A method for determination of three items of urinary calculi by mixed mode chromatography-tandem mass spectrometry without derivatization

Abstract

The application provides a method for determining three items of urinary calculi by mixed mode chromatography-tandem mass spectrometry without derivation, which comprises the following steps: mixing a sample to be measured with a dilution solution, centrifuging, then performing liquid chromatography-mass spectrometry detection, and calculating the content of the three items of urinary calculi in the sample to be measured according to the detection result. The method provided by the application relies on the unique multiple physical and chemical synergistic mechanism of the mixed mode stationary phase and the precise pH elution kinetics regulation, completely solves the stereoisomer interference, significantly improves the accuracy of clinical quantification, effectively avoids the "dead volume" flushing, strongly overcomes the matrix inhibition effect of oxalic acid, resolves the physical and chemical conflicts, takes into account the perfect peak shape of simultaneous detection of special amino acids and organic acids, abandons high-pollution reagents, constructs a "green flow path" for long-life operation of mass spectrometry, greatly simplifies the pretreatment and rapidly elutes the chromatography, and perfectly adapts to the high-throughput automatic pipeline.

Description

Technical Field

[0001] This invention belongs to the field of clinical testing technology, specifically relating to a method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization. Background Technology

[0002] Urinary tract stones (commonly known as kidney stones) are a common and frequently occurring disease in clinical urology. Statistics show that the incidence of urinary tract stones is as high as 5%–10%, and the recurrence rate within 5 years after treatment is as high as 50%. Oxalic acid, cystine, and citric acid (also known as citrate) in 24-hour urine are core biochemical indicators for assessing the risk of kidney stone formation, identifying metabolic etiologies, and guiding individualized dietary and drug interventions. Clinically, they are often referred to as the "three indicators of kidney stones."

[0003] High-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) has gradually become the mainstream method for detecting small molecule metabolites in clinical laboratories due to its high specificity and sensitivity. However, because the physicochemical properties of the three urinary stone markers (oxalic acid, cystine, and citric acid) differ drastically, traditional liquid chromatography separation and mass spectrometry detection face the following significant technical bottlenecks: Oxalic acid is extremely polar and has a very small molecular weight: Oxalic acid (MW 90) has almost no retention on traditional reversed-phase chromatography columns (such as C18 columns) and easily elutes with the dead volume. This makes oxalic acid highly susceptible to severe ionic inhibition (matrix effect) from high concentrations of inorganic salts, urea, creatinine, and other strongly polar matrices in urine, seriously affecting quantitative accuracy and detection limits.

[0004] Cystine exhibits extremely low solubility and amphoteric dissociation characteristics: As an amino acid containing disulfide bonds, cystine has very low solubility under specific pH conditions, readily precipitating in sample processing and the chromatographic column flow path. Simultaneously, its charge state in acid-base systems is complex, making it difficult to achieve good peak shapes in the same mobile phase as the other two organic acids. In traditional methods using acidic mobile phases suitable for organic acids, cystine's solubility is drastically reduced, and it carries a positive charge, leading to adsorption or tailing at the column head. Conversely, in alkaline systems suitable for cystine, oxalic acid and citric acid are completely lost on traditional columns due to excessive dissociation. This conflict arising from the conflict between target charge properties and solubility makes it impossible for existing one-dimensional chromatographic methods to simultaneously achieve perfect peak shapes and high selective retention for all three in a single injection.

[0005] Citric acid faces significant isomer interference: endogenous isocitric acid exists in urine, which has the exact same molecular formula, molecular weight (MW 192), and extremely similar mass spectrometric collision fragments (MRM ion pairs are completely identical) as citric acid. This means that the mass spectrometric signals of the two cannot be distinguished in a mass analyzer, and high-resolution chromatographic separation is required to achieve "baseline separation".

[0006] To address the aforementioned issues, current technologies reported in the field of clinical mass spectrometry mainly employ the following strategies, but all have significant drawbacks: Option 1: Derivatization method (such as reversed-phase chromatography combined with butylation or amidation derivatization) Existing patents and literature often employ n-butanol or specific derivatizing reagents to chemically modify the carboxyl or amino groups in urine, thereby reducing the polarity of oxalic acid and citric acid and increasing the retention of isomers. Examples include butyl esterification and amidation, which are introduced to increase the retention of highly polar small molecules (such as oxalic acid and citric acid) on traditional reversed-phase chromatography columns. Its drawbacks are as follows: the derivatization process is extremely cumbersome, usually requiring offline operations such as high-temperature heating and vacuum drying, involving multiple manual steps and typically taking several hours. This becomes a decisive "bottleneck" limiting clinical laboratories from conducting fully automated, large-scale sample screening, making it extremely difficult to achieve automated high-throughput operations. Furthermore, the derivatization reaction is often incomplete, and the composition of the urine matrix is ​​extremely complex. Differences in the matrix between samples often lead to inconsistent derivatization reaction efficiency (incomplete reaction or side reactions), resulting in the appearance of byproducts and residual derivatizing reagents. This introduces complex chromatographic peaks and chemical noise into the mass spectrum, severely interfering with the accurate extraction and quantification of the target. Moreover, the high concentration of residual derivatizing reagents and byproducts enter the mass spectrometer with the mobile phase, which can easily cause high-temperature carbon deposition and crystallization blockage at the nozzle and cone of the electrospray ionization (ESI) source, leading to rapid attenuation of the mass spectrometry signal and contamination of the mass spectrometry ion source. This significantly increases the frequency of instrument maintenance and clinical testing costs, and shortens the instrument maintenance cycle.

