WO2011097933A1

WO2011097933A1 - Kit for detecting glycochenodeoxycholate-3-sulfate and glycochenodeoxycholate in blood

Info

Publication number: WO2011097933A1
Application number: PCT/CN2010/080483
Authority: WO
Inventors: 王文昭; 谈冶雄; 尹佩源; 洪毅; 路鑫; 汤靓; 王红阳; 许国旺
Original assignee: 中国科学院大连化学物理研究所; 中国人民解放军第二军医大学东方肝胆外科医院
Priority date: 2010-02-10
Filing date: 2010-12-30
Publication date: 2011-08-18
Also published as: CN101995441A

Abstract

A kit for detecting glycochenodeoxycholate-3-sulfate and glycochenodeoxycholate in blood is provided. The kit consists of isotope standard of leucine enkephalin (Lep) or bile acid as the inner standard, ammonium acetate or ammonium formate or ammonium bicarbonate as the buffer solution with pH 4.0-8.0, and acetonitrile or methanol as the eluent.

Description

Kit for detecting blood 3-glycine chenodeoxycholic acid and glycine chenodeoxycholic acid

The invention relates to the field of analytical chemistry and medicine, and is an ultra performance liquid chromatography mass spectrometry method for detecting human blood based on a kit for detecting blood 3-glycine chenodeoxycholic acid and glycine chenodeoxycholic acid (GCDCS and GCDCA). A combination of high-temperature and high-speed analysis for rapid quantitative determination of GCDCS and GCDCA in blood. Background technique

Liver cancer is one of the common malignant tumors in China. Liver cancer either originates from the liver itself or is derived from the metastatic effects of other cancer cells in the body. Primary liver cancer refers to a malignant tumor of liver cells or intrahepatic cholangiocarcinoma cells, and the incidence of primary liver cancer from the liver has been the first in liver malignant tumors. It ranks third only to gastric cancer and esophageal cancer in malignant tumor mortality, and second in rural areas in some areas, second only to gastric cancer. China has died of about 110,000 liver cancers per year, accounting for 45% of the world's liver cancer deaths. In recent years, the incidence and mortality of primary liver cancer in China have increased, while in rural areas it is significantly higher than in urban areas.

In China, liver cancer is mainly associated with diseases such as hepatitis B, hepatitis C virus infection and cirrhosis. The early symptoms of liver cancer are often not obvious. Most patients are asymptomatic, and the physical examination lacks the signs of the tumor itself. This period is called the sub-clinical stage. Once the symptoms of liver cancer appear, it means that most of the patients have entered the middle and late stages. Currently, there is a lack of effective treatment for advanced liver cancer. Therefore, early diagnosis of liver cancer is very important, such as active comprehensive treatment, which can significantly improve the five-year survival rate of liver cancer. General imaging examinations and physical examinations have limited effects. They are often found and confirmed in the middle and late stages, and lack of early warning value. With the continuous development of molecular biology, a variety of new markers have been continuously discovered, which has improved the early diagnosis rate of liver cancer in the subclinical stage and improved the therapeutic effect. The most commonly used liver cancer serum marker is alpha-fetoprotein (AFP). However, alpha-fetoprotein (AFP) serum levels have the disadvantage of being less sensitive and less specific in the diagnosis of liver cancer, and the detection rate when used alone in diagnosis is generally only about 50%-75%. Therefore, in order to improve the sensitivity and specificity of clinical diagnosis, it is imperative to develop new detection methods.

