**1. Introduction**

Members of the plant genus *Salvia* have a long and rich history of use as both culinary and medicinal herbs [1,2]. In general, the perennial shrubs have long stems which can reach heights of 50–100 cm. Although found throughout the world, most *Salvia* species grow in the Mediterranean region, Southeast Asia, and Central and South America [2]. Alluding to the importance of this plant's medicinal properties, the word "*Salvia*" is derived from the Latin word "salvere", meaning "to save" [2]. Members of the genus *Salvia* have been purported to possess a wide range of pharmacological properties, including anti-inflammatory, anti-dementia, anti-nociceptive, anti-hypertensive, anti-lipidemic, anti-mutagenic, antihyperglycemic, and anti-ischemic effects [1–5]. In addition to these purported properties, members of this genus have also been reported to possess anti-microbial and anti-oxidative activities [2–5]. These pharmacological properties vary among *Salvia* genus members.

**Citation:** Lee, J.; Wang, M.; Zhao, J.; Avula, B.; Chittiboyina, A.G.; Li, J.; Wu, C.; Khan, I.A. Chemical Authentication and Speciation of *Salvia* Botanicals: An Investigation Utilizing GC/Q-ToF and Chemometrics. *Foods* **2022**, *11*, 2132. https://doi.org/10.3390/ foods11142132

Academic Editors: Mourad Kharbach and Samuli Urpelainen

Received: 14 June 2022 Accepted: 13 July 2022 Published: 19 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Perhaps one of the most well-known members, *Salvia officinalis*, is utilized both as a culinary and a medicinal herb. This evergreen plant, native to southern Europe, is also cultivated in the United States and Central Asia [5]. As a medicinal herb, both the British Pharmacopoeia and the German Commission E have recognized its use to treat oral cavity and stomach ailments [5,6].

Another genus member, *Salvia apiana* (white sage), also has a rich history of use as a medicinal herb. This drought-resistant shrub, native to California and Baja California, can grow up to 1–3 m high. Traditionally, the herb has been used for its purported diuretic, anxiolytic, and anti-microbial properties [7]. In addition to its medicinal use, the plant is also an important part of traditional Native American religious and healing ceremonies [7].

*Salvia mellifera* (black sage), native to California and parts of Mexico, has also been used as a traditional healing herb. An infusion comprised of the aerial portions of the plant has traditionally been used as a drink to relieve muscle aches and pains [8].

As a popular ingredient in many traditional Chinese medicine (TCM) preparations, the red rhizomes of *Salvia miltiorrhiza* contain a unique group of compounds known as tanshinones, which have been reported to possess a broad spectrum of pharmacological activities [3,9]. The natural habitat of *S. miltiorrhiza* includes the hilly regions of China, Japan, Korea, and Mongolia; however, due to growing demand, most plant material is typically obtained from commercial farming [9]. Commonly referred to as "Danshen" or "Tanshen" in China, the rhizomes are purported to be beneficial for a number of disorders, including hyperlipidemia, vascular diseases, stroke, arthritis, and hepatitis [9].

*Salvia divinorum* is the only member of the *Salvia* genus to contain salvinorins, a group of neoclerodane diterpenes [10,11]. The plant, which can grow up to 1.5 m high, is native to southern Mexico. Traditionally, the Mazatec people would chew the herb or prepare an infusion using water and portions of the herb to take advantage of its psychoactive compounds. One compound, in particular, salvinorin A, possesses psychoactive properties and is a highly selective kappa-opioid receptor agonist [6,11]. Due to its high abuse potential, many local jurisdictions and countries have begun or are considering regulating the herb and/or salvinorin A as a controlled substance [6,11].

With nearly 900 species included in the genus *Salvia*, identifying plant materials and products can be a daunting task [4]. Thus, chemical fingerprint analyses represent a comprehensive approach for the quality assessment of *Salvia* botanicals and their finished products. Clearly, this is an important task given the wide range of pharmacological properties found in members of this genus. A range of methods have been developed to aid in the species identification of *Salvia* plant material [6,11–17]. Perhaps one of the most popular analytical techniques utilized for the identification and quality control of *Salvia* species is liquid chromatography/mass spectrometry (LC/MS) [6,12,13,15–19]. A brief literature search can yield numerous studies concerning this subject [6,12,13,15–19]. In addition to traditional LC/MS, techniques utilizing liquid chromatography/quadrupole time-of-flight (LC/Q-ToF) and liquid chromatography/triple quadrupole mass spectrometry (LC/TQ) have also been described [6]. While valuable to researchers, LC/MS/MS instruments are not often used by botanical industries for quality control purposes due to the cost of the instrument and the necessary technical skills required to develop and operate such tools. Due to the physical separation characteristics of LC/MS, the vast majority of previous research has focused on the non-volatile, LC-amendable polar compounds present in *Salvia* species [6,12,13,15,16,18,19]. Although polar compounds of *Salvia* are pharmacologically important, volatile constituents have also been implicated with bio-active properties and could be useful for establishing species-specific chemical fingerprinting [4,7,20,21].

