FWD 2 Botanical Adulterants Program: Saw Palmetto Extract Lab Guidance Document




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Saw Palmetto Extract Laboratory Guidance Document

By Stefan Gafner, PhD*

American Botanical Council, Austin, TX 78723
*Correspondence: email

Keywords: Adulteration, animal fatty acids, canola oil, coconut oil, palm oil, saw palmetto, Serenoa repens, sunflower oil, vegetable oil

Citation (JAMA) style: Gafner S. Saw palmetto extract laboratory guidance document. Austin, TX: ABC-AHP-NCNPR Botanical Adulterants Prevention Program. 2019. 

CONTENTS

1. Purpose

2. Scope

3. Common and Scientific Names

3.1 Common name

3.2 Other common names for saw palmetto

3.3 Latin binomial

3.4 Synonyms

3.5 Botanical family

4. Botanical Description and Geographical Range

5. Adulterants and Confounding Materials

Table 1. Scientific Names, Family, and Common Names of Plants Used as Sources of Vegetable Oils Known as Saw Palmetto Fruit Extract Adulterants

6. Identification and Distinction using Macroanatomical Characteristics

7. Identification and Distinction using Microanatomical Characteristics

8. Organoleptic Identification

Figure 1: Color of authentic saw palmetto ethanol extracts (1,2) and CO2 extracts (3,4); adulterated ingredients labeled as saw palmetto extract (5-8). Image provided by Euromed, SA (Mollet del Vallès, Spain).

Figure 2: UV/Vis spectrum of a 1% ethanolic solution of authentic saw palmetto CO2 extract (green line) and adulterated ingredients labeled as saw palmetto extract. Image provided by Euromed, SA (Mollet del Vallès, Spain).

9. Genetic Identification and Distinction

10. Physicochemical Tests

11. Chemical Identification and Distinction

11.1 Chemistry of Serenoa repens and potential adulterants

Figure 3: Major Fatty Acids and Phytosterols in Saw Palmetto

Table 2: Relative Fatty Acid (free and bound fatty acid) Composition (in %) of Saw Palmetto and Adulterating Vegetable Oils

11.2 Laboratory methods

11.2.1 High-Performance Thin-Layer Chromatography

11.2.2 Infrared spectroscopy

Figure 4: Saw palmetto extract analysis by FT-IR. One adulterated sample (turquoise line) has a distinct spectrum different from authentic saw palmetto. Spectra were acquired by direct application of the samples (without any dilution) onto an ATR probe (ATR-FTIR Spectrum Two™, Perkin Elmer). Image provided by Euromed, SA (Mollet del Vallès, Spain).

11.2.3 High-performance liquid chromatography

11.2.4 Gas chromatography

Table 3: Comparison among GC Methods to Determine Fatty Acids in Saw Palmetto Extracts.

Figure 5: Saw palmetto fatty acid analysis after conversion into methyl esters by GC-FID. Conditions as detailed in the United States Pharmacopeia. Image provided by Valensa International (Eustis, FL).

11.2.5 Nuclear magnetic resonance

Figure 6: 1H NMR spectrum of saw palmetto (ethanol extract) in deuterated chloroform. The insert shows the characteristic α/α' and β-protons of triglycerides at 4.20 and 5.24 ppm, respectively, in coconut oil. The α/α' protons at 4.15 ppm in the saw palmetto extract overlap with the β-protons of 1,3-diacylglycerides. Saw palmetto NMR spectrum provided by Indena SpA (Milan, Italy).

11.2.6 Stable isotope ratio

Table 4. Comparison among the Different Chemical Methods to Authenticate Saw Palmetto Extract

12. Conclusion

13. References

1. Purpose

There is documented evidence of the adulteration of saw palmetto fruit extracts with a number of vegetable oils, such as canola (Brassica napus ssp. napus, Brassicaceae), coconut (Cocos nucifera, Arecaceae), olive (Olea europaea, Oleaceae), palm (Elaeis guineensis, Arecaceae), peanut (Arachis hypogaea, Fabaceae), and sunflower (Helianthus annuus, Asteraceae) oils. The partial or complete substitution of saw palmetto fruit extracts with mixtures of fatty acids of animal origin was first documented in 2018,1 and seems particularly common in materials sold as saw palmetto originating from China. This Laboratory Guidance Document (LGD) presents a review of the various analytical technologies used to differentiate between authentic saw palmetto extracts and ingredients containing adulterating materials. This document can be used in conjunction with the Saw Palmetto Botanical Adulterants Bulletin, rev. 3, published by the ABC-AHP-NCNPR Botanical Adulterants Prevention Program in 2018.2

