CN109613277B - Method for simultaneously detecting vitamin D and vitamin K - Google Patents
Method for simultaneously detecting vitamin D and vitamin K Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Abstract
The invention belongs to the technical field of detection, and particularly relates to a method for simultaneously detecting vitamin D and vitamin K. The invention provides a method for simultaneously detecting vitamin D and vitamin K, which comprises the following steps: a) extracting vitamin D3 and vitamin K2 from a to-be-detected object by using a mixed solution of n-hexane and absolute ethyl alcohol, b) detecting the to-be-detected object vitamin D3 and vitamin K2 by using a liquid chromatography to obtain liquid chromatography data, and obtaining the content of the to-be-detected object vitamin D3 and vitamin K2 according to a liquid chromatography standard working curve of vitamin D3 and vitamin K2. The experimental result shows that the method can simultaneously detect the vitamin D3 and the vitamin K2, the detection accuracy is high, the method for detecting the vitamin D3 and the vitamin K2 only needs 8.5 hours for each batch of samples, and the detection efficiency is greatly improved.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for simultaneously detecting vitamin D and vitamin K.
Background
At present, vitamin D and vitamin K are detected separately, for the detection of vitamin D3, sample processing needs 4 hours, and the operation is complex and tedious, and comprises sample saponification, extraction, concentration, washing, volume fixing and filtration, the sample set-up time is 6 hours, and the total time for detecting vitamin D3 of a batch of samples needs 10 hours; for the detection of vitamin K2, the sample processing needs 1.5h, the sample processing time is 7h, and the total time for detecting vitamin K2 of a batch of samples needs 8.5 h; the total time for completing the detection of vitamin D3 and vitamin K2 of a batch of samples is 18.5 hours, which is not favorable for the efficient and rapid detection of products. In addition, in the detection samples of vitamin D3 and vitamin K2, a large amount of potassium hydroxide, petroleum ether, ethyl acetate and other reagents which are harmful to human bodies are required to be used in the treatment process, and the health of detection personnel is not facilitated.
Disclosure of Invention
In view of the above, the invention provides a method for simultaneously detecting vitamin D and vitamin K, which is used for solving the problems that the operation process for detecting vitamin D3 and vitamin K2 is complicated, the time is long, and the detection personnel health is not facilitated in the prior art.
The specific technical scheme of the invention is as follows:
a method for simultaneously detecting vitamin D and vitamin K comprises the following steps:
a) extracting vitamin D3 and vitamin K2 from the substance to be detected by using a mixed solution of n-hexane and absolute ethyl alcohol,
b) and detecting the vitamin D3 and the vitamin K2 to be detected by adopting a liquid chromatography to obtain liquid chromatography data, and obtaining the content of the vitamin D3 and the content of the vitamin K2 to be detected according to the liquid chromatography standard working curve of the vitamin D3 and the vitamin K2.
According to the invention, the mixed solution of n-hexane and absolute ethyl alcohol is adopted to extract vitamin D3 and vitamin K2 from the object to be detected so as to process the sample, so that a solvent with high toxicity is avoided, the health of detection personnel is facilitated, and the experimental result shows that the method can be used for simultaneously detecting vitamin D3 and vitamin K2, the detection accuracy is high, the method is used for detecting vitamin D3 and vitamin K2, each batch of samples only needs 8.5 hours, and the detection efficiency is greatly improved.
Preferably, the substance to be detected is a calcium vitamin D vitamin K soft capsule.
Preferably, the volume ratio of n-hexane to absolute ethyl alcohol in the mixed solution of n-hexane and absolute ethyl alcohol is (10-20): (80-90).
More preferably, the volume ratio of n-hexane and absolute ethyl alcohol in the mixed solution of n-hexane and absolute ethyl alcohol is 20: 80.
preferably, the concentration of the vitamin D3 in the standard working curve of the liquid chromatography is 0.128 to 1.28 mu g/ml;
the concentration of the vitamin K2 in the standard working curve of the liquid chromatography is 0.513 mu g/ml-5.13 mu g/ml.
