Method for detecting contents of various components in levocarnitine oral solution by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and discloses a method for detecting the contents of various components in a levocarnitine oral solution by using an HPLC (High Performance Liquid Chromatography) method. The chromatographic conditions of the HPLC method are as follows: octadecylsilane chemically bonded silica is used as a filler, a 0.008-0.012 mol / L diammonium hydrogen phosphate buffer solution with the pH value adjusted to 2.6-2.8 by phosphoric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B for gradient elution, the flow velocity is 0.8-1.2 ml / min, and the column temperature is 25-35 DEG C. According to the detection method disclosed by the invention, a chromatographic column is a conventional C18 column, a mobile phase is a conventional phosphate buffer solution, the preparation is simple, the specificity is good, the levocarnitine, the DL-malic acid, the sodium benzoate and the saccharin sodium salt can be simultaneously detected, the separation degree of each component is good, and the detection is accurate.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting the contents of multiple components in a L-carnitine oral solution. Background Art

[0002] L-Carnitine is a drug used to treat a series of complications caused by secondary carnitine deficiency in patients with chronic renal failure and long-term hemodialysis. It is mainly used to treat cardiomyopathy, skeletal myopathy, arrhythmia, hyperlipidemia, as well as hypotension and muscle spasm during dialysis. Compared with solid preparations, L-Carnitine oral solution has fast absorption, rapid action, good efficacy, and is easy to take and carry, and is widely used clinically.

[0003] The methods for detecting the content of L-carnitine disclosed at present all have certain disadvantages. Since L-carnitine is an ammonium hydroxide inner salt and is alkaline in water, in order to improve the distribution coefficient value of the separated components and ensure that the sample is completely ionized during the detection process, an ion pair reagent is generally added to the mobile phase to improve the tailing phenomenon. This method is not only complicated in configuration, but also requires a long time to balance the chromatographic system, which is easy to damage the chromatographic column. For example, the import registration standard of L-carnitine oral solution (No.: JX20070226) and the USP Pharmacopoeia standard of L-carnitine oral solution both use 0.055% sodium heptane sulfonate-phosphate buffer (pH2.4)-methanol (980:20) as the mobile phase. The ion pair reagent sodium heptane sulfonate is added to this mobile phase system, and the peak time of sodium benzoate is late under this chromatographic system (retention time is about 51.5min), the running time is long, and the work efficiency is low; at the same time, the USP Pharmacopoeia standard of L-carnitine oral solution uses the internal standard method, which requires the additional purchase of internal standards, and the test solution needs to be processed through a disposable stationary phase extraction column. This method is cumbersome to operate, high in cost, and low in efficiency. "Song Xingfa, Sun Zengxian. Determination of L-carnitine content for injection by UPLC [J]. Strait Pharmacy, 2017, 29 (1): 50-51.)" Acetonitrile-diammonium hydrogen phosphate buffer solution (take 13.2g diammonium hydrogen phosphate and 0.5g disodium ethylenediaminetetraacetate, add 1000mL water to dissolve, add 20mL triethylamine, and adjust the pH value to 6.9±0.1 with phosphoric acid) (16:84) as the mobile phase, and the ion pair reagent disodium ethylenediaminetetraacetate is also added. "Ding Zhaomeng. Determination of L-carnitine content in oral solution by UPLC [J] Medical Information, 2011, 24 (10): 6725-6726." Although the mobile phase uses 0.05mol / L phosphate buffer-acetonitrile (35:65), phosphoric acid is used to adjust the pH to 4.3. It does not contain ion pair reagents, but requires the use of MC-PackNH2 chromatographic columns. Amino columns are not as universal as C18 columns, are more fragile, and have a service life far shorter than that of C18 columns.

