Method for measuring four deoxycholic acid isomers through charge state modulation UPLC-MS / MS

Abstract

The invention provides a method for measuring four deoxycholic acid isomers by charge state modulation UPLC-MS / MS. The method comprises the following steps: promoting cholic acid to be homogenized into a carboxylic acid anion form by adopting an alkaline mobile phase (8mmol / L ammonia water), combining a UPLC-MS / MS technology, carrying out gradient elution by adopting a C18 column under a chromatographic condition, and monitoring m / z 391.57 by adopting an anion SIM mode in a mass spectrum, and high-sensitivity and high-resolution rapid separation of four deoxycholic acid isomers (UDCA, CDCA, HDCA and DCA) is realized. According to the method, the detection limit is as low as 0.10-0.22 ng / mL, the analysis time is shortened to 8 minutes, and the method is suitable for synchronous quantitative analysis of four cholic acid isomers in a biological sample.

Description

Technical field

[0001] The present invention relates to the technical field of biomedical detection, and in particular to a method for determining four deoxycholic acid isomers using charge state modulation UPLC-MS / MS. [Background Technology]

[0002] Ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and deoxycholic acid (DCA) are dihydroxylated bile acid isomers (chemical structures see Figure 1 ), share a molecular weight of 392.57 Da, and play key biological roles in lipid digestion, cholesterol homeostasis, and hepatobiliary signaling. These compounds have diverse clinical applications: UDCA, known for its choleretic and cytoprotective properties, is approved for the dissolution of gallstones and treatment of cirrhosis, while CDCA and HDCA show therapeutic potential in cholestatic diseases and gastrointestinal motility regulation. As biomarkers, blood bile acids provide insights into pathological processes and drug pharmacokinetics.

[0003] Since these isomers have high structural similarity and extremely low concentrations (pg to ng / mL) in biological matrices, accurate identification and precise quantification of them remain challenging. As far as I know, most of the methods reported in the literature can only determine one or more of the four isomers individually. Xue et al. established a method for the simultaneous quantification of these four isomers of bile acid. However, this method still has serious drawbacks: the detection limit is unsatisfactory (>1 ng / mL) and the determination time is long (more than 50 minutes). This is mainly due to the fact that the analytical environment is not conducive to the determination of bile acid by UPLC-MS / MS, which has the following disadvantages: (1) incomplete deprotonation of the analyte leads to poor ionization efficiency; (2) the coexistence of multiple species of the analyte leads to differences in chromatographic elution, resulting in peak broadening or splitting; (3) the similar polarity between the analyte and the C18 stationary phase enhances the retention capacity of the chromatographic column and prolongs the elution time.

[0004] In addition, the Chinese patent application number 202010656649.4 provides a biological sample analysis method for deoxycholic acid compounds. This method adopts a simple organic solvent protein precipitation method to pre-treat the biological sample. Based on the peak area ratio of the analyte to the internal standard, a standard curve is obtained by normalization, thereby quantitatively obtaining the concentration of deoxycholic acid compounds in the biological sample. And the Chinese patent application number 202410325938.4 provides a bioanalysis method for determining the concentration of ursodeoxycholic acid and its metabolites. During the chromatographic separation, the mobile phase A in the chromatographic conditions is a 2mM ammonium acetate aqueous solution; the mobile phase B is a methanol-acetonitrile solution with a volume ratio of 1:1. That is, the mobile phase environment of the existing determination method is mostly acidic or neutral, which makes it difficult to simultaneously determine multiple isomers of bile acid, and the fundamental reason for the low sensitivity and long analysis cycle.

[0005] Therefore, there is an urgent need to develop a rapid, sensitive and high-resolution method for the simultaneous quantification of four bile acid isomers to assist in pharmacokinetic studies and quality control in animal health and pharmaceutical applications. [Summary of the invention]

[0006] The technical problem to be solved by the present invention is to provide a method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS. The method has simple pretreatment, is easy to operate, has a short analysis cycle, high detection sensitivity, and good repeatability. It can realize the simultaneous qualitative and quantitative detection of UDCA, CDCA, HDCA and DCA in biological fluid samples such as pigs and poultry and related products.

[0007] The present invention is achieved in that:

[0008] A method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS comprises the following steps:

[0009] (1) Analysis of four deoxycholic acid standards: A series of standard solutions of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), and deoxycholic acid (DCA) were prepared and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The qualitative parameters of UDCA, CDCA, HDCA, and DCA were determined, and standard working curves of UDCA, CDCA, HDCA, and DCA were drawn with the standard concentration as the abscissa and the quantitative ion peak area as the ordinate.

