CN113219097B - Method for splitting and measuring carnitine enantiomer in infant formula milk powder - Google Patents
Method for splitting and measuring carnitine enantiomer in infant formula milk powder Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Abstract
The application relates to a residue detection method of infant formula milk powder, in particular to a method for splitting and determining a carnitine enantiomer in the infant formula milk powder based on an ultra-efficient synthetic phase chromatography technology. The milk powder sample is extracted by hydrochloric acid solution, saponified by potassium hydroxide solution water bath, purified by a cation exchange solid phase extraction column, and subjected to derivatization, and then the derivatization solution is taken for testing. The separation is carried out by adopting an Acquity Trefoil CEL1 chiral chromatographic column (3.0 mm multiplied by 150 mm, 2.5 mu m), supercritical carbon dioxide and 1% ammonia-methanol solution are used as mobile phases, and gradient elution is carried out at the flow rate of 1.0 mL/min. The results show that the two carnitine enantiomers have good linear relation within the mass concentration range of 0.25-10.0 mg/L, the quantitative limits of the methods of the L-carnitine and the D-carnitine are 50 mg/kg and 25 mg/kg respectively, the recovery rate is 92.0-110%, and the relative standard deviation is 3.9-7.2%. The method has the characteristics of good separation effect, high analysis speed, low organic solvent consumption and the like, and can be used for simultaneously determining the content and the purity of the L-carnitine in the infant formula milk powder.
Description
Technical Field
The application relates to a method for separating carnitine enantiomer and a method for detecting the content of carnitine in infant formula milk powder, in particular to a method for separating and quantitatively determining the carnitine enantiomer in the infant formula milk powder based on an ultra-high performance synthetic phase chromatography technology.
Background
Carnitine (Carnitine), the chemical name of which is beta-hydroxy-gamma-trimethylaminyl butyric acid, is a kind of amino acid, belongs to a quaternary ammonium cation complex, can be synthesized from two amino acids, namely lysine and methionine by a biosynthesis method, and is related to fat metabolism into energy in vivo. Carnitine has 1 chiral carbon atom, and has one pair of enantiomers, namely dextrorotatory enantiomer (D-carnitine) and levorotatory enantiomer (L-carnitine). The difference in biological activity between different enantiomers of carnitine is large, wherein D-carnitine has no efficacy and is harmful to human even in large dose[1]L-carnitine has effects, is essential in vivo natural substance in energy metabolism of mammals, and can promote lipid metabolismFat is converted into energy. L-carnitine is poorly synthesized in infants and fails to meet normal metabolism, so L-carnitine needs to be supplemented in infant formula. At present, the L-carnitine used in commercially available infant formulas is mainly obtained from industrial chemical synthesis methods, but the purity of L-carnitine produced by the method is generally not required, so that the infant formula marked in a label to contain L-carnitine contains D-carnitine. The purity of the added L-carnitine directly influences the quality safety of the infant formula milk powder, so that a method for measuring the content and analyzing the purity of the L-carnitine in the infant formula milk powder is urgently needed.
At present, the detection method of carnitine enantiomer mainly comprises high performance liquid chromatography-tandem mass spectrometry[2-5]High performance liquid chromatography[6-9]Spectrophotometric method[10-12]However, these methods generally have problems such as a large amount of organic reagent consumed, poor resolution, and a long separation time. Ultra-high performance phase-locked chromatography (UPC) in recent years2) Because supercritical carbon dioxide and a small amount of organic solvent (absolute ethyl alcohol, methanol and the like) are used as a mobile phase, the density and polarity of the mobile phase are changed by adjusting the system backpressure, the chromatographic column temperature and the proportion of the organic solvent, so that the separation effect of a target object is precisely controlled, and the chiral separation method has obvious advantages and is used in chiral separation. Current UPC2The technology has been successfully applied to bisphenol compounds[13]Triazole pesticide[14]Phthalic acid esters[15]3-chloro-1, 2-propanediol fatty acid ester[16]Analysis of isostructural analogs and isomers, and UPC2The application of the technology in the resolution and content determination of carnitine enantiomer is not reported.
Disclosure of Invention
In order to solve the technical problems, the present application aims to provide a method for separating two enantiomers of carnitine and determining the content of the enantiomers in infant formula milk powder by ultra-high performance synthetic phase chromatography. The method has the characteristics of less consumption of organic solvent, short analysis time, good separation degree and the like, and can meet the requirements of L-carnitine content determination and purity analysis in infant formula milk powder.
