A method and a kit for multiplexed 2nd tier test application in newborn screening

Abstract

The present disclosure relates to analytical LC-MS / MS methods for second tier testing in Newborn Screening Program (2ndtier test or 2TT) for the simultaneous measuring of organic acids, acylcarnitines, aminoacids derivatives and acylglicines in dried blood spot samples.

Description

[0001] “A method and a kit for multiplexed 2ndtier test application in Newborn Screening”

[0002] DESCRIPTION

[0003] The present disclosure relates to analytical LC-MS / MS methods for second tier testing in Newborn Screening Program (2ndtier test or 2TT) for the simultaneous measuring of organic acids, acylcarnitines, aminoacids derivatives and acylglycine in dried blood spot samples.

[0004] Newborn Screening Program (NBS) allows nowadays the early detection of more than 60 rare hereditary inborn disorders, in order to promptly start specific therapies to prevent the onset of very serious permanent disabilities and early mortality. The list of diseases subjected to newborn screening includes inborn errors of metabolism, endocrine conditions and many other pathologies [1-2] that are updated periodically thanks to the better understanding of the pathogenic basis of diseases and to the scientific progresses about therapeutic approaches efficacy, discovery of novel potential biomarkers and technological innovations [3-6], The efficiency of the NBS is based on the integration of a first tier (1st) test in all newborn samples consisting of a disk of dried blood spot (DBS), with the execution, only in selected cases, of a more specific 2ndtier test, to be performed on the same neonatal sample.

[0005] Second-Tier testing is crucial to maximize performance metrics in NBS improving the positive predictive value (PPV%), avoiding unnecessary newborns recalls and expediting the diagnosis.

[0006] 1sttier Newborn Screening test is designed to avoid false negatives by enhancing diagnostic sensitivity rather than diagnostic specificity. This approach leads to a low PPV% and to a significant increase of false positive results due to lack of specificity of the primary target (transient alterations, overlap of newborn disease and normal ranges, primary target sharing by different diseases, interfering substances indistinguishable by the 1sttier test technique). In case of positives results the standard operating procedure involves the newborn recalling to collect a second DBS sampling and / or for biochemical / clinical diagnostic assessment through further investigations on different matrices (plasma and urine). This practice leads to a substantial economic and social burden caused by the further samples collection for the execution of additional diagnostics analysis when 1sttier test results show altered biomarker levels suggesting a presumptive positivity. The recall procedure raises up to supplementary time consuming laboratory workload, redundant hospitalization and anxiety in whole family groups [7-8],

[0007] High analytical method selectivity and sensitivity are important features for enhancing 2TT performance in defining the biochemical risk with high specificity and differential diagnosis. In addition, the rapid application of the second tier test is crucial for defining diseases with high risk of acute metabolic decompensation that need immediate consideration [9], Among different 2TT strategies, the hyphenated technique Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) fit for purpose allowing the rapid simultaneous measurement of a broad panel of more specific, or pathognomonic biomarkers with high sensitivity and selectivity distinguishes numerous structural isomers, stereoisomers and isobaric compounds.

[0008] However, despite the fundamental need of 2TT assays to date there are no commercial kits available for such applications.

[0009] 2TT by LC-MS / MS can exploit the multiplexed approach, but method development is often challenging and complex due to several reasons starting from the lack of commercial kits, standards and certified quality control materials. Moreover, literature shows that it takes efforts to separate isomers and isobars and additional interfering molecules are often not well resolved and some of well-known or potential biomarker are not always included.

[0010] Depending on the first-tier alterations, published in house 2TT protocols consist of separate methods with specific analytical extractions and different chromatographic approaches, that can be sequentially applied when needed in different run batches, leading to a fragmented laboratory workload due to several and distinct 2TT assays [10-12],

[0011] The expansion of NBS panels ever more boosts the need to gather all the existing and potential 2TT biomarkers in a single analytical test, to standardize and develop a simplified operative procedure minimizing turnaround time (TAT), laboratory workload and most of all diagnostic and therapeutic times.

[0012] The largest panels reported in the literature currently allow the evaluation of several disorders by resolving and measuring some limited 2TT biomarker clusters through laborious and time consuming processes [13-14], In fact, these analytical protocols include several steps for the sample preparation such as incubation, concentration through evaporation and reconstitution, purification by filtration and centrifugation, requiring the use of additional instruments and consumables that significantly lengthening the NBS test and boost laboratory equipment costs. Furthermore, these redundant processes make the analytical workflow complicated and slow-down final screening results representing a critical aspect both for low and high throughput laboratories.

[0013] The prior-art publications of HONG XINYING ET AL: "A highly multiplexed biochemical assay for analytes in dried blood spots: application to newborn screening and diagnosis of lysosomal storage disorders and other inborn errors of metabolism", GENETICS IN MEDICINE, vol. 22, no. 7, 20 April 2020 (2020-04-20), pages 1262-1268, ISSN: 1098-3600, DOI: 10.1038 / S41436-020-0790- 9; & Hong Xinying ET AL: "Supplementary Information: A highly multiplexed biochemical assay for analytes in dried blood spots: application to newborn screening and diagnosis of lysosomal storage disorders and other inborn errors of metabolism", 20 April 2020 (2020-04-20), pages 1-10 discloses an assay for the newborn screening of lysosomal storage disorders, which comprises the analysis of four Dry Blood Spots and seven incubations and extractions. The first punch was used for the NeoLSD MSMS Kit (PerkinElmer), the second punch was used for measuring biotinidase and GALT activity, the third punch was extracted with water and the blood extract was split into three ways between assays for 8 conditions and the fourth punch was for biomarker analysis and was extracted with methanol containing the internal standards. The enzymatic assays were quenched and combined together, followed by a liquid-liquid extraction purification step. The purified sample was combined with the methanol extract before analyzing by multiple reaction monitoring (MRM) on a mass spectrometer coupled with UPLC system.

[0014] The prior-art publication PARK JUNG BAE ET AL: "Direct measurement of active thiol metabolite levels of clopidogrel in human plasma using tris(2-carboxyethyl)phosphine as a reducing agent by LC-MS / MS", JOURNAL OF SEPARATION SCIENCE, vol. 36, no. 14, 21 June 2013 (2013- 06-21 ), pages 2306-2314, discloses the use of TCEP, without formic acid, for the quantification of one single analyte represented by a metabolite of the platelet aggregation inhibitor clopidogrel in a plasma sample for carrying out pharmacokinetic studies. The sample preparation procedure comprises the steps of centrifugation and deproteinization.

[0015] The prior-art publication of KILGORE MATTHEW B. ET AL: "Development of a Universal Second-Tier Newborn Screening LC-MS / MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids", ANALYTICAL CHEMISTRY, vol. 95, no. 6, 1 February 2023 (2023-02-01 ), pages 3187-3194, ISSN: 0003-2700, DOI: 10.1021 / acs.analchem.2c03098; & Kilgore Matthew B ET AL: "Supporting Information: Development of a Universal Second-Tier Newborn Screening LC- MS / MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids", 1 February 2023 (2023-02-01 ), pages S1-S26, discloses the use of TCEP only in the development of a method for the quantification of t-HCY in DBS, which is added after the step of extraction. TCEP is not used as a reducing agent and it is said to causes a problematic decline in the t-Hcy peak area compared to DTT.

[0016] Summary of the invention

[0017] The inventors of the present patent application have surprisingly developed a rapid highly- multiplexed 2TT UHPLC-ESI-MS / MS assay and kit for the analysis of a flexible innovative biomarkers panel for measuring with a single one-step preparative procedure and within a single mass spectrometric chromatographic run 60, but not limited to, relevant non-derivatized metabolites among acylcarnitine, acylglycine, organic acids and aminoacids derivatives. The present method has the ability to simultaneously define the biochemical risk of a comprehensive panel of conditions.

[0018] Object of the invention

[0019] In a first object, the present invention discloses an analytical method for the analysis of a biological sample. In a second object, there is disclosed a kit for performing the method of the invention.

[0020] In a third object, the present invention discloses a method for the identification of a VLCAD (Very Long Chain Acyl-CoA Dehydrogenase) deficiency condition and of other pathological conditions.

[0021] In a fourth object, the present invention discloses the use of an extraction solution (ES) for the preparation of an analyte solution from an isolated biological sample.

[0022] In a further object, the present invention discloses the use mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid for the analysis of n analytes in a solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

[0023] In a further object, the present invention discloses an elution path mobile of a phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid for the analysis of n analytes in a solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization for the separate and simultaneous identification of 5-cis-Tetradecenoylcarnitine and 9-cis-Tetradecenoylcarnitine.

[0024] In a further object, the elution path of the present invention allows also to identify and separate Trans-2-C14:1.

[0025] In a further object, the present invention discloses the use of TCEP in the analytical method for the quantification of the total homocysteine (t-HCY).

[0026] In a further object, the present invention discloses an extraction solution (ES).

