阅读说明：本技术 一种uplc-q-tof-ms快速筛查甘草中活性成分的方法 (Method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS ) 是由 李婷 韩建勋 付萌 张雅莉 刘浩 李捷 于 2020-11-11 设计创作，主要内容包括：本发明涉及药品检测领域,公开了一种超高效液相色谱-四级杆飞行时间串联质谱(UPLC-Q-TOF-MS)快速筛查甘草中活性成分的方法。采用75％乙醇溶液提取甘草中活性成分,有机滤膜过滤,UPLC-Q-TOF-MS检测,结合UNIFI软件平台,利用目标化合物特征离子的精确质量数、二级碎片信息和保留时间进行数据匹配,筛查甘草及甘草制品中的活性成分。该方法快速、准确、分析通量高,可以为甘草及甘草制品的产地溯源、真假鉴别、质量分级和市场监管提供重要的方法依据。(The invention relates to the field of medicine detection, and discloses a method for rapidly screening active ingredients in liquorice by using ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UPLC-Q-TOF-MS). Extracting active ingredients in liquorice by using a 75% ethanol solution, filtering by using an organic filter membrane, detecting by using UPLC-Q-TOF-MS, matching data by using the accurate mass number, secondary fragment information and retention time of characteristic ions of a target compound in combination with a UNIFI software platform, and screening the active ingredients in the liquorice and liquorice products. The method is rapid and accurate, has high analysis flux, and can provide important method basis for tracing the origin of the liquorice and liquorice products, identifying true and false, grading quality and monitoring market.)

1. A method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS is characterized by comprising the following steps:

a) sample pretreatment: crushing a sample, sieving the sample by a 60-mesh stainless steel sieve, weighing about 0.5g of the sample in a centrifuge tube, adding 50mL of 75% ethanol for dissolution, and uniformly mixing the solution by vortex; ultrasonic extracting for 10min, centrifuging at 8000r/min for 5min, filtering the supernatant with 0.22 μm organic filter membrane, and collecting filtrate to obtain sample solution;

b) separating the sample solution by UPLC, collecting data by Q-TOF-MS, and establishing a screening spectrum library based on the cracking rule of each active component.

2. The method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS as claimed in claim 1, wherein the separation conditions of the ultra performance liquid chromatography are chromatographic column Acquity UPLC HSS T3, 100mm x 2.1mm, 1.8 μm; the mobile phase A is aqueous solution containing 0.1 percent of formic acid, the phase B is acetonitrile, and gradient elution is carried out; the gradient elution ratio is 0-10 min, and A is 90% → 70%; 10-30 min, wherein A is 70% → 50%; 30-40 min, wherein A is 50% → 10%; 40-53 min, wherein A is 10%; 53-55 min, wherein A is 10% → 90%; 55-60 min, wherein A is 90%; the flow rate was 0.5mL/min, the amount of sample was 5. mu.L, and the column temperature was 45 ℃.

3. The method for rapidly screening active ingredients in liquorice according to claim 1, wherein Q-TOF-MS conditions are ESI ion source and negativeAn ionic mode; the ion source temperature is 100 ℃; the temperature of desolventizing gas is 550 ℃; the desolventizing air flow rate is 600L/h; collision energy: low collision energy off and high collision energy of 20-40V; capillary voltage 2.0 kV; the taper hole voltage is 40 eV; the air flow rate of the taper hole is 50L/h; the scanning mode is a sensitivity mode, and the scanning range m/z is 100-1200; the scanning time is 1.5 s; data acquisition mode is MS^EMode(s).

