CN104007189B - A kind of test kit detecting perchlorate in food and assay method thereof - Google Patents

Abstract

The invention provides and a kind of detect the test kit of perchlorate in food, including perchlorate standard solution, 18O labelling perchlorate's solution, acetic acid solution, acetonitrile, ammonium acetate solution and Solid-Phase Extraction C18 post.Present invention also offers and a kind of detect the method for perchlorate in food: (1) sample extraction；(2) purify；(3) use anti-phase high separating power, the analysis method of high s/n ratio carries out accurate qualitative and quantitative.This test kit uses acetic acid solution as sample extraction solvent；Use Solid-Phase Extraction column purification, there is the preferable response rate and clean-up effect；Employing Soft ionization techniques is measured, inner mark method ration, reaches the effect of high selectivity, high sensitivity, applied range, has extremely strong specificity, is widely used in the mensuration of perchlorate in food.

Description

A kind of test kit detecting perchlorate in food and assay method thereof

Technical field

The present invention relates to the physico-chemical analysis field of chemical substance, be specifically related to a kind of detect perchlorate in food Test kit and assay method thereof.

Background technology

Perchlorate refers to containing perchlorate ([ClO₄]^-) salt, existing naturally occur, also have synthetic.High chlorine Acid ion has the electric charge similar to iodide ion and ionic radius, and the transport protein to iodide ion has higher more affine than iodine Power, it is thus possible to the absorption of suppression iodine, weakens thyroid function, causes mankind's metabolic function to wad a quilt with cotton disorderly, affect fetus and baby The neural normal growth of youngster and growth, the subthyroidism that anemia of pregnant woman is serious can cause the cretinism of fetus.Due to height The high of chlorate dissolves low absorbability, irrigates easily by water source, plant absorption, accumulation, is ultimately transferred to the mankind.Its pollution is asked Topic has caused the very big attention of environmentalist and hygienist.The external research worker that has is to drinking water, water source, milk, vegetables Perchlorate content in dish and supplementary etc. is investigated, China also have research worker to Beijing's drinking water and Water source perchlorate contaminated situation is detected.In April, 2009, American Centers for Disease Control and Prevention research worker report baby Youngster's milk powder, by the problem of perchlorate contaminated, causes people and pays close attention to greatly.Therefore, set up in rapid and accurate determination food The method of perchlorate content is extremely the most urgent.

At present to perchlorate assay method can briefly be divided into the chromatography of ions, surface enhanced raman spectroscopy Method, chromatography of ions-mass spectrometric hyphenated technique and High Performance Liquid Chromatography/Mass Spectrometry/mass spectrography etc..Although chromatography of ions-conductance detection Can be used to measure the perchlorate of underwater trace, but the retention time of chromatograph is not the feature of unique ion, also Further experiment is needed to determine completely.And electric conductivity detector lacks selectivity, when sample contains other ions Time, easily there is false positive in the method.Surface enhanced raman spectroscopy method is to realize the former of perchlorate in water environment with a kind of Position, real-time and highly sensitive detection and the analysis method that grows up.But the repeatability of this technology is poor causes quantitative analysis Difficulty.Few about the liquid chromatography-mass spectrography of perchlorate in food/Mass Spectrometry detection method document report both at home and abroad, inspection Survey object be mainly single bottled water, milk or milk powder substrate, there is not yet can detect include bottled water, milk, milk powder, Liquid chromatography-mass spectrography/the Mass Spectrometry detection method of perchlorate and system in the food such as corn, aquatic products, animal tissue.

Summary of the invention

It is an object of the invention to the defect for above-mentioned prior art, it is provided that a kind of detect perchlorate in food Test kit and assay method thereof, the method is applicable to the foods such as bottled water, fruit, milk, milk powder, corn, aquatic products, animal tissue Product, and methodological science is reasonable, highly sensitive, selectivity is high, applied range and have extremely strong specificity, fully meets domestic The needs of outer detection limitation.

To achieve these goals, technical scheme is as follows:

The a kind of of the present invention detects the test kit of perchlorate in food, including perchlorate standard solution,¹⁸O marks Note perchlorate's solution, acetic acid solution, acetonitrile, ammonium acetate solution and Solid-Phase Extraction C₁₈Post.

Preferably, the CAS No of described perchlorate standard solution is 7601-89-0, and purity is 100%, and concentration is 1000mg/L。

Preferably, described¹⁸O labelling perchlorate's solution is containing Cl¹⁸O₄ ^-Solution, described¹⁸O labelling perchlorate's solution Purity be 90%, concentration be 100 μ g/L.

Preferably, described acetic acid solution is made up of acetic acid and water, and described acetic acid is 1:100 with the volume ratio of described water.

Preferably, described ammonium acetate solution comprises ammonium acetate and water, and the molar concentration of described ammonium acetate solution is 0.1mol/ L.Weigh 7.708g ammonium acetate, be settled to 1000mL with water and get final product or the solution of identical proportioning preparation.

Preferably, described Solid-Phase Extraction C₁₈The model of post is waters, and amount of filler is 200mg, 3mL or suitable person.

Present invention also offers a kind of high separating power, high sensitivity, high selectivity, use mass number to carry out qualitative and fixed Quantitative analysis method measures the perchlorate in food, carries out as follows:

(1) sample preparation and preservation；

(2) described in preparation-obtained sample in step (1) is added¹⁸O labelling perchlorate's solution,

If described food is bottled water, the most directly carries out liquid chromatography-mass spectrography/mass spectrograph and measure；

If described food is non-bottled water, then adds described acetic acid solution and extract；

(3) purify: draw the extraction supernatant obtained in 1.0mL step (2) and cross Solid-Phase Extraction C₁₈Post, discards effluent, Draw 1.5mL supernatant and cross Solid-Phase Extraction C₁₈Post, collects effluent, vortex oscillation 30s, surveys for liquid chromatography-mass spectrography/mass spectrograph Fixed；

(4) detection:

Liquid phase chromatogram condition:

A) chromatographic column: IC-Pak^TMAnion HR post, 6 μm, 4.6mm × 75mm (internal diameter) or quite person；

B) flowing phase: acetonitrile+0.1mol/L ammonium acetate solution=(60+40, V₁/V₂)；

C) flow velocity: 700 μ L/min；

D) column temperature: 40 DEG C；

E) sample size: 10 μ L；

Mass Spectrometry Conditions:

A) ion source: electric spray ion source (ESI)；

B) scan mode: anion scans；

C) monitoring pattern: multiple-reaction monitoring (MRM)；

D) gas curtain gas (CUR): 209kPa；

E) atomization gas (GS1): 414kPa；

F) auxiliary adds steam (GS2): 483kPa；

G) collision gas (CAD): high；

H) electron spray voltage (IS) :-1000V；

I) ion source temperature (TEM): 550 DEG C；

J) monitor ion pair, collision energy, go a bunch voltage, acquisition time and retention time see table:

(5) qualitative, quantitative measures and judges.

