CN113030320A - Separation and identification method and application of low molecular weight aldehyde - Google Patents
Separation and identification method and application of low molecular weight aldehyde Download PDFInfo
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Abstract
The invention discloses a separation and identification method and application of low molecular weight aldehyde, which belong to the technical field of analytical chemistry, a substance to be detected is subjected to derivatization treatment to obtain an MBTH-aldehyde derivative, an alkaline solution is added to adjust the pH value to 6-9, the filtering is carried out, Z-type and E-type isomers of the MBTH-aldehyde derivative are measured by adopting a liquid chromatography-tandem mass spectrometry method, the quantification is respectively carried out by adopting an MRM method, the quantification results of the Z-type and E-type isomers are respectively calculated through respective standard curves, then the amounts of the Z-type and E-type isomers are combined to obtain the total amount of acetaldehyde, propionaldehyde and butyraldehyde, the Z-type and E-type isomers of the MBTH derivative of the acetaldehyde, propionaldehyde and butyraldehyde can be used for separating and identifying the Z-type and E-type isomers of the MBTH derivative of the acetaldehyde, propionaldehyde.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a separation and identification method and application of low molecular weight aldehyde.
Background
Low molecular weight aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde are important raw materials in chemical industry, and can be used for preparing or producing dyeing auxiliaries, crease-resistant finishing agents, printing adhesives and the like, but the low molecular weight aldehydes have strong irritation and certain toxicity, and a small amount of residual low molecular weight aldehydes can cause respiratory tract and skin inflammation through breathing or skin contact, generate a poisoning phenomenon, and have potential hazards such as sensitization, carcinogenesis and mutagenesis. Therefore, the research on the detection method of the low molecular weight aldehyde residue has very important practical significance for guaranteeing the health of human bodies.
The low molecular weight aldehyde reacts with a phenol reagent (3-methyl-2-benzothiazolone hydrazone hydrochloride, MBTH) under an acidic condition to generate a colored MBTH-aldehyde derivative with a C ═ N double bond, and the MBTH-aldehyde derivative has two isomers of a Z type and an E type (shown in figure 1), and can be measured by a spectrophotometric method, a liquid chromatography-tandem mass spectrometry method and the like. However, the ratio of the two isomers Z-form and E-form of the MBTH-aldehyde derivative to be produced varies greatly depending on the reaction conditions (e.g., MBTH concentration, pH of the reaction solution, reaction time, reaction temperature, etc.). When the spectrophotometry is used for measurement, because Z-type and E-type isomers cannot be separated, MBTH-acetaldehyde derivatives, MBTH-propionaldehyde derivatives and MBTH-butyraldehyde derivatives cannot be separated, the total amount of various low molecular weight aldehydes is measured, and the sensitivity is low and the accuracy is poor. The high performance liquid chromatography or the liquid chromatography-tandem mass spectrometry has better separation capability, but MBTH-aldehyde derivative isomers are difficult to separate, chromatographic peak overlapping can be caused if the separation conditions are slightly improper, meanwhile, due to the isomerization phenomenon of the molecular structure, the peak intensity ratio of fragment ions obtained by mass spectrometry cracking has larger difference, when the mass spectrometry MRM method is used for detection, the accuracy of a quantitative result is low, the repeatability is poor, and at present, no separation literature report or measurement literature report exists for Z-type and E-type isomers of the MBTH-aldehyde derivative. Therefore, it is necessary to provide a method capable of separating and identifying both Z-type and E-type isomers of MBTH-aldehyde derivatives.
Disclosure of Invention
The present invention has been made to solve the above-mentioned problems occurring in the prior art, and an object of the present invention is to provide a method capable of separating and identifying Z-type and E-type isomers of MBTH-aldehyde derivatives.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for separating and identifying low molecular weight aldehyde, which comprises the steps of taking an MBTH solution as a derivatization reagent, performing derivatization treatment on a substance to be detected to obtain an MBTH-aldehyde derivative, adding an alkaline solution to adjust the pH value to 6-9, filtering, measuring Z-type and E-type isomers of the MBTH-aldehyde derivative by adopting a liquid chromatography tandem mass spectrometry method, quantifying respectively by adopting an MRM method, calculating the quantitative results of the Z-type and E-type isomers through respective standard curves, and then combining the amounts of the Z-type and E-type isomers to obtain the total amount of acetaldehyde, propionaldehyde and butyraldehyde.
Further, the derivatization treatment comprises the following steps: placing a substance to be detected in a reaction container, adding a phosphoric acid solution with the volume concentration of 2.0%, performing ultrasonic extraction for 10-15 min, performing centrifugal precipitation, sucking a supernatant into the reaction container, adding a derivatization reagent, wherein the volume ratio of the supernatant to the derivatization reagent is 4: 1, shaking up, and then placing in a water bath at 5-35 ℃ for derivatization reaction for 5-30 min.
Further, the alkaline solution comprises one of ammonia, sodium hydroxide, sodium carbonate, ethylenediamine or triethylamine.
