CN112379009A - Method for detecting steroid hormone in biological sample and kit used by method - Google Patents

Abstract

The invention relates to the field of biological detection, in particular to a method for detecting steroid hormone in a biological sample and a kit used by the method. The method comprises the following steps: enriching the steroid component in the biological sample; performing derivatization treatment on the steroid component by taking hydroxylamine with the molecular weight of less than 100 as a derivatization reagent; the concentration of the hydroxylamine is 200 mM-400 mM, the reaction temperature is 50-70 ℃, and the reaction time is 40-80 min; purifying the single component after the derivatization treatment by using an SPE chromatographic column; detection of steroid hormones was performed using MALDI-MS. The MALDI-MS method based on HA derivatization is used for quantitative detection of steroid hormones in a complex biological sample for the first time, and HAs the advantages of high specificity, accurate method, simple and convenient operation and high sensitivity.

Description

Method for detecting steroid hormone in biological sample and kit used by method

Technical Field

The invention relates to the field of biological detection, in particular to a method for detecting steroid hormone in a biological sample and a kit used by the method.

Background

Steroid hormones are a class of cyclopentane polyhydrophenanthrene derivatives with multiple carbonyl and hydroxyl substituents. In the organism, steroid hormones regulate various physiological processes such as water salt balance, glucose metabolism, protein metabolism, lipid metabolism and inflammation, immune function. Imbalances in steroid hormone metabolism can lead to diseases such as primary aldosteronism, cushing's syndrome and congenital adrenal hyperplasia. Accurate quantification of hormone levels in biological samples is of great clinical significance. The important sex hormones estrone, testosterone and progesterone greatly influence sexual function, vascular function and energy homeostasis. Accurate quantification and monitoring of the dynamic changes of these sex hormones is of great importance in the clinical diagnosis of diseases associated with sex hormone disorders, such as precocious puberty, infertility, hypogonadism, cardiovascular diseases and type 2 diabetes.

The major methods for clinical identification and quantification of steroid hormones in general are radioimmunoassay, enzyme-linked immunosorbent assay and chemiluminescent immunoassay. However, the lack of antibodies of appropriate specificity and cross-reactivity of antibodies reduces the accuracy and specificity of the reaction, making the measurements inconsistent from immunoassay to immunoassay. In addition, the concentration of certain hormones in plasma is lower than the LOD of the immunoassay. Therefore, the sensitivity and accuracy of steroid hormone detection methods should be improved.

High information content, high sensitivity and accurate mass spectrometry MS is a label-free method for directly measuring analytes without the use of immunoreagents. Gas chromatography-mass spectrometry GC-MS and liquid chromatography-mass spectrometry LC-MS have been used to quantify steroid hormones in plasma or serum samples with sufficient sensitivity and high accuracy, indicating that mass spectrometry is a reliable method. In the field of clinical mass spectrometry, MALDI-MS is a high-throughput and automatic MS detection method, has the advantages of high sensitivity, good stability, short experimental period and low sample and manpower consumption, and has potential value for clinically quantifying steroid hormones. However, the main problem of mass spectrometric detection of steroid hormones is that the lack of polar groups leads to low ionization efficiency of hormone molecules, which greatly reduces the sensitivity of MS detection. The advent of derivatization methods introduced cationic charges into the molecule to improve detection sensitivity. Various derivatization reagents for steroid hormone labeling have been reported, including Girard reagent T (GT), Girard reagent P (GP), N-methylpyridine-3-sulfonyl (NMPS), and bis (trimethylsilyl) trifluoroacetamide (BSTFA), to impart a stable charge to carbonyl derivatives.

Disclosure of Invention

In order to overcome the disadvantages of the existing methods, the invention provides a novel Hydroxylamine (HA) derivatization (HA-D)/MALDI-MS method for determining steroid hormones in plasma, especially in low concentration.

The invention relates to a method for detecting steroid hormone in a biological sample, which comprises the following steps:

a) purifying and enriching the steroid component in the biological sample;

b) performing derivatization treatment on the steroid component by taking hydroxylamine with the molecular weight of less than 100 as a derivatization reagent;

the concentration of the hydroxylamine is 200 mM-400 mM, the reaction temperature is 50-70 ℃, and the reaction time is 40-80 min;

c) purifying the single component after the derivatization treatment by using an SPE chromatographic column;

d) detection of steroid hormones was performed using MALDI-MS.

