I'd like to hear your opinion concerning internal standard choice for steroid analyses. There are two kinds of IS available, those labelled with deuterium and those labelled with carbon-13. My first thought was that carbon-13 labelled substances are more stable. This is because I suspect that deuterium labelled compounds can exchange deuterium atoms with "ordinary" hydrogen atoms when dissolved. What do you think?
I cannot judge about the above comment since I don't speak polish, so please forgive me if I repeat something! Both deuterium and 13-C should be stable, except if D atoms are located on exchangeable sites such as -OD. An example is salicylic acid which can be bought as D6 but two of the six D rapidly exchange in solution. The D4 form is then perfectly stable.
If you have the choice, better use a 13-C standard since it will better "mimic" natural steroids.
I cannot judge about the above comment since I don't speak polish, so please forgive me if I repeat something! Both deuterium and 13-C should be stable, except if D atoms are located on exchangeable sites such as -OD. An example is salicylic acid which can be bought as D6 but two of the six D rapidly exchange in solution. The D4 form is then perfectly stable.
If you have the choice, better use a 13-C standard since it will better "mimic" natural steroids.
Essentially, D or 13C do the same job. If, however, you are going to use a very high-resolution separation technique, you may prefer 13C as deuterium is notorious for causing the "deuterium" effect, i.e. it may slightly influence retention.
Some trick from our reference lab in Berlin: they used Methanol-1-d as solvent for deuterium labelled compounds. In they opinion such compound are stable in this. What do you think about it?
Yes, that would an option if you are forced to use deuterated standards. But, from the discussion above, it seems that 13C compounds are easier to work with. I just checked prices of isotope labelled testosterone. There's no real difference between deuterium and 13C compounds.
In English: I think that it doesn’t really matter. In my lab I use both: deuterated derivatives and C13. Even in such sensitive method as UHPLC-Q-TOF-MS I haven’t notice the formation of “substituted” derivative after dissolution. I haven’t noticed that even when the sample was in high concentration.
Personally I would choose cheaper standard.
I think that the C13 can be less stabile.
I think that it`s better to use C13 standards albeit their higher price, because of the possible deprotonization (acidic protons) or other losses during fragmentations, ion-molecular reactions and ion exchange reactions.
It depends. For LC either should co-elute with the analyte but, using gc/ms if you use the deuterated standards they don't always co-elute like the 13C.
As pointed out above, 13C (or 15N) labelled internal standards are clearly preferable to deuterated standards - the difference between 1H and 2D is large enough to induce a slight shift in LC retention. If there is no co-elution, the ISTD cannot be used for correction of matrix effects.. There is a very good paper on that topic from Stokvis et al. in Rapid Communications of Mass Spectrometry (published 2007 IIRC)
One word of caution: you should be very careful as to where the compound is labeled. We had a problem with a C13 label that fell off with EI in the MS source and the quantitation ions ended up the same for both internal standard and native compound. We had to go with a multiply deuterated analog that was labeled on the major fragment.
You are absolutely correct about the H/D exchange. Under certain conditions, and certain extraction conditions, you can lose your deuterium. We frequently find no toluene-d8 in our volatiles analyses if the samples are oxidizing (free chlorine).
C13 seem more stable, but we have also seen ionization issues like Joachim mentions. Many of the C13 analogs are quite expensive, so we have a tendency to go with deuterated analogs where we can with the understanding that we have to watch our extractions.
Berg et al published a nice paper in 2011 (Journal of Chromatography A, 1218) showing that 13C is to prefer since it won't give a RT shift as deuterium will when using UPLC.
I can t add much except to agree with Gaetan. Deuterium can exchange if left in solution for long enough. Some seemingly stable Deuterium can exchange under the right conditions so 13C would be a better choice.
If you are using APCI for LC/MS of steroids, then H/D exchange can occur in the gas phase. The degree to which this happens depends on the site of D substitution, as has been pointed out, and also on solvent composition. Even some "stable" protons can exchange under the right conditions. The older papers in the CI literature by McEwen and Hunt might be relevant if you're interested. We use d-labeled standards for DART quantitation with good results, but that is a different experiment.
I do not disagree to the ideas mentioned above. However, here is another thought: depending on the isotopic signature of your molecule you may at least want to be 2 masses away from your original siganl (considering you only have C as isotope signals) if sulfur, silicone or cl/br get tinto the game you certainly want to be away from those. so if you have two cl atoms in the molecule you want to be at least 5-6 masses away from your Molecular ion. (Meaning you would need D6 or 13C6) If this is not clear enough, please do not hesitate to contact me
I have worked with fully deuterated fatty acids (FAs) as internal standards for GC/MS analysis. Samples required derivatization with 5% acetyl chloride in methanol at elevated temperature (75C) for 3 hrs to convert the FAs to their corresponding methyl esters (FAMEs). The dueterated FAMEs eluted earlier than the corresponding normal FAMEs and were shown to be stable under those conditions.
Obviously there are a lot of benefits to matching retention time and getting the structure and features (ionization) as close as possible, but remember that you will likely see more variation from sample extraction and handling compared to anything else. In this way the D/C13 issue is really splitting hairs (though with the catches people have mentioned previously): any analyte with similar physical properties will make a good internal standard if it's added to the sample as soon as possible.
For example, you're probably better off using a steroid with a few extra -CH3 groups if you add it to a homogenate before protein depletion and organic extraction, compared to a perfectly matched C13 compound which is only doped in just before it goes into the autosampler vial.
In our lab we will almost always choose a 13C- or 15N-labeled compound over a D-labeled compound if the 13C- or 15N-labeled compounds are available. Deuterium labeled compounds are often subject to chromatographic shifts relative to the target compound, especially in GC, but also in LC. The problem is worse if you have lots of deuterium in the internal standard. Non-coelution of the internal standard can cause significant error in the quantitative analysis.
This is aside from the issue of H/D exchange.
Also, I want to re-emphasize one point that was made about isotopic interference. You should choose an internal standard that moves the mass of the internal standard well away from any naturally occuring isotopic peak of your target compound. If this is not possible then consult a recent paper by Rule, Clark, Yue, and Rockwood in Analytical Chemistry. The paper showed how to build calibration functions that take isotopic interferences into account.
By the way, Hi Chip.
Elaborating a bit on the issue of non-coelution, if you are doing analysis by LC-MS or LC-MS/MS, and depending on the details of your sample preparation method, you can get a significant amount of ion suppression of your analyte peak, especially if phospholipids coelute. in that case an imperfect match in retention time between your internal standard and your analyte can cause quantitative errors, and these errors can be pretty big. One paper in the literature demonstrated about 40% error in one example. Deuterium labeling raises this risk because it often causes chromatographic shifts relative to the target analyte.
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