Docstoc

metodo analitico- allegato II

Document Sample
metodo analitico- allegato II Powered By Docstoc
					                                            Brussels,
                                      12, June, 2009
                                                        DG- SANCO




                       STANDING COMMITTEE
                       STANDING COMMITTEE
                        ON THE FOOD CHAIN
                        AND ANIMAL CHAIN
                        ON THE FOODHEALTH
                        AND ANIMAL HEALTH

                  AMADEO R. FERNÁDEZ-ALBA
Michelangelo Anastassiades and Amadeo R. Fernández-Alba   CRL-FV
   Nicotine Analysis




Michelangelo Anastassiades and Amadeo R. Fernández-Alba
NICOTINE-Uses
Has been used as a pesticide since at least the 15th century.
• Insecticide with predominantly respiratory action, but also slight
  contact and stomach action
• Was used on a range of crops, including fruit, vines, vegetables, and
  ornamentals to control aphids, thrips, whitefly, and other insects
• Its main use was in greenhouses or enclosed spaces, where a smoke
  canister was set and allowed the nicotine to fill the space
• In the past approved in EU (also for organic farming).
• Non-inclusion in Annex I (10 January 2009).phased out by June 2010
• In US registrations for food use were cancelled in 1994


       Michelangelo Anastassiades and Amadeo R. Fernández-Alba
                        NICOTINE-TOXICOLOGY
                        •High levels of nicotine, when ingested,
                        are very toxic and consuming greater than
                        60mg (the amount contained in four
                        cigarettes) is potentially lethal in adults.

                        NICOTINE-REGULATIONS
                        396/2005/EC : Default at 0.01 mg/kg
                        But, 99% of wild mushrooms do not meet
                        this level ...

NOTE: cross-contamination of low levels of nicotine within
the lab to be considered when evaluating residues!
      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
     Nicotine in Mushrooms
     Background Information




Michelangelo Anastassiades and Amadeo R. Fernández-Alba
Nicotine in Mushrooms - Background Information:
• In 2008 high levels of Nicotine detected in mushrooms at CVUA Sigmaringen
• CVUA Stuttgart was consulted for confirmation (LC-TOF , LC-MS/MS  GC-MS )
• Since then many findings by various labs in various mushrooms mainly from China
• Most affected dried porcini (Boletus edulis) but also truffles and chanterelles
• Porcini are reported to be not cultivable
• China is by far biggest producer (Yunnan) and biggest exporter to EU (~80% of imports)
• Chinese authorities say : tobacco is also widely cultivated in Yunnan
• Nicotine is a naturally occurring alkaloid in tobacco (Nicotiana tabacum) where it occurs
  at concentrations ranging from 2% to 8%
• A possible link between tobacco and mushrooms has to be checked, both are dried so
  cross-contamination in drying/packing sites may be an issue
• Chinese authorities report lower levels than those found in Europe
• Intentional use of nicotine as pesticides is also speculated
• Knowledge exchange Anastassiades and Amadeo R. COM
        Michelangelo of labs is initiated by Germany & Fernández-Alba
Risk assessment
In 2008 German authorities (BfR) issue a first risk assessment report concerning nicotine in
mushrooms following a request from the Federal State of Baden Württemberg
The Confed. of the Food and Drink Industries (CIAA) informs COM that wild mushrooms do
not meet MRL (176 samples with levels between 0.21 - 9.9 mg/kg; mean 0,23 mg/kg).
COM asks EFSA to analyze the risks of eating wild mushrooms (27 April 2009)
EFSA issues a risk assessment statement (08 may 2009):
http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902521498.htm
EFSA: ARfD : 0.0008 mg/kg body weight; ADI: 0.0008 mg/kg b.w. per day
Mean conc. In mushrooms of 0.23 mg/kg exceeds ARfD for Italian consumers 2-fold (Italy
has highest consumption of wild mushrooms, especially porcini)
EFSA : The safe concentration of nicotine in fresh mushrooms (highest level not exceeding
ARfD) was calculated to be 0.036 mg/kg (proposed MRL) = 0.32 mg /kg in dried mushrooms.
EFSA:”further monitoring was needed to set precise safe levels”.
EFSA: "Any effects of eating contaminated wild mushrooms are likely to be mild and would
be short term, possibly including increased heart rate, dizziness and headaches,"

         Michelangelo Anastassiades and Amadeo R. Fernández-Alba
NICOTINE PROPERTIES:                    (K. Chamberlain et al., Pestic. Sci., 47, 265 (1996)
• Basic:
pKa1 = 3.1; pKa2 = 8.2
(i.e. predominantly protonated at pH<8.2 and double protonated at pH<3.1)
• Polar:
logP = 0.93 (25 °C/unionised) , the lower the pH the lower the logP
• Volatile:
 Pvap = 5.6 Pa (25 °C). Evaporation losses reduced at low pH (ionized)


