Method for determining content of dimethyl sulfoxide in cell injection by high performance liquid chromatography-tandem mass spectrometry
The DMSO content in cell injection was detected by high-performance liquid chromatography tandem mass spectrometry, which solved the problems of low detection sensitivity and complex pre-processing of existing gas chromatography methods, and achieved accurate and reliable DMSO detection.
Patent Information
- Application Number
- CN202411518352.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-12-29
- Filing Date
- 2024-10-29
- Publication Date
- 2025-02-11
AI Technical Summary
When the existing gas chromatography detects the DMSO content in cell injection, it is greatly affected by the sample morphology, requires complex pretreatment, has a long cycle and low sensitivity, and cannot meet the detection requirements.
The cells were broken by high-performance liquid chromatography tandem mass spectrometry by tissue grinding, and the supernatant was taken for detection after high-speed centrifugation. Combined with the peak area ratio of DMSO and DMSO-d6, the DMSO content was calculated by linear fitting.
It realizes accurate and reliable detection of DMSO content in cell injection, simplifies the pre-processing steps, improves detection sensitivity, and meets detection requirements.
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Abstract
Description
Technical Field
[0001] The present invention relates to the fields of biology, chemistry and mass spectrometry detection, and in particular to an analytical method for detecting the content of dimethyl sulfoxide (hereinafter referred to as DMSO) in a cell injection solution by liquid chromatography-tandem mass spectrometry. Background Art
[0002] DMSO is a highly polar, high boiling point, thermally stable, non-proton, water-miscible solvent. It is a colorless, odorless, transparent liquid at room temperature. It is hygroscopic and is known as the "universal solvent". It is widely used as a solvent and reaction reagent and has a high selective extraction capacity. In addition, DMSO itself has anti-inflammatory, analgesic, diuretic, and sedative effects. In the pharmaceutical industry, it can be directly used as a raw material and carrier for certain drugs. It is known as a "panacea" and is often added to drugs as an active ingredient in analgesics.
[0003] DMSO is also an important permeable cell cryopreservation agent, which can prevent cells from being destroyed when they are stored at deep low temperatures (minus 196 degrees). During the cryopreservation process, DMSO can quickly penetrate the cell membrane and enter the cell, lowering the freezing point and delaying the cryopreservation process. At the same time, it increases the intracellular ion concentration and reduces the formation of ice crystals in the cell, thereby reducing cell damage. However, DMSO has serious toxic effects. It can react with the hydrophobic groups of proteins, causing protein denaturation and thus destroying the cell structure. At deep low temperatures, the toxicity of DMSO to cells is inhibited, but after recovery, the toxicity of DMSO to cells will become apparent. Most current studies recommend that the concentration of DMSO should be controlled below 0.1% after cell recovery.
[0004] Determining the DMSO content in the cell injection solution is an important indicator for studying whether the cell storage environment meets the requirements. It is also helpful for monitoring the residual DMSO after cell recovery, thereby ensuring that the remaining DMSO does not affect the activity of the cells. At present, the main test method for DMSO is gas chromatography-tandem mass spectrometry. However, this detection method is greatly affected by the sample morphology. Generally, the aqueous phase cannot be directly injected to detect DMSO in the cell solution. It needs to be treated with solutions such as dilution or liquid-liquid extraction. The pre-treatment of the method is complex, the cycle is long, and the sensitivity is low. When using the headspace injection method for detection, the detection sensitivity is low due to the high boiling point of DMSO. Therefore, gas chromatography has major drawbacks in sensitivity and actual operation, and cannot meet the detection requirements of DMSO content in cell injection solution.
[0005] Among all the methods reported so far, there is no report that can directly detect the DMSO content in cell injection solution by high performance liquid chromatography tandem mass spectrometry without complicated treatment. Summary of the invention
[0006] The purpose of the present invention is to provide a method for detecting DMSO in cell injection by high performance liquid chromatography tandem mass spectrometry, wherein a certain volume of cell injection is subjected to a tissue grinder to break the cells, separated by high-speed centrifugation, and the supernatant is taken for detection. This method can accurately and reliably detect DMSO in the injection.