In order to achieve the above object, the present application adopts the following technical solutions:
the method for splitting and determining carnitine enantiomer in infant formula milk powder based on the ultra-high performance synthetic phase chromatography technology comprises the steps of sample extraction and standard solution preparation, wherein the sample extraction comprises the steps of extraction, purification and derivation; the method is characterized in that the method also comprises the steps of respectively carrying out ultra-performance synthesis chromatography analysis on the standard solution and the sample solution, making a standard curve and calculating the content and the purity of L-carnitine and D-carnitine in the sample solution through the standard curve;
the conditions of the ultra-high performance phase-combination chromatographic analysis are as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 5-dimethylphenyl carbamate);
mobile phase: a is CO2B is 1% (v/v) ammonia-methanol solution;
gradient elution procedure: 0-7 min, and 90% (v/v) A-10% (v/v) B of mobile phase; 7-9 min, and 90-72% (v/v) A-10% -28% (v/v) B of mobile phase; 9-12 min, and 72% (v/v) of mobile phase A-28% (v/v) B; 12-13 min, 72-90% (v/v) A-28% -10% (v/v) B of mobile phase; 13-14 min, and 90% (v/v) A-10% (v/v) B of mobile phase; flow rate: 1.0 mL/min;
and (3) system backpressure: 13.8 MPa; column temperature: 40 ℃; sample introduction amount: 5 muL; detection wavelength: 244 nm.
In a preferred embodiment, the extraction steps of the present invention are as follows:
weighing 5 g of milk powder sample in a 50 mL volumetric flask, adding 20 mL of 0.1 mol/L hydrochloric acid solution, uniformly mixing in a vortex manner for 1min, carrying out ultrasonic treatment for 5min to fully dissolve the sample, adding 5mL of 1 mol/L potassium hydroxide solution, uniformly mixing in a vortex manner for 1min, and fixing the volume to 50 mL by using 0.1 mol/L hydrochloric acid solution; and (3) putting 1 mL of the extracting solution into a 15 mL centrifuge tube, adding 9 mL of acetonitrile, uniformly mixing for 1min in a vortex mode, centrifuging for 5min at the speed of 15000 r/min, and purifying the obtained supernatant.
In a preferred embodiment, the purification steps of the present invention are as follows:
putting 1 mL of solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with 3 mL of 10 mmol/L hydrochloric acid aqueous solution, discarding eluent, vacuum drying for 1min, eluting with 2 mL of 4% ammonia-methanol solution, collecting eluent in a 15 mL centrifuge tube, drying with nitrogen at 40 ℃, and collecting residue to be derived.
In a preferred embodiment, the derivatization steps of the present invention are as follows:
adding 0.5mL of derivatization reagent into the obtained residues, dissolving the residues by ultrasonic treatment for 1min, uniformly mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, uniformly mixing in a vortex manner for 3 min, standing at 20 ℃ for derivatization reaction for 60 min, adding 2.0 mL of reaction termination solution, uniformly mixing in a vortex manner for 1min, centrifuging at 5000 r/min for 5min, transferring the upper-layer water phase into a 100 mL round-bottom flask, concentrating to be nearly dry, adding 1 mL of absolute ethanol into a concentration bottle, fully dissolving in a vortex manner, and feeding the mixture into a sample injection bottle through a 0.22 mu m filter membrane;
the derivatization reagent is 4.5 g/L L-alanine-beta naphthylamine acetonitrile solution, the catalyst I is 5.0 g/L triethylamine trichloromethane solution, the catalyst II is 5.0 g/L butyl chloroformate trichloromethane solution, and the reaction termination solution is 50 mmol/L sodium bicarbonate aqueous solution.
In another preferred embodiment, the extraction step of the present invention is as follows:
weighing 5.00 g of milk powder sample (accurate to 0.01 g), placing the milk powder sample in a 50 mL volumetric flask, adding 20 mL of 0.1 mol/L hydrochloric acid solution, carrying out vortex mixing for 1min, carrying out ultrasonic treatment for 5min to fully dissolve the sample, adding 5mL of 1 mol/L potassium hydroxide solution, carrying out vortex mixing, carrying out water bath saponification at 60 ℃ for 30 min, cooling to room temperature, adding 5mL of 1 mol.L-1 hydrochloric acid solution, carrying out vortex mixing for 1min, and carrying out constant volume to 50 mL by using 0.1 mol/L hydrochloric acid solution; and (3) putting 1 mL of the extracting solution into a 15 mL centrifuge tube, adding 9 mL of acetonitrile, uniformly mixing for 1min in a vortex mode, putting a part into a 2 mL centrifuge tube, centrifuging for 5min at 15000 r/min, and allowing the obtained supernatant to be purified.
In a preferred embodiment, the purification steps of the present invention are as follows:
putting 1 mL of solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with 3 mL of 10 mmol/L hydrochloric acid aqueous solution, discarding eluent, vacuum drying for 1min, eluting with 2 mL of 4% ammonia-methanol solution, collecting eluent in a 15 mL centrifuge tube, drying with nitrogen at 40 ℃, and collecting residue to be derived.
