CN114235995A - Method for detecting 15 kinds of bile acids in serum - Google Patents
Method for detecting 15 kinds of bile acids in serum Download PDFInfo
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- CN114235995A CN114235995A CN202111467327.6A CN202111467327A CN114235995A CN 114235995 A CN114235995 A CN 114235995A CN 202111467327 A CN202111467327 A CN 202111467327A CN 114235995 A CN114235995 A CN 114235995A
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- acid
- sample
- detecting
- acetonitrile
- bile acids
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Images
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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
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Abstract
The invention discloses a method for detecting 15 bile acids in serum, which comprises the following steps: 1) sample treatment: taking a sample to be detected, adding a precipitator into the sample, uniformly mixing the mixture by vortex, and centrifuging the mixture; taking supernatant, drying the supernatant under nitrogen, redissolving the supernatant by using redissolution, carrying out vortex oscillation and centrifugation, and taking the supernatant as a test sample; wherein the redissolution is a mixed solution of acetonitrile and water; 2) and detecting the test sample by adopting a liquid chromatography-tandem mass spectrometry device. According to the method for detecting 15 kinds of bile acids in serum, provided by the invention, the steps of re-dissolving after nitrogen blowing and re-centrifuging are added in sample treatment, and a scheme that methanol acetonitrile is used as a precipitator and acetonitrile water is used as a re-dissolving solution is matched, so that the purity of a sample before testing and the service life of a chromatographic column are greatly improved, meanwhile, the response of a target substance can be effectively improved, the limit of quantitation is reduced, and the detection accuracy is improved; the invention can accurately detect different types of bile acid at the same time by one-time sample introduction, and has short analysis time and high analysis speed.
Description
Technical Field
The invention relates to the technical field of in-vitro detection, in particular to a method for detecting 15 bile acids in serum.
Background
The conventional detection method of bile acid is a circulating enzyme method, the operation is simpler, however, the enzyme method is used for determining the total bile acid level, so that the difference between different subtypes cannot be determined, the structural difference between the bile acid and a conjugate thereof is large, the concentration level in certain matrixes is lower, a plurality of isomers possibly exist at the same time, and the difficulty in simultaneously separating and detecting the bile acid is large; the GC-MS has high selectivity and resolution when analyzing bile acid isomers, but the sample preparation is complex, and derivatization, especially analysis can be carried out only by hydrolyzing conjugated bile acid into a non-conjugated form; the high performance liquid chromatography can be used by a series fluorescence detector, but the fluorescence detection method needs a complex derivatization technology and cannot be popularized and applied.
Therefore, there is a need to provide a more reliable solution.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a method for detecting 15 bile acids in serum, aiming at the above-mentioned deficiencies in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for detecting 15 bile acids in serum, the 15 bile acids comprising: cholic acid CA, deoxycholic acid DCA, chenodeoxycholic acid CDCA, ursodeoxycholic acid UDCA, glycocholic acid GCA, glycodeoxycholic acid GDCA, glycochenodeoxycholic acid GCDCA, glycoursodeoxycholic acid GUDCA, taurocholic acid TCA, taurodeoxycholic acid TDCA, taurochenodeoxycholic acid TCDCA, tauroursodeoxycholic acid TUDCA, lithocholic acid LCA, glycolithocholic acid GLCA and taurocholic acid TLCA, wherein the method comprises the following steps:
1) sample treatment: taking a sample to be detected, adding a precipitator into the sample, uniformly mixing the mixture by vortex, and centrifuging the mixture; taking supernatant, drying the supernatant under nitrogen, redissolving the supernatant by using redissolution, carrying out vortex oscillation and centrifugation, and taking the supernatant as a test sample; wherein the redissolution is a mixed solution of acetonitrile and water;
2) and detecting the test sample by adopting a liquid chromatography-tandem mass spectrometry device.
Preferably, the precipitant is a mixture of methanol and acetonitrile.
Preferably, the volume ratio of methanol to acetonitrile in the precipitant is 1: 1.
Preferably, the volume fraction of acetonitrile in the double solution is 20-40%.
Preferably, the volume fraction of acetonitrile in the double solution is 30%.
