A method for detecting glucocorticoids in a cell culture medium and biomass

Abstract

The application provides a cell culture medium and a detection method of glucocorticoids in biomass, and relates to the technical field of food detection. The detection method first extracts glucocorticoids in the cell culture medium and the biomass by using ethyl acetate, then purifies the cell culture biomass through an HLB solid-phase extraction column, and adopts ultra-high performance liquid chromatography-tandem mass spectrometry detection, so that the simultaneous detection of 40 kinds of glucocorticoids in the cell culture medium and the biomass can be realized. The method successfully separates and accurately quantifies 7 pairs of difficult-to-separate isomers including dexamethasone and betamethasone, and has the remarkable advantages of simple operation, high accuracy and wide applicability.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for detecting glucocorticoids in cell culture medium and biomass. Background Technology

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

[0003] However, there is currently a lack of relevant standards for the detection of glucocorticoids in matrix cell culture media and biomass for cell-cultured meat products, which need to be further developed and improved. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for detecting glucocorticoids in cell culture media and biomass. By optimizing the pretreatment process and employing high-sensitivity detection technology, it achieves simultaneous and accurate quantification of target hormones in complex matrices, enabling the accurate identification of 40 glucocorticoids. This provides a crucial quality and safety evaluation and detection technology for the emerging cell-cultured meat industry, filling the technological gap in systematically screening for trace residual hormones before product launch. It is of great significance for promoting the standardized development of this industry and establishing a comprehensive safety standard system.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for detecting glucocorticoids in cell culture medium and biomass, the method comprising the following steps:

[0007] S1. Vortex extract the cell culture medium and biomass with an organic solvent, centrifuge to collect the supernatant, blow dry with nitrogen and then reconstitute to obtain the enrichment solution.

[0008] S2. The solution to be enriched is processed using an HLB solid-phase extraction column to obtain the solution to be tested;

[0009] S3. The solution to be tested is detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and the internal standard method is used for quantification to obtain the residual amount of glucocorticoids.

[0010] The liquid chromatography conditions were as follows: column: BEH Phenyl, 2.1 mm (inner diameter) × 100 mm, 1.7 µm; column temperature: 25-45℃; injection volume: 2-10 µL; mobile phase: 0.1% formic acid aqueous solution and acetonitrile; mobile phase flow rate: 0.2-0.8 mL / min.

[0011] The gradient elution program is 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.

[0012] Preferably, the glucocorticoid is prednisone, cortisone, hydrocortisone, methylprednisolone, fluocinolone, dexamethasone, dexamethasone acetate, betamethasone, prednisolone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, fluocinolone acetate, methylprednisolone acetate, fluocinolone acetate, fluocinolone acetate, budesonide, hydrocortisone butyrate, triamcinolone, fluocinolone acetonide, dexamethasone pentahydrate, or hydrocortisone pentahydrate. The following are combinations of one or more of the following: ester, halcinonide, clobetasol propionate, triamcinolone acetate, clobetasol butyrate, prednisone ester, difluralasone diacetate, acecinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, memetasone, fluocinolone acetonide, and aclomethasone dipropionate.

[0013] Preferably, the specific operation in step S1 is as follows: add deuterated glucocorticoid internal standard solution to cell culture medium or biomass, vortex to mix, add 1.0 mL of ethyl acetate and vortex, extract by sonication in an ice-water bath for 15 min, centrifuge at 10000 r / min at 4℃ for 10 min, take the supernatant, blow dry with nitrogen, and reconstitute with 5% v / v methanol aqueous solution to obtain the enrichment solution.

[0014] Preferably, in step S2, the HLB solid-phase extraction column is activated with ethyl acetate, methanol, and water before use.

[0015] Preferably, the mass spectrometry detection conditions in the liquid chromatography-mass spectrometry (LC-MS) in S3 are as follows: analysis mode: simultaneous scanning mode of positive and negative ions via electrospray ionization, multiple reaction monitoring; ion source temperature: 250-450℃; 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℃; interface temperature: 150-300℃; desolvation tube temperature: 100-350℃.

