WO2023073588A1 - Utilisation de l'abondance isotopique naturelle de composés pour étendre la plage dynamique d'un instrument - Google Patents

Utilisation de l'abondance isotopique naturelle de composés pour étendre la plage dynamique d'un instrument Download PDF

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Publication number
WO2023073588A1
WO2023073588A1 PCT/IB2022/060307 IB2022060307W WO2023073588A1 WO 2023073588 A1 WO2023073588 A1 WO 2023073588A1 IB 2022060307 W IB2022060307 W IB 2022060307W WO 2023073588 A1 WO2023073588 A1 WO 2023073588A1
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sample
isotopic
ion transition
analyte
abundance
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PCT/IB2022/060307
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English (en)
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Scott Daniels
Subhasish Purkayastha
Yongquan LAI
Aaron STELLA
Michal Weinstock
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Dh Technologies Development Pte. Ltd.
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Publication of WO2023073588A1 publication Critical patent/WO2023073588A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • Analytical samples can often provide critical analytical information.
  • compounds of a related group i.e., drugs and metabolites, hormones in a pathway, biomarkers, and/or peptides from a particular biologic drug
  • these samples often possess analytes of interest, metabolites, and biomarkers in concentration ranges that are orders of magnitude higher than other related compounds in a panel. This makes measuring and quantifying the entire panel difficult, and often requires running two or more different assays for one sample.
  • the inventors have recognized the need to increase the dynamic range of an instrument to measure and quantify samples that possess analytes of interest in varying concentration ranges.
  • utilizing natural isotopic abundance and monitoring single reactions single reaction monitoring or “SRM”) or multiple reactions (multiple reaction monitoring or “MRM”) of a predefined precursor ion and a product ion.
  • SRM single reaction monitoring
  • MRM multiple reaction monitoring
  • One aspect of the disclosure relates a method for quantifying at least one analyte in a sample by mass analysis, the method comprising: ionizing the sample; monitoring, by mass spectrometry, at least one product ion transition for the at least one analyte and at least one isotopic ion transition for the at least one analyte; wherein if the product ion transition meets a condition, the method further comprises determining the intensity and/or abundance of at least one isotopic ion transition ; and quantifying the at least one analyte present in the sample using the intensity and/or abundance of the at least one isotopic ion transition.
  • the condition is ionization saturation, detector saturation, product ions generated near a peak apex, peak shape, a threshold intensity and/or a threshold abundance.
  • product ion transition has an intensity and/or abundance of about 100%.
  • the product ion transition is the most intense and/or abundant isotope of the at least one analyte.
  • the intensity and/or abundance of the isotopic ion transition is less than about 100%, alternatively less than about 50%, alternatively less than about 25%, alternatively less than about 15%, alternatively less than about 10%, alternatively less than about 5% than a precursor and/or the product ion transition.
  • determining the intensity and/or abundance of the isotopic ion comprises obtaining mass data corresponding to the at least one isotopic ion transition.
  • a computer program comprising a non-transitory and tangible computer-readable storage medium is used to determine the intensity and/or abundance of the isotopic ion transition.
  • At least two analytes are quantified, alternatively at least three analytes are quantified, alternatively at least four analytes are quantified, alternatively at least five analytes are quantified, alternatively at least six analytes are quantified, alternatively at least seven analytes are quantified, alternatively at least eight analytes are quantified, alternatively at least nine analytes are quantified, alternatively at least ten analytes are quantified.
  • At least two product ion transitions are monitored, alternatively at least three product ion transitions are monitored, alternatively at least four product ion transitions are monitored, alternatively at least five product ion transitions are monitored, alternatively at least six product ion transitions are monitored, alternatively at least seven product ion transitions are monitored, alternatively at least eight product ion transitions are monitored, alternatively at least nine precursor-product ion transitions are monitored, alternatively at least ten precursor-product ion transitions are monitored.
  • At least two isotopic ion transitions are monitored, alternatively at least three isotopic ion transitions are monitored, alternatively at least four isotopic ion transitions are monitored, alternatively at least five isotopic ion transitions are monitored, alternatively at least six isotopic ion transitions are monitored, alternatively at least seven isotopic ion transitions are monitored, alternatively at least eight isotopic ion transitions are monitored, alternatively at least nine isotopic ion transitions are monitored, alternatively at least ten isotopic ion transitions are monitored.
  • the method further comprises quantifying at least a second analyte in the sample using a product ion transition or isotopic ion transition of the second analyte.
  • quantifying the at least one analyte present in the sample comprises using the intensity and/or abundance of the at least one isotopic ion transition to calculate a ratio of the at least one isotopic ion transition to the corresponding analyte and/or a precursor, and quantifying the at least one analyte in the sample using the calculated ratio.
  • the calculated ratio is used to calculate an isotopic dilution factor (IDF).
  • IDF is used as a multiplier to compensate for abundance differences between the isotopic ion transition and the corresponding analyte and/or the precursor.
  • a computer program comprising a non-transitory and tangible computer-readable storage medium is used to calculate the ratio of the at least one isotopic ion transition to the corresponding analyte and/or the precursor, and/or quantifying the at least one analyte in the sample using the calculated ratio.
  • the mass spectrometry is conducted using a mass spectrometer.
