METHOD FOR ANALYTICAL SEPARATION OF INOSITOL PHOSPHATES

Abstract: Method for analytical separation of inositol phosphates and/or derivatives thereof, whereby a quarternary amine is mixed with an aqueous solution containing one or more inositol phosphates and/or derivatives thereof. The aqueous solution obtained is fed to an analytical column containing a solid phase of non-polar kind and the different constituents are detected in a suitable way. The invention also comprises a means for analytical detection of inositol phosphates and/or derivatives thereof.

Description: METHOD FOR ANALYTICAL SEPARATION OF INOSITOL PHOSPHATES 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 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 reproducable measurement of the present, 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. SUBSTITUTE SHEET One detection method is based on determination of radio-actively labelled inositol phosphates. For natural reasons this determination method is limited to experi¬ ments which can be carried out in test tubes and of course it is not possible to use at 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 quartemary amine is mixed with an aqueous solution containing one or more inositol phosphates and/or derivatives thereof. 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. At the admixture of a quartemary amine as mentioned above, positively charged ions are obtained which form non-polar ion pairs together with inositol phosphates and/or derivatives thereof. SUBSTITUTESHEET 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 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 used. For instance, if the ionic strength is caused by perchlorate ions the amount of these can be decreased by addition of potassium hydroxide. According to another method the aqueous solution can be fed through a column for example of ion exchange type. If the eluation is carried out in a suitable way for example with an eluent which is highly volatile a subsequent evaporation can eliminate a too high ionic strength. Sometimes a preconcentration of the aqueous solution containing the inositol phosphates and/or derivatives thereof can be carried out to get a smaller volume containing a certain amount of the substance to pass the column. This process decreases a band broadening which will otherwise arise. Such a band broadening will decrease the sensitivity of the analytical separation system. SUBSTITUTESHEET The preconcentration technique can consist of bringing an ion pair formed between the quartemary amine and the inositol phosphates to fasten to a small, for example 10 mm x 2 mm silicon based column of a non-polar type before an eluation to the analytical column takes place for separation of the constituents present. The content of inositol phosphates and/or derivatives thereof in the original aqueous solution that can be analytically separated according to the present invention is between 0.01 ppb and 100 ppm, preferably between 0.1 ppb and 50 ppm and most preferably between 0.5 ppb and 20 ppm. When a preconcentration step is used the content of inositol phosphates and/or derivatives thereof in the solution fed into the analytical column is between 0.05 ppb and 500 ppm, preferably between 0.5 ppb and 250 ppm and most preferably between 2.5 ppb and 100 ppm. According to the present invention an improvement of the analytical separation can be obtained by using an eluent with a pH between 5 and 9, preferably between 6 and 8 for elution of aqueous solution through the column. For example the quartemary amine used according to the invention can consist of tetrabutyl ammonium ion, tetrapentyl ammonium ion or tetrahexyl ammonium ion. Moreover, possibly other positively charged ions such as benzalconium ion or the like can be used. Suitably, the proportion of the quartemary amine is 0.1 mM to 20 mM, preferably 0.5 mM to 10 mM. SUBSTITUTESHEET According to one embodiment of the invention another solution containing one or more low molecular anions is added to the aqueous solution used before the passage through the analytical column. The anions have a mole¬ cular weight less than 200 g/mol and can consist of sulphate, phosphate, pyrophosphate or the like. This addition improves the analytical separation ability of the solid phase whereby a more efficient separation of the constituents is brought about. According to the present invention the constituents are detected in a suitable way after the passage through the column. Suitably the detection can be brought about by refractive index measurement, by detection of the constituents by means of light scattering technique, by carrying out measurements in the ultra-violet area after an addition of complex forming metal ions such as copper ions or by fluorescence technique. The fluorescence technique meant here relates to the following principle. After the analytical separation of the constituents by passage through the analytical column, a reagent solution (so-called post-column reaction) , consisting of a metal ion complex, comprising in addition to a metal ion also a substance which has fluorescent properties when present unbonded to metal ions, is continuously added to the solution leaving the column. When the substance is bonded to metal ions the complex has no fluorescent properties. The metal ion complex is selected in such a way that the metal ions at the contact with inositol phosphates and/or derivatives thereof are transferred to a metal ion inositol phosphate complex, whereby the fluorescent substance is released. The stoichiometry and the SUBSTITUTE SHEET