Bioequivalence and Bioavailability Forum • About developpement of bioanalytical methods LIQ /IS

Developper bioanalyste
☆

Algeria,
2019-05-01 19:29
(2200 d 17:45 ago)

Posting: # 20264
Views: 5,657

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
★★★

Vienna, Austria,
2019-05-02 14:20
(2199 d 22:55 ago)

@ Developper bioanalyste
Posting: # 20265
Views: 4,947

LLOQ ≤5% Cmax

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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 C_max 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 C_max in any subject.
- Validate also dilution with blank matrix or analysis of an aliquot in case you find concentrations >ULOQ.
LLOQ
- AUC_0–t/AUC_0–∞ ≥80%.
- ≤5% (individual!) C_max.
(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. translate is not that bad. ;-)

—
Dif-tor heh smusma 🖖🏼 Довге життя Україна!
Helmut Schütz

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

Algeria,
2019-05-02 15:52
(2199 d 21:23 ago)

@ Helmut
Posting: # 20266
Views: 4,838

LLOQ ≤5% Cmax

Post reply

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
★★★

Vienna, Austria,
2019-05-02 17:26
(2199 d 19:49 ago)

@ Developper bioanalyste
Posting: # 20267
Views: 4,801

LLOQ ≤5% Cmax

Post reply

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.

875 mg (+125 mg clavulanic acid), 16 subjects
C_max 12.4 µg/mL (8.27–20.7 µg/mL)
t_½ 55 min (38–90 min)
Extrapolated AUC 1.43% (0.70–3.05%)
500 mg (+125 mg clavulanic acid), 15 subjects
C_max 8.82 µg/mL (4.21–14.3 µg/mL)
t_½ 55 min (42–75 min)
Extrapolated AUC 1.88% (1.05–4.11%)
400 mg (+57 mg clavulanic acid), 16 subjects
C_max 8.61 µg/mL (4.36–11.7 µg/mL)
t_½ 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 🖖🏼 Довге життя Україна!
Helmut Schütz

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