A method for rapid detection of neopterin, biopterin, pterin and 7-biopterin in urine

Abstract

The present invention belongs to the field of biological detection technology, and discloses a method for rapid detection of neopterin, biopterin, pterin and 7-biopterin in urine. Specifically disclosed is the application of perfluorobutyric acid in improving the detection effect of pterin spectrum in urine based on liquid chromatography. The present invention uses perfluorobutyric acid for the determination of pterin spectrum in urine. As an ion pair reagent of hydrophobic anions, it can significantly enhance the separation effect of each pterin spectrum (such as biopterin, neopterin, pterin and 7-biopterin), and can significantly improve the detection accuracy of pterin spectrum.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a method for rapidly detecting neopterin, biopterin, pterin and 7-biopterin in urine. Background Art

[0002] Pterins are derivatives of pteridine and play an important supporting role in cellular metabolism. They are widely distributed in human body fluids, including blood, urine, and cerebrospinal fluid, but the concentrations are very low. However, when the immune system is activated by certain diseases, such as cancer, or when metabolic defects are caused by congenital diseases, pterin levels in the body can change significantly.

[0003] Hyperphenylalaninemia (HPA) is a common autosomal recessive inherited metabolic disorder that can lead to severe intellectual disability. HPA is divided into two main categories based on the enzyme deficiency: phenylalanine hydroxylase (PAH) deficiency and tetrahydrobiopterin (BH4) deficiency, the HPA coenzyme. BH4 is a coenzyme for enzymes such as phenylalanine, tyrosine, and tryptophan hydroxylase. Congenital defects in certain enzymes during BH4 synthesis or metabolism lead to metabolic disorders of certain aromatic amino acids, impairing neurotransmitter synthesis in the brain and causing severe neurological damage and intellectual disability in affected children. BH4 deficiency can be classified into the following five categories based on the defective enzyme: (1) 6-pyruvoyl tetrahydropterin synthase (PTPs) deficiency (most common, about 96%); (2) dihydropteridine reductase (DHPR) deficiency (2.4%); (3) guanosine triphosphate cyclohydrolase (GTPCH) deficiency; (4) sepiapterin reductase (SR) deficiency; and (5) pterin-4a-carbinolamine dehydratase (PCD) deficiency. Patients with BH4 deficiency also have significant changes in the pterin profile of their body fluids.

[0004] Different types of HPA have distinct treatment options and outcomes. Currently, the primary method for differentiating and classifying these diseases is liquid chromatography analysis of changes in urine pterin profiles. Sensitive, specific, and accurate urine pterin profile analysis is crucial for HPA screening, treatment, and subtype screening and identification.

[0005] Pterins have high melting points and poor solubility in organic solvents, but are soluble in sodium hydroxide and dilute hydrochloric acid solutions. Some pterins are chemically unstable and easily oxidized or reduced in air. Most naturally occurring pterins exist in body fluids in a reduced form and need to be oxidized into fluorescent substances before liquid chromatography analysis. Fully oxidized pterin compounds are blue or yellow fluorescent compounds. The pterin spectrum in urine mainly includes neopterin (NP), biopterin (BP), BH4, dihydrobiopterin (BH2), pterin (PT), 7-biopterin (7-BP), etc. Among them, neopterin, biopterin, 7-biopterin, and the fully oxidized forms of pterin are blue fluorescent compounds. Currently, the commonly used method for urine pterin detection involves oxidizing reduced BH2 and BH4 in the urine of HPA patients using acidic iodine to NP and BP. The levels of NP and BP in HPA patients' urine are then measured using high-performance liquid chromatography (HPLC) with fluorescence detection. Only NP and BP pterins are measured. 7-Biopterin is a screening test for pterin-4-carbinolamine dehydratase (PCD) deficiency and serves as a characteristic metabolite that distinguishes it from other types. PCD is a benign condition that can be treated with BH4 or without treatment. Therefore, 7-biopterin detection is crucial for screening and typing HPA patients.

