Method for simultaneously detecting uropyridin, deoxypyridin and creatinine by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting uropyridin, deoxypyridin and creatinine by LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry). According to the present invention, by optimizing the derivatization reaction, the Pry and the DPD generate the corresponding derivatives, the ion response of the detection target object on the mass spectrum is indirectly improved by the derivatives, and the derivatives have good retention and high peak response value on the chromatography; secondly, after derivation, ethyl acetate is used for extracting a target object, through nitrogen blowing concentration, interferents are remarkably removed, baseline noise is reduced, the signal-to-noise ratio is increased, high-accuracy and high-sensitivity detection of Pry, DPD and Cr in a urine sample is achieved, and clinical requirements are met.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a method for simultaneously detecting urine pyridinoline, deoxypyridinoline and creatinine by LC-MS / MS. Background Art

[0002] Pyridinoline (Pry) and deoxypyridinoline (DPD) are molecules that stabilize the bonds between collagen. During bone resorption, the bonds between collagen molecules are broken by proteolysis, and Pry and DPD are released into the urine. Clinically, most biochemical markers of bone metabolism exhibit circadian rhythms, with their levels reaching their highest level after midnight and their lowest level in the afternoon. Generally speaking, the diurnal variation in bone formation markers is approximately 10%, while the variation in bone resorption markers can reach 20%. This variation is smaller for Pry and DPD (approximately 12%). Furthermore, Pry and DPD are excreted directly into the urine without further degradation by the liver, unaffected by diet, and unmetabolized internally. As specific indicators of bone resorption, the bone turnover markers Pry and DPD are clearly defined as specific indicators of bone resorption in clinical guidelines such as the "Guidelines for the Clinical Application of Bone Turnover Biochemical Markers," the "Guidelines for the Diagnosis and Treatment of Primary Osteoporosis (2022)," and the "Expert Consensus on the Clinical Application of Bone Metabolism Biochemical Markers (2023 Revised Edition)," and thus have important clinical significance.

[0003] In primary osteoporosis and various metabolic bone diseases with increased bone resorption, urine Pry and DPD levels are significantly elevated. Urine specimens are usually corrected for creatinine (Cr) and expressed as BTM (units) / mmol Cr.

[0004] Accurate quantification of Pry and DPD in urine is challenging due to the low concentrations of free Pry and DPD in human urine and the compounds' inherent polarity (i.e., difficulty retaining on stationary phase materials or poor solubility in organic phases). Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection of Pry and DPD often involves the use of solid-phase extraction (SPE) plates to purify and concentrate the analytes in urine samples prior to detection. This method is complex, has high consumable costs, and exhibits low extraction yields. Alternatively, ion-pairing reagents are used as mobile phase additives to improve chromatographic retention. However, these reagents can cause significant and irreversible contamination of the mass spectrometer. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine by LC-MS / MS, which has high sensitivity, low detection cost and high accuracy, and can simultaneously detect Pry, DPD and Cr in human urine without using ion pair reagents.

[0006] Specifically, in preliminary research, the present invention screened a large number of substances that could theoretically introduce strong UV-absorbing or fluorescent groups into Pry and DPD through reactions, such as Girard reagent P, 2,4-dinitrophenylhydrazine, and o-phenylenediamine. Mass spectrometry tuning Q1 mass scan results showed that these derivatization reagents could not undergo derivatization reactions with Pry and DPD. Through extensive research and testing, the present invention ultimately discovered that under specific pH conditions, 6-aminoquinoline-N-hydroxysuccinimidylcarboxylate (AQC) can be used as a derivatization reagent to undergo a derivatization reaction with the primary amine groups in Pry and DPD. The resulting derivatives indirectly improved the ion response of the target detected on the mass spectrometer, and the derivatives exhibited good retention and high peak response values ​​on the chromatography. This may be because AQC has hydrophobic and easily ionizable groups, which impart hydrophobic and easily ionizable properties to Pry and DPD after reaction. At the same time, the present invention further discovered that using ethyl acetate for liquid-liquid extraction after derivatization can significantly remove impurities and improve the signal-to-noise ratio compared to other conventional extraction solvents (such as n-hexane, methyl tert-butyl ether, etc.), thereby greatly improving the detection accuracy of Pry and DPD in real urine samples.

