Method for determining dextrocarnitine in levocarnitine product by using high performance liquid chromatography

Abstract

The invention discloses a method for determining dextrocarnitine in a levocarnitine product by using high performance liquid chromatography, and the chromatographic conditions are as follows: cellulose-tri-(3, 5-dimethyl phenyl carbamate) is used as a filler for a chromatographic column, a hexafluorophosphate buffer solution-acetonitrile is used as a mobile phase, the volume ratio is 77: 23-69: 31, the column temperature is 15-25 DEG C, the flow velocity is 0.3-0.5 ml / min, and the detection wavelength is 246nm. The method disclosed by the invention can be used for rapidly and accurately detecting the dextrocarnitine in the levocarnitine product and calculating the content of the dextrocarnitine, is simple to operate and good in reproducibility, can better realize quality control of the levocarnitine preparation and an intermediate product thereof, and provides guarantee for synthesis and preparation process optimization.

Description

Technical Field

[0001] The invention relates to the field of drug analysis and detection, and in particular to a method for determining dextrocarnitine in a dextrocarnitine product by using high performance liquid chromatography. Background Art

[0002] L-carnitine, also known as L-carnitine, has a chemical name of (3R)-3-hydroxy-4-(trimethylammonium) butyrate, and a molecular formula of C7H 15 NO3, its chemical structure is as follows:

[0003]

[0004] L-Carnitine is a natural substance in the body that is essential for mammalian energy metabolism. It is a special amino acid that is widely present in body tissues. Its main function is to promote lipid metabolism. L-Carnitine supplementation can alleviate fat metabolism disorders, skeletal muscle and myocardial dysfunction caused by its deficiency in the body. It has the effects of improving renal anemia in patients with kidney disease, improving myocardial ischemia, improving cardiac function, protecting the liver, and treating male infertility. It is widely used in clinical practice. L-Carnitine oral solution is a type of preparation of this type of drug.

[0005] There is a chiral center in the structure of L-carnitine, so there is an enantiomer of L-carnitine, namely D-carnitine. D-carnitine has a competitive inhibitory effect on carnitine acetyltransferase (CAT) and carnitine lipoyltransferase (PTC), and produces toxic side effects in the biochemical process. Therefore, considering the safety of medication, the content of D-carnitine needs to be strictly controlled.

[0006] The chemical name of dextrocarnitine is (D-3-carboxy-2-hydroxypropyl) trimethylammonium salt, and its molecular formula is C7H 15 NO3, the structural formula is as follows:

[0007]

[0008] Among the statutory standards for L-carnitine, only the United States Pharmacopoeia (USP2024) controls the enantiomers in L-carnitine, but the derivatization reagents used in this method are not easy to obtain and are relatively expensive; the statutory standard for L-carnitine oral solution does not yet include a method for controlling the right-handed isomer, so it is necessary to develop a method for detecting the content of D-carnitine in L-carnitine products. The quality standard for L-carnitine oral solution (YBH09082022) controls D-carnitine. The sodium perchlorate monohydrate and perchloric acid used in the perchlorate buffer in the chromatographic conditions are both explosive reagents, which are controlled reagents and are not easy to obtain.

[0009] Therefore, the present invention aims to provide an economical, safe and effective method for determining D-carnitine in a L-carnitine product, so as to achieve quality control of L-carnitine raw materials and preparations thereof. Summary of the invention

[0010] The purpose of the present invention is to provide a safe and effective method for determining dextrocarnitine in a dextrocarnitine product, which can be used for the preparation process of dextrocarnitine raw materials and dextrocarnitine preparations and the quality control of the final product.

[0011] The method of the present invention comprises the following steps:

[0012] (1) Prepare system suitability solution;

[0013] (1.1) Take appropriate amounts of L-carnitine reference substance and D-carnitine reference substance and add solvent to prepare a mixed solution containing 2 mg / ml of L-carnitine and 4 μg / ml of D-carnitine;

[0014] (1.2) Derivatization: Take 10 ml of the above mixed solution and place it in a 100 ml flask. Add 1 drop of phenolphthalein indicator solution, 1-2 drops of 1 mol / L potassium hydroxide solution, and then add 100 mg of 2-bromoacetophenone. Shake well, stir and heat under reflux for 30 min.