[0010] The chromatographic conditions are:

[0011] Mobile phase: Mobile phase A is an alkaline solution with a pH between 7.5 and 12.5, and mobile phase B is acetonitrile;

[0012] Mass spectrometry conditions are:

[0013] ESI electrospray ionization source, negative ion mode, selected ion monitoring SIM, monitoring ion pair m / z 391.57;

[0014] (2) extracting deoxycholic acid from the sample;

[0015] (3) Analysis of four deoxycholic acids in the extract: UPLC-MS / MS was performed on the deoxycholic acids extracted from the sample; the UPLC-MS / MS analysis conditions were consistent with those in step (1), and the mass chromatogram information of the monitoring ion (391.57) was recorded;

[0016] (4) Referring to the qualitative parameters and standard working curves in the standard, the contents of UDCA, CDCA, HDCA, and DCA in the samples were calculated respectively.

[0017] Furthermore, in step (1), the concentration range of the UDCA, CDCA, HDCA, and DCA standard solutions is 0.30 to 4000 ng / mL.

[0018] Furthermore, the chromatographic conditions in step (1) are specifically:

[0019] Chromatographic column: ACQUITY UPLC BEH C18, size: 2.1 × 100 mm, 1.7 μm;

[0020] Mobile phase: Mobile phase A is an alkaline solution with a pH between 7.5 and 12.5, mobile phase B is acetonitrile, mobile phase flow rate is 0.3 mL / min, column oven temperature is 40°C, sample tray temperature is 15°C, injection volume is 2 μL;

[0021] Gradient elution conditions: 0 min, 15% B; 4.0 min, 22% B; 6.0 min, 30% B; 8.0 min, 40% B; 8.1 min, 15% B; 10.0 min, 15% B.

[0022] Furthermore, the pH value of mobile phase A is 9.9-10.3, which enables bile acid to exist in the form of completely deprotonated carboxylate anions.

[0023] Furthermore, the mobile phase A is a 2-10 mmol / L ammonia solution.

[0024] Furthermore, the mobile phase A is 8 mmol / L ammonia solution.

[0025] Furthermore, the mass spectrometry conditions in step (1) are specifically as follows:

[0026] ESI electrospray ion source, negative ion mode, selected ion monitoring SIM, monitoring ion pair m / z 391.57, atmospheric pressure ionization API gas is nitrogen, purity >95%; collision gas is argon, purity >99.999%; capillary voltage is 3.0 kV, cone voltage is 30 V, source offset voltage is 50 V, desolvation temperature is 450 ° C, desolvation gas flow rate is 650 L / h, cone gas flow rate is 150 L / h, and nebulizer pressure is 7.0 Bar.

[0027] Furthermore, the specific steps of extracting deoxycholic acid from the sample in step (2) are as follows: weigh livestock and poultry products, 2 mL of blood sample, 0.2 mL of bile, 0.1 g of concentrated bile product and saponified bile product, place them in a 50 mL clean polypropylene centrifuge tube, add 20 mL of methanol, homogenize at 10,000 rpm for 1 minute, ultrasonically extract in a 40 kHz, 200 W ultrasonic bath for 20 minutes, then centrifuge the centrifuge tube at 4,500 rpm for 5 minutes, pass the supernatant through a 0.22 μm organic filter membrane, and wait for loading on the machine.

[0028] The present invention has the following advantages:

[0029] 1. A novel charge state homogenization strategy was used to improve the ionization efficiency and significantly enhance the detection sensitivity of isomeric bile acids, with a detection limit ranging from 0.10 to 0.22 ng / mL.

[0030] 2. The alkaline mobile phase increases the polarity of isomeric bile acids, reduces their interaction with the non-polar C18 column, and significantly shortens the determination time to 8 minutes.

[0031] 3. Complete deprotonation of the analytes yielded a single species (deoxycholic acid carboxylate anion), preventing peak broadening and bifurcation, thus playing a key role in the efficient separation of the four isomeric bile acids.

[0032] 4. The validated method has excellent analytical properties and shows good application prospects in bile acid-based drug development and its pharmacokinetic studies.

[0033] In summary, the present invention solves the problems of the prior art in that UDCA, CDCA, HDCA and DCA have the same molecular weight and similar structure, resulting in the generation of identical fragment ions in liquid chromatography tandem mass spectrometry, making separation and identification difficult, and leading to poor detection sensitivity and long analysis time.

