CN110514491A - The pre-treating method of organic pollutant in a kind of analysis urine sample - Google Patents
The pre-treating method of organic pollutant in a kind of analysis urine sample Download PDFInfo
- Publication number
- CN110514491A CN110514491A CN201910711460.8A CN201910711460A CN110514491A CN 110514491 A CN110514491 A CN 110514491A CN 201910711460 A CN201910711460 A CN 201910711460A CN 110514491 A CN110514491 A CN 110514491A
- Authority
- CN
- China
- Prior art keywords
- urine sample
- treating method
- organic pollutant
- supernatant
- urine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to urine detection technical fields, disclose a kind of pre-treating method for analyzing organic pollutant in urine sample.Specifically includes the following steps: (1) reacts to obtain pretreated urine sample after mixing urine, rate of recovery indicant, ethyl sodium buffer solution with GRD beta-glucuronidase；(2) urine sample obtained by acetone and step (1) is mixed to occurring precipitating and solution becomes colorless state；(3) desiccant is added in step (2) described solution to continues to mix, separates solid sediment, takes supernatant A；Then acetone is added in obtained solid sediment to continues to mix, supernatant B is taken after filtering, then supernatant A and supernatant B are mixed.The present invention can analyze the organic pollutants such as nicotinic insecticide anabasine, Fipronil, carbamate and its metabolite in urine simultaneously, and when spiked levels are 2~100 μ g/L, the rate of recovery is 69.1 ± 1.1%~114.9 ± 6.2%.
Description
Technical field
The invention belongs to urine detection technical field, in particular to the preceding place of organic pollutant in a kind of analysis urine sample
Reason method.
Background technique
With the development of industrial society, problem of environmental pollution becomes increasingly conspicuous, and people's health requirement also increases day by day.Ring
Pollutant in border can be through breathing, skin contact, and the approach such as drinking-water and food intake enter human body.The overwhelming majority enters human body
Organic contamination in different forms or can measure discharge in urine, from parent to metabolin, from a small quantity to almost all.This
Outside, urine specimen is easier to acquire than the other biologicals material such as blood or tissue.Therefore small organic molecule is that one kind has in urine
The biomarker of reaction human body exposure in environmental pollution of future.In order to show that pollutant is overall sudden and violent in crowd
Dewiness condition first has to carry out comprehensive screening to the pollutant in urine, now with the development of instrument analysis, may be implemented substantially
Simultaneously to the analysis detection of multiple pollutant, however solid phase extraction is used for urine sample pre-treatment, and the operating time is long, cost
Height, the matrix such as extraction efficiency and removal urinary pigment are limited, these factors hinder the efficiency of entire analyte detection process.Therefore, have
Necessity develops new sample-pretreating method, and to meet simultaneously quickly, low cost is suitable for Some Organic Pollutants and metabolin
Sample-pretreating method.
Summary of the invention
In order to overcome the shortcomings and deficiencies of the prior art described above, the purpose of the present invention is to provide a kind of analysis urine samples
The pre-treating method of middle organic pollutant.
The purpose of the present invention is realized by following proposal:
The pre-treating method of organic pollutant in a kind of analysis urine sample, specifically includes the following steps:
(1) it is reacted after mixing urine, rate of recovery indicant, ethyl sodium buffer solution with GRD beta-glucuronidase
To pretreated urine sample；
(2) urine sample obtained by acetone and step (1) is mixed to occurring precipitating and solution becomes colorless state；
(3) desiccant is added in step (2) described solution to continues to mix, separates solid sediment, takes supernatant A；So
Acetone is added in obtained solid sediment afterwards to continues to mix, supernatant B is taken after filtering, then supernatant A and supernatant B are mixed
It closes, obtains premenstrual treated urine sample.
Step (1) rate of recovery indicant is Acetamiprid-d3, the spiked levels in urine are 2~100 μ g/L.
The concentration of step (1) the ethyl sodium buffer solution is 0.2~2M, and pH is 5.0~5.4；Preferably, concentration
For 1M, pH=5.2.
The concentration of step (1) described GRD beta-glucuronidase is 200,00~200,000U/mL, preferably 100,000U/
mL。
The volume ratio of step (1) urine, ethyl sodium buffer solution and GRD beta-glucuronidase be 0.5~
4mL:0.05~0.4mL:15~50 μ L, the μ of preferably 1~2mL:0.1~0.2mL:15~50 L.
