NON-MAMMALIAN METABONOMICS: AN ANALYSIS OF COFFEE BEAN EXTRACTS

Jennifer H. Granger, Robert S. Plumb, and James N. Willis Waters Corporation, Milford, MA , USA

LC ConditionsLC System: Waters® ACQUITY UPLC™ System Column: 2.1 x 100 mm, 1.7 µm ACQUITY

UPLC BEH C18 ColumnFlow Rate: 0.600 mL/min. (split to ~0.150

mL/min. into mass spectrometer)Gradient: 50–95% B in 30 min., where

A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile)

Injection Volume: 10 µL

MS ConditionsMS System: Waters Micromass® LCT Premier™

Mass Spectrometer Ionization Mode: Positive ion Acquisition Range:100–950 m/z with Woptics™

resolutionCone Voltage: 60 VCapillary Voltage:3000 VScan Time: 0.3 sInterscan Delay: 0.05 sLockSpray™: Leucine Enkephalin 25 fmol/µL at

30 µL/min.

Data AnalysisAll UPLC/MS(TOF) data were processed using Waters MarkerLynx™ Application Manager for MassLynx™ Software. Partial least squares (PLS) analysis was performed using Pirouette Software (InfoMetrix) and an exported peak list from MarkerLynx.

RESULTS AND DISCUSSION

Representative total ion current chromatograms for an extract of Sumatran coffee beans is presented in Figure 1.

INTRODUCTION

Metabonomics and Food ScienceThe science of metabonomics has evolved into a useful strategy to facilitate the differentiation of small molecule profiles in complicated biological matrices such as plasma and urine. This methodology consists of four basic steps: 1) an information-rich analysis, such as LC/MS(TOF), 2) data reduction and alignment to convert acquired spectra into a “statistics-friendly”format, 3) multivariate statistical analysis (e.g., principal components analysis (PCA) or partial least squares (PLS)), and 4) user review and interpretation of chemometric results. However, the utility of this approach is not limited to the biological arena. In fact, the pattern recognition algorithms that are at the heart of metabonomics can be applied to several areas including industrial batch syntheses, cosmetics and food sciences. Additionally, the superior chromatographic and mass spectrometric resolution and sensitivity achievable with Ultra Performance LC™

(UPLC™) coupled with the Waters Micromass® LCT Premier™ orthogonal time-of-flight mass spectrometer ensures that the maximum amount of information contained in a sample is extracted. In this example, we will implement a UPLC/MS(TOF)-based metabonomics strategy to probe the regional differences of roasted coffee beans.

EXPERIMENTAL CONDITIONS

Sample PreparationCoffee beans from five geographic regions (Africa, Indonesia, and Central, South and North America) were selected for analysis. Five beans from each region were individually extracted with acetonitrile for twelve hours. The resulting extracts were diluted 1:4 with deionized water prior to analysis by UPLC/MS(TOF).

Probing Reasons for Regional DifferencesOne of the key ions contributing to the observed clustering in that of caffeine, m/z = 195.08. The abundances of this ion in each of the coffee bean extracts can be visualized from the trends plot for caffeine in Figure 3.

Figure 3. Trend line for m/z = 195.08 across all of the samples.

From this trend line, it can be noted that coffee beans from Sumatra and Kenya appear to have the highest caffeine content. The mass spectrum of caffeine from a Kenyan coffee bean extract is given in Figure 4. Both the calculated and observed exact mass value for caffeine (MH+ C8H11N4O2, m/z = 195.0882) are identical resulting in a mass error of zero.

Figure 4. Exact mass spectrum for caffeine.

Figure 1. Representative total ion current chromatogram of an extract of Sumatran coffee beans.

Chemometric AnalysisAlthough differences between each of the chromatograms in Figure 1 can be ascertained via visual inspection, a more complete comparison can be made through the use of a pattern recognition algorithm such as PLS. The resulting scores and loadings plots are presented in Figures 2a and 2b.

Figure 2. (a) scores and (b) loadings plots resulting from PLS analysis of UPLC/MS(TOF) data from coffee bean extracts.

Factor1

Factor2Factor3

Sumatra4

Kenya4

Panama2Panama4Panama5

Sumatra2

Sumatra3Sumatra5

Kenya1Kenya2Kenya3

Kenya5

Kona1

Kona2Kona3

Kona4

Brazil1

Brazil2Brazil3

Brazil4Brazil5

Panama1

Factor3

Factor1

Factor2.3_100.114.4_109.080.6_118.0668.2_569.4474.8_570.335.4_122.087.3_130.5305.1_570.42519.7_571.408.6_133.079.7_137.072.4_140.0779.4_572.4811.2_573.40119.9_143.061.6_149.0659.3_574.40419.7_575.41413.4_575.504.4_152.105.3_157.0839.4_576.449

12.0_577.425.4_160.084.9_163.0773.1_578.4113.1_579.4073.1_580.419.4_167.104.4_169.9787.0_581.42217.4_583.435.3_171.086.4_174.095.4_177.0936.1_585.4157.6_586.489.5_179.098

.7_181.1306.1_586.50315.9_587.448.6_187.089.6_191.092.5_193.10310.2_588.5031.2_590.425.3_195.034.6_195.1549.2_591.4059.2_592.4123.1_593.371.3_200.036.5_203.1139.6_593.42013.1_593.471.5_205.100.6_207.15513.8_593.5159.7_594.42813.4_594.516.3_212.019.3_214.0055.1_595.3877.4_595.437.5_216.110.6_219.113.6_222.1376.8_596.44019.8_597.457.4_224.095.3_226.14518.8_597.4539.7_598.448.7_229.139.6_231.117.5_234.15313.2_599.34510.2_599.492.9_236.116.4_238.01613.5_599.5217.8_600.49414.2_601.521.5_243.157.6_246.1245.1_602.4435.1_603.448.5_248.128.5_251.1633.5_605.4342.8_609.41419.8_610.186.4_255.129.5_257.1084.8_610.46615.4_610.542.6_258.12219.3_262.21714.0_611.5424.9_612.3462.1_263.2392.0_265.25113.5_612.54616.9_613.481.4_267.157.6_269.1261.2_270.26415.9_614.5246.2_615.414.5_272.130.6_274.14315.2_615.48613.3_615.50015.2_616.442.7_278.1751.0_279.17513.9_616.49915.7_618.45015.1_280.181.6_281.19015.8_619.4543.5_620.4513.5_621.455

