Detection method of alpha-methyl-D-mannoside

Abstract

The invention provides a detection method of alpha-methyl-D-mannoside, and belongs to the field of pharmaceutical analytical chemistry. Specifically, on the basis of liquid chromatography-tandem mass spectrometry, chromatographic separation is carried out in a reversed-phase mode by adopting a PFPP chromatographic column, and alpha-methyl-D-mannoside in a sample is detected by adopting a triple quadrupole liquid chromatography-mass spectrometry. The method has the advantage of high sensitivity, is particularly suitable for detecting trace alpha-methyl-D-mannoside residues in recombinant protein drugs (such as recombinant protein vaccines), and is beneficial to quality control or quality monitoring of the drugs.

Description

Technical Field

[0001] The invention provides a method for detecting α-methyl-D-mannoside, and belongs to the field of pharmaceutical analytical chemistry. Background Art

[0002] Recombinant protein technology has been widely used in preventive biological products such as hepatitis B vaccines, influenza vaccines, herpes zoster vaccines, COVID-19 vaccines, and HPV vaccines. These vaccines are collectively referred to as recombinant protein vaccines. Recombinant protein vaccines are vaccines produced by using genetic recombination technology to incorporate genes encoding protective antigens from pathogenic microorganisms into expression vectors. After culture and proliferation, the expressed protective antigens are extracted and purified. These expression vectors can be expressed in bacterial (such as Escherichia coli), yeast, insect cells, plant cells, or mammalian cells. When expressed in eukaryotic expression systems (such as insect cells or mammalian systems), the target protein obtained may exhibit varying degrees of post-translational modifications on its surface, such as glycosylation (referred to as a glycoprotein).

[0003] Purification methods vary depending on the physicochemical properties and characteristics of the target protein. Affinity chromatography media, such as those based on lentil lectin, are the most commonly used. Lectins are proteins that interact with certain carbohydrate residues in a specific and reversible manner, allowing them to capture molecules containing carbohydrate groups (such as glycoproteins) through affinity. Therefore, lectin affinity chromatography is a primary method for the separation and purification of glycoproteins. Lectin affinity chromatography utilizes the affinity between lectins and specific sugar groups to selectively bind and purify the target protein, offering advantages such as high production efficiency and cost-effectiveness.

[0004] During lectin affinity chromatography, large amounts of α-methyl-D-mannoside (CAS No. 617-04-9) are typically used as an eluent to elute the target glycoprotein from the affinity column. Residual α-methyl-D-mannoside in recombinant protein vaccine products may affect vaccine safety or efficacy. Excessive intake of α-methyl-D-mannoside in humans may cause side effects such as headaches, nausea, and diarrhea. According to the "General Introduction to Human Vaccines" in the current edition of the Pharmacopoeia of the People's Republic of China, the production process for subunit vaccines, polysaccharide vaccines, and protein vaccines requires the removal of not only culture medium or cellular components, but also other non-target antigenic components of the bacteria or virus itself, as well as reagents added during the process. Therefore, when α-methyl-D-mannoside is used as an eluent during target protein purification, it should be removed and monitored as an additional reagent to ensure that its residual level in the vaccine remains within a safe range.

[0005] α-Methyl-D-mannoside is a carbohydrate substance with strong polarity. Quantitative analysis of carbohydrates is usually performed by gas chromatography (GC), liquid chromatography (LC), or triple quadrupole liquid chromatography-mass spectrometry (LC-QQQ-MS). Among them, (1) gas chromatography requires sample derivatization before analysis, but the derivatization method is cumbersome and prone to interference, affecting the accuracy of the analysis. (2) Liquid chromatography combined with a differential detector or evaporative light scattering detector can directly measure carbohydrate substances (without derivatization treatment), or the sample can be derivatized and then measured using an ultraviolet detector or fluorescence detector. However, direct determination methods are usually less sensitive and therefore cannot meet the needs of trace analysis of α-methyl-D-mannoside in recombinant protein vaccine samples. Derivatization also has the above-mentioned cumbersome operation problem. (3) Triple quadrupole liquid chromatography-mass spectrometry (LC-MS / MS) is a highly sensitive and selective analytical technique that combines liquid chromatography (LC) with triple quadrupole mass spectrometry (QQQ-MS). It is a subcategory of liquid chromatography-tandem mass spectrometry (LC-MS / MS) and is suitable for the qualitative and quantitative analysis of trace compounds in complex matrices. It has the characteristics of high sensitivity and good selectivity and can perform quantitative analysis of sugars in complex matrix samples without derivatization. The chromatographic column usually uses an amino column, and the liquid phase separation is performed using hydrophilic interaction chromatography (HILIC) mode to enhance the retention of sugars.

