CA2423260C - Automatic identification of compounds in a sample mixture by means of nmr spectroscopy - Google Patents

Abstract

A process for quantitative and qualitative analysis for identifying compounds in a sample mixture involves the identification of a set of reference spectra selected according to a measured condition, e.g. pH, of the sample, which collectively define a composite spectrum which best matches a spectrum produced from the sample. The compounds associated with respective reference spectra of the identified set are the compounds that are determined to be likely to be present in the sample. Quantities of the compounds may be determined from the intensities of certain representative peaks associated with the compounds, relative to the intensity of a peak associated with a reference compound which is unaffected by the measured condition of the sample. Thus, given a test spectrum of a sample and given a set of reference spectra, the process can identify and quantify compounds present in the sample.

Description

AUTOMATIC IDENTIFICATION OF COMPOUNDS IN A SAMPLE MIXTURE
BY MEANS OF NMR SPECTROSCOPY

BACKGROUND OF THE INVENTION
1. Field of Invention This invention relates to qualitative and quantitative chemical analysis, and more particularly to processes, apparatus, media and signals for automatically identifying compounds in a sample.

2. Description of Related Art The field of biometric identification has grown tremendously over the recent decade both from its relevance to medical diagnostics and to its application as a way to uniquely identify a person or an animal, for example. As diagnostic tools have become more sophisticated, complex liquid mixtures, such as human blood or urine for example, can now be analyzed to identify or search for particular compounds that can provide important diagnostic information to a medical technician or a doctor.

Generally, the separation and characterization of mixtures is fundamental to nearly every aspect of analytical chemistry and biochemistry. Most approaches to identify and quantify biological compounds in liquid mixtures require an initial compound separation (chromatographic or physical separation) step to separate a particular compound or set of compounds from the mixture. For example, gas chromatography, electrophoresis, and liquid chromatography are used to separate pure chemical components/compounds, for example, from a mixture before analysis is performed. Initial compound separation is required because most spectral identification processes, such as mass spectrometry or infrared, visible, and ultraviolet spectroscopy, require relatively pure samples in order to minimize noise and increase the accuracy of the measuring device. Spectral identification processes are expensive, manually intensive and require a great deal of technical expertise to be performed properly in an accurate, timely manner.
Nuclear magnetic resonance (NMR) has recently been shown to be an alternative approach to identify and quantify biological compounds without chromatographic separation. In this approach, radio frequency (RF) electromagnetic radiation is applied to a mixture of organic compounds to extract and measure a characteristic RF absorption spectrum of nuclei belonging to each specific organic compound. A large number of compounds are associated with well-defined peaks in the absorption spectrum and knowing which peaks are associated with certain compounds makes it possible to manually identify some of the compounds in the liquid mixture without resorting first to chromatographic separation. However, this process is still quite slow and requires a great deal of a priori information that relates each peak to a given compound. It can take a number of years for experts in NMR spectroscopy to acquire the knowledge required to analyze NMR
spectra to accurately identify and quantify compounds in sample mixtures.
Therefore what is desired is a process and apparatus for quickly, accurately and automatically identifying a number of compounds which may be present in complex liquid mixtures without involving chromatographic separation and without requiring people who are experts in NMR techniques.
SUMMARY OF THE INVENTION
Overall Process The embodiments of the invention disclosed herein provide for automated, accurate analysis of a test spectrum obtained from a sample, to quantitatively and qualitatively identify compounds present in the sample.

In accordance with one aspect of the invention there is provided a computer-implemented process for automatically identifying compounds in a sample mixture. The process involves receiving a representation of a measured condition of the sample mixture, using the representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in the sample mixture, from a library of reference spectra, and receiving a representation of a test spectrum having peaks associated with compounds therein, the test spectrum being produced from the sample mixture under the measured condition. The process also involves combining reference spectra from the set of reference spectra to produce a matching composite spectrum having peaks associated with at least some of the suspected compounds, that match peaks in the test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being indicative of the compounds in the sample mixture, and storing a representation of the matching composite spectrum.

The process may involve identifying compounds associated with the representative reference spectra.
Identifying compounds may involve identifying quantities of the compounds.
Identifying compounds may involve identifying concentrations of the compounds.
The process may involve identifying a peak associated with a calibration compound, in the test spectrum.

Identifying the peak may involve identifying a peak meeting a set of criteria that associate the peak with the calibration compound.

Identifying the peak may involve producing Lorentzian line shape parameters to represent the peak.

The process may involve receiving a measured condition value representing the measured condition of the sample.

The process may involve producing the measured condition value.

Producing the measured condition value may involve measuring pH of the sample.

Producing the measured condition value may involve producing the condition value from the test spectrum.

Producing the condition value may involve identifying in the test spectrum a peak position associated with a condition reference compound.

Identifying a peak position may involve identifying a peak meeting a set of criteria that associate the peak with the condition reference compound.
Producing the condition value may involve producing the condition value as a function of the peak position and parameters of a sample solvent.

Producing the measured condition value may involve determining a pH value for the sample, from the test spectrum.

Determining a measured pH value may involve determining from the test spectrum, the location of a peak associated with a pH reference compound, in relation to a peak associated with a calibration reference compound.
Producing the condition value may involve producing the condition value as a function of the peak location and parameters of a sample solvent.

The process may involve adjusting a set of base reference spectra according to the measured condition value, to produce the set of reference spectra.
Adjusting the set of base reference spectra may involve adjusting parameters of the base reference spectra according to a pH of the sample.

The process may involve producing a derived reference spectrum in response to the measured condition value and a reference spectrum.

Producing the derived reference spectrum may involve identifying a reference spectrum that is associated with a condition value nearest to the measured condition value.

Producing the derived reference spectrum may involve deriving a value from at least one reference spectrum that is associated with a condition value nearest to the measured condition value.

Producing the derived reference spectrum may involve performing mathematical operations on parameters of a reference spectrum to produce new parameters for use as parameters of the derived reference spectrum.

The process may involve identifying in the test spectrum a peak associated with a calibration compound and producing Lorentzian line shape parameters, including a line width parameter, to represent the peak.
The derived reference spectrum may be represented by at least one set of Lorentzian line shape parameters including a line width parameter, the process may further involve calibrating the line width parameter associated with the derived reference spectrum relative to the line width parameter associated with the calibration compound.

-5a-Identifying representative reference spectra may involve adjusting a parameter of the at least one derived reference spectrum until the at least one derived reference spectrum best aligns with the test spectrum.

Identifying representative reference spectra may involve producing a cluster position indicator for the derived reference spectrum, which causes the positions of peaks in the derived reference spectrum to match corresponding peaks of the test spectrum.

Identifying representative reference spectra may involve producing an upper bound concentration estimate of a quantity of a compound associated with the derived reference spectrum.

Producing an upper bound concentration estimate may involve selecting as the upper bound concentration estimate, a lowest concentration value selected from a plurality of concentration values computed for respective peaks in the test spectrum.

Producing the upper bound concentration estimate may involve finding the height of a peak in the test spectrum that corresponds to a peak in the reference spectrum.

Producing the upper bound concentration estimate may involve determining a concentration value for a peak as a function of the height of the peak.
Producing the upper bound concentration estimate may involve predicting whether the height of a peak in the test spectrum is greater than a threshold level and not determining a concentration for the peak when the height is less than the threshold level.
The process may involve adjusting the relative positions of peaks associated with one of the compounds according to pre-defined criteria.

-5b-Identifying representative reference spectra may involve determining scaling factors for peaks in a plurality of reference spectra such that the sum of the peaks scaled by the scaling factors optimally matches the test spectrum.

The process may involve determining concentrations of compounds associated with the reference spectra as a function of the scaling factors.

