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Chemical Standardization of Milk Thistle (Silybum marianum L.) Extract Using UHPLC-MS/MS and the Method of Standard Addition
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  • Ruth N. Muchiri
    Ruth N. Muchiri
    Linus Pauling Institute, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 2900 SW Campus Way, Corvallis, Oregon 97331, United States
    More by Ruth N. Muchiri
  • Richard B. van Breemen*
    Richard B. van Breemen
    Linus Pauling Institute, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 2900 SW Campus Way, Corvallis, Oregon 97331, United States
    *Tel.: 541-737-5078. Fax: 541-737-5077. Email: [email protected]
    More by Richard B. van Breemen
    Orcidhttps://orcid.org/0000-0003-2016-0063
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Journal of the American Society for Mass Spectrometry

Cite this: J. Am. Soc. Mass Spectrom. 2024, 35, 8, 1726–1732
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https://doi.org/10.1021/jasms.4c00125
Published July 2, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Extracts prepared from the seeds of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] are widely used as dietary supplements due to anti-inflammatory, antitumor, and hepatoprotective effects. Called silymarin, the main components of lipophilic extracts of milk thistle seeds are flavonoids and flavonolignans including silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, taxifolin, and 2,3-dehydrosilybins. The aim of this study was to develop a method based on UHPLC-MS/MS for the chemical authentication and standardization of milk thistle silymarin. Validation included the method of standard addition to account for the lack of a blank matrix. Potential matrix effects were investigated by analyzing silymarin standards dissolved only in the initial UHPLC mobile phase. Measurements of six flavonolignans and taxifolin in the milk thistle extract using UHPLC-MS/MS with standard addition or external standard calibration produced similar results for all analytes except silydianin and 2,3-dehydrosilybin B, which showed significant peak enhancement during negative ion electrospray due to botanical matrix effects. The UHPLC-MS/MS-based method of standard addition requires <10 min per injection and is suitable for the standardization of silymarin from milk thistle in support of preclinical and clinical studies of safety and efficacy.

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Introduction

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Milk thistle (Silybum marianum L. Gaertn.) is native to the Mediterranean and is cultivated widely as a medicinal plant and dietary supplement. (1) Extracts of milk thistle seeds have been used for over 2000 years, primarily for liver problems and hepatoprotection, and are under investigation for anti-inflammatory and antitumor activities. (2−5) Milk thistle extract consists of >60% silymarin, (6) which is a mixture of flavonolignans and flavonoids including silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin, 2,3-dehydrosilybin B, and taxifolin (Figure 1). Milk thistle is the 23rd most popular botanical dietary supplement in the United States, (7) and the worldwide market for milk thistle extracts was $103 million in 2022. (8)

Figure 1

Figure 1. Chemical structures of common milk thistle constituents in silymarin and deuterated internal standards.

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Despite being the subject of over 70 clinical trials listed on clinicaltrials.gov alone, the therapeutic benefits of silymarin from milk thistle remain inconclusive, in part due to inadequately standardized test material. (9) Without chemical standardization of the botanical being tested, dosages of the active compounds are unknown, results can vary from batch to batch, and preclinical as well as clinical studies cannot be reproduced. (10,11) Variations in the composition of silybin from milk thistle results from different growth conditions (12,13) and variations in extract preparation and handling. (13−15) Therefore, a validated analytical method for the measurement and standardization of silymarin constituents in test materials is essential for the support of pharmacological and toxicological studies both in vitro and in vivo and to enable the correlation of activities with dosage.
Although there are many analytical methods for measuring silymarin constituents and metabolites in human serum (16) and urine, (17) mostly utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), few methods have been reported for the standardization of silymarin compounds in milk thistle extracts. Analytical methods to measure constituents of silymarin have included thin layer chromatography, HPLC with UV absorbance detection, HPLC with electrochemical detection, HPLC-MS, (18) and UHPLC-MS/MS. (19)
Due to the complexity of the botanical matrix, suppression or enhancement of MS signals can occur when using electrospray. (20) To help compensate for these matrix effects, stable isotope-labeled analogues of each analyte (surrogate standards) can be added to the samples. Even if available, surrogate standards often do not completely correct for quantitation error caused by matrix effects during electrospray MS. One solution to this problem is to enhance the chromatographic separation such that all analytes are completely resolved from interfering matrix constituents using approaches such as ultrahigh-pressure liquid chromatography (UHPLC). (21) Another solution to address this problem is to use a blank matrix when preparing standard curves that contain all components except for the analytes to be measured. Because surrogate standards for silymarin compounds and blank milk thistle extract matrix were unavailable, we used UHPLC instead of HPLC and the method of standard addition (22) to validate a quantitative method based on UHPLC-MS/MS. In this approach, each milk thistle extract served as its own matrix for standard curve preparation. For comparison, external standard curves were also prepared using silymarin compounds dissolved in the initial UHPLC mobile phase. The method of standard addition has been used for the chemical standardization of other botanical extracts including extracts of licorice, (23) garlic, (24) and botanical mixtures. (25) Here, we report the development and standardization of a quantitative assay for six milk thistle flavonolignans plus taxifolin in milk thistle extract using electrospray UHPLC-MS/MS that is fast, sensitive, and accurate.

Methods

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Materials and Reagents

Silychristin A (96.5% purity; DTXSID50187512), silydianin (96.2% purity; DTXSID70858696), and taxifolin (92.8% purity; DTXSID8022450) were purchased from ChromaDex (Los Angeles, CA). Isosilybin A (98.4% purity; DTXSID20453675), isosilybin B (≥88.7% purity; DTXSID80447055), and 2,3-dehydrosilybin B (99.3% purity; DTXSID601102493) were purchased from MilliporeSigma (St. Louis, MO). Silybin B (≥98.0% purity; DTXSID30858697), daidzein-d4, and genistein-d4 (≥99.0% purity) were purchased from Cayman Chemicals (Ann Arbor, MI). An ethanolic extract of milk thistle seed (90.6% silymarin compounds) was supplied by the Botanical Safety Consortium and had been purchased from Euromed S.A. (Barcelona, Spain). HPLC-MS grade acetonitrile, methanol, and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA). Ultrapure water was prepared using a MilliporeSigma Milli-Q water purification system. All other reagents and solvents were reagent grade or better and were purchased from VWR (Visalia, CA).

