JPS6314303B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to quaternary ammonium salts used in clinical control test compositions. In recent years, various devices have been developed to automatically analyze human serum. Such a device is capable of quickly and simultaneously analyzing and measuring various components contained in each of a series of serum samples continuously introduced into the device on a precise quantitative basis. Biochemical measurements are performed on the following components in a serum sample using, for example, such a device. Namely, albumin, alkaline phosphatase, bilirubin, calcium, chloride, cholesterol, carbon dioxide content, creatinine, glucose, lactate dehydrogenase, inorganic phosphorus, potassium,
sodium, total protein, transaminase,
The objects to be measured are nitrogen in urea, uric acid, and creatine phosphokinase. To operate the device for making these measurements, a reference standard serum containing each component with precisely predetermined values is used as the clinical subject test composition. Such reference standard serum must be of a type that does not deteriorate even if stored for a long period of time. To this end, it has been conventional practice to freeze-dry those reference standard sera. Also,
In order to recover the carbon dioxide lost from the serum during this freeze-drying process, the reference standard serum is reconstituted prior to its use, for example using another aqueous solution containing ammonium bicarbonate. This is also done through technology. This restoring solution is disclosed in U.S. Pat.
It is disclosed in the specification of No. The quaternary ammonium salts of this invention are used as reference substances to determine the amount of carbon dioxide present in the blood by thawing lyophilized serum with a known amount of carbonate or bicarbonate to reconstitute the serum. Although their primary purpose is to enable the apparatus to be operated for measurements, the quaternary ammonium salts of this invention are also useful in a variety of other chemical analyses. As is well known, the second targeting material performs two important functions in analytical chemistry testing. These substances are used as standard substances for performing instrumental measurements, and are also used to test the accuracy and precision of analytical results. These second standards typically contain a large number of substances, so that the majority of laboratory analyzes can be performed with a single solution.
Furthermore, reference substances for quality control of complex substances are essential for the use of chemical analyzers for complex analysis. In formulating these standards, the concentration of one or more anions present in the material must be adjusted to the desired level while ensuring that the essential cations do not interfere with other laboratory analyses. It is often difficult to determine the A very important example of this problem, as mentioned above, is determining the total carbon dioxide content (bicarbonate plus carbonate) in quality control or reference standard serum used in clinical chemistry laboratories. . The bicarbonates that have been used to determine the total carbon dioxide content of these various serums include sodium, potassium, lithium, rubidium, tris(hydroxymethyl)aminomethane (also simply called TRIS), and bicarbonates of ammonia. Sodium, potassium, and lithium bicarbonates are unsuitable for this purpose. The reason is that they are important analytical components and lithium is commonly used in flame photometric measurements for sodium and potassium.
This is because it is also used as an internal standard in determination. Rubidium bicarbonate (concentration 25mM) is also not suitable as a reference substance for total carbon dioxide measurements. This is because this compound increases the measured concentration of sodium by about 2 mM, probably due to matrix effects within the flame photometer. The reference material based on Tris bicarbonate disclosed in the above-mentioned US Pat.
However, it was found to have the major drawback of being unsuitable for the purpose of such measurement standards, as it suppresses protein content by about 15% and increases protein measurement results by about 10%. The influence of TRIS on urea measurement results was reported by Gips and Reitsema in "Clin Chem.Ac ta."
