Vitamin D Content of Australian Native Food Plants and Australian-Grown Edible Seaweed

Abstract

Vitamin D has previously been quantified in some plants and algae, particularly in leaves of the Solanaceae family. We measured the vitamin D content of Australian native food plants and Australian-grown edible seaweed. Using liquid chromatography with triple quadrupole mass spectrometry, 13 samples (including leaf, fruit, and seed) were analyzed in duplicate for vitamin D₂, vitamin D₃, 25-hydroxyvitamin D₂, and 25-hydroxyvitamin D₃. Five samples contained vitamin D₂: raw wattleseed (Acacia victoriae) (0.03 µg/100 g dry weight (DW)); fresh and dried lemon myrtle (Backhousia citriodora) leaves (0.03 and 0.24 µg/100 g DW, respectively); and dried leaves and berries of Tasmanian mountain pepper (Tasmannia lanceolata) (0.67 and 0.05 µg/100 g DW, respectively). Fresh kombu (Lessonia corrugata) contained vitamin D₃ (0.01 µg/100 g DW). Detected amounts were low; however, it is possible that exposure to ultraviolet radiation may increase the vitamin D content of plants and algae if vitamin D precursors are present.

Full text

nutrients
Article
Vitamin D Content of Australian Native Food Plants
and Australian-Grown Edible Seaweed
Laura J. Hughes 1, Lucinda J. Black 1, Jill L. Sherriff 1ID , Eleanor Dunlop 1ID , Norbert Strobel 2,
Robyn M. Lucas 3,4 ID and Janet F. Bornman 5,*
1School of Public Health, Curtin University, Bentley WA 6102, Australia;
laura.j.hughes@postgrad.curtin.edu.au (L.J.H.); lucinda.black@curtin.edu.au (L.J.B.);
j.sherriff@curtin.edu.au (J.L.S.); eleanor.dunlop@postgrad.curtin.edu.au (E.D.)
2National Measurement Institute, 1/153 Bertie Street, Port Melbourne VIC 3207, Australia;
norbert.strobel@measurement.gov.au
3National Centre for Epidemiology and Population Health, Research School of Population Health,
The Australian National University, Canberra ACT 2600, Australia; robyn.lucas@anu.edu.au
4Centre for Ophthalmology and Visual Science, University of Western Australia, Perth WA 6009, Australia
5School of Veterinary and Life Sciences, Murdoch University, Murdoch WA 6150, Australia
*Correspondence: janet.bornman@murdoch.edu.au; Tel.: +61-478-473-439
Received: 24 May 2018; Accepted: 3 July 2018; Published: 6 July 2018


Abstract: Vitamin D has previously been quantified in some plants and algae, particularly in leaves
of the Solanaceae family. We measured the vitamin D content of Australian native food plants
and Australian-grown edible seaweed. Using liquid chromatography with triple quadrupole mass
spectrometry, 13 samples (including leaf, fruit, and seed) were analyzed in duplicate for vitamin D
2
,
vitamin D
3
, 25-hydroxyvitamin D
2
, and 25-hydroxyvitamin D
3
. Five samples contained vitamin D
2
:
raw wattleseed (Acacia victoriae) (0.03
µ
g/100 g dry weight (DW)); fresh and dried lemon myrtle
(Backhousia citriodora) leaves (0.03 and 0.24
µ
g/100 g DW, respectively); and dried leaves and berries
of Tasmanian mountain pepper (Tasmannia lanceolata) (0.67 and 0.05
µ
g/100 g DW, respectively).
Fresh kombu (Lessonia corrugata) contained vitamin D
3
(0.01
µ
g/100 g DW). Detected amounts were
low; however, it is possible that exposure to ultraviolet radiation may increase the vitamin D content
of plants and algae if vitamin D precursors are present.
