A method for determining potential genotoxic impurities in levocarnitine by GC-MS
The GC-MS method was optimized to detect genotoxic impurities in L-carnitine, which solved the problem of insufficient detection sensitivity in the existing technology, and achieved efficient and accurate impurity control, ensuring that the product quality meets the standards.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- 南京红太阳医药研究院有限公司
- Filing Date
- 2022-12-19
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies are insufficient for efficiently and accurately detecting the potential genotoxic impurities 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine, resulting in an inability to effectively control drug quality.
The GC-MS method, combined with specific chromatographic and mass spectrometric conditions, was used to prepare test solutions and reference solutions, and the external standard method was used for detection. The extraction and separation process was optimized to ensure complete dissolution of L-carnitine, and the selected ion monitoring mode was used for quantitative and qualitative analysis.
It enables efficient, rapid, and sensitive detection of genotoxic impurities in L-carnitine, ensuring that their content is controlled within 0.5 ppm, thus effectively controlling the quality of L-carnitine.
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Figure CN116223652B_ABST
Abstract
Description
A method for GC-MS determination of potential genotoxic impurities in L-carnitine Technical Field
[0001] This invention belongs to the field of material detection technology, and relates to a method for determining potential genotoxic impurities in L-carnitine by GC-MS (gas chromatography-mass spectrometry), and particularly to a method for determining 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine by GC-MS. Background Technology
[0002] L-carnitine is a drug used to treat a range of complications caused by secondary carnitine deficiency in long-term hemodialysis patients with chronic renal failure. It is mainly used to treat conditions such as cardiomyopathy, skeletal myopathy, arrhythmia, hyperlipidemia, as well as hypotension and muscle spasms during dialysis.
[0003] L-carnitine, chemically known as (3R)-hydroxy-(trimethylammonium)butyrate inner salt, may introduce potentially genotoxic impurities during its chemical synthesis: 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol.
[0004] L-carnitine is the main active ingredient in L-carnitine-related formulations. The acceptable limits for genotoxic impurities are generally calculated based on the threshold for toxicological concern (TTC) limits.
[0005]
[0006] Table 1. TTC Limits
[0007] Treatment period ≤1 month 1–12 months 1–10 years ≥10 years Daily intake (μg / d) 1 20 20 10 1.5 surface
[0008] As the main active ingredient in the injectable form, the maximum daily dose of L-carnitine is 2g. Based on the strictest daily intake of 1.5μg / d, the acceptable limit for genotoxic impurities is 0.75ppm.
[0009] As the main active ingredient in oral formulations, the maximum daily dose of levocarnitine is 3g. Based on the strictest daily intake of 1.5μg / d, the acceptable limit for genotoxic impurities is 0.5ppm.
[0010] L-carnitine is controlled according to the strictest standards, with 0.5 ppm as the acceptable limit for potential genotoxic impurities. Summary of the Invention
[0011] The purpose of this invention is to establish a method for determining the potential genotoxic impurities of levocarnitine, 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol. The inventors first developed and pre-validated a method using commonly used GC (gas chromatography) for 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol. During the pre-validation process, it was found that the sensitivity of GC was insufficient due to the low limits of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol. Therefore, the GC method was adjusted to GC-MS for validation. The results show that GC-MS can effectively and accurately detect 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol.
[0012] The purpose of this invention is to establish a GC-MS method for determining the potential genotoxic impurities 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine, so as to better control the quality of L-carnitine.
[0013] The objective of this invention is achieved through the following technical solution:
[0014] A method for GC-MS determination of potentially genotoxic impurities in levocarnitine includes:
[0015] Preparation of test solution: Weigh approximately 0.2–3 g of L-carnitine accurately, place it in a centrifuge tube, add 1–2 ml of water, shake to dissolve the sample, add 5 ml of acetone, shake thoroughly to extract, then seal and centrifuge, and take the supernatant, which is the test solution.
[0016] Preparation of reference solution: Weigh appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol accurately, and dilute with acetone to prepare a mixed solution containing approximately 0.02–0.2 μg of 2,3-dichloro-1-propanol and approximately 0.02–0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution;
[0017] The reference solution and the test solution were analyzed by GC-MS, and the contents of impurities 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine were determined by external standard method.
