Patent Application
20240420939
Analytical samples, including biological samples and bodily liquids, can often provide critical analytical information. Presently, compounds of a related group (i.e., drugs and metabolites, hormones in a pathway, biomarkers, and/or peptides from a particular biologic drug) are generally measured together. However, 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.
Direct acoustic ionization mass spectrometry is suitable for high-throughput screening of samples, but because there is no separation (i.e., chromatography, ion mobility) performed on the sample that is ejected into the mass spectrometer, this workflow is not optimal for obtaining a specific, quantitative result (definitive testing). For example, when analyzing samples with analytes of varying concentrations, detector saturation may occur with higher concentrated analytes presenting a signal intensity that exceeds the detector capacity. Additionally, as direct acoustic ionization mass spectrometry requires concentrated samples, the samples must be prepared a second time after screening for quantification via definitive testing. Due to the required concentration differences between platforms, a dilution step would be necessary when transferring samples from a direct acoustic ionization mass spectrometry plate to an LC vial. This additional preparation is time-, reagent-, and vial-consuming, requires human intervention, and is prone to errors.
The inventors have recognized the need to combine direct sampling from a container initially used for a first analysis, such as screening, with a quantitative mass analyzer. Relative quantification of an analyte using a quantitative mass analyzer typically requires labeling the analyte (or sample) with an isotopic, isobaric, or metal tag. However, label-free quantification using multiple reaction monitoring (MRM) of natural isotopic abundance enables high throughput quantitative analysis without any physical dilution.
One aspect of the disclosure relates to a sample processing system comprising at least one sample introduction device, wherein the at least one sample introduction device is configured to receive a sample; a mass analyzer coupled to the sample introduction device; a control system configured to at least control the at least one sample introduction device and/or the mass analyzer, wherein the mass analyzer is configured to perform a first mass analysis on the sample, wherein the first mass analysis is mass screening for an analyte of interest in the sample, and wherein if the analyte of interest is detected in the sample, the mass analyzer is configured to perform a second mass analysis, wherein the second mass analysis is a quantitative 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; determining intensity and/or abundance of the at least one product ion transition and/or 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 at least one product ion transition and/or isotopic ion transition.
In an aspect, the product ion transition has an intensity and/or abundance of about 100%. In another aspect, the product ion transition is the most intense and/or abundant isotope of the at least one analyte. In another aspect, 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. In yet another aspect, determining the intensity and/or abundance of the at least one product ion transition and/or isotopic ion transition comprises obtaining mass data corresponding to the at least one product ion transition and/or isotopic ion transition.
In an aspect, 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. In another aspect, 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. In another aspect, 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. In another aspect, the sample processing system further comprises quantifying at least a second analyte in the sample using a product ion transition and/or isotopic ion transition of the second analyte. In another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to determine the intensity and/or abundance of the isotopic ion transition.
In an aspect, if the product ion transition meets a condition, selecting the most intense and/or abundant isotopic ion transition that does not meet the condition, and quantifying the at least one analyte present in the sample using the intensity and/or abundance of said isotopic ion transition. In another aspect, the condition is ionization saturation, detector saturation, product ions generated near a peak apex, peak shape, a threshold intensity and/or a threshold abundance.
In an aspect, quantifying the at least one analyte present in the sample comprises performing a separation step on the sample prior to ionization, 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. In another aspect, the calculated ratio is used to calculate an isotopic dilution factor (IDF). In a further aspect, 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. In yet another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to calculate the ratio of the at least one product ion transition and/or 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.
In an aspect, the sample introduction device comprises an acoustic droplet ejector (ADE), a solid phase extraction system, liquid-liquid extraction, protein precipitation, a liquid aspiration system, a microinjector, a nanoinjector, an inkjet printer nozzle, a chromatography instrument, microflow system, solid phase extraction system, differential mobility spectrometer, a trap-and-elute workflow, an open port interface, or direct flow injection. In another aspect, the sample introduction comprises acoustically ejecting the liquid sample into a mobile phase at an open port interface (OPI) using the acoustic droplet ejector (ADE). In another aspect, 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.
In an aspect, the mass analyzer is a tandem mass spectrometer. In another aspect, 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. In another aspect, the mass analyzer comprises a detector. In a further aspect, the detector is an ion detector. In yet a further aspect, 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.
