CN111650314A - Kit and method for detecting genetic metabolic disease related substances by non-derivation method tandem mass spectrometry - Google Patents
Kit and method for detecting genetic metabolic disease related substances by non-derivation method tandem mass spectrometry Download PDFInfo
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The application discloses a kit and a method for detecting substances related to genetic metabolic diseases by non-derivation tandem mass spectrometry. The kit comprises a mixed isotope internal standard substance and a quality control substance; the mixed isotope internal standard comprises isotope standard of related substances of genetic metabolic diseases of amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine; the quality control product is a whole blood sample containing a quality control analyte of the substance associated with the genetic metabolic disease at a known concentration. The kit can detect amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine and various genetic metabolic disease related substances, and increases the types of detection of the genetic metabolic disease related substances, so that screening or auxiliary screening of more metabolic diseases in one experiment can be realized, and the using requirement of screening of the neonatal genetic metabolic disease is better met.
Description
Technical Field
The application relates to the field of genetic metabolic disease detection, in particular to a kit and a method for detecting genetic metabolic disease related substances by non-derivatization tandem mass spectrometry.
Background
Inherited Metabolic Diseases (IMDs), also known as Inborn Errors of Metabolism (IEMs), refer to a group of diseases that cause biochemical metabolic disorders of the body due to enzyme deficiency, abnormal cell membrane function or receptor deficiency caused by gene mutation, resulting in accumulation of intermediate or bypass metabolites, or end metabolite deficiency, leading to a series of clinical symptoms, and are frequently developed in childhood, with progressive exacerbations, irreversible nervous system damage, or even death. The development of neonatal disease screening can provide good opportunities for early detection of congenital genetic metabolic diseases, early preventive intervention and slowing down of disease progression. Early neonatal screening for inherited metabolic diseases has been popularized in the united states in the 60 s, and neonatal screening in 7 cities in the last 90 s of the century in China has been developed nationwide at present. Through the technical development and improvement for many years, 48 examination items are achieved, including three major types of genetic metabolic diseases, namely organic acidemia type genetic metabolic diseases, fatty acid oxidation deficiency type genetic metabolic diseases and amino acid metabolism disorder type genetic metabolic diseases. Newborn screening has profound significance in improving population quality, reducing and reducing children's mental retardation.
The infant health-care law of the people's republic of China issued in 1994 proposes ' gradually developing neonatal disease screening ' for the first time, so that the development of neonatal disease screening has a fundamental legal guarantee. The 2009 recorded "neonatal disease screening management approach", which clearly stipulates the responsibilities of various levels of health administration departments, neonatal disease screening centers and medical institutions. In order to implement the method for screening and managing neonatal diseases and further standardize the work of screening the neonatal diseases and practically improve the screening quality, the Ministry of health revises the technical specification of screening the neonatal diseases published in 2004 to form the technical specification of screening the neonatal diseases (2010 version). From 1998, the clinical laboratory testing center of Ministry of health performed comparative tests on the capability of newborn disease screening laboratories for 18 newborn disease screening centers in 16 provinces and cities in China, and quality control was performed. In 2012, screening of newborn inherited metabolic diseases has been developed in 30 provinces, cities and autonomous regions. According to the data of the national maternal and child hygiene monitoring office in 2013, the neonatal disease screening center 211 is available in the whole country at present.
Currently, internationally, neonatal screening for inherited metabolic diseases has been developed as a Tandem Mass Spectrometry (TMS) -centered screening. TMS is adopted to detect the levels of dozens of amino acids, free carnitine and acyl carnitine in the neonatal filter paper dried blood, and screen a plurality of inherited metabolic diseases such as amino acid metabolic disturbance, organic acidemia, fatty acid oxidative metabolic disturbance and the like, and the TMS is widely applied to the prevention and treatment of neonatal birth defects. By using the tandem mass spectrometry technology with hypersensitivity, high specificity, high selectivity and rapid detection, dozens of metabolites can be analyzed in 2min for one specimen, namely dozens of genetic metabolic diseases can be detected simultaneously, and the requirement of detecting various diseases by one experiment is met.
In order to standardize each link of the laboratory technology for screening the neonatal diseases by using the tandem mass spectrometry in China, the expert of the neonatal disease tandem mass spectrometry screening technical expert committee organization in the neonatal disease screening laboratory of the clinical examination center of Ministry of health refers to the latest domestic and overseas documents and guidelines, and establishes 'the consensus of the neonatal disease tandem mass spectrometry screening technical experts' for supervising and urging each laboratory to strengthen the quality management and providing standardized screening services so as to promote the standardized application of the tandem mass spectrometry technology in the neonatal disease screening.
