LLOQ ≤5% Cmax [Bioanalytics]

posted by Helmut Homepage – Vienna, Austria, 2019-05-02 15:26  – Posting: # 20267
Views: 1,094

Hi Developper bioanalyste,

» […] for the first question i meant that actually i calculate my LIQ basing on formula of 5% of lower Cmax observed in bibliography i try to go lower than that on my HPLC to cover residual concentrations if observed later, …

Good.

» … we haven't yet a pharmacologue in our CRO (we are just team of scientists working on amoxicilline bioéquivalence), …

Why not – I’m a chemist by training as well and just an interested amateur of pharmacokinetics and biostatistics. Make yourself familiar with the basics of PK. It’s fun. :-D
Examples of my studies: Sampling for eight hours, washout one week, LLOQ 250 ng/mL, no pre­dose concentrations >LLOQ in any of the 94 profiles.
  1. 875 mg (+125 mg clavulanic acid), 16 subjects
    Cmax 12.4 µg/mL (8.27–20.7 µg/mL)
    t½ 55 min (38–90 min)
    Extrapolated AUC 1.43% (0.70–3.05%)
  2. 500 mg (+125 mg clavulanic acid), 15 subjects
    Cmax 8.82 µg/mL (4.21–14.3 µg/mL)
    t½ 55 min (42–75 min)
    Extrapolated AUC 1.88% (1.05–4.11%)
  3. 400 mg (+57 mg clavulanic acid), 16 subjects
    Cmax 8.61 µg/mL (4.36–11.7 µg/mL)
    t½ 59 min (43–86 min)
    Extrapolated AUC 2.52% (1.31–4.65%)
Even taking the slowest half-life observed in any subject of 90 minutes into account a washout of one day is sufficient (i.e., ours was much too long). Sampling for more than eight hours is futile.

» … we have been taught that there is a formul to calculate LIQ from AUC ?

I would be very interested in such a formula! Who ever told you this was wrong.

» 2/My question about concentration of internal standard: actually i use cefadroxil as described in articles as IS most recomended, …

In your first post you wrote

» … hplc and lc ms …

If you are using HPLC you could also try ampicillin. We used post-column derivatization with fluorescamin and FL-detection at 395/485 nm. Fluram is not cheap but extremely stable (0.005% in CH3CN for at least nine years). I would not recommend off-line derivatization: Labor-intensive, higher percentage of CH3OH/CH3CN in the mobile phase, faster run-times but worse separation. If you want to go that way consider keeping a low percentage of organic modifier but move from C18 to C8 (or even C2).
If you are using LC/MS I strongly recommend a stable-isotope amoxicillin internal standard. Otherwise you may be punished by matrix effects.

» … in articles they describe different concentrations, i nedd to know how can we fix exactly this concentration, is it in relation with analyte concentration wich is amoxicilline at ULOQ or i have to test different concentrations of IS then analyte/IS later confused: ?

Wait a minute. The calibration range (LLOQ–ULOQ) is based on the expected concentrations depending on the administered dose. It doesn’t make sense to use a ULOQ which is too high for a study of a low dose. In the worst case your high QC-sample is above any concentration measured in the study. Not a good idea and a deficiency letter approaching.
When it comes to the concentration of the IS: ~150% of the ULOQ of the analyte is used by many.

» i hope receiving responses frome other member of forum, i dont know HOW !

We are posting in our free time. There is no guarantee that you will get a reply by anybody…

Cheers,
Helmut Schütz
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