[0007] Option 2: Traditional reversed-phase chromatography (C18, etc.) Because oxalic acid (molecular weight 90) is extremely polar, it exhibits almost no thermodynamic retention on conventional reversed-phase C18 columns and typically elutes with the dead volume (solvent front). Meanwhile, highly polar interfering substances abundant in urine, such as endogenous high-concentration inorganic salts (e.g., sodium chloride, sulfate), urea, and creatinine, also elute concentrated at the dead volume. The simultaneous nebulization and ionization of oxalic acid and these high-concentration matrix components within the ion source results in extremely severe matrix suppression of the oxalic acid mass spectrometry signal. This directly leads to a deterioration of the limit of detection (LOD) and limit of quantitation (LOQ), making it impossible to accurately measure low-concentration samples.

[0008] Option 3: Ion-pair chromatography To artificially enhance the retention of oxalic acid and citric acid on reversed-phase columns without derivatization, some methods add highly volatile ion-pairing reagents such as tetrabutylammonium (TBA) to the mobile phase to improve the retention of strongly polar organic acids on reversed-phase columns. The drawbacks are: ion-pairing reagents have extremely strong column and tubing adsorption properties, making them very difficult to remove completely through routine cleaning. Once used, they remain in the system for a long time, making thorough cleaning impossible and preventing the system from being switched to other clinical tests (i.e., "poisoning the column"). Furthermore, the three urinary stone markers have higher ionization efficiency in negative ion mode (ESI-), while commonly used cation-pairing reagents form ion-pair complexes with negatively charged targets in the gas phase, drastically inhibiting the ionization efficiency of the targets. This results in a sharp drop in overall detection sensitivity, severely reducing the overall detection sensitivity of mass spectrometry.

[0009] Option 4: Pure Hydrophilic Interaction Chromatography (HILIC) This method utilizes a high proportion of organic phase to retain oxalic acid and citric acid on a hydrophilic column. Its drawbacks are as follows: the HILIC column is extremely sensitive to high concentrations of inorganic salts and water in urine, resulting in drastic matrix effect fluctuations. The aqueous layer on the stationary phase surface is easily affected by fluctuations in urine salt concentration, leading to retention time drift. Furthermore, the column equilibration time is extremely long, making it unsuitable for the needs of rapid clinical testing. More importantly, the HILIC mechanism struggles to establish sufficient selectivity to stably and completely separate citric acid and isocitrate at baseline. Citric acid and its endogenous interfering compound, isocitrate, have completely identical molecular weights and mass spectrometric collision fragments (MRM ion pairs are completely overlapping). The HILIC column primarily relies on a partition mechanism for retention, lacking sufficient steric exclusion and stereoselectivity for these two tricarboxylic acid compounds with very similar structures, leading to peak overlap. Clinically, if isocitrate cannot be completely separated at baseline (resolution Rs < 1.5), it will directly result in a false increase in the quantitative result of citric acid, leading to clinical misdiagnosis.

[0010] In summary, existing analytical methods generally suffer from drawbacks such as cumbersome sample preparation, reliance on harmful reagents, severe instrument contamination, short column life, inability to simultaneously target three different targets, or inability to effectively eliminate isomer interference. These limitations make it difficult to meet the demands of clinical laboratories for high-throughput, low-cost, and highly stable fully automated whole blood / urine analysis. Therefore, developing a derivatization-free, flow-path-clean mass spectrometry method capable of simultaneously achieving highly selective baseline separation of the three urinary calculi markers is a pressing technical solution for the clinical mass spectrometry community. Summary of the Invention

[0011] To address the shortcomings of existing technologies, the present invention aims to provide a derivatization-free mixed-mode chromatography-tandem mass spectrometry method for the determination of three urinary calculi. The method provided by this invention relies on the unique multiple physicochemical synergistic mechanisms of the mixed-mode stationary phase and precise pH elution kinetics control, completely eliminating stereoisomer interference, significantly improving the accuracy of clinical quantification, effectively avoiding "dead volume" erosion, strongly overcoming the matrix inhibition effect of oxalic acid, resolving physicochemical conflicts, ensuring perfect peak shapes for the simultaneous detection of specific amino acids and organic acids, eliminating highly polluting reagents, constructing a "green flow path" for long-life mass spectrometry operation, and featuring extremely simplified pretreatment and ultra-fast chromatographic elution, perfectly adaptable to high-throughput automated production lines.