HA can react with the ketocarbonyl group on steroid molecules to convert steroid hormones into the corresponding oximes, which are prone to [ M + H ] formation]⁺The adduct greatly improves the sensitivity of mass spectrum detection of steroid hormones.

In order to optimize the analytical performance of the HA-D/MALDI-MS method, the present invention systematically investigated the HA concentration, derivatization conditions, and purification steps. The result shows that the established HA-D/MALDI-MS method can be used for clinically measuring steroid hormones in complex biological samples.

According to a further aspect of the invention, the invention also relates to a kit for the detection of steroid hormones in a biological sample, said kit comprising a volatile hydroxylamine as defined above and an SPE chromatography column.

The invention has the beneficial effects that:

1) high specificity and accurate method. The existing immunoassay methods have the problems of lack of specific antibodies, cross reaction and the like. Certain hormones are present in low amounts in plasma and are difficult to detect using immunoassays. Interferents whose structure is closely related to the steroid hormone to be tested may reduce the specificity of the results, making the values measured by different immunoassay methods divergent. And the immunoassay can only detect one steroid hormone at a time. The method provided by the invention does not need immune reagents such as antibodies and the like, only needs HA derivatization and MALDI-MS detection ion information to simultaneously quantify multiple steroid hormones in plasma, and HAs sufficient sensitivity, specificity and higher accuracy.

2) The sample preparation is simple and clear, the method is simple and convenient, the running time of the instrument is short, and the automation degree is high. Existing GC-MS and LC-MS methods require complex methods to set up, optimize and validate, require strong chromatographic and mass spectral background knowledge and professional operators, many clinical institutions that require the determination of steroid hormones in biological samples do not have such conditions, and because steroid hormones are a class of nonpolar molecules, the ionization efficiency of underivatized hormones is not high. The method provided by the invention HAs the advantages that the HA derivatization steroid hormone improves the ionization efficiency, and the sensitivity of MALDI-MS detection is improved. Compared with complicated chromatographic separation procedures such as GC and the like, the method only needs simple SPE purification, and MALDI-MS is easy to operate, has short turnaround time and is easy to realize high-throughput detection of biological samples.

3) The sensitivity is high. We analyzed steroid hormone standards at the same concentrations by derivatization with HA and GT, a conventional derivatization reagent for steroid hormones, respectively, and compared the derivatization performance of the two by MALDI-MS. HA-D HAs higher sensitivity and lower LOD than GT derivative. The detection sensitivity of MALDI-MS is improved by HA-D.

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.

FIG. 1 shows the derivatization of (a) E1, (b) T and (c) Prog with HA followed by a dehydroxylation step. Due to the lack of polar groups in the steroid hormone molecule and the difficulty of forming [ M + H ]]⁺Or [ M + Na]⁺Adducts and therefore have low ionization efficiency in MS detection of unlabeled steroid hormones. In order to improve the efficiency of steroid hormone detection, complex chromatographic separation steps are usually required to pre-enrich steroid hormones in complex biological samples. In this study, plasma samples using the HA-D/MALDI-MS method can improve ionization efficiency and achieve sensitive detection of steroid hormones without the need for complex chromatographic separation. In theory, HA readily reacts with the carbonyl group of steroid hormones to form structurally unstable schiff bases, and the dehydroxylation of this compound can lead to the formation of dehydroxylated oximes (oximes for short). Oximes with high polarity are prone to form [ M + H ] compared to unlabeled carbonyl groups]⁺Adducts, may greatly increase ionization efficiency. The E1 and T molecules have only one carbonyl group and are converted to dehydroxymonooxime, and although Prog HAs two carbonyl groups and reacts with two HA molecules, it eventually forms a dehydroxygenoxime.

FIG. 2 shows the mass spectra of HA added to steroid hormone standards and methanol, respectively, and not derived from steroid hormones, corresponding to (a) E1, (b) T and (c) Prog, respectively, at concentrations of 37.02nM, 34.70nM and 31.82. mu.M. The oxime derivative is mainly used for detecting [ M + H ] through HA derivatization]⁺Ions, and m/z is indicated.

