Environmental Science and Pollution Research
https://doi.org/10.1007/s11356-018-3483-z

REVIEW ARTICLE

Per- and polyfluoroalkyl substances in human breast milk and current
analytical methods
Linda R. Macheka-Tendenguwo 1

&

Joshua O. Olowoyo 1 & Liziwe L. Mugivhisa 1 & Ovokeroye A. Abafe 2

Received: 22 May 2018 / Accepted: 16 October 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Per- and polyfluoroalkyl substances (PFASs) have since become a major health concern as they have been reportedly found in
human tissues, blood and breast milk. The main aim of the study was to review the current data on PFASs in human breast milk,
including the challenges of analysis as well as the possible modes of transfer from maternal blood. In this paper, previously
published data on the concentrations of PFASs in human breast milk from around the world were reviewed and summarised.
Eligible studies with reference lists published before 1 June 2017 were included by searching several databases (including
Scopus, ScienceOpen and SciFinder). From this search, studies with the number of participants in each study ranging from 2
to 1237 were identified. The review indicated that based on the structural profiles and concentration levels, there was variation in
the geographical distribution of these compounds in breast milk. Although there are no recorded investigations on the modes of
transfer from maternal blood to breast milk, literature suggests that the PFASs tend to be transferred through binding to various
proteins. The review also examined the different sample preparation and analytical methods employed to measure the concentrations of PFASs in human breast milk. This showed that solid phase extraction was the most common extraction method. After
extraction, liquid chromatography coupled with tandem mass spectrometry was the most common analysis method. Since several
of these methods were initially dedicated to monitoring PFASs in food and water, they demonstrate some limitations with regard
to specificity and sensitivity to human fluids. Additionally, there are currently no published records of certified reference
materials and/or proficiency scheme devoted to standardising PFAS concentrations in breast milk.
Keywords Perfluoroalkyl substances . Perfluorooctanoic acid . Perfluorooctane sulphonate . Biomonitoring . Human breast milk

Introduction
Per- and polyfluoroalkyl compounds (PFASs) belong to a
group of fluorinated substances manufactured in the 1950s
for application in several commercial and industrial applications (Kissa 2001). PFASs can be defined as artificial organic
compounds, having carbon chains of different lengths where
their hydrogen atoms are completely (perfluorinated) or partially (polyfluorinated) replaced by fluorine atoms, except for
those hydrogen atoms whose replacement would alter the
Responsible editor: Hongwen Sun
* Linda R. Macheka-Tendenguwo
misslindamacheka@gmail.com
1

Sefako Makgatho Health Sciences University, Pretoria, South Africa

2

Agricultural Research Council-OVR, Pretoria, South Africa

nature of the functional groups, forming a sturdier carbon–
fluorine (C–F) bond (Alexander et al. 2003). The high electronegativity of fluorine atoms strongly attracts shared electrons resulting in a compound with high chemical and thermal
stability. The C–F bond is considered one of the strongest in
nature, thereby making the synthesised product highly resistant to biological degradation (Mabury 2005). In addition,
under suitable conditions, polyfluorinated compounds can be
biotically or abiotically transformed into perfluorinated compounds in the environment (Buck et al. 2011).
PFASs are manufactured through two key processes, namely telomerization and electrochemical fluorination (ECF) (Fujii
et al. 2007; Beesoon et al. 2011). The two processes yield two
distinct types of products; whereas ECF produces short-chain
PFASs and perfluorinated sulphonates with branched isomers,
telomerization produces even numbered carbon chains, particularly perfluorocarboxylates (Fujii et al. 2007). By definition,
short-chain PFASs are perfluoroalkyl carboxylic acids with
seven carbons or less and, perfluoroalkane sulphonates with

 Environ Sci Pollut Res

five carbons and less (Buck et al. 2011; Xiao et al. 2018).
Moreover, it has been noted that the same PFAS produced by
either process can be analytically distinguished, for instance,
telomerised perfluorooctanoic acid (PFOA) is a linear isomer
(Kissa 1994; Beesoon et al. 2011) and ECF PFOA is a combination of both branched and linear isomers (Loveless et al.
2006; Beesoon et al. 2011).
The unique properties of PFASs (Table 1), which enhance
their ability to repel water, fat and dust as well as their high
resistance to aggressive chemicals, make them popular among
modern manufacturers (Kärrman et al. 2007). The table below
shows some physico-chemical properties of perfluorooctanoic
acid (PFOA), perfluorooctane sulphonate (PFOS),
perfluorononanoic acid (PFNA), perfluorohexane sulphonic
acid (PFHxS) and perfluorodecanoic acid (PFDA); which
are some of the most frequently detected PFASs in human
breast milk.
The physicochemical properties of PFASs are generally not
well documented in literature and there remain inconsistencies
among the available studies. In addition, much of the available
data has either been predicted or calculated rather than derived
experimentally and this is due to the limited number of pure
PFASs available for such study (Ding and Peijnenburg 2013).
The chemical structures of PFASs, for example PFOA and
perfluorooctane sulphonate (PFOS) (Figs. 1 and 2), enable
them to possess a combination of both hydrophobic properties
from their perfluorinated structures, and hydrophilic properties from their functional groups (-COO− and -SO3−) (Fujii
et al. 2007). These specific properties make PFASs to be used
as surfactants, emulsifiers and performance chemicals.
Currently, PFASs are widely used in several applications,
ranging from fire extinguishing foams, raincoats, non-stick
cookware, medical implants, insecticides to military and aviation technology (Kärrman et al. 2007; German Federal
Environmental Agency 2009; Whitworth et al. 2012).
However, over the past few decades, these compounds have
become a major health concern as they have been found in

human tissues, blood, urine and breast milk (Harada et al.
2003; Hanssen et al. 2010; Lankova et al. 2013), with health
impacts ranging from cancer, high cholesterol, obesity and
allergies (Liu et al. 2010; Steenland et al. 2009; Stahl et al.
2011). In addition to being present in human tissues and
fluids, prenatal exposure of foetuses has intensified concerns
over the persistence of PFASs in the environment. Preliminary
work has established that these compounds can cross the placenta from maternal blood, thus putting the foetus at risk,
where exposure can lead to low birth weight and later life
disorders such as increased risk to heart disease, developmental toxicity and Alzheimer’s disease (Slotkin et al. 2008;
Steenland et al. 2009; Hanssen et al. 2010; Whitworth et al.
2012). Moreover, the persistence of PFASs in breast milk has
been documented, thereby increasing the risk of exposure
even after childbirth (Gu et al. 2010).
The first recommended food for a new-born is normally the
mother’s milk as its components are important for the baby’s
nutritive requirements. Breast milk has cellular, immunochemical and biochemical constituents that can protect a
new born from infections, diarrhoea and malnutrition, and is
recommended from birth to at least 6 months (NICUS 2006).
However, it has been acknowledged that lactation may be a
significant pathway of PFAS exposure for neonates through
breast milk, thus often used as a bio-indicator to estimate the
body-load of some PFASs in infants (Rodriguez et al. 2009;
Gu et al. 2010; Lankova et al. 2013). Additionally, based on
body mass, infants are more vulnerable and are exposed to
comparably elevated levels of chemicals than adults (Liu
et al. 2011).
The two most studied and detected forms of PFASs are
perfluorooctane sulphonate (PFOS) and perflurooctanoic acid
(PFOA) which tend to have elimination half-lives of 5.4 years
and 3.8 years respectively in human blood (Olsen et al. 2007).
However, there is no documented information on their halflives in human breast milk. A study by Haug et al. (2011)
estimated that the total PFOA and PFOS in infants would

Table 1

Physicochemical properties of selected PFASs

Property

PFOS

PFOA

PFNA

PFHxS

PFDA

References

Molecular weight (g/mol)

500.13

414.07

464.08

400.12

514.09

Giesy et al. 2010; ATSDR 2015

Melting point (°C)

> 400

54.3

62

274

79

Boiling point (°C)
Vapour pressure (mmHg) at
25 °C
Solubility in water (mg/L) at
25 °C
logKow at 25 °C
pKa at 25 °C

249
192
218
239
218
2.9 × 10−3 3.16 × 10−2 8.3 × 10−2 4.6 × 10−3 1.01

ATSDR 2015; Haynes 2015; ChemicalBook 2012; ECHA
2015
Kosswig 2000; Haynes 2015; Savu 2000
Bhhatarai and Gramatica 2010; EPA 2012; Ding and
Peijnenburg 2013
ATSDR 2015

