CN114594170B - An in vivo drug analysis method using magnetic solid phase extraction combined with rapid in situ derivatization - Google Patents

Abstract

The present invention belongs to the technical field of analytical chemistry, and relates to an in vivo drug analysis method combining magnetic solid phase extraction with rapid in situ derivatization. In the method of the present invention, titanium dioxide-coated magnetic ferroferric oxide microspheres are synthesized, and the microspheres are used as materials for dispersed magnetic solid phase extraction for loading. In the elution step, a derivatization reagent is mixed with a specific elution solvent, so that the elution and derivatization of the target analyte are combined into one step. The eluate is concentrated by nitrogen blowing and redissolved, and then analyzed by high performance liquid phase tandem mass spectrometry to determine the content of bisphosphonates in a small amount of biological samples. The method combines the traditional solid phase extraction step with the pre-column derivatization step of a specific drug into one step, shortens the sample derivatization time and reduces solvent consumption. The method is used in conjunction with mass spectrometry to accurately determine the content of bisphosphonates in biological samples.

Description

An in vivo drug analysis method using magnetic solid phase extraction combined with rapid in situ derivatization

Technical Field

The present invention belongs to the technical field of analytical chemistry, and relates to an in vivo drug analysis method combined with magnetic solid phase extraction and rapid in situ derivatization, and specifically to a pretreatment method for enriching bisphosphonate drugs in biological samples using magnetic solid phase extraction materials and performing rapid in situ derivatization on the surface of the material.

Background technique

The prior art discloses that the pretreatment of biological samples is the key point and difficulty of in vivo drug analysis. How to efficiently and quickly remove interfering impurities in samples and enrich target analytes has always been a hot topic of concern to those skilled in the art. Generally, sample pretreatment is an indispensable operation step in the analysis of complex samples, which directly affects the sensitivity, selectivity, reliability, analysis speed, etc. of the analytical method. Therefore, sample pretreatment of biological samples has become one of the important research fields of in vivo drugs today.

Studies have shown that some commonly used drugs do not have ultraviolet and fluorescent absorption, and there is a lack of detection methods for their monomers, so it is necessary to introduce derivatization methods. According to the relative position relationship between the derivatization reaction and the pretreatment step, the traditional pre-column derivatization method can be divided into pre-extraction derivatization, post-extraction derivatization, in-situ derivatization and injection port derivatization. Traditional solid phase extraction requires the analyte to be eluted from the adsorbent and the analyte and the solid phase extraction adsorbent are completely separated before the derivatization reaction; while in-situ derivatization is carried out during the elution of the sample solid phase extraction. In-situ derivatization combines the derivatization pretreatment operation required by the traditional method into the sample elution process, which has the advantages of short solid phase extraction time and low solvent consumption, as well as the advantages of high sensitivity and selectivity of the derivatization method. In addition, since the transfer process of the analyte between the two solution environments of elution and derivatization is omitted, the method error can be reduced. At present, the in-situ derivatization method has been used for the in vivo analysis of drugs containing carboxyl, phenolic, amino and carbonyl groups.

Magnetic solid phase extraction is a sample pretreatment technology developed based on the rapid movement of magnetic materials under the action of a magnetic field. The difference between magnetic solid phase extraction and traditional solid phase extraction is that the filler of the solid phase extraction adsorbent does not need to be loaded into the solid phase extraction column, but is dispersed in the solution or suspension of the sample. The target analyte is fully mixed with the dispersed magnetic adsorbent and adsorbed by the groups on the surface of the magnetic adsorbent. Under the action of an external magnetic field, the target analyte is separated from the sample matrix along with the magnetic adsorbent. Compared with traditional solid phase extraction, magnetic solid phase extraction has the following advantages: (1) Magnetic solid phase extraction does not require a pressurizing device such as a solid phase extraction cylinder and a vacuum pump. The adsorbent material and the sample solution can be separated only by applying an external magnetic field. (2) Magnetic solid phase extraction eliminates the complicated and time-consuming column loading process and only requires the magnetic material to be dispersed in the sample matrix. (3) The adsorbent material is in the form of dispersed particles in the sample solution. The adsorbent can fully contact with the target analyte, which helps to improve the mass transfer efficiency, shorten the extraction time, and increase the enrichment multiple of the analyte. (4) When processing complex samples, it is not easy to cause adsorbent clogging. (5) The amount of magnetic solid phase extraction material used is often less than the amount of filler used in solid phase extraction columns, and the required elution solvent volume is also smaller, which is suitable for trace analysis. Based on the above advantages, magnetic solid phase extraction is not only widely used in environmental analysis and food safety, but also shows application prospects in in vivo drug analysis.