FIG. 3 shows the workflow of the method for the quantification of the plasma steroid hormone HA-D/MALDI-MS. IS: internal standard, SPE: and (4) solid phase extraction. First, proteins are precipitated using methanol, and small molecule metabolites (including hormones) are extracted from plasma. Then, an HA reagent is added to the extracted ingredients to derivatize the steroid hormone. The reaction product was SPE purified to remove matrix and excess HA and finally MALDI-MS analyzed.

FIG. 4 shows experimental conditions for optimized HA-D/MALDI-MS analysis of hormones in plasma. The MS abundance of HA derivatives of E1, T and Prog under different conditions was compared. Different concentrations of ha (a), incubation time (b), SPE column type (c). H: oasis HLB, P: oasis PRIME, P-H: oasis PRIME HLB-Oasis HLB. MALDI-MS obtains the intensity of the peak in positive ion mode. The highest peak intensity in each mass spectrum was normalized to 1. Error bar represents SEM of three measurements.

FIG. 5 shows MALDI-MS spectra of 17.35, 69.39nM and 6.94mM standard T, respectively, analyzed by derivatization of HA (a) and GT (b). An increase in ionization efficiency and detection limit was observed for HA derivatization compared to GT derivatization. The derivatives were mainly detected as [ M + H]⁺Ion: [ T-Oxime + H]⁺288.2, [ T-hydrazone + H]⁺＝402.3。

FIG. 6 shows the linear relationship between the addition concentration of steroid hormone (a) E1, (b) T, (c) Prog and the signal-to-noise ratio of hormone to IS.

Fig. 7 shows the correlation of the concentrations of (a) E1, (b) T, (c) Prog in plasma determined by HA-D/MALDI-MS with the plasma steroid hormone concentrations determined by LC-ESI MS/MS method (n-10).

FIG. 8 shows MALDI-MS spectra of steroid hormone oximes obtained by HA derivatization of (a) A2, (B) DOC, (c) Prog17, (d) B and (E) E hormone standards at 34.94, 30.28, 28.88 and 27.76 μ M, respectively.

FIG. 9 shows MALDI-MS mass spectra of 18.51nM, 74.03nM and 7.40mM E1 standard derivatized by HA derivatization (a) and GT derivatization (b). The derivatives were mainly detected as [ M + H⁺]Ion: [ E1-Oxime + H⁺]270.2, [ E1-hydrazone + H⁺]＝384.3。

FIG. 10 shows MALDI-MS mass spectra of 15.91nM, 63.65nM and 6.36mM Prog standards by HA derivatization (a) and GT derivatization (b). The derivatives are mainly detected as [ M + H⁺]Ion: [ Prog-oxime + H⁺]329.2, [ Prog-hydrazone + H⁺]＝428.3。

FIG. 11 shows MALDI-MS mass spectra of various concentrations of hormone standards analyzed by HA derivatization. E1 is 1110.45, 740.30, 185.08, 74.03, 37.02, 18 respectively51, 3.70, 0.19 and 0nM, T1040.91, 693.94, 346.97, 173.48, 69.39, 3.45, 0.35, 0.17, 0.03 and 0nM, Prog 954.73, 636.49, 318.24, 159.12, 63.65, 15.91, 3.18, 0.32, 0.16, 0 nM. With a fixed concentration of D₄-E1([M+H⁺]＝274.2)，¹³C3-T([M+H⁺]291.2) and¹³C3-Prog([M+H⁺]＝332.2)。

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 method for detecting steroid hormone in a biological sample, which comprises the following steps:

a) purifying and enriching the steroid component in the biological sample;

b) performing derivatization treatment on the steroid component by taking hydroxylamine with the molecular weight of less than 100 as a derivatization reagent;

the concentration of the hydroxylamine is 200 mM-400 mM, the reaction temperature is 50-70 ℃, and the reaction time is 40-80 min;

c) purifying the single component after the derivatization treatment by using an SPE chromatographic column;

d) detection of steroid hormones was performed using MALDI-MS.

Unlike other labeling reagents that introduce a pre-charged group into the carbonyl group, Hydroxylamine (HA) derivatization and oxime formation improve ionization efficiency by introducing groups that are easily ionized and protonated. The advantage of derivatization of HA as steroid hormone compound is high reaction efficiency due to the small size and high reactivity of HA.

The MALDI-MS method based on HA derivatization is used for quantitative detection of steroid hormones in a complex biological sample for the first time, and HAs the advantages of high specificity, accurate method, simple and convenient operation and high sensitivity.

