WO2018174858A1

WO2018174858A1 - Clinical method for the population screening of adult metabolic disorder associated with chronic human disease

Info

Publication number: WO2018174858A1
Application number: PCT/US2017/023447
Authority: WO
Inventors: Bruce Xuefeng Ling; Limin Chen; Shiying Hao
Original assignee: Mprobe Inc.
Priority date: 2017-03-21
Filing date: 2017-03-21
Publication date: 2018-09-27

Abstract

A robust, rugged, cost-effective, and high throughput analytical method has been developed for the population screening of adult metabolic disorders associated with chronic human diseases using flow injection chromatography interfaced with electrospray tandem mass spectrometry. This method allows the simultaneous quantitative-based profiling of a big panel of targeted primary metabolites from a variety of human specimens, including serum, plasma, and dried blood spot, at the clinical level for the population health condition management in respect to metabolic risk factors associated with chronic disorders. The integration of enhanced quality control and quality assurance protocols in conjunction with the automatic sample handling platform maximizes the throughput at which the samples are analyzed while accurately and precisely maintaining the quality of the quantitation for clinical diagnosis.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention describes a high throughput, cost-effective, and robust analytical method for the population screening of adult metabolic disorders associated with chronic human diseases using high performance liquid chromatography interfaced with electrospray tandem mass spectrometry. This method integrates the capability of simultaneously assaying a broad range of metabolites with superior specificity and sensitivity to a stationary sample preparation automation platform for clinical screening against a huge population across a panel of chronic human diseases. The efficient sample preparation method allows the subsequent medical diagnosis to be implemented in a timely and consistent manner.

2. Description of Prior Art

Chronic disease is a condition where the disease state is persistent and long-lasting in its effects over the longterm development. As denoted by the term chronic, the symptoms of the disease tend to irregularly persist for a time window' ranging to months to years, causing devastating clinical outcomes on patients suffering from those disorders. Among multiple physiological consequences, the abnormality of endogenous metabolism has been found to be one of the most characteristic hallmarks in relation to the disease pathogenesis and progression, which can not only be indicative to the severity of ongoing disease, but also predictive to the occurrence of upcoming syndrome before the symptoms are clinically noticed and diagnosed. See Sommer et al.. Increased Prevalence of the Metabolic Syndrome in Patients with Moderate and Severe Psoriasis, Arch Dermatol Res (2006), 298:321-328. Therefore, measuring and monitoring the metabolism profile of a certain population of people is of primary importance as means of globally evaluating the metabolic risk factors associated with a variety of chronic disorder for the disease prevention to benefit the population healthcare management.

In last several decades, the cutting-edge mass spectrometry technology has made significant contributions to the advancement of clinical diagnosis of metabolic syndrome, giving rise to the confident diagnosis of different metabolic disorders of great medical interest based on levels of a variety of endogenous metabolites. With the emerging of the advanced tandem mass spectrometry instrument, the tandem mass spec-based analytical platform that is capable of simultaneously screening over a big panel of metabolites has been established to clinically diagnose the occurrence of different metabolic syndromes, typically inherited inborn errors of metabolism, on a routine basis. See Chace et al., Neonatal Screening for Inborn Errors of Metabolism by Automated Dynamic Liquid Sewary Ion Tandem Mass Spectrometry New Horizons in Neonatal Screening, 1994. In consideration of the specificity and sensitivity of tandem mass spectrometer, the quality of obtained results can be consistently maintained in terms of accuracy and precision.

Electrospray ionization is a classic technique which utilizes a tiny spray needle equipped with high electrical voltage and high gas pressure supplies to continuously convert the incoming liquid flow into fine charged aerosol and eventually gaseous phase ion with minimal introduction of the in-source fragmentation of the molecules during the process. While interfaced to the tandem mass spectrometer, the electrospray ionization provides the practical ability to transform the injected sample that originally presented as liquid into the mass spectrometrically detectable particle as gaseous ion, thus paving the way for the development of a high-throughput screening approach. See ashed et al., Clinical Chem. 43 :7, 1 129-1141. In addition, owing to the high ionization efficiency of the electrospray ionization to majority of the metabolites, the quantities of endogenous metabolites required by the electrospray ionization to obtain confident level of sensitivity are quite small, allowing the screening to be implemented even in case of extreme sample limitation. See U.S. Pat. No. 5,352,891.

