Chemistry: analytical and immunological testing – Phosphorus containing – Of inorganic phosphorus compound in body fluid
Patent
1992-02-19
1993-07-06
Housel, James C.
Chemistry: analytical and immunological testing
Phosphorus containing
Of inorganic phosphorus compound in body fluid
436103, 436161, 422 70, 514 6, G01N 33483, G01N 3352
Patent
active
052253490
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for analytical separation of inositol phosphates and/or derivatives thereof and a means for analytical detection of inositol phosphates and/or derivatives thereof.
The research and development during the last few years relating to biologically active substances has to a certain extent been focused on inositol phosphates. These substances play an active role on the cellular level. At the same time there is a great potential to develop inositol phosphates and/or derivatives thereof to pharmaceutical substances. In order to optimally evaluate the reactions and the metabolism of inositol phosphates in cellular processes and to follow their resorption, mechanism of action and degradation when they are added to humans and animals it is necessary to have access to analytical methods of determination which make possible a careful and reproducible measurement of the inositol phosphates and/or derivatives thereof, in most cases, very low proportions of these compounds.
So far the development has resulted in chromatographic separation methods which however have serious limitations when low proportions of the compounds are to be separated. A non-specific absorption to glass, metal objects and certain materials of chromatography columns results in a non-existent possibility to analytically separate the substances.
The detection of inositol phosphates or derivatives thereof has also constituted a big problem since such compounds cannot be detected with conventional detectors such as UV detectors or electrochemical detectors.
One detection method is based on the determination of radio-actively labelled inositol phosphates. For natural reasons this determination method is limited to experiments which can be carried out in test tubes and of course it is not possible to use such tests on humans.
According to the present invention it has quite unexpectedly been possible to solve the above problem. The invention relates to a method for analytical separation of inositol phosphates and/or derivatives thereof. Then a quaternary amine is mixed with an aqueous solution containing one or more inositol phosphates and/or derivatives thereof.
FIG. 1 illustrates the analytical separation of inositol trisphosphate, inositol tetraphosphate, inositol pentaphosphate and inositol hexaphosphate obtained in Example 1.
FIG. 2a shows the proportion of inositol trisphosphate detected in Example 2 when the starting solution contained 36 ng/ml of inositol trisphosphate.
FIG. 2b shows the proportion of inositol trisphosphate detected in Example 2 when the starting solution contained 180 ng/ml of inositol trisphosphate .
The solution obtained is fed into an analytical column containing a solid phase of a non-polar kind, whereby a separation of the different constituents of the fed solution is obtained since the constituents pass through the column at different velocities and are then detected in a suitable way.
The invention makes a separation of the constituents possible also when these are present at very low proportions since the method disclosed eliminates the previously known problems with a non-specific absorption.
From the admixture of a quaternary amine as mentioned above, positively charged ions are obtained which form non-polar ion pairs together with inositol phosphates and/or derivatives thereof.
The aqueous solution can consist of water containing samples of biological origin. Here blood-plasma, blood-serum, blood, urine, faeces, tissue extracts or the like can be mentioned. Sometimes the water containing samples should be pretreated or purified to minimize the interference of proteins, lipids and organic and inorganic substances.
This pretreatment can include a precipitation step for example by the addition of perchloric acid to decrease disturbances from proteins according to known techniques. In certain cases the ionic strength of the aqueous solution can restrict the analytical separation from working optimally. Here different techniques for decreasing the ionic strength of the sample can be
REFERENCES:
Prestwich, S. A., Measurement of Picomole amounts of any inositol phosphate isomer separable by H.P.L.C. by means of a biol. assay., Biochemical J. (3)274, 1991, pp. 663-672.
Taylor, G. S. H.P.L.C. Analysis of Radiolabeled Inositol Phospaates, Anal. Biochemistry, 188, 1990 (1) pp. 118-120.
Wregett, K. A., A Rapid Sepa. Method for inositol phosphates and their Isomers, Biochem. J., 245 (3), 1987, pp. 655-660.
Dialog Inf. Service, file 351: World Patent Index, Acc. No. 3235145 & JP, A, 58109849 (Ashai Chemical IND KK) 30 Jun. 1983, Abstract.
Chemical Abstracts, vol. 107, No. 23, 7 Dec. 1987, D. B. McKay et al.: "Detection of polyols and sugars by cuprammonium ion in the presence of strong Base", pp. 302, Acc. No. 214459h, & Anal. Biochem, 1987, 165(2), 392-398.
Irth Hubertus
Kornfeldt Ruth
Persson Lars
Cano Milton I.
Housel James C.
Perstorp AB
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