On the Influence of Additives and Modifiers on the Chiral HPLC Separation of the Enantiomers of Nicotine

Chirality · 2025-01-26 · 4 citations

The influence of additives and modifiers on the chiral HPLC separation of the nicotine enantiomers using UV/Vis detection is discussed. Selected alcohols as modifiers and selected amines as additives were found to have a significant effect on the resolution and retention times of nicotine enantiomers even to the point of eliminating component elution. Systematic variations in the concentration of ethanol, methanol, and isopropanol, as modifiers, along with variations in the concentration of diethylamine, triethylamine, tributylamine, ethylenediamine, isopropylamine, as additives, revealed that the average resolution (R) of the nicotine enantiomers ranged from 2.9 to 7.57, using a mobile phase flow rate of 0.80 mL/min. The average retention times of the nicotine enantiomer pairs ranged from 7.64 and 8.34 min to 13.47 and 14.97 min, with the S(-) enantiomer eluting first. As expected, faster flow rates of 1.0 mL/min reduced retention times by approximately 1-2 min, with a slight decrease in the R values. The %RSD values for both resolution and retention times consistently remained below 2%. The detection limits for the enantiomers were approximately 5 μg/mL. The optimized method successfully detected one part in 100 for the minor R(+) enantiomer in the presence of the dominate S(-) enantiomer and adhered to all established QuEChERS method protocols.

High-Performance Liquid Chromatography Using Ultraviolet Detection for Separation of Terephthalic Acid and Associated Impurities from Recycled Materials

Journal of Chromatographic Science · 2025-05-01 · 2 citations

An optimized rapid and precise high-performance liquid chromatography method has been developed to separate, identify and quantitate impurities contained within terephthalic acid (TPA) produced from recycled materials. Using a Waters X-Select HSS T3 column for the separation of recycled TPA and eight impurities, and a mobile phase of H2O/CH3CN containing 0.1% trifluoroacetic acid (TFA) with detection by UV at 250 nm, baseline separation of all compounds was achieved in ˂17 min. Four different mobile phase acidic additives were tested, the results indicating that mobile phases with lower pH provided better separation of the compounds with accompanying improved peak shapes. Among all the acidic additives tested in this study, a mobile phase containing 0.1% TFA demonstrated the highest resolution and the best peak shapes. It was concluded that by suppressing the ionization of the acidic compounds contained in the samples through increased acidity of the mobile phase, better peak shapes and higher resolution could be achieved. The amount of increased mobile phase acidity was based in part on the pKa values of the analytes and adjusted so as to eliminate as much as possible any dissociation of the organic acids. A comparison of a standard addition method versus an external calibration curve approach for the quantification of impurities showed similar results, even for relatively low-concentration analytes at levels below 10 ppm. The aspects often linked with validated analytical methods were addressed including linearity, limit of detection, limit of quantitation, sensitivity and selectivity, addressing the criteria set forth in the acronyms of the QuECHERS method description, that is quick easy, cheap, effective, rugged and safe.

Qualitative and Quantitative Analyses of the Enantiomers of Nicotine and Related Alkaloids Employing Chiral Supercritical Fluid Chromatography in Commercial Nicotine Samples and in E-Cigarette Products

Contributions to Tobacco & Nicotine Research · 2023-07-01 · 3 citations

Summary Several commercial sources of tobacco-derived nicotine (TDN) and synthetic nicotine (SyN) and a variety of e-cigarette liquids employing either TDN or SyN have been evaluated to determine the enantiomer distributions of R- and S-nicotine and R- and S-nornicotine by chiral supercritical fluid chromatography (chiral-SFC) with UV diode array detection (DAD-UV). The data generated are used to test the mismatched vs . matched hypothesis of C heetham et al. as a means to distinguish products containing TDN from products with SyN. Two sets of experiments were conducted in this study. The first experiment was conducted on a series of 11 commercial nicotine samples (three characterized as tobacco-derived and eight characterized as synthetic nicotine). The commercial nicotine samples were either from a tobacco-derived nicotine (TDN) source or were synthetic nicotine (SyN). Some of the commercial nicotine samples were nicotine salts. The second experiment was conducted on e-liquids from a set of 11 e-cigarettes. The nicotine in the e-liquids was either from TDN or SyN. The e-liquid samples were differentiated based on the advertised information on the internet or from printed information on the e-cigarette packaging. None of the three commercial TDN samples in the first experiment could be unequivocally characterized as coming from a tobacco source. Five of the eight commercial SyN samples were correctly characterized as SyN based on the matched vs . mismatched nicotine and nornicotine hypothesis of C heetham et al. In the second experiment, none of the e-liquids characterized as being from TDN sources could be unequivocally characterized as coming from a tobacco source. All of the e-liquids characterized as being from SyN sources were either characterized as equivocal or of uncertain origin based on the matched vs . mismatched nicotine and nornicotine hypothesis of C heetham et al. These sets of experiments represent an excellent example of the difficulty that the United States Food and Drug Administration is having in trying to determine if TDN or SyN is being used in tobacco products. Even highly advanced chromatographic methods such as chiral-SFC were not able to unequivocally distinguish products with TDN from products with SyN 100% of the time. Other analytical methods such as 14 C quantitation of nicotine samples by accelerator mass spectrometry offer a more reliable determinate of nicotine source (TDN vs . SyN) and can be used to identify misbranded products labelled as containing SyN, even though this methodology is more expensive and offered in limited locations.

