Liquid chromatography-mass spectrometry detection method for aminolevulinic acid hydrochloride

Abstract

The invention relates to the technical field of substance detection, in particular to an aminolevulinic acid hydrochloride liquid chromatography-mass spectrometry detection method. The method comprises the following steps: (1) pretreating a to-be-detected sample containing aminolevulinic acid hydrochloride to obtain a test solution; and (2) carrying out liquid chromatography-mass spectrometry detection on the test solution obtained in the step (1) to determine the content of aminolevulinic acid hydrochloride in the sample to be detected. The method effectively solves the problem of content detection of the aminolevulinic acid hydrochloride in the sample, especially the problem of analysis of the content of the aminolevulinic acid hydrochloride in a permeation stock solution, a recovery stock solution and a skin retention sample stock solution in in-vitro transdermal research of compatibility of the aminolevulinic acid hydrochloride and a solvent; the specificity, the system applicability, the linearity, the detection limit, the quantitation limit, the accuracy, the precision, the solution stability and the durability of the method all meet the requirements, in addition, the flux of the method is high, rapid detection can be completed within 10 minutes, and a technical basis is provided for industrial automatic production and scientific research.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a liquid chromatography-mass spectrometry detection method for aminolevulinic acid hydrochloride. Background Art

[0002] Aminolevulinic Acid Hydrochloride (ALAHCl), its chemical name is 5-amino-4-ketovaleric acid hydrochloride. Existing topical preparations of aminolevulinic acid mainly include Biofrontera Inc's aminolevulinic acid hydrochloride gel (used for actinic keratosis and basal cell carcinoma), and Shanghai Fudan Zhangjiang Biopharmaceutical Co., Ltd.'s aminolevulinic acid hydrochloride topical powder (used to treat condyloma acuminatum). Aminolevulinic acid hydrochloride is a photosensitizer commonly used in photodynamic therapy (PDT). During the treatment process, it can enter the skin lesion site through transdermal absorption and be converted into photosensitivity protoporphyrin IX (PpIX). Transdermal absorption research mainly focuses on how to improve the transdermal efficiency of drugs and the conversion effect of drugs in the skin.

[0003] Studies have shown that 5-aminolevulinic acid, without derivatization, passes through a UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) and enters mass spectrometry detection. The lower limit of quantification of the method is 1 μg / mL, which is used to evaluate the release characteristics of oral tablets in the colon in vitro. In this method, 5-aminolevulinic acid has no obvious retention on the chromatographic column, and the peak is released at the dead time, with low separation. In addition, studies have been conducted to determine the content of 5-aminolevulinic acid in biological matrices by removing proteins and derivatizing samples after adding stable isotope-labeled internal standards. However, there is no LC-MS detection method for topical preparations of aminolevulinic acid hydrochloride in the prior art. The main difficulties include: (1) the matrix of the in vitro transdermal permeation solution, recovery solution and retention sample of aminolevulinic acid hydrochloride and solvent compatibility is different and the matrix composition is complex. There are interference peaks near the main peak, making it difficult to determine the peak position of the main peak; (2) the main peak of aminolevulinic acid hydrochloride contains -NH2 and -COOH in the chemical structure, which has strong polarity, weak retention and weak ultraviolet absorption; (3) for topical preparations, the detection amount of permeation solution, recovery solution and retention sample is large, and the detection between samples is easily affected. Therefore, developing an effective LC-MS detection method for topical preparations of aminolevulinic acid hydrochloride is of great significance for practical application and will greatly improve the efficiency of detection. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide a.

[0005] In a first aspect of the present invention, the present invention provides a method for detecting aminolevulinic acid hydrochloride. According to an embodiment of the present invention, the method comprises: (1) pre-treating a sample containing aminolevulinic acid hydrochloride to obtain a test solution; (2) performing liquid chromatography-mass spectrometry on the test solution obtained in step (1) to determine the content of aminolevulinic acid hydrochloride in the sample.

[0006] The specificity, system applicability, linearity, detection limit and quantification limit, accuracy and precision, solution stability and durability of the method provided by the present invention all meet the relevant requirements, and the method is a high-throughput detection method that can quickly complete the detection within 10 minutes. The method according to the embodiment of the present invention can stably and effectively detect the aminolevulinic acid hydrochloride in the sample to be tested containing aminolevulinic acid hydrochloride, and the method has high stability and precision.

[0007] According to an embodiment of the present invention, the above method for detecting aminolevulinic acid hydrochloride may further include at least one of the following additional technical features:

[0009] According to the embodiment of the present invention, the conditions for the liquid chromatography detection are: using a Waters Atlantis T3 chromatographic column, using a 0.1% formic acid solution as mobile phase A, and using a methanol solution as mobile phase B for gradient elution. Thus, the liquid chromatography can effectively elute and separate the aminolevulinic acid hydrochloride in the sample, and the detection result is accurate.

[0010] According to the embodiment of the present invention, the conditions of the gradient elution are shown in Table 19, flow rate 0.5-0.7 mL / min, column temperature 35-45° C., injection volume 5 μL. The conditions were obtained by the inventors after a large number of screening and optimization.

[0011] According to an embodiment of the present invention, the flow rate is 0.6 mL / min, the column temperature is 40° C., and the injection volume is 5 μL.

[0012] According to the embodiment of the present invention, the conditions of mass spectrometry detection are: ion source type: ESI, ion source mode: Positive, ESI source temperature: 280°C to 320°C, preferably 300°C, carrier gas flow rate: 5L / min, sheath gas heating temperature: 250°C, sheath gas heating flow rate: 11L / min, nebulizer pressure: 45Psi, capillary voltage: 3500V, qualitative ion pair, quantitative ion pair, fragmentation voltage, collision energy, and dwell time information are shown in Table 20. Therefore, the results of mass spectrometry detection of the content of aminolevulinic acid hydrochloride in the sample are accurate.

[0013] According to an embodiment of the present invention, the concentration of aminolevulinic acid hydrochloride in the sample is 50 to 1000 ng / mL.

[0014] According to an embodiment of the present invention, the sample includes but is not limited to at least one of the following: blood, serum, plasma, feces, urine, sweat, saliva, cells, tissues, osmotic fluid, recovery fluid and skin retention fluid. The method according to an embodiment of the present invention is not particularly limited to the type of sample to be detected, and any sample containing aminolevulinic acid hydrochloride can be detected.

[0015] According to an embodiment of the present invention, the pretreatment includes but is not limited to at least one of the following: dilution, grinding and centrifugation. The sample can be subjected to different conventional pretreatments in the art according to different samples and experimental requirements.

[0016] According to an embodiment of the present invention, the pretreatment method of the penetration stock solution includes: diluting the penetration stock solution with a 0.1% formic acid solution, wherein the dilution multiple is 1000 to 1500 times, preferably 1200 times, and the penetration stock solution is obtained within 2 to 24 hours of the aminolevulinic acid hydrochloride transdermal experiment. The inventor screened the diluents and found that the 0.1% formic acid solution had a better use effect than other diluents.

[0017] According to an embodiment of the present invention, the pretreatment method of the recovered stock solution includes the following operations in sequence: diluting the recovered stock solution 20 to 60 times with physiological saline, then diluting 80 to 120 times with 0.1% formic acid solution, and then diluting 5 to 20 times with 0.1% formic acid 1% physiological saline solution. Herein, an exemplary preparation method of 0.1% formic acid 1% physiological saline solution includes: 0.1ml formic acid, 1ml physiological saline solution and 100ml water are mixed, and those skilled in the art can scale the volume according to this ratio.

[0018] According to an embodiment of the present invention, the pretreatment method of the recovered stock solution includes the following operations in sequence: diluting the recovered stock solution 50 times with physiological saline, then diluting it 100 times with 0.1% formic acid solution, and then diluting it 11 times with 0.1% formic acid 1% physiological saline solution.

[0019] According to an embodiment of the present invention, the recovered liquid stock solution is the supernatant obtained by transferring the remaining aminolevulinic acid hydrochloride and the skin into physiological saline after the aminolevulinic acid hydrochloride transdermal experiment, vortexing and standing.

[0020] According to an embodiment of the present invention, the pre-treatment method of the skin retention sample stock solution includes: diluting the skin retention sample stock solution with a 0.1% formic acid solution, wherein the dilution multiple is 10 to 150 times.

[0021] According to an embodiment of the present invention, the retention sample stock solution is the supernatant obtained by grinding and centrifuging the skin after the aminolevulinic acid hydrochloride transdermal experiment.