In another aspect, the sample is a biological sample. In another aspect, the biological sample is selected from the group consisting of urine, blood, oral fluid, plasma, tissue, bone marrow, and tumor samples. In yet another aspect, the biological sample is dissolved in a solvent, introduced into a solution, or mixed with a matrix material.
In an aspect, quantifying the at least one analyte present in the sample is based on a calibration curve generated for at least one calibration standard. In another aspect, the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
In another aspect, the sample processing system further comprises at least one sample preparation station configured to receive and/or prepare a sample; and at least one container, wherein the sample preparation station is configured to dispense the prepared sample into the at least one container. In another aspect, the sample processing system further comprises: at least one container transport device; sample introduction device is configured to receive the at least one container from the sample preparation station via the at least one container transport device. In yet another aspect, the sample processing system further comprises at least one aliquoting station, wherein the at least one aliquoting station is configured to aliquot a portion of the sample to and/or from the at least one container. In a further aspect, the at least one aliquoting station is housed in the sample preparation station or the at least one sample introduction device. In another aspect, the sample processing system comprises at least two aliquoting stations, wherein a first aliquoting station is housed in the sample preparation station and a second aliquoting station is housed in the at least one sample introduction device. In another aspect, the at least one container is at least one sample plate comprising a plurality of sample wells. In yet another aspect, the sample well comprises the sample.
Another aspect of the disclosure is a sample processing system comprising at least one sample introduction device, wherein the at least one sample introduction device is configured to receive a sample; a first mass analyzer coupled to the sample introduction device; wherein the first mass analyzer is configured to perform a first mass analysis of the sample, wherein the first mass analysis is a mass screening; a second mass analyzer; wherein if an analyte of interest is detected in the sample, the second mass analyzer is configured to perform a second mass analysis; 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; determining the intensity and/or abundance of the at least one product ion transition and/or 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 product ion transition and/or isotopic ion transition; and a control system configured to at least control the at least one sample introduction device, the first mass analyzer, and/or the second mass analyzer.
In an aspect, the product ion transition has an intensity and/or abundance of about 100%. In another aspect, the product ion transition is the most intense and/or abundant isotope of the at least one analyte. In another aspect, 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. In yet another aspect, determining the intensity and/or abundance of the product ion transition and/or isotopic ion transition comprises obtaining mass data corresponding to the product ion transition and/or isotopic ion transition.
In an aspect, 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. In another aspect, 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. In another aspect, 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. In yet another aspect, the sample processing system further comprising quantifying at least a second analyte in the sample using a product ion transition and/or isotopic ion transition of the second analyte. In another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to determine the intensity and/or abundance of the isotopic ion transition.
In an aspect, if the product ion transition meets a condition, the sample processing system further comprises selecting the most intense and/or abundant isotopic ion transition that does not meet the condition, and quantifying the at least one analyte present in the sample using the intensity and/or abundance of said isotopic ion transition. In another aspect, the condition is ionization saturation, detector saturation, product ions generated near the peak apex, peak shape, a threshold intensity and/or a threshold abundance.
In an aspect, quantifying the at least one analyte present in the sample comprises performing a separation step on the sample prior to ionization, 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. In another aspect, the calculated ratio is used to calculate an isotopic dilution factor (IDF). In further aspect, 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. In another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to calculate the ratio of the at least one product ion transition and/or 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.
In an aspect, the sample introduction device comprises an acoustic droplet ejector (ADE), a solid phase extraction system, liquid-liquid extraction, protein precipitation, a liquid aspiration system, a microinjector, a nanoinjector, an inkjet printer nozzle, a chromatography instrument, microflow system, solid phase extraction system, differential mobility spectrometer, a trap-and-elute workflow, an open port interface, or direct flow injection. In another aspect, the sample introduction comprises acoustically ejecting the liquid sample into a mobile phase at an open port interface (OPI) using the acoustic droplet ejector (ADE). In another aspect, 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.
In an aspect, the first mass analyzer is a single-stage mass spectrometer or a tandem mass spectrometer. In another aspect, the single-stage mass spectrometer is selected from the group consisting of magnetic sector, quadrupole, time-of-flight (TOF), and ion traps. In an aspect, the second mass analyzer is a tandem mass spectrometer. In another aspect, 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.