Although the use of ESI/MS/MS has been demonstrated to be able to provide a quick screening method with desired specificity and sensitivity, based on the stringent demand of large sample throughput from the population screening, the method itself, however, should still be complemented with new protocols, comprising efficiency sample preparation, automatic sampling, and optimized mass spec scan function, to accommodate as many samples as needed in a single batch, thereby maximizing the analytical throughput while maintaining the precision and accuracy.

Sample preparation in support of the population screening of metabolic syndrome associated with human chronic disease for the use in mass spectrometry is seen in the art. The method of preparing sample for adult metabolism testing is standardized among available sample types, including serum, plasma, and dried blood spot, by using the newly developed methyl esterification method consists of processing the samples in 96-well plate. To each well of 96- well plate containing one measured sample/quality control, a methanolic solution containing isotope-labeled internal standards at desired concentrations is added automatically to extract metabolites of interest. The list of isotope-labeled internal standards might encompass amino acids, acylcarnitines, creatinine, urea, and succinylacetone combined to generate a mixture with desired concentrations to provide the optimal cost-effectiveness. Following vortex and centrifuge, the sample extracts are transferred into another microplate where the extracts are dried under nitrogen stream. To the residue of each well, methanolic HC1 solution is added followed by heating to complete the derivatization. The microplate containing final samples is placed onto the autosampler tray for automatic sample injection.

The incorporation of isotope- labeled internal standards using isotope dilution technique provides the normalizing factor to correct variations of extraction recovery and ionization efficiency as well as a quantitative reference to determine the level of specific metabolite in each sample. The concentrations of individual internal standards labeled with isotopes are optimized based upon their own linear ranges in the sample matrices to balance between the signal to noise ratio and cost effectiveness.

Quality assurance & control protocol is the procedure of primary importance in the clinical diagnostic environment to determine the degree of acceptance on sample-to-sample and batch- to-batch variations on the long-term basis, allowing the significant systematic errors to be appropriately identified and eliminated in a timely fashion before further interpretative actions are taken.

The advantages of electrospray mass spectrometry over alternative methods are its ability to perform absolute quantification by using isotope dilution technique in a precise and accurate manner in conjunction with its amenability to automation. See Chace et al, Clinical Chem. Vol. 39, No. l, 1993. By integrating the electrospray tandem mass spectrometry instrumentation into the platform, the present invention satisfies the high accuracy, precision, throughput, and cost-effectiveness requirements of population screening for metabolic syndrome associated with chronic disorder in a clinical setting. With present invention, quantitative result and diagnostic report will be generated on basis of assured quality in a timely manner. 3. Prior Art

U.S. Pat. No. 5, 206, 50, Apr. 27, 1993 describes a tandem mass spectrometric system which is capable of extracting tandem mass spectra for each parent ion without separating from other parent ions with different masses. This system would in addition provide the capability to select a specific parent ion prior to excitation.

U.S. Pat. No. 5, 352, 891, Oct. 4^th, 1994 (Mooning et al.) demonstrates the production of mass spectra of molecules with high molecular weights can be improved by generating an enhanced mass spectrum from the observed mass-to-charge ratio spectrum. Signal to noise ratio can, in some cases, be further improved by combining the isotopic envelops from different charged states into a single monoisotopic peak.

SUMMARY OF THE INVENTION

The primary objective of this invention is to introduce a novel clinical method to screen adult metabolic disorders associated with chronic human diseases over a wide range of population to benefit and facilitate the public healthcare management by implementing efficient sample preparation protocols in combination with ESI tandem mass spectrometric analysis. High analytical throughput is obtained from the combination of efficient sampling and fast turnaround period of ESI tandem mass spectrometer and a complementary' platform that handles heavy sampling duties in the most cost-effective manner. Quick diagnosis with high accuracy and precision will be consistently provided based on the measured values.

Elecuospray ionization coupled to tandem mass spectrometry is a well-accepted classic instrumental setup for the quantitative analysis of metabolites. By virtues of its high sensitivity and specificity, individual metabolites of interest can be resolved based on mass to charge ratio by using multiple parent-to-daughter mass transitions, each of which is characteristic and unique to one metabolite. With the addition of stable isotope-labeled internal standards, the extraction variation and ionization bias are effectively eliminated, and endogenous levels of a wide group of metabolites can be simultaneously determined with confident level of accuracy and precision.

The secondary objective of this invention is to improve the sampling throughput by implementing the primary steps, comprising liquid handling, shaking, and heating, of sample preparations in a 96-well plate format with a robust platform through an established automation program, thereby allowing the sample extraction prior to the analysis while eliminating the introduction of potential human errors.

