Detection method of N-nitroso desloratadine

Abstract

The invention relates to the technical field of pharmaceutical analysis, and relates to a method for detecting nitrosamine impurity N-nitroso desloratadine in desloratadine. The detection method is a liquid chromatography-mass spectrometry method, and the chromatographic conditions of liquid chromatography are as follows: purified water is taken as a mobile phase A, methanol is taken as a mobile phase B, and gradient elution is performed; the flow velocity is 0.35 mL / min to 0.42 mL / min, and the column temperature is 20 DEG C to 30 DEG C. The gradient elution procedure is as follows: 0.00-(0.50-1.00) min, and the ratio of a mobile phase A to a mobile phase B is 9: 1; in the (1.00-2.00)-3.00 min, the ratio of the mobile phase A to the mobile phase B is 5: 5; in 4.50-5.50 min, the ratio of the mobile phase A to the mobile phase B is 2: 8; in 6.00-6.50 min, the ratio of the mobile phase A to the mobile phase B is 9: 1. The mass spectrum adopts a triple quadrupole, an ESI ion source, positive ion detection and an SRM mode, the temperature of the ion source is 330 DEG C, the spray voltage is 3500 V, the sheath gas is 10, and the auxiliary gas is 5. The method has the advantages of being simple, convenient, stable, high in precision, good in reproducibility and the like, the impurity N-nitroso desloratadine in the desloratadine raw material medicine can be rapidly and accurately detected, and the whole operation process is reliable and controllable.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and relates to a method for detecting nitrosamine impurity N-nitroso-loratadine in desloratadine. Background Art

[0002] Desloratadine, molecular formula C 19 H 19 N2Cl, as a third-generation antihistamine, has affinity and selectivity for peripheral H1 receptors, has the advantages of rapid onset, lasting effect, no central nervous system sedation, little cardiotoxicity and drug interactions, etc. It has been widely used in the treatment of seasonal allergic rhinitis, urticaria, etc.

[0003] In 2018, after the outbreak of the nitrosamine carcinogenic impurity NDMA (nitrosodimethylamine) in sartan drugs, regulatory agencies such as the FDA and EMA have been emphasizing the necessity of quality control of nitrosamine impurities in the production process of raw materials and finished drugs. The FDA's regulatory requirements for nitrosamine impurities can be roughly divided into two periods: In the initial stage, due to the influence of NDMA contamination in sartan and other drugs, the FDA's attention was mainly focused on the management of such simple nitrosamine impurities. In the second stage, the focus of the FDA and EMA shifted to the so-called NDSRI (Nitrosamine drugsubstance related impurities). Unlike simple nitrosamine impurities, NDSRI refers to the type of drug molecules themselves that participate in the formation of nitrosamine impurities. These drugs usually contain secondary amine (including tertiary amine) structures, which provide amino units for the formation of nitrosamines. Compared with simple nitrosamine impurities such as dimethylnitrosamine (NDMA) and N-nitrosodiethylamine (NDEA), the upper limit values ​​of nitrosated impurities related to these products are specified to be very low.

[0004] On September 1, 2020, the FDA issued the "Control of Nitrosamine Impurities in Human Drugs" guideline, which recommends steps that API and drug manufacturers should take to detect and prevent substandard nitrosamine impurity levels in drugs. The FDA recommends that manufacturers take measures to quantify the nitrosamine content in their drugs and reduce or remove these impurities when they exceed the temporary limit. According to the FDA's information on the update of Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs) on April 18, 2024, the structural formula and AI value (400ng / day) of N-nitroso-loratadine were announced. It can be seen from the drug instructions of desloratadine that its maximum daily intake is 5mg. According to the limit calculation formula (limit = AI / maximum daily intake), the limit of N-nitroso-loratadine can be calculated to be 80ppm.

[0005] The prior art does not involve a method for detecting the nitrosamine impurity N-nitroso-loratadine in desloratadine. Therefore, developing a method for detecting N-nitroso-loratadine is of great significance to drug safety and quality control of desloratadine.

