Nicotine - Science topic

Harris B. Shumacker

The discussion on cigarettes smoking causing migraine has no definite answer, yes or no. Some factors have to be considered before confirming or refuting such assertion. One of the factors is individual differences which remains a significant factor on how individuals respond to certain stimuli.

For now I want to submit that the link between smoking and migraine is speculative.

Otu O. Essien, Ph.D

University of Uyo, Akwa Ibom State,

Nigeria.

Hello Yogi. Thanks for the questions. The history of the beginning of tobacco smuggling in Bhutan is centuries old. Rebecca Sherry and I documented this in our article:

The modern 21st-century version of Bhutan's tobacco smuggling has yet to generate conclusive evidence regarding who is involved. I agree with what you write that a likely culprit is TTCs. Further research is needed. The tobacco company with the most market share in Bhutan has been British American Tobacco--likely due to the history and legacy of British colonialism and post-colonialism in this area.

Nevertheless, the documented history of robust black market tobacco smuggling continued from 2004 to 2021, when Bhutan's ban on most tobacco promotion, use, and production was in force. Why smuggling? 'Tobacco free' and neo-prohibitionist policies are supply oriented. They do not ordinarily include the demand for tobacco. And how to counter that demand in medically appropriate and humane ways. So, I would think one lesson learned is not to deemphasize scientifically proven and humane ways (not stigmatizing addicted smokers, in my view) to reduce demand. The other factor, I think, is that eradicating any addictive substance has a poor to non-existent track record. Unfortunately, some will use these substances for whatever reason. I believe one needs to factor this hard reality (over ideology and belief) into any scientifically proven tobacco mitigation and control efforts. Michael

Dear Anandhu Gopan,

The easiest way to compute a pKa value in Gaussian is to use the isodesmic method. This allows you to avoid any complex thermodynamic cycles. In this method you use a reference with known pKa (e.g. formic acid) to compute the pKa of interest. Please be also aware that the implicit solvation models implemented in Gaussian are far from perfect. Fortunately it fails in a predictable manner so you can scale the obtained raw pKa value in order to obtain a reasonable guess. Using this method you can get pKa values with an accuracy of roughly 1 pKa unit with minor computational costs.

A detailed description of the method, its accuracy and suitable scaling relations can be found here:

Best wishes

Michael

Since polyphenols from plant extract are commonly contaminated with quinones, it would be difficult to obtain a colorless product. You can try to reduce quinones by ascorbic acid

Hi Maria,

You may make an inquiry at Alfa Chemistry, they offer kinds of good-quality chemicals.

Dear Dr. Md Maruf Ahmed ,

regarding this subject and a correct preparation of standard samples, I suggest you to have a look ath the following, interesting paper:

- Rapid identification of nicotine in electronic cigarette liquids based on surface-enhanced Raman scattering

Jun-Yi Chien, Yong-Chun Gu, Hsin-Mei Tsai, Chun-Hao Liu, Chia-Yuan Yen, Yuh-Lin Wang, Juen-Kai Wang, Chi-Hung Lin

JOURNAL OF FOOD AND DRUG ANALYSIS, 28:302e308 (2020)

Available at: https://www.jfda-online.com/cgi/viewcontent.cgi?article=1064&context=journal

My best regards, Pierluigi Traverso.

Hi: I hear questions similar to yours every week from investigators. There are no single or simple answers to your questions. You are hearing "mixed answers" to your questions, because of a profound level of missing information needed to make constructive suggestions. A 'universal' extraction method will not be sample specific (often leaving behind actual sample or even changing or degrading it). None exist. There is no "single" analytical method which is applicable to the range of compounds you list (and please keep in mind that to detect and measure such compounds, they will always be in mixtures with many other chemical compounds too, perhaps hundreds or thousands). There is no "best" solution to use for LC-MS analysis as to make the detector useful, one first has to develop a high quality, sample specific, HPLC method for use (millions of possible methods. Experience and experimentation helps an expert narrow down the choices).

