Quantitative Quantum Mechanical NMR Analysis : the Superior Tool for Analysis of Biofluids

Almost automate quantitative analysis of biofluids is now behind a few clicks, from sample to EXCEL table after minimal sample preparation, without separations, calibration and reference materials, even for unknown compounds! Each organic compound with protons gives a highly diagnostic and unique spectrum which is practically identical with any spectrometer operating at certain field. A distinctive feature of the 1D 1H NMR spectra is that even the most complex spectrum of a compound can be described by a few spectral parameters within experimental accuracy, employing the quantum mechanical theory. The NMR spectral parameters offer also a very efficient way to store artefact free spectra in Adaptive Spectral Libraries (ASL), instead of variable quality experimental spectra. Once spectra have been measured and modelled in one magnetic field strength using Quantum Mechanical Spectral Analysis (QMSA), the spectra can be simulated in every detail in any other field and mixtures – to be used in quantification of the mixtures with ChemAdder software (see http://chemadder.com). The software is described and its application to analyses of serum, volatile fatty acids from biowaste and slaughterhouse waste are used as examples in our presentation.


ABOWE project
Movable ABOWE Pilot biorefinery unit for industry wastes, in Poland for potato industry and restaurant biowaste, and in Sweden for slaughterhouse wastes.The unit was constructed in Savonia University of Applied Sciences, Kuopio, Finland, under supervision of Adjunct Professor Elias Hakalehto.Photo: Mika Ruotsalainen.

Propionate
A structure (or a part of it) be also identified from splittings (coupling constants) of multiplets: the couplings do not depend on instrument or sample.

Results and discussion (Part 2)
Quantum Mechanical Spectral Analysis (QMSA) The chemical shifts depend slightly (0.001-0.05 ppm) on sample, but in qQMSA they can be recognized effectively from their coupling patterns …this forms a problem in the (non-QM) methods based on experimental model spectra.
Even the most complex NMR spectra obey strict quantum mechanical rules and can be simulated in very details Even the most complex NMR spectra obey strict quantum mechanical rules and can be simulated in very details Testosterone: Adaptive Spectrum Libraries (ASL): Analyze spectrum at one (magnetic) field, then the spectrum at any other field and line-shape can be then simulated !Also variations in the chemical shifts can be taken into account.
• SpinAdder: the new generation of QM spin engine.
• Smart chemical shift permutator for complex spectra.
• Graphics & data: No limitation in number of spectra treated simultaneously.
• Fast essential metabolite search from ASL's using FZZY tool: takes advantage from the multispectral data.• Up to 100 or more (?) metabolites.
• Targeted ASL (Adaptive Spectral Library): metabolite libraries for each sample type -any field -any line-shape.• Output in TXT or CSV (EXCEL) format, in mg/ml or, also for unknown compounds, in mmol/ml.• Wizarded protocols.
• Tailored protocols (MENUs) and default profiles for sample types.
• Maximal information using combination of QM spectra, structures and prior knowledge (= any information that can be written for iterator in form of linear equation).
• Tools for preparation of ASL spectra even from very poor spectra (with bad baseline and solvent suppression artefacts) or even from peak lists.• QMSA oriented tools for examination of 1D and 2D spectra.

ChemAdder user interface:
Simultaneous analysis of a set of spectra: extra information for metabolite search from ASL's !GALACTOSE ( and forms) gives a very crowded spectrum of 5 protons within 0.1 ppm (60 Hz): the prediction of the chemical shifts is impossible with such an accuracy and the 2D spectra are almost useless because of strong couplings which also make the multiplets of individual protons unrecognizable and very sensitive to the shifts !Solution is the smart shift permutator, which goes through the combinations of shift order by a try-and-learn algorithm: • ChemAdder & SpinAdder allow analysis of very poor spectra (with bad baseline, solvent suppression artefacts and impurities) or even from peak lists.

SMART PERMUTATOR and preparation of ASLs
SpinAdder: the new generation QMSA engine: non-QM signals can be described by structures

Observed-Calculated Difference
STRUCTURES can be regular or non-regular multiplets

STRUCTURES
Even the smallest details of spectrum can be described by the combination of QM spectra and the structures !ignored ignored

=>
weight point of multiplet Coupling constant (J) difference of two lines => fine structure THE PARAMETERS ARE INDEPENDENT OF INSTRUMENTATION ..The problem with signals in MS, GC and HPLC !! Chemical shift (n) = weight point of multiplet Coupling constant (J) difference of two lines => fine structure THE PARAMETERS ARE INDEPENDENT OF INSTRUMENTATION ..The problem with signals in MS, GC and HPLC !! QMSA Observed spectrum Quantum Mechanical NMR Spectral Analysis: Math NMR intensity spectrum I( ) is sum of spectra of chemical components S( ), background B( ) & noise( ) I( ) = x n S n ( ) + B( ) + noise( ) where is frequency.Each spectrum S( ) is a function of spectral parameters S n ( ) = F n ( , w, J, R, , Line-shape) Where w = chemical shifts, J = coupling constants, R =response factors ( 1.0), = line-widths and line-shape.Structure analysis: I( ) => w & J => structure Quantitative NMR: I( ) => x n (populations) A non-linear mathematical inverse problem -solved iteratively !! (F n is a non-explicit function the values of which can be calculated and differentiated by using matrix formalism) If chemical shifts, coupling constants & line-shape are given, spectrum can be simulated quantum mechanically !If chemical shifts, coupling constants & line-shape are given, spectrum can be simulated quantum mechanically !Model spectra for quantitative analysis -and ASL => Model spectra for quantitative analysis -and ASL Simulated spectrum

25000 transitions ! > 25000 transitions !
Large Spin-networks can be now simulated (by automate splitting into sub-systems)