pore size of hplc reversed phase materials and insulin quantification.doc

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

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Nurjaya Sumiran, Mohammad Tarmizi Mohd Mokhtar, Aminah Kadir and Wong Tin

Wui*,

Particle Design Research Group, Faculty of Pharmacy, Non-Destructive Biomedical andPharmaceutical Research Centre,Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia.

*[email protected] Conference and Exhibition on Pharmaceutical, Nutraceutical andCosmeceutical 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 peptidechains 21 and 30 amino acid residues acknowledged as the A-chain and B-chainrespectively, attached together by disulfide bonds.1-2

Figure 1 Insulin Schematic Diagram

The quantification of insulin was primarily by means of reversed-phase high performanceliquid chromatography with an extensive range of parameters.3-7 Method of high

performance liquid chromatography assay for insulin presented in the United StatesPharmacopoiea uses buffers as mobile phases that predisposed the HPLC system tocontamination of salt precipitations and normally demanded long run times.7-8 Wemodified a reversed-phase high performance liquid chromatography to eliminate the risksof contamination from buffers and reduce the run time. In a development ofpharmaceuticals, analytical techniques and the determination of their qualitycharacteristics have to be validated.9 Researchers have reported vast methods that weredeveloped and validated for quantification of insulin in various solvent utilizing different


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pore size reversed-phase materials. Therefore, we proceed to report the suitability of twodifferent 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 liquidchromatography (Agilent 1100 Series, USA) The run proceeded with a gradient elution of(A) 0.03 % trifluoroacetic acid (TFA) in 90 % deionized water (H 2O) with 10 % of acetonitrile(ACN) and (B) 0.03 % TFA in 10 % H2O with 90 % ACN at 80:20 (A:B) ratio over 5 minfollowed by isocratic run at 20:80 ratio over 10 min. The flow rate, column temperature andsample volume were 0.5 ml/min, 20C 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 and0.01M 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 realconditions.

3.3.2 Linearity

Linearity is determined by calculating the regression line using a mathematical treatmentof 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 isapplied to multiple sampling of a homogenous sample.


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3.3.5 Sensitivity

LOD is the minimum concentration of analyzed substance in the sample that can bedetected; meanwhile LOQ is the minimum concentration of analyzed substance that canbe 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. Theacceptance criterion was 2% for the percent relative standard deviation (%RSD) for thepeak area and retention times of the sample.

3.3.7 Stability

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

4. Results

(a)

80, RT = 8.06 0.02(b)

300 , RT = 8.74 0.01

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

mAU

mAU


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Table 1 Linearity and between-day precision of calibration for insulin by using (a) 80 and (b) 300 pore size column.

Regression Day 1 Day 2 Day 3 Mean of 3 Standard Between-day

equation1

, (n = 3) (n = 3) (n = 3) consecutive deviation precisiony = mx + c days (SD%) (CV%)

(a) m 33228.49 29119.84 34302.68 32217.00 2735.47 8.49

c -73.76 -128.12 227.83 8.65 191.75 2216.78

R2 0.9998 0.9998 0.9995 0.9997 0.00 0.02

(b) m 30990.32 35159.36 32395.39 32848.36 2121.11 6.46

c -146.43 53.74 -1.88 -31.52 103.32 -327.77

R2 0.9994 0.9998 0.9996 0.9996 0.00 0.021

1 Linear regression analysis with regression equation y = mx + c, in which y = area underthe curve, x = concentration in mg/ml, m = slope, c = intercept and R2 = correlationcoefficient.

Table 2 Accuracy and precision of insulin by using (a) 80 and (b) 300 pore sizecolumn.