                           N           CH3
                                       N
                               H

      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
LC-TOF-MS             Identification of Nicotine in mushroom extract spiked at 10 µg/kg

            ESI +                               Total ion chromatrogram




                                 Nicotine      Extracted ion chromatrogram
                                                    163.1229 ± 0.1 m/z




                [M+H]+    163.1229

                                 Nicotine

            Nicotine F1
              132.0808
                   [C9H10N]+



      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
LC-TOF-MS        Identification of Nicotine-methyl d3 in mushroom extract spiked at10 µg/kg


                                                             Total ion chromatrogram
            ESI +




                                                           Extracted ion chromatrogram
                                     Nicotine-methyl d3
                                                                166.1418 ± 0.1 m/z


                                        m/z: 166.0859
                                        Interference




                  [M+H]+ 166.1418            Nicotine-methyl d3
                 [C10H12 D3N2]+

             Nicotine-methyl d3 F1
                     132.0810




      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
x104
   7    1                                                                                                                      1

  6.5
   6                                    Nicotina
  5.5                                   Area:1.06x106
   5
  4.5
   4
  3.5
   3
  2.5
   2
  1.5                                                                   Cotinina
   1                                                                    Area:3.25x103
  0.5

            1   2   3   4   5   6   7     8   9    10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
                                                                  Counts vs. Acquisition Time (min)




            Michelangelo Anastassiades and Amadeo R. Fernández-Alba
QuEChERS – Optimisation of pH at extraction/partitioning step
                           Recovery of Nicotine with QuEChERS - at different pH
                         Recovery for Nicotine from fresh mushrooms pH-dependant

               100
                                               (spiked residues)
               90

               80
                               Natural pH
               70
Recovery (%)




               60
               50
               40

               30
               20

               10
                                                      LC-QToF, ISTD TPP
                0
                     6               7            8                9        10     11

                                            pH-Value before extraction
Using Nicotine D3 as ISTD losses were compensated (rec. 93 - 120 %)
Nicotine D3 was added at 100ppb and contained 1.8 % unlabeled Nicotine.
Thus ca. 2ppb Nicotine also added to sample
This should be kept in mind to avoid false positive interpretations !!!
                         Michelangelo Anastassiades and Amadeo R. Fernández-Alba
Extraction of incurred residues from Dried Porcini
(QuEChERS pH 10, variation of Temperature and Time)
                 Extraction rates of Nicotine from Porcini using QuEChERS
                                      (incurred residues)
area/area ISTD

 2,0


 1,5


 1,0


 0,5


 0,0
        1 min (RT)     10 min (RT)   30 min (RT)   1 min Ultra-   20 min (60°C)   20 min (90°C)
                                                   Turrax (RT)
                                                                           extraction time


         Michelangelo Anastassiades and Amadeo R. Fernández-Alba
Extraction of incurred residues from Dried Porcini
(QuEChERS, variation of pH)
             Extraction rates of Nicotine from Porcini using QuEChERS
                                  (incurred residues)
 area/area ISTD
 2,5


  2


 1,5


  1


 0,5


  0
          natural         8             9             10            11
                                                                   pH-Value

       Michelangelo Anastassiades and Amadeo R. Fernández-Alba
                                       10 g Fresh mushrooms or
   Nicotine                         2 g dried sample + 10 mL water
QuEChERS
  QuEChERS
*2.5% NH3 in the standar solution         Bring pH to 10-11
     by CRL-SRM                        by addition of NaOH 5N

                                      Add 4 g MgSO4+ 1g NaCl

            Shake 1 min by hand
                                          Centrifugue 5 min.
                                            at 3500 r.p.m.
                                                                      Take alicuot
                                      Add 150 mg MgSO4 anh.
                                     + 50 mg PSA per mL extract
                Shake for 30s
                                          Centrifugue 5 min.             Validation (n=5)
                                            at 3500 r.p.m.           using Champignon 10 g
                                                                     Mean Recovery: 106%
                                       Take aliquot and acidify
                                      w. 5% Formic Acid to pH~5            RSD: 4,1%
                                        Analysis via GC or LC

        Michelangelo Anastassiades and Amadeo R. Fernández-Alba
Impact of d-SPE Cleanup with PSA

         Amount of co-extractives following extract evaporation (mg/mL)
                       (Extract of dried porcini 2g/10 mL)
 mg/mL
5
4
3
2
1
0
                Raw Extract                        After Cleanup w. d-SPE




