CN110568114B - Solid phase microextraction-high performance liquid chromatography on-line combined detection method for zoledronic acid and risedronic acid - Google Patents

Solid phase microextraction-high performance liquid chromatography on-line combined detection method for zoledronic acid and risedronic acid Download PDF

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CN110568114B
CN110568114B CN201910805620.5A CN201910805620A CN110568114B CN 110568114 B CN110568114 B CN 110568114B CN 201910805620 A CN201910805620 A CN 201910805620A CN 110568114 B CN110568114 B CN 110568114B
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王家斌
戴永伟
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Fuzhou Jiachen Biological Technology Co ltd
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Abstract

The invention discloses a solid phase microextraction-high performance liquid chromatography online combined detection method for zoledronic acid and risedronic acid. The invention firstly prepares a hydroxyapatite functionalized monolithic column by an in-situ mineralization method, then uses the monolithic column as a solid phase micro-extraction monolithic column, and combines a solid phase micro-extraction-high performance liquid chromatography online combined system to establish a solid phase micro-extraction-high performance liquid chromatography online combined detection method for zoledronic acid and risedronic acid. The invention realizes the high-efficiency extraction and enrichment of zoledronic acid and risedronic acid by utilizing the specific electrostatic interaction between hydroxyapatite on the surface of the monolithic column and a P-C-P structure carried by an analysis object, and eliminates the interference of impurities in a sample matrix on analysis and detection. The method is simple, the process is ingenious, the required instruments are high in popularity and easy to popularize, the efficient extraction and enrichment of trace zoledronic acid and risedronic acid in a complex actual sample can be realized, and the related high-sensitivity detection requirements are met.

Description

Solid phase microextraction-high performance liquid chromatography on-line combined detection method for zoledronic acid and risedronic acid
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a solid-phase microextraction-high performance liquid chromatography online combined detection method for zoledronic acid and risedronic acid.
Background
Osteoporosis, is a metabolic bone disorder characterized by a reduction in the amount of bone tissue per unit volume. At present, osteoporosis is promoted to the seventh common disease and frequently-occurring disease in China, the incidence rate is increased year by year, and the condition is not optimistic. According to statistics, the current 8800 ten thousand people in China suffer from osteoporosis, and the total disease rate is 12.4%; among them, the prevalence rate of the elderly over 65 years old is over 50%, and the incidence rate of fracture is nearly one third. Drugs clinically used for treating osteoporosis are mainly classified into basic supplements, bone resorption-inhibiting drugs and bone formation-promoting drugs according to the occurrence of diseases. Bisphosphates represented by zoledronic acid and risedronic acid are widely used clinically as bone resorption inhibiting agents because of their good therapeutic effects. However, administration of bisphosphonates also causes side effects such as vomiting, diarrhea, dizziness, and if it is taken excessively for a long period of time, diseases such as deterioration of renal function may occur. Therefore, the content detection of the medicines in the human body is very important for ensuring the treatment effect of the osteoporosis and ensuring the medication safety.
Hydroxyapatite (HAP) is the main mineral component of natural bone tissue and teeth, is a crystalline form of calcium phosphate, and has a structural formula of Ca10(PO4)6(OH)2Consisting of a positively charged pair of crystalline calcium ions (C site) and six negatively charged clusters of oxygen atoms (P site). HAP, as a biological separation and purification material, dates back to 1956 for the first time, and the action mechanism thereof comprises anion exchange at C site, cation exchange at P site and coordination of calcium ion. Because the chemical structures of the diphosphonate substances such as zoledronic acid, risedronic acid and the like are provided with structures in which two phosphate groups are connected with one carbon atom (P-C-P), the substances can generate strong specific anion exchange interaction with calcium ion pairs (namely C sites) on HAP, which is not only the pharmacodynamic action mechanism of the medicaments, but also provides a new idea for developing selective extraction and enrichment materials of the medicaments. Firstly, preparing a hydroxyapatite functionalized monolithic column by an in-situ mineralization method; then the monolithic column is used as a solid phase micro-extraction monolithic column, the specific electrostatic interaction between the HAP on the surface of the monolithic column and the P-C-P structures carried by the zoledronic acid and the risedronic acid is utilized to realize the high-efficiency extraction and enrichment of the zoledronic acid and the risedronic acid, and the interference of impurities in a sample matrix on analysis and detection is eliminated; and an online combined detection method of the zoledronic acid and the risedronic acid is established by combining a solid phase microextraction-high performance liquid chromatography online combined system. At present, no relevant report of on-line enrichment extraction of zoledronic acid and risedronic acid by using HAP functionalized monolithic column exists.
