阅读说明：本技术 一种血浆样品中度他雄胺含量的检测方法 (Method for detecting dutasteride content in plasma sample ) 是由 卢念红 陈廷琼 杨再香 严紫薇 谢辉 于 2019-11-26 设计创作，主要内容包括：本发明公开了一种血浆样品中度他雄胺含量的检测方法,所述检测方法为采用二维液相串联质谱法测定血浆样品中度他雄胺含量。其中血浆样品前处理采用液液萃取法,萃取剂为甲基叔丁基醚。有机相为甲醇：乙腈=1:1,水相为5mM乙酸铵水溶液。首先液相“1”对磷脂类化合物与度他雄胺及其同位素内标化合物进行有效分离,然后由液相“2”将度他雄胺及其同位素内标化合物切入分析柱,并在分析柱上对度他雄胺及其同位素内标化合物进行进一步分离纯化并载入质谱检测,与此同时液相“1”加大流速和有机相比例冲洗预柱。通过有效分离磷脂类化合物与度他雄胺及其同位素内标化合物,从而避免了磷脂类化合物对度他雄胺及其同位素内标响应的抑制。本方法的检测限可以达到12.5pg/ml,灵敏度高,血浆用量少,检测快速。(The invention discloses a method for detecting the content of dutasteride in a plasma sample, which is to determine the content of dutasteride in the plasma sample by adopting a two-dimensional liquid tandem mass spectrometry. Wherein the plasma sample pretreatment adopts a liquid-liquid extraction method, and the extractant is methyl tert-butyl ether. The organic phase is methanol: acetonitrile =1:1 and the aqueous phase was 5mM ammonium acetate in water. Firstly, the phospholipid compound, the dutasteride and the isotope internal standard compound thereof are effectively separated by the liquid phase 1, then the dutasteride and the isotope internal standard compound thereof are cut into an analytical column by the liquid phase 2, the dutasteride and the isotope internal standard compound thereof are further separated and purified on the analytical column and are loaded into a mass spectrum for detection, and meanwhile, the flow rate of the liquid phase 1 is increased and the pre-column is washed by an organic phase. The phospholipid compound is effectively separated from the dutasteride and the isotope internal standard compound thereof, so that the inhibition of the phospholipid compound on the dutasteride and the isotope internal standard response thereof is avoided. The detection limit of the method can reach 12.5pg/ml, the sensitivity is high, the plasma dosage is small, and the detection is rapid.)

1. A method for detecting the content of dutasteride in a plasma sample is characterized in that the detection method is a two-dimensional liquid phase tandem mass spectrometry method; the two-dimensional liquid phase consists of a liquid phase '1' and a liquid phase '2'; the two-dimensional liquid phase tandem mass spectrometry comprises a liquid phase 1, a liquid phase 2, a pre-column, an analytical column and a mass spectrum; the analysis method comprises the following steps:

1) in the state of a flow path 1, a liquid phase 1 is communicated with the pre-column; the liquid phase "2" is in communication with the analytical column and mass spectrometer; liquid phase "1" loads analyte into pre-column;

2) in the state of the flow path 2, the liquid phase 1 is not communicated with the pre-column; the liquid phase 2 is communicated with a pre-column, an analytical column and a mass spectrum; liquid phase "2" cuts analyte into the analytical column;

3) under the state of a flow path 1, separating and purifying the analyte by using a liquid phase 2 and loading the analyte into mass spectrometry for detection;

the analyte is a sample comprising dutasteride and an internal standard;

the liquid phase 1 consists of an aqueous phase 1 and an organic phase 1; the liquid phase "2" is composed of an aqueous phase 2 and an organic phase 2.