Pick one, then research what types of other materials may also be found in the same sample which need to be extracted and/or resolved from. Get an idea of how much material (concentration) might be expected. Determine what solvents these compounds are soluble in and under what conditions they may become unstable.

*Your teachers are in the best position to help you. Ask them to help you simply the project and also get help from experienced scientists. Focus on how you can specify detailed goals and then manage the process of finding the answers. 'We' do not need to do all of the work ourselves (critical to learn as a student). Where possible, hire or utilize other experts to work on the parts of the project that you do not have a decade of extra time to study. To develop just a basic level of proficiency in using HPLC and/or developing methods takes a minimum of five years (emphasis on "basic" level, as in after 5 years you will NOT have enough experience to develop reliable methods). HPLC coupled to MS (a quad or Orbitrap) requires far more hands-on training and industrial experience. Save time by finding local experts with industrial experience in using these techniques for the sample types YOU are most interested in. Make sure the proposed techniques really are 'best' for your research. HPLC and LC-MS may not be the right tools. This way you can spend your time collecting and researching samples and the analytical experts can work out the details needed to extract and measure the compounds.

Thank you very much, I will definitely read them and do some tests!

. They report that smoking (nicotine) dampens hallucinations. This is really a health staff hallucination and delusion. I was so disappointed when I saw the staff and smoking with patients in b South-west of Finland. It is known that there are 43 toxins in a cigarette. Does not a healthy body matter if you have a mental disorder?

Hi,

Maybe some of these references are of use to you:

Kovács I, Richman MJ, Janka Z, Maraz A, Andó B. Decision making measured by the Iowa Gambling Task in alcohol use disorder and gambling disorder: a systematic review and meta-analysis. Drug Alcohol Depend. 2017 Dec 1;181:152-161. doi: 10.1016/j.drugalcdep.2017.09.023

Grassi G, Makris N, Pallanti S. Addicted to compulsion: assessing three core dimensions of addiction across obsessive-compulsive disorder and gambling disorder. CNS Spectr. 2020 Jun;25(3):392-401. doi: 10.1017/S1092852919000993

Brière M, Tocanier L, Allain P, Le Gal D, Allet G, Gorwood P, Gohier B. Decision-Making Measured by the Iowa Gambling Task in Patients with Alcohol Use Disorders Choosing Harm Reduction versus Relapse Prevention Program. Eur Addict Res. 2019;25(4):182-190. doi: 10.1159/000499709

that is also referred to as third hand smoking..- where the residues of smoke continuously remain on objects for years.

I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link:

Dear Veno!

You do not observe cell proliferation with nicotine because you are using inappropriate assessment methods.

For example,

Nicotine can promote proliferation, invasion and survival pathways in A549 NSCLC cells. (A) Nicotine can induce S-phase entry in A549 NSCLC cells in a dose-dependent manner, as measured by BrdU assays. (B) Nicotine can induce anchorage-independent growth of A549 cells in soft-agar assay. Nicotine increased the size of the individual colonies but had little effect on the total number of colonies. (C) Nicotine can promote migration and invasion of A549 cells in Boyden Chamber assays, in a dose dependent manner, the maximal effect being observed at 1µM nicotine. (D) Nicotine can confer resistance to anoikis of SAECs. SAECs were treated with 1µM nicotine and plated on polyhema coated slides. Apoptosis was measured by TUNEL assays. (E) Treatment of A549 cells with 1µM nicotine caused morphological changes, similar to VEGF on 3D collagen gel assays.

I am assuming that your compound was administered with nicotine during the induction phase of the CPP study. Nonetheless, I agree with the comment above that I don’t think you can really say much about how the drugs are interacting at a cellular pharmacological level if you are administering the drugs systemically. They may be acting at very different sites, cellular targets or a whole gamut of other possibilities. This doesn’t negate what sounds like an interesting finding but detailed pharmacological studies, possibly in vitro, may be required to get to the bottom of what is going on.

