KR101125871B1 - Process for the preparation of serum certified reference material for measuring glucose and serum certified reference material for measuring glucose prepared therefrom - Google Patents

Abstract

The present invention relates to a method for preparing a wide range of blood glucose measurement standard certified serum, including all ranges of low blood sugar to high blood sugar, and a blood glucose measurement serum standard prepared according to the present invention, comprising the steps of: (1) adding an antibiotic to a raw serum ; (2) first filtering the serum obtained in step (1) to remove solid components; And (3) the blood glucose measurement serum authentication standard prepared according to the method of the present invention comprising the step of filtering the first filtered serum in step (2) with a 0.1 to 0.8 ㎛ sterilizing filter is a low blood sugar hyperglycemia In addition to being able to measure the full range of blood glucose, microbial contamination is eliminated, and thus stability and homogeneity may be usefully used in the art.

Blood glucose measurement, serum certification standard, filtration

Description

Method for preparing serum-certified serum standard for blood glucose measurement and serum-certified serum standard for blood glucose measurement prepared according thereto

The present invention relates to a method for the preparation of a wide range of blood sugar measurement standard for serum glucose, including all ranges from low blood sugar to high blood sugar, and a blood serum measurement standard for blood glucose prepared accordingly.

A reference material is a widely used material for the calibration of measuring instruments, for the evaluation of measuring methods, and for assigning material true values.It is not only necessary to maintain the accuracy and precision of analytical measurements, This is important to avoid inaccurate results. In recent years, as analytical techniques improve, the importance of certified standards in the context of clear traceability information is growing.

Serum certified reference material (serum CRM) means a serum reference material with an accredited certificate and for each specified quantity containing a certification value, measurement uncertainty, and a clearly defined metrological traceability loop. .

Accordingly, the present inventors continue to develop a wide range of blood glucose measurement serum certification standards that cover the entire range of hypoglycemia to hyperglycemia, while adding antibiotics to raw material serum, and preparing the first glucose and second filtration The present invention was completed by discovering that the serum standard for measuring serum can measure the blood sugar of all ranges from low blood sugar to high blood sugar, as well as the microbial contamination is eliminated, which is excellent in stability and homogeneity.

It is an object of the present invention to provide a wide range of blood glucose measurement standards for the standard of blood glucose, which is excellent in stability and homogeneity and includes all ranges of low blood sugar to high blood sugar.

Another object of the present invention is to provide a blood glucose measurement standard material for measuring glucose prepared according to the above method.

In order to achieve the above object, the present invention provides a method for preparing a blood sugar measurement serum certification standard comprising the following steps:

(1) adding antibiotics to the raw serum;

(2) first filtering the serum obtained in step (1) to remove solid components; And

(3) secondary filtration of the first filtered serum in step (2) with a 0.1 to 0.8 μm sterile filter.

In order to achieve the above another object, the present invention provides a blood glucose measurement serum authentication standard prepared according to the method.

Serum authentication standard for measuring blood glucose prepared according to the present invention can measure the blood sugar of all ranges from low blood sugar to high blood sugar, as well as microbial contamination is removed and excellent in stability and homogeneity can be usefully used in the art.

The technical terms and scientific terms used in the present invention can be construed as meaning ordinary meanings understood by those of ordinary skill in the art without departing from the scope of the present invention.

Serum authentication standard for blood glucose measurement of the present invention comprises the steps of: (1) adding an antibiotic to the raw material serum; (2) first filtering the serum obtained in step (1) to remove solid components; And (3) it can be prepared by a method comprising the step of secondary filtration of the serum filtered first in step (2) with 0.1 to 0.8 ㎛ sterilizing filter.

The present invention is 35, 75, 100, 120, 290mg / dL blood glucose measurement certified standard material containing all the range of low blood sugar to high blood sugar based on the normal range of 70 to 120 mg / dL in the serum It is aimed at development.

Glucose certified standards include a process of preparing in an easy-to-preserve form because there is a high risk of deterioration by microbial contamination. This blood glucose certification was determined by isotope dilution mass spectrometry, the highest measurement of organic matter analysis. In the present invention, by adding an antibiotic and firstly removing solids such as fibrin from the raw serum material and passing through a filter that can remove microorganisms, the microorganisms in the serum are completely removed and super-processed by the second filtration. It was possible to prepare a serum-certified serum standard for blood glucose measurement with homogeneity.

The most important part in the development of blood glucose measurement standards is to secure serum raw materials. In the present invention, a raw material for preparing a serum certified standard material was procured through the Seoul Institute of Science, an in vitro diagnostic test institution.

In order to secure a normal serum raw material, the Seoul National Institute of Science chose to include diagnostic tests in small pools (tens of mL) and to include them in large pools only if they were negative.

