Applications of NMR Spectroscopy in Pharmaceutical ...

1 Chapter 12 Applications of NMR Spectroscopy in Pharmaceutical SpectroscopyNuclear magnetic resonance (NMR) Spectroscopy hasevolved asoneofthemost powerfulanalyticaltechniques. It allows thevisualizationof single atoms and molecules in various media insolution as well as in solid state. NMR is nondestructive and givesmolar response that allows structure elucidation and quantificationsimultaneously. magnetic interactions between NMR-active nucleialong covalentbondsresult in spin spin can be detected using the nuclear Overhauser effect(NOE).Boththeseinteractionsfacilit ate thethree-dimensionalstructure dimensional and two dimensional NMRdata can be collected. The 1D NMRexperimentsare1H,13C,31P,19F, 1D NMR techniques will give information regarding thechemical shifts, spin-spin couplings and intensities.

2 The chemicalshifts will give the information regarding environment of the which are close to one another exert an influence on eachother's effective magnetic field. This effect shows up in the NMRspectrum when the nuclei are nonequivalent. If the distance betweennon-equivalent nuclei is less than or equal to three bond lengths, thiseffectis observable. This effect is calledindirect spin-spin intensities will give the relative number of protons under 2D NMR experiments are COSY, TOCSY, HSQC, HMBC,Chapter 13 NOESY, ROESY, etc. These 2D experiments provide informationregarding through bond or through space intensities of the resonance signals1 HNMR Signal IntensitiesTheintensityor the integral of a signal is considered to be thearea under that comparison of the signal intensities in aspectrum will give the ratios of the protons in thereare multiplets in a spectrum, the whole group of peaks should like the chemical shifts and indirect spin-spincouplings, the signal intensities are also important for the signalintensitieswill help in the quantification Signal IntensitiesIn principle,theintensities ofcarbon-13signalscan also beusedto infer the number of carbons responsible for the.

3 The low abundance and sensitivity of the carbon-13isotope will affect the quantification of number of carbons in tothisreason the carbon signals aregenerallynotintegrated in13C NMR quantification of carbon-13 signalcan be madepossible with highdigital resolution, suppression of NOE, a pulse repetition rate that isnot too fastand small spectral width and high pulse NMR SpectroscopyThe one dimensional NMR spectra have twodimensions, theabscissa and the abscissa corresponds to the frequencyaxis and the ordinate gives the signal ,in two-dimensional (2D) NMR spectra, both the abscissaandthe ordinaterepresent frequencyaxes;the third dimension gives the the 2D J-resolved NMR spectrum, the chemical shifts will beplotted along one of the axes and the coupling constants along theother both axes are chemical shifts, then it is called 2D(shift) correlated oftentheshift correlated 2 DNMR dataisused instructure correlations could behomonuclear (1H-1H)or hetero nuclear (1H /13C).

4 J. Jeener has proposed the 2D NMR Spectroscopy in ,of R. R. Ernst andR. Freeman,have madethe 2D variants of 2D NMR experiments were developedby these two NMR Experiments BriefDescriptionsTwo-dimensional NMR experiments will provide valuableinformation which is difficult to obtain from 1D NMR 2D NMR experiments provide additional information by spreadingpeaks out in the second frequency dimension by correlating 15 Homonuclear Correlation ExperimentsThese are the most common 2D NMR experimentsthat provideinformation J-coupling or COSY experiment givescorrelations between directly couple protonsthrough bonds, whereasa NOESY experiment will give correlation between protons throughnuclear Overhauser Spectroscopy (COSY)The simplest and most often used 2D NMR experiment is theCOSY peaks in a COSY spectrum arise from theprotons that are usually observed betweengeminal or vicinal protons (2-or 3-bonds) with coupling constants inthe range of 2 to range couplings also be seen in COSY (DQF-COSY)The DQF-COSY experiment provides information that isidentical to that of DQF-COSY experiment is generally runin phase-sensitive mode, generating narrower peaks than to distinguish the cross peaks near the diagonal of DQF-COSY experiment eliminates or reduces thesinglet peaks in the Correlation Spectroscopy (TOCSY)

5 The information in TOCSY spectra is similar to COSY and DQF-COSY spectra, do not simply display cross peaksbetween pairs ofJ-coupled protons, but also display cross peaksbetween pairs of protons that are not directlyJ-coupled but are in thesameJ-coupling network or spin system by relaying coherence fromone proton to the next within the short mixing time will give correlations between the protonsof 2-3 bonds and the longer mixingtimes will give correlationsbetween 4+ information is very useful if a molecule iscomposed of several spin systems and their protons are TOCSY experiment is commonly used in the peptide andprotein structures helps in the assignments ofindividual amino acids in peptides and Overhauser Effect Spectroscopy (NOESY)NOESY spectra shows cross peaks between the protons whichhave NOE interactions in a NOEs are because of thedipolar cross-relaxation between nuclei in a close spatial cross peaks appearif the protons are less than 5 apart in protons in different parts of the molecule can showChapter 17 NOE information is useful in the determination Frame nuclear Overhauser EffectSpectroscopy(ROESY)