In an aspect, the first and/or second mass analyzer comprises a detector. In another aspect, the detector is an ion detector. In another aspect, 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.
In an aspect, sample is a biological sample. In another aspect, the biological sample is selected from the group consisting of urine, blood, oral fluid, plasma, tissue, bone marrow, and tumor samples. In yet another aspect, the biological sample is dissolved in a solvent, introduced into a solution, or mixed with a matrix material.
In an aspect, quantifying the at least one analyte present in the sample is based on a calibration curve generated for at least one calibration standard. In another aspect, the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
In an aspect, the sample processing system further comprises at least one sample preparation station configured to receive and/or prepare a sample; and at least one container, wherein the sample preparation station is configured to dispense the prepared sample into the at least one container.
In an aspect, the sample processing system further comprises at least one container transport device; sample introduction device is configured to receive the at least one container from the sample preparation station via the at least one container transport device. In another aspect, the sample processing system further comprises at least one aliquoting station, wherein the at least one aliquoting station is configured to aliquot a portion of the sample to and/or from the at least one container. In yet another aspect, the at least one aliquoting station is housed in the sample preparation station or the at least one sample introduction device. In another aspect, the sample processing system comprises at least two aliquoting stations, wherein a first aliquoting station is housed in the sample preparation station and a second aliquoting station is housed in the at least one sample introduction device. In a further aspect, the at least one container is at least one sample plate comprising a plurality of sample wells. In another aspect, the sample well comprises the sample.
Another aspect of the disclosure is a method for quantifying at least one analyte in a sample using a sample processing system, wherein the sample processing system comprises at least one sample introduction device, wherein the at least one sample introduction device is configured to receive a sample; a mass analyzer coupled to the sample introduction device; a control system configured to at least control the at least one sample introduction device and/or the mass analyzer, wherein the mass analyzer is configured to perform a first mass analysis on the sample, wherein the first mass analysis is mass screening for an analyte of interest in the sample, and wherein if the analyte of interest is detected in the sample, the mass analyzer is configured to perform a second mass analysis, wherein the second mass analysis is a quantitative 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; determining the intensity and/or abundance of the at least one product ion transition and/or 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 product ion transition and/or isotopic ion transition.
In an aspect, the product ion transition has an intensity and/or abundance of about 100%. In another aspect, the product ion transition is the most intense and/or abundant isotope of the at least one analyte. In another aspect, 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. In yet another aspect, determining the intensity and/or abundance of the product ion transition and/or isotopic ion transition comprises obtaining mass data corresponding to the product ion transition and/or isotopic ion transition.
In an aspect, 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. In another aspect, 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. In yet another aspect, 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.
In another aspect, the method further comprises quantifying at least a second analyte in the sample using a product ion transition and/or isotopic ion transition of the second analyte. In another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to determine the intensity and/or abundance of the isotopic ion transition.
In an aspect, if the product ion transition meets a condition, selecting the most intense and/or abundant isotopic ion transition that does not meet the condition, and quantifying the at least one analyte present in the sample using the intensity and/or abundance of said isotopic ion transition. In another aspect, the condition is ionization saturation, detector saturation, product ions generated near a peak apex, peak shape, a threshold intensity and/or a threshold abundance.
In an aspect, quantifying the at least one analyte present in the sample comprises: performing a separation step on the sample prior to ionization, 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. In another aspect, the calculated ratio is used to calculate an isotopic dilution factor (IDF). In yet a further aspect, 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. In another aspect, the control system comprises a non-transitory and tangible computer-readable storage medium is used to calculate the ratio of the at least one product ion transition and/or 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.
In an aspect, the sample introduction device comprises an acoustic droplet ejector (ADE), a solid phase extraction system, liquid-liquid extraction, protein precipitation, a liquid aspiration system, a microinjector, a nanoinjector, an inkjet printer nozzle, a chromatography instrument, microflow system, solid phase extraction system, differential mobility spectrometer, a trap-and-elute workflow, an open port interface, or direct flow injection. In another aspect, the sample introduction comprises acoustically ejecting the liquid sample into a mobile phase at an open port interface (OPI) using the acoustic droplet ejector (ADE). In another aspect, 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.