Studies have reported that transition metal ions represented by titanium ions have extremely high selectivity and affinity for phosphate groups. Titanium ion-modified iron oxide magnetic spheres have been successfully used for the selective enrichment of phosphorylated peptides and isopentenyl pyrophosphate in biological matrices. The ability of titanium ion-modified materials to enrich compounds containing phosphate groups has been fully confirmed.

Based on the foundation and current status of the prior art, the inventors of the present application intend to provide a new, simpler and faster method for enriching and derivatizing bisphosphonates in in vivo samples. The method combines magnetic solid phase extraction technology with in situ derivatization technology, and operationally combines the analyte derivatization and magnetic solid phase extraction elution operations into one step, which can significantly reduce the manual operations required for the solid phase extraction and derivatization processes, effectively shorten the pre-treatment time for in vivo analysis of bisphosphonates, and improve the sensitivity of the analysis.

Summary of the invention

The purpose of the present invention is to provide a new, simpler and faster method for enriching and derivatizing bisphosphonates in in vivo samples based on the foundation and status of the prior art, specifically to an in vivo drug analysis method combining magnetic solid phase extraction with rapid in situ derivatization.

In the method of the present invention, two existing sample pretreatment technologies, namely magnetic solid phase extraction and analyte in situ derivatization technology, are combined, inheriting the respective advantages of the two technologies, and providing a simpler and faster method for enriching and rapidly derivatizing bisphosphonates in in vivo samples. Experimental verification shows that the method is simple, rapid, has high extraction efficiency, good reproducibility, consumes less solvent reagents, is safe and environmentally friendly, and can accurately determine the content of bisphosphonates in in vivo samples.

The present invention is based on the special properties of titanium dioxide-coated ferroferric oxide magnetic microspheres, such as large specific surface area, strong adsorption capacity, and strong stability, and applies them to the pretreatment of complex in vivo samples. It is mainly based on the principle of solid phase extraction technology, and the extraction method is simplified to two steps: loading and elution (and in situ derivatization). Compared with the traditional technology of solid phase extraction followed by derivatization, it is simpler and faster.

More specifically, the purpose of the present invention is achieved through the following technical solutions:

Titanium dioxide-coated ferroferric oxide magnetic microspheres were used as magnetic solid phase extraction adsorbents to load plasma and urine samples containing bisphosphonates. A mixed solvent containing a derivatization reagent was used for in situ derivatization and elution to complete the enrichment and derivatization process of bisphosphonates in biological samples. The samples were concentrated by nitrogen blowing and re-dissolved in a certain solvent before high performance liquid phase tandem mass spectrometry analysis to determine the content of bisphosphonates in in vivo samples.

In the present invention, the factors affecting the extraction efficiency of the titanium dioxide-coated ferroferric oxide magnetic microsphere adsorbent include material dosage, sample solution acid concentration, extraction time, derivatization time and derivatization temperature, etc., which are selected and determined;

In the present invention, in the above-mentioned magnetic solid phase extraction in-situ derivatization method, the amount of magnetic material used is 100-800 μg.

In the present invention, in the above magnetic solid phase extraction in situ derivatization method, the concentration of trifluoroacetic acid in the loading solution is 0.5%-8%.

In the present invention, in the above-mentioned magnetic solid phase extraction in-situ derivatization method, the extraction time is 0.5min-12min.

In the present invention, in the above-mentioned magnetic solid phase extraction in situ derivatization method, the elution solvent and the derivatization reagent are mixed in a certain volume ratio, the volume of the mixed solvent is 100-500 μl, and the mixing volume ratio of the two solvents is 3:1 to 1:1.

