CN111337602A - Method for determining neurosteroid hormone by high performance liquid chromatography tandem mass spectrometry - Google Patents
Method for determining neurosteroid hormone by high performance liquid chromatography tandem mass spectrometry Download PDFInfo
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- 238000004885 tandem mass spectrometry Methods 0.000 title description 4
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a method for determining neurosteroid hormone by a high performance liquid chromatography-tandem mass spectrometry method, and relates to the field of animal in-vitro hormone determination methods. The method comprises the following steps: weighing neurosteroid standard substance to prepare standard solution; weighing a sample to be detected, adding acetonitrile and MT into the sample to be detected, carrying out vortex and centrifugation, taking a supernatant, adding the supernatant into an EMR solid-phase extraction small column which is cleaned by acetonitrile in advance, and filtering to obtain a purified sample solution; and (3) detecting and analyzing the standard solution and the sample solution by using a high performance liquid chromatography-tandem mass spectrometer. According to the invention, interference components such as protein and the like are removed by an EMR (electromagnetic radiation) column method after acetonitrile precipitated protein is extracted, so that the problems that an AP standard solution is not linear and the response is extremely low are solved; meanwhile, each neurosteroid measured has better recovery rate.
Description
Technical Field
The invention relates to an animal in-vitro hormone determination method, in particular to a method for determining neurosteroid hormone by high performance liquid chromatography tandem mass spectrometry.
Background
Neurosteroids refer to steroids synthesized in the nervous system and peripheral steroids and metabolic derivatives thereof that enter the nervous system via the blood-brain barrier to function. These neurosteroids can modulate neural function by binding to different neural receptors (e.g. GABAA, Sigma receptors). Neurosteroid behavior is closely related to the exertion of normal central nervous system function. Studies have shown that the content of neurosteroids in the various brain regions varies significantly under different physiological and pathological conditions. Neurosteroids are also involved in the regulation of anxiety and depression. Because the content of the neurosteroid in an organism is extremely low, the interference of a matrix is much, and the determination of the endogenous neurosteroid in the organism is very difficult, the development of a method for quickly and accurately detecting the content of the neurosteroid in the organism is particularly important.
The traditional detection method of the neurosteroids comprises a Radioimmunoassay (RIA) and a gas chromatography-mass spectrometry (GC-MS), but the radioimmunoassay has poor specificity and potential safety hazard; although the gas chromatography-mass spectrometry combined method has high sensitivity, the sample pretreatment step is complex, and high-throughput analysis cannot be realized.
In the prior art, a detection method using high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry and using methyltestosterone as an internal standard is adopted to rapidly and simultaneously determine the content of neurosteroids (Liujia, Zhang Miao, Wanyi, etc.) in different brain regions of a rat;
however, it was found that the standard solution of AP (allopregnanolone) was not linear and the response was very low when tested according to the methods of the aforementioned documents; and the matrix effect of PREG (pregnenolone) and PROG (progesterone) is obvious; meanwhile, the first recovery test has general results. It is therefore desirable to consider improving some of the steps in the assay process to increase its recovery.
Disclosure of Invention
The invention aims to solve the problems in the prior art and aims to provide a method for determining neurosteroid hormone by high performance liquid chromatography-tandem mass spectrometry.
In order to realize the purpose of the invention, the method is realized by the following technical scheme:
a method for measuring neurosteroid hormone by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
(1) preparation of a standard solution: weighing neurosteroid standard substance to prepare standard solution;
(2) sample pretreatment: weighing a sample to be detected, adding acetonitrile and MT, carrying out vortex and centrifugation, and taking a supernatant; adding the supernatant into an EMR solid-phase extraction column which is cleaned by acetonitrile in advance, and filtering to obtain a purified sample solution;
(3) high performance liquid chromatography-tandem mass spectrometry: and (3) detecting and analyzing the standard solution and the sample solution by using a high performance liquid chromatography-tandem mass spectrometer.
Preferably, in step (3), the high performance liquid chromatography conditions are:
XDB-C18a chromatographic column; the mobile phase A is ammonium acetate solution, and the mobile phase B is methanol; a gradient elution mode was used:
when 0-2.5min, the mobile phase A and the mobile phase B are 40% and 60%
When the time is 2.5-5.5min, the mobile phase A and the mobile phase B are 40%, 60% -60% and 95%
5.5-7.0min later, the ratio of mobile phase A and mobile phase B is 5% to 95%
7.0-7.1min later, the mobile phase A, the mobile phase B, is 5%, 40% -95%, and 60%
When 7.1-10.0min, the ratio of mobile phase A and mobile phase B is 40%: 60%
The flow rate is 0.5mL/min, the injection volume is 10 μ L, and the column temperature is 45 ℃.