Preferably, the step 1) is specifically: taking 100 mu L of serum sample to be detected, adding 10 mu L of internal standard working solution and 200 mu L of precipitator into the serum sample, uniformly mixing the internal standard working solution and the precipitator by vortex at 2500rpm for 5min, and centrifuging the mixture at 14000rpm at 4 ℃ for 10 min; drying 200 μ L of supernatant under nitrogen at 40 deg.C, redissolving with 100 μ L of redissolution, and vortex shaking for 5 min; the cells were centrifuged at 14000rpm for 10min at 4 ℃ and 100. mu.L of the supernatant was used as a test sample.
Preferably, in the step 2), the mobile phase a in the liquid chromatography is an aqueous solution; the mobile phase B is acetonitrile solution; the needle washing liquid is acetonitrile water solution with 50 percent of volume fraction.
Preferably, in the step 2), the gradient elution procedure adopted is as follows: 0-1min, 10% B, 1-1.5min, 17% B, 1.5-3.5min, 25% B, 3.5-4.6min, 30% B, 4.6-6.5min, 30% B, 6.5-6.6min, 35% B, 6.6-8.5min, 35% B, 8.5-8.6min, 10% B, 8.6-10min, 10% B; the flow rate was 0.6 mL/min.
Preferably, in the step 2), the chromatographic column is Kinetex C18.
Preferably, in the step 2), the mass spectrometry conditions are as follows: in the negative ion mode, an ESI source is adopted, collision gas is 12psi, air curtain gas is 16psi, atomization gas is 50psi, auxiliary heating gas is 60psi, the spraying voltage is-4500V, and the atomization temperature is 500 ℃.
The invention has the beneficial effects that: according to the method for detecting 15 kinds of bile acids in serum, provided by the invention, the steps of re-dissolving after nitrogen blowing and re-centrifuging are added in sample treatment, and a scheme that methanol acetonitrile is used as a precipitator and acetonitrile is used as a water re-solution is matched, so that the purity of a sample before testing and the service life of a chromatographic column are greatly improved, meanwhile, the response of a target substance can be effectively improved, the limit of quantitation is reduced, and the detection accuracy is improved; the invention can accurately detect different types of bile acid at the same time by one-time sample introduction, and has short analysis time and high analysis speed.
Drawings
FIG. 1 is a diagram of a bile acid standard according to the present invention;
FIGS. 2-16 are standard curves of 15 bile acids constructed in the present invention;
FIGS. 17A to 17B and 18A to 18C are the results of comparison of the optimization of the nitrogen blowing step in example 2 of the present invention;
FIGS. 19A-19B, 20A-20E, 21A-21B are comparison results for the optimization of the reconstituted solution in example 2 of the present invention;
FIGS. 22A-22C are comparative results of optimization of precipitant optimization in example 2 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
This example provides a method for detecting 15 bile acids in serum.
Reagent, instrument description
(1) The standard substances (cholic acid CA, deoxycholic acid DCA, chenodeoxycholic acid CDCA, ursodeoxycholic acid UDCA, glycocholic acid GCA, glycodeoxycholic acid GDCA, glycochenodeoxycholic acid GCDCA, glycoursodeoxycholic acid GUDCA, taurocholic acid TCA, taurodeoxycholic acid TDCA, taurochenodeoxycholic acid TCDCA, tauroursodeoxycholic acid TUDCA, lithocholic acid LCA, glycolithocholic acid GLCA and taurocholic acid TLCA) are purchased from Shanghai Yan quasi-Biotech Co., Ltd;
(2) internal standard products (cholic acid-d 4 CA-d4, deoxycholic acid-d 4 DCA-d4, chenodeoxycholic acid-d 4 CDCA-d4, ursodeoxycholic acid-d 4 UDCA-d4, glycocholic acid-d 4 GCA-d4, glycodeoxycholic acid-d 4 GDCA-d4, glycochenodeoxycholic acid-d 4 GCDCA-d4, glycodeoxycholic acid-d 4 GUDCA-d4, taurocholic acid-d 4 TCA-d4, taurodeoxycholic acid-d 4 TDCA-d4, taurochenodeoxycholic acid-d 4 TCDCA-d4, taurodeoxycholic acid-d 4 LCA-d4, glycolithocholic acid-d 4, GLCA-d4, taurocholic-d 4-TLD 4) from Haematococcus Biotech Co., Ltd;
(3) methanol: using Fisher Chemical company No. 67-56-1 reagent, the purity is LC/MS grade, and storing in a sealed manner at normal temperature;
(4) acetonitrile: using Fisher Chemical 75-05-8 reagent, the purity is LC/MS grade, and storing at normal temperature;
(5) water: meets the national standard GB/T6682-.
