1 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
Keywords: Adulteration, animal fatty acids, canola oil, coconut oil, palm oil, saw palmetto, Serenoa repens, sunflower oil,
Citation (JAMA) style: Gafner S. Saw palmetto extract laboratory guidance document. Austin, TX: ABC-AHP-NCNPR
Botanical Adulterants Prevention Program. 2019.
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
Saw Palmetto Extract
Laboratory Guidance Document
By Stefan Gafner, PhD*
American Botanical Council, PO Box 144345, Austin, TX 78714
*Correspondence email Serenoa repens
Photo ©2019 Steven Foster
Various analytical methods are reviewed here with the
specific purpose of identifying their strengths and limita-
tions 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 analy-
sis. 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 Inter-
national, International Organization for Standardization
(ISO), World Health Organization (WHO), and Interna-
tional 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.
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org2
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 (karliko-
vaya palma, “dwarf palm”), пальма cереноа, co пальметто
Spanish: Sabal, palma enana americana3,4
3.3 Latin binomial: Serenoa repens (W. Bartram) Small
3.4 Synonyms: Chamaerops serrulata Michx., Corypha
obliqua W. Ba rtr am, Corypha repens W. B art ram, Diglos-
sophyllum 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
See Table 1 below.
6. Identification and Distinction using
Botanical descriptions of saw palmetto fruit have been
published in a number of pharmacopeial monographs
Tab l e 1. Scientific Names, Family, and Common Names of Plants Used as Sources of Vegetable Oils Known
as Saw Palmetto Fruit Extract Adulterants*
SpeciesaSynonym(s)aFamily Common namebOther common namesc
Arachis hypogaea L. A. nambiquarae Hoehne Fabaceae Peanut Groundnut
Brassica napus L. Brassica napus ssp. napus Brassicaceae CanoladColza, rape, rapeseed
Cocos nucifera L. Calappa nucifera (L.) Kuntze
Cocos indica Royle
C. nana Griff.
Arecaceae Coconut Coconut palm
E. dybowskii Hua
(Jum. & H. Perrier) Becc.
E. melanococca Gaertn.
Arecaceae African oil palm Oil palm
Helianthus annuus L. H. aridus Rydb.
H. jaegeri Heiser
H. lenticularis Douglas
H. macrocarpus DC
H. ovatus Lehm.
Olea europaea L. Oleaceae Olive
aThe Plant List and the Kew Medicinal Plant Names Services database.8,9 A comprehensive list of synonyms can be accessed through both
bAmerican Herbal Products Association’s Herbs of Commerce, 2nd ed.3
cAmerican Herbal Products Association’s Herbs of Commerce, 2nd ed.,3 and the USDA GRIN database.10
dAccording to the Canadian Food Inspection Agency,11 canola oil may be obtained from Brassica rapa and B. juncea as well.
*Note: Species other than those listed in Table 1 that are used for production of edible fatty oils are also known to be used as adulterants.
In addition, admixture or substitution of saw palmetto extracts with “designer blends,” mimicking the saw palmet to fatty acid composition,
which include fatty acids from animal sources have been described.1,12
The few repor ts 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 adultera-
tion 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
3 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
and books.4,17-19 Criteria to distin-
guish 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 identify-
ing saw palmetto to species. For obvi-
ous reasons, macroscopic identifica-
tion is not applicable to saw palmetto
7. Identification and Distinction
Microscopic descriptions of saw
palmetto are found in the pharma-
copeias of Europe and the United
States, and the American Herbal
Pharmacopoeia’s textbook on micro-
scopic 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 micros-
copy 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 chloro-
phyll with these solvents. Low pressure
CO2 extracts have a yellow to orange-
brown color. While organoleptic evalua-
tion does not provide sufficient safeguard
against adulteration, the extract color
and the very characteristic odor are help-
ful in assessing the authenticity of a saw
9. Genetic Identification and
Several authors have looked into differ-
ences among nucleotide sequences of
various gene regions for saw palmetto
and closely related palm species, includ-
ing dwarf palm and Everglades palm. In
most cases, the purpose was to determine
phylogenetic relationships.23-26 Nucleo-
tide sequences of the chloroplast (atpB,
matK, ndhF, rbcL, r ps16 intron, trnD-
trnT, trnL-trnF, trnQ-rps16) and nucleus
(18S, ITS, ms, prk, rpb2) were used to establish the rela-
tionship 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 abun-
dant. 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
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).
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).
