Spiramycin (SPM), produced by Streptomyces ambofaciens, is a mixture of sixteen-membered macrolide antibiotics with spiramycin I as the main component. HPLC analysis of commercial SPM samples revealed the presence of, besides spiramycins I, II, III and neospiramycins (NSPM) I, II, III (corresponding to spiramycins I, II, III without the neutral sugar mycarose), two unknown compounds (1 and 2) with contents of approximately 0.35% and 0.25% (by normalization), respectively. Compounds 1 and 2 were isolated via open column chromatography (mobile phase: CH₂Cl₂-CH₃OH-25% NH₄OH, 97:3:0.2, v/v/v) followed by semi-preparative HPLC. Their structures were elucidated using FTIR, ¹³C NMR, and Liquid Secondary Ion Mass Spectrometry (LSI-MS). Both compounds retain three unaltered sugar moieties. Compound 1 features an aldehydic group conjugated with a methylene group (FTIR absorption at 1674 cm⁻¹), with ¹³C NMR showing an aldehydic carbonyl at 195.7 ppm (C-18) and methylene carbons at 147.9 and 139.4 ppm (C-17); its LSI-MS spectrum exhibits a (M+H)⁺ ion at m/z 855. Compound 2 lacks the aldehydic group but has an additional methyl group, with a ¹³C NMR signal at 12.5 ppm (C-18, methyl) and a (M+H)⁺ ion at m/z 829 in LSI-MS. Compared to spiramycin I (molecular weight 842), compound 1 has a 12-unit higher mass and compound 2 a 14-unit lower mass. Based on NMR and mass spectrometry data, compound 1 is deduced to be 17-methylenespiramycin, and compound 2 to be 18-deoxy-18-dihydrospiramycin.