Kit for simultaneously detecting multiple steroid hormones in dried blood spots based on liquid chromatography-mass spectrometry and application of kit

Abstract

The invention provides a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MSMS and application of the kit. The kit comprises an eluent, an isotope internal standard substance, an extracting agent, a calibrator, a quality control substance and a reconstitution fluid. According to the kit for detecting the multiple steroid hormones in the dried blood spots, the purpose of simultaneously analyzing and detecting 17 steroid hormones and 17 D / 13C labeled isotope hormones in one sample is achieved by adopting a liquid chromatography-tandem mass spectrometry technology, the sample detection type of the kit is blood spots, the blood sampling requirement is reduced, storage and transportation are convenient, and the kit is suitable for popularization and application. Meanwhile, the kit provides a calibrator and a quality control product for blood spot steroid hormone detection, the method is quick, simple and convenient to operate, multiple in detection variety, high in flux, high in specificity and low in cost, and 17 indexes of metabolic pathways of steroid hormones in blood spots can be effectively monitored and researched.

Description

Technical Field

[0001] The present invention relates to the technical field of steroid hormone detection, and in particular to a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS and an application thereof. Background Art

[0002] Steroid hormones play an important regulatory role in human metabolism, growth, development, reproductive behavior, and maintaining the stability of the internal environment.

[0003] Congenital adrenal hyperplasia (CAH) is an autosomal recessive hereditary disease characterized by impaired cortisol synthesis due to a deficiency of an enzyme in the steroid hormone synthesis metabolism. CAH is divided into 6 types according to enzyme deficiency. 21-hydroxylase deficiency (21-OHD, MIM201910) is the most common type of CAH, accounting for 95%; followed by 11-β-hydroxylase deficiency (11β-OHD, MIM202010), accounting for about 3% to 5%; 17α-hydroxylase and 17,20-lyase deficiency and 3β-hydroxylase deficiency, accounting for about 1% respectively; other types are even rarer. If children are not treated early, they are prone to fatal adrenal crisis. In addition to reducing this risk, early intervention can also minimize the damage to childhood linear growth caused by hyperandrogenism and the impact on long-term reproductive function and hormone metabolism function.

[0004] Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease, which often develops during adolescence. Clinically, irregular menstruation is the main symptom, and there may be various clinical manifestations such as infertility, hirsutism, obesity, and acanthosis nigricans. There are many male hormones in women, among which the serum level of testosterone (T) is higher than that of other male hormones, and it is the main pathogenic male hormone in PCOS. In addition, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), 17-hydroxyprogesterone (17-OHP), and dihydrotestosterone (DHT) are also important types of male hormones in the human body. Some studies have shown that the serum levels of these hormones are different between PCOS patients and normal people, which can provide a reference for the diagnosis of PCOS. 17-OHP is an intermediate substance in the steroid synthesis pathway and is also a precursor of mineralocorticoids and androgens. Many relevant professional societies recommend it for the diagnosis of PCOS patients to exclude Non-classic CAH.

[0005] At present, for the detection of steroid hormones, most clinical laboratories mainly use fluorescent immunoassay to measure the level of 17-hydroxyprogesterone (17-OHP) in neonatal DBS samples as a primary screening method for neonatal CAH. The cross-reaction, matrix interference, relatively low sensitivity and specificity of this detection method will lead to a high false positive rate. Moreover, only one hormone can be measured in one test, which increases the amount of samples required and the cost of detection. Steroid hormones have low content in organisms, similar structures, and a wide variety, so accurate quantification of steroid hormones has high requirements for the sensitivity of the instrument and the accuracy of the method. High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has the advantages of simultaneous detection of multiple targets, wide linear range, high sensitivity and specificity, and can effectively reduce false positives in screening diagnosis by measuring the steroid hormone spectrum in DBS. In recent years, some laboratories at home and abroad have applied LC-MS / MS technology to the detection of steroid hormone spectrum, but these studies mostly collect serum or serum as test samples, which is not conducive to specimen collection (especially for newborns), delivery and preservation. Summary of the invention

[0006] In view of the problems in the detection of steroid hormones in the prior art, the first object of the present invention is to provide a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS, which reduces the demand for blood collection and facilitates the collection, delivery and preservation of specimens.

