Method for detecting content of polyethylene glycol substances

Abstract

The invention belongs to the technical field of medical analysis, and particularly relates to a method for detecting the content of polyethylene glycol substances, the LC-MS method adopted by the detection method has the advantages that the use amount of a sample is small, the response of each selected ion is high, the peak pattern is good, proper ions can be screened out, and the detection accuracy is high. The problem of content detection of polyethylene glycol substances with similar molecular weights and similar structures is effectively solved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and specifically relates to a method for detecting the content of polyethylene glycol substances. Background Art

[0002] Polyethylene glycol (PEG) is a compound formed by the polymerization of ethylene glycol units, and the molecular weight of each unit is 44Da. As a commonly used pharmaceutical excipient, PEG has the characteristics of good water solubility, strong water absorption, stable properties, and good safety at normal doses. Low molecular weight polyethylene glycol is mainly used as a solvent or surfactant to adjust drug solubility or system viscosity; high molecular weight polyethylene glycol is usually used in solid preparations or new drug preparations, and can also be made into a sustained-release matrix with other high molecular polymers.

[0003] At present, there are few methods for detecting the content of polyethylene glycol substances, and they are not included in the pharmacopoeias of various countries. The available high-performance liquid chromatography-evaporative light scattering detection method cannot detect the content of polyethylene glycol substances with similar molecular weight and similar structure. The present invention develops a new method for detecting the content of polyethylene glycol substances on the basis of the existing technology, which can detect the content of polyethylene glycol substances with similar molecular weight and similar structure with a small amount of sample, laying a foundation for drug research, especially the research on the content of polyethylene glycol substances in drugs. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting the content of polyethylene glycol substances on the basis of the prior art, which can realize the research on the content of polyethylene glycol substances with similar molecular weight and structure by using a small amount of sample, find the specific fragment ions of polyethylene glycol substances under an electrospray ion source (ESI), and the response value thereof can meet the sensitivity requirement, so as to develop an LC-MS method for determining the content of polyethylene glycol substances.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for detecting the content of polyethylene glycol substances, the method adopts LC-MS, the chromatographic column is a C18 column, ammonium formate solution-acetonitrile is used as the mobile phase for gradient elution, the pH value of the ammonium formate solution is 6.0-6.6, detection is performed by a mass spectrometry ion source, an internal standard is used, and the polyethylene glycol substance is polyethylene glycol monomethyl ether 350 and / or PEG-400.

[0007] Furthermore, the chromatographic column described in the present invention is a chromatographic column with octadecylsilane bonded silica gel as a filler, preferably an ACE Excel 3C18-PFP chromatographic column, and more preferably a chromatographic column with a specification of 100×2.1 mm, 3 μm.

[0008] Furthermore, the detection wavelength is 210-220 nm, preferably 215 nm.

[0009] Furthermore, the flow rate of the chromatographic column is 0.1 to 0.5 ml / min, preferably the flow rate is 0.27 to 0.33 ml / min, and more preferably the flow rate is 0.3 ml / min.

[0010] Furthermore, the column temperature is 20 to 40°C, preferably the column temperature is 25 to 35°C, and more preferably the column temperature is 30°C.

[0011] Furthermore, the injection volume is 0.2-1.5 μl, preferably the injection volume is 0.5-1.7 μl, and more preferably the injection volume is 1.0 μl.

[0012] Furthermore, the sample pan temperature is 2-15°C, preferably the sample pan temperature is 5°C.

[0013] Furthermore, the content of ammonium formate in the ammonium formate solution is 0.01% to 0.1% (w / w).

[0014] Furthermore, the content of ammonium formate in the ammonium formate solution is 0.03% to 0.08% (w / w).

[0015] Furthermore, the ammonium formate content in the ammonium formate solution is 0.06% (w / w).

[0016] Furthermore, the pH value of the ammonium formate solution is 6.3.

