阅读说明：本技术 用于分析物的自动量化的方法以及用于实施该方法的nmr-测量设备 (Method for the automatic quantification of an analyte and NMR measuring device for carrying out the method ) 是由 T.霍伊斯勒 于 2018-05-16 设计创作，主要内容包括：提出一种用于自动地量化测试样品中的分析物的方法，其至少包括：提供1D?NMR?频谱和2D?NMR?频谱；提供关于至少一种待量化的分析物的至少一个信息；使用至少一种所提供的信息，从测得的2D?NMR?频谱求取待量化的分析物的NMR?信号的化学位移；求取待量化的分析物的NMR?信号的预期波峰?位置；从测得的1D?NMR?频谱求取测得的波峰?位置；使用预期波峰?位置和实际上的波峰?位置来求取干扰信号?波峰?位置；使用所求取的干扰信号?波峰?位置，使用所求取的化学位移，以及使用至少一个所提供的信息，对1D?NMR?频谱建模；对建模的1D?NMR?频谱进行积分；通过内部的或外部的参考物来量化分析物。还提出一种用于实施该方法的NMR?测量设备。(A method for automatically quantifying an analyte in a test sample is presented, comprising at least: providing a 1D-NMR-spectrum and a 2D-NMR-spectrum; providing at least one information about at least one analyte to be quantified; using at least one of the provided information, determining from the measured 2D-NMR-spectrum a chemical shift of the NMR-signal of the analyte to be quantified; determining the expected peak position of the NMR signal of the analyte to be quantified; determining the measured peak position from the measured 1D-NMR spectrum; using the expected peak-to-position and the actual peak-to-position to find an interferer-peak-to-position; modeling the 1D-NMR-spectrum using the determined interference signal-peak-position, using the determined chemical shift, and using at least one of the provided information; integrating the modeled 1D-NMR-spectrum; the analyte is quantified by an internal or external reference. An NMR measuring device for carrying out the method is also proposed.)

1. A method for automatically quantifying an analyte in a test sample (34), in particular a liquid test sample, characterized by at least the following method steps:

providing a 1D-NMR-spectrum (30) (method step 100);

providing a 2D-NMR-spectrum (32) (method step 102), in particular a JRES-spectrum, an HMBC-spectrum, an HSQC-spectrum, a COSY-spectrum and/or a DOSY-spectrum;

providing at least one information about at least one analyte to be quantified (method step 106);

using the at least one provided information, determining a chemical shift (42) of the NMR-signal of the analyte to be quantified from the 2D-NMR-spectrum (32) (method step 108);

determining the expected peak positions of the NMR signal of the analyte to be quantified, in particular of the singlet state and/or the multiple state of the analyte to be quantified (method step 110);

determining the actual peak position (44) from the 1D-NMR spectrum (30) (method step 112);

using the expected peak-to-position and the actual peak-to-position (44) to find an interferer-peak-to-position (method step 114);

using the determined interference signal peak position, using the determined chemical shift (42), and using the at least one provided information, modeling the 1D-NMR spectrum (30) (method step 116);

integrating the modeled, in particular interference-free, signal-peak 1D-NMR spectrum (50) (method step 118);

the analyte is quantified by an internal or external reference (method step 120).

2. The method according to claim 1, characterized in that at least one analyte to be quantified is specified in a further method step (method step 104).

3. The method according to any of the preceding claims, characterized in that at least one information about at least one analyte to be quantified is provided by means of a database access.

4. The method according to any of the preceding claims, characterized in that at least one of the information provided specifies the chemical shift range, the number of evaluable signals, the multiplicity of evaluable signals and/or the correlation constant of the evaluable signals with at least one analyte to be quantified.

5. Method according to one of the preceding claims, characterized in that the expected peak positions of the NMR signal of the analyte to be quantified, in particular of the analyte to be quantified, in the singlet state and/or the multiple state, are calculated using the determined chemical shifts (42) and at least one provided information, in particular the provided multiplicity of the evaluable signal and the provided correlation constant of the evaluable signal.

6. Method according to one of the preceding claims, characterized in that the expected peak positions of the NMR signal of the analyte to be quantified, in particular of the analyte to be quantified, in the singlet state and/or the multiple state, are determined from the 2D NMR spectrum (32), in particular the JRES spectrum, using the determined chemical shifts (42) and at least one provided information, in particular the provided multiplicity of the evaluable signal.

7. Method according to any of the preceding claims, characterized in that the interference signal-peak-position is determined from the difference between the expected peak-position and the actual peak-position (44).

8. The method according to one of the preceding claims, characterized in that the 1D-NMR spectrum (30) is modeled by means of a line fitting algorithm on the basis of the determined interference signal peak position and the determined chemical shift (42) while optimizing fitting parameters with respect to Lorentzian Gaussian distribution, with respect to line width, with respect to intensity, with respect to correlation constants and with respect to the roof effect influence, wherein the interference signal peak is modeled as a singlet state (52).

9. The method according to any of the preceding claims, characterized in that the analyte quantification result is compared with a limit value and the comparison result is output.

10. An NMR-measuring device (10), in particular a hand-held NMR-measuring device, having at least one NMR-sensor (24), a control device (26) for controlling the NMR-measuring device (10) and for analyzing a measurement signal provided by the NMR-sensor (24), an output device (16, 16') for outputting the ascertained information, and an energy supply device, in particular a battery, characterized in that the NMR-measuring device (10) is designed for carrying out the method according to any one of claims 1 to 9.