2.0_282.275

.5_284.13215.0_622.41115.2_623.4103.3_285.303.6_289.125.5_292.15015.9_624.9272.9_625.458.6_295.2271.3_296.27914.1_625.53414.5_626.4901.9_297.291.5_299.1991.2_300.20611.4_627.52111.3_628.5251.6_301.296.4_306.1354.9_630.3446.2_631.41916.9_631.490.5_309.208.6_311.1544.0_632.4503.0_635.4173.0_312.29213.9_313.27319.8_635.54313.9_636.5543.0_637.463.5_317.1375.4_321.31615.7_638.4647.7_639.3732.8_322.3125.4_324.3281.7_326.30613.7_640.98710.0_642.481.5_328.1523.3_329.17819.7_644.46119.4_645.470.4_330.176.6_331.196.5_333.13712.5_648.4285.2_649.420.4_335.2202.9_336.2892.1_649.46313.7_650.53416.0_651.441.7_340.163.5_342.1403.3_652.4612.8_654.470.9_342.3051.1_345.2634.2_655.4707.7_657.36910.0_658.5022.2_348.322.5_350.17811.8_660.38413.5_662.9971.8_351.3124.0_352.336.5_355.15019.7_666.4933.0_668.4571.2_356.2811.2_357.27915.9_669.4404.2_674.445.5_358.2383.5_359.2785.0_361.2763.9_677.48512.4_679.3668.1_362.306.5_366.1609.2_680.46013.3_683.4843.0_684.495.5_369.1603.7_371.3163.0_686.4773.3_691.420.5_373.1942.1_375.30213.9_693.44015.7_695.3852.1_698.4572.1_379.2005.4_380.3746.5_701.3972.3_705.4648.5_381.38514.3_383.365.9_384.3852.2_714.46211.9_717.365.9_388.2126.3_391.33415.4_719.39016.0_722.3972.3_394.29915.1_395.3697.1_397.3857.2_728.3783.0_743.3921.0_398.40814.1_399.2952.1_750.3751.6_762.4761.1_766.5268.2_403.33413.7_407.2741.4_776.56711.3_792.65813.9_408.77412.5_410.74815.1_797.5993.3_804.5594.8_827.5362.1_415.2241.8_416.3382.0_850.82515.2_858.09313.1_417.3711.8_419.3332.0_423.2274.9_861.6661.6_875.72014.8_423.4322.1_425.23619.3_888.70511.9_893.0891.4_426.42715.3_429.374.6_431.3293.0_903.6783.0_912.612.6_432.24113.1_433.36713.9_917.22814.7_934.641

4.8_941.777

13.1_436.3043.0_437.2534.8_943.8524.9_947.7873.0_438.2353.0_440.2391.9_442.3522.9_444.3681.9_446.3443.2_450.260.6_453.16714.7_454.25719.3_454.3351.9_455.335.9_457.28013.1_457.393.8_459.3593.0_461.2122.1_462.2233.0_463.2243.1_464.2493.0_465.3474.0_467.3302.1_469.2574.3_470.2412.3_471.2325.7_471.3955.1_472.400.9_473.3753.0_474.3775.7_475.3895.1_476.3929.7_477.41413.1_479.37313.1_480.3781.1_482.3703.1_484.3361.4_485.1154.9_485.3761.3_486.380.6_487.3531.6_487.3911.2_488.3922.1_489.3424.9_489.4103.0_490.3913.0_491.3418.0_492.313.6_493.2878.1_494.3034.9_495.2208.2_495.3921.0_497.3425.0_498.4185.2_499.3928.2_499.428

7.7_500.426

.9_501.3688.1_501.4384.8_502.4274.5_503.3868.2_503.4237.7_504.42511.8_505.41411.8_506.41710.9_507.3843.0_509.3365.4_509.40011.4_510.4014.9_511.343

4.9_512.4132.3_513.2617.6_513.4075.3_513.4234.0_514.3743.1_514.4365.4_515.3864.9_515.4291.6_516.424

4.9_516.451

9.4_517.4373.3_518.41714.8_519.1404.9_519.4374.9_520.3927.7_521.408

7.7_522.4131.1_523.362.9_524.3551.5_525.3756.1_526.4388.2_527.4229.1_528.4193.0_529.3405.0_529.4463.0_530.37411.3_530.46319.8_531.3684.8_531.4387.6_532.4211.9_533.3893.7_533.3994.9_534.4045.1_535.406

14.6_535.41414.5_536.1634.9_536.41014.8_537.1666.2_537.39815.7_537.6706.1_538.40914.8_539.16615.7_539.43815.8_540.44714.9_541.1554.6_541.4264.6_542.4315.1_543.38610.2_543.45211.4_544.47110.2_545.431.9_546.4268.1_547.41510.2_547.4802.1_549.3849.9_550.6275.1_551.4106.1_552.42411.3_553.4349.6_554.46415.7_555.44212.7_556.4589.5_557.42014.8_558.10510.2_558.464

12.0_559.398

11.1_559.4164.8_559.48311.1_560.4204.9_560.48112.6_561.4283.1_561.43311.9_562.43213.1_562.4351.6_563.40515.4_563.4392.0_563.55112.6_564.4382.0_564.55719.5_565.4493.2_566.41919.8_567.4664.8_569.3386.8_569.420.4_104.10714.8_569.42214.2_569.4596.1_570.423.4_123.0851.0_131.05115.2_570.4336.5_571.429.4_134.077.5_137.110.4_141.10511.3_572.51315.6_573.463.4_144.069.4_149.08817.5_574.39711.4_575.450.3_151.047.4_153.103.3_158.11111.4_576.45113.5_577.507.5_161.073