  • the mass spectrometry is conducted using a mass spectrometer and the mass spectrometer comprises a detector.
  • the detector is an ion detector.
  • the ion detector is selected from the group consisting of an electron multiplier, a Faraday cup, a photomultiplier conversion dynode detector, an array detector, and a charge detector.
  • the mass spectrometer is a tandem mass spectrometer.
  • the tandem mass spectrometer is selected from the group consisting of a triple quadrupole, a quadrupole-linear ion trap, a quadrupole TOF, and a TOF-TOF.
  • the sample is a biological sample.
  • the biological sample is selected from the group consisting of urine, blood, oral fluid, plasma, tissue, bone marrow, and tumor samples.
  • the biological sample is dissolved in a solvent, introduced into a solution, or mixed with a matrix material.
  • quantifying the at least one analyte present in the sample is based on a calibration curve generated for at least one calibration standard.
  • the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
  • the sample is in a container.
  • the container is a sample plate comprising a plurality of sample wells.
  • the sample well comprises the sample.
  • the sample is separated, enriched, and/or desalted using a chromatography instrument, microflow, solid phase extraction, liquid-liquid extraction, protein precipitation, a trap-and- elute workflow, phospholipid removal, filtration, organic solvent extraction, dilution, desalting, hydrolysis, magnetic based purification, or isoelectric point precipitation.
  • the chromatography instrument is a high performance liquid chromatography (HPLC) instrument, an ultra high performance liquid chromatography instrument (UPLC), Micro liquid chromatography, or Nano liquid chromatography.
  • the sample prior to ionization, is acoustically ejected into a mobile phase at an open port interface (OPI) using an acoustic droplet ejector (ADE) or directly injected into an ionization source.
  • OPI open port interface
  • ADE acoustic droplet ejector
  • the method is used to prepare the sample for clinical analysis.
  • the clinical analysis is used to screen for drugs of abuse or peptide markers for disease states.
  • the drugs of abuse are selected from the group consisting of amphetamines, methamphetamines, benzodiazepines, barbiturates, marijuana, cocaine, PCP, methadone, opioids (narcotics), fentanyl, norfentanyl, gabapentin, and pregabalin.
  • the method is used to extend the dynamic range of the mass spectrometer. In another aspect, the method prevents reanalysis of the sample and/or allows for the selective dilution of several analytes.
  • Another aspect of the disclosure relates to a computer- implemented method for quantifying at least one analyte in a sample by mass analysis, comprising: receiving, using a processor, a data set comprising at least one product ion transition for the at least one analyte and at least one isotopic ion transition for the at least one analyte; calculating, using a processor, the intensity and/or abundance of the product ion transition and/or isotopic ion transition; and quantifying, using a processor, the at least one analyte present in the sample using the intensity and/or abundance of the product ion transition and/or isotopic ion transition.
  • the data set comprises at least one product ion transition and at least one isotopic ion transition for at least two analytes, alternatively at least three analytes, alternatively at least four analytes, alternatively at least five analytes, alternatively at least six analytes, alternatively at least seven analytes, alternatively at least eight analytes, alternatively at least nine analytes, alternatively at least ten analytes.
  • the data set comprises at least two product ion transitions, alternatively at least three product ion transitions, alternatively at least four product ion transitions, alternatively at least five product ion transitions, alternatively at least six product ion transitions, alternatively at least seven product ion transitions, alternatively at least eight product ion transitions, alternatively at least nine product ion transitions, alternatively at least ten product ion transitions.
  • the data set comprises at least two isotopic ion transitions, alternatively at least three isotopic ion transitions, alternatively at least four isotopic ion transitions, alternatively at least five isotopic ion transitions, alternatively at least six isotopic ion transitions, alternatively at least seven isotopic ion transitions, alternatively at least eight isotopic ion transitions, alternatively at least nine isotopic ion transitions, alternatively at least ten isotopic ion transitions.
  • the method further comprises determining, using a processor, if the product ion transition meets a condition.
  • the method further comprises generating an alert in response to determining the product ion transition meets a condition.
  • the alert generated is selected from the group consisting of sounding an alarm, displaying a notification in a browser-based dashboard, sending an email to a user, sending a text message to a user, sending a push notification through an application to a mobile device, and displaying a notification through an application on a mobile device.
  • the condition is ionization saturation, detector saturation, product ions generated near a peak apex, peak shape, a threshold intensity and/or a threshold abundance.
  • the at least one analyte is a known analyte and the data set comprises at least one product ion transition and at least one isotopic ion transition specific to said known analyte.
  • the at least one product ion transition and/or at least one isotopic ion transition is used to quantify the known analyte.
  • the product ion transition has an intensity and/or abundance of about 100%.
  • the product ion transition is the most intense and/or abundant isotope of the at least one analyte.
  • the intensity and/or abundance of the isotopic ion transition is less than about 100%, alternatively less than about 50%, alternatively less than about 25%, alternatively less than about 15%, alternatively less than about 10%, alternatively less than about 5% than a precursor and/or product ion.
  • quantifying the at least one analyte present in the sample comprises using the intensity and/or abundance of the at least one product ion transition and/or the at least one isotopic ion transition to calculate a ratio of the at least one product ion transition and/or the at least one isotopic ion transition to the corresponding analyte and/or precursor, and quantifying the analyte in the sample using the calculated ratio.
