Analytical Method To Detect and Quantify Avocatin B in Hass Avocado

Article Cite This: J. Nat. Prod. 2018, 81, 818−824

pubs.acs.org/jnp

Analytical Method To Detect and Quantify Avocatin B in Hass Avocado Seed and Pulp Matter Nawaz Ahmed,† Richard W. Smith,‡ Juan J. Aristizabal Henao,§ Ken D. Stark,§ and Paul A. Spagnuolo*,† †

Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada Department of Chemistry, University of Waterloo Mass Spectrometry Facility, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada § Department of Kinesiology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada ‡

S Supporting Information *

ABSTRACT: Avocatin B, an avocado-derived compound mixture, was demonstrated recently to possess potent anticancer activity by selectively targeting and eliminating leukemia stem cells. Avocatin B is a mixture of avocadene and avocadyne, two 17-carbon polyhydroxylated fatty alcohols (PFAs), first discovered in avocado seeds; their quantities in avocado pulp are unknown. Analytical methods to detect avocado seed PFAs have utilized NMR spectroscopy and GCMS; both of these lack quantitative capacity and accuracy. Herein, we report a sensitive LC-MS method for the quantitation of avocadene and avocadyne in avocado seed and pulp. The method has a reliable and linear response range of 0.1−50 μM (0.03−17.2 ng/μL) for both avocadene and avocadyne (r2 > 0.990) with a lower limit of quantitation (LLOQ) of 0.1 μM. The intra- and interassay accuracy and precision of the quality control (QC) samples at LLOQ showed ≤18.2% percentage error and ≤14.4% coefficient of variation (CV). The intra- and interassay accuracy and precision for QC samples at low and high concentrations were well below 10% error and CV. This method was successfully applied to quantify avocadene and avocadyne in total lipid extracts of Hass avocado pulp and seed matter. ashman et al. in 1969 were the first to discover C17 to C21 carbon-chain polyhydroxylated fatty alcohols (PFAs) in avocado seeds (Persea americana Mill.; Lauraceae).1,2 Avocadoseed-derived PFAs are a group of lipids with a long aliphatic chain, having a terminal unsaturation of either an olefinic (alkene) or an acetylenic (alkyne) nature and multiple hydroxylations on the opposing end. Since their discovery, avocado PFAs have been used in topical cosmetic formulations for skin care products3,4 and as food additives due to their insecticidal, antimicrobial, and spore-inhibiting properties.5−7 Our laboratory previously determined that avocatin B, a mixture of avocadene and avocadyne (Figure 1A), possesses novel anticancer activity by accumulating in mitochondria and selectively inducing apoptosis of leukemia and leukemia stem cells.8 Mechanistically, avocatin B inhibited fatty acid oxidation (FAO), a cellular process utilizing fat for energy, which was determined recently to be critical in acute myeloid leukemia.9 Our interest in avocatin B is highlighted by its potency, exhibiting bioactivity at concentrations 10 times that of etomoxir,8 a standard compound used to explore the FAO pathway. The potential clinical utility of avocatin B is further highlighted by its ability to enhance the anticancer activity of cytarabine synergistically.10 Studies to date have not clearly identified efficient extraction and analytical methods to specifically detect and quantify

K

© 2018 American Chemical Society and American Society of Pharmacognosy

Figure 1. Chemical structure of avocadene and avocadyne (A) and their acetate forms (acetogenins) (B).

avocadene and avocadyne. Currently, gas chromatography− mass spectrometry (GC-MS) is the only analytical technique reported in the patent literature for the quantification of PFAs such as avocadene and avocadyne in avocado seed and pulp extracts,3,4,11−13 but specific details of this procedure have not been outlined to allow comparisons with existing GC Received: October 31, 2017 Published: March 22, 2018 818

DOI: 10.1021/acs.jnatprod.7b00914 J. Nat. Prod. 2018, 81, 818−824

Journal of Natural Products

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Table 1. Summary of Key Molecular Ion Species Detected when Avocatin B Standard Is Directly Infused into a QE-MS in the + ESI mode theoretical mass from m/z value abundant molecular ion species detected [M [M [M [M [M [M

