Ms 8030

Crocin analysis was done as described by Moraga et al. [12 (link)]. For extract preparation, 0.5 mg tissue from tepals, anthers and stigma (collected at three different stages) was crushed with a micropestle in 700 μl Tris–HCl (50 mM, pH 7.5 containing 1 M NaCl), and incubated for 10 minutes on ice. This was followed by addition of 700 μl of chloroform. The extract was then incubated on ice for an additional 10 min. Centrifugation at 3000 g for 5 min at 4°C was done to separate the phases. The lower chloroform phase was evaporated and the dried residues were stored together with the upper aqueous phases at −80°C until high-performance liquid chromatography (HPLC) analysis. The LCMS apparatus of Nexera UHPLC (130 MPa) equipped with MS-8030 (Shimadzu) was used for the Study and data was generated using lab solutions software. Enable RP-C18 column (250 mm × 4.6 mm, 5 μm) was used. The injection volume was 5 μl and flow rate 0.3 ml/min. Mobile Phase A (Water and Acetonitrile ratio 1:1) and mobile phase B (0.1% Acetic acid in water) were used in a linear gradient flow and column temperature was set at 75°C initially.

The LC-MS-system (Shimadzu, Kyoto, Japan) consisted of a CBM-20A controller unit, pumps (LC-20AD), a SIL-20A_HT auto sampler, a CTO-10AS column oven, a valve unit FCV-20AH₂ and an MS-8030. The mobile phase was 0.2% HCOOH. MeOH was added in the following gradient: 0–3.0 min 1% MeOH, 3.1–6.0 min 15% MeOH, 6.1–10.0 min 1% MeOH. The flow rate was set to 1 mL/min. A C18 column (Kinetex 4.6 × 100 mm 2.6 µm 100 Å) was kept at 50 °C. Samples (10 µL) were auto-injected.
For MS detection, electrospray ionization (ESI) was applied as the ion source, while the combination of a triple-quadrupole and an electron multiplier were used as analyzer and detector. Detection times were set from 3.0 min to 10.0 min.
PTCA and TTCA were measured using the positive selected ion monitoring mode in the third quadrupole (Q3-SIM), while PDCA and TDCA were measured using the negative Q3-SIM mode. PDCA and PTCA were identified both by reference substances and mass-to-charge ratio (m/z) (PDCA 154.0; PTCA 200.0). TDCA and TTCA were identified by mass-to-charge ratio (TDCA 172.0; TTCA 218.0) only. The latter were not commercially available. The area under the curve (AUC) obtained by MS analysis was normalized to the total count of all events in the range of 0.5 µm to 50 µm (see Section 4.6). The AUC per count (AUC/count) was used to compare relative amounts of specific degradation products.