{"id":64057,"date":"2025-03-31T11:20:07","date_gmt":"2025-03-31T11:20:07","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=64057"},"modified":"2025-04-18T06:46:10","modified_gmt":"2025-04-18T06:46:10","slug":"box-behnken-design-approach-to-method-development-for-irinotecan-hydrochloride-trihydrate-by-using-rp-hplc-method","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol18no1\/box-behnken-design-approach-to-method-development-for-irinotecan-hydrochloride-trihydrate-by-using-rp-hplc-method\/","title":{"rendered":"Box -Behnken Design Approach to Method Development for Irinotecan Hydrochloride Trihydrate by Using RP-HPLC Method"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n

Analytical Quality by Design (A QbD) is a systematic and risk-based approach to developing analytical methods in the field of pharmaceuticals and other regulated industries. It draws inspiration from the principles of Quality by Design (QbD) and applies them specifically to analytical methods1\u20133<\/sup>. ICH Q8(R2) Pharmaceutical Development and provides further clarification of key concepts outlined in the core guideline4<\/sup>. Moreover, Applying of QbD is important in developing a new method it fit for the purpose assure the quality. The result it could be organized by CQA, risk assessment and outlining of design space it set for the future investigations. 5,6<\/sup>.<\/p>\n

Irinotecan is primarily used in the treatment of Metastatic colorectal cancer. This is semi synthetic derivative of Camptotheca accuminata <\/em>which contains Comptothecin alkaloid belongs to Nyssaceae family7,8<\/sup>. There are the several methods are available in the irinotecan hydrochloride trihydrate injections using QbD. But in this study describes about the implementation of QbD impact the outcome of analytical process development and validation. Understanding about critical quality attributes & critical method, material attributes. In this present work, a novel RP-HPLC method was developed & validated.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Materials<\/strong><\/p>\n

Irinotecan reference standard (99.5%) was purchased in the Reputed pharmaceutical company. The sample injection of Irinotecan was purchased in the market. Acetonitrile analytical grade was purchased in Spectro hem Pvt. Ltd., Mumbai KH2<\/sub>PO4<\/sub> Buffer Purchased in Merck Pvt. Ltd., Mumbai.<\/p>\n

Instruments<\/strong><\/p>\n

A Shimadzu HPLC system equipped with dual pump. UV-Vis detector was used in this present work. The PDA detector used for determination of proper wavelength of irinotecan hydrochloride trihydrate 222 nm was found wave length.<\/p>\n

Experimental Procedure <\/strong><\/p>\n

\u00a0standard stock solution preparation<\/strong><\/p>\n

\u00a0acquired quantity (5 mg) of Irinotecan API was transferred in to a 25 ml calibrated volumetric flask.\u00a0 \u00a0To this 15 ml of mobile phase (ACN: KH2<\/sub>PO4<\/sub>) was added and sonication was done. Finally, it was filled up to the mark with mobile phase. It was used for further analysis.<\/p>\n

Preparation of analyte<\/strong><\/p>\n

Pipette out 0.25 ml of irinotecan injection which is equivalent to 5 mg of drug is transfer in to 25 mal of volumetric flask and made up with mobile phase. From this various concentration there prepared for calibration and validation parameter.<\/p>\n

Method optimization<\/strong><\/p>\n

The mobile phase was optimized according to the ICH-guidelines Q2<\/sub>(R2<\/sub>) using a 3-factor, 3-level Box-Behnken design analyzed with ANOVA software. For this study, we set acetonitrile as factor X, buffer concentration as Y, and buffer pH as Z. Capacity factor (K1<\/sub>) was set as an independent variable, resolution (RS12<\/sub>), and elution time-tR1<\/sub>-was considered as the response. The surface analysis was targeted at the location of the various independent variables’ effects based on observations. Table 1 shows the Box-Behnken-generated experimental design of 20 runs that have been subjected to show the observed and expected responses.<\/p>\n

The results were evaluated using the ANOVA method, besides the desirability function to select optimum conditions after the procedure of optimization was over. The advantage of using a Box-Behnken design lies in the fact that it optimizes experimental designs that contain at least three dependent variables or factors, and several responses can be compared against CCD and full factorial design. This experimental design contains three factors-straight line, quadratic, and interaction terms-which can be expressed by the model Y0<\/sub>.<\/p>\n

