CN111766322B - Method for rapidly extracting progestogen from liquid silica gel and detection method - Google Patents
Method for rapidly extracting progestogen from liquid silica gel and detection method Download PDFInfo
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- CN111766322B CN111766322B CN202010632800.0A CN202010632800A CN111766322B CN 111766322 B CN111766322 B CN 111766322B CN 202010632800 A CN202010632800 A CN 202010632800A CN 111766322 B CN111766322 B CN 111766322B
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The application belongs to the field of quality control of progestogen products, and particularly relates to a method and a kit for quickly extracting progestogen and a method for quickly detecting progestogen. A method for rapidly extracting progestogen comprises adding liquid silica gel mixed with progestogen into a silicone oil extraction reagent, wherein the viscosity of the silicone oil extraction reagent is 10-100cps, dissolving completely, centrifuging with a high speed centrifuge, and discarding supernatant to obtain progestogen precipitate. Then, the HPLC external standard method is adopted for detection, and the progestogen in the liquid silica gel can be qualitatively and quantitatively determined. The method is simple to operate, high in recovery rate, free of high-risk operation links and suitable for general laboratory detection.
Description
Technical Field
The application belongs to the field of quality control of progestogen products, and particularly relates to a method and a kit for quickly extracting progestogen and a method for quickly detecting progestogen.
Background
The progestogen, most commonly progesterone, the major target organ of which is the reproductive organ. Progesterone acts primarily on the endometrium in females, on the one hand stimulating and maintaining endometrial and uterine gland growth and on the other hand reducing the receptor levels of the three hormones progesterone, estradiol and OT (oxytocin) on the endometrium. Progesterone inhibits uterine estradiol and OT receptors, leaving the uterus in a quiescent state; progesterone inhibits autoreceptors on the uterus, so that the above-mentioned effects of progesterone on the uterus gradually decline as the luteal phase continues.
The technology is widely applied to batch production of cows and sows at present and is the most important technology in livestock breeding, and since the birth of the 20 th century 60 th generation, a plurality of methods are derived, and the main principle is that the progestogen and the analogues thereof are applied, the ovarian suppression effect of the drugs disappears in 2-6 days after the drugs are stopped, and the cows can be in estrus at the same time. After the progestogen is stopped, gonadotropin or estrogen can be injected in a matching way, so that the estrus performance of the treated female animals is obvious, and the aim of ovulation is fulfilled.
The progestogen veterinary drug product is produced by mixing the progestogen and silica gel (liquid silica gel) according to a certain proportion by using a matrix of silica gel, and adopting a molding process, wherein the progestogen vaginal sustained-release agent has the highest technical content (development difficulty, production difficulty and detection difficulty). The products are usually placed in vagina or implanted for use, during the use period, the progestogen or the like can be slowly released from the silica gel layer (according to a specific release rate) so as to exert the drug effect (artificially simulating luteal phase, namely inhibiting the oestrus of the female animals), after the progestogen is withdrawn or is stopped, the inhibiting effect disappears, and the female animals are oestrous again (the follicle on the ovary is mature to the ovulation at the same time).
A progestogen product in the same estrus technology, in particular to a slow releasing device (slow releasing agent) of a progestogen silica gel, relates to a product quality control link (liquid silica gel (high viscosity, water viscosity of 1cps) is mixed with the progestogen according to a certain proportion, how to detect whether the progestogen in the liquid silica gel is uniformly mixed, how to quickly and accurately detect the accurate content of the progestogen in the liquid silica gel, which is a problem existing all the time (if the progestogen can not be uniformly mixed in the silica gel, the content uniformity of the product molded by adopting a mold can not be ensured, the content of partial product can exceed an upper limit, the content of partial product is lower than a lower limit, the product difference between batches is larger, and the problems of unstable drug effect, incapability of repeating and the like in clinical practice)).
For example, the Chinese patent application (application No. CN201711083232.8) discloses a method for simultaneously detecting multiple progestogens in an environmental water body, which comprises the following steps: 1) water sample pretreatment; 2) enriching and concentrating the novel solid phase extraction adsorbent Oasis PRIME HLB; 3) UPLC-MS/MS determination of progestogen concentration; 4) drawing a standard curve and performing on-machine determination, wherein a novel solid-phase extraction adsorbent Oasis PRIME HLB is used as a pretreatment technology of an environmental water sample, and a combined ultra-high liquid-mass spectrometer is used as a detection and quantification tool, so that 11 different progestogens in a water environment are simultaneously determined.
The above-mentioned method can bind a progestogen with high selectivity and high sensitivity, but the method is complicated in operation. If the content of the progestogen in the liquid silica gel can be directly and quickly extracted and detected, the method has simple operation and high recovery rate, and is also suitable for general laboratory detection.
Disclosure of Invention
In order to solve the difficult problems of rapid and accurate qualitative and quantitative determination of the content of the progestogen in the liquid silica gel added with the progestogen, the application provides a method for rapidly extracting the progestogen, and the method has the advantages of simple operation, high recovery rate and no high-risk operation link, and is suitable for general laboratory detection.
In order to solve the technical problem, the following technical scheme is adopted in the application:
a method for rapidly extracting progestogen comprises adding liquid silica gel mixed with progestogen into a silicone oil extraction reagent, wherein the viscosity of the silicone oil extraction reagent is 10-100cps, dissolving completely, centrifuging with a high speed centrifuge, and discarding supernatant to obtain progestogen precipitate.
Preferably, the silicone oil extraction reagent is formed by mixing at least silicone oil with the viscosity of 1-50cps and silicone oil with the viscosity of 100-500 cps.
Preferably, the silicone oil extraction reagent is one or more of methyl silicone oil, ethyl silicone oil, phenyl silicone oil, methyl hydrogen-containing silicone oil, methyl phenyl silicone oil, methyl chlorophenyl silicone oil, methyl ethoxy silicone oil, methyl trifluoro propyl silicone oil, methyl vinyl silicone oil, methyl hydroxyl silicone oil, ethyl hydrogen-containing silicone oil and hydroxyl hydrogen-containing silicone oil.
Preferably, the silicone oil extraction reagent is prepared by mixing two kinds of dimethyl silicone oil with different viscosities.
Preferably, the centrifugation conditions are as follows: 10000-.
Preferably, the progestogen includes one or more of progesterone, fluogesterone acetate, tetraethenone, medroxyprogesterone acetate, melengestrol acetate and levonorgestrel.
The method is characterized in that the progestogen extracted by the method is diluted and dissolved completely by absolute ethyl alcohol and then the volume is determined, thus the progestogen in the liquid silica gel can be qualitatively and quantitatively detected.
Preferably, HPLC external standard method is used for detection.
Preferably, the silicone oil extraction reagent is formed by mixing at least silicone oil with the viscosity of 1-50cps and silicone oil with the viscosity of 100-500 cps.
Preferably, the silicone oil extraction reagent is one or more of methyl silicone oil, ethyl silicone oil, phenyl silicone oil, methyl hydrogen-containing silicone oil, methyl phenyl silicone oil, methyl chlorophenyl silicone oil, methyl ethoxy silicone oil, methyl trifluoro propyl silicone oil, methyl vinyl silicone oil, methyl hydroxyl silicone oil, ethyl hydrogen-containing silicone oil and hydroxyl hydrogen-containing silicone oil.
Preferably, the silicone oil extraction reagent is prepared by mixing two kinds of dimethyl silicone oil with different viscosities.
Preferably, the progestogen includes one or more of progesterone, fluogesterone acetate, tetraethenone, medroxyprogesterone acetate, melengestrol acetate and levonorgestrel.
According to the application, the progestogen is insoluble in the silicone oil extraction reagent, the precipitate appears, the liquid silicone gel can be dissolved in the silicone oil extraction reagent, so that the progestogen in the liquid silicone gel is completely dispersed and precipitated, then the progestogen can form the precipitate in a centrifugal tube through high-speed centrifugation, a clear solution mixture formed by the silicone oil extraction reagent and the liquid silicone gel is a supernatant, and after the centrifugation is finished, the supernatant is discarded, and the precipitate is reserved. Then dissolving the progestogen in absolute ethyl alcohol to form a progestogen-ethyl alcohol solution, finally diluting the solution to a certain volume, and measuring by adopting an HPLC external standard method to obtain the progestogen-ethyl alcohol solution.
The beneficial effect of this application lies in: the extraction method and the detection method can be used for quickly extracting and detecting the progestogen in the liquid silica gel, and can be used for qualitatively and quantitatively determining the progestogen in the liquid silica gel, thereby ensuring the quality stability of the progestogen product.
Drawings
Fig. 1 is a progesterone control content chromatogram.
FIG. 2 is a chromatogram of the progesterone content in the liquid silica gel A component.
FIG. 3 is a chromatogram of the progesterone content in liquid silica gel fraction B.
Fig. 4 is a chromatogram of the content of progesterone in progesterone control filtrate a.
Fig. 5 chromatogram of content of progesterone in filtrate b of progesterone control.
FIG. 6 methodology verifies-Standard Curve.
