#1
I split one lyophilized sample into three vials and sent them to two independent labs, plus ran it on a university HPLC. Lab A: 98.6% area, Lab B: 96.8%, in-house: 98.1%. Same week, same batch, all C18, 214 nm. The COA I was shown says 99.2% but only gives a peak table, no chromatogram. I'm trying to figure out if that 1.8% spread is method drift or real degradation. Anyone seen a gap this wide on a split sample?
#2
1.8% is a lot for a split sample. What mobile phase?
#3
TFA vs formic acn shift area. Ask what both labs used.
#4
Both at 214 nm? If one ran 220, area shifts.
#5
I had a peptide standard that gave 98.4% at one lab and 97.1% at another. The lower lab integrated a shoulder at 12.4 min that the other missed. Once both used the same baseline settings, the gap closed to 0.4%. Your 96.8 vs 98.6 could easily be integration, not degradation. I would ask for the raw chromatogram, the integration table with start and end times, and the blank injection. Also check whether either lab used a different wavelength or bandwidth. The number on a COA is not magic; it is just what one analyst drew with one method on one day.
#6
That COA with no chromatogram is the bigger red flag to me. Peak tables alone are weak.
#7
What's the water content? Hygroscopic samples can drop a percent or two after a week.
#8
Water content wouldn't explain a 1.8% area gap if both labs ran the same vial. Method transfer is my suspect.
#9
Method transfer is likely. Different C18 lots and gradient delay can move that 12.4 min shoulder into the main peak.
#10
Yep. I won’t sign off on a COA without the chromatogram and integration table. Peak table alone is just a rumor with decimals.
#11
We had a method transfer once where a different C18 lot split the main peak. Same sample, same vial, 1.2% swing. Raw data settled it.
#12
Ask for the blank. Always ask for the blank.
#13
I keep a notebook of every lab’s baseline settings. Sounds obsessive, but it saved me an argument over 0.9% last month.
#14
Water content can move results, sure, but only if sampling and vial prep differed. Same vial rules that out. I'd still ask Karl Fischer.
#15
At 214 vs 220, peak areas can shift unevenly. I learned that after a two-lab comparison went sideways. Bandwidth matters too.
#16
Integration settings are like handwriting. Everyone thinks theirs is normal. Overlay the chromatograms and the disagreement shows up.
#17
I’d request raw .raw/.d files, injection sequence, and column lot. If they won’t share, that tells you more than the COA.
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