A method for detecting nitrogen mustard-containing sophoridine chlorophenyl derivatives

Abstract

The application belongs to the technical field of analytical testing, and discloses a detection method of a nitrogen mustard-containing sophoridine chlorophenyl derivative. The purity of the derivative to-be-detected substance is detected by adopting an HPLC-DAD method combined with ion pair reagents. The control conditions of the HPLC-DAD include that the chromatographic column is a C18 column, the detection wavelength is 210-220 nm, the mobile phase A is 0.003-0.008 M sodium heptanesulfonate aqueous solution, and the mobile phase B is methanol. The peak type of R1 and R2 is best, the response value is high, and the baseline separation can be achieved with adjacent impurity peaks, the specificity is good, the detection limit is low, the precision is good, the stability is high, and the detection can be quickly and accurately realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical testing technology, and more particularly relates to a detection method of a nitrogen mustard-containing sophoridine chlorophenyl derivative. BACKGROUND

[0002] The nitrogen mustard-containing sophoridine chlorophenyl derivative (chloro(pyridino[3,2,1-ij][1,6]naphthyridin-1-yl)butyl bis(2-chloroethyl)carbamate derivative) is a new compound for tumor or cancer treatment independently developed. The synthesis method and use of the compound are disclosed in 【1】 (A Class of Nitrogen Mustard-Containing Sophoridine Derivatives and Their Preparation Method and Use, ZL201710319723.1) and 【2】 (Sophoridine Derivatives Induce Apoptosis and Autophagy to Suppress the Growth of Triple-Negative Breast Cancer through Inhibition of mTOR Signaling [J]. ChemMedChem, 2022, 17: e202100434). We found through research that the compound shows strong activity in inhibiting the proliferation of various tumor cells such as leukemia, cervical cancer, liver cancer, and breast cancer in in vitro and in vivo experiments, and has low toxicity to normal cells. The compound has good application prospects as a drug, especially for tumor or cancer treatment. However, since no purity detection method has been established for the compound, it cannot be quantified, so the quality cannot be effectively controlled and the drug efficacy cannot be guaranteed, which seriously affects the drug conversion process of the compound. Therefore, establishing a rapid, accurate and effective compound purity detection method has dual evaluation values in terms of quality and efficacy, and is of great significance for promoting the conversion of the compound into an efficient and low-toxicity antitumor drug and effectively ensuring clinical efficacy.

[0003] Currently, there is no suitable quality evaluation method for chloro(pyridino[3,2,1-ij][1,6]naphthyridin-1-yl)butyl bis(2-chloroethyl)carbamate derivatives. Since the compound has high polarity, it is not easy to be retained on a reverse system chromatography column with a C18 chromatography column and an amino chromatography column as the main detection means, and it is difficult to detect by HPLC. The maximum absorption wavelength of the compound is similar to that of the solvent, and the absorption of the solvent will affect the accuracy of the detection results, making it difficult to accurately quantify. The series of derivative compounds have similar structures and similar polarities, making separation difficult. In addition, unknown impurities that are not easy to separate from the compound exist in the sample, making it difficult to establish an effective and accurate quality analysis method, affecting the quality control and subsequent conversion application of the compound.

[0004] Therefore, how to provide a quick, accurate, and low detection limit detection method for nitrogen mustard-containing sophoridine chlorophenyl derivatives is a problem that those skilled in the art need to solve. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies in the prior art, the present application provides a detection method for nitrogen mustard-containing sophoridine chlorophenyl derivatives.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A detection method for nitrogen mustard-containing sophoridine chlorophenyl derivatives adopts HPLC-DAD method combined with ion pair reagent to detect the purity of the derivative to be tested;

[0008] The structure of the derivative is as follows:

[0009]

[0010] Preferably, the derivative is a mixture of one or two of R1 and R2;

[0011] The structure of R1 and R2 is as follows:

[0012]

[0013] R1: 4-((1R,3aR,3a 1 S,10aS)-2-((3-chlorophenyl)sulfonyl)decahydro-1H,4H-pyrido[3,2,1-ij][1,6]naphthyridin-1-yl)butyl bis(2-chloroethyl)carbamate;

[0014] R2: 4-((1R,3aR,3a 1 S,10aR)-2-(4-chlorobenzyl)decahydro-1H,4H-pyrido[3,2,1-ij][1,6]naphthyridin-1-yl)butyl bis(2-chloroethyl)carbamate.