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org4
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 authen-
tication 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 perox-
ide 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 authentic-
ity. Since saw palmetto extracts contain a large concentra-
tion 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 infor-
mation 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 adultera-
tion.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 specifica-
tions 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) meth-
ods, either analyzing the fatty acids directly, or after conver-
sion into fatty acid esters. In addition to GC, thin-layer
chromatography (TLC) is an integral part for identity test-
ing in pharmacopeial monographs. Other methods of chem-
ical 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 impor-
tant saw palmetto constituents and their significance in
authentication are discussed below. The overview of poten-
tial adulterants is based on published literature. The chemi-
cal composition of vegetable oils is dependent on the refin-
ing 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 adulter-
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 carot-
enoids.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 contain 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 myris-
tic (10-12%), palmitic (8.5-9.2%), and linoleic (4.3-6.0%)
acids.34-36 Marti et al. reported the presence of hydroxyl-
ated 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 adultera-
tion more difficult. The main phytosterols are β-sitosterol
(68-72% of total sterols), campesterol (20-23%), and stig-
masterol (8-9%).36,37 Δ5-avenasterol, Δ7-avenasterol, cleros-
terol, 24-methylenecholesterol, and Δ7-stigmasterol have
been reported present in minor amounts.1,38 Fatty alcohols
include octacosanol, hexacosanol, tetracosanol, and triac-
Arachis hypogaea oil: Peanut oil contains approximately
96% triglycerides, with oleic, linoleic, and palmitic acids
as the main fatty acids (Table 2).40 In addition, peanut oil
contains approximately 0.50% phospholipids and 0.30%
phytosterols (β-sitosterol, campesterol, stigmasterol, and
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
5 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
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 brassi-
casterol 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 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 corre-
sponding 1,2-diacylglycerols.41,44 Palm oil has approxi-
mately 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
Figure 3: Major Fatty Acids and Phytosterols in Saw Palmetto
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org6
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 devel-
oped, 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 adul-
teration 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 corre-
sponding (Δ8 ,14)- and Δ14-sterols. Depending on the extent
of refinement, sunflower oil has one of the highest concen-
trations 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 campes-
terol (up to 4%). Numerous unusual phytosterols (e.g.,
Δ7-avenasterol, Δ7-stigmastenol) are present at low concen-
trations.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 oleuro-
pein (≤ 0.035%), oleocanthal (0.004-0.021%), and oleacein
(0.002-0.48%) as the most abundant.49,50
Table 2: Relative Fatty Acid (free and bound fatty acid) Composition (in %) of Saw Palmetto and Adulterat-
ing Vegetable Oils27,34-36,43,46,47
Fatty acid Saw
CanolaaCoconut Olive Palm Peanut SunflowerbSunflowerc
Caprylic C8:0 2.0-2.8 n.d. 4.6-10 n.d. n.d. n.d. n.d. n.d.
2.7-3.2 n.d. 5.0-8.0 n.d. n.d. n.d. n.d. n.d.
26-32 n.d. 45-53 n.d. ≤ 0.2 ≤ 0.1 ≤ 0.1 n.d.
10-12 ≤ 0.2 17-21 < 0.1 0.5-2.0 ≤ 0.1 ≤ 0.2 ≤ 0.1
8.5-9.2 2.5-7.0 7.5-10 7.5-20 39-48 8.0-14 5.0-7.6 2.6-5.0
1.7-2.1 0.8-3.0 2.0-4.0 0.5-5.0 3.5-6.0 1.0-4.5 2.7-6.5 2.9-6.2
30-35 51-70 5.0-10 55-83 36-44 35-69 14-39 75-90
4.3-6.0 15-30 1.0-2.5 3.5-21 9.0-12 12-43 48-74 2.1-17
0.7-1.2 5.0-14 ≤ 0.2 ≤ 1.0 ≤ 0.5 ≤ 0.3 ≤ 0.3 ≤ 0.3
< 0.1 0.2-1.2 ≤ 0.2 ≤ 0.6 ≤ 1.0 1.0-2.0 0.1-0.5 0.2-0.5
0.2 0.1-4.3 ≤ 0.2 n.d. ≤ 0.4 0.7-1.7 ≤ 0.5 0.1-0.5
< 0.1 ≤ 0.6 n.d. ≤ 0.2 ≤ 0.2 1.4-4.5 ≤ 0.7 0.5-1.6
< 0.1 ≤ 0.3 n.d. ≤ 0.2 n.d. 0.5-2.5 ≤ 0.5 ≤ 0.5
aLow erucic acid canola oil
bRegular (= linoleic-type) sunflower oil
cHigh-oleic acid sunflower oil
7 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
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, 2 9 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 concentra-
tions of triglycerides. However, HPTLC is not the method
of choice to detect admixture of vegetable oils, or the pres-
ence 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 spectros-
copy 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 evalu-
ated 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 separating the analytes of interest, and
their lack of a chromophore.
Phytosterol analysis: Three HPLC methods evaluated as
part of this laboratory guidance document, were devel-
oped for the analysis of phytosterols using either a RP-18
or a phenyl column, with mass spectrometric (MS) detec-
tion. 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. Quanti-
tative results with the APCI MS detector were comparable
to gas chromatography with flame-ionization detection
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).
