Patent Application: US-22586107-A

Abstract:
a method of obtaining pure component mass spectra or pure peak elution profiles from mass spectra of a mixture of components involves estimating number of components in the mixture , filtering noise , and extracting individual component mass spectra or pure peak elution profiles using blind entropy minimization with direct optimization . the method may be applied to deconvolution of pure gc / ms spectra of overlapping or partially overlapping isotopologues or other compounds , separation of overlapping or partially overlapping compounds in proteomics or metabolomics mass spectrometry applications , peptide sequencing using high voltage fragmentation followed by deconvolution of the obtained mixture mass spectra , deconvolution of maldi mass spectra in the separation of multiple components present in a single solution , and specific compound monitoring in security and / or environmentally sensitive areas .

Description:
this example illustrates the general applicability of blind entropy minimization to the extraction of unknown component mass spectra from the mass spectra of unresolved or partially resolved systems . in this example , blind entropy minimization was conducted in a manner described in zhang , 2003 using a stochastic optimization technique . chemicals . the following reagents were used : nabh 4 pellets ( alfa aesar , word hill , mass . ); nabd 4 pellets ( 99 % d , cambridge isotope laboratories , ma ); 37 %, dcl in d 2 o ( 99 . 5 % d , aldrich ); 30 % naod in d 2 o ( 99 % d , aldrich ) and d 2 o ( 99 % d , aldrich ). a solution of nabh 4 prepared in h 2 o , was stabilized by adding naoh up to 0 . 1 m final concentration . a solution of nabd 4 ( 0 . 25 m ), prepared in d 2 o was stabilized by adding naod up to 0 . 1 m final concentration . an enriched isotopic standard solution of 260 μg ml − 1 82 se ( iv ) in hno 3 aqueous media ( oak ridge national laboratory , oak ridge , tenn .) was used to spike the reaction media in septum - sealed vials for cold vapour generation of their corresponding hydrides . all other reagents were of analytical grade . the following reagents were used : nabh 4 pellets ( alfa aesar , word hill , mass . ); nabd 4 pellets ( 99 % d , cambridge isotope laboratories , ma ); 37 % dcl in d 2 o ( 99 . 5 % d , aldrich ); 30 % naod in d 2 o ( 99 % d , aldrich ) and d 2 o ( 99 % d , aldrich ). a stock solution of nabh 4 ( 1 . 0 m ) in h 2 o was stabilized by adding naoh up to 0 . 5 m . a stock solution of nabd 4 ( 1 . 0 m ) in d 2 o was stabilized by adding naod up to 0 . 5 m . working solutions of pure 0 . 2 m nabh 4 , 0 . 2m nabd 4 as well as mixed 0 . 2 m ( nabh 4 + nabd 4 ) were prepared before experiments by adding 4 ml of h 2 o to x μl of 1 . 0 m nabh 4 + y μl of 1 . 0 m nabd 4 ( x = 1000 , 900 , 750 , 500 , 250 , 100 , and 0 μl ; x + y = 1000 μl ). the resulting concentration of naoh in the final solutions is 0 . 1 m naoh . a 0 . 2 m nabd 4 solution in d 2 o and 0 . 1 m naod were prepared for hydride generation in fully deuterated reaction medium . cold vapor generation of hydrides was performed in septum - sealed vials using solutions of 500 μg ml − 1 as ( iii ) and bi ( iii ), and 100 - 300 μg ml − 1 ( in acidified aqueous media ) of 123 sb ( iii ), 73 ge ( iii ) and 118 sn ( iv ) ( oak ridge national laboratory , oak ridge , tenn .). all other reagents were of analytical grade . apparatus . a hewlett - packard 6890 gas chromatograph operated in the splitless mode and equipped with a hewlett - packard 5973 mass selective detector was fitted with a db - 1 capillary column ( 30 m × 0 . 25 mm i . d .× 1 μm ; vallobond vb - 1 ). a gas - tight hamilton syringe ( 5 ml ) was employed for sampling headspace gases from reaction vials . screw cap reaction vials fitted with ptfe / silicone septa ( 5 - 10 ml , pierce chemical co .) were used according to experimental requirements . the gc was operated under the following conditions : injector temperature 150 ° c . ; oven temperature 35 ° c . ( isothermal ). the carrier gas was he at 1 . 2 ml min − 1 . hydride generation procedure . the 10 ml reaction vial containing 2 ml of acid ( 1 m hcl or dcl for as , sb and bi and 0 . 01 m hcl or dcl for ge and sn ), approx . 2 - 10 μg of the element ions and a teflon ™ coated stir - bar was capped and two stainless steel needles were inserted through the septum . vigorous stirring of the solution was started and nitrogen was then introduced through one needle in order to purge the atmospheric oxygen from the headspace of the vial . the two needles were then removed and 1 ml of 0 . 