33.257. (1971), it is thought that this may be related to interference with the Bertello reaction by a number of nitrogen-containing compounds. Reference materials reconstituted with ammonium bicarbonate, which is the most widely used reference material for total carbon dioxide measurements, cannot be used in most urease-based methods for urea measurements due to ammonium ion interference. It is possible. Also, ammonium ions significantly poison certain electrodes used for potassium measurements. As mentioned above, currently available reference materials for measuring total carbon dioxide are unsuitable as reference materials for several other important analytes for chemical analysis. In addition to the analytes that are subject to the interference of total carbon dioxide reference materials, laboratories performing total carbon dioxide measurements must prepare two different types of lyophilized serum reference materials. One was quenched by the addition of water, and the other was quenched by the addition of a bicarbonate solution. This duplication of effort is particularly burdensome when a single multi-channel instrument is used to perform the above-described tests. The reason for this is that standardization and quality control operations have to be performed twice as many times as normal. The applicant has discovered that when the quaternary ammonium salt of the present invention is used as a reference material for measuring the total amount of carbon dioxide, there is no need for repeated operations and costs are reduced. This is because the quaternary ammonium salts according to the invention have no effect on the levels of analytes relevant to clinical chemistry, other than the total amount of carbon dioxide. Another advantage of this invention is that several different laboratories participating in a regional quality control program, where all laboratories participating in the program use the same lyophilized serum stock. ) can be directly compared even if a control test is performed in one of the laboratories using water-de-dried serum instead of bicarbonate-de-dried serum. It is. The present invention describes the following types of substances which can be used to determine the concentration of anions present in reference substances for analytical chemistry and which, when used for this purpose, do not cause any interference with other chemical analytes. and the use of salts selected from the compounds. The substance according to the invention used by the applicant for standardizing and monitoring the quality of clinical chemical analytes is
Contains salts of grade ammonium compounds. Such compounds contain at least one tetra-substituted nitrogen, and some of the substituents include, for example:
They may be combined to form heterocyclic compounds such as 1-alkylpyridine and 1,1-dialkylpiperidine, or polymerized through crosslinking of the substituents. The substituent must have minimal interference in clinical measurements, such as the groups described below, and the compound itself must be free of sodium, potassium, chloride, calcium, iron, and ammonium. It must be virtually free of such contaminants. The amounts of each of these ions that may be present in a commercially viable diluent are 2 mmol, 0.25 mmol, 3.0 mmol, 0.5 mg/100 ml, 3.0 μg/100 ml, and 3.0 μ, respectively.
g/100ml, and more preferable values are 1.0 mmol, 0.2 mmol, and 0.2 mmol, respectively.
2.0 mmol, 0.2 mg/100 ml, 2.0 μg/100 ml and 2.0 μg/100 ml. Each of these quantities does not cause interference in the measurement. Furthermore, the substituents must not be such that they render the compound insoluble so that particles of the compound preclude optical measurements of the reference substance. Very importantly, the substituents must not introduce significant buffering capacity into the compound. Examples of such substituents include saturated or unsaturated heterocyclic compounds such as furan or tetrahydrothiophene groups, cycloalkenyl groups such as phenol, cycloalkyl groups such as cyclohexyl and cyclopropyl, methyl, ethyl, propyl, Branched or normal alkenyls include butyl, pentyl, octyl, hexene, isobutyl, and neohexyl. These groups further listed include oxygen, thioethers, ketones, esters, halides,
It can be additionally substituted with or contain nitro or hydroxyl. However, since branched chains, normal alkyls and alkenyls are the least chemically inert substituents, it is preferable to use these, with methyl being the best. Each of the four substituents may be the same as or different from one or more of the remaining groups. The anion that can be used to form the quaternary ammonium salt is at least one anion selected from carbonate and bicarbonate. The preferred tetra-substituted ammonium carbonates or tetra-substituted ammonium bicarbonates of this invention are used to reduce total carbon dioxide levels in serum or other biological fluids to desired levels without conflicting with other chemical methods. It can be adjusted conveniently. Analytes for which no interference was found include: sodium, potassium, chloride,
glucose, nitrogen in blood urea, creatinine,
Calcium, phosphorus, total protein, alkaline phosphatase, total bilirubin, iron, total iron binding capacity, cholesterol, triglyceride, uric acid, alanine aminotransferase (transferase), lactate dehydrogenase, hydroxybutyrate dehydrogenase Enzymes, gamma glutamyl transferase, lactate dehydrogenase, acid phosphatase, alkaline phosphatase, thyroid hormone scavengers, total thyroid hormone, and amylase. Bicarbonate substances do not color and therefore do not affect colorimeter measurements. It does not cause other components in the control test material to precipitate and occlude the device. Additionally, the PH of the control test solution can be adjusted by changing the ratio of bicarbonate to carbonate in the tetra-substituted ammonium salt. An important feature of this invention is that the tetra-substituted ammonium ion has no buffering capacity and, as a result, a minimum amount of this ion is required to determine the carbon dioxide content in the reference material, regardless of the pH value. It's good to have it. On the other hand, Tris has a pH value of 8 and exhibits a buffering effect.