In the present invention, in the above-mentioned magnetic solid phase extraction in-situ derivatization method, the derivatization time is 1 min-16 min, and the derivatization temperature is 25°C-60°C.

Experimental results show that the magnetic solid phase extraction in situ derivatization method of the present invention uses an experimental material that integrates extraction, concentration, and in situ derivatization: titanium dioxide-coated ferroferric oxide magnetic microspheres, which makes the sample pretreatment of bisphosphonates in in vivo samples simple, accurate, reliable, rapid, reproducible, safe and environmentally friendly; it has the advantages of good selectivity, strong specificity, rapidity and convenience, and can be used to accurately determine the concentration of bisphosphonate drugs in in vivo samples.

The present invention provides a simpler and faster method for enriching and derivatizing bisphosphonates in in vivo samples. The magnetic solid phase extraction in situ derivatization technology therein can provide technical support for in vivo analysis and research of drugs that require pre-column derivatization technology. At the same time, the present invention provides new ideas and methods for developing complex sample pretreatment technology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram showing the structure and synthesis of titanium dioxide-coated magnetic ferroferric oxide microspheres.

FIG. 2 is a transmission electron microscope photograph of titanium dioxide-coated ferroferric oxide magnetic microspheres.

FIG3 is a schematic diagram of the operation steps of the magnetic solid phase extraction in situ derivatization technology.

Detailed ways

The following examples are provided to further illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1 Magnetic solid phase extraction in situ derivatization method for detection of alendronate sodium in plasma samples

1) Synthesis of titanium dioxide-coated magnetic ferroferric oxide microspheres:

(1) Synthesis of nano-iron tetroxide: Fe ₃ O ₄ magnetic spheres were synthesized using a solvothermal method ^[9] . The specific synthesis steps were as follows: After fully grinding FeCl ₃ ·6H ₂ O into fine powder, 1.35 g of the powder was dispersed in 75 mL of ethylene glycol and stirred for a period of time until the solution was clear. 3.6 g of sodium acetate was slowly added to the solution and continued to stir until it was uniform. The resulting suspension was transferred to a 200 mL reactor and heated at 200 °C for 16 h. The reactor was then cooled to room temperature. The product was washed alternately with water and ethanol three times and dried in vacuo.

(2) Synthesis of ferroferric oxide-dopamine magnetic microspheres: 40 mg of dried Fe ₃ O ₄ powder was dispersed in 40 mL of Tris aqueous solution (10 mM, pH = 8.5), and 80 mL of ethanol was added and mixed. 160 mg of dopamine hydrochloride was weighed and dissolved in 60 mL of deionized water. The dopamine hydrochloride solution was added to the above reaction system and stirred at room temperature for 12 h. Finally, the product (Fe ₃ O ₄ @PD) was washed alternately with water and ethanol for 3 times and dried in vacuum.

(3) Synthesis of ferroferric oxide-titanium dioxide magnetic microspheres: 40 mg of dried Fe ₃ O ₄ @PD microspheres were added to 200 ml of 50 mM titanium sulfate solution, stirred at room temperature for 4 h, washed with water three times, and vacuum dried. After drying, the material was calcined at 400 °C for 2 h. After complete cooling, the desired material was obtained;

2) Detection of alendronate sodium in rat plasma by magnetic solid phase extraction and in situ derivatization

(1) Instruments: Agilent 1200 high performance liquid chromatograph; AB SCIEX 4000 triple quadrupole mass spectrometer; nitrogen blowdown device; Eppendorf ThermoMixer C oscillator; Eppendorf 5418R desktop centrifuge; magnetic separation rack;

(2) Materials: alendronate sodium standard; alendronate sodium-d6 standard; trimethylsilyldiazomethane solution; methanol; deionized water; ammonium acetate; acetonitrile; trifluoroacetic acid; titanium dioxide-modified ferrosoferric oxide magnetic microspheres; rat plasma samples;