2 m reducing solution ( nabh 4 , nabd 4 or nabh 4 and nabd 4 mixture ) was injected using a plastic syringe fitted with a stainless steel needle . headspace gases ( 2 - 3 ml ) were subsequently sampled with a gas tight syringe and injected into the gc / ms . h 2 se generation procedure . the reaction vial ( 10 ml ) containing 2 ml of 1 m hcl , about 10 μg of 82 se ( iv ) and a teflon ™ coated stir - bar was capped and two stainless steel needles were inserted into the septum . vigorous stirring of the solution was started and nitrogen was then introduced through one needle in order to purge atmospheric oxygen from the headspace of the vial . the two needles were then removed and 1 ml of 0 . 25 m nabh 4 solution was injected using a plastic syringe fitted with a stainless steel needle . headspace gases ( 2 - 3 ml ) were subsequently sampled with a gas tight syringe and injected into the gc / ms . mass spectrum of pure h 2 se was obtained . d 2 se generation procedure . generation of pure d 2 se was attempted using fully deuterated reaction media . a procedure similar to that described for h 2 se generation was adopted with the following modifications . in a 5 ml vial containing 0 . 5 ml of 1 m dcl spiked with 0 . 05 ml of 82 se ( iv ) aqueous standard solution , atmospheric oxygen was degassed and 0 . 5 ml of 0 . 25 m nabd 4 solution was injected . h / d exchange experiments . pure h 2 se was prepared as described above . two aliquots of the h 2 se headspace gas ( each of 3 - 5 ml volume ) were collected in a rapid sequence . the first aliquot was injected into a 5 ml reaction vial ( the exchange vial ) containing 1 ml of 0 , 3 and 6 m dcl in d 2 o and continuously shaken throughout the experiment . the second h 2 se headspace aliquot was injected into the gc / ms to verify the isotopic composition of the synthesized h 2 se . the isotopic composition of the injected hydride in the headspace of the exchange vial was checked at regular intervals by gc / ms . previous experiments in which h 2 se was injected onto a column pre - treated with dcl vapors have demonstrated that the h / d exchange does not take place in the gc capillary column ( d &# 39 ; ulivo , 2005 ). computations were carried out using the commercially available mathcad v . 12 . 0 package ( mathsoft engineering & amp ; educ ., inc .). reconstruction of the mass spectra was done using the statistical mass balance method and the weighted two - band - target entropy minimization algorithm . concentration profiles of the individual isotopologues were obtained using a least squares isotope pattern reconstruction as described elsewhere . a non - negative least squares minimization routine was used for the reconstruction of more complex ge and sn hydride mass spectra to avoid the occurrence of negative contributions ( lawson , 1974 ). spectral contrast angle between the two mass spectra was calculated as follows : cos ⁡ ( a ) = å i ⁢ a i × b i ( 3 ) where a i and b i are the ion intensities on the i th m / z channel . for spectral comparison purposes , all the mass spectra were normalized with respect to their l 2 - norm , i . e . σa i 2 = σb i 2 = 1 , unlike the conventional l 1 - norm ( σa i = σb i = 1 ) for the reasons discussed elsewhere ( alfassi , 2004 ). mass spectra of pure components can be reconstructed using entropy minimization as recently outlined by zhang , 2003 . in essence this approach is a pattern discovery ( recognition ) via entropy minimization . the objective function to be minimized thus is the entropy of the extracted mass spectrum . in information theory entropy is a measure of the average amount of information required to describe the distribution of some variable of interest . the most commonly used measure of entropy was introduced by shannon ( s = p · inp ) ( shannon , 1949 ). this classical entropy definition was previously used in reconstruction of continuous nmr , raman and ir spectra where p has been quantitatively linked with the first or higher order derivatives ( guo , 2005 ; chew , 2002 ; widjaja , 2002 ; widjaja , 2003 ). however , such an entropy definition ( smoothness of the spectra ) cannot be transferred in the context of mass spectrometry where the individual masses are discrete by their nature , thus a modified non - logarithmic entropy definition is used in this model recently developed by the garland group . an overall weighted two band - target entropy minimization algorithm can be outlined in the following steps . first , a mass spectral data matrix i k × n is compiled from k mass spectra each having n mass - to - charge channels ( k ≧ number of possible isotopologues ). each mass spectrum is