Nuclear Magnetic Resonance

4 mins
Author name: Thomas Gold

Determination of Trifluoroacetic Acid in Human Urine

Determination of Trifluoroacetic Acid in Human Urine using 19F Nuclear Magnetic Resonance (NMR), After Inhalation of 1,1,1,2-Tetrafluoroethane or Dichlorodifluoromethane
Thomas B. Gold, Ph.D.¹ and George A. Digenis, Ph.D.²
¹Metrics, Inc., 1240 Sugg Parkway, Greenville, NC 27834.
²University of Kentucky College of Pharmacy, Pharmaceutical Sciences Dept., 789 S. Limestone St., Lexington, KY 40536

Determination of trifluoroacetic acid (TFA) levels in human urine, using 19F Nuclear Magnetic Resonance (NMR).

The chlorofluorocarbons (CFCs) have in the past been vital to such industries as heating/air conditioning, cleaning solvents, and aerosol propellants. In 1974, studies conducted by Crutzen (1) and Johnston (2) on the interaction of stratospheric ozone and chlorine showed that CFC’s have a harmful effect on the ozone. Molina and Rowland (3) investigated the susceptibility of the CFCs to ultraviolet light-induced production of chlorine radicals. Collective research let to regulation of CFC usage in the United States. The reduction and eventual elimination of CFC usage and production promoted the search for a viable alternative to CFCs. The hydrofluoroalkanes (HFAs) constitute a new class of compounds with similar physical characteristics as the CFCs, without the ozone depleting properties. The EU endorsed 1,1,1,2-trifluoroethane, HFA-134a, in 1994, with the FDA committing to expedited review of DMF’s as clinical data became available. HFA-134a is oxidized by cytochrome P450 to trifluoroethanol, with eventual conversion to TFA (4-7).

One method highly sensitive for the detection of HFA-134a metabolism is 19F NMR. Pharmaceutical applications of fluorine detection in human urine were investigated by Monte et al. (7). These researchers used 19F NMR to detect TFA levels as low as 10 ηg/mL in human urine with acquisition times of 2.25 h per sample. The present work uses this method, and by conducting T1 relaxation time experiments with TFA in order to optimize 19F NMR experimental parameters such as flip angle (expressed as pulse width) and line width, much lower detection limits of TFA in human urine were achieved.

Materials & Methods

  • Seventy five clinical samples were received from 3M Pharmaceuticals, St. Paul, MN
  • The clinical studies were conducted by 3M Pharmaceuticals

Sample Preparation:
NMR tubes (5 mm, Wilmad, Vineland, NJ) were silanized by immersion in a 10% solution of dichlorodimethylsilane (Sigma, St. Louis, MO) in toluene for 12 hours. Tubes were rinsed with methanol and dried with compressed nitrogen prior to use. Aliquots (0.500 mL) of the urine samples were placed into polypropylene tubes, after which 220 µL of 10M sodium hydroxide was added, along with 80 µL of deuterium oxide (Norrell, Mays Landing, NJ). The tubes were gently vortexed, and 750 µL of the solution was transferred to the NMR tube.

Calibration standards were prepared , over the range of 2.5 – 50 ηg/mL, by diluting a standard sample of TFA (Aldrich, Milwaukee, WI) in water with control human urine. A blank control of human urine was also prepared.

All spectra were obtained at 20°C on a Varian VXR-300 spectrometer (Varian, Palo Alto, CA), operating at 282.217 MHz with a 5 mm probe. Spectral width was set to 5,000 Hz (with transmitter offset of 8500 Hz) containing 7,000 data points. After conducting T1 experiments of TFA in distilled water, it was determined that a flip angle of 17 µsec was reasonable. For each sample, 12,000 transients were acquired, and the TFA peak at 2387.06 Hz was integrated using limits of 2281.69 Hz and 2492.53 Hz, keeping the vertical and integral scales identical for all calibration standards and blank, as well as samples (vertical scale = 3 x 106). The integral value was plotted against the knowing concentration of TFA in the standards.

Results & Discussion
The calibration graph obtained by regressing the integral values from the 19F NMR signal corresponding to TFA onto the known TFA standard concentrations is shown in Figure 1.

1. P.J. Crutzen. J. Geophys. Res. 76, 7311-7327 (1971).
2. H.S. Johnston. Science. 173, 517-522 (1971).
3. M.J. Molina and F.S. Rowland. Nature. 249, 810-812
4. M.J. Olson, C.A. Reidy, J.T. Johnson and T.C. Pederson. Drug Metabol. Dispos. 18, 992-998 (1990)
5. M.J. Olson, S.G. Kim, C.A. Reidy, J.T. Johnson and R.F. Novak. Drug Metabol. Dispos. 19, 298-304 (1991)
6. S.E. Surbrook, Jr. and M.J. Olson. Drug Metabol. Dispos. 20, 518-524 (1992)
7. S.Y. Monte, I. Ismail, D.N. Mallett, C. Matthews and R.J.N. Tanner. J. Pharm. Biomed. Anal. 12, 1489-1493 (1994).

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