CA2475622A1 - Method and apparatus for measuring chemical compounds using scattered light spectroscopy - Google Patents

Abstract

The invention provides a method of determining the concentration of a compound of interest in a sample using scattered light spectroscopy. The method comprises providing a scattered light spectrometer comprising an algorithm developed for the compound of interest and introducing radiation of about 585nm to about 1635 nm to the sample. The radiation is collected after interaction with the sample, and the concentration of the compound of interest is determined using the algorithm. The present invention also provides an apparatus for scattered light spectroscopy.

Description

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USING SCATTERED LIGHT SPECTROSCOPY
The present invention relates to measuring chemical compounds using scattered light spectroscopy. More specifically this invention pertains to measuring absorbing and non-absorbing chennical compounds using scattered light spectroscopy.
BACKGROUND OF THE I1~1VENTION
There is a need for the development of instruments and associated methods for rapid analysis of blood and bodily fluids without application of chemical reagents.
Preferably, such methods and instrumentation for medical diagnosis would involve non-invasive spectroscopic methods. To date the success of non-invasive spectroscopic methods is limited, with Oxy-meters being an exception.
Current spectroscopic methods for non-invasive analysis of bodily fluids is performed using the long wavelength part of the near infrared spectrum (1,b00nm to 2,550nrn - LNIR) [1-6] or middle infrared spectrum (2,550nm to 11,OOOnm- MIR) [6]. Radiation in these spectral ranges usually can be measured with either InGaAs or lead sulfde detectors. However, radiation in the l,&00nm to 2,550nm, or 2,550nm to 11,000nm ranges is known to have shallow tissue penetration. Therefore potential for the application of these wavelengths for in-vivo medical analysis is very limited.
Radiation with shorter wavelengths has deeper tissue penetration. However, the application of infrared spectroscopy to the analysis of certain analytes in bodily fluids, for example electrolytes, is lacking due to a lack suitable features in the infrared absorbance spectrum of these electrolytes. Detection of electrolytes can be _2_ partially resolved using several methods, for example flame spectrophotometry (for potassium and sodium) or by chemical binding of electrolytes to some other species, whose spectral properties are modified in the process,.and these modified substances are used as indicators of the original substance concentration using ordinary spectrophotometry, [7,8].
All substances are able to leave their imprint on electromagnetic radiation through specimen specific, spectrally dependent absorbance, and also through specimen specific, wavelength dependent variations of propagation affected by refractive index. While the first imprint is widely used for spectaal identification of samples, and for analysis of their chemical composition, the second imprint for purposes of chemical analysis is practically non-existent mainly because of lack of suitable instrumentation and associated methods.
US 5,529,065 provides a method and apparatus for measuring internal information of a scattering medium using a light source having specific wavelengths and two or more detectors. The detectors are positioned on the surface of the scattering medium and receive light scattered by the medium and emitted from a light source of specific wavelength.
WO 00/ 20843 teaches a method and apparatus for determining optical parameters of turbid media. The apparatus includes the use of a probe containing both transmitting and receiving optics for delivering light to the media, and receiving back-scattered light from the media. This apparatus and method are directed to measuring spatially-resolved reflectance of the turbid media.
WO 02/40971 discloses an apparatus for determining optical parameters of turbid media. The apparatus comprises a plurality of spatial and angular detectors that resolve spatially diffuse Light, and angular resolved diffuse Light, respectively, from the sample. These detectors are located at different positions around the sample.

US 6,075,610 teaches a method and apparatus for determining optical parameters of a sample. The apparatus comprises a plurality of detectors positioned around the surface of the sample used to obtain measured values of the sample.
The mean of the measured values is used to determine a reference value indicative of an internal property of the sample.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.

SUMMARY OF TIC INVENTION
T'he present invention relates to measuring chemical compounds using scattered light spectroscopy. More specifically this invention pertains to measuring absorbing and non-absorbing chemical compounds using scattered light spectroscopy.
According to the present invention there is provided a method of determining the concentration of a compound of interest in a sample using scattered light spectroscopy comprising, i) providing a scattered light spectrometer comprising an algorithm developed for the compound of interest;
ii) introducing radiation of about 585nm to about 1635nm to the sample;
iii) measuring collected radiation after interaction with the sample;
iv) determining the concentration of the compound of interest using the algorithm.
The present invention pertains to the method described above, wherein the compound of interest does not exhibit a measurable variability in absorbance within the wavelengths of about 585nm to about 1635nm, and where the refractive index of the compoand of interest changes in a wavelength specific manner over at least a portion of the wavelengths of about 585nrn to about 1635nm.
Furthermore, the invention is directed to the method described above, wherein the step of measuring (step (iii)) involves measuring both scattered and absorbed radiation.
The compound of interest may be selected from the group consisting of protein, albumin, bilirubin, creatine, cholesterol, triglycerides, glucose, urea, intralipid, chloride, potassium, sodium, phosphorous, calcium, magnesium, manganese, iron, sulphur, zinc, aluminium, silicon, copper, nickel, arsenic, nitrogen, fluorine, lithium, selenium, bromine, cadmium iodine, mercury, gold, or other ion or compound that exhibits the property of a refractive index that changes with wavelength, and the sample is a body part, a liquid sample, or a gas sample.

The present invention also provides an apparatus for determining the concentration of a compound of interest in a sample using scattered light spectroscopy comprising, - a radiation source that emits radiation from about 585nm to about 1635nm, - a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation leaving the first optical transmission element to produce scattered radiation, and for directing the scattered radiation to the sample holder;
- the sample holder comprising two or more than two windows;
- a second optical transmission element for receiving the scattered radiation after interaction with the sample, and for directing the scattered radiation to one or more than one scattered radiation processing system;
-the one or more than one scattered radiation processing system comprising a diffraction grating, and a radiation detection system comprising one or more than one algorithms for determining the concentration of the compound of interest.
The present invention further embraces the apparatus described above, wherein the radiation detection system comprises a first and second set of lenses, the first set of lenses focusing the scattered radiation through .a slit, and the second set of lenses, positioned to receive the scattered radiation after passing through the slit, and comprising the diffraction grating, placed between the lenses in the second set of lenses. Furthermore, the second optical transmission element may be bifurcated and splits the scattered radiation into a first and a second scatta~ed radiation beam path.
The first scattered radiation beam path, a$er passing through the second set of lenses may be directed onto a photo diode array capable of detecting radiation from about 585nm to about 1635nm, and wherein the second scattered radiation beam path, after passing through the second set of lenses may be directed onto a photo diode array capable of detecting radiation from about 900nm to about 1635nm.

