Development, validation and transfer of a hydrophilic interaction liquid chromatography/tandem mass spectrometric method for the analysis of the tobacco‐specific nitrosamine metabolite NNAL in human plasma at low picogram per milliliter concentrations

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Abstract
A highly sensitive bioanalytical method based on a simple liquid/liquid extraction and hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC/MS/MS) analysis has been developed, validated and transferred for the determination of 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL), a tobacco‐specific nitrosamine metabolite. Deuterated NNAL (NNAL‐d4) was synthesized and used as the internal standard. This method can be used for the analysis of free and total NNAL (free NNAL plus NNAL‐gluc) in K3‐EDTA human plasma. Free NNAL and NNAL‐d4 are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K3‐EDTA human plasma is treated with β‐glucuronidase to deconjugate the NNAL‐gluc; the total NNAL and internal standard are then extracted using liquid/liquid extraction. After drying down under nitrogen, the residue is reconstituted with acetonitrile and analyzed using positive ion electrospray and HILIC/MS/MS at a flow rate of 1.0 mL/min. The chromatographic run time is 1.0 min per injection, with retention time for both NNAL and NNAL‐d4 of 0.75 min with a capacity factor (k') of 2. The standard curve range for this assay is from 5.00–1000 pg/mL for both free and total NNAL, using a total plasma sample volume of 1.0 mL. The interday precision and accuracy of the quality control (QC) samples demonstrated <7.6% relative standard deviation (RSD) and <3.3% relative error (RE) for free NNAL. For total NNAL, the interday precision and accuracy of the QC samples demonstrated <11.7% RSD and <2.8% RE. Optimization of enzyme hydrolysis of NNAL‐gluc is discussed in detail. The overall recoveries for free and total NNAL and IS were 68.2 and 71.5% (free) and 70.7 and 65.5% (total). No adverse matrix effects were noticed for this assay. Copyright © 2004 John Wiley & Sons, Ltd.

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