Clinical Chemistry 45: 85-91, 1999;
(Clinical Chemistry. 1999;45:85-91.)
© 1999 American Association for Clinical Chemistry, Inc.
A New Gas Chromatography–Mass Spectrometry Method for Simultaneous Determination of Total and Free trans-3'-Hydroxycotinine and Cotinine in the Urine of Subjects Receiving Transdermal Nicotine
Allena J. Ji1,2,a,
George M. Lawson1,
Rodger Anderson1,
Lowell C. Dale2,
Ivana T. Croghan2 and
Richard D. Hurt2
1
Department of Laboratory Medicine and Pathology and
2
Nicotine Research Center, Mayo Clinic, Rochester, MN 55905.
a Author for correspondence. Fax 913-268-1497; e-mail ALLENAJI{at}aol.com.
 |
Abstract
|
|---|
trans-3'-Hydroxycotinine (THOC) has been recognized
as the most abundant metabolite of nicotine. In an attempt to assess
THOC and cotinine (COT) concentrations during nicotine transdermal
therapy, we developed a new quantitative gas chromatography–mass
spectrometry (GC–MS) method for simultaneous determination of total
and free THOC and COT in human urine. The method utilizes the
following: (a) hydrolysis of conjugated THOC and COT by
ß-glucuronidase; (b) basic extraction of THOC and COT
with mixed dichloromethane and n-butyl acetate; (c)
derivatization of THOC with bis(trimethylflurosilyl)acetamide; and
(d) separation and identification by GC–MS with selective
ion monitoring. Lower limits of quantification for the assay were 50
and 20 µg/L for THOC and COT, respectively. The intra- and interassay
CVs were 4.4% and 11% for THOC, and 3.9% and 10% for COT at 1000
µg/L. The results from six consecutive 24-h urine collections in 71
subjects administered daily transdermal nicotine doses of 11, 22, and
44 mg showed that, on average, free THOC was 76% of total THOC and
free COT was 48% of total COT in all subjects. THOC is the major
metabolite of nicotine and constitutes 20% of total nicotine intake at
steady state, whereas urinary nicotine and COT excretion were 8% and
17%, respectively. The method is useful for simultaneous determination
of free and total THOCand COT and can be used to assess the urinary excretion of these
metabolites during transdermal nicotine therapy.
|
Introduction
|
|---|
trans-3'-Hydroxycotinine
(THOC)1
was first
recognized as a metabolite of nicotine in the urine of animals
(1)(2) and humans (3) 38 years ago.
The exact structure of THOC was identified by Dagne et al.
(4). THOC was not recognized as the major urinary metabolite
of nicotine until 1987 (5)(6)(7)(8) because it is too polar to be
extracted from water in a routine nicotine or cotinine (COT) assay.
There have been several liquid chromatographic methods for the
determination of THOC in serum and urine published in recent years
(6)(9)(10)(11)(12)(13). Two gas chromatography–mass
spectrometry (GC–MS) methods for free THOC, using derivatized or
nonderivatized procedures, have been reported
(14)(15). These methods did not measure
conjugated forms of THOC and COT; both methods had long derivatization
procedures or showed peaks of nonderivatized THOC in our GC–MS system.
In the present study, we developed a new GC–MS selective ion
monitoring assay for the determination of total and free THOC
simultaneously with total and free COT in urine. The assay was applied
to 497 urine specimens obtained from 71 smokers admitted to a
smoke-free inpatient treatment program at the Mayo Clinic. For 6 days
the subjects received transdermal nicotine therapy at doses of 0
(placebo), 11, 22, or 44 mg of nicotine per 24 h. Total 24-h
excretion of free nicotine and free COT were determined in a previous
report (16) in an attempt to relate the total nicotine
"burden" delivered by the transdermal nicotine relative to the
total burden while the subject was actively smoking. Because free
nicotine and COT account for only about one-fourth of the nicotine
administered, we combined urinary free nicotine results, using the
method developed in our laboratory (17), with urinary total
and free THOC and COT to assess the percentage of urinary excretion in
terms of transdermal nicotine dose administered. By measuring these
additional metabolites, recovery of the administered nicotine dose is
substantially higher. The study was conducted in accordance with the
Declaration of Helsinki, and the protocol was approved by the Mayo
Clinic Institutional Review Board. Written informed consent was
obtained from each subject before participation in the study.
