Measurement of nicotine in hair by reversed-phase high-performance liquid chromatography with electrochemical detection

doi:10.1016/S0378-4347(00)00540-5

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 753, Issue 2, 5 April 2001, Pages 179-187

https://doi.org/10.1016/S0378-4347(00)00540-5 Get rights and content

Abstract

We have developed an assay for nicotine in hair based on reversed-phase HPLC with electrochemical detection. The method uses a low-metal, high-purity silica reversed-phase column. We have investigated the washing, digestion and extraction procedures and discuss the important points in the HPLC method development. The assay is presented as an application in a population of exposed and non-exposed children. Analytical parameters are satisfactory with linearity, recoveries, limit of quantitation and precision all suitable for epidemiological studies involving environmental tobacco smoke exposure assessment.

Introduction

Accurate assessment of exposure to tobacco smoke in various population groups is an aim of interested epidemiologists. Self reports of environmental tobacco smoke exposure from others have been found to be unreliable [1]. Measurements of plasma and urine cotinine have been used extensively as biochemical markers of exposure (biomarkers). However, these measurements can at best only reflect the last few days exposure to tobacco smoke and samples may be difficult to obtain, especially in young children and infants. Therefore, the development of a reliable biomarker for tobacco smoke exposure reflecting more than 1 week’s exposure and using non-invasive samples, would confer significant benefit to researchers studying environmental tobacco smoke exposure. Nicotine in hair has recently been validated as such a marker [2], [3], [4], [5], [6], and therefore we set out to develop a relatively easy and inexpensive assay for measuring nicotine in hair.

To date gas chromatography–mass spectrometry (GC–MS) [3], [5], [8], [9], [10], GC [7], [9] and radioimmunoassay (RIA) [11], [12] have all been used to measure nicotine in hair. GC, without MS, is prone to interferences [9]. GC–MS is expensive and, as with RIA, not so readily available to a wide range of laboratories. RIA measurement of nicotine also has the problem of cross reactivity, especially with cotinine [2]. As a consequence, other investigators have turned to high-performance liquid chromatography (HPLC), mostly with UV detection [14], [15] as a cheaper and easier means of measuring nicotine in hair. We chose reversed-phase HPLC coupled to electrochemical detection because it has previously been used to measure nicotine in plasma [17] and should give better quantitation limits and selectivity than UV detection.

In a recent study [6] non-exposed infants have been shown to have levels from less than 0.1 to 4.3 ng mg⁻¹ of hair while exposed infants had levels from about 2.5 to 25 ng mg⁻¹. We show that in our assay, linearity and recoveries are satisfactory, limits of detection and quantitation are more than sufficient to assess these levels, and precision is good enough (<10%) to be of use in epidemiological studies, for example, the investigation of the relationship between environmental tobacco smoke exposure and asthma.

Section snippets

Chemicals

Dichloromethane, methanol and acetonitrile were HPLC grade purchased from Lab-Scan (Dublin, Ireland). Nicotine ditartrate was purchased from the Sigma (St. Louis, MO, USA). 2-Phenylimidazole was from Aldrich (Milwaukee, WI, USA), and all other chemicals were AnalaR grade purchased from BDH (Poole, UK). Type 1 water was obtained from a Barnstead Nanopure water purification system (Barnstead/Thermolyne, Dubuque, IA, USA).

Standard preparation, calibration and quality control samples

Stock solutions of 2 g l⁻¹ nicotine ditartrate and 2-phenylimidazole

HPLC and sample preparation

It was found necessary to increase the detector 2 voltage from +0.75 V [16], [17] to +0.9 V in order to accommodate the higher oxidation voltage of 2-phenylimidazole (Fig. 1). We confirmed that cotinine was not electrochemically active in this system [16], and as found previously [16], analytical results were more consistent if the system was allowed to run for 10 to 12 h before making quantitative analyses.

A good internal standard should be chemically similar, and have preparation

Conclusion

The nicotine in hair assay we have developed has good analytical parameters with excellent between batch precision, recoveries, and similar or better quantitation limits to other HPLC hair nicotine assays. Linearity and specificity are good. We have presented evidence that data generated by this assay gives good discrimination between groups of children living in exposed and non-exposed households and therefore is appropriate for its intended use in epidemiological assessment of environmental

Acknowledgements

We wish to thank Dr. Russell Cooke and Dr. Michael Crooke, Laboratory Services, Capital Coast Health Limited, Wellington Hospital, Wellington, New Zealand for their support during the development work and for their helpful advice while writing the manuscript.

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¹: Current address: Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA.

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Measurement of nicotine in hair by reversed-phase high-performance liquid chromatography with electrochemical detection

Abstract

Introduction

Section snippets

Chemicals

Standard preparation, calibration and quality control samples

HPLC and sample preparation

Conclusion

Acknowledgements

Forensic Sci. Int.

J. Chromatogr.

Forensic Sci. Int.

J. Chromatogr.

J. Pharm. Sci.

Forensic Sci. Int.

J. Chromatogr. B

Am. J. Epidemiol.

Ther. Drug. Mount.

J. Occup. Environ. Med.

J. Expo. Anal. Environ. Epidemol.

Int. J. Epidemol.

Ther. Drug. Monit.