equilibrium of the reaction is selected in such a manner that a specific amount of inositol phosphate or a derivative thereof results in a corresponding amount of the substance with fluorescent properties. At a preferred method for analytical separation of inositol phosphates and/or derivatives thereof according to the invention, the detection is made by the fluorescent technique. The invention also relates to a means for analytical detection of inositol phosphates and/or derivatives thereof. The means consists of a metal ion complex which per se has no fluorescent properties but at the contact with a solution of inositol phosphates or derivatives thereof causes a bonding between the metal ions and the inositol phosphates and/or derivatives thereof. The substance which originally formed a complex with the metal ions is then released and it has then in free form, i.e. not as a metal ion complex fluorescent properties. Preferably, the detection is carried out in such a way that the means according to the invention is continuously added to the liquid stream which has passed the analyti¬ cal column which makes a separation of the constituents possible. This so-called post-column reaction must meet certain fundamental requirements in order to work at detection of inositol phosphates and/or derivatives thereof. The means must have such a reactivity that a complete reaction is attained within a very short period. All components of SUBSTITUTESHEET the means must be compatible with the components which are to be separated at the passage through the column. The metal ion complex which is present in the means must consist of components which are rather strongly bonded to each other to avoid the formation of a background fluorescence which decrease the sensitivity of the method. A general desire is that the complex constant of the Q complex should amount to at least 10 . The means should be selected in such a way that a considerably stronger metal ion inositol phosphate complex and/or a complex to a derivative of the inositol phosphate is formed as compared to the original metal ion complex in order to get a rapid and complete yield of the component which is bonded to the metal ions. The metal ions which bind strongly to inositol phosphate and/or derivatives thereof can be found in the group of metal ions which have a charge of three or more. Also some divalent metal ions bind so strongly to inositol phosphates and/or derivatives thereof that they are applicable according to the invention. Accordingly, Fe (III) , Cr (III) , Al (III) , Pb (II) and Ni (II) constitute examples on metal ions which have properties that meet the above requirements. The selection of so-called functional groups included in the so-called ligands that preferably constitute the second part of the metal ion complex is mainly directed to carboxy and hydroxy groups. However, also other metal ion bonding functional groups can be present. SUBSTITUTE SHEET pH is a critical parameter in a solution where the components present should be detected by fluorescence. To get optimal reaction conditions and a maximum measuring signal pH should be between 4 and 9, preferably 5 and 8. Thus, in respect of bondings to Fe (III), for instance mono-hydroxy compounds are usually not relevant since their p a values are often higher than 8. On the other hand many dihydroxy compounds and acetate containing compounds such as imino diacetate compounds are not useful since they form a complex which binds Fe (III) stronger than inositol phosphates and/or derivates thereof. According to the invention it has quite unexpectedly turned out that N-alkylimino acetate containing compounds meet the properties disclosed above. N-alkylimino compounds containing other carboxy functions than acetate are also useful according to the invention. Also sulfo- salicylic acid or chromotropic acid or derivatives thereof are applicable according to the invention. Among the N-alkylimino acetate containing substances, N-methylimino acetate containing compounds such as methylcalcein and methylcalcein blue are preferred. According to a preferred embodiment of the invention the means for analytical detection of inositol phosphates and/or derivatives thereof consists of Fe (III) and methylcalcein blue (MCB) . SUBSTITUTESHEET A temperature above 40 β C and a pH above 5 are preferred to get optimum reaction conditions. Often the metal ion complex is added as a solution containing water and organic solvents such as tetrahydro-furan, methanol, ethanol or acetonitrile. The concentration of the metal ion complex and the proportion between metal ion and the second component of the complex can vary. When the metal ion complex consists of Fe (III)/MCB the concentration of MCB can be between 0.5 μM and 10 mM, preferably between 1 μM and l mM and most preferably between 2 μM and 0.5 mM. The content of Fe (III) can vary from one tenth of the content of MCB to ten times the content of MCB calculated on molar basis. The means according to the invention is used for analyti¬ cal detection of inositol phosphates or derivatives thereof where the content of these substances amounts to 0.01 ppb to 100 ppm, preferably 0.1 ppb to 50 ppm and most preferably 0.5 ppb to 20 ppm. The invention is illustrated further in the embodiment examples below but these examples do not constitute a limitation of the invention. Example 1 relates to an analytical separation of inositol phosphates by means of an ion pair system while example 2 illustrates a detection of analytical amounts of inositol phosphates. SUBSTITUTESHEET Example 1 One ml of an aqueous solution