Quantification of previously undetectable quantities

Abstract

The present invention is directed to a novel "molecular amplification spike," which is a mixture of two components: an aliquot of a quantity of a molecule, composition, compound, or element of interest ("target") in its natural isotopic state, and an aliquot of an isotopically enriched form of the same molecule, composition, compound, or element. The molecular amplification spike contains 20% natural isotopes and the remainder enriched isotopes. The molecular amplification spike can optionally contain more than 20% natural abundance isotopes, thereby reducing the balance of enriched isotopes. Such a mixture spike, when added to a sample prior to mass analysis of that sample, significantly improves the error rate and quantification or confirmation of the absence of the target molecule, composition, compound, or element in the sample.

Description

Technical Field

[0001] <CROSS-REFERENCE TO RELATED APPLICATIONS> This patent application is a non-provisional application based on U.S. Provisional Patent Application No. 63 / 093,881, filed on October 20, 2020. The priority of the provisional patent application is claimed, and the provisional patent application is incorporated herein by reference.

[0002] <Technical Field> The technology of the present invention, in relation to mass spectrometry, enables the quantification for the first time of molecules, compositions, compounds or elements of interest that were previously present in amounts below the LLOQ, i.e., in amounts below the Lower Level of Quantification (in the past), using a novel molecular amplification spike. The Lower Level of Quantification may also be referred to as the Limit of Quantification (LOQ) in samples in medical, biological, environmental, industrial or any other setting. The human body has a concentration gradient of about 10-23 in the concentration difference of molecular concentrations, and many biomarkers are at low concentrations and below the LLOQ of mass spectrometers.

Background Art

[0003] Mass spectrometry still has inherent limitations despite its versatility and popularity in both research and medical laboratories. The current quantification methods rely on calibration curves, and due to the compound expansion of the inherent limitations of mass spectrometry, they do not function well in mass spectrometry at trace levels near or below the LLOQ. In fact, the use of mass spectrometry in medical applications in the life sciences has been limited so far due to the difficulty of quantification and the generally unpredictable variation in percent error. Even when the same mass spectrometer is used to analyze samples at different time intervals, or when different operators use the same mass spectrometer to analyze the same sample, it can still be difficult today to obtain reliable mass spectrometry results. The sensitivity of mass spectrometers related to the LOQ is based on type, adjustment, the physics of operating conditions, and ionization methods.

[0004] Alongside the challenges arising from the limitations of mass spectrometry, the entire control process (sample collection, transport, storage, and evaluation) often contributes to inconsistent results for numerous reasons, including sample instability and errors in sample preparation. For example, medical diagnostics continue to move towards "molecular biomarkers," as biomarkers (and imaging diagnostics) are more directly linked to medical care than ever before. However, legal and practical barriers often exist to medical sample collection. For instance, when blood samples need to be transported across geographical boundaries, there are laws and regulations, both international and sometimes local, that prevent such transport. Apart from laws and regulations, the transport of viable biological samples such as blood and tissue samples also has practical limitations, including, but not limited to, weather issues, unsuitable ambient temperatures, transport delays, packaging issues, and the need to avoid sample degradation during transport. Many populations in remote areas require medical sample evaluation despite the lack of local access to refrigeration, rapid transport, etc., and children or other patients in such locations require qualified diagnoses despite these infrastructure challenges. These challenges are not simply that medical, industrial, environmental, and other types of samples may require evaluation on a routine or emergency basis. Such samples must reach the testing laboratory from their source without degradation. In the field of mass spectrometry, there has long been a need for methodological innovation to identify and quantify components present in any sample, even in undetectable amounts that were previously unquantifiable, thereby enabling the use of easily transportable, stable, and small-volume samples and avoiding the sample degradation problems caused by conditions associated with a series of controls.

