This paper aimed to develop a simple, sensitive, and rapid chromatographic procedure for the simultaneous analysis of human insulin and its main decomposition product using isocratic RP-HPLC/UV. A column type RP-C18 (100 × 4.6？mm, 3？μm particle size, and pore size 130？？) was used. o-Nitrophenol was used as internal standard. The eluent consists of 62% KH2PO4 buffer (0.1？M), 26% ACN, and 12% MeOH. The final pH was adjusted to 3.1. The eluent was pumped at a flow rate of 1.0？mL/min and the effluent was monitored using DAD detector at 214？nm. The method produces a linear response over the concentration range of 0.0106 to 0.6810？mg/mL with detection limit of 0.0029？mg/mL. Considering the specifications of this method, the system was found to be suitable for rapid, direct routine analysis and stability studies of insulin. 1. Introduction Since insulin was discovered in 1921 by Frederick Banting and Charles Best, it has become one of the most thoroughly studied molecules in scientific history. Insulin is a pancreatic hormone that treats diabetes by controlling the amount of sugar in the blood. This peptide hormone is composed of 51 amino acids distributed between two peptides chains, one comprising 21 amino acids (chain A) and the other 30 amino acids (chain B). The two chains are joined together by two disulfide bonds between two cysteine residues, where a disulfide bond is the linkage bond between two sulfurs [1]. Like most proteins, insulin is not stable in aqueous solutions. Its degradation mainly goes through hydrolytic reactions (deamidation) or polymerization mechanisms. Deamidation at asparagine amino acid in chain A of insulin (A21) is considered as the main degradation product of insulin at low pH values, while in neutral or alkaline medium, deamidation at asparagine B3 occurs [2]. Polymerization may also occur in neutral and alkaline medium through transamidation reactions [3]. As a diabetes medicine, the purity of recombinant insulin must be more than 98%, with the insulin-related compound A21 desamido insulin being less than 2% [4, 5]. Most commercial insulin preparations are containing low concentrations of phenol and/or m-cresol as preservatives from bacterial contaminations. Separation of insulin and its related compounds is a difficult mission due to the insignificant differences in chemical structures. Many immune and nonimmune methods have been reported for determination of human insulin. Radioimmunoassay [6–9], enzyme immunoassay [10–13], luminescent immunoassay [14], capillary electrophoresis [15–17], and high performance liquid
References
[1]
J. Espinal, Understanding Insulin Action: Principles and Molecular Mechanisms, Ellis Horwood, Chichester, UK, 1989.
[2]
J. Brange, L. Langkjaer, S. Havelund, and A. Volund, “Chemical stability of insulin. 1. Hydrolytic degradation during storage of pharmaceutical preparations,” Pharmaceutical Research, vol. 9, no. 6, pp. 715–726, 1992.
[3]
J. Brange, S. Havelund, and P. Hougaard, “Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during storage of pharmaceutical preparations,” Pharmaceutical Research, vol. 9, no. 6, pp. 727–734, 1992.
[4]
British Pharmacopoeia, H.M. Stationary Office, London, UK, 2012.
[5]
USP 35-NF 30, Philadelphia, Rockville, MD, USA, 2012.
[6]
M. U. Jars, A. Hvass, and D. Waaben, “Insulin aspart(aspB28 human insulin) derivatives formed in pharmaceutical solutions,” Pharmaceutical Research, vol. 19, no. 5, pp. 621–628, 2002.
[7]
R. S. Yalow and S. A. Berson, “Assay of plasma insulin in human subjects by immunological methods,” Nature, vol. 184, no. 4699, pp. 1648–1649, 1959.
[8]
L. E. M. Miles and C. N. Hales, “Labelled antibodies and immunological assay systems,” Nature, vol. 219, no. 5150, pp. 186–189, 1968.
[9]
J. F. Dezier, A. M. Jouanolle, M. Le Reun, and J. Y. Poirier, “Comparison of two methods to measure microalbuminuria: immunoephelemetry and radioimmunology,” Annales de Biologie Clinique, vol. 45, no. 1, pp. 78–84, 1987.
