A novel method for prenylquinone profiling in plant tissues by ultra-high pressure liquid chromatography-mass spectrometry
© Martinis et al; licensee BioMed Central Ltd. 2011
Received: 19 May 2011
Accepted: 21 July 2011
Published: 21 July 2011
Prenylquinones are key compounds of the thylakoid membranes in chloroplasts. To understand the mechanisms involved in the response of plants to changing conditions such as high light intensity, the comprehensive analysis of these apolar lipids is an essential but challenging step. Conventional methods are based on liquid chromatography coupled to ultraviolet and fluorescence detection of a single or limited number of prenylquinones at a time. Here we present an original and rapid approach using ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-quadrupole time-of-flight mass spectrometry (UHPLC-APCI-QTOFMS) for the simultaneous profiling of eleven prenylquinones in plant tissues, including α-tocopherol, phylloquinone, plastochromanol-8 and plastoquinone-9.
Results and discussion
Mass spectrometry and chromatography parameters were optimized using pure standards. Sample preparation time was kept to minimum and different extraction solvents were evaluated for yield, ability to maintain the redox state of prenylquinones, and compatibility with chromatography. In addition to precise absolute quantification of 5 prenyllipids for which standards were available, relative quantification of 6 other related compounds was possible thanks to the high identification power of QTOFMS. Prenylquinone levels were measured in leaves of Arabidopsis grown under normal and high light intensities. Quantitatively, the obtained results were consistent with those reported in various previous studies, demonstrating that this new method can profile the full range of prenylquinones in a very short time.
The new profiling method proves faster, more sensitive and can detect more prenylquinones than current methods based on measurements of selected compounds. It enables the extraction and analysis of twelve samples in only 1.5 h and may be applied to other plant species or cultivars.
In this paper we propose a novel method for prenylquinone analysis using an optimized sample preparation procedure followed by ultra-high pressure liquid chromatography-atmospheric pressure chemical ionization-quadrupole time of flight mass spectrometry (UHPLC-APCI-QTOFMS). Compared to conventional HPLC, UHPLC uses sub-2 μm particle supports which allows for higher efficiency and optimal velocity [16, 17]. Consequently, high throughput separations can be obtained by reducing column lengths and increased flow rates. The QTOF mass spectrometer is particularly well adapted to coupling with UHPLC thanks to its rapid scanning rate . Moreover its high mass accuracy gives access to the determination of molecular formula, an essential feature for reliable compound identification. Different solvents were tested for their extraction yield, their ability to maintain the redox state of molecules as well as their compatibility with reverse-phase UHPLC as injection solvent. Chromatography and mass spectrometry parameters were optimized for speed, selectivity and sensitivity. The applicability of the developed method was illustrated with the simultaneous quantification of several prenylquinones in Arabidopsis thaliana grown under normal and high light conditions.
Results and Discussion
Optimization of MS conditions
While there have been several publications on the LC-MS analysis of tocopherols [19, 20] and vitamin K homologues [21, 22] as they are essential vitamins for human metabolism, we are not aware of any report for plastoquinone-9 (PQ-9) and plastochromanol-8 (PC-8). Thus different QTOFMS parameters were evaluated to obtain maximal sensitivity for these latter molecules together with α-tocopherol (α-T) and phylloquinone (K). Electrospray and APCI were compared in both positive and negative ionization modes using standard solutions at 1 μg/mL. For PQ-9, the oxidized form (PQ) was used as standard. APCI was found largely superior to electrospray for all four compounds. K, PQ and PC-8 gave similar responses in positive and negative APCI but α-T was much better ionized in negative mode (ca. 8-fold), which was thus selected for further experiments. We then tested the effect of the probe temperature on the ionization efficiency. While α-T and K gave a higher signal at lower temperatures (350-500°C), PC-8 and PQ were best ionized at higher temperatures (475-600°C). A temperature of 475°C was selected as the best compromise for the detection of the four compounds. The source cone voltage was varied from 15 to 50 V and the highest response was obtained at 40 V. Other source parameters such as corona current (18 μA), source temperature (120°C) and desolvation gas flow (800 L/hr) had a less important effect on the MS signal. The influence of the mobile phase composition on the ionization efficiency was evaluated. Methanol (MeOH), acetonitrile (ACN) and tetrahydrofuran (THF) were compared and MeOH gave the best signal for all compounds. For α-T and K, the intensity of MS responses in MeOH was 2-5 fold higher than in ACN or THF. For PQ and PC-8 it was 10-40 fold higher. MeOH was thus selected as the organic solvent of choice for further LC method development.