Preparation of Calibration Standards and Internal Standards

Stock solutions of milk thistle flavonolignan and taxifolin standards (1 mg/mL each) were prepared in methanol, aliquoted, and stored at −20 °C until use. The working solutions for the calibration standards were prepared by diluting the stock solution in methanol/water (1:1, v/v). Internal standard stock solutions containing d4-daidzein and d4-genistein (1 mg/mL) were prepared by dissolving accurately weighed aliquots in dimethyl sulfoxide. Note that only d4-daidzein was used for data normalization. The stock solutions were diluted in methanol/water (1:1, v/v) to obtain working solutions (125 ng/mL) of each deuterium-labeled standard for addition during sample pretreatment. Since isotope-labeled milk thistle flavonolignans were not commercially available, d4-daizein and d4-genistein were used as internal standards to correct for variabilities during sample preparation and analysis as they displayed similar solubility and extraction efficiency.

Standard Addition Method and External Calibration Curve

Milk thistle extract was weighed and dissolved in methanol to produce a 1 mg/mL stock solution, which was diluted to 1 μg/mL with 50% aqueous methanol. Known amounts of flavonolignan and taxifolin standards were spiked into each sample to generate solutions for a standard addition calibration curve. For comparison, an external calibration curve was prepared by spiking different working solutions of flavonolignan and taxifolin standards in neat solvent to give a concentration range of 0.4–512 ng/mL for isosilybin A, isosilybin B, silybin B, 2,3-dehydrosilybin B, and taxifolin and 1.2–512 ng/mL for silychristin and silydianin.

UHPLC-MS and UHPLC-MS/MS of Milk Thistle Flavonolignans and Taxifolin

Chromatographic separation of the milk thistle flavonolignans and taxifolin was carried out using a Shimadzu (Kyoto, Japan) Nexera UHPLC system fitted with a Waters (Milford, MA) Cortecs UPLC C18 column (1.7 μm, 130 Å, 2.1 × 150 mm). The mobile phase consisted of a gradient from water (A) to methanol (B) both containing 0.01% formic acid as follows: 0–2 min 50% B, 2–4.5 min 50–55% B, 4.5–5.5 min 55–60% B, and 6–8 min 60–75% B. The column was reconditioned in 50% methanol for 2 min between injections. The flow rate was 0.3 mL/min, the injection volume was 5 μL, and the column and autosampler temperatures were 40 and 10 °C respectively. The total UHPLC analysis time per sample for the separation of milk thistle flavonolignans and taxifolin was 10 min.
High-resolution mass spectra and tandem mass spectra (using data-dependent acquisition to acquire tandem mass spectra of all major constituents) of flavolignans and taxifolin in milk thistle extract were obtained using a Shimadzu (Kyoto, Japan) 9030 Q-ToF tandem mass spectrometer interfaced with a Shimadzu Nexera UHPLC system. The electrospray ionization interface temperature was 300 °C, and the voltage was −3.5 kV for negative ion mode. The heat block and desolvation line temperatures were 400 and 250 °C, respectively. Nitrogen was used as a drying gas at a flow rate of 10 L/min, for nebulization at 3 L/min, and as a heating gas at 10 L/min. Mass spectra and product ion tandem mass spectra were acquired every 100 ms over the mass range m/z 70–700. During data-dependent acquisition, six dependent events were chosen at an intensity threshold of 3000, and the product ion tandem mass spectra were obtained using a collision energy of 35 V with an energy spread of 17 V.
For quantitative analysis, the UHPLC system was coupled to a Shimadzu LCMS-8060 triple quadruple mass spectrometer equipped with negative ion electrospray. Nitrogen was used as drying gas at a flow rate of 5 L/min and for nebulization at 3 L/min. The interface and desolvation line temperatures were set to 400 and 300 °C, respectively. The milk thistle flavonolignans and taxifolin were measured using collision-induced dissociation with selected reaction monitoring (SRM). The SRM dwell time for each transition was 15 ms, and the collision gas pressure was 230 kPa. Data acquisition, integration, and linear standard curves fitting were carried out using Shimadzu Lab Solutions software version 5.7. The concentrations of flavonolignans and taxifolin in the milk thistle extract were calculated using Microsoft Excel software (Seattle, WA).

Results and Discussion

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Chemical Authentication of Milk Thistle Extract

The goal of this investigation was to develop and validate an accurate and efficient assay for the chemical authentication and standardization of milk thistle extracts for use in preclinical and clinical studies of safety and efficacy. (26,27) This assay helped support the authentication and standardization of the milk thistle extract used by the Botanical Safety Consortium, (28) which is an international group of experts working to identify fit-for-purpose assays for the evaluation of the safety of botanicals used as dietary supplements or medicinal plants. (29,30)
As a first step in this process, reversed phase UHPLC with high-resolution mass spectrometry was used to separate the major constituents in silymarin (Figure 2 and Supplemental Figures 1 and 2) and confirm their elemental compositions (Supplemental Table 1). Most flavonolignans in silymarin are constitutional isomers with the molecular formulas C25H22O10 (Figure 1), and the measured masses of these compounds in this silymarin preparation were within 10 ppm of the expected values (Supplemental Table 1). The major flavanonol taxifolin (molecular formula C15H12O7) was also detected and measured in this extract. Data-dependent high-resolution tandem mass spectra of the major silymarin constituents were acquired and compared with those of standards. Based on identical tandem mass spectra (Supplemental Figures 3–21), identical accurate mass measurements, and identical retention times during UHPLC as standards (Supplemental Table 1), the milk thistle constituents silychristin, silydianin, silybin A, silybin B, isosilybin A, isosilybin B, 2,3-dehydrosilybin B, and taxifolin were identified, additional constituents were characterized, and the extract was chemically authenticated as silymarin from milk thistle. Although no pure commercial standard was available for silybin A, a commercially available equimolar silybin A and silybin B mixture was used to isolate silybin A as described previously. (16) The abundant peak eluting at 19.6 min had an elemental composition (C25H22O10), tandem mass spectrum (Supplemental Figure 10), and retention time that were consistent with silybin A. Also, the peak at 12.8 min in the UHPLC-MS/MS chromatogram (Figure 2) had an elemental composition (C25H22O10), tandem mass spectrum, and retention time consistent with neusilychristin, a constitutional isomer of silychristin (18) (Supplemental Figure 6).

Figure 2

Figure 2. High-resolution negative ion electrospray UHPLC-MS total ion chromatogram survey of milk thistle extract (silymarin) showing separation and detection of major silymarin constituents plus the internal standard daidzein-d4. The corresponding high-resolution Q-ToF positive ion electrospray chromatogram is shown in Supplemental Figure 2. The most abundant milk thistle constituents (labeled in red font) were then measured using a faster UHPLC-MS/MS method on a triple quadrupole mass spectrometer.