[0005] Therefore, the following two challenges need to be addressed simultaneously: firstly, the ability of mass spectrometry to quantify previously undetectable molecules, compositions, compounds, or elements that require quantification in biological, environmental, industrial, or other samples; and secondly, the rationalization of sample collection, stabilization, and transport of the minimum amount of material to be analyzed. Ideally, such a technique would be possible using conventional mass spectrometry, enabling quantification of quantities two orders of magnitude lower than previously achievable in terms of sensitivity and quantification. [Overview of the project]

[0006] To satisfy this requirement, the present invention provides for obtaining one or more (generally small) samples containing one or more molecules, compositions, compounds, or elements known or suspected to be present in amounts below a previously recognized LLOQ (Limit of Quantification). The samples are obtained by reducing the sample size (10 to 30 microliters) or stabilizing them (e.g., dry liquid on a carrier such as a simple blood spot on a card). These small samples are then treated with (contacted with) a molecular amplification spike and analyzed by mass spectrometer. The molecular amplification spike is a mixture of two components, i.e., an aliquot of a certain amount of the molecule, composition, compound, or element to be quantified in its natural isotopic state, mixed with an aliquot of the same molecule, composition, compound, or element in its isotopic-enriched form. The molecular amplification spike contains 20% natural isotopes and the remainder enriched isotopes. The molecular amplification spike may optionally contain more than 20% natural isotopes, which may result in a decrease in the balance of enriched isotopes. Typically, though not always, natural isotopes are present in molecular amplification spikes at concentrations of 90% or less, more preferably 70% or less. When the sample being analyzed is brought into contact with a certain amount of molecular amplification spikes prior to mass spectrometry of the sample, the combination of amplification provided by natural isotopes and isotopic shift tracking enabled by the presence of isotopic-enriched portions of the spikes allows for both mass spectrometry signal generation and the calculation of the initial amount of natural isotopes originally present in the collected sample. Unstable species that transition to other species during measurement are addressed by Speciated Isotope Dilution Mass Spectrometry (SIDMS). SIDMS also benefits from the present invention, as the same premixed molecular amplification spikes of the present invention can be used in SIDMS, since important unstable species such as drugs and biomarkers are quantitatively and definitively evaluated near or at the LOQ of the mass spectrometer. (The instability of the substance (analyte) is when the molecular amplification spikes of the present invention "make SIDMS shine even brighter than before.")This is because SIDMS already allows for the simultaneous tracking of interconversion between two variants and analysis in a single test, and can report the most accurate and precise concentrations of both the analyte and variant in the sample immediately before collection (when interconversion begins). While the inverse calculation is well known to those skilled in isotope dilution mass spectrometry and similar techniques and can be easily automated, no one had previously considered adding natural isotopes to an isotope enrichment spike to generate the molecular amplification spike of the present invention. The effect of adding the molecular amplification spike eliminates the need for expensive transport in significantly large liquid masses and weights of substances that are restricted by state, federal, or international regulations, or that are susceptible to degradation due to weather, temperature, or time, and are otherwise biologically hazardous, either as a liquid or as dry spots on a carrier (e.g., a simple cellulose curd, an inert polymer, or any solid matrix of any kind), either as a liquid or as dry spots. [Brief explanation of the drawing]

[0007] [Figure 1] Figure 1 is a bar graph illustrating how the present invention, colloquially known as "Thor's Hammer," achieves signal amplification by adding METASPIKE® molecular amplification spikes to a sample before mass spectrometry. [Modes for carrying out the invention]

[0008] In the world of analysis, there are countless elusive molecules, compositions, compounds, and elements that need to be quantified, not just discovered and verified. These include, but are not limited to, suspected food or drug contaminants, toxins, blood or bodily fluid metabolites, drug residues including those of abused substances, decomposition contaminants in industrial lubricants, air pollutants from industry, and agricultural pollutants from pesticides; the list is endless. When these substances are elusive, that is, present in small but devastating quantities, the present invention is directed toward their quantification. For the first time, by using the techniques described herein, molecules, compositions, compounds, and elements of all kinds and properties can be identified and quantified from samples of any aspect of medical, biological, environmental, industrial, or other relevant sources of investigation.

[0009] The first problem addressed in this invention was the identification and quantification of low levels of curcuminoids in cerebrospinal fluid (CSF). The need to do this arose because neurologists were studying these turmeric extracts that could cross the blood-brain barrier, attempting to find bioactive agents that could halt demyelination and the formation of troublesome amyloid plaques, the main causes of Alzheimer's disease, for the purpose of researching treatments for Alzheimer's disease. In their investigation of curcuminoids, researchers were able to observe the presence of several forms of curcumin in the CSF, but were unable to quantify the amount present. By using a molecular amplification spike containing 20% ​​natural curcumin isotopes and the remaining isotopically concentrated curcumin, along with the method of this patent specification, it is now possible to quantify curcuminoids in CSF, even at low concentrations. Quantifying the amount of curcumin and other substances in CSF is an important measurement, necessary to determine effective dosages of substances intended to cross the blood-brain barrier and not in levels high enough to cause side effects.