Bioaccumulation factor
(L/kg)

3.2 × 10−3 3.3 × 103

1.8 × 10−1 6.2

4.49
0.14

5.48
3.16
2.90
EPA 2012; Ding and Peijnenburg 2013
− 0.17
0.14
2.61
ATSDR 2015; Ding and Peijnenburg 2013
3.98 × 103 5.01 × 102 7.94 × 103 Martin et al. 2003; Furdui et al. 2007; Houde et al. 2008

3.2 × 10−1

4.81
1.33
3.8 × 10−2

2.8 × 10−2

 Environ Sci Pollut Res

Fig. 1 Chemical structure of perfluorooctanoic acid (PFOA)

contribute > 83% and > 94% respectively, of exposure within
6 months through lactation. Such high exposure rates may in
turn lead to numerous disorders later in life such as attention
deficit hyperactivity disorder, high cholesterol and cardiovascular disease (Steenland et al. 2009; Ode et al. 2014). Some
documented reports have associated the presence of PFOS in
the human body to liver and bladder cancer, as well as contributing to reproductive and developmental toxicity during
growth (Haddow et al. 1999; Thibodeaux et al. 2003; Inoue
et al. 2004; Apelberg et al. 2007).
PFOA toxicity has been associated with accumulation in
the blood, pancreas and liver (Olsen et al. 2007; ATSDR
2015) as well as damage to the thyroid and immune system,
which may increase chances of allergies (Haddow et al. 1999).
Nevertheless, conflicting results were reported by Okada et al.
(2014), who concluded that there were no relationships between exposure to both PFOS and PFOA with allergies and
infections in infants of 18 months. Research in rodents has
also suggested that exposure to PFOA in toxic amounts leads
to adverse developmental effects, which might also be exhibited in humans (Wolf et al. 2007; Rodriguez et al. 2009).
Another PFAS linked with detrimental health effects in
infants is perfluorononanoic acid (PFNA). It is a characteristic
persistent, non-volatile perfluoroalkyl found to target the liver,
causing it to increase in weight (Wolf et al. 2010; Fang et al.
2008). In the USA, it has been reported that children between
the ages of 12 and 15 years having elevated levels of PFNA,
among other PFASs, were at a higher risk of attention deficit
hyperactivity disorder (ADHD) (Hoffman et al. 2009). In a
separate study, high PFNA levels indicated a positive correlation with increased cholesterol levels in USA adults aged between 20 to 80 years (Nelson et al. 2010). On the other hand,
Steenland et al. (2009) mentioned that although high PFASs
may lead to high cholesterol, which in turn predisposes children to higher risk of heart failure and obesity in the future,
many of the epidemiological studies are cross-sectional and

therefore fail to clearly establish the underlying relationships
between levels of exposure to PFASs and human health effects. Consequently, several regulatory and advisory bodies on
environmental toxins, including the US Environmental
Protection Agency’s (US EPA) Science and Advisory Board,
International Agency for Cancer Research (IACR) and US
National Toxicology Program (NTP), have classified PFASs
such as PFOA as potential carcinogens (Biegel et al. 2001;
Lau et al. 2006; ATSDR 2015).
Numerous investigations have attempted to establish the
main exposure pathway of PFAS in humans, but differing
viewpoints make the results inconclusive (Harada et al.
2003; Fujii et al. 2007; Tittlemier et al. 2007; Scheringer
et al. 2007; Hölzer et al. 2008; Stahl et al. 2009). While most
studies have resolved that contaminated water and food play a
key role, Ericson et al. (2008) investigated the relationship
between exposure through contaminated food and acknowledged that a correlation between dietary intake and blood
levels of PFASs did exist but however, concluded that diet
was not the main route of exposure.
For infants, determining the concentrations of these organic
compounds in the mother’s milk may aid in estimating the level
of exposure through their diet. To date, no experimental data
exists that has established the routes of transfer of PFASs to
breast milk from the mother’s blood, where they are known to
bind to serum proteins (Kärrman et al. 2010; Liu et al. 2010). The
present review summarises the current data on PFASs in human
breast milk, including the challenges of analysis as well as the
possible modes of transfer from maternal blood to breast milk.

Structure of the review
Search strategies
Previously published data on the concentrations of PFASs in
human breast milk from across the globe were summarised in
this review. Suitable studies with reference lists published before 1 June 2017 were incorporated into the review. Literature
searches were done using Scopus, PubMed, ScienceOpen,
EuropePMC and SciFinder. Searched keywords included
perfluorinated compounds, perfluorochemicals, breast milk,
lactation, postnatal transfer, analysis and other associated
terms. The titles and abstracts were first evaluated to determine those that qualified for a full-text review. The reference
lists of the articles were also checked to find further literature.

Eligibility criteria

Fig. 2 Chemical structure of perfluorooctane sulphonate (PFOS)

Articles that met the following criteria were eligible for the
review: (1) those where the concentrations of PFASs in human
breast milk were quantified, (2) those where the analytical
method employed for the determination of PFASs was

 Environ Sci Pollut Res

specified and (3) where the participants were not occupationally exposed to PFASs.
Data extraction and assessment Data from the investigations
that fell within the inclusion criteria were extracted and summated. PFAS concentrations were converted to pg/ml for ease
of comparison and summarised into an evidence table
(Table 2), which also included the following information:
the region and country where the study was carried out, the
number of participants, sample collection year and the first
author and year of publication. Information obtained on the
possible modes of transfer from maternal blood to breast milk
was also summarised.
To assess the different analytical methods used to identify
and quantify the concentrations of PFASs in breast milk, data
such as the technique used for the analysis, the sample pretreatment method including extraction and cleanup methods,
the mobile and stationary phases used, the percentage recovery obtained, and the first author and year of publication were
extracted and summarised in Table 2.

Results and discussion
Evidence of PFASs in human milk
Despite the fact that PFASs have been synthesised for more
than 60 years, their presence in human breast milk have only
been studied in Asia, Europe and North America, with increased intensity over the past decade (Apelberg et al. 2007;
Hoffman et al. 2009; Liu et al. 2010). The first published incidence of PFASs found in human breast milk was in 2004
(Kuklenyik et al. 2004), though other investigators later published PFAS concentrations from frozen breast milk samples
collected as far back as 1996 and 1999 (Völkel et al. 2007;
Tao et al. 2008a, b). Since then, several other studies have
followed, thereby increasing concerns of exposure through lactation (Bernsmann and Fürst 2008; Kärrman et al. 2010; Liu
et al. 2010; Lankova et al. 2013). It has been observed that the
most xfrequently detected PFASs in humans include
perfluorohexanesulphonic acid (PFHxS), PFOA and PFOS
(Kärrman et al. 2007; Sündstrom et al. 2011).
Currently, while there are numerous reports of PFASs in the
environment and in serum, no information is available for
their occurrence in breast milk from Africa, Antarctica,
Australia and South America (Table 2). Published data suggests a large variation in the geographical distribution of these
compounds based on their structural profiles and their concentration levels. In North America, PFASs in breast milk have
been quantified in Canada and the USA (Table 2) (Kubwabo
et al. 2013; Kuklenyik et al. 2004; Tao et al. 2008a, b).
In America, high levels of perfluorohexanoic acid
(PFHxA) and perfluoropeptanoic acid (PFPePA) in two

