From cis-Lobeline to trans-Lobeline: Study on the Pharmacodynamics and Isomerization Factors

Abstract

Lobeline is an alkaloid derived from the leaves of an Indian tobacco plant (Lobelia inflata), which has been prepared by chemical synthesis. It is classified as a partial nicotinic agonist and has a long history of therapeutic usage ranging from emetic and respiratory stimulant to tobacco smoking cessation agent. The presence of both cis and trans isomers in lobeline is well known, and many studies on the relationship between the structure and pharmacological activity of lobeline and its analogs have been reported. However, it is a remarkable fact that no studies have reported the differences in pharmacological activities between the two isomers. In this article, we found that different degrees of isomerization of lobeline injection have significant differences in respiratory excitatory effects in pentobarbital sodium anesthetized rats. Compared with cis-lobeline injections, the respiratory excitatory effect was significantly reduced by 50.2% after administration of injections which contained 36.9% trans-lobeline. The study on the influencing factors of isomerization between two isomers shown that this isomerization was a one-way isomerism and only converted from cis to trans, where temperature was the catalytic factor and pH was the key factor. This study reports a new discovery. Despite the widespread use of ventilators, first-aid medicines such as nikethamide and lobeline has retired to second line, but as a nonselective antagonist with high affinity for a4b2 and a3b2 nicotinic acetylcholine receptors (nAChRs). In recent years, lobeline has shown great promise as a therapeutic drug for mental addiction and nervous system disorders, such as depression, Alzheimer disease and Parkinson disease. Therefore, we suggest that the differences between two isomers should be concerned in subsequent research papers and applications.

1. Introduction

Isomerism is widespread in organic substances; the discovery of isomerism and its theoretical clarification play an important role in the development of material composition and structural theory [1]. It opens up the study of structural chemistry and promoted the development of organic chemistry. Cis-trans isomerism, also known as geometric isomerism, which refers to the diastereoisomerism of the compounds in the molecule due to the restriction of free rotation, is a stereoisomerism in isomerism [2,3]. Generally, trans isomers are more stable than cis isomers. Isomerization of the cis-trans isomer [4] is a dynamic equilibrium process which is usually divided into three types: photoisomerization [5], thermoisomerization [6], and catalytic isomerization [7], but is not limited thereto. The differences in physical, chemical, and biological activities due to isomerization are hotspots in organic chemistry research and an important source of new drug discovery.

Lobeline, an alkaloid found in the stems of Lobelia inflata, a plant indigenous to North America [8], is white or microstrip yellow crystal or granular powder, odorless, bitter taste and discoloration in case of light and heat. The molecular formula is C₂₂H₂₇NO₂, it contains both cis and trans isomers [9]. The chemical formula and structural formula are shown in Figure 1. The computed properties of cis-lobeline, such as XLogP3-AA, hydrogen bond donor count, hydrogen bond accept count, topological polar surface area, were 3.8, 1, 3, and 40.5 Ų, respectively. Detailed information could be found in the PubChem database [10].

Evidence suggests that lobeline was a nonselective antagonist with a high affinity for a4b2 and a3b2 nicotinic acetylcholine receptors (nAChRs) [11]. It was used as an emetic and respiratory stimulant in clinical practice since the 19th century and is now prepared by chemical synthesis. As a respiratory stimulant, it is characterized by selective stimulation of the carotid body and aortic chemoreceptors (N-receptors), reflexively exciting the respiratory center, the vagus nerve center, and the vascular movement center, leading to the breath becoming deeper and faster. However, this stimulus lasts only a few minutes. According to the reports in the literature, after intravenous injection, the duration of action usually maintained at 20 min, and the LD₅₀ of intravenous injection in mice is 39.9 mg/kg [12]. It was used for neonatal asphyxia and carbon monoxide asphyxiation, anesthetic poisoning such as ether and chloroform, central inhibitors (such as opioids, barbiturates) poisoning, and respiratory failure caused by various diseases such as pneumonia [13] and diphtheria. However, it is not advisable for the respiratory arrest and respiratory weakness caused by progressive respiratory failure.

In recent years, extensive research has shown that lobeline has a multi-directional mechanism of action, including acetylcholine N receptors, opioid receptors, and monoamine transporters. In particular, it has shown great promise as a therapeutic drug for mental addiction and nervous system disorders, such as Alzheimer’s syndrome [14], Parkinson’s syndrome, and depression [15,16]. As a partial nicotine agonist, it has been used in a variety of commercially available preparations to help stop smoking [17].

