Material and Methods


LC-MS grade acetonitrile, methanol, water, and 2-propanol were purchased from Biosolve Chimie (Dieuze, France). Potassium hydroxide, n-hexane reagent grade, and

2-propanol HPLC grade were purchased from Carlo Erba (Milan, Italy). Ammonium formate was obtained from Alfa Aesar GmbH & Co KG (Karlsruhe, Germany). Standards of trinonanoin (C9C9C9), triundecanoin (СиСцС,,), tritridecanoin (C13Ci3C13), tripen- tadecanoin (С15С15С15), triheptadecanoin (Ci7Ci7Ci7), trinonadecanoin (C19C19C19), and a C4-C24 even saturated fatty acid methyl ester (FAME) 1000 mg/L hexane solution were purchased from MilliporeSigma (Bellefonte, PA, USA).


Samples of unprocessed whole pistachio nuts from different geographical origins and varieties (Bronte, California prime quality, California second quality, Iran normal, Iran perfect green, Turkey mawardi, Turkey red, Turkey perfect green, Greece) were kindly provided by Pisti S.r.L. (Italy, Bronte, CT). A minimum of five production lots were collected for each variety.

Sample Preparation for REIMS Analysis

The pistachio nut outer shell and skin were removed by hand. Equal portions of the different production lots were combined and the nuts were subsequently crushed using a domestic coffee grinder device. A conductive paste was prepared via the addition of 200 pL deionized water per 1 g of crushed nut material. Approximately 1 g of the pistachio paste was placed on a wet tissue on top of the return electrode plate prior to iknife sampling.

REIMS Analysis

The iknife hand-held sampling device (Waters Corporation, Wilmslow, UK) was used to apply a localized high- frequency electric current to the surface of each sample, which instantly vaporizes molecules from the latter. It consisted of a monopolar cutting device with a shortened knife blade approximately 6 mm long and was applied in auto-cut mode in combination with a diathermy electrosurgical generator (Erbe VIO 50 C) (Erbe, Tuebingen, Germany) at a power of 30 W. Sampling was carried out for 3 to 5 s and, for each sample, technical replicates were analyzed, thus taking into account repeatability of the analysis. Mass spectrometric analysis was performed using a Xevo G2 XS QTOF instrument equipped with an REIMS source comprising a helical coiled ribbon collision surface heated by a constant current power supply set to 4.5 A and 4.2 V (Kanthal D 1.0 x 0.1 mm) (Waters Corporation, Wilmslow, UK). All analysis was performed in REIMS TOF MS sensitivity mode with continuum data acquisition. A matrix of 2-propanol was infused directly into the REIMS source at a constant flow rate of 200 pL min-1 to promote the ionization of lipid species and maintain source cleanliness. The mass resolution was approximately 20,000 FWHM over the mass range of interest. The cone voltage was set at 100 V. MS analysis was performed in negative ionization mode over a mass range of 50-1200 mlz with a (scan) acquisition time of 1 s/scan. Prior to use, the instrument was calibrated using sodium formate in 2-propanol and was infused via the matrix inlet on the REIMS source.

For quality control purposes, the endogenous matrix ion mlz 255.2330, corresponding to the deprotonated molecule of palmitic acid (C16H3102), was used for internal lock- mass correction. At least 20 replicate measurements were collected for each sample in two different laboratories (Waters-Wilmslow and Chromaleont-Messina) in order to obtain some inter-laboratory repeatability data and explore the possibility of transferring the statistical model between different geographical locations. Furthermore, replicate burns of a QC sample (porcine liver) were collected at the start and end of the analytical batch. The intensity of the base peak ion at mlz 699.497 was recorded and plotted for quality control monitoring. The iknife blade was cleaned using methanol after every 15 samples, and the 3-m-long transfer tubing and venturi air pump were cleaned in an ultrasonic bath using methanol at the end of each day.

An untargeted MS analysis was performed to discriminate between pistachio nuts grown in different geographical origins (Turkey, Greece, Iran, Sicily, and USA). In the case of pistachio nuts from the Turkey and Iran samples, more than one variety was present. The training samples were analyzed in two different laboratory locations on different days to take into account technical variation originating from intra- and interlaboratory reproducibility. This step thus generated a total of 360 mass spectra (64 spectra for Sicilian Bronte 32 for Greek normal, 84 for Iranian normal and perfect green, 64 for USA prime and second quality, and 116 for Turkish mawardi, red, and perfect green) that were used to train the database. In addition, samples from a different production lot were collected and analyzed in triplicate and used as an independent validation challenge set.

The mass spectrometer was operated in REIMS MS/MS acquisition mode to generate CID fragments for ions identified as discriminatory features during the multivariate statistical analysis. Precursor ions were isolated in the resolving quadrupole region of the instrument. Argon was used as the collision gas, and collision energy was optimized on a compound by compound basis. The following MS/MS specific settings were applied, LM and HR resolution 10 and 14.5, respectively, prefilter 2.0 V, and ion energy 0.8 V.

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