Optimization of LC conditions
Detection of prenylquinones in plant samples
Prenylquinones identified from UHPLC-APCI-QTOFMS data acquired in negative and positive ionization modes.
Optimization of sample preparation
Spontaneous variations in PQ/PQH2 ratio under standard working conditions were determined in the five solvents using a purified PQ standard. No spontaneous reduction was detected (data not shown). Moreover, when spiking PQ in leaf extracts, no increase in PQH2 was observed, suggesting that the reducing agents present in plant tissues are not concentrated enough to promote reduction. However, when ascorbic acid (30 mM) or butyl hydroxytoluene (BHT, 0.05%) were added to PQ solutions prepared in the different solvents, a slight reduction of PQ was observed (about 3% and 1% of the original amount of PQ using ascorbic acid or BHT, respectively). This phenomenon has previously been observed . While anti-oxidant agents are often added to extraction experiments to prevent lipid peroxidation, this procedure does not appear useful for the determination of the redox state of PQ since reduction of the oxidized form may occur. When the PQ standard was chemically reduced with sodium borohydride and then exposed to air at room temperature, an increase of the oxidized form was detected for all solvents starting from 5-7 h after reduction. As a consequence, for the solvent to be effective in maintaining the original PQ/PQH2 ratio, it needs to oxidize the PQH2 present in plant tissues as little as possible. Using CHCl3/MeOH (30:70, v/v), IPA and THF, a PQH2/PQTOT ratio ≥ 0.5 was found in plants exposed to HL conditions, while EtAc led to a PQH2/PQTOT ratio of only 0.3 (Figure 4D). This may be due to lower extraction yield for PQH2 in EtAc rather than oxidation of PQH2 into PQ. Interestingly, when plants grown under normal light intensity (150 μE·m-2·s-1) were extracted in IPA, a complete oxidation of PQH2 was observed, while this did not happen in samples extracted by CHCl3/MeOH (30:70, v/v) and THF (data not shown). This may be explained by the higher amount of antioxidants (tocopherols or other lipid-soluble molecules) naturally present in thylakoid membranes under HL conditions, which could have protected PQH2 from spontaneous oxidation in IPA. A similar profile was observed for UQ-9 (Figure 4F).
Overall, THF represented a good alternative to CHCl3/MeOH (30:70, v/v) since it provided high and reproducible extraction yields, best maintained the redox state of PQ-9 and UQ-9, and was chromatographically compatible. It was thus selected as the solvent of choice for the extraction of biological samples. To determine whether the volume of extraction solvent (500 μL THF for 100 mg of fresh leaf material) was sufficient for extracting most of the prenylquinones, we extracted leaves exposed to HL in 1500 μL or 500 μL (for the latter, the extract was further diluted three times) and extraction yields were compared (n = 3). No significant difference was found, confirming that a volume of 500 μL is sufficient for the extraction of 100 mg of fresh leaves. We noticed that the oxidation of PQH2 became significant about 2 and 5 hours after extraction for plants grown under normal and high light conditions respectively. For this reason, we took good care to always prepare and analyze samples within a maximum period of 1.5 hours. Given the speed of both extraction and analysis, 12 samples can be processed during that period of time. By performing sample preparation and analyses in parallel, a throughput of 100 samples in 8 h could be potentially achieved.
Extraction recovery, matrix effects and limits of quantification
To evaluate sample preparation recovery, two experiments were carried out: first pure standards of α-T, K, PC and PQ were submitted to the extraction procedure. Recovery greater than 95% was obtained for all molecules. To determine if the plant matrix had an impact on the recovery, decyl-plastoquinone was used as non-endogenous structural analogue and spiked before and after extraction of plant samples at identical concentration. Again a recovery greater than 95% was obtained.
APCI is usually less prone to matrix effects than electrospray because ionization occurs in the gas phase. We nevertheless checked if prenyllipids were subjected to suppression or enhancement effects from the Arabidopsis extract. Since these molecules are endogenous in Arabidopsis, a THF extract was prepared and an aliquot was spiked with standard solutions of each prenyllipid. The control extract (C), the standard solutions (SS) and the spiked extract (SE) were injected and the obtained area compared. For all the ions, the area of C+SS were equivalent to those of SE with a variation inferior to 5%. In other words, no significant matrix effect was observed for the analyzed compounds.
