CN103175716B - The pre-treating method of a kind of nail/toenail toxicological analysis - Google Patents
The pre-treating method of a kind of nail/toenail toxicological analysis Download PDFInfo
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- CN103175716B CN103175716B CN201110429247.1A CN201110429247A CN103175716B CN 103175716 B CN103175716 B CN 103175716B CN 201110429247 A CN201110429247 A CN 201110429247A CN 103175716 B CN103175716 B CN 103175716B
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Abstract
The invention provides the pre-treating method of a kind of nail/toenail toxicological analysis, comprising: (1) is by nail/toenail to be tested and hit son and be together placed in grinding container, grinds under the grinding container of good seal being placed in the environment lower than-150 DEG C; (2) ground nail/toenail to be tested and extractant are mixed to form potpourri, form potpourri and utilize ultrasound wave to extract.The pre-treating method of nail provided by the invention/toenail toxicological analysis, freeze grinding and ultrasonic technology are applied in the pre-treatment to nail/toenail, the release efficiency of pretreatment process can be improved, and ensure the poisoning medicine stability of pretreatment process, avoid the impact of the chemical treatment methods such as traditional soda acid on the poisoning medicine of nail/toenail.
Description
Technical field
The present invention relates to a kind of pre-treating method to exhibit identification and analysis, particularly relate to a kind of pre-treating method be applied in nail/toenail qualification in the judicial identification of poison.
Background technology
The poisoning Pharmaceutical Analysis of nail/toenail is just becoming strong one of auxiliary other biological sample evidence, and is expected to play its strong effect in some in the past insoluble medicine thing (case) part of being involved in drug traffic.The cytotoxic drug be embedded in nail/toenail need be discharged for instrumental analysis through pretreatment process.
" referring to the present Research of (toe) first in drug abuse is analyzed and application forecast " that the people such as Chen Hang deliver on 03 phase in 2003 " Journal of Forensic Sciences " in a literary composition, enumerates the way of hydrolysis/digestion process in the conventional nail process comprising acid hydrolysis, alkali digestion and alcohol lixiviate.
Alkali digestion is by the thorough disorganize of thermokalite and eucaryotic cell structure, and can discharge the cytotoxic drug of embedding in it completely, high temperature and strong alkali environment can make unstable compound decompose.Acid-hydrolysis method energy open texture structure, but due to most of cytotoxic drug be alkalescence, the Protonation effect of sour environment can help to be embedded in in-house object and enter extract.There is the collagen level that a large amount of procollagen is formed in nail, procollagen structure meets acid can sex change and crosslinked deposition rapidly, maintains and solidifies form.Therefore, the nail transversal section hierarchical structure after acid hydrolysis is more obvious than untreated nail, more tight before surfaces externally and internally is also untreated.Form fine and close net after procollagen is cross-linked deposition and stop the release wherein embedding cytotoxic drug on the contrary.Methyl alcohol lixiviate relies on the swelling effect of nail tissue and the dissolubility of methyl alcohol to cytotoxic drug itself to extract, as Electronic Speculum finding: because swelling effect surfaces externally and internally all has certain cracking, the hierarchical structure of transversal section is also destroyed due to the power of swelling effect generation.But it is consuming time long, be difficult to the forensic science field being adapted to need quick obtaining result.
Comprehensive conventional disposal route at present, applies mechanically hair analysis pretreatment process mostly to the pretreatment process of nail/toenail analysis.Different due to hair and nail/toenail the Nomenclature Composition and Structure of Complexes aspect, simple method is applied mechanically can not the rationality of support method and applicability.Simultaneously, the pretreatment process of nail/toenail analysis adopts the classic methods such as acid, alkali treatment or organic solvent immersion to reach extraction effect mostly, it is many at different conditions respectively to there is impurity effect in these class methods, soda acid, heat-labile target compound are not suitable for, release, the shortcoming such as extraction efficiency is low.
Summary of the invention
The present invention is directed to Problems existing in the pre-treating method of existing inspection nail/toenail, a kind of full disposal route of brand-new nail/toenail toxicological analysis is provided, improvement can the micromechanism of nail/toenail, increased substantially release efficiency, avoiding the violent chemical methodes such as traditional soda acid may on the impact of the poisoning medicine of nail/toenail.
In order to realize above-mentioned object, the pre-treating method of a kind of nail/toenail toxicological analysis is provided, comprise following sequential steps: step 1: by nail/toenail to be tested with hit son and be together placed in grinding container, grind under the grinding container of good seal being placed in the environment lower than-150 DEG C; Step 2: ground nail/toenail to be tested and extractant are mixed to form potpourri, form potpourri and utilize ultrasound wave to extract.
