LLOQ ≤5% Cmax [Bioanalytics]

posted by Helmut Homepage – Vienna, Austria, 2019-05-02 15:26 (1195 d 06:25 ago) – Posting: # 20267
Views: 3,103

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, …


» … 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…

Dif-tor heh smusma 🖖 [image]
Helmut Schütz

The quality of responses received is directly proportional to the quality of the question asked. 🚮
Science Quotes

Complete thread:

UA Flag
 Admin contact
22,289 posts in 4,666 threads, 1,585 registered users;
online 7 (0 registered, 7 guests [including 4 identified bots]).
Forum time: Tuesday 21:51 CEST (Europe/Vienna)

The existing scientific concepts cover always only
a very limited part of reality,
and the other part that has not yet
been understood is infinite.    Werner Heisenberg

The Bioequivalence and Bioavailability Forum is hosted by
BEBAC Ing. Helmut Schütz