A method for improving the sensitivity of liquid chromatography-mass spectrometry for detecting vitamin K1 in serum

Abstract

The application provides a method for improving the sensitivity of liquid chromatography-mass spectrometry in detecting vitamin K1 in serum, which comprises the following steps: preparing a sample solution of vitamin K1 to be detected by using a liquid-liquid extraction method to obtain an initial sample solution; and redissolving the initial sample solution by using an aqueous solution containing at least isopropanol as a redissolving agent to obtain a sample solution to be detected; and adding ammonium fluoride after an LC-MS column to improve the mass spectrometry detection sensitivity of the vitamin K1 sample. By using the technical scheme of the application, isopropanol or isopropanol+methanol is used as a redissolving agent to replace methanol in the prior art as a redissolving agent, the mass spectrometry detection sensitivity of VK1 is improved, the recovery rate is improved, the detection method has good stability, and the clinical detection requirements can be met; meanwhile, the ammonium fluoride in the liquid chromatography can be completely replaced, the system pressure of the liquid chromatograph is reduced, and the service life of the chromatographic column is prolonged.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a method for improving the sensitivity of liquid chromatography-mass spectrometry in detecting vitamin K1 in serum. Background Technology

[0002] Vitamin K1, also known as phylloquinone, is essential for the liver to synthesize prothrombin, promoting the conversion of prothrombin precursors into prothrombin. Vitamin K deficiency causes hypoprothrombinemia, leading to coagulation disorders. Clinical manifestations of vitamin K deficiency typically include petechiae, ecchymosis, and mucosal bleeding; persistent bleeding after trauma or surgery; hematuria and gastrointestinal bleeding may also occur. In cases of accidental ingestion of rodenticides or overdose of coumarin-based drugs, bleeding symptoms are often more severe and widespread. Long-term vitamin K deficiency can also cause arterial calcification. Vitamin K antagonists are commonly used to prevent thromboembolic diseases. In bone tissue, dicumarol-induced vitamin K deficiency can reduce bone formation, leading to low bone mass and osteoporosis. Vitamin K1 plasma concentrations may be affected by dietary intake and may also be interfered with by triglycerides. The average plasma concentration of vitamin K1 ranges from 0.22 to 8.88 nmol / L (0.099 to 3.99 ng / mL), but generally, the concentration of vitamin K1 during studies is below 2 nmol / L (0.90 ng / mL). LC-MS / MS is the gold standard for the detection of many common small molecules in clinical practice. The separation by liquid chromatography combined with the high specificity of mass spectrometry allows for accurate quantification of many common small molecule compounds. However, the clinical concentration of VK1 is low and the concentration range is narrow, posing a significant challenge to accurate quantitative analysis. A highly sensitive mass spectrometer is usually required to meet detection needs. Due to its low polarity, the APCI ionization source is commonly used for detection in mass spectrometry. Compared to the ESI ionization source, the APCI ionization source is used less frequently in laboratory applications, has a narrower applicability, and requires more frequent maintenance.

[0003] In existing technologies, the lowest concentration of vitamin K1 in serum is generally around 0.1 ng / mL. Quantitative detection using LC-MS / MS is typically difficult. In practice, it either relies on the instrument itself, such as HPLC or even ultra-high performance liquid chromatography (UHPLC), by upgrading to a more sensitive instrument, or by optimizing the detection method to improve the detection limit. For example, Chinese invention patent CN114778719A discloses a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for vitamin K1 detection based on an electrospray ionization source. This method involves adding 10 mM ammonium fluoride to both mobile phase A and mobile phase B to improve the sensitivity and precision of VK1 detection under ESI. Clearly, to lower the detection limit of VK1, the method of adding ammonium fluoride to the mobile phase, thereby separating VK1 from other interfering components through the interaction of the mobile phase and ammonium fluoride, while simultaneously improving the sensitivity of VK1 detection, has been widely used in the field of liquid chromatography-tandem mass spectrometry. This method is also applicable to the detection of various trace components in serum.

