CN113433257B - Method for separating and measuring carnitine enantiomer in health food - Google Patents
Method for separating and measuring carnitine enantiomer in health food Download PDFInfo
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Abstract
The invention relates to a method for separating carnitine enantiomer and a method for detecting the content of carnitine in health-care food, in particular to a method for measuring the content of L-carnitine and D-carnitine in the health-care food. The method adopts ultra-high performance synthetic phase chromatography to resolve carnitine enantiomers, and determines the residual quantity of L-carnitine and D-carnitine enantiomers in the health food. The sample is subjected to ultrasonic extraction by absolute ethyl alcohol, derivatization is carried out, then, Acquity Trefoil CEL1 chiral chromatographic column separation is carried out, gradient elution is carried out by taking cosolvents such as supercritical carbon dioxide-1% (v/v) ammonia water methanol and the like as mobile phases, and quantification is carried out by an external standard method. The method has the characteristics of rapidness, accuracy, high separation efficiency, high repeatability, good stability and the like, can be used for simultaneously determining the content and the purity of the L-carnitine in the health-care food, and provides scientific support for development and use of chiral drugs and formulation of related regulations.
Description
Technical Field
The invention relates to a separation method of carnitine enantiomer and a carnitine content detection method in health food, in particular to a method for determining the content of L-carnitine and D-carnitine in health food.
Background
Carnitine has the chemical name β -hydroxy- γ -trimethylaminebutanoic acid, which contains 1 chiral carbon atom in the molecule (see fig. 1) and has two stereoisomers: including the D-carnitine (D-carnitine) enantiomer and the L-carnitine (L-carnitine) enantiomer. Among them, L-Carnitine (also called Carnitine or L-Carnitine), which is a kind of amino acid that promotes the conversion of fat into energy, is widely distributed in liver organs [1] And is also an essential substance for the metabolism of fatty acid in mitochondria, and the deficiency of the substance can directly cause the synthesis deficiency of energy, and the symptoms of easy fatigue, myasthenia and the like appear. At present, the L-carnitine used in the commercial health care products is mainly obtained from an industrial chemical synthesis method, but the purity of the L-carnitine produced by the method is generally not up to the requirement, so that the L-carnitine health care products contain D-carnitine [2] . D-carnitine is not only ineffective, but even toxic. When D-carnitine is taken into human body, the muscle is damaged, and the symptoms of myasthenia, myalgia, amyotrophy and the like are caused [3] . Therefore, there is an urgent need to establish an analytical method for determining the content and purity of L-carnitine in health food.
The existing L-carnitine detection method mainly comprises a spectrophotometry [4-5] High Performance Liquid Chromatography (HPLC) [6-7] High performance liquid chromatography-tandem mass spectrometry [5,8-10] And the like. However, these reported methods are basically general methods for detecting the content of L-carnitine, and there are few reports on the resolution of two enantiomers of D-carnitine and L-carnitine and the purity analysis of L-carnitine. Zhai Xufeng [11] And congratulating on great joy [2] And the baseline separation of the L-carnitine and the D-carnitine is realized by adopting reverse high performance liquid chromatography, but the methods generally have the problems of long analysis time, large consumption of organic reagents and the like. In recent yearsUltra-high performance phase-locked chromatography (UPC) 2 ) The technology is widely concerned, and the technology supplements the technical characteristics of Ultra Performance Liquid Chromatography (UPLC) and the traditional Supercritical Fluid Chromatography (SFC) and improves various hardware of the traditional SFC [12] The supercritical carbon dioxide and a small amount of organic solvent (acetonitrile, methanol, isopropanol and the like) are used as the mobile phase, and the required system resolution can be obtained more accurately by adjusting and controlling the temperature of a tiny chromatographic column, the system backpressure and the change of the mobile phase, so that the retention time and the separation degree of the substance to be detected are accurately regulated and controlled, and the precision, the stability and the reliability of the system are improved [13] . The study shows that UPC 2 The technology is more suitable for analyzing structural analogues and isomers which are difficult to process by the traditional liquid chromatography, and has been successfully used for triazole pesticides [14] Phenolic acid compound [15] Pigment, pigment [16] Phenol essential oil [17] And the like. Currently, UPC 2 The application of the technology to the resolution and content determination of carnitine enantiomer is only reported.
