CN106918667B

CN106918667B - Pressurized micro-extraction equipment, pressurized micro-extraction method and application thereof

Info

Publication number: CN106918667B
Application number: CN201510993937.8A
Authority: CN
Inventors: 姜勇; 宋月林; 屠鹏飞; 陈金凤; 宋青青
Original assignee: Peking University; Beijing University of Chinese Medicine
Current assignee: Peking University; Beijing University of Chinese Medicine
Priority date: 2015-12-25
Filing date: 2015-12-25
Publication date: 2020-08-21
Anticipated expiration: 2035-12-25
Also published as: CN106918667A

Abstract

The invention provides a pressurized micro-extraction device, which comprises an extraction column and a pressurizing device, wherein the extraction column and the pressurizing device are connected by a pipeline; the extraction column is a liquid chromatograph pre-column sleeve and a pre-column core which is arranged in the liquid chromatograph pre-column sleeve and filled with a sample to be detected and inert fillers. The invention also provides application of the pressurized micro-extraction equipment in extraction, qualitative detection and/or quantitative analysis of plant chemical components, in particular application in component detection of safflower and cistanche. The pressurized micro-extraction equipment provided by the invention can realize rapid, micro and accurate qualitative and quantitative detection of a plant sample, is more environment-friendly and faster than a traditional extraction mode, and is particularly suitable for detection of precious traditional Chinese medicinal materials.

Description

Pressurized micro-extraction equipment, pressurized micro-extraction method and application thereof

Technical Field

The invention belongs to the field of analytical chemistry and natural product chemistry, and particularly relates to a pressurized micro-extraction device, a pressurized micro-extraction method based on the device and application of the pressurized micro-extraction device in component detection and quality control of medicinal plants.

Background

The active ingredients of medicinal plants (including traditional Chinese medicines) are all present in plant tissues, and researches on chemical structure identification, activity detection, quality control and the like can be carried out only by extracting the active ingredients from the plant tissues. Therefore, extraction technology is the basic and key technology for medicinal plant research, especially component analysis and quality control.

In the field of traditional Chinese medicine component analysis and quality control, the traditional extraction methods include a Soxhlet extraction method, an ultrasonic extraction method, a reflux extraction method, an immersion method and the like. Although these extraction methods are widely used, one extraction is as short as tens of minutes and as long as several hours; the extracted herbs are also in grams. Above-mentioned traditional extraction method is not only consuming time and wasting power, and is all great to the use amount of medicinal material and organic solvent moreover, is particularly not suitable for the analysis of famous and precious medicinal material, is unfavorable for environmental protection. In addition, the structure of many active ingredients is unstable, and long-time high-temperature extraction can cause degradation or conversion of the ingredients, thereby influencing or even misleading the detection result. Under the condition that the detection means is mature day by day, extraction is obviously the key for realizing rapid microanalysis. Obviously, the traditional extraction method can not meet the requirements of the quality control and research of the traditional Chinese medicine aiming at rapidness, trace quantity, accuracy and the like.

In recent years, new extraction techniques, such as a pressurized solvent extraction technique, a supercritical fluid chromatography technique, and the like, have been developed. The Chinese patent application with the name of 'a novel pressurized extraction device' and the publication number CN104857739A (published as 2015, 8 and 26) discloses a pressurized extraction device, which comprises a small extraction tank and a flat-top tank cover, wherein the tank cover is fixed at the top end of the small extraction tank by bolts, a sealing ring is arranged between the tank cover and a tank opening, the tank cover is provided with a pressure gauge, a safety valve, a compressed air inlet and control valve, a temperature measuring sensor, a sampling guide pipe, a sampling control valve, a liquid feeding guide pipe and a liquid feeding control valve, the outer side of the tank body is provided with an oil bath or water bath heating device, the heating device is externally provided with a heat preservation layer, and the tank bottom is provided with an electric heating pipe, a temperature control sensor and a PID. Although this apparatus can perform sampling at any time during the pressure extraction, it requires a special pressure extraction apparatus and cannot be miniaturized.

Therefore, there is a need to develop a method that consumes less medicinal materials and less time, and particularly can realize the linkage of on-line extraction and detection.

Disclosure of Invention

In view of the above problems, it is an object of the present invention to provide a pressurized micro-extraction apparatus and a pressurized micro-extraction and on-line detection method using the same. The equipment and the method can be realized based on the existing liquid chromatograph, are simple and quick, and take several minutes to finish extracting the sample only in milligram level once; not only greatly reduces the consumption of medicinal materials and solvents, is particularly suitable for the quality control and component research of rare medicinal materials, but also greatly reduces the probability of chemical component degradation and structural change, thereby ensuring that the detection result is more real.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

a pressurized micro-extraction apparatus comprising an extraction column and a pressurizing device, the extraction column and the pressurizing device being connected by a pipeline; the extraction column is a liquid chromatograph pre-column sleeve and a pre-column core which is arranged in the liquid chromatograph pre-column sleeve and filled with a sample to be detected and inert fillers.

Preferably, the inert filler is selected from one or more of diatomaceous earth, normal phase silica gel, octadecyl bonded silica gel, octane bonded silica gel, and cellulose.

Preferably, the sample to be detected is crushed, and the particle size is less than or equal to 0.5 mm.

Preferably, the pressurizing device is a high-pressure infusion pump of the liquid chromatograph.

Preferably, the pressurized micro-extraction device further comprises a temperature control system; during extraction, the extraction column is placed in the temperature control system.

More preferably, the temperature control system is a column oven of a liquid chromatograph.

The invention also aims to provide the application of the pressurized micro-extraction equipment in the extraction, qualitative detection and/or quantitative analysis of the plant chemical components, which comprises the steps of filling a plant sample to be detected and an inert filler in an extraction column, allowing an extraction solvent to enter the extraction column through the pressurized equipment, collecting effluent liquid to obtain a plant extract to be detected, and then carrying out chemical component separation, qualitative detection and/or quantitative analysis.

Preferably, the detector for qualitative detection and/or quantitative analysis is selected from the group consisting of UV detector, fluorescence detector, electrochemical detector, refractive index detector, diode array detector, evaporative light scattering detector, mass spectrometry detector, and the like, as is conventional in the art.