[0004] Therefore, in practical applications, ammonium fluoride is used as an electrolyte in the mobile phase of liquid chromatography columns to improve vitamin separation, thereby increasing recovery rates and sensitivity in mass spectrometry detection. However, the addition of ammonium fluoride significantly increases the system pressure in liquid chromatography, and the damage to the column caused by ammonium fluoride is irreversible, thus greatly shortening the column's lifespan and increasing detection costs. Even gradient elution or reducing the elution rate cannot completely avoid column damage. Therefore, from the perspective of protecting the column and reducing detection costs, it is best to avoid introducing ammonium fluoride into the liquid chromatography column to protect it.

[0005] Therefore, based on existing technologies, how to use the electrolyte method to maintain the recovery rate and detection sensitivity of vitamins while reducing the damage of electrolytes to the chromatographic column to reduce detection costs is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] To address the technical problems existing in the prior art, the present invention aims to provide a method for improving the sensitivity of liquid chromatography-mass spectrometry (LC-MS) in detecting vitamin K1 in serum, and to improve the sensitivity of mass spectrometry detection of VK1 and increase the recovery rate by replacing ammonium fluoride with isopropanol, while reducing the system pressure of the LC instrument to extend the service life of the chromatographic column.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for improving the sensitivity of liquid chromatography-mass spectrometry (LC-MS) for detecting vitamin K1 in serum, comprising: preparing an initial sample solution of vitamin K1 by liquid-liquid extraction; redissolving the initial sample solution using an aqueous solution containing at least isopropanol as a redissolving solvent to obtain the sample solution to be tested; and combining the method with LC-MS to improve the mass spectrometry detection sensitivity of vitamin K1 samples.

[0008] Preferably, the volume concentration of isopropanol in the complex solvent is 10-70%.

[0009] Preferably, the complex solvent further includes methanol, with a volume concentration of 20-60%.

[0010] Preferably, the complex solvent is an aqueous solution of isopropanol or isopropanol + methanol, wherein, as one of the most preferred embodiments, the isopropanol volume content in the aqueous solution of isopropanol is 70%.

[0011] Preferably, the method further includes adding NH4F as an additive to the mobile phase effluent from the chromatographic column of the liquid chromatography.

[0012] Preferably, the additive is added to and mixed with the mobile phase effluent from the chromatographic column via a methanol solution.

[0013] Preferably, the amount of additive added is 2-20 mM. As a preferred embodiment, the amount of additive added is 5-10 mM, and most preferably, the amount added is 10 mM.

[0014] Preferably, the methanol solution containing the additive is added to the mobile phase at a rate of 5 to 20 μL / min. As a preferred embodiment, the post-column addition concentration is 5 to 10 μL / min, most preferably 5 μL / min.

[0015] Preferably, the specific steps of the method for improving the sensitivity of mass spectrometry detection of vitamin K1 in serum by adding ammonium fluoride after the column include:

[0016] (1) Preparation of the sample to be tested: The extracted sample solution was prepared by liquid-liquid extraction method;

[0017] (2) Preparation of the sample solution to be tested: The extracted sample solution obtained in step (1) is added to isopropanol for redissolution to obtain the sample solution to be tested;

[0018] (3) Pre-column separation: The sample solution to be tested prepared in step (2) is separated by liquid chromatography, and the mobile phase flows out through the chromatographic column;

[0019] (4) Post-column detection: A methanol solution containing ammonium fluoride is continuously added to the mobile phase that flows out in step (2) using a sample pump, so that the additive merges with the mobile phase after the chromatographic column and flows into the mass spectrometer for detection.

[0020] Preferably, step (1) includes liquid-liquid extraction, which involves placing a serum sample into a centrifuge tube, adding methanol containing the internal standard compound VK1-d7 for protein precipitation, shaking for 3 minutes, adding n-hexane, shaking and centrifuging, and then transferring the sample to be tested into the upper organic solvent. The upper organic solvent is then transferred for later use, awaiting the next step.

[0021] Preferably, in step (3), a 0.1% formic acid aqueous solution is used as mobile phase A, and a methanol solution containing 0.1% formic acid is used as mobile phase B for gradient elution.

