Introduction

As the use of traditional medicines in developed countries is exponentially growing, the concern associated with the human health risk by exposure of Pyrrolizidine alkaloids (PAs) has gradually increased because raw plant materials widely distributed in the world are used for medicinalpurposes such as dietary supplements and traditional medicines. PAs are secondary plant metabolites, produced naturally for protection against herbivores in some plant families [1]. Exposure of several PAs over longer periods has been known to cause hepatotoxicity, genotoxicity and carcinogenic potential in humans and animals for decades [2). However, unlike othr herbal medicines, there was difficulty in performance of the validation study of the two herbal medicines. That is, we found that Farfarae Flos contained 3 PAs including Senecionine, Senecionine N-oxide, Senkirkine, whereas Lithospermi Radix had 5 PAs such as Echimedine, Echimedine N-oxide, Intermedine, and Intermedine N-oxide, Lycopsamine N-oxide through the preliminary test.

The blank samples that do not contain the analytes are required for measuring the precise recovery in the validation study. Due to their extremely high content, they occurred easily saturation at LC–MS/MS analysis that quantifies the trace amount in samples to deviate quantitative range so that their recoveries could not be quantified simultaneously with those of the most PAs. To overcome such problem, this study developed a new extraction method for these PAs contained with high content in the herbal medicine samples. The new extraction was to a strategy inducing dilution effect. That is, it was a simple extraction that small amount sample (0.1 g) was extracted with a large amount (20–200 mL) of 50% MeOH without the purification using cation-ion cartridge (Fig. 2B).

Lebada et al. [36] explain that the best method to extract senkirkine and senecionine from Tussilago farfara was a combination comprised of refluxing with 50% methanol acidified and purification using solid-phase extraction on diol‐phase cartridges. The content of senkirkine in coltsfoot leaves was measured using capillary zone electrophoresis and its concentration was 2.5–11.2 ppm. Such a large variation of the amount of PAs seems to be ascribed to a different origin of plant materials, its preparation (raw material or dried samples), extraction methods (with various solvents), and quantification analytical methods (capillary zone electrophoresis, gas, or liquid chromatography) [36, 44]. To quantify the PAs in herbal medicines, the selection of appropriate solvent for extraction of PAs and analytical method is considered to be an important work. In addition, as PAs contained with a large amount in herbal medicines can occur easily saturation, the analysis methods used in practice should be validated to ensure its reliability prior to quantification analysis.

Matrix effects

As most herbal medicines are natural plants that contain various chemical compounds, they may influence the quantification analysis of pyrrolizidine alkaloids. Therefore, this study evaluated the matrix effects of 28 PAs in Farfarae Flos and Lithospermi Radix. The matrix effects (ME) were evaluated by the following categories: (i) high signal suppression (− 50% > ME) and moderate signal suppression (− 50% < ME >  − 20%); (ii) no matrix effect (− 20% < ME > 20%); (iii) moderate signal enhancement (20% < ME > 50%), and high signal enhancement (ME > 50%) [45]. As Farfarae Flow contained 3 PAs such as Senecionine, Senecionine N-oxide, and Senkirkine and Lithospermi Radix included Intermedine, and Intermedine N-oxide with high content. Matrix effects of these PAs could not be successfully evaluated because the coefficient deviations (r2) value of standard in these PAs curve could been not acquired as more than 0.99 so that their quantification analysis could not be accomplished.

As shown in Fig. 4 the matrix effects of the other PAs indicated as follows; In Farfarae Flow, 5 PAs such as Echimidine-N-oxide (EmN), Europine (Eu), Lasiocarpine (Lc), Lasiocarpine N-oxide (LcN), and Retrorsine N-oxide (ReN) indicated strong ion enhancement, whereas 4 PAs including Echimidine (Em), Erucifoline N-oxide (ErN), Jacobine (Jb), Lycopsamine N-oxide (LcN) occurred strong ion suppression. In Lithospermi Radix, 4 PAs such as Echimidine-N-oxide (EmN), Erucifoline N-oxide (ErN), Heliotrine N-oxide (HnN), Lycopsamine (La) showed strong ion enhancement, while 7 PAs like Erucifoline occurred ion suppression. Considering these results, quantification analysis against most of the analytes seemed to be influenced by various components originated from herbal medicine samples so that this study conducted the matrix-matched analysis to avoid or reduce the interfering of various materials contained in the herbal medicine samples.

