Food Chemistry 138 (2013) 1042–1047

Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Vitamin D and 25-hydroxyvitamin D determination in meats by LC–IT-MS

Norbert Strobel a,⇑, Saman Buddhadasa a, Paul Adorno a, Katherine Stockham a, Heather Greenfield b
a
National Measurement Institute (NMI), 1/153 Bertie Street, Port Melbourne, Victoria 3207, Australia
b
University of Sydney, New South Wales 2006, Australia

a r t i c l e i n f o a b s t r a c t

Article history: This paper reports a method for the rapid, sensitive and simultaneous analysis of vitamin D (Vit D) and
Received 12 December 2011 25-hydroxyvitamin D (25OH-Vit D) in meats. Samples were saponified and underwent solid phase
Received in revised form 1 August 2012 extraction with analysis by normal phase liquid chromatography (LC) with ion trap mass spectroscopy
Accepted 18 August 2012
(IT-MS), using positive polarity atmospheric pressure chemical ionisation (APCI). Limits of detection
Available online 30 August 2012
(LOD) and quantification (LOQ) for Vit D and 25OH-Vit D were 0.03 and 0.05 lg/100 g respectively. Deu-
terium labelled Vit D and 25OH-Vit D internal standards were added as surrogates prior to saponification,
Keywords:
correcting for extraction inefficiencies and potential MS matrix enhancement or suppression effects.
Quantification
Vitamin D3 (cholecalciferol)
Recoveries using internal/surrogate standard correction ranged from 80% to 100% for all vitamers. Mea-
Vitamin D2 (ergocalciferol) surement uncertainty ranged from 6% to 15% for all vitamers in this method. This process required only
25-Hydroxyvitamin D3 (25- 7.5 g of sample per extraction and a batch of 28 extractions could be completed in six hours.
hydroxycholcalciferol) Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
25-Hydroxyvitamin D2 (25-
hydroxyergocalciferol)
Meat
Liquid chromatography (LC)
Ion trap (IT)
Mass spectrometry (MS)
Collision induced dissociation,
Tandem mass spectrometry (CID-MS/MS)

1. Introduction
The importance of vitamin D in calcium homeostasis is well-
documented as it promotes calcium absorption and together with
parathyroid hormone (PTH) affects skeletal deposition and mobili-
sation. Vitamin D also plays a central role in phosphate homeosta-
sis and therefore is essential for the proper development and
maintenance of bone (Bell, Demay, & Burnett-Bowie, 2010; Holick,
2004; Truswell, 1990). The two main forms of vitamin D are chole-
calciferol (D3) and ergocalciferol (D2). These compounds are
metabolised in the liver to their respective 25-hydroxyvitamin D
forms which are the main forms circulating in blood. 25-Hydrox-
yvitamin D is further metabolised in the kidneys to the active form
1,25-dihydroxyvitamin D.
In humans vitamin D3 is produced in the skin on exposure to
UVB radiation, while vitamin D3, vitamin D2, and their 25-hydroxy
forms can also be obtained from dietary sources. There is contin-
uing discussion regarding the relative bioactivity of the various
forms (Jakobsen, 2007), but the bioactivity conventionally allo-
cated to 25-hydroxyvitamin D3 is five times that of vitamin D3
⇑ Corresponding author. Tel.: +61 3 96444888; fax: +61 3 96444999.
E-mail address: norbert.strobel@measurement.gov.au (N. Strobel).
(Tanaka, Frank, & DeLuca, 1973), therefore, complete dietary stud-
ies of vitamin D in foods require the measurement of all four forms.
While few foods are rich in vitamin D, foods such as meats, which
may be heavily consumed, need to be analysed for D vitamers.
Jakobsen, Clausen, Leth, and Ovesen (2004) described a method
for the determination of vitamin D3 and 25-hydroxyvitamin D3 in
meat (pork), in order to contribute new information for the food
composition tables. This method was closely followed at the Na-
tional Measurement Institute (NMI) laboratories, with the modifi-
cation of using 25-hydroxyvitamin D2 as the internal standard for
25-hydroxyvitamin D3. It was found to give satisfactory results;
however the low sample throughput and lengthy extraction time
meant that the method was not viable for a commercial laboratory.
Heudi, Trisconi, and Blake (2004) quantified vitamins A, E and
D3 in fortified infant formulae by liquid chromatography–mass
spectrometry (LC–MS). Replacing the cumbersome and time-con-
suming liquid–liquid extraction by separating funnel (De Leenheer,
Lambert, & De Ruyter, 1985) with Chromabond XRT (diatomaceous
earth) solid phase extraction (SPE) was more rapid and convenient,
and there were also considerable reductions in solvent usage per
extraction. As diatomaceous earth is a natural product there may
be subtle variations in performance between individual cartridges.
Therefore it is essential that an internal standard with a chemical