The process may involve producing an indication of compounds associated with reference spectra having peaks that when scaled by the scaling factors have a height greater than a threshold.

The process may involve outputting a value representing at least one of the concentrations.

The process may involve receiving the test spectrum from a spectrum measurement device.

The process may involve doping the sample with a condition indicator.
Doping may involve doping the sample with a pH indicator.

The process may involve doping the sample with a chemical shift reference compound.

The process may involve employing Nuclear Magnetic Resonance (NMR) to produce free induction decay data operable to be transformed into an NMR
spectrum operable to be used as the test spectrum.
The process may involve receiving Nuclear Magnetic Resonance (NMR) free induction decay (FID) data and processing the FID data to produce a -5c-representation of a measured spectrum having well-defined Lorentzian lines, a flat baseline and peaks that have positive well defined areas.

The may involve roducin the test s ec rum from h process p g p t t e measured spectrum.

Producing the test spectrum may involve producing a conditioned spectrum.
Producing the conditioned spectrum may involve producing a baseline corrected spectrum from the measured spectrum.

In accordance with another aspect of the invention there is provided a computer-readable medium for providing computer readable instructions for directing a processor circuit to identify compounds in a sample, the instructions. The computer readable medium includes a set of codes operable to cause the processor circuit to receive a representation of a measured condition of the sample mixture, a set of codes operable to cause the processor circuit to use the representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in the sample mixture, from a library of reference spectra, and a set of codes operable to cause the processor circuit to receive a representation of a test spectrum, produced from the sample mixture under the measured conditions. The computer readable medium further includes a set of codes operable to cause the processor circuit to combine reference spectra from the set of reference spectra to produce a matching composite spectrum having peaks representing at least some of the suspected compounds, that match peaks the test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture, and a set of codes operable to cause the processor circuit to store a representation of the matching composite spectrum.

-5d-In accordance with another aspect of the invention there is provided an apparatus for identifying compounds in a sample mixture. The apparatus includes a processor circuit programmed to receive a representation of a measured condition of the sample mixture, use the representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in the sample mixture, from a library of reference spectra, and receive a representation of a test spectrum, produced from the sample mixture under the measured conditions. The processor circuit is also programmed to combine reference spectra from the set of reference spectra to produce a matching composite spectrum having peaks representing at least some of the suspected compounds, that match peaks the test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture.

In accordance with another aspect of the invention there is provided an apparatus for identifying compounds in a sample mixture. The apparatus includes provisions for receiving a representation of a measured condition of the sample mixture, provisions for using the representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in the sample mixture, from a library of reference spectra, and provisions for receiving a representation of a test spectrum, produced from the sample mixture under the measured conditions.
The apparatus also includes provisions for combining reference spectra from the set of reference spectra to produce a matching composite spectrum having peaks representing at least some of the suspected compounds, that match peaks the test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture.

-5e-Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying Figures.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the invention, Figure 1 is a system for determining the quantity of compounds in a test sample, according to a first embodiment of the invention;

Figure 2 is a flow chart illustrating an automatic process for conditioning a measured spectrum, as implemented by a workstation shown in Figure 1;

Figure 3 is a pictorial representation of a measured spectrum produced by the workstation shown in Figure 1;

Figure 4 is a flow chart of a routine executed on the workstation shown in Figure 1, for conditioning the measured spectrum to suppress a peak caused by a solvent in a sample for which the measured spectrum is produced;

Figure 5 is a flow chart of a process for identifying compounds executed by a spectrum analysis apparatus shown in Figure 1;
Figure 6 is a pictorial representation of a reference spectrum associated with lactic acid at pH of 5.10;

Figures 7A and 7B are a tabular representation of an Extensible Markup Language (XML) file representation of the reference spectrum of Figure 6;

Figure 8 is a flow chart of a process by which base reference spectrum records such as shown in Figures 7A and 7B may be produced;
Figure 9 is a process executed by the spectrum analysis apparatus shown in Figure 1 to identify a peak associated with a calibration compound in a test spectrum;

Figures IOA and 10B are a flow chart of the process for identifying compounds, shown in Figure 5, in greater detail;

Figure 11 is a flow chart of a process executed by the spectrum analysis apparatus for determining a pH value from the test spectrum;
Figure 12 is a flow chart of a process executed by the spectrum analysis apparatus for producing a derived reference spectrum;

Figures 13A and 13B are a tabular representation of a base reference spectrum record associated with lactic acid at a pH of 5.45;

Figures 14A and 14B are a tabular representation of a derived reference spectrum record associated with lactic acid at a pH of 5.28;
Figures 15A and 15B are a tabular representation of a generic type of derived reference record in which equations specify center Parts Per Million (PPM) values for peak clusters, according to one embodiment of the invention;

Figures 16A and 16B are a tabular representation of a derived record comprising look-up table links to center PPM values according to another embodiment of the invention;

Figures 17 is a flow chart of a process for determining an upper bound concentration estimate;

Figure 18 is a flow chart of a least squares fitting routine referenced by Figure 10B;

DETAILED DESCRIPTION
Referring to Figure 1, a system, according to a first embodiment of the invention, for determining the quantity of compounds in a test sample is shown generally at 10. The system includes a spectrum producing apparatus 12 and a spectrum analysis apparatus shown generally at 14. In this embodiment, the spectrum producing apparatus 12 is a Nuclear Magnetic Resonance (NMR) System provided by Varian Inc. of California, U.S.A.
Generally, the system is operable to receive a specially prepared liquid biological test sample and produce a data file comprised of a plurality of (x,y) values which define a measured NMR spectrum. This measured NMR
spectrum is then supplied to the spectrum analysis apparatus 14, where a process according to another aspect of the invention is carried out to provide an indication of the quantities of certain compounds in the specially prepared biological test sample.
The system 10 is suitable for use with biological samples, for example blood or urine, in which the solvent is water, for example. Such samples may be "prepared" by doping them with a small quantity of a condition indicator compound, also referred to as a condition reference compound, and a chemically inert chemical shift calibration standard compound also referred to as a calibration compound. The condition indicator may be trimethylsilyl-l-propanoic acid or Imidazole, where the distortion factor is pH, for example.
Alternatively, the sample itself may have a naturally occurring, inherent condition indicator such as glycine, creatinine, urea, citrate, or trimethylamine-N-oxide, for example. The chemical shift calibration standard compound may be 3-[trimethylsilyl]-1-propanesulfonic acid, also known as DSS, for example.
Alternatively, the chemical shift calibration standard may be dimethylsulphoxide (DMSO), acetone, or tetramethylsilane (TMS), for example.
In this embodiment, the spectrum producing apparatus 12 is comprised of a computer workstation 16, an auto sampler 18, a test chamber 20, and a console 22. The workstation is a Sun Workstation with a 400 MHz UltraSPARC Ili CPU with 2 MB level 2 cache, 128 MB RAM, on-board PGX24 graphics controller, 20 GB 7200 r.p.m. EIDE hard disk 48x CD-ROM drive 1.44 MB floppy drive and 17" flat screen color monitor. The workstation runs Varian VNMR software which includes routines for controlling the auto sampler 18 and the console 22 to cause the specially prepared biological liquid sample to be received in the test chamber 20 and to cause the console to acquire and provide to the workstation Free Induction Decay (FID) data representing the free induction decay of electromagnetic radiation absorptions produced by protons in the compounds of the liquid sample as a result of changes in magnetic properties of the protons due to a nuclear magnetic resonance process initiated in the test chamber 20 by the console 22.

