Abstract
Purpose
Development and validation of a selective analytical method to accurately and precisely quantify nicotine and cotinine levels in rat's plasma after exposure to tobacco cigarettes and tobacco water-pipe.
Methods
An easy HPLC-Photodiode-Array Detection (PDA) method was developed and validated for simultaneous determination of nicotine and cotinine levels in plasma of 15 rats (10 rats after tobacco products exposure and 5 control rats). Nicotine and cotinine were extracted in one step from plasma using acetonitrile and concentrated to lowest volume using nitrogen stream.
Results
The developed method offered a rapid analysis time of 14 min with single step of analytes extraction from rat's plasma with recovery percentage range between 93 and 95% and excellent linearity with correlation factor more than 0.994 with analytical range between 50 and 1000 ng mL−1 and LOD of 25 ng mL−1 and 23 ng mL−1 for nicotine and cotinine, respectively. The analysis of rat's plasma after 28 days of exposure to tobacco cigarettes and tobacco water-pipe revealed that the average concentrations of 376 ng mL−1 for cotinine and 223 ng mL−1 for nicotine were obtained after tobacco cigarettes exposure, and 220 ng mL−1 for cotinine and 192 ng mL−1 for nicotine after tobacco water-pipe exposure.
Conclusion
Higher nicotine and cotinine levels were found in plasma after tobacco cigarettes exposure than water-pipe exposure which may have potential undesirable effects on passive smokers in both cases.
1 Introduction
Nicotine, a pyridine alkaloid most abundant in tobacco leaves, is known as a highly addictive neurotoxin [1, 2]. The main source of nicotine is still tobacco smoking, which is listed as the main cause of cancer in many organs, especially in the respiratory system [3]. Tobacco smoking is associated with other chronic diseases including heart diseases, and hypertension [4, 5], as well as linked with high severity of COVID-19 infection [6]. In 2019, the WHO has reported that nearly third of the adults globally are habitually exposed to tobacco smoke [7]. In the same year, Jordan has been ranked among the world highest rate of smokers with 66% of males over 18 years were smoking cigarettes and/or shisha [8, 9]. Shisha, known locally as Argile or Nargile, is a water-pipe tobacco mixture mixed with fruit flavors [10]. Argile has recently grown in popularity among women and teenagers and led to a huge expansion in the number of waterpipe cafes [11]. Since argile supports nicotine dependence [12], it is necessary to determine the levels of nicotine and cotinine in plasma after argile exposure and compare it to tobacco cigarettes levels to evaluate which is more addictive and also influencer to both direct and passive smokers.
Cotinine is the major metabolite of nicotine [13, 14]. Almost 80% of inhaled nicotine, resulting from tobacco smoke exposure, is transformed into cotinine by CYP2A6 enzyme [15]. Due to its long half-life, cotinine is regularly determined in biological fluids including plasma as a biomarker of tobacco smoke exposure [16, 17].
Several methods, including immunoassays [18, 19] and spectroscopic techniques [20] have been used for the determination of nicotine and cotinine levels in physiological fluids. However, separation techniques have provided more selective approaches especially in complex matrices like plasma. Capillary electrophoresis is a powerful technique with high resolution power and selectivity particularly for metabolites determination in complex samples [21–23], and has been employed successfully for nicotine and cotinine quantification [24–26]. However, higher sensitivity has been provided by chromatographic techniques which are the most frequent methods used for quantification of both nicotine and cotinine in biological fluids. Gas chromatography has been employed widely for simultaneous determination of nicotine and cotinine in plasma [27–33]. Nevertheless, lengthy derivatization steps are usually required. Liquid chromatography (LC) is a preferable technique, as derivatization is now superfluous [34]. LC-MS [35, 36], and LC-MS/MS [37–40] have combined high sensitivity, simple extraction and rapid analysis. Yet, higher operational cost is still one of the main limitations of their use. HPLC-PDA can provide good sensitivity, rapid analysis and low running costs [41, 42], and has been employed efficiently for simultaneous determination of nicotine and cotinine in plasma [43–52].
