Detection method for chiral purity of carnitine

Abstract

The invention belongs to the field of analytical chemistry, and particularly discloses a method for detecting chiral purity of carnitine. The detection method provided by the invention comprises the following steps: respectively reacting a carnitine sample and a DL carnitine standard substance with a novel chiral derivatization reagent (S)-(-)-1, 2, 4-triazole; the method comprises the following steps: carrying out a derivatization reaction on (R)-(-)-1, 1 '-binaphthalene-2, 2'-dibasic phosphoryl chloride or (R)-(-)-1, 1 '-binaphthalene-2, 2'-dibasic phosphoryl chloride in an organic solvent to generate a diastereomer, separating and detecting derivatization products through reversed-phase high performance liquid chromatography, determining the retention time of the carnitine derivative according to the retention time of the DL carnitine derivative, and determining the content of the DL carnitine derivative according to the retention time of the DL carnitine derivative. And calculating the chiral purity through an area normalization method. Compared with the traditional method, the method provided by the invention has the advantages that the cost is reduced by more than 50%, the analysis accuracy is equivalent, the sensitivity is high, the chiral purity of carnitine can be accurately measured, and the market demand on an efficient and economic detection method is met.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting the chiral purity of carnitine. Background Art

[0002] As a key fatty acid metabolic carrier, L-carnitine is widely used in pharmaceuticals and health supplements. Its chiral purity directly impacts drug safety and efficacy. D-carnitine competitively inhibits carnitine acetyltransferase (CAT) and carnitine lipoyltransferase (PTC), producing toxic side effects in biochemical processes. Therefore, its content must be strictly controlled.

[0003] For the chiral purity of L-carnitine, what adopts in national standard now is to measure its specific rotation, though this method is simple to operate, but its accuracy is relatively poor.Use liquid chromatography to detect carnitine and can greatly improve the accuracy of detection.Can use chiral column to directly detect its chiral purity (CN104914183A, CN104698101A, CN113804790A) of carnitine, but need use expensive chiral liquid phase column, and because carnitine does not have ultraviolet absorption, detection wavelength need be below 220nm, and the precision of detection is affected.Another method is to use chiral derivatizing agent that carnitine is derivatized, obtain diastereomer, use conventional achiral liquid phase column just can detect, and derivatizing agent has stronger ultraviolet absorption usually, therefore, detection wavelength is all at 245nm and above, and the precision of detection is better. For example, the literature (J. Pharm. Biomed. Anal. 30 (2002) 209-218) reports a method for determining the chiral purity of L-carnitine using HPLC in combination with a derivatization reagent ((+)-FLEC(+)-1-(9-fluorenyl)-ethyl chloroformate). However, this chiral derivatization reagent is expensive, making it difficult to use for routine testing in actual production.

[0004] Therefore, it is urgent to develop a low-cost, simple and efficient method for determining the chiral purity of L-carnitine. Summary of the Invention

[0005] In response to the shortcomings of the aforementioned prior art, the present invention aims to provide a method for detecting the chiral purity of carnitine. This method provides a novel chiral derivatization reagent that binds to the β-hydroxyl group of carnitine to form diastereomers. This reagent, combined with conventional reversed-phase high-performance liquid chromatography (HPLC), achieves efficient separation, thus addressing the high cost and equipment requirements of the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for detecting the chiral purity of carnitine comprises the following steps:

[0008] A carnitine sample to be tested and a DL-carnitine standard are respectively subjected to a derivatization reaction with a chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride or a chiral derivatization reagent (R)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride in an organic solvent to generate diastereomers. The resulting derivatized products are then separated and detected by reversed-phase high performance liquid chromatography. The retention time of the carnitine derivative product to be tested is determined based on the retention time of the DL-carnitine derivative product, and the chiral purity (i.e., enantiomeric excess percentage) thereof is calculated by an area normalization method.

[0009] Furthermore, the carnitine is subjected to a derivatization reaction with a chiral derivatization agent under organic base conditions in an organic solvent at 20-30° C. for 7.5-8.5 h, preferably at 25±2° C. for 8 h.

[0010] Furthermore, the organic base is any one of pyridine and triethylamine.

[0011] Furthermore, the organic solvent is any one of dichloromethane, ethyl acetate, and acetonitrile.

[0012] Furthermore, the molar ratio of carnitine, chiral derivatization reagent and organic base is 1:1-2:3-5.

[0013] Furthermore, the ratio of carnitine to organic solvent in the carnitine sample is 1 mmol:10-20 mL.

[0014] Furthermore, the derivatization reaction was completed by TLC monitoring, and water was added to quench the reaction. The layers were separated, and the aqueous phase was extracted with dichloromethane. The organic phase after extraction was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the derivatization product.