  • the calculated ratio is used to calculate an isotopic dilution factor (IDF).
  • the IDF is used as a multiplier to compensate for abundance differences between the product ion transition and/or isotopic ion transition and the corresponding analyte.
  • quantifying the at least one analyte present in the sample is based on a calibration curve generated for at least one calibration standard.
  • the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
  • FIG. 1 shows an extracted-ion chromatogram (XIC) for a conventionally diluted sample.
  • FIG. 2 shows a comparison between the most abundant transition and 3rd most abundant isotopic ion transition.
  • FIG. 3 shows glutamine as an example of natural isotopic MRM transitions.
  • FIGs. 4A-4E shows various extracted-ion chromatogram (XIC) for an undiluted sample.
  • FIG. 4A shows XIC of aTRAQ labeled amino acids mixture calibrator on Microflow M5-QTRAP 5500 Plus.
  • FIGs 4B-4E illustrate XIC of representative amino acids.
  • FIGs. 5A-5E shows various extracted-ion chromatogram (XIC) for a sample diluted by using natural isotopic abundance MRM.
  • FIG. 5A shows a XIC of aTRAQ labeled amino acids mixture calibrator on Microflow M5-QTRAP 5500 Plus.
  • FIGs 4B-4E illustrate XIC of representative amino acids.
  • FIG. 6 depicts various calibration curves.
  • FIG. 6A shows a calibration curve for gabapentin without isotopic dilution.
  • FIG. 6B shows a calibration curve for gabapentin with 155x isotopic dilution.
  • FIG. 6C shows a calibration curve for norbuprenorphine without isotopic dilution.
  • FIG. 7 depicts a calibration curve using product ion and isotopic ion MRM for norbuprenorphine and gabapentin, respectively.
  • FIGs. 8A-8C depicts a calibration curve constructed using glutamine.
  • FIG. 8A shows a calibration curve using 100% isotopic abundance MRM.
  • FIG. 8B shows a calibration curve using 8.08% isotopic abundance MRM.
  • FIG. 8C shows the use of 8.08% MRM isotopic abundance only for the highest calibrator level.
  • FIG. 9 shows isotopic MRMs for gabapentin.
  • FIG. 10 shows peaks from samples injected using 155x isotopic dilution factor.
  • FIG. 11 shows a gabapentin calibration using isotopic MRM.
  • FIG. 12 shows a 1,25-OH Vitamin D3 Isotopic MRM Calculator
  • FIG. 13 shows a 25-OH Vitamin D3 Isotopic MRM Calculator.
  • FIG. 14 illustrates simultaneous detection and measurement of 1,25-dihydroxyvitamin D3
  • FIGs. 15A and 15B show the calibration from the simultaneous measurement of 1,25- dihydroxyvitamin D3 and 25 -hydroxyvitamin D.
  • FIG. 15A shows a calibration curve of calibration points (closed blue circles) and quality control samples (open blue circles).
  • FIG. 15B shows the standards and quality samples analyzed for 1,25-dihydroxyvitamin D3 and 25 -hydroxyvitamin D.
  • the sample may be a biological sample.
  • Biological samples may be biological fluids, which may include, but are not limited to, blood, plasma, serum, oral fluid, or other bodily fluids or excretions, such as but not limited to saliva, urine, cerebrospinal fluid, lacrimal fluid, perspiration, gastrointestinal fluid, amniotic fluid, mucosal fluid, pleural fluid, sebaceous oil, exhaled breath, and the like.
  • the biological sample may also be tissue (including tissue biopsies), bone marrow, tumor samples, and other biological samples and materials derived therefrom.
  • the sample may also be a chemical sample.
  • Chemical samples may include any type of sample including chemicals, including, but not limited to, water samples.
  • the sample may also be an environmental sample. Non-limiting examples of environmental samples may include air, soil, and wastes (liquids, solids or sludges).
  • the sample may also be a food sample and the food sample may be solid, semisolid, viscous, or liquid.
  • the food sample may also be used to test for food safety, including microbial or bacterial analysis.
  • the sample may also be dissolved in solvent.
  • the solvent may be a liquid, a solid, a gas, or a supercritical fluid.
  • the solvent may be a polar or nonpolar solvent.
  • the solvent may be organic solvent.
  • the solvent may be water, including deionized water.
  • the sample may be mixed with a matrix material. A non-limiting example of a matrix material includes crystalline compounds.
  • the sample may also be dissolved into a solution, incorporated into a liquid, or a component in a homogenous system.
  • An analyte may include a substance whose presence, absence, or concentration is to be determined according to methods of the present disclosure.
  • Typical analytes may include, but are not limited to, organic molecules, hormones (such as thyroid hormones, estradiol, testosterone, progesterone, estrogen), metabolites (such as glucose or ethanol), proteins, lipids, carbohydrates, and sugars, steroids (such as Vitamin D), peptides (such as procalcitonin), nucleic acid segments, biomarkers (pharmaceuticals such as antibiotics, benzodiazepine), drugs (such as immunosuppressant drugs, narcotics, opioids, etc.), molecules with a regulatory effect in enzymatic processes such as promoters, activators, inhibitors, or cofactors, microorganisms (such as viruses (including EBV, HPV, HIV, HCV, HBV, Influenza, Norovirus, Rotavirus, Adenovirus, etc.), bacteria (H.