The aim of this study was to develop and validate an easy, reliable and straightforward HPLC-PDA method for simultaneous determination of nicotine and cotinine content in rat plasma after tobacco cigarettes and water-pipe exposure.
2 Materials and methods
2.1 Chemicals
All reagents were analytical grade reagents obtained from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise stated. All standard solutions were prepared exploiting Milli-Q water (Millipore, Bedford, MA, USA) and filtered using 0.22 μm syringe filter.
2.2 Instrumentation and HPLC–PDA analytical conditions
Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector (Milford, MA, USA) was used for method development, validation and samples analysis. The analytical column used was C8- Widepore Aeris (4.6 × 250 mm, 3.6 µm) (Phenomenex, Torrance, CA, USA). The mobile phase consisted of potassium dihydrogen orthophosphate 0.272 gm and hexane sulphonic acid 0.182 gm in 1000 mL water and pH value was 3.2 adjusted with orthophosphoric acid: methanol (95%: 5%) in isocratic conditions and ambient temperature and was delivered at a flow rate of 1 mL min−1. Nicotine, cotinine and the IS (gallic acid) were identified at UV wavelengths between 210 and 400 nm and quantifications was carried out at 254 nm.
2.3 Calibration standards, quality control (QC) and samples preparations
Calibration curves (n = 3) were constructed for nicotine and cotinine measurement from seven standard solutions specifically: 50, 100, 200, 400, 600, 800, and 1000 ng mL−1. The standard solutions were prepared by serial dilution of proper amount from stock standard solutions (5 μg mL−1) with methanol (99.8%) and then were evaporated under nitrogen stream then reconstituted in 120 μl mobile phase, filtered using 0.22 μm syringe filter, and then 100 μl were injected into the HPLC column. Gallic acid was added as internal standard (IS) with conc. of 5 μg mL−1.
Following the aforementioned procedure, using concentrations of 50, 100, 200, 400, 600, 800, and 1000 ng mL−1 for both nicotine and cotinine, another calibration curves (n = 3) were constructed from 50, 100, 200, 400, 600, 800, and 1000 ng mL−1 for both nicotine and cotinine in rat plasma. Gallic acid was added as IS with conc. of 5 μg mL−1. The prepared standards were centrifuged at 15,000 rpm at 4°C, then the supernatants were taken and evaporated under nitrogen stream then reconstituted in 120 μl mobile phase, filtered using 0.22 μm syringe filter, and then 100 μl were injected. Moreover, QC's samples of nicotine and cotinine were prepared at 3 levels as QCL low (300 ng mL−1), QCM medium (700 ng mL−1), and QCH high (900 ng mL−1) in methanol and plasma.
For real sample preparations (control rats and rats exposed to tobacco cigarettes and water-pipe smokes), 100 μl of each sample was added to 500 μl acetonitrile (99.9%) and 5 μg mL−1 gallic acid, vortexed for 2 min. Then centrifuged at 15,000 rpm at 4°C then the supernatant were taken and evaporated under nitrogen stream then reconstituted in 120 μl mobile phase and then 100 μl was injected into the separation column.
2.4 Method validation
The developed analytical method was validated as follow:
2.4.1 Selectivity
Method selectivity is essential to discriminate nicotine, cotinine and gallic acid from endogenous substances and other compounds in rat plasma. The selectivity of the method was evaluated using a prepared rat plasma with no previous exposure to tobacco smoke, to ensure zero content of nicotine and cotinine, by comparing the peak signals at the target analyte retention times in blank samples with the peak signals at the target analyte retention time at limit of quantification (LOQ) samples.
2.4.2 Precision and accuracy
Inter-day and intra-day accuracy and precision were evaluated at 3 replicates of 3 QCs levels in one analytical run and three consecutive days respectively.
