CN109946412B - Kit for detecting spectrum of humoral pterin and use thereof - Google Patents
Kit for detecting spectrum of humoral pterin and use thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting a humoral pterin spectrum and application thereof. The kit comprises a pterin mixed standard substance and creatinine.
Description
Technical Field
The present invention relates to the fields of chemical analysis and clinical medicine and medicine. In particular to a new pterin metabolite combination marker and application thereof in preparing a kit for screening hyperphenylalaninemia subtypes.
Background
Pterin compounds are derivatives of pteridine, and are widely distributed in human body in body fluids such as blood, urine, cerebrospinal fluid and the like, but the content of pteridine compounds is very small. Pterin compounds have important auxiliary functions in the process of cell metabolism. The pterin compound in the body fluid is low and stable, and when the immune system is activated by some diseases, such as cancer (kidney cancer, breast cancer) and the like, the pterin compound content is changed remarkably. Hyperphenylalaninemia (HPA) is a common autosomal recessive inherited metabolic disorder that can lead to severe intellectual disability. The differences in genetic variation sites have led to the division of HPA into subtypes, mainly including Phenylketonuria (PKU) and tetrahydrobiopterin (BH4) deficiency, where BH4 deficiency in turn encompasses different subtypes, with distinct prognoses and treatment of HPA for different subtypes. Among them, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is a major domestic subtype of BH4 deficiency, and thus, it is not particularly stated in the present invention that BH4 deficiency is a PTPS deficiency subtype. BH4 is a coenzyme for phenylalanine, tyrosine, tryptophan hydroxylase, where the absence of any synthetase or reductase in anabolism results in insufficient or absent BH4 production. Thus, the humoral pterin spectrum of BH 4-deficient patients also changes significantly.
At present, the HPA screening is mainly carried out by measuring the blood phenylalanine concentration by adopting a fluorescence method or tandem mass spectrometry at home, and the contents of Neopterin (NEO) and Biopterin (BIO) in the urine sample after oxidation and the proportion of BIO are measured by adopting a high performance liquid chromatography-fluorescence detection method for judgment. However, the spectrum of pterin in urine samples is quite complex, and besides the above-mentioned NEO and BIO, there are various pterin substances such as dihydroneopterin (7,8-dihydroneopterin, NH2), dihydrobiopterin (7,8-dihydrobiopterin, BH2), Isoxathopterin (ISO), Pterin (PT), etc., and their mutual relationship relates to the action of multiple enzymes. Therefore, it is difficult to accurately discriminate all subtypes of BH4 deficiency based only on the NEO and BIO contents of the oxidized urine sample. On the other hand, methods based on HPLC-fluorescence detection are currently mainly used for the determination of NEO and BIO, but are not highly sensitive or specific for other pterin-based substances in complex urine samples. In body fluids such as urine, biopterin mainly exists in three different forms of BH4, BH2 and BIO, and BH4 and BH2 can be oxidized into BIO in a certain proportion by the currently common acidic oxidation method. However, under certain conditions in vivo or in vitro, BH4 may be oxidized to PT, and PT may be further oxidized to ISO.
Therefore, in both the HPA screening stage and the treatment stage, a more sensitive, specific and accurate method for analyzing the profile of urine and other humoral pterins is needed for HPA subtype screening and auxiliary differential diagnosis.
Disclosure of Invention
The invention aims to provide a sensitive, specific and accurate method for measuring the spectrum of the humoral pterin.
In a first aspect of the invention, a kit for detecting a profile of humoral pterin is provided, the kit comprising a mixed standard of pterin and creatinine.
In another preferred embodiment, the kit further comprises an internal standard, an oxidizing agent, an acidifying agent, and a mobile phase.
In another preferred embodiment, the composition of the pterin mixed standard is neopterin, biopterin, isoxate, pterin.
In another preferred embodiment, the internal standard is 6-methylpterin.
In another preferred embodiment, the oxidizing agent is selected from manganese dioxide, iodide, potassium permanganate; the acidifying agent is hydrochloric acid.
In another preferred example, the mobile phase comprises a methanol-water system and an acetonitrile-water system.
In another preferred embodiment, the body fluid is selected from urine, blood, cerebrospinal fluid.
In a second aspect of the invention, there is provided the use of a kit as described above according to the invention for the determination of a profile of pterin.