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Chemical Standardization of Milk Thistle Extract

A UHPLC-MS/MS quantitative assay was developed for the measurement of six flavonolignans and taxifolin in milk thistle extract. The SRM transitions used for MS/MS quantitative analysis of each analyte are shown in Table 1. Optimized for speed, baseline separation of each analyte was achieved in <10 min using a C18 UHPLC column (Figure 3). This separation is comparable in speed to a previous UHPLC-MS/MS method (19) for the measurement of flavonolignans in milk thistle extract but considerably faster than an HPLC-MS method that required 40 min per analysis. (31) Because no blank matrix is available for milk thistle extract, standards for external standard curves were prepared in 50% methanol. The external calibration curves for silybin B, isosilybin A, isosilybin B, and taxifolin were linear over 3 orders of magnitude (0.4–512 mg/g dry extract), and the calibration curves for silychristin and silydianin were linear from 1.2 to 512 mg/g dry extract (Table 1). Note that the dynamic range of this method is approximately 10-fold greater than that of a previous UHPLC-MS/MS method. (19)

Figure 3

Figure 3. Negative ion electrospray UHPLC-MS/MS selected reaction monitoring chromatograms of flavonolignans, flavanonol taxifolin, and deuterated internal standards daidzein and genistein. (A) Standards at 256 ng/mL in 50% aqueous methanol; (B) internal standards at 100 ng/mL; and (C) silymarin (10 μg/mL) extracted from milk thistle seed.

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Table 1. UHPLC-MS/MS Negative Ion Electrospray Retention Times (RT), MS/MS SRM Transitions, Collision Energies (CE), Concentrations (ng/mL) of Standards Spiked into Dilute Milk Thistle Extract, and Coefficients of Determination (R2) for External Calibrants in Neat Solvent and for the Method of Standard Addition
analyteRT (min)SRM transitions m/zCE (V)spiked concentrations (ng/mL)R2 neat solventR2 std addn
taxifolin1.7303 → 285a120.42481632641282565120.99910.9999
303 → 12522
silychristin2.0481 → 125311.22481632641282565120.99840.9988
481 → 32521
silydianin2.5481 → 179291.22481632641282565120.99630.9956
481 → 12526
silybin B4.4481 → 125280.42481632641282565120.99920.9996
481 → 30120
isosilybin A5.4481 → 125280.42481632641282565120.99940.9997
481 → 45317
isosilybin B5.7481 → 125270.42481632641282565120.99910.9998
481 → 45319
2,3-dehydrosilybin B8.4479 → 299180.42481632641282565120.99680.9959
479 → 27135
d4-daidzein3.5257 → 21229            
257 → 13639
d4-genistein5.0273 → 16330            
273 → 13731
a

The first SRM transition for each analyte was used as the quantifier while the second SRM transition was used as a qualifier in the UHPLC-MS/MS assay.

To test for matrix effects during UHPLC-MS/MS, which can interfere with quantitative measurements of constituents in complex botanical extracts, the standard addition method of quantitation was also used for the determination of levels of constituents in the milk thistle extract. An aliquot of milk thistle extract was diluted to produce UHPLC-MS/MS signals for the endogenous flavonolignans and taxifolin with signal-to-noise ratios of ∼10:1. Different concentrations of milk thistle standards were spiked into this extract as indicated in Table 1. After UHPLC-MS/MS with SRM analysis, the internal standard normalized peak area for each flavonolignan or taxifolin spiked sample was plotted on the y-axis while the known concentration of each spiked standard was plotted on the x-axis. The resulting linear trendline was extrapolated to the zero-peak area, and the absolute point of interception of the abscissa was the endogenous concentration of the flavonolignan or taxifolin in the sample (as an example, see the method of standard addition curve for silychristin in Figure 4).

Figure 4

Figure 4. Using the method of standard addition, the concentration of the analyte silychristin was determined by extrapolating the calibration curve to 0 on the negative side of the x-axis (in the equation, solve for x when y = 0). A constant amount of internal standard was added to each aliquot of milk thistle extract immediately before UHPLC-MS/MS analysis. The peak area of silychristin, spiked with different amounts of silychristin standard, was normalized by dividing with the internal standard peak area. This area ratio was plotted versus the amount of silychristin spiked into the extract.

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Peak suppression or enhancement during electrospray mass spectrometry that might be caused by milk thistle matrix was evaluated by comparing the concentrations obtained using the standard addition method to that of an external calibration curve prepared in neat solvent. Ion suppression can occur when coeluting compounds compete for ionization during the saturable electrospray process. Peak enhancement can occur when coeluting compounds, such as isomers of the analyte, contribute to the signal or when coeluting compounds potentiate the ionization of the analyte, perhaps through charge exchange. Although both analytical methods produced linear standard curves (Table 1) and nearly identical values for most analytes, matrix effects produced significant ion suppression that was observed for the milk thistle compounds silydianin (10.8%) and 2,3-dehydrosilybin B (28%) (Table 2). Therefore, only the method of standard addition provided accurate measurement for the chemical standardization of milk thistle extracts in this investigation.
Table 2. Quantitative Analysis of Flavonolignans in Milk Thistle Extract Using UHPLC-MS/MS with the Method of Standard Addition or an External Standard Curve Prepared Using Neat Solvent
 milk thistle extract (mg/g extract)
milk thistle compoundstandard addition (n = 3)neat solvent standard curve (n = 3)
taxifolin37.48 ± 2.5135.04 ± 0.64
silychristin158.7 ± 7.6159.0 ± 4.5
silydianin13.95 ± 3.1012.45 ± 0.63
silybin B263.1 ± 13.3242.3 ± 8.3
isosilybin A35.17 ± 1.3435.65 ± 0.75
isosilybin B19.75 ± 1.1320.18 ± 1.91
2,3-dehydrosilybin B3.060 ± 0.5102.210 ± 0.065
Due to superior speed, sensitivity, and selectivity, UHPLC-MS/MS-based assays are widely used for the quantitative analysis of natural products from foods, dietary supplements, and medicinal plants in serum, plasma, urine, and tissues. For those matrices, blank specimens are readily available that contain possible interfering substances but not the natural product analytes. Therefore, during the development of assays for these matrices utilizing electrospray MS, which is highly susceptible to matrix effects like peak enhancement or suppression, potential matrix interference can be detected. Once identified, the analytical method can be changed to mitigate or eliminate matrix interference. However, measurement of endogenous natural products in samples such as botanicals and botanical extracts poses a special problem due to the absence of blank botanical material.
Matrix effects resulting in signal enhancement or suppression can be mitigated but not always eliminated by using stable isotope-labeled surrogate standards of each analyte. However, surrogate standards are expensive and unavailable for many natural products such as the flavonolignans in milk thistle. Another approach used to minimize matrix effects is to optimize chromatographic separation of analytes from interfering matrix compounds. In this investigation, UHPLC was used instead of HPLC for improved analyte separation, but matrix interference still occurred. Slower gradient separations might have overcome these matrix effects but at the cost of longer analysis times and possibly loss of sensitivity due to broader peak shape. Note that a previous UHPLC-MS/MS method did not detect any matrix effects but did not test for them using the method of standard addition. (19)

