Method for simultaneous detection of kynurenine, kynurette and creatinine by LC-MS / MS

Abstract

The present application relates to the technical field of analytical detection, and particularly relates to a method for simultaneously detecting urinopyridine, deoxy-pyridinoline and creatinine by LC-MS / MS. The present application generates corresponding derivatives of Pry and DPD by optimizing a derivatization reaction, which indirectly improves ion response of the detection target in mass spectrometry, and the derivatives have good retention and high peak response value in chromatography. Secondly, the target is extracted by using ethyl acetate after derivatization, and is concentrated by nitrogen blowing, so that interfering substances are significantly removed, baseline noise is reduced, signal-to-noise ratio is improved, high-accuracy and high-sensitivity detection of Pry, DPD and Cr in urine samples is realized, and the requirement of clinic is met.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for simultaneous detection of urinary pyridinium phosphate, deoxypyridinium phosphate and creatinine by LC-MS / MS. Background Technology

[0002] Pyridinium (Pry) and deoxypyridinium (DPD) are molecules that stabilize the bonds between collagen molecules. During bone resorption, these bonds are broken down by protein hydrolysis, releasing Pry and DPD into the urine. Clinically, most biochemical indicators of bone metabolism exhibit diurnal rhythms, peaking after midnight and bottoming out in the afternoon. Generally, the diurnal variation in bone formation indicators is about 10%, while the variation in bone resorption indicators can reach 20%. This variation is smaller for Pry and DPD (around 12%). Furthermore, Pry and DPD are not further degraded by the liver and are directly excreted in the urine, unaffected by diet and not internally metabolized. As specific indicators of bone resorption, Pry and DPD are clearly defined in clinical guidelines such as the "Clinical Application Guidelines for 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 Indicators (2023 Revised Edition)," representing specific indicators of bone resorption and possessing significant clinical value.

[0003] In primary osteoporosis and various metabolic bone diseases with increased bone resorption, urinary Pry and DPD levels are significantly elevated. Urine specimen tests 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 their low concentrations and the strong polarity of the compounds themselves (i.e., difficulty in retention on the stationary phase or poor solubility in the organic phase). In liquid chromatography-tandem mass spectrometry (LC-MS / MS) methods for Pry and DPD detection, solid-phase extraction (SPE) plates are commonly used to purify and concentrate the analytes in urine samples before detection. This method is complex, has high consumable costs, and exhibits low extraction rates. Alternatively, ion-pairing reagents can be used as mobile phase additives to improve the retention of compounds in chromatography; however, ion-pairing reagents can cause significant and irreversible contamination to the mass spectrometer. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a method for the simultaneous detection of urinary pyridinium phosphate, deoxypyridinium phosphate, and creatinine using LC-MS / MS. This method offers high sensitivity, low detection cost, and high accuracy, and can simultaneously detect Pry, DPD, and Cr in human urine without the use of ion-pairing reagents.

[0006] Specifically, in the preliminary research, this invention screened a large number of substances that could theoretically introduce strong UV-absorbing or fluorescent groups onto Pry and DPD through reactions, such as Girard's reagent P, 2,4-dinitrophenylhydrazine, and o-phenylenediamine. Mass spectrometry Q1 mass scanning results showed that these derivatizing reagents could not undergo derivatization reactions with Pry and DPD. Through extensive research and experiments, this invention finally discovered that under specific pH conditions, 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) can undergo derivatization reactions with the primary amine groups in Pry and DPD. The resulting derivative indirectly improves the ionic response of the target analyte on the mass spectrometer, and the derivative exhibits good retention and a high peak response value on the chromatography. This may be because AQC possesses hydrophobic and easily ionized groups, which impart hydrophobic and easily ionized properties to Pry and DPD after reaction. Furthermore, this invention has found 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 solution: In a first aspect, the present invention provides an LC-MS / MS method for the simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine, comprising: S1: The sample to be tested, the mixed internal standard, and the borate solution with a pH of 6.5-9 are mixed to obtain a first mixed solution; preferably, the pH of the borate solution is 8-9; S2: Mix the first mixed solution with the derivatizing reagent and derivatize at 35~65℃ for 10~70 minutes. After derivatization, a second mixed solution is obtained. The derivatizing reagent is 6-aminoquinoline-N-hydroxysuccinimide carbamate. S3: Mix the second mixed solution and the extractant, shake and centrifuge, take the supernatant and dry it with nitrogen, reconstitute it with the reconstituted solution and then perform instrumental testing; the extractant includes ethyl acetate.