Process for Producing a Measured Spectrum The FID data is received and stored in memory at the workstation 16. Then, in this embodiment, a process according to an embodiment of another aspect of the invention, is carried out to cause the workstation to produce a measured spectrum for use by the spectrum analysis apparatus 14. Instructions for directing the workstation to automatically carry out the process for producing the measured spectrum are embodied in computer readable codes 24. These computer readable codes 24 may be provided to the workstation 16 in a variety of different forms including a file or files on a computer readable medium such as a CD-ROM 26, or floppy disk 28, for example, or as a file received as a signal from a communications medium such as an internet 30, extranet or intranet, electrical 32, Radio Frequency (RF) 34, or optical medium 36 or any other medium by which a file comprised of said codes may be provided to the workstation 16 to enable the workstation to be directed by the codes to execute the process described herein to produce a measured spectrum.
Autoprocessing Generally, an automatic computer-implemented process for producing a measured spectrum from NMR data, may involve operating on free induction decay (FID) data produced by a spectrometer to produce a trace file comprised of intensity and frequency values representing a measured spectrum having a flat baseline and well defined peaks that have positive, well-defined areas, for use in a computer-implemented spectrum analysis process such as the process described herein. In particular, the process may involve performing a Fourier Transform on Free Induction Decay (FID) data to produce an initial spectrum, filtering a selected region of the initial spectrum to produce a filtered spectrum and phasing the filtered spectrum to produce a measured spectrum having a flat baseline and well defined positive peaks.
Referring to Figure 2, a flowchart depicting functional blocks implemented by the codes to cause the workstation to execute a specific process for producing a measured spectrum is shown generally at 50. The process begins with a first block 52 that causes the workstation 16 to read and perform an initial Weighted Fourier Transform on the FID data to produce an initial measured spectrum representing signal intensity (i) versus frequency (F).
Then block 54 causes the workstation 16 to produce parameters for use in a later-executed Fourier Transform performed on the FID data to produce a representation of a measured spectrum having well defined Lorentzian lines with a flat baseline and peaks that have positive, well-defined areas. Thus, the result of block 54 is a set of parameters that controls Fourier Transforms later performed on the FID data to produce a representation of a measured spectrum.

Block 56 directs the workstation to save the set of parameters in association with the FID data. Block 58 directs the workstation 16 to perform a Fourier Transform on the FID data, using the parameters produced by block 54 to produce a trace file, which is a file comprised of a plurality of (x,y) values that represent a trace of the measured spectrum, representing intensity versus frequency. Block 59 then causes the workstation to save the trace file for transmission to the spectrum analysis apparatus 14 shown in Figure 1.

An example of a measured spectrum is shown generally at 41 in Figure 3.
The spectrum is a plot of intensity versus frequency. The x-axis 43 is referenced to parts per million (ppm) and depicts a window of the overall spectrum, the window containing relevant information or features for identifying compounds in the sample. The y-axis 39 is referenced to a zero value and the spectrum has a baseline 37 representing a noise level from which a plurality of peaks 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 associated with various compounds in the sample extend. For example peaks 45 and 47 are associated with Imidazole, peak 49 is associated with Urea and peaks 51 and 53 are associated with Creatinine. Peaks 55 and 57 form a first cluster associated with citric acid and peaks 59 and 61 form a second cluster associated with that compound. Peak 63 is associated with DSS, the calibration compound.

Referring back to Figure 2, block 54 which processes the FID data, is shown in greater detail. Block 54 includes sub-functional blocks including a Fourier Transform block 60, a filter selected and/or solvent region block 66 and an automatic phasing block 68, each of which is automatically executed in turn, in the order shown. The process may include an optional spectral window setting block 62 and an optional drift correction block 64, to further process the spectrum, for example.

The Fourier Transform block 60 has an optional sub-block 70 that causes the workstation to perform a weighted Fourier Transform with weights that provide for enhancement of the initial spectrum. These weights may perform a line broadening function to the initial spectrum, for example. To do this in this embodiment, block 70 causes the workstation to set signal enhancement parameters for use in a subsequently executed weighted Fourier Transform block 72. Such signal enhancement parameters may effect line broadening, line narrowing, or gaussian sine-bell conditioning, for example, to the resulting spectrum produced by the Fourier Transform block 72. In the Varian VNMR
software, this is effected by setting a line broadening variable "Ib" to a specified value, which may be 0.5, for example. Also in the VNMR software, the weighted Fourier Transform may be executed by calling the VNMR macro "wft" to perform a weighted Fourier Transform on the FID data, using the lb parameter value set at block 70. This has the effect of broadening the lines or peaks of the spectrum and averaging the spectrum to produce a measured spectrum with a better signal to noise ratio than would be produced without averaging. It also has the effect of eliminating glitches to produce a measured spectrum of better quality.

In this embodiment optional block 62 causes the workstation 16 to define a window on the initial spectrum and this may involve scaling the initial spectrum. It is desirable to set the spectral window to a preset size, i.e. a pre-defined range of frequency, to enable the acquisition of repeatable data and for all useful data to be in a pre-defined window and to scale the spectrum such that the height of its maximum peak is a percentage of the height of the window. In this embodiment, this is effected through the VNMR
software by executing three sub-functional blocks 74, 76 and 78 that cause the workstation 16 to call the VNMR macros "f', "full', and the VNMR
command "vsadj", respectively, in the order shown. The `f' macro sets display parameters "sp" and "wp" for a full display of a 1 D spectrum, the `full' macro sets display limits for a full screen so that the spectrum can be seen as wide as possible in the window, and the `vsadj' command sets up automatically the vertical scale "vs" in the absolute intensity mode "ai", so that the largest peak is of the required height. Effectively this provides for scaling of the spectrum so that the highest peak is 90% of the total window height.

Optional block 64 causes the workstation to produce parameters that perform drift correction on the spectrum to correct the measured spectrum for drift effects, effectively setting the two extremes of the baseline of the spectrum, i.e. the left and right sides of the spectrum to have zero slope. In this embodiment, using the Varian VNMR software, this is achieved by block 80 which causes the workstation 16 to call the "dc" macro of the VNMR software.
Effectively the "dc" macro calculates a linear baseline correction. The beginning and end of a straight line to be used for baseline correction are determined from the display parameters "sp" and "wp". The "dc" command applies this correction to the spectrum and stores the definition of the straight line in the parameters "9vl" (level) and "tlt" (tilt) of the VNMR software.
(cdc resets the parameters "Ivl" and "tlt" to zero.) Block 66 causes the workstation to filter a selected region of the spectrum to adjust the intensity of the spectrum in that region. Filtering may involve applying a notch filter to a selected or solvent region, for example, to suppress a peak associated with a contaminant or solvent in the contaminant or solvent region. This ensures that the solvent region or contaminant region of the spectrum is correctly phased with the rest of the spectrum so that the entire spectrum can be properly phased later. In order to permit the entire spectrum to be phased, the solvent or contaminant residual must be in phase with the rest of the spectrum, ideally reducing the solvent or contaminant region to zero. The solvent region is the region of the spectrum in which solvent compounds in the sample may be found. For example the solvent may be water, in which case the region around the peak in the measured spectrum associated with the compound H20 is considered to be the solvent region.
The contaminant region is a region of the spectrum where peaks associated with contaminants are present.
Referring to Figure 4, a routine for filtering the selected region is shown generally at 66 and involves a first block 92 that causes the workstation 16 to apply a notch filter to the selected region to suppress a peak in that region.
A
set of initial notch filter parameters specifying the attenuation, width and position of the notch filter is used.

