Improving derivatization efficiency of BMAA utilizing AccQ-Tag<Superscript>®</Superscript> in a complex cyanobacterial matrix

Two different assays have been developed and used in order to investigate the optimal conditions for derivatization and detection of acid beta-N-methyl-amino-L-alanine (BMAA) in a cyanobacterial sample. BMAA was extracted from cyanobacterial cultures both from the cytosolic ("free") fraction and in the precipitated ("protein") fraction using a newly developed extraction scheme and the sample matrix was standardized according to protein concentration to ensure the highest possible derivative yield. A rapid and sensitive HPLC method for fluorescence detection of the non-protein amino acid BMAA in cyanobacteria, utilizing the Waters AccQ-Tag chemistry and Chromolith Performance RP-18e columns was developed. Using this new method and utilizing a different buffer system and column than that recommended by Waters, we decreased the time between injections by 75%. The limit of quantification was determined to be 12 nmol and limit of detection as 120 fmol. The linear range was in the range of 8.5 nmol-84 pmol. Accuracy and precision were well within FDA guidelines for bioanalysis.     

Determination of urinary 5-hydroxytryptophol glucuronide by liquid chromatography-mass spectrometry

5-Hydroxytryptophol glucuronide (GTOL) is the major excretion form of 5-hydroxytryptophol (5-HTOL), a minor serotonin metabolite under normal conditions. Because the concentration of 5-HTOL is markedly increased following consumption of alcohol, measurement of 5-HTOL is used as a sensitive biomarker for detection of recent alcohol intake. This study describes the development and evaluation of a liquid chromatography-electrospray ionization mass spectrometry (LC-MS) procedure for direct quantification of GTOL in human urine. Deuterium labelled GTOL (GTOL-(2)H(4)) was used as internal standard. GTOL was isolated from urine by solid-phase extraction on a C(18) cartridge prior to injection onto a gradient eluted Hypurity C(18) reversed-phase HPLC column. The detection limit of the method was 2.0 nmol/L and the measuring range 6-8500 nmol/L. The intra- and inter-assay coefficients of variation were <3.5% (n=10) and <6.0% (n=9), respectively. The new LC-MS method was highly correlated with an established GC-MS method for urinary 5-HTOL (r(2)=0.99, n=70; mean 5-HTOL/GTOL ratio=1.10). This is the first direct assay for quantification of GTOL in urine. The method is suitable for routine application.     

Validated HPLC assay for iron determination in biological matrices based on ferrioxamine formation

A simple, robust and reproducible HPLC method has been developed and validated for iron determination in biological matrices. It is based on chelation with desferrioxamine (DFO) and the measurement of the chelate ferrioxamine (FO). The method was developed to permit monitoring of iron bio-kinetics and estimation of iron status in experimental animals. The chromatography was performed on a stainless steel XTerra MS C18 column (Waters; 250 mm x 4.6 mm i.d., 5 microm) using a gradient of Tris-HCl buffer (10mM, pH 5) and acetonitrile. The method was validated in terms of selectivity, linearity (0.3-80 nmol on-column), limit of detection (0.2 nmol on-column), low limit of quantification (0.3 nmol on-column), recovery (91-102%), intra- and inter-day reproducibility, stability, and robustness. The method's universal applicability was illustrated by monitoring plasma and heart iron kinetic profiles in rats after a single intraperitoneal (i.p.) injection of 200mg/kg iron dextran.     

Development of a urinary free cortisol assay using solid-phase extraction–capillary electrophoresis

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE–CE method for the rapid analysis of UFC. UFC was concentrated using SPE C₁₈ cartridges (3M Empore) under a vacuum and eluted with acetonitrile–SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile–SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10–15 min with a recovery of 89–94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 μg/l with SPE compared to 500 μg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.     