In the above-mentioned pre-treating method provided, also comprise precooling treatment before wherein said grinding, the described precooling treatment time preferably controlled between 2 ~ 10 minutes.
In the above-mentioned pre-treating method provided, the time of hitting bulbec mill in wherein said process of lapping is 1 ~ 5 minute, and collision frequency is 3 ~ 15 s
-1.Process of lapping circulation carries out 2 ~ 5 times, preferred cycle 2 process of lapping.
In the above-mentioned pre-treating method provided, wherein said hyperacoustic frequency is 30 ~ 40KHz.
In the above-mentioned pre-treating method provided, during wherein said extraction, environment temperature is 10 ~ 30 DEG C.
In the above-mentioned pre-treating method provided, the time of wherein said extraction process is 1 ~ 2 hour.
In the above-mentioned pre-treating method provided, wherein said extractant is one or more potpourris in methyl alcohol, formic acid, ether, acetonitrile, ammonium acetate, ethyl acetate, chloroform, or the extraction solvent configured according to the actual requirements.Wherein, preferred extract is the potpourri that acetonitrile and ammonium acetate/formic acid form with the volume ratio of 70:30, and described ammonium acetate/formic acid is made up of 20mmol/L ammonium acetate and 0.1% formic acid.
The pre-treating method of nail provided by the invention/toenail toxicological analysis, freeze grinding and ultrasonic technology are applied in the pre-treatment to nail/toenail, the release efficiency of pretreatment process can be improved, and ensure the poisoning medicine stability of pretreatment process, avoid the impact of the chemical treatment methods such as traditional soda acid on the poisoning medicine of nail/toenail.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope diagram of nail structure after freeze grinding.
Fig. 2 is the nail structure scanning electron microscope diagram after ultrasound wave process.
Fig. 3 is the nail structure scanning electron microscope diagram after acid hydrolysis process.
Fig. 4 is the nail structure scanning electron microscope diagram after methyl alcohol immersion treatment.
Fig. 5 is the nail particle ultrasound wave scanning electron microscope diagram before treatment that scissors rubs.
Fig. 6 is the scanning electron microscope diagram after the nail particle ultrasound wave process of scissors rubbing.
Embodiment
The invention provides the pre-treating method of a kind of nail/toenail toxicological analysis, comprising: (1) is by nail/toenail to be tested and hit son and be together placed in grinding container, grinds under the grinding container of good seal being placed in the environment lower than-150 DEG C; (2) ground nail/toenail to be tested and extractant are mixed to form potpourri, form potpourri and utilize ultrasound wave to extract.
Freeze grinding is the sample preparation technology using special freeze grinding instrument to carry out, and its processing procedure is sample is loaded airtight grinding container to immerse liquid nitrogen, and take electromagnetism as power, drive is hit son and ground sample comminution under liquid nitrogen temperature environment.The more easy grinding because, sample becomes fragile at low temperatures, in the sample simultaneously under low temperature, contained composition information and biologically active are better retained.
Ultrasound wave process refers to the technology using ultrasonic washing instrument to process sample, produce the Mechanical Longitudinal Waves that frequency is greater than 20KHz by ultrasonic generator and transducer make the liquid flow in instrument tank, its huge energy produced can be used for several samples process.
Be described further the pre-treating method of nail provided by the invention/toenail toxicological analysis by the following examples, better to understand the content of the invention, but the content of embodiment does not limit the claimed scope of the invention.
sample collection
Because nail is identical with toenail material, structure and other character, therefore only using nail as checked object in the present embodiment.Nail to be collected from this laboratory without disease of nail, healthy volunteers without drug abuse history, uses clean steel nail nipper to get its daily nail, pruning garbage collection in the centrifuge tube of cleaning, lucifuge normal temperature storage.
the pre-treating method of sample toxicological analysis
The nail samples sodium dodecyl sulfate solution obtained is cleaned 3 times, and with deionized water concussion, acetone soln rinses.Following distinct methods is adopted to carry out analysis pre-treatment to cleaned nail respectively.And the nail relic (or residue) after following various method process and machinery are shredded with freezing ball milling after nail powder, to be put in fuming cupboard clean surface dry under normal temperature.Dried each sample is placed in spread on conducting resinl sample stage and fixes, and with metal spraying plated film instrument metal spraying, is placed in scanning electron microscope working bin, sets short operating distance as far as possible, observe and make respective record under high vacuum environment.
embodiment 1
Get a certain amount of nail to be tested and stainless steel according to the size of grinding pipe to hit son and insert in grinding pipe (nail consumption is between 5 ~ 50mg), and with supporting sealing of lid, the grinding pipe of good seal is placed in freeze grinding instrument, and adds liquid nitrogen.Precooling is after 3 ~ 5 minutes, and ball milling 2 minutes, the collision frequency of hitting son controls at 3 ~ 15s
-1between, and circular grinding process 2 ~ 3 times.