The present invention pertains to the apparatus a described above, wherein the diffraction grating is a volume diffraction grating.
The present invention also provides a method of determining the concentration of a compound of interest in a sample comprising, i) introducing scattered radiation to the sample using an apparatus comprising a) a radiation source that emits radiation from about 585nm to about 1635nn~
b) a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation leaving the first optical transmission element to produce scattered radiation, and for directing the. scattered radiation to the sample holder;
c) the sample holder comprising two or more than two windows;
d) a second optical transmission element for receiving the scattered radiation after interaction with the sample, and for directing the scattered radiation to one or more than one scattered radiation processing system;
e) the one or more than one scattered radiation processing system comprising a diffraction grating, and a radiation detection system comprising one or more than one algorithms for determining the concentration of the compound of interest ii) measuring radiation collected after interaction with the sample;
iii) determining the concentration of the compound of interest.
The present invention provides a spectrophotometer, which is sensitive to spectral variation of the absorption, and also spectral variation of refractive index. As a result, this spectrophotometer can be used for spectrophotometric measurements of chemical components that are characterized as not demonstrating a measurable variability of absorbance, but whose refractive index changes with wavelength in a substance specific manner. Applicability of this spectrophotometer was tested on liduid samples in the form of different matrices including samples prepared by mixing sera extracted from different animals. Good measurability was obtained for a range of compounds in serum.
This summary of the invention does not necessarily describe all necessary features of the invention but that the invention may also reside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
Figure 1 shows a schematic of operation principles of radiation scattering system Figure 2. shows a schematic of a spectrometer.
Figure 3 shows prediction of several compounds in water detected using radiation scattered spectroscopy. Figure 3a shows prediction of albumin in water with intralipid and glucose. Figure 3b shows prediction of intralipid in water with albumin and glucose. Figure 3c shows prediction of glucose in water with albumin and intralipid.
Figure 4 shows prediction of glucose in water using scattered light spectroscopy.
Figure 4a shows prediction of glucose in water with albumin and intralipid on the worst batch of 100 samples. Figure 4b shows prediction of glucose in water with albumin and intralipid on the best batch of 100 samples Figure 5 shows correlations between compounds in water, in the presence or absence of intralipid, using scattered light spectroscopy. Figure Sa shows correlation between concentrations of chemical components in the water samples not containing intralipid.
Figure Sb shows correlation between concentrations of chemical components in the water samples containing intralipid Figure 6 shows prediction of several compounds is water, in the absence of intralipid using scattered light spectroscopy. Figure 6a shows prediction of glucose in water and other analytes without intraiipid. Figure 6b shows rediction of albumin in water and other analytes without intralipid. Figure 6c shows prediction of sodium in water and other analytes without intralipid. Figure 6d shows prediction of chloride in water and other analytes without intralipid. Figure 6e shows prediction of phosphoraus in water and other analytes without intralipid. Figure 6f shows prediction of potassium in water and other analytes without intralipid.
Figure 7 shows prediction of several compounds is water, in the presence of intralipid using scattered light spectroscopy. Figure 7a shows prediction of glucose in water and other analytes with intralipid. Figure 7b shows prediction of albumin in water and other analytes with intralipid. Figure 7c shows prediction of sodium in water and other analytes with intralipid. Figure 7d shows prediction of chloride in water and other analytes with intralipid. Figure 7e shows prediction of phosphorous in water and other analytes with intralipid. Figure 7f shows prediction of potassium in water and other analytes with intralipid. Figure 7g shows prediction of intralipid in water with other analytes.
Figure 8 shows prediction of several compounds in animal serum using scattered light spectroscopy. Figure 8a shows prediction of glucose in animal serum.
Figure 8b shows prediction of albumin in animal serum. Figure 8c shows prediction of cholesterol. Figure 8d shows prediction of proteins. Figure 8eshows prediction of triglycerides. Figure 8f shows prediction of urea. Figure 8g shows prediction of sodium. Figure 8h shows prediction of potassium. Figure 8i shows prediction of chlorides. Figure 8j shows prediction of carbon dioxide. Figure 81~ shows prediction of phosphorous. Figure 81 shows prediction of creatinine. Figure 8m shows prediction of bilirubin.
Figure 9 shows prediction of several other compounds in animal serum using scattered light spectroscopy. Figure 9a shows prediction of magnesium. Figure 9b shows prediction of HDL .

DESCRIPTION OF PREFERRED EMBODIMENT
The present invention relates to measuring chemical compounds using scattered light spectroscopy. More specifically this invention pertains to measuring absorbirg and non-absorbing chemical compounds using scattered Light spectroscopy.
The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.
The present invention provides a spectrophotometer, which is sensitive to the spectral variation of refractive index of a compound in a sample. This invention also provides a method for determining the concentration of compound of interest in a sample. The spectrophotometer can be used for spectrophotometric measurements of chemical components that are characterized as not demonstrating a measurable variability of absorbance, but whose refractive index changes with wavelength in a substance specific manner. The spectroscopic system, and a method for using this spectroscopic system, may be used for spectral characterization of radiation transported through a sample, either biological or non-biological samples, and over a spectral range from about 585nm to about 1635nm.
The spectrophotometer, used for determining the concentration of a compound of interest in a sample comprises, - a radiation source that emits radiation from about 585nrn to about 1d35nm, - a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation leaving the first optical transmission element to produce scattered radiation, and for directing the scattered radiation to the sample holder;
- the sample holder comprising two or more than twowindows;

