Peak fronting in acetylsalicylic acid assay

[Xisiqomelir]

Hey everyone. I work at a pet nutritional products company and we are preparing a line of dog aspirin as a new product. I'm trying to develop an assay method based on the USP monograph for buffered aspirin tablets, but there's been significant peak fronting in all of our trial assays of the plain standard.

The USP method uses a mobile phase of 85% H20, 15% ACN, 2 g sodium heptanesulfonate and acetic acid for titration to pH 3.4.

The samples are dissolved in a solution of 99% ACN and 1% formic acid.

We have a Shimadzu UFLC and run Empower 2 as our data system.

The chromatograms look like this:



Is there a superior method for obtaining a symmetrical peak?

[Vlad Orlovsky]

Try to reduce concentration of your analyte by diluting it or injecting less. Sometimes you get fin-type of peaks due to overloading of the column or operating at wrong pH
This is second post for aspirin and IP method. Who are people submitting to USP ion-pairing method with acidic IP reagents used for acidic compounds??? The only thing I can think about is that if RP column contains amide groups as polar-embedded groups and IP is used to mask residual basic group on the surface (precursor of amides)

[Bruce Hamilton]

I assume the sodium heptane sulfonate concentration is at 2g / Liter in the combined 85:15 water:acetonitrile mobile phase, and that you ensured it was fully dissolved in the 85:15 mobile phase before attempting to adjust the pH to 3.4 with glacial acetic acid.
I also assume you are giving the column adequate time to come to equilibrium before injecting, at least 30 minutes.

The concentration of aspirin should be about 0.5 mg/ml in the 99:1 acetonitrile : formic acid. I would also ensure that samples and standards are fully dissolved, and that you are reading at 280 nm.
If every thing is as above, I'd also try increasing the column temperature slightly and observe what happens to peak shape and retention.

IIRC, some of the claims about the use of SHS in this method were that it reduced peak fronting, possibly inhibited hydrolysis, as well as improving resolution of some impurities from other components - presumably all important when the method was first developed, perhaps on columns using Type A silica.

I assume that many other analysts were also able to use the method without problems, and I don't recollect any major issues, or severe peak fronting - such as shown here.

I would also check whether the method is still approved in the Current USP for buffered aspirin tablets as they may have moved on.

Bruce Hamilton

[Uwe Neue]

The fronting is due to the fact that the sample is dissolved in acetonitrile. You can do several things:
1. dissolve the sample in water (this may create stability issues; others may know more about this)
2. Inject about 10x less sample volume, maybe with an increase in analyte concentration, if this is possible.
3. Put a pipeline (= a larger i.d. tubing) between the injector and the column (reason: UPLC-type systems have less bandspreading than classical systems. With a classical system, the sample is diluted with mobile phase by the time it reaches the column. This may not happen with a UPLC-grade system.)

[Bruce Hamilton]

I would not be surprised if adding water to the sample solvent caused the tablet solution to fail the free salicylic acid test - which uses the same sample solution and HPLC conditions as the assay.

One concern of aqueous solvents would be that the method may become time sensitive with respect to sample preparation. From memory, methanol also increases the rate of degradation - hence the use of acetonitrile.

As usual, Uwe has identified the problem, the sample isn't dissolved or in mobile phase by the time it reaches the column on UPLC-type systems.

Bruce Hamilton

[Consumer Products Guy]