Disclosure of Invention
In order to solve the problems existing in the resolution of enantiomers by the conventional liquid chromatography, the research establishes a method for resolving two enantiomers of carnitine by the ultra-high performance synthetic phase chromatography and determining the content of the enantiomers in health-care food. The stability of derivative products of two standard carnitine enantiomer products is examined through experiments, the main parameters of the extraction method, the derivative condition, the instrument chromatographic separation condition and the like of the carnitine enantiomer in the health-care food are optimized, and the established optimization method is utilized to analyze and measure the standard carnitine racemate and the commercially available health-care food. Meanwhile, the content of the dextro-carnitine in the health-care food sample is low in consideration of being a toxic byproduct; the L-carnitine is the main content and has high content, so the LOQ of the L-carnitine is set to be 10mg/kg, and the LOQ of the L-carnitine is increased to 50 mg/kg. The method has the characteristics of high analysis speed, good separation effect, low consumption of organic solvent, high sensitivity and the like, and provides a reference method for determining the content and the purity of the L-carnitine in the health food.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method comprises the steps of extracting a health food sample, preparing a standard solution and deriving, respectively carrying out ultra-high performance synthetic chromatography analysis on the derived standard solution and the sample solution, making a standard curve and calculating the content and purity of L-carnitine and D-carnitine in the sample solution through the standard curve; the conditions for performing the ultra-performance combined chromatography analysis on the standard solution and the sample solution are as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 5-dimethylphenyl carbamate);
mobile phase: a is CO 2 B 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 a 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;
and (3) system backpressure: 13.8 MPa; flow rate: 1.0 mL/min; sample introduction amount: 5 mu L of the solution; column temperature: 40 ℃; detection wavelength: 244 nm.
Preferably, the sample extraction steps are as follows: taking 20 health product tablets, capsules or granules, grinding, accurately weighing 1.00g (accurate to 0.01g) of sample, placing the sample in a 25mL volumetric flask, adding a proper amount of absolute ethyl alcohol, carrying out ultrasonic extraction for 20min, cooling to room temperature, adding absolute ethyl alcohol to a constant volume, taking a proper amount of constant solution to a centrifugal tube, carrying out high-speed centrifugation for 5min, and obtaining supernatant to be further derived to prepare sample solution.
Preferably, the standard solution is prepared by the following steps: 0.5mL of mixed working solution of L-carnitine and D-carnitine enantiomers is transferred into a 10mL centrifuge tube and further derivatized to prepare standard solutions with the concentrations of 0.20, 0.40, 1.00, 2.00, 5.00, 10.00 and 20.00 mg/L.
Preferably, the derivatization step is as follows:
taking 0.5mL of supernatant or mixed working solution into a centrifuge tube, adding 0.5mL of derivatization reagent, carrying out vortex mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, carrying out vortex mixing for 3min, derivatizing at 20 ℃ for 60min, adding 2.0mL of reaction termination solution, carrying out vortex mixing for 1min, centrifuging for 5000r/min and 5min, transferring the upper-layer water phase into a 100mL round-bottom flask, concentrating to be nearly dry, adding 1mL of absolute ethyl alcohol into a concentration bottle, carrying out vortex dissolving fully, and putting into a sample injection vial through a 0.22 mu m filter membrane;
the derivatization reagent is 0.45g/100mL of L-alanine-beta-naphthylamine-acetonitrile solution, the catalyst I is 0.50g/100mL of triethylamine-trichloromethane solution, the catalyst II is 0.50g/100mL of butyl chloroformate-trichloromethane solution, and the reaction termination solution is 50mmol/L of sodium bicarbonate-water solution.
Preferably, the recovery rate of two carnitine enantiomers, L-carnitine and D-carnitine, is in the range of 86.0% to 110%, and the relative standard deviation (RSD, n ═ 6) is in the range of 4.3% to 7.0%.
Further, the invention also discloses a kit for separating and determining carnitine enantiomers in health food based on the ultra-high performance synthesis chromatography technology, wherein the kit comprises a derivatization reagent, a catalyst I, a catalyst II and a reaction termination solution; the derivatization reagent is 0.45g/100mL of L-alanine-beta-naphthylamine-acetonitrile solution, the catalyst I is 0.50g/100mL of triethylamine-trichloromethane solution, the catalyst II is 0.50g/100mL of butyl chloroformate-trichloromethane solution, and the reaction termination solution is 50mmol/L of sodium bicarbonate-water solution.
The invention establishes a method for simultaneously measuring the content and the purity of L-carnitine in a health-care product, and carries out analysis and measurement on a health-care product sold in the market and a racemate carnitine standard product. The method has the characteristics of rapidness, accuracy, high separation efficiency, good repeatability, good stability and the like, and provides a reference method for determining the content and the purity of the L-carnitine in the health food.
Drawings
FIG. 1 is a chemical structural formula of carnitine.
FIG. 2 is a stability study (anhydrous ethanol solution) of standard solutions of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) after derivatization for 60 days.
FIG. 3 is a graph comparing the separation effect of 3 columns on two carnitine enantiomers (1% ammonia-methanol solution, 13.8 MPa).
FIG. 4 shows the effect of derivatization temperature and time on the derivatization reaction (D-carnitine and L-carnitine).
FIG. 5 is a graph showing the effect of different cosolvents on the separation of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) (13.8MPa, 40 ℃ C.).
FIG. 6 is a graph showing the effect of different system back pressures on the separation of D-carnitine (D-carnitine) and L-carnitine (L-carnitine) (1% (v/v) in aqueous ammonia methanol, 40 ℃ C.).
FIG. 7 is a graph showing the effect of column temperature of different systems on the separation effect of D-carnitine and L-carnitine (L-carnitine) (1% (v/v) ammonia methanol solution, 13.8 MPa).
Detailed Description
Experimental part
1.1 instruments, materials and reagents
Ultra-high performance phase-compatible chromatographs (wawter, usa); bench centrifuge (Thermo corporation, usa); r215 rotary evaporator (Buchi, switzerland); AE260 electronic balance (Mettler, switzerland); MS2 vortex mixer (Shanghai medical instrument factory); ultra-pure water purification system (Elga, uk); microfiltration membrane (0.22 μm, organic phase); nitrogen blowing apparatus (tokyo physical & chemical company, japan).
Methanol, acetonitrile, absolute ethanol (chromatographically pure, Scharlau, spain); sodium bicarbonate, ammonia water, chloroform, triethylamine and butyl chloroformate (guaranteed reagent); 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.
Carnitine racemate (purity 98% or more, SIGMA company, usa).
Two enantiomers: l-carnitine (99.0% purity, Dr.E., Germany), D-carnitine (99.8% purity, CATO, USA).
Standard solution and reagent preparation
1.2.1 racemate standard solution carnitine stock (1.0 g/L): 0.01g (accurate to 0.1mg) of the carnitine racemate standard is accurately weighed respectively, dissolved by absolute ethyl alcohol and subjected to constant volume to 10mL, so as to prepare 1.0g/L standard stock solution.
Enantiomeric standard solutions
Two carnitine enantiomer stocks (1.0 g/L): 0.01g (accurate to 0.1mg) of L-carnitine and D-carnitine standard substance are accurately weighed respectively, dissolved by absolute ethyl alcohol and subjected to constant volume to 10mL, and then 1.0g/L standard stock solution is prepared.
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 0.40, 0.80, 2.00, 4.00, 10.00, 20.00, and 40.00 mg/L.
