阅读说明：本技术 监测与控制用于最佳离子发射的带电液滴的组成的系统 (System for monitoring and controlling the composition of charged droplets for optimal ion emission ) 是由 T·R·科维 刘畅 S·A·泰特 于 2020-02-03 设计创作，主要内容包括：一种产生带电液滴的装置,所述带电液滴的组成被优化以通过电喷雾产生离子,所述装置包括：输送装置,其可操作以将样品组分从液体样品输送到处理室；通过所述处理室的流动的液体流,所述样品沉积到所述处理室中；控制器机构,其可操作以控制输送的样品的量；输送管,通过所述输送管将含有所述样品的所述流动液体引导到电喷雾发射器,所述电喷雾发射器在所述出口处具有高电场；膨胀气体流围绕所述电喷雾发射器,在所述出口处产生压降；以及质谱仪,其用于测量从所述发射器散发的所述带电液滴产生的离子的数量；其中所述样品在所述处理室和传输流体中的稀释度为100至10,000倍。(An apparatus for generating charged droplets whose composition is optimized for generating ions by electrospray, comprising: a delivery device operable to deliver sample components from the liquid sample to the process chamber; a flowing liquid stream through the process chamber into which the sample is deposited; a controller mechanism operable to control the amount of sample delivered; a delivery tube through which the flowing liquid containing the sample is directed to an electrospray emitter having a high electric field at the outlet; an expanding gas stream surrounding the electrospray emitter creating a pressure drop at the outlet; and a mass spectrometer for measuring the number of ions generated from the charged droplets emanating from the emitter; wherein the dilution of the sample in the process chamber and transport fluid is 100 to 10,000 times.)

1. An apparatus for generating charged droplets whose composition is optimized for generating ions by electrospray, comprising:

a sample delivery device operative to transfer a sample component from a liquid sample to a process chamber,

the flow of the flowing liquid through the process chamber where the sample is deposited,

a controller operable to control the amount of sample transferred,

a transport tube through which a flowing liquid containing a sample is directed to an electrospray emitter having a high electric field at an outlet,

the expanding gas flow around the electrospray emitter creates a pressure drop at the outlet, and,

a mass spectrometer for measuring the number of ions generated from the charged droplets emanating from the emitter;

wherein the dilution of the sample in the process chamber and the transport fluid is between 100 and 10,000 times.

2. The device of any one of the preceding claims, which varies the volume of sample droplets directed into a process chamber.

3. The device of any one of the preceding claims, which varies the frequency of sample droplet generation directed into a process chamber.

4. The device of any one of the preceding claims, which varies the amount of sample introduced into the process chamber from the solid surface by controlling the time spent by the solid surface in the liquid sample.

5. The device of any one of the preceding claims, which varies the amount of sample introduced into the process chamber from the solid surface by controlling the time the solid surface spends in the process fluid.

6. The device of any one of the preceding claims, which varies the amount of sample introduced into the process chamber from the solid surface by controlling the composition of the process fluid in contact with the solid surface in the process chamber.

7. The device of any one of the preceding claims, which varies the flow rate of fluid in the treatment chamber.

8. Apparatus according to any preceding claim which varies the flow rate of atomising gas.

9. The apparatus of any preceding claim, further operative to determine a relationship between the amount of sample injected and the signal generated in the mass spectrometer and compare it to a known normal relationship.

10. The device of claim 9, further operative to adjust the amount of sample introduced into the process chamber to another value if the relationship between the amount of sample and the signal is different from normal.

11. The device of claim 9, further operative to adjust the sample droplet frequency to another value if the relationship between sample amount and signal is different from normal.

12. The apparatus of claim 9, further operative to adjust the transmission flow rate to another value if the relationship between the sample volume and the signal is different from normal.

13. Apparatus according to claim 9 further operative to adjust the atomising gas flow to another value if the relationship between the sample volume and the signal is different from normal.

14. The apparatus of claim 9, further operative to adjust an amount of time that the solid sample is in contact with the fluid in the process chamber to another value if the relationship between the sample amount and the signal is different from normal.

15. The apparatus of claim 9, further operative to adjust the composition of the solvent in the process chamber in contact with the solid sample to another value if the relationship between the amount of sample and the signal is different from normal.

16. A method for adjusting the composition of a charged droplet that produces gas phase ions to compensate for a sample whose composition is outside the range of compositions required for optimal ion generation, by:

a droplet of the sample is generated from the liquid sample,

introducing the sample droplet into a flowing liquid stream,

diluting the sample droplet in the flowing liquid stream by a factor of 100 to 10,000, an

Introducing the flowing liquid stream and the diluted sample droplets into an electrospray ionization mass spectrometer to obtain a signal representative of a composition of the sample.

17. The method of claim 16, which increases the amount of sample introduced by increasing the sample droplet volume and determines the relationship between the amount of sample introduced and the mass spectrometer signal.

18. The method of claim 16, which determines the relationship between the amount of sample introduced and the mass spectrometer signal by increasing the frequency of sample droplet introduction to increase the amount of sample introduced.

19. A method according to claim 17 or 18 which compares the relationship between the amount of sample introduced and the mass spectrometer signal with a calibration curve of predetermined sample amount versus signal under solution composition conditions for ideal ion generation from charged droplets.

20. The method of claim 16, which reduces the amount of sample introduced by reducing the droplet volume until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of sample amount versus signal.

21. The method of claim 16, which reduces the amount of sample introduced by reducing the frequency of sample droplet introduction until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of sample amount versus signal.

22. The method of claim 16, wherein the transport fluid flow rate is increased until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of sample amount versus signal.

Technical Field

The present invention relates generally to sample analysis and methods, and more particularly to those measured by electrospray mass spectrometry.

Background

The generation of ions from liquid solutions by electrospray processing has been a successful method of sample introduction for measurement by mass spectrometry. Using this approach, organic and inorganic molecules and atoms in liquid solutions from a wide range of chemical species can be converted into unbroken gas phase ions. Ion generation is a very efficient low detection limit that enables both molecular mass determination and quantitative measurement of the number of such molecules under the correct conditions.

However, electrospray mass spectrometry of biological and other samples can be slow, complex, and sometimes prohibited by the presence of high concentrations of solutes in the sample, which consist of endogenous compounds, and in some cases the analyte itself. In these cases, the high concentration of solute results in a condition known as "ion suppression" in which the electrospray process is ineffective in completely converting the incoming liquid solution into unbroken gas phase ions. Ion suppression is a key reason for the risk of largely preventing direct analysis of biological samples without the need for sample preparation to dilute and purify the biological sample and contamination of the mass spectrometer.

There is a need for systems and methods that allow analysis of biological and other samples while reducing initial sample preparation and purification requirements prior to introduction into an analysis device.

Disclosure of Invention

In one embodiment, a system and method for: i) detecting when conditions in the ion emitting droplet cause ion suppression to occur; and ii) correcting for ion suppression by adjusting the concentration of solutes in the droplets to reduce or eliminate ion suppression. In some aspects, systems and methods are provided to adjust the solute concentration in ion-emitting droplets to ensure that the analytical response of a receiving analytical device is within the linear dynamic range region of the analytical device. In some aspects, the systems and methods are operable to perform detection and correction in a near real-time manner without operator intervention. In some aspects, the systems and methods are operable to perform detection and apply corrections to the system in less than 5 seconds, and more preferably in less than 1-2 seconds.

In one embodiment, a system and method for: i) detecting when conditions in the ion emitting droplet cause ion suppression to occur; and ii) correcting ion suppression by evaluating a detected reference signal of the reference standard included in the ion emitting droplet based on an expected reference signal of the reference standard and adjusting a detected analyte signal of an analyte in the ion emitting droplet based on the evaluation.

In one embodiment, a system and method for detecting when conditions in an ion-emitting droplet cause ion suppression to occur is provided by evaluating a detected reference signal of a reference standard included in the ion-emitting droplet based on an expected reference signal of the reference standard and identifying ion suppression when the detected reference signal deviates from the expected reference signal. In some aspects, the systems and methods are further operable to correct for detected ion suppression by adjusting a detected analyte signal of an analyte in the ion emitting droplet based on a deviation of the detected reference signal from the expected reference signal.