[0007] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides an LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine, comprising: S1: mixing a sample to be tested, a mixed internal standard, and a borate solution with a pH value of 6.5 to 9 to obtain a first mixed solution; preferably, the pH value of the borate solution is 8 to 9; S2: mixing the first mixed solution with a derivatization reagent, and performing derivatization at 35-65° C. for 10-70 minutes to obtain a second mixed solution; the derivatization reagent is 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate; S3: mixing the second mixed solution and the extractant, shaking and then centrifuging, taking the supernatant and blowing it dry with nitrogen, re-dissolving it with a re-solution solution and then performing on-machine testing; the extractant includes ethyl acetate.

[0008] According to the LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the derivatization temperature is preferably 50~60°C, specifically any value among 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C, or a numerical range with any two of the above values ​​as endpoints.

[0009] According to the LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the derivatization time is preferably 20 to 40 minutes, specifically any value among 20 minutes, 25 minutes, 30 minutes, 35 minutes, and 40 minutes, or a numerical range with any two of the above values ​​as endpoints.

[0010] According to an LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the volume ratio of the sample to be tested and the derivatization reagent is 1:1~4; specifically, it can be any ratio such as 1:1, 1:2, 1:3 or 1:4, or a ratio range with any two of the above ratios as endpoints.

[0011] According to the present invention, a LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine is provided, wherein the volume ratio of the sample to be tested and the extractant is 1:12~20; specifically, it can be any ratio such as 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20, or a ratio range with any two of the above ratios as endpoints.

[0012] According to the LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the reconstitution solution is a 0wt% to 60wt% methanol aqueous solution; preferably, the reconstitution solution is water.

[0013] According to an LC-MS / MS method for simultaneously detecting urine pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the internal standards of pyridinoline and deoxypyridinoline are both L-lysine-d4, and the internal standard of creatinine is creatinine-d3.

[0014] According to the present invention, a LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine is provided. The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05wt% to 0.15wt% formic acid aqueous solution, and mobile phase B is methanol.

[0015] Preferably, the gradient elution conditions of the mobile phase include:

[0016] “%” represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1.

[0017] According to an LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the gradient elution conditions of the mobile phase include:

[0018] “%” represents volume percentage.

[0019] According to the present invention, a LC-MS / MS method for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine is provided. The liquid phase detection conditions also include: the chromatographic column is a C18 column, preferably Eclipse Plus C18; the mobile phase flow rate is 0.5~0.7ml / min; the column temperature is further optimized to be 40°C, the injector temperature is 10°C, and the injection volume is 15μL.

[0020] According to the LC-MS / MS method provided by the present invention for the simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine, the mass spectrometry detection conditions include: The ion source was electrospray ionization (ESI) in positive ion mode, and the scanning mode was multiple reaction monitoring mode. The ion source parameters were as follows:

[0021] The mass spectrometry parameters are as follows: .

[0022] In a second aspect, the present invention provides a kit for simultaneously detecting urinary pyridinoline, deoxypyridinoline, and creatinine by LC-MS / MS, comprising: a sample to be tested, an internal standard, a derivatization reagent, an extractant, and a borate solution; The derivatization reagent includes 6-aminoquinoline-N-hydroxysuccinimide formate; the extractant includes ethyl acetate; and the pH value of the borate solution is 6.5-9.

[0023] The present invention creates derivatization conditions within a specific pH range by using a borate solution, uses 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate to carry out a derivatization reaction with a sample to be tested, and generates corresponding derivatives of Pry and DPD. These derivatives indirectly improve the ion response of the detection target on the mass spectrum, and the derivatives have good retention and a high peak response value on the chromatography. Secondly, after derivatization, the target is extracted with ethyl acetate and concentrated by nitrogen blowing, which significantly removes interfering substances, reduces baseline noise, and improves the signal-to-noise ratio, thereby achieving high-accuracy and high-sensitivity detection of Pry, DPD, and Cr in urine samples to meet clinical needs. The invention also protects the instrument and the chromatographic column from contamination, reduces the frequency of instrument maintenance, and improves the life of the chromatographic column.