Technical Field

The invention relates to a method for the automatic quantification of analytes and to an NMR measuring device, in particular a mobile, preferably handheld NMR measuring device, having a nuclear magnetic resonance sensor for carrying out the method.

Background

Methods for the quantification of analytes, particularly in liquids, are numerous, for example in the form of separation methods, such as Gas Chromatography (GC) or High Performance Liquid Chromatography (HPLC), titration, and the like. However, these methods involve high preparation costs and large consumption of solvents. Infrared spectroscopy allows for the detection of large amounts of substances in a sample with little expense. However, due to the significant superposition of the signals, the quantization can only be done indirectly and rather inaccurately. Furthermore, the matrix effect plays a decisive role in infrared spectroscopy, so that the concentration range to be detected is often not sufficient to be able to accurately quantify analytes in different liquids.

The quantification of analytes in liquids by means of nuclear magnetic resonance (hereinafter referred to as NMR) is already state of the art. For example, DE 102014203721 a1 discloses a method for determining the concentration of a substance in a sample by means of nuclear magnetic resonance spectroscopy.

DE 102014218375 a1 and DE 102014218371 a1 disclose mobile NMR measuring devices each with a sensor arrangement, wherein the sensor arrangement has at least one nuclear magnetic resonance sensor (NMR sensor) which is provided for determining a moisture value or for detecting and/or analyzing and/or distinguishing material characteristic values of a workpiece to be examined.

Disclosure of Invention

A method is proposed for automatically quantifying an analyte in a test sample, in particular a liquid test sample, in particular by means of an NMR measuring device. The method according to the invention is characterized by at least the following method steps:

providing a 1D-NMR spectrum measured, in particular, by means of an NMR sensor of an NMR measuring device;

providing a 2D NMR spectrum, in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum, measured by means of an NMR sensor of an NMR measuring device;

providing at least one information about at least one analyte to be quantified;

using at least one of the provided information, the chemical shift of the NMR-signal of the analyte to be quantified (i.e. the exact frequency in the fourier transform spectrum) is found from the 2D-NMR-spectrum;

determining the expected peak position (i.e. in particular the frequency value in the fourier transform spectrum) of the NMR-signal of the analyte to be quantified, in particular of the singlet state and/or the multiple state of the analyte to be quantified;

determining the actual peak position from the 1D-NMR spectrum;

using the expected peak-position and the actual peak-position to find the interferer-peak-position;

using the determined interference signal peak position, using the determined chemical shift, and using at least one of the provided information, to model the 1D-NMR spectrum;

integrating the modeled, in particular interference-free signal-peak 1D-NMR spectrum;

quantification of the analyte by internal or external reference.

In one embodiment of the method, the method steps may be performed in the order described. Alternatively, the order of the method steps performed in the method according to the invention may also differ from the preceding order.

In one embodiment of the method, the method is provided for being carried out by an NMR measuring device or using an NMR measuring device. A corresponding NMR measuring device, in particular a mobile, preferably handheld NMR measuring device, has for this purpose at least one NMR sensor (the terms "nuclear magnetic resonance sensor" and "NMR sensor" are used synonymously in this context), a control device for controlling the NMR measuring device and for analyzing the measurement signals provided by the nuclear magnetic resonance sensor, an output device for outputting the ascertained information, and an energy supply device, in particular a battery. Furthermore, the NMR measuring device, in particular its control device, is provided and designed for carrying out the method according to the invention.

A "handheld NMR measuring device" is understood here to mean, in particular, an NMR measuring device which can be transported without the aid of a transport mechanism, using only one hand, and which can be guided to and/or along different test samples, in particular even during the measuring process. The mass of the handheld measuring device of in particular less than 20kg, preferably less than 10kg and particularly advantageously less than 2kg contributes to this.

In one embodiment of the hand-held NMR measuring device, the components of the NMR measuring device, in particular the NMR sensor control device and the energy supply device of the NMR measuring device, are at least partially accommodated in a housing of the NMR measuring device. In particular, these components are arranged in the housing of the NMR-measuring device in more than 50%, preferably more than 75% and particularly preferably 100% of their total volume.

In one embodiment, the NMR measuring device is realized as an energy-autonomous NMR measuring device. By "energy-autonomous" is meant that the NMR measuring device can be operated at least temporarily, preferably at least during the taking of measurements and analysis, independently of the power grid, i.e. in particular wirelessly. For this purpose, the NMR measuring device has an energy supply in the form of an energy store, in particular a battery, preferably a rechargeable battery, which is independent of the electrical network. The energy supply device is provided for supplying electrical energy to the NMR measuring device for operation and during operation. In one embodiment, the grid-independent energy store can be realized as a fuel cell, a capacitor, a hybrid supercapacitor or another energy store or a combination/summation thereof which appears to be advantageous to a person skilled in the art. The storage battery with the chemical cell, which provides a high power density and/or energy density, is particularly suitable for the energy supply of the NMR measuring device. The high power and/or energy density allows an improved, i.e. long-term, energy supply of the NMR measuring device, which is adapted to the large power requirements of the NMR sensor. This is currently the case for batteries, for example, comprising lithium and lithium-ion chemical cells, in particular lithium-iron phosphate, lithium-manganese oxide, lithium-nickel-cobalt-manganese oxide, over-lithiated lithium-nickel-cobalt-manganese oxide, lithium-sulphur, lithium-polymer and lithium-oxygen batteries.