Keywords:
liquid chromatography with triple quadrupole mass spectrometry (LC-QQQ); liquid
chromatography; triple quadrupole; vitamin D; serum 25-hydroxyvitamin D (25(OH)D); plants; algae
1. Introduction
Approximately a quarter of Australian adults are deficient in vitamin D (serum 25-hydroxyvitamin
D (25(OH)D) < 50 nmol/L) [
1
]. There is seasonal variation in the prevalence of vitamin D deficiency,
with 14% of the adult population deficient in summer and 36% in winter [
1
]. Certain population groups,
such as people with dark skin, those wearing covering for religious or cultural reasons, and people
living largely indoors, are at greater risk of deficiency due to inadequate sun exposure, particularly in
winter months [
2
]. Although the major source of vitamin D for humans is cutaneous synthesis of
vitamin D
3
following exposure of the skin to solar radiation [
3
], when sun exposure is inadequate to
maintain vitamin D status, dietary sources make a small but useful contribution [4].
In the Australian food supply, fish, meat, eggs, dairy, and fortified margarine are known sources
of vitamin D
3
, while mushrooms are mainly a source of vitamin D
2
and small amounts of vitamins D
3
and D
4
[
5
,
6
]. Vitamin D
3
has been found in shiitake mushrooms, a few algae, and several species of
Angiosperms (flowering plants) [
7
] belonging to the plant families of Cucurbitaceae, Fabaceae, Poaceae,
Nutrients 2018,10, 876; doi:10.3390/nu10070876 www.mdpi.com/journal/nutrients

Nutrients 2018,10, 876 2 of 9
and Solanaceae (Table 1). However, although D
3
is found in several plants, previous results have come
mostly from non-edible leaves rather than the edible portions of the different species, such as fruits
or seeds. The hydroxylated metabolite of vitamin D
3
, 25(OH)D
3
, is found in animal products [
8
–
10
]
and likely has a greater biological activity than vitamin D
3
itself [
11
,
12
]. There is some evidence that
vitamin D3taken orally is more effective than vitamin D2at increasing levels of 25(OH)D [13,14].
Vitamin D regulates and maintains critical levels of calcium and phosphates in the skeleton of
vertebrates by promoting absorption of these nutrients from the gastrointestinal tract. Rickets, the
softening of bones in children (or osteomalacia in adults) due to vitamin D deficiency, has been
increasing globally [
15
,
16
]. Vitamin D deficiency has also been linked to a number of other
health conditions besides bone health, including reduced muscle function, autoimmune disease,
cardiovascular disease, and some cancers [17–19].
The anti-rachitic properties of plants were originally discovered by animal feeding studies [
20
–
22
],
although the active compound in these early studies was later identified as vitamin D
2
produced from
fungal contamination, rather than endogenous to the plants. More recently, high-performance liquid
chromatography–ultraviolet (HPLC–UV) with mass spectrometry or liquid chromatography–tandem
mass spectrometry (LC–MS/MS) have been used to measure vitamin D metabolites directly in the
plant matrix [23–25].
Table 1.
Content of vitamin D
2
, vitamin D
3
, and 25-hydroxyvitamin D
3
in plants, microalgae, and
macroalgae derived from previously published literature.
Common Name
(Botanical Name) Plant part Vitamin D2
(µg/100 g)
Vitamin D3
(µg/100 g)
25(OH)D3
(µg/100 g)
Plants
Tomato Leaf Not tested 78 (DW) a2 (DW) a
(Lycopersicon esculentum) Leaf Not tested 110 (FW) b1.5 (FW) b
Leaf Not tested 0.17 (DW) cn/d c
Waxy leaf nightshade Leaf Not tested 0.32 (DW) c0.08 (DW) c
(Solanum glaucophyllum)
Cell culture derived from leaf material
Not tested 220.00 (FW) d100.00 (FW) d
Potato Leaf Not tested 15 (FW) bn/d b
(Solanum tuberosum)
Bell pepper Leaf Not tested n/d cn/d c
(Capsicum annuum)
Day blooming jasmine Leaf Not tested 10 (DW) e10 (DW) e
(Cestrum diurnum)
Zucchini Leaf Not tested 23 (FW) bNot tested
(Cucurbita pepo)
Alfalfa/Lucerne Leaf 4.8 DW) f0.06 (DW) fNot tested
(Medicago sativa)
Rimu
(Dacrydium cupressinum)Fruit 70 (DW) g11.5 (DW) gNot tested
Algae
Microalgae
Phytoplankton Whole algae 1.9–4.3 (DW) h2.2–14.7 (DW) hNot tested
5.3 (DW) i80.4 (DW) iNot tested
72.4 (DW) i271.7 (DW) iNot tested
(Pavlova lutheri) Whole algae 3900 (DW) jNot tested Not tested
(Tetraselmis suecica) Whole algae 1400 (DW) jNot tested Not tested
Marine centric diatom
(Skeletonema costatum)Whole algae 1100 (DW) jNot tested Not tested
(Isochrysis galbana) Whole algae 500 (DW) jNot tested Not tested
(Chaetoceros calcitrans) Whole algae n/d jNot tested Not tested
Macroalgae
Japanese Wireweed
(Sargassum muticum)Not specified 90 (DW) jNot tested Not tested
Decimal places are reported as per the original reference. n/d, not detected; DW: dry weight; FW: fresh weight
a[26]b[24]c[25]d[23]e[27]f[28]g[29]h[30]i[31]j[32].