[0022] According to an embodiment of the present invention, before the liquid chromatography-mass spectrometry detection, the reference substance is preliminarily subjected to the aminolevulinic acid hydrochloride content detection, and the reference substance is a sample containing aminolevulinic acid hydrochloride of known concentration. It is known to those skilled in the art that before each LC-MS analysis run, the reference substance can be used to perform an adaptability experiment on the liquid chromatography system to ensure that the performance of the chromatography system and the mass spectrometry system meets the analysis requirements, including parameters such as the number of theoretical plates, separation, and tailing factor, but if the system has been debugged, this operation may not be performed.

[0023] According to an embodiment of the present invention, the concentration of aminolevulinic acid hydrochloride in the reference substance is 50-1000 ng / mL. Therefore, the system can accurately detect it at this concentration, and then effectively determine whether the performance of the chromatography system and the mass spectrometry system meets the analysis requirements.

[0024] According to an embodiment of the present invention, the concentration of aminolevulinic acid hydrochloride in the reference substance is preferably 500 ng / mL.

[0025] According to an embodiment of the present invention, before performing the liquid chromatography-mass spectrometry detection, a standard curve between the aminolevulinic acid hydrochloride content in the linear solution and the chromatographic peak area is pre-drawn.

[0026] According to an embodiment of the present invention, the linear solution is a plurality of aminolevulinic acid hydrochloride samples containing different known concentrations, and the concentration of aminolevulinic acid hydrochloride in the linear solution is 50 to 1000 ng / mL.

[0027] According to an embodiment of the present invention, the content of aminolevulinic acid hydrochloride is determined based on the chromatographic peak area of ​​the sample to be tested detected by liquid chromatography-mass spectrometry.

[0028] According to an embodiment of the present invention, the content of aminolevulinic acid hydrochloride is determined based on the standard curve and the chromatographic peak area of ​​the sample to be tested detected by liquid chromatography-mass spectrometry.

[0029] In a second aspect of the present invention, the present invention provides a method for detecting aminolevulinic acid hydrochloride. According to an embodiment of the present invention, the method comprises: (1) pre-treating a sample containing aminolevulinic acid hydrochloride to obtain a sample solution; (2) performing liquid chromatography-mass spectrometry detection on the sample solution obtained in step (1) to obtain a spectrum of the sample to be tested, wherein the conditions for the liquid chromatography detection are: using a Waters Atlantis T3 chromatographic column, with 0.1% formic acid solution as mobile phase A, and methanol solution as mobile phase B for gradient elution. The conditions of the gradient elution are shown in Table 19, flow rate 0.6 mL / min, column temperature 40°C, injection volume 5 μL; the conditions of the mass spectrometry detection are: ion source type: ESI, ion source mode: ESI+, ESI source temperature: 280°C to 320°C, preferably 300°C, carrier gas flow rate: 5 L / min, sheath gas heating temperature: 250°C, sheath gas heating flow rate: 11 L / min, nebulizer pressure: 45 Psi, Capillary voltage: 3500V, qualitative ion pair, quantitative ion pair, fragmentation voltage, collision energy, and residence time information are shown in Table 20; (3) Determine the content of aminolevulinic acid hydrochloride based on the chromatographic peak area of ​​the sample to be tested detected by the liquid chromatography-mass spectrometry; wherein: the sample to be tested in step (1) includes at least one of the following: blood, serum, plasma, feces, urine, sweat, saliva, cells, tissues, infiltration stock solution, recovery stock solution and skin retention sample stock solution, the pretreatment includes dilution, grinding, centrifugation, and the diluent is formic acid and / or saline.

[0030] The specificity, system applicability, linearity, detection limit and quantification limit, accuracy and precision, solution stability and durability of the method provided by the present invention all meet the relevant requirements, and the method is a high-throughput detection method that can quickly complete the qualitative and quantitative detection of aminolevulinic acid hydrochloride in a sample within 10 minutes.

[0031] According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

[0032] According to an embodiment of the present invention, before performing step (2), a system suitability experiment is performed in advance using a reference substance, wherein the concentration of aminolevulinic acid hydrochloride in the reference substance is 500 ng / mL.

[0033] According to an embodiment of the present invention, after performing the system suitability experiment and before step (2), the linear solution is subjected to liquid chromatography-mass spectrometry detection, and a standard curve between the aminolevulinic acid hydrochloride content in the linear solution and the chromatographic peak area is drawn, wherein the concentrations of aminolevulinic acid hydrochloride in the linear solution are 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively.

[0034] In the third aspect of the present invention, the present invention provides a kit for detecting aminolevulinic acid hydrochloride in a sample. According to an embodiment of the present invention, the kit includes: (A) a plurality of aminolevulinic acid hydrochloride standards, wherein the concentration of aminolevulinic acid hydrochloride in the standards is 50 to 1000 ng / mL; (B) an aminolevulinic acid hydrochloride reference substance, wherein the reference substance is an aminolevulinic acid hydrochloride solution containing a known concentration; and (C) instructions for use, wherein the instructions record the method described in the first aspect or the second aspect. The kit according to an embodiment of the present invention can effectively process aminolevulinic acid hydrochloride in a sample according to the method described in the specification and the reagents provided therein, and perform rapid and accurate detection by liquid chromatography-mass spectrometry.

[0035] According to an embodiment of the present invention, the above-mentioned kit may further include at least one of the following additional technical features:

[0036] According to an embodiment of the present invention, the above-mentioned kit further comprises: (D) a diluent, wherein the diluent is formic acid and / or physiological saline; and (E) a mobile phase additive, wherein the mobile phase additive comprises formic acid.

[0037] In a fourth aspect of the present invention, the present invention provides a method for quality control of aminolevulinic acid hydrochloride content in a production process. According to an embodiment of the present invention, the method comprises: using the method for detecting aminolevulinic acid hydrochloride described in the first aspect or the second aspect to detect the aminolevulinic acid hydrochloride content of the quality control sample, and comparing the detected aminolevulinic acid hydrochloride content with the target aminolevulinic acid hydrochloride content value to determine whether the quality control sample meets the standard.

[0038] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0040] Figure 1 is a spectrum of a blank solution according to an embodiment of the present invention;

[0041] Figure 2 is a spectrum of a system suitability solution according to an embodiment of the present invention, wherein the peak time of 5-ALA-HCl is 2.520 min;

[0042] Figure 3 A spectrum of a detection limit solution (LOD) according to an embodiment of the present invention;

[0043] Figure 4is a spectrum of a limit of quantitation solution (LOQ) according to an embodiment of the present invention;

[0044] Figure 5 is a spectrum of a blank solution of a permeate matrix according to an embodiment of the present invention;

[0045] Figure 6 is a spectrum of a 10% limit spike solution of a permeate according to an embodiment of the present invention;

[0046] Figure 7 is a spectrum of a 100% limit spike solution of a permeate according to an embodiment of the present invention;

[0047] Figure 8 is a spectrum of a 200% limit spike solution of a permeate according to an embodiment of the present invention;

[0048] Fig. 9 is a spectrum of a blank solution of a recovery liquid matrix according to an embodiment of the present invention;

[0049] Fig.10 A spectrum of a 10% limit spike solution of a recovery solution according to an embodiment of the present invention;

[0050] Fig.11 A spectrum of a 100% limit spike solution of a recovery solution according to an embodiment of the present invention;

[0051] Fig.12 A spectrum of a 200% limit spike solution of a recovery solution according to an embodiment of the present invention;

[0052] Fig.13 is a spectrum of a retention sample matrix blank solution according to an embodiment of the present invention;

[0053] Fig.14 A spectrum of a 10% limit spike solution of a retention sample according to an embodiment of the present invention;

[0054] Fig.15 is a spectrum of a 100% limit spike solution of a retention sample according to an embodiment of the present invention;

[0055] Fig.16 This is a chromatogram of a 200% limit spike solution of a retention sample according to an embodiment of the present invention. DETAILED DESCRIPTION

[0056] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0057] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0058] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0059] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0060] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0061] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0062] Aminolevulinic Acid Hydrochloride (ALAHCl), chemical name is 5-amino-4-ketovaleric acid hydrochloride, molecular formula: C5H9NO3·HCl, molecular weight: 167.59, its chemical formula is shown in formula I):

[0063]

[0064] In this article, the "transdermal test", also known as the in vitro transdermal absorption test, is an important experimental method for evaluating the degree of absorption of substances through the skin barrier. It is widely used in the fields of drugs, cosmetics and food additives for safety assessment and effect prediction.