[0006] However, there are no reports in the prior art regarding the detection and analysis of residual α-methyl-D-mannoside. While the HILIC-LC-MS / MS method (an analytical method combining hydrophilic interaction chromatography (HILIC) and liquid chromatography-mass spectrometry (LC-MS / MS)) has been used in the prior art to analyze carbohydrates, its application to the detection of residual α-methyl-D-mannoside has not been reported. Furthermore, the applicant's previous research found that the HILIC-LC-MS / MS method is not suitable for the detection of trace amounts of α-methyl-D-mannoside in recombinant protein vaccines: such vaccine samples typically contain high concentrations of buffer salts, which can affect HILIC separation, resulting in retention time shifts, peak loss, and poor reproducibility. Consequently, the sensitivity of detecting α-methyl-D-mannoside in spiked samples using this method is low. Removing the buffer salts through pretreatment inevitably increases analytical costs and may also result in loss of the target substance.

[0007] In summary, regarding the residual amount of α-methyl-D-mannoside in vaccine samples, the existing technology has not yet disclosed how to detect it. Moreover, due to unfavorable factors such as its low content as an impurity and the presence of high concentration of buffer salt interference in the measured samples, the detection of this impurity is relatively difficult and requires a very high sensitivity of the analytical method. Summary of the Invention

[0008] In view of the above problems existing in the prior art, the present invention provides a method for detecting α-methyl-D-mannoside, which is used for trace detection of this substance in drug samples.

[0009] In a first aspect, the present invention provides a method for detecting α-methyl-D-mannoside based on liquid chromatography tandem mass spectrometry (LC-MS / MS), characterized in that a PFPP chromatographic column is used for chromatographic separation in reverse phase (RP) mode.

[0010] Preferably, the chromatographic column is a Shimadzu Shim-pack Velox PFPP chromatographic column. Optionally, the chromatographic column has a size of 100 mm x 2.1 mm ID or 50 mm x 2.1 mm ID, with 100 mm x 2.1 mm ID being preferred. Optionally, the chromatographic column packing has a particle size of 1.8 μm, 2.7 μm, or 5 μm, with 1.8 μm being preferred.

[0011] Preferably, in liquid chromatography, the mobile phase (phase A) is ultrapure water, and the organic phase (phase B) is acetonitrile.

[0012] Preferably, gradient elution is used in liquid chromatography; further preferably, the gradient program is: 0-1 min, 0% B; 1-2 min, 0% B-30% B; 2-3 min, 30% B-90% B; 3-4 min, 90% B; 4-4.01 min, 90% B-0% B; 4.01-7 min, 0% B; wherein B represents phase B, i.e., the organic phase acetonitrile.

[0013] Preferably, in liquid chromatography, the injection volume is 1 to 20 μL, more preferably 1 to 5 μL.

[0014] Preferably, in liquid chromatography, the column temperature is 35°C.

[0015] Preferably, in liquid chromatography, the flow rate is 0.4 mL / min.

[0016] Preferably, the mass spectrometer uses a Shimadzu triple quadrupole liquid chromatography-mass spectrometer LCMS-8050.

[0017] Preferably, the ionization mode of the mass spectrometer is ESI negative ion mode.

[0018] Preferably, the temperature of the heating module of the mass spectrometer is 400°C.

[0019] Preferably, the DL temperature of the mass spectrometer is 300°C.

[0020] Preferably, the interface voltage of the mass spectrometer is 3 kV.

[0021] Preferably, the scanning mode of the mass spectrometer is MRM.

[0022] Preferably, the precursor ion (parent ion) characteristic of the mass spectrometer is: a mass-to-charge ratio (m / z) of 193.15.

