Method for detecting substances in body after psoralea corylifolia administration

Abstract

The invention discloses a method for detecting in-vivo substances after psoralea corylifolia administration, which is characterized in that 58 in-vivo substances of psoralea corylifolia can be simultaneously detected within 8 minutes by combining positive ion and negative ion scanning modes and combining reference substance quantification and virtual quantification by utilizing a liquid chromatography-mass spectrometry technology, and 11 groups of isomers can be simultaneously detected. Analysis of a mouse plasma sample shows that the method can resist matrix effect interference, and the quantitative lower limit of most compounds can reach 15.6 nM. The method provided by the invention is sensitive and reliable, and can powerfully support biological sample analysis after psoralea corylifolia administration.

Description

Technical Field

[0001] This invention belongs to the field of in vivo substance analysis of traditional Chinese medicine. Specifically, this invention relates to an analytical method for simultaneously detecting 56 in vivo substances in Psoralea corylifolia. Background Technology

[0002] Psoralea corylifolia is a commonly used but toxic traditional Chinese medicine; overdose can lead to hepatotoxicity. Currently available patented analytical methods for Psoralea corylifolia only focus on a few primary components, establishing quantitative methods for Psoralea corylifolia components in medicinal materials or preparations, as shown in patent documents 1-6 below.

[0003] Patent Document 1: CN202311243224.0 A method for establishing the fingerprint spectrum of a compound drug: only psoralen and isopsoralen in psoralen were detected.

[0004] Patent Document 2: CN202311131659.6 A method for determining the components in Zhuchun capsules: only psoralen and isopsoralen in psoralen were detected.

[0005] Patent Document 3: CN202211438964.5 Fingerprint Spectrum Construction Method and Identification Method of Psoralea corylifolia and Salt-Psoralea corylifolia Traditional Chinese Medicine Formula Granules: Only psoralen and isopsoralen in Psoralea corylifolia were detected.

[0006] Patent Document 4: CN202010831085.3 A method for detecting volatile components in a pharmaceutical preparation: only isopsoralen, psoralen, and psoralenol were detected.

[0007] Patent Document 5: CN201710650981.8 A detection method for Psoralea corylifolia medicinal materials based on a single test and multiple evaluation method: The content of psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavone, psoralen A, psoralen B, psoralen dihydroflavonoid methyl ether, corylifol A, and psoralenol in the medicinal materials was detected.

[0008] Patent Document 6: CN201410389089.5 Method for establishing fingerprint spectrum of Sishen tablets: only isopsoralen and psoralen were detected.

[0009] Existing quantitative analysis methods only focus on a few primary components or establish qualitative analysis methods for metabolites. The toxicological basis of this poisonous traditional Chinese medicine is not yet fully understood, and there is an urgent need for a method to simultaneously detect all major exposed substances in in vivo biological samples to support the subsequent identification of toxic substances. Summary of the Invention

[0010] Based on the problems existing in the prior art, the purpose of this invention is to provide a method for detecting substances in the body after administration of psoralen. This method utilizes liquid chromatography-mass spectrometry (LC-MS) to combine positive and negative ion scanning modes, and combines reference standard quantification with virtual quantification, which can simultaneously detect 58 substances in the body of psoralen within 8 minutes.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A method for detecting substances in the body after administration of psoralen, comprising removing proteins from an individual's body fluid sample and then performing detection using HPLC-MS / MS;

[0013] in,

[0014] Chromatographic conditions include:

[0015] Chromatographic column: C18 column;

[0016] Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: methanol containing 0.1% formic acid;

[0017] Flow rate: 0.2–0.4 mL / min;

[0018] Gradient elution program: 0–0.3 min 8% B; 0.3–5.5 min 8% → 95% B; 5.5–6.5 min 95% B; 6.6–8.0 min 8% B;

[0019] Column temperature: room temperature;

[0020] Mass spectrometry conditions include:

[0021] ESI+ mode: Ejection voltage: 4500V; MRM (Multiple Reaction Monitoring) detection; Ion source temperature: 500℃;

[0022] ESI Mode: Ejection Voltage: 4500V; MRM Detection; Ion Source Temperature: 500℃.

[0023] In a specific embodiment, the substances in the body after administration of psoralen include original components and metabolites, and the HPLC-MS / MS can detect one or more or all of the following substances:

[0024] Original components: psoralen (1), isopsoralen (2), psoralen glycoside (3), psoralen glycoside (4), isopsoralen glycoside (5), isopsoralen dihydroflavonoid (6), psoralen A (7), psoralen B (8), psoralen glycoside (9), psoralen dihydroflavonoid methyl ether (10), paratocarpin K (11), 3'-methoxycoumarin (12), corylifol A (13), psoralen chromene chalcone (14), isopsoralen chromene chalcone (15), 8-isopentenyl daidzein (16), corylifol C(17), daidzein(18), shampodophyllin(19), isoshampodophyllin(20), neopsoralen isoflavone(21), 8-geranioloxypsoralen(22), vitexin(23), 6-isopentenenaringenin(24), 4′-O-methylpsoralenchalcone(25), genistein(26), daidzein(27), astragalin(28), genistein(29), coumarin(31), 13-hydroxypsoralen(32), 3-hydroxypsoralen(33), psoralen(34), p-hydroxybenzoic acid(35), citric acid(36), quinic acid(37), isoquercitrin(38);

