Patent Application
20250044305
This application is based upon and claims priority to Chinese Patent Application No. 202310982084.2, filed on Aug. 4, 2023, the entire contents of which are incorporated herein by reference.
The present invention relates to the technical field of detection, and particularly to a Liquid Chromatography Tandem-mass Spectrometry (LC-MS/MS) analysis method for detecting 11 vitamins D in blood.
Vitamin D is a lipid soluble vitamin, which plays an important role in bone homeostasis and immune regulation. Vitamin D deficiency is related to cardiovascular disease, kidney disease, cancer and other diseases. In addition, vitamin D supplementation can reduce the risk of fracture, especially in postmenopausal women and elderly men. There are two natural forms of vitamin D, comprising vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 can be synthesized by ultraviolet radiation, while vitamin D2 cannot be synthesized in vivo, so vitamin D2 can only be obtained from fortified foods and dietary supplements. Vitamin D is metabolized into 25-(OH)D by 25-hydroxylase in the liver, and 25-(OH)D is considered as the best biomarker to reflect the status of vitamin D in vivo. 25-(OH)D is metabolized into 1,25-(OH)2D in kidney, which is called active vitamin D, and can promote bone health by maintaining calcium and phosphate levels in a steady state. In addition, 25-(OH)D can be metabolized into 24,25-(OH)2D by 25-(OH)D-24-hydroxylase (CYP24), and its concentration is 2%-20% of the total 25-(OH)D. Previously, 24,25-(OH)2D was considered as inactive vitamin D. With the further study of 24,25-(OH)2D, it has been reported that serum 24,25-(OH)2D can predict the level of vitamin D more than 25-(OH)D. In addition, a ratio between 24,25-(OH)2D and 25-(OH)D is called a vitamin D metabolite ratio, and the vitamin D metabolite ratio has been suggested as a candidate biomarker of vitamin D status and a functional biomarker of bone health as the vitamin D metabolite ratio has no significant difference between races.
As is known, all major vitamin D metabolites can be epimerized at C3 position, and the amount of C3 epimers is higher in pregnant women and newborns. The C3 epimers and the vitamin D metabolites have the same precursor ions and product ions, which may lead to false positive results if they are not effectively separated. A study on infants and adults showed that 3-epi-25-(OH)D3 caused 90/(less than one year) and 3% (1 to 94 years old) patients to be wrongly diagnosed as vitamin D deficiency in the total concentration of 25-(OH)D. A previous study showed that if the epimers were not effectively separated by analytical methods, these interferences might account for 14%-55% of the total vitamin D level, so it is necessary to effectively separate the epimers in order to accurately monitor the vitamin level in vivo.
At present, a commonly used method to determine the content of the vitamin D in serum is by using LC-MS/MS. However, most of the reported literatures only detect 25-(OH)D, but not the key metabolites and epimers of the whole metabolic pathway of vitamin D, and most of the detection methods have some problems, such as complicated pre-treatment operation, large sample demand and long analysis time, which are not suitable for high-throughput sample detection.
In view of this, the present invention is provided.
In order to solve the above-mentioned technical problems, the present invention provides an LC-MS/MS analysis method for detecting 11 vitamins D in blood. Specifically, the 11 vitamins D and metabolites comprise: D2, D3, 1,25-(OH)2D2, 1,25-(OH)2D3, 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25(OH)D3, 24,25-(OH)2D2, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3.
The present invention provides a method for simultaneously detecting 11 vitamins D by LC-MS/MS, wherein the 11 vitamin comprises D2, D3, 1,25-(OH)2D2, 1,25-(OH)2D3, 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D2, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3, and the method at least comprises the following steps of:
Optionally, S1 comprises:
Optionally, the derivatization reagent is selected from 50-500 μg/mL 4-Phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD) solution, and the derivatization lasts for 20-60 min; the reconstitution solution is selected from an aqueous solution containing 0.05%-0.2% formic acid by volume and 60%-100% methanol by volume; the protein precipitant is selected from methanol or anhydrous ethanol; and the extractant is a mixed solvent of normal hexane and methyl tert-butyl ether with a volume ratio of 2:1-4:1.
Optionally, in S12, a volume ratio of the standard working liquid I to the internal standard working liquid I is 1:1-2:1; a volume ratio of the internal standard working liquid I to the derivatization reagent is 1; 10-1:20; a volume ratio of the standard working liquid I to the standard working liquid II is 1:1; and a volume ratio of the standard working liquid II to the internal standard working liquid II and the reconstitution solution is 2:1:7.
