Article

Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX pubs.acs.org/JAFC

Chemical Composition of Commercial Cow’s Milk

Aidin Foroutan,†,‡ An Chi Guo,† Rosa Vazquez-Fresno,† Matthias Lipfert,† Lun Zhang,† Jiamin Zheng,†
Hasan Badran,# Zachary Budinski,# Rupasri Mandal,† Burim N. Ametaj,‡ and David S. Wishart*,†,#
†
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
‡
Department of Agricultural, Food and Nutritional Science, Edmonton, Alberta, Canada T6G 2P5
#
Department of Computing Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E8
Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 04/17/19. For personal use only.

ABSTRACT: Bovine milk is a nutritionally rich, chemically complex biofluid consisting of hundreds of different components.
While the chemical composition of cow’s milk has been studied for decades, much of this information is fragmentary and very
dated. In an effort to consolidate and update this information, we have applied modern, quantitative metabolomics techniques
along with computer-aided literature mining to obtain the most comprehensive and up-to-date characterization of the chemical
constituents in commercial cow’s milk. Using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography−mass
spectrometry (LC−MS), and inductively coupled plasma−mass spectrometry (ICP−MS), we were able to identify and quantify
296 bovine milk metabolites or metabolite species (corresponding to 1447 unique structures) from a variety of commercial milk
samples. Through our literature analysis, we also found another 676 metabolites or metabolite species (corresponding to 908
unique structures). Detailed information regarding all 2355 of the identified chemicals in bovine milk have been made freely
available through a Web-accessible database called the Milk Composition Database or MCDB (http://www.mcdb.ca/).
KEYWORDS: milk, metabolomics, NMR, LC−MS, ICP−MS, literature review, chemical composition

■
J. Agric. Food Chem.

INTRODUCTION carbohydrates.11 At the macronutrient level, bovine milk is

Milk is often called the “perfect food”. Produced from the
mammary glands of all periparturient female mammals, milk is
rich in key nutrients such as carbohydrates, proteins, fats,
minerals, and vitamins that are dynamically adjusted to meet
the specific developmental needs of growing newborns.1,2 Milk
not only plays a key role in nourishment and hydration, it also
has an essential role in establishing essential gut microflora and
priming the immune system in all newborn mammals.1 While
milk is normally a species-specific biofluid consumed by young
mammals belonging to that species, humans uniquely consume
milk produced by other species and continue to consume milk
into adulthood.2 Global milk production is dominated by five
animal species with 83% of total milk production coming from
cows, followed by buffaloes with 13%, goats 2%, sheep 1%,
camels 0.4%.3
The abilities of humans to both extract milk from domestic
animals and to consume it into adulthood have played a key
role in the evolution of agriculture, the establishment of civil
societies, and the development of many widely used food
products.4 Indeed, the ability to collect and use bovine milk
has been fundamental to the health, growth, migration, and
success of the human species over the past 10 000 years. Even
today, milk is one of the most widely consumed beverages in
the world (811 million tonnes of milk produced in 2017). It
serves as the feedstock for not only liquid milk but also
flavored milk, ice cream, cheese, butter, yogurt, casein powder,
and many other dairy products.5
Given the significant economic and nutritional importance
of milk, and especially cow’s milk, it is not surprising to learn
that bovine milk has been the subject of detailed chemical or
nutrient analyses for many years.6 These include comprehen-
sive studies on milk vitamins,7 minerals,8 fats,9 proteins,10 and
typically composed of water (85−87%), fats (3.8−5.5%),
proteins (2.9−3.5%) and carbohydrates (5%). At a micro-
nutrient level, bovine milk contains many bioactive compounds
including vitamins, minerals, biogenic amines, organic acids,
nucleotides, oligosaccharides, and immunoglobulins.12 The
precise nature and relative abundance of these compounds is a
function of many internal and external factors.13 These factors
include the metabolic activity within the cow’s mammary
tissues, general udder health conditions, the type of feed given
to the cow, the activity and abundance of certain microbes in
the cow’s ruminal fluid, as well as the microbial activity and
enzymatic reactions occurring within the raw milk.14 Milk
composition also varies with the cattle breed (i.e., Holstein,
Jersey, Brown Swiss, etc.), stage of lactation, level of parity,
number of viable pregnancies, as milk quality control and
processing procedures after milk collection.15,16
Historically, most milk composition studies have been
performed using targeted chemical analyses aimed at character-
izing specific classes of compounds (i.e., sugars only, fats only).
While targeted analytic approaches are very accurate, they
require considerable skill, are rather limited in their chemical
scope, and often require a great deal of time and manual effort.
With the development of quantitative, targeted metabolomics
approaches, it has been possible to achieve far more
comprehensive chemical coverage of foods, biofluids, and
tissues.17 Metabolomics is a branch of “omics” sciences
concerned with the high-throughput, comprehensive character-
Received: January 9, 2019
Revised: March 16, 2019
Accepted: March 17, 2019