2.4.3 Limit of detection (LOD) and limit of quantification (LOQ)
The LOQ is the lowest reliable concentration in the calibration curve that could be quantified by the analytical method. In order to further validate the LOQ of the method experimentally, the analyte signal at the analyte retention time of a blank matrix was compared to the analyte signal at the same retention time of an LOQ sample prepared from the same matrix.
2.5 Tobacco cigarettes and water-pipe exposure to rats
2.5.1 Animals
Fifteen male Sprague-Dawley rats at the age of 10–12 weeks and weighing 180–250 g were inbred in Al-Zaytoonah University of Jordan (ZUJ). The room temperature was kept at 21°C ± 2°C and 50% ± 2% humidity with a 12-h light-dark cycle. All experiments were carried out during the light cycle. Sawdust was used as bedding and replaced regularly for hygienic purposes. The experimental and housing procedures were approved by the Institutional Animal Care and Use Committee at ZUJ, and conducted in accordance with the Helsinki guidelines for animal research [53]. Animals were randomly assigned into three groups. Fresh air or control group (n = 5), whole body cigarette exposed group (n = 5), and whole body waterpipe exposed group (n = 5). All groups had free access to water and food throughout the experiment. Control group was exposed to exposed to room air throughout the experiment. Tobacco cigarette and waterpipe groups were exposed to cigarette and waterpipe smoke, respectively for 2-h session/day for five days week−1, using whole body exposure apparatus.
2.5.2 Tobacco products used for exposure
LD blue cigarettes cruise (Liggett Ducat, 0.6 mg of nicotine, 0.8 mg of tar, and 0.01 mg of carbon monoxide), and Two-Apples flavor tobacco (Mazaya brand, Bahrain), which was purchased from local marketplace in Amman city, Jordan.
2.5.3 Cigarettes whole-body exposure (CE)
The timeline for control, cigarettes whole-body exposure, and waterpipe whole-body exposure, is illustrated in Fig. 1.
The timeline for control, cigarettes whole-body exposure, and waterpipe whole-body exposure groups used for rat exposure experiments
Citation: Acta Chromatographica 35, 1; 10.1556/1326.2022.01054
Cigarette whole-body exposure was achieved by placing the rats within an exposure chamber. The exposure chamber is made of acrylic and has dimensions of (40 × 40 × 40 cm), as shown in Fig. 2A. It includes a door in the upper part that allows the rats to be placed inside. For air circulation, the door contains three slots. Furthermore, the exposure chamber contains a cigarette inlet. The exposure chamber is connected to one pump, which has been modified and set to allow smoke from the cigarettes to enter the chamber via smoke tubes. A timer is linked to the exposure chamber to control the time for puffs (when the pump pulls cigarette smoke into the exposure chamber) and inter-puffs (during which the pump stop pulling the cigarette smoke into the exposure chamber). In this experiment, we defined a 3-s puff with a 30-s inter-puff, with a cycle that would be repeated throughout the duration of the exposure. Two cigarettes were used to saturate the exposure chamber after the rats were placed inside, and then the cigarettes were used in an orderly manner.
Scheme of chambers utilized for rat exposure to tobacco products showing the smoke inlets, pumps, and air inlets at the top. A: chamber used for rat exposure to tobacco cigarettes, B: chamber used for rat exposure to water-pipe
Citation: Acta Chromatographica 35, 1; 10.1556/1326.2022.01054
During the 2-h exposure period, twelve cigarettes were consumed. An electrochemical sensor (Monoxor II, Bacharach Inc. New Kensington, USA) was used to detect carbon monoxide (CO) levels in the exposure chamber. To manage the exposure process, the CO level was kept around 700 ppm. As a result, if the CO meter falls below 500 ppm, additional cigarettes were consumed, and if the CO meter rises beyond 899 ppm, the pump was turned off for 1–2 min to return the CO meter to the required range. In this experiment, the CO meter values varied from 500 to 800 ppm to ensure that the rats received an adequate dose of nicotine without suffocating, the average for each hour is presented in Table 1.
