High pressure liquid chromatograph ic separation of C18 AND C19 steroids

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140

TDROXD=

A high pressure liquid chromatograph, Model 6000 (Waters Associates, Boston, Mass.) equipped with a U6K injector, a differential UV detector (Model ALC/GPC 202) 10 ~1 cell volume, or a Model 440 high sensitivity absorbance detector, 8 ~1 cell volume, a differential refractometer R401, and a dual Omniscribe recorder (Houston Instruments) was used. The androgens and their metabolites were separated on a prepacked stainless steel 0.4 x 30 cm microoorasil (a freely porous silica; particle size % 10 pm) column (Waters Associates, Cat. No. 27447) using a solvent system chloroform:isooctane, 6:4. After mixing the solvents, they were passed through a Millipore Solvinert filter (UHWP 04700, 0.5 pm) and thoroughly degassed under reduced pressure prior to pumping through the column at a rate of 3 ml/min. The steroids to be separated were dissolved in the same solvent mixture. Estrogens were separated on a reverse phase Cl8 micro Bondapak (organosilane bonded to silica; particle size 'L 10 urn) 4 mm x 30 cm stainless steel prepacked column (Waters Associates, Cat. No. 27324), using the solvent system acetonitrile:water, 4:6. Ultraviolet absorbance was measured at 280 nm.

RESULTS AND DISCUSSION

A reproducible

baseline resolution of mixtures of T, A, DHT, DHEA,

A, 3a DIOL and 3~ DIOL was achieved within 30 minutes, as is evident from the HPLC tracings shown in Fig. 1. Figure 2 shows a HPLC profile of the estrogens El, E2 and E3. The relative retention times (RRT) and the elution volumes of the androgens and the estrogens are given under Tables 1 and 2, respectively. There was no adsorption of the injected steroids as seen from the quantitative

recoveries of radioactive

14C-T and 3H-~ (Table 3).

The detector responses, expressed as the height of the peak for different concentrations Fig. 3, A and B.

of the androgens separated are presented in

Employing the ALC/GPC 202 UV detector, T and A could

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141

TBEOLD-

CHC13: !sooctone (614)

~porosll Flow rate 3mllmin

UVx 64 1

I

I

IO

20

30

TIME IN MINUTES

Fig. 1.

High pressure liquid chromatographic tracings of T, A, DHEA and their metabolites separated on a microporasil column and solvent system chloroform:isooctane,6:4.

be measured in quantities as low as 350 ng and a linear relationship was obtained between 350 ng and 2.8 pg (Fig. 3A).

With the Model 440

absorbance detector concentrations of A as low as 5 ng could be measured. At the highest sensitivity (0.005 AUF), 5 ng gives a peak height response of 9 mm; there was a linear response between 5 and 100 ng (Fig. 4).

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142

I

'J?BEOIDI

i

I

1

I

5

IO

15

TIME IN MINUTES

Fig. 2.

Estrone, estradiol and estriol separation by HPLC on micro Cl8 Bondapak and solvent system acetonitrile:water, 4~6. Table 7

Relative Retention Times and Elution Volumes of CTQ Steroids Steroid Androstenedione 5~-Dihydrotestosterone Dehydroepiandrosterone Androsterone Testosterone 5x-Androstane 3@,178-diet 5a-Androstane 3a,17&diof

I j 1 1 / i \ i

RRT 1.00 1.17 1.28 1.60 2.55 3.00 3.24

I I

Elution volume (ml) 18-20 22-24 24.5-26.5 30-33 48-52 57-63 66-70

143 Table 2 Relative Retention Times and Elution Volumes of Estrogens Separated by HPLC Steroid

RRT

Elution volume (ml)

Estriol

0.32

4.0-6.0

Estradiol

1.00

13.0-16.0

Estrone

1.47

20.0-23.0

Table 3 Recovery of Radioactivity from HPLC Separation of a Mixture of 3H-~ and 14C-T

Exp. #

Isotope

Injected (cpm)

O-18 ml

15

1

1% 3H

1440 1820

2

3H 1%

1320 810

Recovered (cpm) 18-20 ml 20-18 ml (A)

1420 1340

10 7

48-52 ml (T)

18;O 840

Much higher amounts of steroids are required for a reliable response with the RI detector. Linearity of the RI response to varying amounts of T and A is shown in Fig. 3B. We have utilized this system routinely in our studies on the synthesis and metabolism of androgens by immature and mature testes (1).

In our superfusion studies of the testes with isotopically labeled T and A, carrier steroids (100 pg) were added to the superfused tissue

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144

T=EOIDI3

UV RESPONSE

RI RESPONSE

I

u

.

/

n

I

I3

n .

I i l

/

./

-

I/

0

T

HX

l

-

xHX

0 0

II

II

I

I

0 0.7 1.4

I

I

2.8

(pd STEROID

Fig. 3.

I NJECTED

Ultraviolet absorbance detector (ALC/GPC 202, 254 nm) and differential refractometer (R401) response to various amounts of steroids injected.

and superfusates;

the steroids were initially separated by TLC before

they were further purified by HPLC.

Relative retention times corres-

ponded to those of standards and no losses in the column were observed. This rapid and reproducible

HPLC system with simultaneous quantitation

has also been used in our laboratory for the study of metabolism androgens in human prostate

(2).

of

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TDEOIDI

145

UV RESPONSE

“0

IO 20 30 40 50 A INJECTED (ng)

Fig. 4.

Response of the highly sensitive absorbance detector (Model 440, 254 nm) to nanogram amounts of androstenedione injected. ACKNOWLEDGEMENTS

This work was supported by grants HD 07197 and CA 15648 of the National Institutes of Health. REFERENCES 1.

Satyaswaroop, P.G., and Gurpide, E.: In HORMONAL REGULATION OF SPERMATOGENESIS (French, F.S., Hansson, V., Ritzen, E.M., and Nayfeh, S.N., Eds) Plenum Press, New York (1975) p. 165.

2.

Malathi, K., and Gurpide, E.:

J. STEROID BIOCHEM. 8: 141, (1977).