Method and composition for absolute quantification of analytes using stable isotope-labeled standards in mass spectrometry

Abstract

This invention introduces a method for the absolute quantification of analytes in samples using mass spectrometry Two or more stable isotope-labeled (SIL) standards of the analyte known and different concentrations are added to a sample. The analyte and SIL standard signals are measured simultaneously in a single injection. The calibration curve generated from these signals enables precise analyte quantification without the need for multiple injections or complex external calibration curves. The SIL standards can be loaded onto a solid phase extraction column. A known volume of sample can be processed on the column by capture, wash and elute. In some embodiments of the invention two or more SIL standards are placed or eluted directly into the sample. This approach is used for both direct and ambient mass spectrometry techniques and for LC-MS methods.

Description

METHOD AND COMPOSITION FOR ABSOLUTE QUANTIFICATION OF ANALYTES USING STABLE ISOTOPE-LABELED STANDARDS IN MASS SPECTROMETRYFIELD OF THE INVENTION

[0001] The present invention relates to methods, devices and kits for performing mass spectrometry-based quantification of an analyte in a sample using stable isotope-labeled (SIL) standards and single analysis by mass spectrometry measurement.BACKGROUND OF THE INVENTION

[0002] Mass spectrometry (MS) is extensively utilized in both scientific research and industrial applications due to its ability to accurately, specifically, sensitively, and reproducibly detect and quantify a wide array of analytes.

[0003] The use of MS for quantification purposes has surged, with over 50 million experiments conducted annually in the US alone. The advantages offered by ambient MS technology include tabletop affordable instruments and portable ease of use. Innovations like Bruker’s stripe technology, Waters’ glass rod, or Advion’s open port technology provide excellent opportunities for direct semi-quantitative analyses.

[0004] However, quantification accuracy can vary significantly depending on the instrument model, compound type, sample type, and run-to-run consistency. MS quantification is highly susceptible to signal magnitude errors due to matrix effects such as ion suppression. To compensate, normalization techniques using analyte-similar internal standards are used.

[0005] External calibrations require the creation of a highly accurate and reproducible analyte matrix that matches the sample matrix. In many cases, this cannot be achieved. In addition, an external calibration technique requires multiple introductions of identical volumes of sample into the instrument which is difficult to achieve.

[0006] In another method, internal standards are spiked into each sample at known concentrations. This process corrects for sample response variations and sample preparation errors. The ratio of an internal standard to an analyte within a sample can be used to calculate an analyte’s concentration. However, this calculation becomes progressively more inaccurate as the difference between the internal standard concentration and the analyte concentration increases. Samples at different concentrations relative to the internal standards have a different mass spectrometer response factor. If chromatography is used, there may be(different) matrix effects from slightly different standard and sample retention times.Therefore, almost all assays are based on using an internal standard and the sample amount or concentration is corrected using an external multipoint calibration curve. The concentration of the samples spiked with an internal standard can be calculated by direct comparison with the external calibration curve of standards. There may be scatter in the calibration curve because of (different) matrix effects for each of the calibration points, but these are corrected for by drawing a straight line or a best fit line for the calibration curve. The addition of multiple internal standards requires a process of multiple introductions of identical volume sample into a mass spectrometry instrument.

[0007] Mass spectrometry (MS) is essential for quantitative analysis in diverse scientific and industrial fields. Existing methods are hampered by challenges like matrix effects, variability in sample preparation, and the complexity of external multipoint calibration. These factors introduce errors and inefficiencies in analyte quantification.

[0008] There exists a need for simple, reliable and absolute mass spectrometry quantification. There exists a need to improve the accuracy and reliability of mass spectrometry -based quantification in the various scientific and industrial applications. There exists a need for mass spectrometry quantification performed without multiple introductions of the sample while still using internal standards.SUMMARY OF THE INVENTION

[0009] This invention addresses these challenges through a novel methodology using SIL standards. It minimizes procedural variability and enhances accuracy by eliminating the need for external calibration and / or repeated sample injections.

[0010] This invention is a method for the absolute quantification of defined analytes of interest in samples using mass spectrometry measurements. In the invention, two or more different stable isotope labelled (SIL) standards are added to the sample with each SIL standard at known and different concentrations. Each of the SIL standards added to the analyte sample are comprised of an isotope that is distinct from the other SIL standard or other SIL standards. In some embodiments of the invention, one or more of the SIL standards added are isotopes of the analyte desired to be quantified. In some embodiments of the invention, one or more of the SIL standards added are closely related to the analyte desired to be quantified. Closely related means that SIL standard or some fragments of the SIL standard are chemically identical or close in mass to charge ratio and chemical characteristics to the desired analyte.

[0011] The mass to charge ratio of each stable isotope labelled standard, or mass fragment, or adduct ion, and the sample analyte are measured by a mass spectrometer together in one single injection and a single mass spectrometer analysis.

[0012] The two or more SIL standards can be present in the sample in known and different concentrations in the spiked sample. Analysis can be performed using a single mass spectrometry sample introduction. All standards and analytes can be introduced into the instrument simultaneously. This SIL standard can be introduced into the instrument in the same aliquot as the sample.

[0013] A calibration curve is generated from the data collected and the sample analyte concentration is determined. This calibration curve can be constructed or calculated from one injection or sample introduction into the mass spectrometer. The sample analyte concentration or mass amount can be calculated with a single measurement. In some embodiments of the invention multiple measurements of the sample can be taken and an average calculated.

