Developper bioanalyste
☆    

Algeria,
2019-05-01 15:29

Posting: # 20264
Views: 850
 

 About developpement of bioanalytical methods LIQ /IS [Bioanalytics]

hello sir helmutz, being a novice in bioanalytical development, i would like to know how to calculate my LIQ frome un infernal Cmax, without taking into account the formula of 5%, is there any other mathematic relation between Cmax as pk parrameter and LIQ as analytical parrameter.

2/ i would like to know how can i calculate the internal standard concentration used in validation from an ULOQ ?

3/IAM working acctually on hplc and lc ms ms dosage methode of amoxicilline in human plasma, iam juste in begining and i need some of your precious recommandations if it is possible ?

N//B excuse me for my english iam doing my best because i usually communicate in frech.

Think you
Helmut
★★★
avatar
Homepage
Vienna, Austria,
2019-05-02 10:20

@ Developper bioanalyste
Posting: # 20265
Views: 754
 

 LLOQ ≤5% Cmax

Bonjour chimiste!

» hello sir helmutz,

Not interested in opinions of other members of the forum?

» […] i would like to know how to calculate my LIQ frome un infernal Cmax, without taking into account the formula of 5%, is there any other mathematic relation between Cmax as pk parrameter and LIQ as analytical parrameter.

You should be able to quantify 5% (or lower) of Cmax in order to demonstrate that there are no residual concentrations of previous administrations in periods >1. In a crossover study you have to avoid carry-over which requires a sufficiently long washout.
It is the job of the pharmacokineticist (not yours) to design the study. Hint: Never plan with the average half-life from the literature. Always assume longer ones (see this presentation, slides 65/66).
The bioanalytical method should be reliable and reproducible for the intended use. Sometimes people forget the last part and reach too far. It’s not necessary to come up with a “perfect” method – it must only be fit for purpose. Goalposts:
  • ULOQ
    • Highest expected Cmax in any subject.
    • Validate also dilution with blank matrix or analysis of an aliquot in case you find concentrations >ULOQ.
  • LLOQ
    • AUC0–t/AUC0–∞ ≥80%.
    • ≤5% (individual!) Cmax.
  • (In)accuracy/(im)precision ≤20% at the LLOQ and ≤15% above.

» 2/ i would like to know how can i calculate the internal standard concentration used in validation from an ULOQ ?

Not sure what you mean. Can you reword/explain?

» 3/IAM working acctually on hplc and lc ms ms dosage methode of amoxicilline in human plasma , iam juste in begining and i need some of your precious recommandations if it is possible ?

Some hints from the method we used in my CRO (250 ng/mL – 31 µg/mL):
  • Blood sampling / stability
    • Anticoagulant EDTA(K3), Na-, or Li-heparin.
    • No stabilization necessary.
    • Centrifuge within 15 minutes after sampling or
      put in an ice bath and centrifuge within one hour.
  • Long-term storage
    • At -20 ℃ stable for two months.
    • At -20 ℃ degradation ~25% after nine months.
    • At -70 ℃ stable for nine months.
  • Stability of plasma at room temperature
    • Stable for four hours.
    • 6% degradation after 24 hours.
  • Stable in three freeze/thaw-cycles.
  • Stability of extracts (in our SFE-method 50% CH3OH)
    • At room temperature for 48 hours.
    • At -20 ℃ for at least one week.
  • Aqueous stock solution (1 mg/mL) at -70 ℃ stable for at least three months.
Good luck in developing/validating your method!

» N//B excuse me for my english iam doing my best because i usually communicate in frech.

No problem. Few native speakers here. Google translate is not that bad. ;-)

Cheers,
Helmut Schütz
[image]

The quality of responses received is directly proportional to the quality of the question asked. ☼
Science Quotes
Developper bioanalyste
☆    

Algeria,
2019-05-02 11:52

@ Helmut
Posting: # 20266
Views: 717
 

 LLOQ ≤5% Cmax

Think you for your explanations, 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), we have been taught that there is a formul to calculate LIQ from AUC ?

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



i hope receiving responses frome other member of forum, i dont know HOW !
Helmut
★★★
avatar
Homepage
Vienna, Austria,
2019-05-02 13:26

@ Developper bioanalyste
Posting: # 20267
Views: 701
 

 LLOQ ≤5% Cmax

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
[image]

The quality of responses received is directly proportional to the quality of the question asked. ☼
Science Quotes
Activity
 Thread view
Bioequivalence and Bioavailability Forum |  Admin contact
19,694 posts in 4,181 threads, 1,355 registered users;
online 11 (0 registered, 11 guests [including 6 identified bots]).
Forum time (Europe/Vienna): 11:35 UTC

A central lesson of science is that to understand complex issues
(or even simple ones), we must try to free our minds of dogma and
to guarantee the freedom to publish, to contradict, and to experiment.
Arguments from authority are unacceptable.    Carl Sagan

The BIOEQUIVALENCE / BIOAVAILABILITY FORUM is hosted by
BEBAC Ing. Helmut Schütz
HTML5