[0004] In addition, L-carnitine oral solution contains auxiliary materials such as DL-malic acid, sodium benzoate and sodium saccharin. For example, the marketed product: L-carnitine oral solution (ALFASIGMA SPA), in addition to L-carnitine as the active ingredient of this product, its content must be strictly controlled to ensure the safety and effectiveness of the drug. It is also necessary to control the dosage of other ingredients, so it is necessary to determine their content: DL-malic acid is used as a pH regulator and antioxidant for oral solution, which makes the active ingredient easier to absorb by the body and improves the antibacterial property of the oral solution. According to ICHQ6A, the content of antioxidants in oral liquid preparations should be controlled, so the pH regulator and antioxidant (DL-malic acid) used in this product need to be determined; sodium saccharin is used as a flavoring agent to adjust the taste and improve patient compliance. Under compliant application conditions, there is generally no adverse effect on the human body. If the product is not produced in compliance with regulations and excessive intake is taken, there may be hazards such as loss of appetite, developmental effects, and allergic reactions. In particular, this product can be used by infants and children, and there should be stricter restrictions on excipients to avoid safety risks. According to the "Guidelines for Pharmaceutical Development of Children's Drugs (Chemical Drugs) (Trial)", "the qualitative and quantitative composition of all components of flavoring agents should be provided as much as possible", it is necessary to conduct quantitative testing of saccharin sodium; sodium benzoate is a preservative for this product, which improves the antibacterial property of the oral solution. According to ICH Q6A, the content of preservatives in oral liquid preparations should be controlled, so the preservative (sodium benzoate) used in this product also needs to be measured; the existing technology only discloses methods for separately detecting L-carnitine, DL-malic acid, sodium benzoate and sodium saccharin, and no method for simultaneously detecting multiple components in L-carnitine preparations is disclosed. Summary of the invention

[0005] The invention provides a feasible and simple method capable of simultaneously detecting levocarnitine and other components whose contents need to be controlled in a levocarnitine oral solution.

[0006] The method disclosed by the present invention is an HPLC method, and the chromatographic conditions are: octadecylsilane bonded silica gel is used as a filler, 0.008-0.012 mol / L diammonium hydrogen phosphate buffer solution whose pH value is adjusted to 2.6-2.8 with phosphoric acid is used as a mobile phase A, acetonitrile is used as a mobile phase B for gradient elution, the flow rate is 0.8-1.2 ml / min, and the column temperature is 25-35° C.

[0007] The invention uses the most commonly used C18 column, controls the chromatographic conditions such as mobile phase composition, gradient elution ratio, column temperature, etc., and does not need to add ion pair reagent to the mobile phase, thus overcoming the tailing phenomenon of L-carnitine, and the peak time is suitable, and DL-malic acid, sodium benzoate and saccharin sodium can be accurately detected at the same time.

[0008] The preferred chromatographic column specifications are 4.6 mm × 250 mm, 5 μm; the preferred concentration of diammonium hydrogen phosphate in mobile phase A is 0.010 mol / L, and the pH is 2.7. The preferred gradient elution conditions are:

[0009]

[0010] Preferably, the wavelengths 210 nm and 272 nm are detected simultaneously, 210 nm is used to detect DL-malic acid, sodium saccharin and L-carnitine, and 272 nm is used to detect sodium benzoate.

[0011] The preferred mobile phase flow rate is 1.0 ml / min; the column temperature is 30°C, and the injection volume is 10 μl.

[0012] The invention is used for detecting multiple components of L-carnitine oral solution, wherein the multiple components are L-carnitine, DL-malic acid, sodium benzoate and saccharin sodium.

[0013] The method disclosed in the present invention for detecting the contents of multiple components in a L-carnitine oral solution by HPLC may comprise the following steps:

[0014] a. Preparation of reference solution:

[0015] a1: DL-malic acid reference solution: Take about 30 mg of DL-malic acid reference, accurately weigh it, put it in a 20 ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, then accurately measure 1 ml of the above solution, put it in a 10 ml volumetric flask, dilute to the scale with mobile phase A, shake well, and you will get a solution containing about 0.15 mg of DL-malic acid per 1 ml.

[0016] a2: Saccharin sodium reference solution: Take about 4 mg of saccharin sodium reference, accurately weigh it, put it in a 20 ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, then accurately measure 1 ml of the above solution, put it in a 10 ml volumetric flask, dilute to the scale with mobile phase A, shake well, and you will get a solution containing about 20 μg of saccharin sodium per 1 ml.

[0017] a3: Sodium benzoate reference substance stock solution: Precisely weigh about 10 mg of sodium benzoate reference substance and place it in a 10 ml volumetric flask. Add mobile phase A to dissolve and dilute to the scale. Shake well to obtain a solution containing about 1 mg per 1 ml.

[0018] a4: L-carnitine + sodium benzoate reference solution: accurately weigh about 20 mg of L-carnitine reference substance and place it in a 5 ml volumetric flask. Accurately measure 1 ml of sodium benzoate reference substance stock solution and place it in the above volumetric flask. Dissolve and dilute to the scale with mobile phase A and shake well to obtain a solution containing about 4 mg of L-carnitine and 0.2 mg of sodium benzoate per 1 ml.