Qualitative and Quantitative Analyses of the Enantiomers of Nicotine and Nornicotine Employing Chiral Supercritical Fluid Chromatography

Journal of Chromatographic Science · 2023 · 5 citations

• Chemistry
• Chromatography
• Organic chemistry

An optimized method employing chiral supercritical fluid chromatography with diode array UV-VIS detection has been developed for the quantitative analysis of nicotine and nornicotine enantiomer distributions. The method parameters that were optimized included: column type (stationary phases, Chiralpak IG-3), column temperature (40°C), modifier types and concentration (isopropyl alcohol, 10%), additive types and concentrations (diethylamine, 0.2%), elution times (<6 min, flow rate 3 mL/min) and resolution factor (>1.2). These optimized conditions led to nicotine and nornicotine enantiomer detection limits of ~5 ng/μL with accompanying %RSD values of <2% from the analyses of commercially available nicotine-containing formulations.

Qualitative and Quantitative Analysis of Nicotine, Nicotine Derivatives, and Nicotine-Related Alkaloid Optical Isomers: A Review

Contributions to Tobacco & Nicotine Research · 2022 · 7 citations

• Computer Science
• Chemistry
• Chromatography

Summary During the last 35 years technologies and hardware for the separation of enantiomers have advanced tremendously. Today, complete resolution of the enantiomers of nicotine and related compounds can effectively be attained within a few seconds and most often less than 10 minutes. In some unique cases, enantiomeric separation for nicotine-related compounds having a wide variety of functional groups has been demonstrated. Particularly, the successful wedding of short HPLC and SFC columns containing very small particle size materials bonded to chiral stationary phase materials coupled with information-rich detectors like electrospray ionization mass spectrometry/mass spectrometry has facilitated this remarkable transition. Taken collectively the HPLC and SFC technologies will allow for very rapid (seconds), reliable, and reproducible (% RSD routinely &lt; 5%) for nicotine and related alkaloid enantiomers.

Separation optimization and quantitative analysis of phytoestrogens employing reverse-phase high-performance liquid chromatography with UV-VIS detection

Journal of Liquid Chromatography &amp Related Technologies · 2021 · 6 citations

• Chemistry
• Chromatography
• Organic chemistry

The effect of different acidic modifiers on the separation of seven selected phytoestrogens by reverse-phase high-performance liquid chromatography has been described. Variation in the hydrocarbon chain length of organic acids as represented by acetic acid, propanoic acid, iso-butyric acid, and n-butyric acid as additives to the mobile phase had minimal effect on the separation of phytoestrogens but notably reduced sample turnaround times relative to the turnaround time linked with formic acid. Two different C18 columns were evaluated in attempts of optimize the phytoestrogen separations. Formic acid as a modifier in the mobile phase and an Atlantis C18 column provided the best overall separation at optimum gradient conditions. A higher acid concentration in the mobile phase did not improve the phytoestrogen separation. Higher column temperature (40 °C) provided an improved separation. Finally, the optimized method was used to quantify different phytoestrogens in selected different botanical samples. For this purpose, both internal and external standard methods were compared to obtain the quantitative results.

Optimization of α-Hydroxyketone and Pyrazine Syntheses Employing Preliminary Reactions of Glucose and Buffer Solutions

Beiträge zur Tabakforschung international · 2019-12-01

Summary Glucose and selected phosphate buffers have been reacted employing systematic variations in reaction temperature and time (150–160 °C for 60–90 min) to optimize the yield of acetol. This mixture was reacted further with NH 4 OH, systematically varying reaction conditions and reagent ratios to optimize pyrazine yield. The highest yield of pyrazine was obtained when 1 g of glucose was reacted with 25 mL of buffer at 150–160 °C for 60 min, which in turn was reacted with 1 mL of concentrated aqueous NH 4 OH at 120–130 °C for 17–18 h. Higher temperatures and higher concentrations of glucose caused a decrease in the yield of pyrazines. The addition of hydrolyzed tobacco-derived F1 protein as a secondary source of nitrogen increased the yield of pyrazines by 2–10% depending on F1 protein concentration. Furthermore, the addition of any α-hydroxyketone, similar in structure to acetol, as a pure reagent to the reaction mixture not only increased the yields of pyrazine by ranging from 25–100 % depending on the reagent concentration, but also significantly altered the qualitative and quantitative distribution of the pyrazines. With all of the reaction parameters examined (reaction time, temperature, reagent ratios, etc.) the most significant impacts on both pyrazine yield and distribution were noted when: 1) glucose was pre-reacted with buffer, 2) hydrolyzed F1 protein was added as a second nitrogen source, and 3) when pure α-hydroxyketones were employed as co-reagents. Use of these reaction parameters was found to dramatically shift the pyrazine distribution toward higher molecular weight resulting in a pyrazine array having more desirable physical and sensory attributes.