In a preferred embodiment, the derivatization steps of the present invention are as follows:
adding 0.5mL of derivatization reagent into the obtained residues, dissolving the residues by ultrasonic treatment for 1min, uniformly mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, uniformly mixing in a vortex manner for 3 min, standing at 20 ℃ for derivatization reaction for 60 min, adding 2.0 mL of reaction termination solution, uniformly mixing in a vortex manner for 3 min to terminate the reaction, centrifuging at 5000 r/min for 5min, transferring the upper-layer water phase into a 100 mL round-bottom flask, concentrating to be nearly dry, adding 1 mL of absolute ethyl alcohol into a concentration bottle, fully dissolving in a vortex manner, and introducing a sample into a small bottle through a 0.22 mu m filter membrane;
the derivatization reagent is 4.5 g/L L-alanine-beta naphthylamine acetonitrile solution, the catalyst I is 5.0 g/L triethylamine trichloromethane solution, the catalyst II is 5.0 g/L butyl chloroformate trichloromethane solution, and the reaction termination solution is 50 mmol/L sodium bicarbonate aqueous solution.
As a preferred mode, the standard solution of the present invention is prepared by the following steps:
1) 1.0 g/L two carnitine enantiomer stocks were prepared: respectively and accurately weighing 0.01 g of dextro-carnitine and levo-carnitine standard substance to be accurate to 0.1 mg; dissolving with absolute ethyl alcohol, and diluting to a constant volume of 10 mL to prepare a standard stock solution of 1.0 g/L;
2) preparation of mixed working solutions of two carnitine enantiomers: accurately sucking a certain amount of standard solution, and gradually diluting with anhydrous ethanol to obtain mixed working solution of 0.50, 1.00, 2.00, 5.00 and 20.00 mg/L;
3) preparation of standard working solutions: respectively and precisely transferring 0.5mL of mixed working solution of two carnitine enantiomers into a 10 mL centrifuge tube, drying by nitrogen, and deriving according to the method to prepare standard solutions with the concentrations of 0.25, 0.50, 1.00, 2.50 and 10.00 mg/L, which are equivalent to the samples containing 25, 50, 100, 250 and 1000 mg/kg of two carnitine enantiomers.
A milk powder sample is extracted by a hydrochloric acid solution, saponified by a potassium hydroxide solution in a water bath, purified by a cation exchange solid phase extraction column, separated by an Acquity Trefoil CEL1 chiral chromatographic column (3.0 mm multiplied by 150 mm, 2.5 mu m) after derivatization, and subjected to gradient elution by taking supercritical carbon dioxide and 1% ammonia-methanol solution as mobile phases at a flow rate of 1.0 mL/min. The result shows that the two carnitine enantiomers have good linear relation within the mass concentration range of 0.25-10.0 mg/L, the quantitative limit of the L-carnitine and the D-carnitine is 25 mg/kg, the recovery rate is 93.0-110%, and the relative standard deviation is 3.9-5.5%. The method has the characteristics of good separation effect, high analysis speed, low organic solvent consumption and the like, and can be used for simultaneously determining the content and the purity of the L-carnitine in the infant formula milk powder.
Drawings
FIG. 1 is a diagram showing the purification effect of different purification columns.
FIG. 2 Effect of derivatization temperature on derivatization reactions (D-carnitine and L-carnitine): a. 4 deg.C, b, 20 deg.C, c, 40 deg.C and d, 60 deg.C.
FIG. 3 Effect of derivatization time on derivatization reactions (D-carnitine and L-carnitine): a. 10 min, b, 30 min, c, 60 min and d, 90 min.
FIG. 4 is a graph comparing the effect of 3 columns on the separation of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) (1% ammonia-methanol solution, 13.8 MPa).
FIG. 5 is a graph showing the effect of column temperature of different systems on the separation effect of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) (1% ammonia-methanol solution, 13.8 MPa).
FIG. 6 resolution of carnitine racemate: a. a carnitine racemate; b. d-carnitine (D-carnitine); c. l-carnitine (L-carnitine).
Detailed Description
Experimental part
1.1 instruments, materials and reagents
Ultra-high performance phase-compatible chromatographs (wawter, usa); ultra-pure water purification system (Elga, uk); bench centrifuge (Thermo corporation, usa); MS2 vortex mixer (Shanghai medical instrument factory); r215 rotary evaporator (Buchi, switzerland); AE260 electronic balance (Mettler, switzerland); nitrogen blowing instrument (tokyo physical and chemical company, japan); microfiltration membrane (0.22 μm, organic phase).
Acetonitrile, methanol, absolute ethanol (chromatographically pure, Scharlau, spain); ammonia, hydrochloric acid, potassium hydroxide, sodium bicarbonate, trichloromethane, triethylamine and butyl chloroformate (guaranteed purity); l-alanine- β -naphthylamine (shanghai' an spectrum); the water is ultrapure water; the reagents used in other experiments were analytically pure except for the special instructions. A mixed cation exchange solid phase extraction column (Oasis MCX 3 mL, 60 mg or equivalent, which was activated with 3 mL methanol, 3 mL water, and 3 mL 10 mmol/L hydrochloric acid in that order prior to use).
Carnitine racemate (CAS number: 461-05-2, purity 98% or higher, SIGMA corporation, USA).
Two enantiomers:Dcarnitine (CAS number: 541-14-0, 99.8% purity, CATO USA),LCarnitine (CAS No. 541-15-1, purity 99.0%, dr.e, germany).