[0024] According to the LC-MS / MS kit provided by the present invention for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine, the kit also contains a mobile phase, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a 0.05wt%~0.15wt% formic acid aqueous solution, and the mobile phase B is methanol.

[0025] In the present invention, the formic acid concentration of mobile phase A can be any value among 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.10wt%, 0.11wt%, 0.12wt%, 0.13wt%, 0.14wt%, and 0.15wt%, or a numerical range in which any two of the above values ​​are endpoint values.

[0026] The present invention has found that the use of the above-mentioned mobile phase can increase the response value of the target object and further improve the detection sensitivity.

[0027] According to the LC-MS / MS kit for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the pH value of the borate solution is 8-9.

[0028] According to the LC-MS / MS kit for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the kit further contains a reconstitution solution, which is a 0wt% to 60wt% methanol aqueous solution.

[0029] In the present invention, the concentration of methanol in the reconstituted solution can be any value between 0wt% and 60wt%, such as pure water, or a methanol aqueous solution having a concentration of 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 20wt%, 30wt%, 40wt%, 50wt% and 60wt%.

[0030] More preferably, the reconstitution solution is water.

[0031] According to the LC-MS / MS kit for simultaneously detecting urinary pyridinoline, deoxypyridinoline and creatinine provided by the present invention, the sample to be tested includes: one or more of urine, quality control products and calibrators.

[0032] According to the LC-MS / MS kit for simultaneously detecting urine pyridinoline, deoxypyridinoline and creatinine provided by the present invention, in the quality control product, the concentration of pyridinoline is 60-480 ng / mL, the concentration of deoxypyridinoline is 15-120 ng / mL, and the concentration of creatinine is 300-2400 ng / mL; in the calibrator, the concentration of pyridinoline is 20-640 ng / mL, the concentration of deoxypyridinoline is 5-160 ng / mL, and the concentration of creatinine is 100-3200 ng / mL; And / or, the internal standard for pyridinoline and deoxypyridinoline is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

[0033] The present invention provides an LC-MS / MS method for simultaneously detecting Pry, DPD, and Cr in urine samples. First, through a derivatization reaction, Pry and DPD generate corresponding derivatives, which indirectly improve the ion response of the detection target on the mass spectrum, and the derivatives have good retention and high peak response values ​​on the chromatography. Secondly, after derivatization, the target is extracted with an organic solvent and concentrated by nitrogen blowing, which significantly removes interfering substances, reduces baseline noise, and improves the signal-to-noise ratio. The quantitative limits of Pry, DPD, and Cr are 5 ng / mL, 2 ng / mL, and 50 μg / mL, respectively. In addition, the method has a spiked recovery rate of 99-115% for the analytes, achieving high-accuracy and high-sensitivity detection of Pry, DPD, and Cr in urine samples, meeting clinical needs. The method can also protect the instrument and chromatographic column from contamination, reduce the frequency of instrument maintenance, and increase the life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is the LQC chromatogram of the low-point quality control (LQC) of pyridoline (Pry) in Example 1 provided by the present invention.

[0036] Figure 2 This is the LQC chromatogram of the deoxypyridinoline (DPD) low-point quality control in Example 1 provided by the present invention.

[0037] Figure 3 This is the LQC chromatogram of the low-point quality control of creatinine (Cr) in Example 1 provided by the present invention.

[0038] Figure 4 This is a quality control chromatogram of the low point of creatinine (Cr) when 20% methanol water is used as the reconstitution solution in Example 6 provided by the present invention.