In an aspect, the mass analyzer is a tandem mass spectrometer. In another aspect, 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. In another aspect, the mass analyzer comprises a detector. In another aspect, the detector is an ion detector. In a further aspect, 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.
In an aspect, the sample is a biological sample. In another aspect, the biological sample is selected from the group consisting of urine, blood, oral fluid, plasma, tissue, bone marrow, and tumor samples. In yet another aspect, the biological sample is dissolved in a solvent, introduced into a solution, or mixed with a matrix material.
In an aspect, quantifying the at least one analyte present in the sample is based on a calibration curve generated for at least one calibration standard. In another aspect, the calibration curve may be generated using at least one product ion transition, at least one isotopic ion transition, or a combination of both.
In an aspect, the sample processing system further comprises at least one sample preparation station configured to receive and/or prepare a sample; and at least one container, wherein the sample preparation station is configured to dispense the prepared sample into the at least one container. In another aspect, the sample processing system further comprises at least one container transport device; sample introduction device is configured to receive the at least one container from the sample preparation station via the at least one container transport device. In another aspect, the sample processing system further comprises at least one aliquoting station, wherein the at least one aliquoting station is configured to aliquot a portion of the sample to and/or from the at least one container. In yet another aspect, the at least one aliquoting station is housed in the sample preparation station or the at least one sample introduction device. In a further aspect, the sample processing system comprises at least two aliquoting stations, wherein a first aliquoting station is housed in the sample preparation station and a second aliquoting station is housed in the at least one sample introduction device. In another aspect, the at least one container is at least one sample plate comprising a plurality of sample wells. In a further aspect, the sample well comprises the sample.
In an aspect, the method is used in a clinical analysis workflow. In another aspect, the clinical analysis is used to screen for drugs of abuse or peptide markers for disease states. In a further aspect, 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.
In an aspect, the method is used to extend the dynamic range of the mass analyzer. In another aspect, the method prevents reanalysis of the sample and/or allows for selective dilution of several analytes.
Aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:
It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the present disclosure or the appended claims.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.
The term “about” is used in connection with a numerical value throughout the specification and the claims denote an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such an interval of accuracy is +/−10%.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.
Aspects of this disclosure include a sample processing system. Aspects of sample processing systems of this disclosure are illustrated in
The sample may be prepared based on the type of analysis desired. In some aspects, this may include, for example, chromatography, 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.
In some aspects, the sample introduction device 210 can be separate from or integrated with a sample preparation system 202. As illustrated in
The container transport device 206 can be any apparatus used to handle or transport a container. Non-limiting examples of suitable container transport devices include, but are not limited to, pick-and-place devices, such as pick-and-place transfer gantrys, transfer shuttles, such as extended linear reaction shuttles, or combinations of pick-and-place transfer gantrys and extended linear reaction shuttles. In some aspects, the sample introduction device 210 may include an aliquoting station (not shown).
In an aspect, the sample introduction device 210 is coupled to an analytical instrument, such as mass analyzer 208. In some aspects, including those illustrated in
In some aspects, the sample preparation station 202 may include an aliquoting station 400. In some aspects, the aliquoting station 400 is configured to aliquot a portion of the sample to and/or from the at least one container. The aliquoting station 400 may comprise at least one aliquotter or pipettor. In some aspects, the sample preparation station 202 or the aliquoting station 400 is further configured to hold at least one reagent pack needed for the first and/or second analysis.
The sample preparation system 202 is further configured to dispense the prepared sample into at least one container. The container may be any suitable container for holding samples, including, but not limited to test tubes, centrifuge tubes, vials, cups, or bottles. In some aspects, 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.
An analyte may include a substance whose presence, absence, or concentration is to be determined according to 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. pylori, Streptococcus, MRSA, C. diff, Ligionella, etc.), fungus, parasites (plasmodium, etc.), cells, cell components (such as cell membranes), spores, nucleic acids (such as DNA and RNA), etc. 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).
