Nicotine, cotinine, and norNicotine were purchased from Sigma-Aldrich (St. Louis, MO) as standards. PseudooxyNicotine, nicotyrine and 6-hydroxyNicotine were obtained from Toronto Research Chemicals (Toronto, ON, Canada). All analytical and high-performance liquid chromatography (HPLC) grade reagents were bought from Merck China (Beijing, China).
Nornicotine
Manufactured by Merck Group
Sourced in United States, Germany
Nornicotine is a laboratory reagent used in various research and analytical applications. It is a chemical compound derived from nicotine. The core function of Nornicotine is to serve as a standard or reference material for analysis and quantification purposes in scientific research, without making claims about its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Cotinine, by Merck Group (7 mentions)
Anabasine, by Merck Group (7 mentions)
Nicotine, by Merck Group (5 mentions)
Formic acid, by Thermo Fisher Scientific (4 mentions)
Nicotinamide, by Merck Group (3 mentions)
Anatabine, by Cayman Chemical (3 mentions)
Agilent 1200 series hplc system, by Agilent Technologies (2 mentions)
Mecamylamine hydrochloride, by Merck Group (2 mentions)
Cytisine, by Merck Group (2 mentions)
Choline, by Merck Group (2 mentions)
Anatabine, by LGC (2 mentions)
Nicotine bitartrate, by Merck Group (2 mentions)
Myosmine, by Merck Group (2 mentions)
Ammonium acetate, by Merck Group (2 mentions)
6 hydroxynicotine, by LGC (1 mentions)
Nicotyrine, by LGC (1 mentions)
Chemstation software, by Phenomenex (1 mentions)
Buspirone hydrochloride, by Bio-Techne (1 mentions)
Acetylcholine bromide, by Merck Group (1 mentions)
Nicotine free base, by Merck Group (1 mentions)
14 protocols using nornicotine
1
Nicotine and Metabolite Standards
2
Quantification of Nicotine and Metabolites
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All reagents were of analytical grade unless otherwise indicated. (-)-Nicotine, (±)nornicotine, (±)-anabasine, (-)-cotinine and methanol CHROMASOLV® for HPLC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Radiolabeled nicotine, DL-[Nmethyl-14 C] nicotine (50-60 mCi/mmol or 1.85-2.22 Gbq/mmol), was obtained from American Radiolabeled Chemicals, Inc. (MO, USA). All solutions and dilutions were prepared with double distilled de-ionised water, produced by an ELGA PURELAB Ultra water purification system from Veolia Water Systems Ltd. (High Wycombe, UK) unless otherwise stated.
3
HPLC Analysis of Alkaloid Compounds
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The Agilent-1200-Series HPLC system equipped with a diode array detector (G1315D), the Agilent ChemStation software and a Phenomenex Gemini-NX 5 μ C18 110A 150 mm × 4.6 mm column (Phenomenex, http://www.phenomenex.com/ ) was used at a column temperature of 35 °C and a flow rate of 1.0 ml·min−1. The injection volume was 20 μl for cell extracts and 30 μl for medium extracts; peaks were quantified at 260 nm. UV spectra were collected over the wavelength range from 200 to 700 nm. Eluent A contained 10% acetonitrile in 20 mM ammonium formate, adjusted to pH 8.7, and eluent B consisted of 100% acetonitrile. A gradient program was employed, composed of a sequence of linear gradients with an initial step of 100% A to 80% A and 20% B over the first 10 min, followed by a second gradient to 10% A and 90% B over the next 10 min, and a final step to 100% B from 21 min after injection till the end of the run at 30 min after injection (Trehy et al. 2011 (link)). The reference alkaloids nicotine and nornicotine were purchased from Sigma-Aldrich (Munich, Germany) and used for sample spiking to verify the identified peaks (Online Resource 1 ). Data represent mean values and standard errors from three independent experimental series. The significance of the observed differences was probed with the non-parametrical Friedman test.
4
Synthesis and Characterization of Nicotinic Compounds
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Acetylcholine bromide (CAS No. 66-23-9), (–)-nicotine free base (CAS No. 54-11-5), (–)-cotinine free base (CAS No. 486-56-6), and ( ±)-nornicotine (CAS No. 5746-86-1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). ( +)-Anabasine hydrochloride (CAS No. 53912-89-3), buspirone hydrochloride (CAS No. 33386-08-2), and metanicotine oxalate (CAS No. 220662-95-3) were purchased from Tocris Bioscience (Bio-Techne®, Minneapolis, MN, USA). ( ±)-Anatabine free base, AZD1446, and (S)-N-methylanatabine dihydrochloride were custom synthesized by WuXi AppTec (all: purity ≥ 95%; Shanghai, China).