Preferably, the qualitative judgement described in step (5) is carried out as follows: the maximum allowable offset of relative ion abundance Less than the scope of following provisions, then can determine whether sample exists corresponding determinand:

If relative ion abundance is ＞ 50%, then the relative deviation allowed is ± 20%；

If relative ion abundance is ＞ 20%～50%, then the relative deviation allowed is ± 25%；

If relative ion abundance is ＞ 10%～20%, then the relative deviation allowed is ± 30%；

If relative ion abundance is≤10%, then the relative deviation allowed is ± 50%.

Preferably, the rational judgment described in step (5) is with mass spectrometric data datatron or by formula (1) calculating sample Chinese medicine Content, result of calculation need to deduct blank value:

$X_i=\frac{c\×c_i\×A\×A_{\mathrm{si}}\×V}{c_{\mathrm{si}}\×A_i\×A_s\×m}\×\frac{1000}{1000}...\left(1\right)$

In formula:

X_iThe content of target compound in sample, unit is ng/kg (μ g/kg)；

The concentration of analysans in c standard working solution, unit is nanograms per milliliter (ng/mL)；

c_iThe concentration of internal standard substance in sample, unit is nanograms per milliliter (ng/mL)；

The peak area of analysans in A sample；

A_siThe peak area of internal standard substance in standard working solution；

V sample constant volume, unit is milliliter (mL)；

c_siThe concentration of internal standard substance in standard working solution, unit is nanograms per milliliter (ng/mL)；

A_iThe peak area of internal standard substance in sample solution；

A_sThe peak area of analysans in standard working solution；

The sample weighting amount of m sample, unit is gram (g).

Food described in said method includes in bottled water, fruit, milk, milk powder, corn, aquatic products and animal tissue One or more.

Compared with prior art, the invention has the beneficial effects as follows:

The present invention uses the purification style of Solid-Phase Extraction to the perchlorate separating in food.¹⁸O labelling perchlorate Ion dilution mass spectrography refers to target compound¹⁸O labelling adds in testing sample, is sufficiently mixed with target compound, logical Cross add the amount of labelling and the target compound of Mass Spectrometer Method and¹⁸The abundance ratio of O labelling calculates target compound content Method.Target compound and¹⁸O, in sample carries out a series of pre-treatment operating process, even if losing, also will not Change¹⁸O labelling forms, and avoids the measurement error thus brought.

Method uses acetic acid solution to extract, C₁₈Solid-Phase Extraction column purification, establishes high performance liquid chromatography-tandem mass method and surveys Determine the perchlorate in food.Introduce during analysis¹⁸O labelling perchlorate (Cl¹⁸O₄ ^-) as label, for dividing Analysis overall process causes object loss give to correct completely, improve the precision of method.This experimental applications is in food The detection of perchlorate and confirmation, have preferable sensitivity and precision.

The inventive method is high be applicable to the food such as bottled water, fruit, milk, milk powder, corn, aquatic products, animal tissue The mensuration of chloranion content, and this method is scientific and reasonable, highly sensitive, selectivity is high, applied range and have extremely strong Specificity, fully meet the needs of detection limitation both at home and abroad.

Accompanying drawing explanation

Fig. 1 be perchlorate standard solution and¹⁸The multiple-reaction monitoring chromatogram of O labelling perchlorate, wherein, (A) (B) is the multiple-reaction monitoring chromatogram of perchlorate standard solution, and (C) is¹⁸O labelling perchlorate how anti- Chromatogram should be monitored；

Fig. 2 is the perchlorate MRM figure separated through synergi4 μM of AX-RP80A post of phenomenex；

Fig. 3 is the perchlorate MRM figure separated through Waters IC-PakTM Anion HR post；

Fig. 4 be the ion concentration of ammonium bicarbonate soln be the MRM color of perchlorate standard solution during 5.0mmol/L Spectrogram；

Fig. 5 be the ion concentration of ammonium bicarbonate soln be the MRM color of perchlorate standard solution during 10mmol/L Spectrogram；

Fig. 6 be the ion concentration of ammonium bicarbonate soln be the MRM color of perchlorate standard solution during 20mmol/L Spectrogram；

Fig. 7 be the ion concentration of ammonium bicarbonate soln be the MRM color of perchlorate standard solution during 25mmol/L Spectrogram；

Fig. 8 be the ion concentration of ammonium bicarbonate soln be the MRM color of perchlorate standard solution during 30mmol/L Spectrogram；

When Fig. 9 is PH > 10, the MRM chromatogram of perchlorate standard solution；

Figure 10 be PH < when 10, the MRM chromatogram of perchlorate standard solution；

Figure 11 is the MRM chromatogram that sample standard is added；

Figure 12 be the ion concentration of ammonium acetate solution be the MRM chromatograph of perchlorate standard solution during 50mmol/L Figure；

Figure 13 be the ion concentration of ammonium acetate solution be the MRM color of perchlorate standard solution during 100mmol/L Spectrogram；

Figure 14 be the ion concentration of ammonium acetate solution be the MRM color of perchlorate standard solution during 200mmol/L Spectrogram；

Figure 15 is the ammonium acetate solution-acetonitrile system MRM chromatogram when volume ratio 5+5；