The quantification of prenyllipids was based on internal standard calibration. Decyl-plastoquinone was found suitable as an internal standard: it was structurally close to the studied prenylquinones, was readily detected in negative APCI with high selectivity (M- ion at m/z 276.2087), eluted within the chromatographic gradient (retention time 0.44 min), and no matrix effect was observed. For α-T, PQ and PC-8, five calibrations points were used (0.05, 0.2, 1.0, 2.0, and 10.0 μg/mL for α-T; 0.05, 0.2, 1.0, 2.0, and 5.0 μg/mL for PQ-9 and PC-8). When PQ was completely reduced using sodium borohydride, the peak corresponding to PQH2 had an identical area to that of PQ. As a result, PQH2 could be quantified based on PQ calibration curve. For K, whose concentration does not significantly change after HL treatment, only four calibration points were used (0.1, 0.25, 1.0, and 2.5 μg/mL). For all compounds the response was linear over the range of the chosen concentrations with coefficients of determination > 0.99. A signal-to-noise ratio (s/n) of 10 was defined as limit of quantification (LOQ). For α-T, K and PC-8, the LOQ was 20 ng/mL. For PQ-9, an LOQ of 10 ng/mL was attained. The other identified prenyllipids (see Table 1) for which no pure standard was available were relatively quantified.
Effect of high light on prenylquinone profile
Concerning plastoquinone-9, 4-week-old A. thaliana plants grown under normal light conditions showed a PQH2/PQTOT ratio of about 0.25, in agreement with a previous study by Szymanska et al.  that used plants of similar age and a different method for prenyllipids extraction. After continuous HL exposure, plastoquinone-9 total content (oxidized + reduced) did not seem to be significantly altered. Yet, the redox state of the electron acceptor pool changed, with a significant accumulation of the reduced form (PQH2), leading to a PQH2/PQTOT ratio of 0.6 (Figure 6D). This result suggests that the accumulation of PQH2 under HL may not be due to the de novo synthesis of the latter but on the reduction of the already available PQ pool. While these findings are distinct from the increase in PQH2 synthesis reported by Szymanska et al. , this discrepancy may well be attributed to the different growth and light conditions or to the different reference units employed (μg/g fresh weight versus μg/mg chlorophyll).
Among the other prenyllipids identified, plastochromanol-8 levels did not significantly change when plants were exposed to HL (Figure 6E), while total ubiquinone-9 content increased about 1.5-fold. Moreover, the UQH2/UQTOT ratio increased in response to the change in light conditions (Figure 6F), as previously observed by Yoshida et al. .
The presented method introduces for the first time the use of UHPLC-APCI-QTOFMS for simultaneously profiling several prenylquinones in plants. It proves to be fast, reliable, very selective and sensitive for the analyzed molecules, and consume less solvent than conventional methods. By combining it with simple and rapid sample preparation, a single plant can be extracted and analyzed in less than 15 min and twelve samples can be processed in 90 min. Moreover it allows for the detection and tentative identification of molecules for which no pure standard is available. The developed method will be used to profile prenylquinones in various Arabidopsis mutants as well as in other commercially relevant crop species.
The solvents used for extraction were methanol (MeOH, HPLC grade, Chromanorm), chloroform (CHCl3, analytical grade, Normapur) and tetrahydrofuran (THF, analytical grade, Normapur) from VWR (Leuven, Belgium), isopropanol (IPA, HPLC grade) and ethylacetate (EtAc, analytical grade) from Acros Organics (Geel, Belgium). ULC/MS grade MeOH and water from Biosolve (Valkenswaard, The Netherlands) were used for the UHPLC-APCI-QTOFMS analyses.
α-T and K standards of HPLC grade (≥ 99.5%) were purchased from Sigma-Aldrich (Steinheim, Germany). Decyl-plastoquinone (~75%) was obtained from Sigma-Aldrich. PQ-9 and PC-8 standards were provided by Jerzy Kruk (Jagiellonian University, Kraków, Poland). The oxidized and reduced PQ-9 standards were obtained as described in  with slight changes. Briefly, an excess (1 μg) of sodium borohydride (Fluka, Buchs, Switzerland) was added to the oxidized PQ standard (100 ng) to completely reduce it to PQH2. The retention time of both forms was then determined by UHPLC-APCI-QTOFMS. Ascorbic acid was purchased from Carl Roth (Karlsruhe, Germany) and butyl hydroxytoluene (BHT) from Sigma-Aldrich.
