Method for determining content of radix morindae officinalis oligosaccharide 5-glycan in blood plasma

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for determining the content of radix morindae officinalis oligosaccharide 5-glycan in blood plasma. According to the method, octadecylsilane chemically bonded silica is taken as a filling agent, an aqueous solution containing 45-55 mmol / L ammonium acetate and 0.08-0.12% ammonia water is taken as a mobile phase A, acetonitrile containing 0.08-0.12% ammonia water is taken as a mobile phase B for gradient elution, and interference of endogenous substances in a sample body is eliminated under a specific elution procedure, so that effective separation of oligosaccharides, pentaglycans, internal standard substances and interferents of radix morindae officinalis is realized; the detection accuracy of the radix morindae officinalis oligosaccharide 5-glycan is remarkably improved, and the precision and the accuracy of the method are not influenced by a plasma anticoagulant.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and particularly relates to a method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma. Background Art

[0002] Morinda officinalis oligosaccharide capsule is currently the only antidepressant Chinese medicine with oligosaccharide monomers as the main ingredient. It is mainly used to treat mild and moderate depression, with clear clinical efficacy and good safety. The oligosaccharide component in Morinda officinalis is mainly inulin-type oligosaccharide (GFn), which is a linear straight-chain oligosaccharide formed by (1→2)-β-D-fructofuranose glycosidic bond. One end of it is sucrose, and the sugar chain growth group is β-furanose. The end carries a glucose residue, and the degree of polymerization is usually 2 to 60. Although the degree of polymerization increases, the growth group and the connection mode remain unchanged. Morinda officinalis oligosaccharide penta-glycan (1F-fructofuranosyl nastose) is one of them and is also one of the effective substances of Morinda officinalis oligosaccharide capsule. Morinda officinalis oligosaccharide capsule usually needs to be taken twice a day for 6 consecutive weeks. Therefore, in order to delay the release time, improve the therapeutic effect and reduce toxic and side effects, pharmaceutical workers are committed to developing new processes for Morinda officinalis oligosaccharide capsules.

[0003] The establishment of relevant component detection methodology is the premise and necessary step for pharmacokinetic and toxicokinetic studies, and plays a vital role. Oligosaccharides are difficult to separate and quantify due to their strong polarity and lack of ultraviolet and fluorescence absorption characteristics; biological samples are more complex, and often contain pigments, proteins, soluble pectin, starch, organic acids, amino acids, tannins and other substances, which result in poor separation, poor peak shape and severe tailing in analytical determination; the above problems make the determination of the content of Morinda officinalis oligosaccharide 5 glycan in biological samples extremely challenging, and no relevant research methods have been reported so far, which hinders the study of pharmacokinetics and toxicokinetics of Morinda officinalis oligosaccharide capsules. Therefore, the pharmacokinetic and toxicokinetic studies of Morinda officinalis oligosaccharide drug preparations such as Morinda officinalis oligosaccharide capsules in vivo require the development of a method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma that is specific to the analyte and can exclude the interference of endogenous substances in the sample with sufficient accuracy and precision. Summary of the invention

[0004] To this end, the technical problem to be solved by the present invention is that the concentration detection method in the prior art has the phenomena of poor separation, poor peak shape and severe tailing, resulting in a high limit of quantification and low sensitivity, and is unable to accurately determine the concentration of low-content Morinda officinalis oligosaccharide 5 glycan in plasma samples, thereby providing a method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma.

[0005] In order to solve the above technical problems, the present invention provides a method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma, comprising the following steps:

[0006] (1) Mixing blank plasma and standard working solutions of Morinda officinalis oligosaccharide 5 glycans of different concentrations to prepare calibration standards; mixing the plasma sample to be tested and the calibration standard with the internal standard in the presence of an acetonitrile aqueous solution, and then performing solid-liquid separation respectively, and taking the liquid to obtain a plasma solution to be tested and a standard plasma solution;

[0007] (2) using a liquid chromatography-mass spectrometry method to detect the test plasma solution and the standard plasma solution, and calculating the content of Morinda officinalis oligosaccharide 5-glycan in the test plasma sample;

[0008] The chromatographic conditions of the LC-MS include:

[0009] Octadecylsilane bonded silica gel was used as filler, an aqueous solution containing 45-55 mmol / L ammonium acetate and 0.08-0.12% ammonia was used as mobile phase A, and acetonitrile containing 0.08-0.12% ammonia was used as mobile phase B for gradient elution. The gradient elution procedure was as follows:

[0010] 0→0.5min, the volume percentage of mobile phase A is 25%, and the volume percentage of mobile phase B is 75%;

[0011] 0.5min→3min, the volume percentage of mobile phase A increased from 25% to 45%, and the volume percentage of mobile phase B decreased from 75% to 55%;

[0012] 3min→4min, the volume percentage of mobile phase A is 45%, and the volume percentage of mobile phase B is 55%;

[0013] 4min→4.1min, the volume percentage of mobile phase A decreased from 45% to 25%, and the volume percentage of mobile phase B increased from 55% to 75%;

[0014] 4.1min→6min, the volume percentage of mobile phase A is 25%, and the volume percentage of mobile phase B is 75%.

[0015] Furthermore, the plasma sample to be tested is a plasma sample of a patient taking a pharmaceutical preparation containing Morinda officinalis oligosaccharide;

[0016] Preferably, the dosage form of the pharmaceutical preparation is capsule, solution, suspension, lyophilized powder, granules or tablets;

[0017] Preferably, the plasma sample is a rat plasma sample.

[0018] Furthermore, the chromatographic conditions of the LC-MS also include: a flow rate of 0.4-0.6 mL / min, a column temperature of 45-55° C.; and / or, a chromatographic column with a specification of 2.1 mm×100 mm, 1.7 μm.

[0019] Furthermore, the solid-liquid separation is centrifugation.

[0020] Furthermore, the mass spectrometry conditions of the LC-MS / MS include:

[0021] Mass analyzer: electrospray ionization source, ion source temperature of 150℃, capillary voltage of 2kV, desolvation gas temperature of 550℃, desolvation gas flow rate of 1000L / Hr, cone gas flow rate of 150L / Hr, nebulizer gas pressure of 7.0Bar, collision gas argon flow rate of 0.15mL / min, cone voltage of Morinda officinalis oligosaccharide 5-glycan and internal standard of 84V and 18V respectively; secondary collision energy of 36eV and 16eV respectively; negative ion detection; scanning mode of multiple reaction monitoring.

[0022] Furthermore, the internal standard is acarbose; and / or the volume percentage of the acetonitrile aqueous solution is 75%-90%.

[0023] Furthermore, the ion reactions used for quantitative analysis were: Morinda officinalis oligosaccharide 5 glycan: m / z 827.22→178.95; internal standard: m / z 644.152→320.109.

[0024] In step (1), the plasma sample to be tested and the calibration standard are respectively mixed with an internal standard in the presence of an acetonitrile aqueous solution. Preferably, the concentration of the acetonitrile aqueous solution containing the internal standard is 50 to 200 ng / mL.

[0025] Furthermore, in step (1), the volume ratio of blank plasma to standard working solutions of different concentrations of Morinda officinalis oligosaccharide 5 polysaccharide is 15 to 25:5.

[0026] Furthermore, in step (1), the volume ratio of the plasma sample to be tested to the solution containing the internal standard is 25:70-80; and the volume ratio of the calibration standard to the solution containing the internal standard is 25:70-80.

[0027] Furthermore, the concentration of Morinda officinalis oligosaccharide 5-glycan in the calibration standard is 5-1000 ng / mL.

