# A Miniaturized QuEChERS Method Combined with Ultrahigh Liquid Chromatography Coupled to Tandem Mass Spectrometry for the Analysis of Pyrrolizidine Alkaloids in Oregano Samples

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Chemicals, Reagents and Standard Solutions

_{4}), sodium chloride (NaCl), sodium citrate tribasic dehydrate, sodium citrate dibasic sesquihydrate and primary-secondary amine (PSA) sorbent were purchased from Scharlab (Barcelona, Spain). Formic acid and ammonium acetate LC-MS grade were supplied by Fluka (Busch, Switzerland). Water (resistivity 18.2 MΩ cm) was obtained from a Millipore Milli-Q-System (Billerica, MA, USA). Standards of the target PAs and related PANOs were all high purity grade (≥90%) and were acquired from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany), except retrorsine, which was from Sigma-Aldrich (St. Louis, MO, USA). Individual solutions of each compound (1000 μg/mL) were prepared in MeOH, except for retrorsine, intermedine, lycopsamine, senecionine, seneciphylline, heliotrine, heliotrine N-oxide, europine and europine N-oxide, which were prepared in ACN/DMSO (4/1, v/v) due to their solubility. A multicomponent standard solution (1 μg/mL) containing a mixture of the 21 compounds was prepared in water. Working standard solutions at different concentration levels were prepared by appropriate dilution of the multicomponent standard solution with water to develop and optimize the analytical procedure. All solutions were stored at −20 °C.

#### 2.2. Samples and Extraction Procedure

_{4}, NaCl, sodium citrate tribasic dehydrate and sodium citrate dibasic sesquihydrate in proportion 4:1:1:0.5) was added. The mixture was vortexed for 1 min, followed by ultrasound agitation for 5 min and centrifuged 10 min at 6000 rpm. An aliquot from the upper part of the extract corresponding to the ACN fraction was transferred into an Eppendorf containing 150 mg of MgSO

_{4}and 25 mg of PSA. The mixture was vortexed for 1 min and centrifuged for 5 min at 10,000 rpm. Then, the supernatant of the purified extract was filtered through a 0.45 μm PTFE filter membrane and injected in the UHPLC-MS/MS system.

#### 2.3. UHPLC-MS/MS Analysis

#### 2.4. Statistical Analysis

## 3. Results and Discussion

#### 3.1. Optimization of the Chromatographic Separation

#### 3.2. Extraction Procedure

_{4}) per mL of extract are recommended [33]. Therefore, as the miniaturized procedure led to 1 mL of organic sample extract, these amounts of sorbents were used in the clean-up step. Figure 3a shows the scheme of the μ-QuEChERS procedure proposed.

#### 3.3. Method Validation and Analysis of Samples

^{2}) values ≥0.999. In addition, the linearity coefficient (Cm) was calculated as (1 − (Sd/m)) × 100, where Sd is the standard deviation of the calibration slopes obtained on different days and m is the average slope, which ranged from 92 to 100 among all the analytes (Table 2), successfully accomplishing the criteria established on the guidelines (≥al 92%) [34,35].

#### 3.4. Analysis of Samples

- -
- Heliotrine-type: including europine, heliotrine, lasiocarpine and their N-oxides.
- -
- Senecionine-type: including erucifoline, jacobine, retrorsine, senecionine, seneciphylline, senecivernine, their N-oxides and senkirkin.
- -
- Lycopsamine-type: including echimidine, indicine, intermedine, lycopsamine and their N-oxides.
- -
- Monocrotaline-type: including monocrotaline, monocrotaline N-oxide and trichodesmine.

## 4. Conclusions

## Supplementary Materials

## Author Contributions

## Funding

## Conflicts of Interest

## References

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**Figure 2.**Extracted ion chromatograms and mass spectra (MS

^{2}) of: intermedine (1), lycopsamine (2), intermedine N-oxide (3), lycopsamine N-oxide (4), seneciphylline (5), senecivernine (6), senecionine (7), seneciphylline N-oxide (8), senecivernine N-oxide (9) and senecionine N-oxide (10) in a standard solution containing 100 µg/L of each analyte. Chromatographic conditions as Table 1.

**Figure 3.**(

**a**) Schematic representation of the µ-QuEChERS extraction procedure and (

**b**) recovery percentages obtained from the analysis of three spiked replicates of an oregano sample (100 μg/kg of each analyte) extracted by the µ-QuEChERS procedure. Error bars represent the standard deviation of sample replicates (n = 3).

**Figure 4.**Total content of PAs/PANOs (µg/kg) found in the different oregano samples analyzed by the µ-QuEChERS method proposed. In the sample identification code, the first letter indicates: W for wild farming, O for organic farming and C for conventional farming. Samples ending with an A or B indicate same trademark samples with different lot number.

**Figure 5.**Extracted ion chromatograms and mass spectra (MS

^{2}) of (

**a**) lasiocarpine in sample C-S10, (

**b**) lasiocarpine N-oxide in sample C-S10, (

**c**) europine N-oxide in sample O-S9 and (

**d**) echimidine N-oxide in sample C-S14 after µ-QuEChERS extraction. Chromatographic conditions as Table 1.

**Table 1.**Retention time and mass spectrum parameters of the targeted analytes using the UHPLC-IT-MS/MS method developed.

