Kit for liquid-phase detection of cystine and liquid-phase detection method of cystine

Abstract

The invention relates to the technical field of detection, in particular to a kit for liquid-phase detection of cystine and a liquid-phase detection method of cystine. The kit for liquid-phase detection of cystine comprises a derivatization reagent and a protective agent, the derivatization reagent comprises one or more of 2, 3-naphthalene dialdehyde, benzaldehyde, p-tolualdehyde and fluorescent amine; the protective agent comprises 3-mercaptopropionic acid. According to the present invention, by introducing the derivatization reagent and the protective agent, the peak shape of the chromatographic peak of the cystine during the liquid phase detection can be good, the derivative product is stable, the yield is not low, and the test sensitivity and the test accuracy of the trace cystine are significantly improved. The method provided by the invention is relatively low in cost and simple and convenient in steps, cystine in urine can be well separated, the method can meet the requirements on the aspects of precision, accuracy, matrix effect and the like in a verification process, and the detection method can realize an accurate detection effect under the condition of not introducing an internal standard, and has a good application prospect. The method plays an important role in evaluating the content of cystine in urine.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, in particular to a liquid phase cystine detection kit and a liquid phase cystine detection method. Background Art

[0002] Cystine (L-Cystine) is a sulfur-containing amino acid and one of the essential amino acids for the human body. Its main functions in the human body include promoting cell oxidation-reduction, promoting liver metabolism, protecting the liver, and enhancing the detoxification capacity of the metabolic system. It plays an important role in maintaining health and preventing diseases. Keratin is one of the most abundant proteins in the skin and hair, which contains a large amount of cystine. Therefore, exogenous supplementation of cystine helps to strengthen hair and improve the symptoms of hair loss.

[0003] Cystine testing in urine has important clinical application value for diagnosing specific diseases, evaluating treatment effects, and monitoring kidney health. For example, cystine testing in urine can be used to monitor the treatment effects of patients with cystinuria. By adjusting diet and drug treatment, the excretion of cystine in urine can be reduced, thereby reducing the risk of stone formation. It can also be used to diagnose cystinuria, assess the risk of kidney stones, and assess kidney damage.

[0004] Currently, the main methods for detecting cystine are: ion chromatography amperometry, high performance liquid chromatography with fluorescence detection (HPLC-FL), liquid chromatography-tandem mass spectrometry (LC-MS / MS), urine ratio of S-methyl-L-ergothioneine to L-ergothioneine, microscopic examination of crystals and stone powder or qualitative reaction of cyanide nitrocyanate. Microscopic examination of crystals and stone powder or qualitative reaction of cyanide nitrocyanate is considered outdated due to its relatively low sensitivity and the need to use unstable and dangerous reagents. Ion chromatography amperometry and LC-MS / MS require professional equipment and technicians, and the cost is relatively high. HPLC-FL and urine ratio of S-methyl-L-ergothioneine to L-ergothioneine need to be standardized to account for individual differences, and additional biochemical analysis steps may be required.

[0005] CN106770779A discloses a method for detecting L-cystine in food, which uses hydrochloric acid to extract the supernatant and then dilutes it with pure water to filter the sample solution, and uses an electrospray ion source and a triple quadrupole tandem mass spectrometer for detection. The above method has the problems of cumbersome instrument operation, high professional ability requirements for users, and high cost.

[0006] Therefore, how to provide a method for accurately detecting cystine in urine with low cost and simple steps has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0007] Since cystine has no ultraviolet or fluorescent response, in order to improve the sensitivity of analysis and detection and separation selectivity, the present invention considers derivatizing the amino acid, connecting it with a luminescent or chromogenic group, and then performing qualitative and quantitative analysis.

[0008] During the preliminary research process, the present invention tried a large number of derivatization reagents commonly used in the field. The results showed that the by-products formed by some derivatization reagents during the derivatization of cysteine ​​would interfere with the chromatographic separation to a certain extent, and sometimes seriously affect the repeatability of the derivatization reaction; the yield of the derivative products after derivatization with some derivatization reagents was extremely low and could not be detected; the derivative products of some derivatization reagents were very unstable and needed to be analyzed immediately after derivatization.

