Patent Description:
The present disclosure relates to the field of a composition of a chemical compound and more particularly to the field of a composition of a high concentration of bilirubin stock. The invention is directed to a composition of bilirubin stock and to a method of preparing a bilirumin stock.

Bilirubin is a compound that forms a part of the metabolic pathway in human beings. Bilirubin is a degradation product of heme component of hemoglobin that is formed during the catabolism of red blood cells (RBC). Bilirubin may be one of the essential components that acts as an interferent in hemolysis studies. Bilirubin solution forms an essential part of a calibration process in hemolysis detection. Bilirubin solution may be used as standard samples of varying known concentrations, so as to calibrate a device configured to determine hemolysis in a whole blood sample. Due to photosensitive nature of bilirubin, it is essential that the handling of bilirubin be performed in dark or low light exposure conditions and variability in the prepared solution be minimal. Commercially available unconjugated form of bilirubin is soluble in organic solvents such as chloroform or dimethyl sulfoxide (DMSO). Such unconjugated bilirubin is insoluble in water. Organic solvents may compromise the analysis of the sample, for example, by damaging the sample and corroding the equipment used in the analysis. The fumes emitted by the organic solvent may also alter the surrounding environment. As organic solvents are mostly hazardous, hygroscopic and volatile in nature, the sample handling process may become more difficult. There are a variety of prior art documents dealing with compositions comprising bilirubin. <CIT> discloses multi-item composite quality control liquor for urine analysis. The multi-item composite quality control liquor comprises a buffering solution, a surface active agent, <NUM>-pyridinethiol <NUM>-oxide sodium salt, MgC12, urea, sodium chloride, poloxamer, anhydrous dextrose, bovine serum albumin, esterase, a ketone body substitute, a bilirubin substitute, nitrite, creatinine, a urobilinogen substitute, anhydrous calcium chloride, red blood cell particles, white blood cell particles and crystals. <CIT> is directed to a device and a method for preparing a bilirubin-containing control solution. The device comprises a carrier vehicle having incorporated with it unconjugated bilirubin and certain dyes. Upon contact with a predetermined quantity of water a control solution results, which will simulate a pathological specimen of conjugated bilirubin-containing solution, upon analysis. <CIT> discloses a control solution for analyzing body liquids comprising bilirubin, albumin and carbonate. The disclosed calibration composition comprises bilirubin, sodium carbonate, human albumin and acetic acid. The calibration solution has to be used immediately after preparation. Scientific article<NPL> deals with inhibiting calcium carbonate precipitation. According to this article, the addition of a bilirubin-albumin solution to a supersaturated solution of calcium chloride and sodium bicarbonate entirely blocked precipitation of calcium carbonate from solution. The scientific article <NPL> discloses a method based on serial ultrafiltration that successively removes impurities in [<NUM>C]bilirubin until a stable binding affinity is achieved, and then it is to assess the effect of albumin concentration and buffer composition on binding. It has been found that binding may be considerably less avid at physiological albumin concentrations than previously believed. Further, Bilirubin binding experiments are disclosed in <NPL>. The disclosed bilirubin solution prepared by dissolving few crystals of bilirubin in <NUM> sodium carbonate solution contains <NUM> EDTH. In order to study the binding of bilirubin at different pH, the bilirubin solution was added to an albumin solution in Tris/HCl buffer having pH between <NUM> and <NUM>, wherein the ratio of bilirubin : albumin is between <NUM> and <NUM>.

The object of the invention is achieved by a composition of a bilirubin stock, a method, a process, and a kit for preparation of a bilirubin stock.

A composition of a bilirubin stock is disclosed, wherein the bilirubin stock comprises:.

Further, a method of preparing a bilirubin stock is disclosed, wherein the method comprises:.

In other instances, well known materials or methods have not been described in detail in order to avoid unnecessarily obscuring embodiments of the present disclosure. While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. Conventionally, bilirubin solution is prepared in small volumes with low concentration such that the stability of the bilirubin is maintained. Therefore, the object of the invention is to provide a composition of a high concentration bilirubin stock and a method of preparing thereof that is stable and can be diluted with less or no variation in consistency.

<FIG> illustrates a flowchart of an embodiment of a method <NUM> of preparing a base solution. The base solution includes one or more buffering agents that may naturally occur in a human body. These buffering agents enable dissolution of bilirubin, mimicking bilirubin dissolution in the human body. In an embodiment, the base solution is a bilirubin buffer. The one or more buffering agents may be, for example, one or more chloride salts; urea; and one or more phosphate salts. The chloride salts may include, for example, sodium chloride. The phosphate salts may include, for example, potassium phosphate, monobasic. In an embodiment, at step <NUM>, the buffering agents are dissolved in <NUM> of purified water in the ratio of <NUM>:<NUM>:<NUM> respectively. Therefore, the base solution is an aqueous solution. For example, a <NUM> of the base solution is prepared by dissolving <NUM>/dL of sodium chloride, <NUM>/dL of urea and <NUM>/dL of potassium phosphate monobasic in <NUM> of purified water in a volumetric flask. At step <NUM>, the pH of the base solution is adjusted to <NUM>±<NUM>. Once the buffering agents are dissolved, the volume is made up to <NUM>. The pH may be adjusted based on the actual pH of the prepared solution by adding 1N sodium hydroxide or 1N acetic acid to the base solution. In an embodiment, one or more preservatives may be added to the base solution so as to prevent bacterial or fungal growth in the base solution. The preservative that may be used includes, for example Supelco ProClin® <NUM>, in the range of <NUM>µL to <NUM>µL.