[0019] b. Preparation of test solution: Accurately measure 1 ml of L-carnitine oral solution (containing 0.1 g of L-carnitine) and place it in a 25 ml volumetric flask. Dissolve and dilute to the mark with mobile phase A and shake well to obtain the solution.

[0020] c. Detection: Accurately measure the reference solution and the test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content by the peak area according to the external standard method.

[0021] Compared with the prior art, the detection method disclosed in the present invention has a chromatographic column that is a conventional C18 column, a mobile phase that is a conventional phosphate buffer, a simple configuration, good specificity, and can simultaneously detect L-carnitine, DL-malic acid, sodium benzoate and saccharin sodium, with a short detection time, good separation of the components and accurate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Specificity overlay HPLC spectrum of the embodiment of the present invention;

[0023] Figure 2 HPLC spectrum of the reference solution of the embodiment of the present invention;

[0024] Figure 3 HPLC spectrum of the test solution of the embodiment of the present invention;

[0025] Figure 4 The HPLC spectra of the reference substance and the test substance solution of Comparative Example 1 of the present invention are shown. DETAILED DESCRIPTION

[0026] The technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the 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.

[0027] The following test substance L-carnitine oral solution was purchased from ALFASIGMA SPA, batch number: 200537; the reference substances were all purchased from China Food and Drug Inspection Institutes, L-carnitine reference substance, batch number: 101041-202204; sodium benzoate reference substance, batch number: 100433-202103; sodium saccharin reference substance, batch number: 520031-201501; DL-malic acid reference substance, batch number: 520031-201501.

[0028] Example 1

[0029] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler (4.6 mm × 250 mm, 5 μm); 0.01 mol / L diammonium hydrogen phosphate (pH adjusted to 2.7 with phosphoric acid) was used as mobile phase A, acetonitrile was used as mobile phase B, and eluted according to the gradient program in the following table; detection wavelengths were 210 nm (DL-malic acid, sodium saccharin, L-carnitine), 272 nm (sodium benzoate); flow rate was 1.0 ml / min, column temperature was 30°C; injection volume was 10 μl.

[0030]

[0031] Preparation of test solution: Accurately measure 1 ml of the test L-carnitine oral solution (containing 0.1 g of L-carnitine), place it in a 25 ml volumetric flask, dissolve it with mobile phase A and dilute to the scale, shake well, and obtain.

[0032] Preparation of reference solution:

[0033] (1) DL-Malic Acid Reference Solution: Accurately weigh about 30 mg of DL-Malic Acid Reference Solution and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with mobile phase A and shake well. Accurately measure 1 ml of the above solution and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well to obtain a solution containing about 0.15 mg of DL-malic acid per 1 ml.

[0034] (2) Saccharin sodium reference solution: Take about 4 mg of saccharin sodium reference, accurately weigh it, place it in a 20 ml volumetric flask, add mobile phase A to dissolve and dilute to the mark, shake well, then accurately measure 1 ml of the above solution, place it in a 10 ml volumetric flask, dilute to the mark with mobile phase A, shake well, and you will get a solution containing about 20 μg of saccharin sodium per 1 ml.

[0035] (3) Sodium benzoate reference stock solution: Accurately weigh about 10 mg of sodium benzoate reference and place in a 10 ml volumetric flask. Add mobile phase A to dissolve and dilute to the mark. Shake well to obtain a solution containing about 1 mg per ml.

[0036] (4) L-carnitine + sodium benzoate reference solution: Accurately weigh about 20 mg of L-carnitine reference and place it in a 5 ml volumetric flask. Accurately measure 1 ml of the sodium benzoate reference stock solution and place it in the above volumetric flask. Dissolve and dilute to the mark with mobile phase A. Shake well to obtain a solution containing about 4 mg of L-carnitine and 0.2 mg of sodium benzoate per 1 ml.

[0037] The above method was validated by methodology, including specificity, linearity and accuracy. The validation results are as follows:

[0038] (1) Exclusivity

[0039] Inject DL-malic acid reference solution, saccharin sodium reference solution, sodium benzoate reference solution (1 ml of sodium benzoate reference stock solution, placed in a 5 ml volumetric flask, dissolved and diluted to the mark with mobile phase A, shake well), L-carnitine reference solution (about 20 mg of L-carnitine reference, accurately weighed, placed in a 5 ml volumetric flask, dissolved and diluted to the mark with mobile phase A, shake well) and test solution. Figure 1 ) It can be seen that the blank solvent does not interfere with the detection of DL-malic acid, saccharin sodium, sodium benzoate and L-carnitine; the peak purity of each component is greater than 990, indicating good specificity.