[0010] When molecules, compositions, compounds, or elements exist beyond LLOQ, conventional or known processes for quantifying them, such as isotopic dilution mass spectrometry or type isotopic dilution mass spectrometry, can be used, generally speaking. A variety of prior art techniques are disclosed and described in the inventors' own prior patents, including, but not limited to, U.S. Patents 6,790,673, 8,383,420, 9,869,684, and 10,962,556. However, with respect to the purpose of the present technology, the inventors of the present invention claim a patent for the use of molecular amplification spikes to achieve identification and quantification of molecules, compositions, compounds, and elements that previously existed below LLOQ, as a specific focus of the present technology and the problems it can solve. The aforementioned patents do not implicitly address any method for evaluating and quantifying substituents that existed below LLOQ prior to the present invention.

[0011] In addition to examining, identifying, and quantifying molecules, compositions, compounds, and elements that previously existed at LLOQ levels, it is also important to be able to detect low concentrations of molecules, compositions, compounds, and elements before they are converted into their variant molecules. Tracking reduced glutathione (natural form) versus oxidized glutathione is crucial, and various elusive substances such as heroin must be detected and quantified before they are converted. For example, heroin in blood or bodily fluid samples is often rapidly converted into morphine and can no longer be identified as heroin. Other substances that are easily converted and therefore need to be evaluated quickly include, but are not limited to, 6-methamphetamine, cocaine, benzoylecgonine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, norbuprenorphine, gabapentin, celecoxib, naloxone, normaloxone, noroxymorphone, fentanyl, norfentanyl, oxycodone, noroxycodone, tetrahydrocannabinol, cannabidiol, and loperamide. Similarly, persistent organic pollutants need to be identified before they are converted into other forms in environmental samples for analysis. These contaminants include, but are not limited to, naphthalene, acenaphthene, fluorene, phenanthrene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluorantene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenzo[ah]anthracene, cyclolodiphenyltrichloroethane, dichlorodiphenyldichloroethylene, and dichlorodiphenyldichloroethane. Perhaps of higher priority substances that must be identified and quantified are hazardous contaminants and toxins in food and drugs, such as, but are not limited to, mercury (in various forms), polydimethylsiloxane (PDMS), chromium, lead, and arsenic, but the list of potential food contaminants is in the thousands. Serious and persistent health concerns permeate the entire nutritional supplement industry.Consumers and manufacturers also want to know if any contaminants are present in vitamins, minerals, herbs, and other nutritional supplements that they expect to be healthy if the contaminants are absent. Various homeland security initiatives require the quantification of fugitive agents and chemical weapons. Authentication of products, valuables, and legal documents requires indestructible molecular coding that needs to be evaluated at trace levels to prevent counterfeiting. Thus, as described here, the list of substances requiring low levels of incidental contamination and quantification is literally endless.