Bile acid is directly synthesized from cholesterol in the mitochondria of the liver parenchyma cells. It is concentrated in the gallbladder after transport through the bile duct. It is a general term for a large class of choline acids present in bile. As a major product of cholesterol metabolism, bile acids have similar sterol core structures and branched structures of different combinations. The human body mainly contains 5 kinds of free bile acids and 10 kinds of bound bile acids which are combined with glycine and taurine; meanwhile, the human body also contains a small amount of monosulfated bile acids combined with 3 hydroxyl groups. Bile acid has been considered to only promote the digestion and absorption of lipids, maintain the dynamic balance of bile acid secretion, inhibit the precipitation of cholesterol in bile to form gallstones, and assist in the diagnosis of diseases such as viral hepatitis and biliary obstruction. . In recent years, bile acid has attracted more and more attention, and it is considered to be a multifunctional and important signal molecule in energy dynamic balance (Document 1: Watanabe, M.; Houten, SM; Mataki, C; Christoffolete, MA ; Kim, BW; Sato, H.; Messaddeq, N.; Harney, JW; Ezaki, 0.; Kodama, T.; Schoonjans, K.; Bianco, A. C; Auwerx, J. Nature 2006, 439, 484 -489. Literature 2: Baxter, JD; Webb, P. Nature 2006, 439, 402-403.), phospholipid metabolism (Document 3: Martin, FPJ; Dumas, ME; Wang, YL; Legido-Quigley, C; Yap, IKS; Tang, H..; Zirah, S.; Murphy, GM; Cloarec , 0.; Lindon, J. C; Sprenger, N.; Fay, LB; Kochhar, S.; van Bladeren, P.; Holmes, E.; Nicholson, JK Molecular Systems Biology 2007, 3, 16. Bilz, S.; Samuel, V.; Morino, K.; Savage, D.; Choi, CS; Shulman, GI American Journal of Physiology-Endocrinology and Metabolism 2006, 290, E716-E722.), Glucose Metabolism (Document 5 : Nguyen, A.; Bouscarel, B. Cellular Signalling 2008, 20, 2180-2197. Document 6: Brinton, EA Diabetes Obesity & Metabolism 2008, 10, 1004-1011. ) and cell proliferation or apoptosis (Reference 7: Metalli , VD; Mancino, M. G; Mancino, A.; Torrice, A.; Gatto, M.; Attili, AF; Al ini, G.; Alvaro, D. Digestive and Liver Disease 2007, 39, 654-662. Document 8: Ramalho, . M.; Viana, . JS; Low, W. C; Steer, CJ; odrigues, CMP Trends in Molecular Medicine 2008, 14, 54-62. ) plays an important regulatory role in integration regulation. In recent years, as people's cognition of oncology continues to deepen, malignant tumors are more regarded as a systemic chronic disease. Taking primary liver cancer as an example, it is often developed from chronic diseases such as hepatitis cirrhosis. When the human body suffers from hepatobiliary diseases, especially malignant tumors, the metabolism of the liver's basic unit, the liver parenchyma cells, will be stimulated by cancer to change the metabolism of bile acids. Although serum total bile acid (TBA) content has been widely used in the auxiliary diagnosis of chronic diseases such as viral hepatitis and cirrhosis, its role in the diagnosis of liver cancer is extremely small. And because the serum total bile acid content is determined based on the 3 hydroxysterol method, it does not reflect the changes in the body's bile acids when the body suffers from primary liver cancer. In view of this, measuring the changes in the content of bile acids in the blood is beneficial to us to understand the metabolic changes of hepatocytes in order to find effective potential markers to develop and establish new methods for detecting liver cancer.

