The development and evaluation of an HPLC method of analysis for nicotine and its major metabolites in urine
This dissertation details the development and evaluation of
an HPLC method of analysis for nicotine and the metabolites cotinine,
nicotine-1'-N-oxide and 3' hydroxycotinine in urine samples.
The significance of nicotine, its absorption, metabolism and
excretion in man and other animals have been described in Chapter 1.
Chapter 2 deals with the development of the HPLC method of analysis
using both isocratic and gradient elution with W detection. A
selection of packing materials/mobile phases covering different
retention mechanisms was investigated. A separation of nicotine,
cotinine, nicotine- 1'-N-oxide and 3' hydroxycotinine and two chromatographic
standards, N' acetyl nornicotine and 2-methyl-6-(3-pyridyl)-
tetrahydro-(1,2)-oxazine was achieved on a Resolve C18 5μ radially
packed cartridge using gradient elution under reverse phase partition
conditions. N' acetyl nornicotine was later discarded in favour of
2-methyl-6-(3-pyridyl)-tetrahydro-(1,2)-oxazine which could be used
as an internal standard.
The statistical analysis of the instrument response to nicotine
and its metabolites in standard solutions was examined in Chapter 3-
A comparison of the measurement parameters peak height and peak area
was made. Within-run and between-run precision were calculated.
Calibration curves were constructed with Working-Hotelling 95%
confidence bands and 95% confidence bounds for 90% of future
observations. The limit of detection values were also statistically
calculated. Precision was found to be low for some of the components
and this was reflected in unacceptably high values of the limit of
The clean-up of urine samples and the extraction of the components
of interest were investigated in Chapter 4. Clean-up and extraction
proved to be very difficult and analyses of smokers' urine samples
underlined the need for an effective clean-up procedure, efficient
chromatography and a sensitive and selective method of detection.
It was concluded that the developed HPLC method of analysis was
inadequate for quantitative analysis of nicotine and its metabolites