5
Competitive ELISA for Nicotine Selectivity
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The selectivity of the nic•mAbs 5G4, 7A8, 12F5, and 8D1 for S-(–)-nicotine was determined by competitive ELISA using 3’AM-S-(–)-Nic-pGlu-coated plates essentially as described above. Serial dilutions (in a blocking/diluent buffer of PBS + 1% non-fat dry milk) of competitors were added to the coated and blocked plates. Subsequently, the nic•mAb was added at a fixed concentration yielding a non-saturating A450 level in control wells without competitor. After 45 min incubation at 37°C, the plates were washed, and detection of bound nic•mAb was done as previously described. Small molecule competitors included S-(–)-nicotine, acetylcholine chloride, β-nicotinamide adenine dinucleotide (NAD), bupropion, S-(–)-cotinine, cytisine, dopamine (3-hydroxytyramine) hydrochloride, mecamylamine hydrochloride, nicotinamide, (±)-norepinephrine (+)-bitartrate, (±)-nornicotine, serotonin hydrochloride, and varenicline tartrate (all from Sigma). These compounds include neurotransmitters, nicotine metabolites, structurally similar compounds, and smoking cessation drugs. The cross-reactivity was calculated from the ratio of the IC50 values as follows: (IC50, nicotine/IC50, competitor) × 100%.
6
Pharmacological Compounds for In Vitro Studies
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Choline, nicotine, cotinine, nornicotine, methyllycaconitine citrate (MLA) and α-bungarotoxin (BTX) were from Sigma Aldrich (St. Louis, MO, USA). Choline, MLA, and BTX were dissolved in distilled water. nicotine, cotinine, and nornicotine were dissolved in ethanol. YM 254890 and pertussis toxin (PTX) were purchased from Tocris-Bio-Techne Corporation (Minneapolis, MN, USA) and dissolved in DMSO and distilled water, respectively. α-Conotoxin RgIA (CTX) was obtained from Alomone Labs (Jerusalem, Israel) and dissolved in distilled water.
7
HPLC Analysis of Tobacco Alkaloids
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The Agilent-1200-Series HPLC system equipped with a diode array detector (G1315D), Agilent ChemStation software and a Phenomenex Gemini-NX 5μ C18 110A 150 mm x 4.6 mm column (Phenomenex, Aschaffenburg, Germany) was used at a column temperature of 35°C and a flow rate of 1.0 ml/min. The injection volume was 20 μl for cell extracts and 30 μl for medium extracts, peaks were quantified at 260 nm. UV spectra were collected over the wavelength range from 200 nm to 700 nm. Eluent A contained 10% acetonitrile in 20 mM ammonium formate adjusted to pH 8.7, and eluent B consisted of 100% acetonitrile. A gradient program was employed composed of a sequence of linear gradients with an initial step of 100% A to 80% A and 20% B over the first 10 min, followed by a second gradient to 10% A and 90% B over the next 10 min, and a final step to 100% B from 21 min after injection till the end of the run at 30 min after injection [40 ]. The reference alkaloids nicotine, anatabine, anatalline and nornicotine were purchased from Sigma aldrich (Munich, Germany), and used for sample spiking to verify the identified peaks (S1 Fig ).
8
Subcutaneous Administration of Nicotinic Compounds
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The compounds (−)-nicotine bitartrate, (+/−)nornicotine, and (+/−)anabasine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The compound (+/−)anatabine was obtained from Toronto Research Chemicals, Inc. (North York, ON, Canada). All drugs were dissolved into sterile saline. The pH of all solutions was adjusted to 7.4 using dilute NaOH or HCl. All drugs were administered subcutaneously (sc) in a volume of 1 mL/kg. All drug doses are expressed as the base. The dose of each drug was as follows, nicotine: 0.50 mg/kg; nornicotine: 6.00 mg/kg; anatabine 3.00 mg/kg; anabasine 3.00 mg/kg. The doses selected were based on those used in our previous study evaluating the effects of the MTAs on deprivation-induced food intake [44 (link)]. The range of doses used in the previous investigation was narrowed down in the present study to a single dose of each drug that had previously produced reductions in food intake, without significant compensatory increases in food intake later in the day.
9
Quantitative Analysis of CBD and 7-OH-CBD Metabolism
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CBD and 7-OH-CBD were purchased
from Cayman Chemicals (Ann Arbor, MI) or Sigma-Aldrich (St. Louis,
MO). Pooled human liver microsomes (HLM) [n = 50,
mixed-gender (21 female and 29 male), race (42 Caucasian, 4 Hispanic,
2 African American, and 2 Asian), and age (5–77 years old)]
were obtained from Sekisui Xenotech, LLC (Lenexa, Kansas). NADPH-regenerating
system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, and 0.4 U/mL glucose
6-phosphate dehydrogenase) was obtained from Corning (Bedford, MA).