12.0_578.42910.2_579.39419.4_580.448.3_167.983.4_170.08915.9_581.45417.0_583.480.7_171.150.7_174.0972.4_584.4735.1_585.4157.8_586.495.7_179.107.4_182.10817.4_586.51811.4_587.489.5_188.100.5_191.092.6_193.1187.8_588.51411.4_590.465.5_195.2311.2_591.42914.1_592.421.5_197.104.5_200.106.6_204.11311.4_593.42114.2_593.477.3_208.03913.4_593.51511.3_594.43113.8_594.518.4_212.116.4_214.0924.6_595.42512.6_595.438.4_216.120.5_220.11615.9_595.50115.4_596.44615.9_597.453.4_224.126.9_226.21910.3_597.4549.5_598.446.5_235.12112.0_599.4107.7_599.4921.5_236.117.6_238.12613.8_599.5227.7_600.495.5_241.113.5_244.117.5_246.15015.7_602.47517.4_603.541.6_248.131.7_251.1872.8_605.4414.3_609.4152.8_610.4191.7_255.214.6_257.0943.0_610.48515.7_610.542.5_258.174.3_261.1302.8_611.42313.8_611.5424.3_612.4621.6_263.2403.2_265.25319.3_613.18415.2_613.916.8_267.174.5_269.1272.2_270.28410.2_614.53114.1_615.429.6_272.1351.6_275.0342.1_615.4894.3_615.520.6_276.146.5_279.13115.2_279.17613.7_616.49915.8_618.44815.3_280.18216.1_281.1927.4_619.458.7_620.4631.5_621.472.5_283.127.6_284.13415.2_622.41215.8_623.427.5_286.138.5_289.13415.2_623.91315.7_624.92814.3_625.4851.3_295.276.5_297.13015.6_625.92414.3_626.491.5_298.141.6_299.1971.0_300.20816.5_627.5384.8_629.332.5_307.1224.8_630.4123.3_631.419.8_308.2011.6_309.292.5_311.22717.4_632.5601.1_635.4552.2_312.29913.7_313.2773.0_636.46215.4_636.55414.5_637.530.5_317.206.5_322.1233.6_638.4751.2_639.415.5_323.142.5_325.1447.7_640.37819.4_641.2138.2_642.5241.7_328.294.6_329.21219.4_644.46811.9_645.91215.5_330.221.6_331.227.5_333.1762.1_648.4564.0_649.4275.0_335.2781.6_336.2942.5_649.51513.5_650.5341.6_337.279.4_340.2295.5_342.1645.3_652.4594.2_654.472.5_343.1572.0_345.29119.8_655.4762.1_657.4344.3_658.5163.8_348.3281.9_350.3109.7_660.44613.7_663.0024.0_351.339.5_353.1392.5_664.4803.5_667.4342.1_668.5111.6_356.2902.4_357.3001.8_670.4663.1_674.4683.5_358.2763.9_359.3181.6_361.2921.7_678.3309.3_679.4457.3_363.2871.2_366.2272.5_681.5153.0_683.497.5_367.1551.2_369.3423.3_371.3193.1_686.54315.2_693.37311.3_373.244.5_376.1981.4_693.48015.7_696.3903.0_699.4711.0_379.295.6_381.1533.1_701.48510.3_705.473.5_382.16514.9_383.366.9_708.2952.1_715.3362.1_717.4591.7_388.2821.1_391.34012.6_719.39119.3_722.4166.5_394.726.5_396.17816.7_397.3871.7_731.5383.0_743.41115.7_398.74516.4_399.3903.0_751.3831.7_763.4781.7_401.4107.3_404.23413.5_407.77618.5_777.54911.3_793.6281.6_409.2064.0_411.26718.6_798.60818.6_804.5691.6_832.6071.8_415.33413.1_416.36613.1_853.70315.2_858.5952.1_418.2262.1_420.22715.7_860.61815.7_862.1154.9_883.6511.6_423.7701.3_425.23915.3_888.81311.9_893.5876.0_427.35810.2_429.3749.7_431.3543.0_905.6637.7_912.7043.0_432.25415.3_434.24619.3_917.66814.7_935.6768.1_434.32615.4_436.3275.1_437.3064.8_944.7884.8_948.7893.0_438.2653.0_440.2531.2_442.353.5_445.2092.9_446.3623.1_451.2643.0_453.2555.5_454.25914.8_454.3378.1_455.4013.0_457.2848.1_457.4088.1_459.394.6_461.2693.2_462.2652.1_463.2265.0_464.2813.9_465.3493.0_467.3362.9_469.2933.0_470.336.6_471.3201.7_471.3965.3_472.3993.3_473.385.9_474.3815.3_475.3914.9_476.393.6_478.2191.4_480.1611.4_481.1601.9_482.3744.0_484.3562.1_485.2121.3_485.3784.9_486.381.8_487.3553.0_487.3931.1_488.392.8_489.3542.9_489.4611.1_490.3993.0_491.3635.0_492.3668.1_493.3163.0_494.3671.6_495.2291.6_496.2274.9_497.4037.7_498.4226.1_499.3949.3_499.4281.1_501.368.5_502.2857.6_502.4344.9_503.3894.9_503.43113.1_504.4267.6_505.4241.2_507.3336.1_507.3945.0_509.35111.4_509.4003.1_510.4051.1_511.3802.1_512.4163.9_513.3217.7_513.4075.1_513.4246.8_514.4109.6_514.4402.0_515.4203.1_515.4406.1_516.4253.0_517.3675.1_517.4501.6_518.4249.4_519.4421.6_520.4197.6_521.4087.6_522.4133.0_523.3651.5_524.3717.7_525.41411.8_526.4679.0_527.4223.1_528.4231.4_529.3962.1_529.4434.8_530.3897.7_530.4641.3_531.40911.4_531.4533.8_532.4471.3_533.3945.4_533.4385.1_534.4036.8_535.38915.7_535.41514.8_536.1645.1_536.4112.1_537.3386.1_537.40514.5_538.16415.2_538.4293.0_539.35714.8_540.1638.1_540.453.5_541.29910.2_541.43714.2_541.46111.3_542.4428.5_543.4217.8_543.461

7.7_544.481

9.4_545.43711.8_546.4387.6_547.4247.7_547.48117.4_549.4309.4_550.62810.7_551.42117.9_552.4465.0_553.4359.4_554.4698.1_555.4599.4_556.47117.5_557.4203.6_558.4047.7_558.4768.5_559.4629.4_559.4995.3_560.4209.4_560.48215.4_561.429

19.3_561.4753.1_562.4327.6_562.4354.4_563.40915.5_563.44014.7_564.1987.9_564.43914.7_565.20015.9_565.45411.4_566.4204.9_568.3426.1_569.4185.1_569.42119.4_104.970.7_110.06416.5_569.42215.9_569.4666.5_570.424.3_124.082.6_132.07714.9_570.4347.2_571.430.4_135.080.9_138.06411.3_571.4897.7_572.5134.9_573.467.3_145.052.4_150.0921.4_574.47713.1_575.458.6_151.082.4_154.0854.8_159.07014.0_576.50513.8_577.509.4_161.108.4_163.0866.1_578.43010.2_579.402.4_165.110.6_168.076.3_170.10219.4_581.45616.9_583.486.3_171.154.4_174.1044.9_584.4836.1_585.46411.8_586.494.3_180.037.3_182.9871.1_587.31510.2_587.498.6_188.108.5_191.1556.0_588.38510.3_589.4331.0_591.355

.7_195.087

.3_196.02011.3_591.4651.0_592.436.3_198.034.7_201.0974.3_593.42214.4_593.486.6_206.118.5_208.10814.0_593.51612.0_594.433.3_210.031.3_213.019.3_215.0155.1_595.42915.4_595.438.5_217.110.6_220.124.9_596.31415.4_596.45410.0_597.451.3_225.017.5_227.12212.4_597.46310.0_598.4531.3_229.143.6_232.11815.9_598.4724.9_599.4327.8_599.4921.3_236.117.5_238.13413.3_599.52213.7_600.523.6_241.106.6_244.129

.5_247.119

2.3_602.4873.5_604.429.3_249.068.6_252.1332.1_605.4586.2_609.4171.3_253.1961.3_255.253.6_257.1373.0_610.51415.1_610.542.5_261.1581.9_611.42913.5_611.5426.6_612.46513.7_263.2402.4_265.25319.7_613.18519.7_614.180.9_267.2444.3_269.1936.5_614.45019.2_615.1761.6_615.443.5_273.1182.3_275.03515.7_615.49414.3_615.5302.3_277.0341.3_279.166.6_279.2302.1_617.42715.6_618.4521.6_280.26412.6_281.19215.7_619.4621.5_620.4671.4_281.268.6_283.1342.0_284.2897.0_622.44615.7_623.427.6_286.140.7_289.26015.7_623.92612.0_625.41514.1_625.4861.6_295.282.6_297.1839.7_626.41814.0_626.493.6_298.1391.2_299.20112.5_626.9211.1_628.33615.8_629.418.3_302.990.5_308.1314.7_630.4595.8_631.4603.0_308.2022.4_309.2992.2_311.3073.0_633.4151.3_635.470.5_313.1332.3_313.2771.1_636.45515.8_636.556.5_315.1901.0_317.289.5_322.1632.7_638.47719.8_639.495