[0015] (1.3) Extraction: Cool, add 50 ml of ethyl acetate for extraction, take 2 ml of the aqueous phase and place it in a 10 ml volumetric flask, dilute to scale with the solvent, shake well, and obtain the above system suitability solution;

[0016] (2) Prepare test solution and blank solution;

[0017] (2.1) taking an appropriate amount of the L-carnitine sample to be tested, and adding the solvent to prepare a L-carnitine stock solution containing 2 mg / ml of L-carnitine;

[0018] (2.2) Derivatization and extraction: Take 10 ml of the above-mentioned L-carnitine stock solution and solvent and follow steps (1.2) and (1.3) to obtain the above-mentioned test solution and blank solution;

[0019] (3) Detection using high performance liquid chromatography

[0020] The blank solution, the system suitability solution and the test solution were injected into the high performance liquid chromatograph respectively, and the chromatogram was recorded. The content of D-carnitine in the D-carnitine test sample was calculated by the peak area normalization method. The calculation formula is:

[0021]

[0022] Where A X -----Peak area of ​​the dextrocarnitine derivative peak in the test solution;

[0023] A R -----Peak area of ​​the L-carnitine derivative peak in the test solution.

[0024] The conditions for HPLC were

[0025] Chromatographic column: Cellulose-tris-(3,5-dimethylphenylcarbamate) was used as filler;

[0026] Mobile phase: hexafluorophosphate buffer-acetonitrile, volume ratio 77:23-69:31;

[0027] Column temperature: 15-25°C;

[0028] Flow rate: 0.3~0.5ml / min;

[0029] Detection wavelength: 246nm.

[0030] Preferably, the volume ratio of the mobile phase hexafluorophosphate buffer to acetonitrile is 72:28, and the elution is isocratic.

[0031] Preferably, the hexafluorophosphate is sodium hexafluorophosphate with a concentration of 0.05 mol / L and a pH value of 3.8.

[0032] Preferably, the chromatographic column has a specification of 4.6 mm×250 mm and a filler particle size of 5 μm.

[0033] Preferably, the above-mentioned chromatographic column is a CHIRAL MD(2)-RH-5U chromatographic column.

[0034] Preferably, the L-carnitine sample to be tested is any one selected from the group consisting of a L-carnitine raw material, a L-carnitine oral solution intermediate and a L-carnitine oral solution.

[0035] The "d-carnitine" mentioned in the present invention refers to the dextrorotatory isomer of levocarnitine.

[0036] The present invention determines the chromatographic conditions for effectively separating L-carnitine and D-carnitine by exploring the chromatographic conditions, and verifies that the method of the present invention has the advantages of good specificity, good separation degree and good reproducibility, which is of great significance to product quality control and stability sample detection.

[0037] The method of the present invention is applicable to the detection of L-carnitine raw materials, L-carnitine preparations including L-carnitine oral solution intermediates and L-carnitine oral solution. The detection method of the present invention is accurate, simple to operate, and the reagents are readily available. It can fully meet the separation requirements of L-carnitine and D-carnitine, better control the content of D-carnitine in the sample to be tested, ensure product quality, and has strong practicality in actual quality control work. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The chromatogram of the system suitability solution of No. 1 in Table 2 of Example 1;

[0039] Figure 2 This is the chromatogram of the dextrocarnitine control solution 1 of sequence number 1 in Table 2 of Example 1;

[0040] Figure 3 This is the system suitability solution chromatogram of sequence number 2 in the comparison of mobile phase buffer salt in Example 1;

[0041] Figure 4 This is the chromatogram of the dextrocarnitine reference solution 2 of sequence number 2 in the comparison of the mobile phase buffer salt in Example 1;

[0042] Figure 5 This is the system suitability solution chromatogram of sequence number 3 in the comparison of mobile phase buffer salt in Example 1;

[0043] Figure 6 This is the blank solution chromatogram with a mobile phase ratio of 72:28 in Example 1;

[0044] Figure 7 This is the chromatogram of the blank auxiliary material solution in Example 1 with a mobile phase ratio of 72:28;

