CN117330684A - Detection method and kit for simultaneously detecting citric acid, oxalic acid and cystine in urine - Google Patents

Abstract

The invention relates to the technical field of analysis and detection, in particular to a detection method and a kit for simultaneously detecting citric acid, oxalic acid and cystine in urine. The detection method comprises the following steps: mixing a sample to be tested with an acidulant, and centrifuging to obtain a first supernatant; mixing the first supernatant with a derivatizing agent for derivatization; the derivatization agent is a mixed solution of n-butanol and trimethylchlorosilane; and mixing the derived mixed solution with water to terminate the derivation, and centrifuging to obtain a second supernatant for liquid chromatography detection. By adopting the detection method, the citric acid, oxalic acid and cystine in the urine sample have good derivatization effect, so that the three components have good peak type and separation degree in the subsequent chromatographic detection process, meanwhile, the reagent type used in the derivatization reaction is less, the derivatization temperature is low, and the detection method is suitable for preparing the kit for detection and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like.

Description

Detection method and kit for simultaneously detecting citric acid, oxalic acid and cystine in urine

Technical Field

The invention relates to the technical field of analysis and detection, in particular to a detection method and a kit for simultaneously detecting citric acid, oxalic acid and cystine in urine.

Background

Calcium oxalate is the most common stone, mainly due to an imbalance between calcium and oxalic acid levels in the body and/or the lack of sufficient crystallization inhibitors. The causes of hyperoxaluria or hyperoxaluria can be categorized into primary (rare) and secondary (common) hyperoxaluria according to the etiology and severity of clinical manifestations. The detection method of oxalic acid (OX) mainly comprises liquid chromatography, liquid chromatography mass spectrometry, chromogenic test paper method, ion chromatography, etc.

Citrate is a potent inhibitor of calcium salt crystallization. Potassium citrate treatment is effective in primary or secondary hypocitrate patients and in dysacidification patients. Hypocitrate is considered to be the cause of kidney stones formation. The detection method of Citric Acid (CA) mainly includes liquid chromatography, liquid chromatography mass spectrometry, and the like.

Cystiuria is an autosomal recessive or incomplete genetic disorder. This disease disrupts the transport of cystine and other dibasic amino acids in the proximal tubules of the kidney, leading to repeated formation of kidney stones. Defects in reabsorption of cystine by the renal proximal tubule cells can result in excessive excretion of cystine into the urine, promoting the precipitation of cystine in the distal tubules. And the solubility of cystine in urine is low, so that patients have high tendency to form cystine stones. The detection method of Cystine (CYS) mainly comprises a liquid chromatography-mass spectrometry method, a gas chromatography-mass spectrometry method and the like.

Methods for simultaneously detecting citric acid, oxalic acid and cystine have been disclosed in the prior art, for example, CN115389684a discloses a method for simultaneously detecting oxalic acid, citric acid and cystine in 24h urine, but multiple reagents such as benzyl alcohol, trimethylchlorosilane, tetramethylammonium fluoride and acetonitrile are adopted in the derivatization process, the derivatization reaction temperature is up to 80 ℃, and the derivatization reaction time is 75min. As another example, CN116381104a discloses a method for detecting organic acid and application thereof, in which derivatization treatment is not performed in pretreatment, and after chromatographic separation, the separation degree and peak type of each substance are poor, and the detection method is greatly affected by environmental pH and cannot be applied to detection of preparation kit.

Therefore, how to provide a detection method which is simple and rapid, has few derivatization reagents and high detection accuracy and is suitable for preparing a kit becomes a technical problem to be solved in the field.

Disclosure of Invention

Firstly, the invention provides a detection method for simultaneously detecting citric acid, oxalic acid and cystine in urine, which comprises the following steps:

mixing a sample to be tested with an acidulant, and centrifuging to obtain a first supernatant;

mixing the first supernatant with a derivatizing agent for derivatization; the derivatization agent is a mixed solution of n-butanol and trimethylchlorosilane;

mixing the derived mixed solution with water to terminate the derivation, and centrifuging to obtain a second supernatant for liquid chromatography detection.

According to the invention, a large number of derivatization reagents are screened, and when n-butanol and trimethylchlorosilane are selected to be matched as the derivatization reagents, citric acid, oxalic acid and cystine in a urine sample have good derivatization effects, so that the three reagents have good peak type and separation degree in the subsequent chromatographic detection process, meanwhile, the derivatization reaction uses few types of reagents, the derivatization temperature is low, and the detection method is more suitable for preparing a kit for detection.

