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Analysis of 16 amino acids in tobacco by ion pair LC ESI MS MS without derivatization M. Bouzige, L. Barbet, V. Ramon, F. Bourcier, B. Vidal, B. Duméry ALTADIS France Introduction Amino acids (AA) are chemical markers of tobacco type, maturity, origin... Two drawbacks for their analysis : lack of chromophoric agents and polarity Derivation before their detection by UV or fluorescence : This step is often time consuming and can lead to extra errors No derivation by using ionic exchange or ion pair chromatography : problems of specificity Mass spectrometry can overcome this problem TSRC 2003 - Norfolk LC MS MS analysis of Amino Acids Work of the Organic and Analytic Chemistry Institute of Orléans University (ICOA) Determination of 20 underivatized proteinic amino acids by ion pairing chromatography and pneumatically assisted electrospray mass spectrometry. • P Chaimbault, K. Petritis, C. Elfakir, M. Dreux. • J. Chromatogr. A 855 (1999) 191 - 202. Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry. • K. Petritis, P. Chaimbault, C. Elfakir, M. Dreux. • J. Chromatogr. A 896 (2000) 253 - 263. TSRC 2003 - Norfolk Purpose Adapt the method to tobacco Extraction Separation Ion pair HPLC Detection ESI MS MS Study its feasibility, performance Compare its results with our traditional method This method allows to analyze minor amino acids we don’t currently analyze Study the interest of these new amino acids for the characterization of tobaccos TSRC 2003 - Norfolk MS MS Conditions Apparatus : Perkin Elmer Sciex API 300 triple quadrupole Ionization : electrospray, positive mode Curtain and collision gas : Nitrogen According to the publications, we verified MS parameters for ionization Fragmentation products for each AA Best collision energy suitable for all the AA : 20 eV Scan mode : Multiple Reaction Monitoring Compounds Alanine (ALA) Arginine (ARG) Asparagine (ASN) Aspartic Acid (ASP) Glutamic Acid (GLU) Glutamine (GLN) Lysine (LYS) Histidine (HIS) Leucine (LEU) Isoleucine (ILE) Methionine (MET) Phenylalanine (PHE) Proline (PRO) Serine (SER) Threonine (THR) Tryptophane (TRP) Tyrosine (TYR) Valine (VAL) Q1/Q3 90/44 175/175 133/87 134/88 148/130 147/130 156/110 132/86 150/104 166/120 116/70 106/60 120/74 205/188 182/165 118/72 TSRC 2003 - Norfolk Amino acids H 2N CH 2 CONH 2 H2N CH COOH Asparagine (ASN) 133 / 87 CH2CH2COOH CH CH3 Aspartic acid (ASP) 134 / 88 H2N COOH CH CH COOH H2N NH2 CH2COOH Alanine (ALA) 90 / 44 CH2CH2CONH2 CH COOH N H COOH Glutamic Ac. (GLU) 148 / 130 Glutamine (GLN) 147 / 130 COOH Proline (PRO) 116 / 70 NH H2N COOH CH2OH CH COOH H2N NH CH N H 2 COOH Serine (SER) 106 / 60 Phenylalanine (PHE) 166 / 120 Arginine (ARG) 175 / 175 TSRC 2003 - Norfolk NH2 Amino acids (2) H2N CH(CH3)2 CH H2 C H N 2 H2N HCH2CH3 C CH COOH COOH Valine (VAL) 118 / 72 NH2 CH N CH COOH H2N N Histidine (HIS) 156 / 110 H2N CH2CH(CH3)2 CH CH3 Isoleucine (ILE) 132 / 86 H2N CH COOH COOH Lysine (LYS) 147 / 130 Leucine (LEU) 132 / 86 N H COOH Tryptophane (TRP) 205 / 188 OH NH2 COOH C H2 H2N CH2CH2SCH3 CH COOH CH H2N CH CH3 COOH Glycine (GLY) 76 / 30 Methionine (MET) 150 / 104 Threonine (THR) 120 / 74 TSRC 2003 - Norfolk LC Conditions Column : C18 silica. Lichrocart (Purospher) 125 x 2 mm, 5 µm Mobile phase : A = Heptafluorobutyric acid (HFBA) 0.1 % / ACN 97.5 / 2.5 B = ACN Flow rate : 0.2 mL/min, split 1/10 before ion source Gradient elution 120 100 100 Necessity to add a washing step with pure ACN between two runs % B 80 60 40 30 30 Equilibrating time 30 min 20 0 0 5 10 15 20 T (min) 25 30 35 TSRC 2003 - Norfolk Sample preparation Standards Tobacco Necessity to add HFBA in the sample to improve the formation of ion pair of HIS, ARG, LYS at the injection Extraction 1 g of tobacco powder in 50 mL of water Internal Standard : choice of AA not detected in a series of tobaccos : methionine & threonine Dilution of the extract by 2 Addition of HFBA : 0.3 % of HFBA are necessary to obtain symmetric peaks for HIS, ARG, LYS. Methionine chosen as Internal Standard. Threonine was rejected due to matrix effect on tobacco. TSRC 2003 - Norfolk Analysis of a Burley tobacco Superposition of the 18 SRM Nicotine PRO PHE ASP ASN MET GLN SER ALA GLU HIS VAL LYS TRP ILE LEU ARG TYR Time min Specificity of MS MS allows to decrease the separation TSRC 2003 - Norfolk Matrix Effect Tobacco (Burley, FlueCured, Oriental) FC