[0045] Figure 8 This is the system suitability solution chromatogram of the mobile phase ratio of 72:28 in Example 1;

[0046] Fig. 9 UV spectrum of the L-carnitine derivative in the detection wavelength determination of Example 1;

[0047] Fig.10 UV spectrum of the dextrocarnitine derivative in the determination of the detection wavelength in Example 1;

[0048] Fig.11 This is the chromatogram of the blank solution in the specificity test of Example 2;

[0049] Fig.12 This is the chromatogram of the auxiliary material control solution in the specificity test of Example 2;

[0050] Fig.13 The chromatogram of the system suitability solution in the specificity test of Example 2;

[0051] Fig.14 This is the chromatogram of the test solution of L-carnitine oral solution in Example 3;

[0052] Fig.15 The chromatogram of the test solution of L-carnitine bulk drug of Example 3 is shown. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] In the following specific implementations, the instruments, reagents, samples and reference substances used are as follows:

[0055] Instrument: Agilent 1260 series high performance liquid chromatograph;

[0056] Reagents: sodium hexafluorophosphate (analytical grade), triethylamine (chromatographic grade), acetonitrile (chromatographic grade), anhydrous ethanol (chromatographic grade), phenolphthalein indicator solution, potassium hydroxide (analytical grade), 2-bromoacetophenone (GC), ethyl acetate (analytical grade);

[0057] Chromatographic column: CHIRAL MD(2)-RH-5U, specification: 4.6 mm × 250 mm, 5 μm;

[0058] Table 1 Samples

[0059]

[0060] Chemical name, structural formula and source of standard products:

[0061] L-Carnitine:

[0062] Source: China National Institute for Food and Drug Control, Content: 100%, Batch No.: 101041-202204;

[0063] Chemical name: (3R)-3-hydroxy-4-(trimethylammonium)butyric acid inner salt

[0064] Structural formula:

[0065]

[0066] Dextrocarnitine:

[0067] Source: CATO, Content: 91.3%, 98.8%, Batch No.: 0522-RE-0103, 1113-RE-0006 Chemical Name: (D-3-carboxyl-2-hydroxypropyl) trimethylammonium salt

[0068] Structural formula:

[0069]

[0070] [Example 1] Determination of HPLC Chromatographic Conditions

[0071] 1. Screening of mobile phase buffer

[0072] 1.1.1 Preparation of test solution

[0073] L-Carnitine stock solution: Take about 100 mg of L-Carnitine raw material, weigh accurately, place in a 50 ml volumetric flask, add appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain.

[0074] Dextrocarnitine stock solution: Take about 4 mg of dextrocarnitine reference substance, weigh accurately, place in a 50 ml volumetric flask, add appropriate amount of solvent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0075] Dextrocarnitine control solution 1: Take 1 ml of dextrocarnitine stock solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, accurately measure 10 ml, place it in a 100 ml round-bottom flask, add 1 drop of phenolphthalein indicator solution, 1-2 drops of 1 mol / L potassium hydroxide solution to make the purple-red color not fade, add 100 mg of 2-bromoacetophenone, shake well, place it on a magnetic stirring heater and heat under reflux for 30 minutes, keep the liquid slightly boiling, cool, add 50 ml of ethyl acetate for extraction, take 1 ml of the aqueous phase, place it in a 5 ml volumetric flask, dilute to the mark with water, shake well, and the solution is ready.

[0076] R-carnitine control solution 2: Take about 100 mg of R-carnitine (source: Aladdin, batch number: B2314233), accurately weigh, place in a 50 ml volumetric flask, add appropriate amount of solvent to dissolve and dilute to the scale, shake well, accurately measure 10 ml, and start from “place in a 100 ml round-bottom flask”. The preparation method is the same as R-carnitine control solution 1.

[0077] System suitability solution: Take 1 ml of D-carnitine stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with L-carnitine stock solution. Shake well. Accurately measure 10 ml and start from “place in a 100 ml round-bottom flask”. Prepare in the same way as D-carnitine control solution 1.

[0078] 1.1.2 Preparation of mobile phase buffer

[0079] The mobile phase buffer was prepared according to Table 2.

