A method for determining the concentration of icodextrin in rat plasma, tissue by LC-MS / MS

Abstract

This invention relates to a method for determining the concentration of ectoine in rat plasma and tissues, belonging to the field of bioanalytical technology. The method employs high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) to detect the concentration of ectoine in rat plasma and tissue samples. A 5% acetonitrile aqueous solution is used as the tissue homogenate, and 0.1% trifluoroacetic acid-acetonitrile is used as the precipitation solvent. The mobile phase in the liquid chromatography consists of trifluoroacetic acid, ammonium acetate, water, and acetonitrile, with isocratic elution. Tolbutamide is used as an internal standard for ectoine sample determination. The LC-MS / MS method for determining ectoine in biological samples established in this invention is simple to operate, highly sensitive, requires small sample volumes, and provides rapid analysis. It is suitable for pharmacokinetic studies of ectoine in vivo and meets the analytical requirements for biological samples in terms of accuracy, precision, and selectivity, as outlined in the "Technical Guidelines for Non-Clinical Pharmacokinetic Studies of Chemical Drugs."

Description

Technical Field

[0001] This invention relates to a method for determining the concentration of ectoine in rat plasma and tissues, belonging to the field of bioanalytical technology. Background Technology

[0002] Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an amino acid derivative derived from extremophilic halophilic bacteria. Ectoine possesses significant anti-inflammatory effects, significantly reducing the production of inflammatory cytokines, thereby alleviating post-organ transplant inflammatory responses and opening new treatment options for pathological conditions such as inflammatory bowel disease and chronic obstructive pulmonary disease. Ectoine can protect cellular macromolecules, reduce inflammation, and prevent apoptosis. Preclinical and clinical data have confirmed that ectoine can increase skin hydration, improve skin barrier function, and reduce inflammation, effectively relieving symptoms of atopic dermatitis. Furthermore, ectoine can be used on the nasal mucosa, significantly relieving sinusitis symptoms such as nasal congestion and mucosal irritation, without the adverse reactions associated with commonly used decongestants. Ectoine-containing eye drops can moisturize, stabilize the tear film, and relieve irritation and itching, making them a viable option for treating ocular symptoms of allergic conjunctivitis and for long-term treatment of dry eye.

[0003] Currently reported methods primarily focus on determining ectoine content in cosmetics, with no reports on methods for analyzing ectoine concentrations in biological samples such as plasma and tissues. Existing methods suffer from limitations including long retention times, high detection limits, large sample volumes required for processing, and the inability to eliminate matrix effects in biological samples, making them unsuitable for analyzing trace amounts of ectoine concentrations. Given the promising applications of ectoine in the pharmaceutical field, developing a simple, highly sensitive, low-sample-volume, and rapid method to accurately measure ectoine concentrations in SD rat plasma and tissues, and to elucidate the pharmacokinetic characteristics of ectoine in vivo, is of great significance for the application of ectoine in the pharmaceutical field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the concentration of ectoine in plasma and tissue biological samples of SD rats.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A method for determining the concentration of ectoine in rat plasma and tissue biological samples, the method comprising the following steps: (1) Plasma sample processing: Take 10 μL of plasma sample, add internal standard working solution to precipitate protein, centrifuge, and take the supernatant and mix it with 0.1% trifluoroacetic acid water.

[0006] (2) Tissue sample processing: add homogenizing solution to tissue sample at a ratio of 1:5 (w / v), homogenize tissue in cooling mode, take 10 μL of sample after homogenization, add internal standard working solution to precipitate protein, centrifuge, take supernatant and mix with 0.1% trifluoroacetic acid water.

[0007] (3) Chromatographic conditions: HILIC column, mobile phase A (trifluoroacetic acid, 10 mol / L ammonium acetate: water: acetonitrile = 1:1:950:50 (v / v)), mobile phase B (trifluoroacetic acid, 10 mol / L ammonium acetate: water: acetonitrile = 1:1:50:950 (v / v)), mobile phase ratio A:B = 35:65, column temperature: 40 ℃; injection chamber temperature: 5 ℃; injection volume: 1 µL; sample detection time: 4 min.