[0006] In addition to the commonly used high-performance liquid chromatography (HPLC) fluorescence detection method, urine pterin profiling also includes gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC / MS-MS). Current domestic and international detection methods have the following problems: 1. Urine contains a large amount of water-soluble, highly polar impurities such as salts. Neopterin, biopterin, and 7-biopterin are highly susceptible to hydrolysis and have poor storage stability, making them unsuitable for large-sample analysis in HILIC mode with a high organic phase ratio. 2. Traditional methods often use a 25cm reversed-phase C18 column, which enhances retention and separation but does not effectively separate biopterin and 7-biopterin. 3. Methods using a reversed-phase bonded silica column with methanol and ammonium acetate buffer as the mobile phase, while gradient elution shortens the analysis time, only detect neopterin and biopterin, not 7-biopterin. 4. HPLC fluorescence detection can only partially separate biopterin and 7-biopterin, but the analysis time is long, which does not meet the requirements of rapid, high-throughput clinical testing. 5. Although LC / MS-MS in HILIC mode can detect neopterin, biopterin, and 7-biopterin, the expensive LC / MS instrumentation and high detection costs hinder its widespread use. 6. GC-MS is undoubtedly a sensitive and specific method for elucidating the structure of unknown pterins. However, it requires iodination, solid-phase extraction, elution, nitrogen purging, and derivatization, making it more time-consuming. It also only detects neopterin and biopterin, not 7-biopterin. Biopterin and 7-biopterin are isomers. Failure to effectively separate them will affect the accuracy of their detection and will also hinder the differentiation between PTPs and PCDs in BH4 deficiency, thereby affecting the precise treatment of patients. Pterin's structural similarity to biopterin and 7-biopterin can interfere with their detection, thus requiring effective separation. Therefore, how to effectively separate biopterin, neopterin, pterin and 7-biopterin and improve the detection accuracy of pterin spectrum is very important for the screening and typing of HPA patients.

[0007] Reversed-phase ion-pair chromatography offers advantages such as ease of use and high separation efficiency. Currently, anionic surfactants such as alkyl sulfonates are widely used. There are reports of using sodium octyl sulfate as an ion-pairing reagent to separate biopterins and pterins, but this involves post-column derivatization, which is complex and time-consuming. Compared to alkyl sulfonates, perfluorocarboxylic acids have the advantages of lower surface activity and a lower boiling point. Perfluorobutyric acid (HBFA) is increasingly used in amino acid analysis due to its ability to increase retention time in a single step and its shortened equilibrium time. Currently, there are no reports of its use in pterin spectrum analysis.

[0008] Developing a method suitable for the clinical detection of neopterin, biopterin, and 7-biopterin to obtain accurate data accurately, rapidly, and with high throughput will help differentiate phenylalanine hydroxylase (PAH) deficiency from BH4 deficiency and various BH4 types, which is particularly important in clinical practice for early screening and early use of appropriate treatment to improve prognosis. Summary of the Invention

[0009] The purpose of the first aspect of the present invention is to provide the use of perfluorobutyric acid in improving the detection effect of pterin spectrum in urine based on liquid chromatography.

[0010] The second aspect of the present invention aims to provide a method for detecting pterin spectrum in urine.

[0011] The third aspect of the present invention aims to provide use of the method of the second aspect of the present invention in differentiating PAH deficiency from BH4 deficiency.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides the use of perfluorobutyric acid in improving the detection effect of pterin spectrum in urine based on liquid chromatography.

[0014] Preferably, the pterin spectrum includes one or more of neopterin, biopterin, dihydrobiopterin, pterin and 7-biopterin.

[0015] A second aspect of the present invention is to provide a method for detecting a pterin spectrum in urine, comprising the following steps: preparing a calibrator solution, preparing a test solution, and analyzing and detecting the calibrator solution and the test solution using liquid chromatography; the pterin spectrum includes neopterin, biopterin, pterin and 7-biopterin.

[0016] Preferably, the mobile phase A in the liquid chromatography is a perfluorobutyric acid aqueous solution, and the mobile phase B is a methanol solution.

[0017] Preferably, the liquid mobile phase A is a 1% to 5% perfluorobutyric acid aqueous solution, and the mobile phase B is a 0 to 5% methanol solution.

[0018] Further preferably, the mobile phase A is a 2% to 5% perfluorobutyric acid aqueous solution, and the mobile phase B is a 0 to 2% methanol solution.

