Metabolic Profiling of Piper Species by Direct Analysis Using Real Time Mass Spectrometry Combined with Principal Component Analysis

Plant Material and Chemicals

Plant materials (fruit, leaf and root of Piper nigrum, P. chaba and P. longum) were collected from the plants grown in the herbal garden of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (TBGRI) Palode, Thiruvananthapuram, India. Voucher specimens, P. nigrum TBGT-57944, P. chaba TBGT-51838 and P. longum TBGT-50930, were deposited in TBGRI, Palode, Thiruvananthapuram, India. For direct analysis in real time mass spectrometry (DART-MS) measurements, crude plant parts were used directly as samples. The plant samples were thoroughly washed with tap water and distilled water in order to remove any foreign particle attached to its surface and kept in oven to dry at 40°C.

Optimization of DART-MS Analysis

The mass spectrometer used was a JMS-100 TLC (AccuTof) atmospheric pressure ionization time-of-flight mass spectrometer (JEOL, Tokyo, Japan) fitted with a DART ion source. The mass spectrometer was operated in positive-ion mode with a resolving power of 6000 (full-width at half-maximum). The orifice 1 potential was set to 28 V, resulting in minimal fragmentation. The ring lens and orifice 2 potentials were set to 13 and 5 V, respectively. Orifice 1 was set to a temperature of 100°C. The RF ion guide potential was 300 V. The DART ion source was operated with helium gas flowing at approximately 4.0 L/min. The gas heater was set to 300°C. The potential on the discharge needle electrode of the DART source was set to 3,000 V; electrode 1 was set to 100 V and the grid 250 V. Freshly cut pieces of plant samples were positioned in the gap between the DART source and mass spectrometer for measurements. Data acquisition was from m/z 10 to 1050. Fifteen repeats for each sample were run to ascertain the reproducibility of the analysis. Exact mass calibration was accomplished by including a mass spectrum of neat polyethylene (PEG) glycol (1:1 mixture of PEG 200 and PEG 600) in the data file. /и-Nitrobenzyl alcohol was also used for calibration. The mass calibration was accurate to within 0.002u. Using the Mass Center software, accurate mass measurement and the elemental compositions could be determined on selected peaks. PCA was carried out using Statistica windows version 7.0 (Stat Soft, Inc., Tulsa, OK, USA) statistical analysis software.

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