Original

Chromatography, Vol.25 No.1 (2004)

Original

Simultaneous Detection of Endocrine Disruptors of 4−Nonyl Phenols and
4−Octyl Phenols in Commodity Products Using Positive Chemical
Ionization−Gas Chromatography/Mass Spectrometry
Tomoo Nakagawa*, Naeko Sugaya*, Katsumi Sakurai*, Junichi Nakagawa**,
Koichi Usukura***, Nobuhiko Onda*** and Koichi Oguma*****Yokohama City Institute of Health, 1−2−17 Takigashira, Isogo−ku, Yokohama, 234−0011, Japan**Tokyo Metropolitan Research Laboratory of Public Health, 3−24−1 Hyakunincho, Shinjuku−ku, Tokyo, 169−0073, Japan***PerkinElmer Japan, 2−8−4 Kitasaiwai, Nishi−ku, Yokohama, 220−0004, Japan****Department of Materials Technology, Faculty of Engineering, Chiba University, 1−33 Yayoi−cho, Inage−ku, Chiba, 263−8522, Japan
Received for review December 20, 2003. Revised manuscript received February 2, 2004. Accepted February 3, 2004

Abstract
Alkyl phenol is the general name for phenol compounds with different length or branched alkyl groups. Among the alkyl phenol group, a series of chemicals called nonyl phenols and octyl phenols that have C 9 or C 8 alkyl chains at the para position relative to the phenolic hydroxyl group are used for additives of synthetic resin products and raw materials for non−ionic surfactants, are classified as the endocrine disruptors. In this study a rapid, highly selective and sensitive analytical method using positive chemical ionization (PCI)−GC/MS has been established to determine trimethylsilyl derivatives of nonyl phenols and octyl phenols. Using this method, 18 isomers of 4−nonyl phenol and 8
isomers of 4−octyl phenol were separated and confirmed for the commercial resin products.
Keywords: endocrine disruptor; nonyl phenols; octyl phenols; positive chemical ionization; GC/MS; commodity products

additives designed to improve the function of the material, theIntroduction
Nonyl phenols and an octyl phenol (4−(1,1,3,3−tetramethyl-chemical analysis becomes complicated and time−consuming.

butyl)−phenol) are compounds identified as endocrine disrupting

Therefore, a rapid and high selective analytical method has beenchemicals by the Japanese Ministry of the Environment [1]. Thoserequired.

alkyl phenols are the compounds that have various alkyl groups

Of the analytical methods that have been used to detect alkyl

with different length or branched alkyl chains at the para position

phenol compounds, some have utilised gas chromatography separa-

relative to the phenolic hydroxyl group [2−3]. Among them, thosetion with hydrogen flame ionization detector (FID) [6] or electronthat have C 9 and C 8 alkyl chains are used as additives in syn-capture detector (ECD) [7], high performance liquid chromatogra-

thetic resin products. Nonyl phenols are deliberately added as anphy (HPLC) with ultra violet / visible absorption (UV) or fluores-

antioxidant or corrosion inhibitor, or present as an impurity of sta-cence (FL) detector [8]. However, gas chromatography/mass spec-

bilising agents, tris(nonyl phenyl) phosphite (TNP), or heat decom-

trometry (GC/MS) is a superior method, with high sensitivity andposition products formed during the production process [4−5].

selectivity [9]. In our previous publication [10], the chromatograms

Since the synthetic resin products contain numerous numbers ofwith better shaped peaks were obtained by derivatisation of the al-

To whom correspondence should be addressed.
Tel: +81−45−314−8381, Fax: +81−45−314−5142
E−mail: nobuhiko.onda@perkinelmer.com―２５―

Chromatography, Vol.25 No.1 (2004)

kyl phenol hydroxy group into trimethylsilyl (TMS) derivatives,

the FT−IR analysis a model Spectrum One FT−IR combined with aand at the same time the molecular ions and unique fragments were

Universal ATR (PerkinElmer, Shelton, CT, USA) was used.

found by “soft ionization” using positive chemical ionization
Leaching of alkyl phenols from resin sample(PCI).
In this publication, we report the separation and confirmation

Leaching was performed by the leaching method of Pharma-of 4−nonyl phenol isomers from synthetic resin products, and also

ceutical Society of Japan for commodity tests (Methods of Analy-

4−octyl phenols that have not previously been reported, were foundsis in Health Science) [11] : a sample (2 cm x 5 cm) were washedin the leaching followed by use of TMS derivatisation and PCI−GC

with purified water and then air−dried. As shown in Fig.1, the sample specimen was placed in 20 mL of n−heptane at 25℃ for 1 hour.

