Drug Testing

and Analysis

Correspondence case report

Received: 15 October 2014

Revised: 2 March 2015

Accepted: 1 April 2015

Published online in Wiley Online Library

(www.drugtestinganalysis.com) DOI 10.1002/dta.1808

Determination of formaldehyde in hair creams

by gas chromatography-mass spectrometry
Fabiana A. Lobo,a* Talysson M. O. Santos,b Karla M. Vieira,b
Vanessa M. Osrioc and Jason Guy Taylora
Introduction
The perfume and cosmetics industry are one of outstanding industrial sectors in Brazil. With an average annual growth of 11%
over the last ten years, Brazil has achieved third place in the
world ranking for consumption of cosmetics. Hair products are
amongst the most consumed cosmetics and the market is growing every day.[1]
The active ingredients in hair straightening creams vary according
to the product and usually include glutaraldehyde, sodium hydroxide, calcium hydroxide, formaldehyde, pyrogallol, thioglycolic
acid, and others. Currently, the most widely used chemical compound in hair products to alter the protein structure of hair to affect
smoothing is formaldehyde.[2]
Four international research institutions have assessed the carcinogenic potential of formaldehyde: the International Agency for
Research on Cancer (IARC), the Environmental Protection Agency
(EPA), the US National Toxicology Program, and the Occupational
Safety and Health Association (OSHA). These studies demonstrated that exposure to formaldehyde can cause cancer of the
pharynx and nasopharynx, increase the chance of pneumonia,
dermatitis and induce allergic reactions (irritation of eyes, nose
and throat, insomnia, chest pains, rashes, asthma attacks, among
others).[35]
In 2001, the National Health Surveillance Agency (ANVISA), which
is a branch of the Brazilian Ministry of Health, issued a resolution to
control the use of formaldehyde, restricting it to a concentration of
up to 0.2 % in cosmetics.[2] However, the maximum (and safe)
amount of formaldehyde (0.2%) permitted in cosmetic formulations is insufficient to produce straightened hair and in addition,
is a health violation. Hair products for children have been identified
for the presence of formaldehyde thus highlighting the importance
for regulatory control in the cosmetic industry.
The literature discusses some methodologies and techniques for
the analysis of formaldehyde in various samples such as blood, food
samples, air, and cosmetics amongst others.[611] Techniques such
as spectrophotometry, capillary electrophoresis, and electrochemistry were used to determine formaldehyde.[1216] However, most of
these techniques involve a series of tedious and labour-intensive
extraction steps and they also suffer from low limits of detection
(LOD) and quantification (LOQ). High performance liquid chromatography (HPLC) is the most widely applied method for the
determination of formaldehyde. Gas chromatography coupled to
mass spectrometry (GC-MS) is a versatile analytical technique and
attempts have been made to apply GC-MS to the determination
of formaldehyde with limited success.[1719] Unfortunately, the
thermal instability makes direct quantification of formaldehyde by

Drug Test. Analysis (2015)

GC-MS difficult and the method is therefore prone to robustness

issues. Therefore, the aim of this study was to develop a GC-MS
method for the quantification of formaldehyde in hair straightening
creams using 2,4-dinitrophenylhydrazine as a derivatizing reagent.

Materials and methods

Materials
Instruments and accessories
The analytical instrument used in this study consists of a Shimadzu
gas chromatograph coupled to single quadrupole mass spectrometer
equipped with a HP-5MS (Agilent, Santa Clara, CA, USA) capillary
column (30 m 0.25 mm id 0.25 m film) containing 5% diphenyl
and 95% dimethylpolysiloxane.
Reagents, solutions and samples
The solvents used were HPLC grade: acetonitrile (Merck, Rio de
Janeiro, Brazil), derivatizing reagent 2,4-dinitrophenylhydrazine
(2,4-DNPH), perchloric acid HClO4 (Vetec, Duque de Caxias,
Brazil); formaldehyde-2,4-dinitrophenylhydrazone standard solution
(formaldehyde - DNPH) 99.99% SUPELCO37 (SUPELCO, So Paulo,
Brazil), 37% aqueous solution of formaldehyde (Lahas, Vila Velha,
Brazil); Methyl alcohol HPLC grade (Merck); Dichloromethane HPLC
grade (Isofar, Duque de Caxias, Brazil) and n - hexane 99 % PA
(Vetec). Helium gas (White Martins, Joo Monlevade, Brazil) with a
purity level of 99.999 % was utilized for GC-MS analysis.
All hair straightening cream samples were collected at various
salons in the city of Joo Monlevade in Brazil. Samples were
enumerated 13 (first collection, period AugustSeptember 2011),
47 (second collection, period JuneDecember 2012), and 911
(third collection period MayJune 2013) as shown in Table 1.