Detailed Description
In order to further understand the technical features of the present application, the present application is described in detail with reference to specific embodiments below. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present application, as many insubstantial modifications of the invention, which would be apparent to those skilled in the art upon reading the present application, are intended to be covered by the present application.
Example 1 Rapid extraction of Progesterone from Progesterone-liquid silica gel mixture
1g of A, B parts of liquid silica gel (Dow Corning, Q7-4840) mixed with 10% (W/W) of progesterone is taken and placed into a 50ml centrifuge tube, 10ml of silicone oil extraction reagent (10cps dimethicone: 200cps dimethicone, V/V4: 1) is added, ultrasonic dissolution is carried out at 35 ℃, and cooling is carried out; placing the mixture in a high-speed centrifuge (pre-balancing, plus or minus 0.5 percent) and centrifuging the mixture for 10 minutes at the rotating speed of 12000 rmp; the supernatant was discarded and the precipitate was progesterone. The whole extraction process takes 20-30 min.
Example 2 HPLC external Standard assay for Progesterone in Progesterone-liquid silica gel mixture
Adding 30ml of absolute ethanol into the progesterone (precipitate) extracted in example 1, and dissolving by ultrasonic wave; the solution was transferred to a 100ml measuring flask, the centrifuge tube was washed 5 times with ethanol, the solution was incorporated into the measuring flask, diluted to the mark with absolute ethanol, shaken up and filtered.
HPLC detection conditions: octadecylsilane chemically bonded silica is used as a filling agent; taking water-methanol (25:75) as a mobile phase; flow rate 1.0mL per minute: the column temperature is 35 ℃; the detection wavelength was 254 nm.
Precisely measuring 10uL of the filtrate, injecting the filtrate into a liquid chromatograph, and recording a chromatogram; taking about 100mg of progesterone working standard substance, precisely weighing, placing in a 100ml measuring flask, adding 30ml of ethanol, performing ultrasonic treatment for 5 minutes, adding ethanol for dilution to scale, shaking up, and measuring by the same method. And calculating the peak area according to an external standard method to obtain the content of the progesterone in the liquid silica gel. The results are shown in FIGS. 1 to 3. Example 3
Precisely weighing about 0.1g of progesterone control, adding the progesterone control into 10mL of silicone oil extraction reagent (10cps dimethicone: 200cps dimethicone, V/V is 4:1), stirring, performing ultrasonic treatment, then placing the mixture into a high-speed centrifuge, centrifuging for 10 minutes at 12000 r/min, separating supernatant, placing the supernatant into a beaker for later use, dissolving precipitate with 30mL of absolute ethyl alcohol, diluting to 100mL, filtering, marking filtrate as a solution a, taking another 5mL of supernatant, diluting with absolute ethyl alcohol to 100mL, filtering, and marking the filtrate as a solution b. The liquid a and the liquid b were each detected under the HPLC detection conditions in example 2. The detection results are shown in fig. 4 to 5.
Example 4 method verification
1) Linear (as shown in fig. 6).
2) Accuracy (recovery) was determined using a mixture of components containing known amounts of the test substance, and the results are shown in table 1.
TABLE 1 repeatability (intermediate precision) results
3) The reproducibility (intermediate precision), the results of the analyses by different analysts a, b, c using the extraction method of example 1 and the detection method of example 2 are shown in table 2.
TABLE 2 repeatability (intermediate precision) results
Comparative example 1
Collecting A, B g of liquid silica gel (Dow Corning, Q7-4840) mixed with about 10% (W/W) progesterone, placing in a 50ml centrifuge tube, adding 30ml of n-hexane solution, ultrasonic dissolving at 35 deg.C, and cooling; placing the mixture in a high-speed centrifuge (pre-balancing, plus or minus 0.5 percent) and centrifuging the mixture for 10 minutes at the rotating speed of 12000 rmp; the supernatant was discarded to obtain a precipitate.
The precipitate was analyzed by detection according to example 2, and the results showed that the extraction step took about 5 hours, the recovery rate of the liquid silica gel fractions A and B was less than 70%, and the recovery rate was poor.
Comparative example 2
Respectively placing A, B g of liquid silica gel (Dow Corning, Q7-4840) mixed with about 10% (W/W) progesterone into 50ml centrifuge tubes, adding 10ml of silicone oil solution with viscosity of 5cps, ultrasonic dissolving at 35 deg.C, and cooling; placing the mixture in a high-speed centrifuge (pre-balancing, plus or minus 0.5 percent) and centrifuging the mixture for 10 minutes at the rotating speed of 12000 rmp; the supernatant was discarded to obtain a precipitate.