Carbon monoxide (CO) monitoring throughout the experiments days of exposures to tobacco cigarettes and water-pipe using
| CO level (ppm) during the tobacco cigarettes exposure days (excluding days without exposure) | CO level (ppm) during the water-pipe exposure days (excluding days without exposure) | |||
| Day no. | 1st hour | 2nd hour | 1st hour | 2nd hour |
| Day 1 | 510 | 509 | 567 | 939 |
| Day 2 | 710 | 799 | 727 | 855 |
| Day 3 | 667 | 663 | 744 | 877 |
| Day 4 | 656 | 648 | 483 | 844 |
| Day 5 | 616 | 620 | 755 | 857 |
| Day 6 | 790 | 733 | 637 | 817 |
| Day 7 | 806 | 742 | 731 | 836 |
| Day 8 | 560 | 600 | 671 | 863 |
| Day 9 | 557 | 610 | 835 | 895 |
| Day 10 | 630 | 579 | 759 | 840 |
| Day 11 | 613 | 610 | 867 | 921 |
| Day 12 | 606 | 632 | 773 | 938 |
| Day 13 | 637 | 646 | 830 | 919 |
| Day 14 | 660 | 662 | 903 | 905 |
| Day 15 | 669 | 700 | 755 | 862 |
| Day 16 | 602 | 620 | 654 | 954 |
| Day 17 | 614 | 790 | 74 | 859 |
| Day 18 | 637 | 740 | 769 | 867 |
| Day 19 | 639 | 700 | 808 | 937 |
| Day 20 | 555 | 612 | 875 | 848 |
| Average | 636.7 | 660.8 | 744.2 | 881.6 |
2.5.4 Water-pipe whole-body exposure (WPE)
Whole-body exposure to waterpipe was accomplished in special box designed in our lab, which were linked to pre-programmed pumps. In this experiment, we defined a 3-s puff with a 17-s inter-puff, with a cycle that would be repeated throughout the duration of the exposure in the exposure chamber, as shown in Fig. 2B. During smoke exposure, ten grams of “Two Apples” flavor Bahraini maassel tobacco with 0.05% nicotine content and charcoal briquettes were put on the aluminum wrapped ceramic head, also 900 mL tap water was used in the waterpipe's water jar. Carbon monoxide (CO) levels in the exposure chamber were monitored using the Monoxor II electrochemical sensor, and the level of CO was maintained at 816.28 ± 91.73 ppm by either opening the box to fresh air and shutting down the exposure for 3–4 min when the levels reached about 999 ppm, or increasing the number of charcoal pieces or adding a new one if the CO levels were low about 600 ppm, the average for each hour is presented in Table 1. Refreshing charcoal was changed after an hour of exposure.
2.5.5 Blood sampling
Retro-orbital bleeding (ROB) tube was used to obtain three milliliters of blood which were collected in a heparinized tube, as previously mentioned [54]. After blood collection, the blood was kept at room temperature for 45 min before being centrifuged to extract plasma. The HERMIL, Z 230 A centrifuge (Hermil Labor Technik, Wehingen, Germany) was used for the centrifugation. The centrifuge method was carried for 35 min at 5500 rpm to separate the plasma, which was subsequently utilized for nicotine-cotinine concentration measurements using HPLC.
2.5.6 Statistical analysis
Data were compiled as means and standard errors of the means (SEM). One-way ANOVA followed by Tukey's multiple comparisons was used to investigate nicotine-cotinine plasma levels. All statistical analyses were done using Prism-GraphPad 9.0 and were based on a P < 0.05 level of significance.