= \u03b20<\/sub> + \u03b21<\/sub> X1<\/sub> + \u03b22<\/sub> X2<\/sub> + \u03b23<\/sub> X3<\/sub> + \u03b212<\/sub> X1<\/sub> X2<\/sub> + \u03b213<\/sub> X1<\/sub> X3 + \u03b223 X2 X3 + \u03b211 X 2 + \u03b222 X 2 + \u03b233 X 2<\/p>\n

Y0 <\/sub>= response to be modeled, \u03b20 = constant, \u03b22<\/sub>, \u03b23<\/sub>, \u03b24 <\/sub>= linear coefficients,<\/p>\n

\u03b213<\/sub>, \u03b214<\/sub>, \u03b224 <\/sub>= interaction coefficients, \u03b211<\/sub>, \u03b222<\/sub>, \u03b233 <\/sub>= quadratic coefficients.<\/p>\n

The statistical parameter given in table 2.<\/p>\n

Method validation <\/strong><\/p>\n

\u00a0Method was validated as per the ICH Q2<\/sub>(R2<\/sub>) guidelines.\u00a0<\/p>\n

System Condition<\/strong><\/p>\n

\u00a0 The system condition was obtained by the mirroring the analysis of six sample standard injections at 80 \u00b5g\/ml and finally get optimize chromatogram.\u00a0 From chromatogram calculate the system condition parameters such as tailing factor, theoretical plate count and reproducibility (% RSD of analyte retention time and area of the six replicates).<\/p>\n

Specificity <\/strong><\/p>\n

\u00a0 The ability of a developed analytical method accurately measures the samples in the presence of the other residues or compounds. the major peak was used studied for the peak purity and developed method it should effectively separates the analytes and denatured products.<\/p>\n

Linearity<\/strong><\/p>\n

\u00a0 \u00a0The linearity verifies by minimum 5 different concentration standard solution was prepared at the different concentration range from 50 to150% to showing that assay concentrations. Weighed accuratlyb5 mg of standard dissolve into 10ml of mobile phase into 25 ml of volumetric flask. Sonicate, make up to the marked volume.<\/p>\n

Precision<\/strong><\/p>\n

\u00a0 Take accurately weighed 5mg of standard irinotecan dissolved in 15ml of mobile phase in to 25ml of volumetric flask and sonicate and make up the volume up to the mark. Accurately take two ml of above solution was transferred to 5ml of volumetric flask and the volume was make up with mobile phase. 6 such preparations were made.<\/p>\n

Accuracy <\/strong><\/p>\n

\u00a0The accuracy was performed by percentage of recovery conducted at the different concentration range 80,100 and 120%. Two ml of standard solution into 5ml of volumetric flask make up to volume with mobile phase solution. 1.6 ml and 2ml of test solution pipette out transfer into two different 5ml volumetric flask make up to the volume to the mark with mobile phase.\u00a0\u00a0<\/p>\n

LOD and LOQ<\/strong><\/p>\n

\u00a0 Serial dilutions of Irinotecan were prepared from standard test solution in the range of 1%, 2 % and 3% to target assay concentration (80ppm). The samples were analyzed by HPLC replicated for 5 times. Standard deviation was calculated in each 5 injections concentration.<\/p>\n

LOD = (3.3 X S.D\/slope)<\/p>\n

LOQ = (10 X S.D\/slope)<\/p>\n

Ruggedness<\/strong><\/p>\n

Ruggedness of this method was confirming by the performing the following variations.<\/p>\n

Analyst person to analyst person variation, 2. Different Column variation, 3. Day to days variation. This mentioned parameter was studied to estimate the percentage assay on few different days using different analytical columns. Sample analysis was carried out by two different analysts, varying one parameter each time. Analysis was carried out in duplicate.<\/p>\n

Robustness<\/strong><\/p>\n

\u00a0\u00a0Robustness test was performed deliberately changes in mobile phase to the developed HPLC conditions viz., Flow rate change into (\u00b1 0.2 ml\/min), Composition change into (\u00b1 5 ml):\u00a0 and buffer pH adjusted in to (\u00b1 0.2) as per method validation protocol.\u00a0<\/strong><\/p>\n

Results and Disscussion<\/strong><\/p>\n

Table 1: Box \u2013 Behnken design arrangement and responses<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Run<\/strong><\/p>\n<\/td>\n