Despite extensive studies, a comprehensive analysis of the pharmacological activities and the influencing factors of the isomerization of trans-lobeline is missing. The presence of cis and trans isomers in lobeline is well known, and many studies on the relationship between the structure and pharmacological activity of lobeline and its analogs have been reported [18,19,20]. However, the differences in pharmacological activities and the influencing factors of isomerization between the two isomers have not been reported. Due to our limited understanding of trans-lobeline, we may underestimate the potential safety risk caused by the trans isomer or miss an opportunity to discover a new medicine.

In pharmaceutical research, the potential safety risks and lack of efficacy caused by the simultaneous presence of two or more isomers in a drug have been a challenge for drug quality control. Here, based on the fact that lobeline was used as respiratory stimulants, we investigated the differences in the ability of cis and trans isomers to excite breathing. The results shown that when the degree of isomerization was 38.7% (the percentage of trans-lobeline was 36.9%), the ability to excite breathing decreased by 50.2% compared with cis-lobeline. Furthermore, when the degree of isomerization was 67.7% (the percentage of trans-lobeline was 22.5%), the ability to excite breathing decreased by 63.0% compared with cis-lobeline. It was suggested that the cis and trans isomers have significant differences in the ability to excite breathing. In addition, considering the necessity for quality control and providing reliable data for pharmacological activity studies, we optimized and established two new HPLC-based methods, which can determine both isomers in a single run for the study of influencing factors of isomerization and the quality evaluation of samples containing lobeline. The results of the isomerization influencing factors experiments showed that pH is the key factor of isomerization, and temperature is the acceleration factor of isomerization. In the solutions containing lobeline, pH should not exceed 2.6 and the temperature in its lifespan should not exceed 40 °C.

2. Results

2.1. Determination of trans-Lobeline Content

Please see Supplementary Materials File S1. LC-MS/MS for the trans-lobeline structure analysis.

2.1.1. Optimized HPLC Conditions Were Feasible

The optimized HPLC conditions enables efficient separation of cis and trans isomers in processed lobeline hydrochloride solutions (Figure 2). Plate ≥ 8000, both of selectivity, specificity, precision, and accuracy, met the requirements; the extraction and recovery were between 95–105% and the calibrations were linear over a certain range from 30 to 240 ng/mL in all samples with a correlation coefficient (R²) larger than 0.9999.

2.1.2. Determination of trans-Lobeline Percentage Content

It is no possible to quantify the exact content of trans-lobeline due to a lack of reference standards. However, based on the area normalization method, it is also acceptable to use the percentage of the peak area to represent the content of each component in the sample. The percentage content of trans-lobeline in the three samples described previously are shown in Table 1.

As shown in Table 1, freshly prepared lobeline solution was trans-lobeline free; after treated at 105 °C for 15 min, the percentage content of trans-lobeline raised to 38.7%, and this value became to 67.7% when it was treated at 121 °C for 120 min.

2.2. Effects of Two Isomers on Stimulation of Respiratory Are Significantly Different

The promotion of respiratory rate of anesthetized mice was used to evaluate whether the ability of three samples to excite respiration is different. As shown in Figure 3 and Table 2, the samples processed by 105 °C for 15 min and 121 °C for 120 min have a significantly different ability to stimulation of respiratory compared with the freshly prepared solutions. The freshly prepared solutions increased the respiratory rate by 22.7 ± 4.44, while the samples which processed by 105 °C for 15 min and 121 °C for 120 min only increased by 11.3 ± 2.21 and 8.4 ± 1.07, respectively. The ability of respiratory stimulation decreases as the percentage content of trans-lobeline increases.

2.3. Method Validation in the Experiment on Influencing Factors of Isomerization

2.3.1. Selectivity and Specificity

As shown in Figure 4, after treatment by all influencing factors involved in this study, there is no interference between the major target matrices (cis- and trans-lobeline isomers) and newly generated impurities and adjacent peaks reached baseline separation, while the resolution was greater than 1.5. As a part of the method specificity evaluation, peak purity in each sample met the requirements.

2.3.2. Linearity and LLOQ

The standard curve, correlation coefficient of cis-lobeline in the selected range and LLOQ were shown in Table 3. The calibrations were linear over a certain range in all samples with a coefficient (R²) larger than 0.999.

2.3.3. Precision and Accuracy

Obtained accuracy and precision data were summarized in Table 4. The accuracies ranged from 96.25% to 99.67%, along with precision ranging from 0.27% to 0.63%. These data suggested that the method was accurate and reproducible for the quantification of analytes in the samples.