Figure 16 is the ammonium acetate solution-acetonitrile system MRM chromatogram when volume ratio 4+6；

Figure 17 is that ammonium acetate solution-acetonitrile system is at the MRM chromatogram that volume ratio is during 3+7

Figure 18 is that ammonium acetate solution-acetonitrile system is at the MRM chromatogram that volume ratio is during 2+8

Figure 19 is multiple-reaction monitoring (MRM) chromatogram of perchlorate standard solution (1.0 μ g/L)；

Figure 20 is the first mass spectrometric figure of perchlorate；

Figure 21 is¹⁸O labelling perchlorate ([³⁵Cl¹⁸O₄]^-) first mass spectrometric figure；

Figure 22 be perchlorate ([³⁵ClO₄]^-) second order ms figure；

Figure 23 be perchlorate ([³⁷ClO₄]^-) second order ms figure；

Figure 24 is¹⁸O labelling perchlorate ([³⁵Cl¹⁸O₄]^-) second order ms figure；

Figure 25 is perchlorate standard solution linear relationship chart；

Figure 26 is the MRM chromatogram of bottled water host solvents；

Figure 27 is the MRM chromatogram of bottled water substrate；

Figure 28 is the MRM chromatogram that bottled water extraction standard adds (pitch-based sphere 0.5 μ g/kg)；

Figure 29 is the MRM chromatogram of Eugenia javanica Lam host solvents；

Figure 30 is the MRM chromatogram of Eugenia javanica Lam substrate；

Figure 31 is the MRM chromatogram that Eugenia javanica Lam extraction standard adds (pitch-based sphere 1.0 μ g/kg)；

Figure 32 is the MRM chromatogram of milk substrate solvent；

Figure 33 is the MRM chromatogram of milk substrate；

Figure 34 is the MRM chromatogram that milk substrate standard adds (pitch-based sphere 1.0 μ g/kg)；

Figure 35 is the MRM chromatogram of milk powder host solvents；

Figure 36 is the MRM chromatogram of milk powder substrate；

Figure 37 is the MRM chromatogram that milk powder extraction standard adds (pitch-based sphere 3.0 μ g/kg)；

Figure 38 is the MRM chromatogram of rice substrate；

Figure 39 is the MRM chromatogram that rice extraction standard adds (pitch-based sphere 3.0 μ g/kg)；

Figure 40 is the MRM chromatogram of flesh of fish substrate；

Figure 41 adds the MRM chromatogram of (pitch-based sphere 3.0 μ g/kg) for flesh of fish extraction standard；

Figure 42 is the MRM chromatogram of Hepar Gallus domesticus substrate；

Figure 43 is the MRM chromatogram that Hepar Gallus domesticus extraction standard adds (pitch-based sphere 3.0 μ g/kg).

Detailed description of the invention

The invention will be further described with specific embodiment below in conjunction with the accompanying drawings, but not as a limitation of the invention.

In embodiment, all reagent are chromatographically pure, and water is to meet the one-level water that GB/T6682 specifies, utensil used is both needed to Soaking 8 hours with 30% salpeter solution, deionized water is rinsed well repeatedly, standby after drying.

1, reagent and material:

(1) ammonium acetate, top grade is pure.

(2) perchlorate standard solution (sodium perchlorate): CAS No:7601-89-0, purity: 100%, dense Degree 1000mg/L.

(3)¹⁸O labelling perchlorate solution (perchloric acid, sodium salt,¹⁸O₄): purity: 90%, Concentration 100 μ g/mL.

(4) Standard Stock solutions: 10mg/L.Accurately draw appropriate perchlorate standard solution, be placed in 100mL volumetric flask In, it is settled to scale with water.This solution is in 4 DEG C of preservations.¹⁸O labelling perchlorate's stock solution: 10mg/L.Accurately draw appropriate 's¹⁸O labelling perchlorate's standard solution, is placed in 10mL volumetric flask, is settled to scale with water.This solution is in 4 DEG C of preservations.Standard Working solution: draw a certain amount of standard use solution and¹⁸O labelling perchlorate uses solution, is configured to series concentration with water Standard working solution, concentration in the range of 0～10 μ g/L,¹⁸O labelling perchlorate's concentration is 2.0ng/mL, and the same day prepares.

(5) acetic acid solution: pipette 10.0mL acetic acid in 1L volumetric flask, add water to scale, shake up standby.

(6) 0.1mol/L ammonium acetate solution: weigh 7.708g ammonium acetate, be settled to 1000mL with water.

(7) Solid-Phase Extraction C₁₈Post: 200mg, 3mL.With front successively with 3mL acetonitrile, the activation of 3mL acetic acid solution, keep pillar Moistening.

2, instrument and equipment

Tissue pulverizer, homogenizer, horizontal oscillator tube, turbula shaker, solid-phase extraction device.

High Performance Liquid Chromatography/Mass Spectrometry/mass spectrograph: distribution esi ion source (ESI).

Analytical balance: sensibility reciprocal is 0.01g.

High speed centrifuge: maximum (top) speed 12000r/min.

Tool plug polypropylene centrifuge tube: 50mL.

Liquid-transfering gun: 10 μ L～100 μ L, 100 μ L～1000 μ L.

Embodiment 1: a kind of of the present invention detects the test kit of perchlorate in food

The present embodiment a kind of detect the test kit of perchlorate in food include perchlorate standard solution,¹⁸O marks Note perchlorate's solution, acetic acid solution, acetonitrile, ammonium acetate solution and Solid-Phase Extraction C₁₈Post.The CAS of perchlorate standard solution No:7601-89-0, purity: 100%, concentration 1000mg/L.¹⁸O labelling perchlorate's solution is concentration: 100 μ g/L, purity: 90% Solution, its preparation can method as described in mentioned reagent and material part be prepared.Described¹⁸O labelling perchlorate's solution is for containing (Cl¹⁸O₄-) solution.Described acetic acid solution is made up of acetic acid and water, and the described volume ratio for acetic acid Yu described-water is 1:100.Institute State Solid-Phase Extraction C₁₈Post be amount of filler be 200mg, 3mL.

