CN113848264A

CN113848264A - Method for monitoring collection quality of dried blood slice sample

Info

Publication number: CN113848264A
Application number: CN202111073150.1A
Authority: CN
Inventors: 李琰; 彭兴
Original assignee: Shenzhen Maitu Precision Technology Co ltd
Current assignee: Shenzhen Maitu Precision Technology Co ltd
Priority date: 2021-09-14
Filing date: 2021-09-14
Publication date: 2021-12-28

Abstract

The invention discloses a monitoring method for the collection quality of a dried blood slice sample, and belongs to the technical field of metabonomics dried blood slice sample quality control. Taking the relative content of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the dried blood slice sample as a quality inspection index, and when the relative content of glycochenodeoxycholic acid is less than X1 represented by a formula (I) and/or the relative content of triglyceride TAG56:4 is less than X2 represented by a formula (II), the collection quality of the dried blood slice sample meets the requirement. The invention also provides an evaluation method of the metabonomics detection result of the dry blood slice sample, which can quickly and accurately judge and monitor the collection quality of the metabonomics dry blood slice sample and ensure the accuracy and reliability of the metabonomics detection result of the dry blood slice sample.

Description

Method for monitoring collection quality of dried blood slice sample

Technical Field

The invention belongs to the technical field of dry blood slice sample collection quality control in blood (especially finger tip blood) sample collection, and particularly relates to application of Glycochenodeoxycholic acid (Glycochenodeoxycholic acid) and/or triglyceride TAG56:4 serving as a quality test indicator for judging the dry blood slice sample collection quality, a monitoring method for the dry blood slice sample collection quality and an evaluation method for the dry blood slice sample metabolism detection result, wherein the monitoring method is used for judging by using the relative content of the Glycochenodeoxycholic acid and/or the triglyceride TAG56:4 in the dry blood slice sample as an index.

Background

Blood is the most commonly used biological sample in research, diagnosis, health and drug monitoring, and is also a common sample in metabolomics research, and the detection of metabolites therein has become an effective means for diagnosing diseases, evaluating health level, and the like. With the expansion of various metabonomics application scenes such as accurate medical treatment, personalized treatment, health management and the like, application requirements of various blood sample collection modes are provided, and the application requirements mainly comprise common sample collection modes of blood tests such as peripheral blood collection, venous blood collection, arterial blood collection and the like, wherein the venous blood collection is most common, and the peripheral blood collection operation is simplest. As a way of peripheral blood collection, the sampling of dry blood sheets from fingertips has many advantages compared with venous blood collection: 1. the minimally invasive surgery is low in cost; 2. the sample demand is small; 3. is stable in the conventional environment and is convenient to transport and store; 4. the operation is simple, the training can be carried out simply, and the possibility is provided for home detection; 5. suitable for sampling in areas with insufficient medical conditions or remote areas and large-queue research of a plurality of centers.

Although the sampling operation of the finger tip blood dried blood sheet is simple, the finger tip blood dried blood sheet is more easily influenced by the environment, individual difference (bleeding amount), diet, tissue fluid, intracellular fluid, and the like compared with other blood sampling methods. Therefore, the collection of the fingertip blood dried blood sample needs to be standardized, and quality control key points are grasped to ensure the quality and stability of the sample. However, there is no method for quality monitoring in fingertip dry blood sample collection aiming at blood metabonomics research, and particularly, there is no evaluation method and judgment standard for whether metabonomics detection results in samples are available or not based on dry blood sample collection quality.

Disclosure of Invention

In view of one or more of the problems in the prior art, an aspect of the present invention provides a method for monitoring the collection quality of a dried blood slice sample, which includes detecting the signal intensities of glycochenodeoxycholic acid or/and triglyceride TAG56:4 and respective additional calibration substances (represented by peak area or peak height detected by one of liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS)) in the collected dried blood slice sample, using the relative content (M) of glycochenodeoxycholic acid in the dried blood slice sample represented by the signal intensity ratio of glycochenodeoxycholic acid and its calibration substance gibberellic acid, or/and the relative content (N) of triglyceride TAG56:4 in the dried blood slice sample represented by the signal intensity ratio of triglyceride TAG56:4 and its calibration substance phosphatidylethanolamine PE (17:0/17:0) as monitoring indexes, and when M or/and N meet a certain value, judging that the collection quality of the dried blood slice sample meets the requirement.

In the monitoring method, when the relative content M of glycochenodeoxycholic acid in the dried blood sample is less than X1 represented by the following formula (I), preferably not more than X2 represented by the following formula (II), and further preferably in the range of X3 represented by the following formula (III), the collection quality of the dried blood sample is satisfactory (for example, the subject used for collecting the dried blood sample is in a fasting state, preferably at least fasted for 12 hours):

formula (I) wherein K1 ═ 0.413; in formula (II), K2 ═ 0.168; in the formula (III), K3 is 0.051-0.168;

in the formulae (I), (II) and (III), Ra is 0.84. mu.g/mL, and R1 represents the on-machine final concentration (detection concentration) of gibberellic acid in the sample at the time of actual on-machine detection, and the unit is. mu.g/mL;

preferably, when a liquid chromatography-mass spectrometry (LC-MS) is used for determining the relative content of glycochenodeoxycholic acid in the dry blood slice sample, the method specifically comprises the following steps:

t1) placing the dried blood slice sample into a cold (methyl tert-butyl ether: methanol, volume ratio 3:1) solution (in which gibberellic acid is added in advance), and vortex and mixing uniformly to extract a blood sample to obtain a first sample extracting solution;

t2) adding a (methanol: water, volume ratio 3:1) solution into the first sample extracting solution, and carrying out vortex and centrifugal separation;

t3) separating the sample, taking the clear part of the lower aqueous phase into a centrifuge tube, adding ice methanol into the centrifuge tube to precipitate protein, centrifuging the protein after precipitation, taking the supernatant, transferring the supernatant into a new centrifuge tube, and drying the supernatant overnight;

t4) adding water into the centrifuge tube dried in the step T3), and incubating;

t5), uniformly mixing the mixture by vortex, and centrifuging after ultrasonic treatment;

t6) taking the supernatant fluid obtained after the centrifugation in the step T5) as a water phase, and performing on-machine detection to obtain mass spectrum data of glycochenodeoxycholic acid and a calibration substance gibberellic acid thereof;

t7) calculating the relative content of glycochenodeoxycholic acid in the dry blood slice sample by taking the peak area or the peak height in the mass spectrum data obtained by the machine detection in the step T6) as the detection signal intensity.