2.3.4. Extraction Recovery

As shown in Table 5, the extraction rate for cis-lobeline was ideal, which ranged from 98.38% to 100.51%.

2.3.5. Stability

As mentioned in the previous paragraph, lobeline was unstable to heat, light, acid, and base, and its stability was conditional. Among the influencing factors of the isomerization experiments, low temperature (2–8 °C) and pH (do not exceed 3.0) were proven to have no effect on the stability of cis-lobeline in 60 days.

2.4. Influencing Factors of Isomerization

2.4.1. Temperature Is the Catalytic Factor of Isomerization

As shown in Figure 5 and Table 6, cis-lobeline solution, which placed at 2–8 °C for 60 days, did not generated any impurities and the content was stable. Placed at 40 °C for 60 days, impurities were significantly increased due to the conversion of cis-lobeline to trans-lobeline. However, when cis-lobeline solution was treated at 121 °C for 120 min, most of the cis-lobeline was destroyed, part of it was converted to trans-lobeline, and the other part became unknown impurities.

2.4.2. pH Is the Key Factor of Isomerization

As shown in Figure 6 and Figure 7 and Table 7, the stability of cis-lobeline decreased with increased pH. The higher the pH, the easier it was converted to trans-lobeline. When the pH was 2.6, it was an inflection point of isomerization.

According to the test of influencing factors of isomerization, we can conclude that pH is the key factor of isomerization, as it determines the final content of trans-lobeline. The cis-lobeline solution formulated with low pH solvent can withstand high temperature-induced catalysis of isomerization. Temperature is the catalytic factor of isomerization; high temperature can either catalyze the isomerization process or it can destroy cis-lobeline directly.

3. Materials and Methods

3.1. Drugs and Reagents

Cis-lobeline chemical reference substance (CRS): Sigma-Aldrich (LOT: 14109JBV). Pentobarbital was purchased from Sigma-Aldrich (CAS: 57-33-0). Water was prepared by double distillation. HPLC-grade acetonitrile was supplied by Fisher Scientific Company Inc. (Boston, MA, USA). All other reagents were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China).

3.2. Animals, Animal Management, and Experimental Design

Pharmacodynamic study of trans-lobeline isomers was conducted in the apparently healthy adult Wistar rats (180–200 g, male). Animals were purchased from the Beijing Keyu Animal Breeding Center (Production license: SCXK (Jing)-2010-0010). All animals were housed in an environmentally controlled breeding room (temperature: 22 ± 2 °C, humidity: 50 ± 5%, dark/light cycle: 12/12 h). The animals were provided standard laboratory food and water. The experimental protocols were performed in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals. Prior to each experiment, all animals were kept under laboratory conditions for a period of 3 days or more for acclimatization.

3.3. Determination of cis-Lobeline and trans-Lobeline Content

Due to the lack of lobeline (both cis and trans isomer) reference standards, we cannot accurately determine the absolute content of these two isomers. However, based on the area normalization method, it is also an acceptable way to use the percentage of the peak area to represent the content of each component in the samples, and cis-lobeline CRS allows us to measure the content of cis-lobeline in the sample relatively accurately. Therefore, a HPLC method was optimized in order to relative quantify the content of cis-lobeline and trans-lobeline in the samples to be tested in the pharmacodynamic study of trans-lobeline isomers.

Chromatographic analysis was performed on an Agilent 1100 Series (Agilent Technologies, St. Clara, CA, USA) LC system containing a quaternary pump, an online degasser, an autosampler, and a thermostatic column compartment set at 30 °C. Chromatographic separation was conducted on a Waters SymmetryShield RP18 column (5 μm, 250 × 4.6 mm) (Milford, MA, USA). Elution was carried out by isocratic elution. The mobile phase consisted of a mixture of 50 mM phosphate buffer (pH 3.0) (A) and acetonitrile (B), where the ratio of A was 80%. The flow rate was 1.0 mL/min. UV detection wavelength was 246 nm.

Our method was different from the isocratic elution method used in the pharmacodynamic study. For more sensitive detection of the impurities in the samples to be tested in the isomerization influencing factors experiment, a new gradient elution method was established. This new method has higher detection rate and lower limits of quantification (LLOQ). Chromatographic analysis was performed on the same Agilent 1100 Series LC system. The mobile phase consisted of a mixture of 50 mM phosphate buffer (pH 3.0) (A) and acetonitrile (B). Gradient elution was performed as follows: (1) mobile phase A was at 85% at 0 min, (2) an isocratic elution was maintained at 85% A from 0 min to 10 min, (3) a linear gradient was decreased to 82% A from 10 min to 30 min, and (4) a linear gradient was decreased to 72% A from 30 min to 70 min. The flow rate was 1.0 mL/min. UV detection wavelength was 210 nm.