Conclusions

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By comparing electrospray UHPLC-MS/MS measurements of constituents in the milk thistle extract silymarin using the method of standard addition to those obtained using an external standard curve with standard dissolved in neat solvent, matrix effects were observed that suppressed the signals for two flavonolignans, silydianin and 2,3-dehydrosilybin B. Although both methods were fast (<10 min per UHPLC-MS analysis) and produced linear standard curves over a wider dynamic range than previous methods, only the method of standard addition corrected for the matrix effects and produced accurate values for all analytes. This method of standard addition was determined to be fit-for-purpose for use in the chemical standardization of milk thistle extracts intended for preclinical and clinical investigation.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.4c00125.

  • Peak assignments for the high-resolution UHPLC-MS analyses of milk thistle extract; tandem mass spectra of all milk thistle analytes measured in this study (PDF)

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Author Information

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  • Corresponding Author
  • Author
    • Ruth N. Muchiri - Linus Pauling Institute, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 2900 SW Campus Way, Corvallis, Oregon 97331, United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors would like to thank Shimadzu for providing the LCMS-9030 Q-ToF system used in this study. This work was supported by the Health and Environmental Sciences Institute (HESI) Botanical Safety Consortium, funded by the U.S. FDA, HHS, NIEHS, and HESI via the DOI FCG under Blanket Purchase Agreement Order 140D0421F0068. Additional support came from the NIH Intramural Research Program, NIEHS projects ZIC ES103391-01 and ZIC ES103373-02, and contracts HHSN273201400027C (Battelle) and HHSN273201400020C (MRIGlobal). The HESI BSC initiative is primarily funded by in-kind contributions of time, expertise, and experimental effort from public and private sector participants, supplemented by direct support for program infrastructure and management from HESI’s corporate supporters. A list of supporting organizations is available at https://botanicalsafetyconsortium.org/.

References

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    Recent advances in the analysis of flavonolignans of Silybum marianum
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    Journal of Pharmaceutical and Biomedical Analysis (2016), 130 (), 301-317CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    A review. Exts. of milk thistle (Silybum marianum, Asteraceae) have been recognized for centuries as remedies for liver and gallbladder disorders. The active constituents of milk thistle fruits are flavonolignans, collectively known as silymarin. Flavonolignans in S. marianum are structurally diverse, 23 constituents have been isolated from purple- and white-flowering variants. Flavonolignans have a broad spectrum of bioactivities and silymarin has been the subject of intensive research for its profound pharmacol. activities. Silymarin is extd. from the seeds, commercialized in standardized form, and widely used in drugs and dietary supplements. The thorough anal. of silymarin, its constituents and silymarin-contg. products has a key role in the quality control of milk thistle-based products. Due to the low concn. of analytes, esp. pharmacol. and pharmacokinetic studies require more and more selective and sensitive, advanced techniques. The objective of the present review is to summarize the recent advances in the chem. anal. of S. marianum exts., including the chem. compn., isolation and identification of flavonolignans, sample prepn., and methods used for qual. and quant. anal. Various anal. approaches have been surveyed, and their resp. advantages and limits are discussed.
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    A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts
    Graf, Tyler N.; Cech, Nadja B.; Polyak, Stephen J.; Oberlies, Nicholas H.
    Journal of Pharmaceutical and Biomedical Analysis (2016), 126 (), 26-33CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
    Validated methods are needed for the anal. of natural product secondary metabolites. These methods are particularly important to translate in vitro observations to in vivo studies. Herein, a method is reported for the anal. of the key secondary metabolites, a series of flavonolignans and a flavonoid, from an ext. prepd. from the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. This report represents the first UHPLC MS-MS method validated for quant. anal. of these compds. The method takes advantage of the excellent resoln. achievable with UHPLC to provide a complete anal. in less than 7 min. The method is validated using both UV and MS detectors, making it applicable in labs. with different types of anal. instrumentation available. Lower limits of quantitation achieved with this method range from 0.0400 μM to 0.160 μM with UV and from 0.0800 μM to 0.160 μM with MS. The new method is employed to evaluate variability in constituent compn. in various com. S. marianum exts., and to show that storage of the milk thistle compds. in DMSO leads to degrdn.
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    A quant. method is described for the detn. of allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester) in garlic, using std. addns. of alliin (L-(+)-S-allylcysteine sulfoxide) in conjunction with supercrit. fluid extn. (SFE) and HPLC anal. with UV-vis absorbance detection. Optimum CO2-SFE conditions provided 96% recovery for allicin with precision of 3% (RSD) for repeat samples. The incorporation of an internal std. (allyl Ph sulfone) in the SFE step resulted in a modest improvement in recovery (99%) and precision (2% RSD). Std. addns. of alliin were converted to allicin in situ by endogenous alliinase (L-(+)-S-alk(en)ylcysteine sulfoxide lyase, EC 4.4.1.4). Complete conversion of the spiked alliin to allicin was achieved by making addns. after homogenization-induced conversion of the naturally occurring cysteine sulfoxides to thiosulfinates had taken place, thus eliminating the likelihood of competing reactions. Concn. values for allicin detd. in samples of fresh garlic (Allium sativum and A. ampeloprasum) and com. available garlic powders (A. sativum) by std. addn. of alliin were found in all cases to be in statistical agreement (95% confidence interval) with values detd. using a secondary allicin std. (concn. detd. using published extinction coeffs.). This method provides a convenient alternative for assessing the amt. of allicin present in fresh and powd. garlic, as alliin is a far more stable and com. prevalent compd. than allicin and is thus more amenable for use as a std. for routine anal.
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    Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry
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    A sensitive method for the simultaneous quantitation of six active constituents in com. silymarin standardized exts. was developed based on liq. chromatog. (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely sepd. and quantified by LC/MS. Silymarin obtained from Sigma-Aldrich Co. was evaluated and used as std. ref. material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in com. standardized silymarin exts., resp. Significant variation was found between different com. silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in com. milk thistle exts. The calibration curves were over the range from 0.25 to 100 μg/mL for silychristin and silydianin, and from 0.10 to 100 μg/mL for silybin A, silybin B, isosilybin A and isosilybin B, resp. (r 2 ≥ 0.9958). For all six active constituents, the overall intra-day precision values, based on the relative std. deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodol. could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.