[0023] Preferably, the product ion (daughter ion) characteristics of the mass spectrometer are: the m / z of the quantitative ion and the qualifier ion are 101.00 and 59.05, respectively.

[0024] In a second aspect, the present invention provides the use of the aforementioned method for detecting α-methyl-D-mannoside in drug quality control or quality monitoring. Preferably, the drug is a recombinant protein drug. Further preferably, the drug is a recombinant protein vaccine.

[0025] In a third aspect, the present invention provides a method for drug quality control or quality monitoring, which employs the aforementioned method for detecting α-methyl-D-mannoside to detect the residual amount of α-methyl-D-mannoside in a drug. Preferably, the drug is a recombinant protein drug. Further preferably, the drug is a recombinant protein vaccine.

[0026] The detection method and application of α-methyl-D-mannoside provided by the present invention offer the advantages of simple pretreatment, sensitivity, rapidity, accuracy, and reliability. They are suitable for quality control or monitoring of drugs containing this impurity (e.g., recombinant protein vaccines). Compared with existing technologies, this discovery has the following beneficial effects:

[0027] (1) The mass spectrometer uses ESI negative ion mode for signal acquisition, which is more conducive to the stability of sample detection.

[0028] (2) The present invention has good detection precision and high sensitivity, with a detection limit of 0.16 ng / mL and a quantification limit of 0.49 ng / mL. It can be used to detect trace amounts of α-methyl-D-mannoside, which is beneficial to the quality control of drugs and thus ensures the safety of drug use.

[0029] (3) Compared with the existing technology of using HILIC-LC-MS / MS to analyze carbohydrates, the reversed-phase PFPP column preferred by this method is more durable than the HILIC column, the reversed-phase chromatography method is more robust, and the analysis cost is lower. Therefore, after experimental comparison, this method chose to use the reversed-phase PFPP column for analysis. The bonded phase of this column is pentafluorophenyl, which has better retention for polar compounds than the C18 column. The sample only needs to undergo a simple acetonitrile protein precipitation treatment and can be analyzed on the machine after high-speed centrifugation without removing the buffer salt in the sample. The pretreatment of this method is simple, sensitive, rapid, accurate, and reliable, and is suitable for the detection of α-methyl-D-mannoside residues in recombinant protein drugs such as recombinant protein vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : MRM spectra of α-methyl-D-mannoside obtained under different mobile phase compositions;

[0031] Figure 2 : MRM spectra of α-methyl-D-mannoside obtained under different DL temperature conditions;

[0032] Figure 3 : MRM spectra of α-methyl-D-mannoside obtained under different interface voltage conditions;

[0033] Figure 4 : MRM spectrum of α-methyl-D-mannoside obtained using Diol-HILIC column in HILIC mode;

[0034] Figure 5 : MRM spectrum of α-methyl-D-mannoside obtained using Amide column in HILIC mode;

[0035] Figure 6 : MRM spectrum of α-methyl-D-mannoside obtained using PFPP column in RP mode;

[0036] Figure 7 : MRM spectra of α-methyl-D-mannoside obtained under different gradient time program conditions;

[0037] Figure 8 :Specificity of LC-MS / MS analysis of α-methyl-D-mannoside;

[0038] Figure 9 :Calibration curve of α-methyl-D-mannoside analyzed by LC-MS / MS;

[0039] Figure 10 : Overlay of MRM profiles of α-methyl-D-mannoside in negative vaccine sample and spiked sample (20 ng / mL). DETAILED DESCRIPTION

[0040] Example 1: Experimental preparation

[0041] (1) Solution preparation

[0042] (1) Standard solution: Weigh an appropriate amount of α-methyl-D-mannoside and dilute it to 1 mg / mL with water to obtain a standard stock solution (1 mg / mL); take an appropriate amount of the above standard stock solution and dilute it to a certain degree with ultrapure water to obtain a standard dilution solution; take 100 μL of the above standard dilution solution, add 100 μL of normal saline (or no normal saline), and then add acetonitrile to make up the total volume of the solution to 1000 μL, mix well, and prepare a certain concentration of α-methyl-D-mannoside standard solution (1 ng / mL~10 μg / mL). If 100 μL of normal saline is added to the standard solution, it contains 10% normal saline, otherwise it is a salt-free sample.