Detection of endogenous ethanol and other compounds in the breath by gas chromatography with on-column concentration of sample

A new method is described for collecting and concentrating volatile compounds in the breath, in order to facilitate their assay by gas chromatography. Breath was collected into sealed Mylar bags containing an internal standard (isopropyl alcohol). The sample was pumped through a cooled gas chromatograph column, where the volatile compounds were concentrated by adsorption onto the resin packing (Porapak Q) at 35 degrees C. The column was then heated, and the volatilized sample was separated for assay by flame ionization detection. The assay was highly sensitive for ethanol (detecting at least 4.0 nmol) and linear up to 20 nmol (r2 = 0.98). Accuracy and precision were determined by assaying nine replicates of a sample containing 12.0 nmol ethanol; a mean value of 12.18 nmol ethanol was obtained with a coefficient of variation of 10.26%. In a group of normal volunteers, endogenous breath ethanol concentrations ranged from 2.23 to 6.51 nmol/liter. This assay provided a number of advantages over previously described methods: The use of breath collection bags enabled the collection of samples outside the laboratory. The use of an internal standard in the collection bag reduced errors that might have resulted from leakage of the specimen. An on-column concentration of the sample in the gas chromatograph eliminated the need for an additional preconcentration device, such as a cryogenic or adsorptive trapping apparatus.     

Urinary metanephrines by liquid chromatography tandem mass spectrometry: using multiple quantification methods to minimize interferences in a high throughput method

Determination of urinary metanephrines is requested frequently for the differential diagnosis and monitoring of pheochromocytoma. Although numerous methods have been developed, interferences are common and hinder most available assays. This study describes the development, validation and implementation of a reliable high-throughput LC-MS/MS method for the measurement of metanephrine and normetanephrine in urine. Metanephrine and normetanephrine were isolated from urine samples subjected to acid hydrolysis using solid phase extraction on a mixed mode cation exchange sorbent in 96-well format. The extracts were injected directly onto a Restek perfluorophenyl column and separated isocratically in 0.2% formic acid in 5% methanol with a gradient cleanout step to 50% methanol. Detection was accomplished using an API 3200 triple quadrupole mass spectrometer with electrospray ionization in positive mode. Data were acquired in multiple reaction monitoring mode. Three transitions were monitored for metanephrine and its deuterated internal standard; two transitions were monitored for normetanephrine and its deuterated internal standard. Two quantification methods were used to address metanephrine interferences without reducing throughput. The method was linear to 15,000 nmol/L. The limits of detection and quantification were 2.5 and 10 nmol/L, respectively. Within run, between-day and total imprecision values were at or below 1.9%, 2.5% and 2.7% for both analytes. The method correlated well with our previously used GC-MS method. Injection-to-injection time was 6 min. The validated LC-MS/MS method for measurement of metanephrine and normetanephrine in urine specimens was placed into service in August 2010 and has performed exceptionally well.     

Quantitative determination of total and unbound paclitaxel in human plasma following Abraxane treatment

A simple, rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) assay was developed and validated for the quantification of both unbound and total paclitaxel in plasma following treatment with Abraxane (ABI-007) or Taxol. Accurate and reproducible analysis of ABI-007, an albumin nanoparticle formulation of paclitaxel could not be achieved using previously published methodology designed for Taxol. The final validated method involved protein precipitation followed by vacuum filtration, in a 96-well format for rapid processing. The 4min run employed gradient elution on a Waters SymmetryShield C8 (2.1mmx50mm, 3.5microm) column, followed by tandem mass spectrometric detection, in electrospray positive mode. Calibrator samples were prepared daily with paclitaxel and analyzed with both ABI-007 and paclitaxel quality control samples. To measure unbound drug, sample preparation was preceded by ultrafiltration. The assay was linear over the range of 10-2500ng/mL, with dilution providing measurement up to 50,000ng/mL. Within-run and between-run precision for all QC samples was less than 5.0% and 10.4%, respectively. Accuracy was high, with deviation of less than 6.1% for all QCs. Measurement of unbound paclitaxel was precise (BRP and WRP <10%).     

Bioluminescence immunoassay for cortisol using recombinant aequorin as a label

The analysis of hormones in saliva is a powerful tool in the assessment of a patient's endocrine function, since it allows multiple noninvasive samplings. Since salivary levels of most hormones are 10 to 50 times lower than plasma levels, accurate and highly sensitive assays are needed for saliva measurements. Herein, we describe the development of a solid-phase competitive immunoassay for cortisol in saliva, in which a mutant of the photoprotein aequorin has been used as a label. We have chemically conjugated cortisol to aequorin at different molar ratios. The various cortisol-aequorin conjugates were characterized in terms of bioluminescent activity and affinity for the anti-cortisol antibody. The conjugate that gave the best analytical performance was used for the development of the immunoassay and the analysis of cortisol in saliva samples. The conjugates were stable for at least 6 months when stored at 4 degrees C. The method fulfilled all the standard requirements of precision and accuracy. The optimized immunoassay gave a detection limit of 300 fmol/tube, corresponding to 3 nmol/L, with a linear dynamic range of 10-1000 nmol/L. Therefore, cortisol can be detected down to 0.1 ng in 100 microl of saliva sample using this assay, without any sample pretreatment. This detection limit is almost one order of magnitude lower than the physiological levels of salivary cortisol, which are reported to be 10-25 nmol/L. This allows the quantification of salivary cortisol to be performed in the linear range of the calibration curve, which is most reliable for quantification purposes.     

Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

New method for HPLC separation and fluorescence detection of malonaldehyde in normal human plasma

A new method for the detection of free and total malonaldehyde (MDA) in human plasma samples based on the derivatization of MDA with 9-fluorenylmethoxycarbonyl hydrazine (FMOC-hydrazine) in an acidic medium was developed. Derivatization was achieved after 4 h at 50 degrees C. The derivatized samples were analyzed by HPLC using a reversed-phase C18 column with fluorescence detection (Ex=270 nm, Em=310 nm). The benefit of this direct injection of deproteinized plasma is to avoid the use of an internal standard. The detection limit was 0.1 pmol (4.0 nmol/L). The recovery of MDA spiked in different human plasma samples was 95.3% (n=25; R.S.D. 5.1%) for the hydrolysation procedure. The total and free MDA in plasma of 15 healthy male volunteers are 426+/-29.8 nmol/L and 153+/-9.6 nmol/L, respectively.     

Simultaneous determination of dexamethasone and 6 beta-hydroxydexamethasone in urine using solid-phase extraction and liquid chromatography: applications to in vivo measurement of cytochrome P450 3A4 activity

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6 alpha- and 6 beta-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6 beta-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6 beta-hydroxydexamethasone using 6 alpha-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C(18) analytical column (4 microm, 300 mm x 3.9 mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6 beta-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6 beta-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99-109%. This method was applied to in vivo measure of the CYP3A4 activity.     

Serum cortisol concentrations during low dose dexamethasone suppression test to screen for Cushing's syndrome.

Forty four subjects (23 obese controls, 11 patients with possible Cushing''s syndrome, and 10 patients with definite Cushing''s syndrome) underwent low dose (0 X 5 mg every six hours for two days) dexamethasone suppression tests during which serum cortisol concentration at 0800 and excretion of urinary free cortisol over 24 hours were measured. Serum cortisol concentration fell to below 60 nmol/1 (2 X 2 micrograms/100 ml) in 31 subjects and remained above 250 nmol/1 (9 X 1 micrograms/100 ml) in the 13 others. Excretion of urinary free cortisol showed a similar response, falling to below 110 nmol (40 micrograms)/24 h in 31 and remaining above 180 nmol (65 micrograms)/24 h in the 13 others. There was complete concordance between the two variables in terms of the pattern of response. Serum cortisol concentration fell to below 60 nmol/1 (2 X 2 micrograms/100 ml) in at least 97% (31 of a possible 32) of subjects without Cushing''s syndrome. On the other hand, a serum cortisol concentration of above 250 nmol/1 (9 X 1 micrograms/100 ml) after low dose dexamethasone gave a false positive diagnosis of Cushing''s syndrome in at most only one of 13 patients (7 X 7%). Measurement of serum cortisol concentration during the low dose dexamethasone test is simpler than, and as accurate and reliable as, measurements of urinary steroids.     

Simple method for the quantitative analysis of endogenous folate catabolites p-aminobenzoylglutamate (pABG) and its acetamido (apABG) derivative in human serum and urine by liquid chromatography-tandem mass spectrometry

