A method for detecting multiple solvent residues in a sample using headspace gas chromatography

Abstract

The present invention discloses a method for detecting multiple solvent residues in a sample by headspace gas chromatography, the method comprising: (1) preparing a marking solution and a separation solution: preparing a mixed solvent standard solution of methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, n-hexane, N-N dimethylformamide, n-heptane and tetrahydrofuran, and diluting the mixed solvent standard solution with DMSO into 20 times, 200 times, 1000 times, 2000 times and 10000 times of marking solution; wherein the solution diluted 20 times with DMSO is also used as a separation solution; (2) dissolving the sample to be detected with DMSO to prepare a sample solution to be detected; and using headspace gas chromatography to detect the marking solution and the sample solution to be detected in sequence. The method of the present invention has good specificity between each solvent peak, and there is no interference from other impurity peaks at the peak position of each target solvent peak, which provides an accurate and efficient detection method for simultaneously detecting nine residual solvents, filling the defects of the prior art.

Description

Technical Field

[0001] The invention relates to a method for detecting residual solvents in a sample, and in particular to a method for detecting residual solvents in a sample by using headspace gas chromatography. Background Art

[0002] Phosphoramidite monomers play an important role in the synthesis of small nucleic acid APIs. Their purity requirement is usually ≥99.0%, and due to their complex structure, quality control is difficult. At present, there are residual solvents in products such as phosphoramidite monomers and phosphates. The main methods for detecting residual solvents are nuclear magnetic resonance, high performance liquid chromatography and headspace gas chromatography.

[0003] However, the accuracy of NMR judgment in the above method is relatively poor. High performance liquid chromatography can only detect some solvents with ultraviolet absorption wavelengths between 210 and 400 nm. Solvents without ultraviolet absorption require replacement of detectors, such as ELSD and CAD detectors. In addition, peaks will appear in the sample, which may be affected by impurities and the main peak.

[0004] At present, the products we produce, such as phosphoramidite monomers, delivery system GalNAc series and fluorescent markers, are likely to use more than 4 kinds of solvents, such as methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, n-hexane, N-dimethylformamide (hereinafter referred to as DMF), n-heptane and tetrahydrofuran. If the existing methods are used, there are some problems with nuclear magnetic resonance spectroscopy or liquid chromatography with ELSD or CAD detectors, as follows:

[0005] (1) The position of the solvent peak in the H NMR spectrum is often interfered by the sample peak, and there is a large deviation between the theoretical chemical shift and the actual hydrogen chemical shift;

[0006] (2) Use ELSD or CAD detectors to detect solvent residues. Because these two types of detectors have very low sensitivity, they cannot accurately determine the solvent residue in the product. In addition, the sample and impurities in the sample will also have peaks, and it cannot be guaranteed that the solvent peak can be completely separated from the sample and impurity peaks. In addition, because the solvent residue in the sample is generally between 0.05% and 15%, it is necessary to inject the sample at a high concentration (generally 100 mg / mL), which can easily cause damage to the equipment.

[0007] Therefore, the use of the original method has brought many difficulties to the accurate and rapid detection of residual solvents in samples, which is a difficult problem that needs to be solved urgently.

[0008] As for headspace gas chromatography, existing literature has similar methods for detecting solvent residues, but generally only one, or two or three solvent residues can be detected at the same time, and it is difficult to detect multiple solvent residues at the same time. Summary of the invention

[0009] The purpose of the present invention is to provide a method for detecting multiple solvent residues in a sample by headspace gas chromatography, which solves the problem that the existing method cannot simultaneously detect the residues of nine solvents, namely methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, n-hexane, N-dimethylformamide, n-heptane and tetrahydrofuran, in the sample. The method can provide a lower detection limit, and the specificity between the solvent peaks is good. The peak position of each target solvent peak is not interfered by other impurity peaks. This provides an accurate and efficient detection method for simultaneously detecting nine residual solvents, and fills the defects of the prior art.

[0010] In order to achieve the above object, the present invention provides a method for detecting multiple solvent residues in a sample by headspace gas chromatography, the method comprising:

[0011] (1) Prepare the marking solution and separation solution

[0012] Prepare a mixed solvent standard solution of methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, n-hexane, N-dimethylformamide, n-heptane and tetrahydrofuran, and dilute it with DMSO to 20 times, 200 times, 1000 times, 2000 times and 10000 times mark line solutions; wherein, the solution diluted with DMSO to 20 times is also used as the separation solution;

[0013] (2) Use headspace gas chromatography to test the sample

[0014] The sample to be tested is dissolved in DMSO to prepare a sample solution to be tested; the marking solution and the sample solution to be tested are detected in sequence by headspace gas chromatography and a detector; after the gas chromatography detection of the marking solution, a standard curve of the peak area-concentration of each solvent is calculated; the solvent residues of the sample to be tested are respectively introduced into the corresponding standard curves to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested to obtain the relative percentage content of the solvent in the sample.