Preferably, the sample to be tested is 24h urine.

Preferably, in the derivatizing agent, the volume ratio of n-butanol to trimethylchlorosilane is (1-10): 1.

more preferably, in the derivatizing agent, the volume ratio of n-butanol to trimethylchlorosilane is (1.5-2.5): 1.

preferably, the volume ratio of the first supernatant to the n-butanol is 1: (1-20).

Preferably, the derivatization temperature is below 90 ℃.

More preferably, the derivatization temperature is 65 ℃ or less.

More preferably, the derivatization temperature is 62 ℃ or less.

Preferably, the acidulant is at least one of hydrochloric acid, sulfuric acid and nitric acid.

When the acidulant is hydrochloric acid, the acidulation effect of the urine sample is better, and the acidulated urine sample can be better subjected to the derivatization reaction after being mixed with the derivatization reagent.

Preferably, 10-100 mu L of acidulant is added to each 1mL of sample to be tested.

Preferably, centrifugation at 4000rpm or more; more preferably 10000rpm or more.

Preferably, the second supernatant is mixed with a sample diluent, centrifuged to obtain a third supernatant, and the third supernatant is detected by liquid chromatography.

Preferably, the sample diluent is the same as the reagents of the liquid chromatography mobile phase.

Preferably, the sample diluent is a mixture of methanol and an aqueous ammonium formate solution.

Preferably, the second supernatant is mixed with a sample diluent, shaken at 1000rpm or more, and centrifuged at 3000 rpm or more to obtain a third supernatant, which is detected by liquid chromatography.

Preferably, the conditions for liquid chromatography detection include:

a C18 chromatographic column;

mobile phase a: a mixed solution of ammonium formate, formic acid and water;

mobile phase B: a mixed solution of ammonium formate, formic acid and methanol;

in the mobile phase A and the mobile phase B, the concentration of ammonium formate is 0.005-0.02 mol/L; formic acid accounts for 0.05% -0.20% of the total volume of the mobile phase A;

the sum of the volume percentages of the mobile phase A and the mobile phase B in the elution process is 100 percent;

the elution conditions were:

。

preferably, the C18 column is a Shim-pack Velox SP-C18, having a specification of 2.7 μm, 2.1X150 mm.

Preferably, the C18 column is a Shim-pack Velox C18, having a size of 2.7 μm, 2.1X150 mm.

Preferably, the flow rate of the mobile phase is 0.1-0.6 mL/min.

Preferably, the detection method further comprises quantitative detection (LC-MS detection) using mass spectrometry after the liquid chromatography detection.

In some embodiments, the ion information is as shown in table 1 when quantitative detection is performed using mass spectrometry.

TABLE 1

In the table of the present invention,indicating the quantitative ion.

In some embodiments, the detection method further comprises: and detecting after the same treatment is carried out on the calibrator and/or the quality control product and the sample to be detected.

In some embodiments, the first supernatant is derivatized after shaking by adding an internal standard working solution, n-butanol, and trimethylchlorosilane.

Further, the present invention provides a kit for realizing the detection method of any one of the above embodiments, comprising:

the device comprises a calibrator, a quality control product, an internal standard working solution, a derivatization agent n-butanol, a derivatization agent trimethylchlorosilane, an acidulant and a mobile phase reagent for liquid chromatography detection.

Preferably, the calibrator comprises C1-C6, wherein C1 comprises 5-15 mug/mL oxalic acid, 35-45 mug/mL citric acid and 2-6 mug/mL cystine; c2 comprises 18-25 mug/mL oxalic acid, 75-85 mug/mL citric acid and 8-14 mug/mL cystine; c3 comprises 35-45 mug/mL oxalic acid, 150-170 mug/mL citric acid and 15-20 mug/mL cystine; c4 comprises 70-80 mug/mL oxalic acid, 250-350 mug/mL citric acid and 25-35 mug/mL cystine; c5 comprises 100-200 mug/mL oxalic acid, 500-700 mug/mL citric acid and 55-65 mug/mL cystine; c6 comprises 220-280 mug/mL oxalic acid, 800-1200 mug/mL citric acid and 80-120 mug/mL cystine.

Preferably, the quality control product comprises Q1-Q3, wherein Q1 comprises 25-35 mug/mL oxalic acid, 100-150 mug/mL citric acid and 10-15 mug/mL cystine; q2 comprises 100-150 mug/mL oxalic acid, 450-550 mug/mL citric acid and 40-60 mug/mL cystine; q3 comprises 160-180 mug/mL oxalic acid, 600-800 mug/mL citric acid and 65-85 mug/mL cystine.

