New RP‐HPLC Method for the Estimation of 6‐Mercaptopurine in Rat Plasma and Various Tissue Homogenates

Abstract

A reversed‐phase high performance liquid chromatography (HPLC) method was developed to determine 6‐mercaptopurine (6‐MP) in plasma and tissue homogenates of rats in the present study. A known quantity of the drug and internal standard (metronidazole) was spiked in rat plasma and tissue extracts in a range of 50–1000 ng/mL and 100–1000 ng/mL. The spiked plasma samples were deproteinized using a methanol and acetonitrile mixture (1∶1, v/v) and centrifuged. The supernatant was collected and analyzed for the drug content. A Shimadzu system with Thermosil^® C₁₈ (5 µm, 25 cm×4.6 mm i.d.) column was used for the analysis. A mixture of 0.01 M KH₂PO₄ buffer∶acetonitrile (80∶20, v/v) was used as a mobile phase at a flow rate of 1 mL/min. Each sample containing 20 µL was injected through a Rheodyne injector, and the effluent was monitored at 325 nm.

The method was applied to determine pharmacokinetic parameters and biodistribution of a drug in tissues. The retention times were 4.0 min and 6.2 min for drug and internal standard, respectively. The plot of ratio of area of 6‐MP and IS vs. concentration of 6‐MP in ng/mL was found linear in the range of 50–1000 ng/mL in plasma and 100–1000 ng/mL in all tissues. High correlation coefficients were observed with plasma (0.998), liver (0.999), lung (0.999), kidney (0.999), heart (0.993), and spleen (0.990). The limit of detection in plasma was 38 ng/mL and 66–89 ng/mL in various tissues. The performed “t” test for the estimated concentration in recovery studies indicate no significant difference between the added and estimated concentration proving the accuracy and low % RSD values indicate the precision of the method. On the basis of total 6‐MP plasma concentrations as determined by HPLC, the drug showed the AUC of 2098.76±351.2 ng · h/mL, C_max of 1437.27±214.84 ng/mL with T_max of 30 min, Vss of 0.27 mL/kg, and total body clearance of 2.32 mL/h/kg in pharmacokinetic studies, by treating rats with 6‐MP at a dose of 5 mg/kg intravenously. The biodistribution studies showed that the drug was more concentrated in the liver and kidney than other organs, with a general rank order of liver>kidney>heart>spleen>lung. The above study showed great potential of the reported method to estimate 6‐MP in plasma and tissues of rats and may be a substitute for other methods, which are complex, time consuming, and consume high quantities of organic solvents for estimation.

Keywords:

• 6‐Mercaptopurine
• HPLC method
• Pharmacokinetic and biodistribution studies

Acknowledgments

The authors are thankful to TIFAC‐CORE for providing the infrastructure, Dabur Therapeutics Ltd., India, for free gift samples of 6‐Mercaptopurine, to AICTE for financial support (as National Doctoral Fellowship to Manish Umrethia), and to Mercury Laboratories Ltd., India for HPLC analysis.