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Dear Group Members,
I am working on HPLC analysis of camptothecin analoques (Irinotecan
and Topotecan). As known these compounds undergo pH-dependent lactone-
carboxylate conversion. Literature indicates that at pH 3.0, hundred
percent conversion to lactone from occurs, whereas at pH 9.0 only the
carboxylate form exists.
I am getting 100% lactone at pH 3.0 but at pH 9.0 both forms exist
(even after leaving it overnight).
How do we get pure acid form?
My next question is if I do not get the pure carboxylate form, then
can I prepare the standard curve by plotting sum of areas (lactone
and acid) versus concentration and quantitate my samples (taking the
sum of acid and carboxylate again) using this standard curve. I would
ensure that pH, matrix, etc. is same for both calibration curve
standards and samples.
Thanks in advance
Regards
Tripta
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Dear Tripta,
Have a look through the following article; you may get valuble info.
Determination by liquid chromatography with fluorescence detection of
total 7-ethyl-10-hydroxy-camptothecin (SN-38) in beagle dog plasma
after intravenous administration of liposome-based SN-38 (LE-SN38) *
ARTICLE
Journal of Chromatography B, Volume 791, Issues 1-2, 5 July 2003,
Pages 85-92
Reversed-phase high-performance liquid chromatography method for the
simultaneous quantitation of the lactone and carboxylate forms of the
novel natural product anticancer agent 10-hydroxycamptothecin in
biological fluids and tissues * ARTICLE
Journal of Chromatography B: Biomedical Sciences and Applications,
Volume 686, Issue 2, 15 November 1996, Pages 257-265 Yu-Feng Li and
Ruiwen Zhang
Santosh Tata
Bioanalytical Division
Apotex Inc, Bangalore
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The following message was posted to: PharmPK
Dear Tripta: Are you actually at pH 3 or did you calculate pH 3.0? Also
how stable is /are the molecules at pH 1.0 or 2 and is the lactone to
acid conversion entirely reversible? What is the pH of your
mobile phase? That could account for the changes you are seeing. Can
you derivatize (last resort) to stabilize either the acid or lactone?
Ed O'Connor, PhD
Technical Director, Immunoanalytical
Tandem Laboratories
115 Silvia Street
West Trenton, New Jersey
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The following message was posted to: PharmPK
Dear Tripta,
It is indeed difficult to convert all of the lactone into carboxylate
form at pH 9. Also use of pH higher than 9 is not practicable as silica
based columns are not compatible.
Having said that, I am wondering what the application of your HPLC
method is? If it is for assay of drug powder or formulation, you can use
acidified methanol (1N HCl in methanol) to convert the entire drug to
lactone form and assay in terms of lactone.
If it is for analysis of PK samples (per-clinical or clinical) you don't
have to actually quantitate carboxylate form directly (which needs
separate calibration curve for carboxylate form), but can be done
indirectly based on the literature evidence that CPT lactone-carboxylate
inter-conversion is reversible and complete. If the concentration of
active CPT form (lactone) and the total CPT (lactone+carboxylate) can be
estimated, concentration of carboxylate form can be obtained form the
difference between the two values.
Active form can be estimated by transferring an aliquot of plasma in to
ice cold (-20oC) methanol (which prevents any lactone-carboxylate
inter-conversion) immediately upon collection of blood sample and
separation of plasma - which can be stored at -20oC until analysis (how
long? depends on the camptothecin (CPT) analogue which you need to
validate) - processing method-1.
Also analyse another aliquot of the same plasma sample using acidified
methanol - this is processing method-2. Quantitate lactone form from
samples processed using method 1 and 2 using calibration curve using
acidified methanol. Using method 1 you get to know the concentration of
lactone form (molar) of CPT present in the sample at the time of plasma
sample collection. Using method 2 you get the total CPT (lactone +
carboxylate) in the plasma sample. The difference between the molar
concentrations of these two would be the concentration of carboxylate
form.
If the method is for conducting Bio-equivalence study, I think, all you
have to do is to process samples using acidified methanol alone which
allows estimation of total CPT exposure in terms of lactone.
For more details on the analytical method you can look at the following
publications and the references therein:
1. Quantitative determination of DRF-1042 in human plasma by HPLC:
validation and application in clinical pharmacokinetics.Upreti VV,
Mamidi RN, Katneni K, Srinivas NR. Biomed Chromatogr. 2003
Sep;17(6):385-90.
2. Safety, tolerability, pharmacokinetics, and pharmacodynamics of an
orally active novel camptothecin analog, DRF-1042, in refractory cancer
patients in a phase I dose escalation study.Chatterjee A, Digumarti R,
Mamidi RN, Katneni K, Upreti VV, Surath A, Srinivas ML, Uppalapati S,
Jiwatani S, Subramaniam S, Srinivas NR. J Clin Pharmacol. 2004
Jul;44(7):723-36.
Hope this helps.
Kasiram.
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The following message was posted to: PharmPK
Please refer to the following two publications for analysis of
irinotecan and topotecan. They both present detail preparation of
standards for lactone and carboxylate calibration curves. Make sure your
base solutions are fresh- CO2 will affect the pH of low molarity basic
slns and you will see both forms. Typically, pH 10 or higher will work
for preparation of the carboxylate stocks.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 May 5;788(1):65-74.
High-performance liquid chromatographic assay with fluorescence
detection for the simultaneous measurement of carboxylate and lactone
forms of irinotecan and three metabolites in human plasma.Owens TS,
Dodds H, Fricke K, Hanna SK, Crews KR.
J Pharm Sci. 2004 Sep;93(9):2284-95. Microbore HPLC method with online
microdialysis for measurement of topotecan lactone and carboxylate in
murine CSF.Leggas M, Zhuang Y, Welden J, Self Z, Waters CM, Stewart CF.
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