\n

Type<\/strong><\/p>\n<\/td>\n

\n

ACN<\/strong><\/p>\n

concentration X (%vol\/vol)<\/strong><\/p>\n<\/td>\n

\n

Buffer<\/strong><\/p>\n

Concentration Y(Milli Mole)<\/strong><\/p>\n<\/td>\n

\n

Buffer Strength <\/strong>(pH) Z\u00a0 <\/strong><\/p>\n<\/td>\n

\n

Capacity <\/strong>factor (K1<\/sub>)<\/strong><\/p>\n<\/td>\n

\n

Resolution (Rs1,2<\/sub>)<\/strong><\/p>\n<\/td>\n

\n

Retention<\/strong><\/p>\n

time <\/strong>(tR1<\/sub>)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

55.00<\/p>\n<\/td>\n

\n

45.00<\/p>\n<\/td>\n

\n

3.16<\/p>\n<\/td>\n

\n

0.78<\/p>\n<\/td>\n

\n

6.90<\/p>\n<\/td>\n

\n

5.25<\/p>\n<\/td>\n<\/tr>\n

\n

7<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

65.00<\/p>\n<\/td>\n

\n

35.00<\/p>\n<\/td>\n

\n

3.84<\/p>\n<\/td>\n

\n

0.78<\/p>\n<\/td>\n

\n

10.46<\/p>\n<\/td>\n

\n

5.10<\/p>\n<\/td>\n<\/tr>\n

\n

14<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

55.59<\/p>\n<\/td>\n

\n

45.00<\/p>\n<\/td>\n

\n

3.00<\/p>\n<\/td>\n

\n

0.96<\/p>\n<\/td>\n

\n

4.68<\/p>\n<\/td>\n

\n

4.55<\/p>\n<\/td>\n<\/tr>\n

\n

18<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

55.00<\/p>\n<\/td>\n

\n

48.36<\/p>\n<\/td>\n

\n

3.00<\/p>\n<\/td>\n

\n

0.86<\/p>\n<\/td>\n

\n

11.46<\/p>\n<\/td>\n

\n

3.36<\/p>\n<\/td>\n<\/tr>\n

\n

19<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

57.00<\/p>\n<\/td>\n

\n

41.64<\/p>\n<\/td>\n

\n

4.00<\/p>\n<\/td>\n

\n

0.85<\/p>\n<\/td>\n

\n

14.60<\/p>\n<\/td>\n

\n

2.45<\/p>\n<\/td>\n<\/tr>\n

\n

17<\/p>\n<\/td>\n

\n

Axial<\/p>\n<\/td>\n

\n

57.41<\/p>\n<\/td>\n

\n

43.00<\/p>\n<\/td>\n

\n

3.00<\/p>\n<\/td>\n

\n

0.94<\/p>\n<\/td>\n

\n

4.92<\/p>\n<\/td>\n

\n

6.50<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Center<\/p>\n<\/td>\n

\n

60.00<\/p>\n<\/td>\n

\n

40.00<\/p>\n<\/td>\n

\n

3.20<\/p>\n<\/td>\n

\n

1.0<\/p>\n<\/td>\n

\n

11.68<\/p>\n<\/td>\n

\n

2.16<\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

60.00<\/p>\n<\/td>\n

\n

45.00<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

0.91<\/p>\n<\/td>\n

\n

11.68<\/p>\n<\/td>\n

\n

8.63<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

55.00<\/p>\n<\/td>\n

\n

53.00<\/p>\n<\/td>\n

\n

2.50<\/p>\n<\/td>\n

\n

0.92<\/p>\n<\/td>\n

\n

8.86<\/p>\n<\/td>\n

\n

9.53<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

58.00<\/p>\n<\/td>\n

\n

42.00<\/p>\n<\/td>\n

\n

2.50<\/p>\n<\/td>\n

\n

0.96<\/p>\n<\/td>\n

\n

8.98<\/p>\n<\/td>\n

\n

6.56<\/p>\n<\/td>\n<\/tr>\n

\n

9<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

58.00<\/p>\n<\/td>\n

\n

42.00<\/p>\n<\/td>\n

\n

2.50<\/p>\n<\/td>\n

\n

0.82<\/p>\n<\/td>\n

\n

10.88<\/p>\n<\/td>\n

\n

6.56<\/p>\n<\/td>\n<\/tr>\n

\n

12<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

58.00<\/p>\n<\/td>\n

\n

42.00<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

0.80<\/p>\n<\/td>\n

