• Recovery: 70-120% at three concentrations (0.2, 1.0 and 5.0 ng/mL) • Repeatability: ≤20% at each level • Linearity: R2
≥0.99 with residuals ≤±20% • LOQ: <0.05 ng/mL
Results and Discussion: Method validation for SPE followed by HPLC-MS/MS
Table 2: Linearity, LOQ and matrix effects for chamomile, peppermint and green tea infusions, following SPE and HPLC-MS/MS analysis.
Table 2 shows the linearity, LOQ and matrix emect (ME) for atropine and scopolamine for each of the herbal infusions validated.
Atropine Herbal Infusion
Chamomile Peppermint
Green tea
Linearity (ng/mL)
R2
0.5 - 50 0.5 - 50
0.5 - 50
0.992 0.996
0.991 LOQ
(ng/mL) 0.010
0.010 0.025
ME (%)
-17 -14
-12 R2
0.995 0.992
0.993 LOQ
(ng/mL) 0.010
0.010 0.025
ME (%)
-38 -24
-22
Each herbal infusion matrix-matched calibration showed good linearity from 0.5 to 50 ng/mL, with R2 values between 0.991 - 0.996. Additionally, all calibration levels had residuals of <±20%, which indicated that the method met the specifi cation for linearity.
Table 2: Linearity, LOQ and matrix eGects for chamomile, peppermint and green tea infusions, following SPE and HPLC-MS/MS analysis.
Each herbal infusionmatrix-matched calibration showed good linearity from 0.5 to 50 ng/mL, with R2
values between 0.991 - 0.996. Additionally, all calibration levels had
LOQ values for each herbal infusion were below the LOQ specifi cation of 0.05 ng/mL. Chamomile and peppermint teas both had an LOQ of 0.010 ng/mL for atropine and scopolamine. Green tea had a slightly higher LOQ concentration of 0.025 ng/mL, due to the nosier baseline around the two analytes, which caused a lower signal to noise level.
residuals of <±20%, which indicated that the method met the specification for linearity.
Matrix effect was evaluated for all three herbal infusions. All samples showed a negative matrix effect (ion suppression), with atropine having a lower matrix effect than scopolamine. SANTE/11312/2021 guidelines state that in the case of >±20% matrix effect, this would need to be addressed in calibration. Hence for recovery and repeatability testing, matrix-matched standards were used to calibrate for both atropine and scopolamine.
LOQ values for each herbal infusion were below the LOQ specification of 0.05 ng/mL. Chamomile and peppermint teas both had an LOQ of 0.010 ng/mL for both atropine and scopolamine. Green tea had a slightly higher LOQ concentration of 0.025 ng/mL, due to the nosier baseline around the two analytes, which caused a lower signal to noise level.
Matrix emect was evaluated for all three herbal infusions. All samples showed a negative matrix emect (ion suppression), with atropine having a lower matrix emect than scopolamine. SANTE/11312/2021 guidelines16
state that in the case of >±20% matrix emect, this would need to be addressed in calibration. Hence for recovery and
repeatability testing, matrix-matched standards were used to calibrate for both atropine and scopolamine.
Scopolamine
Tea Infusion Sample
Chamomile-1 Chamomile-2 Chamomile-3 Chamomile-4 Chamomile-5 Chamomile-6 Peppermint-1 Peppermint-2 Peppermint-3 Green tea-1 Green tea-2 Green tea-3
Atropine <LOQ
<LOQ
0.013 ± 0.0035 <LOQ
0.59 ± 0.0070 <LOQ <LOQ <LOQ <LOQ <LOQ ND
<LOQ 9
Figure 3: Overlaid chromatograms (n=8) showing the intra-day repeatability data for atropine and scopolamine in peppermint infusion at 0.2 ng/mL. Retention time is 1.85 min for scopolamine and 3.43 min for atropine.
Table 4: Concentration values (± standard deviation, n=4) for atropine, scopolamine and sum of both analytes in herbal infusions prepared using the SPE and HPLC-MS/MS method. ND = Not detected, due to signal to noise being less than 3:1.
