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An analytical method employing hydrophobic interaction chromatography (HIC) combined with parallel detectors, provided a quantitative method for the determination of soluble carbohydrates in conifer needle tissues. Evaporative light-scattering detection (ELSD), in parallel with atmospheric pressure chemical ionization mass spectrometry (APCIMS), yielded excellent quantitative and spectral data. Non-linear detector responses of the ELSD were overcome by performing a simple exponential transformation of the detector response data. The transformation allowed for single-point calibrations that yielded quantitative results with excellent accuracy and precision. Method recovery and precision were determined from maltose-fortified conifer tissues. Maltose recovery was 101.7% [relative standard deviation (RSD) ¼ 9.74%] for homogenized Douglas-fir (Pseudotsuga menziesii) needle tissue fortified at 1.01 mg/g and 107.8% (RSD ¼ 2.47%) for tissue fortified at 10.1 mg/g. Maltose recovery from fortified western red cedar (Thuja plicata) tissues (7.60 mg/g) was 93.0% (RSD ¼ 3.0%). The method limit of detection (MLOD) was 0.24 mg/g for maltose in Douglas-fir. The method was employed for the quantitative analysis of soluble carbohydrates in Douglas-fir needles, collected from new and previous-year’s growth for 7 weeks after the emergence of new growth (bud break). Needles collected from dormant trees were also analyzed. The concentrations of fructose and glucose were consistently greater in previous-year’s growth vs. new and dormant growth.