[0015] Preferably, the control conditions of the HPLC-DAD include:

[0016] The chromatographic column is a C18 column;

[0017] The detection wavelength is 210-220 nm;

[0018] The mobile phase A is 0.003-0.008 M sodium heptanesulfonate aqueous solution;

[0019] The mobile phase B is methanol;

[0020] The specific operation of gradient elution is:

[0021] 0~T1 min, mobile phase A content is A1, mobile phase B content is 100%-A1, flow rate is 0.3~0.8 mL / min;

[0022] T1~T2 min, mobile phase A content is A2, mobile phase B content is 100%-A2, flow rate is 0.6~1.2 mL / min;

[0023] T2~T3 min, mobile phase A content is A3, mobile phase B content is 100%-A3, flow rate is 0.3~1.0 mL / min;

[0024] Wherein, 7≤T1≤8, 9≤T2≤20, 12≤T3≤45; 2%≤A1≤7%, 4%≤A2≤10%, 2%≤A3≤8%, and A1≤A3<A2.

[0025] Preferably, the mobile phase A is 0.005M sodium heptanesulfonate aqueous solution.

[0026] The above technical solution has the beneficial effects that: by selecting the mobile phase, R1 and R2 have good separation effect and can be completely separated.

[0027] Preferably, the detection wavelength is 214nm.

[0028] The above technical solution has the beneficial effects that: R1 and R2 have strong absorption at 214nm wavelength and have no solvent interference. The DAD detector detects the peak type of R1 and R2 at 214nm wavelength to be the best, the response value is high, and baseline separation can be achieved with adjacent impurity peaks, which confirms the correctness of the wavelength selection.

[0029] Preferably, it further includes sample pretreatment, and the specific operation is as follows:

[0030] (1) Activate 200-300 mesh silica gel at 105°C for 12h, wet column with eluent petroleum ether-ethyl acetate 1:1, then wash the column with 4 times the column volume of petroleum ether-ethyl acetate 1:1, weigh the derivative synthesis crude product, sample with silica gel, elute with eluent, collect the target sample, remove the solvent by low-temperature rotary evaporator, and vacuum dry to remove the solvent residue to obtain a purified sample that can be directly administered, i.e. the test substance;

[0031] (2) Add methanol to dissolve the test substance, make up to volume, shake well, and prepare a test sample solution. According to the high-low concentration comparison method, first prepare a 100% concentration test sample solution, then dilute it 20 times to obtain a 5% concentration solution as a control solution, and calculate the purity of the test sample.

[0032] According to the above technical solution, compared with the prior art, the application provides a detection method for nitrogen mustard-containing sophoridine chlorophenyl derivative, which has the following beneficial effects:

[0033] (1) The application adopts HPLC-DAD method combined with ion pair reagent for detection, the peak type of R1 and R2 is best, the response value is high, and baseline separation can be achieved with adjacent impurity peaks, the specificity is good, the detection limit is low, the precision is good, the stability is high, and the detection can be realized quickly and accurately.

[0034] (2) The prior art only uses HPLC method to analyze R1 and R2 samples with strong ionization ability, and the retention time of the sample on the reversed-phase chromatographic column is very short or even not retained. Compared with pure HPLC, the application adopts HPLC-DAD method combined with ion pair reagent for detection, which can combine the ions on the analyte to form molecules with retention on the column, thereby increasing the retention time and improving the peak type, so that the sample can be detected.

[0035] (3) R1 and R2 have similar structure and similar polarity, and are not easy to separate. Compared with HPLC combined with other detection reagents, the application adopts HPLC-DAD combined with ion pair reagent method, which can change the polarity of R1 and R2 and their separation properties on the chromatographic column by introducing ion pairs, so that they can be completely separated.

[0036] (4) The application adopts HPLC-DAD method combined with ion pair reagent for detection, which effectively avoids the interference of solvent absorption on the measured compound, and eliminates the reverse peak phenomenon caused by the polarity difference between the sample and the solvent. Moreover, the method can separate ionized and non-ionized substances at the same time, more accurately separate the measured compound and unknown impurities, and ensure the accuracy of the purity detection result of the measured substance. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0038] Figure 1 For experiment 1, Agilent Zorbax Eclipse XDB-C18 type chromatographic column, parallax detector, methanol-water 70:30 as mobile phase, flow rate 1.0 mL / min, detection of R1 chromatogram.