[0015] Among them, the conditions of the headspace gas chromatography are: the chromatographic column uses an ultra-high inert gas capillary chromatographic column; the column temperature is programmed to rise to 45~55°C, maintained for 4~10 min, then increased to 130~140°C at 10~12°C / min, maintained for 0~5 min, and then increased to 200~220°C at 15~20°C / min, maintained for 2~5 min; the carrier is an inert gas; the injection volume is 1~5 μL; the headspace equilibrium oil bath temperature is 80~160°C; the valve box temperature is 150°C; the injection port split ratio is 15~20:1; the injection port temperature is 150~220°C; the detector temperature is 200~250°C; and the headspace injector pipeline temperature is 160°C.

[0016] Preferably, the filler of the ultra-high inert gas capillary chromatography column is selected from: phenyl-methyl-polysiloxane, trifluoropropyl-methyl-polysiloxane, cyanopropyl-phenyl-dimethylpolysiloxane or dimethylpolysiloxane.

[0017] More preferably, the filler of the ultra-high inert gas capillary chromatography column is selected from: 50% phenyl and 50% dimethylpolysiloxane, 6% cyanopropylphenyl and 94% dimethylpolysiloxane.

[0018] Preferably, the ultra-high inert gas phase capillary chromatography column is selected from CP-Volamine or ZB-1.

[0019] Preferably, the inert gas is selected from high-purity nitrogen or high-purity helium.

[0020] Preferably, the flow rate of the carrier is 1-2 mL / min.

[0021] Preferably, in the headspace gas chromatography conditions, the sample preheating equilibrium time is 25 to 32 min.

[0022] Preferably, in the conditions of the headspace gas chromatography, the injection time is 45 s.

[0023] Preferably, the equipment used in the headspace gas chromatography is Agilent 7890B, and the headspace sampler used is Agilent G1888 headspace sampler; or, the equipment used in the headspace gas chromatography is Thermo TRACE1600, and the headspace sampler used is TriPlus500 headspace sampler.

[0024] Preferably, the detector is selected from a FID, ECD, NPD or FPD detector.

[0025] The method of detecting multiple solvent residues in a sample by headspace gas chromatography of the present invention has the following advantages:

[0026] (1) The method of the present invention can provide a lower detection limit, wherein methanol: 6.028ppm; ethanol: 6.074ppm; acetonitrile: 6.012ppm; dichloromethane: 10.03ppm; ethyl acetate: 2.978ppm; n-hexane: 3.03ppm; n-heptane: 1.55ppm; tetrahydrofuran: 4.93ppm; DMF: 3.00ppm, the quantitative limits of the solvent content of the sample to be tested are respectively methanol: 0.003%; ethanol: 0.003%; acetonitrile: 0.003%; dichloromethane: 0.005%; ethyl acetate: 0.0015%; n-hexane: 0.0015%; n-heptane: 0.0008%; tetrahydrofuran: 0.0025%; DMF: 0.0015%, and the results are accurate and reliable;

[0027] (2) The method of the present invention has good specificity between the solvent peaks, and there is no interference from other impurity peaks at the peak position of each target solvent peak, which provides an accurate and efficient detection method for the simultaneous detection of nine residual solvents, filling the defects of the prior art;

[0028] (3) The method of the present invention can detect the above nine residual solvents in products such as phosphoramidite monomers, phosphoramidite raw materials, delivery systems, nucleic acids, phosphorus reagents and fluorescent markers;

[0029] (4) All materials involved in the method of the present invention are commercially available, easy to obtain, with a short synthesis route and simple operation. They are suitable for simultaneous detection of most solvents, saving manpower and equipment costs. Currently, there is no method that can simultaneously detect multiple conventional solvent components. This method has platform significance for the research and application of simultaneous detection of multiple components in the detection industry, and also provides a new idea for the detection industry. This method can simultaneously detect multiple components + other substrates. Currently, there is no literature or patent reporting on this method. It is suitable for various industries such as chemistry, food, and traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a chromatogram of the marking solution of Example 1 of the present invention.

[0031] Figure 2 This is a sample detection diagram of Example 1 of the present invention.

[0032] Figure 3 This is a chromatogram of the marking solution of Example 2 of the present invention.

[0033] Figure 4 This is a sample detection diagram of Example 2 of the present invention.

[0034] Figure 5 This is the chromatogram of the marking solution of Example 3 of the present invention.