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.9b00204

J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
ization of large numbers of small molecule metabolites.18
Thanks to significant advances in analytical techniques such as
mass spectrometry (MS) and NMR, metabolomics methods
are able to routinely identify and quantify hundreds of
compounds from a single sample. Indeed, metabolomics has
already enabled the determination of extensive inventories of
small molecule metabolites for a range of organisms, cell types,
and biofluids.16−20
Over the past 10 years, several comprehensive metabolomic
studies of bovine (and other livestock) milk samples have been
performed.15,16,21−23 The study by Boudonck et al.22 identified
(but unfortunately did not quantify) 93 bovine milk
metabolites including amino acids, short peptides, carbohy-
drates, lipids, vitamins, nucleotides, and enzyme cofactors
using a combination of both liquid chromatography−tandem
mass spectrometry (LC−MS/MS) and gas chromatography−
mass spectrometry (GC−MS). In a later study by Klein et al.,23
NMR and LC−MS were combined to look at how the
chemical composition of bovine milk varied between early and
late lactation in two dairy cattle breeds, Brown Swiss and
Simmental. These authors identified and quantified 44 milk
constituents including many amino acids, sugars, fatty acids,
and organic acids. A comparative metabolomic study of milk
from different dairy animals (Chinese Holstein, Jersey, yak,
buffalo, goat, camel, and horse) was completed by Yang et al.15
This study compared the milk metabolite profile of Chinese
Holstein and Jersey cows with other dairy animals using NMR
and LC−MS. The results showed that a subset of 68, 74, 54,
58, 77, and 91 metabolites were significantly different between
the milks produced by Holstein and Jersey, buffalo, yak, goat,
camel, and horse, respectively. Unfortunately, this study did
not provide a list of the identified metabolites or their
measured concentrations. In a recent metabolomic study
reported by Mung and Li, more than 2500 metabolites were
putatively identified in cow’s milk using chemical isotope
labeling LC−MS techniques.24,25 However, fewer than 80
compounds were positively identified, and none of the
compounds were actually quantified.23,24 The most recent
quantitative metabolomic study done on bovine milk was
described by O’Callaghan et al.16 These authors used NMR
spectroscopy to determine how the chemical composition of
bovine milk varied among different feeding systems. This study
resulted in the identification and quantification of 49 bovine
milk metabolites.
As described above, many studies looking into bovine milk
composition have been conducted, but none have attempted to
comprehensively identify and quantify bovine milk using more
than two analytical techniques. Furthermore, many of these
studies have not made their reported experimental findings
publicly available. In addition, none of these metabolomic
studies attempted to integrate previously published informa-
tion regarding bovine milk composition to extend or validate
their results.
To facilitate further research into milk chemistry and milk
micronutrients, we believe it is critical to comprehensively
characterize the chemical composition of bovine milk using
multiple, fully quantitative metabolomic techniques. Such an
undertaking would benefit livestock researchers, food scien-
tists, nutritionists, and consumers as it would create a
centralized, comprehensive, and electronically accessible data-
base of all detected or detectable metabolites/chemicals found
in bovine milk. To create such a resource, we combined both
experimental metabolomic techniques with computer-aided
B DOI: 10.1021/acs.jafc.9b00204
Article
text mining to compile essentially all of the known chemicals
(endogenous and exogenous) that can be detected in bovine
milk along with their respective concentrations. Experimen-
tally, we used multiple quantitative metabolomics techniques
including high-resolution NMR spectroscopy, liquid chroma-
tography coupled with high-resolution mass spectrometry
(LC−HRMS), LC−MS/MS, and ICP−MS methods to
identify, quantify, and validate 296 bovine milk metabolites
or metabolite species (which corresponds to 1316 lipids and
131 nonlipids). To further enhance our metabolic profiling
studies, we conducted an extensive literature survey and
extracted metabolite data from nearly 150 journal articles that
we identified through computer-aided literature searches. This
“bibliomic” effort yielded data for another 676 metabolites or
metabolite species (which corresponds to 292 lipids and 616
nonlipids). The resulting data is now housed in the Milk
Composition Database (MCDB), a comprehensive Web-
accessible source containing concentration data, physicochem-
ical data, and reference data for 972 metabolites or metabolite
species, which corresponds to 2355 unique compound
structures, found in bovine milk. Here we defined “metabolite
species” as those molecules with nonunique chemical formulas
or masses (such as lipids) while “unique compound structures”
correspond to compounds with a unique and clearly defined
chemical structure and a unique chemical name.
Overall, the intent of this study was to help consumers, milk
producers, nutritional chemists, and the dairy research
communities address four key questions: (1) What kinds of
compounds and nutrients are present in bovine milk? (2) What
is the approximate variation in the concentration of
metabolites in different kinds of commercial bovine milk?
(3) What fraction of the milk metabolome can be identified
and/or quantified using targeted, quantitative metabolomic
techniques? and (4) What analytical methods (NMR, LC−
HRMS, LC−MS/MS, ICP−MS) are best suited for
comprehensively profiling milk? Answering these questions
will provide a common foundation and a more appropriate
baseline for both ongoing and future milk composition studies.
■
MATERIALS AND METHODS
Milk Sample Collection. Four different types of commercially
available bovine milk of varying fat content were analyzed in this
study. These included commercial skim or skimmed milk (<0.2% milk
fat), 1% milk, 2% milk, and 3.25% milk, all of which were fortified
with vitamin D3. All commercial bovine milk samples used in this
study were purchased from local supermarkets in Edmonton, Canada.
While no details regarding the exact type or breed of dairy cows are
available, the vast majority (>95%) of dairy cows in Western Canada
are Holsteins. Following sample acquisition, 1.0 mL samples were
pipetted into 1.5 mL Eppendorf tubes and stored in a freezer at −20
°C until the time of analysis. A total of 16 milk samples (four
replicates from each type of milk) were analyzed by four different
metabolomic techniques.
NMR Spectroscopy. Milk proteins and lipoproteins can seriously
compromise the quality of 1H NMR spectra though the generation of
intense, broad lines that interfere with the identification and
quantification of lower abundance metabolites. Deproteinization can
eliminate these peaks. Deproteinization of the milk samples was done
by centrifugation and ultrafiltration using 3-kDa cutoff centrifuge filter
units (Amicon Micoron YM-3; Sigma-Aldrich, St. Louis, MO),
following a previously reported deproteinization procedure.20 It is
important to note that the 3-kDa filter eliminates not only proteins
but also lipids and reduces the concentrations of certain (known)
hydrophobic compounds. The deproteinized milk samples (280 μL)
were then transferred to a 1.5 mL Eppendorf tube, to which an
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
additional 70 μL of the standard NMR buffer solution (1 mM DSS
(disodium-2,2-dimethyl-2-silapentane-5-sulfonate), in 10% D2O, 150
mM sodium phosphate buffer, pH 7.0) was added. These samples (a
total volume of 350 μL) were then transferred to 3 mm NMR tubes
for spectral analysis. All 1H NMR spectra were collected on a Bruker
Avance III Ascend 700 MHz spectrometer equipped with a 5 mm
cryo-probe (Bruker Biospin, Rheinstetten, Germany). 1H NMR
spectra were collected at 25 °C using the first transient of a nuclear
Overhauser effect spectroscopy (NOESY)-presaturation pulse se-
quence. This pulse sequence was selected based on its excellent
quantitative accuracy.25 NMR spectra were acquired with 128 scans
employing a 4 s acquisition time and a 1 s recycle delay.
NMR Compound Identification and Quantification. Prior to
spectral deconvolution, all free induction decays (FIDs) were zero-
filled to 240 k data points and a 0.5 Hz line broadening function was
applied. The methyl singlet of the added DSS (set to 0.00 ppm)
served both as an internal chemical shift referencing standard and as
an internal standard for quantification. All 1H NMR spectra were
processed using the Chenomx NMR Suite 8.1 software package
(Edmonton, Canada) for quantification as previously described.26 A
minimum of two experienced NMR spectroscopists processed and
analyzed the spectra to eliminate compound identification and
quantification errors. Sample spike-in experiments were also used to
confirm the identity of a number of compounds suspected to be
present in our NMR spectra. A spike-in experiment involves adding
50−500 μM of the presumptive compound to selected milk samples
to test if the corresponding 1H NMR signals changed as expected. All
milk samples were also assessed over multiple time periods (up to 24
h after the first acquisition) to ensure that there were no significant
changes in metabolite concentrations.
LC−HRMS Compound Identification and Quantification. A
targeted, fully quantitative metabolite profiling approach was
employed that combined direct flow injection (DFI)−mass
spectrometry with reverse-phase LC−HRMS to determine concen-
trations of amino acids, biogenic amines, monosaccharides,
acylcarnitines (ACs), diglycerides (DGs), triglycerides (TGs),
phosphatidylcholines (PCs), lysophosphatidylcholines (LysoPCs),
sphingomyelins (SMs), ceramides (Cers), and cholesteryl esters
(CEs) in our milk samples. These analyses were enabled by a newly
released quantitative metabolomics kit (AbsoluteIDQ p400 HR),
available from Biocrates Life Sciences AG (Innsbruk, Austria). This
kit, when used with a QExactive HF OrbiTrap mass spectrometer, can
identify and quantify up to 408 compounds (365 lipids and 43 small
molecules) covering 11 metabolite classes. The absolute quantifica-
tion of amino acids and biogenic amines is ensured by using two
separate UPLC injections, while sugars and lipids are measured by
two column-free, DFI methods. While primarily designed for human
metabolomic studies, the measurable ranges of metabolite concen-
trations available through the p400 kit match very closely with the
known or expected metabolite concentrations in bovine milk.
The detection of each metabolite relies almost solely on measuring
the exact mass of molecular ions or adducts without further
fragmentation. The kit incorporates both isotope-labeled internal
standards and other quality control (QC) standards into its 96-well
plate filter to ensure accurate compound quantification. The kit was
originally developed to assess plasma or serum samples but it also
performs very well for related biofluids (such as milk). The first 14
wells in the kit’s 96-well plate are used for QC and internal
standardization, while the other 82 wells are used for sample analysis.
Altogether, 16 commercial milk samples were metabolically profiled
using the AbsoluteIDQ p400 HR protocol described in the user
manual.
In brief, milk samples were first thawed on ice, vortexed, and then
spun in a centrifuge at 13 000 rpm for 5 min. A volume of 10 μL of
each commercial milk sample was carefully pipetted into an
appropriate sample well of the upper 96-well filter plate and dried
using a stream of nitrogen gas. Amino acid derivatization was done by
adding 50 μL of a 5% solution of phenyl-isothiocyanate (PITC) and
incubating the solution for 20 min. After incubation and PITC
derivatization, the samples were dried down using an evaporator. The
C DOI: 10.1021/acs.jafc.9b00204
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metabolites were extracted by adding an ammonium acetate/
methanol mixture (5 mM ammonium acetate dissolved in 300 μL
methanol) to the upper 96-well filter plate and then centrifuging the
plates so that the extract bled into the lower 96-deep well plate. The
resulting extract was split for LC−HRMS (50 μL) and DFI−MS (10
μL) analyses followed by a dilution step with 450 μL of 40% methanol
for LC−MS analysis and with 490 μL of the kit’s MS running solvent
for DFI−MS analysis. LC−HRMS analyses were conducted on a
Thermo Scientific QExactive HF OrbiTrap mass spectrometer from
Thermo Scientific (Mississauga, Canada) equipped with a Thermo
Scientific Vanquish ultra-high-performance liquid chromatography
(UHPLC) system. The Biocrates MetIQ software (Innsbruk, Austria)
controls the entire assay’s workflow. This includes sample registration,
automated metabolite concentration calculation, and data export to
other data analysis programs.
Characterization of Milk Free Fatty Acids Using LC−MS/MS.
Free fatty acids in the commercial milk samples were analyzed using a
previously described LC−MS/MS method,27 with some modifica-
tions, including a 3-nitrophenylhydrazine (NPH) derivatization step.
The isotopically labeled 13C6-3-nitrophenylhydrazine (which was used
for derivatization and quantification) was purchased from Cayman
Chemical (Ann Arbor, MI). All other reagents and solvents, including
all the fatty acid standards, were acquired from Sigma-Aldrich
(Oakville, Canada).
In conducting the fatty acid assay, 50 μL of the samples (PBS for
the “blank” control samples, seven calibrants numbered from Cal 1 to
Cal 7, and the milk samples) were pipetted into individual 1.5 mL
Eppendorf tubes, then 150 μL of ice-cold methanol was added to each
Eppendorf tube, mixed thoroughly, and left in a −20 °C freezer
overnight for protein precipitation. After the precipitation step, all the
tubes were centrifuged at 13 000 rpm for 20 min, whereupon 50 μL of
the supernatant was pipetted to each well of the Nunc 96 DeepWell
plate. To each well 25 μL of each of the following three solutions was
added: (1) EDC (150 mM in methanol), 3-NPH (250 mM in 50%
methanol), and pyridine (7.5% in 75% methanol). A volume of 50 μL
of the calibration curve standard mixture solution with the highest
concentration (i.e., Cal 7) was again pipetted into the center of an
empty well, followed by the addition of 25 μL each of 13C6-labeled 3-
NPH, EDC, and pyridine solution, which was used as isotope-labeled
internal standard (ISTD). The whole 96-well plate was placed on a
shaker (400 rpm) at 20 °C for 2 h to complete the derivatization
reaction. After the derivatization step, 25 μL of the BHT solution (2
mg/mL in methanol) and 350 μL of water were added to each well in
the plate. This was done to dilute and stabilize the final solution.
Finally, 90 μL of each sample solution was transferred to another
Nunc 96 DeepWell plate and mixed well with 10 μL of the ISTD
solution. In total, 20 μL of the final mixture solution was injected into
a HPLC-equipped QTRAP 4000 mass spectrometer for LC−MS/MS
analysis.
Characterization of Milk Vitamins Using LC−MS/MS. Both
water-soluble and fat-soluble vitamins found in milk were analyzed via
a targeted LC−MS/MS method. Water-soluble vitamin standards
including vitamin B1, vitamin B2, vitamin B3-amide, vitamin B5,
vitamin B6, vitamin B7, vitamin B9, vitamin B12, and vitamin C were
purchased from AccuStandard (Chromatographic Specialties, Brock-
ville, Canada). Fat-soluble vitamin standards including retinol
(vitamin A), cholecalciferol (vitamin D3), calcifediol (25-hydrox-
yvitamin D3 or vitamin OH D3), and α-tocopherol (vitamin E) were
purchased from Sigma-Aldrich (Oakville, Canada). Isotopically
labeled water-soluble vitamin standards 4,5,4-methyl-13C3-thiamine
chloride, d4-nicotinamide, 13C3,15N-pantothenic acid, ring-6,6-d2-
biotin, and 13C-ascorbic acid were bought from Cambridge Isotope
Laboratories, Inc. (Tewksbury, MA); 13C4,15N2-riboflavin was
purchased from IsoSciences (Ambler, PA); d2-pyridoxine hydro-
chloride was acquired from C/D/N Isotopes Inc. (Quebec, Canada);
and d4-folic acid was bought from Santa Cruz Biotechnology, Inc.
(Dallas, Texas). The fat-soluble vitamin standards retinyl acetate,
cholecalciferol-d3, and tocopheryl acetate were bought from Sigma-
Aldrich (Oakville, Canada). HPLC-grade methanol, water, trichloro-
acetic acid (TCA), hexane, zinc sulfate, and ammonium formate were
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
bought from Sigma-Aldrich (Oakville, Canada). LC−MS grade
formate was acquired from Fisher Scientific (Ottawa, Canada).
Individual stock solutions of each of the above-mentioned water-
soluble vitamin standards (2 mM) and isotopically labeled standards
(3 mM) were prepared in HPLC-grade water. Individual standard
stock solutions were then mixed together to prepare 7 calibrants with
concentration ranges as follows: 0.01 to 1 μM for vitamin B1, vitamin
B2, vitamin B6, vitamin B7, vitamin B9, and vitamin B12; 0.05 to 5 μM
for vitamin B3-amide and vitamin B5, and 0.4 to 40 μM for vitamin C.
Similarly, individual internal standard stock solutions were mixed to
make the internal standard mixture solution with a final concentration
of each water-soluble internal standard as follows: 4,5,4-methyl-13C3-
thiamine chloride (3 μM), 13C4,15N2-riboflavin (3 μM), d4-
nicotinamide (30 μM), 13C3,15N-pantothenic acid (15 μM), d2-
pyridoxine hydrochloride (3 μM), ring-6,6-d2-biotin (3 μM), d4-folic
acid (3 μM), and 13C-ascorbic acid (120 μM). To quantify the water-