[0009] After extensive research, the present invention finally screened out several compounds as derivatization reagents for cystine, and at the same time, 3-mercaptopropionic acid needs to be combined as a protective agent. The present invention found that 3-mercaptopropionic acid can prevent degradation during high-temperature derivatization and analysis, and 3-mercaptopropionic acid has a reducing property that can reduce oxidized substances to reducing substances, thereby protecting amino acids from possible oxidative damage; at the same time, 3-mercaptopropionic acid can form hydrogen bonds and ion pairs with cystine, and provide additional protons to cystine molecules, so that cystine can be better dissolved in the solvent in high-performance liquid chromatography analysis. Therefore, 3-mercaptopropionic acid can protect cystine and improve solubility in cystine detection.

[0010] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a liquid phase cystine detection kit, comprising a derivatization reagent and a protective agent; the derivatization reagent comprises one or more of 2,3-naphthalenedialdehyde, benzaldehyde, p-methylbenzaldehyde and fluoresceinamine; the protective agent comprises 3-mercaptopropionic acid.

[0011] The present invention finds that by introducing the above-mentioned derivatization reagent and protective agent, the peak shape of the chromatographic peak of cystine during liquid phase detection can be improved, the derivative product is relatively stable and the yield is not low, and the test sensitivity and accuracy of trace cystine are significantly improved.

[0012] Preferably, the derivatization reagent also includes a solvent, and the solvent includes one or more of methanol, ethanol and diethyl ether.

[0013] Preferably, the derivatization reagent comprises a mixture of 2,3-naphthalenedialdehyde and methanol.

[0014] In the present invention, the derivatization reagent can be a mixed methanol solution of 2,3-naphthalenedialdehyde, or 2,3-naphthalenedialdehyde and methanol packaged separately. When used, 2,3-naphthalenedialdehyde is dissolved in methanol to obtain a mixed solution. The protective agent is also the same, and the specific form is not limited here.

[0015] Specifically, when the aromatic aldehyde compound in the derivatization reagent is 2,3-naphthalenedialdehyde, the solution may be heated in a constant temperature water bath or heated and ultrasonically dissolved in an ultrasonic cleaner, which is not limited here.

[0016] Preferably, the kit also includes standard substances and / or quality control substances.

[0017] More preferably, the concentration of the standard is 4-100 μg / mL; preferably, the concentration of the quality control is 12-85 μg / mL.

[0018] In the present invention, artificial urine is used as a diluent in the preparation process of the quality control product and / or standard product.

[0019] In a second aspect, the present invention provides a liquid phase detection method for cystine, characterized in that it includes: mixing a sample to be tested with a protective agent, then adding a derivatization reagent, derivatizing at 50-60° C., and performing liquid phase detection after the derivatization is completed; the sample to be tested includes one or more of urine, quality control products and standard products; the protective agent includes 3-mercaptopropionic acid; the derivatization reagent includes one or more of 2,3-naphthalene dialdehyde, benzaldehyde, p-methylbenzaldehyde and fluorescent amine.

[0020] As a preferred embodiment of the present invention, the ratio of 2,3-naphthalenedialdehyde dry powder to methanol is (1-10):1 in terms of mass volume ratio.

[0021] Preferably, the volume ratio of the sample to be tested to the protective agent is 1:(1-3).

[0022] Preferably, the volume ratio of the sample to be tested to the derivatization reagent is 1:(1-10).

[0023] Preferably, the mobile phase of the liquid phase detection includes mobile phase A and mobile phase B; the mobile phase A is an aqueous solution of ammonium acetate, and the pH value of the mobile phase A is adjusted to 2-4 with acetic acid; and the mobile phase B is acetonitrile.