(3) Sample pretreatment: Add 200 μl of plasma and 200 μl of trifluoroacetic acid solution to a 2 ml centrifuge tube containing titanium dioxide-modified ferroferric oxide magnetic microspheres, shake for 4 min, separate the materials in a magnetic field, and discard the supernatant; add 400 μl of cleaning solvent, shake for 30 s, separate the materials in a magnetic field, and discard the supernatant; add 200 μl of methanol and 100 μl of trimethylsilyldiazomethane solution, shake at 50°C for 2 min, separate the materials in a magnetic field, and transfer the supernatant to another clean centrifuge tube. Dry the solution in this centrifuge tube with nitrogen at 40°C, reconstitute with 100 μl of mobile phase, and perform LC-MS/MS injection analysis;

(4) LC-MS/MS conditions

Chromatographic column: Agilent ZORBAX Eclipse Plus-C18 (150×2.1mm, 5μm) + (12.5×2.1mm, 5μm) guard column;

Mobile phase: Mobile phase A: 10 mM ammonium acetate solution, pH = 4, mobile phase B: acetonitrile;

Injection volume: 10 μL; column temperature: 30°C; flow rate: 0.5 mL/min; elution time: 6.5 min;

Gradient elution program setting: 0-0.5min maintain A:B = 90:10 ratio, 0.5-1min adjust to A:B = 70:30, 1-2min maintain A:B = 70:30, 2-3min adjust to A:B = 90:10, 3-6.5min maintain A:B = 90:10;

Mass spectrometry conditions:

Ionization mode: ESI (+) ion source: Turbo Ionspray, curtain gas pressure (curtain gas, CUR): 30psi, source temperature (source temperature): 550℃, carrier gas pressure (nebulizer gas, GS1): 45psi, heater gas (GS2): 50psi, ion spray voltage: 4500V, entrance potential (entrance potential, EP: 10V, collision cell exit potential (collision cell exit potential, CXP: 10V;

(5) Accuracy, precision and matrix effect:

The spiked plasma with four different concentrations (lower limit of quantitation, low, medium and high) was processed by the same magnetic solid phase extraction in situ derivatization method, and then analyzed by LC-MS/MS. The accuracy and precision were calculated according to the fitted quantitative curve. The intra-batch accuracy and precision were 100.5-102.7% and 0.2-4.0% (n=5), respectively; the inter-batch accuracy and precision were 100.7-105.3% and 1.1-5.3% (n=15), respectively.

Matrix Effects:

The low and high concentrations of the reference substances were first derivatized with trimethylsilane and diazomethane to prepare the derivatized reference substance solutions; the blank plasma samples were treated with the magnetic solid phase extraction in situ derivatization method, and the solution obtained after treatment was mixed with the derivatized reference substance solution, and the mixed matrix effect sample and the derivatized reference substance solution were injected separately, and the interference of endogenous components in plasma on analyte detection was evaluated by comparing the coefficient of variation of the ratio of the peak area response values of the matrix effect sample and the derivatized reference substance solution of the same concentration; the results showed that the matrix effects of rat plasma at the two concentrations were 7.3% and 0.7%, respectively (n=6).

Claims (2)

1. A method for in vivo drug analysis combining magnetic solid phase extraction with rapid in situ derivatization, characterized in that: The method comprises: 1) synthesizing titanium dioxide-coated ferroferric oxide magnetic microspheres; 2) using the magnetic microspheres as dispersed magnetic particles; The solid phase extraction adsorbent can quickly enrich bisphosphonates in biological samples. 3) The magnetic solid phase extraction The elution and sample derivatization are combined into one, and heating is introduced to increase the reaction rate, achieving rapid derivatization of the analyte. Rapid elution and derivatization; In the method, the magnetic solid phase extraction material used is a core-shell structure magnetic microsphere coated with titanium dioxide, and the amount of magnetic solid phase extraction material used for each sample is 100-800 μg; In the method, the shaking incubation time during sample loading is 2-10 min; In the method, the derivatization reaction time is 2-10 min; In the method, the elution solvent and the derivatization reagent are mixed in a volume ratio of 3:1 to 1:1, and the volume of the solvent after mixing is 100 - 500 μl; In the method, heating is performed during the derivatization process, and the temperature of the derivatization process is controlled in the range of 25°C-60°C. 2. The method according to claim 1, characterized in that, in the method, the biological sample is plasma Sample or urine sample, the sample loading volume is 200-400 μl.