[0039] Figure 5 This is a quality control chromatogram of the low point of creatinine (Cr) when 50% methanol water is used as the reconstitution solution in Example 6 provided by the present invention. DETAILED DESCRIPTION

[0040] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0041] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0042] Example 1 This embodiment provides a LC-MS / MS kit for the simultaneous detection of urinary pyridinoline, deoxypyridinoline, and creatinine as follows: Table 1

[0043] Detection methods include: 1. Sample preparation 1. Preparation of Calibrators and Quality Control Samples Pry, DPD, and Cr standards were prepared as mixed solutions to serve as stock solutions for standard working solutions and quality control working solutions. The stock solution of the standard working solution was mixed with artificial urine at a volume ratio of 1:9, and the stock solution of the quality control working solution was mixed with mixed blank urine at a volume ratio of 1:9. Calibration curves and quality control samples were prepared, respectively. Six concentration series (C1 to C6) were prepared for the calibration curve, and two concentration series (LQC and HQC) were prepared for the quality control samples, as shown in Tables 2 and 3, respectively.

[0044] Table 2 Six concentration series (C1-C6) of Pry, DPD, and Cr in the calibration curve

[0045] Table 3 Two series of spiked concentrations of Pry, DPD, and Cr in quality control samples (LQC and HQC)

[0046] 2. Preparation of internal standard working solution (1) Preparation of internal standard working solution Pipette a certain volume of L-lysine-d4 and creatinine-d3 concentrated stock solutions, dilute with pure water and make up to 10 mL, and mix evenly. The concentrations of L-lysine-d4 and creatinine-d3 are 40 μg / mL and 500 μg / mL, respectively, which are the internal standard working solutions.

[0047] (2) Preparation of internal standard working solution Add 5 mL of internal standard working solution to 100 mL of borate solution (pH = 8.6) to prepare the internal standard working solution.

[0048] 2. Sample Pretreatment Human urine samples, calibrators, and quality control materials were pre-treated as follows: (1) Take 50 μL of calibrator / quality control / human urine sample to be tested and add it to a 2 mL centrifuge tube; add 200 μL of internal standard working solution and shake at 2000 rpm for 1 minute; (2) Add 100 μL of derivatization agent, vortex for 1 minute, and derivatize at 55°C for 30 minutes; (3) Add 800 μL of extraction agent, shake at 2000 rpm for 5 min, and then centrifuge at 10000 rpm for 3 min; (4) Take the supernatant and blow it to dryness with nitrogen at 55°C; (5) Finally, re-dissolve with 100 μL of reconstitution solution and test on the instrument.

[0049] 3. Sample Testing Liquid chromatography tandem mass spectrometry system: AB SCIEX 4500MD liquid chromatography tandem mass spectrometry detection system, chromatographic column: Eclipse Plus C18 (3.5 μm, 3.0×100 mm), mobile phase A: water (0.1% FA), mobile phase B: methanol, initial flow rate: 0.6 ml / min, column temperature: 40°C, injector temperature: 10°C, injection volume: 15 μL.

[0050] The gradient elution conditions are shown in Table 4 below.

[0051] Table 4 Gradient elution conditions

[0052] Mass spectrometry detection conditions: The ion source was electrospray ionization (ESI) in positive ion mode. The ion source parameters are shown in Table 5. The scanning mode was multiple reaction monitoring (MRM) mode.

[0053] Table 5 Ion source parameter information

[0054] The mass spectrometry parameters of the compounds and internal standards are shown in Table 6: Table 6 Mass spectrometry parameters of compounds and internal standards

[0055] 4. Data Processing and Analysis Standard curves were drawn: Linear fit was performed using the concentrations of the Pry, DPD, and Cr calibrants as the abscissa and the peak area ratios of Pry, DPD, and Cr to their respective internal standards as the ordinate to obtain a linear regression equation. The linear equation and correlation coefficient r are shown in Table 7. Good linearity was observed for Pry, DPD, and Cr within the concentration ranges of 20-640 ng / mL, 5-160 ng / mL, and 100-3200 μg / mL, respectively, with correlation coefficients r greater than 0.9900.