[0008] (4) Mass spectrometry conditions: Ion source: ESI source; positive ion mode; curtain gas pressure: 40 psi; collision gas: 8 psi; ionization voltage: 5500 psi; ion source temperature: 600 ℃; spray gas: 70 psi; auxiliary heating gas: 75 psi; multiple reaction monitoring (MRM) mode; detected ions: m / z 143.1→m / z 97.2 (ecodyne, CE is 22 v, DP is 90 v), m / z 271.1→m / z 155.1 (tolbutamide, CE is 23 v, DP is 100 v).

[0009] (5) Preparation of standard curve: Ectoin standard solutions of different concentrations were added to the homogenates of blank SD rat plasma and tissue samples, respectively. The samples were processed according to steps (1) and (2), respectively. The supernatant was taken and analyzed according to steps (3) and (4). The calibration curve was determined by plotting the relationship between peak area ratio and concentration, with a weighting factor of 1 / x. 2 The least squares regression is used to fit the data.

[0010] Preparation of the ectoine standard solution: Accurately weigh an appropriate amount of ectoine reference standard, place it in a brown glass bottle, add a certain volume of 50% methanol, dissolve it by sonication, and dilute it to a stock solution of 5.0 mg / mL. Take a certain amount of the ectoine stock solution and dilute it sequentially with 50% methanol to prepare the working solution for the standard curve.

[0011] Preparation of the internal standard working solution: Accurately weigh a certain amount of tolbutamide, place it in a brown glass bottle, and add a certain volume of acetonitrile to prepare a 1.0 mg / mL internal standard stock solution. Pipette the tolbutamide internal standard stock solution and dilute it with 0.1% trifluoroacetic acid acetonitrile to prepare a 2 μg / mL internal standard working solution.

[0012] The tissue samples included the heart, liver, spleen, lungs, kidneys, stomach, intestines (large intestine and small intestine), gonads (ovaries of female animals and testes of male animals), brain, fat, skeletal muscle, and peritoneal system (greater omentum).

[0013] The method described in this invention can be used for pharmacokinetic studies of ectoine.

[0014] The beneficial effects of this invention are as follows: This invention establishes for the first time an analytical method for ectoin in plasma and tissue samples of SD rats based on LC-MS / MS, and successfully applies it to the detection of ectoin concentration in plasma and tissues of SD rats after intravenous administration. This method has good reproducibility, simple operation, high sensitivity, and requires a small sample volume, making it suitable for large-scale sample detection. It also meets the analytical requirements for biological samples in the "Technical Guidelines for Non-Clinical Pharmacokinetic Studies of Chemical Drugs". Attached Figure Description

[0015] Figure 1 Chromatogram of double-blank samples in blank plasma of SD rats Figure 2 Chromatogram of blank plasma samples with internal standard added from SD rats Figure 3 Chromatogram of SD rat blank plasma sample with added ectoine (LLOQ concentration) Figure 4 Chromatogram of SD rat blank plasma sample with added ectoine (ULOQ concentration without internal standard) Figure 5 Standard curve of ectoine in plasma of SD rats Figure 6 Plasma drug concentration-time curve after intravenous administration in SD rats Figure 7 Chromatogram of double-blank samples in blank liver tissue of SD rats Figure 8 Chromatogram of blank liver tissue from SD rats with internal standard added Figure 9 Chromatogram of ectoine-added sample in blank liver tissue of SD rats (LLOQ concentration) Figure 10 Chromatogram of ectoine sample added to blank liver tissue of SD rats (ULOQ concentration without internal standard). Figure 11 Standard curve of ectoine in liver tissue of SD rats Figure 12 Ectocin exposure in various tissues of SD rats after intravenous administration Detailed Implementation

[0016] To make the objectives and technical solutions of the present invention clearer, the methods of the present invention are further described below. The methodological investigation of the detection method takes SD rat plasma samples and liver tissue samples as examples, and the specific implementation takes the pharmacokinetic experiment of injecting SD rats with ectoine preparations as an example. However, those skilled in the art should know that the further elaboration and examples are only used to illustrate and explain the present invention and do not limit the patent protection scope of the present invention in any way.