[0022] Technical effects of the present invention:

[0023] 1. By adopting the technical solution of this invention, based on LC-MS technology, isopropanol or isopropanol + methanol is used as the redissolver instead of methanol in the prior art. This overcomes the technical bias of using only the mobile phase as the redissolver in the prior art. At the same time, it increases the amount of VK1 dissolved in human serum entering the liquid chromatograph, thereby improving the mass spectrometry detection sensitivity of VK1, increasing the recovery rate, and ensuring good stability of the detection method, which can meet the requirements of clinical testing. In addition, it can completely replace the role of ammonium fluoride in liquid chromatography, improve the separation of VK1 from interfering substances, improve the separation efficiency of liquid chromatography, and reduce the system pressure of the liquid chromatograph, thus extending the service life of the chromatographic column.

[0024] 2. By adopting the technical solution of this invention, combined with the method of adding ammonium fluoride solution after the column, high-precision detection of VK1 can be achieved. The detection and analysis of VK1 in human serum can be performed with the lowest limit of quantitation. During the mass spectrometry detection process, the sample is ionized by an ESI ionization source. By adding ammonium fluoride solution after the column to the mobile phase, the impact on the pressure of the chromatographic system is greatly reduced. The electrolyte does not need to pass through the chromatographic column, which extends the service life of the chromatographic column and maximizes the extension and improvement of the signal-to-noise ratio of low concentration VK1 detection.

[0025] 3. The technical solution and method adopted in this invention are simple. VK1 in human serum only requires liquid-liquid extraction for pretreatment, without derivatization. The sample volume requirement is small. In particular, this invention can directly improve the sensitivity of detecting VK1 in human serum by adding ammonium fluoride solution to the mobile phase effluent after the column, based on existing analytical techniques. It can be combined with various practical situations and is applicable to different types of liquid chromatography and mass spectrometry. It has low requirements for instrument performance and can be selected and used based on the current equipment. It has a wide range of applications and is easy to promote and use.

[0026] 4. By adopting the technical solution of the present invention, sample detection can be performed using an ESI ionization source, which has high sensitivity, low maintenance frequency, and no need to replace the ion source. It can accurately quantify VK1 in serum and has great clinical application value.

[0027] 5. The analytical method for improving sensitivity provided by this invention has the advantages of simple pretreatment, good linearity (R>0.999), serum spike recovery rate within ±15%, and high accuracy; in particular, it also has the advantages of being easy to operate, low cost, short time consumption, and easy to automate.

[0028] 6. This invention uses isopropanol as a redissolving solvent, overcoming the conventional use of liquid chromatography mobile phase as a redissolving solvent in existing technologies. By replacing isopropanol with isopropanol as the redissolving solvent, the amount of VK1 incorporated into the redissolving solvent is increased. At the same time, it has a synergistic effect with ammonium fluoride, which greatly improves the mass spectrometry detection signal, thereby meeting the needs of clinical trials for VK1 detection. Moreover, it does not have high requirements for the performance of liquid chromatography and mass spectrometry equipment, which can promote the widespread use of this detection method and greatly reduce detection costs and requirements. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the process for detecting VK1 in human serum samples according to the present invention.

[0030] Figure 2 This is the LC-MS chromatogram of the 0.1 ng / mL calibrator of Example 1 of the present invention.

[0031] Figure 3 The LC-MS chromatogram of the 0.1 ng / mL calibrator of Example 1 of this invention.

[0032] Figure 4 This is the standard curve for the spiked recovery of the VK1 simulated matrix in Example 1 of the present invention.

[0033] Figure 5 This is an LC-MS chromatogram of a serum sample with a concentration of 0.1 ng / mL for quantitative detection of VK1 in Example 1 of the present invention.

[0034] Figure 6 This is a comparison chart showing the effect of adding different concentrations of ammonium fluoride after column loading on the VK1 signal in Examples 1-5 and Comparative Examples 1-3 of the present invention.

[0035] Figure 7 The graph shows a comparison of the effects of adding ammonium fluoride solution at different flow rates after the column in Examples 1 and 6-8 on the VK1 signal.

[0036] Figure 8 This is a comparison chart showing the effect of adding and not adding ammonium fluoride on the VK1 signal under different complex solvent conditions in Examples 1, 12 and Comparative Examples 3-4 of the present invention.

[0037] Figure 9 This is the LC-MS chromatogram of the calibrator of Comparative Example 4 of this invention.

