Available online on 15.04.2025 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2025 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access Full Text Article   Research Article

UV Spectrophotometric Method for Estimation of Pure Nicotine

Vatsal Navapariya *, Milind M Thosar, Madhav Zinzuvadiya , Dharmesh Rathava , Dharmil Pathak 

Faculty of Pharmacy, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India

 

 

INTRODUCTION:

Nicotine (1-methyl-2-(3-pyridyl)pyrrolidine) is a drug obtained from the plant Nicotiana tabacum. 

 

Figure 1: Structure of Nicotine

It is a colourless to pale yellow oily liquid with a tobacco-like odour. It is a highly addictive alkaloid found in all parts of the tobacco plant.¹ Specifically, Nicotine acts on nicotinic cholinergic brain receptors as well as other parts of the nervous system primarily by releasing or facilitating the production of a variety of neurotransmitters including dopamine, norepinephrine, serotonin, acetylcholine, vasopressin and beta endorphin.² The presence of both a pyrrolidine and a pyridine nitrogen means that nicotine is dibasic with pKa of 7.84 and 3.04 at 25 °C.³There are Very few spectrophotometric methods are available for the determination of nicotine. Smith and Cooke ⁴ They used di-ethyl-thio-barbituric acid as a spectrophotometric reagent for tobacco alkaloids. The absorption At 536 nm was measured and the method applied to the determination of nicotine in urine samples.⁵ In this paper, a simple, UV spectrophotometric method is described for the determination of pure nicotine.

Mechanism of Action:

Nicotinic acetylcholine receptors are ligand-gated cation channels that are widely distributed throughout the nervous system and body.⁶ but this section will focus on neuronal nAChRs.⁷ Nicotinic acetylcholine receptors are pentameric ligand-gated cation channels consisting of five different subunits that form a central aqueous pore which has an entry for cation inflow when activated by the receptor.^7,9 Each subunit has an extracellular N-terminal end, the part which takes part in ligand binding; three hydrophobic transmembrane domains termed M1 to M3, an intracellular loop, a fourth hydrophobic transmembrane domain termed M4 and an extracellular C-terminus. The activation of nAChRs is achieved by the binding of the endogenous neurotransmitter acetylcholine or exogenous ligands like nicotine.^10,11

MATERIAL AND METHOD

Material 

Nicotine API was supplied by Nico Orgo Privet Ltd, Dakor, Gujrat, India as a gift sample. For standard plot Ultra Violet(UV) spectroscopy was used of double beam UV-1900i: SHIMADZU spectrophotometer. Finnpipette™ F3 1(0-100μL), UV chamber(ohmetron), TLC plate (Silica gel 60 F₂₅₄)

Method

Characterization of drug:

We have received COA from the supplier for our Nicotine API that includes all the confirmatory data related to API 

TLC (thin layer chromatography):

A thin-layer chromatography (TLC) method was employed for the alkaloid test using Dragendorff's reagent. The nicotine sample was applied to a TLC plate, and chloroform:methanol (19:1) was used as the mobile phase. After development, the plate was dried and sprayed with Dragendorff's reagent. The presence of nicotine was confirmed by the appearance of an orange or brown spot, indicating a positive result for alkaloids.

Figure 2: UV active image of spots of nicotine

Figure 3: Dragendorff’s active spots of nicotine

Infrared Spectroscopy

The IR technique is used to measure the absorption of various infrared radiations by the target material, to produce an IR spectrum that can be used to identify functional groups and molecular structure in the sample. Then the FTIR spectra were recorded between 4000 and 400 cm^-1. The resolution was 2 cm^-1.

 

 

Figure 4: Infrared Spectra of Nicotine

 

Method 

Stock solution

10 microliters pure (equivalent to 10 mg nicotine) Nicotine was accurately weighed and transferred into a 100 mL volumetric flask this will form 100 ppm stock solution.  

Determination of maximum wavelength

Nicotine 50 µg/ml solution was scanned in UV spectrophotometer within the wavelength range of 200–400 nm. Methanol solvent was used as blank.

Figure 5: Maximum wavelength at 261nm.

Figure 6: Standard calibration curve of Nicotine

Preparation of standard plot/Calibration curve

• 0.4 mL of the stock solution was transferred to another volumetric Flask and volume made up to 10 ml with methanol in Achieve the outcome solution that equals 4 µg/ml.
• Dilutions were then prepared from the stock solution to Get 2 µg/ml, 4 µg/ml, 6 µg/ml, 8 µg/ml, 10 µg/ml 12 µg/ml and solution concentration.
• methanol was taken as blank and absorbance of various dilutions these were measured at 261 nm.

 

 

Analytical method validation

Validation can be said to be (ICH) setting documented proof, that gives a high assurance that a selected activity can systematically produce the desired result or product which meets its pre-set specifications and quality characteristics. The following parameters were evaluated for method validation.¹²

Linearity and range

Using such an analytical methodology that offers linearity enabled it examine data that are directly proportional to the concentration of an analyte in simples placed within a given range towards determining the predicted methodology's linearity, 2-12 µg/ml of the Standard solution of Nicotine was prepared by using stock solution and examined. Every measurement was carried out three times.¹³ 

Table 1: Standard Calibration Curve of Nicotine

 

Precision

The system precision is a measure of the Differences in method. It was estimated by performs three replicate analysis of the same working solutions, precision method was Demonstrated by intraday and interday variation studies. The intra-precision of the developed UV The method was determined by preparing the sample of Same batch in a decision with three Concentrations: 2,4,6 μg /ml and three replicates (n=3) each on same day at 1hours, 3hours, 6hours. The percent RSD of the results was used to evaluate the method precision. The interday was Determined by assaying the sample in triplicate (n=3) a day for three consecutive days. 

Table 2 Repeatability results in precision test for 4 µg/ml solution.

 

Table 3 Repeatability results in precision test for 6 µg/ml solution.

 

Table 4 Repeatability results in precision test for 8 µg/ml solution.

Conc.( µg/ml)	Absorbance	Statistical Analysis
8	0.701	Mean = 0.64016 SD = 0.003933 %RSD = 0.74%
8	0.705
8	0.708
8	0.510
8	0.509
8	0.708

 

 

Table 5: Intra-day Precision study

 

Table 6: Inter-day Precision study

 

 

Accuracy 

The Accuracy of Method Was determined using the standard addition method at three various levels: 50%, 100%, and 150%. Standard addition may be done through the standard amount addition to the sample for each level. Starting concentration is the standard stock of 100 μg/ml; 0.4 ml was taken then diluted to 10 ml to produce 4μg/ml solution. From this solution, 5ml was taken and from prepared 8 μg/ml solution 5ml was taken and added to the first 5ml of 4μg/ml thus resulting in a concentration of 6μg/ml, which is equivalent to 50% addition of the original 4μg/ml solution. 100% addition was performed by combining it with the original 4 μg/ml solution to result in a 12μg/ml solution. For the addition of 150%, a 16μg/ml solution was added to the 4μg/ml solution.

 

 

Table 7 Accuracy study results

Conc.(µg/ml) Initial	Initial Absorbance for 4 µg/ml	Level addition(in%)	After addition Absorbance	Altered concentration (µg/ml)	Average % recovery
4	0.256	50	0.380	5.937	98.95
4	0.256	100	0.508	7.953	99.41
4	0.256	150	0.656	10.09	100.9

 

 

 

 

LOD and LOQ: 

The smallest amount of an analyte that exists in the sample, and can be detected by analysis, is termed the limit of detection (LOD). The smallest amount of an analyte present that could be quantitated with acceptable precision and accuracy is termed the limit of quantification (LOQ). The LOQ and LOD are calculated as shown below using the following equations:

LOQ = 10 s/m

LOD = 3 s/m

In these equations, "s" is the standard deviation of the response, and "m" is the slope of the corresponding calibration curve.

 

 

Ruggedness:

Table 8 Ruggedness study

Conc.( µg/ml)	Analyst 1	Analyst 2	Statistical Analysis
4	0.264	0.268	Mean = 0.3923 SD = 0.00354 %RSD = 0.926%
6	0.403	0.398
8	0.513	0.507

 

 

The reproducibility of a test result under typical expected operating conditions varies from instrument to instrument and analyst to analyst, and this is measured by ruggedness. Experiment was carried out employing several approaches to ascertain the method's robustness. 

Specificity: 

Specificity is the ability to accurately and precisely quantify the analyses of interest in the laboratory in the presence of other foreseen components within the sample matrix. It determines the size of the population. Among the other active drugs are confounding variables which include degradation, contaminants, and additives. Here we have used DMSO (Dimethyl Sulfoxide) as a confounding variable for specificity study in which 5ml volume of 4ppm nicotine sample with another 5ml with 2% DMSO and then after we haven’t observed any major changes in absorbance reading as that taken earlier.

Table 9 Robustness study results

 

Robustness: 

Different concentrations of Nicotine solution like (4 µg/ml,6 µg/ml,8 µg/ml) was analysed at different wavelength that includes 259nm, 260nm, 261nm, 262nm, 263nm to determine robustness of the method.