[0028] Furthermore, calculating the content of Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested includes: collecting the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard in the standard plasma solution and the plasma solution to be tested, and performing linear regression on the ratio of the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard obtained from the standard plasma solution and the concentration of Morinda officinalis oligosaccharide 5 glycan in the calibration standard to generate a standard curve and a linear regression equation; substituting the ratio of the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard obtained from the plasma solution to be tested into the linear regression equation to obtain the content of Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested.

[0029] Correcting the weighed weight with the content of the reference substance means: obtaining the correction weight according to the content of the reference substance in the standard substance instructions, and the volume diluted to the scale is the corresponding correction volume. For example, if the weighed standard substance has a content of 99%, then the correction weight of weighing 100 mg is 99 mg, and 99 mL of volume is required to prepare a 1 mg / mL solution.

[0030] 1. The above technical scheme of the present invention has the following advantages over the prior art: the method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma of the present invention comprises mixing blank plasma and standard working solutions of Morinda officinalis oligosaccharide 5 glycan with different concentrations to prepare calibration standards; taking a plasma sample to be tested and a calibration standard and mixing them with an internal standard in the presence of an acetonitrile aqueous solution, respectively, and then performing solid-liquid separation respectively, taking liquids to obtain a plasma solution to be tested and a standard plasma solution; using a liquid-mass spectrometry method to detect the plasma solution to be tested and the standard plasma solution, and calculating the content of Morinda officinalis oligosaccharide in the plasma sample to be tested. 5 glycan content; and by using octadecylsilane bonded silica gel as a filler, an aqueous solution containing 45-55mmol / L ammonium acetate and 0.08-0.12% ammonia water as mobile phase A, and acetonitrile containing 0.08-0.12% ammonia water as mobile phase B for gradient elution, and under a specific elution program, the interference of endogenous substances in the sample body is eliminated, and the effective separation of Morinda officinalis oligosaccharide 5 glycan, internal standard and interference is achieved, and the accuracy of Morinda officinalis oligosaccharide 5 glycan detection is significantly improved, and the precision and accuracy of the method are not affected by plasma anticoagulants. The test method established by the present invention has good selectivity, stability, injection reproducibility, accuracy and precision, and the residual and matrix effect effects meet the requirements. The established liquid phase and chromatographic conditions can determine the Morinda officinalis oligosaccharide 5 glycan in the quantitative range of 10ng / mL to 1000ng / mL. The method has a good linear relationship, and the sample is diluted 10 or 100 times without affecting the measurement results.

[0031] 2. The selected Morinda officinalis oligosaccharide 5-glycan of the present invention has the largest content in the total sugars of Morinda officinalis oligosaccharides, is the quality marker and main active ingredient of Morinda officinalis oligosaccharide pharmaceutical preparations, and has certain representativeness. Taking it as an investigation indicator, detecting its concentration in rat plasma can better reflect the accumulation of Morinda officinalis oligosaccharide capsules and related preparations containing Morinda officinalis oligosaccharides in rat plasma, and can be applied to the research on the metabolism, pharmacokinetics and toxicokinetics of related drugs, to evaluate the exposure degree of Morinda officinalis oligosaccharide capsules and related preparations containing Morinda officinalis oligosaccharides at different dosage levels in rats and their pharmacokinetic and toxicokinetic characteristics, and to investigate the effectiveness and safety of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0033] Figure 1 is the chromatogram of the blank sample;

[0034] Figure 2 The chromatogram of the standard plasma solution prepared after processing the calibration standard (CAL STD6);

[0035] Figure 3 The chromatogram of the plasma solution to be tested prepared by processing the plasma sample to be tested collected 2 hours after administration;

[0036] Figure 4 This is the chromatogram of the standard plasma solution prepared after LLOQ sample treatment;

[0037] Figure 5 The chromatogram of Morinda officinalis oligosaccharide 5-glycan obtained after selective sample S2 treatment;

[0038] Figure 6 This is the ion full scan of Morinda officinalis oligosaccharide 5 glycan;

[0039] Figure 7 This is the full scan of the ions of the internal standard;

[0040] Figure 8 is the relationship curve between the content of Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested and time;

[0041] Fig. 9 is the chromatogram of the first mobile phase;

[0042] Fig.10 is the chromatogram of the second mobile phase;

[0043] Fig.11 This is the chromatogram after multiple pre-tests of the second mobile phase;

[0044] Fig.12 This is the chromatogram after multiple pre-tests of the third mobile phase.

[0045] in, Figure 1-4 , Figure 9-11 In the figure, the upper figure is the chromatogram of Morinda officinalis oligosaccharide 5-glycan; the lower figure is the chromatogram of the internal standard. DETAILED DESCRIPTION

[0046] The technical features of the present invention are further described below in conjunction with specific embodiments.

[0047] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0048] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0049] 1. Instruments and reagents

[0050] 1) LC-MS / MS: Waters, USA, ACQUITY UPLC I-Class PLUS system including binary solvent manager, sample manager-Flow Through Needle autosampler, column heater-A, Xevo TQ-XS tandem quadrupole mass spectrometer, ZSPRAY ESCI ion source, MassLynx 4.2;

[0051] 2) Liquid dispenser: Eppendorf, Switzerland, 10μL, 50μL, 100μL, 200μL, 1000μL, 5000μL; various specifications of pipette tips;

[0052] 3) Centrifuge: Eppendorf 5415R;

[0053] 4) Ultrapure water machine: Millipore Milli-Q Advantage A10;

[0054] 5) Electronic balance: AX 205 (Max 220g), PB 203-N (Max 210g), Mettler Toledo, Switzerland MSA36S-0CE-DH (Max 31g), Sartorius, Germany;

[0055] 6) Vortex instrument: Scientific Industries, USA Vortex-GENIE2 G560EVortex-GENIE2Digital SI-A246;

[0056] 7) Measuring cylinder 100mL, 500mL;

[0057] 8) Ordinary refrigerator: Haier BCD-651WDCHU1(BX-040);

[0058] 9) Ultra-low temperature refrigerator: Haier DWL-86L728J (BX-033), Haier DWL-86L626 (BX-001);

[0059] 10) Electronic temperature and humidity recorder: ZOGLAB DSR-TT LA Standard Version 2;

[0060] Morinda officinalis oligosaccharide 5-polysaccharide (1F-Fructofuranosyl nystose); molecular formula: C30H52O26; molecular weight: 828.72; content: 94.2%; batch number: 111965-201501; packaging specification: 20 mg / piece; storage conditions: store in a cold place (2-10℃); manufacturer: China Food and Drug Inspection Institutes.

[0061] Internal standard (IS), name: Acarbose; molecular formula: C25H43NO18; molecular weight: 645.6; content: 98.3%; batch number: 100808-202106; packaging specification: 200 mg / tube; storage conditions: protected from light, sealed, stored at 10-30°C; manufacturer: China Food and Drug Inspection Institutes.

[0062] SD rat blank individual plasma: prepared by our center, blood was collected from SD rats under blank anesthesia, anticoagulated with heparin sodium, and separated by centrifugation at 4000g for 10 min. The plasma was stored at -70℃ or below for a long term and stored at -20℃ for a short term after aliquoting.

[0063] SD rat blank mixed plasma: 23 male SD rats and 6 female rats were selected, blood was collected under anesthesia, anticoagulated with heparin sodium, and plasma was separated by centrifugation at 4000g for 10 min. The plasma from different rats was mixed to prepare blank mixed plasma.