[0025] Metabolite components: Psoralen oxidation metabolites (psoralen + O, M1) O1 ), psoralen hydrolysate (psoralen + H2O, M1) H1 ), psoralen hydrolysis + reduction metabolites (psoralen + H2O + 2H, M1) H-R1 ), psoralen hydrolysis + oxidation metabolites (psoralen + H2O + O, M1) H-O1 ), isopsoralen oxidation metabolite (M2) O1 ), isopsostemin metabolite (M2) H1 ), psoralen glucuronide metabolite (psoralen + Glucose, M7) G1 ), psoralen glucuronide metabolite (psoralen + Gluc, M8) G1 ), Psoralen glucuronide metabolite (Psoralen + Glucose, M9) G1 ), Psoralen dihydroflavonoid methyl ether glucuronidated metabolite (Psoralen dihydroflavonoid methyl ether + Gluc, M10) G1 ), psoralen dihydroflavonoid methyl ether oxidation + glucuronidation metabolite (psoralen dihydroflavonoid methyl ether + O + Gluc, M10) O-G1 Corylifol A glucuronidated metabolites (corylifol A+Gluc, including corylifol A glucuronidated metabolite 1 (M13)) G1 Corylifol A glucuronidated metabolite 2 (M13)G2 Neopsoralen isoflavone glucuronidated metabolites (neoporalen isoflavone + Glu, including neopsoralen isoflavone glucuronidated metabolite 1 (M21)) G1 ), New psoralen isoflavone glucuronide metabolite 2 (M21) G2 ), psoralen oxidation metabolites (psoralen + O, M34) O1 ), psoralen glucuronide metabolite (psoralen + gluc, M34) G1 ), psoralen oxidation + glucuronidation metabolites (psoralen + O + Gluc, including psoralen oxidation + glucuronidation metabolite 1 (M34) O-G1 ), psoralen oxidation + glucuronidation metabolite 2 (M34) O-G2 ), psoralen oxidation + glucuronidation metabolite 3 (M34) O-G3 ), psoralen oxidation + oxidation + glucuronidation metabolites (psoralen + O + O + Gluc, M34) O-O-G1 The structures of the above metabolites are shown below:

[0026]

[0027] Wherein, Glu represents glucuronic acid; M34 O-G1-3 Representative psoralen oxidation + glucuronidation metabolite 1 (M34) O-G1 ), psoralen oxidation + glucuronidation metabolite 2 (M34) O-G2 ) and psoralen oxidation + glucuronidation metabolite 3 (M34) O-G3 ).

[0028] In a specific implementation, the HPLC-MS / MS quantitative method uses the external standard method for quantitative analysis; specifically, the original components are quantified using the standard curves established by their respective reference standards, and the metabolites without reference standards are semi-quantitatively analyzed using the standard curves of their respective original compounds. For example, the metabolites of psoralen are semi-quantitatively analyzed using the standard curve of psoralen (34). Because feed also contains some Chinese medicine ingredients, it may interfere with the detection of target substances in biological samples. For example, vitexin (26), citric acid (36), and quinic acid (37) interfere in blank plasma, and vitexin (23), citric acid (36), quinic acid (37), daidzein (18), and genistein (26) interfere in blank urine. Therefore, the blood and urine concentrations of vitexin (23) were semi-quantitatively determined using the standard curve of genistein (29), the blood and urine concentrations of citric acid (36) and quinic acid (37) were semi-quantitatively determined using the standard curve of p-hydroxybenzoic acid (35), and the urine concentrations of daidzein (18) and genistein (26) were semi-quantitatively determined using the standard curve of 8-isopentyldaidzein (16).

[0029] In a specific implementation, the individual is a mammalian individual, such as a human or a mouse. The individual's bodily fluid samples include plasma samples, urine samples, etc.

[0030] In a specific embodiment, the method for removing proteins from an individual's body fluid sample includes: mixing the body fluid sample with methanol, centrifuging to obtain the supernatant, which is the sample to be tested; wherein the volume ratio of methanol to body fluid sample is 1:2 to 5, for example, 1:3; the mixing can be carried out by shaking, with parameters of shaking at 1000 to 2000 rpm for 5 to 15 minutes, for example, shaking at 1400 rpm for 10 minutes; the centrifugation conditions are: centrifuging at 12000 to 18000 rpm for 5 to 15 minutes, for example, centrifuging at 14800 rpm for 10 minutes.

[0031] In a specific implementation, the method for detecting substances in vivo after administration of psoralen includes the preparation of standard plasma samples, standard urine samples, quality control plasma samples, or quality control urine samples by mixing blank plasma or urine with a working solution containing one or more reference standards for substances in vivo after administration of psoralen to be detected, in order to prepare a series of standard plasma samples, standard urine samples, quality control plasma samples, or quality control urine samples.

[0032] In a specific embodiment, the working solution containing one or more reference standards for in vivo substances following administration of psoralen is a dimethyl sulfoxide solution containing the one or more reference standards for in vivo substances following administration of psoralen.