[0007] The second object of the present invention is to provide an application for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS. The kit can be used to simultaneously detect multiple steroid hormones in dried blood spots by LC-MS / MS, thereby achieving the purpose of simultaneously analyzing and detecting 17 steroid hormones and 17 D / 13C / 15N labeled isotope hormones in a sample. It has high specificity and high accuracy, and can be further applied to the detection of steroid content for non-medical treatment purposes.

[0008] In order to achieve the above-mentioned object, the present invention provides a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MSMS, comprising a calibrator, a quality control product, an eluent, an isotope internal standard, an extractant and a reconstitution solution;

[0009] The calibrator includes a mixed standard and a calibration line substitute matrix; the quality control product includes a mixed standard and a human blood matrix; the human blood matrix is ​​human whole blood after sufficient cleaning treatment.

[0010] The calibrator is prepared by adding a mixed standard solution of seven concentrations to a calibration line substitute matrix and diluting the mixed standard working solution;

[0011] The calibration line substitute matrix is ​​a blank blood matrix; the dilution multiples of the mixed standard solution of seven concentrations corresponding to the working solution at the calibration line high point D7 are 1, 2, 5, 10, 25, 50, and 100 times respectively;

[0012] The mixed standard includes progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dexamethasone, 18-hydroxycorticosterone, 18-hydroxycortisol and 11β-ketotestosterone.

[0013] The kit based on LC-MS / MS technology of the present invention realizes the simultaneous analysis of 17 steroid hormones and 17 isotope-labeled hormones in dried blood spots by combining efficient separation with high-sensitivity detection. Its advantage is that liquid chromatography provides precise separation capability, while tandem mass spectrometry ensures high sensitivity and selectivity of detection, and isotope internal standards improve the accuracy of quantification. The use of dried blood spot samples reduces the amount of blood collected, which is convenient for storage and transportation. The addition of calibrators and quality control products ensures the standardization and quality control of the test results. The overall design ensures comprehensive, rapid and reliable detection of multiple steroid hormones, greatly improving the efficiency and accuracy of scientific research.

[0014] At the same time, the kit of the technical solution of the present invention uses a human blood matrix mixed with a mixed standard as a quality control product with a lower base value.

[0015] As a preferred solution, the eluent consists of eluent A and eluent B; the eluent A is a methanol aqueous solution containing ammonium fluoride; and the eluent B is a pure methanol solution.

[0016] As a preferred embodiment, the isotope internal standard comprises progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-D5, D4-cortisol, cortisone-D7, dehydroepiandrosterone sulfate sodium salt-D6, androstenedione-D7, D4-testosterone, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, and 11β-ketotestosterone-D3.

[0017] As a preferred solution, the extractant is composed of ammonia water, acetonitrile and water. The extractant of the present invention can effectively extract a variety of steroid hormones from dry blood spots, and the inventors have found that when the extractant is a pure organic phase, there are multiple granular insolubles after redissolution, and there will be interference peaks. The extractant combination selected in the present invention can not only ensure the response values ​​of a variety of steroid hormones, but also reduce the appearance of interference peaks and ensure the peak shape of the target. It is further preferred that the volume ratio of acetonitrile and water is (70-80)%: (20-30)%, and the amount of ammonia water added is 0.05-0.2% of the total volume of acetonitrile and water.

[0018] As a preferred solution, the reconstitution solution consists of ammonium fluoride, methanol and water.

[0019] The calibration line substitute matrix of the present invention is a blank blood matrix synthesized according to the method described in CN116793787A and a dried blood piece prepared by dropping the blank blood matrix, which can effectively simulate human whole blood and has an ultra-low matrix effect.

[0020] As a preferred embodiment, at the high point D7 of the correction line, the concentrations of progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dexamethasone, 18-hydroxycorticosterone, 18-hydroxycortisol, and 11β-ketotestosterone are 5000, 500, 5000, 4000, 25000, 5000, 4000, 1000, 100000, 20000, 1000000, 4000, 2500, 5000, 2000, 1000, and 1600 ng / dl, respectively.

[0021] As a preferred embodiment, the isotope internal standard includes progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-D5, D4-cortisol, cortisone-D7, dehydroepiandrosterone sulfate sodium salt-D6, androstenedione The target concentrations of keto-D7, D4-testosterone, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, and 11β-ketotestosterone-D3 were 500, 50, 500, 400, 2500, 500, 400, 100, 10000, 2000, 10000, 400, 250, 5000, 200, 100, and 160 ng / dl, respectively.