- a second optical transmission element for receiving the scattered radiation after interaction with the sample, and for directing the scattered radiation to one or more than one scattered radiation processing system;
the one or more than one scattered radiation processing system comprising a diffraction grating, a radiation detection system, and comprising one or more than one algorithm for determining the concentration of the compound of interest.
Also provided is a method of determining the concentration of a compound of interest in a sample. The method comprising, i) introducing scattered radiation to the sample using an apparatus comprising a) a radiation source that emits radiation from about 585nm to about 1635nm, b) a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation leaving the first optical transmission element to produce scattered radiation, and for directing the scattered radiation to the sample holder;
c) the sample holder comprising two or more than two windows;
d) a second optical transmission element for receiving the scattered radiation after interaction with the sample, and for directing the scattered radiation to one or more than one scattered radiation processing system;
e) the one or more than one scattered radiation processing system comprising a diffraction grating, a radiation detection system, and comprising one or more than one algorithm for determining the concentration of the compound of interest;
ii) measuring radiation collected after interaction with the sample;
iii) determining the concentration of the compound of interest using the one or more than one algorithm.
The compound of interest may be characterized as not exhibiting a measurable variability in absorbance within the wavelengths of about 585nm to about 1635nm. In this case the refractive index of the compound of interest changes in a wavelength specific manner over at least a portion of thewavelengths of about 585nm to about 1635nm. However, spectral variation of the absorption, associated with the compound of interest may also be used if any spectral variation is available.
By compounds or chemical components that are characterized as not demonstrating a measurable variability of absorbance, it is meant that the absorbance of the compound or component does not exhibit variability over a spectral range from about 585nm to about 1635nm. There may be an absorbance associated with the compound, but the associate absorbance does not change in in~nsity to any .
significant degree, or the absorbance is otherwise not measurable.
By a refractive index that changes with wavelength in a substance specific manner, it is meant that one or more than one of the wavelengths of the radiation comprise a change in refractive index as a result of interacting with a compound in a sample.
Physical Background In the simplest case of a plane electromagnetic wave [9,10] the electrical component E(Z~t), which is mostly responsible for a signal registered by photo detectors, measured in direction of wave propagation at the distance ~ from the origin at some moment t , can be expressed mathematically in the form:
~ (Z~t) = E~ei(cp + kz -Wit) (1)~
where:
E~, is the amplitude of vibrations of the electrical vector;
~ , is the phase of vibration at the origin of the coordinate system at the moment;
t=a~
Z, is the distance from the origin in the direction of the wave propagation;
~, is the circular frequency ofvibrations; and k, is the complex propagation constant, consisting of real, ~ , and imaginary parts, a k~m k = kRe + ikIm = ~x _ -2~(n + i -~'-Q-a ) (2) 4~
where:
1C' , is the complex index of refraction;
~,~ , the wavelength of the wave in vacuum;
Yt , the index of refraction; and Gl,' , the absorption coefficient.
The real part of propagation constant, k Re ' thus is related to refractive index yl kRe _= (cv )~ - 2~ ~ (3) a ~o and its imaginary part, ~ , to the absorption coefficient, GL , of the radiation in the m medium:
~rx m 2 In the absence of scattering, intensity, I (Z) , of a radiation beam at a distance, Z, averaged over the time significantly longer than vibration period of the electromagnetic field, can be found by calculation of the squared module of electrical component in expression (1):) ~(Z) 12 i ~(Z)~* (Z) , which leads to relation:
I (Z) = I (0)e_aZ (5) with j ( 0 ) being an initial intensity of the beam at origin of the coordinate system.
The above expression (5) clearly shows that in absence of scattering, the intensity of the radiation beam, which is directly measurable with radiation detectors, depends on the imaginary part of complex refractive index only (the absorbance coefficient), and does not depend on its real part i.e. on the refractive index. Hence, only substances, which absorb radiation can be identified from attenuation measurement.
In physical media both the refractive index and the absorption coefficient take different values for radiation with different frequencies (wavelengths or wave numbers) [9-10]. This relationship is a result of the atomic and molecular structure of matter, due to the dynamic redistribution of electrical charges in atoms or in molecules enforced by an imposed electromagnetic field. According to nuclear theory, atoms consist of a positively charged nucleus surrounded by a cloud of electrons with coincident centers of gravity, resulting in an electrically neutral atom.
Molecules consist of a set of electrically interacting atoms, which, as a result of interaction, produce a certain joint distribution of positive and negative charges. in space. According to quantum mechanics theory, there are limits on the variety of spatial distributions, which both charges can take in space. This gives rise to particular spatial structures (usually called states but for clarity, referenced here as nodes, by association with vibration nodes) that can be permanently maintained in a state of equilibrium. In an equilibrium state, the centers of gravity of both positive and negative charges can be coincident, producing a neutral particle, or be spatially separated resulting in a polarized dipole-like structure. Since spatial distribution of charges for each node is different, relative potential energy of the charges in each node also differs and the transition from one node to another either requires energy, which has to be delivered from outside when particle is transferred from a node with lower potential energy to anode with higher potential energy, or energy is~released When a transition occurs in the opposite direction. One of several possible mechanisms for such a transition includes an energy exchange with an external electromagnetic field from which energy either can be taken, in the act of absorption, or released, in the form of a radiated photon. Once the particles (atoms or molecules) reach a state of equilibrium, the charges may perform certain periodical vibrations around the center of equilibrium. This state of equilibrium defines physical properties of externally non-disturbed matter.
The situation changes when an external, periodically changing electromagnetic field is applied. The field distuxbs the fine equilibrium between the charges and enforces their periodical spatial redistribution, resulting on average in an additional polarization effect, for example the creation of a mean electrical dipole. The mean dipole value for each particle (atom or molecule) depends on vibration frequency of incident electromagnetic wave and other factors such as mass, charge, spatial distribution of charges in space (node) and so on. The mean dipole value therefore depends on molecular and atomic structure of the particles under consideration and their physical state. Hence each kind of particle, each in a different state (node) produces a different, sample-spedfic, and node-specific, impact on the electromagnetic field. The strength of this impact can be additionally influenced by the presence and state of other particles in neighborhood. The dipoles interact with an incident electromagnetic wave affecting its propagation, and the strength of this impact depends on all these parameters, which determine thevalue of the dipole.
The total effect of the induced dipoles on an electromagnetic wave is proportional to number of dipoles per unit volume interacting with the wave, hence is dependent on the spatial density of each kind of particle in each state present in the sample. Their impact on the propagation velocity of the electromagnetic wave is expressed by the refractive index of the medium. The refractive index represents the collective impact of each type particle in all possible states per unit volume on the propagation velocity of the wave. Hence, on a macroscopic scale, the impact is expressed as a sum normalized to unit volume of all partial contributions of each type of particle for all possible states in the medium under consideration. The contribution for a single type of particle in a single state can be expressed mathematically in terms of a complex function, resembling that used for the damped harmonic oscillator in the presence of an external harmonic excitation. The total effect represented byrefractive index is obtained by summation of contributions of each type of particle in all possible states contained in unit volume.
The final complex refractive index is a surn of contributions made by all substances and its squared value 1~ 2 can be expressed as:
z x2 c~ =1+ Nlel far ~ 2 ~~ 2 _i .
l 0 Jr ~r y .1 r Jr (6) where:
l , is the number of different component present in the sample;
NI , the number of atoms or molecules of the given substance per unit volume;
a , the charge of electron or ion in a molecule;

