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BY 4.0 license Open Access Published by De Gruyter Open Access March 17, 2023

Determination of nicotine content in locally produced smokeless tobacco (Shammah) samples from Jazan region of Saudi Arabia using a convenient HPLC-MS/MS method

  • Hassan A. Alhazmi EMAIL logo
From the journal Open Chemistry


Shammah is one of the forms of smokeless tobacco (SLT) prepared by mixing tobacco powder with other ingredients to increase its acceptability. Nicotine (NT) is the main alkaloid present in tobacco and is a precursor for carcinogenic metabolites including tobacco specific nitrosamines (TSNAs). In this study, eight varieties of Shammah samples, collected from Jazan region of Saudi Arabia, were analyzed for NT content by a validated high performance liquid chromatography-mass spectrometry/mass spectrometry method. Electrospray ionization was used with the multiple reaction monitoring in the positive mode for data acquisition. The method was fast and short retention times (RT) of 1.4 and 3.1 min were observed for NT and lapatinib used as internal standard (IS), respectively. The inter- and intra-day accuracy and precision results met the acceptance criteria of United States Food and Drug Administration and International Council for Harmonisation validation guidelines. The developed method was successfully applied for the detection of NT contents in various Shammah samples. NT concentration was found to be in the range of 6.94 ± 0.16 to 31.69 ± 0.79% with the maximum level detected in special Shammah from Ahad Al Masarihah and the lowest level in Khadrah Shammah from Samtah city. The results of this investigation have warranted further research to determine the minor Shammah contents including additive materials and assessment of associated health effects.

1 Introduction

The smokeless tobacco (SLT) is a non-combustible form of tobacco which is chewed, snuffed, or dipped [1] and is extremely popular in South and South-East Asian countries. Globally, there are more than 28 varieties of SLT, mainly consumed through oral or nasal routes and called by different names in different regions, such as Shammah, Maras, Neffa, Toombak, Snus, and Nass. Shammah is one of the SLT products locally produced by mixing the powdered tobacco leaves with lime, black pepper, ash, volatile oils as well as flavoring agents and consumed in Middle Eastern regions including Saudi Arabia and Yemen [2,3,4]. SLT may contain dependence producing levels of nicotine (NT) and hence it is equally addictive as smoked tobacco [5]. Data suggest that the absorption of NT through SLT usage is 2–3 times higher than what someone gets from a cigarette and the amount of NT received from 8–10 dips or chews of SLT per day equals smoking 30–40 cigarettes per day [6].

In Saudi Arabia, the trade and importation of SLT products is prohibited and legislation related to consumption of SLT appeared in 1990. The Ministry of Health, Saudi Arabia launched a National Tobacco Control Program in the year 2002 and the efforts further intensified after it became a member of World Health Organization (WHO) Framework Convention for Tobacco Control (FCTC) in the year 2005 [7,8]. A regional survey conducted in Jeddah among middle school students reported that 2% male students used locally produced Shammah [9]. According to a review report, Shammah use has been frequently observed in southern region (Jazan province) of Saudi Arabia, owing to its proximity to Yemen where the usage and trade of SLT is still legalized. More importantly, Shammah use by children between 10 and 13 years of age in Jazan region was also reported, which is thought to be the beginning age for its consumption. However, its use is not restricted to Jazan province; rather, its use has been observed in other regions as well [10].

The health problems associated with SLT use include cardiovascular disorders, weight loss, stillbirth, enhanced levels of creatinine and blood urea, high blood pressure, receding of gums, tooth caries, and oral sub-mucous fibrosis [3,11,12]. Approximately, 26% of all the neck and head cancer cases recorded annually in Saudi Arabia are oral cavity related tumors. Several studies reported strong correlation of increased oral cancer incidence in the country to frequent usage of SLT (Shammah). An investigation showed that 49% of oral cancer patients reported prior usage of Shammah [13,14,15,16]. Several studies reported relatively increased incidence of oral cancer in south-eastern part of Saudi Arabia, which is more predominant in Jazan province and its frequency is related to Shammah use by its residents. According to a review report of Tumor Registry record of 20 years from King Faisal Hospital and Research Center, Riyadh, almost 35.4% of all cases of oral cancer were only from Jazan region [24]. A report prepared by the Oral Maxillofacial Surgery Unit working at the Armed Forces Hospital, Riyadh indicated that out of all cancer forms, the oral cavity related tumors are the most frequent form of cancer in Jazan region, representing 72% of all head and neck cancer and 13% of total cancer cases recorded annually. The report also suggested that the higher incidence of oral cancer in the region is mainly attributed to the habit of Shammah consumption among the population [17]. The harmful effects of all SLT products including Shammah are mainly due to the presence of NT alkaloid.

