A METHOD FOR PREPARATION OF A UNITARY DRY REAGENT SYSTEM CONTAINING HETEROGENEOUS CHEMICALS

Abstract: Method for the preparation of a stable, unitary reagent system, in a solid form, containing heterogeneous reagents. The reagent system prepared according to this invention effectively isolates heterogeneous reagents from one another by selectively encapsulating one or more such reagents prior to their formation in a unitary granule or tablet form. The solid reagent systems prepared in the above fashion are suitable for the performance of various clinical chemistry assays, including creatinine analysis via a Jaffe reaction.

Description: A METHOD FOR PREPARATION OF A UNITARY DRY REAGENT SYSTEM CONTAINING HETEROGENEOUS CHEMICALS BACKGROUND OF THEINVENTION Field of the Invention; This invention is directed to a method, a composition of matter and a process. More specifically, the invention described hereinafter is directed to a method for the isolation of heterogeneous reagents by encapsulation of one or more of such reagents; a unitary solid reagent system prepared by the foregoing method; and a process for conducting an assay of biological fluid sample utilizing the foregoing reagent system. Description of Prior Art; In the field of clinical chemistry, the assay of a patient's body fluids has traditionally involved the addition of certain reagents, in specific portions, to an aliquot of patient's sample and thereafter monitoring the patient's sample for the presence of a reaction product whose concentration can be correlated to the analyte of interest. Because of analytical instrument design, these reagents have generally taken the form of liquids. Ordinarily, such reagents are sold in liquid form, or, if available in solid form, are reconstituted with fluid prior to their use. In the event that the reagent system employs two or more heterogeneous reagents, most often those reagents must be effectively isolated from one another to prevent their premature- interaction. Such interactions generally take the form of reduced shelf-life, and in extreme cases, in the rendering of one of the reagents ineffective for performance of a particular assay. One such reagent system which is susceptible to this type of interaction is used in the determination of creatinine levels via a Jaffe reaction. As is well know, the Jaffe reaction requires the use of an alkali hydroxide and an alkali salt of picric acid in the performance of a creatinine assay. These two reagents react with one another in solution and, thus, any solution formed from these materials is of. limited stability. Generally, these reagents are isolated from one another until immediately prior to use and, even then, they are generally added to the sample sequentially. In typical commercially available automated systems which employ reagent systems for performance of creatinine assay via a 3affe reaction, the reagents, notably the alkali hydroxide and picric acid or picrate salt, are initially isolated from one another by storage in separate containers. If there were a failure to take such preventative measures, it would result in the type of instability alluded to previously. In the Abbott ABA 100 clinical analyzer, and the DuPont ACA Clinical Analyzer, the alkali hydroxide and picric acid solutions are prepared as two separate liquids and these liquids stored in separate dispensing stations, thus requiring coordination of handling and precision on-site mixing. Once these two solutions are combined, their shelf life is limited, generally less than 14 days. In the Technicon SMAC Clinical Analyzer, three (3) separate liquids are used for performance of a creatinine assay via Jaffe reaction. This introduces a further variable into the system. All of the foregoing clinical systems described hereinabove require careful monitoring in order to insure that the reagents are still active; and, that precise calibration of the dispensing mechanism for delivery of required amounts is achieved. The introduction of solid reagent systems for clinical assay has at¬ tempted to address the problems and inconveniences with liquid based reagent systems described hereinabove. Typically, where such reagent systems were composed of heterogeneous reagents, separate tablets, each one containing one of the heterogeneous reagents would be required to ensure stability of the reagent systems. These solid reagents were generally more stable than comparable liquid systems; however, they have up to now been incompatible with assays requiring the presence of heterogeneous reagents in a unitary solid. Thus, current analytical systems requiring the use of heterogeneous reagents in a solid form also suffer rom shortcomings of their similar liquid counterparts. Where solid reagent systems are used, they require the solid be reconstituted as a liquid prior to performance of the assay. The need to