[0038] Figure 10 This is the LC-MS chromatogram of the calibrator of Comparative Example 5 of this invention. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0040] This invention provides a method for improving the sensitivity of mass spectrometry detection of vitamin K1 in serum by adding ammonium fluoride after column chromatography. Specifically, it includes using liquid chromatography-tandem mass spectrometry (LC-MS) to add NH4F to the mobile phase effluent from the chromatographic column of the liquid chromatograph and to add NH4F solution to the mass spectrometer to improve the detection sensitivity of vitamin K1 and perform quantitative analysis.

[0041] The instruments used in this invention are a Shimadzu LC-20 chromatograph and an Agilent Proshell SBC18 column (2.7um, 2.1*50mm).

[0042] Mass spectrometer: Sciex API4000, ESI ionization source; Ion source type: Turbo Spray; Ion source temperature: 400.0℃; Curtain Gas: 27.00; Ionspray: 5500.00; Gas 1: 50.00; Gas 2: 60.00; Acquisition mode: MRM; VK1 quantitative ion pair settings: m / z 451.4-m / z 187.3, DP90, CE40; VK1 qualitative ion pair settings: m / z 451.4-m / z 225.3, DP70, CE29; VK1-d7 quantitative ion pair settings: m / z 458.4-m / z 194.5, DP90, CE35.

[0043] The VK1 detection procedure for human serum samples using the above-mentioned instrument is described in the attached document. Figure 1 A gradient elution was performed using water containing 0.1% formic acid as mobile phase A and methanol containing 0.1% formic acid as mobile phase B. The flow rate was 0.3 mL / min, the injection volume was 20 μL, and the single-needle run time was 11.5 min. A 96% methanol solution containing 0.1–20 mM NH4F (methanol to water volume ratio of 96:4) was continuously added to the mobile phase effluent from the column at a flow rate of 5–10 μL / min using a peristaltic injection pump. This allowed the NH4F additive to merge with the mobile phase after the column and flow together into the mass spectrometer for detection.

[0044] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0045] Example 1

[0046] This embodiment provides a method for improving the sensitivity of mass spectrometry detection of vitamin K1 in serum by adding ammonium fluoride after column chromatography. The specific steps include:

[0047] Preparation of the test sample solution: Liquid-liquid extraction procedure: Take 200 μL of calibrator and serum sample into 2 mL centrifuge tubes respectively, add 200 μL of methanol containing internal standard compound VK1-d7 for protein precipitation, shake for 3 min, add 1 mL of n-hexane, shake for 10 min, centrifuge at 10000g for 10 min, transfer 800 μL of the upper organic solvent into a new centrifuge tube, blow dry with nitrogen at 40℃, and then reconstitute with 100 μL of isopropanol aqueous solution (70% isopropanol + 30% pure water) to obtain the test sample solution.

[0048] Liquid chromatography analysis: Mobile phase A was water containing 0.1% formic acid, and mobile phase B was methanol containing 0.1% formic acid. Gradient elution was performed at a flow rate of 0.3 mL / min, an injection volume of 20 μL, and a single-needle run time of 11.5 min. The gradient elution program is shown in Table 1.

[0049] Table 1 Gradient elution procedure for samples in liquid chromatography analysis.

[0050] Time / min B / ％ 0.01 75 1 75 3 96 8 96 8.1 75 11.5 75

[0051] Mass spectrometry analysis: A methanol solution containing 0.1 mM NH4F was continuously added to the eluting mobile phase at a flow rate of 5 μL / min using a peristaltic pump. This allowed the NH4F additive to merge with the mobile phase after the column and flow into the mass spectrometer for detection. It should be noted that 96% methanol solution refers to a methanol-water aqueous solution with a volume ratio of 96:4. NH4F was dissolved in this 96% methanol solution to obtain a 0.1 mM NH4F solution.

[0052] The calibrators are biological matrix samples containing different concentrations of VK1 prepared from blank bovine serum, and do not contain internal standard compounds. For example... Figure 2 The image shows the chromatogram of a calibrator containing 0.1 ng / mL VK1, with a retention time (Rt) of 7.49 min. The signal-to-noise ratio (S / N) at the VK1 peak in the chromatogram is 18. Clearly, adding ammonium fluoride solution post-column can improve the mass spectrometry detection signal for any concentration of VK1. The reason is speculated to be that ammonium fluoride forms ammonium and fluoride ions within the electrospray ionization source; the gaseous ammonium ions are acidic and can act as [M+H] ions. + A potential proton source with enhanced ion signal, while fluoride ions can capture potential Na+. + Ionic contaminants, thus blocking [M+Na] + Formation of adducts.