Preferably, the conditions of the liquid chromatography are as follows:
the chromatographic column is a C18 chromatographic column;
the temperature of the chromatographic column is 30-50 ℃;
the detection wavelength is 264 nm;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 95% methanol-water solution, and the mobile phase B is isopropanol;
the flow rate of the mobile phase is 0.5mL/min to 1.5 mL/min.
More preferably, the column temperature of the chromatographic column is 40 ℃;
the flow rate of the mobile phase was 1 mL/min.
Preferably, the length of the C18 chromatographic column is 250nm, the inner diameter of the C18 chromatographic column is 4.6mm, and the particle size of the packing of the C18 chromatographic column is 5 μm.
In the invention, the C18 chromatographic column is selected from Agilent C18250X 4.6mm, 5 μm; CNW (Shanghai' an Spectrum Co., Ltd.) C18250X 4.6mm, 5 μm; dimand (Diamond) Plus C18250X 4.6mm, 5 μm or Shimadzu Wondasil C18250X 4.6mm, 5 μm. The C18 column is preferably an Agilent C18250X 4.6mm, 5 μm.
Preferably, the liquid chromatography employs gradient elution.
Preferably, in the gradient elution, the volume ratio of the mobile phase A to the mobile phase B is (98): 2; 20.01-38.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 50: 50; 38.01-42.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 0: 100, respectively; 42.01-45.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 50: 50; 45.01-53.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 98: 2.
preferably, the extraction is ultrasonic extraction.
Preferably, the frequency of the ultrasonic extraction is 20 KHz-60 KHz;
the temperature of ultrasonic extraction is 55-65 ℃;
the ultrasonic extraction time is 25-35 min.
More preferably, the frequency of ultrasonic extraction is 40 KHz;
the temperature of the ultrasonic extraction is 60 ℃;
the ultrasonic extraction time is 30 min.
In the invention, a mixed solution of normal hexane and absolute ethyl alcohol is adopted to extract vitamin D3 and vitamin K2 from a to-be-detected object, constant volume is carried out after extraction, vitamin D3 and vitamin K2 are separated by adopting reverse phase chromatography, an ultraviolet detector is used for detection, and an external standard method is used for quantification to obtain the content of vitamin D3 and vitamin K2 in the to-be-detected object.
In summary, the present invention provides a method for simultaneously detecting vitamin D and vitamin K, comprising the following steps: a) extracting vitamin D3 and vitamin K2 from a to-be-detected object by using a mixed solution of n-hexane and absolute ethyl alcohol, b) detecting the to-be-detected object vitamin D3 and vitamin K2 by using a liquid chromatography to obtain liquid chromatography data, and obtaining the content of the to-be-detected object vitamin D3 and vitamin K2 according to a liquid chromatography standard working curve of vitamin D3 and vitamin K2. The experimental result shows that the method can simultaneously detect the vitamin D3 and the vitamin K2, the detection accuracy is high, each batch of samples only needs 8.5 hours when the method is used for detecting the vitamin D3 and the vitamin K2, the detection efficiency is greatly improved, in addition, the mixed solution of normal hexane and absolute ethyl alcohol is used for extracting the vitamin D3 and the vitamin K2 from the object to be detected for sample treatment, the adoption of a solvent with high toxicity is avoided, and the method is favorable for the health of detection personnel.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a liquid chromatogram of a blank solution in an example of the present invention;
FIG. 2 is a liquid chromatogram of a working solution in an example of the present invention;
FIG. 3 is a vitamin D3 standard operating curve of a liquid chromatography standard operating curve in an embodiment of the present invention;
FIG. 4 is a vitamin K2 standard working curve of a liquid chromatography standard working curve in an example of the present invention.
Detailed Description
The invention provides a method for simultaneously detecting vitamin D and vitamin K, which is used for solving the problems of complex operation process, long time and inconvenience for the health of detection personnel in the prior art for detecting vitamin D3 and vitamin K2.
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For a further understanding of the invention, reference will now be made in detail to the following examples.