samples were quantified to be 820 pg/ml and 1560 pg/ml
respectively (Kuklenyik et al. 2004). It is difficult to explain
the high levels of these two PFASs as there is no information
available on the donors, sampling method or exact sampling
location. Belgium, on the other hand, had one of the highest
levels of PFASs recorded in literature, with peak levels of
PFOS, PFOA and PFHxS being 28,200 pg/ml, 3500 pg/ml
and 5300 pg/ml respectively (Roosens et al. 2010). In this
study, the authors proposed that although more work was
needed on the mode of transfer from maternal blood to human
milk, the PFAS profiles and concentration levels suggested
that sulphonates were easier to transfer to human milk compared to carboxylates (Roosens et al. 2010).
The highest concentrations of PFASs have been detected in
breast milk samples collected from areas of high economic
development and much industrial activity (Environmental
Directorate OECD 2005; Tao et al. 2008a, b; Liu et al.
2010). For instance, in China, PFAS concentrations were
quantified across 12 provinces, in both rural and urban areas.
The results indicated elevated concentrations of PFOA
(814 pg/ml), PFUnDA (196 pg/ml) and PFOS (100 pg/ml)
in urban Shanghai where there is a high level of
industrialisation. In contrast, the least concentrations were
from rural Ningxia, one of the least developed areas in
China, with concentrations values ranging from below the
limit of detection (< LOD) for PFOA and PFUnDA to 6 pg/
ml for PFOS (Liu et al. 2010).
Evidence from literature suggests a relationship between
concentrations of PFASs and industrial development. Tao
et al. (2008a, b) substantiated that individuals from countries
with a high gross domestic product (GDP) had elevated concentrations of PFOS than those from countries with a low
GDP. For instance, PFAS levels in samples from Cambodia,
India and Vietnam were found to be 40–50% less than those
reported in more developed countries such as China and
Germany (Tao et al. 2008a, b). The levels of PFASs reported
in samples from Asia showed that PFOS was the prime compound in human breast milk, with an estimated 85% occurrence in samples from India and 100% in other sampled countries (Tao et al. 2008a, b).
The highest PFOS concentrations ever recorded were
from Ehime in Japan, with a mean of 232 pg/ml. These
levels were attributed to the high usage and production of
consumer goods containing these contaminants (Tao et al.
2008a, b). In more recent investigations, PFAS levels
from other regions in Japan were at much lower concentrations than those from Ehime, although significantly still
on the high end (Nakata et al. 2009; Fujii et al. 2012).
These samples were collected from Hokkaido and Kyoto,
with peak PFAS concentrations found for PFOA as high
as 89 pg/ml and 93.5 pg/ml respectively.
Other high levels of PFOA in breast milk have been observed. In Germany, Suchenwirth et al. (2006) measured 12

 30

Not specified

Muttermilch

North Rhine–Westphalian Sauerland

Munich

Munich

Leipzig
Baviere

Gyor
Bologna

Barcelona

Valencia

Murcia

Catalonia

Uppsala

France

Germany

Germany

Germany

Germany

Germany
Germany

Hungary
Italy

Spain

Spain

Spain

Spain

Sweden

12

10

67

10

20

13
37

38
20

19

201

203

103

50

11 provinces

Belgium

Sample
number
22

Czech
Olomouc
Republic

Region

2004

2007

2014

2012

2008

1996–1997
2010

2006
2010

2006

2007, 2009

2006

PFOS
PFOA
PFOS
PFOA
PFHxS
PFOA
PFOS
PFOS
PFOS
PFOA
PFHxS
PFOS
PFOA
PFOS
PFOA
PFOS
PFNA
PFDA
PFOA
ipPFNA
PFNA
PFOS
PFDA
PFOA
PFNA
PFDA
PFHxS
PFOS
PFHxS
PFOS
PFOSA
PFOA

Sample collection PFASs quantified
year
above LOD
2006
PFOS
PFOA
PFHxS
2010
PFOA
L-PFOS
Br-PFOS
PFNA
PFDA
PFHxS
2010
PFOS
PFOA
2006
PFOA

Reported data of frequently detected PFAS concentrations in human breast milk (pg/ml)

Country

Europe

Table 2

93
176
–
–
–
–
116
126
–
–
–
317
60
47
–
–
–
–
177
14
4
50
43
54
41
24
40
120
85
201
13
–

Mean
(pg/ml)
–
–
–
50
33
20
–
–
–
78
59
–
50–284
80–610
< 30–110
< 150–250
< 20–30
< 201–460
28–239
28–309
< 30–195
> 150
> 20
96–639
24–241
15–288
< 15.2–907
28–865
< 11.5
< 85.5–1095
12–980
139–139
2–21
5–246
1.4–306
< 10–211
15–70
< 10–34
20–110
70–220
31–172
60–470
< 7–30
< 209–492

Range
(pg/ml)
< 400–28,200
< 300–3500
< LOQ–5300
12–128
7–114
< 10–63
< 6–15
< 6–12
< 6–22
24–171
18–102
4100–12,700

7.15
–
–
–
–
–
–
–
–

–
–
–
–
94.8 ng kg − 1 per day
(18.5 of PFOA, 12.2 of PFOS
and 64.1 of the rest of PFASs)

–
–
–
–
–
–
–
–
–
–
–
–
–

Estimated daily intake
(ng/kg body weight/day)
–
–
–
–
–
–
–
–
–
–
–
–

Kärrman et al. 2007

Kärrman et al. 2010

Guzmán et al. 2016

Lorenzo et al. 2016

Völkel et al. 2007
Barbarossa et al.
2013
Lorca et al., 2010

Völkel et al. 2007
Mosch et al. 2010

Völkel et al. 2007

Suchenwirth et al.
2006
Bernsmann and Fürst
2008
Fromme et al. 2010

Kadar et al. 2011

Lankova et al. 2013

Roosens et al. 2010

Reference

Environ Sci Pollut Res

 Phnom Penh

Beijing

Zhoushan

12 Provinces

Jinhu

Chidambaram,Kolkata, Chennai

Jakarta,Purwakarta

Kyoto

Ehime

Hokkaido

Not Specified

Seoul

Cambodia

China

China

China

China

India

Indonesia

Japan

Japan

Japan

Korea

Korea

Europe

Table 2 (continued)

30

17

51

24

30

20

39

50

1237

19

30

24

2010

2007

Not
Specified

1999

2010

2001

2002, 2004,2005

2009

2007

2004

2008–2009

2000

Asia
PFOA
PFOS
PFNA
PFHxS
PFOA
PFNA
PFDA
PFOA
PFOS
PFNA
PFDA
PFHxS
PFOA
PFOS
PFNA
PFDA
PFHxS
PFOS
PFOA
PFNA
PFDA
PFOA
PFOS
PFNA
PFHxS
PFOA
PFOS
PFNA
PFHxS
PFOA
PFNA
PFDA
PFOA
PFOS
PFNA
PFHxS
PFOA
PFNA
PFOS
PFHxS
PFOA
PFOS
PFHxS
PFOA
PFNA

PFNA
–
–
–
–
51.6
15.3
< 15
106.3
120.79
18.1
7.2
20.47
116.0
16.2
9.9
37.6
56
181
26
20
–
–
–
–
83.6
–
–
–
93.5
32.1
21.3
77.7
232
–
7.55
89
35
71
10
41
61
7.2
64.5
14.7

17
< 42.5–132
17.2–327
< 8.82–12.3
< 1.66–18.6
< 40–122
< 10–47
< 15–29
47–210
45–360
6.3–62
3.8–15
4.0–100
< 14.15–814
6–137
6–76
< 1.44–63
< 0.69–15
9–198
25–1440
5–95
< 1–70
< 42.5–335
< 11.0–120
< 8.82
< 1.66–13.3
< 42.5
25.4–256
< 8.82–135
< 1.66–6.23
< 40–194
< 10–72
< 15–65
< 42.5–170
140–523
< 8.82–23.9
< 1.66–18.2
16–270
8–124
46–98
< 4–20
< 43–77
32–130
0.83–16
< 40–173
< 10–41

< 5–20

Tao et al. 2008b

ΣPFOS, PFOA, PFHxS = 13.2
ΣPFOS, PFOA, PFHxS, PFNA = 14.9

–
–
–
–
4.7
0.8
6.9
5.3
1.2

Fujii et al. 2012

Kim et al. 2011

Nakata et al. 2009

Tao et al. 2008b

Fujii et al. 2012

Tao et al. 2008b

ΣPFOS, PFOA, PFHxS = 10.1
ΣPFOS, PFOA, PFHxS, PFNA = 11.0

7.7
2.6
1.8
ΣPFOS, PFOA, PFHxS = 39.2
ΣPFOS, PFOA, PFHxS, PFNA = 40.5

Liu et al. 2011

Liu et al. 2010

So et al. 2006

Fujii et al. 2012

Tao et al. 2008b

–

4.2
1.3
0.6
–
–
–
–
–
88.4
1.4–15.9

ΣPFOS, PFOA, PFHxS = 11.7
ΣPFOS, PFOA, PFHxS, PFNA = 13.1

–

Environ Sci Pollut Res

 Quezon

Hanoi, Ho Chi Minh

Malaysia

Philippines

Vietnam

USA

45

2

USA

Information of
location not
available
Massachusetts

13

40

24

13

264

North America
Canada
Ontario

Korea

Group 1; n = 215 (Seoul, Chungcheong,
Honam, Youngnam)
Group 2; n = 50 (Suwon, Wonju,
Daegu,Gwangju, Jeju)
Penang

Europe

Table 2 (continued)