2. Scope

Various analytical methods are reviewed here with the specific purpose of identifying their strengths and limitations in differentiating saw palmetto fruit extracts from potentially adulterating materials. Less emphasis is given to the authentication of whole, cut, or powdered saw palmetto fruit and distinguishing it from potential confounding materials, e.g., the Everglades palm (Acoelorrhaphe wrightii, Arecaceae), by macroscopic, microscopic or genetic analysis. Analysts can use this review to guide their selection of appropriate analytical authentication techniques. The suggestion of a specific analytical method for testing saw palmetto materials in their particular matrix in this LGD does not reduce or remove the responsibility of laboratory personnel to demonstrate adequate method performance in their own laboratories using accepted protocols. Such protocols are outlined in the United States Food and Drug Administration’s Good Manufacturing Practices (GMPs) rule (21 CFR Part 111) and those published by AOAC International, International Organization for Standardization (ISO), World Health Organization (WHO), and International Conference on Harmonisation (ICH), and national pharmacopeial bodies, as may be applicable, depending on the regulatory requirements of the country in which the saw palmetto extract is being offered for sale, re-sale, and/or processing into finished consumer products.

3. Common and Scientific Names

3.1 Common name: Saw palmetto

3.2 Other common names for saw palmetto

English: Scrub-palmetto, sabal palm, saw palmetto berry

Chinese: Ju zonglu (锯棕榈)

French: Sabal, palmier nain, palmier scie

German: Sabal, Sägepalme, Zwergpalme

Italian: Palma nana, cavolo di palma

Russian: Сереноя ползучая (Serenoa repens), Сабаль пильчатый (Sabal serrulata), карликовая пальма (karlikovaya palma, “dwarf palm”), пальма cереноа, co пальметто

Spanish: Sabal, palma enana americana3,4

Swedish: sågpalmetto

3.3 Latin binomial: Serenoa repens (W. Bartram) Small

3.4 Synonyms: Chamaerops serrulata Michx., Corypha obliqua W. Bartram, Corypha repens W. Bartram, Diglossophyllum serrulatum (Michx.) H. Wendl. ex Drude, Sabal serrulata (Michx.) Nutt. Ex Schult. & Schult. f., Serenoa serrulata (Michx.) G. Nicholson5

3.5 Botanical family: Arecaceae

4. Botanical Description and Geographical Range

Saw palmetto grows as a small shrub, occasionally a small tree with creeping, horizontal, branched stems, usually to a height of 2-7 feet (0.6-2.1 m), although it may reach up to 25 feet (7.5 m). The stem systems run parallel to the soil surface, eventually branching beneath the substrate to form rhizomes. Saw palmetto leaves are fan-shaped, evergreen and about 3 feet (1 m) wide. The margins of the petioles are lined with sharp spines that have given saw palmetto its common name. The flowers are cream-colored and fragrant, with three petals at the end of stalked panicles that grow from the leaf axils. The fruit is a drupe, green or yellow at immature stages, and black when ripe (between August and October), resembling black olives in size and shape.6,7 The plant is endemic to the southeastern United States, growing from the coastal plains of Louisiana across the Florida peninsula and up to South Carolina.6

5. Adulterants and Confounding Materials


The few reports of adulteration of saw palmetto berries with berries from related species, i.e., dwarf palmetto (Sabal minor),13,14 queen palm (Syagrus romanzoffiana),14 and Everglades palm (Acoelorrhaphe wrightii)15 of the palm family (Arecaceae) seem to suggest that such adulteration is rare. Fruits of dwarf palmetto (6-12 mm) and the Everglades palm (10-15 mm) are smaller and spherical compared to saw palmetto fruit, which is oval-shaped, of ca. 15 mm width and 12-25 mm length.4,13,14,16 Compared to saw palmetto, the fruit of queen palm is larger (20-25 mm) and heavier.14

6. Identification and Distinction using Macroanatomical Characteristics

Botanical descriptions of saw palmetto fruit have been published in a number of pharmacopeial monographs and books.4,17-19 Criteria to distinguish saw palmetto fruits from other fruits in their whole form have been published by many authors.16,20,21 The macroscopic assessment is the method of choice to distinguish unripe (green) from semi-mature or mature (black) berries and is sufficient for identifying saw palmetto to species. For obvious reasons, macroscopic identification is not applicable to saw palmetto extracts.

7. Identification and Distinction using Microanatomical Characteristics

Microscopic descriptions of saw palmetto are found in the pharmacopeias of Europe and the United States, and the American Herbal Pharmacopoeia’s textbook on microscopic characterization of botanical medicines.17,18,22 Details of the fruit anatomy of saw palmetto, Everglades palm, and dwarf palmetto have been published by Zona;21 however, no clear differentiation criteria for the fruits of these palms using botanical microscopy were provided.