The instrument adopted in the embodiment of the invention comprises a high performance liquid chromatograph (containing an ultraviolet detector), an ultrasonic instrument and an electronic balance, and the adopted reagents comprise n-hexane (AR), methanol (chromatographically pure), isopropanol (chromatographically pure), absolute ethyl Alcohol (AR), ethyl Acetate (AR), a vitamin D3 reference substance (source: DR, batch number: 100656, purity: 99.9%), a vitamin K2 reference substance (source: USP, batch number: R059X0, purity: 99.8%), and the substance to be detected is a Tangchen Beijian calcium vitamin D vitamin K soft capsule.
Example 1
This example was carried out to prepare vitamin D3 stock solutions and vitamin K2 stock solutions.
Preparation of vitamin D3 stock solution: precisely weighing 3.06mg of a vitamin D3 standard substance, placing the vitamin D3 standard substance in a 500ml brown volumetric flask, dissolving with absolute ethyl alcohol, fixing the volume, shaking up to obtain a vitamin D3 stock solution, and measuring the absorbance by using an ultraviolet spectrophotometer to obtain the measured concentration of 6.4091 mug/ml. Preparation of vitamin K2 stock solution: precisely weighing 10.30mg of vitamin K2 standard substance, placing in a 100ml brown volumetric flask, dissolving with ethyl acetate, fixing volume, shaking to obtain vitamin K2 stock solution, and measuring absorbance by using an ultraviolet spectrophotometer to obtain the concentration of 100 mug/ml. The vitamin D3 stock solution and the vitamin K2 stock solution are stored in a refrigerator at the temperature of 18 ℃ below zero for later use.
Example 2
This example was carried out for the preparation of a standard working solution.
Precisely sucking 10.00ml of vitamin D3 stock solution and 2.50ml of vitamin K2 stock solution, placing the vitamin D3 stock solution and the vitamin K2 stock solution into a 25ml brown volumetric flask, adding absolute ethyl alcohol to a constant volume to scale, shaking up to obtain a standard working solution, precisely sucking 1 mul, 2 mul, 5 mul, 8 mul and 10 mul of the working solution respectively under the following liquid chromatography conditions: the chromatographic column is Agilent C18250 multiplied by 4.6mm, 5 mu m; the temperature of the chromatographic column is 40 ℃; the detection wavelength is 264 nm; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 95% methanol-water solution, and the mobile phase B is isopropanol; the flow rate of the mobile phase is 1 mL/min; gradient elution is adopted for 0.01-20.00 min, and the volume ratio of the mobile phase A to the mobile phase B is 98: 2; 20.01-38.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 50: 50; 38.01-42.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 0: 100, respectively; 42.01-45.00 min, the volume ratio of the mobile phase A to the mobile phase B is 50: 50; 45.01-53.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 98: 2, performing analytical measurements, drawing a liquid chromatography standard working curve, a vitamin D3 standard working curve and a vitamin K2 standard working curve, referring to FIG. 3 and FIG. 4, respectively. Wherein the sample amount of the liquid chromatography detection is 20 mul.
Example 3
Precisely weighing 1.1000g (1 g-3 g) of a substance to be detected, placing the substance in a 10ml (or 25ml) brown volumetric flask, adding n-hexane: placing into a mixed solution of anhydrous ethanol (20: 80), performing ultrasonic extraction in an ultrasonic instrument at 60 deg.C for 30min, taking out, standing at room temperature, extracting with n-hexane: the mixed solution of absolute ethyl alcohol (20: 80) is fixed to the scale, shaken and filtered through an organic filter membrane with the diameter of 0.45 mu m to obtain a sample solution, the sample amount for liquid chromatography detection is 20 mu l (10 mu l-30 mu l), and the conditions of the liquid chromatography are the same as those of the liquid chromatography in the example 2.
And calculating the content of the vitamin D3 or the vitamin K2 in the object to be tested according to the formula X-V multiplied by C/M multiplied by K.
In the formula: x is the content of vitamin D3 or vitamin K2 in the object to be detected, and the unit is mu g/particle;
c is the concentration of vitamin D3 or vitamin K2 in the test solution, and the unit is mug/mL;
m is the mass of the object to be detected, and the unit is g;
v is the volume of dilution of the test substance in mL.