[0006] Summary of the invention

[0007] The invention provides a method for detecting N-nitroso-loratadine, a nitrosamine impurity in desloratadine. The detection method has excellent sensitivity, precision and linearity, and can meet the requirements for accurate detection of N-nitroso-loratadine in desloratadine raw materials.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for detecting N-nitroso-loratadine, the method comprising liquid chromatography-mass spectrometry, and at least the following steps:

[0010] Step 1: Prepare test solution and reference solution:

[0011] Preparation of reference solution: Take N-nitroso-loratadine sample and prepare reference solution with methanol;

[0012] Preparation of test solution: Take a desloratadine sample and prepare a test solution with methanol;

[0013] Step 2: Detecting the test solution and the reference solution by liquid chromatography-mass spectrometry;

[0014] The chromatographic conditions of the liquid chromatography are:

[0015] A Nanochrom C18 chromatographic column (2.1×50mm×1.8μm) was used with purified water as mobile phase A and methanol as mobile phase B for gradient elution; the flow rate was 0.35mL / min~0.42mL / min, the column temperature was 20℃~30℃, and the injection volume was 1μL.

[0016] The procedure of the gradient elution is as follows:

[0017] 0.00~(0.50-1.00)min, mobile phase A:B=9:1;

[0018] (1.00-2.00)~3.00min, mobile phase A:B=5:5;

[0019] 4.50-5.50 min, mobile phase A:B=2:8;

[0020] 6.00-6.50min, mobile phase A:B=9:1.

[0021] The following gradient elution program is preferred:

[0022] 0.00~(0.50-0.80)min, mobile phase A:B=9:1;

[0023] (1.00-1.50)~3.00min, mobile phase A:B=5:5;

[0024] 4.50-5.50 min, mobile phase A:B=2:8;

[0025] 6.00-6.50min, mobile phase A:B=9:1.

[0026] The following gradient elution program is preferred:

[0027] 0.00~0.72min, mobile phase A:B=9:1;

[0028] 1.00~3.00min, mobile phase A:B=5:5;

[0029] 4.50-5.50 min, mobile phase A:B=2:8;

[0030] 6.00-6.50min, mobile phase A:B=9:1.

[0031] Furthermore, the present invention preferably adopts the following chromatographic conditions:

[0032] A Nanochrom C18 chromatographic column (2.1×50mm×1.8μm) was used with purified water as mobile phase A and methanol as mobile phase B for gradient elution; the flow rate was 0.40mL / min, the column temperature was 25°C, and the injection volume was 1μL.

[0033] Gradient elution has a significant effect on the separation effect and peak shape of the detection target. Different gradient elution sequences and different mobile phase compositions may affect the separation effect and peak shape of the target. The gradient elution sequence and mobile phase composition of the present invention can not only separate N-nitroso-loratadine from desloratadine with excellent separation effect, but also take into account detection, short analysis time and high efficiency.

[0034] The mass spectrometer adopts triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330° C., spray voltage of 3500 V, sheath gas of 10, and auxiliary gas of 5.

[0035] The quantitative ion of N-nitroso-loratadine is 266.1 m / z. The quantitative ion enables N-nitroso-loratadine to have a higher response value under the detection conditions, which can improve the accuracy of the detection.

[0036] The procedure of the mass spectrometer switching valve is as follows:

[0037] 0.00~3.7min, discharged; 3.7~5.9min, connected; 5.9~6.5min, discharged.

[0038] The peak time of desloratadine API is about 2.26min, and the peak time of N-nitroso-loratadine API is about 4.90min. The retention time of desloratadine API is excluded, and the retention time of N-nitroso-loratadine is connected. This can avoid damage to the instrument due to high concentration of API, and can also perform qualitative and quantitative analysis of N-nitroso-loratadine.

[0039] The detection method of N-nitroso-loratadine provided by the present invention has the following advantages:

[0040] The detection method of N-nitroso-loratadine provided by the present invention realizes the quantitative and qualitative analysis of N-nitroso-loratadine in a desloratadine raw material medicine. The method has strong specificity and low detection limit. The detection limit of N-nitroso-loratadine is 0.5 ng / mL and the quantitative limit is 1 ng / mL, which meets the detection requirements of N-nitroso-loratadine in a desloratadine raw material medicine. Moreover, the method has good linear relationship and high repeatability. The correlation coefficients within the linear range of the standard curve are all greater than 0.999. In addition, the detection method also has the advantages of fast analysis time and small sample amount, and can greatly improve the sample analysis efficiency.

[0041] The present invention adopts a liquid chromatography-mass spectrometry method to detect N-nitroso-loratadine in desloratadine, and has the advantages of being simple, stable, high in precision, good in reproducibility, etc., and can quickly and accurately detect the impurity N-nitroso-loratadine in the desloratadine raw material medicine. The whole operation process is reliable and controllable, suitable for practical application and promotion, and has broad application prospects.

[0042] The flow rate and column temperature of the present invention can make N-nitroso-loratadine peak in a short time, thus reducing the analysis time, and have excellent separation effect and good peak shape.