reconstitute the solids as liquids which must then be mixed in certain proportions introduces the potential for variability into the assay as mentioned above. One possible alternative for elimination of human error and the variability which flows from such manipulation would be to have all reagents premeasured in a single granular or tabletted form. The reagent could then be simply introduced in the reaction vessel, either prior to or subsequent to diluent, followed by dissolution and the addition of patient sample. Under certain conditions, the sample itself may serve as the diluent. This system would eliminate or reduce the potential for variability in measurement and/or dispensing of reagents and, thus, increase the reliability and repeatability. An automatic clinical analyzer of advanςed design which is adapted to make use of a unitary dry reagent system is the PARAMAX Automated Clinical Analyzer developed by American Dade Division of American Hospital Supply Corporation. The PARAMAX instrument system (to be commercially available around January 1, 1984) uses a tabletted reagent for the perfor¬ mance of clinical assays on patient's samples. The specifications of tabletted reagents used in this system are that they must be stable up to six (6) months; capable of dispensation from a container of a type described in U.S. Patent application 284,980 (30-DA); and, be capable of rapid dissolution to form an optically clear solution. In all but one of the 32 reagent systems described for the PARAMAX instrument to date, virtually all reagents for the performance of a specific assay are incorporated within a single tablet. The simplicity and advantages of such a system are immediately apparent. Because of the specifications for such a tabletted reagent for the PARAMAX analyzer, the use of heterogeneous constituents presented a rather unique set of problems. For example, how could heterogeneous chemicals be incor¬ porated within a unitary solid, compacted in tabletted form and still be capable of rapid dissolution to form a optically clear reagent solution? Because of the uniqueness of the PARAMAX system, these problems have not, up to now, been addressed. Where these problems had been confronted in the past, the traditional approach was to modify the instrument to accommodate the limitations of the reagents. Because this traditional approach was unacceptable to designers of the PARAMAX instrument, new techniques had to be developed to accommodate such heterogeneous chemi¬ cals in a unitary solid. OBJECTS OF THE INVENTION * Accordingly, the object of this invention is to remedy the above as well as related deficiencies in the prior art. More specifically, it is the principal object of this invention to provide a method for the preparation of a unitary, solid reagent system containing heterogeneous ingredients or reactants for use in a Jaffe reaction system. It is another object of this invention to provide a unitary, solid reagent system containing heterogeneous ingredients in which each ingredient is in precise relationship to other ingredients also contained within the solid. It is yet a further object of this invention to provide a dry tabletted reagent system in which a pair of heterogeneous reagents are separated from one another by encapsulation of one or both such reagents. It is still yet another object of this invention to provide a simplified technique for conducting a clinical chemistry assay utilizing a unitary dry tabletted reagent system. SUMMARY OF THE INVENTION The above and related objects are achieved by providing a method for preparation of a unitary dry solid from a heterogeneous reagent system. The method of this invention can be exemplified with the reagents which are used in the conduct of the creatinine assay via a Jaffe reaction. In the preparation of a solid reagent system for a creatinine assay, via a Jaffe reaction, an alkali salt of picric acid is isolated in a solid form. Similarly, lithium hydroxide is isolated in the solid form. For the purpose of this disclosure, the phrase "solid reagent system" is inclusive of not only the reactive components of the solid but also tabletting aids (i.e., hulking agents, lubricants and the like). Either one of the foregoing reagents is thereafter coated with an encapsulation effective amount of an agent which is otherwise compatible with the physical specifications of the solid (i.e., tablet) and the chemical constituent of the sample to be analyzed. Typically, this agent can ( OMP [A WIP be another reagent of the reagent system which is inert relative to the encapsulated component; or a bulking agent used in the formation of the solid; or a combination of reagent and bulking agent. The purpose and extent of such encapsulation is to effectively isolate the encapsulated material and thereby prevent its premature interaction with another of the reagents