2004

2003

2003–2004

2000, 2001

2000, 2004

2003

2013

PFOA
PFNA
PFOS
PFHxS
PFDA

PFOA
PFNA
PFDA
PFHxS
PFOS
PFHxA

PFOA
PFOS
PFNA
PFHxS
PFOA
PFOS
PFNA
PFHxS
PFOA
PFOS
PFNA
PFBS
PFHxS
PFHpA

PFDA
PFOA
PFOS

43.8
7.26
131
14.5
–

178
34
0.4
26
26
820

–
121
–
6.45
–
97.7
–
15.8
75.8
6.81

< 15
71
49

< 30.1–161
< 5.2–18.4
< 32.0–617
< 12.0–63.8
< 7.72–11.1

–
–
–
–
–
–

< 42.5–90.4
48.7–350
< 8.82–14.9
< 1.66–13.3
< 42.5–183
27.0–208
< 8.82–25.0
< 1.66–58.9
< 42.5–89.2
16.9–393
< 8.82–10.9
< 1.11–3.98
< 1.66–26.8
< 4.45–6.88

< 15–19
52–110
31–77

Tao et al. 2008b

ΣPFOS, PFOA, PFHxS = 13.1
ΣPFOS, PFOA, PFHxS, PFBS, PFHpA,
PFNA = 14.1

1.7
–
14.7
–
–

Tao et al. 2008a

Kuklenyik et al.
2004

Kubwabo et al. 2013

Tao et al. 2008b

ΣPFOS, PFOA, PFHxS = 19.8
ΣPFOS, PFOA, PFHxS, PFNA = 21.2

–
–
–
–
–
–

Tao et al. 2008b

Kang et al. 2016

ΣPFOS, PFOA, PFHxS = 19.7
ΣPFOS, PFOA, PFHxS, PFNA = 20.9

0.6
5.0–11.0
3.4–7.5

Environ Sci Pollut Res

 Environ Sci Pollut Res

pooled samples from 103 participants and presented the
highest levels of breast milk PFOA in literature, which ranged
from 4100 to 12,700 pg/ml. These findings were even higher
than levels reported in the North Rhine–Westphalian
Sauerland region, with known PFAS pollution from industrial
wastes and contamination by a recycling company
(Bernsmann and Fürst 2008). The results from the pooled
samples were unusually distributed and distinctly higher than
their matching PFOS levels and thus, should be interpreted
with care. No explanation was given concerning these high
levels. In the USA, a decrease in the levels of PFASs has been
reported in both humans and the environment as a result of the
voluntary phasing out of the manufacturing of PFOS bulk
material in 2001 by the 3 M Company, a known leading producer of PFOS (Kannan et al. 2004; Olsen et al. 2007).
However, PFOA levels have not declined as anticipated, with
records showing that PFOA serum levels remained constant
between April 2003 and August 2007 and may apparently be
increasing (Olsen et al. 2008; Beesoon et al. 2011).

Nursing history and PFAS concentration
A study by Tao et al. 2008b showed that previous breast feeding may lower the concentrations of PFOA in breast milk.
Corresponding to this, numerous studies have found that
mothers who were nursing for the first time had a higher load
of PFASs compared to those who had breast fed before
(Thomsen et al. 2010; Kadar et al. 2011; Barbarossa et al.
2013; Guerranti et al. 2013; Guzmán et al. 2016).
Thomsen et al. (2010) showed that the levels of PFASs in
breast milk of non-occupationally exposed multiparous women who breast fed were comparatively lower than those who
were breast feeding for the very first time (Thomsen et al.
2010). The mean concentrations of PFOS and PFOAwere less
by 44% and 59% respectively in women who had previously
nursed compared to those who were nursing for the first time.
In a similar study in Italy, PFOS levels were quantified above
the LOD in 90% of the breast milk samples from primiparas
women, while they were quantified in 62% of breast milk
from multiparous mothers (Barbarossa et al. 2013). In these
samples, the concentration levels of PFASs in primiparas
mothers ranged from 15 to 288 pg/ml (mean 57 pg/ml) and
from 15 to 116 pg/ml (mean 36 pg/ml) in multiparous women.
In Spain, the overall concentrations of PFASs ranged from
13 to 397 pg/ml (mean 96 + 101 pg/ml) in women who were
nursing for the first time, while in those who had previously
nursed, the values ranged from 13 to 167 pg/ml (mean 40 pg/
ml + 31 pg/ml) (Guzmán et al. 2016). In addition, PFOA
levels above the LOD were detected in all the samples from
primiparas participants, while only 42% of the samples from
multiparous donors contained PFOA. The literature data
above, including other studies (Hamm et al. 2010; Mondal
et al. 2014; Kishi et al. 2017), suggest that breast milk acts

as the prime exposure pathway of environmental PFASs for
breast fed infants whilst acting as a route for progressive elimination of these compounds from the mother’s body.
Noting the significant relationship that may exist between
concentrations of PFASs and the number of previous pregnancies, it is possible to propose that first-born children would
likely experience higher exposure dose of PFASs compared to
subsequent siblings. However, this would only be applicable
if the exposure of the mother remains the same over a stated
period. Should exposure of the mother increase (body burden)
after the first birth, there is a likelihood of corresponding increase in the birth that would follow. Whitworth et al. (2012)
stated that the intervals between pregnancy also had a significant influence on the body burden of PFASs, suggesting that
should there be a prolonged gap between births, contamination of breast milk may end up being as high as that of the first
lactation. To date, this hypothesis has neither been opposed
nor confirmed in the scientific community.
A number of preliminary studies have indicated that PFAS
concentrations are often gender specific in non-occupationally
exposed populations, where females generally have lower
concentrations compared to men (Olsen et al. 2003; Calafat
et al. 2006; Kärrman et al. 2007). Depuration of these
chemicals through long-term breast feeding could be one of
the ways responsible for this observation.

Possible mode of transfer from maternal blood
to breast milk
PFASs have been found to persist in various organs and other
matrices of the human body, including the liver, spleen, urine
and serum (Lau et al. 2007; Olsen and Zobel 2007; Fromme
et al. 2010). Levels of PFASs in breast milk have been reported to be lower than PFAS levels in maternal plasma and serum
in matched samples collected from the same donors
(Hinderliter et al. 2005; Kärrman et al. 2007; Liu et al.
2010). In an animal study, the concentrations of PFOA in rats’
plasma were reported to be ten times higher than those in the
matched milk samples (Hinderliter et al. 2005). On the other
hand, PFOS concentrations in human breast milk were found
to be 1.6% and 0.5% of the levels in blood from Spanish
nursing mothers (Kärrman et al. 2010) and Chinese mothers
(So et al. 2006), respectively. Although matching blood and
milk samples from the same donors has been an attempt to
establish the transfer efficiency and pathway of different
PFASs from maternal blood to breast milk, the mechanism
of transfer has not been clearly documented.
While the mode of transfer of PFASs is still unclear and
very debatable, no experimental data is currently available to
provide insights on the systemic circulation of PFASs in
humans. Furthermore, recent publications have also noted that
the profiles of some PFASs in breast milk differ from those in
blood samples from the same donors (Kim et al. 2011; Fujii