[0043] In the step 1 of the present invention, methanol is selected as the solvent, which not only ensures that the substances other than N-nitroso-loratadine can be extracted as little as possible, but also can extract N-nitroso-loratadine in the desloratadine raw material as much as possible, and has no interference with the detection of liquid chromatography and mass spectrometry, thereby ensuring that the detection result of the liquid chromatography-mass spectrometry method provided by the present invention is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention.

[0045] Figure 1 is a liquid chromatogram of desloratadine in method 1 of embodiment 1 of the present invention;

[0046] Figure 2 is a liquid phase chromatogram of N-nitroso-loratadine in method 1 of embodiment 1 of the present invention;

[0047] Figure 3 is a liquid chromatogram of desloratadine in method 2 of embodiment 1 of the present invention;

[0048] Figure 4 is a liquid phase chromatogram of N-nitroso-loratadine in method 2 of Example 1 of the present invention;

[0049] Figure 5 is a liquid chromatogram of desloratadine in method 3 of embodiment 1 of the present invention;

[0050] Figure 6 is a liquid phase chromatogram of N-nitroso-loratadine in method 3 of Example 1 of the present invention;

[0051] Figure 7 is a liquid chromatogram of desloratadine in method 4 of embodiment 1 of the present invention;

[0052] Figure 8 is a liquid phase chromatogram of N-nitroso-loratadine in method 4 of Example 1 of the present invention;

[0053] Fig. 9 is an ion chromatogram of detecting N-nitroso-loratadine according to method 5 of embodiment 1 of the present invention;

[0054] Fig.10 This is an ion chromatogram of detecting N-nitroso-loratadine according to method 6 of embodiment 1 of the present invention;

[0055] Fig.11 is an ion chromatogram of detecting N-nitroso-loratadine according to method 7 of embodiment 1 of the present invention;

[0056] Fig.12 is an ion chromatogram of detecting N-nitroso-loratadine according to method 8 of embodiment 1 of the present invention;

[0057] Fig.13 is the ion chromatogram of N-nitroso-loratadine in the final detection method of Example 1 of the present invention;

[0058] Fig.14 is an ion chromatogram of a blank solution for detecting N-nitroso-loratadine according to Example 2 of the present invention;

[0059] Fig.15 is a high performance liquid chromatogram of a test solution for detecting N-nitroso-loratadine according to Example 2 of the present invention;

[0060] Fig.16 is an ion chromatogram of a reference solution for detecting N-nitroso-loratadine according to Example 2 of the present invention;

[0061] Fig.17 is an ion chromatogram of a 100% test sample spiked solution for detecting N-nitroso-loratadine according to Example 2 of the present invention;

[0062] Fig.18 is a high performance liquid chromatogram of a 100% test sample spiked solution for detecting N-nitroso-loratadine according to Example 2 of the present invention;

[0063] Fig.19 Graph 1 is the linear regression curve of N-nitroso-loratadine of Example 7 of the present invention. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0065] Example 1

[0066] The present application provides a variety of methods for developing a method for detecting nitrosamines in desloratadine, wherein the method comprises the following steps:

[0067] 1. Effect of different gradient elution procedures on separation effect in liquid chromatography

[0068] Method 1: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.40mL / min, column temperature of 25°C, injection volume of 1μL, wherein the gradient elution procedure is as follows:

[0069] 0.00~1.00min, mobile phase A:B=7:3;

[0070] 1.00~3.00min, mobile phase A:B=5:5;

[0071] 4.00-5.00 min, mobile phase A:B=3:7;

[0072] 6.00-6.50min, mobile phase A:B=7:3.

[0073] Figure 1 This is the liquid chromatogram of desloratadine.

[0074] Figure 2 This is the liquid chromatogram of N-nitroso-loratadine.

[0075] Method 2: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.40mL / min, column temperature of 25°C, injection volume of 1μL, wherein the gradient elution procedure is as follows:

[0076] 0.00~1.00min, mobile phase A:B=9:1;

[0077] 2.00-3.00 min, mobile phase A:B=5:5;

[0078] 4.50-5.50 min, mobile phase A:B=3:7;

[0079] 6.00-6.50min, mobile phase A:B=9:1.

[0080] Figure 3 This is the liquid chromatogram of desloratadine.

[0081] Figure 4This is the liquid chromatogram of N-nitroso-loratadine.

[0082] Method 3: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.40mL / min, column temperature of 25°C, injection volume of 1μL, wherein the gradient elution procedure is as follows:

[0083] 0.00~0.50min, mobile phase A:B=9:1;

[0084] 1.00~3.00min, mobile phase A:B=5:5;

[0085] 4.50-5.50 min, mobile phase A:B=3:7;

[0086] 6.00-6.50min, mobile phase A:B=9:1.