3.4. Method Validation

The method used to determine content in pharmacodynamic studies was adopted from reference [21]. In the article, the mobile phase consisted of a mixture of 50 mM phosphate buffer (pH 3.0) (A) and methanol (B), where the ratio of A was 55%. UV detection wavelength was 210 nm. However, in this manuscript, since methanol has terminal absorption at 210 nm, which will affect the accuracy of content determination, acetonitrile was used to replace methanol. Moreover, the proportion of acetonitrile was adjusted to 20% in order to make cis-lobeline and trans-lobeline better separated. The adjustment of the method was slight and there was no method inapplicability in the subsequent content determination. We did not perform method validation as we just optimized the pH and ratio of mobile phase. However, we performed method validation in the experiment on influencing factors of isomerization because of a big change in approach. These parameters include selectivity and specificity, linearity and LLOQ, precision and accuracy, extraction recovery and stability.

3.5. Pharmacodynamic Study of trans-Lobeline Isomer

The experiments were performed on Wistar rats, anesthetized with sodium pentobarbital solution (60 mg/kg) via the tail vein. The anesthetized rats were fixed on multi-channel electrophysiological apparatus (RM6240E, Chengdu Instrument Factory, Chengdu, China), and their respiratory rate and heart rate were observed and recorded. According to the changes in the respiration rate, pentobarbital sodium (0.1 mL) was injected every 1–2 min until the respiration rate was reduced to 50 bpm and maintained for 3–5 min. Respiration rate and heart rate were collected every 5 min. In 45 min, all rats were injected with injectable freshly prepared lobeline hydrochloride solution, lobeline hydrochloride solution sterilized for 15 min at 105 °C, and lobeline hydrochloride solution sterilized for 120 min at 121 °C at a dosage of 2 mg/kg. Heart rate and respiratory rate were recorded at an interval of 5 min. Mean respiratory rate five minutes before and after administration and elevated respiratory rate after administration were calculated for each rat.

3.6. Study on the Influencing Factors of Isomerization

As lobeline is unstable to heat, light, acid, and base, studies on the isomerization influencing factors were focused on temperature and Pondus Hydrogenii (pH).

3.6.1. Investigation of the Relationship between Temperature and Isomerization

We mainly investigated sterilization temperature and storage temperature. cis-Lobeline CRS (trans-lobeline free) was dissolved in water and pH was adjusted to 3.0 by using 0.1 mol/L hydrochloric acid. Then, we treated these samples and collected data according to the study overview, which is shown in Figure 8. The prepared samples were diluted with mobile phase into a solution containing 120 μg/mL of cis-lobeline for HPLC analysis.

3.6.2. Investigation of the Relationship between pH and Isomerization

Hydrochloric acid solutions of different pH were prepared, and the pH was determined to be 1.97, 2.04, 2.18, 2.32, 2.64, 2.87, 2.97, 3.10, 3.24, 3.43, 3.63, 3.90, and 4.27. A total of 3 mg/mL cis-lobeline hydrochloride solution was prepared by using the different pH solutions as solvent, and sealed by melting the glass ampoules. To improve experiment efficiency, 105 °C and 15 min autoclaving was used to accelerate the isomerization process. The prepared samples were diluted with mobile phase into a solution containing 120 μg/mL of lobeline for HPLC analysis.

3.7. Statistical Analysis

SAS version 9.2 (SAS Inc., Cary, NC, USA) was used to perform the statistical analyses. All data were expressed as the mean ± SD. For comparisons between two groups, Student’s t-test was performed. For comparisons among three or more groups, the data were analyzed using a one-way analysis of variance (ANOVA). For all analyses, a p-value of < 0.05 was considered to indicate statistical significance, and a p-value of < 0.01 was considered to indicate extreme statistical significance. GraphPad prism (version 8.0, GraphPad Software, San Diego, CA, USA) was used to draw the statistical figures.

