Application of serum meso-diaminopimelic acid detection method or detection product in sepsis based on liquid chromatography-mass spectrometry

Abstract

The invention discloses an application of a serum meso-diaminopimelic acid detection method based on liquid chromatography-mass spectrometry or a detection product in sepsis, and relates to the technical field of biological detection. The detection method comprises the following steps: serum sample treatment, derivatization of 6-aminoquinoline-N-hydrogen succinimide carbamate, chromatographic separation and mass spectrometric detection. The detection kit comprises a meso-DAP standard substance, a Lys (Lys-d4) internal standard substance, a borate buffer solution and 6-aminoquinoline-N-succinimide methyl ester. The diagnosis efficacy AUC of meso-DAP is equal to 0.835, compared with a PCT detection method, the meso-DAP has better supplementary diagnosis efficacy, and compared with levo-DAP and dextro-DAP and total DAP, the sepsis diagnosis efficacy of meso-DAP is greatly improved. In addition, the method provided by the invention can also monitor the illness state to a certain extent, and also has a certain indication effect on prognosis.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a serum meso-diaminopimelate detection method based on liquid chromatography-mass spectrometry or an application of a detection product in sepsis. Background Art

[0002] Diaminopimelane (DAP) is an important component of bacterial cell walls and is widely present in most Gram-negative bacteria and some Gram-positive bacteria. DAP exists in three isomers: levorotatory, dextrorotatory, and meso. Among them, meso-DAP (meso-DAP) is a specific ligand for the NOD-like receptor NOD1 and plays an important role in activating innate immunity and inflammation. Our research team has previously established a detection method for serum diaminopimelane. This time, we specifically established a detection method for meso-DAP, hoping that it can be used for the early diagnosis of sepsis and significantly improve its efficacy in diagnosing sepsis. Summary of the Invention

[0003] To address the above technical issues, the present invention provides a method or product for detecting meso-diaminopimelane acid in serum based on liquid chromatography-mass spectrometry (LC-MS / MS) for use in sepsis. This method demonstrates a diagnostic efficacy of meso-diaminopimelane (DAP) of 0.835, demonstrating superior complementary diagnostic efficacy compared to PCT detection and significantly improving the diagnostic efficacy of both L- and D-DAP and total DAP for sepsis. Furthermore, this method can also monitor the condition to a certain extent and provide a prognostic indicator.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] One of the purposes of the present invention is to provide a method for detecting serum meso-diaminopimelate based on liquid chromatography-mass spectrometry for use in the preparation of products for diagnosis, disease monitoring and / or prognosis evaluation of sepsis.

[0006] Preferably, the detection method comprises: serum sample processing, 6-aminoquinoline-N-hydrogen succinimide carbamate (AQC) derivatization, chromatographic separation and mass spectrometry detection.

[0007] Preferably, the serum sample processing comprises: adding the serum sample to a methanol extract containing Lys-d4 internal standard, vortex mixing, centrifugation, and drying the supernatant.

[0008] More preferably, the internal standard content in the methanol extract is 18-22 ng / mL.

[0009] Preferably, the AQC derivatization comprises: adding borate buffer and AQC to the dried sample, vortex mixing, and then incubating and cooling on ice; the volume ratio of the borate buffer to the AQC is (6-8): (1-3).

[0010] Preferably, the chromatographic separation is achieved by using an Acquity UPLC BEH C18 column (1.7 μm, 50×2.1 mm, Waters, US).

[0011] Preferably, the mass spectrometry detection is performed using a triple quadrupole mass spectrometer, wherein the chromatographic mobile phase A is 0.08% to 0.12% formic acid / ultrapure water, and the mobile phase B is 0.08% to 0.12% formic acid / acetonitrile.

[0012] The second object of the present invention is to provide a method for detecting serum meso-diaminopimelate based on liquid chromatography-mass spectrometry, wherein the detection method includes the method in the application.

[0013] The third object of the present invention is to provide a serum meso-diaminopimelate detection product based on liquid chromatography-mass spectrometry, the product including a kit, and its use method including the method in the application.

[0014] Preferably, the kit comprises: an extraction reagent and a derivatization reagent, wherein the extraction reagent comprises a meso-DAP standard and a Lys-d4 internal standard, and the derivatization reagent comprises a borate buffer and 6-aminoquinoline-N-succinimide methyl ester.

