CN117213940A - Creatine, guanidinoacetic acid and amino acid component standard substance with dried blood spots as sample matrix, preparation method thereof and UPLC-ID-MS/MS detection method - Google Patents
Creatine, guanidinoacetic acid and amino acid component standard substance with dried blood spots as sample matrix, preparation method thereof and UPLC-ID-MS/MS detection method Download PDFInfo
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Abstract
The invention relates to the technical field of chemical standard substance preparation, in particular to a creatine, guanidinoacetic acid and amino acid component standard substance taking dried blood spots as a sample matrix, a preparation method thereof and a UPLC-ID-MS/MS detection method. The creatine, guanidinoacetic acid and amino acid component standard substance using the dried blood spots as the sample matrix has good uniformity and stability, and fills the blank that no creatine, guanidinoacetic acid and amino acid component standard substance using the dried blood spots as the sample matrix exists in China.
Description
Technical Field
The invention relates to the technical field of chemical standard substance preparation, in particular to a creatine, guanidinoacetic acid and amino acid component standard substance taking dried blood spots as a sample matrix, a preparation method thereof and a UPLC-ID-MS/MS detection method.
Background
The creatine, guanidinoacetic acid and amino acid levels in the dried blood spots are of great significance for diagnosing the creatine deficiency syndrome, and the development of the creatine, guanidinoacetic acid and amino acid component standard substances taking the dried blood spots as sample matrixes is a basis for ensuring accurate and reliable clinical detection data of related metabolites. The whole blood sample is usually stored and transported in a form of dry blood spots in clinic, and as the preparation process of the dry blood spots is influenced by a plurality of factors such as sample sources, filter paper materials, preparation processes and the like, each metabolite in the dry blood spots is unevenly distributed, and the stability is difficult to control. For the investigation of uniformity and stability in dry blood spots, the analytical method using liquid chromatography-tandem mass spectrometry is currently the most reliable and effective means. At present, there is an amino acid mixed solution standard substance (GBW (E) 100062) in China, but the creatine, guanidinoacetic acid and amino acid component standard substances taking dry blood spots as sample matrixes are blank, so that development of the creatine, guanidinoacetic acid and amino acid component standard substances taking dry blood spots as sample matrixes is needed to be used for quality control and method evaluation in clinical laboratories.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a creatine, guanidinoacetic acid and amino acid component standard substance taking dried blood spots as a sample matrix, a preparation method thereof and a UPLC-ID-MS/MS detection method, so as to fill the blank of the creatine, guanidinoacetic acid and amino acid component standard substance in the dried blood spots in the aspect of detection in China, and play a role in metering and guaranteeing the accuracy and reliability of relevant metabolite clinical detection data.
The specific technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for preparing creatine, guanidinoacetic acid and amino acid component standard substances using dry blood spots as a sample matrix, comprising:
1) Mixing human whole blood with a protease inhibitor to obtain a whole blood solution;
2) Measuring the range of target components such as creatine, guanidinoacetic acid, amino acid and the like in the whole blood solution;
3) Respectively mixing creatine, guanidinoacetic acid and amino acid with a solvent to respectively obtain a creatine base body standard substance working solution, a guanidinoacetic acid base body standard substance working solution and an amino acid base body standard substance working solution with the concentration of 0.8-1.2 mg/L; then mixing the working solution of the matrix standard substance with the whole blood solution to obtain a labeled whole blood solution; wherein, 8-12 mu L of creatine and amino acid matrix standard substance working solution and 1-3 mu L of guanidinoacetic acid matrix standard substance working solution are added into each milliliter of whole blood;
4) And (3) transferring the marked whole blood solution to a dried blood spot collecting card at the temperature of 35-38 ℃ to prepare a dried blood spot sample.
The preparation method provided by the invention can ensure that the creatine, guanidinoacetic acid and amino acid component standard substances taking the dried blood spots as sample matrixes have good uniformity and stability, fills the blank that the creatine, guanidinoacetic acid and amino acid component standard substances in the dried blood spots are not yet found in China, and can completely trace the constant value result to the international SI unit system.
Preferably, the ratio of the protease inhibitor to the human whole blood in step 1) is 120 to 130mg of the protease inhibitor per 50mL of the whole blood, and the ratio can better inhibit the protease activity in the whole blood.