Currently, there are two methods for sample pretreatment for TMS detection of amino acids, free carnitine and acyl carnitine, namely a derivatization method and a non-derivatization method. The non-derivatization method does not need to use n-butyl alcohol hydrochloride to derivatize the substance to be detected, so that potential negative ions are eliminated, methanol is used for extraction and then the detection is directly carried out on a computer, the experimental process is simplified, the experimental time is saved, and the method is particularly suitable for the screening work of large-scale samples. According to the statistics of the Centers for Disease Control and Prevention (CDC), in the near 1100 laboratories participating in the indoor quality evaluation of acylcarnitine detection held by the laboratories, the laboratories using non-derivatization pretreatment showed an increasing trend year by year compared to the laboratories using derivatization pretreatment. Furthermore, there are related documents that the tandem mass spectrometry derivation method and the non-derivation method have no influence on clinical judgment of diseases. Therefore, the non-derivatization pretreatment method is the development direction of a newborn genetic metabolic disease screening laboratory.
In the research aspect of detecting related substances of genetic metabolic diseases by non-derivatization mass spectrometry, the detection indexes of the detection kit comprise 10 amino acids and 13 carnitine in the patent application CN 108152380' detection kit for multiple amino acids and carnitine by the tandem mass spectrometry of national development of International Biotechnology gardens of Shandong. In patent application CN203965385 of Fenghua bioengineering Co., Ltd, Guangzhou, an amino acid and carnitine and acylcarnitine non-derivatization tandem mass spectrometry detection kit, the detection indexes comprise 11 amino acids and 31 carnitine. The CFDA has completed the registration of medical devices, and there is internationally the "underivatized multiple amino acid, carnitine and succinylacetone assay kit (tandem mass spectrometry)" developed by PerkinElmer, usa; the method is characterized in that a succinylacetone and non-derivatization multiple amino acid and carnitine determination kit (tandem mass spectrometry) developed by Fenghua bioengineering limited company in Guangzhou city is provided at home; "succinylacetone and amino acid, carnitine assay kit (tandem mass spectrometry)" developed by Jinan Yingsheng Biotechnology Limited.
The detection method and the detection kit, both international and domestic, have the following defects: 1. only partial amino acid, carnitine and succinylacetone can be detected at one time, and the types of diseases which can be screened are limited; 2. for inherited genetic metabolic disorders: x-linked adrenoleukodystrophy (X-ALD), Creatine Deficiency Syndrome (CDS) -GAMT deficiency and neonatal severe combined immunodeficiency disease (ADA-SCID), the existing kit can not be detected simultaneously, needs to sample and detect independently, and needs to take blood of a person to be detected twice; however, the physical and psychological effects on the subject can be caused by taking a blood sample for a plurality of times.
Moreover, with the increase of knowledge on hereditary metabolic diseases, the types of screened diseases are increased continuously, and detection indexes are required to be increased synchronously, so that the screening of the types of diseases can be expanded, and the screening efficiency and the screening specificity are improved. Therefore, there is a need to develop a new detection scheme capable of rapidly screening a wider variety of genetic metabolic disease-related substances to meet the use requirements of screening a wider variety of genetic metabolic diseases.
Disclosure of Invention
The application aims to provide a novel kit and a novel method for detecting substances related to genetic metabolic diseases by non-derivation tandem mass spectrometry.
The following technical scheme is adopted in the application:
one aspect of the application discloses a kit for detecting substances related to genetic metabolic diseases by non-derivation method tandem mass spectrometry, which comprises a mixed isotope internal standard substance and a quality control substance; wherein the mixed isotope internal standard comprises isotope standard of related substances of genetic metabolic diseases of amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine; the quality control product is a whole blood sample containing a quality control analyte of the substance associated with the genetic metabolic disease at a known concentration.
The kit can realize the simultaneous detection of amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine and various genetic metabolic disease related substances, and can quickly screen more various genetic metabolic disease related substances, thereby meeting the use requirements of more various genetic metabolic disease screening or auxiliary screening.