[0018] The inventors based their work on the fact that L-carnitine is an ionic compound, readily soluble in water but insoluble in acetone. Since water or substances with high water content cannot be used in GC-MS, L-carnitine was dissolved in a small amount of water, extracted with acetone, and then analyzed by GC-MS. Controlling the amount of water used within the scope of this invention ensures effective separation and complete sample dissolution. Excessive water usage leads to failure to separate the sample, resulting in acetone and water miscibility and precipitation of L-carnitine. Insufficient water usage prevents complete dissolution of the L-carnitine sample, thus hindering the effective extraction of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol from the undissolved portion of the L-carnitine sample.
[0019] Preferably, the test solution is prepared as follows: Accurately weigh approximately 2g of L-carnitine, place it in a centrifuge tube, add 1-2ml of water, shake to dissolve the sample, add 5ml of acetone, extract by shaking thoroughly, then seal and centrifuge, and collect the supernatant. Alternatively, the test solution can be prepared as follows: Accurately weigh approximately 0.2-3g of L-carnitine, place it in a centrifuge tube, add 1-1.4ml of water, shake to dissolve the sample, add 5ml of acetone, extract by shaking thoroughly, then seal and centrifuge, and collect the supernatant.
[0020] More preferably, prepare the test solution: accurately weigh about 2g of L-carnitine, place it in a centrifuge tube, add 1-1.4ml of water, shake to dissolve the sample, add 5ml of acetone, shake thoroughly to extract, then seal and centrifuge, and take the supernatant.
[0021] Preferably, the reference solution is prepared by accurately weighing appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, and quantitatively diluting them with acetone to prepare a mixed solution containing 0.2 μg of 2,3-dichloro-1-propanol and 0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
[0022] The gas chromatography conditions were as follows: a capillary column with polyethylene glycol as the stationary phase was used as the chromatographic column, the initial column temperature was 40-120℃, maintained for 2-10 minutes, the temperature was increased to 180-220℃ at a rate of 20℃ / min, and maintained for 5-15 minutes, the injection port temperature was 200℃, the detector temperature was 230℃, and the injection volume was 0.1-2 μl.
[0023] Preferably, the chromatographic column is a capillary column with polyethylene glycol as the immobilizer (column length 30m, inner diameter 0.25mm, membrane thickness 0.25μm).
[0024] Preferably, the gas chromatography conditions are as follows: DB-WAXert column (column length 30m × inner diameter 0.25mm, film thickness 0.25μm), initial column temperature 100-120℃, maintained for 2 minutes, then increased to 200℃ at a rate of 20℃ / min, maintained for 5 minutes, injection port temperature 200℃, detector temperature 230℃, and injection volume 1μl.
[0025] In GC-MS detection, the split ratio affects the sensitivity. The split ratio is between zero and 50:1, with 5:1 being the preferred ratio.
[0026] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0027] The beneficial effects of this invention are:
[0028] This invention employs GC-MS to determine the potential genotoxic impurities 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine. This method features high separation efficiency, fast analysis speed, and high detection sensitivity. By detecting these potential genotoxic impurities, the levels of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine can be controlled to be no greater than 0.5 ppm, effectively controlling the quality of L-carnitine. Attached Figure Description
[0029] Figure 1 shows the preparation of the test sample solution in Example 1.
[0030] Figure 2 shows the GC-MS spectrum of the blank solvent in Example 1.
[0031] Figure 3 shows the GC-MS spectrum of the reference solution in Example 1 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0032] Figure 4 shows the GC-MS spectrum of the test solution in Example 1.
[0033] Figure 5 shows the GC-MS spectrum of the detection limit solution in Example 2 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0034] Figure 6 shows the GC-MS spectrum of the limit of quantitation solution in Example 3 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0035] Figure 7 shows the GC-MS spectrum of the reference solution in Example 4 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0036] Figure 8 shows the preparation of the test solution in Example 5.
[0037] Figure 9 shows the preparation of the test sample solution in Example 6.
[0038] Figure 10 shows the GC-MS spectrum of the 50% accuracy solution in Example 7 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0039] Figure 11 shows the GC-MS spectrum of the 100% accuracy solution in Example 7 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0040] Figure 12 shows the GC-MS spectrum of the 150% accuracy solution in Example 7 (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol). Detailed Implementation
[0041] The technical solution of the present invention is further illustrated by the following embodiments, but the embodiments are not intended to limit the present invention.
