CN112198265A - Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample - Google Patents
Pretreatment method, detection method and kit for simultaneously detecting multiple steroid hormones in blood sample Download PDFInfo
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Abstract
The invention relates to a pretreatment method, a detection method and a kit for simultaneously detecting multiple steroid hormones in a blood sample. The pretreatment method comprises 1) using methyl tert-butyl ether to primarily extract the steroid hormone in the serum sample; 2) centrifuging, and taking supernatant to be sufficiently washed by alkali with alkaline solution; 3) and (3) centrifuging after alkaline washing, taking the supernatant, freezing and drying the supernatant, and redissolving the obtained solid components to obtain the solution to be detected for simultaneously detecting the multiple steroid hormones. The method can at least simultaneously separate the five steroid hormones of 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticosterol and 11-deoxycorticosterol, and has good separation effect and simple operation.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a pretreatment method, a detection method and a kit for simultaneously detecting multiple steroid hormones in a blood sample.
Background
Congenital adrenal cortical hyperplasia (CAH) is a group of diseases caused by enzyme deficiency in the adrenal cortical hormone synthesis pathway, and belongs to autosomal recessive genetic diseases. Enzyme deficiencies include 21-hydroxylase, 11 β -hydroxylase, 3-steroid dehydrogenase, 17-hydroxylase deficiency, etc., with 21-hydroxylase deficiency (21-OHD) being the most common type of CAH (90% to 95%), most of which is due to mutations in the CYP21a2 gene encoding 21-hydroxylase. Secondly, 11 beta-hydroxylase deficiency (11 beta-OHD), accounts for 5% -8%, and others are very rare.
CAH mainly affects cortisol and aldosterone production, and cortisol affects sex hormone secretion, causing virilization in children. Aldosterone acts by reabsorbing sodium through the kidneys, maintaining water and sodium balance. About 75% of CAH children suffer from salt loss, growth retardation, hypovolemic shock and the like due to aldosterone deficiency, and hormone imbalance can also cause wrong sex judgment of male women and the problems of hirsutism, sterility and the like of adult women, so the timely diagnosis, discovery and treatment of CAH are very important.
For the secondary screening or differential diagnosis of common CAH (21-OHD and 11 beta-OHD), it is suggested to simultaneously determine multiple steroid hormones such as 17-hydroxyprogesterone (17-OHP), androstenedione (A4), cortisol (cortisol), 11-deoxycorticol (11-DF), and 21-deoxycorticol (21-DF). Especially, the simultaneous determination of 21-deoxycorticosterol is very necessary and significant in the differential diagnosis of difficult 21-hydroxylase deficiency patients, 21-deoxycorticosterol is an intermediate product in a glucocorticoid synthesis pathway, and under normal conditions, 17-hydroxyprogesterone, a main substrate of glucocorticoid synthesis, is hydroxylated by 21 hydroxylase to generate 11-deoxycorticosterol, and finally, cortisol is generated; however, a very small fraction of 17-hydroxyprogesterone is converted to 21-deoxycorticosterol by 11 β -hydroxylase. In CYP21A2 heterozygous patients, no clinical symptoms are routinely apparent due to the only diminished or partial loss of 21-hydroxylase activity. The prenatal genetic counseling aiming at the part of patients is difficult to find through clinical manifestations or counseling, a large amount of 17-hydroxyprogesterone cannot be metabolized by 21-hydroxylase (with weakened or partially-lost enzyme activity) in a short time through a challenge test, 11 beta-hydroxyprogesterone is normal, 21-deoxycorticosterol is accumulated (increased), and if only 17-hydroxyprogesterone, androstenedione, cortisol and 11-deoxycorticosterol are easily missed, the normal population and CYP21A2 heterozygote patients are difficult to accurately identify; if 21-deoxycorticosterol, 17-hydroxyprogesterone, androstenedione, cortisol and 11-deoxycorticosterol can be measured simultaneously, CYP21A2 heterozygote patients and normal population can be distinguished almost in hundred percent, and differential diagnosis can be carried out on difficult 21-hydroxylase deficiency patients.
Steroid hormones mainly exist in a protein-bound form, and human blood contains a great number of high-affinity binding proteins, the components are complex, the content difference of different hormones is large, and more structural analogues and isomers exist, so that the steroid hormones are easy to interfere with each other. To realize accurate quantification, high requirements on sensitivity and accuracy are required, and a proper sample pretreatment mode needs to be selected to enrich a target substance, so that the sensitivity is improved, and simultaneously matrix interference is eliminated or reduced. At present, the steroid hormone in blood is mostly determined by an immunological method in China, and a large number of documents prove that the immunological method is often interfered by a structural analogue and a substrate when the steroid hormone is determined, so that the problems of inaccurate determination result, high quantitative limit, narrow linear range and the like are caused. Meanwhile, each immunization method can only measure one steroid hormone, and the detection time is long.