Embodiment 2: with the multiple-reaction monitoring experiment of the test kit detection perchlorate of the present invention

By following condition, perchlorate is carried out liquid chromatography-mass spectrography/mass spectrum multiple-reaction monitoring to test.Perchlorate Standard solution and¹⁸O labelling perchlorate's solution is use solution, the concrete visible mentioned reagent of its configuration and material partial content Described.

Liquid phase chromatogram condition:

A) chromatographic column: IC-Pak^TMAnion HR post, 6 μm, 4.6mm × 75mm (internal diameter) or quite person；

B) flowing phase: acetonitrile+0.1mol/L ammonium acetate solution=(60+40, V₁/V₂)；

C) flow velocity: 700 μ L/min；

D) column temperature: 40 DEG C；

E) sample size: 10 μ L；

Mass Spectrometry Conditions:

A) ion source: electric spray ion source (ESI)；

B) scan mode: anion scans；

C) monitoring pattern: multiple-reaction monitoring (MRM)；

D) gas curtain gas (CUR): 209kPa；

E) atomization gas (GS1): 414kPa；

F) auxiliary adds steam (GS2): 483kPa；

G) collision gas (CAD): high；

H) electron spray voltage (IS) :-1000V；

I) ion source temperature (TEM): 550 DEG C；

J) monitor ion pair, collision energy, go a bunch voltage, acquisition time and retention time see table:

Under above-mentioned chromatograph, Mass Spectrometry Conditions, the reference retention time of perchlorate is 4.61min, perchlorate With¹⁸The standard solution multiple-reaction monitoring chromatogram of O labelling perchlorate is shown in Fig. 1, and wherein, (A) (B) is perchlorate standard The multiple-reaction monitoring chromatogram of solution, (C) is¹⁸The multiple-reaction monitoring chromatogram of O labelling perchlorate.

Embodiment 3: the condition optimizing experiment of the detection method of the present invention

1, the selection of Extraction solvent

Perchlorate has the high low absorbability that dissolves, most of soluble in water.The present embodiment is adopted in extracting test respectively With 1% acetic acid solution and water as Extraction solvent, find interpolation test the indistinction of solvent, but sample (the present embodiment is with milk powder As a example by) be added testing after purified treatment, the results are shown in Table 1, table 1 is the Extraction solvent comparative result on the impact of the response rate Table, this result shows to use acetic acid solution, and described acetic acid solution contains acetic acid and water, the concentration of volume percent of described acetic acid solution It is 1%, selects this acetic acid solution to reclaim as the interpolation of Extraction solvent and be better than adopting and use water as Extraction solvent.Therefore, volume is selected Percent concentration is that 1% acetic acid solution is as Extraction solvent.

Table 1

For bottled water substrate, we have employed the method for directly interpolation Isotopic Internal Standard and detect；And fruit, cattle The food such as milk, milk powder, corn, aquatic products, animal tissue then need to carry out purified treatment.Fruit sample need to remove the color in substrate Element, can use Solid-Phase Extraction column purification；It is many for the complex matrices such as milk, milk powder, corn, aquatic products, animal tissue contain Planting organic principle, such as protein, fat, saccharide etc., protein can be removed with acetonitrile precipitation, the water insoluble-acetonitrile system of fat, Will not be extracted.The volume ratio having investigated 1% acetic acid solution-acetonitrile is respectively 1+1；1+2；To in milk powder during 1+3 and 1+4 The impact of perchlorate extraction effect, wherein, the volume ratio that 1% acetic acid solution contains acetic acid and water, acetic acid and water is 1: 100,.Experimental result is shown in Table 2, and table 2 is the comparison of the impact of the different volumes comparison response rate.Result shows, 1% acetic acid solution-second When the volume ratio of nitrile is 1+1, impurity interference is relatively big, and extracting solution is muddiness；The volume ratio of 1% acetic acid solution-acetonitrile is respectively 1+2； During 1+3 and 1+4, impurity interference is less, and fat, albumen precipitation effect are preferable, extracting solution clear.But along with acetonitrile volume ratio Increase, the extraction efficiency of perchlorate is gradually lowered, and considers each influence factor, selects 1% acetic acid solution-acetonitrile Volume ratio 1+2.

Table 2

In theory, when perchlorate extracts, the most extraction effects of consumption of aqueous solution are the best.Investigate same amount of sample Adding the aqueous solution impact on extraction effect of different volumes, result shows that the volume ratio of aqueous solution is extraction when 1,2,5 and 6 times Efficiency, without being clearly distinguished from, the results are shown in Table 3, and table 3 is the different extraction with aqueous solution volume comparative result tables on the impact of the response rate.Examine Considering to factors such as matrix effect and subsequent purification, in this experiment, fruits sample selects the aqueous solution volume of 5 times to extract, milk The aqueous solution volume using 1 times extracts；Milk powder, corn, aquatic products and animal tissue select the aqueous solution volume of 2 times to extract.

Table 3

2. the selection of solid-phase extraction column

Using aqueous solution to carry out extracting target compound, the impurity extracted is more, by above-mentioned acetonitrile precipitation Method removes the fat in sample, albumen etc., and removing other impurity is important purifying step.In recent years, Solid-Phase Extraction becomes The main purification style of medicine food analysis, we use conventional C₁₈Solid-phase extraction column, Oasis HLB solid-phase extraction column and graphitic carbon Black post to perchlorate and¹⁸The purification efficiency of O labelling perchlorate has carried out comparative study.Experimental result is shown in Table bright, C₁₈The recovery test of solid-phase extraction column is more satisfactory, and the impurity such as pigment, a small amount of fat and albumen is retained on pillar removal, and high chlorine Hydrochlorate and¹⁸O labelling perchlorate does not retain on post, flows out with effluent；Oasis HLB solid-phase extraction column and stone Perchlorate is not retained by ink carbon black post, but to it¹⁸O labelling perchlorate has absorption, is also difficult to water be washed out, Therefore C is selected in this experiment₁₈Solid-phase extraction column is made a return journey the removal of impurity.

