Determination of cortisol and associated glucocorticoids in serum and urine by an automated liquid chromatographic assay

Clin Biochem, Vol.26, pp. 153-158, 1993

0009-9120/93 $6.00 + .00 Copyright ©1993 The Canadian Societyof ClinicalChemists.

Printed in the USA.All rights reserved.

Determination of Cortisol and Associated Glucocorticoids in Serum and Urine by an Automated Liquid Chromatographic Assay GHASSAN J. SAMAAN, DOMINIQUE PORQUET, JEAN-FRANQOIS DEMELIER, and DANIEL BIOU Service de Biochimie-Hormonologie, H6pital R. Debre, 48, Bd. Serurier, 75019 Paris, France We describe a method for the determination of urinary free cortisol and glucocorticoids in plasma, used in the diagnosis of adrenal disorders, based on automated reverse-phase highperformance liquid chromatography (HPLC). The within-day and day-to-day CVs were less than 5.5 and 8.0%, respectively. The calibration curves for cortisol and 11-deoxycortisol were linear up to 2000 nmol/L. Cortisol concentrations as low as 3.5 nmol/L in 1 mL of plasma or urine can be measured. Correlation of HPLC results for 40 plasma specimens with those by radioimmunoassay showed r = 0.965. This method is sensitive and free from the interference habitually encountered in immunoassays, and can thus be proposed for research and as a potential reference method.

K E Y W O R D S : a d r e n a l disorders; c o m p e t i t i v e p r o t e i n binding; corticosterone; cortisol; dexamethasone; lldeoxycortisol; fludrocortisone; glucocorticoids; highperformance liquid chromatography; radioimmunoassay.

Introduction

he measurement of urinary free cortisol (UFC), plasma cortisol, and other glucocorticoids, espeT cially ll-deoxycortisol, has been shown to facilitate the diagnosis of adrenal-associated glucocorticoid disorders such as Cushing's syndrome, adrenal hyperplasia, and adrenal tumours (1-4). Methods for measuring glucocorticoids in biological fluids are often based on radioimmunoassay (RIA) or competitive protein-binding radioassays (1,4,5-7). When used to measure cortisol, these immunological methods show moderate imprecision. Accuracy also depends on the specificity of the antibody and on the metabolites present in the urine. Immunoassays often overestimate the concentration of UFC when compared with chromatographic assays. Recently, the development of fully automated immunoassay systems, particularly microparticle capture enzyme immunoassay (MEIA) has improved precision, but the problem of interfering metabolites in urine often remains unresolved (8). The lack of specificity is caused by the reaction of the antiserum with various compounds such as synthetic corticoCorrespondence: Dr. Ghassan J. Samaan. Manuscript received September 4, 1992; revised November 4, 1992; accepted December 23, 1992. CLINICAL BIOCHEMISTRY, VOLUME 26, J U N E 1993

steroids and metabolites with chemical configurations similar to that of cortisol (5,9). High-performance liquid chromatography (HPLC) techniques for measuring cortisol in plasma and urine are potentially free from interference (9-12). Dexamethasone, fludrocortisone, and n-propylparaben are currently proposed as internal standards (11,12) but are not suitable for urine and plasma glucocorticoid assays by HPLC because of interference by other peaks, particularly in urinary extracts. The use of prednisone for plasma and corticosterone for urine samples overcomes these difficulties. HPLC techniques usually involve glucocorticoid extraction with organic solvents followed by laborious washing and transfer steps with NaOH and water (9,11). Our aim was to develop a simplified, highresolution technique by: (i) suppressing the water wash step; (ii) selecting a flow rate and mobile phase composition to accelerate the elution; (iii) reducing sample and reagent volumes. Materials and Methods

H P L C APPARATUS The HPLC system (SPECTRA-PHYSICS, San Jose, CA 95134, USA) consists of an SP 8800 pump, an SP 8450 UV/visible detector set at 254 nm and 0.01 absorbance units (AU) full scale, an SP 4290 integrator, and an SP 8775 autosampler. An EpsonPC microcomputer (Epson America Inc., Torrance, CA 90505, USA) utilizing MS-DOS is used to control the HPLC system. The analytical column is a 250 x 4.6 m m (i.d.) column packed with 5-~m particles of Nucleosil-C18 purchased from the French Society of Columns and Chromatography (S.F.C.C, Eragny, France). REAGENTS