The third objective of this invention is to improve the instrumental throughput by injecting the sample extracts into the LC loop with the flow injection method from an autosampler, delivering the injected samples with intended mobile phases, and detecting the signal responses from metabolites of interest with selected reaction monitoring. With this novel instrumental setup, the analytical timeframe will be significantly shortened into a 2-min period to screen through a large panel of metabolites for a single sample.

The fourth objective of this invention is to improve the detection sensitivities of low abundance metabolites of interest at the MS/MS level by derivatizing them with methanol and generating their corresponding methyl esters to achieve the optimal cost-effectiveness. Compared to the classical butyl esterification protocol, methyl esterification method has been demonstrated to provide greater signal responses on less abundant metabolites after derivatization with simplified sample preparation protocol. Therefore, comparable quantitative results could be obtained with this method using 5-fold less amounts of isotope-labeled standards added per sample to optimize the cost-effectiveness.

The fifth objective of this invention is to resolve the detection issues associated with the presence of isobaric metabolites (e.g. C3DC&C40H carnitine) and overlapping signals (e.g. C2 carnitine butyl ester &C6 carnitine) at the MS/MS level by esterifying the sample extracts with methanol instead of butanol prior to the analysis. Following methyl esterification, certain group of metabolites, especially acylcarnitines, will be fragmented into distinctive patterns which are completely different from the pre-derivatized ones, making this method superior to the classical butyl esterification protocol.

BRIEF DESCRIPTION OF THE DRAWINGS

The presented method generally consists of a series of principal steps as described in greater detail below:

Upon receiving multiple seram/plasma/dried blood spot samples, individual samples are divided into appropriate aliquots and immediately placed into freezer before use. Thereafter, a methanol-based extraction buffer containing known concentrations of a combination of amino acid and carnitine/acylcarnitine standards labeled with different stable isotopes is manually prepared and automatically added to extract metabolites of interest from the individual samples on the 96-well plate through a programmed automatic station. In parallel, quality control samples, encompassing normal, abnormal, and standard controls in duplicate, are prepared by following the identical protocol to ascertain the assay performance on samples from each plate. Afterwards, prepared samples are analyzed by high performance liquid chromatography system in flow injection configuration interfaced to electrospray tandem mass spectrometry in selected reaction monitoring mode, and the quality control samples are matched against their nominal values and thresholds to determine whether the acquired results from unknown samples on that plate will be accepted or rejected. With this system, the quality of obtained data can be confidently and consistently assured for further clinical evaluation and medical diagnosis to be accurately implemented.

Fig.1 is a simplified block diagram showing the overall makeup of the methodology. Five primary processes are inter-correlated from sample preparation to final diagnosis.

Fig.2 is a block diagram showing the details regarding steps involved in the sample preparation flowchart.

Fig.3 shows the roster of individual internal standards labeled with stable isotopes, their intended concentrations in the sample extraction buffer, and the metabolites to which each internal standard act as a normalizer and quantifier.

Fig. 4 is a block diagram showing the details regarding steps involved in the automated analysis flowchart.

Fig. 5 is an example of the extract ion chromatogram on amino acids and carnitine/acylcarnitines showing the pertinent peak area for each monitored parent-to-daughter mass transition and their thresholds.