Preparation of derivatizing agent solution and stop solution
Derivatization reagent (L-alanine- β naphthylamine): 0.45g L-alanine-. beta.naphthylamine was weighed, dissolved with acetonitrile and made to volume of 100 mL. Catalyst I: 0.50g of triethylamine is weighed, chloroform is added and mixed evenly, and the volume is adjusted to 100 mL. Catalyst II: 0.50g of butyl chloroformate is weighed, and chloroform is added to the butyl chloroformate, the mixture is mixed evenly and the volume is adjusted to 100 mL. Reaction stop solution (50mmol/L sodium bicarbonate solution): 0.42g of sodium bicarbonate is weighed and dissolved in water to a volume of 100 mL.
Sample pretreatment
1.3.1 sample extraction
Taking 20 health product tablets, capsules or granules, grinding, accurately weighing 1.00g (accurate to 0.01g) of sample, placing the sample in a 25mL volumetric flask, adding a proper amount of absolute ethyl alcohol, carrying out ultrasonic extraction for 20min, cooling to room temperature, adding absolute ethyl alcohol to a constant volume, taking a proper amount of constant solution to a centrifugal tube, carrying out high-speed centrifugation for 5min, and obtaining supernatant for further derivatization.
1.3.2 derivatization
Taking 0.5mL of supernatant into a centrifuge tube, adding 0.5mL of derivatization reagent, carrying out vortex mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, carrying out vortex mixing for 3min, derivatizing at 20 ℃ for 60min, adding 2.0mL of reaction termination solution (50mmol/L sodium bicarbonate aqueous solution), carrying out vortex mixing for 1min, centrifuging for 5000r/min and 5min, transferring the upper-layer water phase into a 100mL round-bottom flask, concentrating to be nearly dry, adding 1mL of anhydrous ethanol into a concentration bottle, carrying out vortex dissolving fully, and feeding into a sample injection vial through a 0.22 mu m filter membrane.
1.3.3 conditions of analysis
A chromatographic column: acquisty Trefoil CEL1(3.0 mm. times.150 mm, 2.5 μm, filler cellulose-tris (3, 5-dimethylphenylcarbamate), Waters corporation, USA); mobile phase: a is CO 2 B is 1% (v/v) ammonia 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); and (3) system backpressure: 13.8 MPa; flow rate: 1.0 mL/min; sample introduction amount: 5 mu L of the solution; column temperature: 40 ℃; detection wavelength: 244 nm.
Preparation of Standard Curve
0.5mL of mixed working solution of two carnitine enantiomers is transferred into a 10mL centrifuge tube, and then derivatization is carried out according to the method of 1.3.2 to prepare standard solutions with the concentrations of 0.20, 0.40, 1.00, 2.00, 5.00, 10.00 and 20.00 mg/L.
Results and discussion
2.1 examination of stability
Accurately transferring 7 parts of 0.5mL mixed working solution of 4mg/L carnitine enantiomer into 7 centrifuge tubes, derivatizing, transferring into 7 sample injection vials sealed with aluminum caps, sealing with sealing films, storing at-20 deg.C, transferring to UPC with scratch one by one before testing, and transferring to UPC with scratch 2 In dedicated injection vials. The content of standard solutions of carnitine enantiomers immediately after derivatization and that of standard solutions of carnitine enantiomers derivatized and stored for 1, 3,5, 7, 14, 30, 60d, respectively, were plotted and compared. The results show that the content change of 2 carnitine enantiomers after derivatization is less than 10% within 30d after standing at-20 ℃, and the content change is more than 10% after standing for 60 days (figure 2), which shows that the solution after derivatization of 2 carnitine enantiomers is relatively stable within 30 d.