Step (1) it is described reaction at 25~50 DEG C reaction 8~for 24 hours, 12 h are preferably reacted at 37 DEG C.
The volume ratio of step (2) acetone and urine sample is 5~20:1；Preferably 8~15:1；
Step (3) desiccant is at least one of magnesium sulfate, sodium sulphate and calcium chloride.
The mass ratio of step (3) desiccant and step (2) described urine sample is 1~3:1；More preferably 1.5~2:
1。
The mass volume ratio of step (3) desiccant and acetone is 2g:2~10mL；Preferably 2g:4~7mL.
Step (3) is described supernatant A and supernatant B are mixed after, further include that nitrogen blows constant volume step；Preferably, the nitrogen
Blowing constant volume is to be settled to 0.1~1.5mL.
Mixing for the first time described in step (2) and step (3), second of mixing and third time mixing independently are ultrasound 5
~30min, preferably 10min.
Preferably, the organic pollutant in urine sample of the present invention is imidacloprid, chlordene niacin, imidacloprid-guanidine salt
At least one of hydrochlorate, imidacloprid-urea, Fipronil, fluorine formonitrile HCN, Fipronil sulfone and Fipronil sulfoxide.
The present invention compared with the existing technology, have the following advantages and the utility model has the advantages that
1) present invention makes the Stromal Precipitations such as urinary pigment and albumen in urine using acetone, and the method for opposite Solid Phase Extraction operates
More easy, cost is lower.
2) present invention can analyze organic dirt such as nicotinic insecticide anabasine, Fipronil, carbamate in urine simultaneously
Object and its metabolite are contaminated, when spiked levels are 2~100 μ g/L, the rate of recovery is 69.1 ± 1.1%~114.9 ± 6.2%.
Detailed description of the invention
Fig. 1 is analyte rate of recovery figure when spiked levels are 2 μ g/L in embodiment 1.
Fig. 2 is analyte rate of recovery figure when spiked levels are 20 μ g/L in embodiment 1.
Fig. 3 is analyte rate of recovery figure when spiked levels are 100 μ g/L in embodiment 1.
Fig. 4 is spiked levels when being 2ppb, and this method and commercialization HLB carry out the rate of recovery comparison of pre-treatment to urine sample
Figure.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
Agents useful for same can routinely be bought unless otherwise specified from market in embodiment.
1 urine anabasine of embodiment, the measurement of fipronil insecticide and its metabolin recovery of standard addition
By imidacloprid, chlordene niacin, imidacloprid-guanidine hydrochloride, imidacloprid-urea, Fipronil, fluorine formonitrile HCN, Fipronil sulfone
With 2 μ g/L, 20 μ g/L and 100 μ g/L concentration, in synthetic urine, (purchase is in LEAGENE, number CZ0350- with Fipronil sulfoxide
Mark-on is carried out in 500mL), to verify this method low, in, the rate of recovery under high concentration.Three parts of Duplicate Samples, concrete operation step
It is as follows:
Synthetic urine 1.0mL is taken, rate of recovery indicant (Acetamiprid-d is added3, target in concentration and rate of recovery experiment is added
Analyte is identical), 0.1mL acetic acid-sodium acetate buffer solution (1M, pH=5.2) and 15 μ L GRD beta-glucuronidases are added
(100,000U/mL), 37 degree of lower reaction 12h.Obtain pretreated urine sample.10mL acetone is added to pretreated urine
In liquid, ultrasonic 10min, until solution precipitates, solution becomes achromaticity and clarification state.2.0g anhydrous slufuric acid is added into solution
Magnesium continues ultrasound 10min.Centrifugation collects upper layer organic extract liquid, then adds 5mL acetone into magnesium sulfate, ultrasound
10min extract liquor will merge twice, and nitrogen blows constant volume to 1.0mL.Internal standard (imidacloprid-d is added4It is dense with 13C-15N Fipronil
Degree is 2 μ g/L), 0.22 μm of organic system pin hole filter membrane is crossed, 5500 is surpassed using Shimadzu LC-30-AD and AB Sciex QTRAP
High liquid chromatography mass spectrometer measures compound concentration.
