Method for stabilizing hemoglobin a2 and hemoglobin f, method of producing hemoglobin solution, and hemoglobin solution

Abstract

To provide a method for stabilizing hemoglobin A2 and hemoglobin F, which is capable of stabilizing hemoglobin A2 and hemoglobin F in a hemoglobin solution; a method of producing a hemoglobin solution; and a hemoglobin solution.SOLUTION: The present invention provides methods for stabilizing hemoglobin A2 and hemoglobin F, methods of producing a hemoglobin solution, and a hemoglobin solution, the methods including preparing a solution containing a buffer, sucrose, and hemoglobin, wherein the solution has a pH of from 6.0 to 9.6, the concentration of the buffer in the solution is from 5 to 150 mM, the concentration of the hemoglobin in the solution is from 5 to 20 g / L, and the hemoglobin contains at least hemoglobin A2 and hemoglobin F.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for stabilizing hemoglobin A2 and hemoglobin F, a method for producing a hemoglobin solution, and a hemoglobin solution. [Background technology]

[0002] In clinical testing, quality control materials are used to guarantee the quality of test results. Quality control materials are usually sold in a freeze-dried state, and are dissolved in purified water or the like when used. Any remaining quality control materials are stored in a refrigerator and used at a later date. Therefore, quality control materials in a liquid state are required to be stable for a long period of time.

[0003] Patent Document 1 describes a control sample of hemoglobin (hereinafter also referred to as "Hb") F, which is characterized in that it contains sucrose as a stabilizer for a mixed hemolysate of human red blood cells and human umbilical cord blood. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2016-075504 A Summary of the Invention [Problem to be solved by the invention]

[0005] Patent Document 1 describes that since the molecular charge of Hb varies depending on the type, some Hb is stabilized by sucrose and some is not stabilized. Therefore, it is considered difficult to simultaneously stabilize different types of Hb by simply adding sucrose.

[0006] The present disclosure relates to providing a method for stabilizing HbA2 and HbF, a method for producing an Hb solution, and an Hb solution, which are capable of stabilizing HbA2 and HbF in an Hb solution. [Means for solving the problem]

[0007] Means for solving the above problems include the following aspects. <1> preparing a solution comprising a buffer, sucrose, and hemoglobin; The pH of the solution is 6.0 to 9.6; The concentration of the buffer in the solution is 5 to 150 mM; The concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. A method for stabilizing hemoglobin A2 and hemoglobin F. <2> The concentration of the sucrose in the solution is 5 to 20% (w / v); <1> A method for stabilizing hemoglobin A2 and hemoglobin F according to claim 1. <3> preparing a solution comprising a buffer, sucrose, and hemoglobin; The pH of the solution is 6.0 to 9.6; The concentration of the buffer in the solution is 5 to 150 mM; The concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. A method for producing a hemoglobin solution. <4> The concentration of the sucrose in the solution is 5 to 20% (w / v); <3> A method for producing the hemoglobin solution according to claim 1. <5> A hemoglobin solution comprising a buffer, sucrose, and hemoglobin, The pH of the solution is 6.0 to 9.6; The concentration of the buffer in the solution is 5 to 150 mM; The concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. Hemoglobin solution. <6> The concentration of the sucrose in the solution is 5 to 20% (w / v); <5> 2. The hemoglobin solution according to claim 1 . <7> Used as a quality control material, <5> or <6> 2. The hemoglobin solution according to claim 1 . Effect of the Invention

[0008] According to the present disclosure, there are provided a method for stabilizing HbA2 and HbF, a method for producing an Hb solution, and an Hb solution, which are capable of stabilizing HbA2 and HbF in an Hb solution. [Brief description of the drawings]

[0009] [Figure 1] 1 shows a chromatogram of the high performance liquid chromatography performed in Example 1 under the conditions of 100 mM Tris-HCl (pH 8.2) and a Hb concentration of 10 g / L. [Diagram 2] 1 shows a chromatogram of the high performance liquid chromatography performed in Example 1 under conditions of 250 mM phosphate buffer (pH 6.0) and Hb concentration of 5 g / L. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, the form for carrying out the embodiment of the present disclosure will be described in detail. However, the embodiment of the present disclosure is not limited to the following embodiment. In the following embodiment, the components (including element steps, etc.) are not essential unless specifically stated. The same applies to the numerical values ​​and their ranges, and they do not limit the embodiment of the present disclosure.