This paper focuses on some of the Australian native foods in an effort to detect possible new
sources of vitamin D in plants and seaweed. The commercial production of native foods across
Australia is estimated to have a gross value of more than 15 million Australian dollars [
33
]. In a recent
stocktake of the Australian native plant industry, it was found that production of lemon myrtle and

Nutrients 2018,10, 876 3 of 9
mountain pepper dominated over other native species, with cultivated supplies of most species [
33
].
A majority of native food production is used as raw material for value-added products [33].
Given the emerging interest in Australian native food plants for local consumption and export [
33
],
we measured vitamin D
2
, vitamin D
3
, 25-hydroxyvitamin D
2
(25(OH)D
2
), and 25(OH)D
3
in a selection
of Australian native food plants and Australian-grown edible seaweed. Because the metabolisms
of calcium and vitamin D are closely linked in animals and there is a potential link between
calcium and vitamin D metabolism in plants [
34
], we selected plants and seaweed with known
high calcium (Ca) content. These included Acacia victoriae (wattleseed; 434 mg Ca/100 g, seeds [
35
]),
Tasmannia lanceolata (Tasmanian mountain pepper; 495 and 148 mg Ca/100 g, dried leaves and berries,
respectively [
35
]), Backhousia citriodora (lemon myrtle; 1583 mg Ca/100 g, dried leaves [
35
]), and the
seaweeds, Lessonia corrugata (kombu; 706 mg Ca/100 g, dried [
36
]) and Undaria pinnatifida (wakame;
1100–3000 and 150 mg Ca/100 g, dried and fresh, respectively [36,37]).
Relatively little detailed research has been carried out on Australian native foods, although the
existing studies have shown many of the traditional foods to have high nutrient content. For example,
the edible seed of the wattleseed tree (from the family Leguminosae) has a strong nutty or coffee-like
flavor and has been included in sweet dishes or as a coffee substitute [
33
]. It is also a good source of
calcium, magnesium, and zinc [
35
]. The lemon myrtle tree, family Myrtaceae, produces leaves with
an intense lemon/lemongrass flavor. These leaves are used fresh or dried as a culinary herb, as a
tea, or for use in cosmetics and food flavoring agents in the form of extracted oil [
33
]. The lemon
myrtle leaves are high in calcium, vitamin E, and antioxidants [
35
]. Mountain pepper (from the
family Winteraceae), a shrub native to Tasmania, is a versatile plant with both the berries and leaves
being used as a food source [
33
]. The fresh and dried berries are used as an alternative to traditional
pepper, and fresh and dried leaves are used as a culinary herb [
33
]. Both the leaves and berries have
been used as therapeutic agents and as a preservative [
33
]. The plant is high in vitamin E, folate,
and antioxidants with moderate levels of calcium [
35
]. Finally, two types of kelp seaweeds were
selected: kombu (family Lessoniaceae), a native of Tasmania [
38
]; and wakame (family Alariaceae),
an introduced kelp species [
38
,
39
], hand-harvested in the wild, mostly in Tasmania. Both kombu and
wakame are high in calcium [36,37].