Analyte | Retention Time (min) | Ionization Mode | Precursor Ion (m/z) | Fragmentation Amplitude | MS^{2}. Product Ions ^{a} (m/z) |
---|---|---|---|---|---|

Intermedine | 5.6 | ESI (+) | 299 | 0.70 | 138 *, 120 |

Europine | 5.7 | ESI (+) | 329 | 0.80 | 253 *, 138 |

Lycopsamine | 5.8 | ESI (+) | 299 | 0.70 | 138 *, 120 |

Europine N-oxide | 6.2 | ESI (+) | 345 | 0.80 | 327 *, 171.5 |

Intermedine N-oxide | 6.4 | ESI (+) | 315 | 0.80 | 225, 171.5 * |

Lycopsamine N-oxide | 6.5 | ESI (+) | 315 | 0.80 | 171.5 *, 138 |

Retrorsine | 6.8 | ESI (+) | 351 | 0.80 | 323 *, 275 |

Retrorsine N-oxide | 7.0 | ESI (+) | 367 | 0.90 | 339 *, 245 |

Seneciphylline | 7.2 | ESI (+) | 333 | 0.80 | 305 *, 120 |

Heliotrine | 7.2 | ESI (+) | 313.5 | 0.70 | 138 *, 120 |

Seneciphylline N-oxide | 7.5 | ESI (+) | 350 | 0.80 | 321 *, 118 |

Heliotrine N-oxide | 7.6 | ESI (+) | 329 | 1.00 | 171 *, 136 |

Senecivernine | 7.9 | ESI (+) | 335 | 0.80 | 307 *, 120 |

Senecionine | 7.9 | ESI (+) | 335 | 0.80 | 307 *, 120 |

Senecivernine N-oxide | 8.1 | ESI (+) | 351 | 0.80 | 323 *, 219.5 |

Senecionine N-oxide | 8.3 | ESI (+) | 352 | 1.00 | 220, 118 * |

Echimidine | 8.7 | ESI (+) | 398 | 0.60 | 220, 120 * |

Echimidine N-oxide | 8.7 | ESI (+) | 413 | 0.70 | 395 *, 351 |

Senkirkin | 9.1 | ESI (+) | 365 | 0.80 | 167.5 *, 150 |

Lasiocarpine | 9.8 | ESI (+) | 411 | 0.70 | 335 *, 219.5 |

Lasiocarpine N-oxide | 10.4 | ESI (+) | 428 | 0.80 | 409 *, 352 |

^{a}Predominant product ion. * Ions used for quantification. Isolation width (m/z) is 4. Chromatographic conditions with the optimized gradient elution: 5% B (0–0.5 min), 5–50% B (0.5–7 min), 50% B (7–7.5 min), 50–100% B (7.5–11 min), 100% B (11–12 min), 100–5% B (12–14 min). Water containing 0.2% formic acid and 5 mM ammonium acetate as mobile phase A and methanol containing 10 mM ammonium acetate as mobile phase B. The flow rate was 0.25 mL/min.

**Table 2.**Validation parameters of the µ-QuEChERS method for the determination of the target PAs/PANOs in oregano samples.

Analytes | Linear Range (µg/kg) | Matrix-Matched Calibration R^{2}/cm | Accuracy | Precision | MDL (µg/kg) | MQL (µg/kg) | ME (%) | ||
---|---|---|---|---|---|---|---|---|---|

Recovery (% ± sd) | Mean Recovery (% ± sd) | Intra-Day Precision (RSD%) | Inter-Day Precision (RSD%) | ||||||

Intermedine | 25.0–500.0 | y = 2035x + 5771 0.999/93 | 78 ± 7 ^{a} | 77 ± 3 | 5 ^{a} | 6 ^{a} | 7.5 | 25.0 | 23 |

74 ± 2 ^{b} | 7 ^{b} | 10 ^{b} | |||||||

80 ± 6 ^{c} | 3 ^{c} | 3 ^{c} | |||||||

Europine | 10.0–500.0 | y = 4482x + 58,782 0.999/98 | 83 ± 4 ^{a} | 83 ± 7 | 4 ^{a} | 7 ^{a} | 3.0 | 10.0 | 23 |

77 ± 2 ^{b} | 5 ^{b} | 6 ^{b} | |||||||

90 ± 11 ^{c} | 4 ^{c} | 5 ^{c} | |||||||

Lycopsamine | 22.0–500.0 | y = 1559x + 8916 0.999/99 | 95 ± 7 ^{a} | 90 ± 13 | 7 ^{a} | 12 ^{a} | 6.7 | 22.0 | 18 |

75 ± 7 ^{b} | 5 ^{b} | 8 ^{b} | |||||||

99 ± 7 ^{c} | 1 ^{c} | 4 ^{c} | |||||||

Europine N-oxide | 7.5–500.0 | y = 6816x + 43,628 0.999/100 | 100 ± 5 ^{a} | 88 ± 13 | 6 ^{a} | 8 ^{a} | 2.2 | 7.5 | 16 |

75 ± 6 ^{b} | 5 ^{b} | 9 ^{b} | |||||||

90 ± 5 ^{c} | 3 ^{c} | 5 ^{c} | |||||||

Intermedine N-oxide | 7.5–500.0 | y = 2111x + 75,308 0.999/93 | 78 ± 3 ^{a} | 80 ± 11 | 8 ^{a} | 12 ^{a} | 2.2 | 7.5 | 17 |