[0040] (2) Accuracy

[0041] Sample configuration:

[0042] Mixed control stock solution: take about 40 mg of L-carnitine reference substance, about 30 mg of DL-malic acid reference substance, about 4 mg of saccharin sodium reference substance, and about 20 mg of sodium benzoate, place in a 20 ml volumetric bottle, add mobile phase A to dissolve and dilute to scale, shake well. Prepare 3 parts to obtain mixed control stock solution 1 (containing 40.96 mg of L-carnitine, 30.336 mg of DL-malic acid, 4.072 mg of saccharin sodium, and 20.076 mg of sodium benzoate), mixed control stock solution 2 (containing 40.94 mg of L-carnitine, 30.188 mg of DL-malic acid, 4.092 mg of saccharin sodium, and 20.200 mg of sodium benzoate) and mixed control stock solution 3 (containing 41.06 mg of L-carnitine, 30.234 mg of DL-malic acid, 4.068 mg of saccharin sodium, and 20.322 mg of sodium benzoate).

[0043] Mixed reference solution: Accurately measure 1 ml of mixed reference stock solution 1 and 2 respectively, place them in 10 ml volumetric flasks, dilute to the scale with mobile phase A, shake well, and prepare 2 portions, namely mixed reference solution 1 and mixed reference solution 2.

[0044] 50% accuracy test solution: Accurately measure 1 ml each of mixed control stock solutions 1, 2, and 3 and 0.4 ml of blank auxiliary material (excluding DL-malic acid, sodium saccharin, and sodium benzoate), place them in 20 ml volumetric flasks, add 450 μl of phosphoric acid solution to each, dilute to the scale with mobile phase A, shake well, and obtain.

[0045] 100% accuracy test solution: Accurately measure 1 ml each of mixed control stock solutions 1, 2, and 3 and 0.4 ml of blank auxiliary material (excluding DL-malic acid, sodium saccharin, and sodium benzoate), place them in 10 ml volumetric flasks, add 425 μl of phosphoric acid solution to each, dilute to the scale with mobile phase A, shake well, and obtain.

[0046] 150% accuracy test solution: Accurately measure 1.5 ml each of mixed control stock solutions 1, 2, and 3 and 0.6 ml of blank auxiliary material (excluding DL-malic acid, sodium saccharin, and sodium benzoate), place them in 10 ml volumetric flasks, add 625 μl of phosphoric acid solution to each, dilute to the scale with mobile phase A, shake well, and obtain.

[0047] Inject the mixed reference solution and each accuracy test solution. Calculate the concentration of each component by external standard method.

[0048] The results showed that the average recovery of L-carnitine at each concentration was between 99.9% and 101.1%, the average of 9 recoveries was 100.3%, and the RSD was 0.8%; the average recovery of DL-malic acid at each concentration was between 100.2% and 100.8%, the average of 9 recoveries was 100.4%, and the RSD was 0.6%; the average recovery of saccharin sodium at each concentration was between 98.9% and 101.3%, the average of 9 recoveries was 100.2%, and the RSD was 1.1%; the average recovery of sodium benzoate at each concentration was between 99.8% and 101.0%, the average of 9 recoveries was 100.4%, and the RSD was 0.6%, indicating good accuracy, as shown in Table 1.

[0049] Table 1 Accuracy results

[0050]

[0051]

[0052] (3) Linear results

[0053] Sample configuration:

[0054] 20% linear test sample: Accurately measure 31 ml of the mixed control stock solution prepared in (2) and place it in a 50 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0055] 50% linear test sample: Accurately measure 1 ml of the mixed control stock solution prepared in (2) and place it in a 20 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0056] 80% linear test sample: Accurately measure 0.8 ml of the mixed control stock solution 3 prepared in (2) and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0057] 100% linear test sample: Accurately measure 1 ml of the mixed control stock solution prepared in (2) and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0058] 120% linear test sample: Accurately measure 1.2 ml of the mixed control stock solution 3 prepared in (2) and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0059] 150% linear test sample: Accurately measure 1.5 ml of the mixed control stock solution 3 prepared in (2) and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and shake well.

[0060] Inject each linearity solution.