  • aspects of the disclosure can also allow for the simultaneous analysis of multiple analytes in the same class or different classes (e.g., simultaneous analysis of metabolites and proteins).
  • the method is used to prepare the sample for clinical analysis.
  • the clinical analysis can be used to screen peptide markers for disease states.
  • the clinical analysis can also be used to screen for drugs of abuse.
  • Non-limiting examples of drugs of abuse include amphetamines, methamphetamines, benzodiazepines, barbiturates, marijuana, cocaine, PCP, methadone, opioids (narcotics), fentanyl, norfentanyl, gabapentin, and pregabalin.
  • the clinical analysis is a clinical urine test or a urinalysis, and the analysis is used to screen for drugs of abuse.
  • Urine is a common biological sample used in testing for drugs of abuse. A urinalysis or clinical urine test can detect the presence of a drug of abuse after the drug effects have worn off.
  • the method further includes providing at least one container, wherein the at least one container comprises the sample.
  • the container may be any container for holding samples, including, but not limited to test tubes, centrifuge tubes, vials, cups, or bottles.
  • the container is a sample plate, the sample plate comprising a plurality of sample wells.
  • the sample wells may contain the sample. Depending on the analysis desired, the sample wells may contain several different samples and/or calibrants.
  • the sample may be separated, enriched, and/or desalted using a chromatography instrument, microflow, solid phase extraction, liquid-liquid extraction, protein precipitation, a trap-and-elute workflow, phospholipid removal, filtration, organic solvent extraction, dilution, desalting, hydrolysis, magnetic based purification, or isoelectric point precipitation.
  • a chromatography instrument include a high performance liquid chromatography (HPLC) instrument, an ultra high performance liquid chromatography instrument (UPLC), microLC, or nanoLC.
  • the method further includes ionizing the sample.
  • the sample prior to ionization, is separated, enriched or desalted.
  • the sample may be acoustically ejected into a mobile phase at an open port interface (OPI) using an acoustic droplet ejector (ADE) prior to ionization or directly injected into an ionization source.
  • OPI open port interface
  • ADE acoustic droplet ejector
  • the sample may be ionized using an ionization method known in the art.
  • ionization methods include chemical ionization (CI), electron impact ionization (El), fast atom bombardment (FAB), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), laser ionization (LIMS), matrix assisted laser desorption ionization (MALDI), plasmadesorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), and thermal ionization (TIMS).
  • CI chemical ionization
  • El electron impact ionization
  • FAB fast atom bombardment
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • LIMS laser ionization
  • MALDI matrix assisted laser desorption ionization
  • PD plasmadesorption ionization
  • RIMS resonance ionization
  • SIMS secondary ionization
  • TMS thermal ionization
  • mass spectrometers that have the ability to select and fragment molecular ions
  • tandem mass spectrometers i.e., mass spectrometers that have two mass separators with an ion fragmentor disposed in the ion flight path between the two mass separators.
  • mass separators include, but are not limited to, quadrupoles, RF multipoles, ion traps, time-of-flight (TOF), and TOF in conjunction with a timed ion selector.
  • Non-limiting examples of ion fragmentors include, but are not limited to, those operating on the principles of collision induced dissociation (CID, also referred to as collisionally assisted dissociation (CAD)), photoinduced dissociation (PID), surface induced dissociation (SID), post source decay, by interaction with an electron beam (e.g., electron induced dissociation (EID), electron capture dissociation (ECD)), interaction with thermal radiation (e.g., thermal/black body infrared radiative dissociation (BIRD)), post source decay, or combinations thereof.
  • CID collisionally assisted dissociation
  • PID photoinduced dissociation
  • SID surface induced dissociation
  • post source decay by interaction with an electron beam (e.g., electron induced dissociation (EID), electron capture dissociation (ECD)), interaction with thermal radiation (e.g., thermal/black body infrared radiative dissociation (BIRD)), post source decay, or combinations thereof.
  • tandem mass spectrometry systems for mass analysis include, but are not limited to, those which comprise one or more of a triple quadrupole, a quadrupole- linear ion trap (e.g., QTRAP® System), a quadrupole TOF (e.g., TripleTOF® System), and a TOF- TOF.
  • a triple quadrupole e.g., QTRAP® System
  • a quadrupole- linear ion trap e.g., QTRAP® System
  • a quadrupole TOF e.g., TripleTOF® System
  • the method further includes monitoring, by mass spectrometry, at least one product ion transition for the at least one analyte and at least one isotopic ion transition for the at least one analyte.
  • the monitoring of the product ion transition and/or isotopic ion transition for the at least one analyte may also include monitoring an isotopic abundance for at least one ion pair of the at least one analyte.
  • m/z mass-to-charge ratio
  • two analytes are quantified, alternatively at least three analytes are quantified, alternatively at least four analytes are quantified, alternatively at least five analytes are quantified, alternatively at least six analytes are quantified, alternatively at least seven analytes are quantified, alternatively at least eight analytes are quantified, alternatively at least nine analytes are quantified, alternatively at least ten analytes are quantified.