In addition, it is easy to conceive that the present invention also relates to a pretreatment method for MALDI-MS for steroid hormone detection, comprising:

a) purifying and enriching the steroid component in the biological sample;

b) performing derivatization treatment on the steroid component by taking hydroxylamine with the molecular weight of less than 100 as a derivatization reagent;

the concentration of the hydroxylamine is 200 mM-400 mM, the reaction temperature is 50-70 ℃, and the reaction time is 40-80 min;

c) purification of the single component after derivatization treatment was performed using an SPE chromatography column.

Due to the complex matrix effect and the low content of steroid hormones in plasma, purification of the derivatized product is required in order to be able to detect plasma with higher sensitivity and efficiency. The key points of the process, including hydroxylamine concentration, derivatization conditions and SPE column type, were optimized by the present invention for excellent analytical performance.

In some embodiments, the hydroxylamine is present in a concentration of 250mM to 350mM, at a reaction temperature of 55 ℃ to 65 ℃, and for a reaction time of 50min to 70 min.

In some embodiments, the hydroxylamine is present in a concentration of 280mM to 320mM, at a reaction temperature of 57 ℃ to 62 ℃, and for a reaction time of 55min to 65 min.

In some embodiments, the hydroxylamine concentration is 300mM, reaction temperature is 60 ℃, reaction time is 60 min.

In a preferred embodiment, the hydroxylamine is a volatile hydroxylamine. Compared with GT/GP reagent, the derivative of steroid hormone oxime and redundant chemical reagent can be easily separated after the derivatization of micromolecule and more volatile HA reagent, thereby simplifying the purification step and improving the analysis efficiency.

"volatile hydroxylamine" refers to hydroxylamine-based materials that change from solid or liquid to gas at ambient temperature and pressure. In the present application, to ensure the volatility of hydroxylamine, hydroxylamine having a molecular weight of less than 100, such as less than 90, 80, 70, etc., is preferred.

In some embodiments, the volatile hydroxylamine is hydroxylamine hydrochloride.

In some embodiments, step a) further comprises removing the high-abundance hetero-protein from the biological sample.

In some embodiments, in step a), the method of enrichment is a methanol precipitation protein method.

In some embodiments, the SPE chromatography column is a chromatography column comprising N-vinylpyrrolidone and divinylbenzene-reactive groups, further preferably an Oasis HLB chromatography column or a reverse phase separation chromatography column.

In some embodiments, step c) specifically comprises:

activating an SPE chromatographic column by using methanol, loading a sample after the column is balanced, washing the sample by using 3-7 v/v% (or 5 v/v%) methanol aqueous solution, and eluting the sample by using the methanol;

the eluate is collected and the solid phase thereof is dissolved in 40 to 60 v/v% (or 50 v/v%) of an aqueous methanol solution.

In some embodiments, the steroid hormone comprises at least one of estrone (E1), testosterone (testosterone, T), and progesterone (progesterone, Prog).

In some embodiments, the detection is a quantitative detection.

In some embodiments, the quantitative detection is quantified by an internal standard method.

In some embodiments, the internal standard method employs an internal standard that is an isotopically labeled analog of the steroid hormone.

The internal standard may be selected from¹⁵N, deuterium (D),¹³C is an isotope.

Typically, the heavy isotopes contained in analogues of steroid hormones contain more than one heavy isotope. It may be preferable to incorporate a higher number of heavy isotopes because it provides greater mass transfer. In another preferred embodiment, the combination of heavy isotopes comprises¹³C₆、¹³C₆ ¹⁵N₄、¹³C₆ ¹⁵N₄D₇、¹⁵N₄D₇、¹⁵N₄、¹³C₆ ¹⁵N₂、¹⁵N₂、¹³C₆、¹³C₆ ¹⁵N₂D₉、¹⁵N₂D₉、D₄、¹³CD₃、¹³C₉、¹⁵N and¹³C₉ ¹⁵n, and the like. Such heavy isotopically labeled compounds are well known in the art and are available from a variety of manufacturers.

In some embodiments, the isotopically labeled analog of the steroid hormone is added to the biological sample or standard solution prior to step b); in some embodiments, the isotopically labeled analog of the steroid hormone is pre-added to the biological sample or standard solution prior to step a) (i.e., prior to enrichment). The steroid hormone analogues on the one hand help to quantify steroid hormones in a sample and on the other hand explain the loss of target during the experiment. Because of the matrix effect of proteins, the ionization efficiency of steroid hormones in mass spectrometry may be reduced.