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

[0009] According to the LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine provided by the present invention, the derivatization time is preferably 20~40 min, specifically any value among 20 min, 25 min, 30 min, 35 min, and 40 min, or a range of values ​​with any two of the above values ​​as endpoints.

[0010] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine is provided, wherein the volume ratio of the test sample to the derivatizing reagent is 1:1 to 4; specifically, it can be any ratio such as 1:1, 1:2, 1:3 or 1:4, or any ratio range with any two of the above ratios as endpoints.

[0011] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine is provided, wherein the volume ratio of the sample to extractant is 1:12 to 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 any ratio range with any two of the above ratios as endpoints.

[0012] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinium porphyrin, deoxypyridinium porphyrin and creatinine is provided, wherein the reconstitution solution is a 0wt%~60wt% methanol aqueous solution; preferably, the reconstitution solution is water.

[0013] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine is provided, wherein the internal standard for pyridinine and deoxypyridinine is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

[0014] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine is provided. The mobile phase for liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05wt%~0.15wt% aqueous formic acid 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 the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine, and creatinine is provided, wherein the gradient elution conditions of the mobile phase include:

[0018] "%" represents volume percentage.

[0019] According to the LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine provided by the present invention, the liquid phase detection conditions further include: a C18 column, preferably an Eclipse Plus C18; a mobile phase flow rate of 0.5~0.7 ml / min; and further optimized column temperature of 40℃, injector temperature of 10℃, and injection volume of 15 μL.

[0020] According to the present invention, an LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine, and creatinine is provided, wherein 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 (MRM). The ion source parameters are as follows:

[0021] The mass spectrometry parameters are shown below:

[0022] In a second aspect, the present invention provides a kit for simultaneous LC-MS / MS detection of urinary pyridinium, deoxypyridinium and creatinine, comprising: a sample to be tested, an internal standard, a derivatizing reagent, an extractant and a borate solution; The derivatizing agent includes 6-aminoquinoline-N-hydroxysuccinimide carbamate; the extractant includes ethyl acetate; and the pH of the borate solution is 6.5-9.

[0023] This invention creates derivatization conditions within a specific pH range using borate solution. 6-aminoquinoline-N-hydroxysuccinimide carbamate is used to derivatize the sample, generating corresponding derivatives of Pry and DPD. These derivatives indirectly improve the ionic response of the target analytes on mass spectrometry, and exhibit good retention and high peak response values ​​on chromatography. Secondly, after derivatization, ethyl acetate is used to extract the target analytes, followed by nitrogen blowing concentration to significantly remove interfering substances, reduce baseline noise, and improve the signal-to-noise ratio. This achieves high accuracy and sensitivity detection of Pry, DPD, and Cr in urine samples, meeting clinical needs. Furthermore, it protects the instrument and column from contamination, reduces instrument maintenance frequency, and extends column life.

[0024] The LC-MS / MS kit for simultaneous detection of urinary pyridinium, deoxypyridinium and creatinine provided by the present invention further comprises a mobile phase, wherein the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05wt%~0.15wt% aqueous formic acid solution and mobile phase B is methanol.

[0025] In this 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 range of values ​​where any two of the above values ​​are endpoints.

[0026] This invention has found that using the above-mentioned mobile phase can improve the response value of the target analyte and further enhance the detection sensitivity.

[0027] According to the LC-MS / MS kit provided by the present invention for simultaneous detection of urinary pyridinium phosphate, deoxypyridinium phosphate and creatinine, the pH value of the borate solution is 8-9.

[0028] The LC-MS / MS kit for simultaneous detection of urinary pyridinium, deoxypyridinium and creatinine provided by the present invention further comprises a reconstitution solution, wherein the reconstitution solution is a 0wt%~60wt% aqueous methanol solution.

[0029] In this invention, the concentration of methanol in the complex solution can be any value from 0wt% to 60wt%, such as pure water, or methanol aqueous solutions with concentrations of 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 20wt%, 30wt%, 40wt%, 50wt%, and 60wt%.

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

[0031] The LC-MS / MS kit for simultaneous detection of urinary pyridinine, deoxypyridinine and creatinine provided by the present invention includes one or more of the following samples: urine, quality control samples and calibrators.

