Cell culture fluid and method for detecting glucocorticoid in biomass

Abstract

The invention provides a cell culture fluid and a method for detecting glucocorticoid in biomass, and relates to the technical field of food detection. According to the detection method, ethyl acetate is used for extracting glucocorticoids in a cell culture fluid and biomass, then the cell culture biomass is purified through an HLB solid-phase extraction column, ultra-high performance liquid chromatography-tandem mass spectrometry is adopted for detection, and simultaneous detection of 40 glucocorticoids in the cell culture fluid and biomass can be achieved. The method successfully separates and accurately quantifies seven pairs of isomers difficult to separate including dexamethasone and betamethasone, and has the remarkable advantages of simplicity and convenience in operation, high accuracy, wide applicability and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a method for detecting glucocorticoids in cell culture fluid and biomass. Background Art

[0002] Glucocorticoids, also known as adrenocortical hormones, have anti-inflammatory and immunosuppressive effects. In the production of cultured meat, they help maintain cell culture stability, promote cell proliferation, regulate gene expression, enhance immune function, and inhibit apoptosis. However, excessive intake of glucocorticoids can lead to obesity, hypertension, osteoporosis, and other conditions. Currently, countries and regions such as China, Singapore, Australia, and the European Union have established maximum residue limits for glucocorticoids in animal-derived foods. my country has also designated some glucocorticoids as banned drugs. For example, GB 31650-2019, "Maximum Residue Limits of Veterinary Drugs in Food," stipulates a maximum residue limit of 0.3-2 μg / kg for betamethasone and dexamethasone in muscle, liver, and kidney of cattle, pigs, and horses. Hydrocortisone is permitted for use in food-producing animals and does not require a residue limit.

[0003] However, there is currently a lack of standards for the detection of glucocorticoids in the matrix cell culture medium and biomass of supporting cell-cultured meat products, which need further development and improvement. Summary of the Invention

[0004] In response to the shortcomings of existing technologies, the present invention provides a method for detecting glucocorticoids in cell culture fluid and biomass. By optimizing the pretreatment process and high-sensitivity detection technology, it can achieve synchronous and accurate quantification of target hormones in complex matrices, and can accurately identify 40 glucocorticoids; it provides key quality and safety evaluation detection technology for the emerging cell-cultured meat industry, filling the technical gap in systematic screening of trace residual hormones before products are put on the market, and is of great significance to promoting the standardized development of the industry and establishing a complete safety standard system.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for detecting glucocorticoids in cell culture fluid and biomass, comprising the following steps: S1. Vortex-extract the cell culture medium and biomass with an organic solvent, centrifuge to obtain the supernatant, blow dry with nitrogen, and then redissolve to obtain a solution to be enriched; S2. Using an HLB solid phase extraction column to treat the solution to be enriched to obtain a solution to be tested; S3. Detecting the test solution by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry and quantifying by internal standard method to obtain the residual amount of glucocorticoid; The liquid chromatography conditions were as follows: column: BEH Phenyl, 2.1 mm (inner diameter) × 100 mm, 1.7 µm; column temperature: 25-45°C; injection volume: 2-10 µL; mobile phase: 0.1% formic acid in water and acetonitrile; mobile phase flow rate: 0.2-0.8 mL / min; The gradient elution program was as follows: 0-6.6 min, 25%-30% acetonitrile; 6.6-9.5 min, 30%-45% acetonitrile; 9.5-12 min, 45%-60% acetonitrile; 12-13 min, 60%-90% acetonitrile; 13-14 min, 90% acetonitrile; 14-14.1 min, 90%-25% acetonitrile; 14.1-16 min, 25% acetonitrile.

[0006] Preferably, the glucocorticoid is prednisone, cortisone, hydrocortisone, methylprednisolone, fluorometholone, dexamethasone, dexamethasone acetate, betamethasone, prednisone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, flumethasone, methylprednisolone acetate, fluorometholone acetate, fludrocortisone acetate, budesonide, hydrocortisone butyrate, triamcinolone acetonide, fludrocortisone, deflazacort, hydrocortisone valerate, A combination of one or more of acetonide, halcinonide, clobetasol propionate, triamcinolone acetonide acetate, clobetasone butyrate, prednicarbate, diflorasone diacetate, amcinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, mometasone, fluocinonide acetate, and alclomethasone dipropionate.