Second, detection scheme
1. Reagent preparation
(1) Preparing a bile acid standard substance working solution group SW, accurately weighing 1mg of the standard substance, fully dissolving the standard substance by using 1mL of methanol to obtain 1mg/mL of mother solution, and diluting the mother solution to obtain the working solution group SW with the following concentration:
(2) preparation of biological Standard Curve samples
Precisely absorbing 90 mu L of blank matrix (bovine serum), adding 10 mu L of working solution SW1-SW8 of standard substance, uniformly mixing by vortex, and preparing a biological standard curve sample group S1-S8 by a pretreatment process, wherein the concentration is as follows:
(3) preparing internal standard working solution ISW
Diluting 1mg/mL of internal standard mother liquor to obtain internal standard working solution ISW with the following concentration:
2. sample processing and standard curve construction:
(1) sample preparation
Sucking 100 mu L of each serum sample to be detected, and freezing and storing for later use;
(2) sample treatment:
taking out a sample to be detected for unfreezing, taking a biological standard curve sample, carrying out the following operations on the biological standard curve sample and the sample to be detected, and during detection, firstly detecting the biological standard curve sample, constructing a standard curve, and then detecting the sample to be detected:
adding 10 μ L of internal standard working solution, adding 200 μ L of precipitant (volume ratio, methanol: acetonitrile 1:1), and mixing with 2500-rotation vortex for 5 min; centrifuging at 14000rpm at 4 deg.C for 10 min; taking 200 mu L of supernatant, and drying at 40 ℃ under nitrogen; redissolving with 100 μ L of a redissolution (volume ratio, acetonitrile: water ═ 3:7), vortexing and shaking for 5 min; finally, centrifuging at 14000rpm for 10min at 4 ℃; 100. mu.L of the supernatant was taken as a test sample.
3. Detection on machine
The test samples were detected in a liquid chromatography (Kerui) -tandem mass spectrometry (Tianjin national HTQ2020) apparatus. A multi-reaction monitoring (MRM) scanning mode is adopted, and chromatographic test conditions are as follows: the mobile phase A is aqueous solution; the mobile phase B is acetonitrile solution; the needle washing liquid is acetonitrile water (1: 1); the chromatographic column was Kinetex C18, 50 × 3mm, 2.6 μm; the column temperature is 50 ℃; the detection time is 10 min; the sample injection amount is 10 mu L; gradient elution conditions: 0-1min, 10% B, 1-1.5min, 17% B, 1.5-3.5min, 25% B, 3.5-4.6min, 30% B, 4.6-6.5min, 30% B, 6.5-6.6min, 35% B, 6.6-8.5min, 35% B, 8.5-8.6min, 10% B, 8.6-10min, 10% B; the flow rate was 0.6 mL/min. The mass spectrum condition is a negative ion mode, an ESI source is adopted, 12psi of collision gas, 16psi of air curtain gas, 50psi of atomization gas, 60psi of auxiliary heating gas, the spraying voltage is-4500V, and the atomization temperature is 500 ℃. The mass spectrum parameters of the compound are as follows:
referring to fig. 1, a bile acid standard graph is shown; FIGS. 2-16 are standard curves for the 15 bile acids constructed
Example 2 comparative experiment
In the invention, the detection effect can be obviously improved by optimizing the sample processing steps, the reagent and the like.
1. Effect of nitrogen blowing step optimization
(1) The response of the sample with the same concentration is obviously improved:
the response after reconstitution recentrifugation step at feed concentration (250ng/mL DCA, 500ng/mL CDCA, 250ng/mL UDCA) without nitrogen blow (before optimization) is shown in FIG. 17A; the response after the reconstitution recentrifugation step after addition of nitrogen purge (after optimization) is shown in figure 17B.
The results of the response comparison are shown in table 1 below:
TABLE 1
According to data comparison, by taking response comparison before and after optimization of deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA) as an example, the response of a target object can be effectively improved by the steps of re-dissolving after nitrogen blowing and then centrifuging, the strength is improved by more than 20 times, and the peak area is improved by more than 15 times. In the invention, the step of re-dissolving after nitrogen blowing is added can play a role of concentrating the sample, and in addition, the optimized re-dissolving solution is used for sample injection instead of directly taking the supernatant for sample injection, so that the proportion of the liquid phase mobile phase can be more perfectly matched, and the peak type is obviously improved compared with that before optimization.