[0032] According to the LC-MS / MS kit for simultaneous detection of urinary pyridinine, deoxypyridinine, and creatinine provided by the present invention, the quality control contains pyridinine at a concentration of 60-480 ng / mL, deoxypyridinine at a concentration of 15-120 ng / mL, and creatinine at a concentration of 300-2400 ng / mL; the calibrator contains pyridinine at a concentration of 20-640 ng / mL, deoxypyridinine at a concentration of 5-160 ng / mL, and creatinine at a concentration of 100-3200 ng / mL. And / or, the internal standard for pyridinine and deoxypyridinine is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

[0033] This invention provides an LC-MS / MS method for the simultaneous detection of Pry, DPD, and Cr in urine samples. First, through derivatization, Pry and DPD generate corresponding derivatives, which indirectly improve the ionic response of the target analytes on mass spectrometry. These derivatives also exhibit good retention and high peak response values ​​on chromatography. Second, after derivatization, the target analytes are extracted using an organic solvent, followed by nitrogen blowing concentration to significantly remove interfering substances, reduce baseline noise, and improve the signal-to-noise ratio. The limits of quantitation for Pry, DPD, and Cr are 5 ng / mL, 2 ng / mL, and 50 μg / mL, respectively. Furthermore, this method achieves a spiked recovery rate of 99–115% for the analytes, enabling high accuracy and sensitivity detection of Pry, DPD, and Cr in urine samples, meeting clinical needs. It also protects the instrument and column from contamination, reduces instrument maintenance frequency, and extends column life. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0035] Figure 1 The LQC chromatogram of pyridinium (Pry) in Example 1 of this invention is shown.

[0036] Figure 2 The LQC chromatogram of deoxypyridinium (DPD) in Example 1 provided by the present invention.

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

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

[0039] Figure 5 The low-point quality control chromatogram of creatinine (Cr) in Example 6 of this invention, when 50% methanol-water is used as the reconstitution solution. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, all 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 kit for simultaneous LC-MS / MS detection of urinary pyridinium, deoxypyridinium, and creatinine, as shown below: Table 1

[0043] The detection methods include: I. Sample Preparation 1. Preparation of calibrators and quality control samples Pry, DPD, and Cr standards were prepared into mixed solutions as stock solutions for the standard working solution and the quality control working solution. 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 separately. Six series of concentrations (C1 to C6) were prepared for the calibration curves, and two series of concentrations (LQC and HQC) were prepared for the quality control samples, as shown in Tables 2 and 3 below.

[0044] Table 2. Six series of concentrations (C1–C6) of Pry, DPD, and Cr in the calibration curves.

[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 Take a certain volume of concentrated L-lysine-d4 and creatinine-d3 stock solution, add pure water to dilute and bring the volume to 10 mL, mix well. The concentrations of L-lysine-d4 and creatinine-d3 are 40 μg / mL and 500 μg / mL, respectively, which is the internal standard working solution.

[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 obtain the internal standard working solution.

[0048] II. Sample Pretreatment The urine samples, calibrators, and quality control samples were pretreated using the following method: (1) Take 50 μL of calibrator / quality control / test subject urine sample 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 derivatizing agent, vortex for 1 minute, and then derivatize at 55°C for 30 minutes; (3) Add 800 μL of extraction solvent, shake at 2000 rpm for 5 min, and then centrifuge at 10000 rpm for 3 min; (4) Take the supernatant and blow it dry with nitrogen at 55°C; (5) Finally, reconstitute with 100 μL of reconstitution solution and test on the instrument.

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

[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).

[0053] Table 5 Ion source parameter information table

[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] IV. Data Processing and Analysis Standard curves were plotted: The standard curves were plotted with the concentrations of Pry, DPD, and Cr calibrators on the x-axis and the ratio of the peak areas of Pry, DPD, and Cr to their respective internal standards on the y-axis. Linear fitting was performed to obtain linear regression equations. The linear equations and correlation coefficients r are shown in Table 7. Pry, DPD, and Cr showed good linearity in 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 for all of them.

[0056] Table 7 Linear Equations and Correlation Coefficients r

[0057] The method for simultaneous detection of Pry, DPD, and Cr in urine samples provided in this embodiment achieves detection limits of 5 ng / mL, 2 ng / mL, and 50 μg / mL for Pry, DPD, and Cr, respectively, with a precision of 0.43–7.84%. The results are shown in Table 8. Furthermore, spiked recovery experiments were conducted on Pry, DPD, and Cr in urine samples at low and high point levels; the spiked recovery results are shown in Table 9.

[0058] Table 8. Limit of Quantitation (LOQ) data for Pry, DPD, and Cr methods

[0059] Table 9. Recovery rates of Pry, DPD, and Cr in high and low quality control spikes in the samples.

[0060] The low-point quality control (LQC) chromatogram for pyridinium (Pry) is shown below. Figure 1 The low-point quality control (LQC) chromatogram for deoxypyridinium (DPD) is shown below. Figure 2 The LQC chromatogram for low-point creatinine (Cr) is shown below. Figure 3 .