yy~l, the mass of l -th component;
g0, the permittivity of free space; , ~ , , the j-th resonant frequency for l th particle;
.~l .~ the sample specific oscillator strength for, ~ the resonant frequency;
~Jr .Ir the sample specific frictional canstant for the ~ frequency.
~Jr .Ir The above expression implies that both the absorption coefficient and the refractive index are sample specific, and dependent on the frequency of radiation {or equivalently said, dependent on the waveiength or wave number). Since both the absorption coefficient and the refractive index demonstrate strong frequency dependent behavior, they may be used for spectral analysis.
The imaginary part, which expresses absorption of radiation in a medium, can be measured with relative ease and. is widely used in spectroscopy for analysis of the chemical composition of substances. ~iowever, the spectral dependence of the refractive index on wavelength of radiation is seldom used for this purpose.
The measurement of the spectral dependence of absorbance, which is directly related to the extinction coefficient, consists in comparing the intensity of the radiation incident on the sample with that affected by the sample. Compound-specific spectral variation of absorption is expressed by Beer's law, and dependence of absorption on.the concentration ofthe compound is commonly used in spectroscopic analysis. Conversely, spectral dependence of the refractive index is more difficult to measure, since refractive index variations influence only the radiation group velocity, and the propagation direction in a medium. Refractive index variations do not normally affect the intensity of radiation. Furthermore, there is no simple relation between changes in one of these two parameters (i.e. radiation group velocity, and propagation direction in a medium) and concentration, as is the case with Beer's law.
However, with an appropriate signal, multivariate regression may be used to find correlations between refractive index information and a corresponding concentration of an analyte. With this approach, measurement of the concentration of an analyte is determined using information contained in the spectrum of radiation that is reflected or back scattered by the sample. This approach is possible when the registered signals depend not only on the absorption coefficient but also on refractive index of the sample.
Reflected and backscattered signal are very weak, especially if they are taken from boundary layer between two media of comparable refractive index, such as the boundary between a cuvette wall and liquid inside the cuvette. The signal may be increased, for example by increasing the multiple total internal reflection from a boundary surface, to a degree sufficient to measure the concentration of components, that are otherwise not measurable with standard spectroscopic methods, because of an insufficient absorption signal.
There are two physical properties, which can be used to transform information -lg-related to refractive index into measurable intensity variations. One consists of an apparent distance change when a remote object is observed through a medium whose refractive index varies. The second consists of a dependence of the refraction angle of radiation on the refractive index of a prism. Both properties can be used b translate variability of the refractive index into intensity variability associated with scattered light interacting with a sample.
When non-collimated scattered radiation is transmitted through a scattering or non-scattering media, the intensity of radiation collected by an optical system with a limited field of view and a limited numerical aperture (i.e. a limited angular collecting capability) strongly depends on the refractive index of the medium. As a result, the intensity of radiation transported through this media is both absorption and refractive index dependent, and contains more information about sample than spectral dependence of absorption alone. This capability can be demonstrated with reference to the apparatus shown in Figure 1. In this apparatus, a collimated beam of radiation (BR; 20), is produced by an illumination optical system (IOS; 10). The beam illuminates a strongly light scattering diffuser (D1; 40), which may simultaneously acts as a window for cuvette (C; 30), containing a sample, S, with a refractive index, y~ : The cuvette is closed with another diffuser (D2; 50). Due to the refraction of light in the medium, the refractive index of the sample influences the optical distance between the diffusers. The optical distance decreases proportionally to the refractive index of the medium between the diffusers.
For an infinitely large incident beam and an infinitely large La~mbertian diffuser, the radiation intensity on the second diffuser would not depend on the refractive index of the medium between diffusers, and the radiation collecting system (RCS; 60), which delivers radiation to the detector (PD; 70), would provide the same signal regardless of refractive index of the substance between them. If these conditions are not fulfilled, as is the case in a typical system, the radiation intensity on the second diffuser (50) depends on the optical distance from the first diffuser (40), and hence on the refractive index of the medium between diffusers, and the amount of radiation captured by the radiation collecting system {60) and delivered to the photodetector (?0) depends on refractive index of the medium between diffusers. The signal is also affected by absorption of the sample medium, therefore the measured signal contains information related to both factors: absorption and refractive index of the sample medium placed between diffusers.
The situation becomes more complex, when the sample medium between diffusers {40, 50) contains scattering centers in the form of snail, suspended particles.
In this case, the scattering efficiency of suspended particles depends on the ratio of their size, to the wavelength of the interacting radiation, and on relative refractive index of the particles in relation to the surrounding medium. Therefore, these particles contribute additional information on refractive index of the medium.
As is readily apparent, the scattering of radiation by the first diffuser (40) and by particles suspended in the medium, different photons may travel different distances before they reach the second diffuser (50), and eventually the detector (?0). The chance that a photon is absorbed varies depending on traveling path, which may also depend on the refractive index.
Variation of the refractive index on the intensity of the collected radiation, can be further enhanced by using a wedge shaped sample holder (30) and a wedge shaped sample. However, rectangular or circular shaped sample holders, and sample, may also be used as required. A wedge shaped sample introduces variability due to optical distance variation, and also due to variability in the refraction of radiation {hence the capability to collect radiation with different wavelengths) that also becomes dependent on the refractive index, and takes full advantage of changes caused by refractive index variability. The variability of the refractive index, obtained using a wedge-shaped sample, can be then registered by the detecting system (80). The registered signal also depends on properties of beam forming system, optical properties of the diffusers and the collecting capability of the light collecting system.
Therefore, contrary to ordinary spectrometers, the results obtained with radiation scattering samples are typically instrument dependent. As aresult, the simple relation between the absorbance of a chemical compound to the concentration of chemical compound in sample, is not readily obtained.
However, as described in more detail below, a suitable calibration model can be developed using techniques of multivariate regression, for example but not limited to, partial least squares regression (PLSR, PCAR), supervised neural network or other techniques that would be known to one of skill in the art, for samples of a given shape and volume. Furthermore, substances, which do not posses characteristic absorption bands may still be measured, due to compound specific dependence of refractive index on wavelength.
Therefore, the present invention provides a spectrophotometer, which is sensitive to spectral variation of the absorption, and also spectral variation of refractive index of a sample. The spectrophotometer can be used for spectrophotometric measurements of chemical components that are characterized as not demonstrating a measurable variability of absorbance, but whose refractive index changes with wavelength in a substance specific manner. The spectroscopic system, and a method for using this spectroscopic system, was developed for spectral characterization of radiation transported through a sample, for example but not limited to biological and non-biological samples including a body part, for example but not limited to a human finger, an ear lobe and the like. A spectral range from about 585nm to about 1635nm may be used for spectral characterization of the sample.
Therefore, the present invention provides a method of determining the concentration of a compound of interest in a sample using scattered light spectroscopy comprising, i) providing a scattered light spectrometer comprising analgorithm developed for the compound of interest;
ii) introducing radiation of about 585nrn to about 1635 nm to the sample;
iii) measuring collected radiation after interaction with the sample;
iv) determining the concentration of the compound of interest using the algorithm.

Furthermore, the compound of interest may be selected from the group consisting of protein, albumin, bilxrubin, creatine, cholesterol, triglycerides, glucose, urea, intralipid, chloride, potassium, sodium; phosphorous, calcium, magnesium, manganese, iron, sulphur, zinc, aluminium, silicon, copper, nickel, arsenic, nitrogen, fluorine, lithium, selenium, bromine, cadmium iodine, mercury, gold, or other ion or compound that exhibits the property of a refractive index that changes with wavelength. The sample may be a gas or liquid sample, or a body part, for example but not limited to a finger, ear lobe or other body part where radiation may be passed through.
Description of the measurement system.
With reference to Figure 2 there is shown a system for the spectral characterization of radiation scattering samples. Visible and infrared radiation produced by a suitable light source, for example a halogen (HL; 90}, xeon, metal halide, fluorescent lamp is collected by means of an elliptical mirror (EM;
100) and optionally passes through a heat rejection filter (HRF; 110), before it enters a radiation delivering optical system, also referred to as a first optical transrriission element. The optical transmission element may consist of one or two radiation guiding elements (R.GE 1; 120; and RGE2; 140), with a shutter (SH; 130} placed between them.
However, the shutter may be placed elsewhere, as desired. The shutter (130) is able to block radiation completely from entering a sample holder (SAH; 30) through the second radiation-guiding element (RGE2; 140). The radiation guiding elements (the first optical transmission element) may be any suitable element for transmitting radiation, for example but not limited to one or mare than one fiber optic bundles, or radiation guiding rods, for example but not limited to fused silica rods. In this arrangement, no additional beam-forming elements such as lenses or mirrors need to be used, however, beam- forming elements may be used if desired. To further increase production of random scattered radiation, the end face of the second guiding element (140) acts as an artificial, spatially extended radiation source producing within its emission cone randomly scattered radiation, which enters the sample holder (30).

The sample holder (30) may be of any shape for holding a liquid or solid sample, it may be a receptor adapted to receive a body part, as is known in the art, for example but not limited to US 5,631,758; which is incorporated herein by reference.
A non-limiting example of a sample holder, and an enlarged view of the holder (30), adapted for receiving a finger and having the cross-sectional shape is shown in Figure 2. The body of the sample holder (30) may be made of a nontransparent material with two holes, each enclosed by windows (Wl; 170, and W2; 180), to deliver radiation to, and collect it from, the sample (150,160) within the holder (30). The windows may also be modified to act as diffusers, as indicated above. The sample may be a body part, for example but not limited to a finger, an. ear lobe, a body part where radiation may be passed through, or an artificial member (150, 170). A non-limiting example of an artificial member is disclosed in W~ 01/115597 (which is incorporated herein by reference). The artificial member has a sample container {SC, 150) with an axial bore containing a liquid sample (LS, 160), or other desired medium. The sample container (150) is made of highly scattering, for example, but not limited to PTFE.
For reference measurements the sample container is replaced by a thermally stable radiation attenuator (32), consisting of a set of diffusers (D; 36) and, placed between them, a metallic mesh with an array of fine holes (M, 38). The density and size of the holes in the mesh {38), as well as the number of diffusers (36) in the attenuator are selected to obtain optical characteristics comparable to the sample and to achieve the attenuation optimal for the best signal to noise, S/N, ratio of the reference measurement. A~ alternative sample holder (34) contains at least one diffuser (D, 36) on each side of standard spectrometric cuvette or vial (CU, 39).
Furthermore, highly mixed divergent radiation delivered to the sample holder (30) by the radiation delivering optical system is additionally scattered either by the sample surface, for example skin in the case of a body part, the radiation scattering container (150, 34) or diffusers (e.g. 170, 180, or 36), before entering the sample. The sample itself may also contain radiation scattering centers.