[0007] The present invention provides the following solutions: Take appropriate amount of DMSO and DMSO-d6 and prepare reference substance and internal standard solution with water to a certain concentration; Take the cell injection solution, add the internal standard solution, break the cells and centrifuge them, and take the supernatant as the test solution; A series of DMSO standard curve solutions (including internal standard) were prepared, and the relationship between the DMSO / DMSO-d6 peak area ratio and concentration was obtained by LC-MS / MS detection, and a linear fit was performed; The test solution was tested by LC-MS / MS to obtain the DMSO / DMSO-d6 peak area ratio, and the DMSO content in the test solution was calculated by the linear fitting equation; The chromatographic conditions for LC-MS / MS detection in the present invention are as follows: the chromatographic column is Ultimate HILIC Amide, the specification is 4.6*150mm, 3μm; the column temperature is 20-40°C; the flow rate is 0.5-1.5 ml / min; the running time is 10 min; the injection volume is 2-20 μl; the mobile phase A is a 0.1% TFA aqueous solution, and the mobile phase B is an isopropanol solution; the gradient elution program is 0.0min→4.0min (mobile phase A is 2.0%→35.0%), 4.0min→7.0min (mobile phase A is 35.0%→35.0%), 7.0min→7.1min (mobile phase A is 35.0%→2.0%), 7.1min→10.0min (mobile phase A is 2.0%→2.0%); The mass spectrometry conditions for LCMSMS detection in the present invention are as follows: the ion source is an electrospray ion source (ESI+); the scanning mode is a multiple reaction monitoring (MRM) mode; the ion source temperature is 300-500°C; the collision gas is medium; the curtain gas is 35 psi; the ionpray voltage is 5000 V; the ion source gas1 is 50 psi; the ion source gas2 is 60 psi; the DMSO MRM acquisition parameters are 78.9 / 64.0 and 78.9 / 46.9, wherein the CE voltages are 20 V and 33 V, respectively, and the DP voltages are 70 V and 70 V, respectively; the DMSO-d6 MRM acquisition parameters are 84.8 / 67.0 and 84.8 / 50.0, wherein the CE voltages are 21 V and 35 V, respectively, and the DP voltages are 70 V and 70 V, respectively.
[0008] In the chromatographic conditions of the present invention, the column temperature is preferably 30°C, the flow rate is preferably 1.0 ml / min, and the injection volume is preferably 2 μl.
[0009] Furthermore, the present invention provides a method for detecting DMSO in a cell injection solution.
[0010] Furthermore, the present invention uses trifluoroacetic acid and isopropanol as mobile phases to detect DMSO in the cell injection solution. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Attached Figure 1 This is the calibration curve for DMSO detection.
[0012] Attached Figure 2 This is the MRM chromatogram for DMSO detection.
[0013] Attached Figure 3 This is the MRM chromatogram of DMSO-d6 detection.
[0014] Attached Figure 4 Specific spectra for DMSO and DMSO-d6. DETAILED DESCRIPTION
[0015] 1. Instruments and Equipment The instrument used was AB Sciex 6500 UPLC-MS / MS, which consisted of a Shimadzu 3000 ultra-high performance liquid chromatograph and a QTrap6500 mass spectrometer; the grinder used was a Precellys Evolution instrument equipped with an automatic cooling system; and the high-speed centrifuge used was a Thermo small centrifuge.
[0016] 2. Chromatographic conditions The chromatographic column was Ultimate HILIC Amide (4.6*150mm, 3μm); mobile phase A was 0.1% TFA aqueous solution; mobile phase B was isopropanol solution; column temperature was 30 ℃; flow rate was 1.0 ml / min; running time was 10 min; injection volume was 2μl; elution mode was gradient elution, the gradient is shown in Table 1.
[0017]
[0018] 3. Mass spectrometry conditions The ion source was an electrospray ion source; the ionization mode was a positive ion mode; the operation mode was multiple reaction monitoring; the ion source temperature was 500 °C; the collision gas was Medium; the curtain gas was 35 psi; the ionpray voltage was 5000 V; the ion source gas 1 was 50 psi; and the ion source gas 2 was 60 psi. Other acquisition parameters are shown in Table 2.
[0019]
[0020] 4. Solution Preparation Preparation process of reference solution: Weigh appropriate amount of DMSO and DMSO-d6 respectively, prepare standard stock solution with water, and store in a refrigerator at 4℃. Then prepare DMSO standard intermediate solution with water, and then prepare the required standard calibration solution (including internal standard) with a concentration range of 0.3μg / ml -10.0μg / ml. The internal standard concentration of DMSO-d6 in the standard calibration solution is 5.0μg / ml.
[0021] 5. Sample processing method Take an appropriate amount of sample, add internal standard and grinding beads, break the cells with a tissue grinder, centrifuge, take the supernatant, and inject for analysis.
[0022] 6. Experimental Results Calibration curve: The isotope internal standard method is used for quantification. The concentration of the standard substance is used as the x-axis, and the peak area ratio of the standard substance to the internal standard substance is used as the y-axis to establish the internal standard method calibration curve. Figure 1 It can be seen that the linear fit of DMSO in the concentration range of 0.3 μg / ml-10.0 μg / ml is good, and the linear correlation coefficients are all above 0.995.
