A method and kit for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS

Abstract

The present invention discloses a detection method and kit for ornithine aminoformyltransferase activity in human serum based on HPLC-MS / MS, wherein the sample to be tested reacts with a reagent, ornithine is converted into citrulline, HPLC-MS / MS detection is adopted before and after the reaction, and the content of citrulline before and after the reaction is obtained by using a citrulline standard curve; the ornithine aminoformyltransferase activity value is calculated by using the formula: ornithine aminoformyltransferase activity value = (citrulline after reaction-citrulline before reaction) / reaction time. The method calculates the ornithine aminotransferase activity by detecting the content of citrulline in the sample, has strong specificity, can eliminate interference, and uses a derivative method for citrulline detection, so the result is more stable, the detection time is short, and it can be completed within 3 minutes; taking an in vitro sample as the detection object, the activity of the corresponding enzyme in the liver is reflected by detecting the activity of ornithine aminotransferase in the blood, and traumatic liver puncture sampling is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a detection method and a kit for ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS. Background Art

[0002] Ornithine carbamoyltransferase is an aminotransferase that is mainly located in the mitochondria of hepatocytes and is a liver-specific enzyme. The normal value of the enzyme is 8~20u / L. Its activity is reduced: common in premature infants, ornithine carbamoyltransferase deficiency, etc. Increased activity: obvious increase is seen in patients with acute viral hepatitis, liver cancer, liver cell necrosis, acute liver damage caused by drugs or acute pancreatitis, severe shock, etc.; mild or moderate increase is seen in cirrhosis, gallstones, chronic alcoholism, chronic hepatitis, liver abscess, primary or secondary liver cancer, liver trauma, various post-surgery, post-radiotherapy, acute enteritis, etc. Monitoring its activity is of great significance.

[0003] At the same time, detecting its activity is also a huge difficulty. The difficulties are as follows:

[0004] 1. Enzyme activity cannot be detected directly, and detection is difficult.

[0005] 2. Amino acids need to be derivatized, and direct detection results are prone to instability.

[0006] 3. Non-specific reactions in the reaction interfere with the experiment.

[0007] The main function of ornithine aminotransferase is to catalyze ornithine to produce citrulline in the circulation of ornithine in liver cells, which is a key physiological step in the production of urea in the human liver. Usually, liver biopsy can almost measure all defective enzymes in the urea cycle, such as CPS, OCT, AS, AL and arginase, but liver biopsy is traumatic to patients in clinical practice and has great limitations. Summary of the invention

[0008] In order to solve at least one of the above problems, the present invention provides a method and kit for detecting ornithine aminotransferase activity in human serum based on HPLC-MS / MS. The detection method uses mass spectrometry with very strong specificity. The activity of the corresponding enzyme in the liver is reflected by detecting the ornithine aminotransferase activity in the blood sample in vitro, thus avoiding liver puncture sampling.

[0009] In order to achieve the above object, the present invention adopts the following technical means:

[0010] The synthesis of citrulline is catalyzed by ornithine carbamoyltransferase (OCT), which transfers the carbamoyl group of carbamoyl phosphate to the δ-NH2 of ornithine to generate citrulline. This reaction is reversible.

[0011] The reaction formula is as follows:

[0012]

[0013] The first aspect of the present invention provides a method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS, comprising the following steps:

[0014] (1) Add the reaction reagent for ornithine to citrulline to the sample to be tested, react at 37°C for 20 min, and then terminate the reaction with a frozen internal standard-stop solution. Then, perform a derivatization reaction with a derivatization reagent. After derivatization, blow dry the sample with nitrogen gas and then re-dissolve it with a re-solution solution to obtain a test sample.

[0015] (2) preparing a citrulline standard curve by the internal standard method; detecting the sample to be tested before and after the reaction in step (1) by HPLC-MS / MS, and obtaining the citrulline content of the sample to be tested before and after the reaction by using the citrulline standard curve;

[0016] (3) The activity of ornithine carbamoyltransferase was calculated using the formula: ornithine carbamoyltransferase activity value = (citrulline after reaction - citrulline before reaction) / reaction time.