[0017] Furthermore, the mass spectrometry ion source is an electrospray ion source, preferably the ion type is a positive ion, and more preferably the scanning mode is a SIM mode.

[0018] Furthermore, the mass spectrometry drying gas temperature is 300-400°C, preferably the drying gas temperature is 320-370°C, and more preferably the drying gas temperature is 350°C.

[0019] Furthermore, the mass spectrometry drying gas flow rate is 5.0 to 20 L / min, preferably the drying gas flow rate is 10.0 to 15.0 L / min, and more preferably the drying gas flow rate is 12.0 L / min.

[0020] Furthermore, the mass spectrometer nebulizer pressure is 20 to 50 psi, preferably the nebulizer pressure is 30 to 40 psi, and more preferably the nebulizer pressure is 35 psi.

[0021] Furthermore, the mass spectrometer capillary voltage is 2000-4000V, preferably the capillary voltage is 2500-3500V, and more preferably the capillary voltage is 3000V.

[0022] Furthermore, the mass spectrometry solvent delay time is 0 to 10 min, preferably the solvent delay time is 0 to 5 min, and more preferably the solvent delay time is 1.5 min.

[0023] Furthermore, the mass spectrometer MS stop time is 1 to 30 minutes, preferably the MS stop time is 1 to 20 minutes, and more preferably the MS stop time is 14 minutes.

[0024] Furthermore, the internal standard is 2-[2-(2-ethoxyethoxy)ethoxy]ethanol (Ethoxytriglycol).

[0025] Furthermore, polyethylene glycol monomethyl ether 350 selects ions with m / z of 270, 314, 358, 402, 446, and 490 as quantitative ions.

[0026] Furthermore, PEG-400 selects ions with m / z of 300, 344, 388, 432, 476, 520, and 564 as quantitative ions.

[0027] Furthermore, the ion of m / z 196 of the internal standard 2-[2-(2-ethoxyethoxy)ethoxy]ethanol (ethoxytriglycol) was selected as a quantitative ion.

[0028] Furthermore, the detection step includes:

[0029] (1) Solution preparation: prepare internal standard stock solution, blank solution, reference substance stock solution, reference substance solution stock solution, reference substance solution, and test substance solution respectively;

[0030] (2) Determination method: After the system is stable, inject blank solution, test solution and reference solution respectively and record the chromatogram.

[0031] Further, the solution preparation method is as follows:

[0032] (1) Internal standard stock solution: Dissolve 2-[2-(2-ethoxyethoxy)ethoxy]ethanol in methanol and dilute to scale. Shake well to obtain the internal standard stock solution.

[0033] (2) Blank solution: Pipette methanol and ultrapure water into a beaker, add the internal standard stock solution, shake well, and prepare as needed;

[0034] (3) Reference substance stock solution 1: Take polyethylene glycol monomethyl ether 350 and / or PEG-400, accurately weigh, transfer to a volumetric flask, dissolve and dilute to scale with blank solution, and shake well to obtain;

[0035] (4) Reference substance stock solution: Pipette reference substance stock solution 1 into a volumetric flask, dilute to scale with blank solution, and shake well.

[0036] (5) Reference solution: Pipette the reference solution stock solution into a volumetric flask, dilute to scale with blank solution, and shake well.

[0037] (6) Test solution: Take the test sample, accurately weigh it, put it into a volumetric flask, dissolve it with blank solution and dilute it to the mark, and shake well.

[0038] After the baseline is balanced, each solution is tested according to the chromatographic conditions described in the method, and the chromatogram is recorded.

[0039] Further, the solution preparation method is as follows:

[0040] (1) Internal standard stock solution: Take about 50 mg of 2-[2-(2-ethoxyethoxy)ethoxy]ethanol, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it in methanol and dilute it to the mark, and shake well to obtain the internal standard stock solution;

[0041] (2) Blank solution: Pipette 1500 ml of methanol and 1500 ml of ultrapure water into a 5 L beaker, add 3.0 ml of internal standard stock solution, shake well, and prepare as needed;

[0042] (3) Reference stock solution 1: Take about 647 mg of polyethylene glycol monomethyl ether 350 and about 225 mg of PEG-400, accurately weigh them, put them into a 50 ml volumetric flask, dissolve them with blank solution and dilute to the scale, shake well to obtain;

[0043] (4) Reference substance stock solution: Pipette 4.0 ml of reference substance stock solution into a 10 ml volumetric flask, dilute to scale with blank solution, and shake well.