[0056] Table 7 Linear equation and correlation coefficient r

[0057] The method provided in this example for the simultaneous detection of Pry, DPD, and Cr in urine samples achieved limits of quantitative detection of 5 ng / mL, 2 ng / mL, and 50 μg / mL for Pry, DPD, and Cr, respectively, with precisions ranging from 0.43% to 7.84%. The results are shown in Table 8. In addition, spike recovery experiments were conducted at low and high levels for Pry, DPD, and Cr in urine samples. The spike recovery results are shown in Table 9.

[0058] Table 8 Quantitation limit detection data of Pry, DPD and Cr methods

[0059] Table 9 High and low quality control spike recovery results of Pry, DPD and Cr in samples

[0060] Pyridinoline (Pry) low point quality control LQC chromatogram is shown in Figure 1 , the LQC chromatogram of deoxypyridinoline (DPD) low point quality control is shown in Figure 2 , creatinine (Cr) low point quality control LQC chromatogram is shown in Figure 3 .

[0061] Example 2 Effects of different pH environments in the reaction system on the test results The sample preparation and pretreatment conditions were completed according to the steps provided in Example 1. The pH of the reaction system was changed to pH = 6.5 and pH = 8.6, respectively. The low-point urine quality control (LQC) was taken as the test sample. The test results of the sample analytes under various pH conditions were compared, as shown in Table 10.

[0062] Table 10 Comparison of Pry, DPD and Cr detection results under different pH conditions

[0063] From Table 10, when the pH of the reaction system is 6.5, the detection peak areas of Pry, DPD and Cr in the test sample are significantly lower than those in Example 1, that is, the reaction condition of pH 8.6.

[0064] Example 3 Effect of different reaction temperatures on detection results The sample preparation and pretreatment conditions were completed according to the steps provided in Example 1. Different reaction temperatures were set at 40°C, 55°C, and 60°C, respectively. Low-point urine quality control (LQC) was used as the test sample. The test results of the sample analytes Pry and DPD at each reaction temperature were compared, as shown in Table 11.

[0065] Table 11 Comparison of Pry and DPD detection results at different reaction temperatures

[0066] As shown in Table 11, at a reaction temperature of 40°C, the detection response values ​​for the sample analytes Pry and DPD were lower than those in Example 1 (reaction temperature of 55°C), indicating that the higher the reaction temperature, the greater the intensity of the Pry and DPD derivatization reactions. However, when the reaction temperature was increased to 60°C, the detection peak areas of the analytes Pry and DPD did not increase significantly compared to Example 1, indicating that the derivatization reaction intensity reaches its peak at 55°C, and further increasing the reaction temperature has no significant effect on the derivatization reaction. Therefore, the optimal temperature for the derivatization reaction in this patent is 55°C.

[0067] Example 4 Effect of different reaction times on derivatization results The sample preparation and pretreatment conditions were completed under the steps provided in Example 1. Different reaction times of the derivatization reaction were investigated, namely 15 min, 30 min, and 60 min. The low-point urine quality control (LQC) was taken as the test sample, and the detection results of the sample analytes Pry and DPD at each reaction time were compared, as shown in Table 12.

[0068] Table 12 Effects of different reaction times on the detection results of Pry and DPD

[0069] As shown in Table 12, when the reaction time is 15 min and 30 min, the longer the reaction time, the larger the detection peak area of ​​Pry and DPD in the analyte, indicating that the intensity of the derivatization reaction increases with the extension of the reaction time; when the reaction time is extended to 60 min, the detection peak area of ​​the analytes Pry and DPD does not show an increasing trend, indicating that the derivatization reaction has reached its strongest level after 30 min, so the reaction time of 30 min is selected in this patent.

[0070] Example 5 Effect of different derivatization doses in the reaction system on the derivatization results The sample preparation and pretreatment conditions were completed according to the steps provided in Example 1, wherein the uniform derivatization agent concentration was 2.85 mg / L. Different derivatization agent volumes in the derivatization reaction system were investigated, as shown in Table 13. The low-point urine quality control (LQC) was taken as the test sample, and the detection results of the sample analytes Pry and DPD at different derivatization doses were compared, as shown in Table 13.

