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Ultrasonic extraction and capillary gas

chromatography determination of nicotine in
pharmaceutical formulations

Article in Analytica Chimica Acta · November 2004

Impact Factor: 4.51 · DOI: 10.1016/j.aca.2004.09.035

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 Analytica Chimica Acta 526 (2004) 35–39

Ultrasonic extraction and capillary gas chromatography

determination of nicotine in pharmaceutical formulations
Yuegang Zuo∗ , Liliang Zhang, Jingping Wu, Johnathan W. Fritz,
Suzanne Medeiros, Christopher Rego
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA

Received 23 July 2004; received in revised form 14 September 2004; accepted 14 September 2004
Available online 18 October 2004

Abstract

A simple, rapid and accurate analytical method was developed for the determination of nicotine in pharmaceutical formulations. The method
comprises a fast ultrasonic extraction (UE) with heptane as a solvent followed by direct capillary gas chromatography (GC) separation and
quantitation. The application of ultrasound significantly accelerated the analyte extraction. For example, in the conventional method each
extraction step takes up to 24 h whereas with the ultrasonic extraction method developed it took less than 20 min to achieve the same extraction
efficiency. The ultrasonic extracts were directly chromatographed on an Alltech ECTM -5 capillary GC column and a base line separation
was achieved within 10 min. The consumption of environmental harmful organic solvent in this developed UE-capillary GC method is much
lower than in conventional extraction-HPLC methods. The UE developed uses only 1/6 of organic solvent needed in conventional extraction.
The subsequent GC analysis does not consume organic solvent as mobile phase while HPLC does. This green analytical method has been
successfully applied to determine the nicotine content in both chewing and transdermal systems. Standard calibration curves were linear over
the concentration range 1.00–500.0 ␮g/mL. Within-day and day-to-day relative standard deviations less than 1.9 and 2.0%, respectively.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Nicotine; Ultrasonic extraction; Gas chromatography; Pharmaceutical formulation; Green analytical chemistry

1. Introduction effects. These include increased heart rate, blood pressure,

free fatty acids and glucose. The addictive nature of nico-
Nicotine, 1-methyl-2(3-pyridyl)pyrrolidine (Fig. 1), is a tine is due to the effects of nicotine on the neurotransmitter
major alkaloid found in tobacco plants. It is generally ac- dopamine, which plays a significant role in the pleasure cen-
knowledged to be the principal agent motivating tobacco ter of the brain [1,2]. As a result of addiction to nicotine,
smoking and the main impediment to cessation. As an ad- about 47 million adults (∼23% of the adult population) in
dictive substance, nicotine mimics the effects of neurotrans- the United States and 2 billion people worldwide currently
mitter acetylcholine [1,2]. The similarity of the structures of smoke [3]. More than 4 million people died in the world annu-
nicotine and acetylcholine allows nicotine to activate cholin- ally from smoking attributable diseases such as lung cancer,
ergic receptors naturally stimulated by acetylcholine. These chronic obstructive, pulmonary disease, cardiovascular dis-
receptors are located in the brain, heart, muscles, adrenal ease and stroke. Cigarette smoking is a worldwide pandemic
glands and other peripheral nervous systems. These recep- that is completely avoidable.
tors play an important role in breathing, heart rate, learning With the growing awareness of the harmful effects of
and memory. Nicotine, however, does not produce the same smoking, the number of smokers trying to quit is also increas-
effect as acetylcholine, resulting in abnormal physiological ing. However, due to the severe smoking withdrawal symp-
toms, more than 93% of smokers who tried to quit could not
∗ Corresponding author. Tel.: +1 508 999 8959; fax: +1 508 999 9167. succeed in quitting smoking [4,5]. In recent years, several
E-mail address: yzuo@umassd.edu (Y. Zuo). different nicotine replacement products (NRPs), including

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2004.09.035
36 Y. Zuo et al. / Analytica Chimica Acta 526 (2004) 35–39

2. Experimental

2.1. Chemicals

Fig. 1. Chemical structure of nicotine. Nicotine and N-ethylaniline were purchased from Acros
Organics (New Jersey, USA). Nicotine polacrilex gum was
from Pharmacia AB (Stockholm, Sweden) and nicotine trans-
chewing gum and transdermal patches, have been marketed dermal systems from Rite Aid Corporation (Harrisburg, PA,
as smoking cessation aids in the United States and many other USA). n-Heptane was obtained from Pharmco Products Inc.
countries. All of these systems can significantly improve quit- (Brookfield, CT, USA). All solution preparations were made
ting rates when compared with placebo [6]. In addition to the using doubly-distilled and then deionized water. All the other
clear benefits derived from nicotine replacement products, chemicals were of analytical reagent grade and were used
nicotine, the most addictive component of cigarette smoke, without further purification.
however, is delivered to the blood circulation, via means other
than cigarette smoke, to reduce craving. The level of nicotine 2.2. Apparatus
in the NRPs is critical in the efficacy and safety of the drugs.
An accurate and rapid analytical method for monitoring nico- A Shimadzu GC-17A gas chromatograph equipped with a
tine in NRPs is needed for the quality control. split/splitless injector, a flame ionization detector (FID) and
Although various high-performance liquid chromatogra- a Shimadzu AOC-20i GC auto-injector (Shimadzu Scientific
phy (HPLC) [7–10], capillary electrophoresis [11] and gas Instruments, Columbia, MD, USA) was used for all analyses.
chromatography (GC) methods [12–19] have been reported The GC is coupled with a Gateway E-4200 computer that
for the analysis of nicotine and its metabolites in tobacco utilizes CLASS-VP Chromatography Data System Version
leaves, human blood and urine, only HPLC methods have 4.2.
been appeared in the literature for the analysis of nicotine in VWR Signature Ultrasonic Cleaner Model 75D (Power,
chewing gum and transdermal formulations [7–9]. All these 90 W) with digital timer, heat and power (VWR, S. Plainfield,
reported HPLC methods employed expensive and environ- NJ, USA) was employed for all ultrasonic extractions.
mentally harmful organic solvents, and required complex and
time-consuming extraction procedures. Before HPLC deter- 2.3. Extraction of nicotine from pharmaceutical
mination, the gum and transdermal patch have to be first formulations
dissolved in a non-polar or weakly polar solvent to release
nicotine from the formulations. The nicotine can then be ex- A nicotine gum was weighed and cut into small pieces
tracted into an acidic or a basic aqueous phase which can and ground after the small pieces were frozen with liquid ni-
be directly analyzed by reversed-phase or ion-pair HPLC trogen in a mortar. Samples of the crushed gum were placed
formulations [7,8]. However, this procedure requires 100% separately in glass vials containing 10 mL heptane and the
extraction efficiency, which cannot be achieved in a sin- capped vial was sonicated for 20, 40, and 60 min at 37 ◦ C in
gle conventional extraction. It needs generally two or more an ultrasonic bath, respectively. After the separation of the
extractions to achieve a quantitative recovery of nicotine supernatant by centrifugation, the supernatant liquid was de-
into aqueous phase. Each of these conventional extractions canted into a 25 mL volumetric flask and additional solvent
takes up to 24 h. Recent studies have shown that ultrasonica- was used to rinse the gum residues. The rinses were added
tion of samples in organic solvents represents an alternative to the solution to make up to the mark. An aliquot of ex-
technique for speeding up sample extraction [20–22]. Ul- tract and 1.0 mL of 1.0 × 103 mg/L internal standard (I.S.)
trasound waves, when imparted to solutions, causes acous- N-ethylaniline were added into a 10 mL volumetric flask, di-
tic cavitation, that is bubble formation and subsequent im- luted and mixed for the GC analysis. This extraction and
plosion. The collapse of bubbles created by the sonication analysis process was repeated on the residue.
of solutions results in the generation of high local energy For nicotine transdermal systems, a nicotine patch without
and a high contact between solvent and solute [23], and the protecting liners was weighed and cut into small pieces.
can thus greatly increase the sample extraction efficiency. Accurately weighed small patch pieces (∼1/8 patch) were
Gas chromatography can directly separate an analyte mix- immersed in 10 mL heptane, sonicated and analyzed in the
ture in volatile organic solvents. The application of capil- same manners as those for the nicotine gum. All experiments
lary GC coupled with an efficient ultrasonic extraction tech- were performed in triplicates.
nique could avoid the time-consuming multiple extraction
procedures and speed up the analysis. In this study, we have 2.4. Gas chromatography analysis
aimed to develop a fast, accurate, robust and environmen-
tally friendly ultrasonic extraction and capillary GC method GC analysis was carried out on a Shimadzu GC-17A
for the determination of nicotine in pharmaceutical formula- gas chromatograph equipped with a flame ionization detec-
tions. tor (FID) and a Shimadzu AOC-20i GC auto-injector. Sam-
 Y. Zuo et al. / Analytica Chimica Acta 526 (2004) 35–39 37

ples were separated on a 30 m × 0.32 mm i.d., 1.00 ␮m film

ECTM -5 capillary column (Alltech Inc., Deerfield, IL, USA).
The stationary phase is made up of 5% phenyl- and 95%
dimethyl-polysiloxane mixture. Helium was employed as
carrier gas and linear velocity of He was 27 cm s−1 . N2 make
up gas and H2 and compressed air were used for the FID.
A split/splitless injector was used under splitless mode. The
injector volume was 2.0 ␮L. The column temperature was
initially held at 85 ◦ C for 2 min, then programmed to 180 ◦ C
at a rate of 20 ◦ C/min, held there for 1 min, then ramped
up from 180 to 280 ◦ C at a rate of 40 ◦ C/min, with a final
hold time of 8 min. The injector and detector temperature
were maintained at 240 and 280 ◦ C, respectively. The peak
identification and purity verification was performed by em-
ploying a Hewlett-Packard (HP) model GC 5890 Series II
gas chromatograph coupled with an HP 5971 series mass se-
lective detector and an HP 7673 GC autosampler [24,25].
The characteristic ions at m/z 162 (M+ ), 133, and 84 in the Fig. 2. Effect of temperature on the extraction efficiency of nicotine. Ex-
mass spectrum have been employed to identify nicotine in the traction time: 60 min.
sample.
ditions, leading to undesired oxidation of the analytes [26].
2.5. Calibration To test this aspect, solutions of 6.5 × 102 mg/L of nicotine
in heptane with and without formulation extracts were soni-
Calibration was made by plotting the ratio of the peak areas cated at 37 ◦ C in the ultrasonic bath and the concentration
of nicotine to that of the internal standard as a function of the of nicotine was measured at various time intervals up to
nicotine concentration of the standards. This ratio for the 90 min, no detectable degradation of nicotine has been ob-
samples is then used to obtain their nicotine concentrations served.
from a calibration curve. All the calibration standards and To extract nicotine from the pharmaceutical formulations,
pharmaceutical samples were run in triplicates. the ground chewing gum sample or small pieces of trans-
dermal patch was placed in 15 mL glass vial containing
10 mL heptane and sonicated 20, 40, and 60 min at 37 ◦ C
3. Results and discussion in the ultrasonic bath. The extraction efficiency of each ex-
tract in various formulations is given in Table 1. The re-
3.1. Extraction of nicotine from nicotine chewing gum sults indicate that the recovery of nicotine increases with
and transdermal system patch increasing sonication time. A complete extraction of nico-
tine was obtained in less than 20 min from the transder-
Since nicotine was available either as a chewing gum mal patch and 60 min from the chewing gum. Ultrasonic
or as a transdermal system patch, extraction of nicotine extraction reduced the extraction time from 24 h required
from these pharmaceutical formulations was essential prior by applying conventional cold extraction technique to less
to chromatographic analysis of nicotine. In previous stud- than 20 min. The extraction time of 30 min for the nicotine
ies, a conventional liquid extraction method was used for transdermal system patch and 60 min for the chewing gum
the extraction of pharmaceutical formulations [7,8]. The lat- has been chosen for the determination of nicotine in these
est studies have shown that ultrasonication can accelerate formulations.
sample extraction [20–23]. To examine the possible acceler-
ative effect of ultrasound on nicotine extraction, the nico- Table 1
tine chewing gum samples in 10 mL heptane were ultra- Relative extraction efficiency of nicotine with 10 mL heptane
sonicated at three different temperatures for 60 min. The Formulations Sonication First extract Second extract
results in Fig. 2 demonstrated that a 100% extraction ef- time (min) (% extracted)a (% extracted)a
ficiency of nicotine from the chewing gum samples was
achieved at 37 and 50 ◦ C within 1 h; while with the con- Chewing gum 20 95.9 ± 1.4 4.09 ± 0.24
ventional liquid extraction three 24 h extractions are needed 40 98.0 ± 1.2 1.96 ± 0.13
to achieve the same extraction efficiency [7]. Nevertheless, 60 100.0 ± 0.8 0.00 ± 0.01
the application of ultrasound to the extraction of organic an- Transdermal patch 20 99.9 ± 0.8 0.07 ± 0.02
alytes from sample matrix must be further carefully eval- 40 100.0 ± 0.6 0.00 ± 0.01
uated because the acoustic cavitation may produce reac- 60 100.0 ± 0.6 0.00 ± 0.01
tive transients such as free radicals and the pyrolytic con- a Mean ± S.D.; n = 3.
38 Y. Zuo et al. / Analytica Chimica Acta 526 (2004) 35–39

