Degerming and bacteriostasis method of water matrix standard substance

Abstract

The invention provides a degerming and bacteriostasis method of a water matrix standard substance, and relates to the field of degerming and bacteriostasis, and the degerming and bacteriostasis method of the water matrix standard substance comprises the following steps: adding an amino acid standard solution, a glucose standard solution and a sodium cyclamate standard solution into sterilized deionized water, stirring, standing, and filtering to obtain a filtrate; the preparation method comprises the following steps: adding methanol and acetonitrile filtered by a sterile filter membrane, stirring or carrying out ultrasonic bath to promote dissolution to obtain a mixed solution, transferring the mixed solution into a volumetric flask, adding sterilized deionized water to carry out constant volume operation, shaking the volumetric flask to uniformly mix the solution to obtain a finished product, subpackaging the finished product into sterile bottles, and carrying out sealed storage. The added methanol and acetonitrile account for 4% of the total volume of the finished product, and according to the degerming and bacteriostasis method of the water matrix standard substance provided by the invention, the added methanol and acetonitrile account for 4% of the total volume of the finished product, so that the water matrix standard substance prepared in the manner has relatively good uniformity and stability.

Description

Technical Field

[0001] The invention relates to the field of sterilization and bacteriostasis, and in particular to a sterilization and bacteriostasis method for a water matrix standard. Background Art

[0002] A reference substance is a substance or material that has been determined to have one or more sufficiently uniform characteristic values. As a "measuring tool" in the analytical measurement industry, it plays an indispensable role in calibrating measuring instruments and devices, evaluating measurement and analysis methods, measuring the characteristic values ​​of substances or materials, assessing the operational technical level of analysts, and controlling the quality of products during the production process. Matrix reference substances are usually prepared and certified by national or international authoritative organizations and can be used as reference substances to compare and determine the accuracy and reliability of analytical results.

[0003] After being prepared and left for a period of time, water-based standards are prone to breed fungi. Fungal growth is the main reason for the short shelf life of water-based standards, especially small organic molecules such as amino acids, sugars, and cyclamate in water. In order to reduce the growth of fungi, water-based standards generally need to be sterilized. However, the sterilization method directly under high temperature and high pressure conditions makes the compounds very easy to degrade, which to a certain extent affects the normal use of water-based standards.

[0004] Therefore, it is necessary to provide a new method for sterilization and inhibition of water matrix standards to solve the above technical problems. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for sterilizing and inhibiting bacteria of a water matrix standard.

[0006] The method for sterilizing and inhibiting bacteria of a water matrix standard provided by the present invention comprises the following steps:

[0007] S1: Use a sterile filter membrane to filter methanol and acetonitrile to sterilize them;

[0008] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and sodium cyclamate standard solution to sterilized deionized water, stir, and eliminate the introduction of bacterial spores into the preparation environment

[0009] S3: adding methanol and acetonitrile filtered through a sterile filter membrane, stirring or dissolving in an ultrasonic bath to obtain a mixed solution;

[0010] S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume;

[0011] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0012] S6: Pack the finished product into sterile bottles and seal them for storage.

[0013] The added methanol and acetonitrile accounted for 4% of the total volume of the finished product.

[0014] Preferably, when using a sterile filter membrane to filter methanol and acetonitrile, first use a filter membrane with a pore size of 0.45 microns for filtration, and then use a filter membrane with a pore size of 0.22 microns for filtration after filtration.

[0015] Preferably, when adding amino acid standard solution, glucose standard solution and cyclamate standard solution and stirring, the stirring is carried out at a low speed first and then at a high speed, wherein the rotation speed is 30-50rpm and the time is 10-15min when stirring at a low speed, and the rotation speed is 300-450rpm and the time is 10-20min when stirring at a high speed.

[0016] Preferably, when adding aseptically treated methanol and acetonitrile and performing ultrasonic bath dissolution, an ultrasonic bath with a frequency of 35-40 kHz is used, a power setting of 50-100 W, and a time of 10-30 min.

[0017] Preferably, when the sealed storage is carried out, the storage temperature is 4°C and refrigerated.

[0018] Preferably, the deionized water is sterilized by high-pressure ultraviolet sterilization.