Thus, a Tris-bicarbonate solution contains more Tris than bicarbonate since some of the Tris is unsaturated. Additionally, buffering capacity interferes with titrimetric analytical methods for measuring carbon dioxide. EXAMPLES As an example of the application of this invention, the synthesis and use of tetramethylammonium bicarbonate (TMA- _HCO3 ) as a total carbon dioxide content reference material is described below (see also reaction scheme). Analytically pure tetramethylammonium bromide at the desired concentration (e.g. 0.15M) was converted to tetramethylammonium hydroxide by passing it through a chromatographic column containing a strongly basic ion exchange resin in the form of hydroxide. . (1st step) 0.15M (CH ₃ ) ₄ N ⁺ BR ^- (1st step) 0.15M (CH ₃ ) ₄ N ⁺ OH ^- (2nd step) 0.15M (CH ₃ ) ₄ N ⁺ HCO ₃ ^- water Tetramethylammonium oxide TMA−
Conversion to HCO ₃ was carried out by bubbling a stream of CO ₂ gas into the stirred solution until the PH value was 8. The PH value of the solution can be changed by changing the amount of CO ₂ added. The resulting TAM-HCO ₃ solution was adjusted to the desired total carbon dioxide content (e.g. 30 m
M). This TMA-HCO ₃ solution was then used for reconstitution of the control test substance in previously lyophilized serum. Alternatively, the bicarbonate or carbonate salt of TMA may be added to a control sample of lyophilized serum prior to reconstitution with distilled water. These control specimens reconstituted with TMA-HCO ₃ were analyzed by known chemical analysis methods. The control test serum reconstituted in this way was analyzed in parallel with the control test serum reconstituted only with distilled water, but none of the analytes using the control test material, including those mentioned above, were analyzed. However, no significant difference was observed between these two control test sera. EXAMPLE To demonstrate the effectiveness of the quaternary ammonium carbonate and quaternary ammonium bicarbonate of this invention in adding carbon dioxide to a serum reference material, freeze-drying of the same lot was performed. Two samples were taken from the serum. Each sample consisted of as much serum as could be obtained by lyophilizing 10 ml of serum. 10 ml of distilled water was added to the first sample and the bottle containing the sample was agitated to ensure complete dissolution of the serum. The contents of this sample were then analyzed using known equipment for serum analysis. A second sample of lyophilized serum was reconstituted by adding 10 ml of tetramethylammonium bicarbonate at a concentration of 22 mM. The mixture was then slightly stirred again to ensure that the serum was completely dissolved in the tetramethylammonium bicarbonate solution. A second sample was then analyzed using the same equipment used for the first sample. The following table shows the test results for the ingredients tested.
Typical ranges for the ingredients are also shown.

[Table] Reviewing the above test results, it appears that, with the exception of carbon dioxide, the addition of tetramethylammonium bicarbonate does not affect or interfere with the measurement of any of the other serum components. Not, also,
It can be seen that none of the other components of the serum serve to cause changes in the component concentrations in the serum. As far as carbon dioxide is concerned, the above analysis shows that the concentration of carbon dioxide was reduced to 3meq/in the serum during the freeze-drying process. This is a significant decrease compared to the normal numerical range of 24 to 32 meq/. However, by mixing the lyophilized serum with a tetramethylammonium bicarbonate solution, the concentration of carbon dioxide increased to 25 meq/2, which is within the normal numerical range. Similar results were obtained in subsequent tests. All quaternary ammonium salts useful in this invention are aqueous solutions. Furthermore, when used with serum, these quaternary ammonium salts are also readily soluble in serum. It is also easily soluble in chemical mixtures used in clinical trials. Of course, the quaternary ammonium salt should not interfere with any of the clinical analytes. Compositions containing quaternary ammonium salts must be sterile. This sterilization state can be achieved by a known method that does not affect the quaternary ammonium salt. This may be accomplished, for example, by filtering a solution of a quaternary ammonium salt through a filter that captures particles on the order of the size of microorganisms. Cations that can be used to form the quaternary ammonium salts of this invention include the following. TrimethylhydroxyethylammoniumdimethylchloromethylphenylammoniumtetraethylammoniumtetramethylammoniumtetrapropylammoniumtetrabutylammoniumNN-dimethyl-N-ethyl-p-nitrophenylammoniumNN-dimethyl-N-fluoromethyl-N-
