Chromatography Forum

Hello,

Would anyone be able to tell me the best way to approach seperating Isobaric Peaks ( hydroxy estrogens and methoxy estrogens) using LC-MS/MS i.e, LC Gradient elusion. I'm not able to get good resolution of these ISOBARIC peaks.

hydroxy estrogens: 2-hydroxy and 4-hydroxy estrogens

2-hydroxy and 4-hydroxy estradiol (2-OHE2 and 4-OHE2) : 755/521
2-hydroxy and 4-hydroxy estrone (2-OHE1 and 4-OHE1): 753/170

methoxy estrogens: 2-methoxy and 4-methoxy estrogens

2-MeOE2 and 4-MeOE2 : 536/171

2-MeOE1 and 4-MeOE1 : 534/171


Internal Standard: Ethinyl Estradiol (EE2) : 530/171

Dansyl chloride derivatization


Mobile Phase; Water with 0.1% FA (A) and
ACN with 0.1% FA (B)



GRADIENT: Time Flow %B
0.00 500 uL/min 00.00
10.00 500 40.00
12.00 500 65.00
18.00 500 55.00
22.00 500 40.00
22.50 500 00.00
25.00 500 00.00

RP C18 Column: 25 cm x 4.6 mm, 5 um

Flow rate : 500 uL/min ; Total run time is 25 min.

Injection volume : 10 uL

MS: ESI- Positive Ion Mode

Please suggest me a good resolution method.



Thanks

With regards

RajaSekar

C18 is clearly not the column of choice to separate those closely related compounds. Dealing with good separation may be painful, and not worth it.

Another thing you can do first is to see if they have different major fragmentation ions with which you can have different MRMs (theoretically yes, but I am not familiar with those compounds, cannot say for sure). It doesn't hurt to use same MRM for confirmation if you cannot find more than one different MRM.

Good luck

Hello,

It is possible to separate these compounds with a PFP column e.g. from phenomenex. Not all, but most of them.

Peter

First: do you use gradient or isocratic elution?

You have some option:
1. use methanol instead of ACN
2. use column with smaller particle diameter (3 um, 1.8 um)
3. use fused-core technology columns (Kinetex form Phenomenex, Ascentis from Supelco) - it can give you better separation.

Dear All,

Thank you very much for your reply.

Yes, I'm using Gradient:

Mobile Phase; Water with 0.1% FA (A) and

ACN with 0.1% FA (B)


GRADIENT: Time Flow uL/min %B

0.00 500 00.00
10.00 500 40.00
12.00 500 65.00
18.00 500 55.00
22.00 500 40.00
22.50 500 00.00
25.00 500 00.00


Only 2-OHE2 and 4-OHE2 having different fragment ion

2-hydroxy and 4-hydroxy estradiol (2-OHE2 and 4-OHE2) : 755/521



2-hydroxy and 4-hydroxy estrone (2-OHE1 and 4-OHE1): 753/170




methoxy estrogens: 2-methoxy and 4-methoxy estrogens

2-MeOE2 and 4-MeOE2 : 536/171
2-MeOE1 and 4-MeOE1 : 534/171



Internal Standard: Ethinyl Estradiol (EE2) : 530/171


Dansyl chloride derivatization

C18 Column (ODS) : 25 cm x 4.6 mm, 5 um



With kind regards

Rajasekhar

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