4. The method for rapid screening of active ingredients in licorice according to claim 1, wherein the library contains 20 kinds of excimer ions of active ingredients, mass-to-charge ratio information of secondary fragments, retention time, etc., wherein m/z of ferulic acid excimer ion is 193.0501, m/z of secondary fragments is 147.0452, 163.0401, 135.0452, 145.0295, 134.0373, retention time is 1.69 min; the m/z of isoliquiritin apioside excimer ion is 549.1608, the m/z of secondary fragment is 135.0088, 119.0502, 297.0768, 255.0663 and 399.1085, and the retention time is 4.57 min; the liquiritin excimer ion m/z is 417.1186, the secondary fragment m/z is 255.0663, 135.0088, 119.0502, 297.0768 and 399.1085, and the retention time is 4.57 min; the m/z of glycyrrhizin excimer ion is 549.1608, the m/z of secondary fragment is 255.0663, 135.0088, 297.0768, 119.0502 and 417.1191, and the retention time is 4.59 min; the neoliquiritin excimer ion m/z is 417.1186, the secondary fragment m/z contains 255.0663, 135.0088, 119.0502, 150.0322 and 269.0456, and the retention time is 7.58 min; the isoliquiritigenin excimer ion m/z is 255.0657, the secondary fragment m/z is 119.0502, 135.0088, 149.0244, 150.0322 and 117.0346, and the retention time is 8.07 min; the formononetin excimer ion m/z is 267.0657, the secondary fragment m/z is 252.0428, 251.0350, 132.0217, 135.0088 and 119.0502, and the retention time is 9.33 min; the formononetin excimer ion m/z is 429.1186, the secondary fragment m/z is 252.0428, 251.0350, 254.0585, 132.0217 and 135.0452, and the retention time is 10.89 min; the m/z of glycyrrhizin excimer ion is 255.0657, the m/z of secondary fragment is 119.0502, 135.0088, 117.0346, 213.0557 and 109.0295, and the retention time is 12.64 min; the glycyrrhizic acid excimer ion m/z is 821.3960, the secondary fragment m/z is 351.1057, 193.0354, 759.3961, 803.3859 and 175.0248, and the retention time is 16.51 min; the m/z of the javanine B excimer ion is 355.1545, the m/z of the secondary fragment is 323.1289, 203.0714, 283.0612, 135.0452 and 254.0585, and the retention time is 19.74 min; the m/z of the demethyl anhydroicaritin excimer ion is 353.1025, the m/z of the secondary fragment is 297.0405, 284.0326, 269.0456 and 281.0456227.0350, and the retention time is 20.58 min; the m/z of the licoricone excimer ion is 381.1338, the m/z of the secondary fragments is 279.0299, 335.0925, 366.1109, 311.0561 and 308.0326, and the retention time is 21.90 min; glabridin excimer ion m/z is 323.1283, secondary fragment m/z is 135.0452, 323.1289, 201.0921, 253.0506 and 187.0764, and retention time is 24.05 min; the Sophora Moorcroftiana isoflavone A excimer ion m/z is 351.0869, the secondary fragment m/z is 333.0768, 335.0561, 199.0164, 215.0714 and 203.0714, and the retention time is 25.55 min; psoralea corylifolia fixes excimer ions m/z of 335.0919, secondary fragments m/z of 319.0612, 317.0819, 293.0456, 176.0115 and 161.0244, and retention time of 27.46 min; the m/z of licochalcone A excimer ion is 337.1440, the m/z of secondary fragments is 297.1132, 307.0976, 201.0921 and 149.0608, and the retention time is 32.00 min; the molecular ion m/z of the sangrinone C excimer is 421.1651, the secondary fragment m/z is 365.1031, 407.1864, 309.0405, 337.1082 and 323.0561, and the retention time is 32.71 min; the glycyrrhetinic acid excimer ion m/z is 469.3318, the secondary fragments m/z are 201.0121, 219.1027, 425.3425, 221.1183 and 205.0870, and the retention time is 33.69 min; the m/z of the deoxyglycyrrhetinic acid excimer ion is 455.3525, the m/z of the secondary fragments is 251.2380, 135.0451, 429.3374, 161.0972 and 389.3061, and the retention time is 36.77 min. The compound is determined by that the screening meets the condition that the retention time is limited within +/-0.5 min, the accurate mass deviation is 5mDa, the ionization form selects a + H mode, and more than 3 pieces of fragment information are matched.

Technical Field

The invention belongs to the field of medical analysis, and relates to a method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS.

Background

The Glycyrrhrizae radix is dried root and rhizome of Leguminosae plant Glycyrrhrizae radix, radix Glycyrrhizae Inflatae or Glycyrrhrizae radix. The skin has different elasticity, the surface is reddish brown or gray brown, the smell is slight, and the taste is sweet and special. The liquorice has the effects of clearing away heat and toxic materials, eliminating phlegm and stopping cough, tonifying spleen and qi, relieving spasm and pain, harmonizing the drugs and the like, has obvious drug effect and very high clinical use frequency, and plays an important role in the prevention and treatment process of the novel global coronavirus pneumonia which is outbreak at the end of 2019. The liquorice in China is mainly distributed in Ningxia, Xinjiang, Neimang, Gansu and other places, and the types and content differences of active ingredients in the liquorice in different production places or varieties are large, so that the active ingredients in the liquorice can be accurately and qualitatively identified and quantitatively detected, and the method is very important for tracing the production places and grading the quality of related products.