[0024] The present invention finds that when the pH value of the mobile phase A is controlled to be 2-4, the problems of cystine liquid phase peak tailing, peak splitting, etc. can be effectively solved.

[0025] Preferably, each 1 L of the mobile phase A contains 0.5-1.3 ml of acetic acid and 0.7-1.6 ml of ammonium acetate.

[0026] Preferably, the liquid phase detection adopts the following gradient elution program:

[0027] The sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

[0028] Preferably, the column temperature of the chromatographic column is 38-42° C.; and / or the flow rate of the mobile phases A and B is 0.6-1.2 mL / min.

[0029] Preferably, the chromatographic column for liquid phase detection is a PFP chromatographic column or a C18 chromatographic column.

[0030] As an embodiment of the present invention, the chromatographic column for liquid phase detection is a ChromCore PFP chromatographic column, and its specifications are 3 μm and 4.6×150 mm.

[0031] As an embodiment of the present invention, the chromatographic column for liquid phase detection is a Pursuit XRs 5 C18 chromatographic column, and its specification is 150×4.6 mm.

[0032] Since cystine itself absorbs in the ultraviolet light wavelength region of 200-220 nm, but the absorption is weak, it is easily interfered by other substances, resulting in inaccurate detection results. Therefore, it is preferred to use a method of detecting derivative products with an ultraviolet detector to detect cystine. Preferably, the wavelength of the ultraviolet detector is 245-274 nm.

[0033] Preferably, before derivatization, the method further includes: adjusting the pH value of the sample to be tested to 7.5-9.5 using a borate buffer.

[0034] In the present invention, the pH value of the sample to be tested is adjusted to 7.5-9.5 using a borate buffer before derivatization, which helps to better separate cystine in urine.

[0035] Preferably, the liquid phase detection method of cystine comprises: S1: Pre-treatment: The sample to be tested is mixed with a borate buffer to obtain a first mixed solution with a pH value of 7.5 to 9.5; the first mixed solution is mixed with a protective agent to obtain a second mixed solution, the second mixed solution is mixed with a derivatization reagent, and derivatized at 50 to 60° C. for 5 to 10 minutes, and liquid phase detection is performed after the derivatization is completed; wherein the volume ratio of the sample to be tested to the protective agent is 1: (1 to 3); the volume ratio of the sample to be tested to the derivatization reagent is 1: (1 to 10); S2: Liquid phase detection: The mobile phase of the liquid phase detection includes mobile phase A and mobile phase B; the mobile phase A is an aqueous solution of ammonium acetate, and the pH value of the mobile phase A is adjusted to 2-4 with acetic acid; the mobile phase B is acetonitrile; The liquid phase detection adopts the following gradient elution program:

[0036] The sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1; The chromatographic column for liquid phase detection is a PFP chromatographic column or a C18 chromatographic column.

[0037] The liquid phase detection kit for cystine and the liquid phase detection method for cystine provided by the present invention can make the peak shape of the chromatographic peak of cystine in liquid phase detection good by introducing the derivatization reagent and the protective agent, the derivative product is relatively stable and the yield is not low, and the test sensitivity and accuracy of trace cystine are significantly improved. The method provided by the present invention has low cost, simple steps, and can well separate cystine in urine. The method can meet the requirements of precision, accuracy and matrix effect in the verification process, and the detection method of the present invention can achieve the effect of accurate detection without introducing an internal standard. The method plays an important role in evaluating the cystine content in urine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 It is a liquid chromatogram of the cystine calibrator C3 in Example 1 provided by the present invention.

[0040] Figure 2 It is a liquid chromatogram of the cystine calibrator C3 in Example 4 provided by the present invention.

[0041] Figure 3 It is a liquid chromatogram of the cystine calibrator C3 in Comparative Example 1 provided by the present invention.