\n

6.94<\/p>\n<\/td>\n

\n

6.30<\/p>\n<\/td>\n<\/tr>\n

\n

13<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

58.00<\/p>\n<\/td>\n

\n

43.00<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

0.80<\/p>\n<\/td>\n

\n

6.98<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n<\/tr>\n

\n

15<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

58.00<\/p>\n<\/td>\n

\n

43.00<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

0.88<\/p>\n<\/td>\n

\n

8.12<\/p>\n<\/td>\n

\n

6.25<\/p>\n<\/td>\n<\/tr>\n

\n

17<\/p>\n<\/td>\n

\n

Fact<\/p>\n<\/td>\n

\n

60.00<\/p>\n<\/td>\n

\n

42.00<\/p>\n<\/td>\n

\n

3.50<\/p>\n<\/td>\n

\n

0.76<\/p>\n<\/td>\n

\n

10.86<\/p>\n<\/td>\n

\n

6.56<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table 2: Surface response models and parameters<\/strong><\/p>\n\n\n\n\n
\n

\u00a0<\/strong><\/p>\n

\u00a0<\/strong><\/p>\n<\/td>\n

\n

Regression model<\/strong><\/p>\n<\/td>\n

\n

Adjusted<\/strong><\/p>\n

R2<\/sup><\/strong><\/p>\n<\/td>\n

\n

Model P value<\/strong><\/p>\n<\/td>\n

\n

%<\/strong> C.V<\/strong><\/p>\n<\/td>\n

\n

Adequate Precision<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

K1<\/sub><\/p>\n

\u00a0<\/p>\n

Rs1,2<\/sub><\/p>\n

\u00a0<\/p>\n

T R1<\/sub><\/p>\n<\/td>\n

\n

+1.07-2.68E-005A-0.013B-0.022 C+0.042AB- 0.037A2<\/sup>-0.071B2<\/sup>-0.098C2<\/sup><\/p>\n

+12.14-0.79A+0.41C-2.39A2<\/sup>-1.01 C2<\/sup><\/p>\n

+4.41+1.48A-0.17B+0.28AB+0.76 A2<\/sup>+0.17B2<\/sup>–<\/p>\n

3.07\u00a0\u00a0\u00a0\u00a0 AB2<\/sup><\/p>\n<\/td>\n

\n

0.9110<\/p>\n

0.7209<\/p>\n

\u00a0<\/strong><\/p>\n

0.8455<\/p>\n<\/td>\n

\n

<0.0001<\/p>\n

<0.0001<\/p>\n

\u00a0<\/strong><\/p>\n

<0.0001<\/p>\n<\/td>\n

\n

3.61<\/p>\n

5.19<\/p>\n

\u00a0<\/strong><\/p>\n

8.52<\/p>\n<\/td>\n

\n

14.975<\/p>\n

11.601<\/p>\n

\u00a0<\/strong><\/p>\n

15.082<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Trail Chromatogram<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Blank Chromatogram.<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

\u00a0Figure 3: Standard Chromatogram<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 4: Optimized Chromatogram<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 5: Irinotecan loaded PEGylated liposomes using novel method supercritical fluid and condition optimized by Box\u2013Behnken design<\/strong>\u00a0<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Estimated response surfaces were generated under the following fixed conditions: pressure drop (a, d, g) at 22.5 MPa, flow rate (b, f, h) at 1.2 mL\/min, and temperature (c, e, i) at 22.5 \u00b0C, for the mean size, count, and EE,\u00a0respectively.\u00a0<\/strong><\/p>\n

Method Validation<\/strong><\/p>\n

System Suitability<\/strong><\/p>\n

Table 3: System condition parameters<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Injection<\/strong><\/p>\n<\/td>\n

