Measurement of calcium ions

Abstract: A method for determining the concentrations of total calcium and at least one monovalent ion in a sample includes the steps of mixing the sample with a diluent that has a pH within the range pH 6.5 to 7.0 and includes 2-amino-2-hydroxymethyl-1,3 propanediol phosphate and is free of the monovalent ion. An aliquot of the diluted sample is concurrently contacted with a calcium-specific ion selective electrode and an ion selective electrode specifically responsive to the monovalent ion, the response of the calcium-specific ion selective electrode is measured as an indication of the concentration of total calcium in the sample, and the response of the monovalent ion specific ion selective electrode is measured as an indication of the concentration of the monovalent ion in the sample.

Description: This invention relates to measuring total calcium in biological samples. In biological samples, calcium ions are present either as free ions or bound to sample components such as proteins. To determine the calcium content of the sample accurately, it is necessary to measure the contributions from both forms (i.e. the "total calcium"). Total calcium has been measured colorimetrically using an acidic indicator dye, e.g. cresolphthalein complexone, whose color is sensitive to calcium ion concentration and which releases bound calcium ions. Dye and buffer solution are added to the sample separately (2 point calibration method) or combined and added together (1 point calibration method). In the latter method, the dye-buffer reagent must be used within a short period of time following combination because the dye has only limited stability in the alkaline environment of the buffer solution. Colorimetric measurement of total calcium is not compatible with the measurement of monovalent ions, e.g. sodium, potassium or chloride, because these ions cannot be measured colorimetrically. Another way of measuring total calcium has been to use atomic absorption spectroscopy (AAS). AAS, however, is too expensive and time-consuming for routine sample analysis. Total calcium has also been measured using an ion sensitive electrode containing a calcium ionophore. In one such method (Anal. Chem. 1981, 53 , 1970-1974), the sample is acidified to release bound calcium. The calcium ionophore then binds the released ions, thereby changing the electrode's output voltage; the change is related to the calcium ion concentration. Because sodium ions cannot be measured accurately at pH values less than 6.0, this method of measuring calcium is incompatible with measurement of sodium ions. Moreover, conventional calcium ionophores are unstable in acidic conditions (pH < 6) because they hydrolyze and thereby lose their ability to bind calcium ions, a special chemically modified calcium ionophore that is stable under acidic conditions is used in such known methods. A method for the measurement of ionized calcium in a sample which makes use of a buffer containing 2-amino-2 hydroxymethyl-1,3-propanediol phosphate and having a pH of 7.39 is disclosed in Clin. Chem., vol. 31/2, 1985, pages 267-270. The invention provides a method for determining the concentrations of total calcium and least one monovalent ion in a sample which may contain protein-bound calcium, comprising the steps of mixing said sample with a diluent to provide a diluted sample, contacting an aliquot of said diluted sample with a calcium-specific ion selective electrode and an ion selective electrode specifically responsive to said monovalent ion, measuring the response of said calcium specific ion selective electrode as an indication of the concentration of total calcium in said sample, and measuring the response of said monovalent ion specific ion selective electrode as an indication of the concentration of said monovalent ion in said sample, characterized in that said diluent comprises 2-amino-2-hydroxymethyl-1,3-propanediol phosphate, has a pH of 6.5 to 7.0 and is free of said monovalent ion. Preferably, the monovalent ion is sodium, potassium or chloride, and in a particular embodiment, the diluent is free of ions of calcium, sodium, potassium and chloride. Preferably, the diluent further comprises a reducing agent and a surfactant, and in a particular embodiment, the reducing agent is an aldehyde and the surfactant is non-ionic. Preferably, the volume to volume ratio of the sample to the diluent is between 1:30 and 1:60, and the concentration of the 2-amino-2-hydroxymethyl-1,3-propanediol phosphate in the diluent is at least 0.1, preferably between 0.1 and 0.2, millimol per liter. In a preferred process, the calcium-specific ion selective electrode and the monovalent responsive ion selective electrode are contacted with a reference solution that has the same chemical composition as the diluent except that the reference solution also contains calcium ion and the monovalent ion sources. The invention provides a simple, effective, inexpensive method for concurrently measuring the concentration of total calcium, sodium, chloride, and potassium in a biological sample, e.g. serum or urine. Accurate total calcium measurements are obtained using only a single reagent (the diluent). Because the diluent is stable, it can be conveniently stored for long periods of time. In addition, because its pH is in the 6.5 - 7.0 range, it does not hydrolyze the calcium ionophore of the ion sensitive electrode. Thus, conventional commercially available electrodes, rather than special chemically modified electrodes, can be used. Furthermore, a single aliquot of the sample can be analyzed for both calcium and monovalent ions (e.g. sodium, potassium, and chloride) because the diluent does not interfere with the monovalent ion measurements. The sample diluent for measuring total calcium includes a transfer agent, 2-amino-2-hydroxymethyl-1, 3-propanediol (tris base), capable of rendering calcium that is bound to sample components freely available for detection by a calcium ionophore containing electrode. The term "transfer agent" reflects a belief that the transfer agent reversibly binds calcium ions in the pH range 6.5 to 7.0 and thus effectively "transfers" the calcium ions from the sample components to the calcium ionophore on the electrode. The transfer agent contains one or more heteroatoms (i.e. nitrogen, oxygen, or sulfur atoms) having unpaired electrons that confer calcium binding ability on the transfer agent. The transfer agent can be provided in neutral (i.e. uncharged) form or in the form of a salt in which the heteroatom is positively charged (e.g., tris phosphate). A single aliquot of a sample of, e.g. urine or serum can be concurrently analyzed for total calcium, sodium, potassium, and chloride ions in an analyzer of the type disclosed in EP-A-299659. When chloride ions are being measured it is desirable to include a reducing agent (preferably one or more aliphatic aldehyde) and a surfactant (preferably a nonionic surfactant) to the transfer solution to minimize interaction of protein with the chloride electrode. The use of a nonionic surfactant with tris phosphate, also improves calcium ion transfer. The transfer solution can also contain stabilizers and microbicides to extend its shelf life. The sample diluent (pH 6.8) has the following chemical composition: 2-amino-2-hydroxymethyl-1, 3-propanediol (tris base) 0.15M Phosphoric Acid 4.8 ml/l Crotonaldehyde 0.49 ml/l Acetaldehyde 0.90 ml/l Nonionic surfactant (TWEEN® 20) 1.0 g/l Stabilizer (Giv-Gard DXN, C₈H₁₄O₄) 2.0 g/l A reference solution used to calibrate the ion selective electrodes has the following chemical composition: 2-amino-2-hydroxymethyl-1, 3-propanediol (tris base) 0.15M Phosphoric Acid 4.8 ml/l Calcium Nitrate 0.0147 g/l Sodium Chloride 0.158 g/l Potassium Chloride 0.0107 g/l Sodium Sulfate 0.0913 g/l Crotonaldehyde 0.49 ml/l Acetaldehyde 0.90 ml/l Nonionic surfactant (TWEEN® 20) 1.0 g/l Stabilizer (Giv-Gard DXN, C₈H₁₄O₄) 2.0 g/l The chemical composition of the sample diluent is identical to that of the reference solution except that the reference solution also contains calcium nitrate, sodium chloride, potassium chloride, and sodium sulfate, as ion sources for calibration of calcium ion, sodium ion, potassium ion and chloride ion, respectively. The concentrations of calcium, sodium, potassium, and chloride ions in a urine or serum sample are determined as follows. Eleven microliters of the sample is combined with 440 microliters of transfer solution (volume-to-volume ratio of sample to mixture = 1:41). The mixture is then contacted with electrodes capable of selectively sensing sodium, potassium, chloride, and calcium ions, respectively. Readings from each electrode are collected in millivolts and then converted to concentration values using standard techniques. After the sample readings have been taken, the mixture is removed, and the electrodes are then contacted as above with the reference solution in order to provide sample reading reference values.