Preferably, in step (3), the mass spectrometry conditions are:
an ion source: an electrospray ion source;
the scanning mode is as follows: positrons;
scanning mode: monitoring multiple reactive ions;
ionization voltage: 5500V;
241.33kpa of air curtain air;
spraying gas under 413.7 kPa;
auxiliary heating gas: 413.7 kPa;
ion source temperature: at 100 ℃.
Further preferably, in the step (2), the concentration of MT is 1. mu.g/mL.
Further preferably, in the step (2), the centrifugation condition is 15000r/min for 4 minutes.
Further preferably, in the step (1), the neurosteroid standard includes at least one of deoxycorticosterone, dehydroepiandrosterone, methyltestosterone, pregnenolone, progesterone and allopregnanolone
According to the invention, interference components such as protein and the like are removed by an EMR (electromagnetic radiation) column method after acetonitrile precipitated protein is extracted, so that the problems that an AP standard solution is not linear and the response is extremely low are solved; meanwhile, each neurosteroid measured has better recovery rate.
Drawings
FIGS. 1-6 are standard curves for DOC, DHEA, MT, PREG, PROG, and AP, respectively, in a first embodiment;
FIGS. 7-12 are chromatograms of DOC, DHEA, MT, PREG, PROG, AP, respectively, according to examples.
FIG. 13 is an AP chromatogram of example six.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The drawings are only for purposes of illustration and are not intended to be limiting, and are merely schematic and non-limiting.
Instruments and reagents:
analytically pure ammonia water, chromatographic grade ammonium acetate, mass spectrum grade methanol, mass spectrum grade acetonitrile, bovine serum albumin, sodium chloride, analytically pure hydrochloric acid, standard allopregnanolone, standard allophytestosterone, and standard allopregnanolandrone;
MCX solid phase extraction column, EMR solid phase extraction column, and HLB columnPhase extraction column, disposable syringe, disposable filter membrane, Thermo Accucore C182.6 μm 2.1 x 100mm chromatography column
The other instruments, reagents or samples which are not listed are the same or higher precision same/substitute products listed in the high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for measuring the neurosteroid compounds in the rat brain tissue (Liujia, Zhang Miao, Wanyi, etc.. high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for measuring the neurosteroid compounds in the rat brain tissue [ J ]. analytical chemistry, 2015,43(08): 1118-.
EXAMPLES pretreatment of samples and preparation of Standard Curve
Preparing standard stock solution with mass concentration of 1mg/mL by using methanol for each standard substance, and diluting the standard stock solution into mixed standard solution with proper mass concentration by using methanol-water (1:1, V/V) according to needs, and storing the mixed standard solution at the temperature of-20 ℃.
Preparing 5% BSA-0.7% NaCl solution, adding AP, PREG, DOC, THDOC, PROG and DHEA standard substances with different concentrations, wherein the concentration range is 0.1-100 ng/mL (the AP is 10-200 ng/mL), adding an internal standard substance MT with the same concentration (0.3pg/mL), oscillating and mixing uniformly, and then carrying out liquid-liquid extraction. Adding 1.2mL ethyl acetate-n-hexane (9:1, V/V), vortexing for 5min, centrifuging at 12000r/min for 5min, and transferring the upper organic phase to another clean centrifuge tube. Nitrogen blow-drying, 100. mu.L methanol-water (1:1, V/V, 0.1% formic acid) redissolution. Centrifuging at 12000r/min for 15min, and subjecting the supernatant to HPLC-MS/MS analysis.
The results are shown in FIGS. 1 to 6, which are the DOC, DHEA, MT, RPEG, RPOG, and AP standard curves, respectively. Except for AP, the correlation coefficient r is 0.9999; the detection limit can reach 0.1ng/ml, and the signal-to-noise ratio is more than 3: 1; the quantitative limit can reach 0.5ng/ml, and the signal-to-noise ratio is more than 10: 1.
Examples comparative Experimental chromatographic analysis
Rat brain tissue: rats were anesthetized with isoflurane, blood was drawn after decapitation, the medial prefrontal cortex (mPFC), Hippocampus (HF) and thalamic retroventral nucleus (VP) were separated by ice bath, stored in liquid nitrogen, and weighed (accurately called to 0.1 mg). To each tissue, 0.49mL of ultrapure water, 10. mu.L of 15pg/mLMT (methyltestosterone) was added, homogenized by an electric homogenizer, and sonicated. The method adopts two methods of liquid-liquid extraction and solid-phase extraction.