\n

Retention time (min)<\/strong><\/p>\n<\/td>\n

\n

Area of analyte<\/strong><\/p>\n<\/td>\n

\n

Plate count<\/strong><\/p>\n<\/td>\n

\n

Tailing factor<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

2.158<\/p>\n<\/td>\n

\n

8370847<\/p>\n<\/td>\n

\n

2295<\/p>\n<\/td>\n

\n

1.22<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

2.142<\/p>\n<\/td>\n

\n

8372813<\/p>\n<\/td>\n

\n

2289<\/p>\n<\/td>\n

\n

1.21<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

2.167<\/p>\n<\/td>\n

\n

8376715<\/p>\n<\/td>\n

\n

2320<\/p>\n<\/td>\n

\n

1.22<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

2.156<\/p>\n<\/td>\n

\n

8371638<\/p>\n<\/td>\n

\n

2290<\/p>\n<\/td>\n

\n

1.23<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

2.154<\/p>\n<\/td>\n

\n

8371824<\/p>\n<\/td>\n

\n

2323<\/p>\n<\/td>\n

\n

1.21<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

2.159<\/p>\n<\/td>\n

\n

8372438<\/p>\n<\/td>\n

\n

2315<\/p>\n<\/td>\n

\n

1.24<\/p>\n<\/td>\n<\/tr>\n

\n

Mean<\/p>\n<\/td>\n

\n

2.156<\/p>\n<\/td>\n

\n

8372713<\/p>\n<\/td>\n

\n

2305.33<\/p>\n<\/td>\n

\n

1.22<\/p>\n<\/td>\n<\/tr>\n

\n

S D<\/strong><\/p>\n<\/td>\n

\n

0.0081<\/p>\n<\/td>\n

\n

2075.068<\/p>\n<\/td>\n

\n

15.68<\/p>\n<\/td>\n

\n

0.01169<\/p>\n<\/td>\n<\/tr>\n

\n

% RSD<\/strong><\/p>\n<\/td>\n

\n

0.375<\/p>\n<\/td>\n

\n

0.024<\/p>\n<\/td>\n

\n

0.680<\/p>\n<\/td>\n

\n

0.958<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Specificity<\/strong><\/p>\n

Table 4: Result of Specificity<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

\u00a0<\/p>\n<\/td>\n

\n

\u00a0<\/p>\n<\/td>\n

\n

Temp\/
intensity<\/strong><\/p>\n<\/td>\n

\n

Time<\/strong><\/p>\n<\/td>\n

\n

Analyte Rt (min)<\/strong><\/p>\n<\/td>\n

\n

Impurities Rt (min)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Physical degradation<\/p>\n<\/td>\n

\n

Heat stress<\/p>\n<\/td>\n

\n

105\u00ba C<\/p>\n<\/td>\n

\n

6 hrs<\/p>\n<\/td>\n

\n

2.15<\/p>\n<\/td>\n

\n

4.5<\/p>\n<\/td>\n<\/tr>\n

\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Chemical degradation<\/p>\n<\/td>\n

\n

Base degradation (1 N NaOH)<\/p>\n<\/td>\n

\n

60 \u00ba C<\/p>\n<\/td>\n

\n

60 min<\/p>\n<\/td>\n

\n

2.15<\/p>\n<\/td>\n

\n

1.9, 2.7<\/p>\n<\/td>\n<\/tr>\n

\n

Acid degradation (1 N HCl)<\/p>\n<\/td>\n

\n

60 \u00ba C<\/p>\n<\/td>\n

\n

60 min<\/p>\n<\/td>\n

\n

2.15<\/p>\n<\/td>\n

\n

4.6<\/p>\n<\/td>\n<\/tr>\n

\n

Oxidation degradation
(3% w\/v H2<\/sub>O2<\/sub>)<\/p>\n<\/td>\n

\n

60 \u00ba C<\/p>\n<\/td>\n

\n

60 min<\/p>\n<\/td>\n

\n

2.15<\/p>\n<\/td>\n

\n

—<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Linearity<\/strong><\/p>\n

Table 5:\u00a0 Result of Linearity<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Injection<\/strong><\/p>\n<\/td>\n

\n

Con. (%)<\/strong><\/p>\n<\/td>\n

\n

Con. of sample (\u00b5g\/ml)<\/strong><\/p>\n<\/td>\n

\n

Response<\/strong><\/p>\n<\/td>\n

\n

% Purity<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

25<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

220675<\/p>\n<\/td>\n

\n

98.8<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

50<\/p>\n<\/td>\n

\n

40<\/p>\n<\/td>\n

\n

439351<\/p>\n<\/td>\n

\n

98.8<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

75<\/p>\n<\/td>\n

\n

60<\/p>\n<\/td>\n

\n

635989<\/p>\n<\/td>\n

\n

98.89<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

80<\/p>\n<\/td>\n

\n

837084<\/p>\n<\/td>\n

\n

99.6<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

125<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

1025839<\/p>\n<\/td>\n

\n

99.8<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

150<\/p>\n<\/td>\n

\n

120<\/p>\n<\/td>\n

\n

1228245<\/p>\n<\/td>\n

\n

99.8<\/p>\n<\/td>\n<\/tr>\n

\n

Correlation coefficient not more than 1.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0
Slope: 1.016E \u00b1 05.<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n
\"\"<\/td>\n\n