Further, chromatographic and mass spectrometric conditions were as follows:
a chromatographic column: a C18 chromatography column; mobile phase: phase A is acid solution or alkali solution, phase B is acetonitrile, gradient elution procedure: 0-2min, 30-85% of B; 2.0-5.5min, 85% B; 5.5-6.5min, 85-30% of B; 6.5-7.5min, 30% B, flow rate: 0.2-0.4 mL/min; sample introduction amount: 10.0-20.0 mu L; column temperature: 30-50 ℃;
an ionization source: electrospray positive ion mode; the ion spray voltage is 4.0 kV; the ion source temperature is 450 ℃; the collection mode is as follows: monitoring a positive ion mode by multiple reactions; atomizer pressure 50.0 psi; auxiliary air pressure 50.0 psi; air curtain pressure 35.0 psi; the collision gas pressure was 7.0 psi; scanning time is 50 ms; the outlet voltage of the collision chamber is 10.0V; collision cell entrance voltage 10.0V; Q1/Q3 ion pairs, collision energy and declustering voltage are shown in Table 1,
TABLE 16 multiple reaction monitoring of MBTH-aldehyde derivatives Positive ion mode Mass Spectrometry parameters
Note: MBTH 3-methyl-2-benzothiazolone hydrazone hydrochloride; (1) and (4) quantifying ions.
Further, the C18 chromatography column comprises Diamonsil C18, 150 × 2.1mm, 2.2 μm; platisil ODS, 150X 4.6mm, 5 μm; xbridge C18 column, 100 mm. times.2.1 mm,3.5 μm; zorbax SB C18 column, 150mm × 2.1mm,2.1 μm; a Shim-pack XR-ODSII column, 150 mm. times.2.0 mm,2.2 μm or Zorbax extended-C18 column, 150 mm. times.2.1 mm,2.1 μm.
Further, the phase A comprises one of water, trifluoroacetic acid aqueous solution, ammonium acetate-formic acid aqueous solution or ammonia aqueous solution.
Further, the trifluoroacetic acid is at a concentration of 0.1%, 0.2%, 0.5%, 1.0%, 1.5%, or 2.0% by volume.
The method takes a modified C18 chromatographic column as a separation chromatographic column and acetonitrile-0.5% trifluoroacetic acid mixed solution as a mobile phase, and separates and identifies the Z-type and E-type isomers of the MBTH-aldehyde derivatives of acetaldehyde, propionaldehyde and butyraldehyde by a liquid chromatography tandem mass spectrometry. The separation detection method can effectively separate Z-type and E-type isomers of MBTH-aldehyde derivatives of acetaldehyde, propionaldehyde and butyraldehyde, and further accurately control the residual quantity of the acetaldehyde, the propionaldehyde and the butyraldehyde in the textile and the quality of the textile product. The detection method has the characteristics of high accuracy and high precision, and can be used for detecting low molecular weight aldehyde in the textile field.
The invention also provides application of the separation and identification method of the low molecular weight aldehyde in separation and identification of Z-type and E-type isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde.
The invention discloses the following technical effects:
firstly, the baseline separation problem of Z-type and E-type isomers of acetaldehyde, propionaldehyde and butyraldehyde-MBTH derivatives is solved, the invention adopts a common C18 column, trifluoroacetic acid with the volume concentration of 0.5% is added in an acetonitrile-water mobile phase as a mobile phase modifier, the peak shape is improved, the phenomenon of chromatographic peak trailing is reduced, the separation degree is greatly improved, and the Z-type and the E-type of the MBTH derivatives of the acetaldehyde, the propionaldehyde and the butyraldehyde are enabled to achieve baseline separation.
Secondly, a proper reaction terminator is selected. When aldehyde reacts with phenol reagent, reaction terminator ammonia is added. The reaction of aldehyde and phenol reagent needs to be carried out in a strong acid environment (pH is about 1), and is limited by reaction conditions such as reactant concentration, reaction temperature, reaction time and the like, the reaction efficiency in the specified reaction time may not reach 100%, the problem of incomplete derivatization exists, for example, the reaction efficiency of the solution measured at different times can have obvious difference without stopping the reaction, the reaction efficiency of the solution measured later can be higher than that of the solution measured earlier, and errors are brought to the quantitative detection results of acetaldehyde, propionaldehyde and butyraldehyde. When the reaction is carried out for 10min, a proper amount of reaction terminator ammonia water is added into the reaction solution, the pH is adjusted to be about 8, and the reaction is terminated. The consistency of the reaction time and the reaction efficiency of the reaction solution when the reaction solution enters the liquid chromatography separation and measurement is ensured.
