Metabolite profiling

2.10.1 Carotenoid extraction, partition and saponification

Carotenoids were extracted from 25 mg of freeze dried flower petal tissue using 1 mL of carotenoid extraction buffer [acetone:methanol, 7:3] with 200 mg/mLCaCO3 (Appendix 1). The mixture was then vortexed and held at room temperature for 1 h. The insoluble matter was pelleted and the supernatant removed to a fresh foil-covered 15 mL tube and held at room temperature, while the pellet was then re-extracted four more times using carotenoid extraction buffer without CaCO3. Three of the incubation times were again for 1 h while one extended to overnight.

The five extracted supernatants were combined to give a total extractant volume of ca 4.6 mL and then partitioned with diethyl ether according to the following procedure. A 2 mL aliquot of 10% NaCl (w/v) and then 2 mL of diethyl ether was sequentially added to the extractant with inversion after each addition. If the solution did not partition, then 1 mL of water was also added to aid separation. The top acetone:methanol fraction of the partition was collected to a glass tube. The partition procedure was repeated twice more or until the acetone:methanol phase was colourless. The combined diethyl ether fractions were then dried under O2-free N2 and the carotenoids dissolved in 2 mL of 0.8% BHT:acetone (w/v) as described by Ampowmah-Dwamena et al. (2009). The total carotenoid extractant for each petal sample was stored in the dark at 4 °C.

An aliquot (400 µL) of the 2 mL total carotenoid extractant was then saponified by mixing with 10% potassium hydroxide:methanol (w/v). The mixture was incubated at 4 °C overnight and then cleaned up by re-partitioning as described above, that then the combined diethyl ether fraction were re-dissolved in 400 µL of 0.8% BHT:acetone (w/v).

2.10.2 Spectrophotometric determination of total carotenoid concentration

The total carotenoid concentration in the carotenoid extractant was determined spectrophotometrically (Jasco V-530 UV/Vis spectrophotometer, Jasco, Tokyo, Japan) using chloroform diluted samples. Aliquots of 5 μL or 10 μL of the carotenoid extractants were

diluted with 995 μL or 990 μL respectively of chloroform and read against a chloroform blank. Readings were taken at 480nm, 648 nm and 666nm. The total carotenoid concentration of the extractant was calculated using Wellburn’s equation (Wellburn 1994).

Total carotenoid Cx+c=(1000A480-1.42Ca-46.09Cb)/202 =µg/mL x Dilution factor =µg/mL x (volume of extract) ml =µg x 1000 = mg/weight (tissue) g DW =mg/gDW Cx +c=Total carotenoid Ca=Chlorophyll a=10.91A666-1.2A648 Cb =chlorophyll b=16.36A648-4.5A666

Table 2.4:

Elution gradients of HPLC (A) and LCMS (B) analyses A. HPLC Solvent Time (min) A MeOH B H2O:MeOH (20:80) containing 0.2% ammonium acetate C

MTBE Flow rate (mL/min) 0-2 95% 5% N/A 1 2-10 80% 5% 15% 1 30 30% 5% 65% 1 40 25% 5% 70% 1 45 95% 5% N/A 1 B. LCMS Solvent Time (min) A MTBE B

MeOH Flow rate (µL/min)

0 5% 95% 200

33 62.7% 37.3% 200

33.5-36 90% 10% 300

3.10.3 HPLC analysis of petal carotenoid profile

Samples (50 μL) were separated by HPLC using a Dionex Ultimate 3000 solvent delivery system fitted with a YMC RP C30 column (5 μm, 250 x 4.6 mm), coupled to a 20 x 4.6 C30 guard column (YMC Inc. Wilmington, North Carolina, USA) and a Dionex 3000 PDA detector. The column elution rate was 1.0 mL/min and with a column temperature of 25 oC. Elution was performed using solvent A [MeOH], solvent B [H2O:MeOH (20:80) containing 0.2% ammonium acetate] and solvent C [tert-butyl methyl ether (MTBE)] as a linear gradient starting with 95% A and 5% B, decreasing to 80% A, 5% B and 15% C between min 2 and 10, decreasing to 30% A, 5% B and 65% C by min 30, decreasing to 25% A, 5% B and 70% C at min 40 and returning to 95% A and 5% B by min 45 (Table 2.4 A). This is a modified version of the elution gradient described by Fraser et al. (2000). An aliquot (50µL) of the sample was injected on the HPLC. The majority of carotenoids were detected at 450 nm, although other carotenoids and carotenoid precursors were monitored at 430 nm and 350 nm. The carotenoid content was determined as β-carotene equivalents/gDW of tissue. The β- carotene, and lutein were identified in the extracts by comparison of retention times and on- line spectral data with standard samples. Trans-β-carotene was obtained from Sigma Chemicals (St Louis, Missouri, USA). Other carotenoids were tentatively identified by comparison with reported retention times and spectral data (Fraser et al., 2000; Burns et al., 2003; Xu et al., 2006; Kammfer et al., 2010; Lee et al., 2001) and by comparison with carotenoids present in a spinach sample which was also extracted using the same procedures described (Section 2.10.1).

3.10.4 LCMS analysis of petal carotenoid profile

LCMS was performed by Dr Joyce at Plant & Food Research, Lincoln. The LCMS system comprised a Thermo Electron Corporation (San Jose, CA, USA) Finnigan Surveyor MS pump, Thermo Accela Open Auto sampler (PAL HTC-xt with DLW), Finnigan Surveyor PDA plus detector and a ThermaSphere TS-130 column heater (Phenomenex, Torrance, CA, USA). Each prepared extract (5 μL) was separated using a mobile phase consisting of methyl-

tert-butyl-ether (MTBE) (A) and MeOH (B) by reverse phase chromatography (YMC30, 250 x 2.1 mm), which was maintained at 30 °C with a flow rate of 200 µL/min from time 0 to 33.5 min and follow by a flow rate of 300 µL/min from 33.5 to 39 min (Table 2.4. B). A gradient was applied: tmin/A%/B% as t0/5/95, t33/62.7/37.3, t33.5-36/90/10, t36.5-39.50/5/95 (Table 2.4 B)

and eluent scanned by PDA (400-700nm) and API-MS (LTQ, 2D linear ion-trap, Thermo- Finnigan, San Jose, CA, USA) with atmospheric pressure chemical ionisation (APCI) in the positive mode. Data were acquired for parent masses from m/z 350–1500 amu with fragmentation down to MS3. Data were processed with the aid of Xcalibar®2.10 (Thermo Electron Corporation).

CHAPTER THREE