K is a unit conversion coefficient.
The results are shown in Table 1 and Table 2, which are data of vitamin D3 and vitamin K2 detected by the method of the present invention.
TABLE 1 data of vitamin D3 test substance by the method of the present invention
TABLE 2 data of vitamin K2 test substance by the method of the present invention
Comparative example 1
Determination of vitamin D3 according to the fourth method of GB5009.82-2016, comprising the following steps:
(1) preparing standard solution of vitamin D3
Accurately weighing 3.5mg of vitamin D3 standard substance, dissolving with chromatographic pure anhydrous ethanol, and metering to 500mL to obtainThe standard vitamin D3 stock solution is stored in a refrigerator at-20 deg.C under sealed condition, and has an effective period of 3 months. Putting 100 mu L of vitamin D3 standard stock solution into a 10mL brown volumetric flask, using absolute ethyl alcohol to fix the volume to the scale, mixing uniformly to obtain vitamin D3 standard solution, using 1cm quartz cuvettes respectively, using absolute ethyl alcohol as blank reference, and performing formula CVitamin D3The concentration of vitamin D3 standard solution was calculated as ax10000/E.
In the formula: cVitamin D3The concentration of the vitamin D3 standard solution is measured in mu g/mL;
a is the average ultraviolet light absorption value of the vitamin D3 standard solution;
10000 is a conversion coefficient;
e is vitamin D31% specific color light coefficient.
(2) Treatment of test objects
Saponification: weighing 1.0g of a to-be-detected object (accurate to 0.01g) and homogenizing the to-be-detected object in a 150mL flat-bottomed flask, adding 1.0g of ascorbic acid and 0.1g of BHT, uniformly mixing, adding 30mL of absolute ethyl alcohol, adding 10mL to 20mL of potassium hydroxide solution, shaking while adding, uniformly mixing, then carrying out reflux saponification on a constant-temperature magnetic stirrer at 80 ℃ for 30min, and immediately cooling the saponified mixture to room temperature by using cold water to obtain a saponified solution.
Extraction: transferring the saponified solution into 250mL separating funnel with 30mL water, adding 50mL petroleum ether, shaking for extraction for 5min, transferring the lower layer solution into another 250mL separating funnel, adding 50mL petroleum ether, extracting again, and combining ether layers.
Washing: the ether layer was washed with about 150mL of water and repeated 3 times until the ether layer was washed to neutrality (the lower solution was checked for pH using pH paper) and the lower aqueous phase was removed.
Concentration: filtering the washed ether layer into a 250mL rotary evaporation bottle or a nitrogen concentration tube by using anhydrous sodium sulfate (3g), flushing a separating funnel and the anhydrous sodium sulfate by using 15mL petroleum ether for 2 times, merging the flushing funnel and the anhydrous sodium sulfate into the evaporation bottle, connecting the flushing funnel and the anhydrous sodium sulfate to a rotary evaporator or a gas concentrator, carrying out reduced pressure distillation or gas flow concentration in a water bath at 40 ℃ until 2mL ether is left in the bottle, taking down the evaporation bottle, blowing nitrogen to dry, using normal hexane for fixing the volume to 10mL, filtering by using a 0.22 mu m organic filter membrane for semi-preparation of a semi-preparation normal phase high performance liquid chromatography system, and purifying the liquid to be detected.
The conditions for the reverse phase liquid chromatography detection were as follows:
a chromatographic column: a C18 column with a length of 250mm, an inner diameter of 4.6mm and a particle size of 5 μm or a chromatographic column with the same performance;
mobile phase: methanol + water is 95+ 5;
flow rate: 1 mL/min;
detection wavelength: 264 nm;
column temperature: 35 +/-1 ℃;
according to formula XVitamin D3＝ρ×VVitamin D3The vitamin D3 content in the test substance was calculated at XfX 100/m, see Table 3.