[0017] 1. Experimental materials 1.1 Drugs and reagents Ectoine (content: 99.97%, batch number: F009240201), internal standard tolbutamide (purity: ≥99%, batch number: C12512164, Shanghai Macklin Biochemical Co., Ltd.), acetonitrile (chromatographic grade, Thermo Fisher Scientific Inc.), methanol (chromatographic grade, Thermo Fisher Scientific Inc.), formic acid (chromatographic grade, Thermo Fisher Scientific Inc.), ammonium acetate (chromatographic grade, Shanghai Macklin Biochemical Co., Ltd.), trifluoroacetic acid (chromatographic grade, Shanghai Macklin Biochemical Co., Ltd.), water (self-made ultrapure water).

[0018] 1.2 Instruments SCIEX Exion LC-TQ4500 triple quadrupole tandem mass spectrometry system (SCIEX, USA), AUW-120D electronic analytical balance (Shimadzu, Japan), Sorvall 21R high-speed refrigerated centrifuge (Thermo Fisher Scientific Inc.), Vortex-3 vortex mixer (IKA, Germany), SCI-M96 microplate mixer (Dalong Xingchuang Experimental Instrument Co., Ltd.), M10UPU+ ultrapure water machine (Chongqing Aode Instrument Equipment Co., Ltd.), JXFSTPRP-24 automatic sample rapid grinder (Shanghai Jingxin Industrial Development Co., Ltd.).

[0019] 1.3 Animals SPF-grade SD rats, half male and half female, with a body weight of 300 ± 50 g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2021-0011. Breeding conditions: control the temperature at 20 °C to 26 °C, the daily temperature difference ≤ 4 °C, control the relative humidity at 30% to 70%. Provide about 12 / 12 hours of artificial lighting with light and dark alternation every day. If necessary for the experiment, lighting can be provided during the dark period.

[0020] 2. Experimental methods 2.1 Liquid chromatography-mass spectrometry conditions Chromatographic conditions: Nano Chrom ChromCore HILIC-Silica (3 μm, 4.6*150 mm) column; mobile phase A (trifluoroacetic acid, 10 mol / L ammonium acetate: water: acetonitrile = 1:1:950:50 (v / v)); mobile phase B (trifluoroacetic acid, 10 mol / L ammonium acetate: water: acetonitrile = 1:1:50:950 (v / v)); mobile phase ratio A:B = 35:65; column temperature: 40 ℃; injection chamber temperature: 5 ℃; injection volume: 1 µL; sample detection time: 4 min.

[0021] Mass spectrometry conditions: AB SCIEX 4500 triple quadrupole tandem mass spectrometer system; ion source: ESI source; positive ion mode; curtain gas pressure: 40 psi; collision gas: 8 psi; ionization voltage: 5500 psi; ion source temperature: 600 ℃; spray gas: 70 psi; auxiliary heating gas: 75 psi; multiple reaction monitoring (MRM) mode; detected ions: m / z 143.1→m / z 97.2 (ectoine, CE 22 V, DP 90 V), m / z 271.1→m / z 155.1 (tolbutamide, CE 23 V, DP 100 V).

[0022] 2.2 Solution Preparation 2.2.1 Preparation of Standard Solutions Accurately weigh an appropriate amount of ectoine reference standard, place it in a brown glass bottle, add a certain volume of 50% methanol, and dissolve it by sonication to prepare a stock solution of 5.0 mg / mL. Take a certain amount of the ectoine stock solution and dilute it sequentially with 50% methanol to prepare standard curve working solutions of 2000, 1600, 800, 400, 160, 50, 20, and 10 μg / mL, and high, medium, low, and lower limit of quantitation quality control working solutions of 1500, 600, 30, and 10 μg / mL.

[0023] 2.2.2 Preparation of internal standard solution Accurately weigh a certain amount of tolbutamide and place it in a brown glass bottle. Add a certain volume of acetonitrile to prepare a 1.0 mg / mL internal standard stock solution. Pipette 2 mL of the tolbutamide internal standard stock solution and dilute it with 1000 mL of 0.1% trifluoroacetic acid acetonitrile to prepare a 2 μg / mL internal standard working solution.