0308-8146/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.08.041
 N. Strobel et al. / Food Chemistry 138 (2013) 1042–1047
character identical (or at least very similar) to that of the analyte of
interest be added to the sample prior to saponification to correct
for any extraction inefficiencies. The paper by Gören, Bilsel, and
Bilsel (2007), described the analysis of 25-hydroxyvitamin D2 and
25-hydroxyvitamin D3 in human serum by LC–MS/MS. While the
matrix analysed was quite removed from food extracts, this paper
demonstrated the suitability of LC–MS/MS for the analysis of 25-
hydroxyvitamin D.
Since the 2004 publications there have been advancements
in both extraction techniques and instrumentation. The aim of
this study was the development of a commercially viable
analytical method for the simultaneous and routine analysis of
vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvi-
tamin D3 with reduced time and complexity of extraction, to-
gether with increased selectivity and specificity of instrumental
detection.
2. Materials and methods
2.1. Reagents
Vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydrox-
yvitamin D3 were purchased from Sigma–Aldrich Co., Sydney,
Australia. The deuterated standards, vitamin D3 [2H3], 25-hydrox-
yvitamin D3 [2H3], vitamin D2 [2H3] and 25-hydroxyvitamin D2
[2H3], were purchased from IsoSciences Co., King of Prussia, Phila-
delphia (PA), USA. All other chemicals and reagents were AR or
HPLC grade. Chromabond XTR (diatomaceous earth) was pur-
chased from Micro Analytix Pty. Ltd., Taren Point, Australia, and
the SPE cartridges were assembled in the laboratory
2.2. Preparation of standards
All standard solutions were stored in labelled amber screw-
topped glass bottles/vials at less than 10 °C. Stock solutions
were prepared in 2-propanol. They included stock vitamin D
standards at 500 lg/mL and 25-hydroxyvitamin D standards
and deuterated vitamin standards at 100 lg/mL. From the stock
standards intermediate standards were prepared in both ethanol
and heptane each at a concentration of 10 lg/mL. The individual
10 lg/mL ethanol solutions could be periodically standardised
knowing the relevant molar extinction coefficient by measuring
the ultraviolet absorption at 265 nm. The vitamin D and 25-
hydroxyvitamin D mixed spike-recovery standard (for sample
spiking) was prepared at 200 ng/mL in ethanol and mixed cali-
bration standard was prepared at 100 ng/mL in heptane. The
25-hydroxyvitamin D3 [2H3] 500 ng/mL and vitamin D3 [2H3]
100 ng/mL mixed deuterated internal standard was prepared in
ethanol for sample spiking and in heptane for calibration stan-
dard preparation.
2.3. Apparatus
The analyses were done using an Agilent (Varian) 500 MS IT
Mass Spectrometer system comprising of two nano pumps (Varian
212-LC), a cooled (5 °C) autosampler (CTC Analytics, PAL System), a
column heater (Varian ProStar), a Prevail Silica LC column
5 lm  250 mm  4.6 mm ID LC (Alltech) and an ion trap
mass spectrometer. The 500 MS IT Mass Spectrometer was
equipped with an atmospheric pressure chemical ionisation (APCI)
chamber.
The 500 MS is an ion trap based system which operates by gen-
erating gas phase ions from solution at atmospheric pressure, in
this instance, using APCI. Both electrospray ionisation (ESI) and
APCI are described as soft ionisation techniques because they pro-
1043
duce minimal fragmentation. Typically singly charged ions are pro-
duced and these pseudo-molecular ions are ideal precursors for
MS/MS. The ions are transported through two vacuum interfaces
