A new simple, precise and accurate high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of irinotecan (CPT-11) and two related compounds viz., 7-ethyl-10 hydroxycamptothecin (SN-38) and camptothecin (CPT) in pharmaceutical dosage forms. Chromatography was accomplished using a reversed-phase C18 column and ultraviolet (UV)detection and an isocratic mobile phase consisting of 3 % v/v triethylammonium acetate buffer (pH 3) and acetonitrile (70:30 v/v). The linear range of quantitation for all the compounds was 0.1-10 μg/mL. The limit of quantitation for all the compounds ranged between 0.01-0.05 μg/mL. The method has the requisite accuracy, selectivity, sensitivity and precision to assay of CPT-11 and related compounds in pharmaceutical dosage forms and bulk API

Atyabi, F

Dinarvand, R

Esmaeili, F

Mohammadi, A

Walker, Roderick

Mohammadi, A.

Esmaeili, F.

Dinarvand, R.

Atyabi, F.

Walker, R.B.

SEALS Digital commons

Simultaneous determination of irinotecan hydrochloride and its related compounds by high performance liquid chromatography using ultraviolet detection.

Mohammadi, Ali

Esmaeili, Farnaz

Dinarvand, Rassoul

Atyabi, Fatemeh

Walker, Roderick B

South East Academic Libraries System (SEALS)