Brief Description of the Drawings

[0034] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] Figure 1 The chemical structural formulas of four deoxycholic acids are shown below;

[0036] Figure 2 The electrospray ionization mass spectra of four bile acid isomer standards in the examples of the present invention are shown in Figure 1, where A: UDCA, B: CDCA, C: HDCA, and D: DCA.

[0037] Figure 3 This is a flow chart showing the principle of the method for determining four deoxycholic acid isomers by pH modulation UPLC-MS / MS in an embodiment of the present invention;

[0038] Figure 4 This is a mass chromatogram of four bile acid isomer standards in the examples of the present invention;

[0039] Figure 5 Schematic diagram showing the effect of mobile phase pH on the sensitivity and column retention of (A) UDCA, (B) HDCA, (C) CDCA, and (D) DCA in an embodiment of the present invention;

[0040] Figure 6 Schematic diagram of the effect of different concentrations of ammonia solution mobile phase on the separation effect of UDCA, HDCA, CDCA and DCA in an embodiment of the present invention.

[0041] Figure 7 This is the mass chromatogram of the pig bile saponification product in the embodiment of the present invention. [Specific implementation method]

[0042] The following will be combined with the Figure 1-7 The technical solutions of the present invention are clearly and completely described in the following and in detail. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention. If specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0043] Example 1: Establishment of a pH-modulated UPLC-MS / MS method for the determination of four deoxycholic acid isomers

[0044] 1. Principle of the Method

[0045] Charge state homogenization strategy using pH modulation: In ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) methods, retention time, mass-to-charge ratio (m / z) of the analyte, and its signal abundance are often used for qualitative analysis. However, during mass spectrometry analysis, the four free bile acids—UDCA, CDCA, HDCA, and DCA—produce identical molecular ions upon ionization. Even with optimized collision energy, characteristic product ions cannot be generated, making them indistinguishable based solely on their mass-to-charge ratio. Figure 2 Their electrospray ionization mass spectra in negative ion mode are shown.

[0046] Therefore, within this analytical framework, the present invention adopts a charge state homogenization strategy to achieve chromatographic separation of the four deoxycholic acids, thereby distinguishing these bile acids and achieving optimal sensitivity, resolution and shorter analysis time.

[0047] The present invention modulates the pH value of the mobile phase to ensure that bile acids are in the same species in the analytical environment, facilitating UPLC-MS / MS analysis, thereby achieving highly sensitive, high-resolution, and rapid measurement and analysis. The present invention first theoretically deduces the species characteristics of various bile acids in different pH environments, then compares and screens specific mobile phase buffer salts. The optimal method is then methodologically validated for sensitivity (limit of detection (LOD) and limit of quantification (LOQ), linear range, precision, and recovery), and finally evaluated and analyzed using actual samples. Figure 3 The principle flow chart of the UPLC-MS / MS method for the determination of four deoxycholic acid isomers at pH 2.3 and 9.9 respectively; Figure 3 The working principle of this method is as follows: the four free bile acids are determined using UPLC-MS / MS. First, the pH value of the mobile phase is adjusted (pH Regulation), the analytes are injected and chromatographically separated (Liquid Chromatographic Separation), then ionized by the mass spectrometer ion source (Mass Spectrometry Ionization), and finally the mass chromatogram of each bile acid is acquired using a mass spectrometer detector (Data Acquisition).

[0048] 2. Method Optimization

[0049] To achieve efficient separation using pH-driven charge state control, key factors were optimized sequentially: selection of organic mobile phase, optimization of modifier concentration, screening of chromatographic column stationary phase, and optimization of gradient elution procedure, thereby improving separation resolution, enhancing detection sensitivity, and shortening analysis time.

[0050] The optimal instrument analysis conditions were obtained through optimization. The mass chromatograms of the four bile acids are shown in Figure 2. Figure 4 As shown in Figure 2, the retention time of the four bile acid chromatographic peaks in one analysis cycle is less than 8 minutes. The specific instrument analysis conditions are as follows:

[0051] Chromatographic conditions: The column was an ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm). The mobile phase (phase A) consisted of 8 mmol / L ammonia solution, and the organic phase (phase B) was acetonitrile. The mobile phase flow rate was 0.3 mL / min, the column oven temperature was 40°C, the sample tray temperature was 15°C, and the injection volume was 2 μL. The gradient elution program was: 0 min, 15% B; 4.0 min, 22% B; 6.0 min, 30% B; 8.0 min, 40% B; 8.1 min, 15% B; 10.0 min, 15% B.