Thirdly, the detection result is more accurate and reliable. Because the invention adopts liquid chromatography separation and tandem mass spectrometry determination, the Z-type isomer and the E-type isomer of the MBTH derivative of acetaldehyde, propionaldehyde and butyraldehyde reach baseline separation, and accurate quantitative determination is respectively carried out by adopting a standard curve method, thereby avoiding the determination error caused by the difference of mass spectrometry response intensity when the Z-type isomer and the E-type isomer are simultaneously determined, which is reported by literature methods.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a diagram of the reaction process of low molecular weight aldehydes with MBTH under acidic conditions to form two isomers, Z-and E-isomers;
FIG. 2 is a graph of MRM of Z-and E-isomers of the MBTH-acetaldehyde derivative in example 1, wherein peak 1 is Z-MBTH-acetaldehyde derivative and peak 2 is E-MBTH-acetaldehyde derivative;
FIG. 3 is a MRM plot of the Z and E isomers of the MBTH-propanal derivative of example 1, wherein peak 3 is the Z form of the-MBTH-propanal derivative and peak 4 is the E form of the-MBTH-propanal derivative;
FIG. 4 is a MRM plot of the Z and E isomers of the MBTH-butyraldehyde derivative of example 1, wherein peak 5 is the Z-MBTH-butyraldehyde derivative and peak 6 is the E-MBTH-butyraldehyde derivative.
FIG. 5 is a graph of MRM of Z-and E-isomers of MBTH-acetaldehyde derivatives in example 2, wherein peak 1 is Z-MBTH-acetaldehyde derivative and peak 2 is E-MBTH-acetaldehyde derivative;
FIG. 6 is a MRM plot of the Z and E isomers of the MBTH-propanal derivative of example 2, wherein peak 3 is the Z form of the-MBTH-propanal derivative and peak 4 is the E form of the-MBTH-propanal derivative;
FIG. 7 is a MRM plot of the Z and E isomers of the MBTH-butyraldehyde derivative of example 2, wherein peak 5 is the Z-MBTH-butyraldehyde derivative and peak 6 is the E-MBTH-butyraldehyde derivative;
FIG. 8 is a MRM chart of MBTH-acetaldehyde derivatives in example 3, wherein peak 1 is Z-type-MBTH-acetaldehyde derivative and peak 2 is E-type-MBTH-acetaldehyde derivative;
FIG. 9 is a MRM plot of the MBTH-propionaldehyde derivative of example 3, wherein peak 3 is the MBTH-propionaldehyde derivative;
fig. 10 is a MRM plot of MBTH-butyraldehyde derivative of example 3, wherein peak 4 is MBTH-butyraldehyde derivative;
FIG. 11 is a MRM chart of MBTH-acetaldehyde derivatives in example 4, wherein peak 1 is Z-type-MBTH-acetaldehyde derivative and peak 2 is E-type-MBTH-acetaldehyde derivative;
FIG. 12 is a MRM plot of the MBTH-propionaldehyde derivative of example 4, wherein peak 3 is the MBTH-propionaldehyde derivative;
fig. 13 is the MRM plot of MBTH-butyraldehyde derivative of example 4, wherein peak 4 is the MBTH-butyraldehyde derivative;
FIG. 14 is a MRM chart of MBTH-acetaldehyde derivatives in example 5, wherein peak 1 is Z-type-MBTH-acetaldehyde derivative, and peak 2 is E-type-MBTH-acetaldehyde derivative;
FIG. 15 is a MRM plot of the MBTH-propionaldehyde derivative of example 5, wherein peak 3 is the MBTH-propionaldehyde derivative;
fig. 16 is the MRM graph of the MBTH-butyraldehyde derivative of example 5, wherein peak 4 is the MBTH-butyraldehyde derivative;
FIG. 17 is a MRM chart of MBTH-acetaldehyde derivatives in example 6, wherein peak 1 is Z-type-MBTH-acetaldehyde derivative and peak 2 is E-type-MBTH-acetaldehyde derivative;
FIG. 18 is a MRM plot of the MBTH-propionaldehyde derivative of example 6, wherein peak 3 is the MBTH-propionaldehyde derivative;
FIG. 19 is the MRM chart of MBTH-butyraldehyde derivative of example 6, wherein peak 4 is MBTH-butyraldehyde derivative;
FIG. 20 is a MRM chart of MBTH-aldehyde derivatives in example 7, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 21 is a MRM chart of MBTH-aldehyde derivatives in example 8, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 22 is a MRM chart of MBTH-aldehyde derivatives in example 9, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 23 is a MRM chart of MBTH-aldehyde derivatives in example 10, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 24 is a MRM chart of MBTH-aldehyde derivatives in example 11, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 25 is a MRM chart of MBTH-aldehyde derivatives in example 12, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 26 is a MRM chart of MBTH-aldehyde derivatives in example 13, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 27 is a MRM chart of MBTH-aldehyde derivatives in example 14, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 28 is a MRM chart of MBTH-aldehyde derivatives in example 15, in which peak 1 is Z-type-MBTH-acetaldehyde derivative, peak 2 is E-type-MBTH-acetaldehyde derivative, peak 3 is Z-type-MBTH-propionaldehyde derivative, peak 4 is E-type-MBTH-propionaldehyde derivative, peak 5 is Z-type-MBTH-butyraldehyde derivative, and peak 6 is E-type-MBTH-butyraldehyde derivative;
FIG. 29 is a graph of the effect of different reaction terminators of examples 16-19 on the mass spectrometric matrix effect of acetaldehyde, propionaldehyde and butyraldehyde derivatives;
FIG. 30 is a graph of the effect of derivatization reaction time on the derivatization efficiency of acetaldehyde, propionaldehyde, and butyraldehyde in examples 28-34;
FIG. 31 is a graph of the effect of derivatization reaction temperature on the derivatization efficiency of acetaldehyde, propionaldehyde, and butyraldehyde in examples 35-37;
figure 32 is a graph of the effect of residence time on the stability of derivatized reaction products of acetaldehyde, propionaldehyde and butyraldehyde in example 1.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The AB SCIEX 4500 series triple quadrupole tandem mass spectrometer (AB SCIEX company, USA) and the Analyst 1.6.2 data processing system, UlltiMate 3000 standard liquid chromatograph (DIONEX company, USA); SHZ-88B water bath constant temperature oscillator (tai cang venhui instruments), DQS-200E ultrasonic cleaner (kunshan ultrasonic instruments ltd), XE240 mettler toledo electronic analytical balance (mettler toledo switzerland). Milli-Q pure water system (Millipore, USA); graduated tubes (10mL) are stoppered.