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  • Abstract

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    Figure 1

    Figure 1. Chemical structures of common milk thistle constituents in silymarin and deuterated internal standards.

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    Figure 2

    Figure 2. High-resolution negative ion electrospray UHPLC-MS total ion chromatogram survey of milk thistle extract (silymarin) showing separation and detection of major silymarin constituents plus the internal standard daidzein-d4. The corresponding high-resolution Q-ToF positive ion electrospray chromatogram is shown in Supplemental Figure 2. The most abundant milk thistle constituents (labeled in red font) were then measured using a faster UHPLC-MS/MS method on a triple quadrupole mass spectrometer.

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    Figure 3

    Figure 3. Negative ion electrospray UHPLC-MS/MS selected reaction monitoring chromatograms of flavonolignans, flavanonol taxifolin, and deuterated internal standards daidzein and genistein. (A) Standards at 256 ng/mL in 50% aqueous methanol; (B) internal standards at 100 ng/mL; and (C) silymarin (10 μg/mL) extracted from milk thistle seed.

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    Figure 4

    Figure 4. Using the method of standard addition, the concentration of the analyte silychristin was determined by extrapolating the calibration curve to 0 on the negative side of the x-axis (in the equation, solve for x when y = 0). A constant amount of internal standard was added to each aliquot of milk thistle extract immediately before UHPLC-MS/MS analysis. The peak area of silychristin, spiked with different amounts of silychristin standard, was normalized by dividing with the internal standard peak area. This area ratio was plotted versus the amount of silychristin spiked into the extract.