+ + + + + +

H]+ H − H2O]+ H − 2H2O]+ H − 3H2O]+ Na]+ K]+

elemental composition

uncertainty (delta) (ppm)

avocadene

avocadyne

avocadene

avocadyne

avocadene

avocadyne

287.25807 269.24749 251.23703 233.22737 309.24004 325.21404

285.24241 267.23183 249.22137 231.21084 307.22438 323.19838

C17H35O3 C17H33O2 C17H31O C17H29 C17H34O3Na+ C17H34O3K+

C17H33O3 C17H31O2 C17H29O C17H27 C17H32O3Na+ Cl7H32O3K+

−0.1 −0.01 1.21 1.14 −1.14 1.43

−0.06 −0.18 0.09 0.12 −0.21 0.16

Figure 2. (A) Representative LC-MS chromatogram of 100 μM avocatin B standard injected in the described analytical method. (B) Typical standard curve for serial dilutions of avocatin B standard injected into the LC-MS method. (C) Avocatin B extracted ion chromatograms at lower limit of quantitation (LLOQ) (0.1 μM to 0.03 ng/μL of avocadene or avocadyne) employing an extraction window of 10 mDa.

methods.14 Simple HPLC-based methods for PFAs have also been reported in the literature that rely on UV/vis6,15 or photodiode (PDA) detectors.16,17 Quantifying PFAs such as avocadene and avocadyne using existing GC- and HPLC-based methods requires extensive extraction and sample preparation procedures where identified eluted peaks need to be validated using surrogate methods such as 1H NMR and Fouriertransform IR spectroscopy. Kashman and colleagues also discovered acetogenins in avocado seeds, specifically avocadene acetate and avocadyne acetate (Figure 1B), for which quantitative HPLC-PDA16 and HPLC-MS16,18 methods do exist. While structurally similar, the bioactivity of acetogenins and PFAs are quite distinct;19−21 thus the objective of this study was to specifically develop an analytical method to quantify the amounts of avocadene and avocadyne in Hass avocado seed and pulp matter. To develop a simple and effective analytical method, a reversed-phase chromatographic separation coupled to a highresolution quadrupole-orbitrap mass spectrometer (high-

resolution LC-MS) was utilized for sensitive and complete quantitation in which no extensive extraction steps, sample preparation, or additional structure validation would be required. A commercially available standard of avocatin B was used to ascertain the exact masses of the avocadene and avocadyne molecular ions (MS) and their subsequent fragmentations (MS/MS) in the positive electrospray ionization mode (ESI) by high-resolution quadrupole-orbitrap mass spectrometry. The fragmentation peaks [M + H − H2O]+ of avocadene and avocadyne were the most intense and enabled the development of a sensitive high mass resolution UHPLCMS method for quantification. For efficient extraction of avocadene and avocadyne from avocado pulp and seeds, the total lipid extraction method of Folch et al.22 was utilized. Saponification of both the total lipid extracts as well as direct seed and pulp matter was also carried out to measure the release of bound PFAs from complex ester linkages. This work also demonstrated that avocadene and avocadyne are found in appreciable quantities in the pulp and seeds of Hass avocados, 819



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Figure 3. Avocadyne (A) and avocadene (B) content expressed in mg per g dried weight (DW) of Hass avocado pulp or seed material subject to total lipid extraction with varying exposures to extraction solvent (24−72 h). Each box plot represents means ± standard deviation of two biological replicates * = p < 0.05, **** = p < 0.0001.

× 107x + 1.442 × 108, where y represents the peak area under the curve and x represents the concentration of avocatin B. For avocadene, a typical regression equation for calibration curves was y = 1.232 × 108x + 2.214 × 108. The assay sensitivity of 0.1 μM avocatin B (0.03 ng/μL avocadene or avocadyne) (Figure 2C) was determined by the analysis of lower limit of quantitation (LLOQ) triplicate samples on different analytical days (n = 6). The interassay comparison for LLOQ samples yielded acceptable accuracies of 18.2% percentage error for avocadene and 13.3% percentage error for avocadyne (Table S1, Supporting Information). The interassay precision for LLOQ was also acceptable at 13.8% CV for avocadene and 14.4% CV for avocadyne (Table S1, Supporting Information). The intra- and interassay precisions for avocadene and avocadyne low and high quality control (QC) samples were ≤7.5% CV, and accuracies (percentage error) ranged from −0.5 to 9.3% (Table S1, Supporting Information). Both precision and accuracy for all QC samples were found to be within the predefined acceptance criteria, suggesting the developed LC-MS method was suitable for the quantification of avocadene and avocadyne. The assay sensitivity increased to 5 nM avocatin B (0.001 ng/μL avocadene or avocadyne) when the LC-MS method described was run in a high-resolution selected-ion monitoring (SIM) mode, which enabled only the avocadene and avocadyne fragment ions, [M + H − H2O]+, to be detected (Figure S4, Supporting Information). Given the relatively high abundance of avocadene and avocadyne in seed and pulp matter, the added sensitivity provided by the LC-SIM-MS method was not required for this study. Since there are no commercially available internal standards of avocatin B, the specificity and selectivity of the LC-MS method were assessed using the postextraction spike method.24 The percentage recovery of spiked avocadene and avocadyne (from 5 μM avocatin B standard) was assessed using two total lipid extracts of Hass avocado seed and pulp, respectively. Table S2 (Supporting Information) demonstrates that the percent recovery of avocadene and avocadyne from the total lipid extracts of seed and pulp were well within acceptance limits, further suggesting that endogenous compounds in the pulp or seed did not interfere with the ionization of the target analytes (106.5 ± 10.0% and 104.4 ± 2.8% from the pulp and seed for