soluble vitamins in our milk samples, 7-point calibration curves were
generated by adding 20 μL of the isotopically labeled internal
standard mixture to 100 μL of the calibration solutions in Eppendorf
tubes. Milk samples were also prepared by adding the isotopically
labeled internal standard mixture to 100 μL of milk. A total of 120 μL
of an aqueous TCA solution (50 mg/mL) was pipetted to each
Eppendorf tube containing the calibrants or milk samples. Each tube
was vortexed for 30 s for thorough mixing and then left on ice for 1 h.
After cooling, each tube was centrifuged at 13 000 rpm for 20 min,
and 200 μL of the supernatant was transferred to a new HPLC vial. A
volume of 10 μL was injected for LC−MS/MS analysis.
An Agilent 1100 series HPLC system (Agilent Technologies, Palo
Alto, CA) coupled with an AB Sciex QTRAP 4000 mass spectrometer
(Sciex Canada, Concord, Canada) was used to analyze water-soluble
vitamins in our commercial milk samples. An Agilent reversed-phase
Zorbax Eclipse XDB C18 column (3.0 mm × 100 mm, 3.5 μm
particle size, 80 Å pore size) coupled to a Phenomenex (Torrance,
CA) SecurityGuard C18 precolumn (4.0 mm × 3.0 mm) was used for
the separation of all water-soluble vitamins in the milk samples. A
solution gradient separation was performed using two solvents: (1) 5
mM ammonium formate and 0.1% (v/v) formate in water (solvent A)
and (2) 5 mM ammonium formate and 0.1% (v/v) formate in
methanol (solvent B). The gradient, with a flow rate of 400 μL/min,
started at 0% (solvent B) for 1 min, then ramped up from 0% to 30%
(solvent B) in 1.5 min, then from 30% to 60% (solvent B in 2 min,
then from 60% to 75% (solvent B) in 1.5 min, and held at 75% for 0.5
min. After that, the column was equilibrated back to 0% (solvent B)
for 3 min before the next injection. The column oven temperature was
maintained at 40 °C. The positive electrospray ionization MRM
(multiple reaction monitoring) mode was used for the MS/MS
analysis. The IonSpray voltage was set to 5500 V while the ion source
temperature was set to 450 °C. The curtain gas (CUR), ion source
gas 1 (GAS1), and ion source gas 2 (GAS2) were set at 20, 40, and
60, respectively, and collision gas (CAD) parameter was set as
medium.
Individual stock solutions of each above-mentioned fat-soluble
vitamin standards (5 mM) and internal standards (5 mM) were
prepared using HPLC-grade methanol. Individual standard stock
solutions were then mixed together to prepare 7 calibrants with
concentration ranges as follows: vitamin A (0.313−20 μM), vitamin
D3 (0.0156−1 μM), vitamin 25-hydroxyvitamin D3 (0.00625−0.4
μM), and vitamin E (1.25−80 μM). Individual internal standard stock
solutions were mixed to make the internal standard mixture solution
with a final concentration as follows: retinyl acetate (5 μM),
cholecalciferol-d3 (0.2 μM), and tocopheryl acetate (5 μM). For the
LC−MS/MS analysis of fat-soluble vitamins, 50 μL of calibration
solutions or milk samples were pipetted into glass vials, followed by
adding 50 μL of internal standard mixture solution. Subsequently, a
300 μL of methanol and 0.2 M ZnSO4 mixture solution (1:1 v/v) was
added to precipitate the milk proteins and to facilitate the release of
25-hydroxyvitamin D3 from vitamin D binding protein. After this
precipitation step, 1 mL of hexane was added to every sample to
extract the fat-soluble vitamins. All the samples were vortexed for 10
min and centrifuged at 13 000 rpm for 20 min. Subsequently 650 μL
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Article
of the hexane layer was then transferred to a new HPLC vial for
evaporation under nitrogen gas at 40 °C until dried. Finally, 200 μL of
methanol was added to each dried sample to reconstitute the analytes,
and 10 μL was injected for LC−MS/MS analysis.
An Agilent 1100 series HPLC system (Agilent Technologies, Palo
Alto, CA) coupled with an AB Sciex QTRAP 4000 mass spectrometer
(Sciex Canada, Concord, Canada) was used to analyze the fat-soluble
vitamins in all commercial milk samples. A Phenomenex Kinetex C18
column (3.0 mm × 100 mm, 2.6 μm particle size, 100 Å pore size)
connected to a Phenomenex SecurityGuard C18 precolumn (4.0 mm
× 3.0 mm), was used to separate the fat-soluble vitamins. A solvent
gradient was run as follows: (1) 0.1% (v/v) formate in water (solvent
A) and (2) 0.1% (v/v) formate in methanol (solvent B). The
gradient, at a flow of 800 μL/min, started with 85% (solvent B), then
moved from 85% to 100% (solvent B) in 1.8 min where it was
maintained at 100% for 2.2 min. The column was then equilibrated
back to 85% (solvent B) for 3 min before the next injection. The
column oven temperature was kept at 40 °C. Positive electrospray
ionization MRM mode was used for the MS/MS analysis. The
IonSpray voltage was set to 5500 V, and ion source temperature was
set to 400 °C. The CUR, GAS1, and GAS2 parameters were set at 30,
40, and 50, respectively, and the CAD parameter was set as medium.
Data analysis for the water-soluble and fat-soluble vitamins was done
using Sciex Analyst 1.6.2.
Trace Elemental Analyses Using ICP−MS. ICP−MS is a
powerful and sensitive approach for measuring metal ions and other
trace elements in biological samples. For this study, all trace elemental
analysis done on the commercial milk samples was performed on a
PerkinElmer Sciex Elan 6000 quadrupole ICP−MS (Woodbridge,
Canada), operating in a dual detector mode using previously
described methods.28,29
Milk Metabolites in the Literature. We also conducted an
extensive literature review of known bovine milk metabolites and their
concentrations by using several in-house text-mining software
packages that were originally developed for the Human Metabolome
Project (HMP) and the Human Metabolome Database (HMDB).17
Two of the most useful programs were PolySearch 30 and
PolySearch2.31 These programs are able to take simple keywords
(i.e., “milk”, “bovine”, etc.) as input and to rapidly create hyperlinked
lists of abstracts and papers from PubMed (and other data sources)
containing information about milk metabolites and their correspond-
ing concentration data. PolySearch2 was able to compile a ranked list
of milk metabolites by measuring word co-occurrence frequency using
terms such as “cow’s milk”, “milk”, “dairy”, “bovine” and “cattle” in
conjunction with words such as “concentration”, “identification”,
“quantification”, “mM”, or “micromol”. PolySearch2 also extracted key
sentences from the abstracts, then labeled and hyperlinked the
metabolites mentioned in the text. This led to the identification of
∼150 papers, abstracts, and textbooks with relevant chemical
information on bovine milk. These PolySearch results were
supplemented by additional data retrieval from various online
National food composition tables such as the United States
Department of Agriculture [USDA] Food Composition Databases
[FCD] (https://ndb.nal.usda.gov/ndb/), The Frida Food Data
database (https://frida.fooddata.dk/), FooDB (http://www.foodb.
ca), as well as Phenol-Explorer (www.phenol-explorer.eu).32−34
All literature-derived compounds, along with their concentrations
and references, were compiled, compared, and their names
“normalized” to match HMDB,17 CAS, and PubChem identifiers.
The manually derived compound data was further annotated using an
in-house program called DataWrangler17 which automatically
generates names, synonyms, descriptions, structures, chemical
taxonomies, physical property data, and bioavailability. The
information generated by DataWrangler was manually checked by
three different scientists with postgraduate degrees in biochemistry,
physiology, and/or animal sciences. After the manual check was
complete, the data were then entered into the Milk Composition
Database (MCDB). Concentrations were cross-checked manually to
identify large discrepancies (>3×) between entered values. Those that
exceeded this threshold were reanalyzed to see if data entry errors had
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
been made. For highly discrepant values, a “majority wins” scheme
was used to select the best or most likely value. On the other hand, if
our experimental data matched best with one of the discrepant values,
then that value was selected over other reported value(s). The
resulting list of 1030 literature-derived milk metabolites (including
122 overlapping experimentally derived metabolites), along with their
concentration data (when available), helped to confirm many of the
metabolites and metabolite concentrations previously found in our
experimental analyses.
■
RESULTS AND DISCUSSION
This study’s central aim was to identify and quantify all the
chemicals (both endogenous and exogenous) detectable in
commercial cow’s milk using both a comprehensive,
quantitative experimental approach and a literature mining
approach. All the detected/identified/quantified compounds
have been deposited into a freely accessible database called the
MCDB. The MCDB contains a complete list of milk chemicals
including their names, their structures, their reference spectra
(NMR, GC−MS, and LC−MS), along with literature citations
for all (to the best of our knowledge) of the milk compounds
that have ever been identified, quantified, or reported either in
this paper or in the existing scientific literature.
The MCDB is structured and designed to be very popular,
like online metabolomic databases such as the HMDB.
Clicking on the browse button (on the MCDB navigation
panel) generates a tabular view that allows users to casually
scroll through the database or resort its contents by compound
name or mass. Users can select the “Metabolites” view to
facilitate their browsing. Each milk compound entry in the
MCDB is hyperlinked to an individual metabolite description
table (called a MetaboCard) that, when clicked, brings up
additional information on that particular chemical. As a Web-
enabled, electronic database, the MCDB is fully searchable and
supports a variety of text, mass, spectral, and structure searches.
Currently, the MCDB contains information on 2355 detectable
milk compounds with unique, well-defined structures and
names.
Note that the total number of chemical compounds in
MCDB is not a number that will remain unchanged. Certainly,
as technology and instrument sensitivity improve, it is
anticipated that this number will increase as other, lower
abundance metabolites will be detected and will be added to
future versions of the MCDB.
Chemical Composition of Bovine Milk. Inspection of
the MCDB’s data reveals that the chemical composition of
commercial cow’s milk is dominated by carbohydrates
(primarily lactose, glucose, and galactose), inorganic ions
(potassium and calcium), organic acids (citrate), and amine-
containing compounds (creatinine, choline, and urea). Lesser
quantities of vitamins, triglycerides (the dominant lipid
components), di- and monoglycerides, fatty acids, short
chain fatty acids, amino acids, and other small bioactive
compounds are also evident. In addition, milk also contains a
number of macromolecules, including DNA, RNA, and several
proteins such as the bovine casein peptides, β-lactoglobulin
and α-lactalbumin.35,36 While the measurement of macro-
molecules was not an objective of this study, they are included
in the MCDB for completeness. A more detailed description of
both our literature and experimental findings is given in the
following five sections covering (1) literature review/text
mining, (2) NMR, (3) LC−HRMS, (4) LC−MS/MS, and (5)
ICP−MS.
E DOI: 10.1021/acs.jafc.9b00204
Article
Literature Survey of Bovine Milk Metabolites. The
MCDB provides both concentration averages and concen-
tration ranges for 497 quantified milk metabolites or
metabolite species. This corresponds to 1318 lipids with
unique structures as well as 334 nonlipid compounds. In
addition to the experimentally derived values obtained from
the quantitative metabolomic methods described earlier, the
MCDB also contains literature-derived data (including
reported concentrations) of bovine milk metabolites or
chemical constituents with references to the source literature
(PubMed IDs or to non-PubMed journals and textbooks). In
many cases, multiple concentration values are given for
“normal” conditions. This is done to provide users/readers
with a better estimate of the potential concentration variations
that different laboratories or technologies may measure.
Overall, we found very good agreement between the results
reported for most methods and most laboratories. A number of
questionable or profoundly different literature-derived concen-
tration values of milk compounds were eliminated through the
curation process after being deemed mistaken, disproven (by
subsequent published studies), mis-typed or physiologically
impossible. Much of the curation process involved having
multiple curators carefully reading and double-checking the
primary literature to annotate the concentration unit type, to
perform unit conversions, and catch typographical incon-
sistencies.
Other than lactose (98−153 mM) and a variety of inorganic
ions or minerals such as potassium (31−43 mM), calcium
(26−32 mM), phosphorus (19−23 mM), sodium (17−28
mM), and magnesium (4−6 mM), the most abundant organic
metabolites reported in milk are citrate (3.0−9.8 mM),
creatine (312−543 μM), D-glucose (246−478 μM), choline
(152−479 μM), and myo-inositol (122−588 μM). The least
abundant compounds in milk include several vitamins, such as
vitamin D3 (0.1−0.3 nM), vitamin D2 (0.1−2.5 nM), vitamin
OH D3 (0.5 nM) as well as several trace elements including
neodymium (Nd) (0.3−4 nM), lanthanum (La) (0.2−4 nM),
cerium (Ce) (2.4−7 nM), and thallium (Tl) (3−4 nM). A
number of other low-abundance compounds detected in
bovine milk include veterinary antimicrobial agents such as
tetracycline (3.76 μM), lincomycin (0.19 μM), tylosin (0.06
μM), amoxicillin (0.052 μM), penicillin G (0.036 μM), and
oxolinic acid (0.036−0.058 μM).37,38 Antibiotics are com-
monly given to dairy cattle to treat diseases such as mastitis.
Hence, varying amounts of antibiotic residues can be found in
milk. In a comprehensive screening of bovine milk for
antibiotics, Freitas and colleagues36 detected more than 30
different antibiotics from 5 different antibiotic classes when
analyzing bovine, ovine, and caprine milk samples. The
reported concentrations ranged from 0.00001 to 0.0102 μM.
Other low-abundance compounds found in milk include
pesticide and herbicide residues.39 These agricultural con-
taminants are an obvious public safety concern. Therefore, the
European Union (EU) and United States Environmental
Protection Agency (USEPA) have legislated a restriction on
the maximum levels of herbicide and pesticide residues in
human food.40 These contaminants can be found in cow’s milk
if used for weed and grass treatment in the feedstuff used for
animal consumption.41 Some of the herbicide and pesticide
residues reported in bovine milk include triazine herbicides
such as atrazine (0.009−0.019 μM), cyanazine (<0.0083 μM),
prometryn (<0.0083 μM), simazine (<0.0099 μM) and
terbutryn (<0.0083 μM), as well as pesticides such as
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Figure 1. 700 MHz 1H NMR spectrum of the identified metabolites in commercial bovine milk.
dichlorodiphenyltrichloroethane (DDT), hexachlorocyclohex-
ane (HCH), endosulfan, aldrin, endrin, and others.42,43 More
details regarding their concentrations and frequency of
occurrence in bovine milk samples are provided in the
MCDB. The type and abundance of these compounds may
differ depending on the region, the decade of the reported
measurement (1980s, 1990s, 2000s) and the level of pesticide
application.39
Compound Identification and Quantification by
NMR. A representative high-resolution 700 MHz NMR
spectrum of bovine milk is shown in Figure 1. More than
95% of all visible peaks could in this spectrum be assigned to a
specific, fully identifiable compound. Furthermore, nearly all of
the spectral area seen in the spectrum could be fully fitted
using the Chenomx NMR Suite (version 8.1) spectral
deconvolution software. These data suggest that the level of
NMR spectral assignment for commercial cow’s milk is
essentially complete. Commercial milk samples typically have
a total of 59 water-soluble compounds that can be positively
identified and quantified. All 59 metabolites were identified in
F DOI: 10.1021/acs.jafc.9b00204
Article
all milk types (skim, 1%, 2%, and 3.25%) but with differing
concentrations. When analyzed over all milk varieties, the most
abundant compounds were lactose (114−122 mM), citrate
(4.8−5.8 mM), urea (1.1−1.2 mM), glycerophosphocholine
(0.6−0.7 mM), orotic acid (0.5−0.6 mM), creatine (0.49−
0.54 mM), galactose (0.3 mM), and glucose (0.2−0.3 mM).
The lowest concentration that could barely be detected using
NMR was 1−2 μM, for isovaleric acid, isobutyric acid,
isopropanol, and propionic acid. Other low abundance
compounds included the amino acids tryptophan, tyrosine,
histidine, and isoleucine (3−5 μM) as well as leucine and
asparagine (5−9 μM).
Note that these NMR measurements only measured free
amino acid concentrations. Most literature estimates of the
amino acid content in milk include the contribution from
hydrolyzed protein. We distinguish the two (free amino acids
vs total amino acid content) in the MCDB. The complete list
of NMR-derived compound concentrations (including aver-
ages, standard deviations, and the frequency of their
occurrence) is shown in Table 1. This table also shows the
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 1. List of Milk Metabolites Detected by NMR Spectroscopy along with Their Measured or Reported Concentrations and
Their Standard Deviations and/or Ranges (As Measured in Micromolar)