[0011] In the present disclosure, a numerical range indicated using "~" includes the numerical values ​​before and after "~" as the minimum and maximum values, respectively.

[0012] In the numerical ranges described step by step in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical range described in other step-by-step descriptions. Further, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples.

[0013] In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0014] In the present disclosure, the total Hb amount in the solution is measured by the cyanmethemoglobin method. Specifically, for example, it can be measured using Hemoglobin Test Wako (FUJIFILM Wako Pure Chemical Corporation).

[0015] In the present disclosure, the ratios of HbA2 and HbF in Hb are measured by the peak area ratio in high performance liquid chromatography.

[0016] In the description of the embodiments of the present disclosure, unless otherwise specified, pH refers to the pH at 25°C.

[0017] <Method for stabilizing HbA2 and HbF> The method for stabilizing HbA2 and HbF of the present disclosure includes preparing a solution containing a buffer, sucrose, and Hb, wherein the pH of the solution is 6.0 to 9.6, the concentration of the buffer in the solution is 5 to 150 mM, the concentration of the Hb in the solution is 5 to 20 g / L, and the Hb contains at least HbA2 and HbF. In the present disclosure, the solution containing a buffer, sucrose, and Hb prepared is also referred to as an "Hb solution".

[0018] The present inventors have found that by preparing an Hb solution under the above-mentioned specific conditions, both HbA2 and HbF can be stabilized even if the Hb solution is stored for a long period of time. Although it is assumed that the charge of Hb differs depending on the type, and the stable conditions also differ, it is assumed that both can exist stably for a long period of time in a solution in which the pH, buffer concentration, and Hb concentration are within the above-mentioned ranges and further sucrose is contained, since the charge state of both HbA2 and HbF is unlikely to change. This makes it possible to produce, for example, a quality control material containing HbA2 and HbF that is stable for a long period of time. In addition, for example, the quality control material can be stored in a refrigerator after preparation (dissolution) until it is used up. Therefore, in carrying out quality control on a daily basis, the cost of quality control can be reduced, and the process of preparation at the time of use and subdivision and freezing and storage after preparation can be omitted, thereby improving the ease of handling. However, the embodiment of the present disclosure is not limited to the above-mentioned mechanism of action. In this disclosure, HbA2 and HbF being "stable" means that there is little change in the ratio (%) of HbA2 and HbF to total Hb before and after storage of the Hb solution, i.e., within a predetermined range.

[0019] Patent Document 1 describes that sucrose showed a stabilizing effect on HbA1c and HbF, but did not show a practical stabilizing effect on HbA1A. It also describes that the charge of Hb varies depending on the type, that the difference in charge has a large effect on the detection state of high performance liquid chromatography, and that one condition for an Hb stabilizer is that it is difficult to affect the Hb charge state, but that it is difficult to predict from the correlation between the charge state of Hb and the sugar molecule used as the stabilizer. On the other hand, the inventors have focused on two types of Hb, HbA2 and HbF, and have studied a method for stabilizing both for a long period of time. The method disclosed herein is particularly useful in that it can stabilize both HbA2 and HbF for a long period of time.

[0020] (hemoglobin) The Hb solution contains at least HbA2 and HbF, and may or may not contain other Hbs such as HbA0, HbA1, etc. Typically, the Hb solution contains other Hbs such as HbA0, HbA1, etc. derived from blood.

[0021] The concentration of Hb in the Hb solution (i.e., the total Hb concentration) is 5 to 20 g / L. From the viewpoint of better stabilizing HbA2 and HbF, the concentration of Hb in the Hb solution is preferably 10 to 20 g / L, more preferably 10 to 18 g / L. In particular, when the buffering agent described below is a phosphate buffer, the Hb concentration is preferably 10 to 20 g / L, more preferably 10 to 18 g / L. When the buffering agent is a Tris buffer, the Hb concentration is preferably 10 to 20 g / L. When the buffering agent is a carbonate-bicarbonate buffer, the Hb concentration is preferably 10 to 20 g / L.