[0052] Mass spectrometry conditions: ESI electrospray ionization source; negative ion mode; selected ion monitoring (SIM), monitoring ion m / z 391.57; atmospheric pressure ionization (API) gas (nitrogen, purity >95%); collision gas (argon, purity >99.999%); capillary voltage 3.0 kV; cone voltage 30 V; source offset voltage 50 V; desolvation temperature 450 °C; desolvation gas flow rate 650 L / h, cone gas flow rate 150 L / h, and nebulizer pressure 7.0 Bar.

[0053] 3. Method Validation

[0054] The sensitivity of the four bile acids was verified by measuring the signal intensity of serially diluted standard solutions of the analytes. A signal-to-noise ratio (S / N) of 3 was designated as the LOD, and a signal-to-noise ratio (S / N) of 10 was designated as the LOQ. The LOD, LOQ, linear range, equation, and retention time of the present invention are shown in Table 1. Following the recommendations of the "Guidelines for Industry Validation of Bioanalytical Methods," seven determinations were performed on the same day and three determinations were performed on three consecutive days. The accuracy and precision of the proposed method were evaluated at four concentration levels: the lower limit of quantification (LLOQ), three times the lower limit of quantification (LOW), the mid-range (MID), and the high-range (HIGH). The intra-day percentage recoveries and relative standard deviations for the four bile acids ranged from 82.9% to 104.0% and 0.6% to 8.9%, respectively, while the inter-day values ​​ranged from 83.3% to 107.0% and 1.0% to 9.6%, respectively.

[0055] Table 1 Linear range, calibration curve, detection limit, quantification limit and retention time of four bile acids

[0056]

[0057] Example 2: Comparison of the Effect of Mobile Phase pH on Analytical Method Performance

[0058] The ionization form of bile acids varies with the composition and pH of the mobile phase, affecting UPLC-MS / MS sensitivity, resolution, and column retention. To achieve optimal sensitivity, resolution, and fastest measurement time for the four bile acids, the present invention optimizes and modulates the mobile phase composition and pH.

[0059] First, we theoretically deduced the species characteristics of various bile acids at different pH environments. The results showed that within the pH range of 0 to 14, the species can be divided into five regions: 0-1.6, 1.6-2.5, 2.5-7.5, 7.5-12.5, and 12.5-14. Under strongly acidic conditions (pH: 0-1.6), UDCA, CDCA, and DCA all appear as complex species with three or more ionization states. Within the moderately acidic range (pH: 1.6-2.5), the four bile acids exist exclusively in unionized, neutral forms. Between pH 2.5 and 7.5, bile acids are equilibrium mixtures, gradually transforming from unionized states to carboxylate anions. Between pH 7.5 and 12.5, each bile acid exists as a completely deprotonated carboxylate anion. At a strong alkaline pH of 12.5, UDCA, CDCA, and DCA still exist as carboxylate anions, while HDCA exhibits multiple forms. In terms of species abundance, these structurally homogeneous neutral or homogeneous ionic species may be beneficial for improving the detection sensitivity and separation resolution of bile acids.

[0060] Secondly, the present invention verifies the above results by using mobile phases with different pH values. The mobile phase consists of acetonitrile and water with a pH regulator. The aqueous phase contains 0.5% formic acid (pH 2.3), 0.01% formic acid-5mmol / L ammonium acetate (pH 4.8), 2mmol / L ammonium bicarbonate (pH 8.1), and 2mmol / L ammonia water (pH 9.9). The ratio of acetonitrile phase to aqueous phase is 1:1 (volume ratio). The gradient elution program is set as follows: 0 min, 15% B; 2.0 min, 50% B; 6.0 min, 85% B; 7.0 min, 15% B; 7.1 min, 15% B; 10.0 min, 15% B (solvent B: acetonitrile). Other instrument parameters are the same as above.