2. The detection method according to claim 1, wherein each of the aqueous phase 1 and the aqueous phase 2 is independently selected from one of an aqueous ammonium acetate solution, an aqueous ammonium formate solution, an aqueous formic acid solution, an aqueous acetic acid solution, water and an aqueous ammonia solution, and the aqueous phase 1 and the aqueous phase 2 are preferably an aqueous ammonium acetate solution or an aqueous ammonium formate solution; wherein optionally the aqueous ammonium acetate solution is selected from 1-50mM aqueous ammonium acetate solution, preferably 3-20mM aqueous ammonium acetate solution, further preferably 5-10mM aqueous ammonium acetate solution; wherein optionally the aqueous ammonium formate solution is selected from 1-50mM aqueous ammonium formate solution, preferably 3-20mM aqueous ammonium formate solution, further preferably 5-10mM aqueous ammonium formate solution.

3. The detection method according to claim 1, wherein the organic phase 1 and the organic phase 2 are each independently selected from one of methanol, acetonitrile, and a methanol acetonitrile mixed solution, and the organic phase 1 and the organic phase 2 are preferably methanol acetonitrile mixed solutions; wherein, optionally, the methanol acetonitrile mixed solution is methanol: acetonitrile =1:9 to 9:1 (volume ratio), preferably 2:8 to 8:2 (volume ratio), and more preferably 4:6 to 6:4 (volume ratio).

4. The detection method according to claim 1, characterized in that the initial organic phase 1 proportion of the liquid phase "1" is 45-60% (vol%), wherein the detailed gradient elution procedure is:

。

5. the detection method according to claim 1, characterized in that the initial organic phase 2 proportion of the liquid phase "2" is 65-80% (vol%), wherein the detailed elution procedure is:

。

6. the detection method according to any one of claims 1 to 5, wherein the flow rate of the liquid phase "1" is 0.6-1.0 ml/min.

7. The detection method according to any one of claims 1 to 5, characterized in that the flow rate of the liquid phase "2" is 0.6-1.0 ml/min.

8. The detection method according to any one of claims 1 to 5, characterized in that the analytical column is selected from a chromatographic column packed with an octadecylsilane bonded silica filler; optionally, the pre-column is selected from a guard column packed with octadecylsilane bonded silica filler.

9. The detection method according to any one of claims 1 to 5, wherein the column temperature in the two-dimensional liquid phase tandem mass spectrometry is 35 to 45 ℃ and the sample tray temperature is 4 to 15 ℃.

10. The detection method according to any one of claims 1 to 5, wherein the plasma sample is subjected to a pretreatment by a liquid-liquid extraction method, a protein precipitation method, or a solid-phase extraction method; optionally, the liquid-liquid extraction method comprises the steps of extracting dutasteride and an internal standard from a plasma sample containing an internal standard solution to obtain a mixed extract of the dutasteride and the internal standard, drying the mixed extract of the dutasteride and the internal standard, adding a complex solvent for redissolution, and performing two-dimensional liquid-phase tandem chromatography analysis and detection;

optionally, the extracting agent is selected from methyl tert-butyl ether, n-hexane, a mixed solvent of methyl tert-butyl ether and dichloromethane, a mixed solvent of methyl tert-butyl ether and n-hexane, and a mixed solvent of methyl tert-butyl ether and dichloromethane, and the extracting agent is preferably selected from methyl tert-butyl ether, a mixed solvent of methyl tert-butyl ether and dichloromethane, and a mixed solvent of methyl tert-butyl ether and n-hexane; wherein the ratio of methyl tert-butyl ether in the mixed solvent of methyl tert-butyl ether and dichloromethane is: dichloromethane =10: 0.5-10: 3 (volume ratio), preferably 10: 1.5-10: 2.5 (volume ratio); wherein, the ratio of methyl tert-butyl ether to n-hexane in the mixed solvent of methyl tert-butyl ether and n-hexane is as follows: n-hexane =1:9 to 7:3 (volume ratio), preferably 1:9 to 3:7 (volume ratio); the internal standard is selected from an isotope internal standard and a non-isotope internal standard, and the internal standard is preferably an isotope internal standard; the solvent of the internal standard solution is selected from methanol and acetonitrile, and the solvent of the internal standard solution is preferably methanol; the dutasteride internal standard mixed extracting solution needs to be frozen, dried and redissolved by a re-solvent; the double solvent is selected from acetonitrile water solution, methanol water solution, acetonitrile formic acid water solution, acetonitrile acetic acid water solution, methanol formic acid water solution, methanol acetic acid water solution, methanol acetonitrile formic acid water solution and methanol acetonitrile acetic acid water solution, and the double solvent is preferably acetonitrile water solution.

