DD293432A5 - METHOD FOR DETERMINING ANDROSTENONE AND OTHER DELTA 16 STEROIDS IN BIOLOGICAL MATERIAL - Google Patents

Abstract

The invention relates to a method for the determination of androstenone and other D16 steroids in biological material, in particular bats of boars, to allow a possible utilization for human consumption. The object of the invention is the development of a fast, easy-to-use and safe analysis method. According to the invention, the determination of androstenone and other D16 steroids is carried out after saponification of fat samples by means of methanolic potassium hydroxide or enzymatic hydrolysis of blood plasma or saliva, by extraction of the steroids with n-hexane or methanol and subsequent purification of the extracts. After concentrating the extracts, a high-performance thin-layer chromatography on silica gel 60 is carried out. After development of the HPTLC precast plates and subsequent irradiation with UV light of the wavelength 254 nm, a semiquantitative determination is carried out. The detection limits of D16 steroids are between 5 and 150 ng / ml or g sample. {D16 Steroid Screening Test; boar taint; TLC; Androstenone detection; Schlachtkoerperuntersuchung; biological material; objective sensory quality check}

Description

Field of application of the invention

The invention relates to a method for the determination of androstenone and other Δ16 steroids in biological material, esp. Of carcasses of boars, to allow a possible utilization for human nutrition.

Characteristic of the known state of the art

Biological material from the boar contains a more or less strong species-specific sexual odor, which is described differently: sweaty, onion, rancid.

Patterson 1968, at Claus, R (1970) Diss TH Munich isolated from the fatty tissue of odoriferous boar carcasses a specific substance, which causes this gangrene significantly. After the structural elucidation of this chemical compound, the 16-steroid (5a-androst-16en-3on) was synthesized and offered as an indicator substance for the boar odor.

Boar carcasses undergo sensory quality control at the slaughterhouses. A trained jury of experts examines the ascending milling when heating boar fat, whether they have the typical boar odor. The result of this so-called soldering iron sample is subject to strong subjective influences. Especially after testing an odorous sample, the flexibility of the human nose is disturbed. The members of the jury can only examine a small number of samples per day.

In search of objective research methods, the boar taint was analyzed and analyzed by Claus, Vienna, Tierärztl. Mschr. 65, pp. 381-388 (1978) as a combination of 21 different chemical compounds. Investigations on the percentage of individual chemical compounds in boar taint showed that androstenone (5a-androst-16en-3one) had the highest concentrations (Bonneau, A review Livestock Prod., Amsterdam 9 [6], p. 687-705 [1982]). Correlation coefficients of the andiostenone concentration rat ions with the associated sensory test results make it possible to regard androstenone as an indicator substance for the mountain ash. Only under this condition, it was possible to make an objective assessment of the odor intensities of the sample material via the content of androstenone in the fat, by means of a biochemical analysis method. If the organoleptic perception of boar taint is assigned to the corresponding levels of androstenone, the recommendation of Claus 1978 results. Thereafter, the perception threshold for boar odor is 0.3 mg androstenone / g fat. The European Association for Animal Production (ETV) recommends 0.5 \ ig androstenone / g fat than permissible limit Voss and Holtz, breeders 25 (11), S.462-465 (1982).

By means of the androstenone determination, it is possible to objectively assess the boar's carcass on its sensory quality.

Brooksbank u. Haslewood, Biochem. J. 80,488 (1961) and Brooksbank, J. Endocrin, 24, 435 (1962) report a colorimetric assay for androstenone. The low sensitivity of 1 % of Androstenone stimulated the further development of more sensitive androstenone detection methods. Thin-layer chromatography has provided a viable way to isolate the Δ16 steroids from boar fat. A real analytical step was achieved by gas chromatography, coupled to the thin layer chromatography Höbe u. Co-worker, Arch, exper. Vet med., Leipzig 36 (5), pp. 739-750 (1982). Although this coupling allowed the study of biological material, but this combination is too expensive for routine analysis and requires highly qualified personnel. The radioimmunoassay (RIA), as a highly sensitive detection method, can not be used for routine quality control of sensory quality for economic reasons. Claus and co-workers, Arch. F. Lebensmittelhyg. 39 (4), p. 87 (1988), an enzyme immunological measurement method on microtiter plates (MTE) is possible. AB Mortensen (DD Ap 202762 Int Cl I. GO1N 33/12) proposes to obtain an indication of the sensory quality via the skatole content.