[0027] In a further object, the present invention discloses the use of the invention analytical method for biochemical diagnostic confirmation within a screening program in the recall step, for diagnostic evaluation based on clinical evidences including non-newborns patients, for follow-up of patients under treatment.

[0028] Brief description of the figures

[0029] Figure 1 reports the concentration of the internal standards (IS) in the solution of the internal standards (IS MIX) and in the extraction solution (ES) according to an embodiment of the present invention.

[0030] Figure 2 reports the validation results and cut-off.

[0031] Figure 3A reports the ion chromatogram of the 60 analytes according to an embodiment of the present invention. Figure 3B reports the ion chromatogram of some of the analytes with similar sensitivity to facilitate visualization.

[0032] Figure 3C reports the EIC of the highest sensitivity to facilitate visualization.

[0033] Figure 4A shows the differentiation of VLCADD patients (compound heterozygous) and carries and false positive.

[0034] Figure 4B shows the results of 2TT multiplex assay applied on 100 cases flagged as presumptive positive for VLCADD by 1sttier test; PA / : Compound Heterozygous with a variant of uncertain (or unknown) significance (VUS); MADD-Like: Nutritional Riboflavin (vitB2) deficiency; PI: Carriers Ketosis : High 3-hydroxybutyric acid (3HBA) due to fasting.

[0035] Figure 5A shows the results of the improvements in the positive predictive values using the method.

[0036] Figure 5B shows the results of 6 years of application of the 2TT multiplexed assay.

[0037] Figure 6 shows a customization example for a limited panel of 5 analytes.

[0038] Figure 7 shows the results of a clinical validation for the propionate and methionine disorders using41 DBS.

[0039] Figure 8A-E show the chromatograms of 60 analytes according to the invention.

[0040] Figure 9 shows the outline form of a MSUD neonatal case reported in the experimental section where the results of the computational interpretative tool are reported.

[0041] Detailed description of the invention

[0042] “2TT” is used to indicate the second tier testing in Newborn Screening Program.

[0043] “NBS” is the short form for Newborn Screening.

[0044] For the purposes of the present invention, Table 1 lists the analytes that can be screened:

[0045] Table 1

[0046]

[0047] For the purposes of the present invention, the following isomers\isobars are resolved:

[0048]

[0049] According to a first object, there is disclosed an analytical method for the analysis of an isolated biological sample.

[0050] In a preferred embodiment, said sample is isolated from a newborn.

[0051] For the purposes of the present invention, a “newborn” is intended to be a baby between 48thand 72th hours after birth.

[0052] The biological sample may be represented by blood, serum, plasma, urine, liquor, dried blood spot.

[0053] In a preferred embodiment, the sample is represented by blood.

[0054] In a particular embodiment, the sample of blood is isolated and dried on Guthrie filter paper.

[0055] For instance, the sample may be collected and then absorbed and dried and a 3.2 mm size disk is punched.

[0056] According to the present invention, the method can be used to detect the present and optionally to quantify the presence of a number n of analytes in the isolated sample.

[0057] The analytes that can be detected may be between 1 and 200, preferably of between 1 and 100 and even more preferably of between 1 and 60.

[0058] According to a particular embodiment of the present invention, the analytes that can be detected are one or more and preferably all of the analytes of Table 1 above.

[0059] According to the present invention, the method comprises the steps of:

[0060] 1 ) extraction thus obtaining an analytes solution,

[0061] 2) analysis of the analytes solution. As per step 1 ), the extraction is performed by incubating the isolated biological sample with an extraction solution (ES).

[0062] In particular, the extraction solution (ES) comprises:

[0063] For the purposes of the present invention, the final concentration of formic acid in the extraction solution (ES) is 0.1 % (v / v).

[0064] In an embodiment of the invention, the isolated biological sample is incubated with 100 pl of the extraction solution (ES).

[0065] More in detail, the “IS MIX” represents a mixture of an equal volume vis of one or more internal standards (IS) (in other words, any internal standard (IS) is used with the same volume vis).

[0066] Therefore, the volume of each internal standard (IS) is diluted by a factor proportional to the total number of internal standards (IS).

[0067] For instance, if a volume of 100 pl is used for each internal standard (IS) and the total number of internal standards is 25 then the total volume of the “IS MIX” solution is 2500 pl.

[0068] A volume of 100 pl for each internal standard (IS) is for instance used in low-throughput laboratory.

[0069] For instance, if a volume of 500 pl is used for each internal standard (IS) then the total volume of the IS MIX solution is 12500 pl.

[0070] A volume of 500 pl (or even more) for each internal standard (IS) is for instance used in high- throughput laboratory.

[0071] For a practical application, the internal standards solution (IS MIX) may be prepared in large volume and then stored, possibly in aliquots of 50 or 150 pl.

[0072] Storing is performed at suitable temperature such as -20°C.

[0073] For the purposes of the present invention up to and preferably 25 internal standard (IS) can be used.

[0074] For the purposes of the present invention a volume of 0.1 pl of each internal standard (IS) is used for the analysis of one biological sample. Figure 1 reports the concentration of each internal standard (IS) in the solution mixture of all the 25 internal standards (IS) in the extraction solution (ES) according to an embodiment of the present invention wherein 25 internal standards are used (m=25) to detect 60 analytes (n=60).

[0075] For the purposes of the present invention ultrapure water is used.

[0076] The solution of ultrapure water is added with formic acid up to a concentration of 0.1 % (v / v).

[0077] As per the solution of TCEP, it can be prepared directly at the desired working concentration in ultrapure water 0.1 % (v / v) of formic acid (pH~2.5) or starting from a concentrated solution in ultrapure water or adjusted at different pH, which can be suitably diluted with ultrapure water 0.1 % (v / v) of formic acid up to the desired working concentration (pH^5).

[0078] For instance, the solution of TCEP may be prepared from a solution of TCEP 0.5 M in ultrapure water or at pH adjusted to 7, for instance with ammonium hydroxide.

[0079] Alternatively, a solution of TCEP 12.5 mM in ultrapure water can be used.

[0080] The working solution of TCEP comprise formic acid 0.1 % (v / v).

[0081] The final concentration of formic acid in the extraction solution (ES) for the extraction of the sample shall be 0.1 % (v / v).

[0082] The TCEP working solution prepared in acidic conditions according to the present invention retains its reducing power over long-term storage by preventing oxidation.

[0083] It adjusts the pH of the extraction solution (ES) to a pH ~2.3-3, which keeps the extracted dried blood spot (DBS) sample in the well to pH ~4.5.

[0084] At this pH, TCEP is highly effective at reducing disulfide bonds while minimizing the interference with 3OHPA and 5HT IS.

[0085] Additionally, the suppressive effect on the homocysteine (HCY) molecule is prevented.

[0086] Furthermore, this acidic pH provides optimal peak shape and maximizes the recovery for several analytes, including MMA, total HCY (t-HCY), and MCA.

[0087] For the purposes of the present invention, the extraction step 1 ) is carried out at a temperature of 25-45°C.

[0088] Preferably, the extraction step 1 ) is carried out at a temperature of 37°C.

[0089] For the purposes of the present invention, under the above conditions, the extraction step 1 ) may be carried out for a period of 15 minutes to 1 hour.

[0090] Preferably, the extraction step 1 ) is carried out for a period of 25 minutes. An extended incubation beyond 1 hour does not enhance recovery but increases the turnaround time (TAT); higher temperatures may cause accelerate degradation of certain analytes, compromising the stability of the sample.

[0091] Alternatively, similar conditions may be used in terms of time and temperatures with less efficient results in terms of recovery and turnaround time (TAT).

[0092] According to the embodiment of the present invention above reported, the “IS MIX” is comprised in a volume of 2.5% in the extraction solution (ES).

[0093] For the purposes of the present invention, the % volume of the “IS MIX” is generally defined as x= [(1 / 10)*m] wherein m is the number of internal standards used; the volume of the other components of the extraction solution is accordingly adjusted.

[0094] Therefore, the method of the invention for the simultaneous detection of a number n of analytes in an isolated biological sample comprises the steps of:

[0095] 1 ) extraction by incubating said isolated biological sample with an extraction solution (ES) thus obtaining an analytes solution,

[0096] 2) analysis, wherein said extraction solution (ES) has a concentration of 0.625 mM TCEP and comprises a m number of internal standards, wherein said extraction solution is adjusted to 100% (volume) with a solution of ultrapure water 0,1 % (vol / vol) formic acid.

[0097] For the purposes of the present invention, the extraction step 1 ) is the sole extraction step performed of the analytes from the isolated biological sample.

[0098] As per step 2), the analysis is performed with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

[0099] For the purposes of the present invention, the analysis step 2) is performed with UHPLC / MS- MS.