Asian Journal of Chemistry Vol. 22, No. 5 (2010), 3966-3972Simultaneous Determination of Irinotecan Hydrochlorideand its Related Compounds by High Performance LiquidChromatography Using Ultraviolet DetectionALI MOHAMMADI*†, FARNAZ ESMAEILI‡, RASSOUL DINARVAND‡,FATEMEH ATYABI¶ and RODERICK B. WALKER§Department of Drug and Food Control, Faculty of Pharmacy,Tehran University of Medical Sciences, P.O. Box: 14155-6451, Tehran-14174, IranFax: (98)(216)461178; Tel: (98)(216)482607; E-mail: alimohammadi@tums.ac.irA new simple, precise and accurate high performance liquid chromato-graphy (HPLC) method was developed and validated for the simulta-neous determination of irinotecan (CPT-11) and two related compoundsviz., 7-ethyl-10-hydroxycamptothecin (SN-38) and camptothecin (CPT)in pharmaceutical dosage forms. Chromatography was accomplishedusing a reversed-phase C18 column and ultraviolet (UV) detection andan isocratic mobile phase consisting of 3 % v/v triethylammonium acetatebuffer (pH 3) and acetonitrile (70:30 v/v). The linear range of quantitationfor all the compounds was 0.1-10 µg/mL. The limit of quantitation forall the compounds ranged between 0.01-0.05 µg/mL. The method hasthe requisite accuracy, selectivity, sensitivity and precision to assay ofCPT-11 and related compounds in pharmaceutical dosage forms andbulk API.Key Words: Irinotecan, Camptothecin, High performance liquidchromatography-ultra violet, 7-Ethyl-10-hydroxycamptothecin.INTRODUCTIONCamptothecin (CPT) is a natural, water-insoluble quinoline alkaloid producedby the plant Camptotheca acuminata that is indigenous to China. Camptothecin hasbeen chemically modified to produce other anticancer agents including irinotecan(CPT-11)1. Irinotecan is essentially a prodrug and is hydrolyzed by carboxylesterasesto form the active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38)2. Thesecompounds exit as two coexisting forms viz., the active lactone form and an inactivecarboxylate form which has no topoisomerase I inhibitive activity. The lactone form†Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran.‡Nanotechnology Research Centre, Tehran University of Medical Sciences, Tehran, PO Box 14155-6451, Iran.¶Department of Pharmaceutics, Faculty of Pharmacy, Novel Drug Delivery Systems Laboratory,Tehran University of Medical Sciences, Tehran, P.O. Box: 14155-6451, Tehran-14174, Iran.§Department of Pharmaceutics, Faculty of Pharmacy, Rhodes University, Grahamstown 6140, SouthAfrica.has a closed α-hydroxy-δ-lactone ring, which can be hydrolyzed to form an open-ring hydroxylic acid (carboxylate form) (Fig. 1). Under acidic conditions (pH < 5)the lactone structure predominates, whereas at pH values above 8 the carboxylateis present exclusively3. High-performance liquid chromatography coupled with fluoro-metric1-11 detection and mass spectrometry12,13 have been used for the quantitationof these compounds as the intact lactone-carboxylate or as a total form in bufferedsolutions and in biological matrices including plasma, red blood cells, saliva, urine,faeces, microsomes and in tumor tissues. A review of these methods when focusingon CPT-11 and its major metabolite, SN-38 as compounds of interest reveal importantdifference including the matrices from which they are isolated, separation modei.e., isocratic or gradient elution, buffer type, sample preparation, stationary phase,additives and/or ion pairing agents. The in vitro analysis of CPT-11 in IV solutionsis necessary to ensure that products of suitable quality are manufactured and madeavailable to the public. Furthermore in vitro analytical methods for CPT-11 arevital for the evaluation of new formulations of the CPT-11. Such methods may benecessary for determining encapsulation efficacy or the in vitro release characteristicsof particulate delivery systems of CPT-11. Therefore there is a need for a simpleanalytical method for the determination of CPT-11 and its related compounds duringproduct development studies. In this study we report the development and validationof a simple isocratic HPLC method for the determination of the lactone form of theCPT-11 and its main related compounds (SN-38 and CPT) in commercial productsusing UV detection.Compound R1 R2 CPT-11  C2H5 CPT H H SN-38 OH C2H5   Fig. 1. Structure of the lactone and carboxylate forms of CPT-11, CPT and SN-38Vol. 22, No. 5 (2010)       Determination of Irinotecan HCl and its Related Compounds by HPLC  3967EXPERIMENTALIrinotecan (CPT-11) hydrochloride trihydrate, 7-ethyl-10-hydroxycamptothecin(SN-38) and camptothecin (CPT) were purchased from Aurisco Pharmaceutical(Shanghai, China). HPLC-grade acetonitrile was purchased from Merck (Darmstadt,Germany). Triethylammounium acetate (TEAA) buffer was purchased from Fluka(Switzerland). Citric acid and boric acid were purchased from Merck (Darmstadt,Germany). Camptosar® injectable solutions containing 100 mg/5 mL CPT-11 HClwas obtained from the Sobhan Chemotherapeutics Company (Rasht, Iran). Waterwas purified using a Milli-Q® UV plus System (Millipore, Bedford, MA, USA).All other reagents were at least of analytical reagent grade and used without furtherpurification.Chromatographic analyses were performed using an Agilent® HPLC system(Agilent Technologies, USA) that was comprised of a degasser (Model G1322A),quaternary pump (Model G1311A), autosampler system (Model G1329A) and adiode-array detector (Model G1315B). Chemstation® software (Rev.B.02.01-SR1[260]) was used for data monitoring and analysis. The