[0016] This invention provides a method for detecting glucocorticoids in cell culture medium and biomass, which has the following advantages compared with the prior art:

[0017] This invention addresses the lack of a suitable detection method for the novel protein, cultured meat. Focusing on cell culture medium and biomass samples in cultured meat production, the detection method of this invention utilizes a solid-phase extraction column to purify the biomass to be tested, resulting in a higher recovery rate of 80%-120%. Furthermore, the detection method of this invention for cell culture medium eliminates the need for cumbersome pretreatment processes, offering a simple, rapid, high-recovery, and highly sensitive method. Attached Figure Description

[0018] Figure 1 Ion chromatograms of standard solutions of 40 glucocorticoid drugs;

[0019] Figure 2 The total ion chromatograms of 40 glucocorticoid drugs on three types of columns are shown; (a) is HSS T3; (b) is BEH-C18; and (c) is BEH Phenyl.

[0020] Figure 3 The images show liquid chromatograms of isomers of dexamethasone and betamethasone in different columns. The left image (a) shows the BEH C18 column, and the right image (b) shows the BEH Phenyl column.

[0021] Figure 4 MRM chromatograms for the multiple reaction detection of 40 glucocorticoids. Detailed Implementation

[0022] This invention provides a method for determining 40 glucocorticoid drugs in cell culture medium and biomass, comprising the following steps:

[0023] 1) Each glucocorticoid drug standard and the mixed deuterated glucocorticoid internal standard were mixed with methanol to obtain each standard stock solution. Each standard stock solution was then diluted stepwise 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 detected by liquid chromatography-mass spectrometry, and the corresponding linear equation was obtained.

[0024] 2) After adding internal standard to cell culture medium and biomass respectively, mix with ethyl acetate, vortex to mix, and extract by shaking to obtain cell culture medium and biomass extract respectively.

[0025] 3) Centrifuge the cell culture medium extract, take the supernatant and blow it with nitrogen until nearly dry, add methanol to reconstitute, filter through a microporous membrane to obtain the sample solution;

[0026] 4) Centrifuge the biomass extract, and purify the supernatant with liquid nitrogen to obtain the sample solution;

[0027] 5) Detect glucocorticoids in the sample solution using liquid chromatography-mass spectrometry, and obtain the content of each glucocorticoid by combining linear equations.

[0028] Glucocorticoids include prednisone, cortisone, hydrocortisone, methylprednisolone, flumethonone, dexamethasone, dexamethasone acetate, betamethasone, prednisolone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, flumethasone, methylprednisolone acetate, flumethonone acetate, fludrocortisone acetate, budesonide, hydrocortisone butyrate, triamcinolone, fludrocortisone, difflux, and hydrocortisone pentamethasone. Acetates, Halcinonide, Clobetasol Propionate, Triamcinolone Acetate, Clobetasol Butyrate, Prednisolone, Difluralasone Diacetate, Ancinonide, Betamethasone Dipropionate, Beclomethasone Dipropionate, Fluticasone Propionate, Triamcinolone Diacetate, Prednisolone, Prednisolone Acetate, Betamethasone Valate, Betamethasone Acetate, Momethasone, Fluocinolone Acetate, Aclomethasone Dipropionate, Total ion chromatogram see Figure 1 ;

[0029] In this 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 measured under the detection conditions of this invention. A standard curve was plotted with mass concentration as the abscissa (X, ng / mL) and peak area of ​​the analyte as the ordinate (Y). The linear equations for 40 glucocorticoid drugs are shown in Table 1 below.

[0030] Table 1

[0031]

[0032] The sample solution was detected by liquid chromatography-mass spectrometry. The peak areas of the 40 glucocorticoid drugs were recorded as Y and substituted into the linear equation to obtain the corresponding mass concentration X of each glucocorticoid drug.

[0033] In this invention, the sample matrix consists of cell culture medium and biomass.

[0034] In this 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.

