CN112684089A - Method for separating and determining carnitine enantiomer in health food based on ultra-high performance synthetic phase chromatography technology - Google Patents
Method for separating and determining carnitine enantiomer in health food based on ultra-high performance synthetic phase chromatography technology Download PDFInfo
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Abstract
The invention relates to a method for detecting residues of health-care food, in particular to a method for measuring the residual quantity of L-carnitine and D-carnitine enantiomers in the health-care food. The method adopts ultra-high performance synthetic phase chromatography to split carnitine enantiomers, and determines the residual quantity of L-carnitine and D-carnitine enantiomers in 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 results show that the linear correlation coefficient of the two compounds is greater than 0.999 within the range of 0.5-10.0 mg/L, the quantification limit is 50mg/kg, the recovery rate is 89.2-110%, and the relative standard deviation is 4.3-6.9%. 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 method for detecting residues of health-care food, in particular to a method for measuring the residual quantity of L-carnitine and D-carnitine enantiomers in the health-care food.
Background
Carnitine has the chemical name of beta-hydroxy-gamma-trimethylamine-butyric acid, and has 1 chiral carbon atom in its molecule and L-type (levorotatory) and D-type (dextrorotatory) enantiomers. Wherein L-Carnitine (L-Carnitine), also known as Carnitine or L-Carnitine, is widely present in most mammalian tissues, and can promote fatty acid transmission to mitochondria to achieve the effect of oxidative decomposition of fat[1]. L-carnitine deficiency interferes with the metabolism of fatty acids to cause insufficient energy synthesis, and symptoms such as easy fatigue, muscle weakness, palpitation and the like appear, and serious patients have metabolic disorder, coma and even death. The D-carnitine has no physiological activity, even has toxicity, and is harmful to human and animals under the condition of large dose[2]. At present, L-carnitine used in commercially available health products is mainly prepared by a chemical synthesis method with low production cost, the produced carnitine is generally a mixture of L-carnitine and D-carnitine, and the D-carnitine is separated from the L-carnitine by a separation method to obtain the L-carnitine, which is difficult to completely separate the D-carnitine[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]However, few chromatographic conditions have been reported for resolution and content determination of L-carnitine and D-carnitine enantiomers. Due to the large polarity of carnitine molecules, the carnitine molecules are difficult to reserve on a chromatographic column, the enantiomer separation degree is poor, and chromophoric groups are lacked, so that the carnitine molecules have extremely weak absorption in an ultraviolet visible light region, low sensitivity and difficulty in being used in the commonDirect detection in liquid chromatography is used, so chiral resolution technology is required for separating carnitine enantiomer. Zhai Xufeng[11]And the like, L-alanine-beta-naphthylamine is taken as a derivatization reagent to successfully resolve the acetyl carnitine enantiomer, but the methods generally have the problems of low separation efficiency, long analysis time, low reproducibility and the like. Ultra-high performance phase-locked chromatography (UPC) in recent years2) Have received a great deal of attention. UPC2Based on the traditional liquid chromatography, the technology uses supercritical CO2The main mobile phase is a supercritical fluid between gas and liquid, has low viscosity, high diffusivity and fast mass transfer, thereby providing higher separation speed and higher system resolution for the liquid chromatography. The study shows that UPC2The technology is more suitable for analyzing the isomers and structural analogues which are difficult to process by the traditional liquid chromatography, and has been successfully used for separating and detecting various compounds, such as vitamins[12]Phthalic acid esters[13]And an ultraviolet absorber[14]And the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for separating and determining carnitine enantiomer in health-care food based on an ultra-high performance synthetic phase chromatography technology. Research results show that the method has the characteristics of rapidness, accuracy, high separation efficiency, high repeatability, good stability and the like, and can meet the requirements of rapid quantification and purity analysis of L-carnitine in health-care food.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for separating and determining carnitine enantiomer in health food based on ultra-high performance synthetic phase chromatography technology comprises the following steps:
firstly, sample extraction
Taking health product tablets, capsules or granules, grinding, accurately weighing 1.0g of sample, placing the sample in a 50mL 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 further deriving the obtained supernatant to prepare a sample solution;
second, preparation of Standard solution
Transferring 0.5mL of mixed working solution of L-carnitine and D-carnitine enantiomers into a 10mL centrifuge tube, and further deriving to prepare standard solutions with the concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 mg/L;
the derivation in the first step and the second 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 0.42g/100mL of sodium bicarbonate-water solution;
thirdly, respectively carrying out ultra-high performance combined chromatography analysis on the standard solution and the sample solution
The analysis conditions were as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 4-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;
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;
fourthly, making a standard curve and calculating the content and the purity of the L-carnitine and the D-carnitine in the sample solution through the standard curve.
