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The aim of this study was to improve analysis of nonpolar lipidomics sample extracts using reversed phase (RP) chromatography. A 4/3/3 (v/v/v) mixture of methanol/methyl tert-butyl ether/chloroform (MeOH/MTBE/CHCl 3 , MMC) was chosen for sample extraction solvent based on its proven extraction capability for several lipid classes. To avoid carry over, loss of analytes and peak distortion the loops and all capillaries of the presented LC system were flushed and filled up with methanol until the analytical column. The choice of methanol was due to its weak elution strength and being infinitely miscible with MMC and several other nonpolar solvents. This allowed injection of a 100 μl sample that was 20 μl nonpolar extraction solvent diluted fivefold with methanol. All lipids of 25 lipid classes were transferred quantitatively to the column head where the online dilution of methanol was carried out with aqueous eluent for focusing the lipid analytes. The weak elution strength of methanol prevented peak distortions. The consecutive reversed phase elution resulted in remarkably narrow peaks (full width at half maximum was 0.07-0.08 min typically) and enhanced sensitivity (limit of detection usually in sub nM region) because of increased sample injection volume and narrow peaks. Calibration and quality control samples made by diluting commercial lipid standards 200-50000 times confirmed the applicability of this approach both for targeted lipid quantification and for untargeted quantitative comparison of lipids from different sources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

24-hour urinary free cortisol (UFC) is considered as the first-line test for screening and diagnosis of Cushing's syndrome. Although 24-hour UFC assay has been extensively studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS), an accurate assay coupled with a reliable sample preparation procedure and a method-specific reference interval would be very important for reasonable diagnosis. In this study, a simple dilute and shoot method has been proposed for UFC determination by LC-MS/MS. Namely, 50 µL of urine sample was mixed with 200 µL of a 50 % methanol/water solution containing the internal standard cortisol- 13 C3. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. This method was validated with wide linear range from 0.625 to 500 ng/ml with coefficients of variation (CVs) ≤ 3.64 %, excellent precision (intra-day CVs ≤ 5.70 % and inter-day CVs ≤ 5.33 %) and good recovery in the range of 93.3-109 %. The preservatives were further evaluated for urine storage. It was recommended that no preservatives could be used in collection of 24-hour urine for good detecting peaks. The investigation of reference interval and diagnostic performance finally confirmed the potential usage of this LC-MS/MS assay in routing clinical testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)