[0053] Plot a standard curve (linear regression curve) for the VK1 simulated matrix with the calibrator concentration (ng / mL) on the x-axis and the ratio of the peak area of ​​the VK1 chromatogram to the peak area of ​​its internal standard compound on the y-axis, with a linear range of 0.1-20 ng / mL. (See reference...) Figure 4 The standard curves plotted for different calibrator concentrations were fitted to obtain the regression equation: y = 0.182x + 0.00477, with a regression coefficient R = 0.9996.

[0054] See Figure 5 Figure 1 shows the LC-MS spectrum obtained from the human serum sample to be tested. As shown in the figure, this is the LC-MS spectrum of human serum with a VK1 concentration of 0.1 ng / mL in this embodiment. It can be seen from the figure that the retention time of the VK1 peak is Rt = 7.47 min, and the peak shape is symmetrical, indicating a suitable response and excellent separation effect.

[0055] The concentration of VK1 in the serum to be tested is calculated based on the regression equation obtained by fitting and the peak area in the spectrum obtained by detecting the serum sample.

[0056] Example 2

[0057] The difference between this embodiment and Embodiment 1 is that the methanol solution added after the column contains 2 mM NH4F.

[0058] Example 3

[0059] The difference between this embodiment and Embodiment 1 is that the methanol solution added after the column contains 5 mM NH4F.

[0060] Example 4

[0061] The difference between this embodiment and Example 1 is that the methanol solution added after the column contains 10 mM NH4F.

[0062] Example 5

[0063] The difference between this embodiment and Embodiment 1 is that the methanol solution after column addition contains 20 mM NH4F.

[0064] Example 6

[0065] The difference between this embodiment and Example 1 is that a methanol solution containing 10 mM NH4F was added to the mobile phase eluting from the column at a flow rate of 10 μL / min.

[0066] Example 7

[0067] The difference between this embodiment and Example 1 is that a methanol solution containing 10 mM NH4F was added to the mobile phase eluting from the column at a flow rate of 15 μL / min.

[0068] Example 8

[0069] The difference between this embodiment and Example 1 is that a methanol solution containing 10 mM NH4F was added to the mobile phase eluting from the column at a flow rate of 20 μL / min.

[0070] Example 9

[0071] The difference between this embodiment and Example 1 is that in the sample preparation step, the liquid-liquid extraction method includes taking one 200 μL sample containing 20 ng / mL of VK1 calibrator (same as in Example 1) into a blank centrifuge tube, adding 200 μL of methanol containing the internal standard compound VK1-d7 for protein precipitation, shaking for 3 min, adding 1 mL of n-hexane, shaking for 10 min, centrifuging at 10000g for 10 min, transferring 800 μL of the upper organic solvent to a new centrifuge tube, drying under nitrogen at 40°C, and then reconstituted using 100 μL of a mixed isopropanol solution (50% isopropanol + 20% methanol + 30% water) as the reconstitution solvent. Other operations are the same. See [link to relevant documentation]. Figure 3 The image shows the chromatogram of the calibrator containing 20 ng / mL VK1 in this embodiment. The detection signal of VK1 is 7.3E+04, and the retention time Rt of the calibrator is 7.46 min.

[0072] Example 10

[0073] The difference between this embodiment and Embodiment 9 is that, in the resolution step, the resolvent used is a mixed solution of isopropanol (30% isopropanol + 40% methanol + 30% water).

[0074] Example 11

[0075] The difference between this embodiment and Embodiment 9 is that, in the resolution step, the resolvent used is a mixed solution of isopropanol (10% isopropanol + 60% methanol + 30% water).

[0076] Comparative Example 1

[0077] The difference between this comparative example and Example 1 is that the methanol solution added after the column contains 0.5 mM NH4F.

[0078] Comparative Example 2

[0079] The difference between this comparative example and Example 1 is that the methanol solution added after the column contains 1 mM NH4F.

[0080] Comparative Example 3

[0081] The difference between this comparative example and Example 1 is that the methanol solution after column addition contains 0.5 mM NH4F.