via a metal capillary tube, a skimmer cone and ion optics to the
mass analyser. The ion optics consists of a hexapole ion guide
and a series of focussing lenses. The mass analyser consists of
two components an ion trap followed by an ion detector. Ion stor-
age, fragmentation and mass analysis occur in the three dimen-
sional trap. The detector consists of a conversion diode and an
electron multiplier (Varian Inc., 2006).
Other general laboratory equipment included four- and five-
figured analytical balances (Sartorius), a shaker-water-bath (Ratek),
a rotary evaporator (Büchi), a gas manifold (made in-house), a
refrigerated centrifuge (Spintron) and a mini–spin centrifuge
(Eppendorf).
3. Procedure
Each sample for the analysis of vitamin D and 25-hydroxyvita-
min D was spiked with the mixed internal standard prepared in
ethanol, to correct for the relative extraction inefficiencies. There
was a minimum of one duplicate analysis per batch and one recov-
ery analysis per batch where the selected sample was spiked with
the mixed standard at a concentration of approximately 0.5 lg/
100 g of sample matrix. If the sample was known to have a high
concentration of vitamin D or 25-hydroxyvitamin D then the spike
was at concentration of the order of the expected sample level.
Vitamin D and 25-hydroxyvitamin D are sensitive to UV radia-
tion and oxidising reagents. Therefore this analysis was conducted
under subdued lighting, with selected wavelength ‘yellow’ fluores-
cent lighting used in the sample extraction areas. A pure source of
inert gas (high purity nitrogen) was used to flush the headspace of
the saponification mixture and at any stage where the subsequent
extracts needed to be reduced to dryness. This was critically
important with respect to the 25-hydroxyvitamin D where instru-
ment response had been demonstrated to diminish by as much as
70% when the nitrogen gas source was adulterated by a few parts
per million of air (this adulteration did not result in any significant
loss of instrumental response for vitamin D).
3.1. Saponification and extraction
Fifty millilitre of screw topped falconÒ tubes were used as the
reaction vessels. Into each tube was weighed 7.5 g of lean meat
mixed with 0.5 g of the antioxidant sodium ascorbate. Where the
approximate total fat for the sample was known this weight was
adjusted to ensure that the fat analysed did not exceed 1.7 g.
400 lL of the mixed deuterated internal standard, 10 mL of deion-
ised water, 25 mL of ethanol and 7.5 g of potassium hydroxide
were then added sequentially. The headspace of the reaction vessel
was immediately flushed with nitrogen gas prior to securing the
cap, hand shaken to ensure that all the materials were free-flowing
and then placed laterally in a shaker bath at 25 °C for 15 h
(overnight).
The entire saponified extract was quantitatively transferred
onto a 50 mL Chromabond XTR cartridge and allowed 15 min to
absorb before being eluted with six aliquots of 30 mL of petroleum
ether (with 0.02 g of BHT per litre). The eluent was collected into a
250 mL round bottomed flask allowing 30 min after the addition of
the last aliquot of petroleum ether.
The eluent was reduced to between 5 and 10 mL by rotary evap-
oration then blown to dryness under nitrogen. The residues were
quantitatively transferred to a 15 mL falconÒ tube by the addition
of 5 mL n-heptane with vortex mixing followed by a 2 mL n-hep-
tane wash. The falconÒ tubes were centrifuged to pelletise any
1044 N. Strobel et al. / Food Chemistry 138 (2013) 1042–1047

