Extraction and Analysis of Nicotine from the Saliva of Active

Smokers using UV Spectroscopy

Muhammad Taufik

, Desi Ardilla

, Mariany Razali

, Endang Susilawati

, Afniwati

and Nurul Fadillah

Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Department of Agricultural Technology, Universitas Muhammadiyah Sumatera Utara, Medan, Indonesia

Department of Pharmacy, Universitas Tjut Nyak Dhien, Medan, Indonesia

Department of Nursing, Poltekkes Kemenkes Medan, Medan, Indonesia

Keywords: Saliva, FTIR, Nicotine, Uv-Visible Spectrophotometer, Extraction.

Abstract: An accurate and simple method of extraction and analysis needs to be developed in the context of

investigating nicotine in the saliva of active smokers. This study aims to prepare, extract and analyse

nicotine from the saliva of active smokers. The preparation process is carried out from the location where

the samples were taken. Extraction was carried out using sonication coupling maceration for 15 minutes.

Qualitative analysis used spot test with Cyanogen bromide reagent and quantitative analysis used UV

spectrophotometer. Ultra Violet (UV) spectrophotometer analysis at the optimum wavelength of 260 nm

resulted in a sample concentration of Saliva A = 1.4 ppm and Saliva B = 1.6 ppm.

1 INTRODUCTION

Nicotine (Nicotiana tobacum) is the most important

ingredient in tobacco leaves. Nicotine has the

molecular formula C

(Fidrianny 2004).

Nicotine is a clear, slightly yellow liquid that has an

oil-like appearance, dissolves in water and is also

soluble in organic solvents in general, such as

ethanol, petroleum ether, and chloroform (Taufik et

al. 2017). Nicotine is an alkaloid compound that is

widely contained in plants with the genus

Solanaceae (Rahmat Nur Hidayat, Adam M.

Ramadhan 2016). One of them is the type of tobacco

(Nicotiana). Nicotine with the chemical name 1-

Methyl-2- (3-pyridyl) pyrrolidine; β-pyridyl-α- N-

methylpyrolidine or with the molecular formula

or C

NCH

(Clayton et al. 2013).

Identification for nicotine can be done in urine,

hair and including saliva (Taufik, Susilawati, et al.

2021). Saliva is the first biological fluid to be

exposed to cigarette smoke in the oral cavity

(Kunutsor et al. 2018). Cigarette smoke contains a

variety of chemicals that can cause functional and

structural changes in saliva which can reduce the

flow of saliva, causing dry mouth and halitosis

(Fidrianny 2004) (Kunutsor et al. 2018).

Saliva has 99% water and 1% organic and

inorganic components (Jahed, Hamidi, and

Galehassadi 2020). Inorganic components of saliva

include: Sodium, Calcium, Potassium, Magnesium,

Bicarbonate, Chloride, Rodanide and Thiocynate

(CNS), Phosphate, Potassium and Nitrate. While the

organic components in saliva include proteins in the

form of the enzyme amylase, maltase, serum

albumin, uric acid, cretinin, mucin, vitamin C,

several amino acids, lysosime, lactate, and several

hormones such as testosterone and cortisol (Jahed,

Hamidi, and Galehassadi 2020).

The preparation of smoker's saliva which is the

initial stage of work that must be carried out in

various analyzes for sample preparation (Kunutsor et

al. 2018). The process of separating the material

from the mixture is carried out using the appropriate

solvent (Alfian et al. 2018). Sample preparation is

carried out in sample conditions where there are

specific techniques for sampling in order to obtain a

representative sample (Sisco, Najarro, and Burns

2018). This preparation aims to eliminate various

annoyances (Kondeti, Mulpuri, and Meruga 2014).

Sonication maceration is a liquid-liquid

extraction method that utilizes ultrasonic waves with

a frequency of 42 kHz which can accelerate the

contact time between the sample and the solvent

616

Tauﬁk, M., Ardilla, D., Razali, M., Susilawati, E., Afniwati, . and Fadillah, N.

Extraction and Analysis of Nicotine from the Saliva of Active Smokers using UV Spectroscopy.

DOI: 10.5220/0010614700002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 616-620

ISBN: 978-989-758-556-2

even at room temperature (Taufik 2016). Sonication

relies on wave energy that causes the cavitation

process, which is the process of forming small

bubbles due to the transmission of ultrasonic waves

to assist solvent diffusion into the sample (Alfian et

al. 2018). The sonication extraction method is also

efficient and shortens the extraction time

(Delmifiana and Astuti 2013).

Nicotine analysis is needed to determine the

nicotine content of human metabolites (Rahmat Nur

Hidayat, Adam M. Ramadhan 2016). The qualitative

analysis of nicotine was carried out in several ways,

such as the spot test using the Cyanogens bromide

test reagent until an orange color was obtained

which indicated positive nicotine (Paci et al. 2018).

Where the analysis of the Cyanogen bromide test is

carried out with the extraction results dropping 2

drops into the spot test, then dropping 2-3 drops of

Cyanogen bromide until it dissolves in the spot test,

and observing the orange color that occurs then

compared with the standard and differentiated into +

(slightly), + + (moderate), +++ (abundant) (Taufik,

Cahyady, et al. 2021).

Nicotine analysis is needed to determine the

nicotine content of human metabolites (Paci et al.

2018). The qualitative analysis of nicotine was

carried out in several ways, such as the spot test

using the Cyanogens bromide test reagent until an

orange color was obtained which indicated positive

nicotine (Taufik 2017). Where the analysis of the

Cyanogen bromide test is carried out with the

extraction results dropping 2 drops into the spot test,

then dropping 2-3 drops of Cyanogen bromide until

it dissolves in the spot test, and observing the orange

color that occurs then compared with the standard

and differentiated into + (slightly), + + (moderate),

+++ (abundant) (Rahmat Nur Hidayat, Adam M.

Ramadhan 2016) (Taufik et al. 2017).

The quantitative analysis of nicotine was carried

out through UV spectroscopy based on the

interaction of the sample with UV light. UV light

has a wavelength of 190 - 380 nm as a light source,

deuterium lamps can be used. Deuterium, also

known as heavy hydrogen, is a stable isotope of

hydrogen that is abundant in the ocean and land. The

nucleus of deuterium has one proton and one

neutron, while hydrogen has only one proton and no

neutrons (Clayton et al. 2013). This study aims to

extract by maceration the sonication coupling of

nicotine contained in saliva samples of active

smokers and to analyze it using UV spectroscopy.

2 METHODS

2.1 Materials

The materials used in this study were saliva, pure

nicotine (sigma Aldrich), methanol, chloroform (p a

merck), Cyanogen bromide reagent (Sigma Aldrich),

and aquadest.

2.2 Nicotine Standard Solution

Preparation

Nicotine standard solution (sigma aldrich) was

prepared by varying the concentration of nicotine,

respectively, 0.5 ppm, 1 ppm, 1.5 ppm, 2 ppm, 2.5

ppm.

2.3 Preparation

The saliva sample of active smokers was measured

10 ml each added 10 ml of chloroform solvent, then

put it into each separating funnel, then shaken it, and

let it stand for a moment, there were 2 layers (the top

layer of the remaining saliva and the bottom layer of

nicotine).

2.4 Extraction

The saliva used as the sample was sonication

process at a frequency of 42 KHz for 10 minutes.

The result of maceration was taken from the lower

layer of nicotine and then diluted with the addition

of 10 ml methanol. The sonication process was

carried out again for 5 minutes at a frequency of 42

kHz in a sonication bath. Note: comparator saliva

(not smokers) is carried out in the same manner

2.5 Spot Test Analysis

Spot test analysis was carried out using Cyanogens

bromide reagent until an orange color was obtained

which indicated a positive nicotine. The analysis

procedure for the Cyanogen bromide test is carried

out by:

1. The sample is added 2 drops of cyanogen bromide

in the spot test.

2. Observed the orange color that occurs and

differentiated into + (slightly), ++ (moderate),

+++ (abundant).

Extraction and Analysis of Nicotine from the Saliva of Active Smokers using UV Spectroscopy

617

2.6 Analysis using UV Spectroscopy

The analysis of the extracted nicotine was carried

out using a UV spectrophotometer with the

following procedure:

1. Turn on the Uv-Vis spectrophotometer on the

back of the instrument, wait for 10-15 minutes,

then connect it to the computer, and start the

Windows 7 Short-cut application.

2. The cuvette used was a glass cuvette with a

thickness of 10 mm, a square cuvette.

3. Inserted a blank into the UV spectrophotometer,

measured the wavelength of the blank.

4. The maximum wavelength is determined.

5. Sample analysis is performed.

3 RESULTS

3.1 Collecting Samples

Saliva of smokers, and saliva of non-smokers from

male volunteers obtained at Jl. Arca Gang Jawa

Medan Building. All saliva samples were collected

between 08:00 and 11:00 WIB. The saliva was

collected in the morning by instructing the

volunteers not to eat and drink at the time of

collection, to let the saliva go down, to let the foam

on the saliva be left for a while so that the foam

would go down. The sample that has been collected

is put into a beaker.

3.2 Preparation

Smoker's saliva preparation was carried out in the

laboratory of the University of North Sumatra,

Medan. The samples used were 10 ml of active

smoker's saliva with the addition of chloroform

solvent, in the smoker's saliva there is a nicotine

compound that comes from cigarette consumption,

which is directly exposed to cigarettes and cigarette

smoke through the mouth where saliva is contained.

Smoker's saliva has 2 layers perfectly where the

bottom layer of nicotine and the top layer of the

saliva remains. Non-smoker's saliva does not have a

perfect 2-layer separation so it takes 5 minutes to let

the saliva and solvent split into two layers.

3.3 Extraction

Saliva samples of active smokers that have been

added with chloroform and macerated sonication,

the nicotine and the remaining saliva are separated.

Obtained cloudy white nicotine extract. The lower

layer of the sonication maceration process was

taken, then left for a while and added methanol, then

re-macerated sonication with a frequency of 42 kHz

and put into a vial bottle. The same was true for the

comparator saliva (sample of non smokers).

3.4 Spot Test

The results of the spot test for saliva from active

smokers and saliva from non-smokers can be seen in

Table 4.1 below:

Table 3.1 Spot Test Result Data

Sample abundan

Colour Infor

mation

Nicotine

standard

+++ Orange Positive

Nicotine

Saliva A +++ Orange Positive

Nicotine

Saliva B +++ Orange Positive

Nicotine

Comparison

sample

- - Negative

Nicotine

Table 3.1 shows that the saliva samples of active

smokers from the spot test results with the cyanogen

bromide test reagent produce an orange color which

indicates positive for nicotine, and in the saliva of

non-smokers there is no color change (negative).

3.5 UV Spectroscopy Analysis

3.5.1 Determination of the Optimum

Wavelength

The results of determining the optimum wavelength

of nicotine can be seen in Figure 3.1. the following :

Figure 3.1. Optimum wavelength of nicotine

Figure 3.1 shows the highest peak at a wavelength of

260 nm with an absorbance value of 0.048. At a

wavelength of 258 nm, an absorbance value of 0.028

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

618

was obtained, a wavelength of 259 with an

absorbance value of 0.039, a wavelength of 261 with

an absorbance value of 0.041 and a wavelength of

262 with an absorbance value of 0.037. So that the

optimum wavelength of nicotine is at a wavelength

of 260 nm.

3.5.2 Nicotine Standard Curve

The standard nicotine curve can be seen in Figure

3.2. the following :

Figure 3.2. Nicotine standard curve

Figure 3.2 shows a straight line equation, namely r =

0.995 with a value of y = 0.0054x + 0.0345. Based

on the figure, it shows that the relationship between

the concentration of nicotine samples and the

response to ultraviolet spectroscopy is proven to be

linear.

3.5.3 Concentration of Nicotine

Saliva samples that have been extracted are inserted

into the cuvette to measure the absorption and

concentration of each sample at a wavelength (λ) of

260 nm. It can be seen in Table 3.2. below:

Table 3.2. Saliva Sample Concentration

o. Sampel Concentration (ppm)

1. Saliva A 1,4

2. Saliva B 1,6

Table 3.2 shows that the concentration of saliva

samples is saliva A = 1.4 ppm) and saliva B = 1.6

ppm.

4 CONCLUSION

Extraction of saliva from active smokers by

sonication maceration for 10 minutes with the

addition of chloroform solvent at 42 KHz.

Qualitative analysis of the saliva of active smokers

was compared with nicotine standards, which

resulted in an orange color. Ultra Violet (UV)

spectrophotometer analysis at the optimum

wavelength of 260 nm resulted in a sample

concentration of Saliva A = 1.4 ppm and Saliva B =

1.6 ppm.

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