metabolites skim milk 1% milk 2% milk 3.25% milk literature values

2-oxoglutarate 109 ± 6 104 ± 7 112 ± 7 103 ± 3 37−15644
3-hydroxybutyrate 30 ± 3 26 ± 3 29 ± 2 26 ± 5 12−12144
acetic acid (C2:0) 33 ± 3 28 ± 2 30 ± 2 31 ± 2 13−11316,44
acetone 20 ± 2 22 ± 1 23 ± 1 22 ± 1 9−49716,44
L-acetylcarnitine 50 ± 5 48 ± 3 48 ± 2 48 ± 5 37−6516
L-alanine 34 ± 3 32 ± 3 33 ± 2 32 ± 1 18−7816,44
L-arginine 21 ± 3 27.5 ± 7.2 27 ± 12 31.4 ± 10.3
L-asparagine 6±2 6±1 7±1 8±1
L-aspartate 21 ± 3 20 ± 1 19 ± 3 19 ± 3 13−4216
betaine 71 ± 5 73 ± 5 81 ± 4 79 ± 4 33−11516
butyric acid (C4:0) 42 ± 4 33 ± 5 37 ± 2 41 ± 3 20−5745
capric acid (C10:0) 25 ± 1 20 ± 3 19 ± 6 23 ± 2 5−2045
caprylic acid (C8:0) 14 ± 4 16 ± 3 11 ± 2 12 ± 1 3−1445
L-carnitine 62 ± 3 61 ± 6 68 ± 2 68 ± 5 57−10316
choline 214 ± 17 206 ± 14 242 ± 10 235 ± 8 152−47916,44
cis-aconitate 26 ± 5 22 ± 3 22 ± 5 21 ± 5 28−15716,44
citrate 5808 ± 643 5099 ± 409 5219 ± 168 4793 ± 260 3692−743544
creatine 510 ± 42 491 ± 40 537 ± 30 534 ± 16 312−54316
creatine-1-phosphate 59 ± 4 50 ± 3 46 ± 4 30 ± 1 0−10216
creatinine 66 ± 2 66 ± 7 70 ± 6 71 ± 4 36−12516,44
cytidine 12 ± 3 15 ± 1 18 ± 1 17 ± 2 2.4−11.654
dimethyl sulfone 26 ± 3 23 ± 2 31 ± 2 33 ± 2 10−5916
dimethylamine 17 ± 2 15 ± 1 17 ± 1 17 ± 1 7−1716
ethanolamine 62 ± 1 53 ± 15 66 ± 3 66 ± 5 56−16616
formate 16 ± 1 15 ± 1 16 ± 1 15.6 ± 0.3 3.3−46
L-fucose 25 ± 3 35 ± 9 23 ± 11 28 ± 2 6−4116
fumarate 14 ± 1 13 ± 1 13 ± 1 13 ± 1 6−2316,44
D-galactose 310 ± 39 300 ± 18 334 ± 15 318 ± 19 86−196016
D-glucose 250 ± 41 288 ± 32 261 ± 29 235 ± 49 246−47816
glucose-1-phosphate 55 ± 3 58 ± 2 43 ± 3 43 ± 11 0−21616
L-glutamate 271 ± 21 259 ± 10 263 ± 15 260 ± 23 111−74016,44
glycerophosphocholine 705 ± 45 669 ± 53 659 ± 8 646 ± 21 291−121716,44
hippurate 67 ± 4 65 ± 5 69 ± 9 72 ± 5 79−26716
L-histidine 4±2 3±1 2±1 4±1
isobutyric acid 1.2 ± 0.4 1.3 ± 0.1 1.0 ± 0.4 1.3 ± 0.2 0−3616
L-isoleucine 4.7 ± 0.7 4.4 ± 0.5 4.2 ± 0.2 3.9 ± 0.6 2−716
isopropanol 1.9 ± 0.3 1.7 ± 0.1 1.7 ± 0.1 1.8 ± 0.1
isovaleric acid 0.8 ± 0.4 2±1 1.0 ± 0.6 0.9 ± 0.3
L-lactate 59 ± 4 61.4 ± 0.3 57 ± 3 55 ± 1 0−16716
lactose 122036 ± 8571 117931 ± 5096 120445 ± 4407 113883 ± 3432 98357−15321616,44
L-leucine 6.3 ± 1.4 6.8 ± 0.5 4.9 ± 0.4 5.5 ± 0.5 2−616
L-lysine 25 ± 2 18 ± 9 23 ± 3 24 ± 5 13−3055
malate 144 ± 15 151 ± 19 141 ± 23 142 ± 17 55−12216
methanol 29 ± 3 32 ± 1 30 ± 4 32 ± 4 10−4916
N-acetylglucosamine 351 ± 34 238 ± 90 302 ± 76 278 ± 94 127−37156
niacinamide 7±1 5.7 ± 0.7 5.4 ± 0.4 5±2 5.2−7.357,58
O-phosphocholine 120 ± 1 122 ± 8 123 ± 7 116 ± 7 0−94116,44
orotic acid 565 ± 49 511 ± 45 542 ± 18 536 ± 19 208−100244
pantothenic acid 12 ± 3 11 ± 1 13 ± 1 11.2 ± 0.5 14.6−16.459,60
L-proline 27 ± 5 25 ± 1 30 ± 1 27 ± 3 14−3955
propionic acid (C3:0) 2±1 1.9 ± 0.3 3±1 2.3 ± 0.1 0−1516
pyruvate 20 ± 2 17 ± 1 20 ± 2 17 ± 1 0−5416,55
succinate 14 ± 1 14 ± 2 15 ± 1 20 ± 1 16−3016
L-tryptophan 3±1 3±1 3±2 3±2 2−455
L-tyrosine 4±1 4±1 2.6 ± 0.3 3±1 3−1116,55
urea 1104 ± 136 1117 ± 175 1174 ± 115 1115 ± 30 119−44716
uridine 30 ± 3 31 ± 3 27 ± 2 28 ± 2 9−2116
valeric acid (C5:0) 6±3 9±2 4.9 ± 0.3 5±1 2−1016
L-valine 13 ± 1 13 ± 1 13 ± 1 13 ± 1 6−1516

G DOI: 10.1021/acs.jafc.9b00204
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 2. List of Milk Metabolites Detected by LC−HRMS along with Their Measured or Reported Concentrations and Their
Standard Deviations and/or Ranges (As Measured in Micromolar)
metabolites skim milk 1% milk 2% milk 3.25% milk
Acylcarnitines
AC(0:0) 67 ± 1 72.7 ± 0.3 77 ± 1 79 ± 2
AC(12:0) 3.4 ± 0.1 3.75 ± 0.03 4.3 ± 0.1 4.43 ± 0.04
AC(12:1) 0.250 ± 0.003 0.26 ± 0.03 0.28 ± 0.01 0.30 ± 0.01
AC(14:0) 0.10 ± 0.01 0.124 ± 0.003 0.149 ± 0.003 0.171 ± 0.004
AC(14:1) NDa 0.021 ± 0.001 0.032 ± 0.002 0.047 ± 0.003
AC(14:2-OH) 0.019 ± 0.001 0.19 ± 0.01 0.33 ± 0.01 0.47 ± 0.01
AC(16:2-OH) 0.025 ± 0.001 0.137 ± 0.004 0.23 ± 0.01 0.33 ± 0.01
AC(18:1-OH) 0.018 ± 0.001 0.022 ± 0.002 0.031 ± 0.002 0.033 ± 0.002
AC(2:0) 41 ± 1 37 ± 1 39 ± 2 37 ± 1
AC(3:0) 3.93 ± 0.02 4.1 ± 0.1 4.4 ± 0.1 4±1
AC(4:0) 3.99 ± 0.04 4.4 ± 0.2 5.8 ± 0.1 6.6 ± 0.1
AC(4:0-OH) 0.305 ± 0.004 0.33 ± 0.01 0.49 ± 0.01 0.55 ± 0.01
AC(5:0) 2.8 ± 0.1 3.0 ± 0.1 4.1 ± 0.1 4.5 ± 0.1
AC(6:0-DC) 0.149 ± 0.002 0.132 ± 0.004 0.144 ± 0.002 0.153 ± 0.001
AC(4:0-DC) 0.23 ± 0.01 0.202 ± 0.004 0.213 ± 0.003 0.214 ± 0.004
AC(5:1) 0.068 ± 0.002 0.066 ± 0.002 0.086 ± 0.002 0.116 ± 0.001
total ACs 123.28 ± 0.14 126.43 ± 0.11 136.59 ± 0.22 137.9 ± 0.3
Lysophosphatidylcholines
LysoPC(15:0) 0.038 ± 0.003 0.045 ± 0.003 0.041 ± 0.001 0.045 ± 0.002
LysoPC(16:0) 1.00 ± 0.04 0.97 ± 0.03 1.20 ± 0.03 1.25 ± 0.02
LysoPC(18:1) 0.366 ± 0.002 0.366 ± 0.004 0.47 ± 0.01 0.481 ± 0.004
LysoPC(18:2) 0.181 ± 0.002 0.164 ± 0.004 0.19 ± 0.01 0.185 ± 0.004
total LysoPCs 1.59 ± 0.01 1.55 ± 0.01 1.9 ± 0.01 1.96 ± 0.01
Phosphatidylcholines
PC(28:1) 0.54 ± 0.01 0.67 ± 0.01 0.84 ± 0.01 0.89 ± 0.01
PC(29:0) 0.238 ± 0.004 0.28 ± 0.01 0.34 ± 0.01 0.50 ± 0.01
PC(30:0) 5.68 ± 0.04 5.4 ± 0.1 6.7 ± 0.7 7.1 ± 0.1
PC(30:1) 0.29 ± 0.01 ND ND ND
PC(31:0) 1.0 ± 0.1 1.2 ± 0.1 1.27 ± 0.01 1.57 ± 0.02
PC(31:1) 0.175 ± 0.003 0.21 ± 0.01 0.33 ± 0.01 0.416 ± 0.004
PC(31:2) 0.075 ± 0.004 0.15 ± 0.01 0.23 ± 0.01 0.33 ± 0.01
PC(32:0) 10.2 ± 0.3 10.4 ± 0.4 12.5 ± 0.5 15.1 ± 0.3
PC(32:1) 3.18 ± 0.02 3.51 ± 0.03 4.2 ± 0.1 5.2 ± 0.1
PC(32:2) 0.61 ± 0.01 0.63 ± 0.01 0.83 ± 0.02 1.00 ± 0.05
PC(32:3) 0.161 ± 0.003 0.165 ± 0.002 0.20 ± 0.01 0.25 ± 0.01
PC(33:0) 0.70 ± 0.01 2.0 ± 0.1 1.75 ± 0.03 3.0 ± 0.1
PC(33:1) 0.92 ± 0.01 1.45 ± 0.04 2.2 ± 0.1 4.1 ± 0.1
PC(33:2) 0.39 ± 0.01 0.70 ± 0.01 1.41 ± 0.02 1.68 ± 0.03
PC(34:0) 2.26 ± 0.03 2.45 ± 0.04 4.7 ± 0.1 5.2 ± 0.1
PC(34:1) 16.1 ± 0.5 17.0 ± 0.2 22 ± 1 26 ± 1
PC(34:2) 6.9 ± 0.1 7.7 ± 0.1 9.5 ± 0.5 11.0 ± 0.4
PC(34:3) 0.74 ± 0.01 0.84 ± 0.01 0.98 ± 0.04 1.3 ± 0.1
PC(34:4) 0.074 ± 0.004 0.081 ± 0.004 0.10 ± 0.01 0.13 ± 0.01
PC(35:0) 0.20 ± 0.01 0.229 ± 0.004 0.63 ± 0.01 0.88 ± 0.07
PC(35:1) 0.45 ± 0.01 1.1 ± 0.1 1.61 ± 0.03 2.0 ± 0.1
PC(35:2) 0.258 ± 0.003 0.48 ± 0.02 0.808 ± 0.003 0.947 ± 0.004
PC(35:3) 0.067 ± 0.002 0.10 ± 0.01 0.18 ± 0.01 0.29 ± 0.01
PC(36:0) ND ND 0.66 ± 0.01 0.74 ± 0.01
PC(36:1) 5.23 ± 0.03 5.46 ± 0.02 6.5 ± 0.1 8.6 ± 0.1
PC(36:2) 5.48 ± 0.03 5.82 ± 0.04 7.0 ± 0.2 9.5 ± 0.1
PC(36:3) ND 3.24 ± 0.04 4.0 ± 0.1 5.3 ± 0.1
PC(36:4) 1.06 ± 0.01 1.20 ± 0.02 1.63 ± 0.04 1.86 ± 0.03
PC(36:5) 0.200 ± 0.002 0.23 ± 0.01 0.280 ± 0.003 0.349 ± 0.003
PC(37:0) ND ND 0.55 ± 0.01 0.89 ± 0.01
PC(38:2) 0.14 ± 0.01 0.26 ± 0.01 0.34 ± 0.01 0.49 ± 0.01
PC(38:3) 0.226 ± 0.004 0.25 ± 0.01 0.320 ± 0.003 0.375 ± 0.001
PC(38:4) ND ND 0.55 ± 0.01 0.58 ± 0.01
PC(38:5) 0.419 ± 0.003 0.45 ± 0.01 0.537 ± 0.003 0.73 ± 0.01
PC(38:6) 0.115 ± 0.002 0.151 ± 0.003 0.177 ± 0.002 0.22 ± 0.01