analysis of a series of nicotine samples. The relative stan-

dard deviation (R.S.D. %) was found to be 0.01 and 0.02%
for N-ethylaniline and nicotine, respectively. The precision
in the peak area was better than 1.9% for eight consec-
utive injections of the same nicotine sample. Good peak
area precision was achieved without adding any internal
standard.
Day-to day precision was also evaluated by performing
five injections of standard solutions and formulation ex-
tracts each day on four different days within 2 weeks pe-
riod. Day-to-day precision (R.S.D.) on the basis of retention
time and peak area were better than 0.03 and 2.0%, respec-
tively.
Repeatability of the method was performed by three ana-
Fig. 3. GC chromatogram of nicotine and ethylaniline as internal standard lysts (five determinations by each analyst) using the proposed
(I.S.). method and the same instrumentation. The results showed no
significant differences: R.S.D. % = 0.87.

3.2. Chromatographic separation of nicotine in 3.4. Analysis of pharmaceutical formulations

standard mixtures and pharmaceutical formulations
The method developed was applied to the determination of
Various GC stationary phases, both polar [14–17] and non- nicotine level in pharmaceutical formulations. The amounts
polar [12,13,18,27], have been previously employed to sep- of nicotine determined were 1.96 ± 0.06 mg/gum for twelve
arate nicotine and other tobacco alkaloids. In early methods, 2 mg nicotine chewing gums and 52.2 ± 3.6 mg/patch for six
a polar stationary phase of Carbowax and potassium hydrox- 52.5 mg nicotine transdermal system patches. These results
ide was frequently used. Later, some studies [13,19] reported
problems of adsorption and tailing of more basic alkaloids in
polar stationary phases. After multiple preliminary assays, an
ECTM -5 capillary column with a non-polar stationary phase
of 5% phenyl- and 95% dimethyl-polysiloxane mixture was
selected in this study. Fig. 3 shows a representative separation
of a standard mixture of nicotine and the internal standard.
No interfering peaks were observed in the chromatogram.
Fig. 4A and B illustrates the chromatograms obtained from
the nicotine chewing gum and nicotine transdermal system
patch, respectively. In both chromatograms, a baseline sepa-
ration between nicotine and internal standard and other com-
ponents was achieved in 10 min using the method described.
GC–MS experiments further confirmed the determinations
of nicotine in the chewing gum and the transdermal patches
without any interference.

3.3. Method validation

Calibration curves were obtained for nicotine using a se-

ries of standard solutions over the concentration range from
1.00 to 5.0 × 102 ␮g/mL. Three replicate injections of stan-
dards at each concentration were performed. All calibration
curves were linear over the concentration ranges tested with
correlation coefficients r2 > 0.998. The determination limit
was 0.25 ␮g/mL, which was calculated as the concentration
of nicotine that gives rise to peak height with a signal-to-noise
ratio of 3.
The reproducibility of the retention time of the inter-
nal standard N-ethylaniline (7.7 min) and nicotine (9.6 min) Fig. 4. Chromatograms of extracts of nicotine chewing gum (A) and nicotine
was determined from 25 consecutive injections during an transdermal system patch (B).
 Y. Zuo et al. / Analytica Chimica Acta 526 (2004) 35–39 39

are in good agreement with those determined using conven- References

tional extraction HPLC methods [7,28]. According to U.S.P.,
the gum formulation should contain not less than 95% (w/w) [1] J. Le Houezec, Int. J. Tuberc. Lung Dis. 7 (2003) 811.
and not more than 110% (w/w) of the labeled amount of nico- [2] D. Yildiz, Toxicon 43 (2004) 619.
[3] CDC, Cigarette smoking among adults in the United States, Mor-
tine [8]. The nicotine transdermal system must contain not bidity Mortality World Rep. 51 (2002) 300–303.
less than 90% (w/w) and not more than 110% (w/w) of the la- [4] J.P. Pierce, E. Giplin, A.J. Farkas, J. Natl. Cancer Inst. 87 (1992)
beled amount of nicotine [29]. Both these formulations were 87.
found to satisfy the U.S.P. standards. [5] C.T. Orleans, New Jersey Med. 85 (1998) 116.
[6] M. Law, J.L. Tang, Arch. Int. Med. 155 (1995) 1933.
[7] A.K. Dash, S.-T. Wong, J. Chromatogr. A 749 (1996) 81.
[8] First Supplement to The United States Pharmacopeia, XXIII Revi-
4. Conclusion
sion, The United States Pharmacopeial Convention, Rockville, MD,
1995, pp. 2485–2486.
A rapid, sensitive, and accurate ultrasonic extraction and [9] B. Sellergren, A. Zander, T. Renner, A. Swietlow, J. Chromatogr. A
capillary GC method has been developed for the determina- 829 (1998) 143.
tion of nicotine in pharmaceutical formulations. The results [10] E.F. Domino, M. Hariharan, T. VanNoord, T. Demana, Med. Sci.
Res. 20 (1992) 859.
obtained in this study indicate that ultrasound is a reliable
[11] A. Marsh, B.J. Clark, K.D. Altria, Electrophoresis 25 (2004)
tool for the fast extraction of nicotine in pharmaceutical 1270.
formulations. The ultrasonic extraction can shorten the [12] J. Cai, B. Liu, P. Lin, Q. Su, J. Chromatogr. A 1017 (2003) 187.
extraction time from 24 h required by conventional cold [13] M.D.G. Lourenço, A. Matos, M.C. Oliveira, J. Chromatogr. A 898
extraction technique to less than 20 min. The solvent con- (2000) 235.
[14] A.H. Beckett, E.J. Triggs, Nature 211 (1966) 1415.
sumption is six times lower in ultrasonic extraction than in
[15] C. Feyerabend, A.E. Bryant, M.J. Jarvis, M.A.H. Russell, J. Pharm.
conventional extraction methods. Capillary GC can directly Pharmacol. 38 (1986) 917.
separate and quantitate the components in ultrasonic extracts [16] D.T. Burns, E.J. Collin, J. Chromatogr. 133 (1977) 378.
without subsequent clean-up step, which usually required [17] M.V. Djordjevic, L.P. Bush, S.L. Gay, H.R. Burton, J. Agric. Food
by using reversed-phase HPLC methods. The analysis Chem. 38 (1990) 347.
[18] R.A. Davis, M.F. Stiles, J.D. DeBethizy, J.H. Reynolds, Food Chem.
time is considerably reduced. In addition, GC methods do
Toxic. 29 (1991) 821.
not use environmentally harmful organic mobile phase as [19] R.F. Severson, K.L. McDuffie, R.F. Arrendale, G.R. Gwynm, J.F.
HPLC methods. This method has been successfully used to Chaplin, A.W. Johnson, 211 (1981) 111.
determine the nicotine content in both nicotine chewing gum [20] T.S. Koh, Anal. Chem. 55 (1983) 184.
and transdermal patches. The described technique provides [21] M. Baas, R. Pancost, B. van Geel, J.S. Simmingh Dausté, Org.
Geochem. 31 (2000) 535.
an example of green analytical chemistry and can also be
[22] G.M. Greenway, Q.J. Song, J. Environ. Monitor. 4 (2002) 950.
used as an ideal analytical method in the quality control [23] K.S. Suslik, Science 247 (1990) 1439.
process for other tobacco products. [24] Y. Zuo, C. Wang, J. Zhan, J. Agric. Food Chem. 50 (2002) 3789.
[25] K. Zhang, Y. Zuo, J. Agric. Food Chem. 52 (2004) 222.
[26] E. Taylor Jr., B.B. Cook, M.A. Tarr, Ultrason. Sonochem. 6 (1999)
175.
Acknowledgement
[27] J.I. Seeman, J.A. Fournier, J.B. Paine III, B.E. Waymack, J. Agric.
Food Chem. 47 (1999) 5133.
This research was partly supported by the Department of [28] A.V. Gore, Y.W. Chien, Clin. Dermatol. 16 (1998) 599.
Chemistry and Biochemistry, University of Massachusetts [29] Pharmacopeial Forum, vol. 21, The United States Pharmacopeial
Dartmouth. Convention, Rockville, MD, 1995, pp. 585–589.