Pyrolytic methylation-gas chromatography (GC) technique was developed to determine the spectinomycin in fermentation broth without using any pretreatment procedures.With this on-line pyrolytic methylation-GC in the presence of tetramethylammonium hydroxide ((CH3)4NOH, TMAH), spectinomycin was converted into its corresponding methyl ether derivatives in the injector at 300℃.Thus, fermentation broth samples containing spectinomycin could be well determined by direct-injection in GC inject block.As shown in Fig.1, sharp peaks of methyl ether of actinamine and methyl ether of spectinomycin were clearly observed as a result of methylation of hydroxyl groups by the pyrolytic methylation.Important factors such as the temperature of GC inject block, the amount of TMAH to spectinomycin were investigated.As a result, pyrolytic methylation temperature at 300℃ and blending the sample with the TMAH (25%, v/v) methanol solution in a 1∶1 ratio, and injection of 1 μ1 bleding sample were selected as the optimum condition.The achieved calibration curve exhibited good linearity with regression coefficient of 0.999 at the concentration range of 0.1-10 g L-1.High precision with the R.S.D.within 4% (n=5) at 3 g L-1 concentration and low limit of detection with 0.01 g L-1 were achieved.