Nicotine is a dibasic amine consisting of pyrrolidine and pyridine heterocyclic nucleus (Figure 1). Absorption and availability of NT in the biological system is pH dependent and is greater at basic pH. At higher pH values nicotine exists as non-ionic lipophilic molecule, and can easily cross biological membranes. The absorption of NT from SLT takes place through oral mucosa and small intestine [18]. Through consumption of SLT, NT shows slower rate of absorption in comparison to smoked form, resulting in gradual delivery to the bloodstream and taking more time to build up the plasma concentration and produce lesser pharmacological effects [18]. Although, International Agency for Research on Cancer (IARC) did not list NT as carcinogen, there are several reports supporting its carcinogenic potential [19]. NT is a precursor of tobacco specific nitrosamines (TSNAs) in the oral cavity through nitrosation, which can also result in the production of N-nitrosonornicotine and 4-(methyl nitrosamino)-1-(3- pyridyl)-1-butanone, that are strong carcinogens [20].

Figure 1 
               Chemical structures of (a) nicotine and (b) lapatinib (IS).
Figure 1

Chemical structures of (a) nicotine and (b) lapatinib (IS).

A number of analytical methods using various analytical techniques have been reported for detection and quantification of NT in tobacco and tobacco products. These methods include, titrimetric [21], HPLC-UV [22], ion-pair reversed phase HPLC [23], GC-MS [24], and high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) [25,26]. The NT content in biological samples is detected mainly by LC-MS/MS method and since the HPLC-MS/MS is highly sensitive, specific, and a workhorse technique, it is conveniently used for samples with complex matrices and fast analysis. In this study, HPLC-MS/MS technique was chosen to screen the Shammah samples for NT content using lapatinib (Figure 1) as internal standard (IS). Furthermore, according to literature, no HPLC-MS/MS method has been employed for NT estimation in Shammah samples from this region. A simple and single-step procedure with limited quantity of solvent was used for NT extraction from Shammah matrix. Validation of the developed analytical method was performed as per the guidelines provided by United States Food and Drug Administration (USFDA) [27] and the general recommendation of International Council for Harmonisation (ICH) [28].

2 Material and methods

2.1 Chemicals and reagents

NT, acetonitrile (HPLC grade), formic acid (HCOOH), ammonia solution, dimethyl sulfoxide (DMSO), and chloroform were purchased from Sigma-Aldrich (West Chester, PA, US). Lapatinib was procured from LC Laboratories (Woburn, MA, USA) and used as an IS. Ultrapure water was produced in our lab using Milli-Q plus water purification system (Millipore, Bedford, US).

2.2 Collection of Shammah samples

Shammah is produced by local manufacturers who mix the powdered tobacco leaves with a number of other ingredients such as coloring and flavoring agents, lime, volatile oils, black pepper, ash etc., to increase the potency and acceptability of their products among consumers. In the current study, 21 Shammah samples were procured from local suppliers in Jazan, Sabya, Ahad Al Masarihah, Samtah, and Abu Arish localities of the Jazan region of Saudi Arabia. Eight different varieties of Shammah, including Arishi, Khadrah, Special, Adani, Adani cold, Adani hot, Suhail, and Sudani Shammah were collected. The collected samples were of different colors including yellow, black/gray, white, and brown depending on the types of additives added. The samples were stored in refrigerator at 2–8°C until analysis.

2.3 Instrumentation

A HPLC-MS/MS system coupled with Agilent 6410 QqQ mass spectrometer (Agilent Technologies, CA, USA) attached to Agilent HPLC 1200 Series system (Agilent Technologies, CA, USA) was utilized for the analysis of NT and IS. The mass spectrometer was equipped with an electrospray ionization (ESI) interface. The HPLC system consisted of G1316A binary pump, G1316 temperature regulated column chamber, G1316A degasser, and G1316B autosampler. The instrument was monitored and data acquisition was performed using Mass Hunter software (Agilent Technologies, CA, USA).