[0035] In this invention, the standard stock solution is preferably stored at -18 °C and has a shelf life of 12 months.

[0036] In this invention, in step 2), the mass-to-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.

[0037] In this invention, in step 2), the mass-to-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.

[0038] In this 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.

[0039] In this invention, the homogenization is performed on a homogenizer.

[0040] In this invention, in step 3), the centrifugation speed is 5000~12000 r / min, preferably 8000~11000 r / min, more preferably 10000 r / min, and the centrifugation time is 3~15 min, preferably 8~12 min, more preferably 10 min.

[0041] In this invention, in step 3), the purification is achieved by sequentially passing through solid-phase extraction and filtration;

[0042] In the solid-phase extraction, the eluent is acetonitrile, ethyl acetate, and a 1:1 ratio of acetonitrile and ethyl acetate, preferably ethyl acetate and a 1:1 ratio of acetonitrile and ethyl acetate, more preferably 1:1 acetonitrile and ethyl acetate; the eluent volume is 4 to 10 mL, preferably 5 to 8 mL, more preferably 6 mL.

[0043] The filtration process uses a nylon organic filter membrane with a thickness of 0.15~0.3 μm, preferably 0.20~0.27 μm, and more preferably 0.22 μm.

[0044] In this invention, in step 5), the liquid chromatography detection conditions in the liquid chromatography-mass spectrometry are as follows:

[0045] Chromatographic Column: The glucocorticoids in this invention include 40 substances in 7 groups of isomers. Some isomers are extremely difficult to separate, such as betamethasone and dexamethasone, which were often quantified together in previous studies. Furthermore, budesonide, which has R- and S-type differential isomers, cannot be separated. Some isomers have identical parent ions, daughter ions, cone voltage, and collision energies, making them indistinguishable under mass spectrometry alone and requiring liquid chromatography for separation. Therefore, the choice of chromatographic column is crucial. In previous studies, C18 reversed-phase columns have been widely used to separate 7-25 glucocorticoids, including hydrocortisone and prednisolone. In this invention, the separation of 40 glucocorticoids was compared using HSS T3, C18, and BEH Phenyl columns. The total ion chromatogram shows that the separation effect and response of HSS T3 and C18 columns for the 40 glucocorticoids are inferior to those of the BEH Phenyl column. Figure 2 As shown. We further evaluated the separation of isomers using BEH C18 and BEH Phenyl columns. Figure 3 As shown, the C18 column could not achieve complete baseline separation of the six isomers (such as cortisone acetate and prednisolone acetate, difluralasone diacetate and fluocinolone acetonide), while BEH Phenyl achieved complete baseline separation of all isomers with the highest sensitivity. The preferred BEH Phenyl column is 2.1 mm × 100 mm, 1.7 µm. Figure 3 As shown;

[0046] 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;

[0047] The mobile phase flow rate is 0.2~0.8 mL / min, preferably 0.2~0.5 mL / min, and more preferably 0.3 mL / min;

[0048] Column temperature: 25–45 °C, preferably 30–40 °C, more preferably 35 °C;

[0049] Injection volume: 2–10 µL, preferably 4–8 µL, more preferably 5 µL;

[0050] The preferred gradient elution program is 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.

[0051] In this invention, in step 5), the mass spectrometry detection conditions in the liquid chromatography-mass spectrometry are as follows:

[0052] Analysis modes: simultaneous scanning of positive and negative ions via electrospray ionization (ESI+ / ESI-), multiple reaction monitoring (MRM).

[0053] Ion source temperature: 250–450 °C, preferably 300–400 °C, more preferably 350 °C;

[0054] The atomizing gas flow rate is 2.5–4.0 L / min, preferably 2.8–3.5 L / min, and more preferably 3.0 L / min.

