Combined Liquid Chromatography-mass Spectrometry and Next-generation DNA Sequencing Detection of Adulterants and Contaminants in Analgesic and Anti-inflammatory Herbal Medicines.

INTRODUCTION: Methods for assessing the quality of herbal medicine preparations have advanced significantly in recent years in conjunction with increases in herbal medicine use and reports of adulteration and contamination. OBJECTIVE: This study examined the quality of analgesic and anti-inflammatory herbal medicine preparations available on the Australian market by detecting the presence of listed ingredients, adulterants and contaminants. METHODS: Forty-nine analgesic and anti-inflammatory herbal medicine preparations were randomly sourced from Australian capital cities. They were audited using a dual approach of liquid chromatography-mass spectrometry (LC-MS) combined with next-generation DNA sequencing. Once screened, a comparison of listed ingredients with verified ingredients was conducted to determine the accuracy of labelling, and the extent of adulteration and contamination. RESULTS: Twenty-six of 49 (53%) herbal medicines were adulterated or contaminated with undeclared ingredients. LC-MS revealed the presence of pharmaceutical adulterants including atropine and ephedrine. DNA sequencing uncovered concerning levels of herbal substitution, adulteration and contamination, including the use of fillers (alfalfa, wheat and soy), as well as pharmacologically relevant species (Centella asiatica, Panax ginseng, Bupleurum and Passiflora). Pig/boar and bird DNA was found in some preparations, inferring substandard manufacturing practices. Of the 26 contaminated samples, 19 (73%) were manufactured in Australia, and 7 (27%) were imported from other countries (6 from China, 1 from New Zealand). In 23 of 49 (47%) herbal medicine samples, no biological ingredients were detected at all. These were predominantly pain and anti-inflammatory preparations such as glucosamine and eicosapentaenoic and docosahexaenoic acids found in krill and fish oils, so DNA would not be expected to survive the manufacturing process. CONCLUSION: The high level of contamination and substitution of herbal medicine preparations sourced from Australian dispensaries supports the need for more stringent pharmacovigilance measures in Australia and abroad.

Toxicological screening and DNA sequencing detects contamination and adulteration in regulated herbal medicines and supplements for diet, weight loss and cardiovascular health.

Use of herbal medicines and supplements by consumers to prevent or treat disease, particularly chronic conditions continues to grow, leading to increased awareness of the minimal regulation standards in many countries. Fraudulent, adulterated and contaminated herbal and traditional medicines and dietary supplements are a risk to consumer health, with adverse effects and events including overdose, drug-herb interactions and hospitalisation. The scope of the risk has been difficult to determine, prompting calls for new approaches, such as the combination of DNA metabarcoding and mass spectrometry used in this study. Here we show that nearly 50% of products tested had contamination issues, in terms of DNA, chemical composition or both. Two samples were clear cases of pharmaceutical adulteration, including a combination of paracetamol and chlorpheniramine in one product and trace amounts of buclizine, a drug no longer in use in Australia, in another. Other issues include the undeclared presence of stimulants such as caffeine, synephrine or ephedrine. DNA data highlighted potential allergy concerns (nuts, wheat), presence of potential toxins (Neem oil) and animal ingredients (reindeer, frog, shrew), and possible substitution of bird cartilage in place of shark. Only 21% of the tested products were able to have at least one ingredient corroborated by DNA sequencing. This study demonstrates that, despite current monitoring approaches, contaminated and adulterated products are still reaching the consumer. We suggest that a better solution is stronger pre-market evaluation, using techniques such as that outlined in this study.

Evaluation of an early twentieth century Afghan herbalist's preparations.

Mahomet Allum was a flamboyant philanthropist and herbalist who worked in South Australia in the early part of last century, whose herbal therapies generated some controversy at the time. Two of his preparations have survived to the present day, a general tonic and a treatment for liver and kidney dysfunction. Given the frequent use of pharmaceutical drugs in "tonics" at the time, toxicological analysis was undertaken at Forensic Science SA, Adelaide with liquid chromatography/quadrupole-time-of-flight mass-spectrometer (LC-QTOF MS), liquid-chromatography/ diode array detector (LC/UV) and gas chromatography/ nitrogen phosphorous- detector/mass-spectrometer (GC-NPD/MS), to look for common drugs. In addition DNA analysis was also undertaken at Trace and Environmental DNA (TrEnD) Laboratory (Curtin University) to evaluate the types of plant products used to make these remedies. The general tonic contained genera from the Triticeae (wheat) family as well as the Medicago family (includes alfalfa), possibly as fillers. Other genera found included Utrica (nettle) and Passiflora (passion flower). The preparation for liver and kidney disease also contained genera from the Medicago family as well as genera Arctostaphylos (bear berry) which has traditionally been used for the treatment of dysuria and bladder stones. No common drugs were found. Thus it appears that the two treatments prepared by Mahomet Allum contained only herbal substances and not adulterant pharmaceutical agents. The herbals identified provide an insight into herbalist practices in the early twentieth century.

Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.