At present, tandem mass spectrometry is adopted to measure multiple steroid hormones, and the following sample pretreatment methods are generally adopted to process blood samples: firstly, the liquid-liquid extraction is carried out on the sample after the protein precipitation, in order to improve the extraction efficiency, a liquid transfer device is needed to transfer the upper-layer liquid after the extraction as much as possible, so that the transfer operation difficulty is increased, the extraction loss is caused, and the requirement on personnel is higher; secondly, the sample is subjected to derivatization after protein precipitation and then liquid-liquid extraction, so that although the sensitivity of the object to be detected can be improved, the operation is complicated, the requirement on personnel is high, and the derivatization experiment needs to be carried out, so that the time consumption is long; thirdly, the sample is subjected to protein precipitation and then is subjected to a solid phase extraction method, the pretreatment method has high requirements on personnel, not only are multiple operation steps required, but also the investment is large (consumables such as a solid phase extraction column and the like are expensive).
Because the content difference of the same or different steroid hormones in different human bodies is large, the detection method is required to have a wide linear range, and the content of certain compounds (such as 21-deoxycorticol) in the human bodies is extremely low, so that the sensitivity requirement is higher for accurately distinguishing the measured values of normal personnel and abnormal personnel. If the content of compounds (such as cortisol) is extremely low and the content of compounds (such as cortisol) is high, the ionization efficiency is different, and the detector is saturated (the content of the compounds is high), so that the method for measuring the compounds with high content needs to be adjusted and optimized for various mass spectrum parameters. In order to realize accurate quantification and avoid the influence of fragment ion channels or co-outflow and the like existing in structural analogs or isomers, chromatographic separation needs to be realized, and interference is avoided as much as possible.
Since steroid hormones are used as endogenous hormones and are distributed in human bodies or animal bodies to a certain extent, if human blood or animal blood is selected as a substitute substrate, the blood can be used after complex, tedious and time-consuming pretreatment is carried out to remove background, and meanwhile, a commercialized blood substrate is difficult to obtain and has higher cost.
Disclosure of Invention
The invention relates to a pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample, wherein the multiple steroid hormones comprise 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticol and 11-deoxycorticol, and the pretreatment method comprises the following steps:
1) primarily extracting the steroid hormone in the blood based sample using methyl tert-butyl ether;
2) centrifuging, and taking supernatant to be sufficiently washed by alkali with alkaline solution;
3) and (3) centrifuging after alkaline washing, taking supernatant, freezing and drying, and redissolving the obtained solid component to obtain the solution to be detected for simultaneously detecting the multiple steroid hormones.
In addition, the invention also provides a method for simultaneously detecting multiple steroid hormones in a blood sample, which is characterized by comprising the following steps:
treating according to the pretreatment method to obtain a solution to be detected;
introducing the solution to be detected into a high performance liquid chromatography, and performing gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is a methanol solution containing 0.1-0.5 v/v% of formic acid, and the mobile phase B is an aqueous solution containing 0.1-0.5 v/v% of formic acid;
and detecting the components separated by the high performance liquid chromatography by combining with a mass spectrum, and calculating the content of each steroid hormone by adopting a calibration curve quantitatively established by an isotope internal standard method.
According to the invention, through a sample pretreatment method and a chromatographic method which are reasonably optimized, five steroid hormones can be simultaneously separated from serum, the separation purity is high, and the separation method is simple and easy to operate.
Further, the present invention relates to a kit for detecting steroid hormones in a blood sample, comprising:
steroid hormone internal standards and standards, aqueous solutions containing 30 v/v% to 45 v/v% methanol, methyl tertiary butyl ether, alkaline solutions as described above, methanol solutions containing 0.1 v/v% to 0.5 v/v% formic acid, and aqueous solutions containing 0.1 v/v% to 0.5 v/v% formic acid;
wherein the steroid hormones are 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticosterol, and 11-deoxycorticosterol.
Compared with the prior art, the invention has the beneficial effects that:
(1) the separation method provided by the invention adopts a sample pretreatment purification method which is simple to operate and low in price, can effectively remove the interference of acidic impurities in the blood sample on the measurement of the object to be measured, does not need complicated operations such as derivatization, pipettor transfer, solid phase extraction and enrichment and the like, has low requirement on the operation of personnel, high efficiency and low cost of used reagents and consumables, and is suitable for pretreatment of different types of blood samples (such as dried blood spots, serum, plasma and the like).
(2) Due to the fact that the sample extraction efficiency is high, the sampling amount of the sample is small, the sampling amount can be saved by more than 50% compared with the sampling amount of the existing method, the sample is subjected to an optimized pretreatment step, the chromatographic sampling amount is small, equipment can be protected to the maximum degree, the maintenance frequency of the equipment can be reduced, and the service life of a chromatographic column can be shortened.
(3) Through the pretreatment step and the selection and optimization of chromatographic mass spectrometry conditions, the detection method provided by the invention can realize the chromatographic mass spectrometry separation of more than ten steroid hormones, has strong specificity, and can accurately quantify 17OHP, A4, corrisol, 21-DF and 11-DF.
(4) The method of the invention can not only avoid the condition of certain compounds responding saturation, but also improve the response of low-concentration compounds by optimizing the mass spectrum conditions of multiple steroid hormones, can provide extremely wide dynamic linear range, high sensitivity (lower limit of quantitation), and simultaneously gives consideration to accurate quantitation of multiple hormones with different concentrations.
