Role of mass spectrometry in establishing safety and efficacy of botanical dietary supplements

Highlights

• •Mass spectrometry expedites studies of supplement safety and efficacy.
• •Preclinical studies of MS include standardization and mechanisms of action.
• •Clinical applications of MS include safety, pharmacokinetics and efficacy studies.

Abstract

As in drug discovery and development, mass spectrometry has become essential at all stages for establishing the safety and efficacy of botanical dietary supplements. Applications of mass spectrometry to the development of botanical dietary supplements include preclinical studies of the mechanisms of action (e.g., proteomic target identification and validation); identification of active natural products using high resolution tandem mass spectrometry; chemical standardization using UHPLC-MS/MS; and studies of metabolism, absorption and toxicity of active compounds using high resolution and UHPLC-MS/MS. Clinical applications of mass spectrometry include evaluation of the potential for drug-botanical interactions; investigation of the pharmacokinetics of active compounds; and quantitative analysis of biomarkers of efficacy during Phase I and II and clinical trials of safety and efficacy of botanical dietary supplements.

1. Introduction

Traditional medicines, such as botanical dietary supplements, are often primary sources of health care in developing countries, while these products are primarily used for health maintenance in the United States and Europe. The worldwide market for botanical dietary supplements exceeded $33 billion in 2010 and is projected to reach $140 billion by 2024 [1]. In the United States, the market for botanical dietary supplements has grown steadily for over 10 years, experiencing 9.4% growth from 2017 to 2018, and reaching an estimated $8.8 billion in 2018 [2]. Given the enormous market for these products, consumer safety is a major concern, which prompted the U.S. FDA to require that botanical dietary supplements under its jurisdiction be prepared using current good manufacturing practice (cGMP) [3]. Mass spectrometry, especially GC–MS and LC-MS [4], is being used for cGMP testing of botanical dietary supplements for contamination by pesticides or herbicides, as well as for botanical authentication [5]. The requirement of cGMP complements advertising and labeling regulations that are also in effect in the United States, the European Union and elsewhere. However, clinical testing of botanical dietary supplements for safety and efficacy is only required in the United States when health claims are made, and for this purpose, mass spectrometry can provide essential information.

2. Clinical applications of mass spectrometry to botanical dietary supplements research

Consumers expect a safe and effective product. Just as in the development of new drugs, the speed, sensitivity and specificity of mass spectrometry can provide valuable data in support of preclinical and clinical studies of safety and efficacy. As shown in Fig. 1, proteomic and metabolomic applications of mass spectrometry may be used to identify targets and mechanisms of action of botanical dietary supplements (step 1). As an example of mechanism of action determination, mass spectrometry-based lipidomics (a subset of metabolomics) was used to measure changes in serum lipids resulting from chronic coffee consumption. Overall, 58 lipids belonging to multiple classes decreased and 3 lysophosphatidylcholines decreased significantly [6]. As an example of target identification, bottom-up proteomic mass spectrometry has been used to determine that lycopene might prevent prostate cancer in men by enhancing cytoprotection pathways, inhibiting androgen receptor signalling, and inducing apoptosis [7]. When specific pharmacological targets are identified, affinity based mass spectrometric screening may be used to identify the active compounds within botanical dietary supplements (step 2). For example, estrogenic botanical dietary supplements have been screened using pulsed ultrafiltration (PUF) mass spectrometry and magnetic microbead affinity selection mass spectrometry to find ligands to the estrogen receptors-α and -β, such as 8-prenylnaringenin in hops [8]. Quantitative LC-MS/MS may then be used to standardize botanical dietary supplements based on the chemical composition of active compounds (step 3). Chemical standardization is essential to produce a reproducible supplement that can provide consumers with consistent and safe effects [9].

Fig. 1.

Beginning with the identification of targets and active natural products and extending through the clinical evaluation of safety and efficacy, mass spectrometry expedites every step in the development and clinical evaluation of the safety and efficacy of botanical dietary supplements. Proteomics for target identification (step 1) as well as identification of active compounds (step 2) and their metabolites (step 4) typically utilize LC-MS with data dependent MS/MS on high resolution tandem mass spectrometers such as Q-ToF or Orbitrap instruments. Quantitative analyses (steps 3, 5 and 6) usually use triple quadruple LC-MS/MS instruments. (PUF-MS, pulsed ultrafiltration-mass spectrometry; MagMASS, magnetic microbead affinity selection screening).

Once identified, the active compounds contained in botanical dietary supplements should be tested for bioavailability using preclinical models including assays of intestinal permeability, such as human Caco-2 cell monolayers, and assays of metabolic stability and metabolic conversion to active, inactive or possibly toxic constituents using human liver and intestinal microsomes, human hepatocytes, and recombinant drug metabolizing enzymes (step 4). Importantly, the dietary supplement product should be tested in these experiments to determine if other botanical constituents in the mixture affect the bioavailability of the pharmacologically active compounds.