[0012] To achieve this objective, the present invention adopts the following technical solution: This invention provides a method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization, the method comprising the following steps: The sample to be tested was mixed with the diluted reconstituted solution and centrifuged, followed by liquid chromatography-mass spectrometry detection. The content of the three substances related to urinary stones in the sample was calculated based on the detection results.

[0013] The liquid chromatography-mass spectrometry detection was performed using a bifunctional composite stationary phase.

[0014] The three indicators for urinary stones include citric acid, oxalic acid, and cystine.

[0015] The above method relies on the unique multiple physicochemical synergistic mechanisms of mixed-mode stationary phases and precise pH elution kinetics control, which completely solves the interference of stereoisomers, significantly improves the accuracy of clinical quantification, effectively avoids "dead volume" erosion, strongly overcomes the matrix inhibition effect of oxalic acid, resolves physicochemical conflicts, takes into account the perfect peak shape for the simultaneous detection of special amino acids and organic acids, eliminates highly polluting reagents, and constructs a "green flow path" for long-life mass spectrometry operation. The extremely simplified pretreatment and ultra-fast chromatographic elution are perfectly adapted to high-throughput automated production lines.

[0016] Citric acid and isocitrate are a pair of isomers that are mutually blind zones in mass spectrometry. Traditional techniques generally consider that they cannot be separated in one-dimensional conventional reversed-phase or hydrophilic systems without derivatization to alter their spatial volume or the use of extremely slow special rare-earth isomer separation columns. This invention utilizes the unique steric exclusion effect of a mixed-mode stationary phase, combined with the microscopic ion intensity changes that occur with increasing organic content in the mobile phase. By leveraging the extremely small differences in pKa and functional group spatial orientation between the two, a "knife-cut" baseline separation is achieved, directly plugging the technical loophole of misdiagnosis caused by isocitrate interference in clinical practice. Without altering the molecular structure (no derivatization), complete baseline separation of citric acid and endogenous isocitrate is achieved in an extremely short time (less than 5 minutes) (resolution Rs≥1.5).

[0017] Preferably, the diluted reconstitution solution is a buffer solution containing an isotopic internal standard.

[0018] Preferably, the isotopic internal standard includes 13C2-oxalic acid, d4-cysteine, and d4-citric acid.

[0019] Preferably, the buffer solution is an ammonium formate buffer or an ammonium acetate buffer.

[0020] Preferably, the concentration of the buffer solution is 20-50 mmol / L, and the pH is 4-5. The concentration can be 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, or 50 mmol / L, etc., and the pH can be 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5, etc., but is not limited to the values ​​listed above. Other values ​​not listed within the above range are also applicable.

[0021] Preferably, the bifunctional composite stationary phase is a stationary phase with silica gel as the matrix and alkyl and amino functional groups bonded to its surface. The amino group includes any one or a combination of at least two of primary amines, secondary amines, tertiary amines or quaternary ammonium groups, i.e., a reversed-phase / weak anion exchange (WAX / RP) bifunctional composite stationary phase.

[0022] Conventional reversed-phase chromatography cannot retain highly polar oxalic acid, causing it to elute with dead volume and suffer severe matrix inhibition; while HILIC columns are extremely sensitive to fluctuations in water and inorganic salts in urine. This invention cleverly utilizes the positive charge of the WAX ​​functional group to generate a strong electrostatic attraction (ion exchange) with the carboxyl group of oxalic acid, while simultaneously using the RP alkyl chain to provide moderate retention, significantly increasing the capacity factor of oxalic acid and completely offsetting the elution time from the matrix interference zone. This synergistic mechanism is not a simple performance additive effect, but rather breaks the technical prejudice of "no retention" of polar small molecules in conventional chromatography, overcoming the single mechanism limitation of traditional chromatography, and resolving the contradiction between polarity and retention through a dual-mechanism synergy.

[0023] Preferably, in the liquid chromatography-mass spectrometry detection, the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium formate and / or ammonium acetate, and mobile phase B is acetonitrile or methanol.

[0024] Preferably, the total concentration of ammonium formate and / or ammonium acetate in the mobile phase A is 5-30 mmol / L, and the pH of the mobile phase A is adjusted to 4.5-5.2.