Methanol, trifluoroacetic acid, triethylamine, acetonitrile (chromatographically pure) were purchased from Merck, Germany, and hydrochloric acid and ammonia (guaranteed reagent) were purchased from Shanghai pharmaceutical Agents group. Acetaldehyde, propionaldehyde and butyraldehyde standard solutions (1.00mg/mL) were purchased from Chem Service, a standard stock solution of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde (1.00mg/mL, in terms of aldehyde) in types Z and E.
The preparation process of the solution is as follows:
acetaldehyde, propionaldehyde and butyraldehyde mixed standard solution (10.0 mg/L): 1.00mL of standard stock solutions of formaldehyde, acetaldehyde and propionaldehyde (1.00mg/mL) were each accurately pipetted into a 100mL volumetric flask, diluted with water and brought to volume to the scale.
Mixed standard solutions of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde of types Z and E (10.0mg/L, calculated as aldehyde): accurately sucking 1.00mL of MBTH derivative standard stock solution (1.00mg/mL) of acetaldehyde, propionaldehyde and butyraldehyde into a 100mL volumetric flask, diluting with water and fixing the volume to the scale.
MBTH solution (1.0 g/L): accurately weighing 0.10g of phenol reagent, dissolving with water, adding 10mL of concentrated hydrochloric acid, diluting to 100mL with water, storing in a brown container, and refrigerating at 4 deg.C in a refrigerator.
MBTH solution (10.0 mg/L): 10.0mL of phenol reagent solution is put into a 1000mL volumetric flask, then 100mL of water is added, 10mL of concentrated hydrochloric acid is added, and then water is used for dilution and constant volume is carried out to the scale mark.
Preparation of standard solution series: appropriate amounts of mixed standard solutions (10.0mg/L in terms of aldehyde) of Z-type and E-type acetaldehyde, propionaldehyde and MBTH derivatives of butyraldehyde are transferred into 7 10mL volumetric flasks to prepare standard series solutions with concentrations respectively corresponding to 1.0. mu.g/L, 2.0. mu.g/L, 5.0. mu.g/L, 10.0. mu.g/L, 50.0. mu.g/L, 100.0. mu.g/L and 200.0. mu.g/L of acetaldehyde, propionaldehyde and butyraldehyde.
Example 1
1.0g of the cut textile sample is accurately weighed into a 50mL colorimetric tube with a plug, 10mL of 2.0% phosphoric acid solution is added, and ultrasonic extraction is carried out for 10 min. After centrifugal precipitation, sucking 4.0mL of supernatant into a 10mL colorimetric tube with a plug, adding 1.0mL of MBTH solution (10.0mg/L) as a derivatization reagent, shaking up, placing in a water bath at 15 ℃ for derivatization reaction for 10.0min, then adding ammonia water with the volume concentration of 5% to adjust the pH to 8.0, diluting to a scale with distilled water, filtering with a 0.22 mu m water-phase microporous filter membrane, sucking 10 mu L of sample injection for analysis. A sample is put on a C18 chromatographic column, acetonitrile-0.5% trifluoroacetic acid aqueous solution is used as a mobile phase for gradient elution, then a tandem mass spectrometry is adopted to determine Z-type and E-type isomers of the MBTH-aldehyde derivative, an MRM method is adopted to respectively quantify, the quantification results of the Z-type and E-type isomers are respectively calculated through respective standard curves, and then the amounts of the Z-type and E-type isomers are combined to obtain the total amount of acetaldehyde, propionaldehyde and butyraldehyde. The chromatographic mass spectrometry conditions were as follows:
a chromatographic column: diamonsil C18 (150X 2.1mm, 2.2 μm); mobile phase: phase A is 0.5% (V/V) trifluoroacetic acid aqueous solution, and phase B is acetonitrile. Gradient elution procedure: 0-2min, 30-85% of B; 2.0-5.5min, 85% B; 5.5-6.5min, 85-30% of B; 6.5-7.5min, 30% B. Flow rate: 0.35 mL/min; sample introduction amount: 10.0 μ L; column temperature: at 40 ℃.
An ionization source: electrospray positive ion mode; the ion spray voltage is 4.0 kV; the ion source temperature is 450 ℃; the collection mode is as follows: multiple-reaction monitoring (MRM) positive ion mode; atomizer pressure 50.0 psi; auxiliary air pressure 50.0 psi; air curtain pressure 35.0 psi; the collision gas pressure was 7.0 psi; scanning time is 50 ms; the outlet voltage of the collision chamber is 10.0V; the collision cell entrance voltage is 10.0V. Collision cell entrance voltage: 10.0V; the Q1/Q3 ion pair, collision energy, and declustering voltage are shown in Table 1.
TABLE 16 multiple reaction monitoring of MBTH-aldehyde derivatives Positive ion mode Mass Spectrometry parameters
Note: MBTH 3-methyl-2-benzothiazolone hydrazone hydrochloride; (1) quantitative ion
In this example, the detection limits of Z-type, E-type and E-type isomers of acetaldehyde, propionaldehyde and butyraldehyde were 0.05. mu.g/kg, 0.07. mu.g/kg, 0.2. mu.g/kg for Z-type and 0.3. mu.g/kg for E-type. The average recovery of acetaldehyde, propionaldehyde and butyraldehyde for this example was 93.4%, 93.5% and 93.6%, respectively. The average recovery rates of acetaldehyde, propionaldehyde and butyraldehyde which are not separated are respectively 87.7%, 89.7% and 87.4%, the average precision of the acetaldehyde, propionaldehyde and butyraldehyde are respectively 4.8%, 5.1% and 5.8%, and the average precision of the acetaldehyde, propionaldehyde and butyraldehyde which are not separated are respectively 4.8%, 5.3% and 6.8%.
Example 2
The only difference from example 1 is that the column is Platisil ODS (150X 4.6mm, 5 μm).
Example 3
The only difference from example 1 is that the column was an Xbridge C18 column (100 mm. times.2.1 mm,3.5 μm).
Example 4
The only difference from example 1 is that the column was a Zorbax SB C18 column (150 mm. times.2.1 mm,2.1 μm),
example 5
The same as example 1 except that the column was a Shim-pack XR-ODSII column (150 mm. times.2.0 mm,2.2 μm).
Example 6
The only difference from example 1 is that the column was a Zorbax extended-C18 column (150 mm. times.2.1 mm,2.1 μm).
The results of the separation and identification under different chromatographic column conditions in the above examples are shown in FIGS. 5 to 19. The Z and E isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde gave better baseline separation on a Diamonsil C18 (150X 2.1mm, 2.2 μm) column, overlapping of the isomeric chromatographic peaks of butyraldehyde on a Platisil ODS column, overlapping of the isomeric chromatographic peaks of butyraldehyde on an Xbridge C18 column (100mm X2.1 mm,3.5 μm), a Zorbax SB C18 column (150mm X2.1 mm,2.1 μm), a Shim-pack XR-ODSII column (150mm X2.0 mm,2.2 μm) and a Zorbax extended-C18 column (150mm X2.1 mm,2.1 μm). A Diamonsil C18 column (150X 2.1mm, 2.2 μm) was used as the optimal analytical column in the method of the present invention. The separation chromatograms of the phenol reagent derivatives of acetaldehyde, propionaldehyde, and butyraldehyde in example 1 are shown in fig. 2 to 4.
Example 7
The only difference from example 1 is that the mobile phase is acetonitrile-water.
Example 8
The only difference from example 1 is that the mobile phase is acetonitrile-5 mmol/L aqueous ammonium acetate.
Example 9
The only difference from example 1 is that the mobile phase is acetonitrile-5 mmol/L ammonium acetate-0.1% aqueous formic acid.
Example 10
The same as example 1 except that the mobile phase was acetonitrile-0.5% aqueous ammonia solution.
Example 11
The only difference from example 1 is that the trifluoroacetic acid concentration in the mobile phase is 0.1% by volume.
Example 12
The only difference from example 1 is that the trifluoroacetic acid concentration in the mobile phase is 0.2% by volume.
Example 13
The only difference from example 1 is that the trifluoroacetic acid concentration in the mobile phase is 1.0% by volume.
Example 14
The only difference from example 1 is that the trifluoroacetic acid concentration in the mobile phase is 1.5% by volume.
Example 15
The only difference from example 1 is that the trifluoroacetic acid concentration in the mobile phase is 2.0% by volume.