[0033] In a specific embodiment, the standard plasma sample or standard urine sample contains one or more of the following components: psoralen (1), isopsoralen (2), psoralen tincture (3), psoralen glycoside (4), isopsoralen glycoside (5), isopsoralen dihydroflavonoid (6), psoralen A (7), psoralen B (8), psoralen nitrate (9), psoralen dihydroflavonoid methyl ether (10), paratocarpin K (11), 3'-methoxycoumarin (12), corylifol A (13), psoralen chromene chalcone (14), isopsoralen chromene chalcone (15), 8-isopentenyl daidzein (16), corylifol C(17), daidzein(18), shampodoside(19), isoshampodoside(20), neopsoralen isoflavone(21), 8-geranioloxypsoralen(22), vitexin(23), 6-isopentenenaringenin(24), 4′-O-methylpsoralenchalcone(25), genistein(26), daidzein(27), astragalin(28), genistein(29), coumarin(31), 13-hydroxypsoralen(32), 3-hydroxypsoralen(33), psoralen(34), p-hydroxy Benzoic acid (35), citric acid (36), quinic acid (37), isoquercitrin (38); among them, the series concentration of psoralen is in the range of 390.625 to 50000 nmol / L, and the series concentration of the other substances is in the range of 15.625 to 2000 nmol / L; in the corresponding quality control plasma samples or quality control urine samples, the series concentration of psoralen is in the range of 390.625 to 37500 nmol / L, and the series concentration of the other substances is in the range of 15.625 to 1500 nmol / L.

[0034] In a specific implementation, the C18 column can be selected from Agilent Eclipse Plus C18, Agilent Poroshell 120 EC-C18, and CAPCELL PAK C18, preferably an Agilent Eclipse Plus C18 column.

[0035] In a specific implementation, the flow rate is 0.3 mL / min.

[0036] In a specific implementation, the chromatographic conditions also include an injection volume of 3 μL.

[0037] In a specific implementation, the mass spectrometry conditions also include: ESI+ mode or ESI- mode: curtain gas pressure: 20 psi, collision gas pressure: 9 psi, nebulizer gas pressure: 50 psi, auxiliary gas pressure: 50 psi.

[0038] In a specific implementation, the MRM parameters of each analyte during mass spectrometry analysis in the method for detecting substances in vivo after administration of psoralen are as follows:

[0039]

[0040]

[0041] Note: The subscript Gluc in the metabolite indicates glucuronic acid metabolism, O indicates oxidative metabolism, H indicates hydrolysis, and R indicates reductive metabolism.

[0042] DP represents the declustering voltage, EP represents the inlet voltage, CE represents the collision energy, and CXP represents the collision chamber outlet voltage.

[0043] Beneficial effects

[0044] This invention utilizes liquid chromatography-mass spectrometry (LC-MS) to simultaneously detect 58 substances within psoralen bodies within 8 minutes by combining positive and negative ion scanning modes and standard quantification with virtual quantification. It enables the simultaneous detection of 11 isomers. When applied to mouse plasma samples, the method demonstrates resistance to matrix effects, with most compounds achieving a limit of quantification (LOQ) of 15.6 nM. This high-throughput analytical method is sensitive, reliable, and strongly supports the analysis of biological samples after psoralen administration. Attached Figure Description

[0045] Figure 1 This is a chromatogram of psoralen components in mouse plasma.

[0046] Figure 2 This is a chromatogram of metabolites in the plasma of mice after oral administration of Psoralea corylifolia extract.

[0047] Figure 3 It represents the relative exposure of psoralen in mouse plasma and urine samples.

[0048] Figure 4 This is the chromatogram of the standard plasma sample detected using the method corresponding to chromatographic conditions 1 in Comparative Example 1.

[0049] Figure 5 This is the chromatogram of the standard plasma sample detected using the method corresponding to chromatographic conditions 2 in Comparative Example 1.

[0050] Figure 6 This is the chromatogram of the standard plasma sample detected using the method corresponding to chromatographic conditions 3 in Comparative Example 1.

[0051] Figure 7 This is the chromatogram of the standard plasma sample detected using the method corresponding to chromatographic condition 4 in Comparative Example 1.

[0052] Figure 8 This is the chromatogram of the standard plasma sample detected using the method corresponding to chromatographic condition 5 in Comparative Example 1. Detailed Implementation

[0053] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0054] Unless otherwise specified, the raw materials, reagents, and methods used in the embodiments are all conventional raw materials, reagents, and methods in the art.

[0055] Reagents:

[0056] Organic solvents: Chromatographic grade methanol (analytical grade) purchased from Beijing Bailingwei Technology Co., Ltd.; chromatographic grade 95% ethanol and acetonitrile purchased from Sinopharm Chemical Reagent Co., Ltd.; chromatographic grade formic acid (analytical grade) purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; dimethyl sulfoxide (analytical grade) purchased from Sigma-Aldrich; ultrapure water was prepared using a Millipore Mill-Q Integral 3 system water purifier.

[0057] 95% ethanol and 75% ethanol refer to aqueous solutions of ethanol and water with volume ratios of 95:5 and 75:25, respectively.

[0058] The reference standards for psoralen components were purchased from Shanghai Shidande Standard Technology Co., Ltd., Sichuan Weikeqi Biotechnology Co., Ltd., and Wuhan Zhongbiao Technology Co., Ltd., as detailed in the table below.