H DOI: 10.1021/acs.jafc.9b00204
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 2. continued
metabolites skim milk 1% milk 2% milk 3.25% milk
Phosphatidylcholines
PC(40:2) 0.025 ± 0.002 0.026 ± 0.003 0.032 ± 0.003 0.038 ± 0.003
PC(40:3) ND ND ND 0.043 ± 0.003
PC(40:4) ND ND 0.044 ± 0.001 0.056 ± 0.003
PC(44:1) 1.16 ± 0.04 1.39 ± 0.02 2.25 ± 0.03 3.6 ± 0.1
PC(46:1) 0.36 ± 0.01 2.1 ± 0.1 3.1 ± 0.1 3.6 ± 0.1
PC(46:2) 0.9 ± 0.1 1.7 ± 0.1 2.5 ± 0.4 5.0 ± 0.1
PC-O(30:0) 0.425 ± 0.002 0.439 ± 0.004 0.53 ± 0.01 0.63 ± 0.01
PC-O(32:1) 0.20 ± 0.01 0.20 ± 0.01 0.288 ± 0.003 0.323 ± 0.004
PC-O(32:2) 0.123 ± 0.004 0.11 ± 0.01 0.17 ± 0.01 0.189 ± 0.001
PC-O(34:0) 1.02 ± 0.02 1.09 ± 0.01 1.29 ± 0.04 1.54 ± 0.04
PC-O(34:1) 1.19 ± 0.01 1.18 ± 0.02 1.45 ± 0.03 1.88 ± 0.02
PC-O(34:2) 0.40 ± 0.01 0.416 ± 0.002 0.541 ± 0.004 0.66 ± 0.01
PC-O(34:3) 0.184 ± 0.004 0.19 ± 0.01 0.24 ± 0.01 0.30 ± 0.01
PC-O(36:0) 0.696 ± 0.003 0.783 ± 0.002 0.87 ± 0.02 1.19 ± 0.04
PC-O(36:1) 2.59 ± 0.04 2.5 ± 0.1 3.1 ± 0.1 3.7 ± 0.1
PC-O(36:2) 0.596 ± 0.003 0.60 ± 0.01 0.83 ± 0.01 1.016 ± 0.04
PC-O(36:3) ND ND 0.25 ± 0.01 0.275 ± 0.001
PC-O(36:4) 0.077 ± 0.001 0.085 ± 0.004 0.134 ± 0.004 0.148 ± 0.004
PC-O(36:5) 0.041 ± 0.003 0.041 ± 0.003 0.062 ± 0.003 0.066 ± 0.002
PC-O(38:0) 0.273 ± 0.004 0.256 ± 0.001 0.32 ± 0.01 0.37 ± 0.01
PC-O(38:3) 0.078 ± 0.002 0.074 ± 0.003 0.088 ± 0.004 0.102 ± 0.004
PC-O(38:5) 0.038 ± 0.004 0.039 ± 0.003 0.065 ± 0.002 0.064 ± 0.003
PC-O(40:5) ND ND 0.035 ± 0.003 0.048 ± 0.004
PC-O(40:6) ND ND 0.043 ± 0.004 0.042 ± 0.001
PC-O(42:1) ND 0.046 ± 0.002 0.045 ± 0.001 0.057 ± 0.003
PC-O(44:4) ND ND ND 0.041 ± 0.002
PC-O(44:5) ND ND 0.048 ± 0.002 0.063 ± 0.003
total PCs 74.45 ± 0.03 87.08 ± 0.03 114.18 ± 0.07 143.56 ± 0.06
Sphingomyelins
SM(33:1) 1.47 ± 0.02 1.35 ± 0.03 1.48 ± 0.02 1.82 ± 0.03
SM(35:1) 0.60 ± 0.01 0.542 ± 0.004 0.626 ± 0.004 0.701 ± 0.002
SM(41:1) 5.47 ± 0.02 4.8 ± 0.1 5.3 ± 0.1 6.5 ± 0.1
SM(41:2) 0.72 ± 0.01 0.68 ± 0.01 0.66 ± 0.01 0.89 ± 0.01
SM(43:1) 0.369 ± 0.004 0.347 ± 0.003 0.377 ± 0.002 0.487 ± 0.003
SM(34:1) 10.1 ± 0.2 8.9 ± 0.1 10.1 ± 0.4 12.1 ± 0.2
SM(34:2) 0.32 ± 0.01 0.28 ± 0.01 0.34 ± 0.01 0.39 ± 0.01
SM(36:1) 1.13 ± 0.01 1.04 ± 0.02 1.09 ± 0.03 1.19 ± 0.02
SM(36:2) 0.181 ± 0.003 0.163 ± 0.002 0.17 ± 0.01 0.21 ± 0.01
SM(38:1) 6.1 ± 0.1 8.3 ± 0.1 10.2 ± 0.3 15.3 ± 0.8
SM(39:1) 6.7 ± 0.1 7.4 ± 0.1 13.5 ± 0.5 18.2 ± 0.6
SM(40:1) 10 ± 1 16.0 ± 0.5 20 ± 1 21 ± 1
SM(40:2) 1.5 ± 0.1 2.4 ± 0.1 3.2 ± 0.1 4.0 ± 0.1
SM(42:1) 3.57 ± 0.02 3.1 ± 0.1 3.5 ± 0.1 4.34 ± 0.02
SM(42:2) 1.16 ± 0.02 1.00 ± 0.01 1.08 ± 0.04 1.4 ± 0.1
SM(44:1) 0.074 ± 0.003 0.057 ± 0.003 0.065 ± 0.003 0.085 ± 0.003
total SMs 49.5 ± 0.1 56.4 ± 0.1 71.7 ± 0.2 88.6 ± 0.2
Cholesteryl Esters
CE(16:0) 2.4 ± 0.1 5.5 ± 0.1 7.6 ± 0.2 8.7 ± 0.2
CE(18:1) 4.1 ± 0.1 7.6 ± 0.1 8.5 ± 0.2 8.82 ± 0.03
CE(18:2) 20 ± 1 55 ± 3 102 ± 8 110 ± 5
CE(20:5) 60 ± 1 85 ± 3 105 ± 3 162 ± 8
CE(22:5) 23 ± 2 ND ND ND
total CEs 109.5 ± 0.8 153.1 ± 1.2 223.1 ± 2.3 289.5 ± 2.6
Diglycerides
DG(32:1) 3.5 ± 0.1 12.3 ± 0.4 16.5 ± 0.8 20.7 ± 0.9
DG(32:2) 0.83 ± 0.02 2.90 ± 0.11 4.0 ± 0.1 5.8 ± 0.2
DG(34:1) 10 ± 1 33 ± 1 42 ± 1 55 ± 2
DG(34:3) 0.96 ± 0.03 2.3 ± 0.2 3.3 ± 0.1 4.6 ± 0.3
DG(36:2) 2.6 ± 0.1 25 ± 2 55 ± 4 118 ± 3
DG(36:3) 1.16 ± 0.1 12.9 ± 0.2 32 ± 3 69 ± 1

I DOI: 10.1021/acs.jafc.9b00204
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 2. continued
metabolites skim milk 1% milk 2% milk 3.25% milk
Cholesteryl Esters
DG(38:0) 1.6 ± 0.1 6.1 ± 0.1 38 ± 1 57 ± 1
DG(39:0) 2.5 ± 0.1 9.7 ± 0.2 32 ± 3 35 ± 2
DG(41:1) 1.8 ± 0.1 8.9 ± 0.2 45 ± 4 82 ± 2
DG(42:0) 0.7 ± 0.1 4.3 ± 0.1 10.1 ± 0.4 36 ± 1
DG(42:1) 0.12 ± 0.01 0.16 ± 0.01 0.26 ± 0.01 0.33 ± 0.02
DG(42:2) 1.5 ± 0.1 ND ND ND
DG(44:3) 0.7 ± 0.1 1.9 ± 0.1 29 ± 4 41 ± 1
DG-O(34:1) 16 ± 1 301 ± 15 448 ± 16 800 ± 5
DG-O(36:4) 0.70 ± 0.02 ND ND ND
total DGs 44.7 ± 0.2 420.5 ± 1.3 755.2 ± 2.5 1324.4 ± 1.3
Triglycerides
TG(44:1) 20 ± 1 331 ± 13 735 ± 28 2028 ± 48
TG(44:2) 6.5 ± 0.3 187 ± 9 275 ± 7 1095 ± 64
TG(44:4) 0.65 ± 0.03 14 ± 1 21 ± 1 109 ± 7
TG(46:2) 6.8 ± 0.2 200 ± 10 296 ± 12 993 ± 51
TG(48:1) 32 ± 2 421 ± 12 534 ± 11 2239 ± 86
TG(48:2) 11 ± 1 296 ± 15 439 ± 14 1355 ± 13
TG(48:3) 1.6 ± 0.1 76 ± 4 124 ± 13 573 ± 9
TG(49:1) 5.2 ± 0.1 73 ± 54 169 ± 4 383 ± 11
TG(49:2) 1.6 ± 0.1 43 ± 3 63 ± 3 196 ± 4
TG(50:1) 43 ± 1 498 ± 9 715 ± 9 2078 ± 111
TG(50:2) 20 ± 1 498 ± 7 845 ± 7 2030 ± 64
TG(50:3) 5.5 ± 0.1 108 ± 8 242 ± 7 815 ± 9
TG(50:4) 2.6 ± 0.1 73 ± 3 98 ± 8 407 ± 9
TG(51:1) 2.0 ± 0.1 56 ± 1 79 ± 3 302 ± 7
TG(51:2) 2.7 ± 0.1 69 ± 3 77 ± 4 354 ± 25
TG(51:3) 0.76 ± 0.02 19 ± 1 30 ± 4 103 ± 11
TG(51:4) 0.21 ± 0.02 8.6 ± 0.3 20 ± 2 71 ± 8
TG(51:5) 0.54 ± 0.02 41 ± 1 308 ± 397 450 ± 9
TG(52:2) 26 ± 4 668 ± 6 1072 ± 59 3094 ± 208
TG(52:3) 9.8 ± 0.1 275 ± 16 431 ± 7 1299 ± 30
TG(52:4) 3.0 ± 0.2 103 ± 6 167 ± 13 659 ± 23
TG(52:5) 0.29 ± 0.01 24 ± 1 79 ± 4 252 ± 6
TG(53:3) 0.58 ± 0.02 12.4 ± 0.4 24 ± 4 117 ± 5
TG(53:4) 0.23 ± 0.03 10.0 ± 0.3 20.4 ± 0.6 32 ± 2
TG(53:5) 1.2 ± 0.1 41 ± 2 100 ± 6 336 ± 15
TG(53:6) 2.9 ± 0.1 141 ± 6 399 ± 7 1090 ± 76
TG(54:2) 4.6 ± 0.1 77 ± 1 103 ± 5 252 ± 6
TG(54:3) 7.6 ± 0.2 164 ± 12 230 ± 16 738 ± 17
TG(54:4) 3.3 ± 0.1 85 ± 4 102 ± 7 496 ± 6
TG(54:5) 1.3 ± 0.1 39 ± 3 91 ± 3 314 ± 11
TG(54:6) 0.26 ± 0.02 7.5 ± 0.3 20 ± 1 89 ± 4
TG(55:6) 1.7 ± 0.1 59 ± 5 136 ± 9 616 ± 5
TG(55:7) 2.6 ± 0.1 127 ± 11.0 284 ± 42 1093 ± 74
TG(55:8) 1.5 ± 0.1 87 ± 30 286 ± 16 1054 ± 65
TG(55:9) 0.55 ± 0.03 32 ± 3 67 ± 1 378 ± 14
TG(56:6) 0.25 ± 0.01 6.4 ± 0.1 14 ± 1 85 ± 2
total TGs 230.3 ± 0.4 4969.9 ± 7.3 8695.4 ± 20.4 27575 ± 31
Ceramides
Cer(34:0) ND ND 0.125 ± 0.001 0.155 ± 0.003
Cer(34:1) 0.064 ± 0.001 0.13 ± 0.01 0.171 ± 0.004 0.22 ± 0.01
Cer(38:1) 0.07 ± 0.01 0.12 ± 0.01 0.144 ± 0.002 0.22 ± 0.01
Cer(40:1) 0.125 ± 0.003 0.227 ± 0.004 0.35 ± 0.01 0.45 ± 0.01
Cer(41:1) 0.199 ± 0.003 0.30 ± 0.01 0.472 ± 0.001 0.729 ± 0.003
Cer(42:1) 0.235 ± 0.002 0.26 ± 0.01 0.46 ± 0.01 1.08 ± 0.04
Cer(42:2) 0.052 ± 0.002 0.262 ± 0.004 0.357 ± 0.002 0.47 ± 0.01
total Cers 0.745 ± 0.003 1.299 ± 0.007 2.079 ± 0.004 3.324 ± 0.012
Amino Acids and Biogenic Amines
alanine 31 ± 3 29 ± 2 33 ± 1 31 ± 1
arginine 19 ± 2 21 ± 2 30 ± 2 21 ± 1