[0015] Furthermore, the conditions for performing reverse phase high performance liquid chromatography are:

[0016] Chromatographic column: C18 column;

[0017] Mobile phase: a mixture of triethylamine phosphate buffer solution and acetonitrile in a volume ratio of 3:1, with a pH of 2.6±0.2; isocratic elution;

[0018] Detector: UV detector 254nm;

[0019] Flow rate: 1 ± 0.1 mL / min;

[0020] Column temperature: 30±2℃.

[0021] Taking the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride as an example, it reacts with carnitine. The reaction formula for the reaction of the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride with L-carnitine is as follows:

[0022]

[0023] The reaction formula of the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride and D-carnitine is as follows:

[0024]

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] (1) The present invention derivatizes carnitine using a novel chiral derivatization reagent, and utilizes the phosphorus oxychloride group (-POCl) of the novel chiral derivatization reagent to undergo an esterification reaction with the β-hydroxyl group (-OH) of carnitine to accurately detect the chiral purity of carnitine, which is different from existing chiral derivatization reagents.

[0027] (2) The present invention utilizes a novel chiral derivatization reagent containing a binaphthyl structure to react with carnitine to generate diastereomeric derivatives with strong ultraviolet absorption, breaking the traditional reliance on chiral column detection methods. Conventional reversed-phase chromatography columns combined with ultraviolet detectors can be used for separation and detection, reducing costs and improving universality.

[0028] (3) Compared with traditional methods, the method of the present invention reduces the cost by more than 50%, and the analytical accuracy is comparable. At the same time, it has high sensitivity and can accurately determine the chiral purity of carnitine, meeting the market demand for efficient and economical detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a high-resolution mass spectrum of the L-carnitine derivative in Example 1 of the present invention.

[0030] Figure 2 The liquid chromatogram and peak area data of the DL-carnitine derivative product in Example 1 of the present invention are shown.

[0031] Figure 3 The liquid chromatogram and peak area data of the L-carnitine derivative product in Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0032] To make the purpose and technical solutions of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In the following examples, the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride was prepared with reference to the supporting material of Adv.Funct.Mater.2024,34,2409681, which uses S-1,1'-binaphthol as a raw material to prepare and purify (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride.

[0034] In the following examples, the L-carnitine standard used was purchased from Wuhan Tianzhi Biotechnology Co., Ltd., with a carnitine content of ≥99% and an enantiomeric excess percentage of 99.48%.

[0035] In the following examples, the DL-carnitine standard used was purchased from Wuhan Tianzhi Biotechnology Co., Ltd., and its carnitine content was ≥99%.

[0036] Example 1: A method for detecting the chiral purity of L-carnitine, comprising the following steps:

[0037] 1. Derivatization reaction

[0038] L-carnitine standard (1.0 mmol, 0.161 g) was added to a 100 mL three-necked flask containing dichloromethane (10 mL). After cooling to 0–5°C in an ice-salt bath, triethylamine (4.4 mmol, 0.62 mL) was slowly added dropwise, maintaining the temperature at ≤5°C (to neutralize HCl generated during the subsequent reaction). Subsequently, a chiral derivatization reagent, (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride (1.0 mmol, 0.367 g) in dichloromethane (5 mL), was slowly added dropwise to the reaction mixture via a constant pressure dropping funnel (approximately 30 minutes), maintaining the system temperature below 10°C. After the addition was complete, the ice-salt bath was removed, and the mixture was heated to 25±2°C with stirring for 8 hours. Completion of the chiral derivatization reaction was confirmed by TLC (developing solvent: dichloromethane / methanol, volume ratio = 10:1). The reaction solution was recooled to 0°C, quenched by adding pre-cooled deionized water (10 mL) dropwise and stirred vigorously. After separation, the aqueous phase was extracted with dichloromethane (3×10 mL). The combined organic phases were dried over anhydrous sodium sulfate for 30 minutes, filtered and concentrated under reduced pressure to obtain 0.391 g of a pale yellow crude product (TM1) with a yield of 79%. The product was sent for analysis and was analyzed by high-resolution mass spectrometry (see Figure 1) characterization, the results show that calcd.for C 27 H 26 NO6P+H + 492.1576,found 492.1560.

[0039] Similarly, DL-carnitine standard was substituted for L-carnitine standard to carry out derivatization reaction, and finally 0.4 g of pale yellow crude product (TM2) was obtained with a yield of 81%.