* Correspondence to: Fabiana A. Lobo, UFOP- Universidade Federal de Ouro Preto,

Instituto de Cincias Exatas e Biolgicas, Departamento de Qumica, Bauxita,
Ouro Preto-MG, 35400-000. E-mail: fabiana@iceb.ufop.br
a UFOP- Universidade Federal de Ouro Preto, Instituto de Cincias Exatas e
Biolgicas, Departamento de Qumica, Bauxita, Ouro Preto-MG, 35400-000
b UFOP- Universidade Federal de Ouro Preto, Instituto de Cincias Exatas e
Aplicadas, Departamento de Cincias Exatas e Aplicadas, Loanda, Joo
Monlevade-MG, 35931-008
c UFES- Universidade Federal do Esprito Santo, Centro de Cincias Agrrias,
Departamento de Qumica e Fsica, Alto Universitrio, Alegre-ES, 29500-000

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Drug Testing
and Analysis

F. A. Lobo et al.

Procedure for the preparation of standards

Preparation of standards
A stock solution of formaldehyde-2,4-dinitrofenylhydrazone
(1482 ug/mL) was prepared in acetonitrile and used further for
working standard solutions (Table 2).

of 10 C min 1 up to 290 C and finishing with an increase of

10 C min 1 up to 300 C over 2 min. The total run time was
31 min and helium was used as carrier gas at a constant flow of
1.5 mL min 1. Other typical mass spectrometer parameters were
as follows: electron ionization at 70 eV and for quantification, SIM
mode was used to analyze the molecular ion M+ (m/z 210) of the
target analyte at a source temperature of 200 C.

Sample preparation
Initially, 75 mg of DNPH was weighed and dissolved in 50 mL of acetonitrile, acidified with approximately 0.5 mL of 9.2 M HClO4 to provide solution 1. Next, 200 L of this solution was diluted in 9.8 mL of
acetonitrile (Solution 2). Solution 2 was then used to solubilize
300.00 mg of cream sample (Solution 3). Finally, 1000 L of Solution
3 was diluted in 9 mL of acetonitrile, forming the final sample which
was injected into the GC-MS. This procedure was also performed
with all samples shown in Table 2.

Optimization studies for the determination of formaldehyde

in creams
Two procedures were evaluated for determination of formaldehyde
in the sample creams. Quantification was achieved by either using
external calibration (see tables 2 and 3 and description above) or by
the use of the standard addition method.
The analytical characteristics of the procedure include the LOD,
the LOQ, the estimated standard deviation (SD), and the relative
standard deviation (RSD). The SD and RSD values are based on
three replicate injections for each individual sample.

Chromatographic conditions
The operational conditions employed are based on a previous
method reported by Osrio and Cardeal[20] but with the following
modifications. The injector was set in the splitless mode (for
2 min) at a temperature of 250 C. The oven temperature program was as follows: 60 C with an increment of 10 C min 1
up to 120 C (holding time 2 min), and then after, an increment

Table 1. Origin and collection period of the cream samples within the
study
Samples
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11

Salon

Collection period

Salon 1
Salon 2
Salon 3
Salon 4
Salon 5
Salon 6
Salon 7
Salon 8
Salon 9
Salon 10
Salon 11

August-September 2011
August-September 2011
August-September 2011
June-December 2012
June-December 2012
June-December 2012
June-December 2012
June-December 2012
May-June 2013
May-June 2013
May-June 2013

Calibration procedure using the method of analyte addition

Although the use of an external calibration is common, it is very important to evaluate the difference in sensitivity between traditional
calibration standards and calibration standards prepared in the
samples with added analyte to check for compatibility. Thus, for
the 11 samples of creams that were evaluated, the influence of
the sample matrix was also studied. The characteristics of the samples are shown in Table 1.
Calibration curve
Five standards were prepared with different concentrations of
formaldehyde - DNPH, as shown in Table 3. We chose cream number A3 for the preparation of the medium.
Test for analyte recovery
Due to a lack of certified material creams, addition and recovery
tests were also performed in order to determine the accuracy and
precision of the method, using the standard addition method itself.