Applying a notch filter may further involve producing an adjusted set of notch filter parameters and applying a notch filter employing the adjusted set of notch filter parameters to the selected region. The set of notch filter parameters may be adjusted to produce an adjusted set of notch filter parameters that may be applied to the notch filter to filter the selected region until a sum of the absolute values of areas defined by peaks above and below a baseline of the initial spectrum is minimized. In this embodiment this is done by block 94 which causes the workstation to adjust the set of initial notch filter parameters and re-apply the notch filter until the sum of the absolute values of the areas of the spectrum in the selected region, is minimized. One quick way of doing this and minimizing the number of iterations of application of the notch filter is to employ numerical methods to successive values produced. For example, in this embodiment, using the Varian VNMR
software, the parameter "sslsfrq" specifies a notch filter value that affects the minimization of the sum of the areas above and below the baseline. Brent's method, as described in Brent, R.P. 1973, Algorithms for Minimization without Derivatives (Englewood Cliffs, NJ: Prentice-Hall), Chapter 5, [1], for example may be used to find an optimum value for "sslsfrq".

Referring back to Figure 2, after filtering the selected region block 68 is invoked to automatically phase the entire spectrum and make the peaks as symmetrical as possible. This may be done iteratively, for example, by adjusting the real and imaginary components of the transformed FID data until the resulting spectrum has positive, well defined peaks. In this embodiment, employing the Varian VNMR software, this is achieved by invoking block 84 which calls the "aphO" command of the VNMR software. Some versions of the VNMR software may require more than one successive execution of the aphO command.

After automatic phasing parameters of the spectrum have been produced, optionally, a baseline correction block 69 may be executed to flatten out the baseline of the spectrum. Alternatively, baseline correction may be performed later. Baseline correction may be done by analysing the spectrum to determine areas with peaks and areas devoid of peaks and setting areas devoid of peaks to have a common intensity value such as zero, for example.
An example of baseline correction available at www.acdlabs.com/publish/nmr-ar.htmi published by Advanced Chemistry Development Inc. of Toronto, Ontario, Canada.

Block 56 then causes the workstation 16 to save parameters produced by the various sub-processes of block 54 in association with the FID data and text, if desired. With the Varian VNMR software this may be achieved using the `svf($savefid)' command.

Block 58 then directs the workstation 16 to produce a trace file comprised of (x,y) values representing intensity versus frequency, by performing a Fourier Transform on the FID data, using the parameters produced as described above and associated with FID data. The trace file is then transferred or transmitted to the spectrum analysis apparatus 14 or is stored for later transfer to that apparatus.

Spectrum Analysis Apparatus In the embodiment shown, the spectrum analysis apparatus (SAA) 14 is a separate component and includes a Linux workstation configured to receive the trace file representing the measured spectrum, from the spectrum producing apparatus 12. The spectrum analysis apparatus 14 is configured to receive and execute instructions embodied in computer readable codes to carry out a process for identifying compounds in a sample according to an embodiment of another aspect of the invention. The codes may be provided to the spectrum analysis apparatus through any of the media described above including the CD-ROM 26, Floppy disk 28, internet 30, extranet, intranet, electrical 32, RF 34, and optical 36 media and/or any other media capable of providing codes to the spectrum analysis apparatus.

It will be appreciated that the workstation 16 may alternatively be configured with both the codes to effect the process for producing a measured spectrum shown in Figure 2 and the codes to effect the process for identifying compounds, or either of these. It is desirable however, to execute the process for identifying compounds at a computer other than the workstation 16, to enable the process for identifying compounds to be executed while another sample is being subjected to the NMR process, for example.

Process for Identifying Compounds Referring to Figure 5, generally, the process for identifying compounds involves identifying representative reference spectra from a set of reference spectra associated with detectable compounds and selected according to a condition of the sample, which collectively define a composite reference spectrum having features matching a set of features in a test spectrum produced from the sample. Once the representative reference spectra have been identified, compounds with which they are associated may be identified.
The compounds associated with respective reference spectra of the identified set are the compounds that may be expected to be present in the sample.
Quantities of the compounds may be determined from the intensities of certain representative peaks in the test spectrum which are associated with the compounds, relative to the intensity of a peak associated with the chemical shift calibration standard compound which is unaffected by the condition of the sample. A condition may be the pH of the sample, for example, and an accurate measurement of pH can be obtained from the test spectrum. Thus, given a test spectrum of a sample and given a set of reference spectra, the process can identify and quantify compounds present in the sample. Alternatively, the condition may be temperature, osmality, salt concentration, chemical composition, or solvent, for example.
Reference Spectra Before the process for identifying compounds can be carried out, a set of reference spectra for compounds to be detected in the sample must be made available to the SAA 14. This can be done by storing data relating to reference spectra associated with respective compounds and allowing the SAA 14 access to the data. An exemplary reference spectrum for a given compound may initially be represented in the form of intensity versus frequency (x,y) values, which may be represented graphically. A reference spectrum for lactic acid is shown in Figure 6, for example. It will be appreciated that such a spectrum may have a plurality of peaks and/or clusters of peaks 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170 superimposed upon a featureless background, such as noise 172. The resolution along the x axis is dependent upon the frequency of the Magnet used in the Nuclear Magnetic Resonance Process employed to acquire the sample. The peaks that are associated with lactic acid are found in first and second clusters 166 and 154. These clusters are centered at 1.322 ppm and 4.119 ppm respectively. The first cluster is comprised of two peaks and the second cluster 154 is comprised of four peaks.

A reference spectrum of the type shown in Figure 6 can be represented in various formats including mathematical representations such as Lorentzian equations which may specify peaks associated with the compound the spectrum is intended to represent. Such equations have the form:

f(x) = a w2 w2 + 4(x-c)2 where: a represents amplitude of the peak w represents width of the peak; and c represents the center of the peak Thus, for example, the two peaks associated with the cluster centered on 1.322 ppm may be specified by two sets of Lorentzian line shape parameters a, w and c.

The Lorentzian line shape parameters for each peak associated with a given compound may be stored in a base reference spectrum record embodied in an XML file as shown in Figures 7A and 7B, for example. Such a file may have fields 200, 202 and 204, for example, for storing compound information, experiment information and cluster/peak information respectively. The compound information field may include sub-fields for storing the name of the compound with which the record is associated, and the molecular weight of the compound, for example. The experiment field may have sub-fields for storing information about the experiment, such as conditions under which the peak information about the compound was collected. This may include the pH of the solution that was analyzed, the temperature of the solution, the calibration reference compound ratio, the concentration of the compound in the solution, a timestamp, a sourcefile name, the frequency of the magnet used in the NMR process, and the spectral width of the entire spectrum, for example. The cluster/peak information fields may include separate fields 206 and 208 for each cluster (166 and 154 in Figure 6).

Each cluster field 206 and 208 may include sub fields 210, 212, 214, 216 and 218 for representing information relating to the proton number of the cluster, the quantification of the cluster, the Lorentzian line width adjustment of the cluster and first and second peak subfields respectively. The first and second peak subfields may include fields 220, 222, and 224 for representing offset center information, height information and proton ratio information relating to a respective peak in the cluster, respectively.

Effectively, the Lorentzian line shape parameters (a) and (c) for each peak may be stored in the height and offset center fields 222 and 220 respectively and each peak in a given cluster is considered to have the same width (w) which is specified by the contents of the Lorentzian line width adjust field associated with the cluster.

Referring to Figure 8, a process by which base reference spectrum records may be produced is shown generally at 230. The process begins with block 232 representing the preparation of a liquid solution containing a reference compound such as lactic acid, a calibration compound such as DSS and a condition indicator compound such as Imidazole. The liquid solution is prepared to a carefully calibrated concentration of the calibration compound at a carefully controlled temperature and pH. This step is carried out in a laboratory, by a human or by a mechanized process, for example.

Once the liquid solution containing the reference compound has been produced, as shown in block 234, it is subjected to the NMR process carried out by the apparatus 12 shown in Figure 1 to produce FID data.

At block 236, the apparatus 12 subjects the FID data produced by the NMR
process to the process shown in Figure 2, to produce a measured reference spectrum.