Best,

Niall

I would suggest this article by Hassanzadah et al. (2019) as potentially helpful. Although, especially in schizophrenia, nicotine may be helpful as "self-medication" for negative symptoms, other work suggests that chronic nicotine use may lead to increased anxiety and increased catecholamine levels with tachycardia and hypertension. This paper references most of the leading authors in the field. Best,

Steve Mann

Good Answer Joe Graymer

Use a nicotine (alkaloid) selective, or a general extraction procedure, concentrate your extracts, weigh them all carefully, prepare a sample of known concentration (mg/ml) for injection, serially dilute to required levels of concentration (check GC-MS analysis of nicotine procedure, sample pre and injected micro litre sample quantity), the weight of the extract in the injectable volume is a must here! Run your sample, if need be use spiking techniques, record GC and (GC-MS comes later, MS another confirmation for Nicotine, but just don't rely on this info only, compare and see in known nicotine spiking to the injected sample!), identify the nicotine peak from the known column, concentration, and RI (not the RT- Retention Time) values, so the spiking for nicotine confirmation is necessary. Get total peak are, nicotine peak are, calculate the peak % for the nicotine, and correlate with your sample quantities. Repeat for all extracts, and get to know your concentration of the nicotine in the sample - each extract! Precaution: record all weight, volumes, procedure in stepwise manner, including GC-Conditions!

The following link describes a simplified way to extract nicotine.

Nicotine has good MS response. But why do you do headspace analysis? I suggest direct injection (e.g. solvent extracts after LLE/DLLME etc.) of Your sample using split/splitless injection mode (of course it depends on Your matrix).

Best regards - Mateusz

Dear Mohamad-Hani Temsah

Greetings!!

Its a very important topic indeed. Nicotine/Tobacco usage especially among children and teenagers is on a rise, globally. Cancer is a global problem, and regular, indiscriminate consumption of tobacco and related products is thought to be a crucial factor for development of oral malignancies. A lot of factors play in the initial urge among the teenagers/children for using tobacco. It may be due to their curiosity; may be due to bad company of friends; may be due to attractiveness towards the flashy ads of various tobacco products in either television or internet; or may be due to less knowledge of the harmful effects due to long-term exposure. To curve this global menace, the solution has to be solved both at an individual level and as well as strong policies should be taken by the concerned government health agencies. Necessary educative course material regarding harmful implications should be introduced at school/college level to discourage tobacco usage. Practice of healthy lifestyle should be encouraged among the youth/children. Respective governments worldwide on their side can increase the taxes on tobacco related products, which might discourage its usage among the public.

As, you have said and i agree that ".... Several tobacco promotions may reach children in early age, either through social media or peers....". Yes, in this age of high speed internet connectivity this children are more addicted to their computers/mobiles and get inspired from the flashy ads/promos. To solve this regular internet ads, forums etc with a strong focus on chronic harmful effects will be a step towards curving this menace. Health advisory symbol/image in the packaging of all tobacco and its associated marketed products should be strongly implemented.

Check our page 23 of this report on e-cigarette use in Asia -https://seatca.org/dmdocuments/SEATCA_Ecig%20Report_Final.pdf

Hope that it helps.

Thanks JA, actually I am not looking at the crystallinity of the material but the physical structure such as rod like, platelet or spherical in shape.

Dear Richard, there are many ways of analyzing gradients. However, I need to know what are the dependent and independent variables, and how many types of variables are there. Based on the dataset we can decide on the best method of analysis.

Hi Dr Ammar Bader

I have found some information about it

Alpha7 is tricky because kinetics are super fast and desensitization to low concentrations of agonist are a problem. Additionally, they require high concentration of ligand to activate alpha7 simultaneously so you can see anything (for cultured neurons I used a valve-driven system because currents were terrible with normal flow systems). I would suggest using ACh instead of nicotine (all the AChE keeps it from accumulating) at a high concentration (~500 uM). I assume you’re using a picospritzer?

Hi Chitra,

There are multiple things you might need to investigate here.

1) Are the area counts of your heavy labeled IS (Nicotine in this case) constant throughout the batch (<5% CV), or do they vary with the different concentration levels of the analyte of interest?

