Cortisol and cortisone analysis in serum and plasma by atmospheric pressure photoionization tandem mass spectrometry

OBJECTIVES: Cortisol metabolism is controlled by 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isoenzymes, which interconvert cortisol and cortisone. Accurate measurement of the cortisol and cortisone concentrations and their ratio provide useful information about 11beta-HSD activity. METHODS: Cortisol and cortisone were extracted with methyl-tert-butyl ether from 100 microl of serum or plasma. The extract was evaporated, reconstituted with mobile phase, and analyzed by tandem mass spectrometry using a photoionization interface. The transitions monitored were: m/z 363 to 121 and 363 to 97 for cortisol, 361 to 163 and 361 to 105 for cortisone. RESULTS: Within-run and between-run coefficients of variation were less than 6% and 12%; 14% and 22%; 11% and 21% for cortisol, cortisone, and their ratio, respectively. The limit of detection was 1 microg/l for cortisol and 5 microg/l for cortisone. Normal ranges for cortisol and cortisone concentration and for their ratio in plasma (n = 120) determined as the central 95% were 33-246 microg/l for cortisol, 8-27 microg/l for cortisone, and 0.081-0.301 for the cortisone/cortisol ratio. CONCLUSIONS: We developed a simple sensitive method for cortisol and cortisone analysis in plasma and serum that uses a small sample volume. The method is very specific, fast, does not have any known interference, and is useful for diagnosis of variety of disease and pathologic conditions.     

Determination of steroid hormones in a human-serum reference material by isotope dilution--mass spectrometry: a candidate definitive method for cortisol

We report a method, based on isotope dilution--mass spectrometry, for determining cortisol in a pooled specimen of human serum. Isotopically labeled cortisol is added to 5.0 mL of serum so that the molar concentrations of labeled cortisol and unlabeled cortisol are approximately equal. The specimen and two calibration standards are extracted with dichloromethane, and the extracted cortisol is converted to the methoxime-trimethylsilyl ether derivative. Samples and standards are analyzed by gas chromatography--mass spectrometry by monitoring the peak areas for m/z 605 and 608. The cortisol concentration is calculated by linear interpolation between the two bracketing standards. Variances of data collected during six weeks showed that the overall coefficient of variation (CV) was 0.69% (n = 32); the within-vial CV, 0.63%; the among-vial CV, 0.22%; and the among-day CV, 0.15% (means = 3.973 nmol/vial). Method specificity was demonstrated by liquid chromatographic as well as C8 mini-column cleanup of samples before derivation, by alternative ion monitoring at m/z 636 and 639, and by negative-ion chemical ionization at m/z 459 and 462. Derivatives of all observed degradation products of cortisol under basic, neutral, and acidic conditions did not interfere.     

Analytical and clinical performance of two cardiac troponin I immunoassays.

Cardiac troponin I assays for Axsym (Abbott Diagnostics, Abbott Park, IL, USA) and Immuno 1 (Bayer Corporation, Tarrytown, NY, USA) analysers were evaluated. Heparin plasma or serum could be used for both assays. Samples were stable for 24 h at ambient temperature, 3 days at 4-8 degrees C and 3 months at -20 degrees C. After 10 months' storage at -80 degrees C, the recoveries were well above 100% by both assays. Total coefficients of variation for Axsym assay were 9.0%, 5.8% and 5.3% at concentrations of 2.6 microg/l, 9.83 microg/l and 34.3 microg/l respectively; for Immuno 1 these were 4.4 %, 1.6% and 1.8% at 2.3 microg/l, 6.27 microg/l and 44.35 microg/l respectively. It was > or =20% at concentration of < or =0.5 microg/l for Axysm assay and < or =0.15 microg/l for Immuno 1 assay. Recoveries were < or =90% at < or =0.22 microg/l on Axsym and at < or =1.47 microg/l on Immuno 1. Neither method showed significant interference with haemoglobin, bilirubin, triglycerides or rheumatoid factor. Correlation between the two methods was excellent (r = 0.997, Y (Axsym) = 4.2X (Immuno 1) +3.2). The highest concentrations detected in 50 healthy subjects were 0.3 microg/l and 0.1 microg/l by Axsym and Immuno 1 methods, respectively. Twelve out of 43 renal failure patients had troponin I 0.13-0.9 microg/l using Axsym method and 4 had levels of 0.07-0.13 microg/l using Immuno 1. In muscle trauma patients, troponin I was undetectable.     

Determination of homovanillic acid in urine by stable isotope dilution and electrospray tandem mass spectrometry

We have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of homovanillic acid (HVA), a biochemical marker for catecholamine and neurotransmitter metabolism. Urine specimens are spiked with 5 microg of a stable-isotope labeled internal standard, 13C(6)18O-HVA, and prepared by automated solid phase extraction. Residues were dissolved in acetonitrile: 0.05% aqueous acetic acid and analyzed by MS/MS in the selected reaction monitoring mode (HVA: m/z 181 to m/z 137; 13C(6)18O-HVA: m/z 189 to m/z 145) after separation using a Discovery RP Amide C16 column. Consecutive calibrations (n=7) between 0.52 and 16.7 mg/l exhibited consistent linearity and reproducibility. At a urine concentration of 0.51 mg/l, the signal-to-noise ratio for HVA was 21:1. Inter- and intra-assay CVs ranged from 0.3% to 11.4%, at mean concentrations ranging 1.8 to 22.7 mg/l. Recovery of HVA added to urine ranged between 94.7% and 105% (1.25 mg/l added), 92.0% and 102% (5.0 mg/l), and 96.0% and 104% (10 mg/l). LC-MS/MS is well suited to replace an HPLC method for routine HVA determination, by providing positive identification, faster turn around time, virtually no repeat analyses and a 44% reduction of personnel necessary to perform the testing.     

Analytical evaluation of an automated immunoassay for cardiac troponin I: the Vidas Troponin I assay.

Cardiac troponin I (cTnI) is a sensitive and specific biochemical marker of myocardial damage. We assessed the analytical performance of the Vidas Troponin I assay (Biomerieux). Controls and serum pools were used to determine the precision, analytical sensitivity and linearity; 97.5 and 99.5 percentiles concentrations were determined from the reference population. Fifty corresponding samples of serum and plasma (lithium-heparin) were tested and the results compared. The in vitro stability of serum and plasma samples was assessed at 20 degrees C, 4 degrees C and -20 degrees C, respectively. Samples of serum were used to assess the agreement between the Vidas Troponin I method and the revised Dimension RxL cTnI method (Dade-Behring). The total imprecision (CVs) was 13.1-5.2% for concentrations ranging between 0.25 and 19.8 microg/l cTnI. The lower detection limit was <0.1 microg/l. The upper reference limit (97.5 and 99.5 percentiles) was 0.11 microg/l and 0.12 microg/l, respectively (CV > 10%). The assay was linear up to 21 microg/l. The concentrations in lithium-heparin plasma were higher compared to those of the matched serum samples. The study of the agreement between the Vidas and Dimension RxL cTnI assays showed a total concordance of 96% with a bias value of -0.042. The Vidas Troponin I test is a fast, precise and sensitive method for the determination of cTnI.     

Primary hypoadrenalism assessed by the 1 microg ACTH test in hospitalized patients with active pulmonary tuberculosis

Primary hypoadrenalism, assessed by 250 microg ACTH stimulation, is uncommon in patients with active pulmonary tuberculosis (PTB). Since 1 microg ACTH produces an equivalent +30 min cortisol response to 250 microg in control subjects, the 250 microg dose is supraphysiological and may lack sensitivity for the diagnosis of hypoadrenalism. Furthermore, the impact of coexistent HIV infection on the prevalence of primary hypoadrenalism in PTB is uncertain. We thus determined the cortisol response to an intravenous bolus of 1 microg ACTH in 21 controls, 18 HIV-positive (BMI 19.5+/-0.9 kg/m(2), albumin 24+/-1.4 g/l, CD4 count 192+/-47/mm(3)) and 22 HIV-negative (BMI 19.3+/-0.8 kg/m(2), albumin 29+/-1 g/l, CD4 count 652+/-76/mm(3)) patients with active PTB. The mean basal cortisol was greater in patients than in controls (559 vs. 373 nmol/l, p=0. 0009). The mean cortisol after 1 microg ACTH stimulation did not, however, differ significantly when comparing either patients and controls or patients who were HIV-positive and -negative (p>0.05). Using the minimum +30 min cortisol derived from the 21 controls as a marker of normal adrenal function (414 nmol/l), a single patient was classified as hypoadrenal. In conclusion, primary hypoadrenalism, as assessed by the 1 microg ACTH test, is uncommon in a cohort of ill, hospitalized patients with active PTB, irrespective of HIV status.     