[0071] Table 13 Effects of different derivatization doses on Pry and DPD detection results

[0072] As shown in Table 13, when the volume of the derivatizing agent is 50~100μL, the larger the derivatizing agent dosage, the more obvious the growth trend of the detection peak area of ​​Pry and DPD in the analyte, indicating that the intensity of the derivatization reaction increases with the increase of the derivatization agent dosage; and when the volume of the derivatizing agent is increased to 200μL, the detection response values ​​of the analytes Pry and DPD no longer increase, indicating that the reaction is basically at the maximum intensity after the amount of the derivatizing agent added reaches 100μL, so this patent chooses to add a derivatizing agent volume of 100μL.

[0073] Example 6 Comparison of the detection results of Pry, DPD and Cr in the sample using different reconstituted solutions The sample preparation and pretreatment conditions were completed under the steps provided in Example 1. The supernatant after ethyl acetate extraction was nitrogen-blown and redissolved. Different redissolving solutions were investigated, namely pure water, 20% methanol water, and 50% methanol water. The low-point urine quality control (LQC) was used as the test sample. The effects of different redissolving solutions on the detection results of the sample analytes Pry and DPD were compared, as shown in Tables 14 and 14. Figure 4-Figure 5 .

[0074] Table 14 Effects of different reconstitution solutions on the Pry and DPD test results of the tested samples

[0075] From Table 14, for the analytes Pry and DPD, using pure water and 50% methanol water as reconstitution solutions, the peak area of ​​low-point urine quality control (LQC) detection is equivalent to and better than 20% methanol water. Figure 4-Figure 5 It can be seen that the chromatographic peak shape of creatinine in pure water is optimal and the detection sensitivity is sufficient, so pure water is selected as the reconstitution solution for the sample in this patent.

[0076] Comparative Example 1 Comparison of the results of simultaneous detection of Pry and DPD in urine samples using different pretreatment methods Different pretreatment methods in this comparative example (concentration and enrichment using a solid phase extraction plate pretreatment, other derivatization reagents, and the pretreatment method of Example 1) were used to test high-quality control (HQC) urine samples. Table 15 shows that the peak areas of the analytes Pry and DPD detected in samples pretreated using Example 1 were significantly higher than those using the solid phase extraction method.

[0077] Table 15 Comparison of high-point quality control (HQC) of urine samples using different pretreatment methods

[0078] As can be seen from Table 15, by comparing methods 1 and 2, compared with the solid phase extraction plate pretreatment, the method of Example 1, i.e., AQC derivatization followed by liquid-liquid extraction with ethyl acetate, increased the detection peak area of ​​the analytes Pry and DPD by 15 to 16 times, greatly improving the detection sensitivity. Therefore, the pretreatment method of Example 1 was adopted.

[0079] Comparative Example 2 Comparison of the detection results of Pry and DPD in urine samples using different extraction agents The sample preparation and pretreatment conditions were completed under the steps provided in Example 1. Among them, liquid-liquid extraction was performed after AQC derivatization. Conventional extraction agents were investigated, namely n-hexane, methyl tert-butyl ether, and ethyl acetate. Low-point urine quality control (LQC) was taken as the test sample, and the effects of different extraction reagents on the detection results of the sample analytes Pry and DPD were compared, as shown in Table 16.

[0080] Table 16 Effects of different extraction reagents on Pry and DPD detection results

[0081] As shown in Table 16, the liquid-liquid extraction after AQC derivatization and the use of different solvents to extract the analytes have different degrees of effect. In particular, when using n-hexane for extraction, Pry and DPD are basically undetectable on the same instrument. However, when using methyl tert-butyl ether for extraction, the detection peak areas of the analytes Pry and DPD are already lower than the detection limit of quantification of Example 1. When using ethyl acetate as the extraction solvent of the present invention, the detection peak areas of the analytes Pry and DPD are significantly better than those of the other two conventional extraction solvents.

