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Determination of Nectar Nicotine Concentration in N. attenuata
烟草花蜜中尼古丁含量的测定   

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Abstract

In this protocol, the determination of the nicotine concentration in nectar of Nicotiana attenuata is described. This method is applicable for the investigation of small amounts of nectar (above 1 μl). It is a high-throughput protocol optimized and streamlined for one skilled person to process approximately 100 nectar samples per day.

Materials and Reagents

  1. Nicotiana attenuata
  2. Nicotine-D3 (Cambridge Isotope laboratories, catalog number: DLM-1818-0.5 )
  3. Ammonium hydroxide 25% (Fluka, catalog number: 44273-100 ML-F )
  4. Methanol (Merck KGaA, catalog number: 1.06007.2500
  5. Solvent A (see Recipes)
  6. Solvent B (see Recipes)

Equipment

  1. Varian 1,200 triple quad LC-MS system
  2. Phenomenex Gemini NX 50 x 2 mm column, 3 μm C18 (Phenomenex, catalog number: 593381-9 )
  3. 1.5 ml Eppendorf tubes
  4. 1.5 ml GC vials (www.wicom.de)
  5. 25 μl glass capillary (BLAUBRAND®, intraMark) (BRAND GMBH + CO KG, Wertheim, catalog number: 708722 )

Procedure

  1. The best time to collect nectar from N. attenuata is in the morning between 4:00 and 7:00 am, as the maximum nectar accumulation is reached around 4:00 am and remains stable until the sun rises or the lights in the glasshouse are switched on. As flowers of N. attenuata remain open for two nights, one must make sure that the same floral stage is used for collections. Usually newly opened flowers are used, which requires removal of all open flowers 24 h before the actual sampling in order to be sure to collect floral nectar from the same floral stage-- one- and two-day-old flowers are hard to tell apart from each other.
  2. Nectar from flowers is collected by inserting a clean 25 μl glass capillary into the corolla tube until it reaches the base of the nectaries (Figure 1). With practice, a complete nectar sample can be obtained by holding the capillary with one hand and the corolla tube with the other and removing the tube against the counter-pressure of the inserted capillary (Video 1). This technique requires some training in order to avoid damage to the ovary and contamination of the nectar. Alternatively, the complete corolla can be detached from the rest of the flower by simply pulling on the corolla tube. The nectar remains at the base of the tube from which it can be collected with a 25 μl glass capillary (Video 2).

    Figure 1. Floral nectar collection with a glass capillary

    Video 1. Nectar collection from N. attenuata flowers - method 1
    Video 2. Nectar collection from N. attenuata flowers - method 2
  3. Nectar of single flowers is collected separately in 1.5 ml Eppendorf tubes. 2 μl or 1.5 μl (for flowers with low nectar volume) of nectar are transferred into a new tube containing 400 μl water and 20 ng of the internal standard nicotine-D3.
  4. Particles are removed by centrifugation (10 min at 12,000 x g, at 4 °C) and the supernatant is transferred into 1.5 ml GC vials.
  5. 10 μl of the solution are analyzed using a Varian 1,200 triple quad LC-MS system (http://www.varianinc.com) connected to an ESI source with a capillary voltage of 35 V, solvent A ; Solvent B. The gradient (min/% B): 0/5; 0.5/5; 2/80; 6.5/80; 8.5/5; 10/5 (Figure 2) is used with a Phenomenex Gemini NX 5 x 2 mm column, particle size 3 μm. The pH of the mobile phase is the most important parameter which determines nicotine's retention on the column. The starting conditions focus nicotine on the column, while sugars and salts are eluted. The steep gradient guarantees sharp nicotine peaks. The time for reconditioning the column is strongly instrument dependent. The short reconditioning time in our program is adapted to accommodate our slow auto sampler. The transition of the precursor ion nicotine [M + H]+ = 163 and nicotine-D3 [M + H]+ = 166 to the fragment (m/z) = 130 at a collision energy of 14.5 V is recorded for quantification. Quantification was achieved by isotope dilution and can be calculated by the following formula: amount nicotine (ng/μl nectar) = area of targeted compound/area ISD (internal standard = nicotine - D3) x amount ISD (ng/μl nectar).


    Figure 2. Methanol gradient.

Recipes

  1. Solvent A
    Add 1 ml of 25% ammonium hydroxide solution to 1 L Milipore H2O
    Mix carefully, adjust pH to 10 [according to the pka of Nicotine, which is 8.05 (Fujita et al., 1971) the pH should be 10 or higher, to maintain nicotine in its neutral form. The upper pH is limited by the column chemistry and may be adjusted with concentrated ammonia or a few drops of 1:10 diluted formic acid].
  2. Solvent B
    Methanol

Acknowledgments

This work was supported by the Max Planck Gesellschaft. The protocol was adapted from the publication: Kessler et al. (2012).

References

  1. Fujita, T., Nakajima, M., Soeda, Y. and Yamamoto, I. (1971). Physicochemical properties of biological interest and structure of nicotine and its related compounds. Pesticide Biochem Physiol 1(2): 151-162.    
  2. Kessler, D., Bhattacharya, S., Diezel, C., Rothe, E., Gase, K., Schottner, M. and Baldwin, I. T. (2012). Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata. Plant J 71(4): 529-538.    