normalized to unity total ion intensity . second , a singular value decomposition of i k × n matrix is performed . this procedure represents any k × n matrix as a product of three matrices ( u k × n and v n × n are orthonormal , e . g . u t u = v t v = 1 ): this leads to the diagonal singular value matrix σ and the right singular value matrix v t . the obtained singular value decomposition is used further for noise reduction . the main idea is that the experimental i k × n matrix contains components of large and low variation . it can be shown that the small singular values in σ mainly represent the noise . this noise is eliminated by keeping only the first j non - zero singular values ( j ≦ k ). it is worth mentioning that the σ matrix rang ( which equals j ) represents the number of individual compounds whose spectra are to be deconvoluted . the filtered ( truncated ) representation of spectral matrix i j × n now becomes i j ′ n = u j ′ j ′{ dot over ( a )} j ′ j ′ v j ′ n t ( 5 ) the pure component mass spectra can be obtained using the transformation matrix t : a 1 ′ n = t 1 ′ j ′( { dot over ( a )} j ′ j ′ v j ′ n t ) ( 6 ) the presence of matrix σ in this equation serves as a weighting procedure making the model more robust . mass spectral reconstruction of j independent components now becomes a problem of finding all t vectors . there are certain restrictions any candidate t vector must comply with . first , it has to produce a non - negative pure component spectrum estimate a 1 × n . choosing the two target bands ( m / z channels ) a x and a y is the crucial step of the method . during this step a 1 × n matrix is normalized with respect to the total intensity of the target bands : depending on the targeted m / z channels , mass spectra of different isotopologues are extracted . for example , targeting the m / z = 18 and 20 yields a mass spectrum of d 2 o , targeting m / z = 17 and 18 yields a h 2 o mass spectrum and targeting m / z = 17 and 19 or 18 and 19 results in retrieval of the hod spectrum . second , component contribution ( concentration ) in each of the k mass spectra also has to be non - negative : c k ′ 1 = i k ′ n ′ a * n ′ 1 t ′( a * 1 ′ n ′ a * n ′ 1 t ) − 1 ( 8 ) the objective function ( entropy ) to be minimized is simply the sum of the channel intensities in the reconstructed mass spectrum : minimization of the objective function ( in terms of t vector ) is usually optimized using the simulated annealing algorithm which is a good choice for a global minimum search in many dimensions . in this situation the number of dimensions is small ( three for the h 2 o / hod / d 2 o system ) therefore an exhaustive random search was used . as mentioned above , the model seeks the lowest entropy ( most simple ) mass spectra within the non - negativity constraints . in a particular h 2 o / hod / d 2 o example , the three component concentration and mass spectral information is condensed into five spectral channels therefore the system is undetermined and an infinite number of hod mass spectra can be obtained . since one of such possible solutions is a hod spectrum with no fragment ions , this is the obvious minimum entropy result ( the simplest mass spectrum ). in other words , additional contextual information is needed to separate the mathematical solution from the chemically meaningful solution of the hod mass spectrum . the same applies to the reconstruction of the hdse mass spectrum . the blind deconvolution lowest entropy result for the hod mass spectrum is ( 0 . 000 ; 0 . 000 ; 0 . 000 ; 1 . 000 ; 0 . 000 ). this result clearly has no chemical meaning , especially considering the experimentally measured mass spectra of h 2 o and d 2 o . when the abundance of the hod molecular ion is set within the range of h 2 o and d 2 o molecular ions , the reconstructed mass spectrum of hod using the weighted two - band target entropy minimization algorithm ( targeting m / z = 18 and 19 ) with the above mentioned restriction of molecular ion intensity is ( 0 . 017 ; 0 . 056 ; 0 . 126 ; 0 . 801 ; 0 . 