[0064] Morinda officinalis oligosaccharide capsules, active ingredient content: each capsule contains 150 mg of Morinda officinalis oligosaccharides, other ingredients and content: each capsule contains 150 mg of excipient microcrystalline cellulose, properties: off-white to light yellow granules, specifications: each capsule contains 0.3g (containing 150 mg of Morinda officinalis oligosaccharides), packaging: plastic blister packaging, 10 capsules / plate × 2 plates / box, batch number: 22161005, production date: 2022.4.19, valid until: 2024.3, manufacturer: Tong Ren Tang Pharmaceutical Factory, Beijing Tong Ren Tang Co., Ltd. Morinda officinalis oligosaccharide suspension preparation: measure a certain volume of ultrapure water into a beaker, draw a line at the liquid surface, pour out all the ultrapure water, weigh a certain amount of Morinda officinalis oligosaccharide capsules into the beaker, add a certain amount of ultrapure water to the line (stir with a glass rod while adding water), and then stir with a magnetic stirrer for about 10 minutes to mix. The stirring speed can be adjusted according to the volume and concentration of the preparation. The judgment standard is no bubbles and visually uniform stirring. The test preparations with concentrations of 3.33, 10.7, and 33.3 mg / mL (calculated according to Morinda officinalis oligosaccharide content) were obtained.

[0065] 2. General method of data representation

[0066] 1) The peak area data should be rounded to 2 decimal places, the peak area ratio data should be rounded to 3 decimal places, and the retention time data should be rounded to 2 decimal places;

[0067] 2) The unit of concentration or content of plasma samples is ng / mL, and the data should be kept to 3 digits after the decimal point;

[0068] 3) Deviation and coefficient of variation data are expressed as percentages, with two decimal places;

[0069] The correlation coefficient r2 of the calibration curve is retained to 6 significant figures, and a and b are retained to 6 significant figures.

[0070] Example 1

[0071] Collection of plasma samples to be tested: 30 rats were randomly divided into three groups and given Morinda officinalis oligosaccharide suspension preparations by gavage. The dosages of Morinda officinalis oligosaccharides were 50, 160, and 500 mg / kg body weight, respectively, once a day for 6 consecutive weeks.

[0072] Blood was collected alternately in two groups, on the first day of administration and the last day of administration after 6 weeks of administration (2 times in total). No fasting was allowed before blood collection, and drinking water was free. Under isoflurane anesthesia, blood was collected from the retro-orbital venous plexus before administration, 0.25h±3min, 0.5h±5min, 1h±3min, 2h±3min, 4h±5min, 6h±5min, and 24h±10min after administration (8 time points), and the blood collection site was gently pressed to stop bleeding. At the first administration, because the animals were young and had low body weight, about 0.3mL / animal was used each time; at the last administration, about 0.6mL / animal was used each time. The collected blood samples were placed in anticoagulation tubes containing heparin anticoagulation, and gently inverted to mix for anticoagulation. After blood sample collection, the centrifuge tube was placed on an ice bag or crushed ice, and the blood collection time was recorded in time. The sample was centrifuged as soon as possible within 2h after blood collection (4℃, 4000g, 10min) to separate plasma. The separated plasma samples can be temporarily stored in a -20°C refrigerator. After the samples are collected on the same day, they can be transferred to a -70°C refrigerator until measurement.

[0073] The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma described in this embodiment specifically comprises the following steps:

[0074] (1) Preparation of calibration standards (CAL STDs): Accurately weigh about 10 mg of Morinda officinalis oligosaccharide 5-glycan reference substance, place it in a 10 mL volumetric flask, calibrate the weight with the reference substance content, add an appropriate amount of 10% methanol aqueous solution, shake and sonicate to dissolve, dilute to scale, sonicate, add 10% methanol aqueous solution to calibrate the volume, mix well, and obtain Morinda officinalis oligosaccharide 5-glycan stock solution SS1 with a concentration of 1 mg / mL. Accurately measure an appropriate amount of Morinda officinalis oligosaccharide 5-glycan stock solution SS1, and serially dilute it with 75% acetonitrile aqueous solution according to the table below to obtain a series of standard working solutions with concentrations of 25 ng / mL, 50 ng / mL, 250 ng / mL, 375 ng / mL, 500 ng / mL, 2500 ng / mL, 3750 ng / mL and 5000 ng / mL (respectively recorded as WSA8 to WSA1 solutions).

[0075]

[0076] Accurately measure appropriate amounts of blank mixed plasma of rats and standard working solution of Morinda officinalis oligosaccharide 5 polysaccharide series to prepare a series of calibration standards, the concentrations of which are 5, 10, 50, 75, 100, 500, 750 and 1000 ng / mL (respectively denoted as CAL STD8 to CAL STD1). Prepare freshly before use.

[0077]

[0078] (2) Preparation of internal standard working solution (solution containing internal standard): Accurately weigh about 10 mg of internal standard acarbose reference substance, place in a 15 mL centrifuge tube, calibrate the weighed weight with the reference substance content, add an appropriate amount of 10% methanol aqueous solution, shake to dissolve, dilute to scale, sonicate, then add 10% methanol aqueous solution to calibrate the volume, mix well, and obtain an internal standard stock solution IS SS with a concentration of 1 mg / mL. Accurately measure an appropriate amount of internal standard stock solution IS SS, dilute with an internal standard diluent 90% acetonitrile aqueous solution, and obtain an internal standard working solution ISWS2 with a concentration of 100 ng / mL (ISWS2 solution containing internal standard substance).

[0079] (3) Plasma sample processing method: Take 25 μL of each calibration standard and add 75 μL of ISWS2 solution containing internal standard. Vortex all samples for 2 min, centrifuge at 13200 rpm for 15 min, and take 70 μL of supernatant to prepare standard plasma solution.

[0080] Take 25 μL of the plasma sample to be tested, add 75 μL of ISWS2 solution containing the internal standard, vortex mix for 2 minutes, centrifuge at 13200 rpm for 15 minutes, take 70 μL of the supernatant to prepare the plasma solution to be tested.

[0081] Take 25 μL of blank plasma from rats, add 75 μL of 90% acetonitrile aqueous solution without internal standard, vortex mix for 2 minutes, centrifuge at 13200 rpm for 15 minutes, and take 70 μL of supernatant to prepare blank sample.

[0082] Take 25 μL of blank plasma from rats, add 75 μL of 90% acetonitrile aqueous solution containing internal standard, vortex mix for 2 minutes, centrifuge at 13200 rpm for 15 minutes, and take 70 μL of supernatant to prepare zero concentration sample (Zero sample).

[0083] (4) Detecting the test plasma solution and the standard plasma solution by liquid chromatography-mass spectrometry, and calculating the content of Morinda officinalis oligosaccharide 5 glycan in the test plasma sample:

[0084] The detection conditions of the ultra-high performance liquid chromatography are: using Waters ACQUITY BEH Amide chromatographic column (2.1mm×100mm, 1.7μm), equipped with 0.2μm online filter; mobile phase A is 50mmol / L ammonium acetate-0.1% ammonia aqueous solution, mobile phase B is 0.1% ammonia acetonitrile solution, gradient elution, conditions are as shown in the table below, column temperature is 50℃, flow rate is 0.5mL / min, injector temperature is 15℃, injection volume is 10μL. The entire analysis time is 6.0min. The needle washing solution is 50% acetonitrile aqueous solution.