71 ± 2 ^{b} | 7 ^{b} | 11 ^{b} | |||||||

92 ± 4 ^{c} | 2 ^{c} | 4 ^{c} | |||||||

Lycopsamine N-oxide | 12.5–500.0 | y = 2017x + 29,990 0.999/93 | 94 ± 3 ^{a} | 86 ± 13 | 5 ^{a} | 8 ^{a} | 3.7 | 12.5 | 13 |

71 ± 1 ^{b} | 4 ^{b} | 5 ^{b} | |||||||

92 ± 3 ^{c} | 7 ^{c} | 10 ^{c} | |||||||

Retrorsine | 5.5–500.0 | y = 1091x + 12,358 0.999/97 | 91 ± 4 ^{a} | 82 ± 9 | 7 ^{a} | 11 ^{a} | 1.7 | 5.5 | 13 |

73 ± 5 ^{b} | 4 ^{b} | 12 ^{b} | |||||||

82 ± 5 ^{c} | 2 ^{c} | 4 ^{c} | |||||||

Retrorsine N-oxide | 3.5–500.0 | y = 543x + 13,424 0.999/95 | 79 ± 1 ^{a} | 83 ± 5 | 6 ^{a} | 8 ^{a} | 1.0 | 3.5 | 18 |

81 ± 8 ^{b} | 3 ^{b} | 7 ^{b} | |||||||

89 ± 6 ^{c} | 5 | 7 | |||||||

Seneciphylline | 2.0–500.0 | y = 2244x + 25,457 0.999/94 | 88 ± 7 ^{a} | 90 ± 4 | 7 ^{a} | 9 ^{a} | 0.7 | 2.0 | 12 |

95 ± 5 ^{b} | 3 ^{b} | 10 ^{b} | |||||||

87 ± 7 ^{c} | 4 ^{c} | 5 ^{c} | |||||||

Heliotrine | 4.0–500.0 | y = 4771x + 9413 0.999/92 | 80 ± 9 ^{a} | 91 ± 9 | 3 ^{a} | 8 ^{a} | 1.3 | 4.0 | 17 |

96 ± 4 ^{b} | 7 ^{b} | 9 ^{b} | |||||||

96 ± 12 ^{c} | 2 ^{c} | 6 ^{c} | |||||||

Seneciphylline N-oxide | 1.0–250.0 | y = 2856x – 29,835 0.999/95 | 95 ± 4 ^{a} | 88 ± 10 | 3 ^{a} | 4 ^{a} | 0.4 | 1.0 | 34 |

77 ± 5 ^{b} | 6 ^{b} | 9 ^{b} | |||||||

93 ± 7 ^{c} | 2 ^{c} | 7 ^{c} | |||||||

Heliotrine N-oxide | 3.0–500.0 | y = 684x + 13,923 0.999/95 | 81 ± 7 ^{a} | 87 ± 1 | 8 ^{a} | 9 ^{a} | 1.0 | 3.0 | 3 |

82 ± 5 ^{b} | 8 ^{b} | 12 ^{b} | |||||||

99 ± 10 ^{c} | 5 ^{c} | 12 ^{c} | |||||||

Senecivernine | 0.5–500.0 | y = 7802x + 480,780 0.999/96 | 98 ± 7 ^{a} | 89 ± 13 | 4 ^{a} | 6 ^{a} | 0.1 | 0.5 | 25 |

74 ± 3 ^{b} | 4 ^{b} | 9 ^{b} | |||||||

95 ± 8 ^{c} | 2 ^{c} | 4 ^{c} | |||||||

Senecionine | 0.5–500.0 | y = 7830x + 339,046 0.999/95 | 99 ± 4 ^{a} | 88 ± 12 | 4 ^{a} | 7 ^{a} | 0.1 | 0.5 | 18 |

76 ± 6 ^{b} | 4 ^{b} | 7 ^{b} | |||||||

90 ± 11 ^{c} | 4 ^{c} | 6 ^{c} | |||||||

Senecivernine N-oxide | 6.0–250.0 | y = 2287x + 4421 0.999/97 | 95 ± 2 ^{a} | 96 ± 4 | 6 ^{a} | 10 ^{a} | 1.8 | 6.0 | 39 |

100 ± 6 ^{b} | 6 ^{b} | 7 ^{b} | |||||||

93 ± 6 ^{c} | 4 ^{c} | 8 ^{c} | |||||||

Senecionine N-oxide | 3.0–500.0 | y = 1208x + 146,398 0.999/98 | 96 ± 8 ^{a} | 92 ± 4 | 8 ^{a} | 13 ^{a} | 0.9 | 3.0 | 28 |

93 ± 8 ^{b} | 3 ^{b} | 5 ^{b} | |||||||

88 ± 5 ^{c} | 6 ^{c} | 8 ^{c} | |||||||

Echimidine | 7.0–500.0 | y = 4704x + 167,863 0.999/94 | 78 ± 4 ^{a} | 82 ± 10 | 5 ^{a} | 5 ^{a} | 2.0 | 7.0 | 17 |

75 ± 2 ^{b} | 6 ^{b} | 12 ^{b} | |||||||

94 ± 11 ^{c} | 4 ^{c} | 6 ^{c} | |||||||

Echimidine N-oxide | 7.5–250.0 | y = 1005x – 48,988 0.999/96 | 87 ± 2 ^{a} | 86 ± 2 | 8 ^{a} | 12 ^{a} | 2.0 | 7.5 | 111 |