2) If you try to quantify the analyte without any internal standard, are your lower points within acceptable limits?

I would recommend understanding why the corresponding IS isnt working, rather than skipping to a different IS to make your assay meet the criteria. Your labeled IS variation tells you a story, and is specially important in clinical samples where you cannot predict the variability of the patient population and how different diet, disease conditions etc will affect the results for your drugs of interest. The heavy labeled standard most closely mimicks your analyte and therefore should hopefully correct for these variations.

There might be ion suppression/enhancement or other phenomenon which may be the root cause of the inconsistency and it might be worth investigating it to ensure a more robust assay in the long term.

This study might be helpful.

HCl should be highly soluble in ethanol - nearly as soluble as it is in water. Some references suggest that as much as 78 parts per 100 parts ethanol are soluble at 0 C, decreasing to 69 parts at 20 C.

I suspect your problem could be (a) too high of temperature, (b) too large of bubbles, or (c) insufficient contacting time to absorb the HCl.

You say that the volume of your cuvette is 3mls - is it a semi-micro cuvette with a maximum capacity of 3mls, or is that the minimum volume that you can measure? You should check the height of your light path (can be done with water), so that you know the minimum volume that you can use to measure your samples without risk of interference by the air-liquid interface at the surface of your sample. The construction of the standard curve, to give your maximum standard concentration is easy, if you just want it to be around 24mg/ml: a 1 in 4 dilution of 98mg/ml nicotine would give you 24.5mg/ml - so you could dilute 1.5mls of your stock with 4.5mls HPLC grade water, then make further stepwise dilutions of the resulting 6mls of 24.5mg/ml nicotine. If your cuvettes are semi-micro ones, then you should be able to make each subsequent dilution up to a total volume of 1000ul, mix well, then add 800ul to your cuvette (excluding bubbles) to obtain your absorbance reading. Alternatively, you could alter the dilution to give 'nicer' numbers for your standard curve, eg: 25mg/ml (1.5mls of 98mg/ml nicotine stock, made up to a total of 5.88mls with water) or 20mg/ml (1.5mls of 98mg/ml nicotine stock, made up to a total of 7.35mls with water)for your maximum concentration, with dilutions then possible at

1-5mg/ml steps.

It might also be useful to convert your concentrations to molarity:-

Mwt Nicotine=162.236 g/mol; so 1M=162.236mg/ml;

Therefore, your 98mg/ml divided by 162.236 = 0.6041M

I hope this is of some help.

The direct contribution of nicotine to tobacco-associated diseases is unclear, as it is inhaled along with many other substances in tobacco smoke. The role of nicotine is to maintain the addiction and other substances in tobacco smoke, particularly tar and some of the gaseous components such as carbon monoxide, are the main direct causes of disease .

There are two pillars in Nicotine Dependence Treatment: medication and cognitive-behavioral therapy

The term ‘‘tobacco constituent’’ is defined as a substance naturally present in tobacco. The term ‘‘tobacco ingredient’’ is defined as a substance (except water) that is added to tobacco during the manufacturing process and having a specific function on the final tobacco product. Tobacco ingredients are classified as flavours and additives. In 1960, a little over 200 chemical constituents had been identified in tobacco leaf of all types and less than 450 had been reported in smoke. Today, approximately 3000 have been identified and characterized in tobacco leaf and some 4000 in smoke. Estimates are that the total number of chemical constituents in leaf exceeds 4000 and there are over 6000 in tobacco smoke. Mixture tobacco for cigarette consist flue-cured tobacco- virginia, air-cured tobacco-burley, sun-cured tobacco. reconstituted tobacco (RECON) and expanded rib which differ according to the chemical composition. The smoke composition is depend from composition of tobacco mixture.

best regards

thank you both of you #Ramsey Sir and #Nermina Madam

Dear Andrew,

Thank you for your reply. Will you please provide me references or paper supporting above.

Thanks!

Abhilasha

Thanks all!