Having obtained a measured reference spectrum, a process as shown in block 238 is initiated to identify the calibration compound and obtain calibration parameters. This process is shown in greater detail at 238 in Figure 9. Referring to Figure 9, the codes direct the SAA 14 to derive from the measured reference spectrum a characterization of the calibration compound contained in the sample. This involves identifying a position of a peak of the measured reference spectrum that meets a set of criteria that associate the peak with the calibration compound and further involves producing parameters for a mathematical model of the peak, that best represents the peak. Thus, in this embodiment the characterization is a list of Lorentzian line shape parameters (w, c and a) representing width, peak position and center amplitude respectively of a Lorentzian curve that best describes a feature, that is, a peak, of the measured reference spectrum, that is associated with the calibration compound. It will be appreciated that other characterizations could be used, such as those produced by peak picking, linear least squares fitting, the Levenberg-Marquardt method, or a combination of these methods.
To find a peak associated with the calibration compound and to produce a list of Lorentzian line shape parameters that characterize it, the SAA 14 is programmed with codes that include a first block 250 that directs the SAA 14 to determine a noise level at a pre-defined area of the measured reference spectrum. In this embodiment, it is known that an area on the x-axis (frequency) corresponding to positions 64,000 and 65,000 for example can be expected to be void of peaks and contain only noise. The standard deviation of the y-value (signal intensity) over this region of the measured reference spectrum is representative of the noise level of the entire spectrum and provides a measure of the noise level.

Next block 252 directs the SAA 14 to scan the measured reference spectrum in the negative x-direction beginning at the higher order end of the spectrum, to find a y-value that meets a certain criterion. For example, the criterion may be that the y-value must exceed the noise level by a pre-determined amount, such as a factor of 10, at the top of a peak. A y-value meeting this criterion is assumed to be associated with an x-value that represents the position of a peak associated with the calibration compound.

Block 254 then directs the SAA 14 to employ the x-value representing the approximate position of the calibration peak in the test spectrum in a fitting algorithm that fits a curve to the calibration peak and specifies width, height and position values. For example a Lorentzian line shape-fitting algorithm may be employed to produce Lorentzian line shape parameters (a, w and c) that define a Lorentzian line shape that best matches the calibration peak.

Referring back to Figure 8, having calculated Lorentzian line shape parameters that identify and characterize the calibration compound, block 240 is carried out to associate other input data with the measured reference spectrum. Other input data may include information associated with the name and experiment fields 200 and 202 and information such as the number of protons (proton number in XML file) for each cluster and the proton ratio for each peak, for example.

Next at block 242, the measured reference spectrum is characterized by employing the well-known Conjugate Gradient method to determine Lorentzian line shape parameters (a, w and c) for each peak or to determine sets of such parameters that define a mathematical model or models of peaks that best fits the important peaks of the measured reference spectrum.

At block 244, a base reference spectrum record of the type shown in Figures 7A and 7B is produced from the other input data and the characterization of the spectrum. At block 246, the base reference spectrum record is stored in a reference record library, which effectively includes a plurality of reference records for various different reference compounds. For example, the reference record library may include base reference spectrum records for: L-phenylaianine, L-Threonine , Glucose, Citric Acid, Creatinine, Dimethylamine, Glycine, Hippuric acid, L-alanine, L-Histidine, L-Lactic Acid, L-Lysine, L-Serine, Taurine, Trimethylamine, Trimethylamine-N-Oxide, Urea, L-Valine, and Acetone.

Reference records may include base reference records or derived reference records. Base reference spectrum records may be produced by empirical processes as described above. New records known as derived reference records may be produced by operating on data from base reference records, and represent derived reference spectra. Operating on data may include interpolation and/or performing mathematical operations, and/or using a lookup table, for example. Thus, for example, a limited set of base reference spectrum records can be produced, including a record representing the spectrum for lactic acid at a pH of 5.1, and a record for lactic acid at a pH
of 5.45, for example. A derived reference record representing the spectrum of lactic acid in a solution having a pH of 5.28, for example, can then be produced by performing mathematical operations on the Lorentzian line shape parameters specified by the base records associated with solutions at pH 5.1 and pH 5.45 to interpolate values for a solution at a pH of 5.28. Thus, a derived set of reference records can be produced for solutions of any pH, within a reasonable range, when required, thereby avoiding a priori production of base reference records for every pH condition. As will be appreciated below, this feature may be exploited by determining the pH value of a sample under test and using the determined pH value to produce a set of derived reference records for use in identifying compounds present in the sample. In other words, reference records for use in the process for identifying and quantifying compounds are selected from existing base reference records or are "selected" by producing derived reference records, according to a condition of the sample. In this embodiment, the condition is pH.
Process for Identifying Compounds After having produced a reference library of base reference spectrum records, the process of identifying and quantifying compounds in a test sample can be carried out.

Process for Identifying and Qualifying Compounds The process is shown generally at 300 in Figure IOA and 10B and begins with an optional first block of codes 302 that cause the SAA to perform a spectrum conditioning step.
Spectrum Conditioning If the measured NMR spectrum of the test sample is of sufficient quality, it can be used directly in subsequent operations of the process disclosed herein.
However, usually, the measured spectrum will not be of sufficient quality and will require further processing to condition it for later use. This further conditioning may involve baseline correction as described earlier, for example, to produce a conditioned spectrum.

Thus the following description will refer to a test spectrum, which may be the measured spectrum described above, if such measured spectrum is of sufficient quality or it may be a conditioned spectrum. A measured spectrum having a corrected baseline, for example, would be an example of a measured spectrum that would not need to be subjected to further processing to condition it. Usually however the process will involve producing a test spectrum from the measured spectrum.

Calibration Determination After being provided with, or after producing, a test spectrum of the type described, the process involves block 304 to produce a characterization of a calibration compound in the sample or block 306 to determine a representation of a condition of the sample. These two functions can be done independently or the determination of the condition of the sample can be determined after first characterizing the calibration compound.

The process of characterizing the calibration compound generally involves identifying a peak associated with a calibration compound, in the test spectrum. This may involve identifying a peak meeting a set of criteria that associate the peak with the calibration compound. The peak associated with the calibration compound may be characterized by producing Lorentzian line shape parameters to represent the peak.

Block 304 relating to characterizing the calibration compound involves a call to the process shown in Figure 9 to cause the SAA 14 to produce a set of Lorentzian values (a, w and c) which best represent the peak associated with the calibration compound in the test spectrum.

Condition Factor Determination Optionally, as shown by block 308, a separate measuring device may be used to measure the selected condition of the test sample. In this embodiment, the measured condition is pH which may be measured by a separate pH meter to produce a pH condition value that may be supplied to the SAA as indicated at "C" in Figure 10, for use in later functions of the process.

If the condition value has not already been obtained desirably the condition value can be derived from the test spectrum itself as shown at block 306.
This is possible where the measured condition is pH because the identification of a peak associated with a pH indicator compound in a sample can be readily determined from the test spectrum and the Lorentzian line shape values that characterize the representation of the calibration compound in the test spectrum.

Referring to Figure 11, a process for determining a pH condition value from the test spectrum is shown generally at 310. Basically, the process involves identifying a position, height and width of a peak associated with a condition reference compound in the test spectrum and this may involve identifying a peak meeting a set of criteria that associate the peak with the condition reference compound. Once the peak is identified the measured condition value may be produced as a function of the peak position and parameters of the sample medium, the parameters being the parameters that define the calibration compound.

To achieve this, in this embodiment, the codes include a block 312 which directs the SAA 14 to employ the Lorentzian line shape parameter (c) associated with the calibration compound to locate a window in the test spectrum, where a peak associated with the pH indicator compound is expected to be. The window is then scanned along the x-axis (frequency) from left to right, for example, for a y-value (intensity) that is greater than the amplitude value specified by the Lorentzian line shape parameter (a).