[0022] As a preferred solution, the kit further includes at least one of a chromatographic column, a 96-well sample collection plate and a 96-well sealing plate.

[0023] The second object of the present invention is to provide an application of a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS, wherein a dried blood sample containing steroid hormones and an isotope internal standard and an extractant in the kit are added to a 96-well sample collection plate, sealed with a silica gel cover for incubation and extraction, the supernatant is removed and blown dry with nitrogen, the reconstitution solution in the kit is added, incubated for full dissolution, and the supernatant is centrifuged to obtain a sample to be tested, and the sample to be tested is detected by LC-MS / MS.

[0024] The purpose of incubating the dried blood spots in the present invention is to allow the analyte in the dried blood spots to be fully dissolved in the extract.

[0025] As a preferred solution, the conditions of LC-MS / MS are:

[0026] Chromatographic conditions:

[0027] Chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; column temperature: 40 ° C; eluent A: methanol-water solution containing ammonium fluoride; eluent B: pure methanol solution, flow rate 0.35 mL / min, injection volume, 10 μL; gradient elution: 0-0.5 min, 0% B; 0.5-0.6 min, 0-28.5% B; 0.6-5.1 min, 28.5-30% B; 5.1-5.2 min, 30-3 9%B; 5.2~7.4min, 39~40%B; 7.4~7.5min, 40~60%B; 7.5~8.9min, 60%B; 8.9~9.5min, 60~85%B; 9.5~9.6min, 85~100%B; 9.6~10.2min, 100%B; 10.2~10.25min, 100~0%B; 10.25~11.5min, 0%B;

[0028] Mass spectrometry conditions: in electrospray positive and negative ion switching mode; using multiple reaction monitoring mass spectrometry scanning mode, capillary voltage: 1.5 kV; desolvation gas temperature: 650 ° C; desolvation gas flow rate: 800 L / Hr; simultaneous monitoring of target and isotope internal standard.

[0029] As a preferred scheme, after the detection is completed, a standard curve is prepared using the calibrator solution and isotope internal standard in the kit, with the concentration of the calibrator and the isotope internal standard as the X-axis and the peak area of ​​the calibrator and the isotope internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, and the peak area of ​​the corresponding test substance is substituted into the regression equation to calculate the concentration of the steroid hormone test substance in the blood spot sample.

[0030] Compared with the prior art, the beneficial effects of the present invention include but are not limited to:

[0031] (1) The kit provided by the present invention can achieve the purpose of simultaneously analyzing and detecting 17 steroid hormones and 17 D / 13C / 15N-labeled isotope hormones in a sample when used. The operation is fast and simple, and solid phase extraction and derivatization treatment are not required. The kit has a wide range of detection types, high throughput, high specificity, and low cost. It can effectively monitor 17 indicators of the steroid hormone metabolic pathway in dried blood spots.

[0032] (2) The sample detection type of the kit of the present invention is dried blood spots, which reduces the demand for blood collection and is conducive to the collection (especially for newborns), delivery and storage of specimens.

[0033] (3) The detection method used in the kit of the present invention has high specificity and high accuracy. First, by optimizing the chromatographic conditions, the isomers and other interfering substances are baseline separated, effectively avoiding the influence of the isomers and other interfering substances on the index determination results; secondly, the treated analyte is directly detected according to the inherent mass / charge ratio, rather than the traditional indirect detection of derivatives, and the method has strong specificity; finally, 17 D / 13C labeled isotopes are introduced as internal standard quantification, which reduces the influence of the matrix effect between different matrices on the quantitative results.

[0034] (4) The kit of the present invention also provides a method for preparing calibrators and quality control products for detecting steroid hormones in dried blood spots. This compounding method can not only effectively reduce the matrix effect, but also improve the response of the calibrators and quality control products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 LC-MS / MS ion chromatogram of the kit provided in Example 1 for simultaneously detecting 17 steroid hormones in dried blood spots.

[0036] Figure 2 Figure 2 is the ion flow diagram of isomers Aldo and CT.

[0037] Figure 3 Figure 2 is the ion flow diagram of isomers 18OH-CORT and COR.

[0038] Figure 4 The ion current diagrams of isomers 21-DOC, 11-DOC and CORT.

[0039] Figure 5 Ion chromatograms of isomers 17a-OHP4 and 21-OHP.

[0040] Figure 6 This is a standard curve diagram of 17a-OHP4 established based on the standard substance according to the example of the present invention.