Preferably, the temperature is controlled to be 2-8 ℃ in the mixing process in the step 1) and the step 3), and the mixing time is 8-12 h.
Preferably, the solvent in the step 3) comprises 0.05 to 0.15mol/L of dilute hydrochloric acid or 0.05 to 0.15wt% of formic acid aqueous solution; more preferably, the solvent in step 3) is 0.1wt% formic acid in water.
Preferably, the diameter of the collecting ring of the dried blood spot collecting card in the step 4) is not smaller than 12mm. Further, the step 4) comprises the steps of placing the marked whole blood solution in a constant-temperature oscillation incubator, continuously transferring 50-55 mu L of the whole blood solution by using an automatic pipetting gun under the condition of heating and oscillation to prepare a dried blood spot sample, and uniformly spreading the whole blood in a dried blood spot collecting card.
Preferably, the preparation method further comprises sequentially drying, collecting and freezing and preserving the dried blood spot sample.
Further, the drying comprises the steps of placing the dried blood spot sample on an air drying rack for laying flat, and naturally drying in the shade for 8-12 h. In the invention, natural shade drying refers to spreading a dried blood spot collecting card on an air drying frame and airing under the condition that the indoor natural environment is not directly irradiated by sunlight.
Further, the collecting comprises the steps of vacuum split charging the dried blood spot sample by using aluminum foil composite bags, and adding 1 part of drying agent into each bag; preferably, the desiccant is a silica gel desiccant
Further, the cryopreservation includes preserving the collected dried blood spot sample at-70 ℃.
The invention further provides a creatine, guanidinoacetic acid and amino acid component standard substance taking the dried blood spots as a sample matrix, which is prepared by the preparation method.
The invention also provides a UPLC-ID-MS/MS detection method of creatine, guanidinoacetic acid and amino acid component standard substances by taking the dried blood spots as sample matrixes, which comprises the following steps:
1) Sample pretreatment;
mixing the dried blood spot sample with 90% methanol aqueous solution containing creatine, guanidinoacetic acid and amino acid isotope internal standard, swirling for 20-30 s, and vibrating at constant temperature of 23-27 ℃ and 900-1200 r/min for 20-25 min; transferring the supernatant, centrifuging for 10-15 min at 11000-13000 r/min, and taking the supernatant for heat drying; finally, re-dissolving the hot dry sample by using a mixed solution of 2wt% acetonitrile aqueous solution and 0.8wt% formic acid aqueous solution, and waiting for on-machine detection;
2) UPLC-ID-MS/MS detection.
Preferably, in the liquid chromatography condition, the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.8wt% of formic acid, and the mobile phase B is an acetonitrile solution containing 0.8% of formic acid; the elution condition is isocratic elution, and the flow rate is 0.2-0.3 mL/min.
The invention discovers that the liquid chromatography condition can obviously improve the detection accuracy of creatine, guanidinoacetic acid and amino acid component standard substances taking dry blood spots as sample matrixes; the method uses an isotope dilution liquid chromatography tandem mass spectrometry as a main reference measurement program, can effectively correct the recovery rate in sample preparation and instrument analysis, evaluates the uniformity, stability and uncertainty of creatine, guanyl acetic acid and amino acid component standard substances taking dry blood spots as sample matrixes according to national measurement technical specifications of quantitative value, uniformity and stability evaluation of standard substances (JJF 1343-2022), ensures that the creatine, guanyl acetic acid and amino acid component standard substances taking dry blood spots as sample matrixes have good uniformity and stability, fills up the blank that creatine, guanyl acetic acid and amino acid component standard substances in dry blood spots are not yet available in China, and ensures that a fixed value result can be completely traced to the international SI unit system, thereby being applicable to the requirements of marker diagnosis, laboratory detection method verification, quality control and the like of neonatal screening.
Further, the liquid chromatography conditions include: chromatographic column: waters HSS T3 column (100 mm. Times.2.1 mm. Times.1.7 μm); sample injection volume: 5. Mu.L; column temperature: 40 ℃.