The radiation transported through the sample is further scattered on exit, by a similar set of components (e.g. skin, diffusing walls, diffusing windows or diffuser), before directing the scattered radiation to a scattered radiation processing system.
The scattered radiation processing system may comprise a second oprical transmission element (190), a set of first lenses (200, 220), optionally a shaping filter (210), a slit (230), a second set of lenses (240, 260), a diffiaction grating (250), one or more than one detector (e.g. 2?0, 280), optionally one or more than one camera (e.g.
290, 300), and a computer for processing the received data (310, 80).
After leaving the sample, the scattered radiation enters a second optical transmission element, for example but not limited to a bifurcated fiber optic bundle (BFB, 190) whose radiation collecting end is placed as close to the sample holder (30) as possible to secure e~cient collection of light and to reduce or eliminate a need for additional radiation collecting elements such as lenses or mirrors. The bundle (190) collects radiation from the sample holder (30) and, if desired, divided, for example into two or more than two parts. However, the scattered radiation processing system may comprise one detection system, and the second optical transmission element may be undivided, delivering the scattered radiation to one detection system. The number of optical transmission elements the scattered radiation may be divided into can be modified as desired depending upon the application and the number of detector systems used in the scattered radiation processing system. In the present example, which is not to be considered limiting in any manner, the radiation is delivered to two separate spectrophotometers. Also, in the present example, the spectrophotometers of the scattered radiation processing system are array-based. However, other spectrophotometers, as would be known to one of skill in the art, may be used in place of an array-based system.
In the present example, one spectrophotometer {EC1, 290) contains a silicon photodiode array (Si PDA, 280) and covers spectral range from about 585nm to about 1180nm with resolution of about l4nm, while the other spectrophotometer (EC2, 300) contains an InGaAs photodiode array (InGaA PDA, 270) and covers spectral range from about 900nm to about 1635nm'with resolution of about ll.Snm. Variations in the type of arrays used, the spectral range of the array, and the resolution of the array, as can be determined by one of skill in the art are, and these variations are considered to be included within the scope of the present inventi~n. Both spectrophotometers may otherwise be similar.
If a bifurcated bundle (190, the second optical transmission element) is used in the scattered radiation processing system, then radiation from the each leg of the bifurcated fiber optic bundle (190) is captured by lens (L1, 200) located at a distance equal to the focal length of the lens. For example, which is not be considered limiting, the f# close to 2.2 may be used for L 1 (200). Radiation then optionally passes through a closely placed, long wavelength transmission-shaping filter (SF, 210) of the scattered radiation processing system. Filter (210) eliminates second order of di~action of grating (DG, 250) by blocking short wavelength radiation in the working range of the spectrophotometer. When the sample is placed in the sample holder, filter (210) equalizes the response of the system across the spectral range of the spectrometer. The shaping filter (210) for example, may be designed to equalize response of the spectrophotometer for samples with optical characteristics of the human finger. This secures a uniform dynamic range for all wavelengths of the spectrophotometer in the presence of the sample. A second lens (L2, 220) is placed as close to the filter (210) as possible, and captures the radiation transmitted through the filter (210) and creates an image of the fber optic bundle end in a slit plane (S, 230).
The width of the slit (230) assists in determining resolution of the spectrophotometer.
The radiation transmitted through the slit (230) is captured by another lens (L3, 240) of similar f# but longer focal length as lens L1 (200). Lens L3 (240) is placed at the distance equal to the focal length of the lens.
The beam of radiation from lens L3 (240), is directed, and passes through, a high efficient volume holographic transmission grating (DG, 250). The diffraction grating may be any suitable high efficiency grating, for example but not limited to that disclosed in WO 01/37014 (which is incorporated herein by reference). However, an alternate device may be used to decompose the radiation and obtain its corresponding spectrum, for example but not limited to a prism. The radiation dispersed by the diffraction grating is collected by another lens (L4, 260) that is similar to L3 (240), and is focused to create spectrally dispersed images on the slit on a photosensitive surface of a suitable detector, fox example a linear photodetector array, either Si PDA
(280) for the first spectrophotometer, or InGaAs PDA (270) for the second spectrophotometer. In both cases the resolution of spectrophotometers is far from the diffracted limited for the applied optics and is determined by the slit width used in spectrometers as can readily be determined by one of skill in the art. Non-limiting examples of slit widths to be used in the system presently described are about 0.3mm and about 0.2mm for the spectrometers with silicon (280) and InGaAs arrays (270), respectively.
The arrays may be cooled, for example a thermoelectrically cooled, 256 element arrays, with O.OSmm wide pixels, produced by Hlamamatsu. A silicon detector array may be used in the spectrophotometer predestined to work in the about 585nm to about 1180nm range, while a InGaAs array may be applied in the spectrophotometer for about 900nm to about 1635nm spectral range. The signals captured by the arrays are extracted using standard electronic cameras (ECI, 290; and EC2, 300), for example but not limited to an electronic camera obtained from Hamamatsu, and digitized using an AID converter, for example but not limited to a National Instrument I6bits AID converter. Data is then stored in a computer memory for further processing. Further processing may include development of a calibration algorithm as is known in the art, for example using MATLAB.
Software tools used for developing calibration algorithms comprises of the following: MathlabTM used to create programs for smoothing absorbances and derivative of absorbances; StafViewTM used to create algorithms by "step-wise multiple linear regression." PirouetteTM may be used to create calibration algorithms by Partial Least Squares (PLS) or Principal Component Analysis (PCA). It will be appreciated however that other software tools may also be used. It will also be appreciated that any statistical technique may be used, for example, which should not be considered limiting in any way, simple linear regression, multiple linear regression, and multivariate data analysis. Examples of multivariate data analysis, which should not be considered limiting in any way, are Principal Component Analysis (PCA), Principal Component Regression (PCR), Partial Least Squares regression (PLS), and Neural networks.
The above-described spectrophotornetric system, was used for the analysis of solutions comprising various analytes in water and animal serum. A first example included the analysis of concentration of glucose, albumin and intralipid in water, to verify the measurability of non-correlated analytes in the presence of a radiation scattering media. Other determinations were performed to demonstrate the usability of the instrument for the measurement of various analytes, including these without clear absorbance bands within applied spectral range.
As described in more detail in the examples below, scattered light transmission spectrometry in long wavelength visible and short wavelength NIR
parts of the spectrum (far example but not limited to 580nm to 1380nm) may be used to measure physiologically important chemical components, including those normally considered immeasurable with direct spectroscopic methods. lVieasurement of these compounds may be carried out in various matrices, for example but not limited to animal serum, biological fluids or a non-biological sample, and without any chemical treatment of the sample. Other chemical components, not disclosed in the examples, can also be measured with the apparatus described herein. Non-limiting examples of such chemical components include, but are not limited to protein, albumin, bilirubin, creative, cholesterol, triglycerides, glucose, urea, intralipid, chloride, potassium, sodium, phosphorous; calcium, magnesium, inangane~, iron, sulphur, zinc, aluminium, silicon, copper, nickel, arsenic, nitrogen, fluorine, lithium, selenium, bromine, cadmium iodine, mercury, gold, or other ion that exhibits the property of a refractive index that changes with wavelength, or other compound that exhibits the property of a refractive index that changes with wavelength Furthermore, measurement precision can be further improved, as required, by optimizing the apparatus described herein so that a large number of serum analytes may be analyzed. Since the present method does not require the use of additional, consumable reagents, expense associated with each analysis is less than that of present multifunctional analyzers used in medical laboratories.
Therefore, the present invention provides a spectrophotometer, which can be used for spectrophotometric measurements of chemical components that are characterized as not demonstrating a measurable variability of absorbance, but whose refractive index changes with wavelength in a substance specific manner. The apparatus may be used for determining the concentration of a compound of interest in a sample using scattered light spectroscopy. The apparatus comprising, - a radiation source that emits radiation from about 538nm to about 1635nm, - a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation lea~ring the f rst optical transmission element to produce scattered radiation, and for directing the scattered radiation to the sample holder;
- the sample holder comprising two or more than two windows;
- a second optical transmission element for receiving the scattered radiation after interaction with the sample, and for directing the scattered radiation to one or more than one scattered radiation processing system;
-the one or more than one scattered radiation processing system comprising a diffraction grating, a radiation detection system, and comprising one or more than one algorithm for determining the concentration of the compound of interest.
The above description is not intended to limit the claimed invention in any manner, furthermore, the discussed combination of features might not be absolutely necessary for the inventive solution.