[0061] The mass chromatograms of UDCA, HDCA, CDCA and DCA are shown in Figure 2. Figure 5As shown in Figures A, B, C, and D, the corresponding aqueous mobile phase additives are formic acid (blue), formic acid-ammonium acetate (orange), ammonium bicarbonate (brown), and ammonia (green), respectively. The results show that the pH of the mobile phase significantly affects the detection sensitivity and column retention of bile acids. The peak areas of each bile acid under alkaline conditions are much larger than those under acidic conditions. Specifically, the peak area generated by the ammonia system (pH 9.9) is 1-3 times larger than that of ammonium bicarbonate (pH 8.1) and 3-9 times larger than that of the acidic modifier. At pH 9.9, bile acids exist as completely deprotonated carboxylate anions, with retention times significantly shorter than those of the molecular form observed at pH 2.3. For example, at pH 9.9, deoxycholic acid (DCA) eluted at 2.89 minutes, 50% faster than its retention time of 5.73 minutes in the pH 2.3 system. All analytes exhibited similar retention time shifts. The mechanism is that the change in polarity alters the interaction between bile acids and the stationary phase: in 2 mM ammonia (pH 9.9), the fully deprotonated carboxylate anions exhibit a weakened hydrophobic interaction with the nonpolar C18 stationary phase compared to their weakly polar molecular state at pH 2.3, as their increased molecular polarity reduces their affinity for the hydrophobic stationary phase. This effect significantly shortens retention times while preserving peak capacity, enabling efficient chromatographic separation of bile acids with minimal loss of resolution. These results demonstrate that, under alkaline mobile phase conditions, particularly when prepared with 2 mM ammonia, the four bile acids maintain a uniform charge state. This, firstly, significantly improves electrospray ionization (ESI) efficiency, eliminates inconsistent elution behavior, and thus enhances detection sensitivity. Secondly, the change in polarity leads to reduced hydrophobic retention of bile acids on the column, enabling rapid and high-resolution separation by reducing interactions with the stationary phase. Therefore, when the mobile phase is 2 mmol / L ammonia water, UPLC-MS / MS analysis of the four bile acids can achieve better sensitivity, better resolution and faster measurement cycle time.

[0062] Finally, the concentration of ammonia solution was optimized, and the separation effects of ammonia solution with concentrations of 1mM, 2mM, 5mM, 8mM, and 10MM on UDCA, CDCA, HDCA, and DCA were as follows: Figure 6 As shown in the figure, when the concentration of ammonia water is 8mmol / L, the separation effect is the best.

[0063] Therefore, when the mobile phase is 8 mmol / L ammonia water and the chromatographic conditions such as gradient elution are optimized, the UPLC-MS / MS analysis of the four bile acids can achieve the best sensitivity, the best resolution and the fastest determination cycle time.

[0064] Example 3: Determination of bile acid content in pig and poultry blood and bile and their products

[0065] Preparation of blood and bile samples: Poultry and pig blood, bile, concentrated bile products and saponified bile products were purchased from a local slaughterhouse and stored at -20°C until used for preparation and analysis. Take 2 mL of blood sample, 0.2 mL of bile, 0.1 g of concentrated bile product and saponified bile product, place them in 50 mL fresh polypropylene centrifuge tubes, and add 20 mL of methanol to each. The sample was homogenized at 10,000 rpm for 1 minute. Ultrasonic extraction was performed in an ultrasonic bath (40 kHz, 200w) for 20 minutes, and then the centrifuge tube was centrifuged at 4,500 rpm for 5 minutes. The supernatant was filtered through a 0.22 μm organic filter membrane and analyzed by UPLC-MS / MS. The mass chromatogram of pig bile saponification is shown below. Figure 7 shown.

[0066] UPLC-MS / MS analysis conditions used an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). The mobile phase (phase A) consisted of 8 mmol / L ammonia solution, and the organic phase (phase B) consisted of acetonitrile. The mobile phase flow rate was 0.3 mL / min, the column oven temperature was 40°C, the sample tray temperature was 15°C, and the injection volume was 2 μL. The gradient elution program was: 0 min, 15% B; 4.0 min, 22% B; 6.0 min, 30% B; 8.0 min, 40% B; 8.1 min, 15% B; and 10.0 min, 15% B. Mass spectrometry conditions: ESI electrospray ionization source; negative ion mode; selected ion monitoring (SIM), monitoring ion m / z 391.57; atmospheric pressure ionization (API) gas (nitrogen, purity >95%); collision gas (argon, purity >99.999%); capillary voltage, 3.0 kV; cone voltage, 30 V; source offset voltage, 50 V; desolvation temperature, 450°C; desolvation gas flow rate, 650 L / h, cone gas flow rate, 150 L / h, and nebulizer pressure, 7.0 Bar.

