INTRODUCTION:

Nicotine is the most abundant of the volatile alkaloids in the tobacco leaf. The primary commercial source of nicotine is by extraction from the plant Nicotinia tabacum and Nicotinia rustica. Nicotine acts on nicotinic cholinergic receptors, affects most organ systems in the body and is a highly addictive drug ^[1]. Nicotine normally makes up about 5 percent of a tobacco plant, by weight. Cigarettes contain 8 to 20 milligrams (mg) of nicotine (depending on the brand), but only approximately 1 mg is actually absorbed in the human body. Smoking harms nearly every organ of the body, causing many diseases and reducing the health of smokers in general. In case, one thinks of quitting smoking, it has immediate as well as long terms benefits. At any age, quitting confers substantial and immediate health benefits including reduced cardiovascular disease risks^[2], improved lipid profiles and platelet reactivity and reduced risk of stroke and smoking-attributable cancers^[3]. Thus smoking cessation and treatment of tobacco dependence can have great impact in reducing the burden of disease and improving population health. There are numerous behavioral smoking cessation treatments available, including self-help manuals, community-based programs, and minimal and intensive clinical interventions.

In clinical settings, pharmacological treatments, including nicotine replacement therapies (NRT) and bupropion, have become much more widely available in recent years in high-income countries.^[4,5]

Considering the addictive nature of tobacco, tobacco control interventions have increased for the tobacco addiction treatments, such as the nicotine replacement therapy products (NRTs). Nicotine replacement therapy (NRT) aims to replace the nicotine from cigarettes by other means of delivery nicotine skin patches, chewing-gum, lozenges, sublingual tablets, inhalators or nasal spray. NRT provides a background level of nicotine that reduces craving and withdrawal. Nicotine Polacrilex Gum sugar-free chewing pieces provide nicotine to the body system and works as a temporary aid to quit smoking by reducing nicotine withdrawal symptoms. Nicotine Polacrilex Gum provides a lower level of nicotine to ones blood than cigarettes, and allows to gradually do away with body’s need for nicotine. Because Nicotine Polacrilex Gum does not contain the tar or carbon monoxide of cigarette smoke, it does not have the same health dangers as tobacco. However, it still delivers nicotine, the addictive part of cigarette smoke. Nicotine can cause side effects such as headache, nausea, upset stomach, and dizziness.

The amount of information regarding nicotine content in cigarettes is not much and only a few studies have been done. The aim of our study was to develop and validate a rapid and simple HPLC method for quantification of nicotine in a large number of NRT based Nicotine Polyacrilex gum tablets. This investigation describes the most important references for analytical method validation, the validation parameters, acceptance criteria, quality control procedures for routine analysis and required documentation.

The RP-HPLC method reported in this study was validated in accordance with the International Conference on Harmonization (ICH) guideline^[6] and best practice ^[7-9]. Specificity, Linearity, precision, accuracy, limit of detection and limit of quantitation were evaluated.

MATERIALS AND METHODS:

A Thermo Fisher Scientific Dionex grade 3000 Ultimate UHPLC was coupled to a Dionex multisolvent delivery system, in-line degasser AF, auto sampler , a Rheodyne injector with sample loop of 20 μL, and a Dionex variable wavelength UV-Vis detector. A Hypersil BDS Phenyl C18 column (4.6 mm i.d. × 250 mm, 5 μm particle diameter) was used. Nicotine Bitartrate Dihydrate USP makes with 99.9 % purity was used as standard reference material. 0.25 M Sodium 1 decane sulfonate was prepared from AR grade chemicals obtained from Sigma chemicals. Acetonitrile used was HPLC grade obtained from Merck, India.

Preparation CRM Standards:

Accurately weighed 12.5 mg of USP Nicotine bitartrate dihydrate in 10 ml of solvent ( 785 ml water, 200ml acetonitrile, 0.25 sodium 1 decane sulfonate and 25 ml acetate buffer).1 ml of this solution was transferred to a 10 ml volumetric flask and diluted with the solvent and thoroughly mixed.

Sample Preparation:

A piece of nicotine polacrilex gum was accurately weighed in a flask. 50 ml of n Hexane and 50 ml of solvent( A mixture of water, acetonitrile,0.25 M sodium 1- decanesulfonate, and acetate buffer) were added. A stirring bar was inserted and flask was stoppered. The solution was stirred vigourously for about 30 minutes till the test specimen is dispersed. Remove from the stirring mechanism, and allow the solution to stand for about 30 minutes till the phases are separated. An aliquot of the lower layer was taken carefully. The clear filtrate was used for assay preparation.

Method of Analysis:

50 µl of standard and samples were injected into the Liquid chromatograph equipped with the Hypersil C18 column. Mobile phase was a mixture of 685 ml of water, 200ml of acetonitrile, 75 ml of acetate buffer and 0.25M sodium 1 decane sulfonate. A flow rate of 1.5 ml/min was maintained. Standard Nicotine bitartrate dehydrate in the concentration range of 1.25µg/ml to 125 µg/ml was injected in to the system and chromatogram obtained at 254 nm. Figure 1and Figure 2 summarises the result of reference standard of nicotine bitartrate dehydrate and the sample.

A calibration curve of the reference standard for Nicotine was developed in the concentration range 1.25 to 125 µg/ml. A linear curve with coefficient of correlation of 0.9970 was obtained as shown in Figure 3. Nicotine concentration on anhydrous basis was calculated as follows:

RESULTS AND DISCUSSION:

Method Validation Process:

The analytical method protocol was validated in the laboratory by conducting experiments with nicotine samples and standards that were similar to unknown samples analyzed routinely. The experimental procedure meets all the criteria for validation process. A good preparation work was carried out for efficient experiment execution. All the reagents, reference standards, were accurately weighed and checked for exact composition and purity as per specifications. Other consumables such as glasswares were calibrated to make sure that it meets the functional and performance specifications as required for the analytical method.

A sequence was developed based on our laboratory experience with the liquid chromatograph. The validation consisted of Linearity, specificity, detection limits, and recovery of samples.

Linearity

A stock solution of nicotine was prepared by dissolving standard nicotine bitartrate dehydrate in the concentration range of 1.25µg/ml to 125 µg/ml .The samples were filtered through 0.45 um filter and injected into the liquid chromatograph. Areas for all the injected samples were determined and the results plotted to obtain calibration curve. A linear curve with coefficient of correlation of 0.9970 was obtained as shown in Figure 3.

Selectivity/Specificity

For selectivity/specificity, analysis of blank samples of the appropriate sample matrix was obtained. Each blank sample was tested for interference, and selectivity was ensured at the lower limit of detection. Blank sample analysis showed no interference.

Detection limits

Accuracy was determined by replicate analysis of samples containing known amounts of the analyte. Accuracy was determined using a minimum of seven determinations per concentration. The deviation of the mean from the true value serves as the measure of accuracy.

Table 1 : Accuracy and detection limits determination

Name

Test 1

Ug/ml

Test 2

Ug/ml

Test 3

Ug/ml

Test 4

Ug/ml

Test 5

Ug/ml

Test 6

Ug/ml

Test 7

Ug/ml

RSD

Mean

MDL

ug/ml

LOQ

ug/ml

Nicotine

23.3

6.214

6.195

6.171

6.234

6.400

6.259

6.284

0.075

1.214

6.251

0.2587

0.7589

Table 2 : Recovery studies of nicotine gum from samples with known concentration

Sample	% Conc. At specifications level	Amount added , mg	Area	Amount found, mg	Statistical levels	Recovery %
Nicotine Gum	50 %	2.00	18.61 18.60 18.61	2.00 1.99 2.00	Mean: 1.996 SD : 0.00577 RSD: 0.289	99.5
Nicotine Gum	100 %	4.00	35.70 35.49 35.37	4.01 4.03 3.95	Mean:3.99 SD: 0.043 RSD:1.09	99.75
Nicotine Gum	150 %	6.00	54.32 54.09 54.04	6.01 6.02 5.90	Mean:5.97 SD: 0.0665 RSD:1.11	99.5