[0035] Figure 6 This is a sample detection diagram of Example 3 of the present invention.

[0036] Figure 7 This is a chromatogram of the marking solution of Example 4 of the present invention.

[0037] Figure 8 This is a sample detection diagram of Example 4 of the present invention.

[0038] Fig. 9 This is a sample detection diagram of Example 5 of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Example 1 Detection of Solvent Residues in Phosphoramidite Monomer Products

[0041] The residual solvent in the phosphoramidite monomer product was detected by headspace gas chromatography using an external standard method, as follows:

[0042] (1) Preparation of phosphoramidite monomer solution

[0043] Accurately weigh 100.02 mg of phosphoramidite monomer (DMT-2'-OMe-G(dmf)-CE-Phosphoramidite) and place it in a 20 mL headspace bottle. Use a calibrated pipette to pipette 1 mL of DMSO and add it to the phosphoramidite monomer to dissolve the phosphoramidite monomer for standby use.

[0044] (2) Preparation of marking solution and separation solution

[0045] Prepare the mother solutions of each reagent (10 mL): 60.28 mg / mL methanol, 60.74 mg / mL ethanol, 60.12 mg / mL acetonitrile, 100.27 mg / mL dichloromethane, 29.78 mg / mL ethyl acetate, 30.27 mg / mL n-hexane, 30.01 mg / mL N-dimethylformamide, 15.49 mg / mL n-heptane, and 49.34 mg / mL tetrahydrofuran.

[0046] Prepare the standard solution: Take 0.1 mL of the reagent mother solution of the above concentration, add it to a 10 mL volumetric flask, dilute it to the scale with DMSO, shake it well, and use it as the mother solution of the mixed solvent standard solution, and then dilute it step by step. The specific dilution method is as follows:

[0047] Dilution 20X (also used as separation solution): Take 1 mL of the mother solution of the above mixed solvent standard solution, place it in a 20 mL volumetric flask, and then dilute it to the scale with DMSO to obtain a diluted 20X solution;

[0048] Dilution 200X: Place 1 mL of the diluted 20X solution in a 10 mL volumetric flask, and then dilute to the mark with DMSO to obtain a diluted 200X solution;

[0049] Dilution 1000X: Place 2 mL of the 200X diluted solution in a 10 mL volumetric flask, and then dilute to the mark with DMSO to obtain a 1000X diluted solution;

[0050] Dilution 2000X: Place 1 mL of the diluted 200X solution in a 10 mL volumetric flask, and then dilute to the mark with DMSO to obtain a diluted 2000X solution;

[0051] Dilution 10000X: Place 1 mL of the diluted 1000X solution in a 10 mL volumetric flask, and then dilute to the mark with DMSO.

[0052] (3) Determination of residual solvent in phosphoramidite monomers

[0053] The conditions of headspace gas chromatography as shown in Table 1 were used to detect the calibration solution and the sample solution to be detected in sequence. The equipment was Agilent 7890B, and the headspace sampler used was Agilent G1888 headspace sampler.

[0054] Table 1 shows the headspace gas chromatography conditions in Example 1 of the present invention.

[0055]

[0056] After the gas chromatography detection of the marked solution, the peak area-concentration standard curve of each solvent is calculated; the solvent residue of the sample to be tested is respectively inserted into the corresponding standard curve to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested, which is the relative percentage content of the solvent in the sample.

[0057] Through the above detection, the information of the standard curve of each reagent is obtained as follows:

[0058] 1) Standard curve of methanol (F0002):

[0059] Retention time: 2.784min;

[0060] Correlation coefficient: 0.99996;

[0061] Residual standard deviation: 1.78785;

[0062] Standard curve formula: y = m*x, where m=132.82658, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0063] 2) Standard curve of ethanol (F0005):

[0064] Retention time: 3.961min;

[0065] Correlation coefficient: 0.99995;

[0066] Residual standard deviation: 2.46785;

[0067] Standard curve formula: y = m*x, where m=162.05153, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0068] 3) Standard curve of acetonitrile (F0008):

[0069] Retention time: 4.364min;

[0070] Correlation coefficient: 0.99998;

[0071] Residual standard deviation: 2.02513;

[0072] Standard curve formula: y = m*x, where m=222.42860, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0073] 4) Standard curve of dichloromethane (F0001):

[0074] Retention time: 6.039min;

[0075] Correlation coefficient: 0.99998;

[0076] Residual standard deviation: 2.53309;

[0077] Standard curve formula: y = m*x, where m=153.29732, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0078] 5) Standard curve of ethyl acetate (F0007):

[0079] Retention time: 8.300min;

[0080] Correlation coefficient: 1.00000;