000 ) as shown in fig1 . although these two results are quantitatively different , nevertheless , they are nearly identical when compared using the spectral contrast angle ( 99 % similarity ). it is clear that the spectral contrast angle is not a good choice for comparing the experimental and the reconstructed spectra due to its insensitivity to low intensity ions . this was also recently pointed out by zhang et al . ( 2003 ) who proposed using the ratio of the geometric and the arithmetic means as a similarity measure between the mass spectra . using this criterion , the similarity between constrained and unconstrained hod spectra becomes 89 %. in the first unconstrained band - target entropy minimization reconstruction of mass spectra , the garland group achieves 76 - 92 % similarity ( calculated using the ratio of geometric and arithmetic means ) between the reference and measured spectra in a four component mixture ( ethanol , acetone , hexane and toluene in the m / z = 10 - 100 range ). it is evident that when dealing with underdetermined overlapping isotopologue systems , ion intensities in the extracted pure component mass spectra can be biased if no contextual feedback is provided . as has been seen , although such bias might be of little importance for spectral recognition , it is , nevertheless , significant for quantitative purposes . reconstruction of selenium hydride mass spectra is a problem similar to that of the water example above . the difficulty of pure component spectra reconstruction , however , is increased due to the fact that the d 2 se mass spectrum is difficult to obtain even by reaction of trace amounts of se ( iv ) with nabd 4 in deuterium - only medium ( dcl + nabd 4 , x ( d )& gt ; 97 % at .) as discussed previously in the materials and methods section . accordingly , alternating least squares cannot be used to reconstruct the mass spectrum of hdse since no experimental estimate of d 2 se is available . in spite of this , fig2 shows the entropy minimization estimates of h 2 se , hdse and d 2 se mass spectra ( containing only 82 se isotope ). the h 2 se mass spectrum was recovered by targeting the m / z = 82 and 84 , hdse by targeting m / z = 82 and 85 and the d 2 se mass spectrum was recovered by targeting the m / z = 82 and 86 . to reduce the bias in recovered ion intensities , the molecular ion in the hdse mass spectrum was forced to be in the range of h 2 se and d 2 se molecular ion intensities . one can see that the reconstructed h 2 se mass spectrum agrees well with the experimental measurements ( fig2 ). h 2 o / hod / d 2 o . mixtures of h 2 o and d 2 o are characterized by the equilibrium h 2 o + d 2 o = 2hod and isotopic self - exchange reaction of water in gaseous or liquid phases has been the subject of numerous investigations and debates over the past decades . although seemingly simple system , estimates of the h 2 o / d 2 o isotope exchange equilibrium constant ( in both gaseous and liquid phases ) are range between 3 . 41 and the geometric mean of 4 . 0 . this value has been recently ( re ) estimated using ir and nmr techniques . using the above described hod reconstruction / deconvolution algorithm , the equilibrium constant can be calculated from the amount fractions of h 2 o , hod and d 2 o . replicate measurements in the range of x ( d )≈ 50 - 70 % lead to the average value of k = 3 . 85 ± 0 . 03 (± 2s ) using the entropy minimization model . this is in good agreement with the “ best ” theoretical value of 3 . 85 . the two most recent liquid phase equilibrium constant measurements ( at 298 k ) are 3 . 86 ± 0 . 07 and 3 . 86 ± 0 . 01 ( libnau , 1995 ; duplan , 2005 ). previous direct mass spectrometric measurements ( carried out in the 1960 &# 39 ; s ) have led to similar the values ; however , these measurements have been performed at electron accelerating voltages of 3 ev to eliminate any fragment formation ( friedman , 1966 ). although this classical approach avoids the need for deconvolution , it is clearly not of practical use for a variety of analytical purposes due to the deterioration of method sensitivity and robustness . h 2 se / hdse / d 2 se . various gaseous mixtures of h 2 se / hdse / d 2 se were obtained when h 2 82 se ( generated from 82 seo 3 2 − and 2 m hcl / nabh 4 ) was introduced in the headspace of d 2 o / dcl environment . in such conditions rapid h / d exchange takes place resulting in a rise of hd 82 se and d 2 82 se concentrations in the headspace . the resulting mixtures were then analyzed using gc / ms and their composite mass spectra were decomposed using the weighted two band - target entropy minimization algorithm . the relative amount fractions of isotopologues as obtained from both models are in good agreement and are not biased . based on reaction purities of reagents , the estimated h atom fraction in the reaction solution was & lt ; 7 %. the obtained mixture contained 43 % h 2 se and 44 % hdse and only 13 % d 2 se ( 63 % atom fraction of h ). decreasing the volume of the spike of 82 se ( iv ) aqueous standard solution to 0 . 