Quantification of sirolimus by liquid chromatography-tandem mass spectrometry using on-line solid-phase extraction.

Quantification of the new immunosuppressant sirolimus (syn. rapamycin; Rapamune) in whole blood by chromatography is essential for its clinical use since no immunoassay is available although monitoring is mandatory. Here we report on a rapid and convenient liquid chromatography (LC-tandem mass spectrometry method and describe our practical experience with its routine use. Whole blood samples were hemolyzed and deproteinized using an equal volume (150 microl) of a mixture of methanol/zinc sulfate solution containing the internal standard desmethoxy-rapamycin. After centrifugation, the clear supernatants were submitted to an on-line solid-phase extraction procedure using the polymeric Waters Oasis HLB material, with elution of the extracts onto the analytical column in the back-flush mode by column switching. For analytical chromatography a RP-C18 column was used with 90/10 methanol/2 mM ammonium acetate as the mobile phase. A 1:10 split was used for the transfer to the mass spectrometer, a Micromass Quattro LC-tandem mass spectrometry system equipped with a Z-spray source and used in the positive electrospray ionization mode. The following transitions were recorded: sirolimus, 931>864 m/z, and desmethoxy-rapamycin (I.S.), 901>834 m/z. The analytical running time was 5 min, including on-line extraction. The method has a linear calibration curve (r>0.99; range 1.6-50 microg/l) and is rugged and precise with monthly CVs <7% at a sirolimus concentration of 13.1 microg/l in routine use; the instrumentation proved to be reliable and required minimal maintenance. Clin Chem     

Development and evaluation of a simple, direct, solid-phase radioimmunoassay of serum cortisol from readily available reagents

A simple, rapid solid-phase radioimmunoassay for serum cortisol was developed using cortisol antibody distributed by the Scottish Antibody Production Unit and commercially available radioiodinated cortisol ligand. The assay involves a 1-h incubation at ambient temperature, using the antibody covalently linked by the easily performed carbonyldiimidazole method, to microcrystalline cellulose. A detailed comparison of the accepted 0.125 mol/l citrate, pH 4.0, and an alternative 0.1 mol/l phosphate/8-anilinonaphthalene sulphonic acid, pH 7.4, diluent demonstrated similar precision (within-assay 10% to 5%; between-assay 11% to 6% over the range 100-1000 nmol/l cortisol) and recovery (median recoveries: phosphate 98.6%, citrate 99.5%). Linear regression analysis of human serum cortisol concentrations in comparison with the 'Amerlex' cortisol RIA kit gave the following results: citrate = 1.029. 'Amerlex' - 3.5 nmol/l (n = 80, r = 0.967); phosphate = 1.017, 'Amerlex' + 26.2 nmol/l (n = 80, r = 0.961). Phosphate, pH 7.4 diluent was adopted as the diluent of choice, since it was economical of antibody and maintained good precision over a wider working range of cortisol concentration.     

Historical trends in human serum levels of perfluorooctanoate and perfluorooctane sulfonate in Shenyang, China