[0007] Preferably, the specific operation in step S1 is: adding deuterated glucocorticoid internal standard solution to the cell culture medium or biomass, vortex mixing, adding 1.0 mL of ethyl acetate and vortexing, ultrasonic extraction in an ice water bath for 15 min, centrifuging at 10000 r / min at 4 ° C for 10 min, taking the supernatant, blowing dry with nitrogen, and re-dissolving with 5% v / v methanol aqueous solution to obtain the solution to be enriched.

[0008] Preferably, the HLB solid phase extraction column in step S2 is activated with ethyl acetate, methanol and water before use.

[0009] Preferably, the mass spectrometry detection conditions in the liquid chromatography-mass spectrometry in S3 are: analysis mode: electrospray positive and negative ion simultaneous scanning mode, multiple reaction monitoring; ion source temperature: 250-450°C; nebulizing gas flow rate: 2.5-4.0 L / min; drying gas flow rate: 8.0-12.0 L / min; heating gas flow rate: 8.0-12.0 L / min; heating module temperature: 250-450°C; interface temperature: 150-300°C; desolvation tube temperature: 100-350°C.

[0010] The present invention provides a method for detecting glucocorticoids in cell culture fluid and biomass, which has the following advantages over the prior art: The present invention targets a detection method for cell-cultured meat, a novel protein lacking a supporting matrix. Focusing on the cell culture fluid and biomass samples used in cell-cultured meat production, the present detection method utilizes a solid-phase extraction column to purify the biomass to be tested, resulting in a higher recovery rate of between 80% and 120%. Furthermore, the detection method of the present invention eliminates the need for cumbersome pretreatment procedures for cell culture fluid, making it simple, rapid, and highly sensitive with a high recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Extracted ion chromatograms for 40 glucocorticoid drug standard solutions; Figure 2 The total ion currents of 40 glucocorticoid drugs on three types of chromatographic columns; (a) is HSS T3; (b) is BEH-C18; (c) is BEH Phenyl; Figure 3 The liquid chromatograms of dexamethasone and betamethasone isomers in different chromatographic columns. The left figure (a) is a BEH C18 column, and the right figure (b) is a BEH Phenyl column. Figure 4 The MRM chromatograms for the multiple reaction detection of 40 glucocorticoids are shown. DETAILED DESCRIPTION

[0012] The present invention provides a method for determining 40 glucocorticoid drugs in cell culture fluid and biomass, comprising the following steps: 1) Each glucocorticoid drug standard and mixed deuterated glucocorticoid internal standard were mixed with methanol to obtain individual standard stock solutions. Each standard stock solution was then serially diluted with methanol and mixed to obtain a mixed standard intermediate solution. Finally, the mixed standard intermediate solution was diluted with methanol to obtain a series of mixed standard working solutions. The content of each glucocorticoid drug in the mixed standard working solutions was determined by liquid chromatography-mass spectrometry to obtain the corresponding linear equation. 2) After adding the internal standard to the cell culture fluid and biomass, the mixture was mixed with ethyl acetate, vortexed, and extracted by oscillation to obtain the cell culture fluid and biomass extract, respectively; 3) Centrifuge the cell culture extract, take the supernatant and blow it down with nitrogen until it is nearly dry, add methanol to reconstitute it, and filter it through a microporous membrane to obtain the sample solution; 4) Centrifuging the biomass extract, taking the supernatant and purifying it with liquid nitrogen to obtain a sample solution; 5) Detect glucocorticoids in the sample solution using liquid chromatography-mass spectrometry, and calculate the content of each glucocorticoid using a linear equation.

[0013] Glucocorticoids include prednisone, cortisone, hydrocortisone, methylprednisolone, fluorometholone, dexamethasone, dexamethasone acetate, betamethasone, prednisone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, flumethasone, methylprednisolone acetate, fluorometholone acetate, fludrocortisone acetate, budesonide, hydrocortisone butyrate, triamcinolone acetonide, fludrocortisone, deflazacort, hydrocortisone valerate Acid ester, halcinonide, clobetasol propionate, triamcinolone acetonide acetate, clobetasone butyrate, prednicarbate, diflorasone diacetate, amcinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, mometasone, fluocinolone acetate, alclomethasone dipropionate, total ion current is shown in Figure 1 ; In the present invention, mixed standard working solutions with mass concentrations of 5 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, 100.0 μg / L, and 200.0 μg / L were taken and measured under the detection conditions of the present invention. The mass concentration was used as the abscissa (X, ng / mL) and the analyte peak area was used as the ordinate (Y). A standard curve was drawn to obtain the linear equations of 40 glucocorticoid drugs as shown in Table 1 below: Table 1

[0014] The sample solution was detected by liquid chromatography-mass spectrometry, and the peak areas of the 40 glucocorticoid drugs measured were recorded as Y, which were substituted into the linear equation to obtain the corresponding mass concentration X of each glucocorticoid drug.