[0080] Table 2 Comparison of mobile phase buffer salts

[0081]

[0082] 1.1.3 High performance liquid chromatography determination

[0083] The above solution was collected and measured according to the following chromatographic conditions.

[0084] Chromatographic conditions: The chromatographic column was CHIRAL MD(2)-RH-5U, with a size of 4.6 mm × 250 mm and a particle size of 5 μm;

[0085] The mobile phase was buffer-acetonitrile, with a volume ratio of 76:24;

[0086] The flow rate was 0.4 ml / min, the column temperature was 20 °C, the detection wavelength was 235 nm, and the injection volume was 10 μl.

[0087] 1.1.4 Experimental results

[0088] The test results are shown in Table 2 and Figures 1 to 5 As shown. The results show that when using phosphate buffer, the peak of L-carnitine derivatives is affected by the solvent peak, and in the D-carnitine control solution 1, the chromatographic peak of D-carnitine derivatives is not detected; when using fluoroborate buffer, since the retention time of the peak of L-carnitine derivatives is very close to that of D-carnitine derivatives, D-carnitine is not accumulated in the chromatogram of the system suitability solution, which will interfere with each other in the actual sample detection process, and the peak of L-carnitine derivatives is close to the solvent peak, so the integration is easily affected; when using hexafluorophosphate buffer, the separation between the peak of L-carnitine derivatives and the peak of D-carnitine derivatives in the chromatogram of the system suitability solution is greater than 4.0, and both are not affected by the solvent peak, so sodium hexafluorophosphate can be used as a buffer salt for preparing the mobile phase of D-carnitine in this product.

[0089] 2. Adjustment of mobile phase ratio and specificity

[0090] 1.2.1 Preparation of test solution

[0091] Blank solution: Accurately measure 10 ml of solvent and place it in a 100 ml round-bottom flask. Prepare it in the same way as the D-carnitine control solution 1 in 1.1.1 above.

[0092] Blank excipient solution: accurately measure 1 ml of blank excipient and place it in a 50 ml volumetric flask, dilute to the mark with solvent, shake well, accurately measure 10 ml, and start from “place in a 100 ml round-bottom flask”. The preparation method is the same as that of the dextrocarnitine control solution 1 in 1.1.1 above.

[0093] 1.2.2 High performance liquid chromatography

[0094] According to the chromatographic conditions in 1.1.3 above, adjust the mobile phase ratio as shown in Table 3, take 10 μl each of the blank solution, blank excipient solution and the system suitability solution in 1 above, inject them into the liquid chromatograph, and record the chromatogram.

[0095] Table 3 Comparison results of mobile phase ratio

[0096]

[0097] 1.2.3 Experimental results

[0098] The test results are shown in Table 3 and Figures 6 to 8 shown.

[0099] The results showed that when the mobile phase ratio was adjusted to hexafluorophosphate buffer-acetonitrile 72:28, the resolution between the peaks of D-carnitine derivatives and L-carnitine derivatives was greater than 4.0, with no decrease, and the peak height of the D-carnitine derivatives increased slightly. At this time, the blank solvent and blank excipient peaks did not interfere with the sample determination, so the mobile phase ratio was determined to be hexafluorophosphate buffer-acetonitrile 72:28.

[0100] 3. Selection of detection wavelength

[0101] 1.3.1 High performance liquid chromatography

[0102] According to the conditions determined in the above one and two, sodium hexafluorophosphate is used as the buffer salt for preparing the mobile phase of the dextrocarnitine of the product, and the ratio of the mobile phase is hexafluorophosphate buffer-acetonitrile 72:28, which is used as the chromatographic detection condition.

[0103] Take 10 μl of the system suitability solution in 1.1.1 above, inject it into the liquid chromatograph, and record the chromatogram.

[0104] 1.3.2 Experimental Results

[0105] The test results are shown in Table 4 and Fig. 9 , 10 shown.