In the above monitoring method, wherein when the relative content N of the triglyceride TAG56:4 in the dried blood slice sample is less than Y1 represented by the following formula (IV), preferably not more than Y2 represented by the following formula (V), and further preferably within the range of Y3 represented by the following formula (VI), the collection quality of the dried blood slice sample is satisfactory (for example, the subject used to collect the dried blood slice sample is in a fasting state, preferably at least 12 hours fasted):

in formula (IV), L1 ═ 0.0295; in formula (V), L2 ═ 0.0253; in the formula (VI), L3 is 0.0129-0.0253;

in the formulae (IV), (V) and (VI), Rb is 2.33. mu.g/mL, and R2 represents the final on-machine concentration (detection concentration) of phosphatidylethanolamine PE (17:0/17:0) in the sample at the time of actual on-machine detection, and the unit is. mu.g/mL;

preferably, when a liquid chromatography-mass spectrometry (LC-MS) is used for determining the relative content of the triglyceride TAG56:4 in the dry blood slice sample, the method specifically comprises the following steps:

s1) placing the dried blood slice sample into a cold (methyl tert-butyl ether: methanol, volume ratio 3:1) solution (in which a calibration substance phosphatidyl ethanolamine PE (17:0/17:0) of triglyceride TAG56:4 is added in advance), and performing vortex mixing to extract a blood sample to obtain a second sample extracting solution;

s2) adding a (methanol: water, volume ratio 3:1) solution into the second sample extracting solution, and carrying out vortex and centrifugal separation;

s3), layering the sample, and putting the upper organic phase into a centrifuge tube;

s4) drying the organic phase, adding a solution (acetonitrile: isopropanol, volume ratio 3:1), incubating, whirling, mixing uniformly, and centrifuging after ultrasonic treatment;

s5) taking the supernatant fluid obtained after the centrifugation in the step S4) as an organic phase, and performing on a computer to detect to obtain mass spectrum data of triglyceride TAG56:4 and a calibration substance thereof, namely phosphatidylethanolamine PE (17:0/17: 0);

s6) calculating the relative content of triglyceride TAG56:4 in the dried blood sample by taking the peak area or peak height in the mass spectrum data obtained by the computer detection in the step S5) as the detection signal intensity.

In the above monitoring method, wherein when the relative content M of glycochenodeoxycholic acid in the dried blood sample is less than X1 represented by formula (I), preferably not more than X2 represented by formula (II), further preferably in the range of X3 represented by formula (III), and

the relative amount N of the triglyceride TAG56:4 in the dried blood sample is less than Y1 represented by formula (IV), preferably not more than Y2 represented by formula (V), and further preferably in the range of Y3 represented by formula (VI), the quality of the dried blood sample collected is satisfactory (e.g., the subject used to collect the dried blood sample is in a fasting state, preferably fasted for at least 12 hours);

preferably, when a liquid chromatography-mass spectrometry (LC-MS) is used for determining the relative content of the glycochenodeoxycholic acid and the triglyceride TAG56:4 in the dried blood slice sample, the method specifically comprises the following steps:

1) placing the dried blood slice sample into a cold (methyl tert-butyl ether: methanol, volume ratio 3:1) solution (in which gibberellic acid serving as a calibration substance of glycochenodeoxycholic acid and phosphatidylethanolamine PE (17:0/17:0) serving as a calibration substance of triglyceride TAG56:4 are added in advance), and performing vortex mixing to extract a blood sample so as to obtain a third sample extracting solution;

2) adding a (methanol: water, volume ratio of 3:1) solution into the third sample extracting solution, and carrying out vortex and centrifugal layering;

3) obtaining the relative content M of glycochenodeoxycholic acid in the dried blood sample according to the steps T3) to T7) described above, respectively, and obtaining the relative content N of triglyceride TAG56:4 in the dried blood sample according to the steps S3) to S6) described above.

The invention also provides application of glycochenodeoxycholic acid or/and triglyceride TAG56:4 as a quality test indicator for judging the collection quality of the dried blood slice sample, and the collection quality of the dried blood slice sample is judged by taking the relative content of the glycochenodeoxycholic acid or/and triglyceride TAG56:4 in the dried blood slice sample as a monitoring index.

In a further aspect, the invention provides an application of gibberellic acid or/and phosphatidylethanolamine PE (17:0/17:0) as a calibration substance for determining the collection quality of a dried blood slice sample, wherein the gibberellic acid is used as a calibration substance of glycochenodeoxycholic acid, and the phosphatidylethanolamine PE (17:0/17:0) is used as a calibration substance of triglyceride TAG56:4, and the collection quality of the dried blood slice sample is determined by taking the relative content M of the glycochenodeoxycholic acid to the gibberellic acid in the dried blood slice sample or/and the relative content N of the triglyceride TAG56:4 to the phosphatidylethanolamine PE (17:0/17:0) in the dried blood slice sample as monitoring indexes.

In another aspect of the present invention, a method for evaluating metabonomics detection results of dry blood slice samples is provided, which comprises:

detecting a plurality of metabolic compounds in the dry blood slice sample to obtain a metabonomics detection result of the dry blood slice sample, and an additional gibberellic acid detection result serving as a calibration substance of glycochenodeoxycholic acid in the metabolic compounds, or/and a phosphatidylethanolamine PE (17:0/17:0) detection result serving as a calibration substance of triglyceride TAG56:4 in the metabolic compounds;

calculating the ratio a of the detection signal intensity of glycochenodeoxycholic acid to the detection signal intensity of gibberellic acid or/and the ratio b of the detection signal intensity of triglyceride TAG56:4 to the detection signal intensity of phosphatidylethanolamine PE (17:0/17:0) from the obtained detection results;

and evaluating whether the metabonomics detection result of the dry blood sample is available according to whether the ratio a or/and the ratio b meet a certain value.

