The novel assay method for nicotine metabolism to cotinine using high performance liquid chromatography

Article abstract of DOI:10.1248/cpb.59.295

Nicotine is the primary psychoactive component in tobacco. It is taken into the body by tobacco smoking, and mainly metabolized to cotinine in the hepatic cytochrme P450 (CYP) 2A6. The objective of this study was to develop a sensitive method for the determination of nicotine metabolism to cotinine using HPLC. The internal standard, trans-4′-carboxycotinine methyl ester was synthesized with a simple method. The nicotine and cotinine were separated completely and detected by C₁₈ 5- μm analytical column (L-column Octa decyl silyl (ODS), 150mmx4.6 mm i.d.) equipped with a C₁₈ 5- μm guard column (L-column ODS, 10mmx4.6 mm i.d.) and ultraviolet detection at 260 nm. The detection limit of the assay was 0.05 μM for cotinine (n=5, R.S.D) and 0.1 μM for nicotine. Thus the present results provided a sensitive and useful method for the determination of nicotine metabolism catalyzed by CYP2A6.

Full text of DOI:10.1248/cpb.59.295

March 2011
Regular Article
Chem. Pharm. Bull. 59(3) 295—297 (2011)
295
The Novel Assay Method for Nicotine Metabolism to Cotinine Using High
Performance Liquid Chromatography
a
Mitsuo MIYAZAWA,*,a Yumi KAWAUCHI,a Yoshiharu OKUNO,b and Yoshimitsu ODA
a Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University; 3–4–1 Kowakae, Higashiosaka,
b
Osaka 577–8502, Japan: and Department of Strategic Surveillance for Functional Food and Comprehensive Traditional
Medicine Wakayama Medical University; 811–1 Kimidera, Wakayama, Wakayama 641–0012, Japan.
Received June 8, 2010; accepted December 14, 2010
Nicotine is the primary psychoactive component in tobacco. It is taken into the body by tobacco smoking,
and mainly metabolized to cotinine in the hepatic cytochrme P450 (CYP) 2A6. The objective of this study was to
develop a sensitive method for the determination of nicotine metabolism to cotinine using HPLC. The internal
standard, trans-4ꢀ-carboxycotinine methyl ester was synthesized with a simple method. The nicotine and cotinine
were separated completely and detected by C18 5-mm analytical column (L-column Octa decyl silyl (ODS),
150 mmꢁ4.6 mm i.d.) equipped with a C18 5-mm guard column (L-column ODS, 10 mmꢁ4.6 mm i.d.) and ultra-
violet detection at 260 nm. The detection limit of the assay was 0.05 mM for cotinine (nꢂ5, R.S.D) and 0.1 mM for
nicotine. Thus the present results provided a sensitive and useful method for the determination of nicotine me-
tabolism catalyzed by CYP2A6.
Key words trans-4ꢀ-carboxycotinine methyl ester; nicotine; metabolism; inhibition; cytochrome P450 2A6
Tobacco smoking is the single most preventable cause of with a simple method as an internal standard in this assay.
several adverse health effects to both active and passive
Experimental
smokers in the world today. The health consequences of
Chemicals S-(ꢂ)-Nicotine and S-(ꢂ)-cotinine were obtained from
Sigma (St. Louis, MO, U.S.A.), and trans-4ꢀ-carboxycotinine was obtained
from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
Enzyme Preparation Human recombinant CYP2A6 in a baculovirus
system co-expressing human reduced nicotinamide adenine dinucleotide
phosphate (NADPH)-P450 reductase and pooled human liver cytosol were
purchased from BD Gentest Corporation (Woburn, MA, U.S.A). Nicoti-
namide adenine dinucleotide phoshate (NADPꢃ), glucose 6-phosphate, glu-
cose 6-phosphate dehydrogenase were obtained from Oriental Yeast Co.,
Ltd. (Tokyo, Japan).
Metabolism of Nicotine to Cotinine Nicotine C-oxidation activities
were determined by the method of Yamazaki et al. with slight
modifications.6) The standard incubation mixture (final volume of 250 ml)
contained recombinant human CYP2A6 (20 pmol/ml), liver cytsol (0.3 mg/
ml), 50 mM potassium phosphate buffer (pH 7.4), an NADPH-generating
system consisting of 0.5 mM NADPꢃ, 5 mM glucose 6-phosphate, 0.5 unit/ml
glucose 6-phosphate dehydrogenase, and 50 mM nicotine. Incubations were
carried out at 37 °C for 30 min, and then the reaction was terminated by
adding 50% acetonitrile/methanol. trans-4ꢀ-carboxycotinine methyl ester
was added as an internal standard to this solution at final concentration of
smoking include respiratory, cardiovascular and cerebrovas-
cular disorders and cancer.1—3) The most well-established
association between smoking and disease is that for cancer,
which is also the most widespread smoking-related disease.4,5)
Nicotine is the primary psychoactive component in tobacco
responsible for the addictive properties of cigarettes.2,6,7) The
major pathway of nicotine metabolism in humans is a C-oxi-
dation to form cotinine, which is via two-step process. The
first step is catalization by cytochrome P450 (CYP) 2A6 to
produce the intermediate nicotine-D1ꢀ(5ꢀ)-iminium ion, which
is further oxidized to cotinine by cytosolic aldehyde oxi-
dase5—10) (Fig. 1).
So far, detection for nicotine metabolism was carriedout
using high-performance liquid chromatography (HPLC),11—16)
radiometric high-performance liquid chromatography,12) and
gas chromatography-mass spectrometry.17,18) In this study, we
report a simple and sensitive method for the determination of 25 mM.
Preparation of trans-4ꢀ-Carboxycotinine Methyl Ester To 30 mg of
nicotine metabolism to cotinine using new column ((L-column
octa decyl silyl (ODS), 150 mmꢁ4.6 mm i.d., Chemicals
Evaluation and Research Institute, Japan) equipped with a
C18 5-mm guard column (L-column ODS, 10 mmꢁ4.6 mm,
Chemicals Evaluation and Research Institute)), on HPLC.
The assay method using high-performance liquid chromatog-
raphy, Ketamine had been used as an internal standard be-
fore.2,3) However, it was omitted from drug designation in
Japan in 2007 (Ministry of Health, Labour and Welfare,
Japan). Thus, the novel internal standard was needed, and we
newly synthesized trans-4ꢀ-carboxycotinine methyl ester
trans-4ꢀ-carboxycotinine was dissolved in 20 ml of acetone and added 2 ml
of diazomethane. The mixture was made to react for 20 min at the room tem-
perature. After distilling away the mixture, 39 mg of trans-4ꢀ-carboxycoti-
nine methyl ester was obtained (Fig. 2). Electron ionization-mass spectra
(EI-MS) m/z (rel. int.): 234 (100), 219 (19), 206 (40), 191 (73), 175 (97),
147 (92), 121 (69), 119 (96), 97 (16), 91 (19), 78 (32), 68 (34), 55 (50), 42
1
(64). H-NMR (400.00 MHz, in CDCl3, tetramethylsilane (TMS) as internal
standard): d: 2.89 (3H, s), 2.80 (2H, dd, Jꢄ9.3, 17.6 Hz), 2.88 (2H, dd,
Jꢄ8.3 17.6 Hz), 3.05 (1H, ddd, Jꢄ6.4, 8.0, 9.3 Hz), 4.83 (1H, d, Jꢄ6.4 Hz),
7.37 (1H, dd, Jꢄ4.7, 7.7 Hz), 7.57 (1H, ddd, Jꢄ2.4, 2.4, 7.7 Hz), 8.56 (1H,
d, Jꢄ2.4 ), 8.63 (1H, dd, Jꢄ2.4, 4.7 Hz).
HPLC System After terminating the enzyme reaction by adding 50%
acetonitrile/methanol, the denatured protein was removed by centrifugation
Fig. 1. Metabolic Pathway of Nicotine by CYP2A6 and Aldehyde Oxy-
dase
Fig. 2. The Reaction of trans-4ꢀ-Carboxycotinine Methyl Ester with Dia-
zomethane in the Presence of Acetone at Room Temperature for 30 min
∗ To whom correspondence should be addressed. e-mail: miyazawa@apch.kindai.ac.jp
© 2011 Pharmaceutical Society of Japan

