Concurrent analysis of a fluid sample for calcium and a monovalent ion

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.

BACKGROUND OF THE INVENTION 
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, the sample is treated 
with a dilute acidic solution (pH=4.0 to 5.5) 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&lt;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. 
SUMMARY OF THE INVENTION 
In general, the invention features a method for determining the 
concentrations of total calcium and at least one monovalent ion in a 
sample which may contain protein-bound calcium, the method including the 
steps of admixing the sample with a diluent, the diluent comprising 
2-amino-2-hydroxymethyl-1,3-propanediol phosphate and having a pH within 
the range pH 6.5 to pH 7.0 and being free of the monovalent ion. An 
aliquot of the diluted sample is 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. 
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. Preferrably, 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 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. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof, and from the 
claims.

DESCRIPTION OF TICULAR EMBODIMENT 
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 7and 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 copending application Ser. No. 074,942 
entitled LIQUID HANDLING (Case 316) filed concurrently herewith, the 
disclosure of which is specifically incorporated by reference. When 
chloride ions are being measured it is desirable to include a reducing 
agent (preferably one or more aliphatic aldehydes) 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) 0.15 M 
Phosphoric Acid 4.8 ml/l 
Crotonaldehyde 0.49 ml/l 
Acetaldehyde 0.90 ml/l 
Nonionic surfactant (Polysorbate 20 - TWEEN .TM. 20) 
1.0 g/l 
Stabilizer (Dimethoxane-Giv-Gard .TM. DXN, 
C.sub.8 H.sub.14 O.sub.4) 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) 0.15 M 
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 (Polysorbate 20-TWEEN .TM. 20) 
1.0 g/l 
Stabilizer (Dimethoxane DXN, 
C.sub.8 H.sub.14 O.sub.4) 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 admixture=1:41). 
The admixture 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 admixture is removed, and 
the electrodes are then contacted as above with the reference solution in 
order to provide sample reading reference values. 
Other embodiments are within the following claims.