 Environ Sci Pollut Res

et al. 2012), thus adding more ambiguity to their transfer
mechanism. However, many researchers have agreed that
once in the body, PFASs do not accumulate in lipids but rather
in protein-rich organs such as the kidneys, blood and liver
(Martin et al. 2004; Kannan et al. 2004; Völkel et al. 2007;
MacManus-Spencer et al. 2010). In blood, these contaminants
have been noted to form strong bonds with the protein albumin, accounting for their persistence in the serum (Jones et al.
2003; Butenhoff et al. 2006; Liu et al. 2011).
Serum albumin is considered the most abundant protein in
human blood as its concentrations range from 35 to 50 gL−1
(Peters Jr 1985), making it the preferable mode of transport to
distribute biological components, such as hormones, as well
as PFASs to various organs and tissues in the body (Jones et al.
2003; MacManus-Spencer et al. 2010). A study by Han et al.
(2003) has reported that albumin in the serum has a substantial
binding capacity for PFOA, where more than 90% of the
compound in blood binds to the protein. This study further
demonstrated that albumin has 6–9 binding sites per molecule
available for PFOA, thereby leaving an estimated < 5% of the
compound as a free fraction in the plasma (Han et al. 2003). In
a separate study on the interactions between PFOS and
proteins, Jones et al. (2003) noted that PFOS had the potential
to displace lipids and steroid hormones from the binding sites
on serum proteins, thus causing the contaminants to be classified as an endocrine disruptor. This study concluded that
PFOS has a high propensity for albumin, making it unavailable for interaction with other proteins or molecules until all
the accessible binding sites on serum albumin have been occupied (Jones et al. 2003).
Andersen et al. (2008) indicated that the modes of action
for PFASs are largely determined by both their chemical and
physical properties. In this regard, these compounds simulate
fatty acids in both their structure and chemistry and are thus
most likely transferred to the mammary gland by binding to
albumin the same way fatty acids do (Peters Jr 1985;
MacManus-Spencer et al. 2010). The structural similarity of
PFASs to fatty acids suggests the possibility of PFASs to pass
across the mammary epithelial membrane the same way fatty
acids do. McManaman (2014) outlined the different processes
involved in milk production, as well as the transport of fatty
acids needed for milk lipid synthesis. The outline indicated
that natural long-chain fatty acids in humans are first hydrolysed by lipoprotein lipase and then transported to the mammary gland by serum triglycerides or carrier proteins, including albumin. Once at the external plasma membrane, they are
then transferred through regulation of fatty acid transporters in
mammary membranes. These transporters include CD36,
acyl-coA synthetase (ACSL), fatty acid translocase (FAT)
and fatty acid transport proteins (FATP) (McManaman
2014). An example of structural similarities between PFASs
and naturally occurring fatty acids is PFOA (Fig. 1) as previously described, and caprylic acid (octanoic acid) (Fig. 3), an

eight-carbon saturated fatty acid found naturally in the fat
component of milk from many mammals (Beare-Rogers
et al. 2001).
The credit of the protein-binding mechanism is that it explains why PFAS concentrations in milk are lower than those
in serum. However, the limitation of this protein-binding
mechanism is that the high binding efficiency of PFASs to
serum protein limits the possibility of these contaminants to
be passed through to breast milk and ideally should not be
persistent in the same. However, the high levels found in milk
samples in some preliminary data such as 820 pg/ml of
PFHxA in USA (Kuklenyik et al. 2004) and 317 pg/ml of
PFOS in Belgium (Völkel et al. 2007) propose that PFASs
do accumulate in high amounts in breast milk. As aforementioned, once PFASs are bound to albumin in serum, there is
very limited interaction of these compounds with other molecules, and therefore would suggest further limited possibility
of their interaction with fatty acid transporters in mammary
membranes. This then suggests that the method of transfer
should therefore allow for large amounts to pass to breast milk
without being restricted by binding to serum albumin. In this
regard, a membrane-transport mechanism could be responsible for the transfer of unbound PFASs from maternal blood to
breast milk (Figs. 4 and 5).
Transport of chemicals into human breast milk involves
the passage of low molecular weight chemicals through
the mammary epithelium membrane which is semipermeable (Black 1996). While smaller molecules can
easily pass through this membrane through diffusion, larger compounds can be transferred into the alveolar cells by
the process of pinocytosis, where particles or liquids are
engulfed and released back into breast milk through apocrine secretion (Casey 2005). This could account for the
prevalence of short-chain PFASs in breast milk compared
to long-chain PFASs (Andersen et al. 2008; Kim et al.
2011; Fujii et al. 2012; Kang et al. 2016). Short-chain
carbons are more soluble and have a lower molecular
weight, which makes them more readily available to pass
through the mammary epithelial membrane and thus contaminate breast milk. This suggests that chain length plays
a key role in the transfer of these contaminants from the
vascular system to breast milk and influences the pharmacokinetic properties of PFASs, where smaller molecules
are eliminated faster, even via depuration (Ohmori et al.
2003). In addition, Fujii et al. (2012) showed that among

Fig. 3 Chemical structure of perfluorononanoic acid (PFNA)

 Environ Sci Pollut Res

Sample collection and storage

Fig. 4 Chemical structure of perfluorohexane sulphonic acid (PFHxS)

the long-chain compounds (C10–C13) found in breast milk
of mothers in three Asian countries, odd numbered PFASs
were more common than even numbered PFASs, apart
from PFDA in Japanese samples. The reason for this observation is still unclear.
The challenge with the membrane-transport mechanism is
that PFASs are generally lipophobic and do not accumulate in
lipids, thus they are likely not to be transported through serum
lipids across the mammary membrane as other chemicals do.
However, the fact that PFASs are similar to fatty acids, suggests a strong possibility that they can be transported to breast
milk through the same mechanism.
A conclusive preference mode of transfer between these
two mechanisms cannot be established based on the current
data. However, it can be opined that both mechanisms may
work together, to varying degree, to ensure transfer of PFASs
from the maternal vascular system to breast milk.

Analytical challenges for the determination of PFASs
in breast milk
The complexity in the physical and chemical properties of
PFASs presents various challenges during analysis, particularly for complex biological matrices such as breast
milk. The analytical procedures for PFASs usually follow
the general strategies for the analysis of complex organic
compounds in biological matrices, with a common extraction and cleanup steps followed by chromatography and
MS detection. In this review, we emphasised on methods
focussing on breast milk as a matrix. Table 3 presents
different methods reported in the literature for the analysis
of PFASs covering the period 2004 to 2017.

Fig. 5 Chemical structure of perfluorodecanoic acid (PFDA)

Contamination can occur during collection and storage of
breast milk samples for analysis, particularly where collection
bottles have not been pre-treated or the wrong bottle material
is used (Martin et al. 2004; Okáľová 2014). PFASs have been
noted to attach to container surfaces due to their ionic properties and this has been a major challenge for both sampling and
storage, such that they are unavailable for analysis at trace
levels (Reagen et al. 2008). Glass containers have been deliberated as suitable options for collection, but it has also been
argued that ionic PFASs similarly adsorb onto the glass surfaces (Martin et al. 2004; Van Leeuwen and de Boer 2007).
The use of polypropylene containers for the collection and
storage of breast milk for PFAS analysis is very popular in the
scientific literature. This is due to the fact that polypropylene
containers have lower affinity for surface adsorption compared to other materials (Bernsmann and Fürst 2008;
Fromme et al. 2010; Barbarossa et al. 2013). Prior to use,
sample collection containers are usually rinsed with polar or
semi-polar solvents such as methyl tert-butyl ether, methanol
and acetone to reduce contaminations (van Leeuwen and de
Boer 2007).
Sample extraction and cleanup
The complexity of the sample treatment is related to potential
matrix interference and the separation technique used, usually
tandem LC MS (Trojanowicz et al. 2011). Similarly, the
physiochemistry of the analyte, mostly the polarity of
PFASs, have to be considered. Recent advances in extraction
methods together with the parallel improvement of the techniques of analysis has resulted in the reduction in the complexity of the sample pre-treatment and has increased the accuracy and precision of the analysis of PFASs. The matrix in
which specific compounds to be measured exist need to be
considered for suitable sample preparation and cleanup.
Human breast milk is a combination made up of different
constituents which vary depending on the maturity of the
milk. Colostrum is the first milk produced immediately after
birth, and its composition differs from that of mature breast
milk in that its protein content is higher and the carbohydrate
content lower. The fat content remains consistent during lactation but slightly increases with each nursing (Jennes 1979).
Many researchers use mature milk samples to measure the
levels of PFASs present.
The composition of breastmilk usually makes it a very
complex matrix for the analysis of trace contaminants. The
binding of PFASs to proteins, particularly albumins, has
meant that numerous investigators employ protein precipitation using organic solvents such as methanol and acetonitrile
or by freeze-drying for sample pre-treatment (Fromme et al.
2010; Mosch et al. 2010; Thomsen et al. 2010). In their

 Freeze-drying

–

Hydrolysis of protein and fat using Protease Type SPE
XIV (Sigma L 1754-5G) and Lipase Type VII (Strata X SW-SPE
(Sigma P 5147-1G)
column)
(adapted from Karrman
et al. 2004)

HPLC-ESI-MS/MS

HPLC-ES- MS/MS

LC-ESI-MS/MS

SPE
(Waters Oasis WAX)
(Adapted from Taniyasu
et al. 2005)

SPE (Oasis HLB)
(adapted from
Kuklenyik et al. 2004
with modifications)

SPE (60 mg/3 mL Oasis
HLB column)

Formic acid added, vortex mixed and sonicated

HPLC-TIS-MS/MS

Extraction/cleanup
(cartridge used)

Sample pre-treatment

Analytical methods reported in the literature for the determination of PFASs in breast milk

