DEVELOPMENT AND VALIDATION METHOD FOR QUANTITATIVE DETERMINATION OF CIPROFLOXACIN IN HUMAN PLASMA AND ITS APPLICATION IN BIOEQUIVALENCE TEST
Abstract
ABSTRACT
Objective: This study aimed to develop and validate an high-performance liquid chromatography method coupled with an ultraviolet detection for
determination of ciprofloxacin in spiked human plasma and subsequently used for bioequivalence (BE) study.
Methods: The chromatographic separation was achieved on an RP-C18 column (Lachrom Hitachi, 250×4.6 mm, 5 μm), utilizing a mobile phase of
phosphate buffer/acetonitrile (87:13, v/v, pH 3.0±0.1) at a flow rate of 1.5 ml/minutes. Detection is carried out at 276.6 nm using a spectrophotometer.
The developed method is statistically validated for the linearity, accuracy, limit of detection (LOD), limit of quantitation, precision, and specificity. The
specificity of the method is ascertained by comparing chromatograms of blank plasma and plasma containing ciprofloxacin.
Results: Plasma matrix and ciprofloxacin can be separated very well using the validated method. The LOD and limit of quantification (LOQ) were
0.07 µg/ml and 0.26 µg/ml, respectively. The regression curve of the standard was linear (R>0.999) over a range concentration of 0.01-3.00 µg/ml.
The mean recovery of the method ranged between 95.27% and 104.45%. Both intra- and inter-day precision data showed reproducibility (relative
standard deviation ≤8.0, n=9). The validated method is successfully applied in pharmacokinetic and BE study of ciprofloxacin tablet in a small
number of subjects. The mean values of C
max
, T
max
, area under curve (AUC)
(0-12)
, and AUC
for the test (generic) formulation of ciprofloxacin were
2.25±0.29 µg/ml, 1.0 hr, 8.60±0.69 µg/ml/h, and 9.52±0.70 µg/ml/h, respectively. Meanwhile, for the reference formulation (ciproxin tablet), the
values were 2.30±0.27 µg/ml, 1.0 hr, 8.54±0.85 µg/ml/h, and 9.38±0.89 µg/ml/h, respectively. From BE study, the 90% confidence intervals for the
test formulation/reference formulation ratio for the logarithmic transformations of C
max
(0-∞)
, AUC
(0-12)
, and AUC
were within the BE limit of 80-125%
(96.08%, 101.00%, and 102.02%, respectively).
(0-∞)
Conclusion: According to the validation result, the method was rapid, simple, and reliable. It can be used for routine analysis of ciprofloxacin in human
plasma and BE study. The test formulation (tablet Xâ€) met the regulatory criteria for assuming BE in healthy subjects.
Keywords: Validation, High-performance liquid chromatography, Ciprofloxacin, Plasma, Bioequivalence.
Downloads
References
REFERENCES
Ball P. Quinolone generations: Natural history or natural selection? J
Antimicrob Chemother 2000;46 Suppl T1:17-24.
Oliphant CM, Green GM. Quinolones: A comprehensive review. Am
Fam Physician 2002;65(3):455-64.
Katzung BG. Basicand Clinical Pharmacology, Pharmacokinetics &
Pharmacodynamics: Rational Dosing & The Time Course of Drug
Action. 9
ed. New York: McGraw-Hill Medical; 2004.
Amini M, Khanavi M, Shafiee A. Simple high-performance liquid
th
chromatographic method for determination of ciprofloxacin in human
plasma. Iran J Pharm Res 2004;2:99-101.
Molinaro M, Corona G, Fiorito V, Spreafico S, Bartoli AN, Zoia C.
Bioavailability of two different oral formulations of ciprofloxacin in
healthy subjects. Drug Res 1997;47(II):1406-10.
Noman AM, Kadi H. High performance liquid chromatography assay
with UV detection for determination of ciprofloxacin in human plasma.
Greener J Phys Sci 2012;2(1):020-6.
Mandell GL, Pertri WA. Penicillins, cephalosporins and other β-lactam,
antibiotics. In: Goodman & Gilman’s - The Pharmacological Basis of
Therapeutics. 11
ed. New York: McGraw-Hill; 1996. p. 1073.
Manorajan S, Venkateshwarlu G, Mahesh S, Patro SK, Kirankumar B,
th
Saidulu D. Stability indicating UV-spectrophotometer determination of
(0-∞)
ciprofloxacin dosage forms. Int J Pharm Chem Res 2012;2(1):1-8.
Sarr SO, Ndiaye SM, Fall D, Diedhiou A, Diop A, Ndiaye B. Development
and validation of a simple and economical spectrofluorometric method
for estimation of ciprofloxacin in pharmaceutical dosage forms. Int J
Bio Anal Chem 2013;3(3):72-7.
Karunasree A, Thejomoorthy K, Jaffer M, Reddy YP, Ramalinga P.
In vitro protein binding study of cipro floxac in by new UVSpectrophotometric
method. Int J PharmTech
Res 2010;2(2):1150-4.
Turkey SN, Khudhair AF. Determination of ciprofloxacin HCl in
pharmaceutical formulation by continuous flow injection analysis via
turbidimetric (T180°) and scattered light effct at two opposite position
(2N 90°) using ayah 4Sw-3D-T180-2N90-Solar CFI analyser. J Kerbala
Univ 2013;11(4):153-69.
British Pharmacopoeia Commission. British Pharmacopoeia. Vol. III.
London, England: Stationery Office; 2014.