[0042] Figure 4 It is a liquid chromatogram of the cystine calibrator C3 in Comparative Example 3 provided by the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0045] Example 1 This embodiment provides a method for detecting cystine in urine, the steps are as follows: 1. Instrument Table 1

[0046] 2. Reagents 2.1 Preparation of compound and internal standard stock solutions Table 2

[0047] 2.2 Preparation of compound working solution and quality control working solution Table 3

[0048] In this embodiment, artificial urine was purchased from Jiangsu Addison Biotechnology Co., Ltd., item number: ADS083CZ0.

[0049] Take 40 μL of Sub-C6 solution and add it to 960 μL of artificial urine to obtain C1 working solution. C2, C3, C4, C5, LQC, MQC and HQC are prepared in sequence according to the proportions in Table 4.

[0050] Table 4

[0051] 2.3 Preparation of other reagents Prepare a borate buffer standard solution with a pH value of 8.6.

[0052] Weigh 50 mg of 2,3-naphthalenedialdehyde and dissolve it in 10 mL of methanol by heating as a derivatization reagent.

[0053] 3-Mercaptopropionic acid was used as a protective agent.

[0054] 2.4 Sample pretreatment (1) Add sample: Pipette 20 μL of the sample to be tested into 2 mL centrifuge tubes, add 140 μL of borate buffer standard solution, and adjust the pH value of the sample to 8.6; (2) Add protective agent: Pipette 20 μL of protective agent and add it to the corresponding centrifuge tubes above; (3) Add derivatization reagent: Pipette 40 μL of derivatization reagent and add it to the corresponding centrifuge tubes above; (4) Incubation at room temperature: After vortexing, leave at room temperature for 3 min. (5) Heating derivatization: Derivatization in a constant temperature water bath at 55°C for 10 min; (6) Detection: After derivatization, transfer the sample to a 96-well plate, place the 96-well plate in a liquid chromatography instrument, and inject 10 μL for detection. The liquid chromatogram of cystine calibrator C3 is shown in Figure 1 .

[0055] 2.5 Chromatographic conditions The chromatographic conditions are shown in Table 5.

[0056] Table 5

[0057] Test example 1. Precision The present invention evaluates the precision of the detection method of Example 1.

[0058] 1. Intra-batch precision Verification method: Artificial urine was used to prepare the standard curve, and quality control samples prepared with low and high concentrations of artificial urine (cystine concentrations were 12 μg / mL and 85 μg / mL, respectively) were taken for pretreatment; 5 samples were run in parallel at each concentration level.

[0059] Acceptance criteria: Precision was assessed using the coefficient of variation (%CV), which should be within 15.0%.

[0060] 2. Batch-to-batch precision Verification method: Artificial urine was used to prepare the standard curve, and quality control samples prepared with low and high concentrations of artificial urine (cystine concentrations were 12 μg / mL and 85 μg / mL, respectively) were taken for pretreatment; 5 samples were run in parallel at each concentration level, and the verification cycle was 5 days, with one batch per day.

[0061] Acceptance criteria: Precision was assessed using the coefficient of variation (%CV), which should be within 15.0%.

[0062] 3. The experimental results of cystine are shown in Table 6.

[0063] Table 6

[0064] 4. Conclusion The coefficient of variation (%CV) of intra-assay and inter-assay precision at low and high concentrations were both within 15%, meeting the acceptance criteria.

[0065] 2. Accuracy The present invention evaluates the accuracy of the detection method of Example 1.

[0066] Verification method: Artificial urine was used to prepare the standard curve. Low- and high-concentration quality control samples (cystine concentrations were 12 μg / mL and 85 μg / mL, respectively) were prepared with urine for pretreatment. Three samples were run in parallel at each concentration level. The verification period was 5 days, with one batch per day.

[0067] Acceptance criteria: Deviation from target value within 15.0%.

[0068] The experimental results of cystine are shown in Table 7.

[0069] Table 7 Conclusion: The deviations of all concentration points from the target values ​​were within ±15%, meeting the acceptance criteria.

[0070] 3. Linear Evaluation The present invention evaluates the linearity of the detection method of Example 1.

[0071] Verification method: Take a certain amount of compound working solution prepared with artificial urine (concentrations are: C1, C2, C3, C4, C5, C6) for pretreatment for 5 consecutive days.

[0072] Acceptance criteria: The accuracy of all concentration points should be within 85%-115%, and the CV should be within 20.0%. Linear regression: r ≥ 0.99.

[0073] The experimental results of cystine are shown in Table 8.

[0074] Table 8 Conclusion: The deviations of all concentration points were within ±15.0%, meeting the acceptance criteria; linear regression: r>0.99, meeting the acceptance criteria.

[0075] Matrix Effects The present invention evaluates the matrix effect of the detection method of Example 1.

[0076] Verification method: Artificial urine was used to prepare the standard curve. High and low concentration quality control samples were prepared with urine (cystine concentrations were 12 μg / mL and 85 μg / mL, respectively). Artificial urine with the same concentration and mixed liquid quality control samples of artificial urine and urine were also prepared. Each concentration was measured three times and the mean concentration of the samples was calculated.

[0077] Acceptable standard: matrix deviation (%) = (mean value of artificial urine + mean value of urine samples) ÷ (mean value of 2×mixed solution) (%) -1, matrix deviation < ±20%.

[0078] The experimental results of cystine are shown in Table 9.

[0079] Table 9

[0080] Conclusion: The matrix effect was <±20%, meeting the acceptance criteria.

[0081] In summary, for cystine detection, the coefficient of variation (%CV) of the intra-batch precision of this method was between 0.36% and 5.87%, and the coefficient of variation (%CV) of the inter-batch precision was between 2.19% and 4.20%; the recoveries of high and low urine samples were both between 94% and 104%; the detection linearity was 4 μg / mL to 100 μg / mL, and the deviations of all points were within 15%, and the linear regression was r>0.99; for the comparison between urine and artificial urine, the matrix effect was 1.58% to 3.64%.

[0082] Example 2 This embodiment provides a method for detecting cystine in urine, which differs from Embodiment 1 in that the derivatization reagent is replaced by an equal amount of benzaldehyde.

[0083] The experimental results are shown in Table 10. Only when the linear low point is 20 μg / mL, the deviation and linear regression of all concentration points meet the acceptance criteria.

[0084] Table 10 Conclusion: The linear evaluation detection range of Example 2 is smaller than that of Example 1.

[0085] Example 3 This embodiment provides a method for detecting cystine in urine, which differs from Embodiment 1 in that the volume ratio of the sample to be tested to the protective agent is 2:1.

[0086] The experimental results are shown in Table 11. Although the precision is within the required range, the deviation range is large.

[0087] Table 11

[0088] Conclusion: The precision of Example 3 is poorer than that of Example 1.

[0089] Example 4 This embodiment provides a method for detecting cystine in urine, which differs from Embodiment 1 in that: in mobile phase A, acetic acid is used to adjust the pH value of mobile phase A to 5.

[0090] The experimental results are as follows Figure 2 As shown, the results show that the peak time is delayed and the target peak separation is deteriorated; at the same time, the linear evaluation detection limit is higher than that of Example 1, as shown in Table 12.

[0091] Table 12 Conclusion: The peak graph deteriorated and it was not easy to distinguish the peak graph. The deviation and linear regression met the acceptance criteria when the linear range was 25-100 μg / mL.

[0092] Comparative Example 1 This comparative example provides a method for detecting cystine in urine, which differs from Example 1 in that the derivatization is performed at room temperature.

[0093] The experimental results show that the target peak can only be clearly seen when the derivatization time is long (≥1.5h). Figure 3 ), and the peak graph shows distortions such as tailing, fronting or peak splitting, which makes quantitative analysis impossible.

[0094] Comparative Example 2 This comparative example provides a method for detecting cystine in urine, which differs from Example 1 in that no protective agent is added.