Claims: A method for determining the concentrations of total calcium and least one monovalent ion in a sample which may contain protein-bound calcium, comprising the steps of mixing said sample with a diluent to provide a diluted sample, contacting an aliquot of said diluted sample with a calcium-specific ion selective electrode and an ion selective electrode specifically responsive to said monovalent ion, measuring the response of said calcium specific ion selective electrode as an indication of the concentration of total calcium in said sample, and measuring the response of said monovalent ion specific ion selective electrode as an indication of the concentration of said monovalent ion in said sample, characterized in that said diluent comprises 2-amino-2-hydroxymethyl-1,3-propanediol phosphate, has a pH of 6.5 to 7.0 and is free of said monovalent ion. A method according to claim 1 wherein the volume to volume ratio of said sample to said diluent is between 1:30 and 1:60. A method according to claim 1 or 2 wherein the concentration of 2-amino-2-hydroxymethyl-1,3-propanediol phosphate in said diluent is at least 0.1 millimol per liter. A method according to claim 3 wherein the concentration is between 0.1 and 0.2 millimol per liter. A method according to any one of the preceding claims wherein said monovalent ion is selected from sodium, potassium and chloride. A method according to any one of the preceding claims wherein said diluent further comprises a reducing agent. A method according to any one of the preceding claims wherein said diluent further comprises a surfactant. A method according to any one of the preceding claims wherein said aliquot of diluted sample is contacted concurrently with said calcium-specific ion selective electrode and with chloride-specific, sodium-specific and potassium-specific ion selective electrodes. A method according to any one of the preceding claims further comprising the steps of contacting said ion selective electrodes with an aliquot of a reference solution, said reference solution comprising 2-amino-2-hydroxymethyl-1,3-propanediol phosphate and a known concentration of said monovalent ion, and measuring the response of said ion selective electrodes to said known concentration of said monovalent ion as a calibration of said electrode. A method according to claim 9 wherein each of said diluent and said reference solution further comprises an aldehyde reducing agent and a non-ionic surfactant.

Inventors: BERGKUIST CAROLYN, MANZONI ANGELO, MUSACCHIO JOHN, PREMOLI PIETRO, BERGKUIST, CAROLYN, MANZONI, ANGELO, MUSACCHIO, JOHN, PREMOLI, PIETRO
Applicants: INSTRUMENTATION LAB SPA, INSTRUMENTATION LABORATORY S.P.A.
Application year: 1988
Main classification: G01N33
IPC codes: G01N33