When a y-value meeting the above criteria is found, block 314 causes the SAA 14 to execute a characterization algorithm to produce at least a center value (c) representing the center of the peak associated with the pH reference compound. For example a Lorentzian curve algorithm may be used to produce Lorentzian parameters a, w and c defining the peak associated with the pH reference compound.

Block 316 then directs the SAA 14 to execute a modified pH titration Equation as shown below, on the center value c and to use certain parameters of the sample solvent, in the equation, to produce a condition value representing pH
of the sample:

pH = pKa - logL &bs CSA
&HA - & bs where: Sabs is the observed chemical shift (center c);
8A is the chemical shift of the conjugate base;
8HA is the chemical shift of the conjugate acid; and PKA is an association constant for the conjugate base.

Assume that no matter what method of determining pH is used, a pH value of 5.28 is obtained for the sample. Referring Back to Figure 10B block 320 directs the SAA to receive the condition value either produced externally, such as by measurement or produced internally such as by using the test spectrum as described above, to produce a derived reference record representing a derived reference spectrum for use in later functions of the process. Separate derived reference records may be produced from corresponding base reference spectrum records associated with corresponding compounds expected to be in the sample. Thus, in effect a representation of a set of derived reference spectra may be produced from a set of reference spectra and the measured condition value. In general, a process for producing a representation of a spectrum for a hypothetical solution containing a compound, for use in determining the composition of a test sample, involves producing a position value for at least one peak of a reference spectrum as a function of the measured condition of the test sample and a property of the at least one peak in a base reference spectrum. The property may be a position of a peak, amplitude of the peak or width of the peak for example. In this embodiment, a derived reference record is used to represent a representation of a spectrum for the hypothetical solution.
Referring to Figure 12, producing a derived reference record may involve accessing a pre-defined record specifying peaks in a reference spectrum and adjusting a position value in the record, the position value being the position value of the at least one peak. This may be done by block 322 which causes the SAA to identify a base reference spectrum record that is associated with a condition nearest to the measured condition of the sample and to use such reference spectrum as the derived reference spectrum.

Producing a position value for a peak may involve interpolating a position value from position values associated with base reference spectra associated with condition values above and below the measured condition value associated with the sample. For example, block 324 may be employed to cause the SAA 14 to produce a position value by calculating the position value as a function of pH of the sample and to effectively produce or interpolate a derived reference spectrum.

To interpolate a derived reference spectrum, assume that at block 322 a base reference record for lactic acid at a pH of 5.10 is located as being the base reference spectrum record for lactic acid that is nearest to the pH of the sample, 5.28. Such a record is shown in Figures 7A and 7B. Referring back to Figure 12, block 324 may direct the SAA 14 to find another base reference spectrum record for lactic acid that is associated with a pH value greater than the pH of the sample. Assume that it locates a base reference spectrum record associated lactic acid at a pH of 5.45. A record of this type is shown in Figures 13A and 13B. On locating this second base reference spectrum record, block 324 directs the SAA 14 to create a new derived reference spectrum record for lactic acid at a pH of 5.28. To do this the SAA 14 is directed to make a copy of the base reference spectrum record associated with a pH of 5.45 and then to replace the frequency values for the center position of each cluster shown in that record, with interpolated values. A
simple linear interpolation is used to find the value 1.3202 for the first cluster and the value 4.1149 for the second cluster. Figures 14A and 14B show the resulting derived reference spectrum record for a pH of 5.28, for lactic acid, produced using this method. Similarly, derived reference spectrum records are produced for each compound in the reference library to produce derived reference records for a pH of 5.28 for each compound represented in the library.

Alternatively; adjusting the position of a peak may involve locating a measured condition value dependent function in a base reference record, or pre-defined record, producing the position value from the function and associating the position value with the pre-defined record. Associating may involve storing the position value in the pre-defined record, for example. To effect this method of adjusting the position of a peak, a generic type of derived record may be kept, in which equations, effectively specifying the centerPPM
values for the two clusters as a function of pH may be provided in the field associated with the centerPPM value for each cluster, as shown in Figures 15A and 15B. Then, whenever a pH value is found from a sample, a copy of the record can be made and the pH value may be used in the equations in the copied record to produce centerPPM values. These center PPM values can then be substituted for the respective equations that produced them, in the copied record, thereby producing a new derived record for use in later calculations.
Alternatively, producing a position value may involve producing the position value by addressing a lookup table of position values with the measured condition value of the sample. For example the position value of a peak may be adjusted by locating, in a pre-defined record, a link to a lookup table specifying peak positions for various condition values, retrieving the position value from the lookup table and associating the position value with the pre-defined record. To do this a second generic type of derived record may be kept, in which lookup table links, effectively specifying links to lookup tables (not shown) that return centerPPM values for input pH values may be provided in the field associated with the centerPPM value for each cluster, as shown in Figures 16A and 16B. Then, whenever a pH value is found from a sample, a copy of the record can be made and the pH value may be used to address the lookup tables associated with the links specified in the record to produce centerPPM values. These center PPM values can then be substituted for the respective links that produced them, in the copied record, thereby producing a new derived record for use in later calculations.

Referring back to Figure 10B, after having produced a derived reference spectrum for each compound that is likely to be in the sample, block 326 causes the SAA 14 to calibrate the Lorentzian line width values for the derived reference spectrum relative to the test spectrum to provide for a better fit to the test spectrum. To do this, block 326 may direct the SAA 14 to calibrate to the (a, c and w) values associated with the calibration compound in the sample, the spectral linewidths of peaks associated with each of the reference compounds. In this embodiment block 326 may direct the SAA 14 to employ the contents of the Lorentzian width adjust field 214 of each derived reference spectrum record to produce respective absolute values representing actual linewidths relative to the calibration compound Iinewidth. These modified spectral line widths may be associated with respective peaks in the same cluster of each reference compound, by storing these modified spectral line widths in an internal data structure (not shown) that associates modified spectral information with derived reference records.

Still referring to Figure 10B, optionally, compound specific adjustments as shown by block 328 may be made to the contents of the fields of the derived reference records, where it is known, for example that certain effects occur when certain reference compounds are present in the test sample. For example, the shift of peaks associated with citrate is affected by the presence or absence of certain divalent cations and therefore the process may include a compound-specific adjustment to compensate for shifts known to occur when the presence of such divalent cations is known. Other compound-specific adjustments may be made to compensate for shifts due to temperature, chemical interactions, dilution effect and other ligand effects.
Cluster Centering Still referring to Figure 10B the process may further involve a cluster centering step as shown at 330 for shifting the derived reference spectrum in frequency (x-direction) to better align it with the test spectrum. This may involve producing a cluster position indicator for a derived reference spectrum, which causes the positions of peaks in the derived reference spectrum to match corresponding peaks in the test spectrum. A cluster position indicator already associated with the derived reference spectrum may be used or a cluster position indicator that produces a match of the derived reference spectrum to the test spectrum to a defined degree may be derived from the cluster position indicator already associated with the derived reference spectrum. In the embodiment shown, producing a cluster center indicator is achieved by attempting to fit the cluster to the test spectrum. To do this, cluster center values around the cluster center value already associated with the derived reference spectrum are assigned to the derived reference spectrum and used to effectively shift the derived reference spectrum to the left and right of the current cluster center value. For example, cluster center values +/- 0.001 ppm points are successively assigned to the derived reference spectrum to successively shift the center of the derived reference spectrum at successive points in a window extending -0.003 ppm to +0.003ppm from the currently assigned cluster center. At each point, the derived reference spectrum is used in a Levenberg-Marquardt (LM) fitting algorithm that determines a correlation value for each position of the center of the derived reference spectrum in the window. The center position that causes the LM fitting algorithm to produce the best correlation value is then associated with the derived reference spectrum correlation value and is used in later calculations.
Thus in effect, the derived reference spectrum is "wiggled" into alignment with the test spectrum. This wiggling is done independently for each cluster of peaks in the derived reference spectrum.
Upper Bound Concentration Estimates Still referring to Figure 10B, in this embodiment, the process for identifying and quantifying further involves block 332 which causes the SAA 14 to produce an upper bound estimate of a quantity of a compound associated with a derived reference spectrum, for use in a least squares algorithm later in the process. In general, producing an upper bound concentration estimate comprises selecting as the upper bound concentration estimate, a lowest concentration value selected from a plurality of concentration values calculated from respective peaks in the test spectrum. This may involve finding the height of a peak in the test spectrum that corresponds to a peak in the reference spectrum and determining a concentration value for the peak as a function of its height. Prior to determining a concentration estimate for a peak, the process may involve predicting whether the height of a peak in the test spectrum is greater than a threshold level and deciding not to determine a concentration for the peak when the height is less than the threshold level.