[0015] In the present invention, the sample matrix is ​​cell culture fluid and biomass.

[0016] In the present invention, in step 1), the mass concentration of each standard stock solution is independently 0.01 to 0.5 mg / mL, preferably 0.05 to 0.2 mg / mL, and more preferably 0.1 mg / mL.

[0017] In the present invention, the standard stock solution is preferably stored at -18°C and has a shelf life of 12 months.

[0018] In the present invention, in step 2), the mass volume ratio of the biomass to ethyl acetate is 0.05-0.5 g:0.5-2 mL, preferably 0.1-0.3 g:0.5-1.5 mL, and more preferably 0.2 g:1 mL.

[0019] In the present invention, in step 2), the mass volume ratio of the cell culture medium to ethyl acetate is 0.05-0.5 mL:0.5-2 mL, preferably 0.1-0.3 mL:0.5-1.5 mL, and more preferably 0.2 mL:1 mL.

[0020] In the present invention, in step 2), the homogenization speed is 800-12000 r / min, preferably 900-11000 r / min, more preferably 10000 r / min, and the homogenization time is 0.5-3 min, preferably 0.8-1.5 min, more preferably 1 min.

[0021] In the present invention, the homogenization is performed on a homogenizer.

[0022] In the present invention, in step 3), the centrifugal speed is 5000-12000 r / min, preferably 8000-11000 r / min, more preferably 10000 r / min, and the centrifugal time is 3-15 min, preferably 8-12 min, more preferably 10 min.

[0023] In the present invention, in step 3), the purification is sequentially performed by solid phase extraction and filtration; In the solid phase extraction, the eluent is acetonitrile, ethyl acetate, and 1:1 acetonitrile and ethyl acetate, preferably ethyl acetate and 1:1 acetonitrile and ethyl acetate, more preferably 1:1 acetonitrile and ethyl acetate; the elution volume is 4-10 mL, preferably 5-8 mL, more preferably 6 mL.

[0024] The filtration uses a nylon organic filter membrane, and the thickness of the nylon organic filter membrane is 0.15-0.3 μm, preferably 0.20-0.27 μm, and more preferably 0.22 μm.

[0025] In the present invention, in step 5), the liquid chromatography detection conditions in the liquid chromatography-mass spectrometry are: Chromatographic column: The glucocorticoids in the present invention include 7 groups of isomers, totaling 40 substances. Some isomers are extremely difficult to separate, such as betamethasone and dexamethasone. In previous studies, they were often quantified together; and the different isomers of budesonide, which exist in R and S forms, cannot be separated. Some isomers have exactly the same parameters as their parent ions, daughter ions, cone voltages, and collision energies. They cannot be distinguished by mass spectrometry conditions alone and can only be separated by liquid phase conditions. Therefore, the choice of chromatographic column is particularly important. In previous studies, C18 reverse phase columns have been widely used to separate 7-25 glucocorticoids including hydrocortisone and prednisolone. In the present invention, the separation of 40 glucocorticoids by HSS T3, C18, and BEH Phenyl chromatographic columns was compared. The total ion current diagram shows that the separation effect and response of HSS T3 and C18 columns for 40 glucocorticoids are not as good as those of BEHPhenyl columns. Figure 2 We further evaluated the separation of isomers using BEH C18 and BEH Phenyl columns. Figure 3 As shown in Figure 1, the C18 column cannot achieve complete baseline separation for six groups of isomers (such as cortisone acetate and prednisolone acetate, diflorasone diacetate and fluocinonide acetate), while the BEH Phenyl column achieves complete baseline separation of all isomers and has the highest sensitivity. The preferred BEH Phenyl column is 2.1 mm × 100 mm, 1.7 µm, such as Figure 3 As shown; Mobile phase: 0-1% formic acid aqueous solution and acetonitrile, preferably 0-0.5% formic acid aqueous solution and acetonitrile, more preferably 0.1% formic acid aqueous solution and acetonitrile; Mobile phase flow rate: 0.2~0.8 mL / min, preferably 0.2~0.5 mL / min, more preferably 0.3 mL / min; Column temperature: 25 to 45 ° C, preferably 30 to 40 ° C, more preferably 35 ° C; Injection volume: 2-10 µL, preferably 4-8 µL, more preferably 5 µL; The gradient elution program is preferably: 0-6.6 min, 25-30% acetonitrile; 6.6-9.5 min, 30-45% acetonitrile; 9.5-12 min, 45-60% acetonitrile; 12-13 min, 60-90% acetonitrile; 13-14 min, 90% acetonitrile; 14-14.1 min, 90-25% acetonitrile; 14.1-16 min, 25% acetonitrile.