Preferably, the internal standard working solution comprises 80-120 mug/mL of oxalic acid internal standard (OX-IS), 30-70 mug/mL of citric acid internal standard (CA-IS) and 30-70 mug/mL of cystine internal standard (CYS-IS).

Preferably, the acidulant is 1-12 mol/L hydrochloric acid solution.

Preferably, the mobile phase reagent comprises: 1-10 mol/L ammonium formate aqueous solution, formic acid and methanol.

The present invention provides a detection method for diagnosing and treating urinary calculus, which is well known in the art, and the content of oxalic acid, citric acid and cystine in a sample to be detected does not mean that the result can be directly directed to the diagnosis result of related diseases such as urinary calculus.

Compared with the prior art, the invention has the beneficial effects that:

the detection method is suitable for preparing the kit for detection, can simultaneously meet the requirements of linearity, repeatability, batch-to-batch difference, accuracy and the like of citric acid, oxalic acid and cystine, can simultaneously detect the citric acid, the oxalic acid and the cystine, and has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like.

Drawings

FIG. 1 is a chromatogram of citric acid, oxalic acid, and cystine of example 1.

FIG. 2 is a chromatogram of citric acid, oxalic acid and cystine for the CN116381104A assay.

FIG. 3 is a graph showing the peak shape of cystine in comparative example 4.

FIG. 4 is a graph of the peak shape of cystine according to example 1.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The examples are not intended to identify the particular technology or conditions, and are either conventional or are carried out according to the technology or conditions described in the literature in this field or are carried out according to the product specifications. The reagents and instruments used, etc. are not identified to the manufacturer and are conventional products available for purchase by regular vendors.

Because background-free human source samples are difficult to obtain, the following examples use artificial urine as a surrogate matrix for the human body to formulate various concentration points and quality controls of a standard curve.

Example 1

The embodiment provides a detection method for simultaneously detecting citric acid, oxalic acid and cystine in urine and a kit matched with the detection method.

The composition of the kit is shown in Table 2.

TABLE 2

The urine is detected by adopting the kit, and the steps are as follows:

1. preparing a detection reagent:

mobile phase A is prepared: 1000. 1000 mL water was accurately removed by a measuring cylinder, 2mL of ammonium formate solution (5 mol/L) and 1mL of formic acid were added, and after mixing, filtration and ultrasonic degassing were performed for use.

And (3) preparing fluidity B: 1000. 1000 mL methanol is accurately removed by a measuring cylinder, 2mL of ammonium formate solution (5 mol/L) and 1mL of formic acid are added, and after uniform mixing, filtration and ultrasonic degassing are carried out for later use.

Preparing a needle washing liquid: accurately transferring 500 mL methanol and 500 mL water by using a measuring cylinder, uniformly mixing, filtering and ultrasonically degassing for later use.

2. Pretreatment of

Before testing, the kit and the sample are restored to room temperature;

urine acidification: adding 50 mu L of acidulant into 1mL of urine, and uniformly mixing;

urine centrifugation: centrifuging urine with a high-speed centrifuge at 10000rpm for 2min;

and (3) derivatization: accurately transferring 20 mu L of centrifuged urine sample by using a pipette, respectively adding 10 mu L of internal standard working solution into the urine sample, the calibrator and the quality control product, respectively adding 200 mu L of derivatizing agent A and 100 mu L of derivatizing agent B, putting the two into a 2mL centrifuge tube, covering a cover, putting the centrifuge tube on a multitube vortex mixer for 30 seconds, putting the centrifuge tube on a centrifuge tube floating tube, putting the centrifuge tube into a water bath, and derivatizing the centrifuge tube for 60 minutes at 60 ℃;

termination and extraction: adding 0.3mL of water into each centrifuge tube after the derivatization is finished, oscillating for 1min on a multitube vortex mixer, and centrifuging for 2min at 10000rpm by using a high-speed centrifuge;

and (3) transferring: 20 mu L of supernatant is removed and placed in a 96-well plate, 200 mu L of sample diluent is added, and the mixture is vibrated for 5min under the condition of 1500rpm of a micro-plate constant temperature vibrator and then placed in a high-capacity refrigerated centrifuge for centrifugation for 5min under the condition of 4000 rpm.