[0065] In this article, the "Franz cell" is a device used for in vitro transdermal absorption experiments. It simulates the process of drug transdermal absorption and is often used in the study of skin permeability of drug preparations. The Franz cell mainly consists of the following parts: (1) Donor Chamber: used to place drug donor solution or preparation. The drug contacts the skin model through the donor chamber. The donor chamber can be designed to be sealed to prevent the drug from volatilizing or being affected by the external environment. (2) Receiver Chamber: used to receive the drug solution that passes through the skin. The receiver chamber usually contains a certain volume of receiving fluid (such as physiological saline or simulated body fluid) to dissolve and preserve the drug solution that passes through the skin. The receiving fluid can be sampled at regular intervals to determine the amount of drug that passes through the skin. (3) Skin model: a skin sample is placed between the donor chamber and the receiver chamber. The skin sample can be an isolated animal skin or an artificially synthesized membrane, which serves as a barrier for drug transdermal absorption. (4) Diffusion interface: the donor chamber and the receiver chamber are connected by a diffusion interface. The drug needs to penetrate from the donor chamber to the receiver chamber through this interface.

[0066] Herein, the "limit of detection" refers to the minimum concentration or amount of the analyte that can be detected from the background noise by the analytical method.

[0067] As used herein, the "limit of quantitation" refers to the lowest concentration or amount at which an analytical method can reliably quantify an analyte. It is usually higher than the detection limit and requires the analytical results to have appropriate accuracy and precision. The determination of the limit of quantitation is usually based on the precision and accuracy of the method. A common approach is to limit quantitation to the concentration at which the signal-to-noise ratio (S / N) is 10, which means that the signal intensity is at least 10 times the background noise. The limit of quantitation can also be determined by analyzing a series of samples of different concentrations and determining at which concentration level the method can provide acceptable accuracy and precision.

[0068] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0069] Example 1 Development of LC-MS Detection Methodology

[0070] This example uses hydrochloric acid aminolevulinic acid gel as a sample for research, and the sample is purchased from Biofrontera Bioscience GmbH, with a production batch number of 147S02, Ameluz. The hydrochloric acid aminolevulinic acid gel is treated for the development of liquid chromatography-mass spectrometry detection methodology to obtain the optimal liquid chromatography-mass spectrometry detection method and parameters. The experimental materials used in the pretreatment and the specific operation steps of the permeation test and retention test are the same as those in parts 1 to 3 of Example 2 to obtain the permeate stock solution, the recovery liquid stock solution, the retention sample stock solution, and the solution obtained by diluting the permeate stock solution, the recovery liquid stock solution, and the retention sample stock solution in sequence. The specific dilution multiple is also referred to the solution preparation related content in part 3 of Example 2. In addition, the solutions with corresponding names in this example and in Example 2 are prepared by the same method unless otherwise specified. The detailed preparation method is shown in Example 2, which will not be repeated here.

[0071] 1. Mass spectrometry parameter optimization:

[0072] The aminolevulinic acid hydrochloride sample was diluted with water to a concentration of about 4 μg / mL to obtain a reference solution. 5 μL of the aminolevulinic acid hydrochloride reference solution was measured and injected into a liquid chromatography-mass spectrometer (Agilent 1260 / 1290LC+6470MS liquid chromatography-mass spectrometer, both models are acceptable) for analysis, wherein the parameters are optimized as follows.

[0073] 1. SCAN

[0074] SCAN(+): response value 3600989; SCAN(-): response value 98608. The response of positive ion mode is about 36 times that of negative ion mode, so the positive ion mode was selected for the next study.

[0075] 2. Frag (V)

[0076] First, the applicant tested the parameter Frag in the range of 0V to 150V. The results are shown in Table 1. Among them, when Frag (V) is 50, the response value is the highest. Therefore, 41V to 59V is selected for detailed research.

[0077] Table 1:

[0078] Frag(V) Peak area Frag(V) Peak area Frag(V) Peak area Frag(V) Peak area 0 32338 40 139728 80 139472 120 26434 10 57746 50 151974 90 127228 130 10236 20 86587 60 148752 100 98218 140 4577 30 115391 70 143669 110 57786 150 3201

[0079] The research results of 41V~59V voltage are shown in Table 2. Among them, when Frag(V) is 47, the response value is the highest, so Frag(V) is selected as 47V.

[0080] Table 2:

[0081] Frag(V) Peak area Frag(V) Peak area Frag(V) Peak area Frag(V) Peak area 41 204273 46 208892 51 207641 56 203369 42 206400 47 210193 52 207129 57 202661 43 207639 48 209511 53 206284 58 201333 44 208796 49 209383 54 205176 59 200832 45 209102 50 208696 55 203903

[0082] 3. CE(V)

[0083] The applicant screened and optimized the CE(V) parameters, and the results are shown in Table 3. Among them, when CE(V) is 5, the response value of m / z113.9 is the highest, and when CE(V) is 10, the response value of m / z 86.0 is the highest. Therefore, 1V~14V is selected to study m / z 86.0 and m / z 113.9.

[0084] Table 3:

[0085] CE(V) m / z86.0 peak area m / z113.9 peak area CE(V) m / z86.0 peak area m / z113.9 peak area 0 829 6991 40 0 0 5 8230 29350 45 0 0 10 23853 22191 50 0 0 15 21888 7238 55 0 0 20 11036 0 60 0 0 25 3176 0 65 0 0 30 878 0 70 0 0 35 0 0 75 0 0

[0086] The results of the effects of 1V to 14V on m / z 86.0 and m / z 113.9 are as follows: Figure 4 As shown in Table 4, when CE(V) is 6, the response value of m / z 113.9 is the highest, and when CE(V) is 13, the response value of m / z 86.0 is the highest.

[0087] Table 4:

[0088] CE(V) m / z86.0 peak area m / z113.9 peak area CE(V) m / z86.0 peak area m / z113.9 peak area 1 1548 10472 8 18532 29528 2 2274 15755 9 21000 25657 3 4119 20113 10 23028 22056 4 5639 24994 11 24126 18890 5 8729 29640 12 24595 15740 6 12085 30582 13 26543 12637 7 15281 30275 14 25231 9604

[0089] Based on the screening and optimization of the above parameters, the mass spectrometry parameters are determined as shown in Table 5.

[0090] Table 5:

[0091]

[0092] Optimization of HPLC parameters

[0093] Accurately measure 5 μL of each blank solution (i.e., various dilutions) used in the following optimization process and inject them into the liquid chromatography-mass spectrometry system (Agilent 1260 / 1290 LC+6470MS liquid chromatography-mass spectrometer, both models are acceptable) for analysis, wherein the following parameters are optimized.

[0094] 1. Selection of diluent, mobile phase and chromatographic column

[0095] (1) Mobile phase A1: 0.1% formic acid-10 mmol / l ammonium acetate solution, mobile phase A2: 0.1% formic acid solution; mobile phase B1: acetonitrile solution, mobile phase B2: 0.1% formic acid acetonitrile solution.

[0096] Method MRM07 (+): 0-4 min, 80% A1-20% B1; 4.01-8 min, 5% A1-95% B1; 8.01-10 min, 80% A1-20% B1;

[0097] Method MRM08(+): Based on method MRM07(+), mobile phase A1-B1 was adjusted to mobile phase A1-B2;

[0098] Method MRM09(+): Based on method MRM07(+), mobile phase A1-B1 was adjusted to mobile phase A2-B1;

[0099] Method MRM10(+): Based on method MRM07(+), mobile phase A1-B1 was adjusted to mobile phase A2-B2.

[0100] (2) Chromatographic column: Waters Atlantis T3, 5 μm, 4.6*150 mm.

[0101] The chromatographic column Waters Atlantis T3, 5μm, 4.6*150mm was used to detect the dilutions in Table 6. Among them, ① the API response values ​​of dilutions 2, 4, 8, 10, and 14 were relatively high, all greater than 3000; ② MRM09(+) and MRM10(+) had higher responses than MRM07(+) and MRM08(+), and 0.1% formic acid solution was selected as the aqueous phase; ③ The comparison between dilutions 4 and 14 showed that the increase of 0.1% saline reduced the API response value; ④ The comparison between dilutions 5-6, 7-8, 9-10, 11-12, and 13-14 showed that the concentration of saline was relatively high, which also reduced the API response value; ⑤ The comparison between dilutions 8 and 10 showed that the API response values ​​were similar when 0.1% formic acid and 1% formic acid were added.