01 ml ( h atom fraction in reaction solution & lt ; 3 %) improved the purity of the d 2 82 se , but it was unsuccessful in the production of pure d 2 se : the composition of the resulting mixture was 10 % h 2 se , 26 % hdse and 64 % d 2 se . total hydrogen incorporation in this mixture is 23 % which is far from the isotopic distribution of the solvent ( 3 % h and 97 % d ). the present findings contradict the previous hypothesis which assumed that the isotopic composition of hydrogen selenide is similar to that of the solvent due to the rapid h / d exchange ( as a consequence of the strongly acid nature of h 2 se and d 2 se ). deconvolution of eh3 / eh 2 d / ehd2 / ed 3 mass spectra . one of the most prominent features of band target entropy minimization is the selection of the target m / z channels . given a mass spectra with n channels there are ½ · n ( n + 1 ) combinations of two - band targets ( including situations where both bands represent the same m / z channel ). in the case of arsenic hydride mixtures , 28 different targets over a seven channel mass spectra ( m / z = 75 - 81 ) leads to four distinct mass spectra . an important aspect for retrieving a component mass spectrum using this technique is the existence of a unique m / z channel that bears the highest intensity ion among all the other component spectra . ash 3 has such an ion at m / z 78 , ash 2 d at m / z 79 , ashd 2 at m / z 80 and asd 3 at m / z 81 . two of the four mass spectra are in agreement with the experimental spectra of ash 3 and asd 3 ; however , closer inspection of the remaining two spectra showed abnormally large molecular ions . this is not consistent with the observed ion intensity relationships in isotopologue mass spectra since it is reasonable to assume that the molecular and elemental ion intensities of isotopologues have to range between the intensities of fully hydrogenated and deuterated compounds . when such contextual knowledge was included in the entropy minimization algorithm the obtained ash 2 d and ashd 3 mass spectra were in good agreement with a standard statistical model . deconvolution of eh 4 / eh 3 d / eh 2 d 2 ehd 3 / ed 4 mass spectra . aqueous phase reaction of borohydride , bh 4 − , with ge ( iv ) and sn ( iv ) species forms geh 4 and snh 4 respectively . however , in the presence of deuterated borohydride , bd 4 − , four more hydrides can form : eh 3 d , eh 2 d 2 , ehd 3 , and ed 4 ( e = 73 ge , 118 sb ) depending on the bh 4 − / bd 4 − ratio . also , various partially deuterated hydrides can be obtained when geh 4 / ged 4 and snh 4 / snd 4 are subjected to h / d exchange reactions at different ph . germanium and tin hydride mass spectra are different from the as , sb and bi hydride in a sense that the molecular ion is practically absent . also , the number of isotopologues is larger by one than in the case of group v element hydrides . for these reasons the ge and sn hydride isotopologue mass spectra substantially overlap and the retrieval of all five isotopologue mass spectra using the entropy minimization model is impossible . geh 4 , ged 4 and snh 4 , snd 4 mass spectra can be easily recovered using the restrictions analogous to as , sb and bi spectra . alternating the band - targets for germanium hydride mixtures , for example , leads to the retrieval of only geh 4 , ged 4 and gehd 3 spectra . accurate ion intensities , however , cannot be ensured for geh 3 d , geh 2 d 2 , gehd 3 and their sn analogues for two reasons . first , no feasible verification of the ion intensities can be done ( due to the absence of the molecular ions ) and , secondly , there is no safe way to ensure that pure spectra ( and not linear combinations of several spectra ) are retrieved from the entropy minimization . from this it is clear that entropy minimization cannot be used to extract the reference spectra of geh 3 d / geh 2 d 2 and snh 3 d / snh 2 d 2 . despite the difficulties of implementing the entropy minimization model , it has an important advantage in retrieval of fully deuterated hydride reference spectra . due to the high reactivity of ged 4 and snd 4 , the h / d exchange process makes it extremely hard to obtain mass spectra with no impurities from the first exchange products gehd 3 and snhd 3 . validation of the reconstructed mass spectra . although it is practically impossible to obtain pure mass spectra of the partially deuterated species ( unless scrupulous separation is performed ), nevertheless , it is possible to obtain experimental evidence of certain ion ratios for the ashd 2 , sbhd 2 and bihd 2 mass spectra from rather simple experiments . when 75 as , 123 sb and 209 bi hydrides are generated in an environment that contains & gt ; 90 % deuterium ( as bd 4 − ), it is reasonable to assume