sample injection volumewas 20 µL and separation of the compounds of interest was achieved using aNucleosil® RP-18 (5 µm, 250 mm × 4 mm) analytical column protected by a C18Nucleosil® guard column (5 µm, 4 mm × 4 mm) and a mobile phase consisting of 3 %v/v triethylammonium acetate (TEAA) buffer (pH 3) and acetonitrile (70:30 v/v) ata flow rate of 1 mL min-1. All samples and the column compartment were maintainedat an ambient temperature of 25 °C and the eluant was monitored using UV detectionat 254 nm. The mobile phase was filtered through a 0.45 µm Chrom. Tech. Nylon-66filter prior to use.Preparation of stock and standard solutions: Stock solutions (100 µg/mL)of CPT-11, SN-38 and CPT were prepared in ethanol (99.9 % v/v). Standard solutions(0.1, 1, 2, 5 and 10 µg/mL) of each of the compounds were prepared by pipettingappropriate aliquots of the relevant stock solution and making up to volume withthe TEAA buffer solution.Preparation of injections for analysis: Samples of commercial product wereprepared for analysis by emptying the contents of 10 Camptosar® vials into a volum-etric flask after which the solution was mixed well. An aliquot of the resultantsolution equivalent to the volume of one injection was added into each of six 100 mL.A grade volumetric flasks made up to the volume with the TEAA buffer solutionand mixed for a further 2 min. A 50 microlitre aliquot of the dilute solution wastransferred to a 10 mL A grade volumetric flask and made up to the volume withthe TEAA buffer solution to yield an approximate concentration of CPT-11 of 5 µg/mLwhich falls in the previously described range of linearity for this method.RESULTS AND DISCUSSIONHPLC method development and optimization: The retention times for CPT-11,SN-38 and CPT using the previously described chromatographic conditions were5.7 ± 0.14, 11.9 ± 0.12 and 14.7 ± 0.10 min (n = 40), respectively. Representative3968  Mohammadi et al. Asian J. Chem.chromatograms for CPT-11, SN-38 and CPT are shown in Fig. 2. The retention ofall compounds was dependant on the strength of the buffer used to prepare themobile phase and the pH of that buffer. In an attempt to control peak tailing, due tointeraction between the analytes and underivatized silanols on the column wall,triethylammonium acetate was used. At pH values above 3.5 silanols act as weakacids react strongly with basic compounds, thereby delaying elution of the peaks ofinterest and resulting in peak broadening and tailing. Fig. 2. A representative chromatogram of a standard solution containing CPT-11, SN-38and CPTValidation: The method was validated with respect to the followng parametersincluding linearity, limit of quantitation and limits of detection (LOQ and LOD),precision, accuracy, selectivity and recovery.Linearity: Peak area of the compounds of interest were plotted versus concen-tration and least squares linear regression analysis performed on the resultant curve.The calibration curves constructed for CPT-11, SN-38 and CPT were found to belinear over the concentration range of 0.1-10 µg/mL with mean correlation coeffi-cients (r) > 0.999 with % RSD values ranging from 0.2 to 3.5 % obtained followinglinear regression analysis.LOQ and LOD: LOQ was determined based on signal-to-noise ratios andwere selected using an analytical response of 10 times the background noise14. TheLOQ was found to be 10, 50 and 25 ng/mL for CPT-11, SN-38 and CPT, respectively.The LOD was determined based on signal-to-noise ratios and was selected basedon analytical responses of 3 times the background noise. The LOD was found to be4, 15 and 9 ng/mL for CPT-11, SN-38 and CPT, respectively.Precision: The precision of the assay was investigated with respect to bothrepeatability and reproducibility. Repeatability was assessed by injecting replicatesamples (n = 9) of each of the 0.1, 1 and 10 µg/mL standards. Inter-day precisionwas assessed by injecting the same standards (n = 3) on three consecutive days.Within- and inter-day precision data are summarized in Table-1.0          2          4          6          8         10         12       14        16        18        20MinutesmAU302520151050ACPT-11SN-38CPTVol. 22, No. 5 (2010)       Determination of Irinotecan HCl and its Related Compounds by HPLC  3969TABLE-1 PRECISION AND ACCURACY OF THE HPLC METHOD IN MEASURING  KNOWN CONCENTRATIONS OF CPT-11, SN-38 AND CPT With within-day (n = 6) Between-day (n = 6) Compound Target concentration (µg mL-1) Observed concentration (mean ± SD) RSD (%) Accuracy (%) Observed concentration (mean ± SD) RSD (%) Accuracy (%) 0.1 0.098 ± 0.003 3.06 98.00 0.102 ± 0.003 2.94 102.00 1.0 1.023 ± 0.01 0.97 102.30 0.985 ± 0.012 1.22 98.50 CPT-11 10.0 9.82 ± 0.15 1.53 98.20 9.790 ± 0.05 0.51 97.90 0.1 0.101 ± 0.002 1.98 101.10 0.1015 ± 0.0025 2.46 101.50 1.0 1.016 ± 0.02 1.96 101.60 1.022 ± 0.021 2.05 102.20 SN-38 10.0 10.02 ± 0.10 1.00 100.20 10.0 ± 0.03 0.30 100.00 0.1 0.103 ± 0.003 2.90 103.00 0.10± 0.0025 2.50 100.00 1.0 0.997 ± 0.002 0.23 97.00 1.03 ± 0.015 1.45 103.00 CPT 10.0 10.17 ± 0.22 2.16 101.72 10.25 ± 0.035 0.34 102.50  Accuracy: The accuracy of the assay was assessed by interpolation of replicatepeak areas of three accuracy standards (0.1, 1 and 10 µg/mL) of each compoundfrom a calibration curve prepared as previously described. In each case, the per centrelative error was calculated and is summarized in Table-1. Accuracy was determinedas the per cent error by which the mean concentration of the replicate samplesdeviated from the