Optimization of extraction method
Acetonitrile is commonly used in literature reports [6-7] Anhydrous ethanol [4] Methanol, methanol [10] Carnitine is extracted. The extraction effect of the 3 reagents is considered, 3 identical L-carnitine-containing health-care product samples are selected, extracted by the 3 different reagents respectively, derived according to a method of 1.3.2, and subjected to on-machine detection. The experimental result shows that the extraction efficiency of acetonitrile, absolute ethyl alcohol and methanol is adopted13.4%, 98.5% and 70.3% respectively. Therefore, the invention adopts absolute ethyl alcohol for extraction. In addition, the experiment compares the extraction modes of oscillation and ultrasonic wave, 2 same samples of the L-carnitine-containing health-care product are selected, extracted by adopting the extraction modes of oscillation and ultrasonic wave respectively, derived according to the method of '1.3.2', and then subjected to machine-on detection. The results showed that the oscillation extraction efficiency was 54.5% and the ultrasonic extraction efficiency was 96.8%, so ultrasonic extraction was selected for this experiment. Meanwhile, the extraction efficiency of 10min, 20min, 30min and 60min of ultrasonic extraction time is also considered, 4 same samples of the L-carnitine-containing health-care product are selected, extracted by ultrasonic for different time, derived according to the method of 1.3.2 and detected by a machine. The result shows that the ultrasonic extraction is adopted for 10min, the extraction efficiency of the L-carnitine is 70.0%, the ultrasonic extraction time is prolonged to 20min and 91.0%, the ultrasonic extraction time is continuously prolonged to 30min and 60min, the extraction efficiency is respectively 91.8% and 93.2%, and the extraction efficiency of the L-carnitine is not obviously increased, so that the experiment determines that the ultrasonic extraction time of 20min is the best extraction condition.
Optimization of derivatization conditions
Domestic and foreign literature reports ethyl chloroformate [4] Chloroformic acid butyl ester [18] Can be used as a reaction derivatization agent, and butyl chloroformate is selected as the reaction derivatization agent because ethyl chloroformate has low boiling point and strong toxicity and is not easy to purchase. With 0.5mL of derivatizing agent, the derivatization product increased linearly with increasing concentrations of the carnitine enantiomer (0.5, 1.0, 2.0, 5.0, 10.0mg/L), indicating that 0.5mL of derivatizing agent was also completely reactive with the high concentration of carnitine enantiomer, so 0.5mL of derivatizing agent was used.
The invention inspects the influence of the derivatization temperature on the derivatization product, selects the derivatization time to be 30min, and inspects the influence of different derivatization temperatures (4, 20, 40 and 60 ℃) on the derivatization reaction of two carnitine enantiomers, and the result is shown in figure 4. 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 process therefore selects 20 ℃ as the derivatization reaction temperature.
The effect of different derivatization times (10, 30, 60, 90min) on the derivatization of the two carnitine enantiomers was further investigated at room temperature, 20 ℃, and the results are shown in fig. 3. The carnitine enantiomer derivative product gradually increased with the increase of the derivative time, and the derivative product did not increase obviously when the derivative time exceeded 60 min. Therefore, 60min was selected as the derivatization reaction time in the present invention. This experiment compared the effect of the number of extractions of the two carnitine enantiomer derivatization reaction termination solutions (i.e., the extraction reagents) on recovery. 2.0mL of reaction termination solution (i.e., extraction reagent) is sequentially added into the derivatization reaction solution for 2 times of extraction, and experimental results show that compared with the derivatization product extracted for 1 time, the two carnitine enantiomer derivatization products extracted for 2 times are not obviously increased, so that 2mL of derivatization reaction termination solution is adopted for 1 time of extraction.