As a preferred embodiment, the invention provides a method for the pressurized micro-extraction of safflower, which comprises the following specific operations:

I. filling an extraction column: crushing a safflower medicinal material, sieving the crushed safflower medicinal material by a sieve of 60-80 meshes, filling 1.0mg of the crushed safflower medicinal material into a pre-column core without filler, filling the pre-column core with 4.0-6.0 mg of inert material diatomite, and then filling the pre-column core into a pre-column sleeve and placing the pre-column sleeve into a column incubator of a liquid chromatograph;

II, pressurized micro-extraction: the temperature of a column incubator is 55-65 ℃, a methanol aqueous solution with the volume percentage concentration of 25% or a formic acid aqueous solution with the volume percentage concentration of 0.01% is used as an extraction solvent, a liquid-phase high-pressure pump is started, the flow rate is 3-5 mL/min, and the extraction time is 20-120 seconds; and collecting effluent liquid.

Preferably, 25% methanol aqueous solution by volume is used as extraction solvent, the flow rate is 5mL/min, and the extraction time is 27 seconds.

The invention also aims to provide a method for pressure micro-extraction and off-line detection of hydroxysafflor yellow A of safflower, which comprises the following specific operations:

II, pressurized micro-extraction: the temperature of a column incubator is 55-65 ℃, a 25% methanol aqueous solution in volume percentage concentration is used as an extraction solvent, a liquid phase high-pressure pump is started, the flow rate is 3-5 mL/min, and the extraction time is 20-120 seconds; collecting effluent, and mixing to obtain Carthami flos extract;

and III, taking 5 mu L of the safflower extract obtained in the step II, and injecting the safflower extract into a high performance liquid chromatograph to detect the content of the hydroxysafflor yellow A.

Preferably, the chromatographic conditions in step III above are:

a chromatographic column: UPLC HSS T3, 100mm × 2.1mm,1.8 μm;

column temperature: 35 ℃;

mobile phase: gradient elution was performed with 0.01% formic acid (vol.%) water as phase a and 0.01% formic acid (vol.%) acetonitrile as phase B according to the following procedure:

0-5min, 0% → 23% B (volume percent), the balance being A;

5-8min, 23% → 100% B (volume percent), the balance being A;

flow rate: 0.4 mL/min;

a detector: and a photodiode array detector with a detection wavelength of 403 nm.

Preferably, the step III further comprises injecting 5 μ L of a control solution into the hplc, wherein the control solution is prepared by the following method:

weighing a hydroxysafflor yellow A standard substance, adding 25% methanol to dissolve to prepare a stock solution with the concentration of 6.925 mg/mL; diluting the stock solution with 25% methanol solution to obtain standard solution with hydroxysafflor yellow A concentration of 35 μ g/mL.

In addition, the invention also aims to provide a method for pressurized micro-extraction and online detection, which comprises the steps of connecting the pressurized micro-extraction equipment and a detection system, leading extraction solvent to enter the extraction column through the pressurized equipment, and leading effluent to enter the detection system directly or after treatment.

Preferably, the detection system comprises a liquid chromatography column and a detector.

Preferably, the pressurized micro-extraction device is connected with the liquid chromatography column through a multi-way valve; during extraction, the extraction column is connected with the liquid chromatographic analysis column in series, after the pressurizing device is started, an extraction solvent flows into the extraction column, and chemical components in an effluent liquid are enriched at the front end of the liquid chromatographic analysis column; after extraction is finished, the pressurizing device is directly communicated with the liquid chromatographic column, so that a mobile phase directly enters the liquid chromatographic column, elution and separation are carried out on enriched chemical components, and detection is carried out on eluent by a detector.

Preferably, a solid phase extraction column is further arranged between the pressurized micro-extraction device and the detection system, and pressurized extraction liquid enters the detection system after being purified by the solid phase extraction column.

As a preferred embodiment, the invention provides a method for the pressurized micro-extraction and online detection of safflower, which comprises the following specific operations:

connecting a pressurized micro-extraction device and a detection system: the extraction column, the liquid phase high-pressure pump and the analytical chromatographic column are connected to the six-way valve, and the analytical chromatographic column is connected with the detector;

pressure micro-extraction: switching a six-way valve to sequentially connect the liquid phase high-pressure pump, the extraction column and the analytical chromatographic column in series; the temperature of a column incubator is 50-60 ℃, a formic acid aqueous solution with the volume percentage concentration of 0.01% is used as an extraction solvent, a liquid phase high-pressure pump is started, the flow rate is 1mL/min, and the extraction time is 3 minutes;

and IV, online detection: and immediately switching the six-way valve after extraction to directly communicate the liquid phase high-pressure pump with the analytical chromatographic column for elution and detection.

Preferably, in the step I, the filling amount of the diatomite is 6.0 mg.

Preferably, in the step II, the temperature of the column oven is 55 ℃.

Preferably, in the step IV, the chromatographic conditions are:

a chromatographic column: waters CSH column, (150 mm. times.4.6 mm,3.5 μm),

column temperature: at a temperature of 35 c,

mobile phase: taking a formic acid aqueous solution with the volume percentage concentration of 0.01 percent as a phase A, and taking a formic acid acetonitrile solution with the volume percentage concentration of 0.01 percent as a phase B; gradient elution was performed according to the following procedure:

0-3min, 0% → 12% B (volume percent), the balance being A,

3-20min, 12% → 30% B (volume percent), the balance being A,

23-26min, 30% → 100% B (volume percent), the balance being A;

flow rate: 1 mL/min.

Preferably, in the step IV, the detector is a mass spectrometer, and the mass spectrometric detection conditions are as follows:

an ion source: an electric atomizing ion source is arranged on the device,

detection mode: the detection is carried out in a negative ion mode,

ion ejection voltage: -4500V,

temperature: at a temperature of 650 c,

source gas 1 and gas 2: all nitrogen, all at 65psi,

gas curtain gas: nitrogen, at a pressure of 40.0psi,

the scanning mode is as follows: and (4) detecting multiple reactions.