Mass screening allows for the confirmation of a target or suspect analyte and identifying unknown analytes. Single mass analyzers (also known as single-stage), such as the magnetic sector, quadrupole, time-of-flight (TOF), and ion traps, are commonly used for mass screening. Quantitative mass analysis allows for the selection and fragmentation of molecular ions using, for example, 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. Examples of suitable 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. Suitable 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. When operated in single stage, tandem mass spectrometers can be used for qualitative mass analysis. Examples of suitable 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. However, any suitable mass analyzer known in the art may be coupled to the sample introduction device 210.
In an aspect, the sample may be transferred from the sample introduction device 210 to the mass analyzer 208 using transfer techniques generally known in the art such as, for example, techniques comprising a microinjector, a nanoinjector, an inkjet printer nozzle, an acoustic droplet ejector (ADE), a solid phase extraction system, or a liquid aspiration system. While the transferred volumes may vary, typical volumes of transferred solutions fall within a range of about 2.5 nL to about 500 nL.
The sample introduction device 210 may be coupled to the mass analyzer 208 through a connector 501. The sample introduction device 210 may introduce the sample solution to the ion source 160 through the connector 501. The ion source 160 can be controlled by an ion source power supply 506 through a signal line 503. Ions from the analytes of interest, which are generated by the ion source 160, are introduced to a mass analysis region 512, detected by an ion detector 504, and mass analyzed using a data processing unit 510. The data processing unit 510 may be a separate unit or may be part of the control system. In an aspect, the sample solution may be introduced to the ion source by acoustically ejecting the sample into a mobile phase at an open port interface (OPI) using the acoustic droplet ejector (ADE).
In
The acoustic droplet ejection device 11 includes at least one container. In some aspects, the container is a sample plate with a plurality of sample wells. In
The ADE 11 comprises an acoustic ejector 33, which includes acoustic radiation generator 35 and focusing means 37 for focusing the acoustic radiation generated at a focal point 47 within the liquid sample, near the liquid surface. As shown in
The acoustic droplet ejector 33 may be in either direct contact or indirect contact with the external surface of each sample well. With direct contact, in order to acoustically couple the ejector to a sample well, it is preferred that the direct contact be wholly conformal to ensure efficient acoustic energy transfer. That is, the ejector and the sample well should have corresponding surfaces adapted for mating contact. Thus, if acoustic coupling is achieved between the ejector and sample well through the focusing means, it is desirable for the sample well to have an outside surface that corresponds to the surface profile of the focusing means. Without conformal contact, efficiency and accuracy of acoustic energy transfer may be compromised. In addition, since many focusing means have a curved surface, the direct contact approach may necessitate the use of sample wells that have a specially formed inverse surface.
Optimally, acoustic coupling is achieved between the ejector and each of the sample wells through indirect contact, as illustrated in
In operation, sample well 13 and optional sample well 15 of the device are filled with first and second liquid samples 14 and 16, respectively, as shown in
The structure of OPI 51 is also shown in
Liquid flow within the OPI 51 carries the analyte-solvent dilution through a sample transport capillary 61 provided by inner capillary tube 73 toward sample outlet 63 for subsequent transfer to an analytical instrument. For example, a qualitative mass analyzer. The acoustic droplet ejector may also eject or transfer droplets to an analytical instrument via direct flow injection or using a trap and elute system (e.g., a trap and elute system which includes two pumps and a 6-port switching valve).
A sampling pump (not shown) can be provided that is operably connected to and in liquid communication with the sample transport capillary 61, to control the output rate from outlet 63. In an exemplary aspect, a positive displacement pump is used as the solvent pump, e.g., a peristaltic pump, and, instead of a sampling pump, an aspirating nebulization system is used so that the analyte-solvent dilution is drawn out of the sample outlet 63 by the Venturi effect caused by the flow of the nebulizing gas introduced from a nebulizing gas source 65 via gas inlet 67 (shown in simplified form in
The solvent transport capillary 59 and sample transport capillary 61 are provided by outer capillary tube 71 and inner capillary tube 73 substantially co-axially disposed therein, where the inner capillary tube 73 defines the sample transport capillary, and the annular space between the inner capillary tube 73 and outer capillary tube 71 defines the solvent transport capillary 59.