3 Results and discussion
3.1 HPLC method development and validation
The development and validation of an analytical method for quantification of nicotine and cotinine in rat plasma samples has met the acceptance criteria of FDA guidelines [55]. In which the sample processing and preparation involved only a simple and effective one extraction step and dilution procedure where no carry over was reported of analytes. Moreover, the method was selective with no interfering peaks at the retention time of nicotine, cotinine and gallic acid were observed. Figure 3A demonstrates the chromatogram of analyzed rat plasma spiked with target analytes showing gallic acid, cotinine and nicotine peaks at 5.820, 9.583 and 11.713 min, respectively. Whereas, Fig. 3B presents the chromatogram of control rat plasma without spiking with any of target analytes. Likewise, the method showed excellent linearity with correlation factor equal to 0.9947 and 0.9951 over the analytical range of 100–1000 ng mL−1 with LOD of 25 ng mL−1 and 23 ng mL−1, and LOQ of 76 ng mL−1 and 71 ng mL−1 for nicotine and cotinine, respectively.
A: Chromatogram of rat plasma spiked with the target analytes (5000 ng mL−1 Gallic acid IS tR = 5.820 min, 400 ng mL−1 Cotinine tR = 9.583 min, and 400 ng mL−1 Nicotine tR = 11.713 min). B: chromatogram of control not-spiked rat plasma. The following chromatographic condition were applied: injection volume: 100 μL; column: C8- Widepore Aeris (4.6 X 250 mm, 3.6 µm); detector: UV wavelengths between 210 and 400 nm; and quantifications carried out at 254 nm
Citation: Acta Chromatographica 35, 1; 10.1556/1326.2022.01054
The accuracy of the method was performed by comparing the calibration curves in methanol to the calibration curves spiked in the rat plasma to calculate recovery % and study the matrix effect. The mean % recovery for spiked samples for nicotine was 94.83% and for cotinine was 93.34%. Moreover, intra-day and inter-day precisions were 3.324%, 4.005% and 2.006%, 2.322% for both nicotine and cotinine, respectively. Validation parameters were calculated and summarized in Table 2. This indicates that the developed method is reliable, accurate and reproducible.
Summarized results of chromatographic method validation parameters including, retention times (tR), Calibration curve equations, limit of detection (LOD), limit of quantification (LOQ), linearity (R2), analytes concentration within specific linearity range (ng mL−1), inter-day and intra-day accuracy and precision (RSD %) for multi number of injections (n) and percent recovery for nicotine and cotinine for HPLC with PDA detector
| Parameter | Nicotine value | Cotinine value |
| Standards tR average (min) ± SD | 11.713 ± 0.21 | 9.583 ± 0.15 |
| Calibration curve equation | y = 82.82× − 2578.10 | y = 100.58× − 6551.80 |
| Determination coefficient (R2) | 0.9947 | 0.9951 |
| LOD | 25 ng mL−1 | 23 ng mL−1 |
| LOQ | 76 ng mL−1 | 71 ng mL−1 |
| Recovery | 94.83% | 93.34% |
| Intra-day accuracy | 98.61% | 98.34% |
| Intra-day precision | 3.324% | 2.006% |
| Inter-day accuracy | 97.07% | 97.38% |
| Inter-day precision | 4.005% | 2.322% |
3.2 Nicotine and cotinine concentrations in rat's plasma
This study focuses on development and validation of a simple and straightforward HPLC method for selective determination of nicotine and cotinine content in plasma of experimental rats. Table 3 presents the description and labeling of rats, exposure type, duration and date of exposure, and cotinine and nicotine plasma levels after exposure to tobacco cigarettes and water-pipe. Several studies have been conducted for simultaneous determination and quantification of nicotine and cotinine using HPLC-PDA. The developed methods validation findings are summarized in Table 4. The general protocol for previous methods included several steps for extraction of nicotine and cotinine from plasma samples. For example, Shaik et al. [44] and Massadeh et al. [47] followed the following the protocol by which an aliquot of plasma was firstly alkalinized with NaOH, and then vortexed and centrifuged before extraction with dichloromethane-diethyl ether and then followed by vortex and centrifuging. After that, the organic layer was mixed with HCl and then evaporated under a stream of nitrogen until dryness and later reconstituted in mobile phase before injection into HPLC for analysis [44, 47]. Moreover, Nakajima et al. used the same protocol steps with deferent solvents [50]. Other methods proposed additional solid-phase extraction steps which increased the costs as well as time of analysis [45, 46]. In this study, the developed involved minimal extraction steps, only vortexing and centrifuging of plasma samples followed by evaporating and reconstituting of the supernatant with mobile phase before injection into C8 column in the HPLC system are required to obtain accurate and selective quantification, which provide a cost-effective and straightforward way. Moreover, the cost of all solvents and chemicals were selected to cut the cost of the method to obtain highly selective simultaneous analysis of nicotine and cotinine in plasma samples. The developed method have shown comparable validation results in terms of LODs, analysis time, recovery, and column type with other methods. The simplicity and speed of the developed method can be considered as advantages for its use for routine analysis of nicotine and cotinine in plasma samples.