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  • References

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      Polyak, Stephen J.; Morishima, Chihiro; Lohmann, Volker; Pal, Sampa; Lee, David Y. W.; Liu, Yanze; Graf, Tyler N.; Oberlies, Nicholas H.
      Proceedings of the National Academy of Sciences of the United States of America (2010), 107 (13), 5995-5999, S5995/1-S5995/5CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Silymarin, also known as milk thistle ext., inhibits hepatitis C virus (HCV) infection and also displays antioxidant anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, the authors evaluated the hepatoprotective actions of the 7 major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-α-induced NF-κB transcription, virus-induced oxidative stress, and T-cell proliferation. All compds. were well tolerated by Huh7 human hepatoma cells up to 80 μM, except for isosilybin B, which was toxic to cells above 10 μM. Select compds. had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compds. inhibited JFH-1 NS5B polymerase but only at concns. above 300 μM. Silymarin suppressed TNF-α activation of NF-κB dependent transcription, which involved partial inhibition of IκB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compds. blocked JFH-1 virus-induced oxidative stress, including compds. that lacked antiviral activity. The most potent compds. across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixt. of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compds. may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clin. endpoints are urgently needed.
    5. 5
      Saller, R.; Brignoli, R.; Melzer, J.; Meier, R. An Updated Systematic Review with Meta-Analysis for the Clinical Evidence of Silymarin. Forsch. Komplementärmedizin 2008, 15, 9– 20,  DOI: 10.1159/000113648
      There is no corresponding record for this reference.
    6. 6
      Berman, J. 50-Complementary and Alternative Medicines for Infectious Diseases. In Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 8th ed.; Bennett, J. E., Dolin, R., Blaser, M. J., Eds.; W. B. Saunders: Philadelphia, PA, 2015; p 597.
      There is no corresponding record for this reference.
    7. 7
      Smith, T.; Bauman, H.; Resetar, H. US Sales of Herbal Supplements Decline Slightly in 2022. HerbalGram 2024, 139, 52– 69
      There is no corresponding record for this reference.
    8. 8
      Milk Thistle Supplements Market. https://www.factmr.com/report/662/milk-thistle-supplements-market (accessed 2024–05–28).
      There is no corresponding record for this reference.
    9. 9
      Ball, K. R.; Kowdley, K. V. A Review of Silybum marianum (Milk Thistle) as a Treatment for Alcoholic Liver Disease. J. Clin. Gastroenterol. 2005, 39, 520– 528,  DOI: 10.1097/01.mcg.0000165668.79530.a0
      9
      A review of Silybum marianum (milk thistle) as a treatment for alcoholic liver disease
      Ball Karen R; Kowdley Kris V
      Journal of clinical gastroenterology (2005), 39 (6), 520-8 ISSN:0192-0790.
      Silybum marianum (milk thistle) and its derivatives have been used for centuries for the treatment of liver disease. This review focuses exclusively on published literature pertaining to the potential use of Silybum marianum or its derivatives for the treatment of alcoholic liver disease. Clinical studies have varied greatly in quality, with the majority limited by inadequate sample size, lack of uniformity in the population treated, lack of standardization of preparations studied, variability in dosing regimens, inconsistent outcome measures, and lack of information on concurrent use of alcohol during the treatment period. While Silybum marianum and its derivatives appear to be safe and the available evidence on the mechanisms of action appears promising, there are currently insufficient data from well-conducted clinical trials to recommend their use in patients with alcoholic liver disease.
    10. 10
      Tamayo, C.; Diamond, S. Review of Clinical Trials Evaluating Safety and Efficacy of Milk Thistle (Silybum marianum [L.] Gaertn.). Integr. Cancer Ther. 2007, 6, 146– 157,  DOI: 10.1177/1534735407301942
      There is no corresponding record for this reference.
    11. 11
      Fenclova, M.; Novakova, A.; Viktorova, J.; Jonatova, P.; Dzuman, Z.; Ruml, T.; Kren, V.; Hajslova, J.; Vitek, L.; Stranska-Zachariasova, M. Poor Chemical and Microbiological Quality of the Commercial Milk Thistle-Based Dietary Supplements May Account for Their Reported Unsatisfactory and Non-Reproducible Clinical Outcomes. Sci. Rep. 2019, 9 (1), 11118,  DOI: 10.1038/s41598-019-47250-0
      There is no corresponding record for this reference.
    12. 12
      Chambers, C. S.; Holečková, V.; Petrásková, L.; Biedermann, D.; Valentová, K.; Buchta, M.; Křen, V. The Silymarin Composition. . . and Why Does It Matter???. Food Res. Int. 2017, 100, 339– 353,  DOI: 10.1016/j.foodres.2017.07.017
      There is no corresponding record for this reference.
    13. 13
      Booker, A.; Suter, A.; Krnjic, A.; Strassel, B.; Zloh, M.; Said, M.; Heinrich, M. A Phytochemical Comparison of Saw Palmetto Products Using Gas Chromatography and 1H Nuclear Magnetic Resonance Spectroscopy Metabolomic Profiling. J. Pharm. Pharmacol. 2014, 66, 811– 822,  DOI: 10.1111/jphp.12198
      There is no corresponding record for this reference.
    14. 14
      Harkey, M. R.; Henderson, G. L.; Gershwin, M. E.; Stern, J. S.; Hackman, R. M. Variability in Commercial Ginseng Products: An Analysis of 25 Preparations. Am. J. Clin. Nutr. 2001, 73, 1101– 1106,  DOI: 10.1093/ajcn/73.6.1101
      There is no corresponding record for this reference.
    15. 15
      Duan, L.; Carrier, D. J.; Clausen, E. C. Silymarin Extraction from Milk Thistle Using Hot Water. Appl. Biochem. Biotechnol. 2004, 114, 559– 568,  DOI: 10.1385/ABAB:114:1-3:559
      There is no corresponding record for this reference.
    16. 16
      Muchiri, R. N.; van Breemen, R. B. Single Laboratory Validation of UHPLC-MS/MS Assays for Six Milk Thistle Flavonolignans in Human Serum. J. AOAC Int. 2021, 104, 232– 238,  DOI: 10.1093/jaoacint/qsaa110
      There is no corresponding record for this reference.
    17. 17
      Lazzeroni, M.; Petrangolini, G.; Legarreta Iriberri, J. A.; Pascual Avellana, J.; Tost Robusté, D.; Cagnacci, S.; Macis, D.; Aristarco, V.; Bonanni, B.; Morazzoni, P.; Johansson, H.; Riva, A. Development of an HPLC-MS/MS Method for the Determination of Silybin in Human Plasma, Urine and Breast Tissue. Molecules. 2020, 25 (12), 2918,  DOI: 10.3390/molecules25122918
      There is no corresponding record for this reference.
    18. 18
      Csupor, D.; Csorba, A.; Hohmann, J. Recent Advances in the Analysis of Flavonolignans of Silybum marianum. J. Pharm. Biomed. Anal. 2016, 130, 301– 317,  DOI: 10.1016/j.jpba.2016.05.034
      18
      Recent advances in the analysis of flavonolignans of Silybum marianum
      Csupor, Dezso; Csorba, Attila; Hohmann, Judit
      Journal of Pharmaceutical and Biomedical Analysis (2016), 130 (), 301-317CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      A review. Exts. of milk thistle (Silybum marianum, Asteraceae) have been recognized for centuries as remedies for liver and gallbladder disorders. The active constituents of milk thistle fruits are flavonolignans, collectively known as silymarin. Flavonolignans in S. marianum are structurally diverse, 23 constituents have been isolated from purple- and white-flowering variants. Flavonolignans have a broad spectrum of bioactivities and silymarin has been the subject of intensive research for its profound pharmacol. activities. Silymarin is extd. from the seeds, commercialized in standardized form, and widely used in drugs and dietary supplements. The thorough anal. of silymarin, its constituents and silymarin-contg. products has a key role in the quality control of milk thistle-based products. Due to the low concn. of analytes, esp. pharmacol. and pharmacokinetic studies require more and more selective and sensitive, advanced techniques. The objective of the present review is to summarize the recent advances in the chem. anal. of S. marianum exts., including the chem. compn., isolation and identification of flavonolignans, sample prepn., and methods used for qual. and quant. anal. Various anal. approaches have been surveyed, and their resp. advantages and limits are discussed.