  • Product ion transition monitoring is a technique in which the m/z range of a first mass separator is specifically selected to transmit a molecular ion (often referred to as “the parent ion” or “the precursor ion”) to an ion fragmentor to produce fragment ions (often referred to as “daughter ions” or “product ion”).
  • the transmitted m/z range of a second mass separator is selected to transmit one or more product ions to a detector that measures the product ion signal.
  • the observed m/z ratio (and may also referred to as “mass data”) of a parent (or precursor) ion and its corresponding product (or daughter) ion is a product ion transition.
  • This ion transition may also be referred to as a precursor-product ion transition or a product-daughter ion transition.
  • MRM multiple reaction monitoring
  • two or more transitions are monitored, each corresponding to a different fragment or product ion.
  • the parent ion of morphine is 286, and the most intense ions created by the fragmentation of 286 are 201 , 181, and 165.
  • the three product ion transitions for morphine are 286 — 201, 286 — 181, and 286 —>165.
  • At least two product ion transitions are monitored, alternatively at least three product ion transitions are monitored, alternatively at least four product ion transitions are monitored, alternatively at least five product ion transitions are monitored, alternatively at least six product ion transitions are monitored, alternatively at least seven product ion transitions are monitored, alternatively at least eight product ion transitions are monitored, alternatively at least nine product ion transitions are monitored, alternatively at least ten product ion transitions are monitored.
  • the product ion has an intensity and/or abundance of about 100%.
  • the product ion is the most intense and/or abundant isotope of the at least one analyte.
  • Isotopic MRM isotopic multiple reaction monitoring
  • the workflow utilizes the ion transitions based on the natural isotopic abundance of analytes.
  • This ion transition may also be referred to as a precursor-isotopic ion transition or isotopic ion transition.
  • there are two stable isotopes of chlorine chlorine 35 (75.8 % natural abundance) and chlorine 37 (24.2 % natural abundance).
  • each natural isotopic ion’s relative abundance value is different from another in a proportionally decreasing fashion.
  • the intensity and/or abundance of the isotopic ion is less than about 100%, alternatively less than about 50%, alternatively less than about 25%, alternatively less than about 15%, alternatively less than about 10%, alternatively less than about 5% than a precursor and/or the product ion.
  • the natural isotopic daughter ion acts as an internal standard, allowing for quantification of the analyte without requiring the addition of a calibrant or stable-isotope labeled analyte.
  • Extending the linear dynamic range for a given analyte can be done by using a natural isotopic MRM for quantification, which yields an abundance range compatible with other analytes in a given panel and using a less abundant isotopic ion transition decreases peak intensity and avoids detector overload.
  • At least two isotopic ion transitions are monitored, alternatively at least three isotopic ion transitions are monitored, alternatively at least four isotopic ion transitions are monitored, alternatively at least five isotopic ion transitions are monitored, alternatively at least six isotopic ion transitions are monitored, alternatively at least seven isotopic ion transitions are monitored, alternatively at least eight isotopic ion transitions are monitored, alternatively at least nine isotopic ion transitions are monitored, alternatively at least ten isotopic ion transitions are monitored.
  • the method further comprises determining the intensity and/or abundance of the at least one isotopic ion transition; and quantifying the at least one analyte present in the sample using the intensity and/or abundance of the isotopic ion transition.
  • a condition include ionization saturation, detector saturation, product ions generated near the peak apex, peak shape, a threshold intensity and/or a threshold abundance.
  • FIG. 1 shows a conventional 50x water dilution of the highest calibrator level. Low intensity amino acids such as homocytruline and hydroxyproline would not be detected in the lowest calibrator. Using isotopic MRM dilutions prevents the requirement for this manual dilution. Furthermore, monitoring these less abundant isotopic ion transitions could extend the dynamic range by up to lOOx, without the need for reanalysis.
  • the intensity, or area, of a product ion transition or isotopic ion transition depends on several factors including concentration, ease of ionization, and fragmentation efficiency.
  • the peak areas for product ion transitions or isotopic ion transitions are integrated as measures of product ion or isotopic ion abundance and serve as the basis for quantitative comparisons.
  • the signal intensity or relative abundance of the product ion and/or isotopic ion is compared to, for example, a standard curve constructed using one or more standard compounds (i.e., calibration curve) and/or by calculating a ratio of at least one isotopic ion transition to the corresponding analyte, and quantifying the amount of the analyte in the sample using the calculated ratio.
  • the calculated ratio may be used to calculate an isotopic dilution factor (“IDF”). This IDF may be used as a multiplier to compensate for abundance differences between the naturally occurring abundant isotopes and the corresponding analyte and/or precursor.
  • the precursor is most abundant isotope.
  • the isotope MRM conditions may be auto-optimized. For example, the theoretical isotope intensity ratio of both precursor and isotopic ions, and the desired intensity level is used to choose the isotope MRM conditions.
  • the most abundant transition peak is the peak that is used to quantify the majority of the samples.
  • the isotopic ion transition peak is a peak of lower abundance that can be used as a dilution factor. The factor difference between the quantifying transition and isotopic ion transition can be easily determined and applied to samples which require quantification using the isotopic ion transition.
  • Table 1 shows a comparison of isotopic dilution factor (IDF) testosterone values from male subjects against the quantifying transition.
  • IDF isotopic dilution factor
  • Another example of an isotopic MRM workflow is the quantification of amino acids in a panel of 45 analytes.