[0012] Therefore, the key to identifying and quantifying low-level molecules, compositions, compounds, and elements that are difficult to capture by other methods lies in the ability of the molecular amplification spike of the present invention to make detectable (and quantifiable) by mass spectrometry substances that, without the amplification spike, would otherwise be included within the "noise" level of the output of a spectroscopic measurement. This molecular amplification spike, i.e., METASPIKE®, is the most important aspect of the present invention, in which a certain proportion of naturally occurring abundance isotopes is combined with enriched isotopes to create a hybrid spike (the spike is an adduct) containing both naturally occurring abundance and enriched isotopes of the same substance for which identification and quantification are sought. As described above, the present invention obtains one or more small samples containing one or more molecules, compositions, compounds, or elements that are known or suspected to be present in amounts less than previously recognized LLOQ. The samples are obtained by reducing the sample size (10 to 30 microliters) and / or stabilizing them (e.g., a dry liquid on a carrier such as a mere blood spot on a filter sealed in a card). These small samples are then treated with (contacted with) a molecular amplification spike and analyzed by mass spectrometer. A molecular amplification spike is a mixture of two components, i.e., an aliquot of a certain amount of the molecule, composition, compound, or element to be quantified in its natural isotopic state, mixed with an isotopic enriched form of the same molecule, composition, compound, or element. To achieve clinically acceptable accuracy and quantification, the molecular amplification spike contains 20% natural isotopes of the same substance of interest and the remainder enriched isotopes. The molecular amplification spike may optionally contain more than 20% natural isotopes, which may result in a reduced balance of enriched isotopes. (For the purposes of this invention, "containing 20%" applies, for example, to a real-world spike containing 22%, which would naturally empirically contain 20% of naturally occurring isotopes). Typically, but not always, natural isotopes are present in the molecular amplification spike at a concentration of 90% or less natural isotopes.When the sample to be analyzed is equilibrated by contacting a certain amount of molecular amplification spikes before mass spectrometry of the sample, the combination of amplification induced by natural isotopes and isotopic shift tracking made possible by the presence of isotopic-enriched portions of the spikes allows for both mass spectrometry signal generation and inverse calculation of the initial amounts of molecules, compositions, compounds, or elements originally present in the collected sample. While these inverse calculations are well known to those skilled in isotopic dilution mass spectrometry and can be easily automated, no one had before considered adding natural isotopes to the isotopic-enriched spikes to generate molecular amplification spikes that could be optimized to achieve desired quantitative levels below LLOQ. The effect of adding molecular amplification spikes eliminates the need for expensive transport of otherwise biologically hazardous substances in significantly large liquid masses and weights, either as a liquid or as dry spots on a carrier (e.g., simple cellulose fiber, inert polymer, or any kind of solid matrix), which would otherwise be restricted by state, federal, or international regulations or susceptible to decomposition due to weather, temperature, or time. In practice, it should be kept in mind that the use of quantitative dry blood spot cards (DBS) generally reduces the sample and spike signals by one-fifth. Therefore, without the molecular amplification spike of the present invention, for example, 10-20 microliters (uL) of dry blood spots would make quantification of GSSG (glutathione oxidized) and MMA (methylmalonic acid), whose LLOQs are 42 ug / g and 12 ug / g (micrograms / gram), respectively, impossible. These quantified measurements were just above the LOQ on an Agilent 6460 triple quadrupole mass spectrometer using electrospray jet streams. Using the molecular amplification spike of the present invention in conjunction with dry blood spot cards is particularly important because without the mixed spike of the present invention, many of the dry blood spot components in question are simply below the LLOQ level, resulting in wide variability and large percentage errors. The practical and economic benefits of collecting blood samples as dry blood spots on cards rather than in tubes using the spike of the present invention are enormous and potentially transformative.

[0013] The automation of sample evaluation in partially similar fields to isotope dilution mass spectrometry (IDMS) is well known in the art. Conventional IDMS relies on the reliable phenomenon that most elements have two or more stable isotopes, and that their abundances in nature remain constant. When a known amount of enriched stable isotope is added to the sample being analyzed, this reliable "constant" phenomenon adjusts the isotopic ratios after a short period of equilibration. After equilibration, the ratios between isotopes can be measured by mass spectrometry, and working in reverse, it is possible to determine the original concentrations of elements (or other substances) in the sample by mathematically "backing out" the isotopic shifts that occurred during equilibration. Spectrometers and computers have been able to "communicate" with each other for decades, and the feedback mechanism that "runs" a sample prepared under controlled reaction conditions (in the mass spectrometer) over time is already a mature technology. In this invention, it is known that adding a 20% natural abundance isotope / residual isotope enrichment spike to a sample enhances the signal generation of the "unknown analyte" to be identified and quantified. Therefore, it is well within the realm of the art to provide an automated continuous sample analysis that continuously analyzes a sample using spikes with increased natural isotope content as needed, for the purpose of tracking and recording the signal generation of the mass spectrometer for multiples of otherwise identical (pre-spiking) samples. Given this background, it is easy to understand why this invention always functions with 20% natural abundance isotopes in the mixed natural abundance isotope / enrichment isotope combination spike. However, to empirically optimize signal generation, continuous spike analysis can also be performed with increased natural abundance isotope content (compared to the isotope enrichment spike fraction). In other words, including 20% ​​natural abundance isotopes in the molecular amplification spike of this invention is always beneficial, and very beneficial. Furthermore, generating a calibration curve that can identify the analyte containing the additional amount of natural abundance isotopes, and thus yielding even better results, is well within the scope of the art of this field. This is by no means a speculative suggestion.The key to this technology is the inclusion of some (20%) natural isotopes. While conventional methods used enriched isotope spikes, in this invention, knowing that it is a mixed natural abundance isotope / enriched isotope spike, the incidental "tweaking" that occurs when more natural isotopes are selectively added becomes a matter of routine. This is why up to 90% natural abundance isotopes / 10% enriched isotopes can be combined in the molecular amplification spike of this invention, because such spikes usually work without any trial and error. Nevertheless, the 20% inclusion of natural isotopes in the mixed natural abundance / enriched isotope spike is key to this invention, and it works for any molecule, composition, compound, and element of interest when the analyte in question is prepared and isolated with a mixed spike containing 20% ​​of its natural isotopic form and the remainder in its enriched isotopic form. Simply put, the molecular amplification spike of the present invention, when added to a sample analyzed by mass spectrometry, facilitates the identification and quantification of the substance or analyte being sought by "pulls up out of the weeds"—signals that would otherwise have attenuated below the spectrometer's data-to-noise ratio.