/MS method.

After removing the specimen, the leaching solution was transferredExperimental

to a round bottom flask, and concentrated by rotary evaporation,

Samples and Reagentsdried under blowing nitrogen gas and then was dissolved with 0.5

Thirty one (31) home and office use products made of syn-mL of dichloromethane. Thereafter, 0.2 mL of TMS derivatisation

thetic resin, such as small plastic cases, stickers, pen and card cases

reagent (BSTFA) was added to the solution and then made up towere purchased from a market in February, 2003. The resin major

exactly 1 mL with dichlomethane. After a vigorous shake the solu-

materials on the label of these samples were confirmed by Fouriertion was left at room temperature for 1 hour for the complete deri-transform infrared analysis (FT−IR).

vatisation.

Alkyl phenol standards: 4−t−butyl phenol, 4−n−pentyl phenol, 4−n−hexyl phenol, 4−n−heptyl phenol, 4−t−octyl phenol (4−(1,1,3,3−tetramethylbutyl)−phenol), 4−n−octyl phenol, nonyl phenol mixture (environmental analysis grade) and TMS derivatisation
reagent of Bis (trimethylsilyl) trifluoroacetamide (BSTFA) wereobtained from Kanto−Kagaku (Tokyo, Japan). As a leaching solvent, n−heptane (phthalic acid analysis grade) and dichloromethane (pesticide residue analysis grade) from the same reagent supplier were used without any purification. Highly pure nitrogen gas(purity 99.999%) and pure isobutane gas for the chemical ionization were from Takachiho Chemicals (Tokyo, Japan).
Instruments
For the measurement of PCI−GC/MS and EI (Electron Ionization)−GC/MS, a model Clarus 500 Gas Chromatography/Mass
Figure 1. Sample preparation for the determination of 4−alkyl
phenols BSTFA: Bis (trimethylsilyl) trifluoroacetamide)

Spectrometer of PerkinElmer (Shelton, CT, USA) was used. The
capillary column used was a PTE−5 of Supelco (Bellefonte, PA,
USA). The analytical condition of GC/MS is shown in Table 1. For

Preparation of blank and standard solutions

Table 1. Experimental parameters for GC / MS measurements

A sample blank solution was prepared as described aboveGC

without sample material. The standard solution was prepared as

Column: PTE−5 (30 m × 0.25 mm I. D., df: 0.25 µm)
Temperature: 60℃ (1 min) −10℃/min−280℃ (5 min)Carrier gas: helium
Injector: 280℃, Splitless

follows: 100 mg each of alkyl phenol standards was added with dichloromethane to make a 1 mg/mL standard stock solution which
was further diluted with dichloromethane to create several different
concentrations of standard solutions.

PCI−MS
Sample: 2 µl
Reagent gas: isobutane (99.999%)
Ionization Energy: 70 eV
Ion source temperature: 150℃
Mass range: 60−400 m/e

GC/MS measurements
Monitored ions of the target compounds measured by PCI
method are shown in Table 2. The calibration curve was constructed by injecting 1 µL of standard solutions into PCI−GC/MS―２
６―

Chromatography, Vol.25 No.1 (2004)

Tomoo Nakagawa, Naeko Sugaya, Katsumi Sakurai, Junichi Nakagawa,
Koichi Usukura, Nobuhiko Onda and Koichi Oguma

Table 2. Monitor ions of TMS 4−alkyl phenols
CompoundM.W.