[0087] Figure 5 This is the liquid chromatogram of desloratadine.

[0088] Figure 6 This is the liquid chromatogram of N-nitroso-loratadine.

[0089] Method 4: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.40mL / min, column temperature of 25°C, injection volume of 1μL, wherein the gradient elution procedure is as follows:

[0090] 0.00~0.80min, mobile phase A:B=9:1;

[0091] 1.50~3.00min, mobile phase A:B=5:5;

[0092] 4.50-5.50 min, mobile phase A:B=3:7;

[0093] 6.00-6.50min, mobile phase A:B=9:1.

[0094] Figure 7 This is the liquid chromatogram of desloratadine.

[0095] Figure 8 This is the liquid chromatogram of N-nitroso-loratadine.

[0096] in conclusion:

[0097] From the chromatographic conditions of method 1, it can be seen that desloratadine has no obvious absorption peak under this condition ( Figure 1 ), while N-nitroso-loratadine has an absorption peak at 4.902min ( Figure 2 ). To solve this problem, based on method 1, the original 0.00-1.00 min, mobile phase A:B = 7:3, was changed to 0.00-1.00 min, mobile phase A:B = 9:1, the proportion of water was increased, and the end gradient was changed accordingly; the original 1 min gradient change time was extended to 2 min, and the 4 min gradient change time was extended to 4.5 min, adjusted to method 2.

[0098] From the chromatographic conditions of method 2, it can be seen that although desloratadine has an absorption peak at 2.651min ( Figure 3 ), N-nitroso-loratadine has an absorption peak near 4.025min ( Figure 4 ), but it affected the peak shape of N-nitroso-loratadine. The biggest adjustment in Method 2 is the proportion of water. It is very likely that desloratadine has an absorption peak because of the adjustment of the proportion of the mobile phase. Therefore, the proportion of the mobile phase remains unchanged for the time being. The time is adjusted first, from 0.00-1.00min, mobile phase A:B=9:1 to 0.00-0.50min, mobile phase A:B=9:1, shortening the time of the first gradient and then extending the time of the second gradient, adjusting to Method 3.

[0099] According to the chromatographic conditions of method 3, desloratadine has an absorption peak at 1.786 min ( Figure 5 ), while N-nitroso-loratadine has an absorption peak at 4.706min ( Figure 6 ), and the separation degree is good, so it is determined as the basic chromatographic condition, and the optimal chromatographic condition is found on this basis.

[0100] The conditions of chromatographic method 4 are similar to those of method 3. Figure 7 and Figure 8 The results and Figure 5 and Figure 6 The results are similar and can also be used as basic chromatographic conditions for subsequent research.

[0101] 2. Effect of liquid phase conditions on peak shape in ion chromatograms

[0102] Method 5: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.25mL / min, column temperature of 25°C, injection volume of 5μL, wherein the gradient elution procedure is as follows:

[0103] 0.00~0.72min, mobile phase A:B=9:1;

[0104] 1.00~3.00min, mobile phase A:B=5:5;

[0105] 4.00~5.50min, mobile phase A:B=4.5:6.5;

[0106] 6.00-6.50min, mobile phase A:B=9:1;

[0107] The mass spectrometer uses a triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330°C, spray voltage of 3500V, sheath gas of 10, and auxiliary gas of 5. The quantitative ion of N-nitroso-loratadine is 266.1m / z. The reference solution is fully added.

[0108] Fig. 9 This is the ion chromatogram of N-nitroso-loratadine.

[0109] Method 6: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.40mL / min, column temperature of 25°C, injection volume of 5μL, wherein the gradient elution procedure is as follows:

[0110] 0.00~0.72min, mobile phase A:B=9:1;

[0111] 1.00~3.00min, mobile phase A:B=5:5;

[0112] 4.00~5.50min, mobile phase A:B=4.5:6.5;

[0113] 6.00-6.50min, mobile phase A:B=9:1;

[0114] The mass spectrometer uses a triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330°C, spray voltage of 3500V, sheath gas of 10, and auxiliary gas of 5. The quantitative ion of N-nitroso-loratadine is 266.1m / z. The reference solution is fully added.

[0115] Fig.10 This is the ion chromatogram of N-nitroso-loratadine.

