INTRODUCTION:

Camptothecin (CPT) is (S)-4-ethyl-4-hydroxy-1H pyrano[3',4':6,7] indolizino [1,2-b] quinoline-3,14-(4H, 12H)-dione.¹ The CPT is a naturally occurring alkaloid that was extracted from the Chinese medicinal plant Camptotheca acuminate.² It inhibits the growth of cancer by preventing DNA replication by binding to DNA topoisomerase I during the S stage of mitosis.³ Poor solubility and adverse effects, such as diarrhea, hemorrhagic cystitis, etc. have restricted the clinical applications of CPT.⁴US Food and Drug Administration has approved CPT derivatives to treat ovarian and colon cancer.⁵

Additionally, their anticancer potential on additional malignancies, including lung, breast, pancreatic, lymphoma, glioma, and leukemia, has been reported.⁶

The majority of chemical constituent classes can be quantitatively analyzed using chromatographic methods. There are several advantages to using these techniques, including the potential to develop rapidly and with great precision, accuracy, specificity, and sensitivity. Instrumentation and methodology should be established early in the process of developing the analytical procedure, following the objective and scope of the analytical technique. Precision, specificity, linearity, limits of detection (LOD), and limits of quantitation (LOQ) are only a few of the characteristics that must be considered while developing a technique.⁷Validation of analytical methods is the process of ensuring that an analytical procedure is suitable for its intended purpose. The International Conference on Harmonization (ICH) Q2 (R1) provides the most authoritative advice on validation characteristics for analytical techniques.⁸

Several methods have been described for the measurement of camptothecin and its analog using UV spectroscopic methods, high-performance liquid chromatography (HPLC), and high-performance thin layer chromatography (HPTLC).^9-11 There are few reports on the RP-HPLC estimation of Camptothecin in pharmaceutical formulations. Hence the present work aimed to develop a new simple, precise, accurate, and sensitive RP-HPLC method for the determination of CPT in API and micellar pharmaceutical formulation. To ensure the reliability, reproducibility, and sensitivity of the method, the developed analytical method was validated and statistically analyzed rigorously for various parameters following ICH guidelines.

Figure 1.Chemical structure of Camptothecin

MATERIALS AND METHODS:

Materials: The reference sample of camptothecin (API) was obtained from Clearsynth Pvt. Ltd., Goa, India. Pluronic F108 (PF108) and TPGS were purchased from Sigma-Aldrich, HPLC grade acetonitrile, and water was purchased from Spectrochem Pvt. Ltd., Mumbai, India. 1100 Infinity LC and 1220 Infinity LC, Agilent technologies HPLC was used for the measurement. Digital Electronic Balance (Model BL-220H, Contech & Citizen, Mumbai, India) was used for weighing purposes.

Methods:

Determination of absorption maxima:

A Camptothecin Solution (10 μg/mL in methanol) was scanned in the UV range via a UV-visible spectrophotometer. The recorded λ_max was compared with the literature value.^12,13

Equipment and chromatographic conditions:

Analysis was performed on RP-HPLC (Agilent 1100) using mobile phase: acetonitrile: water (90:10 v/v), Inertsil ODS 3V C18 column (250.0 ˟ 4.6 mm, 5.0 µm, Agilent) at flow rate: 1.0 mL/min, column temperature 30 ℃ with injection volume 20 µL and diode Array Detector (DAD) at λ_max: 219nm with run time 5 min and retention time 3.066 min.

Camptothecin-loaded mixed micelles (CPT MMs) Preparation:

The camptothecin-loaded with TPGS and PF108 MMs were prepared using a solvent evaporation technique. Camptothecin (2.5 mg), TPGS (12.2 mg), and PF108 (414 mg) were added to 1 mL of methanol and the resultant mixture was sonicated for 5 min. The resultant solution was added to a beaker containing 10 mL distilled water in a drop wise manner and further stirred at 500 rpm until the evaporation of total methanol. The resultant camptothecin MMs were centrifuged for 10 min at 5000 rpm and from that supernatant was collected for further analysis.^14,15

Preparation of standard solution:

To create camptothecin stock solutions, 10 mg of the Camptothecin was dissolved in 10 mL of acetonitrile (ACN): water (90:10 v/v) to get a concentration of 1000 µg/mL. To completely dissolve the Camptothecin, the solution was immersed in an ultrasonic bath for 5 min.

Preparation of working standard solution:

Solution of concentrations of 20, 40, 60, 80, and 100 μg/mL were prepared using 1000 μg/mL stock solution. All solutions were filtered using a 45 m membrane before analysis.