Current methods for detecting bile acids include: enzymatic, nuclear magnetic resonance, gas chromatography, and liquid chromatography, as well as ultraviolet absorption, fluorescence scattering, mass spectrometry, and the like. The clinical enzymatic method can rapidly detect total bile acids, but cannot accurately quantify the content of each individual bile acid (Article 9 Mashige, R; Tanaka, N.; Maki, A.; Kamei, S.; Yamanaka, M. Clinical Chemistry 1981, 27, 1352-1356.: ); Although NMR technology has the characteristics of fast and unbiased, it is too low sensitivity and costly compared with other technologies; the pretreatment process of gas chromatography and mass spectrometry is tedious, and Bound bile acids are readily decomposed into free bile acids (Reference 10: Keller, S.; Jahreis, G. Journal of Chromatography B- Analytical Technologies in the Biomedical and Life Sciences 2004, 813, 199-207. Document 11: Roda, A.; Piazza, E; Baraldini, M. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 1998, 717, 263-278. Document 12: Makino, I.; Shinozak.K; Nakagawa, S.; Mashimo , K. Journal of Lipid Research 1974, 15, 132-138. ); Conventional liquid chromatography techniques have shortcomings such as low detection sensitivity or complex derivatization processes (Document 11: Roda, A.; Piazza, F.; Baraldini, M . Journal of Chr Omatography B-Analytical Technologies in the Biomedical and Life Sciences 1998, 717, 263-278. Document 13: Sakakura, H.; Kimura, N.; Takeda, H.; Komatsu, H.; Ishizaki, K.; Nagata, S Journal of Chromatography B 1998, 718, 33-40. ). With the development of liquid chromatography-mass spectrometry, high sensitivity And high resolution characteristics continue to improve, widely used in complex body fluid analysis testing of bile acids (Ref. 14: Alnouti, Y.; Csanaky, IL; Klaassen, CD Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2008, 873, 209-217. Literature 15 Bobeldijk, L; Hekman, M.; de Vries-van der Weij, J.; Coulier, L.; Ramaker, R.; Kleemann, R.; Kooistra, T.; Rubingh , C; Freidig, A.; Verheij, E. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2008, 871, 306-313.: Document 16: Tagliacozzi, D.; Mozzi, A. R; Casetta, B.; Bertucci, P.; Bernardini, S.; Di Ilio, C; Urbani, A.; Federici, G. Clinical Chemistry and Laboratory Medicine 2003, 41, 1633-1641. Document 17: Ye, L.; Liu, S. Y; Wang, M.; Shao, Y; Ding, M. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2007, 860, 10-17. Document 18: Ando, M.; Kaneko, T. Watanabe, R.; Kikuchi, S.; Goto, T.; Iida, T.; Hishinuma, T.; Mano, N.; Goto, J. Journal Of Pharmaceutical and Biomedical Analysis 2006, 40, 1179-1186. Document 19: Burkard, L; von Eckardstein, A.; Rentsch, KM Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2005, 826, 147-159. Especially; ultra-high performance liquid chromatography technology is favored because of its higher resolution, resolution and shorter analysis time. However, these techniques can only detect 15 kinds of bile acids in the human body and cannot detect any monosulfated bile acids. However, there are few methods for detecting monosulfated bile acid, and the pretreatment process is cumbersome. The multi-step SPE column is used to separate the sulfide bile acid, and has a large ion suppression effect and low resolution, and can only be used for detecting monosulfation. Disadvantages such as bile acid (see references: Ikegawa S, Yanagihara T, Murao N, etc. JOURNAL OF MASS SPECTROMETRY. 1997, 32, 401-407.). At present, there is no fast and efficient method to detect both conventional bile acids and sulfated bile acids in humans, and no one has linked the changes in bile acids to primary liver cancer. (Document 20: Administration, F. a. D. 2001. )

The invention provides a kit for rapidly detecting blood 3-glycystase chenodeoxycholic acid and glyco-eodeoxycholic acid. The kit does not require complicated SPE pretreatment process, and not only detects blood 3-sulfuric acid. Glycine chenodeoxycholic acid/time flux is high, and it has the characteristics of low detection cost, good repeatability and high stability. Summary of the invention

The object of the present invention is to develop a kit for detecting 3-glycine chenodeoxycholic acid and glycine chenodeoxycholic acid in blood. The method for detecting 3-glycylic acid chenodeoxycholic acid and glycine chenodeoxycholic acid in the blood sample has the characteristics of low detection cost, good repeatability, high stability, fast and high efficiency; only one 19 is needed for determining an actual sample. Minutes, suitable for clinical applications.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

The kit consists of three parts: (1) an internal standard, an isoleucine phenanthride or a bile acid isotope standard; (2) a buffer of ammonium acetate, ammonium formate or ammonium hydrogencarbonate at a pH of 4.0 to 8.0; 3) Eluent - acetonitrile or methanol. This kit uses isoleucine phenanthroline as an internal standard, which can reduce the analysis cost and enhance the detection accuracy. The pH of 5.0 buffer ammonium acetate, ammonium formate or ammonium bicarbonate, eluent acetonitrile system can be obtained. Good separation effect and rapid separation and analysis of 3-glycylic acid chenodeoxycholic acid and glycine chenodeoxycholic acid. The present invention has also been developed based on the use conditions of the test kit and has been applied in normal control, hepatitis, cirrhosis and liver cancer samples.