4−t−butyl phenol
4−n−pentyl phenol
4−n−hexyl phenol
4−n−heptyl phenol
4−t−octyl phenol
4−n−octyl phenol
nonyl phenols

222
236
250
264
278
278
292

Quantification of nonyl phenols
The chromatographic peaks detected for m/z of 293 were

Base peakMajor
fragment ion fragment ion
223
237
251
265
279
279
293

summed into a peak area, and this peak was used for the calibration
curve.

207
179 (279)
179 (293)
179 (307)207
179 (321)
127

Results and Discussion
There are some reports that 4−alkyl phenol compounds canleach from wrapping films for food products, and synthetic resin
food plates [12−13]. The concerned compounds leached from syn-( ) is the adduct ion (m/z) with CI gas of isobutane.

thetic resin products can be easily transferred into food. Kawamura
et al. studied the leaching tests of nonyl phenol residues in syn-system and from the measured peak areas. An 1 µL of the leachingthetic resin products for food such as PVC wrapping film andsample solutions and the blank solution were injected into PCI−GC

gloves, and plastic toys using n−heptane as a pseudo food oil [14−/MS using the splitless mode and analysed.

15]. The leaching method doesn’t report the total amount of nonyl

Table 3. Nonyl phenols released from household utensils(#) Sample name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31

Mat
Mat
Sheet
Sheet
Sheet
Sheet
Bag
Bag
Bag
Letter bag
Carry bagPen case
Pen case
Soft case
Soft card case
Soft card case
Soft card case
Card case
Pass case
Card holder
Card holder
Card holder
Card holder
Seal
Seal
Seal
Header label
Frame
Rain coatRain wear (trousers)
Frost cover

Nonyl phenols

Sampleweight (g)µg/cm2µg/g

Phthalic acid
ester

Plastics

C8 /C9*

1.48
1.37
0.05
0.03
2.46
3.00
0.40
0.52
0.24
0.24
0.19
0.49
0.36
0.35
0.48
0.48
0.38
0.31
0.83
0.06
0.18
0.20
0.04
0.11
0.39
0.05
0.08
0.24
0.06
0.09
0.06

0.20
2.89
0
0
0.31
0
5.60
0
0
2.80
0
0
0
10.2
0
0
0
0
0.35
0
0
0
0
0
0
0
0
1.95
0
0
0

3
42
0
0
3
0
280
3
9
233
0
0
0
581
0
0
0
0
8
0
0
0
0
0
0
0
0
162
0
7
0

＋＋
＋＋
＋
±
＋＋
＋＋
＋＋
＋＋
＋
＋＋
＋
＋＋
＋＋
＋＋
＋＋
＋＋
＋＋
−
＋＋
±
−
−
−
−
＋＋
＋＋
＋
＋
−
＋＋
−

PVC
PVC
PE
PE
PVC
PVC
PVC
PVC
PVC
PVC
PP
PVC
PVC
PVC
PVC
PVC
PVC
PP
PVC
PP
PP
PP
PP
PET
PVC
PET
−
PVC
EVA
PVC
PE

0
0.20
0
0
0
0
0.05
0
0.05
0
0
0.03
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

PVC: Polyvinyl chloride
PE : Polyethylene
PET : Polyethylene terephthalate
EVA: Polyvinyl acetate* Peak height ratio of octyl phenols to nonyl phenols (not calibrated)―２
７―

PP : Polypropylene

Chromatography, Vol.25 No.1 (2004)

Detection of 4−octyl phenols by PCI−GC/MS

phenol in the specimen, but it is easy and simple to apply as a rapid

Among the octyl phenols, both of 4−n−octyl phenol and 4−t−quantitative method. The samples shown in Table 3 were applied tooctyl phenol (4−(1,1,3,3−tetramethylbutyl)−phenol), were not de-this test in this study.

tected from the samples. However, for the samples of 2, 7, 10, 14
in Table 3, 8 isomers of 4−octyl phenols were detected: in Fig.2 theSeparation of 4−alkyl phenols and the mass spectrum
The soft synthetic resin products contain a lot of plasticizer

chromatogram of the sample #10 (synthetic resin letter bag) and in

and antioxidants. The leaching method for those requires a clean−Fig.4 the mass spectrum for each peak are shown. Octyl phenols

up procedure before the measurements. After leaching, Isobe et al.