|
Materials and Methods
|
|---|
materials
THOC was obtained from American Health Foundation (Valhalla, New
York). Deuterated THOC (D7-THOC) was provided by San Francisco General
Hospital, Nicotine Research Group (San Francisco, CA). The following
reagents were all research grade or ACS grade and were purchased from
commercial sources: COT (purity, 99%), deuterated COT (D3-COT; purity,
98%), anhydrous potassium carbonate, sodium acetate, acetic acid, and
bis(trimethylsilyl)trifluoroacetamide (BSTFA) from Sigma Chemical
Co.; sodium metabisulfite from Fisher Scientific; EDTA disodium salt
from J. T. Baker; dichloromethane and n-butyl acetate (HPLC grade)
from EM Science; NEE-154 glusulase (10 000 kilounits/L) from DuPont;
and ethyl acetate from Mallinckrodt Chemical. Sodium acetate (2.0
mol/L), pH 6.0, was prepared with 544 g of sodium acetate,
1.0 g of sodium metabisulfite, and 1.6 g of EDTA in ~2 L of
distilled water; the pH was adjusted using 2.0 mol/L acetic acid.
study design and urine collection
The study design and subject recruitment have been reported in
detail in previous papers (16)(18). Briefly, 71
subjects were recruited into three categories based on self-reported
smoking rates: light (10–15 cigarettes/day), moderate (16–30
cigarettes/day), or heavy (>30 cigarettes/day) smokers. From each of
these categories, smokers were assigned randomly to a placebo or to
11-, 22-, or 44-mg nicotine patch doses per 24 h. Thus, there were
12 groups separated according to baseline smoking rate and patch dose,
each group comprising six subjects. A baseline 24-h urine specimen was
collected while the subjects were still smoking. Within 2 weeks of
collection of the baseline specimens, the subjects were admitted to a
special unit at Saint Mary's Hospital. Transdermal patches delivering
the assigned dose of nicotine or a placebo were applied on the
afternoon of admission and were replaced with fresh patches between
0700 and 0800 daily on each of the 6 inpatient days. Consecutive 24-h
urine collections were obtained beginning from the time of admission
and continuing through the sixth inpatient day.
A nonsmoker urine pool was collected in the Drug Laboratory at Mayo
Clinic and used as the matrix for urine calibrators and blank.
calibration
Twenty microliters of 1.0 mg/L THOC and COT calibrator solutions
and 50–3000 µL of 10.0 mg/L THOC and COT calibrator solutions in
methanol were added to 15-mL conical tubes and dried down in a 45 °C
water bath under nitrogen. Aliquots of 1.0 mL of negative urine
obtained from a nonsmoker were added to the above tubes to establish
the urine-based calibrator. A calibration curve of 20–30 000 µg/L
was used for evaluation of linearity. Because most urine samples had
COT and THOC concentrations in the range of 500-5000 µg/L, a
calibration curve of 500-5000 µg/L was used in most batches. The
above urine calibrators were then treated as "free" THOC and COT
urine samples as described below.
sample preparation
The process involved hydrolysis of conjugated COT and
THOC with ß-glucuronidase, extraction of COT and THOC in a basic
solution with organic solvent, and derivatization of THOC with BSTFA.
Briefly, 60 µL of D7-THOC and 40 µL of D3-COT at concentrations of
10 mg/L were added to each of two 15-mL conical tubes containing 1 mL
of a subject's urine; the tubes were labeled free and total. A series
of urine calibrators were also included in each batch and labeled as
free. Five hundred microliters of 2.0 mol/L sodium acetate buffer, pH
6.0, was added to each of the above tubes. Fifty microliters of
glusulase (10 000 kilounits/L) was added to the tubes labeled total.