[0017] In some specific embodiments of the present invention, the sample to be tested is a blood or serum sample.

[0018] In some embodiments of the present invention, the reaction reagents for ornithine to generate citrulline include ornithine, a phosphate buffer system, and carbamoyl phosphate. The phosphate buffer system is a phosphate buffer solution with a pH value of 7.2-7.4, which is formed by mixing sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate; in some embodiments, 337 mg of ornithine is dissolved in 20 mL of sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate, and the pH value of the solution is 7 to prepare reagent 1. Reagent 1 reacts with the sample to detect the non-specific product of the sample; in some embodiments, 5 mg of carbamoyl phosphate is dissolved in 1.5 mL of reagent 1 to form reagent 2, and reagent 2 is added with water to form a blank control to exclude citrulline interference; in some embodiments, the test sample and reagent 2 are subjected to a citrulline generation reaction for the detection and calculation of the target product. In some specific embodiments of the present invention, the sample or water and the added reagent are in a volume ratio of 1:1.

[0019] During the citrulline generation reaction between the test sample and the reaction reagent 2, the reaction is terminated with an internal standard-stop solution. The frozen internal standard-stop solution has a better termination effect. In an embodiment of the present invention, the internal standard-stop solution is a methanol solution of 160nmol / mL citrulline internal standard. The product is derivatized with a derivatization reagent. In an embodiment of the present invention, the derivatization reagent is a hydrochloric acid n-butanol solution, 30mL n-butanol, and 10mL 11.9mol / L hydrochloric acid is accurately added to prepare it. The derivatized sample is dried with nitrogen at 40°C and then redissolved for easy detection. In an embodiment of the present invention, the redissolved solution is an acetonitrile formic acid aqueous solution: 128mL water, 32mL acetonitrile, and 160μL formic acid are mixed.

[0020] In some specific embodiments of the present invention, the process of the citrulline generation reaction between the test sample and the reaction reagent 2 is as follows:

[0021] 100μL of reagent 2 was added to 100μL of the sample to be tested. When reagent 2 was added, the timing started. The samples were placed at 37 degrees for 20 minutes. Then 100μL of the standard curve and 100μL of the quality control product were added to 500μL of the cold internal standard-stop solution, respectively, and the reaction was stopped. The sample was shaken for 5 minutes, centrifuged at 12000 rpm for 5 minutes at 4℃, and 100μL of the supernatant was aspirated and placed in a clean 96-well plate. The sample was blown dry with nitrogen at 40 degrees, and 100μL of the derivatization reagent was added. The sample was derivatized on a shaking table at 60 degrees for 20 minutes. The sample after derivatization was blown dry with nitrogen at 40 degrees, and 200μL of the reconstitution solution was added. The sample was shaken for 3 minutes, and centrifuged at 12000 rpm for 5 minutes at 4℃. The supernatant was aspirated and placed in a clean 96-well plate for injection.

[0022] In some embodiments of the present invention, the internal standard method is used to prepare the citrulline standard curve, and the process is as follows:

[0023] (1) Preparation of W1, the highest point of the working curve: Add 20 μL of 50 umol / mL citrulline standard solution to 4.98 mL of blank human serum to prepare citrulline W1 with a concentration of 200 nmol / mL.

[0024] (2) Using blank human serum matrix and the highest point W1 of the working curve, 8 different concentrations of standard curve working solutions (W2-W9) were obtained by stepwise dilution for preparing the standard curve. The preparation process and concentration of the standard curve are shown in the following table:

[0025]

[0026] (3) Using blank human serum matrix and the highest point W1 of the working curve, three quality controls (LQC, MQC, and HQC) were prepared. The quality control sample preparation process and the final concentration of the quality control products are as follows:

[0027]

[0028] In some embodiments of the present invention, HPLC-MS / MS is used for detection to obtain a citrulline standard curve and the detection results of the sample to be tested.

