*2.6. Extraction Procedure*

Ultra-pure water (2 mL) was added to each 0.1 g powdered sample of in vitro culture, 3-month-old in vitro derived bulblets, and 3-year-old wild type commercial bulbs before vortexing for 1 min and letting stand for 5 min. After centrifugation at 4000 rpm for 5 min, 0.8 mL of supernatant was collected. Then, 8 µL internal standard (50 µg/mL), 50 µL ammonia solution, and 140 µL Fe3O4@C18 were added into the supernatant with a vortex treatment for 30 s then left to stand still for 5 min. With the help of an external magnet, the analyte-adsorbed Fe3O4@C18 was rapidly separated from the supernatant and vortexed with 100 µL acetonitrile for 30 s to elute the analytes. After that, the external magnet was used to settle the magnetic adsorbent while the elution solution was filtered through a 0.22 µm PTFE (polytetrafluoroethylene) membrane for LC-MS/MS analysis.

#### *2.7. LC-MS*/*MS Conditions 2.7. LC-MS/MS Conditions*

The analysis was performed with a Surveyor LC-MS/MS system (Thermo Scientific, Waltham, CA, USA). The chromatographic separation was achieved by using a Thermo Scientific Accucore C18 column (2.1 × 100 mm, 2.6 µm) at a constant column temperature of 30 ◦C with a flow rate of 0.2 mL/min. The mobile phase A was 10 mM ammonium formate aqueous solution containing 0.1% ammonia solution, and mobile phase B was methanol. The gradient elution program was started at 80% A for 1 min, dropped to 35% A in 1 min, decreased to 20% A in 2 min, held for 5 min, decreased to 10% A within 2 min, held for 3 min, resumed at 80% A within 1 min, and kept constant for 5 min. Figure 2 shows the typically extracted ion chromatograms of the mixed standard solution of peimisine, sipeimine, peiminine, and peimine at the concentration of 0.5 µg/mL. The analysis was performed with a Surveyor LC-MS/MS system (Thermo Scientific, Waltham, CA, USA). The chromatographic separation was achieved by using a Thermo Scientific Accucore C18 column (2.1 × 100 mm, 2.6 μm) at a constant column temperature of 30 °C with a flow rate of 0.2 mL/min. The mobile phase A was 10 mM ammonium formate aqueous solution containing 0.1% ammonia solution, and mobile phase B was methanol. The gradient elution program was started at 80% A for 1 min, dropped to 35% A in 1 min, decreased to 20% A in 2 min, held for 5 min, decreased to 10% A within 2 min, held for 3 min, resumed at 80% A within 1 min, and kept constant for 5 min. Figure 2 shows the typically extracted ion chromatograms of the mixed standard solution of peimisine, sipeimine, peiminine, and peimine at the concentration of 0.5 μg/mL.

*Plants* **2020**, *9*, x FOR PEER REVIEW 5 of 15

**Figure 2.** Extracted ion chromatograms of isosteroidal alkaloid standards (0.5 μg/mL): (**a**) Peimisine, **Figure 2.** Extracted ion chromatograms of isosteroidal alkaloid standards (0.5 µg/mL): (**a**) Peimisine, (**b**) Sipeimine, (**c**) Peiminine, and (**d**) Peimine.

(**b**) Sipeimine, (**c**) Peiminine, and (**d**) Peimine. The mass spectrometer was equipped with an electrospray ionization (ESI) interface operating in positive ion mode. The selected reaction monitoring (SRM) mode was used to acquire the mass spectrometric data. The full width at half maximum (FWHM) of Q1 and Q3 was 0.7 for both. The optimal ESI source parameters were set as follows: the capillary temperature was 300 °C; spray voltage was 4600 V; the pressure of sheath gas, aux gas, and ion sweep gas was maintained at 45, 10, and 10 arb units, respectively. The ion transitions and optimal collision energy of selected reaction monitoring chosen for quantitative analysis were as follows: m/z 428→m/z 114 (44 eV) for peimisine; m/z 430→m/z 138 (48 eV) for sipeimine; m/z 430→m/z 412 (38 eV) for peiminine; m/z 432→m/z 414 (40 eV) for peimine. The retention time, protonated molecule ions (represented as [M+H]+), and the analytical parameters of the developed method for analysis of four alkaloids are listed in Table 1, and The mass spectrometer was equipped with an electrospray ionization (ESI) interface operating in positive ion mode. The selected reaction monitoring (SRM) mode was used to acquire the mass spectrometric data. The full width at half maximum (FWHM) of Q1 and Q3 was 0.7 for both. The optimal ESI source parameters were set as follows: the capillary temperature was 300 ◦C; spray voltage was 4600 V; the pressure of sheath gas, aux gas, and ion sweep gas was maintained at 45, 10, and 10 arb units, respectively. The ion transitions and optimal collision energy of selected reaction monitoring chosen for quantitative analysis were as follows: m/z 428→m/z 114 (44 eV) for peimisine; m/z 430→m/z 138 (48 eV) for sipeimine; m/z 430→m/z 412 (38 eV) for peiminine; m/z 432→m/z 414 (40 eV) for peimine. The retention time, protonated molecule ions (represented as [M+H]+), and the analytical parameters of the developed method for analysis of four alkaloids are listed in Table 1, and chemical structures are shown in Figure 3.