[0044] (5) Reference solution: Pipette 1.0 ml of the reference solution stock solution into a 100 ml volumetric flask, dilute to the mark with the blank solution, and shake well.

[0045] (6) Test solution: Take about 100 mg of the test sample, accurately weigh it, transfer it to a 10-ml volumetric flask, dissolve it with blank solution and dilute it to the mark, shake well; transfer 1.0 ml of the above solution to a 100-ml volumetric flask, dilute it to the mark with blank solution, shake well, and prepare 2 portions in parallel.

[0046] After the baseline is balanced, take each solution and record the chromatogram according to the chromatographic conditions described in the above method.

[0047] Further, the mobile phase gradient elution procedure is as follows:

[0048]

[0049]

[0050] Preferably, the mobile phase gradient elution procedure is as follows:

[0051] Time (minutes) Mobile phase A (volume %) Mobile phase B (volume %) 0 90 10 10 85 15 13 85 15 14 20 80 19 20 80 .

[0052] Furthermore, the detection method is used to detect the content of polyethylene glycol substances in raw materials or pharmaceutical preparations.

[0053] Furthermore, the drug substance or pharmaceutical preparation is a midazolam drug substance or preparation.

[0054] Furthermore, the polyethylene glycol substance is polyethylene glycol monomethyl ether 350 and / or PEG400.

[0055] Beneficial Effects

[0056] The technical solution of the present invention has the following advantages:

[0057] The LC-MS method adopted by the present invention uses less sample amount, and each selected ion has a higher response and a better peak shape, and can screen out suitable ions, effectively solving the problem of detecting the content of polyethylene glycol substances with similar molecular weight and structure, which is of great significance to drug research. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is the linear graph of polyethylene glycol monomethyl ether 350 in Table 5 of the present invention.

[0059] Figure 2 This is the residual graph of polyethylene glycol monomethyl ether 350 in Table 5 of the present invention.

[0060] Figure 3 This is the linear graph of PEG-400 in Table 5 of the present invention.

[0061] Figure 4 This is the PEG-400 residual graph in Table 5 of the present invention. DETAILED DESCRIPTION

[0062] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technical schemes implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0063] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0064] Example 1 System suitability test

[0065] (1) Instruments: liquid chromatography-mass spectrometry, electronic analytical balance, pH meter, etc.

[0066] (2) Solution preparation:

[0067] Internal standard stock solution: Take about 50 mg of 2-[2-(2-ethoxyethoxy)ethoxy]ethanol, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it in methanol and dilute it to the scale, shake well to obtain;

[0068] Blank solution: Pipette 1500ml of methanol and 1500ml of ultrapure water into a 5L beaker, add 3.0ml of internal standard stock solution, shake well and prepare 1 portion as needed;

[0069] Reference stock solution 1: Take about 647 mg of polyethylene glycol monomethyl ether 350 and about 225 mg of PEG-400, accurately weigh, put into a 50 ml volumetric flask, dissolve and dilute to the scale with blank solution, shake well to obtain;

[0070] Reference substance stock solution: Pipette 4.0 ml of reference substance stock solution 1 to a 10 ml volumetric flask, dilute to scale with blank solution, and shake well;

[0071] Reference solution: Pipette 1.0 ml of reference solution stock solution into a 100 ml volumetric flask, dilute to scale with blank solution, shake well, and prepare 1 portion;

[0072] Test solution: Take about 100 mg of the test sample, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it with blank solution and dilute it to the scale, shake well; transfer 1.0 ml of the above solution to a 100 ml volumetric flask, dilute it to the scale with blank solution, shake well, and prepare 2 portions in parallel.