[0014] In some embodiments of the invention, at least one of the SIL standards of the invention is present in the spiked sample at a concentration lower than the analyte concentration. In some embodiments of the invention, at least one of the SIL standards of the invention is present at a concentration higher than the analyte concentration. In some embodiments of the invention, the concentration of the SIL standards bracket the unknown analyte concentration with higher and lower concentrations of the SIL standards. In some embodiments of the invention, a portion of the volume of SIL standards and sample solution are analyzed by mass spectrometer in a single measurement. In some embodiments of the invention, the entire volume SIL standards and sample are analyzed by mass spectrometer in a single measurement.

[0015] In this invention, a calibration curve is calculated using two or more SIL standards having different isotopes and different masses. They can be injected or introduced into the mass spectrometer at the same time as the sample analyte. The concentration of the sample analyte is calculated based on this calibration curve. The two or more SIL standard compounds or fragment signals or adduct ions form a calibration curve, and the signal of the analyte compound of interest is used to quantify the analyte in a single analysis.

[0016] In some embodiments, the two SIL method of the invention is able to quantify more than one analyte in a single analysis. All that is needed is two different SIL standards at different concentrations for each specific analyte type to be quantified.

[0017] The SIL standards used in this invention possess chemical and physical properties closely matching the analyte. In many embodiments, the SIL standards are isotopically labeled forms of the analyte (e.g.,13C or15N-labeled molecules). These standards behave identically to the analyte during ionization and detection, ensuring accurate quantification. In some embodiments of the invention, the SIL standards are closely related to the target analyte or analytes.

[0018] Each SIL standard is distinguishable by its unique mass-to-charge ratio (m / z). This property enables the construction of a calibration curve directly from a single sample injection, eliminating volume-dependent errors.

[0019] In some embodiments of the invention, SIL standard mass spectrometry parent ion peaks are used to generate the calibration curve. In some embodiments of the invention, SIL mass spectrometry fragment peaks are used to generate the calibration curve. In some embodiments of the invention, the parent ion peaks are used to generate the calibration curve and quantify the analyte compound. In some embodiments of SIL standard mass spectrometry of the invention, the first, second or third isotope peaks are used to generate the calibration curve and quantify the analyte compound. In some embodiments of the invention, adduct ion signals are used to generate the calibration curve and quantify the analyte compound.

[0020] The invention provides a method for absolute quantification in mass spectrometry. The process involves:1. Spiking the sample with two or more SIL standards at known mass amounts and / or known concentrations.2. Performing a single injection into a mass spectrometer to simultaneously measure the analyte and SIL standard signals. Either area or peak height measurements are suitable. Optionally, separation with chromatography prior to introduction into the mass spectrometer.3. Generating a calibration curve from the SIL standard signals. Using the signal from the analyte to determine the analyte concentration.

[0021] This approach is particularly suited for direct or ambient MS techniques, offering simplicity, reliability, and expanded dynamic range for calibration.DEFINITIONS

[0022] SIL standard: A stable isotope labelled (SIL) standard having different isotopes in one or more atoms of the molecule when compared to the analyte. For example, deuterium may be substituted in one or more positions of the molecule increasing the molecular weight of the target molecule. Deuterium is heavier than protium (ordinary hydrogen). These mass differences are detected by mass spectrometer provided the identical molecule or molecule fragment is compared. The most common isotope of carbon is carbon-12 (12C), which contains six neutrons in addition to its six protons. The next heaviest carbon isotope, carbon-13 (13C), has seven neutrons and can substitute for carbon-12 in a SIL standard. Nitrogen, phosphorus and other elements have isotopes that can be synthesized into a SIL standard compound. In the invention, two or more different SIL standard compounds are used each having a different number of elemental isotopes or a combination of different types of elemental isotopes or both.

[0023] SIL standard mass spectrometry parent peak: Peak on the mass spectrum that represents the unfragmented molecule, also known as the molecular ion (M+), which is used to determine the molecular weight of a compound. Essentially, it's the highest mass peak of significance in the spectrum that corresponds to the intact molecule before fragmentation occurs.

[0024] In addition to the primary molecular ion (parent peak), adduct ions of both the standards and the analyte may also be used for calibration and quantification. The species formed from mass spectrometry ionization are strongly dependent on the ionization environment and solvent composition. Table 1 shows example adducts for ESI ionization.Table 1

[0025] In addition to the parent peak or fragments of the molecule, lower abundance naturally occurring isotope peaks might be used for generation of the calibration graph and for quantification.

[0026] SIL standard mass spectrometry fragment peak: Peak on the mass spectrum that represents a fragmented molecule. In this invention, calibration curves drawn from SIL standard mass spectrometry signals from a parent peak or a fragment peak are equivalent provided the ability to prepare an accurate calibration curve is validated. However, fragment peak magnitudes always depend on ionization efficacy, ion strength, etc. and all matrix effects. So, signals are similar but not identical, whereas the isotope peaks are identical or close to being identical.

[0027] In some embodiments of the invention, a non-isotope mass spectrometry standard may be used as the internal standard. However, the non-isotope internal standard must have some essential elements and characteristics. The mass to charge ratio of the internal standards to the target analytes must be within the 90% - 110% range. In addition, the nonisotope internal standard, as used in this invention, contains at least 90% of the structural components of the target analyte. For the purposes of this invention, non-isotope internal standards are defined as SIL internal standards provided the standards perform as defined below.