Fig. 6 is a bar graph showing the detection sensitivity differences on less-abundant metabolites between the methyl esterification method described here and the classical butyl esterification method at the MS/MS level. Fig. 7 is a block diagram showing the details regarding the steps involved in the post- acquisition data processing and interpretation in relation to the medical diagnosis and clinical decision-making.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The method will now be described in detail in relation to a preferred embodiment and implementation thereof, which is exemplary in nature and descriptively specific as disclosed. As is customary, it will be understood that no limitation of the scope of the invention is thereby intended. The invention encompasses such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention illustrated herein, as would normally occur to persons skilled in the art to which the invention relates. Fig. 1 represents a general overview of the five major steps involved in the workflow of population screening adult metabolic disorders associated with human chronic diseases in the clinical setting, each of which are important factors determining the rapidness, consistency, and accuracy of the analysis. A robust sample preparation method with good efficiency and reproducibility is the fundamental basis to ensure the accuracy and consistency of the analysis across different samples. The preparation is implemented by employing the synthetic standards labeled with stable isotopes as internal standards to correct potential variabilities on extraction recovery, derivatization yield, as well as ionization efficiency during the analysis. In the presence of internal standards labeled with stable isotopes, quantitative information can also be provided for individual metabolites in the specific sample. Following the sample preparation, the samples are injected into the sample loop by autosampler and delivered to the electrospray tandem mass spectrometer by a constant flow of mobile phase with no column present. When the injected sample arrives at the electrospray ionization source, the tandem mass spectrometer automatically scans over each parent-to- daughter mass pair dedicated for each metabolite of interest to ensure the speed of the analysis. The acquired data from the samples is processed along with quality controls by assigned software to obtain concentration values of individual metabolites generated from the scans of mass spectrometer, and the concentration values are reformatted into a spreadsheet to undergo further inspections on the calculations as a means of ascertaining the performance of high quality. Determined concentrations are then interpreted by an assisted diagnostic interpretation system which correlates the results to the specific disorder based on any noted peaks. This process, operated in conjunction with software, enables the daily monitoring of data output to assist the necessary decision-making for further action, including follow^r-up and retesting. To assure the diagnostic accuracy and consistency, the calculated concentration values from all samples are subject to additional quality verification step by employing a set of quality controls, comprising normal, abnormal, and standard, in duplicate for each 96-well plate and setting up a stringent cutoff to determine the acceptance/rejection of a particular batch. Additionally, periodic system integrity and performance checks are also included as part of the quality control scheme. The combination of above steps maximizes the rate at which samples are screened for metabolic disorders and facilitates the decision-making in the clinical setting. Fig.2 displays an overview of primary steps involved in the sample preparation procedure. A standard sample login is initiated by barcoding each sample and thus associating the code to the specific location at which the sample is placed on the 96-well plate. Depending on various physical forms presented by different sample types (serum/plasma/dried blood spot), either 10 uL of serum/plasma or 3mm dried blood spot punch is prepared and placed into the designated well on the plate. Aftenvards, extraction buffer containing internal standards labeled with stable isotopes is prepared by diluting the stock solutions with methanolic solution, which is then added into each of the sample-containing well by the programmed liquid handling through an automated laboratory station. After programmed shaking and centrifuge, the supernant is transferred to a new 96-well plate and dried under a nitrogen stream. Thereafter, the dried sample extracts are reconstituted with derivatization buffer containing methanol accompanied by hydrochloric acid and chemically modified into the corresponding methyl ester under heating at moderate temperature. The derivative is subsequently placed into the autosampler for direct injection into the mass spectrometer. The plate is sealed to avoid any solvent evaporation prior to the analysis.

Fig.3 the 1^st column to the left shows the roster of individual internal standards labeled with stable isotopes, which details not only the molecular position of the stable isotope but also the type of isotope substituent, including ²Di and ¹ C, presented at a specific molecular spot. In this method, the methanol serves as a solvent extraction medium while the isotope-labeled internal standards act as both normalizer and quantifier for correction of the experimental variations as well as determination of the metabolite concentrations, respectively, in different sample matrices. The preparation of isotope-labeled internal standard-containing extraction buffer is implemented by diluting 6 stock mixes, encompassing 12 amino acids, 13 carnitine/acyl carnitine, and 5 other metabolites, with methanol at a ratio of 1 : 1000 (v/v) to obtain daily working concentration as the extraction buffer, which plays multiple universal functions, such as extraction medium, normalization, and quantification, as listed in the 2^nd column to the left in Fig.3 with more details. As limited by the availability of isotope-labeled internal standards, the metabolites of interest with no available standards labeled with stable isotope are quantified in the sample matrix based on the isotope-labeled analog with closest structural homology. Thus, a single isotope-labeled internal standard could possibly be serving as both normalizer and quantifier for multiple metabolite candidates. The related information detailing individual metabolites to which each internal standard acts as a normalizer and quantifier is summarized in the 3^rd column to the left in Fig.3.

