Nurjaya Sumiran, Mohammad Tarmizi Mohd Mokhtar, Aminah Kadir and Wong Tin Wui*, Particle Design Research

Group, Faculty of Pharmacy, Non-Destructive Biomedical and Pharmaceutical Research Centre, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia. *wongtinwui@salam.uitm.edu.my
International Conference and Exhibition on Pharmaceutical, Nutraceutical and Cosmeceutical Technology, Nov, 2012.

PORE SIZE OF HPLC REVERSED PHASE MATERIALS AND INSULIN QUANTIFICATION

1.0 Introduction

Composed of 51 amino acid residues with a molecular weight of 5808 Da, insulin is represented by the molecular formula C257H383N65O77S6 exists in the form of two peptide chains 21 and 30 amino acid residues acknowledged as the A-chain and B-chain respectively, attached together by disulfide bonds. 1-2

Figure 1 Insulin Schematic Diagram The quantification of insulin was primarily by means of reversed-phase high performance liquid chromatography with an extensive range of parameters.3-7 Method of high performance liquid chromatography assay for insulin presented in the United States Pharmacopoiea uses buffers as mobile phases that predisposed the HPLC system to contamination of salt precipitations and normally demanded long run times.7-8 We modified a reversed-phase high performance liquid chromatography to eliminate the risks of contamination from buffers and reduce the run time. In a development of pharmaceuticals, analytical techniques and the determination of their quality characteristics have to be validated.9 Researchers have reported vast methods that were developed and validated for quantification of insulin in various solvent utilizing different

pore size reversed-phase materials. Therefore, we proceed to report the suitability of two different pore size materials for the determination of insulin by validation process. 2.0 Objective Validation of two different pore size reversed phase materials on insulin quantification method. 3.0 Research methodology

3.1 HPLC Instrumentation Chromatographic analysis was performed with a reversed-phase high performance liquid chromatography (Agilent 1100 Series, USA) The run proceeded with a gradient elution of
(A) 0.03 % trifluoroacetic acid (TFA) in 90 % deionized water (H2O) with 10 % of acetonitrile (ACN) and (B) 0.03 % TFA in 10 % H2O with 90 % ACN at 80:20 (A:B) ratio over 5 min followed by isocratic run at 20:80 ratio over 10 min. The flow rate, column temperature and sample volume were 0.5 ml/min, 20 C and 20 l respectively with UV detector set at 215 nm.

3.2 Standards and calibration curves Bovine insulin was dissolved in a solvent, mixture of USP phosphate buffer pH 6.8 and 0.01M HCl, to obtain a concentration of 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml. 3.3 Validation 3.3.1 Robustness Stability with respect to small variations of the system parameters possible under real conditions. 3.3.2 Linearity

Linearity is determined by calculating the regression line using a mathematical treatment of the results versus analyte concentration. 3.3.3 Accuracy

Measure of the closeness of experimental value to the true value. 3.3.4 Precision

The degree of agreement among individual test results obtained when the method is applied to multiple sampling of a homogenous sample.

3.3.5

Sensitivity

LOD is the minimum concentration of analyzed substance in the sample that can be detected; meanwhile LOQ is the minimum concentration of analyzed substance that can be determined quantitatively at an acceptable precision and accuracy. 3.3.6 System suitability

The system suitability was assessed by triplicate analyses of standard insulin sample. The acceptance criterion was 2% for the percent relative standard deviation (%RSD) for the peak area and retention times of the sample. 3.3.7 Stability

Stability study was performed by measuring the changes in concentration of insulin standard samples that were stored at 4C overnight. 4. Results (a)

mAU

80, RT = 8.06 0.02 (b)

mAU

300 , RT = 8.74 0.01 Fig.2 HPLC chromatogram of insulin by using different column of pore size (a) 80 and (b) 300 .