To gain the exact amount of nicotine absorbed is not an easy study, but you have two solutions (and each has its own limitations): 

You can mimic smokers behaviours on a smoking machine,

or you can do a pharmacokinetic study on the level of nicotine after smoking one cigarette in each participant. 

The first approach will give you information on the amount of nicotine inhaled from one cigarette, the second will give you information on the dose of nicotine taken (with some assumptions). 

But, these solutions are ONLY for short exposure (one cigarette, two etc). If you need something for measure nicotine intake for a day, week or month, you need to look for another solution. You can take into account number of cigarettes smoked, nicotine equivalent in biomarkers etc...

Please where can I send my molecularly imprinted samples for pore size, pore volume and surface area analysis using BET method?

You have said most. I agree.

Will human beings continue with a personally satisfying and, if properly controlled, relatively minor noxious habit (s)? Yes, I think so.

My original question just stressed the relevance of excluding market interest from public health. I hope no one finds the “Winerette” to avoid the glass of Rioja you may drink on Saturday evenings …

Sorry, It is not area of my investigation. Best regard.

I agree with my colleagues.

Best regard-

Hi, in addition of the comments above, I think you can predict your compound by using some on line data base, as example you can try the attached link, I think there are many other free MS on line data bases, best regards

Dear Yash,

 HPLC was born in 1970 after this date until this moment there are more than 5000 flavonoids have been identified based on HPLC. It  seems like HPLC born for flavonoids.  I would recommend that to use HPLC, doing spectrum or HPLC/MS. You say you have noticed that nicotine come up with 2 peaks. how do you know that was nicotine?  If nicotine appeared to me I can determine that from HPLC system; retention time, from spectrum study and definitely from MS system . If you got two peaks from one compounds there is contamination in your standards, what solution did you  use? what solvent used dissolve your sample and standards? what type of antioxidant used in you solvent? 

Best regards

Aly

Validation is not considered necessary for large-scale trials in which the adolescents would not have a clear motivation to lie-- for example, tracking studies which have no cessation component.  Research generally indicates self-reports are adequately valid in this situation.

In France at least tobacco extracts were used on crops to kill insects but they were banned decades ago. Be aware that nicotine may be very toxic for mammals and other vertebrates. Nicotine is absorbed through the skin (see the Center for Disease Control and Prevention fact sheet at http://www.cdc.gov/niosh/ipcsnfrn/nfrn0519.html), so in vitro effects on ticks are not indicative of a potential use for tick control.

Please read this publication.....  

This simplest method for extraction of nicotine and cotinine in plasma. 

The human astrocyte cell line SVGA may be a possibility

Thank you so much sir for your timely help. 

Dear Colleagues: Smokers and those who does not start smoking yet:

The label on a pack of cigarettes reads:  Nicotine 0.4 mg/cig;  TAR :  4 mg/cig.

The ratio TAR/Nicotine is always close to 10. The above example

4.0 Tar/0.4 Nicotine and it is for weakest cigarettes.  Very strong, young  guys prefer (and/or sustain) cigarettes with high concentration of both of components i.e. : TAR and Nicotine.

Of course - both the components sediment on - everything, leaving a reddish thin film which is very difficult to remove. In other words: the smog of cigarettes is "surface addictive"

[I am not writing here about the Pleasure of Smoking. But My own strong opinion is that smokers are addicted to TAR rather to Nicotine].

With Best Regards,                                      Leonid

Yes, Paul, I agree.  I was being a bit hyperbolic and put "silver bullet" in quotes for that very reason.  Thanks for the links; I read one of them previously.  I have read much on this and chatted with someone very active in research in this area.  The question still stands:  Why is this going nowhere?

you should contact Prof. Dirk Selmar at Braunschweig University in this matter: d.selmar@tu-bs.de

Dear Virginia

The answer to your question is "No". But I must admit there are no studies that have investigated your question because it is somewhat counterintuitive. Waterpipes combined a boring process of charcoal with the heating of tobacco with a number of additives and the result is an inhaled mixture that contains many more toxic substances than smoke from tobacco cigarettes. Both include nicotine, the main addictive substance in tobacco addiction. 