[0019] Compared with the related art, the bacteriostasis and sterilization method of the water matrix standard provided by the present invention has the following beneficial effects:

[0020] 1. In the present invention, methanol is added, because methanol can combine with fatty acids on the cell membrane, so that fatty acid molecules are piled together, affecting the permeability of the cell membrane, thereby causing the destruction of the cell membrane. This destructive effect seriously affects the living environment of microorganisms, thereby inhibiting their growth and reproduction.

[0021] 2. As an organic solvent, acetonitrile molecules can penetrate into microbial cells and interact with substances within the cells, thereby affecting the metabolism and reproduction of microorganisms.

[0022] 3. In the preparation of water-based standard substances, the present invention adds methanol and acetonitrile, and the added methanol and acetonitrile account for 4% of the total volume of the finished product, so that the water-based standard substances prepared in this way have better uniformity and stability. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the embodiments.

[0024] Embodiment 1

[0025] A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that it comprises the following steps:

[0026] S1: Methanol and acetonitrile are sterilized by filtering with a sterile filter membrane, and deionized water is sterilized by high-pressure ultraviolet sterilization;

[0027] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and cyclamate standard solution to the sterilized deionized water, stir to eliminate the introduction of bacterial spores into the preparation environment, add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed, wherein the speed is 30-50rpm and the time is 10-15min for low-speed stirring, and the speed is 300-450rpm and the time is 10-20min for high-speed stirring.

[0028] S3: Add methanol and acetonitrile filtered by a sterile filter membrane, stir or dissolve in an ultrasonic bath to obtain a mixed solution, add aseptically treated methanol and acetonitrile, and dissolve in an ultrasonic bath with a frequency of 35-40 kHz, a power of 50-100 W, and a time of 10-30 min;

[0029] S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume;

[0030] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0031] S6: The finished product is packaged into sterile bottles and sealed for storage. During the sealed storage, the storage temperature is 4° C. and refrigerated.

[0032] Among them, methanol accounts for 1% of the total volume of the finished product, and acetonitrile accounts for 3% of the total volume of the finished product;

[0033] When using a sterile filter membrane to filter methanol and acetonitrile, first use a filter membrane with a pore size of 0.45 microns for filtration. After filtration, use a filter membrane with a pore size of 0.22 microns for filtration.

[0034] Embodiment 2

[0035] A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that it comprises the following steps:

[0036] S1: Methanol and acetonitrile are sterilized by filtering with a sterile filter membrane, and deionized water is sterilized by high-pressure ultraviolet sterilization;

[0037] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and cyclamate standard solution to the sterilized deionized water, stir to eliminate the introduction of bacterial spores into the preparation environment, add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed, wherein the speed is 30-50rpm and the time is 10-15min for low-speed stirring, and the speed is 300-450rpm and the time is 10-20min for high-speed stirring.

[0038] S3: Add methanol and acetonitrile filtered by a sterile filter membrane, stir or dissolve in an ultrasonic bath to obtain a mixed solution, add aseptically treated methanol and acetonitrile, and dissolve in an ultrasonic bath with a frequency of 35-40 kHz, a power of 50-100 W, and a time of 10-30 min;

[0039] S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume;

[0040] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0041] S6: The finished product is packaged into sterile bottles and sealed for storage. During the sealed storage, the storage temperature is 4° C. and refrigerated.

[0042] Among them, methanol accounts for 2% of the total volume of the finished product, and acetonitrile accounts for 2% of the total volume of the finished product;

[0043] When using a sterile filter membrane to filter methanol and acetonitrile, first use a filter membrane with a pore size of 0.45 microns for filtration. After filtration, use a filter membrane with a pore size of 0.22 microns for filtration.

[0044] Embodiment 3

[0045] A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that it comprises the following steps:

[0046] S1: Methanol and acetonitrile are sterilized by filtering with a sterile filter membrane, and deionized water is sterilized by high-pressure ultraviolet sterilization;

[0047] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and cyclamate standard solution to the sterilized deionized water, stir to eliminate the introduction of bacterial spores into the preparation environment, add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed, wherein the speed is 30-50rpm and the time is 10-15min for low-speed stirring, and the speed is 300-450rpm and the time is 10-20min for high-speed stirring.

[0048] S3: Add methanol and acetonitrile filtered by a sterile filter membrane, stir or dissolve in an ultrasonic bath to obtain a mixed solution, add aseptically treated methanol and acetonitrile, and dissolve in an ultrasonic bath with a frequency of 35-40 kHz, a power of 50-100 W, and a time of 10-30 min;

[0049] S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume;

[0050] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0051] S6: The finished product is packaged into sterile bottles and sealed for storage. During the sealed storage, the storage temperature is 4° C. and refrigerated.

