*3.5. Stationary Phase and Selection of Chromatographic Conditions*

The major obstacle in vitamin separation is the segregation of isomers. With this in mind, as a starter setup, we used a mobile phase based on MeOH and water using an eight carbon-based alkyl stationary phase (0.75 mL min−<sup>1</sup> , Eclipse Plus C8, 4.6 mm ID × 150 mm, 3 µm, Agilent Technologies). As the resolution was insufficient, a C<sup>18</sup> column was selected, and only flow was modified (1 mL min−<sup>1</sup> , Eclipse Plus C18, 4.6 mm ID × 150 mm, 3 µm, Agilent Technologies). Then we substituted the column for a C30, removed water, and used a less polar solvent in acetonitrile and 2-propanol, reducing yet again the solvent flow (0.5 mL min−<sup>1</sup> ) (Table 1). Finally, retaining a similar proportion of MeOH, we substituted acetonitrile and isopropanol for MTBE. The C<sup>30</sup> column was kept as it already had excellent capabilities reported for highly lipophilic compounds (e.g., carotenoids) [39].

#### *3.6. Chromatographic Conditions*

All assays performed using an Agilent Technologies LC/MS system equipped with 1260 infinity quaternary pump (61311C), column compartment (G1316A), automatic liquid sampler modules (ALS, G7129A) and a 6120-single quadrupole mass spectrometer with electrospray ionization ion source (Agilent Technologies, Santa Clara, CA, USA). Gradient elution was used to separate all the compounds. The solvent gradient was optimized using MeOH (solvent A) and MTBE (solvent B), both acidified with formic acid (0.1 mL/100 mL). Solvent proportions were set as follows: at 0 min 80% A, at 5 min 80% A, at 7 min 73% A, at 15 min 62.5% A, at 20 min 62.5% A, at 30 min 45% A, at 35 min 10% A, at 40 min 10% A, at 45 min 80% A and 50 min 80% A. Flow rate was kept constant at 0.6 mL min−<sup>1</sup> . Injection volume was held at 10 µL. The column compartment was held at a temperature of 10.0 ± 0.8 ◦C. Considering the need for the separation of structurally similar compounds, a 30-carbon alkyl chain based chromatographic column was used to achieve the analytical separation (YMC Carotenoid, 4.6 mm ID × 150 mm, S-3 µm, YMC Co., Ltd., Kyoto, Japan).

#### *3.7. MS Detection System Conditions*

The fragmentor was initially cycled to assess the voltage (from 20 to 300 V) that rendered the highest sensitivity for the compounds; omitting column interaction (Figure 6A). Afterward, total ion chromatographs (TIC) allowed us to obtain the MS spectra for each of the compounds (scan mode using a mass range and detector gain set to 50–750 *m*/z, and 10.00, respectively) (Figure 6B,C). Each TIC was used to identify the molecular ion signal. Drying gas, nebulizer pressure, drying gas temperature, and capillary voltage was set, respectively, to 12.0 L min−<sup>1</sup> , 50 psi, 350 ◦C, 4000 V for positive ion mode electrospray ionization (ESI+). Selected ion monitoring was used to corroborate each compound identity, remove interferences and improve sensitivity (SIM mode with peak width and cycle time set to 0.05 min, and 0.30 s cycle−<sup>1</sup> , respectively) (Table 2, Figure 6D). *Molecules* **2019**, *24*, x FOR PEER REVIEW 12 of 16 using a mass range and detector gain set to 50–750 *m*/z, and 10.00, respectively) (Figure 6B,C). Each TIC was used to identify the molecular ion signal. Drying gas, nebulizer pressure, drying gas temperature, and capillary voltage was set, respectively, to 12.0 L min−1, 50 psi, 350 °C, 4000 V for positive ion mode electrospray ionization (ESI+). Selected ion monitoring was used to corroborate each compound identity, remove interferences and improve sensitivity (SIM mode with peak width and cycle time set to 0.05 min, and 0.30 s cycle−1, respectively) (Table 2, Figure 6D).