[0061] The results showed that the r value of L-carnitine was 0.9999, the linear equation was y=0.9063x+25.484, the Y-axis intercept was 0.01 at 100% response value, the RSD of the response factor of each concentration was 1.5%, and the linearity of L-carnitine was good in the concentration range of 809.36μg / ml to 5994.9μg / ml; the r value of DL-malic acid was 0.9999, the linear equation was y=3.001x+0.574, the Y-axis intercept was 0.1% at 100% response value, The RSD of the response factor of each concentration was 0.4%, and DL-malic acid had good linearity in the concentration range of 30.2331μg / ml to 226.7481μg / ml; the r value of saccharin sodium was 0.9998, the linear equation was y=371.547x+156.098, the Y-axis intercept was 2.0% at 100% response value, the RSD of the response factor of each concentration was 1.7%, and saccharin sodium had good linearity in the concentration range of 4.067μg / ml to 30.5025μg / ml. Combined with the accuracy test results, the concentration range of DL-malic acid is 75.8389μg / ml~226.4048μg / ml, the concentration range of saccharin sodium is 10.2300μg / ml~30.5025μg / ml; the r value of sodium benzoate is 0.9999, the linear equation is y=20701.687x+0.475, the Y-axis intercept is 0.01% at 100% response value, the RSD of each concentration response factor is 0.4%, and sodium benzoate has good linearity in the concentration range of 0.0406mg / ml~0.3048mg / ml. Combined with the accuracy test results, the concentration range of sodium benzoate is 0.1016mg / ml~0.3011mg / ml. It can meet the test product detection needs, and the specific results are shown in Table 2-5.

[0062] Table 2 Linearity results of L-carnitine (210nm)

[0063]

[0064] Table 3 DL-malic acid (210nm) linear results

[0065]

[0066] Table 4 Saccharin sodium (210nm) linear results

[0067]

[0068] Table 5 Sodium benzoate (272nm) linear results

[0069]

[0070] (4) Test results of the test product

[0071] Accurately measure each reference solution and test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content by peak area according to the external standard method.

[0072] The test results of the test sample are as follows: L-carnitine: 104.0%, DL-malic acid: 3.4 mg / ml, saccharin sodium: 0.43 mg / ml, sodium benzoate: 4.7 mg / ml.

[0073] This method has a short detection time, and all components can be detected within 25 minutes, and the separation degree of each component is good (see Figure 2 and Figure 3 ).

[0074] Comparative Example 1: Testing method for import registration standard of L-carnitine oral solution

[0075] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; 0.055% sodium heptane sulfonate-phosphate buffer (pH 2.4) (measure 11.5 ml of phosphoric acid, add 1900 ml of water, adjust the pH value to 2.4 with about 100 ml of 1 mol / L sodium hydroxide, add 1.1 g of sodium heptane sulfonate, and shake to dissolve)-methanol (980:20) was used as mobile phase; the detection wavelength was 225 nm; the flow rate was 1 ml / min, and the column temperature was room temperature; the injection volume was 20 μl.

[0076] Reference solution: Take an appropriate amount of L-carnitine reference substance, dissolve it in water and prepare a solution containing approximately 0.2 mg of L-carnitine per 1 ml.

[0077] Test solution: Accurately measure an appropriate amount of the test sample (approximately equivalent to 0.2 g of L-carnitine) and place it in a 100 ml volumetric flask. Add water to dilute to the mark and shake well.

[0078] The sodium benzoate peak time of this method is about 51.5min, and the peak shape is poor (the peak width is about 8min, see Figure 4 ), the overall detection time is long, which affects the efficiency. At the same time, this mobile phase system is an ion pair, and the preparation process is complicated, so this method is not used.

[0079] Comparative Example 2 USP Standard Test Method for L-Carnitine Oral Solution

[0080] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler (μBondapak C18, 3.9mm×30cm, 10μm); 0.05mol / L phosphate buffer (pH2.4) (measure 11.4ml of phosphoric acid, add 1900ml of water, adjust the pH value to 2.4 with about 100ml of 1mol / L sodium hydroxide, measure 980ml of the above solution, add 555mg of sodium heptane sulfonate, and shake to dissolve)-methanol (980:20) was used as mobile phase; the detection wavelength was 225nm; the flow rate was 1.5ml / min, and the column temperature was room temperature; the injection volume was 40μl; the running time was 30min.

[0081] Internal standard solution: Take about 2 mg of p-aminobenzoic acid reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add water to dissolve and dilute to the scale to obtain a solution containing about 0.02 mg per 1 ml.

