Journal of Pharmaceutical and Biomedical Analysis

32 (2003) 441 /450 www.elsevier.com/locate/jpba

A validated high performance liquid chromatographic method

for analysis of nicotine in pure form and from formulations
Kaustubh R. Tambwekar, Ritesh B. Kakariya, Sanjay Garg *
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar,
Punjab 160 062, India

Received 22 April 2002; received in revised form 12 November 2002; accepted 5 January 2003

Abstract

A reverse phase HPLC method using C18 column has been developed for the quantitative estimation of nicotine in
the bulk material and formulations (extended release and immediate release dosage forms). The method is specific to
nicotine (RT
/4.64 min, asymmetry
/1.75), and can resolve analyte peak from excipient interferences. It is linear
(coefficient of variation
/0.9993), accurate (average recovery
/100%), and passed all the system suitability
requirements. Applicability of the method was evaluated in analysis of drug-excipient compatibility samples,
commercial dosage form (such as nicotine gum) and two novel in-house formulations.
# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Nicotine; HPLC method; Bulk analysis; Extended release formulations; Immediate release formulations; Dissolution;
Analysis of nicotine gum

1. Introduction ability, anxiety, and decrease in concentration

which forces smoker to continue smoking [2].
Smoking is one of the largest single preventable Nicotine replacement therapy (NRT), aimed at
causes of ill health in the world [1], which leads to reducing withdrawal symptoms, has a greater
loss of personal as well as national income through success potential than any other methodology
various associated diseases. Nicotine is the princi- known so far. NRT helps the smoker to overcome
ple alkaloid in tobacco and is responsible for these withdrawal symptoms by providing nicotine
causing dependence due to its psychoactive prop- in therapeutic doses in a tapering manner over a
erties and capacity to induce self-administration period of time [3,4]. Currently, NRT consists of
behavior. Nicotine abstinence results in precipita- four nicotine products (gum, patch, spray, and
tion of various withdrawal symptoms like irrit- inhaler), which have specific advantages. Novel
formulations of nicotine have been developed in
the investigators laboratory, which are targeted at
* Corresponding author. Tel.:
/91-172-21-4682 /87; fax:
/
smoking cessation [5].
91-172-21-4692. Nicotine (Fig. 1) is a weak, diacidic base having
E-mail address: gargsanjay@yahoo.com (S. Garg). two pK a values and is highly soluble in solvents
0731-7085/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0731-7085(03)00236-X
442 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450

