Quantitation of major tobacco alkaloids by high-performance liquid chromatography

Saunders, James A.; Blume, Denise E.

doi:10.1016/s0021-9673(00)81822-1

Cited by 100 publications

(46 citation statements)

References 17 publications

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“…After heating at 60°C for 15 min, the dansylated amines were extracted by adding 0.5 ml of toluene followed by vortex mixing for 30 s. After an aliquot (400 l) of the toluene was removed, the residue was dried and resuspended in a fixed volume of acetonitrile, which was then injected into the HPLC. The chromatographic conditions for the separation of dansylated N-methylputrescine were as described before (29). The retention time of N-methylputrescine was 24 min.…”

Section: Methodsmentioning

confidence: 99%

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Zhang

Ding

Chai

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

245

126

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Scopolamine is a pharmaceutically important tropane alkaloid extensively used as an anticholinergic agent. Here, we report the simultaneous introduction and overexpression of genes encoding the rate-limiting upstream enzyme putrescine N-methyltransferase (PMT) and the downstream enzyme hyoscyamine 6 ␤-hydroxylase (H6H) of scopolamine biosynthesis in transgenic henbane (Hyoscyamus niger) hairy root cultures. Transgenic hairy root lines expressing both pmt and h6h produced significantly higher (P < 0.05) levels of scopolamine compared with the wild-type and transgenic lines harboring a single gene (pmt or h6h). The best line (T 3) produced 411 mg͞liter scopolamine, which was over nine times more than that in the wild type (43 mg͞liter) and more than twice the amount in the highest scopolamine-producing h6h single-gene transgenic line H 11 (184 mg͞liter). To our knowledge, this is the highest scopolamine content achieved through genetic engineering of a plant. We conclude that transgenic plants harboring both pmt and h6h possessed an increased flux in the tropane alkaloid biosynthetic pathway that enhanced scopolamine yield, which was more efficient than plants harboring only one of the two genes. It seems that the pulling force of the downstream enzyme (the faucet enzyme) H6H plays a more important role in stimulating scopolamine accumulation in H. niger whereas the functioning of the upstream enzyme PMT is increased proportionally. This study provides an effective approach for large-scale commercial production of scopolamine by using hairy root culture systems as bioreactors.Agrobacterium ͉ hyoscyamine 6␤-hydroxylase ͉ putrescine N-methyltransferase ͉ scopolamine ͉ transformation

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Section: Methodsmentioning

confidence: 99%

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Zhang

Ding

Chai

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

245

126

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“…The most common methods include thin layer chromatography [3], capillary electrophoresis [4][5][6], gas chromatography [7][8][9][10], and high performance liquid chromatography (HPLC) [11][12][13][14][15][16][17]. Each of these methods has its advantages.…”

Section: Introductionmentioning

confidence: 99%

Determination of nicotine and three minor alkaloids in tobacco by hydrophilic interaction chromatography-tandem mass spectrometry

Taujenis¹,

Olšauskaitė²,

Padarauskas³

2015

Acta Chromatographica

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Summary.A reliable, sensitive and rapid method for determination of nicotine and three minor alkaloids (cotinine, anabasine and nornicotine) in tobacco by ultra-high performance liquid chromatography in hydrophilic interaction chromatography mode coupled with tandem mass spectrometry (HILIC-MS/MS) has been established. HILIC separation was performed on a BEH HILIC column using isocratic elution at 0.5 mL/min with acetonitrile:water (85:15, v/v) mobile phase containing 5 mmol/L ammonium acetate (pH 5.00). Separated analytes were determined by electrospray ionization MS/MS in the positive ion mode using multiple reaction monitoring. Alkaloids from tobacco were extracted in an ultrasonic bath for 10 min with acetonitrile:water mixture (8:2, v/v) containing 5 mmol/L ammonium acetate (pH 5.00). Limits of quantification were 10 µg/g for cotinine, 20 µg/g for anabasine and nornicotine, and 30 µg/g for nicotine. Mean recoveries from tobacco ranged between 94.8% and 104.1% for different analytes with relative standard deviations within 5%. The performance of the proposed method was tested for the extraction and determination of the four alkaloids in cigarette tobacco fillers, and satisfactory results were achieved.

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“…Photosynthetic (17). The content of alkaloids (mainly nicotine) was determined from dried leaves according to the method described by Saunders and Blume (25). Leaves were ground and 500 mg of dry powder was extracted with 10 mL of 25 (20,uL injection volume), which had the same retention time of authentic nicotine (Merck), were integrated by comparison with an external standard calibration curve (r = 0.995).…”

mentioning

confidence: 99%

“…The content of alkaloids (mainly nicotine) was determined from dried leaves according to the method described by Saunders and Blume (25). Leaves were ground and 500 mg of dry powder was extracted with 10 mL of 25 (20,uL injection volume), which had the same retention time of authentic nicotine (Merck), were integrated by comparison with an external standard calibration curve (r = 0.995). Stomatal density and index were counted from several randomly chosen fields (400-fold magnification) on epidermal strips obtained from both the adaxial and abaxial tobacco leaf surfaces.…”

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confidence: 99%

Leaf Photosynthesis and Respiration of High CO₂-Grown Tobacco Plants Selected for Survival under CO₂ Compensation Point Conditions

Delgado¹,

Azcón-Bieto²,

Aranda³

et al. 1992

Plant Physiol.

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Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO2 atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO2 levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO2 assimilation rate per unit area measured in the growth room at high CO2 was not higher in the selected plants. The CO2 assimilation rate versus intercellular CO2 curve and the CO2 compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO2 compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The altemative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO2 survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.The relationship between leaf photosynthesis rate and plant productivity is difficult to establish even though dry matter accumulation obviously reflects the efficiency of the plant photosynthetic processes (14,23). It is well accepted that total canopy photosynthesis during the growth period is closely related to yield, as has been reported in several species (4,29). However, many attempts to improve leaf photosynthesis per unit area by genetic selection did not result in any substantial increase of crop productivity (23,24,27 Haploids were obtained from ethylmethylsulfonate-treated anthers. These selected haploid plants showed a higher growth rate, greater leaf area, and plant production under greenhouse conditions. The floral buds of surviving haploids were treated with colchicine, and diploid fertile flowers were developed. After self-pollination, viable seeds were obtained from five selected lines.A series of field assays showed consistent advantage of selected SP lines2 in plant dry matter production in comparison with the unselected source cultivar (20). These field assays also showed a consistent increase in plant leaf area in those lines. Nevertheless, CO2 assimilation rate of detached leaves did not show substantial differences among selected lines and the control one (12,13

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Quantitation of major tobacco alkaloids by high-performance liquid chromatography

Cited by 100 publications

References 17 publications

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Determination of nicotine and three minor alkaloids in tobacco by hydrophilic interaction chromatography-tandem mass spectrometry

Leaf Photosynthesis and Respiration of High CO₂-Grown Tobacco Plants Selected for Survival under CO₂ Compensation Point Conditions

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Quantitation of major tobacco alkaloids by high-performance liquid chromatography

Cited by 100 publications

References 17 publications

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures

Determination of nicotine and three minor alkaloids in tobacco by hydrophilic interaction chromatography-tandem mass spectrometry

Leaf Photosynthesis and Respiration of High CO2-Grown Tobacco Plants Selected for Survival under CO2 Compensation Point Conditions

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Leaf Photosynthesis and Respiration of High CO₂-Grown Tobacco Plants Selected for Survival under CO₂ Compensation Point Conditions