Many people think that water pipe smoking is less harmful because the smoke is "filtered" through water. The main effect of the water is the cooling and humidification of the inhaled smoke. The other aspect which may suggest it may be a method to quit is that it is used less frequently and mostly in a social context. But real nicotine addicts will hardly be able to reduce nicotine consumption by purely changing to conventional water pipe use.

Regards

Macé

Thanks Prof  Lewis，Prof  Susana  and Prof John！

Hi

Register in the google window:  baromêtre sante tabac en France

Y'll obtain survey of tobacco consumption every 5 years 2005/2010/2015

INPES website:  www.tabac-info-service.fr

Best regards.

Jean.

I was thinking about an experiment with tobacco smoke, to be simple and visual for a class of sudents or pupils. I think the best is to light a cigarette and puss the smoke through a strong light source so that everybody can see the tiny smoke particles, filling the air of the room, which when breathed go into the lungs and  because  they are of the PM2.5 size are trapped in the pulmonary alveoli. Also,  hold a paper filter  (white , coffe filter or chemistry filters) in front of smoke , 6-7 smoke phases are trapped on the paper and give it a black colouration from tar. Collect tar in a vial to show it how black it is, . Then you can say that this smoke particles contain PAH, carcinogenic chemicals, nitro-PAH,  VOCs, benzene, CO, etc. Also, they have polymeric black colour carbonated particulates that trap stable free radicals producing continuously hydroxyl radical which can penetrate into cells and can cause DNA damage. or oxidize lipids in cell membranes. These pictures can be found in books, and projectd on backboard. See my papers on tobacco smoke. in Researchgate. Also, passive smoking can be explained with a smoking a cigarette and allowing to produce secondary smoke which is more dangerous because of lower temperature and pyrolytic burning  in the tip (the smoke covers the cigarette tip prohibiting oxygen to do the oxidizing burning, and in this way is producing more carcinogens. 

Valavanidis A, Professor

Jeff

A more specific compound is 1 acetyl methl pyrazine commonly used in E Cigarettes to suppress cravings and urges to smoke conventional brands. .1 % no self reported effects, .6% effective  and 1% too high a dose based on 100 plus postings on E Cigaretteforum.com. Good qualitative data only.  

  The research points to stimulation of the olfactory nerve and neurological signals through the bulb to different parts of the brain. Somehow these signals reinforce perceived reward (suppression or urges) associated with nicotine withdrawal. Using a E cigarette Vapor device (one that can modify additive content) one could remove and add pyrazines and do self reported craving scores or even fMRI's. I believe Gold in Florida does this for reduced sugars. Since pyrazines act on the olfactory nerve a dietary model will not work easily but an inhalation model will. The best pleasure from food is odor not gustation.     

Pyrazines are formed from the Mallaird Browning process of heating protein, NH3 and reduced sugar and have always made food taste wonderful. Doubt id there are specific receptors but who knows.

Greg Connolly    

Please have a look at this: 

There is no established method, It would be feasible to develop one but it would be fairly lengthy and inefficient.

I concur. To handle this, our lab made a chart of % nicotine inhaled with each concentration of "juice", and we ask participants how quickly they go through refill cartridges. We then compare this to how much nicotine they would get from a normal/average cigarette. Hookahs/water pipes are harder though..we try to just quantify that with hours of use and divide it by the number of people sharing the hookah. 

Hi,

Nicotine exposure may have detrimental effects on body storage of vitamin D. I attach information.

Best regards,

Faustino

Adherent PC-12 Adh ATCC ® CRL-1721.1 works better than the normal floating PC-12 ATCC ® CRL-1721. You may try with adherent version.

Please, take some advices according NICOTINE from Levin ED et al, 2007 - "Zebrafish placed in a novel tank spent the majority of time at the bottom third of the tank during the first minute of a 5-min session and then show a gradual decrease in time spent at the tank bottom. Nicotine treatment at 100 mg/l for 3 min by immersion before testing caused a significant decrease in diving throughout the session, while 50 mg/l was effective during the first minute when the greatest bottom dwelling was seen in controls". Another paper by Braida D et al., 2014 "The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index".