[0025] Oxalic acid and citric acid are polybasic organic acids that do not dissociate and have no charge under strong acid conditions, but completely dissociate under alkaline conditions. Cystine, as an amphoteric amino acid with disulfide bonds, has high solubility under strong acid or strong base conditions, but it easily precipitates in slightly acidic environments (near neutral) and has a complex charge state, resulting in severe peak tailing. This invention introduces a volatile ammonium salt buffer system to lock the pH at 4.5–5.2. Under this specific microenvironment, cystine maintains sufficient solubility, avoids column head crystallization, and achieves a sharp and symmetrical peak shape through phase reversal and weak charge balance; oxalic acid and citric acid are in a moderately dissociated state, allowing for controlled ion exchange with the WAX ​​stationary phase. This design, which resolves the physicochemical conflicts of multiple targets through a precise pH window, possesses high methodological ingenuity, simultaneously addressing targets with diametrically opposed physicochemical properties in a single injection, thus resolving the physicochemical conflicts between polybasic organic acids and amphoteric amino acids.

[0026] Preferably, the liquid chromatography-mass spectrometry detection is performed using a gradient elution program, which is specifically as follows: From 0 to 1.5 min, the volume fraction of mobile phase A is 93-97%, and the remainder is mobile phase B; Between 1.5 and 3.5 minutes, the volume fraction of mobile phase A changes uniformly from 93-97% to 37-43%, with the remainder being mobile phase B. Between 3.5 and 3.51 min, the volume fraction of mobile phase A changed uniformly from 37-43% to 7-13%, with the remainder being mobile phase B; From 3.51 to 4 min, the volume fraction of mobile phase A was 7-13%, with the remainder being mobile phase B; From 4 to 4.01 min, the volume fraction of mobile phase A changed uniformly from 7-13% to 93-97%, with the remainder being mobile phase B; After 4.01 min, the volume fraction of mobile phase A was 93-97%, with the remainder being mobile phase B.

[0027] Preferably, in the liquid chromatography-mass spectrometry detection, the column temperature is 35-45℃, such as 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0028] Preferably, in the liquid chromatography-mass spectrometry detection, the mobile phase flow rate is 0.3-0.5 mL / min, such as 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min or 0.5 mL / min, but not limited to the values ​​listed above. Other unlisted values ​​within the above range are also applicable.

[0029] Ion-pairing reagents have irreversible adsorption-destructive effects on chromatographic columns and mass spectrometers (memory effect), while derivatization suffers from drawbacks such as cumbersome operation, reagent residue, and high-temperature carbon buildup clogging the mass spectrometer nozzle. This invention eliminates the need for any non-volatile or strongly adsorbing reagents throughout the entire process, abandoning the cumbersome derivatization steps and systemically toxic ion-pairing reagents (such as tetrabutylammonium salts) relied upon in existing technologies. It employs a completely volatile ammonium salt elution system, ensuring both ultra-high resolution and a highly clean flow path system. During continuous injection of large batches of clinical samples, the mass spectrometer ion source cone exhibits no carbon buildup or salt precipitation, demonstrating excellent method reproducibility. This significantly reduces instrument maintenance and operating costs in laboratory departments, creating a green and environmentally friendly flow path with "zero derivatization and zero ion pairs," and substantially extending the clinical lifespan of the mass spectrometer.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for the determination of three urinary calculi using mixed-mode chromatography-tandem mass spectrometry without derivatization. Relying on the unique multiple physicochemical synergistic mechanisms of the mixed-mode stationary phase and precise pH elution kinetics, it completely eliminates stereoisomer interference, significantly improves the accuracy of clinical quantification, effectively avoids "dead volume" erosion, strongly overcomes the matrix inhibition effect of oxalic acid, resolves physicochemical conflicts, and ensures perfect peak shapes for the simultaneous detection of specific amino acids and organic acids. It eliminates highly polluting reagents, constructs a "green flow path" for long-life mass spectrometry operation, and features extremely simplified pretreatment and ultra-fast chromatographic elution, perfectly adapting to high-throughput automated production lines. Specifically, it includes: (1) Completely eliminates stereoisomer interference and significantly improves the accuracy of clinical quantification. This invention utilizes a hybrid WAX / RP column with size exclusion selectivity and precise ionic strength gradient elution to achieve a resolution Rs≥1.5 (achieving perfect baseline separation) between citric acid and its endogenous isomer (isocitric acid) without damaging or altering the molecular structure. This completely eliminates the "blind zone interference" of isomers in mass spectrometry detection, avoiding false increases in citric acid detection results caused by the inability to distinguish isocitric acid in traditional methods. It provides accurate and reliable data support for the clinical classification of urinary tract stone etiology and the diagnosis of hypocituria.