[0116] Method 7: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.25mL / min, column temperature of 25°C, injection volume of 5μL, wherein the gradient elution procedure is as follows:

[0117] 0.00~0.72min, mobile phase A:B=9:1;

[0118] 1.00~3.00min, mobile phase A:B=5:5;

[0119] 4.00-5.00 min, mobile phase A:B=2:8;

[0120] 6.00-6.50min, mobile phase A:B=9:1;

[0121] The mass spectrometer uses a triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330°C, spray voltage of 3500V, sheath gas of 10, and auxiliary gas of 5. The quantitative ion of N-nitroso-loratadine is 266.1m / z. The reference solution is fully added.

[0122] Fig.11 This is the ion chromatogram of N-nitroso-loratadine.

[0123] Method 8: The chromatographic conditions of liquid chromatography are: using a Nanochrom C18 column with a specification of 2.1×50mm×1.8μm, 0.1% formic acid water as mobile phase A, methanol as mobile phase B, gradient elution, flow rate of 0.25mL / min, column temperature of 25°C, injection volume of 1μL, wherein the gradient elution procedure is as follows:

[0124] 0.00~0.72min, mobile phase A:B=9:1;

[0125] 1.00~3.00min, mobile phase A:B=5:5;

[0126] 4.00-5.00 min, mobile phase A:B=2:8;

[0127] 6.00-6.50min, mobile phase A:B=9:1;

[0128] The mass spectrometer uses a triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330°C, spray voltage of 3500V, sheath gas of 10, and auxiliary gas of 5. The quantitative ion of N-nitroso-loratadine is 266.1m / z. The reference solution is fully added.

[0129] Fig.12 This is the ion chromatogram of N-nitroso-loratadine.

[0130] in conclusion:

[0131] After the chromatographic conditions were basically determined by method 3, the mass spectrometer was connected and it was found that the peak shape of N-nitroso-loratadine in the mass spectrum was not good. Methods 5-8 were to adjust several conditions in the peak shape process, and the peak shape was improved by changing the gradient time, flow rate and injection volume. However, the peak shape obtained by changing the liquid phase conditions was Figure 9-12 Two sharp peaks appeared near the N-nitroso-chlorpheniramine base, which might be caused by the addition of formic acid, an ionizing agent, into the water.

[0132] After trying to remove formic acid according to the conditions of method 3 or method 4, the two sharp peaks did not appear. Therefore, the composition of the mobile phase was changed to purified water as mobile phase A and methanol as mobile phase B.

[0133] The optimal chromatographic conditions for liquid chromatography were finally determined to be:

[0134] Purified water was used as mobile phase A, methanol was used as mobile phase B, gradient elution was performed, the flow rate was 0.40 mL / min, the column temperature was 25°C, and the injection volume was 1 μL. The gradient elution procedure was as follows:

[0135] 0.00~0.72min, mobile phase A:B=9:1;

[0136] 1.00~3.00min, mobile phase A:B=5:5;

[0137] 4.50-5.50 min, mobile phase A:B=2:8;

[0138] 6.00-6.50min, mobile phase A:B=9:1.

[0139] Its ion chromatogram is as follows Fig.13 shown.

[0140] Under the above gradient conditions, the flow rate of the liquid chromatography is 0.35mL / min~0.42mL / min, the column temperature is 20℃~30℃, or C18 chromatographic columns of different specifications have no significant effect on the separation results.

[0141] Example 2 Detection of N-nitroso-loratadine

[0142] The detection method comprises the following steps:

[0143] Step 1: Prepare blank solution, reference solution and test solution:

[0144] The blank solution was methanol solvent;

[0145] Preparation of reference substance stock solution: Take 0.0050 g of N-nitroso-loratadine sample, accurately weigh it, place it in a 5 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well; then transfer 0.05 mL of the above solution to a 5 mL volumetric flask, add methanol to dilute to the scale, shake well, and use it as reference substance stock solution a; then transfer 0.05 mL of the above reference substance stock solution a to a 5 mL volumetric flask, add methanol to dilute to the scale, shake well, and use it as reference substance stock solution b;

[0146] Reference solution: Pipette 0.04 mL of the reference stock solution a into a 5 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare the reference solution.