3. LC-MS detection

Liquid phase conditions:

chromatographic column: shim-pack Velox SP-C18.7 μm,2.150mm；

Column temperature: 40 ℃;

sample injection volume: 3 μL;

sample manager temperature: 10 ℃;

the elution conditions are shown in Table 3.

TABLE 3 Table 3

Mass spectrometry test conditions:

the ion source parameters are shown in table 4 and the ion information is shown in table 1.

TABLE 4 Table 4

4. Detection result

1. Linear range test data

1.1, verification method: and detecting the solutions of the calibrator C1-C6 according to the detection method, and repeating the test for 3 times for each concentration. And calculating a correlation coefficient r of the linear regression by referring to the formula, wherein the correlation coefficient r is more than or equal to 0.990.

；

r: linear regression correlation coefficients;

x _i : the concentration of C1-C6;

y _i : the peak area ratio of the calibrator in the corresponding concentration solution to the internal standard is the average value.

1.2, acceptance criteria: the correlation coefficient r of citric acid, oxalic acid and cystine regression is more than or equal to 0.990.

1.3 experimental results

Linear range: oxalic acid linear range 10-250 mug/mL, citric acid 40-1000 mug/mL, cystine 4-100 mug/mL.

Table 5 citric acid linearity data

TABLE 6 oxalic acid linearity data

TABLE 7 cystine linearity data

1.4, conclusion: the correlation coefficient r of citric acid and oxalic acid and cystine regression is more than or equal to 0.990, and meets the acceptance standard.

2. Repeatability test data

2.1, verification method: and (3) processing the calibrator C1-C6 solution and the quality control product according to the detection method, taking the actual human sample test background as a low value, adding a sample of a high-concentration standard sample as a high value, repeatedly measuring each sample for 5 times, and performing three batches of tests. And calculating a repeated variation Coefficient (CV) according to a reference formula, wherein the CV of the quality control product is less than or equal to 15%.

CV: a coefficient of variation of repeatability;

: average of 5 measurements;

s: standard deviation of 5 measurements.

2.2, acceptance criteria: the coefficient of variation CV of the low-value quality control product is less than or equal to 15 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent.

2.3 experimental results

TABLE 8 precision between citric acid lots

TABLE 9 precision between oxalic acid lots

TABLE 10 within-lot precision of cystine lot

2.4, conclusion: the coefficient of variation CV of the low-value quality control product is less than or equal to 15 percent, and the coefficient of variation CV of the high-value quality control product is less than or equal to 15 percent, thereby meeting the acceptance standard.

3. Evaluation of matrix Effect

3.1 Experimental procedure

Taking 6 independent humanized matrix samples, a solvent sample, a matrix sample and a solvent sample and matrix sample 1:1, and detecting peak areas of all samples. The average value of the area ratio of the analyte of the solvent sample to the internal standard peak is A, the area ratio of the analyte of the matrix sample to the internal standard peak is BP, and the solvent sample to the matrix sample 1:1 with a CP ratio of mixed sample analyte to internal standard peak area.

3.2, accepted Standard

Matrix deviation (%) = (CP- (a+bp)/2)/((a+bp)/2), matrix deviation is less than or equal to 20%.

3.3 experimental results

TABLE 11 results of citrate matrix effect

TABLE 12 oxalic acid matrix effect

TABLE 13 cystine matrix effects

3.3 experimental summary

The substrate deviation is less than or equal to 20 percent, and meets the requirements.

4. Accuracy of

4.1 test procedure

The standard adding recovery method is adopted, 20 mu L of low and high concentration accuracy working solution samples (the concentration is respectively the background value)0.9+LQC, background value +.>0.9+hqc) for pretreatment; 3 samples per concentration level were parallel.

4.2 acceptance criteria

Accuracy: 85% -115%.

TABLE 14 citric acid accuracy

TABLE 15 oxalic acid accuracy

TABLE 16 cystine accuracy

4.3 experimental summary

Accuracy is satisfied: 85% -115% of the requirements.

In summary, according to the detection method disclosed by the invention, the oxalic acid linear range is 10-250 mug/mL, the citric acid is 40-1000 mug/mL, the cystine is 4-100 mug/mL, and the correlation coefficient r is more than or equal to 0.990; the Coefficient of Variation (CV) of the repeatability of the low-value quality control is less than or equal to 15 percent, the Coefficient of Variation (CV) of the repeatability of the median quality control is less than or equal to 15 percent, and the Coefficient of Variation (CV) of the repeatability of the high-value quality control is less than or equal to 15 percent; the relative substrate deviation is less than or equal to 20 percent, and the accuracy is 85-115 percent.