  • glutamine is shown as an example of isotopic ion transitions, where the isotopic ion transition 294.2 — >120.2 is being used to extend the dynamic range of the instrument.
  • each amino acid can be measured within a linear dynamic range if the isotopic abundance MRM workflow approach is implemented.
  • Table 2 shows that
  • FIGs 4A-4E when an undiluted sample is analyzed the amino acids saturate the detector (FIGs. 4B and 4C) or possess lower and desired intensity for quantitation (FIGs., 4D and 4E). However, when the sample is diluted by using isotopic ion transition (FIG. 5A), the detector is no longer saturated (FIGs., 5B and 5C), allowing acceptable peak shape for reliable quantitation and the amino acids possess lower intensity and remain intact. (FIGs. 5D and 5E).
  • the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
  • norbuprenorphine (15.6-1000 ng/mL) was measured using product ion transitions and gabapentin (780 - 50000 ng/mL) was measured using isotopic ion transitions.
  • This ability to use both product ion transitions and isotopic ion transitions allows for the measurement of two different analytes of interest across greater than 4 orders of magnitude.
  • FIGs. 8A-C depict another calibration curve based on glutamine.
  • the 100% abundant isotopic ion transition is used across all calibrator levels.
  • the 8.08% abundant isotopic ion transition is used across all calibrator levels.
  • the 8.08% abundant isotopic ion transition only for the highest calibrator level is used to extend the instrument's dynamic range and an isotopic dilution factor of 12.4 is applied.
  • a response factor of 0.3 was applied to the highest calibration point to account for response factor differences.
  • the sample data from the MRM and/or isotopic MRM workflow is processed using a computer program comprising a non-transitory and tangible computer-readable storage medium.
  • computer-readable storage medium refers to any media that participates in providing instructions to a processor for execution. Such a medium may take many forms, including but not limited to, non-volatile media, volatile media, and transmission media.
  • Non-volatile media includes, for example, optical or magnetic disks, such as storage device.
  • Volatile media includes dynamic memory, such as memory 106.
  • Transmission media includes coaxial cables, copper wire, and fiber optics, including the wires that comprise bus.
  • Common forms of computer-readable media or computer program products include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.
  • the computer program can be used to determine the intensity and/or abundance of the isotopic ion transition, calculate the ratio of the at least one isotopic ion transition to the corresponding analyte and/or precursor, and/or quantifying the amount of the analyte in the sample using the calculated ratio, and/or automatically calculate and compare MRM ratios for compound identification and quantification and highlight concentrations above a specified maximum residue level.
  • a computer- implemented method can be used to quantify at least one analyte in a sample by mass analysis.
  • the computer-implemented method includes a processor.
  • Processor can be, but is not limited to, a computer, a microprocessor, a single processing core, multiple processing cores, one or more general-purpose processors (e.g., ARM-based processors), a Digital Signal Processor (DSP), a Programmable Logic Device (PLD), an Application-Specific Integrated Circuit (ASIC), a Field-Programmable Gate Array (FPGA), or any device capable of sending and receiving control signals and data.
  • DSP Digital Signal Processor
  • PLD Programmable Logic Device
  • ASIC Application-Specific Integrated Circuit
  • FPGA Field-Programmable Gate Array
  • the processor can receive a data set that comprises at least one product ion transition and at least one isotopic ion transition for an analyte (or analytes) to be quantified.
  • the processor can calculate the intensity and/or abundance of the product ion transition and/or isotopic ion transition; and quantify at least one analyte present in the sample using the intensity and/or abundance of the product ion transition and/or isotopic ion transition.
  • the processor can also determine if the product ion transition meets a condition (e.g., ionization saturation, detector saturation, product ions generated near the peak apex, peak shape, a threshold intensity and/or a threshold abundance) and is the condition is met, generating an alert.
  • a condition e.g., ionization saturation, detector saturation, product ions generated near the peak apex, peak shape, a threshold intensity and/or a threshold abundance
  • the alert include, sounding an alarm, displaying a notification in a browser-based dashboard, sending an email to a user, sending a text message to a user, sending a push notification through an application to a mobile device, and displaying a notification through an application on a mobile device.
  • the processer can select the most intense and/or abundant isotopic ion transition that does not meet the condition, and quantify at least one analyte present in the sample using the intensity and/or abundance of said isotopic ion transition.
  • the data set can comprise at least one product ion transition and at least one isotopic ion transition specific to said known analyte which can then be used to quantify the analyte.
  • Example 1 Extending the linear dynamic range using isotopic MRMs.
  • Urine samples can be collected according to the SAMHSA guidelines (Substance Abuse and Mental Health Services Administration Center for Substance Abuse Prevention), available at https://www.samhsa.gov/sites/default/files/specimen-collection-handbook-2014.pdf. Urine specimens are typically submitted to certified laboratories within 24 hours after collection.
  • the sample is prepared using reagent grade water and mass spectrometry HPLC reagents.
  • the liquid sample undergoes HPLC and tandem mass spectrometry to select the isotopic ion transitions of interest.
  • the isotopic ion transition that is selected for quantification is one that produces a robust signal within the analytical measuremnt range, but also one that is not interfered with by other substances.