that the generated hydride mixtures will contain mostly 75 asd 3 , 123 sbd 3 , 209 bid 3 , some amounts 75 ashd 2 , 123 sbhd 2 , 209 bihd 2 , and virtually no other species such as ash 3 or ash 2 d . the same principle is also observed , for example , in a h 2 o / hod / d 2 o system . in such mixtures ions with even m / z ratios originate from the 75 ashd 2 , 123 sbhd 2 , and 209 bihd 2 and have no contributions from the fully deuterated compounds . thus , the even m / z ion ratios can be compared to the theoretical estimates from entropy minimization models as shown in table 1 . hydrides for this experiment were generated using a 90 : 10 mixture of equimolar nabd 4 and nabh 4 solutions . it is evident from these data that unconstrained ( blind ) entropy minimization may lead to biased ion ratios . nevertheless , the blind entropy minimization leads to mass spectra that are in agreement with experimental evidence . applying restrictions on particular ion intensities is , perhaps , feasible , only in isotopologue systems where partial knowledge is always present ( since the molecular formulas are known ). similarly , it is possible to obtain accurate spectral intensities from the band - target entropy minimization algorithm when certain m / z channels are forced to zero ( as in the case of the ged 4 mass spectra ). in practice , such restriction can be useful when the task is to extract the mass spectrum of a particular component . for example , when a mass spectrum of ash 3 is required , the intensities of the channels 75 & gt ; m / z & gt ; 78 can be set to zero . concentration profiles of isotopologues . the ultimate aim of this study was to be able to obtain accurate concentration profiles for the deuterated element hydrides . to achieve this , various ratios of bh 4 − / bd 4 − were used to generate the arsenic hydrides and the collected normalized mass spectra ( m / z = 75 - 81 ) of the hydride mixtures were then inspected . amount fraction of each compound in the analyzed gas mixtures was calculated from the observed composite mass spectra using the isotope pattern reconstruction . in this procedure , the amount fractions of all four arsenic hydride isotopologues are obtained from the least squares fit of the following equation : the left side of the equation is the average mixture mass spectrum ( from n = 4 experiments ) and right side contains the four mass spectra of the individual isotopologues . least squares fitting of this mass spectrum leads to the relative isotopologue abundances 0 . 18 , 0 . 43 , 0 . 31 and 0 . 09 for ash 3 , ash 2 d , ashd 2 and asd 3 respectively . fig3 shows the deconvoluted concentration profiles of the arsenic hydride isotopologues generated using various amounts of nabd 4 in the mixture of nabd 4 and nabh 4 ( from pure nabh 4 to pure nabd 4 ). from these profiles one can see that the sequential hydrogen and deuterium incorporation into the metal hydrides approximately follows the binomial probability distribution : x ashh 3 - i ⁢ d i = 3 ! i ! × ( 3 - i ) ! × x d i × x h 3 - i ( 11 ) where x d and x h are the deuterium and hydrogen amount fractions in the borohydride mixtures ( bd 4 − / bh 4 − ). any deviations from this equation are the result of kinetic effects of h and d transfer . increasing the amount of deuterium in the borohydride results in the systematic increase of deuterated arsenic hydrides : starting from the ash 2 d , then followed by ashd 2 and ultimately , nabd 4 produces almost pure asd 3 . thus , band - target entropy minimization can extract eh 2 d and ehd 2 mass spectra from a mixture of isotopologues with unknown concentration . accurate ion intensities can be predicted for these compounds . using the predicted mass spectra it is possible to estimate individual isotopologue concentration in unknown mixtures . example 1 demonstrates the utility of blind entropy minimization for the extraction of unknown component mass spectra from the mass spectra of unresolved or partially resolved real - world systems . example 2 describes the use of blind entropy minimization in the context of extracting individual mass spectra of a simulated two - component pesticide mixture . specifically , example 2 demonstrates the properties of entropy surface landscape and its advantages in mass spectral deconvolution . in an arbitrary chosen system of two components , the extracted mass spectra ( not necessarily pure ) is a function of the t vector ( containing two variables a and b ): hence , the entropy landscape of the extracted mass spectra , a , can be explored as a two - variable function : this two component system entropy surface