[0022] The origin of HbA2 and HbF is not particularly limited, and includes human and non-human animal blood, preferably human blood. Non-human animals include non-human mammals (monkeys, dogs, cats, mice, rats, rabbits, cows, horses, pigs, sheep, etc.), birds (chickens, quails, etc.), etc.

[0023] HbA2 may be HbA2 obtained from human peripheral blood, for example. HbF may be HbF obtained from human peripheral blood or human umbilical cord blood, which is rich in HbF, for example. HbA2 and HbF may be derived from the same sample or from different samples. It is simple to prepare an Hb solution containing HbA2 and HbF from the same sample. In addition, by mixing HbA2 and HbF obtained from different samples, it is easy to adjust the concentration ratio of HbA2 and HbF as desired.

[0024] For example, HbA2 and HbF may be obtained by hemolyzing the blood of the human or non-human animal and extracting Hb. Hemolysis may be performed, for example, by adding EDTA to the precipitate obtained by centrifuging whole blood obtained by blood collection, and freezing and thawing the precipitate. In addition, HbA2 and HbF may be obtained by purifying the solution obtained by hemolysis as described above by dialysis or the like.

[0025] (Buffer) The Hb solution contains a buffer, such as a phosphate buffer, a Tris buffer (e.g., Tris-HCl, TBS, TBS(-)-T, TAE, TBE, TE, etc.), a carbonate / bicarbonate buffer, etc., and from the viewpoint of better stabilizing HbA2 and HbF, a Tris buffer is preferred, and Tris-HCl is more preferred.

[0026] The concentration of the buffer in the Hb solution is 5 to 150 mM, and from the viewpoint of better stabilizing HbA2 and HbF, it is preferably 5 to 100 mM, more preferably 5 to 50 mM, and even more preferably 5 to 30 mM. In particular, when the buffer is a phosphate buffer, the concentration of the buffer is preferably 5 to 100 mM, and more preferably 5 to 50 mM. When the buffer is a Tris buffer, the concentration of the buffer is preferably 5 to 100 mM. When the buffer is a carbonate / bicarbonate buffer, the concentration of the buffer is preferably 5 to 100 mM, more preferably 5 to 50 mM, and even more preferably 5 to 30 mM.

[0027] (sucrose) The Hb solution contains sucrose. Sucrose functions as a stabilizer for Hb. The concentration of sucrose in the solution is preferably 5 to 20% (w / v), more preferably 6 to 18% (w / v), and even more preferably 7 to 15% (w / v).

[0028] The amount of sucrose is preferably 5 to 20 times, more preferably 6 to 18 times, and even more preferably 7 to 15 times, the mass ratio of Hb in the Hb solution.

[0029] (Hb solution) The pH of the Hb solution is 6.0 to 9.6, and from the viewpoint of better stabilizing HbA2 and HbF, it is preferably 7.0 to 9.2, and more preferably 7.5 to 9.0. From the viewpoint of better stabilizing HbA2 and HbF, it is preferable that the pH of the Hb solution is 7.0 to 9.2 (more preferably 7.0 to 9.0), the buffer concentration is 5 to 150 mM (preferably 5 to 100 mM, more preferably 5 to 50 mM), and the Hb concentration is 10 to 20 g / L (preferably 10 to 18 g / L).

[0030] The method for stabilizing HbA2 and HbF of the present embodiment includes preparing a solution containing a buffer, sucrose, and Hb. The preparation method is not particularly limited, and the buffer, sucrose, and Hb may be mixed by any method. In one embodiment, the Hb solution can be prepared by mixing a buffer, a sucrose solution, and a solution containing Hb including HbA2 and HbF. In a further embodiment, the Hb solution can be prepared by mixing a buffer, a sucrose solution, a solution containing HbA2, and a solution containing HbF. The order of mixing is not particularly limited.