Optionally, in S2, the internal standard working liquid I, the internal standard working liquid II, the sample to be detected and the protein precipitant are mixed, and a volume ratio of the internal standard working liquid I to the internal standard working liquid II is 1:1; a volume ratio of the sample to be detected to the protein precipitant is 1:1-2:1; a volume ratio of the supernatant for derivatization to the remaining part of supernatant is 1:1; and a volume ratio of the reconstitution solution to the sample to be detected is 1:1-1:2.
Optionally, in S2, the extracting twice with the extractant twice, comprises: a volume ratio of the sample to be detected to the extractant being 2:6.5-1:5; and a volume ratio of the extractant used during the second extraction to the extractant used during the first extraction being 0.8-1:1.
Compared with the prior art, the technical solutions provided by the embodiments of the present invention has the following advantages.
the detection method of the present invention detects the derivative products of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3, and the other 7 vitamins can be directly detected without derivatization, thus ensuring that th11 vitamins D can be extracted with one injection, and ensuring the pg-level sensitivity of the detection method.
Because the substance to be detected in the present invention is endogenous, the preparation of the standard curve of the present invention does not add matrix, and meanwhile, a matrix-free effect is ensured, and a recovery rate is good.
In preferred embodiments, the chromatographic conditions of the present invention can ensure that the 11 vitamins D have good chromatographic resolution, ensure the absolute separation of epimers and isomers, and meanwhile, the derivative products will not interfere with the separation of non-derivatives.
In another preferred technical solution, a standard dissolving reagent can ensure the solubility and storage stability of the standard.
Reference numeral 1 is a chromatographic peak of 25-(OH)D2, with a retention time of 5.92 min; reference numeral 2 is a chromatographic peak of 3-epi-25-(OH)D2, with a retention time of 6.09 min; reference numeral 3 is a chromatographic peak of 25-(OH)D3, with a retention time of 5.64 min; reference numeral 4 is a chromatographic peak of 3-epi-25-(OH)D3, with a retention time of 5.92 min; reference numeral 5 is a chromatographic peak of 24,25-(OH)2D3, with a retention time of 3.98 min; reference numeral 6 is a chromatographic peak of 3-epi-24,25-(OH)2D3, with a retention time of 4.15 min; reference numeral 7 is a chromatographic peak of 24,25-(OH)2D2, with a retention time of 4.45 min; reference numeral 8 is a chromatographic peak of D3, with a retention time of 6.82 min; reference numeral 9 is a chromatographic peak of D2, with a retention time of 6.82 min; reference numeral 10 is a chromatographic peak of 1,25-(OH)2D3, with a retention time of 3.42 min; and reference numeral 11 is a chromatographic peak of 1,25-(OH)2D2, with a retention time of 3.64 min;
In order to better understand the above objects, features and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, in case of no conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other. In the following description, many specific details are set forth in order to fully understand the present invention, but the present invention may be implemented in other ways different from those described herein. Obviously, the embodiments described in the specification are merely a part of, rather than all of, the embodiments of the present invention.
The embodiments of the present invention provide a method for simultaneously detecting 11 vitamins D by LC-MS/MS. The 11 vitamins D comprise D2, D3, 1,25-(OH)2D2, 1,25-(OH)2D3, 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D2, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3, and the method at least comprises the following steps of:
The sample to be detected in the embodiments of the present invention is a serum sample or a plasma sample, and the derivative products of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 are detected by ingenious design of the pre-treatment steps in the embodiments of the present invention, and the other 7 vitamins D can be directly detected without derivatization, so that the 11 vitamins D can be simultaneously detected by one injection. The detection process is simple and fast, the experimental cost is reduced, and the analysis time is short, which is more conducive to the detection of large-volume samples.
In the research process of the embodiments of the present invention, it is found that if D2 and D3 are directly detected without derivatization, a matrix inhibition effect is too strong, and the pg-level sensitivity of the detection method cannot be guaranteed. Meanwhile, as 1,25-(OH)2D2 and 1,25-(OH)2D3 have low contents in blood, and are not easy to ionize, a response value during detection is low, and the pg-level sensitivity of the detection method cannot be guaranteed.
Therefore, the embodiments of the present invention choose to detect the derivative products of these four substances, so as to ensure that the detection sensitivity of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 can reach the pg-level.
The reason why the derivative products of all substances are not completely detected in the present invention is that the analytical method needs to separate three groups of epimers, which are 25-(OH)D2, 3-epi-25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D3, 24,25-(OH)2D3, and 3-epi-24,25-(OH)2D3. After derivatization, a polarity difference of the three groups of epimers becomes smaller, and under the existing gradient elution conditions, the three groups of epimers cannot achieve baseline separation, so the pre-treatment without derivatization is adopted in the present invention to ensure the separation of the three groups of epimers.