[0106] Table 4 Detection wavelength confirmation

[0107] Compound Name Maximum absorption wavelength L-Carnitine derivatives 198nm、246nm Dextrocarnitine derivatives 198nm、246nm

[0108] After derivatization, the maximum absorption wavelengths of L-carnitine and D-carnitine are basically the same, with maximum absorption at the ultraviolet end absorption and 246nm wavelength. Therefore, the detection wavelength of D-carnitine in this product is set at 246nm.

[0109] 4. Post-derivation solvent screening

[0110] Before the solvent screening, the solvents in the above examples are: the solvent used in the derivatization and the steps before it is anhydrous ethanol-water with a volume ratio of 33:67, and the solvent used after the derivatization step is water. Because it was found that the sample to be tested is unstable in water, this example compares the stability of the system suitability solution prepared by using anhydrous ethanol-water and water as two solvents in the steps after the derivatization and the L-carnitine control solution.

[0111] 1.4.1 Preparation of test solution

[0112] Preparation of system suitability solution: Take about 40 mg of L-carnitine reference substance, weigh it accurately, place it in a 20 ml volumetric flask, add anhydrous ethanol-water with a volume ratio of 33:67 to dissolve it, add 1 ml of the D-carnitine stock solution in 1.1.1 above, dilute it to the mark with anhydrous ethanol-water, shake well, accurately measure 10 ml, and prepare it from "place it in a 100 ml round-bottom flask" to "add 50 ml of ethyl acetate for extraction" in the same way as the D-carnitine reference solution 1 in 1.1.1 above. Take 2 ml of the aqueous phase, place it in a 10 ml volumetric flask, dilute it to the mark with water and anhydrous ethanol-water respectively, shake well, and the solution is ready.

[0113] Preparation of L-carnitine control solution: Accurately measure 10 ml of the L-carnitine stock solution in 1.1.1 above, place it in a 100 ml round-bottom flask, and add 50 ml of ethyl acetate to extract. The preparation method is the same as that of the D-carnitine control solution 1 in 1.1.1 above. Take 1 ml of the aqueous phase and place it in a 200 ml volumetric flask, dilute to the scale with water and anhydrous ethanol-water respectively, shake well, and the solution is ready.

[0114] 1.4.2 High performance liquid chromatography

[0115] According to the chromatographic conditions determined in 1 to 3 above, accurately measure 10 μl of the above system suitability solution and L-carnitine control solution, inject them into the liquid chromatograph at 0 h, 4 h, 8 h, 12 h, and 16 h respectively, and record the chromatograms.

[0116] Table 5 Post-derivation solvent screening results - system suitability solutions

[0117]

[0118] Table 6 Results of solvent screening after derivatization - L-carnitine control solution

[0119]

[0120] 1.4.3 Experimental Results

[0121] The test results are shown in Tables 5 and 6.

[0122] The results showed that there was no significant difference in the stability of the system suitability solutions when water and a mixed solution of anhydrous ethanol and water with a volume ratio of 33:67 were used as the post-derivation solvents, respectively. However, no main peak was detected in the L-carnitine control solution using water as the solvent at 16 hours, and its stability was worse than that of the L-carnitine control solution using anhydrous ethanol-water as the solvent. Therefore, anhydrous ethanol-water was determined as the post-derivation solvent, that is, anhydrous ethanol-water was used as the solvent before and after the derivatization step, and water was no longer used as the derivatization solvent.

[0123] V. Conclusion

[0124] In summary, the present invention adopts the following chromatographic conditions to detect the content of D-carnitine in L-carnitine products: the chromatographic column is CHIRAL MD(2)-RH-5U (4.6 mm×250 mm, 5 μm), the mobile phase is 0.05 mol / L sodium hexafluorophosphate buffer-acetonitrile, the volume ratio is 72:28, the flow rate is 0.4 ml / min, the column temperature is 20°C, the detection wavelength is 246 nm, the injection volume is 10 μl, and the solvents used before and after derivatization are all anhydrous ethanol-water, the volume ratio is 33:67.

