The Ayurvedic Pharmacopoeia of India defines Ashwagandha as the dried mature root and gives a macroscopic and microscopic description together with limits for foreign matter, total ash, acid-insoluble ash and alcohol- and water-soluble extractives (Government of India, 2001). The monograph of the World Health Organization on the root is consistent with it and adds chemical identity tests (WHO, 2009). The definition matters because the plant part is the point at which most disputes arise. Roots and leaves of the species both contain withanolides, but in different proportions: comparative analyses show withaferin A to be far more prominent in the leaf, while the root has a distinct profile in which withanolide A and related compounds are characteristic (Chaurasiya et al., 2008; Mirjalili et al., 2009).

Published analysis of commercial ashwagandha preparations in India found wide variation in withanolide content between products (Sangwan et al., 2004). Variation of that kind has several possible causes, and they call for different tests. The forms considered in this protocol, with the way each arises and the test best suited to revealing it, are set out in Table 1. Contamination with heavy metals is included although it is not adulteration in the strict sense, because surveys of Ayurvedic products sold in and exported from South Asia have repeatedly found lead, mercury or arsenic in a proportion of them (Saper et al., 2004; Saper et al., 2008).

The population is single-ingredient ashwagandha root powder offered for retail sale in a named set of cities in North India during a stated period. Multi-ingredient formulations, extracts, capsules and tablets are excluded, since each raises different analytical questions. Within each city the outlets are listed by type: traditional herb and grocery dealers selling loose powder, Ayurvedic pharmacies selling branded packs, and general retailers. The list is built by walking the main herb markets and from trade directories, and it is the frame from which outlets are drawn.

Sampling is stratified by city and outlet type, with outlets selected at random within each stratum. The number of samples follows from the precision wanted for the estimated proportion, using the standard results for stratified sampling (Cochran, 1977), and is fixed before purchasing begins. A survey that simply buys what is convenient can show that adulteration exists but cannot say how common it is.

Samples are bought as an ordinary customer would buy them, in a quantity sufficient for all tiers and a retained portion. Price, pack details, batch number where present, any licence number and the claims on the label are recorded at the time of purchase. Each sample is sealed, given a code that conceals its source from the analysts, and logged. Storage is cool, dry and dark until analysis, and the interval between purchase and testing is recorded.

Every comparison depends on authentic root. Reference material is collected from cultivated plants identified by a taxonomist, with voucher specimens deposited in a recognised herbarium, and is dried and milled in the laboratory. Leaf and stem from the same plants are kept separately so that deliberate mixtures of root with aerial parts can be prepared as positive controls. Because withanolide content varies with chemotype and origin (Mirjalili et al., 2009), reference root from more than one growing area is included.

Colour, odour and texture are noted, and the powder is examined under the microscope after clearing and after staining for starch and lignin. Root powder has a characteristic assemblage of starch grains, vessels and parenchyma; epidermal fragments with stomata, covering trichomes and palisade tissue indicate leaf or herbaceous stem. Foreign matter, total ash, acid-insoluble ash and extractive values are determined by the pharmacopoeial procedures (Government of India, 2001) and the general methods of the World Health Organization (WHO, 2011). High acid-insoluble ash suggests sand or soil, and low extractives suggest exhausted material.

Each sample is run by high-performance thin-layer chromatography beside reference root, reference leaf and withanolide standards on the same plate, following established practice for herbal drug identification (Reich and Schibli, 2007). Selected withanolides are then quantified by reversed-phase liquid chromatography with ultraviolet detection. Published methods separate withaferin A, withanolide A, withanone and related compounds in a single run (Ganzera et al., 2003; Chaurasiya et al., 2008). The method chosen is verified in the laboratory for specificity, linearity, precision and recovery before any market sample is assayed. Interpretation rests on the profile as much as on the total: a powder with an unusually high proportion of withaferin A relative to the root markers is flagged as possibly containing aerial parts or leaf extract.

Where microscopy or chromatography indicates material that is not Withania, DNA is extracted and the ITS2 region is amplified and sequenced, this region having been validated for medicinal plant identification (Chen et al., 2010). The limits of the approach are respected. A barcode cannot distinguish root from leaf of the same species, and published compilations of market studies show that sequencing results on processed material need cautious reading (Ichim, 2019). Failure to amplify is recorded as inconclusive.

Lead, cadmium, mercury and arsenic are determined by atomic spectrometry after acid digestion, and microbial quality by standard enumeration methods. Results are compared with the limits set for Ayurvedic drugs in India and with the guidance of the World Health Organization on contaminants in herbal medicines (WHO, 2007).