[0067] The bile acid contents detected in different animals are shown in Table 3. The experimental results showed that HDCA and CDCA were detected in pig blood, bile and their products, while only CDCA was detected in chicken and duck blood and bile; the bile acid content in animal blood and bile was low, while the content in concentrated products was high.

[0068] Table 2. Bile acid content in different biological samples

[0069]

[0070] Note: “-” means the compound was not detected in the sample and the concentration was below the detection limit.

[0071] In summary, when the method of the present invention is used to detect the four deoxycholic acid isomers, sample pretreatment is simple and easy to operate, and the four bile acid isomers in pig and poultry biological fluids and their products can be quickly and accurately identified and quantified simultaneously. The method of the present invention has the advantages of high sensitivity, high resolution of various bile acids, and short analysis time.

[0072] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for the determination of four deoxycholic acid isomers by charge state modulation UPLC-MS / MS, characterized in that: The following steps are involved: (1) Analysis of four deoxycholic acid standards: A series of standard solutions of ursodeoxycholic acid (UDCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), and deoxycholic acid (DCA) were prepared and analyzed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The qualitative parameters of UDCA, CDCA, HDCA, and DCA were determined, and standard working curves of UDCA, CDCA, HDCA, and DCA were drawn with the standard concentration as the abscissa and the quantitative ion peak area as the ordinate. The chromatographic conditions are: Mobile phase: Mobile phase A is an alkaline solution with a pH between 7.5 and 12.5, and mobile phase B is acetonitrile; Mass spectrometry conditions are: ESI electrospray ionization source, negative ion mode, selected ion monitoring SIM, monitoring ion pair m / z 391.57; (2) extracting deoxycholic acid from the sample; (3) Analysis of four deoxycholic acids in the extract: UPLC-MS / MS was performed on the deoxycholic acids extracted from the sample; the UPLC-MS / MS analysis conditions were consistent with those in step (1), and the mass chromatogram information of the monitoring ion (391.57) was recorded; (4) Referring to the qualitative parameters and standard working curves in the standard, the contents of UDCA, CDCA, HDCA, and DCA in the samples were calculated respectively.

2. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: The concentration range of the UDCA, CDCA, HDCA and DCA standard solutions in step (1) is 0.30 to 4000 ng / mL.

3. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: The chromatographic conditions in step (1) are specifically: Chromatographic column: ACQUITY UPLC BEH C18, specifications: 2.1 × 100 mm, 1.7 μm; Mobile phase: Mobile phase A is an alkaline solution with a pH between 7.5 and 12.5, mobile phase B is acetonitrile, mobile phase flow rate is 0.3 mL / min, column oven temperature is 40°C, sample tray temperature is 15°C, injection volume is 2 μL; Gradient elution conditions: 0 min, 15% B; 4.0 min, 22% B; 6.0 min, 30% B; 8.0min, 40%B; 8.1min, 15%B; 10.0min,15%B.

4. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: The pH value of mobile phase A is 9.9-10.3, which enables bile acid to exist in the form of completely deprotonated carboxylate anions.

5. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: Mobile phase A is 2-10 mmol / L ammonia solution.

6. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 5, characterized in that: Mobile phase A was 8 mmol / L ammonia solution.

7. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: The mass spectrometry conditions in step (1) are specifically as follows: ESI electrospray ion source, negative ion mode, selected ion monitoring SIM, monitoring ion pair m / z 391.57, atmospheric pressure ionization API gas is nitrogen, purity >95%; collision gas is argon, purity >99.999%; capillary voltage is 3.0 kV, cone voltage is 30 V, source offset voltage is 50 V, desolvation temperature is 450 ° C, desolvation gas flow rate is 650 L / h, cone gas flow rate is 150 L / h, and nebulizer pressure is 7.0 Bar.

8. The method for determining four deoxycholic acid isomers by charge state modulation UPLC-MS / MS according to claim 1, wherein: The specific steps of extracting deoxycholic acid from the sample in step (2) are as follows: weigh livestock and poultry products, 2 mL of blood sample, 0.2 mL of bile, 0.1 g of concentrated bile product and saponified bile product, place them in a 50 mL clean polypropylene centrifuge tube, add 20 mL of methanol, homogenize at 10,000 rpm for 1 minute, ultrasonically extract in a 40 kHz, 200 W ultrasonic bath for 20 minutes, then centrifuge the centrifuge tube at 4,500 rpm for 5 minutes, pass the supernatant through a 0.22 μm organic filter membrane, and wait for loading on the machine.

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