86 ± 10 ^{b} | 7 ^{b} | 9 ^{b} | |||||||

84 ± 11 ^{c} | 4 ^{c} | 12 ^{c} | |||||||

Senkirkin | 7.5–500.0 | y = 1327x + 17,984 0.999/97 | 74 ± 5 ^{a} | 83 ± 15 | 3 ^{a} | 7 ^{a} | 2.0 | 7.5 | 8 |

75 ± 3 ^{b} | 4 ^{b} | 7 ^{b} | |||||||

101 ± 2 ^{c} | 5 ^{c} | 7 ^{c} | |||||||

Lasiocarpine | 25.0–500.0 | y = 282x + 4928 0.999/92 | 96 ± 5 ^{a} | 94 ± 2 | 7 ^{a} | 10 ^{a} | 7.5 | 25.0 | 18 |

95 ± 3 ^{b} | 3 ^{b} | 10 ^{b} | |||||||

92 ± 10 ^{c} | 5 ^{c} | 7 ^{c} | |||||||

Lasiocarpine N-oxide | 10.0–500.0 | y = 6252x + 2254 0.999/100 | 101 ± 6 ^{a} | 92 ± 10 | 5 ^{a} | 12 ^{a} | 3.0 | 10.0 | 13 |

93 ± 7 ^{b} | 8 ^{b} | 10 ^{b} | |||||||

82 ± 7 ^{c} | 5 ^{c} | 5 ^{c} |

^{a}Low spiked level (10 µg/kg);

^{b}Medium spiked level (100 µg/kg);

^{c}High spiked level (1000 µg/kg).

**Table 3.**Content of the target PAs/PANOs (µg/kg) quantified in the different oregano samples analyzed by the µ-QuEChERS method proposed.

Analytes (µg/kg) | C-S1 | C-S2 | C-S3 | C-S4 | C-S5 | C-S6 | C-S7 | C-S8 | C-S9 | C-S10 | C-S11 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|

Intermedine | n.d. | n.d. | n.d. | <MQL | <MQL | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | |

Europine | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | <MQL | |

Lycopsamine | n.d. | n.d. | n.d. | n.d. | <MQL | <MQL | n.d. | <MQL | n.d. | n.d. | n.d. | |

Europine N-oxide | 14 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | <MQL | <MQL | <MQL | 25 ± 5$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 15 ± 4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 14 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | <MQL | 16 ± 6$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a}\end{array}$ | 15 ± 4$\begin{array}{c}\mathrm{d},\mathrm{e}\\ \mathrm{a}\end{array}$ | 8 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a}\end{array}$ | |

Intermedine N-oxide | 93 ± 4$\begin{array}{c}\mathrm{g}\\ \mathrm{b}\end{array}$ | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | 138 ± 4$\begin{array}{c}\mathrm{f}\\ \mathrm{c}\end{array}$ | 235 ± 5$\begin{array}{c}\mathrm{g}\\ \mathrm{d}\end{array}$ | n.d. | n.d. | |

Lycopsamine N-oxide | 111 ± 5$\begin{array}{c}\mathrm{h}\\ \mathrm{e}\end{array}$ | n.d. | <MQL | n.d. | 39 ± 2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b}\end{array}$ | <MQL | <MQL | 165 ± 7$\begin{array}{c}\mathrm{g}\\ \mathrm{f}\end{array}$ | 261 ± 11$\begin{array}{c}\mathrm{h}\\ \mathrm{h}\end{array}$ | <MQL | 17 ± 2$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | |

Retrorsine | 10 ± 2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | <MQL | n.d. | n.d. | 17 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{e},\mathrm{f}\end{array}$ | 10 ± 5$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 6.7 ± 0.4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | <MQL | 12 ± 4$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{c},\mathrm{d}\end{array}$ | <MQL | <MQL | |

Retrorsine N-oxide | 5 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 6 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | <MQL | n.d. | 41 ± 12$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{g}\end{array}$ | 12 ± 4$\begin{array}{c}\mathrm{a}\\ \mathrm{b},\mathrm{c},\mathrm{d},\mathrm{e}\end{array}$ | 55 ± 5$\begin{array}{c}\mathrm{f}\\ \mathrm{h}\end{array}$ | 19 ± 3$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{e}\end{array}$ | 10 ± 3$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | <MQL | 31 ± 4$\begin{array}{c}\mathrm{d}\\ \mathrm{f}\end{array}$ | |

Seneciphylline | n.d. | n.d. | n.d. | n.d. | 12 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b}\end{array}$ | n.d. | <MQL | n.d. | n.d. | 5 ± 4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | n.d. | |

Heliotrine | 5 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 5 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 7.9 ± 0.3$\begin{array}{c}\mathrm{a}\\ \mathrm{b},\mathrm{c}\end{array}$ | <MQL | 22 ± 5$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{e}\end{array}$ | 8 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{b},\mathrm{c}\end{array}$ | 36 ± 5$\begin{array}{c}\mathrm{e}\\ \mathrm{g}\end{array}$ | <MQL | <MQL | 9.3 ± 0.4$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b},\mathrm{c}\end{array}$ | 6.1 ± 0.5$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b}\end{array}$ | |