[0014] Further information regarding the preparation and use of known prior art for isotopic spikes is largely available in the inventors' own previous patents (e.g., the patents cited above). However, the technique of using spikes in mass spectrometry is not as complex as it may seem, if one knows what they are and how to apply them. In most direct terms, when a mass spectrometry sample is prepared, in order to add spikes, one simply chooses to add the spike material to the sample in the order of (e.g.) 10 ppm or 100 ppm, as in the specific example for the GSSG described above on an Agilent Model 6460 mass spectrometer, while tracking the amount of spike added (and in this case, the ratio of the natural isotopic abundance of the sample to the isotopic enriched isotopes in the spike). The initial abundance and amount of the same material, which also appears as a natural isotopic spike component, can then be easily calculated by observing the expected isotopic shifts that are present everywhere in nature, using standard IDMS techniques. More specifically, when analyzing a 10-microliter sample, regardless of whether the 10-microliter sample is in liquid form or dried after collection, the amount of pre-mixed spike added would be 70–100 picograms (pg) when analyzed using an ESI-QQQ-MS mass spectrometer known in the art. If the target substance in the 10-microliter sample is suspected to be present at a level of approximately 1 nanogram or less, 70 picograms of spike are used. If the target substance in the sample is suspected to be present at a level of less than 0.1 nanograms, 100 picograms of the mixed spike (of the present invention) are added, again exemplified by ESI-QQQ-MS. The spike may be added directly to the sample or pre-fixed to a sample collection device such as a card, tube, or other collection container or vehicle. The inventors have repeatedly demonstrated that when the disclosed amounts of mixed spike are added to the sample and equilibrated before analysis, it is possible to detect and quantify substances that cannot be detected or reliably quantified by conventional mass spectrometry. Equilibrium can occur in as little as one minute, but in practice, it is necessary to allow the sample to equilibrium for at least 30 minutes to an hour before mass spectrometry.Equilibrium formation generally occurs at ambient temperature and pressure. When the collection medium of the present invention (such as a card, matrix, or liquid tube) is pre-spiked with the mixing spike of the present invention and the sample to be tested is collected in or on them, equilibrium of the sample occurs naturally during the normal transport time of the sample to the laboratory.

[0015] The above explains, in general terms, how important the technology of the present invention is. However, there are specific applications where the technology of the present invention is literally irreplaceable. One such environment is the Food and Drug Administration's (FDA) requirement for high precision and low error rates for the measurement of small amounts of substances, and the ability of the present invention to perform measurements within the FDA's requirements. Specifically, the FDA requires precision and accuracy of 15% error or less at a 95% confidence interval. The U.S. FDA not only actively encourages the use of biomarkers in biomedical practice, anticipating a wide range of biomarker applications in drug development, but also "turns a blind eye" if biomarkers or other measurements do not meet their error specifications. As one example among many, when the present invention's technique is used to identify and quantify important biomarkers GSSGs (glutathione variants important for diagnosis and prognosis), the technique can (empirically) achieve an error reduction of 130% to 231% compared to conventional methods for quantifying GSSGs. Specifically, in the example mass spectrometer, with all other conditions kept the FDA accuracy and precision below 15%, a difference of 1.5 orders of magnitude was maintained compared to the mass spectrometer's LOQ.