With this initial study, we highlight the possibility of using the selected plants and algae as a
natural and additional source of vitamin D for reducing the incidence of vitamin D deficiency in
vulnerable population groups. For the vitamin D analyses of the selected species, we used liquid
chromatography with triple quadrupole mass spectrometry (LC-QQQ), which has been validated for
the detection of low levels of vitamin D metabolites in biological samples [
40
], modified to suit the
complex matrices of plants and algae.
2. Materials and Methods
2.1. Sample Acquisition
Samples of Australian native food plants (wattleseed (Acacia victoriae), lemon myrtle (Backhousia
citriodora), and Tasmanian mountain pepper (Tasmannia lanceolata)) and Australian-grown edible
seaweeds (wakame (Undaria pinnatifida) and kombu (Lessonia corrugata)) were sourced from commercial
growers or wild harvesters. The selected plants were identified as commonly consumed and
commercially available in the Australian food supply [33,35].
The samples were shipped directly from the growers and harvesters to the National Measurement
Institute of Australia (NMI), Port Melbourne, Victoria, for preparation and analysis. To maintain their
integrity, fresh samples were shipped in an insulated box containing cooler bricks. The details of
quantity and source of samples, along with any processing by the growers and harvesters, are outlined
in the Supplementary Materials (Table S1).

Nutrients 2018,10, 876 4 of 9
2.2. Sample Preparation
Upon arrival at NMI, dried samples were stored at room temperature, and fresh samples were
stored at <5
◦
C. Dried and freeze-dried samples were homogenized. Fresh samples were prepared as
follows: the leaves were cut to 1 cm squares, and fresh fruit was blended; the weight was recorded; the
samples were frozen overnight at
−
70
◦
C and then freeze dried for 48 h to
−
50
◦
C and <10 mTorr; the
weight was recorded again; and the freeze-dried factor was determined. Each dried and fresh sample
yielded one analytical sample. The prepared samples were stored between
−
16
◦
C and
−
20
◦
C until
extraction and analysis.
2.3. Sample Analysis
The instrumentation used was similar to that of Jäpelt et al. [
25
]. Extraction procedures were
derived from published methodology [
25
,
41
]. An equivalent deuterated internal standard was added
for each vitamin D analogue under investigation: 100
µ
L of a mixed internal standard solution
was added to each sample. This contained 100 ng/mL each of vitamin D
3
[
2
H
3
], vitamin D
2
[
2
H
3
],
25(OH)D3[2H3], and 25(OH)D2[2H3] (Iso Sciences/PM Separations).
The samples were homogenized with 1 g ascorbic acid, 10 mL deionized water, 30 mL absolute
ethanol, 2 g potassium hydroxide pellets, and 100
µ
L of 100 ng/mL deuterated internal standard mix
and made to 50 mL with deionized water. The headspace was flushed with nitrogen gas, capped, and
placed in a shaker for saponification overnight. The samples underwent centrifugation and 10 mL
of the ethanol layer was extracted onto diatomaceous earth Solid Phase Extraction (SPE) cartridges
(ChemElute Agilent). The organic soluble compounds were washed off with two 30 mL aliquots
of petroleum spirits. The washes were collected into 80 mL glass EPA vials and then evaporated
to dryness under high purity nitrogen gas. The residue was reconstituted into 400
µ
L heptane and
transferred to a LC vial containing a 400
µ
L glass insert. The prepared extracts were stored at
−
20
◦
C.
The reagent, PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) reacts non-specifically with dienes under
a reaction mechanism called the Diels–Alder Reaction. While in theory, excess PTAD is added
to derivatize the vitamin D analogues, samples with high diene content may limit the amount
of PTAD available for derivatization. Therefore, where samples were determined to have high
diene content, extract clean-up via normal phase chromatography fraction collection was performed.