**Table 1.** LC-MS/MS based method development and validation for four standards.

**Regression Equation b**

**Linearity and Range Sensitivityc**

0.0630 0.9923 0.1–40 0.01 0.04

**Linear Range (μg/g)**

**LOD (ng/g)**

**LOQ (ng/g)**

**Correlation Coefficient (r2)**

chemical structures are shown in Figure 3.

**tRa (min)** **Mass/Charge (m/z)**

Peimisine 5.78 428.316 y = 0.0396x +

**Marker Compounds**


**Table 1.** LC-MS/MS based method development and validation for four standards. *Plants* **2020**, *9*, x FOR PEER REVIEW 6 of 15

a tR: Retention time. <sup>b</sup> The regression equations are presented as y = mx+c, and y and x are defined as peak area and concentration of the compound, respectively. <sup>c</sup> LOD: Limit of detection, S/N = 3; LOQ: limit of quantification, S/N = 10. peak area and concentration of the compound, respectively. c LOD: Limit of detection, S/N = 3; LOQ: limit of quantification, S/N = 10.

**Figure 3.** Chemical structures of four isosteroidal alkaloids: (**a**) Peimisine, (**b**) Sipeimine, (**c**) Peimine, (**d**) Peiminine. **Figure 3.** Chemical structures of four isosteroidal alkaloids: (**a**) Peimisine, (**b**) Sipeimine, (**c**) Peimine, (**d**) Peiminine.

#### *2.8. Statistical Analysis 2.8. Statistical Analysis*

Software SAS 9.1 was used for statistical analysis. Data were subjected to the least significant difference (LSD) test at a 5% probability level (*p* < 0.05) wherever possible. In Tables 2 and 3, the number of replicates is 3 (three bottles under each LED light treatment). In Table 4, the number of replicates is 21 (each bottle had 7 explants, and each treatment had 3 bottles). The experiments were repeated three times, including LC-MS/MS analysis. Software SAS 9.1 was used for statistical analysis. Data were subjected to the least significant difference (LSD) test at a 5% probability level (*p* < 0.05) wherever possible. In Tables 2 and 3, the number of replicates is 3 (three bottles under each LED light treatment). In Table 4, the number of replicates is 21 (each bottle had 7 explants, and each treatment had 3 bottles). The experiments were repeated three times, including LC-MS/MS analysis.

*Plants* **2020**, *9*, 1351


**Table 2.** Influence of different LED lights on the growth and development of embryogenic callus cultures \*.

**Table 3.** LC-MS/MS analysis of four isosteroidal alkaloids in in vitro cultures exposed to eight different LED lights, in vitro derived bulblets (3 months old), and commercial *Fritillaria cirrhosa* D. Don bulbs (wild type, three years old).


\* ND: Not detected. \*\* Mean ± standard error. Means within each column followed by the same letter(s) are not significantly different at 5% level by Fisher's protected LSD test.

**Table 4.** Development of bulblets in single embryo, a cluster of five embryos, and single embryo with cotyledonary leaf (3–6 cm long) in *Fritillaria cirrhosa* D. Don.


\* Culture medium MSBM supplemented with 2% sucrose, 0.4% GPP. Observations recorded after three months of incubation. \*\* Mean ± standard error. Means within each column followed by the same letter(s) are not significantly different at 5% level by Fisher's protected LSD test.