Standard solution and reagent preparation
1.2.1 Standard solution of racemate
Carnitine stock (1.0 g/L): 0.01 g (accurate to 0.1 mg) of the carnitine racemate standard is accurately weighed respectively, and is dissolved by absolute ethyl alcohol, the volume is determined to be 10 mL, and a standard stock solution of 1.0 g/L is prepared.
Enantiomeric standard solutions
Two carnitine enantiomer stocks (1.0 g/L): 0.01 g (accurate to 0.1 mg) of dextro-carnitine and levo-carnitine standard substance are respectively and accurately weighed, dissolved by absolute ethyl alcohol and subjected to constant volume of 10 mL to prepare 1.0 g/L standard stock solution.
Mixed working solutions of two carnitine enantiomers: accurately sucking a certain amount of standard solution, and gradually diluting with anhydrous ethanol to obtain mixed working solution of 0.50, 1.00, 2.00, 5.00, and 20.00 mg/L.
Preparation of derivatizing agent solution and stop solution
Derivatizing reagent (L-alanine- β -naphthylamine): 0.45 g L-alanine-beta-naphthylamine was weighed, dissolved in acetonitrile and brought to 100 mL. Catalyst I: 0.50 g of triethylamine is weighed, chloroform is added and mixed evenly, and the volume is adjusted to 100 mL. Catalyst II: 0.50 g of butyl chloroformate is weighed, added with chloroform, mixed evenly and metered to 100 mL. Reaction stop solution (50 mmol/L sodium bicarbonate solution): 0.42 g of sodium bicarbonate is weighed and dissolved in water to a volume of 100 mL.
Sample pretreatment
1.3.1 sample extraction
Weighing 5.00 g (accurate to 0.01 g) of milk powder sample into a 50 mL volumetric flask, adding 20 mL of 0.1 mol/L hydrochloric acid solution, mixing uniformly by vortex for 1min, fully dissolving the sample by ultrasonic treatment for 5min, adding 5mL of 1 mol/L potassium hydroxide solution, mixing uniformly by vortex, saponifying in a water bath at 60 ℃ for 30 min, cooling to room temperature, adding 5mL of 1 mol.L-1And (3) uniformly mixing the hydrochloric acid solution in a vortex mode for 1min, and metering to 50 mL by using 0.1 mol/L hydrochloric acid solution. And (3) putting 1 mL of the extracting solution into a 15 mL centrifuge tube, adding 9 mL of acetonitrile, uniformly mixing for 1min in a vortex mode, putting a part into a 2 mL centrifuge tube, centrifuging for 5min at 15000 r/min, and allowing the obtained supernatant to be purified.
Purification
Putting 1 mL of solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with 3 mL of 10 mmol/L hydrochloric acid aqueous solution, discarding eluent, vacuum drying for 1min, eluting with 2 mL of 4% ammonia-methanol solution, collecting eluent in a 15 mL centrifuge tube, drying with nitrogen at 40 ℃, and collecting residue to be derived.
Derivatisation
Adding 0.5mL of derivatization reagent (4.5 g/L L-alanine-beta naphthylamine acetonitrile solution) into the obtained residues, dissolving the residues by ultrasonic treatment for 1min, uniformly mixing, sequentially adding 0.5mL of catalyst I (5 g/L triethylamine trichloromethane solution) and 0.5mL of catalyst II (5 g/L butyl chloroformate trichloromethane solution), uniformly mixing for 3 min by vortex, standing at 20 ℃ for derivatization reaction for 60 min, adding 2.0 mL of reaction stop solution (50 mmol/L sodium bicarbonate aqueous solution), uniformly mixing by vortex for 1min, centrifuging for 5min at 5000 r/min, transferring the upper aqueous phase into a 100 mL round-bottomed flask, concentrating to near dryness, adding 1 mL of absolute ethyl alcohol into a concentration bottle, fully dissolving by vortex, and feeding the mixture into a sample injection bottle through a 0.22 mu m filter membrane.
Conditions of analysis
A chromatographic column: acquisty Trefoil CEL1(3.0 mm multiplied by 150 mm, 2.5 mu m, filler is fiber)Vitamin-tris (3, 5-dimethylphenylcarbamate), Waters corporation, usa); mobile phase: a is CO2B is 1% ammonia water-methanol solution; gradient elution procedure: 0-7 min (10% B), 7-9 min (10% -28% B), 9-12 min (28% B), 12-13 min (28% -10% B), 13-14 min (10% B); flow rate: 1.0 mL/min; and (3) system backpressure: 13.8 MPa; column temperature: 40 ℃; sample introduction amount: 5 muL; detection wavelength: 244 nm.
Preparation of Standard working solutions
Respectively and precisely transferring 0.5mL of mixed working solution of two carnitine enantiomers in 1.2.2 into a 10 mL centrifuge tube, drying by nitrogen, and deriving according to a 1.3.3 method to prepare standard solutions with the concentrations of 0.25, 0.50, 1.00, 2.50 and 10.00 mg/L, which are equivalent to two carnitine enantiomers with the concentrations of 25, 50, 100, 250 and 1000 mg/kg in a sample.