[0100] For the purposes of the present invention, step 2) is the sole analysis step performed with LC-MS / MS and preferably with UHPLS / MS-MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

[0101] In particular, elution is performed using:

[0102] Mobile phase A of 0.1 % (v / v) formic acid in ultrapure water and

[0103] Mobile phase B of acetonitrile 0.1 % (v / v) formic acid. According to the present invention, the elution path comprises:

[0104] -a first elution path phase wherein mobile phase A is highly predominant,

[0105] -a second elution path phase wherein mobile phase A is predominant over mobile phase B,

[0106] -a third elution path phase wherein mobile phase A is highly predominant.

[0107] Highly predominant shall be intended as >60-70% volume.

[0108] Predominant shall be intended as around 40-60% volume.

[0109] In particular, the elution path comprises three main phases:

[0110] -a first elution path using a mobile phase A in concentration from 95-100%, and preferably from 100%, to 72%,

[0111] -a second elution path using a mobile phase A in concentration from 55 to 43%,

[0112] - a third elution path using a mobile phase A in concentration of 100%.

[0113] According to a preferred embodiment:

[0114] -the first elution path using a mobile phase A in concentration from 95-100%, and preferably from 100%, to 72% for 6 minutes,

[0115] -the second elution path using a mobile phase A in concentration from 55 to 43% for 1 or 2 minutes, -the third elution path using a mobile phase A in concentration of 100% for 2 or 3 minutes.

[0116] The second elution path wherein the mobile phase A is lowered for 2 minutes has the purpose of detecting and possibly quantifying the cis-5 tetradecenoylcarnitine and cis 9- tetradecenoylcarnitine.

[0117] According to a first embodiment, the elution path may be the following:

[0118] According to a second embodiment, the elution path may be the following:

[0119] According to an embodiment of the present invention, the first period of 2 minutes using 100% volume mobile phase A can be omitted in case some of the analytes are not investigated.

[0120] Alternatively, the first period of 2 minutes may be performed using 95% volume mobile phase A.

[0121] The elution path of the present invention allows also to identify and separate Trans-2-C14:1.

[0122] As per an aspect of the present invention, the n analytes may be quantified with the stable isotope dilution technique using a single-point or a multipoint calibration curve calibration curve.

[0123] More in particular, the n analytes may be detected according to the following parameters (Table 2):

[0124] Table 2

[0125]

[0126]

[0127]

[0128] According to an embodiment of the invention, the n analytes may be detected according to the following parameters (Table 3): able 3

[0129]

[0130]

[0131]

[0132]

[0133] For the purposes of the present invention, the following calibration parameters may be used (Table 4): able 4

[0134]

[0135]

[0136] According to a second object, the present invention discloses a kit for performing the method of the invention.

[0137] According to a first embodiment, the kit of the invention may comprise:

[0138] -TCEP in ultrapure water 0.1 % (v / v) of formic acid,

[0139] -ultrapure water 0.1 % (v / v) formic acid.

[0140] According to an optional embodiment, the kit of the invention may further comprise:

[0141] -a solution of m internal standards (IS),

[0142] -a solution of mobile phase A of ultrapure water containing 0.1 % (v / v) formic acid and a solution of mobile phase B of acetonitrile containing 0.1 % (v / v) formic acid,

[0143] -instructions for using the kit.

[0144] In particular, the instruction to use the kit of the invention may comprise one or more of the following information of each one of the n analytes and m internal standards (IS):

[0145] -the transition,

[0146] -the retention time,

[0147] -the polarity,

[0148] -the window,

[0149] -the dwell time,

[0150] -calibration.

[0151] The calibration information and control materials may comprise one or more of: range, low quality control, medium quality control, high quality control.

[0152] According to an embodiment of the invention, the TCEP solution in the kit may comprise:

[0153] -a solution of TCEP 12.5 mM in ultrapure water 0.1 % (v / v) formic acid (pHS5), or

[0154] -a stock solution of concentrated TCEP to be suitably diluted with ultrapure water 0.1 % (v / v) formic acid up to a desired concentration (pH<5).

[0155] According to the present invention, each of the above disclosed kit components, may be independently from one another, in a liquid or in a lyophilized form. According to a third object, the present invention discloses a method for the identification of a VLCAD (Very Long Chain Acyl-CoA Dehydrogenase) deficiency in a patient.

[0156] For the purposes of the present invention, the patient is a newborn.

[0157] The method of the invention in fact may be used for the diagnosis of a VLCAD (Very Long Chain Acyl-CoA Dehydrogenase) deficiency condition; in other words, the method of the invention may be used to identify a condition of VLCAD deficiency.

[0158] The ratio Cis-5-C14:1 / Cis-9-C14:1 or Cis-9-C14:1 / Cis5-C14:1 may be considered a useful and / or potential indicator to differentiate physiologic elevations of C14:1 related to fasting vs. VLCAD deficiency.

[0159] More in particular, said method comprises the steps of: performing the method of the invention as above discloses, identifying the analytes represented by 5-cis-Tetradecenoylcarnitine and 9-cis- Tetradecenoylcarnitine determining the ratio 5-cis-Tetradecenoylcarnitine / 9-cis-Tetradecenoylcarnitine.

[0160] The ratio Cis-5-C14:1 / Cis-9-C14:1 or Cis-9-C14:1 / Cis5-C14:1 may be considered a useful and / or potential indicator to differentiate physiologic elevations of C14:1 related to fasting vs. VLCAD deficiency.

[0161] In particular, the ratio Cis-5-C14:1 / Cis-9-C14:1 or Cis-9-C14:1 / Cis-5-C14:1 may be indicative of a diagnosis of VLCAD.

[0162] According to the present invention, the analytical method disclosed can be used for the diagnosis in a patient of one or more of the following conditions: 2-methylbutyryl-CoA dehydrogenase deficiency (2-MBG), beta-ketothiolase deficiency (BKT), cobalamine disorders (CblA / B and CbIC / D), glutaric aciduria type I (GA 1 ), 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG), 3-methylglutaconyl- CoA hydratase deficiency (3MGCA), isovaleric acidemia (IVA), malonic acidemia (MAL), multiple carboxylase deficiency (MCD), methylmalonyl-CoA mutase deficiency (MMA-MUT), propionic acidemia (PA), 2-methyl-3-hydroxybutyryl-coa dehydrogenase deficiency (HSD10 or 2M3HBA), 3- methylcrotonyl-CoA carboxylase deficiency (3MCC), 3-methylglutaconyl-CoA hydratase deficiency (3MGCA), isobutyrylglycinuria (IBG), cystathionine beta-synthase deficiency (CBS), methylenetetrahydrofolate reductase deficiency (MTHFR), glycine N-methyltransferase deficiency (GNMT), methionine adenosyltransferase deficiency (MATI / II I), S-adenosylhomocysteine hydrolase deficiency (SAHH), adenosine kinase deficiency (ADK), maple syrup urine disease (MSUD), ornithine transcarbamylase deficiency (OTC), citrullinemia type I (CIT I), argininosuccynic aciduria (ASA), glutaric acidemia type II (GA 2, or multiple acyl-CoA dehydrogenase deficiency, MADD), medium / short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), medium chain acyl-CoA dehydrogenase deficiency (MCAD), short-chain acyl-CoA dehydrogenase deficiency (SCAD), aromatic l-amino acid decarboxylase (AADC), adenosine deaminase deficiency (ADA-SCID), ethylmalonic encephalopathy (EE), 3-hydroxyisobutyryl-CoA hydrolase deficiency (HIBCH), shortchain enoyl-CoA hydratase deficiency (SCEH), methylmalonate semialdehyde dehydrogenase deficiency (MMSDH), ATP-specific succinyl-coa synthetase, subunit (SUCLA2), combined malonic and methylmalonic aciduria (CMAMMA), very long chain acyl-CoA dehydrogenase (VLCAD).

[0163] According to a fourth object, the present invention discloses the use of an extraction solution (ES) comprising ultrapure water 0.1 % (v / v) formic acid in an extraction step of an isolated biological sample to be analyzed with LC-MS / MS.

[0164] In a preferred embodiment, the extraction solution (ES) also comprises TCEP at a suitable concentration.

[0165] In a further object, the present invention discloses an extraction solution (ES) comprising TCEP 0.625 mM, a “IS MIX” solution of x % volume, wherein x= [(1 / 10)*m] wherein m is the number of internal standards, said extraction solution (ES) being adjusted to 100% (volume) with a solution of ultrapure water 0,1 % (vol / vol) formic acid.

[0166] In preferred embodiments, the use of the extraction solution (ES) is according to the above disclosure.

[0167] For instance, the extraction solution (ES) is used the extraction of n analytes from an isolated biological sample selected from an isolated sample of blood, Dried Blood Spot (DBS), serum, plasma, urine, liquor.

[0168] In a further object, the present invention discloses the use mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid for the analysis of n analytes in a solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

[0169] In a preferred embodiment, said analysis is performed using an elution path comprising:

[0170] -a first elution path phase using mobile phase A >60-70% volume,

[0171] -a second elution path phase using mobile phase A 40-60% volume,

[0172] - a third elution path phase using mobile phase A > 60-70% volume.