Preferably, in the kit of the present application, the amino acids include 14 amino acids, the carnitine includes 36 carnitines, the ketone is succinylacetone, the lysophosphatidylcholine includes 4 lysophosphatidylcholine, and 59 genetic metabolic disease-related substances in total; 59 genetic metabolic disease-related substances, the isotope standard substance and the quality control analyte are shown in Table 1,
TABLE 1
Wherein arginine and argininosuccinic acid adopt the same isotope standard substance and quality control substance, arginine is adopted as quality control substance for both, and isotope standard substances for both are13C6-Arg; leucine, isoleucine and hydroxyproline, wherein the leucine is adopted as a quality control product, and the leucine is adopted2H3-Leu as an isotope standard; malonyl carnitine, 3-hydroxy-butyryl carnitine and butyryl carnitine, all of which adopt butyryl carnitine as quality control substances, all of which adopt butyryl carnitine2H3-C4 as an isotope standard; succinyl carnitine, 3-hydroxy-isovaleryl carnitine, isovaleryl carnitine and prenyl carnitine, wherein isovaleryl carnitine is adopted as a quality control product, and isovaleryl carnitine is adopted2H9-C5 as an isotope standard; glutaryl carnitine, 3-hydroxy-caproyl carnitine and adipoyl carnitine, wherein the adipoyl carnitine is adopted as a quality control product, and the adipoyl carnitine is adopted2H9-C5DC as an isotope standard; octanoyl carnitine and octenoyl carnitine, which are used as quality control substances, are used2H3-C8 as an isotope standard; decanoyl carnitine, deceneAcyl carnitine and decadienyl carnitine, namely decanoyl carnitine is adopted as a quality control product, and decanoyl carnitine is adopted as a quality control product2H3-C10 as an isotope standard; the dodecenoylcarnitines and dodecenoylcarnitines both adopt dodecenoylcarnitine as quality control substances and both adopt dodecenoylcarnitine as quality control substances2H3-C12 as an isotope standard; tetradecylcarnitine, and 3-hydroxy-tetradecylcarnitine, which all employ tetradecylcarnitine as a quality control material, are provided2H3-C14 as an isotope standard; the hexadecanoyl carnitine, 3-hydroxy-hexadecanoyl carnitine and 3-hydroxy-hexadecanoyl carnitine all adopt hexadecanoyl carnitine as quality control substances2H3-C16 as an isotope standard; octadecanoylcarnitines, octadecenoylcarnitines, octadecadienylcarnitines, 3-hydroxy-octadecanoylcarnitines, 3-hydroxy-octadecenoylcarnitines and 3-hydroxy-octadecadienylcarnitines, which all adopt octadecanoylcarnitines as quality control substances, and octadecanoylcarnitines as quality control substances2H3-C18 as an isotope standard; the eicosanoic carnitine, docosanoic carnitine, tetracosanoic carnitine and hexacosanoic carnitine are all adopted as quality control products and are all adopted2H3-C26 as an isotope standard; the twenty-six carbon lysophospholipids are adopted as quality control products and are adopted2H4-C26:0-LPC as isotope standard.
It should be noted that the kit of the present application can simultaneously analyze and detect 59 genetic metabolic disease-related substances, and the correspondence between partial metabolic diseases and detection indexes is shown in table 2.
TABLE 2 correspondence between partial metabolic diseases and detection indices
Preferably, in the kit of the application, the whole blood sample of the quality control product contains enzyme inhibitors, and the enzyme inhibitors are adenosine deaminase inhibitors and arginase inhibitors;
preferably, the dosage of the adenosine deaminase inhibitor is 50-60 mu mol/L, and the dosage of the arginase inhibitor is 95-100 mu mol/L.
In the present application, the quality control product contains 33 quality control standards, and in order to ensure the stability of all quality control standards, an enzyme inhibitor is preferably added to the quality control product; for example, the whole blood contains adenosine deaminase, ADO and D-ADO can be degraded, so that the detection of quality control products ADO and D-ADO prepared in each batch is unstable, and the adenosine deaminase inhibitor can be added to effectively avoid the degradation of ADO and D-ADO, so that the stability of the quality control products is improved. The same whole blood contains arginase, which can degrade Arg, so that the detection of Arg in the quality control product is unstable.