[0041] Figure 7This is a standard curve diagram of 11-DOC based on standards according to an embodiment of the present invention.

[0042] Figure 8 This is a standard curve diagram of 21-DOC based on standards according to an embodiment of the present invention.

[0043] Fig. 9 This is a standard curve diagram of T based on standard substances in an embodiment of the present invention.

[0044] Fig.10 This is a standard curve diagram of COR based on standards according to an embodiment of the present invention.

[0045] Fig.11 2 is a comparative ion flow diagram of a normal person and a 21-OHD sample according to an embodiment of the present invention.

[0046] Fig.12 1 is a comparative ion current diagram of a normal person and a 11βOHD sample according to an embodiment of the present invention.

[0047] Fig.13 1 is a comparative ion flow diagram of a normal person and a 17-OHD sample according to an embodiment of the present invention.

[0048] Fig.14 This is a comparative ion flow diagram of a normal person and a sample with abnormal androgen metabolism according to an embodiment of the present invention.

[0049] Fig.15 1 is a comparative ion flow diagram of a normal person and a sample with abnormal cortisol metabolism according to an embodiment of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0052] The method and kit of the present invention are further described by the following examples in conjunction with the accompanying drawings, but these examples do not constitute any limitation to the present invention.

[0053] The abbreviations of various free fatty acids involved in the present invention are shown in Table 1 below.

[0054] Table 1 Abbreviations of free fatty acids

[0055]

[0056]

[0057] Example 1

[0058] This embodiment is a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MSMS, which is composed of a calibrator, a quality control product, an eluent, an isotope internal standard, an extractant, a reconstitution solution, a 96-well collection plate, and a 96-well silica gel cover plate. The components are configured as follows:

[0059] 1. Preparation of Calibrators

[0060] The dissolution of the standard and the preparation of the working solution HB-1 are shown in Table 2. The substances are mixed and then 110 μl of 80% methanol is added to the solution to obtain 1 ml of the working solution HB-1.

[0061] Seven different concentration points were set for the calibrator, which was prepared by adding the calibration line substitute matrix to the mixed standard working solution HB-1 for dilution. The calibration line substitute matrix was a blank blood matrix solution, which was prepared by weighing 1200 mg of hemoglobin into a centrifuge tube, and measuring 300 μl of phosphate buffered saline solution, 150 mg of BSA, and 2700 μl of ultrapure water in turn, and mixing them evenly to obtain a 3 ml blank blood matrix solution as shown in Table 3.

[0062] The mixing scheme of the working solution HB-1 at each concentration point and the calibration line substitute matrix is ​​shown in Table 4. The final concentrations of various steroid hormones in the 7 calibrators are shown in Table 5.

[0063] Table 2 Concentrated stock solutions of steroid hormones and the concentrations of each substance in the working solution

[0064]

[0065]

[0066] Table 3 Composition of blank blood matrix solution

[0067]

[0068] Table 4 Preparation of blank blood matrix and mixed standard at each concentration point

[0069] Serial number Concentration point (μl) HB-1 / 50 HB-1 / 5 HB-1 Synthetic blood volume 1 S1 30 - - 2970 2 S2 60 - - 2940 3 S3 - 12 - 2988 4 S4 - 30 - 2970 5 S5 - - 12 2988 6 S6 - - 30 2970 7 S7 - - 60 2940

[0070] Note: HB-1 / 50, HB-1 / 5 and HB-1 in the table represent the solution of working solution HB-1 diluted 50 times, the solution of working solution HB-1 diluted 5 times and working solution HB-1 respectively.

[0071] Table 5 Concentrations of various steroid hormones in 7 calibrators

[0072] serial number ng / dl D1 D2 D3 D4 D5 D6 D7 1 P4 50 100 200 500 1000 2500 5000 2 21-OHP 5 10 20 50 100 250 500 3 CORT 50 100 200 500 1000 2500 5000 4 Aldo 40 80 160 400 800 2000 4000 5 17a-OHP5 250 500 1000 2500 5000 12500 25000 6 17a-OHP4 50 100 200 500 1000 2500 5000 7 21-DOC 40 80 160 400 800 2000 4000 8 11-DOC 10 20 40 100 200 500 1000 9 COR 1000 2000 4000 10000 20000 50000 100000 10 CT 200 400 800 2000 4000 10000 20000 11 DHEAS 10000 20000 40000 100000 200000 500000 1000000 12 4-AD 40 80 160 400 800 2000 4000 13 T 25 50 100 250 500 1250 2500 14 DXMS 50 100 200 500 1000 2500 5000 15 18OH-CORT 20 40 80 200 400 1000 2000 16 18OH-COR 10 20 40 100 200 500 1000 17 11-KT 16 32 64 160 320 800 1600