Fig. 1 is analyte rate of recovery figure when spiked levels are 2 μ g/L in embodiment 1.Fig. 2 is spiked levels in embodiment 1
Analyte rate of recovery figure when for 20 μ g/L.Fig. 3 is analyte rate of recovery figure when spiked levels are 100 μ g/L in embodiment 1.Mark-on
Test result show the rate of recovery of test analyte under different spiked levels 69.1 ± 1.1%~114.9 ± 6.2% it
Between.
2 matrix effect of embodiment
It 1) is solvent respectively by imidacloprid, chlordene niacin, imidacloprid-guanidine hydrochloride, imidacloprid-urea, fluorine using pure methanol
Worm nitrile, fluorine formonitrile HCN, Fipronil sulfone and Fipronil sulfoxide are configured to the series of standards solution that concentration is 0.02~50 μ g/L, internal standard
Compound is imidacloprid-d4With 13C-15N Fipronil, concentration is 2 μ g/L.
2) as the methanol solution of matrix to be solvent through treated the artificial urine of embodiment 1, respectively by imidacloprid, chlordene
Niacin, imidacloprid-guanidine hydrochloride, imidacloprid-urea, Fipronil, fluorine formonitrile HCN, Fipronil sulfone and Fipronil sulfoxide are configured to concentration
For the series of standards solution of 0.02~50 μ g/L, internal standard compound is imidacloprid-d4, concentration is 2 μ g/L.
3) it is obtained using 5500 superelevation liquid chromatography mass combined instrument of Shimadzu LC-30-AD and AB Sciex QTRAP above-mentioned
The standard curve of two each target compounds of class series standard solution.
4) matrix effect (matrix effect, ME) of each target compound is calculated by formula (2).
ME=(Km-Ks)/Ks
KmIt is target compound as the methanol solution of matrix to be its in solvent through treated the artificial urine of embodiment 1
The slope of standard curve；KsFor the slope of target compound its standard curve in methanol solvate.
As a result: the matrix effect value of each test analyte is as shown in table 1.As known from Table 1, majority of compounds shows as base
Matter weakens effect, and only Fipronil sulfone, Fipronil sulfoxide shows as matrix enhancement effect, and the matrix effect range of compound is-
40.76~25.68%.
Each analyte matrix effect value of table 1
Comparative example 1
The specific steps of HLB progress sample pre-treatments: (1) by urine, rate of recovery indicant, ethyl sodium buffer solution
(add in substance additional amount and concentration and embodiment 1 with react to obtain pretreated urine sample after GRD beta-glucuronidase mixing
It marks identical when concentration is 2 μ g/L)；(2) take the solid-phase extraction column (60mg, 3mL) equipped with HLB filler in solid-phase extraction device,
Extraction column is activated with 3mL methanol and 5mL ultrapure water respectively.(3) the pretreated urine sample for obtaining step (1) adds
Be downloaded to it is activated after solid-phase extraction column in.(4) solid-phase extraction column in step (3) is eluted with 5mL methanol.Nitrogen blows concentration
To 1mL.
Fig. 4 is spiked levels when being 2ppb, and this method and commercialization HLB carry out the rate of recovery comparison of pre-treatment to urine sample
Figure.The rate of recovery of analyte is wanted figure 4, it is seen that this research method is able to satisfy with commercialization HLB solid-phase extraction column
It asks.But pre-treatment is carried out to urine sample with this method, relative standard deviation (RSD%) is relative to HLB solid phase between Duplicate Samples
Extraction is smaller, i.e., this method has better stability than solid phase extraction.In addition, this method is easy to operate, quickly, it is not necessarily to
Using solid-phase extraction device and filler, can effectively save personnel's training on operation and material cost, improve the preceding place of urine sample
Manage efficiency, the batch samples pre-treatment suitable for Some Organic Pollutants and metabolin.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. the pre-treating method of organic pollutant in a kind of analysis urine sample, which is characterized in that specifically includes the following steps:
(1) it reacts to obtain after mixing urine, rate of recovery indicant, ethyl sodium buffer solution with GRD beta-glucuronidase pre-
Treated urine sample；
(2) urine sample obtained by acetone and step (1) is mixed to occurring precipitating and solution becomes colorless state；
(3) desiccant is added in step (2) described solution to continues to mix, separates solid sediment, takes supernatant A；Then exist
Acetone is added in obtained solid sediment to continues to mix, supernatant B is taken after filtering, then supernatant A and supernatant B are mixed, obtained
To premenstrual treated urine sample.
2. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that:
The concentration of step (1) the ethyl sodium buffer solution is 0.2~2M, and pH is 5.0~5.4；
The concentration of step (1) described GRD beta-glucuronidase is 200,00~200,000U/mL；
Step (1) rate of recovery indicant is Acetamiprid-d3, the spiked levels in urine are 2~100 μ g/L.
3. the pre-treating method of organic pollutant in analysis urine sample according to claim 1 or 2, it is characterised in that:
The volume ratio of step (1) urine, ethyl sodium buffer solution and GRD beta-glucuronidase is 0.5~4mL:0.05
The μ of~0.4mL:15~50 L.
4. the pre-treating method of organic pollutant in analysis urine sample according to claim 1 or 2, it is characterised in that:
The volume ratio of step (2) acetone and urine sample is 5~20:1.
5. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that: step
(3) desiccant is at least one of magnesium sulfate, sodium sulphate and calcium chloride.
6. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that:
The mass ratio of step (3) desiccant and step (2) described urine sample is 1~3:1；
The mass volume ratio of step (3) desiccant and acetone is 2g:2~10mL.
7. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that:
The mass ratio of step (3) desiccant and step (2) described urine sample is 1.5~2:1；
The mass volume ratio of step (3) desiccant and acetone is 2g:4~7mL.
8. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that: step
(1) it is described reaction at 25~50 DEG C reaction 8~for 24 hours.
9. the pre-treating method of organic pollutant in analysis urine sample according to claim 1, it is characterised in that:
Step (3) is described supernatant A and supernatant B are mixed after, further include that nitrogen blows constant volume step；
Mixing for the first time described in step (2) and step (3), second mixing and third time mixing independently be ultrasound 5~
30min。
10. the pre-treating method of organic pollutant in analysis urine sample according to claim 9, it is characterised in that:
It is to be settled to 0.1~1.5mL that step (3) described nitrogen, which blows constant volume,.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910711460.8A CN110514491A (en) | 2019-08-02 | 2019-08-02 | The pre-treating method of organic pollutant in a kind of analysis urine sample |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910711460.8A CN110514491A (en) | 2019-08-02 | 2019-08-02 | The pre-treating method of organic pollutant in a kind of analysis urine sample |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110514491A true CN110514491A (en) | 2019-11-29 |
Family
ID=68624221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910711460.8A Pending CN110514491A (en) | 2019-08-02 | 2019-08-02 | The pre-treating method of organic pollutant in a kind of analysis urine sample |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110514491A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009057A (en) * | 2020-10-22 | 2021-06-22 | 重庆医科大学 | Method for detecting neonicotinoid insecticides and metabolites in urine by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN114813991A (en) * | 2022-03-09 | 2022-07-29 | 上海交通大学医学院 | Method for detecting neonicotinoid insecticide in human urine |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106483230A (en) * | 2016-03-16 | 2017-03-08 | 南方医科大学南方医院 | A kind of method for quick of urine Hydroxyl Polycyclic Aromatic |
| CN108414631A (en) * | 2018-02-08 | 2018-08-17 | 浙江省食品药品检验研究院 | The detection method of 4 kinds of pesticide residues in a kind of spring bamboo |
| CN109507335A (en) * | 2018-12-29 | 2019-03-22 | 上海交通大学医学院附属新华医院 | Utilize the method for a variety of different type environmental contaminants in LC-MS-MS high throughput detection urine |
-
2019
- 2019-08-02 CN CN201910711460.8A patent/CN110514491A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106483230A (en) * | 2016-03-16 | 2017-03-08 | 南方医科大学南方医院 | A kind of method for quick of urine Hydroxyl Polycyclic Aromatic |
| CN108414631A (en) * | 2018-02-08 | 2018-08-17 | 浙江省食品药品检验研究院 | The detection method of 4 kinds of pesticide residues in a kind of spring bamboo |
| CN109507335A (en) * | 2018-12-29 | 2019-03-22 | 上海交通大学医学院附属新华医院 | Utilize the method for a variety of different type environmental contaminants in LC-MS-MS high throughput detection urine |
Non-Patent Citations (5)
| Title |
|---|
| SEMIHOTLES著: "《食物成分与食品添加剂分析方法》", 31 August 2008 * |