[0028] The non-isotope mass spectrometry internal standard should have a MS ionization efficiency similar to the target analyte under the same MS conditions. This ensures that the internal standard and analyte respond proportionally to variations in ionization, improving quantification accuracy.

[0029] The internal standard must be chemically stable under the sample preparation, storage, and analytical conditions to avoid degradation or reactivity that could affect quantification.

[0030] Finally, the signal of the standard must be proportional to the signal / concentration of the target analyte.

[0031] SIL internal standards work well as used in this invention because their mass spectrometry ionization and mass detection profiles are very similar or identical to those of the sample analyte. However, a non-isotope internal standard can also perform well as an internal standard of this invention provided the structure and mass to charge ratio correlate with the target analyte. The correlation can be confirmed by preparing a solution containing identical or known concentrations of the internal standard and target analyte and then performing a mass spectrometry analysis. The correlation is confirmed if the known and measured concentration of the analyte are the same. In this way, the concentration of the target analyte in a sample can be accurately determined.

[0032] SPE columns, cartridges or media: For this invention, the terms columns, cartridges, pipette tip columns, discs and media are considered equivalent.

[0033] Mixing sample analyte and SIL standards: For this invention, adding SIL standards to the sample or analyte by spiking, adding sample to dried SIL standards, preloading SIL standards onto SPE columns, cartridges or media and eluting, or mixing in solution prior to sample introduction and measurement are considered equivalent.

[0034] Spiking refers to the act of adding a known amount of SIL standard to a sample.

[0035] Dried SIL standards: For this invention, dried SIL standards in a vial, container, on filter paper or on an SPE solid or stationary phase are considered equivalent.

[0036] SIL standard concentrations of the invention: Since the SIL standards used in the invention have a different molecular weight compared to the analyte, the concentration of each SIL standard may be measured in moles per unit volume. In some embodiments of the invention, the mass amount per unit volume may be used if corrected for molecular weight. In some embodiments of the invention, the differences of the molecular weights of the SIL standards and analyte are determined to be insignificant and mass per unit volume concentrations are used.

[0037] Analyte concentrations of the invention: Concentration of the analyte may be calculated as moles per unit volume or mass per unit volume, depending on the reporting requirements.

[0038] Calibration curve of the invention: Calibration curve constructed or calculated fromthe detector response of a single injection of one or more SIL standards.

[0039] Single shot mass spectrometry analysis. An analysis by mass spectrometry analysis of sample and SIL standards in a single analytical measurement run.

[0040] Analytes: Analytes are the target molecule in a sample of which the concentration or amount is measured. Analytes can be any type of molecule or compound including, but not limited to molecules in pharmaceuticals, food safety compounds, clinical biomarkers, or environmental contaminants.

[0041] Solid phase extraction columns: Solid phase extraction columns include columns or systems from the group comprised of packed columns, monolithic columns, discs and / or automated online systems. Media in 96-well plates and similar or any multiplexed media may be used for this invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Embodiments of the invention are described in further detail below in conjunction with the accompanying drawings, in which:

[0043] Fig. l is a schematic illustration of an SPE column connected in series to a preloaded SPE standard device for simultaneous elution of sample analytes and standards;

[0044] Fig. 2 is a flowchart shows steps in a method for the quantification of an analyte in a sample using mass spectrometry that is an embodiment of the invention;

[0045] Fig. 3 is an elution of preloaded SIL standards and prepared sample with analysis by LC / MS. Selected LC / MS ion monitoring trace 0.1 ppm diazepam-d3 and 0.3 ppm diazepam-d8 SIL standards and 0.3 ppm diazepam prepared analyte;

[0046] Fig. 4 is a two-point calibration curve from the elution of preloaded SIL standards and sample and analysis by LC / MS chromatogram shown in Fig. 1. Selected LC / MS Ion Monitoring Trace 0.1 ppm diazepam-d3 and 0.3 ppm diazepam-d8 SIL standards and prepared 0.3 ppm diazepam analyte; and

[0047] Fig. 5 is a two-point calibration curve, from the elution of preloaded SIL standards and direct MS analysis with no HPLC. Selected Ion Monitoring Trace 0.1 ppm diazepam-d3 and 0.3 ppm diazepam-d8 SIL standards and prepared 0.3 ppm diazepam analyte.DETAILED DESCRIPTION OF THE INVENTIONStable Isotope-Labeled (SIL) Standards

[0048] The SIL standards used in this invention possess chemical and physical properties closely matching the analyte. In most embodiments, the SIL standards are isotopically labeled forms of the analyte (e.g.,13C or15N-labeled molecules). These standards behave identically to the analyte during ionization and detection, ensuring accurate quantification. The mass of the analyze and fragments used for the analysis behave almost identically to the analyte during ionization and detection. Any SIL standard can be used as long as the standard meets this characterization.

[0049] Each SIL standard is distinguishable by its unique mass-to-charge ratio (m / z). This property enables the construction of a calibration curve directly from a single sample injection, eliminating volume-dependent errors.

[0050] In the analysis, the mass spectrometer signal is traceable to the SIL standard and its corresponding concentration in the spiked sample. The concentration is known for each SIL standard and is distinguished by their mass-to-charge ratio (m / z). Therefore, the calibration curve can be calculated, and the analyte concentration is determined.