In the formula: xVitamin D3The content of vitamin D3 in the test substance is presented in the unit of mu g/granule;
rho is the concentration of vitamin D3 in the object to be detected calculated according to the standard working curve, and the unit is mug/mL;
Vvitamin D3The volume is determined by the volume of normal hexane, and the unit is mL;
f is the dilution multiple of the liquid to be detected in the dilution process;
100 is a conversion coefficient calculated by every 100 grams of the medium amount of the object to be detected;
m is the sample weight of the substance to be detected and the unit is gram.
Table 3 data of vitamin D3 as a test substance detected by GB5009.82-2016 fourth method
Comparative example 2
The determination of vitamin K2 was performed with reference to TCJC-SOP/B-589-2016, comprising the following steps:
(1) preparing standard solution of vitamin K2
Weighing 5mg of vitamin K2 standard substance to 0.01mg accurately, adding into a 50mL volumetric flask, adding ethyl acetate, ultrasonically dissolving, fixing the volume, shaking up, and filtering with a 0.45 μm filter membrane to obtain the final product.
(2) Treatment of test objects
And (3) uniformly mixing the contents of 20 to-be-detected substances, weighing about 1.5g of the substances, putting the substances into a 25ml volumetric flask, adding ethyl acetate, carrying out ultrasonic treatment at 35 ℃ (power of 500W and frequency of 50kHz) until the substances are completely extracted (15min), taking out the substances, cooling the substances, fixing the volume, shaking the substances uniformly, filtering the substances through a filter membrane of 0.45 mu m to obtain a to-be-detected liquid, and carrying out liquid chromatography detection.
The conditions for the liquid chromatography detection were as follows:
a chromatographic column: octadecylsilane chemically bonded silica gel as filler (column length 25cm, inner diameter 4.6mm, particle diameter 5 μm) or chromatographic column with the same performance;
column temperature: 40 ℃;
detection wavelength: 270 nm;
mobile phase: 50% mobile phase A: methanol: 0.1% acetic acid-water solution 95:5
50% mobile phase B: isopropanol (I-propanol)
Flow rate: 1.0 ml/min;
according to formula XVitamin K2＝VVitamin K2×CVitamin K2/MVitamin K2×KVitamin K2The content of vitamin D3 in the test substance was calculated, and the results are shown in table 4.
In the formula: xVitamin K2The content of vitamin K2 in the object to be detected is in mu g/granule;
Cvitamin K2The concentration of vitamin K2 in the test solution is presented in the unit of mu g/mL;
Mvitamin K2The unit is the mass of the object to be measured and is g;
Vvitamin K2Volume of dilution of the test solution, mL.
KVitamin K2Is a unit conversion coefficient.
TABLE 4 data of vitamin K2 for the test substance with reference to TCJC-SOP/B-589-2016
Example 4
This example compares the data for vitamin D3 test of example 3 with the data for vitamin D3 test of comparative example 1, and compares the data for vitamin K2 test of example 3 with the data for vitamin D3 test of comparative example 2. See tables 5 and 6 for results. The result shows that the detection method is scientific and effective in content determination of vitamin K2 and vitamin D3, and can achieve the purpose of quality control of the content of vitamin K2 and vitamin D3 in the double-calcium vitamin D and vitamin K soft capsule.
Table 5 comparison of data for testing vitamin D3 for example 3 and comparative example 1
Table 6 comparison of data for testing vitamin K2 in example 3 and comparative example 2
Example 5
This example was validated for methodology.
(1) Specificity test
Referring to fig. 2, which is a liquid chromatogram of a working solution according to the liquid chromatography conditions of example 2 in the present invention, fig. 2 shows that the working solution contains a vitamin D3 peak and vitamin K2. The blank solution was processed by weighing the sample in the same manner as in example 3, and the blank solution was measured under the conditions of liquid chromatography as in example 2, and the results are shown in fig. 1, and compared with the time at which the vitamin D3 and vitamin K2 control solutions appeared, the results showed that the blank solution had no absorption peak at the time at which the vitamin D3 and vitamin K2 appeared, indicating that the blank had substantially no interference with the results of the vitamin D3 peak and vitamin K2 measurements.
