PG analysis by UPLC/MS/MS

MikeT

<p>Hello </p><p></p><p>I have been trying to develop a method to measure <a name="Acyl" title="Acyl"/>Phosphatidylglycerol (PG) in Amniotic fluid which I've been working on for over a year. While there is a lot of lipid analysis by LCMSMS it appears I'm the first to undertake this on amniotic fluid. Historically it is qualitatively measured by TLC or immunologic agglutination. It has been a one step forward, two back, process and I've had little help figuring what is going on with the method. Instrument is a UPLC/TQD.</p><p></p><p>PG is a phospholipid that is measured in amniotic fluid to determine lung maturity in newborns. The one I'm trying to measure is 16:0-18:1 PG and it looks like this:</p><p></p><p>http://www.avantilipids.com/ProductStructures.asp?n=840457</p><p></p><p>I've developed a MRM method in neg ESI measuring the precursor at 747 and the two main product ions of 255 (R1) and 281 (R2), the two FA chains. Specimens are extracted in chloroform which I dry down and reconstitute with mobile phase of ammonium acetate:ACN.</p><p></p><p>It seems to work well but my results do not match enzymatic methods developed in the past. One thing I've discovered is the pure standards from Avanti lipids are much more concentrated than the egg yoke PG purchased from sigma. Which standard is correct? If I use the Avanti standard my patients are ridiculously low. If I use the sigma PG derived from egg yoke my results are more consistence with previous reported values. So use the sigma PG. But I have yet another conundrum. </p><p></p><p>That is the ion ratios of product ions (R1 and R2 FA's) for amniotic fluid PG, Avanti PG and the sigma egg yoke PG are not the same. I've done daughter scans of all three and all three shows the same PG fragmentation but the ion intensity for each are different. The main product ions I'm looking at is R1 (255), R2 (281) and 153 the PO4-OH group.</p><p></p><p>And so while the IR for R2:R1 of the PG in amniotic fluid is closer to that of the Avanti pure standard, the values are too low. And while my values are closer to what they should be using the sigma egg yoke PG the IR's are 25 to 30% too low.</p><p></p><p>Why would I be getting more R2 fragment (18:1 FA chain) in one type specimen over another? Does that mean I'm not measuring the same substance?</p><p></p><p>Maybe I should forget about measuring the ion ratio's? But how else can I insure adequate identification?</p><p></p><p>Anyway I thought I'd just post these questions in case there is anyone in the community that understands phospholipid LC mass spectrometry and wouldn't mind sharing ideas.</p><p></p><p>TIA</p><p></p><p>Mike</p>

KWB

Hello Mike,

I spoke with Peter Lee, a Principle Applications Scientist in Milford, and he has the following comment. I hope it is helpful.

"The abundances of the fragment ions are governed by the steric configuration of phosphatidylglycerol (PG). For example the regioisomers,16:0/18:1-PG (m/z 747) and 18:1/16:0-PG (m/z 747), have very different of ion ratio. According to the data reported in J. Am. Soc. Mass Spectrom. 12 (2001), 1036-1043, the ion ratio of 255/281 is about 0.5 for 16:0/18:1-PG and about 2 for 18:1/16:0-PG.

According to the information provided, I think perhaps the specimen and standards have different regioisomer ratios. I think he can determine isomeric differentiation of specimen by creating an ion ratio calibration curve with isomeric pure standards." - Peter Lee

Ken Blakeslee

MikeT

Thanks for the reply, I'm going to try and get that article.

Unfortunately it doesn't explain my situation as while I am getting about 0.59 IR for 16:0/18:1 PG (Avanti pure standard), I'm getting 0.38 IR using egg yoke PG. And Avanti doesn't make 18:1/16:0 PG so I can't compare the two.

I'll get the article and see if I can glean something from that.

regards

Mike

KWB

Peter Lee responds:

The 0.38-IR of 16:0/18:1 PG (from egg yoke PG) is very similar to the reported result (Figure 1a in the attachment). According to the theory reported in J. Am. Soc. Mass Spectrom. 12 (2001), 1036-1043, the samples having high IR might contain some regioisomer 18:1/16:0-PG. It is possible to estimate the ratio of 18:1/16:0-PG in the samples using the data in Figure 1a and 1b if the regioisomers 16:0/18:1-PG and 18:1/16:0-PG are co-eluted.

MikeT

Thank-you very much kind sir .

And thank-you for posting the article.

Regards

Mike