[0102] Table 6:

[0103] serial number Dilution 1 Methanol 2 50% methanol 3 Mobile phase A1 4 Mobile phase A2 5 1% saline-50% mobile phase A1-50% methanol solution 6 0.1% saline-50% mobile phase A1-50% methanol solution 7 1% saline-mobile phase A2-100% methanol solution 8 0.1% saline-mobile phase A2-100% methanol solution 9 1% saline-1% formic acid-100% methanol solution 10 0.1% saline-1% formic acid-100% methanol solution 11 1% saline solution-mobile phase A1 12 0.1% saline solution-mobile phase A1 13 1% saline solution-mobile phase A2 14 0.1% saline solution-mobile phase A2

[0104] (3) Chromatographic column: Waters XBridgeAmide, 3.5μm, 4.6*250mm

[0105] The dilutions in Table 6 were detected using a chromatographic column Waters XBridgeAmide, 3.5μm, 4.6*250mm. The experimental results showed that for MRM09(+): ① the API response values ​​of dilutions 2, 4, 8, 10, and 14 were relatively high, all greater than 3000, but the peak shapes of 8 and 10 were poor; ② the chromatographic column had strong retention (nearly 5min); ③ MRM09(+) and MRM10(+) had higher responses than MRM07(+) and MRM08(+), and 0.1% formic acid solution was selected as the aqueous phase; ④ the comparison between dilutions 4 and 14 showed that the increase of 0.1% saline reduced the API response value; ⑤ the comparison between dilutions 5-6, 7-8, 9-10, 11-12, and 13-14 showed that the concentration of saline was relatively high, which also reduced the API response value; ⑥ the comparison between dilutions 8 and 10, and the API response values ​​were similar when 0.1% formic acid and 1% formic acid were added. MRM10(+): ① For dilutions 2, 4, and 14, the API response values ​​were relatively high, all greater than 2900, but the peak shape was poor; ② The column had strong retention (nearly 5 minutes).

[0106] (4) Mobile phase A1: 0.1% formic acid solution; mobile phase B1: methanol solution; mobile phase B2: 0.1% formic acid methanol solution.

[0107] Method MRM11 (+): 0-4 min, 80% A1-20% B1; 4.01-8 min, 5% A1-95% B1; 8.01-10, 80% A1-20% B1;

[0108] Method MRM12(+): Based on method MRM11(+), mobile phase A1-B1 was adjusted to mobile phase A1-B2.

[0109] Chromatographic columns: Waters Atlantis T3, 5 μm, 4.6*150 mm (T3) and Waters XBridge Amide, 3.5 μm, 4.6*250 mm (Amide).

[0110] Liquid chromatography was performed using the above materials and the diluents in Table 6. The experimental results showed that the T3 column: MRM11 (+): ① The API response values ​​of diluents 1, 2, 4, 8, 10, and 14 were relatively high, all greater than 7700; ② The retention of the chromatographic column was weak (nearly 2.4 min); the effects of diluents 3-5 were the same as those of 4-6. Next, the specificity and recovery rates of diluents 8, 10, 14, 0.1% saline + 100% methanol, and 0.1% saline + 50% methanol were investigated. MRM12 (+): ① The API response values ​​of diluents 1, 2, 4, 8, 10, and 14 were relatively high, all greater than 7800; ② The retention of the chromatographic column was weak (nearly 2.4 min); the effects of diluents 3-5 were the same as those of 4-6.

[0111] Amide column: MRM11(+): ① API response values ​​of diluents 1, 2, 4, 6, 8, 10, 14 were relatively high, all greater than 5000, but the peak shapes of 4 and 14 were poor (with small packets), and the peak shapes of diluents 1, 8, 10 were relatively broad; ② The column had strong retention (nearly 5 min); 3-5 were the same as 4-6. MRM12(+): ① API response values ​​of diluents 1, 2, 4, 6, 8, 10, 14 were relatively high, all greater than 4800, but the peak shapes of 4 and 14 were poor (with small packets), and the peak shapes of diluents 4 and 6 were relatively poor, and the peak shapes of diluents 1, 8, 10 were relatively broad; ② The column had strong retention (nearly 5 min); the effects of diluents 3 to 5 were the same as 4 to 6.

[0112] Based on the experimental data of the above liquid chromatography, 0.1% formic acid solution was selected as the aqueous mobile phase, and methanol was selected as the organic mobile phase.

[0113] 2. Specificity and linearity

[0114] This experiment uses diluent 1: 50% methanol + 1% saline; diluent 2: 0.1% formic acid + 1% saline. The preparation methods of each solution used are the same as the preparation methods of the solutions with corresponding names in Example 2, wherein 1-KB-ST and 2-KB-ST are diluent groups 1 and 2, respectively, and 1-SYS and 2-SYS are system adaptability solutions prepared with diluent 1 and diluent 2, respectively.

[0115] (1) Amide column (MRM12(+))

[0116] Liquid chromatography was performed using the above diluents 1 and 2 and an Amide column (MRM12(+)). The results are shown in Table 7, wherein the response value of the blank (matrix) / reference solution was greater than 10%, indicating poor specificity.

[0117] Table 7:

[0118] name Response value Blank (matrix) / system suitability solution 1-KB-ST 14232 37.7 1-SYS 37758 / 2-KB-ST 8638 29.9 2-KB-HS 25104 86.8 2-KB-ZL 161431 557.9 2-SYS 28938 /

[0119] (2) T3 column (MRM12(+))

[0120] Liquid chromatography was performed using the above diluents 1 and 2 and a T3 column (MRM12(+)). The results are shown in Table 8. Under the chromatography-mass spectrometry conditions, the recovery rate was between 115.4% and 137.7%, and further optimization of the parameters was required.

[0121] Table 8:

[0122] name Response value Concentration value (ng / ml) Blank (matrix) / system suitability solution Recovery rate (%) 2-KB-ST / / 0 / 2-1% SPK-ST / / / / 2-50% SPK-ST 8187 72.4164 / 135.3 2-100% SPK-ST 15294 130.4706 / 121.9 2-KB-HS / / 0 / 2-1% SPK-HS / / / / 2-50% SPK-HS 8341 73.6743 / 137.7 2-100% SPK-HS 16598 141.1225 / 131.9 2-KB-ZL / / 0 / 2-1% SPK-ZL / / / / 2-50% SPK-ZL 7371 65.7508 / 122.9 2-100% SPK-ZL 14445 123.5355 / 115.4

[0123] (3) T3 column (MRM11(+))

[0124] Liquid chromatography was performed using the above diluents 1 and 2, and a T3 column (MRM11(+)). The results are shown in Table 9. Under the chromatography-mass spectrometry conditions, the recovery rate was between 115.0% and 138.9%, and further optimization of parameters was required.

[0125] Table 9:

[0126] name Response value Concentration value (ng / mL) Blank (matrix) / system suitability solution Recovery rate (%) 2-KB-ST / / 0 / 2-1% SPK-ST / / / / 2-50% SPK-ST 8568 74.3477 / 138.9 2-100% SPK-ST 15209 128.7678 / 120.3 2-KB-HS / / 0 / 2-1% SPK-HS / / / / 2-50% SPK-HS 8428 73.2004 / 136.8 2-100% SPK-HS 16722 141.1662 / 131.9 2-KB-ZL / / 0 / 2-1% SPK-ZL / / / / 2-50% SPK-ZL 7429 65.0141 / 121.5 2-100% SPK-ZL 14510 123.0398 / 115.0

[0127] Based on the above data, we can see that ① Linearity: 1 ng / mL concentration was too low to be detected, 10 ng / mL and 30 ng / mL had poor responses, and 50 ng / mL (LOQ); ② After the permeate and the retained sample were diluted 100 times, they were both within the range of 50 ng / mL to 1000 ng / mL; ③ The linear range was adjusted to 50 ng / mL-1000 ng / mL; ④ SYS: 500 ng / mL was selected as SYS, that is, 10% SPK, 100% SPK, and 200% SPK were set as the recovery range, and the method needs to be further optimized; ⑤ Dilution: 0.1% formic acid 1% saline solution; ⑥ Chromatographic column: Waters Atlantis T3, 5um, 4.6*150mm.

[0128] 3. Recovery rate

[0129] The solutions and methods used in this experiment are as follows:

[0130] The solutions shown in Tables 10 to 14 were used. The preparation method of each solution was the same as the preparation method of the corresponding solution in Example 2 unless otherwise specified.

[0131] Mobile phase: mobile phase phaseA1 (0.1% formic acid solution); mobile phase phaseB1 (methanol solution), mobile phase: phaseB2 (0.1% formic acid methanol solution).