[0016] Another area of ​​innovation that relies on this invention is the diagnosis and treatment of lysozyme storage diseases. Currently, more than 50 types of lysozyme storage diseases (LSDs) are known, and tens of thousands of people are deficient in some enzyme. On average, these patients die before reaching the age of 30. Finding treatments and effective treatments for these diseases requires the ability to quantify the presence of replacement enzymes, which are generally effective at levels too low to be quantified by conventional diagnostic methods and cannot be administered at any high levels. Since enzymes are proteins, the technology of this invention is naturally best suited for quantifying enzyme levels, especially at very low levels in cerebrospinal fluid (CSF), by using a corresponding spike containing the natural abundance isotope of the enzyme in question, mixed with an isotopically enriched version of the same enzyme.

[0017] Because the present invention can identify and quantify low-level substances of interest, it naturally enables quantification even with very small sample volumes compared to prior art identification techniques. In typical blood collection, for example, a phlebotomist may collect blood into 2 to 4 standard tubes, each containing 6 to 10 ml of patient blood. Therefore, with up to 40 ml of blood in 4 tubes, the mass and volume are considerable, raising challenges regarding transportation, shipping, storage stability, and biohazards. In contrast, the technique of the present invention allows for the easy analysis of 10 to 30 microliter blood samples, such as 10 to 30 microliters of dried blood spots deposited on cellulose fiber, polymer, or any other type of inert solid matrix carrier and easily stabilized by drying. A typical card of this type may have four locations for four dried blood spots, each inoculated with 10 to 30 microliters of blood (from a simple finger "stick"). Indeed, the ability of mass spectrometers to analyze samples given to a solid matrix is ​​already well known, see, for example, U.S. Patent No. 8,383,420, which is above and is part of this specification by reference. In remote locations or where the transport of biological materials presents challenges, cards with dried blood spots or similarly deposited and dried samples (including, but not limited to, biological samples) become highly practical and low-cost sample collection devices with virtually limitless applicability to every corner of the globe. Indeed, even from a cost management standpoint alone, the cost difference between shipping blood collection tubes and literally mailing cards with blood spots (if necessary) is significant, demonstrating how the technology of the present invention greatly reduces the transportation costs and complexity of sample collection before analysis.

[0018] As described above, a typical card with dried blood spots usually contains four blood spots and is easily collected from an animal or human patient with a single "finger stick" using a lancet known in the art. The card or matrix may be ordinary cellulose, or a pure cellulose matrix such as "Whatman paper," or another inert polymer or composite card that does not react with the biological sample and can be dried without degrading the biological sample (including but not limited to blood). This type of cellulose fiber, card, or solid matrix may be used to collect, store, and transport dryable biological, environmental, or industrial materials, including, but not limited to, blood, urine, plasma, saliva, bone marrow, cerebrospinal fluid, or, but not limited to, water, oil or gas resources, industrial fluids, and other existing interesting materials. Typically, the card contains four pre-assigned locations for four spots of sample, each spot capable of holding about 10 to 30 microliters of sample before drying and transport. When analyzing spots on the card or another matrix by mass spectrometer, a typical protocol includes the following further steps. If the collection card or matrix has not been pre-treated with the molecular amplification spike of the present invention, 70 to 100 picograms of the pre-mixed molecular amplification spike of the present invention are added to each blood or specimen spot on the card to equilibrate. (If the card has been pre-treated, it is pre-treated with the same amount of the pre-mixed spike of the present invention.) Equilibration means incubation at ambient temperature at rest for at least one minute or several minutes, or for one hour, 24 hours, or longer. After incubation, each blood or specimen spot is typically excised from its carrier using an 8 mm punch tool (8 mm diameter), and each excised blood or specimen spot is then usually further cut into four separate pieces. These pieces are typically placed in a microcentrifuge tube containing 70% water and 30% acetonitrile (extraction medium), and the tube is typically vortexed and sonicated at 60°F for one hour.The sonicated sample is centrifuged, and the liquid layer is transferred to a new microtube. The sample in the new tube is dried for 3 hours in a "SpeedVac" (known in the art) to obtain a pellet sample. The pellet is then reconstituted and analyzed by mass spectrometry according to a known protocol, and the results are integrated and used for quantification. In a common manner, for molecules (including, but not limited to, drugs, proteins, and enzymes), compositions, compounds, and elements, the analysis performed as described above can identify and quantify substances in a sample that cannot be quantified without the addition of the mixed "molecular amplification spike" of the present invention, METASPIKE®.