[0147] Preparation of the test solution: Take 0.0050 g of desloratadine sample, accurately weigh it, put it into a 5 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as the test solution;

[0148] 100% test sample spike solution: Take 0.0050g of desloratadine sample, weigh accurately, put it into a 5mL volumetric flask, accurately add 0.04mL of reference substance stock solution a, then add methanol to the mark, shake well, and use this as the 100% test sample spike solution;

[0149] Step 2: Detect N-nitroso-loratadine in desloratadine by using liquid chromatography-mass spectrometry to detect the blank solution, the test solution, the reference solution and the 100% test solution spiked with the test solution, and record the spectrum. The chromatogram is as follows: Figure 14-18 As shown;

[0150] The chromatographic conditions of the above liquid chromatography are:

[0151] A Nanochrom C18 column with a specification of 2.1×50mm×1.8μm was used, with purified water as mobile phase A and methanol as mobile phase B, gradient elution, a flow rate of 0.40mL / min, a column temperature of 25°C, and an injection volume of 1μL. The gradient elution procedure was as follows:

[0152] 0.00~0.72min, mobile phase A:B=9:1;

[0153] 1.00~3.00min, mobile phase A:B=5:5;

[0154] 4.50-5.50 min, mobile phase A:B=2:8;

[0155] 6.00-6.50min, mobile phase A:B=9:1;

[0156] The mass spectrometer adopts triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330°C, spray voltage of 3500V, sheath gas of 10, auxiliary gas of 5, and the quantitative ion of N-nitroso-loratadine is 266.1m / z.

[0157] The procedure for switching the valve is as follows:

[0158] 0.00~3.7min, discharge: 3.7~5.9min, access; 5.9~6.5min, discharge.

[0159] from Figure 16-Figure 17 It can be seen that the chromatographic peak of N-nitroso-loratadine reference substance appears at a retention time of about 4.90 min, and the chromatographic peak separation is good.

[0160] from Figure 14-18 It can be seen that the methanol solvent and the desloratadine raw material have no interference with the detection of N-nitroso-loratadine, indicating that the liquid chromatography-mass spectrometry method provided by the present invention has good specificity.

[0161] Example 3 Repeatability

[0162] Repeatability of N-nitroso-loratadine: Take the same batch of samples and prepare 6 100% test sample spiked solutions in parallel. Detection is carried out by liquid chromatography-mass spectrometry and the spectra are recorded. The specific conditions of liquid chromatography and mass spectrometry are as described in Example 2. The results are shown in Table 1.

[0163] The preparation method of the above-mentioned 100% test sample spiked solution is as follows: take 0.0050 g of N-nitroso-loratadine sample, accurately weigh it, put it in a 5 mL volumetric flask, accurately add 0.04 mL of reference substance stock solution a, then add methanol to the scale, shake well, and use it as the 100% test sample spiked solution.

[0164] Table 1 Repeatability results

[0165]

[0166] It can be seen from Table 1 that the RSD values ​​of the contents of N-nitroso-loratadine in the 6 analytical repeatability solutions are 3%, all less than 15.0%, indicating that the detection method provided by the present invention has good repeatability.

[0167] Example 4 Injection Precision

[0168] Injection precision solution: Example 2 reference solution is the impurity reference solution (100% concentration), and 6 injections are made continuously. Liquid chromatography-mass spectrometry is used for detection, and the spectra are recorded. The specific conditions of liquid chromatography and mass spectrometry are as described in Example 2, and the results are shown in Table 2.

[0169] Table 2 Injection precision results

[0170]

[0171]

[0172] As can be seen from Table 2, the RSD of the peak area of ​​6 consecutive injections is ≤15%, and the injection time is less than 5%, indicating that the instrument has good injection precision.

[0173] Example 5 Intermediate Precision

[0174] Intermediate precision solution: Different personnel took the same batch of samples at different times, and prepared two batches of 6 100% test sample spiked solutions in parallel. The same instrument was used for LC-MS detection in the same laboratory. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2. The spectra were recorded and the results are shown in Table 3.

[0175] The preparation method of the above-mentioned 100% test sample spiked solution is as follows: take 0.0050 g of N-nitroso-loratadine sample, accurately weigh it, put it in a 5 mL volumetric flask, accurately add 0.04 mL of reference substance stock solution a, then add methanol to the scale, shake well, and use it as the 100% test sample spiked solution.

[0176] Table 3 Intermediate precision results

[0177]

[0178] It can be seen from Table 3 that the RSD of the content of each analyte in the two batches of 6 intermediate precision solutions is ≤15.0%, indicating that the intermediate precision of the detection method provided by the present invention is good.

[0179] Example 6 Limit of Quantitation and Limit of Detection

[0180] Quantitative limit solution: Pipette 0.05 mL of the reference substance stock solution b prepared in Example 2 into a 5 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare the solution. Pipette as the quantitative limit solution. The above quantitative limit solution was tested by liquid chromatography-mass spectrometry, and the sample was injected 6 times continuously. The spectra were recorded. The results are shown in Table 4 below, where the specific conditions of liquid chromatography and mass spectrometry are as described in Example 2.