Methanol (RPE-ACS, analytical grade) and chloroform (RS-HPLC) were from Carlo-Erba (Milan, Italy).All other chemicals were of analytical grade (Sigma, St. Louis, MO, USA). Distilled,sterilewater 153

SAMAAN, PORQUET, DEMELIER, AND BIOU

for hospital use (Central Hospital Pharmacy, Nanterre, France) was used for all reagent preparations. The internal standard (IS1) for the urinary free cortisol assay was a 2 tLmol/L solution of corticosterone in methanol. The internal standard (IS2) for the plasma corticosteroid assay was a 2 tLmol/L solution of prednisone in methanol. Three commercial sera, Seronorm, Patho. H. and Patho. L. (Mycomed Laboratory, Oslo, Norway), and two spiked control urine pools were used for the quality control of the plasma and urinary glucocorticoid assays, respectively. Urine and plasma samples were obtained from 12 children (age range: 1-12 years) hospitalized in a general ward for adrenal disorders and 135 children without adrenal disorders undergoing routine biological investigations (age range: 6 m o n t h s - 1 8 years). All standards, quality control preparations, and specimens were stored at - 2 0 °C until use. CHROMATOGRAPHIC CONDITIONS

The mobile phase is methanol/tetrahydrofuran/ water (55/0.5/44.5, v/v), filtered through a 0.45-~m (pore size) nylon filter (Millipore Corporation, USA) and degassed with helium before use. A flow rate of 1.2 mL/min is used. PROCEDURES

To measure cortisol and associated glucocorticoids we used a modification of the HPLC method proposed by Canalis et aI. (9) for the determination of glucocorticoids in both plasma and urine: 1 mL of urine or plasma is mixed with 100 ~LL of internal standard (IS1 for urine and IS2 for plasma). Two mL of chloroform is added and the mixture is shaken for 5 min then centrifuged for 3 min at 2000 x g at 4 °C. After removing the aqueous layer, the organic layer is washed with 2 mL of 0.3 M NaOH by vortexing for 2 min, then evaporated under a stream of nitrogen. The residue is dissolved in 200 ~L of the mobile phase, then 20 ~L is injected into the column. Analyte concentrations are calculated from the ratios of their peak surfaces to that of the internal standard, the peaks being identified by their retention times. The RIA used was the "Magic" Cortisol RIA (Ciba-Corning Laboratory, Medfield, USA) for the assay of plasma cortisol according to the manufacturer's instructions. Results

Retention times were 6.7 min for prednisone (n = 35); 7.5 min for cortisone (n = 35); 10.0 min for cortisol (n = 52); 13.8 min for ll-deoxycortisol (n = 35); and 16.0 min for corticosterone (n = 35). Figure l(I) shows the chromatogram of an aqueous standard of prednisone (IS2); cortisone (peak A); cortisol (peak B); ll-deoxycortisol (peak C); and corticosterone (peak D). Figure l(II) shows the chromatogram of a urine sample obtained from a healthy 154

child of 12 years and supplemented with corticosterone as internal standard (IS1). Cortisol and internal standard peaks were well separated from the interfering peaks usually detected in urine from subjects with adrenal hyperplasia [Figure l(III)]. Figure I(IV) shows the chromatogram of a plasma sample obtained from a child with adrenal hyperplasia, and supplemented with prednisone as internal standard. This child was not receiving medication known to affect the pituitary-adrenal axis. The cortisol precursors present in high amounts in plasma and urine from patients with adrenal hyperplasia were not eluted within the run time of the chromatogram, because they are all much less polar than cortisol, and therefore do not interfere with the quantification. Similarly, testosterone, progesterone, and androstenedione, known to cross-react with antisera used in the RIA of cortisol, are eluted after the corticosterone peak (retention time greater than 22.0 min). Aldosterone does not interfere with cortisol (peak B) or 11-deoxycortisol (peak C) determination, and is eluted in 5.2 min. Cortisol metabolites present in the urine sample from a patient with adrenal hyperplasia, that is, 20 a-dihydrocortisol and 6~-hydroxycortisol, were eluted from the column in 7.0 and 4.5 min, respectively, and consequently did not interfere with cortisol (peak B). The urinary cortisol result is not affected by conditions that elevate plasma corticosterone such as congenital a d r e n a l hyperplasia. Corticosterone is rapidly metabolized in the liver, and under normal circumstances, plasma concentrations above 500 nmol/L (approximately 15-fold normal value) do not affect the urinary cortisol result determined by HPLC (results not shown). Glucocorticoid determination in plasma containing high amounts of prednisone can be achieved by injecting the plasma extract without adding prednisone (blank), then substracting the blank from the IS peak. Using blank substraction, the standard error is less than 2.0% (n = 15). The mean (+-SD) plasma cortisol levels evaluated by means of HPLC in samples from 100 children (age range: 1 month-18 years) without adrenal disorders was 288.8 nmol/L (+-147.2) giving a normal range of 30-520 nmol/L. There was no significant difference between the mean values for boys (n = 62) and girls (n = 38): 296.4 nmol/L (-+144.1) and 297.2 nmol/L (+-143.3), respectively. The normal plasma ll-deoxycortisol range in the 100 children was 6-55 nmol/L. The mean (+-SD) urinary excretion of cortisol evaluated by HPLC in samples from 35 apparently healthy children (age range 6 months-16 years) was 43.3 +- 37.9 nmol/24 h; range 15-180 nmol/24 h. Mean (+-SD) urinary excretion of cortisol in the 23 boys and 12 girls were respectively, 47.4 nmol/24 h (+-41.4) and 34.7 nmol/24 h (+-27.2). This difference was not statistically significant. In the group of children (age range 1-12 years) with adrenal hyperplasia (n = 12), urinary free cor-