[0132] Liquid chromatography was performed according to the above mobile phase and the following methods. The experimental results are shown in Tables 10 to 14, where the concentration range of the linear solution (L-1 to 5) was adjusted to 50 ng / mL-1000 ng / mL; 500 ng / mL was selected as SYS, that is, 10% SPK, 100% SPK, and 200% SPK were set as the recovery range; diluent: 0.1% formic acid 1% saline solution; chromatographic column: Waters Atlantis T3, 5um, 4.6*150mm: Method 19 and Method 20 were selected, and the recovery range was 95.5% to 112.9%. Considering the gradient elution procedure and API peak time, Method 19 was selected, and the concentrations of each solution and the test results are as follows:

[0133] (1) Method: MRM11(+): phaseA1-B1; MRM13(+): phaseA1-B1 (90% A-10% B).

[0134] Table 10:

[0135]

[0136] (2) Method: MRM15(+): phase A1-B1 (70% A-30% B); MRM17(+): phase A1-B1 (60% A-40% B)

[0137] Table 11:

[0138]

[0139] (3) Method: MRM19(+): phaseA1-B1 (95%A-5%B); MRM20(+): phaseA1-B2 (95%A-5%B)

[0140] Table 12:

[0141]

[0142] (4) Method: MRM12(+): phaseA1-B2 (95%A-5%B); MRM14(+): phaseA1-B2 (90%A-10%B)

[0143] Table 13:

[0144]

[0145]

[0146] (5) Method: MRM21(+): phaseA1-B1 (98%A-2%B); MRM22(+): phaseA1-B2 (98%A-2%B)

[0147] Table 14:

[0148]

[0149] 4. Linear

[0150] Based on the previously optimized parameters, the applicant attempted to broaden the concentration range of the linear solution (L1-L6). The experimental results are shown in Table 15. The linear maximum point of 10000 ng / mL did not fit well. In subsequent use, the concentration range of the linear solution can be selected according to demand.

[0151] Table 15:

[0152]

[0153] Example 2 LC-MS Detection Method Verification

[0154] 1. Experimental Materials

[0155] The experimental materials used in this example are as follows:

[0156] (1) The aminolevulinic acid hydrochloride gel used is shown in Table 16.

[0157] Table 16:

[0158] Material Name batch number Manufacturer Remark Aminolevulinic acid hydrochloride gel 147S02 Biofrontera Bioscience GmbH Ameluz

[0159] (2) Physiological saline: Weigh 18 g of sodium chloride, add pure water to 2000 mL and dissolve it completely.

[0160] 2. Penetration test

[0161] The pigskin from a one-month-old Bama Xiang pig was washed and soaked in the above-prepared physiological saline for 0.5 h, then taken out and dried with filter paper. The pigskin was then clamped between the sample supply pool and the receiving pool of the Franz diffusion cell (automatic transdermal diffusion instrument, equipment model: DHC-6TD, manufacturer: Logan), and the receiving pool was filled with about 15 mL of physiological saline (diffusion cell volume 12 mL). The test amount (about 300 mg) of product 1 (aminolevulinic acid hydrochloride external powder) was added to the pigskin, the stirring speed was 600 rpm, and the temperature was 32°C. 1.5 mL of the receiving solution was taken at 2h, 4h, 8h, 12h, 16h, 20h, and 24h after the addition of the drug as the original permeate solution. After sampling in the full emptying mode, 15 mL of isothermal blank physiological saline was added.

[0162] After the test, the remaining drug and pig skin were transferred to a 50 mL centrifuge tube, 50 mL of normal saline was added, vortexed for 1 min, and 1 mL of supernatant was taken for testing after standing, as the recovery solution. The weighing of the above-mentioned aminolevulinic acid hydrochloride external powder sample is shown in Table 17. The weight before loading is: injection needle weight + sample loading amount, and the weight after loading is: injection needle weight.

[0163] Table 17:

[0164] product Before loading (mg) After loading (mg) Sample amount (mg) 1(Aminolevulinic acid hydrochloride powder for external use) 2873.4 2630.2 243.2

[0165] 3. Retention test

[0166] Take out the pigskin used for the penetration test, use a pipette to transfer 1mL of saline to rinse the residual drug solution, and repeat 3 times. After absorbing the residual liquid on the pigskin with filter paper, cut out the drug-applied part with a mold, weigh the weight, cut it into thin strips and place it in a grinding tube, add 1mL of saline, add grinding steel balls (2 large and 1 small), and grind it with a homogenizer for 30 minutes. The grinding conditions are 2°C, frequency 70Hz, run 400s, interrupt 6s, add 0.5mL of saline after grinding, vortex, and then centrifuge at 8000rpm for 10min. After centrifugation, take the supernatant for testing, which is the retained sample stock solution. The skin weighing situation is shown in Table 18:

[0167] Table 18:

[0168] sample Batch number of aminolevulinic acid hydrochloride powder for external use Pigskin weight after penetration test (mg) Pigskin 147S02 138.9

[0169] (1) Solution preparation:

[0170] Physiological saline solution: 9g / L sodium chloride solution.

[0171] Dilution solution (blank original solution / blank solution, KB): 0.1% formic acid 1% saline solution (0.1 ml formic acid + 1 ml saline solution + 100 ml water, mix well, and the ratio can be enlarged or reduced according to this ratio).

[0172] Permeate matrix blank solution (KB-ST): 0.1% formic acid 1% permeate matrix blank stock solution (0.1ml formic acid + 1ml permeate blank solution + 100ml water, mix well, and the ratio can be enlarged or reduced according to this ratio).

[0173] Recovery liquid matrix blank solution (KB-HS): 0.1% formic acid 1% recovery liquid matrix blank stock solution (0.1ml formic acid + 1ml recovery liquid blank solution + 100ml water, mix well, and the ratio can be enlarged or reduced according to this ratio).

[0174] Retention sample matrix blank solution (KB-ZL): 0.1% formic acid 1% retention sample matrix blank stock solution, filter with 0.22μm filter membrane, take the filtrate (0.1ml formic acid + 1ml retention sample blank solution + 100ml water, mix well, and the ratio can be enlarged or reduced according to this ratio).

[0175] The sample weight of 5-ALA-HCl reference substance is 10.97 mg, and the theoretical concentration of 5-ALA-HCl system suitability solution is 543.0150 ng / mL.

[0176] 5-ALA-HCl stock concentrated solution (5-ALA-HCl concentration is about 100 μg / mL): Take about 10 mg of 5-ALA-HCl, accurately weigh it, put it in a 100 mL volumetric flask, add the above diluent to dissolve and dilute to the scale, completely dissolve and shake well.

[0177] 5-ALA-HCl stock solution ① (5-ALA-HCl concentration is about 10 μg / mL): Accurately measure 10 mL of 5-ALA-HCl concentrated stock solution, place it in a 100 mL volumetric flask, dilute to the scale with diluent, completely dissolve and shake well.

[0178] 5-ALA-HCl stock solution ② (5-ALA-HCl concentration is about 1000 ng / mL): Accurately measure 10 mL of 5-ALA-HCl stock solution ① and place it in a 100 mL volumetric flask. Dilute to the scale with diluent, completely dissolve and shake well. It can also be used as L-5 linear solution.

[0179] 5-ALA-HCl system suitability solution (SYS, 5-ALA-HCl concentration is about 500 ng / mL): Accurately measure 10 mL of 5-ALA-HCl stock solution②, place it in a 20 mL volumetric flask, dilute to the scale with diluent, completely dissolve and shake well.

[0180] (2) Specificity experiment

[0181] Accurately measure 5 μL of the blank solution, permeate matrix blank solution, recovery matrix blank solution, retentate matrix blank solution, and system suitability solution prepared above, and inject them into the LC-MS system (Agilent 1260 / 1290LC+6470MS LC-MS, both models are acceptable) for analysis. The conditions and parameters used in the LC-MS system are as follows:

[0182] LC-MS parameters:

[0183] Chromatographic column: Waters Atlantis T3, 150 mm × 4.6 mm, 5 μm

[0184] Column temperature: 40°C

[0185] Injection volume: 5 μl

[0186] Sample plate temperature: 10°C

[0187] Flow rate: 0.6mL / min

[0188] Mobile phase: 0.1% formic acid solution (mobile phase A), methanol solution (mobile phase B)

[0189] Elution mode: gradient elution. The gradient elution conditions are shown in Table 19.