4. Discussion

As described in the results, the isomerization of lobeline is affected by temperature and pH. The higher the temperature, the higher the degree of isomerization, and the lower the pH, the lower degree of isomerization. Among the existing literatures that can be searched, there is no similar article describing the factors of lobeline isomerization. However, as described in the introduction section, thermoisomerization is one of the types of transformation of cis-trans isomers. Isomerization due to temperature changes is a very common phenomenon [22,23]. As shown in Figure 3, after being treated by different temperatures, there are significant differences in the excitatory effects of lobeline solutions with different degrees of isomerization on respiration. The data show that when the degree of isomerization is 38.7%, the respiratory stimulation effect is only half of that of fresh solution, and when the degree of isomerization is 67.7%, the respiratory stimulation effect is only 37% of that of fresh solution.

However, 105 °C and 121 °C are the sterilization processes that were often used in lobeline hydrochloride injection production. We further detected the content of cis- and trans-isomers in lobeline hydrochloride injections which are currently commercially available; the results also confirmed that both isomers were detected simultaneously. However, the samples from different companies have slightly different degrees of isomerization. The above results suggest that lobeline hydrochloride injection has defects in the efficacy of excitatory breathing. We also hope to provide a warning for the use of lobeline through this manuscript, and hope that users know and will manage this risk.

Another type of transformation of cis-trans isomers is catalytic isomerization. Noble metal catalysts supported by a solid acid, such as platinum-alumina, platinum-molecular sieve, palladium-alumina, etc., are common catalysts for catalytic isomerization [24]. Such catalysts consist of bifunctional catalysts in which the metal component acts as a hydrogenation and dehydrogenation and the solid acid acts as an isomerization. When such catalysts were used, the reaction needs to be carried out in the presence of hydrogen, so it is also called a hydroisomerization catalyst. However, these catalysts were often used for gas phase isomerization.

There are also differences in the pharmacological activities of the two isomers for respiratory excitatory effects. However, unfortunately, there is currently no pure trans-lobeline, so it is impossible to directly compare the differences in the pharmacological activities of the two isomers. An indirect comparison can only be made through the trans-lobeline produced by isomerization. For lobeline hydrochloride injection, the difference in isomer activity is a side effect. However, for those two isomers alone, this is an opportunity. Analogs [11] such as meso-Transdiene (MTD), lobelane, nor-lobelane, carboxylic acid, and sulfonic acid ester are being investigated currently as a clinical candidate for the treatment of psychostimulant abuse [25]. These analogs might lack nicotinic receptor affinity [24], retain affinity for vesicular monoamine transporter 2 (VMAT2) [20], or enhance affinity for dopamine (DA) and serotonin transporters. In summary, for chemical receptors that interact with lobeline, the specificity of activity between different analogs were inconsistent, which provides a reference for the new clinical application study of trans-lobeline.

How to carry out future research is something we have been considering. Here, we believe that the following issues should be focused on. First, the proper mechanism by which pH induced isomerization should be revealed. Secondly, the study of isomer activity should not be limited to nAChRs receptors, nor should it be restricted to the excitatory respiration effect [26]. Last but not the least, the study of the structure–activity relationship of trans-lobeline should be considered in subsequent studies.

Author Contributions

Conceptualization, J.-F.Y. and H.-H.X.; methodology, H.-H.X. and Z.-H.J.; validation, H.-H.X., L.Y., M.-X.T., A.-P.Y. and B.-D.T.; formal analysis, H.-H.X. and Z.-H.J.; writing—original draft preparation, H.-H.X.; writing—review and editing, H.-H.X., L.Y. and Z.-H.J.; supervision, J.-F.Y.; funding acquisition, J.-F.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Ethics Committee Jiangxi University of Chinese Medicine, (protocol code 20220228003 and date of approval 28 February 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated and analyzed in the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Not applicable.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. The chemical structure of cis-lobeline ((−)-lobeline) and trans-lobeline ((+)-lobeline). (A): 2-[(2R,6S)-6-[(2S)-2-Hydroxy-2-phenylethyl]-1-methylpiperidin-2-yl]-1-phenylethanone ((−)-lobeline); (B): 2-[(2S,6R)-6-[(2R)-2-hydroxy-2-phenylethyl]-1-methylpiperidin-2-yl]-1-phenylethanone ((+)-lobeline); Molecular formula: C22H27NO2, MW:337.

Figure 2. Representative chromatograms to evaluate the separation effect of isocratic elution methods. 1 = cis-lobeline, 2 = trans-lobeline, 3 = Intermediate.

Figure 3. Effects of lobeline injection with different isomerization levels on excited respiration in rats with respiratory depression. (A): treated at 121 °C for 120 min, (B): treated at 105 °C for 15 min, (C): fresh, (D): respiration rate. “***” represents compared with the Fresh group, p ≤ 0.001, “##” represents compared with the 105 °C 15 min group, p ≤ 0.01.