non soluble components and the extract decanted into a clean
15 mL falconÒ tube. The pelletised solid was analysed for chicken
meat samples and found to contain 70–80% fatty acids, 3.3% cho-
lesterol and BHT (added with the petroleum ether).
The extract was evaporated to dryness under nitrogen and
reconstituted in 400 lL of n-heptane. It was not necessary to evap-
orate for long periods to total dryness, as the internal standards
would compensate for any slight volume variations between
samples. The concentrated extract was then filtered by micro
centrifugation through a 0.2 lm single-use filter and transferred
to a HPLC vial. 50 lL was injected onto the LC–MS.
3.2. Calibration and check standards
Additions of 50, 100, 150, 200, 250, 300, 350, and 400 lL of the
mixed calibration standard in heptane were evaporated to dryness
under nitrogen, reconstituted with 400 lL of the mixed internal
standard (in heptane), then transferred to HPLC vials. These

Fig. 1. Chromatographic and spectral data from a single analytical analysis of a calibration standard. Included are the ‘Total Ion Count’ and ‘Selected Ion’ chromatograms and
the CID-MS/MS spectra for vitamin D3, vitamin D2, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3.
 N. Strobel et al. / Food Chemistry 138 (2013) 1042–1047 1045

solutions were analysed by the LC–MS to establish calibration
curves ranging from 12.5 to 100 ng/mL for each of vitamin D3, vita-
min D2, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2. Typi-
cally, the nine point calibrations (included origin) had quadratic
curve fits with coefficients of determination (r2) greater than 0.998.
A check standard with a component concentration of 50 ng/mL
was used to monitor the stability of the mixed spike recovery stan-
dard and the mixed deuterated internal standard (in ethanol) with
respect to the instrument calibration. Aliquots of 100 lL of the
mixed spike recovery standard and 400 lL of the mixed deuterated
internal standard (in ethanol) were mixed, evaporated to dryness
under nitrogen and reconstituted in 400 lL of n-heptane.
MS/MS or tandem mass spectrometry is the isolation of a pre-
cursor ion formed during ionisation followed by collision induced
dissociation (CID) of this precursor ion by applying energy in the
presence of an inert gas. The resultant product ions can then be
trapped and scanned. Multiple reaction monitoring (MRM) can
be performed for multiple precursor ions, where analytes and/or
deuterium-labelled internal standards co-elute.
Fig. 1 illustrates the CID-MS/MS spectra of a calibration stan-
dard. For vitamin D3 the 385.4 Da precursor ion is dissociated into
product ions measured over an instrument set range of 118–
395 Da. Similarly for vitamin D2 the 397.4 Da precursor ion disso-
ciation provides product ion data over a set range of 121–407 Da;
for 25-hydroxyvitamin D2 the 395.4 Da precursor ion dissociation
provides product ion data over a set range of 120–405 Da; and for
25-hydroxyvitamin D3 the 383.4 Da precursor ion dissociation
provides product ion data over a set range of 118–393 Da.
3.3. Instrument settings
A normal phase, liquid chromatography gradient profile (Table
1) on a PrevailÒ Silica 5 lm  250 mm  4.6 mm column (Alltech)
was used to achieve the required chromatographic separation. The
LC column was mounted in a column oven set at 35 °C.
A five component valve just prior to the APCI chamber diverted
the majority of the chromatographic effluent to waste. Only com-
pounds at or about the expected retention of vitamin D and 25-
hydroxyvitamin D entered the APCI chamber and were analysed.
The MS/MS scan time was set to 0.5 s per scan, the spray chamber,
drying gas, and vaporising gas temperatures were 50, 350 and
350 °C, respectively and the nebuliser, drying gas and vaporiser
Table 1
Gradient profile with mobile phase ‘A’ 10% (v/v) isopropyl alcohol in n-heptane and
mobile phase ‘B’ n-heptane.
gas pressures were 50, 25 and 30 psi respectively. The CID-MS/
MS settings for the ion trap are detailed in Table 2.
4. Results & discussion
4.1. Method validation
Chicken meat was selected as the initial matrix for method
validation. Vitamin D3 and 25-hydroxyvitamin D3 were assessed,
vitamin D2 and 25-hydroxyvitamin D2 were spiked to determine
extraction efficiencies and deuterated vitamin D3 and deuterated
25-hydroxyvitamin D3 were the internal standards, added after
the extraction, just prior to instrumental analysis. The selection
of this matrix was influenced by the expectation that neither vita-
min D2 nor 25-hydroxyvitamin D2 were present (since vitamin D2
is not utilised by poultry (National Research Council, 1994)) thus
making it possible to assess the respective extraction efficiencies
independently of the internal standards.
In general the extraction efficiency for vitamin D2 (average 95%)
was higher than that of the 25-hydroxyvitamin D2 (average 49%).
This was expected as 25-hydroxyvitamin D is more polar than vita-
min D; therefore its extraction into an essentially non-polar sol-
vent (petroleum ether) from an aqueous saponification mixture
would be comparatively diminished. A second observation was
that the recoveries were variable and if left uncorrected would
be a major source of uncertainty. This was also expected since
the solid phase extraction medium, diatomaceous earth, varies in
its particulate diameter and absorptive capacity leading to a varia-
tion in extraction efficiencies between cartridges (Table 3).
Recoveries for vitamin D3 and 25-hydroxyvitamin D3 corrected
for their respective extraction efficiencies, as determined from
vitamin D2 and 25-hydroxyvitamin D2, were within an acceptable
range of 100 ± 20%. This supported literature sources where vita-
min D2 had been used as an internal standard for vitamin D3 and
25-hydroxyvitamin D2 as an internal standard for 25-hydroxyvita-
min D3 (Indyk & Woollard, 1985; Mattila, Piironen, Uusi-Rauva, &
Koivistoinen, 1995). As the extraction process was validated with
a chicken matrix further validation studies had the addition of
the deuterated vitamin D3 and deuterated 25-hydroxyvitamin D3
internal standards added prior to extraction. The deuterated inter-
nal standards automatically corrected for extraction inefficiencies,
instrument injection variability and the possibility of any matrix
enhancement or suppression effects. Thus vitamin D2, D3 and their
respective 25-hydroxylated metabolites could be analysed in a sin-
gle analytical run.

