Journal of AOAC INTERNATIONAL, 104(2), 2021, 368–383

doi: 10.1093/jaoacint/qsaa154
Advance Access Publication Date: 7 December 2020
Review Paper

ENVIRONMENTAL CHEMICAL CONTAMINANTS

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Synthetic Musk Compounds in Human Biological
Matrices: Analytical Methods and Occurrence—A Review
Guru Prasad Katuri,1 Xinghua Fan,1 Ivana Kosarac,2 Shabana Siddique,1 and
Cariton Kubwabo1,*

1
Environmental Health Science and Research Bureau, Health Canada, Ottawa Ontario, Canada, 2Science
Division, Tobacco Control Directorate, Health Canada, Ottawa Ontario, Canada

*Corresponding author’s e-mail: Cariton.Kubwabo@canada.ca

Abstract
Extensive use of synthetic musk compounds (SMs) in numerous consumer and personal care products has resulted in direct
human exposures via dermal absorption, inhalation of contaminated dust and volatilized fragrances, and oral ingestion of
contaminated foods and liquids. SMs and their metabolites are lipophilic, hence commonly detected in various biological
matrices such as blood, breast milk, and adipose tissue. Appropriate analytical techniques are needed to detect and
quantify SMs in biological matrices to assess their potential effects on human health. Different methods to process and
analyze SMs in biological matrices, including sample-pretreatment, solvent extraction, cleanup, and instrumental analysis,
are presented in this review. The concentration levels of selected musk compounds in biological samples from different
countries/regions are summarized. Finally, research gaps and questions pertaining to the analysis of SMs are identified and
suggestions made for future research studies.

Synthetic musk compounds, or simply synthetic musks (SMs),
are widely used as fragrances in various consumer and personal
care products such as perfumes, soaps, body lotions, shampoos,
fabric softeners, laundry detergents, and air fresheners (1). SMs
are produced to replace expensive natural musk compounds
extracted from a gland of the musk deer or musk ox but are
structurally and chemically different from natural musks. Their
physico-chemical properties are similar to some synthetic
chemicals such as polychlorinated biphenyls (PCBs), organo-
chlorine pesticides (OCs), and flame retardants (e.g., polybromi-
nated diphenyl ethers or PBDEs), which are known to be
persistent in the environment and biomagnify through the food
chain. Synthetic musk compounds also display the tendency to
bioaccumulate (2). They are mainly divided into two types: nitro
musks and polycyclic musks. Nitro musks include musk xylene
(MX), musk ketone (MK), musk ambrette (MA), musk moskene
(MM), and musk tibetene (MT). Polycyclic musks include
Galaxolide (HHCB), Tonalide (AHTN), Celestolide (ADBI),
Phantolide (AHMI), Traseolide (ATII), and Cashmeran (DPMI).
The physico-chemical properties and chemical structures of
these SMs are listed in Table 1 and their chemical structures il-
lustrated in Figure 1.
Extensive use of synthetic musk compounds has resulted in
their ubiquity in the environment. Several nitro musks (e.g., MX
and MK) were first reported in water, fish, and mussels from
Tokyo in 1981 (6), which provided the first evidence of their
presence in the aquatic environment, food chain, and potential
exposure for humans. Since then, synthetic musk compounds
have been detected in various environmental samples and ma-
rine ecosystems, such as in freshwater rivers (7), lakes (8), sedi-
ment (9), soil (10), sewage sludge (11), effluent from wastewater
treatment plants (12), and in the dust and air (13, 14). Due to
their lipophilic nature (e.g., logKow values of 5.4–6.3; Table 1),
they have also been reported in human biological matrices in-
cluding serum, umbilical cord blood (15, 16), breast milk (16, 17),
and adipose tissues (18, 19).

Received: 6 August 2020; Revised: 16 September 2020; Accepted: 20 October 2020

C AOAC INTERNATIONAL 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com
V

368
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 369

Table 1. Chemical and physical properties of synthetic musks

Molecular Vapor
Trade name mass, pressure, Log
(abbreviation) Chemical CAS RN Formula g/mol Pa Kow Reference

Cashmeran (DPMI) 6,7-dihydro-1,1,2,3,3-pentamethyl- 33704-61-9 C14H22O 206.3 0.530 6.4 (3)

4[5H]indanone
Celestolide (ADBI) 4-acetyl-1,1-dimethyl-6-tert- 13171-00-1 C17H24O 244.3 0.019 5.4 (4)
butylindane
Phantolide (AHMI) 6-acetyl-1,1,2,3,3,5- 15323-35-0 C17H24O 244.3 0.020 5.8 (4)
hexamethylindane

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Galaxolide (HHCB) 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hex- 1222–05-5 C18H26O 258.4 0.073 5.9 (4)
amethylcyclopenta-c-2-benzopyran
Traesolide (ATII) 5-acetyl-1,1,2,6-tetramethyl-3- 68140-48-7 C18H26O 258.4 0.009 6.3 (4)
isopropylindane
Tonalide (AHTN) 7-acetyl-1,1,3,4,4,6- 1506-02-1 C18H26O 258.4 0.061 5.8 (4)
hexamethyltetrahydronaphthalene
Musk ambrette (MA) 4-tert-butyl-2,6-dinitro-3- 83-66-9 C12H16N2O5 268.2 0.002 4.2 (3)
methoxytoluene
Musk ketone (MK) 4-tert-butyl-2,6-dimethyl-3,5- 81-14-1 C14H18N2O5 294.3 0.00004 4.3 (4)
dinitroacetophenone
Musk moskene (MM) 1,1,3,3,5-pentamethyl-4,6- 116-66-5 C14H18N2O4 278.3 0.00022 5.4 (3)
dinitroindane
Musk xylene (MX) 1-tert-butyl-3,5-dimethyl-2,4,6- 81–15-2 C12H15N3O6 297.3 0.00003 4.9 (4)
trinitrobenzene
Musk tibetene (MT) 1-tert-butyl-3,4,5-trimethyl-2,6- 145–39-1 C13H18N2O4 266.3 0.0064 5.2 (3)
dinitrobenzene
Musk T 1,4-Dioxacycloheptadecane-5,17- 105–95-3 C15H26O4 236.2 0.00006 4.7 (5)
dione
Muscone 3-Methylcyclopentadecanon 10403–00-6 C16H30O 238.4 0.03 5.9 (5)
Habanolide Oxacyclohexadec-12-en-2-one, (12E)- 34902–57-3 C15H26O2 238.4 0.02 – (5)