Seneciphylline N-oxide | 18.7 ± 0.8$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 82 ± 3$\begin{array}{c}\mathrm{f}\\ \mathrm{e}\end{array}$ | 20.5 ± 0.7$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 26 ± 1$\begin{array}{c}\mathrm{c}\\ \mathrm{b}\end{array}$ | 88 ± 18$\begin{array}{c}\mathrm{d}\\ \mathrm{e}\end{array}$ | 19 ± 4$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 65 ± 7$\begin{array}{c}\mathrm{g}\\ \mathrm{d}\end{array}$ | 100 ± 13$\begin{array}{c}\mathrm{e}\\ \mathrm{f}\end{array}$ | 19 ± 2$\begin{array}{c}\mathrm{d},\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 53 ± 4$\begin{array}{c}\mathrm{f}\\ \mathrm{c}\end{array}$ | 49 ± 6$\begin{array}{c}\mathrm{e}\\ \mathrm{c}\end{array}$ | |

Heliotrine N-oxide | 13.2 ± 0.9$\begin{array}{c}\mathrm{c}\\ \mathrm{b},\mathrm{c}\end{array}$ | n.d. | n.d. | n.d. | 47 ± 1$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{e}\end{array}$ | n.d. | 15 ± 6$\begin{array}{c}\mathrm{c}\\ \mathrm{c}\end{array}$ | 6.6 ± 0.4$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | n.d. | n.d. | n.d. | |

Senecivernine | 11 ± 4$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | 154 ± 3$\begin{array}{c}\mathrm{h}\\ \mathrm{e}\end{array}$ | 102 ± 7$\begin{array}{c}\mathrm{d}\\ \mathrm{d}\end{array}$ | 105 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{d}\end{array}$ | 1027 ± 58$\begin{array}{c}\mathrm{f}\\ \mathrm{i}\end{array}$ | 385 ± 6$\begin{array}{c}\mathrm{e}\\ \mathrm{g}\end{array}$ | 66 ± 3$\begin{array}{c}\mathrm{g}\\ \mathrm{b},\mathrm{c}\end{array}$ | 6 ±$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ 5 | 6 ± 3$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 225.4 ± 0.4$\begin{array}{c}\mathrm{i}\\ \mathrm{f}\end{array}$ | n.d. | |

Senecionine | 24 ± 3$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 166 ± 4$\begin{array}{c}\mathrm{i}\\ \mathrm{e}\end{array}$ | 120 ± 5$\begin{array}{c}\mathrm{e}\\ \mathrm{d}\end{array}$ | 121 ± 8$\begin{array}{c}\mathrm{f}\\ \mathrm{d}\end{array}$ | 1103 ± 60 | 385 ± 15$\begin{array}{c}\mathrm{e}\\ \mathrm{g}\end{array}$ | 81.8 ± 0.8$\begin{array}{c}\mathrm{h}\\ \mathrm{c}\end{array}$ | 24 ± 5$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | 24 ± 3$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 231 ± 1$\begin{array}{c}\mathrm{j}\\ \mathrm{f}\end{array}$ | 2.4 ± 0.3$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | |

Senecivernine N-oxide | 8 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 17 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{d},\mathrm{e}\end{array}$ | 10.9 ± 0.3$\begin{array}{c}\mathrm{a}\\ \mathrm{b},\mathrm{c}\end{array}$ | 8 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 138 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{j}\end{array}$ | 11 ± 1$\begin{array}{c}\mathrm{a}\\ \mathrm{b},\mathrm{c}\end{array}$ | 24 ± 6$\begin{array}{c}\mathrm{d}\\ \mathrm{f}\end{array}$ | 53 ± 3$\begin{array}{c}\mathrm{d}\\ \mathrm{h}\end{array}$ | 9 ± 3$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 10.8 ± 0.7$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 70 ± 4$\begin{array}{c}\mathrm{g}\\ \mathrm{i}\end{array}$ | |

Senecionine N-oxide | n.d. | 16 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{b}\end{array}$ | n.d. | n.d. | 91 ± 9$\begin{array}{c}\mathrm{d}\\ \mathrm{c}\end{array}$ | n.d. | 9 ± 5$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | n.d. | n.d. | n.d. | n.d. | |

Echimidine | <MQL | <MQL | <MQL | <MQL | 19 ± 5$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | <MQL | <MQL | <MQL | n.d. | <MQL | n.d. | |

Echimidine N-oxide | 160 ± 2$\begin{array}{c}\mathrm{i}\\ \mathrm{e},\mathrm{f}\end{array}$ | 100 ± 6$\begin{array}{c}\mathrm{g}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 194 ± 2$\begin{array}{c}\mathrm{f}\\ \mathrm{h},\mathrm{i}\end{array}$ | 185 ± 2$\begin{array}{c}\mathrm{g}\\ \mathrm{g},\mathrm{h}\end{array}$ | 140 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{d}\end{array}$ | 148 ± 1$\begin{array}{c}\mathrm{d}\\ \mathrm{d},\mathrm{e}\end{array}$ | 106 ± 7$\begin{array}{c}\mathrm{i}\\ \mathrm{c}\end{array}$ | 253 ± 6$\begin{array}{c}\mathrm{h}\\ \mathrm{k}\end{array}$ | 302 ± 7$\begin{array}{c}\mathrm{i}\\ \mathrm{l}\end{array}$ | 202 ± 11$\begin{array}{c}\mathrm{h}\\ \mathrm{i},\mathrm{j}\end{array}$ | 267 ± 10$\begin{array}{c}\mathrm{h}\\ \mathrm{k}\end{array}$ | |

Senkirkin | 10.32 ± 0.06$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | n.d. | 8 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | n.d. | 27 ± 5$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{e}\end{array}$ | <MQL | 10 ± 1$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | <MQL | <MQL | <MQL | <MQL | |