Results and discussion

Optimization studies
The first attempts at GC-MS analysis of unreacted formaldehyde
were in good accordance with earlier studies,[1719] and indicated

Table 2. Preparation of standards Formaldehyde - DNPH

Standard

1
2
3
4
5
6
7
8
9

[FormaldehydeDNPH - Final]
1
g mL

Final Volume
(mL)

1.48
3.70
7.40
11.11
14.82
37.04
74.08
111.11
148.15

2
2
2
2
2
2
2
2
2

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Table 3. Preparation of Formaldehyde - DNPH standards using the

analyte addition method for analyte samples of creams
Volumetric flask N
1
2
3
4
5

[Formaldehyde- DNPH]
L 1
final g

Volume final, (mL)

3.70
7.41
14.82
37.04
74.08

1,0
1,0
1,0
1,0
1,0

Where: * Va - sample volume added (100 L) in the form of preparation

as 2.2.1.

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Drug Testing
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Using GC-MS to enforce legislation concerning formaldehyde limits

the need for derivatization. The reaction that forms formaldehydeDNPH is shown in Scheme 1.
The pH is an important factor in this reaction due to competition
between nucleophilicity and basicity of 2,4-DNPH. For the analyte in
question, the compound that is formed is called Formaldehyde-2,4dinitrophenylhydrazone but is referred to herein as Formaldehyde DNPH. Figure 1 shows the peak obtained, and Figure 2 illustrates
the mass spectrum for the analyte in question.
A well-defined peak was identified in the chromatogram
(Figure 1), eluting at ~17.95 min and its mass spectrum (Figure 2)
corresponded to the reference spectrum of NIST (National Institute
of Standards and Technology) database. This peak has the exact
same retention time as the standard and presents a molecular ion
(m/z = 210) and characteristic fragmentation pattern that confirms
the identification of Formaldehyde DNPH. The mass spectrum for
the sample is the superimposition of the reference spectrum found
in the NIST database. Specifically, fragment peaks m/z 210 (80%),
180 (15%), 122 (20%), 106 (5%), 79 (100%), 78 (75%), 63 (95%) and
51 (60%) are present in both spectra with similar relative intensities.
The optimized method produced a calibration curve with the
following equation: y = ax + b; and R2 = 0.9950 (Figure 3).
The LOD and LOQ for external calibration were calculated. The
standard deviation (sd) for 10 measurements of the blank was
calculated and values for the LOD and LOQ were then

Scheme 1. Reaction of formaldehyde with DNPH in the formation of 2,4

dinitrophenylhydrazone.

Figure 3. Analytical curve for formaldehyde-DNPH standard (concentration

1
range 3.70383 to 74.076 mg mL ).

calculated, LOD = (3SD)/K and LOQ = (10sd)/K, where K is the slope

of the line.
The values found using the external calibration for the LOD and
LOQ were 0.0197 and 0.066 mg L 1, respectively. The standard
deviation and relative standard deviation obtained for the LOQ
were 14.79 and 22.4 %.
The curves for external calibration and standard addition method
are shown in Figure 4, to allow for comparison of method sensitivities.
Interference from the sample matrix is quite noticeable for the
standard addition curve (decreases the sensitivity) and for this
reason, the standard addition method was chosen to quantify
formaldehyde in the hair cream samples.
The sensitivity values are significantly different, which was
expected due to the different means of standard preparation that
were adopted. However, in both cases the quantity of the standard
in the samples did not deviate outside of 95% of nominal. Calibration curves for all samples (data not shown) were performed.
Determination of formaldehyde by standard addition method
Analyte addition is accomplished by the addition of set volumes of
a solution with a known concentration of analyte. This method is
especially appropriate when the sample composition is unknown

Figure 1. Chromatogram of formaldehyde-DNPH standard.