[0019] Generally, the present invention does not function effectively (i.e., accurately or precisely) when applied to attempt to identify and quantify substances beyond two orders of magnitude (quantitative improvement range) of the previous lower limit of quantification (LLOQ) of currently available mass spectrometers. Broadly speaking, the present invention is suitable for identifying and quantifying substances present in a sample at amounts of 0.01 to 1 nanogram per 10 microliters of sample. Other techniques are suitable for quantifying substances present at amounts greater than 1 nanogram per 10 microliters of sample, and to the best of the inventors' knowledge, there are currently no techniques for quantifying substances present at amounts less than 0.01 nanograms per 10 microliters of sample. Therefore, in a non-limiting example of MMA, substances are typically present in human blood at approximately 1.9 micrograms per gram (ranging from 0.0 to 0.35 micrograms per gram), and the signal loss from liquid blood to a dry blood spot is approximately one-fifth. This means that it is impossible to quantify methylmalonic acid from a 10-microliter dry blood spot without adding a naturally occurring abundance / isotope enrichment spike pre-mixed with the corresponding methylmalonic acid to the blood spot to actually amplify the methylmalonic acid signal. This is important and representative for any other substance to be evaluated according to the present invention, as it is present in a similar concentration range to GSH, GSSG, and MMA. The inventors achieved an error of less than 15% at a 95% confidence interval in their data that meets US FDA (Food and Drug Administration) standards, and it would be impossible to meet this error limit level without the molecular amplification spike of the present invention. However, the fact that this specification refers to MMA and GSSG should not be understood as limiting the present invention to the quantification of specific substances. The present invention can actually quantify any low concentration of indicator metabolites resulting from cancer chemotherapy, for example, by identifying them at early low levels before the chemotherapy patient experiences the recognizable negative effects that the indicator foreshadows. Finding lead, mercury, and arsenic in patient and environmental samples is as important as finding biological markers in diagnostic samples, and the present invention enables the quantification of all important molecules, compositions, compounds, and elements that are often hidden in small amounts.

[0020] That said, are there any limitations on what substances can be candidates for quantification by the use of the molecular amplification spikes of the present invention within the scope of the full operation of the present invention? As described throughout this specification, the present invention applies to all molecules, compositions, compounds and elements. "Molecules, compositions, compounds and elements" means, but is not limited to, bioactive agents, peptides, proteins, enzymes, vitamins, drug metabolites, elements, organic solvents, insecticides, anesthetics, lipids, sugars, polysaccharides, growth factors, biological markers, antigens, antibodies, growth factor receptors and antigen receptor markers, etc. Since all of these substances can be equilibrated with isotopically enriched analogs, according to the present invention, each of them can be quantified using "spikes" which are pre-mixed for the purpose of molecular signal amplification, containing both the naturally occurring abundance isotope (of the substance to be measured in the sample) and an isotopically enriched version of the substance. Such pre-mixed molecular amplification spikes, containing 20% ​​natural isotopes as described above, can be used for the identification and quantification of substances present in a sample at amounts of 0.01 to 1 nanogram per 10 microliters of sample. For those unfamiliar with IDMS and related techniques, it is important to remember that currently available isotope enrichments are not radioactive isotopes. The key to isotope enrichment when preparing pre-mixed spikes is to provide a combined natural abundance / enriched isotope mixture for substantially any substance whose quantification is desired. Therefore, the present invention is suitable for investigating, discovering, and quantifying substantially any target substance that may be present in any type of sample at amounts of 0 to 0.01 nanograms per 10 microliters of sample.