<FIG> illustrates a flowchart of an embodiment of a method <NUM> of preparing the bilirubin stock. At step <NUM>, a first solution is created by dissolving a carbonate salt in the base solution. The carbonate salt may be, for example, sodium carbonate. At step <NUM>, the pH of the first solution is controlled such that a pH range of <NUM> to <NUM> is achieved. At step <NUM>, bilirubin is dissolved in the pH controlled first solution so as to form a second solution. In an embodiment, the bilirubin is in an unconjugated form. Bilirubin being acidic in nature reduces the pH of the first solution. Therefore, an alkaline pH is necessary is required to achieve dissolution of bilirubin in the first solution. Therefore, the pH of the first solution is increased so as to easily dissolve bilirubin in the first solution. At step <NUM>, human serum albumin is dissolved in the base solution to create a third solution. Human serum albumin is found in human blood plasma and serum. Human serum albumin acts as a carrier molecule for unconjugated bilirubin, thereby enabling transportation of bilirubin. At a high pH of the second solution, bilirubin may precipitate. This is avoided by the presence of human serum albumin. At step <NUM>, the pH of the second solution is controlled so as to be in the range of <NUM> to <NUM>. The pH of the second solution may be reduced because human serum albumin may not be stable at an alkaline pH. At step <NUM>, the third solution is added to the second solution, thereby stabilizing the bilirubin and achieving the bilirubin stock. Therefore, in a <NUM> of the base solution, the carbonate salt, bilirubin and human serum albumin is present in the ratio of <NUM>:<NUM>:<NUM>. In an embodiment, filtered plasma may be added to the bilirubin stock. Addition of filtered plasma extends the stability of the bilirubin stock. Such filtered plasma may be added in the ratio of <NUM>:<NUM> to the bilirubin stock.

<NUM> of sodium carbonate per mg of bilirubin is dissolved in <NUM> of the base solution, at a pH of <NUM>±<NUM>, thereby creating the first solution. The pH of the first solution is controlled so as to be in the range of <NUM>±<NUM>. The pH may be controlled, for example, using 1N sodium hydroxide. <NUM> of bilirubin is added to the first solution to create a second solution. On addition of bilirubin, the solution is stirred at a range of <NUM> to <NUM> revolutions per minute (rpm) using a magnetic stirrer, for a time period of <NUM> to <NUM> minutes. Constant stirring of the solution enables dissolution of bilirubin in the first solution, thereby forming the second solution. The pH of the first solution is maintained at <NUM>±<NUM> until bilirubin is dissolved completely. <NUM>/mL human serum albumin is dissolved in <NUM> of the base solution at pH <NUM>±<NUM>, such that the concentration of human serum albumin is <NUM>/mL. This forms the third solution. The third solution may be sterilized, for example, using a <NUM> of syringe filter. The pH of the third solution may be maintained at <NUM>. In the next step, the pH of the second solution is controlled from <NUM>±<NUM> to <NUM>±<NUM>, for example, using 1N acetic acid. One mL of the third solution is added to the pH controlled second solution such that a final concentration of <NUM>/mL of human serum albumin is achieved. The volume may be made up to <NUM> using the base solution with a pH range of <NUM>±<NUM> using, for example, 1N acetic acid. This pH range of <NUM>±<NUM> mimics the physiological pH of a human being. The concentration of the bilirubin in the bilirubin stock may be in the range of <NUM> to <NUM>/dL. Equal volume of the bilirubin stock may be diluted with equal volume of plasma so as to obtain stocks of varied concentrations.

<FIG> illustrates a flowchart of an embodiment of a process <NUM> of preparing a bilirubin stock. At step <NUM>, a pH of the first solution is controlled to be in the range of <NUM> to <NUM>. In an embodiment, the first solution includes a carbonate salt dissolved in the base solution. The carbonate salt may be, for example, sodium carbonate. The pH of the first solution may be controlled, for example, using 1N sodium hydroxide or 1N acetic acid. At step <NUM>, bilirubin is dissolved in the first solution so as to obtain a second solution. Dissolution of bilirubin in the first solution may be achieved by constant stirring, for example, using a magnetic stirrer. The pH of the first solution may be maintained in the range of <NUM> to <NUM> until the bilirubin dissolves completely to form the second solution. At step <NUM>, human serum albumin is dissolved in the base solution so as to obtain a third solution. At a highly alkaline pH, the bilirubin may precipitate. The presence of human serum albumin may control or eliminate precipitation of bilirubin. However, human serum albumin may be unstable at an alkaline pH. Therefore, at step <NUM>, the pH of the second solution is controlled so as to be in the range of <NUM> to <NUM>. The pH of the second solution may be controlled, for example, using 1N acetic acid. Once the pH of the second solution is controlled to be in the neutral range, at step <NUM>, a portion of the third solution is added to the second solution to obtain the bilirubin stock.