[0190] Table 19:

[0191] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 95 5 4.00 95 5 4.01 5 95 8.00 5 95 8.01 95 5 10.00 95 5

[0192] The mass spectrometry analysis conditions were:

[0193] Ion source type: ESI

[0194] Ion source mode: Positive

[0195] ESI source temperature: 300°C

[0196] Carrier gas flow rate: 5L / min

[0197] Sheath gas heating temperature: 250℃

[0198] Sheath gas heating flow rate: 11L / min

[0199] Sprayer pressure: 45Psi

[0200] Capillary voltage: 3500V

[0201] The qualitative and quantitative ion pairs, collision gas energy and declustering voltage are shown in Table 20.

[0202] Table 20:

[0203]

[0204] 5 μL of the above solution was injected into an ultra-high performance liquid phase mass spectrometer for qualitative analysis using retention time and mass-to-charge ratio and quantitative analysis using peak area.

[0205] The results in Table 21 show that the peak area ratios of the blank solution, permeate matrix blank solution, recovery matrix blank solution, retentate matrix blank solution and 5-ALA-HCl system suitability solution (SYS) are all 0, which meets the requirements. Figure 1 The spectrum of the permeate matrix blank solution is shown in Figure 5 As shown, the blank solution spectrum of the recovery solution matrix is ​​as follows Fig. 9 As shown, the chromatogram of the matrix blank solution of the retention sample is as follows Fig.13 shown.

[0206] Table 21:

[0207]

[0208]

[0209] (3) System suitability test

[0210] Accurately measure 5 μL of the above system suitability solution (SYS) and inject it into the liquid-mass spectrometry system for analysis, where SYS selects 6 parallel samples (SYS-1 to 6) in the same injection vial for instrument stability detection, and other system suitability solutions (SYS-S1 to S4) at different time points are used to examine the stability of the reference solution. The results in Table 22 show that the RSD% of the peak area of ​​5-ALA-HCl in the 6-injection system suitability solution is 0.7%; the RSD% of the peak area of ​​5-ALA-HCl in the multi-injection system suitability solution (n≥7) is not more than 4.4%, which meets the requirements.

[0211] System suitability solution spectrum (peak time of 5-ALA-HCl is 2.520min) Figure 2 shown.

[0212] Table 22:

[0213]

[0214] (4) Calculation and fitting of linear equations

[0215] The preparation method of 5-ALA-HCl linear solution is as follows: accurately pipette an appropriate volume of the above 5-ALA-HCl stock solution ② (expressed as L-5), place it in a 20mL volumetric flask, dilute to the scale with diluent, and shake well.

[0216] The preparation method of the linear solutions (L-1 to 4) used in this experiment is shown in Table 23 below, wherein L-1 in Table 23 is taken as an example. The preparation method of L-1 is: take 1 mL of L-5 transfer solution, dilute it to a 20 mL volumetric flask, and obtain 5-ALA-HCl with a final concentration of 50 ng / mL. The preparation methods of the remaining groups of solutions are similar. In addition, the linear equations and their correlation coefficients obtained after testing the solutions prepared in Table 23 are shown in Table 24.

[0217] Table 23:

[0218]

[0219] Table 24:

[0220]

[0221] The results in Table 24 show that 5-ALA-HCl has a linear relationship with the response value in the range of 54.3015 ng / mL to 1086.0300 ng / mL, and the correlation coefficient R 2 The value is 1.00, which meets the requirement.

[0222] (5) Limit of detection and limit of quantification

[0223] The solution preparation method for this experiment is as follows:

[0224] The sample weight of 5-ALA-HCl reference substance is 10.97 mg, the theoretical concentration of 5-ALA-HCl detection limit solution is 27.1508 ng / mL, and the theoretical concentration of 5-ALA-HCl quantification limit solution is 54.3015 ng / mL.

[0225] Detection limit solution (LOD, 5-ALA-HCl concentration is about 25 ng / mL): Accurately measure 0.5 mL of 5-ALA-HCl stock solution②, place it in a 20 mL volumetric flask, dilute to the scale with diluent, fully dissolve and shake well.

[0226] Limit of quantitation solution (LOQ, 5-ALA-HCl concentration of about 50 ng / mL): Accurately measure ②1 mL of 5-ALA-HCl stock solution, place it in a 20 mL volumetric flask, dilute to the scale with diluent, fully dissolve and shake well.

[0227] Use the system suitability solution to establish system suitability, accurately measure 5 μL of the prepared detection limit solution (LOD) and quantification limit solution (LOQ), inject them into the liquid mass spectrometer for analysis and record the spectrum. Among them, LOQ is selected from 6 replicates (LOQ1 to 6) in the same injection vial. The detection spectrum of the detection limit solution (LOD) is as follows: Figure 3 As shown, the detection spectrum of the quantitation limit solution (LOQ) is as follows Figure 4 shown.

[0228] Table 25:

[0229]

[0230] The experimental results in Table 25 show that the detection limit concentration of 5-ALA-HCl is 27.1508 ng / mL, and the S / N is 15.39, which meets the requirements. The quantitative limit concentration of 5-ALA-HCl is 54.3015 ng / mL, and the S / N is 28.46-30.95, and the peak area RSD% of the 6 quantitative limit solutions is 0.6%, which meets the requirements.

[0231] (6) Accuracy

[0232] The preparation methods of the solutions used in this experiment are as follows:

[0233] Permeate 10% limit spike solution (5-ALA-HCl concentration is about 50 ng / mL): Accurately measure 1 mL of 5-ALA-HCl stock solution ② and place it in a 20 mL volumetric flask. Dilute to the mark with the above permeate matrix blank solution (KB-ST), completely dissolve and shake well. Prepare 3 portions in parallel and label them as 10% SPK-ST-1 to 3.

[0234] Permeate 100% limit spike solution (5-ALA-HCl concentration about 500 ng / mL): Accurately measure 1 mL of 5-ALA-HCl stock solution and place it in a 20 mL volumetric flask. Dilute to scale with the above permeate matrix blank solution, completely dissolve and shake well. Prepare 3 portions in parallel and label them as 100% SPK-ST-1 to 3.

[0235] 200% limit spike solution of permeate (5-ALA-HCl concentration is about 1000ng / mL): Accurately measure 5-ALA-HCl stock solution①2mL, place in a 20mL volumetric flask, dilute to scale with the above permeate matrix blank solution (KB-ST), completely dissolve and shake well. Prepare 3 portions in parallel and label them as 200%SPK-ST-1~3.

[0236] Recovered liquid 10% limit spike solution (5-ALA-HCl concentration is about 50ng / mL): Accurately measure 5-ALA-HCl stock solution ②1mL, place in a 20mL volumetric flask, dilute to scale with the above-mentioned recovered liquid matrix blank solution (KB-HS), completely dissolve and shake well. Prepare 3 portions in parallel and mark them as 10%SPK-HS-1~3 respectively.

[0237] Recovery solution 100% limit spike solution (5-ALA-HCl concentration about 500ng / mL): Accurately measure 1mL of 5-ALA-HCl stock solution, place in a 20mL volumetric flask, dilute to scale with the above recovery solution matrix blank solution (KB-HS), completely dissolve and shake well. Prepare 3 portions in parallel, labeled as 100% SPK-HS-1 to 3.

[0238] Recovery solution 200% limit spike solution (5-ALA-HCl concentration about 1000ng / mL): Accurately measure 5-ALA-HCl stock solution①2mL, place in a 20mL volumetric flask, dilute to scale with the above recovery solution matrix blank solution (KB-HS), completely dissolve and shake well. Prepare 3 portions in parallel, labeled as 200%SPK-HS-1~3 respectively.

[0239] Retention sample 10% limit spike solution (5-ALA-HCl concentration is about 50ng / mL): Accurately measure 5-ALA-HCl stock solution ②1mL, place in a 20mL volumetric flask, dilute to scale with the above retention sample matrix blank solution (KB-ZL), completely dissolve and shake well, filter with a 0.22μm filter membrane, and take the filtrate. Prepare 3 portions in parallel and mark them as 10%SPK-ZL-1~3 respectively.

[0240] Retention sample 100% limit spike solution (5-ALA-HCl concentration about 500ng / mL): Accurately measure 1mL of 5-ALA-HCl stock solution ①, place in a 20mL volumetric flask, dilute to scale with the above retention sample matrix blank solution (KB-ZL), shake well, filter with a 0.22μm filter membrane, and take the filtrate. Prepare 3 portions in parallel and mark them as 100% SPK-ZL-1~3 respectively.

