Combination of the scanning methods (viz. product-ion, precursor-ion, and neutral-loss scanning) allowed identifying five different anthocyanins present in wine residues. The results thus obtained have been validated by complementary analysis of the extracts using LC-TOF/MS in high-resolution mode. Quantitation of the monitored compounds was supported on selected reaction monitoring (SRM) and calibration curves run with standards of anthocyanidins and proanthocyanidins.

Integrated identification/confirmatory and targeted analysis of epoxyeicosatrienosic acids in human serum by LC-TOF MS and automated on-line SPE-LC-QqQ MS/MS.

A combined strategy is here proposed for qualitative/quantitative targeted analysis of epoxyeicosatrienoic acids (EETs) in human serum. Identification of EET regioisomers was initially carried out by LC-TOF MS in high accuracy mode under optimum conditions for chromatographic separation of the four isomers with an isocratic method using 40:40:20 (v/v/v) methanol-acetonitrile-water containing 0.02% acetic acid. Confirmatory analysis was supported on MS/MS experiments using the hybrid QqTOF mass analyzer by targeted fragmentation of the precursor ion fitting with the molecular formula C20H32O3 (319.2279 m/z). Identification of selective fragment ions in high accuracy mode enabled the localization of the epoxy functional group and, therefore, the assignation of chromatographic peaks to each EET isomer. After qualitative analysis, an automated method was developed for analysis of EETs in human serum by direct analysis using an on-line platform based on SPE-LC-QqQ MS/MS in selected reaction monitoring. Recovery factors estimated with a dual-cartridge configuration were above 87% for all metabolites either using non-spiked and spiked serum at three different concentrations. Precision, calculated as within-laboratory repeatability and expressed as relative standard deviation, ranged from 2.5 to 9.9% with detection limits below 0.15 ng mL(-1). The optimization of the method was completed with a stability study under different conditions to assess the suited conditions for analysis of EET intermediate metabolites. Finally, concentration ranges of EETs were measured in nine healthy individuals.

Automated method for determination of olive oil phenols and metabolites in human plasma and application in intervention studies.

The interest for olive oil phenols (OOPs) is a growing trend thanks to their contribution to prevent or improve diseases associated to oxidative damage. OOPs ingested in the diet are found at low concentrations in blood either as free forms (e.g. hydroxytyrosol, tyrosol, vanillin, ferulic acid, coumaric acid) or conjugated as sulfate and glucuronide derivatives. Therefore, the identification/quantitation of OOPs in plasma to study their biological effects and elucidate their metabolism requires selective and sensitive methods. The present research describes the development, validation and application of an automated method based on on-line coupling of solid-phase extraction and liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for quantitation of conjugated and free OOPs in human plasma. This approach minimizes sample handling-thus reducing analyte losses and degradation by contact with the atmosphere-and increases analysis throughput, which is crucial in intervention studies dealing with cohorts formed by numerous individuals. The fundamental of the approach is the retention of OOPs and metabolites in an SPE anionic cartridge with subsequent on-line elution to an LC-MS/MS system. Quantitative analysis of OOPs (relative quantitation for conjugated OOPs) was carried out by selected reaction monitoring mode that reported relative limits of detection and quantitation between 0.02-0.28 ng/mL (16.6-232 pg on-column) and 0.05-0.83 ng/mL (41.5-689 pg on-column), respectively. The accuracy of the method, estimated as recovery factor, ranged from 84.2 to 99.4%, and precision, expressed as relative standard deviation, was below 3.8%. The resulting method has been applied to the determination of OOPs and metabolites in plasma samples from individuals who ingested a breakfast prepared with virgin olive oil. The proposed method has an excellent potential for high-throughput use in both clinical and research laboratories.

Comparison of sample preparation approaches for phospholipids profiling in human serum by liquid chromatography-tandem mass spectrometry.

Fractionation of biological fluids for proper analysis by LC-MS of low-abundance metabolites in the presence of high-abundant endogenous metabolites is of interest, particularly when the latter cause undesirable phenomena such as ionization suppression, as is the case with phospholipids (PLs). An exhaustive study and comparison of different protocols for sample preparation based on deproteinization, liquid-liquid extraction (LLE) and solid-phase extraction (SPE) has been carried out with the main aim of implementing metabolomics analysis of PLs within a global metabolite profiling of serum. After selecting LLE and SPE as the best strategies for sample preparation, the fractions from each approach were analyzed by LC-TOF/MS in positive and negative ionization modes, and the highest number of molecular features was found by the latter strategy (2667 versus 1911 in the positive ionization mode). SPE also allows obtaining a clean elution of PLs which made possible tentative identification of these metabolites in both ionization modes by matching to the Human Metabolome Database. Confirmatory analysis to assess PLs identity was supported on MS/MS experiments at different collision energies.

On-line coupling of automatic solid-phase extraction and HPLC for determination of carotenoids in serum.