Application of the method on herbal tea infusions
Concentration (ng/mL) Scopolamine ND
<LOQ <LOQ ND
0.35 ± 0.012 ND
<LOQ <LOQ
0.011 ± 0.0014 ND ND ND
Sum of Atropine and Scopolamine
<LOQ <LOQ
0.013 ± 0.0035 <LOQ
0.94 ± 0.013 <LOQ <LOQ <LOQ
0.011 ± 0.0014 <LOQ ND
<LOQ
The validated SPE and HPLC-MS/MS method was applied to twelve herbal infusions of chamomile, peppermint and green teas, with each infusion prepared in quadruplicate. Most samples contained low or undetectable levels of atropine and scopolamine. However, one chamomile sample (chamomile-5) signifi cantly exceeded EU regulatory limits for atropine at 0.59 ng/mL (~3 x EU limit of 0.2 ng/mL), scopolamine detected at 0.35 ng/mL (exceeds the EU limit), with a sum of atropine and scopolamine concentrations of 0.94 ng/mL (~5 x EU limit).
Table 4: Concentration values (± standard deviation, n=4) for atropine, scopolamine and sum of both analytes in herbal infusions prepared using the SPE and HPLC-MS/MS method. ND = Not detected, due to signal to noise being less than 3:1.
The validated SPE and HPLC-MS/MS method was applied to twelve herbal infusions of chamomile, peppermint and green teas, with each infusion prepared in quadruplicate. Most samples contained low or undetectable levels of atropine and scopolamine. However, one chamomile sample (chamomile-5) significantly exceeded EU regulatory limits for atropine at 0.59 ng/mL (~3 x EU limit of 0.2 ng/mL), scopolamine detected at
Figure 2: Chromatogram showing the LOQ of atropine and scopolamine at 0.010 ng/mL (n=8). Retention time is 1.85 min for scopolamine and 3.43 min for atropine. The resolution between atropine and the peak at 3.20 minutes is 5.0.
Table 3: Recovery and repeatability data for chamomile, peppermint and green tea infusions using the combined SPE and HPLC-MS/MS method for the analysis of atropine and scopolamine. Intra-day repeatability (n=8, in one day) and inter-day repeatability (n=16, on two days) SD = standard deviation.
Figure 2:Chromatogram showing the LOQ of atropine and scopolamine at 0.010 ng/mL (n=8). Retention time is 1.85 min for scopolamine and 3.43 min for atropine. The resolution between atropine and the peak at 3.20 minutes is 5.0.
Herbal Infusion Sample
Chamomile - 0.2 ng/mL Chamomile - 1 ng/mL Chamomile - 5 ng/mL Peppermint - 0.2 ng/mL Peppermint - 1 ng/mL Peppermint - 5 ng/mL Green Tea - 0.2 ng/mL Green Tea - 1 ng/mL Green Tea - 5 ng/mL
Recovery (% ± SD)
93 ± 1.7 99 ± 1.6 98 ± 2.0 87 ± 0.88 95 ± 0.78 92 ± 1.6 78 ± 2.7 96 ± 1.2 96 ± 0.66
Atropine Intra-day
Repeatability (%RSD)
1.8 1.6 2.1 1.0 0.8 1.7 3.4 1.3 0.7
Inter-day
Repeatability (%RSD)
6.9 9.8 2.4 2.4 4.5 2.2
15.8 5.1 3.9
Recovery (% ± SD)
84 ± 3.1 94 ± 2.4 99 ± 2.1 85 ± 1.8 96 ± 1.6 95 ± 1.2 78 ± 2.5 93 ± 2.4 96 ± 1.4
Scopolamine Intra-day
Repeatability (%RSD)
3.6 2.6 2.2 2.1 1.7 1.3 3.2 2.6 1.5
The recovery and repeatability results showed good recovery for each herbal infusion at every concentration tested, ranging from 78 – 99% for both atropine and scopolamine. The intra-day repeatability showed excellent results, ranging from 0.7 - 3.4 %RSD for atropine and 1.3 - 3.6 %RSD for scopolamine. Inter-day repeatability had a higher %RSD value, ranging from 2.2 - 15.8 %RSD for atropine and 2.1 - 10.8 %RSD for scopolamine which was within specifi cation. This was expected to be higher due to additional variance when testing on different days. This demonstrated that all herbal infusions met specifi cation for recovery and reproducibility.
Table 3 - Recovery and repeatability data for chamomile, peppermint and green tea infusions using the combined SPE and HPLC-MS/MS method for the analysis of atropine and scopolamine. Intra-day repeatability (n=8, in one day) and inter-day repeatability (n=16, on two days) SD = standard deviation.