简介

在该方案中,描述了烟草的花蜜中尼古丁浓度的测定。 这种方法适用于少量花蜜(高于1微升)的调查。 它是一种高通量协议,对于技术人员每天处理大约100种花蜜样品而言是优化的和简化的。

材料和试剂

  1. Nicotiana attenuata
  2. 尼古丁-D3(剑桥同位素实验室,目录号:DLM-1818-0.5)
  3. 氢氧化铵25%(Fluka,目录号:44273-100ML-F)
  4. 甲醇(Merck KGaA,目录号:1.06007.2500)
  5. 25μl玻璃毛细管(BLAUBRAND ,intraMark)(BRAND GMBH + CO KG,Wertheim,德国,目录号:708722)
  6. 溶剂A(参见配方)
  7. 溶剂B(参见配方)

设备

  1. Varian 1200三重四极杆LC-MS系统
  2. Phenomenex Gemini NX 50×2mm柱,3μmC18(Phenomenex,目录号:593381-9)
  3. 1.5ml Eppendorf管
  4. 1.5 ml GC小瓶( www.wicom.de
  5. 25μl玻璃毛细管(BLAUBRAND ,intraMark)(BRAND GMBH + CO KG,Wertheim,目录号:708722)

程序

  1. 从 N收集花蜜的最佳时间。衰减在上午4:00和7:00之间是上午,因为最大的花蜜积聚在上午4:00左右达到,并保持稳定,直到太阳升起或玻璃房中的灯打开。作为 N的花。 attenuata 保持开放两晚,必须确保相同的花卉舞台用于收藏。通常使用新打开的花,其需要在实际取样之前24小时去除所有开花,以确保从相同的花阶段收集花蜜 - 一天和两天的花难以区分开彼此。
  2. 花蜜通过将一个干净的25微升玻璃毛细管插入花冠管收集,直到它到达蜜饯的基地(图1)。在实践中,可以通过保持获得完整的花蜜样品 一只手的毛细管和另一只手的花冠管,并克服插入的毛细管的反压力移除管(视频1)。这种技术需要一些训练,以避免损害卵巢和污染的花蜜。或者,通过简单地拉动花冠管,可以将完整的花冠从花的其余部分分离。花蜜保留在管的基部,其可以用25μl玻璃毛细管收集(视频2)。

    图1.带有玻璃毛细管的花蜜集合

    视频1.从 N。 attenuata 花 - 方法1
                                                                  
    视频2.从 N的花蜜收集。 attenuata 花 - 方法2
                                                                           
  3. 单花的花蜜分别收集在1.5ml Eppendorf管中。将2μl或1.5μl(对于具有低花蜜体积的花)的花蜜转移到含有400μl水和20ng内标尼古丁-D3的新管中。
  4. 通过离心(在12,000×g,在4℃下10分钟)除去颗粒,并将上清液转移到1.5ml GC小瓶中。
  5. 使用Varian 1,200三重四极杆LC-MS系统( http://www.varianinc.com)分析10μl的溶液,连接到具有35V毛细管电压的ESI源,溶剂A;溶剂B.梯度(min /%B):0/5; 0.5/5; 2/80; 6.5/80; 8.5/5; 10/5(图2)与Phenomenex Gemini NX 5×2mm柱一起使用,粒径为3μm。流动相的pH是确定尼古丁在柱上的保留的最重要的参数。起始条件将尼古丁聚焦在柱上,而糖和盐被洗脱。陡峭的梯度保证了尖锐的尼古丁峰。重新调整色谱柱的时间与仪器有很大关系。我们的程序短的修复时间适应我们的慢自动采样器。前体离子烟碱[M + H] + sup = 163和尼古丁-D3 [M + H] + sup/+ = 166的跃迁到片段(m/z )= 130,在14.5V的碰撞能量下记录用于定量。通过同位素稀释实现定量并且可以通过下式计算:量尼古丁(ng /μl花蜜)=目标化合物的面积/面积ISD(内标=尼古丁-D3)×量ISD(ng /μl花蜜) br />

    图2.甲醇梯度。

食谱

  1. 溶剂A
    将1ml 25%氢氧化铵溶液加入1L Milipore H 2 O中 小心混合,将pH调节至10 [根据尼古丁的pka(其为8.05(Fujita等人,1971)),pH应当为10或更高,以维持尼古丁的中性形式。 上部pH由柱化学限制,并且可以用浓氨或几滴1:10稀释的甲酸调节。
  2. 溶剂B
    甲醇

致谢

这项工作得到了马克斯普朗克协会的支持。该方案改编自出版物:Kessler等人(2012)。

参考文献

  1. Fujita,T.,Nakajima,M.,Soeda,Y。和Yamamoto,I。(1971)。 尼古丁及其相关化合物的生物学利益和结构的物理化学性质。 Pesticide Biochem Physiol 1(2):151-162。    
  2. Kessler,D.,Bhattacharya,S.,Diezel,C.,Rothe,E.,Gase,K.,Schottner,M.and Baldwin,I.T。 花蜜尼古丁的不可预测性促进了蜂鸟在烟草中的突变 。 Plant J  71(4):529-538。    
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Rothe, E., Schöttner, M., Kessler, D. and Baldwin, I. . (2013). Determination of Nectar Nicotine Concentration in N. attenuata. Bio-protocol 3(8): e451. DOI: 10.21769/BioProtoc.451.
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