The present invention will be further illustrated in the following examples.
However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.
Examples Description of the measurement system.
The measurement system is as described above, using custom built spectrophotometers. The first spectrophotometer contains a silicon photodiode array and covers spectral range from 585nm to 1180nm (280) with resolution about l4nm, while the second one contains an InGaAs photodiode array (270) and covers spectral range from 900nm to 1635nm with resolution about 11.Snm. Slit width used in spectrometers is 0.3mm and 0.2mm, for the spectrometers with silicon and InGaAs arrays, respectively. Both arrays are thermoelectrically cooled, 256 elements arrays (Hamamatsu), with 0.05mm wide pixels. The signals captured by the arrays are extracted using standard electronic cameras (Hamamatsu), and digitized with a standard National Instrument l6bits A/,D converter, and stored in a computer memory for further processing.
Example 1: Measurement technique Two kinds of data were collected during each measurement process: a reference and sample measurement. 1~ or reference measurements, a stable diffusing reference member (32), consisting of volume diffusers (36, Figure 2) and mesh radiation attenuator (38), whose radiation transporting capability is as close as possible to that of the sample, is placed in the sample holder (30) for reference measurement. The shutter (140) is then opened, and the system takes measurements of the signal at all pixels, identifies the pixel with the largest signal and sets integration time in such a way that reading at this pixel reaches approximately 85% of the possible maximum. Having the integration time defined, the system collects a number of spectra, which can be averaged to improve, signal to noise ratio (SNR).
After the collection of the reference signal is completed, the shutter blocks the optical path of the radiation and a set of reference dark measurements with the same integration time as reference signal is collected. For sample measurements, the diffuse reference attenuator (32) is then replaced with a sample (150), and the process of signal reading, determining integratian time, light and dark signals collecting is repeated for the signal affected by the sample.
The mean dark signals are subtracted from corresponding mean light signals and, similar to an absorbance calculation, a negative logarithm of ratio of radiation transported through the sample to that transported through the reference member is calculated at each wavelength. The value calculated in such a way is further referenced as a relative diffuse absorbance. The relative diffuse absorbance, tagether with the data on the concentration of all chemical compounds, gives the starting point for evaluation of the instrument applicability for measurements of non-absorbing samples.
Since the relative diffuse absorbance depends not only on optical properties of the sample and reference member, but also on collecting capabilities of the optical system, a simple relation between the relative diffuse absorbance and concentration of the compound in the sample cannot be established. Rather, a relationship between the relative diffuse absorbance and concentration of the compound in the sample can be modeled for each instrument applying techniques of multivariate regression [11-13], as described below.
A large number of samples (several hundred in some cases) with statistically non-correlated concentrations distribution of all analytes were prepared and measured.
This number depends on complexity of the samples, variability range of all non-correlated components in matrix, system noise and required precision, so that a distribution of concentration across the variability range is obtained.
Naturally available samples were normally distributed in terms of their analyte concentration. A
calibration sample set was created by spiking some samples with selected analytes, and mixing some samples. Care was taken to destroy any possible correlation between concentrations of different analytes in the samples used for model development. As well, care was made to insure that there were no correlations in sample order.
The concentration of the compounds of interest in the samples was determined (calculated from mixing proportions of samples with known concentrations of the compounds in the base samples, and, by standard analytical measurements of the concentration in the final sample). In the case of discrepancy between values obtained with these methods, a spectrum of the sample was collected and used to identify sources of discrepancy between measurements, and not for model development and verification.
After sufficient number of samples with known concentration of analytes of interest had been produced and spectra had been collected, the obtained data set was analyzed to identify evident outliers. Remaining samples were divided into two subsets containing comparable numbers of samples, using an interleaved division. In this method, the spectrum of every second sample was assigned to a separate subset, producing two statistically independent subsets, so that the two interleaved set did not have sample repeats in common. ~ne interleaved set is used for model development using PLSR method, while the second interleaved set is used for model verification.
The performance difference between both sample sets was statistically negligible.
Example 2: Measurement of glucose, albumin and intralipid in water The first test was performed using a mixture of three analytes: glucose, intralipid; and albumin, in non-correlated quantities, in water. The glucose variability range extended from Ommol/L to 5~mmol/L, the albumin range extended from 4g/dl.

to 8gIdL (with a few points in 3g/dL to 4g/dL range) and the intralipid range extended from 0.4g/dL to 0.8g/dL. A small amount of preservative was added to prevent sample deterioration in time between its production and characterization was carried out with traditional analytical method. The concentrations of each analyte in each sample within their respective variability ranges were selected accordingly to the numbers produced by a random number generator, being a part of MATLAB software environment, and samples were produced by weighing and mixing of particular analytes accordingly to generated numbers in quantities proportional to the intended volume of the final solution. Initially these quantities were dissolved in limited volume of steam distilled water and after dissolving, steam distilled water was replenished to produce predetermined volume of the solution.
After samples were produced, concentration of each analyte was independently verified using standard analytical methods and correlation between concentrations of all three analyzes were recalculated. In the produced set of samples the correlation coefficient between glucose and albumin concentrations was 0.03 ~, between glucose and intralipid 0.053 and between albumin and intralipid 0.035.
The coefficients are close to values observed for two sets of random numbers containing similar number of elements varying in similar ranges.
Samples were kept in a refrigerator for cooling and storage. The first spectrum was collected shortly after the sample was taken from the refrigerator, and four more spectra immediately ore after another (each spectrum consisting of a number of averaged raw measurements at a fixed integration time). One measurement (light and dark readings for reference and sample) lasted about two minutes, with a total measurement.time for a given set of 5 measurements lasting about ten minutes.
During this time the temperature of samples increased from that of refrigerator (about 4°C) to room temperature (about 20-24°C), or higher, due to sample heating by both environment and the radiation used for testing. All spectra are to be considered as taken at random temperatures. The spectra of the same sample were treated as a single block, and to avoid prediction on itself, the block was assigned to the same selection for model development and prediction. Errors in predictions, for spectra taken at different temperatures, were not correlated to the measurement order.
Therefore, the models developed do not make use of temperature dependent features to a signif cant degree. The presented results are mean predicted values of all five measurements taken during the rise the sample temperature.
Altogether over 500 samples were prepared. Five spectral measurements were taken for each sample at variable temperatures, resulting in a total of 2500 measurements. Spectra obtained were visually tested for evident outliers and after elimination of bad measurements, 499 samples (2495 measurements) were selected far analysis. The samples and associated measurements were divided into five consecutive batches (four containing 100 and one containing 99 samples}. To avoid prediction on itself all spectra of the same sample were treated as a single block and always assigned to the same batch. The blocks of spectra of a given selection (batch or all samples together) were divided into two independent subsets of comparable size applying either random pickup or interleaved selection, as described before.
Standard PLSR procedures were applied to one of these subsets to build three sLparate sets of prediction models (one set for each compound}. These models were then used to predict the concentrations in the second subset of respective spectral measurement and the standard error of prediction (SEP) was calculated for each subset. The models providing the smallest SEP were selected for performance comparison of models build on sets with different numbers of elements. From this comparison it was concluded that setseontaining 100 samples divided into two equal subsets for model development and prediction are sufficient for preliminary evaluation of measurability of particular compounds with instrumentation described above.
The above described process was repeated on the same subsets in the reversed order (second subset used for model development and first for model selection).