8. Organoleptic Identification

Saw palmetto berries are initially sweet, then pungent, acrid, and saponaceous. The aroma is strongly aromatic and foul, reminiscent of foul-smelling socks. Saw palmetto extracts also have a distinct aromatic and foul odor. Color can provide an indication to detect adulterated ingredients (Figures 1 and 2). Ethanol, hexane, and high pressure CO2 extracts of saw palmetto from ripe berries typically have a dark green-brown color due to the extraction of chlorophyll with these solvents. Low pressure CO2 extracts have a yellow to orange-brown color. While organoleptic evaluation does not provide sufficient safeguard against adulteration, the extract color and the very characteristic odor are helpful in assessing the authenticity of a saw palmetto extract.



9. Genetic Identification and Distinction

Several authors have looked into differences among nucleotide sequences of various gene regions for saw palmetto and closely related palm species, including dwarf palm and Everglades palm. In most cases, the purpose was to determine phylogenetic relationships.23-26 Nucleotide sequences of the chloroplast (atpB, matK, ndhF, rbcL, rps16 intron, trnD-trnT, trnL-trnF, trnQ-rps16) and nucleus (18S, ITS, ms, prk, rpb2) were used to establish the relationship among members of the palm family.25,26 Genome skimming was applied to assemble the entire chloroplast nucleotide sequence in leaf samples of 29 palm species, including saw palmetto and the Everglades palm.23 Genetic data on commercial saw palmetto products are less abundant. Nevertheless, Little and Jeanson15 investigated the authenticity of 37 commercial saw palmetto products containing dried, cut and sifted plant material using mini-barcodes from the matK and the rbcL regions. Amplifiable DNA was obtained for 34 samples (92%), with 29 (85%) samples containing saw palmetto. In three samples, only the matK mini-barcode sequence was obtained, which was insufficient to distinguish between saw palmetto and its closest relative, the Everglades palm. Two products were made from fruit of other palm trees; one was made solely from Everglades palm fruit, while for the other, the exact species could not be determined.15

Comments: Based on the report described above,15 the use of DNA mini-barcoding is a suitable means for authentication of crude saw palmetto fruit materials.27 However, the use of genetic techniques to determine the authenticity of saw palmetto extracts is not appropriate because fatty oils are generally devoid of DNA of appropriate quality to permit reliable identification.28

10. Physicochemical Tests

The European Pharmacopoeia (Ph. Eur.) monograph for saw palmetto extract includes specifications for the relative density, refractive index, acid value, iodine value, and peroxide value in saw palmetto extracts.29 Among these tests, the determination of the acid value is the most important for the establishment of saw palmetto extract authenticity. Since saw palmetto extracts contain a large concentration of free fatty acids, the acid value is much higher for saw palmetto than for other vegetable oils.27,30 While this simple test is helpful in detecting saw palmetto adulteration, the acid value assay must be used in combination with an appropriate chemical test to rule out adulteration with some of the materials mentioned in section 5.

The peroxide value is a function of fatty acid oxidation (rancidity), and therefore does not provide any helpful information for authenticity determination. The relative density and refractive index of saw palmetto and common vegetable oils do not differ substantially, and thus these analytical measurements are also not useful in establishing adulteration.27 With the exception of palm oil, the iodine value of most vegetable oils is either above or below the value of saw palmetto extract specified by the Ph. Eur.27 However, a material that is compliant with the iodine value specifications of saw palmetto oil in Ph. Eur. is easily obtained by using a suitable mixture of vegetable oils.

11. Chemical Identification and Distinction

Chemical authentication of saw palmetto extracts has long been dominated by gas chromatographic (GC) methods, either analyzing the fatty acids directly, or after conversion into fatty acid esters. In addition to GC, thin-layer chromatography (TLC) is an integral part for identity testing in pharmacopeial monographs. Other methods of chemical authentication are less common, although a number of additional techniques have been investigated and are discussed below. Distinction based on the phytochemical profile requires detailed knowledge of the constituents of saw palmetto and its likely adulterants. Some of the important saw palmetto constituents and their significance in authentication are discussed below. The overview of potential adulterants is based on published literature. The chemical composition of vegetable oils is dependent on the refining processes. Refining usually leads to a substantial loss of phytosterols, tocopherols, and phospholipids. However, there are numerous other vegetable oils that may be used as undeclared substituents of saw palmetto extracts.