In order to ensure safety, only three samples were tested for acquired immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis. The serum pool thus collected was stored at all times in an ultra-cold freezer maintained at -75 ° C until use. In addition, the raw material serum used to prepare a serum certification standard is preferably obtained by centrifugation for 10 to 50 minutes at 5000 to 15000 rpm at 4 to 30 ℃.

Particular attention should be paid to the preparation of CRM for blood sugar calibration. To this end, this study sterilized all used containers and minimized contact with air by flowing inert gas to prevent microbial contamination in the air.

Summarizing the manufacturing process of the serum glucose standard for measuring blood glucose of the present invention is as follows.

First, all containers used for preparation are prepared, washed, sterilized in a sterilizer and opened immediately before use.

Since blood glucose is a nutritional source of microorganisms, the concentration of blood glucose continuously decreases when infected with serum, which is not suitable in terms of stability of certified standards. . The amount of the antibiotic to be added is not limited so long as it exhibits an antibiotic effect, but the concentration is usually 50 μg / mL.

Subsequently, primary filtration is performed to remove solids such as fibrin in the serum, for example using a Miracloth filter. This process is suitable for filtering filter paper at the level of particles because it is intended to remove large solids.

Serum from which the solid component has been removed through the primary filter is secondary filtered using a peristaltic pump with a sterile filter (Millipore). At this time, high-purity nitrogen gas flows during filtration to prevent contact with air. The pore size of the micropores of the sterilizing filter used in the present invention is preferably 0.1 to 0.8 μm, more preferably 0.2 μm. In general, bacteria or microorganisms have a minimum particle size of 1 µm or more, so that the microorganisms can be completely removed by filtration with a 0.8 µm, preferably 0.2 µm, sterile filter. When the pore size of the fine pores is less than 0.1 μm, there is a problem that the time required for filtration may be too large and inefficient, and when it exceeds 0.8 μm, the microorganism may not be completely filtered.

High concentration CRM after filtration can be prepared by adding a glucose solution to a constant concentration.

Dispense the serum by purging the inside of the sterile ampoule with argon and dispensing the serum. In addition, nitrogen is flowed into the serum pool to minimize contact with air, and the sealing process is performed immediately after dispensing.

Immediately after dispensing, the inlet is sealed with a vitreous gas burner. Subsequently, the label is labeled, stored in a rack, and stored in a -75 ° C freezer.

That is, when the serum certified standard material according to the present invention is stored under the temperature condition of -75 ° C, the certified value of each component is stably maintained for at least 3 years within the uncertainty range of the initial certified value. It can be used as a certified standard material with better stability compared to the material.

In addition, the serum certification standard is preferably 0.8% or less of the standard deviation of the concentration for each component in the serum, more preferably 0.5% or less, most preferably 0.2% or less.

Serum authentication standard for measuring blood glucose prepared according to the present invention can measure the blood sugar of all ranges from low blood sugar to high blood sugar, as well as microbial contamination is removed and excellent in stability and homogeneity can be usefully used in the art.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following specific examples are only for illustrating the present invention and the scope of the present invention is not limited thereto.

Example 1 Preparation of Glucose Certification Standards

(Step 1) Preparation of Equipment and Containers

All containers used for preparation were prepared, washed and sterilized in a sterilizer (Tomy SX-500, 121 ° C, 20 minutes). The sterilized container was dried and stored in a sealed plastic bag and then opened immediately before use.

(Step 2) Addition of Antibiotic

Since blood glucose is a nutritional source of microorganisms, a phenomenon in which blood glucose concentration decreases continuously when infected with serum, which is inadequate in terms of stability of certified standards, has added gentamicin sulfate antibiotics in the present invention.

First, distilled water and 25 ml vials were sterilized, and then a stock solution was prepared at a concentration of 10 mg / mL. The serum of the freezer was then thawed and the approximate volume was measured to add a gentamicin sulfate stock solution to a concentration of 50 μg / mL. After addition, the solution was returned to form a homogeneous serum while being returned without foaming.

(Step 3) Removing Solid Components of Serum Raw Material

In order to remove a solid part such as fibrin in the serum, it was filtered using a Miracloth filter using a Phenomenex filtration kit. This process is suitable for filtering filter paper at the level of particles because it is intended to remove large solids.

(Step 4) Preparation of Serum by Sterile Filtration

Serum from which the solid component was removed through the primary filter was filtered using a peristaltic pump with a sterilizing filter (Millipore, 0.2 μm) and collected in a 2 L container. In this case, the filter used was sterilized and commercially available, and thus, no further sterilization process was performed. High purity nitrogen gas was flowed during filtration to minimize contact with air. In general, bacteria or microorganisms have a minimum particle size of 1 μm or more, and the microorganisms can be completely removed by filtration with a 0.2 μm sterile filter. Since the serum contains a lot of actual proteins or lipids, the filter replacement cycle was about 200 mL when filtered through a 0.2 μm filter.