[0059]

[0060]

[0061] Example 1. Method for detecting substances in the body after administration of psoralen.

[0062] 1. Preparation of the sample to be tested

[0063] (1) Preparation of 75% ethanol extract of Psoralea corylifolia:

[0064] Pulverization and Soaking: Psoralea corylifolia (purchased from Beijing Tongrentang (Anguo) Chinese Herbal Pieces Co., Ltd., batch number 2022060705, origin: Henan) was pulverized and passed through a No. 1 sieve. 200g of the powder was transferred to a 2L three-necked flask, and 1L of 75% ethanol was added to completely submerge the powder. After stirring, the flask was stoppered and sonicated for 5 minutes. The mixture was then soaked overnight at room temperature in the dark.

[0065] Heating and reflux: Set the heating mantle temperature to 80-81℃, and start timing 1.5h when the herb begins to boil and reflux. After timing, stop heating and cool to room temperature. Filter the extract using filter paper and a Buchner funnel, and collect the filtrate. Add 1L of 75% ethanol to the filter residue and heat and reflux again for 1.5h. Repeat this process 3 times, taking 1mL of the filtrate from each batch and retaining it. Combine the three filtrates.

[0066] Rotary evaporation and freeze-drying: Repeat the above operation once more, using a total of 400g of raw Psoralea corylifolia herb and heating under reflux in 6L of 75% ethanol (w / v: 1 / 15), and combine the extracts. The combined extract (5.4L) is then rotary evaporated to remove the organic solvent ethanol, yielding 2.8L of concentrated extract. The concentrated extract is frozen solid at -80℃ and then rapidly transferred to a freeze dryer for freeze-drying, finally obtaining 56.7g of freeze-dried powder of the 75% ethanol extract of Psoralea corylifolia. The yield is 14.185%.

[0067] The lyophilized powder was reconstituted in methanol and the resulting solution was used for pharmacokinetic experiments in mice.

[0068] (2) Mouse pharmacokinetics experiment:

[0069] Seventy-two male Kunming mice were randomly divided into 12 groups (n=6) and administered 4.2 g / kg of lyophilized psoralen extract powder (corresponding to 30 g / kg of raw psoralen) by gavage. Blood samples were collected as completely as possible from the retro-orbital venous plexus at 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, and 48 h after administration into heparinized centrifuge tubes. The plasma was immediately collected after centrifugation at 10,000 rpm for 2 min at 4 °C, aliquoted into 30 μl portions, and stored at -70 °C.

[0070] Twenty-four male Kunming mice were randomly divided into six groups of four, housed in the same metabolic cage (34×22×15cm³). Each mouse was administered 4.2 g / kg of lyophilized psoralen extract powder by gavage (corresponding to 30 g / kg of raw psoralen). Urine samples were collected at 0 h, 0-4 h, 4-8 h, 8-24 h, 24-48 h, and 48-72 h post-administration, and the volume of each sample was measured. After centrifugation at 10,000 rpm for 2 min at 4 °C, the supernatant was collected, aliquoted into 50 μl containers, and stored at -70 °C.

[0071] 2. Preparation and pretreatment of standard samples and quality control samples:

[0072] (1) Preparation of standard working solution and quality control working solution:

[0073] Each reference monomer compound (compounds 1-38) was prepared into a 10 mM solution using DMSO. 20 μl of each of the 10 mM stock solutions of the compounds other than psoralen (compound 34) was added to an appropriate amount of methanol to obtain a mixed standard solution with a concentration of 200 μM.

[0074] Add 40 μl of the above-mentioned 200 μM mixed standard solution and 340 μl of methanol to 20 μl of 10 mM psoralen DMSO stock solution to obtain mixed standard working solution ①. The concentration of psoralen in standard working solution ① is 500 μM, and the concentration of other compounds is 20 μM. Then, prepare standard working solutions ② to ⑧ according to the table below.

[0075]

[0076] Add 60 μl of the above-mentioned 200 μM mixed standard solution and 710 μl of methanol to 30 μl of 10 mM psoralen DMSO stock solution to obtain mixed quality control working solution ①. The concentration of psoralen in quality control working solution ① is 375 μM, and the concentration of other compounds is 15 μM. Then, prepare quality control working solutions ② to ⑥ according to the table below.

[0077]

[0078] (2) Preparation of standard plasma samples: 27 μl of blank mouse plasma + 3 μl of the above 10× standard working solution were used to obtain standard plasma samples of various concentrations. The concentrations of compounds 1-33 and 35-38 were 2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 nmol / L, respectively, and the concentrations of psoralen (compound 34) were 50000, 25000, 12500, 6250, 3125, 1562.5, 781.25 and 390.625 nmol / L, respectively.

[0079] (3) Preparation of quality control plasma samples: 27 μl of blank mouse plasma was mixed with 3 μl of the above 10× quality control working solution to obtain quality control plasma samples of various concentrations. The concentrations of compounds 1-33 and 35-38 were 1500, 800, 40, 31.25, 20, and 15.625 nmol / L, respectively, and the concentrations of psoralen (compound 34) were 37500, 20000, 1000, 781.25, 500, and 390.625 nmol / L, respectively. Six parallel batches of quality control plasma samples were prepared.