There have been several toxicity studies on nitro musks. In
vitro tests on human MCF-7 breast cancer cells have shown the
estrogenic activity of MK, MX, and a major metabolite of MX (p-
amino-MX) (20, 21); on the contrary, their anti-estrogenic activ-
ity has been reported in fish (22). MX and MK are also reported
to be strong inducers of phase I liver enzymes and may in-
crease/influence the genotoxicity of other chemicals (e.g., ben-
zo[a]pyrene) in a species-specific manner (23, 24). MX was
prohibited in 2009 by the international fragrance association
(IFRA) since it was identified as a persistent and bioaccumula-
tive compound (25). In Regulation (EC) No 1223/2009 of the
European Parliament and of the Council of November 30, 2009,
MK was added to the list of substances prohibited in cosmetic
products (26). Owing to its neurotoxicity and potential photo-
sensitivity in humans, the use of musk ambrette (MA) was
phased out and finally banned by the European commission in
1995 (27). The use of other nitro musks, including MM and MT in
fragrant products, have been also banned by IFRA because of
adverse outcomes (23). However, nitro musks are not banned in
the United States and Canada. In the United States, the produc-
tion volume for MX was reported to be about 250 tons from 1986
to 2006 (28), whereas the production volume of MK was 12.5–50
tons in 2014 and less than 12.5 tons in 2015 (29). In Canada, MX
and MK were both imported in quantities between 1.1 and 11
tons in 2008, respectively. In the same year MX and MK were
also manufactured in quantities less than 0.11 tons and be-
tween 0.11 and 1.1 tons, respectively (30). According to a recent
Canadian draft screening assessment of nitro musks, these
chemicals were not considered to be harmful to the environ-
ment at the current levels of exposure and occurrence (30). The
same screening assessment also specifies that there is a
potential of increasing risk to the environment with the in-
crease in levels of nitro musks in Canada. Therefore, the
Canadian government is currently considering follow-up activi-
ties to document usage levels and resulting exposure of nitro
musks, specifically MX and MK.
Concerns regarding the toxicity and persistence of nitro
musks gradually led to increasing use of polycyclic musks, since
the latter were believed to be less toxic and easily degradable in
the environment (24). However, ATTN showed strong neurotoxic
effects (31), therefore its production was discontinued from 1980
(32). The most widely used polycyclic musks are HHCB and
AHTN. Global production of HHCB and AHTN in 2004 was about
1000 tons and 5000 tons, respectively (33). The consumption of
HHCB and AHTN in Europe in 2004 was 1307 tons and 247 tons,
respectively (34, 35). The production volume of HHCB in the
United States was up to 4000 tons per year during 2012–2015,
which was up to 3 times higher than the volume in 2011 (36).
AHTN is the second highest volume polycyclic musk in terms of
global production: in North America in 2011, reported use vol-
umes were in the range of 100–150 tons (37). However, polycyclic
musks have demonstrated to have some adverse health effects.
For example, in vitro and/or in vivo studies in zebra fish, rats,
and humans, HHCB, AHTN, AHMI, and ATTN have displayed en-
docrine disrupting properties and can affect sexual development
(38–40). Exposure to these compounds during pregnancy may
pose a risk to the developing child because of the susceptible
window of exposure. The adverse outcomes may range from ter-
atogenicity, low birth weight, and delayed neurological develop-
ment (41). Furthermore, HHCB and AHTN along with other
polycyclic and nitro musks are listed on the US EPA’s Toxic
Substances Control Act (TSCA) Inventory (42).
 370 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
O O2N NO 2
O2N NO 2
NO 2
MA
MX
O
O
AHTN
HHCB
O
O
O
O O
Musk T
Figure 1. Chemical structures of nitro, polycyclic, and macrocyclic musks compounds.
Because of the concerns regarding toxicity of nitro and poly-
cyclic musks, new types of SMs such as macrocyclic and alicy-
clic musks were developed and gradually entered into the
market. Macrocyclic musks are easily degradable and thus con-
sidered to be safer than both nitro and polycyclic musks; how-
ever, due to the high production cost, their use is limited (43).
Limited biomonitoring data is available for Muscone, musk T,
and Habanolide. The physico-chemical properties of these three
macrocyclic compounds are listed in Table 1 and their chemical
structures shown in Figure 1. The newest group, the alicyclic
musks, were introduced as alternatives to other musk com-
pounds and are considered to be cost-effective and presumed to
be biodegradable (44). They are considered the fourth genera-
tion of musk compounds; however, data on their use is still very
scarce, and thus not discussed in this review.
There is scarcity of data on metabolism and distribution of
musk fragrances in humans. The metabolism of musks is typi-
cally the result of biotic or abiotic degradation of the parent
compound, which often occurs in wastewater treatment plants,
in water and sediments of receiving surface waters and in
aquatic organisms. Four musk metabolites have been studied in
environmental samples: 2- and 4-amino musk xylene, 2-amino-
MK, and HHCB-lactone (45, 46). Dermal absorption and deposi-
tion study of nitro musks revealed benzylic alcohol metabolites
of MA and MX, which are formed by the oxidation of aryl methyl
in humans (47). Riedel et al. (1999) investigated 4-amino-MX
metabolite in human urine, the binding of which to hemoglobin
in blood samples was used as a biomarker for MX human expo-
sure studies (48). Quantification of polycyclic musk metabolites
in human samples is only limited to the HHCB-lactone.
Biomonitoring of environmental contaminants (e.g., SMs) is
important to assess their direct human exposure. The most
commonly used matrices for biomonitoring are milk and blood
(41, 49, 50). Adipose fat also gives an accurate measure but col-
lection of adipose tissue is more invasive when compared to
blood or milk (51). SMs are often not included in routing biomo-
nitoring programs and thus their biomonitoring data are rather
limited. It might be due to the lack of standardized analytical
O2N NO 2
O2N O2N NO 2
NO 2 O
MT MK
MM
O O
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O
AHMI ATII DMPI
O ADBI
O
O
Muscone Habanolide
methods, although different analytical methods have been de-
veloped worldwide, trying to accurately measure the levels of
SMs in biological matrices. A few papers have reviewed the ana-
lytical methods in general for the measurements of environ-
mental contaminants (including musks and other compounds
of emerging concern) in different environmental and biological
matrices (44, 52–55). The occurrence data of SMs in biological
samples are sporadically summarized with other environmen-
tal contaminants such as plasticizers, preservatives, and surfac-
tants (53, 55). This paper attempts to review recent advances in
analytical methods (including sample preparation, instrumen-
tal analysis, and challenges associated) and concentration lev-
els of selected SMs in biological matrices including milk, blood,
and adipose tissues. Research gaps in analytical methods will
be identified and suggestions made for future biomonitoring re-
search on SMs.
Analytical Methods
The compositions of biological samples (e.g., milk, blood, and
adipose tissues) are complicated and levels of SMs in biological
samples are low, thus posing several analytical challenges such
as low and/or variable recoveries, poor selectivity and sensitiv-
ity, and difficulty in chromatographic separation. The general
procedures for biomonitoring include extraction of analytes
from the sample matrices, sample cleanup to remove interfer-
ing compounds, and finally separation and detection by an in-
strument such as gas chromatogram (GC) coupled with mass
spectrometry (MS).
Sample Extraction
Human samples (e.g., blood, serum, and plasma) often contain
large amounts of protein. To disrupt the binding of protein with
other compounds and to prevent clogging during subsequent
sample cleanup by solid phase extraction (SPE), protein precipi-
tation (PPT) using different solvents (e.g., acetonitrile, ethanol,
and acetone) is a common practice prior to solvent extraction of
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
analytes from biological samples (56–59). Acidic precipitation
using formic acid has also been employed (60–62). For milk and
tissue samples, sodium sulfate (51, 63–67) and potassium oxa-
late with ethanol and diethyl ether (68–73) have been used to
grind and further disintegrate the samples during the extraction
process. Specific details of PPT have been presented in Table 2.
Extraction of analytes from biological samples can be per-
formed in many ways depending on sample matrices, such as
liquid-liquid extraction (LLE), Soxhlet extraction, pressurized
liquid extraction (PLE), microwave-assisted extraction, solid
phase microextraction (SPME), and stir bar sorptive extraction
(SBSE). LLE is one of the most commonly used sample extraction
technique for SMs in biological matrices due to its simplicity
and versatility. Non-polar solvents such as pentane, hexane,
and heptane prove to be a reasonable choice for LLE as has been
shown in many studies with acceptable recoveries and low de-
tection limits (16, 57, 58, 60, 68, 69). Detailed information on LLE
is summarized in Table 2. However, it is labor-intensive and
consumes large amount of solvents. Soxhlet extraction is an-
other extraction technique with high extraction efficiency. For
example, Soxhlet extraction was used to extract nitro and poly-
cyclic musks from serum, umbilical cord serum, milk and tissue
samples using hexane and DCM (18, 41, 51, 67, 75). The recover-
ies of 90–108% and detection limits of 1.0 ng/g in serum, 0.5 ng/g
in milk and 1–5.0 ng/g in tissue were achieved. However, the col-
lection vessels (e.g., round flask bottles), condensers, and
extractors must be thoroughly cleaned for reuse, which is not
only time-consuming but may introduce potential contamina-
tion as well; therefore, Soxhlet extraction is not suitable for
trace analysis of many samples. In order to minimize cross-
contamination, use of disposable glassware (e.g., extraction
tubes, collection vessels, and vials) is preferred. Pressurized liq-
uid extraction (PLE) (also called accelerated solvent extraction,
or ASE), is one advanced technique for solvent extraction. The
extraction is performed at temperatures above the boiling point
of a solvent at high pressure, thus greatly reducing the extrac-
tion time and solvent consumption. For example, Wang et al.
(2011) used ASE to extract nitro and polycyclic musks from milk
samples with hexane/DCM and achieved quantification limits
of 0.6–5.4 ng/g and recoveries of 82.4–112%, which were compa-
rable with normal LLE approaches (78). In addition, ASE can be
fully automated; therefore, this technique could be a better
choice for extraction of musks from biological matrices.
However, this technique has not found widespread applications
probably due to the high cost of the equipment.
Direct extraction without solvent or even without sample
cleanup, such as solid phase microextraction (SPME) or stir bar
sorptive extraction (SBSE), can also be used for the analysis of
SMs in biological samples. SPME is simple, does not use any sol-
vent, and can be fully automated. However, SPME is not an ex-
haustive extraction, but based on the distribution equilibrium
between the fiber coating and the headspace or liquid phase of
a sample. Therefore, an appropriate internal standard should be
used, preferably the isotope-labelled compounds (i.e., either
deuterated or 13C-labelled). In case of musk compounds, 13C-la-
belled musk compounds are not commercially available. As of
now, only two deuterated compounds, DMX-d15 and AHTN-d3
are commercially available. However, they are not very suitable
internal standards for musk analysis using SPME, since they
could undergo thermal degradation and exchange between pro-
ton (H) and deuterium (D), as reported by several studies (87,
88). In addition, all adsorbed compounds are introduced into GC
injection liner for desorption, thus potentially contaminating
the GC/MS system (e.g., GC injection liner, GC column, and MS
| 371
sources). One study measured polycyclic musks in serum and
milk samples by SMPE using a bonded PDMS/DVB fiber in the
headspace of the sample extracts, which were first cleaned up
by an automated solid phase extraction (SPE on a Bond Elute C8
cartridge) (62). The first SPE step significantly removed many in-
terfering compounds. If the first SPE step was not performed,
the SPME results could be compromised. The whole process was
automated, and good recoveries were achieved for HHCB,
AHTN, ADBI, and MM in the range of 78–90%; the detection lim-
its were in the range 0.03–0.3 ng/mL (62). But two important ni-
tro musks (i.e., MX and MX) were not included in that study,
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probably because the headspace SPME is not suitable for MX or
MK due to their low vapors. The estimated vapor pressures of
MX and MK are 0.00003 Pa and 0.00004, respectively, 3 orders of
magnitude lower than those of HHCB (0.073 Pa) and AHTN
(0.061) (Table 1). Another limitation of SPME is its low capacity
due to its thin coating of the adsorbent, often in the range of a
few micrometers (mm) to a few hundred mm (e.g., 7–100 mm). One
technique based on SPME, referred to as stir bar sorptive extrac-
R
tion (SBSE) (marketed as Gerstel TwisterV), was developed by
Gerstel Inc. (Gerstel GmbH & Co. KG, Germany). The magnetic
stir bar is coated with the same adsorbent used for SPME but the
thickness is on the order of millimeter (e.g., 0.5–1 mm) and the
length a few centimeters (e.g., 1–2 cm), thus offering the capac-
ity 100–1000 times larger than an SPME fiber coating. The SBSE
is magnetically stirred to extract organic compounds from an
aqueous sample, which could be suitable for biological samples
such as serum and blood. So far, only one study has reported
using SBSE for the analysis of four musks (i.e., MX, MK, HHCB,
and AHTN) in blood samples. Blood sample was diluted with
water in a 20 mL vial and a SBSE bar (1 cm long, coated with
0.5 mm of PDMS) was stirred in the solution for 60 min at 20 C.
After extraction, the bar was removed, rinsed with distilled wa-
ter, and dried with a tissue paper, and then placed in a thermal
desorption unit for subsequent GC/MS analysis (83). Very low
detection limits of 0.01–0.04 ng/mL and good recoveries in the
range of 70–130% were achieved. The process of SBSE can be
fully automated, however it can only be performed on GerstelV
R
thermal desorption unit. SBSE has larger capacity and thus
higher sensitivity than SPME but could introduce more interfer-
ing compounds into GC/MS system, compromising GC separa-
tion, generating high background, and potentially
contaminating GC/MS system. Other extraction methods are
not frequently used for the analysis of SMs in biological samples
and thus not discussed in detail here, but they are listed in
Table 2.
Sample Cleanup
Solvent extraction of musk compounds from biological samples
can also co-extract lipids and other unwanted compounds,
compromising the subsequent instrumental analysis; therefore,
additional cleanup procedures such as SPE or gel-permeation
chromatography (GPC) are implemented in many studies.
SPE technique is the most conventional and frequently used
method for extraction of target analytes in biological matrices.
SPE has the advantages of being simple and rapid, using less
solvent, having high recovery, as well as scope of automation
and on-line usage (89, 90). Normal phase SPE packed with
adsorbents such as silica, florisil, and silica/alumina are often
used to clean up sample extracts (e.g., blood, milk, and tissue)
for musk analysis. Nitro musks have been purified from milk
samples for the first time on a silica gel eluting with 150 mL of
petroleum ether/DCM (74). The high purity and neutral pH of
 372 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