Preferably, in the mass spectrometry conditions, the mass spectrometry conditions of creatine, guanidinoacetic acid and amino acids and isotopically characteristic ions thereof include:
further, the mass spectrometry conditions include:
ion source: electrospray ion source (ESI);
ionization mode: a positive ion mode;
scanning mode: multiple Reaction Monitoring (MRM) mode;
air curtain gas: 20psi;
ion source high pressure: 5500V;
ion source temperature: 550 ℃;
atomized Gas1:55psi;
auxiliary Gas2, 50psi;
according to the conditions of the liquid chromatography tandem mass spectrometry, the single sample injection time is 8.5min.
Based on the technical scheme, the invention has the beneficial effects that:
according to the creatine, guanidinoacetic acid and amino acid component standard substance taking the dried blood spots as the sample matrix, the creatine, guanidinoacetic acid and amino acid component standard substance taking the dried blood spots as the sample matrix is evaluated according to the national measurement technical specification (JJF 1343-2022) on the basis of the standard substance's fixed value, uniformity and stability', the creatine, guanidinoacetic acid and amino acid component standard substance taking the dried blood spots as the sample matrix has good uniformity and stability, the blank that the creatine, guanidinoacetic acid and amino acid component standard substance taking the dried blood spots as the sample matrix does not exist in China is filled, the fixed value result can be completely traced to the international SI unit system, and the method is suitable for marking diagnosis, laboratory detection method verification, quality control and other requirements of neonatal screening.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is an ion extraction chromatogram of creatine, guanidinoacetic acid, and amino acid standards;
FIG. 2 is an extraction ion chromatogram of creatine, guanidinoacetic acid and amino acid component standard substances prepared in example 1 and using dry blood spots as sample matrix;
FIG. 3 is an extraction ion chromatogram of a sample of creatine, guanidinoacetic acid, and amino acid component standard substances with dried blood spots as a sample matrix in the detection method of comparative example 1 of the present invention;
FIG. 4 is an extraction ion chromatogram of a sample of creatine, guanidinoacetic acid, and amino acid component standard substances with dried blood spots as a sample matrix in the detection method of comparative example 2 of the present invention;
fig. 5 is a flow chart of the preparation method of the creatine, guanidinoacetic acid and amino acid component standard substance using dry blood spots as a sample matrix.
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 with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, 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.
Unless otherwise indicated, all of the starting materials used in the examples were commercially available conventional starting materials, and the technical means used were conventional means well known to those skilled in the art.
Example 1
The embodiment provides a creatine, guanidinoacetic acid and amino acid component standard substance with dried blood spots as a sample matrix, and the preparation method comprises the following steps:
1) Screening a matrix standard substance raw material: human whole blood is collected from hospitals for screening of matrix standard substance raw materials;
2) Measuring the range of target components such as creatine, guanidinoacetic acid, amino acid and the like in the whole blood solution;
3) Adding a protease inhibitor: mixing whole blood with a protease inhibitor to obtain a whole blood solution; wherein 125mg protease inhibitor is added to each 50ml whole blood for inhibiting protease activity in the matrix;
4) Mixing evenly: placing the whole blood solution into a mixer for mixing uniformly for 12 hours at the ambient temperature of 4 ℃;
5) And (3) marking: creatine, guanidinoacetic acid and amino acid are respectively mixed with 0.1% formic acid aqueous solution to respectively obtain creatine base body standard substance working solution, guanidinoacetic acid base body standard substance working solution and amino acid base body standard substance working solution with the concentration of 1 mg/L; then mixing the working solution of the matrix standard substance with the whole blood solution to obtain a labeled whole blood solution; wherein, 10 mu L of creatine and amino acid matrix standard substance working solution and 2 mu L of guanidinoacetic acid matrix standard substance working solution are added into each milliliter of whole blood;
6) Homogenizing: placing the labeled whole blood solution into a mixer for mixing uniformly for 12 hours at the ambient temperature of 4 ℃;
7) And (5) subpackaging: placing the homogenized standard-added whole blood solution in the step (5) in a constant-temperature oscillation incubator, continuously transferring 50 mu L of the whole blood solution by using an automatic pipette under the conditions of 37 ℃ and oscillation speed of 1000r/min to prepare a dried blood spot sample, and uniformly spreading the whole blood in a dried blood spot collecting card with the diameter of a collecting ring of 12 mm;
8) And (3) drying: and (3) placing the dried blood spot sample in the step (6) on an air drying rack in a flat manner, and placing the air drying rack in a room. The dried blood spot collecting card is dried for 12 hours under the condition of natural environment without direct sunlight;
9) And (3) collecting: collecting the dried blood spot sample in the step (7) by using aluminum foil composite bags, vacuum packaging, wherein each bag contains 1 part of dried blood spot collecting card, adding 1 part of silica gel drying agent, and collecting 500 parts in total;
10 Cryopreservation of: and (3) storing the dried blood spot sample collected in the step (8) in a refrigerator at the temperature of-70 ℃.
