The methods and apparatus described may be used for the discrimination of two closely spaced spectral lines. In certain embodiments the methods and apparatus may be used for the detection of isotopes and isotopic ratios by Raman spectroscopy. In certain embodiments the conversion of a labeled substrate to a labeled product may be detected. In particular embodiments the conversion of a 13C labeled substrate to a 13C labeled product may be detected. In other embodiment the conversion of 13C urea to 13CO2 is detected. Embodiments described include the diagnostic determination of 13CO2/12CO2 ratio in a test sample.