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Objective <p>The rapid proliferation of smokeless tobacco (SLT) products in India has occurred without sufficient awareness of their toxicological risks and addiction potential. This study developed and validated a robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the accurate quantification of nicotine in smokeless tobacco products (SLT) includes Tobacco, Snus, Pan masala, Zarda and Supari. The method was optimized for simplicity, accuracy, stability, and rapid analysis, enabling precise determination of nicotine concentrations at microgram-per-gram (ppm) levels. Diverse commercial and locally sourced smokeless tobacco samples from five Indian states (Uttar Pradesh, West Bengal, Hyderabad, Assam and Gujrat) were analysed.</p> Method <p>Optimal chromatographic separation was achieved using an Inertsil C18 column with an isocratic mobile phase of 1.1% triethylamine (TEA) buffer and acetonitrile (75:25, pH 7.0) at a flow rate of 1.0 mL/min. UV detection was performed at 254&#xa0;nm. The method was validated according to ICH Q2(R1) guidelines.</p> Results <p>The developed HPLC assay demonstrated a nicotine peak purity of 99.62%. Validation parameters met ICH guidelines, with system, method, and intermediate precision RSD values of 0.03%, 0.18%, and 0.12%, respectively. Mean recovery was 99.18%, and linearity was established over a 2.5–150% range (R² = 0.9993). Nylon and PVDF filters were deemed suitable for nicotine analysis. Methanol diluent stability was confirmed for 20&#xa0;h for standard solutions and 12&#xa0;h for test solutions. Forced degradation studies revealed significant nicotine instability under acidic, basic, and oxidative conditions. Analysis of 84 smokeless tobacco samples yielded nicotine concentrations ranging from 24.0 to 55296.8&#xa0;µg/g in West Bengal, 17.2 to 35778.2&#xa0;µg/g in Uttar Pradesh, 56.3 to 2239.0&#xa0;µg/g in Gujarat, 7239.0 to 22768.2&#xa0;µg/g in Himachal Pradesh, and 52.2 to 27192.0&#xa0;µg/g in Hyderabad.</p> Conclusion <p>Quantitative analysis of smokeless tobacco products across India revealed substantial inter-product variability in nicotine concentrations. These findings highlight the potential public health risks associated with smokeless tobacco use, particularly concerning the addictive nature of nicotine.</p>

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RP-HPLC Analysis of Nicotine Content in Diverse Smokeless Tobacco Matrices: A Comparative Study of Commercial and Local Products from India

  • 期刊论文

  • 发表日期：

    2025年10月6日

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  • Ashu Rathi,
  • Arbab Husain,
  • Afreen Khanam,
  • Mohd Farhan,
  • Mohammad Aatif,
  • Rajeev Singh Raghuvanshi,
  • Gaurav Pratap Singh,
  • Anirudh Malik

摘要

Objective

The rapid proliferation of smokeless tobacco (SLT) products in India has occurred without sufficient awareness of their toxicological risks and addiction potential. This study developed and validated a robust reversed-phase high-performance liquid chromatography (RP-HPLC) method for the accurate quantification of nicotine in smokeless tobacco products (SLT) includes Tobacco, Snus, Pan masala, Zarda and Supari. The method was optimized for simplicity, accuracy, stability, and rapid analysis, enabling precise determination of nicotine concentrations at microgram-per-gram (ppm) levels. Diverse commercial and locally sourced smokeless tobacco samples from five Indian states (Uttar Pradesh, West Bengal, Hyderabad, Assam and Gujrat) were analysed.

Method

Optimal chromatographic separation was achieved using an Inertsil C18 column with an isocratic mobile phase of 1.1% triethylamine (TEA) buffer and acetonitrile (75:25, pH 7.0) at a flow rate of 1.0 mL/min. UV detection was performed at 254 nm. The method was validated according to ICH Q2(R1) guidelines.

Results

The developed HPLC assay demonstrated a nicotine peak purity of 99.62%. Validation parameters met ICH guidelines, with system, method, and intermediate precision RSD values of 0.03%, 0.18%, and 0.12%, respectively. Mean recovery was 99.18%, and linearity was established over a 2.5–150% range (R² = 0.9993). Nylon and PVDF filters were deemed suitable for nicotine analysis. Methanol diluent stability was confirmed for 20 h for standard solutions and 12 h for test solutions. Forced degradation studies revealed significant nicotine instability under acidic, basic, and oxidative conditions. Analysis of 84 smokeless tobacco samples yielded nicotine concentrations ranging from 24.0 to 55296.8 µg/g in West Bengal, 17.2 to 35778.2 µg/g in Uttar Pradesh, 56.3 to 2239.0 µg/g in Gujarat, 7239.0 to 22768.2 µg/g in Himachal Pradesh, and 52.2 to 27192.0 µg/g in Hyderabad.

Conclusion

Quantitative analysis of smokeless tobacco products across India revealed substantial inter-product variability in nicotine concentrations. These findings highlight the potential public health risks associated with smokeless tobacco use, particularly concerning the addictive nature of nicotine.