[0031] (2) Effectively avoids the erosion of "dead volume" and strongly overcomes the matrix inhibition effect of oxalic acid. In conventional reversed-phase chromatography, highly polar oxalic acid cannot be retained (k′≈0) and is easily suppressed by strong ions from high-concentration background components such as inorganic salts, urea, and creatinine eluting with the dead volume in urine. This invention cleverly utilizes the strong electrostatic attraction between a weak anion-exchange stationary phase and oxalic acid molecules, resulting in a capacity factor k′>1.5 for oxalic acid. This ensures a stable and consistent elution time, completely avoiding matrix interference. Experimental data show that the matrix effect of oxalic acid in mass spectrometry is controlled between 85% and 115%, significantly improving the detection sensitivity and quantitative accuracy limit for low-concentration oxalic acid. Its limit of detection (LOD) can be as low as 1 / 10 of that of conventional reversed-phase chromatography.

[0032] (3) It resolves physicochemical conflicts and achieves perfect peak shapes for the simultaneous detection of special amino acids and organic acids. Amphoteric amino acids (cystine) and polyprotic organic acids (oxalic acid, citric acid) exhibit a natural conflict in solubility and charged state on traditional single-mechanism chromatographic columns. This invention precisely locks the mobile phase system within the "golden key window" of pH 4.5–5.2. In this specific microenvironment, cystine maintains excellent solubility (completely avoiding the technical risk of column head crystallization and clogging) while also achieving a moderate reverse-phase partitioning with the stationary phase through its spatial dipole moment, resulting in sharp and symmetrical peak shapes (tailing factor Tf ≤ 1.2). Simultaneously, oxalic acid and citric acid maintain moderate ionization, ensuring the method's versatility and high reproducibility.

[0033] (4) By eliminating highly polluting reagents, a "green flow path" for long-life mass spectrometry operation was constructed. This invention completely eliminates the cumbersome and chemically noisy derivatization reagents, and also avoids non-volatile ion-pairing reagents (such as tetrabutylammonium salts) that have an irreversible "toxic memory effect" on mass spectrometry systems. The entire mobile phase uses only a highly volatile, mass spectrometry-friendly low-concentration ammonium formate / ammonium acetate buffer system. In large-scale clinical sample testing (continuous injections >1000 injections): No high-temperature carbon deposits or inorganic salt crystallization were observed at the nozzle and high-pressure cone orifice of the electrospray ionization (ESI) mass spectrometry source, and the stability fluctuation of the mass spectrometry response was <5%; It greatly extends the lifespan of the chromatographic column (the column can withstand more than 3 times the injection volume), and significantly extends the hardware maintenance cycle of the clinical mass spectrometer from the usual once a week to more than once a month, thus significantly reducing the operating and human maintenance costs of clinical departments.

[0034] (5) The pretreatment is extremely simplified and the chromatographic elution is extremely fast, making it a perfect fit for high-throughput automated production lines. Thanks to the superior selectivity and interference resistance of the chromatographic end, the pretreatment end has successfully achieved "burden reduction": The sample does not need to undergo any evaporation, derivatization, high-temperature incubation or cumbersome solid-phase extraction (SPE). It can be directly loaded with just one simple step of "dilution-acidification red dissolution-centrifugation". The pretreatment time has been reduced from several hours to less than 5 minutes.

[0035] With the high-flow-rate gradient elution program, the overall chromatographic acquisition (analysis) time for a single injection is controlled within 5.0 minutes.

[0036] It greatly shortens the clinical report turnaround time (TAT) and can be seamlessly integrated into the fully automated small molecule mass spectrometry testing lines of hospitals and third-party medical laboratories (ICLs). Attached Figure Description

[0037] Figure 1 This is a graph showing the detection results of Example 1; Figure 2 This is a graph showing the test results for Comparative Example 1; Figure 3 The results are the mass spectrometry signal attenuation results of 1000 consecutive injections of real urine samples using the method in Example 1. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0039] In the following examples and tests, the purity of the reference standards oxalic acid, cystine, citric acid, and isocitrate was ≥99.0% (purchased from Sigma-Aldrich).

[0040] Isotope internal standards: 13C2-oxalic acid, d4-cystine, and d4-citric acid, all with a purity ≥98.0% (purchased from CIL Company).

[0041] Analytical instruments: Ultra-high performance liquid chromatograph (Waters ACQUITY UPLC) and tandem triple quadrupole mass spectrometer (Sciex Triple Quad 5500+), equipped with an electrospray ionization source (ESI).

[0042] Example 1: Derivatization-free mixed-mode chromatography-mass spectrometry for the detection of mixed standards according to the present invention 1. Reagent preparation Dilution buffer: Weigh 1.54 g of ammonium acetate, dissolve it in 950 mL of pure water, precisely adjust the pH to 4.8 using glacial acetic acid, and add water to bring the volume to 1000 mL to prepare a 20 mmol / L ammonium acetate buffer.