Figure 4. Representative chromatograms to evaluate the separation effect of gradient elution methods. 1 = cis-lobeline, 2 = trans-lobeline, 3 = Intermediate, 4 and 5 = unknown impurities.

Figure 5. Formation of impurities in the cis-lobeline reference solution after it was treated by different temperature. 1 = cis-lobeline, 2 = trans-lobeline, 3 = Intermediate, 4 and 5 = unknown impurities.

Figure 6. Formation of impurities in the cis-lobeline reference after it was processed by different pH solutions and autoclaving at 105 °C for 15 min. 1 = cis-lobeline, 2 = trans-lobeline, 3 = Intermediate, 4 and 5 = unknown impurities.

Figure 7. Relationship between pH and degree of isomerization (n = 3).

Figure 8. The experiment program of sample preparation in the temperature and isomerization test.

Table 1

The percentage content of trans-lobeline in the samples used to pharmacodynamic study (n = 3).

Sample Name	cis-Lobeline (%)	trans-Lobeline (%)	Degree of Isomerization (%)
Fresh	100%	0	0
105 °C 15 min	61.3 ± 0.45%	36.9 ± 0.68%	38.7%
121 °C 120 min	32.3 ± 0.47%	22.5 ± 1.05%	67.7%

Note: Degree of isomerization = 1 − Average of cis-lobeline (%).

Table 2

Relationship between increased respiratory rate and degree of isomerization (n = 10).

Sample Name	Degree of Isomerization (%)	Increased Respiratory Rate (bpm)
Fresh	0	22.7 ± 4.14
105 °C 15 min	38.7%	11.3 ± 2.21 ***
121 °C 120 min	67.7%	8.4 ± 1.07 ***, ##

Note: “***” means compared with the Fresh group, p ≤ 0.001, “^##” means compared with the 105 °C 15 min group, p ≤ 0.01.

Table 3

Standard curves, correlation coefficients, linear range, and LLOQ of cis-lobeline.

Name	Standard Curves	R 2	Linear Range (μg/mL)	LLOQ (μg/mL)
cis-lobeline	y = 21.426x − 8.0696	0.9998	30–240	0.36

Table 4

Intra-/interday accuracy and precision of the developed assay (n= 6).

Name	QC Conc.(μg/mL)	Intraday			Interday
		Calc Conc. (μg/mL)	RSD(%)	Accuracy(%)	Calc Conc.(μg/mL)	RSD (%)	Accuracy(%)
cis-lobeline	60.52	60.08 ± 2.13	0.27	99.67 ± 0.28	61.03 ± 1.96	0.38	99.15 ± 1.06
	121.48	120.61 ± 3.27	0.50	98.79 ± 1.02	120.57 ± 2.69	0.50	98.76 ± 0.94
	242.55	245.37 ± 4.95	0.69	97.34 ± 2.06	240 ± 3.54	0.63	96.25 ± 3.21

Table 5

Recovery of the developed assay (n = 9).

Group	Sample Intake (mg)	Reference Material Added (mg)	Content Determination (mg)	Recovery (%)	Average (%)	RSD (%)
80%	2.122	1.960	4.016	98.37	98.38	0.51
	2.122	1.960	3.997	97.89
	2.122	1.960	4.037	98.89
100%	2.653	2.450	5.119	100.31	100.51	0.61
	2.653	2.450	5.165	101.20
	2.653	2.450	5.105	100.03
120%	3.184	2.941	6.138	100.23	100.27	0.42
	3.184	2.941	6.117	99.88
	3.184	2.941	6.168	100.71

Table 6

Relationship between temperature and degree of isomerization (n = 3).

Sample Name	cis-Lobeline (%)	trans-Lobeline (%)	Degree of Isomerization (%)
Fresh	100	0	0
4 °C 60 days	99.2 ± 0.39	0.6 ± 0.08	0.8
40 °C 60 days	93.2 ± 0.63	6.7 ± 0.49	6.8
105 °C 15 min	61.3 ± 0.45	36.9 ± 0.68	38.7
121 °C 120 min	32.3 ± 0.47	22.5 ± 1.05	67.7

Note: Degree of isomerization = 1 − Average of cis-lobeline (%).