# Time (min:sec) %A %B Flow (lL/min) Comments

4.2. Uncertainty determination
1 0:00 1 99 1000
2 20:00 25 75 1000 Gradient 1
The measurement uncertainty calculation determined the limi-
3 28:00 86 14 1000 Gradient 2 tations of this methodology, allowing results to be compared to
4 28:01 100 0 1000 Column clean up those of existing techniques. The uncertainty calculation included
5 32:00 100 0 1000 a linear regression analysis of the D vitamer calibration curve,
6 32:01 1 99 1000 Column stabilisation
cause and effect diagrams of standards preparation and the analy-
7 35:00 1 99 1000
sis of duplicates and recoveries. If the relative standard deviation

Table 2
The ion trap CID-MS/MS settings for each vitamin analysed. These settings were optimised to maximise product ion response.

Compound Precursor Polarity Capillary RF loading Excitation Quantitation Qualifier product

ion (Da) voltage (Volts) (%) amplitude (Volts) product ion (Da) ions (Da)
Vitamin D2 397.4 +ve 90 93 0.89 379.4 271.3, 309.3
Vitamin D3 385.4 +ve 45 97 1.51 367.3 259.3, 255.3, 287.3
Deuterated vitamin D3 388.4 +ve 60 84 0.85 370.4 259.3, 371.3
25-OH vitamin D2 395.4 +ve 72 92 1.40 377.4 269.2, 378.4
25-OH vitamin D3 383.4 +ve 79 89 0.72 365.4 257.2, 271.2, 255.4
Deuterated 25-OH vitamin D3 386.4 +ve 75 84 0.76 368.2 257.4, 232.2
1046 N. Strobel et al. / Food Chemistry 138 (2013) 1042–1047

