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c/hplc·posted 2 years ago by u/sofia_petrescu

[Question] how do you actually verify LC-MS

Question Clean Column ×9 Slow Clap ×2 Long Haul ×1

Right, the integration question again, but with numbers this time.

I have 13 data points over 6 weeks. The pattern is consistent enough that I do not think it is chance, and boring enough that nobody is going to screenshot it, which is usually a good sign.

The thing I would flag for anyone new: the effect I am describing showed up at week 3, not week 52. People give up long before the interesting part.

Interested in whether this matches other people's logs or whether I am an outlier.

12,780 up / 4,707 down73% upvoted41 commentsid t7uqp59 Jul 2024

41 comments

29 in this archive, depth 5

best — the order this archive was captured in

u/coa_janitormod · c/coa-26 points·2 years ago

A shoulder on the main peak that 5 analysts integrated 6 different ways. Same trace, 98.4 spread.

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u/freya_baptista419 points·2 years ago

area% and mass% are not the same and this whole post conflates them

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u/erez_yilmaz122 points·2 years ago·edited

Not convinced. The evidence does not support that reading.

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u/first_hundred66 points·2 years ago

No. This is the kind of confident post that gets copied into a screenshot and repeated for years. Where is the evidence.

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[removed]307 points·2 years ago

[removed by moderator]

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u/careful_gradient75 points·2 years ago

chromatogram or it did not happen

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u/rina_bergstrom109 points·2 years ago

That is net peptide content, not purity. Different number, different meaning.

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u/blunt_coldbox_2024224 points·2 years ago

Strongly agree. Largest single impurity tells a different story than total purity.

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u/quiet_reader_99282 points·2 years ago

chromatogram or it did not happen

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u/sofia_petrescuOP167 points·2 years ago

Respectfully this is a sample of one presented as a finding.

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u/b12_baseline55 points·2 years ago·edited

Strongly agree. Largest single impurity tells a different story than total purity.

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u/mass_spec_maggieMS149 points·2 years ago

What was the column and what gradient?

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u/two_mil_or_one229 points·2 years ago

related substances are where the story lives, not the main peak

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u/ewan_zielinski155 points·2 years ago·edited

Yeah, the chromatogram profile matters more than the headline number.

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u/nora_lundgren86 points·2 years ago

area% and mass% are not the same and this whole post conflates them

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u/sofia_petrescuOP44 points·2 years ago

area% and mass% are not the same and this whole post conflates them

Disagree on that part. 98.7% and 98.9% on the same vial is normal.

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u/clara_danquah48 points·2 years ago

Yeah, the chromatogram profile matters more than the headline number.

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u/rasmus_petrescu21 points·2 years ago

co-elution is real and purity is usually not enough to resolve it

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u/step_count_stan11 points·2 years ago

integration decision on a shoulder changes your number by half a point on its own

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u/santiago_villalobos5 points·2 years ago

No. This is the kind of confident post that gets copied into a screenshot and repeated for years. Where is the evidence.

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u/sofia_petrescuOP4 points·2 years ago

The related substances profile is way more interesting than the main peak %. A 98% with 8 peaks is different from a 98% that is one 33% impurity.

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u/hamza_weiss5 points·2 years ago

integration decision on a shoulder changes your number by half a point on its own

Adding to this: integration is doing more work than the comment implies.

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u/week_four_wall41 points·2 years ago

Did they post the raw trace or just the summary?

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u/ferran_krastev11 points·2 years ago

Small correction: the trial was 5 weeks, not 34. Does not change your point but people will quote it.

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u/marisol_frisk7 points·2 years ago

Not to be pedantic but chromatogram and method development are being used interchangeably and they are not interchangeable in real life.

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u/rina_bergstrom5 points·2 years ago

Strongly agree. Largest single impurity tells a different story than total purity.

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u/enzo_petrescu1 point·2 years ago

related substances are where the story lives, not the main peak

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u/phase_two_pete21 points·2 years ago

Yeah, the chromatogram profile matters more than the headline number.

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u/sig_figs_sammod · analytical5 points·2 years ago

Detection wavelength? purity and that changes at different wavelengths.

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The analytical deep end: RP-HPLC methods, gradient design, integration decisions, LC-MS confirmation, amino-acid analysis, water content, and why two labs can report 98.1% and 96.4% on the same vial and both be right. Post the chromatogram, not the summary line.

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