[0042] All reagents used in the examples are commercially available or synthesized using existing methods.
[0043] GC-MS (Gas Chromatograph-Mass Spectrometer) Model:
[0044]
[0045] The chromatographic columns used in Examples 1-6 were DB-WAXert (30m × 0.25mm, 0.25μm).
[0046] Reagents used:
[0047]
[0048] Example 1
[0049] Methods for GC-MS determination of potentially genotoxic impurities in L-carnitine include:
[0050] Preparation of the test solution: Weigh approximately 2g of L-carnitine accurately, place it in a centrifuge tube, add 1.4ml of water, shake to dissolve the sample, extract with 5ml of acetone (shake thoroughly), seal and centrifuge (see Figure 1), and collect the supernatant.
[0051] Preparation of reference solution: Take appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, weigh them accurately, and dilute them quantitatively with acetone to prepare a mixed solution containing approximately 0.2 μg of 2,3-dichloro-1-propanol and 0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
[0052] The reference solution and the test solution were detected by GC-MS, respectively.
[0053] The chromatographic conditions for GC-MS detection were as follows: a DB-WAXert column (30m×0.25mm, 0.25μm) was used, the initial column temperature was 100℃, maintained for 2 minutes, the temperature was increased to 200℃ at a rate of 20℃ / min, and maintained for 5 minutes. The injection port temperature was 200℃, the detector temperature was 230℃, the split ratio was 5:1, and the injection volume was 1μl.
[0054] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0055] Figure 2 shows the GC-MS spectrum of the blank solvent in this embodiment. Figure 3 shows the GC-MS spectrum of the reference solution in this embodiment, where peak 1 represents 1,3-dichloro-2-propanol and peak 2 represents 2,3-dichloro-1-propanol. Figure 4 shows the GC-MS spectrum of the test solution in this embodiment, which does not contain 1,3-dichloro-2-propanol or 2,3-dichloro-1-propanol.
[0056] Example 2
[0057] Detection limit solution: Weigh appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol accurately, and dilute quantitatively with acetone to prepare a mixed solution containing approximately 0.005 μg of 2,3-dichloro-1-propanol and 0.005 μg of 1,3-dichloro-2-propanol per 1 ml.
[0058] The solution was detected by GC-MS.
[0059] The chromatographic conditions for GC-MS detection were as follows: a DB-WAXert column (30m×0.25mm, 0.25μm) was used, the initial column temperature was 100℃, maintained for 2 minutes, the temperature was increased to 200℃ at a rate of 20℃ / min, and maintained for 5 minutes. The injection port temperature was 200℃, the detector temperature was 230℃, the split ratio was 5:1, and the injection volume was 1μl.
[0060] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0061] The GC-MS spectrum of the detection limit solution in this embodiment is shown in Figure 5. The detection limit of this method is 0.01 ppm, which is equivalent to 2% of the limit of 0.5 ppm. This indicates that the detection sensitivity of this method is very high.
[0062] Example 3
[0063] Limit of Quantification Solution: Weigh appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol accurately, and dilute quantitatively with acetone to prepare a mixed solution containing approximately 0.01 μg of 2,3-dichloro-1-propanol and 0.01 μg of 1,3-dichloro-2-propanol per 1 ml.
[0064] GC-MS was used to detect the solution at the limit of quantitation.
[0065] The chromatographic conditions for GC-MS detection were as follows: a DB-WAXert column (30m×0.25mm, 0.25μm) was used, the initial column temperature was 100℃, maintained for 2 minutes, the temperature was increased to 200℃ at a rate of 20℃ / min, and maintained for 5 minutes. The injection port temperature was 200℃, the detector temperature was 230℃, the split ratio was 5:1, and the injection volume was 1μl.
[0066] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0067] The GC-MS spectrum of the solution at the limit of quantitation in this embodiment is shown in Figure 6. The limit of quantitation for this method is 0.03 ppm, which is 6% of the limit of 0.5 ppm. This indicates that the quantitative sensitivity of this method is very high.
[0068] Example 4
[0069] Preparation of reference solution: Take appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, weigh them accurately, and dilute them quantitatively with acetone to prepare a mixed solution containing approximately 0.2 μg of 2,3-dichloro-1-propanol and 0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
[0070] The reference solution was analyzed by GC-MS.