Optimization of separation conditions
The two carnitine enantiomers resolved in the research are not easy to separate due to very similar structures. Therefore, the separation effect of two carnitine enantiomers was examined by selecting 3 chiral separation columns of Acquity Trefoil AMY1(3.0 mm. times.150 mm, 2.5 μm), Acquity Trefoil CEL1(3.0 mm. times.150 mm, 2.5 μm) and Acquity Trefoil CEL2(3.0 mm. times.150 mm, 2.5 μm) with the same specification. Transferring 0.5mL of 3 parts of mixed working solution of 10mg/L carnitine enantiomer with the highest linear point into 3 centrifuge tubes, derivatizing according to a method of '1.3.3', and detecting a derivative product on a machine. The results show that, when the chiral chromatographic columns of Acquity Trefoil AMY1 and Acquity Trefoil CEL2 were used for separation, the two carnitine enantiomers could not be completely separated and the chromatographic peak shapes were poor, whereas when the chiral chromatographic column of Acquity Trefoil CEL1 was used for separation, the separation degree was good and the chromatographic peak shapes were good (fig. 3). Therefore, the application selects an Acquity Trefoil CEL1 chiral chromatographic column to separate carnitine enantiomers.
Optimization of co-solvents in mobile phase
UPC 2 Supercritical carbon dioxide is adopted as a main mobile phase, a small amount of organic cosolvent is usually added to adjust the polarity of the mobile phase so as to enhance the dissolving capacity and the eluting capacity of a target substance, and different cosolvents have important influences on the separation degree and the peak-off time of the target substance [12] . In order to obtain good separation effect and peakIn the present invention, the co-solvent is examined. An Acquity Trefoil CEL1 chiral chromatographic column is selected, and the influence of 3 cosolvents with different polarities, such as acetonitrile, methanol and isopropanol, on the separation effect of carnitine is compared under the conditions of 13.8MPa of backpressure and 40 ℃ of column temperature. The results show that when acetonitrile, isopropanol and methanol are used, the separation of the two carnitine enantiomers is not good and the broadening is significant, although the separation of methanol as co-solvent is slightly better. The carnitine contains hydroxyl and belongs to a compound with stronger polarity, and the peak shape of the target compound can be obviously improved by adding ammonia water. The separation effect of 0.1% (v/v) ammonia water methanol solution, 0.5% (v/v) ammonia water methanol solution, and 1% (v/v) ammonia water methanol solution as the co-solvent was examined herein. As shown in FIG. 5, the peak shapes and separation effects of both carnitine enantiomers were significantly improved as the concentration of ammonia was increased. Therefore, 1% (v/v) ammonia methanol solution was chosen as a cosolvent in this experiment.
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 is to control the carbon dioxide to be in a supercritical fluid state in the whole operation process. Since the temperature of carbon dioxide exceeds 31 ℃ and the pressure exceeds 7.38MPa, CO 2 The supercritical carbon dioxide state is entered. Therefore, the present invention uses 1% (v/v) ammonia methanol solution as a cosolvent, and examines the effects of separating two carnitine enantiomers at a back pressure of 10.3, 13.8, 17.2 and 20.7MPa respectively at a column temperature of 40 ℃ (FIG. 6). The results show that the analyte peak time is advanced as the system backpressure increases. When the backpressure is 10.3MPa, the broadening of the peak shape of the L-carnitine is obvious; when the backpressure is increased to 13.8MPa, the peak shapes of two carnitine enantiomers are good, and good baseline separation is realized within 11 min; when the back pressure is increased to 17.2MPa, the separation degree of the two carnitines is reduced; when the back pressure is continuously increased to 20.7MPa, the system gives an overpressure alarm. The analysis speed and the separation effect are comprehensively considered, and 13.8MPa is selected as the optimal system backpressure.
Selection of column temperature
Column temperature is another important factor affecting supercritical fluids of carbon dioxide. Followed byThe temperature of the coloring chromatographic column is increased, the viscosity of the carbon dioxide supercritical fluid is reduced, the density is reduced, the solvating capacity is reduced, the dissolving and exchanging capacity of the supercritical fluid for the target compound is weakened, and the retention time of the target compound is increased. Considering that the highest recommended operating temperature of the Acquity Trefoil CEL1 chiral chromatographic column is 40 ℃, carbon dioxide needs to exceed 31 ℃ and the pressure exceeds 7.38MPa, CO 2 The supercritical carbon dioxide state is entered, so the invention examines the influence of the temperature of the chromatographic column in the range of 31-40 ℃ on the separation of the target object under the condition that the system backpressure is 13.8MPa (figure 7). The results show that the retention time of the target is gradually prolonged as the column temperature is increased. When the column temperature is 31 ℃, the separation degree of two carnitine enantiomers is poor; when the column temperature is raised to 35 ℃, the system gives an overpressure alarm; when the column temperature is continuously increased to 40 ℃, the peak shapes of two carnitine enantiomers are good, and good baseline separation is realized within 11 min. Therefore, the optimum column temperature was selected to be 40 ℃.