2.0_323.3031.2_325.2957.6_640.3787.6_641.3799.7_643.4251.1_328.3212.0_329.3181.4_644.49211.9_646.40811.0_330.2242.1_331.28215.3_647.9133.0_648.45112.5_649.429.7_335.2913.6_336.32712.5_649.93115.9_650.942.5_338.1495.4_340.350.6_342.2114.9_653.3704.9_654.473.3_343.1804.8_345.71513.5_655.4914.4_657.515.5_347.1472.1_349.2313.1_350.3434.4_660.50213.9_663.0003.3_351.3392.2_353.3172.1_665.4723.0_667.44610.7_668.5271.7_356.3541.7_357.3573.2_671.5293.0_674.506.5_358.3677.7_359.7324.6_676.45412.5_678.3949.2_679.4571.0_363.3211.7_366.3013.0_682.3552.3_683.5421.2_367.231.6_370.190.5_372.2092.1_687.44414.9_693.3753.5_373.2942.1_376.19415.2_694.32315.8_696.3922.9_377.3162.8_379.3692.1_381.1973.0_702.4405.0_705.5164.9_382.29914.6_383.36719.8_708.4922.1_715.4666.3_717.4875.1_389.320.5_392.1833.2_719.5061.1_722.5023.0_395.19015.2_396.36819.7_725.4771.6_732.5454.8_743.54917.1_399.1973.0_400.220.9_752.4974.0_764.4174.0_402.2242.1_405.26513.8_407.7761.4_777.5782.9_794.5423.0_409.2661.7_411.36018.6_799.6114.8_805.6081.4_412.4164.4_415.3342.1_417.22213.1_854.70615.2_859.0932.1_419.2233.1_420.24315.8_860.6244.9_863.6724.9_884.6563.0_424.2423.0_425.24415.5_888.82316.0_893.65614.5_427.3976.8_429.37412.4_896.6182.9_906.69819.8_912.76313.1_432.35615.8_434.2473.0_917.7283.0_937.6613.3_434.3268.1_436.33017.0_437.4494.9_945.74513.7_949.71316.4_438.4091.4_440.3372.9_442.355.6_445.3342.9_447.3593.0_451.3105.0_453.2753.0_454.28711.3_454.34015.4_455.415.6_457.3072.9_458.344.6_460.2713.2_461.268.6_462.2663.2_463.2733.9_464.3355.0_466.2682.1_467.3643.0_469.3352.3_470.3762.1_471.3615.3_471.396

4.9_472.398

5.1_473.3964.9_474.4025.1_475.39110.6_476.8652.1_478.236.9_480.2675.1_481.2833.0_483.3581.3_484.390.7_485.3352.9_485.3942.9_486.3991.1_487.3886.2_487.4113.8_488.3931.3_489.3731.2_490.2626.1_490.4081.1_491.3854.9_492.3403.0_493.36110.9_494.3633.0_495.2691.0_496.34210.5_497.38519.3_498.452

7.7_499.414

8.5_499.4298.5_500.4313.1_501.369.9_502.3727.7_502.4355.0_503.3918.1_504.4238.5_504.4287.7_505.4293.1_507.3403.0_508.3724.9_509.3533.0_510.3363.3_510.4243.1_511.4083.0_512.4173.0_513.3338.1_513.4093.0_513.4417.7_514.4109.4_514.446

1.6_515.420

9.5_515.4463.1_516.4311.1_517.3727.7_517.4475.0_518.4305.0_519.34714.7_520.146.5_521.3358.1_521.4101.6_522.4321.3_523.39211.9_524.4586.5_525.4453.0_527.1399.4_527.4229.4_528.4287.6_529.4024.9_529.4471.3_530.4066.1_530.467.7_531.4137.8_531.4686.1_532.45710.8_533.3963.8_533.4413.7_534.4124.9_535.40615.2_535.6393.0_536.368

15.2_536.418

15.3_537.34315.2_537.42214.8_538.16515.0_538.4311.6_539.4109.6_540.41510.7_541.39111.3_541.43813.4_541.47210.2_542.4422.7_543.42619.3_543.5084.9_545.4817.6_546.4757.7_547.4241.6_548.41211.4_549.4399.7_550.63211.4_551.44716.0_552.49016.0_553.4903.0_555.33912.8_555.45311.8_556.5163.1_557.4329.2_558.4047.8_558.48111.4_559.4107.6_559.46412.0_559.66011.8_560.4203.7_561.38215.1_561.4302.1_561.47912.6_562.43315.6_562.43510.2_563.419

19.4_563.445

1.6_564.40912.5_564.4412.1_565.33115.7_565.45515.9_566.4594.8_568.4386.5_569.418

15.2_569.421.3_105.111.3_111.06515.5_569.42314.8_569.474.3_118.071.4_125.083.4_132.0957.0_570.45815.7_571.437.6_135.09315.7_572.4567.9_572.5171.6_573.469.3_146.118.7_150.09611.4_574.50313.9_575.499.4_151.090.6_155.11913.1_575.50413.1_576.5064.9_578.353.3_162.038.3_164.07112.5_578.4356.1_579.436.6_165.125.4_168.081.3_170.9818.0_581.46811.8_583.492.3_172.130.5_175.095

4.0_585.41311.3_585.508.6_177.099.4_183.0944.0_587.42311.3_587.505.5_189.106.5_192.110

2.7_588.43719.7_589.4645.1_591.402.9_195.08914.1_591.47514.3_592.474.3_198.112.5_201.1044.6_593.41814.2_593.43415.6_593.494.6_207.066.3_208.1281.0_594.3721.0_594.441.5_210.124.5_213.109.6_215.12014.7_595.43611.9_595.441.3_217.108.5_221.1006.7_596.37012.5_596.457.5_222.157.5_225.110.7_227.12217.1_597.48810.8_598.457.5_230.0893.0_598.4765.1_599.43819.8_599.507.9_237.117.5_239.11911.9_600.41711.4_600.550.5_242.115.5_245.12214.4_601.5223.1_602.4887.2_604.459.5_249.122.9_252.13811.9_607.4054.8_609.443.6_254.121.5_256.115.6_257.17015.0_610.54019.3_611.184.6_259.131.5_262.1354.2_611.43315.4_611.545.5_263.097.5_264.086.3_266.0604.9_613.35119.5_614.182.5_268.1201.2_269.2605.1_614.47719.4_615.1841.6_615.4832.9_273.212.5_275.12913.4_615.4963.6_616.396.5_277.136.7_279.16815.3_616.45615.2_617.43819.8_618.5161.4_280.26415.7_281.1923.0_619.48715.3_621.395.5_282.1231.0_283.1993.3_284.2991.5_622.4757.0_623.448.5_287.091.6_290.1389.2_624.3989.7_625.416.5_293.140.6_296.1351.0_297.1872.8_626.4412.1_626.5131.0_298.1911.0_299.20212.4_627.4261.3_628.4197.4_629.458.5_303.138.7_308.1314.0_630.4721.3_631.461.5_308.210.5_310.14217.5_631.5763.3_633.4603.0_635.470.5_314.1394.7_636.46314.2_636.5561.0_315.197.5_318.1562.1_322.2754.5_638.4773.0_639.495.5_324.1412.0_325.3157.1_640.4937.7_641.3803.2_326.327.6_328.3231.1_329.33011.9_645.41119.9_646.480.5_330.2282.3_331.28612.5_647.9272.5_648.4963.0_649.4551.6_335.3064.2_336.3273.6_650.44019.5_650.942