Example 2
This example provides a UPLC-ID-MS/MS detection method of creatine, guanidinoacetic acid and amino acid composition standard substance prepared in example 1 using dried blood spot as sample matrix, comprising:
1) Sample pretreatment:
mixing the dried blood spot sample with 90% methanol aqueous solution containing creatine, guanidinoacetic acid and amino acid isotope internal standard, swirling for 20s, and oscillating for 10min at constant temperature of 25 ℃ and 1000 r/min; transferring supernatant, centrifuging at 12000r/min for 10min, and taking supernatant for heat drying; finally, re-dissolving the hot dry sample by using a mixed solution of 2wt% acetonitrile aqueous solution and 0.8wt% formic acid aqueous solution, and waiting for on-machine detection;
a piece of dried blood spot sample is taken and placed into a 2mL centrifuge tube, 90% methanol aqueous solution containing creatine, guanidinoacetic acid and amino acid isotope internal standard is added for extraction, vortex is carried out for 20s, and the dried blood spot sample is placed into a constant temperature oscillation incubator, and oscillation is carried out for 20min at the constant temperature of 1000r/min at the temperature of 25 ℃. The supernatant was removed and placed in a 2mL centrifuge tube, centrifuged at 12000r/min for 10min, 300. Mu.L of supernatant was removed, and dried by heat. The mixture of 2wt% acetonitrile in water and 0.8wt% formic acid in water was reconstituted to 1mL and checked on the machine.
2) UPLC-ID-MS/MS detection:
instrument: ultra-high performance liquid chromatography (AB Sciex, usa); API4000 mass spectrometer (AB Sciex, usa);
the liquid chromatography conditions included:
chromatographic column: waters HSS T3 column (100 mm. Times.2.1 mm. Times.1.7 μm);
mobile phase: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is an aqueous solution containing 0.8wt% of formic acid, and the mobile phase B is an acetonitrile solution containing 0.8wt% of formic acid;
flow rate: 0.25mL/min;
sample injection volume: 5. Mu.L;
column temperature: 40 ℃;
elution conditions: isocratic elution.
The mass spectrometry conditions included:
ion source: electrospray ion source (ESI);
ionization mode: a positive ion mode;
scanning mode: multiple Reaction Monitoring (MRM) mode;
air curtain gas: 20psi;
ion source high pressure: 5500V;
ion source temperature: 550 ℃;
atomized Gas1:55psi;
auxiliary Gas2:50psi.
Other mass spectral parameters are shown in table 1.
Table 1 mass spectrometry conditions for targets and their isotopically characteristic ions
According to the conditions of the liquid chromatography tandem mass spectrometry, the single sample injection time is 8.5min.
The extraction ion chromatogram of the standard substance of creatine, guanidinoacetic acid and amino acid is shown in figure 1, and the extraction ion chromatogram of the standard substance of creatine, guanidinoacetic acid and amino acid with dry blood spots as sample matrix is shown in figure 2.
In fig. 1 and 2, there are 12 chromatographic peaks including each compound and the isotope internal standard, and the peak outlet time of each compound and the isotope internal standard are consistent, and the sequence of the retention time is as follows: guanidinoacetic acid and its isotope internal standard, creatine and its isotope internal standard, valine and its isotope internal standard, isoleucine and its isotope internal standard, leucine and its isotope internal standard, phenylalanine and its isotope internal standard.