Preferably, the quality control product comprises a low-concentration whole blood sample, a medium-concentration whole blood sample and a high-concentration whole blood sample; wherein the concentrations of the quality control analytes in the low-concentration whole blood sample, the medium-concentration whole blood sample and the high-concentration whole blood sample are shown in Table 3,
TABLE 3
It should be noted that the low-concentration whole blood sample, the medium-concentration whole blood sample, and the high-concentration whole blood sample shown in table 3 are only quality control materials specifically used in one implementation manner of the present application, and may be adjusted based on experimental requirements, for example, the concentration of the quality control analyte is increased or decreased according to the experimental requirements, and is not limited in particular.
Preferably, the whole blood sample of the quality control product of the present application is in the form of a dried plaque tablet.
Preferably, the kit of the present application further comprises a mobile phase solution, and the mobile phase solution is composed of methanol, oxalic acid and water.
Preferably, the concentration of the methanol is 75-80%, and the concentration of the oxalic acid is 1-2 mmol/L.
It should be noted that, in order to detect 59 kinds of targets simultaneously and stably, the present application optimizes and improves the mobile phase solution, and finally finds that the effect of the mobile phase solution composed of methanol, oxalic acid and water is the best; in particular, in one embodiment of the present invention, a mobile phase solution having a methanol concentration of 75% and an oxalic acid concentration of 1.5mmol/L is preferably used.
Preferably, the kit of the present application further comprises an extraction liquid.
Preferably, the extract is 85% to 90% methanol.
Preferably, the kit of the present application further comprises a succinylacetone treatment solution.
The succinylacetone treatment solution is used only when succinylacetone needs to be detected, and if succinylacetone is not detected, the succinylacetone treatment solution does not need to be added.
Preferably, the kit of the present application further comprises a 96-well microplate and a corresponding aluminum foil sealing film, and instructions for operation.
In the kit of the present application, the mobile phase solution may be configured according to the description of the present application, the extract and the succinylacetone treatment solution are also conventional reagents for non-derivatization tandem mass spectrometry, and the 96-well microplate and the aluminum foil sealing film may be purchased separately, so that these may be selectively added to the kit of the present application as required.
In another aspect of the application, the use of the kit of the application for the detection of substances associated with genetic metabolic diseases for non-diagnostic therapeutic purposes is disclosed.
It should be noted that the kit of the present application is mainly used for detecting the substances related to the genetic metabolic diseases, although it can be used for genetic metabolic disease screening or auxiliary screening; however, their use for non-diagnostic therapeutic purposes is not excluded.
The application further discloses a method for detecting genetic metabolic disease related substances by non-derivation tandem mass spectrometry for non-diagnosis and treatment purposes, which comprises the step of detecting a sample to be detected by adopting the kit.
Preferably, in the method of the present application, the detecting the sample to be detected includes performing liquid phase gradient elution with a mobile phase solution according to the conditions in table 4;
TABLE 4
| Time (minutes) | Flow rate (mL/min) |
| initial | 0.1 |
| 0.10 | 0.05 |
| 0.40 | 0.3 |
| 0.50 | 0.8 |
| 0.80 | 0.8 |
| 0.85 | 0.1 |
| 1.20 | 0.1 |
。
It should be noted that, in the kit and the detection method of the present application, in order to simultaneously and stably detect 59 targets, not only the mobile phase solution is optimized and improved, but also the liquid phase elution gradient is studied and improved, thereby ensuring the stability and accuracy of the detection of the 59 targets.
The beneficial effect of this application lies in:
the kit can detect amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine and various genetic metabolic disease related substances, and increases the types of detection of the genetic metabolic disease related substances, so that screening or auxiliary screening of more metabolic diseases in one experiment can be realized, and the using requirement of screening of the neonatal genetic metabolic disease is better met.
Drawings
FIG. 1 is a graph showing the results of detection of lysophospholipid C20:0-LPC using a conventional mobile phase in the examples of the present application;
FIG. 2 is a graph showing the results of detection of arginine using a conventional mobile phase in the examples of the present application;
FIG. 3 is a graph showing the results of detection of carnitine C0 using a conventional mobile phase in the present example;
FIG. 4 is a graph showing the results of detection of lysophospholipid C20:0-LPC using modified mobile phase solution in the examples of the present application;
FIG. 5 is a graph showing the results of arginine detection using a modified mobile phase solution in the examples of the present application;
FIG. 6 is a graph showing the results of carnitine C0 assay using a modified mobile phase solution according to the present example;
FIG. 7 is a graph showing the change in the results of ADO and D-ADO detection of a spot at normal temperature in the example of the present application;
FIG. 8 is a graph showing the change in the results of ADO and D-ADO detection of spots at low temperatures in the examples of the present application.