11.1 Chemistry of Serenoa repens and potential adulterants

Serenoa repens: Ripe saw palmetto fruit contains 15-20% lipids, primarily free fatty acids, fatty acid esters, triglycerides, and sterols (Figure 3). The fruit is also rich in acidic polysaccharides. Additional compounds include phenolic acids such as 4,5-di-O-caffeoylshikimic acid, 4,5-di-O-caffeoylquinic acid, and gallic acid, as well as flavonoids (rutin, isoquercitrin, and astragalin), and carotenoids.4,13,31 Based on Peng et al., immature berries contain lower concentrations of fatty acids than mature berries, and approximately the same amounts of lauric and oleic acids (compared to mature berries, where oleic acid concentrations are higher than those of lauric acid).14 The main compounds in saw palmetto oil are free fatty acids (70-95%), followed by glycerides (mono-, di-, and triglycerides [5-6%]), fatty acid methyl esters (ca. 2%), phytosterols (0.20-0.50%) and fatty alcohols (0.15-0.35%).29,32,33 Ethanol extracts are distinct from hexane and CO2 extracts by the relatively high concentration of phosphorylated glycerides. Hexane extracts contained a higher proportion of free fatty acids, but lower amounts of mono-, di-, and triglycerides.32 The fatty acid composition (numbers in parentheses refer to the percentage of fatty acid compared to total [free and bound] fatty acids in a CO2 extract) is dominated by oleic (30-35%) and lauric (26-32%) acids, followed by myristic (10-12%), palmitic (8.5-9.2%), and linoleic (4.3-6.0%) acids.34-36 Marti et al. reported the presence of hydroxylated fatty acids (12-hydroxy-5,8,10,14-eicosatetraenoic acid, 10,11-dihydro-12-oxo-15-phytoenoic acid, corchorifatty acid F) in commercial saw palmetto extracts.32 The fatty acid composition can be used to distinguish saw palmetto extracts from most vegetable oils (Table 2), although some oil blends are designed to mimic the authentic saw palmetto fingerprint in order to make the detection of adulteration more difficult. The main phytosterols are β-sitosterol (68-72% of total sterols), campesterol (20-23%), and stigmasterol (8-9%).36,37 Δ5-avenasterol, Δ7-avenasterol, clerosterol, 24-methylenecholesterol, and Δ7-stigmasterol have been reported present in minor amounts.1,38 Fatty alcohols include octacosanol, hexacosanol, tetracosanol, and triacontanol.33,39



Arachis hypogaea oil: Peanut oil contains approximately 96% triglycerides, with oleic, linoleic, and palmitic acids as the main fatty acids (see Table 2).40 In addition, peanut oil contains approximately 0.50% phospholipids and 0.30% phytosterols (β-sitosterol, campesterol, stigmasterol, and Δ5-avenasterol).41

Brassica napus oil: Canola oil contains 94.9-99.1% triglycerides, with oleic acid making up over 60% of the total fatty acids. Other major fatty acids in canola oil are palmitic and linoleic acids (Table 2).40,42 Commercial canola oil used in products on the market is usually low in erucic acid (< 2%). There are some high erucic acid oils, which are most often referred to as rapeseed oils (although the name sometimes is used interchangeably with canola oil), with erucic acid content over 50% of total fatty acids. Unique to canola oil is the presence of sulfur-containing fatty acids (epithiostearic acids). The concentration of sterols varies between 0.70-1.00%, mainly represented by β-sitosterol, campesterol, and brassicasterol. Since brassicasterol is unique to Brassica oils, it can be used as a marker compound to detect adulteration with canola oil.41 The content of phospholipids is between 0.10-2.50%, depending on processing, with water- or acid-degummed oils having lower contents (0.10-0.60%).

Cocos nucifera oil: The triglyceride content in coconut oil is reported to be approximately 97%. One of the features of coconut oil is that it contains mainly saturated fatty acids. Lauric acid (45.1-53.2% of total fatty acids) represents the most abundant fatty acid, followed by myristic, palmitic, and oleic acids (Table 2).27,43 The sterol content is 0.04-0.12%, dominated by β-sitosterol (32-51% of total sterols), Δ5-avenasterol (20-41%), and stigmasterol (11-16%).27,41

Elaeis guineensis oil: Fatty acids in palm oil exist mainly as triglycerides (92-96%) and of diglycerides (4-7%). The latter are represented primarily by palmitoyloleoyl-, dioleoyl- and dipalmitoyl-glycerols, and can be used to differentiate palm oil from other vegetable oils. In commercial palm oils, the 1,3-diacylglycerols are more abundant than the corresponding 1,2-diacylglycerols.41,44 Palm oil has approximately equal amounts of unsaturated and saturated fatty acids. The fatty acid composition is dominated by palmitic and oleic acids, with lesser amounts of linoleic and stearic acids (Table 2).27,41 Minor components of palm oil include 0.03-0.07% sterols (consisting of 55-67% β-sitosterol and 19-28% campesterol), 0.10-0.30% glycolipids, and 0.02-0.10% tocopherols. The red color of crude palm oil is due to the presence of carotenoids (0.05-0.07%).27,41