Comparative example 1
This comparative example is substantially identical to the detection method of example 2, except that: the mobile phase A is an aqueous solution containing 0.1wt% of formic acid, and the mobile phase B is an acetonitrile solution containing 0.1% of formic acid. The extraction ion chromatogram of the creatine, guanidinoacetic acid and amino acid component standard substance sample with dry blood spot as sample matrix is shown in figure 3.
As a result, when formic acid was added in an amount of 0.1wt% to the mobile phase, the time for the chromatographic peak to appear was long, and the overall detection time was prolonged.
Comparative example 2
This comparative example is substantially identical to the detection method of example 2, except that: the organic phase component used in mobile phase B was methanol.
The extraction ion chromatogram of the creatine, guanidinoacetic acid and amino acid component standard substance sample with dry blood spot as sample matrix is shown in figure 4.
The results show that when methanol is used as the organic phase for mobile phase B, the time to peak of the chromatographic peak is prolonged and the peak separation degree is poor. In contrast, the detection method of example 2 can shorten the detection time and obtain good peak shape with higher accuracy.
Test examples
1. The uniformity and stability of the creatine, guanidinoacetic acid and amino acid component standard substance samples prepared by the method taking the dry blood spots as the sample matrix are inspected, and the constant value and uncertainty are assessed;
1) And (3) uniformity test:
and (3) carrying out uniformity test on the standard substance according to the requirements of national measurement technical Specification (fixed value and uniformity and stability evaluation of standard substance) (JF 1343-2022). The study randomly extracted 11 packaging units according to the front, middle and rear of the whole packaging process, randomly extracted samples are numbered from 1 to 11, and 3 sub-samples are taken in parallel for detection from each randomly extracted unit. The result is statistically analyzed by an analysis of variance method, and is judged by comparing the F test value with the F critical value.
The results of the uniformity test of the present invention obtained by the above uniformity test are shown in Table 2.
TABLE 2 evaluation results of uniformity of creatine, guanidinoacetic acid and amino acid component standards prepared in example 1 and using dried blood spots as a sample matrix
According to the detection result, due to F<F a No significant difference between the groups was noted and the samples were uniform.
2) Stability investigation
According to the requirements of national measurement technical Specification (fixed value, uniformity and stability evaluation of Standard substance) (JF 1343-2022), the short-term stability of Standard substance is inspected and the characteristic value of Main evaluation Standard substance is changed or influenced by the change of ambient temperature in the transportation process, and the long-term stability is inspected and the characteristic value of Main evaluation Standard substance is changed or influenced by the change of ambient temperature in the storage process. The study used short term stability monitoring at days 0, 1, 3, 5, 7, respectively, by placing randomly drawn samples in an environment of 4deg.C, room temperature (25deg.C) and 50deg.C. Long-term stability randomly drawn samples were stored at-80 ℃ and monitored at months 0, 1, and 3, respectively. The short-term stability measurement method is the same as long-term stability monitoring, and trend analysis is adopted to carry out statistical analysis on the monitored data.
The stability was evaluated by measuring the characteristic value of the standard substance at different times, and drawing the relationship between the characteristic value and time with the time as the X axis and the characteristic value as the Y axis.
The stability assessment base model can be expressed as:
Y＝β 0 +β 1 X；
wherein:
β 0 ，β 1 -regression coefficients;
x-time;
y-the characteristic value of the standard substance candidate;
beta for stable standard substance 1 Is zero.
Assume that there are n observations of X, Y (X i ,Y i ) Y on each analog line i Can use formula Y i ＝β 0 +β 1 X i A representation;
wherein:
X i -the ith time point;
Y i -a characteristic value of the standard substance candidate corresponding to the ith time point.
The slope can be calculated using the following equation:and (5) estimating.
Wherein:
X i -the ith time point;
Y i -an i-th time point observation;
-average of all time points;
-average of all observations.
Intercept available formulaCalculation of
β 1 Standard deviation S (. Beta.) of (2) 1 ) From the formulaAnd (5) calculating.
Wherein: s in the formula, the standard deviation of each point on the straight line is represented by the formulaAnd (5) calculating.