used in the conduct of the particular assay. Subsequent to the encapsulation procedure, various reagents, including the encapsulated material, are com¬ bined in the relative proportions into a unitary solid form. The solid can take the form of free flowing material (i.e., powder or granule) or be compressed in a tablet of precise dimension and weight. DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS This invention describes a method for preparing a stabilized dry reagent system for use in bioassays requiring the isolation of one or more dry components to prevent their premature interaction. A representative rea¬ gent system requiring isolation of their key chemical component from one another are the chemical constituents used in the Jaffe analytical protocol for creatinine determination. In brief, the Jaffe analytical protocol involves the following reaction: 2,4,6-trinitrophenol + creatinine excess adduct LiOH adduct = a Janowsky-type adduct (a colored complex of picrate + creatinine) Following, or concurrent with interaction of the above reagents and sample, the absorption of the solution is monitored by conventional spectro- photometry at a predetermined wavelength. It has been observed experimen¬ tally that picric acid and lithium hydroxide interact with one another to an extent proportional to reactants concentration and temperature. The solu- tion of picrate and lithium hydroxide containing both these reagents is stable for only a relatively short period of time; thus, these two reagents must be maintained apart from one another until combined, at the appropriate phase of the analytical procedure, in the presence of the patient sample. The basic tendency of these two (2) components of the Jaffe reagent system to interact is also prevalent when they are combined with one another in the solid form. ** * Their interaction is manifested as abbreviated stability or shelf life of the solid. This invention permits the formation of such unitary dry reagent system into a stable solid composition from a complement of interactive (hereinafter "heterogeneous") reagents. 0 As applied to the Jaffe reagent system, the procedure involves provid¬ ing an alkali salt of picric acid in solid form as one of the reagents. Subsequently, lithium hydroxide, also in solid form, is twice-coated with an encapsulating agent. This agent can preferably be any nonpolymeric binding agent compatible with a tabletted system, i.e., one which retains its optical 5 clarity in reconstituted solution, permits a rapid dissolution capability; and, which is non-reactive in the solid form with respect to the other components in the reaction system. This initial encapsulation process can be followed, in one of the preferred embodiments of this invention, by further encapsulation sequence with a detergent/surfactant. The above encapsulation procedure 0 effectively prevents the interaction of the lithium hydroxide with the picrate when placed in a unitary solid state. After encapsulation of the lithium hydroxide, the picrate salt and the coated lithium hydroxide can then be combined. Formulation of a unitary dry solid in the foregoing manner will maintain the stability of the reagent system by preventing their interaction, 5 while retaining the requisite optical clarity of the reconstituted solution. Alternatively, this procedure is equally applicable to isolation of the picrate salt by coating it rather than the lithium hydroxide. In this particular application, sucrose is used as a bulking agent for Q tabletting purposes. Picric acid must be neutralized by forming the alkali salt so as to prevent the acid catalyzed hydrolysis of the sucrose to produce glucose and fructose. The picric acid normally acts as a catalyst for this reaction, both in aqueous solution and the solid phase. Glucose is a substance which interferes with the Jaffe reaction and the elimination of this is 5 accomplished by using the neutralized picrate. The alkali salt ^ of picric acid can be formed by introducing lithium hydroxide to an aqueous solution of picric acid, followed by the addition of sucrose to the picrate solution and subsequent lyophilization of the resultant mixture. The lyophilization step is preferably conducted in stainless steel or glass trays .since the picrate OMP solution is reactive towards certain materials (i.e., reacts with aluminum to produce intensely red product). Coated lithium hydroxide can be obtained by preparing a solution of lithium hydroxide in isopropanol and to the stirred solution is added an aqueous solution of sucrose. Adding sucrose dropwise, by fine spraying • throughout the bulk of the suspension, or any other metered controlled addition technique is acceptable so long as the even distribution of sucrose solution throughout the lithium hydroxide is accomplished. Because the sucrose is not soluble in isopropanol, it crystallizes