Thank you for the extra info and revisions. Comments follow:

• Detection settings sound good, but I sure hope you are not using the Reference Wavelength feature (that would cause the problem)! *I can not see your chromatograms very clearly, but use of the Reference Wavelength would invalidate all of your data. Many people have never received any formal chromatography training and develop invalid methods. For those unfamiliar with the "Reference Wavelength", please turn it OFF as default and please don't use a DAD/PDA without first getting some training in how to use it properly (please click on the link below to obtain some basic HPLC DAD training for free). Please get proper training before you use HPLC to develop methods.
• "4.6 mm" is not a standard ID anymore...
• Please delete the TEA (something we stopped doing in the ~1980's) and use a high quality column (you still did not say what column you are using)  with a gradient.
• The chromatogram provided does show signs of sample overload and/or inappropriate injection solution. What is the injection solution? Try diluting all stds down to 1:10 and see if the peak shape/sym improve. Please make sure you are using the mobile phase as the injection solution and don't forget (if you choose to use an isocratic method, which I do not recommend) to wash down the column with 80% ACN (vs acidified water) and also some 80/20 (MeOH/Water) after those isocratic runs to avoid column fouling.

http://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

What column brand and type did you use?

What is the column ID ?

What are the wavelength & bandwidth used? *Its max is 242 nm.

Why did you add an acid and base to your mobile phase??? Why did you use nitric acid?

Tailing is common with this compound (but you appear to have another issue) and one of the best solutions is to us a modern ultra high purity metal free column to reduce. Additionally, a simple GRADIENT of ACN and acidified water (use formic or fresh acetic acid) will resolve this compound out with good peak sym and a proper K prime of > 1.5. *Warning, many of the published methods for this sample are trash as they show little or no retention of the compound on the column (it elutes at or near the solvent front). Those methods are unscientific and should not be used.

Thank you Bill. The column id is pretty standard: 4,6 mm. The detector is set a 242 nm, and the bandwidth employed is 16 nm. The system is an Agilent 1100

Sorry it wasn't nitric acid, but phosphoric acid....I've edited my message to correct it. I use acid in the mobile phase because at neutral pH, this compound is not retained at all. TEA is a sacrificial base, commonly used in HPLC, to link with the free silanol groups of the C18 avoiding therefore polar interactions. The final pH of this mobile phase is 2,5.

Thank you for the extra info and revisions. Comments follow:

• Detection settings sound good, but I sure hope you are not using the Reference Wavelength feature (that would cause the problem)! *I can not see your chromatograms very clearly, but use of the Reference Wavelength would invalidate all of your data. Many people have never received any formal chromatography training and develop invalid methods. For those unfamiliar with the "Reference Wavelength", please turn it OFF as default and please don't use a DAD/PDA without first getting some training in how to use it properly (please click on the link below to obtain some basic HPLC DAD training for free). Please get proper training before you use HPLC to develop methods.
• "4.6 mm" is not a standard ID anymore...
• Please delete the TEA (something we stopped doing in the ~1980's) and use a high quality column (you still did not say what column you are using)  with a gradient.
• The chromatogram provided does show signs of sample overload and/or inappropriate injection solution. What is the injection solution? Try diluting all stds down to 1:10 and see if the peak shape/sym improve. Please make sure you are using the mobile phase as the injection solution and don't forget (if you choose to use an isocratic method, which I do not recommend) to wash down the column with 80% ACN (vs acidified water) and also some 80/20 (MeOH/Water) after those isocratic runs to avoid column fouling.

http://hplctips.blogspot.com/2011/03/reference-wavelengths-as-used-in-hplc.html

Thanks a lot for the info, Bill. Here i answer you and complete the situation:

- Reference wavelength: in this particular case, i think there's no difference in using or not a reference wavelength. The peaks look exactly the same with or without it. I guess it would be more critical in a gradient separation, But i'll follow your suggestion and study in depth the use of it. For my experience in the past, i've never seen the utility of it, maybe because of the simple applications i use to need: Allways one or three components in isocratic elution with little sample preparation.

- 4,6 mm is the column size i've been allways using, i remark, for simple applications. But i'm sure i can be a bit outdated as i allways try to follow the rule:" Does it work?-->Don't Touch It!!" If i don't have a problem, i try not to create it,...:)))

- The column is an Ultrabase C18, from a local HPLC consumables supplier. I mean, it's from a generic manufacturer, not an international brand like Agilent, Waters, etc.

- I don't use the TEA normally. But this time it was a simple option i've tried to see if it helps with the symmetry. I've tried the same separation without the TEA and there's no difference at all. Nonetheless, the conditions suggested in the European Pharmacopeia include the TEA and a buffer at pH 4.

- Maybe you've hitted the bell with the injection solvent: i've used water to prepare my standards, i'm going to try with the mobile phase and see if this is the point. I knew it's allways recommendable to use the mobile phase as solvent, but never seen such an impact in the symmetry. I'll post the results. Thank you!

- I'm reluctant to use a gradient if i can do it with isocratic separation, with adequate resolution and reasonable time...again the principle of the KISS ("Keep It Simple, Stupid!!!") (Of course the "stupid" is by no means related to you...:))))))

Thank you VERY MUCH for your help.

This is not tailing but is "fronting" as a result of column overload. I would inject less to see if it will have a good peak shape at lower level.  Show me the overlay of chromatogram at low levels.

I think you need something stronger than just 75% ACN.  Try some IPA too!

@Wahab..  You are absolutely correct, I apologize and please ignore my recommendation.

Thanks a lot Narong, Bruce and M. Farooq. Finally i think i can conclude there was two factors affecting this: the solvent and the column overload. When i've changed the solvent of the standars, the symmetry of the most diluted standard (15 ppm) was 1,024. But as the concentration grows, the symmetry degrades to 0,64. I post the same standards, dissolved in the mobile phase. If i have time, i'll change the injection volume from 20 to 5 microlitres, and see if the symmetry gets ok. But for the moment, the peaks in the samples are well resolved, and i think there's not a big problem with this overload. Do you think the same?

Thanks a lot everybody!!

While you are checking your injection volume, check your concentration too!  As a rule of thumb I never went above 10 uL for a 100 ug/mL standard (but who knows!).

As stated earlier, the problems were most likely related to "possible overload and/or inappropriate injection solution". I always start with the most likely and basic causes for these types of issues. Most of the time, some fundamental guidelines are not being followed and this leads to poor quality chromatography and inaccurate data.  The more basic training you acquire in the fundamentals of chromatography, the easier these things will get for you.Good luck with your project and keep learning!

*Please also try reducing the sample concentration or inj volumes as suggested too.

Thanks a lot Bill and M. Farooq. I'll put in practice this value information you've provided asap!!

This is a typical example of the column overloading. In this case it could be from high standard concentrations and the presence of TEA, as well. The chromatogram will be OK provided that TEA is omitted from the mobile phase and the concentration of standards are decreased.