Internal standard ratios fluctuating

Discussions about GC and other "gas phase" separation techniques.

6 posts Page 1 of 1
Hi to all,
I have the same problem as this links,


I wonder if someone could give me some suggestions. I am using 2 internal standars. I found that the ratios of these two internal standards fluctuates at different calibration solution levels. For samples, the caculated results will be different based on using different internal standards. Could anyone give me some suggestions. Thank you.
Hi to all,

I also have a problem with fluctuations of perylene-d12 signal. The problem with the perylene-d12 signal (m/z 264) affects the analytical calibration (internal standard calibration) and also the two control standards analyzed after the calibration standards (one control standard closer to instrumental quantitation limit of 0.1 µg/mL and the second control standard with concentration at the middle of instrumental working range (1 µg/mL)). I use the signal of perylene-d12 (264 as quantitation ion and 260 as confirmation ion) to calibrate the PAH benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, indeno(1,2,3-cd)pyrene, dibenzo(a,h,)anthracene and benzo(g,h,i)perylene. When I calibrate these PAH using chrysene-d12, the calibration works very well and I have also seen improvements in the calibration of these PAH when I use as internal standard the perylene-d12 quantifyed by m/z 260.
I tried, without any improvements in the perylene-d12 signal, in the calibration and in the analysis of control standards:
- changing the guard column. The injection mode used is on column (1 µL injected volume);
- cutting around 50 cm of column (side attached to guard column);
- conditioning again the column (60ºC to 325ºC - 10ºC/min and 325ºC for 20 min);
- injecting a standard with only target PAH (without internal standards - deuterated PAH: naphtalene-d8, acenaphtelene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12) to verify any interference of the these PAH in the signal of perylene-d12;
- changing the column (DB-5MS 30m x 0.25 mm x 0.25µm) to another one new (DB-5MS 60m x 0.25 mm x 0.25µm);
- analyzing new calibration and control standards prepared from a new standard ampoule, opened for this purpose and within the validity. The ampoule contained naphtalene-d8, acenaphtelene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12;
- changing acquisition conditions (final temperature of the oven program changed from 310ºC to 320ºC; ion source temperature from 230ºC to 250ºC).
- cleaning ion source

- Calibration and control standards prepared in n-hexane with any problems in the past with the same column type, acquisition method and GC-MS instrument;
- GC- 6890N Agilent; MS - 5975B Agilent
- Acquisition method:
- injection: on column (1 µL)
- with guard column (fused silica deactivated 0,53mm ID)
- column: DB-5MS 30m x 0.25 mm x 0.25µm
- helium: 1.1 mL/min
- oven program: 60ºC (1min); to 100ºC at 9ºC/min; to 310ºC at 5ºC/min; 310ºC (30min)
- interface: 300ºC
- ion source: 230ºC
- quadrupole: 150ºC
- selected ion mode

Could anyone give me some suggestions to identify and solve what is happening? Thank you all.
Hey catarina, thanks for bringing up this topic, interesting!

I don't have a solution or explanation (sorry), all I can say is we have the exact same issue with fluctuation of the perylene-d12 signal in the PAH analysis of food. This is with GC-MS/MS. We see that it (sometimes) increases the recovery of most notably benzo(b)fluoranthene in standard addition samples and control standards at the end of the sequence over 120%. Changing the internal standard for this analyte during quantification to chrysene-d12 gives a good recovery.

Like some of the posters in the old topic linked in the OP, I've been thinking about loss/exchange of deuterium but it's all guessing. In my opinion, not using perylene-d12 as internal standard is not a bad solution. If your calibration and QCs are better and more stable with a different internal standard, I see no reason to keep using it. Unless you measure perylene itself as well (which we don't do): it seems a bit sketchy to not use the deuterated analogue.. Do you have problems with perylene?