Detailed Description
The existing kit and method for detecting the genetic metabolic disease related substances by non-derivation tandem mass spectrometry can detect various genetic metabolic disease related substances, but the number of types which can be detected is limited; moreover, with the progress and development of genetic metabolism research, the types of diseases and related substances to be screened are increasing; therefore, the existing kit and method cannot meet the use requirement.
For the above reasons, the present application has developed a novel kit and method capable of simultaneously detecting 59 genetic metabolic disease-related substances. Based on the kit of the application, 14 amino acids, 36 carnitines, 1 ketone, 4 lysophosphatidylcholine, 2 acids and 2 adenosine can be detected, which are detailed in table 1; thereby realizing the screening of more than 50 metabolic diseases, and part of the metabolic diseases are shown in the table 2.
It is stated that although tandem mass spectrometry detection has a high throughput, not all substances can be detected in one test, the present application creatively combines isotope standards of 14 amino acids, 36 carnitines, 1 ketone, 4 lysophosphatidylcholine, 2 acids, and 2 adenosines with a quality control standard to realize the detection of 59 genetic metabolic disease-related substances, and the quality control product, the detection method, and the conditions are improved and optimized, so that the 59 genetic metabolic disease-related substances can be detected simultaneously and stably.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Examples
Kit for detecting genetic metabolic disease related substances by non-derivation method tandem mass spectrometry
The kit of this example consists of two major parts, namely, kit A, which contains the non-liquid part, and kit B, which contains the solution, as shown in Table 5.
TABLE 5 kit Components
Wherein the mixed isotope internal standard comprises 13 amino acids, 14 carnitine, 1 ketone, 1 lysophosphatidylcholine, 2 acids and 2 adenosine, and the total number of the 33 isotope internal standard products; the quality control product is a dried blood spot sample containing 33 quality control analytes with known concentration; the 59 genetic metabolic disease-related substances, isotope standards and quality control analytes are shown in Table 1.
Table 159 genetic metabolic disease related substances and isotope standard substances and quality control substances thereof
The quality control product in this example was a dried plaque piece of whole blood including a low concentration whole blood sample (L-QC), a medium concentration whole blood sample (M-QC), and a high concentration whole blood sample (H-QC); the concentrations of the quality control analytes in the low-concentration whole blood sample, the medium-concentration whole blood sample, and the high-concentration whole blood sample are shown in table 3.
TABLE 3 concentration of each control analyte in the control
The mobile phase solution of this example consisted of methanol, oxalic acid and water, wherein the concentration of methanol was 75% and the concentration of oxalic acid was 1.5 mmol/L.
The extract in this example was 90% methanol. The succinylacetone treatment solution is a succinylacetone treatment solution which is conventionally used. The 96-well micro-porous plate is a 350-mu L V-shaped bottom 96-well plate.
Second, kit using method
The specific procedures and conditions for detecting the substances related to the genetic metabolic diseases by using the kit of the present example are as follows:
1. preparation of mixed isotope internal standard solution: adding 0.7mL of extract into a mixed isotope internal standard bottle, and thoroughly mixing the liquid until the liquid is completely dissolved, wherein the time is usually 60-70min, and if necessary, ultrasonically promoting the solution.
2. Preparing an extraction working solution: mixing the mixed isotope internal standard solution, the extraction liquid and the succinylacetone treatment liquid according to the volume ratio of 1:2.5:96.5 to prepare extraction working liquid.
3. Sample pretreatment: using a 3mm punch, 1 blood spot was punched on the blood spot and placed in 1 well of a 96-well plate. Adding 100 μ L of the extractive solution, sealing membrane, mixing at 750rpm and 37 deg.C for 30min, and centrifuging. Take 75. mu.L of supernatant into a new 96-well plate and seal the membrane.
If succinylacetone is detected and the sample is allowed to stand at room temperature for 2h to ensure adequate derivatization of succinylacetone, this step can be omitted if not detected. And (4) detecting and analyzing the sample supernatant by using a tandem mass spectrum.
4. Shimadzu LCMS-8040CL liquid chromatography mass spectrometer
The analysis mode is as follows: FIA flow injection mode
(1) Chromatographic conditions
Temperature of the sample cell: 8 deg.C
Sample introduction volume: 1.0 μ L
The liquid phase gradient elution conditions are shown in table 4.