Helianthus annuus oil: As with other vegetable oils, sunflower oil is composed mainly of triglycerides (up to 97%). Using selective breeding techniques, sunflower seeds with different fatty acid compositions have been developed, with regular, high-oleic and mid-oleic types being the most common. The fatty acid compositions of regular and high-oleic acid sunflower oils are presented in Table 2. The mid-oleic acid type is the most popular sunflower oil in the US retail market, with approximately 55-75% oleic, 15-35% linoleic, 5% stearic, and 4% palmitic acids.41,43,45 The sterol content in sunflower seed oil is between 0.17-0.52%. Of this, 42-70% is represented by β-sitosterol, 5-13% by stigmasterol and campesterol, respectively, and up to 9% Δ7-stigmastenol. The latter has not been reported in saw palmetto and could be used as a marker for adulteration with sunflower oil. While Δ7-stigmastenol can be eliminated by heat treatment or bleaching, these treatments result in a conversion of Δ7-stigmastenol into the corresponding (Δ8,14)- and Δ14-sterols. Depending on the extent of refinement, sunflower oil has one of the highest concentrations of α-tocopherol (0.04-0.11%) of all vegetable oils, and contains 0.72-0.86% phospholipids (the latter are not found in refined sunflower oil).27

Olea europaea oil: Olive oil is composed mainly of triglycerides (ca. 99%), with oleic, linoleic, and palmitic acids as the most abundant fatty acids (Table 2).46-48 The sterol content is between 0.1-0.2%, composed of a mainly β-sitosterol (75-90%), Δ5-avenasterol (5-20%), and campesterol (up to 4%). Numerous unusual phytosterols (e.g., Δ7-avenasterol, Δ7-stigmastenol) are present at low concentrations.48 The content of squalene, a phytosterol precursor, is between 0.02-0.75%. Olive oil also contains pigments such as pheophytin, α- and β-carotene, and lutein.48 While the phenolics content is low, some of these molecules are rather unique and can be used as specific markers for olive oil. Of particular interest are the secoiridoids, with oleuropein (≤ 0.035%), oleocanthal (0.004-0.021%), and oleacein (0.002-0.48%) as the most abundant.49,50

11.2 Laboratory methods

Table 4, which appears at the end of this section, provides a summary comparison of different methods of analysis of saw palmetto oil.

11.2.1 High-Performance Thin-Layer Chromatography

Methods from the following sources were evaluated in this review: Ph. Eur. 9.1,17,29 the HPTLC Association,51 and Halkina and Sherma.52

Comments: The HPTLC conditions in both documents include a relatively non-polar mobile phase combination with 1% acetic acid to prevent peak tailing on silica gel plates. The detection is carried out with anisaldehyde17,29 or phosphomolybdic acid52 reagent. The conditions employed by Halkina and Sherma provide a rough separation into compound categories: triglycerides, free fatty acids, and phytosterols.52 Identification of target analytes in the Ph. Eur. is not provided, although Melzig et al.4 suggest that the method identifies the presence of lauric acid, oleic acid, and β-sitosterol, which is not sufficient for the detection of adulteration. Images of representative saw palmetto extract HPTLC fingerprints using the Ph. Eur. conditions can be viewed on the website of the HPTLC Association,51 since the conditions are the same as in the Ph. Eur. Based on the paper by Halkina and Sherma, total substitution with vegetable oils can be determined by the larger concentrations of triglycerides. However, HPTLC is not the method of choice to detect admixture of vegetable oils, or the presence of designer blends with a similar fatty acid profile to saw palmetto extract.

11.2.2 Infrared spectroscopy

Two methods, Hanson et al.53 and Villar and Mulà,54 to detect saw palmetto extract adulteration using infrared spectroscopy were identified for this review.

Comments: Hanson et al. evaluated the authenticity of 16 retail samples of saw palmetto products by infrared (IR) spectroscopy and subsequent chemometric analysis using principal component analysis (PCA). The addition of vegetable oils was readily detected due to the higher content of triglycerides.53 Similarly, Villar and Mulà presented the results of an analysis of 28 saw palmetto samples using FT-IR (Figure 4) followed by PCA. The method clearly distinguished between authentic and adulterated extracts.53,54 Based on these investigations, IR spectroscopy combined with appropriate statistical methods may be suitable for detection of palmetto extract adulteration with vegetable oils. However, adulterants with low triglyceride content may be missed.