[0015] Compared with the prior art, the present invention has the following technical effects:

[0016] The detection method of the present invention comprises serum sample preparation, derivatization with 6-aminoquinoline-N-hydrosuccinimidyl carbamate, chromatographic separation, and mass spectrometric detection. The detection kit of the present invention comprises a meso-DAP standard, a Lys-d4 internal standard, a borate buffer, and 6-aminoquinoline-N-succinimidyl methyl ester. The diagnostic efficacy of meso-DAP in the present invention is an AUC of 0.835, demonstrating superior complementary diagnostic efficacy compared to PCT detection methods and significantly improving the diagnostic efficacy of both levorotatory and dextrorotatory DAP and total DAP for sepsis. Furthermore, the method of the present invention can monitor disease progression to a certain extent and provide a prognostic indicator. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To verify the accuracy and precision of the meso-DAP detection method established in Example 1;

[0018] Figure 2To verify the recovery and stability of the meso-DAP detection method established in Example 1;

[0019] Figure 3 The serum meso-DAP test results of the discovery set and validation set in Example 2;

[0020] Figure 4 The serum meso-DAP test results of the longitudinally sampled patients in Example 3;

[0021] Figure 5 This is the comparative result of the diagnostic efficacy of L+DAP, total DAP and meso-DAP in sepsis in Comparative Example 2. DETAILED DESCRIPTION

[0022] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. The reagents, products and instruments used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0023] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0024] Example 1

[0025] This embodiment provides a method for detecting serum meso-diaminopimelate based on liquid chromatography-mass spectrometry, comprising: serum sample preparation, 6-aminoquinoline-N-hydrogen succinimide carbamate (AQC) derivatization, chromatographic separation, and mass spectrometry detection.

[0026] Specifically:

[0027] A 1.00 mg / mL stock solution was prepared by accurately weighing meso-DAP (Macklin, C14919383), and ten levels of working solutions were prepared by serial dilution as follows: 500, 250, 100, 50, 25, 10, 5, 2.5, 1, and 0.5 ng / mL (for method validation). 100 μL of each serum sample or working solution was then added to 400 μL of methanol (20 ng / mL) containing Lys-d4 (Sigma, 616192) as an internal standard (IS). After vortex mixing for 30 seconds, the mixture was centrifuged at 12,000 g for 10 minutes (4°C), and 400 μL of the supernatant was collected and dried using a vacuum centrifugal concentrator (Thermo, US). Derivatization was performed using the AccQ-Tag™ Ultra Derivation Kit (Waters, USA) as directed: 70 μL of borate buffer and 20 μL of the derivatization reagent, AQC (MCE, HY-117695), were added to the dried sample and vortexed for 30 seconds. The tube was then incubated at 55°C for 10 minutes, and the sample was cooled on ice for 1 minute. Finally, the sample was centrifuged at 12,000 g for 10 minutes, and 5 μL of the supernatant was injected for LC-MS analysis. UHPLC-MS analysis was performed on a 4500MD liquid chromatograph using a triple quadrupole mass spectrometer (QTRAP4500 MD, AB Sciex, US) using an electrospray ionization (ESI) source. Separation was achieved using an Acquity UPLC BEH C18 column (1.7 μm, 50 × 2.1 mm, Waters, US). The column temperature was set to 50°C, and the autosampler temperature was maintained at 10°C. The mobile phase consisted of 0.1% formic acid in water (as phase A) and 0.1% formic acid in acetonitrile (as phase B), with a flow rate of 0.3 mL / min. The gradient elution for sequence analysis was as follows (A:B): 95:5 at 0.01 min; 90.9:9.1 at 4.00 min; 90.10 at 5.00 min; 5:95 at 7.00 min; 5:95 at 8.00 min; 95:5 at 8.5 min; and controller stop at 10.00 min. Multiple reaction monitoring (MRM) mode was used for quantification, with m / z 266.0 → 171.1 (DP: 60, CE: 22) for mid-DAP and 321.0 → 171.1 (DP: 60, CE: 22) for IS (Lys-d4). The mass spectrometer parameters were as follows: curtain gas, 35 psi; collision gas, 9 psi; ion spray voltage, 5.5 kV; interface temperature: 550° C.; and ion source gas, 50 psi.

[0028] Method validation: The linear range and lower limit of quantification of meso-DAP were 0.5-500 ng / mL (r² = 0.99146) and 0.1 ng / mL, respectively. Intra-day and inter-day accuracy and precision were determined at four levels (LLOQ, 0.5 ng / mL; LQC, 1 ng / mL; MQC, 25 ng / mL; and HQC, 500 ng / mL). Figure 1 The recovery and stability of the assay were evaluated using three levels of spiked serum samples. The recoveries of meso-DAP in human serum samples ranged from 96-101%, indicating negligible endogenous interference with the assay ( Figure 2 Meso-DAP levels were stable (92-105%) under both conditions, including storage at 4°C for 12 and 24 hours after preparation. In the validation analysis, all data were within the required range, demonstrating that the quantification was accurate and reproducible and could be used to detect meso-DAP in real samples.

[0029] Example 2

[0030] This example is a clinical multicenter study: the discovery set includes 108 non-septic patients and 140 septic patients; the validation set includes 111 non-septic patients and 92 septic patients. The serum meso-DAP levels of these patients were detected using the method described in Example 1. The septic patient group was subgroup analyzed based on negative and positive blood culture, and with and without shock. The results are shown in Figure 1. Figure 3 As shown. Among them: Figure 3 A. Comparison of serum meso-DAP levels between non-septic and septic patients; Figure 3 B. Comparison of serum meso-DAP levels between patients with and without shock in sepsis cases; Figure 3 C. Comparison of serum meso-DAP levels between patients with negative and positive blood cultures in the sepsis case series; Figure 3 D. Receiver operating characteristic (ROC) curve of meso-DAP for diagnosis of sepsis was found. Figure 3 E. Comparison of serum meso-DAP levels between non-septic and septic patients in the validation set; Figure 3 F. Comparison of serum meso-DAP levels between patients with shock and those without shock in the validation set of sepsis cases; Figure 3 G. Comparison of serum meso-DAP levels between patients with negative and positive blood cultures in the validation set of sepsis cases; Figure 3 H. ROC curve of meso-DAP for diagnosing sepsis in the validation set.

[0031] The results of both the discovery set and the validation set showed that the serum meso-DAP level in patients with sepsis was significantly higher than that in patients without sepsis ( Figure 3A, E; Mann-Whitney U test p < 0.001). This biomarker showed strong discriminatory power for septic shock cases, with levels significantly higher in both cohorts than in non-shock sepsis patients ( Figure 3 In addition, meso-DAP concentrations were significantly increased in patients with positive blood cultures compared with those with negative blood cultures in both the discovery and validation sets ( Figure 3 C, G), indicating that this indicator is correlated with pathogen load. Finally, ROC analysis showed that serum meso-DAP had a strong ability to discriminate sepsis, and the area under the curve (AUC) value of the cohort was found to be 0.878 (95% CI, 0.820-0.930) ( Figure 3 D), the AUC value of the validation cohort was 0.835 (95% CI, 0.769-0.893) ( Figure 3 H).

[0032] Example 3

[0033] In this embodiment, longitudinal sampling is performed on patients. Note that it is not suitable to take samples from patients multiple times a day. In this embodiment, the time points are set to be taken three times a week (T1 is within 24 hours of admission, T2 is 2-3 days after admission, and T3 is 4-7 days after admission). The longitudinal time point detection results are as follows: Figure 4 As shown. Among them: Figure 4 A. Dynamic changes of meso-DAP in sepsis patients longitudinally sampled at 28 days between the surviving and non-surviving groups; Figure 4 B. Comparison of serum meso-DAP levels between the improvement and deterioration groups of sepsis patients according to SOFA score; Figure 4 C. Receiver operating characteristic (ROC) curve of meso-DAP for differentiating between improvement and deterioration of sepsis.

[0034] The results showed that among patients sampled longitudinally throughout the study period, although there was a slight trend towards a decrease in treatment response in both the 28-day survival and non-survival groups, meso-DAP levels were significantly higher in the non-survival group than in the survival group at different sampling time points in both cohorts ( Figure 4 A), showing the association between meso-DAP levels and disease progression. In addition, in our longitudinal cohort analysis, patients were divided into two groups: improvement (decreased SOFA score) or worsening (increased SOFA score). Compared with the improvement group, the change in meso-DAP levels (ΔT2-T1) was significantly increased in the worsening group (p < 0.001, Figure 4 B). This also reflects the trend that when the disease worsens, meso-DAP levels increase; and when the disease improves, meso-DAP levels decrease. Longitudinal studies found that meso-DAP showed significant discriminative ability for disease changes, with an AUC of 0.779 (95% CI 0.585-0.935, Figure 4 C). This shows that meso-DAP can monitor the disease to a certain extent and also has a certain indication of prognosis (28-day survival).

[0035] Comparative Example 1

[0036] This comparative example is the PCT detection method, which is the only indicator currently approved by the U.S. FDA for sepsis detection. PCT can be monitored daily for patients with severe infections. However, PCT may also have false negatives. For example, in the clinical samples collected by the present invention, there were 34 and 21 sepsis patients in the discovery set and validation set, respectively, whose PCT was <2 ng / mL. This situation is clinically considered to have a low risk of sepsis. However, the meso-DAP detection method in the embodiment of the present invention is as described above, with a discovery set AUC of 0.85 and a validation set AUC of 0.89. Therefore, the present invention has a good supplementary diagnostic efficacy for the PCT indicator.

[0037] Comparative Example 2

[0038] This comparative example is based on the previous research described in the background technology, that is, the comparative experiment set up by the authorized patent 202110736789.7. In addition to meso-dapoxetine, levorotatory and dextrorotatory substances also exist in human serum. We found two peaks of DAP during the early detection in the authorized patent 202110736789.7. After comparison with the standard, we found that one peak was meso-dapoxetine and the other peak was the sum of levorotatory and dextrorotatory substances. The authorized patent 202110736789.7 added the two peaks together in application, which is equivalent to the total DAP level. The present invention specifically analyzes meso-dapoxetine. Based on the above situation in the authorized patent 202110736789.7, the comparative example experiment newly supplemented in this comparative example has the following sample numbers: 111 non-septic patients and 92 septic patients. The test results are as follows Figure 5 shown.

[0039] The results showed that the diagnostic efficacy of L-DAP and D-DAP for sepsis was AUC = 0.685, while the diagnostic efficacy of meso-DAP in the present invention was AUC = 0.835, significantly improving the diagnostic efficacy of L- and D-DAP. Furthermore, the diagnostic efficacy of total DAP was AUC = 0.792, which was also significantly inferior to that of meso-DAP in the present invention.

[0040] Comparative Example 3

[0041] This comparative example is the detection method in the "Non-targeted metabolomics study of sepsis model in mice infected with Klebsiella pneumoniae". The model in this method is a bacteremia model caused by direct tail vein injection of Klebsiella pneumoniae into the blood, and the amount of bacteria injected is large (5×108 CFU / cell), which is actually a relatively extreme modeling method. Strictly speaking, this model should be considered bacteremia (the presence of live bacteria in the blood), but sepsis is not limited to bacteremia and also includes other conditions (the latest definition of sepsis is organ dysfunction caused by infection, and bacteremia, sepsis, and septicemia are currently collectively classified as sepsis). The sepsis patients in this invention have different etiologies and different pathogens (blood cultures can be negative or positive). The article also detects the total DAP content, not specifically meso-DAP, while the present invention specifically detects meso-DAP. Furthermore, the article uses a standard non-targeted metabolomics assay, which is a relative quantitative analysis method. This method cannot detect DAP in the sepsis patients of this invention. However, the present invention utilizes a derivatization method, combined with an isotopic internal standard and a meso-DAP standard, to achieve precise quantitative detection.

[0042] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of a method for detecting serum meso-diaminopimelate based on liquid chromatography-mass spectrometry in the preparation of products for diagnosis, disease monitoring and / or prognosis assessment of sepsis.

2. The use according to claim 1, characterized in that The detection method comprises: serum sample processing, 6-aminoquinoline-N-hydrogen succinimide carbamate (AQC) derivatization, chromatographic separation and mass spectrometry detection.

3. The use according to claim 2, characterized in that The serum sample processing comprises: adding the serum sample to a methanol extract containing Lys-d4 internal standard, vortex mixing, centrifugation, and drying the supernatant.

4. The use according to claim 3, characterized in that The internal standard content in the methanol extract is 18-22 ng / mL.

5. The use according to claim 2, characterized in that The AQC derivatization comprises: adding borate buffer and AQC to the dried sample, vortex mixing, incubating, and cooling on ice; the volume ratio of the borate buffer to the AQC is (6-8): (1-3).

6. The use according to claim 2, characterized in that The chromatographic separation was achieved using an Acquity UPLC BEH C18 column (1.7 μm, 50×2.1 mm, Waters, US).

7. The use according to claim 2, characterized in that The mass spectrometry detection is performed using a triple quadrupole mass spectrometer, wherein the chromatographic mobile phase A is 0.08% to 0.12% formic acid / ultrapure water, and the mobile phase B is 0.08% to 0.12% formic acid / acetonitrile.

8. A method for detecting serum meso-diaminopimelate based on liquid chromatography-mass spectrometry, characterized in that: The detection method includes the method in any one of claims 1 to 7.

9. A serum meso-diaminopimelate detection product based on liquid chromatography-mass spectrometry, characterized in that: The product includes a kit, and the method of using the kit includes the method in any one of claims 1 to 7.

10. The detection product according to claim 9, characterized in that: The kit comprises: an extraction reagent and a derivatization reagent, wherein the extraction reagent comprises a meso-DAP standard and a Lys-d4 internal standard, and the derivatization reagent comprises a borate buffer and 6-aminoquinoline-N-succinimide methyl ester.

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