Preferably, the recovery rate of two carnitine enantiomers, L-carnitine and D-carnitine, is in the range of 89.2% to 110%, and the relative standard deviation (RSD, n ═ 6) is in the range of 4.3% to 6.9%.
Furthermore, the invention also discloses a kit for separating and determining carnitine enantiomer in health food based on the ultra-high performance combined phase chromatography technology, which 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 0.42g/100mL 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, high 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 shows the stability of two standard solutions of derivatized acylated carnitine enantiomers in absolute ethanol over 60 days (D-carnitine and L-carnitine).
FIG. 2 shows the effect of derivatization temperature and time on the derivatization reaction (D-carnitine and L-carnitine).
FIG. 3 shows the effect of different cosolvents on the separation of the two carnitine enantiomers (13.8MPa, 40 ℃).
FIG. 4 shows the effect of different system back pressures on the separation of the two carnitine enantiomers (1% (v/v) in aqueous ammonia in methanol, 40 ℃ C.).
FIG. 5 shows the effect of column temperature on the separation of two carnitine enantiomers (1% (v/v) in ammonia in methanol, 13.8MPa) in different systems.
Detailed Description
1 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 corporation, 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, 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.
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).
1.2 Standard solution and reagent preparation
1.2.1 Standard solution of racemate
Carnitine stock (1.0 g/L): respectively and accurately weighing appropriate amount of the standard carnitine racemate, dissolving with anhydrous ethanol, and metering volume to obtain 1.0g/L standard stock solution.
1.2.2 enantiomeric Standard solutions
Two carnitine enantiomer stocks (1.0 g/L): respectively and accurately weighing appropriate amount of L-carnitine enantiomer and D-carnitine enantiomer standard substances, dissolving with anhydrous ethanol, and metering volume to obtain 1.0g/L standard stock solution.
Mix standard working solution (for assay of health food sample residue): accurately sucking a certain amount of standard solution, and gradually diluting to 1.0, 2.0, 4.0, 10.0 and 20.0mg/L standard working solution with anhydrous ethanol.
1.2.3 preparation of derivatizing agent solution and stop solution
Derivatizing 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.
1.3 sample pretreatment
1.3.1 sample extraction
Taking health product tablets, capsules or granules, grinding, accurately weighing 1.0g of sample, placing the sample in a 50mL 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 carrying out further derivatization on the obtained supernatant.
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.
1.4 conditions of analysis
A chromatographic column: acquisty Trefoil CEL1(3.0 mm. times.150 mm, 2.5 μm, filler cellulose-tris (3, 4-dimethylphenylcarbamate), Waters corporation, USA); mobile phase: a is CO2B 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.
1.5 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, so as to prepare standard solutions with the concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 mg/L.
2 results and discussion
2.1 examination of stability
The carnitine enantiomer was stored at-20 ℃ after derivatization. The content of carnitine enantiomers immediately after derivatization and storage of 1, 3, 5, 7, 14, 30, 60d, respectively, is plotted for comparison. The results show that the content change of the 2 carnitine enantiomer after derivatization and acylation is less than 10% within 30d at-20 ℃, and the content change is more than 10% within 30 days (figure 1), which indicates that the solution after derivatization and acylation of the 2 carnitine enantiomer is relatively stable within 30 d.