Figure 6: Linearity Curve.<\/strong><\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Precision<\/strong><\/p>\n

Table 6: Result of Precision<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Injection<\/strong><\/p>\n<\/td>\n

\n

Peak area<\/strong><\/p>\n<\/td>\n

\n

% Assay<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

8345201<\/p>\n<\/td>\n

\n

99.69<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

8342215<\/p>\n<\/td>\n

\n

99.65<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

8343136<\/p>\n<\/td>\n

\n

99.66<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

8343217<\/p>\n<\/td>\n

\n

99.66<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

8343328<\/p>\n<\/td>\n

\n

99.67<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

8344307<\/p>\n<\/td>\n

\n

99.68<\/p>\n<\/td>\n<\/tr>\n

\n

Mean<\/p>\n<\/td>\n

\n

8343567<\/p>\n

\u00a0<\/p>\n<\/td>\n

\n

99.66<\/p>\n<\/td>\n<\/tr>\n

\n

Standard deviation<\/p>\n<\/td>\n

\n

1040.27<\/p>\n<\/td>\n

\n

0.01472<\/p>\n<\/td>\n<\/tr>\n

\n

%RSD<\/p>\n<\/td>\n

\n

0.012<\/p>\n<\/td>\n

\n

0.014<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Accuracy<\/strong><\/p>\n

Table 7: Accuracy results<\/strong><\/p>\n\n\n\n\n
\n

Compound<\/strong><\/p>\n<\/td>\n

\n

Concentration<\/strong><\/p>\n

(%)<\/strong><\/p>\n<\/td>\n

\n

Amount added<\/strong><\/p>\n

(mg)<\/strong><\/p>\n<\/td>\n

\n

Amount found<\/strong><\/p>\n

(mg)<\/strong><\/p>\n<\/td>\n

\n

Mean<\/strong><\/p>\n

Recovery (%)<\/strong><\/p>\n<\/td>\n

\n

SD<\/strong><\/p>\n<\/td>\n

\n

RSD<\/strong><\/p>\n

(%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Irinotecan<\/p>\n<\/td>\n

\n

80<\/p>\n

100<\/p>\n

120<\/p>\n<\/td>\n

\n

0.064<\/p>\n

0.080<\/p>\n

0.096<\/p>\n<\/td>\n

\n

0.06374<\/p>\n

0.07864<\/p>\n

0.09542<\/p>\n<\/td>\n

\n

\u00a0<\/strong><\/p>\n

99.1<\/p>\n<\/td>\n

\n

\u00a0<\/strong><\/p>\n

0.458<\/p>\n<\/td>\n

\n

\u00a0<\/strong><\/p>\n

0.462<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Result of Limit of Detection and Limit of Quantification<\/strong><\/p>\n

LOD and LOQ were calculated by the method which was based on the standard deviation of the response and slope of calibration curve at the level approximately Limit of Detection and Limit of Quantification was found to be 0.8ppm and 2ppm respectively.\u00a0<\/strong><\/p>\n

Robustness<\/strong><\/p>\n

Table 8: Results of robustness study<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Parameter<\/strong><\/p>\n<\/td>\n

\n

% RSD of % purity<\/strong><\/p>\n<\/td>\n

\n

Tailing factor<\/strong><\/p>\n<\/td>\n

\n

Plate count<\/strong><\/p>\n<\/td>\n

\n

% Purity<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Flow rate \u00b1 0.2<\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 (ml)<\/p>\n<\/td>\n