Plant material and treatments
Arabidopsis thaliana (Columbia-0) plants were grown on soil under standard growth conditions (150 μE·m-2·s-1, 8/16 h light/dark period, 21/18°C, 55% relative air humidity) according to the protocol described in  with slight modifications. HL treatment was performed on 4- to 5-week-old rosettes by exposure to continuous HL conditions (500 μE·m-2·s-1, 21°C, 55% relative air humidity) for 1 week in a PGC 6HID growth chamber (Percival Scientific, Boone, IA) equipped with 400 W metal halide lamps (Philips).
Arabidopsis leaves from 4- to 5-week-old rosettes were ground in a mortar with liquid nitrogen. Approximately 100 mg of leaf material was then exactly weighed, transferred to a 1.5 mL microcentrifuge tube (Eppendorf, Hamburg, Germany) and swiftly re-suspended in five volumes of the selected solvent (e.g. 500 μL for 100 mg) containing decylplastoquinone at 2 μg/mL as internal standard. Care was taken that no thawing occurred before the solvent was added. Glass beads of about 1 mm of diameter (Assistent, Sontheim, Germany) were added and samples were further homogenized for 3 min at 30 Hz in a tissue lyser (Retsch MM 300, Haan, Germany). Tubes were centrifuged on a benchtop centrifuge (14,000 × g for 3 min at 4°C) and 400 μL of supernatant was then transferred to an appropriate glass vial for immediate UHPLC-QTOFMS analysis.
Liquid chromatography-mass spectrometry analysis
The LC-MS system consisted of a Waters Acquity UPLC™ (Milford, MA) coupled to a Waters Synapt G2 MS QTOF equipped with an atmospheric pressure chemical ionization (APCI) source. Prenyllipids were separated on an Acquity BEH C18 column (50 × 2.1 mm, 1.7 μm) under the following conditions: Solvent A = water; Solvent B = MeOH; 90-100% B in 1.5 min, 100% B for 2.5 min, re-equilibration at 90% B for 0.5 min. The flow rate was 800 μL/min and the injection volume was 2.5 μL. The temperature of the column was set to 60°C and the autosampler chamber was kept at 15°C. Data were acquired with a scan time of 0.4 s over an m/z range of 225-1200 in the negative ion MS mode. The corona current was set to 18 μA and the cone voltage to 40 V. The source temperature was maintained at 120°C and the APCI probe temperature at 475°C. The desolvation gas flow was set to 800 L/hr. The mobile phase was diverted to waste for 0.3 min at the beginning of the gradient. Accurate mass measurements were obtained by infusing a 400 ng/mL solution of the small peptide leucin-enkephalin at a flow rate of 10 μL/min through the Lock Spray™ probe. For the identification of prenyllipids, positive and negative ion MS/MS experiments were carried out using a fixed collision energy of 40 eV and argon as collision gas at a flow of 2.1 mL/min. The quadrupole LM resolution was 4.7, and the HM resolution was 15. MS/MS product ion spectra were acquired over the m/z range 50-1200. Absolute quantities of prenyllipids were determined using standard curves obtained from standard compounds. The concentrations of the calibration points for α-T were 0.05, 0.2, 1.0, 2.0, and 10.0 μg/mL, for PQ-9 and PC-8 0.05, 0.2, 1.0, 2.0, and 5.0 μg/mL. For K, the concentrations were 0.1, 0.25, 1.0, and 2.5 μg/mL. All standard solutions contained decylplastoquinone (internal standard) at a concentration of 2 μg/mL.
Data were processed using Masslynx v4.1 (Waters). Multivariate analysis was carried out using MarkerLynx XS™ (Waters). The following parameters were used: initial and final retention times 0.7-3.0 min, mass range m/z 225-1200 Da, mass tolerance 0.03 Da, retention time window 0.10 min, automatic peak width detection, intensity threshold 1000 counts. The deisotope filtering function was applied. Non-normalized peak areas were generated. Variables were UV-scaled before applying PCA.
We thank Jerzy Kruk for kindly providing purified plastoquinone-9 and plastochromanol-8 standards. GG acknowledges support from the Swiss Plant Science Web. FK was supported by UniNE, SystemsX PGCE, NCCR Plant Survival and SNF 31003A_127380.
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