Peak fronting in acetylsalicylic acid assay

[grzesiek]

"I think TFA has some UV absorbance. " - sure it has

"Is the higher result because of higher efficiency of the column?" - are you making new calibration or using the one from the older method?

[mijnadarian]

The answer is no-
I have made new sets of standards-

[mijnadarian]

Here is the summary of two recent methods
Calibration curves are

y = 1.2462x + 1.08 for 2.84% average assay and R square value of 0.9999
and
y= 1.2944x + 2.32 for 2.96% average assay and R square value of 0.9999

I don't think it's a significant difference, I'm just curious whether the data is reliable on the new column.
The SST for second test is passing-The first one gave me very very low Theoretical Plate counts
That's why I decided to go to second method with retention time of 4.3 minutes.

Both baseline integration seems to be ok. The instrument goes to Autozero mode every time before the injection

The intercept in the second equation is twice as large- And ; the coefficient of the slope is slightly larger-
Assuming the larger value assay is because of the slightly larger slope for the second equation; why these two curves are so different?

Thanks guys

[Uwe Neue]

Are these the results from the tests where the plate counts where 200 and 7000 respectively? You would have had a roughly 6 times lower peak height in the first test, if this is correct...

[grzesiek]

"The SST for second test is passing-The first one gave me very very low Theoretical Plate counts " - you can't say SST is passing or not before method was validated

"y = 1.2462x + 1.08 for 2.84% average assay and R square value of 0.9999
and
y= 1.2944x + 2.32 for 2.96% average assay and R square value of 0.9999" - these numbers say absolutely nothing, raw data would be useful

[mijnadarian]

Hello there guys

Uwe – Thank you for the feedback sir

The heights on the 1st method are about 2.5 to 3 times lower than the second test.
These are the same samples, with same set of standards ran on the same Phenomenex new column on the same instrument-
The only thing that was different was the mobile phase. I just don’t get what has caused this.
Grzesiek-

I was giving a theoretical plate count-The old method on Chromeleon is set to give a summary page with SST figures-
With regards to equations-
I’m just trying to see if, anyone can understand and troubleshoot the problem here
-You asked for Raw Data
This is the first test-

Sample Name Ret.Time Area Height Amount
min mAU*min mAU ug/mL
SALICYLIC ACID SALICYLIC ACID SALICYLIC ACID SALICYLIC ACID
UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1
STD A CONDITIONING 1.687 28.3141 158.8333 21.8533
STD A CONDITIONING 1.637 31.0944 112.0938 24.0843
STD A CONDITIONING 1.633 31.1954 111.395 24.1654
STD A 1.633 31.111 110.9687 24.0976
STD A 1.633 31.2156 111.2357 24.1816
STD A 1.637 31.214 111.5311 24.1804
STD B 1.64 60.5743 216.0677 47.7398
STD B 1.64 60.5816 220.5307 47.7457
STD B 1.63 60.6572 220.054 47.8064
STD C 1.647 118.4764 427.8461 94.2021
STD C 1.653 118.5276 438.1781 94.2431
STD C 1.643 118.8991 428.7186 94.5413
STD D 1.71 145.5133 768.7655 115.8972
STD D 1.653 147.9849 543.0502 117.8805
STD D 1.647 148.3637 545.1422 118.1844
Sample 1 1.63 68.7136 247.3062 2.8015
Sample 1 1.63 72.0692 263.0577 2.9405
Sample 2 1.633 77.6144 276.4491 2.7976
Sample 2 1.637 77.5249 279.1761 2.7943
Sample 3 1.627 72.2428 266.1038 2.8546
Sample 3 1.633 72.5645 261.8911 2.8675
This is the second test

Sample Name Ret.Time Area Height Amount
min mAU*min mAU ug/mL
SALICYLIC ACID SALICYLIC ACID SALICYLIC ACID SALICYLIC ACID
UV_VIS_1 UV_VIS_1 UV_VIS_1 UV_VIS_1
STD A CONDITIONING 4.382 32.7968 358.4948 23.5434
STD A CONDITIONING 4.365 34.553 321.7749 24.9001
STD A CONDITIONING 4.355 33.6217 309.1319 24.1806
STD A 4.352 33.615 309.7702 24.1755
STD A 4.353 33.6318 309.257 24.1885
STD A 4.347 33.683 309.9012 24.228
STD B 4.34 65.5131 591.5538 48.8179
STD B 4.347 65.8654 599.5605 49.09
STD B 4.345 66.6388 606.8394 49.6875
STD C 4.337 124.7443 1106.632 94.5761
STD C 4.34 124.5351 1107.396 94.4144
STD C 4.333 124.5819 1110.046 94.4506
STD D 4.332 153.3235 1347.815 116.6544
STD D 4.345 153.477 1347.138 116.7731
STD D 4.34 153.0249 1342.318 116.4238
Sample 1 4.335 75.2051 679.0194 2.9065
Sample 1 4.347 77.5771 699.6472 3.0011
Sample 2 4.34 86.5754 776.8715 2.9651
Sample 2 4.345 86.5173 781.6997 2.963
Sample 3 4.345 79.6425 718.7284 2.9861
Sample 3 4.352 57.1179 614.801 2.9316

[grzesiek]

before we dig into it I want to clarify some things first

1. is it coincidence that STD A number 1 and 3 have results with only 3 decimals? (there is only one more result with 3 decimals
2. what is this Amount column?
3. What are the STDs concentrations?
4. How is it possible that Sample results with higher area have lower amount?
5. Have you tried to repeat calibration curve with new set of standards?
6. How do you go about performing the calibration?

and additional one: what is your t0 and how far is your peak from it?