[0061] Example 2: The effect of different pH environments in the reaction system on the detection results Under the sample preparation and pretreatment conditions 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, and the detection results of the test analytes in the samples under each pH condition were compared, as shown in Table 10.

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

[0063] Table 10 shows that when the pH of the reaction system is 6.5, the peak areas of Pry, DPD and Cr in the sample to be tested are significantly lower than those in Example 1, i.e., the reaction condition with pH 8.6.

[0064] Example 3: Effect of different reaction temperatures on detection results Under the sample preparation and pretreatment conditions provided in Example 1, different reaction temperatures were set at 40°C, 55°C, and 60°C. The lowest-temperature urine quality control (LQC) sample was used as the test sample. The detection results of the test analytes Pry and DPD in the samples 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] Table 11 shows that at a reaction temperature of 40°C, the detection response values ​​of the analytes Pry and DPD were lower than those in Example 1 (reaction temperature 55°C), indicating that the higher the reaction temperature, the greater the derivatization reaction intensity of Pry and DPD. However, when the reaction temperature was increased to 60°C, compared with Example 1, the detection peak areas of the analytes Pry and DPD did not increase significantly, indicating that the derivatization reaction intensity was at its peak at 55°C, and further increasing the reaction temperature had no significant effect on the derivatization reaction. Therefore, this patent selects 55°C as the optimal temperature for the derivatization reaction.

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

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

[0069] Table 12 shows that 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. However, when the reaction time is extended to 60 min, the detection peak area of ​​Pry and DPD in the analyte does not show an increasing trend, indicating that the derivatization reaction reaches its strongest level after 30 min. Therefore, this patent selects a reaction time of 30 min.

[0070] Example 5: Effect of different derivatization doses on derivatization results in the reaction system Under the sample preparation and pretreatment conditions provided in Example 1, where the uniform derivatizing agent concentration was 2.85 mg / L, different derivatizing agent volumes in the derivatization reaction system were investigated, as shown in Table 13. Low-point urine quality control (LQC) samples were used as test samples, and the detection results of analytes Pry and DPD under different derivatizing doses were compared, as shown in Table 13.

[0071] Table 13 Effect of different derivative doses on Pry and DPD detection results

[0072] Table 13 shows that when the volume of the derivatizing agent is 50~100μL, the larger the derivatizing agent dosage, the greater the peak area of ​​Pry and DPD in the analyte, indicating that the intensity of the derivatization reaction increases with the increase of the derivatizing agent dosage. However, when the volume of the derivatizing agent is increased to 200μL, the detection response values ​​of Pry and DPD no longer increase, indicating that the reaction is basically at its maximum intensity after the amount of derivatizing agent added reaches 100μL. Therefore, this patent selects a volume of 100μL for the added derivatizing agent.

[0073] Example 6 Comparison of detection results of Pry, DPD and Cr in test samples with different reconstitution solutions Under the sample preparation and pretreatment conditions provided in Example 1, the supernatant after ethyl acetate extraction was reconstituted under nitrogen blowing. Different reconstitution solutions were investigated: pure water, 20% methanol-water, and 50% methanol-water. Low-quality control urine (LQC) samples were used as test samples to compare the effects of different reconstitution solutions on the detection results of the analytes Pry and DPD. See Table 14 for details. Figures 4-5 .

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

[0075] Table 14 shows that for the analytes Pry and DPD, using pure water and 50% methanol-water as the reconstitution solution, the low-point urine quality control (LQC) peak area was comparable to and better than that of 20% methanol-water. Furthermore, [the following text appears to be incomplete and requires further context: "from..."] Figures 4-5 It can be seen that creatinine has the best chromatographic peak shape in pure water and the detection sensitivity is sufficient. Therefore, pure water was chosen as the sample reconstitution solution for this patent.

[0076] Comparative Example 1: Comparison of results from simultaneous detection of Pry and DPD in urine samples using different pretreatment methods Different pretreatment methods (solid phase extraction followed by concentration and enrichment, other derivatization reagents, and the pretreatment method of Example 1) were used to detect high-point quality control (HQC) of urine samples. Table 15 shows that the peak areas of analytes Pry and DPD in the sample after pretreatment using Example 1 were significantly higher than those using solid phase extraction.

[0077] Table 15 Comparison of High-Point Quality Control (HQC) for Urine Samples Detected Using Different Pretreatment Methods

[0078] As can be seen from Table 15, comparing methods 1 and 2, compared with the solid phase extraction plate method for pretreatment, using Example 1, i.e., AQC derivatization followed by ethyl acetate liquid-liquid extraction, 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 is adopted.