[0181] Table 4 Quantitation limit results

[0182]

[0183]

[0184] Detection limit solution: Pipette 0.025 mL of the reference substance stock solution b prepared in Example 2 into a 5 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare the solution. Pipette as the detection limit solution. The above detection limit solution was tested by liquid chromatography-mass spectrometry, injected once, and the spectrum was recorded. The results are shown in Table 5 below, where the specific conditions of liquid chromatography and mass spectrometry are as described in Example 2.

[0185] Table 5 Detection limit results

[0186]

[0187] The results showed that in the method of the present invention, the detection limit of N-nitroso-loratadine was 0.5 ng / mL and the quantification limit was 1 ng / mL.

[0188] Example 7 Linear Relationship

[0189] Linear solution: Accurately pipette different volumes of reference substance stock solution a and reference substance stock solution b as shown in Table 6 below into different volumes of containers, dilute to scale with methanol, and shake well to obtain a series of linear solutions of different concentrations. The preparation method of the reference substance stock solution is the same as that in Example 2.

[0190] Table 6 Linear solutions

[0191]

[0192] The linear solution prepared above was detected by liquid chromatography-mass spectrometry, and the spectrum was recorded. Each concentration was injected once, wherein the specific conditions of liquid chromatography and mass spectrometry were as described in Example 2, and the spectrum was recorded. The concentration (ppb) of N-nitroso-loratadine was used as the abscissa and the peak area was used as the ordinate to draw a standard curve, and the regression equation was calculated. The results are shown in Table 7, and the linear relationship diagram is shown in Fig.19 .

[0193] Table 7 Linearity results

[0194]

[0195] It can be seen from the results in Table 7 that N-nitroso-loratadine has a good linear relationship within the concentration range of 1 ppb-100 ppb.

[0196] Example 8 Accuracy

[0197] Accuracy solutions: Includes 1.25% (LOQ) spike solution, 50% spike solution, 100% spike solution, and 125% spike solution.

[0198] 1.25% (LOQ) spiked solution: Take 0.0050 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.05 mL of the reference substance stock solution b prepared in Example 2, dilute to the scale with methanol, shake well, and prepare three replicates in parallel.

[0199] 50% spiked solution: Take 0.0050 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.02 mL of the reference substance stock solution a prepared in Example 2, dilute to the scale with methanol, shake well, and prepare three times in parallel.

[0200] 100% spiked solution: Take 0.0050 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.04 mL of the reference substance stock solution a prepared in Example 2, dilute to the scale with methanol, shake well, and prepare three replicates in parallel.

[0201] 125% spiked solution: Take 0.0050 g of the test sample, accurately weigh it, and place it in a 5 mL volumetric flask. Add 0.05 mL of the reference substance stock solution a prepared in Example 2, dilute to the scale with methanol, shake well, and prepare three replicates in parallel.

[0202] The accuracy solution prepared above was detected by liquid chromatography-mass spectrometry, and the spectrum was recorded. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2. The recovery rate results are shown in Table 8.

[0203] The recovery rate was calculated according to the following formula:

[0204] Recovery rate (%) = measured amount / theoretical amount × 100%

[0205] Table 8 Accuracy results

[0206]

[0207] As can be seen from Table 8, the single value range of the recovery rate of N-nitroso-loratadine is 97.66% to 114.76%, all between 70% and 125%, and the RSD is less than 10%, which shows that the detection method of nitrosamines in desloratadine provided in the present application has good accuracy.

[0208] Example 9 Solution Stability

[0209] The reference solution and the test solution prepared in Example 2 were injected once at 0 h, 2 h, 5 h, 8 h, 12 h, and 24 h, respectively, and detected by liquid chromatography-mass spectrometry, and the spectra were recorded. The specific conditions of liquid chromatography and mass spectrometry were as described in Example 2. The results are shown in Table 9 below.

[0210] Table 9 Solution stability results

[0211]

[0212] As can be seen from Table 9, the ratio of the peak area of ​​each analyte to the 0h area in the reference solution is between 1.01 and 1.07, which indicates that the reference solution provided by the present application has good stability. No peak of N-nitroso-loratadine was detected in the test sample, which indicates that the test solution provided by the present application has good stability.