 

 

Table 10: Robustness study results

Conc.(µg/ml)	Absorbance at 259nm	Absorbance at 260nm	Absorbance At 262nm	Absorbance at 263nm	Statistical Analysis
4	0.207	0.212	0.210	0.208	Mean = 0.3406 SD = 0.003091 %RSD = 0.93%
6	0.335	0.341	0.338	0.334
8	0.472	0.481	0.480	0.478

 

 

RESULTS AND DISCUSSION:

From dissolution point of view, attempt was made to dissolve the Nicotine in methanol. The calibration curve of Nicotine plotted at 261 nm (Figure no-1) a linear relationship was obtained between 2-12 µg/ml. The accuracy of the method was determined by calculating mean percentage recovery it was found to be 98–101 % (Table no-7). Further precision was calculated as repeat ability, inter and intraday variations and %RSD was less than one (Table-4). The LOD value was found to be 0.422 µg/ml and LOQ value was found to be 2.513 µg/ml.

CONCLUSION: 

In this paper Accurate, economical, and convenient methodology for the nicotine in pure API form using UV spectrophotometric method was established. The % RSD values for Intraday and Interday was found to be less than 2 which is very significant. the limit of detection and limit of quantification of the developed strategy ware found to be 0.422 µg/ml and 0.513 µg/ml this indicating that developed method is sensitive. Developed method was also robust and rugged as the %RSD for both the tests was found to be less than 2%.

REFERENCES:

1. Benowitz NL, Jacob P 3rd, Fong I, Gupta S. Nicotine metabolic profile in man: comparison of cigarette smoking and transdermal nicotine. J Pharmacol Exp Ther. 1994;268(1):296-303. https://doi.org/10.1016/S0022-3565(25)38479-X PMid:8301571

2. Miller EI, Norris HR, Rollins DE, Tiffany ST, Wilkins DG. A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. J Chromatogr B. 2010 Mar 15;878(9-10):725-37. https://doi.org/10.1016/j.jchromb.2009.12.018 PMid:20097626 PMCid:PMC2975562

3. Yildiz D. Nicotine, its metabolism and an overview of its biological effects. Toxicon. 2004 May 1;43(6):619-32. https://doi.org/10.1016/j.toxicon.2004.01.017 PMid:15109883

4. Smith CL, Cooke M. Evaluation of diethylthiobarbituric acid as a spectrophotometric reagent for tobacco alkaloids. Analyst. 1987;112(11):1515-8. https://doi.org/10.1039/an9871201515 PMid:3439600

5. Al-Tamrah SA. Spectrophotometric determination of nicotine. Anal Chim Acta. 1999 Jan 11;379(1-2):75-80. https://doi.org/10.1016/S0003-2670(98)00517-0

6. Papke RL. Merging old and new perspectives on nicotinic acetylcholine receptors. Biochem Pharmacol. 2014 May 1;89(1):1-1. https://doi.org/10.1016/j.bcp.2014.01.029 PMid:24486571 PMCid:PMC4755309

7. Cooper GF, Herskovits E. A Bayesian method for constructing Bayesian belief networks from databases. In: Uncertainty Proceedings 1991. Morgan Kaufmann; 1991. p. 86-94. https://doi.org/10.1016/B978-1-55860-203-8.50015-2 PMid:1998825 PMCid:PMC225491

8. Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. Annu Rev Pharmacol Toxicol. 2007 Feb 10;47(1):699-729. https://doi.org/10.1146/annurev.pharmtox.47.120505.105214 PMid:17009926

9. Morales-Perez CL, Noviello CM, Hibbs RE. X-ray structure of the human α4β2 nicotinic receptor. Nature. 2016 Oct 20;538(7625):411-5. https://doi.org/10.1038/nature19785 PMid:27698419 PMCid:PMC5161573

10. Karlin A. Structure of nicotinic acetylcholine receptors. Curr Opin Neurobiol. 1993 Jun 1;3(3):299-309. https://doi.org/10.1016/0959-4388(93)90121-E PMid:8369624

11. Wittenberg RE, Wolfman SL, De Biasi M, Dani JA. Nicotinic acetylcholine receptors and nicotine addiction: A brief introduction. Neuropharmacology. 2020 Oct 15;177:108256. https://doi.org/10.1016/j.neuropharm.2020.108256 PMid:32738308 PMCid:PMC7554201

12. Warule PS, Patel VP, Gosavi SA. Development and validation of UV spectrophotometric method for the estimation of curcumin in an Ayurvedic formulation Haridrakhand. Int J Pharm Drug Anal. 2017;5(5):193-7.

13. ICH I. Q2 (R1): Validation of analytical procedures: text and methodology. In: International Conference on Harmonization, Geneva; 2005 Nov 6.

14. Sharma S, Goyal S, Chauhan K. A review on analytical method development and validation. Int J Appl Pharm. 2018 Nov 7;10(6):8-15. https://doi.org/10.22159/ijap.2018v10i6.28279