[0052] Among them, methanol accounts for 3% of the total volume of the finished product, and acetonitrile accounts for 1% of the total volume of the finished product;

[0053] When using a sterile filter membrane to filter methanol and acetonitrile, first use a filter membrane with a pore size of 0.45 microns for filtration. After filtration, use a filter membrane with a pore size of 0.22 microns for filtration.

[0054] Comparative Example 1

[0055] A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that it comprises the following steps:

[0056] S1: Methanol is sterilized by filtering it with a sterile filter membrane, and deionized water is sterilized by high-pressure ultraviolet sterilization;

[0057] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and cyclamate standard solution to the sterilized deionized water, stir to eliminate the introduction of bacterial spores into the preparation environment, add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed, wherein the speed is 30-50rpm and the time is 10-15min for low-speed stirring, and the speed is 300-450rpm and the time is 10-20min for high-speed stirring.

[0058] S3: Add methanol filtered through a sterile filter membrane, stir or dissolve in an ultrasonic bath to obtain a mixed solution, add aseptically treated methanol and acetonitrile, and dissolve in an ultrasonic bath with a frequency of 35-40 kHz, a power of 50-100 W, and a time of 10-30 min;

[0059] S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume;

[0060] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0061] S6: The finished product is packaged into sterile bottles and sealed for storage. During the sealed storage, the storage temperature is 4° C. and refrigerated.

[0062] Of these, methanol accounts for 4% of the total volume of the finished product;

[0063] When using a sterile filter membrane to filter methanol, first use a filter membrane with a pore size of 0.45 microns for filtration. After filtration is completed, use a filter membrane with a pore size of 0.22 microns for filtration.

[0064] Comparative Example 2

[0065] A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that it comprises the following steps:

[0066] S1: Deionized water is sterilized by high pressure UV sterilization;

[0067] S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and cyclamate standard solution to the sterilized deionized water, stir to eliminate the introduction of bacterial spores into the preparation environment, add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed, wherein the speed is 30-50rpm and the time is 10-15min for low-speed stirring, and the speed is 300-450rpm and the time is 10-20min for high-speed stirring.

[0068] S3: Transfer the solution treated in S2 to a volumetric flask and add sterilized deionized water to make up the volume;

[0069] S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product;

[0070] S6: The finished product is packaged into sterile bottles and sealed for storage. During the sealed storage, the storage temperature is 4° C. and refrigerated.

[0071] It should be noted that, in each embodiment and comparative example, 25 ml of amino acid standard solution (1000 mg / L), 30 ml of glucose standard solution (1000 mg / L) and 35 ml of cyclamate standard solution (1000 mg / L) were added, and finally, the volume was fixed to 5 L with ion water.

[0072] In Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 3, 5 sterile bottles were respectively selected, each sterile bottle was measured 3 times, and the uniformity test data within and between sterile bottles were statistically analyzed by variance analysis method.

[0073] When 1% methanol and 3% acetonitrile, amino acid uniformity evaluation table:

[0074]

[0075] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 2.314 is less than 3.107, it proves that the difference is not significant.

[0076] When 2% methanol, 2% acetonitrile, amino acid uniformity evaluation table:

[0077]

[0078] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 2.319 is less than 3.107, it proves that the difference is not significant.

[0079] When 3% methanol, 1% acetonitrile, amino acid uniformity evaluation table:

[0080]

[0081] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 2.159 is less than 3.107, it proves that the difference is not significant.

[0082] When 4% methanol, no acetonitrile, amino acid uniformity evaluation table:

[0083]

[0084] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 3.69 is greater than 3.107, the difference is significant.

[0085] When there is no methanol, no acetonitrile, amino acid uniformity evaluation table

[0086]

[0087] When 1% methanol and 3% acetonitrile, glucose uniformity evaluation table:

[0088]

[0089] When 2% methanol, 2% acetonitrile, glucose uniformity evaluation table:

[0090]

[0091] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 1.25 is less than 3.107, it proves that the difference is not significant.

[0092] When 3% methanol, 1% acetonitrile, glucose uniformity evaluation table:

[0093]

[0094] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 1.182 is less than 3.107, it proves that the difference is not significant.