[0051] As the calibration standards are measured together with the compound of interest, the injected sample amount / volume does not affect the result of quantification, thus making the technology ideal for direct MS technology. This single analysis method is similar to ambient MS or open port systems which up until now, can only be used for qualitative or semi-quantitative analysis in complex procedures.Instrumentation and Applicability

[0052] Any mass spectrometer capable of producing a concentration-dependent analyte signal is compatible with this method. The signal does not require a specific slope or zero intercept for calibration, allowing flexibility across different MS systems and sample introduction methods.

[0053] Direct MS techniques, including ambient MS and open-port systems, benefit significantly from this approach. Previously limited to qualitative or semi-quantitative analyses, these systems can now achieve absolute quantification without complex sample preparation.

[0054] Any type or model of mass spectrometer employing any type of sample introduction may be used for the method of the invention provided the mass spectrometer is able to acquire a concentration-dependent signal from the analyte of interest. A concentrationdependent analyte signal means the magnitude of the mass of the analyte signal is dependent on the concentration of the unknown analyte in the sample. However, the concentration -signal dependency does not have to have a graphical slope of 1 nor does the intercept of a calibration curve have to go through 0.

[0055] The invention is a methodology which uses two or more stable isotope labelled analytical standards. In some embodiments of the invention, the SIL standards can be provided as solution or in dry form. In some embodiments of the invention, the SIL standards can be loaded onto a trapping media including a stationary phase, a solid phase extraction (SPE) column, solid phase extraction pipette tip column or any solid phase extraction column or disc. For high throughput, multi-well solid phase plates can be used. The use of solid phase trapping media is useful for sample enrichment or clean-up.

[0056] In some embodiments of the invention, the SIL standards are provided as a solid in a vial or container or loaded onto media, filter paper, solid or solid phase extraction columns. In some embodiments of the invention, the SIL standards are provided as a liquid spiked into the sample during any stage of the analysis. SIL standards may be added to the sample as a solid, deposited on a solid or as a liquid.

[0057] In one embodiment of the invention, a specified volume of sample is processed on a solid phase extraction column that has the SIL standards pre-loaded. The method steps are capture, wash, elute and analyze. In some embodiments of the invention, the SIL standards are placed directly into the sample.

[0058] A further benefit of the methodology described is the opportunity to increase the linearity of calibration range. Using the first, second and third isotope peak of a specific measured compound detected by MS-technology based on the naturally occurring isotopes, the linear range of the calibration can be increased by several orders of magnitude. In some embodiments of the invention, the SIL standard compounds used may be based on naturally occurring isotopes of the sample analyte albeit at different and known concentrations.

[0059] In a hypothetical analyte, a cluster of 10 carbon atoms analyzed by mass spectrometry results in a mass spectrum. C-13 isotopes have a natural probability of 0.56%. Deuterium is a heavy isotope of hydrogen with an abundance of 0.015%.

[0060] Using high-concentrated SIL standards, the linear range can be extended by a factor of 100 or more.

[0061] The method of the invention can be used for determining whether a controlled analyte is within the allowed specification in a product. In this case, no external calibration curve is needed.

[0062] Fig. 2 is a flowchart that illustrates the steps of a method 200 according to an embodiment of the invention. The method comprises: a first step 202 of providing a samplethat contains an analyte; a second step 204 of providing two or more SIL standards at known concentrations, wherein the SIL standards are isotope labeled forms of the the analyte, wherein each SIL standard has a different mass to charge ratio; a thrid step 206 of mixing the sample with the SIL standards to create a mixture; a fourth step 208 of introducing the mixture into a mass spectrometer; a fifth step 210 of measuring the signals of the SIL standards to generate a calibration curve; and a sixth step 212 of determining the analyte concentration using the calibration curve. Further detail of these steps is discussed below.

[0063] In one embodiment of the invention, the steps of the invention are:

[0064] Step 1 : Preparation of the Sample

[0065] The sample is provided in its natural matrix or diluted by a known factor.

[0066] Two or more SIL standards are added to the sample to create a mixture. In some embodiments, the SIL standards can be added at predetermined concentrations, typically encompassing a range above and below the expected analyte concentration.

[0067] Step 2: Introduction of SIL standards

[0068] The SIL standards can be added in liquid form or preloaded onto a solid-phase extraction (SPE) column for automated addition.

[0069] When using SPE, the sample is passed through the column, capturing the analyte and co-eluting it with the SIL standards. In other embodiments, the SIL standards can be eluted from an SPE column, cartridge or media and mixed with the analyte. In these embodiments, the analyte is not subjected to solid phase extraction.

[0070] The SIL standards can be provided in dry form and mixed with the sample analyte.

[0071] Step 3 : Mass Spectrometry Analysis

[0072] The mixture of the sample and the SIL standards can be injected or introduced into the mass spectrometer.

[0073] Signals corresponding to the analyte and SIL standards are simultaneously measured.

[0074] A calibration curve is constructed from the SIL standard signals, and the analyte concentration is determined.

[0075] In some embodiments of the invention, the analyte and SIL standard signals are measured simultaneously by direct introduction (without chromatography sample preparation) into a mass spectrometer.

[0076] In some embodiments of the invention, chromatography is performed on the sample and standards prior to introduction into a mass spectrometer. The chromatography effluent is directed to the mass spectrometer used in the invention.

[0077] Direct injection or ambient mass spectrometry techniques may be used with the invention or any mass spectrometer. See the list below.