In some embodiments, in step d), a standard curve is plotted based on the MS signal-to-noise ratio of the hormone standard and internal standard and the concentration of the added hormone, and the concentration of the steroid hormone in the biological sample is calculated.

In another embodiment of the invention, for the purpose of calibration curve plotting, the ratio of the signal to noise ratio of the hormone/IS MS IS the y-axis and the concentration of the added hormone IS the x-axis in a matrix simulating the plasma fraction, corresponding hormone standards and isotope analogues of steroid hormones (IS) are added, indicating a linear relationship between the signal to noise ratio of the HA-D/MALDI-MS method and the concentration of steroid hormone standards.

In some embodiments, the biological sample is a blood-based sample.

In some embodiments, the blood-based sample comprises dried blood spots, blood, serum, or plasma.

In one embodiment of the present invention, the method comprises the following specific steps:

1) addition of IS D to plasma samples to a final concentration of 50ng mL-1₄-E1、¹³C₃-T and¹³C₃prog and mix well.

2) Aspirate 400. mu.L of sample, add 800. mu.L of methanol to precipitate protein, vortex for 1min, centrifuge at 4 ℃ 3500rpm for 15 min.

3) Pipette 1mL of the supernatant into a fresh tube, dry in a lyophilizer, then resuspend in 500. mu.L of 300mM 50% methanol-solubilized HA HCl, incubate for 1h at 60 ℃.

4) After treatment with the derivatizing agent, the reaction product is subjected to Solid Phase Extraction (SPE). The HLB Oasis SPE cartridge was first activated with 1mL of methanol, equilibrated with 1mL of water, and the sample applied to the SPE cartridge, then washed with 1mL of 5% methanol, and finally eluted with 100% methanol. The eluate was drained using a lyophilizer. The dried product containing the steroid oxime derivative was redissolved in 20 μ L of 50% methanol.

5) Take 1.5 μ L of the solution and 1.5 μ L of DHB [ acetonitrile/water/trifluoroacetic acid (1: 1: 0.002, v/v/v), 10mg mL-1], and pipetted 1.5. mu.L of the solution onto the surface of the stainless steel target plate. The steroid hormone derivatives were quantitatively analyzed by MALDI-MS, and the steroid hormone was analyzed in a reflectance mode using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer. MALDI mass spectra were obtained by accumulating and averaging 400 shots in the positive ion mode with laser intensity of 100-120. Spectrogram acquisition and processing was performed using Shimadzu Biotech Launchpad software (version 2.9).

According to a further aspect of the invention, the invention also relates to a kit for the detection of steroid hormones in a biological sample, said kit comprising a volatile hydroxylamine as defined above and an SPE chromatography column.

In some embodiments, the kit further comprises a component selected from i) to iii):

i) extracting and/or purifying a desired agent from a biological sample;

ii) at least one of a column activation column, a column equilibrium solution, a column washing solution and a column eluent required for SPE chromatographic column purification;

iii) isotopically labeled analogues of said steroid hormones.

Embodiments of the present invention will be described in detail with reference to examples.

Example 1

Feasibility analysis of steroid hormones in plasma by HA-D/MALDI-MS. The method comprises the following steps:

1) the steroid hormone standard was prepared to 10. mu.g mL with methanol^-1(E1, T and Prog 37.02, 34.70 and 31.82. mu.M, respectively), 500. mu.L of the solution was taken, and an equal volume of the methanol solution was taken at the same time, and dried in a lyophilizer.

2) To the hormone standard and methanol was added 500. mu.L of 300mM HA hydrochloride dissolved in 50% methanol, respectively. At the same time, 500. mu.L of 50% methanol was added to the same hormone standard sample which was drained. After 1h at 60 ℃, the derivative product was drained using a lyophilizer. The dried product containing the steroid oxime derivative was redissolved in 20 μ L of 50% methanol. The derivatization of the hormone, followed by a dehydroxylation step, is shown in FIG. 1.