Preparation of sample solution:

Sample preparation was done by adding 4 mL of the supernatant in a 10 mL volumetric flask and volume was made up to 10 mL with mobile phase ACN: Water (90:10). The sample was sonicated for 5 min in an ultrasonic bath. Lastly, the sample was filtered before analysis using a 45 µm membrane.

Validation of method:

The validation of the analytical methodology was carried out by the guidelines of the ICH (International Council for Harmonization).The method was validated for specificity, linearity, detection limit (LOD), quantification limit (LOQ), precision, accuracy, robustness, and ruggedness.

Specificity:

Excipient interference in CPT determination was assessed to gauge the method's specificity. A comparison of camptothecin mixed micelles formulation (CPT MMs) and without the drug (Blank MMs) was carried out by comparing the chromatograms of the two formulations to confirm excipients interference with drug measurement.

Linearity:

The concentrations of the CPT standard solutions were 20, 40, 60, 80, and 100 µg/mL. By plotting concentration against the corresponding peak area, the calibration curve was created, further, the least square regression approach was used to determine the linearity. Linear regression was used to determine the line's equation. Slope, Y-intercept, and correlation coefficient of the regression line were calculated.¹⁶

LOD and LOQ determination:

The detection limit (LOD) and quantification limit (LOQ) were used to determine the method's sensitivity. LOD is the lowest analyte concentration that reliably produces a reaction but cannot always be precisely quantified. The lowest concentration of an analyte at which an accurate result can be obtained is known as the LOQ. The following formula was used to determine the LOD and LOQ using the standard deviation of response (SD) and slope (S) methods. At least three separate analyses were done for each study.¹⁷

3.3* SD

LOD = ------------ .... (1)

10* SD

LOQ = ------------ .... (2)

Precision:

The precision of the procedure was evaluated by executing intra-day (repeatability) and inter-day (intermediate) analyses using triplicate injections of the CPT MMs. To accomplish the intra-day precision, the CPT MMs solution was examined three times on the same day (Day 1, n = 3), while to perform the inter-day precision, these solutions were examined three times on three different days (Day 1, n = 3; Day 2, n = 3; Day 3, n = 3). The relative standard deviation (%RSD) of a set of measurements was used to express the precision data.¹⁶

Accuracy (recovery):

The accuracy of the procedure was evaluated by comparing the known drug concentration in the blank MMs sample. Known concentrations of CPT standard solutions were added to a blank mixed micelles solution to generate concentrations of 8, 10, and 12 µg/mL. The preparation of each sample was done in triplicate, and the recovery rate was assessed.¹⁶

Ruggedness:

Employing subtle and intentional changes to the chromatographical conditions allowed for an evaluation of the method's robustness and ruggedness. The effects of changing the flow rate, column temperature, and percentage of organic solvent on retention time and CPT content percentage were evaluated.¹⁷

RESULTS AND DISCUSSION:

The developed method was validated as per ICH guidelines.

Determination of Absorption Maxima:

The 10 μg/mL concentration of Camptothecin solution was scanned in the UV range using a UV spectrophotometer. The recorded spectrum showed the absorbance maxima (λ_max) at 219 nm as shown in Fig. 2.⁹

Figure 2. λ_max for Camptothecin in ACN: Water (90:10)

Method Validation:

Specificity:

The method's specificity was determined by testing its ability to identify the drug in a mixture with other formulation excipients. Figure 3 shows the chromatogram of camptothecin, Figure 4 shows the chromatogram of blank CPT MMs, and Figure 5 shows the chromatograms for CPT MMs. Because none of the peaks showed the same RT of CPT, the specificity studies amply demonstrated the absence of any other excipient interference. As a result, the perfect separation of drugs from MMs justified the specificity and demonstrated that there was no interaction with the quantitative determination of CPT from MMs components.¹⁷

Figure 3. Chromatogram of Camptothecin (100 µg/mL)

Figure 4. Chromatogram obtained from Blank MMs

Figure 5. Chromatogram obtained from CPT MMs

Linearity:

The method was found to be linear in the range of 20-100 µg/mL for CPT. The linearity determination coefficient (R²) for CPT was found to be 0.9995.

Figure 6. Linearity curve of Camptothecin at 219 nm

Table 1. Result of Camptothecin regression analysis

Parameter	Result
λ_max	219 nm
Beers law limit (µg/mL)	20-100
Regression Equation Data
Slope	62.287
Intercept	+393.98
Correlation coefficient	0.9995

Accuracy:

The % recoveries of the findings show that the recoveries are well within the acceptability range (%RSD<2), indicating that the procedure is accurate according to the established standards (ICH, 2005).