out using the lithium hydroxide particles as nuclei around which to grow, hence the coating of lithium hydroxide. The resulting product is washed, .dried and sieved in preparation for a second layer of sucrose to be applied by the same procedure. A spray-drying apparatus can be employed to effect the controlled addition of sucrose wherein the solid lithium hydroxide is suspended by air currents in a spray-drying chamber and an aqueous solution of the appropriate solute is sprayed into the chamber at a controlled rate and droplet size. This results in the condensation of the sucrose around the lithium hydroxide, and the product is followed by rapid drying. In a similar manner, the encapsulated lithium hydroxide can be added to warmed methanol and to the suspension which is formed is added dodecyl sodium sulfate (hereinafter "SDS"), followed by evaporation of the methanol leaving the product which is then dried and sieved. The SDS is an ionic detergent that reduces protein interference during the Jaffe reaction, and acts as a clarifying agent keeping the protein in solution. Furthermore, the SDS acts as a lubricant and mold-releasing agent reducing the amount of sticking of the mixture to the equipment used in the tabletting process. The tablet can be formed from this free-flowing solid material prepared by the above manner, blending the lyophilized picrate and coated lithium hydroxide with sucrose or similar substance (as a bulking agent) in the proper relative proportions followed by a compression of the blend into wafers which are then granulated and tabletted. "g JRE OMPI This procedure can be adapted to any reagent system which contains two or more heterogeneous reagents which must be kept isolated from one another during storage. An example of another such reagent system are the heterogeneous chemicals used to measure magnesium levels in blood serum. In the analytical protocol for magnesium quantitation Calmagite (available from Eastman Chemicals, Tennessee, which has the generic name of 1-(1- hydroxy-4-methyl 2-phenylaza)-2-napthal-4-sulfonic acid), in an ex-cess alka¬ line environment, reacts to yield a colored complex which can be spectropho- tometrically measured. In this procedure, the alkaline reagent used is tribasic sodium phosphate. The problem with "this reagent is that it has the tendency to absorb water, which decreases the linearity of measurements observed and also decreases the reagent system stability. By coating the sodium phosphate with mannitol or some such similar bulking agent, the hygroscopic tendency of this alkaline reagent is reduced, thereby the reagent system can be contained in a single unitary dry solid for use in an automatic chemical analyzer. EXAMPLES The Examples which follow further describe, define and illustrate a number of different embodiments of this invention. The apparatus and techniques used in the conduct of this method, in the evaluation of the unitary dry systems produced thereby, are standard of hereinabove described. Parts and percentages appearing in such Examples are by weight unless otherwise indicated. Example 1 The procedure for manufacturing a single solid reagent involves three basic steps: O PΓ 1. Preparation of a lyophilized lithium picrate Prepare a lithium picrate solution by adding 62g of A.C.S. grade picric acid (85%) to nine (9) liters of water at room temperature. Add 6.61g of lithium hydroxide to this with mixing, until a homogeneous solution results. Dissolve 463g sucrose in the picrate solution and bring the volume to ten (10) liters by adding 10 water. Lyophilize the liquid reagent in stainless steel trays. Sieve the resulting product through a 40 mesh (U.S. standard) 1 stainless steel sieve and collect it. 2. Preparation of coated-lithium hydroxide 20 Suspend 65g of lithium hydroxide in 1000 ml of isopropanol, while stirring vigorously and keeping at a temperature of 40-50C. Prepare a sucrose solution by dissolving 26g of sucrose in 45 ml of warm water, added dropwise to the above suspension, while stirring. Collect the precipitated product by vacuum filtration 25 washing with isopropanol, and dry. Sieve this product through a 40 mesh stainless steel screen and continue with the second layer of sucrose in the same manner described above, using 86g sucrose- coated lithium hydroxide with a solution of 24g of sucrose in 40 ml water. Add 108.6g of dried sucrose-double-coated lithium 30 hydroxide (about 54% w/w lithium hydroxide) to 1000 ml warmed methanol; add to this suspension 108.3g powdered dodecyl sulfate (SDS). Stir the mixture vigorously in a suitable mixer (e.g., Hobart "Kitchen aide") until all the methanol has evaporated. Dry « and sieve the product through a 40 mesh stainless steel screen. 35 . 