Chromatography Forum

hi all, i am working in a Pharmacokinetics laboratory, we are developing methods in plasma for generics using HPLC-PDA, many time the LOQ we found for these considerably higher than has been reported in some of the research articles even if we follow the reported conditions\? my question how reliably should we take data from reported stuides? also i have seen papers describing LOQ as low as 5 ngm, in our lab in my expereince getting an LOQ of 25 ngm is difficlut, ?

please help me on this

thank you

LOD and LLOQ specifications in methods are notoriously unreliable because they are ultimately based on signal/noise ratio (see the Bruce Hamilton's comment in the thread on baseline noise measurement:
http://www.sepsci.com/chromforum/viewtopic.php?t=3798 ).

In my experience, baseline noise can vary by more than an order of magnitude on the same instrument, depending on things like lamp age, degassing effectiveness, reagent/buffer/solvent/glassware cleanliness, and temperature fluctuations.

Furthermore, noise is, well noisy. You should expect +/- 30% in your noise measurement and another +/- 30% in your peak height at LOD for a combined uncertainty of +/- 40% in the S/N ratio. This is before you suffer with inter-instrument and inter-laboratory variabilities.

well, a very popular process called evidence based decision-making(evidence based medicion, etc) can be used to assess the relaibility of reported scientific data. For examples, in clinical application, a meta-analysis is more reliable than a randomized controlled trial, the latter is more reliable than a single case study, and so..on. Furthermore, the more reputable the journal, the more reliable the data published (just common sense, not apply to many recent scandals).

In my opinion, if the claim 5ng is true, I don't think you have problem to achieve a 25ng LOQ (10X sigma or s/n>6-9). However, in many occations, the LOQ reported is the lowest that could be got in many tries.

My suggestion is to look in more reputable journals or more similar articles to find the common groud among them, and you may be able to get a solution.

Good luck

i am not saying we are achieving LOQs of 25 ngm on our wates system, but i am adviced by my senoirs not to go below 20 ngm, as our requirement is so, but still what i am asking is using a stanard HPLC-UV is it possibe to get LOQ of 5ngm or so, repeatedally over hunderads of injection, as usually in our system area for 25 ngm comes abous 4500, so 5 ngm would be lost in the noise i guess with area about 900, what do u thinK? should we go for lower LOQ or better reprodcubility? i must say i am just a beginer in chromatography, so i might sound kiddish, my apologies for that

thank you,

Aniket

using a stanard HPLC-UV is it possibe to get LOQ of 5ngm or so, repeatedally over hunderads of injection, as usually in our system area for 25 ngm comes abous 4500, so 5 ngm would be lost in the noise i guess with area about 900,

That's a different question from the one you originally asked

LOD and LOQ are defined by signal/noise ratio. They can be improved (lowered) in one of two ways: more signal; less noise.

The signal will depend on the UV absorbance and molecular weight of the analyte (that's why it's impossible to say whether you will be able to quantitate 5 ng unless you know details about the analyte), on the dimensions of the column (smaller columns result in less dilution), on the k' of the peak (smaller k' = narrower peak = less dilution), and on the flow cell path length.

The noise will depend on things like wavelength, lamp age, flow variations, temperature stability, and mobile phase purity.

Area counts are useless in an absolute sense, because what's actually measured will depend on the data system. What's really important here is the height of the analyte peak compared to the noise (units don't matter so long as the same units are used for both).

Final answers:
- is LLOQ < 5ng possible with UV detection? Yes
- is LLOQ < 5ng possible for your compound with UV detection? Can't tell without much more information.

Sorry to jump in, but for bioanalysis, LOQ is not defined by signal to noise ratio's. Look at the FDA document: Guidance for Industry, bioanalytical method validation. The LOQ is defined by accuracy and precision and so, rather objective. This is also the case in the EMEA guidelines. The relevance of LOD is subject to discussion and in my opinion, not relevant.

Regards Bert

One usually has to run a blanc somewhwere so you sort of have a built in S/N in "bio..." Of course, the officially allowed ranges are usually quite large, as many of the clinical chemistry determinations are estimations or guesses (a very commonly accepted value of SD = 10% boils down to an allowed variation of ~60% for all values).