Disclosure of Invention
The invention aims to provide a solid phase microextraction-high performance liquid chromatography online combined detection method for zoledronic acid and risedronic acid. Firstly, preparing a hydroxyapatite functionalized monolithic column by an in-situ mineralization method; then the monolithic column is used as a solid phase micro-extraction monolithic column; the specific electrostatic interaction between the C site on the HAP particles generated by in-situ mineralization on the surface of the monolithic column and the P-C-P structure is benefited to realize the efficient extraction, enrichment and online purification of trace zoledronic acid and risedronic acid in a complex biological sample; and then, establishing an online combined detection method for the zoledronic acid and the risedronic acid by combining a solid phase microextraction-high performance liquid chromatography online combined system.
In order to achieve the purpose, the invention adopts the following technical scheme:
1) the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method comprises the following steps:
first step, preparing a matrix monolithic column: cleaning a polytetrafluoroethylene empty tube with chromatographic pure methanol for half an hour, and then placing the polytetrafluoroethylene empty tube in a 60 ℃ oven for drying; 1g/mL urea aqueous solution, 37% formaldehyde solution and 0.1mol/L hydrochloric acid solution are mixed according to the weight ratio of 4: 5: 1, putting the mixture into a polytetrafluoroethylene tube which is cleaned in advance, and reacting in a water bath at the temperature of between 45 and 65 ℃ for 10 to 120 minutes to prepare a urea-formaldehyde resin matrix monolithic column;
and secondly, coating polydopamine: washing the urea resin matrix monolithic column with water for 1 hour by a micro-infusion pump, and removing unreacted substances in the column; meanwhile, 5mL of 10mmol/L Tris-HCl solution with the pH value of 8.0 is taken, 10mg of dopamine is added, and the solution is dissolved, uniformly mixed and then kept stand for 5 minutes to prepare dopamine solution; then, a trace infusion pump is used for filling the urea-formaldehyde resin matrix monolithic column with the dopamine solution; then, standing for 3 hours without closing the two ends of the monolithic column; continuously repeating the operation twice to obtain a urea-formaldehyde resin integral column coated with the polydopamine on the surface;
thirdly, in-situ mineralization generation of hydroxyapatite: firstly, 354.5mg of NaCl and 15.9mg of NaHCO were weighed3、16.8mg KCl、16.1mg Na2HPO4、6.4mg MgCl2、12.5mg CaCl2、3.2mg Na2SO4Dissolving in 30mL of deionized water to prepare simulated body fluid; filling the monolithic column with the polydopamine coated surface by using a micro-infusion pump; sealing two ends of the monolithic column by using gaskets, and placing the monolithic column in a water bath at 37 ℃ for incubation for 3 days; then, the whole column is taken out, water is taken as a mobile phase, and a micro-infusion pump is used for flushingWashing for 1 hour; and continuously repeating the operation twice to obtain the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method. The monolithic column is balanced with methanol for half an hour before use.
Wherein the polytetrafluoroethylene tube has a length of 10cm and an inner diameter of 750 μm.
2) The construction and operation of the solid phase micro-extraction-high performance liquid chromatography online combined system are as follows:
the on-line combined system of solid phase micro-extraction-high performance liquid chromatography comprises a ten-way valve, a six-way valve, a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method, a liquid chromatography infusion pump-pump A, a 0.5mL PEEK tube quantitative ring, a liquid chromatography infusion pump-pump B, a 0.2mL PEEK tube quantitative ring, a liquid chromatography analysis column and a detector.