Table 7

Relationship between pH and degree of isomerization (n = 3).

pH	Hydrogen Ion Concentration (μmol/L)	Degree of Isomerization (%)
1.97	10,715.2	3.084
2.04	9120.1	4.04
2.18	6606.9	4.629
2.32	4786.3	6.316
2.64	2290.9	10.237
2.87	1349.0	12.132
2.97	1071.5	13.537
3.1	794.3	16.236
3.24	575.4	21.66
3.43	371.5	26.61
3.63	234.4	27.134
3.9	125.9	34.796
4.27	53.7	45.824

Note: Degree of isomerization = 1 − Average of cis-lobeline (%).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27196253/s1, File S1. LC-MS/MS for the trans-lobeline structure analysis: Figure S1. LC-MS/MS chromatogram of lobeline; Figure S2. Secondary mass spectrum of trans-lobeline; Figure S3. Secondary mass spectrum of cis-lobeline; Table S1. Fragment of cis-lobeline; Table S2. Fragment of trans-lobeline.

References

1. Malcolmson, S.J. Racing against unwanted isomerization. Science; 2021; 371, pp. 345-346. [DOI: https://dx.doi.org/10.1126/science.abf8851] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33479139]

2. Ringle, M.L.; Loomba, R.; Dykes, J.C.; Khan, D.; Schidlow, D.; Wernovsky, G. The multisystem nature of isomerism: Left isomerism complicated by Abernethy malformation and portopulmonary hypertension. Cardiol. Young; 2021; 31, pp. 532-540. [DOI: https://dx.doi.org/10.1017/S1047951121000809] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33731242]

3. Sengupta, A.; Rognoni, L.E.; Merkel, U.; Žoldák, G.; Rief, M. SlyD Accelerates trans-to-cis Prolyl Isomerization in a Mechanosignaling Protein under Load. J. Phys. Chem. B; 2021; 125, pp. 8712-8721. [DOI: https://dx.doi.org/10.1021/acs.jpcb.1c03648] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34279937]

4. Ganguly, S.; Murugan, N.A.; Ghosh, D.; Narayanaswamy, N.; Govindaraju, T.; Basu, G. DNA Minor Groove-Induced cis-trans Isomerization of a Near-Infrared Fluorescent Probe. Biochemistry; 2021; 60, pp. 2084-2097. [DOI: https://dx.doi.org/10.1021/acs.biochem.1c00281]

5. Gai, F.; Hasson, K.C.; McDonald, J.C.; Anfinrud, P.A. Chemical dynamics in proteins: The photoisomerization of retinal in bacteriorhodopsin. Science; 1998; 279, pp. 1886-1891. [DOI: https://dx.doi.org/10.1126/science.279.5358.1886]

6. Tian, X.Q.; Chen, T.; Matsuoka, L.; Wortsman, J.; Holick, M. Kinetic and thermodynamic studies of the conversion of previtamin D3 to vitamin D3 in human skin. J. Biol. Chem.; 1993; 268, pp. 14888-14892. [DOI: https://dx.doi.org/10.1016/S0021-9258(18)82416-4]

7. Delidovich, I.; Palkovits, R. Catalytic Isomerization of Biomass-Derived Aldoses: A Review. ChemSusChem; 2016; 9, pp. 547-561. [DOI: https://dx.doi.org/10.1002/cssc.201501577]

8. Meléndez, E.N.; Carreras, L.; Gijón, J.R. New alkaloid from Lobelia portoricensis Urban. J. Pharm. Sci.; 1967; 56, pp. 1677-1680. [DOI: https://dx.doi.org/10.1002/jps.2600561234]

9. Chenevert, R.; Morin, P. Synthesis of (−)-lobeline via enzymatic desymmetrization of lobelanidine. Bioorg. Med. Chem.; 2009; 17, pp. 1837-1839. [DOI: https://dx.doi.org/10.1016/j.bmc.2009.01.055]

10. National Center for Biotechnology Information. PubChem Compound Summary for CID 101616, Lobeline. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/Lobeline (accessed on 12 September 2022).

11. Miller, D.K.; Crooks, P.A.; Zheng, G.; Grinevich, V.P.; Norrholm, S.D.; Dwoskin, L.P.; Rinaldi-Carmona, M.; Barth, F.; Congy, C.; Martinez, S. et al. Lobeline analogs with enhanced affinity and selectivity for plasmalemma and vesicular monoamine transporters. J. Pharmacol. Exp. Ther.; 2004; 310, pp. 1035-1045. [DOI: https://dx.doi.org/10.1124/jpet.104.068098]

12. Kim, H.L. Effects of SKF 525-A, phenobarbital and 3-methylcholanthrene on the toxicity of lobeline sulfate. Vet. Hum. Toxicol.; 1985; 27, pp. 1-2.