Edit. What I believe it boils down to is that PAHs in general are slippery molecules to ionize with standard electron impact. Take a look at the mass of the fragments we detect in SIM, or even MRM, and compare them with the molecular weight of the PAH: the PAH skeleton always stays intact, just a couple of hydrogens (deuteriums) are getting lost. This is logical based on their structure, but not an ideal situation. I'm not implying to use a different technique, i'm actually amazed how well it works.
The size of the guard column may be too large, I always use the same size as my column. Is your sample matrix "bad" enough that you feel you need a guard column?
What was the last inlet maintenance done? The injector body may need cleaning and the split vent line swapped out.
What is your sample prep? Our issues with our d12 compounds show in extracted samples. We warm and vortex the vials to get the d12's into solution as they tend to fall out of solution.
Hi Rndirk,

Sorry for the late answer to your comment about my GC problem.
I’m measuring perylene so I don’t think a good idea change the internal standard perylene-d12 to another one. I’m quantifying all PAHs with molecular mass over 252 (m/z 252 for benzo(b+k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, 276 for indeno(1,2,3-c,d)pyrene and benzo(g,h,i)perylene and 278 for Dibenzo(a,h)anthracene) with perylene-d12 (m/z 264). During my tests I have noted that the problem with perylene-d12 is more obvious for signal quantified in the 264 mass fragmentogram and not so obvious when the signal is quantified in the 260 mass fragmentogram (confirmation ion used for perylene-d12). Eventualy I could use another internal standard, as benzo(e)pyrene or benzo(a)pyrene, but the m/z used to quantify these peaks are the same used for perylene-d12.
You mentioned that changing the internal standard to chrysene-d12 gives a good recovery. I also verified this but in my case I didn't verify in fortified samples but in control standards (good recoveries) and in the analytical calibration (good correlation coefficient).
Hi Bigbear,

I’m sorry also for the late answer.
I’m using a DB-5MS 30 m (Agilent) and I tested also a DB-5MS 60 m (Agilent). I’m using, for guard column, more or less 1 m to 1.5 m of 0,53 µm fused silica deactivated (Agilent). I change the guard column when I observe loss of resolution, for critical pairs of peaks, and peak taling (much more than usual taling because PAHs have always a little of peak taling). I also change the guard column when the criteria for control standard evaluation (20% for relative difference between theorical and determined concentration of PAH in control standard) is exceeded. After that I repeat all the injections made between the analyses of the two control standards who have not complied the quality control.
I’m using on column injection, injection directly to guard column. I don’t think that the problem is related to injector. Could it be if I’m using on column injection?
I make an extraction using pressurized liquid extraction technique (hexane:acetone, 50:50 v/v as solvent) and extract clean up using glass column prepared with 5 g silica gel (5% deactivated with milli-q water), 5 g basic aluminum oxide (5% deactivated with milli-q water) and aprox. 1 g Na2SO4 (to remove any trace of water). The eluent is 50 mL of hexane:dichloromethane 90:10, v/v. After that I concentrate the purified extract to aprox. 1 mL, I add the mixture of 5 PAHs-d (naftalene-d8, acenaftene-d10, phenanthrene-d10, chrysene-d12 and perylene-d12) and I adjust the extract volume rigorously to 1 mL. This extract is analyzed by GC-MS according operational conditions referred in a previous comment. I never tried to warm the standards or samples extracts due to loss of more volatile PAHs. I have some problems with the more volatile PAHs due to the two concentration extract steps in the sample preparation procedure (before clean up and after clean up).
I identify the problem related to perylene-d12 in calibration standards and control standards not in the sample extracts but probably the problem could also affect the sample extracts.
I prepared new calibration and control standards using a new ampule with mixture of PAHs-d that was stored according certificate (room temperature, 15 to 30ºC) to look for some degradation of the calibration and control standards. However, I had the same problem. Probably, at room temperature may also occur precipitation of dissolved perylene-d12 in the ampule of PAHs-d mixture.
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