Solid phase extraction method: StrataC was previously activated in equilibrium with 1mL of methanol and 1mL of water18And (3) a column. The tissue homogenate was centrifuged at 12000r/min for 5min, and the supernatant was added to StrataC18In the column; elution of StrataC with 1mL of 5% methanol as eluent18And (3) a column. After being pumped to near dryness, the mixture was eluted with 1mL of methanol. Collecting the eluate, and eluting with N at room temperature2And (5) drying. 100 μ L of methanol-water (1:1, V/V, 0.1% formic acid) was redissolved. Centrifuging at 12000r/min for 15min, and subjecting the supernatant to UFLC-MS/MS analysis.
And (3) chromatographic analysis:
AgilentXDB-C18a chromatographic column (50mm × 4.6.6 mm, 1.8 mu m, Agilent company, USA) and a mobile phase, wherein the mobile phase comprises 5mmol/L of ammonium acetate solution (A) and methanol (B), a gradient elution mode is adopted, the gradient elution mode is 0-2.5min, 60% of B, 2.5-5.5min, 60-95% of B, 5.5-7.0min, 95% of B, 7.0-7.1min, 95-60% of B, 7.1-10.0min, the flow rate of 60% of B is 0.5mL/min, the sample injection volume is 10 mu L, the sample chamber temperature is room temperature, the column temperature is 45 ℃, and an internal standard method is adopted for quantitative analysis.
The results of the MRM chromatograms are shown in FIGS. 7 to 12, and are chromatograms of DOC, DHEA, MT, RPEG, RPOG and AP, respectively, and the concentration thereof is 10 ng/ml.
Mass spectrum conditions:
electrospray ion source (ESI), positive ion scan mode, multiple reaction monitoring scan (MRM) mode. Ionization voltage (IS) 5500V, air curtain gas (CUR) 241.33kPa, spray gas (GS1) 413.7kPa, auxiliary heating gas (GS2) 413.7kPa, and collision device (CAD) Medium. Since the ammonia adduct of THDOC is easily decomposed by heat, the ion source Temperature (TEM) was set to 100 ℃.
Example three precision test
RSD (relative standard deviation) was calculated according to the experimental method of example two by continuously injecting 6 times with the standard substance concentration of 10ng/ml, and the results are shown in Table 1 below
TABLE 1
| DOC | DHEA | MT | PREG | PROG | AP | |
| RSD％ | 1.3 | 1.2 | 0.6 | 0.9 | 1.4 | 2.5 |
Example four matrix Effect test
Because of the matrix effect, the mass spectral response of the standard in methanol/water solution is different from its response in the biological matrix, so different concentrations of the standard should be added to the blank matrix to make the working curve. The neurosteroids belong to endogenous substances, and blank matrixes cannot be obtained, so that 5% BSA-0.7% NaCl is selected as the blank matrixes. 6 solutions of neurosteroid compounds with the concentration of 0.1, 0.2, 0.5, 1, 2, 510ng/mL are prepared. Then, the matrix effect was calculated according to the method of example two under the condition that the concentration of the standard substance was 10ng/ml, and the results are shown in Table 2.
TABLE 2
| DOC | DHEA | MT | PREG | PROG | AP | |
| Matrix Effect% | 110.1 | 91.2 | 105.3 | 60.5 | 69.4 | 108.9 |
Example five recovery test
According to the experimental method of example two, the sample is continuously and independently injected 3 times at the standard substance concentration of 10ng/ml, and the recovery rate is calculated, and the results are shown in the following table 3.
TABLE 3
| Unit: is based on | DOC | DHEA | MT | PREG | PROG | AP |
| Recovery ratio-1 | 120.1 | 105.4 | 110.1 | 56.3 | 69.0 | / |
| Recovery rate-2 | 40.5 | 37.9 | 41.0 | 21.9 | 28.5 | 15.4 |
| Recovery ratio-3 | 52.5 | 44.8 | 56.2 | 33.7 | 27.9 | 14.5 |
From the first to fifth embodiments, if the experiment is repeated according to the method in "high performance liquid chromatography-triple quadrupole/composite linear ion trap mass spectrometry for determining neurosteroid compounds in rat brain tissue", the standard solution with AP is not linear, the response is extremely low, and the result does not conform to the paper data; meanwhile, the matrix effects of PREG and PROG are obvious. The first recovery test result is general, but is equivalent to the matrix effect test result, which shows that the method or the experiment is not sufficient in purification and has great influence. The present invention therefore improves upon the drawbacks of the previous solutions.