[0029] In some specific embodiments of the present invention, in the detection method, liquid chromatography is used to separate non-specific substances from target detection substances, and the chromatographic conditions are:

[0030] Chromatographic column: Phenomen UPLC C18 (100×2.1 mm, 2.6 μm);

[0031] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile;

[0032] Flow rate: 0.4 mL / min;

[0033] Column temperature: 40 °C;

[0034] Injection volume: 2 μL;

[0035] The gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; where:

[0036] The volume fraction of mobile phase A was maintained at 90% from 0.01 to 1.1 min;

[0037] 1.1-1.2 min The volume fraction of mobile phase A decreased from 90% to 15%;

[0038] 1.2-1.5 min: the volume fraction of mobile phase A was maintained at 15%;

[0039] From 1.5 to 1.51 min, the volume fraction of mobile phase A increased from 15% to 90% and then stopped at 3 min.

[0040] In the detection method, the mass spectrometry conditions are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 450 °C; ion source nebulizer gas (GS1): 50 psi; ion source heating auxiliary gas (GS2): 55 psi; curtain gas (CUR): 40 psi; collision gas (CAD): 8 psi; scanning mode: MRM;

[0041] The MRM quantitative detection product ion parameters are as follows:

[0042]

[0043] The second aspect of the present invention provides a detection kit for ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS, comprising a reaction reagent, an internal standard-stop solution, a derivatization reagent and a reconstitution solution, wherein the reaction reagent comprises ornithine, phosphate buffer and carbamoyl phosphate, the internal standard-stop solution is a frozen citrulline internal standard methanol solution, the derivatization reagent is a hydrochloric acid n-butanol solution, and the reconstitution solution is an acetonitrile formic acid aqueous solution.

[0044] In some embodiments of the invention, the phosphate buffer includes sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate.

[0045] In some embodiments of the present invention, the internal standard-stop solution is a methanol solution of citrulline internal standard at a concentration of 160 nmol / ml, which is frozen and stored at -80°C before use. The internal standard-stop solution is both an internal standard and a reaction stop solution. The citrulline internal standard at a concentration of 160 nmol / ml serves as a calibration solution, and the methanol contained in the solution serves to stop the reaction. The frozen internal standard-stop solution has a better termination effect.

[0046] In some embodiments of the present invention, a citrulline standard, a citrulline internal standard and a mobile phase are also included, wherein mobile phase A is 0.1% formic acid aqueous solution; mobile phase B is 0.1% formic acid acetonitrile.

[0047] In some embodiments, the reaction reagent includes reagent 1 prepared from ornithine and a phosphate buffer system, 5 mg of carbamoyl phosphate is dissolved in 1.5 mL of reagent 1 to form reagent 2, the phosphate buffer system is a phosphate buffer solution with a pH value of 7.2-7.4 mixed with sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate, reagent 1 reacts with the sample to detect the non-specific product of the sample; reagent 1 is prepared by dissolving 337 mg of ornithine in a solution with a pH value of 7 after mixing 20 mL of sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate. Reagent 2 is added with water to form a blank control to exclude citrulline interference; the test sample and reagent 2 are subjected to a citrulline generation reaction for the detection and calculation of the target product. In some specific embodiments of the present invention, the sample or water and the added reagent are in a volume ratio of 1:1.

[0048] During the citrulline generation reaction between the test sample and the reaction reagent, the reaction is terminated with an internal standard-stop solution. In an embodiment of the present invention, the internal standard-stop solution is a methanol solution of 160nmol / ml citrulline internal standard, and the internal standard-stop solution is frozen at -80°C before use. The product is derivatized with a derivatization reagent. In an embodiment of the present invention, the derivatization reagent is a hydrochloric acid n-butanol solution, 30mL n-butanol, and 10mL 11.9mol / L hydrochloric acid is accurately added. The derivatized sample is dried with nitrogen at 40°C and then redissolved for easy detection. In an embodiment of the present invention, the redissolved solution is an acetonitrile formic acid aqueous solution: 128mL water, 32mL acetonitrile, 160μL formic acid mixed.