[0080] (4) Preparation of standard urine samples: 45 μl of blank mouse urine sample + 5 μl of the above 10× standard working solution were used to obtain standard urine samples of various concentrations. The concentrations of compounds 1-33 and 35-38 were 2000, 1000, 500, 250, 125, 62.5, 31.25 and 15.625 nmol / L, respectively. The concentrations of psoralen (compound 34) were 50000, 25000, 12500, 6250, 3125, 1562.5, 781.25 and 390.625 nmol / L, respectively.

[0081] (5) Preparation of quality control urine samples: 45 μl of blank mouse plasma + 5 μl of the above 10× quality control working solution were used to obtain quality control urine samples of various concentrations. The concentrations of compounds 1-33 and 35-38 were 1500, 800, 40, 31.25, 20, and 15.625 nmol / L, respectively, and the concentrations of psoralen (compound 34) were 37500, 20000, 1000, 781.25, 500, and 390.625 nmol / L, respectively. Six parallel batches of quality control urine samples were prepared.

[0082] Protein precipitation was performed by adding 30 μl of blank mouse plasma, 30 μl of standard plasma sample, 30 μl of quality control plasma sample, and 30 μl of mouse plasma sample before and after drug administration to 30 μl of each sample.

[0083] Protein precipitation was performed by adding 3 times the volume (150 μl) of methanol to 50 μl of blank mouse urine sample, 50 μl of standard urine sample, 50 μl of quality control urine sample, and 50 μl of mouse urine sample before and after drug administration.

[0084] The precipitated sample was then shaken at 1400 rpm for 10 min and centrifuged at 14800 rpm for 10 min. 60 μl of the supernatant was then collected into a sample vial.

[0085] 3. LC / MS / MS detection conditions:

[0086] Instruments and equipment:

[0087] The liquid chromatography-mass spectrometry (LC / MS / MS) system consists of an Agilent 1290 Infinity II series liquid chromatograph connected in series with an AB SCIEX API5500 mass spectrometer detector.

[0088] Chromatographic conditions:

[0089] Column: Agilent Eclipse Plus C18 (2.1*50mm, 5μm);

[0090] Mobile phase: Phase A: H2O (containing 0.1% formic acid), Phase B: MeOH (containing 0.1% formic acid);

[0091] Gradient elution program: 0–0.3 min: 8% B; 0.3–5.5 min: 8% → 95% B; 5.5–6.5 min: 95% B; 6.6–8.0 min: 8% B;

[0092] Mobile phase flow rate: 0.3 ml / min;

[0093] Column temperature: room temperature;

[0094] Injection volume: 3 μL;

[0095] Mass spectrometry detection conditions:

[0096] Ion source parameters:

[0097] Due to the differences in the ionization characteristics of various compounds in multi-component detection, an instrument capable of simultaneously detecting positive and negative ions should be selected for the analysis of substances in psoralen.

[0098]

[0099] MRM detection channel:

[0100]

[0101]

[0102]

[0103] Note: The subscript Gluc in the metabolite indicates glucuronic acid metabolism, O indicates oxidative metabolism, H indicates hydrolysis, and R indicates reductive metabolism.

[0104] DP represents the declustering voltage, EP represents the inlet voltage, CE represents the collision energy, and CxP represents the collision chamber outlet voltage.

[0105] The metabolite structure inferred from the mass spectrometry data is as follows:

[0106]

[0107] For the above-mentioned original components, standard curves established using their respective reference standards were used for quantification. However, since some traditional Chinese medicine components in the feed may interfere with the detection of target substances in biological samples, the blood and urine concentrations of vitexin (23) were semi-quantitatively analyzed using the standard curve of genistein (29); the blood and urine concentrations of citric acid (36) and quinic acid (37) were semi-quantitatively analyzed using the standard curve of p-hydroxybenzoic acid (35); and the urine concentrations of daidzein (18) and genistein (26) were semi-quantitatively analyzed using the standard curve of 8-isopentyldaidzein (16). For metabolites without reference standards, the standard curves of their respective original compounds were used for semi-quantitative analysis.

[0108] 4. Methodological Validation

[0109] 4.1 Quantitative analysis of standard curves and linear range

[0110] The standard samples and quality control samples prepared in "2. Preparation and pretreatment of standard samples and quality control samples" were injected and analyzed. By combining the response differences of each compound, standard curves for the quantitative analysis of each compound in mouse plasma and mouse urine samples were obtained, and the quantitative linear range was determined.

[0111] 4.2 Relative matrix effect and extraction recovery

[0112] Preparation of SET1 sample: Mix water and methanol at a ratio of 45:150 (v / v). Dilute 5 μL of 10× quality control working solution with 195 μL of the above mixture (since the actual concentration of the quality control solution varies depending on the concentration of different compounds, use 10× quality control working solutions ①②③⑤, n=6). The peak area of ​​the analyte obtained after injection analysis is Set1.

[0113] Preparation of SET2 samples: Blank matrix (plasma and urine) from 6 different Kunming mice was collected. The blank matrix from each mouse was mixed with methanol at a ratio of 45:150 (v / v). After protein precipitation, 5 μL of 10× control working solution was added. The peak area of ​​the analyte obtained after sample injection analysis is Set2.