Table 3 : Values and uncertainities in nicotine polacrilex gum analysis

S.No.	Source	Value (X)	Standard Uncertainty (Ux)	Relative uncertainty (Ux/X)	(Ux/x)²
A	Purity	99.5	2.886x 10^-4	2.901x10^-5	8.415x10^-10
B	Mass	10	1.0x 10-³	1.0x10^-4	1.00x10^-8
C	Volumetric Flask a ml	10	5.77x10 ^-2	5.77x10^-3	3.329x10^-5
D	Volumetric Flask, b ml	10	5.77x10 ^-2	5.77x10^-3	3.329 x 10.^-5
E	Volumetric Flask, c ml	10	5.77x10 ^-2	5.77x10^-3	3.329x 10^-5
F	Pipette -1	0.2	5.773x10^-3	2.886x10^-2	8.328 x 10^-4
G	Pipette-2	0.1	5.773x10^-4	5.773x10^-3	3.329 x 10 ^-5
H	Corre. Coeff.	0.9980	2.00x10^-3	2.004x10^-3	4.016 x 10 ^-6

Recovery

Accuracy of the method was evaluated by determining recovery of nicotine gum at levels of 50%, 100%, and 150%. Results of recovery studies are shown in the Table 2.The mean recovery data obtained for all the levels were within an RSD of < 2.0% which satisfies the acceptance criteria of this study.

Uncertainty Determinations:

Attempt was also made to estimate the uncertainty associated with the analytical method by applying a bottom-up approach. All data appearing in this study complies with NABL 17025 requirements. It was implemented in our laboratory as a routine method and our laboratory was accredited. The uncertainty of each step was estimated identifying which of them are relevant in the global uncertainty analysis by a cause and effect technique as illustrated in Figure. 4.

Attempt was also made to estimate the uncertainty associated with the analytical method for nicotine polyacrilex gum by applying a bottom-up approach. All data appearing in this study complies with NABL ISO-IEC-17025 requirements. It was implemented in our laboratory as a routine method and our laboratory has been accredited. The uncertainty of each step was estimated identifying which of them are relevant in the global uncertainty. The relevant uncertainty sources in the nicotine gum analysis is illustrated by a cause and effect diagram as shown in Figure 4. The parameters of the measured represented by the main branches in the diagram. Further factors are added to the diagram, considering each step in analytical procedure. The values and uncertainties are shown in Table3.The standard uncertainties associated with each step is quantified by estimating analytes concentration from the calibration curve, calculating recovery of the sample extract. After obtaining the standard uncertainty (u(x)), expressed as a standard deviation, and combined standard uncertainty were determined.

The different aspects explained above for estimating the combined uncertainties have been applied to the nicotine gum analysis. Table 3 summarizes the relevant information for calculating uncertainties associated with the preparation of primary standard solutions, volumetric materials, and analytical balance.

In some cases, it is feasible to use relative uncertainties which represent the value of the uncertainty normalized. It is obtained as the quotient between the standard uncertainty u(x) and the value of x:

The uncertainty estimation was carried as per the following steps:

(1) Specifying the measured. This involved making a clear statement of what is being measured, including the relationship between the measured and the input quantities (Measured quantities, constants and calibration standard values).

(2) Identifying uncertainty sources i.e. listing the possible sources of uncertainty, usually specified in the above step.

(3) Quantifying uncertainty components i.e. estimating the uncertainty component associated with each potential source of uncertainty identified. The different contributions to the overall uncertainty is expressed as standard deviation which is calculated depending on the data available from a standard deviation value (this value is directly used); from the standard deviation of experimental data sets; from a declared purity and uncertainty value (which is given in a certificate of calibration for reference materials) and from a correlation coefficient of calibration curves.