As another preferred embodiment, the present invention provides a method for pressure micro-extraction and on-line detection of cistanche, comprising the following steps:

I. filling an extraction column: crushing cistanche medicinal materials, sieving with a 60-80-mesh sieve, filling 1.0mg of the cistanche medicinal materials in a pre-column core with fillers removed, filling 4.0-6.0 mg of inert material normal phase silica gel, then filling the pre-column core in a pre-column sleeve, and placing the pre-column sleeve in a column incubator of a liquid chromatograph;

connecting a pressurized micro-extraction device and a detection system: the extraction column, the liquid phase high-pressure pump and C₁₈The chromatographic column and the solid phase extraction small column are connected to a six-way valve, C₁₈The chromatographic column, the detector and the solid phase extraction small column are connected to the other six-way valve; said C is₁₈The chromatographic column is connected with the detector;

pressure micro-extraction: adjusting the two six-way valves to enable the liquid phase high-pressure pump and the extraction column solid phase extraction small column to be sequentially connected in series; the temperature of a column incubator is 55-65 ℃, a formic acid aqueous solution with the volume concentration of 0.1% is used as an extraction solvent, a liquid-phase high-pressure pump is started, the flow rate is 2-4 mL/min, and the extraction time is 60-180 seconds;

and IV, online detection: and after extraction is finished, adjusting the two six-way valves to enable the liquid phase high-pressure pump, the solid phase extraction small column and the analysis chromatographic column to be sequentially connected in series for elution and detection.

Preferably, in the step I, the filling amount of the normal phase silica gel is 6.0 mg.

Preferably, in the step III, the temperature of the column oven is 60 ℃.

Preferably, in the step III, the flow rate of the formic acid aqueous solution with the volume concentration of 0.1% is 2mL/min, and the extraction time is 180 seconds.

Preferably, in the step IV, the chromatographic conditions are:

column temperature: at the room temperature, the reaction mixture is mixed,

mobile phase: taking a formic acid aqueous solution with the volume percentage concentration of 0.1 percent as a phase A and taking an acetonitrile solution as a phase B; gradient elution was performed according to the following procedure:

0-32min, 10% → 30% B, the balance being A;

flow rate: the concentration of the active carbon is 0.25mL/min,

a detector: and a photodiode array detector for detecting the wavelength of 278 nm.

Preferably, the step IV further comprises injecting 10 μ L of a control solution into the high performance liquid chromatograph; the control solution was prepared by the following method:

respectively weighing cistanoside E, echinacoside, acteoside, isoacteoside, cistanoside C, 2' -acetylneroside, isocistanoside C, and angionoside B standard substances, and dissolving with DMSO to obtain stock solutions with concentration of 4 mg/mL; and mixing the standard substance stock solutions, and dissolving and diluting the mixture into a mixed standard stock solution with the concentration of each standard substance of 400 mu g/mL by using 50% methanol to obtain the finished product.

The pressure micro-extraction equipment and the pressure micro-extraction and online detection method can be widely applied to various medicinal plants (including traditional Chinese medicinal materials) for micro-extraction and component detection, and are not limited to the safflower and the cistanche. Furthermore, depending on the physicochemical properties of the components contained in the medicinal plants and the purpose of extraction and analysis, suitable extraction solvents, elution solvents, analytical columns and detectors can be flexibly used.

In the prior art, when the high performance liquid chromatography is used for component detection and analysis of medicinal plants (including traditional Chinese medicinal materials), a sample solution to be tested usually adopts a heating reflux extraction method, an ultrasonic oscillation extraction method or a cold soaking method, and in any method, a plurality of grams of samples to be tested and a plurality of tens to hundreds of milliliters of extraction solvent are consumed, so that dozens of minutes to several hours are consumed, while only dozens of microliters are needed for practical analysis, and the residual sample solution can only be discarded. Therefore, the waste of the sample to be detected and the organic solvent is caused, and the environment is easily polluted. In addition, for valuable traditional Chinese medicinal materials such as cordyceps, safflower and the like, the test cost is high due to the fact that the amount of the medicinal materials which need to be consumed for analyzing one sample is relatively large.

The pressurized micro-extraction equipment and the pressurized micro-extraction and on-line detection method based on the pressurized micro-extraction equipment are based on a widely applied high performance liquid chromatograph, under the conditions of high pressure and high temperature, only milligram-grade medicinal materials and a few milliliters of extraction solvent are needed for each extraction, and the extraction time is dozens of seconds but not more than a few minutes. And the extraction example of the safflower and the cistanche shows that the extraction efficiency is higher than that of the ultrasonic extraction in the prior art, the extracted chemical components have more quantity and the content of each component is higher. Therefore, the method of the invention realizes the rapid and trace detection of the medicinal plants containing the Chinese medicinal materials. Compared with the prior art, the consumed organic solvent and the sample amount are greatly reduced, and the detection cost is correspondingly reduced.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a liquid chromatogram of the safflower extract of example 3, wherein the chromatographic peak No. 1 is an absorption peak of hydroxysafflor yellow A.

FIG. 2 is a schematic diagram showing the structure of the apparatus for pressure micro-extraction and on-line detection in example 5, in which FIG. 2A shows the connection of the apparatus during extraction and enrichment, and FIG. 2B shows the connection of the apparatus during analysis. In the figure, 1 is a high-pressure infusion pump, 2 is an extraction column, 3 is an analytical column, 4 is a detector, and 5 is a six-way valve.

FIG. 3 is a chromatogram of the extract obtained by the different extraction methods of safflower in example 5; wherein A is chromatogram obtained by online detection of extract obtained by pressure micro-extraction of safflower, and B is chromatogram of extract obtained by warm-soaking extraction of safflower.

FIG. 4 is a comparison graph of the peak areas of the chromatogram peaks of the extract obtained by pressure micro-extraction of safflower and the extract obtained by warm-dipping extraction of safflower of example 5, in which FIG. 4A shows the comparison result of the peak areas of the chromatogram of the compound C1-C21, and FIG. 4B shows the comparison result of the peak areas of the chromatogram of the compound C22-C43. In the figure, ■ represents pressure micro-extraction, and □ represents warm immersion extraction.

FIG. 5 is a schematic diagram showing the structure of the apparatus for pressure micro-extraction and on-line detection of example 6, wherein FIG. 5A shows the connection of the apparatus during extraction and enrichment, and FIG. 5B shows the connection of the apparatus during analysis. In the figure, 1 is a high pressure liquid pump, 2 is an extraction column, 3 is a solid phase extraction column (SPE column), 4 is an analytical column, 5 is a detector, and 6 is a six-way valve.