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are widespread contaminants in the environment, as well as in wildlife and in humans. The PFOS and PFOA concentrations were determined in historical human serum samples collected in Shenyang, China, in 1987 (n=15), 1990 (n=33), 1999 (n=68) and 2002 (n=119). The serum donors were students, faculty members and university workers. Since the serum PFOA and PFOS levels did not follow a normal or log-normal distribution, a nonparametric method was applied to analyze the historical trends. For the total male and female subjects, the median level of serum PFOA increased significantly from 0.08 microg/l in 1987 to 4.3 microg/l in 2002 (p<0.05), while the median level of serum PFOS also increased significantly from 0.03 microg/l in 1987 to 22.4 microg/l in 2002 (p<0.05). Both the serum PFOA and PFOS levels continued to increase from 1999 to 2002, with remarkable increases observed in females: 6.3-fold increase for PFOA and 13-fold increase for PFOS. In 2002, serum PFOA and PFOS concentrations of female subjects have increased to 4.9 microg/l and 22.4 microg/l in median, respectively, which are comparable to those in U.S.A. and Japan. For male subjects, serum PFOA and PFOS concentrations (1.6 microg/l and 8.3 microg/l in median, respectively) are comparable to those in Italy. The data from this study indicate that females are likely to experience higher exposure to these chemicals.     

Dialyzable free cortisol after stimulation with Synacthen

OBJECTIVES: To compare the changes in free vs. total serum cortisol concentrations after acute stimulation of the adrenal cortex. DESIGN AND METHODS: Paired serum samples of ten individuals taken immediately before and 1 h after stimulation with 250 microg ACTH (1-24) (Synacthen) given i.v. were analyzed. Total cortisol was quantified using liquid chromatography tandem-mass spectrometry with an online sample extraction system and tri-deuterated cortisol as the internal standard. Free cortisol was measured with the same method after equilibrium dialysis. Concentrations of the corticosteroid-binding globulin (CBG) were determined by radioimmuno assay. RESULTS: Total cortisol increased by a mean of 106% (mean basal cortisol 312 nmol/L (SD 140 nmol/L), stimulated 686 nmol/L (SD 163 nmol/L); p < 0.001, paired t-test for differences); no significant change of CBG concentrations was found (874 nmol/L (SD 179 nmol/L) before stimulation, 869 nmol/L (SD 225 nmol/L) after stimulation). The mean increase of free cortisol was 263% (mean basal free cortisol 20.3 nmol/L (SD 13.2 nmol/L), stimulated 73.8 nmol/L (SD 26.7 nmol/L); p < 0.001) and thus substantially more pronounced compared to the increase of total cortisol (p < 0.01). The ratio of free to total serum cortisol was significantly increased after stimulation (6.1% (SD 1.7%) before stimulation, 10.6% (SD 1.9%) after stimulation; p < 0.001). CONCLUSIONS: After acute neuroendocrine stimulation of the adrenal cortex the relative increase of free bioactive cortisol concentrations is substantially more pronounced than the increase of total cortisol concentrations.     

Free serum cortisol during the postoperative acute phase response determined by equilibrium dialysis liquid chromatography-tandem mass spectrometry.

In severely ill patients low concentrations of the corticosteroid binding globulin are typically found; the aim of this study was to quantify directly free bioactive cortisol concentrations in the sera of postoperative cardiosurgical patients. Serum samples of 12 consecutive patients undergoing aortocoronary bypass surgery taken preoperatively and on the postoperative days 1 to 4 were analyzed. Total serum cortisol was quantified using liquid chromatography-tandem mass spectrometry with an on-line sample extraction system and tri-deuterated cortisol as the internal standard, and free serum cortisol was measured after over-night equilibrium dialysis. Whereas on the first postoperative day, the median total serum cortisol concentration was approximately two-fold increased compared to preoperative samples (preoperatively, 245 nmol/l (interquartile range (IQR) 203-293 nmol/l); first postoperative day, 512 nmol/l (IQR 410-611 nmol/l)), median dialyzable free cortisol concentration was almost seven-fold increased (preoperatively, 14.2 nmol/l (IOR 10.9-20.7 nmol/l); first postoperative day, 98.3 nmol/l (IQR 81.3-134 nmol/l)). On the fourth postoperative day, median free cortisol was still significantly increased compared to baseline sampling (p < 0.05), whereas median total cortisol was not. A median of 5.7% (IQR 5.4-7.0%) of total cortisol was found as free cortisol on the preoperative day, 21.2% (IQR 18.9-23.5%) on the first postoperative day and 10.5% (IQR 9.8-14.0%) on the fourth postoperative day. It is concluded that during the postoperative period the free-to-bound ratio of cortisol is highly variable and that during the acute phase response direct quantification of free bioactive cortisol concentrations seems to be biologically more appropriate than the measurement of total cortisol concentrations.     