Specifically, preparation methods of the solutions used in the embodiments of the present invention are as follows:
A preparation method of the standard working liquid 11 is: dissolving 24,25-(OH)2D2 solid standard with 70%-100% methanol aqueous solution or/70%-100% ethanol aqueous solution by volume to obtain a mother liquid, and mixing the mother liquid with 25-(OH)D2, 25-(OH)D3, 3-epi-25-(OH)D2, 3-epi-25-(OH)D3, 24,25-(OH)2D3 and 3-epi-24,25-(OH)2D3 with known concentrations, and continuously diluting with 70%6-100% methanol aqueous solution or 70%-100% ethanol aqueous solution by volume to obtain the standard working liquid II; and
In the preparation process of the standard working liquids, different diluents are selected according to different properties of the standards, thus ensuring the solubility and storage stability of the standards.
The preparation method of the internal standard working liquid I is: diluting commercially available liquid standards of isotopic internal standards of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 with anhydrous ethanol or methanol to obtain the internal standard working liquid I; and
In the preparation process of the internal standard working liquids, different diluents are selected according to different properties of the internal standards, thus ensuring the solubility and storage stability of the internal standards.
As an improvement of the embodiments of the present invention, the derivatization reagent is selected from 50-500 μg/mL PTAD solution, and the concentration of the PTAD solution is preferably 100-200 μg/mL, more preferably 100-150 pg/mL, and may specifically be 100 μg/mL, 120 μg/mL, 125 μg/mL, 130 pg/mL, 135 μg/mL or 140 μg/mL; and more further preferably 125 μg/mL. The selection of this derivatization reagent can better eliminate a matrix effect in the detection of D3 and D2 samples, and greatly improve the detection sensitivity of 1,25-(OH)2D2 and 1,25-(OH)2D3. The derivatization may last for 20-60 min, and may last for 30 min specifically.
As an improvement of the embodiments of the present invention, the reconstitution solution is selected from an aqueous solution containing 0.05%-0.2% formic acid by volume and 60%-100% methanol by volume; further preferably, the reconstitution solution is selected from an aqueous solution containing 0.08%-0.15% formic acid by volume and 65%-75% methanol by volume; and more preferably, the reconstitution solution is selected from an aqueous solution containing 0.1% formic acid by volume and 70% methanol by volume. Using the reconstitution solution can ensure the solubility of the 11 vitamins D and minimize the matrix effect.
As an improvement of the embodiments of the present invention, the protein precipitant is selected from methanol or anhydrous ethanol, which can ensure the subsequent extraction effect of the 11 vitamins D.
As an improvement of the embodiments of the present invention, the extractant is a mixed solvent of normal hexane and methyl tert-butyl ether with a volume ratio of 2:1-4:1, may further be a mixed solvent of the n-hexane to the methyl tert-butyl ether with a volume ratio of 2.5:1, 3:1, 3.5:1 or 4:1, and more preferably, a mixed solvent of the volume ratio of the n-hexane and the methyl tert-butyl ether with a volume ratio of 4:1. Screening experiments show that the extraction effect can be improved and the pg-level sensitivity of endogenous substances detection can be ensured by using the extractant for twice extraction.
As an improvement of the embodiments of the present invention, in S12, a volume ratio of the standard working liquid I to the internal standard working liquid I is 1:1-2:1; a volume ratio of the standard working liquid I to the derivatization reagent is 1:10-1:20; a volume ratio of the standard working liquid I to the standard working liquid II is 1:1; and a volume ratio of the standard working liquid II to the internal standard working liquid 11 and the reconstitution solution is 2:1:7.
As an improvement of the embodiments of the present invention, the conditions of S12 are as follows: the derivatization reagent is added, and after vortex mixing at a rotating speed of 1,500-2,500 rpm for 30 s to 1 min, derivatization treatment is carried out, and after adding the reconstitution solution, vortex mixing is carried out at a rotating speed of 1,500-2,500 rpm for 1-3 min to obtain the sample solution for standard curve.
As one specific embodiment of the embodiments of the present invention, the specific conditions of S12 are as follows: the derivatization reagent is added, and after vortex mixing at a rotating speed of 2,000 rpm for 30 s, derivatization treatment is carried out, and after adding the reconstitution solution, vortex mixing is carried out at a rotating speed of 2,000 rpm for 1 min to obtain the sample solution for standard curve.