"setting" especially means "programming", "designing" and/or "equipping". An object is "provided" for a certain function, which means in particular that the object fulfils and/or performs this particular function in at least one application and/or operating state, or is designed to fulfil this function.

The NMR-measuring device according to the invention has a control device for its control. The control device is connected to other components of the NMR measuring device, in particular the NMR sensor, the output device, the energy supply device, and also, for example, the input device and/or the data communication interface in terms of signals. The control device is provided for communication with these components during operation of the NMR-measuring device. "control device" means in particular a device with at least one electronic control unit, which has means for communicating with other components of the NMR-measuring device, for example means for controlling and/or regulating the NMR-sensor, means for data processing, means for data storage and/or other means which are advantageous to the person skilled in the art. In one embodiment, the electronic control unit of the control device is a processor unit which is connected to a memory unit and to an operating program stored in the memory unit, which is executed during the control process. In particular, the electronics of the control device can be arranged on a circuit board (printed circuit board), for example in the form of a microcontroller. Furthermore, the control device is used for analyzing at least one test signal provided by the NMR sensor, in particular for analyzing a 1D NMR spectrum provided by the NMR sensor and a 2D NMR spectrum provided by the NMR sensor, in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum. Alternatively or additionally, the test signals, in particular the 1D-NMR spectrum and the 2D-NMR spectrum, in particular the JRES spectrum, the HMBC spectrum, the HSQC spectrum, the COSY spectrum and/or the DOSY spectrum, can also be provided to the control device in other ways, for example using a data communication interface of the NMR measuring device. The term "provided for analysis" means, in particular, that the control device has an information input for receiving the test signal, in particular the frequency spectrum, an information processing unit for processing, in particular analyzing, the test signal, in particular the frequency spectrum, and an information output for transmitting the processed and/or analyzed test signal, in particular the frequency spectrum, and/or the analysis result obtained. In one embodiment, the control device has for this purpose components comprising at least one processor, a memory and an operating program with evaluation and calculation routines. In particular, these components may also be referred to as analysis devices and/or analysis devices which are designed separately from the control device.

An "output device" of an NMR-measuring device refers to at least one device which is provided for outputting at least one variable information to an operator acoustically, optically and/or tactually. The output device is used for outputting at least the information obtained by using the NMR-measuring apparatus to an operator of the NMR-measuring apparatus. In particular, the result of the quantification of the analyte can be output by means of an output device. The output can be realized, for example, by means of a display screen, a touch-sensitive display screen, an acoustic signal, a vibration generator and/or an LED display. In one embodiment of the output device, the information can be output graphically or alphabetically as a measurement result of the examination. In one embodiment, the output device is arranged in a housing of the hand-held NMR measuring device. Furthermore, the information or results to be output can also be output to the control device and/or to the data processing system, in particular for increasing user comfort. The latter comprises at least outputting information to external devices, such as smartphones, laptops, PCs and another external data device which appears to be beneficial to a person skilled in the art, which data device is connected to the NMR-measuring device via a data communication interface. In particular, the output device can be arranged directly in the housing of the NMR measuring device and can additionally also be supplemented by an external output device. Using the information output by the output device to the user of the NMR measuring device allows the user of the NMR measuring device to obtain intuitively understandable results after the quantification of the analyte in the test sample.

For the measurement, in particular for the measurement of 1D-NMR spectra and 2D-NMR spectra, in particular JRES spectra, HMBC spectra, HSQC spectra, COSY spectra and/or DOSY spectra, the NMR measuring device has at least one NMR sensor. The mode of operation of the NMR sensor is based on the nuclear physics effect, in which the nuclei in the test sample to be examined are in a first magnetic field-with B₀Indicating an alternating magnetic field that absorbs and emits electromagnetic radiation. Nuclear magnetic resonance is based on the precession of nuclear spins (larmor precession) of atomic nuclei in a test sample to be examined around the field lines of a first magnetic field, which is in particular constant and/or stationary. In particular, the nuclear spins of the atomic nuclei in the volume to be examined are aligned by the first magnetic field. If energy is incident on the nuclei in the form of a second electromagnetic field, in particular an alternating magnetic field, for example a pulsating magnetic field, which energy is in resonance with the larmor precession of the nuclear spins of the nuclei (energy quantum), the nuclei will change their spin orientation relative to the first magnetic field by absorbing this energy. The incident second magnetic field thus serves to excite the nuclear spins, which change their nuclear spin state upon absorption of energy. Equivalently, the emission of an energy quantum, as a result of the excited nuclear spin returning to another, lower energy level, leads to the emission of an alternating electromagnetic field which can be observed by means of the device for detecting a change in the magnetic field, in particular by means of an antenna and/or a coil. Nuclei refer in particular to protons (H) and other nuclear magnetic resonance-active nuclei, such as 13C, 15N, 19F, 31P.

The NMR measuring device according to the invention allows to determine the NMR-spectral information obtained by the at least one NMR-sensor and to derive therefrom a judgment about the properties and in particular the state of the test sample and in particular about the concentration of the analyte contained therein. For example, when the NMR-spectrum is appropriately analyzed, information on the quality (of, for example, a beverage or fuel or a body fluid), the presence state of contents (for example, contents harmful to health, prohibited contents, pesticides, bactericides, hormones, antibiotics), and the like can be obtained. From this analyzed information, the user of the NMR measuring device can thus check and verify in a simple manner the properties of the test sample to be checked, such as quality, origin, authenticity, composition, potential danger, etc. Furthermore, the information obtained can be compared with the manufacturer data and the authenticity of the manufacturer data is checked accordingly.