2.2 optimization of the extraction method
Acetonitrile is commonly used in literature reports[6-7]Anhydrous ethanol[4]Methanol, methanol[10]Carnitine is extracted. The extraction efficiencies of the above solvents were compared, and the results showed that the extraction efficiencies using acetonitrile, absolute ethanol, and methanol were 13%, 98%, and 70%, respectively. Therefore, the invention adopts absolute ethyl alcohol for extraction. In addition, the experiment compares the oscillation and ultrasonic wave extraction modes, and the result shows that the ultrasonic wave extraction efficiency is better. Meanwhile, the ultrasonic extraction time is also considered, and the result shows that the effect of extracting for 20min by adopting ultrasonic waves is optimal, and the extraction efficiency reaches 91%. Therefore, ultrasonic extraction for 20min is determined as the optimal extraction condition.
2.3 optimization of the derivatization conditions
Domestic and foreign literature reports ethyl chloroformate[4]Chloroformic acid butyl ester[15]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 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 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. 2. 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. Therefore, the method optimizes main factors influencing the derivatization reaction, finds that the optimal derivatization reaction condition is reaction at 20 ℃ for 60min, the derivatization reaction of two carnitine enantiomers is sufficient, and the stability of the derivatization product is good.
Under otherwise identical conditions, the recovery rates of the two carnitine enantiomers at 1.0mL, 2.0mL and 3.0mL volumes of the derivatization stop solution were compared. The results of the experiments show that the recovery rate of both carnitine enantiomers added to 2.0mL of derivatization stop solution is more than 95%. The derivatization reaction was therefore stopped here by adding 2mL of sodium bicarbonate solution.
The invention also investigates the influence of the constant volume reagent on the peak shapes of two carnitine enantiomers. The results show that when isopropanol-n-heptane (1:1, v/v) solution, methanol and acetonitrile are used for constant volume, a plurality of miscellaneous peaks appear in the chromatogram. When the volume is determined by using isopropanol and absolute ethyl alcohol, the chromatogram has less miscellaneous peaks, and the chromatogram has a sharper peak shape when the volume is determined by using the absolute ethyl alcohol, so the absolute ethyl alcohol is used as the volume determination liquid.
2.4 optimization of the separation conditions
2.4.1 optimization of chromatography columns
The two carnitine enantiomers which are resolved in the invention are not easy to separate because the structures are very similar. 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. The result shows that after the mobile phase is optimized, when the chiral chromatographic columns of Acquity Trefoil AMY1 and Acquity Trefoil CEL2 are used for separation, the chromatographic peak response is low, and the separation degree and the peak shape are poor; when the chiral chromatographic column of Acquity Trefoil CEL1 is used for separation, the peak shape is sharp and the symmetry is good. Therefore, the subsequent experiment selects an Acquity Trefoil CEL1 chiral chromatographic column to separate the carnitine enantiomers.
2.4.2 optimization of Co-solvent in the Mobile phase
UPC2Supercritical 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 the selection of the cosolvent has important influence on the peak emergence time, the peak emergence sequence and the separation degree of the target substance[12]. In order to obtain good separation effect and peak shape, the invention examines the cosolvent. 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. 3, the peak shapes and separation effects of both carnitine enantiomers were significantly improved as the concentration of ammonia was increased. In consideration of the separation effect of chromatographic peaks and the tolerance of chromatographic columns, the invention does not continue to improve the concentration of ammonia water for investigation, but selects 1% (v/v) ammonia water methanol solution as a cosolvent.
2.4.3 selection of System backpressure
In the ultra-high performance combined phase chromatography, the system back pressure also influences the separationOne of the important factors of the process, the main role of which is to control the carbon dioxide in the supercritical fluid state throughout the operation. Since the temperature of carbon dioxide exceeds 31 ℃ and the pressure exceeds 7.38MPa, CO2The supercritical carbon dioxide state is entered. Therefore, the invention takes 1% (v/v) ammonia water methanol solution as a cosolvent, and examines the influence of backpressure within the range of 10.3-20.7 MPa on the separation effect of two carnitine enantiomers under the condition of column temperature of 40 ℃ (figure 4). The results show that the analyte peak time is advanced as the system backpressure increases. When the backpressure is 10.3 MPa, 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 a high-pressure alarm. Comprehensively considering the analysis speed and the separation effect, the invention selects 13.8MPa as the best system back pressure.