[0049] In some specific embodiments of the present invention, the process of the citrulline generation reaction between the test sample and the reaction reagent 2 is as follows:

[0050] 100μL of reagent 2 was added to 100μL of the sample to be tested. When reagent 2 was added, the timing started. The samples were placed at 37 degrees for 20 minutes. Then 100μL of the standard curve and 100μL of the quality control product were added to 500μL of cold internal standard-stop solution methanol, respectively, for termination. The mixture was shaken for 5 minutes, centrifuged at 12000 rpm for 5 minutes at 4℃, 100μL of the supernatant was aspirated and placed in a clean 96-well plate, dried with nitrogen at 40 degrees, 100μL of the derivatization reagent was added, and the samples were derivatized on a shaker at 60 degrees for 20 minutes. The samples after derivatization were blown dry with nitrogen at 40 degrees, 200μL of the reconstitution solution was added, shaken for 3 minutes, and centrifuged at 12000 rpm for 5 minutes at 4℃. The supernatant was aspirated and placed in a clean 96-well plate for injection.

[0051] In some specific embodiments of the present invention, a citrulline standard and a mobile phase are also included, wherein mobile phase A is 0.1% formic acid aqueous solution; mobile phase B is 0.1% formic acid acetonitrile. A series of standard curve working solutions and quality control products of different concentrations are prepared using the citrulline standard to obtain a standard curve for citrulline, and the content of citrulline before and after the reaction in human serum samples is detected by HPLC-MS / MS, and the change in citrulline content reflects the activity of ornithine aminoformyl transferase.

[0052] The third aspect of the present invention also provides the use of the detection kit in the detection of ornithine carbamoyltransferase activity in human serum, using the detection method described in the first aspect and the kit described in the second aspect to determine the citrulline concentration of the sample, and then using the calculation formula ornithine carbamoyltransferase activity value = (citrulline after reaction - citrulline before reaction) / reaction time to calculate the ornithine carbamoyltransferase activity value, wherein the reaction time is 20 minutes.

[0053] The detection principle of the present invention:

[0054] Ornithine aminotransferase activity mainly depends on the following reaction:

[0055]

[0056] The reaction was carried out at 37°C for 20 minutes to separate the non-specific reactants of citrulline. The concentration of citrulline in each component was accurately quantified using the LC-MS standard curve. The ornithine aminotransferase activity value was calculated using the following formula.

[0057] The calculation formula was optimized as follows: ornithine aminotransferase activity value = (citrulline content after reaction - citrulline content before reaction) / reaction time.

[0058] During the detection of ornithine aminotransferase activity, steps were also performed to eliminate citrulline interference and deduct citrulline background to further eliminate nonspecific effects.

[0059] In the present invention, there are two ways to exclude citrulline interference: 1. Use water matrix instead of serum as blank control, subtract the water matrix result, and exclude citrulline interference. 2. Directly separate nonspecific peaks in liquid phase to exclude the influence of interference. Citrulline background subtraction: Replace the citrulline substrate with blank water matrix to conduct citrulline parallel test, and the value obtained is the citrulline background value. If there is any influence, subtract the result in the calculation.

[0060] Beneficial Effects of the Invention

[0061] Compared with the prior art, the beneficial effects of the present invention are:

[0062] In view of the fact that the activity of ornithine carbamoyltransferase cannot be directly detected and the difficulty of detection is high, we have developed a method for detecting a forward reaction based on ornithine to generate citrulline by HPLC-MS / MS with high specificity (blank matrix will not produce non-specific reaction) to obtain the activity of ornithine carbamoyltransferase. The present invention is the first to propose the use of HPLC-MS / MS detection method to detect the activity of ornithine carbamoyltransferase. In this method, the peak time of 1.18 is citrulline, and the peak time of 1.28 is a non-specific reaction. The non-specific substances produced by the ornithine carbamoyltransferase activity experiment can be separated from the liquid phase and eliminated.

[0063] Compared with traditional experimental methods such as chemiluminescence or ultraviolet methods, HPLC-MS / MS high-specificity recognition is used to replace serum with water, and water is used as a blank control substrate for control reaction. It is detected that there is only a non-specific peak when water is used as a blank control. The citrulline detection result has no effect on the final ornithine aminoformyl transferase activity, eliminating interference, and there is no need to test non-specific reactions, making the citrulline results more accurate. In addition, the liquid phase is used to eliminate the non-specific generation peak of citrulline, so that this reaction is not interfered.