11.2.3 High-performance liquid chromatography

Methods described in the following articles were evaluated in this review: Bedner et al.,55 Fibigr et al.,56 Marti et al.,32 and Al-Achi et al.57

Comments: High-performance liquid chromatography (HPLC) is rarely used for the analysis of saw palmetto due to the challenges in resolving the analytes of interest, and their lack of a chromophore.

Phytosterol analysis: Three HPLC methods evaluated as part of this laboratory guidance document, were developed for the analysis of phytosterols using either a RP-18 or a phenyl column, with mass spectrometric (MS) detection. Bedner et al. developed two isocratic HPLC methods (comparing RP-18 and phenyl columns) for the separation of campesterol, cycloartenol, lupenone, lupeol, β-sitosterol, and stigmasterol. The peak shapes and resolution were better with the phenyl column, but despite the 80-minute run time, campesterol and stigmasterol co-eluted. Quantitative results with the APCI MS detector were comparable to gas chromatography with flame-ionization detection (GC-FID).55 Fibigr et al. achieved acceptable separation of eight phytosterols, including campesterol, β-sitosterol, and stigmasterol, on a narrow-bore RP-18 column in 8.5 minutes. However, the test method was not applied to a saw palmetto extract.56 Establishing the presence of ubiquitous phytosterols such as campesterol, β-sitosterol, and stigmasterol does not provide a definitive means to detect adulteration. However, the assessment of the phytosterol fingerprint as an approach for the detection of saw palmetto adulteration could be useful as a complementary method in evaluation of the extract authenticity. While none of the above methods have measured phytosterols in adulterating vegetable oils, the qualitative and quantitative sterol composition of these adulterating materials is well known. Some of the “saw palmetto” samples containing animal fats have low (< 0.2%) amounts of total sterols, but unusually high content of Δ5,24-stigmastadienol or Δ7-avenasterol. In addition, these samples tend to have a low campesterol/stigmasterol ratio (0.78 – 1.67) compared to authentic saw palmetto extracts (2.22-2.35).1 Compared to most GC-FID methods, HPLC-MS has the advantage of a faster sample preparation since there is no need to silylate the phytosterols after hydrolysis in potassium hydroxide. However, the resolution of the sterols is generally better using GC-FID.

Fatty acid analysis: Al-Achi et al. analyzed fatty acids after a conversion into fatty acid bromophenacyl esters using 2,4’-dibromoacetophenone and dicyclohexano-18-crown-6 as catalyst, allowing the use of an ultraviolet (UV) detector for quantification. Gradient conditions suggest a normal phase separation, but neither the column packing nor the detection wavelength was indicated. The method allowed for quantification of eight fatty acids in commercial saw palmetto products.57 The omission of important method information, lack of a chromatogram to assess the resolution and peak shape, and absence of validation data means that the method cannot be evaluated for its fitness to detect adulteration. Since validated GC methods are available for fatty acid analysis (see below) with comparable time and complexity requirements regarding sample preparation, these validated methods are considered a better option for use in evaluating the authenticity of saw palmetto extracts. In 2019, Marti et al. analyzed 35 samples of saw palmetto extract by ultra high-performance liquid chromatography (UHPLC)-high-resolution MS. In addition to the fatty acids, the authors determined the amounts of mono-, di-, and triglycerides, and phosphorylated glycerides. Ethanol, hexane, and CO2-extracts were readily distinguished using multivariate statistics (PCA, orthogonal partial least squares discriminant analysis [OPLS-DA]).32 No adulterated samples were included in the analysis, but based on the inclusion of a large number of saw palmetto constituents, and the discriminatory power of the assay, this approach may be very useful in the determination of saw palmetto authenticity.

11.2.4 Gas chromatography

Numerous methods described in the following literature were evaluated in this review: Bedner et al.,55 Booker et al.,58 Mikaelian and Sojka,35 Ph. Eur. 9.1,17,29 Penugonda and Lindshield,59 Priestap et al.,60 Sorenson and Sullivan,61 Srigley and Haile,62 de Swaef and Vlietinck,63 USP,18,33,64 and Wang et al.65

Comments: Gas chromatography has been the method of choice to analyze fatty acids, fatty alcohols, and phytosterols in saw palmetto extracts. The determination of the qualitative and quantitative fatty acid content has been the major focus in the analysis of saw palmetto extracts.