In the mixed extractant that the nail powder obtain grinding and 1.0mL are made up of acetonitrile, ammonium acetate and formic acid (in extract, the proportioning of each material is: acetonitrile: 20mmol/L ammonium acetate and 0.1% formic acid solution=70 30 v/v), under extract and nail potpourri are placed in the isoperibol of 20 ± 3 DEG C, be the ultrasound wave of 40KHz by frequency, ultrasonic process 1 hour.
control experiment 1
Nail machinery to be tested is shredded, is immersed in 1mL 10% sodium hydroxide solution, at 80 DEG C, be incubated 20 minutes.
control experiment 2
Nail machinery will be collected shred, be immersed in 0.1mol/L hydrochloric acid, at 45 DEG C, be incubated 18 hours.
control experiment 3
Nail machinery will be collected shred, be immersed in the HPLC level methyl alcohol of 2mL, at 40 DEG C, be incubated 24 hours.
control experiment 4
Nail machinery to be tested is shredded, be immersed in the mixed extractant that 1.0mL is made up of acetonitrile, ammonium acetate and formic acid (in extract, the proportioning of each material is: acetonitrile: 20mmol/L ammonium acetate and 0.1% formic acid solution=70 30 v/v), under extract and nail potpourri are placed in the isoperibol of 20 ± 3 DEG C, be the ultrasound wave of 40KHz by frequency, ultrasonic process 1 hour.
Observe the solution system obtained after above-mentioned each method process, find that in the nail samples solution system of embodiment 1, the disperse of visible white powder is in whole solution system, and nail powder is suspended in the solution, is long placed in slow sedimentation.The nail samples of control experiment 1 is cleared up rapidly in thermokalite, and whole solution is bright yellow, and double swerve solution has the similar glue that rocks to feel.In the nail samples solution system of control experiment 2 ~ 4, there is micro white bottom solution to transparent chip, whole liquid clear.
Nail samples after dry above-mentioned each method process, and by sem observation nail surface situation.Fig. 1 and Fig. 2 is scanning electron microscope diagram in embodiment 1 after freeze grinding before and after the process of nail mealy structure ultrasound wave (enlargement factor be respectively × 1200 and × 1200) respectively.As can be observed from Figure, the nail size of powder particles after freeze grinding is about 10 μm, and nail powder is spherical in shape.After ultrasound wave process, on microcosmic, still basic maintenance is spherical, is fragmented into less fragment or chondritic expansion vestige without powder.
The nail chips of control experiment 1, after alkali digestion, nail microstructure is totally disrupted, and generation colloidal solution system is sprawled after drying up and presents soil shape.Fig. 3 is the nail structure scanning electron microscope diagram after acid hydrolysis process, from left to right respectively illustrates the outside surface of nail, transversal section and inside surface, enlargement factor is respectively × 160, × 600 and × 150.Fig. 4 is the nail structure scanning electron microscope diagram after methyl alcohol immersion treatment, from left to right respectively illustrates the outside surface of nail, transversal section and inside surface, enlargement factor is respectively × 160, × 1200 and × 160.The nail chips of control experiment 2 and control experiment 3, after acid hydrolysis or alcohol are soaked, the outside surface of nail is coarse, and many places surface structure is dug.The wave structure of nail inside surface is destroyed or disappear, and the nail inside surface wherein after alcohol process ftractures as seen.After acid hydrolysis, the structure compared with normal nail of the intra-level formula of nail is more obvious and show slightly and compact, and the hierarchical structure after alcohol soaks is fuzzy is difficult to identification.Fig. 5 and Fig. 6 is the scanning electron microscope diagram before and after the scissors nail particle ultrasound wave process of rubbing respectively, enlargement factor is successively × and 98 and × 98.See from figure, the nail chips of contrast experiment 4 uses scissors mechanicalness to rub, and nail grain size is about diameter 1mm, and particle spherical after ultrasonic process launches in the shape that is crashed to pieces.
In embodiment 1, nail freeze grinding is become and is about 10 μm of spherical particle powder, the specific surface area of effective increase nail powder, more specific surface area means and more to contact with Extraction solvent, increase considerably the chance that the poisoning medicine of nail enters Extraction solvent, release efficiency is increased.Because nail is that protection finger tip preserves from the densification and micro-arcuate structure that exist, make its quality comparatively hard, be difficult to use common scissors to rub to enough granules.When using scissors mechanicalness to rub nail, every part of nail average cost rubs to about 1mm particle and very requires great effort for 10 minutes, and freeze grinding improves nail Specific Surface Area Measurement about 100 times under being about the time of spending same.In addition, machine grinding can ensure the even of grain size, and low temperature effectively can ensure the stability of nail endogenous toxic material medicine in processing procedure simultaneously.