Referring to Figure 17 a process implemented by program codes operating on the SAA 14 of Figure 1, for producing an upper bound concentration estimate is shown generally at 340. A first block 342 causes the SAA 14 to select a reference record. Next block 344 causes the SAA 14 to sort by height those peaks in the reference record that have a quantification value equal to 1.
This causes the process to consider only those peaks that provide reliable concentration estimates. Next, block 346 directs the SAA to address the (next) highest peak of those that have just been sorted at block 344.
Reference is made to the "next" high peak because the peaks are considered in succession. On the first pass through the process however, the highest peak found in the sort is the first peak addressed.

Next block 348 causes the SAA 14 to use the position of the currently addressed peak in the reference spectrum to locate a corresponding peak in the test spectrum. This may involve looking for a peak in a window positioned at a corresponding position in the test spectrum. On finding such a peak, the maximum intensity value (max(y)) associated with that peak is found.

At block 350, the SAA 14 is directed to calculate a concentration value as a function of the max (y) value, using the following equation:
Ct = adjustedwidth * max(y) * dssconcentration * dssprotonratio (17) Dssheight * peakprotonratio Where: Ct is the concentration value for the peak adjustedwidth is the width of the peak as determined from the variable w calculated as shown in Figure 9 and the Lorentzian width adjust value stored in the reference record max(y) is the maximum y-value associated with the corresponding peak in the test spectrum dssconcentration is the concentration of DSS in the sample 0.5mM, for example dssprotonratio is the DSS proton ratio (9, for example) Dssheight is the DSS height value a, calculated as shown in Figure 9 Peakprotonratio is the proton ratio of the peak, as indicated in the reference record.

At block 352 SAA 14 is directed to determine whether the currently calculated concentration value is less than the previously calculated value. If so, then block 354 causes the SAA 14 to set a preliminary upper bound concentration value to the current concentration value. If at block 352, the currently calculated concentration value is not less than the previously calculated value, the preliminary upper bound concentration estimate value remains at its former value. The effect of blocks 352 and 354 is to cause the preliminary upper bound concentration estimate to be set to the lowest concentration value calculated for any of the peaks.

Once the preliminary value has been determined from the current pass, block 356 directs the SAA 14 to determine whether all peaks with quantification values of 1 have been considered. If so, the SAA 14 is directed to optional block 357 in Figure 17. If not, the SAA 14 is directed to block 358 which causes the SAA to calculate the expected height of the next peak associated with the compound, in the test spectrum. To do this equation 17 above is solved for max(y) using the current preliminary concentration estimate, and the Lorentzian width adjust value, and the peak proton ratio of the next highest peak from the list of sorted peaks. Then, block 359 in Figure 17 causes the SAA 14 to determine whether the max(y) value so found is less than the noise level of the spectrum. (noise level was calculated at block 250 in Figure 9). If not, then the next peak is worth considering and the SAA 14 is directed to resume processing at block 346 to address the next highest peak in the sorted list.
If the estimated height of the next highest peak found at block 358 is less than the noise level of the spectrum, the SAA 14 is directed to an optional block 357 which increases the amplitude of the preliminary concentration estimate value by the amplitude of the noise in the test spectrum to produce a true estimate of the upper bound concentration limit for the compound. This is useful where concentration values are very low.

Then, finally, block 355 directs the SAA 14 to associate the true upper bound concentration estimate with the reference record, such as by storing the upper bound concentration estimate value in a field (not shown) of the record, or in a field of a data structure maintained in the SAA 14 to create such associations.
Least squares Fitting Referring back to Figure 10B, the process for identifying and quantifying compounds involves a block 334 which causes the SAA 14 to perform a least squares fitting algorithm using all of the derived reference records and the test spectrum to produce scaling values for each peak in each reference spectrum such that when all peaks from all reference spectra are summed they produce a composite spectrum that best matches the test spectrum.
Referring to Figure 18, the least squares fitting routine includes a first block 360 which causes the SAA 14 to produce "signature" spectra comprised of (x,y) pairs that define a composite spectrum representative of the sum of all Lorentzians in a given derived reference record. A separate signature spectrum is produced for each derived reference record. Thus a separate (x,y) array is produced for each derived reference record.

Block 362 then provides each signature spectrum, upper bound concentrations and the (x,y) array representing the test spectrum to a Linear Least Squares fitting routine, which in this embodiment is LS SOL licensed from Stanford University of California, USA. This routine returns scaling factors for each peak in each applicable reference record, such that when the scaled Lorentzian models specified in all applicable reference records are summed together to make a composite spectrum, the composite spectrum has features matching features in the test spectrum produced from the sample. These scaling factors thus identify representative reference spectra from a set of reference spectra associated with detectable compounds and selected according to the measured condition of the sample.

In this embodiment, an indication of compounds associated with reference spectra having peaks that when scaled by the scaling factors have a height greater than a threshold may be produced. This may involve producing a list of compounds, for example. Thus, scaled peaks having a height less than the threshold may indicate that the presence of the associated compound in the sample is questionable and therefore such compound should not be listed as being present in the sample.
Block 364 then causes the SAA 14 to employ these scaling factors in the following equation to quantify each compound by producing concentration values for each compound represented by a reference record:

Conc. = (DSSRatio * scalingFactor * cdb) / pxDSS

Where: Conc.: concentration of the given compound in the sample DSSRatio: the DSSRatio entry for the given compound (see field 202 in Figure 7A) scalingFactor: the scaling factor of the highest peak in the given compound (from least squares fitting) cdb: the concentration of the given database entry (see field 202 in Figure 7A) pxDSS: the pixel height of DSS in the spectrum (the value a as determined by the process shown in Figure 9) Block 366 then causes the SAA 14 to associate these concentration values with the compounds associated with the derived reference records.

Block 368 then causes the SAA 14 to produce a list or indication of compounds in the sample, along with their associated concentration values.
This list may be printed and/or displayed on a monitor, for example.
Concentration values may be expressed in moles, mmol/L, g/L or moles/mole, for example and absolute quantities may be obtained by a simple equation converting concentration to absolute quantity values, in moles, for example.
While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

Claims (49)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A computer-implemented process for automatically identifying compounds in a sample mixture, the process comprising:

receiving a representation of a measured condition of the sample mixture;

using said representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in said sample mixture, from a library of reference spectra;

receiving a representation of a test spectrum having peaks associated with compounds therein, said test spectrum being produced from the sample mixture under said measured condition;

combining reference spectra from said set of reference spectra to produce a matching composite spectrum having peaks associated with at least some of said suspected compounds, that match peaks in said test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being indicative of the compounds in the sample mixture; and storing a representation of said matching composite spectrum.

