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Hi, i'm following a published method by Humbert et al. (2001) (DOI: 10.1002/jms.185) to derivatise the redox-sensitive tripeptide glutathione (GSH) for analysis for GC-MS. The agents used are ethyl chloroformate and acidic methanol. We spent quite a lot of time optimising the method to work in our lab, it then worked consistently for two days and now is not working at all. I was wondering if someone would mind reading my post and offering any advice or if anyone could make any suggestions? Apologises for my post being so long.
The derivatisation method is:
First, a solution of 10 µmol GSH and 40 µmol DTT in 1 mL 0.2M phosphate buffer (pH 7.5) is prepared. For derivatisation of the amine and thiol groups, 200 µL of ethyl chloroformate is added and the solution shaken for 10 minutes. The pH is adjusted to 1.5 with 1 N HCl, 0.5 g of NaCl is added and the product is extracted twice in 4 mL of diethyl ether. This is evaporated using a rotovap, the residue taken up in 1 mL of methanol, and 1 drop of concentrated HCl added. For esterification of the carboxylic acid groups, the solution is headed to 80 °C for 10 minutes, cooled, and then evaporated using a rotovap. Finally, the residue is taken up in 400 µL of ethyl acetate and 1 µL loaded onto the GCMS.
The GC run is:
Injector 270 °C, helium flux 1 mL/min. Split 5:1. Initial temp 150 °C, raised to 275 °C at 15 °C/min, ramped to 300 °C at 40 °C/min and then held at 300 °C for 3 minutes. This is on a 30m HP-5MS column. The GCMS is an Agilent, I don't have the model number in front of me right now but its not brand new, nor particularly old. We plan to move over the SIM mode once the method is stable, but are currently running a scan.
At first we found the method required quite a bit of optimisation. The reducing agent DTT is used because of the redox-sensitive nature of GSH but we found that this was reacting with the ethyl chloroformate (how was this not in the paper ...) and producing a massive 'side-reaction' peak after about 7 minutes on the chromatogram. We found limited amount of the desired derivatised GSH product after about 10 minutes on the chromatogram, but were concerned that results would not be accurate if all of the ECF was being soaked up by the DTT. We then moved to the slightly weaker reducing agent 2,3-Dimercapto-1-propanesulfonic acid sodium salt (DMPS) as this is reported by Švagera et al. (2012) (DOI: 10.1007/s00216-012-5727-y) to not undergo the side-reaction. I also have some Tris(3-hydroxypropyl)phosphine (THP) on order which is also recommended by Švagera. Using the DMPS really helped as we suddenly started getting a substantial GSH peak at 10 minutes (much higher than with DTT, and a sharper peak too). We found that samples would be fine left in ethyl acetate in the fridge overnight and could be run the next day. Detection only went down to 5 µmol/mL but we were hoping to solve that by moving to splitless.
We were about to start running calibration curves and loading biological samples, but out of the blue the GSH could no longer be detected. There simply isn't a peak on the GC anymore, and no ions of the desired m/z anywhere. Samples that previously gave good peaks are suddenly not showing up anything. The 4 small impurity peaks are still there on the GC and look identical to working runs. We've spent 2 days constantly making new samples and doing GC-MS runs, but getting absolutely nothing. We have no idea what has happened and what we can do about it. Does anyone have any ideas?
(Also, the syringes we use to inject into the GC-MS are getting very sticky, very quickly, and in some cases seizing up completely (we've had to bin 3 syringes). I've never had this before. In one case the syringe was completely seized immediately after injection. We normally clean them right after injection using the ethyl acetate. Does anyone know why this is happening?)
Thanks for reading.
The derivatisation method is:
First, a solution of 10 µmol GSH and 40 µmol DTT in 1 mL 0.2M phosphate buffer (pH 7.5) is prepared. For derivatisation of the amine and thiol groups, 200 µL of ethyl chloroformate is added and the solution shaken for 10 minutes. The pH is adjusted to 1.5 with 1 N HCl, 0.5 g of NaCl is added and the product is extracted twice in 4 mL of diethyl ether. This is evaporated using a rotovap, the residue taken up in 1 mL of methanol, and 1 drop of concentrated HCl added. For esterification of the carboxylic acid groups, the solution is headed to 80 °C for 10 minutes, cooled, and then evaporated using a rotovap. Finally, the residue is taken up in 400 µL of ethyl acetate and 1 µL loaded onto the GCMS.
The GC run is:
Injector 270 °C, helium flux 1 mL/min. Split 5:1. Initial temp 150 °C, raised to 275 °C at 15 °C/min, ramped to 300 °C at 40 °C/min and then held at 300 °C for 3 minutes. This is on a 30m HP-5MS column. The GCMS is an Agilent, I don't have the model number in front of me right now but its not brand new, nor particularly old. We plan to move over the SIM mode once the method is stable, but are currently running a scan.
At first we found the method required quite a bit of optimisation. The reducing agent DTT is used because of the redox-sensitive nature of GSH but we found that this was reacting with the ethyl chloroformate (how was this not in the paper ...) and producing a massive 'side-reaction' peak after about 7 minutes on the chromatogram. We found limited amount of the desired derivatised GSH product after about 10 minutes on the chromatogram, but were concerned that results would not be accurate if all of the ECF was being soaked up by the DTT. We then moved to the slightly weaker reducing agent 2,3-Dimercapto-1-propanesulfonic acid sodium salt (DMPS) as this is reported by Švagera et al. (2012) (DOI: 10.1007/s00216-012-5727-y) to not undergo the side-reaction. I also have some Tris(3-hydroxypropyl)phosphine (THP) on order which is also recommended by Švagera. Using the DMPS really helped as we suddenly started getting a substantial GSH peak at 10 minutes (much higher than with DTT, and a sharper peak too). We found that samples would be fine left in ethyl acetate in the fridge overnight and could be run the next day. Detection only went down to 5 µmol/mL but we were hoping to solve that by moving to splitless.
We were about to start running calibration curves and loading biological samples, but out of the blue the GSH could no longer be detected. There simply isn't a peak on the GC anymore, and no ions of the desired m/z anywhere. Samples that previously gave good peaks are suddenly not showing up anything. The 4 small impurity peaks are still there on the GC and look identical to working runs. We've spent 2 days constantly making new samples and doing GC-MS runs, but getting absolutely nothing. We have no idea what has happened and what we can do about it. Does anyone have any ideas?
(Also, the syringes we use to inject into the GC-MS are getting very sticky, very quickly, and in some cases seizing up completely (we've had to bin 3 syringes). I've never had this before. In one case the syringe was completely seized immediately after injection. We normally clean them right after injection using the ethyl acetate. Does anyone know why this is happening?)
Thanks for reading.