[0241] Retention sample 200% limit spike solution (5-ALA-HCl concentration about 1000ng / mL): Accurately measure 5-ALA-HCl stock solution①2mL, place in a 20mL volumetric flask, dilute to scale with the above retention sample matrix blank solution (KB-ZL), shake well, filter with a 0.22μm filter membrane, and take the filtrate. Prepare 3 portions in parallel and mark them as 200%SPK-ZL-1~3 respectively.

[0242] The permeate blank matrix solution (KB-ST), recovery blank matrix solution (KB-HS), retention blank matrix solution (KB-ZL) and their corresponding spiked solutions were spiked at three concentration levels: low (0.1 times), medium (1.0 times) and high (2 times). Each concentration level was repeated three times, and the recovery rate was calculated based on the background concentration, measured concentration and spiked concentration.

[0243] Use the system suitability solution to establish system suitability, accurately measure 5 μL of the test sample matrix blank solution and each limit spike solution, and inject them into the liquid chromatography-mass spectrometry system for analysis.

[0244] The results in Table 26 show that the recovery of 5-ALA-HCl in the 10% limit spiked solution of the three permeates is 100.6% to 105.7%, and the RSD% (n=3) is 2.5%, the recovery of 5-ALA-HCl in the 100% limit spiked solution of the three permeates is 103.5% to 106.9%, and the RSD% (n=3) is 1.8%, and the recovery of 5-ALA-HCl in the 200% limit spiked solution of the three permeates is 99.7% to 103.2%, and the RSD% (n=3) is 1.9%. The recovery of the spiked sample solutions of the permeates with different limits is in the range of 99.7% to 106.9%, and the recovery RSD% (n=3) is not greater than 2.5%, which meets the requirements.

[0245] Table 26:

[0246]

[0247] The results of Table 27 show that the recovery rates of the 10% limit spiked solutions of the three recovery solutions are 102.3% to 104.2%, and the RSD% (n=3) is 1.0%, the recovery rates of the 100% limit spiked solutions of the three recovery solutions are 104.5% to 106.0%, and the RSD% (n=3) is 0.7%, and the recovery rates of the 200% limit spiked solutions of the three recovery solutions are 102.0% to 104.7%, and the RSD% (n=3) is 1.4%. The recovery rates of the spiked sample solutions of the recovery solutions with different limits are within the range of 102.0% to 106.0%, and the recovery RSD (n=3) is not greater than 1.4%, which meets the requirements.

[0248] Table 27:

[0249]

[0250] The results of Table 28 show that the recovery rates of the 10% limit spiked solution of three retained samples are 107.7% to 110.4%, and the RSD% (n=3) is 1.4%, the recovery rates of the 100% limit spiked solution of three retained samples are 113.3% to 113.8%, and the RSD% (n=3) is 0.3%, and the recovery rates of the 200% limit spiked solution of three retained samples are 106.2% to 108.7%, and the RSD% (n=3) is 1.3%. The recovery rates of the spiked sample solutions of different limit retained samples are within the range of 106.2% to 113.8%, and the recovery RSD% (n=3) is not greater than 1.4%, which meets the requirements.

[0251] Table 28:

[0252]

[0253] (7) Solution stability

[0254] Reference solution: The above system suitability solution (SYS) was used for stability test of reference solution, and the limit spike solution (100% SPK-ST, 100% SPK-HS, 100% SPK-ZL) was used for stability test of test solution. The peak area of ​​the first injection of system suitability and limit spike sample solution was used as the initial point. During the injection process, the system suitability solution and limit spike sample solution were re-injected and compared with the peak area of ​​the initial point.

[0255] Use system suitability solution to establish system suitability. Accurately measure 5 μL of system suitability solution and limit spike solution, inject them into the HPLC system for analysis and record the spectrum.

[0256] The results in Table 29 show that the system suitability solution response value at different time points and the ratio of the initial time are 102.6%-111.8%, which meets the development requirements. The system suitability solution is stable within 28.5 hours.

[0257] Table 29:

[0258] name Solution standing time (h) Peak area Ratio of peak area at different time points to the initial time point (%) SYS-S0 0 254061 / SYS-S1 8.5 260791 102.6 SYS-S2 15.5 266380 104.8 SYS-S3 22 283921 111.8 SYS-S4 28.5 282976 111.4

[0259] The results in Table 30 show that the ratio of the response value of the permeate stability solution to the initial time at different time points is 101.3%-106.0%, which meets the development requirements. The permeate 100% spiked solution is stable within 28.5 hours.

[0260] Table 30:

[0261] name Solution standing time (h) Peak area Ratio of peak area at different time points to the initial time point (%) 100% SPK-ST-S0 0 273874 / 100% SPK-ST-S1 8.5 277452 101.3 100% SPK-ST-S2 15.5 279974 102.2 100% SPK-ST-S3 22 290361 106.0 100% SPK-ST-S4 28.5 286398 104.6

[0262] The results in Table 31 show that the response value of the recovery solution stability at different time points and the ratio of the initial time are 98.9% to 105.3%, which meets the development requirements. The recovery solution 100% spiked solution is stable within 28.5 hours.

[0263] Table 31:

[0264] name Solution standing time (h) Peak area Ratio of peak area at different time points to the initial time point (%) 100%SPK-HS-S0 0 272102 / 100% SPK-HS-S1 8.5 268991 98.9 100% SPK-HS-S2 15.5 277708 102.1 100% SPK-HS-S3 22 283936 104.3 100% SPK-HS-S4 28.5 286652 105.3

[0265] The results in Table 32 show that the ratio of the response value of the retention sample stability solution to the initial time at different time points is 102.5% to 108.9%, which meets the development requirements. The retention sample 100% spiked solution is stable within 28.5 hours.

[0266] Table 32:

[0267] name Solution standing time (h) Peak area Ratio of peak area at different time points to the initial time point (%) 100% SPK-ZL-S0 0 285408 / 100% SPK-ZL-S1 8.5 292679 102.5 100% SPK-ZL-S2 15.5 297532 104.2 100% SPK-ZL-S3 22 310717 108.9 100% SPK-ZL-S4 28.5 308258 108.0

[0268] (8) Durability

[0269] In this experiment, the durability was investigated by changing the flow rate to 0.5 mL / min and 0.7 mL / min, the column temperature to 35°C and 45°C, and the ESI Heater temperature to 280°C and 320°C.

[0270] Use the system suitability solution to establish system suitability, accurately measure 5 μL of the durability solution, inject it into the LC / MS system for analysis and record the spectrum. According to the method described in the previous solution preparation section, prepare 1 part of system suitability solution, 1 part of permeate matrix blank solution and 1 part of permeate 100% limit spike solution, 1 part of recovery matrix blank solution and 1 part of recovery 100% limit spike solution, 1 part of retention sample matrix blank solution and 1 part of retention sample 100% limit spike solution, and inject and analyze them under the above conditions respectively. The results are shown in Table 33.

[0271] Table 33:

[0272]

[0273] Under each durability condition, the 6-pin system suitability solution 5-ALA-HCl peak area RSD% was no more than 2.5%; the 100% limit spike recovery of the permeate was between 101.1% and 107.1%, the 100% limit spike recovery of the recovery solution was between 99.4.0% and 106.3%, and the 100% limit spike recovery of the retention sample was between 107.7% and 113.6%. The results showed that the flow rate, column temperature, and ion source temperature changed slightly, and the durability met the requirements.

[0274] (7) Content determination results

[0275] The permeate, recovery liquid and retention sample samples collected at 2h, 4h, 8h, 12h, 16h, 20h and 24h in Example 1 were processed and analyzed, and the content was calculated based on the peak area, and the recovery rate was calculated.

[0276] Permeate solution (dilution factor: 12*100=1200): Accurately measure 0.1 mL of the permeate stock solution, place it in a 10 mL volumetric flask, dilute it to the scale with diluent (0.1% formic acid), fully dissolve and shake well, and wait for testing. The actual detection value of the permeate solution is shown in Table 34. The content of the permeate stock solution can be obtained by the measured value * dilution factor, which is convenient for further evaluation of the recovery rate.

[0277] Table 34:

[0278]

[0279]

[0280] Recovery solution (dilution factor: 50*100*11=55000): Accurately measure 0.1 mL of the recovery solution, place it in a 10 mL volumetric flask, dilute to the mark with diluent (0.1% formic acid), completely dissolve and shake well for testing.

[0281] Table 35:

[0282] name Detection concentration (ng / mL) 147S02HS 339.8383

[0283] Retention sample solution (dilution factor: 1.5*100=150): Accurately measure 0.1 mL of the retention sample stock solution, place it in a 10 mL volumetric flask, dilute to the scale with diluent (0.1% formic acid), shake well, and wait for measurement.