[10]
H. V. Webster, A. J. Bone, K. A. Webster, and T. J. Wilkin, “Comparison of an enzyme-linked immunosorbent assay (ELISA) with a radioimmunoassay (RIA) for the measurement of rat insulin,” Journal of Immunological Methods, vol. 134, no. 1, pp. 95–100, 1990.
[11]
L. Andersen, B. Dinesen, P. N. Jorgensen, F. Poulsen, and M. E. Roder, “Enzyme immunoassay for intact human insulin in serum or plasma,” Clinical Chemistry, vol. 39, no. 4, pp. 578–582, 1993.
[12]
H. Shen, C. A. Aspinwal, and R. T. Kennedy, “Dual microcolumn immunoassay applied to determination of insulin secretion from single islets of Langerhans and insulin in serum,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 689, no. 2, pp. 295–303, 1997.
[13]
K. Zaitsu, Y. Kimura, Y. Ohba et al., “Heme-undecapeptide labeling on insulin for the immunoassay of insulin with chemiluminescence detection,” Analytical Sciences, vol. 15, no. 9, pp. 871–878, 1999.
[14]
C. Arcelloni, L. Falqui, S. Martinenghi, A. E. Pontiroli, and R. Paroni, “Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms,” Electrophoresis, vol. 19, no. 8-9, pp. 1475–1477, 1998.
[15]
C. Yomota, Y. Matsumoto, S. Okada, Y. Hayashi, and R. Matsuda, “Discrimination limit for purity test of human insulin by capillary electrophoresis,” Journal of Chromatography B: Biomedical Applications, vol. 703, no. 1-2, pp. 139–145, 1997.
[16]
W. Tong and S. Edward, “Determination of insulin in single pancreatic cells by capillary electrophoresis and laser-induced native fluorescence,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 685, no. 1, pp. 35–40, 1996.
[17]
I. German and T. R. Kennedy, “Rapid simultaneous determination of glucagon and insulin by capillary electrophoresis immunoassays,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 742, no. 2, pp. 353–362, 2000.
[18]
A. Hvass and B. Skelbaek-Pedersen, “Determination of protamine peptides in insulin drug products using reversed phase high performance liquid chromatography,” Journal of Pharmaceutical and Biomedical Analysis, vol. 37, no. 3, pp. 551–557, 2005.
[19]
C. Toriumi and K. Imai, “Determination of insulin in a single islet of Langerhans by high-performance liquid chromatography with fluorescence detection,” Analytical Chemistry, vol. 74, no. 10, pp. 2321–2327, 2002.
[20]
A. Pastore, S. Bernardini, L. Dello Strologo, G. Rizzoni, C. Cortese, and G. Federici, “Simultaneous determination of inulin and p-aminohippuric acid in plasma and urine by reversed-phase high-performance liquid chromatography,” Journal of Chromatography B: Biomedical Sciences and Applications, vol. 751, no. 1, pp. 187–191, 2001.
[21]
J. V. O'Connor, “Chromatography of recombinant proteins,” Developments in Biological Standardization, vol. 97, pp. 39–47, 1999.
[22]
G. Khaksa, K. Nalini, M. Bhat, and N. Udupa, “High-performance liquid chromatographic determination of insulin in rat and human plasma,” Analytical Biochemistry, vol. 260, no. 1, pp. 92–95, 1998.
[23]
B. V. Fisher, D. Smith, and J. Pharm, “HPLC as a replacement for the animal response assays for insulin,” Journal of Pharmaceutical and Biomedical Analysis, vol. 4, pp. 377–387, 1986.
[24]
N. Abu Heshmeh, A. Sallam, Y. Mater, and M. Alawi, “A new method for determination of human insulin in aqueous injections,” International Journal of Research in Pharmaceutical and Biomedical Sciences, vol. 4, no. 2, pp. 428–435, 2013.
[25]
B. A. Moussa, F. Farouk, and H. M. E. Azzazy, “A validated RP-HPLC method for the determination of recombinant human insulin in bulk and pharmaceutical dosage form,” E-Journal of Chemistry, vol. 7, no. 1, pp. S449–S457, 2010.
[26]
A. Shrivastava and V. B. Gupta, “Methods for the determination of limit of detection and limit of quantitation of the analytical methods,” Chronicles of Young Scientists, vol. 2, pp. 21–25, 2011.