DNA barcoding is another technique that has been proposed to aid in the species identification of *Salvia*. The authors of one study developed an effective DNA barcoding method to differentiate *S. miltiorrhiza* from other *Salvia* species [22]. Although differentiation was achieved, this technique involved extensive and complex sample preparation which did not lend itself to high-throughput sample analysis. In addition, the authors explained that their method was particularly developed for *S. miltiorrhiza* identification and may not be ideal for other *Salvia* species [22].

A high-performance thin-layer chromatography (HPTLC) fingerprinting method for 20 *Salvia* species was developed by Ciesla and co-workers [23]. The method utilized polar and semi-polar compounds (mostly polyphenols) for identification purposes and was successfully validated. Regarding its applicability as a high-throughput method, the authors estimated that 20 samples could be fully processed within one hour. However, one major limitation of this method was the requirement for a large amount of sample material (around 5 g), which could be problematic if the plant material is difficult to obtain, i.e., *S. divinorum* [23]. Therefore, there is a need to develop efficient and reliable methods.

Gas chromatography/mass spectrometry (GC/MS) is a well-established means of obtaining chemical fingerprints from various plants, primarily by analyzing volatile compounds. For example, this technique has been used to establish the chemical fingerprints of *Salvia* species by Rzepa and colleagues [24]. Based on the number of products sold in the U.S. market and pending botanical drug applications, five *Salvia* species, *viz*. *S. apiana*, *S. divinorum*, *S. mellifera*, *S. miltiorrhiza*, and *S. officinalis*, were selected to conduct a comprehensive and comparative study for quality evaluation and identification purposes. Currently, to the authors' knowledge, a comparative study of the five selected *Salvia* species has not been conducted. Given these five species' extensive history and current use as medicinal herbs, it is important to develop reliable and efficient identification methods for species specificity purposes and to assure the overall quality of various *Salvia*-based finished products.

With this information in mind, our goal is to develop a simple, reliable, and efficient GC method coupled with accurate mass spectrometry to establish species-specific chemical fingerprints of *Salvia*. Chemometric analysis and principal component analysis (PCA) are applied to differentiate between *Salvia* species, as well as to establish a sample class prediction model (SCP) based on partial least square discriminant analysis (PLS-DA) for the quality evaluation of commercial products. Marker and characteristic compounds present in each of the five species are identified. The integration of analytical data with statistical tools and the development of personal compound databases and libraries (PCDL) are anticipated to expedite the rapid evaluation of the quality of *Salvia*-based finished products, including raw materials used in commerce.

#### **2. Materials and Methods**

### *2.1. Plant Material*

Twenty-four authentic plant samples from five *Salvia* species were used for this investigation. The samples included both leaf and aerial portions of the plants from *S. divinorum*, *S. officinalis*, *S. mellifera*, and *S. apiana*, with each having 3, 7, 3, and 5 individual samples, respectively. *S. divinorum* samples were procured from Trish Flaster (Botanical Liaisons, LLC, Boulder, CO, USA) (#578) and cultivated at the Medicinal Plant Garden (University, MS, USA) (#18434, #22491). *S. officinalis* samples were obtained from the Missouri Botanical Garden (St. Louis, MO, USA) (#7917, #7686, 20712), China (#16732), Richters.com (#13095), Trish Flaster (#2852), and Williams Warehouse (USA) (#1523). *S. mellifera* samples were sourced from AHP (Scotts Valley, CA, USA) (#22771, #22772), and SageRageHerb (Montclair, CA, USA) (#22506). *S. apiana* samples were obtained from AHP (#22773), Richters.com (#13096), SageRageHerb (Montclair, CA, USA) (#22502), and commercial sources (#22497, #22498). Six individual samples from the root portion of *S. miltiorrhiza* were also investigated. These samples were procured from Harvard Medical School (Boston, MA, USA) (#9729), the Medicinal Plant Garden (#11750), Beijing Yuke Botanical Development Co. Ltd. (Beijing, China) (#767), Missouri Botanical Garden (#8676, #12535), and a commercial source (#5399). The authenticity of the collected botanical samples was established based on morpho-anatomical and organoleptic properties by Dr. John Adams, a taxonomist at the National Center for Natural Products Research (NCNPR), University of Mississippi. In addition, DNA barcoding was also used for species verification purposes. Voucher samples

of all the botanical material were deposited in the Botanical Repository of the NCNPR. The detailed sample information is given in Table 1.


**Table 1.** Analyzed authenticated *Salvia* samples.

#### *2.2. Chemicals*

Dichloromethane was purchased from Fisher Scientific (Pittsburgh, PA, USA). Both internal standards, tridecane (C13H28) and docosane (C22H46), were obtained from Polyscience Corporation (Niles, IL, USA). The reference standards, α-pinene, β-pinene, 3-carene, eucalyptol, camphor, endo-borneol, β-caryophyllene, viridiflorol, α-bisabolol, tanshinone II, cryptotanshinone, salvinorin A, and salvinorin B, used to confirm compound identification, were purchased from Sigma-Aldrich (St. Louis, MO, USA), Agilent Technologies, Inc. (Santa Clara, CA, USA), or isolated from plant material in-house at the NCNPR.