In a third aspect of the invention, there is provided a method for determining the profile of pterin in a bodily fluid selected from the group consisting of urine, blood, cerebrospinal fluid, comprising the steps of:
(1) preparing a standard substance solution and a creatinine solution; the standard comprises a pterin mixed standard and an internal standard;
(2) adding an oxidizing agent and an acidifying agent to the sample solution to oxidize the sample;
(3) determining the amounts of neopterin, biopterin, isoxapterin, pterin, and creatinine in the standard solution, the creatinine solution, and the oxidized sample by LC-MS; and
(4) values for neopterin/creatinine, biopterin + pterin/creatinine, and isoxathin/creatinine were calculated, respectively.
In another preferred example, the oxidation time in step (2) is 1 to 10 minutes; more preferably 2-5 minutes.
In another preferred example, the mobile phase of the liquid chromatography in the step (3) comprises a mobile phase A and a mobile phase B; the mobile phase A is an acetonitrile solution containing ammonium acetate, ammonia water and water; the mobile phase B is an aqueous solution containing ammonium acetate, ammonia water and acetonitrile.
Therefore, the invention provides a more sensitive, specific and accurate method for analyzing the profile of the urine and other humoral pterins, which can be used for HPA subtype screening and auxiliary differential diagnosis.
Drawings
Figure 1 is a selective ion flow graph of four pterin metabolites, internal standard, and creatinine. The four pterin standards included BIO, NEO, ISO and PT, with 6MPT for Internal Standard (IS) and CRE for creatinine.
Figure 2 is an optimization of urine oxidation conditions. And the mass spectrum response peak areas of four kinds of pterin NEO, BIO, ISO and PT reach a steady state after the oxidation time is 2min, and the oxidation time is determined to be 5 min.
FIG. 3 shows the measured and calculated values of NEO/CRE, ISO/CRE, BIO%, (BIO + PT)% in each experimental group in example 5.
Figure 4 is a graph of the diagnostic results of each of the combination markers of example 5 in the test to identify the healthy control group, PKU group, and BH4 deficiency group. Wherein Prob1 and Prob2 are respectively the probability of distinguishing a healthy control group and a BH4 deficiency group by adopting NEO/CRE, (BIO + PT)/CRE combined markers, NEO/CRE, (BIO + PT)/CRE and ISO/CRE combined markers; prob3 and Prob4 are respectively the probability of distinguishing a healthy control group from a healthy control group and a PKU group by adopting NEO/CRE, (BIO + PT)/CRE combined markers, NEO/CRE, (BIO + PT)/CRE and ISO/CRE combined markers; prob5 and Prob6 are probabilities of discriminating PKU group and BH4 group by using NEO/CRE, (BIO + PT)/CRE combined marker, NEO/CRE, (BIO + PT)/CRE and ISO/CRE combined marker respectively.
Detailed Description
The invention aims to provide a novel pterin metabolite combination marker to solve the problem that various subtypes of hyperphenylalaninemia cannot be distinguished by the existing genetic metabolic disease mass spectrum screening technology and provide reference for clinical diagnosis.
The invention optimizes the pretreatment method and the mass spectrum detection method, and simultaneously detects 4 pterin and urinary creatinine in urine. And according to the mutual transformation relation of pterin and the detection result of the actual sample, performing statistical analysis to determine that NEO, BIO, PT and ISO are used as combined diagnostic markers. A combination of two or three of the following 3 criteria was used: NEO, (BIO + PT) and ISO realize that the health control group, the PKU group and the BH4 deficiency disease group can be effectively distinguished only by pterin spectrum detection for the first time, and an effective detection means is provided for the clinical HPA subtype screening and auxiliary differential diagnosis.
The invention provides a combination of metabolites that can be used to distinguish between various subtypes of HPA, the combination comprising four pterin metabolites of NEO, BIO, PT, ISO. Preferably, the combination comprises a combination of two or three of the following 3 criteria: NEO, (BIO + PT), ISO.