The results of the separation identification of different mobile phase improvers are shown in fig. 20 to 28. The invention verifies the separation and identification effects of alkaline ammonia water, triethylamine, acidic formic acid, acetic acid, trifluoroacetic acid and ammonium acetate. The results show that after ammonia, triethylamine and ammonium acetate are added, the separation degree among all isomers is reduced, formic acid and acetic acid improve the separation effect, but no trifluoroacetic acid is obvious. Trifluoroacetic acid is therefore the most preferred mobile phase modifier in the process of the invention.
The results of the identification of the separation of the different mobile phases are shown in FIGS. 20 to 28. The invention verifies the influence of different varieties of mobile phases on the chromatographic separation of Z-type and E-type isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde, and mainly tests 5 mobile phases (acetonitrile-water, acetonitrile-0.5% trifluoroacetic acid aqueous solution, acetonitrile-5 mmol/L ammonium acetate-0.1% formic acid aqueous solution and acetonitrile-0.5% ammonia aqueous solution). From the above examples, it can be seen that for the Z-and E-isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde, the 5 mobile phases only comprise acetonitrile-0.5% trifluoroacetic acid in water, and after selecting a suitable gradient, the base line separation of Z-and E-isomers is better, the mass spectrum response sensitivity is better, and the overall effect of acetonitrile-0.5% trifluoroacetic acid in water is better.
The results of the effect of different trifluoroacetic acid concentrations (0.1%, 0.2%, 0.5%, 1.0%, 1.5% and 2.0%) on the degree of separation of the Z and E isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde are shown in fig. 2-4 and fig. 24-28. The results show that 0.5% trifluoroacetic acid has the best resolution and moderate sensitivity. 0.1% and 0.2% trifluoroacetic acid, no baseline separation was achieved. Trifluoroacetic acid at 1.0%, 1.5% and 2.0% caused a decrease in sensitivity of the mass spectral response of 10.3%, 15.8% and 38.2% respectively, compared to the mass spectral sensitivity of 0.5% trifluoroacetic acid.
Example 16
The same as example 1 except that 1.0mol/L NaOH was added to adjust the pH.
Example 17
The procedure is as in example 1 except that a 5% sodium carbonate solution is added to adjust the pH.
Example 18
The procedure is as in example 1, except that the pH is adjusted by adding an aqueous ethylenediamine solution having a concentration of 5% by volume.
Example 19
The procedure is as in example 1, except that 5% by volume aqueous triethylamine solution is added to adjust the pH.
The reaction between phenol reagent and low molecular weight aldehyde needs to be carried out rapidly under a stronger acidic condition (pH <2), and the reaction speed can be changed by changing the acidity of the solution. The invention verifies that different alkaline substances are used as reaction terminating agents, such as 5% ammonia water, 1.0mol/L sodium hydroxide, 5% sodium carbonate solution, 5% ethylenediamine solution or 5% triethylamine solution, the intersolubility of the reaction terminating agents and the phenol reagents, the convenience of operation, the matching property of mass spectrometry detection and the like are respectively considered, and the matrix effect results of different reaction terminating agents are shown in figure 29. The results show that the above 5 alkaline substances can stop the derivatization reaction, but sodium hydroxide is strong in alkalinity, is added in a concentrated solution, is not easy to control in quantity, and is easy to generate turbidity of the solution after being excessive, the main reason is that the alkalinity of the sodium hydroxide is too strong, and the pipelines of liquid chromatography and mass spectrometry and the ion source are easy to block due to the non-volatility of sodium salt. Although the end point of the sodium carbonate can be easily judged by bubbles, the addition amount is easy to master, but the pipeline and the ion source are easy to block due to the non-volatility of the sodium salt. The ethylene diamine and the triethylamine are moderate in alkalinity and mutually soluble with the phenol reagent, but the redundant ethylene diamine or triethylamine in the reactant has certain matrix effect, so that the mass spectrum response sensitivity of the aldehyde-MBTH derivative is restricted. The ammonia water has moderate alkalinity, reacts with hydrochloric acid in the phenol reagent solution to generate volatile ammonium chloride at high temperature, has no matrix effect, is easy to obtain and has low cost. The method of the invention takes ammonia water with 5% volume concentration as an optimal reaction terminator.
Example 20
The only difference from example 1 is that the pH was adjusted to 1.0.
Example 21
The only difference from example 1 is that the pH was adjusted to 2.0.
Example 22
The only difference from example 1 is that the pH was adjusted to 3.0.
Example 23
The only difference from example 1 is that the pH was adjusted to 4.0.
Example 24
The only difference from example 1 is that the pH was adjusted to 5.0.
Example 25
The only difference from example 1 is that the pH was adjusted to 6.0.
Example 26
The only difference from example 1 is that the pH was adjusted to 7.0.
Example 27
The only difference from example 1 is that the pH was adjusted to 9.0.