[0019] More preferably, the mobile phase A is a 3% perfluorobutyric acid aqueous solution, and the mobile phase B is methanol.

[0020] Preferably, the test solution is prepared by mixing the sample to be tested with an iodine / potassium iodide solution and hydrochloric acid, reacting the mixture, and adding ascorbic acid to obtain the test solution.

[0021] Preferably, the calibration standard solution is prepared by mixing the calibration standard with an iodine / potassium iodide solution and hydrochloric acid, reacting the mixture, and adding ascorbic acid to obtain the calibration standard solution.

[0022] More preferably, the concentration of the iodine / potassium iodide solution is 1-5 wt %, and the usage amount is 20-50 μL / 100 mL sample.

[0023] More preferably, the reaction is carried out at room temperature in the dark for 40 to 50 minutes.

[0024] More preferably, the concentration of ascorbic acid is 1-2 mg / mL, and the usage amount is 50-70 μL / 100 mL sample.

[0025] Further preferably, the ascorbic acid is added followed by centrifugation and the supernatant is filtered.

[0026] Preferably, the elution in the liquid chromatography adopts a constant flow mode.

[0027] Preferably, the stripping procedure is 95% to 99% solution A and 1% to 5% solution B.

[0028] Preferably, the flow rate of the mobile phase in the liquid chromatography is 0.2 to 1.5 mL / min; further 0.4 to 1 mL / min; further 0.8 to 1 mL / min.

[0029] Preferably, the chromatographic column in the liquid chromatography is Eclipse XDB 80A-C18.

[0030] Further preferably, the chromatographic column is Eclipse XDB 80A-C18, 4.6×250 mm, 5 μm.

[0031] Preferably, the detection conditions in the liquid chromatography are as follows: the excitation light wavelength is 250-300 nm, the emission light wavelength is 400-450 nm, and the injection plate temperature is 2-8°C.

[0032] Preferably, the column temperature in the liquid chromatography is 28-32° C., and the injection volume is 1-2 μL.

[0033] The third aspect of the present invention is to provide use of the method of the second aspect of the present invention in differentiating PAH deficiency and BH4 deficiency.

[0034] Preferably, the BH4 deficiency is one or more of 6-pyruvoyltetrahydropterin synthase deficiency, dihydropteridine reductase deficiency, guanosine triphosphate cyclohydrolase deficiency, septopterin reductase deficiency and pterin-4-carbinolamine dehydratase deficiency.

[0035] The beneficial effects of the present invention are:

[0036] The present invention uses perfluorobutyric acid for the determination of pterin spectra in urine. As an ion pair reagent for hydrophobic anions, perfluorobutyric acid can significantly enhance the separation effect of various pterin spectra (such as neopterin, biopterin, pterin and 7-biopterin), and can significantly improve the detection accuracy of pterin spectra.

[0037] The method provided by the present invention has low cost, simple pre-treatment, can realize batch processing detection, optimizes the chromatographic method to shorten the analysis time, greatly improves the sample throughput, and is particularly suitable for the needs of clinical and daily detection for chromatographic methods.

[0038] The present invention adopts sample pre-column oxidation-liquid chromatography separation-fluorescence detector detection, and effectively separates neopterin, biopterin, pterin and 7-biopterin by optimizing the flow rate and increasing the proportion of organic solvent in the mobile phase, thereby improving the analytical specificity and facilitating the differentiation of phenylalanine hydroxylase deficiency and various types of BH4 deficiency.

[0039] In the method provided by the present invention, perfluorobutyric acid is added to the mobile phase as an ion pair reagent for hydrophobic anions, which can significantly enhance the separation effect of neopterin, biopterin, pterin and 7-biopterin to improve the accuracy of detection. The method provided by the present invention is used to quantitatively detect biopterin, neopterin and 7-biopterin in urine. The results show that biopterin, neopterin and 7-biopterin are within the linear range, the correlation coefficient (R) is greater than 0.999, the detection limit is 0.02-0.04 μmol / L, the quantitative limit is 0.04-0.11 μmol / L, the recovery rate of neopterin is between 95.2% and 104.1%, the recovery rate of biopterin is between 95.9% and 104.3%, and the recovery rate of 7-biopterin is between 96.0% and 104.3%, which meets the standard recovery rate of 85% to 115% for general sample determination. It can be seen that the determination results of this method are accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a chromatogram of the detection of neopterin, biopterin, pterin and 7-biopterin in the standard product of Example 1.