[0043] (2) Test solution (negative vaccine spiked sample): Take an appropriate amount of non-recombinant protein vaccine sample (α-methyl-D-mannoside was not used in the preparation process of this vaccine sample, so it does not contain this substance, that is, "negative vaccine"), add a certain amount of α-methyl-D-mannoside (that is, "spiked"), add acetonitrile with a final concentration of 80%, mix well, centrifuge at 14000 rpm for 10 min, remove the precipitated protein, and take the supernatant for analysis.

[0044] (II) Determination of α-methyl-D-mannoside:

[0045] The standard solution to be tested was taken and α-methyl-D-mannoside was detected by LC-MS / MS. By selecting or setting different parameter conditions, the effects of different process conditions on the detection of α-methyl-D-mannoside content in the sample were investigated, as described in Examples 2 to 5 below.

[0046] Example 2: Investigation of mobile phase composition

[0047] In this example, the effects of different aqueous phase compositions on the analysis of target substances were investigated. The different aqueous phases were: (1) ultrapure water, (2) an aqueous solution containing 0.1% formic acid (prepared with ultrapure water as the solvent), and (3) an aqueous solution containing 10 mM ammonium formate (prepared with ultrapure water as the solvent). The organic phases were all acetonitrile.

[0048] Measured sample: α-Methyl-D-mannoside standard with a concentration of 10 mg / L (10 μg / mL) (without saline); injection volume: 1 μL; chromatographic column: Shim-pack Scepter Diol-HILIC-120 100 mm x 2.0 mm (Diol-HILIC), 1.9 μm.

[0049] The resulting overlapping chromatograms are shown in Figure 1 , where the upper line: the aqueous phase is ultrapure water; the middle line: the aqueous phase is an aqueous solution containing 0.1% formic acid; the lower line: the aqueous phase is an aqueous solution containing 10 mM ammonium formate. Figure 1 It can be seen that if the aqueous phase contains formic acid or ammonium formate buffer salt, the mass spectrometry signal of α-methyl-D-mannoside will be significantly suppressed, reducing the sensitivity. Therefore, the preferred aqueous phase composition is ultrapure water.

[0050] Example 3: Investigation of mass spectrometry interface parameters

[0051] In this example, the effects of different mass spectrometry interface parameters on the analysis of target substances were investigated, wherein the nebulizing gas flow rate, drying gas flow rate, heating gas flow rate, interface temperature, DL temperature, interface voltage, etc. were systematically investigated.

[0052] Measured sample: α-Methyl-D-mannoside standard with a concentration of 10 mg / L (10 μg / mL) (without saline); injection volume: 1 μL; chromatographic column: Shim-pack Scepter Diol-HILIC-120 100 mm x 2.0 mm I.D. (Diol-HILIC), 1.9 μm.

[0053] Through research, it was found that the parameters that have the greatest impact on mass spectrometry signals are DL temperature and interface voltage. Under different DL temperatures (150℃, 200℃, 250℃, 280℃) and different interface voltages (0.5kV, 1 kV, 2 kV, 3 kV, 4 kV, 5 kV), the chromatograms obtained are shown in Figure 2. Figure 2 and Figure 3 .Depend on Figure 2 It can be seen that the higher the DL temperature, the better the mass spectrum response of α-methyl-D-mannoside. The optimal DL temperature is the maximum value of 300°C. Figure 3 It can be seen that the optimal interface voltage is 3 kV, at which time the mass spectrometric response of α-methyl-D-mannoside is the best.

[0054] Example 3: Investigation of chromatographic separation modes

[0055] In this example, the effects of different separation modes and the salt content in the sample on the detection results were investigated.

[0056] (i) HILIC mode, column: Shim-pack Scepter Diol-HILIC-120 100 mm x 2.0 mm I.D. (Diol-HILIC), particle size: 1.9 μm.

[0057] Sample: α-Methyl-D-mannoside standard solution containing 1 mg / L (1 μg / mL). Two types of solutions were prepared, one containing no saline and the other containing 10% saline. Injection volume: 1 μL.