[0064] In the whole reaction, the buffer system is also particularly important, providing suitable buffer conditions and pH for the whole reaction. The buffer system of the present invention uses sodium hydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, mixed in different proportions to achieve the required pH value: 7.2-7.4. No special ions with low human content are introduced, no interference is caused to the results, only sodium and potassium ions are used to simulate the human body environment; no strong acid or strong base is used, and the buffer salt is cleverly used to adjust the pH, which is more conducive to the reaction of the whole system.

[0065] The reaction in this method is a reversible reaction. During the reaction, frozen methanol is used as the stop solution, avoiding the use of general strong acid to stop the reaction. Using general strong acid to stop the reaction will change the acid-base of the reaction system and make the reaction go in reverse. Using methanol to stop the reaction can, on the one hand, deform the protein, and on the other hand, stop the reaction at low temperature without destroying the acid-base system and affecting the reaction process.

[0066] The citrulline test uses a derivatization method, which has more stable results; the gradient changes within 3 minutes and will not block the column. This method uses in vitro samples as the test object, and reflects the activity of the corresponding enzyme in the liver by detecting the activity of ornithine aminotransferase in the blood of in vitro samples, avoiding traumatic liver puncture sampling. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The linear relationship diagram of citrulline in the present invention is shown;

[0068] Figure 2 The figure shows the citrulline detection result of the blank control group in the present invention;

[0069] Figure 3 The figure shows the detection result of the citrulline standard substance in the present invention;

[0070] Figure 4 The figure shows the detection result of citrulline in sample 1 before reaction in the present invention;

[0071] Figure 5 The figure shows the detection result of citrulline after the reaction of sample 1 in the present invention;

[0072] Among them, the retention time of citrulline is 1.18, and the retention time of nonspecific reactants is 1.25. DETAILED DESCRIPTION

[0073] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0075] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0076] Example 1

[0077] 1. Preparation of working curve W1

[0078] Prepare the highest point W1 solution of the citrulline working curve: take 4.98 mL of blank serum without the target substance, add 20 μL of 50 umol / mL citrulline standard solution to obtain a citrulline W1 concentration of 200 nmol / mL.

[0079] 2. Preparation of working curve solution

[0080] The highest point solution W1 of the citrulline working curve and the blank matrix: human serum were used to perform stepwise dilution according to the method recorded in Table 1 to obtain the standard curve working solution.

[0081] Table 1 Configuration of standard curve working solution

[0082]

[0083] 3. Preparation of Quality Control Samples

[0084] W1 was diluted with blank matrix in sequence to obtain low, medium and high quality controls (LQC, MQC and HQC). The preparation process and final concentrations of the quality controls are shown in Table 2.

[0085] Table 2 Quality control sample preparation and final concentration

[0086]

[0087] IV. Sample processing

[0088] 1. Reagent configuration

[0089] Reagent 1: 0.01 M ornithine and phosphate buffer system, pH 7.

[0090] The preparation method is as follows: 337 mg of ornithine is dissolved in 20 mL of a mixed solution of sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate with a pH value of 7.

[0091] Reagent 2: Weigh 5 mg of carbamoyl phosphate and dissolve it in 1.5 mL of reagent 1.

[0092] Internal standard-stop solution: a methanol solution of citrulline internal standard at a concentration of 160 nmol / mL, stored at -80°C for future use.

[0093] Derivatization reagent: 30mL of n-butanol, add 10mL of 11.9mol / L hydrochloric acid accurately;

[0094] Reconstitution solution: 128 mL water, 32 mL acetonitrile, 160 μL formic acid.

[0095] The sample preparation was carried out using a 1.5 mL EP tube. The sample preparation scheme is shown in Table 3.

[0096] Table 3 Sample preparation scheme

[0097]

[0098] According to the operation in the table above, when reagent 2 is added, the timing starts, and the reaction is placed at 37 degrees for 20 minutes. Then, 100 μL of the standard curve and 100 μL of the quality control product are taken, and 500 μL of the frozen internal standard-stop solution are added to stop the reaction. Shake well for 5 minutes, and centrifuge at 12000 rpm for 5 minutes at 4°C.

[0099] 100 μL of supernatant was aspirated and placed in a clean 96-well plate, blown dry with nitrogen at 40 degrees, 100 μL of derivatization reagent was added, and derivatization was carried out on a shaking table at 60 degrees for 20 minutes.

[0100] The derivatized samples were dried with nitrogen at 40°C, 200 μL of reconstitution solution was added, shaken for 3 min, and centrifuged at 12,000 rpm for 5 min at 4°C.

[0101] Pipette the supernatant and place it in a clean 96-well plate for injection.

[0102] The activity of ornithine aminotransferase mainly depends on the following reaction formula. The reaction time is 20 minutes at 37°C, and the ornithine aminotransferase activity value is obtained by subtracting the amount of citrulline generated from the background amount of citrulline.

[0103]

[0104] 5. Testing conditions

[0105] 1. Chromatographic conditions

[0106] Chromatographic column: Phenomen UPLC C18 (100×2.1 mm, 2.6 μm);

[0107] Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile;

[0108] Flow rate: 0.4 mL / min;

[0109] Column temperature: 40 °C;

[0110] Injection volume: 2 μL;

[0111] The gradient elution method was used, and the gradient elution method was as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; the gradient elution time was 3 minutes and then stopped.

[0112] The elution gradient parameter settings are shown in Table 4.

[0113] Table 4 Gradient elution parameter settings

[0114]

[0115] 2. Mass spectrometry conditions:

[0116] Ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 450°C; ion source nebulizer gas (GS1): 50 psi; ion source heating auxiliary gas (GS2): 55 psi; curtain gas (CUR): 40 psi; collision gas (CAD): 8 psi; scan mode: MRM; MRM parameter conditions are set as shown in Table 5.

[0117] Table 5 MRM parameter settings

[0118]

[0119] In the above table, Q1 is the parent ion; Q3 is the daughter ion; DP is the declustering voltage; EP is the injection voltage, CE is the collision voltage; CXP is the collision cell ejection voltage.

[0120] 6. Results

[0121] (1) The standard curve working solution was detected by HPLC-MS / MS, with the chromatographic peak area ratio of the target analyte to the corresponding internal standard as the ordinate and the concentration of the target analyte in the standard curve working solution as the abscissa. The standard curve equations corresponding to the target analyte were fitted as shown in Table 6 below.

[0122] Table 6 Standard curve equations for target analytes

[0123]

[0124] Citrulline linear curve is shown in Figure 1 .

[0125] (2) To further exclude citrulline interference, the blank control was tested using HPLC-MS / MS. Figure 2 shown.

[0126] The results showed that the mass spectrometry method was very specific. No blank control substrate was produced, there was no background interference, and the blank control only had non-specific peaks. The citrulline detection result in the blank control was almost 0, which had no effect on the final calculation result of the ornithine aminoformyl transferase activity value.

[0127] In order to verify the detection effect of the present invention, the citrulline standard solution was detected by HPLC-MS / MS. The detection results are as follows Figure 3 shown.

[0128] The results showed that the retention time of the standard citrulline was 1.18.

[0129] (3) Three different serum samples, sample 1 from a patient with homozygous ornithine aminotransferase deficiency, sample 2 from a patient with heterozygous ornithine aminotransferase deficiency, and sample 3 from a normal person, were tested before and after the reaction using HPLC-MS / MS. The content of the target analyte in the test samples was calculated based on the corresponding standard curve equation, and the ornithine aminotransferase activity value was finally calculated using the following formula: ornithine aminotransferase activity value = (citrulline content after reaction - citrulline content before reaction) / reaction time. The results are shown in Table 7 below.

[0130] Table 7 Test values ​​and calculation results of three samples

[0131]

[0132] The detection of citrulline in sample 1 before reaction is shown in the figure Figure 4 As shown in the figure, the retention time of citrulline is 1.18, the retention time of non-specific peak is 1.25, and there is almost no non-specific peak in the figure. The detection diagram of citrulline after the reaction is as follows Figure 5 As shown, the retention time of citrulline is 1.18, and the retention time of the non-specific peak is 1.25. It can be seen from the figure that citrulline and the non-specific peak have been separated.