[0041] Figure 2 The chromatogram of Example 1 is a chromatogram of detecting neopterin, biopterin and 7-biopterin in normal human urine samples.

[0042] Figure 3 Example 1 is a chromatogram showing the detection of neopterin, biopterin, pterin and 7-biopterin in urine samples from patients with 6-pyruvoyltetrahydropterin synthase (PTPs) deficiency.

[0043] Figure 4Example 1 is a chromatogram showing the detection of neopterin, biopterin, pterin and 7-biopterin in urine samples from patients with pterin-4-carbinolamine dehydratase (PCD) deficiency.

[0044] Figure 5 This is a chromatogram of the detection of neopterin, biopterin, pterin and 7-biopterin in the standard product of Example 2.

[0045] Figure 6 This is a chromatogram of the detection of neopterin, biopterin, pterin and 7-biopterin in the standard product of Example 3.

[0046] Figure 7 This is a chromatogram of the detection of neopterin, biopterin, pterin and 7-biopterin in the standard product of Comparative Example 1.

[0047] Figure 8 This is a chromatogram of the detection standard of neopterin, biopterin, pterin and 7-biopterin in Comparative Example 2.

[0048] Figure 9 This is a chromatogram of the detection of neopterin, biopterin, pterin and 7-biopterin in the standard product of Comparative Example 3.

[0049] Figure 10 This is a chromatogram of the detection standard of neopterin, biopterin, pterin and 7-biopterin in Comparative Example 4.

[0050] Figure 11 This is a chromatogram of the detection standard of comparative example 5 for neopterin, biopterin, pterin and 7-biopterin.

[0051] Figure 12 This is a chromatogram of the detection standard of comparative example 6 for neopterin, biopterin, pterin and 7-biopterin.

[0052] Figure 13 This is the linear regression curve for detecting neopterin using Example 1.

[0053] Figure 14 This is the linear regression curve for detecting biopterin using Example 1.

[0054] Figure 15 This is the linear regression curve for detecting 7-biopterin using Example 1. DETAILED DESCRIPTION

[0055] The present invention will now be described in detail with reference to specific embodiments, but the scope of the present invention is not limited thereto.

[0056] Unless otherwise specified, the materials and reagents used in this example were obtained from commercial sources.

[0057] Example 1

[0058] A method for rapidly detecting neopterin, biopterin, pterin, and 7-biopterin in urine comprises the following steps:

[0059] (1) Preparation of standard solution

[0060] 1) Pterin standards: neopterin, biopterin, pterin, 7-biopterin

[0061] 2) Preparation of standard products:

[0062] ①Standard storage solution

[0063] Accurately weigh 1 mg each of neopterin, biopterin, pterin, and 7-biopterin neopterin standards, first add 100 μL of 0.1 mol / L hydrochloric acid and sonicate for 30 seconds, then add 900 μL of 0.1 mol / L sodium hydroxide to dissolve, and prepare a 1 mg / mL stock solution of each pterin.

[0064] ②Using liquid:

[0065] a. STD3: dilute the standard stock solution 10-fold with artificial urine (without neopterin, biopterin, pterin, and 7-biopterin);

[0066] b. STD2:STD3 solution was diluted 10-fold with artificial urine (without neopterin, biopterin, pterin, and 7-biopterin);

[0067] c. STD1:STD2 solution was diluted 10-fold with artificial urine (without neopterin, biopterin, pterin, and 7-biopterin).

[0068] ③ Preparation of calibration curve (Table 1):

[0069] Table 1 Calibration curve preparation

[0070]

[0071] Neopterin 1 ng / mL = 0.0039 μmol / L, biopterin 1 ng / mL = 0.0042 μmol / L, 7-biopterin 1 ng / mL = 0.0042 μmol / L.