[0082] Comparative Example 4

[0083] The difference between this comparative example and Example 9 is that, in the resolution step, an aqueous methanol solution (70% methanol + 30% water) is used as the resolvent.

[0084] Comparative Example 5

[0085] The difference between this comparative example and Example 1 is that, in the resolution step, an aqueous solution of isopropanol (75% isopropanol + 25% water) is used as the resolvent.

[0086] See Figure 5 The figure shows a comparison of the effects of adding different concentrations (1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 0 mM, 0.1 mM, 0.5 mM) of ammonium fluoride solution after column chromatography on the VK1 signal for Examples 1-5 and Comparative Examples 1-3. Figure 5 As shown, when the concentration of ammonium fluoride added after the column is below 2 mM, the VK1 signal is suppressed; when the concentration is above 2 mM, the VK1 signal gradually increases. Considering the mass spectrometry tolerance concentration, the sensitivity of mass spectrometry detection, and the recovery rate of VK1, adding 10 mM ammonium fluoride solution after the column is the optimal implementation method.

[0087] See Figure 7 The figure shows a comparison of the effect of adding 10 mM ammonium fluoride solution at different flow rates (5 μL / min, 10 μL / min, 15 μL / min, 20 μL / min) after column addition on the VK1 signal in Examples 1 and 6-8. As shown in the figure, the VK1 signal tends to decrease with the change of the post-column addition flow rate. Considering the actual operation and stability of post-column addition, the optimal post-column addition concentration is 5 to 10 μL / min.

[0088] Furthermore, the present invention investigated the proportions of different copolymers. Examples 1, 9-11, and Comparative Examples 4 and 5 investigated the effects of the composition of different copolymers on liquid chromatography.

[0089] See Figure 9-10 The figures are chromatograms of VK1 in different co-solvents.

[0090] Specifically, Comparative Example 4 used 70% methanol + 30% water as the complex solvent, see reference. Figure 9 Clearly, the signal intensity detected by chromatography was only 2284.5 cps, compared to... Figure 3 The detection signal was 7.3E+04 cps. In comparison, the detection signal was significantly reduced when methanol was used as the resolvent. Compared with Comparative Example 4, the resolvent in Example 9 was 50% isopropanol + 20% methanol + 30% water, and all other experimental conditions were the same. This shows that the introduction of isopropanol can significantly improve the chromatographic detection signal, increase the amount of VK1 separated from the sample, and improve the signal intensity of the chromatographic detection.

[0091] Comparative Example 5 used 75% isopropanol + 25% water as the resolvent. (See reference...) Figure 10The peak shape extension and deformation in the chromatogram affects the accuracy of VK1 quantitative detection. Obviously, although the sensitivity of the detection signal is still maintained at a high level (5.4E4cps.) when isopropanol is used instead of methanol as the complex solvent, the addition ratio should not exceed 70% when considering the requirements of quantitative accuracy.

[0092] In existing technologies, the mobile phase of liquid chromatography is typically used as the redistributing solvent to reduce the influence between the solvent and the mobile phase and improve detection stability. However, when methanol is used as the redistributing solvent, the separation effect of the chromatographic peaks is significantly worse compared to isopropanol, thus affecting the sensitivity of the mass spectrometry detection signal. Considering clinical testing requirements, for a limit of quantitation of 0.1 ng / mL, using methanol as the redistributing solvent requires combining it with other techniques to improve sensitivity; otherwise, it will not meet the requirements. In this invention, isopropanol or an aqueous solution of isopropanol and methanol is used as the redistributing solvent, which significantly improves the detection signal in the mixed aqueous solution.

[0093] When the volume content of isopropanol or isopropanol + methanol exceeds 70%, a solvent effect occurs, causing chromatographic peak extension and distortion, which is detrimental to the accuracy of peak area calculation. Therefore, the volume concentration of isopropanol and methanol in the mixed solvent should not exceed 70%. Furthermore, when the isopropanol ratio is between 50% and 70%, the VK1 signal is strong, and the peak shape exhibits a normal distribution, enabling more accurate quantification, providing accurate data support for clinical trials, and showing good recovery. Most preferably, a 70% isopropanol aqueous solution is the optimal mixed solvent ratio.