(5) The method provides an alternative pure solvent blank matrix, has negligible effect on matrix effect, is more easily obtained compared with human blood matrix, is convenient to operate and low in cost, and can save at least 48 hours of matrix pretreatment time.
(6) The kit for detecting the steroid hormone in the blood sample can be used for quickly diagnosing the congenital adrenal cortical hyperplasia (CAH).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIGS. 1 to 5 are diagrams sequentially showing a background measurement spectrum of a Corisol of a BSA aqueous solution of VWF, sigma, caision and Vetec and 35% methanol water in one embodiment of the present invention;
FIGS. 6 to 10 are diagrams sequentially showing a background measurement spectrum of 17-OHP of BSA aqueous solutions of VWF, sigma, caision and Vetec and 35% methanol water in one embodiment of the invention;
FIGS. 11 to 15 are background measurement maps of A4 background of a BSA aqueous solution of VWF, sigma, caision and Vetec and 35% methanol water in sequence according to an embodiment of the present invention;
FIGS. 16 to 20 are diagrams sequentially showing background measurement spectra of 21-DF of BSA aqueous solutions of VWF, sigma, caision and Vetec and 35% methanol water according to an embodiment of the present invention;
FIGS. 21 to 25 are diagrams illustrating background measurement of 11-DF of BSA aqueous solutions of VWF, sigma, caision and Vetec and 35% methanol water according to an embodiment of the present invention;
FIG. 26 is a graph of A4 measurements taken before a sample is subjected to caustic washing in accordance with an embodiment of the present invention;
FIG. 27 is a measurement map of A4 of the same specimen as in FIG. 26 after alkali washing;
FIG. 28 is a 17-OHP profile of a sample prior to caustic wash in accordance with an embodiment of the present invention;
FIG. 29 is a 17-OHP assay profile of the same sample as in FIG. 28 after alkaline washing;
FIG. 30 is a 21-DF chromatogram from an XB-C18 column separation in accordance with an embodiment of the invention;
FIG. 31 is a 21-DF chromatogram from a C18 column according to an embodiment of the present invention;
FIG. 32 is a total ion flow diagram of an XB-C18 column in one embodiment of the present invention;
FIG. 33 is a total ion flow diagram of a C18 column in accordance with an embodiment of the present invention;
FIG. 34 is a total ion flow graph of multiple steroid detection after addition of multiple interfering species in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample, wherein the multiple steroid hormones comprise 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticol and 11-deoxycorticol, and the pretreatment method comprises the following steps:
1) primarily extracting the steroid hormone in the blood based sample using methyl tert-butyl ether;
2) centrifuging, and taking supernatant to be sufficiently washed by alkali with alkaline solution;
3) and (3) centrifuging after alkaline washing, taking supernatant, freezing and drying, and redissolving the obtained solid component to obtain the solution to be detected for simultaneously detecting the multiple steroid hormones.
The preprocessing method provided by the invention ensures the detection precision in the subsequent detection. The advantages of alkaline washing are various, one can remove acidic impurity interferent, avoid affecting the quantification, the second improves the sample cleanness, avoids the interference of complex matrix in blood samples, increases the signal-to-noise ratio, can detect samples with lower concentration, and the third can protect chromatographic columns and detection equipment.
In the present invention, the blood sample may be dried blood spot serum or plasma.
In some embodiments, the volume ratio of the blood sample to the methyl tert-butyl ether is 1 (8-12), and may be 1:9, 1:10 or 1: 11.
In some embodiments, the basic solution in step 2) is an aqueous ammonia solution;
furthermore, the concentration of the ammonia water solution is 0.1 wt% -0.5 wt%, and 0.2 wt%, 0.3 wt%, 0.4 wt% can be selected;
further, the volume ratio of the supernatant to the ammonia water solution is (2.5-4.5): 1, 3:1, 3.5:1 and 4:1 can be selected.
In some embodiments, before the operation of sufficiently alkaline-washing the supernatant with the alkaline solution, the method further comprises freezing the supernatant at-50 ℃ to-80 ℃ for 30min to 60 min;
in some embodiments, in the operation of freeze drying the supernatant, the supernatant is frozen at-50 ℃ to-80 ℃ for 30min to 60min, and then is dried by nitrogen;
freezing at 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C or 75 deg.C; the freezing time can be 35min, 40min, 45min, 50min or 55 min.
In some embodiments, the centrifugation is at 10000rpm to 13000rpm for 3min to 7 min.
In some embodiments, the centrifugation is at 10000rpm for 5 min.
In some embodiments, the volume of reconstitution solution used to reconstitute the resulting solid component is 80 μ L to 120 μ L.
In addition, the invention also relates to a method for simultaneously detecting multiple steroid hormones in a blood sample, which comprises the following steps:
treating the solution to be detected according to the pretreatment method to obtain a solution to be detected;
introducing the solution to be detected into a high performance liquid chromatography, and performing gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is a methanol solution containing 0.1-0.5 v/v% of formic acid, and the mobile phase B is an aqueous solution containing 0.1-0.5 v/v% of formic acid;
and detecting the components separated by the high performance liquid chromatography by combining with a mass spectrum, and calculating the content of each steroid hormone by adopting a calibration curve quantitatively established by an isotope internal standard method.