[0040] 2. High performance liquid chromatography detection

[0041] (1) Chromatographic conditions

[0042] Chromatographic column: Agilent C18 column (250mm×4.6mm, 3.5μm)

[0043] Column temperature: 30°C

[0044] Flow rate: 1 mL / min

[0045] Injection volume: 20 μL

[0046] UV detector: 254nm

[0047] Mobile phase: Thoroughly mix 2000 mL of water and 5 mL of 85 wt% phosphoric acid. While stirring, accurately add 13.6 mL of triethylamine dropwise to prepare a buffer solution. Mix 1500 mL of this buffer solution with 500 mL of acetonitrile. Adjust the solution to pH 2.6 with 50 wt% phosphoric acid. Filter through a 0.4 μm filter and set aside. Perform isocratic elution.

[0048] (2) Sample configuration

[0049] Blank solution: Take 50 mL of mobile phase as blank solution.

[0050] Reference solution 1: Accurately weigh 0.05 g of the pale yellow crude product (TM2, hereinafter referred to as "DL-carnitine derivative product") obtained by derivatization of DL-carnitine standard with the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphinoyl chloride, dissolve it in 50 mL of mobile phase, filter through a 0.4 μm filter membrane, and use the filtrate as reference solution 1.

[0051] Reference solution 2: Accurately weigh 0.05 g of the pale yellow crude product (TM1, hereinafter referred to as "L-carnitine derivative product") obtained by derivatization of L-carnitine standard with the chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphinoyl chloride, dissolve it in 50 mL of mobile phase, filter it through a 0.4 μm filter membrane, and use the filtrate as reference solution 2.

[0052] (3) Measurement process

[0053] Under the chromatographic conditions of (1) above, blank solution, reference solution 1 and reference solution 2 were injected respectively, and the chromatograms and peak areas were recorded. The liquid chromatograms and peak area data of DL-carnitine derivatives are shown in Figure 2 The liquid chromatography and peak area data of L-carnitine derivatives are shown in Figure 3 Based on the elution pattern of the DL-carnitine derivative, the peak times of the two configurations of the carnitine derivative were determined to be 20.239 min and 23.647 min, respectively. Comparing the peak pattern with that of the L-carnitine derivative, the peak with a retention time of 20.2 min was determined to be the L-carnitine derivative. The enantiomeric excess percentage (i.e., chiral purity) was calculated by area normalization, and the enantiomeric excess percentage of the L-carnitine was 99.454%.

[0054] The above chromatographic detection method has been verified by methodological experiments and has good repeatability and high analytical accuracy.

[0055] The above is merely an embodiment of the present invention, and its description is relatively specific and detailed, but it should not be understood as limiting the scope of the present invention. It should be pointed out that those skilled in the art can make a number of modifications and improvements without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention.

Claims

1. A method for detecting the chiral purity of carnitine, characterized in that: The method comprises the following steps: subjecting a carnitine sample to be tested and a DL-carnitine standard to derivatization reaction with a chiral derivatization reagent (S)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride or a chiral derivatization reagent (R)-(-)-1,1'-binaphthyl-2,2'-diylphosphoryl chloride in an organic solvent, respectively; separating and detecting the obtained derivatization products by reversed-phase high performance liquid chromatography; determining the retention time of the carnitine derivative product to be tested based on the retention time of the DL-carnitine derivative product; and calculating the chiral purity thereof by an area normalization method.

2. The detection method according to claim 1, wherein Carnitine is subjected to a derivatization reaction with a chiral derivatization reagent under organic base conditions in an organic solvent at 20-30° C. for 7.5-8.5 hours.

3. The detection method according to claim 2, characterized in that Carnitine was derivatized with a chiral derivatization reagent at 25±2°C for 8 h.

4. The detection method according to claim 2, characterized in that The organic base is any one of pyridine and triethylamine.

5. The detection method according to claim 2, characterized in that The organic solvent is any one of dichloromethane, ethyl acetate and acetonitrile.

6. The detection method according to claim 2, characterized in that The molar ratio of carnitine, chiral derivatization reagent and organic base is 1:1-2:3-5.

7. The detection method according to claim 1, characterized in that After the derivatization reaction was completed as monitored by TLC, water was added to quench the reaction, the layers were separated, the aqueous phase was extracted with dichloromethane, and the organic phase after extraction was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the derivatization product.

8. The detection method according to claim 1, wherein The conditions for reverse phase high performance liquid chromatography were as follows: chromatographic column: C18 column; Mobile phase: a mixture of triethylamine phosphate buffer solution and acetonitrile in a volume ratio of 3:1, with a pH of 2.6±0.2; isocratic elution; Detector: UV detector 254nm; Flow rate: 1 ± 0.1 mL / min; Column temperature: 30±2℃.

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