Fatty acid analysis: Measuring fatty acids by GC is usually done after converting the free and bound fatty acids into fatty acid methyl esters. An exception is one of the methods by Priestap et al., where the fatty acids are determined without derivatization using a nonpolar column (Table 3). While sample preparation is quick and easy, the run time is long and the peaks are broader and less well-resolved than those of the corresponding methyl esters. Conversion into fatty acid methyl esters is done by methanolysis under acidic or alkaline conditions,33,35,58,59,64 or by using specific methylation reagents such as trimethylsulfonium hydroxide,17,29,63 diazomethane,60 or m-trifluoromethylphenyl trimethylammonium hydroxide.65 Methanolysis takes more time since it involves heating the samples for up to two hours to complete the reaction. Some of the methylating reagents represent a convenient alternative, but are considered more hazardous to health. Particular caution should be used when using diazomethane due to its acute toxicity and risk of explosion.

Separation of the fatty acid methyl esters has been done on a number of stationary phases, with methylpolysiloxane-, cyanopropyl-, or polyethylene-coated columns being the most commonly used. Run times vary between 14 minutes35 and 66 minutes59 (see Table 3), not including the time to re-establish initial temperature and column equilibration. Detection is achieved by FID17,18,29,33,59,60,63,64 and/or MS.60,65 Chromatograms were presented in only two publications: de Swaef and Vlietinck have a good separation of all the fatty acid methyl and ethyl esters.63 In the case of Wang et al., the peaks of linolenic and oleic acids overlap, and show an apparent fronting.65

Table 3: Comparison among GC Methods to Determine Fatty Acids in Saw Palmetto Extracts.

aThe sample preparation time is based on the reported duration of various sample preparation steps provided in the experimental section of the corresponding paper and the estimated duration of e.g., weighing, dilution, centrifugation, etc., listed in the ABC-AHP-NCNPR Botanical Adulterants Prevention Program’s Skullcap Adulteration Laboratory Guidance Document.66

Based on thorough validation of GC methodology and easy sample preparation, the Ph. Eur. method is a good choice for the analysis of saw palmetto fatty acids. Sample preparation time in the USP method (Figure 5) is longer, but the shorter GC run time is advantageous. In addition, USP has detailed a specific range for the ratio of nine fatty acids relative to lauric acid, which can be used to detect adulteration with vegetable oils, unless these are mixed in a way to mimic the saw palmetto fatty acid composition. A simple additional sample preparation method for the determination of free fatty acids in saw palmetto extract has been developed and submitted in 2017 to USP as a Saw Palmetto Extract monograph revision.35 This method uses methanolic sodium hydroxide to hydrolyze the mono-, di, and triglycerides in order to selectively provide fatty acids methyl esters from fatty acids bound to glycerin. Conversely, when methanolic sulfuric acid (or other strong acid) is used for the reaction, methyl esters of both free and bound fatty acids are obtained. By calculating the difference between total and glycerin-bound concentrations for each individual fatty acid, the concentration of free fatty acids can be determined.

Designer blends that are made with mixed vegetable oils or fatty acids derived from animal fats may be present when phytosterol or fatty alcohol concentrations are outside the specifications. In other cases, the use of stable isotope measurements has proven helpful to detect such fraud.1,12


Phytosterol analysis: Due to the need for a hydrolysis step (some of the phytosterols occur as fatty acid esters in the extract) with subsequent derivatization with a silylating agent, the sample preparation for sterols is lengthy, involving many manipulations. Hydrolysis is achieved by heating the sample in ~2M potassium hydroxide solution. Sterols are either recovered by partitioning the aqueous solution with toluene55,61 or diethyl ether,62 or by adsorbing the solution onto diatomaceous earth, followed by elution with methylene chloride.29,33 Both Ph. Eur. and USP use the same GC-FID method on a dimethylpolysiloxane column. The AOAC method (Sorenson and Sullivan; Bedner et al.)55,61 and the method by Srigley and Haile62 use a phenylmethylpolysiloxane stationary phase, although AOAC also permits a dimethylpolysiloxane column. Run times are 33-66 minutes. The conditions established by Srigley and Haile, with a run time of 66 minutes, allow quantification of up to 18 common phytosterols. All methods provide a good separation of the saw palmetto phytosterols and have been extensively validated. As mentioned in section 11.2.3 above, the analysis of phytosterols as a stand-alone method is not sufficient to rule out adulteration, but it is an excellent choice as a complementary method since deviations from pharmacopeial specifications (not less than 0.2% total sterols, not less than 0.1% β-sitosterol) are a good indication of ingredient adulteration.

