TABLE 1: Intra and inter-day accuracy and precision of ibuprofen, ibuprofen 2-hydroxyethyl ester, and codrug


QC Samples Intra-day validation, n = 6 Compound


Ibuprofen


Ibuprofen 2-hydroxyethyl ester


Codrug


160 240 15 30


160 240 15 30


160 240


Inter-day validation, n = 18 Compound


Ibuprofen


Ibuprofen 2-hydroxyethyl ester


Codrug


160 240 15 30


160 240 15 30


160 240


Nominal concentration (µg mL-1 15 30


Nominal concentration (µg mL-1 15 30


) Mean Found concentration (µg mL-1 14.8 30.1


160.6 240.7 15.1 30.2


161.2 239.5 15.1 30.2


162.0 240.9


) Mean Found concentration (µg mL-1 14.7 30.2


160.2 240.5 15.0 30.2


160.3 239.2 15.1 30.1


161.3 239.9


) Standard deviation (µg mL-1 0.14 0.16 1.48 0.61


0.063 0.09 1.41 0.32 0.13 0.19 1.33 1.19


) RSD (%) Accuracy % 0.97 0.53 0.92 0.26 0.42 0.31 0.87 0.13 0.84 0.62 0.82 0.49


98.6


100.4 100.4 100.3 100.4 100.8 100.8 99.8


100.9 100.5 101.3 100.4


) Standard deviation (µg mL-1 0.11 0.13 1.19 1.37 0.07 0.11 1.22 0.95 0.09 0.12 0.94 0.95


) RSD (%) Accuracy % 0.75 0.43 0.75 0.57 0.46 0.36 0.76 0.39 0.62 0.41 0.58 0.39


98.2


100.6 100.1 100.2 100.3 100.6 100.2 99.7


100.8 100.4 100.8 99.9


The coefficient of correlation of the linear regression equation R2 for the calibration curves was calculated by the method of least square. Limit of detection (LOD) and limit of quantification (LOQ) are calculated according to signal-to-noise ratio. The acceptable determination limits for estimating LOD and LOQ are a signal-to-noise ratios of 3:1 and 10:1, respectively.


Selectivity and recovery


Selectivity of the method was evaluated and confirmed by analysis of samples’ chromatograms containing IBP, IBP 2-hydroxyethyl ester, and codrug, spiked into supernatant obtained from the human plasma as well as rat liver homogenate. The percentage of recovery of IBP, IBP 2-hydroxyethyl ester, and codrug from human plasma and rat liver homogenate were calculated by comparing the theoretical concentration to that recovered from spiked samples in a solution of equal volume of human plasma or rat liver homogenate, and methanol at two concentration levels, 25 and 100 µg mL-1


.


Accuracy and precision Accuracy of the analytical method was obtained and confirmed by the replicate analysis of four control samples at concentration level of 15, 30, 160, and 240 µg mL-1


for each of the aforementioned


compounds. The samples were injected daily for three days. On the other hand, the intra- and inter-day precision were determined by replicate injection (n = 6) for four different concentrations (15, 30, 160, and 240 µg mL-1 three consecutive days.


038 MEDLAB MAGAZINE ISSUE 1 2012


Freeze-Thaw Stability The freeze–thaw stability of IBP, IBP 2-hydroxyethyl ester, and codrug in both human plasma and rat liver homogenate was evaluated at three concentrations 25, 100, and 160 µg mL-1


over 60 days. These samples


were analyzed in triplicate without being frozen at first and then stored at −15°C, and thawed at room temperature (25°C ±1).


RESULTS AND DISCUSSION Selectivity Figure 2 (a-c) shows chromatograms for IBP, IBP 2-hydroxyethyl ester, and codrug mixtures in aqueous solution, human plasma, and rat liver homogenate respectively. It is evident that there is excellent resolution and lack of interference between the peak of IBP and the peak of its ester or codrug in aqueous solution as well as biological samples. This is important because IBP is the final chemical and enzymatic degradation product from the ester and codrug. This can be further verified by the resolution of the peaks (Rs


) values, where Rs for IBP and its ester, while for IBP ester and codrug, Rs


of 2.0 was obtained had a value


of 6.9. Thus, a chromatographic run time of 10 min was sufficient for sample analysis; allowing the analysis of three components with variant physicochemical properties over a short period of time; saving time and solvents, thus cost effectiveness is enhanced.


) of IBP, IBP 2-hydroxyethyl ester, and codrug for


Linearity, Calibration and Limits of Detection and Quantification Correlation coefficient, R2


, was no less than 0.999 for IBP, IBP 2-hydroxyethyl ester, and codrug over the calibration curve range, 


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