[0114] Preparation of SET3 samples: Blank matrix (plasma and urine) from 6 different Kunming mice, identical to those in SET2, was collected. Quality control samples were prepared according to the method described in "2. Preparation of Standard Samples, Quality Control Samples, and Sample Pretreatment," and then 3 times the volume of methanol was added for sample pretreatment. The peak area of ​​the analyte obtained after injection analysis is Set3.

[0115] Matrix effect = Set2 / average Set1 × 100%;

[0116] Recovery rate = Set3 / average Set2 × 100%;

[0117] The RSD of the matrix effect at each concentration, i.e., the relative matrix effect, should be within 15%.

[0118] 4.3 Precision and Accuracy

[0119] An analytical batch is composed of blank samples, standard samples, and quality control samples (n=6). Three analytical batches are run (over at least 2 days) to examine the intra-batch and inter-batch accuracy and precision of each substance in mouse plasma and urine. The accuracy of each concentration of the quality control samples, i.e., the ratio of the measured concentration to the labeled concentration of each compound in the sample, should be between 85% and 115%, and the LLOQ accuracy should be between 80% and 120%. Precision is expressed as the RSD of six analyses of each concentration of the quality control samples and should not exceed 15% (LLOQ should not exceed 20%).

[0120] 5. Results:

[0121] 5.1 Quantitative analysis standard curve and linear range

[0122] Table 1 below shows the quantitative standard curves and quantitative ranges of the main exposed substances in plasma and urine samples after oral administration of psoralen extract to mice:

[0123] Table 1. Standard curves and linear ranges for the quantitative analysis of major exposed substances in plasma and urine samples.

[0124]

[0125]

[0126]

[0127] 5.2 Relative matrix effect and extraction recovery

[0128] Table 2. Relative matrix effect and extraction recovery rate of psoralen in mouse plasma samples

[0129]

[0130] 5.3 Precision and Accuracy

[0131] Table 3. Precision and accuracy of the analytical method for psoralen in mouse plasma samples

[0132]

[0133]

[0134]

[0135] 5.4 Relative exposure of psoralen in mouse plasma and urine samples

[0136] The chromatogram of plasma elements of psoralen extract in mice after oral administration is shown below. Figure 1 As shown, the chromatogram of the metabolite is as follows: Figure 2 As shown.

[0137] Since the plasma from mice at different time points came from different individuals, the average plasma drug concentration at each time point was used to calculate the area under the plasma drug-time curve (AUC). The relative exposures of psoralen in plasma and urine after oral administration of psoralen extract to mice are shown below. Figure 3 As shown.

[0138] After administration of Psoralea corylifolia, a wide variety of substances with significant differences in properties are produced in the body, and there are many isomers of flavonoids. Therefore, appropriate chromatographic and mass spectrometric conditions are required to achieve effective separation of isomers and other components. Key influencing factors include the elution system (including the selection and ratio of the organic phase in the mobile phase and the adjustment of acidity), the elution program settings, and the selection of the mass spectrometry detection mode and MRM detection channel. The following are some typical detection methods different from the examples provided to illustrate the problems encountered in the detection of substances in vivo after administration of Psoralea corylifolia.

[0139] Comparative Example 1

[0140] Standard plasma samples containing psoralen (1) / isopsoralen (2), psoralen glycoside (4) / isopsoralen glycoside (5), isopsoralen dihydroflavonoid (6) / psoralen A (7) / psoralen B (8), 8-isopentenyl daidzein (16) / neopsoralen isoflavone (21), and psoralen (34) were detected using chromatographic or mass spectrometric conditions different from those described in Example 1 ("3.LC / MS / MS detection conditions").

[0141] Chromatographic conditions 1:

[0142] Mobile phase: Phase A: H2O (containing 0.004% formic acid), Phase B: Acetonitrile (containing 0.004% formic acid);

[0143] Gradient elution program: 0–0.5 min: 10% B; 0.6–2.5 min: 10% B → 55% B; 2.6–6.5 min: 55% B → 95% B; 6.6–7.0 min: 95% B; 7.1–8.0 min: 10% B.

[0144] Chromatographic conditions 2:

[0145] Mobile phase: Phase A: H2O (containing 0.004% formic acid), Phase B: Acetonitrile (containing 0.004% formic acid);

[0146] Gradient elution program: 0–0.5 min: 5% B; 0.6–2.0 min: 5% B → 65% B; 2.1–6.0 min: 65% B → 98% B; 6.1–7.0 min: 98% B; 7.1–8.0 min: 5% B.

[0147] Chromatographic condition 3:

[0148] Mobile phase: Phase A: H2O (containing 0.004% formic acid), Phase B: Acetonitrile (containing 0.004% formic acid);

[0149] Gradient elution program: 0–0.5 min: 10% B; 0.6–2.0 min: 10% B → 65% B; 2.1–6.0 min: 65% B → 98% B; 6.1–7.0 min: 98% B; 7.1–8.0 min: 10% B.