5.4_338.342

8.5_340.3597.1_651.5172.1_653.4371.7_655.339.5_344.156.3_345.91913.7_655.4919.2_658.431.5_347.1841.2_349.277.5_351.1433.1_661.42312.5_663.463.5_352.1605.4_353.35415.9_665.4853.0_667.4681.7_355.286.6_356.354.6_357.3582.8_672.4243.1_675.468.5_359.1627.6_359.7334.4_676.4758.9_678.4431.1_679.480.6_364.1491.2_366.3041.7_682.36917.5_684.2013.4_367.2843.0_684.5052.1_687.4839.5_693.3901.7_373.3195.0_376.30615.2_694.3772.1_696.45013.9_377.7755.4_379.3691.5_381.3243.0_703.44110.3_706.4758.5_382.3896.0_383.36811.3_709.4381.7_715.56414.5_385.37319.4_391.237.5_394.16715.4_720.3885.0_722.5366.6_395.2286.3_396.37114.0_726.5191.7_733.5373.5_744.368.9_399.2461.6_400.3383.0_753.3871.6_764.48111.9_402.339.9_406.1651.1_767.52811.3_782.521.9_796.53712.6_409.41714.5_411.36415.1_801.5511.7_809.4861.3_413.41513.1_415.35612.5_417.22513.1_855.67115.2_859.5933.1_419.2392.1_421.21615.7_861.11819.3_869.761.5_423.2423.0_424.2723.0_425.2543.0_889.70919.3_893.78311.9_428.36515.4_430.34012.5_896.6183.0_907.6993.0_913.6399.7_432.3563.0_434.24819.3_917.78414.7_939.5892.1_435.2343.3_436.3434.8_942.7404.9_945.7784.8_949.7843.0_439.1982.1_440.44016.1_442.4142.9_445.3632.1_448.2403.0_452.2485.3_453.3883.3_454.29415.4_454.4061.6_456.214.9_457.3401.2_458.3473.1_460.2661.3_461.3155.0_462.2805.0_463.2833.0_464.3363.0_466.3493.0_467.3643.9_469.3353.3_470.3831.1_471.3624.9_471.5321.7_472.4054.9_473.40213.1_474.4124.9_475.39110.0_477.36310.5_478.8655.1_480.2863.0_481.3231.9_483.3792.9_484.3904.0_485.3606.1_485.4111.9_486.4121.4_487.3891.2_488.2384.9_488.4083.0_489.4138.0_490.30914.1_490.8891.3_491.3862.9_492.3436.5_493.3749.7_494.3634.9_495.2708.1_496.4268.1_497.42919.1_498.45419.3_499.4558.9_500.4311.3_501.4021.2_502.3748.2_502.4798.1_503.420.9_504.3823.1_505.3403.0_506.3315.0_507.3584.0_508.3553.0_509.3664.9_510.35814.1_510.8951.6_511.3895.3_512.4234.0_513.3901.6_513.4099.4_513.4421.6_514.4112.3_515.2707.8_515.4213.0_516.36013.2_516.4303.7_517.4064.9_517.4559.4_518.4401.1_519.380.5_520.3263.0_521.3441.6_521.4285.1_523.3217.7_523.41612.0_524.45812.0_525.4612.1_527.3333.1_527.4306.1_528.4427.7_529.40311.4_529.4637.7_530.42211.8_530.4847.2_531.4156.1_531.4701.1_532.4475.2_533.39811.3_533.4571.1_534.444

15.2_535.406

15.2_535.6581.1_536.38014.9_536.4197.7_537.38515.7_537.423

4.9_538.39015.7_538.433

9.6_539.4106.3_540.4158.5_540.4524.0_541.39111.8_541.44111.8_541.4767.6_542.4617.7_543.4628.5_544.4254.9_544.4837.8_545.4867.7_546.476.9_547.4306.1_548.416.9_550.3599.2_550.6309.4_551.6321.6_553.3772.4_554.1803.0_555.3589.4_555.47316.0_556.5172.3_557.4323.1_558.4409.4_558.4935.3_559.41410.2_559.4641.7_559.8811.6_560.43611.8_561.4219.4_561.4306.1_561.48112.5_562.4338.1_562.4367.7_563.43715.7_563.4464.3_564.41319.7_564.44511.4_565.41619.4_565.45519.7_566.46417.5_568.4375.4_569.41814.5_569.421.4_106.06413.7_113.93614.1_569.42911.4_569.486.3_119.062.4_126.053.4_133.0774.9_570.49415.1_571.438.4_136.076.6_138.06715.6_572.45010.9_573.381.6_143.058.4_147.093.3_150.1031.0_575.35913.3_575.499.3_151.549.4_155.11911.3_575.520

13.8_576.5061.3_578.389.5_162.094.5_164.10015.4_578.43515.4_579.441.4_166.089.3_168.99312.5_580.4457.0_582.4262.9_584.318.3_173.031.4_175.0983.7_585.4142.1_585.525