[0071] The chromatographic conditions for GC-MS detection were as follows: a DB-WAXert column (30m×0.25mm, 0.25μm) was used, the initial column temperature was 120℃, maintained for 2 minutes, the temperature was increased to 200℃ at a rate of 20℃ / min, and maintained for 5 minutes. The injection port temperature was 200℃, the detector temperature was 230℃, the split ratio was 5:1, and the injection volume was 1μl.
[0072] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0073] The GC-MS spectrum of the reference solution in this embodiment is shown in Figure 7, indicating that the initial column temperature has no significant effect on the detection results.
[0074] Example 5
[0075] Preparation of the test solution: Weigh approximately 2g of L-carnitine accurately, place it in a centrifuge tube, add 1ml of water, shake to dissolve the sample, add 5ml of acetone, shake thoroughly, extract, seal and centrifuge, see Figure 8.
[0076] Compared to the preparation of the test solution in Example 1 (adding 1.4 ml of water), the shaking time in this example is longer, but it still meets the detection requirements. If the amount of water is further reduced, L-carnitine may not dissolve completely, thus preventing the effective extraction of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol from the undissolved portion of the L-carnitine sample, thereby affecting the detection results.
[0077] Example 6
[0078] Preparation of the test solution: Weigh approximately 2g of L-carnitine accurately, place it in a centrifuge tube, add 2ml of water, shake to dissolve the sample, add 5ml of acetone for extraction (shake thoroughly), seal and centrifuge, see Figure 9.
[0079] Compared with the test solution prepared in Example 1 (with 1.4 ml of water added), the lower layer of this example showed turbidity, indicating that as the amount of water increased, acetone and water dissolved each other, causing L-carnitine to precipitate. The volume of the extract also changed, with the extract volume increasing and the sample concentration decreasing, resulting in a lower value in the test results.
[0080] Example 7: Accuracy Example
[0081] Methods for GC-MS determination of potentially genotoxic impurities in L-carnitine include:
[0082] Preparation of the test solution: Weigh approximately 2g of L-carnitine accurately, place it in a centrifuge tube, add 1.4ml of water, shake to dissolve the sample, add 5ml of acetone for extraction (shake thoroughly), seal and centrifuge, and collect the supernatant.
[0083] Preparation of reference solution: Take appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, weigh them accurately, and dilute them quantitatively with acetone to prepare a mixed solution containing approximately 0.2 μg of 2,3-dichloro-1-propanol and 0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
[0084] Impurity reference solution: Take appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, weigh them accurately, and dilute them quantitatively with acetone to prepare a mixed solution containing approximately 20 μg of 2,3-dichloro-1-propanol and 20 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
[0085] 50% accuracy solution: Take 2g of L-carnitine, place it in a centrifuge tube, add 1.4ml of water, and shake to dissolve the sample; accurately measure 25μl of impurity reference solution, place it in the same centrifuge tube, add 5ml of acetone for extraction (shake thoroughly), seal and centrifuge, and take the supernatant.
[0086] 100% accuracy solution: Take 2g of L-carnitine, place it in a centrifuge tube, add 1.4ml of water, and shake to dissolve the sample; accurately measure 50μl of impurity reference solution, place it in the same centrifuge tube, extract with 5ml of acetone (shake thoroughly), seal and centrifuge, and take the supernatant.
[0087] 150% accuracy solution: Take 2g of L-carnitine, place it in a centrifuge tube, add 1.4ml of water, and shake to dissolve the sample; accurately measure 75μl of impurity reference solution, place it in the same centrifuge tube, extract with 5ml of acetone (shake thoroughly), seal and centrifuge, and take the supernatant.
[0088] GC-MS was used to detect the reference solution, test solution, 50% accuracy solution, 100% accuracy solution, and 150% accuracy solution.
[0089] The chromatographic conditions for GC-MS detection were as follows: a DB-WAXert column (30m×0.25mm, 0.25μm) was used, the initial column temperature was 100℃, maintained for 2 minutes, the temperature was increased to 200℃ at a rate of 20℃ / min, and maintained for 5 minutes. The injection port temperature was 200℃, the detector temperature was 230℃, the split ratio was 5:1, and the injection volume was 1μl.
[0090] The mass spectrometry conditions were as follows: electron impact ionization (EI); ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode (SIM) was selected. In full scan mode, the quantitative ion mass-to-charge ratio (m / z) of 1,3-dichloro-2-propanol was confirmed to be 79, and the qualitative ion mass-to-charge ratio (m / z) was 43 and 81; the quantitative ion mass-to-charge ratio (m / z) of 2,3-dichloro-1-propanol was 64, and the qualitative ion mass-to-charge ratio (m / z) was 62 and 63.
[0091] Figure 10 shows the GC-MS spectrum of the 50% accuracy solution (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol); Figure 11 shows the GC-MS spectrum of the 100% accuracy solution (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol); Figure 12 shows the GC-MS spectrum of the 150% accuracy solution (peak 1 is 1,3-dichloro-2-propanol, peak 2 is 2,3-dichloro-1-propanol).
[0092] The contents of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in 50%, 100%, and 150% accuracy solutions were determined using the external standard method. The recoveries of 1,3-dichloro-2-propanol at the 50% accuracy level were 111.00%, 96.12%, and 100.98%; those at the 150% accuracy level were 93.87%, 88.67%, and 90.68%. Therefore, the method of this invention can effectively and accurately reflect the quality of the product.
Claims
1. A method for GC-MS determination of potential genotoxic impurities in L-carnitine, characterized in that: include: Preparation of the test solution: Accurately weigh 0.2–3 g of L-carnitine, place it in a centrifuge tube, add 1–2 ml of water, shake to dissolve the sample, add 5 ml of acetone, shake to extract, then seal and centrifuge, and collect the supernatant; Preparation of the reference solution: Accurately weigh appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, and dilute with acetone to a concentration of 0.02–0.2 μg per ml of solution. A mixed solution of 2,3-dichloro-1-propanol and 0.02–0.2 μg of 1,3-dichloro-2-propanol was prepared. The reference solution and the test solution were analyzed by GC-MS, and the contents of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol in L-carnitine were determined by external standard method. The gas chromatography conditions were as follows: a capillary column with polyethylene glycol as the stationary phase was used; the initial column temperature was 40–120 °C, maintained for 2–10 minutes, then increased to 180–220 °C at a rate of 20 °C / min and maintained for 5–15 minutes; the injection port temperature was 200 °C; the detector temperature was 230 °C; and the injection volume was 0.1–2 µl.
2. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: Preparation of test solution: Weigh 2g of L-carnitine accurately, place it in a centrifuge tube, add 1-2ml of water, shake to dissolve the sample, add 5ml of acetone, shake to extract, then seal and centrifuge, and take the supernatant.
3. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: Preparation of the test solution: Weigh 0.2-3g of L-carnitine accurately, place it in a centrifuge tube, add 1-1.4ml of water, shake to dissolve the sample, add 5ml of acetone, shake to extract, then seal and centrifuge, and take the supernatant.
4. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: Preparation of the test solution: Weigh 2g of L-carnitine accurately, place it in a centrifuge tube, add 1-1.4ml of water, shake to dissolve the sample, add 5ml of acetone, shake to extract, then seal and centrifuge, and take the supernatant.
5. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: Preparation of reference solution: Take appropriate amounts of 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol, weigh them accurately, and dilute them quantitatively with acetone to prepare a mixed solution containing 0.2 μg of 2,3-dichloro-1-propanol and 0.2 μg of 1,3-dichloro-2-propanol per 1 ml of solution.
6. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: The gas chromatography conditions were as follows: DB-WAXert column, 30m×0.25mm, 0.25μm, initial column temperature 100~120℃, maintained for 2 minutes, then increased to 200℃ at a rate of 20℃ / min and held for 5 minutes, injection port temperature 200℃, detector temperature 230℃, and injection volume 1µl.
7. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: In GC-MS detection, the split ratio is no split ratio ~ 50:
1.
8. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 7, characterized in that: In GC-MS detection, the split ratio is 5:
1.
9. The method for determining potential genotoxic impurities in L-carnitine by GC-MS according to claim 1, characterized in that: The mass spectrometry conditions were as follows: electron impact ion source; ion source temperature: 230℃; interface temperature: 200℃; solvent delay time: 4.2 min; acquisition mode: ion monitoring mode was selected, the quantitative ion for 1,3-dichloro-2-propanol was 79 m / z, and the qualitative ion was 43 and 81 m / z; the quantitative ion for 2,3-dichloro-1-propanol was 64 m / z, and the qualitative ion was 62 and 63 m / z.