[0173] In a preferred embodiment, said analysis step is performed using an elution path comprising:

[0174] T1 -a first elution path phase using mobile phase A in concentration from 95-100%, and preferably from 100%, to 72% for about 6 minutes,

[0175] -a second elution path phase using mobile phase A in concentration from 55 to 43% for about 1 or 2 minutes,

[0176] - a third elution path phase using mobile phase A in concentration of 100% for about 2 or 3 minutes.

[0177] In another preferred embodiment, the analysis step is performed using an elution path comprising:

[0178] In another embodiment, said analysis step is performed using an elution path comprising:

[0179] Different techniques of LC / MS-MS may be used adjusting the flow path conditions of time and flow rate, while maintaining the same path steps (volume % of mobile phase A versus mobile phase B).

[0180] In a further object, the present invention discloses the above elution paths for the separate and simultaneous identification of 5-cis-Tetradecenoylcarnitine and 9-cis-Tetradecenoylcarnitine.

[0181] In a particular, embodiment, said analysis step follows the extraction step with any one of the extraction solution (ES) according to the present invention. According to a further object, the present invention discloses the use of TCEP an analytical method for the quantification of the total homocysteine (t-HCY).

[0182] In a preferred embodiment, the analytical method is the method according to the above disclosure.

[0183] In a particular embodiment, TCEP is used at a concentration of about 0.625 mM.

[0184] According to the present invention, TCEP and particularly TCEP at a concentration of 0.625 mM prevents the suppressive and interfering effects caused on other analytes, such as: HCY, HCY IS, 5HT IS, 3OHPA in the analysis step.

[0185] In a further object, the present invention discloses an extraction solution (ES) comprising TCEP 0.625 mM, a “IS MIX” solution of x % volume, wherein x= [(1 / 10)*m] wherein m is the number of internal standards, wherein said extraction solution (ES) is adjusted to 100% (volume) with a solution of ultrapure water 0,1 % (vol / vol) formic acid.

[0186] In a further object, the present invention discloses the use of the analytical method above disclosed for biochemical diagnostic confirmation within a screening program in the recall step, for diagnostic evaluation based on clinical evidences including non-newborns patients, for follow-up of patients under treatment.

[0187] A particular embodiment of the present invention will be further described in the following Experimental Section.

[0188] Solution Preparations

[0189] Stock and working solutions of reference and labelled internal standards (IS) are preferably dissolved in ultrapure water up to fixed concentrations (Figure 1 ) and aliquots are stored at -20°C where stability has been verified over a period of 3 years.

[0190] TCEP reducing agent stock solution (i.e. 0.5 M) in ultrapure water or in pH 7.0 aqueous solution is diluted up to a ready to use working solution of a suitable concentration (i.e. concentration of 12.5 mM) in 0.1 % FA ultrapure water pH <5 and aliquots are stored at -20°C for 6 months.

[0191] An internal standard mix solution (IS MIX) containing 25 labeled internal standards were prepared by diluting the single working solutions by a factor corresponding to the total number of labelled internal standard used, that in this instance is 1 :25. For example for a low-throughput laboratory, 100 pl of every 25 IS working solutions is diluted in the same vial to a total volume of 2500 pl; shifting the IS number to 15, dilution factor is 1 :15 and the final volume is 1500 pl. In case of a greater workload and 25 IS, 500 pL of IS working solutions achieved the final IS MIX volume of 12500 pl or 7500 pl when IS number is reduced.

[0192] The IS MIX solution can be stored into aliquots of 50 or 150 pl which at -20° C where the stability has been verified over a period of 3 months.

[0193] In this teaching the working extraction solution (ES) is a combination of 2.5% IS MIX, 85% of 0.1 % FA in ultrapure water, 7.6% of ultrapure water and necessarily 5% of TCEP 12.5 mM 0.1 % formic acid.

[0194] In some embodiments, the ES can be prepared and stored at 4°C or -20°C for different periods.

[0195] The IS MIX fraction of the ES can vary proportionally to the internal standard number as described above and the content of ultrapure water varies in an inversely proportional way in respect to the IS MIX fraction. Formic acid content can also vary but it must be considered that below 0.1 % the sensitivity is decreased for several analytes and that for contents greater than 0.1 % the background noise of the signal of many analytes is increased.

[0196] Optionally, a volume of organic solvent can be introduced in the extraction mixture in order to provide the extraction of further molecules that are less soluble in water, however it decreases the sensitivity of some other analytes.

[0197] TCEP molecule was chosen as reducing agent to quantify the total homocysteine (t-HYC) since it is more stable in acidified aqueous solutions and more effective in breaking disulphide bonds at low concentrations compared to the commonly DL Dithiothreitol (DTT) reported in the literature

[0016] , Moreover, we verified that it can also provide a positive effect in improving the methylcitric acid analytical sensitivity, thus increasing the peak signal up to 5 folds.

[0198] Biochemically and genetically confirmed newborn and follow-up patient DBS and random control NBS samples were collected on Guthrie filter paper (Ahlstrom, PerkinElmer 226) from Liguria and Lombardy Region neonatal population between 48thand 72ndh after birth and were used to clinically validate the method and to collect biomarkers reference values; informed consent was obtained accordingly to the national legislation about newborn screening program and relative residual samples [9], Results of ongoing and retrospective analyzed samples are reported in Figure 7. For retrospective analysis samples were stored at -20°C. A group of 24 newborn control DBS were included in the analysis and genetically confirmed as negative.

[0199] Sample preparation and analysis One 3.2 mm size disk (~3.2 pl) dried blood spot (DBS) sample is punched into each 96-well microplate. The punched sample can be extracted by incubation with 100 pl of a fresh extraction solution at 37 °C for 25 minutes under stirring at 800 rpm into a T rinest incubation shaker from Revvity (Waltham, MA, USA).

[0200] In some embodiments the method can be used with smaller or greater amounts of DBS punched disk (> or > of 3.2 mm).

[0201] In some embodiments other incubator shaker can be used

[0202] In some embodiments sample extraction can be optionally performed at different temperatures, different time ranges and at different agitation.

[0203] During the incubation an adhesive microplate should be used to minimize the evaporation.

[0204] The extracted sample can be optionally transferred to another microplate to keep the instrumentation cleaner and 5 pL, but not limited to, can be injected in the LC-MS / MS system equipped with Electrospray Source (ESI) operating in multiple reaction mode (MRM) in both positive and negative ionization. For each analyte the pseudomolecular ion and at least one fragment product ion can be monitored.

[0205] MS instrumental source parameter settings (Table 5):

[0206] Table 5

[0207] The optimized MRM transition parameters for analytes involved in this assay are shown in Table 5 above.

[0208] The UHPLC separation can be performed using an Acquity UPLC HSS T3 column (2.1 x 100 mm, 1.8 pm) associated with Acquity HSS T3 VanGuard Pre-column (2.1 x 5 mm, 1.8 pm), from Waters (Milford, USA) with an elution gradient of 11 minutes run to run. The mobile phase solvents of this teaching consist of ultrapure water containing 0.1 % formic acid (mobile phase A) and ACN containing 0.1 % formic acid (mobile phase B). Gradient composition starts from 100% mobile phase A and maintained for 2 minute, then decreased to 80% at 4 min and to 72% at 6 min; at 6.1 min it rapidly can change to 55%, and to 43 at 8.1 min, then it can increase to 100% at 8.2 min, remaining till the end of the analysis. The method flow rate is maintained 0.4mL / min for 6 min than increased to 0.5 mL / min at 6.1 till 9.1 min and returns to 0.4 mL / min at 9.2 min till the end of the analysis. The column temperature was set at 30 °C for the entire assay.

[0209] The method gradient can optionally be changed. For example, chromatographic run can be reduced to 10 minutes by modifying gradient from 55% of mobile phase A by reaching 43% from 6.1 to 7 minutes and then 100% up to 7.1 minutes till the end of the run and reducing the flow rate from 0.5 ml / min to 0.4 at 8.1 minutes. In this way a faster analysis and TAT is achieved but separation of the C14:1 isomers is impaired.

[0210] Optionally, a variety of organic solvent content can be introduced in the gradient starting composition when a content of organic solvent is optionally added in the extraction solution. However, this variation does not guarantee a satisfactory chromatographic resolution and peak shape for some of the analytes.

[0211] This disclosure provides validation on Revvity Qsight 225 MD and SCIEX Triple Quad 6500, but is understandable that any type of LC-MS / MS platform with different configuration is suitable for this method application as well as a variety of UHPLC separation columns. For example, a Dr. Maish column Reprosil Acqua 2.1 x 100 mm can be used.

[0212] In some embodiments, the analytes can be quantified with stable isotope dilution method using a single-point calibration. This is a common used screening quantitation strategy based on the comparison of the peak area of each analyte in the sample to a corresponding stable isotope-labelled internal standard (IS) with very similar physical chemistry behaviour that is added to each sample at the same known concentration. Assuming that it responds identically to the analyte during the measurement, the area ratio (analyte / IS) is used to calculate the analyte unknown concentration in the sample.