In some embodiments, the isotopic internal standard used in the isotopic internal standard method is further preferably D8-17OHP,13C3-A4、D4-cortisol、13C3-11-DF and D8-21-DF.
In some embodiments, the mobile phase A may also be selected from a methanol solution containing 0.2 v/v%, 0.3 v/v%, or 0.4 v/v% formic acid.
In some embodiments, the mobile phase B may also be selected from an aqueous solution containing 0.2 v/v%, 0.3 v/v%, or 0.4 v/v% formic acid.
In some embodiments, the packing material of the reverse phase column for high performance liquid chromatography is octadecyl, preferably Phenomenex Kinetex-C18 or Phenomenex Kinetex-XB-C18 column, and more preferably Phenomenex Kinetex-C18.
In some embodiments, the reverse phase column has a column temperature of 30 ℃ to 60 ℃, and may also be selected from 35 ℃, 40 ℃, 45 ℃, 50 ℃ or 55 ℃.
In some embodiments, the amount of sample is 15. mu.L to 25. mu.L, preferably 20. mu.L, when performing high performance liquid chromatography.
In some embodiments, the parameters of the gradient elution are:
0.01-7.00 min, mobile phase A: 40% -65%; 7.00-7.50 min, mobile phase A: 65 percent; 7.50 min-8.5 min, mobile phase A: 65 to 100 percent; 8.50-8.60 min, mobile phase A: 100% -40%; 8.60-9.00 min, mobile phase A: 100 percent;
wherein the above conditions relate to the time of analysis, the floating range of the respective numerical parameter of the volume percentages of mobile phases A and B being ± 20%, or ± 15%, or ± 10%, or ± 5% of the numerical parameter.
In some embodiments, the gradient elutes at a flow rate of 0.3mL/min to 0.5mL/min, preferably 0.4 mL/min.
In some embodiments, the blood based sample is present in an amount of 80 μ L to 120 μ L, preferably 100 μ L; the sample size of the high performance liquid chromatography is 15-25 mu L, preferably 20 mu L.
In some embodiments, the blank matrix used in the isotope internal standard method is 30 v/v% to 45 v/v% methanol aqueous solution, and 35 v/v% or 40 v/v% methanol aqueous solution can also be used.
The choice of a blank matrix is also important in the establishment of mass spectrometry, and in principle it is preferable to select a matrix homologous to the sample as the blank matrix, but when the homologous matrix is difficult to obtain, a matrix similar thereto may be selected instead. At present, animals (such as bovine serum) are generally selected as a replacement matrix of human serum by using tandem mass spectrometry for detecting steroid hormone, but because the steroid hormone is an endogenous substance, the animal serum often contains partial hormone background residues, so that the dissolved bovine serum needs to be pretreated before use, an activated carbon adsorption method is generally adopted to ensure that the background is clean, the operation of activated carbon adsorption is very complicated and long in time consumption, and at least more than 48 hours are needed for treating a batch of animal serum with clean hormone background. The detection method provided by the invention uses 30 v/v% -45 v/v% methanol aqueous solution to replace the commonly used bovine serum albumin substrate, does not need to be subjected to complicated pretreatment, is simple and easy to obtain, and has a substrate effect meeting the requirement of a blank substrate.
In some embodiments, the method of detecting steroid hormones in a blood based sample does not include a step of activated carbon adsorption.
In some embodiments, the mass spectrum is quadrupole mass spectrometry, provided that it is positive ion mode, and the scanning mode is multiple reaction monitoring ion scanning MRM.
In some embodiments, the collision energies of the mass spectrum for different detection objects are respectively:
17-hydroxyprogesterone: 20V to 24V; androstenedione: 28V-32V; cortisol: 18V to 22V; 21-deoxycorticosterol: 35V to 45V; 11-deoxycorticosterol: 33V to 37V.
By optimizing mass spectrum conditions, the method solves the problems that different steroid hormones have large content difference in human bodies and high-concentration samples are easy to saturate on mass spectra, can realize accurate quantification of the hormones with different human body contents and different concentrations, and has stable project performance.
The method has high extraction efficiency and high sample cleanliness in the pretreatment process, only needs 80-120 mu L of sampling amount and 15-25 mu L of sample amount to measure the multiple steroid hormones, has wide linear range and low quantitative limit, and can adjust the sampling amount and the sample amount according to the clinical application requirement, further expand the linear range and reduce the quantitative limit.
The detection method provided by the invention has excellent performance, and the precision can reach less than 5%. The method can realize the detection of five steroid hormones, namely 17-OHP, A4, Cortisol, 21-DF and 11-DF, can also realize the detection by adding other steroid hormones with similar structures (testosterone, dihydrotestosterone, dehydroepiandrosterone, progesterone, cortisone and corticosterone), and can combine a plurality of detection substances according to the application range.
Further, the present invention relates to a kit for detecting steroid hormones in a blood sample, comprising:
steroid hormone internal standards and standards, aqueous solutions containing 30 v/v% to 45 v/v% methanol, methyl tert-butyl ether, alkaline solutions as described above, methanol solutions containing 0.1 v/v% to 0.5 v/v% formic acid, and aqueous solutions containing 0.1 v/v% to 0.5 v/v% formic acid;
wherein the steroid hormones are 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticosterol, and 11-deoxycorticosterol.