And better to evaluate a paper at Attachment, Please, and all the best. Vladimir

The norepinephrine vials that they use medically can be kept at room temperature (in the dark!) for extended periods of time, so I think it is pretty stable. I would say the same for nicotine: if it survives smoking it takes a lot of activation energy to degrade it.

Aliquotting and freezing is never wrong though.

I would suggest that you wash out the nicotine.  In my own experience with nicotine in water, the longer they sit in the water, the more of an exposure to the nicotine that they have - which depending on the time and dose of nicotine could cause death of the embryos. You can see the protocol that I followed in the paper that I published in PNAS (linked - Petzold, 2009), but I honestly cant remember if the full details of the procedure were listed in there. Incubation of the zebrafish - for me - lasted around 30 seconds to a minute once I had everything figured out, but I used custom made filter dishes (a small dish with a screen on the bottom so that I could dunk the fish into the dish of nicotine). Earlier on in the testing process they were exposed to a longer dose - 5-30 minutes, but I got a similar response as the shorter time period, so I went with a shorter exposure. Whenever they were left in overnight or for more than a few hours, there tended to be a bit more death. Granted I also was looking at 3-5 dpf larvae, so your results could be vastly different if exposing at an earlier time point.

Good luck!

To begin, here are few

Sir, I agree with Isabelle's opinion, because Eventhough nicotine acts  as insecticide, I has got high mammalian toxicity profile as it mimics ACh of mammals. If nicotine would be safe u imagine more than thousands of nicotine based insecticides would be there in the market especially in India , as we have lot availability of tobacco

And, perhaps more relevant is this other paper:

Liu, X., Xu, Y., Wang, X., Barrantes, F.J. y Jiang, H. (2008). Unbinding of nicotine from the acetylcholine binding protein: Steered molecular dynamics simulations J. Phys. Chem. B 112, 4087-4093. DOI: 10.1021/jpc0716738

We also measure cotinine via saliva samples collected in a salavette.  ABA in Nottm charge £25 per sample but I understand that there are other options available.  We collect all our samples then send off in one huge batch which I believe is cheaper.  Hope this is helpful.

As far as I know this drug is not commercially available. It was developed by the scientists from Sanofi-Aventis, France and they still hold the patent rights. You can ask from Sanofi-Aventis for research purposes. Regards.

With no details about the precise targets of your study, detailed advices cannot be provided. However, if you want to evaluate in vivo effects, probably the best option is to use selective agonists (e.g. PHA543613, WAY317538, AR-R17779) or antagonists (e.g., alpha-conotoxin ArIB, α-Bungarotoxin) by means of microinjections to hippocampus. Then you can obtain neurochemical, electrophysiological or behavioral measures by means of a variety of methods. I hope this has been helpful...

Smokeless tobacco use occurs frequently in Sweden but is forbidden in Finland.

Based on the literature it seems to me that  MoCA is suitable relative to tobacco dependency: http://www.nhchc.org/wp-content/uploads/2014/06/feinberg-mahan-kohl-the-use-of-the-moca-as-a-brief-screen-for-cognitive-impairment-montreal-cognitive-assessment.pdf

(http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_013.pdf)

Try: Maritt A M et al., Nicotinic cholinergic receptors in the rat retina: simple and mixed heteromeric subtypes. J Pharm Exp. There 68(2005): 1656-1668.

Can't find it also, is it a publication? It isn't listed at Pubmed. You should ask him directly:

You might have better luck getting a doctor on board if you offer them co-authorship. I know nicotine is given to nonsmokers in research projects, so collect some articles and include them with your proposal to a doctor. Your IRB should have doctors they work with and you should start there. If your institution has a medical school/hospital you should be able to easily find a doctor that needs to get some publications so letting them know they can be a co-author should give you the leverage you need to find someone. I have added an article to get your started...good luck!