Table 1 Linearity and between-day precision of calibration for insulin by using (a) 80 and (b) 300 pore size column. Regression equation1, y = mx + c (a) m c R2 (b) m c R2
1

Day 1 (n = 3)

Day 2 (n = 3)

Day 3 (n = 3)

Mean of 3 consecutive days

Standard deviation (SD%) 2735.47 191.75 0.00 2121.11 103.32 0.00

Between-day precision (CV%) 8.49 2216.78 0.02 6.46 -327.77 0.021

33228.49 29119.84 34302.68 32217.00 -73.76 0.9998 -128.12 0.9998 227.83 0.9995 8.65 0.9997

30990.32 35159.36 32395.39 32848.36 -146.43 0.9994 53.74 0.9998 -1.88 0.9996 -31.52 0.9996

Linear regression analysis with regression equation y = mx + c, in which y = area under the curve, x = concentration in mg/ml, m = slope, c = intercept and R 2 = correlation coefficient. Table 2 Accuracy and precision of insulin by using (a) 80 and (b) 300 pore size column. Standard solution (mg/ml) (a) 0.1 0.2 0.3 0.4 0.5 (b) 0.1 0.2 0.3 0.4 0.5 Between-day Recovery (mg/ml) 0.098 0.201 0.301 0.405 0.496 0.099 0.198 0.303 0.403 0.498 Accuracy Precision (%) CV (%) 98.02 100.28 100.35 101.24 99.16 98.63 98.77 101.14 100.73 99.51 5.876 1.449 0.083 0.255 0.596 4.643 4.273 1.345 0.688 1.197 Within-day Recovery (mg/ml) 0.093 0.197 0.288 0.391 0.470 0.092 0.201 0.290 0.402 0.471 Accuracy (%) 93.37 98.33 96.11 97.63 94.02 93.83 100.40 99.25 100.50 95.85 Precision CV (%) 1.670 2.838 4.839 3.282 1.179 2.225 2.229 1.824 1.491 0.358

Table 3 Limit of detection (LOD) and limit of quantitation (LOQ) of insulin by using 80 and 300 pore size column. LOD 80 300 0.36499 0.02765 LOQ 1.10603 0.08379

Table 4 Stability of insulin by using 80 and 300 pore size column. Concentration (mg/ml) 0.1 0.2 0.3 0.4 0.5 Concentration (mg/ml) 0.1 0.2 0.3 0.4 0.5 80 (area, mAU) 0h 24h 2433.17142.06 2371.47117.85 5239.43175.67 5082.57182.99 7880.38165.02 7641.66171.13 10398.53129.07 10098.83151.80 13181.88183.87 12780.75191.40 Stability (%) 80 300 97.51 1.09 97.00 0.64 96.97 0.35 97.12 0.92 96.96 0.62 99.09 0.25 99.20 0.30 98.83 0.28 99.02 0.14 98.70 0.22 300 (area, mAU) 0h 24h 2636.252.91 5583.925.60 8321.3120.35 10984.4511.86 13832.3531.28 2612.376.76 5539.1517.63 8223.588.73 10876.5014.00 13652.5312.33

Table 5 System suitability of insulin by using 80 and 300 pore size column. Concentration (0.3 mg/ml) (mAUl) Mean (n=24) SD %RSD 8.06 0.0422 0.5239 10156.62 419.80 4.133 8.74 0.0240 0.2749 10427.74 444.32 4.261 80 Retention time Peak area (tR, min) (mAU) 300 Retention time Peak area (tR, min)