**Figure 6.** Example of the voltage cycling for phylloquinone to obtain the most sensitivity, data obtained at 100 μg mL<sup>−</sup>1. (**A**) A parameter was selected where the signal delivered the most area under the curve. (**B**) Mass spectra obtained from a total ion chromatogram for phylloquinone at 100 μg mL<sup>−</sup>1. Fragmentation as follows (molar mass 450.7 g mol<sup>−</sup>1): 473.3 ([M + K]+), 451.4 ([M + H]+), 381.3 ([C26H35O2]•), 353.4 ([C24H30O2]2•), 225 ([C15H13O2]• and partial alkyl isoprenoid chain [C16H33]•), and 186 (quinone ring, [C12H9O2]•) *m*/*z* [60]. (**C**) Chromatogram for phylloquinone was obtained using the selected [M + H]+ 451 *m*/*z* and (**D**) selected ion monitoring (SIM) for the target analyte at 20 μg mL<sup>−</sup>1. **Figure 6.** Example of the voltage cycling for phylloquinone to obtain the most sensitivity, data obtained at 100 µg mL−<sup>1</sup> . (**A**) A parameter was selected where the signal delivered the most area under the curve. (**B**) Mass spectra obtained from a total ion chromatogram for phylloquinone at 100 µg mL−<sup>1</sup> . Fragmentation as follows (molar mass 450.7 g mol−<sup>1</sup> ): 473.3 ([M + K]+), 451.4 ([M + H]+), 381.3 ([C26H35O<sup>2</sup> ] • ), 353.4 ([C24H30O<sup>2</sup> ] 2• ), 225 ([C15H13O<sup>2</sup> ] • and partial alkyl isoprenoid chain [C16H33] • ), and 186 (quinone ring, [C12H9O<sup>2</sup> ] • ) *m*/*z* [60]. (**C**) Chromatogram for phylloquinone was obtained using the selected [M + H]<sup>+</sup> 451 *m*/*z* and (**D**) selected ion monitoring (SIM) for the target analyte at 20 µg mL−<sup>1</sup> .

more dramatic as 100 mg L−1 standard has to be prepared in TIC for a detectable signal while 0.136

**Figure 7.** Calibration curve and sensitivity comparison using (**A**). TIC and SIM modes for vitamin K1 both signals tested at 137.8, 68.9, 34.4, 17.2, 8.61, and 4.31 mg L<sup>−</sup>1 and using a fragmenter of 140 V. No signal is noticeable at 17.2 mg L<sup>−</sup>1 for TIC (blue line in panel (**A**)). Meanwhile, a calibration curve is

easily constructed in SIM mode with the lowest point in 4.31 mg L<sup>−</sup>1 (purple line panel (**B**)).

mg L−1 is still noticeable (Figure 2B), which represents ca. 750-fold in increased sensitivity.

 Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4 mg phylloquinone L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore,

Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4 mg phylloquinone L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore, within curve sensitivity reaches only 24.1 mg L−<sup>1</sup> for TIC while the signal for 4.31 mg L−<sup>1</sup> , in SIM, is still appreciable (i.e., 80471 area under the curve, 0.54 mg L−<sup>1</sup> within curve sensitivity) (Figure 7A,B). Absolute sensitivity to vitamin K increases almost 50 fold (24.1/0.54). For carotenoids, the change is more dramatic as 100 mg L−<sup>1</sup> standard has to be prepared in TIC for a detectable signal while 0.136 mg L −1 is still noticeable (Figure 2B), which represents ca. 750-fold in increased sensitivity. Sensitivity is greatly improved using a SIM targeted scan. For example, the same standard 34.4 mg phylloquinone L-1 in TIC throws 37870 vs. 457785 area under the curve in SIM. Furthermore, within curve sensitivity reaches only 24.1 mg L−1 for TIC while the signal for 4.31 mg L−1, in SIM, is still appreciable (i.e., 80471 area under the curve, 0.54 mg L−1 within curve sensitivity) (Figure 7A,B). Absolute sensitivity to vitamin K increases almost 50 fold (24.1/0.54). For carotenoids, the change is more dramatic as 100 mg L−1 standard has to be prepared in TIC for a detectable signal while 0.136 mg L−1 is still noticeable (Figure 2B), which represents ca. 750-fold in increased sensitivity.

**Figure 6.** Example of the voltage cycling for phylloquinone to obtain the most sensitivity, data obtained at 100 μg mL<sup>−</sup>1. (**A**) A parameter was selected where the signal delivered the most area under the curve. (**B**) Mass spectra obtained from a total ion chromatogram for phylloquinone at 100 μg mL<sup>−</sup>1. Fragmentation as follows (molar mass 450.7 g mol<sup>−</sup>1): 473.3 ([M + K]+), 451.4 ([M + H]+), 381.3 ([C26H35O2]•), 353.4 ([C24H30O2]2•), 225 ([C15H13O2]• and partial alkyl isoprenoid chain [C16H33]•), and 186 (quinone ring, [C12H9O2]•) *m*/*z* [60]. (**C**) Chromatogram for phylloquinone was obtained using the

*Molecules* **2019**, *24*, x FOR PEER REVIEW 12 of 16

using a mass range and detector gain set to 50–750 *m*/z, and 10.00, respectively) (Figure 6B,C). Each TIC was used to identify the molecular ion signal. Drying gas, nebulizer pressure, drying gas temperature, and capillary voltage was set, respectively, to 12.0 L min−1, 50 psi, 350 °C, 4000 V for positive ion mode electrospray ionization (ESI+). Selected ion monitoring was used to corroborate each compound identity, remove interferences and improve sensitivity (SIM mode with peak width

and cycle time set to 0.05 min, and 0.30 s cycle−1, respectively) (Table 2, Figure 6D).