In one embodiment, an apparatus for generating charged droplets is described, the composition of which is optimized for generating ions by electrospray. The device includes: a sample delivery device operable to deliver a sample component from a liquid sample to a process chamber defining a process zone for the sample process component; a flowing liquid stream through the process chamber in which the sample is deposited; a controller operable to control the amount of sample delivered; a transport tube through which a flowing liquid containing a sample is directed to an electrospray emitter having a high electric field at an outlet; an expanding gas flow around the electrospray emitter, which creates a pressure drop at the outlet; and a mass spectrometer for measuring the number of ions generated from the charged droplets emanating from the emitter; wherein the dilution of the sample in the process chamber and the transport fluid is 100 to 10,000 times.

In some embodiments, the device varies the volume of sample droplets directed into the process chamber.

In some embodiments, the device varies the frequency of sample droplet generation directed into the process chamber.

In some embodiments, the device varies the amount of sample introduced into the process chamber from the solid surface by controlling the time the solid surface spends in the liquid sample.

In some embodiments, the device varies the amount of sample introduced into the process chamber from the solid surface by controlling the time the solid surface spends in the process fluid.

In some embodiments, the device alters the amount of sample introduced into the process chamber from the solid surface by controlling the composition of the process fluid in contact with the solid surface in the process chamber.

In some embodiments, the device varies the flow rate of fluid in the process chamber.

In some embodiments, the means varies the flow rate of the atomizing gas.

In some embodiments, the device is further operable to determine a relationship between the amount of sample injected and the signal generated in the mass spectrometer and compare it to a known normal relationship.

In some embodiments, the device is operable to adjust the amount of sample introduced into the process chamber to another value if the relationship between the amount of sample and the signal is different from normal.

In some embodiments, the device is operable to adjust the sample droplet frequency to another value if the relationship between sample amount and signal is different from normal.

In some embodiments, the device is operable to adjust the delivery flow rate to another value if the relationship between the sample volume and the signal is different from normal.

In some embodiments, the device is operable to adjust the atomising gas flow to another value if the relationship between the sample volume and the signal is different from normal.

In some embodiments, the device is further operable to adjust the amount of time that the solid sample is in contact with the fluid in the process chamber to another value if the relationship between the sample amount and the signal is different than normal.

In some embodiments, the device is further operable to adjust the composition of the solvent in the process chamber that is in contact with the solid sample to another value if the relationship between the amount of sample and the signal is different from normal.

In some embodiments, a method is described for adjusting the composition of a charged droplet that produces gas phase ions to compensate for samples whose composition is outside the range of those samples (boundary) required for optimal ion generation, the method comprising: generating sample droplets from a liquid sample, introducing the sample droplets into a flowing liquid stream, diluting the sample droplets in the flowing liquid stream by a factor of 100 to 10,000, and introducing the flowing liquid stream and the diluted sample droplets into an electrospray ionization mass spectrometer to obtain a signal representative of a component of a sample.

In some embodiments, the method increases the amount of sample introduced by increasing the sample droplet volume and determines the relationship between the amount of sample introduced and the mass spectrometer signal.

In some embodiments, the method increases the amount of sample introduced by increasing the frequency of sample droplet introduction, which determines the relationship between the amount of sample introduced and the mass spectrometer signal.

In some embodiments, the method compares the relationship between the amount of sample introduced and the mass spectrometer signal to a calibration curve of predetermined amount of sample versus signal under solution composition conditions for ideal ion generation from charged droplets.

In some embodiments, the method reduces the amount of sample introduced by reducing the droplet volume until the relationship between the amount of sample introduced and the signal of the mass spectrometer is equivalent to the relationship of an ideal calibration curve of the amount of sample versus the signal.

In some embodiments, the method reduces the amount of sample introduced by reducing the frequency of sample droplet introduction until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of sample amount versus signal.

In some embodiments, the method increases the transport fluid flow rate until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of the amount of sample versus the signal.

These and other embodiments are contemplated by the appended claims.

Drawings

Fig. 1A is a system diagram illustrating an embodiment of a mass analysis system.

FIG. 1B is a block diagram illustrating an embodiment of computing resources for booting a quality analysis system.

Fig. 2 is an idealized graph showing the relationship between electrospray sample concentration and mass analyzer response.

Fig. 3 is a simplified diagram showing the relationship between electrospray sample concentration and mass analyzer response.

FIG. 4 is an exemplary embodiment of an apparatus according to the present teachings.

Fig. 5A, 5B, and 5C depict various geographical relationships of components of an apparatus according to the present teachings.

FIG. 6 depicts various embodiments of sample processing regions according to the present teachings.

Fig. 7 depicts various embodiments of a sample introduction method.

Fig. 8 depicts a sequence of events that result in the generation of ions by electrospray.

Figure 9 depicts droplet conditions for different regions.

Fig. 10 is a comparative graph of various verapamil analyses.

FIG. 11 is a data plot showing detection and correction of inhibition in the broth medium.

Fig. 12A is a graph of ion signal during a sampling run, showing the real-time nature of the process.

Fig. 12B is a close-up view of some of the data in fig. 12A.

Fig. 13 is a simplified schematic diagram illustrating an embodiment of a system for detecting ion suppression.

Fig. 14 is a diagram illustrating an embodiment of a method for correcting ion suppression in analysis results.

Detailed Description

In some embodiments, a system for analyzing a sample is provided. In some aspects, the system may be operable to generate charged sample droplets containing sample components for analysis, and to optimize the composition of the generated charged sample droplets to generate sample ions by electrospray.

In some embodiments, a system for analyzing a sample material is provided. The system includes a sample transport component for transporting a measured volume of sample from a sample material to a process chamber. The flowing liquid stream flowing through the process chamber receives and captures the delivered measurement volume of sample. A delivery conduit is provided to deliver a flowing stream of liquid and captured sample to an electrospray emitter having a high electric field at the discharge end. The flowing liquid stream includes a solvent for diluting the captured sample. In some aspects, the systems and methods are operable to dilute the captured sample by a factor of 100 to 10,000. The electrospray emitter is operable to discharge a flowing stream of liquid and diluted sample at a discharge end in the form of charged sample droplets. The expanding gas stream is directed to surround the discharge end of the electrospray emitter, creating a pressure drop at the discharge end and converting the charged sample droplets into sample ions. A mass analysis device is operable to receive and analyze the sample ions and to generate an analysis result representative of the transported sample.

In some aspects, the system further comprises a sample delivery control component for controlling the measured volume of the sample delivered to the process chamber.

In some aspects, the system is further operable to perform a plurality of sampling runs on the sample material, and vary the amount of sample delivered to the process chamber for each sampling run of the plurality of sampling runs. The system is further operable to evaluate the analysis results for the plurality of sample volumes and compare the evaluated analysis results to the expected relationship. If the evaluated analysis results match the expected relationship, the analysis result corresponding to the largest volume of sample delivered for the plurality of sampling runs is identified as the optimal analysis result. If the evaluated analysis result does not match the expected relationship, an additional sampling run is performed that delivers a sample of a smaller volume to generate an additional analysis result representative of the smaller volume, and the additional analysis results are evaluated and compared until the additional analysis result matches the expected relationship and the sample that delivers the largest volume for the additional analysis run is identified as the optimal analysis result.

In some embodiments, the system is operable to vary the volume of the delivered sample by varying the volume of each sample delivered to the process chamber. In some embodiments, the system is operable to vary the volume of delivered samples by varying the frequency of each sample delivered to the process chamber, such that multiple delivered samples can be combined in the flowing liquid stream for higher frequency sample delivery to produce a higher concentration of sample delivered to the electrospray emitter.

In some embodiments, the sample material comprises a liquid sample material and the measurement volume of the sample from the sample material comprises a sample droplet ejected from the liquid sample material.

In some embodiments, the sample material comprises a solid sample material, and the measured volume of the sample from the sample material can be varied by controlling the immersion time for which the solid sample material remains immersed in the solvent. In some aspects, the solvent comprises a liquid flowing through the processing chamber.

In some embodiments, the measurement volume of the sample can be varied by controlling the composition of the flowing liquid stream that is in contact with the sample material in the process chamber. In some embodiments, the measurement volume of the sample can be varied by controlling the flow rate of the flowing liquid stream in contact with the sample material in the process chamber.

In some embodiments, the system is operable to vary the flow rate of the atomizing gas based on the analysis results.

