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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access   Full Text Article                                                       Research Article 

Determination of Irinotecan enantiomer impurity in Irinotecan Hydrochloride API by using reverse-phase liquid chromatography

Vijay Srinivas P¹, Gana Manjusha K²*, Anjaneyulu Vinukonda³

¹ Stira Pharma limited, Kukatpally, Hyderabad, Telangana, India 500072 

² Vignan Institute of Pharmaceutical Technology, Viskahapatnam, Andhra Pradesh, India 530049

³ Stira Pharmaceuticals  LLC, New Jersey, USA

INTRODUCTION

Colorectal cancer is treated with the anti-cancer drug irinotecan (molecular formula C33H38N4O6). It is (S)-10-[4-(-piperidino) piperidinocarbonyl oxoyl]-4,7-diethyl - 4-hydroxy -1H-pyrano [3,4:6,7] indolizino[1,2-b]diethyl-3,14[4H,12H]-dionemono hydrochloride trihydrate chemically (Fig.1).  Irinotecan is a semisynthetic camptothecin derivative available under the brand names Camptosar and Onivyde. ¹

Topoisomerase I is prevented from functioning by irinotecan. Irinotecan binds to the topoisomerase I-DNA complex and prevents the DNA strand from religating. When this ternary complex forms, it disrupts the replication fork, causing it to move slowly and leading to deadly double-stranded DNA breaks. Due to ineffective DNA damage repair, apoptosis (programmed cell death) takes place.

For the treatment of metastatic cancer of the colon or rectum, irinotecan is utilised as a first-line therapy along with fluorouracil and leucovorin^2.

The undesired compounds that remain with active pharmaceutical ingredients (APIs), emerge during formulation, or appear as formulations age are known as impurities in pharmaceuticals. The efficacy and safety of pharmaceutical products may be impacted by the presence of these undesirable substances, even in trace concentrations. Impurities found in APIs are of ever-increasing interest. Purity and impurity profiles have recently become crucial due to numerous regulatory requirements³.

Most of the drug substances single enantiomer is active. In such cases, the inactive enantiomer is considered an impurity.Chiral separation, as well as the determination of the optical purity of chiral pharmaceuticals, has attracted a great deal of attention from the healthcare and pharmaceutical industries⁴.

High-performance liquid chromatography (HPLC) on chiral stationary phases (CSPs), which is widely used and one of the most effective, direct, and simple techniques for the determination of the optical purity and analytical separation of several enantiomeric drugs and pharmaceutical preparations^5–12, is one of the chiral analytical techniques currently used to achieve chiral separation of chiral mixtures. Due to the commercial accessibility of a number of CSPs for the direct separation of enantiomers^5,13, it is now standard practise to utilise HPLC to evaluate the chiral purity of medicines, their synthetic intermediates, and raw materials.

Few HPLC methods for the quantitative determination of R-enantiomer of irinotecan were reported in the literature. Hence the study aimed to develop a sensitive and rapid LC method to estimate the R-enantiomer of irinotecan¹⁴.

A simple sensitive, reliable, robust and rapidhigh-performance liquid chromatography method was developed and validated for the estimation of Irinotecan enantiomer in Irinotecan Hydrochloride trihydrate API using reverse phase chromatography.  Chromatography was accomplished using thechiralPak IC-3 150 x 4.6 mm 3µ column and ultraviolet (UV) detection and gradient mobile phase consisting of 0.1 %V/V Formic acid and Acetonitrile.  The linear range of quantitation for the compound was 0.2 – 8 µg/mL.  RP HPLC method significantly improve the retention behaviour and separation of Irinotecan and irinotecan enantiomer the runtime was no more than 20 minutes. The method has the requisite accuracy, sensitivity and precision in both pharmaceutical dosage forms and bulk API.

Figure 1 Irinotecan Hydrochloride trihydrate

Irinotecan Hydrochloride enantiomer / Irinotecan Impurity D

METHODS

Chemicals and reagents

Formic acid AR grade and Acetonitrile HPLC grade were procured from India. Irinotecan and Irinotecan enantiomer was procured from TLCchemicals.