Table 2. Summary of the analytical methods for the determination of synthetic musks in various biological matrices

Matrix Substances Extraction Detection GC column Recovery, % Detection limit Reference

a
Milk MX, MK, MA (1) LLE: Petroleum ether ECD SE 54-DF NA 0.01 ng/g (74)
and DCM
(2) Cleanup: SPE Silica
column
Milk MX, MK (1) Soxhlet extraction MS-SIM HP-5 NA NA (75)
(2) Cleanup: SPE1-
Aluminum oxide and
SPE2-silica column

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Milk (25 g) HHCB, AHTN, (1) LLE: MS-SIM Rtx-200, and 66.0–76.0 20–30 ng/g (76)
ATII, MX, i. Acetone and petro- DB-17
MT, MM leum ether
ii. Acetonitrile
(2) Cleanup: SPE1-Florisil
cartridge and SPE2-
Amino-cartridge
Milk (30 g) HHCB, AHTN, (1) Add Potassium oxa- HRMS-SIM DB-5MS 90.0–110.0 0.22–5.0 ng/g (71)
ADBI, AHMI, late, ethanol and di-
ATII, MX, ethyl ether
MK, MA, MM, (2) LLE: Pentane
MT, MK (3) Cleanup: GPC & SPE
Florisil column
Milk (10 mL) HHCB, AHTN, (1) Add 8% (w/w) potas- MS-SIM Rxi-5MS 84.1–108.0 2.0–5.0 ng/g (70)
MX, MK, sium oxalate, etha-
HHCB- nol and diethyl ether
lactone (2) LLE: Hexane
(3) Cleanup: GPC & SPE
Silica column
Milk (20 g) MX, MK (1) Lipid-phase ground ECD HP-5 NA NA (63)
with sodium sulfate
(2) Purification: Micro
glass column
(3) Cleanup: GPC with
bio-beads S-X3
Milk (10 g) MX, MA (1) Homogenized with ECD HP-5MS NA 0.5a ng/mL (77)
silica gel
(2) Purification: SPE
Silica column
Milk (10 mL) HHCB, AHTN, (1) Add ethanol and po- MS-SIM NA 95.1–106.1 2.0–9.0a ng/g (73)
ADBI, ATII, tassium oxalate
AHDI, MX, (2) LLE: Hexane, diethyl
MK ether, ethanol
(3) Cleanup: GPC
Milk (5–30 mL) HHCB, AHTN, (1) The cream grinded ECD & MS-SIM PS-088 and OV- 70.0–120.0 0.1–10.0 ng/g (65)
AHMI, MX, with sodium sulfate 1701-OH
MK, (2) LLE: n- Hexane
Habanolide (3) Cleanup: SPE Silica
column
Milk HHCB, AHTN, (1) Add 8% (W/W) potas- MS-SIM HP-5MS 71.0 –118.0 4.0–5.0 ng/g (68)
MX, MK sium oxalate, etha-
nol, and diethyl
ether
(2) LLE: n-Hexane
(3) Cleanup: GPC
Biobeads S-X3 & SPE
silica column
Milk (15 g) HHCB, AHTN, (1) Add Celite GC-HRMS DB-5MS 82.4–112.0 5.4–0.6a ng/g (78)
MK, MT (2) ASE cells packed
glass
(3) Cleanup: GPC & SPE
Florisil column
Milk (5–8 mL) HHCB, AHTN, mECD HP-5MS 68.8–78.1 5.0–15.0 ng/mL (69)
HHCB

(continued)
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 373

Table 2. (continued)

Matrix Substances Extraction Detection GC column Recovery, % Detection limit Reference

lactone, MX, (1) 8% (w/w) Potassium

MK oxalate, ethanol and
diethyl ether
(2) LLE: n-Hexane
(3) Cleanup: GPC
Biobeads S-X3 & SPE
2:1 silica/alumina
Milk (20 g) MX, MK (1) Lipid-phase ground ECD HP-5 or HP-1 NA 1.0–3.0a ng/g (64)

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with sodium sulfate
(2) Cleanup: SPE Silica
column
(3) Further Cleanup: GPC
Milk (10 mL) HHCB, AHTN, (1) Add potassium oxa- MS-SIM DB-5MS 95.3 2.0–5.0a ng/g (72)
MX, MK, MM late, ethanol and di-
ethyl ether
(2) LLE: Hexane
(3) Cleanup: GPC & SPE
silica
Milk (15 g) 12 musks (1) Add Celite GC-HRMS DB-5MS 82.4–112 5.4–6.0a ng/g (17)
(2) ASE cells packed
glass
(3) Cleanup: GPC & SPE
Florisil
Plasma (1 g) MX (1) LLE: Acetone NCI-MS-SIM PS086 86 50.0 fg/g (79)
(2) LLE: n-Hexane
(3) Cleanup: SPE Silica
column
Plasma (2 mL) MX (1) PPT: Formic acid MS-SIM DB-5 NA 0.1 ng/mL (60)
(2) LLE: Heptane
(3) Cleanup: SPE Silica
column
Blood (20 mL) MX, MK, MA, (1) LLE: Polar solvents MS-SIM HP-5MS NA 0.02 ng/mL (80)
MM, MT (2) Cleanup: SPE Silica
column
Plasma MX (1) PPT: Formic acid ECD DB-17 NA 0.1 ng/mL (61)
(2) LLE: Heptane
(3) Cleanup: SPE Silica
column
Blood (8.5– HHCB, AHTN, (1) PPT: Acetonitrile NCI-MS-SIM DB-624 95.1–99.4 0.012–0.062 ng/ (81)
9 mL) AHMI, ATII (2) LLE: n-Pentane mL
(3) Cleanup: SPE1- Silica
& SPE2- aluminium
oxide column
Blood (8.5– HHCB, AHTN, (1) PPT: Acetonitrile CI-MS-SIM DB-624 77.1–99.4 0.029–0.062 ng/ (82)
9 mL) ADBI, AHMI, (2) LLE: Pentane mL
ATII, DPMI, (3) Cleanup: SPE Silica
MX, MK, MA, column
MM, MT
Blood (8.5– HHCB, AHTN, (1) PPT: Acetonitrile NCI-MS-SIM DB-624 57.5–88.3 0.03–0.062 ng/ (56)
9 mL) ADBI, AHMI, (2) LLE: Pentane mL
ATII, DPMI, (3) Cleanup: SPE1-SPE
MX, MK, MA, Silica & SPE2-alumin-
MM, MT ium oxide
Blood (2 mL) HHCB, AHTN, (1) PPT: Ethanol MS-SIM HP-5MS 77.9–118.5 0.15 ng/mL (58)
ADBI, AHMI, (2) LLE: Hexane
ATII, MX, (3) Cleanup: SPE Silica
MK, column and anhy-
drous sodium sulfate
Blood (0.5 mL) HHCB, AHTN, (1) Dilute with water MS-SIM HP-5MS 70.0–130.0 0.01–0.04 ng/ (83)
MX, MK (2) SBSE extraction mL
Blood (2 mL) (1) PPT: Ethanol MS-SIM HP-5MS 62.8–95.7 0.29–0.35 ng/g (57)
(2) LLE: n-Hexane