In some embodiments, the system is further operable to determine a relationship between the amount of sample injected and the signal generated in the mass spectrometer in response to the received sample ions and compare it to a known normal (i.e. expected) relationship. In some aspects, the system is further operable to adjust the amount of sample delivered to the process chamber if the signal does not match a known normal relationship. In some aspects, the system is further operable to adjust the frequency of the sample delivered to the process chamber if the signal does not match a known normal relationship. In some aspects, the sample comprises a liquid sample droplet, and wherein the system is further operative to adjust the frequency of delivery of the liquid sample droplet to the process chamber if the signal does not match a known normal relationship. In some aspects, the system further operates to adjust the flow rate of the liquid stream if the signal does not match a known normal relationship. In some aspects, the system is further operable to adjust the atomizing gas flow rate if the signal does not match a known normal relationship. In some aspects, the system is further operable to adjust the amount of time that the solid sample material is in contact with the flowing fluid stream in the process chamber to another value if the signal does not match a known normal relationship. In some aspects, the system is further operable to adjust the composition of the flowing liquid stream flowing through the process chamber in contact with the solid sample to another if the signal does not match a known normal relationship. In some aspects, the flowing liquid stream comprises a solvent and the composition comprises at least one of a concentration, a temperature, a solvent type, or an additive to the solvent.

In some embodiments, there is provided a method for adjusting the composition of charged droplets producing gas phase ions to compensate for samples having a composition outside of the range of compositions required for optimal ion generation by: generating sample droplets from a liquid sample, introducing the sample droplets into a flowing liquid stream, diluting the sample droplets in the flowing liquid stream by a factor of 100 to 10,000, and introducing the flowing liquid stream and the diluted sample droplets into an electrospray ionization mass spectrometer to obtain a signal representative of a component of a sample.

In some aspects, the method increases the amount of sample introduced by increasing the sample droplet volume and determines a relationship between the amount of sample introduced and the mass spectrometer signal. In some aspects, the method increases the amount of sample introduced by increasing the frequency of sample droplet introduction, which determines the relationship between the amount of sample introduced and the mass spectrometer signal. In some aspects, the method compares the relationship between the amount of sample introduced and the mass spectrometer signal to a calibration curve of predetermined sample amount versus signal under solution composition conditions for ideal ion generation from charged droplets. In some aspects, the method reduces the amount of sample introduced by reducing the droplet volume until the relationship between the amount of sample introduced and the signal of the mass spectrometer is equivalent to the relationship of an ideal calibration curve of the amount of sample versus the signal. In some aspects, the method reduces the amount of sample introduced by reducing the frequency of sample droplet introduction until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of sample amount versus signal. In some aspects, the method increases the transport fluid flow rate until the relationship between the amount of sample introduced and the mass spectrometer signal is equivalent to the relationship of an ideal calibration curve of the amount of sample versus the signal.

In some embodiments, systems and methods for analyzing eluents are provided. In some aspects, the eluent can be delivered from the liquid separator as a metered amount that is delivered continuously during the separation period. In some aspects, the systems and methods include: processing a sample material in a liquid separator having an inlet for receiving the sample material and an outlet for eluting separated components of the sample material; delivering the eluted separated components of the sample material as a sample to a process chamber; a flowing liquid stream flowing through the process chamber, receiving and capturing the delivered sample. A delivery conduit is provided to deliver a flowing stream of liquid and captured sample to an electrospray emitter having a high electric field at the discharge end. The flowing liquid stream includes a solvent for diluting the captured sample. In some aspects, the dilution is in the range of 100 to 10,000 fold. The electrospray emitter is operable to discharge a flowing stream of liquid and diluted sample at a discharge end in the form of charged sample droplets. The expanding gas stream is directed to surround the discharge end of the electrospray emitter, creating a pressure drop at the discharge end and converting the charged sample droplets into sample ions. A mass analysis device is operable to receive and analyze the sample ions and to generate an analysis result representative of the transported sample.

In some aspects, the liquid separator comprises a Liquid Chromatograph (LC) device. In some aspects, the liquid separator comprises a Capillary Electrophoresis (CE) device.

In some aspects, the dilution factor provided by the flowing liquid stream may be pre-calculated based on the expected eluent flow rate from the liquid separator and the composition of the eluent.

In some aspects of the systems and methods, the system is further operable to adjust a flow rate of at least one of the eluted separated components of the sample material and the flowing liquid stream and compare analysis results at different flow rates to a known relationship. In some aspects, if the analysis result does not match the known relationship, the system is further operable to adjust the flow rate of at least one of the eluted separated components of the sample material and/or the flow rate of the flowing liquid stream until the analysis result matches the known relationship. In some aspects, if the analysis result does not match the known relationship, the system may be further operable to decrease the flow rate of at least one of the eluted separated components of the sample material and/or increase the flow rate of the flowing liquid stream until the analysis result matches the known relationship.

Fig. 1A illustrates an exemplary mass analysis system 100 in accordance with various embodiments of the present teachings. The mass analysis system 100 is an electromechanical instrument for separating and detecting ions of interest from a given sample. The quality analysis system 100 includes computing resources 130 to perform control of system components and to receive and manage data generated by the quality analysis system 100. In the embodiment of FIG. 1A, the computing resources 130 are shown as having separate modules: a controller 135 for directing and controlling system components, and a data handler (data handler)140 for receiving and compiling data reports of detected ions of interest. The computing resources 130 may include more or fewer modules than depicted, depending on the requirements, may be centralized, or may be distributed across system components, depending on the requirements. Typically, the detected ion signals generated by the ion detector 125 are formatted in the form of one or more mass spectra based on control information and other process information for various system components. Subsequent data analysis can then be performed on the data report (e.g., mass spectrum) using a data analyzer (not shown in fig. 1A) in order to interpret the results of the mass analysis performed by the mass analysis system 100.

In some embodiments, the mass analysis system 100 may include some or all of the components shown in fig. 1A. For purposes of this application, the mass analysis system 100 can be considered to include all of the illustrated components, but the computing resource 130 may not have direct control or provide data processing to the sample separation/transport component 105.

In the context of the present application, the separation/delivery system 105 includes a delivery system capable of delivering a measurable amount of sample (typically a combination of analyte and accompanying solvent sample fluid) to an ion source 115 disposed downstream of the separation system 105 to ionize the delivered sample. Mass analyzer 120 receives the generated ions from ion source 115 for mass analysis. The mass analyzer 120 is operable to selectively separate ions of interest from generated ions received from the ion source 115 and to deliver the ions of interest to the ion detector 125, which generates a mass spectrometer signal indicative of the detected ions to the data handler 140.

It will also be appreciated that the ion source 115 may have a variety of configurations known in the art. The present application relates generally to ionization sources that operate by ionizing a sample in the form of droplets, such as electrospray processing.

For purposes of this application, the components of the mass analysis system 100 can be considered to operate as a single system. In general, the combination of the mass analyzer 120 and the ion detector 125 and the associated components of the controller 135 and the data handler 140 are commonly referred to as a mass spectrometer, and the sample separation/transport device may be considered a separate component. It should be understood, however, that while some components may be considered "isolated," such as the separation system 105, all components of the mass analysis system 100 operate in concert for analysis of a given sample.

Fig. 1B is a block diagram illustrating an exemplary computing resource 130 on which embodiments of the present teachings, including the quality analysis system 100, may be implemented. The computing resources 130 may comprise a single computing device, or may comprise a plurality of distributed computing devices in operative communication with the components of the quality analysis system 100. In this example, computing resource 130 includes a bus 152 or other communication mechanism for communicating information, and at least one processing element 150 coupled with bus 152 for processing information. As will be appreciated, the at least one processing element 150 may include multiple processing elements or cores, which may be packaged as a single processor or in a distributed arrangement. Further, in some embodiments, multiple virtual processing elements 150 may be provided to provide control or management operations for the quality analysis system 100.

The computing resources 130 also include volatile memory 150, which may be Random Access Memory (RAM) as shown or other dynamic memory component, coupled to the bus 152 for use by the at least one processing element 150. The computing resources 130 may further include static non-volatile memory 160, such as a Read Only Memory (ROM) as shown or other static memory component, coupled to the bus 152 for storing information and instructions used by the at least one processing element 150. A storage component 165, such as a storage disk or storage memory, is provided and coupled to bus 152 as shown for storing information and instructions used by at least one processing element 150. As will be appreciated, in some embodiments, storage component 165 may comprise a distributed storage component, such as a networked disk or other storage resource available to computing resource 130.