In addition, the chromatogram of this example is shown in fig. 1, the chromatogram obtained by the detection method disclosed in CN116381104a is shown in fig. 2, and the results show that when the detection method of the present invention is used, citric acid, oxalic acid and cystine have good separation degree and peak type, and the test results in CN116381104a show that citric acid, oxalic acid and uric acid are basically located at the same position and peak, and the separation degree is obviously deteriorated.

Effect example

The test example is used for exploring the detection response value of oxalic acid, cystine and citric acid by the volume ratio of derivatization reagent n-butanol and trimethylchlorosilane. The detection method is the same as in example 1.

The test results are shown in Table 17.

TABLE 17

The result shows that when the volume ratio of the n-butanol to the trimethylchlorosilane is (1-10): 1, the detection method of the invention has better detection effect and the best volume ratio is 2:1.

Comparative example 1

This comparative example provides a method for detecting citric acid, oxalic acid and cystine in urine, which differs from example 1 only in that:

in the pretreatment step, derivatizing agent A was replaced with an equivalent amount of methanol.

Comparative example 2

This comparative example provides a method for detecting citric acid, oxalic acid and cystine in urine, which differs from example 1 only in that:

in the pretreatment step, derivatizing agent A was replaced with an equal amount of ethanol.

Comparative example 3

This comparative example provides a method for detecting citric acid, oxalic acid and cystine in urine, which differs from example 1 only in that:

in the pretreatment step, derivatizing agent a was replaced with an equivalent amount of propanol.

As a result, it was found that the derivative products and water could not be separated after the termination of the derivatization and the extraction of comparative examples 1 to 3.

Comparative example 4

This comparative example provides a method for detecting citric acid, oxalic acid and cystine in urine, which differs from example 1 only in that:

neither mobile phase a nor mobile phase B was added with ammonium formate solution.

The test results show that the addition of ammonium formate has a significant improvement in the peak shape of cystine. The peak pattern of cystine in this comparative example is shown in FIG. 3, and the peak pattern of cystine in example 1 is shown in FIG. 4.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The detection method for simultaneously detecting citric acid, oxalic acid and cystine in urine is characterized by comprising the following steps of:

mixing a sample to be tested with an acidulant, and centrifuging to obtain a first supernatant;

mixing the first supernatant with a derivatizing agent for derivatization; the derivatization agent is a mixed solution of n-butanol and trimethylchlorosilane;

mixing the derived mixed solution with water to terminate the derivation, and centrifuging to obtain a second supernatant for liquid chromatography detection.

2. The detection method according to claim 1, wherein the derivatization agent comprises n-butanol and trimethylchlorosilane in a volume ratio of (1-10): 1.

3. the method according to claim 2, wherein the volume ratio of the first supernatant to n-butanol is 1: (1-20).

4. The method according to claim 1, wherein the derivatization temperature is 90 ℃ or lower.

5. The method according to claim 1, wherein the acidulant is at least one of hydrochloric acid, sulfuric acid and nitric acid.

6. The method according to claim 1, wherein the second supernatant is mixed with a sample diluent, centrifuged to obtain a third supernatant, and the third supernatant is detected by liquid chromatography.

7. The method according to claim 1, wherein the conditions for liquid chromatography detection include:

a C18 chromatographic column;

mobile phase a: a mixed solution of ammonium formate, formic acid and water;

mobile phase B: a mixed solution of ammonium formate, formic acid and methanol;

in the mobile phase A and the mobile phase B, the concentration of ammonium formate is 0.005-0.02 mol/L; formic acid accounts for 0.05% -0.20% of the total volume of the mobile phase A;

the sum of the volume percentages of the mobile phase A and the mobile phase B in the elution process is 100 percent;

the elution conditions were:

。

8. the method according to claim 7, wherein the C18 column is a Shim-pack Velox SP-C18 having a size of 2.7 μm, 2.1X 50mm;

or the C18 column is a Shim-pack Velox C18, which has a specification of 2.7 μm, 2.1X150 mm.

9. The method of detecting according to claim 7, further comprising: after the liquid chromatography detection, quantitative detection is performed by mass spectrometry.

10. A kit for carrying out the detection method according to any one of claims 1 to 9, comprising:

the device comprises a calibrator, a quality control product, an internal standard working solution, a derivatization agent n-butanol, a derivatization agent trimethylchlorosilane, an acidulant and a mobile phase reagent for liquid chromatography detection.