[0085] Table 1 Mobile phase gradient elution program

[0086] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 25.0 75.0 0.50 25.0 75.0 3.00 45.0 55.0 4.00 45.0 55.0 4.10 25.0 75.0 6.00 25.0 75.0

[0087] Mass spectrometry conditions: electrospray ionization source (ESI), ion source temperature 150℃, capillary voltage 2kV, desolvation gas temperature 550℃, desolvation gas flow rate 1000L / Hr, cone gas flow rate 150L / Hr, nebulizer gas pressure 7.0Bar, collision gas argon flow rate 0.15mL / min. The cone voltages of Morinda officinalis oligosaccharide 5 glycan and internal standard were 84V and 18V, respectively; the secondary collision energies were 36eV and 16eV, respectively; negative ion detection; scanning mode was multiple reaction monitoring (MRM), and the ion reactions used for quantitative analysis were m / z827.22→178.95 (Morinda officinalis oligosaccharide 5 glycan) and m / z644.152→320.109 (IS acarbose). See Figure 6 and 7 shown.

[0088] (5) Calculation of standard curve and sample concentration: 8 plasma calibration standards of Morinda officinalis oligosaccharide 5 glycans at 5 ng / mL to 1000 ng / mL were processed to prepare standard plasma solutions, as well as 1 blank sample and 1 zero concentration sample. Based on the correlation between the labeled concentration of the calibration standard and the measured ratio of the peak area of ​​Morinda officinalis oligosaccharide 5 glycans to the internal standard, the concentration X of Morinda officinalis oligosaccharide 5 glycans in the calibration standard was used as the horizontal coordinate, and the peak area ratio Y of Morinda officinalis oligosaccharide 5 glycans to the internal standard was used as the vertical coordinate. The least square method was used with a weight of 1 / X2 to perform linear regression. The concentration points whose %Dev of the back-calculated concentration was greater than ±15% of the labeled value were deleted. After deletion, the standard curve was refitted to ensure that the %Dev of at least 6 effective concentrations was within ±15% and the %Dev of the LLOQ was within ±20%, and the standard curve and linear equation were obtained.

[0089] The regression equation of the calibration curve is: Y=b+aX (Y: peak area ratio, X: labeled concentration, a: slope, b: y-intercept).

[0090] Deviation (%Dev) and coefficient of variation (%CV) were calculated using MassLynx workstation or Microsoft Excel 2003 or above. Accuracy was evaluated by deviation, and precision was evaluated by coefficient of variation. Mean and standard deviation (SD) were calculated directly using Excel.

[0091]

[0092] Four standard curves were prepared according to the above method, and the batch numbers are shown in the table below. The average r value of the four standard curves of this analytical method was verified to be 0.9971; the % Dev of each concentration point of the four curves within the range of the marking line was -7.78% to 6.47%, that is, the % Dev of all calibration standards of the four marking lines was within ±15%, and the LLOQ deviation was -15.15% to 14.88%, all within ±20%. This shows that the linear relationship of Morinda officinalis oligosaccharide 5 glycans in rat plasma is good within this range.

[0093] The parameters a, b, correlation coefficient r, response values ​​of each concentration, back-calculated values ​​and deviations of the linear equations of all accepted analysis batches (4 analysis batches) in the plasma validation test are detailed in Table 3. The mark sample data, fitting equations and specific data of the 4 methodological validation analysis batches are shown in Table 2.

[0094] Table 2 Summary of standard curve coefficients of Morinda officinalis oligosaccharide 5-glycan in rat plasma

[0095]

[0096] Table 3 Validation of the analytical method for Morinda officinalis oligosaccharide 5 glycans in rat plasma Methodological validation Standard curve Results Summary

[0097]

[0098]

[0099] The verified quantitative range of rat plasma sample analysis is 10 ng / mL to 1000 ng / mL, 10 ng / mL is the lower limit of quantification LLOQ of the plasma sample analysis by this method, and 1000 ng / mL is the upper limit of quantification ULOQ of the plasma sample analysis by this method.

[0100] (6) Substitute the ratio of the peak area of ​​the Morinda officinalis oligosaccharide 5 glycan obtained from the plasma solution to be tested to the peak area of ​​the internal standard into the linear regression equation to obtain the content of the Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested as shown in the following table, and draw a curve of the relationship between the content of the Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested (ng / mL) and time as shown in Figure 8 .

[0101] Table 4 Average blood drug concentration-time curve of Morinda officinalis oligosaccharide 5 glycan in SD rats given oral administration of Morinda officinalis oligosaccharide 50, 160, 500 mg / kg for 6 weeks (n=10)

[0102]

[0103] in, Figure 1 is the chromatogram of the blank sample. Figure 2 This is the chromatogram of the standard plasma solution prepared after processing the calibration standard (CAL STD6). Figure 3 The chromatogram of the plasma solution to be tested is obtained by processing the plasma sample to be tested collected 2 hours after administration. Figure 1-3 It can be seen that the above method has good specificity.

[0104] Example 2 Methodology Verification

[0105] The method of Example 1 was methodologically verified.

[0106] 1. Selectivity

[0107] Blank plasma from 6 SD rats of different origins was taken as S1 to S6 selective samples.

[0108]

[0109]

[0110] Take 25 μL of each selective sample, add 75 μL of 90% acetonitrile aqueous solution without internal standard, vortex mix for 2 min, centrifuge at 13200 rpm for 15 min, take 70 μL of supernatant, and measure under the above LC-MS / MS conditions. Figure 4 This is the chromatogram of the standard plasma solution prepared after LLOQ sample treatment. Figure 5 This is the chromatogram obtained after selective sample S2 treatment.

[0111] The retention time of Morinda officinalis oligosaccharide 5 glycan is about 2.8min. The interference peaks of 6 selective samples at the retention time of Morinda officinalis oligosaccharide 5 glycan are recorded as Morinda officinalis oligosaccharide 5 glycan interference peaks. The retention time of the internal standard is about 2.4min. The interference peaks at the retention time of the internal standard are recorded as internal standard interference peaks.

[0112] The retention time (RT) of the glycan interference peak of Morinda officinalis oligosaccharide 5, the retention time (IS RT) of the internal standard interference peak, the area (Area) of the glycan interference peak of Morinda officinalis oligosaccharide 5, the area (IS Area) of the internal standard interference peak, the peak area of ​​the glycan interference peak of Morinda officinalis oligosaccharide 5 as LLOQ, the peak area of ​​the glycan interference peak of Morinda officinalis oligosaccharide 5 as LLOQ (% Area of ​​LLOQ), and the peak area of ​​the internal standard interference peak as LLOQ as LLOQ internal standard peak area (% IS Area of ​​LLOQ) are shown in Table 5. The LLOQ preparation method is shown in Example 1.

[0113] Table 5 Selectivity of Morinda officinalis oligosaccharide 5-glycan in rat plasma (n=6)

[0114]

[0115] NF: not found

[0116] The peak areas of the interference peaks at the retention time of Morinda officinalis oligosaccharide 5 glycans of the 6 selective samples were 0.42% to 8.13% of the peak area of ​​the LLOQ Morinda officinalis oligosaccharide 5 glycans, all lower than the allowable standard of 20%. The peak areas of the interference peaks at the retention time of the internal standard of the 6 selective samples were 0.00% to 0.06% of the peak area of ​​the LLOQ internal standard, all lower than the allowable standard of 5%. The selectivity of this method is good.

[0117] 2. Carry-over

[0118] When preparing the standard curve in Example 1, after injecting the standard plasma solution treated with ULOQ, a blank sample was injected to estimate the residual, and the result was repeated three times. The peak area of ​​the blank sample Morinda officinalis oligosaccharide 5 glycan was calculated as a percentage of the peak area of ​​the LLOQ Morinda officinalis oligosaccharide 5 glycan (residual percentage) and the peak area of ​​the blank sample internal standard was calculated as a percentage of the peak area of ​​the LLOQ internal standard (IS residual percentage).

[0119] The results showed that the peak areas of the residual blank sample Morinda officinalis oligosaccharide 5 glycan were all less than 20% of the LLOQ Morinda officinalis oligosaccharide 5 glycan peak area, and the peak areas of the internal standards did not exceed 5% of the corresponding peak areas of the LLOQ. The specific results are shown in Table 6.

[0120] Table 6 Residues of Morinda officinalis oligosaccharide 5-glycan and internal standard in plasma samples

[0121]

[0122]

[0123] 3. Accuracy and Precision

[0124] Three analytical batches were performed within three days to investigate the accuracy and precision of the method. Each analytical batch used separately prepared stock solutions to prepare LLOQ, LQC, MQC and HQC quality control samples of Morinda officinalis oligosaccharide 5 glycans at concentrations of 10, 30, 400 and 800 ng / mL, respectively, and five samples were measured in parallel at each concentration within the batch.

[0125] Specifically, accurately weigh about 10 mg of the Morinda officinalis oligosaccharide 5-glycan reference substance, place it in a 10 mL volumetric flask, calibrate the weighed weight with the reference substance content, add an appropriate amount of 10% methanol aqueous solution, shake and sonicate to dissolve, dilute to the scale, sonicate, add 10% methanol aqueous solution to calibrate the volume, mix well, and obtain a Morinda officinalis oligosaccharide 5-glycan reserve solution SS2 with a concentration of 1 mg / mL. Accurately measure an appropriate amount of reserve solution SS2, dilute it in series with a diluent, and obtain quality control working solutions with concentrations of 50 ng / mL, 150 ng / mL, 2000 ng / mL, and 4000 ng / mL.

[0126]

[0127] Accurately measure appropriate amounts of blank mixed plasma from rats and Morinda officinalis oligosaccharide 5-glycan quality control working solution, mix them evenly to obtain quality control (QC) samples with concentrations of 10 ng / mL, 30 ng / mL, 400 ng / mL and 800 ng / mL.

[0128]

[0129] Take 25 μL of each of the above quality control samples, add 75 μL of ISWS2 solution containing internal standard respectively, vortex mix for 2 min, centrifuge at 13200 rpm for 15 min, take 70 μL of supernatant, to prepare quality control sample solution, take 10 μL of quality control sample solution respectively and put it into injection bottle, adopt LC-MS / MS injection analysis, the liquid phase conditions and mass spectrometry conditions are the same as Example 1.

[0130] The results showed that the intra-batch accuracy %Dev of LQC, MQC and HQC of this method was between -10.35% and 6.50%, and the intra-batch precision %CV was between 2.68% and 6.17%, all within ±15%; the inter-batch accuracy %Dev was between -1.78% and 1.29%, and the inter-batch precision %CV was between 3.99% and 8.78%, all within ±15%. The intra-batch accuracy %Dev of LLOQ was between -6.08% and 6.25%, the intra-batch precision %CV was between 9.12% and 17.91%, the inter-batch accuracy %Dev was 1.53%, and the inter-batch precision %CV was 13.11%, all within ±20%. The above data show that the accuracy and precision of this method are good, and the specific results are shown in Table 7.

[0131] Table 7 Intra-day and inter-day accuracy and precision of rat plasma QCs (n=5)

[0132]

[0133] 4. Matrix Effects

[0134] Take appropriate amounts of blank plasma from SD rats from 6 different sources and the above-mentioned Morinda officinalis oligosaccharide 5-glycan quality control working solution to prepare LME and HME matrix effect samples with concentrations of 30 ng / mL and 800 ng / mL, and prepare 3 samples in parallel for each concentration and each batch of plasma.

[0135]

[0136] Take 25 μL of each of the above matrix effect samples, add 75 μL of ISWS2 solution containing internal standard respectively, vortex mix for 2 min, centrifuge at 13200 rpm for 15 min, take 70 μL of supernatant, put it in an injection bottle to prepare a matrix effect sample solution, take 10 μL of matrix effect sample solution respectively and use LC-MS / MS injection analysis, the liquid phase conditions and mass spectrometry conditions are the same as Example 1.

[0137] The results showed that 6 batches of blank plasma from different donors (M1, M9, M13, F5, F9, F10) were used to prepare 30 ng / mL and 800 ng / mL matrix effect samples, and 3 samples were prepared in parallel for each batch of plasma at each concentration. The accuracy %Dev of the 6 batches of plasma samples was within ±15% of the labeled concentration, and the precision %CV of all single source batch matrices was within 0.22% to 12.43%, which was no more than 15%. The specific results are shown in Table 8.

[0138] Table 8 Morinda officinalis oligosaccharide 5 polysaccharide matrix effect sample determination results (n = 3)

[0139]

[0140] 5. Dilution Integrity

[0141] Take WSA solution (preparation method see step (1) of Example 1) and prepare dilution quality control samples (Dilution QC) with a concentration greater than ULOQ according to the following table. Dilute Dilution QC 10 times with SD rat blank mixed plasma to obtain a dilution reliability sample DIS-10 with a concentration of 1000 ng / mL; dilute Dilution QC 100 times with SD rat blank mixed plasma to obtain a dilution reliability sample DIS-100 with a concentration of 100 ng / mL. Prepare 5 samples in parallel for each dilution factor.

[0142]

[0143] Take 25 μL of each of the above diluted reliability samples, add 75 μL of ISWS2 solution containing internal standard respectively, vortex mix for 2 min, centrifuge at 13200 rpm for 15 min, take 70 μL of supernatant to prepare diluted reliability sample solution, take 10 μL of diluted reliability sample solution respectively and use LC-MS / MS injection analysis, the liquid phase conditions and mass spectrometry conditions are the same as Example 1.

[0144] The results showed that the accuracy %Dev of Morinda officinalis oligosaccharide 5 glycans in each sample was -4.06% and 9.89%, and the precision %CV was 3.44% and 12.67%, which met the requirements of accuracy %Dev within ±15% and precision %CV not greater than 15%, indicating that dilution of rat plasma samples did not affect accuracy and precision. Specific results are shown in Table 9.

[0145] Table 9 Dilution reliability of Morinda officinalis oligosaccharide 5-glycan in rat plasma (10, 100 folds) (n = 5)

[0146]

[0147] 6. Stability

[0148] (1) Long-Term Stability in Matrix (LTS)

[0149] Accurately measure an appropriate amount of WSL or WSH solution (see item 3 of this embodiment for preparation method), accurately measure an appropriate amount of blank mixed plasma of SD rats, and mix them to obtain stability quality control samples (LSS and HSS) with concentrations of 30 and 800 ng / mL. The stability quality control samples of 2 concentrations are packaged, and at least 3 samples are placed in a -70°C refrigerator for each concentration level and each placement condition to investigate the long-term stability LTS.

[0150]

[0151] The freshly prepared Morinda officinalis oligosaccharide 5-glycan stability samples were frozen in a -70°C refrigerator and processed and tested according to the following method on the day of preparation and after being stored at -70°C for 50 days, 167 days, 289 days and 343 days. Three samples were tested for each concentration.