The automated method developed for the determination of carotenoids uses 200 µL of serum, which was mixed with 400 µL of tetrahydrofuran, vortexed for 1 min, settled for 10 min, centrifuged for 6 min and the supernatant injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration. A 10% water-acetonitrile mobile phase at 1.5 mL min(-1) eluted the retained compounds and transferred them on-line to a reversed-phase analytical column for individual separation of the target analytes. Visible detection was performed at 450 and 460 nm. The detection limits for the target analytes were between 3 and 30 ng mL(-1); the precision (expressed as relative standard deviation) ranged between 2.83 and 5.06% for repeatability and between 3.80 and 7.40% for within laboratory reproducibility. The total analysis time was 18 min. The proposed method is reliable, robust, and has an excellent potential for high-throughput use in both clinical and research laboratories.

Standard operation protocol for analysis of lipid hydroperoxides in human serum using a fully automated method based on solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring.

Standard operating procedures (SOPs) are of paramount importance in the analytical field to ensure the reproducibility of the results obtained among laboratories. SOPs gain special interest when the aim is the analysis of potentially unstable compounds. An SOP for analysis of lipid hydroperoxides (HpETEs) is here reported after optimization of the critical steps to be considered in their analysis in human serum from sampling to final analysis. The method is based on automated hyphenation between solid-phase extraction (SPE) and liquid chromatography-mass spectrometry (LC-MS). The developed research involves: (i) optimization of the SPE and LC-MS steps with a proper synchronization; (ii) validation of the method-viz. accuracy study (estimated as 86.4% as minimum value), evaluation of sensitivity and precision, which ranged from 2.5 to 7.0 ng/mL (0.25-0.70 ng on column) as quantification limit and precision below 13.2%), and robustness study (reusability of the cartridge for 5 times without affecting the accuracy and precision of the method); (iii) stability study, involving freeze-thaw stability, short-term and long-term stability and stock solution stability tests. The results thus obtained allow minimizing both random and systematic variation of the metabolic profiles of the target compounds by correct application of the established protocol.

Automated method for targeting analysis of prostanoids in human serum by on-line solid-phase extraction and liquid chromatography-mass spectrometry in selected reaction monitoring.

Prostanoids are potent biologically active lipid molecules demanding for analysis methods combining precision, sensitivity and high-throughput for pharmacological and clinical applications. The present research describes the development and validation of an on-line automated method based on solid-phase extraction liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) for the quantification of prostanoids in human serum. This approach overcomes the main limitation of previous methods involving manual protocols, such as analyte losses, metabolites degradation and time-consuming protocols, are minimized. Human serum (100 µL) was directly injected into an automatic solid-phase extraction workstation for cleanup and preconcentration of the target metabolites. The eluate was on-line transferred to a reversed-phase analytical column for chromatographic separation prior to mass spectrometry detection in selected reaction monitoring mode. The detection limits for the target analytes ranged from 2.3 to 63.3 pg on column. The precision (expressed as relative standard deviation) was within 3.30 and 6.15% for repeatability and from 4.16 to 11.11% for within-laboratory reproducibility. Accuracy was evaluated with spiked and non-spiked serum samples to estimate concentration differences that could be affected by matrix effects or inefficient SPE performance. Accuracy, estimated as recovery factor, was from 87.7 to 100% for the target compounds. The proposed method is reliable and has an excellent potential for high-throughput use in both clinical and research laboratories by minimizing analyst intervention.

Evaluation of vitamin D endocrine system (VDES) status and response to treatment of patients in intensive care units (ICUs) using an on-line SPE-LC-MS/MS method.

Vitamin D deficiency is recognized as one of the most common chronic medical conditions in the world. Vitamin deficiency has been associated with increased mortality. The aim of the study here presented was to evaluate the vitamin D endocrine system (VDES) status in healthy blood donors and critically ill patients baseline and in response to treatment during a week with two doses of 1.5 mg of 25-hydroxyvitamin D3 and 2 microg calcitriol (1,25(OH)2D3) IV on alternate days, by monitoring levels in serum of major vitamin D metabolites in critically ill patients. Group 1: healthy blood donors (control group) (n=92), and group 2: critically ill subjects from an intensive care unit (ICU) (n=33). Critically ill patients were divided into three groups: group A (n=12) is the control group; group B (n=11), administration PO 1,5 mg of 25(OH)D3, in days 0 and 4 of treatment; and group C (n=11), administration IV of 2 microg 1,25(OH)2D3 on alternate days. Baseline serum levels of vitamin D2 and 25(OH)D2 were not detected. Vitamin D3 (9.8 vs 26.0 nM) (p<0.05), 25(OH)D3 (13.3 vs 52.3 nM) (p<0.001), and 1,25(OH)2D3 (53.8 vs 120.5 pM) (p<0.01) serum levels were significantly lower in critically ill subjects than in healthy donors. After treatment in group B: 25OHD3 increased to 46.0+/-16.5 ng/ml (p<0.0001) (22.2%<75 nM, 11.1% <50 nM). 1,25(OH)2D3 increased to 121.8+/-61.8 pM<0.01 whereas were slightly decreased in the other groups during the study. 24,25(OH)2D3 serum levels were increased in patients treated with calcitriol 8.5+/-5.3 vs 24.8+/-16.3 nM (p<0.05) while the levels kept stable in group A patients. In summary, critically ill patients have a severe vitamin D deficiency, which can be easily corrected by administration of high doses of 25OHD (PO). The VDES functional deficiency could be probably also corrected through administration of calcitriol (IV). Both treatments could produce an improvement in the general health and probably a reduction in overall mortality risk of the critically ill patients.