296
Vol. 59, No. 3
Fig. 3. UV Spectra of A: trans-4ꢀ-Carboxycotinine Methyl Ester, B: Nico-
tine C: Cotinine
Concentrations of these chemicals examined were all 250 mM.
at 3000 rpm at 4 °C for 10 min and the supernatant was used for HPLC
analysis with a LC-CCPS system (Tosoh, Tokyo, Japan) equipped a detector
UV-8020. The separation was done with a C18 5-mm analytical column (L-
column ODS, 150ꢁ4.6 mm i.d., Chemicals Evaluation and Research Insti-
tute, Japan) equipped with a C18 5-mm guard column (L-column ODS, 5—
4.6 mm, Chemicals Evaluation and Research Institute). The eluent consisted
of a mixture of 10% acetonitrile (v/v) containing 25 mM potassium phos-
phate buffer (pH 7.0). The flow-rate was 1.0 ml/min and the UV detection
was done at 260 nm (Fig. 3). Peak areas thus obtained were integrated with a
Chromatopac Instrument (C-R6A Chromatopac, Shimadzu, Kyoto, Japan).
Inhibitory Effect of (ꢃ)-Menthofuran on Nicotine Metabolism The
standard incubation mixture (final volume of 250 ml) contained recombinant
human CYP2A6 (20 pmol/ml), liver cytsol (0.3 mg/ml), an NADPH-generat-
ing system consisting of 0.5 mM NADPꢃ, 5 mM glucose 6-phosphate,
0.5 unit/ml glucose 6-phosphate dehydrogenase, and 50 mM nicotine, 1 mM
(ꢃ)-menthofuran (dissolved in dimethyl sulfoxide (DMSO)), 50 mM potas-
sium phosphate buffer (pH 7.4). Incubations were carried out at 37 °C for
30 min, and then the reaction was terminated by adding 50% acetonitrile that
were dissolved in methanol (v/v). trans-4ꢀ-Carboxycotinine methyl ester was
added as an internal standard to this solution at final concentration of 25 mM.
Fig. 4. Standard Curve of Cotinine (1) and Nicotine (2) by HPLC Meth-
ods
Cotinine was dissolved in the 10% acetonitrile (v/v) containing 25 mM potassium
phosphate buffer (pH 7.0).
Results
Standard Curve of Cotinine and Nicotine Standard
curve was constructed by plotting peak area (PA) versus con-
centration of the cotinine and nicotine. Within-day precision
of assay was determined by analysis of replicate (nꢄ5) sam-
ples of 5 different concentrations on the same day. Standard
curves for cotinine were liner over the concentration range of
0.05—50 mM and and these for nicotine were 0.1—250 mM.
The cotinine concentration metabolized from nicotine was
determined by comparison to a standard curve. UV intensi-
ties increased linearly between 0.05 and 50 mM cotinine in the
HPLC method (Fig. 4).
Detection of trans-4ꢀ-Carboxycotinine Methyl Ester by
HPLC To detect trans-4ꢀ-carboxycotinine methyl ester by
HPLC, trans-4ꢀ-carboxycotinine was methylated by dia-
zomethane and produced to trans-4ꢀ-carboxycotinine methyl
ester. Both trans-4ꢀ-carboxycotinine and trans-4ꢀ-carboxyco-
tinine methyl ester were dissolved in 50 mM potassium phos-
phate buffer (pH 7.4), and analyzed by HPLC (Fig. 5). The
peaks of trans-4ꢀ-carboxycotinine and trans-4ꢀ-carboxycoti-
nine methyl ester were detected.
Fig. 5. HPLC Analysis for 50 mM trans-4ꢀ-Carboxycotinine Methyl Ester
trans-4ꢀ-Carboxycotinine methyl ester was dissolved in 50 mM potassium phosphate
buffer (pH 7.4).
metabolism was investigated by using this method. The coti-
HPLC Analysis for the Detection of Nicotine Metabo- nine produced by nicotine metabolism was hardly detected
lism by CYP2A6 The cotinine produced from nicotine me- by 1 mM (ꢃ)-menthofuran (Fig. 6).
tabolism was separated from nicotine and trans-4ꢀ-carboxy-
cotinine methyl ester as an internal standard by HPLC. Fur- Discussion
thermore, the inhibitory effect of (ꢃ)-menthofuran, which is
In this work, we showed to be able to quantitatively deter-
known as a major inhibitor of CYP2A6 activity, on nicotine mine nicotine metabolism to cotinine by using C18 5-mm ana-