3) Take 1.5 μ L of the solution and 1.5 μ L of DHB [ acetonitrile/water/trifluoroacetic acid (1: 1: 0.002, v/v/v) dissolved, 10mg mL^-1]Mix and pipette 1.5. mu.L of solution onto the surface of the stainless steel target plate. The steroid hormone derivatives were quantitatively analyzed by MALDI-MS and identified in reflectance mode using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer. MALDI mass spectra were obtained by accumulating and averaging 400 shots in the positive ion mode with laser intensity of 100-120. Spectrogram acquisition and processing was performed using Shimadzu Biotech Launchpad software (version 2.9). The results are shown in FIG. 2.

To demonstrate the effectiveness of HA-D/MALDI-MS, we first used HA derived E1, T and Prog standards, followed by MALDI-MS detection and comparison with blank samples and underivatized steroid hormones. The mass spectrum in figure 2 shows no MS signal when methanol is mixed with HA. In the mass spectrum of underivatized steroid hormones, it is difficult to detect the protonated E1 or Prog signal. Protonated T is observed in the underivatized T mass spectrum. However, the HA derivatized form of E1, T and Prog was observed to have strong mass spectra signals, which initially suggested that HA derivatization of steroid hormones could improve ionization efficiency and sensitivity of MALDI-MS detection.

The E1 and T molecules have only one carbonyl group and are converted to dehydroxymonooximes, as [ M + H ]]⁺The formation of ions is shown as m/z 270.2(E1) and 288.2(T), respectively. Despite the fact that Prog HAs two carbonyl groups and reacts with two HA molecules, a dehydroxylated Prog-oxime is finally formed, with an m/z of 329.2. Similar results were obtained with other different steroid hormones (fig. 8). We derived androstenedione (A2), Deoxycorticosterone (DOC), 17-alpha-hydroxyprogesterone (Prog17), corticosterone (B) and cortisone (E) by HA and characterized the derived products by MALDI-MS. Oxime derivatives of the hormones can be ionized more efficiently by derivatization than their non-derivatized steroid hormone counterparts, and produce strong signals during MALDI-MS analysis and improve detection sensitivity. M/z values for A2, DOC, Prog17, B and E were observed to be 301.2, 345.2, 345.2, 361.2 and 375.2, respectively. These results indicate that HA-D/MALDI-MS can be used to analyze a wide variety of steroid hormones.

Example 2

Optimization of the workflow of steroid hormone analysis in plasma. The method comprises the following steps:

1) plasma samples were taken and mixed in equal volumes to give a pooled plasma sample. mu.L of each plasma sample was taken, 800. mu.L of methanol was added to precipitate the protein, the mixture was centrifuged at 4 ℃ at 3500rpm for 15min, and then 1mL of the supernatant was aspirated and transferred to a new tube and dried in a lyophilizer.

2) To obtain excellent analytical performance, we optimized the key points of the method including the concentration of HA hydrochloride, the derivatization time and the type of SPE column, by adding 500 μ L of 50% methanol-dissolved HA hydrochloride at a derivatization temperature of 60 ℃. HA concentrations were 1, 10, 50, 100, 600mM, respectively; the derivatization time was 1, 2, 3, 4, 5 hours, respectively. After treatment with the derivatizing reagent, the reaction product is subjected to SPE. The SPE columns are H, P, P-H respectively, and represent SPE chromatographic columns Oasis HLB, Oasis PRIME HLB and the combination of the two chromatographic columns respectively. The derivative product was then lyophilized using a lyophilizer. The dried product containing the steroid oxime derivative was redissolved in 20 μ L of 50% methanol.

3) Take 1.5 μ L of the solution and 1.5 μ L of DHB [ acetonitrile/water/trifluoroacetic acid (1: 1: 0.002, v/v/v), 10mg mL-1], and pipetted 1.5. mu.L of the solution onto the surface of the stainless steel target plate. The steroid hormone derivatives were quantitatively analyzed by MALDI-MS and identified in reflectance mode using a SHIMADZU AXIMA RESONANCE MALDI-IT-TOF mass spectrometer. The MALDI mass spectrum is obtained by accumulating and averaging 400 shots under a positive ion mode with the laser intensity of 100-120. Spectrogram acquisition and processing was performed using Shimadzu Biotech Launchpad software (version 2.9).