[0073] 2. Preparation of quality control products

[0074] Quality control spike concentration setting and preparation process: The quality control product contains three levels of low, medium and high concentration quality control dry blood spots. The quality control product is prepared by adding mixed hormone standard substances to human blood matrix. The human blood matrix is ​​a mixture of activated carbon-purified plasma blank samples and fully cleaned red blood cells in proportion. The mixing preparation scheme of working solution HB-1 and quality control matrix is ​​shown in Table 6. The concentrations of various steroid hormones at three levels are shown in Table 7.

[0075] Table 6 Configuration of three levels of concentration

[0076]

[0077]

[0078] Table 7 Concentrations of various steroid hormones in quality control products

[0079] serial number Substance (ng / dl) QC-L QC-M QC-H 1 P4 200 500 2500 2 21-OHP 20 50 250 3 CORT 200 500 2500 4 Aldo 160 400 2000 5 17a-OHP5 1000 2500 12500 6 17a-OHP4 200 500 2500 7 21-DOC 160 400 2000 8 11-DOC 40 100 500 9 COR 4000 10000 50000 10 CT 800 2000 10000 11 DHEAS 40000 100000 500000 12 4-AD 160 400 2000 13 T 100 250 1250 14 DXMS 200 500 2500 15 18OH-CORT 80 200 1000 16 18OH-COR 40 100 500 17 11-KT 64 160 800

[0080] 3. Eluent

[0081] Eluent A: 10 vol% methanol + 90 vol% water + 0.05 mM ammonium fluoride;

[0082] Eluent B: methanol;

[0083] 4. Preparation of isotope internal standards

[0084] Dissolve and prepare the internal standard mixed solution of internal standard according to the preparation steps of standard solution, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2;2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-D5, D4-cortisol, cortisone-D7, dehydroepiandrosterone sulfate sodium salt-D6 , androstenedione-D7, D4-testosterone, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, and 11β-ketotestosterone-D3 were 500, 50, 500, 400, 2500, 500, 400, 100, 10000, 2000, 10000, 400, 250, 5000, 200, 100, and 160 ng / dl, respectively.

[0085] 5. Preparation of extraction agent

[0086] Accurately measure acetonitrile and water solution according to the volume ratio of acetonitrile to water of 80%:20%, and then absorb 0.2 vol% NH3H2O ​​solution and add it into the mixed solution.

[0087] 6. Selection of reconstitution solution

[0088] The reconstitution solution is a mixed solution of eluent A and eluent B in a volume ratio of 1:0.8.

[0089] The following is an application of a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS in this embodiment.

[0090] 1. Sample processing

[0091] 1) Take 5 3.2 mm dried blood pieces of the steroid hormone to be tested into a 400 μl 96-well sample collection plate, add 200 μl of extractant containing internal standard working solution in a fume hood, seal with a 96-well silicone cover plate, and incubate at 40°C and 600 rpm for 30 min;

[0092] 2) Cool to room temperature, transfer the extract to a new 96-well sample collection plate, and remove the solvent under a gentle nitrogen flow.

[0093] 3) The residue was redissolved in 50 μl of reconstitution solution, sealed with a pre-cut silicone cover plate, incubated at 35°C and 600 rpm for 10 min, and loaded after full dissolution, with an injection volume of 10 μl.

[0094] 2. Liquid chromatography and mass spectrometry conditions

[0095] Chromatographic conditions:

[0096] Eluent A: 10% methanol water + 0.05 mM ammonium fluoride;

[0097] Eluent B: methanol;

[0098] Chromatographic column: ACQUITY UPLC BEH C18 column, 50 mm × 2.1 mm, 1.7 μm; flow rate: 0.35 mL / min; column temperature: 40 °C; injection volume, 10 μL; gradient elution, gradient elution program see Table 8.