Technical Field

The invention relates to the technical field of medicines, in particular to a method for detecting the content of dutasteride in a biological matrix sample.

Background

Dutasteride is a steroid 5 a-reductase inhibitor with two types I and II which is developed by Kulansu Schker (GSK) company and approved to be marketed in the United states by FDA in 6 months of 2003, can inhibit the conversion of testosterone into 5 α -dihydrotestosterone, is a novel medicament for treating benign prostatic hyperplasia, and can reduce acute urinary retention and reduce the surgical treatment of the benign prostatic hyperplasia.

Document 1 (Drug research (2018), 68(4), 238-240) discloses a detection method for measuring dutasteride in plasma, which adopts a one-dimensional liquid phase and uses the plasma with the dosage of up to 1 ml. Document 2 (Talanta (2015), 131, 728-. The phospholipid compound is remained in the system due to the fact that the one-dimensional liquid phase cannot effectively remove waste liquid of the phospholipid compound, dutasteride response is inhibited, and dutasteride sensitivity is reduced, so that the defects that the required plasma dosage is large, the sample collection time is long, the pretreatment is complex and the like are overcome.

Disclosure of Invention

In order to solve the problems of large plasma consumption, long sample collection time, complex pretreatment and the like, the invention provides a rapid, sensitive and convenient biological detection method for detecting the content of dutasteride in a plasma sample.

The invention provides a method for detecting the content of dutasteride in a plasma sample, which is a two-dimensional liquid phase tandem mass spectrometry method; wherein the two-dimensional liquid phase consists of a liquid phase "1" and a liquid phase "2"; the two-dimensional liquid phase tandem mass spectrometry comprises a liquid phase 1, a liquid phase 2, a pre-column, an analytical column and a mass spectrum; the analysis method comprises the following steps:

1) in the state of a flow path 1, a liquid phase 1 is communicated with the pre-column; the liquid phase "2" is in communication with the analytical column and mass spectrometer; liquid phase "1" loads analyte into pre-column and carries out coarse separation on the analyte and biological matrix such as phospholipid compound (flow path 1 is shown in figure 1);

2) in the state of the flow path 2, the liquid phase 1 is not communicated with the pre-column; the liquid phase 2 is communicated with a pre-column, an analytical column and a mass spectrum; the liquid phase "2" cuts the analyte into the analytical column (flow path 2 see fig. 2);

3) in the state of a flow path 1, a liquid phase 1 is communicated with the pre-column; the liquid phase "2" is in communication with the analytical column and mass spectrometer; the liquid phase 2 is used for further separating and purifying the analyte and loading the analyte into a mass spectrum for detection;

the analyte is a sample comprising dutasteride and an internal standard;

the mobile phase of the liquid phase 1 is composed of an aqueous phase 1 and an organic phase 1; the mobile phase of the liquid phase 2 is composed of an aqueous phase 2 and an organic phase 2;

the aqueous phase 1 and the aqueous phase 2 are respectively and independently selected from one of ammonium acetate aqueous solution, ammonium formate aqueous solution, formic acid aqueous solution, acetic acid aqueous solution, water and ammonia aqueous solution, preferably ammonium acetate aqueous solution or ammonium formate aqueous solution;

wherein the ammonium acetate aqueous solution is selected from 1-50mM ammonium acetate aqueous solution, preferably 3-20mM ammonium acetate aqueous solution, and further preferably 5-10mM ammonium acetate aqueous solution;

wherein the aqueous ammonium formate solution is selected from 1-50mM aqueous ammonium formate solution, preferably 3-20mM aqueous ammonium formate solution, and more preferably 5-10mM aqueous ammonium formate solution;

the organic phase 1 and the organic phase 2 are respectively and independently selected from one of methanol, acetonitrile and a methanol acetonitrile mixed solution, preferably the methanol acetonitrile mixed solution;