have isomers as well as nonyl phenols, but those have not been re-used 5% H2O (w/w) in activated column chromatography to sepa-

ported to date, with the above exception of 4−n−octyl phenol and 4

rate fractions and purify samples, and then analysed 4−alkyl phe-−t−octyl phenol (4−(1,1,3,3−tetramethylbutyl)phenol). The PCI−

nols by EI−GC/MS (SIM mode) [13]. In the present study a lot of

GC/MS spectra of these 8 isomers have little fragmentation, and an

phthalate esters were detected, but direct separation and determina-

M+1 (m/z 279) ion is detected as the strong fragment of 4−octyltion without clean−up procedure was possible because of selective

phenol derivatives. This mass number agrees with the sum (m/zdetection of the TMS derivatised and of high specificity of PCI279) of molecular weight (206) of 4−octyl phenol increased by amethod: isobutane is “softer” reagent gas, and analysis was donemass of 72 by trimethylsilyl derivatisation and increased by a massby the lesser fragmentation as previously reported [10]. For all ofof 1 by proton transfer. On the other hand, a peak of m/z 207 is anmass spectra of the separated 4−nonyl phenol TMS derivatives, aunique fragment for the tertiary alkylated TMS derivative of phe-strong molecular ion of m/z 293 of M+1 was detected as the basenols [10] and m/z 113 fragment is interpreted as the mass of octyl

peak. Compared with EI−GC/MS method, this is a superior method

group of C8H17+. These results suggest all of the 8 detected com-with less interference of sample contaminants. The fragment withpounds are the structural isomers of 4−octyl phenols. Since somem/z 279 of M+1 was used for the qualitative analysis and determi-

spectra such as (d), (e), and (h) in Fig.3, are contaminated withnation of 4−octyl phenols.

high concentration of 4−nonyl phenol, therefore M+1 ion of 4−oc-

For the 31 tested samples, the results are shown in Table 3.

tyl phenols doesn’t become the base peak. However, quantitation

The compound of 4−nonyl phenols (0.20−10.0 µg/cm ) were de-using the mass chromatograms doesn’t have problem because oftected in 8 samples of stationary such as letter bags, desk mat, andthe difference in mass number. All of the detected 4−octyl phenol

pen case. Also 4−octyl phenols were detected in 4 samples at about

compounds do not exhibit M+43 fragment that is typical for 4−n−2−3 % of 4−nonylphenols as calculated from the peak area ratio.

alkyl phenol, and also m/z 179 fragment ion that is specific to pri-

2mary alkyl phenols [10] are not detected. One can conclude fromthose results that 4−n−octyl phenol doesn’t exist in those 8 com-

Figure 2. Separation of TMS derivatives (a−h) of “octyl phenols” in a sample #10 (PVC)―２８―Chromatography, Vol.25 No.1 (2004)

Tomoo Nakagawa, Naeko Sugaya, Katsumi Sakurai, Junichi Nakagawa,
Koichi Usukura, Nobuhiko Onda and Koichi Oguma

Figure 3. PCI spectrum of the peaks (a−h) in Fig.2.

amount of 4−nonyl phenols was detected in high concentration

pounds.

(2.80 µg/cm2) for sample #10 (synthetic resin letter bag), and 9 of
Separation and determination of 18 compounds of 4−nonyl phenol

new isomers of (1)−(9) in addition to 9 isomers of (a)−(i) that pre-isomers by PCI−GC/MS

viously reported [10] were separated. The spectra obtained from

The quantitation was performed based on the calibration curve

PCI−GC/MS had less fragmentation and M+1 ion of 4−nonyl phe-shown in Fig. 4, where the peak area of 4−nonyl phenols is the sum

nol derivatives as base peak was detected from all of the spectra.

of 9 components separated previously [10]. As shown in Fig.5, theEach peak agreed as well with the sum (m/z 293) of molecular―２９―Chromatography, Vol.25 No.1 (2004)