Technique

Table 3
Stationary phase

Amount
of breast
milk
sample
used

A: 2 mM
ammonium
acetate in
water
B: Methanol
(flow rate of
150 μl/min)

C18 Column (Agilent,
100 × 2.1 mm, 1.8 μm)

Discovery HS C18 Column
A: 2 mM
(50 mm × 2.1 mm inner
ammonium
diameter, 3 μm, 120 Å
acetate in
pore size)
methanol
B: 2 mM
ammonium
acetate in
water
(flow rate of
300 μL/min)

3 ml

1 ml

Betasil C8 column
1 ml
A: 20 mM
(3 × 50 mm, 5 μm)
ammonium
acetate
(pH 4) in
water
B: 20 mM
ammonium
acetate
(pH 4) in
methanol
(flow rate of
300 μL/min)
A: 2 mM
Keystone Betasil C18 column 2 ml
ammonium
(2.1 × 50 m, 5 μm)
acetate
B: Methanol
(flow rate of
300 μL/min)

Mobile phase

PFBS
PFHxS
PFOS
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFBuS
PFHxS
PFOS
THPFOS
PFDS
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFDoDA
PFOSA
PFBS
PFPA
PFHxA
PFHpA
PFHxS
PFHpS
PFOA

PFOSA
PFHxS
PFOS
PFPeA
PFHxA
PFOA
PFNA
PFDeA
PFUA
PFDoA

Analyte

Kuklenyik
et al.
2004

So et al.
2006

82
82
74
80
102
89
80
62
55
26
86
92
93
88
92
92
92
92
94
79
81
83
51
72
80
84
82
77
43
38
39
34
Recovery
at
1.25 μg/l
92.1
80.1
93.0

Bernsmann
and Fürst
2008

Kärrman
et al.
2007

Reference

Average
percentage
recovery

Environ Sci Pollut Res

 Hydrolysis of protein and fat using Lipase Type
VII (Enzyme Commission number: 3.1.1.3;
EC: 232–619-9) and Protease Type XIV (mix
of different enzymes; EC: 232–909-5)

–

LC-TIS-MS/MS

UPLC-ES-MS/MS

On-line SPE
(Oasis HLB 20 × 2.2,
25 μm)
(adapted from Mosch
et al. 2010)
SPE

Ultrasonic extraction
followed by SPE
(Oasis WAX)

–

LC-MS/MS

Internal standards added and
samples sonicated with 90.2%
formic acid

HPLC-ESI-MS/MS

SPE
(Waters Oasis WAX;
6 cc,150 mg) (adapted
from Kuklenyik et al.
2004, with
modifications)
SPE
(Waters Oasis WAX;
6 cc,150 mg)
(adapted from Tao et al.
2008a)
–

Internal standards added and samples
sonicated with 90.2% formic acid

HPLC-ESI-MS/MS

Extraction/cleanup
(cartridge used)

HPLC-ESI-MS/MS Internal standards added and
samples sonicated with 600
(Adapted from Brink
μl acetonitrile
et al., 2006)

Sample pre-treatment

Technique

Table 3 (continued)

Reprosil-Pur C18 AQ, Dr.
Maisch, Ammerbuch,
Germany
(5 μm, 33 mm × 3 mm)

1 ml

0.4 ml

–

–

400 μl
Reprosil-Pur C18 AQ, Dr.
Maisch, Ammerbuch,
Germany
(5 μm, 33 mm × 3 mm)

Betasil® C18 (2.1 × 50 mm,
5 μm)

–

5 ml

Betasil® C18 column
(100 × 2.1 mm, 5 μm)

–

–

PFHxS
PFOS
PFOA
PFNA

–

5 ml

Betasil® C18 column
(100 × 2.1 mm, 5 μm)

101
105
109
94

88.1
103.6
93.0
95.4
98.5
88.2
94.9
57.7
65.8
95.6

PFNA
PFOS
PFDA
PFDS
PFUnA
PFDoA

A: 2 mM
ammonium
acetate
B: Methanol
(flow rate of
300 μL/min)
A: 2 mM
ammonium
acetate
B: Methanol
(flow rate of
300 μL/min)
100% of a
2 mM
ammonium
acetate buffer
solution
(flow rate of
0.6 ml/min)
20 μmol/L
Ammonium
acetate in
water
(flow rate of
0.2 mL/min)
A: 2 mM
ammonium
acetate
B: Methanol

Average
percentage
recovery

Analyte

Amount
of breast
milk
sample
used

Stationary phase

Mobile phase

Fromme
et al.
2010

Nakata
et al.
2009

Völkel
et al.
2007

Tao et al.
2008b

Tao et al.
2008a

Reference

Environ Sci Pollut Res

 Sample pre-treatment

Internal standards added and samples sonicated
with formic acid. Final solution filtered.
(adapted from Liu et al. 2008)

Enzymatic protein hydrolysis
and protein precipitation

Internal standards added and samples sonicated
with formic acid. Final solution filtered.
(adapted from Liu et al. 2008)

Technique

UPLC-ES-MS

LC – ESI-MS/MS

UPLC-ES-MS

Table 3 (continued)

SPE
(Waters Oasis WAX)
(adapted from Liu et al.
2008)

On-line SPE
(Oasis HLB 20 × 2.2,
25 μm)

A: 2 mM
ammonium
acetate
B: Methanol
A: 2 mM
aqueous
ammonium
acetate
solution
B: Methanol
(flow rate of
0.4 mL/min)

A: 2 mM
ammonium
acetate in
methanol
B:2 mM
ammoniu m
acetate in
water
(flow rate of
400 μL/m)
A: 2 mM
ammonium
acetate
B: Methanol
(flow rate of
0.4 mL/min)

(Waters Oasis WAX)

SPE
(Waters Oasis WAX
60 mg/3 mL)

Mobile phase

Extraction/cleanup
(cartridge used)

Amount
of breast
milk
sample
used

Reprosil-Pur C18 AQ, Dr.
Maisch, Ammerbuch,
Germany
(5 μm, 33 mm × 3 mm)
Waters BEH C18 Column
(2.1 × 50 mm; 1.7 μm)

2 ml

0.4 ml

Waters Acquity UPLC® BEH 2 ml
C18 Column
(2.1 × 50 mm; 1.7 μm)

Waters Acquity UPLC® BEH
C18 Column
(2.1 × 50 mm, 1.7 μm)

Stationary phase

Average
percentage
recovery

Kärrman
et al.
2010

Reference

PFHxS
PFOS
PFDS
PFPeA
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUdA
PFDoA
PFTrDA

Recovery
at
0.1 ng/mL
83
94
49
112
87
119
93
97
89

Liu et al.
2011

Liu et al.
Recovery
2010
at
100 pg/mL
114
110
96
136
99
95
110
98
102
57
73–112% of all PFASs Mosch
et al.
2010
PFHxS
PFHpS
PFOS
PFPeA
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUdA

Analyte

Environ Sci Pollut Res

 Sample pre-treatment

Formic acid and water added to samples

–

Protein precipitation and hydrolysis
using proteas and lipase

Freeze-drying

Technique

UPLC-ESI-MS/MS
-

LC-ESI-HRMS

HPLC-ESI-MS/MS

GC-EI-MS

Table 3 (continued)

IPE (adapted from
Taniyasu et al. 2005)
Followed by SPE
(adapted from Tao
et al. 2008a)
(Waters WAX; 6 cc,
150 mg)

LLE followed by a
purification on two
successive SPE
(Oasis HLB and carbon
graphitized Envicarb
cartridges)

SPE
(Waters Oasis WAX)
(adapted from Taniyasu
et al. 2005)

Extraction/cleanup
(cartridge used)

Stationary phase

Amount
of breast
milk
sample
used

J &W DB-5MS column

C18 column

A: Methanol
Gemini C18 reverse phase
B: 20 mM
Column (3 μm,
Ammonium
50 × 2.0 mm)
acetate (flow
rate of
0.6 mL/min)

2 ml

3 ml

Ammonium
ACQUITY BEH C18 column 1 ml
hydroxide in
(2.1 × 50 mm; 1.7 mm,
acetonitrile
Waters, USA)