The results from this analysis are shown in Figures 3a to 3c (Figure 3a for albumin, Figure 3b for intralipid and Figure 3c for glucose), in a form of scatter plots.
Each scatter plot shows the relation between reference value and value predicted on independent samples, together with basic statistical information, which includes the correlation coefficient, r, between predicted and reference values, the slope of the linear regression line for these values, the intercept of the linear regression line with the ordinate and mean percentage error of prediction. These figures also show the identity line (line with slope equal to 1), the linear regression line for predicted versus reference values (ideally it should coincidence with the identity Line) and 20% relative error limit lines. Error was found to be very small in comparison to the mean measured value, the intercept was found to be close to 0 and the slope was found to be close to 1, demonstrating that all three analytes were measured using the apparatus described herein.
Taking into account that these analyt~s are randomly mixed it is unlikely that any, of them, especially glucose, whose volume concentration is much smaller than other components, is measured through a water replacement signal, which would be of limited value owing to its lack of specificity. It is clear that the components can~be measured in selected variability ranges with precision sufficient for majority applications.
These results were obtained using relatively large set of data (about 250 samples far model development and comparable amount for model selection), resulting in long evaluation period. Therefore a test was performed to reduce the data set of I00 samples. Sample size reduction had the largest effect on glucose measurability, and two extreme cases, the worst and the best, are shown in Figures 4a and 4b, respectively. Comparison of results presented in Figures 4a and 4b, with Figure 3c indicates that the difference in performance of models built on I00 or 500 samples is within the. statistical error.
Ezample 3: Measurement of glucose, albumin, sodium, potassium, r chloride and phosphorous without and with intralipid, in water The procedures developed in Example 2 were used for the measurability test of six different analytes: glucose, albumin, sodium, potassium, chloride, and phosphorous, all present in solution of distilled water in the absence and presence of intralipid as a radiation scattering factor. Glucose and albumin were u~d to test system performance, while the other components (excluding intralipid), which do not possess clear absorbance spectra in applied spectral range, were used for testing applicability of the method and instrumentation for concentration measurements of non absorbing substances.
Each of these substances had different molar concentration ranges, and their concentrations were intentionally combined in a way reducing possible correlations between them. The role of intralipid was to introduce scattering into the samples.
Correlation coefficients for water solutions of the selected six components without intralipid, and with intraiipid, as well as correlation of their concentrations to sample number, are shown in Figures 5a and Sb, respectively. The results presented in Figures Sa and Sb demonstrate that the correlation between sample number and concentrations of ail analyzes is comparable to that expected for correlations of similar size of data sets containing the series of ordered natural numbers and equal size set of random numbers, and that the predictability for measurable analyzes is not a result of spurious correlation.
Prediction results from the measurement of the compounds, for samples prepared in the absence of intraiipid are shown in Figures 6a to 6f, and Figures 7a to 7g, for samples prepared in intralipid. Figure 7g shows predictability of intralipid.
'These results presented in Figures 6a to 6d, and 7a to 7d, demonstrate that scattered light spectroscopy may be used to measure physiologically important analytes that do not possess specific absorbance bands in working range of the .". ,_ . ... _,... ~,~,w. ,..~ _~.,. ~~~"-x~~z.~w~.~:,,,~<~~..,.~.,~,~., w.
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y , spectrometric system. Furthermore, scattered light spectroscopy may be used to measure analytes over physiologically important ranges of their concentration.
The analytes potassium and phosphate, which do not exhibit predictability in water (Figures 6e and 6fj, show some predictability in a solution with intralipid (Figures 7e and 7t]. Without wishing to be bound by theory, this result may be explained as a result of their low molar concentrations used, and that for given optical path the concentration of each of these analytes was not sufficient to produce a recognizable signal. In the presence of intralipid, the effective optical path is increased, and these compounds exhibit a refractive index dependent signature.
Adding intralipid increases the mean relative prediction error of analytes which possess absorption bands in applied spectral range, glucose and albumin.
For components that exhibit their presence by light scattering, the error increase.is not as evident.
Example 4: Measurement of a range of compounds in animal serum Nine samples of serum, from horse, goat, sheep, chicken, pig, and bovine, two adult cows and two newly barn calves, were obtained from Sigma-Aldrich. These samples were characterized using a J&J Vitros 250 instrument, and the concentration of the the following analytes: albumin, chloride, cholesterol, carbon dioxide, creatinine, glucose, potassium, sodium, total bilirubin, total protein (including albumin), triglyceride, and urea, determined. Small quantities of each sample were also extracted for spectral characterization, and the remaining sera used for the production of one hundred samples.
The sample set was by mixing basic sera in different proportions and spiking with selected analytes to expand their variability range to fill the extremes expected in human physiology. Five analyzes were used for spiking: albumin, creatinine, glucose, intralipid and urea. The sera used for spiking had the following concentrations of the selected compounds: albumin 20g/dL; creatinine 100mmo1/L; glucose 300mmoI/L;
urea 2,Smo1/L and a 20% standard intralipid solution. Intralipid was used to modify concentration of triglycerides and to modify scattering properties of the samples. High concentration of additives was used so that only a small amount of spiking solution was required to adjust the final concentration of the selected component in the sample, thereby having a minimal impact on concentrations of other components, existing in the spiked serum. Final concentration of a particular analyte in a sample was determined by calculation, and verified using a YSI Model 2300 Scat Plus, for glucose measurements, and a Vitros DT60II with DTE II module for glucose and other analytes.
Two batches of serum were prepared for a first measurement. The first batch contained one hundred samples prepared by mixing and spiking as described above.
After collecting and analyzing the spectra from the fast hundred of the samples and analyzed, a second batch of samples was prepared. The original, non-modified samples of nine animal sera described above, were used as samples of the second batch for model prediction. However, these samples were not used for productDn of calibration model. The remaining 294 samples (403 in total) were produced using six different sera (horse, sheep and four bovine: three cows and one newly born calf] and spiked with albumin, creatinine, glucose, ir~tralipid, urea, sodium (in form of NaO~
and potassium (in form of KH2P04), as described above.
The prepared samples were stored in a refrigerator, from which they were removed just before measurement. The measurement protocol was as described in Example 2. As required, the data were either treated as one large set or divided in different subsets, each subset containing all measurements of the same sample and alternatively used either to build models or to select the model providing the best performance using PLSR procedures. The comparable size of these subsets allowed for comparison of the models build on each of them.