Specific steps are as follows,

1) Select the best composition of the kit: The internal standard is selected as isoleucine phenanthrene (other bile acid isotopes can also be used as internal standards (literatures 14, 15, 18), but they are less expensive because of their high price. In practical applications, it can effectively reduce the analysis cost and enhance the detection accuracy; choose ammonium acetate, ammonium formate or ammonium hydrogencarbonate with pH=5.0 as the buffer; choose acetonitrile system for the eluent (also choose acetic acid with pH 4.0~8.0) Ammonium, ammonium formate or ammonium bicarbonate and acetonitrile (methanol) systems, but these systems are slower to analyze and have poorer separation of bile acids).

2) Kit use conditions: The chromatographic instrument is Waters ultra-high performance liquid chromatography, the column is C8 column, the column temperature is 60 degrees Celsius, the flow rate is 0.5 ml/min (C18 column can also be selected, the column temperature is 25-60 degrees Celsius, the flow rate is 0.2. -1. Om min conditions for the analysis of bile acids, but because of the slower separation of these methods and the poor separation of bile acids have not been used (literature 14-19), the mass spectrometer is a single quadrupole mass spectrometer.

3) Take the isolated serum of normal fasting patients and confirmed hepatitis, cirrhosis and liver cancer patients under the same conditions. After collection, they should be allowed to stand at 4 ° C for half an hour and centrifuged at 9000 g for 15 minutes to take the supernatant (serum). Store immediately in a refrigerator at -80 ° C for later use.

4) Pretreatment of serum samples: The samples were thawed at room temperature when tested, 150 μ ΐ serum was added, 600 μ ΐ acetonitrile, 30 l isoleucine phenanthride concentration was 3.3 g/ml acetonitrile aqueous solution (acetonitrile water volume ratio was 1 : 1 ), oscillation

After 30 seconds, centrifuge at 9000g for 15 minutes at 4°C, then 650 μl of the supernatant was lyophilized, reconstituted with a volume ratio of 1:3 in acetonitrile water, 复 μ 1 , and centrifuged at 9000 g for 15 minutes at 4 ° C. After taking the supernatant, the sample was directly loaded, and the peak area of the internal standard peak, 3-glycine chenodeoxycholic acid and chenodeoxycholic acid was calculated by the total ion current diagram to determine the 3-glycylic acid chenodeoxycholic acid. And the content of glycine chenodeoxycholic acid.

5) Four groups of serum samples were subjected to ultra-high performance liquid chromatography mass spectrometry high-speed high-speed analysis in random order, and the blood bile acid analysis method was marked. Linear equations and single-needle injection of 14 bile acid standards were required. Repeatability, recovery, and daytime and intraday precision are in compliance with US Food and Drug Administration (FDA) testing standards (Ref. 13).

6) In the serum sample test, GCDCS was significantly elevated in the liver cancer group compared with the normal group. The sensitivity of GCDCS for judging liver cancer was 75.56%, and the specificity was 88.89%. The sensitivity of using AFP to judge liver cancer was 62.2% and the specificity was 100%. Among the 45 liver cancer samples, 17 samples with AFP values less than 20 ug/L (false negative) were judged by GCDCS, and 14 of them were found to be greater than 0.21 (positive result), which can be judged as liver cancer. This indicates that GCDCS has good complementarity with AFP and can be used to assist AFP in primary hepatocellular carcinoma screening.