the most cultivated and widely consumed variety of avocados in the world.23

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RESULTS AND DISCUSSION MS Tuning. Direct infusion studies with an avocatin B standard in full-scan positive ESI mode revealed three key molecular ions ([M + H]+, [M + Na]+, and [M + K]+) and three fragment ions ([M + H − H2O]+, [M + H − 2H2O]+, [M + H − 3H2O]+), as outlined in Table 1. Avocadene and avocadyne [M + H]+ ion intensities were relatively low in abundance in comparison to their fragment ions [M + H − H2O]+ and [M + H − 2H2O]+, which showed high intensities. The fragment ions were confirmed by MS/MS to be from the avocadene and avocadyne molecular ions [M + H]+ (Figures S1 and S2, Supporting Information). Sodium adduct peaks, [M + Na]+, were the most abundant for both avocadene and avocadyne in comparison to their potassium adduct peaks and were completely resistant to fragmentation with a range of normalized collision energies (NCE = 10−40) (data not shown). Given that the original source of the molecular adduct ion peaks was unknown, all direct infusion studies revealed that [M + H − H2O]+ ions for both avocadene and avocadyne potentially could be suitable for quantification in an LC-MS method. Method Validation. A typical UHPLC-MS chromatograph for 100 μM avocatin B standard (28.4 and 28.6 ng/μL avocadyne and avocadene, respectively) is presented in Figure 2A. Retention times for avocadyne and avocadene were 5.65 and 7.94 min, respectively. As predicted from direct infusion studies, the intensities of [M + H − H2O]+ ions for both avocadyne and avocadene were consistently 10-fold higher compared to their [M + H]+ molecular ion peaks (Figure 2A) and were thus chosen for quantification. The sodium and potassium adduct peaks for both avocadene and avocadyne did not have any significant intensity in the LC-MS method (Figure S3, Supporting Information) compared to what was observed in direct infusion studies. Linearity was assessed based on the average of three standard curves from three separate LC-MS validation days where acceptable linearity was achieved in the range of 0.1−60 μM avocatin B standard (or 0.03−17.2 ng/μL of avocadyne or avocadene), with correlation coefficients (r2) > 0.990 for all validation batches (Figure 2B). For avocadyne, a typical regression equation for calibration curves was y = 9.036 820



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avocadene, respectively; 100.8 ± 3.1% and 101.5 ± 3.3% from the pulp and seed for avocadyne, respectively). Avocadene and Avocadyne in Total Lipid Extracts of Hass Avocado Pulp and Seed. Total lipid extracts from 1 g of Hass avocado dried seed and pulp samples were prepared and analyzed to determine the total amounts of avocadene and avocadyne. To date, this is the first report in the literature that shows the extraction of avocadene and avocadyne from both the pulp and seed using the Folch et al.22 method. The amount of avocadene and avocadyne extracted from both the pulp and seed increased in proportion to the solvent maceration time (Figure 3A and B). In total lipid extractions after maceration for 72 h, avocadyne was found in the pulp and seed at 0.18 ± 0.04 mg/g dry weight (DW) and 0.41 ± 0.02 mg/g DW, respectively. Similarly, avocadene was extracted in the pulp and seed at 0.22 ± 0.04 mg/g DW and 0.43 ± 0.04 mg/g DW, respectively (Table 2). The concentrations of both avocadene