Buffered Aspirin Tablets
» Buffered Aspirin Tablets contain Aspirin and suitable buffering agents. Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of aspirin (C9H8O4).
Packaging and storage— Preserve in tight containers.
USP Reference standards 11—
USP Aspirin RS .
USP Salicylic Acid RS .
Identification—
A: Crush 1 Tablet, boil it with 50 mL of water for 5 minutes, cool, and add 1 or 2 drops of ferric chloride TS: a violet-red color is produced.
B: Infrared Absorption 197K—
Test specimen— Shake a quantity of finely powdered Tablets, equivalent to about 500 mg of aspirin, with 10 mL of chloroform for several minutes. Centrifuge the mixture. Pour off the clear supernatant, and evaporate it to dryness.
Dissolution 711—
Medium: 0.05 M acetate buffer, prepared by mixing 2.99 g of sodium acetate trihydrate and 1.66 mL of glacial acetic acid with water to obtain 1000 mL of solution having a pH of 4.50 ± 0.05; 500 mL.
Apparatus 2: 75 rpm. [note—Where the Tablet is composed of multiple layers, a stainless steel wire helix may be used, if needed, to hold the Tablet in proper orientation in the apparatus.]
Time: 30 minutes.
Procedure— Determine the amount of aspirin (C9H8O4) dissolved by employing UV absorption at the wavelength of the isosbestic point of aspirin and salicylic acid at 265 ± 2 nm on filtered portions of the solution under test, suitably diluted with Medium, if necessary, in comparison with a Standard solution having a known concentration of USP Aspirin RS in the same Medium. [note—Prepare the Standard solution at the time of use. An amount of methanol not to exceed 1% of the total volume of the Standard solution may be used to dissolve the Reference Standard prior to dilution with Medium.]
Tolerances— Not less than 80% (Q) of the labeled amount of C9H8O4 is dissolved in 30 minutes.
Uniformity of dosage units 905: meet the requirements.
Acid-neutralizing capacity 301: not less than 1.9 mEq of acid is consumed for each 325 mg of aspirin in the Tablets.
Limit of free salicylic acid—
Mobile phase and Diluting solution—Prepare as directed in the Assay.
Standard solution— Dissolve an accurately weighed quantity of USP Salicylic Acid RS in the Standard preparation prepared as directed in the Assay, to obtain a solution having a known concentration of about 0.015 mg of salicylic acid per mL.
Test solution— Use the Stock solution, prepared as directed for Assay preparation in the Assay.
Chromatographic system— Prepare as directed in the Assay. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for salicylic acid and 1.0 for aspirin; the resolution, R, between salicylic acid and aspirin is not less than 2.0; and the relative standard deviation determined from salicylic acid is not more than 4.0%.
Procedure— Proceed as directed in the Assay. Calculate the percentage of salicylic acid (C7H6O3) in the portion of Tablets taken by the formula:
2000(C / QA)(rU / rS)
in which C is the concentration, in mg per mL, of USP Salicylic Acid RS in the Standard solution; QA is the quantity, in mg, of aspirin (C9H8O4) in the portion of Tablets taken, as determined in the Assay; and rU and rS are the peak responses of salicylic acid obtained from the Test solution and the Standard solution, respectively: not more than 3.0% is found.
Assay—
Mobile phase— Dissolve 2 g of sodium 1-heptanesulfonate in a mixture of 850 mL of water and 150 mL of acetonitrile, and adjust with glacial acetic acid to a pH of 3.4.
Diluting solution— Prepare a mixture of acetonitrile and formic acid (99:1).
Standard preparation— Dissolve an accurately weighed quantity of USP Aspirin RS in Diluting solution to obtain a solution having a known concentration of about 0.5 mg per mL.
Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 100 mg of aspirin, to a suitable container. Add 20.0 mL of Diluting solution and about 10 glass beads. Shake vigorously for about 10 minutes, and centrifuge (Stock solution). Quantitatively dilute an accurately measured volume of the Stock solution with 9 volumes of Diluting solution (Assay preparation). Retain the remaining portion of Stock solution for the test for Limit of free salicylic acid.
Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.0-mm × 30-cm column containing packing L1. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0; and the relative standard deviation is not more than 2.0%.
Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of aspirin (C9H8O4) in the portion of Tablets taken by the formula:
200C(rU / rS)
in which C is the concentration, in mg per mL, of USP Aspirin RS in the Standard preparation; and rU and rS are the peak responses of aspirin obtained from the Assay preparation and the Standard preparation, respectively.

[HW Mueller]

Isn´t the L1 column a fairly old normal Waters C-18?
If yes, we seem to be throwing good money after bad when we try to explain the use of the heptanesulfonate. Maybe the originators of this method also got the fronting due to the 99% ACN, threw something in here, maybe also there, and the fronting was gone (due to what we used to call an idiot relationship).
Also, a flow rate of 2mL/min?

[grzesiek]

"4.0-mm × 30-cm column containing packing L1. The flow rate is about 2 mL per minute" - yeah the flow rate is pretty big

"With a classical system, the sample is diluted with mobile phase by the time it reaches the column. This may not happen with a UPLC-grade system.)" - this is probably very good advice, use the older system

"the tailing factor is not more than 2.0" - seems that the criteria are met in case of fronting/tailing

"I'm trying to develop an assay method based on the USP monograph for buffered aspirin tablets" - why would you do that? why would you based it on USP method? why would you use very expensive ion-pair reagent? why would you use such column on that kind of system (UFLC)?

[Xisiqomelir]

Thanks for your responses guys (espec. Bruce Hamilton, really helpful comment about oven temperature).

Problem solved:



Moderators can close the topic if they want.

[Uwe Neue]

If you are using the prescribed column and the prescribed procedure, your peak is unretained now!!!

[Xisiqomelir]

If you are using the prescribed column and the prescribed procedure, your peak is unretained now!!!

Changed the procedure.

[Uwe Neue]

But used a column with the dimensions 4 mm x 30 cm and a flow of 2 mL/min?

[danko]

Hi Xisiqomelir,
I bet you’ll obtain even â€

[Xisiqomelir]

[quote="danko"]Hi Xisiqomelir,
I bet you’ll obtain even â€

[danko]

Hi Xisiqomelir,

Before you mentally close the topic, you might like to calculate the Retention Factor (K’) – just as a confirmation of the statement "problem solved".

Best Regards