[0058] In this mode, the chromatograms corresponding to the samples without saline and with 10% saline are as follows: Figure 4 As shown in the figure, the upper line indicates the sample solvent contains no saline; the lower line indicates the sample solvent contains 10% saline. As can be seen, no α-methyl-D-mannoside peak is observed when the sample solution contains 10% saline. Because recombinant protein vaccine samples often contain buffer salts, Diol-HILIC columns in HILIC mode are difficult to use for analysis of residual α-methyl-D-mannoside in these vaccine samples.

[0059] (ii) HILIC mode, column: Shim-pack GIST AMIDE 150 mm x 2.1 mm ID (AMIDE), particle size: 3 μm.

[0060] Measured sample: α-Methyl-D-mannoside standard solution containing 50 μg / L (50 ng / mL) in 10% saline. Injection volume: 1 μL or 5 μL.

[0061] In this mode, the chromatograms of salt-containing standards at different injection volumes are as follows: Figure 5 As shown in the figure, the upper line indicates an injection volume of 1 μL; the lower line indicates an injection volume of 5 μL. It can be seen that when the sample solution contains 10% saline: (1) α-methyl-D-mannoside peaks normally when 1 μL is injected; (2) α-methyl-D-mannoside peaks do not appear when 5 μL is injected. Figure 5 and Figure 4 The comparison shows that the Amide column performs better than the Diol-HILIC column using the HILIC mode, but it is still sensitive to the salt in the sample, and the development of a robust analytical method is quite challenging.

[0062] (iii) RP mode, column: Shim-pack Velox PFPP 100 mm x 2.1 mm ID (PFPP), particle size: 1.8 μm.

[0063] Samples tested: Sample solutions containing 1 μg / L (1 ng / mL) of α-methyl-D-mannoside. Sample 1 is a standard solution without saline, Sample 2 is a standard solution containing 10% saline, and Sample 3 is a negative vaccine spiked sample (used to assess the suitability and specificity of the assay) without the use of α-methyl-D-mannoside during the manufacturing process. Injection volume: 1 μL for each sample.

[0064] In this mode, the chromatograms of different samples are as follows: Figure 6 As shown, the top line indicates a salt-free sample solution; the middle line indicates a sample solution containing 10% saline; and the bottom line indicates a negative vaccine spiked sample. Overlapping MRM spectra of the salt-free standard, the standard containing 10% saline, and the negative vaccine spiked sample demonstrate that, using the RP mode PFPP column, the chromatographic peaks for α-methyl-D-mannoside essentially overlap, demonstrating minimal influence of the sample matrix on the analysis of α-methyl-D-mannoside, good recovery, and an accurate and reliable method.

[0065] Furthermore, a comparison of the three separation mode experiments revealed that even when the target substance content in the sample was significantly reduced (1000 ng / mL → 1 ng / mL) and contained 10% saline, the detection of α-methyl-D-mannose using the PFPP column in RP mode still achieved excellent results, demonstrating that the selection of RP mode and PFPP column brought unexpected technical benefits to the trace detection of the target substance.

[0066] Example 5: Investigation of gradient elution conditions

[0067] In this example, the effects of different gradient time programs on the analysis were investigated.

[0068] Gradient conditions 1: Phase A: ultrapure water, Phase B: acetonitrile. Flow rate: 0.3 mL / min. Gradient program: 0–3 min, 5% B to 60% B; 3–4 min, 60% B to 90% B; 4–4.01 min, 90% B to 5% B; 4.01–7 min, 5% B.

[0069] Gradient conditions 2: Phase A: ultrapure water, phase B: acetonitrile. Flow rate: 0.4 mL / min. Gradient program: 0% B (0–1 min); 0% B (0% B) to 30% B (1–2 min); 30% B (30% B) to 90% B (2–3 min); 90% B (3–4 min); 90% B (4–4.01 min); 0% B (4.01–7 min).

[0070] Among them, B represents "B phase" (organic phase: acetonitrile), "5%B" means that the proportion of B phase in the mobile phase is 5%, and the remaining 95% is A phase (aqueous phase: ultrapure water), and so on.

[0071] Measured sample: α-methyl-D-mannoside standard with a concentration of 10 mg / L (10 μg / mL) (without normal saline); injection volume: 1 μL; chromatographic column: PFPP.