First, plasma was incubated with various concentrations of HA hydrochloride to select the appropriate derivative concentration. Figure 4a shows that the MS intensity of E1, T and Prog oxime derivatives continued to increase with increasing HA hydrochloride concentration. The three steroid hormones in plasma showed similar trends in the 1-600mM HA range. Although the T-oxime signal was more intense when the HA concentration exceeded 100mM, there was no significant difference in the MS signal of E1 or the Prog derivative. Considering that it may be difficult to remove excess HA, we finally selected 300mM HA for derivatization and analysis of steroid hormones. Furthermore, derivatization reaction times were investigated in plasma samples (fig. 4 b). After 1 hour of derivatization of the three hormones, the yields reached the highest level. Incubation times exceeding 1 hour are not recommended because 1 hour is sufficient to achieve maximum abundance of oxime derivatives, and excessively long reaction times may cause oxime decomposition or redundant reactions to form byproducts. The results show that the HA-D/MALDI-MS method requires less time than the GT derivatization reaction time suggested in the literature, 4 hours. Furthermore, we investigated whether the type of SPE column affected the yield of oxime produced. We tested two commercial SPE chromatography columns, Oasis HLB and Oasis PRiME HLB, which have a unique balance of hydrophobicity and water wettability and are generally able to purify a variety of acidic, basic and neutral compounds from a variety of matrices using a simple general procedure. The Oasis PRIME HLB column, as a high quality reverse phase adsorbent, removes 95% of the common matrix interferences including proteins, salts and phospholipids. Two SPE columns were compared and the combination of SPE columns was used to optimize the purification step. Figure 4c shows that HLB Oasis SPE chromatography column can extract hormones from plasma, producing the strongest oxime signal. Other types of SPE chromatography columns do not produce the same purification capacity due to different purification characteristics and functions. Notably, the combination of two SPE chromatography columns reduced the MS intensity of the hormone oxime compared to the HLB Oasis SPE chromatography column alone, probably because of the loss of target using redundant purification steps.

Finally, 300mM HA hydrochloride was selected, incubated for 1 hour, and purified on an HLB Oasis SPE column as a further condition for the detection of plasma endogenous steroid hormones.

Example 3

The efficiency of derivatization of HA and GT for the carbonyl steroid hormones was compared. Characterized in that HA and GT derivatization were performed at 6.94mM, 69.39nM and 17.35nM T, respectively, HA-D conditions as described above, 300mM HA, incubation for 1 hour, and purification on HLB Oasis SPE column. For GT derivatization, 1mL of 60mM GT was prepared dissolved in 50% methanol, 5% acetic acid, and then incubated at 85 ℃ for 4 hours. To compare the performance of GT-and HA-derivatization, the other experimental procedures were identical.

We analyzed steroid hormone standards at the same concentrations for HA-and GT-derivatization, respectively, and compared the derivatization performance of the two by MALDI-MS. The mass spectra in FIG. 5 show that strong signals were observed at 6.94mM T, 288.2 for T-oxime m/z and 402.3 for T-hydrazone m/z, after HA derivatization (FIG. 5a) and GT derivatization (FIG. 5b), but no GT derivative or ion associated with the GT derivative was observed in the mass spectra after the concentrations were reduced to 69.39 and 17.35nM T, as shown in FIG. 5 b. By comparison, HA derivatization showed MS signals of T-oxime at different concentrations (fig. 5 a). Similar results were obtained when E1 and Prog were derivatized with HA or GT (fig. 9 and 10). For 18.51nM, 74.03nM E1 and 15.91nM, 63.65nM Prog produced MS signals of the oxime derivatives, whereas no signal of the GT derivative was observed. Although Prog has two carbonyl groups, the main peak in the MS spectrum is a monosubstituted derivative of Prog (fig. 10). The conversion of the hormone into oxime or hydrazone after HA or GT treatment is compared, which shows that the detection sensitivity of MALDI-MS is improved by HA derivatization. Possible reasons include the small molecular size, volatility of HA, which may lead to higher reaction efficiency, excess HA can be easily removed from oxime derivatives by SPE purification.