To further investigate the potential of GCDCS in clinical practice and its mechanism in the development of liver cancer, hepatitis GCDCS in the blood of the cirrhosis group, its upstream metabolite GCDCA and its related parameter GCDCS/GCDCA were compared with normal controls and liver cancer groups, and GCDCS was found to be higher in hepatitis and cirrhosis. The changes in the content of GCDCA in the normal control group, hepatitis, cirrhosis and liver cancer group were found to be similar to GCDCS. However, its content in the liver cancer group was higher than that in the normal group but not significant. GCDCS/GCDCA was significantly elevated in the liver cancer group compared to the three groups of non-hepatoma groups. Through ROC curve analysis, only GCDCS/GCDCA and AFP were not significant, indicating that GCDCS/GCDCA has more potential for application. The sensitivity of liver cancer using GCDCS/GCDCA was 68.89%, and the specificity was 86.15%. The sensitivity of using AFP to judge liver cancer was 62.2% and the specificity was 89.2%. Among the 45 liver cancer samples, 17 samples with AFP value less than 20 ug/L (false negative) were judged by GCDCS/GCDCA, and 10 of them were found to be greater than 0.097 (positive result), which can be judged as liver cancer. This indicates that GCDCS/GCDCA has good complementarity with AFP and can be used to assist AFP in the diagnosis of primary liver cancer.

The method of the invention has the following effects: The internal standard isotucine phenanthroline constituting the kit can effectively reduce the analysis cost and enhance the detection accuracy; the ammonium acetate and acetonitrile systems with pH=5.0 can obtain the best buffer. The separation effect not only detects 15 kinds of conventional bile acids in the blood, but also can detect several sulfated bile acids such as 3-glycine chenodeoxycholic acid. The method based on kit development has the following characteristics: The sample collection and storage adopt standardized operation procedures to avoid introducing human error; the pretreatment process is simple, and the analysis process adopts high temperature and high speed method, which not only improves the separation of analytes. The ability to resolve, and greatly shorten the analysis time, can separate the conventional bile acids, and can simultaneously separate several sulfated bile acids. In serum sample testing, GCDCS/GCDCA can assist AFP in the diagnosis of primary liver cancer. The method for diagnosing liver cancer established by this method has the characteristics of safety, reliability, high flux, high sensitivity and specificity, and is suitable for early screening and auxiliary diagnosis of liver cancer. DRAWINGS

Figure 1 TIC diagram of human serum bile acid: 1, leucine brain phenanthrene (internal standard); 2, 3-glycine ursodeoxycholic acid (GUDCS); 3, 3-glycolic acid chenodeoxycholic acid (GCDCS) 4, 3-glycylic acid deoxycholic acid (GDCS); 5, glycine ursodeoxycholic acid (GUDCA); 6, burdock chenodeoxycholic acid (TUDCA); 7, glycocholic acid (GCA); 8, burdock cholic acid (TCA); 9, glycine chenodeoxycholic acid (GCDCA); 10, burdock chenodeoxycholic acid (TCDCA); 11, glycodeoxycholic acid (GDCA); 12, burdock deoxygenation Cholic acid (TDCA); 13, cholic acid (CA); 14, ursodeoxycholic acid (UDCA); 15, glycocholic acid (GLCA); 16, burdock cholic acid (TLCA); 17, goose deoxygenation Cholic acid (CDCA); 18, deoxycholic acid (DCA); 19, lithocholic acid (LCA).

Figure 2 (A) Changes in serum GCDCS levels in serum samples from normal controls, liver cancer, and cirrhosis (mean standard error). (B) ROC curves of GCDCS and AFP in serum samples from normal control group and liver cancer group: AUC values were GCDCS=0.866 and AFP=0.935, respectively. (C) Changes in serum GCDCA levels in serum samples from normal controls, liver cancer hepatitis, and cirrhosis.