(continued)
 374 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

Table 2. (continued)

Matrix Substances Extraction Detection GC column Recovery, % Detection limit Reference

HHCB, AHTN, (3) Cleanup: SPE Silica

ADBI, AHMI, column
ATII, MX, MK
Serum (5 mL) HHCB, AHTN, (1) PPT: Acetonitrile GC-HRMS ZB- 5HT 86.0–105.0 0.04–0.17 ng/ (59)
MX, MK (2) LLE: Pentane mL
(3) Cleanup: SPE Silica
column
Adipose MX, MK (1) LLE: Acetone and pe- ECD DB-5 NA 10.0 ng/g (84)

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tissues troleum ether
(2) Cleanup: GPC & SPE
silica column
Adipose tissues HHCB, AHTN, (1) Homogenization GC-HRMS PS 086 88.0–101.0 0.1–1.0 ng/g (66)
(2.5 g) ADBI, MX, with cyclohexane
MK, MA, MM, and ethyl acetate
(2) Cleanup: Glass col-
umn packed with an-
hydrous sodium
sulphate & GPC
Adipose HHCB, AHTN, (1) LLE: Methanol, di- GC-ECD & MS- CP-Sil 19 CB 72.0–100.0 1.0–2.0 ng/g (32)
tissues ADBI, AHDI, ethyl ether and pe- SIM and DB5
ATII, MX, MK troleum ether
(2) Cleanup: GPC
Adipose tissues HHCB, AHTN, (1) Ground with sodium MS-SIM Rxi-5MS NA 5.0 ng/g (51)
(4 g) HHCB- sulfate
lactone (2) Soxhlet extraction:
DCM and hexane
(3) Cleanup: GPC & SPE
silica column
Adipose fat HHCB, AHTN (1) Ground with sodium MS-SIM DB-5 85.0–98.0 1.0a ng/g (67)
sulfate
(2) Soxhlet extraction:
DCM and hexane
(3) Cleanup: GPC & SPE
Silica column
Adipose tissues HHCB, AHTN, (1) Homogenized with MS-SIM DB-5MS 86.0–95.0 1–2a ng/g (18)
(1–2 g) MK, MX, MM anhydrous sodium
sulfate
(2) LLE: DCM and
hexane
(3) Cleanup: GPC
Blood MX, 4-A-MX Plasma NCI-MS-SIM DB-1 Plasma: 87.0 166.0a fmol/mL (85)
(10 mL),Urine (1) PPT: Acetone Urine: 42.0
(5–10 mL) (2) LLE: n-Hexane
(3) Dried over Na2SO4

Urine
(1) LLE: Ethyl Acetate
(2) Dried over Na2SO4
(3) Cleanup: SPE

Serum, Milk HHCB, AHTN, (1) PPT: Methanol MS-SIM Restek Rxi-5MS Serum: 0.03–0.30 ng/ (62)
(1 mL) MM, ADBI (2) Cleanup: SPE & SPME 78.0–90.0 mL
Milk:
62.0–69.0
Milk (10 g), adi- HHCB, AHTN (1) Soxhlet extraction: MS-SIM BPX-5 (SGE) 99.0, 109.0 Milk: 1.0 & (86)
pose tissue DCM and hexane Tissue:
(1 g) (2) Cleanup: GPC 0.1 ng/g
Serum, HHCB, AHTN, (1) Soxhlet extraction: MS-SIM Rxi-5 MS 89.5–108.0 Serum: 1.0 & (41)
Umbilical, HHCB-lac- DCM and hexane Milk: 0.5 ng/g
Milk tone MX, (2) Cleanup: GPC & SPE
MK, MM silica column
(1) LLE: Hexane MS-SIM HP-5MS 74.0–121.0 3.0–8.0 ng/mL (16)