\n

1.2<\/p>\n<\/td>\n

\n

0.92<\/p>\n<\/td>\n

\n

1.3<\/p>\n<\/td>\n

\n

2345<\/p>\n<\/td>\n

\n

99.2<\/p>\n<\/td>\n<\/tr>\n

\n

0.8<\/p>\n<\/td>\n

\n

1.10<\/p>\n<\/td>\n

\n

1.4<\/p>\n<\/td>\n

\n

2549<\/p>\n<\/td>\n

\n

99.5<\/p>\n<\/td>\n<\/tr>\n

\n

Temperature\u00b1 5 \u00baC<\/p>\n<\/td>\n

\n

20<\/p>\n<\/td>\n

\n

0.64<\/p>\n<\/td>\n

\n

1.2<\/p>\n<\/td>\n

\n

2285<\/p>\n<\/td>\n

\n

99.1<\/p>\n<\/td>\n<\/tr>\n

\n

30<\/p>\n<\/td>\n

\n

0.70<\/p>\n<\/td>\n

\n

1.3<\/p>\n<\/td>\n

\n

2299<\/p>\n<\/td>\n

\n

99.3<\/p>\n<\/td>\n<\/tr>\n

\n

Mobile phase\u00a0\u00a0 composition \u00b1 5 ml<\/p>\n<\/td>\n

\n

45:55<\/p>\n<\/td>\n

\n

1.10<\/p>\n<\/td>\n

\n

1.3<\/p>\n<\/td>\n

\n

2472<\/p>\n<\/td>\n

\n

98.6<\/p>\n<\/td>\n<\/tr>\n

\n

35:65<\/p>\n<\/td>\n

\n

1.30<\/p>\n<\/td>\n

\n

1.2<\/p>\n<\/td>\n

\n

2283<\/p>\n<\/td>\n

\n

98.7<\/p>\n<\/td>\n<\/tr>\n

\n

pH of buffer \u00b1 0.2<\/p>\n<\/td>\n

\n

3.4<\/p>\n<\/td>\n

\n

0.83<\/p>\n<\/td>\n

\n

1.4<\/p>\n<\/td>\n

\n

2457<\/p>\n<\/td>\n

\n

99.5<\/p>\n<\/td>\n<\/tr>\n

\n

3.0<\/p>\n<\/td>\n

\n

1.01<\/p>\n<\/td>\n

\n

1.2<\/p>\n<\/td>\n

\n

2269<\/p>\n<\/td>\n

\n

99.8<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The developed method was then validated according to ICH guidelines and is suitable for estimating the bulk drug in marketed products. This method was applied to the injection Irnocam from the local market to determine the amount of irinotecan in bulk drug formulations. The marketed formulation was analyzed in triplicate, and it was found that the irinotecan content in Irnocam is 19.76 mg\/ml, whereas the claimed value was 20 mg\/ml. The sample purity in percentage was estimated as 98.2%. Since the result for % RSD was 0.132%, the developed RP-HPLC method is accurate and precise for the quantitative determination of irinotecan in bulk dosage forms.<\/p>\n

Conclusion<\/strong><\/p>\n

In the present study, the Box-Behnken design was used to develop a new technique for the estimation of irinotecan from marketed formulations using RP-HPLC. This technique utilized surface methodology to determine the impact of independent variables, namely % mobile phase ratio, flow rate, and triplet levels on chromatographic responses. The chromatographic resolution, tailing factor, and theoretical plate values were the responses measured. The developed technique was then validated according to ICH guidelines Q1(R2) and showed precision, accuracy, and speed. It is specifically designed for the quantitative determination of irinotecan in pharmaceutical formulations and hence suitable for routine analysis at laboratories.<\/p>\n

Acknowledgement<\/strong>\u00a0<\/strong><\/p>\n

We would like to thank our Respected Principal prof. Dr. M. Kumar, VMCP & VMRF (DU) for this overall support to carry out this research in the campus.\u00a0<\/strong><\/p>\n

Conflict of Interest<\/strong><\/p>\n

The author(s) do not have any conflict of interest.<\/p>\n

Data Availability Statement<\/strong><\/p>\n

This statement does not apply to this article.<\/p>\n

Ethics Statement<\/strong><\/p>\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval.<\/p>\n

Informed Consent Statement<\/strong><\/p>\n

This study did not involve human participants, and therefore, informed consent was not required.<\/p>\n

Clinical Trial Registration<\/strong><\/p>\n

This research does not involve any clinical trials\u00a0<\/strong><\/p>\n

Author Contributions<\/strong>\u00a0<\/strong><\/p>\n