All of the tubes (free and total) were covered with
ParafilmTM, vortex-mixed briefly, and incubated in a
50 °C heat block for 2 h. One milliliter of 500 g/L potassium
carbonate and 3.0 mL of n-butyl acetate:dichloromethane (2:1, by
volume) were added to the above tubes. The tubes were then vortex-mixed
on a BIG Vortexer (Glas-Col Apparatus) at 80 rpm for 5 min and then
centrifuged at 3000g for 5 min. The upper organic layer was
transferred to a clean tube and dried completely in a 45 °C water
bath under nitrogen. Ethyl acetate (50 µL) and BSTFA (50 µL) were
added to the dried tubes containing extracted THOC and COT. The tubes
were quickly vortex-mixed, covered with Parafilm, and incubated in a
heat block at 70 °C for 30 min. The above solutions were transferred
immediately to GC sample inserts. The sample vials were purged with
nitrogen to remove moisture and capped tightly. All GC sample vials
were stored at 4 °C for 2-4 h until GC–MS analysis was performed.
gc–ms
Samples were injected into an HP 5890A gas chromatograph (Hewlett
Packard) equipped with a DB-5 MS fused-silica capillary column (15 m x
0.32 mm i.d., 1 µm; J & W Scientific). Samples were injected in the
splitless mode with the purge valve closed for 0.8 min. The oven
temperature started at 85 °C for 0.5 min, followed by a temperature
ramp of 30 °C/min, and stopped at 300 °C. The temperature of the
injection port and the ion source of electron impact were both
250 °C, and the GC interface was 275 °C. The total separation
time was 8 min. The retention times were 5.6–5.8 min for THOC and
4.8–5.1 min for COT at 35 kPa helium pressure. Mass spectrometric
analyses were carried out on an HP 5987 mass-selective detector
(Hewlett Packard) connected to an RTE Operating System using Aquarius
software. During electron impact ionization detection, the selective
ion monitoring device was set to monitor the ions m/z 249,
144, and 116 for THOC; 256, 147, and 119 for D7-THOC; 98, 176, and 118
for COT; and 101,179, and 121 for D3-COT. The first ion listed above
for each analyte was used for its quantification.
|
Results
|
|---|
determination of total and free urinary thoc and cot
Total and free THOC and COT determinations in urine were performed
using the corresponding deuterated compounds as internal standards. The
GC ion chromatograms from a nonsmoker and a smoker are shown in Fig. 1
. The retention times of THOC and COT were 5.67 and 4.95 min,
respectively. The mass spectra of derivatized THOC and D7-THOC are
shown in Fig. 2
.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 1. GC ion chromatograms of urine from a smoker and a
nonsmoker, using the method described in Materials and
Methods.
The ions monitored are as follows: m/z 249, 144, and 116 for
THOC; 256, 147, and 119 for D7-THOC; 98, 176, and 118 for COT; and 101,
179, and 121 for D3-COT.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
Figure 2. Mass spectra of derivatized THOC and D7-THOC from the THOC
peak in Fig. 1
.
Ions m/z 249, 144, and 116 were used to monitor THOC; and
ions m/z 256, 147, and 119 were used to monitor D7-THOC.
|
|
linearity and precision
Urine calibrators were prepared by drying known concentrations of
THOC and COT methanol solution to dryness and redissolving them in
negative urine obtained from a nonsmoker. Linear regression analysis
obtained from eight calibrators at 50–5000 µg/L vs the GC–MS
readings (µg/L) yielded a correlation coefficient
(r2) of 0.998 for THOC and 0.999 for COT. During
GC–MS analysis, different voltage multiplier settings were used for
concentration ranges from 20 to 3000 µg/L and from 3000 to 30 000
µg/L to optimize accuracy at low and high concentrations. The linear
ranges were 50–30 000 µg/L for THOC and 20–15 000 µg/L for COT.
The interassay CVs (shown in Table 1
) were 4.2–12% for THOC and 10–12% for COT.
extraction and analytical recoveries
Absolute extraction recovery was determined by comparing GC–MS
peak areas from extracted urine samples to which known THOC and COT
concentrations had been added with peak areas from nonextracted THOC
and COT in methanol. The drying and derivatization processes were
applied to both extracted and nonextracted samples before analysis on
GC–MS. The average extraction recoveries from 20 determinations were
14% for THOC and 49% for COT at concentrations of 1000 and 3000
µg/L. The analytical recovery (relative recovery) was assessed from
GC–MS readings from extracted THOC and COT urine calibrators divided
by target concentrations of THOC or COT. The analytical recoveries were
95.6–101% for THOC and 91.8–96.2% for COT in a concentration range
of 500-3000 µg/L from eight batches.
lower limits of detection and quantification
Lower limits of detection were determined as three times the
background noise at the appropriate retention times, converting the
peak areas to THOC and COT concentrations. On the basis of 20
determinations, the lower limits of detection were 10 µg/L for THOC
and 5 µg/L for COT. Lower limits of quantification were evaluated
from four batches; the mean analytical recoveries were 109% for THOC
at 50 µg/L and 102% for COT at 20 µg/L. The CVs (imprecision)
among batches were 15.5% for THOC at 50 µg/L and 8.8% for COT at 20
µg/L. Because the accuracy was within 20% of the theoretical
concentrations and the CV was <20% at these concentrations
(19), the lower limits of quantification were 50 µg/L for
THOC and 20 µg/L for COT.
determination of urinary total and free thoc and cot in transdermal
subjects
Urine specimens (n = 497) from 71 smokers were assayed using
the present method. Each smoker submitted seven urine specimens
(baseline and inpatient days 1–6). To determine total urinary THOC and
COT (glucuronides and free), the samples were first adjusted to pH 6.0
by adding 2.0 mol/L sodium acetate and ß-glucuronidase and then
incubated at 50 °C for 2 h. For free THOC or COT, samples were
treated as above except that ß-glucuronidase was not added. The mean
concentrations of total THOC for each dose group at baseline and
inpatient days 1–6 are shown in Fig. 3
. The excretion kinetics of total THOC and COT were similar to
those of urinary free COT and nicotine reported previously on these
same urine samples (16). Steady-state excretion rates were
reached on the third day after beginning nicotine patch therapy. When
mean excretion rates were compared, free COT was ~48% of total COT
and free THOC was ~76% of total THOC in each dose group during
regular smoking (baseline) and at steady state on transdermal nicotine
therapy (Fig. 4
). The percentage of free COT or free THOC was independent of
transdermal nicotine dose and thus independent of the concentration of
total THOC and COT in urine. However, the difference between total THOC
and total COT excretion rates increased with increasing nicotine patch
dose, as shown in Fig. 5
, where the mean excretion rates in the 11-mg and 44-mg patch
groups are plotted at baseline and inpatient days 1–6.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 4. Mean percentage of free COT to total COT (solid
columns) and free THOC to total THOC (hatched columns)
at baseline (BL) and during inpatient days 1–6.
|
|
The percentage of replacement at steady state in each group of subjects
was calculated using the sum in micromoles of free nicotine, total COT,
and total THOC, averaged over inpatient days 3–6 and divided by the
sum in micromoles of free nicotine, total COT, and total THOC excreted
at baseline. The results showed that the average percentages of
nicotine replacement were 27%, 77%, and 146% for the 11-, 22-, and
44-mg nicotine patch dose groups, respectively. The results support
those reported previously (16) because the percentages of
replacement calculated from the excretion rates of free nicotine, total
COT, and total THOC were similar to the percentages of replacement
calculated only from free nicotine and free COT (16).
If the entire dose of nicotine is absorbed transdermally during a 24-h
period, the recovery of nicotine administered in terms of total
excretion can be calculated by using the sum in micromoles of free
nicotine, total COT, and total THOC in urine each day during steady
state and dividing by the transdermal nicotine dose expressed in
micromoles. The average recovery of nicotine metabolites is shown in
Table 2
. Recovery of total nicotine intake was ~45% in each group of
this study, whereas a maximum recovery of 19% has been reported if
only free nicotine and COT (16) are measured. Measurement of
the additional metabolites substantially increases the total recovery
of nicotine-derived metabolites in urine.
|
Discussion
|
|---|
development of gc–ms thoc assay
This study has established a new GC–MS method for simultaneous
determination of total and free THOC and COT and confirmed the
observations that THOC is the major metabolite of nicotine and that
both THOC and COT are excreted in urine in free and conjugated forms
(20). The present assay for simultaneous determination of
total and free THOC and COT has substantially reduced analysis time and
provided a wide concentration range for assays of THOC and COT in the
urine of smokers. Although Voncken et al. (15) reported a
method of simultaneous determination of nicotine, THOC, and COT in
urine that does not require derivatization, the method is suitable only
for samples with very high THOC and COT concentrations. At low
concentrations of THOC, the THOC peak was skewed in our GC, which
reduced accuracy. Jacob et al. (14) reported a method using
derivatization for urinary THOC. Because the method requires two to
three dichloromethane extraction steps and entails a relatively long
derivatization procedure, we did not use it in the present study. Use
of a single n-butyl acetate (15), dichloromethane
(10)(14), or chloroform–methanol mixture
(11) to extract THOC and COT has been reported previously.
The current assay uses n-butyl acetate:dichloromethane (2:1, by volume)
as the extraction solvent for THOC and COT. The advantages of this
solvent are that chromatography is cleaner than when only
dichloromethane is used and requires less drying time (15–20 min) than
when only n-butyl acetate is used (55–65 min). The absolute extraction
recoveries were 14% for THOC and 49% for COT, whereas average
analytical recoveries were 94–99% in the range of 500-3000 µg/L in
eight batches. Unfortunately, our initial attempts to measure COT and
THOC simultaneously with nicotine were not successful. The assay was
unable to measure the urinary nicotine concentration, possibly because
of loss of nicotine in the solvent evaporation step. The free COT
concentrations in those urine samples were also determined using an
HPLC method in our laboratory (21) and compared with the
current GC–MS results. A correlation coefficient of 0.960 was obtained
(data not shown).
A method for assaying urinary glucuronide conjugates of nicotine, COT,
and THOC was reported by Byrd et al. (20). The method
required removal of free THOC by dichloromethane extraction, which was
followed by addition of ß-glucuronidase to the same urine sample for
release of conjugated nicotine metabolites and subsequent measurement
of released nicotine metabolites by liquid chromatography–mass
spectrometry. The current assay is simpler and less time-consuming than
the reported method because the preparation of free and total nicotine
metabolites was the same except for the addition of 50 µL of
ß-glucuronidase to urine samples for total THOC and COT. The
amount of ß-glucuronidase, the incubation time, and the incubation
temperature for completion of the hydrolysis reaction were evaluated
during method development. The condition used in the present
assay leads to complete hydrolysis of THOC and COT glucuronides.
determination of urinary free and total thoc and cot
Although there was variability in the absolute amounts of THOC and
COT in 71 subjects during active smoking (baseline) or transdermal
nicotine therapy (inpatient days 1–6), the present study shows that
free THOC constituted an average of 76% of total THOC and that free
COT was 48% of total COT in all dose groups. These results are
consistent with those that Byrd et al. (20) reported in 11
smokers. The results also confirm that transdermal nicotine therapy and
active smoking have the same metabolic pathway, as Benowitz et al.
(22) reported recently.
THOC has been recognized as the main urinary metabolite in smokers. The
sum of nicotine, COT, and THOC excretion was reported as ~80% of the
total measurable metabolites in nine smokers, and THOC itself
constituted 41–50% of total measurable nicotine metabolites
(5). However, the data from a review paper (23)
showed that the sum of nicotine, COT, and THOC excretions was ~49%
of the total nicotine metabolites. Our observations of 71 smokers
showed that excretion of the sum of nicotine, COT, and THOC in the
steady state was ~45% of the nicotine administered at doses of 11,
22, and 44 mg/24 h. The designated doses of 11, 22, and 44 mg are the
amount of nicotine absorbed in 24 h, on average, as tested by the
manufacturer (24). Total THOC constituted 20%, total COT
constituted 17%, and free nicotine constituted 8% of the nicotine
intake. To our knowledge, this is the largest subject study that
reports absolute THOC excretion recovery in terms of nicotine patch
intake. In our study, the nicotine:THOC molar ratio was 1:2.5, whereas
in another nicotine patch study, the ratios were from 1:2.3 to 1:2.9
(Table 3
) after our calculation. The ratio of nicotine to COT does not
show any consistent relationship. The ratio of nicotine to THOC
probably can be used for estimation of THOC concentration when nicotine
is the only available test. Because THOC has a much longer half-life
than nicotine or COT, its measurement may provide a better screening
test for assessing smoking status.