Standard Between-day Within-daysolution(mg/ml) Recovery Accuracy Precision Recovery Accuracy Precision

(mg/ml) (%) CV (%) (mg/ml) (%) CV (%)

(a) 0.1 0.098 98.02 5.876 0.093 93.37 1.6700.2 0.201 100.28 1.449 0.197 98.33 2.8380.3 0.301 100.35 0.083 0.288 96.11 4.8390.4 0.405 101.24 0.255 0.391 97.63 3.2820.5 0.496 99.16 0.596 0.470 94.02 1.179

(b) 0.1 0.099 98.63 4.643 0.092 93.83 2.2250.2 0.198 98.77 4.273 0.201 100.40 2.2290.3 0.303 101.14 1.345 0.290 99.25 1.8240.4 0.403 100.73 0.688 0 .402 100.50 1.4910.5 0.498 99.51 1.197 0.471 95.85 0.358


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

LOD LOQ

80 0.36499 1.10603300 0.02765 0.08379

Table 4 Stability of insulin by using 80 and 300 pore size column.

Concentration 80 (area, mAU) 300 (area, mAU)(mg/ml) 0h 24h 0h 24h

0.1 2433.17142.06 2371.47117.85 2636.252.91 2612.376.76

0.2 5239.43175.67 5082.57182.99 5583.925.60 5539.1517.630.3 7880.38165.02 7641.66171.13 8321.3120.35 8223.588.730.4 10398.53129.07 10098.83151.80 10984.4511.86 10876.5014.000.5 13181.88183.87 12780.75191.40 13832.3531.28 13652.5312.33

Concentration Stability (%)(mg/ml) 80 300

0.1 97.51 1.09 99.09 0.250.2 97.00 0.64 99.20 0.300.3 96.97 0.35 98.83 0.280.4 97.12 0.92 99.02 0.140.5 96.96 0.62 98.70 0.22

Table 5 System suitability of insulin by using 80 and 300 pore size column.

Concentration 80 300 (0.3 mg/ml) Retention time Peak area Retention time Peak area

(tR, min) (mAU) (tR, min)(mAUl)

Mean (n=24) 8.06 10156.62 8.74 10427.74SD 0.0422 419.80 0.0240 444.32%RSD 0.5239 4.133 0.2749 4.261


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5. Discussion

A modified reversed-phase high performance liquid chromatography method forquantification of bovine insulin from polymeric nano spray-dried particles wassuccessfully developed from previous method for two different pore size materialcolumn. While assessing the suitability of both parameters on the quantification ofinsulin, the difference was chosen as the robustness of validation since it fits as the smallvariation factor of system parameters. The chromatograms of bovine insulin were elutedat 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 timeand in agreement with the previous report. 10 The absence of effect on retention timesuggests that this analytical procedure is robust with respect to different pore sizecolumn.

The calibration curve constructed was evaluated on its correlation coefficient. Thepeak 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 of0.999.9 Thus, it were demonstrated that both of the columns signify a good linearity ofanalytical method (Table 1).

Accuracy and precision of the samples were calculated for between-day andwithin-day. The results were calculated using the equation of observed meanconcentration over estimated concentration for accuracy and standard deviation overmean for precision. Both columns demonstrate a full recovery of insulin over the rangeof 0.1 -0.5mg/ml of insulin (Table 2). The results depicted the close proximity betweenthe obtained and the real results hence, it can be concluded that the analytical method isaccurate.

The column with pore size 300 is more sensitive by exhibiting low LOD andLOQ values with 0.02765 and 0.08379, respectively compared to 80 pore size columnwith LOD and LOQ values of 0.36499 and 1.10603 respectively (Table 3). It shows that300 pore size column can detect lowest amount of insulin and a reliable lower amountof 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 inTable 5. The retention time and peak area %RSD of both 80 and 300 pore sizecolumns are all under the 2% acceptance criterion therefore both are suitable for insulinquantification.

The stability of insulin was investigated by measuring the concentration changesin the standard samples over time. It was assessed by subjecting the insulin samples atroom temperature, 25oC for 0 and 24h. The results showed that analytical system with the


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300 pore size column is more stable with the mean 98.97% recovery compared to97.11% recovery for 80 pore size column is significantly different (Table 4, p


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diameters. J Chrom 360: 353-369

pore size of hplc reversed phase materials and insulin quantification.doc

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