Wherein:
X i -the ith time point;
Y i -an i-th time point observation;
β 1 ，β 0 -regression coefficients;
n-number of measurements;
beta-based 1 The following determination can be made using t-test: if |beta 1 |<t 0.95·n-2 ·s(β 1 ) The slope was not significant and no instability was observed.
According to formula u s Estimate stability-induced uncertainty u =s (β1) ·x s 。
Wherein:
u s uncertainty introduced by stability;
s(β 1 )——β 1 standard deviation of (2);
x-given shelf life;
the results of the short-term stability evaluation of the creatine, guanidinoacetic acid and amino acid component standard substances obtained using dried blood spots as a sample matrix are shown in tables 3 to 5.
TABLE 3 evaluation of short-term stability of creatine, guanidinoacetic acid and amino acid composition Standard substances with dried blood spots as sample matrix (4 ℃ C.)
TABLE 4 evaluation of short-term stability of creatine, guanidinoacetic acid and amino acid composition criteria Using dried blood spots as sample matrix (25 ℃ C.)
TABLE 5 evaluation of short-term stability of creatine, guanidinoacetic acid and amino acid composition criteria Using dried blood spots as sample matrix (50 ℃ C.)
The short-term stability results show that the standard substance value can be kept unchanged for 7 days under the conditions of the ambient temperature of 4 ℃, the room temperature (25 ℃) and 50 ℃, and the standard substance is recommended to have good stability.
The results of evaluation of the long-term stability of the obtained creatine, guanidinoacetic acid and amino acid component standard substances using dried blood spots as a sample matrix are shown in Table 6.
TABLE 6 evaluation of Long-term stability of creatine, guanidinoacetic acid and amino acid composition Standard substances Using dried blood spots as sample matrix (-80 ℃ C.)
The long-term stability result shows that the matrix standard substance of creatine and guanidinoacetic acid in the dry blood spots is stable and reliable in characteristic magnitude within 3 months under the preservation condition of-80 ℃.
3) Constant value
According to the requirements of national measurement technical Specification (standard substance determination and uniformity and stability assessment) (JF 1343-2022), the invention adopts an isotope-labeled-based ultra-high performance liquid chromatography tandem mass spectrometry to perform determination.
The results of the obtained creatine, guanidinoacetic acid and amino acid component standard substances with dried blood spots as sample matrix are shown in Table 7.
TABLE 7 determination of the values of creatine, guanidinoacetic acid and amino acid component criteria with dried blood spots as sample matrix
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4) Uncertainty analysis:
the uncertainty sources of the creatine, guanidinoacetic acid and amino acid component standard substances prepared by the invention and taking the dry blood spots as sample matrixes mainly comprise three parts: i.e. uncertainty of introduction of the standard substance constant value; uncertainty of introduction of uniformity of the standard substance; uncertainty introduced by stability of standard substance. The specific uncertainty components and specific calculation results are shown in Table 8.
TABLE 8 uncertainty results of creatine, guanidinoacetic acid, and amino acid composition criteria for dried blood spots as sample matrices of the invention
The relative standard uncertainty is obtained by multiplying the creatine, guanidinoacetic acid and amino acid component standard substance synthesis relative standard uncertainty of the dry blood spot serving as a sample matrix by the expansion factor k=2, and the obtained final fixed value result and the relative expansion uncertainty result are shown in table 9.
TABLE 9 determination of creatine, guanidinoacetic acid and amino acid composition criteria and relative expansion uncertainty results with dried blood spots as sample matrices
Conclusion: the final values and the expanded uncertainties of the creatine, guanidinoacetic acid and amino acid composition standards of the present invention using dried blood spots as the sample matrix are shown in table 9. The results show that the creatine, guanidinoacetic acid and amino acid component standard substances prepared by the method and using the dried blood spots as sample matrixes can provide standard substances consistent with the matrixes of the samples to be tested for analysis and detection of the creatine, guanidinoacetic acid and amino acid related components in the dried blood spots by institutions such as hospitals, clinical laboratories and the like, and can be used as reference substances for the internal quality control and analysis methods of detection laboratories to confirm and evaluate.
The preparation method of the creatine, guanidinoacetic acid and amino acid component standard substance using the dried blood spots as a sample matrix and the subsequent detection process flow chart are shown in figure 5.