6 The ROESY and NOESY spectra are almost of the ROESY experiment is that NOEs can be seenregardless of molecular ROESY spectra can show TOCSY-like artifacts, while NOESY spectra can have COSY-type Correlation ExperimentsThe 2D NMR experiments involving more than one nucleus arereferred as heteronuclear , one of the nucleuswill be proton and the other can be either nitrogen or two types of heteronuclear correlation experimentsviz, directdetectionexperiments and indirect detection thedirect detection heteronuclear experiments, the heteronucleus isdirectly of these experiments is less duetothelow abundance of heteronucleusatomwhichisdirectly contrary,the indirect detection heteronuclear experiments observethe less abundant hetronuclearatomsthrough more leads tovery highsensitivity of indirect detectionexperimentswhen compared to direct detection heteronuclear 18 Direct Detection nuclear Correlation (HETCOR)The13C-1H correlations between protons and carbons,connected through one bond will give correlation in this experiment, the directly detected nucleus will of low abundance of carbon-13 the experimenttakes more time to get a good HETCOR experiment has beenreplacedby HSQC and HMQC of these experiments arethat the carbons can be assignedeasily from their attached experiment is alsouseful inthe identification of diastereotopic Multiple Quantum Correlation (HMQC)

7 The HMQC is an inverse detection nucleus is directly detected and the carbon nucleus is information we get from HMQC is same as theinformation we get from HETCOR differencebetweenthese two experimentsisthat HMQC will take less Single Quantum Correlation (HSQC)TheHSQC and HMQC experiments give essentially the difference is how the coherence evolves during theevolution HSQC is superior in cases where the13 CChapter 19spectrum is HSQC experiment is often used instead Multiple Bond Correlation (HMBC)In the HMBC experiment, long range1H-13C correlations canbe HMBC experiments are mainly used in the assignmentsof quaternary carbons, where there are no attached correlations extend nitrogen or oxygen atoms for the of NMRin HPLC is used extensively for the analysis of detectors, RI, UV and Fluorescence.

8 Are used to monitorthe separations done by detectors can give only limitedinformation on molecular LC-MS is widely used for themolecular LC-MS is particularly used in thepharmaceutical industry for the identification andquantification ofdrugs and their metabolites and is very difficult to identify the structure of impurities anddegradation products unambiguously by LC-MS is thebest tool for the identification and characterization of thesemetabolites and the identification of the structure ofmetabolites and impurities by NMR we need isolation of these byChapter 110preparative is a time consuming and need lot of LC-NMR has been increasingly used for theidentification ofmetabolites, impurities and degradation products[3-8].Working modes in LC-NMRT here are several ways that the NMR data can be collected inconjugation with NMR data can be collected during thechromatographic this case the separated componentswill flow through the NMR flow cell after passing through UV NMR data will be collected on process of acquiring NMRdata is NMR data can also be collected by stopping the flow whenthe separated component enters into the flow process separated fractions can be stored on loops and thesefractions can be pushed into flow cell after completing the , LC-NMR instrumentshave up to ten is called loop storage and loop LC-NMR is being routinelyused for the identification ofimpurities, drug intermediates, combinatorial chemistry.

9 Separation ofpharmaceutical mixtures 111b. Quantitative NMR spectroscopyNMR Spectroscopy is considered as a primary method for themeasurement of ratios[10&11]. The ratio of substances can bedetermined directly from the proton NMR spectrum. We don t needany substance for referencing. The absolute amountof substance canbe determined by NMR Spectroscopy if we use a reference is possible only in case where the signals ofinterest are well separated. Sometimes it is possible to resolve thechemicals shifts by changing the solvent,the pH value or by addingauxiliary advantage of NMR is the little time it takes for the takes only 10 20 mg of the sample in ofdeuterated qNMR is used for the quantification ofimpurities, residual solvents, isomers, diastereomers andeanntiomeric analysis is generally performed using theintegrated intensities of the signals of interest.

10 In recent times, thechemical shift displacements has been employed for profile study of APIsIdentification and characterization of impurities has becomeessential as per various regulatory organicChapter 112material arises out of synthesis, besides the drug substance oringredients is calledan impurities may form during theformulation or stability ICH has published guidelines for the validation of methods foranalyzing in new drug substances, drug products, residual solventsand impurities[13-16].The isolation and identification of process related impurities anddegradation products are well studied by LC-MS, HR-MS and NMRspectroscopic techniques[17].As per ICH guidelines the impurities are classified as given below[18].a) Common names-By-products-Degradation products-Interaction products-Penultimate intermediates-Intermediates-Transformati on products-Related products-b)ICH terminologyAs per ICH guidelines the impurities in the drug substanceformed during chemical synthesis can be classified into the Impurities (Process and Drug related) SolventsThe storage or manufacturing process may give rise to organicimpurities a given materials or productsGuidelines for impurities limits (ICH)The identification of