Highly sensitive immunoassay of free prostate-specific antigen in serum using europium(III) nanoparticle label technology

BACKGROUND: Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle-antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform. METHODS: Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle-antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer. RESULTS: The detection limit (mean + 2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-microl sample volume. The assay response was linear up to 5 microg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005-0.5 microg/l was 6.4-21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2-2.5 microg/l were 3.4-7.2% and 4.4-7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope = 1.018 +/- 0.018; intercept = 0.012 +/- 0.021 microg/l; S(y/x) = 0.112 microg/l; r = 0.993; n = 51. CONCLUSIONS: The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.     

Increased serum maternal levels of the HER2 oncoprotein p105 ectodomain in preeclampsia.

HER2/ERBB2 protein is a 185 kDa transmembrane growth factor receptor whose extracellular domain, a 105 kDa fragment (p105), can be released from cell surfaces by proteolytic cleavage. The aim of our study was to compare serum p105 concentrations in normal and pathological pregnancies and to determine whether any correlation exists between preeclampsia and p105 levels. Serum p105 was assayed in 96 non-pregnant women and 89 pregnant women (26 normotensive, 14 normotensive with a history of preeclampsia or fetal hypotrophy, 10 with chronic hypertension, 10 with gestational hypertension and 29 with preeclampsia). Median serum p105 levels (median; 95% confidence interval) were higher in the preeclampsia group (13.9 microg/l; 12.8-16.1 microg/l) than in the normotensive (11.7 microg/l; 10.6-13.3 microg/l; p < 0.05) or non-pregnant groups (9.3 microg/l; 8.9-9.6 microg/l; p < 0.001). There were no significant differences between the other pregnancy groups. In the normotensive group, serum p105 was correlated with the number of gestations (r = 0.46; p < 0.05), parity (r = 0.39; p < 0.05) and placenta weight (r = 0.61; p < 0.05). In preeclamptic women, serum p105 correlated with parity (r = 0.46; p < 0.05). Serum p105 concentrations above 11.9 microg/l were associated with a high odds ratio (OR) for onset of preeclampsia (after adjustment for parity OR = 9.0; 95% CI = 2.3-36.0; p < 0.005). Preeclampsia is associated with increased serum p105 concentrations, which may be related to increased fetomaternal cell traffic.     

Saliva collection method affects predictability of serum cortisol

BACKGROUND: Measurement of cortisol is of particular importance for early diagnosis of adrenocorticotropic dysfunction and therapeutic monitoring. Saliva samples clearly present an easily manageable, non-invasive and anytime practicable assessment method of glucocorticoid production. The aims of the present study were to compare salivary cortisol collection method "Salivette" versus "passive drool", salivary cortisol concentrations with calculated free serum cortisol (FSC) and total serum cortisol (TSC) and the prediction of serum cortisol by salivary cortisol. METHODS: Saliva samples were collected by Sarstedt-Salivette(R) and by passive drooling into plastic tubes. Salivary cortisol, TSC measurements and calculation of FSC concentrations as well were carried out simultaneously in 10 healthy volunteers on two consecutive days at six definite points of time (08.00-23.00). RESULTS: Significant differences of salivary cortisol concentrations dependent on the saliva collection method (F-test 6.45; p=0.0317) and points of times were found (F-test 41.19; p<0.0001). Stronger overall correlation was observed in Salivette-salivary cortisol vs. TSC (r=0.813) and FSC (r=0.836) as compared to passive drooling. Specifically, more significant correlations were established in Salivettes vs. TSC (8/12 points of time, 66.7%) and FSC (8/12 points of time, 66.7%) than in passive drooling (TSC: 1/12 points of time, 8.3% and FSC: 2/12 points of time, 16.7%). Importantly, cortisol in Salivettes was a better predictor for FSC and TSC than passive drooling. CONCLUSIONS: Our data indicate that salivary cortisol measurement with Salivettes is a reliable prediction method of total and calculated free serum cortisol levels. Moreover, it is a convenient method for saliva collection, handling and laboratory processing.     