For the measurement, a mobile NMR measuring device, in particular at least one NMR sensor, is arranged in the vicinity of the test sample to be examined, or vice versa. The use of a measuring device allows the test specimen to be inspected without damage, in particular without damage, contamination or the like.

A mobile energy-independent NMR measuring device is a special-purpose measuring device which has very limited functions compared to scientific nuclear magnetic resonance measuring devices, which are optimized for the examination of test samples. In particular, the control device with its analysis routine is suitable for carrying out the method according to the invention. Furthermore, the NMR-measuring device may be adapted to evaluate the obtained information and to prepare it for display and output by means of an output device. When using the measuring device for examining a test sample, the measurement results are provided to the user of the NMR-measuring device inside the device and immediately after the measurement, so that the analytes in the test sample can be quantified quickly, unambiguously and in particular independently of other devices, such as computers or even laboratories, in the field. Advantageously, the measuring device can be operated easily and intuitively, without the special previous experience of the operator being required.

By using a handheld measuring device which is energy-independent and is specifically adapted to the application of the examination of the test specimen, it is possible to achieve an accurate and extensive quantification of the analyte in the test specimen, for example in the case of warehouses, shops, wholesalers, at patient visits, etc., in a rapid and damage-free manner and thus economically particularly cost-effectively, on the spot on a mobile basis.

"quantifying an analyte in a test sample" means in particular determining the concentration of a substance, i.e. an analyte, in a test sample, in particular a liquid test sample. In NMR-spectroscopy, any analyte provides one or more unique frequency-dependent signals (NMR-signals). The integrated intensity of a peak in the 1D-NMR spectrum caused by a certain analyte is substantially proportional to the concentration of the analyte of interest in the sample. Since each proton contributes to the 1D NMR spectrum, a direct and precise quantification is possible. However, due to the superposition of a plurality of peaks in the 1D-NMR spectrum, it is often not easy to quantitatively determine the signal component belonging to a certain substance. If there are multiple different analytes in the test sample whose NMR-signals are superimposed, the representation of the signal set by the individual signals can be achieved by a "line fitting" technique. In this way, quantization can thus be achieved even in the case of various signals being superimposed.

By "NMR-signal" is meant herein a process-independent, physical signal of the test sample during NMR-measurement, generated only as a result of the chemical structure in the test sample, e.g. fourier transform.

In one embodiment of the method, at least one analyte to be quantified is specified in a further method step. In this way, the analyte to be quantified can be selected according to its interest by the user of the method according to the invention. In particular, at least one analyte to be quantified can be specified as a result of a user input or selection, for example by means of an input device or by means of a menu selection or by means of a barcode scanner (for example by scanning a product-specific barcode) or the like. Alternatively, the information can also be provided to the computing unit or the control device in another way, for example by reading out a memory. In one embodiment, the information about at least one analyte to be quantified is static information, i.e. the same analyte is quantified throughout the method according to the invention.

By specifying at least one analyte to be quantified, the information processing, in particular the spectral analysis or the like, can be advantageously adapted to the analyte to be examined. For example, the reference database may be selected according to specifications. Furthermore, in particular in relation to the regulation, the operating program, the control routine, the analysis routine and/or the calculation routine of the control device can be adjusted.

In a method step of the method according to the invention, a 1D-NMR spectrum is provided. The term "providing" is used in the following to mean, in particular, providing the corresponding information or data set to a computing unit, in particular a control device of the NMR measuring device, which carries out the method according to the invention. Such a 1D NMR spectrum is usually obtained when the nuclear spins contained in the analyte align in a strong static magnetic field and are excited in energy (i.e. nuclear magnetic rotation) in high-frequency pulses. The high frequency response of the sample is then recorded as a function of time and the FID-signal (free induction decay signal) is obtained. From the FID signal, a fourier transformation can be used to obtain a spectrum of the sample, in which peaks are contained that are specific to the individual components of the sample, wherein the individual peaks of these components overlap in intensity to a greater or lesser extent.

In one embodiment of the method, 1H-NMR spectroscopy is used to quantify the analytes. In this case, 1H-NMR spectroscopy is used to detect all substances having non-exchangeable protons, such as sugars, ethanol, organic acids and esters, aromatic substances such as aniline or vanillin, etc. In one embodiment of the method, the 1H-NMR spectrum is provided without frequency suppression, in order to be able to quantify the main components such as water and/or ethanol. Alternatively, a 1H-NMR spectrum is provided with selective signal suppression, in particular solvent suppression, in order to be able to quantify analytes in very small concentrations.

In addition, in a method step of the method according to the invention, a 2D NMR spectrum, in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum, is provided. Such a 2D NMR spectrum is usually obtained when the nuclear spins contained in the analyte align themselves in a strong static magnetic field and are excited energetically in high-frequency pulses. By means of a pulse sequence typical for a corresponding 2D NMR spectrum, which has a defined waiting time between the individual high-frequency pulses, a magnetization exchange between two or more nuclear spin-active nuclei takes place. The high frequency response of the sample is recorded as a function of time and the FID-signal is obtained. By means of a fourier transformation, in particular a double fourier transformation, a 2D spectrum of the sample can be obtained from the FID signal, in which spectrum peaks are contained that are specific to the individual components of the sample.