5. Discussion A modified reversed-phase high performance liquid chromatography method for quantification of bovine insulin from polymeric nano spray-dried particles was successfully developed from previous method for two different pore size material column. While assessing the suitability of both parameters on the quantification of insulin, the difference was chosen as the robustness of validation since it fits as the small variation factor of system parameters. The chromatograms of bovine insulin were eluted at 8.06 0.02 and 8.74 0.01 min for 80 and 300 of column pore size respectively (Figure 2). The different pore size brought about little or no effect on the retention time and in agreement with the previous report. 10 The absence of effect on retention time suggests that this analytical procedure is robust with respect to different pore size column. The calibration curve constructed was evaluated on its correlation coefficient. The peak area of the insulin appeared linear in the range of 0.1-0.5 mg/ml for both columns (Table 1). The correlation coefficients exceeded the proposed recommended value of 0.999.9 Thus, it were demonstrated that both of the columns signify a good linearity of analytical method (Table 1). Accuracy and precision of the samples were calculated for between-day and within-day. The results were calculated using the equation of observed mean concentration over estimated concentration for accuracy and standard deviation over mean for precision. Both columns demonstrate a full recovery of insulin over the range of 0.1 -0.5mg/ml of insulin (Table 2). The results depicted the close proximity between the obtained and the real results hence, it can be concluded that the analytical method is accurate. The column with pore size 300 is more sensitive by exhibiting low LOD and LOQ values with 0.02765 and 0.08379, respectively compared to 80 pore size column with LOD and LOQ values of 0.36499 and 1.10603 respectively (Table 3). It shows that 300 pore size column can detect lowest amount of insulin and a reliable lower amount of insulin for quantification. The system suitability was studied by using 0.3mg/ml of insulin concentration. The mean, SD and %RSD of retention time and peak area were calculated as shown in Table 5. The retention time and peak area %RSD of both 80 and 300 pore size columns are all under the 2% acceptance criterion therefore both are suitable for insulin quantification. The stability of insulin was investigated by measuring the concentration changes in the standard samples over time. It was assessed by subjecting the insulin samples at room temperature, 25oC for 0 and 24h. The results showed that analytical system with the

300 pore size column is more stable with the mean 98.97% recovery compared to 97.11% recovery for 80 pore size column is significantly different (Table 4, p<0.05). Conclusion A quantification method of high performance liquid chromatography for bovine insulin was successfully modified and validated for two respective different pore size materials of 80 and 300 columns using a set of common analytical parameters of standard procedure; robustness, linearity, accuracy, precision, specificity, sensitivity, system suitability, stability and specificity. 15The analytical procedure is proven robust with pore size variation. Both columns depicted acceptable linearity, accuracy, and precision. However, 300 pore size column exhibit better sensitivity and stability although both system suitability values well under the acceptance criterion. References 1. 2. 3. 4. 5. 6 Wong TW (2010) Design of oral insulin delivery systems. J Drug Targeting 18(2): 7992. Wong TW (2009) Chitosan and its use in design of insulin delivery system. Recent Pat Drug Deliv Formulat 3(1): 8-25. Cheng K, Lim LY (2004) Insulin-loaded calcium pectinate nanoparticles: effects of pectin molecular weight and formulation pH. Drug Dev Ind Pharm 30(4): 359-367. Leobandung W (2002) Preparation of stable insulin-loaded nanospheres of poly(ethylene glycol) macromers and N-isopropyl acrylamide. J Control Release 80: 357-363. Sonaje K (2010) Enteric-coated capsules filled with freeze-dried chitosan/poly(glutamic acid) nanoparticles for oral insulin delivery. Biomaterials 31: 3384-3394. Xu X, Fu Y, Hu H, Duan Y, Zhang Z (2006) Quantitative determination of insulin entrapment efficiency in triblock copolymeric nanopaerticles by high-performance liquid chromatography. J Pharm Biomed Anal Sarmento B (2006) Development and validation of a rapid reversed-phase hplc method for the determination of insulin from nanoparticulate systems. Biomed Chromatogr 20:898-903 Insulin/Official Monographs, in: United States Pharmacoeia 24/National Formulary 19, United States Pharmacopeial Convention, Inc., Rockville, MD, 1999, pp. 880-882 pshtein NA (2004) Structure of chemical compounds, methods of analysis and process control: validation of hplc techniques for pharmaceutical analysis. Pharm Chem J 38(4): 212-228 Sands BW (1986) Characterization of bonded-phase silica gels with difference pore

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