TABLE 4 gradient elution
(2) Conditions of Mass Spectrometry
The mass spectrometric detection conditions of this example are shown in table 6.
TABLE 6 Mass Spectrometry parameters
| MS method | MRM | Ion source | ESI+ |
| Velocity of atomizing gas | 3L/min | DL temperature | 280℃ |
| Dry air flow rate | 18L/min | Temperature of heating block | 350℃ |
| Interface voltage | 4.5kV |
Test 1 optimization of mobile phase solution
The LC-MS/MS detection method for screening newborn babies generally adopts a column flow method at present, chromatographic column separation is not used, 59 genetic metabolic disease related substances detected in the method have large property difference, and stable and symmetrical chromatographic peaks of all indexes are difficult to obtain simultaneously by using the conventional mobile phase such as methanol/acetonitrile, water and formic acid. Therefore, the present example optimizes the mobile phase, creatively adopts the mobile phase consisting of methanol, oxalic acid and water, and optimizes the dosage of methanol and oxalic acid. The concentration of oxalic acid is 1.0mmol/L, and the response intensity and the peak shape of each index under the conditions of different methanol proportions (75%, 80%, 85% and 90%) are compared; and the concentration of methanol was kept at 75%, and the mobile phase solution in this example was finally determined to be an aqueous solution containing 75% methanol and 1.5mM oxalic acid by comparing the response intensity and peak shape of each index under the conditions of different oxalic acid concentrations (0.5mmol/L, 1.0mmol/L, 1.5mmol/L, 2.0 mmol/L).
In this case, a conventional mobile phase of 4:4:2 water in methanol and acetonitrile, and 0.1% formic acid, respectively, was used; compared with the optimized mobile phase solution in the embodiment, namely an aqueous solution containing 75% of methanol and 1.5mM of oxalic acid, the test materials, the method and the conditions are the same as those of the kit for detecting the substances related to the genetic metabolic diseases by the one-derivation and non-derivation tandem mass spectrometry and the method for using the two-step kit, and the only difference is that the mobile phase solution used by the two-step kit is different.
The detection results of the conventional mobile phase show that the conventional mobile phase is difficult to obtain chromatographic peaks with stable and symmetrical indexes, the peak shapes of partial indexes such as lysophospholipid, Arg and carnitine are poor, the detection is unstable, and partial results are shown in figures 1 to 3. FIG. 1 is a graph showing the results of detection of lysophospholipid C20:0-LPC using a conventional mobile phase, FIG. 2 is a graph showing the results of detection of arginine using a conventional mobile phase, and FIG. 3 is a graph showing the results of detection of carnitine C0 using a conventional mobile phase.
The results of the detection using the mobile phase solution optimized and modified in this example show that the mobile phase solution of this example can stably detect all analytes simultaneously and obtain chromatographic peaks with better peak shapes, and some of the results are shown in fig. 4 to 6, fig. 4 is a graph of the results of the detection using lysophospholipid C20:0-LPC in the modified mobile phase solution, fig. 5 is a graph of the results of the detection using arginine in the modified mobile phase solution, and fig. 6 is a graph of the results of the detection using carnitine C0 in the modified mobile phase solution.
Experiment 2 optimization of quality control
The quality control product process is a major difficulty of the existing kit product, and the newly added indexes ADO and D-ADO in the embodiment are directly added into the whole blood and can be degraded by adenosine deaminase in the whole blood, so that the detection of the quality control products ADO and D-ADO prepared in each batch is unstable. In order to ensure that ADO and D-ADO added into a quality control product can stably exist, the action effects of three adenosine deaminase inhibitors, namely EHNA hydrochloride, Deazadenosine and pentastatin, are investigated by evaluating different enzyme inhibitors and addition amounts, and the action effect of the EHNA hydrochloride is found to be optimal; then, the action and effect of different EHNA hydrochlorides at 25. mu. mol/L, 50. mu. mol/L, 75. mu. mol/L and 100. mu. mol/L were examined, and finally it was determined that the enzyme inhibitor used was EHNA hydrochlorides at 50. mu. mol/L, and ADO and D-ADO could be stably present and detected.