    19. 19
      Graf, T. N.; Cech, N. B.; Polyak, S. J.; Oberlies, N. H. A Validated UHPLC-Tandem Mass Spectrometry Method for Quantitative Analysis of Flavonolignans in Milk Thistle (Silybum marianum) Extracts. J. Pharm. Biomed. Anal. 2016, 126, 26– 33,  DOI: 10.1016/j.jpba.2016.04.028
      19
      A validated UHPLC-tandem mass spectrometry method for quantitative analysis of flavonolignans in milk thistle (Silybum marianum) extracts
      Graf, Tyler N.; Cech, Nadja B.; Polyak, Stephen J.; Oberlies, Nicholas H.
      Journal of Pharmaceutical and Biomedical Analysis (2016), 126 (), 26-33CODEN: JPBADA; ISSN:0731-7085. (Elsevier B.V.)
      Validated methods are needed for the anal. of natural product secondary metabolites. These methods are particularly important to translate in vitro observations to in vivo studies. Herein, a method is reported for the anal. of the key secondary metabolites, a series of flavonolignans and a flavonoid, from an ext. prepd. from the seeds of milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. This report represents the first UHPLC MS-MS method validated for quant. anal. of these compds. The method takes advantage of the excellent resoln. achievable with UHPLC to provide a complete anal. in less than 7 min. The method is validated using both UV and MS detectors, making it applicable in labs. with different types of anal. instrumentation available. Lower limits of quantitation achieved with this method range from 0.0400 μM to 0.160 μM with UV and from 0.0800 μM to 0.160 μM with MS. The new method is employed to evaluate variability in constituent compn. in various com. S. marianum exts., and to show that storage of the milk thistle compds. in DMSO leads to degrdn.
    20. 20
      Furey, A.; Moriarty, M.; Bane, V.; Kinsella, B.; Lehane, M. Ion Suppression; a Critical Review on Causes, Evaluation, Prevention and Applications. Talanta. 2013, 115, 104– 122,  DOI: 10.1016/j.talanta.2013.03.048
      20
      Ion suppression; A critical review on causes, evaluation, prevention and applications
      Furey, Ambrose; Moriarty, Merisa; Bane, Vaishali; Kinsella, Brian; Lehane, Mary
      Talanta (2013), 115 (), 104-122CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      A review. The consequences of matrix effects in mass spectrometry anal. are a major issue of concern to anal. chemists. The identification of any ion suppressing (or enhancing) agents caused by sample matrix, solvent or LC-MS system components should be quantified and measures should be taken to eliminate or reduce the problem. Taking account of ion suppression should form part of the optimization and validation of any quant. LC-MS method. For example the US Food and Drug Administration has included the evaluation of matrix effects in its "Guidance for Industry on Bioanal. Method Validation" (F.D.A. Department of Health and Human Services, Guidance for industry on bioanal. method validation, Fed. Regist. 66 (100) 2001). If ion suppression is not assessed and cor. in an anal. method, the sensitivity of the LC-MS method can be seriously undermined, and it is possible that the target analyte may be undetected even when using very sensitive instrumentation. Sample anal. may be further complicated in cases where there are large sample-to-sample matrix variations (e.g. blood samples from different people can sometimes vary in certain matrix components, shellfish tissue samples sourced from different regions where different phytoplankton food sources are present, etc) and therefore exhibit varying ion-suppression effects. Although it is widely agreed that there is no generic method to overcome ion suppression, the purpose of this review is to: provide an overview of how ion suppression occurs, outline the methodologies used to assess and quantify the impact of ion suppression, and discuss the various corrective actions that were used to eliminate ion suppression in sample anal., that is to say the deployment of techniques that eliminate or reduce the components in the sample matrix that cause ion suppression. This review article aims to collect together the latest information on the causes of ion suppression in LC-MS anal. and to consider the efficacy of common approaches to eliminate or reduce the problem using relevant examples published in the literature.
    21. 21
      Van De Steene, J. C.; Lambert, W. E. Comparison of Matrix Effects in HPLC-MS/MS and UPLC-MS/MS Analysis of Nine Basic Pharmaceuticals in Surface Waters. J. Am. Soc. Mass Spectrom. 2008, 19, 713– 718,  DOI: 10.1016/j.jasms.2008.01.013
      There is no corresponding record for this reference.
    22. 22
      Guidelines for Standard Method Performance Requirements AOAC Official Methods of Analysis. Appendix F. 2016; https://www.aoac.org/wp-content/uploads/2019/08/app_f.pdf (accessed 2024–05–28).
      There is no corresponding record for this reference.
    23. 23
      Li, G.; Nikolic, D.; van Breemen, R. B. Identification and Chemical Standardization of Licorice Raw Materials and Dietary Supplements Using UHPLC-MS/MS. J. Agric. Food Chem. 2016, 64, 8062– 8070,  DOI: 10.1021/acs.jafc.6b02954
      23
      Identification and Chemical Standardization of Licorice Raw Materials and Dietary Supplements Using UHPLC-MS/MS
      Li, Guannan; Nikolic, Dejan; van Breemen, Richard B.
      Journal of Agricultural and Food Chemistry (2016), 64 (42), 8062-8070CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      Defined as the roots and underground stems of principally three Glycyrrhiza species, Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC., and Glycyrrhiza inflata Batalin, licorice has been used as a medicinal herb for millennia and is marketed as root sticks, powders, and exts. Identity tests described in most pharmacopeial monographs enabled the distinction of Glycyrrhiza species. Accordingly, an ultrahigh-performance liq. chromatog.-tandem mass spectrometry (UHPLC-MS/MS) assay using the method of std. addn. was developed to quantify 14 licorice components (liquiritin, isoliquiritin, liquiritin apioside, isoliquiritin apioside, licuraside, liquiritigenin, isoliquiritigenin, glycyrrhizin, glycyrrhetinic acid, glabridin, glycycoumarin, licoricidin, licochalcone A, and p-hydroxybenzylmalonic acid), representing several natural product classes including chalcones, flavanones, saponins, and isoflavonoids. Using this approach, G. glabra, G. uralensis, and G. inflata in a variety of forms including root powders and exts. as well as complex dietary supplements could be differentiated and chem. standardized without concerns due to matrix effects.
    24. 24
      Rybak, M. E.; Calvey, E. M.; Harnly, J. M. Quantitative Determination of Allicin in Garlic: Supercritical Fluid Extraction and Standard Addition of Alliin. J. Agric. Food Chem. 2004, 52, 682– 687,  DOI: 10.1021/jf034853x
      24
      Quantitative Determination of Allicin in Garlic: Supercritical Fluid Extraction and Standard Addition of Alliin
      Rybak, Michael E.; Calvey, Elizabeth M.; Harnly, James M.
      Journal of Agricultural and Food Chemistry (2004), 52 (4), 682-687CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A quant. method is described for the detn. of allicin (2-propene-1-sulfinothioic acid S-2-propenyl ester) in garlic, using std. addns. of alliin (L-(+)-S-allylcysteine sulfoxide) in conjunction with supercrit. fluid extn. (SFE) and HPLC anal. with UV-vis absorbance detection. Optimum CO2-SFE conditions provided 96% recovery for allicin with precision of 3% (RSD) for repeat samples. The incorporation of an internal std. (allyl Ph sulfone) in the SFE step resulted in a modest improvement in recovery (99%) and precision (2% RSD). Std. addns. of alliin were converted to allicin in situ by endogenous alliinase (L-(+)-S-alk(en)ylcysteine sulfoxide lyase, EC 4.4.1.4). Complete conversion of the spiked alliin to allicin was achieved by making addns. after homogenization-induced conversion of the naturally occurring cysteine sulfoxides to thiosulfinates had taken place, thus eliminating the likelihood of competing reactions. Concn. values for allicin detd. in samples of fresh garlic (Allium sativum and A. ampeloprasum) and com. available garlic powders (A. sativum) by std. addn. of alliin were found in all cases to be in statistical agreement (95% confidence interval) with values detd. using a secondary allicin std. (concn. detd. using published extinction coeffs.). This method provides a convenient alternative for assessing the amt. of allicin present in fresh and powd. garlic, as alliin is a far more stable and com. prevalent compd. than allicin and is thus more amenable for use as a std. for routine anal.