[0073] After the baseline is balanced, take each solution and record the chromatogram according to the following method.

[0074]

[0075]

[0076] The injection sequence requires the use of a running control (reference solution). Take the first 5 injections of the reference solution and 1 injection of the running control, and calculate the RSD of the R value (the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard). The RSD must be ≤5.0%. If multiple running controls are performed, the same method should be used for calculation, and the RSD must be ≤5.0%.

[0077]

[0078] After the system is balanced, according to the above chromatographic conditions, inject 2 injections of blank solution and 6 injections of reference solution, and record the chromatogram. Report the peak areas of the analyte and internal standard of 6 consecutive injections of reference solution, the mean value of the peak area ratio R of the analyte and internal standard, and its RSD value.

[0079] Acceptable criteria:

[0080] 1. The reference solution was injected 6 times continuously, and the RSD of the peak area ratio of the analyte to the internal standard was ≤5.0%;

[0081] 2. The RSD of the peak area ratio R value of the first 5 injections of the reference solution and the accompanying control of the analyte to the internal standard is ≤5.0%.

[0082] Table 1 System suitability results

[0083]

[0084]

[0085] in conclusion:

[0086] 1. The maximum RSD of the peak area ratio R value of the analyte and the internal standard after 6 consecutive injections of the reference solution is 1.01% (RSD≤5.0%);

[0087] 2. The maximum RSD of the peak area ratio R value of the analyte to the internal standard in the first 5 injections of the reference solution and the accompanying control was 1.19% (RSD≤5.0%), and the system applicability of the method met the detection requirements.

[0088] Example 2 Specificity Test

[0089] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0090] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0091] Test solution: Prepare 1 portion in the same manner as the test solution in Example 1;

[0092] Test sample spiked solution: Take about 50 mg of the test sample, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it with blank solution, then add 2.0 ml of reference substance stock solution 1, dilute it to the scale with blank solution, and shake well; transfer 1.0 ml of the above solution to a 100 ml volumetric flask, dilute it to the scale with blank solution, shake well, and prepare 1 portion.

[0093] After the system suitability is qualified, take blank solution, reference solution, test solution and test spiked solution according to the chromatographic conditions of Example 1, inject one needle each, record the chromatogram, and report the retention time of the analyte in each solution.

[0094] Acceptable criteria:

[0095] 1. Comparing the chromatogram of blank solution with that of reference solution, the blank solution should have no interference at the peak of the analyte;

[0096] 2. The retention time of the analyte peak in the test solution, reference solution and test spiked solution should be consistent.

[0097] Table 2 Specificity results

[0098]

[0099] in conclusion:

[0100] 1. Compared with the chromatogram of the reference solution, the blank solution has no interference at the peak of the analyte;

[0101] 2. The retention time of the analyte peak in the test solution, reference solution and test spike solution is consistent. The method has good specificity.

[0102] Example 3: Precision

[0103] 3.1 Repeatability

[0104] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0105] Repeatability solution: Prepare 6 replicates in the same manner as the test solution in Example 1.

[0106] After the system suitability is qualified, take 6 duplicate solutions according to the chromatographic conditions of Example 1, inject 1 needle into each, and calculate the content of the analyte in the 6 duplicate solutions according to the average value of the peak area ratio R of the analyte to the internal standard in the system suitability reference solution. Report the peak area and peak area ratio R of the analyte and internal standard, the single value of the content, Mean±SD, RSD value and 95% confidence interval.

[0107] Acceptable criteria:

[0108] The RSD of the analyte content in 6 replicate solutions was ≤5.0%.

[0109] Table 3 Repeatability results

[0110]

[0111] in conclusion:

[0112] The maximum RSD of the analyte content in 6 replicate solutions was 0.57% (≤5.0%).