[0043] Mixed internal standard working solution: Prepare a mixed internal standard solution containing 13C2-oxalic acid (5.0 μmol / L), d4-cysteine ​​(2.0 μmol / L), and d4-citric acid (50.0 μmol / L) using the dilution buffer.

[0044] 2. Sample pretreatment Take 100 μL of the mixed standard (containing 50.0 μmol / L oxalic acid, 20.0 μmol / L cystine, 300.0 μmol / L citric acid, and 100.0 μmol / L isocitrate) and place it in a 1.5 mL centrifuge tube. Add 900 μL of the mixed internal standard working solution and vortex vigorously for 1 min to ensure thorough mixing. Centrifuge at 12,000 rpm for 5 min at 4 °C. Take the supernatant as the test solution and inject it directly into the liquid chromatography-mass spectrometry (LC-MS) system for analysis.

[0045] 3. Analytical conditions for liquid chromatography-mass spectrometry (LC-MS) Column: Waters Atlantis Premier BEH C18 AX (Mixed-Mode WAX / RP) column (2.1 mm × 100 mm, 1.7 μm), column temperature 40℃.

[0046] Mobile phase and gradient: Mobile phase A was a 20 mmol / L ammonium acetate aqueous solution (pH adjusted to 4.8 with acetic acid), and mobile phase B was pure acetonitrile. The elution gradient program is shown in the table below: Elution gradient program of Example 1 Mass spectrometry parameters: negative ion scanning mode (ESI-), multiple reaction monitoring (MRM). Ion source temperature (TEM): 550℃; spray voltage (IS): -4500 V; curtain gas (CUR): 35 psi.

[0047] The quantitative and qualitative ion pair parameters for the three targets and isotopic internal standards are shown in the table below: 4. Detection results and spectral features ( Figure 1 ) The analytical results show that, under the above mixed-mode chromatographic system, all substances exhibited extremely ideal chromatographic behavior: Oxalic acid: retention time was approximately 1.25 min, and its capacity factor k′≈3.1 (the dead volume elution time of the system was 0.30 min), successfully detaching from the dead volume scouring zone.

[0048] Cystine: Retention time is approximately 2.10 min, with a sharp and symmetrical peak shape and no tailing.

[0049] Isocitric acid and citric acid: The retention time of isocitric acid was approximately 2.85 min, and the retention time of citric acid was approximately 3.28 min. The resolution between the two was Rs = 1.85 (greater than 1.5), achieving complete baseline separation. They appeared as two independent, non-overlapping peaks in the extracted ion chromatogram (XIC) of mass spectrometry.

[0050] Example 2: Derivatization-free mixed-mode chromatography-mass spectrometry for detecting real urine samples according to the present invention 1. Sample Source Collect 24-hour urine samples from patients clinically diagnosed with urinary tract stones (provided by clinical collaborating laboratories).

[0051] 2. Sample pretreatment (real urine matrix) Accurately pipette 100 μL of a well-mixed real urine sample into a 1.5 mL centrifuge tube, add 900 μL of mixed internal standard working solution (containing 5.0 μmol / L 13C2-oxalic acid, 2.0 μmol / L d4-cysteine, and 50.0 μmol / L d4-citric acid; the matrix is ​​20 mmol / L ammonium acetate buffer, pH 4.8), and vortex vigorously for 1 min to ensure thorough mixing and acidification. Centrifuge at 12000 rpm for 5 min at 4 °C to precipitate large protein molecules and insoluble particles in the urine. Transfer the supernatant as the test solution and inject it directly into the liquid chromatography-mass spectrometry (LC-MS) system for analysis.

[0052] 3. Analytical Results and Evaluation of Matrix Effects The real urine sample was tested using the same LC-MS / MS conditions as in Example 1. The results showed that, in the complex matrix of real urine, the three endogenous urinary stones (oxalic acid, cystine, and citric acid) and the isomeric isocitric acid all achieved good chromatographic retention and baseline separation, with retention times highly consistent with the mixed standard (Example 1).

[0053] The matrix effect (ME) of real urine on the target site was evaluated using the post-column addition method and matrix matching curve method. Experimental results showed that: Oxalic acid: Due to its significantly delayed retention time (approximately 1.25 min) and complete separation from the high concentration of inorganic salts and endogenous polar impurities in the dead volume (0.30 min), its matrix effect is between 85% and 115% (matrix inhibition rate <15%), successfully overcoming the severe ion inhibition caused by urine matrix in conventional reversed-phase chromatography.