[0081] Residual standard deviation: 0.43008;

[0082] Standard curve formula: y = m*x, where m=548.66070, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0083] 6) Standard curve of n-hexane (F0147):

[0084] Retention time: 8.413min;

[0085] Correlation coefficient: 0.99998;

[0086] Residual standard deviation: 5.38811;

[0087] Standard curve formula: y = m*x, where m=1259.50967, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0088] 7) Standard curve of tetrahydrofuran (F0012):

[0089] Retention time: 8.883min;

[0090] Correlation coefficient: 1.00000;

[0091] Residual standard deviation: 2.01242;

[0092] Standard curve formula: y = m*x, where m=1069.66254, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0093] 8) Standard curve of n-heptane (F0006):

[0094] Retention time: 11.102min;

[0095] Correlation coefficient: 1.00000;

[0096] Residual standard deviation: 1.78831;

[0097] Standard curve formula: y = m*x, where m=1642.15317, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0098] 9) Standard curve of DMF (F0014):

[0099] Retention time: 12.136min;

[0100] Correlation coefficient: 0.99985;

[0101] Residual standard deviation: 3.07273;

[0102] Standard curve formula: y = m*x, where m=23.52080, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0103] The test results are shown in Tables 2~3 and Figure 1~2 .

[0104] Table 2 shows the chromatographic peak information of the marking solution

[0105]

[0106] Table 3 is the chromatographic peak information of phosphoramidite monomer solution

[0107]

[0108] Example 2 Detection of Solvent Residues in Phosphoramidite Monomer Raw Material Products

[0109] The residual solvent in the phosphoramidite monomer raw material product was detected by headspace gas chromatography using an external standard method, as follows:

[0110] (1) Preparation of phosphoramidite monomer raw material product solution

[0111] Accurately weigh 110.22 mg of the phosphoramidite raw material (DMT-2'-O-TBDMS-C(Ac)) and place it in a 20 mL headspace bottle. Use a calibrated pipette to transfer 1 mL of DMSO and add it to the phosphoramidite monomer to dissolve the phosphoramidite monomer for later use.

[0112] (2) Preparation of marking solution and separation solution

[0113] Prepare the mother solutions of each reagent (10 mL): methanol 59.66 mg / mL, ethanol 59.96 mg / mL, acetonitrile 60.22 mg / mL, dichloromethane 100.01 mg / mL, ethyl acetate 29.98 mg / mL, n-hexane 30.06 mg / mL, N-dimethylformamide 30.00 mg / mL, n-heptane 15.26 mg / mL, and tetrahydrofuran 50.32 mg / mL.

[0114] The preparation and dilution of the mother solution of the mixed solvent standard solution are the same as the steps in Example 1, except that the concentrations of the mother solutions of the reagents are different.

[0115] (3) Determination of residual solvent in phosphoramidite monomer raw materials

[0116] The conditions of headspace gas chromatography as shown in Table 4 were used to detect the calibration solution and the sample solution to be detected in sequence. The equipment was Agilent 7890B, and the headspace sampler used was Agilent G1888 headspace sampler.

[0117] Table 4 shows the headspace gas chromatography conditions in Example 2 of the present invention.

[0118]

[0119] After the gas chromatography detection of the marked solution, the peak area-concentration standard curve of each solvent is calculated; the solvent residue of the sample to be tested is respectively inserted into the corresponding standard curve to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested, which is the relative percentage content of the solvent in the sample.

[0120] Through the above detection, the information of the standard curve of each reagent is obtained as follows:

[0121] 1) Standard curve of methanol (F0002):

[0122] Retention time: 3.001min;

[0123] Correlation coefficient: 0.99994;

[0124] Residual standard deviation: 1.13327;

[0125] Standard curve formula: y = m*x, where m=69.41562, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0126] 2) Standard curve of ethanol (F0005):

[0127] Retention time: 4.239min;

[0128] Correlation coefficient: 0.99996;

[0129] Residual standard deviation: 1.27623;

[0130] Standard curve formula: y = m*x, where m=90.83439, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0131] 3) Standard curve of acetonitrile (F0008):

[0132] Retention time: 4.644min;

[0133] Correlation coefficient: 0.99999;

[0134] Residual standard deviation: 0.86389;

[0135] Standard curve formula: y = m*x, where m=123.46858, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0136] 4) Standard curve of dichloromethane (F0001):

[0137] Retention time: 6.311min;

[0138] Correlation coefficient: 1.00000;

[0139] Residual standard deviation: 0.68066;

[0140] Standard curve formula: y = m*x, where m=83.73798, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0141] 5) Standard curve of ethyl acetate (F0007):

[0142] Retention time: 8.541min;