Figure 3 (A), (B) Changes in serum GCDCS/GCDCA and AFP levels in normal controls, hepatitis, cirrhosis, and liver cancer. (C) GCDCS, AFP in serum samples from normal controls, hepatitis, cirrhosis and liver cancer And the ROC curve of GCDCS/GCDCA: AUC values were GCDCS=0.653, AFP=0.875, and GCDCS/GCDCA=0.810, respectively. detailed description

Example 1

Serum sample collection

Before the collection, volunteers were included to sign the informed consent form.

Inclusion criteria for normal controls, hepatitis, cirrhosis, and primary liver cancer: Refer to "Huang Jiayu Surgery (7th Edition)" People's Health Publishing House 2008 Beijing

The serum of 36 normal people, 14 confirmed hepatitis, 15 cases of liver cirrhosis and 45 cases of liver cancer were collected under the same conditions. After collection, they were allowed to stand at 4 ° C for half an hour and centrifuged at 9000 g for 15 minutes. Store in a refrigerator at -80 ° C for use.

2. Analytical methods,

2. 1 serum sample pretreatment

The sample was thawed at room temperature, and 150 μl of serum was added. 600 μl of acetonitrile and 30 μl of isoleucine phenanthride were added to a concentration of 3.3 μg/ml acetonitrile (acetonitrile water volume ratio of 1:1). After shaking for 30 seconds, centrifugation at 9000 g for 15 minutes at 4 °C, 650 μl of the supernatant was lyophilized, and the solution was mixed with acetonitrile in a volume ratio of 1:3, ΙΟΟ μ Ι reconstituted, and centrifuged at 9000 g at 4 ° C. After taking the supernatant for a minute, the sample was directly loaded, and the peak area of the internal standard peak, 3-glycine chenodeoxycholic acid and glycine chenodeoxycholic acid was calculated by the total ion current map to determine the 3-glycolglycane deoxycholate. Acid and glycine chenodeoxycholic acid content.

2. 2 high performance liquid chromatography mass spectrometry high temperature high speed analysis

(1) The liquid phase conditions are: the chromatographic instrument is Waters ultra performance liquid chromatography, the column is Waters ACQUITY UPLCTM BEH C8 column, the mobile phase A is 50 mM ammonium acetate solution with pH=5.0, B is acetonitrile; the gradient elution condition is : 0~0.5 min is 75% A phase, 0.5~3.5 min linearly changes to 70% A phase, 3.5~9 min drops to 45%, 9~11 min drops to 25%, 11~ 13.5 min drops to 15%, 13.5~15 min down to 10%, and keep it for 2 min, 17~17.5 min, increase to 75% phase A and keep it for 1.5 min; flow rate 0.5 mL/min, column temperature 60 V, injection volume 20 μί, post-column effluent Directly enter the mass spectrometry without splitting.

(2) Mass spectrometry conditions are: Mass spectrometer is Waters single quadrupole mass spectrometer, detected by electrospray ion source negative ion mode; desolvation gas and cone gas flow are divided into 700 L/h and 50 L/h, desolvation gas and The cone gas is high purity nitrogen; the desolvation gas temperature is 300 V, the ion source temperature is 120 V; the capillary voltage and the cone voltage are 2800 V and 45 V, respectively; detection by selective ion monitoring mode: 0~2.6 min Detection of ions 528.3 and 554.3, 2.65~3.7 min detection of ions 448.3, 464.3, 498.3 and 514.3, 3.75~6 min detection ions 391.3, 407.3, 448.3 and 498.3, 6.05~19 min detection ions 375.3, 391.3, 432.3 and 482.3; per 0.1 Data is collected once in seconds (Figure 1).