[0095] Glucose uniformity evaluation table when 4% methanol, no acetonitrile:

[0096]

[0097] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 3.19 is greater than 3.107, it proves that the difference is significant.

[0098] When there is no methanol, no acetonitrile, glucose uniformity evaluation table

[0099]

[0100] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 4.99 is greater than 3.107, it proves that the difference is significant.

[0101] When 1% methanol and 3% acetonitrile, the uniformity evaluation table of sodium cyclamate

[0102]

[0103] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 1.409 is less than 3.107, it proves that the difference is not significant.

[0104] When 2% methanol, 2% acetonitrile, sodium cyclamate uniformity evaluation table:

[0105]

[0106] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 2.36 is less than 3.107, it proves that the difference is not significant.

[0107] When 3% methanol, 1% acetonitrile, sodium cyclamate uniformity evaluation table:

[0108]

[0109] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 1.045 is less than 3.107, it proves that the difference is not significant.

[0110] When 4% methanol, no acetonitrile, sodium cyclamate uniformity evaluation table:

[0111]

[0112] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 3.92 is greater than 3.107, the difference is significant.

[0113] When there is no methanol, no acetonitrile, sodium cyclamate uniformity evaluation table:

[0114]

[0115] According to the degrees of freedom and the given significance level α=0.05, under the condition of a confidence level of 0.95, the critical value is 3.107. Since the statistic 3.91 is greater than 3.107, the difference is significant.

[0116] According to the General Principles and Statistical Principles of the Determination of Reference Materials, 6 sampling time points were set to detect the stability of Example 1 that met the uniformity requirements at 4 degrees Celsius, and the detection time and concentration (mg / L) were recorded. A linear model was used for linear fitting, with X representing time and Y representing concentration value, to fit a straight line. The significance of the slope of the straight line was used to judge the stability of the sample.

[0117] Amino acid test results

[0118]

[0119] Calculated: , ,

[0120] Glucose test results

[0121]

[0122] Calculated: , ,

[0123] Cyclamate test results

[0124]

[0125] Calculated: , ,

[0126] According to the data calculated in the above table, no instability was observed in the amino acid detection results, glucose detection results and cyclamate detection results in Example 1 of the present invention.

[0127] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the specification of the present invention, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for sterilizing and inhibiting bacteria of a water matrix standard, characterized in that: The following steps are involved: S1: Use a sterile filter membrane to filter methanol and acetonitrile to sterilize them; S2: Prepare in a sterilized nitrogen-filled glove box, add amino acid standard solution, glucose standard solution and sodium cyclamate standard solution into sterilized deionized water, and stir; S3: adding methanol and acetonitrile filtered through a sterile filter membrane, stirring or dissolving in an ultrasonic bath to obtain a mixed solution; S4: Transfer the mixed solution to a volumetric flask and add sterilized deionized water to make up the volume; S5: Shake the volumetric flask to mix the solution evenly to obtain the finished product; S6: Pack the finished product into sterile bottles and seal them for storage; Among them, the added methanol and acetonitrile accounted for 4% of the total volume of the finished product.

2. The method for sterilizing and inhibiting bacteria of a water matrix standard according to claim 1, characterized in that: When using a sterile filter membrane to filter methanol and acetonitrile, first use a filter membrane with a pore size of 0.45 microns for filtration. After filtration, use a filter membrane with a pore size of 0.22 microns for filtration.

3. The method for sterilizing and inhibiting bacteria of a water matrix standard according to claim 1, characterized in that: Add amino acid standard solution, glucose standard solution and cyclamate standard solution, and stir at a low speed first and then at a high speed. The speed is 30-50 rpm for 10-15 min when stirring at a low speed, and the speed is 300-450 rpm for 10-20 min when stirring at a high speed.

4. The method for sterilizing and inhibiting bacteria of a water matrix standard according to claim 1, characterized in that: When adding aseptically treated methanol and acetonitrile and performing ultrasonic bath dissolution, use an ultrasonic bath with a frequency of 35-40 kHz, a power setting of 50-100 W, and a time of 10-30 minutes.

5. The method for sterilizing and inhibiting bacteria of a water matrix standard according to claim 1, characterized in that: When the sealed container is stored, the storage temperature is 4° C. and the container is refrigerated.

6. The method for sterilizing and inhibiting bacteria of a water matrix standard according to claim 1, characterized in that: The deionized water is sterilized by high pressure ultraviolet sterilization.

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