Nicotine, norNicotine, HPBA, NOX, Nic-Gluc, cotinine, COX, Cot-Gluc,
3HC, and 3HC glucuronide (3HC-Gluc) standards were purchased from
Sigma-Aldrich. Fluconazole, benzydamine hydrochloride, clopidogrel,
trifluoperazine, diclofenac, amitriptyline, and tranylcypromine were
also purchased from Sigma-Aldrich. Optima grade methanol, acetonitrile,
and formic acid were obtained from Fisher Scientific (Waltham, MA).
Ultra-low-binding microcentrifuge tubes, Dulbecco’s modified
Eagle’s medium, Dulbecco’s phosphate-buffered saline,
UDP glucuronic acid (UDPGA), alamethicin, MgCl2, and Geneticin
(G418) were purchased from VWR (Radnor, PA). BCA protein assays were
purchased from Pierce (Rockford, IL); premium-grade fetal bovine serum
(FBS) was purchased from Seradigm (Radnor, PA), and ChromatoPur bovine
serum albumin (BSA) was purchased from MB Biomedicals (Santa Ana,
CA).
from Cayman Chemicals (Ann Arbor, MI) or Sigma-Aldrich (St. Louis,
MO). Pooled human liver microsomes (HLM) [n = 50,
mixed-gender (21 female and 29 male), race (42 Caucasian, 4 Hispanic,
2 African American, and 2 Asian), and age (5–77 years old)]
were obtained from Sekisui Xenotech, LLC (Lenexa, Kansas). NADPH-regenerating
system (1.3 mM NADP, 3.3 mM glucose 6-phosphate, and 0.4 U/mL glucose
6-phosphate dehydrogenase) was obtained from Corning (Bedford, MA).
Nicotine, norNicotine, HPBA, NOX, Nic-Gluc, cotinine, COX, Cot-Gluc,
3HC, and 3HC glucuronide (3HC-Gluc) standards were purchased from
Sigma-Aldrich. Fluconazole, benzydamine hydrochloride, clopidogrel,
trifluoperazine, diclofenac, amitriptyline, and tranylcypromine were
also purchased from Sigma-Aldrich. Optima grade methanol, acetonitrile,
and formic acid were obtained from Fisher Scientific (Waltham, MA).
Ultra-low-binding microcentrifuge tubes, Dulbecco’s modified
Eagle’s medium, Dulbecco’s phosphate-buffered saline,
UDP glucuronic acid (UDPGA), alamethicin, MgCl2, and Geneticin
(G418) were purchased from VWR (Radnor, PA). BCA protein assays were
purchased from Pierce (Rockford, IL); premium-grade fetal bovine serum
(FBS) was purchased from Seradigm (Radnor, PA), and ChromatoPur bovine
serum albumin (BSA) was purchased from MB Biomedicals (Santa Ana,
CA).
10
Quantification of Tobacco Alkaloids and Metabolites
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Nicotine, anabasine, myosmine, nicotinamide, cotinine, norNicotine, norcotinine, nicotinic acid, and their corresponding deuterated or 13C internal standards were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated below. Anatabine was purchased from Cayman Chemicals (Ann Arbor, MI, USA), and Anatabine-d4 and myosmine-d4 were obtained from Toronto Research Chemicals (Ontario, ON, Canada). Solvents used for LC-MS analysis were from Honeywell (Charlotte, NC, USA). Ammonium acetate was from Sigma-Aldrich, and formic acid was from Thermo Fisher Scientific (Waltham, MA, USA).
The following solvents used for microfractionation were purchased from Shanghai Titan Scientific Co. Ltd. (Shanghai, China): butanol (≥99.5%, certified analytical reagent (AR)), dichloromethane (≥99.5%, AR), ethyl acetate (≥99.5%, AR), 95% ethanol (95%, AR), ethanol (≥99.7%, AR), hexane (≥97.0%, AR), and methanol (≥99.5%, AR). Ultrapure water was produced by Bicoll (Planegg, Germany). The pipettes for 96-well microtiter plates were produced by Eppendorf (Hamburg, Germany). Polytetrafuoroethylene membrane filters (0.22 µm) and syringes were purchased from Anpel technologies (Shanghai, China).
The following solvents used for microfractionation were purchased from Shanghai Titan Scientific Co. Ltd. (Shanghai, China): butanol (≥99.5%, certified analytical reagent (AR)), dichloromethane (≥99.5%, AR), ethyl acetate (≥99.5%, AR), 95% ethanol (95%, AR), ethanol (≥99.7%, AR), hexane (≥97.0%, AR), and methanol (≥99.5%, AR). Ultrapure water was produced by Bicoll (Planegg, Germany). The pipettes for 96-well microtiter plates were produced by Eppendorf (Hamburg, Germany). Polytetrafuoroethylene membrane filters (0.22 µm) and syringes were purchased from Anpel technologies (Shanghai, China).
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