The combined system comprises the following specific operation steps:
firstly, a ten-way valve and a six-way valve are both in the LOAD position; the loading liquid passes through a hydroxyapatite functionalized monolithic column prepared by a pump A balance in-situ mineralization method, and the flow rate is 0.2 mL/min; the mobile phase passes through the analytical column directly by a pump B to obtain a stable baseline required by chromatographic separation, and the flow rate is 1.0 mL/min; meanwhile, filling a sample solution into a 0.5mL PEEK tube quantitative ring through a sample injection needle;
when the six-way valve is adjusted to the INJECT position, solid phase micro-extraction starts, a sample in a 0.5mL PEEK tube quantitative ring is brought into a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method through loading liquid, and the six-way valve is adjusted back to the LOAD position after a given time;
thirdly, the solution conveyed by the pump A is changed into eluent from loading liquid, the flow rate is set to be 0.1mL/min, the eluent is utilized to elute the enriched analysis object on the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method, and the enriched analysis object is collected in a 0.2mL PEEK tube quantitative ring; when the elution is completed, the ten-way valve is adjusted to the INJECT position, the collected eluent is sent to a liquid chromatographic column by using the mobile phase for separation, and then the detection is carried out by using a detector.
Wherein, the sample solution and the loading solution consist of: according to the volume fraction ratio, the methanol/water is 70/30; the mobile phase is as follows: methanol/15 mmol/L phosphate buffer solution (containing 5mmol/L tetrabutylammonium bromide) with pH 7.0 of 30/70; the eluent is phosphate buffer solution with the pH value of 7.0 of 15 mmol/L; the analytical column is a C18 liquid chromatographic analytical column; the detector is a diode array detector; the column oven temperature was 40 ℃ and the detection wavelengths were 220nm (zoledronic acid) and 260nm (risedronic acid).
The invention has the following remarkable advantages:
1) the HAP functionalized monolithic column prepared by the in-situ mineralization method is used as a solid-phase micro-extraction medium, the HAP coverage degree of the surface of the monolithic column is high, the action probability between zoledronic acid, risedronic acid and HAP can be improved, the specific interaction between the zoledronic acid and risedronic acid is enhanced, and the specific extraction capability of the monolithic column is improved; meanwhile, the influence of other impurities in the actual sample on the enrichment extraction of the analysis object can be reduced, and the high-efficiency enrichment extraction and high-sensitivity detection of the analysis object are realized.
2) The HAP functionalized monolithic column prepared by the in-situ mineralization method keeps the excellent permeability of the matrix monolithic column, so that higher flow rate can be used in the solid-phase microextraction operation, the operation efficiency of analysis and detection is improved, the cleaning and regeneration of the monolithic column are facilitated, the service life of the monolithic column is prolonged, and the stability and the reproducibility of the method are improved.
Drawings
FIG. 1 is a schematic structural diagram of a solid phase microextraction-high performance liquid chromatography on-line combined system.
FIG. 2 shows that when an HAP functionalized monolithic column prepared by an in-situ mineralization method is used as a solid-phase microextraction medium to construct a solid-phase microextraction-high performance liquid chromatography online combined system, and online enrichment detection is performed on zoledronic acid and risedronic acid, the influence of the methanol content in a loading liquid on the sample enrichment efficiency is realized.
FIG. 3 is a chromatogram of on-line enrichment detection of trace amounts of zoledronic acid and risedronic acid added to a blank serum sample by using an HAP functionalized monolithic column prepared by an in-situ mineralization method as a solid-phase microextraction medium to construct a solid-phase microextraction-high performance liquid chromatography on-line combined system. FIG. 3-a is an on-line system analysis chromatogram of a spiked serum sample; FIG. 3-b is a chromatogram of a conventional liquid chromatography system for measuring a spiked serum sample.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Step one, preparing a hydroxyapatite functionalized monolithic column by an in-situ mineralization method:
(1) preparing a matrix monolithic column: cleaning a polytetrafluoroethylene empty tube with chromatographic pure methanol for half an hour, and then placing the polytetrafluoroethylene empty tube in a 60 ℃ oven for drying; 1g/mL urea aqueous solution, 37% formaldehyde solution and 0.1mol/L hydrochloric acid solution are mixed according to the weight ratio of 4: 5: 1, putting the mixture into a polytetrafluoroethylene tube which is cleaned in advance, and reacting in a water bath at 55 ℃ for 10 minutes to obtain the urea-formaldehyde resin matrix monolithic column.