In the method according to the invention, 2D-NMR spectroscopy, for example JRES spectroscopy, is used to be able to determine the correlation constants and chemical shifts of the peaks of the analyte in the supplied NMR-signal, in particular in the supplied 1D-NMR-spectroscopy, with accuracy. In particular, heteronuclear 2D NMR spectra, such as HMBC or HSQC, can also be used to assign peaks unambiguously. Information about the association with heteronuclear nuclei, such as 13C, is used here.

In particular, these spectra, i.e. the 1D-NMR-spectrum and the 2D-NMR-spectrum, can be supplied to a calculation unit, which carries out the method according to the invention. In one embodiment, these spectra can be provided to the control device of the NMR-measuring device according to the invention. In one embodiment, these spectra can be measured by means of at least one NMR sensor of the NMR measuring device according to the invention and supplied to the control device of the NMR measuring device.

In one embodiment of the method, the 1D-NMR spectrum and the 2D-NMR spectrum are obtained under the same measurement conditions, in particular by means of an NMR sensor of an NMR measuring device. Under these identical measurement conditions, a particularly precise and reliable quantization is possible, since distortions or deviations in the frequency spectrum due to different measurement conditions can be avoided and thus excluded. The consistent measurement conditions relate in particular to the temperature of the test specimen and/or the solvent in the test specimen and/or the magnetic field strength of the static magnetic field during the respective measurement.

In one embodiment of the method, the frequency spectrum is advantageously processed before further processing using known data analysis methods. For example, the 1D NMR spectrum, or correspondingly the fourier transformed FID spectrum, can be multiplied beforehand by a suitably selected exponential function in order to obtain an improved signal-to-noise ratio. Furthermore, it is possible to reduce or eliminate the deviations from spectral symmetry that occur due to uniformity and phase deviations by an automated, so-called "reference deconvolution" -method. Within the scope of this method, phase correction and background correction (base line correction) can also be carried out automatically, as can the frequency spectrum by mathematical methods such as smoothing, filtering, averaging, etc.

In a further method step of the method according to the invention, at least one piece of information is provided about at least one analyte to be quantified. In particular, this information may be provided to a computing unit implementing the method according to the invention. In particular, this information can be provided to the control device of the NMR-measuring device according to the invention. In one embodiment of the method, the at least one provided item of information specifies a chemical shift range, a number of evaluable signals, a multiplicity of evaluable signals and/or a correlation constant of the evaluable signals with the at least one analyte to be quantified. In this way, an especially simple, extensive and accurate quantification of the analyte is possible. In particular, the provided information can be used to analyze the provided 1D-NMR-spectrum and/or the provided 2D-NMR-spectrum in a simple, extensive and precise manner. In an embodiment of the method according to the invention, the information provided may also relate to the chemical shift range of the carbon nucleus associated with the proton. This information is used for identification by 2D NMR spectroscopy and for determining external chemical shifts.

The term "chemical shift range" is understood here to mean the non-hazardous frequency range in which the signal to be detected of the analyte is expected in the provided frequency spectrum.

The "number of signals evaluable" is the number of those signals in the spectrum caused by the analyte. This amount is thus related to the chemical structure of the analyte. In particular, the number of signals that can be evaluated is dictated by the pre-selection of the analyte to be quantified.

The "multiplicity" of the evaluable signal represents the number of those single signals that produce the evaluable signal as the total signal. The splitting of the total signal into single signals is caused by spin-spin coupling. The multiplet of the signal that can be assessed is related to the chemical structure of the analyte. In particular, "multiplicity" denotes the number of linearly independent states that the analyte can be in terms of spin orientation, the so-called spin multiplicity. The multiplicity can be transferred directly to the NMR-spectrum, in which a number of peaks corresponding to the multiplicity is generated in the analyte signal.

The "correlation constant of the evaluable signal" is a constant that determines the strength of the interaction of two associated nuclei. The correlation constant is specific to the corresponding analyte. The correlation constants are used for simulation/modeling of the identified and evaluable signals by a line fitting algorithm.

In one embodiment of the method according to the invention, at least one piece of information about at least one analyte to be quantified is provided by a database access. In this case, the at least one piece of information can be stored in the device internally in a database located on a memory unit, in particular a computing unit of the NMR measuring device or a memory unit of the control device. In an alternative or additional embodiment, the at least one piece of information can also be stored in an external, in particular device-external, advantageously always updated database. For example, the at least one information may be stored in a database located on a computer, a server or another data storage and/or data processing device which appears to be beneficial to a person skilled in the art and provided by the database when carrying out the method according to the invention. In particular, the provision of the at least one piece of information can also be carried out via an internet portal of the NMR measuring device. Alternatively or additionally, the at least one piece of information may be stored internally, in particular internally in the device, in the NMR-measuring device and likewise updated via an internet portal of the NMR-measuring device, for example by comparison with an external reference database.