March 2011
297
results suggest that we have developed a high throughput
method for simultaneous quantification of nicotine and coti-
nine, and this method could be useful to study the effect of
nicotine metabolism to cotinine.19)
This HPLC method gives a good resolution of nicotine
metabolism within a short analysis time (20 min), and the
sample preparation is very simple without need for evapora-
tion. We hypothesized that the administration of chemicals
that strongly and specifically inhibited the nicotine metabo-
lism catalyzed by CYP2A6 activity might result in an inhibi-
tion of nicotine concentration reduction.20,21) Some chemicals
such as (ꢃ)-menthofuran, and tranylcypromine were re-
ported to strongly inhibit CYP2A6.3,8) One of inhibitory
assay, we investigated the ability of (ꢃ)-menthofuran to in-
hibit nicotine metabolism on CYP2A6 using this method. As
a result, the cotinine produced by nicotine metabolism was
hardly blocked by 1 mM of (ꢃ)-menthofuran (Fig. 6). These
results suggest that this method of nicotine metabolic assay
could be useful to study the effect of nicotine metabolism to
cotinine.
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Nicotine is metabolized by CYP2A6 to produce the inter-
mediate nicotine-D1ꢀ(5ꢀ)-iminium ion, which is further oxi-
dized to cotinine by cytosolic aldehyde oxidase. The cotinine
detected at the concentrations range of 0.01—50 mM, and
nicotine was 0.05—250 mM in the HPLC method. Detection
limit of cotinine was 0.05 mM and nicotine was 0.1 mM. These