Fatty Acid Method Development: Carryover Contamination?

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

6 posts Page 1 of 1
Hi all

Long time lurker, new poster.

I have been tasked with developing a new GC/FID method for fatty acid determination in a topical drug product (5% label claim linoleic acid). I have been able to meet LoQ requirements, have good peak shape (tailing factor below 1.3, etc.), and have reproducible results. However, what I also have is a carryover problem. This is my first GC method that is purposefully trying to quantitate and use fatty acids, so maybe I do not have the appropriate method conditions?

Diluent: EtOH (DMSO was assessed, EtOH showed better results)
Internal Standard: Palmitic Acid
Quant. Standard: 1.25 mg/mL linoleic acid and 0.0625 mg/mL palmitic acid
LoQ - 0.1% of nominal
GC: Agilent 7890 with ALS
Column: DB-FFAP (30 m x 0.32 mm x 0.25 µm)
Liner: Agilent 5186-4647 (Low pressure drop, split, deactivated (removed glass wool))
Carrier Gas: He
Injection volume: 0.5 µL
Injection Temp: 230 °C
Mode: Pulsed Split (30 PSI for 0.75 min)
Split: 5:1
Column Flow: 1.5 ml/min
Septum Purge: 3 ml/min
Initial Oven Temp: 120 °C
Ramp: 20 °C/min
Final Oven Temp: 245 °C, hold for 23.75 min (GC run time = 30 min)
FID: 280 °C; 400 ml/min air; 40 ml/min H2; 30 ml/min makeup gas (He))

Anyone have any advise?
IMO this:
Mode: Pulsed Split (30 PSI for 0.75 min)
is the perversion

https://www.restek.com/en/pages/chromat ... /GC_FF1261
Injection
Inj. Vol.: 1 µL split (split ratio 20:1)
My 2 cents, and 2 comments:

1. In my decades of work with fatty acids and soaps, we always made methyl esters for GC analysis.

2. In your instance, I myself would've stayed away from any alcohol solvent (you stated ethanol) just to eliminate any chance of ester formation.
Consumer Products Guy wrote:
My 2 cents, and 2 comments:

1. In my decades of work with fatty acids and soaps, we always made methyl esters for GC analysis.

2. In your instance, I myself would've stayed away from any alcohol solvent (you stated ethanol) just to eliminate any chance of ester formation.


Thanks for the reply!

1) I'm unsure derivatizing a drug product would work (they were hoping to track impurities in the drug product).

2) We tried DMSO and had decent results, but we were getting the same carryover issues. The problem is mostly coming from the drug product solubility, where it is insoluble in hexane and DCM (there is also wax as an excipient).

The samples are being filtered via syringe glass filter prior to vialing, but I'm starting to wonder if some of the drug product isn't sitting in my inlet. When I pulled the septa off, there was a residue on the bottom side. Do you think a "steam cleaning" of the inlet help?

After doing front-end maintenance yesterday (changed septa, liner, and ~3" of inlet-end column, and all rinse solutions), I ran some "no injection" blanks (used a broken syringe with no needle so no penetration of the septa) and the first one showed the carryover chromatographic profile, the second one had greatly reduced peaks, and the third was down to just one small peak (<LOQ).

If I was just evaluating an API, I don't think I would be having these problems (maybe)?
Consumer Products Guy wrote:
My 2 cents, and 2 comments:

1. In my decades of work with fatty acids and soaps, we always made methyl esters for GC analysis.

2. In your instance, I myself would've stayed away from any alcohol solvent (you stated ethanol) just to eliminate any chance of ester formation.



lottt86 wrote:
Internal Standard: Palmitic Acid


3rd cent:
3. I would not use palmitic acid as internal standard, think there could possibly be some in the linoleic acid standard you use to make your Rf mix. I suggest C-13, C15, or C17 fatty acid instead.
free acids are pretty sticky they tend to stick to glass like the autosampler syringe and everything. I agree with CPG. I would esterfy. Transesterfication should work on any sample. I like the mild methanolysis protocol in this paper. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817593/

Also for an internal standard do not use palmitic. It is very likely endogenous in the sample. Use an odd number fatty acid or some odd branched chain not sure if that is available. Most people use C11, C13, C15, or C17 acid.
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