In a method step, at least one of the provided information is used to determine a chemical shift of the NMR signal of the analyte to be quantified from the provided 2D NMR spectrum. For this purpose, the exact position of the peak belonging to the analyte to be quantified is determined from the 2D spectrum, in particular the JRES spectrum. In one embodiment of the method, a displacement range typical for the analyte, i.e. known in advance, is considered as an initial value of the analysis. Preferably, at least one provided piece of information specifying the displacement range is accessed. In this way slight deviations in chemical shifts are tolerated. Based on the initial parameters, the 2D-NMR-spectrum is analyzed for signals, i.e. peaks, which correspond to given parameters, such as the correlation constant, multiplicity or chemical shift of the associated 13C-nuclei. When the peak is consistent with the given parameters from the database, the exact chemical shift of the peak is determined.

Furthermore, the correlation constant and the multiplicity of the analyte can also be determined from the 2D NMR spectrum, in particular the JRES spectrum, in this method step. The determined correlation constant and the determined multiplicity can then be compared with at least one provided item of information, which can likewise relate to the correlation constant and the multiplicity of the analyte.

In a method step, the expected peak positions of the NMR signals of the analytes to be quantified, in particular of the singlet and/or the multiple states of the analytes to be quantified, are determined. In one embodiment of the method, the determined chemical shifts and at least one provided information, in particular the provided multiplicity of the evaluable signal and the provided correlation constant of the evaluable signal, are used to calculate expected peak positions of the analyte to be quantified, in particular of the singlet and/or the multiple NMR-signal of the analyte to be quantified. For this purpose, the chemical shifts of the individual signals are calculated from the multiplicity by adding or subtracting a defined frequency value from a given correlation constant and the determined chemical shift.

In an alternative or additional embodiment of the method, the expected peak positions of the analyte to be quantified, in particular of the singlet and/or the multiple NMR signal of the analyte to be quantified, are determined from the 2D NMR spectrum, in particular the JRES spectrum, using the determined chemical shifts and at least one provided information, in particular the provided multiplicity of the evaluable signal. From the 2D NMR spectrum, in particular the JRES spectrum, the correlation constant of the NMR signal to be analyzed is determined by determining the distance of the peak positions (the so-called signal position or center frequency). The chemical shift of the individual signals is calculated from the multiplicity by adding or subtracting a defined frequency value from the determined chemical shift, based on a correlation constant determined from the 2D NMR spectrum, in particular the JRES spectrum.

In a method step, the actual peak position is determined from the provided, in particular measured, 1D-NMR spectrum. In this case, the peak positions of all peaks whose intensities in the frequency spectrum exceed a given threshold are determined and stored in a temporary memory.

In a method step, an interference signal peak position is determined using the expected peak position and using the determined actual peak position. In one embodiment of the method, the interference signal peak position is determined from the difference between the expected peak position and the actual peak position. "interference signal peak position" is intended in particular to mean such a peak position of an interference signal which is superimposed on the examined frequency spectrum and is not caused by the analyte to be examined. That is, the interference signal-peak-position is not caused by the analyte to be examined.

In a method step, the 1D-NMR spectrum is modeled using the determined interference signal peak position, using the determined chemical shift and using at least one of the provided information. In one embodiment of the method, the 1D-NMR spectrum is modeled by means of a line fitting algorithm on the basis of the determined interference signal peak position and the determined chemical shift, while optimizing fitting parameters with respect to the lorentzian gaussian distribution, with respect to the line width, with respect to the intensity, with respect to the correlation constant and with respect to the roof effect influence, wherein the interference signal peak is modeled as a singlet state.

A "line fitting algorithm" is an algorithm that iteratively adapts a calculated signal to a measured signal. The fitting algorithm is performed in one embodiment for each peak-signal (e.g., for the methanol resonance line), but may also be extended over the entire spectrum, with multiple analyte-induced signals being observed simultaneously.

In a further method step, the modeled, in particular interference-free signal peaks are integrated over the 1D-NMR spectrum.

In one method step, the analyte is quantified by an internal or external reference. For internal reference, a suitable reference reagent must be added to the test sample. Typical reference reagents are for example sodium Trimethylsilylpropionate (TSP) for aqueous solutions or Tetramethylsilane (TMS) for organic solutions.

The external quantification can be carried out in particular by means of the established PULCON method. The PULCON-process is described, for example, in G.wire, L.Dreier, J. Am. chem. Soc. 2006, number 128 (pages 2571 and 2576).

In one embodiment of the method, the result of the quantification is further processed by means of a control device of the NMR measuring device. In one embodiment of the method, the result can be output to an operator of the NMR-measuring device using an output device of the NMR-measuring device. In one embodiment of the method, in particular using a control device of the NMR measuring device or using a computing unit which carries out the method, the analyte quantification result is compared with a limit value and the comparison result is output. The output is carried out in particular by means of an output device of the NMR measuring device.

With the method according to the invention, a plurality of analytes in a liquid can be quantified by means of NMR-spectroscopy quickly, automatically and accurately. In this case, the method allows the processing and analysis of the spectral NMR data in an advantageous manner, so that a quantification of different analytes can be achieved with complete automation of the analysis. The combination of data processing with information integration of different NMR data sets, in particular 1D-NMR spectra and 2D-NMR spectra, enables quantification by means of a reliable line fitting-optimization algorithm, thereby ensuring that a constant accuracy is maintained. Furthermore, the simultaneous quantification of multiple analytes in a test sample can be achieved using the method according to the invention. Advantageously, as a result of each analyte to be quantified, the concentration and the corresponding measurement unreliability may be output. Since no trained specialist is required for analyzing the measurement data, the method can be implemented particularly advantageously in an NMR measuring device designed for use by untrained users.