  • the isotopic MRM transitions monitored were 172.13 — 137.10, 173.14 138.10, 174.14 139.10, 173.14 137.10, 174.14 137.10, and 174.14 ⁇ 138.10.
  • a 155 isotopic dilution factor was calculated for the selected 174.14 — 139.10 transition (0.645% abundance), which was then applied to determine the concentration of calibrators and control samples (FIGs. 10 and 11). In FIG. 11, calibrator 8 was excluded due to analyte saturation at the high-end.
  • vitamin D family of metabolites including vitamin D3, vitamin D2, and metabolites of vitamin D3 and vitamin D2, such as the 25 -hydroxy (25-OH) and the 1,25 dihydroxy (1,25-OH) analogs
  • the analytical range for 25-OH Vitamin D is 20 - 150 ng/mL
  • 1 , 25-OH Vitamin D is 20 - 150 pg/mL making it difficult to detect and measure these analytes simultaneously.
  • only one assay run is required for detection and quantification when using isotopic MRM for 25-OH Vitamin D and traditional MRM for 1,25-OH Vitamin D.
  • the sample is prepared using reagent grade water and mass spectrometry HPLC reagents.
  • the sample is purified via solid phase extraction and then derivatized using Amplifex Diene reagent (SCIEX). 200 pl of sample was dispensed into a microcentrifuge tube. The sample was diluted with 700 .1 of deionized water, vortexed for 30 second and spun down. 20 pl of internal standards were pipetted into the first of two vertically stacked solid phase extraction columns. 900 pl of diluted samples were loaded onto the first stacked column (only 20 pl were loaded for calibrators). The first stacked column was eluted using di-isopropyl ether onto the second stacked column.
  • SCIEX Amplifex Diene reagent
  • the second stacked column was eluted with twice using 4.5ml of 4% isoprpanol in hexanes.
  • the second stacked column was then eluted again using 6ml of 6% isopropanol in hexanes, followed by an elution using 4.5 ml of 30% isopropanol in hexanes.
  • the solvent was dried using a speed vacuum and 300 pl methanol was added to in the dried samples in a glass tube.
  • the tubes were vortexed and dried and 50 pl Amplifex Diene reagent were added to the dried sample and vortexed twice.
  • the mixture was incubated for 30 minutes at ambient temperature and 50 pl of di water were added to the sample tube and vortexed. The contents were then transferred to a vial for prior to HPLC and tandem mass spectrometry to select the isotopic or traditional MRM transitions of interest.
  • a method for quantifying at least one analyte in a sample by mass analysis comprising: ionizing the sample; monitoring, by mass spectrometry, at least one product ion transition for the at least one analyte and at least one isotopic ion transition for the at least one analyte; wherein if the product ion transition meets a condition, the method further comprises determining the intensity and/or abundance of at least one isotopic ion transition ; and quantifying the at least one analyte present in the sample using the intensity and/or abundance of the at least one isotopic ion transition.
  • the condition is ionization saturation, detector saturation, product ions generated near a peak apex, peak shape, a threshold intensity and/or a threshold abundance.
  • determining the intensity and/or abundance of the isotopic ion comprises obtaining mass data corresponding to the at least one isotopic ion transition.
  • any one subparagraphs 1-7 wherein at least two analytes are quantified, alternatively at least three analytes are quantified, alternatively at least four analytes are quantified, alternatively at least five analytes are quantified, alternatively at least six analytes are quantified, alternatively at least seven analytes are quantified, alternatively at least eight analytes are quantified, alternatively at least nine analytes are quantified, alternatively at least ten analytes are quantified.
  • any one subparagraphs 1-8 wherein at least two product ion transitions are monitored, alternatively at least three product ion transitions are monitored, alternatively at least four product ion transitions are monitored, alternatively at least five product ion transitions are monitored, alternatively at least six product ion transitions are monitored, alternatively at least seven product ion transitions are monitored, alternatively at least eight product ion transitions are monitored, alternatively at least nine precursor-product ion transitions are monitored, alternatively at least ten precursor-product ion transitions are monitored.
  • any one subparagraphs 1-9 wherein at least two isotopic ion transitions are monitored, alternatively at least three isotopic ion transitions are monitored, alternatively at least four isotopic ion transitions are monitored, alternatively at least five isotopic ion transitions are monitored, alternatively at least six isotopic ion transitions are monitored, alternatively at least seven isotopic ion transitions are monitored, alternatively at least eight isotopic ion transitions are monitored, alternatively at least nine isotopic ion transitions are monitored, alternatively at least ten isotopic ion transitions are monitored.
  • the method further comprises quantifying at least a second analyte in the sample using a product ion transition or isotopic ion transition of the second analyte.
  • quantifying the at least one analyte present in the sample comprises: using the intensity and/or abundance of the at least one isotopic ion transition to calculate a ratio of the at least one isotopic ion transition to the corresponding analyte and/or a precursor, and quantifying the at least one analyte in the sample using the calculated ratio.
  • the ion detector is selected from the group consisting of an electron multiplier, a Faraday cup, a photomultiplier conversion dynode detector, an array detector, and a charge detector.
  • tandem mass spectrometer is selected from the group consisting of a triple quadrupole, a quadrupole-linear ion trap, a quadrupole TOF, and a TOF-TOF.