wherein the methanol acetonitrile mixed solution is selected from methanol: acetonitrile =1:9 to 9:1 (volume ratio), preferably 2:8 to 8:2 (volume ratio), further preferably 4:6 to 6:4 (volume ratio);

the initial organic phase 1 proportion of the liquid phase "1" is 45-60% (vol%), wherein the detailed gradient elution procedure is:

the initial organic phase 2 proportion of the liquid phase "2" is 65-80% (vol%), wherein the detailed elution procedure is:

the flow rate of the liquid phase 1 is 0.6-1.0 ml/min;

the flow rate of the liquid phase 2 is 0.6-1.0 ml/min;

the analytical column is selected from a chromatographic column filled with octadecylsilane chemically bonded silica filler;

the pre-column is selected from a protective column filled with an octadecylsilane bonded silica filler;

the column temperature in the two-dimensional liquid phase tandem mass spectrometry is 35-45 ℃, and the temperature of the sample injection tray is 4-15 ℃.

The pretreatment method of the plasma sample is selected from liquid-liquid extraction method, protein precipitation method and solid phase extraction method;

the liquid-liquid extraction method comprises the steps of extracting dutasteride and an internal standard from a plasma sample containing an internal standard solution to obtain a mixed extract of the dutasteride and the internal standard, blow-drying the mixed extract of the dutasteride and the internal standard, adding a re-solvent for re-dissolution, and analyzing and detecting by using a two-dimensional liquid-phase series chromatography;

the extractant is selected from methyl tert-butyl ether, n-hexane, a mixed solvent of methyl tert-butyl ether and dichloromethane, preferably a mixed solvent of methyl tert-butyl ether, methyl tert-butyl ether and dichloromethane, and a mixed solvent of methyl tert-butyl ether and n-hexane;

wherein the methyl tert-butyl ether dichloromethane mixed solvent is selected from methyl tert-butyl ether: dichloromethane =10: 0.5-10: 3 (volume ratio), preferably 10: 1.5-10: 2.5 (volume ratio);

wherein the methyl tert-butyl ether n-hexane mixed solvent is selected from methyl tert-butyl ether: n-hexane =1:9 to 7:3 (volume ratio), preferably 1:9 to 3:7 (volume ratio);

the internal standard is selected from an isotope internal standard and a non-isotope internal standard, and preferably an isotope internal standard;

the solvent of the internal standard solution is selected from methanol and acetonitrile, preferably methanol;

after the mixed extract of the dutasteride and the internal standard is frozen and dried, re-dissolving the mixed extract by using a re-solvent;

the double solvent is selected from acetonitrile water solution, methanol water solution, acetonitrile formic acid water solution, acetonitrile acetic acid water solution, methanol formic acid water solution, methanol acetic acid water solution, methanol acetonitrile formic acid water solution, methanol acetonitrile acetic acid water solution, preferably acetonitrile water solution

Wherein the aqueous acetonitrile solution is selected from the group consisting of 20 to 80% (vol%) aqueous acetonitrile solution, preferably 40 to 60% (vol%) aqueous acetonitrile solution.

Drawings

FIG. 1 shows that in the state of flow path 1, liquid phase "2" is communicated with a Mass Spectrometer (MS) through an analytical column, and liquid phase "1" is communicated with a pre-column;

FIG. 2 shows that in the state of flow path 2, the liquid phase "2" is communicated with the analytical column and the Mass Spectrometer (MS) through the pre-column;

FIG. 3 shows samples of LLOQ concentration levels;

FIG. 4 shows a sample at a low concentration level;

FIG. 5 shows samples at medium concentration levels;

figure 6 shows the sample at a high concentration level.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail. The experimental methods of the preferred embodiments, which do not indicate specific conditions, are generally performed according to conventional conditions, and the examples are given for better illustration of the present invention, but the present invention is not limited to the examples. Those skilled in the art can make insubstantial modifications and adaptations to the embodiments described above while remaining within the scope of the invention.