[0054] Cystine and citric acid: The matrix effect in real urine matrix was also in the ideal range of 90% to 110%, with no obvious matrix interference or sudden drop in response observed.

[0055] Example 3: Methodological Performance Verification of the Detection Method of the Invention To verify the scientific validity and reliability of the derivatization-free mixed-mode chromatography-tandem mass spectrometry method described in this invention in clinical quantification, a systematic clinical methodological performance validation was performed on the method. The results of various indicators are as follows: 1. Linear range and limit of detection / limit of quantitation (LOD / LOQ) A series of concentration standard curves for each target were prepared using a blank matrix (simulated urine desalted by activated carbon adsorption; the specific preparation method was as follows: weigh 20.0 g of urea, 0.2 g of uric acid, and 1.1 g of creatinine, dissolve in 900 mL of pure water, add 10 g of activated carbon, stir and adsorb for 2 hours to remove any possible trace amounts of endogenous organic acids, filter to remove the activated carbon, and make up to 1000 mL with distilled water). The target concentration was plotted on the X-axis, and the peak area ratio of the target to the corresponding isotopic internal standard was plotted on the Y-axis (1 / X). 2 Linear regression.

[0056] The results showed that all three targets exhibited excellent linearity within the clinically accepted metabolic concentration range (R² > 0.999). The limit of detection (LOD) was determined using a signal-to-noise ratio (S / N) of 3, and the limit of quantitation (LOQ) was determined using an S / N of 10 with satisfactory accuracy and precision. The data are shown in the table below. Linear equations and sensitivity indices for three target sites 2. Precision and Accuracy Quality control samples (QC) at low, medium, and high concentrations were prepared using a mixed matrix of real urine. Each concentration level was measured six times in parallel on the same day, and intra-batch precision and accuracy were calculated. Measurements were then performed continuously for five days, and inter-batch precision and accuracy were calculated. The detection method was the same as in Example 1. Results showed that both intra-batch and inter-batch coefficients of variation (CV) were strictly controlled within 6%, and the accuracy ranged from 95.2% to 104.8%, demonstrating extremely high quantitative precision.

[0057] Precision and accuracy validation data of the method (n=6) 3. Matrix Recovery Six real clinical urine samples from different sources with known baseline levels were precisely spiked with low, medium, and high levels of standard substances. After subtracting the background, the recovery rate was calculated. Each level was measured in six parallel runs. The results are shown in the table below: Recovery rates of real urine matrix spikes for three targets (n=6) Experimental results show that, due to the spatial exclusion and precise elution of the mixed-mode WAX / RP column, the target sites perfectly avoid interference from the urine matrix. The average matrix spike recoveries of the three targets are all within the ideal range of 95.0% to 105.0%, confirming that this method is not easily affected by the complex matrix differences between individual urine samples.

[0058] Comparative Example 1: Comparative testing using a conventional reversed-phase chromatographic column (C18) To verify the superiority of the mixed-mode chromatographic column and method of this invention, the chromatographic column was replaced with a conventional reversed-phase chromatographic column (Waters ACQUITY UPLC BEH C18, 2.1 mm × 100 mm, 1.7 mm). μ Except for m), the other pretreatment, mobile phase composition, elution gradient (Table 1), and mass spectrometry parameters are completely consistent with those in Example 1.

[0059] Analysis of test results ( Figure 2 (x-axis represents time, y-axis represents detector response) Oxalic acid showed no retention: the retention time of oxalic acid was 0.31 min, almost completely eluting synchronously with the solvent dead volume. When real urine matrix was added, the mass spectrometry signal of oxalic acid encountered extremely severe matrix inhibition, with an inhibition rate as high as 92%, due to the concentration of a large amount of inorganic salts and endogenous polar impurities in the dead volume, making it impossible to accurately integrate and quantify the extracted peak.

[0060] The isomers were completely identical: both isocitric acid and citric acid eluted within the retention time range of 1.10 min to 1.20 min, and their separation degree was... Rs =0. The two peaks merge into a broad, blunt co-eluent peak on the mass spectrum. Because their MRM ion pairs are completely identical, it is impossible to quantify citric acid separately.

[0061] Cystine peak shape deterioration: Cystine exhibits severe tailing and broadening in this conventional reversed-phase system, with tailing factor... Tf >2.5.

[0062] Comparative Example 2: Comparative testing was conducted using a conventional pure hydrophilic interaction column (HILIC). The column was replaced with a conventional hydrophilic interaction column (Waters ACQUITY UPLC BEH Amide, 2.1 mm × 100 mm, 1.7 mm). μ m), the mobile phase system was changed to the high-proportion organic phase elution system commonly used in HILIC (the initial mobile phase B had a pure acetonitrile ratio of 90%, which was linearly reduced to 50% within 1.5 min to 4.0 min to elute strongly hydrophilic substances), and the remaining pretreatment and mass spectrometry parameters were the same as in Example 1.