(continued)
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
Table 2. (continued)
Matrix Substances Extraction
Serum (2– HHCB, AHTN, (2) Cleanup: GPC, SPE1-
4 mL), HHCB-lac- Silica and SPE2-
Umbilical (8– tone, ADBI, Alumina column
10 mL), Milk AHMI, ATII,
(8–10 mL) MX, MK
a
LOQ ¼ Limit of quantitation. Detection limit are stated as LOD unless specified otherwise.
silica gel provide an ideal stationary phase for optimum and re-
producible purification. Despite its simplicity, the method uses
a large volume of organic solvents that needs to be evaporated.
For other methods, different elution solvents (polar, non-polar,
or a mixture of both), including petroleum benzene (60), hexane
and hexane/DCM (57, 58), petroleum ether and DCM (77), and
hexane/DCM and DCM (16), have been used to clean-up the
extracts on SPE cartridges packed with silica and silica/alumina
adsorbents. Different solvents could be used to elute different
classes of musk compounds. For example, Schlumpf et al. (2010)
developed a method based on silica SPE for cleanup of milk and
tissue samples and were able to elute nitro musks and polycy-
clic musks by toluene and subsequently elute slightly polar
macrocyclic musks by mixed solvent (toluene–acetone, v/v,
95:5) from the column. Recoveries of 70–120% and detection lim-
its of 0.1–10.0 ng/g were achieved for nitro, polycyclic, and mac-
rocyclic musks (65).
For complex matrices, one stage of SPE cleanup may not be
sufficient and thus additional SPE is required. For example,
Hutter et al. (2005, 2009, 2010) used two stages of SPE for the
cleanup of sample extracts of plasma (56, 81, 82). The first stage
of clean up was carried out on silica gel, eluted with hexane–
ethyl acetate (4:1), dichloromethane, and ethyl acetate. Another
cleanup step included an aluminum oxide column with hexane
as elution solvent. The two stages of SPE cleanup helped to
greatly reduce the background and achieve an increase in the
signal-to-noise ratio for targeted musk compounds, allowing
the detection of analytes at trace levels. Ott et al. (1999) purified
milk extracts by SPE using aluminum and silica columns for an-
alyzing MX and MK (75). Zehringer and Herrmann (2001) used
two stages of SPE to cleanup milk samples: the first with florisil
for nitro and polycyclic musks and the second on an amino-
cartridge only for polycyclic musks with elution by hexane and
hexane–acetone (76). Recoveries of nitro musks were in the
range of 66–76% while the mean recovery of polycyclic musks
was 72%.
Gel Permeation Chromatography
Biological samples (e.g., milk and adipose tissues in particular)
often contain large molecular compounds such as fat and pro-
tein, which could interfere with sample analysis even after SPE
clean up. Therefore, GPC is often used to remove lipids, pro-
teins, and dispersed macromolecular compounds from milk
and tissue samples. The GPC column used for cleanup of biolog-
ical samples (e.g., milk, tissue) for the analysis of musk com-
pounds are usually packed with copolymers of styrene and
divinylbenzene (e.g., Bio-Beads SX-3 column from Bio-Rad) and
solvent(s) such as hexane or hexane/DCM are used as a mobile
phase to elute musk compounds (32, 63, 64, 66, 71, 73).
Complex biological samples often require additional SPE
cleanup even after GPC cleanup. For example, Wang et al. (2011)
used additional SPE cleanup on florisil for the analysis of
| 375
Detection GC column Recovery, % Detection limit Reference
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selected musk compounds following a GPC cleanup and
achieved absolute recoveries (90–110%) for the majority of the
compounds (e.g., HHCB, AHTN, MK, MT) (78). In studies from
South Korea, GPC was also carried out on a column connected
sequentially to a silica column to purify polycyclic musks from
milk extracts with hexane/DCM (18, 72).
Separation and Detection
GC is often used to separate musk compounds, but the perfor-
mance of each type of GC column is slightly different due to dif-
ferent sample matrices and cleanup methods. For example,
Rimkus et al. (1994) found that MX and MA co-eluted on DB-
1701 (14%-cyanopropylphenyl)-methylpolysiloxane) column but
were well separated on either DB-1 (100% dimethylpolysiloxane)
or DB-5 (5% dipenyl and 95% dimethylpolysiloxane) columns
(84). Detailed GC column information is listed in Table 2.
Synthetic musk compounds can be detected using different
techniques such as electron capture detector (ECD) or MS. GC-
ECD has been used to analyze musk compounds in biological
samples including milk, blood, and adipose tissues (60, 61, 63–
65, 74, 77, 84), but ECD is only sensitive to nitro musks due to
the presence of electronegative nitro functional groups.
However, in addition to nitro musks, polycyclic and macrocyclic
musks were also analyzed with quantification limits of 0.5, 5.0,
and 20.0 ng/g, respectively (65). The method was 10 times more
sensitive for the detection of nitro musks compared to polycy-
clic musks, and 40 times more sensitive compared to macrocy-
clic musks. Overall, the limits of quantification obtained by
coupling GC with ECD were in the range of 0.1–10 ng/g, higher
than those obtained by MS (Table 2).
In addition, ECD cannot give structural information and thus
the identification is obtained by comparing the retention time
of the analyte to that of a standard; therefore, a good selection
of the column and oven temperatures is required in order to
avoid co-elution. Two major musks HHCB and AHTN are often
difficult to separate by GC (15, 59). Therefore, SMs are mostly
detected by MS (Table 2), which can provide structure informa-
tion and high sensitivity, with detection limits down to pico-
gram (pg) or even sub-pg, sufficient for trace analysis of SMs in
the biological samples (Table 2).
MS using electron impact ionization (EI) has been used pre-
dominantly for quantifying SMs in biological matrices. The
overall detection limits of SMs in the biological matrices by GC/
EI-MS with MS operated in SIM mode were in the range of 0.01–
0.15 ng/mL (Table 2). For some complex matrices, GC/MS in SIM
mode may not be able to offer baseline separation due to co-
eluting compounds that have the same ions with high abun-
dance, which are often used for quantitation and confirmation.
For example, HHCB and AHTN are two compounds with the
same molecular formula and thus same molecular weight
(258 g/mol). They elute very close on a ZB-5 column (stationary
phase: 5% phenyl 95% Dimethylpolysiloxane) and their peaks
 376 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
overlap. Their EI mass spectra were similar, with m/z 243 and
m/z 258 being the first and second highest abundance ions,
which were often used for quantitation and confirmation (83).
Therefore, their quantitation would be compromised if either of
these two ions was used for quantitation. With the MS operated
in multiple monitoring mode (MRM), the transition of m/z 243!
213 for HHCB and m/z 243! 187 for AHTN were used for quanti-
tation. In addition, MRM monitors the transition from one pre-
cursor ion to its product ion, which can greatly reduce the
background noise and provide better selectivity as well as sensi-
tivity. For example, Wang et al. (2011) used MRM mode to ana-
lyze nitro and polycyclic musks from milk samples and
achieved the method detection limits (MDLs) down to 2 ng/g
lipid for nitro musks and 5 ng/g lipid for polycyclic musks. In a
more recent study (59), GC-MS/MS was used for the determina-
tion of 10 musk compounds in human serum with MDLs rang-
ing from 0.04–0.17 ng/mL. High-resolution mass spectrometry
(HRMS), which can offer accurate mass and thus enhanced se-
lectively and sensitivity, has also been used for analysis of SMs.
Nitro and polycyclic musks were quantified in human milk with
detection limits in the range of 0.22–5.0 ng/g using GC-EI-HRMS-
SIM method (71). Another research group used a GC-EI-HRMS-
MRM method to quantify nitro and polycyclic musk compounds
in tissue samples with detection limits of 0.1 ng/g for polycyclic
musks and 1.0 ng/g for nitro musks (66).
Studies on determination of SMs by gas chromatography
negative ion chemical ionization mass spectrometry (GC-NCI-
MS) are scarce, although this ionization mode could be more
sensitive for the analysis of electron-capturing compounds
such as nitro musks (56, 79, 81, 82, 85). For example, Hutter et al.
(2010) analyzed 11 SMs in blood sample by GC-NCI/MS using
ammonia as reagent gas with the MS operated single ion moni-
toring (SIM) mode. They achieved high sensitivity for nitro
musks, with MDLs down to 4 ng/L for MK and 6 ng/L for MX;
however, the MDLs for HHCB and AHTN were 62 and 25 ng/L, re-
spectively, significantly higher than those of nitro musk. In ad-
dition, the quantitation ion for two major polycyclic musks,
HHCB and AHTN, was the same (m/z 257.3) and the difference in
the GC retention time (RT) for HHCB (RT: 26.44 min) and AHTN
(RT: 26.65 min) was only about 0.1 min even on a 60-m long DB-
624 column. Such a challenge on GC separation requires rela-
tively clean sample extracts with less interfering compounds. In
the study, Hutter et al. used two stages of SPE cleanup as men-
tioned earlier, which made the cleanup process more tedious
and expensive as well (Hutter et al., 2010). Therefore, the NCI/
MS may be suitable for the detection of nitro musks but may
not be for others (e.g., polycyclic musks) (Table 2).
Occurrence of SMs in Human Biological Matrices
Over the past several years, there has been an increased interest
in using human plasma, serum, milk, and tissue as biomonitor-
ing matrices to assess direct human exposure to environmental
contaminants, including SMs. Reported studies on levels of SMs
in biological matrices are listed in Tables 3–5.
Milk
The levels of SMs in human milk have been reported by numer-
ous studies conducted worldwide. The first study in 1993
reported the presence of nitro musks in milk (n ¼ 391) in
Bavaria, Germany, with the ranges of 10–1220 ng/g (mean: 100,
median: 70 ng/g) for MX, 10–240 ng/g (mean: 40, median: 30 ng/g)
for MK, and 10–290 ng/g (mean: 40, median: 30 ng/g) for MA
(Table 3). MX was detected in all 391 samples analyzed while
MK and MA were detected in 98% and 94% of samples, respec-
tively (74). Rimkus et al. (1994) reported the concentrations of
MX and MK in milk samples (n ¼ 23) collected in 1992 and 1993
from Germany in the range of 20–190 ng/g (median: 60 ng/g) and
10–90 ng/g (median: 20 ng/g), respectively (84). The same re-
search group reported concentrations in samples collected in
1993–1995 for HHCB, AHTN, MX, and MK in the range of 16.0–
108.0, 11.0–58.0, 10–30, and 5–15 ng/g, respectively (32). MA and
MM were also detected but at very low levels and only in a few
samples. The levels of MX and MK measured in this study were
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much lower than those reported in previous two studies.
Another German study conducted on human milk reported
mean concentrations (n ¼ 198) of 41 ng/g for MX and 10 ng/g for
MK in samples collected in 1984, 1990, and 1995 (75). In a study
from Denmark, HHCB and AHTN were detected in all milk sam-
ples (n ¼ 10, collected in 1999), with a higher median concentra-
tion of HHCB (147 ng/g) compared to AHTN (17.5 ng/g) (71). Nitro
musk compounds were detected at median concentrations of
9.4 ng/g for MX, 14.9 ng/g for MK, and <1.3 ng/g for MM in
Denmark study and reported levels lower than previous studies
from Germany (74, 84). Contrary to the belief, a study conducted
in Switzerland reported that no correlation was observed be-
tween mothers’ use of cosmetics and the concentration of SMs
in their milk (76). Muscone, a macrocyclic musk, was also ana-
lyzed in the study but it was not detected in any of the samples.
Raab et al. (2008) analyzed MX and MK in 39 breast milk samples
collected in 2005 in Germany and reported the concentrations
up to 240 ng/g (median: 8 ng/g, detection frequency (DF): 54%)
and 6 ng/g (DF: 21%) for MX and MK, respectively (63). The group
also analyzed 516 samples collected in 2012 and detected MX in
44 samples (DF: 8.5%) with concentrations up to 48 ng/g. MK was
detected in only one samples with concentration of 13 ng/g of
lipid (64). This suggests that MX and MK concentrations and
their detection frequencies decreased significantly from 2005 to
2012, which could be attributed to stringent restriction and
eventually ban on the use of MX and MK in Europe (25, 26). The
median concentrations reported for HHCB, AHTN, MX, and MK,
from 101 samples collected in Sweden between 1996 and 2003,
were 63.9, 10.4, 9.5, and <5 ng/g), respectively (73). The concen-
trations of AHTN and MX declined significantly between 1996
and 2003 in Sweden, suggesting a decrease in their use, whereas
there was no change in HHCB concentrations. The concentra-
tions of ADBI, ATII, AHDI, and MK were below their correspond-
ing LOQs in almost 80% of the samples analyzed. In a large
cohort study conducted in Germany, MX and MA were mea-
sured in milk samples (n ¼ 4314) collected during 1999–2006.
Both MX and MA were detected above their corresponding LOQs
(0.5 ng/mL) in 15.6% and less than 1% of the samples, respec-
tively (77). In milk samples collected in Switzerland between
2004 and 2006 (65), the median concentrations of HHCB, AHTN,
MX, and MK were lower when compared to those in samples
collected in 1998 and 1999 in an earlier Swiss study (76). Similar
to other studies, levels of polycyclic musks in Swiss samples
collected during 2004–2006 were much higher than those of ni-
tro musks (71, 73), hence providing further evidence that use of
polycyclic musk may have been increasing while use of nitro
musks were decreasing.
Relatively higher concentrations of SMs were reported in a
U.S. Study (n ¼ 39), where concentrations ranged from 5 to
917 ng/g, 5 to 144 ng/g, 2 to 150 ng/g, and 2 to 238 ng/g for HHCB,
AHTN, MX, and MK, respectively (70). In contrast to other stud-
ies, the detection frequency and concentration of MK was
higher than MX in this study. In another study from the United
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 377

