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Rajaa Farhan Hussein and Muhammad M. Hammami*


We optimized and validated a rapid, simple, and sensitive HPLC assay for pioglitazone concentration in human plasma and studied the stability of pioglitazone under various clinical laboratory conditions. Pioglitazone and lansoprazole as internal standard (IS) were extracted from 1 ml plasma sample spiked with 4.0 μg in 160 μl mobile phase) as follows: 4.0 ml of tert. butyl methyl ether was added and the sample was vortexed for 5 min then centrifuged for 10 min at 6000 rpm at room temperature. The organic layer was transferred to a clean tube and dried under a gentle stream of nitrogen at 40 ºC, and the residue was reconstituted in 250 μl mobile phase. After centrifugation at 3500 rpm for 2 min, the supernatant was transferred into auto-sampler vials and 100 μl were injected into the HPLC system. Pioglitazone and IS -Pak C18 steel column, using a Guard Pak pre-column module with Nova-Pak C18 4-μm insert, with a run time of 7 min. The mobile phase consisted of a mixture of 0.01 M dibasic sodium hydrogen phosphate and dihydrogen sodium phosphate (pH= 7.0), methanol, and acetonitrile (55:30:15, v:v:v) and was delivered at a flow rate of 1.0 ml/min. The analytes were detected with Waters 2996 photodiode array detector with the wavelength set at 228 nm. The response was linear over the range of 0.05 – 5 μg/ml. Extraction recovery of pioglitazone and inter-run bias and imprecision were ≥ 90%, ≤ 7.2%, and ≤ 5.8, respectively. In human plasma, pioglitazone was stable for at least 24 hours at room temperature (RT) (≥ 100%), 14 weeks at –200C (≥ 99%), and after 3 cycles of freeze at –200C and thaw at RT (≥ 100%). In processed samples, it was stable for at least 24 hours at RT (≥ 97%) and 48 hours at –200C (≥ 96%). Stock solution of pioglitazone (1 mg/ml in methanol) was stable for at least 48 hours at RT (93%) and 14 weeks at –200C (98%). The assay was successfully employed to measure pioglitazone level in samples obtained from a healthy volunteer.

Keywords: Pioglitazone, Lansoprazole, HPLC, Validation, Stability.

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