[0063] Analysis of test results: Isomer separation failed: Although the HILIC column prolonged the retention of oxalic acid (retention time increased to 2.50 min), the separation efficiency between citric acid and isocitric acid, which have extremely similar structures, was only [missing information]. Rs=0.65, indicating severe peak overlap, which fails to meet the requirements for accurate quantitative diagnosis of hypocituria in clinical medicine.

[0064] Poor reproducibility: After 20 consecutive injections of real urine samples, the high concentration of inorganic salts and water in the urine disrupted the water film microenvironment on the surface of the HILIC stationary phase, resulting in a significant forward shift in the retention time of oxalic acid and cystine, with a coefficient of variation (Relative Standard Deviation, RSD) > 4.5%, and extremely slow column equilibration.

[0065] 5. Comprehensive Comparative Analysis of Experimental Data To more intuitively demonstrate the technical effects of the present invention, the key methodological indicators of Example 1, Comparative Example 1, and Comparative Example 2 in detecting real patient urine samples were summarized and compared. The results are shown in the table below. Figure 3 : Conclusion: The experimental data clearly demonstrate that the derivatization-free mixed-mode chromatographic separation method described in this invention not only successfully solves the two major industry bottlenecks of oxalic acid retention and isomer non-separation, but also exhibits surprisingly high mass spectrometry tolerance and system stability. With a green flow path that avoids cumbersome derivatization, after 1000 consecutive injections of real urine, the mass spectrometry signal showed almost no attenuation due to the absence of strong ion-pairing reagents and thorough matrix splitting, fully meeting the methodological requirements for industrial-grade high-throughput clinical screening.

[0066] The applicant declares that this invention illustrates the method for determining three urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0067] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0068] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization, characterized in that, The method includes the following steps: The sample to be tested was mixed with the diluted reconstituted solution and centrifuged, followed by liquid chromatography-mass spectrometry detection. The content of the three urinary stones in the sample was calculated based on the detection results. The liquid chromatography-mass spectrometry detection was performed using a bifunctional composite stationary phase. The three indicators for urinary stones include citric acid, oxalic acid, and cystine.

2. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to claim 1, characterized in that, The diluted reconstitution solution is a buffer solution containing an isotopic internal standard.

3. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to claim 2, characterized in that, The isotopic internal standards include 13C2-oxalic acid, d4-cysteine, and d4-citric acid.

4. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to claim 2 or 3, characterized in that, The buffer solution is ammonium formate buffer or ammonium acetate buffer; Preferably, the concentration of the buffer solution is 20-50 mmol / L and the pH is 4-5.

5. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to any one of claims 1-4, characterized in that, The bifunctional composite stationary phase is a stationary phase with silica gel as the matrix and alkyl and amino functional groups bonded to its surface. The amino group includes any one or a combination of at least two of primary amines, secondary amines, tertiary amines, or quaternary ammonium groups.

6. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to any one of claims 1-5, characterized in that, In the liquid chromatography-mass spectrometry detection, the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium formate and / or ammonium acetate, and mobile phase B is acetonitrile or methanol.

7. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to claim 6, characterized in that, The total concentration of ammonium formate and / or ammonium acetate in the mobile phase A is 5-30 mmol / L, and the pH of the mobile phase A is adjusted to 4.5-5.

2.

8. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to claim 6 or 7, characterized in that, The liquid chromatography-mass spectrometry detection was performed using a gradient elution program, which is as follows: From 0 to 1.5 min, the volume fraction of mobile phase A is 93-97%, and the remainder is mobile phase B; Between 1.5 and 3.5 minutes, the volume fraction of mobile phase A changes uniformly from 93-97% to 37-43%, with the remainder being mobile phase B. Between 3.5 and 3.51 min, the volume fraction of mobile phase A changed uniformly from 37-43% to 7-13%, with the remainder being mobile phase B; From 3.51 to 4 min, the volume fraction of mobile phase A was 7-13%, with the remainder being mobile phase B; From 4 to 4.01 min, the volume fraction of mobile phase A changed uniformly from 7-13% to 93-97%, with the remainder being mobile phase B; After 4.01 min, the volume fraction of mobile phase A was 93-97%, with the remainder being mobile phase B.

9. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to any one of claims 1-8, characterized in that, In the liquid chromatography-mass spectrometry detection, the column temperature is 35-45℃.

10. The method for determining three parameters of urinary stones using mixed-mode chromatography-tandem mass spectrometry without derivatization according to any one of claims 1-9, characterized in that, In the liquid chromatography-mass spectrometry detection, the mobile phase flow rate is 0.3-0.5 mL / min.

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