Table 3
Validation study summary on chicken breast mince. D2 vitamers were added as surrogates directly to the mince prior to extraction and deuterated D3 vitamers were added as
internal standards to the final extract prior to analysis by LC–MS. Note that the ‘raw’ recoveries for vitamin D2 and 25-hydroxyvitamin D2 were 95% and 49%, respectively and that
the corrected recoveries for vitamin D3 and 25-hydroxyvitamin D3 were approximately 80–100%.

n Result (lg/100 g) Spike (lg/100 g) Recovery (%) Standard deviation

Unspiked
Vitamin D2 9 – 0.586 95 8%
25-Hydroxyvitamin D2 – 0.492 49 4%
Vitamin D3 (corrected for vitamin D2 recovery) 0.04 – – 0.01
25-Hydroxyvitamin D3 (corrected for 25-hydroxyvitamin D2 recovery) 0.14 – – 0.01
Spike: 0.1 lg/100 g
Vitamin D3 (corrected for vitamin D2 recovery) 9 – 0.10 92 20%
25-Hydroxyvitamin D3 (corrected for 25-hydroxyvitamin D2 recovery) – 0.11 90 34%
Spike: 0.4 lg/100 g
Vitamin D3 (corrected for vitamin D2 recovery) 9 – 0.40 102 11%
25-Hydroxyvitamin D3 (corrected for 25-hydroxyvitamin D2 recovery) – 0.43 82 11%

(RSD) for recovery determination was set at unity, then the RSD for
calibration was 0.04, the RSD for standards preparation was 0.02
and the RSD for duplicate analysis was 0.01. It was evident that
the recovery determination was by far the major source of uncer-
tainty, as expected, knowing the susceptibility of the 25-hydrox-
yvitamin D to oxidative degradation and the performance
variation between the SPE cartridges. This highlighted the neces-
sity of the internal standards to compensate for variable extraction
efficiencies. The expanded uncertainty was determined with a cov-
erage factor of two to give a 95% confidence interval. For each D
vitamer this uncertainty was determined to vary inversely with
the concentration. Samples with a determined concentration
comparable to the mid calibration range (0.25 lg/100 g) had an
uncertainty of ±11% for vitamin D2, ±6% for vitamin D3, ±12% for
25-hydroxyvitamin D2 and ±15% for 25-hydroxyvitamin D3.
4.3. Standard reference materials
All Standard reference materials (SRMs) sourced were milk
powder matrices with only one D vitamer, vitamin D3, fortified at
levels much higher than occurring naturally in meats (SRMs from
more suitable matrices are not yet available). To maintain an
instrument calibration identical to that when analysing lean meats
and to keep modifications to both the extraction and instrumental
analysis to a minimum, it was necessary to make the following
alterations to sample handling and preparation: For moderately
fortified samples 5–10 mg/100 g, a 0.5 g sample was saponified
and the whole saponification liquor extracted and analysed with-
out further alteration to the methodology. For the more heavily
fortified samples 20 to 30 mg/100 g, where a sample analysis
weight of less than 0.5 g may be considered non representative, a
0.5 g sample was saponified and the resulting liquor made to a vol-
ume of 50 mL. A 10 mL aliquot of the liquor was taken and diluted
to 50 mL with the addition of 40 mL of blank saponification mix-
ture (water, and ethanolic potassium hydroxide). The diluted li-
quor was then extracted and analysed without further alteration
to the methodology.
Despite this increased dependence upon sample homogeneity,
the results for the NIST SRM 1849 (Table 4) were within the certi-
fied value range.

Table 4
Results for NIST SRM 1849 (National Institute of Standards and Technology, 2010). The certified value for vitamin D3 is 25.1 ± 2.7 lg/kg, i.e., the true value is within the range of
22.4–27.8 lg/kg.

Sample Quantitation ion (Da) Result (lg/kg) Average (lg/kg) Uncertainty (lg/kg) Range (lg/kg)
NIST SRM 1849 Extraction 1 367 27.66 27.2 ±1.6 25.6–28.8
259 27.77
Extraction 2 367 26.73
259 26.67

(Note: The result for NIST SRM 1849 was within the certified concentration range and was thus deemed satisfactory.)