The extracts were inspected for cold precipitate: if present, the liquid extract was transferred to a
fresh 400
µ
L glass insert. Of the remaining liquid extract, 200
µ
L were injected onto a normal phase
chromatographic system with a silica column, 1 mL/min 2% isopropyl alcohol in heptane mobile
phase, and a photodiode array detector set to 265 nm. Vitamin D and 25(OH)D fractions were collected.
Fractions of vitamin D and 25(OH)D were combined and evaporated under high purity nitrogen
gas. The dry material was reconstituted in 200
µ
L of dry acetonitrile containing 1 mg/mL of
4-phenyl-1,2,4-triazole-3,5-dione (PTAD) and transferred to a fresh LC vial. Two hours were allowed to
complete derivatization. The sample was evaporated under high purity nitrogen gas. The dry material
was reconstituted in 100
µ
L of methanol and water (70:30), transferred to a fresh 400
µ
L glass insert,
and placed into an LC vial. A limit of quantitation was conservatively set at 0.05
µ
g/100g, which is
half of the ‘spiked’ recovery level of 0.1 µg/100g.
The recoveries were determined for each sample analyzed. The recoveries at the 0.1
µ
g/100 g ‘spiked’
recovery level were as follows: vitamin D
3
, 86–104%; vitamin D
2
, 76–105%; 25(OH)D
3
, 85–114%; and
25(OH)D
2
, 90–114%. The recovery for 25-hydroxyvitamin D
3
in wattleseed (roasted/milled/ground
seed) could not be determined due to a matrix interference.
The samples were analyzed for vitamin D
2
, vitamin D
3
, 25(OH)D
2
, and 25(OH)D
3
using
LC-QQQ (Agilent, San Jose, CA, USA). The calibration samples of vitamin D
2
, vitamin D
3
, 25(OH)D
2
,
and 25(OH)D
3
were prepared. The calibration concentrations (in ng/mL) were 0, 2.5, 5, 7.5, 10, 15,
25, 50, 75, and 100. Each calibration sample also contained 10 ng/mL of deuterated internal standard
for each vitamer (vitamin D analogue) tested. The calibrations and samples were analyzed using
1290 Infinity Series LC System/6460 Triple Quad liquid chromatography–tandem mass spectrometry

Nutrients 2018,10, 876 5 of 9
(LC–MS/MS; Agilent Technologies Mulgrave, Victoria, Australia) fitted with a Jet Stream ESI source in
positive ion mode using a Supelco Ascentis Express C18 10 cm
×
2.1 mm, 2.7
µ
m LC chromatographic
column (Sigma-Aldrich, Sydney Australia).
For each vitamer analyzed, each sample was tested in duplicate, and duplicate values were
averaged to obtain one mean value for each sample. A third sample, spiked with the same vitamer,
was analyzed for each sample tested to provide quality control data. The mean percentage recovery
and mean relative percentage difference were calculated for each vitamer. At the time of writing,
the expected limit of detection, post validation study, is expected to be 0.05
µ
g/100 g (N. Strobel,
email communication, 10 October 2017).
The mean recovery percentage across all samples for vitamin D
2
, vitamin D
3
, 25(OH)D
2
,
and 25(OH)D
3
was 96%, 98%, 101%, and 94%, respectively. Across all samples, the mean relative
percentage difference between duplicate samples for vitamin D
2
, vitamin D
3
, 25(OH)D
2
, and 25(OH)D
3
was 71%, 15%, 50%, and 56%, respectively.
3. Results
Of the 13 samples tested, three contained quantifiable vitamin D metabolites. Vitamin D
2
was
found in dried lemon myrtle leaves (0.24
µ
g/100 g) and the dried leaves and berries of Tasmanian
mountain pepper (0.67 and 0.05
µ
g/100 g, respectively). In addition, three samples contained detectable
vitamin D metabolites at levels below the limit of quantitation. Approximate levels are provided for
indicative purposes only. Vitamin D
2
was detected in raw wattle seed (
≈
0.03
µ
g/100 g) and fresh
lemon myrtle leaves (
≈
0.03
µ
g/100 g). Vitamin D
3
was detected in fresh kombu (
≈
0.01
µ
g/100 g).
There were no detectable vitamin D metabolites in the other samples (Table 2).