[0143] Correlation coefficient: 0.99994;

[0144] Residual standard deviation: 2.54783;

[0145] Standard curve formula: y = m*x, where m=302.36307, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0146] 6) Standard curve of n-hexane (F0147):

[0147] Retention time: 8.652min;

[0148] Correlation coefficient: 0.99918;

[0149] Residual standard deviation: 38.76095;

[0150] Standard curve formula: y = m*x, where m=1242.43325, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0151] 7) Standard curve of tetrahydrofuran (F0012):

[0152] Retention time: 9.123min;

[0153] Correlation coefficient: 0.99999;

[0154] Residual standard deviation: 2.80799;

[0155] Standard curve formula: y = m*x, where m=551.38104, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0156] 8) Standard curve of n-heptane (F0006):

[0157] Retention time: 11.312min;

[0158] Correlation coefficient: 0.99976;

[0159] Residual standard deviation: 11.08198;

[0160] Standard curve formula: y = m*x, where m=1314.61665, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0161] 9) Standard curve of DMF (F0014):

[0162] Retention time: 12.343min;

[0163] Correlation coefficient: 0.99991;

[0164] Residual standard deviation: 1.26103;

[0165] Standard curve formula: y = m*x, where m=12.44128, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0166] The test results are shown in Tables 5~6 and Figure 3~4 .

[0167] Table 5 shows the chromatographic peak information of the marking solution

[0168]

[0169] Table 6 is the chromatographic peak information of the phosphoramidite monomer raw material product solution

[0170]

[0171] Example 3 Detection of Solvent Residues in Drug Delivery System Products

[0172] The residual solvents in the delivery system products were tested by headspace gas chromatography using an external standard method as follows:

[0173] (1) Preparation of delivery system sample solution

[0174] Accurately weigh 99.60 mg of the drug delivery system sample (GalNAc-L96, i.e., triantennary N-acetylgalactosamine) and place it in a 20 mL headspace bottle. Use a calibrated pipette to transfer 1 mL of DMSO to the delivery system sample to dissolve the delivery system sample for later use.

[0175] (2) Preparation of marking solution and separation solution

[0176] Prepare the mother solutions of each reagent (10 mL): 59.92 mg / mL methanol, 60.01 mg / mL ethanol, 60.62 mg / mL acetonitrile, 99.98 mg / mL dichloromethane, 30.20 mg / mL ethyl acetate, 29.92 mg / mL n-hexane, 30.01 mg / mL N-dimethylformamide, 15.03 mg / mL n-heptane, and 50.02 mg / mL tetrahydrofuran.

[0177] The preparation and dilution of the mother solution of the mixed solvent standard solution are the same as the steps in Example 1, except that the concentrations of the mother solutions of the reagents are different.

[0178] (3) Determination of residual solvent in delivery system samples

[0179] The conditions of headspace gas chromatography as shown in Table 7 below were used to detect the mark line solution and the sample solution to be detected in sequence. The equipment was Agilent 7890B, and the headspace sampler used was Agilent G1888 headspace sampler.

[0180] Table 7 shows the headspace gas chromatography conditions in Example 3 of the present invention.

[0181]

[0182] After the gas chromatography detection of the marked solution, the peak area-concentration standard curve of each solvent is calculated; the solvent residue of the sample to be tested is respectively inserted into the corresponding standard curve to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested, which is the relative percentage content of the solvent in the sample.

[0183] Through the above detection, the information of the standard curve of each reagent is obtained as follows:

[0184] 1) Standard curve of methanol (F0002):

[0185] Retention time: 2.810min;

[0186] Correlation coefficient: 0.99993;

[0187] Residual standard deviation: 2.76500;

[0188] Standard curve formula: y = m*x, where m=154.70703, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0189] 2) Standard curve of ethanol (F0005):

[0190] Retention time: 4.000min;

[0191] Correlation coefficient: 0.99994;

[0192] Residual standard deviation: 3.12473;

[0193] Standard curve formula: y = m*x, where m=187.58178, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0194] 3) Standard curve of acetonitrile (F0008):

[0195] Retention time: 4.399min;

[0196] Correlation coefficient: 0.99999;

[0197] Residual standard deviation: 1.74666;

[0198] Standard curve formula: y = m*x, where m=261.94062, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0199] 4) Standard curve of dichloromethane (F0001):

[0200] Retention time: 6.075min;

[0201] Correlation coefficient: 1.00000;

[0202] Residual standard deviation: 0.15659;

[0203] Standard curve formula: y = m*x, where m=178.88165, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0204] 5) Standard curve of ethyl acetate (F0007):

[0205] Retention time: 8.334min;

[0206] Correlation coefficient: 0.99999;