FIG. 6 shows the chromatograms of the control solution, cistanche deserticola and cistanche tubulosa obtained by the pressure micro-extraction and on-line detection method in example 6, wherein A is the chromatogram of the control solution, B is the chromatogram of cistanche deserticola and C is the chromatogram of cistanche tubulosa. In the figure, the No. 1 peak is the absorption peak of cistanoside E, the No. 2 peak is the absorption peak of echinacoside, the No. 3 peak is the absorption peak of verbascoside, the No. 4 peak is the absorption peak of isoacteoside, the No. 5 peak is the absorption peak of cistanoside C, the No. 6 peak is the absorption peak of isocistanoside C, the No. 7 peak is the absorption peak of 2' -acetylnereid, and the No. 8 peak is the absorption peak of tubular anthocyanin B.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Wherein,laboratory apparatus：

Chromatograph: 1) shimadzu high performance liquid chromatograph (pump: LC-20ADx2, autosampler SIL-20A, column oven CTO-20A), ultra pure water purification system, Millipore;

2) a Waters ACQUITY UPLC H-Class system is equipped with a Quaternary Solvent Manager (QSM), a sample manager (SM-FTN), and a photodiode array detector (PDA), Watt technologies, Inc., USA.

Mass spectrometry: the ABCiex QTrap4500 triple quadrupole linear ion trap composite tandem mass spectrometer (equipped with an ion spray ion source, Analysis 1.6.2 data processing system, ABCiex corporation, USA).

A chromatographic column: 1) UPLC HSS T3 column (100 mm. times.2.1 mm,1.8 μm), 2) CSH column (150 mm. times.4.6 mm,3.5 μm), 3) ACE UltraCore super C18 column (150 mm. times.3.0 mm,2.5 μm).

Phenomenex Security Guard with pre-column sleeve^TMCylindrical core Phenomenex cartridge (3.0mm × 4mm i.d.) and RP-C removed₁₈And (4) filling.

Experimental medicinal material：

The Carthami flos is dried flower of Carthamus tinctorius L. The Cistanchis herba is dry succulent stem with scaly leaf of Cistanchis herba Deserticola Y.C.Ma or Cistanchis herba Bulosa (Schrenk) light of Orobanchaceae.

Experimental reagents and drugs:

hydroxysafflor yellow A (HSYA, batch: MUST-14101810, purity HPLC: 98%) was purchased from Khmannist Biotech, Inc.;

cistanoside E (cistanoside E), Echinacoside (Echinacoside), verbascoside (Acteoside), Isoacteoside (Isoacteoside), cistanoside C (cistanoside C), 2 '-acetylpolioside (2' -Acetolpenioside), isocistanoside C (isocistanoside C) and tubular glycoside B (tubular glycoside B) are extracted and separated from cistanche deserticola by the modern research center of Beijing university traditional Chinese medicine, and the purity is more than 95%.

Liquid pure acetonitrile, methanol and formic acid, purchased from Saimer Feishale (China) science and technology Limited; diatomaceous earth, chemically pure, available from west longe chemical gmbh; normal phase silica gel, 100 meshes and 200 meshes, was purchased from Qingdao ocean chemical Co.

Example 1Research and condition optimization of safflower pressure micro-extraction

1. Basic conditions

Index components: hydroxysafflorflavin A (HSYA)

Chromatograph: waters ACQUITY UPLC H-Class system

A chromatographic column: UPLC HSS T3 chromatographic column (100 mm. times.2.1 mm,1.8 μm)

And (3) extraction column: loading 1.0mg of Carthami flos powder (sieved with 60 mesh sieve) into pre-column core, filling with inert material diatomite to obtain extraction column, loading into pre-column sleeve, and placing in column incubator.

Preparation of a reference solution: a proper amount of HSYA standard substance is weighed and dissolved by adding 25 percent by volume of methanol aqueous solution (hereinafter referred to as 25 percent methanol) to prepare 6.925mg/mL stock solution. And mixing the stock solutions of the standard products in proper amount, and diluting the mixture with a 25% methanol solution to obtain a standard solution with the compound concentration of 35 mu g/mL.

The detection mode is as follows: HSYA content determination by UPLC-PDA system

Chromatographic conditions are as follows: a UPLC HSS T3 column (100 mm. times.2.1 mm,1.8 μm); column temperature: 35 ℃; mobile phase: 0.01% formic acid water (a) -0.01% formic acid acetonitrile (B), flow rate: 0.4mL/min, gradient elution: 0-5min, 0-23% B, 5-8min, 23-100% B; the detection wavelength is 403 nm.

2. Safflower pressurization micro-extraction and extraction condition optimization

Connecting the extraction column with a high pressure pump of a chromatograph, delivering the extraction solvent to the extraction column via the high pressure pump, collecting effluent, injecting a proper amount (5 μ L) into a UPLC-PDA system for separation and detection, recording the chromatographic peak area of HSYA, and calculating the content.

2.1 extraction solvent

For comparison with ultrasonic extraction, pressure micro-extraction also uses 25% methanol in water as extraction solvent.

2.2 investigation of extraction flow Rate

The extraction flow rate affects the extraction speed and the pressure during extraction, and is an important factor affecting the extraction efficiency. Different extraction flow rates were therefore investigated. The extraction efficiencies of 3mL/min (pressure: 2400psi) extraction for 3min and 5mL/min (pressure: 3600psi) extraction for 1.8min were examined using 25% methanol at 60 deg.C (column oven temperature) as the basic condition, and the results are shown in Table 1.

Table 1 different extraction flow rate results (n ═ 3)

The extraction flow rate of 5mL/min is preferred because the high HSYA content in the extract liquid indicates that the extraction efficiency of HSYA is better at this flow rate.

2.3 examination of extraction time

The extraction time is too short, and the sample cannot be completely extracted, so that the extraction efficiency is low; the extraction time is too long, the solvent consumption is large, and the sample is easy to degrade at a high temperature, so that the extraction efficiency is low. Different extraction times were investigated at a flow rate of 5mL/min and a temperature of 60 ℃: extraction efficiencies at 27 seconds(s), 54 seconds(s) and 108 seconds(s) and the results are shown in Table 2.