[0113] 3.2 Intermediate precision

[0114] The following operations were performed by different analysts at different times in the same laboratory using the same instrument.

[0115] Solution preparation:

[0116] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0117] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0118] Intermediate precision solution: Prepare 6 portions in parallel using the same method as the test solution in Example 1.

[0119] After the system suitability is qualified, take 6 portions of intermediate precision solution according to the chromatographic conditions of Example 1, inject 1 needle into each, and record the chromatogram. Calculate the content of the analyte in the 6 portions of intermediate precision solution according to the average value of the peak area ratio R of the analyte to the internal standard in the system suitability reference solution. Report the peak area of ​​the analyte to the internal standard and the peak area ratio R, the single value of the content, Mean±SD value, RSD value and 95% confidence interval in the 6 portions of intermediate precision solution. Calculate the RSD value of 12 contents of repeatability and intermediate precision.

[0120] Acceptable criteria:

[0121] 1. The RSD of the analyte content in 6 intermediate precision solutions is ≤5.0%;

[0122] 2. Repeatability and intermediate precision The RSD of the analyte content in 12 solutions is ≤7.5%.

[0123] Table 4 Intermediate precision results

[0124]

[0125]

[0126] in conclusion:

[0127] 1. The maximum RSD of the analyte content in 6 intermediate precision solutions is 0.34% (≤5.0%);

[0128] 2. Repeatability and intermediate precision The maximum RSD of the analyte content in 12 solutions was 1.17% (≤7.5%), and the method had good precision.

[0129] Example 4: Linearity and Range

[0130] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0131] Reference substance stock solution 1: prepare 1 portion in the same manner as the reference substance stock solution 1 in Example 1;

[0132] Reference solution (L-100% STD): Prepare 1 portion in the same manner as the reference solution in Example 1;

[0133] 50% linear solution (L-50% STD): Pipette 2.0ml of reference stock solution 1 into a 10ml volumetric flask, dilute to scale with blank solution, and shake well; Pipette 1.0ml of the above solution into a 100ml volumetric flask, dilute to scale with blank solution, and shake well;

[0134] 75% linear solution (L-75% STD): Pipette 3.0ml of reference stock solution 1 into a 10ml volumetric flask, dilute to scale with blank solution, and shake well; Pipette 1.0ml of the above solution into a 100ml volumetric flask, dilute to scale with blank solution, and shake well;

[0135] 125% linear solution (L-125% STD): Pipette 5.0ml of reference stock solution 1 into a 10ml volumetric flask, dilute to scale with blank solution, and shake well; Pipette 1.0ml of the above solution into a 100ml volumetric flask, dilute to scale with blank solution, and shake well;

[0136] 150% linear solution (L-150% STD): Pipette 6.0 ml of the reference stock solution into a 10 ml volumetric flask, dilute to the mark with the blank solution, and shake well; Pipette 1.0 ml of the above solution into a 100 ml volumetric flask, dilute to the mark with the blank solution, and shake well.

[0137] After the system suitability is qualified, take one injection of each linear solution according to the chromatographic conditions of Example 1, and record the chromatogram. Report the actual concentration of each linear solution, the peak area of ​​the analyte and the internal standard, and the peak area ratio R. Perform a univariate linear regression on the concentration of the analyte and the internal standard peak area ratio R, and calculate the linear correlation coefficient r, the Y-axis intercept and the residual sum of squares, the 95% confidence interval of the Y-axis intercept, and the absolute value of the peak area ratio R corresponding to the Y-axis intercept / 100% concentration.

[0138] Acceptable criteria:

[0139] 1. The linear correlation coefficient of the test object is r≥0.998;

[0140] 2. The ratio of the absolute value of the Y-axis intercept to the peak area corresponding to the 100% limit concentration is ≤ 15.0%.

[0141] Table 5 Linearity results

[0142]

[0143] in conclusion:

[0144] 1. The minimum linear correlation coefficient r value of the test object is 0.9995 (≥0.998);

[0145] 2. The maximum ratio of the absolute value of the Y-axis intercept to the peak area ratio R corresponding to 100% concentration is 3.37% (≤15.0%).

