CN106596817A

CN106596817A - Method for determining content of paraquat in blood with gas chromatography-mass spectrometry

Info

Publication number: CN106596817A
Application number: CN201510668324.7A
Authority: CN
Inventors: 洪霞; 张淑雅; 刘静
Original assignee: Nanjing Yite Biological Technology Co Ltd
Current assignee: Nanjing Yite Biological Technology Co Ltd
Priority date: 2015-10-17
Filing date: 2015-10-17
Publication date: 2017-04-26

Abstract

The invention discloses a method for determining the content of paraquat in blood with a gas chromatography-mass spectrometry. The method comprises a sample treatment step and an assay determination step, in the assay determination step, the gas phase condition of a gas chromatograph is set as follows: an HP-5MS (30 m*250 mu m, 0.25 mu m) capillary chromatographic column is adopted, the column box temperature is kept at 50 DEG C for 1 min, increased to 200 DEG C at 10 DEG C/min, increased to 250 DEG C at 40 DEG C/min and kept at 260 DEG C for 4 min; the interfere temperature of gas chromatography and mass spectrometry is 280 DEG C. The result detection data of the paraquat in blood are accurate, and the method meets the national requirement and is time-saving and efficient. A qualitative and quantitative method that anticoagulant of a patient is detected through mass spectrometry full scanning after simple pretreatment is established, and the method detection limit is 0.2 mg/L; the detection result of the same blood is relatively stable, and the relative standard deviation is smaller than or equal to 10%. In conclusion, the method is simple and quick to operate, the reagent cost is low, and the assay result is reliable.

Description

Using gas chromatogram - The method of the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in mass spectrometric determination blood

Technical field

The present invention relates to a kind of method of the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in employing Gas Chromatography-Mass Spectrometry blood, belongs to clinical economics field.

Background technology

N,N'-dimethyl-.gamma..gamma.'-dipyridylium (paraquat), another name Gramoxone, Aerial gramoxone, prairie fire, hundred, Paraquat etc., chemical name 1,1- dimethyl -4,4- bipyridine cation salt is organic heterocyclic class herbicide.Wherein it is in the great majority with dichloro salt dissolving again.Sterling is white crystal, is highly soluble in water, is slightly soluble in the alcohols of low-molecular-weight, stable in acid, neutral solution, is easily decomposed in alkaline solution.It is orally the main path of poisoning, poisoning case fatality rate is high.N,N'-dimethyl-.gamma..gamma.'-dipyridylium is 5%～15% in gastrointestinal absorbance, is enough to commercially available mL～15 mL of 20% N,N'-dimethyl-.gamma..gamma.'-dipyridylium water preparation 5 by oral administration lethal.At present the main measuring methods of N,N'-dimethyl-.gamma..gamma.'-dipyridylium are spectrophotography, high performance liquid chromatography, but its suitability, accuracy are slightly inadequate.Laboratory is quick, accurately ascertain poisoning material, just can preferably give treatment to patient for clinician and provide safeguard.Meanwhile, the blood of patient is easier to be obtained in the very first time than urine.Intend herein taking the lead in setting up blood samples of patients Jing after simple pre-treatment with the method for quick of gas chromatograph-mass spectrometer (GC-MS) sample introduction analysis.

The content of the invention

It is an object of the invention to deficiency of the prior art, the method that a kind of N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in employing Gas Chromatography-Mass Spectrometry blood is provided, with detection limit it is low, the response rate is high, favorable reproducibility, low cost, it is simple to operate the features such as, the method provides reference for the detection of the N,N'-dimethyl-.gamma..gamma.'-dipyridylium in blood.

To solve above-mentioned technical problem, the technical solution used in the present invention is：A kind of method that employing gas chromatography determines the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in blood, including the analysis determination step of sample, its innovation is：The gas phase condition of gas chromatograph is set to adopt capillary chromatographic column for HP-5MS (30 m × 250 μm, 0.25 μm) in described analysis determination step；Post case temperature:From 50 DEG C, 1min is kept, with 10 DEG C/min 200 DEG C are warming up to, then 40 DEG C/min rises to 250 DEG C, runs afterwards to 260 DEG C, keeps 4 min;Gas chromatogram and mass spectrographic interface temperature are 280 DEG C

Further, the sample analysis step includes sample treatment, the preparation of standard curve and determines and sample determination.