The recovery and repeatability results showed good recovery for each herbal infusion at every concentration tested, ranging from 78 – 99% for both atropine and scopolamine. The intra-day repeatability showed excellent results, ranging from 0.7 - 3.4 %RSD for atropine and 1.3 - 3.6 %RSD for scopolamine. Inter-day repeatability had a higher %RSD value, ranging from 2.2 - 15.8 %RSD for atropine and 2.1 - 10.8 %RSD for scopolamine which was within specification. This was expected to be higher due to additional variance when testing on dimerent days. This demonstrated that all herbal infusions met specification for recovery and reproducibility.
Inter-day
Repeatability (%RSD)
4.8 5.5 2.5 4.1 5.7 2.6
10.8 5.1 2.1
Figure 4: Overlaid chromatograms showing the 0.2 ng/mL EU limit reference standard versus chamomile-5’s chromatograms (n=4) at a mean concentration of 0.35 ng/mL for scopolamine and 0.59 ng/mL for atropine. Retention time is 1.85 min for scopolamine and 3.43 min for atropine.
Conclusions
This work validated a 96-well format SCX solid phase extraction method using a smaller bed weight (10 mg) for the analysis of atropine and scopolamine in three commonly consumed herbal infusions – chamomile, peppermint and green tea. The method met SANTE/11312/2021 validation guidelines with the method showing good linearity, low limits of quantifi cation and high recovery with good repeatability (intra- and inter-day) across three concentrations (0.2, 1.0 and 5.0 ng/mL). The application of the method to commercially available teas confi rmed that most infusions contained low or undetectable levels of atropine and scopolamine. However, one chamomile sample exceeded the regulatory limits for both atropine and scopolamine by almost fi ve times. These results show that a low bed weight SPE method is suitable for the analysis of herbal tea samples and can offer a more sustainable alternative to higher bed weight SPE methods.
References
1: L. González-Gómez, S. Morante-Zarcero, D. Pérez-Quintanilla, I. Sierra, Occurrence and Chemistry of Tropane Alkaloids in Foods, with a Focus on Sample Analysis Methods: A Review on Recent Trends and Technological Advances, Foods, 2022, 11, 407, DOI: 10.3390/foods11030407.
2: P. P. J. Mulder, M. Nijs, M. Castellari, M. Hortos, S. MacDonald, C. Crews, J. Hajslova, M. Stranska, Occurrence of tropane alkaloids in food, EFSA Supporting Publications, 13, DOI: 10.2903/sp.efsa.2016. EN-1140.
3: M. Nijs, C. Crews, F. Dorgelo, S. MacDonald, P. P. J. Mulder, Emerging Issues on Tropane Alkaloid Contamination of Food in Europe, Toxins, 2023, 15, 98, DOI: 10.3390/toxins15020098
4: L. González-Gómez, S. Morante-Zarcero, J. A. M. Pereira, J. S. Câmara, I. Sierra, Improved Analytical Approach for Determination of Tropane Alkaloids in Leafy Vegetables Based on µ-QuEChERS Combined with HPLC-MS/MS, Toxins (Basel), 2022, 14, 650, DOI: 10.3390/toxins14100650.
5: Health for humans, animals & plants – Tropane Alkaloids,
https://www.ages.at/en/human/nutrition-food/ residues-contaminants-from-a-to-z/tropane-alkaloids, (accessed May 2025).
6: Commission Regulation (EU) 2023/915 of 25 April 2023 on maximum levels for certain contaminants in food and repealing Regulation (EC) No 1881/2006,
https://eur-lex.europa.eu/legal-content/EN/ TXT/?uri=CELEX%3A02023R0915-20250101, (Accessed May 2025)
7: ISO 3103: Tea — Preparation of liquor for use in sensory tests, 1980 edition.
8: Analytical Quality Control And Method Validation Procedures For Pesticide Residues Analysis in Food and Feed - Sante 11312/2021 V2,
https://food.ec.europa.eu/system/fi les/2023-11/pesticides_mrl_guidelines_ wrkdoc_2021-11312.pdf, (accessed April 2025).
Figure 3: Overlaid chromatograms (n=8) showing the intra-day repeatability data for atropine and scopolamine in peppermint infusion at 0.2 ng/mL. Retention time is 1.85 min for scopolamine and 3.43 min for atropine.
9: Commission Implementing Regulation (EU) 2023/2783 of 14 December 2023 laying down the methods of sampling and analysis for the control of the levels of plant toxins in food and repealing Regulation (EU)
2015/705,
https://eur-lex.europa.eu/eli/reg_impl/2023/2783/oj, (accessed April 2025).
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