Does anyone have ideas on detecting heme in saliva by LC-MSMS? I cannot seem to elute it out of a Restek Aromax column with higher than 60% aq. My LC starting gradient for detecting other analytes in the method requires 65% aq. Any higher than 60% aq makes heme stick to the column--it does even not elute during the column wash step. It is forced out only after several solvent injections. I am using 10mM formic in both water and acetonitrile as mobile phase. I get good peaks for heme in a separate method with starting gradient of 60% aq but would like to incorporate it into the original method which starts at 65% aq.
I would look online for a LC-MS/MS method for Hemoglobin and set my MS for 'selective ion mode (SIM)' looking for the Fe atom.
You have a problem since a higher amount of ACN will precipitate proteins like Heme.
I would switch to HPLC grade Methanol (with 0.1% formic acid) and try a series of 90%, 80%, 70%, 60%. Hopefully your Heme will elute somewhere and sometime.
ALL proteins prefer a polar or 'water loving' environment. ACN is an aprotic common HPLC organic solvent. Methanol is a protic (thus polar) HPLC organic solvent and is thus more acceptable to proteins.
You probably choose the wrong HPLC method (reverse phase HPLC). Usually proteins are analyzed by 'ion exchange' HPLC or 'electrophoresis'.
Hi Bruce thank you for your replies. I was trying to evaluate if heme will elute under the conditions used for analyzing cortisol --which is the primary test running in the clinical lab. Apparently it does not. If I could even have some sort of a ratio that I could monitor (like it is reported that cortisol to cortisone ratio >1 in saliva) in all samples and then reflex the contaminated samples to test separately for heme that would work too. But that ratio does not seem to be fixed. In some visibly contaminated samples the ratio is
The retention mechanism depends on many things (like time, temperature, target molecule, column, mobile phases...). In short you are setting up a proportionality concentration gradient between the mobile phase and the column (not all C18 columns are alike!). I would use Methanol and go from the highest strength in my series above and add aqueous until you are satisfied with the chromatography. For a 5 cm column with a flow of 1.0 mL/min. the 'best' RT is between 5-10 minutes. For a 25 cm column with a flow of 0.5 mL/min. the 'best' RT is between 10-15 minutes.
Hi Julie,
Is it important that the heme and cortisol be measured in the same method?
Alan
Julie,
Try stitching the online methods for Cortisol and your Heme method together. This is the basis for a gradient HPLC method.
Hi Bruce, if I start with 65% aqueous which is required for Corts, then heme does not elute even in the 95% organic wash step. Something happens to heme inside the column. If I start with 60% or lower aq, then it elutes but it destroys the corts method--which is already a running method in the clinical lab. I want to preserve that method.
Could you use a column switching method with two injections and two columns, one for the heme part of the analysis and one for the cortisol part? You might be able to do that as a way to preserve the cortisol method. It would of course add some complication and time to the overall method, but at least the two could be integrated more tightly than if the methods were separated completely.
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