2. The process of claim 1 further comprising identifying compounds associated with said representative reference spectra.

3. The process of claim 2 wherein identifying compounds comprises identifying quantities of said compounds.

4. The process of claim 2 wherein identifying compounds comprises identifying concentrations of said compounds.

5. The process of claim 1 further comprising identifying a peak associated with a calibration compound, in said test spectrum.

6. The process of claim 5 wherein identifying said peak comprises identifying a peak meeting a set of criteria that associate said peak with said calibration compound.

7. The process of claim 5 wherein identifying said peak comprises producing Lorentzian line shape parameters to represent said peak.

8. The process of claim 1 further comprising receiving a measured condition value representing said measured condition of the sample.

9. The process of claim 8 further comprising producing said measured condition value.

10. The process of claim 9 wherein producing said measured condition value comprises measuring pH of the sample.

11. The process of claim 9 wherein producing said measured condition value comprises producing said condition value from said test spectrum.

12. The process of claim 11 wherein producing said condition value comprises identifying in said test spectrum a peak position associated with a condition reference compound.

13. The process of claim 12 wherein identifying a peak position comprises identifying a peak meeting a set of criteria that associate said peak with said condition reference compound.

14. The process of claim 12 wherein producing said condition value comprises producing said condition value as a function of said peak position and parameters of a sample solvent.

15. The process of claim 9 wherein producing said measured condition value comprises determining a pH value for the sample, from said test spectrum.

16. The process of claim 15 wherein determining a measured pH value comprises determining from said test spectrum, the location of a peak associated with a pH reference compound, in relation to a peak associated with a calibration reference compound.

17. The process of claim 16 wherein producing said condition value comprises producing said condition value as a function of said peak location and parameters of a sample solvent.

18. The process of claim 8 further comprising adjusting a set of base reference spectra according to said measured condition value, to produce said set of reference spectra.

19. The process of claim 18 wherein adjusting said set of base reference spectra comprises adjusting parameters of said base reference spectra according to a pH of the sample.

20. The process of claim 8 further comprising producing a derived reference spectrum in response to said measured condition value and a reference spectrum.

21. The process of claim 20 wherein producing the derived reference spectrum comprises identifying a reference spectrum that is associated with a condition value nearest to said measured condition value.

22. The process of claim 21 wherein producing the derived reference spectrum comprises deriving a value from at least one reference spectrum that is associated with a condition value nearest to said measured condition value.

23. The process of claim 22 wherein producing the derived reference spectrum comprises performing mathematical operations on parameters of a reference spectrum to produce new parameters for use as parameters of said derived reference spectrum.

24. The process of claim 20 further comprising identifying in said test spectrum a peak associated with a calibration compound and producing Lorentzian line shape parameters, including a line width parameter, to represent said peak.

25. The process of claim 24 wherein said derived reference spectrum is represented by at least one set of Lorentzian line shape parameters including a line width parameter, said process further comprising calibrating said line width parameter associated with said derived reference spectrum relative to said line width parameter associated with said calibration compound.

26. The process of claim 25 wherein identifying representative reference spectra comprises adjusting a parameter of said at least one derived reference spectrum until said at least one derived reference spectrum best aligns with said test spectrum.

27. The process of claim 25 wherein identifying representative reference spectra comprises producing a cluster position indicator for said derived reference spectrum, which causes the positions of peaks in said derived reference spectrum to match corresponding peaks of said test spectrum.

28. The process of claim 25 wherein identifying representative reference spectra comprises producing an upper bound concentration estimate of a quantity of a compound associated with the derived reference spectrum.

29. The process of claim 28 wherein producing an upper bound concentration estimate comprises selecting as said upper bound concentration estimate, a lowest concentration value selected from a plurality of concentration values computed for respective peaks in the test spectrum.

30. The process of claim 29 wherein producing said upper bound concentration estimate comprises finding the height of a peak in the test spectrum that corresponds to a peak in the reference spectrum.

31. The process of claim 30 wherein producing said upper bound concentration estimate comprises determining a concentration value for a peak as a function of said height of said peak.

32. The process of claim 31 wherein producing said upper bound concentration estimate comprises predicting whether said height of a peak in the test spectrum is greater than a threshold level and not determining a concentration for said peak when said height is less than said threshold level.

33. The process of claim 25 further comprising adjusting the relative positions of peaks associated with one of said compounds according to pre-defined criteria.

34. The process of claim 25 wherein identifying representative reference spectra comprises determining scaling factors for peaks in a plurality of reference spectra such that the sum of said peaks scaled by said scaling factors optimally matches said test spectrum.

35. The process of claim 34 further comprising determining concentrations of compounds associated with said reference spectra as a function of said scaling factors.

36. The process of claim 35 further comprising producing an indication of compounds associated with reference spectra having peaks that when scaled by said scaling factors have a height greater than a threshold.

37. The process of claim 35 further comprising outputting a value representing at least one of said concentrations.

38. The process of claim 1 further comprising receiving said test spectrum from a spectrum measurement device.

39. The process of claim 1 further comprising doping the sample with a condition indicator.

40. The process of claim 39 wherein doping comprises doping the sample with a pH indicator.

41. The process of claim 40 further comprising doping the sample with a chemical shift reference compound.

42. The process of claim 41 further comprising employing Nuclear Magnetic Resonance (NMR) to produce free induction decay data operable to be transformed into an NMR spectrum operable to be used as the test spectrum.

43. The process of claim 1 further comprising receiving Nuclear Magnetic Resonance (NMR) free induction decay (FID) data and processing said FID data to produce a representation of a measured spectrum having well-defined Lorentzian lines, a flat baseline and peaks that have positive well defined areas.

44. The process of claim 43 further comprising producing said test spectrum from said measured spectrum.

45. The process of claim 44 wherein producing said test spectrum comprises producing a conditioned spectrum.

46. The process of claim 45 wherein producing said conditioned spectrum comprises producing a baseline corrected spectrum from said measured spectrum.

47. A computer-readable medium for providing computer readable instructions for directing a processor circuit to identify compounds in a sample, the instructions comprising:

a set of codes operable to cause the processor circuit to receive a representation of a measured condition of the sample mixture;
a set of codes operable to cause the processor circuit to use said representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in said sample mixture, from a library of reference spectra;

a set of codes operable to cause the processor circuit to receive a representation of a test spectrum, produced from the sample mixture under said measured conditions;

a set of codes operable to cause the processor circuit to combine reference spectra from said set of reference spectra to produce a matching composite spectrum having peaks representing at least some of said suspected compounds, that match peaks said test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture; and a set of codes operable to cause the processor circuit to store a representation of said matching composite spectrum.

48. An apparatus for identifying compounds in a sample mixture, the apparatus comprising a processor circuit programmed to:

receive a representation of a measured condition of the sample mixture;

use said representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in said sample mixture, from a library of reference spectra;

receive a representation of a test spectrum, produced from the sample mixture under said measured conditions; and combine reference spectra from said set of reference spectra to produce a matching composite spectrum having peaks representing at least some of said suspected compounds, that match peaks said test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture.

49. An apparatus for identifying compounds in a sample mixture, the apparatus comprising:

means for receiving a representation of a measured condition of the sample mixture;

means for using said representation of a measured condition of the sample mixture to select a set of reference spectra of compounds suspected to be contained in said sample mixture, from a library of reference spectra;

means for receiving a representation of a test spectrum, produced from the sample mixture under said measured conditions; and means for combining reference spectra from said set of reference spectra to produce a matching composite spectrum having peaks representing at least some of said suspected compounds, that match peaks said test spectrum, the compounds associated with the reference spectra that combine to produce the matching spectrum being the compound in the sample mixture.