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Table 3 Effects of solvents, extraction duration, phase separation and storage on extraction reproducibility and efficiency.

From: Proposal for field sampling of plants and processing in the lab for environmental metabolic fingerprinting

  Duration for first extraction Ξ (mean ± sd)   Number of metabolites (mean ± sd)  
a) Solvents
1:0 CH3OH:CH2Cl2 0 h 0.74 ± 0.02 a 1544 ± 49 A
  1 day 0.85 ± 0.03 b 1980 ± 47 B
  1 week 0.71 ± 0.02 c 574 ± 23 C
3:1 CH3OH:CH2Cl2 0 h 0.78 ± 0.03 d 1677 ± 46 D
  1 day 0.77 ± 0.02 d 647 ± 16 E
  1 week 0.75 ± 0.02 ade 602 ± 23 E
2:1 CH3OH:CH2Cl2 0 h 0.83 ± 0.02 d 1730 ± 52 F
  1 day 0.86 ± 0.05 bef 616 ± 13 CE
  1 week 0.72 ± 0.02 c 605 ± 12 CE
1:1 CH3OH:CH2Cl2 0 h 0.82 ± 0.03 g 1712 ± 52 DF
  1 day 0.88 ± 0.01 f 2040 ± 20 G
  1 week 0.72 ± 0.01 ac 649 ± 25 E
b) pH a
pH 2 - pH 2 - pH 2   0.81 ± 0.06 abc 1501 ± 20 AB
pH 2 - pH 6 - pH 9   0.85 ± 0.16 cd 1509 ± 16 AB
pH 2 - pH 9 - pH 6   0.90 ± 0.03 e 1513 ± 28 AB
pH 6 - pH 6 - pH 6   0.91 ± 0.04 e 1538 ± 29 AB
pH 6 - pH 2 - pH 9   0.93 ± 0.02 e 1545 ± 14 A
pH 6 - pH 9 - pH 2   0.89 ± 0.02 de 1380 ± 226 B
pH 9 - pH 9 - pH 9   0.81 ± 0.26 bc 1498 ± 10 AB
pH 9 - pH 2 - pH 6   0.80 ± 0.05 bc 1510 ± 29 AB
pH 9 - pH 6 - pH 2   0.79 ± 0.02 b 1505 ± 46 AB
c) Phase separation method of three extraction steps
A) Extraction in CH3OH:CH2Cl2, H2O added to pooled supernatant (2:1:1)   0.99 ± 0 a 1662 ± 14 A
B) Extraction in CH3OH:CHCl3, H2O added to pooled supernatant (2:1:1)   0.93 ± 0.02 b 1594 ± 20 B
C) Extraction in CH3OH:CH2Cl2, H2O added to every supernatant (2:1:1)b   0.97 ± 0.01 a 1627 ± 18 AB
D) Extraction in CH3OH:CH2Cl2 (2:1), removal of aqueous phase and addition of new solvent mixture after every extraction stepb   0.97 ± 0 a 1656 ± 23 A
d) Storage c
-80°C   0.85 ± 0.04 a 831 ± 5 A
4°C   0.91 ± 0.01 b 849 ± 25 A
  1. aSubsequent extraction steps with the solvent mixture methanol:dichloromethane 2:1 of different-pH.
  2. Addition of 0.1% formic acid and of 0.1% ammonia solution respectively was used to adjust pH to 2 or 9.
  3. The untreated solvent mixture had a pH between 6 and 6.5.
  4. bTwo parts of H2O were added again to the final pooled supernatant.
  5. cSamples were kept after first extraction in 2:1 methanol:dichloromethane at room temperature for 1 week until further processing. Completely processed extracts were stored at different temperatures before analysis.
  6. Similarities between chromatograms, calculated as Ξ values (pairwise comparisons, formula (2)) and number of metabolites (peaks) (mean ± standard deviation) in chromatograms of replicate extractions (N = 5, for Table 3b: N = 3) of Plantago lanceolata leaf material. Leaf samples were extracted threefold whereat the first extraction step was kept at room temperature for one week before further processing (if not noted otherwise, see a). Samples were analysed by LC-MS. The closer Ξ is to 1, the more similar are the resulting chromatograms. Differences between Ξ values and number of metabolites were calculated by ANOVA followed by Tukey's HSD tests. Different lower case letters indicate significantly different Ξ values (a): F3,16 = 3.24, b) F8,19 = 2.48, c) F3,16 = 3.24, d) F1,8 = 5.32, for all P < 0.05), different upper case letters refer to significantly different numbers of metabolites (a): F3,16 = 11.38, b) F8,19 = 0.96, c) F3,16 = 5.36, d) F1,8 = 1.77, for all P < 0.05).