Table 3. Levels of synthetic musks in human milk

Sample Sampling location,

size (N) Target analytes sampling year Mean (range/max), ng/g Reference

391 MX, MK, MA Bavaria (Germany), 1991– MX: 100 (10–1220), MK: 40 (10–240), MA: (74)
1992 40 (10–290)
198 MX, MK Hesse (Germany), 1984, MX: 41 (NAa), MK: 10 (NA) (75)
1990, 1995
23 MX, MK Schleswig-Holstein MX: NA (20–220), MK: NA (10–90), MA, (84)
(Germany), 1992–1993 MM: NA, MT: NDb
5 HHCB, AHTN, ADBI, Schleswig-Holstein HHCB: NA (16.0–108), AHTN: NA (11–58), (32)

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AHDI, ATII, MX, (Germany), ADBI: NA (1–18), AHDI: ND, ATII: ND,
MK 1993–1995 MX: NA (10–30), MK: NA (5–15)
53 HHCB, AHTN, ATII, Basle (Switzerland), 1998– HHCB: 62c (max: 281), AHTN: 31c (max: (76)
MX, MM, MT 1999 136), ATII: 74c (max: 74), MX: ND (max:
35), MM: ND (max: 170), MT: ND (max:
25)
10 HHCB, AHTN, ADBI, Denmark, 1999 HHCB: 147c (38–422), AHTN: 17.5c (5.6– (71)
AHMI, ATII, MX, 37.9) ADBI: 6c (max: 11.2), AHMI: ND
MK, MM, (max: 9.94), ATII: ND (max: 2.58), MX:
9.4c (max: 46.4), MK: 14.9c (max: 26.9),
MM: ND (max: 30.6), MA, MT: ND
39 MX, MK, HHCB, Massachusetts (USA), 2004 MX: 29.2 (<10–88), MK: 83.3 (<2–212) (70)
AHTN HHCB: 227.0 (<5–917), AHTN: 50.5 (<5–
144), HHCB lactone: ND
(<10–88)
85 MX, MK Bavaria (Germany), 2005 MX: 8.0c (max: 240), MK: ND (max: 6) (63)
42 HHCB, AHTN, MX, Shanghai (China), 2007 HHCB: 12.5c (max: 63.4), AHTN: 1.6c (max: (16)
MK 8.3), HHCB lactone: 8.6c (<LOD–851.3)
ADBI, AHMI, ATII: ND, MX: 10.4c (max:
63.1), MK: 7.3c (max: 478.2)
4314 MX, MA Germany, 1999–2006 NA (77)
101 HHCB, AHTN, ADBI, Uppsala, (Sweden) HHCB: 63.9c (2.8–268), AHTN: 10.4c (<3– (73)
ATII, AHDI, MX, 1996–2003 53), ADBI: <LOD, ATII: <LOD, AHDI:
MK <LOD, MX: <LOD, MK: <LOD
54 HHCB, AHTN, Basel (Switzerland), HHCB: 55.0 (6–309.6), AHTN: 13.9 (4.8– (65)
Habanolide, MX, 2004–2006 28.8), Habanolide: 15.0 (max: 15), MX:
MK 2.9 (0.25–31.6), MK: 1.6 (0.25–12)
100 HHCB, AHTN, MX, Shanghai (China), HHCB: 63c (<5–782), AHTN: 5c (<5–139), (68)
MK 2006–2007 MX: 17c (<5–198), MK: 4c (<4–105)
10 HHCB, AHTN Chengdu (China), HHCB: NA (max: 67.6), AHTN: NA (max : (78)
2009–2010 117.9), HHCB lactone: 40.1 (<LOD-70.6),
MK, MT: ND
26 HHCB, AHTN, ADBI Atlanta (USA), 2004 HHCB: 800 (20.1–131.6), AHTN: 560 (26.4– (62)
41.3), ADBI: 210 (3.4–51.7)
17 HHCB, AHTN, MX, Seoul (South Korea), 2007 HHCB: 220.0 (max: 520), AHTN: 40.0 (41)
MK, MM (max: 90), HHCB lactone: 16 (max: 100),
MX: 30.0 (max: 220), MK: 60.0 (max:
150), MM: 10.0 (max: 350)
46 HHCB, AHTN, MX, Shanghai (China), 2006, HHCB: 21.1c (2.8–180.6), AHTN: 2.3c (1.1– (69)
MK 2008, 2010 6.1), HHCB lactone : 10.8 (<LOD –74.7
ADBI, AHMI, ATII: ND, MX: 11.6c (2.2–
100.6), MK: 3.9c (1.4–35.4)
110 HHCB, AHTN, MK Chengdu (China), 2009 HHCB: 11.5c (>1.1–456.7), AHTN: 16.5c (91)
(0.6–794.3), HHCB lactone: 7.85
(<0.7–258.3), ADBI, AHMI, ATII: ND, MK:
<1.4c (1.4–11), MX, MA, MM, MT: ND
516 MX, MK Bavaria (Germany), MX: 2.0 (<2–48), MK: 3.0 (<3–13) (64)
2007–2008
20 HHCB, AHTN Japan, 2006–2008 HHCB: NA (<50–440), AHTN: NA (86)
(<50–190)
208 HHCB, AHTN, MX, Seoul (South Korea), 2011 HHCB: 299.0 (5–1346), AHTN: 65.1 (5–350), (72)
MM, MK MX: 2.2 (2–73.5), MM: 3.8 (2–130), MK:
12.4 (2–250)
24 pooled (1237) HHCB, AHTN, MK, China, 2007 HHCB: 18.03 (4.42–58.2), AHTN: 10.30 (17)
MA, (3.33–29.1), MK: 4.68 (<1.5–7.5), MA:
< 5.20 (<1.4–10.7)

a
NA ¼ Not available.
b
ND ¼ Not detected.
c
Median.
 378 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021

Table 4. Levels of synthetic musks in human blood

Sampling location and

Sample size, N Target analytes sampling year Mean (range/max concentration), ng/L Reference

a
11 MX Switzerland, NA MX: NA (12–49) (79)
72 MX Germany, NA MX: 240b (100–1120) (60)
41 MX Germany, 1993, 1998 MX (1998): ND (max: 290) (61)
152 MX, MK Heidelberg (Germany) MX: 65.5b (10–1183), MK: 55.5b (10–518), MA, (80)
1994–1996 MM, MT: NDc
100 HHCB, AHTN Vienna (Austria), NA HHCB: 420a (max: 4100), AHTN: ND (max: (81)