Table 2 shows extrapolated amounts of avocadyne and avocadene in the pulp and seed of one Hass avocado. As expected, higher PFA levels were determined in the pulp (5.25 ± 1.15 mg of avocadyne and 6.65 ± 1.25 mg of avocadene) compared to the seed given the larger expected average dry mass of the pulp. Methanolic Saponification of Avocado Pulp and Seed. Methanolic saponification of the total lipid content of the seed and pulp matter or directly on solid seed and pulp matter was carried out to test if avocadene and avocadyne could be released from all bound forms (complex ester linkages). Figure 4 shows that saponification of total lipid extracts or direct saponification of solid pulp and seed material increases the yield of total avocadyne and avocadene by greater than 10-fold compared to amounts quantified in total lipid extracts only (Table 2). No significant differences were seen in the total amounts of PFAs recovered with any of the three methods of saponification. Table 3 summarizes results only from overnight saponification of total lipids with low KOH extractions. Avocadyne was detected in the pulp and seeds at 1.97 ± 0.55 and 6.04 ± 3.02 mg/g DW (p < 0.01), respectively. Similarly, 3.92 ± 1.75 and 7.09 ± 3.17 mg/g DW of avocadene was recovered in the pulp and seeds. Overall, Table 3 shows that far greater amounts of avocadene and avocadyne can be released if complex ester linkages in avocado pulp and seed can be saponified to release the free fatty alcohols present. With solvent-based saponification, 59.05 ± 16.40 and 60.35 ± 30.23 mg of avocadyne can be expected in the dry weight of one Hass avocado pulp and seed, respectively. Similarly, with solvent saponification, 117.50 ± 52.53 and 70.92 ± 31.69 mg of avocadene can be anticipated in the dry weight of the pulp and seed, respectively, from one Hass avocado. It is important to note that patent and literature reports have previously outlined methods of alkaline saponification of cold-pressed avocado pulp oil that yield not only polyhydroxylated fatty alcohols but mainly avocado alkyl furans and other avocado unsaponifiables.4,11,27 Saponification methods utilized in this study are specific to recovering avocadyne and avocadene only and did not allow for total extraction and characterization of avocado unsaponifiable substances.

Table 2. Projected Amounts of Avocadene and Avocadyne in Total Lipid Extract of Seed and Pulp of a Hass Avocadoa avocadyne

pulp seed

avocadene

mean amount per gram dry weight (mg/g ± SD)

projected amount in average dry weight of one Hass avocado (mg ± SD)



0.18 ± 0.04b 0.41 ± 0.02c

5.25 ± 1.15 4.10 ± 0.23

0.22 ± 0.04b 0.43 ± 0.04c

6.65 ± 1.25 4.25 ± 0.44

Mean amount per gram dry weight ± standard deviation of two biological replicates representative of Figure 3 from the 72 h solvent maceration condition. b,cMeans with different letters within the same column are significantly different (p < 0.0001). a

and avocadyne were close to 2-fold higher in the seed when compared to the pulp (p < 0.0001). However, there were no differences between avocadene and avocadyne concentrations within the pulp or seed matter (Table 2). These findings were also reported by Kashman et al.,1,2 where a Soxhlet extraction method was used for the avocado seeds and pulp. Total lipid extractions of pulp and seed were reflective of free avocadyne and avocadene in dried pulp and seed; thus levels below 0.5 mg/g DW were expected. Assuming the dried weight of a typical Hass avocado is 30 g for the pulp and 10 g for seed,25,26

Figure 4. Avocadyne (A) and avocadene (B) content expressed in mg per g dried weight (DW) of Hass avocado pulp or seed material subject to three outlined saponification methods. Each box plot represents means ± standard deviation of two biological replicates; * = p < 0.05, ** = p < 0.01, **** = p < 0.0001. 821



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Table 3. Projected Amounts of Avocadene and Avocadyne in Saponified Extract of Seed and Pulp of a Hass Avocadoa avocadyne

pulp seed

avocadene





1.97 ± 0.55b 6.04 ± 3.02c

59.05 ± 16.40 60.35 ± 30.23

3.92 ± 1.75 7.09 ± 3.17

117.50 ± 52.53 70.92 ± 31.69

Mean amount per gram dry weight ± standard deviation of two biological replicates representative of Figure 4 from the overnight saponification of total lipids−low KOH condition. b,cMeans with different letters within same column are significantly different (p < 0.05).