Table 5
Vitamin D results from the LC–MS analyses of four lean meat matrices (limit of reporting <0.05 lg/100 g).

Vitamin D2 (lg/100 g) 25-Hydroxyvitamin D2 (lg/100 g) Vitamin D3 (lg/100 g) 25-Hydroxyvitamin D3 (lg/100 g)

Beef silverside <0.05 <0.05 0.06 0.10
Standard deviation – – 0.02 0.02
Uncertainty – – ±0.01 ±0.03
Pork loin <0.05 <0.05 0.18 0.17
Standard deviation – – 0.03 0.07
Uncertainty – – ±0.02 ±0.05
Lamb chump chop <0.05 <0.05 <0.05 0.05
Standard deviation – – – 0.02
Uncertainty – – – ±0.02
Skinless chicken breast <0.05 <0.05 0.12 0.21
Standard deviation – – 0.05 0.04
Uncertainty – – ±0.03 ±0.04
 N. Strobel et al. / Food Chemistry 138 (2013) 1042–1047
4.4. Vitamin D in lean meats
Silverside beef, pork loin, lamb chump chops and skinless chick-
en breast were purchased from a local butcher by a representative
of the University of Sydney. These meats were kept at 4 °C until
prepared. Each meat was prepared as purchased with the excep-
tion of the lamb which had bone and visible fat removed. The
meats were homogenised using a domestic blender (Thermomix),
freighted and stored at 20 °C until analysed.
Three samples were analysed for each of the four supplied
matrices – beef, pork, lamb and chicken. Duplicate analyses were
performed on each sample. These results were determined by
CID-MS/MS using the two most prominent product ions for each
analyte. Quantitation was done by alternately assigning one ion
for quantitation and the other ion as a qualifier. Therefore two re-
sults could be obtained for each analyte (one for each ion) which
were then averaged. Where the background made integration of
one ion unsatisfactory, the integration of the second ion was taken
as the result. Care was taken when interpreting results <0.05 lg/
100 g as the method uncertainty would approach 50%.
The results (Table 5) suggest higher levels of vitamin D3 and 25-
hydroxyvitamin D3 were detected in pork and chicken rather than
lamb and beef. This is not totally unexpected as beef and mutton
are generally ‘free range’; while pork and poultry are fed with for-
tified stock feeds.
Of the samples analysed the ‘lamb chump chop’ was the least
uniform. Despite efforts to create a homogeneous matrix, varia-
tions in the proportions of flesh to fat were observed between sam-
ples (although the fat content per sample was not determined).
Chromatographically the lamb samples exhibited a comparatively
poor base line increasing the difficulty of integration and thus
quantitation.
5. Conclusions
A quantitative method has been developed for the simultaneous
determination of vitamin D and 25-hydroxyvitamin D in meat. De-
signed to quantify at the low levels anticipated in these matrices,
the limit of quantification was 0.05 lg/100 g. This method is com-
mercially viable for a food analytical testing laboratory as four
batches of up to twenty-eight extractions can be completed per
week.
Compared to vitamin D, 25-hydroxy vitamin D has a far greater
susceptibility to oxidation. Its quantification is facilitated by a
methodology where there are no excessive delays between sample
extraction and instrumental analysis.
The use of Chromabond XTR (diatomaceous earth) SPE rather
than the more traditional liquid/liquid extractions has the advanta-
ges of reduced extraction time and reduced solvent usage. How-
ever, as there may be variation in the extraction efficiency
between Chromabond XTR cartridges, it is essential that deuter-
ated internal standards be added while preparing the saponifica-
tion. The deuterium labelled vitamin D3 and 25-hydroxyvitamin
D3 internal/surrogate standards corrects for any extraction