(2) Increase the service life of a chromatography column
The initial mixing peak condition is shown in fig. 18A, the peak pattern change of the sample after 20-pin treatment is shown in fig. 18B after redissolving and re-centrifuging steps without nitrogen blowing (before optimization); after addition of nitrogen purge (after optimization), redissolution and recentrifugation steps, the peak profile change of the 20 pin treated samples was tested as shown in fig. 18C. Specific comparative data are shown in table 2 below.
TABLE 2
It can be seen that the response and the peak type after testing the sample treated by 20 needles after optimization are basically consistent, but the result before optimization is poorer, the peak is widened, the intensity is reduced by about 50%, and the peak area is reduced by about 30%. The main reasons are that the optimized pretreatment sample is cleaner, no pollution is caused to the column after the sample enters the chromatographic column, and on the contrary, the cleanliness of the pretreatment method before the optimization is not high, and protein or phospholipid residues exist in the column after the sample introduction, so that the column efficiency is reduced, the peak type is poor and the response is reduced.
2. Optimized effect of complex solution
(1) Selection of organic phase in double solution
In this experiment, 30% acetonitrile water and 30% methanol water were compared, and the response results of 30% acetonitrile water and 30% methanol water are shown in FIG. 19A and FIG. 19B, respectively, when Cholic Acid (CA) was used as an example and the same concentration was injected. Specific comparative data are shown in table 3 below.
TABLE 3
| Compounding ratio of complex solution | Peak intensity of CA | Area of CA peak |
| 30% acetonitrile Water | 5.13e5 | 1.52e6 |
| 30% methanol water | 4.05e5 | 1.59e6 |
From the results, it can be seen that when acetonitrile and methanol are used as the redissolution, the peak area response is basically consistent, but the peak intensity response of cholic acid is higher when acetonitrile is used, the peak shape is narrower and symmetrical when acetonitrile is used, and the peak width and the front edge peak are wide when methanol is used; further, it is theoretically preferable to select a reconstituted solution in the development of a liquid process without introducing a reagent other than a mobile phase, so it is clear that 30% acetonitrile water as a reconstituted solution is more suitable for the process of the present invention than 30% methanol water.
(2) Compounding ratio of complex solution
In this experiment, responses of 10%, 20%, 30%, 40%, 50% double solutions at the same injection concentration were compared, and responses of 10% -50% acetonitrile aqueous solution are shown in fig. 20, in which lithocholic acid (LCA) is used as an example, fig. 20A: 10%, FIG. 20B: 20%, FIG. 20C: 30%, fig. 20D: 40%, FIG. 20E: 50 percent. Specific comparative data are shown in table 4 below.
TABLE 4
| Compounding ratio of complex solution | LCA peak intensity | Area of LCA peak |
| 10% acetonitrile Water | 1.41e5 | 4.77e5 |
| 20% acetonitrile Water | 5.29e5 | 1.91e6 |
| 30% acetonitrile Water | 9.97e5 | 3.75e6 |
| 40% acetonitrile Water | 9.49e5 | 3.44e6 |
| 50% acetonitrile Water | 5.30e5 | 2.23e6 |
The LCA response was found to be strongest and similar using 30% acetonitrile and 40% acetonitrile. On this basis, the response of Cholic Acid (CA) was compared: as shown in FIGS. 21A (30% acetonitrile water) and 21B (40% acetonitrile water), the data for the comparison are shown in Table 5 below.
TABLE 5
| Compounding ratio of complex solution | Peak intensity of CA | Area of CA peak |
| 30% acetonitrile Water | 5.13e5 | 1.52e6 |
| 40% acetonitrile Water | 4.02e5 | 1.38e6 |
It can be seen that the highest response of the target was obtained when 30% acetonitrile water was used as the reconstitution solution.
3. Effect of optimization of precipitant
In this experiment, using tauroursodeoxycholic acid (TUDCA) as an example, responses to methanol, acetonitrile, and methanol acetonitrile (volume ratio, 1:1) as precipitants were tested, and the results of treating samples of the same concentration were shown in fig. 22A for the methanol precipitant, fig. 22B for the acetonitrile precipitant, fig. 22C for the methanol acetonitrile (1:1) precipitant, and the specific data are shown in table 6 below.