Fig.4 illustrates the main steps involved in the automated analysis after the intended sample has been prepared and placed into the autosampler. Before the prepared sample is introduced into the sample loop, the high-performance liquid chromatography and electrospray tandem mass spectrometry system are separately optimized and equilibrated to daily standard working conditions as detailed below, hi respect to the high-performance liquid chromatography system, the solvent channel is purged in switch-valve -on configuration with designated mobile phase at 100-200 system volumes and equilibrated in switch- valve-off configuration with the same mobile phase for an additional 50 system volumes to reach satisfactory working condition. In respect to the electrospray tandem mass spectrometry system, the tandem mass spectrometer is optimized and performance checked by infusing a system tuning solution through a syringe pump, which is then subject to system equilibration with another 20 volumes of mobile phase. Following the automatic injection of prepared sample by the autosampler, the mobile phase is pumped to flush through the sample loop and pushing the injection sample to advance to the electrospray tandem mass spectrometer where a panel of metabolites of interest are sequentially scanned and measured at the MS/MS level. The scans implemented by tandem mass spectrometer are selected reaction monitoring scans where superior sensitivity and specificity can be achieved with individual metabolites in a small timescale, thereby allowing individual metabolites to be quantified accurately and precisely in the presence of their isotope- labeled internal standards. Based on acquired data, an automatic recognition system identifies the samples or quality controls sitting beyond the thresholds and tags those numbers with flag to facilitate the decision-making between sample re-testing and clinical diagnosis.

Fig. 5 shows an example of the extract ion chromatogram on a set of amino acids and carnitme/acylcarnitines with the pertinent peak area for each monitored parent-to-daughter mass transition and their thresholds included. The pertinent peak area associated with each measured metabolite is first divided by the pertinent peak area associated with the designated internal standard to obtain the peak area ratio, which is then multiplied by the working concentration of designated internal standard in conjunction with the sample dilution factor to calculate out the endogenous concentration of each metabolite. The obtained concentration values will later be subject to the systematic recognition and identification based on the known cutoff values to accelerate the data processing and interpretation steps with increased data output.

Fig.6 represents a bar graph showing the detection sensitivity differences on metabolites of low abundance between our methyl esterification method and classical butyl esterification method at the MS/MS level. Based on the graph, several-fold of detection sensitivity enhancement is achieved with the methyl esterification method compared to the classical butyl esterification method while aliquots of one samples are prepared using the identical experimental conditions. Furthermore, by shifting the derivatization reagent from butanol to methanol, the final step of sample preparation, excess derivatization reagent removal from the old protocol, can be bypassed with no found significant impacts on the subsequent analysis. hence further increasing the analytical throughput by reducing the turnaround time of sample preparation.

Fig.7 describes the primary steps involved in the post-acquisition data processing and interpretation in relation to the medical diagnosis and clinical decision-making. Following the acquisition, acquired data sets, containing all peak area values, concentration values, parent- to-daughter transition mass values, and sample codes, are inputted into the software in conjunction with the threshold values to calculated metabolite concentrations and identify abnormal samples as well as unsatisfactory quality controls with marked flag. Thereafter, the processed data is organized into a spreadsheet and inputted into a database module for recognition of file type and sample type, which is then interpreted by assigned parameters to generate the results. For samples show an abnormality or seem to show a revealing peak, interpretation is implemented by using a reference guide and decision tree to correlate the flagged samples with data module used to distinguish abnormal, leading to the final decision of either diagnosis or re-testing. As another means of quality assurance, the quality control mean and trending are recorded on the daily basis to follow the statistic incidence of certain diseases.

Claims

I claim:

1. A method of screening metabolic disorder associated with human chronic disease in adult population, comprising:

a. Preparing samples by using extraction buffer and derivatization;

b. Analyzing sample extracts by using high performance liquid chromatography interfaced to electrospray tandem mass spectrometry;

c. Calculating the concentrations of metabolites by referring to internal standards;

d. Interpreting the results by referencing to known thresholds;

e. Maintaining quality by including quality control sample sets into the above procedure from the beginning and comparing to the known threshold by the end.

2. The method of claim 1 wherein said sample comprises hemolyzed blood, plasma, serum, and dried blood spot.

3. The method of claim 1 witerein said quality control sample set comprises hemolyzed blood, plasma, serum, and dried blood spot.

4. The method of claim 1 wherein said extraction buffer comprises internal standard, methanolic solution, hydrazine hydrate, and 2-mercaptoethanol.

5. The method of claim 4 wherein said internal standard comprises an amino acid standard.

6. The method of claim 5 wherein said amino acid standard comprising ¹³C, ¹⁵N-Glycine, ²D₄- L- Alanine, ²D₈-L- Valine, ²D₃-L-Leucine, ²D₃-L-Methionine, ¹³C6-L-Phenylalanine, ¹³C6- L-Tyrosine, ²D₃-L-Aspai ate, ²D₃-DL-Glutamate, ²D2-L-Ornithine, ²D2-L-Citrulline, and 1 C, ²D₄-L-Arginine-HCl.

