KRA-03

14  A vula et al . : J ournal of AOAC I nternational V ol . 98, N o . 1, 2015

1.8 µm). The mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) at a flow rate of 0.23 mL/min, with the gradient elution program as given in Table 1. Each run was followed by a 5 min wash with 100% B and an equilibration period of 5 min with 85% A/15% B. A 2 μL volume of sample was injected. The column temperature was 35 ° C. The mass spectrometric analysis was performed with a hybrid QToF mass spectrometer (Model No. G6530A, Agilent Technologies) equipped with an ESI source with Jet Stream technology using the following parameters: drying gas (N 2 ) flow rate, 9.0 L/min; drying gas temperature, 250 ° C; nebulizer, 35 psig; sheath gas temperature, 325 ° C; sheath gas flow, 10 L/min; capillary, 3500 V; skimmer, 65 V; Oct radio frequency (RF) V, 750 V; and fragmentor, 125 V. All operations and acquisition and analysis of data were controlled by Agilent MassHunter Acquisition software version A.05.00 and processed with MassHunter Qualitative Analysis software Version B.06.00. Each sample was analyzed in the positive ion mode over the range of m/z = 100–1100 and extended dynamic range (flight time to m/z 1700 at 2 GHz acquisition rate). Accurate mass measurements were obtained by means of reference ion correction using reference masses at m/z 121.0509 (protonated purine) and 922.0098 [protonated hexakis (1 H , 1 H , 3 H -tetrafluoropropoxy) phosphazine or HP-921] in the positive ion mode, while m/z 112.9856 [deprotonated trifluoroacetic acid (TFA)] and 1033.9881 (TFA adducted HP-921) were used in the negative ion mode. The compounds were confirmed in each spectrum. For this purpose, the reference solution was introduced into the ESI source via a T-junction using an Agilent Series 1200 isocratic pump and a 100:1 splitter set at a flow rate of 20 µL/min. For recording ToF mass spectra, the quadrupole was set to pass all ions (RF only mode), and all ions were transmitted into the pusher region of the ToF analyzer where they were mass analyzed with a 1 s integration time. For the ESI-MS/MS collision induced dissociation (CID) experiments, precursor ions of interest were mass selected by the quadrupole mass filter. The selected ions were then subjected to collision with nitrogen in a high pressure collision cell. The collision energy was optimized to afford good product ion signals, which were subsequently analyzed with the ToF mass spectrometer. Analysis was performed in the reflectron mode with a resolving power of about 10,000 at m/z 922. The instrument was set to the extended dynamic range (up to 10 5 with lower resolving power). MS/MS spectra were recorded simultaneously at a rate of 2.0 spectra/s. In order to filter selected precursor ions and their isotopes for MS/MS, an isolation window of 1.3 m/z was set for the quadrupole. MS/MS studies were performed by isolating [M+H] + ions. The fragmentation pattern was obtained Table 1. Mobile phase gradient elution program Time, min A, % B, % 0.00 85 15 20.00 65 35 30.00 0 100 35.00 0 100

Figure 1. Structures of reference compounds.

for detailed structural analysis of alkaloids. This paper describes a method to resolve and characterize 12 indole and oxindole diastereomer alkaloids. The instrumentation consists of an ultra-HPLC (UHPLC) system coupled with a QToF mass spectrometer that can be used for chemical fingerprinting analysis of M. speciosa and is also suitable for the QC of various commercial samples. The fragmentation patterns for 7-hydroxymitragynine [ 1 ], isospeciofoline [ 2 ], isospeciofoleine [ 3 ], isorotundifoline [ 4 ], corynoxine B [ 5 ], corynoxine [ 6 ], 7β-hydroxy-7 H -mitraciliatine [ 7 ], paynantheine [ 8 ], mitragynine [ 9 ], speciogynine [ 10 ], 3-isopaynantheine [ 11 ], and speciociliatine [ 12 ] were studied with proposed structures (Figure 1) for each significant product ion. With this characterization and chromatographic optimization, alkaloidal mixtures containing a large number of diastereoisomers were separated in extracts of M. speciosa leaves. The method offered more information about the chemical constituents of M. speciosa with the diastereomeric alkaloids identified and characterized according to retention times (RTs) and mass spectra. UHPLC/QToF-MS Instrumentation and Conditions The UHPLC system was an Agilent Technologies (Santa Clara, CA) Series 1290 comprising the following modular components: a binary pump, vacuum solvent microdegasser, autosampler with 100-well tray, and thermostatically controlled column compartment. Separation was achieved on an Agilent Zorbax SB-octylsilyl (C8) RRHD column (2.1×100 mm, Experimental