7/30/2019 PORE SIZE OF HPLC REVERSED PHASE MATERIALS AND INSULIN QUANTIFICATION

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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, bothof 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 byexhibiting low LOD and LOQ values 80 pore sizecolumn (Table 3). It shows that 300 pore size columncan 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 80and 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 stablewith the mean 98.97% recovery compared to 97.11%

recovery for 80 pore size column is significantlydifferent (Table 4, p