Mobile phase

Average
percentage
recovery

Reference

–

89
75
36
Ranged between 78% Roosens
et al.
and 101% for PFOS
2010
and PFOA. For
PFNA, PFHxA,
PFBS and PFHxS
averages recoveries
varied between 81%
and 109%.
Recoveries
of PFBA and PFDA
were 70% and 160%,
respectively.
Kadar et al.
Recovery
PFBS
2011
at
PFHxS
750 pg/PFHpS
mL
PFOS
79
PFBA
83
PFPeA
104
PFHxA
89
PFHpA
82
PFOA
61
PFNA
69
PFDA
78
PFUnA
87
PFDoA
78
PFOSi
82
81
102
82
Kim et al.
2011

Analyte

Environ Sci Pollut Res

 –

IPE
(adapted from Guerranti
et al. 2013)

SPE
(HLB glass cartridges)

GC-EI-MS
(Adapted from
Dufková et al.
2009, 2012)

–

LC–ESI-MS/MS

LLE followed by a
purification on two
successive SPE
systems
(Oasis HLB cartridge
then Supelclean™
ENVI-Carb cartridge)

d-SPE
(C18)

Protein precipitation with acetone

UPLC-ESI-MS/MS

Helium carrier J &W DB-35MS column
gas (flow rate
(30 m × 0.25 mm × 0.25
of
μm)
1 mL/min)

0.5 ml

Helium carrier
gas (flow rate
of
1.5 mL/min)
A: Ammonium Waters Acquity UPLC® BEH 3 ml
C18 reversed phase
acetate
column (50 × 2.1 mm,
20 mM
1.7 μm)
aqueous
solution
B: Methanol
(flow rate of
0.5 mL/min)
Two Discovery HS C18
1 ml
A: 20%
columns (7.5 cm 2.1 mm,
acetonitrile
3 μm)
in 2 mM
ammonium
acetate
B: 90%
acetonitrile
in 2 mM
ammonium
acetate
(flow rate of
200 μl/min)
Waters Acquity UPLC HSS 15 ml
A: 5 mM
T3 analytical column
ammonium
(100 mm × 2.1 mm,
acetate
1.8 μm)
B: Methanol
(flow rate of
0.3 mL/min)

Amount
of breast
milk
sample
used

LLE followed by SPE
(Presep-C silica gel
column)

Stationary phase

Mobile phase

Extraction/cleanup
(cartridge used)

UHPLC-ESI-MS/MS Formic acid and acetonitrile
added, hand mixed

Sample pre-treatment

Technique

Table 3 (continued)
Average
percentage
recovery

92
PFBA
91
PFPeA
97
PFHxA
95
PFHpA
104
PFOA
94
PFNA
97
PFDA
99
PFUdA
90
PFDoA
PFTrDA 101
PFTeDA 98
99
PFBS
101
PFHxS
Percentage mean
recovery for all
PFASs was 96.71%

–

Analyte

Guzmán
et al.
2016

Lankova
et al.
2013

Kubwabo
et al.
2013

Barbarossa
et al.
2013

Fujii et al.
2012

Reference

Environ Sci Pollut Res

 Protein precipitation and hydrolysis
using proteas and lipase

HPLC-ESI-MS/MS

UHPLC-ESI-MS/MS Alkaline digestion
(adapted from Lorca et al. 2010)

Sample pre-treatment

Technique

Table 3 (continued)
Mobile phase

Stationary phase

IPE followed by SPE
(adapted from Kim et al.
2011 with
modifications)

YMC-Pack ODS-AQ C18
A: 2 mM
column, (2.0 × 150 mm,
aqueous
3.0 μm)
ammonium
acetate
solution
B: Methanol
(flow rate of
200 μL/min)
SPE
Ammonium
Kinetex 1.7 V XB − C18
(Strata-X 33 m polymeric
hydroxide in
column (50 × 2.1 mm and
reversed phase 60 mg)
methanol
5 μm)

Extraction/cleanup
(cartridge used)

–

Amount
of breast
milk
sample
used

Average
percentage
recovery

Reference

PFBA
PFPeA
PFBS
PFHxA
PFHpA
PFHxS
PFOA
PFHpS
ipPFNA
PFNA
PFOS
ipPFNS
PFDA
PFDS
PFDoDA
PFTrDA
PFTeDA
PFHxDA
PFODA
PFUnDA

Recovery
Lorenzo
at
et al.
75 ng L2016
−1
90
81
91
81
90
98
96
79
89
80
112
107
90
91
91
89
97
98
92
108

Ranged between 80% Kang et al.
and 120%, except for
2016
PFPeA
(72.5–86.1%)

Analyte

Environ Sci Pollut Res

 Environ Sci Pollut Res

methodology, Bernsmann and Fürst (2008) applied a hydrolysis step before protein precipitation, utilising a protease and
a lipase enzyme to break down the milk emulsion and discharge the PFASs bound in the micelles to improve detection
and quantification. The results showing the success of the
hydrolysis were a change in the pH of the samples from 7.5
to 6.9, and a clearly visible breakdown of the emulsion after
incubation (Bernsmann and Fürst 2008).
Different extraction protocols have been reported for
PFASs in breast milk. However, solid phase extraction
(SPE) is more popular owing to high recovery, short analysis
times, simpler procedures and use of less solvents compared
to other techniques such as liquid/liquid extraction (LLE).
Although SPE is a refined alternative to LLE, several authors
employed a combination of LLE and SPE for sample extraction and cleanup, since LLE is efficient in the extraction of
compounds with high molecular weights (Kadar et al. 2011;
Fujii et al. 2012; Barbarossa et al. 2013), this is followed by
SPE cleanup of the extracts (Table 3). LLE on its own is not
effective as an extraction method since some PFASs do not
partition in the presence of LLE organic solvents, which then
results in low recoveries (Jones-Lepp et al. 2004). Moreover,
SPE is preferred as both extraction and cleanup can be performed simultaneously.
SPE cartridges that have been commonly used for PFASs
are hydrophile-lipophile balance cartridges (HLB), carbon-18
(C18) cartridges, silicon cartridges and weak anion exchange
cartridges (WAX) (Hu and Yu 2010; Sun et al. 2017). In some
investigations involving milk as the matrix, reversed phase
SPE cartridges packed with silica have been used to further
clean up extracts and to also reduce interfering compounds
(Hu and Yu 2010; Fujii et al. 2012; Lorenzo et al. 2016).
Results in these studies have reported lower matrix effects.
WAX cartridges, however, have been distinguished as providing the best extraction outcomes during SPE for both the
perfluoroalkyl carboxylic acids and the perfluoroalkyl
sulphonates as they have been noted to reduce matrix effects
(Sun et al. 2017). HLB cartridges, on the other hand, provide
good extraction recovery only for the long-chain PFASs.
While C 18 cartridges have been found to contain trace
amounts of PFASs and are less recommended (Yamashita
et al. 2004; Wang et al. 2014; Sun et al. 2017), their ability
to provide high recovery and suitable peak shapes for certain
PFASs such as PFOS and PFOA have prevented their phaseout (Schiessel and Krepich 2017). In addition, C18 phases
normally require an adjustment of pH to 2 by adding formic
acid so as to increase its capacity to trap the required analytes.
On-line SPE have been employed for extensive biomonitoring which requires large throughput and high sensitivity
(Mosch et al. 2010; Schiessel and Krepich 2017). This extraction technique has been used in milk samples as well as several other aqueous matrices and it has shown several advantages over the conventional SPE methods. Firstly, the