As an improvement of the embodiments of the present invention, in S12, the standard working liquid 1 and the internal standard working liquid 1 are prepared into the standard curve working liquid with gradient concentration by using methanol/anhydrous ethanol.
As one specific embodiment of the embodiments of the present invention, 20 μL of the standard working liquid I and 10 μL of the internal standard working liquid I with gradient concentration are taken to prepare 8 standard curve working liquids with different concentrations.
The standard curve working liquid is blow-dried by nitrogen, added with 200 μL of PTAD with a concentration of 125 μg/mL, subjected to vortex mixing at a rotating speed of 2,000 rpm for 30 s, derived for 30 min, the derived standard substance working liquid is blow-dried in nitrogen, 20 μL of the standard working liquid II and 10 μL of the internal standard working liquid II by using a pipette, added into a centrifugal tube after blow-drying by nitrogen, added with 70 μL of the reconstitution solution, subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min to obtain the sample solution for standard curve for on-machine detection.
As an improvement of the embodiments of the present invention, in S2, the internal standard working liquid I, the internal standard working liquid II, the sample to be detected and the protein precipitant are mixed, and a volume ratio of the internal standard working liquid I to the internal standard working liquid II is 1:1; a volume ratio of the sample to be detected to the protein precipitant is 1:1-2:1; a volume ratio of the supernatant for derivatization to the remaining part of supernatant is 1:1-9:10; and a volume ratio of the reconstitution solution to the sample to be detected is 1:1-1:2.
As an improvement of the embodiments of the present invention, the internal standard working liquid I, the internal standard working liquid 11, the sample to be detected and the protein precipitant are subjected to vortex mixing at a rotating speed of 1,500-2,500 rpm for 3-5 min, extracted twice with the extractant to obtain the total supernatant, the derivatization reagent is added to part of the total supernatant, and derivatization treatment is carried out after vortex mixing at a rotating speed of 1,500-2,500 rpm for 30 s to 1 min, the remaining part of the total supernatant is added after blow-drying, then the mixture is blow-dried again, added with the reconstitution solution, subjected to vortex mixing at a rotating speed of 1,500-2,500 rpm for 1-3 min, centrifuged at a rotating speed of 12,000-14,000 rpm for 5-10 min, and the supernatant is taken as the incoming sample.
As a specific embodiment of S2 of the embodiments of the present invention, the specific conditions of S2 are as follows: the internal standard working liquid 1, the internal standard working liquid II, the sample to be detected and the protein precipitant are subjected to vortex mixing at a rotating speed of 2,000 rpm for 3 min, extracted twice with the extractant to obtain the total supernatant, the derivatization reagent is added to part of the total supernatant, and derivatization treatment is carried out after vortex mixing at a rotating speed of 2,000 rpm for 30 s, the remaining part of the total supernatant is added after blow-drying, then the mixture is blow-dried again, added with the reconstitution solution, subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min, centrifuged at a rotating speed of 14,000 rpm for 5 min, and the supernatant is taken as the incoming sample.
As an improvement of the embodiments of the present invention, in S2, the extractant is used for extraction twice, during the first extraction, a volume ratio of the sample to be detected to the extractant is 2:6.5-1:5; and a volume ratio of the extractant used during the second extraction to the extractant used during the first extraction is 0.8-1:1.
The specific conditions of the extraction in S2 are as follows: during the first extraction, the internal standard working liquid, the sample to be detected and the protein precipitant are mixed uniformly, and then the extractant is added, and the mixture is subjected to vortex mixing at a rotating speed of 1,500-2,500 rpm for 3-5 min, and centrifuged at a rotating speed of 12,000-14,000 rpm for 5-10 min, and the supernatant is taken as supernatant I; the extractant is added into the centrifuged precipitate, subjected to vortex mixing at a rotating speed of 1,500-2,500 rpm for 3-5 min, centrifuged at a rotating speed of 12,000-14,000 rpm for 5-10 min, the supernatant is taken as a supernatant U, and the supernatant I and the supernatant II are combined to obtain the total supernatant.
As a specific embodiment of the extraction method according to the embodiments of the present invention, during the first extraction, the internal standard working liquid, the sample to be detected and the protein precipitant are mixed uniformly, and then the extractant is added, and the mixture is subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, and centrifuged at a rotating speed of 14,000 rpm for 10 min, and the supernatant is taken as supernatant I; the extractant is added into the centrifuged precipitate, and the mixture is subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, centrifuged at a rotating speed of 14,000 rpm for 5-10 min, the supernatant is taken as a supernatant II, and the supernatant I and the supernatant II are combined to obtain the total supernatant.