2.4 HPLC-MS/MS conditions

Agilent eclipsed plus C18 column (50 mm × 2.1 mm internal diameter, 1.8 µm particle size) (Agilent Technologies, CA, USA) was utilized to perform chromatographic separation of NT and IS at a column oven temperature of 22 ± 2°C. The injection volume was 5 µL and elution of the analyte was achieved in isocratic flow mode using a mixture of formic acid (0.1%) in water (70% v/v, pH∼3.2) and acetonitrile (30% v/v) as mobile phase at a flow rate of 0.3 mL/min. Total run time of the current method was 5 min and NT and IS were detected at 1.4 and 3.1 min retention times (RTs), respectively. A QqQ mass spectrometric detector was used for the identification and quantification of NT and IS. The source of ionization was through ESI operated in the positive mode. The multiple reaction monitoring (MRM) mode was used for the detection of analytes by monitoring their transitions. Standard solutions of analytes were injected into the mass spectrometer using a syringe pump and the parameters were optimized to achieve highest [M–H]+ abundance. At a flow rate of 12 L/min, high purity nitrogen gas was employed as a drying gas and as a collision gas at a pressure of 60 psi, capillary voltage was 4,000 V, and the ion source temperature was 350°C. Fragmentor voltage was set at 140 and 145 V with collision energy of 16 and 15 eV for NT and IS, respectively. The molecular ion peaks for NT and IS were observed at m/z 163 and 584, respectively, in the positive MS scan.

2.5 Preparation of working standard solution

Stock solution of NT at a concentration of 1.0 mg/mL was prepared by dissolving NT in the mobile phase. The standard working solution-1 (WS-1, 100 µg/mL) was obtained by diluting the stock solution ten folds using mobile phase. The WS-1 was further diluted ten folds using the mobile phase to obtain a standard working solution-2 (WS-2) in order to obtain a final concentration of 10 µg/mL. The stock solution of IS (100 µg/mL) was obtained by dissolving lapatinib in DMSO and the working solution for IS (2 µg/mL, WI) was then prepared by diluting the stock solution using the mobile phase.

2.6 Preparation of quality control solutions and calibration standards (CSs)

Liquid–liquid extraction using ammonia/chloroform was employed for the preparation of CSs and samples extraction. The CSs were prepared by serially diluting the working standard solution in the mobile phase. WS-2 (10 µg/mL) was diluted to achieve 11 calibration concentrations, 5, 10, 15, 30, 50, 100, 150, 200, 300, 400, and 500 ng/mL, keeping the final volume to at least 10 mL for each. The CSs were re-prepared by transferring 10 mL of each CS to a separating flask followed by addition of 5 mL ammonia solution. The flask was shaken and approximately 25 mL of chloroform was added. Flasks were then closed using a stopper and placed on a mechanical shaker for about 1 h. All the flasks were manually shaken again for another 2 min. The chloroform layer was collected and evaporated under vacuum to obtain a solid residue. The dried residue was reconstituted with 10 mL of mobile phase and 0.1 mL IS solution was added to correct any sudden change in MS detection efficiency (WHO, 2014). The quality control solutions were prepared similarly to obtain low quality control (LQC), medium quality control (MQC), and high quality control (HQC) solutions with nominal concentrations of 15, 150, and 400 ng/mL. Blank solutions with and without IS were prepared by following the same procedure by omitting NT alone and NT and IS both, respectively. Blank solutions were injected in the system to rule out any interference at the RTs of NT and IS.

2.7 Extraction of NT from Shammah powder and preparation of sample solution

One gram of Shammah sample was taken in a separating conical flask and the NT was extracted by following the same procedure used for preparing the CSs. Appropriately measured volume of IS was added after final dilution to achieve sample solutions possessing an analytical concentration within the calibration range with the same final IS concentration as in the CSs. The sample solutions were transferred to autosampler inserts and 5 µL of sample from each vial was injected into HPLC-MS/MS for analysis.