Embodiments of the present invention will be described in detail with reference to examples.
Example 1
The present example provides a method for assaying multiple steroids comprising the steps of:
A. configuring a standard curve:
blank matrix: mixing methanol and water in different proportions to prepare a 35% methanol water solution, fully mixing, and ultrasonically removing bubbles to obtain the product.
High-concentration samples with 17-OHP of 271nmol/L, A4 of 41.7nmol/L, corrisol concentration of 600nmol/L, 21-DF of 38.5nmol/L and 11-DF of 27.1nmol/L are prepared respectively by using 35% methanol aqueous solution as a blank matrix, and are diluted one by one in a double way to obtain curve points with different concentrations.
B. Sample pretreatment method
1) Sucking 100 mu L of sample into a 2.0mL centrifuge tube, adding 50 mu L of internal standard, adding 800 mu L-1200 mu L of extracting solution methyl tert-butyl ether (MTBE), and violently shaking for 5min by vortex;
2) centrifuging at 10000rpm at normal temperature for 5 min;
4) freezing at-70 deg.C for 60min, transferring the upper layer into 2.0mL centrifuge tube, adding 350 μ L0.1 wt% ammonia water, and vortexing vigorously for 5 min;
5) centrifuging at 10000rpm at normal temperature for 5 min;
6) freezing at-70 deg.C for 50min, transferring the upper layer into a 1.5mL centrifuge tube, and blowing with nitrogen gas;
7) adding 100 μ L of the complex solution, vortexing vigorously for 5min, centrifuging at 10000rpm at normal temperature for 5min, and transferring to a 1.5mL sampling bottle;
8) the assay was performed using LC-MS/MS.
C. Liquid phase process
TABLE 1 liquid phase gradient
Mobile phase a ═ 0.3% formic acid, methanol (V/V)
Mobile phase B ═ 0.3% formic acid, water (V/V)
A chromatographic column: phenomenex Kinetex-C18, 2.6 μm, 100X 2.1mm
Column temperature: 45 deg.C
Sample introduction amount: 20 μ L
Flow rate: 0.4mL/min
In the invention, the mobile phase A is 0.1 percent formic acid, methanol (V/V)
In the invention, the mobile phase A is 0.1 percent of formic acid and water (V/V)
D. Conditions of Mass Spectrometry
TABLE 2 Mass Spectrometry parameters
Wherein DP is the declustering voltage, EP is the entrance voltage, CE is the collision voltage
Atomizing gas (source gas 1): 35.0 psi;
auxiliary heating gas (source gas 2): 50.0 psi;
ion source temperature (source temp): at 450 ℃;
air curtain gas (curl gas): 50.0 psi;
collision gas (collision gas): 9 psi;
ionization voltage IS: 4500V.
The detection results are shown in FIG. 33.
The detection method is verified as follows:
(1) linear range and quantitative limit: respectively preparing high-concentration samples with 17OHP of 271nmol/L, A4 of 41.7nmol/L, corrisol concentration of 601nmol/L, 11-DF of 27.1nmol/L and 21-DF of 38.5nmol/L by using 35% methanol aqueous solution as a blank matrix, and carrying out one-to-two dilution to obtain curve points with different concentrations; parallelly treating 2 samples with each concentration every day, measuring for 3 days, and respectively detecting once; and (3) quantifying by using an internal standard curve method, wherein the ratio of the peak area of each hormone to the area of the corresponding internal standard compound is a vertical coordinate, the ratio of the concentration of each hormone to the concentration of the internal standard compound is a horizontal coordinate, the weight is 1/x, and a standard curve is drawn. The results show that: the detection Limits (LOD) of the 17-OHP, A4, Cortisol, 21-DF and 11-DF methods were 0.016nmol/L, 0.010nmol/L, 0.073nmol/L, 0.009nmol/L, 0.003nmol/L, respectively; the limit of quantitation (LOQ) of 17OHP, A4, Cortisol, 11-DF, 21-DF were 0.033nmol/L, 0.020nmol/L, 0.147nmol/L, 0.027nmol/L, 0.038nmol/L, respectively; 17OHP, A4, Cortisol, 11-DF and 21-DF are respectively in the concentration ranges of 0.033-271nmol/L, 0.020-41.7nmol/L, 0.147-601nmol/L, 0.027-27.1nmol/L and 0.038-38.5nmol/L, the linearity is good, and the correlation coefficient R is2>0.99。
(2) Precision: samples of the pooled human serum were collected and subjected to labeling to obtain samples at three different concentration levels, low (L), medium (M) and high (H), with 12 sets of data measured in a short time and 4 sets of data measured in a continuous 5-day batch-to-batch precision using the method of example 1, 20 sets of data were collected and their mean values, SD and RSD were calculated as shown in tables 3-6.
(3) Accuracy: serum samples with low, medium and high concentration levels are taken, the accuracy of the method is verified through a standard recovery rate experiment, the result recovery rates are all between 85% and 115%, and the accuracy of the method meets the requirement, as shown in table 7.