[0284] Table 36:

[0285] name Detection concentration (ng / mL) 147S02ZL 106.8529

[0286] The mass balance calculations of permeate, recovered liquid and retentate are shown in Table 37.

[0287] Table 37:

[0288]

[0289] Note: Penetration 1 (mg) = cumulative permeate sample detection concentration at different sampling time points (ng / ml) * dilution factor (12ml*100) * unit conversion (mg / 1000000ng),

[0290] Holdup 2 (mg) = retention sample detection concentration (ng / ml) * dilution factor (1.5ml*100) * unit conversion (mg / 1000000ng),

[0291] Recycling volume 3 (mg) = recovery solution detection concentration (ng / ml) * dilution factor (50ml*100*11) * unit conversion (mg / 1000000ng),

[0292] Recovery rate 4 =(permeate amount (mg) + retention amount (mg) + recovery amount (mg)) / gel specification / sample amount*100%.

[0293] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0294] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for detecting aminolevulinic acid hydrochloride, characterized in that, include: (1) pre-treating a sample containing aminolevulinic acid hydrochloride to obtain a test solution; (2) performing liquid chromatography-mass spectrometry on the test solution obtained in step (1) to determine the content of aminolevulinic acid hydrochloride in the test sample.

2. The method according to claim 1, characterized in that The conditions for the liquid chromatography detection are: A Waters Atlantis T3 column was used, with 0.1% formic acid solution as mobile phase A and methanol solution as mobile phase B for gradient elution; Optionally, the conditions of the gradient elution are as shown in Table 19; Flow rate 0.5-0.7 mL / min, column temperature 35-45 °C, injection volume 5 μL; Preferably, the flow rate is 0.6 mL / min, the column temperature is 40° C., and the injection volume is 5 μL.

3. The method according to claim 1, characterized in that The conditions for mass spectrometry detection are: Ion source type: ESI, Ion source mode: Positive, ESI source temperature: 280°C to 320°C, preferably 300°C, Carrier gas flow rate: 5L / min, Sheath gas heating temperature: 250°C, Sheath gas heating flow rate: 11L / min, Sprayer pressure: 45Psi, Capillary voltage: 3500V, The qualitative ion pair, quantitative ion pair, fragmentation voltage, collision energy, and dwell time information are shown in Table 20.

4. The method according to claim 1, characterized in that: The concentration of aminolevulinic acid hydrochloride in the sample is 50 to 1000 ng / mL; Optionally, the sample comprises at least one of the following: blood, serum, plasma, feces, urine, sweat, saliva, cells, tissue, permeate stock solution, recovery stock solution and skin retention sample stock solution; Optionally, the pretreatment comprises at least one of the following: dilution, grinding and centrifugation; Optionally, the pretreatment method of the penetration stock solution includes: diluting the penetration stock solution with a 0.1% formic acid solution, wherein the dilution multiple is 1000 to 1500 times, preferably 1200 times, and the penetration stock solution is obtained within 2 to 24 hours of the aminolevulinic acid hydrochloride transdermal experiment; Optionally, the pretreatment method of the recovered stock solution includes the following operations in sequence: diluting the recovered stock solution 20 to 60 times with physiological saline, then diluting it 80 to 120 times with 0.1% formic acid solution, and then diluting it 5 to 20 times with 0.1% formic acid 1% physiological saline solution; Preferably, the pretreatment method of the recovered stock solution includes the following operations in sequence: diluting the recovered stock solution 50 times with physiological saline, then diluting it 100 times with 0.1% formic acid solution, and then diluting it 11 times with 0.1% formic acid 1% physiological saline solution; Optionally, the recovered liquid stock solution is the supernatant obtained by transferring the remaining aminolevulinic acid hydrochloride and the skin into physiological saline after the aminolevulinic acid hydrochloride transdermal experiment, vortexing and standing; Optionally, the pretreatment method of the skin retention sample stock solution includes: diluting the skin retention sample stock solution with a 0.1% formic acid solution, wherein the dilution multiple is 10 to 150 times, preferably 150 times; Optionally, the retention sample stock solution is the supernatant obtained by grinding and centrifuging the skin after the aminolevulinic acid hydrochloride transdermal experiment.

5. The method according to claim 1, characterized in that Before the liquid chromatography-mass spectrometry detection, the aminolevulinic acid hydrochloride content of the reference substance is detected in advance, and the reference substance is a sample containing aminolevulinic acid hydrochloride of known concentration; Optionally, the linear concentration of aminolevulinic acid hydrochloride in the reference substance is 50 to 1000 ng / mL, preferably the 100% limit concentration is 500 ng / mL; Optionally, before performing the liquid chromatography-mass spectrometry detection, a standard curve between the aminolevulinic acid hydrochloride content in the linear solution and the chromatographic peak area is pre-drawn; Optionally, the linear solution is a plurality of samples containing aminolevulinic acid hydrochloride of different known concentrations, and the concentration of aminolevulinic acid hydrochloride in the linear solution is 50 to 1000 ng / mL; Optionally, the content of aminolevulinic acid hydrochloride is determined based on the chromatographic peak area of ​​the sample to be tested detected by liquid chromatography-mass spectrometry; Optionally, the content of aminolevulinic acid hydrochloride is determined based on the standard curve and the chromatographic peak area of ​​the sample to be tested detected by liquid chromatography-mass spectrometry.

6. A method for detecting aminolevulinic acid hydrochloride, characterized in that: include: (1) Pre-treating a sample containing aminolevulinic acid hydrochloride to obtain a test solution; (2) performing liquid chromatography-mass spectrometry on the sample solution obtained in step (1) to obtain a spectrum of the sample to be tested, wherein the conditions of the liquid chromatography detection are: A Waters Atlantis T3 chromatographic column was used, with a 0.1% formic acid solution as the mobile phase A and a methanol solution as the mobile phase B for gradient elution. The conditions of the gradient elution are shown in Table 19. Flow rate 0.6 mL / min, column temperature 40 °C, injection volume 5 μL; The conditions for mass spectrometry detection are: Ion source type: ESI, Ion source mode: ESI+, ESI source temperature: 300°C, Carrier gas flow rate: 5L / min, Sheath gas heating temperature: 250°C, Sheath gas heating flow rate: 11L / min, Sprayer pressure: 45Psi, Capillary voltage: 3500V, The qualitative ion pair, quantitative ion pair, fragmentation voltage, collision energy, and dwell time information are shown in Table 20; (3) determining the content of aminolevulinic acid hydrochloride based on the chromatographic peak area of ​​the sample to be tested detected by the liquid chromatography-mass spectrometry; in: The sample to be tested in step (1) includes at least one of the following: blood, serum, plasma, feces, urine, sweat, saliva, cells, tissues, osmotic fluid, recovery fluid and skin retention sample fluid, the pretreatment includes dilution, grinding and centrifugation, and the diluent is formic acid and / or physiological saline.

7. The method according to claim 6, characterized in that Before performing step (2), a system suitability experiment is performed in advance using a reference substance, wherein the concentration of aminolevulinic acid hydrochloride in the reference substance is 500 ng / mL; Optionally, after performing the system suitability experiment and before step (2), the linear solution is subjected to liquid chromatography-mass spectrometry detection, and a standard curve between the aminolevulinic acid hydrochloride content in the linear solution and the chromatographic peak area is drawn, wherein the concentrations of aminolevulinic acid hydrochloride in the linear solution are 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively.

8. A kit for detecting aminolevulinic acid hydrochloride in a sample, characterized in that: include: (A) a plurality of aminolevulinic acid hydrochloride standards, wherein the concentration of aminolevulinic acid hydrochloride in the standards is 50 to 1000 ng / mL; (B) an aminolevulinic acid hydrochloride reference substance, wherein the reference substance is a solution containing an aminolevulinic acid hydrochloride solution of known concentration; and (C) instructions for use, wherein the instructions describe the method according to any one of claims 1 to 8.

9. The kit according to claim 8, characterized in that Further including: (D) a diluent, wherein the diluent is formic acid and / or physiological saline; (E) A mobile phase additive, wherein the mobile phase additive comprises formic acid.

10. A method for quality control of aminolevulinic acid hydrochloride content in a production process, characterized in that: The method comprises: The aminolevulinic acid hydrochloride content of the quality control sample is detected using the method for detecting aminolevulinic acid hydrochloride according to any one of claims 1 to 7, and the detected aminolevulinic acid hydrochloride content is compared with the target aminolevulinic acid hydrochloride content value to determine whether the quality control sample meets the standard.

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