2.8 Method validation

The proposed method for analysis of NT in Shammah samples was validated by following the analytical method validation guidelines of USFDA [27] and the general recommendation of ICH [28] with respect to sensitivity, linearity, specificity, accuracy, and precision.

2.8.1 Linearity and sensitivity

Linearity of the developed method was evaluated by injecting six replicates of all the CSs. The ratio of peak area obtained for NT and IS against the nominal NT concentrations were used to plot the calibration curve. A linear least-square regression analysis was performed to assess the linearity using appropriate weighting. The detection limit (LOD) and quantitation limit (LOQ), based on the standard deviation of the intercept and slope of the calibration plot, were calculated to determine the method’s sensitivity. It was calculated using equation (1).

(1) LOD or LOQ = mS n ,

where “S” is the standard deviation of the intercept and “n”’ represents the slope of the calibration plot; and ”m” equals 3.3 for LOD and 10 for LOQ.

2.8.2 Precision and accuracy

To establish the repeatability of the current method, inter- and intra-day precision and accuracy were determined. The QC samples were analyzed at LQC, MQC, and HQC concentrations at three different times on the same day to determine the intra-day precision and accuracy, and the analysis was done at three different times on three different days to determine the inter-day precision and accuracy (six determinations). The percent relative standard deviation (% RSD) and percent relative error (% RE) between the measured and nominal values were used to express precision and accuracy, respectively. The % RSD values less than 15% and % RE values within ± 15% were set at acceptance criteria for precision and accuracy, respectively. The % RSD and % RE were calculated using the following formulae (2) and (3):

(2) % RSD = SD Mean × 100 ,

(3) % RE = ( Average back calculated concentration Expected concentration ) Expected concentration × 100 .

2.8.3 Specificity and selectivity

Double blank solution (n = 6; no NT and no IS) and blank solution with IS (n = 6) after analysis of highest calibration standard were analyzed in order to establish the specificity and selectivity of the developed method. Interference responses at the RTs of NT and IS was examined and response less than 20% of the lowest calibration standard and 5% of the mean response of the working solution of IS (WI) were considered as acceptance criterion for NT and IS, respectively.

2.9 Statistical analysis

Chemometric analysis of the collected Shammah samples were performed using NCSS-2021 statistical software and the amount of nicotine present in all the samples were compared and analyzed. A commonly used multivariate technique, hierarchical cluster analysis (HCA) was employed to divide the samples into clusters on the basis of amount of nicotine present as variables and a dendrogram tree was developed to establish similarities between the samples.

3 Results and discussion

3.1 Chromatographic and spectrometric conditions

Chromatographic parameters such as mobile phase composition are extremely important to achieve good chromatographic outcomes including selectivity, sensitivity, symmetric peaks, and short run time. It also affects the ionization characteristics of the analytes in MS detector [29]. Consequently, after several trials, an optimum composition of mobile phase which consisted of formic acid (0.1%) in water (pH ∼ 3.2, 70% v/v) with acetonitrile (30% v/v) using isocratic mode of elution at 0.3 mL/min flow rate offered best peak shape with optimum response and short runtime. Peaks of NT and IS were detected at 1.4 and 3.1 min, respectively, with a total runtime of 5 min. Both NT and IS were found to be resolved properly and no carryover was observed in the blank chromatograms. Representative chromatograms of analytes and blanks are depicted in Figure 2. Lapatinib was chosen as IS for this analysis because it showed closest chromatographic behavior to NT.

Figure 2 
                  Total ion chromatogram MRM chromatograms of (a) NT and lapatinib (IS), (b) blank with IS and (c) double blank (without NT and IS).
Figure 2

Total ion chromatogram MRM chromatograms of (a) NT and lapatinib (IS), (b) blank with IS and (c) double blank (without NT and IS).

To optimize the MS conditions, experiments were conducted by employing positive mode of ionization in order to achieve good detection sensitivity for both precursor and product ions [25,26]. However, higher responses for both NT and IS were achieved in the positive mode in comparison to negative ionization mode. Consequently, positive mode of ionization was finalized for the current HPLC-MS/MS method and optimized to receive the best result. The molecular ion peaks for NT and IS were observed at m/z 163 and m/z 581.1, respectively. Two product ions at m/z 365 and m/z 350 were identified for IS, whereas further fragmentation of NT produced two fragment ions at m/z 130 and m/z 84. In accordance with the established method, these ions were chosen for the MRM of NT and IS. Figure 3 shows the MS/MS spectra obtained for NT and IS.