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org8
(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 ubiq-
uitous 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 compo-
sition 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 addi-
tion, these samples tend to have a low campesterol/stigmas-
terol 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 hydro-
lysis 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 resolu-
tion 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. Etha-
nol, hexane, and CO2-extracts were readily distinguished
using multivariate statistics (PCA, orthogonal partial least
squares discriminant analysis [OPLS-DA]).32 No adulter-
ated 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
Table 3: Comparison among GC Methods to Determine Fatty Acids in Saw Palmetto Extracts.
Run time Column Comments
Booker58 150 min 24 min 5% Phenyl/ 95%
Modified method of the German Society
for Fat Science.
Mikaelian35 210 min 14 min Polyethylene glycol Modified USP sample preparation
Ph. Eur.29 25 min 32 min Poly(dimethyl)siloxane Accepted standard method.
Penugonda59 155 min 66 min Cyanopropyl Validation data is lacking.
Priestap60 Unclear 44 min Polyethylene glycol Validation data is lacking.
Priestap60 10 min 84 min Phenyl arylene Direct (without derivatization) analysis of
fatty acids. Oleic, linoleic, and linolenic
acids not resolved. Broad peak shape.
Validation data is lacking.
De Swaef63 85 min 36 min Cyanopropyl Good separation, validated for lauric acid
and ethyl laurate.
USP33 210 min 17 min Polyethylene glycol Accepted standard method.
Wang65 50 min 41 min 5% Phenyl/ 95%
The peaks of linolenic and oleic acids
overlap, and the peak shape is not
optimal. Validation data available.
aThe sample preparation time is based on the repor ted duration of various sample preparation steps provide d in the experimental sec tion 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
9 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
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 U SP,18 ,33,64
and Wang et al.65
Comments: Gas chromatography has been the method
of choice to analyze fatty acids, fatty alcohols, and phytos-
terols 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 meth-
ods 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 hydrox-
ide,17,29, 6 3 diazomethane,60 or m-trif luoromethylphenyl
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 consid-
ered 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 methylpolysilox-
ane-, 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 equili-
bration. Separation of the fatty acid methyl esters has been
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).
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org10
done on a number of stationary phases, with methylpoly-
siloxane-, cyanopropyl-, or polyethylene-coated columns
being the most commonly used. 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 Vlie-
tinck 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
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 differ-
ence 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, involv-
ing 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 methy-
lene chloride.29,33 Both Ph. Eur. and USP use the same
GC-FID method on a dimethylpolysiloxane column. The
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 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
AOAC method (Sorenson and Sullivan; Bedner et al.)55,61
and the method by Srigley and Haile62 use a phenyl-
methylpolysiloxane 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 excel-
lent 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 stan-
dard 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 load-
ings 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 prin-
cipal 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 instru-
ments 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 publi-
cations 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 photo-
synthetic 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 chromatogra-
phy with an isotope mass spectrometer. In the approach by
Perini et al., the addition of a single-quadrupole mass spec-
trometer 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 read-
ily 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 appro-
priate 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 anal-
ysis may be best suited as an orthogonal assay to confirm
adulteration, and to determine the origin of the adulterant.
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 character-
istic odor is a good indication that the oil is adulterated. In
practice, several assays are needed to confirm the authen-
ticity 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.
Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org12
Table 4. Comparison among the Different Chemical Methods to Authenticate Saw Palmetto Extract
Method Analyte Pro Contra
HPTLC Free fatty acids
Basic systems affordable
Available methods unlikely to detect
sophisticated types of adulteration
HPLC-MS Free fatty acids
Quick sample preparation for
Complicated sample preparation for fatty acid
MS identification of fatty acids not essential
since reference standards are available
Reliance on solely fatty acid or sterol
determination not sufficient for the detection
of mixtures mimicking the saw palmetto fatty
GC-FID Fatty alcohols
Total fatty acids
Free fatty acids
Standard equipment in many
Basic systems affordable
Ability to measure three classes
of compounds provides robust
approach for the detection of
saw palmetto adulteration
Mainly quantitative method
Need for derivatization of target analytes
Total fatty acid analysis is not sufficient for
the detection of mixtures mimicking the saw
palmetto fatty acid profile
GC-MS Free fatty acids Qualitative and quantitative Equipment expensive
Need for derivatization of target analytes
MS identification of fatty acids not essential
since reference standards are available
Not sufficient for the detection of mixtures
mimicking the saw palmetto fatty acid profile
Infrared Fingerprint No derivatization needed for
Little data available data on adulterant
Need to build-up reference library
No derivatization needed for
Ability to detect most/all
Need to build-up reference library
Free fatty acids
Ability to detect most
Analysis offered by limited number of contract
13 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org
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