[0213] Example 10 Durability

[0214] Durability of N-nitroso-loratadine: Take the same batch of samples, prepare 100% test sample spiked solution, use liquid chromatography-mass spectrometry to detect, record the spectrum, the specific conditions of liquid chromatography and mass spectrometry are as described in Example 2, and the liquid chromatography conditions are slightly adjusted:

[0215] The preparation method of the above-mentioned 100% test sample spiked solution is as follows: take 0.0050 g of desloratadine, accurately weigh it, put it in a 5 mL volumetric flask, accurately add 0.40 mL of reference substance stock solution a, then add methanol to the scale, shake well, and use it as the 100% test sample spiked solution.

[0216] Fine-tuning 1. Flow rate adjustment: The flow rate was 0.35 mL / min, and the other conditions of liquid chromatography were the same as those in Example 2. The results are shown in Table 10.

[0217] Fine-tuning 2: Flow rate adjustment: The flow rate was 0.42 mL / min, and the other conditions of liquid chromatography were the same as those in Example 2. The results are shown in Table 10.

[0218] Fine-tuning 3: Column temperature adjustment: The column temperature was 30° C., and the other conditions of liquid chromatography were the same as those in Example 2. The results are shown in Table 10.

[0219] Fine-tuning 4: Column temperature adjustment: The column temperature was 20° C., and the other conditions of liquid chromatography were the same as those in Example 2. The results are shown in Table 10.

[0220] Table 10 Solution durability results

[0221]

[0222] The results of LC-MS detection in fine-tuning 1 to fine-tuning 4 are basically the same as the separation effect of Example 2, and the separation degree of the chromatographic peaks is good, which shows that the fine-tuning of the chromatographic conditions has no effect on the detection of nitrosamines in desloratadine.

[0223] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting N-nitroso-loratadine, characterized in that: The detection method is liquid chromatography-mass spectrometry, and the chromatographic conditions of the liquid chromatography are: Use purified water as mobile phase A and methanol as mobile phase B for gradient elution; the flow rate is 0.35mL / min~0.42mL / min, and the column temperature is 20℃~30℃; The procedure of the gradient elution is as follows: 0.00~(0.50-1.00)min, mobile phase A:B=9:1; (1.00-2.00)~3.00min, mobile phase A:B=5:5; 4.50-5.50 min, mobile phase A:B=2:8; 6.00-6.50min, mobile phase A:B=9:1; The mass spectrometer adopts triple quadrupole, ESI ion source, positive ion detection, SRM mode, ion source temperature of 330° C., spray voltage of 3500 V, sheath gas of 10, and auxiliary gas of 5.

2. The method for detecting N-nitroso-loratadine according to claim 1, characterized in that: The procedure of the gradient elution is as follows: 0.00~(0.50-0.80)min, mobile phase A:B=9:1; (1.00-1.50)~3.00min, mobile phase A:B=5:5; 4.50-5.50 min, mobile phase A:B=2:8; 6.00-6.50min, mobile phase A:B=9:

1.

3. The method for detecting N-nitroso-loratadine according to claim 1 or 2, characterized in that: The procedure of the gradient elution is as follows: 0.00~0.72min, mobile phase A:B=9:1; 1.00~3.00min, mobile phase A:B=5:5; 4.50-5.50 min, mobile phase A:B=2:8; 6.00-6.50min, mobile phase A:B=9:

1.

4. The method for detecting N-nitroso-loratadine according to any one of claims 1 to 3, characterized in that: The procedure of the mass spectrometer switching valve is as follows: 0.00~3.7min, discharged; 3.7~5.9min, connected; 5.9~6.5min, discharged.

5. The method for detecting N-nitroso-loratadine according to any one of claims 1 to 3, characterized in that: The chromatographic conditions of the liquid chromatography are: Purified water was used as mobile phase A, methanol was used as mobile phase B, gradient elution was performed, the flow rate was 0.4 mL / min, the column temperature was 25 °C, and the injection volume was 1 μL.

6. The method for detecting N-nitroso-loratadine according to any one of claims 1 to 5, characterized in that: Before testing, methanol was used to prepare N-nitroso-loratadine reference solution and desloratadine test solution.

7. The method for detecting N-nitroso-loratadine according to any one of claims 1 to 6, characterized in that: The quantification ion of N-nitrosochloridone is 266.1 m / z.

8. The method for detecting N-nitroso-loratadine according to any one of claims 1 to 6, characterized in that: The chromatographic column is a Nanochrom C18 chromatographic column.

9. The method for detecting N-nitroso-loratadine according to claim 8, characterized in that: The specifications of the chromatographic column are 2.1×50 mm×1.8 μm.

10. Use of the detection method according to any one of claims 1 to 9 in detecting N-nitroso-loratadine in desloratadine.

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