[0078] Mass spectrometry sample introduction systems that may be used in this invention include:■ Electrospray Ionization (ESI)+■ Matrix- Assisted Laser Desorption / Ionization (MALDI)■ Chemical Ionization (CI)■ Inductively Coupled Plasma Ionization (ICP)■ Secondary Ion Mass Spectrometry (SIMS)■ Ambient SIMS■ Desorption / Ionization on Solids (DESI)■ Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)

[0079] Table 2 is a list of mass spectrometer types that will accurately analyze analytes in a sample using the multi point SIL calibration method of the invention. Any mass spectrometer system can be used.Table 2&

[0080] In some embodiments of the invention, two or more SIL standards are added to the sample by using a preloaded standard column. Two or more SIL standards previously loaded onto a solid phase extraction (SPE) column can be provided. A known volume of sample is processed using the steps of capture, wash and elute.

[0081] In some embodiments of the invention, two or more SIL standards of the isotope of the analyte are added through a second solid phase extraction column, cartridge or media. A first SPE column, cartridge or media processes the sample as normal through the conditioning, capture and wash steps. A second SPE column, cartridge or media can be placed on the end and then the elution step is performed with both columns connected in series. The sample and SIL standards are then analyzed. Fig. 1 shows the schematic illustration of a SPE column connected in series to a pre-loaded SPE standard device for simultaneously elution of sample and standards. Sample prep SPE column 10 is processed and loaded with sample on SPE bed 20. After loading and washing the sample, and before final elution of the sample analyte, second SPE cartridge 40 that is pre-loaded with SIL standard(s) on SPE bed 50 is attached to first SPE column 10 by a slip luer connection 30.The elution solvent is passed through SPE cartridge bed 20 and SPE cartridge 50 eluting the sample analyte and SIL standards simultaneously. The solution is ready for mass spectrometry analysis.

[0082] In some embodiments the solution containing both analyte and standards solution is dried and reconstituted in a defined volume for further enrichment before introduction into the mass spectrometer.

[0083] In some embodiments in the invention, only one SPE device is used. A single SPE column, cartridge, disc or media can be used to contain and store preloaded SIL standards. In some embodiments of the invention, the sample analyte is in liquid form and acts as an eluent. The sample containing the analyte is passed through the SPE column. This approach for combining the analyte with the SIL standards can also be used with a cartridge or media format. Following elution, the solution is ready for mass spectrometry analysis.

[0084] In some embodiments of the invention, two or more SIL standards which are isotopes of the analyte are added as a spike of liquid into the sample and its matrix. This operation is typically performed at the beginning of sample preparation to compensate for any potential losses or variations during analysis. The concentration or mass amounts of the SIL standards are known. The solution is ready for mass spectrometry analysis.

[0085] The treated sample containing SIL standards at known concentrations is introduced into the mass spectrometer instrument. In some embodiments of the invention, the sample is separated with chromatography prior to introduction into the mass spectrometer. The data are analyzed, and the analyte concentration is calculated.Use of SPE

[0086] Solid phase extraction (SPE) is a technique designed for rapid, selective sample preparation and purification prior to analysis. A liquid sample solvent passes through the SPE adsorbent media and the media retains the sample. Then the appropriate strength solvent flushes away impurities. Finally, a solvent is used to elute and collect the regulated analyte or analytical substance. SPE is widely applied to the analysis of various foods, environmental and biological samples. In one embodiment of the invention, the SIL standard is preloaded onto the SPE media before the sample preparation process.

[0087] Non-limiting examples of solid phase extraction sorbents or media include polymer (polar, aromatic and nonpolar), Cl 8, HLB, WAX, SDVB, C8, SCX, etc. SPE sorbent media can be broken into four general categories, according to their retention mechanism: 1) polar, 2) nonpolar, 3) ion exchange and 4) mixed mode.

[0088] The adsorbents or stationary phases used in normal phase SPE are polar and include media such as silica gel, alumina, silica-silver, Florisil ( MgO ASiCh), etc. to extract polar substances. Non-polar or weakly polar adsorbents include C8, Cl 8, phenyl column, polystyrene, etc. The target compounds extracted using these media are somewhat polar to non-polar. An example of nonpolar media is reversed phase C18. Ion exchange SPE media can be used extract charged target molecules. Examples of ion exchange media include silica-NH2 (aliphatic primary amine bonded silica gel) or silica-SCX (aliphatic sulfonic acid bonded silica gel), etc.

[0089] An example of a mixed mode SPE media is hydrophilic interaction chromatography (EHLIC), a variant of normal phase liquid chromatography that uses a hydrophilic stationary phase and a reversed-phase type eluent (high organic content with a small amount of water) to separate polar and hydrophilic compounds. Any type of media can be mixed with another type to produce a mixed mode media. An example of a commercial mixed phase SPE material is Oasis manufactured and distributed by Waters Corp. Ion exchange or reversed phase stationary phases are built into a hydrophilic polymer substrate.

[0090] Typical applications of the invention with SIL standards preloaded onto SPE media include but are not limited to biological fluid analysis, analysis and monitoring of drinking water, groundwater, and sewage, volatile oils, plant tissues, fruits, vegetables, and grains, liquid drug samples, soils, and sediments, meat, fish, and other animal tissues, tablets and other solid drugs.

[0091] Other applications include the analysis of PFAS, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) for wet environments, pesticide and herbicide residues in fruits, vegetables and food, antibiotic analysis, and clinical drug analysis.

[0092] Prewash, activation or conditioning prior to loading a sample on SIL pre-loaded SPE media.