Judging the model based on mass spectrum detection data: firstly, a model I and a model II for distinguishing a healthy control group from a BH4 deficiency group are established based on the healthy control group and BH4 deficiency group data, and regression equations are as shown in formula 1 and formula 2:
model I
X1＝-19.881+12.057*A-46.727*B
Prob1＝1/(1+e-X1) (Cutoff value (Cutoff) ═ 0.5) (formula 1)
Model II
X2＝-18.504+11.907*A-45.819*B–5.498*C
Prob2＝1/(1+e-X2) (Cutoff value (Cutoff) ═ 0.5) (formula 2)
A, B, C represents the ratio of the pterin metabolite concentration to the urine creatinine concentration in the urine sample from the subject, i.e., NEO/CRE, (BIO + PT)/CRE, ISO/CRE, in mmol/mol. Prob1 and Prob2 represent the probability of judging a patient as having a BH4 deficiency, with a Cutoff value (Cutoff) of 0.5, i.e., when Prob1 or Prob2 values are greater than 0.5, the subject is judged to have BH4 deficiency.
Secondly, establishing a model III and a model IV for distinguishing the healthy control group from the PKU group based on the data of the healthy control group and the PKU group, wherein a regression equation is as shown in a formula 3 and a formula 4:
model III
X3＝-9.528-0.823*A+5.976*B
Prob3＝1/(1+e-X3) (Cutoff value (Cutoff) ═ 0.5) (formula 3)
Model IV
X4＝-13.228-1.469*A+8.148*B–3.99*C
Prob4＝1/(1+e-X4) (Cutoff value (Cutoff) ═ 0.5) (formula 4)
A, B, C represents the ratio of the pterin metabolite concentration to the urine creatinine concentration in the urine sample from the subject, i.e., NEO/CRE, (BIO + PT)/CRE, ISO/CRE, in mmol/mol. Prob3 and Prob4 represent the probability of being judged as a PKU patient, and the Cutoff value (Cutoff) is 0.5, i.e., when Prob3 or Prob4 value is greater than 0.5, the subject is judged to have PKU.
Finally, a model V and a model VI for distinguishing the PKU group and the BH4 deficiency group are established based on the PKU group and the BH4 deficiency group data, and a regression equation is as shown in formula 5 and formula 6:
model V
X5＝9.301+2.789*A–17.809*B
Prob5＝1/(1+e-X5) (Cutoff value (Cutoff) ═ 0.5) (formula 5)
Model VI
X6＝10.617+2.215*A–19.624*B+8.46*C
Prob6＝1/(1+e-X6) (Cutoff value (Cutoff) ═ 0.5) (formula 6)
A, B, C represents the ratio of the pterin metabolite concentration to the urine creatinine concentration in the urine sample from the subject, i.e., NEO/CRE, (BIO + PT)/CRE, ISO/CRE, in mmol/mol. Prob5 and Prob6 represent the probability of judging a patient as having a BH4 deficiency, with a Cutoff value (Cutoff) of 0.5, i.e., when Prob5 or Prob6 values are greater than 0.5, the subject is judged to have BH4 deficiency.
On the other hand, the invention discloses a tandem mass spectrometry pterin detection kit which can be used for screening and distinguishing various subtypes of hyperphenylalaninemia based on the four pterin (NEO, BIO, PT and ISO) marker combinations, and the kit comprises a pterin mixed standard, an oxidant, an acidifier and the like.
In some embodiments, the oxidizing agent comprises manganese dioxide, iodonium triion, potassium permanganate, with manganese dioxide being preferred;
in some embodiments, the acidifying agent comprises hydrochloric acid, trichloroacetic acid, hypochlorous acid, preferably hydrochloric acid;
in some embodiments, the mobile phase is a gradient or isocratic elution, wherein the gradient elution comprises a methanol-water system, an acetonitrile-water system, preferably: mobile phase a was acetonitrile (containing 1mM ammonium acetate and 20mM ammonia, 5% water) and mobile phase B was water (containing 1mM ammonium acetate and 20mM ammonia, 5% acetonitrile).
In some embodiments, the tandem mass spectrometry pterin detection kit further comprises a U-shaped heat resistant 96-well extraction plate, a V-shaped bottom 96-well detection plate, an adhesive microplate envelope, and a detection plate aluminum foil.
In some embodiments, the tandem mass spectrometry pterin detection kit further comprises a pterin mixed standard control solution.
The invention also discloses a using method of the detection kit for the pterin by the tandem mass spectrometry, which comprises the following steps:
1. preparing standard curve series standard solution:
the standard solution comprises 4 mixed standards of pterin standard: neopterin, biopterin, isoxathin, pterin 4 endogenous pterins; 6-methylpterin as an internal standard; creatinine. See table 1 for details.