OBJECTIVE: To develop a routine method for quantitative measurement of the folate catabolites p-aminobenzoylglutamate (pABG) and acetamidobenzoylglutamate (apABG) in serum and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN AND METHODS: Urine, serum and aqueous standards were thawed. Two microliters of d3-glutamic acid (d3-Glu; 1 mmol/L) was added to 200 uL of specimen as internal standard. The samples were acidified with 4 uL 6N HCL, and aliquots were precipitated with 2 volumes (412 uL) of acetonitrile. For urine specimens 30 volumes (6.18 mL) of acetonitrile was used. Samples were centrifuged at 1900 x g for 10 min and the supernatant (10 microL) injected into a Biorad CAT/MET analytical column fitted to the LC-MS/MS. Detection of the catabolites was by selective multiple ion monitoring (multiple SRM) of the respective transitions. Urine and serum samples were analysed in a group of healthy volunteers and in anonymous samples from patients being tested for PTH and urinary catecholamines. RESULTS: pABG and apABG eluted at 5.2 and 4.74 min, respectively while the d3-glutamic acid eluted at around 7 min. Limit of quantitation (LOQ) for both catabolites was 10 nmol/L (which is equivalent to 33.3 fmol for a 10 microL injection). Limit of detection (LOD) was 1 nmol/L based on a signal to noise ratio of 5:1. A linear calibration curve was obtained from 10 to 100 nmol/L for serum specimens and from 10 to 200 micromol/L for urines. Imprecision for spiked serum samples (n=10) was between 2.5 and 20% for apABG and 4.5 and 21% for pABG (at 10 and 100 nmol/L, respectively). Imprecision for spiked urine samples (n=10) was between 2.9 and 4.0% for apABG and 6.0-12.7% for pABG. Recoveries were between 80 and 122% for serum samples and between 92 and 102% for urine specimens. Total folate catabolites in random urine samples from volunteers (n=5) are 2.9+/-2.3 umol/L (mean+/-S.D.). This group also had total serum catabolites of 11.9+/-7.6 nmol/L and serum folate of 35.3+/-5.8 nmol/L. Serum from patients being tested for PTH (n=11) had serum folate levels of 27.0+/-10.4 nmol/L with total serum catabolites of 20.4+/-23.8 nmol/L. Levels of serum folate and total catabolites in pregnant women (n=18) were 33.9+/-22.7 and 11.4+/-8.7 nmol/L, respectively. Mean urinary folate catabolites in patients being tested for urinary catecholamines (n=19) was 581.8+/-368.4 nmol/L. CONCLUSION: A simple, reliable and highly specific method by LC-MS/MS for detecting and quantifying the folate catabolites pABG and apABG was developed. This enables, for the first time, the routine clinical analysis of folate utilization in patients.     

Relationship between MPA free fraction and free MPAG concentrations in heart transplant recipients based on simultaneous HPLC quantification of the target compounds in human plasma

A simple and sensitive HPLC method for the simultaneous analysis of free MPA and free MPAG was developed. Separation was achieved on a X-Terra RP18 column with acetonitrile-40 mM orthophosphoric acid as eluents using a gradient elution mode over 35 min at a flow rate of 1.5 ml/min. The assay was linear in the range 0.005 mg/L (LOQ) to 5mg/L for free MPA and 0.05 mg/L (LOQ) to 200 mg/L for free MPAG. Isolation of free MPA and free MPAG was done by ultrafiltration and the ultrafiltrate was directly injected. A positive correlation between MPA free fractions and free MPAG concentrations was found. Likewise, free MPAG was related to total MPAG concentrations in the seven heart transplant patients.     

Determination of prostaglandin E(2) by on-line solid-phase extraction-liquid chromatography with ultraviolet detection for microsomal prostaglandin E(2) synthase-1 inhibitor screening

A rapid, robust and selective on-line solid-phase extraction-liquid chromatographic method with ultra-violet detection (on-line SPE-LC-UV) for microsomal prostaglandin E(2) synthase-1 (mPGES-1) inhibitor screening was developed and validated. Disrupted A549 cells were used as mPGES-1 source and the formation of prostaglandin E(2) (PGE(2)) out of the substrate prostaglandin H(2) (PGH(2)) was determined at 195 nm. Direct on-line sample clean up was achieved by automated column switch (C18 trap column) prior isocratic separation using a C18 analytical column. The on-line SPE-LC-UV method was accurate, precise and reproducible in the range of 71-1763 ng/ml for PGE(2) and met the generally accepted criteria for bioanalytical methods. The method was successfully applied to determine the IC(50) value of the known mPGES-1 inhibitor NS-398.     

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard? column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters^® Quattro Premier? XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L (r² = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.     