Optionally, computing resource 130 may be coupled via bus 152 to display 170 for displaying information to a computer user. An optional user input device 175, such as a keyboard, may be coupled to bus 152 for communicating information and command selections to the at least one processing element 150. An optional graphical input device 180, such as a mouse, a trackball, or cursor direction keys, is used to communicate graphical user interface information and command selections to the at least one processing element 150. As shown, the computing resources 130 may also include input/output (I/O) components 185, such as serial connections, digital connections, network connections, or other input/output components to allow for intercommunication with other computing components and various components of the quality analysis system 100.

In various embodiments, computing resources 130 may be connected to one or more other computer systems or networks to form a networked system. The network may comprise a private network or a public network such as the internet. In a networked system, one or more computer systems may store data and provide the data to other computer systems. In a cloud computing scenario, the one or more computer systems that store and provide data may be referred to as a server or a cloud. For example, one or more computer systems may include one or more web servers. For example, other computer systems that send and receive data to and from a server or cloud may be referred to as clients or cloud devices. The operation of the distributed computing system may support various operations of the quality analysis system 100.

The computing resource 130 is operable to control the operation of the components of the quality analysis system 100 by the controller 135 and process data generated by the components of the quality analysis system 100 by the data handler 140. In some embodiments, the analysis results are provided by the computing resources 130 in response to the at least one processing element 150 executing instructions contained in memory 160 or 165 and performing operations on data received from the quality analysis system 100. Execution of the instructions contained in the memory 155, 160, 165 by the at least one processing element 150 causes the mass analysis system 100 to be operable to perform the methods described herein. Alternatively, hardwired circuitry may be used in place of or in combination with software instructions to implement the teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

According to various embodiments, instructions configured to be executed by the processing element 150 to perform a method or to make the quality analysis system 100 operable to implement the method are stored on a non-transitory computer-readable medium accessible to the processing element 150.

In some embodiments, systems and methods are described that dynamically measure and adjust physicochemical conditions in charged droplets to optimize gas phase ion generation for electrospray mass spectrometry. Non-optimal ion generation is commonly referred to as ionization suppression. The mass analysis system 100 is operable to detect when ionization suppression occurs, i.e., when the composition of highly charged nano-droplets generated during an electrospray process operating on a delivered sample begins to limit the ion generation rate and introduces nonlinearity in the relationship between the analyte concentration in the sample and the response signal generated by the mass analysis system 100.

In some embodiments, the systems and methods are operable to detect when ions generated by an ionization source are suppressed beyond a predetermined threshold or shut off completely due to conditions in the droplet. The systems and methods may include an alarm condition indicative of detected ion suppression or lack of ion generation, which may be presented in association with mass analysis results produced by the mass analysis system 100.

In some embodiments, the systems and methods are operable to introduce corrective measures to modify the composition of droplets produced in the ionization source during the electrospray process to return the operation of the ionization source to conditions in which a linear relationship between the concentration of the delivered sample and the response produced by the mass analysis system 100 is achieved.

The high concentration of analyte provided in the delivered sample may introduce non-linearity in the quantification process. High concentrations of endogenous substances in the delivered sample, such as those from biological and other sources, particularly those with surface active properties, crowd the surface of the charged droplets produced by the electrospray process, which inhibits or prevents the release of lower concentrations of ions of interest that may be trapped in the internal bulk fluid (bulk fluid). High concentrations of endogenous material in the delivered sample will result in the formation of solid charged residues that effectively capture molecular components of the sample in the droplets and prevent gas phase ion production of those captured molecular components. Due to sub-optimal ion generation, the mass analysis system will not be able to accurately detect and/or characterize all components of the delivered sample, a phenomenon commonly referred to as ion suppression.

Because of this problem, it is standard practice to perform extensive sample preparation prior to delivery in order to ensure that the concentration of endogenous material in the delivered sample does not result in ion suppression during the ionization process due to high solute concentrations. The standard procedure is also to perform sample preparation prior to delivery to purify the sample and remove matrix components that may cause a "matrix effect", i.e. ion suppression due to the nature of the matrix components.

In an embodiment, a system and method for receiving an undoped sample and automatically adjusting the composition of the sample delivered to an ionization source to ensure that ion suppression does not occur during ionization by the ionization source is provided.

In some embodiments, the systems and methods are operable to adjust the solute concentration in the delivered sample prior to delivery to the ionization source such that droplets produced by the ionization source have a sufficiently low solute concentration to avoid ion suppression. In these cases, a droplet with a sufficiently low concentration of solute is provided to a low concentration of analyte proximate to the droplet surface such that a high electric field at the droplet surface induces field ion emission of charged molecules of the low concentration of analyte from the liquid phase to the gas phase for mass spectrometry analysis. In some aspects, the process of detecting and correcting for ionization suppression may occur in a near real-time manner that consumes low nanoliter volumes of sample. Thus, these systems and methods avoid the need to manually dilute and purify the sample prior to introduction into the sample transport component of an electrospray ionization mass spectrometer.

For any chemical measuring device, it is crucial to be able to provide a response that is reproducibly proportional to the amount of material being measured to obtain an accurate and precise determination of the amount of material present. In the case of mass spectrometry, the relationship between ion count at the mass spectrometer detector and the gravimetric mass of the analyte in the electrospray ionized sample has been extensively studied and empirically determined to have a mass of about 10³To 10⁴Linear dynamic range of (2). As the sample concentration becomes lower, the linearity of the relationship is maintained, i.e. the number of molecules available for analysis in the sample is lower, with no limitation on the dynamic range, except that too few molecules are present to be detected. This means that as ion generation, transmission and detection become more efficient (improved sensitivity and signal-to-noise ratio), the linear dynamic range is also improved. Ionization suppression imposes a limit on the ionization process only at the high end of the linear dynamic range.

The fundamental limitation on linear dynamic range is imposed by high concentrations of analytes in the sample solution and/or high concentrations of other foreign chemical species, commonly referred to in the biological disciplines as endogenous or matrix components. This is a result of both the modification of colligative and chemical properties of the fluid entering the ion source in the sample, and both of these reasons are referred to as ionization suppression in terms of impact on the response of the mass analysis system.

Figure 2 is a simplified graph showing a classical understanding of electrospray sample concentration versus signal response for a purified sample fluid without matrix components. FIG. 2 shows an idealized relationship to illustrate that at low analyte concentrations, typically below about 10 in a relatively pure sample (region A)^-5M, the signal response to sample concentration is linear. The linear relationship typically maintains analyte concentrations of about 3 to 4 orders of magnitude, depending on the minimum level of detectable analyte concentration defined by the lowest detection limit of the system. The lower the limit of detection (LoD), the measurableThe wider the linear dynamic range. Thus, the effective linear dynamic range of the mass analysis system can be broadened and extended to the lower quantitative limit (LoQ) where the efficiency of ion transport to the system detector is increased. Up to 10⁴The dynamic range of (a) is obtained with a high efficiency mass spectrometer capable of detecting lower ion signals. In the limit, the sensitivity of the detector is balanced against noise, so in many cases it is still desirable to increase the ion signal to improve the overall S/N.

At a concentration of about 10 for the analyte of interest^-5M, the signal increases with increasing concentration level (region B), i.e. the slope of the signal increase versus the sample concentration decreases. In general, the relationship between signal and concentration in region B may begin to shift into a non-linear relationship as the inhibitory effect increases with increasing concentration. Due to competition with surface sites around the high field ion emitting droplet, the ion signal of the analyte does not increase linearly with a corresponding increase in the sample concentration in region B. This phenomenon can be understood from the equilibrium assignment model of Enke and experimental observations of Bruins.

When the analyte concentration or other competing compound in solution reaches about 10^-5M, the linearity reaches the balance. When the analyte concentration exceeds 10^-4M, enters the region of severe inhibition where its concentration or the concentration of other components continues to increase. Although not shown in fig. 2, in some cases, in the latter part of the region B, the response may remain constant even as the analyte concentration increases. Above about 10^-4At point M, the slope of the calibration curve becomes negative as shown in fig. 2 (region C). In this region of severe inhibition, the signal response decreases with increasing analyte concentration until, at some point, complete inhibition of the response signal occurs.

Fig. 3 is a simplified graph comparing the idealized graph of fig. 2 with a simplified mass analyzer signal response that is subject to ion suppression due to the additive effects of analyzing the sample matrix in the sample. Typically, the presence of the sample matrix will have the effect of reducing the maximum detectable ionic signal for a given sample, and may have additional inhibitory effects. This effect, known as the matrix effect, can occur when the sample typically contains greater than millimolar concentrations of non-volatile sample matrix. The sample matrix completely suppresses the signal due to the formation of solid residues. Surfactant compounds have a particularly severe inhibitory effect at sub-millimolar concentrations due to the complete shielding of the ion-emitting droplets.