[0213] This method provides for the use of 25 stable isotope-labelled internal standard, so that some molecules share the same internal standards as shown in Table 4 above. A relative response factor (RRF) can be applied for quantification of the correct concentration when the stable isotope-labelled internal standard used provides a not identical measurement signal to the analyte response.

[0214] The number of the added stable isotope-labelled internal standard can optionally be tailored, increasing or reducing them as desired, as already explained in this disclosure background.

[0215] Analytes of this method can be optionally quantified by a multi-points calibration curve when the authentic reference standards are commercially available Table 4.

[0216] Two biomarkers (5OH-Hexanoic and Iso-Hexanoylglycine) are only qualitatively evaluated. In some embodiments they can be optionally quantified. In some embodiments the method can involve the measurements of one or more additional analytes or can be also used qualitatively to detect the presence of this 60-plex metabolites panel as well as one or more additional analytes (for example other acylcarnitines, acylglycines, organic acids and aminoacids, creatine, creatinine, guanidinoacetate, steroids and other molecules).

[0217] In some embodiments the method can also be used for quantitative / qualitative detection of this 60- plex metabolites and one or more additional analytes in other biological fluids and any other application for detecting one or more analytes of the panels.

[0218] In some embodiments the method can be used to identify novel biomarkers. For example, some unknown analytes have been found at different retention times in various matrices.

[0219] Data are acquired and processed for quantification with Simplicity 3Q MD® 2.0 (Revvity) and Analyst 1 .7 (Sciex) software. The entire automated workflow is managed by Specimen Gate® Laboratory software (Perkielmer, Waltham, USA).

[0220] In some embodiments data can be processed with different quantification and informatics systems software.

[0221] Validation

[0222] For analytical validation of this method, performance for linearity, limit of detection (LOD), lower limit of quantitation (LOQ), recovery, within -day and between-day imprecision (coefficient of variation %) have been evaluated.

[0223] Linear range has been established using a calibration curve obtained with in-house DBS calibrators Table 4. prepared by spiking a different known amounts of individual reference available standard in aliquots of a whole blood sample from a healthy adult donor adjusting hematocrit to 55%. Each DBS calibrator was generated by spotting 50 pl of each level on a filter paper card and analyzed in duplicate for 5 days. The goal was to obtained r2> 0.98.

[0224] Curve slope and x-intercept and endogenous levels were calculated as the average of the extrapolated x-intercept of these curves by no weight linear regression (1 / x).

[0225] Within-day (n= 10) and between-day (n=10) imprecisions were evaluated by the analysis of in-house Quality Control Samples (QC), prepared as described above, of 3 different levels (Low, Medium and High) covering normal and abnormal concentrations; QC are analyzed by 3 different operators over a period of 2 weeks. The goal was to have a coefficient of variation (CV%) 15%

[0226] The same QC material has been used to calculate the Recovery according to this formula: Recovery % = [(measured concentration - endogenous concentration) / spiked concentration]*! 00 using labeled stable isotope single -point calibration quantitation method. LOD and LOQ defined as the lowest detected and measured concentration respectively were calculated by Taylor methods

[0013] .

[0227] For only 4 analytes of the panel Certified DBS Quality Controls are available. Certified Materials (Lot. N 2114 and 2214 (A,B,C,D), from Center of Disease Control and Prevention (CDC) was used for accuracy evaluation analyzing 5 replicates of each enriched level of two lots. The recovery acceptance criteria were to have an accuracy within ±20% of the expected value. Validation results are reported in Figure 2.

[0228] Since 2020 Proficiency External Quality Assurance Programs DBS (A-F) from European Research Network for Evaluation and Improvement of Screening, Diagnosis and Treatment of Inherited Disorders of Metabolism (ERNDIM) and MSITA (Italian Working group on Mass Spectrometry) from the Italian Society for the Study of Inherited Metabolic Diseases and Newborn Screening (SIMMESN), were used to evaluate the global interpretative performance of the 2TT assay showing 100 % of diagnostic of sensitivity

[0229] When authentic standards are not available (C4OH-iso-S, C4OH-S, C4OH-iso-R, MAAHC, MHBC, iso-Hexagly, Cis-9-C14:1 , 5OH-hexanoic) the analyte peaks have been identified by monitoring the transitions (m / z) reported in literature and running DBS o urine samples from affected patients and / or external quality control materials containing the markers.

[0230] Robustness has been evaluated through the implementation of the method in 2 different screening centers laboratories as already described, in both full or partial format in terms of analytes number and through the routinely method application over a total period of 6 years. Results show very good accordance results.

[0231] Stability of individual stock, working, extractions solutions and reagent was already described in the solutions preparation paragraph.

[0232] Stability of in-house generated QC and calibrators has been verified for a period of 13 months at - 20°C without stabilizers addition. Concentration remained constant for most of the analytes except for HCY and S-ADO-MET that showed a slight decrease.

[0233] Newborn samples stored at room temperature or 4°C for 2 months, showed an increase of GA, 3OH- PA, S-ADO-HCY, SUCCA and probably due to bacterial proliferation. The same results have been obtained from the analysis of newborn samples stored for 1 years and at -20°C for. Gradual decrease of HCY and S-ADO-MET was also observed.

[0234] Cut-off determination

[0235] 500 newborn samples, collected between 48thand 72ndhours of life, at IRCCS Istituto Giannina Gaslini Hospital of Genova, Italy, and 5000 DBS from Functional Genomics and Rare diseases, Buzzi Children's Hospital of Milan, Italy were evaluated with Cutoff Analyzer software (Revvity) to establish cut off at the 99thpercentile and in a few cases at the 0.5thpercentile (Figure 2).

[0236] Validation results

[0237] All analytes proved an excellent linearity with correlation factors (r2) S 0.98.

[0238] Total imprecision for most of the metabolites was < 15% with few exceptions.

[0239] Recovery study showed good results within ± 20% of the nominal concentration. Lower recovery of some analytes may be due to matrix effect that cannot be compensated suppressed by the use of single-point calibration methods based on the area ratio of analyte / IS only. However, considering that this quantitation strategy is the common practice in NBS laboratories, obtained results can be considered acceptable and fit-for-purpose since cut-off values are evaluated with the same quantification method

[0013] . The same QC material has been quantified by DBS home-made 7 points calibration curve, showing excellent accordance with QC nominal concentrations for most of the analytes.

[0240] Data Analysis

[0241] This teaching provides for a novel tool that integrates additional data from an external file ("Cut off value. xlsx") to enrich the analysis. This is achieved by creating a separate sheet for each unique identifier extracted from the original files. Each of these sheets is populated with data from "Cut off value. xlsx," and a comparison is made against the extracted identifiers to validate and crossreference the results.

[0242] The program automates the formatting and organization of data, including adding borders, adjusting cell colors, and performing conditional formatting based on comparison logic. It evaluates the numerical or categorical data according to set conditions (e.g., “>”, or specific threshold values) and highlights discrepancies directly in the generated report.

[0243] This tool provides an automated, structured approach to process large volumes of Excel data, generate structured reports, and conduct cross-comparisons with external reference files. This solution minimizes manual data handling, ensures consistency, and enhances the quality of data presentation and interpretation in the laboratory setting (Figure 9).

[0244] From the above disclosure, the person skilled in the art will immediately understand the advantages offered by the present invention.

[0245] Briefly, the present teaching provides for a unique multiplex analytical 2TT approach applicable to 38 hereditary diseases 27 of which are inborn errors of metabolism (IEM), further 9 pathologies are functional for the differential diagnosis, 1 is a neurometabolic defect and 1 a severe combined immunodeficiency. The last two disorders are both candidates to be included in the national disease lists. The assay can also allow the recognition of 4 non-hereditary I conditions that frequently cause false positive results (Figures 4A and 4B).

[0246] The present method has the ability to simultaneously define the biochemical risk of a comprehensive panel of conditions integrated in both the mandatory Italian Newborn Screening Panel

[0015] and the US Recommended Uniform Screening Panel (RUSP) that are the most extensive worldwide (Figure 3A,3B,3C).

[0247] The present teaching provides the detection of 60, but not limited to, well known and / or potentially useful markers for the evaluation of many hereditary disorders for Newborn Screening Program, comparing the level of the measured analytes with normal levels in order to differentiate abnormal biochemical profiles from physiological ones.

[0248] This 2TT assay has demonstrated to drastically improve the positive predictive value (PPV%) as showed in Figures 5A and 5B thanks to the numerous markers detected for each evaluated disease and to the high performance in resolving various isomers\isobaries. In fact, in this way high diagnostic specificity is achieved for conditions characterized by a wide variability of biochemical findings arising from biochemical and / or clinical phenotypes heterogeneity. Moreover, differential diagnosis can be strengthened in this manner, when primary markers are shared among diseases. This discrimination capability provides also the identification of some currently untreatable incidental findings. This aspect is raising ethical issues worldwide

[0027] , but it certainly has the advantage of reducing parental stress due to the diagnostic odyssey.