Table 2.
New data on the content (dry weight) of vitamin D
2
, vitamin D
3
, 25-hydroxyvitamin D
2
,
and 25-hydroxyvitamin D3in Australian native food plants and edible seaweed.
Common name
(Botanical name)Food Type Part Tested Vitamin D2
(µg/100 g)
Vitamin D3
(µg/100g)
25(OH)D D2
(µg/100 g)
25(OH)D D3
(µg/100 g)
Wattleseed Plant Leaf <0.05 <0.05 <0.05 <0.05
(Acacia victoriae) Raw seed 0.03 * <0.05 <0.05 <0.05
Roasted, milled seed <0.05 <0.05 <0.05 <0.05
Tasmanian mountain
pepper Plant Fresh leaf <0.05 <0.05 <0.05 <0.05
(Tasmannia lanceolata) Dried leaf 0.67 <0.05 <0.05 <0.05
Fresh berries <0.05 <0.05 <0.05 <0.05
Dried berries 0.05 <0.05 <0.05 <0.05
Lemon myrtle Plant Fresh leaf 0.03* <0.05 <0.05 <0.05
(Backhousia citriodora) Dried Leaf 0.24 <0.05 <0.05 <0.05
Wakame Algae Fresh upper leaf and
central stem <0.05 <0.05 <0.05 <0.05
(Undaria pinnatifida)Dried upper leaf and
central stem <0.05 <0.05 <0.05 <0.05
Kombu Algae Fresh leaf <0.05 0.01* <0.05 <0.05
(Lessonia corrugata) Dried leaf <0.05 <0.05 <0.05 <0.05
* This result is below the limit of quantitation and is provided for indicative purposes only.
4. Discussion
We detected low levels of vitamin D
2
in raw wattleseed, dried leaves and fruit of Tasmanian
mountain pepper, and fresh and dried lemon myrtle leaves. Although fungal infection was not
tested for in our study, the vitamin D
2
content found in the plants may have been due to fungal
contamination [
42
]. Vitamin D
2
is considered a marker for fungal contamination in some crops, such as
ryegrass (Lolium perenne L.) and hops (Humulus lupulus L.) [
43
,
44
]. Vitamin D
3
and, in some cases,
25(OH)D
3
have previously been detected in the leaves of tomato [
24
–
26
], waxy leaf nightshade [
23
,
25
],

Nutrients 2018,10, 876 6 of 9
potato [
24
], day blooming jasmine [
27
], zucchini [
24
], and alfafa [
28
]; however, we did not detect these
metabolites in our samples of native Australian plants and detected only very low levels in seaweed.
Recently, the fruit of the rimu tree (Dacrydium cupressinum), a podocarp native to New Zealand,
was found to contain substantial amounts of both vitamin D
2
and D
3
[
29
]. Measured by isocratic
reversed-phase HPLC, the average vitamin D
2
and D
3
contents of rimu berries were reported as
70
µ
g/100g and 11.5
µ
g/100g, respectively, although no quality control data were provided. In another
study, the precursors of vitamin D
2
and D
3
(ergosterol and 7-dehydrocholesterol, respectively) were
detected in plant oils [
45
]. Other studies have found that the vitamin D
3
and 25-hydroxyvitamin D
3
content of leaves and cell cultures of certain plants increases after ultraviolet (UV) irradiation [
25
,
27
,
46
].
For example, exposure to UV radiation increased the vitamin D
3
content of tomato (Solanum
lycopersicum L.) leaves by almost 60 times to 100 ng/g, compared with 1.7 ng/g in non-UV-exposed
leaves [
25
]. Future investigations into other potential plant sources of vitamin D and the effect of
exposure to UV radiation appear warranted by the finding that consumption of plant oils, particularly
UV B-irradiated wheat germ oil, increased 25(OH)D plasma concentration in mice [45].