[0207] Residual standard deviation: 2.08200;

[0208] Standard curve formula: y = m*x, where m=611.67100, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0209] 6) Standard curve of n-hexane (F0147):

[0210] Retention time: 8.447min;

[0211] Correlation coefficient: 1.00000;

[0212] Residual standard deviation: 7.66775;

[0213] Standard curve formula: y = m*x, where m=4315.49487, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0214] 7) Standard curve of tetrahydrofuran (F0012):

[0215] Retention time: 8.917min;

[0216] Correlation coefficient: 1.00000;

[0217] Residual standard deviation: 2.42063;

[0218] Standard curve formula: y = m*x, where m=1095.82555, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0219] 8) Standard curve of n-heptane (F0006):

[0220] Retention time: 11.130min;

[0221] Correlation coefficient: 0.99999;

[0222] Residual standard deviation: 6.90208;

[0223] Standard curve formula: y = m*x, where m=4057.32491, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0224] 9) Standard curve of DMF (F0014):

[0225] Retention time: 12.168min;

[0226] Correlation coefficient: 0.99995;

[0227] Residual standard deviation: 2.03071;

[0228] Standard curve formula: y = m*x, where m=26.37063, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0229] The test results are shown in Tables 8~9 and Figures 5 and 6 .

[0230] Table 8 shows the chromatographic peak information of the marking solution

[0231]

[0232] Table 9 shows the chromatographic peak information of the delivery system sample solution

[0233]

[0234] Example 4 Detection of Solvent Residues in Nucleic Acid Products

[0235] The solvent residues in nucleic acid products were detected by headspace gas chromatography using the external standard method, as follows:

[0236] (1) Preparation of nucleic acid product solutions

[0237] Accurately weigh 110.02 mg of DUTP (deoxyuridine triphosphate) and place it in a 20 mL headspace bottle. Use a calibrated pipette to transfer 1 mL of DMSO to the DUTP sample to dissolve the DUTP sample and set aside.

[0238] (2) Preparation of marking solution and separation solution

[0239] Prepare the mother solutions of each reagent (10 mL): methanol 59.66 mg / mL, ethanol 59.96 mg / mL, acetonitrile 60.22 mg / mL, dichloromethane 100.01 mg / mL, ethyl acetate 29.98 mg / mL, n-hexane 30.06 mg / mL, N-dimethylformamide 30.00 mg / mL, n-heptane 15.26 mg / mL, and tetrahydrofuran 50.32 mg / mL.

[0240] The preparation and dilution of the mother solution of the mixed solvent standard solution are the same as the steps in Example 1, except that the concentrations of the mother solutions of the reagents are different.

[0241] (3) Determination of residual solvents in nucleic acid products

[0242] The conditions of headspace gas chromatography as shown in Table 10 below were used to detect the mark line solution and the sample solution to be detected in sequence. The equipment was Agilent 7890B, and the headspace sampler used was Agilent G1888 headspace sampler.

[0243] Table 10 shows the headspace gas chromatography conditions in Example 4 of the present invention.

[0244]

[0245] After the gas chromatography detection of the marked solution, the peak area-concentration standard curve of each solvent is calculated; the solvent residue of the sample to be tested is respectively inserted into the corresponding standard curve to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested, which is the relative percentage content of the solvent in the sample.

[0246] Through the above detection, the information of the standard curve of each reagent is obtained as follows:

[0247] 1) Standard curve of methanol (F0002):

[0248] Retention time: 3.497min;

[0249] Correlation coefficient: 0.99989;

[0250] Residual standard deviation: 3.39003;

[0251] Standard curve formula: y = m*x, where m=151.49732, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0252] 2) Standard curve of ethanol (F0005):

[0253] Retention time: 4.892min;

[0254] Correlation coefficient: 0.99991;

[0255] Residual standard deviation: 3.72236;

[0256] Standard curve formula: y = m*x, where m=182.78872, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0257] 3) Standard curve of acetonitrile (F0008):

[0258] Retention time: 5.316min;

[0259] Correlation coefficient: 0.99995;

[0260] Residual standard deviation: 3.08897;

[0261] Standard curve formula: y = m*x, where m=211.42772, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0262] 4) Standard curve of dichloromethane (F0001):

[0263] Retention time: 7.016min;

[0264] Correlation coefficient: 0.99982;

[0265] Residual standard deviation: 10.46378;

[0266] Standard curve formula: y = m*x, where m=219.52850, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0267] 5) Standard curve of ethyl acetate (F0007):

[0268] Retention time: 9.206min;

[0269] Correlation coefficient: 0.99999;

[0270] Residual standard deviation: 1.68129;