[0125] [Example 2] Methodological verification

[0126] 1. Exclusivity

[0127] 2.1 Preparation of test solution

[0128] Excipient control solution: Take 1 ml of blank excipient solution prepared according to the prescription, place it in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 10 ml, place it in a 100 ml round-bottom flask, add 1 drop of phenolphthalein indicator solution, 1 to 2 drops of 1 mol / L potassium hydroxide solution to make it purple-red without fading, add 100 mg of 2-bromoacetophenone, shake well, place it on a magnetic stirring heater and heat it under reflux for 30 minutes, keep the liquid slightly boiling, cool, add 50 ml of ethyl acetate to extract, take 2 ml of the aqueous phase, place it in a 10 ml volumetric flask, dilute to the scale with solvent, shake well, filter, and take the filtrate to obtain.

[0129] Blank solution: Accurately measure 10 ml of solvent and place it in a 100 ml round-bottom flask. Prepare it in the same way as the excipient control solution.

[0130] System suitability solution: Take appropriate amount of L-carnitine and D-carnitine reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 2mg of L-carnitine and 4μg of D-carnitine per 1ml. Accurately measure 10ml and start from "placing in a 100ml round-bottom flask" and prepare in the same way as "excipient reference solution".

[0131] 2.2 HPLC detection and its results

[0132] Accurately measure 10 μl of each of the system suitability solution, blank solution and excipient control solution, inject them into the liquid chromatograph, perform the test according to the chromatographic conditions summarized in Example 1, and record the chromatogram. The chromatographic test results are shown in Figure 10 to Figure 12 The results showed that the blank solvent and blank excipients had no chromatographic peaks detected at the corresponding positions of the L-carnitine derivative peak and the D-carnitine derivative peak, and had no interference with the determination of D-carnitine.

[0133] 2. Limit of Detection and Limit of Quantitation

[0134] Take an appropriate amount of dextrocarnitine reference substance, use solvent to make a 0.08mg / ml solution, accurately measure 10ml, and start from "placing in a 100ml round-bottom flask", and prepare the same method as the "excipient reference solution" in 2.1 above. Then dilute with solvent to make a series of solutions with different concentrations, and test according to the chromatographic conditions summarized in Example 1.

[0135] The detection limit was determined by the corresponding concentration when the signal-to-noise ratio was 3:1. The detection limit of dextrocarnitine was 0.5109 μg / ml, which was equivalent to 0.03% of the concentration of the test solution. The quantification limit was determined by the corresponding concentration when the signal-to-noise ratio was 10:1. The sample was injected 5 times and the RSD value was calculated. The quantification limit of dextrocarnitine was 1.0218 μg / ml, which was equivalent to 0.05% of the concentration of the test solution, which was not higher than the reporting limit of L-carnitine oral solution (0.05%). After 5 consecutive injections, the RSD value of the peak area was less than 10%, which met the acceptable standard of the quantification limit. The results are shown in Table 7.

[0136] Table 7 Results of determination of limit of quantitation and limit of detection

[0137]

[0138]

[0139] 3. Linearity and Range

[0140] Accurately weigh appropriate amounts of L-carnitine and D-carnitine reference substances, dissolve and dilute with solvent to prepare solutions containing 2 mg of L-carnitine and 0.1 mg of D-carnitine per 1 ml. Accurately measure 10 ml and prepare them in the same way as the above-mentioned "excipient reference solution" starting from "placing in a 100 ml round-bottom flask" to obtain L-carnitine derivative stock solutions and D-carnitine derivative stock solutions. Prepare 5 solutions of different concentrations with solvent, inject them separately, and perform detection according to the chromatographic conditions summarized in Example 1. Perform linear regression analysis with concentration as the abscissa and peak area as the ordinate to obtain the linear regression equations of L-carnitine and D-carnitine. The results are shown in Table 8.

[0141] Table 8 Linearity and range determination results

[0142]

[0143] The results showed that under this method, L-carnitine and D-carnitine showed a good linear relationship within a certain concentration range.

[0144] 4. Precision and Accuracy

[0145] Take appropriate amount of D-carnitine reference substance and L-carnitine oral solution, add solvent to make a solution containing 2mg of L-carnitine and 2μg of D-carnitine per 1ml, accurately measure 10ml, and prepare it in the same way as the above-mentioned "excipient reference solution" from "placing it in a 100ml round-bottom flask". Prepare 6 portions in parallel, inject them separately, and test them according to the chromatographic conditions summarized in Example 1. Calculate the D-carnitine content and recovery rate. The test results are shown in Table 9.