As one specific embodiment of the embodiments of the present invention, S2 comprises: taking 10 μL of internal standard working liquid I and 10 μL of internal standard working liquid U, adding 200 μL of serum sample to be detected, adding 100 μL of protein precipitant, carrying out vortex mixing at a rotating speed of 2,000 rpm for 3 min, adding 650 μL of extractant, carrying out vortex mixing at a rotating speed of 2,000 rpm for 5 min, centrifuging at a rotating speed of 14,000 rpm for 10 min, taking 600 μL of supernatant I, adding 650 μL of extractant to the centrifuged precipitate, carrying out vortex mixing at a rotating speed of 2,000 rpm for 5 min, centrifuging at a rotating speed of 14,000 rpm for 10 min, taking the supernatant as the supernatant 11, and combining the supernatant I and the supernatant II to obtain the total supernatant. 600 μL of the total supernatant was put into a 1.5 mL centrifuge tube, added with 200 μL of PTAD with a concentration of 125 μg/mL, subjected to vortex mixing at a rotating speed of 2,000 rpm for 30 s, derivatized for 30 min, and then blow-dried by nitrogen. The remaining 600 μL supernatant of the total supernatant is added into the derivatized centrifuge tube, blow-dried with nitrogen, added with 100 μL of reconstitution solution, subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min, centrifuged at a rotating speed of 14,000 rpm for 5 min, and 90 μL supernatant is taken as the solution to be detected.
The five groups of isomers in the substances to be detected in the present invention are: 25-(OH)D2 and 3-epi-25-(OH)D2, 25-(OH)D3 and 3-epi-25-(OH)D3, 24,25-(OH)2D3 and 3-epi-24,25-(OH)2D3, 24,25-(OH)2D3 and 1,25-(OH)2D3 as well as 24,25-(OH)2D2 and 1,25-(OH)2D2. Therefore, it is necessary to realize chromatographic separation of the above substances. Therefore, high-performance liquid phase conditions are studied in the embodiments of the present invention, thereby realizing the complete separation of all substances before and after derivatization, avoiding ion crosstalk and improving the detection accuracy.
As an improvement of the embodiments of the present invention, the high-performance liquid chromatography conditions when using the LC-MS/MS for detection are as follows: the chromatographic column can be pentafluorophenyl chromatographic column with a particle size of 2.6 μm; Specifically, Phenomenex Kinetex F5 100A chromatographic column (2.6 μm, 3.0 mm×50 mm) may be used.
The mobile phases may be as follows: phase A is an aqueous solution containing 0.05%-0.2% formic acid and 1 mM-5 mM ammonium formate or ammonium acetate; and phase B is a methanol solution containing 0.05%-0.2% formic acid and 1 mM-5 mM ammonium formate or ammonium acetate. Further preferably, the phase A is an aqueous solution containing 0.1% formic acid and 1 mM ammonium formate; and phase B is a methanol solution containing 0.1% formic acid and 1 mM ammonium formate.
The gradient elution conditions are as follows:
Preferably, the concentration A1 is selected from 38%-32%, the concentration B1 is selected from 62%-68%, and the concentration A1+the concentration B1=100%;
Further preferably, the gradient elution conditions are as shown in Table 1.
As an improvement of the embodiments of the present invention, the mass spectrum conditions when using the LC-MS/MS for detection are as follows: using electrospray ion source (ESI) and positive ion mode for multi-reaction monitoring under ionspray voltage: 5,000 V-5,500 V, preferably 5,500 V; ion source temperature: 300-400° C., preferably 350° C.; atomizing gas: 45-55 psi, preferably 50 psi; auxiliary gas: 25-35 psi, preferably 30 psi; curtain gas: 20-25 psi, preferably 20 psi; and collision gas: 8-10 psi, preferably 9 psi.
Ion pair parameters are as shown in Table 2.
The embodiment provided a liquid chromatogram analysis method for contents of 11 vitamins D, which comprised the following steps.
D3 and D2 standards were accurately weighed and dissolved with anhydrous ethanol to obtain mother liquids with corresponding concentrations. 1,25-(OH)2D2 and 1,25-(OH)2D3 were liquid standards in the market, and the mother liquid concentrations were known. A certain volume of the mother liquids of the above three vitamins were taken and diluted with anhydrous ethanol to obtain corresponding intermediate liquids. The specific mother liquid/intermediate liquid concentrations were shown in Table 3.