Optimal Synthesis of Substituted and Branched Pyrazines via Reaction of Alpha Hydroxy Ketones with Selected Nitrogen Sources

Beiträge zur Tabakforschung international · 2019-08-01 · 1 citations

Summary Employment of 1-hydroxy-acetone as a carbon source and NH 4 OH as a source of base and nitrogen, has enabled arrays of pyrazines to be synthesized. Reaction conditions such as temperature, time, carbon/nitrogen mole ratios and pH were optimized to maximize the quantity of pyrazines, thereby providing the synthesis of at least 19–20 structurally different pyrazines. Addition of amino acids, selected aldehydes, and hydrolyzed tobacco-derived F1 protein has positively impacted the array of pyrazines from both qualitative and quantitative aspects. Results further showed that by changing the carbon source from 1-hydroxy-acetone to 1-hydroxy-2-butanone and/or 2-hydroxy-3-butanone, control of the type of pyrazines being synthesized could be realized in that the qualitative and quantitative distributions of the pyrazine array were shifted to higher molecular weight derivatives. A relatively large scale reaction (1.5 L) employing optimized parameters yielded &gt; 2 g of a diverse array of pyrazines dominated by multiple dimethylpyrazine derivatives. While systematically varying reaction conditions and reagent mole ratios can predictably alter the distribution and yield of pyrazines, the two most overwhelmingly significant factors governing these two pyrazine product characteristics included the structure of the carbon source and the presence or absence of aldehydes and free amino acids.

Isolation and Purification of Pyrazines Produced by Reaction of Cellulosic-Derived Sugars with NH4OH and Selected Amino Acids

Journal of Chromatographic Science · 2019-07-02 · 5 citations

Various pyrazines have been synthesized via reaction of selected cellulosic-derived sugars, ammonium hydroxide and amino acids at 110°C for 2 hours. Different methods of sample cleanup such as liquid-liquid extraction (LLE), liquid-solid extraction, column chromatography and distillation were employed to isolate pyrazines from the reaction mixture. Effective LLE of pyrazines from aqueous solution using either hexane, methyl-t-butyl ether (MTBE) or ethyl acetate required multiple extraction steps with fresh solvent each time. When hexane was used as the extraction solvent, no imidazole derivatives were extracted with the pyrazines. However, when MTBE or ethyl acetate was employed, 4-methyl imidazole was co-extracted and further cleanup was required. Passing the organic solvent extracts through a column of silica revealed that the silica retained the undesirable imidazoles, such as 4-methyl imidazole. A mixture of 90/10 hexane/ethyl acetate as eluting solvent provided the desirable pyrazines, but it also provided a desirable separation of pyrazines as a function of total alkyl substituent content. Distillation of the aqueous reaction mixture was also used to isolate the pyrazines, leaving the undesirable imidazoles in the undistilled portion of the reaction. Additional chromatographic methods were used to isolate pyrazines from the aqueous distillate including a column packed with C18-bonded silica.

Synthesis of Pyrazines Using Sugar Derived from Tobacco Cellulose and Hydrolyzed Tobacco F1 Protein as an Amino Acid Source

Beiträge zur Tabakforschung international · 2018-08-01 · 1 citations

Summary An array of pyrazines have been synthesized using sugars derived from tobacco cellulose (CDS), ammonium hydroxide, and hydrolyzed tobacco F1 protein as a source of free amino acids (isolated amino acids from F1 hydrolysate, from filtered F1 hydrolysate and from non-filtered F1 hydrolysate). All reactions were performed at 120 °C for 60 min using a 40-mL Parr reaction vessel. Results showed that the addition of hydrolyzed F1 protein as free amino acid source increased the number of pyrazines with branched alkyl chains (for example, 2-butyl-3-methyl pyrazine) compared to when no amino acids were added. However, using isolated amino acids from hydrolyzed F1 protein versus just hydrolyzed F1 protein (filtered or not filtered) did not make a difference in yield or type of branched pyrazines. When non-filtered hydrolyzed F1 protein was used, the solution was much more viscous and contained suspended solid material when compared to the use of filtered hydrolyzed F1 protein. Addition of threonine (THR) to the reaction mixture did not increase the yield of pyrazines but did slightly shift the distribution of pyrazines toward those with three and four carbons attached. Similar but not identical arrays of pyrazines were obtained when somewhat resembling reaction conditions were applied on a larger reaction scale (~1.5 L). A significant 50%-decrease in pyrazine yield was observed when the reaction temperature was reduced from 120 to 100 °C. No noticeable difference in the array of pyrazines from these two reactions was observed. In the majority of cases, the presence of free amino acids resulted in an increase in pyrazine yield coupled with a change in the qualitative array of pyrazines. These results clearly illustrate that sugar prepared from tobacco cellulose (glucose) can be used just like high fructose corn syrup to prepare flavor compounds via Amadori and Maillard reactions. The evidence highlights that hydrolyzed amino acids from F1 tobacco protein can be used via Maillard reactions to produce complementary arrays of pyrazine flavor compounds.