HPLC-NMR spectroscopy

Sidelmann UG, U Braumann, M Hofmann, M Spraul, JC Lindon, JK Nicholson, SH Hansen (1997) Directly coupled 800 MHz HPLC-NMR spectroscopy of urine and its application to the identification of the major phase II metabolites of tolfenamic acid. Anal Chem 69 607-612. 

HPLC with UV detection at 280 nm showed essentially only the required drug compound but a quantitative assay against a reference material showed a level of only 96.6% of that expected. A number of impurities were detected by using HPLC with MS and refractive index detection and the structures of the major components were elucidated by using 500 MHz H HPLC-NMR spectroscopy. 

This shows that directly coupled HPLC-NMR and HPLC-NMR-MS can enhance the ability to characterise impurities in a pharmaceutical product. As such, this approach is potentially significant as a general tool for purity analyses and would be expected to be important in speeding up production chemistry processes and for regulatory affairs. In the fluticasone study, it was shown that HPLC-NMR spectroscopy could detect and characterise impurities below the 0.1% peak level, which is the relevant limit for submissions to regulatory authorities [27]. 

Directly coupled HPLC-NMR spectroscopy has been used in a number of other studies of chemical impurities. An impurity in a bulk drug sample of the glycinamide ribonucleotide transformylase inhibitor AG2034, shown below ... 

Another different area of application where there is a need to characterise low-level impurities occurs in environmental studies. A recent study has used HPLC-NMR spectroscopy and HPLC-MS in the MS mode to identify organic components in the industrial waste-water effluent of a textile company. Generally in the past, GC-MS and HPLC-MS have been used for such analyses where the identity of many of the pollutants are not known beforehand. However, it has recently been shown that HPLC-NMR can provide useful information and if this is used with complementary information from HPLC-MS then this is the most efficient approach. 

Other studies of natural products, which will be described in detail elsewhere in this book, involve the characterisation of isomeric structures, an application for which NMR spectroscopy is particularly powerful. For example, Albert et al. have used HPLC-NMR spectroscopy to determine the structures of isomers of vitamin A acetate [45]. The parent molecule has all-trans double bonds as shown below and by brief heating of this substance a number of isomers with a single cis double bond and one isomer with two cis double bonds can be formed ... 

Figure 3.10 Comparison of lipoprotein standards and HPLC-resolved lipoproteins, (a) Partial 600 MHz HPLC-1 H NMR spectra (80.5-2.0) of the HDL, LDL and VLDL fractions separated by using directly coupled HPLC-NMR spectroscopy at 15.5, 58.9 and 86.6 min, respectively, (b) Partial 600 MHz 1H NMR spectra (8 0.5-2.0) of standard (std) HDL, LDL and VLDL. Abbreviations VLDL1, LDL1 and HDL1 indicate the resonances from the terminal methyl groups of the mobile fatty acid chains bound in the various lipoproteins VLDL2, LDL2 and HDL2 indicate resonances from the methylene groups of the mobile fatty acid chains...

Shockcor, J. P. Unger, S. E. Wilson, I. D. Foxall, J. P. Nicholson, J. K. Lindon, J. C. 1996. Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine. Anal. Chem., 68,4431 1435. 

Lindon JC, Farrant RD, Sanderson PN, Doyle PM, Gough SL, Spraul M, Hofmann M, Nicholson JK, Separation and characterization of components of peptide libraries using on-flow coupled HPLC-NMR spectroscopy, Magn. Reson. Chem., 33 857-863, 1995. 

N. Mistry, I. M. Ismail, M. S. Smith, J. K. Nicholson, and J. C. Lindon, Characterization of impurities in bulk drug batches of fluticasone propionate using directly coupled HPLC-NMR spectroscopy and HPLC-MS, J. Pharm. Biomed. Anal. 16 (1997), 697-705. 

An example for the separation of ecdysteroids with HP-RPC systems was published by Louden et al.119 and related to the separation of an ecdysteroids extract from the plant Lychnis flos-cuculi (Caryopyllaceae). These authors used an analytical 4.6 mm x 100 mm 5 pm C18 silica column with isocratic elution (eluent ACN and DzO 99.8% isotopic purity 20 80 v/v) at 1.0 ml min 1 and DAD between 190 and 360nm. This study also explored the application of HPLC—NMR spectroscopy and HPLC—NMR spectroscopy—MS to these ecdys-teroid-containing plant extracts, showing the advantages and limitations of the use of complex multiply hyphenated detection systems, which incorporate detectors of differing sensitivities. 

To date, HPLC-NMR spectroscopy has been applied to the profiling and identification of the metabolites of a number of drugs and xenobiotics present in biofluids such as plasma, urine and in bile samples from rat and... 

Lindon JC, Nicholson JK, Wilson ID. The development and application of coupled HPLC-NMR spectroscopy. Adv Chromatogr 1996 36 315-382. 

The major (83%) and minor (17%) B12 compounds purified from the Spirulina tablets are identified as pseudo-Bi2 and Bi2, respectively, judging from TLC, HPLC, NMR spectroscopy, ultraviolet-visible spectroscopy, and biological activity data. The Spirulina tablets are not suitable for use as a Bi2 source, especially for vegetarians, because pseudo-Bi2 appears to be inactive for humans. 

Sweatman BC, Farrant RD, Sanderson PN, et al. (1995) Evaluation of the detection limits of directly coupled 600 MHz H AND H- C HPLC-NMR spectroscopy. Journal of Magnetic Resonance Analysis 1 9-12. 

---