Lasiocarpine | 81 ± 3$\begin{array}{c}\mathrm{f}\\ \mathrm{d}\end{array}$ | 45 ± 5$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 76 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 36 ± 2$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 66 ± 4$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 47 ± 4$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 57 ± 7$\begin{array}{c}\mathrm{f}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 50 ± 5$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 65 ± 4$\begin{array}{c}\mathrm{f}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 72 ± 7$\begin{array}{c}\mathrm{g}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 55 ± 7$\begin{array}{c}\mathrm{f}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | |

Lasiocarpine N-oxide | 12 ± 1$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | 25 ± 5$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 18 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 14 ± 1$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 38 ± 3$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 15 ± 4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 27 ± 6$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 14 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 15 ± 4$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 17.9 ± 0.2$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 18 ± 1$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | |

Total | 576 ± 10 | 616 ± 12 | 557 ± 10 | 495 ± 10 | 2940 ± 88 | 1055 ± 19 | 573 ± 19 | 829 ± 19 | 974 ± 18 | 841 ± 15 | 524 ± 15 | |

Analytes (µg/kg) | C-S12 | C-S13 | C-S14-A | C-S14-B | C-S15-A | C-S15-B | C-S16-A | C-S16-B | O-S17 | O-S18 | 0-S19 | W-S20 |

Intermedine | <MQL | 47 ± 3$\begin{array}{c}\mathrm{f}\\ \mathrm{b}\end{array}$ | 27.7 ± 0.8$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | <MQL | <MQL | n.d. | <MQL | n.d. | n.d. | <MQL | n.d. | n.d. |

Europine | <MQL | 3142 ± 4$\begin{array}{c}\mathrm{j}\\ \mathrm{d}\end{array}$ | 170 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{c}\end{array}$ | <MQL | <MQL | 11 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | <MQL | <MQL | <MQL | 25 ± 6$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b}\end{array}$ | <MQL | <MQL |

Lycopsamine | <MQL | 44 ± 5$\begin{array}{c}\mathrm{f}\\ \mathrm{b}\end{array}$ | 25.4 ± 0.8$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | <MQL | <MQL | n.d. | <MQL | n.d. | n.d. | <MQL | n.d. | <MQL |

Europine N-oxide | 9 ± 1$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a}\end{array}$ | 118 ± 7$\begin{array}{c}\mathrm{h}\\ \mathrm{b}\end{array}$ | 737 ± 9$\begin{array}{c}\mathrm{e}\\ \mathrm{c}\end{array}$ | 12.2 ± 0.2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 10.5 ± 0.1$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | 23 ± 4$\begin{array}{c}\mathrm{b}\\ \mathrm{a}\end{array}$ | 16.6 ± 0.8$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a}\end{array}$ | 11.3 ± 0.2$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | 17 ± 1$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | 1195 ± 235$\begin{array}{c}\mathrm{d}\\ \mathrm{d}\end{array}$ | 10.1 ± 0.2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | 10.5 ± 0.7$\begin{array}{c}\mathrm{b}\\ \mathrm{a}\end{array}$ |

Intermedine N-oxide | n.d. | n.d. | 22.8 ± 0.1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | n.d. | n.d. | n.d. | 152 ± 7$\begin{array}{c}\mathrm{i}\\ \mathrm{c}\end{array}$ | 206 ± 1$\begin{array}{c}\mathrm{d}\\ \mathrm{d}\end{array}$ | n.d. | n.d. | 602 ± 8$\begin{array}{c}\mathrm{h}\\ \mathrm{f}\end{array}$ | 305 ± 10$\begin{array}{c}\mathrm{i}\\ \mathrm{e}\end{array}$ |

Lycopsamine N-oxide | 24 ± 4$\begin{array}{c}\mathrm{g}\\ \mathrm{a}\end{array}$ | 18 ± 4$\begin{array}{c}\mathrm{b},\mathrm{c},\mathrm{d}\\ \mathrm{a}\end{array}$ | 78 ± 4$\begin{array}{c}\mathrm{b}\\ \mathrm{c}\end{array}$ | 104 ± 9$\begin{array}{c}\mathrm{h}\\ \mathrm{d},\mathrm{e}\end{array}$ | <MQL | 40 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{b}\end{array}$ | 99 ± 5$\begin{array}{c}\mathrm{h}\\ \mathrm{d}\end{array}$ | 99 ± 6$\begin{array}{c}\mathrm{c}\\ \mathrm{d}\end{array}$ | n.d. | 35.1 ± 0.3$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b}\end{array}$ | 211 ± 10$\begin{array}{c}\mathrm{g}\\ \mathrm{g}\end{array}$ | 163 ± 7$\begin{array}{c}\mathrm{g}\\ \mathrm{f}\end{array}$ |

Retrorsine | 19 ± 2$\begin{array}{c}\mathrm{f}\\ \mathrm{f}\end{array}$ | 12 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{c},\mathrm{d}\end{array}$ | 19 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{f}\end{array}$ | 9 ± 3$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 14 ± 2$\begin{array}{c}a\\ \mathrm{d},\mathrm{e}\end{array}$ | 8 ± 2$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | 11 ± 2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 10 ± 3$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | <MQL | <MQL | <MQL | <MQL |