In the above evaluation method, the evaluation criteria according to the ratio a are:

when the ratio a is not less than X1 represented by formula (I), the dry blood sample metabolomics test result is not available;

when the ratio a is less than X1 represented by formula (I) and greater than X2 represented by formula (II), the metabonomic test result of the dry blood sample is in doubt;

when the ratio a is not more than X2 represented by formula (II), and further preferably in the range of X3 represented by formula (III), the metabonomic test result of the dried blood slice sample is available;

preferably, when the liquid chromatography-mass spectrometry (LC-MS) is used for detecting a plurality of metabolic compounds and gibberellic acid in the dried blood sample, the method specifically comprises the following steps:

LC-MS detection data of each metabolic compound in the aqueous phase and the added gibberellic acid in the dried blood sample are obtained according to the above-described steps T2) to T7) and the LC-MS detection data of each metabolic compound in the organic phase in the dried blood sample are obtained according to the above-described steps S2) to S6), respectively, using the above-described first sample extract.

In the above evaluation method, the evaluation criteria according to the ratio b are:

when the ratio b is not less than Y1 represented by formula (IV), the dried blood sample metabolomics test result is not available;

when the ratio b is less than Y1 represented by formula (IV) and greater than Y2 represented by formula (V), the metabonomic detection result of the dry blood slice sample is in doubt;

when the ratio b is not more than Y2 represented by formula (V), and is further preferably in the range of Y3 represented by formula (VI), the metabonomic test result of the dried blood slice sample can be used;

preferably, when a liquid chromatography-mass spectrometry (LC-MS) is used for detecting a plurality of metabolic compounds and phosphatidylethanolamine PE (17:0/17:0) in a dry blood slice sample, the method specifically comprises the following steps:

using the second sample extract, LC-MS detection data of each metabolic compound in the aqueous phase in the dry blood slice sample are obtained according to the steps T2) to T7) and LC-MS detection data of each metabolic compound in the organic phase in the dry blood slice sample and the added phosphatidylethanolamine PE (17:0/17:0) are obtained according to the steps S2) to S6).

In the above evaluation method, the evaluation criteria according to the ratio a and the ratio b are:

when the ratio a is not less than X1 represented by formula (I), and/or the ratio b is not less than Y1 represented by formula (IV), the dried blood sample metabonomics test result is not available;

the dried blood sample metabonomics test result is in doubt when the ratio a is less than X1 represented by formula (I) and more than X2 represented by formula (II), and/or the ratio b is less than Y1 represented by formula (IV) and more than Y2 represented by formula (V);

when the ratio a is not more than X2 represented by formula (II), and more preferably in the range of X3 represented by formula (III), and the ratio b is not more than Y2 represented by formula (V), and more preferably in the range of Y3 represented by formula (VI), the metabonomic test result of the dried blood slice sample is available;

preferably, when a liquid chromatography-mass spectrometry (LC-MS) is used for detecting a plurality of metabolic compounds in a dry blood sample, gibberellic acid and phosphatidylethanolamine PE (17:0/17:0), the method specifically comprises the following steps:

using the third sample extract, LC-MS detection data of each metabolic compound and gibberellic acid in the aqueous phase in the dried blood sheet sample are obtained according to the above steps T2) to T7), and LC-MS detection data of each metabolic compound and phosphatidylethanolamine PE (17:0/17:0) in the organic phase in the dried blood sheet sample are obtained according to the above steps S2) to S6).

Based on the above technical solution, the present invention uses the relative content of glycochenodeoxycholic acid or/and triglyceride TAG56:4 (wherein 56:4 represents the specific structure of triglyceride, that is, 3 fatty acid chains in triglyceride contain 56 carbon (C) atoms in total, and 4 carbon bonds are unsaturated bonds) in dried blood slice samples (wherein the relative content of glycochenodeoxycholic acid is relative to gibberellic acid in the same sample, and the relative content of triglyceride TAG56:4 is relative to phosphatidylethanolamine PE (17:0/17:0) in the same sample (wherein 17:0/17:0 represents the structure of the substance, that is, 2 fatty acid carbon chains contained in PE (17:0/17:0) each contain 17 carbon (C) atoms and are all saturated carbon bonds)) M or/and N as quality inspection indexes to judge whether the collection quality of dried blood slice samples meets the requirements (for example, whether the subject from whom the dried blood sample was taken is in a fasting state). The present study results show that the quality of dried blood slice sample collection is satisfactory when the relative content M of glycochenodeoxycholic acid in the dried blood slice sample is less than X1 represented by formula (I), preferably not more than X2 represented by formula (II), and further preferably in the range of X3 represented by formula (III), and/or the relative content N of triglyceride TAG56:4 in the dried blood slice sample is less than Y1 represented by formula (IV), preferably not more than Y2 represented by formula (V), and further preferably in the range of Y3 represented by formula (VI), i.e. the subject used for collecting the dried blood slice sample is in a fasting state (e.g. fasted for at least 12 hours) when performing fingertip blood dried blood slice sampling.

By utilizing the application, the invention provides a monitoring method of the collection quality of the metabonomic dried blood slice sample and an evaluation method of the metabonomic detection result of the dried blood slice sample based on the ratio of the detection signal intensity of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the metabonomic dried blood slice sample to the detection signal intensity of each calibration substance in the same metabonomic dried blood slice sample, and the collection quality of the metabonomic dried blood slice sample can be rapidly and accurately judged and monitored by detecting the relative content of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the collected dried blood slice sample, the metabonomic detection result is evaluated, and the accuracy and reliability of the metabonomic detection result of the dried blood slice sample are ensured.

The invention uses M or N as a quality inspection index, the change of the value is directly related to whether a subject is in a fasting state, and experimental data shows that the value range has obvious difference under the condition of fasting state, so that the value has enough representativeness and sensitivity as the quality inspection index. The invention can independently use M or N as a quality inspection index, namely only gibberellic acid or only phosphatidylethanolamine PE (17:0/17:0)) can be added in the dry blood slice sample detection according to the laboratory condition, and whether the dry blood slice sample meets the collection quality requirement is judged according to the numerical value of M or N, so that the collection quality of the dry blood slice sample can be monitored in the simplest mode, unqualified samples can be quickly screened out, and the meaningless labor of further detecting a large amount of biological metabolites in the unqualified samples is avoided. When M and N are used in combination, the distribution (the former group is distributed in a water phase, the latter group is distributed in an organic phase) of glycochenodeoxycholic acid and a calibration substance thereof gibberellic acid, triglyceride TAG56:4 and a calibration substance thereof phosphatidylethanolamine PE (17:0/17:0)) in dry blood slice sample detection and the position and the shape of a detection peak can be fully considered, and the two indexes are consistent to ensure that the monitoring of the dry blood slice sample collection quality is more stable; when the two indexes are inconsistent, the two indexes are applied to the evaluation of the metabonomics detection result, whether the result is doubtful or not can be accurately distinguished, the data is focused during subsequent use, and support is provided for abnormal data elimination and data later analysis.