Adrenal Dysfunction in Hemodynamically Unstable Patients in the Emergency Department

OBJECTIVE: Adrenal failure, a treatable condition, can have catastrophic consequences if unrecognized in critically ill ED patients. The authors' objective was to prospectively study adrenal function in a case series of hemodynamically unstable (high-risk) patients from a large, urban ED over a 12-month period. METHODS: In a prospective manner, critically ill adult patients presenting to the ED were enrolled when presenting with a mean arterial blood pressure < or =60 mm Hg requiring vasopressor therapy for more than one hour after receiving fluid resuscitation (central venous pressure of 12-15 mm Hg or a minimum of 40 mL/kg of crystalloid). Patients were excluded if presenting with hemorrhage, trauma, or AIDS, or if steroids were used within the previous six months. An adrenocorticotropic hormone (ACTH) stimulation test was performed and serum cortisol was measured. Treatment for adrenal insufficiency was not instituted. RESULTS: A total of 57 consecutive patients were studied. Of these, eight (14%) had baseline serum cortisol concentrations of <20 microg/dL (<552 nmol/L), which was considered adrenal insufficiency (AI). Three additional patients (5%) had subnormal 60-minute post-ACTH-stimulation cortisol responses (<30 microg/dL) and a delta cortisol < or =9 microg/dL, which is the difference between the baseline and 60-minute levels. This is functional hypoadrenalism (FH). There were no laboratory abnormalities that distinguished patients with AI or FH from those with preserved adrenal function (PAF). Rates of survival to discharge did not differ between the AI group (7 of 8) and PAF patients (21 of 46; p = 0.052). CONCLUSIONS: Adrenal dysfunction is common in high-risk ED patients. Overall, it has a frequency of 19% among a homogeneous population of hemodynamically unstable vasopressor-dependent patients. The effect of physiologic glucocorticoid replacement in this setting remains to be determined.     

Baseline cortisol levels,cortisol response to corticotropin,and prognosis in late septic shock

The prognostic value of basal and corticotropin-stimulated cortisol concentration in patients with sepsis remains a controversial issue. In a retrospective cohort study, 82 consecutive patients with septic shock underwent a short corticotropin test performed more than 24 h after the onset of vasopressor therapy. Forty-one (50%) patients died within 28 days after the onset of septic shock. The mean (SD) basal cortisol level was 22.7 (10.6) microg/dL. With threshold values of 7 and 9 microg/dL maximal increases in cortisol level, 28 (34%) and 31 (38%) patients were, respectively, classified as nonresponders to the short corticotropin test. On multivariate analysis, a cortisol level >20 microg/dL (P = 0.0002), a maximal response to corticotropin <9 microg/dL (P = 0.044), abnormal lactate values (P = 0.0098), and positive blood cultures (P = 0.004) were independent predictors of 28-day mortality. In conclusion, high basal cortisol and low increase on corticotropin stimulation are predictors of a poor outcome in late septic shock. The underlying mechanisms of these prognostic patterns remain to be elucidated.     

Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone

BACKGROUND: Urinary free cortisol and cortisone measurements are useful in evaluation of Cushing syndrome, apparent mineralocorticoid excess, congenital adrenal hyperplasia, and adrenal insufficiency. To reduce analytical interference, improve accuracy, and shorten the analysis time, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for urinary cortisol and cortisone. METHODS: We added 190 pmol (70 ng) of stable isotope cortisol-9,11,12,12-d(4) to 0.5 mL of urine as an internal standard before extraction. The urine was extracted with 4.5 mL of methylene chloride, washed, and dried, and 10 microL of the reconstituted extract was injected onto a reversed-phase C(18) column and analyzed using a tandem mass spectrometer operating in the positive mode. RESULTS: Multiple calibration curves for urinary cortisol and cortisone exhibited consistent linearity and reproducibility in the range 7-828 nmol/L (0.25-30 microg/dL). Interassay CVs were 7.3-16% for mean concentrations of 6-726 nmol/L (0.2-26.3 microg/dL) for cortisol and cortisone. The detection limit was 6 nmol/L (0.2 microg/dL). Recovery of cortisol and cortisone added to urine was 97-123%. The regression equation for the LC-MS/MS (y) and HPLC (x) method for cortisol was: y = 1.11x + 0.03 microg cortisol/24 h (r(2) = 0.992; n = 99). The regression equation for the LC-MS/MS (y) and immunoassay (x) methods for cortisol was: y = 0.66x - 12.1 microg cortisol/24 h (r(2) = 0.67; n = 99). CONCLUSION: The sensitivity and specificity of the LC-MS/MS method for urinary free cortisol and cortisone offer advantages over routine immunoassays or chromatographic methods because of elimination of drug interferences, high throughput, and short chromatographic run time.     