1047
inefficiencies as well as negating any potential MS matrix enhance-
ment or suppression effects.
Gradient program normal phase liquid chromatography pro-
vided adequate control over the separation of vitamin D and 25-
hydroxyvitamin D from a multitude of co extracted compounds.
The majority of these co extracted compounds could be diverted
away from the detector directly to waste, protecting the detection
system from unnecessary contamination and premature loss of
sensitivity. LC–IT-MS/MS is a highly selective technique which dis-
tinguished the vitamin D and 25-hydroxymitamin D from the
remaining background.
Acknowledgements
The authors acknowledge Professor David Fraser and Mr. Jerry
Liu of the University of Sydney; Associate Professor Jayashree Arcot
from the University of New South Wales; Mr. Jason Chau Lu,
Mr. George Dabos, and Mr. Tim Stobaus of the National Measure-
ment Institute, Australia for their advice and support during this
research project.
References
Bell, T. D., Demay, M. B., & Burnett-Bowie, S. A. M. (2010). The biology and pathology
of vitamin D control in bone. Journal of Cellular Biochemistry, 111(1), 7–13.
De Leenheer, A. P., Lambert, W. E., & De Ruyter, M. G. M. (Eds.). (1985).
Chromatographic analysis of the vitamins. New York: Marcel Dekker Inc..
Gören, A. C., Bilsel, G., & Bilsel, M. (2007). Rapid and simultaneous determination of
25-OH-vitamin D2 and D3 in human serum by LC/MS/MS: Validation and
uncertainty assessment. Journal of Chemical Metrology, 1, 1–9.
Heudi, O., Trisconi, M.-J., & Blake, C.-J. (2004). Simultaneous quantification of
Vitamins A, D3, and E in fortified infant formulae by liquid chromatography-
mass spectrometry. Journal of Chromatography A, 1022, 115–123.
Holick, M. F. (2004). Sunlight and vitamin D for bone health and prevention of
autoimmune diseases, cancers and cardiovascular disease. The American Journal
of Clinical Nutrition, 80(Suppl. 6), 1678S–1688S.
Indyk, H., & Woollard, D. C. (1985). The determination of vitamin D in supplemented
milk powders by HPLC. Incorporation of internal standard. New Zealand Journal
of Dairy Science and Technology, 20, 19–28.
Jakobsen, J. (2007). Bioavailability and bioactivity of vitamin D3 active compounds –
Which potency should be used for 25-hydroxyvitamin D3? International
Congress Series, 1297, 133–142.
Jakobsen, J., Clausen, I., Leth, T., & Ovesen, L. (2004). A new method for the
determination of vitamin D3 and 25-hydroxyvitamin D3 in meat. Journal of Food
Composition and Analysis, 17, 777–787.
Mattila, H., Piironen, V. I., Uusi-Rauva, E. J., & Koivistoinen, P. E. (1995). Contents of
cholecalciferol ergocalciferol and their 25-hydroxylated metabolites in milk
products and raw meat and liver as determined by HPLC. Journal of Agricultural
and Food Chemistry, 43, 2394–2399.
National Institute of Standards & Technology. (2010). Certificate of Analysis
Standard Reference MaterialÒ 1849 Infant/Adult Nutritional Formula.
<https://www-s.nist.gov/srmors/certificates/1849.pdf?CFID=1308830&CFTOKEN=
5570359d0e9bc5ce-F15A963C-A1B7-DE8B-4A80B512A3CE987C&jsessionid=
f03027c915aff00cec9e5e1034753f1d135c>.
National Research Council (1994). Nutrient Requirements of Poultry Ninth Revised
Edition. Washington D.C., National Academy Press.
Tanaka, Y., Frank, H., & DeLuca, H. F. (1973). Biological activity of 1,25-
dihydroxyvitamin D3 in the rat. Endocrinology, 92, 417–422.
Truswell, A. S. (Ed.). (1990). Recommended nutrient intakes: Australian papers
(pp. 149–158). Australian Professional Publications.
Varian Inc. (2006). 500-MS IT Mass Spectrometer – Hardware Operation Manual.
03-954076-00: Rev. 1: 38–52.