[0133] (4) System adaptability, precision and stability

[0134] System suitability: Repeat the injection 6 times to obtain the retention time of the peak area, the CV value of the internal standard retention time and the concentration.

[0135] Precision: Intra-batch precision: Prepare 6 samples of the same concentration and load them onto the machine after pre-treatment. Calculate the precision value between the 6 samples. Inter-batch precision: The intra-batch precision is operated for three consecutive days, and the precision between the three days is calculated, which is called inter-batch precision.

[0136] The results are shown in Table 8.

[0137] Table 8 System suitability, precision and stability verification data

[0138]

[0139] The above results show that the system adaptability, residue and precision of citrulline all meet the requirements of: FDA: Bioanalytical Method Validation-Guidance for Industry; CLSI C62-A: A New Standard for Clinical Mass Spectrometry; Chinese Medical Doctor Association Laboratory Physicians Branch: Development and Validation Guidelines for Liquid Chromatography Tandem Mass Spectrometry Clinical Detection Methods.

[0140] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS, characterized in that: The steps include: (1) Add the reaction reagent for ornithine to citrulline to the sample to be tested, react at 37°C for 20 min, and then terminate the reaction with a frozen internal standard-stop solution. Then, perform a derivatization reaction with a derivatization reagent. After derivatization, blow dry the sample with nitrogen gas and then re-dissolve it with a re-solution solution to obtain a test sample. (2) preparing a citrulline standard curve by the internal standard method; detecting the sample to be tested before and after the reaction in step (1) by HPLC-MS / MS, and obtaining the citrulline content of the sample to be tested before and after the reaction by using the citrulline standard curve; (3) The activity value of ornithine carbamoyltransferase was calculated using the formula: ornithine carbamoyltransferase activity value = (citrulline after reaction - citrulline before reaction) / reaction time; The internal standard-stop solution is a methanol solution of citrulline internal standard; The reaction reagent for ornithine to generate citrulline includes ornithine, a phosphate buffer system and carbamoyl phosphate, wherein the phosphate buffer system is a phosphate buffer solution formed by mixing sodium hydrogen phosphate, sodium dihydrogen phosphate and potassium dihydrogen phosphate with a pH value of 7.2-7.4; In the detection method, liquid chromatography is used to separate nonspecific substances from target substances, and the chromatographic conditions are: Column: Phenomen UPLC C18, 100 × 2.1 mm, 2.6 μm; Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; Flow rate: 0.4 mL / min; Column temperature: 40 °C; Injection volume: 2 μL; The gradient elution method is as follows: the volume fraction of mobile phase A + the volume fraction of mobile phase B = 100%; where: The volume fraction of mobile phase A was maintained at 90% from 0.01 to 1.1 min; 1.1-1.2 min The volume fraction of mobile phase A decreased from 90% to 15%; 1.2-1.5 min: the volume fraction of mobile phase A was maintained at 15%; From 1.5 to 1.51 min, the volume fraction of mobile phase A increased from 15% to 90% and then stopped at 3 min.

2. The method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS according to claim 1, characterized in that: The derivatization reagent is a hydrochloric acid n-butanol solution.

3. The method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS according to claim 1, characterized in that: The reconstitution solution is an acetonitrile formic acid aqueous solution.

4. The method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS according to claim 1, characterized in that: A step of using water matrix as a control to exclude citrulline interference is also included.

5. The method for detecting ornithine carbamoyltransferase activity in human serum based on HPLC-MS / MS according to claim 1, characterized in that: In the detection method, the mass spectrometry conditions are: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature, TEM: 450 °C; ion source nebulizer gas, GS1: 50 psi; ion source heating auxiliary gas, GS2: 55 psi; Curtain gas, CUR: 40 psi; Collision gas, CAD: 8 psi; Scan mode: MRM; The MRM quantitative detection product ion parameters are as follows: Citrulline: Q1, 232.5; Q3, 70.3; DP, 50; EP, 10; CE, 25; CXP, 12; Citrulline internal standard: Q1, 236.2; Q3, 73.9; DP, 70; EP, 10; CE, 17; CXP, 12.

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