Figure 3 
                  The MS/MS spectra of NT and lapatinib (IS) in positive mode.
Figure 3

The MS/MS spectra of NT and lapatinib (IS) in positive mode.

3.2 Method validation

3.2.1 Linearity and sensitivity

HPLC-MS/MS analysis of a series of calibration standards were performed (n = 6) and calibration curve was obtained by plotting the peak area ratio of NT to IS against the nominal concentrations to perform a regression analysis using 1/X 2 weighting factor (MS Excel). Results showed excellent linearity within the range 5–500 ng/mL as indicated by the correlation coefficient value (r 2 > 0.999) which was close to unity. The regression equation of the calibration curve was obtained to be y = 3.06x – 10.39. These results indicated good linearity of the current method over a wide range of concentrations. Additionally, the calibration points were validated in order to generate the calibration curve as shown by the low SD value of the intercept and slope. The LOD and LOQ values were calculated to be 4.36 and 13.23 ng/mL, respectively, demonstrating appropriate sensitivity. After that, the working standard solutions above the LOQ (15–500 ng/mL, r 2 > 0.999, y = 3.06x – 10.99) have been used for routine calibration curve preparation and quantification of NT.

3.2.2 Precision, accuracy, and stability

The intra- and inter-day precision and accuracy of NT in the LQC, MQC, and HQC samples are summarized in Table 1. The intra-day precision (% RSD, n = 6) at all three QC concentrations were ≤3.34% and the accuracy (% RE, n = 6) was in the range of −1.26 to 2.46%; whereas, the inter-day precision showed % RSD (n = 6) values ≤3.03% and the accuracy (% RE) values were within −1.45 to 1.57%. These results were within the acceptable limits as required by the guidelines, demonstrating that the current method was precise, accurate, repeatable, and reliable. Recovery of the analyte in the intra- and inter-day analysis at all three QC concentrations were in the range of 100.9–98.8%, which further proved the accuracy of the present method.

Table 1

Intra- and inter-day precision and accuracy results of the developed method for NT in the quality control samples

Parameters LQC (15 ng/mL) MQC (150 ng/mL) HQC (400 ng/mL)
Intra-day analysis* Inter-day analysis** Intra-day analysis* Inter-day analysis** Intra-day analysis* Inter-day analysis**
Mean observed concentrations (ng/mL) 15.09 15.14 149.11 148.68 396.21 395.34
Percent recovery 100.6 100.9 99.4 99.1 99.1 98.8
Standard deviation (SD) 0.51 0.46 3.47 3.58 5.49 5.51
Precision (% RSD) 3.34 3.03 2.32 2.42 1.39 1.40
Accuracy (% RE) 2.46 1.57 −0.30 −1.13 −1.26 −1.45

*Average of 6 replicates of day 1 analysis. **Average of 6 replicates performed in 3 consecutive days.

The stability of the standard solutions has been evaluated using LQC, MQC, and HQC at normal laboratory temperature for 3 days and refrigerated (2–8°C) for 14 days. The overall recovery percentage was in the range of 100 ± 3% indicating good stability for routine analysis purpose.

3.2.3 Specificity/selectivity

It was evident from the representative chromatograms of double blank (no NT and IS) and blank with IS (no NT) that there was no interference at the RTs of NT (1.4 min) as well as IS (3.1 min) and no carryover effect was present for either main analyte (NT) or IS. Consequently, the method was considered to be specific for the analyte determination. The specificity/selectivity of the method was further proved by excellent recovery of analyte (NT) in the assay of QC samples at LQC, MQC, and HQC levels (Tables 1 and 2).

Table 2

Recovery, precision, and accuracy data of NT in Shammah sample*

Nominal concentration (ng/mL) Mean back calculated concentration (ng/mL) Percent recovery SD Precision (% RSD) Relative accuracy error (RE%) Absolute accuracy error (ng/mL)
15.00 15.38 102.5 0.51 3.34 2.51 +0.38
150.00 149.55 99.7 3.47 2.32 −0.30 −0.45
400.00 395.30 98.8 5.49 1.39 −1.17 −4.70

*Average of six determinations.