TABLE 1 names and abbreviations of metabolites to be tested
| Pterin | English name | Abbreviations |
| Biopterin | Biopterin | BIO |
| New pterin | Neopterin | NEO |
| Pterin | Pterin | PT |
| Isoflavopterin | Isoxanthopterin | ISO |
| 6-Methylpterin | 6-Methylpterin | 6MPT |
| Creatinine | Creatinine | CRE |
Wherein the concentrations of BIO, NEO, ISO and PT in the mixed standard solution of the 4 pterin standard substances are all 10 mu g/mL; the concentration of the internal standard 6MPT mother liquor is 1 mug/mL; the concentration of the creatinine mother liquor is 1 mg/mL.
Preparing standard solutions of different concentrations: diluting and preparing a pterin and creatinine mixed standard working solution, wherein the concentration gradients of BIO, NEO, ISO and PT are as follows in sequence: 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300 and 1000ng/mL, and the concentration gradient of creatinine is 1, 3, 6, 10, 30, 60, 100, 300, 600 and 10000ng/mL in sequence.
2. Weighing an oxidizing agent
And adding the oxidant into corresponding holes of the 96-hole plate in sequence according to the number of samples to be detected.
3. Sampling
And (4) sequencing the blank, the quality control sample and the sample to be detected on a 96-well plate. If the liquid samples are urine and the like, directly adding the liquid samples into a U-shaped bottom heat-resistant 96-hole extraction plate; if the sample is a dry filter paper sheet such as a diaper, the diaper is sequentially punched into a U-shaped bottom heat-resistant 96-hole extraction plate by a 3.2mm puncher.
4. Oxidation by oxygen
Taking a urine sample as an example, adding a proper amount of ultrapure water into a 96-well plate according to different types of samples, so that the total volume of the solution in each well is 200 mu L; add 50. mu.L of 1M hydrochloric acid solution, seal the 96-well plate with an adhesive patch, shake the reaction at room temperature for 5min, and centrifuge at 12000rpm at 4 ℃ for 5 min.
5. Sample on-machine detection
The sealing membrane is opened. Remove 100 μ L of supernatant per well with a pipette and move into a new 96 well plate in order. The 96-well plate was sealed with aluminum foil and placed in a liquid autosampler for mass spectrometric detection.
6. Liquid phase mass spectrometry combined detection
The different samples are body fluids, and the body fluids are one of urine, blood, plasma, serum or cerebrospinal fluid.
The detection kit for the pterin by the tandem mass spectrometry has the advantages of high sensitivity, simple operation and short analysis time, and has important significance for improving the screening level of the genetic metabolic diseases.
The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
The main advantages of the invention are:
1. the invention adopts a tandem mass spectrometry method to detect the urine sample pterin spectrum, and overcomes the defects of low sensitivity and specificity of the liquid chromatography-fluorescence detection method adopted at present.
2. The invention detects 4 kinds of pterin in urine sample and simultaneously detects the urinary creatinine. The detection cost and the detection time are saved.
3. The current detection method detects only two pterins, BIO and NEO, and BIO is poor in stability in body fluid. The invention adds the detection of PT and ISO, and can compensate the content reduction caused by BIO degradation in vivo or in the transportation and storage process.
4. The existing judgment standard is as follows: calculating the content of NEO and BIO in the urine sample, and calculating BIO%: BIO/(NEO + BIO). times.100%. When BH4 synthetase, PTPS, was deficient, urinary NEO increased significantly, BIO decreased significantly, with BIO% < 10%, even < 5% or no detectable.
The above judgment standard only uses NEO and BIO as diagnosis markers, the invention provides an improved judgment standard, and uses NEO, BIO, PT and ISO as combined diagnosis markers.
5. The invention takes NEO, BIO, PT and ISO as the combined diagnosis markers and has several advantages: (A) calculate (BIO + PT)%: the (BIO + PT)/(NEO + BIO + PT) x 100% replaces the existing BIO%, and the false positive rate caused by BIO% less than 10% after BIO degradation can be reduced. (B) A combination of two or three of the following 3 criteria was used: NEO, (BIO + PT), ISO. The diagnosis model constructed by the diagnosis marker has good effect, high sensitivity and good specificity, and is suitable for screening the inherited metabolic diseases.
The invention is further illustrated below with reference to specific examples. 109 samples of neonatal urine were tested using the kit. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Instruments, materials and reagents
Tandem mass spectrometry: waters UPLC ultra high pressure liquid chromatography (Waters, usa) tandem API5500 triple quadrupole mass spectrometer (Sciex, usa). Fully automatic high speed refrigerated centrifuge 5810R (Eppendorf, germany); water purifier Milli-Q (Millipore, USA); one in ten thousand electronic analytical balance (CPA225D, Sartorius, germany).
