Collision Induced Fragmentation of Macrolide Antibiotics
Emery Pharma’s LC-MS spectrometer is built for generality. Our photodiode array can capture all wavelengths from 200 nm to 800 nm simultaneously; our added evaporative light scattering detector (ELSD) is capable of detecting molecules with no UV/Vis absorbance and no ionization; our mass spectrometer (MS) employs a dual electrospray ionization (ESI) / atmospheric pressure chemical ionization (APCI) system to achieve the most general ionization of nearly any molecule. Emery Pharma’s standard setup is optimized to detect readily-ionized (e.g., nitrogen-containing), moderately lipophilic (0 < log D < 6), medium-weight (100 < MW < 1000), drug-like molecules. The setup is amenable to routine LC-MS analysis of synthetic compounds. However, on occasion, we've had to modify standard conditions to examine special characteristics of particular molecules. One such modification is collision-induced fragmentation (CID) of ions. In our machine, ions are generated by ESI or APCI, and are pulled through a capillary, along with neutral fragments, toward the detector. The ions are accelerated through the Q-array and octopole by an electric field and are detected.
Figure 1. Schematic of our mass spectrometer
Because ions and neutral particles are present in the low vacuum space between the capillary and Q-array, an additional voltage difference between the capillary and Q-array will cause ion collisions. Ions will then fragment, and will be sorted by the detector.
Clarithromycin is a macrolide antibiotic with two pendant glycoside groups. LC-MS spectra taken under standard conditions (Q-array DC = 0 V) show the parent molecule, flying as the [MH]+ ion, with m/z = 748. A small peak can be seen for m/z = 590.
Figure 2. Standard mass spectrum of clarithromycin
Adjusting the Q-array DC to +60 V promotes collision-induced fragmentation of the parent ion into two major fragments, one with m/z = 590, and the other with m/z = 158.
Figure 3. Mass spectrum of collision-induced fragments of clarithromycin
These fragment ions are consistent with cleavage of glycosidic bonds at the C3 cladinose and C6 desosamine groups. The fragments of the molecule containing the amine are disproportionately represented in the mass spectrum, as the non-nitrogen-containing species tend to be neutral (and therefore invisible to the detector).
Figure 4. Assignment of collision-induced fragments of clarithromycin
Spiramycin is another macrolide antibiotic with three pendant glycosides. LC-MS spectra taken under standard conditions show a [MH₂]²⁺ ion with m/z = 422 and a methanol adduct ([MH₂+MeOH]²⁺) at m/z = 438. Adjusting the Q-array DC to +40 V promotes collision-induced dissociation to five major fragments with m/z values of 145, 174, 343, 540, and 699. Four of these fragments (145, 174, 540, 699) are consistent with fragmentation at various glycosidic bonds; the fifth fragment (343) corresponds to the loss of the aldehyde on the core structure.
Figure 5. Standard spectrum of spiramycin, collision-induced fragments, and assignment of fragments
The CID method produced by this voltage adjustment is not as efficient as fragmentation in a triple quadrupole mass spectrometer, but it can be used in a similar way:
(a) to elucidate complex molecular structures, or
(b) to identify diagnostic fragments which can be quantitated in complex mixtures.
Using a Q-array voltage bias, we've been able to generate collision-induced fragments of complex macrolide antibiotics, consistent with fragmentation at glycosidic bonds. If you’d like to learn more about collision-induced fragmentation, glycoside fragmentation in LC-MS, or advanced mass spectrometry techniques, contact Emery Pharma today!