In conclusion, a new GC–MS method for the simultaneous
determination of total and free THOC and COT has been developed and may
be useful in monitoring patients undergoing smoking cessation therapy.
When urine concentrations of nicotine, total THOC, and total COT are
used, the total nicotine intake can be estimated. Therefore, the
nicotine patch dose for cessation therapy may be determined more
accurately. The sum of free nicotine, total COT, and total THOC
excretion was 45% of total nicotine intake. On average, THOC
constituted 20% of nicotine transdermal intake, whereas total COT was
17% and free nicotine was 8% of total nicotine transdermal intake.
THOC is the main metabolite of nicotine, and the urinary molar ratio of
free nicotine to total THOC was 1:2.5 on average in all dose groups.
Determination of THOC has substantially increased the recovery of the
total body nicotine burden.
|
Acknowledgments
|
|---|
This study was supported by a grant from Lederle Laboratories,
Pearl, NY. We thank Dr. Peton Jacob at San Francisco General Hospital,
San Francisco, CA and Drs. Shantu Amin and Dhimant Desai, American
Health Foundation, Valhalla, NY for generously providing deuterated and
nondeuterated THOC.
|
Footnotes
|
|---|
2 Present address: Kansas City Analytical Services, 12700 Johnson
Drive, Shawnee, KS 66216. 
1 Nonstandard abbreviations: THOC,
trans-3'-hydroxycotinine; COT, cotinine; GC–MS, gas
chromatography–mass spectrometry; D7-THOC, deuterated
trans-3'-hydroxycotinine; D3-COT, deuterated cotinine; and
BSTFA, bis(trimethylsilyl)trifluoroacetamide.
|
References
|
|---|
-
McKennis H, Turnball LB, Bowman ER, Wada E. Demethylation of cotinine in man. J Am Chem Soc 1959;81:3951-3954.
-
McKennis H, Turnball LB, Schwartz SL, Tamaki E, Bowman ER. Demethylation in the metabolism of (-)-nicotine. J Biol Chem 1962;237:541-546.
[Free Full Text]
-
Bowman ER, McKennis H. Studies on the metabolism of (-)-cotinine in the human. J Pharmacol Exp Ther 1962;135:306-311.
[Abstract/Free Full Text]
-
Dagne E, Castagndi N. Cotinine N-oxide, a new metabolite of nicotine. J Med Chem 1972;15:840-841.
[ISI][Medline]
[Order article via Infotrieve]
-
Neurath GB, Pein FG. Gas chromatographic determination of trans-3'-hydroxycotinine, a major metabolite of nicotine in smokers. J Chromatogr 1987;415:400-406.
[ISI][Medline]
[Order article via Infotrieve]
-
Neurath GB, Dunger M, Orth D, Pein FG. trans-3'-hydroxycotinine as a main metabolites in urine of smokers. Int Arch Occup Environ Health 1987;59:199-201.
[ISI][Medline]
[Order article via Infotrieve]
-
Jacob P, III, Benowitz NL, Shulgin AT. Recent studies of nicotine metabolism in human. Pharmacol Biochem Behav 1988;30:249-253.
[ISI][Medline]
[Order article via Infotrieve]
-
Schere G, Jarczyk L, Heller WD, Biber A, Neurath GB, Adlkofer A. Pharmacokinetics of nicotine, cotinine, and 3'-hydroxycotinine in cigarette smokers. Klin Wochenschr 1988;66:5-11.
-
McManus KT, Debethizy KM, Garteiz DA, Kyerematen GA, Vesell ES. A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids. J Chromatogr Sci 1990;28:510-516.
[ISI][Medline]
[Order article via Infotrieve]
-
Pichini S, Altieri I, Pacifici R, Rosa M. Simultaneous determination of cotinine and trans-3'-hydroxycotinine in human serum by high-performance liquid chromatography. J Chromatogr 1992;577:358-361.
[ISI][Medline]
[Order article via Infotrieve]
-
Rop PP, Grimaldi F, Oddoze C, Viala A. Determination of nicotine and its metabolites in urine by high-performance liquid chromatography. J Chromatogr 1993;612:302-309.
