Purification, characterization and quantitative analysis of swainsonine
4.3. Results and Discussion
electrons can be detected at m a elengths of 200 nm (Gao et al., 2008). Hence, the mUV wavelength (205 nm) was considered for swainsonine detection. The partially purified broth fractions were HPLC analysed using different mobile phases and gradient runs with variable flow rates. The three different mobile phases with gradient proportions (program) were optimized for maximum peak resolution and minimal signal-to-noise ratio (S/N) for chromatographic analysis of swainsonine (Fig.
4.3). Method A resulted in a major peak between 2.12-2.65 min with two minor peaks thereafter between elution time 5.2-5.8 min. The elution of major peak before 3 min of initial run of water (Solvent-B) might have resulted in the poor chromatographic resolution of swainsonine due to its extreme hydrophilicity and polarity (Fig. 4.3a) (Stockigt et al., 2002). Hence, swainsonine elution was further optimized using Method B and Method C, which resulted in a major chromatographic peak between elution time 5.59-6.13 min (Fig. 4.3b) and 5.85-6.23 min (Fig. 4.3c), respectively. The optimal gradient elution of products were obtained with Method C using acetonitrile (solvent-A) and 0.1 % TFA in water (Solvent-B) as mobile phase (Fig. 4.3c). The chromatographic separation of major peaks in partially purified broth fractions was further confirmed with standard swainsonine runs with elution time 5.95-6.52 min followed by MS analysis of the HPLC fractions (Fig. 4.3a-b). The chromatographic separation of swainsonine was further calibrated by reducing the flow rates to 0.25 ml/min from 5.0-10.0 min, to resolve the two separate peaks between te = 5.25-7.10 min. Hence, two separate peaks were resolved at te = 6.10-6.92 min (comparable to the elution time for swainsonine standard) and an unknown peak between 7.24-8.20 min (Fig. 4.4c). The corresponding peak fractions were further characterized using mass spectrometry analysis, with respective MS1 fraction [M]+ 173.04, [M+H]+ 174.30 and [M+2H]+ 175.04 for swainsonine (insets of Fig. 4.4a-c). The HPLC
quantification of swainsonine was performed using the standard curve between swainsonine concentrations (µg/ml) and peak area (mAU.min) with regression equation:
Where Y = peak area for swainsonine, X = swainsonine concentration with regression coefficient R2 = 0.98 (Fig. 4.5).
The average swainsonine concentration was determined to be 7.67±0.33 µg/ml.
The MS-ESI+ characterization of the HPLC fractions provided the sensitive analysis method for swainsonine in Metarhizium fermentation broth. The characteristic MS1 [M+H]+ m/z = 174 was observed in both standard and purified samples, which confirmed the presence of swainsonine.
4.3.3. Liquid chromatography-mass derived purification
The selective mass derived purification can be used directly with excellent recovery and purity without involving the tedious purification strategies (Kagan et al., 2009). Here, the approach is based on unique fragmentation patterns requiring either a precursor MS1 ion scan or a neutral loss scan observed under MS/MS2 to generate the species specific quantification methodology as reported by Chen et al., (2012).
Solvent system A extracted broth was selected for LC-MSD purification. Swainsonine MS1 fraction [M+H]+ 174.36±0.21 was eluted at time (te) 4.91±0.04 min. The MS1 fractions were collected between te = 4.41-4.78 min in vial-1 and 4.80-5.15 min in vial-2. Native mass fractions of (m/z 173) were eluted between te = 5.66-6.14 min in vial-4 but at negligible signal counts (Fig. 4.6). The purified fractions were further analyzed and validated for swainsonine purification with analytical LC-MS system.
characteristic m/z = 174.10 under MS1 scan showing 3.74×105 counts (Fig. 4.7b), which further confirmed the precision of MSD purification method.
4.3.4. LC-MSD ESI-MS+ quantification
LC-MSD purification was simultaneously accompanied with mass derived quantification using the MS1 ion counts [M+H]+ 174.36±0.21 in selected ion monitoring (SIM) mode. The standard curve with regression equation:
Where, Y = MS1 ion counts (intensity) for swainsonine, X = swainsonine concentration (µg/ml) with regression coefficient R2 = 0.99 (inset of Fig. 4.8).
Swainsonine concentration was expressed as an average of three replicate runs with standard deviation values, 7.85±1.59 µg/ml (Fig. 4.8). The lack of any interfering peak under the SIM-chromatogram demonstrated the high selectivity of the method as described by John et al., (2010). Thus, the mass derived technique was found to be more rapid with high accuracy, selectivity and sensitivity for both qualitative and quantitative analyses.
4.3.5. Comparative evaluation of conventional HPLC and mass derived quantitative methods
4.3.5.1. Limit of detection
The lower detection limit in conventional HPLC with mUV (205 nm) detection was as low as 1-2 µg/ml, whereas the same was 0.25-1 µg/ml in MSD-ESI+ mode with significant values of ion count [M+H]+ 174.36±0.21.
4.3.5.2. Linearity
Both HPLC and MSD quantification showed the linear calibration curves in the closed concentration ranges of 1-10 µg/ml (HPLC) and 1-8 µg/ml, respectively.
4.3.5.3. Selectivity and precision
The selectivity in mUV-HPLC method was comparatively lesser with various non-specific peaks, whereas in MSD method only selected mass fractions [M+H]+ 174.36±0.21 were observed in SIM-mode, proving its more specific nature. The methods’ precision was indexed with the relative standard deviation (RSD) values for chromatogram data. The observed RSD values for peak area and MS1 ion counts were 4.31 % and 4.04 % in HPLC and MSD methods respectively. The RSD values for sample elution times were 1.25 % for HPLC and 0.79 % for LC-MSD methods, which implies the higher degree of precision for mass spectrometry based detection procedure.
4.3.5.4. Inter-method accuracy
The concentration determined from the HPLC and MSD based methods were marginally varied with RSD value of 1.63 %. Hence, the data exhibits a higher and admissible degree of accuracy between to swainsonine concentrations determined.
4.3.5.5. Ruggedness
Furthermore, the investigation of ruggedness under a confined range of different HPLC and MSD conditions proved the method to be stable and rugged.