[0055] Drying airflow rate: 8.0–12.0 L / min, preferably 9.0–11.0 L / min, more preferably 10.0 L / min;

[0056] Heating gas flow rate: 8.0–12.0 L / min, preferably 9.0–11.0 L / min, more preferably 10.0 L / min;

[0057] Heating module temperature: 250~450 ℃, preferably 350~450 ℃, more preferably 400 ℃;

[0058] Interface temperature: 150~300 ℃, preferably 200~280 ℃, more preferably 250 ℃;

[0059] The temperature of the desolventizing tube is 100–350 °C, preferably 150–250 °C, and more preferably 200 °C.

[0060] The matrix effect of the determination method of this invention was investigated. This invention uses a standard curve prepared with a blank matrix solution and a standard curve prepared with pure solvent to evaluate the matrix effect by comparing their slopes. A slope ratio of 0.8–1.2 indicates a low degree of matrix interference; a slope ratio of 0.5–0.8 or 1.2–1.5 indicates a moderate matrix interference effect. A slope ratio less than 0.2 or greater than 1.5 indicates a strong matrix interference effect. The results show that the purification method adopted in this invention has a weak matrix effect, with matrix effect values ​​between 0.71 and 1.16, and the influence of the matrix effect can be ignored.

[0061] The technical solutions provided by the present invention will be 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.

[0062] The instruments and reagents used in the examples are as follows: Nexera LC liquid chromatography system (SHIMADZU Corporation, Japan); LC-MS 8050 triple quadrupole tandem mass spectrometer (SHIMADZU Corporation, Japan); CR22 GIII centrifuge (HITACHI Corporation, Japan); KQ-600DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Milli-Q ultrapure water system (Millipore GmbH, Germany); XS105 electronic analytical balance (Mettler Toledo Instruments Ltd.); Vortex-5 vortex mixer (Haimen Qilinbei Instrument Manufacturing Co., Ltd.); DCY-24S water bath nitrogen blowing apparatus (Haimen Qilinbei Instrument Manufacturing Co., Ltd.); JTCQ-24 solid phase extraction apparatus (Beijing Zhongxi Huada Technology Co., Ltd.); Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm); HLB solid phase extraction column (500 mg, 6 mL, Waters Corporation, USA).

[0063] Prednisone, Cortisone, Hydrocortisone, Methylprednisolone, Fluorometasol, Dexamethasone, Dexamethasone Acetate, Betamethasone, Prednisolone Acetate, Cortisone Acetate, Hydrocortisone Acetate, Beclomethasone, Fluorometasol, Methylprednisolone Acetate, Fluorometasol Acetate, Fluorocortisone Acetate, Budesonide, Hydrocortisone Butyrate, Triamcinolone, Fluorocortisone, Difucoxate, Hydrocortisone Valate, Hashi Sinemet, clobetasol propionate, triamcinolone acetate, clobetasol butyrate, prednisolone, difluralasone diacetate, ansinemet, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, memetazone, fluocinolone acetonide acetate, aclomethasone dipropionate, with a purity ≥95%.

[0064] A method for determining 40 glucocorticoid drugs was established using the culture medium and biomass obtained during the production of pork from porcine fat cell culture as representative samples.

[0065] Example 1:

[0066] Preparation of mixed standard solutions: Weigh 10 mg (accurate to 0.1 mg) each of glucocorticoid drug standards and mixed deuterated glucocorticoid internal standard standards, dissolve them in an 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, shake well, and prepare a standard intermediate working solution with a concentration of 10 μg / mL. The mixed deuterated glucocorticoid internal standard solution is serially diluted with methanol to an appropriate concentration. Accurately transfer an appropriate amount of the mixed standard working solution and dilute it 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. The content of each glucocorticoid drug in the mixed standard working solution was determined by liquid chromatography-mass spectrometry (LC-MS). The corresponding standard curves, linear equations, and correlation coefficients are shown in Table 2. Table 2 shows that the linear range of the 40 glucocorticoids was 10–200 ng / mL, exhibiting good linearity, with correlation coefficients (r) all greater than 0.99. When the mixed standard was added to the blank sample, the limit of detection (LOD) was defined as the concentration at which the signal-to-noise ratio (S / N) was ≥3. The LOD for the 40 glucocorticoids in cell culture biomass was 0.3–2.2 μg / kg, and the limit of quantitation (LOQ) in cell culture medium was 0.1–2 μg / L. The limit of quantitation (LOQ) was defined as the concentration at which the signal-to-noise ratio (S / N) was ≥10. The LOQ for the 40 glucocorticoids in cell culture biomass was 0.3–6.1 μg / kg, and the LOQ in cell culture medium was 0.2–70 μg / L. The MRM chromatogram of a mixed standard working solution of 40 glucocorticoid drugs is shown below. Figure 4 .