The system can also include an adjuster 75 coupled to the outer capillary tube 71 and the inner capillary tube 73. The adjuster 75 can be adapted for moving the outer capillary tube tip 77 and the inner capillary tube tip 79 longitudinally relative to one another. The adjuster 75 can be any device capable of moving the outer capillary tube 71 relative to the inner capillary tube 73. Exemplary adjusters 75 can be motors including, but are not limited to, electric motors (e.g., AC motors, DC motors, electrostatic motors, servo motors, etc.), hydraulic motors, pneumatic motors, translational stages, and combinations thereof. As used herein, “longitudinally” refers to an axis that runs the length of the probe 51, and the inner and outer capillary tubes 73, 71 can be arranged coaxially around a longitudinal axis of the probe 51, as shown in
Additionally, as illustrated in
In some aspects, the sample is ionized prior to being transferred to the qualitative or quantitative mass analyzer. Suitable non-limiting ionization methods include chemical ionization (CI), electron impact ionization (EI), fast atom bombardment (FAB), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), laser ionization (LIMS), matrix assisted laser desorption ionization (MALDI), plasma-desorption ionization (PD), resonance ionization (RIMS), secondary ionization (SIMS), and thermal ionization (TIMS).
As shown in
In the depicted aspect, the ionization chamber 112 can be maintained at an atmospheric pressure, though in some aspects, the ionization chamber 112 can be evacuated to a pressure lower than atmospheric pressure. The ionization chamber 112, within which the analyte can be ionized as the analyte-solvent dilution is discharged from the electrospray electrode 164, is separated from a gas curtain chamber 114 by a plate 114a having a curtain plate aperture 114b. As shown, a vacuum chamber 116, which houses the mass analyzer, is separated from the curtain chamber 114 by a plate 116a having a vacuum chamber sampling orifice 116b. The curtain chamber 114 and vacuum chamber 116 can be maintained at a selected pressure(s) (e.g., the same or different sub-atmospheric pressures, a pressure lower than the ionization chamber) by evacuation through one or more vacuum pump ports 118. It will also be appreciated by a person skilled in the art and in light of the teachings herein that the mass analyzer can have a variety of configurations, including a qualitative mass analyzer 208 or a quantitative mass analyzer 212.
In accordance with the aspect and aspects of the disclosure, an acoustic signal can be detected and/or monitored in one or more regions of the sampling system. In an example aspect in accordance with
In some aspects, the combination of open port interface (OPI) and acoustic droplet ejection (ADE) is referred to as Acoustic Ejection Mass Spectrometry (AEMS). Examples of acoustic ejection mass spectrometry systems for qualitative mass analysis include an Echo® MS System.
It will further be appreciated that any number of additional elements can be included in the sample processing system including, for example, an ion mobility spectrometer (e.g., a differential mobility spectrometer for ion selection) that is disposed between the ionization chamber and the mass analyzer and is configured to separate ions based on their mobility through a drift gas in high- and low-fields rather than their mass-to-charge ratio. Additionally, it will be appreciated that the mass analyzer can comprise a detector that can detect the ions which pass through the mass analyzer and can, for example, supply a signal indicative of the number of ions per second that are detected.
In some aspects the sample introduction device 210, and the mass analyzer 208 are individual components in a modular laboratory automation system.
The sample processing system may include a control system 300. The control system may be configured to control the sample preparation station 202, the container transport device 206, the sample introduction device 210, and/or the mass analyzers 208, 208a, 212. In an aspect, there is a plurality of control systems that can control an individual element or a combination of elements. In an aspect, the control system may also include a data processor 302, a computer-readable storage medium 304 (including a computer program), and data storage 306. In an aspect, the sample data from an isotopic MRM workflow is processed using a computer program comprising a non-transitory and tangible computer-readable storage medium.
The term “computer-readable storage medium” as used herein 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. 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.
In an aspect, the mass analyzer 208 or first mass analyzer 208a is used to screen the sample for an analyte of interest. If an analyte of interest is detected in the sample, for example, above a certain qualitative threshold, such as having a signal above background noise, the mass analyzer 208 or second mass analyzer 212 is used to perform a quantitative analysis of the analyte. The quantitative analysis includes performing a separation step on the sample, ionizing the separated components of 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; determining the intensity and/or abundance of the at least one product ion transition and/or 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 product ion transition and/or isotopic ion transition. The separation step can involve one or more than one of several separation techniques including chromatography and ion mobility. For ion mobility, the step can comprise the use of differential mobility spectrometry.