Results and discussion
2.1 purification Condition optimization
The application considers the purifying effects of three purifying columns, namely a mixed weak cation exchange solid phase extraction column (Oasis WCX 3 mL, 60 mg), a polymer solid phase extraction column (Oasis HLB 3 mL, 60 mg) and an HLB mixed strong cation exchange solid phase extraction column (Oasis MCX 3 mL, 60 mg). The addition concentration of the L-carnitine in 3 identical milk powder samples with zero addition is 10 mg-10.2 mL of the enantiomer mixed working solution is extracted according to the method of '1.3.1', then purified by respectively adopting the 3 different solid phase extraction small columns, and then derived according to the method of '1.3.3', and a derived product is detected on a machine. The experimental results show that the chromatogram of the eluent purified by WCX and HLB does not have a carnitine enantiomer chromatographic peak, while the chromatogram of the eluent purified by MCX has a carnitine enantiomer chromatographic peak (figure 1), so that the MCX column is adopted to purify the carnitine enantiomer.
Optimization of derivatization reaction conditions
Domestic and foreign literature reports that chloroformic acid butyl ester[17]Chloroformate[10]Can be used as a reaction derivatization agent, and butyl chloroformate is selected as the reaction derivatization agent because ethyl chloroformate has strong toxicity and low boiling point and is not easy to purchase.The present application examined the effect of the addition of derivatizing agents on the derivatization effect. Transferring 0.5mL of 3 parts of mixed working solution of carnitine enantiomer with 20.0 mg/L of linear peak into 3 centrifuge tubes, drying by nitrogen, correspondingly adding 0.5mL of derivatization reagents (4.5, 9.0 and 13.5 g/L) with different concentrations, respectively, derivatizing according to the method of '1.3.3', and performing on-line detection. The results show that as the concentration of derivatizing reagent increases, there is no significant increase in derivatizing product, indicating that all derivatizing reagents are used in excess, so that a concentration of 4.5 g/L derivatizing reagent is used in the present application.
The application considers the influence of the derivatization temperature on the derivatization product, selects the reaction time of 30 min, and compares the influence of two carnitine enantiomers derivatization reactions at 4 ℃, 20 ℃, 40 ℃ and 60 ℃, and the result is shown in figure 2. The derivatization products increased rapidly with increasing derivatization temperature starting from 4 ℃ for both carnitine enantiomers. Whereas when the derivatization temperature exceeds 20 ℃, the carnitine-derivatized product decreases sharply. The present application therefore selects 20 ℃ as the derivatization reaction temperature.
The derivatization temperature was chosen to be 20 ℃ and the influence of different derivatization reaction times (10, 30, 60, 90 min) on the derivatization of the two carnitine enantiomers was examined further. The results are shown in figure 3, the carnitine enantiomer derivative products increased gradually with increasing derivatization time, and there was no significant increase in derivative products when the derivatization time exceeded 60 min. The present application therefore selects 60 min as the derivatization reaction time. Therefore, the main factors influencing the derivatization reaction are optimized, and the optimal derivatization reaction condition is found to be that the reaction is carried out for 60 min at the temperature of 20 ℃, and the derivatization reaction of two carnitine enantiomers is sufficient.
Optimization of chromatography columns
The two carnitine enantiomers resolved in the research are not easy to separate due to very similar structures. Therefore, 3 chiral separation chromatographic columns of the same specification, Acquity Trefoil AMY1(3.0 mm × 150 mm, 2.5 μm), Acquity Trefoil CEL1(3.0 mm × 150 mm, 2.5 μm) and Acquity Trefoil CEL2 (3.0 mm × 150 mm, 2.5 μm) are selected to investigate the splitting effect of the two carnitine enantiomers. Transferring 0.5mL of 3 parts of mixed working solution of carnitine enantiomer with 20.00 mg/L of the highest linear point into 3 centrifuge tubes, drying by nitrogen, performing derivatization according to a method of '1.3.3', and detecting a derivatization product on a machine. The results show that when the chiral chromatographic columns of Acquity Trefoil AMY1 and Acquity Trefoil CEL2 are used for separation, the two carnitine enantiomers cannot be completely separated, and the chromatographic peak shapes are poor, while when the chiral chromatographic column of Acquity Trefoil CEL1 is used for separation, the separation degree is good, and the chromatographic peak shapes are sharp (figure 4). Therefore, the application selects an Acquity Trefoil CEL1 chiral chromatographic column to separate the carnitine enantiomers.
