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Using the Power of Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry for the Non-Target Screening of Natural Products in Herbal Tea
Michelle Misselwitz, Jack Cochran, Julie Kowalski; Restek Corporation, 110 Benner Circle; Bellefonte, PA, USA
Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS) provides enhanced separation and increased peak capacity compared to a one-dimensional GC analysis.
The extra peak capacity and resolution is beneficial for separating potentially important biologically active analytes in complex herbal tea extracts that would otherwise be masked in matrix interferences in a one-dimensional GC analysis.
Herbal tea, a non-caffeinated drink made from plants, herbs, or spices has been used throughout history for its potential medicinal benefit.
Used frequently in Traditional Chinese Medicine (TCM) different blends of herbal material will be formulated depending on the desired medicinal properties.
A South African tea, Sceletium & Honeybush Tea, contains active alkaloids that have been isolated and formulated into a pill format.
The active mesmebrine-type alkaloids, have been found to reduce stress, increase energy ,and uplift your mood. Other herbal teas contain many natural products that could be beneficial biologically active compounds.
Herbal tea samples were extracted using the QuEChERS method. A split injection of the tea extract with no cleanup was used to screen the samples for natural
products. The use of GCxGC-TOFMS was very beneficial for data mining of non-target analytes in the herbal tea
extracts. We took advantage of the full mass spectra for NIST library searches, and the ordered chromatograms produced by GCxGC.
Introduction Experimental
Sceletia Tea and elev8 pills were purchased in South Africa. Other herbal teas were purchased in the USA at a local grocery store.
The contents of 1 tea bag (~1.5 g) was hydrated in 10 mL of freshly boiled water in a capped glass vial. Two elev8 pills were crushed and soaked in 10 mL of water.
The contents were transferred to a 50 mL centrifuge tube with 3 rinses of acetonitrile (10 mL total). An internal standard was added to every sample. Samples were vortexed for 30 min. EN QuEChERS Method extraction salt formulation packets were added to each sample and shaken for 1
min and subsequently centrifuged for 5 min. An aliquot of the supernatant was transferred to an autosampler vial for GCxGC-TOFMS analysis LECO Pegasus 4D GCxGC-TOFMS
1’ Column: Rxi-5Sil MS, 30 m x 0.25 mm x 0.25 µm 2’ Column: Rtx-200, 1 m x 0.25 mm x 0.25 µm Inlet: 250°C, 1 µL split (10:1), Sky Split Precision Liner with wool Oven: 90°C (1 min) to 310°C (5 min) @ 5°C/min, secondary oven + 5°C offset Carrier: He, corrected constant flow, 2 mL/min Modulation time: 2 sec Acquisition: 45 – 550 u @ 100 spectra/sec
Data were processed using the LECO ChromaTOF software by performing an automated peak find and library search for anything with a S/N of at least 100.
Non-Target Screening Structured Chromatograms
So many peaks... So little time
Process data, automated peak find, library search
Sort by Similarity
Some names area easily recognized.
Others may need further investigation.
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 78
51
122
106
94
Caliper - sample "Mate Lemon Tea extract:1", 610 , 1.420 sec , sec to 610 , 1.420 sec , sec - 400 , 0.000 sec , sec to 400 , 0.000 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 78
51
122
106
94 67
Peak True - sample "Mate Lemon Tea extract:1", peak 559, at 610 , 1.420 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 122
78
106
51
94 66
Library Hit - similarity 912, "Niacinamide"
Be critical with NIST library matches. Is it real? More unique spectrum with abundant high m/z
ions gives better confidence in match.
Recognizing chemical names can sometimes be difficult. Using the synonym list is helpful. “Pyridine, 2-chloro-6-(2-furanylmethoxy)-4-
(trichloromethyl)-” is also known as Pyroxyfur, a fungicide.
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 81
53
107 71
Caliper - sample "Mate Lemon Tea extract:1", 514 , 1.110 sec , sec to 514 , 1.110 sec , sec - 400 , 0.000 sec , sec to 400 , 0.000 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 81
53
107
Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514 , 1.110 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 81
53
Library Hit - similarity 910, "Pyroxyfur"
With less spectral information it is difficult to assign a positive match.
The time-of-flight mass spectrometer can acquire a full mass range without sacrificing sensitivity, making non-target
screening possible.
Niacinamide is part of the vitamin
B group. It has anti-inflammatory,
anti-anxiety properties.
Pyroxyfur is a pyridine fungicide.
Increased Resolution and Spectral Deconvolution
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 135
152
77
63
107
121 169 434 285 399 357 231
Caliper - sample "Pau d'Arco Tea extract:1", 786 , 0.790 sec , sec to 786 , 0.790 sec , sec - 150 , 0.000 sec , sec to 150 , 0.000 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 65
169
141
115
87
47 434 285 356
Peak True - sample "Pau d'Arco Tea extract:1", peak 292, at 786 , 0.790 sec , sec
50 100 150 200 250 300 350 400 450 500 550
200
400
600
800
1000 170
115 141
63 89 39
155
Library Hit - similarity 948, "o-Phenylphenol"
In order to maximize resolution in a GCxGC contour plot two columns with different selectivity must be chosen.
We used a “5% phenyl-type” column in the first dimension and
a trifluoropropylmethyl column in the second dimension.
500 1000 1500 2000 2500
5e+006
1e+007
1.5e+007
2e+007
2.5e+007
3e+007
3.5e+007
4e+007
4.5e+007
1st Time (s)TIC
m/z 204, 218, 289
Flavonoids – polyphenolic compounds that may
reduce the risk of certain cancers.
Phytosterols – may help reduce blood cholesterol levels.
The GCxGC-TOFMS contour plots are ordered which aids in compound identification. Homologous series of compounds and compound classes elute together in bands
across the chromatogram.
The South African Sceletium tortuosum herb has been extracted for the medicinally relevant mesembrine-type alkaloids.
Mesembranol
Mesembrine
Mesembrenol
Mesembrenone
m/z 204, 218, 289
elev8TM
“elevates mood, assists in coping with stress”
Sceletium and Honeybush Tea
“The Cup that Cheers”
The mesebrine-type alkaloids have shown to elevate mood, reduce stress
and promote a sense of calm
The minor mesembrine alkaloid mesembranol was not detected in the tea sample, however many other beneficial compounds (phytosterols, flavonoids) that were present in
the tea were not in the tablet.
A reconstructed 1st dimension separation of the Pau d’Arco tea extract shows what a 1D analysis would
look like. Peak capacity is significantly reduced making non target screening difficult.
Although the “caliper” mass spectra does not show a
close match to the library hit for the fungicide
o-phenylphenol, spectral deconvolution provided a
“peak true” spectra that has a library similarity of 948.
Caliper
Peak True
Library Hit o-Phenylphenol