[0093] In a preloaded SIL standard SPE column or disc, the media can be washed and / or conditioned with an organic solvent. Then the SPE is preloaded with one or more SIL standards, dried and stored. To use the SPE column or disc, the sample is passed through the media.

[0094] In some embodiments of the invention, the sample can be applied directly to SPE media to capture the analyte compounds present in the sample. In some embodiments of the invention, the SPE media is conditioned with solvent to ensure thorough wetting of the media prior to applying the sample. For hydrophobic phases, this SPE conditioning can be carried out with a water-miscible organic solvent such as methanol, acetonitrile, or tetrahydrofuran. To activate SPE cartridges, it is possible to use a 1-50% organic solvent solution, which helps to wet the sorbent. Before using an SPE cartridge, column or disc containing the preloaded SIL standards, conditioning or activating the sorbent material or media can ensure the sorbent is properly hydrated or solvated and ready for sample interaction. The organic conditioning solvent concentration may be in the range of 2-40%, 3-35%, 4-30%, 5-25%, or 10-20%. However, the conditioning process does not remove the preloaded SIL standard(s).

[0095] Any volume of conditioning solvent can be added to the SPE medium prior to the performance of SPE. In some embodiments of the invention, the conditioning solvent can be added in 1 bed volume of liquid. In some embodiments, the conditioning solvent can be added as 1-2 bed volumes of liquid. In some embodiments, the bed volume of media can be approximately 50% of the media (chamber) volume. In some embodiments of the invention, the volume of the conditioning solvent added can be 1-3, 1-4, or 1-5 bed volumes of liquid. In some embodiments, the volume of conditioning solvent added can be 1-10, 2-10, 3-20, 4, or 5 bed volumes of liquid.

[0096] For reversed-phase SPE cartridges, methanol can be for conditioning or activating the sorbent. While a 5% or 10% methanol solution is a common starting point, the specific concentration and volume of methanol used for activation can depend on the type of sorbent material and the specific application. A mixed phase stationary phase may also benefit from a low concentration conditioning solvent. For example, a WAX Oasis type phase which is used for PFAS analysis may be preconditioned with an organic solvent without eluting the SIL standards.

[0097] Depending on the type of SPE cartridge and the nature of the analytes, other solvents like acetonitrile or water-miscible organic solvents can also be used for conditioning.However, it is important to preserve the presence of the preloaded SIL standard that is sorbed to the cartridge, column, disc or media. Proper conditioning ensures that the sorbent is fully hydrated or solvated, and ready to interact with the analytes in the sample and sorb the analytes to the media but does not interfere with the presence of the SIL standard(s).

[0098] In some embodiments of the invention, a kit may be provided to perform the analysis of the invention. A kit may be comprised of SIL standards at known amounts. The standards may be in liquid form or dried form. Dried form standards may be provided on an SPE column, cartridge, disc or media. The kit may also be comprised of vials, glassware, plasticware, to contain and manipulate the sample, standards and analyte prior to analysis. The packaging may include means to protect the SIL standards, for example to prevent contamination or degradation.EXAMPLESExample 1

[0099] A two-point SIL standard concentration calibration and measurement of diazepam in a Gewacalm 2mg tablet via a single HPLC (Thermo Scientific Ultimate 3000) TOF-MS (Bruker Maxis Impact MS) analysis.

[0100] Preloaded Column Standard Preparation. A mixture containing 0.1 ppm diazepam-d3 and 0.3 ppm diazepam-d8 was prepared and loaded onto an SPE column, followed by drying.

[0101] Diazepam Sample Preparation. One tablet of a prescription drug containing 2 mg diazepam was dissolved in 2 mL of methanol. The solution was vortexed, sonicated, and centrifuged for 30 seconds at 20,000 ref. The supernatant was diluted to a concentration of 0.3 ppm in 50% methanol (v / v). This prepared sample was passed through the preloaded SPE cartridge.

[0102] Elution and LC / MS Analysis. The compounds captured on the SPE cartridge (0.1 ppm diazepam-d3, 0.3 ppm diazepam-d8, and 0.3 ppm diazepam) were eluted and introduced into the LC / MS system.

[0103] Data Analysis. Signals from a single LC / MS measurement were recorded. The MS signal intensities of diazepam-d3 and diazepam-d8 were used to construct a two-point calibration graph. The signal intensity of diazepam was then applied to calculate the sample concentration.

[0104] In this example, absolute quantification of diazepam was performed by an uHPLC-hr-qTOF / MS. In the first step, a preloaded solid phase extraction column is prepared withdifferent known amounts of stable isotope labelled (SIL) standards of the compound of interest. The SIL standards are eluted with the sample analyte.

[0105] The sample and SIL standards (0.1 ppm diazepam-d3, 0.3 ppm diazepam-d8 and 0.3 ppm diazepam) that were loaded onto the SPE cartridge were eluted from the column and analyzed by LC / MS. The MS-signal intensities of diazepam-d3 and diazepam-d8 were used to generate a two-point calibration graph, and the signal intensity of diazepam was used to calculate its concentration.

[0106] For the analytical investigation, a Thermo Scientific Dionex UltiMate 3000 HPLC System, in conjunction with a Bruker-maxis Impact Ultra High-Resolution TOF was employed. The chromatographic separation was carried out using an Agilent RRHD Zorbax EclipsePlus C18 column (2.1 x 50 mm, 1.8 pm). The mobile phase consisted of 0.1% formic acid (FA) in milli-Q water (A) and acetonitrile (B). Operational parameters were set as follows: a flow rate of 0.4 ml / min, injection volume of 1 pl, and a column oven temperature maintained at 40 °C. The employed gradient profile was as follows: 0 min, 15% B; 3 min, 40% B; 10 min, 70% B; 10.5 min, 100% B; 13 min, 100% B; 13.5 min, 15% B; 15.0 min, 15% B.