[0099] Table 8 Gradient elution program

[0100] Time (min) Flow rate (ml / min) ％A ％B initial 0.35 100 0 0.5 0.35 100 0 0.6 0.35 71.5 28.5 5.1 0.35 70 30 5.2 0.35 61 39 7.4 0.35 60 40 7.5 0.35 40 60 8.9 0.35 40 60 9.5 0.35 15 85 9.6 0.35 0 100 10.2 0.35 0 100 10.25 0.35 100 0 11.5 0.35 100 0

[0101] Mass spectrometry conditions:

[0102] In the electrospray ionization (ESI) positive and negative ion switching mode; using the mass spectrometry scanning mode of multiple reaction monitoring (MRM), the capillary voltage (Capillary): 1.5kV; the desolvation gas temperature: 650℃; the desolvation gas flow rate: 800L / Hr; the target object and the isotope internal standard were monitored simultaneously.

[0103] The mass spectrometry parameters for the detection of 17 steroid hormones in multiple reaction monitoring mode are shown in Table 9.

[0104] The mass spectrometry parameters for detecting 17 isotope-labeled steroid hormones in multiple reaction monitoring mode are shown in Table 10.

[0105] Table 9 Mass spectrometry parameters of steroid hormones

[0106]

[0107]

[0108] Note: * in the table indicates quantitative ion pairs.

[0109] Table 10 Mass spectrometry parameters of isotope-labeled steroid hormones

[0110]

[0111]

[0112] The LC-MS / MS ion chromatograms of 17 steroid hormones detected in dried blood spots are shown in Figure 1 , the ion flow diagrams of each compound and their isomers are shown in Figure 2 to Figure 5 The ion currents of normal samples were compared with those of 21-OHD samples, 11βOHD samples, and 17-OHD samples. Figure 11 to Figure 13 .

[0113] The method of this embodiment is used to detect normal human samples, and samples with abnormal androgen metabolism and samples with abnormal cortisol metabolism to obtain comparative ion flow diagrams. Fig.14 and Fig.15 .like Fig.14 The ion flow diagram of the normal person and the sample with abnormal androgen metabolism in the embodiment of the present invention is compared. The androgen dehydroepiandrosterone sulfate sodium salt and testosterone in the abnormal sample are both one order of magnitude higher than those in the normal sample. Fig.15 This is a comparative ion flow diagram of a normal person and a sample with abnormal cortisol metabolism according to an embodiment of the present invention. The cortisol in the abnormal sample is one order of magnitude higher than that in the normal sample.

[0114] 3. Method Validation

[0115] After the sample to be tested enters the chromatographic column for separation in the UPLC gradient elution mode, the multiple ion reaction monitoring mode (MRM) of positive / negative ion electrospray ionization (ESI) is used to perform qualitative and / or quantitative MS / MS detection on the test substance. A standard curve is prepared using a mixed standard solution of seven concentrations, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, and the peak area of ​​the corresponding test substance is substituted into the regression equation to calculate the concentration of the test substance in the sample; the measured linear results are shown in Table 11. The standard curves of substances 17α-OHP4, 11-DOC, 21-DOC, T and COR based on the standard are shown in Figure 6 to Figure 10 .

[0116] The accuracy was evaluated through the spike recovery experiment, and the measured spike recovery is shown in Table 12.

[0117] Table 11 Linearity results of the tested substances

[0118]

[0119]

[0120] Using the method of the present invention, the quantitative range of the 17 detection indicators is greater than or equal to 2 orders of magnitude, with a wide quantitative interval, meeting the accurate quantitative requirements of samples of different concentrations. The 7 calibration points set, the linear equation of each indicator r 2 The values ​​are all ≥0.99, with excellent linear performance.

[0121] Table 12 Sample spike recovery results

[0122]

[0123] The present invention prepares quality control products with three levels of concentration, L, M, and H, by adding a standard product of known concentration to a QC matrix containing human blood, and then tests each concentration six times. The theoretical value is the sum of the concentration of the endogenous analyte in the sample and the added standard product. The ratio of the measured value to the theoretical value can be used to evaluate the accuracy. The detected value is within ±15% of the target value, and the method has good accuracy.

[0124] Example 2: Selection and optimization of extraction agent

[0125] Different extractants were compared - methanol, acetonitrile, 50 vol% methanol-50 vol% acetonitrile, methanol-water, acetonitrile-water.

[0126] The results show that when the extractant is a pure organic phase, there are many granular insolubles after re-dissolution, which may block the injection needle and pipeline; from the peak shape, there is a large interference peak after 21-OHP and a peak before 17-OHP, which affects the quantification. When methanol water is used as the extractant, the response of 18-OH-COR will be significantly lower. Therefore, acetonitrile-water is a better choice.