(2) Coating of polydopamine: washing the urea resin matrix monolithic column with water for 1 hour by a micro-infusion pump, and removing unreacted substances in the column; meanwhile, 5mL of 10mmol/L Tris-HCl solution with the pH value of 8.0 is taken, 10mg of dopamine is added, and the solution is dissolved, uniformly mixed and then kept stand for 5 minutes to prepare dopamine solution; then, a trace infusion pump is used for filling the urea-formaldehyde resin matrix monolithic column with the dopamine solution; then, standing for 3 hours without closing the two ends of the monolithic column; continuously repeating the operation twice to obtain a urea-formaldehyde resin integral column coated with the polydopamine on the surface;
(3) in-situ mineralization generation of hydroxyapatite: firstly, 354.5mg of NaCl and 15.9mg of NaHCO were weighed3、16.8mg KCl、16.1mg Na2HPO4、6.4mg MgCl2、12.5mg CaCl2、3.2mg Na2SO4Dissolving in 30mL of deionized water to prepare simulated body fluid; filling the monolithic column with the polydopamine coated surface by using a micro-infusion pump; sealing two ends of the monolithic column by using gaskets, and placing the monolithic column in a water bath at 37 ℃ for incubation for 3 days; then, taking out the monolithic column, taking water as a mobile phase, and flushing for 1 hour by using a micro-infusion pump; and continuously repeating the operation twice to obtain the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method. The monolithic column is balanced with methanol for half an hour before use.
Step two, the construction and operation of a solid phase micro-extraction-high performance liquid chromatography online combined system:
the on-line combined system of solid phase micro-extraction-high performance liquid chromatography comprises a ten-way valve, a six-way valve, a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method, a liquid chromatography infusion pump-pump A, a 0.5mL PEEK tube quantitative ring, a liquid chromatography infusion pump-pump B, a 0.2mL PEEK tube quantitative ring, a liquid chromatography analysis column and a detector.
The online combination system comprises the following specific operation steps:
firstly, a ten-way valve and a six-way valve are both in the LOAD position; the loading liquid passes through a hydroxyapatite functionalized monolithic column prepared by a pump A balance in-situ mineralization method, and the flow rate is 0.2 mL/min; the mobile phase passes through the analytical column directly by a pump B to obtain a stable baseline required by chromatographic separation, and the flow rate is 1.0 mL/min; meanwhile, filling a sample solution into a 0.5mL PEEK tube quantitative ring through a sample injection needle;
when the six-way valve is adjusted to the INJECT position, solid phase micro-extraction starts, a sample in a 0.5mL PEEK tube quantitative ring is brought into a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method through loading liquid, and after 3.5 minutes, the six-way valve is adjusted back to the LOAD position;
thirdly, the solution conveyed by the pump A is changed into eluent from loading liquid, the flow rate is set to be 0.1mL/min, the eluent is utilized to elute the enriched analysis object on the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method, and the enriched analysis object is collected in a 0.2mL PEEK tube quantitative ring; when the elution is completed, the ten-way valve is adjusted to the INJECT position, the collected eluent is sent to a liquid chromatographic column by using the mobile phase for separation, and then the detection is carried out by using a detector.
Wherein, the sample solution and the loading solution consist of: according to the volume fraction ratio, the methanol/water is 70/30; the mobile phase is as follows: methanol/15 mmol/L phosphate buffer solution (containing 5mmol/L tetrabutylammonium bromide) with pH 7.0 of 30/70; the eluent is phosphate buffer solution with the pH value of 7.0 of 15 mmol/L; the analytical column is a C18 liquid chromatographic analytical column; the detector is a diode array detector; the column oven temperature was 40 ℃ and the detection wavelengths were 220nm (zoledronic acid) and 260nm (risedronic acid).