[0072] The obtained chromatogram is shown in Figure 7 , where the upper line (black line) is gradient condition 1 and the lower line (colored line) is gradient condition 2. Figure 7 It can be seen that when the initial mobile phase ratio is 100% ultrapure water and the flow rate is increased to 0.4 mL / min, it is helpful to obtain a good chromatographic peak shape of α-methyl-D-mannoside, which helps to improve sensitivity and repeatability.

[0073] Example 6: Study on the effectiveness of the α-methyl-D-mannoside detection method

[0074] Through Examples 2 to 5 and a series of other parameter explorations, the optimized process conditions for detecting α-methyl-D-mannoside by LC-MS / MS were obtained as follows (Tables 1 to 3):

[0075] Table 1: Chromatographic conditions

[0076]

[0077] Table 2: Mass spectrometry conditions

[0078]

[0079] Table 3: MRM parameters

[0080]

[0081] According to the chromatographic and mass spectrometric detection conditions / parameters shown in Tables 1 to 3, standard solutions and test solutions were taken to further study the effectiveness (specificity, linearity, detection limit, precision, accuracy, etc.) of the α-methyl-D-mannoside detection method.

[0082] The preparation methods of the standard solution and the test solution are as follows:

[0083] Standard solution: (1) Weigh an appropriate amount of α-methyl-D-mannoside and dilute it to 1 mg / mL with water to obtain a standard stock solution; (2) Take an appropriate amount of the above standard stock solution (1 mg / mL) and dilute it to 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 ng / mL with water to obtain a standard dilution solution; (3) Take 100 μL of the above standard dilution solutions of different concentrations, add 100 μL of physiological saline, add 800 μL of acetonitrile, mix well, and prepare standard solutions with final concentrations of α-methyl-D-mannoside of 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 ng / mL for analysis on the instrument.

[0084] Test solution: (1) Negative vaccine sample: Take an appropriate amount of recombinant protein vaccine that does not use α-methyl-D-mannoside in the preparation process, add physiological saline with a final concentration of 10% and acetonitrile with a final concentration of 80%, mix well, centrifuge at 14000 rpm for 10 min, and take the supernatant for analysis on the machine; (2) Negative vaccine spiked sample: Take an appropriate amount of the above negative vaccine, add a certain amount of α-methyl-D-mannoside, then add physiological saline with a final concentration of 10% and acetonitrile with a final concentration of 80%, mix well, centrifuge at 14000 rpm for 10 min, and take the supernatant for analysis on the machine.

[0085] (1) Specificity

[0086] Take 2ng / mL standard solution and solvent blank for determination respectively, and the MRM overlap spectrum of standard solution and solvent blank ( Figure 8 ) showed that there was no interference at the retention time of the target peak, indicating good method specificity.

[0087] (2) Linearity and range

[0088] Different concentrations of standard solutions (0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500 ng / mL) were taken for determination, and the standard curve was drawn with the peak area as the ordinate and the concentration as the abscissa. Figure 9 ), which showed that α-methyl-D-mannoside had good linearity in the concentration range of 0.5~500 μg / L (i.e., 0.5~500 ng / mL), with a correlation coefficient of 0.9992.

[0089] (III) Detection limit and quantification limit

[0090] The detection limit (S / N=3) and quantification limit (S / N=10) were calculated based on the signal-to-noise ratio using LabSolutions software. The detection limit of α-methyl-D-mannoside was 0.16 ng / mL, and the quantification limit was 0.49 ng / mL.

[0091] (IV) Precision

[0092] Six standard solutions at three different concentration levels (2 ng / mL, 20 ng / mL, and 200 ng / mL) were prepared to investigate the precision of the detection method for α-methyl-D-mannoside provided by the present invention, as well as the precision of retention time and peak area. The results are shown in Table 4. The RSDs of retention time and peak area for the three standard concentrations ranged from 0.03% to 0.05% and from 3.69% to 4.83%, respectively, indicating good method precision.