Object of the invention

The objective of the method is to determine biologically material from live animals or carcasses objectively for the content of androstenone and other A16 steroids semiquantitatively, thereby enabling a decision on the usability of boar meat to be made by comparison with the established limit values.

Explanation of the essence of the invention

Biological material from male animals ₍ especially from pigs) may have increased A16 steroid content

Δ16 steroids cause a sex-related sensory influence, whereby the steroid content correlates with the intensity of the influence. In extreme cases, the influence on the sensory quality can increase so much that the biological material is unfit for human consumption. A typical example of this is the carcass of the uncastrated boar.

It is therefore an object of the invention to develop a fast, easy-to-use and secure analysis method.

According to the invention the object is achieved in that fat samples are taken from the slaughtered animal, and after appropriate work-up and extraction chromatographically on HP ^T LC finished plates, the content of the Δ16 steroids

semiquantitatively separated and identified. Surprisingly, it was found that using UV light of wavelength = 254 nm, a sensitivity increase of up to 100 times is achieved compared to the previous methods. The process according to the invention consists of the following individual steps:

The comminuted fat of a mixed sample is saponified by heating with methanolic potassium hydroxide solution for several hours. After cooling the sample is extracted several times with η-hexane. After washing the n-hexane phases with 70% methanol and distilling from water and drying the hexane extract with a suitable desiccant such. For example, anhydrous sodium sulfate is concentrated under vacuum at temperatures up to 4O ⁰ C to dryness.

The residue is dissolved in chloroform and usually applied to the HPTLC finished boards and then developed with Laufmittfilgemisch.

for 5a-androst-16 en-3-or: benzene / isopropanol 98: 2

for 5a-androst-16en-3a-ol: benzene / methanol 98: 2

for 5a-androst-16 en-3β-ol: benzene / methanol 98: 2

The HPTLC precast sheets are air dried and wetted with the spray reagent (glacial acetic acid, sulfuric acid, p-methoxybenzaldehyde). After brief heating to 120 ° C, the finished plate is observed under a UV lamp (wavelength = 254 nm).

The Δ16 steroids fluoresce pink to red-brown, depending on the concentration. The intensity of the color is concentration-dependent; by comparison of running standard substances a rapid assignment and quantitative determination of the quantity is possible.

With there. Methods may also be used to evaluate the content of Δ16 steroids in living animals. Particularly suitable for these investigations are plasma and saliva.

The procedure has a sensitivity of:

<10 ng Δ16 steroids as pure substance

5 ng A16 steroids / ml plasma or saliva 150 ng A16 steroids / g fat.

The high sensitivity guarantees that biological material (carcasses of boars) can be assessed in terms of quality, taking into account an A16 steroid limit value. The method allows automation and can be coupled with other methods. The introduction of the method requires no major expenditure on equipment. It can be carried out in any laboratory equipped for general chemical work, ie directly at the slaughterhouse.

embodiments

Based on two embodiments, the invention will be explained in more detail.

example 1

The examination of a fat sample

Claus 1978 examined boar carcasses on maximum and minimum content of A16 steroids at anatomically different sites. For this purpose fat was taken from 4 boiled body halves in 9 different places. The determined different androstenone concentrations did not significantly secure a preferred site. Therefore, to study the A16 steroid content in fat, it is recommended to use a mixed sample from several carcass donor sites.

5 g of crushed fat of a mixed sample are refluxed for 2 hours in a 100 ml round-bottom flask with 10 ml of n-heptane and 15 ml of 10% methanolic potassium hydroxide solution (Kaufmann et al., 1975). The contents, cooled under running water, are mixed with 20 ml of water and 20 ml of methanol, placed in a separating funnel and shaken out in succession with 30 ml, 25 ml and 20 ml of η-hexane. The collected Hexrnphasen be washed 3x with 5ml of 70% methanol and 3x with 5ml of water. All aqueous and methanolic: η phases are discard. The hexane extract is dried with anhydrous sodium sulfate and the sodium sulfate is washed with approx. 10 ml of η-hexane. The entire hexane is concentrated at 40 ^° C. under a slight vacuum. To reduce Androstenoneverlusten concentration is constant time constant. Under our conditions, for about 80ml of hexane, 16 to 18 minutes were needed. Occurring moisture must be removed. The flask with the dry extract can be stored in a sealed container in the freezer. The residue in the round bottom flask is taken up with chloroform and the volume is quantitatively transferred to an Eppendorf cup with a microliter metering syringe. The chloroform volume should be in the range of 200 to 250 μΙ. From the Eppendorf cup, an aliquot is applied in the form of a column (2-3 mm in diameter) 15 mm from the bottom of the plate onto the nano-TLC plate. For grease samples, sic.i has proven an order volume of 10 to 30 μΙ. In proximity to the starting spots of the samples, selected pure substance concentrations of the Δ16 steroids are placed. Before each sample, several different volumes should be applied to a TLC plate. The addition of a certain amount of pure substance to a smaller sample volume applied makes it easier to estimate fluorescence intensities.