[0094] Referring to Table 2, under the condition of 70% isopropanol + methanol volume concentration, different ratios of isopropanol and methanol were used to directly compare the peak areas of VK1 in liquid chromatography. When the amount of isopropanol added increased from 10% to 70%, the peak area increased (at least 15 times). Obviously, with the increase of isopropanol content in the complex solvent, the peak area of ​​the chromatographic peak gradually increased, indicating that the complex solvent can dissolve more VK1 in the serum sample, and more VK1 can be separated in the chromatography, thereby improving the signal of mass spectrometry detection and improving the recovery rate.

[0095] Table 2. Effects of different complex solvent compositions in Examples 1, 9-11 and Comparative Example 4.

[0096] Complex solvent composition VK1 chromatographic peak area Example 1 70% isopropanol + 30% water 3.90E+05 Example 9 50% isopropanol + 20% methanol + 30% water 2.22E+05 Example 10 30% isopropanol + 40% methanol + 30% water 9.66E+04 Example 11 10% isopropanol + 60% methanol + 30% water 2.80E+04 Comparative Example 4 70% methanol + 30% water 2.6E4

[0097] In summary, using isopropanol as a redistributive solvent in chromatography-mass spectrometry (GC-MS) provides an unexpected technical improvement in the sensitivity of VK1 detection. The reason for this improvement can be analyzed from a theoretical perspective: isopropanol is more nonpolar than methanol, and its polarity is more similar to that of VK1. Therefore, increasing the proportion of isopropanol in the redistributive solvent allows for the dissolution and recovery of more VK1. Simultaneously, isopropanol has a slightly lower surface tension than methanol. Therefore, when using a redistributive solvent containing isopropanol as the solvent in the liquid chromatography, it can mix rapidly with the mobile phase, facilitating better interaction between the target compound VK1 and the stationary phase, thus maintaining a good peak shape. In existing technologies, using methanol as a redistributive solvent results in a detection limit of 1 ng / mL and a quantitation limit of 2 ng / mL for VK1. Clearly, using an aqueous solution of isopropanol + methanol or isopropanol as the redistributive solvent, combined with the addition of ammonium fluoride after the column, can significantly improve the detection limit of VK1, achieving a quantitation limit of 0.1 ng / mL, which fully meets clinical detection requirements.

[0098] See Figure 8 The figure presents the peak area of ​​VK1 detected by mass spectrometry with and without the addition of ammonium fluoride after the column, using 70% methanol aqueous solution and 70% isopropanol aqueous solution as the redistributing solvent, respectively. As shown in the figure, with 70% isopropanol aqueous solution as the redistributing solvent, the signal intensity of VK1 is increased by at least 10 times regardless of whether ammonium fluoride is added after the column. Furthermore, combining the method of adding ammonium fluoride after the column further improves the sensitivity of VK1, with the signal intensity increased by 50% compared to not adding ammonium fluoride.

[0099] Furthermore, to verify the feasibility of using isopropanol to replace ammonium fluoride in the prior art using the method of the present invention, the recovery rate of VK1 was verified using the technical solution of Example 1. Referring to Table 3, 0.1 ng / mL, 1 ng / mL, 2 ng / mL, 8 ng / mL, and 16 ng / mL of VK1 were added to the human serum samples of Example 1, respectively, and the recovery rate was detected. The results showed that within the detection range of 0.1–16 ng / mL of VK1, the recovery rate was between 89% and 104%, the sample detection stability (relative standard deviation) was between 4% and 10%, and the serum spiked recovery rate was within ±15%, indicating that the technical solution of the present invention has good stability and high accuracy in detecting VK1 in human serum.

[0100] Table 3. Recovery rates of human serum samples with different concentrations of VK1.

[0101] VK1 concentration added / g / mL VK1 recovery rate Relative standard deviation Cv (n=9) 0.1 96％ 9％ 1 89％ 10％ 2 101％ 9％ 8 91％ 4％ 16 104％ 6％

[0102] Note: The above values ​​are the results of 9 parallel experiments. Cv is the relative standard deviation calculated from multiple samples. The added concentration is VK1 added to human serum at a known concentration.

