LC-MS/MS analysis of pesticides in Chrysanthemum morifolium and Ophiopogon japonicus following Ch. P 2025 General Rule 2341

Abstract

An UHPLC-MS/MS method was developed and validated for the simultaneous determination of 31 restricted pesticide residues in Chrysanthemum morifolium and Ophiopogon japonicus in accordance with Method VI, “Determination of Multiple Pesticide Residues in Relevant Medicinal Materials and Decoction Pieces,” specified in the General Rule 2341 of the 2025 Chinese Pharmacopoeia. Following matrix-specific sample preparation and solvent extraction, chromatographic separation was achieved using a Purospher^® STAR RP C18 endcapped column (2 μm, Hibar^® HR 100-2.1 mm). Method validation was performed in both herbal matrices by evaluating linearity, recovery, precision, and sensitivity. All target analytes exhibited excellent linearity, with correlation coefficients (R²) equal or greater than 0.995. Mean recoveries ranged from 77.6% to 114.9%, meeting the acceptance criteria specified in the Chinese Pharmacopoeia. Relative standard deviations (RSDs) were below 9.4% at reporting level, demonstrating good method precision. These results demonstrate that the method is suitable for the routine determination of restricted pesticide residues in Chrysanthemum morifolium and Ophiopogon japonicus.

Section overview

• Introduction
• Experimental
• Results and discussion
• Conclusion
• Related products

Introduction

Chrysanthemum morifolium and Ophiopogon japonicus are two medicinal herbs widely used in traditional Chinese medicine (TCM) due to their diverse therapeutic functions.¹ Phytochemical studies have shown that Chrysanthemum morifolium contains flavonoids, volatile oils, and polysaccharides, which contribute to its antibacterial, anti-inflammatory, and antioxidant effects.² Ophiopogon japonicus contains saponins, steroids, carbohydrates, and various amino acids and has been reported to exhibit multiple pharmacological effects, including anti-inflammatory, immunomodulatory, and cardiopulmonary protective activities.^3,4 Owing to their medicinal importance and growing demand, both herbs are cultivated extensively.

The large-scale cultivation of Chrysanthemum morifolium and Ophiopogon japonicus requires effective pest and disease management to maintain crop quality and yield. Consequently, pesticides are often used during cultivation to protect the crops and ensure consistent production. While the appropriate use of pesticides can effectively control pests and diseases, excessive or improper application may result in the accumulation of pesticide residues in plant tissues. These residues can enter the human body through the consumption of herbal products and may pose potential health risks.⁵ Therefore, reliable analytical methods for monitoring pesticide residues in medicinal herbs are essential to ensure product quality, consumer safety, and regulatory compliance.

The analysis of pesticide residues in traditional Chinese medicinal materials received increasing attention since the release of the 2020 edition of the Chinese Pharmacopoeia.^6,7 The 2025 edition further strengthens regulatory oversight through General Rule 2341, which introduces more comprehensive and stringent requirements on pesticide residue determination in Chinese herbal medicines. Compared with the 2020 edition, the list of banned pesticides has been expanded from 33 to 47 compounds, and the maximum residue limits for several highly toxic pesticides, including phorate, carbofuran, dicofol and metsulfuron-methyl, have been lowered. In addition, the 2025 edition establishes a dedicated regulatory framework for restricted pesticides via General Chapter 2341, covering 15 medicinal materials, including Chrysanthemum morifolium and Ophiopogon japonicus, and specifying 41 restricted pesticide residues, thereby filling the gap in the 2020 edition. Among the prescribed analytical procedures, Method VI—"Determination of Multiple Pesticide Residues in Relevant Medicinal Materials and Decoction Pieces”, specifies multi-residue pesticide detection methods for 15 types of medicinal materials, including Chrysanthemum morifolium and Ophiopogon japonicus.⁸ The method includes the determination of 10 pesticide residues by GC-MS/MS and 31 pesticide residues by LC-MS/MS.⁸ Based on Method VI of General Chapter 2341 in the 2025 Chinese Pharmacopoeia, an LC-MS/MS method was developed and evaluated for the determination of 31 restricted pesticide residues in Chrysanthemum morifolium and Ophiopogon japonicus. 

Experimental

In accordance with Method VI of General Rule 2341 in the 2025 edition of the Chinese Pharmacopoeia, the 15 medicinal materials specified in this general chapter are categorized into three groups, each with a dedicated sample pretreatment workflow. Chrysanthemum (Group 1) and Ophiopogon japonicus (Group 2) were selected as representative matrix samples for this study.

Standard preparation

• Pesticide stock solutions (500 μg/mL): 10 mg of each of the 31 pesticide reference standards were accurately weighed into separate 20 mL volumetric flasks. The solutions were diluted to volume with acetonitrile and mixed thoroughly.
• Mixed Standard Stock Solution (500 ng/mL): An aliquot of 10 μL from each of the 31 pesticide stock solutions, was transferred to a single 10 mL volumetric flask. The solution was diluted to volume with methanol and mixed thoroughly.
• Blank Matrix Solution: The blank matrix solution was prepared by processing a blank matrix sample according to the same preparation procedure described below. Note: both matrices showed background of certain target compounds (see below).
• Matrix-Matched Mixed Standard Solutions 1-7 (2.5, 5, 12.5, 25, 60, 125, and 250 ng/mL): Aliquots (2.0 mL) of the blank matrix solution were transferred into eight nitrogen evaporator vials and evaporated to dryness in a 40 °C water bath. The residues were reconstituted with 0.4 mL of methanol and spiked with 10 μL, 20 μL, 50 μL, 100 μL, 240 μL, 500 μL, and 1000 μL of the mixed standard stock solution, respectively. Subsequently, 0.6 mL of mobile phase A was added, and the solutions were brought to a final volume of 2.0 mL with methanol. The resulting solutions were vortex-mixed, filtered through a 0.2 μm PVDF membrane, and collected for LC-MS/MS analysis.