EXAMPLE six
Rats were anesthetized with isoflurane, blood was drawn after decapitation, brains were taken after ice bath, and about 0.4g was weighed. Adding 1.5mL acetonitrile +0.2mL 1 mug/mLMT, whirling for 10min, centrifuging at the rotating speed of 15000r/min for 4 min, taking the supernatant, fixing the volume to 2mL, taking 1mL sample liquid, feeding to an EMR solid phase extraction column with the specification of 1mL (firstly washing by 1mL acetonitrile before use), and filtering to a sample bottle.
Then, the analysis was performed according to the procedure of the chromatographic analysis in example two, and the sample was continuously and independently injected 3 times at a standard substance concentration of 10ng/ml, and the recovery rate was calculated, the results are shown in Table 4 below
TABLE 4
| Unit: is based on | DOC | DHEA | MT | PREG | PROG | AP |
| Recovery ratio-1 | 88.8 | 90.0 | 89.4 | 93.5 | 91.5 | 88.1 |
| Recovery rate-2 | 90.3 | 92.7 | 90.6 | 92.5 | 90.9 | 90.6 |
| Recovery ratio-3 | 92.6 | 93.4 | 87.6 | 86.5 | 93.4 | 93.2 |
From the results in Table 4, it is clear that the method of treating the column with EMR solid phase extraction after the protein extraction by acetonitrile precipitation is more excellent than the method of treating the column with HLBC after the protein extraction by methanol precipitation18A method of solid phase extraction of the cartridge; all neurosteroids can achieve better and more stable recovery rate. The method of the sixth embodiment is proved to be capable of effectively removing other interference components such as protein and the like. However, this method has an interference peak near AP, and requires chromatographic separation, as shown in fig. 13, where the boxed part is the interference peak.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (6)
1. A method for measuring neurosteroid hormone by high performance liquid chromatography-tandem mass spectrometry is characterized by comprising the following steps:
(1) preparation of a standard solution: weighing neurosteroid standard substance to prepare standard solution;
(2) sample pretreatment: weighing a sample to be detected, adding acetonitrile and MT, carrying out vortex and centrifugation, and taking a supernatant; adding the supernatant into an EMR solid-phase extraction column which is cleaned by acetonitrile in advance, and filtering to obtain a purified sample solution;
(3) high performance liquid chromatography-tandem mass spectrometry: and (3) detecting and analyzing the standard solution and the sample solution by using a high performance liquid chromatography-tandem mass spectrometer.
2. The method for measuring neurosteroid hormones by high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein in the step (3), the high performance liquid chromatography conditions are as follows:
XDB-C18a chromatographic column; the mobile phase A is ammonium acetate solution, and the mobile phase B is methanol; a gradient elution mode was used:
when 0-2.5min, the mobile phase A and the mobile phase B are 40% and 60%
When the time is 2.5-5.5min, the mobile phase A and the mobile phase B are 40%, 60% -60% and 95%
5.5-7.0min later, the ratio of mobile phase A and mobile phase B is 5% to 95%
7.0-7.1min later, the mobile phase A, the mobile phase B, is 5%, 40% -95%, and 60%
When 7.1-10.0min, the ratio of mobile phase A and mobile phase B is 40%: 60%
The flow rate is 0.5mL/min, the injection volume is 10 μ L, and the column temperature is 45 ℃.
3. The method for measuring neurosteroid hormones by high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein in the step (3), the mass spectrometry conditions are as follows:
an ion source: an electrospray ion source;
the scanning mode is as follows: positrons;
scanning mode: monitoring multiple reactive ions;
ionization voltage: 5500V;
241.33kpa of air curtain air;
spraying gas under 413.7 kPa;
auxiliary heating gas: 413.7 kPa;
ion source temperature: at 100 ℃.
4. The method for measuring neurosteroid hormones according to any one of claims 1 to 3, wherein in step (2), the concentration of MT is 1 μ g/mL.
5. The method for measuring neurosteroid hormones according to any one of claims 1 to 3, wherein in the step (2), the centrifugation condition is 15000r/min rotation speed centrifugation for 4 minutes.
6. The method for measuring neurosteroid hormones according to any one of claims 1 to 3, wherein in step (1), the neurosteroid standard substance comprises at least one of deoxycorticosterone, dehydroepiandrosterone, methyltestosterone, pregnenolone, progesterone and allopregnanolone.
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