[0271] Standard curve formula: y = m*x, where m=652.85691, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0272] 6) Standard curve of n-hexane (F0147):

[0273] Retention time: 9.330min;

[0274] Correlation coefficient: 1.00000;

[0275] Residual standard deviation: 3.92110;

[0276] Standard curve formula: y = m*x, where m=3729.00764, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0277] 7) Standard curve of tetrahydrofuran (F0012):

[0278] Retention time: 9.808min;

[0279] Correlation coefficient: 1.00000;

[0280] Residual standard deviation: 1.35458;

[0281] Standard curve formula: y = m*x, where m=950.35734, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0282] 8) Standard curve of n-heptane (F0006):

[0283] Retention time: 11.928min;

[0284] Correlation coefficient: 0.99999;

[0285] Residual standard deviation: 5.19280;

[0286] Standard curve formula: y = m*x, where m=3733.15508, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0287] 9) Standard curve of DMF (F0014):

[0288] Retention time: 12.961min;

[0289] Correlation coefficient: 0.99979;

[0290] Residual standard deviation: 3.60669;

[0291] Standard curve formula: y = m*x, where m=23.35782, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0292] The test results are shown in Tables 11~12 and Figure 7-8 .

[0293] Table 11 is the chromatographic peak information of the marking solution

[0294]

[0295] Table 12 shows the chromatographic peak information of nucleic acid product solutions

[0296]

[0297] Example 5 Detection of Solvent Residues in Fluorescent Marker Products

[0298] The residual solvent in the fluorescent marker product was detected by headspace gas chromatography using the external standard method, as follows:

[0299] (1) Preparation of fluorescent marker product solution

[0300] Accurately weigh 110.66 mg of the fluorescent marker sample (Cy5 UTP) and place it in a 20 mL headspace bottle. Use a calibrated pipette to transfer 1 mL of DMSO and add it to the fluorescent marker sample to dissolve the fluorescent marker sample for later use.

[0301] (2) Preparation of marking solution and separation solution

[0302] Prepare the mother solutions of each reagent (10 mL): methanol 60.06 mg / mL, ethanol 60.70 mg / mL, acetonitrile 60.62 mg / mL, dichloromethane 100.11 mg / mL, ethyl acetate 30.09 mg / mL, n-hexane 30.77 mg / mL, N-dimethylformamide 30.22 mg / mL, n-heptane 15.29 mg / mL, and tetrahydrofuran 50.66 mg / mL.

[0303] The preparation and dilution of the mother solution of the mixed solvent standard solution are the same as the steps in Example 1, except that the concentrations of the mother solutions of the reagents are different.

[0304] (3) Determination of residual solvent in fluorescent marker samples

[0305] The conditions of headspace gas chromatography as shown in Table 13 below were used to detect the mark line solution and the sample solution to be detected in sequence. The equipment was Thermo's TRACE1600 with TriPlus500 headspace sampler.

[0306] Table 13 shows the headspace gas chromatography conditions in Example 5 of the present invention.

[0307]

[0308] After the gas chromatography detection of the marked solution, the peak area-concentration standard curve of each solvent is calculated; the solvent residue of the sample to be tested is respectively inserted into the corresponding standard curve to obtain the concentration of the solvent, which is then divided by the mass of the added sample to be tested, which is the relative percentage content of the solvent in the sample.

[0309] Through the above detection, the information of the standard curve of each reagent is obtained as follows:

[0310] 1) Standard curve of methanol (F0002):

[0311] Retention time: 2.672min;

[0312] Correlation coefficient: 1.00000;

[0313] Residual standard deviation: 0.14632

[0314] Standard curve formula: y = m*x, where m=2.7314, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0315] 2) Standard curve of ethanol (F0005):

[0316] Retention time: 3.803min;

[0317] Correlation coefficient: 1.00000;

[0318] Residual standard deviation: 0.18630

[0319] Standard curve formula: y = m*x, where m=3.4144, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0320] 3) Standard curve of acetonitrile (F0008):

[0321] Retention time: 4.203min;

[0322] Correlation coefficient: 0.99996;

[0323] Residual standard deviation: 0.31301

[0324] Standard curve formula: y = m*x, where m=4.9078, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0325] 4) Standard curve of dichloromethane (F0001):

[0326] Retention time: 5.872min;

[0327] Correlation coefficient: 0.99966;

[0328] Residual standard deviation: 0.37647

[0329] Standard curve formula: y = m*x, where m=3.3032, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0330] 5) Standard curve of ethyl acetate (F0007):

[0331] Retention time: 8.158min;

[0332] Correlation coefficient: 0.99993;

[0333] Residual standard deviation: 0.38180

[0334] Standard curve formula: y = m*x, where m=11.3169, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0335] 6) Standard curve of n-hexane (F0147):