Table 2 extraction time study results (n ═ 3)

^a: the extraction efficiency was calculated by the following formula

The extraction efficiency is the measured HSYA amount/the weighed medicinal material amount multiplied by 100% (W/W%)

The results showed that the extraction efficiency was almost unchanged for the extraction time of 27 to 54 seconds, but the extraction efficiency decreased rather as the time was further extended. Therefore, the purpose of complete extraction can be achieved only by extracting for 27-54 seconds under pressure; and the shorter the extraction time, the less reagents are needed; therefore, 27 seconds is preferable.

2.4 examination of extraction temperature

The temperature is too low, and the chemical component dissolution efficiency is low; the high temperature of HSYA is easy to degrade, which also results in low extraction efficiency. Under the basic conditions of extraction flow rate of 5mL/min and extraction time of 27s, five different extraction temperatures of 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C and 70 deg.C were examined, and the results are shown in Table 3.

Table 3 extraction temperature investigation results (n ═ 3)

The data in Table 3 show that the concentration of HSYA in the extract was higher when safflower was extracted at 55-65 deg.C, with the highest concentration at 55 deg.C. Therefore, the extraction temperature is preferably 55 to 65 ℃, and most preferably 55 ℃.

2.5 inspection of diatomaceous Earth Loading

The inert material serves primarily to disperse sample particles and to prevent excessively fine sample particles from clogging the outlet of the extraction tube. Therefore, extraction results loaded with different amounts of diatomaceous earth were examined. The results are shown in Table 4.

Table 4 diatomaceous earth loading investigation results (n ═ 3)

The results in Table 4 show that diatomaceous earth, either 4mg or 6mg, had little effect on extraction efficiency. However, the more the loading, the compacter the sample, the greater the degree of dispersion, and the better the stability between batches, so that the loading of diatomaceous earth is preferably 6.0 mg.

2.6 examination of the Effect of extraction matrix on extraction efficiency

Preparing a blank extraction column: the pre-column core was filled with only 6.0mg of diatomaceous earth without filling safflower.

A control stock solution (2. mu.L; equivalent to 13.85. mu.g of HSYA) at a concentration of 6.925mg/mL was injected through the blank extraction column and the HSYA peak area was detected and reported as "sample peak area". Meanwhile, 2 mu L of a reference substance stock solution with the concentration of 6.925mg/mL is directly injected into a UPLC HSS T3 chromatographic column, and the peak area of HSYA is detected and is marked as the peak area of the reference substance. The results are shown in Table 5.

Table 5 examination of influence of extraction substrate on extraction efficiency (n ═ 6)

^a: the matrix effect is calculated by the following formula:

matrix effect-sample peak area/control peak area × 100%

The results in table 5 show that the average matrix effect is 96.50% and the RSD value is 4.50%; indicating that the diatomite does not influence the efficiency of the on-line pressurized solvent micro-extraction.

2.7 safflower pressure micro-extraction optimization conditions

Through the research, the preferable process conditions of the safflower pressurized micro-extraction are established:

crushing a safflower medicinal material, sieving the crushed safflower medicinal material by a sieve of 60-80 meshes, filling 1.0mg of the crushed safflower medicinal material into a pre-column core without filler, filling the pre-column core with 4.0-6.0 mg of inert material diatomite, and then filling the pre-column core into a pre-column sleeve and placing the pre-column sleeve into a column incubator of a liquid chromatograph; the temperature of the column incubator is 55-65 ℃, 25% volume concentration methanol aqueous solution is used as an extraction solvent, the flow rate is 3-5 mL/min, and the extraction time is 20-60 seconds; and collecting effluent liquid.

The most preferable technological conditions for the safflower pressure micro-extraction are as follows:

pulverizing Carthami flos, sieving with 60 mesh sieve, placing 1.0mg into a pre-column core without filler, filling with 6.0mg of inert material diatomaceous earth, placing into a pre-column sleeve, and placing into a liquid chromatograph column incubator; the temperature of the column incubator is 55 ℃, 25% methanol aqueous solution by volume concentration is used as an extraction solvent, the flow rate is 5mL/min, and the extraction time is 27 seconds; and collecting effluent liquid.

Example 2Study of safflower pressure micro-extraction methodology

The methodology was examined under the most preferred safflower pressure micro-extraction conditions established in example 1, and under the chromatographic conditions described in example 1.

1. Linear range investigation

The reference standard solutions prepared in example 1 were injected into the sample at 0.1, 0.2, 0.5, 1,2.5 μ L, the corresponding chromatographic peak areas are recorded. The sample size X (. mu.g) was linearly regressed by the peak area Y of the control. The regression equation is: Y5196429.6X-3599.9 (R)²1) and the results show that the linear relationship between the sample introduction amount of 3.5 ng and 175ng is good.

2. Precision survey

Taking 1.0mg of safflower medicinal powder, precisely weighing, filling into a pre-column core, filling with 6.0mg of diatomite, and filling into a pre-column sleeve to obtain the extraction column. Placing the extraction column in a column incubator, extracting according to the most preferable pressure micro-extraction condition of the safflower medicinal material established in example 1, carrying out sample injection on the extract for 5 mu L, carrying out continuous sample injection for 6 times, and recording the chromatographic peak area. The results are shown in Table 6.

Table 6 results of precision examination (n ═ 6)

The data in Table 6 show that the instrument precision is good, the RSD value is 0.2%, and the requirement of the analysis method is met.

3. Repeatability survey

Taking 1.0mg of safflower medicinal powder, precisely weighing, filling into a pre-column core, filling with 6.0mg of diatomite, and filling into a pre-column sleeve to obtain the extraction column. 6 samples were prepared in parallel. Placing the extraction column in a column incubator, extracting under the most preferable pressure micro-extraction conditions of Carthami flos material established in example 1, injecting 5 μ L, and calculating HSYA content. The results are shown in Table 7.

Table 7 repeatability test results (n ═ 6)

The results in table 7 show that RSD < 5%, indicating good reproducibility, meets the requirements of the analytical method.