[0039] Figure 2 For experiment 1, Agilent Zorbax Eclipse XDB-C18 type chromatographic column, parallax detector, methanol-water 70:30 as mobile phase, flow rate 1.0 mL / min, detection of R2 chromatogram.

[0040] Figure 3 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0041] Figure 4 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0042] Figure 5 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0043] Figure 5 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0044] Figure 6 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0045] Figure 6 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0046] Figure 7 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0047] Figure 7 The chromatogram of R2 was detected in Experiment 1 using an Agilent Zorbax Eclipse XDB-C18 column, a Parallax detector, methanol-water 95:5 as the mobile phase, and a flow rate of 0.6 mL / min.

[0048] Figure 8 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, mobile phase: acetonitrile-0.005M sodium heptanesulfonate in water 97:3, flow rate: 0.4 mL / min, wavelength: 211 nm, column temperature: 25 °C.

[0049] Figure 8 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, mobile phase: acetonitrile-0.005M sodium heptanesulfonate in water 97:3, flow rate: 0.4 mL / min, wavelength: 211 nm, column temperature: 25 °C.

[0050] Figure 9 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, mobile phase: acetonitrile-0.005M sodium heptanesulfonate in water 97:3, flow rate: 0.4 mL / min, wavelength: 211 nm, column temperature: 25 °C.

[0051] Figure 10 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, mobile phase: acetonitrile-0.005M sodium heptanesulfonate in water 95:5, flow rate: 0.8 mL / min, wavelength: 211 nm, column temperature: 30 °C.

[0052] Figure 11 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, 3D plot of full wavelength scan.

[0053] Figure 12 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, 3D plot of full wavelength scan of R1.

[0054] Figure 13 Column: Agilent Zorbax Eclipse XDB-C18, DAD detector, 3D plot of full wavelength scan of R2.

[0055] Figure 14Figure 3A is a chromatogram of a negative sample detected in Experiment 3 using an Agilent Zorbax Eclipse XDB-C18 column, DAD detector, methanol-0.005 M sodium heptanesulfonate in water 95:5 as mobile phase, 0.8 mL / min flow rate, 214 nm wavelength, and 30 °C column temperature.

[0056] Figure 14 Figure 3B is a chromatogram of R1 and R2 detected simultaneously in Experiment 3 using an Agilent Zorbax Eclipse XDB-C18 column, DAD detector, methanol-0.005 M sodium heptanesulfonate in water 95:5 as mobile phase, 0.8 mL / min flow rate, 214 nm wavelength, and 30 °C column temperature.

[0057] Figure 15 Figure 4A is a chromatogram of R1 and R2 detected in Experiment 4 using an Agilent Zorbax Eclipse XDB-C18 column, DAD detector, methanol-0.005 M sodium heptanesulfonate in water 96:4 as mobile phase, 0.6 mL / min flow rate.

[0058] Figure 15 Figure 4B is a chromatogram of R1 and R2 detected in Experiment 4 using methanol-0.005 M sodium heptanesulfonate in water 97:3 as mobile phase, 0.5 mL / min flow rate.

[0059] Figure 15 Figure 4C is a chromatogram of R1 and R2 detected in Experiment 4 using methanol-0.005 M sodium heptanesulfonate in water 98:2 as mobile phase, 0.4 mL / min flow rate.

[0060] Figure 16 Figure 5 is a chromatogram obtained using gradient elution condition 1) in Experiment 4.

[0061] Figure 17 Figure 6 is a chromatogram obtained using gradient elution condition 2) in Experiment 4.

[0062] Figure 18 Figure 7 is a chromatogram obtained from the multi-wavelength detection of R1 in Section 3.1 of Example 1.

[0063] Figure 19 Figure 8 is a chromatogram obtained from the multi-wavelength detection of R2 in Section 3.1 of Example 1.

[0064] Figure 20 Figure 9 is a chromatogram obtained in Section 3.2 of Example 1.

[0065] Figure 21 Figure 10 is a linear relationship plot obtained in Section 3.3 of Example 1.

[0066] Figure 22 Chromatogram for Rl quantitation limit in Example 1, Section 3.4.

[0067] Figure 23 Chromatogram for R2 quantitation limit in Example 1, Section 3.4.

[0068] Figure 24 Chromatogram for Rl detection limit in Example 1, Section 3.4.

[0069] Figure 25 Chromatogram for R2 detection limit in Example 1, Section 3.4. DETAILED DESCRIPTION

[0070] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0071] Experiment 1 RID detector, chromatographic column investigation

[0072] At first, it is not sure whether it has strong ultraviolet absorption, and it is not sure of the detection wavelength, so the general type of parallax detector (RID) is selected to detect the sample, and the C18 chromatographic column (Agilent Zorbax Eclipse XDB-C18 type chromatographic column, 5 μm, 4.6 mm x 250 mm) is selected as the stationary phase, and methanol-water (70:30) is selected as the mobile phase, and Rl ( Figure 1 ) and R2 ( Figure 2 ) are detected at a flow rate of 1.0 mL / min. Result: When the sample is detected by the differential detector, the baseline fluctuation is large and it is not easy to accurately quantify, the polarity difference between the sample and the solvent will cause the reverse peak phenomenon, and the polarity difference between Rl and R2 is small and they cannot be completely separated.

[0073] In order to improve the separation effect, the mobile phase is changed to methanol-water (95:5), and the flow rate is 0.6 mL / min for detection, and the result shows that the baseline is flat, but the reverse peak phenomenon still exists, Rl ( Figure 3 ) and R2 ( Figure 4 ) still cannot be completely separated, and it is found that the change of the proportion of the mobile phase cannot change the separation effect, and has little effect on the retention time of the sample, and it is likely that the sample is not easy to retain on the chromatographic column, so changing the proportion of the mobile phase and the flow rate cannot effectively separate the series of compounds.

[0074] In order to be able to accurately quantify and improve the reverse peak phenomenon, it is considered to use DAD detector for further investigation. Considering the characteristics of the sample not easy to retain on the C18 chromatographic column, it is proposed to replace the amino column for testing.

[0075] Since R1 and R2 have similar properties, R2 was chosen as the focus of the investigation. The detector and column were initially tested. An amino-based column (Hypersil NH2 HPLC column, 5 μm, 4.6 mm × 250 mm) and a DAD detector were used to investigate the detection method: acetonitrile-water (60:40) was used as the detection medium. Figure 5 Acetonitrile-water (3:97) Figure 6 The mobile phase was 1.0 mL / min, and the wavelength was 254 nm. Figure 5 China A, Figure 6 (middle A) and 211nm ( Figure 5 B, Figure 6 Detection was performed at wavelength B). Results showed that the sample absorbed very little at 254 nm, resulting in a low response value, making it unsuitable for detection. However, the sample exhibited strong UV absorption at 211 nm, with a high detection peak, making it suitable for detection. Furthermore, using a DAD detector resulted in a more stable baseline and prevented peak reversal for further investigation. Changing the mobile phase ratio did not significantly alter the retention time of the sample on the column, indicating that it remains poorly retained on amino columns. Figure 6 The condition for simultaneously detecting R1 and R2 ( Figure 7 It was found that R1 and R2 were still inseparable. Therefore, there was no essential difference in sample analysis between using a C18 column or an amino column (Elite Hypersil NH2 liquid chromatography column, 5μm, 4.6mm×250mm).

[0076] In summary: A DAD detector was selected for sample detection; a C18 column was proposed, and ion-pairing reagents were used as the mobile phase for further investigation.

[0077] Experiment 2 Mobile Phase Selection

[0078] Using an aqueous solution of sodium heptanesulfonate as the mobile phase, and combining it with organic solvents such as acetonitrile and methanol, the following experiments were conducted:

[0079] A C18 column (Agilent Zorbax Eclipse XDB-C18, 5 μm, 4.6 mm × 250 mm) was used as the stationary phase, with acetonitrile-0.005 M sodium heptanesulfonate aqueous solution (97:3). The flow rate was 0.4 mL / min, wavelength 211 nm, and column temperature 25 °C. R1, R2, and the negative sample methanol were detected. Results: The retention times and separation efficiency of R1 and R2 were slightly improved. Figure 8 (B) At this time, the negative sample methanol ( Figure 8 If A) is interfered with, this method is not applicable and needs to be improved.

