Chromatography Forum

Hi there!

I've conducted my senior internship in the Academic Hospital of Maastricht Netherlands. My project is about the identification of hemoglobin variants with LC/MS or LC/MS/MS. Everything went good so far but since November 29th I've a problem.

When I measure blanks, I get a strange peak which elutes at 15 minutes. When I analyze this peak, I mainly receive two m/z values which are 253.1 and 505.0.

HPLC: HP 1100 Series
Column: Zorbax C-18 at 35 Celcius with a C-8 guard column
Flow: 0.2 ml/min
Mobile phase: A - 2mM Ammoniumacetate 0.1% FA
B - Acetonitril 0.1% FA
Gradient: 0-15 min 40%A 60%B
15-16 min 40%A 60%B
16-17 min 98%A 2%B
17-25 min 98%A 2%B

MS: Agilent 6410 QQQ on full-scan because I'm still optimizing my method

It seems that the problem is the MQ water and I changed all mobile phases to ULC/MS water and the peak disappears but now it's back again..

Anyone suggestions

Hi there!

I've conducted my senior internship in the Academic Hospital of Maastricht Netherlands. My project is about the identification of hemoglobin variants with LC/MS or LC/MS/MS. Everything went good so far but since November 29th I've a problem.

When I measure blanks, I get a strange peak which elutes at 15 minutes. When I analyze this peak, I mainly receive two m/z values which are 253.1 and 505.0.

HPLC: HP 1100 Series
Column: Zorbax C-18 at 35 Celcius with a C-8 guard column
Flow: 0.2 ml/min
Mobile phase: A - 2mM Ammoniumacetate 0.1% FA
B - Acetonitril 0.1% FA
Gradient: 0-15 min 40%A 60%B
15-16 min 40%A 60%B
16-17 min 98%A 2%B
17-25 min 98%A 2%B

MS: Agilent 6410 QQQ on full-scan because I'm still optimizing my method

It seems that the problem is the MQ water and I changed all mobile phases to ULC/MS water and the peak disappears but now it's back again..

Anyone suggestions

I assume you work with positive ionization. The 505-signal will be singly charged. If you add a proton you will have a doubly charged molecule which will appear at m/z 253. So i assume you are dealing with just one compound.
In a list which can be found here: "Keller, B.O.; Sui, J.; Young, A.B.; Whittal, R.M. Interferences and contaminants encountered in modern mass spectrometry; Analytica Chimica Acta, 2008." a contaminant signal at 505 is identified as a sodium adduct of polypropyleneglycol, a ubiquitous polyether as they call it.
If you want to go more into detail, you could try to perform a fragmentation on the signal(s). Perhaps you get some additional structural information.

If m/z 505 = [M+Na]+ and m/z 253.1 = [M+Na+H]2+ for MW 482,
then isotope peaks should be at m/z 253.6 and 254.1.

I would suggest m/z 253.1 = [M+H]+ and m/z 505 = [2M+H]+ for
MW 252, then isotope peaks should be at m/z 254.1 and 255.1.

Visual inspection should let you determine which case applies.

Also, MS/MS of m/z 505 = [2M+H]+ should readily give m/z 253 (and possibly lower m/z fragment ions). MS/MS of m/z 253.1 = [M+H]+ should readily give fragment ions.
MS/MS fragmentation of sodium adduct ions is notoriously difficult, so MS/MS of m/z 505 = [M+Na]+ would not be readily achieved.

However, interpretation of limited MS data is like walking in a minefield. For instance, if m/z 253 = [M+Na], then MW = 230; if m/z 253 = [M+NH4]+, then MW = 235.

If you can permanently eliminate the contaminant, then structural ID is not important. As it has returned, you should probably do the
MS/MS experiments.

Do you have a UV spectrum of the contaminant ?

Thank you for your response!

I think I've found the problem. It started to be a big problem at 29th of november... I used MQ water and i just checked the MQ device and saw that it's filter has been changed the 28th of november. MQ filters contain polypropyleneglycol (correct me if i'm wrong). I'm still running some clean steps, I'll keep you informed!

Edit: The samples which were analyzed also contain that MQ water.. so i need to make new reaction buffer with ULC/MS water to perform my restrictions with.

I think you are on the right track there because I believe polypropyleneglycol is sometimes uses as a preservative in those DI packs. Also I think I have seen it used in HPLC columns as a preservative when shipping them.

Another thing to watch, is if you are using PPG(polypropyleneglycol) as a tuning compound, which ABI Sciex uses quite often for mass calibration checks. I know I use a set of PEEK tubing when tuning my ABI3200 that is only used for tuning since it can be difficult to wash the PPG out.