VALIDATION AND DETERMINATION OF
CLARITHROMYCIN AND METRONIDAZOLE IN RAT
PLASMA BY USING HIGH PERFORMANCE LIQUID
(HPLC/MS) IN PRESENCE OF POMEGRANATE JUICE
Haifa Tawfeeq Abu Tbeekh
A Thesis Submitted in Partial Fulfillment of the Requirements for the
Degree of Master of Science in Pharmaceutical Sciences at
University of Petra
Faculty of Pharmacy and Medical Sciences
Dr. Eyad Mallah
Dr. Wael Abu Dayyih
Pomegranate juice has been widely consumed around the world especially in the
Middle Eastern countries from the standpoint of its prevention and treatment of common
diseases. Clarithromycin and Metronidazole are widely and concomitantly used drugs. In
addition, both of them are considered as CYP3A4 metabolic enzyme substrates and
A simple, reproducible and rapid analytical method by using high performance
liquid chromatography-mass spectrometry (HPLC/MS) with high resolution and
sensitivity for simultaneous quantification of Clarithromycin and Metronidazole in
presence of pomegranate juice was validated. A gradient mobile phase system consisting
of methanol and 0.1% of formic acid and ACE 5 C18 column (50 X 2.1 mm, 5μ) were used
with a flow rate of 1.0 ml/ min.
An acceptable recovery was achieved (97.75%) and (96.0%) for Clarithromycin
and Metronidazole, respectively, with good accuracy and precision. Coefficient of
determination (R²) of the standard curves for both drugs ranged between 0.9986 and
According to results obtained, there was no significant effect on pharmacokinetic
parameters of Clarithromycin after pretreatment with single and multiple doses of
pomegranate fresh juice. However, there was two hours-long delay on Tmax after the
single dose of juice which is possibly with no clinically significant. Furthermore, the
changing in t½ and the elimination rate constant of Clarithromycin after single and
multiple pomegranate juice administration give an expectation that pomegranate juice
could affect the hepatic-mediated metabolism of Clarithromycin but with insignificant
Pharmacokinetic parameters of Metronidazole was not affected by single dose
administration of juice, on the other hand, multiple dose pretreatment with juice
significantly elevating the maximum plasma concentration (Cmax) (P˂0.05) and the area
under the curve (AUC) of Metronidazole (P=0.001). Since there was a very slight
changing on the t½ and the elimination rate constant, the hepatic enzymes could not be
affected. In contrast, concomitant administration of Metronidazole and Clarithromycin
showed no significant interaction.
Since there are different enteric metabolic enzymes involved in the orally
administered drug metabolism, further in vitro and in vivo investigations to study the
mechanism of the pomegranate juice effect on the pharmacokinetic parameters of these
drugs should be established.
To my parents, my husband, and my wonderful children.
You are my family which I always proud of. I hope you are proud of me as
well by this work.
First, a great thankful to Allah for helping me without waiting for giving back and all of
people assistances are the grace of him.
I would like to acknowledge my supervisor Dr. Eyad Mallah and co-supervisor Dr. wael
Abu Dayyih for helping me to overcome a lot of difficulties that I faced and urging me to
complete this work.
Special thanks to Prof. Tawfiq Arafat for his keen interest to help all students, without
Special thanks to Dr. Nidal Qinna for all of his help.
Special thanks to Prof. Khalid Matalka for all of his help.
This acknowledgment will be not complete if I don’t give thanks with respect to Prof.
Tawfiq Al hussainy for his wisdom and for giving me hope and confidence.
I am indebted to Jordan Center for Pharmaceutical Research, special thanks to Ahmad
Abu Awad and Hamza Al Horoub for their assistance.
I would like to thank all members of committee for their time to evaluate this work.
Finally, special thanks to my wonderful husband Dr. Eng Qahtan Abu Tbeekh for his
encouragement and trusting in my successes.