[Uwe Neue]

Don't worry about the plate count. You are running a gradient, and the plate count means nothing in a gradient.

You should look at your areas. They are reasonably consistent between both data sets. The peak heights are different, because you are running different gradients. The intercept is related to the noise and the signal height.

[mijnadarian]

Thanks for replying guys- I really appreciate it.
Grzesiek

1. Yes. It is coincidence- In fact I haven't even paid attention to it.
2. The Amount column is showing the concentration of the standards and for your sample amount you can set it to show you the weight percent.
3. The concentration of the Standards are what you can see there under the Amount column. BTW- I tried to put both sets in tables ; I don't know why it didn't paste correctly-
4- I don't know the answer- I really don't know.
5. No- But I will
6. I am performing calibration, with weighing the USP Salicylic Acid standard amount in the calibrated 100 ml volumetric flask., and then proceed with series of dilutions in smaller 25 ml flasks- 1/25, 2/25, 4/25, and 5/25. I am following the SOP we used to have. It has been coming out correct all along.
Some people say , there is no need for 4 point calibration-But I have been following the SOP all along.
7. The T0 is about 1.17 minutes and its 3.18 minutes far from my peak.

Thanks Uwe
So,
What I'm getting is; the difference between 2.8 and 2.9 could've been results of different mobile phases or matrix effects. And; if this is a systematic error, then I can detect this by either standard addition /or the determination of recovery function. Whichever gave better recovery and I stick with that method?( Test 1 or 2)
Am I correct?

[grzesiek]

just a few things more

ad 2. this amount is calculated by software or is it inputed by you?
ad 6. for one (say A for example) standard there are 3 injections or three different solutions - STD A is one solution or three?
ad 7. how come that in set 1 your peak has retention time of 1.6? t0 still about 1.2?

[grzesiek]

some more comments - I don't know how much % are your standards but the difference seems more than 0.1% (between 2.8 and 2.9) from my calculations it seems like about between 2.8 and 3.25%

clearly there is something wrong with the calculations (by software?) - "4- I don't know the answer- I really don't know. "

the curves look good, only this intercept looks like sth not right, do you see anything (peak?) in that place when you inject diluent or make blank run?

there is more to this - sample 1 in both sets looks strange, the range between two results is big comparing to other samples (excluding number 3 from the second set - 79.6425 and 57.1179)

please refer to the questions asked so we can continue

[mijnadarian]

Yes Grzesiek

On your first posting
-this amount is calculated by software or is it inputed by you?
It is calculated by the Chromeleon software
this amount is calculated by software or is it inputed by you?
Yes- Standard A is one solution- So as B,C and D
how come that in set 1 your peak has retention time of 1.6? t0 still about 1.2
Set 1 has run on a different mobile phase- I think T0 for the set one was about 1.07 minutes


On your second postings

I don't know how much % are your standards but the difference seems more than 0.1% (between 2.8 and 2.9) from my calculations it seems like about between 2.8 and 3.25%
This 2nd data on 2.9% is what I got-It is what I got on summary page on Chromeleon.
Very interesting that you mentioned this- - In the beginning I was getting 3.3% on the old column-That's why I decided to change the column thinking maybe it's deteriorating. I was running the sample without the active, and I was getting Salicylic Acid peak- I swear it; it became an Alfred Hitchcock mystery problem.
do you see anything (peak?) in that place when you inject diluent or make blank run?
Haven't run a blank- But in the old method when I was running the sequence ,I would get 3.3% and the sample without the active was giving me the SA peak. Why? I never figured it out- This is when you asked me- Why you didn't trouble shoot the method at first place-
sample 1 in both sets looks strange, the range between two results is big comparing to other samples (excluding number 3 from the second set - 79.6425 and 57.1179)

I don't know the answer -Could've been instrument hiccup?

[grzesiek]

"It is calculated by the Chromeleon software " - so please write the concentrations or weights of the standards (and samples) as your software is doing pretty bad things with your data right now

"I think T0 for the set one was about 1.07 minutes " - I would be focusing on the second set if I were you, there's better retention so potentially less problems in the future

"It is what I got on summary page on Chromeleon. " - you know what I think right now

"I was running the sample without the active, and I was getting Salicylic Acid peak" - so you got a ghost peak? maybe in the new sets as well?

"Haven't run a blank" - how come?

"Why? I never figured it out" - please run your blanks, always

"Could've been instrument hiccup?" - could have in which case you should repeat and try to find the source of the problem

"Some people say , there is no need for 4 point calibration-But I have been following the SOP all along. " - ask the author why? what is this SOP about? is it general or specific?

[mijnadarian]

Yes
Here are the concentrations
Std A 23.6 ug/ml
STD B 47.1 ug/ml
STD C 94.2 ug/ml
STD D 117.8 ug/ml

And the weight of the samples are about .45 to 0.5 grams-

Yes- I agree with you to go with the second analysis.
No- No ghost peaks in either first or second sets of analysis-
With regards to blank runs- Do we do this after numbers of runs; say for example 10 injections? or after every change in standard concentration? Do I have to run blanks in between samples as well?

Thank you again for all the feedback

[grzesiek]

"With regards to blank runs- Do we do this after numbers of runs; say for example 10 injections? or after every change in standard concentration? Do I have to run blanks in between samples as well? " - depends what you want from them, I wannt to see the purity of your mobile phases so you should run them prior to analysis

If you want to see if you have some of the substance remaining in injector then make blank runs betweeen

"No- No ghost peaks in either first or second sets of analysis- " - you are making this observation looking at your blank runs right?