Selection of system backpressure
In ultra-high performance combined phase chromatography, the system back pressure is also one of the important factors influencing the separation process, and the main function of the system back pressure is to control CO2In a supercritical fluid state throughout the operation. Under different system back pressures, CO2Supercritical fluids have different solvating capacities for the sample. When the back pressure is increased, the density of the supercritical fluid is increased, the solvating power is enhanced, and the column pressure is increased. The application takes CO2And 1% ammonia water-methanol solution as mobile phase, and examining the separation effect of two carnitine enantiomers at backpressure of 10.3, 13.8, 17.2 and 20.7 MPa respectively. The results show that as the backpressure increases, the analyte peak time decreases. When the backpressure is 10.3 MPa, the broadening of the peak shapes of two carnitine enantiomers is obvious; when the back pressure is increased to 13.8 MPa, the separation degree of two carnitine enantiomers is good, and chromatographic peak shapes are symmetrical; when the back pressure is increased to 17.2 MPa, the separation degree of two carnitine enantiomers is reduced; when the back pressure is continuously increased to 20.7 MPa, the system gives an overpressure alarm. The dynamic back pressure is selected to be 13.8 MPa according to the application by comprehensively considering the chromatographic peak shape and the separation degree.
Optimization of chromatographic column temperature
Column temperature is another important factor affecting supercritical fluids of carbon dioxide. As the temperature of the chromatographic column increases, the viscosity of the supercritical carbon dioxide fluid decreases, the density decreases, the solvating power decreases, and the dissolving and exchanging capacity of the supercritical carbon dioxide fluid for the target compound may be weakened, so that the retention time of the target compound is increased. Considering that the maximum recommended operating temperature of the Acquity Trefoil CEL1 chiral chromatographic column is 40 ℃, and carbon dioxide needs to enter the supercritical carbon dioxide state when the temperature exceeds 31 ℃ and the pressure exceeds 7.38 MPa, the application examines the influence of the temperature of the chromatographic column being 31 ℃, 35 ℃ and 40 ℃ respectively on the separation of the target object under the condition that the system backpressure is 13.8 MPa (fig. 5). The results show that as the column temperature increases, the retention time of the target substance is gradually prolonged, and the separation degree is improved. Compared with the column temperature of 31 ℃ and 35 ℃, when the column temperature is 40 ℃, the separation degree of two carnitine enantiomers is better, and the chromatographic peak shapes are more symmetrical. Therefore, the optimal column temperature was selected to be 40 ℃ for this application.
Linear range and quantitative limit
The serial mixed standard solutions of derivatized D-carnitine and L-carnitine were analyzed under "1.4" chromatographic conditions. And (5) drawing a standard curve by taking the peak area (Y) of the standard substance as a vertical coordinate and the corresponding mass concentration (X) as a horizontal coordinate, and solving a regression equation and a correlation coefficient. The result shows that the two carnitine enantiomers have good linear relation in the mass concentration range of 0.25-10.0 mg/L, and the correlation coefficient is more than 0.999. The standard substance is added into the infant formula milk powder sample without L-carnitine, the determination is carried out according to the application, the quantitative Limit (LOQ) is calculated according to the signal to noise ratio S/N =10, and the LOQ of the L-carnitine and the L-carnitine is 25 mg/kg.
Recovery, accuracy and precision
The method of adding standard solution into the infant formula milk powder without L-carnitine is adopted to carry out the determination of the addition recovery rate and the determination of the precision of the method, the addition levels of the L-carnitine and the L-carnitine are 25, 50 and 250 mg/kg, the parallel determination is carried out for 6 times, and the addition recovery rate and the Relative Standard Deviation (RSD) are calculated, and the results are shown in Table 1. The recovery rate of two carnitine enantiomers is within the range of 93.0-110%, the relative standard deviation is within the range of 3.9-5.5%, and the recovery rate meets the requirements of SN/T0001-one 2016 [18]The requirement on the analysis result can meet the requirements of L-carnitine content determination and purity analysis in infant formula milk powder samples.
TABLE 1 spiked recovery and relative standard deviation of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) in infant formula samples: (n=6）
2.8 application of the method
2.8.1 actual sample detection
To examine the effectiveness and practicality of the present application, the established methods were used to measure the content of D-carnitine and L-carnitine in 20 commercial infant formulas. As a result, L-carnitine was detected in 20 milk powders, but D-carnitine was not detected. Wherein the L-carnitine is detected in 10 parts of infant formula milk powder without label values, and the detection values are respectively 4.14-19.45 mg/100 g; wherein the content of L-carnitine in 10 parts of infant formula milk powder with label value is 5.95-22.9 mg/100g, which all meet the national standard of food safety GB 28050-2011[19]And GB 13432-2013[20]The requirement that the actual content of the nutrient components is more than or equal to 80 percent of the marked value is specified.
Resolution of racemic standard
The method established herein is adopted to carry out the resolution and the determination of the purchased carnitine racemate standard substance, as shown in figure 6a, 2 carnitine enantiomers realize effective resolution within 11 min, and the retention time sequence of chromatographic peaks sequentially comprises the following steps: d-carnitine and L-carnitine. The respective ratios of the two carnitine enantiomers were calculated with the sum of the areas of the peaks of D-carnitine and L-carnitine as 100%, wherein D-carnitine accounted for 51.2% and L-carnitine accounted for 48.8% of the carnitine racemate.