Figure 2. Mass spectrum of Formaldehyde-DNPH Standard.

Drug Test. Analysis (2015)

Figure 4. Analytical curves for Formaldehyde-DNPH in acetonitrile,

external calibration method (red) and standard addition (green) using
cream A3 in green.

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Drug Testing
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F. A. Lobo et al.

Table 4. [Formaldehyde-DNPH] found using external calibration

method and percentage of formaldehyde in the samples
Samples

Formaldehyde-DNPH]
found in the sample, mg L

A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11

6.59
6.54
6.55
6.63
6.58
6.45
6.46
6.47
<LOD
<LOD
<LOD

Percentage of formaldehyde
in the samples, %
2.34
2.92
2.75
2.20
2.41
1.93
1.75
1.75
<LOD
<LOD
<LOD

or complex, such as samples of creams. In order to minimize interference, quantification of the analyte even when present at low
concentrations is determined with a signal situated within an appropriate range where no other signals reside. Furthermore, this
method is advantageous in that it permits the evaluation of
recovery of the added analyte and therefore validating the sample
preparation procedure.
The LOD and LOQ were calculated for each analytical curve of
each sample. The values found for the LOD and LOQ were less than
or equal to 0.0165 and 0.055 mg L 1, respectively. The standard
deviation and relative standard deviation obtained were lower or
equal to 81.36 and 18.67%, respectively. The recovery of known
amounts of standard from a blank cream sample lay in the range
of 88% to 115%.
Satisfactory results obtained for formaldehyde-2,4 DNPH
standard solutions motivated us to determine formaldehyde in
the samples. These results are shown in Table 4 as a percentage
of total mass composition.
It was possible to determine the concentration of the first eight
samples. Samples collected in 2011 (A1 to A5) have a relatively
higher percentage of formaldehyde than samples collected in
2012 (A6 to A8). This could be explained by the fact the health authorities implemented greater surveillance and inspection in the
city of Joo Monlevade. Unfortunately, all samples that had values
above the LOD, are still above the allowable limits set by Brazilian
law, which is 0.2 %. Samples from 2013 (A9 to A11) showed no
presence of formaldehyde and these results are explicable by the
fact that formaldehyde has recently been in some creams
substituted for other substances such as glycosyl acid or
glutaraldehyde.[2] Upon heating by a dryer or flat iron, these
substances produce formaldehyde which straightens hair.[2]
Although this method has been developed to solve a particularly
Brazilian problem, this new protocol can be applied to other types
of samples in different industrial settings around the world.

Conclusion
In conclusion, formaldehyde is very volatile and thermally unstable
and due to these intrinsic characteristics, it has not been possible to
quantify this analyte by direct injection into GC-MS. Derivatization
of the analyte was therefore essential to determine formaldehyde
and to this end, a new protocol was developed. The method has

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been analytically validated and conveniently employs the readily

available and cheap 2,4-DNPH as derivatizing reagent. Analyte addition method was found to be the best choice for unknown and
complex cream compositions. This method minimizes interference
and allows for quantification of the analyte at low concentrations.
Moreover, this method permits the evaluation of recovery of the
added analyte and therefore validates the sample preparation
protocol. The LOD and LOQ were 0.0165 and 0.055 mg L 1,
respectively. The analyzed samples contain between 1.5 and 2.4%
of formaldehyde. Unfortunately, the level of formaldehyde found
in some hair product cream samples is well above the limits allowed
by Brazilian law, and gives rise to health concerns, for primary users
of adulterated products in hair salons. Noncompliance is still large,
and the developed method could help to establish or propose a
suitable normalization test. As yet, there is no literature and/or
legislation, methods for determination of formaldehyde using
GC-MS in hair straightening creams. This simple method could be
potentially useful for other formaldehyde analyses in other products.
Acknowledgements
The authors thank the University of Ouro Preto (UFOP-PROPP), the
Brazilian research funding agencies FAPEMIG and CNPq for their
financial support and grants.

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