Non Destructive Biomedical and Pharmaceutical Research Centre (NDBPRC)

Particle Design Research Group

Mohammad Tarmizi Mohd Mokhtar, Nurjaya Sumiran, Aminah Kadir, Syed Mohd Al-Azi Syed Othman 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

PORE SIZE OF HPLC REVERSED PHASE MATERIALS AND INSULIN QUANTIFICATION

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

Figure 1 Schematic Diagram of Insulin.

The quantification of insulin is primarily conducted by means of reversed-phase high performance liquid chromatography with an extensive range of parameters.3-7 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 In the development of pharmaceuticals, analytical techniques and the determination of their quality characteristics have to be validated.9 Researchers have reported a vast range of methods that were developed and validated for quantification of insulin. In this study, we report the suitability of reverse phase materials with two different pore sizes for assay of insulin.

RESULTS
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DISCUSSION
The different pore size brought about little or no effect on the retention time (Figure 2) 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, 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 estimated 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 80 pore size column (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).

Figure 2 HPLC chromatogram of insulin determined by using columns of pore size (a) 80 and (b) 300

OBJECTIVE
Validation of suitability of reversed phase materials with different pore sizes on insulin quantification.

RESEARCH METHODOLOGY
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, 20C and 20 l respectively with UV detector set at 215 nm. 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 or 0.5mg/ml. Validation Robustness Stability with respect to small variations of the system parameters possible under real conditions. Linearity Linearity is determined by calculating the regression line using a mathematical treatment of the results versus analyte concentration. Accuracy Measure of the closeness of experimental value to the true value. Precision The degree of agreement among individual test results obtained when the method is applied to multiple sampling of a homogenous sample.
REFERENCES
Wong TW (2010) J Drug Targeting 18(2): 79-92. Leobandung W (2002). J Control Release 80: 357-363. Sarmento B (2006). Biomed Chromatogr 20:898-903. pshtein NA (2004). Pharm Chem J 38(4): 212-228.

CONCLUSION
Analytical procedure is proven robust with pore size variation. Both columns depicted acceptable linearity, accuracy, and precision. However, 300 pore size column exhibits better sensitivity and stability although both system suitability values well under the acceptance criterion.

Wong TW (2009) Recent Pat Drug Deliv Formulat 3(1): 8-25. Cheng K, Lim LY (2004).Drug Dev Ind Pharm 30(4): 359-367. Sonaje K (2010). Biomaterials 31: 3384-3394. Xu X, Fu Y, Hu H, Duan Y, Zhang Z (2006) J Pharm Biomed Anal Insulin/Official Monographs, in: United States Pharmacoeia 24/National Formulary 19, United States Pharmacopeial Convention, Inc., Rockville, MD, 1999, pp. 880-882. Sands BW (1986) J Chrom 360: 353-369.