CLINICAL BIOCHEMISTRY, VOLUME 26, JUNE 1993

I I.S A

mml

II

B

0

T

T _z

r.)

B

i

• -

i

0

I

5

,,,

I

i0

,

15

0

I

|

i

6.6

13.3

I

20 rain

20 min

III

"

IV

0 i 0

I

B

ca

A

C

IS,

1

er

t

J

I

0

5

C

P

I

I

0

5

.

i

i0

"O 4)

L----!

15

min

I

10

1.5 rain

i1|

Figure 1 -- Chromatograms obtained after chloroform extraction and H P L C analysis of."(I) A n aqueous standard of prednisone (IS2),cortisone, cortisol,11-deoxycortisoI,and corticosterone,each at a concentration of 2 ~mol/L. (II)A urine sample from a healthy child (aged 16 years); urinary free cortisol,180 nmol/L (ISi,2000 nmol/L). (HI) A urine sample from a child (aged 12 years) with adrenal hyperplasia; urinary free cortisol,580 nmol/L (IS,, 2000 nmol/L). (IV) A plasma sample from a child (aged 12 years) with adrenal hyperplasia; plasma cortisol,560 nmol/L; 11-deoxycortisol, 430 nmol/L (IS2, 2000 nmol/L); cortisone, 453 nmol/L. Peak A: cortisone; peak B: cortisol;peak C: 11-deoxycortisol; peak D: corticosterone; detector sensitivity: 0.01 A U full scale. CLINICAL BIOCHEMISTRY, VOLUME 26, JUNE 1993

155

SAMAAN, PORQUET, DEMELIER, AND BIOU

tisol and plasma cortisol, determined by HPLC, were always above 200 nmol/24 h and 520 nmol/L, respectively. Using urine specimens, we failed to obtain satisfactory results with RIA, because of nonspecificity of the antibody that cross-reacts with urinary steroid metabolites. In the group of children with adrenal hyperplasia (n = 12), all plasma cortisol levels determined by RIA were above the highest normal value, 500 nmol/L. Otherwise, from 100 plasmas of children without adrenal disorders and tested using RIA, five specimens presented pathological plasma cortisol levels, above 500 nmol/L (specificity: 95%). It was not possible to assess the presence of interfering substances with the RIA because of the difficulty in obtaining plasma from these children (n = 5) and their brief stay in the hospital.

TABLE 2.

Imprecision of Extracted Urinary and Plasma Cortisol Measured by HPLC Within Run (n = 15)

Plasma Urine

Between Run (n = 30)

Mean -+ SD (nmol/L)

CV (%)

Mean +-- SD (nmol/L)

CV (%)

123 - 4.5 248 -+ 13.5 825 -+ 11.4 232 -+ 6.9 472-+ 11.4

3.7 5.4 1.4 2.9 2.4

126.0 253.2 798.3 255 524

5.7 5.9 2.7 6.8 7.8

-+ 7.2 +- 15.0 +- 21.3 - 17.4 -+ 4.1

detector response (-+SD), the data being obtained by injecting the mobile phase twenty times. The detection limit was estimated as 3.5 nmol/L for cortisol, cortisone, ll-deoxycortisol, and corticosterone. This technique can thus be used for biochemical diagnosis of hypocortisolism, which is associated with plasma cortisol levels below 25 nmol/L.