An assay for separating and quantifying four bilirubin fractions in untreated human serum using isocratic high-performance liquid chromatography

BACKGROUND: The quantification of serum bilirubin fractions has been widely performed with both the diazo-method and an enzymatic method; however, the accuracy of these methods has not been evaluated because quantitative fractional high-performance liquid chromatography (HPLC) reference methods have yet to be established. METHODS: Samples were analyzed using HPLC and Shodex Asahipak GS-320HQ columns. Human serum was subjected to HPLC using direct injection, then eluted with acetonitrile: 0.3 mol/l phosphate buffer (pH 6.5) containing 1% Brij 35 and 0.08% sodium ascorbate (30:70, v/v). RESULTS: Serum bilirubin was separated into 4 fractions; retention times of 9.24, 19.92, 24.07, 35.75 min were identified as delta bilirubin, bilirubin diglucuronide, bilirubin monoglucuronide, and unconjugated bilirubin, respectively. Mean recovery was 93.0%-99.2%. Total precision of peak retention time, height and area exhibited <4.26% variation. Detection range was 3.1 to 348 mg/l. Hemoglobin (6 g/l) and immunoglobins produced a small positive interference. beta-carotene (20 mg/l), vitamin-B2 (370 microg/l) and B(12) (9.5 microg/l) did not interfere with this analysis. Results (n=30) with this method were closely correlated to those by Adachi's HPLC method as r=0.9941 to 0.9960, slope=0.88 to 1.27, intercept=-3.2 to +4.9, for each fraction. CONCLUSIONS: Since this method was a precise quantitative HPLC method for serum bilirubin fractionation, it might be used to evaluate the accuracy and the characteristics of various routine methods for bilirubin measurement.     

Increased anxiety level and high salivary and serum cortisol concentrations in patients with recurrent aphthous stomatitis

Recurrent aphthous stomatitis (RAS) is one of the most common oral mucosal disorders. The aim of the study was to determine any association between anxiety levels and concentrations of salivary and serum cortisol in patients with RAS. It has been suggested that stress with its presumed effects on the immune system, constitutes one of the major causative agents of RAS. The concentrations of salivary and serum cortisol were measured in 38 patients with recurrent aphthous stomatitis, and 38 healthy controls. Salivary and serum cortisol levels were measured using a Luminenscent Immunoassay (LIA) method. Anxiety levels were evaluated using Spielberger's State-Trait Anxiety Inventory which measures both trait anxiety as a general aspect of personality (STAI-T) and state anxiety as a response to a specific situation (STAI-S). The salivary cortisol levels were 1.44 (+/- 0.58) microg dl(-1) in RAS patients and 0.91 (+/- 0.56) microg dl(-1) in controls (p = 0.001), while the serum cortisol levels were 3.13 (+/- 1.59) microg dl(-1) in RAS patients and 1.89 (+/- 1.11) microg dl(-1) in controls (p = 0.001). The state anxiety levels (STAI-S) were 48.85 (+/- 9.7) in RAS group and 39.45 (+/- 7.5) in control group (p = 0.001). The trait anxiety levels (STAI-T) were 49.78 (+/- 13.02) in RAS group and 38.49 (+/- 10.31) in control group (p = 0.001). Salivary and serum cortisol concentrations and state and trait anxiety levels in RAS were significantly higher than those in the control group. Our results suggest that stress may be involved in the pathogenesis of RAS.