[0080] The mobile phase was changed to methanol-0.005M sodium heptanesulfonate aqueous solution (97:3) as the mobile phase, the flow rate was 0.4 mL / min, the wavelength was 211 nm, and the column temperature was 25°C. It was found that R1 and R2 were better separated than acetonitrile (Example 1). Figure 9

[0081] The method was further improved. The mobile phase was methanol-0.005M sodium heptanesulfonate aqueous solution (95:5), the flow rate was 0.8 mL / min, the wavelength was 211 nm, and the column temperature was 30°C. The results showed that R1 and R2 could be completely separated (Example 3). Figure 10

[0082] Therefore, methanol and ion-pair reagent were finally selected as the mobile phase.

[0083] Experiment 3: Investigation of detection wavelength

[0084] The sample detection was performed by 3D chromatogram analysis using full wavelength scanning from 190 nm to 300 nm to determine the optimal detection wavelength. The solvent had strong absorption between 190 nm and 214 nm, but the absorption was significantly weakened at 214 nm and approached the baseline (Example 2). Figure 11 The maximum absorption wavelength of R1 was 202 nm, but the solvent absorption peak interfered with the detection results at this wavelength. However, there was no solvent interference when the detection wavelength was greater than or equal to 214 nm (Example 2). Figure 12 The maximum absorption wavelength of R2 was 214 nm, and there was no solvent interference at this wavelength (Example 2). Figure 13 Therefore, the wavelength of 214 nm, at which R1 and R2 had strong absorption and no solvent interference, was selected as the final detection wavelength to improve the accuracy of the detection results.

[0085] Repetition

[0086] The detection wavelength was changed to 214 nm under the conditions of Example 2. It was found that the chromatographic peak shape was sharper at 214 nm compared to 211 nm, the response value of the R2 chromatographic peak was higher, and the response value of the R1 chromatographic peak was similar (Example 3). Figure 10 At 214 nm, there were no chromatographic peaks at the positions corresponding to R1 and R2 in the negative sample, and the baseline fluctuation value mAU was below 4, indicating that there was no interference from the negative sample at this wavelength. Therefore, this method was accurate and reliable (Example 3). Figure 14 Figure 14 Experiment 4: Investigation of mobile phase ratio and flow rate

[0087] The mobile phase was changed to methanol-0.005M sodium heptanesulfonate aqueous solution (95:5), the flow rate was 0.8 mL / min, the wavelength was 211 nm, and the column temperature was 30°C. The results showed that R1 and R2 could be completely separated (Example 3).

[0088] ​​​Using C18 column (Agilent Zorbax Eclipse XDB-C18 column, 5 μm, 4.6 mm x 250 mm) as stationary phase, wavelength 214 nm, column temperature 30 °C, respectively investigate: 1) methanol-0.005 M sodium heptanesulfonate aqueous solution (96:4), flow rate 0.6 mL / min Figure 15 Middle A); 2) methanol-0.005 M sodium heptanesulfonate aqueous solution (97:3), flow rate 0.5 mL / min Figure 15 Middle B); 3) methanol-0.005 M sodium heptanesulfonate aqueous solution (98:2), flow rate 0.4 mL / min Figure 15 Middle C). Each condition can determine R1, R2, but the peak type has tailing phenomenon.

[0089] Further investigate two gradient elution conditions:

[0090] 1) Using C18 column (Agilent Zorbax Eclipse XDB-C18 column, 5 μm, 4.6 mm x 250 mm) as stationary phase, wavelength 214 nm, column temperature 30 °C, gradient elution program as follows. Get Figure 16 .

[0091] Time min Mobile phase A (%) Mobile phase B (%) Flow rate mL / min 0-8 2 98 0.4 8-9 6 94 1.0 9-12 2 98 0.4

[0092] 2) Using C18 column (Agilent Zorbax Eclipse XDB-C18 column, 5 μm, 4.6 mm x 250 mm) as stationary phase, wavelength 214 nm, column temperature 30 °C, gradient elution program as follows. Get Figure 17 .

[0093] Time min Mobile phase A (%) Mobile phase B (%) Flow rate mL / min 0-7 2 98 0.4 7-12 5 95 1.1 12-15 2 95 0.4

[0094] Results: The peak type of the chromatogram is the best using gradient elution condition 2), and the method can quickly and effectively detect the target compound.