Accurately sucking 5mL of MBTH solution (1.0mg/L) into 7 50mL test tubes with plugs, respectively adding 5% ammonia water with different amounts, adjusting the pH value of the solution to be 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 (namely, examples 1 and 20-27), then adding 100ng of acetaldehyde, propionaldehyde and butyraldehyde, and measuring the peak heights of 6 aldehyde-MBTH derivatives and isomers thereof generated by a liquid chromatography tandem mass spectrometry method after reacting for 10min, wherein the results are shown in Table 2. As can be seen from table 2, the peak height of each derivative at different pH values decreased with increasing pH value, and was close to 0 at pH 8.0. The results show that acetaldehyde, propionaldehyde and butyraldehyde hardly react with the phenol reagent at pH 8.0, and pH 8.0 is selected as the pH at which the reaction is terminated in the solution, i.e., the pH is adjusted to 8.0 by adding ammonia.
Table 2 MRM chromatographic peak heights of MBTH-aldehyde derivatives at different pH values (n ═ 3)
Example 28
The only difference from example 1 is that the derivatization time was 1.0 min.
Example 29
The only difference from example 1 was that the derivatization time was 5.0 min.
Example 30
The only difference from example 1 was that the derivatization time was 15.0 min.
Example 31
The only difference from example 1 was that the derivatization time was 20.0 min.
Example 32
The only difference from example 1 was that the derivatization time was 30.0 min.
Example 33
The only difference from example 1 was that the derivatization time was 45.0 min.
Example 34
The only difference from example 1 was that the derivatization time was 60.0 min.
The derivatization reaction efficiency was measured at different derivatization times (1.0, 5.0,10.0, 15.0, 20.0, 30.0, 45.0, 60.0min, i.e., examples 1, 28-34) using 5.0mL of 1.0mg/L phenol reagent solution to perform derivatization reaction on 5.0mL of 20.0 μ g/L acetaldehyde, propionaldehyde, and butyraldehyde, and the results are shown in fig. 30. The result shows that the derivatization reaction of acetaldehyde, propionaldehyde and butyraldehyde with phenol reagent can reach more than 95% in 10.0min under proper conditions.
Example 35
The same as example 1, except that the derivatization temperature was 5 ℃.
Example 36
The same as example 1, except that the derivatization temperature was 25 ℃.
Example 37
The only difference from example 1 is that the derivatization temperature is 35 ℃.
The effect of different derivatization temperatures (5, 15, 25, 35 ℃, i.e., examples 1, 35-37) on the derivatization efficiency of acetaldehyde, propylene, and butyraldehyde was determined, and the results are shown in fig. 31. The results show that the derivatization reaction is faster at higher temperatures in solutions with a pH of 1, the derivatization reaction is relatively slow at 5 ℃, and at 15 ℃ the derivatization reaction can reach 90% or more in 10 min.
Verification example 1 stability of MBTH-aldehyde derivatives
After the derivatization of acetaldehyde, propionaldehyde and butyraldehyde with a phenol reagent is completed, the stability of the derivative directly influences the accuracy of the determination result. The present invention demonstrates the effect of standing at room temperature for various times (0, 1, 3, 6, 12, 24, 36, 72h) on the stability of MBTH-aldehyde derivatives of acetaldehyde, propionaldehyde and butyraldehyde prepared in example 1, and the results are shown in figure 32. The results show that the MBTH-aldehyde derivatives of acetaldehyde, propionaldehyde and butyraldehyde have no change in the measurement results after being placed for 72 hours at room temperature. It was shown that the MBTH-aldehyde derivative remains stable for at least 72h under the conditions of the present invention.
Verification example 2 Linear Range, detection Limit and quantitation Limit
A series of standard working solutions were prepared using a mixed standard solution (10.0mg/L in terms of aldehyde) of the Z-and E-type acetaldehyde, propionaldehyde and the MBTH derivative of butyraldehyde of example 1, in acetonitrile as solvent. So that the concentration ranges of acetaldehyde, propionaldehyde and butyraldehyde are 0.1-100 mu g/L (0.1,0.2,0.5,1.0,5.0,10.0,50.0 and 100.0 mu g/L). The sample was tested under the instrument conditions of example 1. And (3) drawing a standard working curve by taking the concentration of the sample as an abscissa and the response value of the instrument as an ordinate, and quantifying by an external standard method. As shown in table 3, acetaldehyde, propionaldehyde and butyraldehyde showed good linear relationship (r is not less than 0.9990) in the respective measurement ranges, and the detection limit was calculated by the method with the S/N signal-to-noise ratio of 3 and the quantification limit was calculated by the method with the S/N signal-to-noise ratio of 10, and the detection limit of acetaldehyde, propionaldehyde and butyraldehyde was 0.05 to 0.07 μ g/kg, and the quantification limit of acetaldehyde, propionaldehyde and butyraldehyde was 0.2 to 0.3 μ g/kg.