United States Pharmacopeia and National Formulary. USP-30 NF-25.
Asian Edition. Rockville, MD: United States Pharmacopeial Convention;
Indonesian Pharmacopoeia Commission. Indonesian Pharmacopoeia.
ed. Jakarta: Health Ministry of Republic Indonesian; 1995.
Qureshi MN, Rahman IU, Marwat GA. Comparative analysis of
th
ciprofloxacinin different pharmaceutical products by high performance
liquid chromatograph. Sci Tech Dev 2012;31(1):69-73.
Khan KM, Khan FM. Extraction and separation of cipro floxacin by
HPLC from human plasma. Int Chem Pharm Med J 2005;2:267-70.
Pola LM, Sankar DG. Novel isocratic reverse phase high performance
liquid chromatography method development and validation for
simultaneous estimation of ciprofloxacin and phenazopyridine in solid
dosage form. Int J Pharm Pharm Sci 2015;7(4):355-61.
Sachan N, Chandra P, Saraf SK, Gupta RC. Novelbmethod for
simultaneouses timation ofciprofloxacinhydrochlorideandofloxacin by
reverse phase-high performance liquid chromatography (RP-HPLC).
Int J Pharm Sci Res 2010;1(7):100-7.
Manceau J, Gicquel M, Laurentie M, Sanders P. Simultaneous
determination of enrofloxacin and ciprofloxacin in animal biological
fluids by high-performance liquid chromatography. Application in
pharmacokinetic studies in pig and rabbit. J Chromatogr B Biomed Sci
Appl 1999;726(1-2):175-84.
Al-Dgither S, Alvi SN, Hammami MM. Development and validation
of an HPLC method for the determination of gatifloxacin stability in
human plasma. J Pharm Biomed Anal 2006;41:251-5.
International Conference on Harmonization. Validation of Analytical
Procedure Methodology ICH Q2 (R1); 2005.
National Agency of Drugs and Foods Controls in Indonesia.
Methodology Guidelinesfor Bioequivalence Study of Specific Active
Substances. Jakarta: Badan Pengawas Obat dan Makanan RI: 2012.
Food and Drug Administration. Guidance for Industry: Bioanalytical
Method Validation, Department of Health and Human Services. United
States of America; 2001.
US Department of Health and Human Services, (HHS), Food
and Drug Administration (FDA), Center for Drug Evaluation and
Research (CDER). Guidance for Industry. Bioavailability and
Bioequivalence Studies for Orally Administrered Drug ProductsGeneral
Considerations; 2003. Available
from: http://www.fda.gov/
downloads/Drugs/GuidanceComplianceRegulatoryInformation/
Guidance/ucm070124.pdf.
[Last accessed on 2015
Nov
.
The European Agency for the Evaluation of Medicinal Products
(CPMP). Note for Guidance on the Investigation of Bioavailability
and Bioequivalence. Available from: http://www.apps.who.int/
prequal/info_applicants/BE/emea_bioequiv.pdf. [Last accessed on
Nov 23].
The European Medicines Agency. Guideline for Good Clinical Practice
E6(R2). Available from: http://www.ema.europa.eu/docs/en_GB/
document_library/Scientific_guideline/2015/08/WC500191488.pdf.
[Last accessed on 2015 Dec 12].
Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid
Chromatography. 3
ed. United States of America: John Wiley & Sons,
Inc., Publication; 2010.
th
Ahuja S, Dong MW. Handbook of Pharmaceutical Analysis by
HPLCSeparation Science and Technology. 1
ed. Amsterdam: Elsevier;
st
Chan CC, Lam H, Lee YC, Zang XM. Analytical Method Validation
and Instrument Performance Verification.Canada: John Wiley & Sons,
Inc.; 2004. p. 16-24.
AOAC, Official Methods of Analysis, Appendix F. Guidelines for
Standard Method Performance Requirements. San Diego: Academic
Press; 2012. p. 194-206, 9.
Mukti et al.
Asian J Pharm Clin Res, Vol 9, Issue 3, 2016, 89-95
González AG, Herrador MA, Asuero AG. Intra-laboratory assessment
of method accuracy (trueness and precision) by using validation
standards. Talanta 2010;82(5):1995-8.
Vella J, Busuttil F, Bartolo NS, Sammut C, Ferrito V, SerracinoInglott
A, et
al.
A
simple HPLC-UV method for the determination
of ciprofloxacin in human plasma. J Chromatogr B Analyt Technol
Biomed Life Sci 2015;989:80-5.
VybÃralová Z, Nobilis M, Zoulova J, Kvetina J, Petr P. Highperformance
liquid
chromatographic
determination of
ciprofloxacin in
plasma
samples. J
Pharm
Biomed Anal
;37(5):851-8.
Locatelli M, Ciavarella MT, Paolino D, Celia C, Fiscarelli E, Ricciotti G,
et al. Determination of ciprofloxacin and levofloxacin in human sputum
collected from cystic fibrosis patients using microextraction by packed
sorbent-high performance liquid chromatography photodiode array
detector. J Chromatogr A 2015;1419:58-66.
Wagenlehner FM, Kinzig-Schippers M, Sörgel F, Weidner W,
Naber KG. Concentrations in plasma, urinary excretion and bactericidal
activity of levofloxacin (500 mg) versus ciprofloxacin (500 mg) in
healthy volunteers receiving a single oral dose. Int J Antimicrob Agents
;28(6):551-9
Published
How to Cite
Issue
Section
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.