Example 4

To investigate the relative quantitative capacity of our established MS method, we added fixed concentrations of IS, using E1, T and Prog at 0-1110.45, 0-1040.91 and 0-954.73nM, respectively, and compared the MS signal intensity ratio (fig. 11). IS serves to compensate for sample loss during the experiment and accounts for matrix effects, thus facilitating the detection of the target product. We selected stable isotopes of hormonal compounds, labeled E1-IS with four deuterium species and three with three¹³The C isotope labels T-IS and Prog-IS. As shown in FIG. 11, the signal intensity ratio of steroid hormone oxime to IS oxime gradually increased with increasing concentration of spiked hormone and was at E1 (r)²＝0.9582)，T(r²0.9931) and Prog (r)²0.9770) and the concentration of the steroid hormone standard added (fig. 6). These results indicate that the HA-D/MALDI-MS method can quantify plasma hormones. Table 1 shows that the MS signal of HA-D/MALDI-MS shows good reproducibility in samples in the low, medium and high concentration ranges (E1, T and Prog CV% 23.99% -27.96%, 21.08% -22.79% and 3.47% -17.76%, respectively). In addition, E1, T and Prog had LODs of 0.007, 0.021 and 0.067nM, respectively. E1, the recovery rates of T and Prog are 95.05% -119.65%, 98.34% -117.47% and 94.67% -97.39% respectively. These results indicate that HA-D/MALDI-MS can be a promising method for quantitative determination of steroid hormones in complex samples.

TABLE 1

Furthermore, to test whether this method can relatively quantify hormones in plasma, we measured hormone levels in real plasma samples. By analyzing the ratio of the MS intensity of the hormone of interest to its corresponding IS, we calculated the concentrations of E1, T and Prog in plasma and compared the concentrations detected by HA-D/MALDI-MS with the concentrations detected by LC-ESI MS/MS. As shown in FIG. 7, the hormone concentrations of the samples evaluated using the HA-D/MALDI-MS method are well comparable to the LC-ESI MS/MS method, where r is E1, T and Prog²The values are 0.9864, 0.9969 and 0.8442, respectively. The results of the steroid hormone HA-D/MALDI-MS method were consistent with those of the LC-ESI MS/MS method. The above described correlation test for measuring hormones using standards and plasma samples shows that the method of isotope addition, HA derivatization and MALDI-MS detection can be considered as a potential platform for quantitative detection of plasma steroid hormones.

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 (13)

1. A method for detecting a steroid hormone in a biological sample, comprising:

a) purifying and enriching the steroid component in the biological sample;

b) performing derivatization treatment on the steroid component by taking hydroxylamine with the molecular weight of less than 100 as a derivatization reagent;

the concentration of the hydroxylamine is 200 mM-400 mM, the reaction temperature is 50-70 ℃, and the reaction time is 40-80 min;

c) purifying the single component after the derivatization treatment by using an SPE chromatographic column;

d) detection of steroid hormones was performed using MALDI-MS.

2. The method of claim 1, wherein the hydroxylamine is hydroxylamine hydrochloride.

3. The method according to claim 1, wherein in step a), the enrichment method is a methanol precipitation protein method.

4. The method as claimed in claim 1, wherein the SPE chromatography column is an Oasis HLB chromatography column or a reverse phase separation chromatography column comprising N-vinylpyrrolidone and divinylbenzene reactive groups.

5. The method according to claim 4, wherein step c) comprises in particular:

activating an SPE chromatographic column by using methanol, loading a sample after the column is balanced, washing by using 3-7 v/v% methanol aqueous solution, and eluting by using the methanol;

collecting the eluent, and dissolving the solid phase in 40-60 v/v% methanol water solution.

6. The method of claim 1, wherein the steroid hormone comprises at least one of estrone, testosterone, and progesterone.

7. The method according to any one of claims 1 to 6, wherein the detection is a quantitative detection by an internal standard method using an isotopically labeled analogue of the steroid hormone.

8. The method according to claim 7, wherein the internal standard is added to the biological sample or standard solution prior to step a) or b).

9. The method according to claim 7, wherein in step d), the concentration of said steroid hormone in said biological sample is calculated by plotting a calibration curve based on the MS signal to noise ratio of the hormone standard and internal standard and the concentration of the added hormone.

10. The method according to any one of claims 1 to 6 and 8 to 9, wherein the biological sample is a blood sample.

11. The method of claim 10, wherein the blood-based sample comprises dried blood spots, blood, serum, or plasma.

12. A kit for the detection of steroid hormones in a biological sample, comprising a volatile hydroxylamine as defined in any one of claims 1 to 11 and an SPE chromatography column.

13. The kit of claim 12, further comprising a component selected from i) to iii):

i) extracting and/or purifying a desired agent from a biological sample;

ii) at least one of a column activation column, a column equilibrium solution, a column washing solution and a column eluent required for SPE chromatographic column purification;

iii) isotopically labeled analogues of said steroid hormones.

Images