Mobile phase A

Transfer 1 mL of formic acid in 100 ml volumetric flask containing 50 ml of water and make up to the volume with 100 ml of water

Mobile phase

Acetonitrile

Preparation of diluent

Prepare a mixture of 0.1 % formic acid in the water, Acetonitrile and Methanol in A ratio of 60:20:20 (v/v/v).

Conditions of Chromatography

Shimadzu CHT2030 chiral Pak IC3 Cellulose tris (3,5-dichlorophenylcarbamate) immobilised on 3m silica-gel (150 x 4.6 mm, 3) column was used as immobile phase,  mobile phase consisting of 0.1 percent in water and acetonitrile during reverse-phase HPLC analysis. Gradient was provided by the mobile phase at 1.0 mL/min. The UV detection's wavelength was set to 370 nm. 20 L was chosen as the injection volume, and temperatures of 25 C for the column heater and 5 C for the autosampler were employed

Gradient program: Time / B%: 0/5, 10/ 85, 15/5, 16/5, 20/5.

Preparation of blank

Diluent is used as blank

Preparation of Irinotecan Hydrochloride enantiomer stock solution

Precisely weigh and transfer 3mg of irinotecan Hydrochloride enantiomer standard into a 10 ml volumetric flask at 14 mL of Diluent and sonicate For 2 minutes to dissolve and dilute to volume with dilute and mix.

Transfer 2.5 mL of this solution into a 25 mL volumetric flask and dilute the volume with dilute and mix.  Further, transfer 5 mL of the solution into a 50 mL volumetric flask and dilute the volume with dilute and mix.

Preparation of resolution solution

Precisely weigh about 20 mg of irinotecan hydrochloride trihydrate and transfer it to a 10 ml volumetric flask.   Add about 3 mL of diluent and sonicate for 2 minutes to dissolve.  To this solution add 2 mL of irinotecan impurity D stock solution and dilutethe volume with diluent and mix.

Preparation of sample solution (2mg/ml)

Accurately weigh about 100 mg of irinotecan hydrochloride trihydrate and transfer it to a 50 ml volumetric flask.  At about 15 ml of diluent and sonicate for 2 minutes to dissolve and dilute the volume with diluent.

RESULTS

Analytical method Validation:

The validation experimentsdemonstrated system suitability and control sample analysis,  the limit of detection and limit of quantification,   Precision at LOQ  and accuracy,  Precision of the test method,  method  Precision ( repeatability),  intermediate precision (  ruggedness- intra lab)  and linearity of the detector response.  The HPLC method for the determination of irinotecan Enantiomer in irinotecan hydrochloride trihydrate API by HPLC is precise (repeatability and reproducibility), accurate and linear over the range of 0.4 μg/mL -8 μg/mL.

System suitability:

Equilibrate the chromatographic system with mobile phase until a consistent baseline is seen, and then inject a blank, resolution solution, and standard solution in the appropriate order. The results were reported in Table 1 after the system's appropriateness was assessed.

Specificity 

Interference from placebo and a blank

A study was conducted to determine whether placebo and blank effects were present. To determine the aforementioned chromatographic conditions and record the blank and placebo chromatograms, diluent and placebo injections were made into columns. The chromatograms of the blank samples revealed no peak during the retention time of either the enantiomer or the analyte peak of irinotecan. This has shown that the diluent solution used to prepare the sample does not interfere with the measurement of the enantiomer of irinotecan. The Irinotecan enantiomer and Irinotecan analyte peaks did not appear on the chromatogram produced for the placebo solution. This also showed that the placebo used to make the sample solution did not affect how well irinotecan enantiomer impurity were estimated in irinotecan injection.

Precision

System precision

By making blank and standard solutions in accordance with the test procedure and chromatographing them into the HPLC system, system precision was demonstrated. For these system appropriateness injections, the analyte retention time and Pinnacle areas were noted. Retention time's percent RSD was found to be 0.2, and area response's was found to be 1.0.

Method precision

By injecting six sample solutions spiked with irinotecan enantiomer, the precision of the impurity was calculated at the specification level. The samples were made in accordance with the method. Table 2 is a summary of the precision study's findings. For irinotecan Enantiomer, the percent RSD of method precision was discovered to be 0.6 percent.

Limit of detection and quantitation:

Three injections of a solution containing 0.12 μg/ml of irinotecan enantiomer were made. The worst signal-to-noise ratio for each piece in each injection was greater than 3, and peaks were found in all three injections.