[0031] The prepared Hb solution is preferably stable for 21 days or more when stored at 8°C after preparation. For example, in the Hb solution, the fluctuation value of the proportion of HbA2 after 21 days when stored at 8°C after preparation is preferably less than 0.25%, more preferably less than 0.21%. In addition, in the Hb solution, the fluctuation value of the proportion of HbF after 21 days when stored at 8°C after preparation is preferably less than 0.60%, more preferably less than 0.40%, and even more preferably less than 0.36%. The "fluctuation value" of the proportion of HbA2 or the proportion of HbF is calculated as follows. The proportions (%) of HbA2 and HbF in the Hb solution immediately after preparation and after storage are calculated by high performance liquid chromatography. The absolute difference between the proportions after storage and the proportions before storage is calculated, and these are taken as the fluctuation values ​​of the proportions (%) of HbA2 and HbF, respectively. In measuring the fluctuation value, the conditions of high performance liquid chromatography are the method described in Example 1 or a method equivalent thereto.

[0032] <Method of manufacturing hemoglobin solution> A method for producing an Hb solution in one embodiment of the present disclosure includes preparing a solution containing a buffer, sucrose, and Hb, wherein the pH of the solution is 6.0 to 9.6, the concentration of the buffer in the solution is 5 to 150 mM, the concentration of the Hb in the solution is 5 to 20 g / L, and the Hb contains at least HbA2 and HbF. Any of the matters described in the section "Method for stabilizing HbA2 and HbF" can be applied to the details of the buffer, sucrose, Hb, Hb solution, and the preparation method thereof in this embodiment.

[0033] <Hemoglobin solution> The Hb solution in one embodiment of the present disclosure is an Hb solution containing a buffer, sucrose, and Hb, the pH of the solution is 6.0 to 9.6, the concentration of the buffer in the solution is 5 to 150 mM, the concentration of the Hb in the solution is 5 to 20 g / L, and the Hb contains at least HbA2 and HbF. Regarding the details of the buffer, sucrose, Hb, Hb solution, and the preparation method thereof in this embodiment, all of the matters described in the section "Method for stabilizing HbA2 and HbF" can be applied.

[0034] In one aspect, the Hb solution of the present embodiment is used as a quality control material. A quality control material is a sample with a known composition used for quality control of an analysis, and the quality control material of this aspect refers to a sample with a known composition of HbA2 and HbF (i.e., the respective percentages (%) of the total Hb in the quality control material). EXAMPLES

[0035] Next, the embodiments of the present disclosure will be specifically described using examples, but the embodiments of the present disclosure are not limited to these examples.

[0036] 1. Preparation of Hb solution <Hemolysis> (1) Human whole blood was centrifuged (10,000 g, 10 minutes, 4° C.) and the supernatant was discarded. (2) The precipitate was suspended in an amount of saline equal to the amount of whole blood. (3) The suspension from step (2) was centrifuged (10,000 g, 10 minutes, 4° C.) and the supernatant was discarded. (4) Steps (2) and (3) were carried out again. (5) The precipitate was dissolved with 20 mM EDTA (pH 7.0) in a volume 1.3 times the volume of the whole blood. (6) The lysate from step (5) was frozen at -80°C.

[0037] <Refining> (7) After confirming that the lysis solution from step (6) was completely frozen, it was thawed in a water bath at room temperature (about 25°C). This was used as the hemolyzed sample. (8) The hemolyzed sample was enclosed in a dialysis membrane with a molecular weight cutoff of 3,500 and dialyzed using purified water as the external solution. (9) The external solution was replaced (volume of external solution: 85 times or more of the internal solution, 3 hours or more each time, 6 times). (10) The recovered internal solution was centrifuged (40,000 g, 10 minutes, 4° C.), and the supernatant was recovered and designated as solution A. (11) The Hb concentration in solution A was measured using a Hemoglobin Test Wako (FUJIFILM Wako Pure Chemical Industries, Ltd.) (cyanmethemoglobin method). (12) 28% (w / v) sucrose solution, 0.5 M of each buffer solution, and solution A measured in step (11) were mixed so that the final concentrations of each solution were as specified.