[0146] Table 9 Precision test and accuracy test results

[0147] Test product number 1 2 3 44 5 6 average value RSD(%) Dextrocarnitine content (%) 0.092 0.096 0.082 0.085 0.093 0.088 0.09 5.9 Recovery rate (%) 91.62 96.18 82.17 84.88 93.14 88.29 89.4 5.9

[0148] The results showed that the RSD value of the D-carnitine content was less than 10%, indicating that the method had good repeatability; the recovery rate of D-carnitine was 89.4%, and within the range of 80% to 120%, the RSD value of the recovery rate of D-carnitine in each test solution was 5.9%, less than 10%, indicating that the method had good accuracy.

[0149] 5. Durability test

[0150] The durability test was conducted by examining the column temperature change of ±5°C, the flow rate change of ±0.1ml / min, the buffer salt concentration in the mobile phase within the range of -25% to +50%, the mobile phase pH value change of ±0.2, the mobile phase ratio change range of -3% to +5%, and the selection of chromatographic columns with different serial numbers. The system suitability solution was prepared according to the preparation method in 1.1.1 above, 10μl was accurately measured and injected into the liquid chromatograph. The test was carried out according to the chromatographic conditions summarized in Example 1, and the test results are shown in Table 10.

[0151] Table 10 Durability test results

[0152]

[0153] The results showed that when all parameters changed slightly, the separation between the peaks of L-carnitine derivatives and D-carnitine derivatives met the system suitability requirements, indicating that the method had good durability.

[0154] [Example 3] Determination of the content of dextrocarnitine in samples

[0155] 1. Chromatographic conditions

[0156] Chromatographic column: CHIRAL MD(2)-RH-5U, specification: 250mm×4.6mm, 5μm; mobile phase: sodium hexafluorophosphate buffer (take 8.39g of sodium hexafluorophosphate, add 1000ml of water to dissolve, and adjust the pH value to 3.8 with triethylamine)-acetonitrile, volume ratio: 72:28; flow rate: 0.4ml / min; column temperature: 20℃; detection wavelength: 246nm; injection volume: 10μl; solvent: anhydrous ethanol-water, volume ratio: 33:67.

[0157] 2. Preparation of test solution

[0158] 3.2.1 Preparation of blank solution

[0159] Accurately measure 10 ml of solvent, place in a 100 ml round-bottom flask, add 1 drop of phenolphthalein indicator solution, 1 to 2 drops of 1 mol / L potassium hydroxide solution to make the purple-red color not fade, add 100 mg of 2-bromoacetophenone, shake well, heat to reflux on a magnetic stirring heater for 30 minutes, keep the liquid slightly boiling, cool, add 50 ml of ethyl acetate to extract, take 2 ml of the aqueous phase, place in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0160] 3.2.2 Preparation of system suitability solution

[0161] Take appropriate amount of L-carnitine and D-carnitine reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 2mg of L-carnitine and 4μg of D-carnitine per 1ml. Accurately measure 10ml and start from "placing in a 100ml round-bottom flask" and prepare in the same way as the blank solution.

[0162] 3.2.3 Preparation of test solution

[0163] Accurately measure 1 ml of L-carnitine oral solution (source: Jiangsu Guangcheng Pharmaceutical Co., Ltd.; batch number: 2403264), place in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 10 ml, and start from “place in a 100 ml round-bottom flask”. The preparation method is the same as the blank solution; take about 100 mg of L-carnitine raw material (source: Shandong Qidu Pharmaceutical; batch number: 5F240202), place in a 50 ml volumetric flask, dissolve and dilute to the scale with solvent, shake well, accurately measure 10 ml, and start from “place in a 100 ml round-bottom flask”. The preparation method is the same as the blank solution.

[0164] 3. Determination Method

[0165] Accurately measure the blank solution, system suitability solution and test solution respectively, inject them into the liquid chromatograph, detect according to the chromatographic conditions mentioned above, and record the chromatogram.