4. Investigation of sample recovery

Taking 1.0mg of safflower medicinal powder, precisely weighing, filling into a pre-column core, filling with 6.0mg of diatomite, filling into a pre-column sleeve, and paralleling 6 samples. Placing the extraction column in a column incubator, injecting 2 μ L (equivalent to 13.85 μ g HSYA) of 6.925mg/mL control stock solution into the extraction column, extracting under the most preferable pressure micro-extraction conditions of Carthami flos material established in example 1, collecting extractive solution, injecting 5 μ L, measuring and recording peak area, and calculating sample recovery rate of HSYA, with the results shown in Table 8.

Table 8 results of sample recovery rate test (n ═ 6)

The data in Table 8 show that the sample recovery rate of HSYA is 95.71%, the RSD value is 3.64%, and the requirement of the analysis method is met.

The results of this example show that the safflower pressure micro-extraction method established by the invention has good stability and reproducibility.

Example 3A method for micro-extracting Carthami flos under pressure

Pulverizing Carthami flos, sieving with 60 mesh sieve, placing 1.0mg into a pre-column core without filler, filling with 6.0mg of inert material diatomaceous earth, placing into a pre-column sleeve, and placing into a liquid chromatograph column incubator; the temperature of the column incubator is 55 ℃, 25% methanol aqueous solution by volume concentration is used as an extraction solvent, the flow rate is 5mL/min, and the extraction time is 27 seconds; collecting the effluent to obtain the safflower extract.

The liquid chromatogram of the extract was obtained using the detection method and chromatographic conditions of example 1, as shown in FIG. 1.

Example 4Comparison of the pressure micro-extraction method of the present invention with the conventional extraction method

1. Preparation of test solution

A. Online pressurized solvent micro-extraction of safflower medicinal material: loading 1.0mg of Carthami flos powder into a pre-column core, filling with 6.0mg of inert material diatomite, loading into a pre-column sleeve, placing the extraction column in a column incubator, extracting with 25% methanol at 55 deg.C and 5mL/min (pressure: 3600psi), and extracting under pressure for 27 s. Collecting extractive solution, injecting 5 μ L into UPLC for analysis, recording corresponding chromatographic peak area and calculating extraction efficiency.

B. Ultrasonic extraction of safflower medicinal material: extracting according to the method for measuring the content of HSYA in the safflower medicinal material in Chinese pharmacopoeia (2010 version), namely taking about 0.1g of safflower medicinal material powder (passing through a 60-mesh sieve), precisely weighing, then placing in a conical flask with a plug, precisely adding 12.5mL of 25% methanol, weighing, ultrasonically treating (power 300W and frequency 5kHz) for 40 minutes, cooling, weighing again, complementing the lost weight with 25% methanol, shaking up, filtering, taking 0.5 mu L of subsequent filtrate, injecting into UPLC for analysis, recording the corresponding chromatographic peak area and calculating the extraction efficiency.

2. Chromatographic analysis conditions:

the same chromatographic conditions as in example 1.

3. And (3) measuring results: see table 9, samples and solvent amounts, extraction times, etc. for both extraction methods are also listed in table 9 for comparison.

TABLE 9 comparison of on-line pressure solvent microextraction with ultrasonic extraction (n ═ 3)

The data in table 9 show that the pressure micro-extraction method of the present invention has improved extraction rate compared to the ultrasonic extraction method, and can significantly shorten the extraction time and reduce the amount of sample and solvent used, which has significant advantages.

Example 5Method for pressure micro-extraction and online detection of safflower

1. Pressurized micro-extraction and on-line detection equipment

The pressurized micro-extraction and on-line detection device was set up as shown in FIG. 2, where 1 is a high pressure infusion pump, 2 is an extraction column, 3 is an analytical column, 4 is a detector, and 5 is a six-way valve.

2. Detection conditions are as follows:

2.1 chromatographic conditions

And (3) analyzing the column: waters CSH column (150 mm. times.4.6 mm,3.5 μm, cat # 186005270);

column temperature: room temperature;

mobile phase: 0.01% formic acid water (a) -0.01% formic acid acetonitrile (B), room temperature, flow rate: 1 mL/min; the gradient elution procedure is shown in Table 10.

TABLE 10 procedure for on-line pressurized solvent microextraction for trace components extraction and elution of mobile phase from safflower

2.2 detector:

an ABSciex QTrap4500 type triple quadrupole linear ion trap composite tandem mass spectrometer detects trace components in safflower by adopting a multi-reaction detection mode (MRM) of Qtrap4500, and the corresponding mass spectrum conditions are as follows: the ion source is an electric atomization ion source; detecting in a negative ion mode, wherein the ion jet voltage is-4500V; the temperature is 650 ℃; source gas 1 and gas 2: the nitrogen pressure was 65 psi; gas curtain gas: nitrogen pressure was 40.0 psi; the scanning mode is a multiple reaction detection.

3. Extraction and detection

1.0mg of safflower medicinal material powder (sieved by a 60-mesh sieve) is filled in a pre-column core and is filled with 6.0mg of inert material diatomite, the filled pre-column core is filled in a protective column sleeve and is placed in a column incubator, a six-way valve is adjusted to the position shown in figure 2A, and an extraction device and an analytical column are connected in series. The extraction solvent was 0.01% formic acid water, the flow rate was 1mL/min, and the temperature during extraction in the column oven was 55 ℃. The sample is subjected to high temperature, pressure extraction and enrichment on the analytical column. After extraction is complete, the six-way valve is adjusted to the position shown in fig. 2B, the mobile phase is directed through the analytical column, the components retained at the front end of the analytical column are eluted and separated, and injected into the mass spectrum for analysis. The chromatogram is shown in FIG. 3, and the corresponding peak areas are recorded, and the results are shown in FIGS. 4A and 4B.

4. Warm-soaking extraction of safflower medicinal material

Taking about 0.5g of safflower medicinal material powder (sieving by a 60-mesh sieve), precisely weighing, then placing in a conical flask with a plug, precisely adding 5.0mL of 0.01% formic acid aqueous solution, weighing, extracting for 60 minutes at the water bath temperature of 55 ℃, cooling, weighing again, complementing the lost weight with 0.01% formic acid aqueous solution, shaking up, filtering, taking 10 mu L of subsequent filtrate (the sample amount is 1.0mg when being converted to the sample amount, and is the same as the sample amount of the pressurized micro-extraction), injecting into a chromatograph, and eluting and detecting in the same manner as the pressurized micro-extraction and on-line detection in the embodiment. The chromatogram is shown in FIG. 3, and the peak areas are recorded, and the results are shown in FIGS. 4A and 4B.