[0024] 2.3 Animal testing methods SD rats, half male and half female, were administered the drug via intravenous injection at a volume of 5 ml / kg and a dose of 50 mg / kg.

[0025] Plasma sample collection: Blood samples were collected from the jugular vein before medication (0 h) and at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after medication administration. The samples were placed in EDTA-K2 anticoagulant centrifuge tubes and gently shaken to thoroughly mix the blood with the anticoagulant. The blood samples were placed on ice packs and centrifuged within 2 h of collection (4 ℃, 8000 rpm, 10 min). After centrifugation, the collected plasma was temporarily stored in an ultra-low temperature freezer for later analysis.

[0026] Tissue sample collection: The following tissue types were collected: heart, liver, spleen, lung, kidney, stomach, intestines (large and small intestines), gonads (ovaries in females, testes in males), brain, fat, skeletal muscle, and peritoneal system (greater omentum). Experimental animals were anesthetized and sacrificed at 0.5, 1, and 3 hours after drug administration, and whole blood and plasma samples were collected simultaneously. All tissue types were collected from each animal. After collection, the tissues were rinsed with running water, placed in a separate container filled with pre-cooled physiological saline, blotted dry with filter paper, and then placed in a properly labeled container.

[0027] 2.4 Biological Sample Processing 2.4.1 Plasma Sample Processing After thawing the plasma sample, vortex it to mix. Take 10 μL and add 400 μL of internal standard working solution (for blank samples, add 0.1% trifluoroacetic acid acetonitrile without internal standard). Vortex to mix and centrifuge at 3500 rpm and 8℃ for 15 min. Take 150 μL of the supernatant after centrifugation and add it to a 96-well plate. Add 150 μL of 0.1% trifluoroacetic acid water, vortex to mix, and inject for analysis.

[0028] 2.4.2 Tissue Sample Processing After the tissue samples were removed from the refrigerator, homogenizing solution (5% acetonitrile aqueous solution) was added at a ratio of 1:5 (w / v). The tissue was homogenized in a cooling mode, and the homogenized samples were processed as follows.

[0029] 3. Data Processing and Analysis The non-compartmental model of Phoenix WinNonlin 8.1.1.279 software was used to calculate the pharmacokinetic parameters in plasma and various tissues, and the average value and SD value were calculated using Microsoft Office 2010.

[0030] 4. Results and Evaluation 4.1 Methodological Validation of Plasma Samples 4.1.1 Selectivity Blank Samples: Six blank plasma samples from SD rats of different sources were collected and processed according to the sample preparation method in 2.4 (the internal standard solution was replaced with 0.1% trifluoroacetic acid acetonitrile). Zero-Concentration Sample Preparation: Six blank plasma samples from SD rats of different sources were collected and processed according to the sample preparation method in 2.4. ULOQ Sample Preparation: Six blank plasma samples from SD rats of different sources were collected to prepare plasma samples containing ectoin (100,000 ng / mL), and processed according to the sample preparation method in 2.4 (the internal standard solution was replaced with 0.1% trifluoroacetic acid acetonitrile). The interference of the analytes was evaluated. The results are as follows: Figure 1-4 Endogenous substances in rat plasma did not interfere with the determination of ectoine and the internal standard tolbutamide. The retention times of ectoine and tolbutamide were 2.19 min and 1.85 min, respectively.

[0031] 4.1.2 Standard Curve Different concentrations of standard curve working solutions were diluted 20-fold with blank SD rat plasma to prepare standard curve plasma samples with concentrations of 100,000, 80,000, 40,000, 20,000, 8,000, 2,500, 1,000, and 500 ng / mL. These samples were processed according to the sample processing method in section 2.4. The calibration curve was calculated using 1 / x... 2 Weighted by the peak area ratio, a linear regression was performed on the concentration of ectoine using the peak area ratio. Within the plasma concentration range of 500–100,000 ng / mL, ectoine concentration showed a good linear relationship with the peak area ratio (R0). 2 ≥0.9912).