J DOI: 10.1021/acs.jafc.9b00204
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 2. continued
metabolites skim milk 1% milk 2% milk 3.25% milk
Amino Acids and Biogenic Amines
asparagine 5.3 ± 0.2 6.6 ± 0.3 6.5 ± 0.2 6.5 ± 0.3
aspartate 21 ± 2 19 ± 1 20 ± 2 19 ± 1
citrulline 5.0 ± 0.1 4.9 ± 0.2 5.0 ± 0.1 4.7 ± 0.1
glutamine 17 ± 1 17.0 ± 0.1 20 ± 2 10.0 ± 0.3
glutamate 257 ± 25 297 ± 8 260 ± 11 303 ± 9
glycine 65 ± 3 68 ± 3 72 ± 2 77 ± 3
histidine 2.8 ± 0.1 2.0 ± 0.1 3.1 ± 0.2 4.2 ± 0.1
isoleucine 4.8 ± 0.1 3.9 ± 0.1 4.5 ± 0.1 4.5 ± 0.1
lysine 22 ± 2 30 ± 1 21 ± 2 26 ± 1
methionine 1.0 ± 0.1 1.0 ± 0.1 0.9 ± 0.1 1.0 ± 0.1
phenylalanine 1.99 ± 0.01 1.7 ± 0.1 1.9 ± 0.1 1.9 ± 0.1
proline 30 ± 1 27 ± 2 30 ± 2 29 ± 2
serine 15 ± 1 15 ± 1 18 ± 1 15.7 ± 0.2
threonine 8±1 10 ± 1 9±1 8±1
tryptophan 2.1 ± 0.1 2.0 ± 0.1 2.0 ± 0.1 2.18 ± 0.02
tyrosine 2.3 ± 0.1 1.6 ± 0.1 2.30 ± 0.04 2.4 ± 0.1
valine 15 ± 1 13 ± 1 14 ± 1 16.7 ± 0.2
acetyl-ornithine 1.48 ± 0.04 1.5 ± 0.1 1.6 ± 0.1 1.72 ± 0.03
asymmetric dimethylarginine 0.10 ± 0.01 0.08 ± 0.01 0.09 ± 0.01 0.1 ± 0.01
alpha-aminoadipic acid 5.0 ± 0.1 4.3 ± 0.1 6.1 ± 0.1 5.4 ± 0.1
carnosine 0.46 ± 0.01 0.48 ± 0.01 0.50 ± 0.01 0.46 ± 0.01
creatinine 68 ± 2 81 ± 2 87 ± 1 83 ± 2
trans-OH-proline 5.1 ± 0.1 5.1 ± 0.1 4.6 ± 0.1 4.9 ± 0.1
spermidine 0.70 ± 0.03 0.88 ± 0.02 0.89 ± 0.02 0.84 ± 0.02
taurine 37 ± 2 37 ± 2 41 ± 2 38 ± 2
a
Not detected.

agreement between most of the NMR-measured concen-
trations and those reported in the literature and with LC−
HRMS analysis (Table 2). A total of 54 out of the 59 chemicals
identified and quantified by NMR for the four types of
commercial bovine milk had concentration values very close to
previously reported literature values (due to space limitations
not every literature value is presented in Table 2, only the most
representative or most reliable value is reported). More
specifically, a total of 91% (49/54) of these compounds
agreed very well with previously published literature values
(i.e., the average values from our own experiments fell within
one standard deviation of the literature value). The reasons for
the discrepancies for the other 5 compounds could be due to
the choice of sample type (single-source raw milk vs
commercial/pasteurized milk), the choice of the bovine
breed (Jersey vs Holstein), the animal diet, farming methods,
age, and/or stage of lactation of the animals being used. Other
discrepancies may be explained by their inherent volatility
(especially formate), chemical instability of certain compounds
(especially acetic acid), the choice of measurement technique
(NMR vs GC−MS vs enzyme-based colorimetric assays), their
low concentration (i.e., tryptophan), or interactions with the
solvent and metals (for example urea). Another important
consideration is that the majority of reported values in the
literature concerning organic acids or other metabolites are not
usually reported in food composition tables. As a rule, the
determination of metabolite concentrations by NMR are very
accurate since they involve direct measurement of the
compound. In other words, the NMR concentration values
reported in Table 1 are generally more reliable than those
measured via other technologies.
K DOI: 10.1021/acs.jafc.9b00204
LC−HRMS Compound Identification and Quantifica-
tion. As seen in Table 2, each commercially available milk type
yielded an average of 188 metabolites or metabolite species
(16 ACs, 62 PCs, 4 LysoPCs, 16 SMs, 5 CEs, 7 Cers, 15 DGs,
36 TGs, 19 amino acids, and 8 biogenic amines). Note that the
method we employed identifies lipid species (as opposed to
specific lipids) using their total acyl/alkyl chain content (i.e.,
PC(38:4)) rather than their unique structure. Therefore, each
phospholipid species identified by the Biocrates AbsoluteIDQ
p400 HR Kit typically corresponds to 5−10 possible unique
structures. Consequently, the total number of PCs identified
by this method corresponded to 434 unique structures. Similar
calculations for ACs, SMs, CEs, DGs, TGs, and Cers, yielded
29, 22, 5, 46, 768, and 7 unique structures, respectively. For the
four milk types (skim, 1%, 2%, 3.25%), the most abundant
compounds identified by LC−HRMS were TG(52:2) (26−
3094 μM), TG(50:1) (43−2078 μM), and TG(48:1) (32−
2239 μM). While some TGs are particularly abundant in high-
fat milk (2% and 3.25%), they could not be detected by NMR
since these (and other) lipids were removed by the 3-kDa
filtration units. From our results, the lower limit of
quantification by LC−HRMS, based on the AbsoluteIDQ
p400 HR kit, was 18−33 nM for the acylcarnitine AC(18:1-
OH), 19−47 nM for AC(14:2-OH)), and 25−38 nM for the
phosphatidylcholine species PC(40:2). Other low abundance
compounds include PC-O(40:5) (35−48 nM), LysoPC15:0
(38−45 nM), and PC-O(44:4) (41 nM). In addition, some
lipids were only detected in some but not all four types of the
commercial milk such as PC(30:1), CE(22:5), and DG(42:2),
which were found in skim milk but not in 1%, 2%, and 3.25%
milk. This variation is likely due to the fact that these
compounds were at their lower limit of detection/quantifica-
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 3. List of Milk Free Fatty Acids Detected by LC−MS/MS along with Their Measured or Reported Concentrations and
Their Standard Deviations and/or Ranges (As Measured in Micromolar)
metabolites skim milk 1% milk 2% milk 3.25% milk literature values
acetic acid (C2:0) 38 ± 1 37 ± 1 46 ± 1 43 ± 1 13−11316,44
propionic acid (C3:0) 1.0 ± 0.1 2.94 ± 0.12 2.0 ± 0.1 2.0 ± 0.1 0−1516
butyric/isobutyric acid (C4:0)a 37 ± 1 34.9 ± 0.2 24 ± 1 27 ± 1 20−5745
valeric/isovaleric acid (C5:0)b 1.8 ± 0.1 4.0 ± 0.2 4.18 ± 0.03 4.91 ± 0.13 2−1016
caproic acid (C6:0) 12.8 ± 0.2 14.3 ± 0.2 10.5 ± 0.2 11.8 ± 0.3 7−2045
heptanoic acid (C7:0) 0.441 ± 0.014 0.568 ± 0.024 0.448 ± 0.012 0.45 ± 0.02
caprylic acid (C8:0) 10.5 ± 0.4 13.4 ± 0.1 10.2 ± 0.2 8.8 ± 0.1 3−1445
pelargonic acid (C9:0) 0.251 ± 0.001 0.468 ± 0.012 0.568 ± 0.014 0.64 ± 0.02
capric acid (C10:0) 10.1 ± 0.1 14.3 ± 0.1 12.7 ± 0.2 11.4 ± 0.1 5−2045
undecylic acid (C11:0) 0.38 ± 0.01 0.51 ± 0.01 0.516 ± 0.014 0.45 ± 0.02
lauric acid (C12:0) 5.8 ± 0.1 9.8 ± 0.3 9.4 ± 0.3 10.2 ± 0.2 3−2145
tridecylic acid (C13:0) 0.284 ± 0.003 0.388 ± 0.002 0.415 ± 0.012 0.351 ± 0.012
myristic acid (C14:0) 10.5 ± 0.1 14.4 ± 0.3 17.5 ± 0.3 19 ± 1 7−4145
palmitic acid (C16:0) 29 ± 1 104 ± 4 54.3 ± 2.2 47.3 ± 2.3 13−10645
stearic acid (C18:0) 6.53 ± 0.12 11.5 ± 0.2 13 ± 1 17 ± 1 1−3645
oleic acid (C18:1) 11.1 ± 0.2 21.1 ± 0.4 24 ± 1 32 ± 2 5−8745
linoleic acid (C18:2) 7.04 ± 0.14 14 ± 1 14 ± 1 12.4 ± 0.3 3−1145
alpha-linolenic acid (C18:3) 3.78 ± 0.22 4.8 ± 0.2 6.01 ± 0.03 6.4 ± 0.2 2−845
a
Butyric/isobutyric acid (C4:0) could not be separated. bValeric/isovaleric acid (C5:0) could not be separated.