| YAN TAO等: "Urinary monitoring of neonicotinoid imidacloprid exposure to pesticide applicators", 《SCIENCE OF THE TOTAL ENVIRONMENT》 * |
| 张泽鸿主编: "《药学专业知识（一）考点精要》", 31 July 2011 * |
| 李水军等主编: "《液相色谱-质谱联用技术临床应用》", 31 October 2014 * |
| 胡彬等: "用柱切换HPLC测定其中痕量农药", 《药物分析杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113009057A (en) * | 2020-10-22 | 2021-06-22 | 重庆医科大学 | Method for detecting neonicotinoid insecticides and metabolites in urine by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN113009057B (en) * | 2020-10-22 | 2023-11-07 | 重庆医科大学 | Method for detecting neonicotinoid insecticides and metabolites in urine by solid phase extraction-ultra-high performance liquid chromatography-tandem mass spectrometry |
| CN114813991A (en) * | 2022-03-09 | 2022-07-29 | 上海交通大学医学院 | Method for detecting neonicotinoid insecticide in human urine |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ouyang et al. | An ultra-facile and label-free immunoassay strategy for detection of copper (II) utilizing chemiluminescence self-enhancement of Cu (II)-ethylenediaminetetraacetate chelate | |
| Divrikli et al. | Determination of some trace metals in water and sediment samples by flame atomic absorption spectrometry after coprecipitation with cerium (IV) hydroxide | |
| Shabani et al. | On-line solid phase extraction system using 1, 10-phenanthroline immobilized on surfactant coated alumina for the flame atomic absorption spectrometric determination of copper and cadmium | |
| CN110514491A (en) | The pre-treating method of organic pollutant in a kind of analysis urine sample | |
| Shao et al. | Preparation of molecularly imprinted hybrid monoliths for the selective detection of fluoroquinolones in infant formula powders | |
| Peralta et al. | Solid phase extraction using nylon membranes with fluorescence detection as a fast and sensitive method for amiloride and furosemide determination in urine samples | |
| CN104788560B (en) | Ciclosporin A immunogene, anti-Ciclosporin A specific antibody and Ciclosporin A detection reagent | |
| Jin et al. | Determination of bromadiolone in whole blood by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry | |
| He et al. | Facial synthesis of nickel (II)-immobilized carboxyl cotton chelator for purification of histidine-tagged proteins | |
| Muñiz-Bustamante et al. | Drugs of abuse in tap water from eight European countries: Determination by use of supramolecular solvents and tentative evaluation of risks to human health | |
| CN104447984A (en) | Docetaxel immunogen, anti-docetaxel specific antibody and docetaxel detection reagent | |
| CN110045053A (en) | A kind of QuEChERS pre-treating method of the analysis of the amphetamines suitable for blood | |
| CN109336773A (en) | A kind of preparation method and application of fluorescent optical sensor | |
| JP2013541009A (en) | Sample detection method | |
| Wang et al. | A polymeric solid-phase microextraction fiber for the detection of pharmaceuticals in water samples | |
| CN105175531A (en) | Hydroxyproline immunogen, specific antibody and detection reagent, and preparation methods thereof | |
| Tzanavaras et al. | Sequential injection method for the direct spectrophotometric determination of bismuth in pharmaceutical products | |
| Pu et al. | A novel deep eutectic solvent-based ultrasound-assisted dispersive liquid–liquid microextraction coupled with high-performance liquid chromatography for the determination of quinolones in environmental water samples | |
| CN104774256B (en) | Catecholamine immunogene, derivative and synthetic method, specific antibody and detection reagent and preparation method | |
| CN112858690B (en) | Urine albumin/urine creatinine composite quality control product and preparation method thereof | |
| CN110133154A (en) | The measuring method of perfluorochemical precursor substance in Atmospheric particulates sampling membrane | |
| CN117783332A (en) | Method for synchronously detecting multiple types of PPCPs in high-flux multiple matrixes | |
| Wei et al. | Preparation of MIL-101 (Cr)–NH2@ TAPB-DVA-COF based membrane solid-phase extraction for efficient enrichment and sensitive determination of trace aromatic disinfection by-products in juice drinks | |
| Nakaya et al. | Sensitive fluorimetric flow injection analysis for fluoride ion with a novel reagent, 2′, 7′-dichlorofluorescein di-tert-butyldimethylsilyl ether | |
| CN108008033A (en) | A kind of method for measuring water nitrite and nitrous acid content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191129 |