[ISI][Medline]
[Order article via Infotrieve]
-
Moore J, Greenwood M, Sinclair N. Automation of a high performance liquid chromatographic assay for the determination of nicotine, cotinine and 3-hydroxycotinine in human urine. J Pharm Biomed Anal 1990;8:1051-1054.
[ISI][Medline]
[Order article via Infotrieve]
-
Pacific R, Pichini S, Altieri I, Rosa M, Bacosi A, Caronna A, Zuccaro P. Determination of nicotine and two major metabolites in serum by solid-phase extraction and high-performance liquid chromatography-particle beam mass spectrometry. J Chromatogr 1993;612:209-213.
[ISI][Medline]
[Order article via Infotrieve]
-
Jacob P, III, Shulgin AT, Yu L, Benowitz NL. Determination of the nicotine metabolite trans-3'-hydroxycotinine in urine of smokers using gas chromatography with nitrogen-selective detection or selected ion monitoring. J Chromatogr 1992;583:145-154.
[ISI][Medline]
[Order article via Infotrieve]
-
Voncken P, Schepers G, Schafer KH. Capillary gas chromatographic determination of trans-3'-hydroxycotinine simultaneously with nicotine and cotinine in urine and blood samples. J Chromatogr 1989;479:410-418.
[ISI][Medline]
[Order article via Infotrieve]
-
Lawson GM, Hurt RD, Dale LC, Offord KP, Croghan IT, Schroeder DR, et al. Application of urine nicotine and cotinine excretion rates to assess nicotine replacement in light, moderate, and heavy smokers undergoing transdermal therapy. J Clin Pharmacol 1998;38:510-516.
[Abstract]
-
Baskin LB, Anderson RW, Charlson JR, Hurt RD, Lawson GM. Determination of nicotine in serum and urine by isotope dilution using solid-phase extraction and gas chromatography/mass spectrometry with selected ion monitoring. Ann Clin Biochem 1998;35:522-527.
-
Dale LC, Hurt RD, Offord KP, Lawson GM, Croghan IT, Schroeder DR. High-dose nicotine patch therapy, percentage of replacement and smoking cessation. JAMA 1995;274:1353-1358.
[Abstract]
-
Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, et al. Analytical methods validation: bioavailability, bioequivalence, and pharmacokinetics studies. J Pharm Sci 1992;81:309-312.
-
Byrd GD, Chang KM, Green JM, deBethizy JD. Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers. Drug Metab Dispos 1992;20:192-197.
[Abstract]
-
Machacek DA, Jiang NS. Quantitation of cotinine in plasma and saliva by liquid chromatography. Clin Chem 1986;32:979-982.
[Abstract/Free Full Text]
-
Benowitz NL, Jacob P, III, Fong I, Gupta S. Nicotine metabolic profile in main: comparison of cigarette smoking and transdermal nicotine. J Pharmacol Exp Ther 1994;268:296-303.
[Abstract/Free Full Text]
-
Kyerematen GA, Vesell ES. Metabolism of nicotine. Drug Metab Rev 1991;23:3-41.
[ISI][Medline]
[Order article via Infotrieve]
-
Bannon YB, Corish J, Corrigan OI, Devane JG, Kavanagh M, Mulligan S. Transdermal delivery of nicotine in normal human volunteers: a single dose and multiple dose study. Eur J Clin Pharmacol 1989;37:285-290.
[ISI][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
T. P. Moyer, J. R. Charlson, R. J. Enger, L. C. Dale, J. O. Ebbert, D. R. Schroeder, and R. D. Hurt
Simultaneous Analysis of Nicotine, Nicotine Metabolites, and Tobacco Alkaloids in Serum or Urine by Tandem Mass Spectrometry, with Clinically Relevant Metabolic Profiles
Clin. Chem.,
September 1, 2002;
48(9):
1460 - 1471.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W D. Clouse, K. S Rud, R. D Hurt, and V. M Miller
Short-term treatment with transdermal nicotine affects the function of canine saphenous veins
Vascular Medicine,
May 1, 2000;
5(2):
75 - 82.
[Abstract]
[PDF]
|
|
|
Top
Abstract
Introduction
), 11 mg (
),
22 mg (
); 44 mg (
).
, THOC excretion
rate for 44-mg nicotine patch;