[0082] L-Carnitine reference solution: Take about 20 mg of L-Carnitine reference, place in a 10 ml volumetric flask, add 2.0 ml of internal standard solution, dissolve with water and dilute to make a solution containing about 2 mg of L-Carnitine per 1 ml.

[0083] Test sample stock solution: Accurately measure 2 ml of the test sample (containing 200 mg of L-carnitine) and place it in a 20 ml volumetric flask. Add water to make a solution containing approximately 10 mg of L-carnitine per 1 ml.

[0084] Test solution: Take a 10mm×4cm disposable stationary phase extraction column containing 500mg of filler, rinse it with 2 column volumes of dichloromethane, 2 column volumes of methanol and 3 column volumes of water in sequence, then accurately measure 5ml of the test sample stock solution and place it in the disposable column, rinse the column twice with 6ml of water, collect the filtrate and washing liquid in a 25ml volumetric flask, add 5ml of internal standard solution, dilute to the scale with water, and shake well.

[0085] Determination: Accurately measure 40 μl of L-carnitine reference solution and test solution, respectively, inject them into liquid chromatograph and record the chromatogram.

[0086] The chromatographic conditions of this method are consistent with those of Comparative Example 1. This method takes a long time to detect as a whole, and the sample pretreatment is complicated, which greatly affects the work efficiency and wastes manpower and material resources. Therefore, this method is not adopted.

[0087] The above embodiments of the present invention are merely examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for detecting the contents of multiple components in a L-carnitine oral solution by HPLC, characterized in that: The chromatographic conditions of the HPLC method are: using octadecylsilane bonded silica gel as a filler, using 0.008-0.012 mol / L diammonium hydrogen phosphate buffer solution adjusted to a pH value of 2.6-2.8 with phosphoric acid as a mobile phase A, using acetonitrile as a mobile phase B for gradient elution, a flow rate of 0.8-1.2 ml / min, and a column temperature of 25-35°C.

2. The method according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6mm×250mm, 5μm.

3. The method according to claim 1, characterized in that The concentration of diammonium hydrogen phosphate in the mobile phase A is 0.010 mol / L, and the pH is 2.

7.

4. The method according to claim 1, characterized in that The conditions of the gradient elution are:

5. The method according to claim 1, characterized in that: The detection wavelengths are 210 nm and 272 nm.

6. The method according to claim 1, characterized in that The mobile phase flow rate was 1.0 ml / min.

7. The method according to claim 1, characterized in that The column temperature was 30°C and the injection volume was 10 μl.

8. The method according to claim 1, characterized in that: The multiple ingredients in the L-carnitine oral solution are L-carnitine, DL-malic acid, sodium benzoate and saccharin sodium.

9. The method according to claim 1, characterized in that: The following steps are involved: a. Preparation of reference solution: a1: DL-malic acid reference solution: take about 30 mg of DL-malic acid reference, accurately weigh it, put it in a 20 ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, then accurately measure 1 ml of the above solution, put it in a 10 ml volumetric flask, dilute to the scale with mobile phase A, shake well, and you will get a solution containing about 0.15 mg of DL-malic acid per 1 ml; a2: Saccharin sodium reference solution: Take about 4 mg of saccharin sodium reference, accurately weigh it, put it in a 20 ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, then accurately measure 1 ml of the above solution, put it in a 10 ml volumetric flask, dilute to the scale with mobile phase A, shake well, and you will get a solution containing about 20 μg of saccharin sodium per 1 ml; a3: Sodium benzoate reference substance stock solution: weigh about 10 mg of sodium benzoate reference substance accurately and place it in a 10 ml volumetric flask. Add mobile phase A to dissolve and dilute to the mark. Shake well to obtain a solution containing about 1 mg per 1 ml. a4: L-carnitine + sodium benzoate reference solution: accurately weigh about 20 mg of L-carnitine reference and place it in a 5 ml volumetric flask. Accurately measure 1 ml of sodium benzoate reference stock solution and place it in the above volumetric flask. Dissolve and dilute to scale with mobile phase A and shake well to obtain a solution containing about 4 mg of L-carnitine and 0.2 mg of sodium benzoate per 1 ml. b Preparation of test solution: Accurately measure 1 ml of L-carnitine oral solution, place in a 25 ml volumetric flask, dissolve and dilute to scale with mobile phase A, shake well, and obtain; c Detection: Accurately measure the reference solution and the test solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the content by the peak area according to the external standard method.

Images