Retrorsine N-oxide | 12.8 ± 0.8$\begin{array}{c}\mathrm{d},\mathrm{e}\\ \mathrm{c},\mathrm{d},\mathrm{e}\end{array}$ | 46 ± 7$\begin{array}{c}\mathrm{f}\\ \mathrm{g}\end{array}$ | 33 ± 9$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{f}\end{array}$ | 15 ± 3$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{d},\mathrm{e}\end{array}$ | 5.4 ± 0.4$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 4.8 ± 0.8$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | 5 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | n.d. | n.d. | <MQL | n.d. | 7 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ |

Seneciphylline | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |

Heliotrine | 12 ± 2$\begin{array}{c}\mathrm{c},\mathrm{d},\mathrm{e}\\ \mathrm{c}\end{array}$ | 30 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{f}\end{array}$ | 45 ± 10$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{h}\end{array}$ | <MQL | 6.2 ± 0.9$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b}\end{array}$ | n.d. | 12 ± 1$\begin{array}{c}\mathrm{c}\\ \mathrm{c}\end{array}$ | 5 ± 1$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 6.6 ± 0.5$\begin{array}{c}\mathrm{a}\\ \mathrm{b}\end{array}$ | 8.1 ± 0.7$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b},\mathrm{c}\end{array}$ | 7 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b},\mathrm{c}\end{array}$ | 17 ± 1$\begin{array}{c}\mathrm{c}\\ \mathrm{d}\end{array}$ |

Seneciphylline N-oxide | 17 ± 1$\begin{array}{c}\mathrm{e},\mathrm{f}\\ \mathrm{a},\mathrm{b}\end{array}$ | 20 ± 3$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 23 ± 4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 22 ± 3$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 23 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | n.d. | 21 ± 2$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 20 ± 4$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 13.7 ± 0.2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | 19 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 16 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | 20 ± 3$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ |

Heliotrine N-oxide | n.d. | <MQL | 34 ± 4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{d}\end{array}$ | n.d. | n.d. | n.d. | <MQL | 6 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | n.d. | 75 ± 6$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{f}\end{array}$ | 11 ± 4$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b}\end{array}$ | n.d. |

Senecivernine | n.d. | n.d. | 492 ± 78$\begin{array}{c}\mathrm{d}\\ \mathrm{h}\end{array}$ | 94 ± 5$\begin{array}{c}\mathrm{g}\\ \mathrm{c},\mathrm{d}\end{array}$ | n.d. | 8 ± 0.7$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | 19 ± 2$\begin{array}{c}\mathrm{d}\\ \mathrm{a}\end{array}$ | 21 ± 4$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | 115 ± 7$\begin{array}{c}\mathrm{g}\\ \mathrm{d}\end{array}$ | 224 ± 7$\begin{array}{c}\mathrm{c}\\ \mathrm{f}\end{array}$ | 57 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{b}\end{array}$ | n.d. |

Senecionine | 4 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 24 ± 4$\begin{array}{c}\mathrm{d},\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 524 ± 80$\begin{array}{c}\mathrm{d}\\ \mathrm{h}\end{array}$ | 52 ± 4$\begin{array}{c}\mathrm{f}\\ \mathrm{b},\mathrm{c}\end{array}$ | 5 ± 1$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | n.d. | 60 ± 6$\begin{array}{c}\mathrm{f}\\ \mathrm{b},\mathrm{c}\end{array}$ | 62 ± 4$\begin{array}{c}\mathrm{b}\\ \mathrm{b},\mathrm{c}\end{array}$ | 126 ± 8$\begin{array}{c}\mathrm{h}\\ \mathrm{d}\end{array}$ | 240 ± 6$\begin{array}{c}\mathrm{c}\\ \mathrm{f}\end{array}$ | 82 ± 5$\begin{array}{c}\mathrm{f}\\ \mathrm{c}\end{array}$ | 5.7 ± 0.4$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ |

Senecivernine N-oxide | 7.2 ± 0.6$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a},\mathrm{b}\end{array}$ | 17 ± 2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{d},\mathrm{e}\end{array}$ | 11 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{b},\mathrm{c}\end{array}$ | 13 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{c},\mathrm{d}\end{array}$ | 10.8 ± 0.2$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | n.d. | 12 ± 2$\begin{array}{c}\mathrm{c}\\ \mathrm{b},\mathrm{c}\end{array}$ | 20 ± 5$\begin{array}{c}\mathrm{a}\\ \mathrm{e},\mathrm{f}\end{array}$ | <MQL | 6 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 13 ± 2$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{c},\mathrm{d}\end{array}$ | 29 ± 1$\begin{array}{c}\mathrm{d}\\ \mathrm{g}\end{array}$ |

Senecionine N-oxide | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |

Echimidine | n.d. | n.d. | n.d. | n.d. | n.d. | <MQL | n.d. | n.d. | n.d. | n.d. | n.d. | n.d. |

Echimidine N-oxide | 186 ± 3$\begin{array}{c}\mathrm{i}\\ \mathrm{g},\mathrm{h}\end{array}$ | 322 ± 7$\begin{array}{c}\mathrm{i}\\ \mathrm{m}\end{array}$ | 183 ± 11$\begin{array}{c}\mathrm{c}\\ \mathrm{g},\mathrm{h}\end{array}$ | 297 ± 5$\begin{array}{c}\mathrm{i}\\ \mathrm{l}\end{array}$ | 204 ± 4$\begin{array}{c}\mathrm{g}\\ \mathrm{i},\mathrm{j}\end{array}$ | 173 ± 29$\begin{array}{c}\mathrm{d}\\ \mathrm{f},\mathrm{g}\end{array}$ | 182 ± 9$\begin{array}{c}\mathrm{j}\\ \mathrm{g},\mathrm{h}\end{array}$ | 491 ± 11$\begin{array}{c}\mathrm{e}\\ \mathrm{n}\end{array}$ | 102 ± 7$\begin{array}{c}\mathrm{f}\\ \mathrm{b},\mathrm{c}\end{array}$ | 84.6 ± 0.9$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a}\end{array}$ | 87 ± 6$\begin{array}{c}\mathrm{f}\\ \mathrm{a},\mathrm{b}\end{array}$ | 211 ± 8$\begin{array}{c}\mathrm{h}\\ \mathrm{j}\end{array}$ |