[0038] 2. Evaluation [Example 1] <Test conditions> Phosphate buffer: 5 to 250 mM (pH 6.0, 7.0, 7.5, 8.0, 8.2) ·Tris-HCl: 5~250mM (pH7.5, 8.0, 8.2, 9.0) Carbonate / bicarbonate buffer: 5-250 mM (pH 9.2, 9.6) Final sucrose concentration: 7% (w / v) ·Hb concentration: 5~20g / L

[0039] <High performance liquid chromatography> (1) The proportions of HbA2 and HbF in each sample were measured using a calibrated high-performance liquid chromatography device (n=3). (2) The samples were stored at 8°C for 21 days. (3) Using a calibrated high-performance liquid chromatography device, the proportions of HbA2 and HbF in each sample after storage were measured (n=3). (4) The changes in the average measured values ​​(n=3) of HbA2 percentage (%) and HbF percentage (%) before and after storage were calculated.

[0040] <High-performance liquid chromatography device> (1) High-performance liquid chromatography equipment A commercially available cation exchange chromatography column packed with a hydrophilic polymer made of a methacrylic acid ester copolymer was connected to a commercially available high performance liquid chromatography device. An optical detector (specifically, an absorbance meter) for detecting the concentration of Hb flowing through the flow path was attached at a predetermined position in the flow path downstream of the cation exchange chromatography column.

[0041] (2) Eluent Three types of eluents were prepared as aqueous solutions having the compositions shown in Table 1 below.

[0042] [Table 1]

[0043] The eluent A was adjusted to pH 5.08, the eluent B was adjusted to pH 8.0, and the eluent C was adjusted to pH 6.82. With regard to the elution ability of Hb among these eluents, eluent A had the smallest ability, and eluent B had the largest ability.

[0044] (3) Chromatogram The column was equilibrated by passing eluent A through the high performance liquid chromatography device described above in (1). Then, the hemolyzed blood sample was introduced into the column. Then, eluent A was passed for 13 seconds to elute HbA1a, HbA1b, HbF, and HbA1c. Next, a mixture of eluent A and eluent C in a ratio of 1:9 was passed for 5 seconds to elute HbA0. Next, eluent C was passed for 5 seconds to elute HbA2. Then, eluent B was passed for 2 seconds to elute all the Hb remaining in the column, and then eluent A was passed for 5 seconds. During this elution, a chromatogram was created from the absorbance obtained by an optical detector at a detection wavelength of 420 nm.

[0045] The percentage of HbA2 and the percentage of HbF were calculated using the following formula. HbA2(%)=Total area of ​​HbA2 / Total area of ​​all Hb×100 HbF(%)=Total area of ​​HbF / Total area of ​​all Hb×100

[0046] <Calculation of fluctuation value> The percentage of HbA2 was calculated before and after storing the samples at 8°C for 21 days. The absolute difference between the average value after storage (n=3) and the average value before storage (n=3) was calculated and used as the change in the percentage of HbA2 (%). The change in the percentage of HbF (%) was also calculated using the same method.

[0047] <Result> The fluctuation values ​​of the respective percentages (%) of HbA2 and HbF are shown in the table below. In the table, "x" indicates that a measurement was taken but the peak was outside the measurement range and no detection result was obtained. "-" indicates that a measurement was not taken. The reference range for the fluctuation value of the HbA2 percentage (%) is less than 0.25%. The reference range for the fluctuation value of the HbF percentage (%) is less than 0.60%. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0048] An example of a chromatogram is shown in Figure 1. Figure 1 is a chromatogram obtained under conditions of 100 mM Tris-HCl (pH 8.2) and an Hb concentration of 10 g / L. The dashed line shows the chromatogram on day 0 (the day of preparation), and the solid line shows the chromatogram on day 21. Both the HbA2 peak and the amount of HbF are consistent on days 0 and 21, indicating that the condition of the sample is stable.