landscape can be visualized as a 3d scatter plot . for this purpose , an overlapping chromatogram of two - component mixture of two pesticides ( fenac and sulfotep , using ions from m / z = 50 to 300 ) was simulated with added proportional white noise . for this purpose , reference spectra of the pure compounds were obtained from the nist mass spectral library , and an overlapping chromatographic elution profile was then established using exponentially modified gaussian peak profiles ( tailing peaks ) with added noise as shown in fig4 . the entropy landscape surface then was explored by calculating the entropy of arbitrary chosen fragment ion , say m / z = 193 . this ion is found in both component mass spectra so using this ion as target will extract the component with highest concentration , in this case fenac . the entropy surface plot for this ion is shown in fig5 . in fig5 , entropy of the extracted mass spectra using m / z = 193 target ion is plotted as a function of the t vector ( variables t 1 and t 2 ). white area corresponds to small entropy while black areas represent large entropy regions . it is evident that the entropy surface is smooth , regardless of the mass spectral noise . also , there are formally two minima , however , due to the matrix multiplication properties , both yield identical extracted mass spectra . this is due to the fact that both minima correspond to vectors that differ only by the sign : in this example , t ≈(− 0 . 9 , − 0 . 9 ) and t =(+ 0 . 9 , + 0 . 9 ). this means that for practical purposes it does not matter which minima if found since they both carry same physical meaning . unfortunately , visualizing the entropy landscape in more component systems is challenging as is any multidimensional visualization task . in this case , the four components are pesticides aldrin , bifenox , bromophos and captan . chromatographic elution of the four pesticide mixture was simulated in silico . for this purpose , reference spectra of the pure compounds were obtained from the nist database , and an overlapping chromatographic elution profile was then established with added noise . concentration of the each component in this mixture was equal and severe chromatographic overlap was created . the obtained total ion current chromatogram is shown in fig6 . from the chromatogram of fig6 , a mass spectrum was extracted for every given time interval ( scan ) and the band - target entropy minimization was performed without using any prior knowledge of the system . four components were identified and their extracted spectra ( fig7 a ) agree well with the used library spectra ( fig7 b ). the deconvoluted single ion chromatograms of each component ( fig8 a ) also agree well with the values used for the simulation ( fig8 b ). when a mixture of the same four components with added noise was simulated wherein one of the components is a minor component present at the 10 % level , an elution profile as shown in fig9 a is obtained . entropy minimization still yielded the mass spectra of all four pesticide components . the extracted mass spectrum of the minor component ( fig9 b ) agrees well with the experimental spectrum ( fig9 c ). for the system described in example 3 , the fact that the entropy landscape surfaces are monomodal can be proved also by the fact that many simplex minimizations each initialized randomly at different position leads to the same results , identical to the minimum entropy result obtained by exhaustive random search . targeting the ion with the mass - to - charge ratio m / z = 75 ( major ion in bromophos mass spectrum ), leads to the retrieval of bromophos mass spectrum each time . global entropy minimum found by exhaustive random search in this case is close to that found using several directed optimization attempts ; each initiated at random starting point . same quality spectra were obtained using gradient - based directed optimization approaches , such as the steepest descent minimization . results for the above four component system are summarized table 2 . mass spectra obtained from directed and stochastic optimization approaches are identical with except to minor random noise . hence , it is evident that directed downhill optimization strategies can be successfully used to find the pure component mass spectra without resort to general stochastic approaches , such as simulated annealing . one of the main difficulties in applying simulated annealing is that many choices have to be made concerning parameter values ( tuning ), which are not trivial and are quite often very poorly made by trial and error . most crucially , one has to