A certain volume of the mother liquids/intermediate liquids of the above four vitamins D were taken, and continuously diluted with anhydrous ethanol to obtain an L8 point of the standard working liquid I, as shown in Table 4.
Preparation process of L7-L1 point (ng/mL): from L8 point, the standard working liquid I was obtained by gradual dilution with anhydrous ethanol, as shown in Table 5.
A certain amount of 24,25-(OH)2D2 standard were accurately weighed and dissolved with 70% methanol water to obtain mother liquids with corresponding concentrations. The other six vitamins D were all liquid standards in the market with known concentrations. A certain volume of the mother liquids of the above five vitamins were taken and diluted with 70% a methanol to obtain corresponding intermediate liquids. The specific mother liquid/intermediate liquid concentrations were shown in Table 6.
A certain volume of the mother liquids/intermediate liquids shown in Table 6 were taken, and diluted with 70% methanol aqueous solution to obtain an L8 point of the standard working liquid II, which were specifically shown in Table 7.
Preparation process of L7-L1 point: from L8 point, the standard working liquid 11 was obtained by gradual dilution with 70% i methanol, as shown in Table 8.
D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 isotopic internal standards were all commercially available liquid standard standards with known concentrations. A certain volume of the internal standard mother liquids of the above four vitamins were taken and diluted with anhydrous ethanol to obtain corresponding internal standard intermediate liquids. The specific mother liquid/intermediate liquid concentrations were shown in Table 9.
The internal standard intermediate liquid shown in Table 9 was continuously diluted with anhydrous ethanol to obtain the internal standard working liquid 1, and the specific preparation process was shown in Table 10.
The internal standard intermediate liquid shown in Table 11 was continuously diluted 70% methanol aqueous solution to obtain the internal standard working liquid 11, and the specific preparation process was shown in Table 12.
The prepared standard working liquids and internal standard working liquids were marked with names and concentrations, and stored at −80° C., and valid for at least 3 months.
20 μL of standard working liquid I and 10 μL of internal standard working liquid I were respectively taken by a pipette and placed in a 1.5 mL centrifuge tube to prepare eight standard curve working liquids with different concentrations, blow-dried by nitrogen, added with 200 μL of PTAD with a concentration of 125 μg/mL, and subjected to vortex mixing at a rotating speed of 2,000 rpm for 30 s, and derived for 30 min. The derived standard working liquids were blow-dried by nitrogen. 20 μL of standard working liquid II and 10 μL of internal standard working liquid II were respectively taken by a pipette and placed in a centrifuge tube blow-dried by nitrogen, and then added with 70 μL of 70% methanol aqueous solution (0.1% formic acid), and subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min to obtain the sample solution for standard curve.
The sample solution for standard curve was detected by using an LC-MS/MS to obtain standard solution chromatograms of 11 vitamins D (D3, D2, 1,25-(OH)2D3, 1,25-(OH)2D2, 25-(OH)D3, 25-(OH)D2, 3-epi-25(OH)D3, 3-epi-25-(OH)D2, 24,25-(OH)2D3, 24,25-(OH)2D2 and 3-epi-24,25-(OH)2D3) with eight different concentrations. Peak areas of the 11 vitamins D and the internal standards were obtained from the standard solution chromatograms of the above 11 vitamins D and the internal standards respectively. A ratio of the peak areas of the standard solutions of the 11 vitamins D with eight different concentrations to the peak area of the internal standards as an ordinate y of the standard curve equation, and taking a ratio of the concentration of the standard working liquid of the 11 vitamins D to the concentration of the internal standard working liquid as an abscissa x of the standard curve equation, the data of the eight different concentrations detected above were linearly regressed, the standard curve equation was fitted as y=a*x+b, and the linear equation coefficients a and b were obtained.
10 μL of internal standard working liquid I and 10 μL of internal standard working liquid II were respectively taken with a pipette first and then put into a 1.5 mL centrifuge tube, 200 μL of serum sample to be detected was taken by a pipette, added with 100 μL of methanol, subjected to vortex mixing at a rotating speed of 2,000 rpm for 3 min, added with 650 μL of n-hexane: methyl tert-butyl ether=4:1, subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, and centrifuged at a rotating speed of 14,000 rpm for 10 min, 600 μL of supernatant I was added into a clean 1.5 mL centrifuge tube, and then 650 μL of n-hexane: methyl tert-butyl ether=4:1 was added into the 1.5 mL centrifuge tube, subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, and centrifuged at a rotating speed of 14,000 rpm for 10 min to obtain a supernatant II. The supernatant I and the supernatant II were combined to obtain a total supernatant. 600 μL of the total supernatant was put into a 1.5 mL centrifuge tube, added with 200 μL of PTAD with a concentration of 125 μg/mL, subjected to vortex mixing at a rotating speed of 2,000 rpm for 30 s, derivatized for 30 min, and then blow-dried by nitrogen. The remaining 600 μL supernatant of the total supernatant was added into the derivatized centrifuge tube, blow-dried with nitrogen, added with 100 μL of reconstitution solution (water solution containing 0.1% formic acid and 70% methanol by volume) for redissolution, subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min, centrifuged at a rotating speed of 14,000 rpm for 5 min, and 90 μL supernatant was taken as the solution to be detected.