Sample preparation

Preparation of Chrysanthemum morifolium sample solution

1. Chrysanthemum morifolium powder (5.0 g, passed through a size No. 3 sieve), was accurately weighed and transferred to a 100 mL stoppered centrifuge tube.
2. 10 mL of water was added, and the mixture was shaken thoroughly.
3. The mixture was allowed to stand for 30 minutes.
4. 5 g of sodium chloride was added and the mixture was immediately shaken to ensure uniform dispersion.
5. 40 mL of acetonitrile was added, and the sample was homogenized at 12,000 rpm for 2 minutes.
6. The mixture was centrifuged at 4,000 rpm for 5 minutes.
7. The supernatant was collected. Acetonitrile (50 mL) was added to the residue, and the mixture was homogenized for 1 minute.
8. The mixture was centrifuged again at 4,000 rpm for 5 minutes.
9. The two supernatants were combined and diluted to 100 mL with acetonitrile.
10. The resulting solution was mixed thoroughly to obtain the Chrysanthemum morifolium sample solution.
11. An aliquot (2.0 mL) of the above solution was evaporated to dryness under a nitrogen stream at 40 °C. The residue was reconstituted with 0.4 mL of methanol, mixed with 0.6 mL of mobile phase A, and diluted to a final volume of 2.0 mL with methanol. The solution was filtered through a 0.2 μm PVDF membrane, and the filtrate was transferred to an autosampler vial for LC-MS/MS analysis.

Preparation of Ophiopogon japonicus sample solution

1. Ophiopogon japonicus powder (3.0 g, passed through a size No. 3 sieve), was accurately weighed and transferred to a 50 mL stoppered centrifuge tube.
2. A 1% glacial acetic acid aqueous solution (15 mL) was added, and the mixture was vortexed to ensure uniform dispersion.
3. The mixture was allowed to stand for 30 minutes.
4. 15 mL of acetonitrile was added, and the mixture was vortex-mixed thoroughly.
5. The tube was placed on an oscillator and shaken at 500 cycles per minute for 5 minutes.
6. The tube was placed in an ice bath and allowed to stand for 20 minutes.
7. A mixture of anhydrous magnesium sulfate and anhydrous sodium acetate (7.5 g, 4:1) was added, and the mixture was immediately shaken to ensure uniform dispersion.
8. The tube was shaken on the oscillator for 3 minutes.
9. The mixture was centrifuged at 4,000 rpm for 5 minutes.
10. An aliquot (5 mL) of the supernatant was transferred to a volumetric flask, diluted to 20 mL with acetonitrile, and mixed thoroughly to obtain the Ophiopogon japonicus sample solution.
11. An aliquot (2.0 mL) of the above solution was evaporated to dryness under a nitrogen stream at 40 °C. The residue was reconstituted with 0.4 mL of methanol, mixed with 0.6 mL of mobile phase A, and diluted to a final volume of 2.0 mL with methanol. The solution was filtered through a 0.2 μm PVDF membrane, and the filtrate was transferred to an autosampler vial for LC-MS/MS analysis.

Spiked samples -recovery/precision solution (0.25 mg/kg)

Chrysanthemum morifolium

Spiked solutions were prepared in accordance with the blank Chrysanthemum morifolium pretreatment procedure described above. After weighing blank powder in Step 1 and before extraction, 2.5 mL mixed standard stock solution (500 ng/mL) was added to 5.0 g blank powder to obtain a fortification level of 0.25 mg/kg, followed by 30 min equilibration. All subsequent extraction, dilution, nitrogen evaporation, reconstitution and filtration procedures were identical to Steps 1–11 described above.

Ophiopogon japonicus

Spiked solutions were prepared in accordance with the blank Ophiopogon japonicus pretreatment procedure described above. After weighing blank powder in Step 1 and before extraction, 1.5 mL mixed standard stock solution (500 ng/mL) was added to 3.0 g blank powder to obtain a fortification level of 0.25 mg/kg, followed by 30 min equilibration. All subsequent extraction, dilution, nitrogen evaporation, reconstitution and filtration procedures were identical to Steps 1–11 described above.

Spiked samples -recovery/precision solution (0.05 mg/kg)

The reporting limit (RL) spiked samples of Chrysanthemum morifolium and Ophiopogon japonicus were prepared following the same procedure as the 0.25 mg/kg fortified group. Before extraction, 0.5 mL (Chrysanthemum morifolium) and 0.3 mL (Ophiopogon japonicus) of the 500 ng/mL mixed standard stock solution was added to the weighed blank matrix to obtain a spiking level of 0.05 mg/kg.

LC-MS/MS analysis

The matrix-matched calibration standards and samples were analyzed by LC-MS under the chromatographic and mass spectrometric conditions summarized in Tables 2 to 4.

Results

Pesticide residues in Chrysanthemum morifolium and Ophiopogon japonicus were extracted according to the sample preparation procedures described in Method VI in the 2025 edition of General Rule 2341 and quantified by LC-MS/MS using matrix-matched external calibration. Method performance was evaluated through the assessment of matrix-matched calibration curves, precision, recovery, and sensitivity in both Chrysanthemum morifolium and Ophiopogon japonicus samples.

The chromatograms of Chrysanthemum morifolium and Ophiopogon japonicus matrix matched standards containing 31 pesticides at a concentration of 12.5 ng/mL are shown in Figures 1 and 2. Chromatograms of the unspiked Chrysanthemum morifolium and Ophiopogon japonicus samples are shown in Figures 3 and 4, respectively.