TABLE 3 Linear equation, correlation coefficient, linear range, detection limit, and quantitation limit
Verification example 3 precision and accuracy of the method
Adding different amounts of acetaldehyde, propionaldehyde and butyraldehyde standard stock solutions into an MBTH solution (1.0mg/L) to prepare low, medium and high 3 concentration (0.2, 5.0 and 80.0 mu g/L) Quality Control (QC) samples, respectively adopting the method and the liquid chromatography-mass spectrometry method with overlapped isomer chromatographic peaks to carry out 6 times of repeated measurement, and calculating the recovery rate and precision. The measurement results of the present invention were calculated as the sum of the measured amounts of 2 isomers. The result shows that the recovery rate of the method is 90.0-98.4%, the recovery rate of the non-separation method is 82.5-93.2%, the precision of the method is 3.5-7.6%, and the precision of the non-separation method is 3.4-9.0%. The results are shown in Table 4.
Table 4 results of recovery and precision tests with standard addition (n ═ 3)
The non-separation method refers to a liquid chromatography-tandem mass spectrometry method in which isomers are not subjected to chromatographic baseline separation, i.e., a liquid chromatography-tandem mass spectrometry method in which isomer chromatographic peaks overlap. The column was Shim-pack XR-ODS II (100 mm. times.2.0 mm, 2.5 μm); the mobile phase A is water, and the mobile phase B is methanol; the gradient elution procedure was: 0-2min, 35% B-90% B, 2.0-5.5min, 90% B, 5.5-6.5min, 90% B-35% B, 6.5-7.5min, 35% B; the elution flow rate was 0.3 mL/min; the column temperature was 40 ℃.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (7)
1. A separation and identification method of low-molecular-weight aldehyde is characterized by taking an MBTH solution as a derivatization reagent, performing derivatization treatment on a substance to be detected to obtain an MBTH-aldehyde derivative, adding an alkaline solution to adjust the pH value to 6-9, filtering, measuring Z-type and E-type isomers of the MBTH-aldehyde derivative by adopting a liquid chromatography-tandem mass spectrometry method, respectively quantifying by adopting an MRM method, respectively calculating the quantitative results of the Z-type and E-type isomers through respective standard curves, and then combining the amounts of the Z-type and E-type isomers to obtain the total amount of acetaldehyde, propionaldehyde and butyraldehyde.
2. The method for separating and identifying low molecular weight aldehydes of claim 1, wherein the derivatization treatment comprises the following steps: placing a substance to be detected in a reaction container, adding a phosphoric acid solution with the volume concentration of 2.0%, performing ultrasonic extraction for 10-15 min, performing centrifugal precipitation, sucking a supernatant into another reaction container, adding a derivatization reagent, wherein the volume ratio of the supernatant to the derivatization reagent is 4: 1, shaking up, and then placing in a water bath at 5-35 ℃ for derivatization reaction for 5-30 min.
3. The method of claim 1, wherein the chromatographic and mass spectrometric conditions are as follows:
a chromatographic column: a C18 chromatography column; mobile phase: phase A is acid solution or alkali solution, phase B is acetonitrile, gradient elution procedure: 0-2min, 30-85% of B; 2.0-5.5min, 85% B; 5.5-6.5min, 85-30% of B; 6.5-7.5min, 30% B, flow rate: 0.2-0.4 mL/min; sample introduction amount: 10.0-20.0 mu L; column temperature: 30-50 ℃;
an ionization source: electrospray positive ion mode; the ion spray voltage is 4.0 kV; the ion source temperature is 450 ℃; the collection mode is as follows: monitoring a positive ion mode by multiple reactions; atomizer pressure 50.0 psi; auxiliary air pressure 50.0 psi; air curtain pressure 35.0 psi; the collision gas pressure was 7.0 psi; scanning time is 50 ms; the outlet voltage of the collision chamber is 10.0V; collision cell entrance voltage 10.0V; Q1/Q3 ion pairs, collision energy and declustering voltage are shown in Table 1,
TABLE 16 multiple reaction monitoring of MBTH-aldehyde derivatives Positive ion mode Mass Spectrometry parameters
Note: MBTH 3-methyl-2-benzothiazolone hydrazone hydrochloride; (1) and (4) quantifying ions.
4. The method of claim 3, wherein the C18 chromatographic column comprises Diamonsil C18, 150 x 2.1mm, 2.2 μm; platisil ODS, 150X 4.6mm, 5 μm; xbridge C18 column, 100 mm. times.2.1 mm,3.5 μm; zorbax SB C18 column, 150mm × 2.1mm,2.1 μm; a Shim-pack XR-ODSII column, 150 mm. times.2.0 mm,2.2 μm or Zorbax extended-C18 column, 150 mm. times.2.1 mm,2.1 μm.
5. The method of claim 3, wherein the phase A comprises one of water, aqueous trifluoroacetic acid, aqueous ammonium acetate-formic acid, or aqueous ammonia.
6. The method according to claim 5, wherein the trifluoroacetic acid is at a concentration of 0.1%, 0.2%, 0.5%, 1.0%, 1.5% or 2.0% by volume.
7. The use of the method for separating and identifying a low molecular weight aldehyde according to any one of claims 1 to 5 for separating and identifying Z-type and E-type isomers of MBTH derivatives of acetaldehyde, propionaldehyde and butyraldehyde.
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