Conclusion
According to the method, the main parameters such as an optimized pretreatment method, a derivative condition, an instrument chromatographic condition and the like are considered, and a method for separating two enantiomers of carnitine and determining the content of the enantiomer in infant formula milk powder by using an ultra-high performance synthetic phase chromatography is established. Extracting a milk powder sample by using a hydrochloric acid solution, saponifying the milk powder sample by using a potassium hydroxide solution in a water bath, purifying the milk powder sample by using a cation exchange solid phase extraction column, separating the milk powder sample by using an Acquity Trefoil CEL1 chiral chromatographic column after derivatization, performing gradient elution by using a supercritical carbon dioxide-1% ammonia water methanol solution as a mobile phase, and quantifying the milk powder sample by using an external standard method. An addition recovery experiment is carried out within the range of 25-250 mg/kg, the standard recovery rate of 2 carnitine enantiomers is 93.0-110%, the RSD is 3.9-5.5%, the content of the dextro-carnitine and the levo-carnitine in 20 parts of commercially available infant formula milk powder samples is determined by the method, the dextro-carnitine is not detected, and the detected amount of the levo-carnitine is 4.14-22.9 mg/100 g.
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[20] GB 13432-.
Claims (9)
1. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on the ultra-high performance synthetic phase chromatography technology comprises the steps of sample extraction and standard solution preparation, wherein the sample extraction comprises the steps of extraction, purification and derivation; the method is characterized in that the method also comprises the steps of respectively carrying out ultra-performance synthesis chromatography analysis on the standard solution and the sample solution, making a standard curve and calculating the content and the purity of L-carnitine and D-carnitine in the sample solution through the standard curve;
the extraction step comprises: weighing a milk powder sample, adding a hydrochloric acid solution, uniformly mixing in a vortex manner, fully dissolving the sample, adding a potassium hydroxide solution, uniformly mixing in a vortex manner, and fixing the volume by using the hydrochloric acid solution; adding acetonitrile into an extracting solution centrifuge tube, uniformly mixing by vortex, centrifuging, and purifying the obtained supernatant;
the purification step comprises: putting the solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with a hydrochloric acid aqueous solution, removing an eluent, vacuum-drying, eluting with an ammonia-methanol solution, collecting the eluent in a centrifugal tube, drying with nitrogen, and collecting the residue to be derived;
the derivation step: adding a derivatization reagent into the obtained residue, dissolving the residue by ultrasonic, uniformly mixing, sequentially adding a catalyst I and a catalyst II, uniformly mixing by vortex, standing for derivatization reaction, adding a reaction termination solution, uniformly mixing by vortex, centrifuging, transferring an upper aqueous phase, concentrating to be nearly dry, adding absolute ethyl alcohol into a concentration bottle, fully dissolving by vortex, and filtering by a filter membrane into a sample injection bottle; the derivatization reagent is an L-alanine-beta naphthylamine acetonitrile solution, the catalyst I is a triethylamine trichloromethane solution, the catalyst II is a butyl chloroformate trichloromethane solution, and the reaction termination solution is a sodium bicarbonate aqueous solution;
the ultra-high performance phase-combination chromatographic analysis conditions are as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 5-dimethylphenyl carbamate);
mobile phase: a is CO2B is 1% (v/v) ammonia-methanol solution;
gradient elution procedure: 0-7 min, and 90% (v/v) A-10% (v/v) B of mobile phase; 7-9 min, and 90-72% (v/v) A-10% -28% (v/v) B of mobile phase; 9-12 min, and 72% (v/v) of mobile phase A-28% (v/v) B; 12-13 min, and 72-90% (v/v) A-28% -10% (v/v) B of a mobile phase; 13-14 min, and 90% (v/v) A-10% (v/v) B of a mobile phase; flow rate: 1.0 mL/min;
and (3) system backpressure: 13.8 MPa; column temperature: at 40 ℃; sample introduction amount: 5 muL; detection wavelength: 244 nm.
2. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 1, wherein the extraction steps are as follows:
weighing 5 g of milk powder sample in a 50 mL volumetric flask, adding 20 mL of 0.1 mol/L hydrochloric acid solution, uniformly mixing in a vortex manner for 1min, carrying out ultrasonic treatment for 5min to fully dissolve the sample, adding 5mL of 1 mol/L potassium hydroxide solution, uniformly mixing in a vortex manner for 1min, and fixing the volume to 50 mL by using 0.1 mol/L hydrochloric acid solution; and (3) putting 1 mL of the extracting solution into a 15 mL centrifuge tube, adding 9 mL of acetonitrile, uniformly mixing for 1min in a vortex manner, centrifuging for 5min at the speed of 15000 r/min, and purifying the obtained supernatant.
3. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 2, wherein the purification steps are as follows:
putting 1 mL of solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with 3 mL of 10 mmol/L hydrochloric acid aqueous solution, discarding eluent, vacuum drying for 1min, eluting with 2 mL of 4% ammonia-methanol solution, collecting eluent in a 15 mL centrifuge tube, drying with nitrogen at 40 ℃, and collecting residue to be derived.
4. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 3, wherein the derivation steps are as follows:
adding 0.5mL of derivatization reagent into the obtained residues, dissolving the residues by ultrasonic treatment for 1min, uniformly mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, uniformly mixing in a vortex manner for 3 min, standing at 20 ℃ for derivatization reaction for 60 min, adding 2.0 mL of reaction termination solution, uniformly mixing in a vortex manner for 1min, centrifuging at 5000 r/min for 5min, transferring the upper-layer water phase into a 100 mL round-bottom flask, concentrating to be nearly dry, adding 1 mL of absolute ethanol into a concentration bottle, fully dissolving in a vortex manner, and feeding the mixture into a sample injection bottle through a 0.22 mu m filter membrane;
the derivatization reagent is 4.5 g/L L-alanine-beta naphthylamine acetonitrile solution, the catalyst I is 5.0 g/L triethylamine trichloromethane solution, the catalyst II is 5.0 g/L butyl chloroformate trichloromethane solution, and the reaction termination solution is 50 mmol/L sodium bicarbonate aqueous solution.
5. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 1, wherein the extraction steps are as follows:
weighing 5.00 g of milk powder sample to be accurate to 0.01 g, placing the milk powder sample in a 50 mL volumetric flask, adding 20 mL of 0.1 mol/L hydrochloric acid solution, mixing uniformly by vortex for 1min, fully dissolving the sample by ultrasonic for 5min, adding 5mL of 1 mol/L potassium hydroxide solution, mixing uniformly by vortex, saponifying in a 60 ℃ water bath for 30 min, cooling to room temperature, adding 5mL of 1 mol.L-1Hydrochloric acid dissolutionUniformly mixing the solution for 1min in a vortex mode, and fixing the volume to 50 mL by using 0.1 mol/L hydrochloric acid solution; and (3) putting 1 mL of the extracting solution into a 15 mL centrifuge tube, adding 9 mL of acetonitrile, uniformly mixing for 1min in a vortex mode, putting a part into a 2 mL centrifuge tube, centrifuging for 5min at 15000 r/min, and allowing the obtained supernatant to be purified.
6. The method for separating and determining carnitine enantiomer in infant formula based on ultra-high performance synthetic phase chromatography technology according to claim 5, wherein the purification steps are as follows:
putting 1 mL of solution to be purified in an activated MCX cation solid-phase extraction column, loading, eluting with 3 mL of 10 mmol/L hydrochloric acid aqueous solution, discarding eluent, vacuum drying for 1min, eluting with 2 mL of 4% ammonia-methanol solution, collecting eluent in a 15 mL centrifuge tube, drying with nitrogen at 40 ℃, and collecting residue to be derived.
7. The method for splitting and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 6, wherein the derivation steps are as follows:
adding 0.5mL of derivatization reagent into the obtained residues, dissolving the residues by ultrasonic treatment for 1min, uniformly mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, uniformly mixing in a vortex manner for 3 min, standing at 20 ℃ for derivatization reaction for 60 min, adding 2.0 mL of reaction termination solution, uniformly mixing in a vortex manner for 3 min to terminate the reaction, centrifuging at 5000 r/min for 5min, transferring the upper-layer water phase into a 100 mL round-bottom flask, concentrating to be nearly dry, adding 1 mL of absolute ethyl alcohol into a concentration bottle, fully dissolving in a vortex manner, and introducing a sample into a small bottle through a 0.22 mu m filter membrane;
the derivatization reagent is 4.5 g/L L-alanine-beta naphthylamine acetonitrile solution, the catalyst I is 5.0 g/L triethylamine trichloromethane solution, the catalyst II is 5.0 g/L butyl chloroformate trichloromethane solution, and the reaction termination solution is 50 mmol/L sodium bicarbonate aqueous solution.
8. The method for separating and determining carnitine enantiomer in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology of claim 7, wherein the standard solution is prepared by the following steps:
1) 1.0 g/L two carnitine enantiomer stocks were prepared: accurately weighing 0.01 g of standard dextro-carnitine and levo-carnitine respectively to be accurate to 0.1 mg; dissolving with absolute ethyl alcohol, and diluting to a constant volume of 10 mL to prepare a standard stock solution of 1.0 g/L;
2) preparation of mixed working solutions of two carnitine enantiomers: accurately sucking a certain amount of standard solution, and gradually diluting to mixed working solution of 0.50, 1.00, 2.00, 5.00 and 20.00 mg/L by using absolute ethyl alcohol;
3) preparation of standard solution: precisely transferring 0.5mL of mixed working solution of two carnitine enantiomers into a 10 mL centrifuge tube, drying by nitrogen, and derivatizing according to the method of claim 3 to obtain standard solutions with the concentrations of 0.25, 0.50, 1.00, 2.50 and 10.00 mg/L, which are equivalent to samples containing 25, 50, 100, 250 and 1000 mg/kg of two carnitine enantiomers.
9. The method for separating and measuring carnitine enantiomers in infant formula milk powder based on ultra-high performance synthetic phase chromatography technology according to claim 7, wherein the recovery rate of two carnitine enantiomers is in the range of 93.0% to 110%, and the relative standard deviation is in the range of 3.9% to 5.5%.
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