Ionization suppression is intrinsically insidious, since if the composition of a sample changes, its occurrence is unpredictable between samples, which is almost always the case in biological systems. The complete composition of biological and other samples for analysis has never been fully understood a priori. Sample purification methods such as solid phase extraction, liquid-liquid extraction, and liquid chromatography can help reduce but not eliminate their occurrence. This is mainly because in order to remove the troublesome sample components, their chemistry must be understood in advance to selectively optimize the purification process.

When the analyte concentration is outside the linear dynamic range or there are extraneous sample components causing ionization suppression, it is desirable to know whether or not ionization suppression occurs when analyzing the sample, and to account for correction conditions for deviations from linear calibration. The method of doing this is primarily post-hoc, i.e., after the analysis is complete and deviations from linearity are observed, then action is taken and the analysis is repeated to see if corrections to the method improve the accuracy and precision of the analysis.

Ionization inhibition is typically addressed by using a wide range of sample pre-purification protocols including solid phase extraction, liquid-liquid partitioning, antibody affinity pull-down of target components, and high performance liquid chromatography. Determining whether the purification procedure will solve the inhibition problem requires a test experiment using a protocol and iteratively adjusting and fine-tuning the separation protocol until the inhibition problem can be proven to be eliminated. The work of King clearly describes this situation and its implications. He designed a method to determine where areas of high ionization suppression occurred in the HPLC chromatogram due to co-elution of contaminants with the analyte. The method of King bears the assistance of experienced analytical scientists who are customizing sample extraction procedures and chromatographic separation of specific analytes and biological matrices. This method is time consuming, requires expert knowledge, and is empirical in nature, however it represents the state of the art. In many application areas where expertise and time are key determinants, such as clinical hospital laboratories, these types of advanced analysis techniques can be burdensome. Moreover, these methods do not provide a way to analyze the sample directly without pre-purification, which in many cases distorts the chemical composition of the sample to be analyzed in an unknown manner.

If the sample is not sufficiently purified by HPLC or other methods, the linear dynamic range shifts to higher analyte concentrations. At 10^-5In the presence of the matrix at the concentration of M, the signal of the analyte decreases. Competition for surface sites around the high field ion emitting droplet. At concentrations greater than millimolar, the non-volatile sample matrix will completely suppress the signal due to the formation of solid residues. Surfactant compounds have a severe inhibitory effect at < millimolar due to complete shielding of the ion emitting droplets. (see, e.g., equilibrium assignment model by Enke and experimental observations by Bruins).

Although these values vary depending on the chemical nature of the analyte, the presence of other dissolved solutes, and the nature of the supporting solvent, these concentration milestones remain significantly consistent. The root cause of ionization suppression is the chemical nature of the system and its physical state. The surface tension and viscosity of the liquid and the surface activity, solubility and ionic character of the compound will vary from case to case, thereby introducing unpredictable factors into the onset of ionization suppression. The physical state of the sample defining conditions under which ion generation can occur is a constant, including the critical electric field strength and the colligative property that defines whether the system is a solid or liquid state.

The role played by the chemical and physical properties of the system in the ionization suppression phenomenon can be explained from the fundamental share of ion evaporation theory to be described. Components of the sample other than the analyte in solution will reduce the signal from the compound of interest at concentrations in the non-linear range. If the concentration of endogenous compounds is sufficiently high, eradication of all signals from the sample will occur. Compounds with surfactant properties have a dominant effect over all other chemicals.

Embodiments of the present system and method enable analysis of raw material samples that have not been modified by sample purification or chromatographic separation by assessing the degree of inhibition that occurs and taking appropriate action in a timely, real-time manner to correct the conditions of ion generation and to perform sample analysis in an automated, uninterrupted manner. To ameliorate the suppression problem, intervention in the ion generation process must have the net effect of producing optimal ion generation conditions at the stage of ion generation.

Some embodiments of the present systems and methods enable analysis of eluents produced by liquid separators without additional purification procedures. These embodiments may be useful, for example, when LC or CE buffers may be considered incompatible with mass spectrometry. For example, some buffers may contain surfactants, which can result in severe inhibition using conventional techniques.

Mechanism of ionization suppression

Three successive steps are involved in the ionization process. The first step involves charging the bulk liquid with an excess of positive or negative charges, followed by the use of an electric field or pneumatic shear forces to produce an initial aerosol of charged droplets from the liquid. When the local electric field reaches the rayleigh limit, the rapid evaporation of these droplets causes them to break down into smaller charged droplets (step 2). As their size is further reduced to tens of nanometers in diameter, the electric field at the surface exceeds the solvation energy of the compounds in solution, thereby expelling or "evaporating" ions into the gas phase (step 3).

The proper method comprises the following steps: examining the mechanism by which each is driven in detail, and whether or not it is possible to straighten out high concentrations of sample components can alter the course of each in a way that causes ionization suppression.

Phase 1. bulk fluid is charged and initially charged droplets are produced.

Bulk fluid charging and subsequent droplet charging is a process similar to that of an electrolytic cell. When an electric field is generated between the anode and cathode by a power source that transports or removes electrons from the electrodes, ions are formed in large numbers, migrating in the solution bridging them. In the special case of electrospray, there is no continuous fluid between the electrodes, but rather an air gap through which the charge in the form of ions in the droplets must jump. The charge migrates to the anode or cathode within charged droplets containing excess charge that migrates through the air rather than through the continuous fluid between them. The entrance of the mass spectrometer ion optics is selected to be the anode or cathode depending on the polarity of the ions required for analysis and is the location where the vacuum system draws a portion of the migrating ions into the mass analyser. The other is typically a tube through which the liquid flows and from which droplets are produced. The small outer diameter of the tube helps to generate a high electric field from the applied voltage.

To generate excess positive charge in the liquid, a positive voltage, typically a few thousand volts, is applied relative to the mass spectrometer inlet, which extracts electrons and causes oxidation reactions at the metal-liquid interface. For example, water is oxidized into oxygen and hydrogen ions (protons). To produce an excess negative charge, a negative potential is applied and a reduction reaction occurs, such as the reduction of water to hydrogen and hydroxide ions. Electrolytic methods that result in excess charge generation in bulk solutions have been extensively studied by Van Berkel and Kebarle.

The spray current indicates the amount of charge transferred to the bulk liquid, which is typically in the low uA range. As the fluid flow rate increases, the spray current increases approximately proportionally, indicating that the charge density is the same at low and high flow rates and that the solvent has reached charge saturation. When electrolytes or other charge-carrying components, such as endogenous sample components, are added to the fluid, the spray current increases and the charge density of the droplets formed also increases. If ionization suppression occurs at this stage, a decrease in spray current will be observed. Rather, at this stage, the generation of charged droplets is improved by adding a charge carrying component (which may take the form of a salt or any ionizable organic molecule) to the sample. This and other observations provide conclusive evidence that ionization suppression is not caused by a reduction in the charge transferred to the liquid during this initial charged droplet formation phase. In fact, only in a minimal case, the composition of the sample will affect ion generation by inhibiting formation in the first stage of the ion generation process.

Of bulk liquidConductive charging. When a sufficiently high electric field is concentrated at the surface of the liquid, charged droplets are emitted from the protrusions formed at the surface of the location where the field is maximal and travel through the air to the counter electrode of the field. This point of emission from the surface droplet is known as the taylor cone. It is commonly referred to in the art of mass spectrometry that this method is used to generate ions as electrospray ionization.

Of neutral dropletsInductive charging. Some droplets separated from the bulk liquid by forces such as pneumatic atomization, piezoelectricity, or acoustic dispensing have a small net charge during the droplet formation process through statistical fluctuations in the number of charge carriers in different regions of the bulk solution. By this mechanism, gas phase ions are formed in the form of clouds or near waterfalls, and research into this process has led to the elucidation of IE models for ion generation that use an ion mobility analyzer to roughly determine the size of the ions produced. The number of droplets with a net charge can be increased by causing them to drift through a strong electric field generated by a grid or lens at atmospheric pressure. The grid polarizes the charge in the droplets, which when broken down form more droplets with a net charge through this induction process. The method is used for verifying theoretically obtained IE models by using a mass spectrometer to identify generated ions, and then performing prototype analysis as an LC/MS interface. Subsequently, when conductive charging of a liquid is introduced with electrospray and ion spray, the difference in ion generation efficiency between the inductive charging method and the conductive charging method becomes significant. The type of ions produced is the same because the mechanism of ion generation is the same for all three, but the average charge per initial droplet and the number of droplets with any charge are found to be lower than charging by conduction.