Application example 1
Firstly, preparing an HAP functionalized monolithic column by an in-situ mineralization method according to the embodiment, and taking the monolithic column as a solid phase micro-extraction monolithic column; then, a solid phase microextraction-high performance liquid chromatography online combined analysis system is constructed according to the figure 1, and the influence of the methanol content in the loading liquid on the sample enrichment efficiency is examined when online enrichment detection is carried out on zoledronic acid (ZOD) and risedronic acid (RID). The loading liquid consists of methanol and H2O, the sample solution composition is methanol/H2O is 70/30(v/v), the injection flow rate is 0.2mL/min, and the injection volume is 500 muL; the eluent composition was 15mmol/L phosphate buffer solution with pH 7.0, elution flow rate 0.1mL/min, elution volume 200 μ L (collection from 0.5 min to 2.5 min); the mobile phase was separated from methanol/15 mmol/L phosphate buffer solution (containing 5mmol/L tetrabutylammonium bromide) at pH 7.0 30/70(v/v), the separation flow rate was 1mL/min, the column oven temperature was 40 ℃, and the detection wavelengths were 220nm (zoledronic acid) and 260nm (risedronic acid), respectively.
As shown in FIG. 2, the efficiency of zoledronic acid and risedronic acid extraction increases significantly as the methanol content of the carrier liquid increases. During the extractive enrichment of analytes, a key role is played by the specific anion exchange interaction between the P-C-P structure in the analyte and the C site on the HAP. This specific force is enhanced when the methanol content is increased, improving the extraction efficiency, since the water in the loading liquid can compete with the assay for the C sites on the HAP, impairing the interaction. However, when the methanol content is increased from 70% (v/v) to 80% (v/v), the extraction efficiency is rather decreased, because the solubility of the strongly polar substances zoledronic acid and risedronic acid in the solution is decreased due to the excessively high methanol content, resulting in the decrease of the extraction efficiency of zoledronic acid and risedronic acid. Therefore, we chose the loading liquid methanol ratio of 70% (v/v) as the optimum ratio.
Application example 2
Firstly preparing HAP functionalized monolithic column by in-situ mineralization method according to the embodiment, and using the monolithic columnAs solid phase micro extraction monolithic column; then, a solid phase microextraction-high performance liquid chromatography online combined analysis system is constructed according to the figure 1, and trace amounts of zoledronic acid and risedronic acid added in a blank serum sample are enriched and detected on line. The sample injection sample solution and the loading solution consist of methanol/H2O is 70/30(v/v), the injection flow rate is 0.2mL/min, and the injection volume is 500 muL; the eluent composition was 15mmol/L phosphate buffer solution with pH 7.0, elution flow rate 0.1mL/min, elution volume 200 μ L (collection from 0.5 min to 2.5 min); the mobile phase was separated from methanol/15 mmol/L phosphate buffer solution (containing 5mmol/L tetrabutylammonium bromide) at pH 7.0 30/70(v/v), the separation flow rate was 1mL/min, the column oven temperature was 40 ℃, and the detection wavelengths were 220nm (zoledronic acid) and 260nm (risedronic acid), respectively.
FIG. 3 is a chromatogram of the online enrichment detection system for trace amounts of zoledronic acid and risedronic acid added to a blank serum sample. In FIG. 3-a, peak 1 was zoledronic acid and peak 2 was risedronic acid. As can be seen from the figure 3-a, under the condition of the combined system, the HAP functionalized monolithic column prepared by the in-situ mineralization method eliminates the interference of a large amount of impurities such as protein, lipid, carbohydrate and the like in a serum sample matrix on analysis and detection, and realizes the high-efficiency enrichment extraction and high-sensitivity detection of trace amounts of zoledronic acid and risedronic acid added in a blank serum sample. When the same sample is analyzed using the direct sample injection mode of the conventional liquid chromatography system, the impurities in the serum sample matrix cause strong interference with the detection of the analyte and overlap with the absorption peak of the analyte (fig. 3-b). This shows that the HAP functionalized monolithic column prepared by the in-situ mineralization method has excellent impurity removal capability and strong specific extraction performance.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (1)

