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Hi,
I am working on the Pharmacokinetics of a new Fluoroquinolone antibiotic
developed in my lab. I am finding a difficulty in developing an HPLC method
for the compound. Details are as such :
Solubility of compound : DMSO or alkaline methanol (pH:11.5 - 12)
Mobile : Water(0.1%TEA;pH: 7 with orthophosphoric acid) : Acetonitrile
(40:60 v/v)
Detection wavelength : 303 nm.
Flow rate : 1.5 ml/min.
Concentration range : 0.6=B5g/ml - 20=B5g/ml.
Column : C-18, super endcapped.
Since the compound is soluble only in DMSO, I am injecting the sample in
DMSO.
But I am not getting a linear response. Retention time is stable but areas
are going here and there.
COULD ANY ONE SUGGEST ME SOMETHING.
Yati Chugh Ph.D
Sr.Scientist : DMPK
Wockhardt Research Centre.
India.
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[Two replies - db]
From: "Dr. P. M. Murali"
To:
Subject: Re: PharmPK Assay for PK of a new Fluoroquinolone antibiotic
Date: Tue, 6 Apr 1999 21:13:34 +0530
MIME-Version: 1.0
X-Priority: 3
Your problem appears to be that of pH. C18 may not be ok. Some suggestions
is possible if you give more chemistry of your compound.
Dr. Murali
---
From: "R. C. Gupta"
To:
Subject: Re: PharmPK Assay for PK of a new Fluoroquinolone antibiotic
Date: Tue, 6 Apr 1999 17:43:46 +0530
MIME-Version: 1.0
X-Priority: 3
I Had similar problem with one of my compound. When I deleted DMSO as
injection solvent or peek fittings from HPLC system the problem was solved.
R. C. Gupta
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Dear Dr. Chugh:
Your Problem: A new Fluoroquinolone antibiotic is soluble only in
DMSO, I am injecting the sample in
DMSO. But I am not getting a linear
response. Retention time is stable but areas
are going here and there.
I will first look into the pH effect on the solubility of your drug. Most of
the Fluoroquinolinones are soluble in acidic pH (if your drug is not an acid
salt such as HCl). I am suggesting the following generic method give it a
try.
Internal standard: use any 4-fluoroquinolinone (nalidixic acid/norfloxacin,
cipro etc.). I am assuming you are attempting in plasma.
Take plasma adjust the pH to a slightly basic pH (7.0 - 8.0) with phosphate
buffer and extract into an organic solvent such as Ch2Cl2 . Evaporate the
organic layer reconstitute in 2% Acetic acid in Methanol or a
Methanol-sodium sulfate (pH 2.0) mixture and inject onto a regular HPLC
system consisting of a C-18 column.
Have you considered using a fluorescent detector??
Establish absolute recovery (desired recovery above 80%) of your drug with
the extraction solvent. Irreproducible peak shape is due to poor recovery.
Playing around with pH and little up front work will help the over all
performance of your assay method. It appears you are working in
toxicokinetics sort of work. Also consider diluting the matrix with a buffer
prior to the extraction.
Hope this helps and good luck in your projects.
Prasad NV Tata, Ph.D.
Dept. of Clinical Pharmacokinetics, Pharmacodynamics and Drug Metabolism
Otsuka America Pharmaceutical, Inc.
2440 Research Blvd.
Rockville, MD 20850
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eliminating PEEk fittings should effect the system at all since they do not
contact solvent (if properly fitted) PEEK tubing might have a very big effect.
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