[0026] In the present invention, in step 5), the mass spectrometry detection conditions in the liquid chromatography-mass spectrometry are: Analysis mode: electrospray ionization positive and negative ion simultaneous scanning mode (ESI+ / ESI-), multiple reaction monitoring (MRM); Ion source temperature: 250-450°C, preferably 300-400°C, more preferably 350°C; Atomizing gas flow rate: 2.5-4.0 L / min, preferably 2.8-3.5 L / min, more preferably 3.0 L / min; Drying gas flow rate: 8.0-12.0 L / min, preferably 9.0-11.0 L / min, more preferably 10.0 L / min; Heating gas flow rate: 8.0-12.0 L / min, preferably 9.0-11.0 L / min, more preferably 10.0 L / min; Heating module temperature: 250-450°C, preferably 350-450°C, more preferably 400°C; Interface temperature: 150-300°C, preferably 200-280°C, more preferably 250°C; Desolvation tube temperature: 100-350°C, preferably 150-250°C, more preferably 200°C.

[0027] The assay method of the present invention was investigated for matrix effect. The present invention used a standard curve prepared with a blank matrix solution and a standard curve prepared with a pure solvent to compare the slopes to evaluate the matrix effect. When the slope ratio was 0.8 to 1.2, it indicated that the matrix interference was relatively low; when the slope ratio was 0.5 to 0.8 or 1.2 to 1.5, it indicated that there was a medium-intensity matrix interference effect. When the slope ratio was less than 0.2 or greater than 1.5, it indicated that the matrix effect interference was strong. The results showed that the purification method adopted by the present invention had a weak matrix effect, with a matrix effect value between 0.71 and 1.16, and the influence of the matrix effect could be ignored.

[0028] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The instruments and reagents used in the examples are as follows: Nexera LC liquid phase system (Shimadzu, Japan); LC-MS 8050 triple quadrupole tandem mass spectrometer (Shimadzu, Japan); CR22 GIII centrifuge (Hitachi, Japan), KQ-600DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water system (Millipore, Germany); XS105 electronic analytical balance (Mettler Toledo Instrument Co., Ltd.); Vortex-5 vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen); DCY-24S water bath nitrogen blowdown apparatus (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen); JTCQ-24 solid phase extraction apparatus (Beijing Zhongxi Huada Technology Co., Ltd.); Waters BEH C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm); HLB solid phase extraction column (500 mg, 6 mL, Waters, USA).

[0030] Prednisone, cortisone, hydrocortisone, methylprednisolone, fluorometholone, dexamethasone, dexamethasone acetate, betamethasone, prednisone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, flumethasone, methylprednisolone acetate, fluorometholone acetate, fludrocortisone acetate, budesonide, hydrocortisone butyrate, triamcinolone acetonide, fludrocortisone, deflazacort, hydrocortisone valerate, haloperidol Acetonide, clobetasol propionate, triamcinolone acetonide acetate, clobetasone butyrate, prednicarbate, diflorasone diacetate, amcinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, mometasone, fluocinolone acetate, alclomethasone dipropionate, purity ≥ 95%.

[0031] A determination method for 40 glucocorticoid drugs was established using the culture medium and biomass obtained in the production of porcine adipocyte cultured meat as representative samples.

