Interference of Zimmerman reaction by organic solvents

158

Clinica Chimica Acta, 76 (1977) 158-160 0 El sevier/North-Holland Biomedical Press

BRIEF TECHNICAL

NOTE

CCA 8558

INTERFERENCE

BEN RADEMAKER

OF ZIMMERMAN

*, JAN POORTMAN

Department of Endocrinology, Catharijnesingel 101, Uirecht (Received

December

31st,

REACTION

BY ORGANIC

SOLVENTS

and JOS H.H. THIJSSEN

Uniucrsify Hospital, (The Netherlands)

State

University

of Utrecht,

1976)

Introduction Recently we described a semi-automated method for the determination of lldeoxo-17-oxo-steroids (ll-DOOS) in urine [l]. The usefulness of this method stimulated us to investigate the possibility of automating the determination of 17-oxo-steroids (17-0s). The only method described for this automation [ 21 has two major disadvantages: (1) diethyl ether used as extraction solvent is unacceptably dangerous; (2) the solvent exchanger is unsuitable for routine purposes, according to the experience of the manufacturer. To avoid the evaporation step, attempts were made to automate the determination of 17-0s by the use of solvent-solvent partition. Preliminary results show, unexpectedly, that organic solvents interfere with the Zimmerman reaction for 17-oxo-steroids. Materials Steroids : Dehydroepiandrosterone (3P-hydroxy-5-androsten-17-one) was purchased from Ikapharm (Ramat Gan, Israel). The organic solvents, and all other reagents, were purchased from British Drug House Ltd. (U.K.). An Auto-Analyzer II system from Technicon Instruments B.V. (Rotterdam, The Netherlands) was used. Methods Urine without pre-treatment was introduced into the analyzer. Conjugates were hydrolysed with HCl at 9O”C, steroids liberated were extracted with 1,2* To

whom

correspondence

should

be

addressed

159

s

air

KOH +air

reagent . .

g

‘.

dr L 0

I ,:

‘. 1.

,’ /’ A’ /’ 1,2-dichbro-ethane

Fig.

1. Manifold

LT‘-~NKOH --l-+7

flow

I

A

I

”

l%MOB m ethanol

diagram.

dichloroethane [3]. A first attempt was made to perform the Zimmerman reaction by solvent-solvent partition of the free steroids dissolved in the dichloroethane and alkaline dinitrobenzene phases. The manifold flow diagram is given in Fig. 1. No colour developed in the organic solvent phase or the alkaline reagent phase. Therefore the flow was changed to perform the reaction by direct mixing of the extraction phase and the dinitrobenzene, dissolved in an alkaline solution. This flow diagram is shown in Fig. 2. Results

and discussion

The attempts with the manifold as shown in Fig. 1 failed completely, although the hydrolysis step worked very well. It appeared that the extraction solvent i.e. 1,2dichloroethane, interfered with the Zimmerman reaction. Therefore we investigated a wide range of organic solvents.

mair . sample

+air

Calorimeter, 2.

Revised

air

v

sample

o-

3N KOH

hydroxide in ethanol

I-*

Fig.

O-

manifold

flow

diamxm.

160

There are two factors limiting the choice of a suitable solvent: first, the resistance of the pump tube towards the solvent and secondly the polarity of that solvent with respect to the efficiency of the extraction of 17-0s. The following solvents were tested: carbon tetrachloride, toluene, ethylacetate, propylether, xylene, benzene, dichloroethane, amylacetate and dichloromethane. In addition we tested several combinations of the Zimmerman reagent. a. 0.6% dinitrobenzene in ethanol, plus 5 M potassium hydroxide in water, b. 0.5% dinitrobenzene dissolved in 2% Hyamine-1622 solution, plus 5 M potassium hydroxide in water, c. both solutions of dinitrobenzene, but tetramethylammonium hydroxide (20% in water) instead of potassium hydroxide. Besides isooctane, isopentane and hexane, which are unsuitable due to their low polarity, we could not find a solvent with an acceptable extraction efficiency which did not interfere with the Zimmerman colour complex. The attempts to develop the coloured complex directly in an homogeneous system of extraction solvent and dinitrobenzene plus potassium hydroxide or benzyltrimethylammoniumhydroxide dissolved in ethanol (Fig. 2) were also unsuccessful. Again we tried the same organic solvents that are suitable for the extraction of 17-0s. Our results indicate that the solvents tested interfere with the formation of the coloured complex. Manual tests revealed that such solvents have no influence after the formation of the complex. In our opinion it is highly unlikely that automation of the 17-0s estimation for routine purposes will be successful with a solvent-solvent partition or a direct colour development in the extraction solvent. A more promising approach seems to be the estimation of 17-0s by enzymatic conversion to 17-hydroxy-compounds, with hydroxysteroid dehydrogenase (EC 1.1.1.64) and NADH as a cofactor. Research in this direction is under way in our laboratory. References 1

Rademakvr, 349-3

2

B.,

McNeil,

C..

Analvtlral 3

.Jon~cj;ln-Timmrr.

A..

Poortman.

.I. and

Thijssen.

.J.H.H.

(1976)

Clin.

Chim.

Acta

70.

56

Korymlwrsky,

CONTENTS

Berrett,

C.R.,

Chemistry, K.D.,

(continued

Rich,

IV.,

Technicon Stubbs,

from

Gaufin,

S.,

International

K.J).

and

West,

Warr.

M.A.

Congress, H.F.

(1953)

and pp.

Hutchinson, 547~

Lancct

550,

II.

(1971)

Mediad

Inr..

in Automation New

in

York

i, 1276-1281

cover)

Brief tcclzrlical note Interference of the Zimmerman-reaction K. Rademaker, CJ. Poortman and cember31,1976) . .

by organic solvents qJ.II.H. Thijssen (Utrecht,

.

.

.

The

. .

Netherlands)

.

(De-

158