[0152] Take 25 μL of each of the above samples, add 75 μL of ISWS2 solution containing internal standard respectively, vortex mix for 2 min, centrifuge at 13200 rpm for 15 min, take 70 μL of supernatant to prepare long-term stability sample solution, take 10 μL of long-term stability sample solution respectively and use LC-MS / MS injection analysis, the liquid phase conditions and mass spectrometry conditions are the same as Example 1.

[0153] The accuracy of the 5-glycan of Morinda officinalis oligosaccharide was measured to be between -12.21% and 4.11%, which is consistent with the accuracy within ±15%. It is proved that the rat plasma samples of Morinda officinalis oligosaccharide 5-glycan remain stable after being stored at -70°C for 0 days, 50 days, 167 days, 289 days and 343 days. The specific results are shown in Tables 10 and 11.

[0154] Table 10 Concentration values ​​of rat plasma stability samples of Morinda officinalis oligosaccharide 5 glycan on the day of preparation (n=3)

[0155]

[0156] Table 11 Long-term stability of Morinda officinalis oligosaccharide 5 glycan in rat plasma samples (n=3)

[0157]

[0158] (2) Bench-Top Stability (BTS) of rat plasma samples

[0159] Accurately measure the blank mixed plasma of rats and the quality control working solution of Morinda officinalis oligosaccharide 5 glycan, mix them, and store them at room temperature on the test bench for a period of time to obtain the high concentration (BTS H) and low concentration (BTS L) samples of Morinda officinalis oligosaccharide 5 glycan pretreatment stability with concentrations of 30 ng / mL and 800 ng / mL, respectively. Prepare 3 samples in parallel for each concentration.

[0160]

[0161] Freshly prepared Morinda officinalis oligosaccharide 5 polysaccharide pre-treatment stability high concentration and low concentration samples were prepared in parallel for each concentration, and the samples were immediately measured after treatment. The samples before treatment were stored at room temperature in the dark for 5 hours and then treated and measured. The treatment and measurement method is as follows: 25 μL of each of the above samples were taken, 75 μL of ISWS2 solution containing internal standard was added respectively, vortex mixed for 2 minutes, centrifuged at 13200 rpm for 15 minutes, and 70 μL of the supernatant was taken to prepare the pre-treatment stability sample solution. 10 μL of the pre-treatment stability sample solution was taken for LC-MS / MS injection analysis, and the liquid phase conditions and mass spectrometry conditions were the same as in Example 1.

[0162] The measured concentration of each stability QC was calculated as the mean value. The deviation of the two measured results of the sample was -8.27% for the low concentration and -9.44% for the high concentration, which met the requirement of being within the range of ±15%. The specific results are shown in Table 12.

[0163] Table 12 Pretreatment stability of Morinda officinalis oligosaccharide 5 glycan in rat plasma samples (n=3)

[0164]

[0165] (3) Freeze and Thaw Stability (FTS) of Rat Plasma Samples

[0166] Accurately measure an appropriate amount of WSL or WSH solution (see item 3 of this embodiment for preparation method), accurately measure an appropriate amount of blank mixed plasma of SD rats, and mix them to obtain stability quality control samples (LSS and HSS) with concentrations of 30 and 800 ng / mL. The stability quality control samples of 2 concentrations are packaged, and at least 3 samples are placed in a -70°C refrigerator for freeze-thaw stability FTS.

[0167]

[0168] The rat plasma samples containing Morinda officinalis oligosaccharide 5 polysaccharide were stored at -70°C, and after at least 3 freeze-thaw cycles, the samples were kept frozen for at least 12 hours between thawing cycles for processing and determination. The processing and determination methods are as follows: 25 μL of each of the above samples were taken, 75 μL of ISWS2 solution containing internal standard was added respectively, vortexed for 2 minutes, centrifuged at 13200rpm for 15 minutes, 70 μL of supernatant was taken, and freeze-thaw stability sample solution was prepared. 10 μL of freeze-thaw stability sample solution was taken respectively for LC-MS / MS injection analysis, and the liquid phase conditions and mass spectrometry conditions were the same as those in Example 1. The freeze-thaw stability was determined. Three samples were measured for each concentration of the stability quality control sample, and the mean accuracy of the stability QC was: -10.16% and -5.20%, respectively. It meets the requirements within the range of ±15%. The specific results are shown in Table 13.

[0169] Table 13 Freeze-thaw stability of Morinda officinalis oligosaccharide 5 glycan in rat plasma samples (n=3)

[0170]

[0171] (4) Processed Sample Stability (PSS) of Rat Plasma Samples after Extraction

[0172] The QC sample (batch number 20230116) injected on January 16, 2023 was taken, and the freshly prepared standard plasma solution (prepared according to the method of Example 1) and the above QC sample were used for determination on January 17, 2023 to investigate the stability of rat plasma samples after extraction. The mean accuracy of each concentration of QC of the stability sample was 1.12% and -9.80%, respectively, which met the requirement of being within ±15% of the labeled concentration. The total time from treatment to injection the next day was 25.2 hours. The specific results are shown in Table 14.

[0173] Table 14 Stability of Morinda officinalis oligosaccharide 5 glycans after extraction in rat plasma samples (n=5)

[0174]

[0175] (5) Stock Solution Stability (SSS) of Morinda officinalis Oligosaccharide 5

[0176] The Morinda officinalis oligosaccharide 5 polysaccharide stock solution after storage for a period of time is diluted into a quality control working solution WSM-2 with a concentration of 2000ng / mL, and an appropriate amount of WSM-2 is accurately measured, and an appropriate amount of blank mixed plasma of rats is mixed to obtain a Morinda officinalis oligosaccharide 5 polysaccharide stock solution stability sample SSS with a concentration of 400ng / mL. Three samples are prepared in parallel. The above samples are processed and measured, and the processing and measurement method is as follows: take 25μL of each of the above samples, add 75μL of ISWS2 solution containing internal standard respectively, vortex mix for 2min, centrifuge at 13200rpm for 15min, take 70μL of supernatant, prepare a stock solution stability sample solution, take 10μL of the stock solution stability sample solution and use LC-MS / MS injection analysis, and the liquid phase conditions and mass spectrometry conditions are the same as in Example 1.

[0177]

[0178] The percentage changes in the peak area between the Morinda officinalis oligosaccharide 5 glycan stock solution stored at 4°C for 15 and 36 days and the fresh stock solution in the rat plasma analysis batch were 3.23% and -1.41%, respectively, which were less than the allowable range of 5%. It was believed that the Morinda officinalis oligosaccharide 5 glycan stock solution remained stable after being stored at about 4°C for 15 and 36 days, respectively. The specific results are shown in Table 15.

[0179] Table 15 Stability of Morinda officinalis oligosaccharide 5 polysaccharide stock solution (n=3)

[0180]

[0181]

[0182] Note: Change percentage % = (average peak area of ​​stored stock solution - average peak area of ​​fresh stock solution) / average peak area of ​​fresh stock solution * 100%

[0183] (6) Work Solution Stability (WSS)

[0184] Accurately measure the blank mixed plasma of rats and the quality control working solution WSM of Morinda officinalis oligosaccharide 5, mix them well, dilute the internal standard stock solution after storage for a period of time into the internal standard working solution ISWS2 with a concentration of 100 ng / mL, and treat it according to the plasma sample treatment method of step (3) of Example 1 to obtain the internal standard stock solution stability sample ISS with a concentration of Morinda officinalis oligosaccharide 5 and acarbose at 400 ng / mL. Prepare 3 samples in parallel and inject them according to the method of step (4) of Example 1 for testing.