Sargassum, an edible macroalgae [
38
], was first discovered to have anti-rachitic properties when
the lipid fractions of the algae were fed to rats with induced rickets [
47
]. Since then, vitamin D
2
and vitamin D
3
have been found in microalgae and macroalgae using high-performance liquid
chromatography (HPLC) in much larger quantities than were found in our study [
30
–
32
]. Vitamin D
metabolites were largely undetected in macroalgae in our study, with the exception of vitamin D
3
in
kombu (Lessonia corrugata) measured at 0.01
µ
g/100 g. In other studies, Japanese wireweed (Sargassum
muticum) was found to contain 90
µ
g/100 g, while vitamin D
2
and D
3
contents in microalgae ranged
from not detected to 3900
µ
g/100 g and 2.2–271.7
µ
g/100 g, respectively [
30
–
32
]. Ergosterol and
7-dehydrocholesterol have also been found in microalgae [
31
]. As with plants, it has been suggested
that the significant vitamin D content of algae is dependent on exposure to UV radiation [
30
,
48
].
The role of UV radiation has been implicated by the finding that microalgae harvested in summer
have a higher vitamin D
2
and D
3
content than those harvested in autumn and winter [
30
]. Although
we detected only low levels of vitamin D
3
in kombu and no vitamin D
2
or vitamin D
3
in wakame,
the algae tested in our study were harvested in the winter months and were not sundried or exposed
to UV radiation after harvest.
Plant and algal matrices present challenges for the quantification of vitamin D
2
, vitamin D
3
,
25(OH)D
2
, and 25(OH)D
3
, due in part to the presence of interfering compounds such as chlorophyll
and lipophilic pigments [
48
]. Therefore, any method used must be highly sensitive and selective [
48
].
When compared to single mass spectrometry (MS), LC-QQQ has higher sensitivity and selectivity
when applied to the detection of pesticides in water and soil samples [
49
]. To our knowledge, this
method has not been used previously to detect vitamin D metabolites in complex plant and algal
matrices and is a major strength of our study due to the low detection limits of the instrumentation.
The mean recovery from all spiked samples in our study was high, indicating that LC-QQQ is highly
accurate in detecting low levels of vitamin D in plant and algal matrices. All samples were measured
in duplicate, and where possible, we tested the edible portion in addition to the leaf material. However,
we tested only a few species of Australian native food plants and Australian-grown edible seaweed.
Although regional and seasonal variation have been shown to influence the vitamin D content of
plants [
43
,
48
], we analyzed only single samples sourced from single locations and during months of
relatively low UV radiation levels.
In conclusion, this study has demonstrated the high sensitivity of LC-QQQ methodology, which
will be used in future studies in search of natural dietary sources of vitamin D. Our results show that
the selected Australian native plants and algae have very low levels of vitamin D. However, given that
the vitamin D precursors, ergosterol and 7-dehydrocholesterol, have previously been found in both
plants and algae, testing the effect of exposure to UV radiation on the vitamin D content of Australian
native food plants and Australian-grown edible seaweed is warranted. Also, a larger sample size across
a greater number of plant species and habitats will increase the likelihood of identifying nutritionally

Nutrients 2018,10, 876 7 of 9
relevant amounts of vitamin D. An important factor in future studies will be taking the seasonal effect
of vitamin D into account in natural ecosystems, because this will relate back to the levels of solar UV
radiation for potentially increasing vitamin D levels.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/10/7/876/s1,
Table S1: Description of plant and algae samples included in the current study.
Author Contributions:
J.F.B., R.M.L., J.L.S., and L.J.B. conceived of and designed the study; L.J.H. sourced the
samples and wrote the paper; N.S. conducted laboratory analyses; L.J.B., J.L.S., J.F.B., R.M.L., E.D., and N.S.
provided critical revision of the manuscript for important intellectual content. All authors are in agreement, and
this material has not been published elsewhere.
Funding:
This work was supported by the School of Public Health, Curtin University. RML is supported by a
National Health and Medical Research Council of Australia Senior Research Fellowship.
Acknowledgments:
The authors would like to thank the following growers, producers, and wild harvesters for
supplying samples for testing: Australian Rainforest Products, Diemen Pepper Supplies, Kai Ho/Ocean Treasure,
and Mootooroo.
Conflicts of Interest: The authors declare no conflict of interest.
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