Ounds, to possess a percentage worth of the glucuronic acid derivatives of every single compound, a hydrolysis step with -glucuronidase was performed on 20 urine samples (10 males, ten females) in which the free-form concentrations of each compound had been previously detected with no hydrolysis. For the hydrolysis procedure, 500 of ammonium acetate buffer 1 M (pH = 6.1) and 40 L of -glucuronidase were added. Samples have been incubated for 60 min at 55 5 . The extraction methods have been the exact same as described for samples not undergoing deconjugation.Arioli F. et al.LC S parametersThe chromatographic separation was performed at room temperature, in isocratic condition, on a reversed-phase C18 Sunfirecolumn (150 two.1 mm i.d., three.5 particle size; Waters, Milford, MA, USA) equipped having a Sunfire C18 Guard Column(2.1 10 mm i.d., 3.5 particle size; Waters, Milford, MA, USA). The mobile phase consisted of a mixture of aqueous option 0.1 formic acid and acetonitrile (77:23) at a flow price of 0.three mL min-1. An LTQlinear ion trap mass spectrometer equipped with an Electrospray Source (Thermo Fisher, San Jos CA, USA), connected to a Surveyor Autosampler and a Surveyor MS Pump (Thermo Fisher, San Jos CA, USA), was applied. The linear ion trap was operated in negative electrospray ionization mode [ESI ( -)] below the following situations: sheath and auxiliary gas (nitrogen) flow prices of 40 and 20 arbitrary units, respectively; sweep gas was off; spray voltage of 4 kV; ion transfer capillary temperature 275 ; capillary voltage – 5 V; and tube lens – 30 V. Helium was made use of as collision gas. Collision power ranged involving 18 and 25 . As a result of the truth that all compounds considered in the study can form an extremely abundant and stable adduct with formic acid in ESI ( -) mode ([M + HCOO]-), the MS parameters had been optimized applying the cortisol adduct with formic acid (MW = 407 Da) as reference compound, by direct injection of a standard resolution of cortisol (1 mL-1) at a flow rateTable 1 Corticosteroids with their molecular weights (MW), retention times (Rt), m/z values of their adducts with formic acid (employed as precursor ions for MS2 fragmentation), collision energy (CE ) for MS2 fragmentation, solution ion inside the MS2 spectrum utilized like precursor ion for MS3 fragmentation, collision power (CE ) for MS3 Corticosteroid 6-Hydroxicortisol 20-Dihydrocortisol 20-Dihydrocortisone 20-Dihydrocortisol 20-Dihydrocortisone Prednisolone Cortisol Cortisone -Cortolone -Cortolone Allo-tetrahydrocortisol 5-Dihydrocortisol Tetrahydrocortisol Methylprednisolone (IS) Allo-tetrahydrocortisone 5-Dihydrocortisol Tetrahydrocortisone MW 378 364 362 364 362 360 362 360 366 366 366 364 366 374 364 364 364 Rt 1.SHH Protein manufacturer eight 5.PDGF-AA Protein Synonyms 5 6.PMID:23659187 2 six.5 7.1 ten.5 11.1 12.1 13.four 15.three 16.eight 17.eight 18.three 21 22.4 23.8 25.4 Precursor ion [M – HCOO]- 423 409 407 409 407 405 407 405 411 411 411 409 411 419 409 409 409 CE 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20of 20 min-1. Data acquisition and evaluation had been achieved making use of Xcalibursoftware version two.1 (Thermo Fisher, San Jos CA, USA). The compounds, their retention occasions, their molecular weights, the molecular weights of their adducts with formic acid (used as precursor ions), plus the solution ions with the associated molecular weight are shown in Table 1, together together with the collision energies (CE ). The item ions obtained right after the MS2 experiment had been applied as precursor ions for the subsequent MS3 and eventual MS4 steps of evaluation.Approach validationDue for the endogenous natur.