Sciex os 1

Prior to HPLC-ESI-MS analysis, the freeze-dried oligopeptide was rehydrated with 1.0 mL of Milli-Q water. Before being used, the water was boiled for 5 min and then cooled to 4 °C. The rehydrated solution was stored at −20 °C until analysis.
HPLC-ESI-MS was carried out on a SCIEX X500R Q-TOF mass spectrometer (Framingham, U.S.A.). The MS conditions were as follows: ESI-MS analysis was performed using a SCIEX X500R Q-TOF mass spectrometer equipped with an ESI source. The mass range was set at m/z 100–1500. The Q-TOF MS data were acquired in positive mode and the conditions of MS analysis were as follows: CAD gas flow-rate, 7 L min⁻¹; drying gas temperature, 550 °C; ion spray voltage, 5500 V; declustering potential, 80 V. Software generated data file: SCIEX OS 1.0.

Prior to the HPLC-ESI-MS analysis, the lyophilized mixture was rehydrated with 1.0 mL of Milli-Q water. Before being used, the water was boiled for 5 min and then cooled to 4 °C. The rehydrated solution was stored at −20 °C until analysis.
HPLC-ESI-MS was carried out on a SCIEX X500R Q-TOF mass spectrometer (Framingham, U.S.A.). The MS conditions were as follows: ESI-MS analysis was performed using a SCIEX X500R Q-TOF mass spectrometer equipped with an ESI source. The mass range was set at m/z 100–1000. The Q-TOF MS data were acquired in the positive mode, and the conditions of MS analysis were as follows: CAD gas flow-rate, 7 L min⁻¹; drying gas temperature, 550 °C; ion spray voltage, 5500 V; declustering potential, 80 V. Software generated data file: SCIEX OS 1.0.

For ranking the marker ions according to their importance, the VIP (variable importance in the projection) plots were used. To illustrate the role of these ions as markers, trend plots were constructed. Another filtration was performed by MetaboAnalyst open-source software v 5.0 (https://www.metaboanalyst.ca), which was used for receiver operating characteristics (ROC). The area under the ROC curve (AUC), t-test false discovery rate (FDR) adjusted p-value, and fold change (FC) parameters were determined for the created binary models of saffron stigmas and potential plant adulterants. The most important variables with VIP scores > 1 and AUC > 0.8 were chosen.
To verify whether the marker is specific for only one or more adulterants, RAW UHPLC-HRMS/MS data and software SCIEX OS 1.5 (SCIEX, Canada) were used. This software was also used to determine the lowest detectable amount of potential adulterant in a mixture with saffron when using respective marker ions. The detectability of individual marker ions in extracted ion chromatograms was carefully controlled; the lowest detectable amount of respective adulterant in a mixture of saffron stigmas and individual adulterants corresponded to signal-to-noise ratio of 10:1.

To control the authenticity of unknown saffron samples in terms of their dilution by some plant adulterants, a targeted screening of 82 markers shown in Table S1 – Markers of saffron and potential adulterants and their characteristics in Supplementary materials was performed. The internal database of these markers was included in the SCIEX OS 1.5 software. This software enabled the comparison of the retention time, exact mass, isotopic pattern in the MS spectrum, and fragments in the MS/MS spectrum of the marker in the measured sample and in the library. The analyst will then check these results. To confirm that the analyzed sample is authentic saffron, all saffron markers from the internal library should be detected, and their relative abundance should not be significantly different from the values in Table S2– Relative abundance of saffron markers in samples of stigmas and saffron powder in Supplementary materials. A sample would be considered adulterated supposing more than 80% of the markers of one adulterant were detected. The limit of 80% is based on the Pareto principle.

The internal database of markers was created using LibraryView 1.0.3. software (SCIEX, Canada) and was subsequently imported into the SCIEX OS 1.5 software (SCIEX, Canada). The formula or exact measured mass, retention time, and MS/MS spectra of all markers are saved in this database. This software was also used to evaluate unknown samples in which markers from the database were searched.

UHPLC separation was performed on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 1.7 μm) at 45 °C on the SCIEX ExionLC™ AC system. Mobile phases consisted of water (A) and acetonitrile (B), both with 5 mM of formic acid. The LC flow rate was 0.5 mL and the mobile phase eluted under the following linear gradient conditions: (A:B, v/v) isocratic elution at 95:5 for 0.5 min, from 95:5 to 5:95 in 7.5 min, isocratic elution at 5:95 for 0.5 min, and final re-equilibration for 2.5 min to the initial condition before each injection. Total run time was 10 min.
All analyses were performed using a quadrupole time-of-flight SCIEX X500R QTOF mass spectrometer (Sciex, Darmstadt, Germany) equipped with a Turbo VTM ion source operating in electrospray positive-ion mode. MS and MS/MS data were collected for each sample using SWATH™ Acquisition mode [17 (link)]. Data acquisition included a preliminary TOF-MS high-resolution scan followed by SWATH™ Acquisition using variable window setup (12 windows covering mass range from 150 to 465 m/z at 0.025 resolving power), resulting in a final cycle time of 0.564 s. Data were acquired using the SCIEX OS 1.5 Software.