Fig. 4
figure 4

Matrix effects of 28 pyrrolizidine alkaloids in Farfarae Flos and Lithospermi Radix; 1: Echimidine, 2: Echimidine-N-oxide, 3: Erucifoline, 4: Erucifoline-N-oxide, 5: Europine, 6: Europine-N-oxide, 7: Heliotrine, 8: Heliotrine-N-oxide, 9: Intermedine, 10: Intermedine-N-oxide, 11: Jacobine, 12: Jacobine-N-oxide, 13: Lasiocarpine, 14: Lasiocarpine-N-oxide, 15: Lycopsamine, 16: Lycopsamine-N-oxide, 17: Monocrotaline, 18: Monocrotaline-N-oxide, 19: Retrorsine, 20: Retrorsine-N-oxide, 21: Senecionine, 22: Senecionine-N-oxide, 23: Seneciphylline, 24: Seneciphylline-N-oxide, 25: Senecivernine, 26: Senecivernine-N-oxide, 27: Senkirkine, 28: Trichodesmine

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Linearity and sensitivity

Because of matrix effects in herbal medicines, our validation study was performed by matrix-matched analysis. The results of linearity in most of the PAs revealed to have good linearity, indicating r2 ≥ 0.997 in Farfarae Flos or Lithospermi Radix (Tables 3 and 4). The linearity of 3 PAs (Senecionine, Senecionine N-oxide, Senkirkine) contained in Farfarae Flos and 2 PAs ( Intermedine and Intermedine N-oxide) included in Lithospermi Radix revealed to have good linearities, indicating r2 = 0.999. In Farfarae Flos, LOQs of 25 PAs included in the original samples were revealed to have the range of 0.1–24.2 μg/kg (Table 3). In Lithospermi Radix, LOQs of 24 PAs contained in Lithospermi Radix indicated to be the range 0.2–7.2 μg/kg. The LOQs of Seneciphylline and Retrorsin N-oxide indicated to have 23.0 μg/kg, and 17.4 μg/kg in Lithospermi Radix respectively, indicating comparatively higher LOQ than that of the other PAs (Table 4). The improved LC–MS/MS method indicated that Lycopsamine N-oxide had 1.1 μg/kg of LOQ in Farfarae Flos. Through such results, the matrix components seemed to influence LOQs of the PA.

Table 3 Linearities, LODs, LOQs, recoveries, and RSDs obtained by LC–MS/MS analysis method in Farfarae Flos
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Table 4 Linearities, LODs, LOQs, recoveries, and RSDs obtained by LC–MS/MS analysis method in Lithospermi Radix
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Also, we assumed that certain components that contained Lithospermi Radix might occur strong suppression of Lycopasamine N-oxide. As 3 PAs such as Senecione, Senecione N-oxide, and Senkerkine in Farfarae Flos and 5 PAs intermedine, inter medine N-oxide, Echimidine, and Echimidine N-oxide, and Lycopsamine N-oxide in Lithospermi Radix were contained with a large amount in herbal medicines so that the saturation occurs, their LOD and LOQ could not be measured. BfR-PA-Tea method indicated to have 0.5–1.7 μg/kg of LOD and 1.7–6.4 μg/kg of LOQ for 28 PAs. The proposed method revealed to have LOQ values less than 10 μg/kg in most PAs, except of a few PAs. The LOQ values obtained by the proposed method was not largely different from those of BfR method.

Accuracy and precision

The recoveries and relative standard deviations (RSDs) of PAs were evaluated through the matrix-matched analysis. The test solutions were prepared as follows; after the mixed standard solutions were spiked into the samples to be three different concentration levels and then pretreated by the method described above. However, These PAs that contained a large amount in Farfarae Flos or Lithospermi Radix, the recoveries and RSDs were measured by quantification method using standard solution instead of matrix-matched analysis. In test method validation, AOAC guideline requires the reference criteria such as recovery of 70–125%, repeatability precision (intra-day RSD) < 15%, and the reproducibility precision (inter-day RSD) < 32%.

