Area of internal standard increase during calibration..

[Pegry]

Hi everybody, we are developing a method for the analysis of some tryglycerides in one products. We use a GCMS and we make a calibration with 6 standard and we use as internal standard (ISTD) trinonanoin. We have found that the area of nonanoin is increasing after each injection of the calibration standard. In fact we get an RSD value of 9% on the area of the ISTD. We have tried to inject six times the last point of the calibration curve and we have found an RSD value of 1.7% on the area of the ISTD. We have also excluded that our standard contain the ISTD.
Have you any idea about this increase of the area of the ISTD?

Instrumental conditions:
Inj = 340°C
Det = 340°C
Column = RTX-5MS 30m 0.25mm 0.25um
150°C x 1min
40°C/min to 340°C
340°C x 15min.

Thanks in advance for the answer,
have a good day,
Davide

[shalash]

hi

can you tell me about your injection tecniq i mean do you use headspace or Autosampler injection?

Regards,
Shalash

[Pegry]

hi

can you tell me about your injection tecniq i mean do you use headspace or Autosampler injection?

Regards,
Shalash

Dear Shalash, we are analyzing intact tryglycerides and we are using liquid injection by autosampler.
Best regards
Davide

[GasMan]

Pegry,

It looks to me as though you may have some 'active' sites in your system that are absorbing your internal standard. As you re-inject the internal standard, less of it is absorbed, and hence the increase in area. You could try injecting a high concentration of the standard to 'flood' the active sites and prevent further absorption, but you should really try to eliminate these sites. They could be in the inlet, transfer line to the detector or in the detector itself, infact anywhere where the sample travels in your system.

Gasman

[Peter Apps]

Hi Davide

Gasman is probably right, and there are some other possibilities.

When you say the "last point of the calibration curve" is that the highest or lowest concentration of analytes ?.

How are you adding the internal standard to the calibration standards ?, at what stage and by what technique ?

What is the rsd of the analyte peak areas for the replicates of the last point ?

What shape is the curve of analtye area vs concentration, curved ?, and what is the r-squared ?

What is the shape of the analyte/i.s. vs concentration, and the r-squared ?

If you do a blank injection after a standard, do you see any caryyover ?

Peter

[Pegry]

Hi Davide

Gasman is probably right, and there are some other possibilities.

When you say the "last point of the calibration curve" is that the highest or lowest concentration of analytes ?.

How are you adding the internal standard to the calibration standards ?, at what stage and by what technique ?

What is the rsd of the analyte peak areas for the replicates of the last point ?

What shape is the curve of analtye area vs concentration, curved ?, and what is the r-squared ?

What is the shape of the analyte/i.s. vs concentration, and the r-squared ?

If you do a blank injection after a standard, do you see any caryyover ?

Peter

Dear Peter,

I've tried to inject six times both the highest and the lower calibration point but i got always a low RSD (about 3%). I add the internal standard by volumetric pipetting in each vial the same volume of internal standard solution (i.e. 100uL).
The replicates of the area of the analytes are similar to the internal standard (about 3%). The calibration line (analyte/i.s. vs. concentration) is linear with a r-squared of 0.999. I always inject a blank after the higher standard but there isn't carry over.
Is the first time that I face such a reproducibility problem but maybe is related to the poor evaporation of the tryglyceride in the injector? I have to add that before every calibration we do a system suitability with the middle concentration standard and we inject that standard six times with good rsd (<3%).
The strange thing is that the area of the internal standard increase after each injection when doing the calibration but remain constant when doing system suitability...
If you have some cue please tell me because I would like to know how to solve this problem..

Thanks in advance and have a good day
Davide

[Bruce Hamilton]

Try running your calibration from highest standard to lowest standard. See what happens to your Int std peak area. I assume all of your standards and blanks have the same amount of triglyceride added - to match your samples. A blank without TAG might have a high Int Std area.

It may be that your injector is strugging with the triglyceride effect on sample viscosity and volatility. You could check by varying the TAG content of one concentration of standard and seeing if the IS peak area decreases as TAG concentration increases. If it does, you may have to review your protocol.

Please keep having fun,

Bruce Hamilton