# _Mringa oleifera_ Leaf Powder as Functional Additive in Cookies to Protect SH-SY5Y Cells
## 2 Materials and Methods
### Biological Evaluation
#### 2.2.2 HPLC-ESI-TOF-MS Analysis  
For the analysis by HPLC-MS, MLP before and after the thermal treatment was reconstituted in ethanol-water (80:20, _v/v_) at a concentration of 25 mg/mL. In order to avoid solid particles, the samples were passed through a 0.2 \(\upmu\)m regenerate cellulose syringe filter (Millipore, Bedford, MA, USA). The analyses were performed in triplicate on an Agilent 1200 HPLC instrument (Agilent Technologies, Palo Alto, CA, USA) of the Rapid Resolution Series equipped with a binary pump, an online degasser, an auto-sampler and a thermostatically controlled column compartment, as well as a diode array detector. The samples were separated on an Agilent ZorBax Eclipse Plus C18 column (1.8 mm, 4.6 \(\times\) 150 mm). The mobile phase consisted of water with 0.1% formic acid (eluent A) and acetonitrile (eluent B). The gradient elution for resolving the compounds from MLP was as follows: initial conditions 5% B; 10 min, 20% B; 21 min, 28% B; 30 min, 40% B; 35 min, 95% B; 37 min, 5% B; and 42 min, 5% B. The flow rate of mobile phase was 0.5 mL/min, the column temperature was maintained at 25 \({}^{\circ}\)C and the injection volume was 10 mL.  
Detection was performed using a microTOF II mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) within a mass range of 50-1000 m/z operating in negative ion mode. The coupling was performed with an electrospray ionization (ESI) interface (Agilent Technologies, Palo Alto, CA, USA), using a \"T\" type splitter (split ratio 1:3) in order to ensure stable ionization conditions and, consequently, reproducible results. Nitrogen was used as the drying and nebulizing gas. The operating parameters were as follows: drying gas flow rate, 9 L/min; drying gas temperature, 210 \({}^{\circ}\)C; nebulizer, 2.3 bar; capillary, 3500 V; End Plate Offset, \(-\)500 V; Capillary exit voltage, \(-\)120 V; Skimmer 1, \(-\)40 V; Hexapole 1, \(-\)23 V; Hexapole RF, 80 Vpp; Skimmer 2, \(-\)22.5 V; Lens 1 transfer, 50 ms and Lens 1 Pre-Pulse Storage, 3 ms. In order to achieve accurate mass measurements with a precision of 5 ppm, external mass spectrometer calibration was performed with a 5 mM sodium formate solution, prepared in water/2-propanol (1:1, _v/v_) with 0.1% (_v/v_) formic acid. In order to recalibrate the mass spectra, the mixture was injected at the beginning of each run using a 74900-00-05 Cole Palmer syringe pump (Vernon Hills, IL, USA) directly connected to theinterface. Each analysis was calibrated before identification in quadratic plus high-precision calibration (HPC) regression mode.  
All data acquisition and processing operations for chemical characterization of the raw and heat-treated MLP were controlled with HyStar 3.2 and Data Analysis 4.0 software, respectively (Bruker Daltonics, Bremen, Germany). This software provided a list of possible elemental formula by using the Generate Molecular FormulaTM Editor, as well as a sophisticated comparison of the theoretical with the measured isotopic pattern (Sigma Aldrich St. Louis, MO, USA) for increased confidence in the suggested molecular formula. Thus, the tentative compound identification was performed by the molecular formula provided by the software in combination with the information reported in literature [18] and from data bases such as SciFinder, Scopus or SciDirect.