Drawings

FIG. 1 is an extracted ion flow chromatogram of glycochenodeoxycholic acid in fasting and non-fasting group dried blood slice samples;

FIG. 2 is an extracted ion flow chromatogram of triglyceride TAG56:4 in fasting and non-fasting group dried blood slice samples;

FIG. 3 is a graph comparing the relative content differences of glycochenodeoxycholic acid in fasting and non-fasting group dried blood slice samples;

fig. 4 is a graph comparing the relative content differences of the triglyceride TAG56:4 in fasting versus non-fasting dried blood slice samples.

Detailed Description

The invention firstly provides application of glycochenodeoxycholic acid and/or triglyceride TAG56:4 as a quality inspection indicator for judging the collection quality of a dried blood slice sample. Specifically, a liquid chromatography-mass spectrometer (LC-MS) and/or a GAs chromatography-mass spectrometer (GC-MS) can be used as a detection means to measure the relative content of glycochenodeoxycholic acid and/or triglyceride in the collected dry blood slice sample (i.e. the ratio of the detection signal intensity of glycochenodeoxycholic acid and/or triglyceride to the detection signal intensity of each calibration substance (wherein the calibration substance corresponding to glycochenodeoxycholic acid is Gibberellic acid (GA 3)), and the calibration substance corresponding to triglyceride is phosphatidylethanolamine PE (17:0/17:0)), so as to determine whether the collection quality of the dry blood slice sample is qualified or not according to the value, and to evaluate whether the metabonomic detection result is available or not.

On the other hand, the invention also provides a monitoring method of the collection quality of the dried blood slice sample based on the relative content of glycochenodeoxycholic acid and/or triglyceride in the dried blood slice sample, and provides an accurate reference standard and a judgment basis for the correct collection of the dried blood slice sample.

On the other hand, the invention also provides an evaluation method of the dry blood slice sample metabonomics detection result based on the relative content of glycochenodeoxycholic acid and/or triglyceride in the dry blood slice sample, which can be used for carrying out primary screening on abnormal dry blood slice samples (such as diet of a subject before collection of the dry blood slice sample) and providing support for data abnormal rejection and data later analysis.

The present invention will be described in detail with reference to the following detailed description and accompanying drawings.

In the following, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

The methods used in the following examples are conventional methods unless otherwise specified.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure only and should not be limiting as to the source of the biological material of the present invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

Example 1: quality test indicator for determining and judging dry blood sample collection quality

1) Dry blood sample was prepared from the blood of the fingertips of 3 human subjects (age range 30-33 years, signed with informed consent): fingertip blood was collected in the fasting state (fasting not less than 12 hours, i.e., fasting group) and 2 hours after the food (i.e., non-fasting group) in the morning to prepare dry blood chip samples, and 2 samples (taken as fasting group sample and non-fasting group sample, respectively) were collected from each subject for a total of 6 dry blood chip samples;

2) taking 4 blood slices with the diameter of 3 mm from each dried blood slice sample, placing the blood slices as 4 repeats respectively into 700 mu L of precooled (methyl tert-butyl ether: methanol, volume ratio 3:1) solution (in which gibberellic acid with the concentration of 0.45 mu g/mL (standard substance added in the form of aqueous solution of gibberellic acid) and PE with the concentration of 1.0 mu g/mL (17:0/17:0) (standard substance added in the form of dichloromethane solution of PE (17:0/17:0)) and vortex and mixing uniformly to extract respective blood samples to obtain respective sample extracting solutions;

3) adding 350 mu L of (methanol: water, volume ratio of 3:1) solution into the sample extracting solution, carrying out ultrasonic treatment, standing, vortexing, and centrifuging for layering;

4) after layering the sample, taking 350 mu L of the upper layer into a centrifuge tube to obtain an organic phase;

5) after the organic phase was dried, 200. mu.L (acetonitrile: isopropanol, volume ratio 3:1) was added and incubated at room temperature for 15 minutes;

6) after incubation, vortex the tube well, sonicate for 5 minutes, centrifuge the tube at room temperature for 5 minutes (12000 rpm);

7) after centrifugation in step 6), taking 180 μ l of supernatant from the centrifuge tube into a 2mL glass sample vial to obtain an organic phase substance, detecting by a computer (LC-MS) (detecting PE (17:0/17:0) in the organic phase substance to obtain a final concentration of 2.33 μ g/mL before detection by the computer), and detailing in step 13);

8) taking 400 mu L of the lower-layer aqueous phase after centrifugal layering in the step 3) to a centrifuge tube, adding 1100 mu L of glacial methanol into the centrifuge tube, and precipitating protein;

9) after protein precipitation in a centrifuge tube, centrifuging the centrifuge tube, taking 1000 mu L of supernatant, transferring the supernatant into a new centrifuge tube, and drying the supernatant overnight;

10) adding 200 mu L of water into the dried centrifugal tube, and incubating for 15 minutes at room temperature;

11) after incubation, vortex the tube well, sonicate for 5 minutes, centrifuge the tube at room temperature for 5 minutes (12000 rpm);

12) after centrifugation in step 11), taking 180 μ L of supernatant from the centrifuge tube into a 2mL glass sample injection vial as an aqueous phase substance, and detecting on a computer (LC-MS) (detecting that the final concentration of gibberellic acid in the aqueous phase substance is 0.84 μ g/mL before detection on the computer), which is detailed in step 13);

13) and (3) sample on-machine detection: data information was collected using a liquid chromatography-mass spectrometer (LC-MS) consisting of an electrospray ionization source (ESI) and an electrostatic field orbital trap (Orbitrap) mass analyzer (watset) to obtain LC-MS detection data (including mass spectrometry data and liquid chromatography data) for each compound in each set of test samples (organic and aqueous phases).

The method specifically comprises the following steps: mu.l of the aqueous phase obtained in step 12) was injected onto an ACQUITY UPLC HSS T3 (2.1X 100mm, 1.8 μm) column and 2. mu.l of the organic phase obtained in step 7) was injected onto an ACQUITY UPLC HSS BEH C8 (2.1X 100mm, 1.7 μm) column. The elution gradients of the liquid chromatography are listed in tables 1 and 2 below.