Pterin standard substance: neopterin, biopterin, isoxathin, pterin; creatinine, sodium hydroxide, hydrochloric acid; ascorbic acid; a creatinine standard; methanol, acetonitrile, formic acid, ammonium acetate and ammonia water (more than or equal to 25 percent of NH3) are all HPLC grades.
Nunc polystyrene PS transparent 96-well plate and Nunc acrylic acid 96-well plate, polyester sealing film aluminum foil Nunc (Theromo scientific).
The use method of the kit comprises the following steps:
1. preparing standard curve series standard solution:
the standard solution comprises 4 mixed standards of pterin standard: neopterin, biopterin, isoxathin, pterin 4 endogenous pterins; 6-methylpterin as an internal standard; creatinine. See table 1 for details. Wherein the concentrations of BIO, NEO, ISO and PT in the mixed standard solution of the 4 pterin standard substances are all 10 mu g/mL; the concentration of the internal standard 6MPT mother liquor is 1 mug/mL; the concentration of the creatinine mother liquor is 1 mg/mL.
Preparing standard solutions of different concentrations: diluting and preparing a pterin and creatinine mixed standard working solution, wherein the concentration gradients of BIO, NEO, ISO and PT are as follows in sequence: 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300 and 1000ng/mL, and the concentration gradient of creatinine is 1, 3, 6, 10, 30, 60, 100, 300, 600 and 10000ng/mL in sequence.
2. Weighing an oxidizing agent
5mg of manganese dioxide is weighed in each part and is sequentially added into corresponding holes of a 96-hole plate according to the number of samples to be detected.
3. Sampling
And (4) sequencing the blank, the quality control sample and the sample to be detected on a 96-well plate. If the liquid samples are urine and the like, directly adding the liquid samples into a U-shaped bottom heat-resistant 96-hole extraction plate; if the sample is a dry filter paper sheet such as a diaper, the diaper is sequentially punched into a U-shaped bottom heat-resistant 96-hole extraction plate by a 3.2mm puncher.
4. Oxidation of urine samples
Adding a proper amount of ultrapure water into a 96-well plate according to different types of samples, so that the total volume of the solution in each well is 200 mu L; add 50. mu.L of 1M hydrochloric acid solution, seal the 96-well plate with an adhesive patch, shake the reaction at room temperature for 5min, and centrifuge at 12000rpm at 4 ℃ for 5 min.
5. Sample on-machine detection
The sealing membrane is opened. Remove 100 μ L of supernatant per well with a pipette and move into a new 96 well plate in order. The 96-well plate was sealed with aluminum foil and placed in a liquid autosampler for mass spectrometric detection.
6. Chromatographic conditions are as follows: chromatography columns (Hypercarb, 2.1X 30mm, 3 μm, Thermo, USA); column temperature, room temperature; the flow rate is 0.45 mL/min; sample introduction volume, 10 μ L; autosampler temperature, 4 ℃; mobile phase a was acetonitrile (containing 1mM ammonium acetate and 20mM ammonia, 5% water) and mobile phase B was water (containing 1mM ammonium acetate and 20mM ammonia, 5% acetonitrile). Gradient elution (0 to 2.5min mobile phase B increased linearly from 5% to 45%, 2.5 to 3min mobile phase B increased linearly from 45% to 95%, and held for 0.2min, back to 5% holding ratio equilibrium for 0.79min within 0.01 min).
7. Mass spectrum conditions: an ion source: electrospray ion source (ESI); the detection mode is as follows: multiple ion reaction monitoring (MRM); spraying voltage: 4500V; scanning mode: a negative ion mode; ion source Temperature (TEM): at 450 ℃; air curtain pressure (CUR): 30 psi; nebulizer pressure (GS 1): 60 psi; assist gas pressure (GS 2): 60 psi; injection voltage (EP): -10V; collision cell ejection voltage (CXP): -17V. Data collection and processing was done by Analyst 1.5 analysis software (Sciex, usa). Ion pairs and parameters for pterin and internal standard, creatinine are shown in table 2:
TABLE 2 Mass spectrometric parameters of four combination markers, creatinine and internal standards
Example 1
Optimization of urine oxidation conditions
In order to examine the optimal oxidation time of pterin in urine under the condition of acidic manganese dioxide, a certain amount of urine sample is taken according to the operation steps, an oxidant and an acidifier are added to react for 0, 1, 2, 5, 10, 15, 20 and 25min respectively, and the mass spectrum response peak areas of four kinds of pterin NEO, BIO, ISO and PT under different oxidation treatment times are compared. The results show (fig. 1) that the pterin detection value reaches a steady state after the oxidation time is 2min, and the oxidation time is determined to be 5min later in consideration of the convenience of experimental operation.