[0166] IV. Results and Conclusions

[0167] Test results are shown in Fig.14 , 15 The blank solution did not produce any peaks at the corresponding positions of the L-carnitine derivative peak and the D-carnitine derivative peak, which had no interference with the detection; in the system suitability solution chromatogram, the separation degree between the L-carnitine derivative peak and the D-carnitine derivative peak was greater than 2.0, which met the separation requirements; in the test solution chromatogram, D-carnitine was not detected in both the L-carnitine API and the L-carnitine oral solution.

Claims

1. A method for determining D-carnitine in a L-carnitine product by high performance liquid chromatography, characterized in that: The method comprises the following steps: (1) Prepare system suitability solution; (1.1) Take appropriate amounts of L-carnitine reference substance and D-carnitine reference substance and add solvent to prepare a mixed solution containing 2 mg / ml of L-carnitine and 4 μg / ml of D-carnitine; (1.2) Derivatization: Take 10 ml of the mixed solution and place it in a 100 ml flask, add 1 drop of phenolphthalein indicator solution, 1-2 drops of 1 mol / L potassium hydroxide solution, and then add 100 mg of 2-bromoacetophenone, shake well, stir and heat under reflux for 30 min; (1.3) Extraction: Cool, add 50 ml of ethyl acetate for extraction, take 2 ml of the aqueous phase and place it in a 10 ml volumetric flask, dilute to scale with the solvent, shake well, and obtain the system suitability solution; (2) Prepare test solution and blank solution; (2.1) taking an appropriate amount of the L-carnitine sample to be tested, and adding the solvent to prepare a L-carnitine stock solution containing 2 mg / ml of L-carnitine; (2.2) Derivatization and extraction: Take 10 ml of the L-carnitine stock solution and the solvent and follow steps (1.2) and (1.3) to obtain the test solution and blank solution; (3) Detection using high performance liquid chromatography The blank solution, the system suitability solution and the test solution are injected into a high performance liquid chromatograph respectively, and the chromatogram is recorded, and the content of dextrocarnitine in the dextrocarnitine test sample is calculated by the peak area normalization method; the conditions of the high performance liquid chromatography are: Chromatographic column: Cellulose-tris-(3,5-dimethylphenylcarbamate) was used as filler; Mobile phase: hexafluorophosphate buffer-acetonitrile, volume ratio 77:23-69:31; Column temperature: 15-25°C; Flow rate: 0.3~0.5ml / min; Detection wavelength: 246nm.

2. The method according to claim 1, wherein the volume ratio of the mobile phase hexafluorophosphate buffer to acetonitrile is 72:28, and the elution is isocratic.

3. The method according to claim 1, wherein the hexafluorophosphate salt is sodium hexafluorophosphate.

4. The method according to claim 3, wherein the concentration of the sodium hexafluorophosphate buffer is 0.0375 mol / L to 0.075 mol / L, and the pH value is 3.6 to 4.

0.

5. The method according to claim 1, wherein the chromatographic conditions are: Flow rate: 0.4ml / min; Column temperature: 20°C; The hexafluorophosphate buffer has a concentration of 0.05 mol / L and a pH value of 3.

8. The method according to claim 1 , wherein the chromatographic column is a CHIRAL MD(2)-RH-5U chromatographic column.

7. The method according to claim 6, wherein the specification of the chromatographic column is 4.6 mm×250 mm, and the particle size is 5 μm.

8. The method according to claim 1, wherein the solvent is anhydrous ethanol-water, and the volume ratio of anhydrous ethanol-water is 33:

67.

9. The method according to claim 1, wherein the chromatographic conditions are: the chromatographic column is CHIRAL MD(2)-RH-5U (4.6 mm×250 mm, 5 μm), the mobile phase is 0.05 mol / L sodium hexafluorophosphate buffer-acetonitrile, the volume ratio is 72:28, the flow rate is 0.4 ml / min, the column temperature is 20° C., the detection wavelength is 246 nm, the injection volume is 10 μl, and the solvents used before and after derivatization are all anhydrous ethanol-water, the volume ratio is 33:

67.

10. The method according to any one of claims 1 to 9, characterized in that The L-carnitine samples to be tested are L-carnitine raw materials, L-carnitine preparations and intermediate products thereof.

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