[0072] (2) Sample pretreatment

[0073] 1) Sampling: Collect a fresh urine sample from a human, place it at room temperature, mix thoroughly, and transfer 100 μL to a 4 mL brown sample bottle. For calibration, add the sample to a 4 mL brown sample bottle according to Table 1 above.

[0074] 2) Oxidation: Add 50 μL of 1% iodine / potassium iodide solution and 150 μL of 0.2 mol / L hydrochloric acid to all the above samples. Add 700 μL of deionized water to the test samples and shake for 1 min. Add deionized water to the calibration samples according to Table 1 and shake for 1 min. Incubate at room temperature in the dark for 45 min.

[0075] 3) Neutralization: Add 50 μL of 2 mg / mL ascorbic acid to each of the above samples and vortex for 1 min;

[0076] 4) Filtration: For all the above samples, the supernatant was transferred using a 2.0 mL disposable syringe and passed through a 0.22 μm aqueous filter membrane to obtain the sample solution to be tested.

[0077] (3) Detection

[0078] The sample solution to be tested is subjected to liquid chromatography analysis, and the analysis conditions are as follows:

[0079] Liquid phase conditions:

[0080] Chromatographic column: Eclipse XDB 80A-C18 (4.6×250 mm, 5 μm);

[0081] Mobile phase A: 3% perfluorobutyric acid in water;

[0082] Mobile phase B: methanol;

[0083] Detection wavelength: excitation light wavelength is 280nm, emission light wavelength is 444nm;

[0084] Peak width: 74.07HZ;

[0085] Column temperature: 28°C;

[0086] Injection volume: 1 μL;

[0087] Sample plate temperature: 5°C;

[0088] Flow rate (constant flow mode): 0.8 mL / min, 97% solution A, 3% solution B;

[0089] Retention time: NP 6.0 min, BP 11.8 min, PT 12.4 min, 7-BP 13.2 min

[0090] Retention time window: 5%;

[0091] width: 0.1;

[0092] Threshold: 50;

[0093] Detector: Fluorescence detector.

[0094] The retention time and chromatographic peak of the target substance in the sample solution are compared with the chromatogram of the standard NP, BP, PT and 7-BP for qualitative analysis. The target substance standards with different concentration gradients are measured separately, and the corresponding concentrations are regressed by the chromatographic peak area to obtain a standard curve; under the same conditions, the test solution is measured, and the chromatographic peak area of ​​each target substance in the sample solution is measured and substituted into the standard curve for quantitative analysis. Figure 1 The chromatogram of the standard analysis is Figure 2 This is the chromatogram of normal human urine. Figure 3 This is the chromatogram of a patient with 6-pyruvoyltetrahydropterin synthase (PTPs) deficiency. Figure 4 This is a chromatogram from a patient with pterin-4-carbinol dehydratase (PCD) deficiency.

[0095] Example 2

[0096] The other conditions of this example are the same as those of Example 1, with the following differences: the flow rate is 1.0 mL / min. Figure 1 and Figure 5 It can be seen that compared with Example 1 ( Figure 1 ), under the same pretreatment conditions, chromatographic column, and fluorescence detector, increasing the flow rate and shortening the analysis time will result in poor separation of biopterin, pterin, and 7-biopterin ( Figure 5 ).

[0097] Example 3

[0098] The other conditions of this example were the same as those of Example 1, with the following difference: the mobile phase B was 2% solution B.

[0099] Depend on Figure 6 It can be seen that compared with Example 1 ( Figure 1 ) compared to the previous method, which used the same pretreatment conditions, chromatographic column, and fluorescence detector, but reduced the concentration of the organic solvent methanol, the retention time of each component was prolonged, and the separation of pterin and 7-biopterin was better. However, bifurcation occurred in neopterin due to the low methanol content, which affected the accuracy of neopterin detection.

[0100] Comparative Example 1

[0101] The other conditions of this example were the same as those of Example 1, with the following differences: mobile phase A was water, without perfluorobutyric acid.

[0102] The mobile phase has a great influence on the peak shape and chromatographic retention time of the analyte. Figure 7It can be seen that compared with Example 1, when the mobile phase does not contain perfluorobutyric acid, the retention time of each component is shortened, and the separation effect of pterin and 7-biopterin is poor. This is because perfluorobutyric acid acts as an ion pair reagent and reacts with the component to be measured to form a neutral ion pair, which can increase retention.

[0103] Comparative Example 2

[0104] The other conditions of this example were the same as those of Example 1, with the following difference: mobile phase A was a 1% perfluorobutyric acid aqueous solution.

[0105] Depend on Figure 8 It can be seen that compared with Example 1, when the concentration of perfluorobutyric acid in the mobile phase is reduced, the retention time of each component is shortened, and the separation effect of pterin and 7-biopterin is poor. This is because perfluorobutyric acid is an ion pair reagent. The lower the concentration, the weaker the retention of the neutral ion pair formed by the reaction with the component to be measured.

[0106] Comparative Example 3

[0107] Other conditions in this example were the same as those in Example 1, with the following difference: mobile phase A was a 2% perfluorobutyric acid aqueous solution.

[0108] Depend on Figure 9 It can be seen that compared with Example 1, when the concentration of perfluorobutyric acid in the mobile phase is reduced, the retention time of each component is shortened, and the separation effect of pterin and 7-biopterin is poor. This is because perfluorobutyric acid is an ion pair reagent. The lower the concentration, the weaker the retention of the neutral ion pair formed by the reaction with the component to be measured.

[0109] Comparative Example 4

[0110] Other conditions in this example were the same as those in Example 1, with the following difference: the chromatographic column was Eclipse XDB C18 (4.6×150 mm, 3.5 μm).

[0111] Depend on Figure 10 It can be seen that under the same pretreatment conditions, the same mobile phase, and flow rate, using the same filler but with a shorter, smaller particle size column, although the analysis time is shortened, the separation effect of biopterin from pterin and 7-biopterin is poor, affecting the detection accuracy of biopterin and 7-biopterin.

[0112] Comparative Example 5

[0113] The other conditions of this example are the same as those of Comparative Example 4, with the only difference being the flow rate: flow rate: 0.6 mL / min.

[0114] Depend on Figure 11It can be seen that under the same pretreatment conditions, the same fluorescence detector, the same mobile phase, and the same filler with a shorter, smaller particle size chromatographic column, the flow rate was reduced to 0.6 mL / min. Although the elution time of biopterin and 7-biopterin was prolonged, biopterin and pterin were not separated, affecting the accuracy of biopterin detection.

[0115] Comparative Example 6

[0116] The other conditions of this example are the same as those of Comparative Example 4, with the only difference being the flow rate: flow rate: 0.4 mL / min.

[0117] Depend on Figure 12 It can be seen that under the same pretreatment conditions, the same fluorescence detector, the same mobile phase, and the same filler with a shorter, smaller particle size chromatographic column, the flow rate was reduced to 0.4 mL / min. Although the elution time of biopterin and 7-biopterin was prolonged, biopterin and pterin were not separated, affecting the accuracy of biopterin detection.

[0118] Further effect test of Example 1

[0119] 1. Linearity, detection limit, and quantification limit of the detection method

[0120] Standard solutions of different mass concentrations were prepared as described in Example 1. This series of standard solutions was measured (test conditions were the same as in Example 1). A standard curve was plotted with the mass concentration of each target component as the abscissa (x) and the corresponding peak area as the ordinate (y) to determine the linear range, regression equation, and correlation coefficient of the method.

[0121] The results showed that neopterin, biopterin and 7-biopterin showed good linear relationships within the corresponding linear ranges, and the correlation coefficients (R) were all greater than 0.999 ( Figures 13-15 The detection limit and quantification limit of the method in Example 1 are shown in Table 2.

[0122] Table 2 Detection limit, quantification limit and linear range of the method

[0123]

[0124] 2. Spiked recovery of the detection method

[0125] High, medium and low concentration standard solutions were added to the negative sample, which was artificial urine (not containing neopterin, biopterin, pterin and 7-biopterin), and the samples were extracted and further determined according to the sample pretreatment method.

[0126] The results showed that the recovery rates of neopterin were between 95.2% and 104.1%, the recovery rates of biopterin were between 95.9% and 104.3%, and the recovery rates of 7-biopterin were between 96.0% and 104.3%, which met the standard range of 85% to 115% for the spiked recovery of general samples. This shows that the determination results of this method are accurate and reliable.

