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TABLE 2: Recovery of ibuprofen, ibuprofen 2-hydroxyethyl ester, and codrug in human plasma and rat liver homogenate


QC Recovery Samples Human plasma, n = 3 Compound


Ibuprofen


Ibuprofen 2-hydroxyethyl ester


Codrug


Nominal concentration (µg mL-1 25


100 25


100 25


100


Rat liver homogenate, n = 3 Compound


Ibuprofen


Ibuprofen 2-hydroxyethyl ester


Codrug


100 25


100 25


100 0.195 - 400 µg mL-1


Nominal concentration (µg mL-1 25


) )


Recovered concentration (µg mL-1 24.4 96.9 24.2 98.2 23.9 98.2


Recovered concentration (µg mL-1 25.1


100.1 24.8 99.6 24.9 99.5


; indicating excellent linearity. The limits of


detection were between 20 and 24 ng mL-1 were between 60 and 80 ng mL-1


and LOQ = 60 ng mL-1 and limits for quantification . The linear regression equations


for IBP, its ester, and codrug in addition to the limits of detection and quantification are as follows: IBP, y = (28179)x + (40185), R2 LOD = 20 ng mL-1


y = (17230)x + (58266), R2 = 65 ng mL-1


(23400)x + (27671), R2 ng mL-1


.


Accuracy and precision Table 1 shows the intra-assay and inter-assay variability results. The RSD (%) values for IBP, IBP 2-hydroxyethyl ester, and codrug were determined and were less than the maximum allowed limit; % RSD ≤ 1.0% for intra-day precision and ≤ 2% for inter-day precision. This confirms the intra-day and inter-day reproducibility and accuracy of the developed HPLC method across the chosen four concentrations for the three former compounds.


Recovery Recovery for IBP, IBP 2-hydroxyethyl ester, and codrug from human plasma and rat liver homogenate was assessed (n = 3) at 25 and 100 µg mL-1


. As shown in table 2, the recovery for IBP, IBP 2-hydroxyethyl


ester, and codrug were more than 95%, in human plasma and more than 99% in rat liver homogenate. The high plasma recovery for IBP, its ester, and its codrug suggests negligible loss of each of the aforementioned compounds during protein precipitation process using methanol, similarly the extraction efficiency of the intended compounds in rat liver homogenate was greatly efficient as the former. 


FIGURE 1: The chemical structures of ibuprofen, ibuprofen 2-hydroxyethyl ester, and ibuprofen-nicotinic acid codrug


OR OR


OO


Ibuprofen 2-hydroxyethyl ester and nicotinic acid codrug, R’’= Ibuprofen 2-hydroxyethyl ester and nicotinic acid codrug, R'' =


Ibuprofen 2-hydroxyethyl ester, R’= OH Ibuprofen 2-hydroxyethyl ester and nicotinic acid codrug, R'' =


Ibuprofen, R= H Ibuprofen, R=H Ibuprofen 2-hydroxyethyl ester, R' = Ibuprofen 2-hydroxyethyl ester, R' = 040 MEDLAB MAGAZINE ISSUE 1 2012 OH O N OH O O N O Ibuprofen, R= H = 0.9999, LOD = 20 ng mL-1


, IBP 2-hydroxyethyl ester - nicotinic acid codrug, y = = 0.9999, LOD = 24 ng mL-1


= 0.9999,


, IBP 2-hydroxyethyl ester, and LOQ


and LOQ = 80 40


35 30 25 20


15 10 5 0


-5 0 1 2 3 4 5 6 Time (min) 40


35 30 25 20


15 10 5 0


-5 0 1 2 3 4 5 6 Time (min) 40


35 30 25 20


15 10 5 0


-5 0 1 2 3 4 5 6 Time (min) 7 8 9 10 C 7 8 9 10 B 7 8 9 10 A )


Standard deviation (µg mL-1 0.68 2.62 0.55 1.38 0.47 1.72


)


Standard deviation (µg mL-1 0.05 0.12 0.19 0.03


0.15 0.23


) Recovery (%) RSD (%) 2.78 2.71 2.27 1.41 1.97 1.75


97.7 96.9 96.8 98.2 95.4 98.2


) Recovery (%) RSD (%) 0.18 0.12 0.78 0.03 0.59 0.23


100.2 100.1 99.1 99.6 99.5 99.5


FIGURE 2: HPLC chromatogram of ibuprofen, ibuprofen 2-hydroxyethyl ester, and codrug from stability study in (a) aqueous solution, (b) human plasma, (c) rat liver homogenate


mAU


mAU


mAU


Ibuprofen 2-hydroxyethyl Ester Ibuprofen 2-hydroxyethyl Ester Ibuprofen 2-hydroxyethyl Ester Ibuprofen


Ibuprofen


Ibuprofen


Codrug Codrug


Codrug


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