The description of rats, exposure type, duration and date, and cotinine and nicotine plasma levels (all rats were obtained from ZUJ)
| Rat label | Starting date of exposure | Duration of exposure | Type of exposure | Cotinine (ng mL−1) ± SD | Nicotine (ng mL−1) ± SD |
| Rat 1T | 10 Sept, 2021 | 28 Days | Tobacco Cigarettes | 363.87 ± 3.17 | 224.31 ± 14.23 |
| Rat 2T | 10 Sept, 2021 | 28 Days | Tobacco Cigarettes | 267.34 ± 1.27 | 222.19 ± 12.81 |
| Rat 3T | 10 Sept, 2021 | 28 Days | Tobacco Cigarettes | 409.04 ± 27.72 | 219.15 ± 8.94 |
| Rat 4T | 10 Sept, 2021 | 28 Days | Tobacco Cigarettes | 460.61 ± 35.58 | 227.42 ± 8.33 |
| Rat 5T | 10 Sept, 2021 | 28 Days | Tobacco Cigarettes | 379.72 ± 7.41 | 221.84 ± 13.23 |
| Rat 1W | 10 Sept, 2021 | 28 Days | Water pipe | 217.72 ± 6.40 | 189.29 ± 22.28 |
| Rat 2W | 10 Sept, 2021 | 28 Days | Water pipe | 248.28 ± 9.33 | 202.03 ± 10.61 |
| Rat 3W | 10 Sept, 2021 | 28 Days | Water pipe | 203.23 ± 13.19 | 193.21 ± 9.02 |
| Rat 4W | 10 Sept, 2021 | 28 Days | Water pipe | 221.69 ± 20.05 | 178.39 ± 17.11 |
| Rat 5W | 10 Sept, 2021 | 28 Days | Water pipe | 209.97 ± 16.10 | 199.02 ± 11.74 |
| Rat 1C | 10 Sept, 2021 | 28 Days | Control (no exposure) | Below LOQ | Below LOQ |
| Rat 2C | 10 Sept, 2021 | 28 Days | Control (no exposure) | Below LOQ | Below LOQ |
| Rat 3C | 10 Sept, 2021 | 28 Days | Control (no exposure) | Below LOQ | Below LOQ |
| Rat 4C | 10 Sept, 2021 | 28 Days | Control (no exposure) | Below LOQ | Below LOQ |
| Rat 5C | 10 Sept, 2021 | 28 Days | Control (no exposure) | Below LOQ | Below LOQ |
Summary of methods validation findings in the literature for nicotine and cotinine quantification in plasma after exposure to tobacco products using HPLC-PDA
| Literature | Column type | Matrix | LOD of nicotine and cotinine (ng mL−1) | Analysis time (min) | % Recovery nicotine and cotinine | Country |
| Baj et al. [43] | C18 | Plasma | 1.5 and 1.59 | 12 | 102.5 and 100.2 | Poland |
| Shaik et al. [44] | C18 | Plasma | NA | 10 | NA | India |
| Papadoyannis et al. [45] | C8 | Plasma | 10 and 10 | 15 | 94.2 and 93.3 | Greece |
| Dawson et al. [46] | C18 | Plasma | 8 and 13.6 | 20 | 51 and 64 | USA |
| Massadeh et al. [47] | C18 | Plasma | 0.32 and 0.26 | 16 | 96 and 95.8 | Jordan |
| Abu-Qare et al. [48] | C18 | Plasma | 20 and 150 | 17 | 84.7 and 80.1 | USA |
| Abu-Qare et al. [49] | C18 | Plasma | 20 and 30 | 11 | 85.8 and 81 | USA |
| Nakajima et al. [50] | C18 | Plasma | 0.2 and 1 | 20 | 103.3 and 82.1 | Japan |
| Sioufi et al. [51] | C18 | Plasma | 1 and 20 | 18 | 90 and 92 | France |
| Hariharan et al. [52] | C18 | Plasma | 1 and 3 | 10 | 94 and 96 | USA |
| This study | C8 | Plasma | 25 and 23 | 12 | 94.8 and 93.3 | Jordan |
3.3 Effect of whole-body cigarettes exposure and whole-body waterpipe exposure on cotinine and nicotine plasma concentration