7. The method of claim 4 wherein said internal standard comprises a carnitine/acylcarnitine standard.

8. The method of claim 7 wherein said carnitine/acylcarnitine standard comprising ¾9-L- Camitine, ²D₃-0-Acetyl-L-Camitine-HCl, ²D₃-0-Propionyl-L-Carnitine-HCi, ²D₃-0- Butyryl-L-Carnitine-HCl, ²D9-0-Isovaleryl-L-Carnitine-HCl, ²D₃-0-Octanoyl-L- Carnitine-HCl, ²D₉-0-M Tistoyl-L-Carnitine-HCl, ²D₃-0-Palmitoyl-L-Carnitine-HCl, 2D₃-0-Glutaryl-L-Carnitine-C104, ²D₃-3-Hydroxyisovaleryl-L-Carnitine-C104, ¾9-0- Dodecanoyl-L-Carnitine-HCl, ²D₃-0-Octadecanoyl-L-Carnitine-HCl, and ²D -0-DL- Hydroxypalmitoyl-L-Carnitine-C10₄.

9. The method of claim 4 wherein said internal standard comprises a metabolite standard.

10. The method of claim 9 w^rherein said metabolite standard comprising ^LlC5-Succinylacetone, l⁵N₂-Urea, ²D₃-Creatinine, ^l3C₆, ^l3N4-Argininosuccinic Acid-3H₂0, ²D4-L-Homocysteine, and ^l3Cs, ^l5N-L-Proline.

1 1. The method of claim 4 wherein said methanolic solution comprises methanol and water.

12. The method of claim 1 wherein said derivatization comprises:

a. Drying the sample extract under nitrogen purging station;

b. Reconstituting the dried sample with derivatization buffer;

c. Incubating the reaction mix under heating at moderate temperature.

13. The method of claim 12 wherein said derivatization buffer comprises methanol, water, and hydrochloric acid.

14. The method of claim 1 wherem said section b further comprising the steps of:

a. Checking electrospray tandem mass spectrometer performance by infusing tuning solution through a syringe pump;

b. Equilibrating the system by flushing it with certain volumes of mobile phase;

c. Injecting the derivatized sample into the sample loop; d. Eluting the injected sample with a constant flow of mobile phase in flow injection configuration;

e. Ionizing the eluted sample by using electrospray ionization;

f. Determining the peak area value of each metabolite and internal standard by using selected reaction monitoring function of tandem mass spectrometer;

g. Washing and equilibrating the system for next run.

15. The method of claim 1 wherein said section c further comprising the steps of:

a. Inputting the acquired data into the designated processing software;

b. Calculating the concentration value of metabolite based on peak area ratio, internal standard concentration, and dilution factor;

c. Organizing the processed data into a spreadsheet.

16. The method of claim 15 wherein said peak area ratio comprises the ratio obtained by dividing peak area value of metabolite by peak area value of internal standard.

17. The method of claim 16 wherein said ratio comprises the ratio between one metabolite and its exact isotope-labeled standard as well as the ratio between one metabolite and its isotope-labeled analog with closest structural homology.

18. The method of claim 15 wherein said internal standard concentration comprises the working concentration of internal standard in the extraction buffer by diluting the stocks with methanol.

19. The method of claim 15 wherein said dilution factor comprises the fold number to which the pre-extracted sample is diluted prior to the analysis.

20. The method of claim 1 wherein said section d further comprising the steps of: a. Inputting the organized spreadsheet into a database module to assist the data recognition and interpretation;

b. Identifying the sample with value sitting beyond the set threshold;

c. Flagging each identified sample revealed a value above/below the previously determined threshold;

d. Interpreting the flagged sample by using a reference guide;

e. Making decision between sample re-testing and clinical diagnosis.

21. The method of claim 19 wherein said set threshold comprises both upper and lower cutoff values.

22. The method of claim 1 wherein said section e further comprising the steps of:

a. Including quality control sample set into the sample batch before sample preparation. b. Preparing the quality control sample set in along with the measured sample.

c. Analyzing the prepared quality control sample set in conjunction with the measured sample.

d. Calculating the concentration of each metabolite in each analyzed sample.

e. Comparing the concentration value of each metabolite in each quality control sample to individual set threshold.

f. Deciding the acceptance/rejection of the results of analyzed sample based on the performance of quality control sample set.

23. The method of claim 22 wherein said quality control sample set comprises a normal sample as negative control, an abnormal sample as positive control, and a matrix-free standard alone sample as instrumental control.