It addition, this effects C of different amount of filler₁₈Solid-phase extraction column be respectively 60mg/3cc, 200mg/3cc and 500mg/3cc.Experiment finds, after sample liquid crosses the pillar purification of above-mentioned three kinds of different amount of filler, all can get the molten of clear Liquid, but sample liquid is positioned over 4 DEG C, when cold preservation 2 is little after, crossing the sample liquid that amount of filler is 60mg/3cc has White Flocculus to separate out, Tracing it to its cause is pillar overload, and impurity do not removes totally, and the sample liquid of other two kinds of amount of filler no abnormal.It addition, enter Finding after row Instrumental Analysis, the sample after purifying with 200mg/3cc and 500mg/3cc respectively adds recovery nothing and is clearly distinguished from, and sees Table 4, table 4 is the different amount of filler comparative result tables on the impact of the response rate.Therefore this method uses the C of 200mg/3cc₁₈Solid phase Extraction column is decontaminating column.

Table 4

3. the selection of chromatographic column

Perchlorate is a kind of containing strong anion perchlorate ([ClO4]^-) salt, perchlorate exists , being not particularly suited for analyzing, therefore, this seminar puts forth effort on the selection of anion-exchange column on C18 post without reserve.This experiment Synergi4 μM of AX-RP80A post of phenomenex and Waters is investigated respectively in the case of identical flow velocity (0.7mL/min) IC-Pak^TMAnion HR post, 6 μm, the separation situation of 4.6 × 75mm (i.d.).phenomenex synergi4μ MAX- RP80A post uses methanol-0.1% formic acid to be flowing phase, as in figure 2 it is shown, Fig. 2 is through synergi4 μM of AX-of phenomenex The perchlorate MRM figure that RP80A post separates, good separating effect, but peak width, peak shape is poor, and required analysis time is longer. Waters IC-Pak^TMAnion HR post uses volatile ammonium salt solution-acetonitrile system to be flowing phase, as it is shown on figure 3, Fig. 3 For the perchlorate MRM figure separated through Waters IC-PakTM Anion HR post.Good separating effect, peak shape is the most right Claiming, can complete to analyze in 5 minutes, free from admixture disturbs.Therefore, this experimental selection Waters IC-Pak^TMAnion HR post, 6 μ M, 4.6 × 75mm (i.d.) are for analyzing chromatographic column.

4. the selection of the phase that flows

The flowing that anion-exchange column is conventional is NaOH or KOH solution mutually, cannot directly enter mass spectrum, need to be with chemistry The method of suppression converts it into H₂O could enter MS detection, and this needs by suppressor after the incidental post of ion chromatograph Realize.Therefore this experiment uses volatilizable salt ammonium hydrogen carbonate or ammonium acetate solution to make flowing phase.Compare different ions The impact that perchlorate is separated by the ammonium hydrogen carbonate of intensity with ammonium acetate solution, finds that the kind of saline solution is on separating impact Not quite, but ionic strength affect is the biggest.

When the ion concentration of ammonium bicarbonate soln is 5.0mmol/L, the retention time of perchlorate up to 12.4min with On, peak width reaches 1min (Fig. 4);When the ion concentration of ammonium bicarbonate soln be 10,20,25,30,50mmol/L time, perchlorate The retention time of ion gradually decreases, and peak shape is more and more sharp-pointed along with the increase of ion concentration, and sensitivity is become better and better, Peak shape during 25mmol/L is symmetrical sharp-pointed, and retention time is 4.3min, but during 30mmol/L, peak shape is symmetrical, but sensitivity declines, Therefore, 50mmol/L does not continues to test (Fig. 5-Fig. 8) again.It addition, compare under different PH simultaneously, the reservation of perchlorate Time, peak shape, sensitivity.Less than PH10, peak width, retention time is more than 7min(Fig. 9), more than PH10, peak is sharp-pointed, but peak shape is not Symmetry, leans forward, retention time 3.2min(Figure 10).So the ammonium bicarbonate soln selecting ion concentration to be 25mmol/L, PH10, For optimum selection, but in actual applications, for the substrate sample liquid that some are more complicated, the retention time of perchlorate and mark The retention time of quasi-solution has the skew (Fig. 7 and Figure 11) of 0.3min.Therefore, acetonitrile-ammonium bicarbonate soln system is not Good flowing selects mutually.

Relatively ammonium acetate solution be 50,100,200m mol/L time perchlorate separation situation.When ammonium acetate solution is During 50mmol/L, the peak shape of perchlorate is poor, trails (Figure 12)；When ammonium acetate solution is 100m mol/L, high chlorine The peak shape of hydrochlorate is the most symmetrical, sensitivity higher (Figure 13)；When ammonium acetate solution is 200m mol/L, perchlorate Peak shape is preferable, but sensitivity declines (Figure 14).Therefore, selecting ammonium acetate solution is optium concentration when being 100m mol/L.It addition, This experiment also compares the ammonium acetate solution-acetonitrile system situation (Figure 15-Figure 18) in volume ratio 5+5,4+6,3+7,2+8.When Ammonium acetate solution-acetonitrile system is when volume ratio 5+5, and perchlorate retention time is at 6.1min, but peak shape is wider；Work as ammonium acetate Solution-acetonitrile system is when volume ratio 4+6, and perchlorate retention time is at 4.6min, and peak shape is symmetrical sharp-pointed, sensitivity High；But along with the increase of acetonitrile volume ratio, peak shape is the poorest, and baseline noise is the highest.Consider separating degree, retention time and chromatograph The factors such as peak shape, select 100m mol/L ammonium acetate-acetonitrile (volume ratio 4+6) for flowing phase, and flow velocity 0.7mL/min, at this Under part, the retention time of perchlorate is about 4.6min.The MRM chromatogram of perchlorate standard solution is shown in Figure 19, figure 19 is multiple-reaction monitoring (MRM) chromatogram of perchlorate standard solution (1.0 μ g/L).