At present, liquid chromatography tandem mass spectrometry and other methods are mostly adopted for qualitative and quantitative analysis of one or more active ingredients in liquorice in domestic and overseas researches, but relevant research reports for rapidly screening the active ingredients in the liquorice by using UPLC-Q-TOF-MS are few, and no method can realize rapid high-throughput screening of more than 20 active ingredients in the liquorice. However, the traceability, the identification of true and false and the quality grading of liquorice and products thereof all urgently need a high-throughput screening and detecting technology of active ingredients thereof.

Therefore, a high-flux rapid screening and detecting method for active ingredients in liquorice is urgently to be developed.

Disclosure of Invention

In order to solve the problems, the invention provides a method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS. The method takes 75% ethanol as an extraction solvent, has high extraction rate and good stability on each active component in the liquorice, can effectively remove interfering impurities, and simultaneously adopts UPLC-Q-TOF-MS to rapidly screen samples without standard products.

The specific technical scheme of the invention is as follows: a method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS comprises the following steps:

(1) sample pretreatment: crushing a sample, sieving the sample by a 60-mesh stainless steel sieve, weighing about 0.5g of the sample in a centrifuge tube, adding about 50mL of 75% ethanol for dissolution, and uniformly mixing the solution by vortex; ultrasonic extracting for 10min, centrifuging at 8000r/min for 5min, filtering the supernatant with 0.22 μm organic filter membrane, collecting filtrate to be tested;

(2) separating the sample solution by UPLC, collecting data by Q-TOF-MS, and establishing a screening spectrum library based on the cracking rule of each active component.

(3) The separation conditions of the UPLC are chromatographic column Acquity UPLC HSS T3, 100mm multiplied by 2.1mm, 1.8 μm; the mobile phase A is aqueous solution containing 0.1 percent of formic acid, the phase B is acetonitrile, and gradient elution is carried out; the gradient elution ratio is 0-10 min, and A is 90% → 70%; 10-30 min, wherein A is 70% → 50%; 30-40 min, wherein A is 50% → 10%; 40-53 min, wherein A is 10%; 53-55 min, wherein A is 10% → 90%; 55-60 min, wherein A is 90%; the flow rate was 0.5mL/min, the amount of sample was 5. mu.L, and the column temperature was 45 ℃.

(4) The Q-TOF-MS condition is an ESI ion source and a negative ion mode; the ion source temperature is 100 ℃; the temperature of desolventizing gas is 550 ℃; the desolventizing air flow rate is 600L/h; collision energy: low collision energy off and high collision energy of 20-40V; capillary voltage 2.0 kV; the taper hole voltage is 40 eV; the air flow rate of the taper hole is 50L/h; the scanning mode is a sensitivity mode, and the scanning range m/z is 100-1200; the scanning time is 1.5 s; data acquisition mode is MS^EMode(s).