[0107] The LC eluate was directly introduced into the mass spectrometer, operating in Full-Scan-MS mode with mass scanning from 50-1500 m / z and a spectra rate of 4 Hz.Electrospray ionization was utilized in positive ionization mode, negative ionization mode, and MS / MS modes. Additional optimized parameters included a nitrogen nebulizer gas pressure of 3 bar, a drying gas flow rate of 12 l / min, endplate offset at 500 V, and capillary voltage at 5000 V / 5500 V for positive / negative modes. The drying temperature was set to 230 °C, with funnel 1 RF and funnel 2 RF at 350 Vpp and 450 Vpp, respectively. Collision-induced dissociation (CID) energy was maintained at 0 eV, hexapole RF at 100 Vpp, and quadrupole ion energy at 7 eV. Low mass filtering occurred at 50 m / z.

[0108] Collision cell parameters were configured as follows: collision energy at 10 eV / 5 eV for positive / negative modes, collision RF at 650 Vpp, transfer time at 80 ps, and pre-pulse storage at 7 ps. Detection at 280 nm was performed using MWD. For Auto MS / MS, the parameters included a number of precursors set at 3, a threshold at 435 cts, and released after 0.20 min.

[0109] Fig. 3 shows the extracted ion chromatograms of diazepam analyte 70 and SIL standards diazepam-d3, 0.1 ppm 60, and diazepam-d8, 0.3 ppm 80. Minor retention time shifts were observed due to isotopic substitution effects of the extracted ion chromatogram of the two SIL-diazepam standards and the diazepam sample. The minor differences in theretention time were a result of minor changes due to substitution of hydrogen atoms with deuterium, called a kinetic isotope effect.

[0110] The elution times shown in the figure are slightly different for the SIL standards and analyte. SIL standards comprised of different isotopes may exhibit slight variations in retention times in HPLC or GC. Isotopic substitutions can subtly alter physicochemical properties such as polarity or bond strengths. These changes may affect interactions with the stationary and mobile phases. Although the chromatography retention times may differ only slightly, the SIL standard substances and the analytes are subject to different matrix effects, which can lead to errors in quantification. In some embodiments of the invention, the effect may be determined to be minor through pretesting of standards.[OHl] It is beneficial to analyze without chromatography so that both analyte and SIL standards are subjected to identical matrix ion suppression effects.

[0112] The calibration curve is shown in Fig. 4. The calibration curve magnitudes are by signal area of extracted ion chromatograms. The signal intensities of the two signals diazepam-d3 and diazepam-d8 were used to perform a two-point calibration. In this example, the parent ion signals were not used. The second isotope peak was used for all signals (analyte and each standard). Based on the given calibration curve, the concentration of the diazepam sample was calculated to be 0.29 ppm 90. The calculated diazepam concentration matched the known sample concentration within acceptable error limits, validating the method.Example 2

[0113] Absolute Quantification of Diazepam by Direct Compact MS Technology (Advion MS) without chromatographic separation.

[0114] The method of Example 1 was performed exactly as described except without chromatographic separation. Instead, direct ambient mass spectrometry analysis was used.

[0115] Preloaded Column Standard Preparation. A mixture containing 0.1 ppm diazepam-d3 and 0.3 ppm diazepam-d8 was prepared and loaded onto an SPE column, followed by drying.

[0116] Diazepam Sample Preparation. One tablet of a prescription drug containing 2 mg diazepam was dissolved in 2 mL of methanol. The solution was vortexed, sonicated, and centrifuged for 30 seconds at 20,000 ref. The supernatant was diluted to a concentration of 0.3 ppm in 50% methanol (v / v). This prepared sample was passed through the preloaded SPE cartridge.

[0117] Sample recovery and ambient MS Analysis. The compounds captured on the SPE cartridge (0.1 ppm diazepam-d3, 0.3 ppm diazepam-d8, and 0.3 ppm diazepam) were eluted and introduced directly into an ambient MS system for analysis without chromatographic separation. Signals from a single MS measurement were recorded. The MS signal intensities of diazepam-d3 and diazepam-d8 were used to construct a two-point calibration graph. As shown in Fig. 5, the signal intensities of the two SIL standards, diazepam-d3 and diazepam-d8, were used to perform a two-point calibration. Based on this calibration curve, the diazepam concentration in the sample was calculated to be 0.28 ppm 100, matching the expected value within the acceptable limits of accuracy.Example 3

[0118] The method used in Examples 1 and 2 was performed exactly as described except without chromatographic separation and without any preloaded standards on the solid phase extraction column. The SIL standards were combined with the sample in liquid form and introduced into the mass spectrometer by infusion sample introduction (direct injection). Based on the calibration curve, the concentration of the diazepam sample was calculated to be 0.27 ppm matching the expected value within acceptable limits of accuracy.Example 4

[0119] In an approach to improve the accuracy and efficiency of PFAS analysis in environmental samples, PFAS SIL standards are loaded onto an SPE column prior to sample loading. By preloading the standards directly onto the column, the recovery rates of PFAS compounds improve significantly, ensuring more accurate quantification, especially for complex matrices like wastewater or soil. Analysis is performed by LC / MS. A calibration curve is calculated by two or more SIL standards and the concentration of individual PFAS are calculated.