[0336] Retention time: 8.267min;

[0337] Correlation coefficient: 0.99981;

[0338] Residual standard deviation: 0.84669

[0339] Standard curve formula: y = m*x, where m=37.7166, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0340] 7) Standard curve of tetrahydrofuran (F0012):

[0341] Retention time: 8.750min;

[0342] Correlation coefficient: 0.99997;

[0343] Residual standard deviation: 1.08987

[0344] Standard curve formula: y = m*x, where m=20.7324, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0345] 8) Standard curve of n-heptane (F0006):

[0346] Retention time: 10.988min;

[0347] Correlation coefficient: 0.99999;

[0348] Residual standard deviation: 0.79584

[0349] Standard curve formula: y = m*x, where m=59.2723, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0350] 9) Standard curve of DMF (F0014):

[0351] Retention time: 12.053min;

[0352] Correlation coefficient: 0.9996;

[0353] Residual standard deviation: 0.61478

[0354] Standard curve formula: y = m*x, where m=0.4052, x is the sample concentration (mg / mL), and y is the peak area (pA*s).

[0355] The test results are shown in Tables 14~15 and Fig. 9 .

[0356] Table 14 shows the chromatographic peak information of the marking solution

[0357]

[0358] Table 15 shows the chromatographic peak information of the fluorescent marker sample solution

[0359]

[0360] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for detecting multiple solvent residues in a sample using headspace gas chromatography, characterized in that: The method includes: (1) Prepare the marking solution and separation solution Prepare a mixed solvent standard solution of methanol, ethanol, acetonitrile, dichloromethane, ethyl acetate, n-hexane, N, N-dimethylformamide, n-heptane and tetrahydrofuran, and dilute it with DMSO to 20 times, 200 times, 1000 times, 2000 times and 10000 times of marking solution; wherein, the solution diluted with DMSO to 20 times is also used as the separation solution; (2) Use headspace gas chromatography to test the sample Dissolve the sample to be tested in DMSO to prepare a sample solution to be tested; The marking solution and the sample solution to be detected are detected in sequence by headspace gas chromatography and a detector; after the marking solution is detected by gas chromatography, a standard curve of peak area-concentration of each solvent is calculated; the solvent residues of the sample to be detected are respectively introduced into the corresponding standard curve to obtain the concentration of the solvent, and then divided by the mass of the added sample to be detected to obtain the relative percentage content of the solvent in the sample; the sample to be detected is a phosphoramidite monomer DMT-2'-OMe-G(dmf)-CE-Phosphoramidite, a phosphoramidite raw material DMT-2'-O-TBDMS-C(Ac), GalNAc-L96, deoxyuridine triphosphate or Cy5 UTP; Wherein, the conditions of the headspace gas chromatography are: The chromatographic column is an ultra-high inert gas-phase capillary chromatographic column; the ultra-high inert gas-phase capillary chromatographic column is selected from CP-Volamine or ZB-1; The column temperature was programmed to rise from 45 to 55 °C for 4 to 10 min, then to 130 to 140 °C at 10 to 12 °C / min, and then to 200 to 220 °C at 15 to 20 °C / min, and then to 2 to 5 min. The carrier is an inert gas; The injection volume is 1~5 μL; The headspace equilibrium oil bath temperature is 80~160℃; Valve box temperature: 150℃; The split ratio of the injection port is 15~20:1; The injection port temperature is 150~220℃; The detector temperature is 200~250℃; The headspace sampler line temperature was 160°C; The detector is a FID detector.

2. The method for detecting multiple solvent residues in a sample using headspace gas chromatography according to claim 1, characterized in that: The inert gas is selected from high-purity nitrogen or high-purity helium.

3. The method for detecting multiple solvent residues in a sample using headspace gas chromatography according to claim 1, characterized in that: The flow rate of the carrier is 1-2 mL / min.

4. The method for detecting multiple solvent residues in a sample by headspace gas chromatography according to claim 1, characterized in that: In the headspace gas chromatography conditions, the sample preheating equilibrium time is 25 to 32 minutes.

5. The method for detecting multiple solvent residues in a sample using headspace gas chromatography according to claim 1, characterized in that: In the conditions of the headspace gas chromatography, the injection time is 45 s.

6. The method for detecting multiple solvent residues in a sample by headspace gas chromatography according to claim 1, characterized in that: The equipment used in the headspace gas chromatography method is Agilent 7890B, and the headspace sampler used is Agilent G1888 headspace sampler; or, the equipment used in the headspace gas chromatography method is Thermo TRACE1600, and the headspace sampler used is TriPlus500 headspace sampler.

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