(2) Linear range
The test data for example 2 are shown in tables 7 and 8, respectively, i.e. test data for vitamin D3 and vitamin K2. With the peak area as the abscissa and the concentration as the ordinate, the vitamin D3 standard working curve and the vitamin K2 standard working curve were respectively plotted according to the test data of table 7 and table 8, that is, fig. 3 and fig. 4. The results show that the correlation coefficients R2 of vitamin D3 and vitamin K2 are 0.9999206 and 0.9999592 respectively, the method disclosed by the invention is used for determining that vitamin D3 presents good linearity between 0.12818 mu g/ml and 1.28182 mu g/ml and vitamin K2 presents good linearity between 0.51397 mu g/ml and 5.13970 mu g/ml, and the method meets the requirements of GB/T27404-2008 laboratory quality control Specification (GB/T27404-2008 requires that the correlation coefficient R is more than or equal to 0.99).
Table 7 vitamin D3 test data
Table 8 vitamin K2 test data
(3) Detection limit and quantification limit
The detection limit DL and the quantification limit QL of the analytical method are calculated from the signal-to-noise ratio (S/N). DL is defined as the concentration to be analyzed corresponding to S/N of 3, and QL is defined as the concentration to be analyzed corresponding to S/N of 10.
1) Detection limit
When the signal to noise ratio (S/N) is 3, the detection limits of vitamin D3 and vitamin K2 are 0.01156 mug/ml and 0.02685 mug/ml respectively, and when the weighed amount is 1.1g, the detection limits of vitamin D3 and vitamin K2 in the detection method are 10.5072 mug/100 g and 0.2455mg/kg respectively.
2) Limit of quantification
The limit of quantitation of vitamin D3 and vitamin K2 is 0.03853. mu.g/ml and 0.09000. mu.g/ml, respectively, when the signal-to-noise ratio (S/N) is 10, and the limit of quantitation of vitamin D3 and vitamin K2 in the detection method of the present invention is 35.0273. mu.g/100 g and 0.8182mg/kg, respectively, when the weighed amount is 1.1 g.
(4) Precision test
6 parts of the test substance was weighed, the test substance was treated according to the method of example 3, the contents of vitamin D3 and vitamin K2 in the test substance were measured, and RSD (%) thereof was calculated, and the data of vitamin D3 and vitamin K2 are shown in tables 9 and 10, respectively. The result shows that the RSD for detecting the content of vitamin D3 and vitamin K2 of 6 to-be-detected objects is 3.6 percent and 3.3 percent respectively, the detection method has better precision, and meets the requirements of GB/T27404-2008 laboratory quality control Specification (the RSD (%) of GB/T27404-2008 is less than or equal to 7.5 percent).
TABLE 9 data for vitamin D3 in the precision test
TABLE 10 data for vitamin K2 in the precision test
(5) Stability test
Respectively standing vitamin D3 and vitamin K2 test solutions at room temperature for 0h, 1h, 4h, 8h, 12h and 24h, measuring the concentration under 5.1 conditions, and calculating the RSD (%). Data for vitamin D3 and vitamin K2 in the stability test are shown in tables 11 and 12, respectively. The results show that after the test solution is respectively placed at room temperature for 0h, 1h, 4h, 8h, 12h and 24h, the RSD (%) of the vitamin D3 and the vitamin K2 in the test solution is respectively detected to be 1.5% and 4.3%, and the stability of the vitamin D3 and the vitamin K2 in the test solution is good within 24h at room temperature.
TABLE 11 data for vitamin D3 in the stability test
TABLE 12 data for vitamin K2 in the stability test
(6) Accuracy test (recovery rate)
Adding a standard: precisely weighing about 1.1g of a to-be-detected object, wherein the contents of vitamin D3 and vitamin K2 in the to-be-detected object are respectively as follows: 5.0 mug/g, 16 mug/g) 9 parts, divided into 3 groups, each group having 3 parts, each group precisely adding vitamin D3 stock solution (concentration is 6.4091 mug/ml) 0.60ml, 0.80ml, 1.00 ml; then precisely adding 1.10ml, 1.60ml and 1.90ml of vitamin K2 stock solution (with the concentration of 0.0102794mg/ml) respectively to obtain a standard sample. The labeled analyte was then treated as in example 3.