[0249] In some embodiments, the method can be applied to the quantitative and / or quantitative analysis the 60-plex, but not limited to, metabolites in DBS.

[0250] In some embodiments, the method can be applied to the qualitative and / or quantitative analysis of one or more metabolites of the 60-plex panel.

[0251] In some embodiments, the method can be applied to the qualitative and / or quantitative analysis of one or more metabolites of the 60-plex, but not limited to, metabolites panel for any other biochemical matrices (3.2 pl of plasma, serum, urine, liquor) and other applications.

[0252] For instance, the method can be applied without any modification for biochemical diagnostic confirmation within the screening program in the recall step, as well as for diagnostic evaluation based on clinical evidences including non-newborns patients, and for follow-up of patients under treatment.

[0253] The entire analytical protocol - from punching phase to result - lasts less than 45 minutes, and does not employ any evaporation, derivatization, filtration or centrifugation steps. The process velocity was also guaranteed by an accurate standardization study of each method detail (e.g. ideal partitioning of stock and working solutions, customizable formulation according to the needs and workload of the different NBS Laboratory, manufacture of internal quality controls or matrix calibrators).

[0254] The MS and LC settings were optimized using individual reference standards working solution to be infused in MS source and singly chromatographed, to identified specific MRM transitions and the retention times, in order to well resolve several isomers\isobars (Figures A-E), for both NBS test sensitivity and specificity improvement. This feature allows PPV% enhancement and favor differential diagnosis.

[0255] The use of a “one-step” sample preparation procedure suitable for multiple classes of molecules resolved by a unique chromatographic run simplifies workflows and reduce diagnostic timing (TAT) which must be minimized especially for newborns at high risk of metabolic decomposition.

[0256] The extraction step assured a robust recovery / yields for all the molecules listed in this 2TT panel thanks to their water solubility. In fact the use of acidified water as extraction solvent, instead of common mixtures of volatile organic solvents (methanol, or ACN) as well as ensuring safer operative conditions, also allows purification from all those DBS matrix lipophilic organic interfering substances that are not soluble in water; by doing so, interferences were reduced yielding optimal instrumental performances in view of ESI ionization, sensitivity, selectivity and finally also maintenance was simplified extending the spare parts lifetime such as ESI capillaries and chromatographic column.

[0257] In addition, the protocol details and the optimized chromatographic gradient make the method flexible for the introduction of further markers in the already large panel. Method is flexible in configuration by simply adding or removing m / z transitions and / or associated IS volume from the solutions.

[0258] The whole formulation was developed for kit configuration and standardization for both low and high throughput NBS laboratories. The method can be customized to meet the needs of each laboratory by varying the number of transitions, while the integral mass configuration promotes the 2TT protocol harmonization among the various NBS centers.

[0259] Reference

[0260] 1. Rajabi, F. Updates in Newborn Screening. Pediatr Ann. 2018, 47(5):e187-e190. doi: 10.3928 / 19382359- 20180426-01.

[0261] 2. Therrell, B.L.; Padilla, C.D.; Loeber, J.G.; Kneisser, I.; Saadallah, A.; Borrajo, G.J.; Adams, J. Current status of newborn screening worldwide: 2015. Semin Perinatol. 2015, 39(3):171-87.

[0262] 3. Chien, Y.H.; Hwu, W.L. The modern face of newborn screening. Pediatr Neonatol. 2023, 64 Suppl 1 :S22- S29

[0263] 4. Cavarzere ,P.; Camilot, M.; Teofoli, F.; Tato, L. Neonatal Screening for Congenital Adrenal Hyperplasia in North-Eastern Italy: A Report Three Years into the Program. Horm Res 2005, 63:180-186. doi: 10.1 159 / 000085021 Report Three Years into the Program. Horm. Res. 2005, 63, 180-186.

[0264] 5. Burlina, A.; Giuliani, A.; Polo, G.; Gueraldi, D.; Gragnaniello, V.; Cazzorla, C.; Opladen, T.; Hoffmann, G.; Blau, N.; Burlina, A.P. Detection of 3-O-methyldopa in dried blood spots for neonatal diagnosis of aromatic L-amino-acid decarboxylase deficiency: The northeastern Italian experience. Mol Genet Metab. 2021 , 133(1 ):56-62.

[0265] 6. Gragnaniello, V.; Burlina, A.P.; Polo, G.; Giuliani, A.; Salviati, L.; Duro, G.; Cazzorla, C.; Rubert, L.; Maines, E.; Germain, D.P.; Burlina, A.B. Newborn Screening for Fabry Disease in Northeastern Italy: Results of Five Years of Experience. Biomolecules. 2021 , 27;1 1 (7):951 .

[0266] 7. Gurian, E.A.; Kinnamon, D.D.; Henry, J.J.; Waisbren, S.E. Expanded Newborn Screening for Biochemical Disorders: The Effect of a False-Positive Result. Pediatrics 2006, 1 17 (6): 1915-1921. 10.1542 / peds.2005-2294;

[0267] 8. Schmidt, J.L.; Castellanos-Brown, K.; Childress, S.; et al., The impact of false-positive newborn screening results on families: a qualitative study. Genet Med. 2012, 14(1 ):76-80.

[0268] 9. la Marca, G.; Malvagia, S.; Casetta, B.; Pasquini, E.; Donati, M.A.; Zammarchi, E. Progress in expanded newborn screening for metabolic conditions by LC-MS / MS in Tuscany: update on methods to reduce false tests. J Inherit Metab Dis. 2008, 31- (Suppl 2):S395-S404.

[0269] 10. Sinclair, G.B.; Ester, M.; Horvath, G.; van Karnebeek, C.D.; Stockler-lpsirogu, S.; Vallance, H. Integrated Multianalyte Second-Tier Testing for Newborn Screening for MSUD, IVA, and GAMT Deficiencies. Journal of Inborn Errors of Metabolism and Screening. 2016, 4.

[0270] 1 1 . Monostori, P.; Klinke, G.; Richter, S.; Barath, A.; Fingerhut, R.; Baumgartner, M.R.; Kolker, S.; Hoffmann, G.F.; Gramer, G.; Okun, J.G. Simultaneous determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in dried blood spots: Second-tier LC-MS / MS assay for newborn screening of propionic acidemia, methylmalonic acidemias and combined remethylation disorders. PLoS One. 2017, 15;12(9):e0184897.

[0271] 12. Alodaib, A.; Carpenter, K.; Wiley, V.; Sim, K.; Christodoulou, J.; Wilcken, B. An improved ultra performance liquid chromatography-tandem mass spectrometry method for the determination of alloisoleucine and branched chain amino acids in dried blood samples. Ann Clin Biochem. 2011 , 48(Pt 5):468-70.

[0272] 13. Kilgore, M.B.; Platis, D.; Lim, T.; Isenberg, S.; Pickens, C.A.; Cuthbert, C.; Petritis, K. Development of a Universal Second-Tier Newborn Screening LC-MS / MS Method for Amino Acids, Lysophosphatidylcholines, and Organic Acids. Anal Chem. 2023, 14;95(6):3187-3194.

[0273] 14. Pajares-Garcia, S.; Gonzalez de Aledo-Castillo, J.M.; Flores-Jimenez, J.E.; Collado, T.; Perez, J.; Paredes-Fuentes, A.J.; Argudo-Ramirez, A.; Lopez-Galera, R.M.; Prats, B.; Garcia-Villoria, J. Analysis of a second-tier test panel in dried blood spot samples using liquid chromatography-tandem mass spectrometry in Catalonia's newborn screening programme. Clin Chem Lab Med. 2023, 6.