[0049] Another example of the chromatogram is shown in Figure 2. Figure 2 is a chromatogram obtained under conditions of 250 mM phosphate buffer (pH 6.0) and Hb concentration of 5 g / L. The dashed line shows the chromatogram on day 0 (preparation day), and the solid line shows the chromatogram on day 21. The HbA2 peaks do not coincide on days 0 and 21, indicating that the condition of the sample is not stable.

[0050] This example shows that in an Hb solution having a pH of 6.0 to 9.6, a buffer concentration of 5 to 150 mM, and an Hb concentration of 5 to 20 g / L, HbA2 and HbF are stable at 8° C. for 21 days.

[0051] [Example 2] <Test conditions> ·Tris buffer: 25mM (pH8.8) Final sucrose concentration: 7% (w / v) ·Hb concentration: 13~18g / L

[0052] <High performance liquid chromatography> (1) The proportions of HbA2 and HbF in each sample were measured using a calibrated high-performance liquid chromatography device (n=3). (2) The samples were stored at 8°C for 21 days. (3) Using a calibrated high-performance liquid chromatography device, the proportions of HbA2 and HbF in each sample after storage were measured (n=3). (4) The changes in the average measured values ​​(n=3) of HbA2 percentage (%) and HbF percentage (%) before and after storage were calculated.

[0053] <High-performance liquid chromatography device> The high-performance liquid chromatography apparatus used was an ADAMS HA-8180T (Arkray, Inc.). The apparatus used was a column unit 80T (Arkray, Inc.), which is a column packed with 0.45 ml of a hydrophilic polymer made of a methacrylic acid ester copolymer as a packing material. As reagents, 80A was used as eluent A for eluting Hb from the column, 80B was used as eluent B, and 80CT (Arkray, Inc.) was used as eluent C, respectively, and 80H was used as a hemolysis / washing solution. The compositions and pH of 80A, 80B, 80CT, and 80H are as follows. These reagents were passed through the column at a flow rate of 1.7 ml / min. Eluent A (80A): Phosphate buffer (pH 5.35 ± 0.05) Eluent B (80B): Phosphate buffer (pH 8.05 ± 0.20) Eluent C: (80CT): Phosphate buffer (pH 7.0-7.4) Hemolysis / washing solution (80H): Phosphate buffer solution (pH 7.5±0.1) containing surfactant

[0054] <Calculation of fluctuation value> As in Example 1, the fluctuations in the proportions (%) of HbA2 and HbF were determined.

[0055] <Result> The table below shows the variation from the initial values ​​of the HbA2 percentage (%) and HbF percentage (%) of the samples. The reference range for the variation of the HbA2 percentage (%) was less than 0.25%. The reference range for the variation of the HbF percentage (%) was less than 0.60%. It was confirmed that the variation values ​​of both HbA2 and HbF were within the reference range.

[0056] [Table 8]

Claims

1. preparing a solution comprising a buffer, sucrose, and hemoglobin; the pH of the solution is 6.0 to 9.6; the concentration of the buffer in the solution is 5 to 150 mM; the concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. A method for stabilizing hemoglobin A2 and hemoglobin F.

2. The method for stabilizing hemoglobin A2 and hemoglobin F according to claim 1, wherein the concentration of the sucrose in the solution is 5 to 20% (w / v).

3. preparing a solution comprising a buffer, sucrose, and hemoglobin; the pH of the solution is 6.0 to 9.6; the concentration of the buffer in the solution is 5 to 150 mM; the concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. A method for producing a hemoglobin solution.

4. The method for producing a hemoglobin solution according to claim 3, wherein the concentration of the sucrose in the solution is 5 to 20% (w / v).

5. A hemoglobin solution comprising a buffer, sucrose, and hemoglobin, the pH of the solution is 6.0 to 9.6; the concentration of the buffer in the solution is 5 to 150 mM; the concentration of the hemoglobin in the solution is 5 to 20 g / L; The hemoglobin includes at least hemoglobin A2 and hemoglobin F. Hemoglobin solution.

6. 6. The hemoglobin solution of claim 5, wherein the concentration of said sucrose in said solution is 5 to 20% (w / v).

7. The hemoglobin solution according to claim 5 or 6, which is used as a quality control substance.

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