RU2380704C2 - Method for simultaneous detection of clinically significant steroids in human biological fluid - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns method for simultaneous detection of clinically important steroids in human biological fluid used for screening of gynaecologic and oncologic diseases caused by disturbed synthesis and metabolism of steroid hormones. The method involves extraction of steroid conjugates, solid content recovery and cleavage with simultaneous protection of functional groups. Steroid conjugates are fluid-fluid extracted in ethyl acetate medium. Steroid conjugates are cleaved by combined acid hydrolysis at temperature 80°C through reaction of benzene, steroid conjugates and hydrochloric acid diluted to 1:2, with a metal ball added.

EFFECT: higher effectiveness of the method.

3 cl, 1 ex, 1 tbl, 2 dwg

Description

The present invention relates to the field of biochemistry and medicine, and in particular to a method for the simultaneous determination of clinically important steroids in human biological fluid, used to detect a number of gynecological and oncological diseases associated with impaired synthesis and metabolism of steroid hormones, many of which can only be diagnosed by steroid profile (SP). In addition, the joint venture provides information on a number of steroids at the same time, which is especially important for diagnostic purposes.

Studies of hormones in the blood by immune methods can only be used for rough estimates, since the results depend on the momentary state of the human body and, as a result, range from 200 to 500%. Urinalysis is a more accurate, and, most importantly, non-invasive diagnostic method that prevents the patient from becoming infected with AIDS, hepatitis B, etc.

Daily urine, as an object of study, has significant advantages over the immune methods for determining blood hormones that are currently common in clinical medicine, since it eliminates the effect on the results of circadian rhythms and instantaneous fluctuations in hormones in the blood, which are often significant.

A known method for determining the SP in daily urine, which is described in the article Leunissen W.J.J., Thijssen J.H.H., J. Chromat, Biomed Appl., 1978, v.146, p.365.

The disadvantage of this method is enzymatic hydrolysis, using expensive enzymes, the activity of which varies with time; the presence of an additional stage of sample purification before entering the chromatograph using a Lipidex-5000 column, followed by elution of the derivatives with a mixture of derivatives, which greatly complicates the process and makes it extremely cumbersome. To implement the method, mixtures of low-boiling solvents are used, which increases the risk of fire.

The closest method for determining the steroid profile of urine (SPM) (prototype) is described in the review "Profiling steroid hormones and urinary steroids" of the journal "Journal of chromatography biomedical applications", vol. 379, June 20, 1986, C.H.L. Shakelton, pp. 91-156.

In this method, for the extraction of conjugates of steroids, various variations of solid-phase extraction are used, for example, using C24 cartridges, the cost of which is relatively high, and therefore they are unacceptable for research in Russia, in addition, highly toxic methanol (which requires special permission).

On the other hand, the prototype for the hydrolysis of steroid conjugates uses a very expensive enzyme obtained by extraction from a grape snail, the activity of which varies over time, which affects the quality of hydrolysis and requires additional testing of this enzyme. In addition, using this enzyme it is not possible to achieve quantitative hydrolysis, for which another additional stage is introduced - solvolysis, which increases the complexity of the procedure and takes a lot of additional time.

The disadvantage of this method is the two-stage protection of functional groups: silylation and the preparation of methoxy derivatives, which further complicates an already complex and multi-stage process.

The task of the invention is the determination of SP by daily human urine in a simpler and cheaper way.

The solution to this problem lies in the fact that in the method for the simultaneous determination of clinically important steroids in human biological fluid, including extraction of steroid conjugates, isolation of the dry residue of steroid conjugates and their subsequent splitting with simultaneous protection of functional groups, liquid-liquid extraction of steroid conjugates is used "In the environment of ethyl acetate, the decomposition of steroid conjugates is carried out by combined acid hydrolysis at a temperature of 80 ° C by the interaction of benz ol, conjugates of steroids and hydrochloric acid, diluted to a ratio of 1: 2, in the presence of a metal ball, using a metal ball of steel ШХ15 with a diameter of 3.175 mm.

The protection of functional OH groups is carried out in one step by silylation.

Compared to other polar solvents, ethyl acetate has the following advantages: it is a good solvent for both sulfates and steroid glucuronides; has a relatively low boiling point under normal conditions (77.1 ° C); It has low toxicity and low cost.

The task of combined acid hydrolysis (CCG) of conjugates is to transfer free steroids formed in the process of hydrolysis in an aqueous hydrophobic phase to the organic hydrophobic phase as quickly as possible and thereby protect steroids from the destructive effect of a strong ionizing proton medium.

A comparative analysis of the corresponding characteristics of a number of solvents tested in this work allows us to dwell on such an inexpensive and affordable solvent as benzene, which has a boiling point of 80 °, a high ability to dissolve the necessary steroids, less toxic compared to, for example, halogenated hydrocarbons, and most importantly, from Due to the absence of benzene ions, it does not destroy steroids at all. That is why immediately after hydrolysis of the hydrophilic conjugate molecule, the formed hydrophobic steroid molecule quickly passes into the solvent volume and, thereby, is reliably protected in the future from the destructive influence of the acidic medium.

The main criteria by which acid is selected for conducting SCC are the following: firstly, the ability to not dissolve in an organic solvent, secondly - form poorly separated suspensions and emulsions with a solvent, and thirdly, the chemical inertness of the reagent with respect to the solvent , as well as the availability in Russia and the low cost of the reagent. With all this in mind, we chose hydrochloric acid. A comparative experimental verification of a number of reagents showed that in the case of using hydrochloric acid after blowing the organic solvent out with a nitrogen stream, even traces of hydrogen chloride were not found in the precipitate. The study of the kinetics and mechanism of the hydrolysis reaction using hydrochloric acid is carried out using the example of PdGl cleavage.

