Study On The Method Of Liquid Chromatography Tandem Mass Spectrometry For Combined Screening Genetic Metabolic Diseases In Newborns

Research background and purposeGenetic metabolic diseases in newborns are mainly caused by gene mutations leading to enzyme or receptor deficiencies,which in turn cause disorders in the body’s biochemical metabolism processes.Some children may also be life-threatening due to organ failure.Among them,the incidence rate of phenylketonuria(PKU),congenital adrenal hyperplasia(CAH),congenital hypothyroidism(CH)and X-linked adrenoleukodystrophy(X-ALD)is low and seriously threatens the health of infants,and some children cannot be detected before birth,If there are no symptoms of related diseases after birth,and the disease progresses with age,it will cause irreversible damage to the patient’s nervous and vascular system,which can seriously lead to death.The traditional detection techniques for neonatal diseases mainly include Grignard bacterial inhibition assay(BIA),fluorescence assay(FIA),immunoradiometric assay(IRMA),enzyme-linked immunosorbent assay(ELISA),time-resolved immunofluorescence assay(TRFIA),gas chromatography(GC),and liquid chromatography(LC).The main drawbacks of traditional technology in screening for genetic metabolic diseases are the complex and time-consuming sample preparation steps,and poor sensitivity.In addition,traditional technologies can only detect one indicator at a time in an experiment and can only screen for one disease,making the expansion of testing projects complex and expensive.Liquid Chromatography Tandem Mass Spectrometry(LC-MS/MS)uses liquid chromatography as the separation system and mass spectrometry as the detection system.LC-MS/MS reflects the complementary relationship between the advantages of chromatography and mass spectrometry,and has the characteristics of high sensitivity and high throughput.There is still much space for exploration in the application of LC-MS/MS in neonatal disease screening.In order to improve the detection efficiency of neonatal screening,reduce blood collection requirements,and improve detection efficiency,this study developed a method for detecting PKU,CAH,CH,X-ALD biomarkers using the high sensitivity,specificity,and high-throughput characteristics of liquid chromatography-tandem mass spectrometry.Dilute the isotope labeled internal standard with organic solvent and prepare it as an internal standard working solution.Incubate and extract samples of neonatal filter paper dry blood slices,and then use liquid chromatography tandem mass spectrometry to detect the extracted solution.By calculating the concentration of corresponding analytes in the test sample through standard curves,it is possible to simultaneously detect multiple disease markers and improve the efficiency of detection.Simultaneously evaluate the various performance indicators of the method and test a certain number of clinical samples to evaluate its clinical applicability.Method:1 Sample pretreatment research1.1 Preparation of standard and quality control whole blood:Centrifuge sheep blood at room temperature for 10 minutes,discard the supernatant and add the same volume of 0.9%sodium chloride to obtain a simulated blank matrix.Add the prepared analyte concentrate in proportion to the processed blood,dilute it to 5 concentration dose points of whole blood calibration samples,and control the quality of whole blood at low,medium,and high concentrations.1.2 Preparation of filter paper dry blood tablets:Use a pipette to absorb 60%μL prepared blood,drop onto Whatman 903 filter paper.After processing,the filter paper dry blood pieces are left to dry,collected the next day and placed in a plastic bag with desiccant added,and stored at 4℃.1.3 Preparation of internal standard working solution:Add each substance’s internal standard in proportion to the pre prepared 80%methanol solution to prepare the internal standard working solution.1.4 Sample processing:Use a punch to punch a 3mm blood sample into a 96 well microplate,and add 100 μL Incubate with L internal standard working solution at 37℃and 750 rpm for 45 minutes.After incubation,80 μL supernatant transfer for machine testing.1.5 Mass spectrometry,liquid chromatography method1.5.1 Mass spectrometry method:Dilute the prepared analyte standard with 50%methanol and inject it into the mass spectrometer.Adjust the capillary voltage,cone hole voltage,collision energy,desolvent