.5_178.103

.7_180.109.4_184.0942.7_587.4307.7_587.515.6_189.105.6_192.11217.5_588.45916.3_589.459.3_194.0541.1_195.091.6_196.09314.3_591.47514.1_592.476.4_199.030.4_201.1103.6_593.4191.0_593.43916.0_593.494.5_207.069.3_209.0184.6_594.42315.6_594.497.3_211.005.6_213.1272.1_595.32612.4_595.43615.6_595.447.6_221.1267.6_596.37611.1_597.397.5_223.128.7_225.113.5_228.11013.1_598.3469.2_598.457.6_230.123.7_233.1251.3_598.5069.5_599.450.6_235.120.6_240.10815.1_600.46412.5_601.424.3_243.021.6_245.12613.9_601.5233.5_603.4259.2_605.386.5_249.153.5_252.16617.5_608.5014.8_609.4621.3_254.248.6_256.1174.3_610.42314.0_610.54119.8_611.186.4_259.157.6_262.1474.3_611.47015.5_611.545.5_263.128.5_264.148.4_266.1345.0_613.40019.3_614.182.6_268.132.6_270.1233.9_614.4781.6_615.347.5_271.1221.0_273.227.6_275.13113.8_615.4969.2_616.412.6_277.14414.5_279.1726.6_616.491.8_617.4391.0_619.451.6_281.13312.5_281.1923.0_619.5082.1_621.404.5_282.1531.4_283.27515.2_621.9127.7_622.4997.0_623.452.5_287.136.5_290.15111.4_624.40515.9_625.424.4_294.150.5_296.139.6_297.2474.0_626.44116.5_626.5331.9_298.2961.9_299.2952.5_627.4282.5_628.4321.6_300.2911.2_303.219.7_308.1966.5_630.4724.0_631.4692.4_308.2964.0_310.3122.1_632.4021.2_634.4534.9_635.4821.0_313.181.5_314.1793.0_636.50815.5_636.557.5_316.155.5_319.14215.9_637.93115.5_638.5743.0_639.5081.2_324.2903.3_325.3233.0_640.4939.9_641.476.5_327.146.5_329.172.5_330.1404.0_645.4403.0_647.4121.3_330.303.5_332.1414.0_648.42311.1_648.518.7_333.2052.8_335.316.6_337.11619.5_650.44019.4_650.954.5_339.147.5_341.14813.7_651.5375.3_653.4652.1_655.4241.2_344.261.4_346.1651.6_656.3812.1_658.4361.6_347.3245.1_349.2912.1_659.4623.9_661.52113.7_663.9981.6_352.320.5_354.1633.5_666.4352.1_667.4985.4_355.363.5_357.159.5_358.1572.8_673.4274.4_675.469.5_359.228.5_360.17611.9_677.3531.2_678.4792.1_361.3021.0_364.3268.5_366.3731.1_683.43616.3_684.2054.3_367.3681.6_370.3342.3_684.5301.4_688.52313.7_693.4372.1_375.192.7_376.3162.1_694.45015.6_697.390.7_378.138.5_380.1572.1_699.4436.7_703.461.9_707.283.5_383.1548.5_383.3933.0_709.4722.1_716.449.9_385.39016.0_391.2466.5_394.22815.3_720.4041.1_723.5071.8_395.35415.1_396.3737.2_727.37310.3_737.4642.1_398.270.6_399.246.5_401.22914.6_758.5677.8_765.5174.0_402.7252.1_406.23310.3_768.48011.3_782.56915.1_796.59311.9_409.7362.1_412.24315.1_802.5591.1_810.5531.2_414.32210.0_415.35613.1_837.72715.2_856.59315.7_859.62013.0_419.2728.3_421.38215.8_861.11915.3_870.1131.6_423.2701.6_424.2739.0_426.3403.0_890.72411.9_894.0961.8_429.31015.2_430.38112.5_897.1167.6_911.68210.3_431.354.6_433.2349.3_434.27913.8_918.23511.4_939.7133.0_435.25214.2_436.377

4.8_942.777

4.8_945.81413.7_950.7183.0_439.2361.2_441.3251.3_443.3331.9_445.3422.9_448.3623.0_452.26014.3_454.2405.0_454.3281.6_455.2113.0_456.2352.9_457.342.6_459.296.8_460.362.8_461.3701.4_462.3181.2_463.3041.4_465.1313.9_466.3543.0_468.3663.1_469.3793.1_470.3833.1_471.3804.8_471.624.6_473.334.9_474.3082.1_475.30713.1_475.420.9_477.368.9_479.2618.1_480.307.8_481.344

11.9_483.432

3.1_484.4238.1_485.37411.8_485.4406.1_486.4151.3_487.3892.1_488.3332.9_488.4193.7_489.3968.1_490.310.5_491.2343.0_491.415.8_492.3534.9_493.377

5.0_494.3803.1_495.35610.5_496.88110.5_497.8793.0_499.3558.9_499.4247.7_499.6528.1_500.4392.9_501.4154.9_502.3838.2_503.3668.5_503.4213.3_504.3883.0_505.361.9_506.3684.9_507.36210.9_508.3891.3_509.3713.7_510.3713.0_511.1577.1_511.4175.1_512.4233.7_513.4152.1_513.4108.5_513.4427.6_514.4133.0_515.3476.1_515.4211.0_516.3887.8_516.4394.8_517.4083.1_518.3644.9_518.4405.1_519.3883.0_520.35111.8_521.38611.8_522.3913.0_523.3247.6_523.4161.1_525.34711.9_525.4621.2_527.3866.1_527.44011.4_528.4601.3_529.40111.9_529.4926.8_530.4324.0_531.3425.4_531.4167.6_531.47211.4_532.4566.1_533.3981.1_533.4501.1_535.37514.9_535.41114.5_535.8312.1_536.39115.5_536.4203.0_537.3937.6_538.3906.3_539.4115.2_540.4188.9_540.45310.9_541.3918.1_541.45714.8_542.1157.7_542.461

9.4_543.4442.7_544.4338.1_544.483

7.7_545.486

13.9_547.38311.8_547.4447.7_548.4213.8_550.40013.7_551.3575.1_552.4135.4_553.3939.2_554.40813.1_555.41815.4_555.48514.8_557.09811.4_557.4334.9_558.45911.3_558.51912.5_559.4147.7_559.4646.8_560.460

12.5_561.423

8.1_561.4321.6_561.48215.4_562.434

6.1_562.45612.6_563.43715.9_563.445

10.2_564.42315.7_564.45010.2_565.42419.8_565.46119.8_566.46514.2_568.4387.2_569.419Y1.3_107.063.3_114.09217.5_569.43818.5_569.489.4_120.066.4_127.0447.2_570.42411.4_570.49115.8_571.440.6_136.077.4_139.07615.8_572.45317.4_573.393.3_143.0591.0_147.118.9_151.0469.2_575.388