[0274] 15. Ruoppolo, M.; Malvagia, S., Boenzi, S.; Carducci, C.; Dionisi-Vici, C.; Teofoli, F.; Burlina, A.; Angeloni, A.; Aronica, T.; Bordugo, A.; Bucci, I.; Camilot, M.; Carbone, M.T.; Cardinal!, R.; Carducci, C.; Cassanello, M.; Castana, C.; Cazzorla, C.; Ciatti, R.; Ferrari, S.; Frisso, G.; Funghini, S.; Furlan, F.; Gasperini, S.; Gragnaniello, V.; Guzzetti, C.; La Marca, G.; La Spina, L.; Lore, T.; Meli, C.; Messina, M.; Morrone, A.; Nardecchia, F.; Ortolano, R.; Parenti, G.; Pavanello, E.; Pieragostino, D.; Pillai, S.; Porta, F.; Righetti, F.; Rossi, C.; Rovelli, V.; Salina, A.; Santoro, L.; Sauro, P.; Schiaffino, M.C.; Simonetti, S.; Vincenzi, M.; Tarsi, E.; Uccheddu, A.P. Expanded Newborn Screening in Italy Using Tandem Mass Spectrometry: Two Years of National Experience. Int J Neonatal Screen. 2022, 9;8(3):47. Barreira-Santarino, I.; Oliveira, S.C.B.; Oliveira-Brett, A.M. Protein reducing agents dithiothreitol and tris(2-carboxyethyl)phosphine anodic oxidation. Eclectrochemistry Communications. 2012, 23, 114-117. Rizzo, C.; Boenzi, S.; Inglese, R.; la Marca, G.; Muraca, M.; Martinez, T.B.; Johnson, D.W.; Zelli, E.; Dionisi-Vici, C. Measurement of succinyl-carnitine and methylmalonyl-carnitine on dried blood spot by liquid chromatography-tandem mass spectrometry. Clin Chim Acta. 2014, 15;429:30-3. Soeters, M.R.; Serlie, M.J.; Sauerwein, H.P.; Duran, M.; Ruiter, J.P.; Kulik, W.; Ackermans, M.T.; Minkler, P.E.; Hoppel, C.L.; Wanders, R.J.A.; Houten, S.M. Characterization of D-3-hydroxybutyrylcarnitine (ketocarnitine): an identified ketosis-induced metabolite, Metabolism, Volume 61 , Issue 7, 2012, Pages 966-973. Marcadier, J.L.; Smith, A.M.; Pohl, D.; Schwartzentruber, J.; Al-Dirbashi, O.Y.; FORGE Canada Consortium; Majewski, J.; Ferdinandusse, S.; Wanders, R.J.; Bulman, D.E.,; Boycott, K.M.; Chakraborty, P.; Geraghty, M.T. Mutations in ALDH6A1 encoding methylmalonate semialdehyde dehydrogenase are associated with dysmyelination and transient methylmalonic aciduria. Orphanet J Rare Dis. 2013 9;8:98. Peters, H.; Ferdinandusse, S.; Ruiter, J.P.; Wanders, R.J.; Boneh, A.; Pitt, J. Metabolite studies in HIBCH and ECHS1 defects: Implications for screening. Mol Genet Metab. 2015, 115(4): 168-73. Yamada, K.; Aiba, K.; Kitaura, Y.; Kondo, Y.; Nomura, N.; Nakamura, Y.; Fukushi, D.; Murayama, K.; Shimomura, Y.; Pitt, J.; Yamaguchi, S.; Yokochi, K.; Wakamatsu, N. Clinical, biochemical and metabolic characterisation of a mild form of human short-chain enoyl-CoA hydratase deficiency: significance of increased N-acetyl-S-(2-carboxypropyl)cysteine excretion. J Med Genet. 2015, 52(10):691 -8. Pajares, S.; Lopez, R.M.; Gort, L.; Argudo-Ramirez, A.; Marin, J.L.; Gonzalez de Aledo-Castillo, J.M.; Garcia-Villoria, J.; Arranz, J.A.; Del Toro, M.; Tort, F.; Ugarteburu, O.; Casellas, M.D.; Fernandez, R.; Ribes, A. An incidental finding in newborn screening leading to the diagnosis of a patient with ECHS1 mutations. Mol Genet Metab Rep. 2020 2;22: 100553. Huemer, M.; Kozich, V.; Rinaldo, P.; Baumgartner, M.R.; Merinero, B.; Pasquini, E.; Ribes, A.; Blom, H.J. Newborn screening for homocystinurias and methylation disorders: systematic review and proposed guidelines. J Inherit Metab Dis. 2015, 38(6): 1007-19. Flinn, A.M.; Gennery, A.R. Adenosine deaminase deficiency: a review. Orphanet J Rare Dis. 2018, 24;13(1 ):65. Di Carlo, E.; Santagata, S.; Sauro, L.; Tolve, M.; Manti, F.; Leuzzi, V.; Angeloni, A.; Carducci, C. Simultaneous determination of 5-hydroxytryptophan and 3-O-methyldopa in dried blood spot by UPLC- MS / MS: A useful tool for the diagnosis of L-amino acid decarboxylase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci. 2021 , 15; 1185:122999.

Claims

CLAIM1. A method for the simultaneous detection of a number n of analytes in an isolated biological sample, said method comprising the steps of:1 ) extraction by incubating said isolated biological sample with an extraction solution (ES) thus obtaining an analytes solution,2) analysis of the analytes solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization, wherein said extraction solution (ES) has a concentration of 0.625 mM TCEP and comprises a “IS MIX” solution of a m number of internal standards, wherein said “IS MIX” solution has a % volume of x= [(1 / 10)*m], and wherein said extraction solution (ES) is adjusted to 100% (vol / vol) with a solution of ultrapure water 0,1 (vol / vol) formic acid.

2. The method for the simultaneous detection of a number n of analytes in an isolated biological sample according to the preceding claim, said method comprising the steps of:1 ) extraction by incubating said isolated biological sample with an extraction solution (ES) thus obtaining an analytes solution,2) analysis of the analytes solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization, wherein said extraction solution (ES) comprises:wherein the final concentration of formic acid in the extraction solution is 0.1 % (v / v), wherein “IS MIX” is a mixture of 25 internal standard (IS) prepared by mixing together an equal volume vis of each internal standard (IS).

3. The method according to the preceding claim 1 or 2, wherein the isolated biological sample is represented by an isolated sample of blood, Dried Blood Spot (DBS), serum, plasma, urine, liquor.

4. The method according to any one of the preceding claims, wherein said isolated biological sample is represented by Dried Blood Spot (DBS).

5. The method according to any one of the preceding claims, wherein the number n of analytes is between 1 and 200, preferably between 1 and 100 and even more preferably between 1 and 60.

6. The method according to any one of the preceding claims, wherein the volume vis of each internal standard is between 1 pl and 10 ml, preferably between 1 pl and 1 ml or between 10 and 500 pl.

7. The method according to any one of the preceding claims, wherein the extraction step is performed at a temperature of 25-45°C.

8. The method according to the preceding claim, wherein the extraction step is performed at a temperature of about 37°C9. The method according to any one of the preceding claims, wherein the extraction step is performed for a period of about 15 minutes to 1 hour.

10. The method according to the preceding claim, wherein the extraction step is performed for a period of about 25 minutes.

11. The method according to any one of the preceding claims, wherein the analysis step is performed with UHPLC / MS-MS.

12. The method according to any one of the preceding claims, wherein the analysis step is performed using a mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid.

13. The method according to the preceding claim, wherein the analysis step is performed using an elution path comprising-a first elution path phase wherein the mobile phase A is >60-70% volume,-a second elution path phase wherein the mobile phase A is around 40-60% volume,-a third elution path phase wherein the mobile phase A is >60-70% volume.

14. The method according to the preceding claim 12 or 13, wherein the analysis step is performed using an elution path comprising:-a first elution path using the mobile phase A from 95-100%, and preferably from 100%, to 72% volume for about 6 minutes,-a second elution path using the mobile phase A from 55 to 43% volume for about 1 or 2 minutes,- a third elution path using the mobile phase A of 100% volume for about 2 or 3 minutes.

15. The method according to any one of the preceding claims 12 to 14, wherein the analysis step is performed using the elution path comprising:

16. The method according to any one of the preceding claims 12 to 15, wherein the analysis step is performed using the elution path comprising:

17. The method according to any one of the preceding claims, further comprising the step for the quantification of the n analytes with the stable isotope dilution technique.

18. The method according to the preceding claim, wherein the quantification of the n analytes with the stable isotope dilution technique uses a single-point calibration curve or a multipoint calibration curve.

19. The method according to any one of the preceding claims, wherein in the analysis step, the n analytes may be detected according to the following parameters:

20. The method according to any one of the preceding claims, wherein in the analysis step, the n analytes may be detected according to the ollowing parameters:

21. The method according to any one of the preceding claims, wherein the one or more n analytes detected are selected from:

22. The analytical method according to any one of the preceding claims, wherein saidxtraction step is the sole extraction step performed of the n analytes from the isolated biologicalample.