It can be seen that of the results of the tauroursodeoxycholic acid (TUDCA) test with the lowest concentration of 15 bile acid species, the use of methanol acetonitrile (1:1) as the precipitant works best, with a response 79% higher than that of methanol precipitant and 63% higher than that of acetonitrile precipitant, and the TUDCA lower limit can be detected only with methanol acetonitrile precipitant.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (10)
1. A method for detecting 15 bile acids in serum, the 15 bile acids comprising: cholic acid CA, deoxycholic acid DCA, chenodeoxycholic acid CDCA, ursodeoxycholic acid UDCA, glycocholic acid GCA, glycodeoxycholic acid GDCA, glycochenodeoxycholic acid GCDCA, glycoursodeoxycholic acid GUDCA, taurocholic acid TCA, taurodeoxycholic acid TDCA, taurochenodeoxycholic acid TCDCA, tauroursodeoxycholic acid TUDCA, lithocholic acid LCA, glycolithocholic acid GLCA, taurocholic acid TLCA, wherein the method comprises the following steps:
1) sample treatment: taking a sample to be detected, adding a precipitator into the sample, uniformly mixing the mixture by vortex, and centrifuging the mixture; taking supernatant, drying the supernatant under nitrogen, redissolving the supernatant by using redissolution, carrying out vortex oscillation and centrifugation, and taking the supernatant as a test sample; wherein the redissolution is a mixed solution of acetonitrile and water;
2) and detecting the test sample by adopting a liquid chromatography-tandem mass spectrometry device.
2. The method according to claim 1, wherein the precipitating agent is a mixture of methanol and acetonitrile.
3. The method for detecting 15 bile acids in serum according to claim 2, wherein the volume ratio of methanol to acetonitrile in the precipitating agent is 1: 1.
4. The method for detecting 15 bile acids in serum according to claim 3, wherein the volume fraction of acetonitrile in the complex solution is 20-40%.
5. The method for detecting 15 bile acids in serum according to claim 4, wherein the volume fraction of acetonitrile in the complex solution is 30%.
6. The method for detecting 15 bile acids in serum according to claim 1, wherein the step 1) is specifically as follows: taking 100 mu L of serum sample to be detected, adding 10 mu L of internal standard working solution and 200 mu L of precipitator into the serum sample, uniformly mixing the internal standard working solution and the precipitator by vortex at 2500rpm for 5min, and centrifuging the mixture at 14000rpm at 4 ℃ for 10 min; drying 200 μ L of supernatant under nitrogen at 40 deg.C, redissolving with 100 μ L of redissolution, and vortex shaking for 5 min; the cells were centrifuged at 14000rpm for 10min at 4 ℃ and 100. mu.L of the supernatant was used as a test sample.
7. The method for detecting 15 bile acids in serum according to claim 1, wherein in the step 2), the mobile phase A in the liquid chromatography is an aqueous solution; the mobile phase B is acetonitrile solution; the needle washing liquid is acetonitrile water solution with 50 percent of volume fraction.
8. The method for detecting 15 bile acids in serum according to claim 7, wherein the gradient elution procedure adopted in step 2) is as follows: 0-1min, 10% B, 1-1.5min, 17% B, 1.5-3.5min, 25% B, 3.5-4.6min, 30% B, 4.6-6.5min, 30% B, 6.5-6.6min, 35% B, 6.6-8.5min, 35% B, 8.5-8.6min, 10% B, 8.6-10min, 10% B; the flow rate was 0.6 mL/min.
9. The method for detecting 15 bile acids in serum according to claim 8, wherein in the step 2), the chromatographic column is Kinetex C18.
10. The method for detecting 15 bile acids in serum according to claim 9, wherein in the step 2), the mass spectrum conditions are as follows: in the negative ion mode, an ESI source is adopted, collision gas is 12psi, air curtain gas is 16psi, atomization gas is 50psi, auxiliary heating gas is 60psi, the spraying voltage is-4500V, and the atomization temperature is 500 ℃.
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| CN114894926A (en) * | 2022-04-27 | 2022-08-12 | 天津国科医工科技发展有限公司 | Bile acid detection method based on dry blood paper sheet method |
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