CN106596742A

CN106596742A - Urotropine content measured through ultra-high performance liquid chromatography-tandem mass spectrometry

Info

Publication number: CN106596742A
Application number: CN201510665913.XA
Authority: CN
Inventors: 洪霞; 张淑雅; 刘静
Original assignee: Jiangsu Wise Science and Technology Development Co Ltd
Current assignee: Jiangsu Wise Science and Technology Development Co Ltd
Priority date: 2015-10-16
Filing date: 2015-10-16
Publication date: 2017-04-26

Abstract

The invention belongs to the field of bromatology detection, and particularly relates to the measuring method of urotropine content. The measuring method comprises the steps of adopting acetonitrile to extract urotropine in a dried beancurd stick, conducting PXA purification via a solid phase extraction column, using UPLC-MS/MS to conduct analysis, adopting a multi-reaction monitoring mode, and using an external standard method to quantify. The chromatography measuring condition is a chromatography column ACQUITY UPLC BEH HIL IC, 2.1*50mm, and 1.7 microns; as to the moving phase, A is 10 mmol/L of an ammonium acetate solution, and B is acetonitrile; the flow speed is 0.3 mL/min; the column temperature is 40 DEG C; the sample size is 10 microliters. A recovery test of three additive dose levels is conducted on a sample, adding standard recovery is 81.67%-101.67%, RSD measured within many times by the recovery test is 1.84%-3.99%, and degree of accuracy and degree of precision of the method meet the analysis requirement. It is shown that the measuring method of the urotropine content is easy and fast to operate and good in the degree of accuracy and stability. By the application of the method, dried beancurd stick products of 6 brands in markets and supermarkets are detected, and residues of urotropine are not found. Results show that the lowest detection limit is 1 microgram per liter, whether the sample exceeds the standard or not can be directly judged, and the method has important significance on the aspects of detection and monitoring of urotropine in the dried beancurd stick.

Description

Using Ultra Performance Liquid Chromatography - Tandem mass spectrometry determines hexamethylenamine content

Technical field

The invention belongs to bromatology inspection field.The specifically related to assay method of hexamethylenamine content.

Background technology

Dried bean is the traditional soybean product that a kind of people like, but its retention cycle is very short, in order that dried bean appearance is bright and new

, long shelf-life, some illegal producers Chang Feifa addition Boraxs and sodium formaldehyde sulfoxylate during production, rectify through special administration for many years, it is fresh rare to arrive, but their addition maneuver and technology are also increasingly advanced and hidden, current supervision department has been found that hexamethylenamine is added in dried bean as the succedaneum of sodium formaldehyde sulfoxylate.

Hexamethylenamine (urotropine), also known as hexamethylenetetramine (hexamethylenetetramine

), itself is belonging to lower toxicity, is commonly applied to medical and industrial.Used as illegal additive, it is primarily served

Anti-corrosion, brightens, and improves the effect of product appearance, and its principle is that hexamethylenamine can decompose in acid condition generation formaldehyde, and kidney, liver, nervus centraliss, immunologic function, digestive system to human body etc. has infringement, forbids

For in food or food processing process.At present, the standard of hexamethylenamine in dried bean does not also detect in China, and only industry standard SN/T 2226-2008 is applied to Wu Luotuo in Carnis Gallus domesticus, chicken liver, chicken kidney and Carnis Sus domestica

The detection ［ 1 ］ of product.The detection that pertinent literature report is applied in dried bean has at present minority report, there are the methods ［ 6-7 ］ such as laser Raman spectrometry ［ 2 ］, gas chromatography ［ 3-4 ］, liquid chromatography ［ 5 ］ and Liquid Chromatography/Mass Spectrometry, therefore carry out coherent detection and work highly significant.The Ultra Performance Liquid Chromatography and tandem mass spectrum method of dried bean are established herein, and the exploitation of the method can greatly reduce sample analysis time.

The content of the invention

High performance liquid chromatography is to order about chromatographic column of the mobile phase by filling fixing phase using high pressure pump, because each component is variant in property and structure in mixture, it is also variant with the size that fixing phase is had an effect, therefore in the presence of same motive force, holdup time of the different component in fixing phase is different, so as to flow out from fixing phase according to sequencing, reaching carries out detached effect to material, and the method for carrying out quantitative analyses and qualitative analyses.

The present invention is comprised the following steps：

（1）Sample pretreatment

1.0g dried bean (being accurate to 0.01g) (crushed uniform) and 1.0g anhydrous sodium sulfates are weighed in politef centrifuge tube, add 10m L acetonitrile solutions, vortex mixed 2min, ultrasonic 10min, 10000r/min is centrifuged 10min, takes supernatant to be clean.

PXA pillars are activated with 5mL acetonitriles, supernatant is added in post, collect effluent, add 1m L

Acetonitrile, collects effluent, merges effluent, uses N₂Dry up, use 90: 10 acetonitrile：Water dissolution is settled to 1m L, is vortexed and mixes.0.22 μm of filter membrane is crossed, waits to use on the machine.