Simultaneous determination of urinary cortisol, cortisone and corticosterone in parachutists, depressed patients and healthy controls in view of biomedical and pharmacokinetic studies

A rapid and sensitive reversed-phase liquid chromatographic method (RP-LC) with UV detection has been developed for the determination of free cortisol, cortisone and corticosterone in human urine. The assay was performed after a solid-phase extraction procedure (SPE) with dexamethasone as the internal standard. Chromatographic separation was carried out on a Nucleosil 100 C(18) analytical column using a mixture of acetonitrile and water (30 : 70, v/v) as a mobile phase at a flow-rate of 1 mL min(-1). Spectrophotometric detection was performed at 240 nm. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The absolute recoveries of glucocorticoids were above 94.6%. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 2 ng mL(-1), respectively, for all analytes. Linearity was confirmed in the range of 2-300 ng mL(-1) with a correlation coefficient greater than 0.9997 for all steroid hormones. The proposed method was sensitive, robust and specific allowing reliable quantification of steroid hormones. This method was successfully applied for determination of three endogenous glucocorticoid levels in human urine. The studies were performed on 20 sedentary healthy volunteers in comparison to two socially diversified groups, namely 10 parachutists before and after jump and 10 patients with depression. Pharmacokinetic studies performed on these groups indicated that urinary free cortisol and cortisol-to-cortisone ratios can be treated as biomarkers of stress and depressive disorders.     

Chromatographic system for the simultaneous measurement of plasma 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone by radioimmunoassay: reference data for neonates and infants and its application in aldosterone-synthase deficiency

A new chromatographic system for the steroid precursor separation and a sensitive radioimmunoassay system for the subsequent measurement of 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone has been developed. 18-Hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone were extracted with methylene chloride and separated from cross-reacting steroids by Sephadex LH-20 column chromatography. Anti-18-hydroxy-11-deoxycorticosterone and anti-18-hydroxycorticosterone antibodies raised in rabbits were used. The lower detection limit of the assay is 0.03 nmol/l and 0.128 nmol/l for 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone, respectively. Normal values for this assay in 128 healthy neonates and infants aged 0-5 months were established as a basis for the early hormonal diagnosis of aldosterone synthase deficiency types I and II. Its application for the diagnosis of aldosterone synthase deficiency is demonstrated in two patients with homozygous mutation/deletion in the encoding CYP11B2 gene.     

Free malondialdehyde determination in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for quantification of malondialdehyde (MDA) in human plasma. Deproteinized samples were injected onto a Waters carbohydrate analysis column which was eluted with 20% (v/v) 0.03 M Tris buffer, pH 7.4, in acetonitrile. Peak absorbancy was measured at 267 nm. In contrast to data already published, we did not detect any free MDA in normal human plasma. This suggests that the classical thiobarbituric acid test is not suitable for the determination of MDA in human plasma.     

Urinary free cortisol and cortisone excretion in healthy individuals: influence of water loading

The influence of water loading on urinary excretion of free cortisol and cortisone was investigated in healthy men. The results were as follows: water loading tests (intake of 0.25-1.5 L) in a single individual showed that a water load of 1.5 L reliably increased the excretion of urine, free cortisol and cortisone (p < 0.01). Regression analyses gave significant correlations of urine volume with free cortisol and free cortisone, and of free cortisol and free cortisone. Corresponding results were obtained when water loading tests were performed in males who ingested 1.5 L of water (n = 8): the excretion of urine, free cortisol and free cortisone were significantly augmented; correlated was urine volume with free cortisol and free cortisone, and free cortisol with free cortisone. In a third set of tests, volunteers collected one 5 h urine (10:00-15:00 h) after the intake of 3 x 0.1 or 0.5 L at 11:00, 12:00 and 14:00 h. Excretion of urine, free cortisol and free cortisone in males of the low water loading group (3 x 0.1 L) was 0.59 mL/min, and 8.2 or 15.0 microg/5 h; corresponding values in individuals ingesting 3 x 0.5 L of water were 1.5 mL/min (p < 0.01), 12.3 microg/5 h (p > 0.05) and 26.3 microg/5 h (p < 0.02). In summary, urinary free cortisol and cortisone excretion in healthy men depends on urine volume, especially during water diuresis. Thus, interpretation of free cortisol and especially of free cortisone excretion is only possible if subjects strictly control their fluid intake and if urine volume is considered an important pre-analytical parameter-otherwise, interpretation of urinary free cortisol results is difficult and of urinary free cortisone data remains tenuous at best.