[0103] In summary, this invention, using isopropanol or isopropanol + methanol as a redissolver, introduces isopropanol into the detection method of chromatography-mass spectrometry (GC-MS). In the chromatographic stage, it completely replaces the role of pre-column ammonium fluoride, promoting the separation of VK1 from other interfering substances. Simultaneously, in the mass spectrometry stage, it further improves the sensitivity of VK1 detection. In particular, the synergistic effect of isopropanol and ammonium fluoride significantly enhances the VK1 detection signal during mass spectrometry, increasing detection sensitivity and providing technical support for the accuracy of minimum quantitation (LOQ) concentration detection. Moreover, combining this with the method of adding ammonium fluoride post-column not only further improves the sensitivity of VK1 detection in mass spectrometry but also avoids the introduction of ammonium fluoride into the chromatographic column. Isopropanol causes almost no damage to the chromatographic column, obviously reducing the system pressure in liquid chromatography, extending the column's lifespan, and saving on the analytical cost of VK1 detection.

[0104] Clearly, combining isopropanol or isopropanol + methanol as a resolvent and adding ammonium fluoride after column chromatography can significantly improve the detection limit and quantitation limit of VK1, with a minimum quantitation limit concentration of 0.1 ng / mL, which meets clinical testing requirements.

[0105] It should be noted that the lowest limit of quantitation (LOQ) concentration used in this invention is 0.1 ng / mL. This concentration already meets the minimum clinical detection requirements. Therefore, only the spectral results at 0.1 ng / mL are shown. Concentrations below 0.1 ng / mL are not very meaningful in actual clinical applications. Furthermore, the technical solution of this invention is applicable to existing liquid chromatography and mass spectrometry models. For instruments with lower sensitivity, a VK1 concentration below 0.1 ng / mL will result in quantification failure, which is not universal. Therefore, lower concentrations are not within the protection and research scope of this invention.

[0106] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A method for improving the sensitivity of liquid chromatography-mass spectrometry in detecting vitamin K1 in serum, characterized in that, The sample solution containing vitamin K1 was prepared by liquid-liquid extraction to obtain an initial sample solution; the initial sample solution was redissolved using an aqueous solution containing at least isopropanol as a redissolving solvent to obtain the sample solution to be tested; combined with LC-MS, the mass spectrometry detection sensitivity of vitamin K1 samples was improved. In the complex solvent, the volume concentration of isopropanol is 10-70%; The complex solvent also includes methanol, with a volume concentration of 20-60%. The LC-MS method includes: using water containing 0.1% formic acid as mobile phase A and methanol containing 0.1% formic acid as mobile phase B, performing gradient elution at a flow rate of 0.3 mL / min; continuously adding a 96% methanol solution containing 2–20 mM NH4F to the mobile phase eluted from the liquid chromatography column at a flow rate of 5–20 μL / min, and then co-eluting into the mass spectrometer for detection; The chromatographic column used in the liquid chromatography was an Agilent Proshell SB C18 column; The gradient elution program includes: 0.01–1 min, mobile phase B at 75%; 3–8 min, mobile phase B at 96%; 8.1–11.5 min, mobile phase B at 75%. The ion source for the mass spectrometer is an ESI ionization source; the temperature of the ion source is 400.0℃.

2. The method for improving the sensitivity of liquid chromatography-mass spectrometry in detecting vitamin K1 in serum according to claim 1, characterized in that, The specific steps include: (1) Preparation of the sample to be tested: The extract sample solution was prepared by liquid-liquid extraction; (2) Preparation of the sample solution to be tested: The extracted sample solution obtained in step (1) is added to isopropanol for redissolution to obtain the sample solution to be tested; (3) Pre-column separation: The sample solution to be tested prepared in step (2) is separated by liquid chromatography, and the mobile phase flows out through the chromatographic column; (4) Post-column detection: A methanol solution containing ammonium fluoride is continuously added to the mobile phase that flows out in step (2) using a sample pump, so that the methanol solution containing ammonium fluoride merges with the mobile phase after the chromatographic column and flows into the mass spectrometer for detection.

3. The method for improving the sensitivity of liquid chromatography-mass spectrometry in detecting vitamin K1 in serum according to claim 2, characterized in that, In step (1), the liquid-liquid extraction method includes placing the serum sample into a centrifuge tube, adding methanol containing the internal standard compound VK1-d7 for protein precipitation, shaking for 3 minutes, adding n-hexane, shaking and centrifuging, and then transferring the sample to be tested into the upper organic solvent for later use.

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