Linear range and quantitative limit
The series of mixed standard solutions of derivatized L-carnitine and D-carnitine were assayed according to the chromatographic conditions described above. 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 are in a good linear relation within the mass concentration range of 0.2-20.0 mg/L, and the correlation coefficient is larger than 0.999. The standard substance is added into the health-care product sample without carnitine, the determination is carried out according to the method, the quantitative Limit (LOQ) is calculated by taking the signal-to-noise ratio S/N as 10, the LOQ of the obtained dextro-carnitine and levo-carnitine is 10mg/kg, and the content is lower considering that the dextro-carnitine in the health-care food sample is a toxic by-product; the L-carnitine is the main content and has high content, so the LOQ of the L-carnitine is set to be 10mg/kg, and the LOQ of the L-carnitine is increased to 50 mg/kg. .
Recovery, accuracy and precision
The method for adding standard solution into solid health food and liquid health food without carnitine respectively comprises measuring the recovery rate and precision of the method, and adding L-carnitineThe addition levels were 50, 100 and 500mg/kg, respectively, and the addition levels of D-carnitine were 10, 20 and 100mg/kg, respectively, and were measured 6 times in parallel, and the normalized recovery rate and the Relative Standard Deviation (RSD) were calculated, and the results are shown in Table 1. The recovery rates of the two carnitine enantiomers ranged from 86.0% to 110%, and the relative standard deviation (RSD, n ═ 6) ranged from 4.3% to 7.0%. The recovery rate and precision conform to SN/T0001- [19] The requirements of (1) can meet the analysis requirements of samples of different dosage forms, and can be used for detection of daily analysis.
Table 1 spiked recovery and relative standard deviation of 2 carnitine enantiomers in nutraceutical samples (n ═ 6)
2.7 application of the method
2.7.1 testing of actual samples
In order to examine the effectiveness and practicability of the method, the established method was used to determine the content of D-carnitine and L-carnitine in 10 parts of commercially available nutraceutical, wherein 5 parts of tablet nutraceutical, 2 parts of capsule nutraceutical and 3 parts of liquid nutraceutical. The results show that D-carnitine is not detected in 10 health care products, the content of L-carnitine in tablet health care products and capsule health care products is 2.53-27.02 g/100g, the content of L-carnitine in liquid health care products is 1.44g/10mL and 2.40g/15mL respectively, and the content reaches 96-102% of the label value. The European Union stipulates a daily limit of L-carnitine of 2g [20] The dosage of the Chinese L-carnitine is mainly referred to the regulations of European Union, and the recommended dosage of 10 purchased L-carnitine health-care foods is 0.12-1.8 g/day, which meets the requirements of the European Union and the Chinese regulations.
Resolution of racemic standard
The established method is applied to split and measure the purchased carnitine racemate standard substance. The results showed that the carnitine racemate contained both the L-carnitine and D-carnitine enantiomers, 48.8% for L-carnitine and 51.2% for D-carnitine.
Conclusion
The stability of derivative products of two standard carnitine enantiomer products is considered, main parameters such as a pretreatment method, a derivative condition, an instrument chromatographic separation condition and the like of carnitine enantiomers in health food are optimized, and a method for separating the two carnitine enantiomers by using an ultra-high performance synthetic phase chromatography and determining the content of the enantiomers in the health food is established. Meanwhile, the established optimization method is utilized to analyze and measure carnitine racemate standard products and health-care foods sold in the market. . Research results show that the method has the characteristics of high analysis speed, good separation effect, high sensitivity, environmental protection and the like, and can meet the requirements of quick quantification and purity analysis of L-carnitine in health-care food.