Whole-body cigarettes exposure and whole-body waterpipe exposure for 4 weeks have a great effect on cotinine and nicotine plasma concentration compared to control group, as shown in Fig. 4A and 4B, respectively. High cotinine and nicotine plasma concentration was observed in the CE and the WPE groups but not Control group, which were exposed to fresh air room only. Furthermore, cotinine and nicotine plasma concentration in CE group was higher than cotinine and nicotine concentration in WPE group. This pattern of effect was confirmed by One-way ANOVA revealing a significant main effect of Treatment in cotinine plasma concentration [F (2, 12) =100.1, P < 0.0001; Fig. 4A] and a significant main effect of Treatment in nicotine plasma concentration [F (2, 12) = 2299, P < 0.0001; Fig. 4B]. Turkey's multiple comparison showed that there were significant increase in cotinine and nicotine plasma concentration in the CE and WPE groups compared to Control group, also there were a significant increase in cotinine and nicotine plasma concentration in CE group compared to WPE group (***P < 0.001, ****P < 0.0001).
Cotinine and nicotine plasma concentration in male rats (mean ± SEM) after exposure for 4 weeks. A: One-Way ANOVA showed an increase in cotinine plasma concentration in CE, and WPE groups compared to Control group, also there was an increase in cotinine plasma concentration in CE group compared to WPE group (***P < 0.001, ****P < 0.0001), (n = 5 for each group). B: Nicotine plasma concentration in male rats (mean ± SEM) after exposure for 4 weeks. One-Way ANOVA showed an increase in nicotine plasma concentration in CE, and WPE groups compared to Control group, also there was an increase in nicotine plasma concentration in CE group compared to WPE group (***P < 0.001, ****P < 0.0001), (n = 5 for each group)
Citation: Acta Chromatographica 35, 1; 10.1556/1326.2022.01054
Conclusion
The developed HPLC-PDA method was successfully applied accurately and precisely for ridable simultaneous determination of nicotine and cotinine content in plasma samples after exposure to different tobacco products. The validated method was simple, cost effective, and straightforward for determination of nicotine and cotinine in less than 12 min lower nicotine and cotinine concentrations in rat's plasma exist after exposure to water-pipe and tobacco cigarettes. However, the data obtained shows that exposure to both tobacco products is harmful to both direct and passive smokers.
Competing interests
The authors declare no competing interests.
Acknowledgments
This work was supported by the Deanship of Scientific Research at Al-Zaytoonah University of Jordan (2019–2020/23/6).
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