3.3 Method performance in the presence of Shammah ingredients

In order to ensure the performance of the proposed method, recovery of NT was determined in Shammah samples at LQC (15 ng/mL), MQC (150 ng/mL), and HQC (400 ng/mL) levels. The QC samples were prepared in six replicates and the precision and accuracy for analyte were calculated as % RSD and % RE between the back calculated and nominal concentrations of NT, respectively. Results revealed that the current method was precise and accurate for NT analysis in the Shammah samples, as the % RSD values were below 5% and % RE values were in the range of −1.17 to 2.51%. The recoveries at all QC concentrations were within 100 ± 15% (Table 2). The appropriateness of the method was further demonstrated by the lack of any interference from the sample matrix at the NT and IS retention periods. The acceptable recovery results and absence of interference in the chromatograms of analyte and IS indicated the selectivity of the present analytical method.

3.4 Determination of NT content in Shammah samples

Shammah contains remarkable concentrations of NT along with other constituents with known pharmacological and toxicological potentials. After examining the suitability, the validated method was used to estimate the amount of NT present in the collected 21 Shammah samples and the results are shown in Table 3. The NT present in Shammah samples was extracted in chloroform by following the procedure described in Section 2. Samples were injected into the HPLC-MS/MS system for the detection and quantification of NT using the developed method. All the samples were analyzed in triplicate and results were reported as NT content in mg/g on received basis ±% RSD. As evident from Table 3, NT content present in the samples varied considerably from one sample to another and was detected in the range of 6.94 ± 0.16 to 31.69 ± 0.79 mg/g of Shammah, with an average of 16.58 ± 5.00 mg/g. Maximum NT (31.69 ± 0.79 mg/g) was recorded in Special Shammah collected from Ahad Al Masarihah, while the lowest NT content (6.94 ± 0.16 mg/g) was measured in Khadrah Shammah from Samtah.

Table 3

Nicotine contents observed in different Shammah samples collected from Jazan region of Saudi Arabia

Sample code Shammah samples Collection site/city NT content (mg/g)* ± SD**
SLT-1 Special Shammah Ahad Al Masarihah 31.69 ± 0.79
SLT-2 Khadrah Shammah 14.41 ± 0.27
SLT-3 Arishi Shammah 23.26 ± 0.26
SLT-4 Adani cold 15.33 ± 0.21
SLT-5 Adani hot Shammah 20.04 ± 0.13
SLT-6 Special Shammah Sabya 19.42 ± 0.41
SLT-7 Khadrah Shammah 12.39 ± 0.17
SLT-8 Arishi Shammah 16.32 ± 0.18
SLT-9 Adani cold Shammah 15.54 ± 0.14
SLT-10 Sudani Shammah 13.55 ± 0.17
SLT-11 Suhail Shammah 17.61 ± 0.33
SLT-12 Khadrah Shammah Jazan 10.53 ± 0.15
SLT-13 Arishi Shammah 17.89 ± 0.26
SLT-14 Adani cold 18.15 ± 0.19
SLT-15 Special Shammah Samtah 17.69 ± 0.16
SLT-16 Khadrah Shammah 6.94 ± 0.16
SLT-17 Special Shammah Abu Arish 15.46 ± 0.19
SLT-18 Khadrah Shammah 17.82 ± 0.26
SLT-19 Arishi Shammah 11.00 ± 0.10
SLT-20 Adani cold Shammah 17.76 ± 0.23
SLT-21 Adani hot Shammah 15.46 ± 0.13
Mean value ± SD 16.58 ± 5.00

*On as received basis. **Average of three determinations.

Although the type of tobacco used to prepare all Shammah varieties remains similar, the variation in NT content was due to the variability in amounts of tobacco along with other natural and chemical ingredients used in different types and lots. The variation in the NT content in different Shammah samples is obvious because there is no standard protocol in the preparation being followed. Therefore, same Shammah samples obtained from different locations were found to have different levels of NT. The NT content might even vary from one batch of Shammah to another from same manufacturer (lot variation). Therefore, comparison between the products of same type or the products made at different time points may not be generalized. For instance, the NT content detected in Khadrah Shammah collected from Ahad Al Masarihah was 14.41 ± 0.27 mg/g, while those from Sabya, Jazan, Samtah, and Abu Arish were found to contain 12.39 ± 0.17, 10.53 ± 0.15, 6.94 ± 0.16, and 17.82 ± 0.26 mg/g, respectively. With respect to the location of sample collection, highest average NT content was observed in the sample from Ahad Al Masarihah, whereas the minimum content was found in samples from Samtah.