Example 2
Determination of flow partner
The four pterin standard solutions and the actual urine samples are analyzed according to the formula shown in the table 3, and the corresponding chromatographic peak separation degree, the corresponding sensitivity and the corresponding linear range are inspected, so that the detection of the actual urine samples can be completed. Preferably, when a C18 reversed phase chromatographic column is used and the mobile phase adopts the formula 6 in the table 3, the better sensitivity and the wider linear range are obtained.
TABLE 3 optimization of mobile phase formulations
Example 3
Detection kit for pterin by tandem mass spectrometry
3.1A detection kit for pterin by tandem mass spectrometry comprises
Four pterin mixed standards: freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
a pterin standard (internal standard): freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
four pterin mixed standard quality controls (urine-containing matrix): freeze-drying, storing at-20 deg.C in 3 bottles with 3 concentrations;
manganese dioxide powder: drying, 1 bottle (2 g);
hydrochloric acid solution (1M HCl): 1 bottle (50 mL);
mobile phase: 2 vials (1L each), mobile phase a acetonitrile (containing 1mM ammonium acetate and 20mM ammonia, 5% water), mobile phase B water (containing 1mM ammonium acetate and 20mM ammonia, 5% acetonitrile);
u-shaped bottom, heat-resistant microporous plate, 96-hole, 2-plate;
v-shaped bottom cutting, cleaning a microporous plate, 96-hole detection plates and 2 plates;
adhesive microplate envelope: 5 pieces of
Aluminum foil micropore plate sealing sleeve: 5 pieces of
Microplate bar code labeling: 10 portions of
3.2A detection kit for pterin by tandem mass spectrometry comprises
Four pterin mixed standards: freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
a pterin standard (internal standard): freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
four pterin mixed standard quality controls (urine-containing matrix): freeze-drying, storing at-20 deg.C in 3 bottles with 3 concentrations;
manganese dioxide powder: drying, 1 bottle (2 g);
hydrochloric acid solution (1M HCl): 1 bottle (50 mL);
mobile phase: 2 vials (1L each), mobile phase a acetonitrile (20 mM ammonium acetate and 10mM ammonia, 5% water), mobile phase B water (20 mM ammonium acetate and 10mM ammonia, 5% acetonitrile);
u-shaped bottom, heat-resistant microporous plate, 96-hole, 2-plate;
v-shaped bottom cutting, cleaning a microporous plate, 96-hole detection plates and 2 plates;
adhesive microplate envelope: 5 pieces of
Aluminum foil micropore plate sealing sleeve: 5 pieces of
Microplate bar code labeling: 10 portions of
3.3A kit for detecting pterin by tandem mass spectrometry comprises
Four pterin mixed standards: freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
a pterin standard (internal standard): freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
four pterin mixed standard quality controls (urine-containing matrix): freeze-drying, storing at-20 deg.C in 3 bottles with 3 concentrations;
manganese dioxide powder: drying, 1 bottle (2 g);
hydrochloric acid solution (1M HCl): 1 bottle (50 mL);
mobile phase: 2 vials (1L each), mobile phase a acetonitrile (10 mM ammonium acetate and 10mM ammonia, 5% water), mobile phase B water (10 mM ammonium acetate and 10mM ammonia, 5% acetonitrile);
u-shaped bottom, heat-resistant microporous plate, 96-hole, 2-plate;
v-shaped bottom cutting, cleaning a microporous plate, 96-hole detection plates and 2 plates;
adhesive microplate envelope: 5 pieces of
Aluminum foil micropore plate sealing sleeve: 5 pieces of
Microplate bar code labeling: 10 portions of
3.4A detection kit for pterin by tandem mass spectrometry comprises
Four pterin mixed standards: freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
a pterin standard (internal standard): freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
four pterin mixed standard quality controls (urine-containing matrix): freeze-drying, storing at-20 deg.C in 3 bottles with 3 concentrations;
manganese dioxide powder: drying, 1 bottle (2 g);