3. Preparation of final tabletted reagent ** Combine 462.2g sieved, sucrose-lithium picrate mixture with 196.6g of the sieved triple-coated lithium hydroxide and 341.2g of 40 mesh screened and dried sucrose. Blend in a twin shell blender for about 45 minutes. Q Compress the blend into 1.27 cm diameter wafers, granulate through a 20 mesh screen and tablet the granules into 0.397 cm diameter flat face tablets. NOTE: Quantities described include calculated excesses to compensate 5 for processing losses. Example 2 0 Lithium picrate is prepared as described in Example 1. To prepare the coated lithium hydroxide, suspend 65g of lithium hydroxide in 1000 ml of isopropanol, while stirring vigorously at a temperature of 40-50C. Prepare a mannitol solution by dissolving 26g mannitol in 45 ml of warmed water and adding dropwise to the suspension, while stirring. Collect the precipitated 5 product by vacuum filtration, wash with isopropanol and dry. Sieve this product through a 40 mesh stainless steel screen and continue with a second layer of mannitol in the same manner described above, using 86g mannitol- coated lithium hydroxide with a solution of 24g mannitol in 40 ml of water. 0 Add 108.6g of dried mannitol double-coated (54% w/w lithium hydrox¬ ide) to 1000 ml warmed methanol; add to the solution 108.3g powdered SDS. Stir the mixture vigorously in a suitable mixture until all the methanol has evaporated. Dry and sieve the product through a 40 mesh stainless steel screen. Form the tablet by blending and slugging two components as 5 described above. - Example 3 • After the lithium picrate is prepared, a solution of sorbital is used to double-coat the lithium hydroxide, followed by coating SDS and tabletting the blended mixture using the method and proportions as described in Example 1.

Claims: WHAT IS CLAIMED IS 1. * A method for preparing a stabilized dry solid reagent from hetero- -_ geneous reagents for creatinine assay via a Jaffe reaction, comprising: (a) providing an alkali salt of picric acid in solid form; (b) providing lithium hydroxide in solid form; (c) coating at least one of said picric .acid salt or lithium hydroxide by forming a suspension of one of said components in a nona- queous liquid and then adding to said suspension an aqueous solution of a compatible component of said solid reagent system whereby said component is precipitated upon the suspended parti¬ cles and thereby effectively isolates said particle from interac¬ tion, in the solid state with the other components of the reaction system. (d) isolating the coated component of the reagent system; and (e) combining the isolated coated component of said reagent system with the other constituents of ' the Jaffe system in the appropriate relative proportions into a unitary solid. * 2. The method of claim 1, wherein the aqueous solution is contacted with the nonaqueous suspension by spraying said solution into the suspension. 3. The method of claim 1, wherein the aqueous solution is contacted with the nonaqueous suspension by spraying said solution onto the suspension. 4. The method of claim 1, wherein: (a) either the solid lithium hydroxide or alkali salt of picric acid is suspended by air currents in a spray drying apparatus chamber; and (b) the aqueous solution is sprayed into said chamber at a predetermined rate and droplet size such that said aqueous solution contacts said suspended solid and condenses thereon; 5. The method of claim 1, wherein the coating compound is a nonpoly¬ meric substance that does not interfere with the reaction system. 6. A process for performance of analysis of an aqueous sample for the presence of an analyte in an automated clinical analyzer, comprising: (a) providing a stabilized dry solid reagent system containing hetero¬ geneous reagents in tablet or granular form, wherein at least one of said heterogeneous reagents is coated with an encapsulation effective amount of another of the compatible components of the reagent system; (b) adding said solid reagent system and a solvent for said solid to a reaction vessel; (c) dissolving said solid reagent system in said solvent in the reaction vessel; (d) adding to the reaction vessel a sample containing an unknown concentration of creatinine; and (e) monitoring said reaction vessel for the presence of a colored constituent which can be correlated with creatinine concentration of the sample. 7. The process of claim 6, wherein sufficient ultrasonic energy is applied to the solid reagent system to effect its rapid dissolution in the solvent. 8. The method of claim 6, wherein: (a) the analyte is creatinine; and r- (b) the solid reagent system in comprised of lithium hydroxide and an alkali salt of picric acid such that either or both components are isolated from one another. O PI

Inventors: BODDEN ROBERT L, EDWARDS RICHARD B, BODDEN, ROBERT, L., EDWARDS, RICHARD, B.
Applicants: AMERICAN HOSPITAL SUPPLY CORP, AMERICAN HOSPITAL SUPPLY CORPORATION
Application year: 1984
Main classification: G01N21
IPC codes: G01N33