5. And (4) conclusion:

by comparing the peak areas of the corresponding absorption peaks of the extracts obtained by the pressure micro-extraction and on-line detection methods and warm-immersion extraction methods of the present invention, it can be seen that: under the same extraction solvent (0.01% formic acid aqueous solution), extraction temperature (55 ℃) and detection conditions, the absorption peak areas of all components in the extract obtained by the pressure micro-extraction method of the invention are all larger than the corresponding components in the extract obtained by the warm immersion method. The pressurized micro-extraction method disclosed by the invention is proved to improve the extraction efficiency, save the sample and the solvent and be more suitable for extracting and detecting trace components in the safflower.

Example 6Cistanche pressurization micro-extraction and online detection method

1. Pressurized micro-extraction and on-line detection equipment

The pressurized micro-extraction and on-line detection equipment was set up as shown in FIG. 5, where 1 is a high pressure infusion pump, 2 is an extraction column, 3 is a solid phase extraction cartridge (SPE cartridge), 4 is an analytical column, 5 is a detector, and 6 is a six-way valve.

The working process of the pressurized micro-extraction and the online detection is as follows: during extraction, the positions of the two six-way valves 6 are shown in fig. 5A, the extraction column 2 (at this time, the extraction column is placed in the column incubator) is connected in series with the SPE column 3, and a sample is extracted at high temperature under pressure and is enriched on the SPE column 3. After extraction is finished, the positions of the two six-way valves are switched to the positions shown in fig. 5B, the SPE column 3 is connected with the analysis column 4 in series, components remained on the SPE column 3 are backflushed to the analysis column 4 for separation, and are injected into the detector 5 for detection, a chromatogram is recorded, and corresponding calculation is carried out; the extraction column 2 can now be sample exchanged.

2. Test sample preparation

2.1 extraction column

Weighing herba cistanches Deserticolae (sieved by a No. four sieve) 1mg, filling into a pre-column core, filling with normal phase silica gel 6mg, and filling the pre-column core into a pre-column sleeve for later use. The extraction column is placed in a column incubator during extraction.

2.2 preparation of control solutions

Weighing appropriate amount of cistanoside E, echinacoside, acteoside, isoacteoside, cistanoside C, 2' -acetylneroside, isocistanoside C, and tubuloside B, and dissolving with DMSO to obtain stock solution with concentration of 4 mg/mL. And mixing the standard substance stock solutions in proper amount, and dissolving and diluting the mixture by using 50% methanol to obtain a mixed standard stock solution with the concentration of each standard substance of 400 mu g/mL for later use.

3. Extraction and detection conditions:

3.1 extraction conditions

Extracting solvent: 0.1% aqueous formic acid solution

Extraction temperature: 60 deg.C

Flow rate: 2mL/min

3.2 detection conditions

And (3) analyzing the column: ACE UltraCore super C18 chromatographic column (specification: 150 mm. times.3.0 mm,2.5 μm) (cat # CORE-25A-1002U);

detection wavelength: 278 nm;

column temperature: room temperature;

mobile phase: 0.1% formic acid (a) -acetonitrile (B), room temperature; the gradient elution procedure is shown in Table 11.

TABLE 11 Desertliving cistanche pressurizing micro-extraction-solid phase extraction-on-line detection mobile phase program

4. Methodology investigation

4.1 detection and quantitation limits

Filling the pre-column core with only the cleaned normal phase silica gel, and placing in a pre-column sleeve to obtain a blank extraction column. And (3) continuously diluting the mixed standard stock solution with 50% methanol, loading the diluted mixed standard stock solution into a blank extraction column, measuring according to the working processes of pressurized micro-extraction and online detection, and determining the detection line and the quantitative limit of each compound by respectively taking the signal-to-noise ratio of 3 and 10 as standards.

The results are shown in Table 12.

4.2 Standard Curve

Filling the pre-column core with only the cleaned normal phase silica gel, and placing in a pre-column sleeve to obtain a blank extraction column. An appropriate amount of the mixed standard stock solution was dissolved and diluted with 50% methanol to prepare a standard substance having a concentration of 200. mu.g/mL, 100. mu.g/mL, 80. mu.g/mL, 50. mu.g/mL, 25. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, 1. mu.g/mL, respectively. And (3) measuring according to the working process of pressurized micro-extraction and online detection, and drawing a standard curve by taking the peak area of the target compound as a vertical coordinate and the concentration as a horizontal coordinate to obtain a linear regression equation.

As a result: see table 12.

TABLE 12 measurement results of Linear equation, detection Limit and quantitation Limit for Standard

The data in Table 12 show that the compounds of the respective standards have good linear relationship between the detection limit and the quantitative limit concentration, and the method of this example has high sensitivity.

4.3 precision

A. Precision in the day: filling the pre-column core with normal phase silica gel, and placing in a pre-column sleeve to obtain a blank extraction column. Precisely sucking three concentrations of mixed standard solutions of high (80 mug/mL), medium (25 mug/mL) and low (5 mug/mL) and continuously injecting samples for six times respectively. And (4) measuring according to the working processes of pressurized micro-extraction and online detection, recording the peak area of each compound, and calculating the corresponding RSD value.

B. Precision in the daytime: filling the column core with normal phase silica gel, and placing in a pre-column sleeve. Precisely sucking three concentrations of mixed standard solutions of high concentration (80 mug/mL), medium concentration (25 mug/mL) and low concentration (5 mug/mL), injecting samples for three days respectively, and injecting samples for three times every day. And (4) measuring according to the working processes of pressurized micro-extraction and online detection, recording the peak area of each compound, and calculating the corresponding RSD value.

As a result: see table 13.

Table 13 results of measurement of intra-day precision (n ═ 6) and inter-day precision (n ═ 9)

The data in Table 13 show that the process is accurate.