Table 1: HST 3 column elution gradient

	Time (min)	Flow rate (mL/min)	％A	％B	Curve
							1	Initial	0.4	99	1	Initial
2	1	0.4	99	1	6
						3	11	0.4	60	40	6
4	13	0.4	30	70	6
						5	15	0.4	1	99	6
6	18	0.4	1	99	6
						7	19	0.4	99	1	6
8	21	0.4	99	1	6

Note: in Table 1, mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution, which are chromatographically pure

Table 2: HSS BEH C8 chromatography column elution gradient

	Time (min)	Flow rate (mL/min)	％A	％B	Curve
							1	Initial	0.4	45	55	Initial
2	1	0.4	45	55	6
						3	4	0.4	25	75	6
4	12	0.4	11	89	6
						5	15	0.4	0	100	6
6	19.5	0.4	45	55	6
						7	19.51	0.4	45	55	6
8	24	0.4	45	55	6

Note: in Table 2, mobile phase A is an aqueous solution containing 0.1% acetic acid, and mobile phase B is an acetonitrile-n-hexane (volume ratio 7:3) solution containing 0.1% acetic acid and 1% ammonium acetate, all of which are chromatographically pure solutions

14) Data processing: converting LC-MS data of each group of detection samples obtained in the step 13) into an H5 format, analyzing by using Rstudio software (commercial software), and completing according to the instruction operation, wherein the data are screened for multiple times, and the data comprise removal of isotope peaks, ions fragmented in a source, different addition modes of the same substance, repeated compounds and the like. Finally, csv format data are derived, and then are analyzed by a metabonomic analysis website MetabioAnalyst 5.0 (commercial website), and the detection signal intensity of each detected compound (metabolite) is annotated and calculated.

15) The processed data are analyzed for repeatability among independent samples by a t-test method

The detection signal intensity of each compound (metabolite) was annotated and calculated in this example by R studio software and metabolomic database MetaboAnalyst 5.0 (detection signal intensity is expressed as peak area/peak height value of the extracted ion current chromatogram for the corresponding compound). Comparing the intensity of detection signals of the respective compounds in the fasting group and non-fasting group samples, and among the compounds (abnormal metabolites) having higher intensity of detection signals in the non-fasting group samples than in the fasting group samples, two metabolites glycochenodeoxycholic acid and triglyceride TAG56:4 were screened, which have significantly higher intensity of detection signals in the non-fasting group samples than in the fasting group samples, as shown in fig. 1 and fig. 2 (ordinate indicates relative intensities of chromatographic peaks, abscissa indicates retention time ranges of chromatographic peaks, and solid and dotted lines are used to mark heights of chromatographic peaks in the non-fasting group and the fasting group), and showing the extracted chromatogram ion currents of glycochenodeoxycholic acid and triglyceride TAG56:4 in the two groups samples (fasting group and non-fasting group), respectively. The t-test analysis has statistical significance (P is 0.043 and 0.0053 respectively), and the other detected abnormal metabolites have no statistical significance on the change of the detection signal intensity through the t-test analysis, so that the metabolites glycochenodeoxycholic acid and triglyceride TAG56:4 in the dry blood sample can be used as quality detection indicators for judging whether the collection quality of the dry blood sample meets the requirements or not.

In order to quantify the collection quality judgment standard of the dried blood slice sample, the inventor takes the ratio of the detection signal intensity of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the dried blood slice sample to the detection signal intensity of each calibration substance as a quality detection index. Specifically, gibberellic acid or/and phosphatidylethanolamine PE (17:0/17:0) are added in the step 2), and the ratio a of the detection signal intensity of glycochenodeoxycholic acid in the water phase substance to the detection signal intensity of gibberellic acid, and/or the ratio b of the detection signal intensity of triglyceride TAG56:4 in the organic phase substance to the detection signal intensity of phosphatidylethanolamine PE (17:0/17:0) are used as the quality inspection indexes, preferably both are used as the quality inspection indexes.

Specifically, in this example, the on-machine final concentration (i.e., the concentration at the time of on-machine detection) of gibberellic acid used as a labeling substance for glycochenodeoxycholic acid in the aqueous phase substance subjected to on-machine detection was 0.84. mu.g/mL, and the final concentration of phosphatidylethanolamine PE (17:0/17:0) used as a labeling substance for triglyceride TAG56:4 in the organic phase substance subjected to on-machine detection was 2.33. mu.g/mL. As shown in fig. 3 and 4, which show the relative content differences of glycochenodeoxycholic acid and triglyceride TAG56:4 in the fasting group and non-fasting group dried blood slice samples, respectively, it can be seen that the relative content of glycochenodeoxycholic acid and triglyceride TAG56:4 in the non-fasting group samples is higher relative to the fasting group samples. As shown in fig. 3, the relative content of glycochenodeoxycholic acid is not less than 0.413 in the non-fasted group samples, whereas in the fasted group samples, the relative content of glycochenodeoxycholic acid is less than 0.413, more precisely not more than 0.168, between 0.051 and 0.168; as shown in FIG. 4, the relative amount of triglyceride TAG56:4 is no less than 0.0295 in the non-fasted group samples and no less than 0.0295, more precisely no greater than 0.0253, in the fasted group samples, and is between 0.0129 and 0.0253. Therefore, the evaluation criteria for determining the quality inspection indexes are as follows: the quality of the dried blood specimen is satisfactory when the relative content M of glycochenodeoxycholic acid in the dried blood specimen is determined to be less than 0.413, preferably not more than 0.168, and more preferably 0.051 to 0.168, or the relative content N of triglyceride TAG56:4 in the dried blood specimen is determined to be less than 0.0295, preferably not more than 0.0253, and more preferably 0.0129 to 0.0253, or both the relative contents M and N of glycochenodeoxycholic acid and triglyceride TAG56:4 in the dried blood specimen are determined to satisfy the above requirements, i.e., the quality of the specimen in this embodiment is in a fasting state (e.g., at least 12 hours fasting).