[0093] Table 4: Precision test results

[0094]

[0095] (V) Accuracy

[0096] According to the guidelines for validation of analytical methods in the Chinese Pharmacopoeia, the accuracy of the analytical method is verified by the spike recovery rate. It mainly examines whether there are other substances in the sample that interfere with the detection of the target substance, thereby affecting the accuracy of the analytical method. Generally speaking, the closer the recovery rate is to 100%, the better the accuracy.

[0097] In this experiment, a recombinant protein vaccine sample (negative vaccine sample) without α-methyl-D-mannoside in its preparation process was first tested by LC-MS / MS to confirm the presence of the target substance in this sample, with the result being not detected (ND). Next, a certain amount of α-methyl-D-mannoside was added to the negative vaccine sample to prepare three spiked samples (20 ng / mL, 200 ng / mL, and 2000 ng / mL, covering low, medium, and high levels), and the target substance was detected by LC-MS / MS. Figure 10 Overlapping chromatograms of negative vaccine sample and spiked sample (20 ng / mL) are shown in Figure 10 (Blue line: negative sample; green line: spiked sample); Table 5 shows the spike recovery results, showing that the spike recoveries for the three different concentrations ranged from 94.5% to 103.4%. Specifically, when the concentration of α-methyl-D-mannoside was 20–200 ng / mL, the spike recovery ranged from 101.5% to 103.4%, approaching 100%. Even when the concentration of α-methyl-D-mannoside in the spiked sample (2000 ng / mL) was far beyond the linear range (0.5–500 ng / mL), a good spike recovery (94.5%) was still achieved.

[0098] Table 5: Accuracy test results

[0099]

[0100] Note: ND means not detected.

[0101] The above examples show that the detection method of α-methyl-D-mannoside provided by the present invention is suitable for detecting trace amounts of α-methyl-D-mannoside in drugs, which may be recombinant protein drugs, such as recombinant protein vaccines.

[0102] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the spirit and scope of protection of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the scope of protection of the present invention.

Claims

1. A method for detecting α-methyl-D-mannoside based on liquid chromatography tandem mass spectrometry, characterized in that: Chromatographic separation was performed using a PFPP column in reverse phase mode.

2. The method according to claim 1, characterized in that The PFPP chromatographic column is a Shim-pack Velox PFPP chromatographic column.

3. The method according to claim 1, wherein In liquid chromatography, the mobile phase is ultrapure water and the organic phase is acetonitrile.

4. The method according to claim 3, characterized in that Liquid chromatography was performed with gradient elution, and the gradient program was as follows: 0–1 min, 0% B; 1–2 min, 0% B–30% B; 2–3 min, 30% B–90% B; 3–4 min, 90% B; 4~4.01min, 90%B~0%B; 4.01~7min, 0%B; where B represents the organic phase.

5. The method according to claim 1, wherein The injection volume of liquid chromatography is 1~20μL.

6. The method according to claim 1, wherein The column temperature of the liquid chromatography was 35°C.

7. The method according to claim 1, wherein The flow rate of the liquid chromatography was 0.4 mL / min.

8. The method according to claim 1, wherein The mass spectrometer was Shimadzu triple quadrupole liquid chromatography-mass spectrometry (LCMS-8050).

9. The method according to claim 1, wherein The ionization mode of the mass spectrometer was ESI negative ion mode.

10. The method according to claim 1, wherein The heating module temperature of the mass spectrometer was 400 °C.

11. The method according to claim 1, wherein The DL temperature of the mass spectrometer was 300 °C.

12. The method according to claim 1, wherein The interface voltage of the mass spectrometer was 3 kV.

13. The method according to claim 1, wherein The mass spectrometer was scanned in MRM mode.

14. The method according to claim 1, wherein The precursor ion in the mass spectrometer had an m / z of 193.

15.

15. The method according to claim 1, wherein Among the product ions of the mass spectrometer, the m / z of the quantitative ion and the qualifier ion are 101.00 and 59.05, respectively.

16. Use of the method according to any one of claims 1 to 15 in drug quality control or quality monitoring.

17. The use according to claim 16, characterized in that The drug is a recombinant protein drug.

18. A method for drug quality control or quality monitoring, characterized in that: The method according to any one of claims 1 to 15 is used to detect the residual amount of α-methyl-D-mannoside in a drug.

19. The use according to claim 18, characterized in that The drug is a recombinant protein drug.

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