In Farfarae Flos, most PAs that were not contained in Farfarae Flos showed satisfactory recovery as the ranges of 70.4–118.0%. In precision evaluation of Farfarae Flos, most analytes indicated satisfactory intra-day RSD values, however, a few analytes showed to be beyond the criteria in intra-day RSD as follows; Echimidine(18.8%), Retrorsine (20.1%), Seneciphylline (17.6%), Seneciphylline N-oxide (16.9%), Seneciverine (17.8%). In inter-day RSDs, 25 PAs indicated to be less than the criteria recommended at AOAC guideline (Table 3).

In Lithospermi Radix, most of the PAs that were not contained in the Lithospermi Radix indicted the ranges of 70.2–116.5%. Their intra-day RSDs of all analytes except 2 PAs, Europine N-oxide (24.2%) and Jacobine (18.5%), showed to be less than 15% and the inter-day RSDs of 24 PAs indicated to have values less than 32% (Table 3).

Three PAs including senecionine, senecionine N-oxide and senkirkine showed satisfactory recovery in Farfarae Flos, indicating the ranges of 92.8–119.0% and their intra-and inter-day RSDs also indicated to satisfy the recommended criteria. Also, in Lithospermi Radix, 5 PAs considered to have high content in Lithospermi Radix revealed recovery ranges of 108.5–130.3%, even though only Echimidine N-oxide did not satisfy the criteria, indicating 130.2% of recovery. Their intra-and inter-day RSDs had satisfactory values in in Lithospermi Radix (Table 4). On the basis of these results, the proposed method was considered as a great quantification analysis that can determine some PAs with high content such as 3 PAs in Farfare Flos and 5 PAs in Lithospermi Radix as well as most of the PAs used in this study.

Monitoring of PAs

The developed LC–MS/MS method was applied to determine the content of 28 PAs in 20 herbal medicine samples purchased from Korean herbal medicine markets imported from China. Ten herbal medicine samples per matrix were used for the monitoring study. Most PAs among 28 PAs were not detected or indicated to have contents of less than the LOQ in two herbal medicine samples, Farfarae Flos and Lithospermi Radix. However, they revealed to possess a part of PAs with high concentration in. In Farfarae Flos, all samples contained three PAs such as senecionine (6.9–9.5 μg/g), senecionine N-oxide (6.2–13.7 μg/g), and senkirkine (97.4–175.1 μg/g), whereas in Lithospermi Radix, all samples showed to have four PAs such as intermedine (2.3–30.9 μg/g), intermedine N-oxide (27.1–78.5 μg/g), echimedine (0.4–1.6 µg/g), and echimedine N-oxide (0.7–5.5 µg/g) and only one sample contained Lycopsamine (1.73 µg/g) and Lycopsamine N-oxide (7.82 µg/g).

According to the literature, Adamczak et al. [35] quantified the alkaloid content (Senkirkine and Senecionine) in T. farfara using HPLC analysis method. The method employs two-time extraction by using 50% (v/v) methanol acidified with citric acid and dichloromethane and diethyl ether. After evaporating the solvent, the dissolved residual solution was purified by SPE extraction. As a result, their content indicated 0.02–0.58 of Senecionine and 0.02–0.47 μg/g of Senkirkine. Also, Tussilago farfara are reported to contain toxic PAs with various ranges from 0.1 to 368 μg/g [36, 44, 46,47,48]. Also, some researchers claim that dry drugs of Tussilago farfara (Farfarae Flos) contain senkirkine from 0.1 to 150 ppm [49, 50] and senecionine ranging from 0.1 to 10 ppm [50]. In comparing these results, the contents of senkirkine and senecionine obtained by the proposed method seemed to be not largely different from their content reported previously. Therefore, the proposed method was considered to determine well such PAs in herbal medicinal samples. In addition, this study suggests that it is are necessary to monitor their residual amount in the final herbal medicinal products used Farfarae Flos or Lithospermi Radix as well as the herbal medicines through a further study.