[0185]

[0186] The percentage changes in peak area between the internal standard stock solution stored at 4°C and about -20°C for 91 days and the fresh stock solution in the rat plasma analysis batch were 0.37% and -2.81%, respectively, which were less than the allowable range of 5%. It was considered that the internal standard acarbose stock solution remained stable after being stored at 4°C and about -20°C for 91 days. The specific results are shown in Table 16.

[0187] Table 16 Stability of internal standard acarbose stock solution (n=3)

[0188]

[0189] Note: Change percentage % = (average peak area of ​​stored stock solution - average peak area of ​​fresh stock solution) / average peak area of ​​fresh stock solution * 100%

[0190] 7. Injection reproducibility

[0191] The calibration standard sample of the 20230112 analysis batch and the five LQC, MQC and HQC quality control samples were processed according to the method of step (3) of Example 1, and then analyzed by LC-MS / MS injection. The liquid phase conditions and mass spectrometry conditions were the same as those of Example 1. The injection was repeated under the same conditions on the second day to investigate the injection reproducibility. The average accuracy of the three concentrations of QCs of the repeated injection was within the range of -5.22% to -0.83%, all within the range of ±15% of the labeled concentration, and the precision was 5.31% to 12.95%, and the precision was no more than 15%. The specific results are shown in Table 17.

[0192] Table 17 Batch injection reproducibility of Morinda officinalis oligosaccharide 5-glycan rat plasma sample analysis

[0193]

[0194] 8. Effects of anticoagulants in plasma

[0195] Three analytical batches were measured within two days to investigate whether the anticoagulant components in plasma would affect precision and accuracy. Each analytical batch used separately prepared reserve solutions to prepare LLOQ, LQC, MQC and HQC quality control samples of Morinda officinalis oligosaccharide 5 glycans at concentrations of 10, 30, 400 and 800 ng / mL, and 5 samples were measured in parallel at each concentration in the batch. Specifically: According to the method under item 3 of this embodiment, blank mixed plasma of rats anticoagulated with sodium heparin was used, and mixed with the quality control working solution of Morinda officinalis oligosaccharide 5 glycans, respectively, to obtain quality control samples of sodium heparin anticoagulated plasma at concentrations of 10 ng / mL, 30 ng / mL, 400 ng / mL and 800 ng / mL. Five samples were measured in parallel at each concentration. The quality control samples of sodium heparin anticoagulated plasma were processed and measured, and the processing and measurement method was as follows: 25 μL of each of the above samples were taken, 75 μL of ISWS2 solution containing the internal standard was added respectively, vortexed for 2 minutes, centrifuged at 13200 rpm for 15 minutes, 70 μL of the supernatant was taken to prepare an anticoagulated plasma quality control sample solution, and 10 μL of the anticoagulated plasma quality control sample solution was taken for LC-MS / MS injection analysis, and the liquid phase conditions and mass spectrometry conditions were the same as in Example 1.

[0196] The intra-batch accuracy of LQC, MQC and HQC of this method is between -14.63% and 6.60%, and the intra-batch precision is between 0.92% and 7.25%, all within ±15%; the inter-batch accuracy is between -7.32% and -1.55%, and the inter-batch precision is between 6.61% and 11.60%, all within ±15%. The intra-batch accuracy of LLOQ is between 9.06% and 15.08%, the intra-batch precision is between 8.97% and 19.04%, the inter-batch accuracy is 11.58%, and the inter-batch precision is 13.80%, all within ±20%. This shows that the anticoagulant component in plasma will not affect the precision and accuracy of this method. The specific results are shown in Table 18.

[0197] Table 18 Effect of anticoagulants in rat plasma (n=5)

[0198]

[0199] 9. Acceptability of analytical batches

[0200] The method of Example 1 and Example 2 was verified. This verification experiment included 16 analysis batches in total. Each analysis batch or each partial batch included blank samples, zero concentration samples, calibration standards at at least 6 concentration levels, at least 3 concentration level duplicate samples or QCs of at least 5% of the total number of test samples, and the analyzed test samples. All samples in an analysis batch were processed and extracted in the same sample batch in the order in which they were to be analyzed, that is, processed at the same time, by the same analyst, with the same batch of reagents, and processed successively under consistent conditions without time intervals. QCs were distributed throughout the batch to ensure the accuracy and precision of the entire analysis batch.

[0201] Acceptance criteria for plasma analysis batches: The % Dev of QC samples should be within ± 15% of the labeled concentration, at least 67% of the quality control samples, and at least 50% of the samples at each concentration level should meet this standard. If these criteria are not met, the analysis batch should be rejected and the corresponding test samples should be re-extracted and analyzed. Among them, the accuracy and precision (A&P) analysis batches of method validation tests do not need to meet the QC acceptance criteria, and other validation analysis batches need to set up at least 3 concentrations of QC duplicate samples and meet the QC acceptance criteria.

[0202] In addition to the precision and accuracy analysis batches, a total of 13 analysis batches met the acceptance criteria specified in the protocol, and the data of the 13 analysis batches were accepted and can be used. The calibration line fitting results and QCS results are shown in Tables 19 to 26, respectively.

[0203] Table 19 Standard curves of different batches (20230109~20230113)

[0204] 20230109 Y=0.315065*x+-0.039041R^2=0.994566 20230110 Y=0.497477*x+-0.361664R^2=0.997254 20230111 Y=0.531694*x+-0.517261R^2=0.998060 20230112 Y=0.660403*x+0.0780684R^2=0.994885 20230113 Y=0.726042*x+-0.49398R^2=0.988599

[0205] Table 20 Test results of standard curve (20230109~20230113)

[0206]

[0207] Table 21 Standard curves of different batches (20230116~20230304-1)

[0208] 20230116 Y=0.652458*x+-0.700862R^2=0.990969 20230117 Y=0.701723*x+-0.937413R^2=0.994558 20230203 Y=0.363156*x+-0.50027R^2=0.990692 20230303 Y=0.430785*x+-0.387287R^2=0.997757 20230304-1 Y=0.456323*x+0.280201R^2=0.998199

[0209] Table 22 Test results of standard curve (20230116~20230304-1)

[0210]

[0211]

[0212] Table 23 Standard curves of different batches (20230203~20231219)

[0213] 20230304-2 Y=0.435179*x+0.137752R^2=0.995811 20230626 Y=0.262237*x+-0.409609R^2=0.998267 20231027 Y=0.297192*x+0.222036R^2=0.998782 20231219 Y=0.440326*x+0.608784R^2=0.996936

[0214] Table 24 Test results of standard curve (20230203~20231219)

[0215]

[0216] Table 25 QC sample results of Morinda officinalis oligosaccharide 5 glycan plasma sample determination (20230109~20230113)

[0217]

[0218]

[0219] Table 26 QC sample results of Morinda officinalis oligosaccharide 5 glycan plasma sample determination (20230116~20231219)

[0220]

[0221] Experimental Example 1

[0222] The purpose of this experimental example is to investigate the type and concentration of the mobile phase. A standard plasma solution treated with a calibration standard having a polysaccharide concentration of 50 ng / mL of Morinda officinalis oligosaccharide 5 (preparation method is the same as in Example 1) was taken for LC-MS / MS injection analysis. The following mobile phases were used for elution. The elution procedure and other liquid phase conditions and mass spectrometry conditions were the same as in Example 1.