gas flow rate,and collision energy to obtain the optimal signal response values for the parent and daughter ions,and record the corresponding parameters.1.5.2 Liquid chromatography method:Using Waters HSS UPLC T3(2.1*50 mm,1.8 μm)Chromatographic column and Waters HSS UPLC C8(2.1*100 mm,1.7μm)A corresponding elution gradient was designed for the separation of processed samples.The remaining liquid phase conditions include:injection volume of 10 μL and 5 μL;Column temperature 40 ℃;The sample disk temperature is 10 ℃,and the mobile phase A is a 2 mmol/L ammonium acetate solution containing 0.1%formic acid and a 5 mmol/L ammonium acetate solution containing 0.1%formic acid.The mobile phase B is methanol.2.Method validationComprehensively evaluate the performance indicators of self built liquid chromatography tandem mass spectrometry reagents under the optimal reaction system,including linearity,accuracy,precision,detection and quantification limits,accuracy,and analysis of clinical sample determination and screening results.Results:1.Each compound has good linearity,precision,and recovery within a certain content range.The linear regression coefficient is equal to or greater than 0.992,the coeficient of variation is less than 10%,and the recovery rate is between 80%-120%.The linear range of 17-OHP is 6.1-490.2nmol/L,with a coeficient of variation of 2.2%6.0%and a recovery rate of 92.5%-115.4%;The linear range of ASD is 0.7-56.6nmol/L,with a coefficient of variation of 5.0%-7.5%and a recovery rate of 93.0%-95.5%;The linear range of COR is 13.8-1117.3nmol/L,with a coefficient of variation of 2.3%-5.6%and a recovery rate of 100.5%-105.3%;The linear range of PHE is 12.1-980.7nmol/L,with a coefficient of variation of 2.8%-7.2%and a recovery rate of 97.4%-107.3%;The linear range of TYR is 3.7-298nmol/L,with a coefficient of variation of 3.2%-6.7%and a recovery rate of 97.0%-102.5%;The linear range of TT4 is 32.2-2606.6nmol/L,with a coefficient of variation of 2.8%-5.1%and a recovery rate of 97.5%-99.1%;The linear range of C26:0-LPC is 31.5-1258.1nmol/L,with a coefficient of variation of 5.4%-9.2%and a recovery rate of 94.1%-101.7%;The linear range of C24:0-LPC is 32.9-1316.1nmol/L,with a coefficient of variation of 4.4%-7.2%and a recovery rate of 97.5%-106.8%;The linear range of C22:0-LPC is 34.5-1379.8nmol/L,with a coefficient of variation of 3.6%-8.6%and a recovery rate of 95.6%-103.7%;The linear range of C20:0-LPC is 36.2-1450.0nmol/L,with a coefficient of variation of 3.0%-7.4%and a recovery rate of 95.0%-99.8%.2.The standard substances for the fifth generation newborn PHE and 17-OHP provided by the ISNS International Society for Neonatal Screening were measured.After calibration,the average deviation between the two target substances and the set value was less than 15%,which can achieve equivalent consistency.3.The clinical sample testing results showed that the combination of 17-OHP and(17-OHP+ASD/COR)showed higher positive predictive values and lower false positive rates compared to using 17-OHP.The combination of PHE and PHE/TYR for clinical evaluation also showed higher positive predictive values and lower false positive rates compared to using PHE.For CH samples,The false negative rate generated by TT4 screening was 60%,and the sensitivity did not reach 100%.Conclusion:This study established a detection method for screening neonatal PKU,CAH,CH,and X-ALD using liquid chromatography tandem mass spectrometry.Compared with conventional inspection techniques,the advantage of this method lies in overcoming the problems of insufficient sensitivity and cross reaction interference in conventional methods.It only requires a relatively small number of samples and does not require derivative or time-consuming concentration methods,reducing the cost of sample detection.Methodological validation and practical application have shown that the method is fast,accurate,and has good stability,demonstrating the feasibility of joint screening for multiple neonatal genetic metabolic diseases.For clinical screening of CH,further research and development of TSH combined testing can be carried out on the basis of TT4 testing,providing an efficient and accurate approach for CH screening.For X-ALD,LPC can be added to the PKU,CAH,and CH combined detection method in the future to provide an absolute quantification of neonatal genetic metabolic diseases and enrich the types of neonatal disease screening.