Phenylammonium N-cyclohexyl-NN-dimethyl-N-
Isobutylammonium N-ethyl-N-methylpiperidinium NN-dimethyl-N-ethyl-N-vinyl-
Ammonium NN-diethyl-N-(2-methoxyethyl)
-N-n-butylammonium Nn-octyl-NNN-trimethylammonium NNNN′.N′.N′-hexamethylethylenediaminium N-(2-hydroxyethyl)trimethylammonium Usable anions are those specified above. and contains therein at least one anion selected from carbonate and bicarbonate. When obtaining reconstituted serum, a sufficient amount of quaternary ammonium carbonate or quaternary ammonium bicarbonate according to the present invention is added to the lyophilized serum, and about 10 to about 40 meq/carbon dioxide is added to the reconstituted serum. exist. Reference serum can be prepared by mixing a solution of a tetra-substituted ammonium salt with the desired final concentration with previously lyophilized serum.
Alternatively, a predetermined amount of a tetra-substituted ammonium salt may be added to serum before it is lyophilized. To prepare the reference serum of this invention by the latter method, a sufficient amount of the tetra-substituted ammonium salt is added to the pooled serum so that the desired total amount of carbon dioxide is present in the reconstituted serum. . For example, add 1.0 ml of 0.4 M tetraethylammonium bicarbonate to 10.0 ml of pooled serum. This mixture is then freeze-dried by a known freeze-drying process. This lyophilized serum was reconstituted by dissolving it in 10.0 ml of distilled water before its use.
Analyze by known methods. Although the invention has been described in detail, it will be readily apparent that current or future knowledge may be applied to adapt the invention to various conditions.

Claims (1)

[Scope of Claims] 1. At least one anion selected from carbonate and bicarbonate, wherein the amounts of sodium, potassium, calcium, ammonium, chloride and iron ions are 2 mmol and 0.25 mmol, respectively. , 0.5 mg/100 ml, 3.0 μg/100 ml, 3.0 mmol, and 3.0 μg/100 ml or less. 2. The quaternary ammonium salt used in the clinical control test composition according to claim 1, wherein the anion is the carbonate. 3. The quaternary ammonium salt used in the clinical control test composition according to claim 1, wherein the anion is the bicarbonate. 4. The quaternary ammonium salt used in the clinical control test composition according to claim 1, wherein the anion is a mixture of the carbonate and the bicarbonate. 5 The amounts of the sodium, potassium, calcium, ammonium, chloride, and iron ions are:
1.0 mmol, 0.2 mmol, 0.2 mg/100 respectively
ml, 2.0μg/100ml, 2.0mmol, and 2.0μ
g/100ml, claims 1, 2,
A quaternary ammonium salt for use in the clinical control test composition according to any one of Items 3 and 4. 6. A quaternary ammonium salt used in the clinical control test composition according to any one of claims 1, 2, 3, 4, or 5, which is in a sterile state. 7. The carbonate is a quaternary carbonate used in the clinical control test composition of claim 2, wherein the carbonate is at a concentration sufficient to provide a CO ₂ concentration of about 10 to about 40 meq/liter. ammonium salt. 8. The bicarbonate is at a concentration sufficient to provide a _CO2 concentration of about 10 to about 40 meq/liter. grade ammonium salt. 9. The mixture contains quaternary ammonium for use in the clinical control composition of claim 4 at a concentration sufficient to provide a _CO2 concentration of about 10 to about 40 meq/liter. salt. 10 Urea, uric acid, creatinine, glucose, cholesterol, bilirubin, total protein, lactate dehydrogenase, amylase, acid phosphatase, alkaline phosphatase, thyroid hormone traps, and total thyroid hormones that affect analysis values The clinical control test composition according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, or 9, which does not contain Quaternary ammonium salt used.