Table 2. Results of statistical analysis of accuracy

Concentration level (%)	Pre-analyzed sample solution (μg/ml)	Amount of drug added (μg/ml)	% Recovery Mean ± SD	% RSD (%)
80	10	8	101.38 ± 0.19	1.05
100	10	10	100.76 ± 0.53	1.09
120	10	12	101.02 ± 0.98	1.01

Values are mean ±SD, n=3

Precision:

Table 3 below provides the %RSD for intraday and interday variation. The %RSD readings were found to be substantially within the 2% limit in both situations, proving the repeatability of the current method.

Table 3. Results of statistical analysis of intra-day assay and inter-day assay

Sr. No.	Concentration (μg/mL)	Standard solution		Sample solution
Sr. No.	Concentration (μg/mL)	Intra-day precision	Inter-day precision	Intra-day precision	Inter-day precision
1	100	6483.13232	6507.64523	6254.23926	6342.88524
2	100	6490.45801	6518.75488	6288.56241	6375.54962
3	100	6486.29883	6523.02157	6267.98213	6389.64238
4	100	6485.38795	6514.38912	6243.79521	6359.57985
5	100	6492.75123	6509.57921	6259.45981	6385.65892
6	100	6484.87235	6511.95641	6273.96328	6391.58962
Mean		6487.150115	6514.224403	6264.667017	6374.150938
SD		3.673566667	5.79568164	15.74729015	19.33050875
%RSD		0.056628359	0.088969634	0.251366754	0.303264057

Values are mean ±SD, n=3

LOD and LOQ:

To determine the sensitivity of the method, LOD and LOQ were evaluated. The LOD for CPT was found to be 6.35 µg/mL. On the other hand, the LOQ for CPT was found to be 19.25 µg/mL. As per the acquired results, the developed method is sufficiently sensitive to estimate the Camptothecin.

Ruggedness:

Minor changes are made on purpose to test the method's robustness and ruggedness. The outcomes showed that under specific chromatographic circumstances, no obstruction affected drug findings. Peak areas and retention times showed no discernible change. Additionally, the drug content of CPT was close to 100% for all evaluated situations, with an RSD of less than 2%. The collected findings demonstrated the suggested method's robustness and ruggedness under slight variations in chromatographic settings.

Table 4. Results of statistical analysis of ruggedness

Parameter		Quantity of Camptothecin added (μg/mL)	Quantity of Camptothecin detected (Mean ± SD)	% RSD
Change in mobile phase composition	ACN: Water = 88:12	100	99.57 ± 0.38	0.83
	ACN: Water = 90:10	100	100.82 ± 0.51	0.57
	ACN: Water = 92:8	100	100.21 ± 0.79	0.27
Change in column temperature	25 ℃	100	100.14 ± 0.55	0.29
	30 ℃	100	100.66 ± 0.23	0.97
	35 ℃	100	100.78 ± 0.19	0.31
Change in flow rate	0.9 mL/min	100	100.46 ± 0.46	0.18
	1 mL/min	100	99.21 ± 0.62	0.47
	1.1 mL/min	100	100.35 ± 0.85	0.68

Values are mean ±SD, n=3

CONCLUSION:

For the quantitative determination of Camptothecin from bulk and mixed micelles formulations, the HPLC approach was devised in this work. The proposed method of validation for quantification of Camptothecin in mixed micelles formulation has proven to be very specific, linear, accurate, precise, sensitive, and economic. The method exhibits simplicity in terms of short analysis time, isocratic mode of elution of mobile phase, and effective, and clear resolution with low LOD and LOQ values. The proposed method was developed and validated as per the ICH guidelines making it ideal for routine Camptothecin analysis from a medicinal dosage formulation.¹⁸

ABBREVIATIONS:

Ultraviolet spectrophotometer, (UV); International Council for Harmonisation, (ICH); Active pharmaceutical ingredient, (API); %Relative standard deviation, (%RSD); Standard deviation, (SD); Limits of detection, (LOD), Limits of quantitation, (LOQ), Correlation coefficient, (R²); Camptothecine, (CPT).

ACKNOWLEDGMENT:

The authors appreciate Bharati Vidyapeeth College of Pharmacy, Kolhapur, and Tatyasaheb Kore College of Pharmacy, Warnanagar for providing the necessary facilities for carrying out research work.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 14.07.2022 Modified on 17.08.2022

Research J. Pharm. and Tech 2022; 15(9):4248-4252.

DOI: 10.52711/0974-360X.2022.00714