[0150] Chromatographic condition 4:

[0151] Mobile phase: Phase A: H2O (containing 0.004% formic acid), Phase B: MeOH (containing 0.004% formic acid);

[0152] Gradient elution program: 0–0.5 min: 10% B; 0.6–2.5 min: 10% B → 55% B; 2.6–6.5 min: 55% B → 95% B; 6.6–7.0 min: 95% B; 7.1–8.0 min: 10% B.

[0153] The MRM parameters corresponding to chromatographic conditions 1-4 are as follows:

[0154] MRM detection channel:

[0155]

[0156] Chromatographic condition 5:

[0157] Mobile phase: Phase A: H2O (containing 0.004% formic acid), Phase B: MeOH (containing 0.004% formic acid);

[0158] Gradient elution program: 0–0.3 min: 8% B; 0.4–5.5 min: 8% B → 95% B; 5.6–6.5 min: 95% B; 6.6–8.0 min: 8% B.

[0159] The MRM parameters corresponding to chromatographic condition 5 are:

[0160] MRM detection channel:

[0161]

[0162]

[0163] The chromatograms of standard plasma samples detected using chromatographic conditions 1-5 are shown below. Figures 4-8 The results showed that under chromatographic condition 1, with acetonitrile as the organic phase, the separation of psoralen (1) / isopsoralen (2), isopsoralen dihydroflavonoid (6) / psoralen A (7) / psoralen B (8), and 8-isopentenyl daidzein (16) / neopsoralen isoflavone (21) was poor. Figure 4 Based on chromatographic condition 1, chromatographic condition 2 adjusted the mobile phase gradient, which effectively improved the separation of other isomers, but psoralen (1) / isopsoralen (2) could not be effectively separated. Figure 5 After further increasing the initial proportion of acetonitrile to slightly slow down the gradient change of the intermediate mobile phase (chromatographic condition 3), the peak shape of psoralen (4) / isopsoralen (5) deteriorated. Figure 6 The results indicate that the peak shapes of both substances are greatly affected by the initial mobile phase ratio, and careful exploration is needed to achieve better chromatographic peak shapes and resolution. After replacing the organic phase with methanol under chromatographic condition 4, the separation of each substance was improved. Under chromatographic condition 5, after further improving the mobile phase gradient, the MRM detection channel for psoralen isoflavones, and adding the negative ion detection mode for psoralen, the separation of each substance was further improved, indicating that psoralen (34) is highly lipophilic and a high proportion of elution zone is required to avoid incomplete elution.

Claims

1. A method for detecting substances in the body after administration of psoralen, comprising removing proteins from an individual's body fluid sample and then performing detection using HPLC-MS / MS; in, Chromatographic conditions include: Chromatographic column: C18 column; Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: methanol containing 0.1% formic acid; Flow rate: 0.2–0.4 mL / min; Gradient elution program: 0–0.3 min 8% B; 0.3–5.5 min 8% → 95% B; 5.5–6.5 min 95% B; 6.6–8.0 min 8% B; Column temperature: room temperature; Mass spectrometry conditions include: ESI+ mode: Ejection voltage: 4500V; MRM (Multiple Reaction Monitoring) detection; Ion source temperature: 500℃; ESI Mode: Ejection Voltage: 4500V; MRM Detection; Ion Source Temperature: 500℃.

2. The detection method according to claim 1, characterized in that, After administration of psoralen, the substances in the body include the original components and metabolites. The HPLC-MS / MS method detects one or more or all of the following substances: Original components: psoralen (1), isopsoralen (2), psoralen glycoside (3), psoralen glycoside (4), isopsoralen glycoside (5), isopsoralen dihydroflavonoid (6), psoralen A (7), psoralen B (8), psoralen glycoside (9), psoralen dihydroflavonoid methyl ether (10), paratocarpin K (11), 3'-methoxycoumarin (12), corylifol A (13), psoralen chromene chalcone (14), isopsoralen chromene chalcone (15), 8-isopentenyl daidzein (16), corylifol C(17), daidzein(18), shampodophyllin(19), isoshampodophyllin(20), neopsoralen isoflavone(21), 8-geranioloxypsoralen(22), vitexin(23), 6-isopentenenaringenin(24), 4′-O-methylpsoralenchalcone(25), genistein(26), daidzein(27), astragalin(28), genistein(29), coumarin(31), 13-hydroxypsoralen(32), 3-hydroxypsoralen(33), psoralen(34), p-hydroxybenzoic acid(35), citric acid(36), quinic acid(37), isoquercitrin(38); Metabolite components: Psoralen oxidation metabolite (M1) O1 ), psoralen hydrolysate (M1) H1 ), psoralen hydrolysate + reductive metabolite (M1) H-R1 ), psoralen hydrolysis + oxidation metabolites (M1) H-O1 ), isopsoralen oxidation metabolite (M2) O1 ), isopsoralen hydrolysate (M2) H1 ), psoralen glucuronide metabolite (M7) G1 ), psoralen glucuronide metabolite (M8) G1 ), Psoralen glucuronide metabolite (M9) G1 ), Psoralen dihydroflavonoid methyl ether glucuronide metabolite (M10) G1 ), Psoralen dihydroflavonoid methyl ether oxidation + glucuronidation metabolite (M10) O-G1 Corylifol A glucuronidated metabolite 1 (M13) G1 Corylifol A glucuronidated metabolite 2 (M13) G2 ), New psoralen isoflavone glucuronide metabolite 1 (M21) G1 ), New psoralen isoflavone glucuronide metabolite 2 (M21) G2 ), psoralen oxidation metabolite (M34) O1 ), psoralen glucuronide metabolite (M34) G1 ), psoralen oxidation + glucuronidation metabolite 1 (M34) O-G1 ), psoralen oxidation + glucuronidation metabolite 2 (M34) O-G2 ), psoralen oxidation + glucuronidation metabolite 3 (M34) O-G3 ), psoralen oxidation + oxidation + glucuronidation metabolite (M34) O-O-G1 ); The structures of the above metabolites are shown below: Wherein, Glu represents glucuronic acid; M34 O-G1-3 Representative psoralen oxidation + glucuronidation metabolite 1 (M34) O-G1 ), psoralen oxidation + glucuronidation metabolite 2 (M34) O-G2 ) and psoralen oxidation + glucuronidation metabolite 3 (M34) O-G3 ).