1. The solid phase microextraction-high performance liquid chromatography on-line combined detection method of zoledronic acid and risedronic acid is characterized in that: the detection method is characterized in that a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method is used as a solid-phase microextraction monolithic column, and a solid-phase microextraction-high performance liquid chromatography online combined detection method of zoledronic acid and risedronic acid is established by combining a solid-phase microextraction-high performance liquid chromatography online combined system; the method is used for detecting serum samples;
the preparation method of the hydroxyapatite functionalized monolithic column comprises the following steps:
(1) preparing a matrix monolithic column: cleaning a polytetrafluoroethylene empty tube with chromatographic pure methanol for half an hour, and then placing the polytetrafluoroethylene empty tube in a 60 ℃ oven for drying; 1g/mL urea aqueous solution, 37% formaldehyde solution and 0.1mol/L hydrochloric acid solution are mixed according to the weight ratio of 4: 5: 1, putting the mixture into a polytetrafluoroethylene tube which is cleaned in advance, and reacting in a water bath at 55 ℃ for 10 minutes to obtain a urea-formaldehyde resin matrix monolithic column;
(2) coating of polydopamine: washing the urea resin matrix monolithic column with water for 1 hour by a micro-infusion pump, and removing unreacted substances in the column; meanwhile, taking 5mL of 10mmol/L Tris-HCl solution with pH =8.0, adding 10mg of dopamine, dissolving and uniformly mixing, and standing for 5 minutes to prepare dopamine solution; then, a trace infusion pump is used for filling the urea-formaldehyde resin matrix monolithic column with the dopamine solution; then, standing for 3 hours without closing the two ends of the monolithic column; continuously repeating the operation twice to obtain a urea-formaldehyde resin integral column coated with the polydopamine on the surface;
(3) in-situ mineralization generation of hydroxyapatite: firstly, 354.5mg of NaCl and 15.9mg of NaHCO were weighed3、16.8 mg KCl、16.1 mg Na2HPO4、6.4 mg MgCl2、12.5 mg CaCl2、3.2 mg Na2SO4Dissolving in 30mL of deionized water to prepare simulated body fluid; filling the monolithic column with the polydopamine coated surface by using a micro-infusion pump; sealing both ends of the monolithic column by using gaskets, and placing the monolithic column in a water bath at 37 ℃ for incubation for 3 days; then, taking out the monolithic column, taking water as a mobile phase, and flushing for 1 hour by using a micro-infusion pump; continuously repeating the operation twice to obtain a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method; before the monolithic column is used, the monolithic column is balanced by methanol for half an hour;
the combined system comprises the following specific operation steps:
1) firstly, the ten-way valve and the six-way valve are both in the LOAD position; the loading liquid passes through a hydroxyapatite functionalized monolithic column prepared by a pump A balance in-situ mineralization method, and the flow rate is 0.2 mL/min; the mobile phase passes through the analytical column directly by a pump B to obtain a stable baseline required by chromatographic separation, and the flow rate is 1.0 mL/min; meanwhile, filling a sample solution into a 0.5mL PEEK tube quantitative ring through a sample injection needle;
2) when the six-way valve is adjusted to the INJECT position, solid phase micro-extraction starts, a sample in a 0.5mL PEEK tube quantitative ring is brought into a hydroxyapatite functionalized monolithic column prepared by an in-situ mineralization method through a loading liquid, and the six-way valve is adjusted back to the LOAD position after a given time;
3) then, the solution conveyed by the pump A is changed from loading liquid to eluent, the flow rate is set to be 0.1mL/min, the eluent is utilized to elute an analysis object enriched on the hydroxyapatite functionalized monolithic column prepared by the in-situ mineralization method, and the analysis object is collected in a 0.2mL PEEK tube quantitative ring; when the elution is finished, the ten-way valve is adjusted to the INJECT position, the collected eluent is sent into a liquid chromatographic column by utilizing the mobile phase for separation, and then the detector is utilized for detection;
wherein, the sample solution and the loading solution consist of: methanol/water =70/30 by volume fraction ratio; the mobile phase is as follows: methanol/15 mmol/L phosphate buffer solution with pH =7.0 =30/70, wherein the 15mmol/L phosphate buffer solution with pH =7.0 contains 5mmol/L tetrabutylammonium bromide; the eluent is 15mmol/L phosphate buffer solution with pH = 7.0; the analytical column is a C18 liquid chromatographic analytical column.
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