chemical detector (ECD) or hazardous chemicals

like barbituric acid [6,20], potassium cyanide [12],
and bromine [7] have been used in these methods.
Examples of these methods include HPLC coupled
with mass detector [21], HPLC coupled with glassy
carbon detector [22], ion-pair HPLC using various
Fig. 1. Structure of nicotine.
sulphonic acid sodium salts [13,23], and combina-
tion of reverse phase and ion-exchange chromato-
such as water, methanol, acetonitrile, chloroform, graphy [8].
and petroleum ether. Literature reports colori- The methods described above were essentially
metric [6,7], spectrophotometric, and chromato- bioanalytical in nature. They have been used either
graphic methods for analysis of nicotine. Amongst to support pharmacokinetic studies on nicotine,
determination of free base and its metabolites in
these, chromatographic methods have been widely
various physiological fluids such as saliva [24,25],
used [8 /12]. Pichini et al. [13] has reported
plasma/serum [26,27], urine [28], and nicotine
application of solvent optimization software in
determination from tobacco samples etc. These
method development that uses C18 column for
methods though highly specific and sensitive for
isolation and quantitation of nicotine present in
nicotine, may not yield precise and accurate results
serum of smokers as well as spiked serum samples.
during in vitro analysis of nicotine dosage forms.
The method successfully isolated various peaks
Nicotine dosage forms (gum and patch) are
and mobile phase consisted of ion-pairing reagent.
official in USP [15]. USP mentions chromato-
Gas chromatographic methods are cited in litera-
graphic method for nicotine polacrilex gum (assay)
ture, however, Thompson et al. has reported
and transdermal patch (release studies and uni-
inability of such methods to quantitate labile formity of dosage). These methods use mobile
compounds such as nicotine 1?-N-oxide [14]. Gas phase additives such as surfactants (sodium dode-
chromatography is the official method for deter- cyl sulfate), amines (triethylamine, dioctylamine),
mination of chromatographic purity of nicotine in and sulfonic acid esters (sodium 1-decanesulfonate
United States Pharmacopoeia [15]. Ion-pairing and dodecane sulfonate). At the same time,
and ion-suppression methods have been most Pharmacopoeia mentions different chromato-
commonly employed for the analysis of weakly graphic parameters for various analyses involving
basic drugs such as nicotine [16], however, they nicotine products. The method developed in our
have certain disadvantages. Methods based on laboratory has a specific advantage over a USP
ion-pairing principle usually exhibit poor selectiv- method that, it does not use any of the above-
ity, and often result in band broadening due to mentioned mobile phase additives. In addition,
inadequate buffering action or dissociation of ion- single method can be adopted for wide variety of
pairs [17]. Nicotine, being a weak base has a pK a analyses such as in vitro release studies, content
value in the alkaline range. In order to fully uniformity, selection of excipients for a formula-
suppress the ionization of nicotine, mobile phase tion based on drug-excipient analysis etc. that is
pH should be selected in the range of 8/9, at which not the case with the reference method.
most of the silica-based phases are unstable. We used a base-deactivated C18 column wherein
Under the conditions of uncontrolled ionization, the residual silanol groups have been deactivated
a strong polar interaction of nicotine with residual by special procedures. This kind of packing
silanol groups on the silica surface often results in material requires minimal or no mobile phase
band broadening and poor efficiency [18]. Use of additives such as amines, ion-paring agents etc.
triethanolamine is, therefore, suggested by some Base-deactivated silica columns are widely used
workers [19] while others have used both amine worldwide for analyses involving basic drugs such
and ion-pairing agents simultaneously [15]. In as nicotine mainly for superior quality of analysis
addition, costly detector systems such as electro- and economic advantage when compared with
 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450 443

ordinary C18 columns. The proposed method has AG-245 electronic balance, and Millipore Filtra-
been developed and fully validated with a specific tion assembly were used in the study. Water used
aim of having a method, which is simple in throughout the HPLC analysis was prepared by
operation, cost-effective, avoids hazardous chemi- reverse-osmosis using USF ELGA system. Dis-
cals and able to analyze bulk material, uniformity solution studies were conducted in USP 24 dis-
of dosage, in vitro release samples, and to detect solution apparatus (Electrolab, India).
minute changes that may take place during drug-
excipient compatibility study. 2.2. Materials
The spectrum of applications covered by the
method will definitely help readers to use this (-) Nicotine was purchased from Sigma Chemi-
method in routine analysis of nicotine as well as cals, USA (Lot no. 108H1312). Similarly nicotine
conducting various studies on the formulations. hydrogen tartrate was also procured from Sigma
chemicals, USA (Lot no.100K3250). Both nicotine
and nicotine hydrogen tartrate were used after
2. Experimental performing initial assay by non-aqueous method
as given in USP 24 [15].
2.1. Instrumentation Potassium dihydrogen orthophosphate GR was
obtained from Hi Media Laboratories Ltd., Mum-
A Shimadzu HPLC system equipped with LC- bai, India, and HPLC grade methanol was ob-
10ATVP Pump, DGU-14AM on-line degasser, tained from Rankem, Punjab, India.
SIL-10-ADVP auto injector, CTO-10-ASVP col- Two in-house formulations (NIPER ER and
umn oven, and SPD-10AVP-UV-VIS detector was NIPER IR) were evaluated using the present
utilized. The second instrument, Shimadzu HPLC method. NIPER ER contained 8.75 mg of nicotine
system equipped with LC-10ATVP pump, DGU- hydrogen tartrate salt (equivalent to 2.8 mg of
14A on-line degasser, SIL-10-ADVP auto injector, nicotine) per tablet in a matrix of bioadhesive
CTO-10AVP column oven and SPD-M10AVP- polymers and NIPER IR contained 2 mg of
PDA detector was used for determining peak nicotine per tablet along with other excipients [5].
purity. Shimadzu CLASS-VP software (Version The excipients used for the development of NI-
5.03) was used for data acquisition and system PER ER and NIPER IR were obtained from
suitability calculations. The chromatographic con- commercial sources and were used as such. Table 2
ditions are outlined in Table 1. In addition, gives the composition of NIPER ER and NIPER
Branson 3510 ultrasonic bath, Mettler Toledo
Table 2
Table 1 Composition of nicotine in-house formulations
Chromatographic parameters for determination of nicotine
Ingredients
Parameter Condition
NIPER IR NIPER ER
Method Reverse phase high performance liquid
chromatography Nicotine hydrogen tartrate Nicotine hydrogen tartrate
Column Hypersil C18 BDS (Thermo Hypersil, UK) Flowlac Flowlac
250/4.6 mm, and 5 mm particle size Avicel PH-102 Metolose 90SH100
Flow rate 1 ml/minute Pearlitol SD100 Carbopol 974P
Detection UV detector, 259 nm PDA detector, 200 / L- HPC Polycarbophil
800 nm for peak purity testing Magnesium stearate Magnesium stearate
Column tem- 35 8C Polyplasdone Talc
perature Ac-Di-Sol
Injection volume 20 ml Aspartame
Mobile phase Phosphate buffer (pH 6.8; 10 mM):metha- Menthol
nol (35:65% v/v) Aerosil
444 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450

IR formulations. For practical purposes, the in- 2.3.3. Specificity of method for dissolution studies
house formulations were prepared using equiva- To determine the specificity of the method in
lent amount of nicotine hydrogen tartrate dihy- presence of excipients, a polymer matrix (100 mg)
drate. Marketed NRT formulations such as consisting different excipients present in final
nicotine gum (Nicorette† containing 2 mg nico- formulation was prepared in 100 ml of mobile
tine, Lot BL 516, expiry March 2003 and phase. 20 ml of this solution was injected on
Nicorette† containing 4 mg nicotine, Lot BL column after filtration through 0.45 mm nylon
086, expiry April 2003) were purchased from the filter and peak response was recorded.
retail pharmacies.
2.3.4. Accuracy and recovery studies
The developed analytical method was validated
2.3. Method validation for its accuracy in determining the drug content
from solution and from the excipient blend. Two
The developed method was validated for the different procedures were used for analyzing drug
parameters like linearity, range, precision, repro- solution and excipient blend as given below.
ducibility, specificity, accuracy, recovery, filter
validation, and system suitability as described 2.3.4.1. Drug solution (assay). For demonstrating
below. accuracy of an analytical method, three concen-
tration levels of drug solution (80, 100, and 120%
2.3.1. Linearity of assay concentration) were prepared in triplicate
A stock solution of nicotine 500 mg/ml was and analyzed.
prepared by dissolving 156.25 mg of accurately
weighed nicotine hydrogen tartrate dihydrate 2.3.4.2. Recovery from excipient blend (assay by
(equivalent to 50 mg of nicotine) in 100 ml of spiking). Recovery studies from excipients blend
phosphate buffer (pH 6.8). This was labeled as was carried out by spiking a specified amount of
solution ‘‘A’’. Various dilutions were prepared in drug solution (80, 100, and 120% of assay con-
duplicate using solution A in the concentration centration) in 100 mg of excipient matrix in a small
range of 2/40 mg/ml in phosphate buffer (pH 6.8). vial. The solutions were prepared in methanol in
The samples were filtered through 0.45 mm nylon triplicate. The samples were mixed thoroughly
filter and injected on column in duplicate. Areas using vortex mixer and allowed to dry in a dark
for four injections were determined and graph place. The dried blend was then quantitatively
prepared. Slope and intercept were estimated. transferred to 50 ml volumetric flask avoiding
losses during transfer and 35 ml of phosphate
buffer was added. The solution was sonicated for
2.3.2. Precision 30 min in an ultrasound bath. The volume was
adjusted to 50 ml using buffer and the solutions
2.3.2.1. Repeatability. Repeatability of the method were filtered through 0.45 mm nylon filter. 20 ml of
was checked by analyzing six replicate samples of this solution was injected in duplicate. Percentage
nicotine (at the 100% assay concentration i.e. 28 of drug recovered was calculated using a standard
mg/ml) and calculating percent relative standard curve prepared on the same day. Similarly, the
deviation (% R.S.D.). changes, if any, in retention time and peak shape
were also recorded in the presence and absence of
excipient blend.
2.3.2.2. Intermediate precision. Intermediate preci-
sion of the method was checked by repeating the 2.3.5. Filter validation
entire procedure for 3 consecutive days and Filter validation was performed by analyzing
calculating the R.S.D. between 3 days for area, solutions at 2 and 40 mg/ml (the lowest and the
slope, and intercept. highest concentration of the linearity curve). The
 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450 445

solutions were analyzed in duplicate after filtration Samples were dissolved in 50 ml HPLC metha-
through 0.45 mm nylon filter. The results were nol and kept aside for 15 min. Volume was made
compared with the unfiltered sample injected at upto 100 ml with phosphate buffer such that the
the same concentration levels and the amount concentration is approximately 1 mg/ml. This
retained by the filter was calculated. solution was suitably diluted to 50 ml with
phosphate buffer (28 mg/ml). The solutions were
2.3.6. System suitability filtered through 0.45 mm nylon membrane and
Data from five injections (at 100% assay con- areas were determined. Amount of nicotine pre-
centration) was utilized for calculating system sent in the mixtures was determined using stan-
suitability parameters like capacity factor, asym- dard curve.
metry, number of theoretical plates and area using
CLASS-VP software. 3.2. Content uniformity

An accurately weighed tablet was triturated in a

3. Analysis of nicotine formulations mortar and the contents were transferred quanti-
tatively to a 10 ml volumetric flask. The contents
Utility of the proposed method was evaluated were dispersed in 8 ml of methanol with the help of
by performing analysis of drug-excipient compat- sonication for 10 min and the volume was made up
ibility samples. This exercise was carried out to to mark with methanol. 1 ml of this solution was
select various excipients for formulation develop- diluted to 10 ml with phosphate buffer (pH 6.8) in
ment. The developed analytical method was also a 10 ml volumetric flask. The samples, after
tested for analysis of nicotine from the in-house filtration through a 0.45 mm nylon filter were
formulations. An immediate release formulation subjected to HPLC analysis. The experiment was
NIPER IR (2 mg of nicotine per tablet) was repeated on five additional tablets.
evaluated for content uniformity while extended
release formulation NIPER ER containing 2.8 mg 3.3. Dissolution studies
of nicotine per tablet was evaluated for content
uniformity and in vitro dissolution. Dissolution study with NIPER extended release
formulation (n/6) was carried out on a modified
3.1. Drug-excipient compatibility study USP II dissolution apparatus using a rotating
paddle method (50 RPM). Phosphate Buffer pH
Accurately weighed amounts of drug and ex- 6.8 (100 ml) maintained at 379/0.5 8C was used as
cipients in 1:1 ratio were taken in 5 ml glass vials dissolution medium. The samples (5 ml) were
and mixed well. Water equivalent to 5% w/w (10 withdrawn at the predetermined time and replaced
ml) was added and mixed thoroughly. The samples with an equivalent amount of fresh medium. The
then stored at 40 8C and 75% RH in stability samples were filtered through 0.45 mm nylon
chambers for 3 weeks. Similarly, another set of membrane filter and analyzed using validated
samples were prepared, screw capped and stored at HPLC method. The cumulative percent drug
4 8C in the refrigerator. Isothermally stressed release was plotted against time to determine the
samples were visually evaluated after first, second release profile.
and third week of storage to note any physical
change in the mixture and compared with the 3.4. Analysis of nicotine gum
control samples. After 3 weeks, all the samples
(both stressed and control) were quantitatively An accurately weighed nicotine gum was cut
analyzed by the proposed HPLC method. Simi- into four pieces and transferred to 250 ml stop-
larly, another set of vials were prepared without pered conical flask. The contents of the gum were
adding water and subjected to the stress condi- extracted in 50 ml of phosphate buffer (pH 6.8)
tions. and 50 ml of n-hexane under vigorous stirring or
446 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450

till the contents were fully dispersed. The aqueous 4.1. Linearity
layer was separated and washed the precipitate
with additional 50 ml of phosphate buffer (pH Table 3 describes regression statistics obtained
6.8). Combined the washings and filtered through for various analytical tests. The linearity of an
0.45 mm nylon membrane filter. 20 ml of this analytical method is its ability to elicit test results
solution was injected and response recorded. that are directly, or by a well-defined mathema-
Amount of nicotine present per unit of gum was tical transformation, proportional to concentra-
calculated from the standard curve. The analysis tion of analyte within a given range. The linearity
was repeated on five additional units. of the method was observed in the expected
Another set of analysis was conducted on 4 mg concentration range, demonstrating its suitability
nicotine gum as per the USP method [15]. The for analysis. The goodness of fit (R2) was found to
chromatographic conditions used were as given by be 0.9993.
USP except for the replacement of 30-cm column
with 25-cm column. 4.2. Precision

The precision of an analytical method is the

degree of agreement among the individual test
results when the method is applied repeatedly to
4. Results and discussion multiple sampling of homologous sample. Repeat-
ability refers to the use of analytical procedure
Fig. 2 represents chromatogram of nicotine within a laboratory over a short period of time
obtained by the developed method. Nicotine elutes using the same analyte with the same equipment
at retention time of 4.64 min with an asymmetry and is expressed as the percent R.S.D. The method
value of 1.75. passed the test for repeatability as determined by

Fig. 2. Representative chromatogram of nicotine using a developed method.

 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450 447

Table 3
Regression statistics for nicotine

Parameter Target concentration (mg/ml) Range (mg/ml) Goodness-of-fit (R2) Slope Intercept

Assay, release, and compatibility study 28 2 /40 0.9993 4876.6 /174.94

percent R.S.D. (1.38%) of the area of the peaks of the true value. It can be determined by application
six replicates injection at 100% assay concentra- of analytical procedure to an analyte of known
tion. purity (for a drug substance) or by recovery
Intermediate precision involves estimation of studies, where known amount of standard is
variations in analysis when a method is used spiked in the placebo (for the drug product). The
within laboratories, on different days, by different results of accuracy studies from solution and
analysts, and on different equipments. The inter- excipient matrix are shown in Table 5, and it is
mediate precision was studied by preparing the evident that method is accurate within desired
standard curve for 3 different days, and the results range.
of interday variation are given in Table 4. The
method passed the test for intermediate precision 4.5. Filter validation
as percent R.S.D. of the slope and intercept
obtained with 3 different days were within the The R.S.D. obtained at higher concentration
limits of 2%. (0.08%) and lower concentration (1.27%) indicates
suitability of the nylon filter for the filtration of
4.3. Specificity the dissolution sample, as the R.S.D. is less than
2%.
Specificity is the ability of an analytical method
to assess unequivocally the analyte in the presence 4.6. System suitability testing
of components that are present in the sample
matrix. The representative chromatogram (Fig. 3) System suitability test are an integral part of
of excipient blend shows that excipients do not chromatographic methods and are used to verify
interfere with the drug peak indicating specificity that the resolution and reproducibility of the
of the method for nicotine. system are adequate for the analysis to be per-
formed [29]. The results of system suitability are
4.4. Accuracy given in Table 6. All the values for the system
suitability parameters are within the acceptable
The accuracy of an analytical method is the range.
closeness of test results obtained by the method to
4.7. Applicability of method
Table 4
Intermediate precision The developed method was successfully applied
for the assay of nicotine in mixtures containing
Concentration Intra-day variation Inter-day variation
(mg/ml) (% R.S.D.) (% R.S.D.)
various excipients, NIPER extended release, im-
mediate release, and marketed formulations. The
2 2.0 1.99 results of drug-excipient compatibility study re-
4 1.74 2.21 vealed absence of co-elution at the retention time
8 0.95 1.63
16 0.69 0.51
of nicotine (peak purity /0.9997). There was no
32 0.64 1.21 significant degradation of nicotine in mixtures at
40 1.24 0.48 the end of 3-week storage period (average assay /
95%). The content uniformity results are shown in
448 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450

Fig. 3. Chromatogram of excipient blend.

oped method and dissolution profile is shown in

Table 5
Fig. 4.
Accuracy/recovery data for nicotine
The proposed method was also compared with
Parameter Concentration level (mg/ Recovery R.S.D. the reference method [15] with respect to assay of
ml) (%) marketed formulations and linearity. Linearity
Assay 80 101.33 0.37 was determined as per the chromatographic
100 102.14 1.11 method given by USP and the results are given
120 100.89 1.94 in Table 8.
Assay (Spik- 80 97.76 0.55
ing)
100 98.21 1.05
120 100.59 1.50 5. Conclusion