Senkirkin | <MQL | 9.8 ± 0.8$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | 17 ± 2$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{c},\mathrm{d}\end{array}$ | 19 ± 3$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{d}\end{array}$ | 8.6 ± 0.1$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{a}\end{array}$ | 9 ± 2$\begin{array}{c}\mathrm{a}\\ \mathrm{a}\end{array}$ | 16 ± 2$\begin{array}{c}\mathrm{c},\mathrm{d}\\ \mathrm{c}\end{array}$ | 10 ± 3$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 10.5 ± 0.1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 105 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{g}\end{array}$ | 13 ± 1$\begin{array}{c}\mathrm{b},\mathrm{c}\\ \mathrm{b}\end{array}$ | 30 ± 1$\begin{array}{c}\mathrm{d}\\ \mathrm{f}\end{array}$ |

Lasiocarpine | 70 ± 11$\begin{array}{c}\mathrm{h}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 326 ± 1$\begin{array}{c}\mathrm{i}\\ \mathrm{e}\end{array}$ | 986 ± 98$\begin{array}{c}\mathrm{f}\\ \mathrm{f}\end{array}$ | 48 ± 4$\begin{array}{c}\mathrm{e},\mathrm{f}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 36 ± 5$\begin{array}{c}\mathrm{f}\\ \mathrm{a},\mathrm{b}\end{array}$ | 38 ± 9$\begin{array}{c}\mathrm{c}\\ \mathrm{a},\mathrm{b},\mathrm{c}\end{array}$ | 86 ± 3$\begin{array}{c}\mathrm{g}\\ \mathrm{d}\end{array}$ | 67 ± 2$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 32 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{a}\end{array}$ | 80 ± 13$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{c},\mathrm{d}\end{array}$ | 63 ± 10$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 58 ± 2$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ |

Lasiocarpine N-oxide | 15 ± 3$\begin{array}{c}\mathrm{e},\mathrm{f}\\ \mathrm{a},\mathrm{b}\end{array}$ | 63 ± 6$\begin{array}{c}\mathrm{g}\\ \mathrm{c},\mathrm{d}\end{array}$ | 2947 ± 76$\begin{array}{c}\mathrm{g}\\ \mathrm{e}\end{array}$ | 45 ± 4$\begin{array}{c}\mathrm{e}\\ \mathrm{b},\mathrm{c},\mathrm{d}\end{array}$ | 10 ± 2$\begin{array}{c}\mathrm{c}\\ \mathrm{a}\end{array}$ | 25 ± 5$\begin{array}{c}\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 30 ± 8$\begin{array}{c}\mathrm{e}\\ \mathrm{a},\mathrm{b}\end{array}$ | 13 ± 1$\begin{array}{c}\mathrm{a}\\ \mathrm{a},\mathrm{b}\end{array}$ | 27 ± 2$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 31 ± 1$\begin{array}{c}\mathrm{a},\mathrm{b}\\ \mathrm{a},\mathrm{b}\end{array}$ | 27 ± 1$\begin{array}{c}\mathrm{d}\\ \mathrm{a},\mathrm{b}\end{array}$ | 72 ± 4$\begin{array}{c}\mathrm{f}\\ \mathrm{d}\end{array}$ |

Total | 376 ± 13 | 4239 ± 17 | 6375 ± 168 | 730 ± 15 | 334 ± 8 | 340 ± 31 | 722 ± 17 | 1041 ± 16 | 450 ± 14 | 2128 ± 236 | 1199 ± 19 | 928 ± 16 |

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**MDPI and ACS Style**

Izcara, S.; Casado, N.; Morante-Zarcero, S.; Sierra, I.
A Miniaturized QuEChERS Method Combined with Ultrahigh Liquid Chromatography Coupled to Tandem Mass Spectrometry for the Analysis of Pyrrolizidine Alkaloids in Oregano Samples. *Foods* **2020**, *9*, 1319.
https://doi.org/10.3390/foods9091319

**AMA Style**

Izcara S, Casado N, Morante-Zarcero S, Sierra I.
A Miniaturized QuEChERS Method Combined with Ultrahigh Liquid Chromatography Coupled to Tandem Mass Spectrometry for the Analysis of Pyrrolizidine Alkaloids in Oregano Samples. *Foods*. 2020; 9(9):1319.
https://doi.org/10.3390/foods9091319

**Chicago/Turabian Style**

Izcara, Sergio, Natalia Casado, Sonia Morante-Zarcero, and Isabel Sierra.
2020. "A Miniaturized QuEChERS Method Combined with Ultrahigh Liquid Chromatography Coupled to Tandem Mass Spectrometry for the Analysis of Pyrrolizidine Alkaloids in Oregano Samples" *Foods* 9, no. 9: 1319.
https://doi.org/10.3390/foods9091319