[0127] Comparison of different volume ratios of acetonitrile and water - 50% to 80% acetonitrile-water solutions.

[0128] The results show that the multi-index response of 80vol% ACN-20vol% water is higher, and the 18-OH-COR response of 50vol% ACN-50vol% water and 60vol% ACN-40vol% water is significantly lower.

[0129] Selection and optimization of additives in the extractant: When acid is added to the extractant of 80vol%ACN+20vol%water, the response of multiple indicators is low, such as 21-OHP, DHEA, 4-AD, 18OH-CORT, etc. When alkali is added to the extractant of 80vol%ACN+20vol%water, the response of multiple indicators will increase to a certain extent, such as Aldo, etc. The amount of ammonia water in the extractant of 80vol%ACN+20vol%water is 0.05vol%, 0.1vol%, and 0.2vol%, among which the relative response of 0.2vol% ammonia water will be slightly higher, mainly manifested as 4-AD and T.

[0130] Amount of extractant: Compare different amounts of extractant - 150μl, 200μl, 250μl.

[0131] The results show that when the amount of extractant is 250μl, nitrogen blowing takes a long time, about 1h, and it cannot be completely dried. From the response point of view, there is not much difference between 150μl, 200μl, and 250μl of extractant. Therefore, 200μl of extractant is the best choice.

[0132] Therefore, after optimization and comparison, it was found that 200 μl of 0.2 vol% NH3H2O+80 vol% ACN+20 vol% water was the best choice for the extraction agent.

[0133] Example 3: Selection of Calibrator and Control Matrices

[0134] The base values ​​of different animal blood and human blood, the base values ​​of processed animal blood and human blood, the base values ​​of calibrators and quality control matrices were compared. At the same time, 6 batches of dried blood spots from different individuals were investigated. By comparing the signal of the analyte spiked into the sample after extraction and the signal of the analyte spiked into the standard curve matrix after extraction, and calculating the ratio of the peak area response in the presence of matrix (measured by adding analyte and internal standard after extraction of blank matrix) to the corresponding peak area response without matrix (pure solution of analyte and internal standard), the matrix factor of each analyte (low, medium and high quality control concentration) and internal standard was calculated, and the matrix factor normalized by internal standard was calculated. The base values ​​of different blood matrices were statistically obtained and are shown in Table 13.

[0135] Three female samples, three male samples and QC-human blood matrix were randomly selected and the matrix factor normalized to the internal standard relative to the STD-blank blood matrix. The results are shown in Table 14.

[0136] Table 13 Selection of base values ​​for calibrators and quality control matrices

[0137]

[0138] The results show that the animal whole blood has a high base value for multiple indicators. The base value of the treated animal blood is significantly reduced, and only the testosterone content is obvious, which is not suitable as a blank matrix. As shown in the table above, the blank blood matrix and the human blood matrix contain lower base values. According to the content of the base value, the blank blood matrix and the human blood matrix are used as the matrix of the calibrator and the quality control product respectively.

[0139] Table 14 Matrix factor results normalized by internal standard

[0140]

[0141] The matrix factor deviations of the 6 random samples and QC-human blood matrix relative to the STD-blank blood matrix were all within ±10%, and the CV of ME was within 15%. There was no obvious inhibition or enhancement between the blank dry blood matrix and the 6 random samples and QC-human blood matrix samples, and there was consistency in quantification, which fully demonstrated the accuracy of the STD-blank blood matrix and QC-human blood matrix in quantifying steroid hormones in human-derived dry blood films.

[0142] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS, characterized in that: Includes calibrators, quality control products, eluents, isotope internal standards, extractants and reconstitution solutions; The calibrators include mixed standards and calibration line substitute matrices; The quality control products include mixed standards and human blood matrix; The calibrator is prepared by adding a mixed standard solution of seven concentrations to a calibration line substitute matrix and diluting the mixed standard working solution; The calibration line substitute matrix is ​​a blank blood matrix; the dilution multiples of the mixed standard solution of seven concentrations corresponding to the working solution at the calibration line high point D7 are 1, 2, 5, 10, 25, 50, and 100 times respectively; The mixed standard includes progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dexamethasone, 18-hydroxycorticosterone, 18-hydroxycortisol and 11β-ketotestosterone.