[0146] like Figure 1 and Figure 2 As shown, the method shows a linear relationship in the concentration range of polyethylene glycol monomethyl ether 350 from 0.026 mg / ml to 0.077 mg / ml (50% to 150% concentration level); Figure 3 and Figure 4 As shown, the PEG-400 concentration was linear in the range of 0.009 mg / ml to 0.027 mg / ml (50% to 150% concentration level).

[0147] Example 5: Accuracy

[0148] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0149] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0150] Test solution: Prepare 2 portions in parallel in the same manner as the test solution in Example 1;

[0151] Reference substance stock solution 1: prepare 1 portion in the same manner as the reference substance stock solution 1 in Example 1;

[0152] S+75% STD solution: Take about 50 mg of the test sample, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it with blank solution, then add 1.0 ml of reference substance stock solution 1, dilute it to the mark with blank solution, shake well; transfer 1.0 ml of the above solution to a 100 ml volumetric flask, dilute it to the mark with blank solution, shake well, and prepare 3 copies in parallel;

[0153] S+100% STD solution: Take about 50 mg of the test sample, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it with blank solution, then add 2.0 ml of reference substance stock solution 1, dilute it to the mark with blank solution, shake well; transfer 1.0 ml of the above solution to a 100 ml volumetric flask, dilute it to the mark with blank solution, shake well, and prepare 3 copies in parallel;

[0154] S+125% STD solution: Take about 50 mg of the test sample, accurately weigh it, put it into a 10 ml volumetric flask, dissolve it with blank solution, then add 3.0 ml of reference substance stock solution 1, dilute it to the scale with blank solution, and shake well; transfer 1.0 ml of the above solution to a 100 ml volumetric flask, dilute it to the scale with blank solution, shake well, and prepare 3 copies in parallel.

[0155] After the system suitability is qualified, take 2 parts of the test solution and 9 parts of the accuracy solution and inject 1 needle each according to the chromatographic conditions of Example 1, and record the chromatogram. Calculate and report the content of the analyte in the test solution before and after spike addition, the measured addition amount, the theoretical addition amount, the single value of the recovery rate, the average value of the recovery rate, the RSD value and the 95% confidence interval.

[0156] The recovery rate is calculated as follows:

[0157]

[0158] Theoretical addition amount (mg) = WT + s × CT + Cs × V

[0159]

[0160] Where:

[0161] RT+S is the peak area ratio R of the analyte to the internal standard in the spiked solution of the test sample;

[0162] RS is the average value of the peak area ratio R of the analyte to the internal standard in six consecutive injections of the reference solution;

[0163] WT+S is the weight of the spiked solution of the test sample, mg;

[0164] WS is the mass of the substance to be tested weighed to prepare the reference solution, mg;

[0165] DT+S is the dilution factor of the test sample spiked solution;

[0166] DS is the dilution factor of the reference solution;

[0167] CT is the content of the substance to be tested in the test solution, %; calculated according to item 3.6;

[0168] CS is the concentration of the analyte in the reference stock solution added to each spiked solution, mg / ml;

[0169] V is the volume of reference stock solution added to each spiked solution, ml.

[0170] Acceptable criteria:

[0171] 1. The individual recoveries of the analytes in the 9 accuracy solutions ranged from 95.0% to 105.0%;

[0172] 2. The RSD of the overall recovery (n=9) was ≤5.0%.

[0173] Table 6 Accuracy results

[0174]

[0175]

[0176] in conclusion:

[0177] The recovery of polyethylene glycol monomethyl ether 350 in 1.9 accuracy solutions ranged from 98.6% to 101.4% (between 95.0% and 105.0%), and the recovery of PEG-400 ranged from 98.7% to 101.3% (between 95.0% and 105.0%);

[0178] 2. The maximum RSD of the overall recovery (n=9) was 0.92% (≤5.0%), and the method had good accuracy.

