A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

Moghbel, Nahid; Ryu, BoMi; Steadman, Kathryn J.

doi:10.1016/j.jchromb.2015.06.006

Cited by 25 publications

(24 citation statements)

References 29 publications

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“…This is a faster and more efficient HPLC-UV method than any previously available in the literature, and allows quantitative analysis of six alkaloids, nicotine, nornicotine, anatabine, anabasine, myosmine and cotinine, in tobacco plants and products (Moghbel et al, 2015). Also an efficient LC-MS/MS method was adopted from published methods and validated in our lab for quantifying the two important carcinogenic TSNAs, NNN and NNK that stem from tobacco alkaloids during post-harvest processing of tobacco products.…”

Section: Discussionmentioning

confidence: 99%

“…Both fresh and cured freeze-dried leaves (50 mg) were exhaustively extracted for pyridine alkaloids using 40% aqueous methanol containing 0.1% 1N hydrochloric acid following our previously published method (Moghbel et al, 2015) and were quantified for nicotine, nornicotine, anatabine, anabasine, myosmine and cotinine. Quantitative analysis of the extracts was carried out on an Agilent 1100 series high performance liquid chromatography (HPLC) system equipped with a UV detector and on a Zorbax Extend C18 column (Agilent Technologies, Mulgrave, Vic, Australia) with a gradient mobile phase consisting of 15 mM ammonium formate buffer and acetonitrile as described in chapter 2 (Moghbel et al, 2015).…”

Section: Alkaloid Analysismentioning

confidence: 99%

“…Quantitative analysis of the extracts was carried out on an Agilent 1100 series high performance liquid chromatography (HPLC) system equipped with a UV detector and on a Zorbax Extend C18 column (Agilent Technologies, Mulgrave, Vic, Australia) with a gradient mobile phase consisting of 15 mM ammonium formate buffer and acetonitrile as described in chapter 2 (Moghbel et al, 2015).…”

Section: Alkaloid Analysismentioning

confidence: 99%

“…Analysis of the samples was performed using an Agilent Technologies 1200 series High Performance Liquid Chromatography (HPLC) system attached to a binary pump, degasser, an auto sampler, thermostat column compartment and a diode array detector following our previously validated method as mentioned in chapter 2 (Moghbel et al, 2015).…”

Section: Nicotine and Other Alkaloids Analysis With Hplc-uvmentioning

confidence: 99%

“…Chemical analysis of the extracts was carried out on an Agilent 1100 series high performance liquid chromatography (HPLC) system equipped with a UV detector and Zorbax Extend C18 column (Agilent Technologies, Mulgrave, Vic, Australia) with a mobile phase consisting of 15 mM ammonium formate buffer and acetonitrile according to our previously validated method as mentioned in chapter 2 (Moghbel et al, 2015). The powdered extracts were dissolved in 10% aqueous acetonitrile containing 25 mg/ml caffeine as internal standard and quantification was performed using a constructed calibration curve of nicotine, nornicotine, anatabine, anabasine, myosmine and cotinine over a range of concentrations in 10% aqueous acetonitrile.…”

Section: Quantification Of Nicotine and Other Alkaloidsmentioning

confidence: 99%

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Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Moghbel¹

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The Aboriginal population of Central Australia use endemic Nicotiana spp. to make a smokeless tobacco product known as pituri that they chew/suck for nicotine absorption. This thesis describes the relative abundance of nicotine alkaloids amongst Australian Nicotiana spp., with special focus on the molecular characteristics of nicotine to nornicotine conversion.The most popular chewed species, N. gossei, is investigated for nicotine release and cytotoxicity in comparison to similar products to gain insight into potential hazards to pituri users.To analyse the alkaloids of Nicotiana leaves, a HPLC-UV method was developed to separate and quantify six closely related alkaloids (nicotine, nornicotine, anatabine, anabasine, myosmine, cotinine). A C18 column with a mobile phase of ammonium formate buffer (pH 10.5) separated the six alkaloids within 13 min with detection at 260 nm. Linearity, precision and reproducibility were satisfactory. The limit of quantification was 2.8 and 4.8 µg/mL for nornicotine and nicotine, respectively, and below 2 µg/mL for other alkaloids. This method quantifies more alkaloids and in less time than previously reported methods.Tobacco alkaloids are responsible in formation of carcinogenic tobacco specific nitrosamines such as N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To quantify NNN and NNK, a fast LC-MS/MS method was developed using a HILIC column with a triple quadrupole tandem mass spectrometry. The linearity, accuracy, recovery and repeatability for this method were satisfactory, with quantification limit of 2.6 and 4.3 ng/mL for NNN and NNK, respectively. Nornicotine results from demethylation of nicotine, and is associated with negative effects on health. A group of cytochrome P450 genes that are mainly expressed during senescence or drying is involved in this conversion. The conversion loci were amplified in all studied 24 taxa, and sequenced in 6 selected species that contrast in conversion phenotype. Transcript accumulation of the responsible loci in fresh versus dried leaves of low or nonconverter N. gossei, N. excelsior and N. benthamiana maintained a steady level or a slight increase, but increased by 3 fold in cured leaves of the high converter N. goodspeedii, N. velutina and N. cavicola. This indicates the presence of functional loci that are triggered by curing only in high ii converter species and poses a potential risk for chewers of these species due to their greater potential for nornicotine production.The release of nicotine from the leaves of N. gossei was compared to that from a Swedish snus, the CORESTA reference smokeless tobacco (CRP2), and Nicabate chewing gum. A model buccal cavity system was developed and three different chewing conditions, performed manually were tested over 120 minutes: no chewing action, initial chewing and chewing at 15-minute intervals. To simulate the effect of alkaline wood ash, the effect of alkaline pH on the release of nicotine from N. gossei dry leaves was also evaluated. Samples ...

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Section: Discussionmentioning

confidence: 99%

Section: Alkaloid Analysismentioning

confidence: 99%

Section: Alkaloid Analysismentioning

confidence: 99%

Section: Nicotine and Other Alkaloids Analysis With Hplc-uvmentioning

confidence: 99%

Section: Quantification Of Nicotine and Other Alkaloidsmentioning

confidence: 99%

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Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Moghbel¹

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Plant‐derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

Schwestka

Tschofen

Vogt

et al. 2020

Biotech & Bioengineering

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The encapsulation of biopharmaceuticals into micro‐ or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N‐terminal sequence of the maize storage protein, γ‐zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N‐terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration‐based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen‐presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.

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Development of an HPLC‐DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts

Xin

Zhang

et al. 2018

Biomedical Chromatography

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For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC-DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and tetrahydropalmatine), which were the main active chemical constituents of this plant and belong to four kinds of isoquinoline-type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r > 0.9975), precision (intra-day RSD < 4.8% and inter-day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC-DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti-inflammatory and analgesic.

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A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

Cited by 25 publications

References 29 publications

Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

Plant‐derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

Development of an HPLC‐DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts

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