2.4.4 selection of 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 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, CO2The supercritical carbon dioxide state is entered, so the invention examines the influence of the chromatographic column temperature in the range of 31-40 ℃ on the separation of the target object under the condition that the system backpressure is 13.8MPa (figure 5). 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 a high-pressure 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 ℃.
2.5 Linear Range, detection Limit and quantification Limit
The series of mixed standard solutions of the derivatized L-carnitine enantiomer and D-carnitine enantiomer 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 good linear relation within the mass concentration range of 0.5-10.0 mg/L, and the correlation coefficient is more than 0.999. The LOQ of the two carnitine enantiomers was 50mg/kg, obtained by adding a standard to a blank sample of the nutraceutical, measuring according to the method, and calculating the limit of quantitation (LOQ) with a signal-to-noise ratio S/N of 10.
2.6 recovery, accuracy and precision
The method for adding standard solution into blank solid health food and liquid health food is adopted to carry out the measurement of the addition recovery rate and the precision measurement of the method, the addition level is respectively 50, 100 and 500mg/kg, the parallel measurement is carried out for 6 times, the addition standard recovery rate and the Relative Standard Deviation (RSD) are calculated, and the result is shown in the table 1. The recovery rates of the two carnitine enantiomers ranged from 89.2% to 110% with a relative standard deviation (RSD, n ═ 6) ranging from 4.3% to 6.9%. The recovery rate and precision conform to SN/T0001-[16]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 is used for detecting the content of D-carnitine and L-carnitine in 10 commercially available health-care products. The results show that D-carnitine is not detected in 10 health care products, and the content of L-carnitine is 1.44-27.02 g/100 g. The European Union stipulates a daily limit of L-carnitine of 2g[17]The dosage of Chinese L-carnitine is mainlyReferring to the regulations of European Union, the recommended dose of the 10L-carnitine health foods meets the requirements of European Union and China.
2.7.2 resolution of racemic Standard
The established method is applied to split and measure the purchased carnitine racemate standard substance. The results show that the carnitine racemate contains two enantiomers of L-carnitine and D-carnitine, the proportion of L-carnitine is 48.8%, the proportion of D-carnitine is 51.2%, and the proportion between the two enantiomers is as same as that in the literature[3]The reports are in agreement.
3.0 conclusion
The method is developed by inspecting and optimizing main parameters such as pretreatment method, derivatization time, derivatization temperature, instrument chromatographic conditions and the like, and is used for simultaneously determining the content and purity of L-carnitine in health-care food, and analyzing and determining the health-care products sold in the market and racemate carnitine standard products. Research results show that the method has the characteristics of rapidness, accuracy, high separation efficiency, high repeatability, good stability and the like, and can meet the requirements of rapid quantification and purity analysis of L-carnitine in health-care food.
Reference documents:
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Claims (3)
1. A method for separating and determining carnitine enantiomer in health-care food based on an ultra-high performance synthetic phase chromatography technology is characterized by comprising the following steps:
firstly, sample extraction
Taking health product tablets, capsules or granules, grinding, accurately weighing 1.0g of sample, placing the sample in a 50mL 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 further deriving the obtained supernatant to prepare a sample solution;
second, preparation of Standard solution
Remove 0.5mL L-carnitine,D-mixed working solution of two enantiomers of carnitine in 10mL centrifuge tube, further derivatizing to prepare standard solution with concentration of 0.5, 1.0, 2.0, 5.0, 10.0 mg/L;
the derivation in the first step and the second 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, 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 0.42g/100mL of sodium bicarbonate-water solution;
thirdly, respectively carrying out ultra-high performance combined chromatography analysis on the standard solution and the sample solution
The analysis conditions were as follows:
a chromatographic column: acquity Trefoil CEL1, the filler is cellulose-tri (3, 4-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;
and (3) system backpressure: 13.8 MPa; flow rate: 1.0 mL/min; sample introduction amount: 5 muL; column temperature: 40 ℃; detection wavelength: 244 nm;
fourthly, making a standard curve and calculating the content of the sample solution through the standard curveL-carnitine,D-content and purity of carnitine.