Table 4. List of Milk Vitamins Detected by LC−MS/MS along with Their Measured or Reported Concentrations and Their
Standard Deviations and/or Ranges (As Measured in Micromolar)
metabolites skim milk 1% milk 2% milk 3.25% milk literature values
vitamin A 0.265 ± 0.004 0.65 ± 0.02 0.70 ± 0.01 0.55 ± 0.01 0.052−4.8061
vitamin D3 0.015 ± 0.001 0.0147 ± 0.0002 0.019 ± 0.001 0.0435 ± 0.0002 0.00013−0.0002659a
vitamin OH D3 0.0004 ± 0.0001 0.0005 ± 0.0002 0.0006 ± 0.0001 0.0007 ± 0.0001 0.000562
vitamin D2 0.0003 ± 0.0001 0.0003 ± 0.0001 0.0003 ± 0.0002 0.0003 ± 0.0001 0.00013−0.002559
vitamin E 0.5 ± 0.1 0.5 ± 0.1 0.8 ± 0.1 0.9 ± 0.2 0.16−3.0261,63
vitamin K 0.002 ± 0.001 0.002 ± 0.001 0.004 ± 0.001 0.0042 ± 0.0004 0.002−0.0464
vitamin B1 1.53 ± 0.04 1.6 ± 0.1 1.6 ± 0.1 1.8 ± 0.1 0.75−1.7361
vitamin B2 5.8 ± 0.1 6.0 ± 0.1 5.8 ± 0.1 6.3 ± 0.1 2.79−5.1561
vitamin B3-amide 10.3 ± 0.3 10.6 ± 0.2 10.4 ± 0.2 11.3 ± 0.2 5.2−7.357,65
vitamin B5 20.3 ± 0.2 20.3 ± 0.3 20 ± 1 20 ± 1 14.6−16.459,60
vitamin B6 2.0 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 1.8 ± 0.1 2.0−3.061
vitamin B7 0.090 ± 0.002 0.080 ± 0.003 0.097 ± 0.004 0.090 ± 0.003 0.041−0.08659,60
vitamin B9 0.09 ± 0.01 0.087 ± 0.004 0.083 ± 0.001 0.09 ± 0.01 0.11−0.2659
vitamin B12 0.0046 ± 0.0001 0.0037 ± 0.0001 0.0037 ± 0.0001 0.0037 ± 0.0001 0.0026−0.004061
vitamin C 8.2 ± 0.3 34.7 ± 0.2 39 ± 1 34.0 ± 0.3 11.4−85.261
a
The concentration range is measured in milk with no added Vitamin D3.

tion and so there is some inherent “noise” in their reporting.
All of these compounds, along with their corresponding
estimated concentrations have all been entered into the
MCDB.
To the best of our knowledge, this is the first study of milk
conducted using the Biocrates AbsoluteIDQ p400 HR Kit.
Overall, where we found compound overlap, the results agreed
very well with reported literature values. We also found that for
the 13 metabolites that were measured by both LC−HRMS
and NMR, there was a generally good overall agreement with
the concentration values. Additionally, detailed comparisons
with the values obtained in milk with those reported in serum
showed that the lower and upper limits of detection were all
compatible with the kit specifications. This suggests that the
Biocrates p400 system is highly suited for analyzing bovine
milk. Altogether our LC−HRMS and NMR techniques
identified and quantified 234 metabolites or metabolite species,
which corresponds to 1387 unique chemical compounds.
L DOI: 10.1021/acs.jafc.9b00204
Given the propensity for LC−HRMS to measure lipids and
NMR to measure water-soluble metabolites, these two
methods appear to be complementary techniques for the
identification and quantification of small molecules in bovine
milk.
Milk Free Fatty Acids Identification and Quantifica-
tion Using LC−MS/MS. Using an in-house developed assay,
we were able to identify and quantify a total of 18 free fatty
acids in all four varieties of commercial bovine milk (skim, 1%,
2%, and 3.25%). The detected fatty acids, their concentrations,
and literature values are given in Table 3. All measured fatty
acids exhibited good agreement with previously published
literature values.16,44,45 Butyric/isobutyric acid and valeric/
isovaleric acids could not be separated using this method, and
so the combined concentration values are reported in Table 3.
Concentration data for four odd-chain free fatty acids (C7:0,
C9:0, C11:0, C13:0) is reported for the first time.
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 5. List of Milk Trace Elements Detected by ICP−MS along with Their Measured or Reported Concentrations and Their
Standard Deviations and/or Ranges (As Measured in Micromolar)
metabolites skim milk 1% milk 2% milk 3.25% milk literature values
Li 4.0 ± 0.1 3.0 ± 0.3 4±1 4±1 1−7266
B 54 ± 7 55 ± 9 58 ± 15 56 ± 11 17−8767
Na 15926 ± 1396 16345 ± 1257 16560 ± 1174 16197 ± 1765 17000−2800068
Mg 4011 ± 192 3815 ± 255 3857 ± 286 4325 ± 473 4000−600068
Al 147 ± 7 135 ± 23 148 ± 5 155 ± 14 43−15469
P 21317 ± 549 19781 ± 478 25132 ± 1669 22155 ± 2514 19000−2300068
K 33641 ± 811 27763 ± 5593 32800 ± 2591 35991 ± 3736 31000−4300068
Ca 32909 ± 2255 28909 ± 2255 26132 ± 1964 25529 ± 803 26000−3200068
Ti 0.4 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.02−328
V 1.4 ± 0.3 1.1 ± 0.3 1.4 ± 0.4 1.1 ± 0.2 1−270
Cr 1.50 ± 0.03 1.5 ± 0.2 1.5 ± 0.1 1.7 ± 0.2 0.7−369
Fe 4.0 ± 0.5 4.0 ± 0.2 4.0 ± 0.3 4.0 ± 0.3 1−1371
Mn 0.6 ± 0.1 0.5 ± 0.1 0.45 ± 0.02 0.50 ± 0.04 0.7−569
Co 0.03 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.03 ± 0.01 0.01−0.1428
Cu 2±1 1.1 ± 0.1 1.4 ± 0.2 1.3 ± 0.1 0.5−1028
Zn 65 ± 3 54 ± 8 65 ± 5 69 ± 6 15−6328
Ga 0.46 ± 0.01 0.47 ± 0.01 0.42 ± 0.01 0.45 ± 0.01 0.5−171
As 0.11 ± 0.05 0.13 ± 0.01 0.14 ± 0.01 0.14 ± 0.01 0.01−0.1428
Se 0.33 ± 0.04 0.3 ± 0.1 0.35 ± 0.02 0.36 ± 0.03 0.27−0.4970
Rb 17 ± 1 14 ± 2 11 ± 2 12 ± 2 0.5−1228
Sr 5.0 ± 0.1 4.0 ± 0.4 4.0 ± 0.2 4.0 ± 0.3 4−4628
Y 0.005 ± 0.001 0.005 ± 0.001 0.006 ± 0.001 0.005 ± 0.0004 0.001−0.13628
Zr 0.3 ± 0.2 0.020 ± 0.003 0.010 ± 0.004 0.010 ± 0.002 0.001−0.04772
Mo 0.29 ± 0.01 0.26 ± 0.02 0.25 ± 0.01 0.25 ± 0.02 0.006−0.2828
Pd 1.0 ± 0.2 1.3 ± 0.1 2±1 2±1 0.001−0.41372
Cd 0.4 ± 0.3 0.1 ± 0.1 0.5 ± 0.1 0.1 ± 0.1 0.008−0.86773
Te 0.01 ± 0.01 0.010 ± 0.004 0.03 ± 0.04 0.03 ± 0.01 0.004−0.08228
Cs 0.030 ± 0.001 0.010 ± 0.002 0.01 ± 0.01 0.010 ± 0.001 0.01−0.0470
Ba 1.40 ± 0.04 1.3 ± 0.1 1.4 ± 0.1 1.5 ± 0.1 1.3−1.670
La 0.002 ± 0.001 0.0010 ± 0.0002 0.002 ± 0.001 0.0010 ± 0.0002 0.0002−0.00470
Ce 0.007 ± 0.003 0.004 ± 0.001 0.0050 ± 0.0003 0.0040 ± 0.0003 0.0024−0.00770
Nd 0.003 ± 0.001 0.0017 ± 0.0003 0.0020 ± 0.0002 0.002 ± 0.001 0.0003−0.00428
Re 0.004 ± 0.001 0.004 ± 0.001 0.0050 ± 0.0002 0.0050 ± 0.0004 0.005−0.00670
Tl 0.0030 ± 0.0001 0.0050 ± 0.0004 0.005 ± 0.001 0.004 ± 0.001 0.003−0.00470
Pb 0.02 ± 0.01 0.020 ± 0.001 0.02 ± 0.01 0.020 ± 0.002 0.002−0.0971

Vitamin Identification and Quantification Using LC−
MS/MS. Milk is widely considered to be a good source of
vitamins which not only serve as vital enzyme cofactors but
which also play an important role in scavenging free radicals of
reactive oxygen and nitrogen groups.46 While we had no
expectations that our work would reveal anything new
regarding the vitamin content of milk, we used this assay as
an opportunity to assess the reliability of our in-house LC−
MS/MS methods for vitamin analysis. Using this tandem MS-
based vitamin analysis, we were able to identify and quantify a
total of 15 vitamins in all four varieties of commercial bovine
milk (skim, 1%, 2%, and 3.25%). The detected vitamins, their
concentrations, and literature values are given in Table 4.
Except for vitamin D3, all other vitamins exhibited good
agreement with previously published literature values. With
regard to vitamin D3, our LC−MS/MS results showed a
concentration range of 15−44 nM for different varieties of
milk, while the literature range was reported as 0.13−0.26 nM.
We speculate that this ∼100-fold difference is due to the fact
that our commercial milk varieties were fortified with
additional vitamin D3, whereas the reported concentration
range from the literature were related to those milk varieties
which had no added vitamin D3. For the four varieties of milk
M DOI: 10.1021/acs.jafc.9b00204
we analyzed, the most abundant vitamins identified and
quantified were vitamin C (8.2−39 μM), vitamin B5 (20−20.3
μM), vitamin B3-amide (10.3−11.3 μM), and vitamin B2 (5.8−
6.3 μM). As seen in Table 4, our measured values for vitamin
B5 and vitamin B3-amide tended to be slightly higher than
those reported elsewhere. These higher B vitamin levels may
be due to the fact that Canadian dairy cattle are grain fed (as
opposed to grass fed) and the feed is often supplemented with
vitamins. As might be expected, the concentration of the fat-
soluble vitamins in milk (A, D, and E) varied strongly with the
milk fat content of the different milk samples. Typically, the
fat-soluble vitamins including vitamin D3 (0.015−0.044 μM)
and vitamin E (0.5−0.9 μM) are present in higher
concentration in 3.25% milk as compared to skim milk (the
highest and lowest concentrations for vitamin A (0.27−0.70
μM) were related to 2% and skim milk, respectively). The least
abundant vitamins quantified by LC−MS were vitamin D2
(0.0003 μM) and vitamin OH D3 (0.0004−0.0007 μM). As
can be seen from inspection of Table 4, there is an excellent
agreement between our values and those reported in the
literature.
ICP−MS Trace Element Identification and Quantifi-
cation. Milk and other dairy products make an important
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Table 6. Comparison of Concentrations of 18 Metabolites between LC−HRMS and NMR Methods along with Their Measured
or Reported Concentrations and Their Standard Deviations and/or Ranges (As Measured in Micromolar)
LC−HRMS NMR
metabolites skim milk 1% milk 2% milk 3.25% milk skim milk 1% milk 2% milk 3.25% milk average difference (%)
glycine 65 ± 3 68 ± 3 72 ± 2 77 ± 3 LOa LO LO LO
L-alanine 31 ± 3 29 ± 2 33 ± 1 31 ± 1 34 ± 3 32 ± 3 33 ± 2 32 ± 1 5.5
L-arginine 19 ± 2 21 ± 2 30 ± 2 21 ± 1 21 ± 3 27.5 ± 7.2 27 ± 12 31.4 ± 10.3 16.1
L-asparagine 5.3 ± 0.2 6.6 ± 0.3 6.5 ± 0.2 6.5 ± 0.3 6±2 6±1 7±1 8±1 8.1
L-aspartate 21 ± 2 19 ± 1 20 ± 2 19 ± 1 21 ± 3 20 ± 1 19 ± 3 19 ± 3 0
L-glutamine 17 ± 1 17.0 ± 0.1 20 ± 2 10.0 ± 0.3 LO LO LO LO
L-glutamate 257 ± 25 297 ± 8 260 ± 11 303 ± 9 271 ± 21 259 ± 10 263 ± 15 260 ± 23 5.9
L-histidine 2.8 ± 0.1 2.0 ± 0.1 3.1 ± 0.2 4.2 ± 0.1 4±2 3±1 2±1 4±1 7.2
L-isoleucine 4.8 ± 0.1 3.9 ± 0.1 4.5 ± 0.1 4.5 ± 0.1 4.7 ± 0.7 4.4 ± 0.5 4.2 ± 0.2 3.9 ± 0.6 2.9
L-lysine 22 ± 2 30 ± 1 21 ± 2 26 ± 1 25 ± 2 18 ± 9 23 ± 3 24 ± 5 9.5
L-proline 30 ± 1 27 ± 2 30 ± 2 29 ± 2 27 ± 5 25 ± 1 30 ± 1 27 ± 3 6.2
L-serine 15 ± 1 15 ± 1 18 ± 1 15.7 ± 0.2 LO LO LO LO
L-threonine 8±1 10 ± 1 9±1 8±1 LO LO LO LO
L-tryptophan 2.1 ± 0.1 2.0 ± 0.1 2.0 ± 0.1 2.18 ± 0.02 3±1 3±1 3±2 3±2 36.7
L-tyrosine 2.3 ± 0.1 1.6 ± 0.1 2.30 ± 0.04 2.4 ± 0.1 4±1 4±1 2.6 ± 0.3 3±1 45.0
L-valine 15 ± 1 13 ± 1 14 ± 1 16.7 ± 0.2 13 ± 1 13 ± 1 13 ± 1 13 ± 1 12.1
creatinine 68 ± 2 81 ± 2 87 ± 1 83 ± 2 66 ± 2 66 ± 7 70 ± 6 71 ± 4 15.5
taurine 37 ± 2 37 ± 2 41 ± 2 38 ± 2 LO LO LO LO
a
Lactose overlap.