Drawings

The invention will be described in detail in the following description with the aid of embodiments shown in the drawings. The figures, description and claims contain numerous combinations of features. The person skilled in the art can also examine these features individually and make other advantageous combinations depending on the purpose. The same or similar reference numbers in the drawings identify the same or similar elements.

FIG. 1 is a perspective view of one design of a mobile NMR-measuring apparatus according to the invention;

FIG. 2 is a top view of a first housing side of one design of an NMR-measurement apparatus according to the invention;

FIG. 3 is a method flow diagram of one embodiment of a method according to the present invention;

FIG. 4 shows an exemplary 1D-NMR spectrum;

FIG. 5 shows an exemplary 2D-NMR-spectrum and an enlarged part of the 2D-NMR-spectrum;

FIG. 6 shows an exemplary 1D-NMR spectrum after the method steps for determining the actual peak position have been carried out;

fig. 7 shows an exemplary 1D NMR spectrum after the method step of performing the line fitting.

Detailed Description

Fig. 1 and 2 show two views of an exemplary embodiment of a handheld, energy-autonomous NMR measuring device 10 according to the invention in perspective view or in a simplified schematic top view.

The exemplary NMR measuring device 10 has a housing 12. In this housing 12, an input device 14 in the form of an operating element 14' is arranged, which is suitable for switching the NMR measuring device 10 on and off, for starting and configuring the measuring process and for inputting operating parameters. In addition, an output device 16 in the form of a display screen 16' is provided in the housing 12 for outputting the information sought and the operating parameters. The NMR measuring device 10 has a handle 18 for transport and handling thereof. The handle 18, the operating member 14 'and the display 16' are located on a first housing side 20 (also called "front side") of the NMR-measuring device 10, which generally faces the operator when operating the NMR-measuring device.

For the energy supply of the NMR measuring device 10, the NMR measuring device 10 has a recess on a second housing side (not shown in detail here) opposite the first housing side 20 on the device rear side, which recess serves to accommodate a power grid-independent energy store in the form of a rechargeable battery (not shown in detail here). Due to the energy store, which is independent of the electrical network, the NMR measuring device 10 can be at least temporarily energy-autonomous, i.e. operated independently of the electrical network and thus in particular also wirelessly. The exemplary proposed NMR measuring device 10 has a lithium-ion accumulator, the high energy density and power density of which are advantageously suitable for the energy supply of the NMR measuring device 10. In an alternative embodiment, the energy store can also be arranged in the handle 18 of the NMR measuring device 10. Preferably, the energy supply device has a releasable form-fitting and/or force-fitting connection interface, so that the energy accumulator (also usually a plurality of energy accumulators) can be arranged removably and replaceably. Furthermore, the energy store can be supplied with energy from the power grid and charged in and/or outside the NMR measuring device 10.

On the carrier part 22, in particular on a system circuit board or printed circuit board inside the housing 12, further components of the NMR measuring device 10, in particular the NMR sensor 24, a control device 26 for controlling the NMR measuring device 10 and for evaluating the measurement signals provided by the NMR sensor 24, and a data communication interface 28 (see in particular fig. 2) connected to the control device 26 are arranged. The control device 26 is used to evaluate at least one measurement signal provided by the NMR sensor, in particular a measured 1D NMR spectrum 30 (see FIG. 4) and a measured 2D NMR spectrum 32 (see FIG. 5), in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum. Alternatively or additionally, the measurement signals, in particular the 1D NMR spectrum 30 and the 2D NMR spectrum 32, in particular the JRES spectrum, the HMBC spectrum, the HSQC spectrum, the COSY spectrum and/or the DOSY spectrum, can also be supplied to the control device 26 in other ways, for example using the data communication interface 28 of the NMR measuring device 10. The control device 26 has an electronic control which includes means for communicating with other components of the NMR-measuring apparatus 10, such as means for controlling and regulating the NMR-sensor 24, an analysis device separate from the control device 26, etc. The control device 26 comprises in particular a unit with a processor unit, a memory unit and an operating program stored in the memory unit. The control device 26 is provided for adjusting at least one operating function parameter of the NMR measuring device 10 as a function of at least one input by an operator, via the sometimes provided evaluation device and/or via the data communication interface 28. Furthermore, the control device 26 is provided for carrying out the method according to the invention and for this purpose has a memory with an executable operating program stored thereon.

The NMR-sensor 24 is arranged to excite nuclear magnetic resonance in the nuclei of the material of the test sample 34. The NMR sensor 24 is provided in particular for measuring a 1D NMR spectrum 30 and a 2D NMR spectrum 32, in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum, of a test sample 34.

Fig. 3 shows a method program which represents an exemplary embodiment of the method according to the present invention for quantifying analytes in a test sample 34, in particular in a liquid test pattern, in particular by means of the NMR measuring device 10.

In a method step 100, a 1D-NMR spectrum 30 is provided, which is measured by means of the NMR sensor 24 of the NMR measuring device 10. The 1D-NMR spectrum 30, in particular the data on which it is based, is already prepared for analysis in method step 100, for example by smoothing, filtering, etc. An exemplary 1D-NMR spectrum 30 is shown in FIG. 4. The 1D-NMR spectrum 30 has peaks 48 specific to the test sample 34, in particular the analyte contained therein.