2. 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 of the two carnitine enantiomers ranges from 89.2% to 110%, relative standard deviation (RSD,n=6) is in the range of 4.3% to 6.9%.
3. A carnitine enantiomer splitting and measuring kit in health food based on ultra-high performance synthetic phase chromatography technology comprises a derivatization reagent, a catalyst I, a catalyst II and a reaction stopping 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 0.42g/100mL of sodium bicarbonate-water solution.
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| CN202110511088.3A CN113219097B (en) | 2021-01-15 | 2021-05-11 | Method for splitting and measuring carnitine enantiomer in infant formula milk powder |
| CN202110535082.XA CN113433257B (en) | 2021-01-15 | 2021-05-17 | Method for separating and measuring carnitine enantiomer in health food |
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| CN113791145A (en) * | 2021-08-26 | 2021-12-14 | 浙江省检验检疫科学技术研究院 | Clenbuterol enantiomer resolution and determination method based on ultra-high performance combined phase chromatography technology |
| CN113804790A (en) * | 2021-09-18 | 2021-12-17 | 四川新希望畜牧科技有限公司 | Method for simultaneously detecting additive, L-carnitine and D-carnitine |
| CN114858942A (en) * | 2022-05-17 | 2022-08-05 | 浙江省检验检疫科学技术研究院 | Method for rapidly determining fenpropathrin enantiomer residue in pear and product thereof |
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| JP2005221425A (en) * | 2004-02-06 | 2005-08-18 | Nippon Menaade Keshohin Kk | Method for measuring carnitines |
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| CN102539546B (en) * | 2010-12-09 | 2014-10-22 | 北京国立柏林医学科技发展有限公司 | Methods for detecting content of free carnitine or content of total carnitine in body fluid |
| JP6031636B1 (en) * | 2014-12-15 | 2016-11-24 | 積水メディカル株式会社 | Method for detecting amino acid or acylcarnitine |
| CN106885857B (en) * | 2017-03-15 | 2019-08-30 | 上海烟草集团有限责任公司 | The analysis method of nicotine chiral isomer in a kind of tobacco and flue gas |
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| CN108152410B (en) * | 2017-12-19 | 2020-01-31 | 安徽古井贡酒股份有限公司 | method for rapidly detecting chiral triadimenol in grains |
| CN108426972B (en) * | 2018-06-15 | 2020-01-17 | 国家烟草质量监督检验中心 | Method for splitting and measuring chiral pesticide benalaxyl enantiomer by ultra-high performance combined chromatography-tandem mass spectrometry technology |
| CN110658288B (en) * | 2019-10-18 | 2023-01-31 | 贵州省烟草科学研究院 | Method for analyzing nicotine isomer in fresh tobacco based on derivatization purification and back extraction enrichment technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113791145A (en) * | 2021-08-26 | 2021-12-14 | 浙江省检验检疫科学技术研究院 | Clenbuterol enantiomer resolution and determination method based on ultra-high performance combined phase chromatography technology |
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| CN115963169A (en) * | 2021-10-11 | 2023-04-14 | 生物岛实验室 | Detection method and detection kit for carnitine |
| CN115963169B (en) * | 2021-10-11 | 2023-10-13 | 生物岛实验室 | Detection method of carnitine and detection kit |
| CN114858942A (en) * | 2022-05-17 | 2022-08-05 | 浙江省检验检疫科学技术研究院 | Method for rapidly determining fenpropathrin enantiomer residue in pear and product thereof |
| CN114858942B (en) * | 2022-05-17 | 2023-05-23 | 浙江省检验检疫科学技术研究院 | Method for rapidly determining fenpropathrin enantiomer residues in pears and products thereof |
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