In an embodiment a standard curve and linearity of the bilirubin stock may be tested, for example, by obtaining optical density values using light having a wavelength range of <NUM> to <NUM>. Stability studies were performed for the bilirubin stock solution having a concentration of <NUM>/dL at varying temperatures, across days. The stability of the stock was checked at days <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> by obtaining optical density values at wavelength range of <NUM> to <NUM>. <FIG> illustrates a set of graphical representations <NUM>, <NUM>, <NUM> depicting the stability of the bilirubin stock having a concentration of <NUM>/dL. The graph <NUM> depicts the stability of the bilirubin stock at room temperature. The bilirubin stock was prepared strictly in a dark environment or in low light exposure. The bilirubin stock so prepared was stored in dark, amber colored tubes so as to avoid photo-degradation. For purposes of brevity, the graph depicts stability data for days <NUM>, <NUM>, <NUM> and <NUM>. The vertical bars in the graph <NUM> depict the average standard deviation of the optical density at each wavelength, across days.

From the graph <NUM>, it is observed from the standard deviation values that at room temperature, the bilirubin stock remains stable for a period of <NUM> days. Beyond <NUM> days, the bilirubin stock degrades and may be unstable for use. The graph <NUM> depicts the stability of the bilirubin stock at <NUM>. For the purposes of brevity, the graph <NUM> depicts stability data obtained at days <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. The vertical bars in the graph <NUM> depict the average standard deviation of the optical density at each wavelength, across days. From the graph <NUM>, it is observed from the standard deviation values that at <NUM>, the bilirubin stock remains stable for a period of <NUM> days. Beyond <NUM> days, the bilirubin stock degrades and may be unstable for use. The graph <NUM> depicts the stability of the bilirubin stock at -<NUM>. For the purposes of brevity, the graph <NUM> depicts stability data obtained at days <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. The vertical bars in the graph <NUM> depict the average standard deviation of the optical density at each wavelength, across days. From the graph <NUM>, it is observed from the standard deviation values that at -<NUM>, the bilirubin stock remains stable for a period of <NUM> days. Therefore, long storage of bilirubin may be achieved at -<NUM>. The graph <NUM> depicts the stability of the bilirubin stock at -<NUM>. For the purposes of brevity, the graph <NUM> depicts stability data obtained at days <NUM>, <NUM>, <NUM>, <NUM> and <NUM>. The vertical bars in the graph <NUM> depict the average standard deviation of the optical density at each wavelength, across days. From the graph <NUM>, it is observed from the standard deviation values that at -<NUM>, the bilirubin stock remains stable for a period of <NUM> days.

Kits (not according to the invention) are described herein, wherein the kits are designed to expedite performance of the subject methods. Kits serve to expedite the performance of the methods of interest by assembling two or more components required for carrying out the disclosed methods. Kits may contain components in pre-measured unit amounts to minimize the need for measurements by end-users. Kits may include instructions for performing one or more of the disclosed methods and processes. Preferably, the kit components are optimized to operate in conjunction with one another. The kit includes the base solution for dissolving bilirubin and human serum albumin. The kit further includes the first solution. The first solution may include a carbonate salt dissolved in the base solution. The carbonate salt may be, for example, sodium carbonate. The kit further includes bilirubin and human serum albumin. The bilirubin may be dissolved in the base solution to obtain the second solution and the human serum albumin may be dissolved in the base solution to obtain the third solution. A part of the third solution may be added to the second solution to obtain the bilirubin stock. In an alternate embodiment, the kit may further include one or more pH controlling reagents such as sodium hydroxide and/or acetic acid. The pH controlling reagents may be used to control the pH of the first solution, the second solution and/or the third solution.

Claim 1:
A composition of bilirubin stock, wherein the bilirubin stock comprises:
a base solution;
a carbonate salt;
bilirubin; and
human serum albumin
wherein the base solution is an aqueous solution and <NUM> base solution is prepared by dissolving <NUM>/dL of sodium chloride, <NUM>/dL of urea and <NUM>/dL of potassium phosphate monobasic in <NUM> of purified water, after sodium chloride, urea and potassium phosphate monobasic are dissolved, the volume is made up to <NUM>; the base solution comprises sodium chloride, potassium phosphate monobasic, and urea, wherein the sodium chloride, urea, and potassium phosphate monobasic, are in the ratio of <NUM>:<NUM>:<NUM>, wherein the pH of the base solution is adjusted to <NUM> ± <NUM>,
and wherein the composition of sodium carbonate, bilirubin and human serum albumin with respect to the base solution is in the ratio of <NUM>:<NUM>:<NUM>, wherein the given ratios are weight ratios.