Further, the sample treatment is processed according to national standard.

Further, the preparation of the standard curve and measure are prepared standard curve according to national standard.

Further, the sample determination is the blank of the serial operating condition determination sample of bioassay standard and sample, the sample for measuring and the peak height of blank sample or peak area value, and by standard curve or regression equation the concentration of institute's test sample product is obtained.

Beneficial effects of the present invention：This employing gas chromatography of the present invention, is HP-5MS (30 from capillary chromatographic column M × 250 μm, 0.25 μm)；Post case temperature:From 50 DEG C, 1min is kept, with 10 DEG C/min 200 DEG C are warming up to, then 40 DEG C/min rises to 250 DEG C, runs afterwards to 260 DEG C, keeps 4 min；Gas phase color

Spectrum and mass spectrographic interface temperature are 280 DEG C.N,N'-dimethyl-.gamma..gamma.'-dipyridylium result detection data in detection blood accurately, meets national requirements, time-saving and efficiency.It is 80%～90% from the gas Chromatographic Determination response rate, concentration limit is 0.2 mg/L, can meet the detection requirement to the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in blood.

Description of the drawings

Fig. 1 is mark-on blood sample acetonitrile extraction total ions chromatogram.

Specific embodiment

With reference to being embodied as elaborating technical scheme.

Using the method for the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in Gas Chromatography-Mass Spectrometry blood：

1st, instrument：

Agilent gas chromatograph-mass spectrometer 5975B (band automatic sampler, the U.S. Agilent companies)

Table-type high-speed refrigerated centrifuge (CT15RT, Shanghai Tian Mei companies)

High speed homogeneous dispersion machine (German IKA companies)

Nitrogen evaporator

Ultrasonic cleaner

Vortex vortex mixer

Various glass drying ovens etc.

2nd, reagent：

N,N'-dimethyl-.gamma..gamma.'-dipyridylium (two villaumites, purity >=98.8% )

Acetonitrile (chromatographically pure)

Methanol (chromatographically pure)

Ultra-pure water.

3rd, analysis is determined：

Sample pretreatment

Take 1 mL whole bloods to be added dropwise in the 15 mL centrifuge tubes containing 4 mL acetonitriles and 50 μ L formic acid, homogenizer stirs 1 min, 6 DEG C of inner chamber, 15 000 r/min is centrifuged 6 min, the mL of supernatant 2.5 is taken in 5 mL teat glasses, nitrogen is blown to do in 60 DEG C of water-baths, with 0.500 mL acetonitrile constant volumes, is vortexed 30 s, 20 s of ultrasound, 30 s are vortexed again, 15 000 r/min centrifugations 2 Min, moves appropriate supernatant machine test sample on 400 μ L internal lining pipes.

Qualitative and quantitative analysis

Compared with the usual retention time of gas chromatography, add full scan acquisition mass spectrum and compare with the standard spectrogram in NIST storehouses, realized accurately qualitative.

N,N'-dimethyl-.gamma..gamma.'-dipyridylium standard is added to make working curve, the residual quantity being converted in blood using blank blood:Concentration point includes 1mg /L、5 mg /L、10 mg / L、20 mg / L、50 mg / L.It is the true residual concentration in accurate evaluation blood samples of patients in terms of total Ion response peak area of N,N'-dimethyl-.gamma..gamma.'-dipyridylium, actual sample concentration is with close concentration point single-point calibration.

4th, method and result

The optimization of detecting instrument condition

A, B cleanout fluid on automatic sampler is respectively acetonitrile, pure water.Before sample introduction, sample introduction needle 3 times is first cleaned with A;After sample introduction, sample introduction needle 3 times is first cleaned with A, then cleaned with B 3 times.The memory effect of sample introduction needle can so be eliminated.N,N'-dimethyl-.gamma..gamma.'-dipyridylium polarity is bigger than normal, and the optimization of injection port is mainly to maintain the screening of bushing pipe inertia and gasification temperature.Can buy and deactivate bushing pipe, or by this routine processes bushing pipe:Remove glass fibre in bushing pipe, first cleaned with suds immersion ultrasound, again with methanol, ethyl acetate, normal hexane are rinsed successively, then dichloro-dimethyl silicon (silicon) alkane-toluene (1: 9, V/V) the h of solution soaking 2, then cleaned with normal hexane, ethyl acetate, methanol successively, then a night is toasted under 300 DEG C of high temperature, be placed in after taking out in exsiccator and preserve;Few glass fibre is filled in used time bushing pipe.Our experiments show that, the sample introduction in 260 DEG C of gasification temperature, collection of illustrative plates peak area is stable and relatively large.