The tissue space of nail can be torn formation one cavity by the Cavitation effect that ultrasound wave process produces, and this cavity can be filled by the liquid of surrounding or gas instantaneously with the vibration of surrounding medium.If what be filled is liquid, then because powerful attractive force causes liquid knockout nail groups surface of second order to produce a large amount of solid-liquid mass exchange.If what be filled is gas, larger tangential force can be produced by the bulking effect of gas and impel more tissue space to be opened.To relying on dead cell and the stacked nail structure of keratic accumulation force, the larger cut power that Cavitation effect and mechanical effect produce, being enough to tear tissue space and causing particle to be crashed to pieces the situation of expansion.The surface area that tissue space is torn and increases and Cavitation effect, make increasing of solid-liquid mass exchange release efficiency.
The cytotoxic drug molecular motion being embedded in nail inside is accelerated by the thermal effect that ultrasound wave process brings, be conducive to being dissolved into Extraction solvent, but easily cause the water temperature over-high in ultrasonic tank, thermally sensitive cytotoxic drug can produce destruction, affects the low temperature advantage that freeze grinding brings simultaneously.Therefore the environment temperature of ultrasound wave process controls under the isoperibol of 20 ± 3 DEG C in the present embodiment 1.
In sum, freeze grinding is combined with ultrasonic technology in the pre-treatment of nail sample by the present invention, compared with traditional pre-treating method, improves the micromechanism of nail, substantially increase release efficiency, avoiding the violent chemical methodes such as soda acid may on the impact of the poisoning medicine of nail.Whole pretreatment process controls certain temperature simultaneously, can ensure the stability of cytotoxic drug.Freeze grinding combining ultrasonic wavefront treatment technology is more suitable for the needs of the poisoning Pharmaceutical Analysis of nail.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (7)
1. a pre-treating method for nail/toenail toxicological analysis, is characterized in that, comprises following sequential steps:
Step 1: by nail/toenail to be tested with hit son and be together placed in grinding container, grinds under the grinding container of good seal being placed in the environment lower than-150 DEG C;
Step 2: ground nail/toenail to be tested and extractant are mixed to form potpourri, form potpourri and utilize ultrasound wave to extract;
Wherein, the potpourri that extract forms with the volume ratio of 70:30 for acetonitrile and ammonium acetate/formic acid, described ammonium acetate/formic acid is made up of 20mmol/L ammonium acetate and 0.1% formic acid.
2. pre-treating method according to claim 1, it is characterized in that, also comprise precooling treatment before described grinding, the described precooling treatment time is 2 ~ 10 minutes.
3. pre-treating method according to claim 1, is characterized in that, the time of hitting bulbec mill in described process of lapping is 1 ~ 5 minute, and collision frequency is 3 ~ 15s
-1.
4. pre-treating method according to claim 3, is characterized in that, described process of lapping circulation carries out 2 ~ 5 times.
5. pre-treating method according to claim 1, it is characterized in that, described hyperacoustic frequency is 30 ~ 50KHz.
6. pre-treating method according to claim 1, it is characterized in that, during described extraction, environment temperature is 10 ~ 30 DEG C.
7. pre-treating method according to claim 1, is characterized in that, the time of described extraction process is 1 ~ 2 hour.
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| CN105571918B (en) * | 2016-01-18 | 2018-04-10 | 中国农业科学院茶叶研究所 | A kind of sample-pretreating method for the detection of bioactive substance accurate quantification |
| CN106324149B (en) * | 2016-11-18 | 2019-02-19 | 司法部司法鉴定科学技术研究所 | A kind of method of the just special careless main component of quick detection |
| CN110179500A (en) * | 2019-06-26 | 2019-08-30 | 赵绪龙 | A kind of illicit drugs inspection sampler, sampling system and sampling method |
| CN112198315B (en) * | 2020-09-11 | 2023-10-27 | 杭州奥泰生物技术股份有限公司 | Method for collecting and detecting abused drugs in nail or toenail sample |
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Inventor after: Shen Min Inventor after: Chen Hang Inventor after: Xiang Ping Inventor after: Sun Qiran Inventor before: Chen Hang Inventor before: Xiang Ping Inventor before: Sun Qiran Inventor before: Shen Min |
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Address after: No.1347, Guangfu West Road, Putuo District, Shanghai 200061 Patentee after: ACADEMY OF FORENSIC SCIENCE Address before: 200063 Guangfu West Road 1347, Putuo District, Shanghai Patentee before: INSTITUTE OF FORENSIC SCIENCE, MINISTRY OF JUSTICE PRC |