3. Analysis of serum test results and auxiliary diagnostic potential In the serum sample test, the content of GCDCS in the normal control and liver cancer groups is shown in Figure 2A. GCDCS was significantly increased in the liver cancer group. The ROC curves were obtained for GCDCS and AFP, respectively. The AUC values of the ROC curve were GCDCS=0.866 and AFP=0.935, respectively (Fig. 2B). By comparison, we found that the use of GCDCS to diagnose liver cancer not only has higher sensitivity and specificity, but also its combined diagnosis with AFP can improve the ability to detect liver cancer. For example, in 45 cases of liver cancer, 17 samples with AFP values less than 20 ug/L (false negative) were judged by GCDCS, and 14 of them were found to be greater than 0.21 (positive result), which can be judged as liver cancer. It shows that GCDCS has good complementarity with AFP. Table 1 shows the sensitivity and specificity of AFP, GCDCS, and "AFP+GCDCS".

In order to further investigate the clinical application potential of GCDCS and its mechanism in the development of hepatocarcinoma, GCDCS in the blood of hepatitis and cirrhosis, its upstream metabolite GCDCA and its related parameters GCDCS/GCDCA were compared with normal controls and liver cancer groups. GCDCS was found to be more abundant in hepatitis and cirrhosis (Fig. 2A). The changes in the content of GCDCA in the normal control group, hepatitis, cirrhosis and liver cancer group were found to be similar to those of GCDCS (Fig. 2C). Although it was significantly elevated in the hepatitis and cirrhosis groups, its content in the liver cancer group was increased compared with the normal group but was not significant. GCDCS/GCDCA was significantly elevated in the liver cancer group compared to the three groups of non-hepatoma groups (Fig. 3A). We performed ROC curves for GCDCS, AFP, and GCDCS/GCDCA in the liver cancer group and the three non-hepatoma groups. The AUC values of the ROC curve were GCDCS=0.653, AFP=0.875, and GCDCS/GCDCA=0.810 (Fig. 3B). By comparison, we found that the use of GCDCS/GCDCA to diagnose liver cancer not only has higher sensitivity and specificity, but only GCDCS/GCDCA and AFP are not significant, indicating that GCDCS/GCDCA has more potential for application, and its combined diagnosis with AFP is more Helps improve the ability to detect liver cancer. For example, when AFP was used to detect liver cancer, 17 of 45 patients with liver cancer had a negative reaction (AFP<20 ng/L), and when the GCDCS/GCDCA method was used to diagnose these 17 patients, 10 of them were found to be present. Positive reaction (GCDCS/GCDCA>0.097). This indicates that GCDCS/GCDCA has good complementarity with AFP, and Table 2 shows the sensitivity and specificity of GCDCS, AFP, GCDCS/GCDCA, and "AFP+GCDCS/GCDCA".

Table 1 Sensitivity and specificity of AFP and GCDCS in normal control group and liver cancer group

Marker Sensitivity (%) Specificity (%)

GCDCS 75.56 88.89

AFP 62.22 100.00

"AFP+GCDCS" 93.33 100.00

3⁄4 2 GCDCS AFP, GCDCS/GCDCA and "AFP+GCDCS/GCDCA"

Sensitivity and specificity in non-hepatocarcinoma and liver cancer groups

Marker Sensitivity (%) Specificity (%)

GCDCS 75.56 61.54

AFP 62.22 89.23

GCDCS/GCDCA 68.89 86.15

"AFP+GCDCS/GCDCA" 84.44 96.92

Claims

1. A kit for detecting 3-glycylic acid chenodeoxycholic acid and glycine chenodeoxycholic acid in a blood, characterized in that the kit consists of three parts: (1) an internal standard-isoleucine bird phenanthride or Bile acid isotope standard; (2) Buffer solution: ammonium acetate, ammonium formate or ammonium hydrogencarbonate at pH 4.0~8.0; (3) Eluent-acetonitrile or methanol.

2. The method according to claim 1, wherein: the selected kit is labeled as isoleucine phenanthroline, which can effectively reduce the analysis cost and enhance the detection accuracy.

3. The method according to claim 1, wherein: the selected reagent buffer is ammonium acetate, ammonium formate or ammonium hydrogencarbonate with pH=5.0, and the eluent is acetonitrile, which can obtain better separation effect and The purpose of rapid separation analysis of 3-glycylic acid chenodeoxycholic acid and conventional bile acids was achieved.