[0127] 3. Precision of the detection method

[0128] The precision of the detection method of Example 1 was investigated through intra-batch and inter-batch reproducibility experiments. The results are shown in Tables 3 to 5. The precision of the detection method of Example 1 for neopterin, biopterin, and 7-biopterin was less than 20% for both intra-batch and inter-batch coefficients of variation, meeting the requirements.

[0129] Table 3 Precision experimental results of the detection method of Example 1 for neopterin (NP)

[0130]

[0131] Table 4 Precision test results of the detection method of Example 1 for biopterin (BP)

[0132]

[0133] Table 5 Precision test results of the detection method of Example 1 for 7-biopterin (7-BP)

[0134]

[0135]

[0136] 4. Sample storage stability

[0137] Three fresh human urine samples were collected and divided into four equal parts. The urine was protected from light and stored at room temperature, 2-8°C, -20°C, and -70°C, respectively. The levels of neopterin, biopterin, and 7-biopterin in the urine were tested every 0, 1, 2, 5, 8, 15, and 22 days using the same detection method as in Example 1.

[0138] The results are shown in Tables 6 to 17, indicating that urine samples can be stably stored in the dark.

[0139] Table 6 Stability results of neopterin stored in the dark at room temperature

[0140]

[0141] Table 7 Stability results of biopterin stored in the dark at room temperature

[0142]

[0143] Table 8 Stability results of 7-Biopterin stored in the dark at room temperature

[0144]

[0145] Table 9 Stability results of neopterin stored in the dark at 2-8°C

[0146]

[0147] Table 10 Stability results of biopterin stored in the dark at 2-8°C

[0148]

[0149] Table 11 Stability results of 7-Biopterin stored in the dark at 2-8°C

[0150]

[0151] Table 12 Stability results of neopterin stored at -20°C in the dark

[0152]

[0153] Table 13 Stability results of biopterin stored at -20°C in the dark

[0154]

[0155] Table 14 Stability results of 7-Biopterin stored at -20°C in the dark

[0156]

[0157]

[0158] Table 15 Stability results of neopterin stored at -70°C in the dark

[0159]

[0160] Table 16 Stability results of biopterin stored at -70°C in the dark

[0161]

[0162] Table 17 Stability results of 7-Biopterin stored at -70°C in the dark

[0163]

[0164] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Application of perfluorobutyric acid in improving the detection of pterin spectra in urine by liquid chromatography; The conditions for the detection are: The chromatographic column was Eclipse XDB 80A-C18, 4.6 × 250 mm, 5 μm; Mobile phase A was 3% perfluorobutyric acid in water, and mobile phase B was methanol; Elution was performed in a constant flow mode with a flow rate of 0.8 mL / min, 97% mobile phase A, 3% mobile phase B; The detector is a fluorescence detector; The pterin spectrum includes neopterin, biopterin, pterin and 7-biopterin.

2. A method for detecting pterin spectrum in urine, comprising the following steps: preparing a calibrator solution and a test solution, and analyzing and detecting the calibrator solution and the test solution by liquid chromatography; the pterin spectrum includes neopterin, biopterin, pterin and 7-biopterin; The conditions for the detection are: The chromatographic column was Eclipse XDB 80A-C18, 4.6 × 250 mm, 5 μm; Mobile phase A was 3% perfluorobutyric acid in water, and mobile phase B was methanol; Elution was performed in a constant flow mode with a flow rate of 0.8 mL / min, 97% mobile phase A, 3% mobile phase B; The detector is a fluorescence detector.

3. The method according to claim 2, characterized in that The preparation method of the calibration solution and the test solution is as follows: the calibration solution and the sample to be tested are mixed with iodine / potassium iodide solution and hydrochloric acid, reacted, and ascorbic acid is added to obtain the calibration solution and the test solution.

4. The method according to claim 2, characterized in that The detection conditions in the liquid chromatography are as follows: the excitation light wavelength is 250-300 nm, the emission light wavelength is 400-450 nm, and the injection plate temperature is 2-8°C.

5. The method according to claim 4, characterized in that The column temperature in the liquid chromatography was 28-32° C., and the injection volume was 1-2 μL.

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