After the extraction and removal of the solvent, the steroids are at the bottom of the conical flask in the form of sulfates and glucuronides, which is convenient in the subsequent stages of the analysis.

As a rule, in the process of hydrolysis, a system is formed consisting of two phases: the lower one is dilute hydrochloric acid, the upper one is benzene, and at the reaction temperature of 80 ° C the lower phase is stationary, while the upper one is boiling intensively. The existence of a stationary lower phase is a negative factor, since hydrolyzed steroids are in an aggressive environment for a long time and therefore are subject to relatively greater destruction.

To eliminate this drawback, combined acid hydrolysis is carried out in the presence of a metal ball made of steel ШХ15

(03.175 mm). The metal interacts with dilute hydrochloric acid (at a temperature of 80 ° C), as a result of which tiny hydrogen bubbles form at the same time as boiling centers in the system.

In this case, the whole mixture boils intensively, mixing well, which in turn leads to an increase in the number of transfer of steroids during hydrolysis. Thus, the resulting free steroids are much faster transferred to the hydrocarbon phase, where they are reliably protected from the damaging effects of hydrochloric acid.

A series of control experiments showed that the presence of metal does not affect the quality of the hydrolysis.

Example

Clinically important steroids are determined in the biological fluid of a person (pregnant woman).

For this purpose, 4 ml taken from the daily urine of a pregnant woman is placed in a conical flask, 10 ml ethyl acetate is added and liquid-liquid steroid conjugates are extracted with constant shaking for 5-10 minutes, after which ethyl acetate is evaporated and at the bottom of the conical flask remain conjugates of steroids in the form of dry plaque (figure 1).

Then, to break down the conjugates of steroids, acid hydrolysis combined with extraction is carried out, for which 10 ml of benzene and 2 ml of hydrochloric acid diluted in a ratio of 1: 2 are added.

In addition, a metal ball is placed at the bottom (steel grade ШХ15, diameter 3.175 mm) to ensure uniform boiling.

The flask equipped with a reflux condenser is placed in a water bath with a temperature of 80-83 ° C. Hydrolysis lasts 20 minutes, starting from the moment the boil begins. Then the mixture is rapidly cooled, poured into a separatory funnel and the lower layer is discarded.

The remaining extract is washed successively with 2 ml of 0.5 N alkali solution, 4 ml of distilled water and centrifuged. The residue after centrifugation (supernatant) is transferred to a conical flask and evaporated to dryness.

After acid hydrolysis, free steroids obtained as a result of such a procedure cannot be immediately introduced into the chromatograph, since the extract contains a lot of impurities. In the present invention, the method of washing the hydrolyzate with an alkali solution to remove acidic and other residues is selected as the main cleaning method. After alkaline treatment, the extract is washed with distilled water, while the alkali residue is removed. In order to achieve the lowest loss of steroids, the hydrolyzate purification stage is also optimized.

For the analysis of clinically important steroids, a gas chromatographic analysis is performed.

The obtained quantitative characteristics of clinically important steroids and the concentration profile of clinically important steroids in daily urine are presented in the table and figure 2, respectively.

In gas chromatographic analysis, two types of columns are used - traditional packed and highly efficient capillary columns. Since the advantages of capillary columns are high selectivity and durability, they are recommended to be used in the analysis of SP. The optimal conditions for chromatography are as follows: the temperature of a thermostat is 150 ° -300 ° C, an evaporator is 250 ° -300 ° C, and a detector is 250 ° -300 ° C.

Table. Quantification of steroids in the daily urine of a pregnant woman Norms (μmol / 24 hours) Steroid Names and Abbreviations The results are defin. Abs. value % 3.8-15.1 Androsterone (An) 15.4 43.7 3.0-14.8 Ethiocholanolone (Et) 12,2 34.8 0-2.8 Dehydroepiandrosterone (DHEA) 2,4 7.0 0.3-2.7 11-Ketoandrosterone (11-Keto-An) 1,0 2.7 0.4 -2.5 11-Ketoethiocholanolone (11-Keto-Et) 1.3 3.6 2.0-7.5 11c-Hydroxyandrosterone (11-OH-An) 2.2 6.4 0.5-3.1 11c-Hydroxyethiocholanolone (11-OH-Et) 0.6 1.8 9.9-35.5 Amount 17-KS 35,2 100.0 <3.0 The discriminant of van de Calceide 2,8 32.2 - 220 Pregnandiol (Pd) 101.1 2.2-8.0 allo-pregnanediol (allo-pd) 17.4 1.3-21.7 Pregnanolone 32.1 delta 5-pregnanediol (delta 5-Pd) 0.9 Pregnantriol (Pt) 0.5 16alpha-Hydroxy-Et 0,0 4.3-39.5 16alpha-hydroxy-an 0,0 16alpha-Hydroxy-DHEA 0,0 Only for pregnant Estradiol 37.0 Only for pregnant Estron 13.6 5.6-9.8 Cholesterol (Ch) 8.5 0.5-1.8 Daily urine (liters) 2.05

Claims (3)

1. A method for the simultaneous determination of clinically important steroids in human biological fluid, including extraction of steroid conjugates, isolation of the dry residue of steroid conjugates and their subsequent cleavage with simultaneous protection of functional groups, characterized in that liquid-liquid type steroid conjugates are used in extraction ethyl acetate, and the splitting of steroid conjugates is carried out by combined acid hydrolysis at a temperature of 80 ° C by the interaction of benzene, steroid conjugates and hydrochloric acid, diluted to a ratio of 1: 2, in the presence of a metal ball. 2. The method according to claim 1, characterized in that the protection of functional OH groups is carried out in one stage by silylation. 3. The method according to claim 1, characterized in that they use a metal ball made of steel ШХ15 with a diameter of 3.175 mm.

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