Figure 4. Calibration curve for TMS derivative of 4−nonyl phenols
Total peak area: the sum of peak area of 9 isomers of (a)−(i) found in 4−nonyl phenols standard [10]Figure 5. Separation of TMS derivatives (1−9 and a−i) of “nonyl phenols” in a sample #10
Synthetic resin products containing 4−alkyl phenol

weight (220) of 4−nonyl phenol, increase mass 72 by trimethylsilyl
derivatisation, and the increase of mass 1 of proton transfer. There-

The detection of 4−alkyl phenols and plasticizers is shown infore all of 18 compounds separated from sample #10 were con-Table 3. The base materials of synthetic resin products were con-cluded as structural isomers.

firmed by Fourier transform infrared analysis. The major materials

The accurate quantitation of 4−nonyl phenols is limited for thein the 31 samples are polyvinyl chloride (PVC) (18 samples),mixture of the structural isomers, and further there is none of thepolypropylene (PP) (6 samples), polyethylene (PE) (3 samples),single compound standard available except 4−n−nonyl phenol. It

polyethylene terephthalate (PET) (2 samples), and polyvinyl ace-

elutes very late and does not fall in the so−called class of the nonyl

tate (EVA) (1 sample). Many additives such as plasticizer and anti-

phenols [10]. Total peak area of 9 isomers of (a)−(i) in Fig.5 ac-

oxidants etc. have been used in the soft synthetic resin products. In

counts for more than 90% of the peak area found for m/z 293 mass

22 samples the plasticizers (phthalate esters) were detected. Also 4chromatogram.

−nonyl phenols were detected for 8 samples of synthetic resin―３０―Chromatography, Vol.25 No.1 (2004)

Tomoo Nakagawa, Naeko Sugaya, Katsumi Sakurai, Junichi Nakagawa,
Koichi Usukura, Nobuhiko Onda and Koichi Oguma

Figure 6. Separation of TMS derivatives of ”octyl phenols ” (upper) and “nonyl phenols” (lower)

Figure 7. Overlapping of TMS derivatives of “octyl phenols” (m/z 279) and “nonyl phenols” (m/z 293).
The scale of octyl and nonyl phenols are normalized with each largest peak.

products as above mentioned. Four samples (sample 2, 7, 10, andReferences

14) that exhibit the high concentration of 4−octyl phenol contain[1]

Japan Environmental Agency: Environmental Agency’s In-

also 4−nonyl phenols. Mass chromatogram of sample #10 is shown

itiatives to Issues of Exogenous Endocrine Disruptors−Envi-in Fig.6 for each of those 4−alkyl phenols. Fig.7 shows the over-ronmental Hormone Strategic Project SPEED ’98, 1998,

lapping of the elution of isomers of 4−octyl phenol and 4−nonyl

Tokyo, Japan (in Japanese).
[2]

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B. D. Bhatt, J. V. Prasad, G. Kalpana, and S. All, J. Chromatogr. Sci., 1992, 30, 203.

each). The amount of 4−octyl phenol detected was about 0.03−0.20µg/cm of 4−nonyl phenol. As an interesting result, those 4−alkyl
2[3]

T. F. Wheeler, J. R. Heim, M. R. LaTorre, and A. B. Janes,
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phenols detected samples are all made of PVC.
[4]

Y. Kawamura, C. Tagai, T. Maehara, and T. Yamada, Shokuhin Eiseigaku Zasshi, 1999, 40, 274 (in Japanese).

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vatisation of nonyl phenol and octyl phenol in synthetic resin prod-nese).
[6]

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E. Stephanou and W. Giger, Environ. Sci. Technol., 1982,
16, 800.

of 4−nonyl phenol and 8 isomers of 4−octyl phenol were separated[7]

and confirmed. All of 4−octyl phenols were detected from PVC

C. Wahlberg, L. Renberg, and U. Wideqvist, Chemosphere,
1990, 20, 179.

resin products that contain the high concentration of 4−nonyl phe[8]nols. From the soft synthetic resin products, phthalate esters wereK. K . Chee, M. K. Wong, and H. K. Lee, J. Liq. Chrom.
Technol., 1996, 19, 259.

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[9]

―３
１―

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[10] T. Nakagawa, N. Sugaya, K. Sakurai, J. Nakagawa, K.

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