In a method step 102, a 2D NMR spectrum 32, in particular a JRES spectrum, an HMBC spectrum, an HSQC spectrum, a COSY spectrum and/or a DOSY spectrum, is provided which is measured by means of the NMR sensor 24 of the NMR measuring device. The 2D NMR spectrum 32, in particular the data on which it is based, is already prepared for analysis in method step 102, for example by smoothing, filtering, etc. An exemplary 2D NMR spectrum 32, in particular a JRES spectrum, is shown in fig. 5 a.

In a method step 104, at least one analyte to be quantified is specified, for example as a result of an input or selection by a user, for example by means of the input device 14 of the NMR measuring device 10. In this embodiment, the user specifies the analyte to be quantified, for example by choosing the name of the analyte "ethanol" (for example from a given list).

In method step 106, information is provided about the analyte to be quantified, based on the specified name of the analyte. Here, the information specifies at least one chemical shift range 36 (a rough range 42 of expected chemical shifts, represented by range 36 in fig. 5), a plurality of evaluable signals 38 (represented by boxes in fig. 4, respectively), a multiplicity of evaluable signals and a correlation constant of the evaluable signals with the analyte to be quantified. The multiplicity of the evaluable signals and the correlation constants of the evaluable signals, which represent the amplification region of the 2D NMR spectrum 32 shown in fig. 5a, are summarized in fig. 5b as reference symbols 40. In the example shown in fig. 5b, the multiplicity is 2 (dual state) and the correlation constant is about 6.4Hz (corresponding to the distance between two peaks, indicated by reference numeral 40). In this embodiment, the information about the at least one analyte to be quantified is provided by a database query, in particular using an internet connection. The data retrieved from the database 200 (see fig. 1) by means of the data communication interface 28 of the NMR measuring device 10 are transmitted to the control device 26.

In a method step 108, using the at least one provided information, the chemical shift 42 of the NMR signal of the analyte to be quantified is determined from the 2D NMR spectrum 32. Using the chemical shift range 36 of the analyte, the data set of the 2D-NMR spectrum 32 is first searched for the true signal with the appropriate parameters and the "actual" chemical shift 42 is determined therefrom. If a corresponding signal cannot be evaluated in the data set, i.e., using the chemical shift range 36, then a re-query can be initiated for other information in the database 200. Alternatively, an error notification or a notification that the evaluation was not successful can be output to the user of the NMR measurement device 10.

In method step 110, the expected peak positions of the NMR signals of the analytes to be quantified, in particular of the singlet and/or the multiple states of the analytes to be quantified, are determined (not shown in detail here). In this exemplary embodiment, the determined (i.e. actual) chemical shift 42 and at least one provided information, in particular the provided multiplicity of the evaluable signal and the provided correlation constant of the evaluable signal (reference numeral 40), are used to calculate the expected peak position of the NMR-signal, and, in addition, the determined (actual) chemical shift 42 and at least one provided information, in particular the provided multiplicity of the evaluable signal (40), can also be used to determine the expected peak position of the NMR-signal from the 2D-NMR spectrum 32, in particular the JRES-spectrum (not shown in detail here).

In a method step 112, the actual peak position 44 is determined from the 1D NMR spectrum 30. For this purpose, the 1D-NMR spectrum 30 is analyzed, for example, for local maxima. An exemplary 1D NMR spectrum is shown in fig. 6, wherein the actual peak-position 44 is indicated and the values it has are shown.

In method step 114, the interference signal peak position is determined using the expected peak position and the actual peak position 44 from the difference between the expected peak position and the actual peak position 44 (not shown in detail).

In a method step 116, the 1D NMR spectrum 30 is then modeled using the determined chemical shifts 42 and using the at least one provided information using the determined interference signal peak positions. By means of a line fitting algorithm, the model is made on the basis of the determined interference signal peak positions and the determined chemical shifts 42, while fitting parameters are optimized with respect to the lorentzian gaussian distribution, with respect to the line width, with respect to the intensity, with respect to the correlation constants and with respect to the roof effect influence, wherein the interference signal peaks are modeled as singlet states 52. In this way, a pure, in particular interference-free, signal-peak modeled 1D-NMR spectrum 50 is generated. Fig. 7 thus shows an exemplary illustration of a part of the 1D NMR spectrum 30 after/during the execution of method step 116. In the exemplary data set, the dual state of isobutanol (reference numeral 50) -the NMR expected to be quantified for the analyte-is superimposed with the interference signal 52 (singlet state) of methanol. The provided 1D-NMR-spectrum 30 is represented by a curve 54 after performing the line fitting algorithm, which shows the modeled 1D-NMR-spectrum 54. The desired NMR signal of the analyte to be quantified is represented by the modeled 1D NMR spectrum 50 without interfering signal peaks.

In a method step 118, the modeled 1D NMR spectrum 50, in particular of the interference-free signal peaks, is integrated.

In method step 120, the analyte is quantified by an internal or external reference. If more than one signal is present for each analyte, an average of the quantified values can finally be calculated (not shown in detail).

In a method step 122, the result of the quantification is further processed by means of the control device 26 of the NMR measuring device 10, in particular output to an operator of the NMR measuring device 10 by means of the output device 16 of the NMR measuring device 10. In this embodiment, the output indicates a percentage value of the analyte concentration in the test sample 34. Furthermore, the result of the quantification of the analyte is compared with a limit value and the result of the comparison with colored LEDs (red, yellow, green) is output (not shown in detail).