  • chromatography instrument is a high performance liquid chromatography (HPLC) instrument, an ultra high performance liquid chromatography instrument (UPLC), Micro liquid chromatography, or Nano liquid chromatography.
  • HPLC high performance liquid chromatography
  • UPLC ultra high performance liquid chromatography instrument
  • Micro liquid chromatography Micro liquid chromatography
  • Nano liquid chromatography Nano liquid chromatography
  • the sample is acoustically ejected into a mobile phase at an open port interface (OPI) using an acoustic droplet ejector (ADE) or directly injected into an ionization source.
  • OPI open port interface
  • ADE acoustic droplet ejector
  • drugs of abuse are selected from the group consisting of amphetamines, methamphetamines, benzodiazepines, barbiturates, marijuana, cocaine, PCP, methadone, opioids (narcotics), fentanyl, norfentanyl, gabapentin, and pregabalin.
  • a computer-implemented method for quantifying at least one analyte in a sample by mass analysis comprising: receiving, using a processor, a data set comprising at least one product ion transition for the at least one analyte and at least one isotopic ion transition for the at least one analyte; calculating, using a processor, the intensity and/or abundance of the product ion transition and/or isotopic ion transition; and quantifying, using a processor, the at least one analyte present in the sample using the intensity and/or abundance of the product ion transition and/or isotopic ion transition.
  • the data set comprises at least one product ion transition and at least one isotopic ion transition for at least two analytes, alternatively at least three analytes, alternatively at least four analytes, alternatively at least five analytes, alternatively at least six analytes, alternatively at least seven analytes, alternatively at least eight analytes, alternatively at least nine analytes, alternatively at least ten analytes.
  • the computer-implemented method subparagraph 38 or subparagraph 39 wherein the data set comprises at least two product ion transitions, alternatively at least three product ion transitions, alternatively at least four product ion transitions, alternatively at least five product ion transitions, alternatively at least six product ion transitions, alternatively at least seven product ion transitions, alternatively at least eight product ion transitions, alternatively at least nine product ion transitions, alternatively at least ten product ion transitions.
  • the data set comprises at least two isotopic ion transitions, alternatively at least three isotopic ion transitions, alternatively at least four isotopic ion transitions, alternatively at least five isotopic ion transitions, alternatively at least six isotopic ion transitions, alternatively at least seven isotopic ion transitions, alternatively at least eight isotopic ion transitions, alternatively at least nine isotopic ion transitions, alternatively at least ten isotopic ion transitions.
  • quantifying the at least one analyte present in the sample comprises: using the intensity and/or abundance of the at least one product ion transition and/or the at least one isotopic ion transition to calculate a ratio of the at least one product ion transition and/or the at least one isotopic ion transition to the corresponding analyte and/or precursor, and quantifying the analyte in the sample using the calculated ratio.

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Abstract

Les revendications et la technologie décrite par la présente invention concernent des procédés de quantification d'au moins un analyte dans un échantillon par analyse de masse par ionisation de l'échantillon ; de surveillance, par spectrométrie de masse, d'au moins une transition d'ions produits pour le ou les analytes et d'au moins une transition ionique isotopique pour le ou les analytes ; le procédé comprenant en outre les étapes consistant à déterminer l'intensité et/ou l'abondance d'au moins une transition d'ions isotopiques ; et quantifier le ou les analytes présents dans l'échantillon à l'aide de l'intensité et/ou de l'abondance de la ou des transitions d'ions isotopiques. L'invention concerne également des procédés mis en œuvre par ordinateur pour quantifier au moins un analyte dans un échantillon.
PCT/IB2022/060307 2021-11-01 2022-10-26 Utilisation de l'abondance isotopique naturelle de composés pour étendre la plage dynamique d'un instrument WO2023073588A1 (fr)

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US20190029591A1 (en) * 2011-12-07 2019-01-31 Glaxosmithkline Llc Methods for determining total body skeletal muscle mass

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Publication number Priority date Publication date Assignee Title
US20190029591A1 (en) * 2011-12-07 2019-01-31 Glaxosmithkline Llc Methods for determining total body skeletal muscle mass

Non-Patent Citations (3)

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Title
DI RAGO M ET AL: "Ultra-rapid targeted analysis of 40 drugs of abuse in oral fluid by LC-MS/MS using carbon-13 isotopes of methamphetamine and MDMA to reduce detector saturation", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 408, no. 14, 18 March 2016 (2016-03-18), pages 3737 - 3749, XP035747420, DOI: 10.1007/S00216-016-9458-3 *
LIU H ET AL: "Expanding the linear dynamic range for multiple reaction monitoring in quantitative liquid chromatographytandem mass spectrometry utilizing natural isotopologue transitions", TALANTA, vol. 87, 28 September 2011 (2011-09-28), pages 307 - 310, XP028114072, DOI: 10.1016/J.TALANTA.2011.09.063 *
TSUJI M ET AL: "A validated quantitative liquid chromatography-tandem quadrupole mass spectrometry method for monitoring isotopologues to evaluate global modified cytosine ratios in genomic", JOURNAL OF CHROMATOGRAPHY B, vol. 953, 7 February 2014 (2014-02-07), pages 38 - 47, XP028633212, DOI: 10.1016/J.JCHROMB.2014.01.050 *

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