3.5 Hierarchical cluster analysis (HCA)

A cluster-hierarchy was developed using agglomerative hierarchical clustering algorithms represented as a tree diagram also called as dendrogram. Each object was placed in separate clusters and at every step, two most similar clusters were joined into a single new cluster. The HCA of all 21 SLT samples were performed using nicotine content as variable and a dendrogram tree was obtained (Figure 4). The horizontal axis of the dendrogram shows the distance or dissimilarities between the samples and the vertical axis represents the clusters or objects.

Figure 4 
                  Dendrogram tree obtained for the 21 Shammah samples showing clusters of samples and distance indicating similarities between them.
Figure 4

Dendrogram tree obtained for the 21 Shammah samples showing clusters of samples and distance indicating similarities between them.

As evident from Figure 4, all the 21 samples were divided into two distinct clusters which were differentiated by red and blue color lines. The first cluster was the major cluster represented by red horizontal lines consisting of 16 samples with NT content from 11 to 20 mg/g. The other cluster was represented in blue lines and consisted of only three samples SLT-12, SLT-16, and SLT-19 having NT content in range of 6–11 mg/g. Interestingly, two samples, SLT-1 (Special Shammah) and SLT-3 (Arishi Shammah) from Ahad Al Masarihah city could not be placed in any cluster and were different from all other samples since their NT content was more than 20 mg/g. These two samples were separated from cluster – 1 and cluster – 2 with distances more than 1 showing dissimilarities between them. Also, the two samples were separated with a large distance as evident from the figure. This indicated that the tobacco used to prepare these two samples might have different origins than the others as they might be of a grade having higher NT content or a pure NT powder could be added. The dendrogram divides the samples in clusters on the basis of similarities between them. More is the distance between the two samples, more will be the dissimilarity between them. Generally, the distance less than 1 is considered to be significant and two samples separated by distance less than 1 are considered to be similar which might have originated from a single source. Lesser distances will have more probability of getting originated from the same source and these samples share similar characteristics. Fusion of two clusters is represented by splitting of a horizontal line into two horizontal lines on the dendrogram and their position show the dissimilarity between the two clusters.

4 Conclusion

NT is a well-known alkaloid present in tobacco and in addition to its strongly addictive nature it adversely affects almost all organs of the body. Several studies indicated its carcinogenic potential and there are evidences indicating that nitrosation of NT in vivo could lead to formation of known carcinogens including TSNAs. In this research, a fast, sensitive, and selective HPLC-MS/MS method for the detection and quantification of NT was developed and validated. The chromatographic method allowed quick elution of analyte under isocratic mode and applied for determination of NT in the Shammah samples collected from Jazan region of Saudi Arabia. A simple and single-step procedure for the extraction of NT from the SLT matrix was used and the detection was made under MRM using positive mode ESI technique. The present investigation found considerably variable concentrations of NT (6.94 ± 0.16 to 31.69 ± 0.79%) among the tested Shammah samples, which is mainly due to variable amounts of the tobacco used in the mixture from one manufacturer to another. Further research is warranted to measure the contents of minor alkaloids, TSNAs, and other potentially harmful ingredients and to assess the health impact associated with the use of these products. This study would help to enhance the awareness among the common people of the region about the harmful effects associated with the use of Shammah through educational measures and would help the decision makers and government bodies to restrict and minimize its production and sale.


The author extends his appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number: ISP22-12.

  1. Funding information: This work was funded by the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia through the project number: ISP22-12.

  2. Author contributions: Conceptualization, formal analysis, writing – original draft preparation, and writing – review and editing: HAA.

  3. Conflict of interest: Author declares no conflict of interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All final data generated or analysed during this study are included in this published article. The sub-datasets are available from the corresponding author on reasonable request.


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Received: 2022-09-14
Revised: 2023-02-10
Accepted: 2023-02-24
Published Online: 2023-03-17

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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