[0032] Example 1: Preparation of mixed standard solutions: Weigh 10 mg each of glucocorticoid drug standard and mixed deuterated glucocorticoid internal standard drug standard (accurate to 0.1 mg), dissolve with appropriate amount of methanol and dilute to 100 mL in a brown volumetric flask to prepare standard stock solutions with a concentration of 100 mg / L; accurately pipette 1 mL of each glucocorticoid drug standard stock solution into a 100 mL volumetric flask, dilute to the mark with methanol, and shake well to prepare a standard intermediate working solution with a concentration of 10 μg / mL; dilute the mixed deuterated glucocorticoid internal standard standard solution stepwise with methanol to the appropriate concentration; accurately pipette an appropriate amount of the mixed standard working solution and dilute with methanol to prepare a series of standard working solutions with concentrations of 5 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, 100.0 μg / L, and 200.0 μg / L, with each standard working solution containing 80 μg / L of each internal standard. Liquid chromatography-mass spectrometry was used to determine the concentration of each glucocorticoid in the mixed standard working solution. The corresponding standard curves, linear equations, and correlation coefficients are shown in Table 2. As shown in Table 2, the linear range for the 40 glucocorticoids was 10–200 ng / mL, with good linear relationships and correlation coefficients r greater than 0.99. When the mixed standard was spiked into blank samples, the limits of detection (LOD) for the 40 glucocorticoids were determined at a signal-to-noise ratio (S / N) ≥ 3. The limits of detection (LOD) for the 40 glucocorticoids in cell culture biomass were 0.3–2.2 μg / kg, and the limits of quantification (LOQ) for the 40 glucocorticoids in cell culture fluid were 0.1–2 μg / L, respectively. When the signal-to-noise ratio (S / N) ≥ 10 was determined as the limit of quantification (LOQ), the limits of quantification for the 40 glucocorticoids in cell culture biomass were 0.3–6.1 μg / kg, and the limits of quantification for the 40 glucocorticoids in cell culture fluid were 0.2–70 μg / L. The MRM chromatogram of the mixed standard working solution of 40 glucocorticoid drugs is shown in Figure 4 .

[0033] Table 2

[0034] Cell culture medium: Weigh 200 µL (accurate to 0.01 µL) of cell culture medium spiked with each glucocorticoid at a level of 10 µg / kg, vortex mix, and place in a 2 mL plastic centrifuge tube. Add 8 µL of a 1.0 mg / L mixed deuterated glucocorticoid internal standard solution, vortex mix, and then add 1.0 mL of ethyl acetate. Homogenize for 30 s, extract with oscillation for 10 min, and centrifuge at 4°C, 10,000 rpm for 10 min. Transfer the supernatant to another 2 mL centrifuge tube, blow with nitrogen until nearly dry, then add 100 µL of methanol to reconstitute the solution. Pass the tube through a microporous filter membrane for determination.

[0035] Biomass: Weigh 0.2 g (accurate to 0.0001 g) of biomass (spiked at 10 μg / kg of each glucocorticoid) into a 2 mL plastic centrifuge tube. Add 8 µL of a 1.0 mg / L mixed deuterated glucocorticoid internal standard solution, vortex to mix, add 1.0 mL of ethyl acetate, homogenize for 30 seconds, extract in an ice-water bath with ultrasonication for 15 minutes, and centrifuge at 10,000 rpm at 4°C for 10 minutes. Transfer the entire supernatant to another 2 mL centrifuge tube, purge to near dryness with nitrogen, and dilute with 2 mL of ultrapure water to prepare the stock solution. An HLB solid-phase extraction cartridge was activated with 6 mL of ethyl acetate, 6 mL of methanol, and 12 mL of ultrapure water. The stock solution was passed through the cartridge and drained. Upon analysis, elution was performed with 6 mL of elution solution. The eluate was collected, dried with nitrogen, and dissolved in 100 µL of methanol. The cartridge was passed through a 0.22 µm microporous filter membrane and then analyzed. Liquid chromatography-mass spectrometry was used to detect the content of various glucocorticoid drugs in the sample solution.

[0036] The liquid chromatography detection conditions for the glucocorticoid mixed standard working solution are: Column: BEH Phenyl, 2.1 mm (id) × 100 mm, 1.7 µm; Mobile phase: 0.1% formic acid in water and acetonitrile; Mobile phase flow rate: 0.3 mL / min; Column temperature: 35 °C; Injection volume: 5 µL; Gradient elution program: 0-6.6 min, 25-30% acetonitrile; 6.6-9.5 min, 30-45% acetonitrile; 9.5-12 min, 45-60% acetonitrile; 12-13 min, 60-90% acetonitrile; 13-14 min, 90% acetonitrile; 14-14.1 min, 90-25% acetonitrile; 14.1-16 min, 25% acetonitrile.

[0037] The mass spectrometry conditions for the glucocorticoid mixed standard working solution were as follows: positive ion scanning, multiple reaction monitoring (MRM); ion source type: electrospray ionization (ESI); ion spray voltage: 3000 V; ion source temperature: 300°C; nebulizing gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; heating block temperature: 400°C; interface temperature: 250°C; desolvation tube temperature: 200°C; The reference retention times, monitoring ion pairs, and collision energies of the 40 sample solutions are shown in Table 3: Table 3

[0038] The peak areas of the 40 glucocorticoid drugs measured were recorded as Y, and substituted into the linear equation to obtain the corresponding mass concentration X of each glucocorticoid drug.