Follow-up experiments should include Phase I clinical investigations of the pharmacokinetics, metabolism and excretion of the active compounds in the botanical dietary supplement product in order to evaluate acute safety, maximum tolerated dose, and half-lives of active compounds (step 5). Such studies provide important safety information for setting dosage and frequency of administration. For example, the long half-lives of hop prenylated phenols like xanthohumol and 8-prenylnaringenin suggest dosing intervals for hop products of not more than once daily [10]. If predicted by preclinical models, additional Phase I clinical studies of drug-botanical interactions should be carried out in a manner analogous to drug-drug interactions to determine if chronic consumption of botanical dietary supplements has the potential to induce or inhibit drug metabolizing enzymes and transporters. A well-documented example of a drug-botanical interaction is the induction of human cytochrome P450 CYP3A4 and P-glycoprotein by the botanical dietary supplement St. John’s wort, which can produce accelerated metabolism and sub-therapeutic levels of certain drugs [11].

Finally, double-blind and placebo controlled Phase II clinical studies should be carried out using the botanical dietary supplement product to evaluate its chronic safety profile, as well as efficacy (step 6). LC-MS/MS should be used to measure serum and, if appropriate, tissue levels of active compounds, possible biomarkers of toxicity, and biomarkers of efficacy. As an example of measuring active compounds, Fig. 2 shows LC-MS/MS chromatograms for the quantitative analysis of lycopene in human serum and prostate tissue from a Phase II clinical study of prostate cancer prevention in men who consumed a tomato oleoresin containing 30 mg doses of lycopene for 21-days [12]. The exquisite sensitivity and selectivity of mass spectrometry is indicated by the magnitude and simplicity of the LC-MS/MS selected reaction monitoring (SRM) signal for lycopene in serum or extracted from a single biopsy of human prostate (Fig. 2).

Fig. 2.

Negative ion atmospheric pressure chemical ionization LC-MS/MS selected reaction monitoring (SRM) chromatograms obtained using a Thermo (San Jose, CA) TSQ Quantum triple quadrupole mass spectrometer showing the measurement of lycopene extracted from human serum and prostate biopsy tissue [12].

As an example of biomarker measurement in support of clinical trials, LC-MS/MS was used to measure changes in eicosanoids (PGE₂, PGE₃, LTB₄, LTB₅, 5-HETE, 12-HETE, 15-HETE, and 13-HODE) in colon biopsies following consumption of a Mediterranean diet rich in monounsaturated fats from plants. Pro-inflammatory eicosanoids did not change, but PEG₃ levels increased by 50% [13]. In another example, urinary 8-iso-PGF_2α, a biomarker of lipid peroxidation, was measured in biopsies of human liver from patients with hepatitis C using LC-MS/MS to investigate the possible relationship between retinoid and carotenoid levels and oxidative stress, which contributes to liver disease progression. Oxidative stress biomarkers like 8-iso-PGF_2α were inversely correlated with liver levels of retinoids and carotenoids, which are modifiable dietary factors [14]. An example of LC-MS/MS quantitative analysis of both 8-iso-PGF_2α and the DNA oxidation biomarker, 8-oxo-dG, in human urine are shown in Fig. 3.

Fig. 3.

Electrospray LC-MS/MS SRM chromatograms of biomarkers of oxidative stress and their corresponding stable isotope-labeled internal standards extracted from human urine. Top) DNA oxidation biomarker 8-oxo-deoxyguanosine (positive ion); and Bottom) 8-iso-PGF_2α (negative ion), which is a non-enzymatic peroxidation product of arachidonic acid. Both biomarkers were measured during the same analysis using polarity switching on a Shimadzu (Kyoto, Japan) LCMS-8050 triple quadrupole mass spectrometer.

In summary, the same rigorous preclinical and clinical approaches used to develop and evaluate new drugs can be applied, with some modifications, to the evaluation of the safety and efficacy of botanical dietary supplements. Beginning with cGMP, which has already contributed significantly to the safety of these products, the implementation of the additional steps, outlined in Fig. 1, will improve the standardization, reproducibility, safety, and efficacy of botanical dietary supplements. Carefully designed Phase I and adequately powered Phase II clinical studies of the actual botanical dietary supplement products marketed to consumers are needed to ensure the safe and efficacious use of these products.

Funding

This work was supported by grant P50 AT000155 from the NIH Office of Dietary Supplements and the National Center for Complementary and Integrative Health.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: