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Determination of Nicotine in Juices, Soft and Energy Drinks collected from

Libyan Local Markets Using UV-Visible Spectrophotometer

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 Libyan Journal of Basic Sciences (LJBS)
Vol: 16, No: 1, P:79-89 , April. 2022 https://ljbs.omu.edu.ly/eISSN 2707-6261

Determination of Nicotine in Juices, Soft and Energy

Drinks collected from Libyan Local Markets
Using UV–Visible Spectrophotometer
Galal Elmanfe*, Huda Saad and Yasmine Hossin

Department of Chemistry, Faculty of Sciences, University of Omar Al-Mukhtar, El-Beida, Libya

Correspondence authors: galal.elmanfe@omu.edu.ly

Abstract

This study aims to determine the nicotine in juices, soft and

energy drinks using a UV-Visible spectrophotometer. In the presented
study, sixteen samples (four juices, seven soft drinks, and five energy
drinks) are collected from local markets in El-Beida - Libya. A rapid,
simple, and reliable extraction method is developed and validated for
the determination of nicotine in the samples under study using
spectrophotometric methods. The method is validated over a wide linear
range of 1.62–81.06 µg/mL with correlation coefficients being
consistently greater than 0.9999 by UV-Visible Spectrophotometer. The
minimum nicotine level was observed in the juices samples, while the
energy drinks samples showed the highest nicotine content. The
spectrophotometric measurement indicates that the results of nicotine
concentrations in juices samples are in the range of 0.3233 - 0.5238
µg/mL, whereas its concentrations in soft drinks samples are in the
range of 0.7251 - 0.9949 µg/mL, and the concentrations in energy
drinks samples are in the range of 0.8521 - 1.3471 µg/mL. The criteria
considered for validation are linearity, the limit of detection, and the
limit of quantitation. The nicotine contents in all the samples analyzed
in our study were below the allowable limits set by the US Food and
Drugs Administration.
Keywords: Juices, Soft drinks, Energy drinks, Nicotine, Extraction and
UV-Vis Spectrophotometer.
Introduction:
Nicotine, 3-(1-methyl-2-pyrrolidinyl) pyridine present in the leaves of
Nicotiana tabacum (1, 2). It is one of the several thousands of compounds
identified in tobacco, many of which contribute to the flavor, aroma, and
physiological effects. Nicotine appears to be a promising tracer for
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

environmental tobacco smoke, because of its specificity for tobacco (3). It is

also a chemical that is commonly used as an insecticide, as well as being a
highly addictive drug (4). Assessment of nicotine metabolism and disposition
has become an integral part of nicotine dependency treatment programs.
Cotinine is the major primary metabolite of nicotine (5, 6), and it accumulates in
the body with regular smoking. Nicotine and cotinine appear to be metabolized
by the same liver enzyme (6). The enzymes responsible for the metabolism of
nicotine in the human liver to cotinine are cytochrome P4502A6 (CYP2A6) and
cytosolic aldehyde oxidase (7, 8). Cotinine inhibits nicotine metabolism,
resulting in slower nicotine clearance in smokers compared with nonsmokers
(6). Nicotine has a relatively short half-life, approximately 2 h, and cotinine has
a half-life of approximately 20 h (9, 10). Energy drinks generally contain
methylxanthines including caffeine, nicotine, B vitamins, carbonated water, and
high-fructose corn syrup (for non-diet versions) (11). Other commonly used
ingredients are guarana, yerba mate, and taurine, plus various forms of ginseng,
maltodextrin, inositol, carnitine, creatine, glucuronolactone, sucralose, and
ginkgo Biloba (12). Nicotine in all the samples can be detected using different
instrumental techniques. For example, high-performance liquid chromatography
(HPLC) was using UV detector (11, 13-16) or mass spectrometry detector (MS)
(17). Other studies have determined nicotine and/or cotinine concentrations in
smokers’ and nonsmokers’ blood by UV-Visible Spectrophotometer, HPLC,
and/or gas chromatography (GC) - MS (10, 16, 18, 19). In this study, a rapid,
simpl,e and reliable extraction method is developed, based on other methods
recommended by other researchers with some modifications, in order to isolate
and determine nicotine in beverages, soft drinks, and energy drinks (10, 11, 20).
The extraction method used was more rapid and simple compared with other
extraction methods (10, 20). The analyses were developed and validated using
UV-Visible Spectrophotometer.
Materials and Methods:
Chemicals and reagents:
All chemicals, analytical standards, reagents, and solvents used throughout this
study were analytical grade and highly pure. Nicotine was purchased from
(Fluka) with the purity of Assay ≥ 99% (for research and development use
only). Also, other chemicals and solvents were used including Carbon
tetrachloride (CCl4) (Riedel-Dehaen AG Seelze Hannover) with purity 99 % as

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

the extracting solvent; Sodium carbonate (Na2CO3) (Riedel-Dehaen AG Seelze

Hannover), with purity 99.5%); Caffeine (BDH-Analor) with purity 99.2 %.
Chemicals:
a)- Standard nicotine solution (Fluka): 50 mg in 100 mL (0.5 mg/mL)
solution was prepared. Working standards were prepared by appropriate dilution
of the stock. (1.62, 16.21, 48.64, 81.06 and 162.12 µg/mL).
b)- Sodium carbonate (Na2CO3) 20 %: 20 g of Na2CO3 was dissolved in 100
mL distilled water. This solution was used in the extraction procedure.
Instrumentation:
The UV-Visible Spectrophotometer system: UV-VIS Spectrophotometer
(BECKMAN COULTER DU 800).
Samples collection:
Sixteen samples were collected from local markets in El-Beida city- Libya. Four
of these samples were juice samples, seven were soft drinks samples and five
were energy drinks samples. On the other hand, nine of these samples were
Libyan products, and the other samples were imported from Arabic, Islamic and
other countries.
The nicotine extraction procedure:
An aliquot (5 mL) of the sample was drawn with a pipette and placed into a 250
mL separating funnel followed by the addition of 10 mL of distilled water, then
one mL of 20% aqueous Na2CO3 solution and 20 mL of analytical grade
CCl4. The nicotine was extracted by inverting the funnel at least three times,
venting the funnel after each inversion. The non-aqueous CCl4 layer was
removed to a clean 50 mL volumetric flask. Another 20 mL portion of CCl 4
was added to the aqueous solution in the separating funnel and the extraction
procedure was repeated twice more and the CCl4 solvent layers combined. This
volume was made up to 50 mL with the solvent. This procedure was repeated
for all the samples. The absorbance of the resulting solutions was then
measured on UV/Vis spectrophotometer at 258 nm using a quartz cuvette.
Results and Discussion:
Preparation of calibration graph:
The absorbance of each standard solution was measured at an absorption
maximum of 258 nm using a quartz cuvette. The absorbance values were then
Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

plotted against concentrations to generate a standard calibration curve. Figure

(1) shows the spectrum of nicotine of different concentrations (1.62 - 81.06
µg/mL) at 258 nm. Figure 2 shows the calibration curve for the standard
solutions of nicotine of different concentrations (1.62 - 81.06 µg/mL) at 258
nm. The standard linear calibration curve obtained from the analysis of the
standard solution is presented in figure 2. It showed a good linear relationship
between the absorbance and concentrations of the standard solutions.

2
Absorbance

200 250 300 350 400

Wavelength/nm

Figure 1: Spectrum of different concentrations of Nicotine.

2
Absorbance at 258 nm

1.6 y = 0.022x + 0.014

R² = 0.999
1.2

0.8

0.4

0
0 20 40 60 80 100
Concentration of nicotine / ppm

Figure 2: Calibration curve for nicotine, expressed on a linear scale.

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

Determination of nicotine in the studied samples:

The Spectrophotometric measurements indicate that the results of nicotine

contents in the juice samples were in the range of 0.3233 – 0.5238 µg/mL
(ppm), while in the soft drinks samples were in the range of 0.7251 – 0.9949
µg/mL and in the energy drinks samples were in the range of 0.8521 – 1.3471
µg/mL. These results are shown in table 1 and figure 3.
The minimum nicotine level was observed in the juices samples, while the
energy drinks samples showed the highest nicotine content. These results (in
this study) were similar to those obtained in the study of the determination of
caffeine in such samples (11). The results in table 1 and figure 3 show the
minimum nicotine level was observed in the mixed fruits juice of Al-Mazzra
sample from Tripoli - Libya (0.03233 ppm (µg/mL), while the highest nicotine
level was observed in the energy drink, Red Bull sample from Austria (1.3471
ppm (µg/mL)).

Figure 3: levels of nicotine in the samples

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

Table 1: Concentrations of nicotine in the samples under study

Concentration of
N° Sample name (type)
Nicotine / ppm(µg/mL)

S1 Juice Al Rayhan-Benghazi 0.5238

S2 Juice Said-Benghazi 0.5123

S3 Soft drink Mirinda peach-Tripoli 0.8030

S4 Juice Al Mazraa- Mixed fruits-Tripoli 0.3233

S5 Juice Judi mixed fruits- Tripoli 0.4398

S6 Soft drink Maradh Drink-Benghazi 0.7251

S7 Soft drink Seven Up 7 up- Benghazi 0.8437

S8 Soft drink Saada Cola-Benghazi 0.9949

S9 Soft drink Sadaka Pine apple-Drana 0.8188

S10 Soft drink Pine apple Mirinda -Egypte 0.9551

S11 Soft drink Fuzze -Libanon 0.8437

Energy Drink Beck's (Non Alcohol)-

S12 0.8521
Germany

S13 Energy Drink Boom Boom - Austria 1.1087

S14 Energy Drink Red Bull - Austria 1.3471

S15 Energy Drink Big Shock - Czech Republic 1.2088

S16 Energy Drink Bomba - Australia 0.9838

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

Type of the samples:

Juice samples: The minimum nicotine level was observed in the mixed fruits
juice of Al-Mazzra sample from Tripoli - Libya (0.3233 µg/mL), while the
maximum nicotine level was observed in the juice of Al Rayhan from Benghazi-
Libya (0.5238 µg/mL).
Soft drinks samples: The minimum nicotine level was observed in the Maradh
Drink sample from Benghazi - Libya (0.7251 µg/mL), while the maximum
nicotine level was observed in Saada Cola from Benghazi-Libya (0.9949
µg/mL).
Energy drinks samples: The minimum nicotine level was observed in the
Beck's (Non-Alcohol) sample from Germany (0.8521 µg/mL), while the
maximum nicotine level was observed in the Red Bull sample - from Austria
(1.3471 µg/mL).
Source of the samples (country of origin) :
Libyan samples: The minimum nicotine level was observed in the mixed fruits
juice of the Al-Mazzra sample from Tripoli - Libya (0.3233 µg/mL), while the
maximum nicotine level was observed in the Soft drink of Saada Cola from
Benghazi-Libya (0.9949 µg/mL).
Arabic and other countries samples: The minimum nicotine level was
observed in the soft drink Fuzze sample from Libanon (0.8437 µg/mL), while
the maximum nicotine level was observed in the energy drink Red Bull sample
– from Austria (1.3471 ppm (µg/mL)).
The results obtained in this study indicate that the nicotine contents in all the
samples analyzed were below the allowable limits set by the US Food and
Drugs Administration and other studies (4, 11, 21, 22).
Statistic study
Linearity: Examination of calibration curves by computing a linear least-
squares regression analysis on the plot of absorbance of nicotine to the external
standard versus concentrations demonstrated a linear relation over the range
1.62 - 81.06 µg/mL (using five concentration levels) with correlation
coefficients (R2) being consistently greater than 0.9997.
Limit of detection (LOD): Is defined as the concentration of analyte required
to give a signal equal to three times the standard deviation of the blank. The
LOD was calculated using the following equation:

3  sy/ x
LOD 
b
Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

wheres is the average of the standard deviation SDyx . In the presented study,
the limit of detection (LOD) value for nicotine was 2.59 µg/mL.
Limit of quantitation (LOQ): Is defined as the concentration of analyte
required to give a signal equal to ten times the standard deviation of the blank.
The LOD was calculated using the following equation:

10  s y / x
LOQ 
b
The limit of quantitation (LOQ) value for nicotine in this study was determined
to be 8.62 µg/mL.
Conclusion:
The order of nicotine concentration in the samples under study was: Energy
drinks > Soft drinks > Juices samples. The concentrations of nicotine in Libyan
juices and Libyan soft drinks samples were less than other soft and energy
drinks that were imported from the other countries. The concentrations of
nicotine in the energy drink Red Bull sample from Austria were higher than its
concentrations in other energy drinks samples and all the samples under study.
The extraction method used in this study provided a high efficiency. The results
obtained for analysis of nicotine in juices, soft and energy drinks samples using
UV-Visible Spectrophotometer showed that there are differences in the
concentrations of nicotine in these samples and the level of nicotine was below
the allowable limits set by the US Food and Drugs Administration in all the
samples under study. Thus current advise the other researchers to study the
determination of nicotine and caffeine concentrations and their metabolisms in
other samples to complete our study.
References:
1- C. Wu, W. F. Siems, H. H. Hill, R. M. Hannan, Analytical
determination of nicotine in tobacco by supercritical fluid
chromatography-ion mobility detection. Journal of Chromatography A,
811(1-2), 157-161 (1998).
2- J. V. Rodricks, Calculated Risks; Cambridge University Press:
Cambridge, (1992), pp 251-256.
3- F.E. Jones, Toxic Organic Vapors in the Work Place; Lewis Publishers:
London, CRC Press Inc.: New York, (1994).
4- F. Meelad, S. Edrah, H. Alnade, A. Mlitan, H. Elgubi, Spectroscopic
Study on Tubacam Nicotina Leaves and Commercial Tobacco in the