Triboelectric charging of the bulk liquid can be performed without a power source by using the triboelectric effect (which is a kind of triboelectric charging) before the formation of the droplets. In this case, the liquid passes through the surface of the metal conductor (small-bore steel pipe) and is accelerated by the very high velocity gas at the outlet of the pipe. Molecules in solution instantaneously attach to the electrode and exchange charge during this time, which can result in electron loss to the electrode when the molecules are swept away by the high velocity gas before recombination can occur. The net effect may be to generate a high charge density spray similar to the conductive charging method but more difficult to control. A common term for this method is sonic spraying.

Shortly after the bulk liquid is charged, initially charged droplets are formed. The energy gained from the electric field is limited by the electrical breakdown of the surrounding gas at atmospheric pressure. The energy available to generate an aerosol by this method was determined by taylor in his initial paper on the subject, where he described the formation of a fluid cone emanating from the bulk liquid at a point in the field most commonly referred to as the "taylor cone". The cone dispenses charged droplets from its apex with average diameters ranging from microns to slightly sub-microns.

The energy available based on Taylor's equation compared to the surface tension/viscosity of common solvents accounts even for a solution containing 10^-3Those solvents for M solute should also have sufficient energy to generate droplets from even the worst ion-suppressed samples, indicating that this is not where ion suppression is effective.

The energy from the electric field is sufficient to disperse into commonly used solvents such as water and alcohol aerosols, even when their viscosity is mostly at 10^-3The matrix compounds present in M are altered and therefore ionization suppression that prevents the formation of initially charged droplets cannot be expected to occur at this stage unless dissolved solutes reduce the amount of charge that can be deposited in the bulk liquid. This is not the case, as described below.

The effectiveness of this method is chopped by the maximum fluid flow by which a continuous aerosol of droplets can be maintained, which is caused by the frequency and volume limitations of the droplets produced by the electric field that must be kept below the discharge value. In practice, this is about 1uL/min or less in the flow of fluid commonly referred to as nanospray, which severely hampers its analytical applications operating at flow rates between 1-1000 uL/min. For this reason, other energy sources have been investigated to generate droplets, and pneumatic atomization has become the dominant method.

The energy available from the rapidly expanding high pressure gas is enormous and far exceeds any extreme value of the sample viscosity encountered in practice. As the gas expands, the gas accelerates, reaching sonic velocity within about 1mm of the distance from the expansion nozzle. The G force exceeds 200,000 in this region, taking into account the gas acceleration required to achieve this. Any fluid entering this region is instantaneously sheared into droplets of low micron diameter. This method is called ion spraying because it is a mixture of pure electric spraying (direct electric field droplet generation-Fenn-Gall) and ion evaporation (pneumatic droplet generation by inductive indirect charging-Thomson).

No sample conditions can have an effect on the results of the method. Ionization suppression does not play its role at this stage unless, as with the electric field atomization method, the sample composition can reduce the amount of charge deposition in the droplets, which is required for success at later stages of the ionization process.

In the present invention, the force generated by the expansion of the gas is used for a second purpose. Is the driving force to push the moving stream of fluid through the system from the droplet capture point to the charged droplet formation point.

And 2, decomposing the charged droplets. At atmospheric pressure, the charged droplets formed in stage 1 lose neutral solvent by evaporation. The rapidly increasing charge density of each droplet leads to droplet instability because the internal electric field within each droplet exceeds the ability of surface tension to keep a droplet of that size (about 1 micron in diameter) intact. Each of these droplets will have tens of thousands of charges. They will reach the rayleigh stability limit in hundreds of microseconds, at which point the drops will discharge their own excess charge in the form of smaller drops of about one tenth of the original size to be reproduced, containing the necessary amount of charge to relieve strain, in each sibling drop (the approximate hundreds of charges). This process repeats itself under the drive of continuous evaporation of the droplets and subsequent coulomb explosion. As the diameter of the droplet decreases, the electric field at the surface increases with decreasing radius of curvature. The charge density increases as the droplets cascade to ever decreasing diameters.

It is conceivable ifThe concentration of endogenous substances in the sample is sufficiently high that the process will be inhibited by the generation of solid charged residues in which the analyte is captured. However, the concentration of material to achieve this in 100nm diameter droplets is much greater than the observed 10^-4M, furthermore, no strong chemical effect was subsequently observed that allows the high surface active compounds to inhibit ionization at lower concentrations. The surface active component reduces the surface tension, making the rayleigh limit easier to achieve.

Except in extreme cases, there is no clear explanation of how the presence of endogenous materials in the sample that inhibit ion production affects this charged droplet break-up process.

Stage 3. gas phase ion production. The root cause of ionization suppression can be justified from an understanding of the basic principles of ion formation in electrospray processing. Two theories dominate the current scientific literature, the ion evaporation model (IE model) and the charged residue model (CR model). Both models share a key premise that the final event that results in the generation of gas phase or cluster ions from bulk solution occurs on charged droplets with a radius on the order of 10 nm. The effect of the foreign compound on causing an inhibitory event occurs at this stage or during the cascade of events immediately preceding it, so intervention to ameliorate the problem must have its primary effect at this stage.

The solution to the problem described here can be justified from either model, since the method involves controlling the composition of the final ions that produce the droplets and those that immediately cause it to obtain the best ionization efficiency. The IE model more readily explains the common empirically observed situation where ionization suppression occurs, particularly where chemical effects predominate, for example in the presence of surfactants. This model will be used to explain the general approach taken here. Furthermore, it is widely recognized in the scientific literature that the IE model has the greatest advantage for all compounds with molecular weights below several thousand, whereas the CR model has advantages mainly only for very large extensin molecules of several tens to hundreds of thousands of amu, where the physical size of these molecules and their associated solvent clusters is of the order of 10 nm. Since most assays are directed to compounds below several thousand molecular weights, IEM is more suitable for explaining the method of controlling ionization suppression employed in the present invention.

The release of ions from the solution phase to the gas phase requires that the main condition be met is that the local electric field in the droplet exceeds the solvation energy of the analyte molecules, thereby expelling them from the liquid. This can be calculated to occur at field strengths of 1-3V/nm, which requires droplets of about 10nm radius containing about 10 elementary charges. Droplets of this size release their internal coulombic stress by ejecting ions rather than the larger vaporized droplet ejected charged droplets when they reach the rayleigh limit.

Ions are ejected from the surface of the droplet where the electric field is concentrated around the radius. When the surface is fully occupied by analyte ions, an increase in analyte concentration will not result in an increase in the rate of evaporation of ions from the droplet. Given by 10^-5M number of molecules in solution, 5nm per ion on the surface of these droplets was calculated²The space of (a). The average organic ion or molecule with a C-C bond length of 0.15nm has a radius of about 1nm, which occupies a space of about 3 nm. The phenomenon of ionization suppression is explained by competition for space on the surface of ion-emitting droplets having a radius on the order of about 10 nanometers and an internal electric field that exceeds the solvation energy of organic molecules having one or more molecular charges.

Clusters of analyte ions and their neutral compatabilizers occur at the point where the linear dynamic range begins to level off and the molecular ion generation rate slows down as the concentration increases. Analyte ion production decreases with increasing concentration when the surface is fully occupied by endogenous matrix components. Greater than 10^-4The concentration of M ultimately leads to the most severe ion suppression performance, where no signal at all is observed from any component of the solution. This is because the droplet condition of 10nm radius is never satisfied. The evaporated droplets become a solid charged residue, trapping all available ions before reaching the ion emission diameter and field. These so-called "stars" have been observed and measured directly using new mass spectrometry scanning functions.

When the field strength at the surface exceeds the solvation free energy of a charged species in solution, they are atoms or molecules, otherwise known as ions, which are freely expelled into the surrounding gas, usually hydrogen bonded to several solvent molecules and known as ion clusters. This process supports the current understanding of the mechanism by which ions are formed during electrospray. There are some minor variations, but they all depend on small high charge density droplets from which multiple ions in solution are emitted, or if the dissolved ions are large and on the order of the droplet size, only one ion is present in the depleted droplet.