The first analysis was performed using one hundred samples of the first run (each sample consisting of five replicate measurements), and divided into two interleaved subsets for model development and model selection. For all selected analytes up to fifty models were generated each model corresponding to the number of latent variables used. Performance of each model was evaluated, giving rise to a range of models that demonstrated the ability to predict with mean relative errors well below 20% and with a slope of the best fitting regression line within 15% from the slope of the identity line.
The scatter plots of prediction using models built on a complete set of 403 samples (divided into two of comparable sized subsets for model development and model evaluation for ail thirteen components, for which calibration data were available, are presented in Figures 8a to 8m.
Caood predictability was observed with potassium (Figure 8h) over a concentration variability range that was close to that used in experiment with water solution (Example 3). Similarly to that observed with potassium, models were also developed that predicted concentrations of phosphorous (Figure 8k).
The results were confirmed in separate tests. In addition to the majority of analytes analyzed during first test the measurability of two new components, magnesium and I-il)L was successfully verified, and corresponding scatter plots are produced (Figures 9a and 9b).
The results shown in Figures 8h and 8k demonstrate that potassium and phosphate, in serum, are measurable using scatter light spectroscopy.
Existence of many components in the serum gives rise to a refractive index, which is significantly higher than that of water, resulting in higher sensitivity of the system to refractive index associated with analyzes of interest.

J
v 1 All citations are herein incorporated by reference.
The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as described herein.
Z. References [1] R. Jensen, I. Lugan, E. Peuchant, "Biological analysis without reagent.
Myth or reality? Application to determination of serum total lipids". Bull Soc Pharm.
Bordeaux 1986, 125, 43-52 [2] J. W. Hall and A. Pollard; 'mtear-Infrared Spectrophotometry: A New Dimension in Clinical Chemistry", Clin. Chem., 38, (1992), 1623-1631 [3] J. W. Hall and A. Pollard, "Near-Infrared Spectroscopic Determination of Serum Total Proteins, Albumin, Globulins, and Urea", Clin. Biochem., 26, (1993), [4] K. H. Hazen, M. A. Arnold and G. W. Small, "Measurement of glucose and other analytes in undiluted human serum with near-infrared transmission spectroscopy", Anal. Chim. Acta, 371, (1998), 255-267 [5] P. A, da Costa Filho and R. J. Poppi, "Determination of triglycides in human plasma using near-infrared spectroscopy and multivariate calibration methods", Anal. Chim. Acta, 446, (2001), 39-47 [6] R. A. Shaw and H. H. Mantsch, "Infrared Spectroscopy in Clinical and Diagnostic Analysis", Encyclopedia of Analytical Chemistry, Edited by R. A.
Meyers, John Wiley & Sons Ltd, Chichester,.2000, 1-19 [7] M. G. Scott, J. W. Heusel, V. A. LeGrys and O. Siggaard-Andersen, Chapter 31 "Electorlytes and Blood Gases" in Tietz Textbook of Clinical Chemistry, 3~ edition, W.B. Saunders Company, Philadelphia, (1999) [8] L. A. Kaplan, A. J. Pesce and S. C. Kazmierczak, "Clinical Chemistry:
Theory, analysis and correlation" 3~° edition, Mosbay, St Louis, (1996) [9] M, Born and E. Wolf, "Principles of Optics", 6~' edition, Pergamon Press, Oxford, (1980).

[10] M. V. Klein, "Optics", John Willey and Sons, new York, (1970) [1 l] "Chemometrics: Mathematics and Statistics in Chemistry", B. R.
Kowalski, Ed., NATO ASI Series C: Mathematical and Physical Sciences Vol. 138, D. Reidel Publishing Company, Dordrecht, Boston, Lancaster, 1983 [12] H. Martens and T. Naes; "Multivariate Calibration", John Wiley & Sons Ltd, Chichester, 1989 [I3] D. A. Burns and E. W. Ciurczak, "Handbook of Near-Infrared Analysis", Marcel Dekker Inc., New York, Basel, Hong Kong, 1992

Claims (20)

1. A method of determining the concentration of a compound of interest in a sample using scattered light spectroscopy comprising, i) providing a scattered light spectrometer comprising an algorithm developed for the compound of interest;
ii) introducing radiation of about 585nm to about 1635 nm to the sample;
iii) measuring collected radiation after interaction with the sample;
iv) determining the concentration of the compound of interest using the algorithm.

2. The method of claim 1, wherein the compound of interest does not exhibit a measurable variability in absorbance within the wavelengths of about 585nm to about 1635nm, and where the refractive index of the compound of interest changes in a wavelength specific manner over at least a portion of the wavelengths of about 585nm to about 1635nm.

3. The method of claim 1, wherein the step of measuring (step (iii)) involves measuring both scattered and absorbed radiation.

4. The method of claim 1, wherein the compound of interest is selected from the group consisting of protein, albumin, bilirubin, creatine, cholesterol, triglycerides, glucose, urea, intralipid, chloride, potassium, sodium, phosphorous, calcium, magnesium, manganese, iron, sulphur, zinc, aluminium, silicon, copper, nickel, arsenic, nitrogen, fluorine, lithium, selenium, bromine, cadmium iodine, mercury, gold, other ion, and a compound that exhibits the property of a refractive index that changes with wavelength.

5. The method of claim 1, wherein the sample is a body part.

6. The method of claim 4, wherein the sample is a body part.

7. The method of claim 1, wherein the sample is a liquid or a gas sample.

8. The method of claim 4, wherein the sample is a liquid or a gas sample.

9. An apparatus for determining the concentration of a compound of interest in a sample using scattered light spectroscopy comprising, - a radiation source that emits radiation from about 585nm to about 1635nm, - a first optical transmission element for receiving, transmitting and directing the radiation from the radiation source to a sample holder that comprises a sample, the first optical transmission element having a first and second end, the first end positioned to receive the radiation produced by the radiation source, the second end for scattering the radiation leaving the first optical transmission element to produce scattered radiation, and for directing the scattered radiation to the sample holder;
- the sample holder comprising two or more than two windows;
- a second optical transmission element for receiving the scattered radiation after interaction with the sample, and far directing the scattered radiation to one or more than one scattered radiation processing system;
- the one or more than one scattered radiation processing system comprising a diffraction grating, a radiation detection system, and comprising one or more than one algorithm for determining the concentration of the compound of interest.

10. The apparatus of claim 9, wherein the radiation source further comprises an elliptical mirror.

11. The apparatus of claim 9, wherein the windows of the sample holder are diffusers.

12. The apparatus of claim 9, wherein the radiation detection system comprises a first and second set of lenses, the first set of lenses focusing the scattered radiation through a slit, and the second set of lenses, positioned to receive the scattered radiation after passing through the slit, and comprising the diffraction grating, placed between the lenses in the second set of lenses.

13. The apparatus of claim 9, wherein the second optical transmission element is bifurcated and splits the scattered radiation into a first and a second scattered radiation beam path.

14. The apparatus of claim 13, wherein the first scattered radiation beam path, after passing through the second set of lenses is directed onto a photo diode array capable of detecting radiation from ablaut 585nm to about 1180nm, and wherein the second scattered radiation beam path, after passing through the second set of lenses is directed onto a photo diode array capable of detecting radiation from about 900nm to about 1635nm.

15. The apparatus of claim 9, wherein the diffraction grating is a volume diffraction grating.

16. The apparatus of claim 12, wherein the first set of lenses further comprise a filter placed between the lenses.

17. The apparatus of claim 5, wherein a heat rejection filter is placed between the radiation source and the first optical transmission element.

18. The apparatus of claim 9, wherein the first optical transmission element is composed of two optical transmission elements positioned in series, and a shutter is placed between the two optical transmission elements.

19. The apparatus of claim 9, wherein the first and second optical transmission elements are comprised of one or more than one optical fibers or one or more than one radiation guiding rods.

20. A method of determining the concentration of a compound of interest in a sample comprising, i) introducing scattered radiation to the sample using the apparatus of claim 10;
ii) measuring radiation collected after interaction with the sample;
iii) determining the concentration of the compound of interest.