Mass Spectrometry & Spectroscopy


Ambient Ionisation MS and GC-MS Analysis and Identifi cation of Aroma Compounds in Agave Based Alcoholic Beverages


Robert B. Cody, Jeol USA, Inc, Email: cody@jeol.com


Mezcal and tequila are alcoholic beverages produced from the Agave plant. Tequila is produced exclusively from the blue Weber agave (Agave tequilana) while mezcal can be produced by any of approximately 30 different agave species. Agave hearts are roasted in the process of making mezcal, giving it a characteristic smoky fl avor that distinguishes mezcal from tequila. Reposado (‘rested’) tequila is aged for between two months and one year in oak barrels.


Volatile aromas from three different varieties of mezcal and a reposado tequila were analysed by using three different mass spectrometers: a time-of-fl ight mass spectrometer equipped with a Direct Analysis in Real Time (DART) ion source (JEOL AccuTOF-DART 4G), a quadrupole GC-MS system (JEOL Q1500 GC), and a GC- high-resolution time- of-fl ight MS (GC-HRTOFMS) system (JEOL AccuTOF GCX Plus). The different systems provided different, and often complementary, pieces of information. Ambient ionisation rapidly distinguished between the different samples and identifi ed specifi c chemical differences but did not distinguish between isomers. Chemometric analysis (SpectralWorks’ AnalyzerPro XD) found compound-specifi c differences between the samples using data from the quadrupole GC-MS system. The GC-HRTOFMS and data analysis software (JEOL msFineAnalysis) gave the most detailed and specifi c analysis of each sample by integrating data from electron ionisation (EI) and soft ionisation (photoionisation and fi eld desorption).


Experimental Samples


Two single-varietal mezcals were analysed, one from Agave tobala (Agave potatorum) and one from Agave americana var. oaxacensis, sub-varietal arroqueño. The third mezcal (‘Ensamble’) was a blend containing A. tobala, A. arroqueño, and three other agaves. A reposado tequila was also measured for comparison. Two separate bottles of Ensamble from different batches were analysed by AccuTOF-DART and AccuTOF-GCX.


Ambient Ionisation Mass Spectrometer (AccuTOF-DART 4G)


Data were acquired, calibrated, summed and background subtracted with JEOL msAxel software. Oxygen Adduct Formation (OAF) [1] and negative ion measurement was carried out using capillary action to sample each beverage with a narrow-diameter 5 µL glass micropipette. Liquid was vacuum aspirated into the sampling orifi ce of the mass spectrometer (Orifi ce 1) by positioning the tip of the pipette against the orifi ce. Replicate measurements were made for each sample at a rate of 9-10 seconds per sample. The RF ion guide was set to 500V to pass ions larger than approximately m/z 60. The atmospheric pressure interface potentials were Orifi ce 1: -15V, Ring Lens: -5V, Orifi ce 2: -5V and orifi ce 1 was heated to 120°C. Fomblin Y was measured as an external mass reference standard [2]. Mass Mountaineer software (massmountaineer.com) was used for data interpretation and chemometric analysis of the high-resolution mass spectra.


Positive-ion DART mass spectra were measured by inserting a glass tee between the DART ceramic insulator and Orifi ce 1 and sampling the headspace from approximately 200 µL of each liquid in a GC autosampler vial by holding the mouth of the vial against the open end of the tee.


Gas Chromatograph


A PAL SPME Arrow (Restek 1.10 mm: DVB/C-WR/PDMS Cat# 27875) was used to sample beverage headspace for all GC-MS analysis. The gas chromatograph conditions are given in Table 1.


Table 1. GC Conditions. -Gas chromatograph-


Inlet Injector Injector liner Column Carrier gas fl ow Oven temperature program:


Agilent 7890B Split/splitless


260°C, 20:1 split


Restek Topaz Liner (SPME) 1.8 mm x 6.5 x 78.5 Cat# 23280


Restek BAC-1 Plus 30m x 0.32mm ID 1.8 µ df


2 mL/min (He, constant fl ow) 40°C (1 min), 10°C/min to 260°C.


Quadrupole Mass Spectrometer (Q1500 GC)


Data were acquired with JEOL msPrimo software. SpectralWorks’ AnalyzerPro XD software was used for analysis. Three replicate injections were made for each sample. Table 2. Q1500 MS Conditions.


Ionisation: EI (70 eV, 50 µ A) Ion source temperature GC interface temperature Scan


200°C 250°C


m/z 35-500, step 0.1, cycle 460 ms


GC-TOF Mass Spectrometer (AccuTOF-GCX Plus) GC-MS


Samples were measured with the combination electron ionisation/photoionisation (EI/ PI) source. Samples were alternately measured in EI mode and PI mode and some samples were measured using fi eld ionisation (FI) with the combination EI/FI ion source. JEOL msFineAnalysis (version 2) software was used for automatic chromatographic deconvolution and reporting of EI and PI data. The msFineAnalysis report combined exact mass and isotope information with results of searching the NIST 2020 mass spectral database into an integrated report with optional interactive inspection of each assignment.


Table 3. AccuTOF-GCX Plus MS Conditions.


Ionisation (EI/PI) EI (70 eV, 300 µ A) / PI (Deuterium lamp,10.8 eV)


Ionisation (EI/FI) EI (70 eV, 300 µ A) / FI (JEOL FI Emitter) Resolving power


Ion source temperature GC interface temperature Spectral acquisition


Results Ambient ionisation: AccuTOF-DART 4G


Positive-ion DART mass spectra were dominated by the proton-bound dimer of ethanol, with minor contributions from other components. Although solid-phase microextraction (SPME) can improve the positive-ion DART sensitivity for minor components, negative-ion DART was chosen because ethanol is not readily deprotonated.


Negative-ion DART is very sensitive to acidic compounds such as carboxylic acids and phenols. Acidic compounds appeared in the negative-ion mass spectra as deprotonated molecules [M – H]—


detected as even nominal m/z values (Figure 1). Although the ethanol m/z is below the detected range, its oxygen adduct is present at m/z 78.031. The majority of peaks have elemental compositions assigned to deprotonated organic acids or oxygen adducts. Peaks at m/z 60.994 and 70.993 are background peaks for HCO3


with odd nominal m/z values and oxygen adducts [M + O2]—


incompletely subtracted. Lactic acid is present in all samples and acetic acid is present in the mezcal samples, but not in the tequila. The tequila sample showed abundant octanoic acid and decanoic acid peaks at m/z 143.105 and 171.138.


— and HCO4 — that are were


>10,000 (FWHM)


250°C (EI and PI) or 40°C (FI) 250°C


m/z 35-800, 0.4 s cycle


INTERNATIONAL LABMATE - SEPTEMBER 2020


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