3. The detection method according to claim 1, characterized in that, The external standard method was used for quantitative analysis in HPLC-MS / MS. For original components, quantification was performed using standard curves established with their respective reference standards; for metabolites without reference standards, semi-quantitative analysis was performed using standard curves of their respective original compounds.

4. The detection method according to claim 1, characterized in that, The individual is a mammalian individual, preferably a human or a mouse; the body fluid samples of the individual include plasma samples and urine samples.

5. The detection method according to claim 1, characterized in that, The method for removing proteins from an individual's bodily fluid sample includes: mixing the bodily fluid sample with methanol, centrifuging to obtain the supernatant, thus obtaining the sample to be tested; wherein the volume ratio of methanol to the bodily fluid sample is 1:2 to 5, for example 1:3; and / or The mixing is performed by oscillation, with parameters of oscillation at 1000-2000 rpm for 5-15 minutes, for example, oscillation at 1400 rpm for 10 minutes; the centrifugation conditions are: centrifugation at 12000-18000 rpm for 5-15 minutes, for example, centrifugation at 14800 rpm for 10 minutes.

6. The detection method according to claim 1, characterized in that, The preparation of standard plasma samples, standard urine samples, quality control plasma samples, or quality control urine samples includes: mixing blank plasma or urine with a working solution containing one or more in vivo reference standards for substances to be tested after administration of psoralen to prepare a series of concentrations of standard plasma samples, standard urine samples, quality control plasma samples, or quality control urine samples. Preferably, the working solution containing one or more reference standards for in vivo substances following administration of psoralen is a dimethyl sulfoxide solution containing the one or more reference standards for in vivo substances following administration of psoralen.

7. The detection method according to claim 6, characterized in that, The standard plasma sample or standard urine sample contains one or more of the following components: psoralen (1), isopsoralen (2), psoralen tincture (3), psoralen glycoside (4), isopsoralen glycoside (5), isopsoralen dihydroflavonoid (6), psoralen A (7), psoralen B (8), psoralen nitrate (9), psoralen dihydroflavonoid methyl ether (10), paratocarpin K (11), 3'-methoxycoumarin (12), corylifol A (13), psoralen chromene chalcone (14), isopsoralen chromene chalcone (15), 8-isopentenyl daidzein (16), corylifol C(17), daidzein(18), shampodoside(19), isoshampodoside(20), neopsoralen isoflavone(21), 8-geranioloxypsoralen(22), vitexin(23), 6-isopentenenaringenin(24), 4′-O-methylpsoralenchalcone(25), genistein(26), daidzein(27), astragalin(28), genistein(29), coumarin(31), 13-hydroxypsoralen(32), 3-hydroxypsoralen(33), psoralen(34), p-hydroxy Benzoic acid (35), citric acid (36), quinic acid (37), isoquercitrin (38); among them, the series concentration of psoralen is in the range of 390.625 to 50000 nmol / L, and the series concentration of the other substances is in the range of 15.625 to 2000 nmol / L; in the corresponding quality control plasma samples or quality control urine samples, the series concentration of psoralen is in the range of 390.625 to 37500 nmol / L, and the series concentration of the other substances is in the range of 15.625 to 1500 nmol / L.

8. The detection method according to claim 1, characterized in that, The C18 column is selected from one of Agilent Eclipse Plus C18, Agilent Poroshell 120 EC-C18, and CAPCELL PAK C18, preferably an Agilent Eclipse Plus C18 column; and / or The flow rate is 0.3 mL / min; and / or The chromatographic conditions also include an injection volume of 3 μL.

9. The detection method according to claim 1, characterized in that, The mass spectrometry conditions also include: ESI+ mode or ESI- mode: curtain gas pressure: 20 psi, collision gas pressure: 9 psi, nebulizer gas pressure: 50 psi, auxiliary gas pressure: 50 psi.

10. The detection method according to claim 1, characterized in that, In the method for detecting substances in vivo after administration of psoralen, the MRM parameters of each analyte during mass spectrometry analysis are as follows: Wherein, DP represents the declustering voltage, EP represents the inlet voltage, CE represents the collision energy, and CXP represents the collision chamber outlet voltage.

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