ACCURACY

The accuracy of our method was tested by using urine and plasma samples spiked with cortisol and l l - d e o x y c o r t i s o l (500 nmol/L). The r e s u l t s are shown in Table 1. Mean analytical recoveries were between 96% and 110% and the CVs for cortisol and ll-deoxycortisol were 4.0% and 2.6%, respectively.

HPLC-RIA CORRELATION Using plasma samples, a good correlation was found (Figure 3) between HPLC (x) and RIA (y):y = 0.77x + 57.3, r = 0.965, n = 40, with a standard error of -+0.04 and -+14.3 for slope and intercepts, respectively. Using urinary samples, we failed to establish a good correlation between HPLC and RIA (results not shown). Indeed, RIA greatly overestimated UFC levels compared to HPLC in normal children because of the presence of interfering steroids and cortisol metabolites.

IMPRECISON

Imprecision was determined by processing aliquots of control plasma and urine samples containing high, normal, and low concentrations of cortisol (Table 2). The within-run and between-run CVs were less than 10% for both urine and plasma. LINEARITY

Plasma samples containing 100, 200, 400, 800, 1000, a n d 2 0 0 0 n m o l / L of c o r t i s o l a n d 11deoxycortisol were used to test linearity. Each level was tested five times, with a CV of less than 4.2%. R e s u l t s w e r e l i n e a r up to c o r t i s o l a n d l ldeoxycortisol concentrations of 2000 nmol/L (Figure 2).

Discussion We describe a method for assaying the two main glucocorticoids of clinical interest (cortisol and 11deoxycortisol) in urine and plasma by means of automated reverse-phase HPLC. This technique can also be used to determine other plasma glucocorticoids such as cortisone and corticosterone. The technique is suitable for use with pediatric specimens; if used for adult specimens it must be preceded by an

DETECTION LIMIT

The limit of detection for each analyte was calculated as the mean concentration obtained from the

TABLE 1

Analytical Recovery of Cortisol and ll-Deoxycortisol Cortisol

11-Deoxycortisol

Samples

Mean --- SD (nmol/L)

CV (%)

Recovery (%)

Mean -+ SD (nmol/L)

CV (%)

Recovery (%)

Plasma Urine

510 -+ 20.5 480 +-- 18.7

4.0 3.9

102 96

490 -+ 12.8 550 -+ 10.6

2.6 1.9

98 110

Expected value, 500 nmol/L, n = 15. 156

CLINICAL BIOCHEMISTRY, VOLUME 26, JUNE 1993

S E R U M A N D URINE CORTISOL A N D G L U C O C O R T I C O I D S B Y H P L C i0

A i2

8 l0 A

~8

2 2

I

0

I

0

I

,

1000

/

,

2000

0

,

3000

I

i

1000

nmol/L

I

2000

30o0

nmol/L

Figure 2 - - S t a n d a r d curves for (A) cortisol a n d (B) 11-deoxycortisol: absorbance u n i t as a function of concentration. Slopes and intercepts are, respectively: 4.09 (SD -+ 0.05) a n d - 0 . 2 0 4 × 1 0 - 3 (SD -+ 0.04 × 10-3) for cortisol; 5.14 (SD -+ 0.1), a n d - 0 . 4 0 7 × 10 - 3 (SD -+ 0.118 x 10 -3) for ll-deoxycortisol.

evaluation of normal ranges in a healthy adult population. Prednisone and corticosterone are used as internal standards for the plasma and urine assays, respectively, and show no interference with the peaks of glucocorticoids or other eluates [Figure l(II, III,IV)], especially dihydro and tetrahydro compounds. The imprecision and accuracy of the method are within the range commonly obtained by radioimmunoassay or other HPLC methods (10,11,13). Our radioimmunoassay is purchased from the manufacturer for use in routine determination of serum, plasma, and urinary cortisol. In practice, urinary 1200

/

1000

determination is weakly accurate and is not convenient requiring a manual extraction before analysis. Quality control testing showed the satisfactory accuracy and precision of our technique. The mean (-+SD) concentration of urinary free cortisol (43.2 -+ 37.9 nmol/24 h) in healthy subjects was in good agreement with previously reported values (4,9). Furthermore, the HPLC method clearly discriminated (specificity: 100%) between a population of healthy children (plasma cortisol below 520 nmol/L and urinary free cortisol below 200 nmol/24 h) and children with adrenal disorders. The good correlation found between HPLC and RIA is caused by the use of plasma samples containing moderate levels of interfering compounds, especially prednisone and prednisolone, with an error of less than 8.2% when plasma samples are tested with RIA.