**Figure 7.** Calibration curve and sensitivity comparison using (**A**). TIC and SIM modes for vitamin K1 both signals tested at 137.8, 68.9, 34.4, 17.2, 8.61, and 4.31 mg L<sup>−</sup>1 and using a fragmenter of 140 V. No signal is noticeable at 17.2 mg L<sup>−</sup>1 for TIC (blue line in panel (**A**)). Meanwhile, a calibration curve is easily constructed in SIM mode with the lowest point in 4.31 mg L<sup>−</sup>1 (purple line panel (**B**)). **Figure 7.** Calibration curve and sensitivity comparison using (**A**). TIC and SIM modes for vitamin K<sup>1</sup> both signals tested at 137.8, 68.9, 34.4, 17.2, 8.61, and 4.31 mg L−<sup>1</sup> and using a fragmenter of 140 V. No signal is noticeable at 17.2 mg L−<sup>1</sup> for TIC (blue line in panel (**A**)). Meanwhile, a calibration curve is easily constructed in SIM mode with the lowest point in 4.31 mg L−<sup>1</sup> (purple line panel (**B**)).

Retention times and mass spectra were collected by the centroid of the chromatographic peak. Quantitation was carried out by comparing the peak areas found in the samples with those of standard solutions. The identification and quantification of targeted compounds analyzed by LC-ESI+-MS were performed using OpenLab Chemstation C.01.07 (Agilent Technologies) for the processing of MS data sets. Confirmation of target analytes was based on the retention time (± 0.2 min as accepted time deviation), measurement of the molecular ion in a specific timeframe (Table 2).

#### *3.8. Statistical Analysis*

Calibration curves parameters (i.e., slopes and intercepts), coefficients of determination, limits of detection, and standard errors were computed as a linear fit model using SAS JMP 13 (Marlow, Buckinghamshire, England). An ANOVA with a post-hoc Dunnet test was used to assess differences among treatments during the optimization of the conditions during saponification. Concentrations obtained using the conditions 1 h and 1 mmol KOH mL ethanol−<sup>1</sup> at 80 ◦C, were used as the control parameters; the test considered if the data was below the control, with α = 0.05 significance level. The statistical analysis was performed using IBM SPSS®® Statistics 23 (Armonk, NY, USA).

#### **4. Conclusions**

The proposed method was regarded as a greener option by replacing chlorinated solvents and allowed two nutritionally relevant families of bioactive compounds (i.e., carotenoids and fat-soluble vitamins) to be analyzed together (which is not usually the case) and offered an adequate resolution in the case of tocopherol and calciferol isomers to improve their differential quantification. We obtained an accurate, sensitive, robust, and highly specific multi-analyte method that was successfully applied to avocadoes, a fruit of high economic value, of dietary interest, and a staple of Latin-American cuisine. The use of separation based entirely on organic solvents and the C<sup>30</sup> column retention capability rendered a versatile method that can facilitate the incorporation of other pigments that have been reported present in avocado fruit (e.g., neoxanthin, trollichrome, chrysanthemaxanthin) [14,21] to further extend its chemical characterization. Saponification was paramount in the recovery of fat-soluble compounds. Therefore, optimized conditions should be assessed for each matrix to be tested. The method may be extended to evaluate fat-soluble vitamins and carotenoids in other matrices. Mass spectrometry was a crucial tool in enabling the discrimination of structurally related compounds (e.g., all three tocopherol isomers could be easily accounted for in avocado).

**Author Contributions:** Conceptualization, F.G.-C., G.A., and C.C.-H.; methodology, F.G.-C. and C.C.-H.; software, F.G.-C. and C.C.-H.; validation, F.G.-C., G.A., A.C., and C.C.-H.; formal analysis, F.G.-C., G.A., A.C., and C.C.-H.; investigation, F.G-C.; resources, F.G.-C., G.A., and C.C.-H.; data curation, C.C.-H., F.G-C., and G.A; writing—original draft preparation, F.G.-C.; writing—review and editing, F.G.-C., G.A., A.C., C.C.-H.; visualization, F.G.-C.; supervision, F.G.-C. and C.C-H; project administration, F.G.-C. and C.C-H.; funding acquisition, C.C-H.

**Funding:** This research received no external funding except for the APC, which was funded by the Vice Provost Office for Research of the Universidad de Costa Rica.

**Acknowledgments:** Laura Arroyo is acknowledged for acquiring the avocado samples and preliminary integration of a few avocado samples and María Sabrina Sánchez for their suggestions, revising the manuscript and for language editing.

**Conflicts of Interest:** The authors declare no conflict of interest.