[0095] Example 1 R1 and R2 detection

[0096] The detection method described in the application is as follows:

[0097] 1, sample pretreatment

[0098] The 200-300 mesh silica gel is activated at 105 °C for 12 h, and then packed into a column using eluent petroleum ether-ethyl acetate 1:1 by wet method, and then washed with 4 times the column volume of petroleum ether-ethyl acetate 1:1. An appropriate amount of crude synthetic product is weighed, mixed with silica gel, and then packed into a column. The target sample is eluted with eluent, and then collected. After low-temperature rotary evaporation to remove the solvent and vacuum drying to remove the solvent residue, R1 and R2 samples for detection are obtained.

[0099] 2. Sample purity determination

[0100] Instrument: Agilent 1260 high performance liquid chromatograph

[0101] Detector: Agilent DAD detector

[0102] Chromatographic column: C18 bonded phase (Agilent Eclipse Plus C18 4.6*250mm; 5μm)

[0103] Mobile phase A: 0.005M sodium heptanesulfonate aqueous solution was used as mobile phase A.

[0104] Mobile phase B: methanol was used as mobile phase B.

[0105] The gradient elution program is as follows:

[0106] Time min Mobile phase A (%) Mobile phase B (%) Flow rate mL / min 0-7 2 98 0.4 7-12 5 95 1.1 12-15 2 95 0.4

[0107] Wherein:

[0108] Injection volume: 2μL

[0109] 100% test solution: 10mg of R1 and R2 to be tested was accurately weighed into a 5mL volumetric flask, dissolved with methanol, and made up to volume, shaken well, and used as the test solution.

[0110] 5% test solution: 5mL of the above 100% test solution was accurately measured into a 100mL volumetric flask, made up to volume with methanol, shaken well, and used as the 5% test solution.

[0111] 2μL of the above 100% test solution and 5% test solution was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded. The purity of the test sample was calculated according to the high-low concentration method.

[0112] Calculation formula (%) = (A x Ci x D) / (Ai x m) x 100%

[0113] Wherein, A is the peak area of the 100% test solution, Ai is the peak area of the diluted test solution, D is the dilution factor, and m is the mass of the test sample.

[0114] 3. Method validation

[0115] 3.1 Determination of detection wavelength

[0116] In this study, the absorption intensity and peak shape of R1 and R2 were investigated by multi-wavelength detection. The results showed that the DAD detector detected the peak shape of R1 and R2 at a wavelength of 214nm, which was the best and had a high response value, and could be separated from the adjacent impurity peaks, confirming the correctness of the wavelength selection. Figure 18-19).

[0117] 3.2 Specificity Examination

[0118] Mixed sample solutions containing R1 (0.845 mg / mL) and R2 (0.833 mg / mL) and negative sample solutions were prepared using methanol as solvent and diluent and injected into the liquid chromatograph, respectively. The results are shown in the figure. Figure 20 The negative sample showed no interference in the positions of the R1 and R2 peaks in the mixed sample solution, indicating that the method has good specificity.

[0119] 3.3 Linearity and Range

[0120] The purity of the samples was calculated using a high-concentration method, and linearity was verified.

[0121] Table 1 Linearity and Range

[0122]

[0123] Conclusion: R1 is within the range of 0.021 mg / mL to 1.056 mg / mL, i.e., the content is within the range of 0.11 ug to 5.28 ug. 2 The linear relationship was 0.9999 (r = 0.9995); the R² value was in the range of 0.021 mg / mL to 1.041 mg / mL, i.e., the concentration was in the range of 0.11 ug to 5.21 ug. 2 The linear relationship is 0.9983 (r = 0.9991). See Table 1 and... Figure 21 .

[0124] 3.4 Limit of Detection and Limit of Quantification

[0125] The limit of detection is defined as the mass corresponding to a signal-to-noise ratio of 3, and the limit of quantitation is defined as the mass corresponding to a signal-to-noise ratio of 10.

[0126] The chromatogram was determined and recorded according to the method in "Example 1" ( Figure 22 and Figure 23 The quantitation limits for R1 and R2 calculated by this method are 30.00 and 29.50 ng, respectively.