In practical tests, the concentration of gibberellic acid, which is a calibration substance for glycochenodeoxycholic acid, added to the blood sample extraction solution in the on-machine test and the concentration of phosphatidylethanolamine PE (17:0/17:0), which is a calibration substance for triglyceride TAG56:4, in the on-machine test are not limited to the above-mentioned test concentrations, and accordingly, the signal intensity of the detected gibberellic acid and the signal intensity of the phosphatidylethanolamine PE (17:0/17:0) are changed, which in turn results in the calculated relative content of glycochenodeoxycholic acid and the calculated relative content of triglyceride TAG56:4 being changed. Since the detection signal intensities of gibberellic acid and phosphatidylethanolamine PE (17:0/17:0) actually indicate the concentrations of both in the detection sample, the change in the detection signal intensities can directly indicate the change in the detection concentrations and have a linear correlation, for example, assuming that the detection signal intensity of glycochenodeoxycholic acid is represented as P, the original detection concentration of gibberellic acid is represented as Ra (in μ g/mL), the detected signal intensity is represented as P0, the detection concentration of gibberellic acid after the change is represented as R1 (in μ g/mL), and the corresponding detected signal intensity is represented as P1, the following relationship exists: and R1/Ra-P1/P0, the relative content M of glycochenodeoxycholic acid calculated after the detection concentration of the gibberellic acid is changed is P/P1 (P/P0) · (Ra/R1). This relationship also applies to the triglyceride TAG56:4 and phosphatidylethanolamine PE (17:0/17: 0). Thus:

in the case of gibberellic acid, when the original detection concentration Ra is 0.84 μ g/mL in this embodiment, and the value of P/P0 calculated in the fasting group dried blood specimen is K '(according to the above data, the maximum value of K' is set to K1 ═ 0.413, preferably K2 ═ 0.168, and more preferably K3 ═ 0.051 to 0.168), when the detection concentration of gibberellic acid in the fasting group dried blood specimen becomes R1 (i.e., the concentration at the time of actual detection), the calculated relative content M of glycochenodeoxycholic acid is expressed as the following formula (I-I):

M＝P/P1＝(P/P0)·(Ra/R1)＝K’·(Ra/R1) (I-I)

when K' in formula (I-I) is K1, K2 and K3 respectively, the relative content M of the corresponding glycochenodeoxycholic acid is expressed as: X1-K1- (0.84/R1), X2-K2- (0.84/R1), X3-K3- (0.84/R1), wherein R1 is the detection concentration (unit is mu g/mL) of gibberellic acid in an aqueous phase substance sample during actual on-machine detection, K1-0.413, K2-0.168, and K3-0.051-0.168.

Similarly, for phosphatidylethanolamine PE (17:0/17:0), when the detected concentration of phosphatidylethanolamine PE (17:0/17:0) is changed from the original detected concentration Rb (2.33. mu.g/mL) to R2 (i.e., the concentration at the time of actual detection), the calculated relative content N of triglyceride TAG56:4 is represented by the following formula (I-II):

N＝L’·(Rb/R2) (I-II)

in the formula (I-II), L' represents the ratio of the detection signal intensity of triglyceride TAG56:4 in the organic phase material sample in the fasting group on-machine detection to the detection signal intensity of phosphatidylethanolamine PE (17:0/17:0) calculated at the detection concentration of phosphatidylethanolamine PE (17:0/17:0) of 2.33 μ g/mL in the present example, and the maximum value L1 is 0.0295, preferably L2 is 0.0253, and more preferably L3 is 0.0129 to 0.0253. When L' in formula (I-II) is L1, L2, and L3, respectively, the relative content N of the corresponding triglyceride TAG56:4 is expressed as: Y1-L1- (2.33/R2), Y2-L2- (2.33/R2) and Y3-L3- (2.33/R2), wherein R2 is the detection concentration (unit is mu g/mL) of phosphatidylethanolamine PE (17:0/17:0) in an organic phase substance sample during actual on-machine detection.

In conclusion, the relative content of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the dried blood slice sample is used as a quality inspection index to judge whether the collection quality of the dried blood slice sample meets the requirement, and any one of the following judgment standards (1) to (3) is selected according to the type and the quantity of the added calibration substances (gibberellic acid and phosphatidylethanolamine PE (17:0/17: 0)):

(1) when only gibberellic acid is added, the quality of the dried blood sample is satisfactory when the relative content of glycochenodeoxycholic acid in the dried blood sample is less than the value represented by X1, preferably not more than the value represented by X2, and further preferably within the range represented by X3 (in this case, satisfactory quality of collection means that the subject from which the dried blood sample is collected is in a fasting state, for example, at least 12 hours after fasting);

(2) when phosphatidylethanolamine PE (17:0/17:0) alone is added, the collection quality of the dried blood slice sample is satisfactory when the relative content of triglyceride TAG56:4 in the dried blood slice sample is less than the value represented by Y1, preferably not more than the value represented by Y2, and further preferably within the range represented by Y3;

(3) when gibberellic acid and phosphatidylethanolamine PE (17:0/17:0) are added simultaneously, the collection quality of the dried blood sheet sample is satisfactory when the relative content of glycochenodeoxycholic acid in the dried blood sheet sample is less than the value represented by X1 (preferably not more than the value represented by X2, and more preferably in the range represented by X3), and the relative content of triglyceride TAG56:4 in the dried blood sheet sample is less than the value represented by Y1 (preferably not more than the value represented by Y2, and more preferably in the range represented by Y3).

Example 2: verification of glycochenodeoxycholic acid as quality control indicator for judging collection quality of dried blood slice sample

1) Taking fingertip blood dry blood sheet samples sampled by 1 subject for three times at different time as detection samples, taking 3 dry blood sheet samples in total, recording information of the collected three dry blood sheet samples, and listing in the following table 3;

2) taking 4 blood slices with the diameter of 3 mm from each dried blood slice sample, respectively placing the blood slices as 4 repeats in 700 mu L of precooled (methyl tert-butyl ether: methanol, volume ratio of 3:1) solution (gibberellic acid is added in the solution in advance), and performing vortex mixing to extract respective blood samples to obtain respective sample extracting solution;

3) adding a (methanol: water, volume ratio of 3:1) solution into the sample extracting solution, removing an upper organic phase, taking 400 mu L of a lower aqueous phase into a centrifuge tube, adding 1100 mu L of glacial methanol into the centrifuge tube, and precipitating protein;

4) after protein precipitation, centrifuging the centrifuge tube, taking supernatant, transferring the supernatant into a new centrifuge tube, and drying overnight;

5) adding 200 mu L of water into the dried centrifugal tube, and incubating for 15 minutes at room temperature;

7) after the centrifugation in the step 6), taking 180 mu L of supernatant from the centrifuge tube into a 2mL glass sample injection vial as a water-phase substance, and detecting by a computer (LC-MS) according to example 1 to obtain mass spectrum data of glycochenodeoxycholic acid and a calibration substance gibberellic acid thereof;

8) the detection signal intensity information (peak area representation of the extracted ion current chromatogram) of glycochenodeoxycholic acid in each dry blood sample and the detection signal intensity information of gibberellic acid as a calibration substance thereof are obtained from the mass spectrum data according to the data processing method of the embodiment 1, and the relative content of glycochenodeoxycholic acid is calculated.