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Claims (3)
1. A method for separating and measuring carnitine enantiomer in health-care food based on ultra-high performance synthetic phase chromatography includes such steps as extracting the sample of health-care food, preparing standard solution, deriving, respectively carrying out ultra-high performance synthetic phase chromatography on said standard solution and sample solution, preparing standard curve, and calculating the standard curveL-carnitine,D-content and purity of carnitine; the method is characterized in that the conditions for respectively carrying out the ultra-performance phase-combination chromatographic analysis on the standard solution and the sample solution are as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 5-dimethylphenyl carbamate);
mobile phase: a is CO 2 B is 1% v/v ammonia methanol solution;
gradient elution procedure: 0-7 min, and the mobile phase is 90% v/vA-10% v/vB; 7-9 min, and 90-72% v/vA-10% -28% v/v B of a mobile phase; 9-12 min, and 72% v/vA-28% v/vB of mobile phase; 12-13 min, and 72-90% v/vA-28% -10% v/v B of mobile phase; 13-14 min, and the mobile phase is 90% v/vA-10% v/vB;
and (3) system backpressure: 13.8 MPa; flow rate: 1.0 mL/min; sample introduction amount: 5 muL; column temperature: at 40 ℃; detection wavelength: 244 nm;
the steps of sample extraction are as follows: taking 20 health product tablets, capsules or granules, grinding, accurately weighing 1.00g of sample to be accurate to 0.01g, placing the sample in a 25mL volumetric flask, adding a proper amount of absolute ethyl alcohol, carrying out ultrasonic extraction for 20min, cooling to room temperature, adding absolute ethyl alcohol to a constant volume, taking a proper amount of constant solution to a centrifugal tube, carrying out high-speed centrifugation for 5min, and obtaining supernatant to be further derivatized to prepare a sample solution;
the derivatization steps are as follows:
taking 0.5mL of supernatant or mixed working solution into a centrifuge tube, adding 0.5mL of derivatization reagent, carrying out vortex mixing, sequentially adding 0.5mL of catalyst I and 0.5mL of catalyst II, carrying out vortex mixing for 3min, carrying out derivatization at 20 ℃ for 60min, adding 2.0mL of reaction termination solution, carrying out vortex mixing for 1min, centrifuging for 5000r/min and 5min, transferring the upper-layer water phase into a 100mL round-bottom flask, concentrating to be nearly dry, adding 1mL of absolute ethyl alcohol into a concentration bottle, carrying out vortex dissolving sufficiently, and filtering through a 0.22 mu m filter membrane to be placed into a sample injection vial;
the derivatization reagent is 0.45g/100mL of L-alanine-beta-naphthylamine-acetonitrile solution, the catalyst I is 0.50g/100mL of triethylamine-trichloromethane solution, the catalyst II is 0.50g/100mL of butyl chloroformate-trichloromethane solution, and the reaction termination solution is 50mmol/L of sodium bicarbonate-water solution.
2. The method for separating and determining carnitine enantiomer in a health food based on ultra-high performance combined phase chromatography technology of claim 1, wherein the standard solution is prepared by the following steps: remove 0.5mL L-carnitine,DMixed working solutions of two enantiomers of carnitine were further derivatized in 10mL centrifuge tubes to prepare standard solutions with concentrations of 0.20, 0.40, 1.00, 2.00, 5.00, 10.00, 20.00 mg/L.
3. The method for separating and determining carnitine enantiomer in a health food based on ultra-high performance combined phase chromatography technique according to claim 1,L-carnitine,DThe recovery rate of two carnitine enantiomers ranges from 86.0% to 110%, the relative standard deviation ranges from 4.3% to 7.0%, the relative standard deviation RSD,n=6。
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