[0067] Table 2

[0068]

[0069] Cell culture medium: Weigh 10 μg / kg of each glucocorticoid drug and 200 µL (accurate to 0.01 µL) of cell culture medium and vortex mix them into a 2 mL plastic centrifuge tube. Add 8 µL of 1.0 mg / L mixed deuterated glucocorticoid internal standard solution, vortex mix, then add 1.0 mL of ethyl acetate, homogenize for 30 s, shake to extract for 10 min, centrifuge at 4℃ and 10,000 r / min for 10 min, transfer the supernatant to another 2 mL centrifuge tube, blow with nitrogen to near dryness, add 100 μL of methanol to reconstitute, filter through a microporous membrane, and wait for analysis.

[0070] Biomass: Weigh 0.2 g (accurate to 0.0001 g) of biomass at a spiking level of 10 μg / kg for each glucocorticoid drug into a 2 mL plastic centrifuge tube. Add 8 µL of 1.0 mg / L mixed deuterated glucocorticoid internal standard solution, vortex to mix, add 1.0 mL of ethyl acetate, homogenize for 30 s, sonicate in an ice-water bath for 15 min, centrifuge at 4 ℃ and 10000 r / min for 10 min, transfer the supernatant to another 2 mL centrifuge tube, blow dry with nitrogen until nearly dry, and dilute with 2 mL of ultrapure water to prepare the stock solution. Activate the HLB solid-phase extraction column sequentially with 6 mL of ethyl acetate, 6 mL of methanol, and 12 mL of ultrapure water. Pass the biomass stock solution through the column and dry it under vacuum. For analysis, elute with 6 mL of elution solution, collect the eluent, blow dry with nitrogen, and dissolve it in 100 µL of methanol. Filter through a 0.22 µm microporous membrane for analysis. The content of various glucocorticoid drugs in the sample solution was determined by liquid chromatography-mass spectrometry.

[0071] The liquid chromatography detection conditions for the mixed standard working solution of glucocorticoids are as follows:

[0072] Column: BEH Phenyl, 2.1 mm (inner diameter) × 100 mm, 1.7 µm;

[0073] Mobile phase: 0.1% formic acid aqueous solution and acetonitrile;

[0074] Mobile phase flow rate: 0.3 mL / min;

[0075] Column temperature: 35 ℃;

[0076] Injection volume: 5 µL;

[0077] 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.

[0078] The mass spectrometry conditions for the mixed standard working solution of glucocorticoids were as follows: positive ion scan, multiple reaction monitoring (MRM); ion source type: electrospray ionization (ESI); ion spray voltage: 3000 V; ion source temperature: 300 °C; nebulizer gas flow rate: 3.0 L / min; drying gas flow rate: 10.0 L / min; heating gas flow rate: 10.0 L / min; heating module temperature: 400 °C; interface temperature: 250 °C; desolvation tube temperature: 200 °C.

[0079] The reference retention times, monitored ion pairs, and collision energies for 40 sample solutions are shown in Table 3.

[0080] Table 3

[0081]

[0082] The peak areas of 40 glucocorticoid drugs were measured and denoted as Y. Substituting these values ​​into the linear equation, the corresponding mass concentration X of each glucocorticoid drug was obtained.

[0083] The test was repeated 6 times, and recovery rate and precision experiments were performed on the results of each test.

[0084] Example 2:

[0085] The only difference between this embodiment and Example 1 is that the addition level of each glucocorticoid drug in the cell culture medium and biomass is 20 μg / kg.

