Methods
Chemicals
İnsulin (Humulin R, Lilly), PP bags (Polifarma) and PVC infusion sets (Sapphire) were obtained from a pharmaceutical warehouse. All the chemicals were analytical grade on preparing buffers and solutions. Acetonitrile was purchased from Merck (Darmstadt, Germany). Ultrapure water was purchased from the Milli-Q water system (Barnstead, USA).
Analytical Conditions
Chromatographic methods for insulin are included in the Turkish Pharmacopoeia [20]. In this study, insulin was analyzed by reverse phase high performance liquid chromatography (RP-HPLC) which the assay was validated according to international guidelines [21]. Briefly, the LC system (Agilent 1220 Infinity II) consisted of a gradient pump, a degasser, and a UV detector (Thermo Separation Products, USA). The separations were achieved by a C18 Phenomenex column (250 x 4.6 mm, 100Å, 5 µm i.d.) at room temperature. The mobile phase was acetonitrile:phosphate buffer (50:50 v/v, pH:3.0, 10 mM) and, solutions delivered at a flow rate of 1mL/min which gave the optimum chromatographic conditions. Injection volume was 20 μL and UV detection was performed at 214 nm.
Sample Preparation
In the study, solutions containing 1 IU/mL concentration (n = 6) were prepared by adding 100 IU insulin to PP bags containing 100 ml of 0.9% NaCl solution. Also, PVC infusion sets were used. In the experimental setup established in accordance with clinical practice, the infusion rate of the solutions was determined as 2 IU/hour through the infusion pump. 600 µL samples were taken from the bag at the beginning of the infusion (hour 0), as well as from the bag and at the end tip of the infusion set at the end of the 1st, 2nd, 4th, 12th and 24th hours. In each sample taken, insulin quantification was made. This experimental procedure was carried out simultaneously with 6 separate insulin solutions prepared at room temperature. As a result of the analysis of the samples taken from each bag and set at specified times, the adsorption rates of insulin to PP bag and PVC infusion set were evaluated.
Also, to evaluate the stability of insulin solutions at room temperature during infusion, the analysis results of samples taken from insulin solutions stored at room (+25°C) and refrigerator (+4°C) temperatures at 0, 4, 12 and 24 hours were compared.
Statistical analysis
Statistical evaluation was performed using Student’s t-test and thep values ≤0.05 were considered statistically significant. In the beginning (0 hours), insulin solutions in PP bags are the control group.