Adding standard sample to obtain measured amount of the substance to be measured
Percent recovery (%). measured addition/theoretical addition × 100%
The data for vitamin D3 and vitamin K2 in the accuracy test are shown in tables 13 and 14, respectively.
The experimental result shows that the average recovery rates of the vitamin D3 and the vitamin K2 are 97.66% and 99.06% respectively, the Relative Standard Deviation (RSD) is 1.7% and 1.3% respectively, and the requirements of GB/T27404-2008 'laboratory quality control Specification' are met (the required recovery rate of GB/T27404-2008 is 95-105%). In addition, the accuracy of the detection of the vitamin D3 is improved by 2.8 percent (the recovery rate of the vitamin D3 of the fourth method of GB5009.82-2016 is 95 percent, and the recovery rate of the detection method of the invention reaches 97.66 percent)
TABLE 13 data on vitamin D3 in accuracy tests
TABLE 14 data on vitamin K2 in accuracy tests
The above results show that the specificity test, linear range, precision test, stability test, accuracy test, detection limit and quantification limit of the method for simultaneously detecting vitamin D and vitamin K all meet the requirements of GB/T27404-2008 'laboratory quality control Specification', and the content determination method is scientific and effective and can achieve the purpose of quality control on the content of vitamin D3 and vitamin K2 in the soup minister calcium-strengthening vitamin D and vitamin K soft capsules.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method for simultaneously detecting vitamin D3 and vitamin K2, comprising the steps of:
a) carrying out ultrasonic extraction on vitamin D3 and vitamin K2 on a to-be-detected object by adopting a mixed solution of normal hexane and absolute ethyl alcohol;
the frequency of ultrasonic extraction is 20 KHz-60 KHz, the temperature of ultrasonic extraction is 55 ℃ to 65 ℃, and the time of ultrasonic extraction is 25 min-35 min;
the volume ratio of the n-hexane to the absolute ethyl alcohol in the mixed solution of the n-hexane and the absolute ethyl alcohol is 10-20: 80-90;
b) detecting the vitamin D3 and the vitamin K2 to be detected by adopting a liquid chromatography to obtain liquid chromatography data, and obtaining the content of the vitamin D3 and the content of the vitamin K2 to be detected according to a liquid chromatography standard working curve of the vitamin D3 and the vitamin K2;
the substance to be detected is a calcium vitamin D vitamin K soft capsule;
the conditions of the liquid chromatography are as follows:
the chromatographic column is a C18 chromatographic column;
the temperature of the chromatographic column is 30-50 ℃;
the detection wavelength is 264 nm;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a 95% methanol-water solution, and the mobile phase B is isopropanol;
the flow rate of the mobile phase is 0.5 mL/min-1.5 mL/min;
the liquid chromatography adopts gradient elution;
0.01-20.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 98: 2;
20.01-38.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 50: 50;
38.01-42.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 0: 100, respectively;
42.01-45.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 50: 50;
45.01-53.00 min, wherein the volume ratio of the mobile phase A to the mobile phase B is 98: 2.
2. the method according to claim 1, wherein the concentration of vitamin D3 in the standard working curve of liquid chromatography is 0.128-1.28 μ g/ml;
the concentration of the vitamin K2 in the standard working curve of the liquid chromatography is 0.513 mu g/ml-5.13 mu g/ml.
3. The method of claim 1, wherein the length of the C18 column is 250mm, the inner diameter of the C18 column is 4.6mm, and the packing particle size of the C18 column is 5 μm.
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| CN111574348B (en) * | 2020-06-15 | 2022-10-25 | 浙江诚意药业股份有限公司 | Vitamin K1 purification process |
| CN115308338A (en) * | 2022-08-24 | 2022-11-08 | 哈药集团技术中心 | Method for detecting vitamin D3 related substances in vitamin D3 drops |
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