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.

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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

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Peak True - sample "Mate Lemon Tea extract:1", peak 559, at 610 , 1.420 sec , sec

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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.

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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

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Peak True - sample "Mate Lemon Tea extract:1", peak 462, at 514 , 1.110 sec , sec

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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

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1000 135

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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

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Peak True - sample "Pau d'Arco Tea extract:1", peak 292, at 786 , 0.790 sec , sec

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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.

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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