hydrochloric acid solution (1M HCl): 1 bottle (50 mL);
mobile phase: 2 bottles (1L each), mobile phase A is methanol (containing 0.1% formic acid), and mobile phase B is water (containing 0.1% formic acid);
u-shaped bottom, heat-resistant microporous plate, 96-hole, 2-plate;
v-shaped bottom cutting, cleaning a microporous plate, 96-hole detection plates and 2 plates;
adhesive microplate envelope: 5 pieces of
Aluminum foil micropore plate sealing sleeve: 5 pieces of
Microplate bar code labeling: 10 portions of
3.5A kit for detecting pterin by tandem mass spectrometry comprises
Four pterin mixed standards: freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
a pterin standard (internal standard): freeze-drying the powder, and storing the powder at-20 ℃ in 1 bottle;
four pterin mixed standard quality controls (urine-containing matrix): freeze-drying, storing at-20 deg.C in 3 bottles with 3 concentrations;
iodine powder: brown bottle, dry, 1 bottle (3 g);
potassium iodide powder: brown bottle, dry, 1 bottle (5 g);
hydrochloric acid solution (1M HCl): 1 bottle (50 mL);
mobile phase: 2 vials (1L each), mobile phase a acetonitrile (containing 1mM ammonium acetate and 20mM ammonia, 5% water), mobile phase B water (containing 1mM ammonium acetate and 20mM ammonia, 5% acetonitrile);
u-shaped bottom, heat-resistant microporous plate, 96-hole, 2-plate;
v-shaped bottom cutting, cleaning a microporous plate, 96-hole detection plates and 2 plates;
adhesive microplate envelope: 5 pieces of
Aluminum foil micropore plate sealing sleeve: 5 pieces of
Microplate bar code labeling: 10 portions of
Example 4
Evaluation experiment of kit
4.1 Linear Range, minimum detection Limit and minimum quantitation Limit
And analyzing the concentration of the standard curve sample of the substance to be detected by adopting the optimized analysis conditions. The mass concentrations of pterin (NEO, BIO, ISO, PT) and Creatinine (CRE) are in the ranges of 0.1-300ng/mL and 1-3000ng/mL respectively, and the mass spectrum response and the concentration of the pterin show a good linear relationship (r > 0.998). The linear regression equations, minimum detection limits (S/N >3) and minimum quantitation limits (S/N >10) for pterin (NEO, BIO, ISO, PT) and Creatinine (CRE) are shown in Table 4.
TABLE 4 Linear regression equation, minimum detection limit and minimum quantitation limit for pterin (NEO, BIO, ISO, PT) and Creatinine (CRE) in urine samples
4.2 precision and accuracy
And (3) carrying out precision and accuracy examination on the samples with low, medium and high three QC concentrations of pterin within a day (n is 6) and in the day (n is 18) respectively by adopting optimized analysis conditions. As shown in Table 4, the results of the measurements, expressed as relative standard deviations, ranged from 1.44% to 5.61% in intra-day precision and from 3.23% to 9.02% in inter-day precision, respectively (Table 4), indicating good reproducibility of the method. The accuracy of the method is shown by the standard deviation (RE) between the measured concentration and the standard concentration, with an in-day accuracy (RE) in the range of 90.7% to 107% and an in-day accuracy (RE) in the range of 92.8% to 113% (table 5). The precision and accuracy of the method both meet the quantitative detection requirement of the biological sample.
TABLE 5 Intra-day, inter-day of pterin (NEO, BIO, ISO, PT) and Creatinine (CRE) in urine samples
Accuracy and precision.
4.3 matrix Effect
The results of matrix effect examination of pterin and creatinine using the optimized assay conditions are shown in table 5, in which the relative matrix effect 1 (spiked matrix vs. standard concentration solution) of pterin (NEO, BIO, ISO, PT) and Creatinine (CRE) is in the range of 85.8% to 106%, respectively, and the relative matrix effect 2 (urine sample spiked matrix vs. spiked matrix) is in the range of 85.1% to 103%, respectively (table 6).
TABLE 6 matrix Effect of target analytes
Matrix effect 1%, peak area of compound in sample/peak area of compound in adsorption matrix.
Matrix effect 2%, peak area of compound in adsorption matrix/peak area of compound in pure solution.