13. Zernov, N.G. Effect of lobeline and cytiton in pneumonia in infants. Vopr. Pediatrii; 1953; 21, pp. 32-38.

14. Martin, C.A.; Nuzzo, P.A.; Ranseen, J.D.; Kleven, M.S.; Guenthner, G.; Williams, Y.; Walsh, S.L.; Dwoskin, L.P. Lobeline Effects on Cognitive Performance in Adult ADHD. J. Atten Disord.; 2018; 22, pp. 1361-1366. [DOI: https://dx.doi.org/10.1177/1087054713497791]

15. Roni, M.A.; Rahman, S. Lobeline attenuates ethanol abstinence-induced depression-like behavior in mice. Alcohol; 2017; 61, pp. 63-70. [DOI: https://dx.doi.org/10.1016/j.alcohol.2017.01.015]

16. Kulkarni, A.M.; Rampogu, S.; Lee, K.W. Computer-Aided Drug Discovery Identifies Alkaloid Inhibitors of Parkinson’s Disease Associated Protein, Prolyl Oligopeptidase. Evid.-Based Complement. Altern. Med.; 2021; 2021, 6687572. [DOI: https://dx.doi.org/10.1155/2021/6687572]

17. Stead, L.F.; Hughes, J.R. Lobeline for smoking cessation. Cochrane Database Syst. Rev.; 2012; 2012, Cd000124. [DOI: https://dx.doi.org/10.1002/14651858.CD000124.pub2]

18. Zheng, G.; Dwoskin, L.P.; Deaciuc, A.G.; Norrholm, A.S.D.; Crooks, P.A. Defunctionalized lobeline analogues: Structure-activity of novel ligands for the vesicular monoamine transporter. J. Med. Chem.; 2005; 48, pp. 5551-5560. [DOI: https://dx.doi.org/10.1021/jm0501228]

19. Rosecrans, J.A.; Young, R. Discriminative Stimulus Properties of S(−)-Nicotine: “A Drug for All Seasons”. Curr. Top. Behav. Neurosci.; 2018; 39, pp. 51-94.

20. Hojahmat, M.; Horton, D.B.; Norrholm, S.D.; Miller, D.K.; Grinevich, V.P.; Deaciuc, A.G.; Dwoskin, L.P.; Crooks, P.A. Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters. Bioorg. Med. Chem.; 2010; 18, pp. 640-649. [DOI: https://dx.doi.org/10.1016/j.bmc.2009.12.002]

21. Xia, R.; Dai, H.; Che, B.; Zhang, J.; Guo, H. Determiniation of Related substances in Lobeline Hydrochloride Injection by RP-HPLC. Drug Stand. China; 2009; 10, pp. 46-50.

22. Seniutinas, G.; Balčytis, A.; Tomašiūnas, R.; Petruškevičius, R.; Getautis, V. Freezing out all-optical poling dynamics of azophenylcarbazole molecules in polycarbonate. Phys. Chem. Chem. Phys.; 2013; 15, pp. 14219-14228. [DOI: https://dx.doi.org/10.1039/c3cp51918j]

23. Kanda, J.; Egami, N.; Sasamori, T.; Imayoshi, A.; Hosoya, T.; Tsubaki, K. Synthesis of Bridged Indigos and Their Thermoisomerization and Photoisomerization Behaviors. J. Org. Chem.; 2021; 86, pp. 17620-17628. [DOI: https://dx.doi.org/10.1021/acs.joc.1c01726]

24. Mahatthananchai, J.; Dumas, A.M.; Bode, J.W. Catalytic selective synthesis. Angew. Chem. Int. Ed. Engl.; 2012; 51, pp. 10954-10990. [DOI: https://dx.doi.org/10.1002/anie.201201787]

25. Crooks, P.A.; Zheng, G.; Vartak, A.P.; Culver, J.P.; Zheng, F.; Horton, B.D.; Dwoskin, P.L. Design, synthesis and interaction at the vesicular monoamine transporter-2 of lobeline analogs: Potential pharmacotherapies for the treatment of psychostimulant abuse. Curr. Top. Med. Chem.; 2011; 11, pp. 1103-1127. [DOI: https://dx.doi.org/10.2174/156802611795371332]

26. Zheng, Q.; Wang, Y.; Zhang, S. Beyond Alkaloids: Novel Bioactive Natural Products From Lobelia Species. Front. Pharmacol.; 2021; 12, 638210. [DOI: https://dx.doi.org/10.3389/fphar.2021.638210]