Tryptophane (TRP) Int, Ratio 1,6 Filetype FC Standard 1,2 5 spikes 3 repetitions 2 Linearity of the response 3 Comparison with standard 4 Recovery calculation 0,8 0,4 0 0 1 2 3 4 Amino acid concentration /100 (mg/kg of dry tobacco) Blank 1 Linearity of standards 5 concentrations 3 repetitions TSRC 2003 - Norfolk Recoveries AA ASN SER ASP GLU GLN ALA PRO HIS LYS VAL ARG ILE LEU PHE TRP TYR Burley Flue Cured Oriental Recovery (%) Recovery (%) Recovery (%) 98 106 109 87 102 104 103 103 105 99 84 93 98 103 103 94 102 106 97 102 107 108 110 103 103 98 98 81 81 82 102 95 94 89 103 100 86 100 97 100 109 102 105 103 102 86 82 95 Linearity of standards and spiked tobaccos always validated Some matrix effects but acceptable TSRC 2003 - Norfolk Repeatability & Limit of quantification Analysis of a Burley tobacco during 6 weeks over 4 months AA ALA ARG ASN ASP GLN GLU HIS ILE LEU LYS PHE PRO SER TRP TYR VAL CV % 7 15 13 10 18 13 15 9 14 17 15 13 11 13 20 14 Estimation of LOQ, comparison with the mean value observed after analysis of 106 various tobaccos Necessity to calibrate every 3 days Deuterated Internal Standards should improve the repeatability Limits of quantification compatible with the analysis of tobaccos LOQ mg/Kg 6 3 13 3 3 25 4 10 2 3 0.3 2 4 0.5 4 2 Mean 411 149 5424 5443 713 561 271 40 49 192 464 3572 275 276 71 82 TSRC 2003 - Norfolk Comparison with our current method New Method Current Method Extraction Extraction Ion pair HPLC ESI MS MS Analysis of tobaccos Derivation 28 Burleys 38 Flue Cured 13 Oriental Reverse phase HPLC UV or Fluorimetry Asn,Asp, Glu, Gln, Ser, Pro Asn,Asp, Glu, Gln, Ser, Pro Ala, Arg, His, Tyr, Val, Leu, Ile, Trp, Phe, Lys Ala, Arg, His TSRC 2003 - Norfolk Comparison with our current method AA R2 adjusted Global By FC O ASN 91.6 91 81 99 SER 77 84 51 96 ASP 96.6 84 84 99 GLN 80 80 67 75 GLU 79 95 9 93 PRO 91 95 77 64.6 ARG HIS ALA 95 90.9 16.6 92 94 62 58 78 3 18000 Slope Global 1.03 1.13 1.16 1.02 0.94 0.88 96 94 32 1.16 1.05 0.3 1400 Comparison of ASP results 16000 Comparison of HIS results 1200 14000 1000 ESI LC MS MS ESI LC MS MS y = 1.1608x + 10.45 12000 2 R = 0.9667 10000 8000 6000 800 600 y = 1.0524x - 1.3003 400 2 R = 0.9094 4000 200 2000 0 0 200 400 600 800 1000 0 0 2000 4000 6000 8000 10000 Current method 12000 14000 16000 -200 Current method TSRC 2003 - Norfolk 1200 Comparison with our current method Similar results Shorter analysis time no derivation faster separation Lower limits of detection for amino acids : at least a factor 10 Possibility of analyzing new amino acids Reproducibility should be improved TSRC 2003 - Norfolk Application to the characterization of tobaccos Extraction Separation Ion pair HPLC Detection ESI MS MS Analysis of tobaccos 28 Burleys 27 Dark 38 Flue Cured 13 Oriental New AA compared to our current method Do these new AA improve the characterization of tobaccos ? TSRC 2003 - Norfolk Differences between tobacco types Box and Whiskers Box and Whiskers BRU TYPE BUR ORI VIR BUR ORI VIR 0 300 600 900 1200 0 1500 30 HIS 60 90 120 150 ILE 900 Mean DARK Mean BURLEY Mean ORIENTAL Mean FC 800 Concentration in mg/kg of dry tobacco TYPE BRU 700 600 500 400 300 200 100 0 HIS TRP PHE ARG VAL LYS TYR ILE LEU TSRC 2003 - Norfolk Selection of the best amino acids 5.30 Component 2 - Inertia: 23.9% BRU Factorial plane Correlation circle ASP 3.10 BRU BRU 0.90 -1.30 BRU BRU BRU BRU BRUBRU BRU BY BRU BRU BRU BRU BRUBY BRU BRU BRU BRU BY BRU BY BRU BY BRU BY BY BRU BY VIR VIRVIRVIRVIR VIR VIR VIR VIR VIR VIRVIR VIR VIR VIR VIR VIR VIR VI VIR VIR VIR ORI VIR VIRVIR VI VIRR VIR VIR R VIR ORIVIR VIR VIR ORI ORI VIR VIR VIR VIR ORI OR ORI ORI ORI I VIR LYS BRU BY BY BRU BY BY BY BY BY BY BY BY BY BY BY VAL ASN BY BY BY BY BY TRP HIS ALA BY BY ORI PRO ORI ORI ORI -3.50 -3.00 -0.50 2.00 4.50 7.00 Component 1 - Inertia: 53.8% Ala, Trp, His, Val, Lys bring some information AA separate well Burley tobaccos TSRC 2003 - Norfolk Conclusion Amino acids can be analyzed by ESI LC MS MS without derivation in tobaccos Results are comparable to our current method The major amino acids can be analyzed within 6 minutes Some new amino acids can be better detected A lot of minor amino acids are Burley markers Ala, Trp, His, Val, Lys help discriminating tobacco types Study the interest of the minor amino acids for the characterization of tobaccos from the same tobacco type (Burley) TSRC 2003 - Norfolk