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.
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-d(4)) 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 K(3)-EDTA human plasma. Free NNAL and NNAL-d(4) are extracted from human plasma by liquid/liquid extraction. To analyze for total NNAL and the internal standard, a separate aliquot of the K(3)-EDTA human plasma is treated with beta-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-d(4) 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.
Pan
Jiongwei
J
Covance Bioanalytical Chemistry, Madison, WI 53704, USA.
Song
Qi
Q
Shi
Haihong
H
King
Melissa
M
Junga
Heiko
H
Zhou
Shaolian
S
Naidong
Weng
W
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008832:Microchemistry / Q000379:methods*; D009602:Nitrosamines / Q000032:analysis*; D011725:Pyridines / Q000032:analysis*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014026:Nicotiana / Q000737:chemistry*
D009602:Nitrosamines; D011725:Pyridines; C099565:4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol