Determination of urinary free cortisol by "on-line" liquid chromatography.

Abstract

This is a fully automated method for the specific assessment of urinary free cortisol. A 1-mL urine sample is concentrated and prepurified on a reversed-phase precolumn with alkaline, acid, and organic washes. After selective elution, the cortisol-containing organic eluate is "polarized" by admixing water in such a way that cortisol is focused on the top of a second reversed-phase precolumn. From this precolumn, cortisol is desorbed by backflush, separated from the still-remaining related compounds on an analytical column, and finally detected by ultraviolet absorbance. Losses of cortisol throughout the total procedure are negligible and thus external calibration is feasible for quantification. CVs were 4.1% for interassay variability, 2.6% for intra-assay variability. Cortisol concentrations down to 15 nmol/L are assayable, we estimated the median amount of free cortisol excreted daily by normal students, outpatients, hospitalized patients, and patients under intensive care. After stimulation with corticotropin1-24, the median concentration of free cortisol in urine increased from 99 nmol/L to 1238 nmol/L (n = 6). Results by radioimmunoassay for normal persons and hospitalized patients were about fourfold those by this technique. The same method can also be used for free cortisone in urine.

General Methods for the Extraction, Purification, and Measurement of Steroids by Chromatography and Mass Spectrometry

“…The availability of switching valves which can be operated automatically has enabled the use of multiple columns, automatically switching selected peaks from one column to another -so-called "column switching" or "heart cutting". Schoneshofer et al (1983) described such a system for the measurement of triamcinolone in urine and later adapted the procedure for the measurement of urinary free cortisol (Schoneshofer et al, 1985) and for 20-reduced metabolites of cortisol and cortisone in urine (Schoneshofer et al, 1986). Henion and Lee (1990) commented that despite the obvious value of such column switching techniques, they were then not widely used.…”

Bronchitis bei Kindern unter Belastung durch Strassenverkehrslarm und Abgase. Bronchitis in Children Exposed to Road Traffic Noise and Exhaust Fumes

“…Bei den teilnehmenden Kindern wurde nachts um 1 Uhr und morgens der Harn zur Bestimmung der stündlichen Ausscheidungsmengen von freiem Cortisol und Cortisolmetaboliten mittels HPLC [11] sowie Speichelproben [7] gesammelt. Die Verkehrslärm‐ und Abgasbelastung außerhalb der Kinderzimmer wurde zum Teil durch direkte Messungen des nächtlichen Mittelungspegels und der mittleren NO 2 ‐Belastung über 3–4 Tage ermittelt, und ansonsten durch Experten anhand von genauen Straßenkarten sowie der abgefragten Schlafzimmerausrichtung des Kindes abgeschätzt [7].…”

Pharmacokinetics and pharmacodynamics of methylprednisolone after one bolus dose compared with two dose fractions

“…Serum samples were assayed for methylprednisolone concentrations, lymphocytes, glucose, insulin, and cortisol. Methylprednisolone concentrations were measured by a modified high‐pressure liquid chromatographic (HPLC) method (7). Cortisol levels were determined by HPLC simultaneously with methylprednisolone concentrations.…”