    25. 25
      Wen, D.; Li, C.; Di, H.; Liao, Y.; Liu, H. A Universal HPLC Method for the Determination of Phenolic Acids in Compound Herbal Medicines. J. Agric. Food Chem. 2005, 53, 6624– 6629,  DOI: 10.1021/jf0511291
      25
      A Universal HPLC Method for the Determination of Phenolic Acids in Compound Herbal Medicines
      Wen, Dawei; Li, Chenchen; Di, Hao; Liao, Yiping; Liu, Huwei
      Journal of Agricultural and Food Chemistry (2005), 53 (17), 6624-6629CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A universal method to sep. and quantify 13 phenolic acids (gallic acid, chlorogenic acid, gentisic acid, vanillic acid, caffeic acid, syringic acid, sinapic acid, p-coumaric acid, ferulic acid, anisic acid, rosmarinic acid, salicylic acid, and cinnamic acid) in some compd. herbal medicines was established by liq. chromatog. (HPLC). On an Agela XBP-C18 (5 μm, 4.6 mm × 150 mm) column, a multistep binary gradient elution program and a simplified sample pretreatment approach were used in the expt. For all of the phenolic acids, detection limits ranged around 0.01 mg/L. Linear ranges of higher than 2 orders of magnitude were obtained with a correlation coeff. of 0.9991 to 1. Repeatability was 0.39-2.24% (relative std. deviation, RSD) for intraday, 1.17-3.96% (RSD) for interday, and 0.14-5.33% (RSD) for drug sample anal. Recovery, tested by a std. addn. method, ranged from 83.3% to 104.9% for various trace phenolic acids.
    26. 26
      Patel, D.; Sorkin, B. C.; Mitchell, C. A.; Embry, M. R.; Rina-Kong, S.; Adams, R. E.; DeTemple, E. R.; Reddam, A.; Gafner, S.; Kelber, O.; Rider, C. V.; Oketch-Rabah, H.; Roe, A. L.; Marles, R. J.; Dever, J.; Dentali, S. Improving the Rigor and Utility of Botanical Toxicity Studies: Recommended Resources. Regul. Toxicol. Pharmacol. 2023, 144, 105471,  DOI: 10.1016/j.yrtph.2023.105471
      There is no corresponding record for this reference.
    27. 27
      van Breemen, R. B.; Fong, H. H. S.; Farnsworth, N. R. The Role of Quality Assurance and Standardization in the Safety of Botanical Dietary Supplements. Chem. Res. Toxicol. 2007, 20, 577– 582,  DOI: 10.1021/tx7000493
      27
      The Role of Quality Assurance and Standardization in the Safety of Botanical Dietary Supplements
      van Breemen, Richard B.; Fong, Harry H. S.; Farnsworth, Norman R.
      Chemical Research in Toxicology (2007), 20 (4), 577-582CODEN: CRTOEC; ISSN:0893-228X. (American Chemical Society)
      A review. The consumer expects a botanical dietary supplement that is safe for consumption. The essential quality control and quality assurance procedures that the dietary supplement industry should follow to ensure the safety of botanical dietary supplements were described in detail above and ar summarized. Addnl. information was described by Fong et al. and Shilter et al. These procedures include acquiring the botanicals from growers or collectors who use good agriculture and collection practices. To be certain that the correct species was acquired, the material should be authenticated using macroscopic and microscopic botanical examn. Alternative authentication assays include genetic identification using PCR techniques, immunoassays to identify species specific proteins, or chem. anal. for unique marker compds. After processing, the botanical dietary supplement should be assayed for hazardous contaminants such as pesticides, herbicides, heavy metals, mycotoxins, and microbes. In addn., pharmaceutical contamination or adulteration should be ruled out by chromatog. assays designed to detect drugs that might were added either inadvertently or deliberately during processing. Finally, the botanical dietary supplement should be standardized both chem., based on the concn. of active compds. (or marker compds. if active constituents are unknown), and biol., based on bioassays for known or desired pharmacol. and physiol. effects. These final standardization steps will ensure the consumer of a reproducible product. In addn. to these basic steps to ensure the safety of botanical dietary supplements, more advanced toxicity tests that are beyond the scope of this review should be carried out that include preclin. and clin. studies as described by Fong et al. Although these procedures will probably be implemented over a long period of time, they will be essential to help ensure the safety botanical dietary supplements.
    28. 28
      National Toxicology Program. Botanical Safety Consortium–Chemical Analysis. DOI: 10.22427/NTP-DATA-500-007-001-000-3 (accessed 2014–05–28).
      There is no corresponding record for this reference.
    29. 29
      Mitchell, C. A.; Dever, J. T.; Gafner, S.; Griffiths, J. C.; Marsman, D. S.; Rider, C.; Welch, C.; Embry, M. R. The Botanical Safety Consortium: A Public-Private Partnership to Enhance the Botanical Safety Toolkit. Regul. Toxicol. Pharmacol. 2022, 128, 105090,  DOI: 10.1016/j.yrtph.2021.105090
      There is no corresponding record for this reference.
    30. 30
      Waidyanatha, S.; Collins, B. J.; Cristy, T.; Embry, M.; Gafner, S.; Johnson, H.; Kellogg, J.; Krzykwa, J.; Li, S.; Mitchell, C. A.; Mutlu, E.; Pickett, S.; You, H.; van Breemen, R.; Baker, T. R. Advancing Botanical Safety: A Strategy for Selecting, Sourcing, and Characterizing Botanicals for Developing Toxicological Tools. Food Chem. Toxicol. 2024, 186 (3), 114537,  DOI: 10.1016/j.fct.2024.114537
      There is no corresponding record for this reference.
    31. 31
      Lee, J. I.; Narayan, M.; Barrett, J. S. Analysis and Comparison of Active Constituents in Commercial Standardized Silymarin Extracts by Liquid Chromatography–Electrospray Ionization Mass Spectrometry. J. Chromatogr. B 2007, 845, 95– 103,  DOI: 10.1016/j.jchromb.2006.07.063
      31
      Analysis and comparison of active constituents in commercial standardized silymarin extracts by liquid chromatography-electrospray ionization mass spectrometry
      Lee, James I.; Narayan, Mahesh; Barrett, Jeffrey S.
      Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences (2007), 845 (1), 95-103CODEN: JCBAAI; ISSN:1570-0232. (Elsevier B.V.)
      A sensitive method for the simultaneous quantitation of six active constituents in com. silymarin standardized exts. was developed based on liq. chromatog. (LC) in combination with mass spectrometry (MS). The six main active constituents, namely, silydianin, silychristin, diastereoisomers of silybin (silybin A and B), and diastereoisomers of isosilybin (isosilybin A and B) were completely sepd. and quantified by LC/MS. Silymarin obtained from Sigma-Aldrich Co. was evaluated and used as std. ref. material for the six individual constituents in comparing the relative content of silymarin and the relative ratio of each constituent in com. standardized silymarin exts., resp. Significant variation was found between different com. silymarin sources. As a result, this method has proven useful in evaluating and quantifying the six active constituents in com. milk thistle exts. The calibration curves were over the range from 0.25 to 100 μg/mL for silychristin and silydianin, and from 0.10 to 100 μg/mL for silybin A, silybin B, isosilybin A and isosilybin B, resp. (r 2 ≥ 0.9958). For all six active constituents, the overall intra-day precision values, based on the relative std. deviation replicate for four QC levels, ranged from 1.18% to 12.4% and accuracy ranged from 89.4% to 112%. This methodol. could easily be incorporated into standardized testing to assess content uniformity including lot-to-lot variation as part of routine process controls as well as a means to describe cross-product variation among the exiting marketed formulations.

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    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jasms.4c00125.

    • Peak assignments for the high-resolution UHPLC-MS analyses of milk thistle extract; tandem mass spectra of all milk thistle analytes measured in this study (PDF)


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