contribution to the supply of trace elements in the human diet.
Many trace elements or minerals are considered essential for
enzyme function, general metabolism, skeletal growth,
reproduction, and overall health.47 Bovine milk is known to
contain many essential minerals such as calcium, phosphorus,
magnesium, potassium, and sodium. It also contains a wide
range of lower-abundance trace elements including zinc,
copper, iron, and manganese. To experimentally determine
the trace elemental composition of milk samples, we used
ICP−MS. This technique is often considered the “gold
standard” for the characterization of the elemental composition
of biological samples. Our multielemental analysis of bovine
milk using ICP−MS provided quantitative results for 35
metabolites or trace minerals. The results are shown in Table
5. Over all four varieties of milk, the most abundant trace
elements identified and quantified by ICP−MS were potassium
(28−36 mM), calcium (26−33 mM), phosphorus (20−25
mM), and sodium (16−17 mM), while the least abundant
compounds quantified by ICP−MS were lanthanum (1−2
nM), neodymium (2−3 nM), thallium (3−5 nM), and yttrium
at (5−6 nM). All of these trace minerals originate from the
feed and water given to the dairy cows.
All 35 of the trace elements identified and quantified in the
four types of commercial bovine milk we analyzed had
concentration values previously reported in the literature. All
exhibited good agreement with previously published literature
values (i.e., the average values from our own experiments fell
within one standard deviation of the literature value). Where
there are some modest disagreements, we suspect that these
could be due to the same reasons mentioned previously,
including the choice of sample type (single-source raw milk vs
commercial/pasteurized milk), the bovine breed, the diet,
farming methods, age, and/or stage of lactation of the animals
being used. Other differences may arise from the choice of
measurement technique (ICP−MS vs electrode measure-
ment). As a general rule, ICP−MS is considered as a gold
standard for the identification and quantification of trace
metals;18 therefore, we tend to have higher confidence in the
N DOI: 10.1021/acs.jafc.9b00204
values derived via ICP−MS over those measured by other
(older or less sensitive) technologies.
Comparison among Different Metabolomics Plat-
forms. We used four different metabolic profiling methods
to experimentally characterize as much of the chemical
composition of bovine milk as possible: (1) NMR, (2) LC−
HRMS, (3) LC−MS/MS, and (4) ICP−MS. Using these four
experimental techniques, we were able to identify a total of 296
distinct metabolites or metabolite species. This corresponds to
1316 lipids with unique structures and 131 nonlipids with
unique structures. To the best of our knowledge, this
represents the largest number of metabolites or chemicals
experimentally (and quantitatively) measured for any milk
analysis reported to date. The chemical coverage we achieved
was maximized by using as many different platforms as possible
and carefully optimizing the metabolite coverage of each
platform. Some platforms clearly performed better than others.
Over the four milk varieties, 59, 33, and 35 compounds with
unique structures were identified and quantified using NMR
spectroscopy, LC−MS/MS, and ICP−MS respectively, while
LC−HRMS identified and quantified 1316 lipids (with unique
structures) as well as 27 nonlipid compounds. NMR and LC−
HRMS were able to identify a common set of 13 metabolites
while NMR and LC−MS/MS identified eight common
metabolites (niacinamide, pantothenic acid, acetic acid, butyric
acid, capric acid, caprylic acid, propionic acid, and valeric acid).
The differences in metabolite coverage from the different
technologies are largely due to differences in sensitivity,
detection/instrument biases, and separation protocols as well
as differences in compound stability, solubility, volatility, and
other intrinsic chemical factors.
While several pairwise platform comparisons have already
been discussed, it is perhaps instructive to look at how two
different platforms (NMR and LC−HRMS) functioned in the
identification and quantification of the one group of
compounds that both platforms measured: amino acids
(Table 6). Comparison of the free amino acid concentrations
as measured by these two platforms showed that the
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
quantitative results were in very good agreement with most
differences being between 0 and 16% (0% for aspartate, 16%
for arginine). The most significant exceptions were for
tryptophan (36%) and tyrosine (45%) with the concentrations
reported by NMR being 3 μM for tryptophan and 3−4 μM for
tyrosine, whereas the concentrations reported by LC−HRMS
were 2 μM for tryptophan and 2−2.5 μM for tyrosine. These
values are at the lower limit of sensitivity for NMR and so the
differences are likely due to instrumental noise. The most
abundant amino acid detected by both methods was glutamate
(NMR = 271 μM in skim milk and LC−HRMS = 303 μM in
3.25% milk), while the least abundant ones were tryptophan
and tyrosine. Glycine, glutamine, serine, threonine, and taurine
were quantified only by LC−HRMS but not by NMR. This is
because the NMR signals corresponding to these amino acids
were completely covered by the dominant lactate peaks.
Because of their fundamentally different separation and
detection technologies, different metabolomics platforms tend
to target or detect different classes of metabolites. For instance,
NMR is relatively “untargeted” but is biased toward highly
abundant, water-soluble compounds. Other methods were
quite targeted, with ICP−MS being limited only to metal ions
and LC−HRMS (the AbsoluteIDQ p400 HR Kit) being
limited to a preselected set of 408 compounds. While this
study did employ a relatively wide range of metabolomic
platforms (NMR, LC−MS/MS, HRMS, ICP−MS) it did not
use all available detection tools (GC−MS, GC × GC−TOF)
nor did it explore all available separation protocols (e.g., solid
phase extraction and enrichment, immune or ELISA detection,
chemical derivatization, etc.). However, for this particular
study, we wanted to address the question of how well a cross
section of commonly accessible metabolomic technologies or
platforms could perform in identifying and quantifying
metabolites in commercial cow’s milk. Overall, LC−HRMS
appears to be the most suitable method for milk character-
ization, especially in terms of its broad coverage (primarily of
lipids) and its amenability to quantification. It also requires
very little sample volume (10 μL), is relatively inexpensive (on
a per sample basis), largely automated, and offers a high-
throughput route for measuring metabolites. The other
methods, such as LC−MS/MS, NMR, and ICP−MS offer
complementary data to LC−HRMS. However, they do not
provide the breadth of coverage, the level of automation nor
the sensitivity available via LC−HRMS.
Comparison of Bovine Milk to Other Biofluids.
Altogether our combined literature review and experimental
efforts led to the identification and/or quantification of 972
metabolites or metabolite species, corresponding to 2355
unique metabolite structures in bovine milk. The vast majority
of the compounds identified are triglycerides (1053). A
number of compounds were identified or reported for the
first time in bovine milk using our experimental methods.
These include LysoPC(18:2) (0.16−0.19 μM), PC(28:1)
(0.54−0.89 μM), and TG(48:3) (1.6−573 μM). In addition to
the expected endogenous compounds typically found in milk,
our literature review revealed the presence of a large number of
phytochemicals, such as salicylate, equol, daidzein, formono-
netin, and genistein23,48−50 as well as many contaminants or
pesticide residues such as DDT, HCH, and endosulfan.43
Comparison of the bovine milk metabolome to other
published biofluid metabolomes characterized by our lab or
compiled through literature surveys shows that bovine milk is
somewhat simpler than human saliva,51 human serum,20
O DOI: 10.1021/acs.jafc.9b00204
Article
human urine,19 or human cerebrospinal fluid.18 In particular,
the total number of compounds identified for human serum,
urine, saliva, and cerebrospinal fluid were 25 372, 4 236, 1 233,
and 450 compounds, respectively. From a compositional
standpoint, bovine milk also appears to be somewhat simpler
than human milk, especially with regard to its carbohydrate or
oligosaccharide content.52 In addition, human milk is reported
to have a high interindividual variation in its components such
as milk fat, protein, and minerals that may be due to various
environmental factors and dietary patterns specific to the
mother.53
In conclusion, we have conducted a comprehensive
literature review along with a comprehensive, quantitative
metabolomic investigation of four varieties of commercial
cow’s milk. The metabolomic studies used combination of
various analytical techniques, including NMR, LC−HRMS,
LC−MS/MS, and ICP−MS, while the literature review was
facilitated by computer-aided literature mining. A total of 168
compounds or compound species (corresponding to 1285
unique structures) are being reported for the first time,
including 2 LysoPCs, 7 Cers, 15 DGs, 16 SMs, 35 TGs, and 62
PCs and 31 other compounds, all of which have been added to
the MCDB. As might be expected, our work clearly shows that
the concentration of lipid compounds is much higher in high
fat (3.25%) milk compared to lower fat (2%, 1% and skim)
varieties, while the concentration of nonlipid compounds is not
significantly different among the different milk varieties. Our
experimentally acquired data corresponds to a total of 61% of
the total number of milk metabolites/chemicals reported in the
MCDB. This compilation represents the most complete
chemical inventory or chemical assessment of bovine milk
that has been achieved to date. All this information along with
other details regarding concentration ranges, chemical
structures, names, chemical classes, NMR and MS spectra,
other physicochemical properties, and associated references are
publicly accessible in the MCDB.
This study is most certainly not the final word on the
chemical composition of bovine milk. Over the coming years,
we plan to further characterize the bovine milk metabolome
using other metabolomics techniques, i.e., GC−MS, GC ×
GC−MS (to help identify volatile compounds), untargeted
FT−MS, and more extensive targeted LC−MS/MS techniques
to compare and extend the metabolite coverage. Similar to
previous metabolomics studies presented from our lab, one of
our main objectives with this study was to help advance the
field of quantitative metabolomics by demonstrating the power
and potential of these techniques to comprehensively
characterize common biofluids. The results presented here
also have implications far beyond the field metabolomics,
especially given the economic importance of bovine milk in the
food industry and its importance in human and animal
nutrition. We expect these data to serve as a benchmark in
comparing various technologies and assessing future methodo-
logical improvements in bovine milk research. In the
meantime, it is hoped that the MCDB will provide a reliable
source for metabolomics researchers, animal scientists, food
chemists, nutritional scientists, physicians, and consumers by
providing a comprehensive, easy-to-use, and highly centralized
Web-based resource on the chemical composition of
commercial cow’s milk.
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
■ AUTHOR INFORMATION
Corresponding Author
*Phone: 780-492-0383. Fax: 780-492-1071. E-mail: david.
wishart@ualberta.ca.
ORCID
Hasan Badran: 0000-0002-8072-5233
David S. Wishart: 0000-0002-3207-2434
Funding
This research was supported by grants from Canadian
Institutes of Health Research (CIHR) (Grant 111062), Alberta
Innovates−Bio Solutions (AIBS) (Grant QFH-11-045),
Alberta Livestock and Meat Agency (ALMA) Grant
(2012Q009R), and Genome Alberta/Genome Canada
(Grant 12103).
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors would like to thank Dr. Beomsoo Han for
assisting in figure production from the experimental data. We
also thank Seyed Ali Goldansaz, Edison Dong, Fozia Saleem,
and Rahmatollah Rajabzadeh for their help in preparing milk
samples and assisting with the literature review.
■
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R DOI: 10.1021/acs.jafc.9b00204
J. Agric. Food Chem. XXXX, XXX, XXX−XXX