2. A kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 1, characterized in that: The eluent consists of eluent A and eluent B; the eluent A is a methanol aqueous solution containing ammonium fluoride; and the eluent B is a pure methanol solution.

3. A kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 2, characterized in that: The isotopic internal standard includes progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2;2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-D5, D4-cortisol, cortisone-D7, dehydroepiandrosterone sulfate sodium salt-D6, androstenedione-D7, D4-testosterone, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4 and 11β-ketotestosterone-D3.

4. A kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to any one of claims 1 to 3, characterized in that: The extractant consists of aqueous ammonia, acetonitrile and water; The complex solution consists of ammonium fluoride, methanol and water.

5. The kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 1, characterized in that: At the high point D7 of the correction line, the concentrations of progesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 17-hydroxypregnenolone, 17α-hydroxyprogesterone, 21-deoxycortisol, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate, androstenedione, testosterone, dexamethasone, 18-hydroxycorticosterone, 18-hydroxycortisol, and 11β-ketotestosterone are 5000, 500, 5000, 4000, 25000, 5000, 4000, 1000, 100000, 20000, 1000000, 4000, 2500, 5000, 2000, 1000, and 1600 ng / dl, respectively.

6. The kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 3, characterized in that: Among the isotope internal standards, progesterone-D9, D7-11-deoxycorticosterone, corticosterone-D8, D7-aldosterone, [13C2; 2H2]-17α-hydroxypregnenolone, 17α-hydroxyprogesterone-D8, 21-deoxycortisol-D8, 11-deoxycortisol-D5, D4-cortisol, cortisone-D7, dehydroepiandrosterone sulfate sodium salt-D6, androstenedione-D7, The target concentrations of D4-testosterone, dexamethasone-D4, 18-hydroxycorticosterone-D4, 18-hydroxycortisol-D4, and 11β-ketotestosterone-D3 were 500, 50, 500, 400, 2500, 500, 400, 100, 10000, 2000, 10000, 400, 250, 5000, 200, 100, and 160 ng / dl, respectively.

7. A kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 5 or 6, characterized in that: The kit also includes at least one of a chromatographic column, a 96-well sample collection plate and a 96-well sealing plate.

8. Use of a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to any one of claims 1 to 7, characterized in that: A dried blood sample containing steroid hormones and the isotope internal standard and extractant in the kit are added to a 96-well sample collection plate, which is sealed with a silicone cap for incubation and extraction, the supernatant is removed and blown dry with nitrogen, the reconstitution solution in the kit is added, incubated for full dissolution, and the supernatant is centrifuged to obtain a sample to be tested, and the sample to be tested is detected by LC-MS / MS.

9. Use of a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 8, characterized in that: The conditions for LC-MS / MS were: Chromatographic conditions: Chromatographic column: C18 column, 50 mm × 2.1 mm, 1.7 μm; Column temperature: 40°C; eluent A: methanol-water solution containing ammonium fluoride; eluent B: pure methanol solution, flow rate 0.35 mL / min, injection volume, 10 μL; gradient elution: 0-0.5 min, 0% B; 0.5-0.6 min, 0-28.5% B; 0.6-5.1 min, 28.5-30% B; 5.1-5.2 min, 30-39% B; 5.2-7.4 min n, 39~40%B; 7.4~7.5min, 40~60%B; 7.5~8.9min, 60%B; 8.9~9.5min, 60~85%B; 9.5~9.6 min, 85~100%B; 9.6~10.2min, 100%B; 10.2~10.25min, 100~0%B; 10.25~11.5min, 0%B; Mass spectrometry conditions: in electrospray positive and negative ion switching mode; using multiple reaction monitoring mass spectrometry scanning mode, capillary voltage: 1.5 kV; desolvation gas temperature: 650 ° C; desolvation gas flow rate: 800 L / Hr; simultaneous monitoring of target and isotope internal standard.

10. Use of a kit for simultaneously detecting multiple steroid hormones in dried blood spots based on LC-MS / MS according to claim 9, characterized in that: The detection is to use the calibrator solution and isotope internal standard in the kit to prepare a standard curve to quantify the pre-treated sample, wherein the concentration of the calibrator and the isotope internal standard is the X-axis, and the peak area of ​​the calibrator and the isotope internal standard is the Y-axis; a linear regression analysis is performed to obtain a regression equation, and the peak area of ​​the corresponding test substance is substituted into the regression equation to calculate the concentration of the steroid hormone test substance in the blood spot sample.

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