The incoming sample in the step (3) was detected by using an LC-MS/MS to obtain chromatograms of the 11 vitamins D of the incoming sample and the internal standard, and obtain peak areas of the 11 vitamins D and the internal standard from the chromatograms of the 11 vitamins D and the internal standard. Substituting a ratio y of the peak areas of the 11 vitamins D to the internal standard into a standard curve equation that y=a*x+b in the step (2), relative concentrations x of the 11 vitamins D in the incoming sample and the internal standard were obtained by calculation, and the working liquid concentration of the internal standard was known, so the concentrations of the 11 vitamins D in the blood sample to be detected were calculated.
Chromatographic column used for chromatographic analysis was phenomenex Kinetex F5 100A chromatographic column (2.6 μm, 3.0 mm×50 mm). A mobile phase consisted of water (0.1% formic acid+1 mM ammonium formate) and methanol (0.1% formic acid+1 mM ammonium formate), with gradient elution (as shown in Table 1) at a flow rate of 0.3 mL/min, a column temperature of 30° C., and a sample volume of 20 μL; and an analysis time was 8 min.
A mass spectrum detector was SCEIX AB6500 detector, adopting electrospray ion source (ESI), positive ion mode, multi-reaction monitoring (MRM), an ionspray voltage (IonSpray Voltage) of 5,500 V; an ion source temperature (TEM) of 350° C.; an atomizing gas (Gas1) of 50 psi; an auxiliary gas (Gas2) of 30 psi; a curtain gas (CUR) of 20 psi; and a collision gas of 9 psi. Ion pair parameters were shown in Table 2.
The obtained chromatograms were shown in
The technical method in Example 1 was demonstrate as follows:
11 vitamins D were diluted into eight standard curve working liquids with different concentrations in proportion, and detected according to the method of Embodiment 1. With a ratio of a quantitative ion peak area of a target analyte to a peak area of a corresponding isotopic internal standard as an ordinate and a concentration of the standard substance as an abscissa, a standard curve was drawn, and 1/x2 was used for curve weight when fitting the standard curve. An obtained linear range was as shown in Table 13.
It could be seen from Table 13 that linear relationships of the 11 target analytes were good in the respective linear ranges thereof (r>0.9962).
11 vitamin D standard working liquids were prepared into three concentrations-; low, medium and high, and recovery rate and precision experiments were carried out. The samples were determined according to the method in Example 1, and five batches were repeatedly analyzed and determined. The recovery rates and precision of 11 vitamins D were shown in Table 14 respectively.
Note: The isotope internal standard of 3-epi-24,25-(OH)2D3 was not purchased, and 24,25-(OH)2D3-D6 was used as the internal standard thereof in methodological evaluation.
Based on the above verification tests, the average recovery rate of the 11 vitamins D in the range of low, medium and high addition levels was 94.23%-106.88%, and the relative standard deviation was 1.13%-6.15%. The technical indexes such as recovery rate and precision met the requirements. The method for detecting the content of 11 vitamins D in blood had good reproducibility and high recovery rate, which improved the accuracy of the detection results.
The detection limit was estimated by the signal-to-noise ratio (3 times S/N) of the low concentration point of the standard curve; the contents of 11 vitamins D in actual serum samples were determined, diluted with 4% BSA in different proportions according to the estimated detection limit concentration, and carry out detection according to the method in Embodiment 1. Each concentration was repeatedly detected for 6 times, it was satisfied that the relative standard deviation was less than or equal to 20%, and the lowest concentration with signal-to-noise ratio greater than 10:1 was set as the quantitative lower limit, and the experimental results obtained were shown in Table 15.
The embodiment was used to illustrate a comparative experiment of the previous treatment method.