A simple HPLC method using a C18 type

column was developed for the analysis of nicotine
Table 7, which demonstrates the suitability and in bulk material, extended, and immediate release
wide applicability of the developed method. Dis- formulations. The method was specific for nicotine
solution samples are also analyzed using a devel- and was validated with respect to various analy-

Table 6
System suitability parameters

Parameter Maximum Minimum % R.S.D. Status

Asymmetry 1.60 1.58 0.559 Passed

Capacity factor 0.45 0.43 1.853 Passed
Theoretical plates 1871.25 1780.24 1.708 Passed
Area 146 958 146 328 0.179 Passed
 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450 449

Table 7
Content uniformity of nicotine gum and NIPER formulations

Sr. No. Product Nicotine label claim (mg/unit) Amount found (mg) Recovery (%) % R.S.D.

1 NIPER IR 2.0 2.02 101.00 4.83

2 NIPER ER 2.8 2.81 100.35 2.71
3 Nicotine gum 2.0 1.99 99.5 4.01

at the retention time of nicotine under the

proposed method as indicated by peak purity
index. Although, both the methods gave accepta-
ble assay values with percent R.S.D. much lower
for USP method than proposed method, the
goodness of fit (R2) and other linearity parameters
suggested enhanced accuracy and sensitivity of in-
house method.
Further, due to the presence of ion-pair reagent,
Fig. 4. Dissolution profile of NIPER ER formulation. it is expected for reference method to have a longer
column equilibration time (/3 h) in order to
tical parameters. The method was found suitable reduce retention time variation (a normal phenom-
for the analysis of in-house nicotine formulations enon with such methods) while under proposed
as well as marketed dosage forms. method, chromatographic conditions stabilized in
The proposed method had specific advantages less than an hour. In such cases, cost considera-
over the USP method. The elution time of nicotine tions acquire a significant dimension. In addition
under the proposed method is much less than the to this, quaternary mobile phase and a solvent
reference method indicating suitability for rapid mixture of similar composition is mentioned in the
determination of nicotine from bulk/formulations. USP thereby increasing the complexity of the
Though, the theoretical plates for proposed method. Though, the initial cost of special base-
method were less as compared with USP method, deactivated silica column is slightly higher than the
however, they qualified the pharmacopoeial re- conventional C18 reversed phase column, however,
quirement. Also, the peak shape of nicotine was the long term benefits (rapid analysis, simple
reasonably good and principal peak was well chromatographic conditions, accuracy and sensi-
separated from the mobile phase interferences. tivity of system, operational convenience, and cost
There was no co-elution of excipients/tartrate salt reduction) offered by the proposed method are

Table 8
Comparison of proposed method vs. USP24 method

Parameter USP method Proposed method USP limits

Retention time (min) 35.97 4.64 Not given

R.S.D. for replicate injections 1.4% 1.38% NMT 2.0%
Asymmetry (10%) 1.31 1.75 NMT 2.0
Theoretical plates (N) 12127.58 5453.51 NLT 2500
Slope (linearity curve) 33 925 4876.6 Not given
Intercept (linearity curve) 16 567 /174.94 Not given
Goodness of fit (R2) (linearity curve) 0.9983 0.9993 /0.999
% Assay (R.S.D.) 103.5% (0.14) 99.5 (4.0) 90 /120%
450 K.R. Tambwekar et al. / J. Pharm. Biomed. Anal. 32 (2003) 441 /450

significant. Therefore, under the experimental [8] M.R. Carlisle, M.L. Chicone, M.B. Wygant, Int. J. Pharm.
80 (1992) 227 /242.
conditions, the proposed method was found to
[9] L.A. Ciolino, D.B. Fraser, Y. Yit, J.A. Turner, D.Y.
be better than the USP method with respect to Barnett, H.A. McCauley, J. Agric. Food Chem. 47 (1999)
above-mentioned parameters. 3713 /3717.
However, the proposed method should be [10] K.T. McManus, J.D. deBethizy, D.A. Garteiz, G.A.
evaluated for its ability to separate various degra- Kyerematen, E.S. Vesell, J. Chromatogr. Sci. 28 (1990)
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[11] W. Naidong, W. Shou, Y.L. Chen, X. Jiang, J. Chroma-
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Acknowledgements [15] United States Pharmacopoeia/National Formulary, 24th
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Authors wish to thank National Institute of [16] V.R. Meyer, Practical High-Performance Liquid Chroma-
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