[0127] Test detection limit standard solution: Accurately weigh compounds R1 and R2, place them in a 100 mL volumetric flask, add methanol to dissolve them, dilute to the mark, and shake well. Then accurately measure 1 mL and place it in a 100 mL volumetric flask, add diluent and dilute to the mark, and shake well to obtain (R1: 0.0009 mg / mL; R2: 0.0010 mg / mL). Determine according to the method in "Example 1" and record the chromatogram. Figure 24 and Figure 25 The detection limits of this method for R1 and R2 were calculated to be 4.50 and 5.00 ng, respectively.

[0128] 3.5 Precision

[0129] The RSD of the peak area values of R1 and R2 in the mixed sample was 0.49% and 0.65%, respectively, both less than 2%, and the precision was good. The results are shown in Table 2.

[0130] Table 2 Precision determination

[0131]

[0132] 3.6 Stability

[0133] The RSD of the peak area values of R1 and R2 in the mixed sample within 24 h was 0.75% and 0.71%, respectively, both less than 2%, indicating that the test solution was stable at room temperature for 24 h. The results are shown in Table 3.

[0134] Table 3 Stability determination

[0135]

[0136] 3.7 Sample determination

[0137] The sample solution (6 portions) was prepared according to "Example 1", the chromatogram was recorded, and the content of R1 and R2 in the sample was calculated, respectively. The content of R1 in the sample was 85.93%, and the content of R2 in the sample was 96.43%, and the determination results are shown in Table 4.

[0138] Table 4 Sample determination

[0139]

[0140]

[0141] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be mutually referred to. For the solutions disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple, and the relevant parts can be referred to the method part.

[0142] The above description of the disclosed embodiments enables a person skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting a nitrogen mustard-containing sophoridine chlorophenyl derivative, characterized by, The purity of the derivative to be tested is detected by using HPLC-DAD method combined with ion pair reagent; The structural formula of the derivative is as follows: ； The control conditions of the HPLC-DAD include: The chromatographic column is a C18 column; The detection wavelength is 210-220 nm; The mobile phase A is 0.003-0.008 M sodium heptanesulfonate aqueous solution; The mobile phase B is methanol; The specific operation of the gradient elution is as follows: 0~T1min, the content of the mobile phase A is A1, the content of the mobile phase B is 100%-A1, and the flow rate is 0.3~0.8mL / min; T1~T2 min, the content of the mobile phase A is A2, the content of the mobile phase B is 100%-A2, and the flow rate is 0.6~1.2mL / min; T2~T3 min, the content of the mobile phase A is A3, the content of the mobile phase B is 100%-A3, and the flow rate is 0.3~1.0mL / min; Wherein, 7≤T1≤8, 9≤T2≤20, 12≤T3≤45; 2%≤A1≤7%, 4%≤A2≤10%, 2%≤A3≤8%, and A1≤A3<A2; The sample pretreatment is also included, and the specific operation is as follows: (1) 200~300 mesh silica gel is activated at 105℃ for 12h, and then column packing is carried out by using eluent petroleum ether-ethyl acetate 1:1 by wet method, and then the column is washed with 4 times the column volume of petroleum ether-ethyl acetate 1:1; the crude product of the derivative synthesis is weighed, and then the silica gel is mixed with the sample for column packing, and then the sample is eluted by using the eluent, and then the target sample is collected; the solvent is removed by using a rotary evaporator at low temperature, and then the solvent residue is removed by vacuum drying, so that a purified sample which can be directly administered, i.e. the to-be-tested substance, is obtained; (2) methanol is added to dissolve the to-be-tested substance, and then the volume is made up and shaken uniformly to obtain a test solution; according to the high-low concentration comparison method, the test solution with a concentration of 100% is prepared first, and then the test solution is diluted by 20 times to obtain a solution with a concentration of 5% as a control solution, and then the purity of the test solution is calculated.

2. The method for detecting sophoridine alkaloid chlorophenyl derivatives in nitrogen-containing mustard according to claim 1, characterized in that, The derivative is one of R1 and R2 or a mixture of R1 and R2; The structural formula of R1 and R2 is as follows: R1 R2.

3. The method for detecting sophoridine alkaloid chlorophenyl derivatives in nitrogen-containing mustard according to claim 1, characterized in that, The mobile phase A is 0.005M sodium heptanesulfonate aqueous solution.

4. The method for detecting sophoridine alkaloid chlorophenyl derivatives in nitrogen-containing mustard according to claim 1, characterized in that, The detection wavelength is 214 nm.

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