After filtration, a high concentration of CRM was prepared by adding a glucose solution to a constant concentration. The glucose solution was used to prepare a stock solution with sterilized water to a concentration of about 170,000 ppm to prevent serum dilution.

(Step 5) Dispensing Serum

Serum was dispensed in 2 mL portions using a Hamilton dispenser. First, the sterilized ampoule was purged with argon, and then 2 mL of serum was dispensed. The probe of the dispenser was lengthened to prevent the sample from adhering to the ampoule inlet and foaming. At the time of dispensing, the serum pool was returned to the magnetic stirrer to homogenize, and excessive stirring was avoided to prevent foaming. Also, nitrogen was flowed into the serum pool to minimize contact with air, and sealing was performed immediately after dispensing.

(Step 6) Sealing and Labeling

Immediately after dispensing, the inlet portion was hermetically sealed using a chogas burner. After sealing the inlet of the ampoule, the cut glass part was rounded and heat treated to make it easy to handle. After sealing, a label was attached and arranged in a rack and stored in a -75 ° C. freezer (Revco).

(Step 7) Investigation of homogeneity and physicochemical properties

Samples were selected by selecting one sample per 50 according to the manufacturing procedure among the samples, and the authentication, homogeneity, pH and density were measured.

(Step 8) Certification Process and Certification Results

IDMS (Isotopic Dilution Mass Spectrometry) was used to measure glucose in serum. IDMS is a representative analytical method that can secure traceability of organic and inorganic metals analysis in samples with medium. Pre-treatment procedures, including extraction and purification, are essential for the analysis of specific chemical components in the sample with media. Accurate correction of the recovery rate of this pretreatment process and the reaction coefficient of the instrument measurement process is required to ensure traceability of this chemical analysis. IDMS analysis involves pretreatment after adding a certain amount of isotopically labeled compounds with the same physical and chemical properties to the sample as an internal standard. In this pretreatment, the recovery rates of the two components will be the same, so the isotope ratio in the pretreated and purified samples is the same as the isotope ratio before extraction. Therefore, the recovery rate is corrected according to the pretreatment, so it is recognized as the primary analysis method of organic matter analysis. The chromatogram by this method is as shown in FIG.

Table 1 below shows the results of glucose measurement in serum by ID LC / MS.

Serum number Measured value (/ g) One
2
3
4
5
6 316.9
317.6
314.6
316.7
316.0
318.0 Average
Standard Deviation(%) 316.6
1.22

Serum number Measured value (/ g) One
2
3
4
5
6 735.7
732.9
734.6
734.1
728.5
734.1 Average
Standard Deviation(%) 733.3
2.54

Serum number Measured value (/ g) One
2
3
4
5
6 959.5
955.8
956.2
964.9
956.0
962.8 Average
Standard Deviation(%) 959.2
0.41

Serum number Measured value (/ g) One
2
3
4
5
6 1189.5
1201.2
1192.1
1196.2
1197.2
1195.7 Average
Standard Deviation(%) 1195.3
4.1

Serum number Measured value (/ g) One
2
3
4
5
6 2922.2
2921.6
2922.8
2908.5
2935.9
2923 Average
Standard Deviation(%) 2922.3
8.69

Serum authentication standard for measuring blood glucose prepared according to the present invention can measure the blood sugar of all ranges from low blood sugar to high blood sugar, as well as microbial contamination is removed and excellent in stability and homogeneity can be usefully used in the art.

FIG. 1 shows Selection Detection Mode (SIM) mass chromatograms of D-glucose and D-glucose- ¹³ C ₆ in serum.

Claims (9)

Method for producing a blood sugar authentication standard for measuring blood glucose, characterized in that it comprises the following steps: (1) adding antibiotics to the raw serum; (2) first filtering the serum obtained in step (1) to remove solid components; And (3) filtering the first filtered serum in step (2) with a 0.1-0.8 μm sterile filter while flowing high purity nitrogen gas to minimize contact with air. The method of claim 1, Raw material serum is obtained by centrifugation at 5000 to 15000 rpm for 10 to 50 minutes at 4 to 30 ℃ and obtained by maintaining at -75 ℃ before use. The method of claim 1, Wherein the antibiotic in step (1) is 50 μg / mL of gentamycin sulfate. The method of claim 1, Method of using a Miracloth filter in the first filtration process of step (2). The method of claim 1, A process characterized in that in step (3) a 0.2 μm sterile filter is used. delete The method of claim 1, Purging the secondary filtered serum with argon in step (3) and then dispensing. The method of claim 7, wherein And storing the dispensed serum in a -75 ° C. freezer. delete

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