Measurement of cortisol,cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma,plasma ultrafiltrate,urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R² = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R² = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.     

Investigation on the Phytochemical Composition,Antioxidant and Enzyme Inhibition Potential of Polygonum Plebeium R.Br: A Comprehensive Approach to Disclose New Nutraceutical and Functional Food Ingredients

The present work describes medicinal potential and secondary metabolic picture of the methanol extract (PP-M) of Polygonum plebeium R.Br. and its fractions; hexane (PP-H), ethyl acetate (PP-E) and water (PP-W). In total bioactive component estimation, highest contents of phenolic (89.38±0.27 mgGAE/g extract) and flavonoid (51.21±0.43 mgQE/g extract) were observed in PP-E, and the same fraction exhibited the highest antioxidant potential in DPPH (324.80±4.09 mgTE/g extract), ABTS (563.18±11.39 mgTE/g extract), CUPRAC (411.33±15.49 mgTE/g extract) and FRAC (369.54±1.70 mgTE/g extract) assays. In Phosphomolybdenum activity assay, PP-H and PP-E showed nearly similar potential, however, PP-H was the most active (13.54±0.24 mgEDTAE/g extract) in metal chelating activity assay. PP-W was the stronger inhibitor (4.03±0.05 mgGALAE/g extract) of the enzyme AChE, while PP-H was potent inhibitor of BChE (5.62±0.27 mg GALAE/g extract). Interestingly, PP-E was inactive against BChE. Against tyrosinase activity, PP-E was again the most active fraction with inhibitory value of 71.89±1.44 mg KAE/g extract, followed by the activity of PP-M and PP-W. Antidiabetic potential was almost equally distributed among PP-M, PP-H and PP-E. For mapping the chemodiversity of P. plebeium, PP-M was analyzed through UHPLC/MS, which led to the identification of more than 50 compounds. Flavonoids were the main components derived from isovitexin, kaempferol and luteolin however, gallic acid, protocatechuic acid, gingerols and lyoniresinol 9′-sulfate were among important bioactive phenols. These findings prompted to conclude that Polygonum plebeium can be a significant source to offer new ingredient for nutraceuticals and functional foods.     

Does Aconitum septentrionale chemically protect floral rewards to the advantage of specialist bumblebees?

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Identification and quantification of sophorolipid analogs using ultra-fast liquid chromatography–mass spectrometry

An ultra-fast liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (UHPLC/APCI-MS) has been developed for the separation and quantification of sophorolipid analogs produced by the yeast Candida bombicola. The sophorolipid mixture was produced by growing the yeast in the presence of glucose and oleic acid under higher aeration. It was found that more than 95% of the analogs are lactonic sophorolipids and all the produced sophorolipids produced were either mono- or di-acetylated. Also observed was a sophorolipid analog with a tri-unsaturated fatty acid, which has not been reported previously.     

Fast chiral separation of drugs using columns packed with sub‐2 μm particles and ultra‐high pressure

The use of columns packed with sub‐2 μm particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and β‐blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4‐tri‐O‐acetyl‐α‐D ‐arabinopyranosyl isothiocyanate (AITC) and N‐α‐(2,4‐dinitro‐5‐fluorophenyl)‐L ‐alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2–5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)‐(S)‐amphetamine and (+)‐(S)‐methamphetamine. Similar results were obtained with β‐blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several β‐blocker enantiomers was performed in 1 min or less. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

The influence of cytokinins on proliferation and polyphenol accumulation in shoot cultures of Scutellaria altissima L.

S. altissima shoots were cultivated on MS (Murashige and Skoog) solid medium supplemented with IAA (indole-3-acetic acid, 0.57 μM) and various concentrations of cytokinin: zeatin, kinetin, BAP (6-benzylaminopurine) (1, 2, 4, 8 μM) or TDZ (thidiazuron) (0.2, 0.5, 1 μM). The effects on shoot proliferation and their accumulation of pharmacologically valuable phenolic compounds (baicalin, wogonoside, luteolin, luteolin-7-O-glucoside, verbascoside) were evaluated. Ultra-high performance liquid chromatography (UHPLC) was used for quantitative analysis of the compounds accumulated in the plant biomass. The metabolites were identified by comparing their retention time, UV spectra and mass spectra with those of the standard compounds and published data. The highest metabolite contents were recorded in shoots treated with TDZ at a concentration of 1 μM; under these conditions, the total level of all evaluated flavones expressed as the sum of baicalin, wogonoside, luteolin and luteolin-7-O-glucoside (17.35 mg/g dry wt) was more than twice that of those grown on MS cytokinin-free medium (7.55 mg/g dry wt). Verbascoside accumulation was also stimulated by 1 μM TDZ; its level was about six times higher than that found on the control medium (6.2 mg/g dry wt vs. 1.03 mg/g dry wt). The highest number of shoots (5.5–6.4 per explant within five weeks) was achieved with 2–8 μM BAP.