The detection was carried out according to the method of Embodiment 1, with a difference that protein precipitants were different in type and dosage. Specifically, methanol and anhydrous ethanol were selected as the protein precipitants respectively, and ratios of addition volumes of the protein precipitants to a serum volume were 1:2 and 1:1 respectively. Internal standard response values of 11 vitamins of the samples after pre-treatment were compared, and a comparison diagram of the experimental results was shown in
It could be seen from
In actual blood samples, contents of 1,25-(OH)2D3 and 1,25-(OH)2D2 in 11 vitamins D were the lowest, so that a final dosage of the protein precipitant was selected to be that methanol:serum=1:2 under overall consideration.
The embodiment was used to illustrate comparative experiments of extractants and derivation reagents and conditions.
Three extractants were selected, which were n-hexane, a mixed solvent of n-hexane: methyl tert-butyl ether=4:1, a mixed solvent of n-hexane: ethyl acetate=4:1, a mixed solvent of n-hexane: methyl tert-butyl ether=2:1, and a mixed solvent of n-hexane: methyl tert-butyl ether=1:1. Internal standard response values of 11 vitamins of samples after pre-treatment were compared, and the specific results were shown in
It can be seen from
PATD was selected as the extractant to carry out an optimal experiment on dosages of the derivation reagent. The dosages of the derivation reagent were 20 μg/mL, 50 μg/mL, 125 μg/mL and 500 μg/mL respectively, and the specific optimization results were shown in
It could be seen from
In order to give consideration to the overall response of four substances and consider the cost, the concentration of the derivation reagent was finally selected to be 125 μg/mL.
The derivation times of 10 min, 20 min, 30 min and 60 min were investigated respectively, and the specific optimization results of the derivation times were shown in
It could be seen from
The embodiment was used to illustrate comparative experiments of liquid phase conditions.
The obtained chromatogram was shown in
It could be seen from
The obtained chromatogram was shown in
It could be seen from
The obtained chromatogram was shown in
It could be seen from
The obtained chromatogram was shown in
It could be seen from
The obtained chromatograms were shown in
It could be seen from
A pure product, a sample and an L8 point of a standard curve were detected according to the method of Embodiment 1, while the differences were that: (2) the calibration of the standard curve working liquid and (3) the treatment of sample to be detected were carried out by the following methods.
Compared with the calibration method of the standard curve working liquid: 20 μL of standard working liquid I, 10 μL of internal standard working liquid I, 20 μL of standard working liquid II and 10 μL of internal standard working liquid II were respectively taken by a pipette to prepare eight standard curve working liquids with different concentrations, blow-dried by nitrogen, added with 70 μL of 70% methanol aqueous solution (0.1% formic acid), and subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min.
Compared with the pre-treatment conditions: 10 μL of internal standard working liquid I and 10 μL of internal standard working liquid II were respectively taken with a pipette first and then put into a 1.5 mL centrifuge tube, 200 μL of serum sample to be detected was taken by a pipette, added with 100 μL of methanol, subjected to vortex mixing at a rotating speed of 2,000 rpm for 3 min, added with 650 μL of n-hexane: methyl tert-butyl ether=4:1, subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, and centrifuged at a rotating speed of 14,000 rpm for 10 min, 600 μL of supernatant was added into a clean 1.5 mL centrifuge tube, and then 650 μL of n-hexane: methyl tert-butyl ether=4:1 was added into the 1.5 mL centrifuge tube, subjected to vortex mixing at a rotating speed of 2,000 rpm for 5 min, and centrifuged at a rotating speed of 14,000 rpm for 10 min to obtain a supernatant. The supernatant was added with 100 μL of 70% methanol aqueous solution (containing 0.1% formic acid) for redissolution, subjected to vortex mixing at a rotating speed of 2,000 rpm for 1 min, centrifuged at a rotating speed of 14,000 rpm for 5 min, and 90 μL of supernatant was taken as a solution to be detected.
Response values of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 were measured. Meanwhile, the same samples were detected according to the method of Embodiment 1 to measure the response values. Obtained experimental results were as shown in Table 21 and Table 22.
It could be seen from Table 21 and Table 22 that the response values of the four substances of D2, D3, 1,25-(OH)2D2 and 1,25-(OH)2D3 after the derivatization treatment were significantly increased, and matrix inhibition in D2 and D3 samples was significantly improved.
The above are only specific embodiments of the present invention, so that those skilled in the art can understand or realize the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the generic principles defined herein may be embodied in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not to be limited to these embodiments shown herein, but is to be in conformity with the widest scope consistent with the principles and novel features disclosed herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202310982084.2 | Aug 2023 | CN | national |