[0179] Example 6: Solution Stability

[0180] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0181] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0182] Test solution: Prepare 1 portion of the test solution in the same manner as in Example 1.

[0183] The prepared reference solution and test solution are placed in a sample tray (5°C). Without interrupting the system, after the system suitability is qualified, according to the chromatographic conditions of Example 1, the reference solution is injected at 0h, 5.1h, 10.7h and 14.1h, and the test solution is injected at 0h, 3.0h, 8.5h and 11.0h, respectively. The peak area of ​​the analyte and the internal standard and the peak area ratio R value are recorded, and the ratio of the peak area ratio R value at each time point to the 0h peak area ratio R value is calculated.

[0184] Acceptable criteria:

[0185] If the ratio of the peak area ratio R value of the analyte to the internal standard in each solution at each time point to the peak area ratio R value of the analyte to the internal standard at 0 h is within the range of 0.95 to 1.05, then each solution is determined to be stable within the time.

[0186] Table 7 Solution stability results

[0187]

[0188]

[0189] in conclusion:

[0190] 1. The ratio of the peak area ratio R of the analyte to the internal standard in the reference solution and the test solution at each time point to the ratio R of the peak area ratio R of the analyte to the internal standard at 0h is between 0.97 and 1.00 (within the range of 0.95 to 1.05);

[0191] 2. The reference solution is stable for at least 14.1 hours when placed at 5°C; the test solution is stable for at least 11.0 hours when placed at 5°C.

[0192] Example 7: Durability

[0193] The tolerance of the measurement results was investigated by slight changes in the chromatographic condition parameters.

[0194] Blank solution: prepare 1 portion in the same manner as the blank solution in Example 1;

[0195] Reference solution: prepare 1 portion in the same manner as the reference solution in Example 1;

[0196] Test solution: Prepare 2 portions in parallel using the same method as in Example 1.

[0197] Based on the above HPLC-MS conditions, perform single factor changes according to Table 8. After the system is balanced, inject 2 injections of blank solution, 3 injections of reference solution, and 1 injection of each of the 2 test solutions, and record the chromatogram. Report the peak area of ​​the analyte and the internal standard, the peak area ratio R value, the mean R value and its RSD value of the reference solution injected 3 times in a row, and the content of the analyte in the test solution.

[0198] Table 8 Changes in HPLC mass spectrometry conditions

[0199]

[0200] Acceptable criteria:

[0201] 1. The reference solution was injected three times continuously, and the RSD of the peak area ratio of the analyte to the internal standard was ≤5.0%;

[0202] The average content of polyethylene glycol monomethyl ether 350 in the two test solutions should be within the range of 49.21% to 54.39%, and the average content of PEG-400 should be within the range of 17.13% to 18.93%.

[0203] Table 9 Results of Chromatographic Condition Changes

[0204]

[0205]

[0206]

[0207]

[0208] in conclusion:

[0209] 1. The maximum RSD value of the peak area ratio R value of the analyte and the internal standard after three consecutive injections of the reference solution is 0.85% (RSD≤5.0%);

[0210] 2. The average content of the analyte polyethylene glycol monomethyl ether 350 in the 2 test solutions was between 51.92% and 53.06% (within the range of 49.21% to 54.39%), and the average content of PEG-400 was between 17.59% and 17.94% (within the range of 17.13% to 18.93%).

[0211] The experimental results of durability change of chromatographic conditions showed that the chromatographic conditions of this method had good durability within the range of flow rate change of ±0.03 ml / min, column temperature change of ±5°C, and initial proportion change of mobile phase B (organic phase) of ±1%.

[0212] In summary, the system applicability and specificity, precision repeatability, intermediate precision, linearity and range, accuracy, solution stability and durability of this method all meet the requirements, indicating that this method is suitable for the detection of polyethylene glycol content.