[0032] 4.1.3 Accuracy and Precision Different concentrations of quality control working solutions were diluted 20-fold with blank SD rat plasma to prepare standard curve plasma samples with concentrations of 75000, 30000, 1500, and 500 ng / mL. These samples were processed according to the sample preparation method in section 2.4. Six parallel samples were prepared for each concentration, and three analytical batches were analyzed over three days to assess intra-batch and inter-batch accuracy and precision. Table 1 shows that the intra-batch and inter-batch precision and accuracy of this method meet the requirements for quantitative analysis of biological samples.

[0033]

[0034] 4.1.4 Recovery rate Three QC samples at different concentrations were prepared, and six samples were analyzed in parallel for each concentration to obtain the peak area A of the normally extracted sample. Blank plasma was precipitated by adding 0.1% trifluoroacetic acid acetonitrile at a volume ratio of 1:19, vortexed, and centrifuged (3500 rpm, 8℃, 15 min). The supernatant was used as the blank matrix solution. 10 µL of a 20-fold diluted quality control working solution was added, along with 200 µL of 4000 ng / mL internal standard solution. Then, 200 µL of the blank matrix solution was added, vortexed, and centrifuged (3500 rpm, 8℃, 15 min). 150 µL of the supernatant was added to 150 µL of 0.1% trifluoroacetic acid water. Six parallel samples were prepared for each concentration to obtain the peak area B of the sample after blank matrix extraction and standard addition. The ratio of peak area A to B for the same concentration was the recovery rate, as shown in Table 2. The overall RSD of the recovery rate was less than 15%, indicating that this pretreatment method had no effect on the measurement of ectoine concentration in plasma.

[0035]

[0036] 4.1.5 Matrix effect Six blank matrix samples (190 μL each) from different individuals were taken, and 10 μL of low- and high-concentration quality control working solutions were added to each sample. The mixture was vortexed and then processed according to the sample processing method in 3.1. Each individual had three replicates. The results are shown in Table 3. The matrix effect (RE) was within ±15%, and the RSD was less than 15%, indicating that the plasma matrix had no effect on the determination of ectoine concentration.

[0037]

[0038] 4.1.6 Stability The stability of ectoine plasma samples was investigated under various experimental conditions at both low and high concentration levels. The results are shown in Table 4, indicating that the RE of ectoine under various experimental conditions was within ±15%, and the RSD value was less than 15%, indicating that its stability meets the requirements.

[0039]

[0040] 4.2 Methodological Validation of Liver Tissue Samples 4.2.1 Selectivity Blank samples: Six homogenates of blank liver tissue from SD rats of different sources were prepared according to the sample processing method in 2.4 (the internal standard solution was replaced with 0.1% trifluoroacetic acid acetonitrile). Zero-concentration sample preparation: Six homogenates of liver tissue from SD rats of different sources were prepared according to the sample processing method in 2.4. ULOQ sample preparation: Six homogenates of liver tissue from SD rats of different sources were used to prepare plasma samples of ectoin (100,000 ng / mL), which were prepared according to the sample processing method in 2.4 (the internal standard solution was replaced with 0.1% trifluoroacetic acid acetonitrile). The interference of the analytes was evaluated. The results are as follows: Figure 7-10 The response of endogenous substances in rat liver tissue was 20% lower than the limit of quantification for ectoine, but this did not affect the determination of ectoine. The retention times of ectoine and tolbutamide were 2.19 min and 1.85 min, respectively.

[0041] 4.2.2 Standard Curve Different concentrations of standard curve working solutions were diluted 20-fold with SD rat liver tissue homogenate to prepare standard curve liver tissue homogenate samples with concentrations of 100,000, 80,000, 40,000, 20,000, 8,000, 2,500, 1,000, and 500 ng / mL. These samples were processed according to the sample processing method in section 2.4. The calibration curve was calculated using 1 / x... 2 Weighted by the peak area ratio, a linear regression was performed on the concentration of ectoine. Within the matrix, the concentration of ectoine showed a good linear relationship with the peak area ratio in the range of 500–100,000 ng / mL (R0.05). 2 ≥0.9944).