a

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up to 5 mmol/L (5 mM or 2.85 mg/mL) in 1:1 CH3CN−MeOH and was diluted to 10 μM in 1:1 methanol−water and 0.1% formic acid (FA). Standards of 10 μM were directly infused into the QE using a 500 μL Hamilton syringe (Hamilton Company, NV, USA) connected directly to the ESI source via fused-silica tubing at a flow rate of 10 μL/min. By comparing blank runs against PFA standards, the most abundant molecular ions were determined. All ion elemental compositions were confirmed by high-resolution, accurate mass determinations with lock mass correction. Targeted MS/MS experiments on precursor ions ([M + H]+) of avocadene and avocadyne were also performed using the same 10 μM avocatin B standard injections as described above, where mass resolution was the only ESI parameter changed to 35000. MS/MS data were collected at NCE values that ranged from 10 to 40. Ultra-High-Performance Liquid Chromatography and Mass Spectrometry. For LC-MS method development, the QE was coupled to a Dionex UltiMate 3000 UPLC System (Dionex Corporation, Bannockburn, IL, USA) using Chromeleon Xpress (version 7.2; Thermo-Fisher Scientific). For LC-MS, only three MS parameters were altered compared to the direct infusion studies: (i) capillary temperature (300 °C), (ii) spray voltage (3.5 kV), (iii) lock mass (diisooctyl phthalate, m/z ratio 391.28429, as was present in mobile phase). A C18 Zorbax Eclipse Plus UHPLC reversed-phase column was used (2.1 × 5.1 mm, 1.8 μm particle size) (Agilent, Santa Clara, CA, USA) for UHPLC. The mobile phases used for the chromatographic separation of avocatin B was water + 0.1% FA (solvent A) and CH3CN + 0.1% FA (solvent B). The multistep gradient was 40% B for 0−2 min, 100% B for 2−15 min, 100% B hold for 15−19 min, 40% B for 19−20 min, and equilibration at 40% B for 20−30 min. The flow rate was set at 0.2 mL/min, and the column compartment was kept at room temperature. The Thermo Xcalibur QualBrowser (version 2.1; Thermo-Fisher Scientific) was used for extracting ion chromatograms, exporting MS spectra, and integrating peak areas for quantification. In an attempt to increase the sensitivity of the LC-MS method described above, the quadrupole mass filter capabilities of the QOrbitrap were used by specifying the m/z ratios of avocadene and avocadyne fragmentation peaks [M + H − H2O]+ in a targeted inclusion list. Running the LC-MS method described above in this high-resolution SIM mode enabled avocadene and avocadyne fragmentation ions to be detected by the QE and achieved a much lower detection limit compared to the LC-MS method. Method Validation: Preparation of Calibration Standards and Quality Control Samples. On LC-MS analysis days, a stock solution of avocatin B standard was prepared in 1:1 CH3CN−MeOH at 5 mM (2.85 mg/mL). The stock solution was then diluted in 1:1 CH3CN−MeOH to produce a working solution containing 100 μM avocatin B. Eight calibration standards were prepared from the working solution by making serial dilutions at the final concentrations of 0.1, 5, 10, 20, 30, 40, 50, and 60 μM (a standard curve with a range of 0.03−17.2 ng/μL of avocadene or avocadyne injected using the avocatin B standard) in the LC starting gradient (60% water−40% CH3CN with 0.1% FA). QC samples containing 0.1, 5, and 50 μM avocatin B were prepared the same way using a different 5 mM avocatin B stock. A 10 μL amount of all prepared calibration standards and QC samples were injected into the LC-MS method. Standard curves were constructed using least-squares linear regression of peak area under the curve versus nominal standard concentrations. Linearity was assessed by evaluating the slope, intercept, and coefficient of