The example also considers the spot spots and the change of the ADO and D-ADO detection results under different time and temperature conditions. Dividing the prepared whole blood into two groups, placing one group of the whole blood into spots at room temperature, placing the other group of the whole blood into spots on ice at low temperature, placing the two groups of the whole blood into spots at three time points of 0h, 1h and 2h respectively, after the blood sheet is dried in the air, performing pretreatment, and performing on-machine detection, wherein the results are shown in fig. 7 and 8, fig. 7 is a detection result graph of the spots at the normal temperature of about 25 ℃, and fig. 8 is a detection result graph of the spots at the low temperature of about 4 ℃ on ice; in fig. 7 and 8, the results of 0h spotting were 100%. The results of fig. 7 and 8 show that, in spotting at normal temperature, ADO, D-ADO show a tendency of gradual degradation, particularly ADO, as the spotting time interval increases; however, under the condition of low temperature, spots are spotted within 2 hours, and the spots have no obvious influence on ADO and D-ADO.
The quality control product can be used for evaluating the stability of the method, the CV percent is less than or equal to 20 percent to indicate that the method is stable, and the data acquisition is reliable. CV% values of quality control measurement results for the low concentration whole blood sample (L-QC), the medium concentration whole blood sample (M-QC), and the high concentration whole blood sample (H-QC) are shown in Table 7.
TABLE 7 stability of quality control tests (CV% (n 18))
The results in table 7 show that the quality control product of the present example can meet the use requirements, and the method of the present example is stable and the data acquisition is reliable.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Claims (10)
1. A kit for detecting substances related to genetic metabolic diseases by non-derivation method tandem mass spectrometry is characterized in that: comprises a mixed isotope internal standard substance and a quality control substance;
the mixed isotope internal standard comprises isotope standard substances of related substances of genetic metabolic diseases of amino acids, carnitines, ketones, lysophosphatidylcholine, guanidinoacetic acid, creatine, adenosine and deoxyadenosine;
the quality control product is a whole blood sample containing a quality control analyte of the genetic metabolic disease-related substance with a known concentration.
2. The kit of claim 1, wherein: the amino acids comprise 14 amino acids, the carnitines comprise 36 carnitines, the ketones are succinylacetone, the lysophosphatidylcholine comprise 4 lysophosphatidylcholine, and 59 genetic metabolic disease-related substances in total;
the 59 genetic metabolic disease related substances, the isotope standard substances and the quality control analytes are shown in Table 1,
TABLE 1
3. The kit of claim 1, wherein: the whole blood sample of the quality control product contains enzyme inhibitors, and the enzyme inhibitors are adenosine deaminase inhibitors and arginase inhibitors;
preferably, the dosage of the adenosine deaminase inhibitor is 50-60 mu mol/L, and the dosage of the arginase inhibitor is 95-100 mu mol/L.
4. The kit of claim 1, wherein: the quality control product comprises a low-concentration whole blood sample, a medium-concentration whole blood sample and a high-concentration whole blood sample;
the concentrations of the quality control analytes in the low concentration whole blood sample, the medium concentration whole blood sample and the high concentration whole blood sample are shown in Table 3,
TABLE 3
Preferably, the whole blood sample of the quality control product is in the form of a dried blood spot tablet.
5. The kit according to any one of claims 1 to 4, characterized in that: the water-soluble organic solvent is characterized by also comprising a mobile phase solution, wherein the mobile phase solution consists of methanol, oxalic acid and water;
preferably, the concentration of the methanol is 75-80%, and the concentration of the oxalic acid is 1-2 mmol/L.
6. The kit according to any one of claims 1 to 4, characterized in that: also comprises an extraction liquid;
preferably, the extract is 85-90% methanol.
Preferably, the method further comprises a succinylacetone treatment solution.
7. The kit according to any one of claims 1 to 4, characterized in that: also comprises a 96-hole microporous plate and a corresponding aluminum foil sealing film, and an operation instruction.
8. Use of a kit according to any one of claims 1 to 7 for the detection of substances associated with genetic metabolic diseases for non-diagnostic therapeutic purposes.
9. A method for detecting substances related to genetic metabolic diseases by non-derivation method tandem mass spectrometry for non-diagnosis and treatment purposes is characterized in that: comprising the use of a kit according to any one of claims 1 to 7 for the detection of a sample to be tested.
10. The method of claim 9, wherein: the detection of the sample to be detected comprises the steps of carrying out liquid phase gradient elution by adopting a mobile phase solution according to the conditions in the table 4;
TABLE 4
。
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| CN117074589A (en) * | 2023-08-23 | 2023-11-17 | 山东英盛生物技术有限公司 | Method for detecting succinylacetone, amino acid and carnitine in maternal amniotic fluid |
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