As shown in table 3 below, it can be seen that the relative content of glycochenodeoxycholic acid in the dried blood samples No. 1 and No. 3 collected from the same subject at different time points exceeds 0.413, and the relative content of glycochenodeoxycholic acid can be used to judge that the subject is in a non-fasting state, i.e. eating before collecting the sample, which is consistent with the subject information recorded when collecting the dried blood samples. The relative content of glycochenodeoxycholic acid in the detected No. 2 dried blood sample is less than 0.413, and the relative content of glycochenodeoxycholic acid can judge that the subject is in a fasting state, namely, the subject does not eat or eat before the sample is collected, which is consistent with the information of the subject recorded when the dried blood sample is collected. The result proves that the metabolite glycochenodeoxycholic acid in the dried blood slice sample can be really used as a quality test indicator for judging the collection quality of the dried blood slice sample, and the collection quality of the dried blood slice sample can be monitored by detecting the relative content of the glycochenodeoxycholic acid in the dried blood slice sample.

Table 3: different dry blood sample detection results

Example 3: verification of triglyceride TAG56:4 as a quality control indicator for determining quality of dried blood sample collection

1) Using fingertip blood dry blood sheet samples sampled by 1 subject three times at different time as detection samples (the detection samples are the same as the detection samples used in example 2), 3 dry blood sheet samples are obtained in total, and information of the three dry blood sheet samples at the time of collection is recorded and is listed in table 4 below;

2) taking 4 blood slices with the diameter of 3 mm from each dried blood slice sample, placing the blood slices as 4 repeats respectively into 700 mu L of precooled (methyl tert-butyl ether: methanol, volume ratio of 3:1) solution (PE (17:0/17:0) is added into the solution in advance), and performing vortex mixing to extract respective blood samples so as to obtain respective sample extracting solution;

4) after layering the sample, taking 200 mu L of an upper organic phase into a centrifugal tube to obtain an organic phase substance;

7) after the centrifugation in the step 6), taking 180 μ l of supernatant from the centrifuge tube into a 2mL glass sample injection vial as an organic phase substance, and detecting by a computer (LC-MS) according to example 1 to obtain mass spectrum data of the triglyceride TAG56:4 and a calibration substance PE (17:0/17:0) thereof;

8) the information on the detection signal intensity of the triglyceride TAG56:4 in each dried blood sample (peak area representation of the extracted ion current chromatogram) and the information on the detection signal intensity of the labeling substance PE (17:0/17:0) thereof were obtained from the mass spectrum data according to the data processing method of example 1, and the relative content of the triglyceride TAG56:4 was calculated.

The results are shown in Table 4 below, and it can be seen that the relative amount of triglyceride TAG56:4 in the dried blood sample Nos. 1 and 3 taken from the same subject at different time points both exceeded 0.0295, and that the subject was in a non-fasting state, i.e., was on a diet prior to the sample being taken, which is consistent with the subject information recorded at the time of the dried blood sample being taken, as judged from the relative amount of triglyceride TAG56: 4. The relative amount of triglyceride TAG56:4 in the dried blood sample number 2 tested was less than 0.0295, and the subjects were judged to be in a fasting state from the relative amount of triglyceride TAG56:4, i.e., not eating before the sample was taken, consistent with the subject information recorded at the time the dried blood sample was taken. The result proves that the triglyceride TAG56:4 in the dried blood slice sample can be used as a quality detection indicator for judging the collection quality of the dried blood slice sample, and the collection quality of the dried blood slice sample can be monitored by detecting the relative content of the triglyceride TAG56:4 in the dried blood slice sample.

Table 4: different dry blood sample detection results

Example 4: evaluation of metabonomics detection results of dry blood sample

This example utilizes the method of detecting a metabolite compound of example 1 (i.e., the method step of detecting a plurality of metabolite data to obtain a metabolomics detection result), preparing a test sample using a batch of dried blood samples taken from a hospital according to the method of example 1, wherein the relative amounts M of glycochenodeoxycholic acid in the dried blood samples are not more than 0.168 and the relative amount N of triglyceride TAG56:4 in the dried blood samples is not more than 0.0253 as criteria for whether the metabolomics detection result of the dried blood samples is usable, and further wherein the detection data of the compound obtained from the dried blood samples that are not in compliance is judged as a usable metabolomics detection result for further data analysis, and the metabolomics detection result of the dried blood samples that are not in compliance is judged as in doubt (when M0.168 < M < 0.413 and/or 0.53 < N < 0.0295) or is not usable (when M >0.413, and/or N ≧ 0.0295), such data are of particular concern for further data analysis.

The following table 5 shows the evaluation results of the metabonomic detection results of the dry blood slice samples, wherein the relative content (M and N) of glycochenodeoxycholic acid and triglyceride TAG56:4 in the samples 1, 4 and 5 in the dry blood slice samples does not exceed the standard, and the compound detection data obtained from the corresponding dry blood slice samples can be used as metabonomic detection result data; the values of M and N of the sample 2 exceed the standard, M is more than 0.413, N is more than 0.0295, and the compound detection data obtained from the sample 2 cannot be used as metabonomics detection result data; the M value of the sample 3 exceeds the standard, the N value does not exceed the standard, M is more than 0.168 and less than 0.413, the M value of the sample 6 does not exceed the standard, N value exceeds the standard, N is more than 0.0253 and less than 0.0295, whether the compound detection data obtained from the samples 3 and 6 can be used as metabonomic detection result data or not is questionable, but whether the metabonomic detection result data obtained from the samples 3 and 6 are available or not can be judged in combination with other indexes or the samples of the samples are detected again in view of the fact that the samples 3 and 6 are detected completely. It can be seen that the preliminary screening of abnormal dry blood sample (e.g. diet of the subject used for dry blood sample collection before collection) can be performed by detecting the relative content of glycochenodeoxycholic acid and/or triglyceride TAG56:4 in the dry blood sample, thereby providing support for data abnormal rejection and data post-analysis.