[0095] The experimental results show that there are many peaks in the peak diagram, but none of them can meet the requirements of linear regression and the target peak cannot be determined.

[0096] Comparative Example 3 This comparative example provides a method for detecting cystine in urine, which differs from Example 1 in that the derivatization reagent is replaced by an equal amount of OPA-FMOC mixed derivatization solution.

[0097] The experimental results show that when OPA-FMOC mixed derivatization liquid is used as the derivatization agent, the peak elution time is delayed, and there are many impurity peaks near the target peak and the separation is poor (such as Figure 4 ), the results of precision, repeatability, stability, sample recovery rate, etc. cannot meet the requirements.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liquid phase detection kit for cystine, characterized in that: The invention comprises a derivatization agent and a protective agent; the derivatization agent comprises one or more of 2,3-naphthalenedialdehyde, benzaldehyde, p-methylbenzaldehyde and fluorescent amine; and the protective agent comprises 3-mercaptopropionic acid.

2. The liquid phase detection kit for cystine according to claim 1, characterized in that: The derivatization reagent also includes a solvent, and the solvent includes one or more of methanol, ethanol and ether.

3. The liquid phase detection kit for cystine according to claim 1 or 2, characterized in that: The kit also includes standard products and / or quality control products; Preferably, the concentration of the standard is 4-100 μg / mL; preferably, the concentration of the quality control is 12-85 μg / mL.

4. A liquid phase detection method for cystine, characterized in that, include: The sample to be tested is mixed with a protective agent, and then a derivatization reagent is added, and derivatization is performed at 50-60° C. After the derivatization is completed, liquid phase detection is performed; the sample to be tested includes one or more of urine, quality control products and standard products; the protective agent includes 3-mercaptopropionic acid; the derivatization reagent includes one or more of 2,3-naphthalenedialdehyde, benzaldehyde, p-methylbenzaldehyde and fluorescent amine.

5. The liquid phase detection method for cystine according to claim 4, characterized in that: The volume ratio of the sample to be tested to the protective agent is 1:(1-3).

6. The liquid phase detection method for cystine according to claim 4 or 5, characterized in that: The volume ratio of the sample to be tested to the derivatization reagent is 1:(1-10).

7. The liquid phase detection method for cystine according to any one of claims 4 to 6, characterized in that: The mobile phase of the liquid phase detection includes mobile phase A and mobile phase B; the mobile phase A is an aqueous solution of ammonium acetate, and the pH value of the mobile phase A is adjusted to 2-4 with acetic acid; the mobile phase B is acetonitrile.

8. The liquid phase detection method for cystine according to any one of claims 4 to 7, characterized in that: The liquid phase detection adopts the following gradient elution program: The sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

9. The liquid phase detection method for cystine according to any one of claims 4 to 8, characterized in that: The chromatographic column for liquid phase detection is a PFP chromatographic column or a C18 chromatographic column.

10. The liquid phase detection method for cystine according to any one of claims 4 to 9, characterized in that: include: S1: Pre-treatment: Mixing the sample to be tested with a borate buffer to obtain a first mixed solution with a pH value of 7.5 to 9.5; The first mixed solution is mixed with a protective agent to obtain a second mixed solution, the second mixed solution is mixed with a derivatization reagent, and derivatization is performed at 50-60° C. for 5-10 minutes, and liquid phase detection is performed after the derivatization; wherein the volume ratio of the sample to be tested to the protective agent is 1:(1-3); the volume ratio of the sample to be tested to the derivatization reagent is 1:(1-10); S2: Liquid phase detection: The mobile phase of the liquid phase detection includes mobile phase A and mobile phase B; the mobile phase A is an aqueous solution of ammonium acetate, and the pH value of the mobile phase A is adjusted to 2-4 with acetic acid; the mobile phase B is acetonitrile; The liquid phase detection adopts the following gradient elution program: The sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1; The chromatographic column for liquid phase detection is a PFP chromatographic column or a C18 chromatographic column.

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