[0120] This approach optimizes the PFAS analysis process by ensuring accurate quantification and reliable results, especially in complex sample matrices.

[0121] To prepare and perform the invention, SIL standards of the analyte are needed. For example, the following PFAS isotope standards can be acquired from Cambridge Isotope Laboratories, Inc, Tewksbury, MA:■ ES-5640-APFAS SIL Superfund Mixture 1 1.2 mL in methanol.■ Sodium perfluoro-n-butyrate (PFBA) (2,3,4-13C3, 99%)■ Sodium perfluoro-n-pentanoate (PFPeA) (pentanoyl-13C5, 99%)■ Sodium perfluoro-n-hexanoate (PFHxA) (13C6, 99%)■ Sodium perfluoro-n-heptanoate (PFHpA) (13C7, 99%)■ Perfluoro-n-octanoic acid (PFOA) (13C8, 99%)■ Perfluoro-n-nonanoic acid (PFNA) (13C9, 99%)■ Sodium perfluoro-n-decanoate (PFDA) (13C9, 99%)■ Sodium perfluoro-n-undecanoate (PFUA) (13C9, 99%)■ Sodium perfluoro-n-dodecanoate (PFDoA) (13C12, 99%)■ Perfluoro-n-tetradecanoic acid (PFTeDA) (1,2,3,4,5,6-13C6, 99%)■ Sodium perfluoro-n-hexadecanoate (PFHxDA) (1,2,3,4-13C4, 99%)■ Potassium perfluoro- 1 -butanesulfonate (PFBS) (13C4, 99%)■ Potassium perfluoro- 1 -hexanesulfonate (PFHxS) (13C6, 99%)■ Potassium perfluoro- 1 -octanesulfonate (PFOS) (13C8, 99%)■ Sodium lH,lH,2H,2H-perfluoro-l -hexanesulfonate (4:2 FTS) (13C2, 99%; D4, 98%) ■ Sodium 1H, lH,2H,2H-perfluoro-l -octanesulfonate (6:2 FTS) (13C2, 99%; D4, 98%) ■ Sodium 1H, lH,2H,2H-perfluoro-l -decanesulfonate (8:2 FTS) (13C2, 99%; D4, 98%) ■ Sodium lH,lH,2H,2H-perfluoro-l -dodecanesulfonate (10:2 FTS) (13C2, 99%; D4, 98%)■ Perfluorooctanesulfonamide (PFOSA) (13C8, 99%)■ N-Methylperfluorooctanesulfonamide (N-MeFOSA) (D3, 98%)■ N-Methylperfluorooctanesulfonamidoacetic acid (N-MeFOSAA) (D3, 98%)■ N-Ethylperfluorooctanesulfonamidoacetic acid (N-EtFOSAA) (D5, 98%)■ Sodium tetrafluoro-2-(heptafluoropropoxy)propanoate (HFPO-DA) “GenX” (13C3, 99%)

Claims

CLAIMS1. A method for the quantification of an analyte in a sample using mass spectrometry, comprising:(a) providing a sample that contains an analyte;(b) providing two or more SIL standards at known concentrations, wherein the SIL standards are isotope labeled forms of the the analyte, wherein each SIL standard has a different mass to charge ratio;(c) mixing the sample with the SIL standards to create a mixture;(d) introducing the mixture into a mass spectrometer;(e) measuring the signals of the SIL standards to generate a calibration curve; and (f) determining the analyte concentration using the calibration curve.

2. The method of claim 1, wherein step (d) is performed using direct or ambient mass spectrometry.

3. The method of claim 1 or claim 2, wherein step (d) is performed using a single introduction or injection.

4. The method of any one of claims 1 through 3, wherein step (c) is performed by a method selected from the group consisting of spiking, adding the sample to dried SIL standards, and mixing in solution.

5. The method of any one of the preceding claims, wherein following step (a), the analyte is purified from the sample using solid phase extraction, wherein the solid phase extraction is performed using solid phase extraction media, a solid phase extraction column or a solid phase extraction cartridge and wherein the purified analyte is mixed with the SIL standards in step (c).

6. The method of any one of the preceding claims, wherein the SIL standards are preloaded onto solid phase extraction media, wherein step (c) is performed by processing the sample or the purified analyte on the preloaded solid phase extraction media and wherein the analyte and the SIL standards elute together from the solid phase extraction media.

7. The method of claim 6, wherein the preloaded solid phase extraction media is contained in a column, disc or cartridge.

8. The method of any one of the preceding claims, wherein the analyte is selected from a group consisting of pharmaceuticals, food safety compounds, clinical biomarkers, and environmental contaminants.

9. The method of any one of claims 5 through 8, wherein the solid phase extraction is performed using a packed bed column, a cartridge, a monolithic column, a disc, or an automated online system.

10. A kit for performing the method of any one of the preceding claims, wherein the kit is comprised of a container for receiving a sample that contains an analyte and two or more SIL standards.

11. The kit of claim 10, wherein the kit is further comprised of a means to protect the SIL standards from degradation.

12. The kit of claim 10 or claim 11, wherein the SIL standards are liquid, solid or dried on filter paper.

13. The kit of any one of claims 10 through 12, wherein the kit is further comprised of an SPE column.

14. The kit of claim 13, wherein the SIL standards are preloaded on the SPE column.

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