Example 5
Screening and clinical specimen validation
109 samples of neonatal urine were tested using the kit. The NEO/CRE, BIO%, (BIO + PT)/CRE, (BIO + PT)% and ISO/CRE values in the groups are shown in Table 6 and FIG. 3. From the existing diagnostic criteria, the percentage of BIO was calculated: as shown in fig. 3 and table 6, in the healthy control group, the BIO/(NEO + BIO) × 100%, when the BIO% < 10%, the patients were suspected to be deficient in BH4, the BIO% was 9.21% in one case (No.44), and was less than 10%, and the BIO% was 10.24% in the other case (No.3), which was close to the critical value. Calculating the BIO% value alone would yield false positive results. Calculate (BIO + PT)%: the (BIO + PT)/(NEO + BIO + PT). times.100% replaces the existing BIO%, and the values of the (BIO + PT)% of the two examples are 16.88% (No.44) and 29.38% (No.3), respectively, so that the judgment of false positive is avoided. Therefore, the adoption of (BIO + PT)% < 10% as a judgment standard instead of the existing BIO% < 10% standard can reduce the false positive rate caused by the degradation of BIO in vivo or during transportation and storage.
Meanwhile, the probability, probability value and distribution are shown in fig. 4 in the corresponding regression equations (equations 1-6) calculated according to the relative content of each marker (table 7). A cut-off value of 0.5 is used, i.e. a probability greater than 0.5 is considered a positive result. When the model I and the model II are used for distinguishing a healthy control group from a BH4 deficiency disease group, the correct diagnosis rate is 100 percent, and the AUC of the combined marker is 1; when the model III distinguishes a healthy control group from a PKU group, the correct diagnosis rate is 95.7% (45/47) and 93.9% (46/49), and the AUC of the combined marker is 0.997; when the model IV distinguishes a healthy control group from a PKU group, the correct diagnosis rate is 97.9% (46/47) and 95.9% (47/49), and the AUC of the combined marker is 0.998; when the model V and the model VI are used for distinguishing the PKU group and the BH4 group, the correct diagnosis rate is 100%, and the AUC of the combined marker is 1.
TABLE 7 clinical specimen test results
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.
Claims (2)
1. A kit for determining a pterin spectrum in body fluid by liquid mass spectrometry is characterized by comprising a pterin mixed standard, creatinine, an internal standard, an oxidizing agent, an acidifying agent and a mobile phase; the pterin mixed standard substance comprises neopterin, biopterin, isoxate and pterin; the internal standard is 6-methylpterin; the oxidant is selected from manganese dioxide, iodide and potassium permanganate; the acidifying agent is hydrochloric acid; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is an acetonitrile solution containing ammonium acetate, ammonia water and water; the mobile phase B is an aqueous solution containing ammonium acetate, ammonia water and acetonitrile; the kit further comprises a model:
model I
X1＝-19.881+12.057*A-46.727*B
Prob1＝1/(1+e-X1) (Cutoff value (Cutoff) ═ 0.5)
Model II
X2＝-18.504+11.907*A-45.819*B–5.498*C
Prob2＝1/(1+e-X2) (Cutoff value (Cutoff) ═ 0.5)
Model III
X3＝-9.528-0.823*A+5.976*B
Prob3＝1/(1+e-X3) (Cutoff value (Cutoff) ═ 0.5)
Model IV
X4＝-13.228-1.469*A+8.148*B–3.99*C
Prob4＝1/(1+e-X4) (Cutoff value (Cutoff) ═ 0.5)
Model V
X5＝9.301+2.789*A–17.809*B
Prob5＝1/(1+e-X5) (Cutoff value (Cutoff) ═ 0.5)
Model VI
X6＝10.617+2.215*A–19.624*B+8.46*C
Prob6＝1/(1+e-X6) (Cutoff value (Cutoff) ═ 0.5)
A, B, C in the model are the values of neopterin/creatinine, (biopterin + pterin)/creatinine, and isoxathin/creatinine, respectively, all in mmol/mol; prob1 and Prob2 indicate the probability of being judged as tetrahydrobiopterin deficiency, Prob3 and Prob4 indicate the probability of being judged as phenylketonuria, and Prob5 and Prob6 indicate the probability of being judged as BH4 deficiency in hyperphenylalaninemia.
2. Use of a kit according to claim 1 for the determination of a profile of pterin.
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