•

Conclusion 800 .J

600 E 400

2OO

0

i o

i

i soo

i

i

l

Iooo

i

i

i

i

isoo

i 2000

The main advantage of this H P L C method is to avoid frequent overestimation of urinary free cortisol when RIA is used. In the manual mode, the method is time consuming and maintenance costs are more than for RIA. If, however, modern automated equipment is available, one technician can handle about 22 samples within one working day. Furthermore, the assay can be started at any time, because all the analytical steps are preprogrammed. The proposed method is currently used for routine glucocorticoid investigation in our laboratory, and could provide the basis for reference methods.

HPLC (nmol/L)

Figure 3 - - Correlation b e t w e e n p l a s m a cortisol as measured by HPLC (x) a n d RIA (y):y = 0.77x + 57.3, r = 0.965, n = 40 (slope error = -+0.04, intercept error = -+14.3). CLINICAL BIOCHEMISTRY, VOLUME 26, JUNE 1993

Acknowledgements W e thank Mrs. Corinne Burger and Miss Massagbe Cisse for their expert technical assistance. 157

SAMAAN, PORQUET, DEMELIER,AND BIOU

References 1. Murphy BEP. Clinical evaluation of urinary cortisol determination by competitive protein binding radioassay. J Clin Endocrinol Metab 1968; 28: 243-48. 2. Nakamura J, Yakata M. Clinical evaluation of the liquid-chromatographic determination of urinary free cortisol. Clin Chem 1983; 29: 847-51. 3. Bertrand PV, Rudd BT, Weller PH, Day AJ. Free cortisol and creatinine in urine of healthy children. Clin Chem 1987; 33: 2047-51. 4. Huang CM, Zweig M. Evaluation of a radioimmunoassay of urinary cortisol without extraction. Clin Chem 1989; 35: 125-6. 5. Schoneshofer M, Fenner A, Dulce HJ. Interferences in the radioimmunological determination of urinary free cortisol. Clin Chim Acta 1980; 101: 125-34. 6. Schoneshofer M, Fenner A, Altinok G, Dulce HJ. Specific and practicable assessment of urinary free cortisol by combination of automatic high-pressure liquid chromatography and radioimmunoassay. Clin Chim Acta 1980; 106: 63-73. 7. Lantto O. Radioimmunoassay and liquid chromategraphic analysis for free cortisol in urine compared with isotope dilution-mass spectrometry. Clin Chem 1982; 28: 1129-32. 8. Flore M, Mitchell J, Doan T, et al. The Abbott IMX

158

9. 10. 11. 12.

13. 14.

15.

automated Benchtop Immunoassay Analyser System. Clin Chem 1988; 34: 1726-32. Canalis E, Reardon GE, Caldarella AM. A more specific liquid-chromatographic method for free cortisol in urine. Clin Chem 1982; 28: 2418-20. Rose JO, Jusko WJ. Corticosteroid analysis in biological fluids by high performance liquid chromatography. J Chromatogr 1979; 162: 273-80. Nakamura J, Yakata M. Two-cycle liquid-chromatographic quantification of cortisol in urine. Clin Chem 1982; 28: 1497-500. Turnell DC, Cooper JDH, Green B, Hughes G, Wright DJ. Totally automated liquid-chromatographic assay for cortisol and associated glucocorticoids in serum, with use of ASTED sample preparation. Clin Chem 1988; 34: 1816-20. Schoneshofer M, Kage A, Weber B, Leng I, K6ttgen E. Determination of urinary free cortisol by "on-line" liquid chromatography. Clin Chem 1985; 31: 564-8. Zadik Z, De Lacerda L, De Camargo LAH. A comparative study of urinary 17-hydrocorticosteroids, urinary free cortisol, and the integrated concentration of plasma cortisol. J Clin Endocrinol Metab 1980; 51: 1099-101. Westerhuis LW. Measurement of cortisol with EMIT and Amerlite Immunoassays. Clin Chem 1988; 34: 2374 (Tech. Brief).

CLINICAL BIOCHEMISTRY,VOLUME 26, JUNE 1993