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

Uv, Ir Regions And Its phytochemical screening of natural native

tobacco from Libya. International Journal of Agriculture and Plant
Science, 1(3), 04-09 (2019).
5- A.S. Xu, L.L. Peng, J.A. Havel, M.E. Petersen, J.A. Fiene, J.D. Hulse,
Determination of nicotine and cotinine in human plasma by liquid
chromatography–tandem mass spectrometry with atmospheric-pressure
chemical ionization interface. J. Chromatogr. B 682, 249-257 (1996).
6- S. Zevin, P. Jacob, N. Neal Benowitz, Cotinine effects on nicotine
metabolism. Clinical Pharmacology & Therapeutics, 61(6), 649-654
(1997).
7- F. Ceppa, Y. El Jahiri, H. Mayaudon, O. Dupuy, P. Burnat, High-
performance liquid chromatographic determination of cotinine in urine
in isocratic mode. J. Chromatogr. B 746, 115-122 (2000).
8- J.W. Gorrod, J. Wahren, Eds. Nicotine and Related Alkaloids, 1st ed.
Chapman and Hall, Boca Raton, FL, (1993).
9- A. C. Holloway, Nicotine. Encyclopedia of Toxicology (Third Edition),
(2014), pp 514-516.
10- A. M. Massadeh, A. A. Gharaibeh, K.W. Omari, A Single-Step
Extraction Method for the Determination of Nicotine and Cotinine in
Jordanian Smokers’ Blood and Urine Samples by RP-HPLC and GC-
MS. Journal of Chromatographic Science, 47, 170-177 (2009).
11- G. Elmanfe, K. Ali, R. Abd Al zain, Spectrophotometric Determination
of Caffeine in Juices, Soft Drinks and Energy Drinks collected from
some Local Markets in El-Bieda City – Libya. 2nd Libyan Conference
on Chemistry and its Applications (LCCA-2), 09-11 May 2017,
Benghazi - Libya, 90- 93 (2017).
12- B. Meier, Energy Drinks Promise Edge, but Experts Say Proof Is Scant.
The New York Times. New York: NYTC. ISSN 0362-4331 (2013).
13- C. Oddoze, A.M. Pauli, J. Pastor, Rapid and sensitive high performance
liquid chromatographic determination of nicotine and cotinine in
nonsmoker human and rat urines. J. Chromatogr. B 708, 95-101 (1998).
14- G. Elmanfe, S. Omar, A. Eznien, Determination of Nicotine in Libyan
Smokers’ and Nonsmokers' Urine samples by Ultraviolet-Visible
Spectrophotometry. Journal of Pure and Applied Sciences, 19(5), 126-
131 (2020).
15- G. Elmanfe, S. Omar, N. Abdolla, A. Hassan, Determination of
Nicotine in Libyan Smokers’ Urine Compared with that of Nonsmokers
using Reversed Phase-High Performance Liquid Chromatography (RP-
HPLC). Al-Mukhtar Journal of Sciences, 34(3), 172-180 (2019).
16- G. Elmanfe, A. Abdulla, Spectrophotometric Determination of Nicotine
in Some Libyan Smokers’ Blood and Compared with Nonsmokers. Oral
presentation,1st Conference on Chemical Sciences and its Applications,
21-23 April 2014, El-beida-Libya (2014).