The self-built screening spectrum library comprises excimer ions of 20 active ingredients in liquorice, more than 3 pieces of secondary fragment information, retention time and the like, wherein m/z of ferulic acid excimer ions is 193.0501, m/z of secondary fragments is 147.0452, 163.0401, 135.0452, 145.0295 and 134.0373, and the retention time is 1.69 min; the m/z of isoliquiritin apioside excimer ion is 549.1608, the m/z of secondary fragment is 135.0088, 119.0502, 297.0768, 255.0663 and 399.1085, and the retention time is 4.57 min; the liquiritin excimer ion m/z is 417.1186, the secondary fragment m/z is 255.0663, 135.0088, 119.0502, 297.0768 and 399.1085, and the retention time is 4.57 min; the m/z of glycyrrhizin excimer ion is 549.1608, the m/z of secondary fragment is 255.0663, 135.0088, 297.0768, 119.0502 and 417.1191, and the retention time is 4.59 min; the neoliquiritin excimer ion m/z is 417.1186, the secondary fragment m/z contains 255.0663, 135.0088, 119.0502, 150.0322 and 269.0456, and the retention time is 7.58 min; the isoliquiritigenin excimer ion m/z is 255.0657, the secondary fragment m/z is 119.0502, 135.0088, 149.0244, 150.0322 and 117.0346, and the retention time is 8.07 min; the formononetin excimer ion m/z is 267.0657, the secondary fragment m/z is 252.0428, 251.0350, 132.0217, 135.0088 and 119.0502, and the retention time is 9.33 min; the formononetin excimer ion m/z is 429.1186, the secondary fragment m/z is 252.0428, 251.0350, 254.0585, 132.0217 and 135.0452, and the retention time is 10.89 min; the m/z of glycyrrhizin excimer ion is 255.0657, the m/z of secondary fragment is 119.0502, 135.0088, 117.0346, 213.0557 and 109.0295, and the retention time is 12.64 min; the glycyrrhizic acid excimer ion m/z is 821.3960, the secondary fragment m/z is 351.1057, 193.0354, 759.3961, 803.3859 and 175.0248, and the retention time is 16.51 min; the m/z of the javanine B excimer ion is 355.1545, the m/z of the secondary fragment is 323.1289, 203.0714, 283.0612, 135.0452 and 254.0585, and the retention time is 19.74 min; the m/z of the demethyl anhydroicaritin excimer ion is 353.1025, the m/z of the secondary fragment is 297.0405, 284.0326, 269.0456 and 281.0456227.0350, and the retention time is 20.58 min; the m/z of the licoricone excimer ion is 381.1338, the m/z of the secondary fragments is 279.0299, 335.0925, 366.1109, 311.0561 and 308.0326, and the retention time is 21.90 min; glabridin excimer ion m/z is 323.1283, secondary fragment m/z is 135.0452, 323.1289, 201.0921, 253.0506 and 187.0764, and retention time is 24.05 min; the Sophora Moorcroftiana isoflavone A excimer ion m/z is 351.0869, the secondary fragment m/z is 333.0768, 335.0561, 199.0164, 215.0714 and 203.0714, and the retention time is 25.55 min; psoralea corylifolia fixes excimer ions m/z of 335.0919, secondary fragments m/z of 319.0612, 317.0819, 293.0456, 176.0115 and 161.0244, and retention time of 27.46 min; the m/z of licochalcone A excimer ion is 337.1440, the m/z of secondary fragments is 297.1132, 307.0976, 201.0921 and 149.0608, and the retention time is 32.00 min; the molecular ion m/z of the sangrinone C excimer is 421.1651, the secondary fragment m/z is 365.1031, 407.1864, 309.0405, 337.1082 and 323.0561, and the retention time is 32.71 min; the glycyrrhetinic acid excimer ion m/z is 469.3318, the secondary fragments m/z are 201.0121, 219.1027, 425.3425, 221.1183 and 205.0870, and the retention time is 33.69 min; the m/z of the deoxyglycyrrhetinic acid excimer ion is 455.3525, the m/z of the secondary fragments is 251.2380, 135.0451, 429.3374, 161.0972 and 389.3061, and the retention time is 36.77 min. The compound is determined by that the screening meets the condition that the retention time is limited within +/-0.5 min, the accurate mass deviation is 5mDa, the ionization form selects a + H mode, and more than 3 pieces of fragment information are matched.

Has the advantages that:

the invention provides a method for rapidly screening active ingredients in liquorice by UPLC-Q-TOF-MS, which can realize qualitative screening of 20 active ingredients under the condition of no standard substance contrast, provides a new effective method for tracing the origin of the liquorice and products thereof, identifying true and false and grading quality, and has important significance for market supervision and quality assurance.