（2）Upper machine is determined

With Ultra Performance Liquid Chromatography-tandem mass spectrum detection, chromatogram, internal standard peak height are recorded（Hi）With hexamethylenamine peak height（Hs）, use standard curve converted score.

The assay method of aforesaid hexamethylenamine residual quantity, it is characterised in that the method be with BEH HILIC as chromatographic column, with ammonium acetate solution, acetonitrile as mobile phase A, Mobile phase B, the mL of flow velocity 0.3 /min；Column temperature is 40 DEG C；Sample size is 10 L；Detection wavelength is 229nm.

The invention has the beneficial effects as follows：The present invention detects that the hexamethylenamine content in dried bean has preferable veracity and precision using high performance liquid chromatography-tandem mass method.

Description of the drawings

Fig. 1 is the canonical plotting of hexamethylenamine.

Fig. 2 is the mass chromatogram of hexamethylenamine.

Specific embodiment

The specific embodiment of the present invention is described more detail below：

1. instrument and reagent：

Instrument：

Waters ACQULITY UPLC Ultra Performance Liquid Chromatography instruments;Waters Quattro Premier XE

Mass spectrograph Waters, USA;High speed refrigerated centrifuge SIGMA, Germany;Homogenizer;Nitrogen evaporator;ACQULITY UPLC BEH HILIC 2.1 × 50mm of chromatographic column, 1.7 μm.

Reagent：

99% ;100 μ g/m L standard substance storing solutions are dissolved by acetonitrile, are placed in sealing preserve in 4 DEG C of refrigerators;Chromatographic Pure Methanol, acetonitrile and ammonium acetate are purchased from Dikma companies;Experimental water is ultra-pure water;Solid-phase extraction column is purchased from the Pro Elut PXA of DIKMA companies, and specification is 150mg/6m L.

2. method and result

Chromatograph, Mass Spectrometry Conditions and system suitability test

Liquid chromatograph operating condition:Flow velocity: 0.3m L/min;Column temperature:40℃ ;Sampling volume:10μL

;Mobile phase:A is 10mmol/L ammonium acetate solutions, and B is acetonitrile.

Mass ion source selects electric spray ion source (ESI);Scan pattern:Cation scans (ESI+);Detection mode:Many reaction detection;It is dried gas:Nitrogen;Ion source temperature:110 DEG C, desolventizing temperature: 350℃

, desolventizing gas:600L/h, taper hole gas: 50L/h.

The making of standard curve

Working solution is distinguished into the μ L of sample introduction 10, regression equation is obtained (Y is peak area, and X is concentration, μ g/L)

Y=174.564 × X+0.775512, linearly dependent coefficient R is 0.998, into good linear in 1～20 μ g/L concentration ranges.Standard curve is as shown in Figure 1.

Recovery of standard addition and precision

Add the hexamethylenamine of basic, normal, high three contents in dried bean blank sample, response rate experiment is carried out according to method operating procedure, simultaneously each parallel repetition of addition concentration 6 times, determine precision, and the measure lower bound and standard deviation in terms of S/N >=10 the results are shown in Table shown in 1.As can be seen from Table 1, recovery of standard addition is 81.67%～101.67%, and 1.84%～3.99%, the accuracy of method and precision meet analysis and requires RSD that it is repeatedly determined.Add the chromatogram that concentration is 2 μ g/L on dried bean blank sample and see figure

2。

Table 1

Claims

1. the assay method of hexamethylenamine content, it is characterised in that after by testing sample pretreatment, with Ultra Performance Liquid Chromatography-tandem mass spectrometry detection, comprises the following steps：

（1）Sample pretreatment

1.0g dried bean (being accurate to 0.01g) (having crushed uniform) and 1.0g anhydrous sodium sulfates are weighed in politef centrifuge tube, adds 10m L acetonitrile solutions, vortex mixed 2min to surpass Sound 10min, 10000r/min are centrifuged 10min, take supernatant to be clean；PXA pillars are activated with 5mL acetonitriles, supernatant is added in post, collect effluent, add 1mL acetonitriles, collect effluent, merge effluent, use N₂Dry up, use 90: 10 acetonitrile：Water Dissolving is settled to 1m L, whirlpool Rotation is mixed；0.22 μm of filter membrane is crossed, waits to use on the machine；

（2 ）Upper machine is determined

With Ultra Performance Liquid Chromatography-tandem mass spectrum detection, chromatogram, internal standard peak height are recorded（Hi）With hexamethylenamine peak height（Hs）, use standard curve converted score；The assay method of aforesaid hexamethylenamine residual quantity, it is characterised in that the method be with BEH HILIC as chromatographic column, with ammonium acetate solution, acetonitrile as mobile phase A, Mobile phase B, the mL of flow velocity 0.3 /min；Column temperature is 40 DEG C；Sample size is 10 L；Detection wavelength is 229nm.

2. the invention has the beneficial effects as follows：The present invention detects that the hexamethylenamine content in dried bean has preferable veracity and precision using high performance liquid chromatography-tandem mass method.