[0213] The above is a description of a specific embodiment of the present invention. It should be understood that the present invention is not limited to the above embodiment, and the above embodiment and description are only for illustrating the principle of the present invention.

[0214] Various non-substantial changes and improvements of the present invention can be made by those skilled in the art without departing from the concept of the present invention, and all of these fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the content of polyethylene glycol substances, the detection method adopts LC-MS, the chromatographic column is a C18 column, ammonium formate solution-acetonitrile is used as the mobile phase for gradient elution, the pH value of the ammonium formate solution is 6.0-6.6, detection is performed by a mass spectrometry ion source, an internal standard is used, and the polyethylene glycol substance is polyethylene glycol monomethyl ether 350 and / or PEG-400.

2. The detection method according to claim 1, characterized in that: The chromatographic column is a chromatographic column with octadecylsilane bonded silica gel as a filler, preferably an ACE Excel 3C18-PFP chromatographic column, more preferably a chromatographic column with specifications of 100×2.1 mm, 3 μm.

3. The detection method according to any one of claims 1 to 2, characterized in that: Furthermore, the chromatographic conditions include: detection wavelength: 210-220 nm, flow rate: 0.1-0.5 ml / min, column temperature: 20-40° C., injection volume: 0.2-1.5 μl, sample plate temperature: 2-15° C.

4. The detection method according to any one of claims 1 to 3, characterized in that: The content of ammonium formate in the ammonium formate solution is 0.01% to 0.1% (w / w); preferably, the content of ammonium formate in the ammonium formate solution is 0.03% to 0.08% (w / w).

5. The detection method according to any one of claims 1 to 4, characterized in that: The mass spectrometer ion source is an electrospray ion source, the ion type is a positive ion, and the scanning mode is a SIM mode.

6. The detection method according to any one of claims 1 to 5, characterized in that: The detection method also includes mass spectrometry conditions: drying gas temperature: 300-400°C, drying gas flow rate: 5.0~20L / min, nebulizer pressure: 20~50psi, capillary voltage: 2000~4000V, solvent delay time: 0~10min, MS stop time: 1~30min.

7. The detection method according to any one of claims 1 to 6, characterized in that: The detection method also includes mass spectrometry conditions: polyethylene glycol monomethyl ether 350 selects ions with m / z of 270, 314, 358, 402, 446, and 490 as quantitative ions, PEG-400 selects ions with m / z of 300, 344, 388, 432, 476, 520, and 564 as quantitative ions, and the internal standard 2-[2-(2-ethoxyethoxy)ethoxy]ethanol selects an ion with m / z of 196 as a quantitative ion.

8. The detection method according to any one of claims 1 to 7, characterized in that: The preparation methods of the solutions used in the test are as follows: (1) Internal standard stock solution: Dissolve 2-[2-(2-ethoxyethoxy)ethoxy]ethanol in methanol and dilute to scale. Shake well to obtain the internal standard stock solution. (2) Blank solution: Pipette methanol and ultrapure water into a beaker, add the internal standard stock solution, and shake well. (3) Reference substance stock solution 1: Take polyethylene glycol monomethyl ether 350 and / or PEG-400, accurately weigh, transfer to a volumetric flask, dissolve with blank solution and dilute to scale, shake well to obtain; (4) Reference substance stock solution: Pipette reference substance stock solution 1 into a volumetric flask, dilute to scale with blank solution, and shake well; (5) Reference solution: Pipette the reference solution stock solution into a volumetric flask, dilute to scale with blank solution, and shake well. (6) Test sample solution: Take the test sample, accurately weigh it, put it into a volumetric flask, dissolve it with blank solution and dilute it to the mark, and shake well.

9. The detection method according to any one of claims 1 to 8, characterized in that: The mobile phase gradient elution procedure is as follows: Preferably, the mobile phase gradient elution procedure is as follows:

10. Use of the detection method according to any one of claims 1 to 9 for detecting the content of polyethylene glycol substances in raw materials or pharmaceutical preparations.

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