CONCLUSIONS Avocadene and avocadyne are bioactive compounds with potential physiological benefits and were first identified in avocado seed. Although their quantity in avocado pulp has not been reported, their known presence in the seed provided an ideal source to test the presently proposed analytical method. As such, the developed LC-MS method was successfully applied to lipid extracts of Hass avocado seed and pulp matter. An established total lipid extraction method, adopted from Folch et al.,22 was used in tandem with methanolic saponification to extract and quantify the target PFAs. The amounts of avocadene and avocadyne (in mg/g DW) quantified in total lipid extracts were 2-fold higher in the seed compared to the pulp, a finding well supported in literature.7 The more than 10-fold increased recovery of avocadyne and avocadene after methanolic saponification of either total lipid extracts or original starting material further supports previous evidence that fatty alcohols largely exist naturally as compounds bound to complex ester linkages.28,29 While more avocatin B was found in the seeds, the pulp still contained appreciable amounts, suggesting that dietary avocado consumption results in avocatin B intake;30−32 however, future studies will be needed to directly quantify the bioavailability of avocatin B from the ingestion of avocados. In summary, this study presents a simple, sensitive, and reproducible LC/MS method of quantifying avocatin B and can be used to evaluate other methods of extracting polyhydroxylated fatty alcohols available in the literature.3,16,18,33 Future studies can also utilize the described analytical method to further explore differences in the amounts of avocadene and avocadyne present in different avocado cultivars.

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EXPERIMENTAL SECTION

Chemicals and Reagents. HPLC grade solvents acetonitrile (CH3CN), chloroform, methanol (MeOH), and hexane were purchased from Thermo-Fisher Scientific (Napean, ON, Canada). Reagents including monobasic sodium phosphate, formic acid (Optima grade), and 12 M sodium hydroxide were purchased from Sigma (St. Louis, MO, USA). Avocatin B standard was purchased from Microsource Discovery Systems Inc. (CT, USA), where NMR spectroscopy was used to determine that the ratio of avocadyne to avocadene was 1:1. MS Tuning Experiments. Direct infusion studies of avocatin B standard (1:1 mixture of avocadene and avocadyne) into the Thermo Scientific Q-Exactive (QE) quadrupole-orbitrap mass spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA) were performed to record all abundant molecular ion species, adducts, and fragments. For all direct infusion studies, the QE mass spectrometer was operated using the following ESI parameters: heater temperature, 0 °C; sheath gas flow rate, 2; auxiliary gas flow rate, 0; sweep gas, 0; spray voltage, 3.2 kV; capillary temperature, 275 °C; and S-lens 60. Scan parameters were set to full-scan range, m/z 100 to 1000; polarity, positive; mass resolution, 70 000; lock mass, m/z 371.10123 (polysiloxane); automatic gain control (AGC) target, 1 × 106; and maximum injection time (maxIT), 50 ms. Avocatin B standard stock was made 822



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stored at 4 °C until sample preparation was required for LC-MS analysis. This method is referred to as “overnight saponification of total lipidslow KOH”. A variation of this method was also tested where 200 mM KOH was used instead of 100 μM; this method is referred to as “overnight saponification of total lipidshigh KOH”. The third saponification method, termed “direct methanolic saponification”, was applied to seed and pulp material directly to release bound fatty alcohols. Briefly, 1 g of seed or pulp material was saponified under reflux in 50 mL of 1 M KOH for 1 h, after which the solution was left to cool. Unsaponifiable constituents were collected in 40 mL of hexane and discarded as outlined above, and the same acidification step was applied to the methanol phase, which was then evaporated and stored at 4 °C until sample preparation was required. All saponification experiments were performed in duplicate for each of the three avocados. Sample Preparation of Extracts for LC-MS Analysis. All dried extracts were dissolved in 10 mL of 1:1 CH3CN−MeOH and vortexed for several minutes. A 10 μL portion of dissolved extracts was added to 990 μL of LC starting gradient (100-fold dilution) to prepare the unspiked final samples. For each unspiked sample, a postextraction spiked sample was also prepared, which contained 5 μM avocatin B standard. All samples were prepared in duplicates, and 10 μL of each was injected. To minimize degradation of target analyte, samples were injected within 24 h of reconstitution. Statistics. All data were expressed as means ± standard deviations and were analyzed using two-way analyses of variance (ANOVA) and Bonferroni’s post hoc test using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). For histograms, * = p < 0.05, ** = p < 0.01, and **** = p < 0.0001.