     Michelangelo Anastassiades and Amadeo R. Fernández-Alba
        Nicotine                           15 g Sample
QuEChERS
      QuEChERS
                                                                   Add 15 ml acetonitrile
                                                                  Extraction at basic pH
                                                                  with acetic acid 1%(v/v)
*2.5% NH3 in the standar solution                                         (pH=9)
       by CRL-FV                                                 and 200 μl TPP (25 ppm)
                                    Add 6 g de MgSO4 anhidrous
                                        + 2.5 g NaAc·3H2O
            Shake automatically
                 in Agitax
                                         Centrifugue 5 min.
                                           at 3700 r.p.m.
                                                                 Take 5 ml alicuot
                                      Add 750 mg MgSO4 anh.
                                          + 250 mg PSA
                       Shake 20s
                        in Vortex
                                         Centrifugue 5 min.        Reconstitution 100%
                                           at 3700 r.p.m.              H2O milliQ

                                     Evaporation with addition
                                      of Take 500µl alicuote
                                         formic acid (pH=3-4)
                                                                 Evaporate to dryness
Pvap 25ºC Nicotine= 5.6 Pa                                       and reconstitute with
Pvap 25ºC Dimetoate= 0.001 Pa                                       AcN:H2O (1:9)
                                             Analysis

        Michelangelo Anastassiades and Amadeo R. Fernández-Alba
  Nicotine EtAc
      Method
EtAcMethod                        10 g Sample


     Shake vigorously 3 s
                                                          Add 10 at basic
                                                       Extractionml EtAc pH
          by hand
                                                              (pH=10)
                                                     and 200 µL TPP (25 ppm)

                                 Add 1.5 g NaCl +
                               8 g MgSO4 anhidrous


      Shake 15 minutes
     automatically in Agitax

                                Centrifugue 5 min.
                                  at 3700 r.p.m.




                                     Analysis
                                    in GC/MS

    Michelangelo Anastassiades and Amadeo R. Fernández-Alba
                                 Method Validation
                                               LC-TOF-MS                  GC-MS/MS(IT)
                                          Mushroom spikes 10 µg/kg    Mushroom spikes 20 µg/kg

                                                          Nicotine                       Nicotine
                                         QuEChERS                    EtAc Method
                                                         QuEChERS                      EtAc Method
                   Quantification with
                                          54 (±10)        105 (±6)    25 (±11)           80 (±7)
                   external standar


 RECOVERIES        Quantification with
R(±RSD)% (n=4)     intenal estándar
                   (Nicotine methyl-      110 (±8)          No                             No
                           d3)



           LOD (µg/kg)
                                                     3                             4
        Limit Of Determination




       Michelangelo Anastassiades and Amadeo R. Fernández-Alba
         Tobacco Crop
                                                Crop duration: 9 months




                                                Acumulation in soil (mg/kg)
                                                Nicotine          2,6633
                                                Cotinine          0,0102


Concentration of Nicotine in tobacco leaves  ~ 20. 000 mg/kg (Dry weight)


    Michelangelo Anastassiades and Amadeo R. Fernández-Alba
CROMATOGRAMA Y ESPECTRO MS/MS NICOTINA GC-MS/MS(IT)




    Michelangelo Anastassiades and Amadeo R. Fernández-Alba
GC-MS/MS (IT)- PCI with methanol           Identification of nicotine in a mushroom extract at 20 µg/kg



                      Total Ion Chromatogram
              Nicotine Full scan CI
              spectrum                                       Precursor ion
                                                             [M+H]+



                   Extracted Ion Chromatogram          Nicotine
                   m/z 106




                                                Nicotine MS/MS
                                                spectrum




       Michelangelo Anastassiades and Amadeo R. Fernández-Alba
Michelangelo Anastassiades and Amadeo R. Fernández-Alba
LC-TOF-MS             Identification of Nicotine in mushroom extract spiked at 10 µg/kg

            ESI +                               Total ion chromatrogram




                                 Nicotine      Extracted ion chromatrogram
                                                    163.1229 ± 0.1 m/z




                [M+H]+    163.1229

                                 Nicotine

            Nicotine F1
              132.0808
                   [C9H10N]+



      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
LC-TOF-MS        Identification of Nicotine-methyl d3 in mushroom extract spiked at10 µg/kg


                                                             Total ion chromatrogram
            ESI +




                                                           Extracted ion chromatrogram
                                     Nicotine-methyl d3
                                                                166.1418 ± 0.1 m/z


                                        m/z: 166.0859
                                        Interference




                  [M+H]+ 166.1418            Nicotine-methyl d3
                 [C10H12 D3N2]+

             Nicotine-methyl d3 F1
                     132.0810




      Michelangelo Anastassiades and Amadeo R. Fernández-Alba
                              Thank you very much
                                for your Attention!!




Michelangelo Anastassiades and Amadeo R. Fernández-Alba

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:0
posted:3/4/2013
language:Latin
pages:26