[0021] While specific amounts of spikes are disclosed (in ppm or picograms) in the various contexts described above, knowing how much (what amount) of spike to use in various isotope dilution mass spectrometry iterations is well within the scope of the skills of the art. The present invention relates to a type of premixed spike used to provide newly required quantitative results when quantifying low-level analytes, but those skilled in the art will know how much to use in a given application. Optimization of spike ratios for using prior art related to isotope enrichment spike techniques is well known at the time of writing this specification and typically involves introducing error propagation factors understood by those skilled in the art. In fact, it is virtually impossible to perform prior art IDMS (or type isotope dilution mass spectrometry) without using error propagation factors. These same ratios, amounts, and error propagation factors are equally applicable to the present invention, except that the molecular amplified spikes of the present invention contain 20% naturally occurring isotopes and the remaining isotope enrichment isotopes of the analyte, in contrast to the 100% isotope enrichment isotopes present in prior art IDMS and SIDMS spikes. After learning that 20% of the spike material of the present invention is added to the remaining isotopic enriched species, those skilled in the art already know how much spike to add to how much sample in order to perform the necessary mass spectrometry according to the present invention. Another way of understanding the concept of this paragraph is that the molecular amplified spike of the present invention is not a quantification method in itself. Instead, the molecular amplified spike of the present invention amplifies the signal, as its name suggests, thereby facilitating analysis using calibration curves known in IDMS, SIDMS, and (if the signal is sufficiently amplified) mass spectrometry techniques.

[0022] The present invention may be used with any existing or future mass spectrometer or ionization method, including, but not limited to, quadrupole mass spectrometers, triple quadrupole mass spectrometers, time-of-flight mass spectrometers, trap mass spectrometers, orbitrap mass spectrometers, sector mass spectrometers, or any mass spectrometer, using any ionization source or any ionization, such as electrospray, electron ionization, thermal ionization, matrix-assisted laser desorption ionization, laser ablation, or inductively coupled plasma.

[0023] Although the present invention has been described in detail above, it should be limited only to the extent described in the appended claims.

Claims

1. A method for amplifying the mass spectrometry signal of a target substance that is thought to be present in a sample analyzed by a mass spectrometer at a level below the lower limit of quantification (LLOQ) of the mass spectrometer, (a) A step of obtaining a certain amount of the target substance as a natural abundance isotope, (b) A step of obtaining a certain amount of concentrated isotope of the target substance, (c) A step of preparing a mixed molecular amplification spike by mixing the aforementioned amount of natural abundance isotope from step (a) with the enriched isotope from step (b) as the remainder, (d) A step of preparing a spiked sample by mixing an aliquot of the mixed molecular amplification spike with a certain amount of sample to be analyzed, (e) A step of equilibrating the spiked sample for at least one minute to obtain an equilibrated sample, (f) A step of analyzing the equilibrated sample with the mass spectrometer to quantify the amount of the target substance, if present, Methods that include...

2. The method according to claim 1, wherein the certain amount of sample in step (d) is 10 to 30 microliters of human or animal body fluid on a blood card, measured before drying, and the target substance is selected from the group consisting of bioactive agents, peptides, proteins, enzymes, vitamins, drug metabolites, elements, organic solvents, insecticides, anesthetics, lipids, sugars, polysaccharides, growth factors, biological markers, antigens, antibodies, growth factor receptors and antigen receptor markers.

3. The method according to claim 1, wherein the mixed molecule amplification spike contains 20 to 90% of the natural isotope of the target substance.

4. The method according to claim 1, wherein the mass spectrometer is a quadrupole mass spectrometer, triple quadrupole mass spectrometer, time-of-flight mass spectrometer, trap mass spectrometer, orbitrap mass spectrometer, sector mass spectrometer, or any mass spectrometer using any ionization source or any ionization such as electrospray, electron ionization, thermal ionization, matrix-assisted laser desorption ionization, laser ablation, or inductively coupled plasma.

5. The sample is dried blood, The mass spectrometer is an ESI-QQQ-MS mass spectrometer. The amount of the mixed molecular amplification spike added to the sample is between 70 and 100 picograms. The method according to claim 3, wherein the certain amount of sample in step (d) is 10 microliters.

6. The method according to claim 1, wherein the sample is selected from the group consisting of whole blood, serum, urine, cerebrospinal fluid, or body fluid.

7. The method according to claim 1, wherein the sample is a blood sample collected on an inert solid matrix pretreated with aliquots of the mixed molecular amplification spike and dried in situ, or an inert solid matrix to which a pre-mixed sample and metaspikes are added and dried in situ.

8. The method according to claim 1, wherein the sample, after equilibration with the mixed molecular amplification spike, is in any form: dry, liquid, or gas.

9. The method according to claim 1, further comprising the step of quantifying the amount of the target substance at a mass spectrometer below LLOQ using metaspikes.

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