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800), ATII, AHMI: ND (max: up to 100), ND,
ADBI, DPMI: ND
114 HHCB, AHTN, AHMI, MX, Vienna (Austria), NA HHCB: 420a (max: 4100), MX: 11a (max: 800), (82)
MK, MA AHTN: NA (max: 65), AHMI: NA (max: 60),
ADBI, ATII, DPMI: ND, MK: NA (max: 67),
MA: NA (max: 16), MM, MT: ND
204 HHCB, AHTN China, 2007–2008 HHCB: 850a (max: 1630), AHTN: 530a (max: (58)
1290), ADBI, ATII, AHMI: ND, MX, MK: ND
58 HHCB, AHTN AHMI, DPMI, Vienna (Austria), NA HHCB: NA (max: 6900), AHTN: NA (max: (56)
MX, MK, MM, MA 290), AHMI: NA (max: 34), DPMI: NA (max:
89), ATII: ND, MX : NA (max: 190), MK : NA
(max: 280), MM: NA (max: 17), MA: NA
(max: 18), MT: ND
7 HHCB, AHTN, ADBI, MM Atlanta (USA), 2004 HHCB: 1040 (380–1700), ADBI: 160 (40–470), (62)
MM: 120 (50–280)
60 HHCB, AHTN China, 2012 HHCB: 504a (max: 4201), AHTN: 260a (max: (57)
830), ADBI, AHMI, ATII, MX, MK: ND
414 HHCB, AHTN, MX, MK Belgium, 2007 HHCB: 710 (300–1500), AHTN: 120 (20–300), (83)
MX: ND, MK: ND
20 HHCB, AHTN, MX, MK, Seoul (South Korea), 2007 Maternal serum: HHCB: 500 (max: 1400), (41)
MM AHTN: 300 (max: 1400), HHCB lactone:
250 (max: 800), MX: 110.0 (max: 510), MK:
ND (<170), MM: 140.0 (max: 360) ng/gd
Umbilical serum: HHCB: 910 (max: 2700),
AHTN: 560 (max: 2700), HHCB lactone:
640 (max: 2200), MX: ND (<670), MK: ND
(<670), MM: 410 (max: 1000) ng/gd
42 HHCB, AHTN, MX, MK Shanghai (China), 2007 Maternal serum: HHCB: 31.1a (max: 125.9), (16)
AHTN: ND (max: 2.7), HHCB lactone: ND
(max: 1073.3), ADBI, AHMI, ATII: ND, MX:
ND (max: 69), MK: ND (max: 60.6) ng/gd
42 Umbilical serum: HHCB: 56.8a (max: 98.3),
AHTN: ND (max: 39.5), HHCB lactone: 83.3
(max: 3182.8), ADBI, AHMI, ATII: ND, MX:
ND (max: 63.1), MK: ND (max: 478.2) ng/gd
20 HHCB, AHTN, MX, MK, Canada, 2006 HHCB: 570 (max: 820), AHTN: ND, ADBI, (59)
AHMI, ATII: ND, MX, MK, MA, MM, MT:
ND
Canada, 2014 HHCB: 470 (max: 1970), AHTN: ND (max:
120), MX, MK, MA, MM, MT: ND

a
NA ¼ Not available.
b
Median.
c
ND ¼ Not detected.
d
ng/g.

States, 26 milk samples were investigated and concentrations
ranged from 20 to 132 ng/g, 26 to 41 ng/g, and 3 to 52 ng/g for
HHCB, AHTN, and ADBI, respectively (62). MM was detected in
only one sample at a concentration of 7 ng/g. Mean concentra-
tions of HHCB and AHTN were about 5 and 2 times lower than
the previous U.S. study (70). Variations in SMs concentrations in
the two U.S. studies are likely due to the differences among
study participant characteristics in terms of location, use of per-
sonal care products, and dietary intakes.
Milk samples have been analyzed for SMs in different prov-
inces in East and Southwestern China. A study from Sichuan
(China) reported concentrations of HHCB and AHTN in the range
of 12–68 ng/g lipid and 23–118 ng/g lipid, respectively (78). Musk
T, a macrocyclic musk, was also analyzed in this study but it
was not detected in any of the samples. Another study from the
same region reported the concentrations in the range of <1.1 to
456.7 ng/g, 0.6 to 794.3 ng/g, and 1.4 to11.0 ng/g for HHCB, AHTN,
and MK, respectively (91). In milk samples (n ¼ 100) collected
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021 | 379

Table 5. Levels of synthetic musks in adipose tissues

Sampling location and

Sample size, N Target analytes sampling year Mean (range/max concentration), ng/g Reference

32 MX, MK Schleswig-Holstein MX: NAa (20–220), MK: NA (10–220), MA, MM: (84)
(Germany), 1992–1993 NA, MT: NDb
15 HHCB, AHTN, ADBI, MX, Switzerland, 1983, 1994 HHCB: NA (12–171), AHTN: NA (1–23), ADBI: (66)
MK MA, MM NA (0.1–3.5), MX: NA (12–288), MK: NA
(<1–173), MA: NA (<1–67), MM: NA
(<1–42)
49 HHCB, AHTN New York (USA), HHCB: 178.0 (12–798), AHTN: 42.0 (8–134) (67)

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2003–2004
12 HHCB, AHTN, HHCB- Siena (Italy), 2005, 2006 HHCB: 361 (<5 –1435, AHTN: 132 (<5–931), (19).
lactone HHCB-lactone: ND
43 HHCB, AHTN MK South Korea, 2007–2008 HHCB: 81.0 (28–211), AHTN: 12.0 (2–51), MK: (18)
13.0 (2–34), MX, MM: ND
50 HHCB, AHTN, MX, New York (USA), HHCB: 49 (3.4–320), AHTN: 8.4 (0.1–33), MX: (92)
1996–1998 34 (nd-236)
14 HHCB, AHTN, ADBI, AHDI, Schleswig-Holstein HHCB: NA (28–189), AHTN: NA (8–33), ADBI: (32)
ATII, MX, MK (Germany), 1993–1995 NA (2–3), AHDI: NA (1–5), MX: NA (5–50),
MK: NA (5–30)
3 HHCB, AHTN Japan, 2006–2008 HHCB: NA (<10–3), AHTN: NA (<10–13) (86)

a
ND ¼ Not detected.
b
NA ¼ Not available.