This laboratory low-bleed, resistant to elevated temperatures gas chromatogram detached dowel only have HP-5MS, are then performed an analysis post with it, and total ion current collection of illustrative plates shows that the peak width of N,N'-dimethyl-.gamma..gamma.'-dipyridylium is larger, hangover after sample introduction, the min of peak base portion span 1.4, as shown in Figure 1.By first meeting the HP-INNOWax that one section of 1.5 m, internal diameter are all 0.25 mm in injection port, object peak type is improved, the min of peak base portion span 0.4.Connected with glass adaptor in the middle of 2 pillars so that object retention time slightly extends, peak shape good, peak height increases (Fig. 1).

Blood mark-on test evaluation

Carry out the parallel test of 3 same levels with the blood of 11 people respectively, mark-on is horizontally disposed to be shown in Table 1, and as a result statistics is shown in Table 1, table 2.The computing formula of the response rate is:

I.e. plus standard specimen determine liquid object response value divided by N,N'-dimethyl-.gamma..gamma.'-dipyridylium standard solution (acetonitrile as solvents) during theoretical concentration object response value.As a result show, during same blood mark-on same level, response rate stability is preferable, relative standard deviation≤10%; The same level standard addition response rate in different human bloods is slightly different, response rate distribution fluctuation slightly larger, response rate Distribution value Relatively centralized when high-level during low-level;Varying level standard addition, in terms of 11 blood sample totality response rate, the response rate is presented trend and is:Gradually increase with adding scalar to increase the response rate, and the response rate value relative standard deviation between different human bloods is little.Solid content confrontation N,N'-dimethyl-.gamma..gamma.'-dipyridylium in blood has adsorptivity, and consulting literatures are without the related theory for determining.This research speculates that N,N'-dimethyl-.gamma..gamma.'-dipyridylium possesses amine substance analog structure, possesses actively through the ability of the cell membrane for having amine substance movement system, it should be the not high main cause of blood sample low-level plus the timestamp response rate.

Table 1

Table 2

Claims

1. a kind of method that employing adopts the N,N'-dimethyl-.gamma..gamma.'-dipyridylium content in Gas Chromatography-Mass Spectrometry blood, it is characterised in that：The gas phase condition of gas chromatograph is set to adopt capillary chromatographic column for HP-5MS (30 m × 250 μm, 0.25 μm) in described analysis determination step； Post case temperature:From 50 DEG C, 1min is kept, with 10 DEG C/min 200 DEG C are warming up to, then 40 DEG C/min rises to 250 DEG C, runs afterwards to 260 DEG C, keeps 4 min;Gas chromatogram and mass spectrographic interface temperature are 280 DEG C.

2. the method for the N,N'-dimethyl-.gamma..gamma.'-dipyridylium in employing Gas Chromatography-Mass Spectrometry Herba Marsileae Quadrifoliae blood according to claim 1, it is characterised in that：The analysis determination step includes sample treatment, the preparation of standard curve and determines and sample determination.

3. the method for the N,N'-dimethyl-.gamma..gamma.'-dipyridylium in employing Gas Chromatography-Mass Spectrometry blood according to claim 2, it is characterised in that：The sample treatment is processed according to national standard.

4. the method for the N,N'-dimethyl-.gamma..gamma.'-dipyridylium in employing Gas Chromatography-Mass Spectrometry blood according to claim 2, it is characterised in that：The preparation of the standard curve and measure are prepared standard curve according to national standard.

5. the method for the N,N'-dimethyl-.gamma..gamma.'-dipyridylium in employing Gas Chromatography-Mass Spectrometry blood according to claim 2, it is characterised in that：The sample determination is the blank of the operating condition determination sample of bioassay standard series and sample, the sample for measuring and the peak height of blank sample or peak area value, and by standard curve or regression equation the concentration of institute's test sample product is obtained.