known target concentration as described below: (mean concentration/target concentration × 100). The accuracy of the assay ranged from 97-103 % at theconcentrations assessed for all analytes (Table-1).Selectivity: Typical chromatograms developed following the analysis of CPT-11,SN-38 and CPT are shown in Fig. 2 and clearly reveal the selectivity of the methodfor separation of irinotecan and its related compounds. Photodiode array detectionwas used to provide additional evidence of the selectivity of the method, and toevaluate the homogeneity of the each of the peaks of interest. Chromatographicpeak purity data was obtained from the spectral analysis report and peak purityvalues of greater than 0.999 indicated the presence of an homogenous peak for thecompounds under investigation.Recovery: Spiked samples of CPT-11, SN-38 and CPT were prepared by additionof standard solutions to placebo injections at different percentages of the expectedconcentration of each compound in dilute injection solutions. Each vial of Camptosar®injection (100 mg/5 mL) contains 225 mg sorbitol NF powder and 4.5 mg of lacticacid (USP-grade) at a pH of 3.0-3.815. Mean recoveries were found to be above99.65 % at low concentration and above 99.85 % at high concentrations (Table-2).Assay: The proposed method was applied to the determination of CPT-11 inCamptosar® injections. A typical chromatogram obtained following the assay ofCamptosar® injections is depicted in Fig. 3. The results of this analysis yielded102.4 % (% RSD = 1.12 %) of label claim indicating that the method is suitable forthe selective analysis of CPT-11 and is free from interference of excipients used inthis dosage form.3970  Mohammadi et al. Asian J. Chem.TABLE-2 MEAN RECOVERY OF CPT-11, SN-3 AND CPT AT LOW (n = 6)  AND HIGH (n = 6) CONCENTRATIONS OF THE ANALYTES Analyte CPT-11 SN-38 CPT Low Concentration    Concentration (µg mL-1) 0.1 0.1 0.1 Mean recovery (%) 99.65 101.13 98.71 RSD (%) 1.25 2.70 3.01 High Concentration    Concentration (µg mL-1) 10 10 10 Mean recovery (%) 100.18 101.27 99.55 RSD (%) 0.45 1.25 1.05   Fig. 3. Resultant chromatograms following the analysis of a standard solution of (A) CPT-11 (5 µg/mL) and (B) Camptosar® injection showing CPT-11(1)10            2            4             6            8           10           12          14          16           18          20MinutesA0            2            4             6            8           10           12          14          16           18          20MinutesBmAU302520151050mAU302520151050Vol. 22, No. 5 (2010)       Determination of Irinotecan HCl and its Related Compounds by HPLC  3971ConclusionIn summary, a simple HPLC method is developed and validated for simultaneousdetermination of CPT-11, SN-38 and CPT in commercial products of CPT-11. Themethod is fast, efficient and has simple sample preparation procedure. The totalanalytical time for all analytes is less than 15 min which is an advantage for in vitroanalysis. Rapid analyses reduce the risk of degradation and conversion of analytesduring sample preparation and analysis.ACKNOWLEDGEMENTSThe authors would like to acknowledge financial assistance from the Pharma-ceutical Sciences Research Center, as well as the Novel Drug Delivery SystemsLaboratories, Tehran University of Medical Sciences, Tehran, Iran, and SobhanChemotherapeutics Company for providing facilities used throughout this research.REFERENCES1. E. Gravel, P. Bourget, L. Mercier and A. Paci, J. Pharm. Biomed. Anal., 39, 581 (2005).2. E. Gupta, T.M. Lestingi, R. Mick, J. Ramirez, E.E. Vokes and M.J. Ratain, Cancer Res., 54,3723 (1994).3. L. Zufia, A. Aldaz and J. Giraldez, J. Chromatogr. B Biomed., 764, 141 (2001).4. A. Sparreboom, P. Bruijn, M. Jonge, W. J. Loos, G. Stoter, J. Verweij and K. Nooter,  J. Chromatogr.B: Biomed., 712, 225 (1998).5. K. Sano, M. Yoshikawa, S. Hayasaka, K. Satake, Y. Ikegami, H. Yoshida, T Ishikawa, S. Sawadaand S. Tanabe, J. Chromatogr. B Biomed., 795, 25 (2003).6. Z. Hu, X. Yang, X. Chen, E. Chan, W. Duan and S.-F. Zhou, J. Chromatogr. B: Biomed., 850,575 (2007).7. T. Oguma, J. Chromatogr. B Biomed., 764, 49 (2001).8. T. Owens, H. Dodds, K. Fricke, S. Hanna and K. Crews, J. Chromatogr. B: Biomed., 788, 65(2003).9. X. Yang, Z. Hu, Y.C. Sui, B.C. Goh, W. Duan, E. Chan and S. Zhou, J. Chromatogr. B: Biomed.,821, 221 (2005).10. A. Kurita and N. Kaneda, J. Chromatogr. B: Biomed., 724, 335 (1999).11. L.P. Rivory, M. Findlay, S. Clarke and J. Bishop, J. Chromatogr. B: Biomed., 714, 355 (1998).12. S. Ragot, P. Marquet, F. Lachatre, A. Rousseau, E. Lacassie, J.M. Gaulier, J.L. Dupuy and G.Lachatre, J. Chromatogr. B: Biomed., 736, 175 (1999).13. H.M. Dodds , J. Robert and L.P. Rivory, J. Pharm. Biomed. Anal., 17, 785 (1998).14. ICH Draft Guidelines on Validation of Analytical procedures: Definitions and Terminology, FederalRegister, Vol. 60, IFPMA, Switzerland (1995).15. PDR, Thomson PDR publication, Montvale, USA, edn. 60, p. 2602 (2006).(Received: 3 September 2009;          Accepted: 23 January 2010)           AJC-83603972  Mohammadi et al. Asian J. Chem.

Simultaneous determination of irinotecan hydrochloride and its related compounds by high performance liquid chromatography using ultraviolet detection

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Simultaneous determination of irinotecan hydrochloride and its related compounds by high performance liquid chromatography using ultraviolet detection

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