determination (r2) of three different calibration curves produced on separate analytical days. Method Validation: Precision and Accuracy. Intra- and interday accuracy and precision of the developed method were determined by assaying three concentrations of QCs (LLOQ = 0.1 μM, low QC = 5 μM, and high QC = 50 μM) in triplicates on three different analytical days. Precision was reported as percent coefficient of variation (% CV) of replicates within one sample run (intra-assay) or between sample runs (interassay). Intra- and interassay accuracy was reported as percent relative error (% RE) or the percent deviation of QC replicates from nominal concentration. The acceptance limits for accuracy and precision, at low and high QC concentration levels, were set to 15% RE and 15% CV, respectively. For LLOQ, accuracy and precision acceptance limits below 20% RE and 20% CV, respectively, were acceptable in keeping with United States Food and Drug Administration guidelines.34 Method Validation: Specificity and Selectivity. The total lipid profiles of all avocado seed and pulp samples were expected to contain avocadene and avocadyne.1,2 To test if unknown constituents of the extracted samples interfere with the analysis of the target analytes, 5 μM avocatin B postextraction spike control samples were prepared for every unspiked sample.24 Both samples were injected into the method, and percentage recovery calculations were performed to assess selectivity and specificity. For instance, if the concentration of an unspiked sample was reported from the method to be at 2.5 μM avocadene, its respective postextraction spike control sample was expected to contain 7.5 μM ± 15% avocadene. Plant Material Preparation. Three ripe Hass avocados were purchased from Sobey’s Canada (Guelph, ON, Canada) on three separate occasions in July 2016 and prepared separately. The seed and pulp of each avocado was separated from the peel, and 3−5 mm thick slices (4 in. in length) were placed evenly inside a ventilated oven for 2 h at 65 °C. Dried pulp slices were further cut into smaller particles, whereas dried seed slices were pulverized in a high-speed food processor. Dried pulp and seed matter from each of the three avocados were frozen at −80 °C until extraction day. One gram of dried pulp and seed samples from each avocado was used for all solvent extraction methods. Total Lipid Extraction from Plant Material. For total lipid extraction of the prepared dried avocado seed and pulp material, a modified Folch protocol22 was adopted. Briefly, 1 g of sample was macerated in 20 mL of 2:1 chloroform−methanol (v/v) for different periods of time (immediate, 24 h, 48 h, 72 h) at room temperature, away from light. After maceration, 3 mL of 0.2 M sodium-phosphate (NaHPO4) buffer in ddH2O (pH 4.4) was added to induce layer separation. After inversion, samples were centrifuged for 5 min at 1500 rcf. The total lipid-containing organic layer was collected, and an additional 9 mL of chloroform was added to the aqueous layer as a wash step and as an additional round of extraction. The second organic layer was combined with the first, and the buffer layer was discarded. Chloroform extracts were rotary evaporated and stored at 4 °C until sample preparation was required for LC-MS analysis. All total lipid extraction experiments were performed in duplicate for each of the three avocados. Methanolic Saponification of Avocado Pulp and Seed. Literature evidence from environmental and bacterial samples shows that several classes of fatty alcohols are bound to complex ester linkages.28,35 To release free avocadene and avocadyne from all potential bound forms (e.g., wax esters, acetogenins (Figure 1B), or triacylglycerols36) in avocado seed and pulp material, three different saponification methods were tested. First, total lipids were extracted from seed and pulp samples, as outlined above, after which 50 mL of methanol containing 100 μM sodium hydroxide was added and allowed to shake at a moderate speed for 16 h at 37 °C. After saponification, a liquid−liquid extraction was performed with 40 mL of hexane to extract unsaponifiable substances (note that avocadene and avocadyne are not soluble in hexane). The methanol (lower phase) was collected and reacidified with 100 μM HCl to protonate fatty acid salts and any free fatty alcohols that may have been deprotonated in the saponification. The methanol phase was rotary evaporated and

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00914. Spectra, chromatograms, and further details (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 519-824-4120, ext. 53732. E-mail: paul.spagnuolo@ uoguelph.ca. ORCID

Ken D. Stark: 0000-0001-7828-4072 Paul A. Spagnuolo: 0000-0002-2431-4368 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.A. is supported by a Mitacs Accelerate scholarship. J.J.A.H. is supported by a National Sciences and Engineering Research Council of Canada (NSERC) Post-Graduate Doctoral Scholarship. K.D.S. is supported by a Canada Research Chair in Nutritional Lipidomics. P.A.S. is supported by grants from NSERC, the Hass Avocado Board (CA, USA), and University of Guelph. The authors acknowledge the support of Dr. T. Mohamed and Dr. P. N. Rao (University Waterloo School of Pharmacy) for mentoring and planning initial extraction and method development protocols.

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