[0023] Specificity: There is no interference peak in the DMSO color spectrum of the blank solution, there is no interference near the target peak in the DMSO color spectrum of the test solution, and the DMSO color spectrum in the reference solution and the spiked test solution shows the target peak. Figure 2-4 It can be seen that the specificity in the present invention meets the requirements.
[0024] Limit of quantification: The concentration of each substance with a signal-to-noise ratio (S / N) of not less than 10 is used as the limit of quantification of the substance, and the relevant results are given. The limits of quantification of DMSO are shown in Table 3:
[0025] Accuracy: The recovery rate of DMSO in the cell injection solution was tested, and the error (%) between the result and the marked value indicated the accuracy of the measurement result. From the data in Table 4, it can be seen that the error of the marked value at each concentration point was within the range of ±15%.
[0026]
[0027] Intermediate precision test: The RSD of DMSO concentration in 6 spiked test solutions prepared by the same person should not be greater than 15%; the RSD value of DMSO concentration in a total of 12 spiked test solutions prepared by different personnel should not be greater than 20%. The results are shown in Table 5.
[0028]
[0029] The above contents are preferred implementation cases of the present invention and are not intended to limit the scope of the present invention. For relevant detection personnel in this field, the contents and technical solutions of the present invention can be modified, or some characteristic parameters can be replaced by equivalents. Any improvements, equivalent replacements or modifications made within the ideas and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for determining the content of dimethyl sulfoxide in a cell injection solution by high performance liquid chromatography tandem mass spectrometry, characterized in that: The steps include: S1. Take appropriate amounts of DMSO and DMSO-d6 reference substance respectively, use water as diluent, and prepare DMSO reference substance solution and DMSO-d6 internal standard solution of appropriate concentrations; S2, take the cell injection solution, add DMSO-d6 internal standard solution, and prepare the test solution; S3. Prepare a series of standard curve solutions (including internal standard), detect the peak area ratios of DMSO and DMSO-d6 in the standard curve solutions by LC-MS / MS, and perform linear fitting with the concentration to obtain the relevant fitting equation; S4. Take the test solution and detect it by LC-MS / MS to obtain the peak area ratio of DMSO to DMSO-d6, and calculate the DMSO content in the test solution by the internal standard method.
2. The method for determining the content of dimethyl sulfoxide in a cell injection solution by high performance liquid chromatography tandem mass spectrometry as described in claim 1, characterized in that The LC-MS / MS chromatographic method in steps S3 and S4 is as follows: the chromatographic column is Ultimate HILICAmide, with specifications of 4.6*150mm, 3μm; the column temperature is 20~40°C; the flow rate is 0.5~1.5 ml / min; the running time is 10min; the injection volume is 2~20 μl; the mobile phase A is 0.1% TFA aqueous solution, and the mobile phase B is isopropanol solution; the gradient elution program is 0.0min→4.0min (mobile phase A is 2.0%→35.0%), 4.0min→7.0min (mobile phase A is 35.0%→35.0%), 7.0min→7.1min (mobile phase A is 35.0%→2.0%), and 7.1min→10.0min (mobile phase A is 2.0%→2.0%).
3. The method for determining the content of dimethyl sulfoxide in a cell injection solution by high performance liquid chromatography tandem mass spectrometry as described in claim 1, characterized in that The LC-MS / MS mass spectrometry method in steps S3 and S4 is as follows: the ion source is an electrospray ion source (ESI+); the scanning mode is a multiple reaction monitoring (MRM) mode; the ion source temperature is 300-500°C; the collision gas is medium; the curtain gas is 35 psi; the ionpray voltage is 5000 V; the ion source gas1 is 50 psi; the ion source gas2 is 60 psi; the DMSO MRM acquisition parameters are 78.9 / 64.0 and 78.9 / 46.9, wherein the CE voltages are 20 V and 33 V, respectively, and the DP voltages are 70 V and 70 V, respectively; the DMSO-d6 MRM acquisition parameters are 84.8 / 67.0 and 84.8 / 50.0, wherein the CE voltages are 21 V and 35 V, respectively, and the DP voltages are 70 V and 70 V, respectively.
4. The method for determining the content of dimethyl sulfoxide in a cell injection solution by high performance liquid chromatography tandem mass spectrometry as described in claim 1, characterized in that The chromatographic conditions were as follows: column temperature was 30 °C; flow rate was 1.0 ml / min; injection volume was 2 μl.