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- Posts: 2
- Joined: Sat Dec 11, 2004 1:14 am
Dear All,
Have anyone chemically derivatized gentamicin complex before. I'm using o-phthalaldehyde reagent (OPA) to add the chromophore into the 3 components of genta, namely c1, c1a and c2 (determined by injecting water-dissolved gentamicin). For your info, OPA attacks the primary amine groups in genta. The derivatization was done by dissolving the OPA solid in methanol and added into borate buffer with pH=10.4. Subsequently, mercaptopropionic acid was added. The pH was adjusted to 10.4 using KOH. This was then left for 90 minutes before use. After that, 0.5ml of 1mg/ml genta was added into 1ml of methanol and 0.8ml of OPA Reagent. The solution was put into a waterbath at 60C for 15 minutes.
Upon cooling to room temperature, I subjected the derivatized sample to LC/MS but was only able to see the +2 ion of C1(m/z = 546). Somehow, other +2s and the original c1a and c2 peak were absent. No +1 toos. Puzzled!!!I'm wondering what has happened. Could some kind souls out there give some advice?
Have anyone chemically derivatized gentamicin complex before. I'm using o-phthalaldehyde reagent (OPA) to add the chromophore into the 3 components of genta, namely c1, c1a and c2 (determined by injecting water-dissolved gentamicin). For your info, OPA attacks the primary amine groups in genta. The derivatization was done by dissolving the OPA solid in methanol and added into borate buffer with pH=10.4. Subsequently, mercaptopropionic acid was added. The pH was adjusted to 10.4 using KOH. This was then left for 90 minutes before use. After that, 0.5ml of 1mg/ml genta was added into 1ml of methanol and 0.8ml of OPA Reagent. The solution was put into a waterbath at 60C for 15 minutes.
Upon cooling to room temperature, I subjected the derivatized sample to LC/MS but was only able to see the +2 ion of C1(m/z = 546). Somehow, other +2s and the original c1a and c2 peak were absent. No +1 toos. Puzzled!!!I'm wondering what has happened. Could some kind souls out there give some advice?