13.8_575.503

.3_152.046.9_155.1193.6_576.41713.4_576.5074.9_160.078.7_162.094.3_164.1091.0_579.38212.5_579.441.6_166.091.4_169.0799.5_580.45119.4_582.4678.5_584.4461.1_173.089.5_176.0822.6_585.4144.0_586.418.6_178.091.5_181.09210.2_586.4942.9_587.4317.6_587.516.5_192.1536.6_588.46319.4_589.462.6_194.1171.3_195.092.3_197.02014.5_591.47614.5_592.479.5_199.105.5_202.0985.2_593.41915.4_593.456.4_205.019.5_207.112.5_209.1075.2_594.42416.0_594.497.6_211.092.5_213.1701.0_595.3686.8_595.43615.7_595.495.6_218.117.3_222.0197.7_596.37914.2_597.395.6_223.178.3_226.02619.4_597.4527.7_598.38018.8_598.4607.7_598.52115.9_599.474.7_237.119.5_240.11711.9_600.4667.4_601.444.5_243.1181.6_245.22912.4_602.430.9_603.441.5_247.155.5_250.135.6_252.17716.9_608.52416.9_609.5191.4_254.2521.7_256.2176.2_610.43913.8_610.5414.9_611.340.5_260.130.6_262.17916.0_611.48719.4_612.185.6_263.1331.6_264.24313.9_612.5436.5_613.4502.1_614.4041.6_268.266.5_270.12411.4_614.5212.1_615.405.6_271.114.5_274.099.5_276.14014.0_615.4962.1_616.416.6_277.21611.4_279.1741.6_616.49119.8_617.515.5_280.167.7_281.18515.9_281.19214.5_619.50814.9_621.4081.0_282.1952.0_283.28314.9_621.91515.2_622.91319.8_623.524.6_288.130.5_290.20215.8_624.42615.7_625.4281.6_294.2801.0_296.18014.5_625.4865.1_626.44316.9_626.535.5_299.094.6_300.1435.1_627.4454.8_628.456.5_301.128.5_304.149.3_308.1962.8_631.39813.7_631.474.5_309.1233.3_310.3122.8_632.39613.8_634.536.5_312.1447.2_313.181.6_315.12719.9_636.54615.8_636.942.5_316.210.6_321.13519.4_638.21619.3_639.21619.4_640.2173.2_324.292.5_326.14819.8_640.5027.1_641.4941.8_327.310.6_329.17315.6_643.96812.5_645.44412.4_647.428.5_331.134.5_332.17915.5_648.42412.4_648.9285.0_334.276.5_336.143.5_337.1532.9_650.450.7_651.2245.4_339.3471.5_341.17413.5_651.5373.3_653.465.6_342.259.5_345.1641.2_346.2742.1_656.4313.0_658.4431.2_348.2753.8_349.3339.0_659.48712.5_662.46215.9_664.4755.4_352.323.5_354.2133.0_666.46211.3_668.407.5_356.147.7_357.1447.1_668.5432.1_673.4455.4_675.6727.6_359.2295.2_360.2909.7_677.3652.1_678.5041.2_362.211.9_365.139.4_367.06013.8_683.48317.1_684.218.5_368.167.4_371.17317.3_685.2023.2_689.5003.5_372.2935.0_375.3022.1_377.19816.0_694.52215.8_697.4022.1_378.1982.1_380.1923.0_700.43110.0_704.48719.8_707.4852.1_383.1895.4_383.4021.7_709.5531.7_716.571.5_386.1823.0_391.2853.2_718.47915.9_721.39819.8_724.47215.0_395.3681.2_397.2943.0_727.4274.9_737.55516.4_398.386.6_399.2533.9_401.35314.6_759.5747.7_765.5811.7_402.7553.4_406.2961.1_771.47811.3_783.5731.6_408.1924.9_410.3303.9_412.2733.3_803.5531.1_811.55910.0_414.348.6_416.21513.1_838.72315.2_857.09515.7_860.1186.7_419.2732.1_422.21715.6_861.61815.4_870.6087.3_423.3267.3_424.3322.9_885.7203.0_891.70511.9_894.583.7_429.31915.4_430.38212.5_897.6187.7_911.68413.1_431.3543.0_433.2575.1_434.2933.0_918.7234.9_939.7752.1_436.231.6_437.1994.8_943.7833.0_946.588.6_438.1791.4_439.3331.9_441.3492.9_443.36415.3_445.3682.1_449.2485.0_452.27319.3_454.25616.1_454.3343.0_455.2451.8_456.3601.8_457.366.6_459.3211.8_460.3883.8_461.3731.8_462.378.8_463.3535.0_465.280.5_467.1972.1_468.3688.1_469.3815.0_470.386 4.9_471.3842.3_472.2342.9_473.340.7_474.3343.0_475.36113.7_475.8628.1_477.3825.4_479.2953.0_480.3217.1_482.32612.0_483.43311.9_484.4361.1_485.3745.4_485.8612.1_487.3213.8_487.389.8_488.3561.9_489.3291.1_489.3962.1_490.3208.1_491.31310.5_491.8613.0_492.3655.1_493.3774.9_494.3824.9_495.3843.0_497.3311.6_498.239.8_499.3538.1_499.4273.0_500.35710.5_500.8597.7_501.4341.3_502.415.9_503.3777.6_503.4224.9_504.393.9_505.3643.0_506.3723.0_507.3706.1_508.3951.1_509.3721.1_510.3762.1_511.15916.0_511.46316.0_512.4675.6_513.4058.3_513.4119.6_513.4435.0_514.426.9_515.3497.7_515.4225.4_516.3907.7_516.4426.1_517.4201.1_518.3731.6_518.4504.9_519.393.8_520.3553.9_521.39313.1_522.4042.1_523.32913.6_523.4213.3_525.3626.0_526.3715.1_527.387

11.4_527.455

11.9_528.4881.6_529.4014.0_530.2965.0_530.439.9_531.3457.6_531.4195.4_532.4193.0_533.32717.5_533.3972.1_534.3763.0_535.382

15.5_535.4144.9_535.8381.6_536.39614.2_536.4207.6_537.38716.0_537.4307.7_538.39116.1_538.4359.5_539.41415.7_540.4447.4_540.4781.6_541.3978.5_541.454.5_542.3134.9_542.47210.2_543.4449.4_544.4371.6_545.3958.1_545.4891.6_547.40910.2_547.4467.6_548.42517.3_550.4366.8_551.36515.2_552.4189.2_553.40115.2_554.43015.1_555.4285.4_556.4123.6_557.3997.7_557.47911.3_558.4431.7_559.3819.7_559.4151.6_559.4659.7_560.4196.1_560.4779.7_561.42414.8_561.43311.8_562.36115.2_562.4351.6_562.4807.6_563.437

19.8_563.447

15.5_564.42919.4_564.4503.2_565.42411.1_565.46311.0_566.4678.2_568.4476.3_569.419Y2.3_108.081.3_116.1073.1_569.4442.0_569.564.4_121.077.3_128.14414.5_570.4254.9_571.3774.9_571.459.4_137.072.7_139.0774.9_572.46314.2_573.39519.3_143.061.4_148.08411.4_573.5193.6_575.40914.0_575.503.3_156.13512.0_576.4191.3_577.385.7_160.079.4_162.105.5_165.0879.2_579.40410.2_580.397.4_166.115.8_169.97815.9_580.45013.4_582.501.3_171.0361.0_173.097.6_176.0925.7_585.4156.1_586.421.6_178.128.5_181.12911.3_586.5041.1_587.44817.5_587.523.6_190.094.3_193.06710.2_588.49212.0_589.464.4_194.122.4_195.1049.2_591.3995.1_592.4061.0_593.369.3_199.113.6_202.10512.0_593.4194.8_593.468.6_205.098.3_207.155.5_209.1643.6_594.42416.0_594.501.4_211.137.6_213.1961.2_595.3761.0_595.437.4_215.123.5_219.110.5_222.1177.4_596.4397.4_597.443.3_224.010.6_226.09510.8_597.45214.2_598.41119.4_598.465.5_231.116.6_234.1197.6_598.52219.9_599.490

.7_236.114

1.9_237.22314.1_599.51919.9_600.4907.8_601.491.6_243.1194.0_602.4424.0_603.449.5_248.09715.1_251.158.4_253.1073.0_609.37219.4_610.184.7_255.1161.9_256.2658.2_610.44613.4_610.541.5_258.124.6_260.13315.3_262.21213.3_611.54119.8_612.1861.4_263.238.5_265.12913.7_612.5474.3_613.46514.1_614.419.5_269.094.4_270.15814.2_614.5223.6_615.403.5_272.0953.9_615.48615.5_615.497.8_616.435.5_278.14112.0_279.17513.5_616.4981.0_618.449.9_280.1791.0_281.1892.1_281.24914.3_619.51115.2_621.41015.7_282.198.5_284.097.9_622.3869.2_623.396.5_285.129

.5_288.138

.5_291.13115.7_624.4286.2_625.446.5_295.1581.9_296.26114.7_625.48714.5_626.44611.3_626.560.5_299.131.5_300.1404.0_627.4447.4_628.457.6_301.210.5_305.1374.8_629.4602.1_631.39716.5_631.489.7_309.205.5_311.1485.1_632.429