5. the optimization of Mass Spectrometry Conditions

With peristaltic pump with the perchlorate of the flow velocity of 10 μ L/min injection 0.5mg/L the most continuously and¹⁸O labelling height chlorine Acid ion standard solution enters in ESI ion source, owing to the chloride ion in perchlorate exists isotope in nature³⁵Cl and³⁷Cl, carries out first mass spectrometric analysis (Q1 scanning) under anionic textiles mode, obtains perchloric acid perchlorate The quasi-molecular ion peak (m/z99, m/z101) of radical ion and¹⁸The quasi-molecular ion peak of O labelling perchlorate (m/z107, M/z109), Figure 20-Figure 21 is seen.Figure 20 is the first mass spectrometric figure of perchlorate, Tu21Wei¹⁸O labelling perchlorate ([³⁵Cl¹⁸O₄]^-) first mass spectrometric figure.Quasi-molecular ion peak is carried out second mass analysis (daughter ion scanning), obtain fragment from Sub-information, perchlorate and¹⁸The second order ms of O labelling perchlorate see Figure 22-Figure 24, Figure 22 be perchlorate from Son ([³⁵ClO₄]^-) second order ms figure, Figure 23 be perchlorate ([³⁷ClO₄The second order ms figure of]-), Tu24Wei¹⁸O marks Note perchlorate ([³⁵Cl¹⁸O₄]^-) second order ms figure.Ion pair m/z99/ that Response to selection value is high, baseline noise is low 83, m/z101/85 as monitoring ion pair (qualitative ion pair), select the strongest m/z99/83 of signal as quota ion pair, The m/z107/89 conduct that Response to selection value is high¹⁸The characteristic ion pair of O labelling perchlorate.Use MRM type collection number According to, selection residence time is 200ms, and optimize each ion pair removes a bunch voltage (DP), collision gas energy (CE), focus voltage (FP), entrance potential (EP), collision cell exit potential (CXP).Result of the test shows, when spray voltage from-4500v carry to- During 1000v, sensitivity improves three times.Mass spectrometry parameters and the multiple-reaction monitoring condition of each monitoring ion pair see table.

Mass spectrometry parameters	Parameter value
		Atomization gas	60
Gas curtain gas	30
		Auxiliary adds steam	70
Collision gas	high
		Auxiliary adds hot air temperature, DEG C	550
Spray voltage, v	-1000

Note: the ion of band " * " is quota ion.

Embodiment 4: the determination of the range of linearity

Under experiment condition determined by method, take the perchlorate standard solution of a series of variable concentrations, with instrument Device response peak area carries out linear regression to the concentration of perchlorate, and result shows, when the concentration of perchlorate is 0 In the range of μ g/L～10 μ g/L, linear relationship is good, its regression equation y=0.447x+0.0097, correlation coefficient r=0.9999, figure 25 is perchlorate standard solution linear relationship chart.When the perchlorate concentration in sample exceedes this range of linearity Time, can suitably add the extension rate of large sample.

The quantitative limit that the mensuration lower bound of this method is announced with reference to U.S. FDA official website, wherein bottled water is 0.5 μ g/kg, Fruit is 1 μ g/kg, and milk, milk powder, corn and aquatic products etc. are 3 μ g/kg, and the situation using additive process to carry out surveying determines , it is contemplated that baby is Susceptible population, and immunity is more weak, and milk is its Major Foods, for protecting the health of baby, in milk The mensuration lower bound of perchlorate is down to 1.0 μ g/kg.In this method bottled water, the mensuration lower bound of perchlorate is 0.5 μ g/L, In fruit, milk, the mensuration lower bound of perchlorate is 1.0 μ g/kg, high chlorine in milk powder, corn, aquatic products, animal tissue The mensuration lower bound of acid ion radical ion is 3.0 μ g/kg.

Embodiment 5: with the test kit of the present invention to bottled water, Eugenia javanica Lam, milk, milk powder, rice, the flesh of fish and seven kinds of samples of Hepar Gallus domesticus Product are added the response rate and Precision Experiment

With the test kit of the present invention, bottled water, Eugenia javanica Lam, milk, milk powder, rice, the flesh of fish and seven kinds of samples of Hepar Gallus domesticus are carried out The TIANZHU XINGNAO Capsul of three levels and Precision Experiment, each pitch-based sphere is repeated 10 times, and concrete grammar is as follows:

(1) sample preparation and preservation:

1. bottled water, milk, milk powder

Take representative sample about 500g, seal in loading clean container, indicate labelling.In 0 DEG C～4 DEG C preservation.

2. Eugenia javanica Lam, the flesh of fish, Hepar Gallus domesticus

Take representative sample about 500g, its edible part is first shredded, fully smash to pieces uniformly through tissue mashing machine, load clean Seal in clean container, indicate labelling.In less than-18 DEG C freezen protective.

3. rice

Take representative sample about 500g, pulverize through tissue mashing machine and pass through 1.0mm hole sizer, being mixed, load clean container Interior sealing, indicates labelling.Preserve under room temperature.

In the operating process of sample preparation, sample contamination should be prevented or the change of residuals content occurs.

(2) described in preparation-obtained sample in step (1) is added¹⁸O labelling perchlorate's solution extracts:

1. bottled water

Accurately draw sample 980 μ L in test tube, add 20 μ L¹⁸O labelling perchlorate's solution, vortex oscillation 30s, for liquid phase Chromatography-mass spectroscopy/mass spectrograph measures.

2. Eugenia javanica Lam

Weigh sample 5g (being accurate to 0.01g) in 50mL centrifuge tube, add 100 μ L¹⁸O labelling perchlorate's intermediate solution, Adding 25.0mL acetic acid solution, vortex oscillation 2min, 10000r/min is centrifuged 10min, separates supernatant and fills in centrifuge tube in tool, uses second Acid solution is settled to 30mL scale, shakes up, to be clean.

3. milk, milk powder, rice

Weigh sample 5g (being accurate to 0.01g) in 50mL centrifuge tube, add 100 μ L internal standard intermediate solution, add 10.0mL(cattle Milk adds 5.0mL) acetic acid solution, vortex oscillation 2min, add 20.0ml acetonitrile, horizontal oscillator tube vibration 30min, 10000r/min from Heart 10min, separates supernatant and fills in centrifuge tube in tool, be settled to 30mL scale with acetic acid solution, shake up, to be clean.

