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Troubleshooting Recoveries Over 100% In Spiked Placebo

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

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Hello, first time posting here. I'm working on a method to determine the amount of a preservative in a cosmetic skin cream. The analyte of interest is Ethylhexylglycerin.

When I dilute the placebo in a spiking solution that is 0.01 mg EHG / mL IPA, the EHG peak response is greater than when I inject the spiking solution by itself. Hopefully that makes sense. I would expect the EHG concentration to be the same regardless of the presence of placebo.

When the placebo is diluted in pure IPA, there are no peaks interfering with the EHG RT. There are peaks eluting just before and just after where EHG would elute. These peaks remain present in the spiked placebo, so I don't think that this is interference adding to the EHG area count.

I'm using a 35% phenyl (ZB-35HT) 30m x 0.25 um x 0.320mm column.

Initially I thought that derivatizing the solutions with BSTFA w/ 1% TMS would solve my problems but I'm having some issues getting the reaction to go to completion, or find a similar substance to use as an internal standard.

Another thing I tried was using a standard addition technique to quantitate the recovery. This actually worked the first time that I tried it, but then the next time that I tried this, the recoveries were significantly different. I don't feel like this is a very robust method.

I came across some research papers discussing ways to overcome "matrix effect" and one of the papers presented the idea of using "protecting groups" to outcompete the sample matrix for active sites in the GC. They looked at the effect of adding a concentrated spike of very polar molecules to solutions containing pesticide sample matrix. What they found was that the addition of polar molecules caused an increase in response for a lot of their polar analytes. It was hypothesized that the mechanism behind the phenomenon is basically the GC is going to prevent a certain number of polar molecules every injection from reaching the detector. By purposely saturating the solution in polar protecting groups, the less polar analytes of interest are protected from the GC and make it to the detector.

One of the "protecting groups" was Glycerin. I am assuming that what is happening is basically what they described in their research. My EHG is being protected by the Glycerin in my placebo, so the amount of EHG that makes it to the detector is higher than in the spiking solution which contains no placebo and therefore no protecting group.

LLE to remove matrix components has proved challenging as the product is an emulsion.

Any thoughts or similar experiences? I'm hoping someone else went through something similar and has an idea of which thread I should be following.
I worked in the Analytical Chemistry department of a large consumer products company for decades. Developing this type of matches my experience base quite well.
Initially I thought that derivatizing the solutions with BSTFA w/ 1% TMS would solve my problems but I'm having some issues getting the reaction to go to completion, or find a similar substance to use as an internal standard.
If my project, I would start by seeing whether N,N-dimethylformamide or pyridine dissolves ethylhexylglycerin readily. Then I'd dissolve or disperse my sample in which of those two solvents you choose. Then I'd filter (if cloudy) and add an aliquot to an autosampler vial, add BSTFA containing 1% TMCS, cap, and shake. I'd inject on a non-polar or slightly polar capillary, and program the oven to clean out other sample components after the silylated ethylhexylglycerin elutes.

OK: what to use for internal standard? To be honest, I'd do by external standard as that's how we quantitated glycerin, propylene glycol, butylene glycol, triclosan, triethanolamine, diethylene glycol, and similar components in samples. A modern autosampler can readily achieve acceptable precision and accuracy.

If you really feel you want to use an internal standard, then choose one that is available pure that has hydroxy-containing group(s) and similar MW as the ethylhexylglycerin, and that when silylated falls into a region of the chromatogram where no sample components elute.
If my project, I would start by seeing whether N,N-dimethylformamide or pyridine dissolves ethylhexylglycerin readily.
Thank you for your suggestions, I will go ahead and try DMF and pyridine based derivations as you suggested but I have some questions.

Why would DMF or Pyridine be your solvents of choice for derivation? I'm asking because I've tried a derivation I'll describe below in Toluene and the reaction was not completed after 2 hours at 75ºC. Would these other solvents catalyze silylation?

Here's some more info on the product and what I've tried:
My product is 60 wt% water, EHG is 0.1 wt%. I'm analyzing 1 gram of sample which gives me 1 mg of EHG and 600 mg of water. I've heard water can be an issue for derivitizing with BSTFA, so I've been either performing a LLE or evaporation to get rid of the water.

For the LLE, the sample is dispersed in water, acidified w/ 5 mL 1N HCl, 20 mL of Toluene added to extract my analyte, and >32 g of NaCl added to improve the partitioning into Toluene. 0.5 mL of that toluene layer gets transferred to a vial along with 0.5 mL of BSTFA w/ 1% TMCS.

I've tried BSTFA derivation of Ethylhexylglycerin in Toluene at 75ºC for 2 hours. This was injected multiple times and in each injection, the main peak would have a higher area count than in the previous, while a small adjacent peak would have a lower area count than in the previous. To me, this looked like the reaction was still ongoing.
Thank you for your suggestions, I will go ahead and try DMF and pyridine based derivations as you suggested but I have some questions.

Why would DMF or Pyridine be your solvents of choice for derivation? I'm asking because I've tried a derivation I'll describe below in Toluene and the reaction was not completed after 2 hours at 75ºC. Would these other solvents catalyze silylation?
DMF and pyridine are HCl acceptors, and what I used for DECADES in the analytical chem laboratory. These would allow the derivatization reaction to proceed quickly - well within a few minutes - and at room temperature. Both solvents have odor, DMF less so. You can go with my recommendations, or stumble around putting on your underwear, your choice.

When one is derivatizing the tougher -N-H hydrogens of an amine, then I'd only use pyridine as solvent, and would add some heat on steam bath after adding BSTFA w/ 1% TMCS and capping vials.

My product is 60 wt% water, EHG is 0.1 wt%. I'm analyzing 1 gram of sample which gives me 1 mg of EHG and 600 mg of water. I've heard water can be an issue for derivitizing with BSTFA, so I've been either performing a LLE or evaporation to get rid of the water.
60% water - that's all? We assayed products with 85% water without issues. If you decide to listen to my suggestions, dissolve/disperse 0.6 grams well-mixed sample into 25 ml DMF or pyridine; filter if cloudy or precipitate occurs. In an autosampler vial, add 2 parts sample solution with 1 part of BSTFA w/ 1% TMCS, then cap and mix. Take to GC autosampler, inject once vials return to room temperature (the reaction of the BSTFA w/ 1% TMCS and water will produce some heat).

If you want to mix equal amounts of sample solution and the BSTFA w/ 1% TMCS, do so, you'll just use up more BSTFA reagent.
You can go with my recommendations, or stumble around putting on your underwear, your choice.
The DMF worked thank you for your help.
You can go with my recommendations, or stumble around putting on your underwear, your choice.
The DMF worked thank you for your help.
Winner, winner: chicken dinner !!! The CPG was a baby genius and got dumber through the years, but knows his stuff.
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