19.8_634.540

.6_312.1581.4_313.182.6_315.18319.7_636.5442.1_637.3881.0_316.2851.4_321.29215.5_638.4647.6_639.3711.4_322.2922.0_324.3101.3_326.2733.0_640.5151.2_642.4422.0_327.3394.8_329.1773.5_644.4504.0_645.4568.1_647.481.4_331.1812.3_332.2925.1_648.42712.5_648.9292.8_334.312.5_336.2211.4_649.45515.9_650.4412.8_651.444.5_340.1561.6_341.31816.0_651.9531.7_654.3351.6_342.3044.9_345.2146.1_346.7142.8_656.4562.1_658.4785.1_348.291.5_350.1384.4_659.49819.8_662.5742.1_351.220.5_352.3245.4_354.35719.9_666.492.6_668.403.5_356.216.5_357.21211.3_669.4132.1_674.44012.0_676.3517.7_359.2302.1_360.2908.9_677.4421.7_679.3385.0_362.2795.4_365.35312.5_680.37513.5_683.48419.3_684.2401.2_368.316.5_371.2149.2_686.4246.1_690.4381.6_372.3133.2_375.3011.6_377.26815.8_695.3852.1_697.4531.6_378.2741.5_380.3303.1_700.4775.0_704.5115.4_381.3763.0_383.26614.6_384.3725.1_711.3642.1_717.3346.3_387.3493.3_391.2872.1_719.33415.9_722.4081.1_724.5156.3_395.36916.4_397.3837.2_728.3464.8_738.5587.1_398.38715.8_399.2543.0_749.3878.1_760.5711.1_766.4881.2_403.2312.1_407.21719.7_775.47114.7_784.5842.1_408.22112.6_410.42014.5_412.37118.5_803.5631.3_820.595.6_415.2112.1_416.2264.9_850.61315.2_857.59411.8_417.2281.2_419.2793.0_422.28815.7_861.61915.3_871.11716.0_423.40016.0_424.4042.9_886.72711.9_892.59612.5_896.1122.3_429.351.5_431.16615.9_899.7613.0_912.58915.2_431.3822.1_433.26319.3_913.8182.9_930.7704.9_940.7713.0_436.2492.1_437.2114.8_943.8114.9_946.778.6_438.2178.3_439.39316.0_441.410.9_444.312.5_446.2172.9_449.3654.0_452.29413.5_454.25614.0_454.3353.0_455.2628.1_456.4043.8_457.3782.9_459.3588.1_460.4021.8_461.3821.4_463.1331.4_464.1342.1_465.3445.1_467.2741.5_468.3813.3_469.3824.9_470.3885.1_471.3953.1_472.3793.1_473.3742.9_474.34013.7_475.3622.9_476.3534.9_477.4003.0_479.3271.3_480.3633.0_482.33510.5_483.85612.0_484.4365.1_485.3753.0_486.273.8_487.3484.9_487.3903.0_488.3843.0_489.3356.1_489.4063.0_490.335.8_491.3468.1_492.3111.1_492.3891.1_493.3951.0_494.4005.1_495.3882.1_497.3313.8_498.4091.3_499.3892.8_499.428.7_500.3714.9_501.3717.8_501.4342.9_502.4173.3_503.3857.6_504.4253.3_505.3943.9_506.375.9_507.376.5_509.2473.7_509.3723.0_510.3783.0_511.3414.0_512.3874.9_512.6436.8_513.406

4.9_513.422

8.1_513.4634.9_514.4291.1_515.3845.0_515.4265.4_516.4045.1_516.4511.6_517.4268.1_518.4074.8_518.4751.6_519.4165.0_520.39713.1_521.4028.0_522.411.9_523.35111.9_523.4276.0_525.3714.9_526.4366.8_527.40811.9_527.45812.0_528.4891.9_529.4034.0_530.3404.9_530.4443.0_531.3647.7_531.4194.0_532.420.9_533.3615.1_533.3991.3_534.3992.1_535.384

14.2_535.4142.1_535.8386.8_536.39314.6_536.4201.6_537.40415.3_537.4721.6_538.40715.9_538.47715.8_539.438

7.7_540.44614.8_541.13711.1_541.402

7.7_541.454

10.9_542.395.5_543.30811.4_543.4486.1_544.4521.1_545.4251.2_546.4001.2_547.4037.8_547.479.9_549.35511.4_550.4443.7_551.40215.0_552.41915.2_553.42515.7_554.43515.8_555.44115.7_556.4469.2_557.3997.6_557.4806.8_558.4643.6_559.40911.8_559.4156.8_559.46511.4_560.4191.6_560.47811.9_561.42415.7_561.4338.1_562.3937.7_562.4352.1_562.48312.5_563.4386.1_563.4627.7_564.43715.9_564.44916.1_565.44614.7_566.1923.8_567.407

11.4_568.481

7.4_569.420

.5_261.122

7.8_541.455

.4_110.060.5_163.082

.5_195.079

.5_206.110.4_229.140

.5_232.116

.5_302.141

7.8_500.427

.5_519.315

12.0_559.408

.5_204.105.5_259.123

7.8_540.448

7.8_544.482

.4_138.064.5_180.107.5_196.092.5_233.114

.5_313.178

.5_370.194

7.8_499.418

.5_218.108

.5_237.118

15.8_537.428

15.8_538.43312.0_560.418

.5_190.091.5_230.127.5_234.120

.5_236.110

.4_152.073.5_246.121.5_274.1327.7_503.422

Samples5 10 15 20 25

%

0

100

4.470e+004

Kenya

Brazil Kona

Sumatra

Panama

Samples5 10 15 20 25

%

0

100

4.470e+004

Kenya

Brazil Kona

Sumatra

Panama

Other Components in CoffeeAlthough caffeine is one of the more dominant compounds, it is but a small fraction of the other classes of compounds found in coffee bean extracts. Using this metabonomic approach we have been able detect many other markers of geographic origin including plant sterols, an example of which is m/z = 535.40. The trend line for this marker (Figure 5) indicates that, like caffeine, it is present at higher levels in Sumatran coffee.

Figure 5. Trend line for m/z = 535.40, a proposed sterol derivative in coffee bean extracts.

Samples

CONCLUSION

The application of metabonomics to non-mammalian samples has been successfully illustrated by the analysis of whole, roasted coffee bean extracts from different areas of the world. We have demonstrated that the UPLC/MS(TOF)-based approach allows for a rapid and sensitive analysis. The high mass accuracy of the LCT Premier coupled with the superior chromatographic resolution afforded by the ACQUITY UPLC System has facilitated the identification of several markers of interest, such as caffeine and sterol derivatives, which has been successfully confirmed by exact mass.

5 10 15 20 25

%

0

100

2.492e+005

Kenya

BrazilKona

Sumatra

Panama

Samples5 10 15 20 25

%

0

100

2.492e+005

Kenya

BrazilKona

Sumatra

Panama

Waters, Micromass, Ultra Performance LC, UPLC, LCT Premier, Woptics, LockSpray, MarkerLynx, and MassLynx are trademarks of Waters Corporation.All other trademarks are property of their respective owners. ©2004 Waters Corporation Produced in the U.S.A.Nov. 2004 720001058EN KJ-PDF