4. the flesh of fish, Hepar Gallus domesticus

Weigh sample 5g (being accurate to 0.01g) in 50mL centrifuge tube, add 100 μ L¹⁸O labelling perchlorate's intermediate solution, Add 10.0mL1% acetic acid solution, homogeneous extraction 30s, separately take a centrifuge tube, add 20mL acetonitrile washing homogenizer cutter head, united extraction Liquid, horizontal oscillator tube vibration 30min, 10000r/min are centrifuged 10min, separate supernatant and fill in centrifuge tube in tool, use acetic acid solution It is settled to 30mL scale, shakes up, to be clean.

(3) purify:

Draw the extraction supernatant obtained in 1.0mL step (2) and cross Solid-Phase Extraction C₁₈Post, discards effluent, draws 1.5mL Supernatant crosses Solid-Phase Extraction C₁₈Post, collects effluent, vortex oscillation 30s, measures for liquid chromatography-mass spectrography/mass spectrograph；

(4) detection:

Liquid phase chromatogram condition:

A) chromatographic column: IC-Pak^TMAnion HR post, 6 μm, 4.6mm × 75mm (internal diameter) or quite person；

B) flowing phase: acetonitrile+0.1mol/L ammonium acetate solution=(60+40, V₁/V₂)；

C) flow velocity: 700 μ L/min；

D) column temperature: 40 DEG C；

E) sample size: 10 μ L；

Mass Spectrometry Conditions:

A) ion source: electric spray ion source (ESI)；

B) scan mode: anion scans；

C) monitoring pattern: multiple-reaction monitoring (MRM)；

D) gas curtain gas (CUR): 209kPa；

E) atomization gas (GS1): 414kPa；

F) auxiliary adds steam (GS2): 483kPa；

G) collision gas (CAD): high；

H) electron spray voltage (IS) :-1000V；

I) ion source temperature (TEM): 550 DEG C；

J) monitor ion pair, collision energy, go a bunch voltage, acquisition time and retention time see table:

(5) qualitative, quantitative measures and judges

Qualitatively judge and carry out as follows: the maximum allowable offset of relative ion abundance is less than the model of following provisions Enclose, then can determine whether sample exists corresponding determinand:

If relative ion abundance is ＞ 50%, then the relative deviation allowed is ± 20%；

If relative ion abundance is ＞ 20%～50%, then the relative deviation allowed is ± 25%；

If relative ion abundance is ＞ 10%～20%, then the relative deviation allowed is ± 30%；

If relative ion abundance is≤10%, then the relative deviation allowed is ± 50%.

Rational judgment is with mass spectrometric data datatron or by the content of formula (1) calculating sample Chinese medicine, result of calculation needs deduction Blank value:

$X_i=\frac{c\&times;c_i\&times;A\&times;A_{\mathrm{si}}\&times;V}{c_{\mathrm{si}}\&times;A_i\&times;A_s\&times;m}\&times;\frac{1000}{1000}...\left(1\right)$

In formula:

X_iThe content of target compound in sample, unit is ng/kg (μ g/kg)；

The concentration of analysans in c standard working solution, unit is nanograms per milliliter (ng/mL)；

c_iThe concentration of internal standard substance in sample, unit is nanograms per milliliter (ng/mL)；

The peak area of analysans in A sample；

A_siThe peak area of internal standard substance in standard working solution；

V sample constant volume, unit is milliliter (mL)；

c_siThe concentration of internal standard substance in standard working solution, unit is nanograms per milliliter (ng/mL)；

A_iThe peak area of internal standard substance in sample solution；

A_sThe peak area of analysans in standard working solution；

The sample weighting amount of m sample, unit is gram (g).

This method, with bottled water, Eugenia javanica Lam, milk, milk powder, rice, the flesh of fish and Hepar Gallus domesticus as sample, uses standard addition method pair Interpolation sample carries out the response rate respectively and Precision Experiment the results are shown in Table 5-table 7, and table 5 is the bottled water substrate indoor response rate and essence Density experiment result；Table 6 is Eugenia javanica Lam, the milk substrate indoor response rate and Precision Experiment result table；Table 7 is milk powder, rice, fish Meat and the Hepar Gallus domesticus substrate indoor response rate and Precision Experiment result table；Corresponding MRM figure is shown in Figure 26-Figure 43, from figure it can be seen that sample Product substrate does not has the liquid chromatography tandom mass spectrometry determination to perchlorate to interfere.Figure 26 is bottled water host solvents MRM chromatogram；Figure 27 is the MRM chromatogram of bottled water substrate；Figure 28 is that bottled water extraction standard adds (pitch-based sphere 0.5 μ G/kg) MRM chromatogram；Figure 29 is the MRM chromatogram of Eugenia javanica Lam host solvents；Figure 30 is the MRM chromatogram of Eugenia javanica Lam substrate；Figure 31 The MRM chromatogram of (pitch-based sphere 1.0 μ g/kg) is added for Eugenia javanica Lam extraction standard；Figure 32 is the MRM chromatograph of milk substrate solvent Figure；Figure 33 is the MRM chromatogram of milk substrate；Figure 34 is the MRM color that milk substrate standard adds (pitch-based sphere 1.0 μ g/kg) Spectrogram；Figure 35 is the MRM chromatogram of milk powder host solvents；Figure 36 is the MRM chromatogram of milk powder substrate；Figure 37 is milk powder substrate mark The accurate MRM chromatogram adding (pitch-based sphere 3.0 μ g/kg)；Figure 38 is the MRM chromatogram of rice substrate；Figure 39 is rice substrate Standard adds the MRM chromatogram of (pitch-based sphere 3.0 μ g/kg)；Figure 40 is the MRM chromatogram of flesh of fish substrate；Figure 41 is flesh of fish base Matter standard adds the MRM chromatogram of (pitch-based sphere 3.0 μ g/kg)；Figure 42 is the MRM chromatogram of Hepar Gallus domesticus substrate；Figure 43 is Hepar Gallus domesticus Extraction standard adds the MRM chromatogram of (pitch-based sphere 3.0 μ g/kg)；The response rate scope of each pitch-based sphere and relative standard are inclined Difference data is shown in Table 8.Table 8 is pitch-based sphere and the test data table of response rate scope of perchlorate.The above results illustrates, this Bright test kit can preferably detect the perchlorate in bottled water, Eugenia javanica Lam, milk, milk powder, rice, the flesh of fish and Hepar Gallus domesticus.