[0082] Comparative Example 3 Effect of different pH environments in the reaction system on the test results The sample preparation and pretreatment conditions were completed according to the steps provided in Example 1. The pH of the reaction system was changed to pH=3 and pH=8.6, respectively. The low-point urine quality control (LQC) was taken as the sample to be tested. The test results of the sample analytes under various pH conditions were compared, as shown in Table 17.

[0083] Table 17 Comparison of Pry, DPD and Cr detection results under different pH conditions

[0084] From Table 17, the reaction system is in an acidic environment, specifically when the pH is 3, the instrument cannot detect Pry and DPD in the sample to be tested. This may be because the derivatization reaction cannot occur under acidic conditions.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A LC-MS / MS method for the simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine, characterized in that: include: S1: mixing a sample to be tested, a mixed internal standard, and a borate solution with a pH value of 6.5 to 9 to obtain a first mixed solution; preferably, the pH value of the borate solution is 8 to 9; S2: mixing the first mixed solution with a derivatization reagent, and performing derivatization at 35-65° C. for 10-70 minutes to obtain a second mixed solution; the derivatization reagent is 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate; S3: mixing the second mixed solution and the extractant, shaking and then centrifuging, taking the supernatant and blowing it dry with nitrogen, re-dissolving it with a re-solution solution and then performing on-machine testing; the extractant includes ethyl acetate.

2. The LC-MS / MS method for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to claim 1, wherein The volume ratio of the sample to be tested to the derivatization reagent is 1:1-4.

3. The LC-MS / MS method for simultaneously detecting urine pyridinoline, deoxypyridinoline and creatinine according to claim 1 or 2, characterized in that, The volume ratio of the sample to be tested to the extractant is 1:12-20.

4. The LC-MS / MS method for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to any one of claims 1 to 3, wherein The reconstitution solution is a 0 wt % to 60 wt % methanol aqueous solution; preferably, the reconstitution solution is water.

5. The LC-MS / MS method for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to any one of claims 1 to 4, characterized in that The internal standard for pyridinoline and deoxypyridinoline was L-lysine-d4, and the internal standard for creatinine was creatinine-d3.

6. The LC-MS / MS method for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to any one of claims 1 to 5, characterized in that The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05wt% to 0.15wt% formic acid aqueous solution and mobile phase B is methanol; Preferably, the gradient elution conditions of the mobile phase include: "%" represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1.

7. The LC-MS / MS method for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to any one of claims 1 to 6, characterized in that Mass spectrometry detection conditions include: The ion source was electrospray ionization (ESI) in positive ion mode, and the scanning mode was multiple reaction monitoring mode. The ion source parameters were as follows: The mass spectrometry parameters are as follows: 。 8. A kit for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine by LC-MS / MS, characterized in that: include: Test samples, internal standards, derivatization reagents, extraction agents, and borate solution; The derivatization reagent includes 6-aminoquinoline-N-hydroxysuccinimidyl carboxylate; the extractant includes ethyl acetate; the pH value of the borate solution is 6.5-9; preferably, the pH value of the borate solution is 8-9.

9. The LC-MS / MS kit for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine according to claim 8, characterized in that, The kit also contains a mobile phase, which includes a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.05 wt% to 0.15 wt% formic acid aqueous solution, and the mobile phase B is methanol; Preferably, the kit further contains a reconstitution solution, which is a 0 wt % to 60 wt % methanol aqueous solution; more preferably, the reconstitution solution is water.

10. The kit for simultaneous detection of urinary pyridinoline, deoxypyridinoline and creatinine by LC-MS / MS according to claim 8 or 9, characterized in that, The sample to be tested includes: one or more of urine, quality control products and calibration products; and / or, in the quality control product, the concentration of pyridinoline is 60-480 ng / mL, the concentration of deoxypyridinoline is 15-120 ng / mL, and the concentration of creatinine is 300-2400 ng / mL; in the calibrator, the concentration of pyridinoline is 20-640 ng / mL, the concentration of deoxypyridinoline is 5-160 ng / mL, and the concentration of creatinine is 100-3200 ng / mL; And / or, the internal standard for pyridinoline and deoxypyridinoline is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

Images