The examination and analysis of the above-mentioned precipitates with reference to example 2 revealed that the extraction step took about 2 hours, the recovery ratio of progesterone from the liquid silica gel A fraction to the liquid silica gel B fraction was 84.6% and 86.9%, the recovery ratio was less than 90%, and the time was long.
Comparative example 3
Respectively placing A, B g of liquid silica gel (Dow Corning, Q7-4840) mixed with about 10% (W/W) progesterone into 50ml centrifuge tubes, adding 10ml of silicone oil solution with viscosity of 350cps, ultrasonic dissolving at 35 deg.C, and cooling; placing the mixture in a high-speed centrifuge (pre-balancing, plus or minus 0.5 percent) and centrifuging the mixture for 10 minutes at the rotating speed of 12000 rmp; separating supernatant (viscous and poor clarity) to obtain precipitate.
The precipitate was analyzed by assay according to example 2, and the results showed that the extraction step took about 1 hour, the recovery ratio of progesterone in the liquid silica gel fraction a to fraction B was 77.2% and 75.3%, and the recovery ratio was less than 80%; the supernatant was further examined by referring to the method of example 2, and the result showed that the supernatant contained approximately 20% of progesterone. In the method, due to the high viscosity, the progesterone in the silicone oil cannot be completely centrifuged by high-speed centrifugation, so that the recovery rate is poor and the requirement cannot be met.
Claims (5)
1. A method for rapidly extracting progestogen is characterized in that liquid silica gel mixed with progestogen is taken and added into a silicone oil extraction reagent, the viscosity of the silicone oil extraction reagent is 10-100cps, so that the silicon oil extraction reagent is fully dissolved, then a high-speed centrifuge is used for centrifuging, and the supernatant is discarded to obtain a progestogen precipitate;
the silicone oil extraction reagent is formed by mixing silicone oil with the viscosity of 1-50cps and dimethyl silicone oil with the viscosity of 100-500 cps; the progestogen is progesterone.
2. The method for rapidly extracting the progestogen according to claim 1, wherein the centrifugation conditions are as follows: 10000-.
3. A method for rapidly detecting progestogen, which comprises diluting the progestogen extracted by the method of claim 1 or 2 with absolute ethanol, dissolving completely, and then fixing the volume, thereby qualitatively and quantitatively detecting the progestogen in liquid silica gel.
4. The method for rapid detection of progestin according to claim 3 wherein detection is by HPLC external standard method.
5. The extraction method of claim 1 or 2 or the assay of claim 3 for quality control assay of progestogen product.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107375611A (en) * | 2017-08-18 | 2017-11-24 | 福建省中医药研究院(福建省青草药开发服务中心） | A kind of Chinese medicine composition and preparation method with chest enlarge effect |
| CN109187821A (en) * | 2018-11-16 | 2019-01-11 | 北京工商大学 | A method of measurement cosmetics residual sex hormones amount |
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| JP4422430B2 (en) * | 2003-05-14 | 2010-02-24 | 帝國製薬株式会社 | External patch containing estrogen and / or progestogen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107375611A (en) * | 2017-08-18 | 2017-11-24 | 福建省中医药研究院(福建省青草药开发服务中心） | A kind of Chinese medicine composition and preparation method with chest enlarge effect |
| CN109187821A (en) * | 2018-11-16 | 2019-01-11 | 北京工商大学 | A method of measurement cosmetics residual sex hormones amount |
Non-Patent Citations (6)
| Title |
|---|
| A dynamic mechanical method for determining the silicone elastomer solubility of drugs and pharmaceutical excipients in silicone intravaginal drug delivery rings;Malcolm RK 等;《BIOMATERIALS》;20020930;第23卷(第17期);第3589-3594页 * |
| Malcolm RK 等.A dynamic mechanical method for determining the silicone elastomer solubility of drugs and pharmaceutical excipients in silicone intravaginal drug delivery rings.《BIOMATERIALS》.2002,第23卷(第17期),第3589-3594页. * |
| RP-HPLC外标法测定黄体酮含量;周卫军 等;《中南药学》;20050430(第2期);第78-79页 * |
| STUDY OF DIFFUSIONAL PROPERTIES OF SILICONE MATRICES FOR DRUG BIOAVAILABILITY MODULATION;Moreau JC 等;《JOURNAL OF MATERIALS SCIENCE-MATERIALS IN MEDICINE》;19911031;第2卷(第4期);第243-247页 * |
| 复方左炔诺孕酮阴道避孕环的制备及体外释药研究;王幼民 等;《复旦学报(医学版)》;19870930;第14卷(第5期);第353-357页 * |
| 超高效液相色谱-串联质谱法快速测定化妆品中14种性激素;李卓 等;《日用化学工业》;20200229;第50卷(第2期);第137-142页 * |
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