Fig. 4 depicts an embodiment of the present invention showing five main components, each having a different function. The first is a sample delivery device whereby the amount of sample delivered can be controlled. In this embodiment, sample delivery is achieved by a burst of sound waves (burst) that transfers energy to the surface of the fluid sample by ejecting a droplet of known and controllable volume. The amount of sample entering the processing region (i.e., the sample processing chamber of the sample processing component) can be varied by varying the power, frequency, or duration of the sonic pulses. By varying the amount of injection, the dilution of the sample in the treatment area is varied. Other sample transport means are contemplated, including transport of liquid samples by pneumatic or other spraying, liquid injection, liquid transfer under the influence of gravity, flowing liquid transfer, solid sample transfer by physical transport, solid sample transfer by immersion in a liquid stream, and other known means for transporting samples.

The second component is a sample processing region or chamber of the sample processing component in which the sample is received and the concentration of the sample is adjusted to be optimal for electrospray ionization. In this embodiment, the sample processing component includes a fluid delivery pump to provide fluid for sample processing and delivery. The flow rate of the transport fluid into the region may be varied with the pump to vary the degree of dilution and the rate of delivery of the sample. The volume of the sample processing region can also be varied by changing its geometry, which will affect the amount of dilution the sample will encounter. This is an effective way to increase or decrease the dilution ratio, but may require replacement of physical components or additional mechanical linkages, which may not easily accommodate real-time, rapid online modification of the dilution ratio.

The third section comprises an ionization section providing a facility for generating charged droplets from the processed sample, comprising a gas expansion region for creating a pressure drop to attract the sample from the processing region to a charged droplet generation region where a high electric field is applied. Applying a high electric field to the charged droplets converts the discharged sample droplets into sample ions. Control of this gas flow will allow the liquid flow out of the treatment zone to be varied, thereby providing an additional way to vary the degree of dilution in the treatment zone.

The fourth component is an atmospheric pressure ionization mass spectrometer for receiving sample ions, filtering the sample ions by m/z, and measuring the amount of ions generated.

The fifth component is a computer equipped with data and algorithms for interpreting the signals generated and communication links to the sample delivery device, fluid delivery pump and pressurized gas source. After measuring the signal and determining the degree of ionization suppression based on the resulting analysis, appropriate dilution of the sample is performed by adjusting the sample delivery device parameters, fluid delivery pump flow rate, and/or pressurized gas flow rate. In some aspects, if none of these actions is able to fully correct the inhibition, the system can calculate the volume required for the sample processing region to reach the desired dilution, and the sample processing region can be manually replaced.

The embodiment of fig. 4 is useful because of its speed, reproducibility, and accuracy in delivering sample droplets of known and reproducible volumes. In some aspects, this embodiment may further comprise a movement component for moving a sample well plate comprising a plurality of sample wells to position a desired sample well in alignment with the sample processing region. In some embodiments, the time required to locate a sample well and acoustically detonate into a processing chamber is on the order of tens of milliseconds per sample. Individual samples can be stacked in the transfer line between the process chamber and the ion generation site, with their time interval limited only by molecular diffusion in the solution in the conduit, typically on the order of hundreds of milliseconds in the prototype of the invention. This enables the samples to be detonated, their signals detected, compared to a reference to assess inhibition, and re-detonated by an amount to provide appropriate dilution in the process chamber in near real-time to provide the correct conditions in the ion-emitting droplets for a linear analyte response and to avoid inhibitory effects.

Fig. 5A, 5B, and 5C depict different geometric relationships between components. Fig. 5A shows a vertically upward oriented sample processing component and a vertically downward oriented charged droplet generation component. This allows the sample to be deposited in the process chamber of the processing component using gravity or other forces. Fig. 5B shows the same two compartments, which are oriented in opposite vertical directions. Fig. 5C shows two compartments in a horizontal configuration. Any angle between vertical and horizontal may be used if the sample can be introduced into the processing region and the charged droplet generation means oriented such that the ions can somehow reach the entrance aperture of the mass spectrometer.

Fig. 6(A, B, C and D) shows an additional embodiment of a sample processing region having fluid inlet and outlet tubes that are not coaxially arranged. Fig. 6(a) and 6(B) show a single tube or docked 2 tubes, linearly aligned or curved, with an opening to allow sample to enter the processing region. In some aspects, a process chamber may comprise a single tube having an aperture that exposes process fluid flowing through the tube. Fig. 6(C) shows the inlet pipe and the outlet pipe arranged in parallel with each other. Fig. 6(D) shows that the two tubes are arranged co-linearly with a gap between them to then define the resulting sample processing region. In some aspects, a flat groove surface may be provided to confine fluid by coating the surface with a hydrophobic material. In some aspects, the treatment area may not be bounded by walls and is limited only by the surface tension of the pooled liquid on the surface as it is transported between the 2 tubes. Other embodiments of the treatment zone are also contemplated, such as using 2 tubes enclosed in the treatment chamber, either close to or remote from each other. One type of processing chamber is in the form of an open tank having a supply line for supplying a processing fluid and a drain line for draining the processing fluid from the tank.

In one embodiment, the sample droplet generated by the gas pressure pulse forces the sample droplet through an aperture in the sample well into the processing region. Nanoliter volumes of droplets can be dispensed and the volume introduced into the treatment area is controlled by pressure, frequency and pulse duration. Similarly, a syringe driven by a fast response motor or a piezo-based dispenser may be used to deliver and vary the volume of sample entering the processing region. Larger volume dispensing devices, such as pipettes, may also be used, as long as the volume dispensed and the dilution ratio can be controlled in the process chamber.

Fig. 7(A, B, C and D) shows an exemplary embodiment of a method of introducing a sample by a delivery device included in the present invention, and fig. 7(a) shows the launching of droplets into a processing region using acoustic energy transmitted through a sample well and focused on the surface. A drop of sample in the low nanoliter volume range is advanced into this region. Different values of energy, frequency or burst rate may be used to vary the drop volume. Firing successive droplets at a high rate will cause them to coalesce in the treatment region before they are delivered to the ionization region. This is another way of varying the amount of sample delivered to the ionization region.

Fig. 7(B) shows the vertical orientation of the sample processing region to receive a sample droplet introduction system that operates more realistically as a fall in the direction of gravity. One approach is to have a hole in the bottom of the sample containing the well, with a diameter on the order of tens of microns, and expel the sample droplet through the hole aperture with a gas pressure pulse applied to the sample reservoir. Using this method, an array of samples can be provided to a system for analysis, e.g., of samples in microtiter well plates.

Fig. 7(B) also shows a sample delivery option where the sample is dispensed through an aperture of a tube by mechanical force, such as a syringe with a piston driven by a fast stepper motor, or by using vibration of a piezoelectric element to generate a droplet. The droplets may also be generated from the end of a tube held at a voltage such that the inlet to the process chamber is at a sufficiently different voltage to generate a high electric field between the two.

Fig. 7(C) shows the sample transported to the processing region of the solid substrate surface. Disposable sampling devices made of glass, plastic or wood, for example, can have a coating that absorbs components of the liquid sample, such as blood. High porosity beads (beads) may be adhered to these sampling devices or attached with a magnetic force that can adsorb large amounts of target analytes from a relatively large volume of sample. Control of the amount released into the processing region can be accomplished by controlling the amount of time the sample is exposed to the fluid in the processing region. Alternatively, the amount released may be controlled by varying the composition of the fluid in the treatment area. A binary pump that delivers fluid to this region can adjust the composition in a step-wise or gradual gradient manner to control the amount released and separate the matrix components from the analyte, as they will elute from the surface at different solvent compositions.

Figure 7(C) depicts another way in which control of the amount of sample deposited into the process chamber can be achieved when the sample is solid or adsorbed onto a surface. By adjusting the composition of the treatment fluid with 2 or more pumps, the elution intensity can be modified to remove unwanted components and selectively elute target compounds. This effectively reduces the total sample load to the chamber in a controlled manner. A more complex version of this method is to deliver a gradual gradient of elution solvent with composition over time, effectively minimizing sample loading during the ionization process. This has the added benefit that it is particularly important to provide some chromatographic separation of sample components when the same mass of isopyzme is present and cannot be distinguished with a mass spectrometer. All of the above can be altered in response to a signal from the mass spectrometer after comparing it to a sample known that inhibition will not occur.