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 Determination of Nicotine in Juices, Soft and Energy Drinks… Galal Elmanfe et al.,

17- E. Davoli, L. Stramare, R. Fanelli, L. Diomede, M. Salmona, Rapid

solid-phase extraction method for automated gas chromatographic -
mass spectrometric determination of nicotine in plasma. J. Chromatogr.
B 707, 312-316 (1998).
18- J.S. Torano, H.J. Van Kan, Simultaneous determination of the tobacco
smoke uptake parameters nicotine, cotinine and thiogyanate in urine,
saliva and hair, using gas status of recently exposed subjects. Analyst,
128(7), 838-843 (2003).
19- D.L. Heavner, J.D. RichardsonW.T. Morgan, M.W. Ogend, Validation
and application of a method for the determination of nicotine and five
major metabolites in smokers’ urine by solid-phase extraction and liquid
chromatography-tandem mass spectrometry. Biomed. Chromatogr.
19(4), 312-328 (2005).
20- W. Amos-Tauta, M. Bamidele, E.R.E. Diepreye, Ultra-Violet
spectrophotometric determination of caffeine in soft and energy drinks
available in Yenagoa, Nigeria. Adv. J. Food Sci. Technol., 6(2), 155-
158 (2014).
21- FDA (Food and Drug Administration), Food Additives Status List,
(2006). http://www. cfsan.fda.gov/dms/opa-appa.html.
22- S.J. Sheen, Detection of Nicotine in Foods and Plant Materials. Journal
of Food Science, 53(5), 1573-1573 (1988).

Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022
 ‫…‪Determination of Nicotine in Juices, Soft and Energy Drinks‬‬ ‫‪Galal Elmanfe et al.,‬‬

‫تقدير النيكوتين في العصائر والمشروبات الغازية ومشروبات الطاقة جمعت من‬

‫األسواق المحلية الليبية باستخدام مطيافية األشعة المرئية و فوق البنفسجية‬

‫جالل المنفي* ‪ ,‬هدى سعد و ياسمين حسين‬

‫قسم الكيمياء ‪ ،‬كلية العلوم ‪ ،‬جامعة عمر المختار ‪ ،‬البيضاء‬

‫المستخلص العربي‬

‫هدفت هذه الدراسة إلى تقدير النيكوتين في عينات من العصائر والمشروبات الغازية ومشروبات الطاقة‬
‫جمعت من األسواق المحلية الليبية باستخدام الطرق الطيفية‪ .‬في هذه الدراسة تم جمع ستة عشر عينة‬
‫( أربعة عينات عصائر‪ ،‬سبعة عينات مشروبات غازية و خمسة عينات مشروبات الطاقة) من األسواق‬
‫المحلية في البيضاء ‪ -‬ليبيا‪ .‬تم تطوير طريقة استخراج سريعة وبسيطة وموثوقة وتم التحقق من‬
‫صالحيتها وذلك لتقدير النيكوتين في العينات تحت الدراسة باستخدام مطيافية األشعة المرئية وفوق‬
‫البنفسجية‪ .‬تم التحقق من صحة الطريقة على مدى خطي واسع من ‪ 81.06-1.62‬ميكروغرام ‪ /‬مل مع‬
‫معامالت االرتباط تزيد باستمرار عن ‪ 0.9999‬بواسطة جهاز تقنية التحليل الطيفي في منطقة األشعة‬
‫المرئية وفوق البنفسجية‪ .‬الحد األدنى من تركيز النيكوتين لوحظ في عينات العصائر ‪ ،‬بينما أظهرت‬
‫عينات مشروبات الطاقة أعلى محتوى من النيكوتين‪ .‬يشير القياس الطيفي إلى أن نتائج تركيزات‬
‫النيكوتين في عينات العصائر تتراوح بين ‪ 0.5238 - 0.3233‬ميكروغرام ‪ /‬مل‪ .‬وتركيزاته في عينات‬
‫المشروبات الغازية تتراوح بين ‪ 0.9949 - 0.7251‬ميكروغرام ‪ /‬مل‪ .‬بينما تتراوح تركي ازته في عينات‬
‫مشروبات الطاقة بين ‪ 1.3471 - 0.8521‬ميكروغرام ‪ /‬مل‪ .‬محتوي النيكوتين في جميع العينات التي‬
‫تم تحليلها في دراستنا كان أقل من الحدود المسموح بها التي وضعتها إدارة الغذاء والدواء األمريكية‪.‬‬

‫الكلمات المفتاحية‪ :‬العصائر؛ المشروبات الغازية؛ مشروبات الطاقة؛ النيكوتين؛ االستخالص؛ مطيافية‬
‫األشعة المرئية وفوق البنفسجية‪.‬‬

‫‪Libyan Journal of Basic Sciences, Vol: 16, No: 1, P: 79-89, April. 2022‬‬

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