Six injections of a solution containing 0.4 μg/ml of the irinotecan enantiomer standard were made. The average deviation for each standard, the relative SD of the areas, and the deviations of each of the six replicates from the linear regression curve were computed

Limit of detection and quantitation:

Irinotecan Enantiomer limit of quantitation and limit of detection values were obtained and summarised in Tables 3 and 4 within the permissible range.

Linearity

By injecting the solutions in duplicate containing irinotecan impurity-D varying from LOQ to 200 percent of the designated limit, linearity was ascertained.It displayed a concentration versus area graphic. The peak regions were given as an entire number. Four notable figures were reported for the concentrations. The data were subjected to a linear regression analysis (without pushing via the origin). The results are tabulated in Table 5 for the correlation coefficient ®, slope, and percent y-intercept.

Accuracy

The accuracy or recovery samples were prepared by spiking the irinotecan hydrochloride enantiomer standard and hydrochloride control sample using solutions at concentrations spanning from LOQ to 200% to the specified limit.   6 preparations were made at LOQ, 100% Level and three preparations at 50%, 150% and 200%.

Once each solution was injected, it was examined. Calculating the percent recovery for each individual preparation at each level allowed for the determination of the mean percent recovery of the irinotecan hydrochloride enantiomer. The findings were compiled in Table 6. 

Solution stability

Initial injection of the solution into HPLC was followed by measurements of the percent area of irinotecan impurity-D in spiked solution at each interval. The percent area difference from the initial day interval was then computed. Standard and sample solutions were consistent for 48 hours on the benchtop and in cooler (2-8⁰C) conditions. The results are presented in tables 9 and 10. A solution stability parameter was constructed.

Table-1: System suitability results

Table-2: Method precision results

Table-3: LOD and LOQ results

Table 4: LOQ Precision results

Table-5:  Results for Linearity of detector response Study

Figure 3: Linearity plot of Irinotecan Enantiomer

Table 6: Accuracy

Spiked sample Chromatogram

Blank (Diluent)

Figure 4: Spiked sample and Blank (Diluent) Chromatogram

DISCUSSION

A successful reverse phase HPLC process was devised that was simple, affordable, accurate, and precise. On a chiral Pak IC-3 (3 m, 4.6 X 150 mm) column (amylose-based chiral stationary phase), the partition was produced using formic acid concentration of 0.1 percent and an acetonitrile as a mobile phase at a flow rate of 1 ml/min. 25°C for the column temperature, 20 l for the injection volume, 5°C for the sample cooler, and 370 nm for the detection wavelength. The findings were found to be precise and repeatable. According to ICH guidelines and USP 1225>, the new technique was statically certified in terms of selectivity, accuracy, linearity precision ruggedness, and solution stability.

Chromatograms of Irinotecan enantiomer and Irinotecan standard and sample solutions were taken in order to determine the selectivity. The results of this analysis showed that the peaks were properly spaced apart. In order to determine the Irinotecan enantiomer in Irinotecan hydrochloride trihydrate API, the approach was selective. At the Irinotecan enantiomer and Irinotecan peak, there is no interference from diluent or placebo.

The method was found to be linearity over the range of 0.4 g/ml to 8 g/ml with a correlation coefficient value greater than 0.99. The LOD and LOQ for the irinotecan enantiomer standard were 0.1 and 0.4 g/ml, respectively. The result was found to be within the limitations, with the maximum percent recovered shown being between 80 and 120 percent. Consequently, the method's accuracy was determined. Additionally, the recoveries observed for the irinotecan enantiomer have relative SD values that range from 0.03 to 3.6 percent. Six replicate injections were used to determine precision studies. The peak area of irinotecan enantiomer, which was found to be 0.1 percent, was used to calculate the percent RSD. The results originated to be within the acceptance limit, and the acceptance limit should not be greater than 10.

As a result, the chromatography method created for irinotecan hydrochloride trihydrate API was quick, easy, precise, sensitive, and accurate. The suggested method is therefore helpful for the regular analysis of the pharmaceutical active components for the declaration of their quality during formulation.

CONCLUSION

The optimised method is Rapid and sensitive which is practically following the quality control requirements of testing in Irinotecan hydrochloride. 