Table 5

Table 6

Table 7

Table 8

Embodiment 6: laboratory monitoring checking test

This method is recognized through Fujian Entry-Exit Inspection and Quarantine Bureau, Guangzhou quality surveillance detection academy, south China green product Card inspection center, five units checkings such as Zhuhai Entry-Exit Inspection and Quarantine Bureau and Guangzhou agricultural standard and monitoring center etc., sample Add 0.5 or 1.0 or 3.0 μ g/kg, 1.0 or 2.0 or 6.0 tri-levels of μ g/kg, 5.0 or 10.0 or 30.0 μ g/kg, measure knot Fruit is shown in Table 9, and table 9 is the result table between bottled water, Eugenia javanica Lam, milk, milk powder, rice, the flesh of fish and Hepar Gallus domesticus room.Thus result, can See that the response rate between the room of this method, Precision test result meet the requirement of relevant regulations.

Table 9

Embodiment described above, the simply one in the present invention more preferably detailed description of the invention, the technology of this area The usual variations and alternatives that personnel are carried out in the range of technical solution of the present invention all should comprise within the scope of the present invention.

Claims (4)

1. one kind is detected the test kit of perchlorate in food, it is characterised in that include perchlorate standard solution,¹⁸O marks Note perchlorate's solution, acetic acid solution, acetonitrile, ammonium acetate solution and Solid-Phase Extraction C₁₈Post；

The CAS No of described perchlorate standard solution is 7601-89-0, and purity is 100%, and concentration is 1000mg/L；

Described¹⁸O labelling perchlorate's solution is containing Cl¹⁸O₄ ^-Solution, described¹⁸The purity of O labelling perchlorate's solution is 90%, concentration is 100 μ g/L；

Described acetic acid solution is made up of acetic acid and water, and described acetic acid is 1:100 with the volume ratio of described water；

Described ammonium acetate solution comprises ammonium acetate and water, and the molar concentration of described ammonium acetate solution is 0.1mol/L；

Described Solid-Phase Extraction C₁₈The model of post is waters, and amount of filler is 200mg, 3mL；

Described acetonitrile and ammonium acetate solution are by following proportioning mixing conduct flowing phase: acetonitrile+0.1mol/L ammonium acetate solution=60+ 40, V₁/V₂。

2. test kit described in claim 1 is for detecting the assay method of perchlorate in food, it is characterised in that include Following steps:

(1) sample preparation and preservation；

(2) described in preparation-obtained sample in step (1) is added¹⁸O labelling perchlorate's solution,

If described food is bottled water, the most directly carries out liquid chromatography-mass spectrography/mass spectrograph and measure；

If described food is non-bottled water, then adds described acetic acid solution and extract；

(3) purify: draw the extraction supernatant obtained in 1.0mL step (2) and cross described Solid-Phase Extraction C₁₈Post, discards effluent, Draw 1.5mL supernatant and cross described Solid-Phase Extraction C₁₈Post, collects effluent, vortex oscillation 30s；

(4) liquid chromatography-mass spectrography/mass spectrograph measures:

Liquid phase chromatogram condition:

A) chromatographic column: IC-Pak^TMAnion HR post, 6 μm, 4.6mm × 75mm, internal diameter；

B) flowing phase: acetonitrile+0.1mol/L ammonium acetate solution=60+40, V₁/V₂；

C) flow velocity: 700 μ L/min；

D) column temperature: 40 DEG C；

E) sample size: 10 μ L；

Mass Spectrometry Conditions:

A) ion source: electric spray ion source (ESI)；

B) scan mode: anion scans；

C) monitoring pattern: multiple-reaction monitoring (MRM)；

D) gas curtain gas (CUR): 209kPa；

E) atomization gas (GS1): 414kPa；

F) auxiliary adds steam (GS2): 483kPa；

G) collision gas (CAD): high；

H) electron spray voltage (IS) :-1 000V；

I) ion source temperature (TEM): 550 DEG C；

J) monitor ion pair, collision energy, go a bunch voltage, acquisition time and retention time see table:

(5) qualitative, quantitative measures and judges.

Assay method the most according to claim 2, it is characterised in that the qualitative judgement described in step (5) is as follows Carry out: the maximum allowable offset of relative ion abundance is less than following provisions scope, then can determine whether sample exists corresponding treating Survey thing:

If relative ion abundance is ＞ 50%, then the relative deviation allowed is ± 20%；

If relative ion abundance is ＞ 20%～50%, then the relative deviation allowed is ± 25%；

If relative ion abundance is ＞ 10%～20%, then the relative deviation allowed is ± 30%；

If relative ion abundance is≤10%, then the relative deviation allowed is ± 50%.

Assay method the most according to claim 2, it is characterised in that the quantitative result described in step (5) is as follows Calculate:

With mass spectrometric data datatron or the content that calculates sample Chinese medicine by formula (1), result of calculation need to deduct blank value:

In formula:

X_iThe content of target compound in sample, unit is ng/kg (μ g/kg)；

The concentration of analysans in c standard working solution, unit is nanograms per milliliter (ng/mL)；

c_iThe concentration of internal standard substance in sample, unit is nanograms per milliliter (ng/mL)；

The peak area of analysans in A sample；

A_siThe peak area of internal standard substance in standard working solution；

V sample constant volume, unit is milliliter (mL)；

c_siThe concentration of internal standard substance in standard working solution, unit is nanograms per milliliter (ng/mL)；

A_iThe peak area of internal standard substance in sample solution；

A_sThe peak area of analysans in standard working solution；

The sample weighting amount of m sample, unit is gram (g).