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

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

[0081] Table 16 shows that after AQC derivatization, liquid-liquid extraction was performed, and different solvents were used to extract the analytes, resulting in varying degrees of effectiveness. In particular, when hexane was used for extraction, Pry and DPD were almost undetectable on the same instrument. However, when methyl tert-butyl ether was used for extraction, the peak areas of Pry and DPD were lower than the detection limits of Example 1. When ethyl acetate was used as the extraction solvent in this invention, the peak areas of Pry and DPD were significantly better than those of the other two conventional extraction solvents.

[0082] Comparative Example 3: The effect of different pH environments in the reaction system on the detection results Under the sample preparation and pretreatment conditions 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 test sample, and the detection results of the test analytes in the samples under each pH condition were compared, as shown in Table 17.

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

[0084] Table 17 shows that the reaction system is in an acidic environment. Specifically, this invention indicates that when the pH is 3, the instrument cannot detect Pry and DPD in the sample, which 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, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An LC-MS / MS method for simultaneous detection of urinary pyridinine, deoxypyridinine, and creatinine, characterized in that, include: S1: Mix the sample to be tested, the mixed internal standard, and the borate solution with a pH of 8-9 to obtain the first mixed solution; S2: Mix the first mixed solution with the derivatizing reagent and derivatize at 35~65℃ for 10~70 minutes. After derivatization, a second mixed solution is obtained. The derivatizing reagent is 6-aminoquinoline-N-hydroxysuccinimide carbamate. S3: Mix the second mixed solution and the extractant, shake and centrifuge, take the supernatant and dry it with nitrogen, reconstitute it with the reconstituted solution and then perform instrumental testing; the extractant includes ethyl acetate; The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05wt%~0.15wt% aqueous formic acid solution and mobile phase B is methanol; The chromatographic column is a C18 column; Gradient elution conditions for the mobile phase include: "%" represents volume percentage, and the sum of the volume percentages of mobile phase A and mobile phase B is 1; Mass spectrometry detection conditions include: The ion source was electrospray ionization (ESI) in positive ion mode, and the scanning mode was multiple reaction monitoring (MRM). The ion source parameters are as follows: The mass spectrometry parameters are shown below:

2. The LC-MS / MS method for simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine according to claim 1, characterized in that, The volume ratio of the test sample to the derivatizing reagent is 1:1 to 4.

3. The LC-MS / MS method for simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine according to claim 1, characterized in that, The volume ratio of the sample to the extractant is 1:12~20.

4. The LC-MS / MS method for simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine according to any one of claims 1 to 3, characterized in that, The reconstitution solution is a methanol aqueous solution of 0wt% to 60wt%.

5. The LC-MS / MS method for simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine according to any one of claims 1 to 3, characterized in that, The complex solution is water.

6. The LC-MS / MS method for simultaneous detection of urinary pyridinium, deoxypyridinium, and creatinine according to any one of claims 1 to 3, characterized in that, The internal standard for pyridinine and deoxypyridinine is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

7. A kit for detecting urinary pyridinine, deoxypyridinine, and creatinine using the method of claim 1, characterized in that, include: The test sample, internal standard, derivatizing reagent, extraction solvent, and borate solution; The derivatizing agent includes 6-aminoquinoline-N-hydroxysuccinimide carbamate; the extractant includes ethyl acetate; and the pH of the borate solution is 8-9. The kit also contains a mobile phase, which includes mobile phase A and mobile phase B. Mobile phase A is a 0.05wt%~0.15wt% aqueous formic acid solution, and mobile phase B is methanol.

8. The reagent kit according to claim 7, characterized in that, The kit also contains a reconstitution solution, which is a 0wt% to 60wt% aqueous methanol solution.

9. The reagent kit according to claim 7, characterized in that, The kit also contains a reconstitution solution, which is water.

10. The kit according to claim 7, characterized in that, The test sample includes one or more of the following: urine, quality control samples, and calibrators; And / or, in the quality control material, the concentration of pyridinium is 60~480 ng / mL, the concentration of deoxypyridinium is 15~120 ng / mL, and the concentration of creatinine is 300~2400 ng / mL; in the calibrator, the concentration of pyridinium is 20~640 ng / mL, the concentration of deoxypyridinium is 5~160 ng / mL, and the concentration of creatinine is 100~3200 ng / mL. And / or, the internal standard for pyridinine and deoxypyridinine is L-lysine-d4, and the internal standard for creatinine is creatinine-d3.

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