Fig. 7(D) is a variation of fig. 7(C) wherein the solid sampling surface is a film or paper. One common method of sampling and storing blood for analysis is drying on paper. The paper can be used to deliver samples directly to the system and control the amount released by using variable elution times or variable elution solvent composition methods.

Other embodiments for delivering a sample are also contemplated, such as flowing a liquid sample for delivery to a process chamber by a delivery device.

Fig. 8 depicts a sequence of events resulting in the generation of ions by electrospray processing. Electrolytic charging and gas atomization of the bulk fluid produces initially charged droplets. The sample composition has no measurable effect on these processes occurring on the bulk fluid and therefore does not contribute to the inhibition of ion generation at this stage.

These droplets start to evaporate and lose the neutral solvent and other volatile components very quickly, entering the next stage of the process. At the rayleigh limit, the internal electric field in each droplet exceeds the surface tension, causing its fragmentation and satellite charged droplet generation with further evaporation and cascaded coulomb explosions to release the enthalpy stress. The stability of a droplet is related to its radius and chemical composition. At this stage of the process, the surface tension holding the droplets together is lower than the local electric field surrounding each droplet, which is generated by the internal charge and radius. The electric field force available at the rayleigh limit is greatly exceeded and due to the stability of the droplet surface of the dissolved component at 10^-5M concentrations and higher increase their viscosity and surface active properties. Thus, the suppression effect is not due to interference in this coulomb explosion phase of the droplet size reduction process.

FIG. 9(A, B and C) shows the physical state of the ion-emitting droplet during the linear (region A), non-linear (region B) and suppressed portions (region C) of the dynamic range curve in FIG. 2, in the first case the signal scales linearly with the amount, the droplet radius ≦ 10nm, and the electric field at the surface ≦ 10⁹V/m, the surface sites are empty and are occupied as the solute concentration increases. In the nonlinear condition, the surface is crowded, preventing the signal from increasing proportionally with increasing concentration. During severe inhibition, no 10nm drop was obtained. The solute concentration is high enough that a highly charged solid residue is formed and no ions are emitted.

FIG. 9(A) depicts when the linear response between analyte and amount is below 10^-5M, solution conditions in the droplet. The droplet surface has available space for charged molecules to occupy. And mass spectrometer signalThe process chamber, pump and gas in communication with the sampling device are responsible for maintaining this ideal condition during sample analysis.

FIG. 9(B) illustrates a schematic view at 10^-4M, the surface is fully occupied and further increases in the internal concentration of analyte do not trigger increased signals. Surface active compounds are particularly effective in creating a state that creates a barrier impermeable to other types of molecules.

Fig. 9(C) shows that at even higher concentrations, the droplets started to form solid residues before ion emission conditions were reached. Severe inhibition occurs and ion production is reduced and completely eliminated. Large charged residues with m/z ratios beyond the measurable range of the mass spectrometer are produced, but for this reason, characterizing them becomes a problem. Recently, new modifications to tandem quadrupole mass spectrometers have demonstrated the ability to detect and characterize these so-called stars, demonstrate their presence and add a definite credit to the theory surrounding the phenomenon of ionization suppression (Schneider, Yang, Covey).

Figure 10 is a comparative graph comparing experimental analysis runs of verapamil (verapamil) sampled as an analyte in a complex biological sample (plasma in trial a) and the same analyte in pure water in trial B, for this example, the verapamil sample has a dilution factor of 4000x in the treatment zone by the treatment stream (MEOH with 0.1% formic acid).

The data provided in test a shows no signal inhibition from this system in complex biological samples (plasma). In this case, it is expected that direct injection of raw plasma into the electrospray ion source would result in a severe suppression effect. It is common practice to perform extensive purification and/or chromatographic separation in order to analyze plasma using an electrospray ion source. However, unexpectedly, the signal for reserpine is shown in fig. 10 to be unaffected by the plasma matrix (test a) relative to the reference standard in water (test B). The dilution factor in the process chamber and transfer line is about 4000-fold, moving the conditions of droplet ejection from the surface saturation point of the endogenous compounds to the point where surface sites remain unoccupied and available for unimpeded ion ejection from the sample.

In the example of fig. 10 (panel a), multiple sampling runs were conducted with increasing volume of verapamil in the plasma matrix delivered for successive sampling runs. As shown, the ion signal detected for each sampling run increases with a substantially linear response to the increased plasma delivered for each sampling run. By way of contrast, fig. 10 (panel B) shows a number of sampling runs carried out according to the same protocol using verapamil in water. As shown, the ion signal response of the matrix-free sample shown in fig. 10(B) corresponds to the ion signal response generated by the matrix sample shown in fig. 10 (a). If there is a matrix effect caused by the increased concentration in fig. 10(a), it is expected that the analysis results in fig. 10(a) (matrix) will show lower signal levels than the analysis results produced in fig. 10(B) (no matrix).

From these results, it can be observed that by comparing the detected ionic signal of water with the signal from the analyte in plasma, the analyte concentration is in the linear range with no evidence of inhibition.

FIG. 11 is a data plot illustrating detection and correction of inhibition in the broth medium. The expected signal from the 5nL injection of methionine was 40% of that expected for it. A dilution ratio of 1650/1, 1nL (e.g., 20% concentration) reinjected into the treatment area produced a signal comparable to that expected and 50% of the 5nL signal instead of the expected 20%. Lowering the injection to 1nL returned the signal to the linear calibration region with a dilution ratio of 8250/1.

Fig. 12A is a graph of ion signal during a sampling run, showing the real-time nature of the process. The collection rate per sample was < 1 second. Thus, the detection and correction of the suppression occurs within a time frame of a few seconds.

Fig. 12B is a close-up view of the analytical data shown in fig. 12A.

Fig. 13 is a simplified schematic diagram illustrating an embodiment of a system for detecting ion suppression. The system of figure 13 includes means for introducing an inhibition reference standard into the solvent transport stream of the capture probe. The controller is operable to detect the presence of an inhibition reference standard in an analytical signal generated by the mass spectrometer. During operation, as analyte is introduced to the capture probe, the controller is operable to detect ion suppression by evaluating an inhibition reference standard signal. Inhibition of the reference standard signal from the initial value, i.e., the deviation of the signal from the measured signal in the absence of analyte introduction, indicates that the introduced analyte results in ion inhibition. The controller may then be operable to apply a corrective action, such as by decreasing the volume or frequency of analyte introduced to the capture probe, increasing the solvent flow rate through the capture probe, and so forth.

Fig. 14 is a diagram illustrating an embodiment of a method for correcting ion suppression in analysis results. For example, the method of fig. 14 may be performed using a system similar to that of fig. 13, in which the magnitude of the offset of the suppression reference standard signal may be evaluated to obtain a quantitative estimate of how much suppression occurred. Corrective action may then be taken by the controller based on the quantitative estimate.

In some aspects, the corrective action may include, for example, a controller that adjusts one or more operating parameters of the system. For example, sample introduction volume or frequency, solvent flow rate, etc. In an aspect, one or more operating parameters may be selected based on the quantitative estimation. In an aspect, a degree of correction applied to one or more operating parameters may be determined based on the quantitative estimation. For example, for small offsets that suppress the reference standard signal, relatively small adjustments can be made to sample introduction (droplet size or frequency), solvent flow rate, and the like. In the case of large offsets, correspondingly larger adjustments may be made to correct for ion suppression. In this way, the ion suppression condition can be corrected with higher efficiency and with fewer trial and error methods than the blind correction method. In addition, the next measurement with the corrected operating parameters may confirm no or little ion suppression based on the suppression reference standard signal evaluated from the next measurement.

In some aspects, the corrective action may include the controller making a computational measurement adjustment to measurements made by the system. In this embodiment, rather than re-performing the measurement with the adjusted operating parameter, the raw measurement with the detected ion suppression may be adjusted based on the quantitative estimate to produce an adjusted measurement that corrects for the detected ion suppression.

As an example, the negative peak area from the suppressed reference standard signal may be evaluated. The negative peak area can be compared to a calibration curve to determine the number of molecules "missing" from the measurement. This value can then be added back to the measurement signal including the analyte signal to correct for ion suppression. In some aspects, a peak threshold may be provided, wherein the correction is applied only if the detected ion suppression is below the threshold. In the event that the detected ion suppression is above the peak threshold, the measurement may be repeated while the operating parameters are adjusted, as described above.