specify the temperature decrease profile ( annealing schedule ), and the initial temperature parameters . this can be rather sensitive to the minimization speed . it has now been surprisingly found that the use of stochastic approaches such as simulated annealing is not necessary as direct optimization approaches can be successfully used . the gain in computing time of downhill simplex ( a directed optimization approach ) compared to simulated annealing from various tested systems is about 100 times ( two orders of magnitude ). the above outlined entropy surface properties ( i . e . that the entropy landscape is monomodal ( with no local minima )) leading to the usefulness of directed optimization approaches are not obvious a priori . it is possible to use entropy minimization to recognize a specific compound from a complex mixture . three components in a three component mixture have mass spectra as shown in fig1 a - c . a component of interest ( a ) has an m / z 5 and m / z 7 ion ratio of 1 : 1 ( fig1 a ). in this mixture , the component a itself is present in amounts of less than 1 %, or , in other words , 99 % of the signal is noise for spectral identification purposes . the ratio of ions m / z 5 and 7 in the present overlapping system varies from 0 . 54 to 0 . 38 during the elution of these three highly overlapping components . this means that visual detection of the component a is impossible from such system since the correct ion ratio ( i . e . 1 . 00 ) is not observed in any of the individual mass spectrometry scans . targeting the two ions m / z 5 and 7 for entropy minimization , however , can extract almost pure component spectrum as shown in fig1 . in this example , the highest amount of the target compound in the chromatographic elution profile is about 1 . 5 % whereas entropy minimization could purify this component up to 99 %. the m / z 5 and 7 ion ratio in the extracted spectrum is from 0 . 95 to 1 . 03 depending on the actual simulated noise levels . fig1 a is the elution profile of the mixture and fig1 b is the mixture mass spectrum corresponding to the highest amounts of component a present ( at 14 . 5 min elution time ). clearly , no pattern of component a can be recognized from this spectrum . such targeted retrieval of component helps to increase the analysis specificity . as a result , the number of false positive detections is decreased and number of positive detections is increased . the mere presence of correct two ion ratio within a mixture chromatogram does not guaranty the presence of the component with such ion ratios in its pure spectrum ( false positive detection ). this example shows that the presence of certain patterns can be detected by entropy minimization where the commonly used ion ratio approach fails . elution time and m / z scale can be swapped and entropy minimization performed in the time domain . in this way , pure peak elution profiles ( usable for quantification of components ) can be obtained rather than the mass spectra ( usable for identification of components ). in this modification of the entropy minimization algorithm , the time domain is used as the variable space . in other words , the entropy minimization yields no longer pure component mass spectra but pure component chromatographic elution profiles . the modification is achieved by 90 ° rotation of the data matrix and the singular value decomposition is then performed on the rotated matrix . band targeting is now performed for retention time ( spectral scans ). using the three - component system described in example 5 , the noise - free chromatogram ( fig1 , line ) represents the theoretical chromatographic profile used for simulation . to this , line noise was added and so were the other two overlapping components . the extracted chromatogram ( fig1 , dots ) is in good agreement with the actual pure component elution profiles . hence , the concentration of components ( peak area of the pure component chromatogram ) can be estimated rather accurately . alfassi , z . b . on the normalization of a mass spectrum for comparison of two spectra . j . am . soc . mass spectrom . 2004 , 15 , 385 - 387 . brunengraber , h . ; kelleher , j . k . ; des rosiers , c . applications of mass isotopomer analysis to nutrition research . annu . rev . nutr . 1997 , 17 , 559 - 596 . chew , w . ; widjaja , e . ; garland , m . band - target entropy minimization ( btem ): an advanced method for recovering unknown pure component spectra . application to the ftir spectra of unstable organometallic mixtures . organometallics 2002 , 21 , 1982 - 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