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. A preparation method of creatine, guanidinoacetic acid and amino acid component standard substances with dried blood spots as sample matrixes is characterized by comprising the following steps:
1) Mixing human whole blood with a protease inhibitor to obtain a whole blood solution;
2) Determining the ranges of creatine, guanidinoacetic acid and amino acid components in the whole blood solution;
3) Respectively mixing creatine, guanidinoacetic acid and amino acid with a solvent to respectively obtain a creatine base body standard substance working solution, a guanidinoacetic acid base body standard substance working solution and an amino acid base body standard substance working solution with the concentration of 0.8-1.2 mg/L; then mixing the working solution of the matrix standard substance with the whole blood solution to obtain a labeled whole blood solution; wherein, 8-12 mu L of creatine and amino acid matrix standard substance working solution and 1-3 mu L of guanidinoacetic acid matrix standard substance working solution are added into each milliliter of whole blood;
4) And (3) transferring the marked whole blood solution to a dried blood spot collecting card at the temperature of 35-38 ℃ to prepare a dried blood spot sample.
2. The method for preparing the standard substance of creatine, guanidinoacetic acid and amino acid components using dried blood spots as a sample matrix according to claim 1, wherein the ratio of the protease inhibitor to the whole human blood in step 1) is 120-130 mg/50 mL whole blood.
3. The method for preparing the creatine, guanidinoacetic acid and amino acid component standard substance using dried blood spots as a sample matrix according to claim 1 or 2, wherein the temperature is controlled to be 2-8 ℃ in the mixing process in the step 1) and the step 3), and the mixing time is 8-12 h.
4. The method for preparing creatine, guanidinoacetic acid and amino acid composition standard substances using dried blood spots as sample matrix according to any one of claims 1 to 3, wherein the solvent in step 3) comprises 0.05-0.15 mol/L dilute hydrochloric acid or 0.05-0.15 wt% formic acid aqueous solution.
5. The method for preparing creatine, guanidinoacetic acid and amino acid component standard substances using dried blood spots as a sample matrix according to any one of claims 1 to 4, wherein the diameter of the collecting ring of the dried blood spot collecting card in the step 4) is not less than 12mm.
6. The method for preparing the creatine, guanidinoacetic acid and amino acid composition standard substance using dried blood spot as a sample matrix according to any one of claims 1 to 5, wherein the method further comprises sequentially drying, collecting and freeze-preserving the dried blood spot sample.
7. Creatine, guanidinoacetic acid and amino acid component standard substances with dried blood spots as sample matrix, characterized in that they are prepared by the preparation method according to any one of claims 1 to 6.
8. The UPLC-ID-MS/MS detection method of creatine, guanidinoacetic acid, and amino acid component standard substance using dried blood spot as a sample matrix according to claim 7, comprising:
1) Sample pretreatment;
mixing the dried blood spot sample with 90% methanol aqueous solution containing creatine, guanidinoacetic acid and amino acid isotope internal standard, swirling for 20-30 s, and vibrating at constant temperature of 23-27 ℃ and 900-1200 r/min for 20-25 min; transferring the supernatant, centrifuging for 10-15 min at 11000-13000 r/min, and taking the supernatant for heat drying; finally, re-dissolving the hot dry sample by using a mixed solution of 2wt% acetonitrile aqueous solution and 0.8wt% formic acid aqueous solution, and waiting for on-machine detection;
2) UPLC-ID-MS/MS detection.
9. The method for detecting the UPLC-ID-MS/MS using dry blood spots as a sample matrix according to claim 8, wherein the mobile phase comprises a mobile phase A and a mobile phase B in the liquid chromatography condition, wherein the mobile phase A is an aqueous solution containing 0.8wt% of formic acid, and the mobile phase B is an acetonitrile solution containing 0.8% of formic acid; the elution condition is isocratic elution, and the flow rate is 0.2-0.3 mL/min.
10. The method for detecting the UPLC-ID-MS/MS of the standard substances of the creatine, the guanidinoacetic acid and the amino acid components, which take the dried blood spots as sample matrixes, according to claim 8 or 9, wherein the mass spectrometry conditions of creatine, guanidinoacetic acid and amino acid and isotope characteristic ions thereof comprise:
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