4.4 repeatability

Weighing about 1mg of cistanche deserticola sample (passing through a No. four sieve) and filling the sample into a pre-column core, measuring according to the working process of pressurized micro-extraction and online detection, injecting a sample, recording the retention time and the peak area, and calculating the retention time and the RSD value of each compound in the sample.

As a result: see table 14.

4.5 sample application recovery test

Weighing about 0.5mg of cistanche deserticola sample (passing through a No. four sieve) and filling the sample into a pre-column core, taking a mixed standard solution with three concentrations of high (80 mu g/mL), medium (25 mu g/mL) and low (5 mu g/mL), and calculating the recovery rate of each compound according to the measured amount and the added amount.

As a result: see table 14.

Table 148 recovery (n ═ 3) and reproducibility (n ═ 5) for the compounds

The data in Table 14 show that the method has good repeatability and good sample recovery results. The method is proved to be good in accuracy.

5. Determination of content

Respectively taking desert cistanche and cistanche tubulosa of different production areas and batches, respectively loading the desert cistanche and the cistanche tubulosa into a pre-column core, filling 6mg of normal phase silica gel into a pre-column sleeve to obtain an extraction column, placing the extraction column into a column incubator, and establishing a pressurized micro-extraction and online detection device as shown in figure 6. The extraction solvent is 0.1% formic acid water solution, the extraction temperature is 60 ℃, and the extraction flow rate is 2 mL/min. The detection conditions were as described under item 3.2.

During extraction, the positions of the two six-way valves 6 are shown in fig. 5A, the extraction column 2 and the SPE column 3 are connected in series, and a sample is extracted at high temperature under pressure and is enriched on the SPE column 3. After extraction is finished, the positions of the two six-way valves are switched to the positions shown in fig. 5B, the SPE column 3 is connected with the analysis column 4 in series, the components remained on the SPE column 3 are backflushed to the analysis column 4 for separation, and are injected into the detector 5 for detection, the chromatogram is recorded, and corresponding calculation is carried out.

Chromatograms of the control solution, cistanche deserticola and cistanche tubulosa are shown in FIG. 6 (A-C). The results of the content measurement are shown in Table 15.

TABLE 15 measurement of cistanche contents (μ g/mg, n ═ 1)

In the table, compounds No. 1-8 are cistanoside E (1), echinacoside (2), acteoside (3), isoacteoside (4), cistanoside C (5), isocistanoside C (6), 2' -acetylnerchinoside (7) and tubuloside B (8), respectively.

6. This example method compares with ultrasonic extraction method

6.1 ultrasonic extraction of cistanche deserticola

Taking about 0.5g of desert cistanche (screened by a No. four sieve), precisely weighing, placing in a conical flask with a plug, precisely adding 50mL of 50% methanol, weighing, carrying out ultrasonic treatment (power 300W and frequency 5kHz) for 40 minutes, cooling, weighing again, complementing the weight loss by 50% methanol, shaking up, filtering, taking 10 mu L of subsequent filtrate, injecting into UPLC for analysis, recording the corresponding chromatographic peak area and calculating the extraction efficiency. The results are shown in Table 16.

TABLE 16 comparison of on-line pressure solvent microextraction with ultrasonic extraction (n ═ 5)

The results of comparison in the aspects of extraction solvent, solvent dosage, extraction time, medicinal material quantity, extraction efficiency and the like show that the cistanche pressurized micro-extraction method can achieve higher extraction rate, can achieve the extraction effect of organic solvent (50% methanol) on each compound under the condition of using aqueous solution, can obviously shorten the extraction time and reduce the dosage of samples and solvents, and has remarkable advantages in the aspects of economic benefit and environmental protection.

The pressurized micro-extraction and online detection method provided by the embodiment can be used for carrying out rapid, micro and accurate qualitative and quantitative detection on cistanche. In addition, the online SPE cartridge is used for enriching the sample, and the higher extraction efficiency of the compound with lower polarity is still achieved under the condition that the organic solvent is not used for extraction, so that the method is more environment-friendly than the traditional extraction mode.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Claims

1. A method for pressure micro-extraction of safflower comprises the following steps:

2. The method of claim 1, wherein 25% by volume of aqueous methanol is used as the extraction solvent, the flow rate is 5mL/min, and the extraction time is 27 seconds.

3. A method for carrying out pressurized micro-extraction and off-line detection on hydroxysafflor yellow A of safflower comprises the following specific operations:

4. The process according to claim 3, wherein the chromatographic conditions in step III are:

a chromatographic column: UPLC HSS T3, 100mm × 2.1mm,1.8 μm;

column temperature: 35 ℃;

mobile phase: using formic acid water with the volume percentage concentration of 0.01% as an A phase and formic acid acetonitrile with the volume percentage concentration of 0.01% as a B phase, and performing gradient elution according to the following procedures:

0-5min, 0% → 23% by volume of B, and the balance A;

5-8min, 23% by volume → 100% B, and the balance A;

flow rate: 0.4 mL/min;

5. The method of claim 3, wherein step III further comprises injecting 5 μ L of a control solution into the high performance liquid chromatograph, wherein the control solution is prepared by:

6. A method for the pressurized micro-extraction and the online detection of safflower comprises the following specific operations:

7. The method according to claim 6, wherein the loading amount of the diatomite in the step I is 6.0 mg.

8. The method of claim 6, wherein in step III, the column oven temperature is 55 ℃.

9. The method according to claim 6, wherein in step IV, the chromatographic conditions are:

a chromatographic column: a Waters CSH chromatographic column with the specification of 150mm multiplied by 4.6mm and the fixed phase grain diameter of 3.5 μm,

column temperature: at a temperature of 35 c,

mobile phase: taking a formic acid aqueous solution with the volume percentage concentration of 0.01 percent as a phase A, and taking a formic acid acetonitrile solution with the volume percentage concentration of 0.01 percent as a phase B; wherein, the procedures of extracting and eluting the trace components in the safflower comprise the following steps:

0-3min, 0% of B by volume percentage and the balance of A,

3-6min, 0% → 12% by volume of B, and the balance A,

6-23min, 12% → 30% by volume of B, and the balance A,

23-27min, 30% → 100% B by volume, the balance A,

27-29min, volume percent of 100% B,