(4) Combined uncertainty is calculated by combining different contributions to the overall uncertainty according to the appropriate rules.

(5) Expanded uncertainty by applying the appropriate coverage factor.

The combined uncertainty and expanded uncertainty were calculated for the nicotine as follows :

U(c nicotine) = C _nicotine { [ u(P)/P ] ² + [ u (m)/m ]² + [ u (Vflask₁) /Vflask₁ ] ² +

[ u (Vflask₂) /Vflask₂ ] ² + [ u (Vflask₃) /Vflask₃ ] ² + [ u ( V pipet1)/pipet1 ] ² +

[ u ( V pipet2)/pipet2] ² + [ u (Vcalib)/ calib ] ² + ……..} ^1/2

Where, u(P) is the uncertainty in purity of the nicotine standard as quoted in the suppliers certificate, u(m) is the uncertainty in the mass of the nicotine in the certified reference standard solution, u(V_flask ) is the uncertainty in volumetric flasks measurements estimated by considering the influences of calibration, repeatability and temperature effects and determined by measurement of uncertainty in internal volumes and variation in filling volumetric flask to the mark, u(v_pipet1) , and u(V_pipet2) are the uncertainties in volumetric measurements using pipettes, U(calib) is the uncertainty in calibration determined from the coefficient of correlation obtained for the calibration curve. Using the above relation the combined uncertainty obtained was 0.0314.

The expanded uncertainty U(c nicotine) was subsequently determined to develop an interval within which the value of the measured may lie. A factor of 2 was thus used for obtaining a confidence level of 95%.

U(c nicotine) = 2 x 0.0314=0.0628

CONCLUSION:

The RP-HPLC method for the estimation of Nicotine in Nicotine polyacrilex Gum in pharmaceutical dosage form is accurate, precise, linear, rugged, simple and rapid.

Hence the present RP-HPLC method is suitable for the quality control of the raw materials, and formulation studies. The method is validated and uncertainty determined.

ACKNOWLEDGEMENTS:

The authors would like to thank Dr. P. L. Muthal, and Dr. Pranav Nagarnaik for guidance and encouragement in carrying out the above work.

REFERENCES:

1. Benovitz NL, Pharmacology of nicotine addiction and therapeutics, Annu Rev Pharmacol Toxicol, 1996; 36 : 597- 613.

2. Peto R, Lopez AD, Boreham J, Thun M, Heath C Jr. Mortality from smoking in developed countries 1950-2000. Indirect estimation from National Vital Statistics. Oxford (UK): Oxford University Press; 1994.

3. World Bank, Development Report, Entering the 21^st Century, 1999/2000, available at http://www.worldbank.org/wdr/2000/index.html, last accessed 6 October 2004.

4. US Department of Health and Human Services. Reducing tobacco use: a report of the Surgeon General. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control and Prevention, Office on Smoking and Health, 2000. www.cdc.gov/tobacco/sgr_tobacco_use.htm (accessed 3 May 2002)

5. American Psychiatric Association. Diagnostic and statistical manual of mental disorders, 4th ed. Washington, DC: American Psychiatric Association, 1994

6. International Conference on Harmonization, "Q2B: Validation of Analytical Procedures:Methodology; Availability," Federal Register 1997,62 (96), 27463–27467.

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9. Guidance for Industry: Analytical Procedures and Methods Validation: Chemistry, Manufacturing and Controls Documentation; Draft Guidance. Rockville, MD. US FDA. 2000.

10. Analytical Detection Limits. Guidance and Laboratory Guide for Determining Method Detection Limits, Report PUBL-TS-056-96, Wisconsin Department of Natural Resources, Laboratory Certification Program, April, 1996

Received on 10.09.2014 Accepted on 16.10.2014

Asian J. Pharm. Ana. 4(4): Oct. - Dec. 2014; Page 156-161