[0086] The peak areas of 40 glucocorticoid drugs were measured and denoted as Y. Substituting these values ​​into the linear equation, the corresponding mass concentration X of each glucocorticoid drug was obtained.

[0087] The test was repeated 6 times, and recovery rate and precision experiments were performed on the results of each test.

[0088] Example 3:

[0089] The only difference between this embodiment and Example 1 is that the addition level of each glucocorticoid drug in the cell culture medium and biomass is 50 μg / kg.

[0090] The peak areas of 40 glucocorticoid drugs were measured and denoted as Y. Substituting these values ​​into the linear equation, the corresponding mass concentration X of each glucocorticoid drug was obtained.

[0091] The test was repeated 6 times, and recovery rate and precision experiments were performed on the results of each test.

[0092] The recovery rates of 40 glucocorticoid drugs at 3 levels in Examples 1-3 are shown in Tables 4-5:

[0093] Table 4. Average recovery rates of 40 glucocorticoids in biomass matrices in Examples 1-3.

[0094]

[0095] Table 5. Average recovery rates of 40 glucocorticoids in cell culture medium in Examples 1-3.

[0096]

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting glucocorticoids in cell culture medium and biomass, characterized in that, The detection method includes the following steps: S1. The cell culture medium and biomass were extracted separately by vortex extraction with ethyl acetate, the supernatant was collected by centrifugation, dried under nitrogen and then reconstituted to obtain the cell culture medium test solution and the biomass enrichment solution, respectively. S2. The biomass enrichment solution was processed using an HLB solid-phase extraction column to obtain the biomass test solution. S3. The solution to be tested is detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and the internal standard method is used for quantification to obtain the residual amount of glucocorticoids. And the liquid chromatography conditions are: Chromatographic column: BEH Phenyl, 2.1 mm (inner diameter) × 100 mm, 1.7 µm; column temperature: 25-45℃; injection volume: 2-10 µL; mobile phase: 0.1% formic acid aqueous solution and acetonitrile; mobile phase flow rate: 0.2-0.8 mL / min; The gradient elution program is 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, characterized in that, The glucocorticoids mentioned are prednisone, cortisone, hydrocortisone, methylprednisolone, fluocinolone, dexamethasone, dexamethasone acetate, betamethasone, prednisolone acetate, cortisone acetate, hydrocortisone acetate, beclomethasone, fluocinolone, methylprednisolone acetate, fluocinolone acetate, fluocinolone acetate, budesonide, hydrocortisone butyrate, triamcinolone, fluocinolone acetonide, dexamethasone valerate, and hydrocortisone valerate. The following are combinations of one or more of the following: halcinonide, clobetasol propionate, triamcinolone acetate, clobetasol butyrate, prednisone ester, difluralasone diacetate, acecinonide, betamethasone dipropionate, beclomethasone dipropionate, fluticasone propionate, triamcinolone diacetate, prednisolone, prednisolone acetate, betamethasone valerate, betamethasone acetate, memetazone, fluocinolone acetonide, and aclomethasone dipropionate.

3. The detection method according to claim 1, characterized in that, The specific operation in step S1 is as follows: add deuterated glucocorticoid internal standard solution to cell culture medium or biomass, vortex to mix, add 1.0 mL of ethyl acetate and vortex, extract by sonication in an ice-water bath for 15 min, centrifuge at 10000 r / min at 4℃ for 10 min, take the supernatant, blow dry with nitrogen, and reconstitute with 5% v / v methanol aqueous solution to prepare the cell culture medium test solution or biomass enrichment solution.

4. The detection method according to claim 1, characterized in that: 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, characterized in that, The mass spectrometry detection conditions in liquid chromatography-mass spectrometry in S3 are as follows: Analysis mode: simultaneous scanning mode of positive and negative ions via electrospray ionization, multiple reaction monitoring; Ion source temperature: 250-450℃; atomizing 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℃; interface temperature: 150-300℃; desolvation tube temperature: 100-350℃.

Images