Table 5: evaluation of dry blood sample metabonomics detection result

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for monitoring the collection quality of a dried blood slice sample comprises the steps of detecting the signal intensity (represented by the peak area or the peak height detected by one of a liquid chromatography mass spectrum (LC-MS) and a gas chromatography mass spectrum (GC-MS)) of glycochenodeoxycholic acid or/and triglyceride TAG56:4 and an additional respective calibration substance in the collected dried blood slice sample, using the relative content (M) of the glycochenodeoxycholic acid in the dried blood slice sample represented by the signal intensity ratio of the glycochenodeoxycholic acid and the gibberellic acid as a calibration substance, or/and the relative content (N) of the triglyceride TAG56:4 in the dried blood slice sample represented by the signal intensity ratio of the triglyceride TAG56:4 and phosphatidylethanolamine PE (17:0/17:0) as a monitoring index, and when the M or/and the N meet a certain value, and judging that the collection quality of the dried blood slice sample meets the requirement.

2. The monitoring method according to claim 1, wherein the collection quality of the glycochenodeoxycholic acid in the dried blood sample meets the requirement (e.g., the subject used to collect the dried blood sample is in a fasting state, preferably at least fasted for 12 hours) when the relative content M of the glycochenodeoxycholic acid in the dried blood sample is less than X1 represented by the following formula (I), preferably not more than X2 represented by the following formula (II), and further preferably within a range of X3 represented by the following formula (III):

3. A monitoring method according to claim 1, wherein the quality of the dried blood sample collection is satisfactory (e.g. the subject used to collect the dried blood sample is in a fasting state, preferably at least 12 hours fasted) when the relative content N of the triglyceride TAG56:4 in the dried blood sample is less than Y1 represented by the following formula (IV), preferably not more than Y2 represented by the following formula (V), further preferably within the range of Y3 represented by the following formula (VI):

4. The monitoring method according to claim 1, wherein when the relative content M of glycochenodeoxycholic acid in the dried blood sample is less than X1 represented by formula (I) mentioned in claim 2, preferably not more than X2 represented by formula (II), further preferably within the range of X3 represented by formula (III), and

the quality of the dried blood sample collection is satisfactory (for example, the subject used for collecting the dried blood sample is in a fasting state, preferably at least 12 hours fasting) when the relative content N of the triglyceride TAG56:4 in the dried blood sample is less than Y1 represented by formula (IV) mentioned in claim 3, preferably not more than Y2 represented by formula (V), and further preferably within the range of Y3 represented by formula (VI);

3) obtaining the relative content M of glycochenodeoxycholic acid in the dried blood sample according to steps T3) to T7) mentioned in claim 2, respectively, and obtaining the relative content N of the triglyceride TAG56:4 in the dried blood sample according to steps S3) to S6) mentioned in claim 3.

5. Use of glycochenodeoxycholic acid or/and triglyceride TAG56:4 as a quality test indicator for determining the quality of dried blood sample collection, wherein the quality of dried blood sample collection is determined by using the relative content of glycochenodeoxycholic acid or/and triglyceride TAG56:4 in the dried blood sample as a monitoring index as described in any one of claims 1 to 4.

6. Use of gibberellic acid or/and phosphatidylethanolamine PE (17:0/17:0) as a calibration substance for determining the quality of dried blood slice sample collection, wherein gibberellic acid is used as a calibration substance for glycochenodeoxycholic acid, and phosphatidylethanolamine PE (17:0/17:0) is used as a calibration substance for triglyceride TAG56:4, and the quality of dried blood slice sample collection is determined by using the relative content M of glycochenodeoxycholic acid to gibberellic acid in the dried blood slice sample, or/and the relative content N of triglyceride TAG56:4 to phosphatidylethanolamine PE (17:0/17:0) in the dried blood slice sample as a monitoring index.

7. The evaluation method of the metabonomics detection result of the dry blood slice sample comprises the following steps:

8. The evaluation method according to claim 7, wherein the evaluation criterion according to the ratio a is:

when the ratio a is not less than X1 represented by formula (I) mentioned in claim 2, the dry blood sample metabonomics detection result is not available;

when the ratio a is less than X1 represented by formula (I) mentioned in claim 2 and is greater than X2 represented by formula (II), the result of metabonomics detection of the dry blood sample is questioned;

obtaining LC-MS detection data of each metabolic compound and added gibberellic acid in the aqueous phase in the dried blood sample according to steps T2) to T7) mentioned in claim 2, and obtaining LC-MS detection data of each metabolic compound in the organic phase in the dried blood sample according to steps S2) to S6) mentioned in claim 3, respectively, using the first sample extract mentioned in claim 2.

9. The evaluation method according to claim 7, wherein the evaluation criterion according to the ratio b is:

when the ratio b is not less than Y1 represented by formula (IV) mentioned in claim 3, the dried blood sample metabolomics test result is not available;

when the ratio b is less than Y1 represented by formula (IV) mentioned in claim 3 and greater than Y2 represented by formula (V), the dry blood slice sample metabonomics detection result is in doubt;

obtaining LC-MS detection data of each metabolic compound in an aqueous phase in the dried blood sample according to steps T2) to T7) mentioned in claim 2, respectively, using the second sample extract mentioned in claim 3, and obtaining LC-MS detection data of each metabolic compound in an organic phase in the dried blood sample and additionally phosphatidylethanolamine PE (17:0/17:0) according to steps S2) to S6) mentioned in claim 3.

10. The evaluation method according to claim 7, wherein the evaluation criterion according to the ratio a and the ratio b is:

when the ratio a is not less than X1 represented by formula (I) mentioned in claim 2, and/or the ratio b is not less than Y1 represented by formula (IV) mentioned in claim 3, the dried blood sample metabolomics test result is not available;

the dried blood sample metabonomics test result is in doubt when the ratio a is less than X1 represented by formula (I) mentioned in claim 2 and is greater than X2 represented by formula (II), and/or the ratio b is less than Y1 represented by formula (IV) mentioned in claim 3 and is greater than Y2 represented by formula (V);

obtaining LC-MS detection data of each metabolic compound and gibberellic acid in the aqueous phase in the dried blood sample according to steps T2) to T7) mentioned in claim 2, respectively, using the third sample extract mentioned in claim 4, and obtaining LC-MS detection data of each metabolic compound and phosphatidylethanolamine PE (17:0/17:0) in the organic phase in the dried blood sample according to steps S2) to S6) mentioned in claim 3.