Drawings

FIG. 1 Total ion flow diagram of Glycyrrhiza samples

FIG. 2 ion chromatogram and MS of ferulic acid extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 3 ion chromatogram of isoliquiritin apiose extraction and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 4 extraction ion chromatogram and MS of liquiritin^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 5 ion chromatogram and MS of glycyrrhizin extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 6 ion chromatogram of extraction of neoliquiritin and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 7 ion chromatogram of isoliquiritigenin extraction and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 8 extracted ion chromatogram of formononetin and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 9 extraction ion chromatogram and MS of formononetin^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 10 ion chromatogram and MS of liquiritigenin extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 11 chromatogram of extracted ions of glycyrrhizic acid and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 12. white jadeExtracted ion chromatogram of Lanting B and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 13 ion chromatogram and MS of extraction of anhydro-noricaritin^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 14 ion chromatogram for extraction of Glycyrrhiza uralensis and MS^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 15 ion chromatogram and MS of glabridin extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 16 ion chromatogram and MS of Sophora Moorcroftiana isoflavone A extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 17 ion chromatogram and MS of psoralen extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 18 ion chromatogram and MS of licochalcone A^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 19 ion chromatogram and MS of Morusinone C extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 20 ion chromatogram and MS of Glycyrrhizinic acid extraction^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

FIG. 21 ion chromatogram and MS of extraction of deoxyglycyrrhetinic acid^EHigh collision energy channel mass spectrogram and secondary fragment matching condition under mode

Detailed Description

Example 1 active ingredient screening in licorice

1 materials and methods

1.1 materials and instruments

Liquorice, purchased from the institute for food and drug testing, china; acetonitrile, methanol, formic acid LC-MS grade, ThermoFisher Scientific; and (3) standard substance: the purity of ammonium glycyrrhizinate, liquiritin, glycyrrhetinic acid and the like is more than or equal to 93 percent, and the purity of the ammonium glycyrrhizinate, the liquiritin, the glycyrrhetinic acid and the like is shown in China institute for testing and researching biological products of medicines; nylon filter 0.22 μm, Agela Technologies USA.

Waters Xevo G2-XS QTOF/UPLC System electrospray ion source Waters corporation, USA; UNIFI 1.8 software System, Waters corporation, USA; Vortex-Genie 2 Vortex shaker, Scientific in.d. industries, usa; Millipore-Q ultra pure Water purifiers Millipore-Q, USA; AB135-S analytical balance, sensory 0.01g/0.01mg, Mettler TOLEDO, Switzerland; KH-500E ultrasonic cleaner, Kunshan Selengensis ultrasonic Instrument Co., Ltd.

1.2 Experimental methods

1.2.1 chromatographic conditions

Chromatographic column Acquity UPLC HSS T3, 100mm × 2.1mm, 1.8 μm; the mobile phase A is aqueous solution containing 0.1 percent of formic acid, the phase B is acetonitrile, and gradient elution is carried out; the gradient elution ratio is 0-10 min, and A is 90% → 70%; 10-30 min, wherein A is 70% → 50%; 30-40 min, wherein A is 50% → 10%; 40-53 min, wherein A is 10%; 53-55 min, wherein A is 10% → 90%; 55-60 min, wherein A is 90%; the flow rate was 0.5mL/min, the amount of sample was 5. mu.L, and the column temperature was 45 ℃.

1.2.2 Mass Spectrometry conditions

Ion source ESI, negative ion mode; the ion source temperature is 100 ℃; the temperature of desolventizing gas is 550 ℃; the desolventizing air flow rate is 600L/h; collision energy: low collision energy off and high collision energy of 20-40V; capillary voltage 2.0 kV; the taper hole voltage is 40 eV; the air flow rate of the taper hole is 50L/h; the scanning mode is a sensitivity mode, and the scanning range m/z is 100-1200; the scanning time is 0.2 s; the data acquisition mode is an MSE mode.

1.2.3 sample pretreatment

Crushing a sample, sieving the sample by a 60-mesh stainless steel sieve, weighing about 0.5g of the sample in a centrifuge tube, adding about 50mL of 75% ethanol for dissolution, and uniformly mixing the solution by vortex; then ultrasonic extracting for 10min, taking out, centrifuging for 5min at 8000r/min, taking supernatant, filtering with 0.22 μm organic filter membrane, and collecting filtrate to be tested.

2 results and analysis

MS^EAnd (4) collecting samples in a mode, and screening by using a self-built spectrum library by combining a UNIFI software platform. The results show that the licorice sample contains liquiritin and licoriceLess than 3 fragments of 20 active ingredients such as hypochlorous acid, etc., successfully matching, and making mass error within 5mDa, and extracting ion chromatogram and MS of each active ingredient in Glycyrrhrizae radix^EThe mass spectrum of the high collision energy channel in the mode and the matching situation of the secondary fragment are shown in fig. 2 to 21.

The above embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.