HELP! Troubleshooting HPLC. Increased concentrations

[ramzi60]

We are running PCMX/Triclosan on our Varian Pro Star HPLC and are experiencing increased concentrations of both. We changed the cloumn, flow cell, UV lamp, recalibrated numerous times. The method is the same and the same standards are being used that were used before the problem started. We even ran a known of a finished product containing the active ingredients and it also gave spiked concentrations. We are at a complete loss. Can anyone out there shed some light on this? It would be greatly appreciated.

[tom jupille]

same standards are being used that were used before the problem started

What you're seeing is consistent with degraded standards.

[Consumer Products Guy]

Tom is saying that standards lower than the stated value make the samples higher in comparison. That's why the known sample (your control sample) also assayed high.

We assayed samples containing both chloroxylenol and triclosan for years and never had such issues. Make up standards and samples fresh. I assume that you are doing gradient elution so that your triclosan peak isn't too broad, or that your earlier-eluting chloroxylenol is retained adequately.

Waht solvent are you using for samples and standards?

[Consumer Products Guy]

OK, now I re-read:

The method is the same and the same standards are being used that were used before the problem started.

Does this mean that you re-injected the standards or that you used the areas from the past?

We changed the column, flow cell, UV lamp...

Interesting. Because if my peak shapes are areas were essentially the same as I'd always gotten, I don't think I would've changed any of those, as whatever of those that affected my standards would also affect the samples.

Just for "fun", how much higher were the recent assays? When you look at the chromatograms and peak areas, does that confirm your findings, or are you simply relying on electronic data analysis where someone could've made a mistake with a dilution, calibration table, multiplier, etc.?

We've encountered stuff off because someone used a 200 ml flask and entered it as 250ml, and the reverse. Have a second analyst go through with you, and definitely make up fresh standards. I assume that you make a standard containing both API ingredients, and assay in a single gradient run.

[ramzi60]

The solvents we are using are methanol and sodium perchlorate. The peaks are non-linear at the LOQ but had been linear in the past. We are seeing about a %10 increase in PCMX and about %25 increase in Triclosan.

[Consumer Products Guy]

We used water-ACN with a little acetic acid. Sometimes simple is better.