23. The analytical method according to any one of the preceding claims, wherein said analysis step is the sole analysis step performed with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

24. The analytical method according to any one of the preceding claims for the in vitro diagnosis of one or more of the following conditions in a patient: 2-methylbutyryl-CoA dehydrogenase deficiency (2-MBG), beta-ketothiolase deficiency (BKT), cobalamine disorders (CblA / B and CbIC / D), glutaric aciduria type I (GA 1 ), 3-hydroxy-3-methylglutaryl-CoA lyase deficiency (HMG), 3- methylglutaconyl-CoA hydratase deficiency (3MGCA), isovaleric acidemia (IVA), malonic acidemia (MAL), multiple carboxylase deficiency (MCD), methylmalonyl-CoA mutase deficiency (MMA-MUT), propionic acidemia (PA), 2-methyl-3-hydroxybutyryl-coa dehydrogenase deficiency (HSD10 or 2M3HBA), 3-methylcrotonyl-CoA carboxylase deficiency (3MCC), 3-methylglutaconyl-CoA hydratase deficiency (3MGCA), isobutyrylglycinuria (IBG), cystathionine beta-synthase deficiency (CBS), methylenetetrahydrofolate reductase deficiency (MTHFR), glycine N-methyltransferase deficiency (GNMT), methionine adenosyltransferase deficiency (MATI / 111), S-adenosylhomocysteine hydrolase deficiency (SAHH), adenosine kinase deficiency (ADK), maple syrup urine disease (MSUD), ornithine transcarbamylase deficiency (OTC), citrullinemia type I (CIT I), argininosuccynic aciduria (ASA), glutaric acidemia type II (GA 2, or multiple acyl-CoA dehydrogenase deficiency, MADD), medium / short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD), medium chain acyl- CoA dehydrogenase deficiency (MCAD), short-chain acyl-CoA dehydrogenase deficiency (SCAD), aromatic l-amino acid decarboxylase (AADC), adenosine deaminase deficiency (ADA-SCID), ethylmalonic encephalopathy (EE), 3-hydroxyisobutyryl-CoA hydrolase deficiency (HIBCH), shortchain enoyl-CoA hydratase deficiency (SCEH), methylmalonate semialdehyde dehydrogenase deficiency (MMSDH), ATP-specific succinyl-coa synthetase, subunit (SUCLA2), combined malonic and methylmalonic aciduria (CMAMMA), very long chain acyl-CoA dehydrogenase (VLCAD).

25. The analytical method according to any one of the preceding claims, wherein said patient is a newborn.

26. An analytical method for the diagnosis of VLCAD (Very Long Chain Acyl-CoA Dehydrogenase) deficiency in a patient from an isolated sample of dried blood or from Dried Blood Spot from said patient, said method comprising the steps of: performing the method according to any one of the preceding claims, identifying the analytes represented by 5-cis-Tetradecenoylcarnitine and 9-cis- Tetradecenoylcarnitine, and determining a ratio of 5-cis-Tetradecenoylcarnitine / 9-cis-Tetradecenoylcarnitine, or 9-cis-Tetradecenoylcarnitine / 5-cis-Tetradecenoylcarnitine, wherein said ratio of is indicative of a diagnosis or of a condition of VLCAD.

27. The analytical method for the diagnosis of VLCAD (Very Long Chain Acyl-CoA Dehydrogenase) in a patient according to the preceding claim, wherein said ratio of 5-cis- Tetradecenoylcarnitine / 9-cis-Tetradecenoylcarnitine or 9-cis-Tetradecenoylcarnitine / 5-cis-25 is, respectively, Cis-5-C14:1 / Cis-9-C14:1 or Cis-9-C14:1 / Cis5-C14:1 .

28. An analytical method for the diagnosis of VLCAD deficiency in a patient from an isolated sample of dried blood or from Dried Blood Spot from said patient, said method comprising the steps of: performing the method according to any one of the preceding claims 1 to 25, separating and / or quantifying Trans-2-C14:1.

29. A kit for the simultaneous detection of a number n of analytes in an isolated biological sample with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization, said kit comprising the following components:-TCEP in ultrapure water 0.1 % (v / v) of formic acid,-ultrapure water 0.1 % (v / v) formic acid, and optionally:-a solution of m internal standards (IS),-a solution of mobile phase A of ultrapure water containing 0.1 % (v / v) formic acid and a solution of mobile phase B of acetonitrile containing 0.1 % (v / v) formic acid, and further comprising-instructions for using the kit comprising one or more of the following information for each one of the n analytes and m internal standards (IS):-the transition,-the retention time,-the polarity,-the window,-the dwell time,-calibration range, optionally with the low quality control and / or medium quality control and / or high quality control.

30. The kit according to the preceding claim, wherein said isolated biological sample is an isolated sample of blood, Dried Blood Spot (DBS), serum, plasma, urine, liquor.

31. The kit according to the preceding claim 29 or 30, wherein the TCEP solution is:-a solution of TCEP 0.5 M in ultrapure water, preferably at pH=7, to be suitably diluted with ultrapure water 01 % (v / v) formic acid, or-a solution of TCEP 12.5 mM in ultrapure water 0.1 % (v / v) formic acid.

32. The kit according to any one of the preceding claims 29 to 31 , wherein said components are each independently from one another in a liquid or in a lyophilized form.

33. Use of an extraction solution (ES) solution comprising:wherein the final concentration of formic acid in the extraction solution is 0.1 % (v / v), for the extraction of n analytes from an isolated biological sample selected from an isolated sample of blood, Dried Blood Spot (DBS), serum, plasma, urine, liquor.

34. Use of an extraction solution (ES) comprising TCEP 0.625 mM, a “IS MIX” solution of x % volume, wherein x= [(1 / 10)*m] wherein m is the number of internal standards, said extraction solution (ES) being adjusted to 100% (volume) with a solution of ultrapure water 0,1 % (vol / vol) formic acid so that the final concentration of formic acid in the extraction solution is 0.1 % (v / v), for the extraction of n analytes from an isolated biological sample selected from an isolated sample of blood, Dried Blood Spot (DBS), serum, plasma, urine, liquor.

35. Use of the extraction solution (ES) according to the preceding claim 33 or 34, wherein said biological sample is represented by Dried Blood Spot (DBS).

36. Use of the extraction solution (ES) according to any one of claims 33 to 35, for the extraction of one or more of the following n analytes:

37. Use of a mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid for the analysis of n analytes in a solution with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization.

38. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and the mobile phase B of acetonitrile containing 0.1 % formic acid according to the preceding claim, wherein the analysis step is performed using an elution path comprising:-a first elution path phase using mobile phase A >60-70% volume,-a second elution path phase using mobile phase A 40-60% volume,- a third elution path phase using mobile phase A > 60-70% volume.

39. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and the mobile phase B of acetonitrile containing 0.1 % formic acid according to the preceding claim 37 or 38, wherein the analysis step is performed using an elution path comprising:-a first elution path using mobile phase A in concentration from 95-100%, and preferably from 100%, to 72% for about 6 minutes,-a second elution path using mobile phase A in concentration from 55 to 43% for about 1 or 2 minutes,- a third elution path using mobile phase A in concentration of 100% for about 2 or 3 minutes.

40. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and the mobile phase B of acetonitrile containing 0.1 % formic acid according to any one of the preceding claims 37 to 39, wherein the analysis step is performed using the elution path comprising:

41. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid according to any one of the preceding claims 37 to 40, wherein the analysis step is performed using the elution path comprising:

42. Use of a mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization, wherein the analysis step is performed using an elution path comprising:-a first elution path phase using mobile phase A >60-70% volume,-a second elution path phase using mobile phase A 40-60% volume,- a third elution path phase using mobile phase A > 60-70% volume. for the separate and simultaneous identification of 5-cis-Tetradecenoylcarnitine and 9-cis- Tetradecenoylcarnitine, and optionally of Trans-2-C14:1.

43. Use of a mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1 % formic acid with LC-MS / MS with Electrospray Source operating in multiple reaction mode (MRM) in both positive and negative ionization according to the preceding claim, wherein the analysis step is performed using an elution path comprising:-a first elution path using mobile phase A in concentration from 95-100%, and preferably from 100%, to 72% for about 6 minutes,-a second elution path using mobile phase A in concentration from 55 to 43% for about 1 or 2 minutes,- a third elution path using mobile phase A in concentration of 100% for about 2 or 3 minutes,44. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and the mobile phase B of acetonitrile containing 0.1 % formic acid according to the preceding claim 42 or 43, wherein the analysis step is performed using the elution path comprising:

45. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and a mobile phase B of acetonitrile containing 0.1% formic acid according to any one of the preceding claims 42 to 44, wherein the analysis step is performed using the elution path comprising:

46. Use of the mobile phase A of 0.1 % formic acid in ultrapure water and the mobile phase B of acetonitrile containing 0.1% formic acid according to any one of the preceding claims 42 to 45, wherein said n analytes have been extracted from an isolated biological sample with an extraction solution (ES) comprising:wherein the final concentration of formic acid in the extraction solution (ES) is 0.1 % (v / v),47. Use of TCEP in an analytical method for the quantification of the total homocysteine (t- HCY).

48. Use of TCEP in the analytical method according to any one of the preceding claims 1 to 28 for the quantification of the total homocysteine (t-HCY).

49. Use of TCEP at a concentration of TCEP of 0.625 mM in the analytical method according to any one of the preceding claims 1 to 28 for preventing suppressive and interfering effects caused on other analytes such as: HCY, HCY IS, 5HT IS, 3OHPA in the analysis step.

50. An extraction solution (ES) comprising TCEP 0.625 mM, a “IS MIX” solution of x % volume, wherein x= [(1 / 10)*m] wherein m is the number of internal standards, said extraction solution (ES) being adjusted to 100% (volume) with a solution of ultrapure water 0,1% (vol / vol) formic acid.

51. Use of the analytical method according to any one of the preceding claims 1 to 28 for the biochemical diagnostic confirmation within a screening program in the recall step, for diagnostic evaluation based on clinical evidences including non-newborns patients, for follow-up of patients under treatment.

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