The presented method allows the simultaneous isolation and identification of the three Δ16 sterols (5a-androst-16en-3on, 5a-androst-16en-3aol, and 5a-androst-16en-3βol) on an HPTLC precast plate (10 x 10 cm). For the preference of one of the three steroids it is recommended to choose the appropriate eluent. For androstenone a mixture of benzene / iso-propanol 98: 2 and for the 3a and Jß androstenols benzene / methanol 98: 2.

The chromotographed plates are air-dried in a fume hood and wetted uniformly with the spray reagent (48.5 ml of glacial acetic acid, 1.0 ml of sulfuric acid and 0.5 ml of p-methoxybenzaldehyde, Kritchevsky and Trepper 1968). The DC plates should appear transparent in the area of the steroid licks. In a drying oven preheated to 120 ^° C., the sprayed TLC plates are heated for 2 to 3 minutes and observed to develop fluorescence intensities in UV light (254 nm). In UV light, the A16 steroids fluoresce pink to red-brown, depending on the concentration. The color intensity of steroid spots is concentration-dependent. The accompanying standard makes it easier to find the A16 steroids sought and allows the comparison of the individual intensities for a screening test. The detection limit is 150ng A16 steroids / g fat.

Example 2 The examination of a plasma or saliva sample Heparinized blood is centrifuged and the recovered plasma used for the study. Saliva is from

heterogeneous constituents freed and can be used for hydrolysis.

30-50 μM plasma and the same volume of physiological saline are adjusted to pH 5.2 with 1N acetic acid.

Subsequently, the addition of 30 ml of acetate buffer (, oH 5.2) and 0.2 ml of enzyme solution (ß-glucuronidase / arylsulfatase from Helix pomatia). The incubation is carried out for 2 hours at 55 ⁰ C in a sealed Erlenmeyer flask. The hydrolyzate is absorbed

Cooled water and poured over a prepared Florisilsäure. About 1 g of methanol and water washed Florisil is needed to fill the microcolumn. The filled microsulo comes with 100ml physiological

Washed saline and can then be used for the hydrolyzate. After passing through the column is

washed with 10ml 1 / 10N sodium hydroxide solution and 30ml saline and then sucked dry with the water jet pump.

25ml of methanol elute the A16 Stercide from the Florisil directly into a 50ml pointed bottom flask. The methanol extract is concentrated to dryness at 40 ° C under a slight vacuum. The arrears can immediately for the

Thin layer chromatography can be used, whereby the work-up and the use of the extract of plasma and Saliva does not differ from that of fat. The chloroform extracts of plasma and saliva contain less Impurities and can therefore be applied to the nano-TLC plate with a larger volume (recommended Volume 30 to 50 μl. The detection limit is 5 ng A16 steroids / ml plasma or saliva.

Claims (2)

1. A method for the determination of androstenone and other Δ16 steroids in biological material by means of high-performance thin-layer chromatography ', characterized in that after saponification of fat samples with methanolic potassium hydroxide solution and extraction of the steroids by means of η-hexane and subsequent purification of the extract with methanol and water or After enzymatic hydrolysis of blood plasma or saliva, after column chromatography on magnesium silicate and elimination with methanol at reduced pressure until dried, the remainder was dissolved in chloroform and chromatographed on silica gel 60-HPTLC precast plates and developed and subsequently irradiated with UV light. Light of wavelength λ = 254nm is evaluated. 2. The method according to claim!, Characterized in that are used as eluent mixtures of benzene with methanol or isopropanol in the ratio 98: 2.