According to ICH criteria, the suggested reverse phase HPLC technique has been sophisticated and authenticated to be able to quantify the irinotecan enantiomer in irinotecan hydrochloride trihydrate at a trace level concentration. The specificity, exactitude, linearity, and correctness of the method assured its efficacy. As a result, the method is appropriate for the task at hand, may be successfully used for routine laboratory analysis, and is acceptable for quality control.

REFERENCES

1. https://go.drugbank.com/drugs/DB00762

2. https://www.drugs.com/monograph/irinotecan.html

3. Kumar R, Singla RK. Impurities in Pharmaceutical Dosage Form: A Subject Matter of Great Concern. WebmedCentral Pharmaceutical Sciences 2012;3(1):WMC002884. https://doi.org/10.9754/journal.wmc.2012.002884

4. Rebizi, Mohamed &Sеkkoum, Khаlеd&Belboukhari, Nasser &Abdelkrim, Cheriti&Aboul-Enein, Hassan. Chiral separation and determination of enantiomeric purity of the pharmaceutical formulation of cefadroxil using coated and immobilized amylose-derived and cellulose-derived chiral stationary phases. Egyptian Pharmaceutical Journal, 2016; 15: 88-97. https://doi.org/10.4103/1687-4315.190399

5. Aboul-Enein HY, Ali I. Polysaccharide-based chiral stationary phases, Chapter 2 in chiral separation by liquid chromatography and related technologies. New York, NY: Marcel Dekker Inc.; 2003; 21-88. https://doi.org/10.1201/9780203911112.ch2

6. Ravikumar M, Narasimhanaidu M, Srinivasulu K, Raju TS, Reddy MRS, Swamy PY. Enantiomeric separation of docetaxel starting material by chiral LC using amylose-based stationary phase. Chromatographia, 2009; 69:163-167 https://doi.org/10.1365/s10337-008-0837-6

7. Aboul-Enein HY, Ali I. Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantioseparation of nebivolol by HPLC. Journal of Biochemical and Biophysical Methods, 2001; 48(2):175-188. https://doi.org/10.1016/S0165-022X(01)00148-8

8. Ishii K, Minato K, Nishimura N, Miyamoto T, Sato T. Direct chromatographic resolution of four optical isomers of diltiazem hydrochloride on a Chiralcel OF column. Journal of Chromatography, 1994; 686:93-100. https://doi.org/10.1016/S0021-9673(94)89012-9

9. Khan M, Viswanathan B, Rao DS, Reddy R. Chiral separation of frovatriptan isomers by HPLC using amylose based chiral stationary phase. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2007; 846(1-2):119-123. https://doi.org/10.1016/j.jchromb.2006.08.033

10. Nadalini G, Dondi F, Massi A, Dondoni A, Zhang T, Cavazzini A. Highperformance liquid chromatographic separation of dihydropyrimidineracemates on polysaccharide-derived chiral stationary phases. Journal of ChromatographyA, 2006; 1126(1-2):357-364. https://doi.org/10.1016/j.chroma.2006.05.095

11. Chen L, Zhang LF, Ching CB, Ng SC. Synthesis and chromatographic properties of a novel chiral stationary phase derived from heptakis (6-azido6-deoxy-2,3-di-O-phenylcarbamoylated)-beta-cyclodextrin immobilized onto amino-functionalized silica gel via multiple urea linkages. Journal of Chromatography, 2002; 950(1-2):65-74. https://doi.org/10.1016/S0021-9673(02)00043-2

12. Hoffmann CV, Laemmerhofer M, Lindner W. Novel strong cation-exchange type chiral stationary. Journal of Chromatography, 2007; 1161(1-2):242-251. https://doi.org/10.1016/j.chroma.2007.05.092

13. Wang T, Wenslow RM Jr. Effects of alcohol mobile-phase modifiers on the structure and chiral selectivity of amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase. Journal of Chromatography, 2003; 1015(1-2):99-110. https://doi.org/10.1016/S0021-9673(03)01262-7

14. Dhakane VD, UBALE MB, A validated chiral LC method for the enantiomeric separation of irinotecan hydrochloride on immobilized cellulose-based stationary phase. International Journal of Pharmaceutical Sciences Review and Research, 2013; 19(2):66-69.