containing 5 μg of a mixture of inositol phosphates was injected into a chromatographic system where the analytic column used was a Nucleosil RP, 8 column (200 x 4 mm) and the eluent which passed the column consisted of methanol/tetrahydro- furan/5mM trishydroxymethyl aminomethan solution adjusted to pH 7.0 with hydrochloric acid (Tris buffer) and 3 mM tetrapentyl bromide solution: 50/5/45 per cent by volume. A post-column reaction was carried out to bring about a detection system based on fluorescense technique. A solution of methanol/Tris buffer: 95/5 per cent by volume and 10 μM methyl calcein blue and 5 μM iron (III) nitrate was added continuously to the eluent coming out of the column. After the reaction the solution was allowed to pass a fluorescence detector for detection of the inositol phosphates which had been separated in the analytical column. The analytical separation of inositol trisphosphate (IP 3 ) , inositol tetraphosphate (IP 4 ) , inositol pentaphosphate (IP 5 ) and inositol hexaphosphate (IP 6 ) obtained is illustrated in Figure 1. Example 2 Two aqueous solutions contained 180 ng/ml and 36 ng/ l respectively of inositol trisphosphate (IP,). First 0.5 ml of the solution containing 180 ng/ml was injected SUBSTITUTE SHEET into an analytical system where the analytical column used was a Hypersil ODS column (200x4.6 mm) and where the eluent consisted of methanol/tetrahydrofuran/5mM tris¬ hydroxymethyl aminomethan solution adjusted to pH 6.8 with hydrochloric acid (Tris buffer) and 20 mM tetrabutyl ammonium bromide 30/3/67 per cent by volume. After the chromatographic column a solution was added which made a detection by means of fluorescence technique possible. This solution consisted of methanol/Tris buffer: 95/5 per cent by volume and 10 μm methylcalcein blue and 5 μm iron (III) nitrate. The post-column reaction was carried out at 60*C. The excitation wavelength used in the fluorescence detector was 318 nm and the emission wavelength was 440 nm. The above procedure was repeated with 0.5 ml of the aqueous solution containing 36 ng/ml. The detection of these small proportions of IP_ is illustrated in a chromatogram in Figure 2. SUBSTITUTE SHEET

Claims: CLAIMS 1. Method for analytical separation of inositol phosphates and/or derivatives thereof, c h a r a c¬ t e r i z e d in that a quartemary amine is mixed with an aqueous solution containing one or more inositol phosphates and/or derivatives thereof, that 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 consti¬ tuents pass through the column at a different volocity and that the constituents are detected in a suitable way. 2. Method according to claim 1, c h a r a c t e r i¬ z e d in that the aqueous solution containing one or more inositol phosphates and/or derivatives thereof is purified from possible impurities before the mixing with quartemary amine. 3. Method accordng to claim 1 or 2, c h a r a c t e¬ r i z e d in that the content of inositol phosphate and/or derivatives thereof in the original aqueous solution is 0.1 ppb-50ppm, preferably 0.5 ppb-20 ppm. 4. Method according to any one of claims 1-3, c h a r a c t e r i z e d in that the content of inositol phosphate and/or derivatives thereof in the aqueous solution fed to the analytical column is 0.5 ppb-250 ppm, preferably 2.5 ppb-100 ppm. 5. Method according to any one of claims 1-4, c h a r a c t e r i z e d in that the eluent used has a pH of 5-9, preferably 6-8. SUBSTITUTESHEΪT 6. Method according to any one of claims 1-5, c h a r a c t e r i z e d in that the quartemary amine consists of tetrabutyl ammonium ion, tetrapentyl ammonium ion or tetrahexyl ammonium ion. 7. Method according to any one of claims 1-6, c h a r a c t e r i z e d in that the separation in the column of the different constituents is improved by treatment of the solid phase with an aqueous solution containing one or more different anions with a molecular weight less than 200. 8. Method according to any one of claims 1-7, c h a r a c t e r i z e d in that the aqueous solution contains blood-plasma, serum, blood, urine, faeces or tissue extracts. 9. Method according to any one of claims 1-8, c h a r a c t e r i z e d in that a reagent which makes a detection by fluorescence technique possible is added to the solution when it has passed out of the column. 10. Means for analytical detection of inositol phosphates and/or derivatives thereof, c h a¬ r a c t e r i z e d in that it consists of a metal ion complex which per se has no fluorescent properties but when in contact with inositol phosphates and/or derivatives thereof the metal ions are bonded to these whereupon a fluorescent substance is released. SUBSTITUTESHEET 11. Means according to claim 10, c h a r a c t e r i¬ z e d in that the metal ions of the means consists of iron, chromium or aluminum. 12. Means according to claim 10 or 11, c h a r a c t- e r i z e d in that the other part of the metal ion complex consists of sulfosalicylic acid, chromotropic acid or N-methylimino acetate containing substances or derivatives thereof. 13. Means according to any one of claims 10-12, c h a r a c t e r i z e d in that it consists of a complex of Fe (III) and methyl calcein blue. 14. Means according to any one of claims 10-13, c h a r a c t e r i z e d in that it makes a detection of inositol phosphate and/or derivatives thereof possible down to a content of 0.1 ppb. SUBSTITUTESHEET

Inventors: IRTH HUBERTUS, KORNFELDT RUTH, PERSSON LARS, IRTH, HUBERTUS, KORNFELDT, RUTH, PERSSON, LARS
Applicants: PERSTORP AB
Application year: 1990
Main classification: G01N33
IPC codes: C07F9, G01N30, G01N33