[0042] 4.2.3 Accuracy and Precision Different concentrations of quality control working solutions were diluted 20-fold with SD rat liver tissue homogenate to prepare standard curve liver tissue homogenate samples with concentrations of 75000, 30000, 1500, and 500 ng / mL. These samples were processed according to the sample preparation method in section 2.4. Six replicates were prepared for each concentration to assess accuracy and precision. Table 5 shows that the precision and accuracy of this method meet the requirements for quantitative analysis of biological samples.

[0043]

[0044] 4.2.4 Stability The stability of ectoine liver tissue homogenate samples was investigated at both low and high concentration levels at room temperature for 24 hours. The results showed that the residual activity (RE) of the ectoine liver tissue homogenate samples after 24 hours at room temperature was within ±15%, and the relative standard deviation (RSD) was less than 15%, indicating that the stability met the requirements.

[0045] 4.3 Pharmacokinetics in SD Rats The validated method was used to detect ectoine concentrations in plasma and tissues to evaluate its pharmacokinetic characteristics. The plasma drug concentration-time curves are shown below. Figure 6 As shown, the ectocin exposure levels in each tissue are as follows: Figure 12 As shown, the established method has been successfully applied to detect the concentration of ectoine in plasma and tissues, and can accurately depict pharmacokinetic characteristics.

[0046] Although the embodiments of this invention have been disclosed above, they are not intended to limit the invention. Any modifications and improvements made to the above embodiments based on this invention without departing from the spirit of this invention shall fall within the scope of protection claimed in this invention.

Claims

1. A method for determining the concentration of ectoine in rat plasma and tissue biological samples, characterized in that, Specifically, the steps include the following: (1) Plasma sample processing: Take 10 μL of plasma sample, add internal standard working solution to precipitate proteins, centrifuge, and mix the supernatant with 0.1% trifluoroacetic acid water. Perform LC-MS / MS analysis on the mixed solution. The internal standard is tolbutamide; (2) Tissue sample processing: Tissue samples were added to homogenizing solution at a ratio of 1:5 (w / v), and homogenized under cooling mode. After homogenization, 10 μL of the sample was added to the internal standard working solution to precipitate the protein. After centrifugation, the supernatant was mixed with 0.1% trifluoroacetic acid water, and the mixed solution was analyzed by LC-MS / MS. The internal standard was tolbutamide. (3) Chromatographic conditions: HILIC column, mobile phase of trifluoroacetic acid, ammonium acetate, water and acetonitrile, isocratic elution, column temperature: 40℃; injection chamber temperature: 5℃; injection volume: 1 µL; sample detection time: 4 min; (4) Mass spectrometry conditions: Ion source: ESI source; Positive ion mode; curtain air pressure: 40 psi; Collision gas: 8 psi; ionization voltage: 5500 psi; ion source temperature: 600 ℃; spray gas: 70 psi; auxiliary heating gas: 75 psi; multiple reaction monitoring (MRM) mode; detected ions: m / z 143.1→m / z 97.2 (ectoine, CE 22 V, DP 90 V), m / z 271.1→m / z 155.1 (tolbutamide, CE 23 V, DP 100 V); (5) Preparation of standard curve: Ectoin standard solutions of different concentrations were added to blank SD rat plasma and tissue homogenate samples, respectively. The samples were processed according to steps (1) and (2), respectively. The supernatant was taken and analyzed according to steps (3) and (4). The calibration curve was determined by plotting the relationship between peak area ratio and concentration. The weighting factor was 1 / x. 2 The least squares regression is used to fit the data.

2. The method for determining the concentration of ectoine in rat plasma and tissue biological samples as described in claim 1, characterized in that, Take plasma samples and tissue homogenate samples, add 0.1% trifluoroacetic acid acetonitrile at a ratio of 1:40 to precipitate proteins.

3. The method for determining the concentration of ectoine in rat plasma and tissue biological samples as described in claim 1, characterized in that, The homogenate is a 5% acetonitrile aqueous solution.

4. The method for determining the concentration of ectoine in rat plasma and tissue biological samples as described in claim 1, characterized in that, The organs include the heart, liver, spleen, lungs, kidneys, stomach, intestines (large intestine and small intestine), gonads (ovaries in female animals and testes in male animals), brain, fat, skeletal muscle, and peritoneal system (greater omentum).

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