Fatty alcohol analysis: USP is the only compendial standard to measure fatty alcohols in saw palmetto extracts.33 Sample preparation and analysis conditions are the same as for the sterols, which is convenient as both classes of compounds can be measured in a single run. As with the sterol analysis, the determination of fatty alcohols by itself is insufficient to detect adulteration, but it is considered a valuable complementary means of verifying the authenticity of saw palmetto extracts.

11.2.5 Nuclear magnetic resonance

Two methods described in the literature were evaluated in this review: Booker et al.,58 and de Combarieu et al.67 The NMR parameters outlined by de Combarieu et al. were also used by Perini et al.1

Comments: Even though the 1H NMR spectrum of saw palmetto extract is relatively simple compared to extracts of other botanicals, a lot of useful information can be obtained by visual evaluation of the spectrum. Adulteration with vegetable oils can be readily distinguished by the presence of the signals of the α/α' and β-protons of triglycerides (Figure 6), which are much smaller in saw palmetto extracts than in vegetable oils.68 Using data from the PCA loadings plot, Booker et al.58 and Perini et al.1 noticed that the regions between 4.1-4.2 ppm, and between 5.3-5.5 ppm were important for clustering of the samples (commercially available finished products). The assessment of three principal components allowed for authentic saw palmetto to be distinguished, even from animal fat-based ‘designer blends’ matching the saw palmetto fatty acid profile.1 Based on all the data, 1H NMR represents a valuable tool to detect saw palmetto adulteration, but is often not part of the instruments found in a botanical ingredient or dietary supplement manufacturing quality control laboratory.


11.2.6 Stable isotope ratio

Stable isotope analysis for the authentication of saw palmetto extracts has been described in two separate publications by Perini et al.1,12

Comment: Variations in the stable isotopic ratios (SIRs) in plants and animals may occur for a number of reasons. For example, the 2H/1H ratio in plants is influenced by the geographical origin of the local water. The 13C/12C ratio in plants depends on the type of photosynthesis that a plant utilizes. While most plants exclusively use the Calvin cycle, some plants (e.g., corn [Zea mays, Poaceae] or sugar cane [Saccharum officinarum, Poaceae]) have additional photosynthetic pathways, leading to a slightly higher 13C/12C ratio in the latter. The 13C/12C isotopic ratio of animal fats is known to be correlated with their diet, e.g., animals that feed exclusively on corn will have a higher 13C/12C ratio than those that ingest a wider variety of plants. The 18O/16O ratio depends on the temperature, freshwater input, and other climatic factors. Results are expressed as the ratio difference (δ2H, δ13C, δ18O) of the material to be analyzed and a standard with a known isotopic ratio, e.g., the Pee Dee Belemnite (based on a Cretaceous marine fossil from the Pee Dee Formation in South Carolina), which is one of the standards used for the 13C/12C ratio, and the Vienna Standard Mean Ocean Water (VSMOW), which defines the 2H/1H and 18O/16O composition of fresh water.

Isotope ratios can be measured using gas chromatography with an isotope mass spectrometer. In the approach by Perini et al., the addition of a single-quadrupole mass spectrometer allowed identification of individual compounds at the same time as the isotope ratios were measured. While reported stable isotope ratios of some of the vegetable oils overlap with those of saw palmetto, measuring the δ18O may provide valuable information about the possible risk of adulteration since the δ18O of most vegetable oils is lower than the range observed in saw palmetto. Fatty acids derived from animal sources have a δ18O and a δ2H below those reported for saw palmetto, and therefore can be readily detected as adulterants, as evidenced in the publications by Perini et al.1,12

Measuring the stable isotope ratios of bulk fatty oils is a helpful means to detect adulteration, especially when a number of isotopes are measured and analyzed using appropriate statistical tools. Further research needs to be done to verify the ability of SIR analysis to detect other potential adulterants, and to determine the limit of detection of this technique. Due to the availability and ease-of-use of more established methods, the application of stable isotope analysis may be best suited as an orthogonal assay to confirm adulteration, and to determine the origin of the adulterant.


12. Conclusion

Identification of saw palmetto extract adulteration has been achieved using a number of analytical techniques. Macroscopic and organoleptic assessment may provide the first indication of adulteration by observing the color and strongly aromatic and foul odor. Absence of the characteristic is a good indication that the oil is adulterated. In practice, several assays are needed to confirm the authenticity of saw palmetto extract. Gas chromatography for measuring fatty acid, fatty alcohol, and phytosterol profiles, combined with a visual and organoleptic inspection of the liquid and determination of the acid value, provides a robust affirmation of saw palmetto extract authenticity. 1H NMR spectroscopy (with or without chemometric data analysis) provides a suitable option for those companies with access to an NMR instrument.

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