TABLE 3 in-batch precision
TABLE 4 in-batch precision
TABLE 5 precision between batches
TABLE 6 precision between batches
TABLE 7 recovery with addition of standard
By integrating the verification experiments, the linear range, the quantitative limit, the precision, the accuracy and other indexes of the embodiment can meet the requirements of clinical sample detection, and in the method, the 17-OHP batch precision is 1.0-3.5%, and the batch precision is 2.0-3.2%; a4, the precision in batch is 1.0-4.0%, and the precision between batches is 2.0-3.0%; the intrabatch precision of the cortisol is 1.0-3.0%, and the batch precision is 2.0-4.0%; the internal precision of 11-DF is 2.0% -4.0%, and the inter-batch precision is 2.0% -5.5%; the 21-DF precision is 2.0% -9.0% and the batch-to-batch precision is 3.0% -5.0%, and the batch precision and the batch-to-batch precision of each hormone index show very excellent performance. At present, in many methods for measuring multiple steroid hormones in serum, generally, in order to save detection time, only mass spectrum separation (different ion channels are selected) is implemented, and chromatographic separation is not completely implemented, so that the reproducibility of the established method is poor (the precision is between 5% and 15%), and particularly, the precision can be up to 10% to 20% for the measurement of samples with low concentration levels. In the embodiment, the sample is purified by a better pretreatment method, and besides the selection of the mass spectrum ion pair, the method can realize the mass spectrum separation of the substance to be detected and the interference substance with similar structure and the isomer interference substance, and also realizes the complete separation of each compound to be detected in the liquid phase by the selection of the chromatographic column and the optimization of the liquid phase condition, thereby avoiding the influence of co-outflow impurities on the quantification.
Example 2
Because the content difference of different steroid hormones in serum is large, the content of partial steroid hormones is very low, such as 11-deoxycorticosterol, in order to improve the detection sensitivity of an object to be detected, a mode of increasing the sampling quantity or the sampling quantity can be used, the prior method generally adopts the sampling quantity of at least more than 200 mu L and the sampling quantity of 20 mu L-50 mu L, and the method has the advantages of high extraction efficiency and high sample cleanliness degree in the pretreatment process, only 100 mu L of sampling quantity and 20 mu L of sampling quantity are needed for measuring 5 kinds of steroid hormones, wide linear range and low quantitative limit, and the sampling quantity can be adjusted according to the clinical application requirements, so that the linear range is further expanded, the quantitative limit is reduced, and the detection sensitivity is improved.
Example 3
In order to accurately measure low-content hormone (such as 21-deoxycorticol), a high-sensitivity instrument is required, but the problem of detector saturation exists when measuring high-content hormone at the same time, and the measurement concentration range of a high-concentration sample is influenced. The method adopts an AB SCIEX API 5500 triple quadrupole mass spectrometer, enables the response of hormone items with different human body contents on the mass spectrum to be similar through optimizing mass spectrum parameter collision energy, gives consideration to the measurement of high and low concentration samples, and simultaneously, as the collision energy is a voltage parameter which is applied to a collision chamber and enables components of an object to be measured to be cracked in the mass spectrum, the impact on the performance of the items is large, if the adjustment amplitude is too large, the signals of the items in the detection process are possibly unstable, so whether the optimized amplitude is appropriate or not needs to be evaluated.
Through optimization of mass spectrum parameters, the response of the hormone index linear high points with different human body contents on the mass spectrum basically approaches to the same order of magnitude, accurate quantification of high and low concentration levels of hormone items with different concentrations of a human body can be met, and a wide dynamic linear range is provided (as shown in a linear range verification result in embodiment 1). Meanwhile, by monitoring the internal standard of the sample, the project performance after the parameter optimization is stable and the reproducibility is good.
TABLE 8 comparison before and after optimization of Mass Spectrometry parameters
TABLE 9 post-optimization of Mass Spectrometry parameters index stability monitoring
Example 4
Selection of blank matrix
a. Weighing 400mg of 4 bovine serum albumin of different brands respectively, using deionized water to fix the volume to 10mL, and fully dissolving to obtain bovine serum albumin matrix;
b. taking the prepared 4 bovine serum albumin matrixes of different brands and 35% methanol aqueous solution respectively, pretreating the sample according to the method of the embodiment 1, and then testing on a machine;
the Cortisol background determination maps of VWF, sigma, caision, BSA aqueous solution of Vetec and 35% methanol water are shown in the figures 1-5 in sequence; the 17-OHP background determination spectra of the BSA aqueous solution of VWF, sigma, caision and Vetec and the 17-OHP background determination spectra of 35% methanol water are sequentially shown in FIGS. 6-10; a4 background determination spectra of a BSA aqueous solution of VWF, sigma, caision and Vetec and an A4 background determination spectrum of 35% methanol water are sequentially shown in FIGS. 11-15; the 21-DF background determination spectra of the BSA aqueous solution of VWF, sigma, caision and Vetec and the 21-DF background determination spectra of 35% methanol water are sequentially shown in figures 16-20; the 11-DF background determination maps of the BSA aqueous solution of VWF, sigma, caision and Vetec and the 11-DF background determination map of 35% methanol water are sequentially shown in figures 21-25.
From the chromatogram, the cortisol was detected in different degrees in 4 different brands of bovine serum albumin matrix samples without pretreatment (activated carbon adsorption), while the other hormones 17-OHP, a4, 11-deoxycorticosterol and 21-deoxycorticosterol were partially detected, which indicated that the steroid hormone was an endogenous substance and was present in a certain amount in the animal body, and the commercial bovine serum albumin was subjected to pretreatment when the serum was used as the serum substitute for the substrate in the hormone item, whereas the method provided pure solvent blank substrate (35% methanol water), had low background noise and no background detection, and could be prepared without complicated pretreatment, and the preparation method was simple and easy, and the influence of matrix effect was negligible (as shown in the matrix effect evaluation result of example 5).
As bovine serum albumin as a substitute matrix needs to be pretreated, the background can be reduced or eliminated by adopting an activated carbon adsorption mode, the operation is complicated, at least 48 hours are consumed for preparing a batch of available substitute matrices, the pure solvent matrix provided by the method (the solvent components and the proportion can be adjusted according to the situation) can be prepared just in 10 minutes, the operation steps are simple and easy to implement, and the curve prepared by the method can be stable for at least more than half a year through preliminary verification.
TABLE 10 bovine serum albumin and pure solvent matrix configuration procedure and time consuming
Example 5
Matrix effect assessment
Collecting patient samples from six different sources;
b. using a blank matrix (35% methanol-water) for preparing a standard curve to dissolve at least two standard solutions with different concentration levels;
c. respectively mixing the standard solution and the patient matrix samples from 6 different sources according to the proportion of 1:1 and 3:2 to obtain mixed samples;
d. the standard solution, the patient sample and the mixed sample are processed in parallel by 3 samples respectively and are tested on a machine.
The method comprises the following steps: the response value of the mixed sample (analyte/internal standard) should be less than 20% different from the mean response value of the patient sample and the standard solution, indicating no relative matrix effect.
TABLE 11 response values of pre-mix samples (analyte/internal standard)
TABLE 12 sample matrix Effect data (1)
TABLE 13 sample matrix Effect data (2)
From the results, the difference between the response value (analyte/internal standard) of the mixed sample and the response mean value of the patient sample and the standard solution is less than 20%, which shows that the matrix (25-45% methanol aqueous solution) selected by the invention can be used as a human serum substitute matrix for detecting steroid hormone projects, the matrix effect is negligible, the acquisition is simple and easy to obtain, the pretreatment time of at least 48 hours can be saved, the operation requirement of personnel is low, and the superposition effect on curve background caused by incomplete pretreatment when human or animal serum (hormone is removed) is selected as the substitute matrix can be avoided, so that the accurate quantification of the sample is influenced.
Example 6
Randomly selecting 20 samples, and parallelly taking each sample for 2 times, wherein one group is subjected to sample treatment according to the pretreatment method in the embodiment 1 of the invention, the other group is not subjected to the alkali washing step in the invention, and the others are consistent with the pretreatment method in the invention; the results are shown in FIGS. 26 to 29.
The two groups of data are compared in a peak mode, so that the fact that the matrix in part of patient samples is complex, if alkali washing is not carried out for impurity removal, impurity interference can occur near the peak of an object to be detected, the phenomenon of baseline elevation is caused, the accuracy and the stability of sample quantification are influenced, one of the samples subjected to alkali washing can remove acidic impurity interferents, the influence on quantification is avoided, the second step is that the cleanliness degree of the sample is improved, the baseline is reduced, the signal-to-noise ratio is increased, the sample with lower concentration can be detected, and the third step also has a protection effect on a chromatographic column and detection equipment.
Example 7
In order to verify the influence of different C18 chromatographic columns on the separation effect, the method uses two different Phenomenex Kinetex-C18(2.6 mu m, 100 multiplied by 2.1mm) and Phenomenex Kinetex-XB-C18(2.6 mu m, 100 multiplied by 2.1mm) chromatographic columns for sample analysis, and FIG. 30 is an XB-C18 column separation 21-DF chromatogram, which shows that the 21-DF and the interferent with similar structure do not completely realize chromatographic separation and have influence on the measurement result; FIG. 31 is a chromatogram of column separation 21-DF of C18, which can realize the chromatographic separation of 21-DF and structurally similar interferents, and Phenomenex Kinetex-C18(2.6 μm, 100X 2.1mm) has better retention and higher column efficiency on target compounds, can obviously improve the detection sensitivity of the target compounds, and at the same time, the retention time is obviously advanced, and the detection time can be saved by 0.5 min. The total ion flow diagrams of the XB-C18 column and the C18 column are shown in FIG. 32 and FIG. 33, respectively.
The result shows that Phenomenex Kinetex-C18(2.6 mu m, 100 multiplied by 2.1mm) has better separation effect on various steroids and structural analogues thereof, has shorter analysis time, and is beneficial to improving the specificity and sensitivity of detection.
Example 8
a. Taking a serum sample of a mixed patient, respectively adding interferents such as testosterone (TT), Dihydrotestosterone (DHT), Dehydroepiandrosterone (DHEA), Progesterone (Progesterone), cortisone (cortisone), corticosterone (corticosterone) and the like, and observing whether the structural analogue and the isomer interferent can interfere with a to-be-detected object or not, and whether chromatographic separation can be realized or not.
b. From the chromatogram (fig. 34), it is clear that the 5 steroid hormones detected by the method can be separated by chromatography.
In the current methods for measuring multiple steroid hormones, mass spectrometry is generally only performed for saving analysis time, but a common ion channel exists for isomer compounds (such as 11-deoxycorticosterol, 21-deoxycorticosterol and corticosterone), and if chromatographic separation is not performed, co-outflow can cause false increase of results. After the selection of a chromatographic column, the optimization of liquid phase gradient and the selection of mass spectrum ion pairs are carried out, and a plurality of analogues or isomer interferents are added, not only can various hormones to be detected realize chromatographic separation, but also can the added interferents of testosterone, dihydrotestosterone, dehydroepiandrosterone, progesterone, cortisone, corticosterone and other steroid hormones realize chromatographic separation.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (12)
1. A pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample, wherein the multiple steroid hormones include 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticol, and 11-deoxycorticol, the pretreatment method comprising the steps of:
1) primarily extracting the steroid hormone in the blood based sample using methyl tert-butyl ether;
2) centrifuging, and taking supernatant to be sufficiently washed by alkali with alkaline solution;
3) and (3) centrifuging after alkaline washing, taking supernatant, freezing and drying, and redissolving the obtained solid component to obtain the solution to be detected for simultaneously detecting the multiple steroid hormones.
2. The pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 1, wherein the volume ratio of the blood sample to the methyl tert-butyl ether is 1 (8-12).
3. The method for pretreating a blood sample for simultaneously detecting multiple steroid hormones as claimed in claim 1, wherein the alkaline solution in step 2) is an aqueous ammonia solution, the concentration of the aqueous ammonia solution is 0.1 wt% to 0.5 wt%, and the volume ratio of the supernatant to the aqueous ammonia solution is (2.5 to 4.5): 1.
4. the pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 1, wherein said operation of taking the supernatant with an alkaline solution for alkaline washing is further comprised of freezing the supernatant at-50 ℃ to-80 ℃ for 30min to 60 min.
5. The pretreatment method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 1, wherein in the operation of freeze drying the supernatant, the supernatant is frozen at-50 ℃ to-80 ℃ for 30min to 60min, and then is dried by nitrogen.
6. A method for simultaneously detecting multiple steroid hormones in a blood sample, comprising the steps of:
treating the mixture according to the pretreatment method of any one of claims 1 to 5 to obtain a solution to be detected;
introducing the solution to be detected into a high performance liquid chromatography, and performing gradient elution by using a mobile phase A and a mobile phase B, wherein the mobile phase A is a methanol solution containing 0.1-0.5 v/v% of formic acid, and the mobile phase B is an aqueous solution containing 0.1-0.5 v/v% of formic acid;
and detecting the components separated by the high performance liquid chromatography by combining with a mass spectrum, and calculating the content of each steroid hormone by adopting a calibration curve quantitatively established by an isotope internal standard method.
7. The method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 6, wherein the packing material of the reverse phase column for high performance liquid chromatography is octadecyl.
8. The method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 6, wherein the parameters of the gradient elution with mobile phase A and mobile phase B are as follows: 0.01-7.00 min, mobile phase A: 40% -65%; 7.00-7.50 min, mobile phase A: 65 percent; 7.50 min-8.5 min, mobile phase A: 65 to 100 percent; 8.50-8.60 min, mobile phase A: 100% -40%; 8.60-9.00 min, mobile phase A: 100 percent; wherein the above conditions relate to the float of the analysis time, the volume percentage of mobile phases a and B for each numerical parameter within a range of ± 20% of that numerical parameter.
9. The method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 6, wherein the volume of the test solution is 80 to 150 μ L; the sample injection amount of the high performance liquid chromatography is 15-25 mu L.
10. The method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 6, wherein the blank matrix used in the isotope internal standard method is 30-45 v/v% methanol aqueous solution in the operation of calculating the content of each steroid hormone by using the calibration curve quantitatively established by the isotope internal standard method.
11. The method for simultaneously detecting multiple steroid hormones in a blood sample according to claim 6, wherein the mass spectrum is a triple quadrupole mass spectrum, and the collision energies for different detection objects are respectively as follows:
17-hydroxyprogesterone: 20V to 24V; androstenedione: 28V-32V; cortisol: 18V to 22V; 21-deoxycorticosterol: 35V to 45V; 11-deoxycorticosterol: 33V to 37V.
12. A kit for detecting steroid hormones in a blood-based sample, comprising:
steroid hormone internal standards and standards, aqueous solutions containing 30 to 45 v/v% methanol, methyl tert-butyl ether, alkaline solutions as defined in claim 1 or 3, methanol solutions containing 0.1 to 0.5 v/v% formic acid and aqueous solutions containing 0.1 to 0.5 v/v% formic acid;
wherein the steroid hormones are 17-hydroxyprogesterone, androstenedione, cortisol, 21-deoxycorticosterol and 11-deoxycorticosterol.
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