[0223] The first mobile phase: mobile phase A: 0.1% ammonia in water, mobile phase B: 0.1% ammonia in acetonitrile;

[0224] The second mobile phase: 100mmol / L ammonium acetate-0.1% ammonia aqueous solution, mobile phase B: 0.1% ammonia acetonitrile solution;

[0225] The third mobile phase: 50mmol / L ammonium acetate-0.1% ammonia aqueous solution, mobile phase B: 0.1% ammonia acetonitrile solution;

[0226] The results showed that when 0.1% ammonia aqueous solution and 0.1% ammonia acetonitrile solution were used as the mobile phase, the peak of the analyte Morinda officinalis oligosaccharide 5 glycan was prone to tailing ( Fig. 9 The second mobile phase was eluted by adding 100mmol / L ammonium acetate to the aqueous phase. The results showed that after adding ammonium acetate to the aqueous phase, the peak time of the analyte was stable, and the response and peak shape were relatively ideal ( Fig.10). However, after several preliminary tests, it was found that the peak of the analyte showed tailing phenomenon and the response became low, and the content of Morinda officinalis oligosaccharide 5 glycan could not be accurately determined. It was initially believed that the chromatographic column might be damaged due to the excessive concentration of ammonium acetate ( Fig.11 Therefore, the concentration of ammonium acetate was adjusted to 50mmol / L, and the result was ideal ( Fig.12 ), Example 2 confirms that the detection method has good selectivity, stability, injection reproducibility, accuracy and precision, and the influence of residue and matrix effect meets the requirements, and can accurately determine the content of Morinda officinalis oligosaccharide 5 glycan in rat plasma.

[0227] Experimental Example 2

[0228] The purpose of this experimental example is to investigate the effect of the type and concentration of the precipitant on the test results. A standard plasma solution (preparation method is the same as in Example 1) treated with a calibration standard sample with a concentration of 50 ng / mL of Morinda officinalis oligosaccharide 5 polysaccharide was taken for LC-MS / MS injection analysis. The following precipitants were used to replace the 90% acetonitrile aqueous solution, and other process conditions were the same as in Example 1. The ratio of the responsiveness of each precipitant (including the responsiveness obtained when the 90% acetonitrile aqueous solution used in Example 1) to the responsiveness of acetonitrile was calculated based on the responsiveness of acetonitrile as the precipitant. The results of the ratio of the responsiveness obtained by different precipitants to the responsiveness obtained by acetonitrile as the precipitant are shown in the table.

[0229] Table 27 Response results of different precipitants

[0230]

[0231] The results showed that, after comparison, although the sample response after acetonitrile precipitation was higher than that of methanol, it still could not meet the detection requirements. Compared with acetonitrile, the presence of part of the aqueous phase can make the 5-glycan of Morinda officinalis oligosaccharide precipitate better, thereby improving the extraction efficiency, and the best effect was achieved with 90% acetonitrile aqueous solution as the precipitant.

[0232] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma, characterized in that: The steps include: (1) Mixing blank plasma and standard working solutions of Morinda officinalis oligosaccharide 5 glycans of different concentrations to prepare calibration standards; mixing the plasma sample to be tested and the calibration standard with the internal standard in the presence of an acetonitrile aqueous solution, and then performing solid-liquid separation respectively, and taking the liquid to obtain a plasma solution to be tested and a standard plasma solution; (2) using a liquid chromatography-mass spectrometry method to detect the test plasma solution and the standard plasma solution, and calculating the content of Morinda officinalis oligosaccharide 5-glycan in the test plasma sample; The chromatographic conditions of the LC-MS include: Octadecylsilane bonded silica gel was used as filler, an aqueous solution containing 45-55 mmol / L ammonium acetate and 0.08-0.12% ammonia was used as mobile phase A, and acetonitrile containing 0.08-0.12% ammonia was used as mobile phase B for gradient elution. The gradient elution procedure was as follows: 0→0.5min, the volume percentage of mobile phase A is 25%, and the volume percentage of mobile phase B is 75%; 0.5min→3min, the volume percentage of mobile phase A increased from 25% to 45%, and the volume percentage of mobile phase B decreased from 75% to 55%; 3min→4min, the volume percentage of mobile phase A is 45%, and the volume percentage of mobile phase B is 55%; 4min→4.1min, the volume percentage of mobile phase A decreased from 45% to 25%, and the volume percentage of mobile phase B increased from 55% to 75%; 4.1min→6min, the volume percentage of mobile phase A is 25%, and the volume percentage of mobile phase B is 75%.

2. The method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma according to claim 1, characterized in that: The plasma sample to be tested is a plasma sample taken from a patient taking a pharmaceutical preparation containing Morinda officinalis oligosaccharide; Preferably, the dosage form of the pharmaceutical preparation is capsule, solution, suspension, lyophilized powder, granules or tablets; Preferably, the plasma sample is a rat plasma sample.

3. The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma according to claim 1, characterized in that: The chromatographic conditions of the LC-MS also include: a flow rate of 0.4-0.6 mL / min, a column temperature of 45-55° C.; and / or a chromatographic column with a specification of 2.1 mm×100 mm, 1.7 μm.

4. The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma according to claim 1, characterized in that: The mass spectrometry conditions of the LC-MS include: An electrospray ionization source was used with an ion source temperature of 100-200°C, a capillary voltage of 1.8-2.2 kV, a desolvation gas temperature of 500-600°C, a desolvation gas flow rate of 800-1200 L / Hr, a cone gas flow rate of 100-200 L / Hr, a nebulizer gas pressure of 6.0-8.0 Bar, and a collision gas argon flow rate of 0.1-0.2 mL / min. The cone voltages of Morinda officinalis oligosaccharide 5-glycan and internal standard were 80-90 V and 15-20 V, respectively; the secondary collision energies were 30-40 eV and 15-18 eV, respectively; detection was in negative ion mode; and the scanning mode was multiple reaction monitoring.

5. The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma according to claim 1, characterized in that: The internal standard is acarbose; and / or the volume percentage of the acetonitrile aqueous solution is 75%-90%.

6. The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma according to claim 5, characterized in that: The ion reactions used for quantitative analysis were: Morinda officinalis oligosaccharide 5 glycan: m / z827.22→178.95; internal standard: m / z644.152→320.

109.

7. The method for determining the content of Morinda officinalis oligosaccharide 5 glycan in plasma according to claim 1, characterized in that: In step (1), the plasma sample to be tested and the calibration standard are respectively mixed with an internal standard in the presence of an acetonitrile aqueous solution. Preferably, the concentration of the acetonitrile aqueous solution containing the internal standard is 50 to 200 ng / mL.

8. The method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma according to any one of claims 1 to 7, characterized in that: In step (1), the volume ratio of blank plasma to standard working solutions of Morinda officinalis oligosaccharide 5 glycans of different concentrations is 15-25:

5.

9. The method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma according to any one of claims 1 to 8, characterized in that: In step (1), the volume ratio of the plasma sample to be tested to the solution containing the internal standard is 25:70-80; the volume ratio of the calibration standard to the solution containing the internal standard is 25:70-80.

10. The method for determining the content of Morinda officinalis oligosaccharide 5-glycan in plasma according to any one of claims 1 to 9, characterized in that: Calculating the content of Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested includes: collecting the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard in the standard plasma solution and the plasma solution to be tested, performing linear regression on the ratio of the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard obtained from the standard plasma solution and the concentration of Morinda officinalis oligosaccharide 5 glycan in the calibration standard to generate a standard curve and a linear regression equation; substituting the ratio of the peak areas of Morinda officinalis oligosaccharide 5 glycan and the internal standard obtained from the plasma solution to be tested into the linear regression equation to obtain the content of Morinda officinalis oligosaccharide 5 glycan in the plasma sample to be tested.

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