from three cities in Eastern China, the median concentrations
of HHCB, MX, AHTN, and MK were 63 ng/g, 17 ng/g, 5 ng/g, and
4 ng/g, respectively (68). The concentrations reported from this
study were similar to those reported from Sweden (73), but
lower than those from Switzerland (76), Denmark (71), and the
United States (70). In another study from Eastern China, the
median concentrations were reported as 21.1 ng/g for HHCB,
2.3 ng/g for AHTN, 11.6 ng/g for MX, and 3.9 ng/g for MK (69). The
levels of MX and MK were comparable to those from other stud-
ies from the same province, while there was a 2- to 5-fold differ-
ence in the concentration levels for HHCB and AHTN. In a study
from Shanghai, the median concentrations were reported as
12.5, 1.6, 10.4, and 7.3 ng/g for HHCB, AHTN, MX, and MK, respec-
tively (16). A very strong decrease in the levels of HHCB and
AHTN was observed in the study when compared to other stud-
ies from China. Yin et al. (2016) studied 13 musk compounds in
24 pooled milk samples obtained from 1237 individuals in 12
provinces of China (17). The detection frequencies of HHCB,
AHTN, and MK were 100% and the concentrations were in the
range of 4.42–58.20, 3.33–29.10, and <1.40–10.71 ng/g, respec-
tively. Other musk compounds investigated in this study were
below their respective limits of detection.
A study in Japan on 20 milk samples collected between 2006
and 2008 reported the concentrations of HHCB and AHTN in the
range of 50–440 and 50–190 ng/g, respectively (86). In a Korean
study, 17 milk samples collected in 2007 were analyzed and the
concentrations of HHCB, AHTN, HHCB-Lactone, MX, MK, and
MM were in the range of 55–515, 15–91, 15–100, 15–220, 15–150,
and 15–35 ng/g lipid, respectively (41). In another Korean study,
higher levels of SMs were observed in milk samples collected
from four cities, where the concentrations for HHCB, AHTN, MX,
MM, and MK ranged from 5 to 1346 ng/g, 5 to 350 ng/g, 2 to
73.5 ng/g, 2 to 130 ng/g, and 2 to 250 ng/g, respectively (72).
Human Blood
Several studies have been conducted to determine the levels of
musks in human blood. MX was quantified for the first time in a
Swiss study where the reported concentration ranged from 12
to 49 ng/g (79). One study from Germany in 1994 reported a
higher concentration of MX in the range of 100–1120 ng/L (60).
MX levels in males (median: 275 ng/L, n ¼ 30) seemed to be
higher than in females (median: 200 ng/L, n ¼ 42), but this differ-
ence was not statistically significant. The same research group
analyzed 41 plasma samples collected in 1998 and compared
their data with the samples collected during 1993–94 (61) and
found that MX was only detected in 12% of the samples col-
lected in 1998 (median: <100 ng/L, range: <100–290 ng/L). This
remarkable decrease in MX levels in samples collected in 1998
compared to samples collected in 1993–94 was probably due to
discontinued use of MX in detergents in Germany since 1993
(61). Nitro musk compounds were analyzed in 152 blood sam-
ples collected between 1994 and 1996 from women with gyneco-
logical problems in Germany (80); the median concentrations
for MX and MK were 66 ng/L (max: 1183 ng/L) and 56 ng/L (max:
518 ng/L), respectively. In the same study, MX levels were found
to be lower in patients who had not suffered a miscarriage and
had given normal births to children compared to patients who
suffered a miscarriage. The median concentration of MX was
lower compared to the samples collected in 1993 and 1994 and
higher compared to the samples collected in 1998 from
Germany (61). In a study from the United States, serum samples
from 7 lactating women were analyzed (62); the concentrations
of HHCB, ADBI, and MM ranged from 380 to 1700 ng/L, 40 to
470 ng/L, and 50 to 280 ng/L, respectively. AHTN was not
detected in any of the samples in the study. In a study that in-
vestigated musk compounds in 20 maternal and umbilical cord
serum samples from Korea (41), the mean concentrations of
HHCB, AHTN, and MM were 500, 300, and 140 ng/g in serum
samples, and 910, 560, and 410 ng/g in umbilical serum samples.
MM was detected in 35% of maternal serum and 15% of umbili-
cal cord serum samples, while MX was detected in only one se-
rum sample; MK was not detected in any of the samples. In a
study conducted in 2007 in Belgium, HHCB and AHTN were
detected in 92% of blood samples collected from 210 youths and
204 adults; the mean concentrations of HHCB and AHTN were
 380 | Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
710 (range: 301–1539) ng/L and 120 (range: 20–307) ng/L, respec-
tively (83). Nitro musks were detected in 20 samples, but with
concentrations below LODs (19 ng/L for MK and 38 ng/L for MX).
Higher HHCB levels were found in girls (GM: 826, range: 775–
880 ng/L) compared to boys (GM: 645, range: 603–690 ng/L),
whereas no significant difference was observed in the mean lev-
els of AHTN between girls and boys. Similarly, in studies con-
ducted in Austria (n ¼ 100), significantly higher levels of
polycyclic musk compounds were detected in women than in
men (81, 82). HHCB and MX were found with a higher detection
frequency, with median concentrations of 420 and 11 ng/L, re-
spectively. Both MK and AHTN were found only in 17% of the
samples analyzed; moderate decline in levels of MX and a
strong decline in levels of MK were observed in comparison to
the data reported a decade before (60, 79). A study conducted in
2010 in Austria (n ¼ 58) compared levels of SMs in samples col-
lected from elderly women with younger female volunteers.
The levels in elderly women were found to be higher compared
to the younger counterparts, which may be attributed to more
usage years of products containing musk compounds such as
lotions and personal care products by older females (56). In a
study conducted in China on 204 blood samples collected from
11 cities, the median concentrations were reported as 850.0 ng/L
and 530.0 ng/L for HHCB and AHTN, respectively (58). The con-
centrations of the other musk compounds, including MX and
MK, were below their quantification limits (0.15 ng/mL). The
study demonstrated that musk concentrations were not signifi-
cantly related to gender and body weight, but positively corre-
lated with age groups and locations. Another study from China
investigated blood samples (n ¼ 6) of hairdressers and the gen-
eral public to evaluate the risk associated with the occupational
exposure to SMs (57). Interestingly, the study revealed that there
was no difference in the levels between the two groups.
However, no conclusions can be drawn because of the small
sample size. A study from Shanghai reported musks in mater-
nal blood and umbilical cord blood samples (16). The total lipid-
based concentrations were higher in umbilical cord serum
(87 ng/g) compared with maternal serum (71 ng/g). HHCB was
the most abundant musk compounds in maternal serum
(n ¼ 42) and umbilical cord serum (n ¼ 42), with median concen-
trations of 36.1 and 77.3 ng/g, respectively. HHCB concentration
in umbilical cord serum samples collected in Shanghai was
much lower than those in samples collected in South Korea (41).
In a recent study from Canada, SMs were analyzed in 40 blood
samples collected from Canadian women in 2006 and 2014 (59).
The mean serum concentration of HHCB was 520 ng/L in the
Canadian study, a two-fold lower than the levels reported in the
United States, where the mean concentration was 1040 ng/L
(62). The median concentration of HHCB in the Canadian study
was consistent with studies from Austria (56, 81, 82) and China
(57). Musk ketone (MK) was not detected in any of the Canadian
samples collected in 2006 but was detected in 60% of the sam-
ples collected in 2014 with a median concentration of 290 ng/L
(max: 1520 ng/L), higher than the levels reported in Germany
(80) and Austria (56, 82).
Tissues
Only a few studies have reported the occurrence of SMs in hu-
man adipose tissues. A study from Germany reported the resi-
due levels of MX and MK ranging from 20 to 220 ng/g and 10 to
220 ng/g in females, respectively, which were higher than those
in males with the ranges of 20 to 90 ng/g for MX and from 10 to
30 ng/g for MK (84). Another study investigated nitro and
polycyclic musks in 14 adipose tissue samples collected in
Germany in 1993. The concentrations of HHCB, AHTN, ADBI,
MX, and MK in the study ranged from 16 to 108 ng/g, 11 to 58 ng/
g, 1 to 18 ng/g, 10 to 30 ng/g, and 5 to 15 ng/g, respectively (32).
AHDI and ATII were detected in a few samples at low levels.
Nitro musk levels measured in this study were about one order
of magnitude lower than previously reported by the same re-
search group. Adipose tissue samples (n ¼ 15, collected in 1983,
1994) from Switzerland were analyzed for polycyclic and nitro
compounds and the concentrations were reported to be in the
range of 12–171, 12–288, and <1–173 ng/g for HHCB, MX, and MK,
Downloaded from https://academic.oup.com/jaoac/article/104/2/368/6025173 by Xiamen University user on 01 December 2021
respectively (66). MX and MK levels were in agreement with
samples collected in 1994 (84) and about five times higher than
the samples collected in 1996 (32). Kannan et al. (2005) reported
the concentrations of SMs in adipose fat samples collected in
New York (USA) (67). In female adipose fat samples (n ¼ 37) the
median concentrations of HHCB and AHTN were 180 ng/g
(range: 18–798 ng/g) and 31.5 (<8–134 ng/g), respectively, while
in male adipose fat samples (n ¼ 12) the median concentrations
were 90.5 ng/g (12–509 ng/g) and 38.7 ng/g (<8–110 ng/g), respec-
tively (67). Concentration of HHCB was positively correlated
with the gender of donors, but a similar trend was not observed
for AHTN. These values are much higher than those measured
in adipose fat samples collected a decade ago in Germany and
Switzerland (32, 66). This was likely due to the replacement of
nitro musks by polycyclic musks and hence increase in their us-
age. In a study conducted on only 3 adipose tissue samples col-
lected in Japan, the concentrations of HHCB and AHTN ranged
from <11 to 33 ng/g and from <9 to 13 ng/g, respectively (86). In
a study conducted on adipose tissues (n ¼ 43) in 2007–2008 from
Korean women, the mean concentrations of HHCB, AHTN, and
MK were 81 (28–211), 12 (2–51), and 13 (2–34) ng/g, respectively
(18). In this study, HHCB was found in 100% of samples, while
AHTN and MK were detected in 81% of samples. MX and MM
were not detected in any of the samples. This discrepancy could
either be due to the different sources and magnitude of expo-
sure to these compounds. A study from Italy where 12 samples
were collected during 2005–2006 reported the concentrations of
HHCB and AHTN in the range of <5 to 1435 ng/g and <5 to
931 ng/g (19). Very recently, Li et al. (2019) determined the con-
centrations of SMs in breast tissues collected from 50 breast
cancer patients between 1996 and 1998 in New York (92). In this
study, HHCB, AHTN, and MX were found with mean concentra-
tions of 49 (3.4–320), 8.4 (0.1–33), and 34 (0–236) ng/g, respec-
tively. These values were lower than those reported for adipose
fat samples collected from New York City (67), Korean females
(18), and Italian samples (19).
Conclusions
The frequent detection of synthetic musk compounds (e.g.,
HHCB, AHTN, MX, and MK) in biological matrices confirmed
their bioaccumulation in the human body. However, the biomo-
nitoring data of SMs are still scarce. The complex composition
of biological samples, presence of large amounts of macromo-
lecular compounds such as fat and proteins, and very low levels
of SMs pose great challenges associated with biomonitoring of
SMs, which provides more room in the improvement of analyti-
cal methods. Extensive sample cleanup, such as PPT, SPE, and
GPC is required prior to instrument analysis by gas chromatog-
raphy couple with mass spectrometry (GC/MS). An HRMS or a
triple quadrupole mass spectrometer operated in multiple reac-
tion monitoring (MRM) mode, which can provide enhanced se-
lectively and sensitivity, is preferred. Nitro musks
 Katuri et al.: Journal of AOAC INTERNATIONAL Vol. 104, No. 2, 2021
concentrations (MX and MK in particular) were found to be de-
creasing over the last decades as they were gradually replaced
by polycyclic musks such as HHCB and AHTN. Relatively higher
levels of SMs were reported in milk samples in the United States
and Korea, whereas in China levels of SMs were high in serum
samples. More samples, in the order of hundreds or even thou-
sands, are required for biomonitoring data of SMs at regional or
even national scales for a better understanding on their metab-
olism and their impact on human health, which calls for the de-
velopment of sensitive, robust, and high throughput methods
for the SM analysis in biological matrices. Therefore, sample
preparation methods based on automation such as PLE and au-
tomated sample cleanup by SPE are preferred.
Acknowledgments
Many thanks to Arezoo Habibagahi and Jianjun Niu of the
Environmental Health Science Bureau (Health Canada) for their
careful reading of our manuscript and valuable comments and
suggestions.
Conflict of Interest
The authors declare that there are no conflicts of interest.
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