conventional SPE method requires the methanol extract to
be evaporated before reconstitution, whereas on-line SPE bypasses this stage and therefore takes less time to complete
(Schiessel and Krepich 2017). Furthermore, on-line SPE requires a smaller volume of the sample to be used (Table 3) and
offers more sensitivity due to the lower injection volume.
Recent studies have also used ion-pairing extraction (IPE)
either in conjunction with SPE or as the sole extraction method (Kim et al. 2011; Guzmán et al. 2016; Kang et al. 2016).
This method is mainly used because of the acidic nature of
PFASs, where an ion-pairing agent is added to the sample to
improve the extraction efficiency of the analytes (Villaverdede-Sáa et al. 2012). Nevertheless, this process is often followed by successive SPE cleanups, which makes sample preparation tedious and not cost-effective.
Instrumental analysis
Gas chromatography Methods based on gas chromatography
(GC) coupled with mass spectrometer (MS) as the detector
have been used for the identification and quantification of
PFASs in various environmental matrices (Trojanowicz et al.
2011). Although GC was earlier used to study the levels of
PFASs in both indoor and outdoor air, it has also been used in
breast milk analysis as it offers high separation efficiencies for
individual PFASs (Table 3) (Shoeib et al. 2006; Fujii et al.
2012; Trojanowicz and Koc 2013; Guzmán et al. 2016). In
addition, the good peak resolution offered, GC helps to determine branched isomers and their distribution in a sample
(Trojanowicz and Koc 2013). However, they have since become very unpopular due to tedious derivatisation steps
occasioned by the general low volatility of PFASs.
In environmental samples, GC is often used when the target
analytes are PFASs or fluorotelomer alcohols which are more
volatile than the acids. Guzmán et al. (2016), however, quantified both PFAAs and PFASs in breast milk using GC-MS by
first esterifying PFOA, PFNA, PFDA, perfluoroundecanoic
acid and perfluorododecanoic acid using chloroformates to
make them semi-volatile by forming methyl esters. During
sample preparation for GC analysis, derivatisation can be
done using diazomethane; benzyl bromide during LLE; 2,4difluoroaniline; strong anion exchange extraction in conjunction with methyl iodide; or using methanol or butanol (Martin
et al. 2003; Orata et al. 2009; Trojanowicz and Koc 2013).
However, the major challenge with the derivatisation of
PFASs is the formation of unstable derivatives, which makes
it difficult to obtain reproducible data with GC. In addition,
GC methods involve long, multiple sample preparation stages
before analysis, which in turn subject the whole process to
multiple sources of human error (Larsen and Kaiser 2007)
and loss of analytes. Although the use of GC with electron
capture detector (ECD) has been proposed for the analysis of
PFASs, the strong bonds between the fluorine and carbon

 Environ Sci Pollut Res

atoms in the chemical structures of PFASs, renders ECD with
low specificity (Okáľová 2014).
Liquid chromatography methods The primary analytical technique used for the determination of PFASs in breast milk is
liquid chromatography (LC) with various detectors such as
mass spectrometer (MS) tandem electrospray ionisation mass
spectrometry (ESI-MS/MS) and high-resolution mass spectrometer (HRMS) (Table 3). LC methods have been more
popular than GC methods owing to their good selectivity
and high sensitivity for a wider range of PFASs (Yusa et al.
2012). In recent time, ultra-high-performance liquid chromatography (UPLC-MS/MS) is becoming very popular in the
analysis of PFASs due to the use of ultra-small particle size
(such as 1.7 μm) stationary phases and very high pressure
offered by UPLC results in shorter analysis time and better
LOD compared to HPLC-MS (Trojanowicz and Koc 2013).
As shown in Table 3, various columns have been employed
for the separation of PFASs; however, as highlighted above,
C18 and HBL shorter columns with small particle size are
becoming more popular (So et al. 2006; Kärrman et al.
2007; Nakata et al. 2009; Lorenzo et al. 2016). The reduced
internal diameter reduces separation speed and increases analysis time, thereby giving better peak resolution of each PFAS
at trace level. The use of reverse phase chromatography applying standard mobile phases such as acetonitrile, methanol
and water offers great selectivity, sensitivity and good peak
quality, hence its popularity in the analysis of PFASs. To improve the ionisation efficiencies of PFASs in negative ESIMS/MS mode, most authors have included ammonium acetate
buffer to mobile phases (Haug et al. 2009).
Electrospray ionisation in the negative polarity mode has
been the ionisation technique of choice when analysing
PFASs in breast milk by numerous investigators (Tao et al.
2008a, b; Kärrman et al. 2010; Roosens et al. 2010; Kim et al.
2011). The advantage of ESI is that it is significantly sensitive
and offers high selectivity in the measurement of PFASs and
has been esteemed as the first reliable method for analysing
PFASs (Reagen et al. 2008). As PFASs are often required to be
detected at trace levels, ESI becomes the most suitable
ionisation method since it offers low sensitivity usually in
the range 2 to 100 pgml−1 in biological samples (Okáľová
2014; Sun et al. 2017). Furthermore, Table 3 indicates the
popularity of small internal diameter columns among
investigators.
A major challenge of ESI, however, is matrix effect induced by either signal suppression or enhancement (Yusa
et al. 2012). Signal enhancement occurs when matrix components or interferences, particularly lipids from the breast milk,
reduce surface tension during detection and thereby make the
ionisation detection signals stronger (Enke 2007; Sun et al.
2017). Signal suppression, on the other hand, occurs when
co-eluting residual components compete with the target

analytes for charge and in turn reduce the number of analyte
ions available, thus resulting in erroneous detection (Enke
2007). To overcome matrix effect in ESI, most authors have
introduced the use of matrix-matched calibrations and carbon13 labelled stable isotopes or deuterated analogues as internal
standards for the quantitation of PFASs in biological matrices
such as breastmilk (Villagrasa et al. 2006; van Leeuwen et al.
2008; Sun et al. 2017). Similarly, in order to overcome the
shortcomings of ESI, liquid chromatography high resolution
mass spectrometry (LC-HRMS) has been applied for the analysis of PFASs in human milk samples from France (Kadar
et al. 2011). The main strength of this system is that the
high-resolution technique reported minimal matrix effects
hence reducing the risk of either underestimating or
overestimating the levels of PFASs in the samples. Since most
PFASs do not contain any chromophore, analysis of PFASs
with HPLC coupled with ultraviolet-visible spectroscopy
(UV/Vis) detectors is limited.
Quality control
There is an overall struggle for quality with regard to PFASs in
human breast milk samples as there are no standard reference
materials or stable isotope internal standards specifically for
milk samples. The various methods that have been published,
with both their strengths and limitations, are not standardised
and many of these are only validated in-house, hence posing a
loop-hole in data accuracy. Because many of these methods
were initially dedicated to monitoring PFASS in food, water
and blood, they demonstrate some limitations with regard to
their specificity and sensitivity to breast milk. Therefore, there
is need for a validated reference method, which defines the
accuracy, stability, recovery, calibration, precision, sensitivity
and uncertainties of the method.

Conclusion
Intake of breast milk by infants is a major exposure source of
perfluoroalkyl substances through depuration by the mother.
Notable effects of such post-natal exposure have been recorded in literature and these vary from developmental, neurological and reproductive effects. On the other hand, it has also
been noted that breastfeeding acts as an elimination pathway
from the mother as investigations have found that previous
breastfeeding tends to reduce the levels of PFASs in
breastmilk. Although there is strong evidence of the persistence of these contaminants in human milk, many of the published material is based on small sample size and thus should
be interpreted with care. Large gaps still exist in literature of
their geographical profiles and distribution as there are no
recorded documents of these compounds from Africa, South
America and Australia.

 Environ Sci Pollut Res

The mode of transfer from maternal blood to breast milk has
not yet been scientifically established, thus we proposed that
transfer could be through both protein binding and membrane
transport mechanisms. Although a conclusive preferred mode
of transfer between these two cannot be established based on
current data, it can be submitted that both mechanisms may
work together to varying degree. Understanding the mode of
transfer could be the key to limiting transfer of these and other
organic pollutants to infants through breast feeding. In addition,
a lot of attention in the studies of PFASs is more focused on
long chain compounds, yet preliminary studies indicate that
short-chain compounds are predominant in breast milk.
Research on the differences in levels of PFASs in colostrum
and matured milk could also be beneficial in identifying the
most vulnerable breast-feeding stage for infant exposure. In
addition, it has been suggested that depuration of these compounds through breast feeding could be one of the ways responsible for females having lower levels of PFASs than men;
further studies may need to be done to elaborate on this as
menstruation and delivery are also possible excretion methods.
Generally, there have been great advancements in the development of various analytical methods to quantify PFASs in
breast milk, but a standard validated method specific to breast
milk is lacking within the scientific community which is key,
as reliable data are crucial in understanding the toxicity and
the fate of these contaminants. Many challenges in analysis
have been noted when it comes to PFASs, mostly related to
their physicochemical properties. Firstly, these properties limit
the types of methods that can be used to quantify them, for
instance, they do not have chromophores therefore ultravioletvisible spectroscopy cannot be used. They also have low volatility and form unstable derivatives, which in turn limits the
use of GC-MS. One other major challenge of analysis is that
they form strong anions which enable them to bind to laboratory equipment, containers and solvents, making them unavailable for analysis at trace levels.
In conclusion, there remain gaps in literature where more
work focussing on the fate, transfer mechanism and accurate
analytical procedure for the determination of PFASs in breast
milk is needed to further understand the fate, load and exposure mechanisms of PFASs. Generally, literature data suggests
a global decline in PFAS levels in breast milk, therefore the
benefits of breast feeding may outweigh the potential risks
associated thereof.

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