[0039] The test was repeated 6 times, and the recovery and precision experiments were performed on the test results each time.

[0040] Example 2: The only difference between this example and Example 1 is that the addition level of each glucocorticoid drug in the cell culture medium and biomass is 20 μg / kg.

[0041] The peak areas of the 40 glucocorticoid drugs measured were recorded as Y, and substituted into the linear equation to obtain the corresponding mass concentration X of each glucocorticoid drug.

[0042] The test was repeated 6 times, and the recovery and precision experiments were performed on the test results each time.

[0043] Example 3: The only difference between this example and Example 1 is that the addition level of each glucocorticoid drug in the cell culture medium and biomass is 50 μg / kg.

[0044] The peak areas of the 40 glucocorticoid drugs measured were recorded as Y, and substituted into the linear equation to obtain the corresponding mass concentration X of each glucocorticoid drug.

[0045] The test was repeated 6 times, and the recovery and precision experiments were performed on the test results each time.

[0046] The recovery rate experiments of 40 glucocorticoid drugs added at three levels in Examples 1-3 are shown in Tables 4-5: Table 4 Average recovery of 40 glucocorticoids in biomass matrix in Examples 1 to 3

[0047] Table 5 Average recovery of 40 glucocorticoids in cell culture medium in Examples 1-3

[0048] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting glucocorticoids in cell culture fluid and biomass, characterized in that: The detection method comprises the following steps: S1. Vortex-extract the cell culture medium and biomass with an organic solvent, centrifuge to obtain the supernatant, blow dry with nitrogen, and then redissolve to obtain a solution to be enriched; S2. Using an HLB solid phase extraction column to treat the solution to be enriched to obtain a solution to be tested; S3. Detecting the test solution by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry and quantifying by internal standard method to obtain the residual amount of glucocorticoid; And the liquid chromatography conditions are: Chromatographic column: BEH Phenyl, 2.1 mm (id) × 100 mm, 1.7 µm; column temperature: 25-45°C; injection volume: 2-10 µL; mobile phase: 0.1% formic acid in water and acetonitrile; mobile phase flow rate: 0.2-0.8 mL / min; The gradient elution program was as follows: 0-6.6 min, 25%-30% acetonitrile; 6.6-9.5 min, 30%-45% acetonitrile; 9.5-12 min, 45%-60% acetonitrile; 12-13 min, 60%-90% acetonitrile; 13-14 min, 90% acetonitrile; 14-14.1 min, 90%-25% acetonitrile; 14.1-16 min, 25% acetonitrile.

2. The detection method according to claim 1, wherein The glucocorticoids are prednisone, cortisone, hydrocortisone, methylprednisolone, fluorometholone, dexamethasone, dexamethasone acetate, betamethasone, prednisone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, flumethasone, methylprednisolone acetate, fluorometholone acetate, fludrocortisone acetate, budesonide, hydrocortisone butyrate, triamcinolone acetonide, fludrocortisone, deflazacort, hydrocortisone valerate , halcinonide, clobetasol propionate, triamcinolone acetonide acetate, clobetasone butyrate, prednicarbate, diflorasone diacetate, amcinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, mometasone, fluocinonide acetate, and a combination of one or more of alclometasone dipropionate.

3. The detection method according to claim 1, wherein The specific operation in step S1 is as follows: deuterated glucocorticoid internal standard solution is added to the cell culture medium or biomass, vortexed to mix, 1.0 mL of ethyl acetate is added and vortexed, ultrasonic extraction is performed in an ice-water bath for 15 min, centrifuged at 10,000 r / min at 4°C for 10 min, the supernatant is collected, blown dry with nitrogen, and re-dissolved with 5% v / v methanol aqueous solution to prepare the solution to be enriched.

4. The detection method according to claim 1, wherein: In step S2, the HLB solid phase extraction column is activated with ethyl acetate, methanol and water before use.

5. The detection method according to claim 1, wherein The mass spectrometry detection conditions in the liquid chromatography-mass spectrometry in S3 are: Analysis mode: Electrospray positive and negative ion simultaneous scanning mode, multiple reaction monitoring; Ion source temperature: 250-450°C; nebulizer gas flow rate: 2.5-4.0 L / min; drying gas flow rate: 8.0-12.0 L / min; heating gas flow rate: 8.0-12.0 L / min; heating module temperature: 250-450°C; interface temperature: 150-300°C; desolvation tube temperature: 100-350°C.

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