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. As long as the mass-to-charge ratio (m/z) for the analytes of interest and/or corresponding ion pairs do not overlap, an infinite number of ion pairs can be monitored. This allows for the quantification of multiple analytes in one sample. In exemplary methods, 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. In a multiple reaction monitoring (“MRM”) workflow, two or more transitions are monitored, each corresponding to a different fragment or product ion. For example, the parent ion of morphine is 286, and the most intense ions created by the fragmentation of 286 are 201, 181, and 165. As such, the three product ion transitions for morphine are 286->201, 286->181, and 286-165.
In other exemplary methods, 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. In some exemplary methods, the product ion has an intensity and/or abundance of about 100%. In some exemplary methods, the product ion is the most intense and/or abundant isotope of the at least one analyte.
In an isotopic multiple reaction monitoring (“Isotopic MRM”), 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. For example, there are two stable isotopes of chlorine; chlorine 35 (75.8% natural abundance) and chlorine 37 (24.2% natural abundance). In general, each natural isotopic ion's relative abundance value is different from another in a proportionally decreasing fashion. For example, 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.
In other exemplary methods, 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.
In a further aspect, if the product ion transition meets a condition, 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. Non-limiting examples of a condition include ionization saturation, detector saturation, product ions generated near the peak apex, peak shape, a threshold intensity and/or a threshold abundance. When the product ion transition meets this condition, the peak quality and/or detector saturation makes it difficult to quantify the analyte using the product ion transition and an isotopic ion transition is used for quantification.
For example, if the lowest endogenous biomarker level is at the instrument detection limit for a normal “healthy” sample but an affected “sick” sample is above the detector saturation, or meet another condition, instead of reprocessing and diluting the sample, a lower isotopic intensity ion transition may be used. By using less abundant isotopic MRM transitions, it is possible to analyze multiple analytes of varying concentrations without changing the dilution factor of samples, eliminating the need to reprocess and rerun the sample.
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. To quantify an analyte using product ion transitions or isotopic ion transitions, 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. In some aspects, the precursor is most abundant isotope. In some aspects, 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.
Extending the linear dynamic range for a given analyte can be done by using a natural isotopic MRM transition for quantification, which yields an abundance range compatible with other analytes in a given panel and using a less abundant isotopic MRM transition decreases peak intensity and avoids detector overload. Monitoring these less abundant isotopic transitions could extend the dynamic range by up to 100x, without the need for reanalysis.
Instead of running two assays on the same sample, only one assay run is required for quantification when an isotopic MRM workflow for fentanyl and its metabolite, norfentanyl, is employed.
The sample is collected using any specimen collection method known in the art. 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 urine samples are prepared using reagent grade water and mass spectrometry HPLC reagents. In an Echo® 384-well plate, 14 μL of fentanyl-d5 and norfentanyl-d5 internal standards (2000 ng/mL each) in water were added to 36 μL of the urine sample. The plate was then shook on a planar rotator.
Using Echo® MS, the urine samples are screened for the presence of fentanyl and norfentanyl. Once fentanyl and norfentanyl have been positively identified, the sample plate is transferred to an LC-MS/MS system and the fentanyl and norfentanyl undergo definitive testing. As shown in
Calibration curves were also established using several different fentanyl and norfentanyl calibrators and quality control samples. As illustrated in
In another example, the urine sample undergoes the same processing and screening, but different isotopes are used for the fentanyl isotopic MRM (as shown in
Other aspects, advantages, and modifications are described in the following subparagraphs:
While the present disclosure has been described with reference to certain aspects, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present disclosure or appended claims. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present disclosure without departing from its scope. Therefore, it is intended that the present disclosure not be limited to the particular aspects disclosed, but that the present disclosure will include all aspects falling within the scope of the appended claims.
All patents, patent applications, publications, and descriptions mentioned above are herein incorporated by reference in their entirety.
The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/274,352, filed Nov. 1, 2021, the content of which is hereby incorporated by reference in its entirety into this disclosure.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/060304 | 10/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63274352 | Nov 2021 | US |
