SQC7008 ADVANCE LABORATORY SKILL I

1.0 Objective.
1) To develop a HPLC separation method for determination of parabens in cosmetic creams
2) To determine the concentration of parabens in cosmetic creams.

2.0 Introduction

Parabens are groups of the alkyl esters of p-hydroxybenzoic acid widely used in cosmetics,
toiletries, and pharmaceuticals. In this experiment, the 4 different types of paraben that are being
studied are methyl-paraben, ethyl-paraben, propyl-paraben and butyl-paraben. Parabens are
being used as they have good bactericidal and fungicidal properties. The antimicrobial activity
of the parabens increases with increasing carbon number of the ester group. Furthermore, it acts
as an effective preservative for the cosmetics and skin care products. However, parabens may
cause undesirable effects range from mild skin irritation to estrogenic activity. It also could
potentially induce human breast tumors. Advantages have bactericidal and fungicidal properties
( Antimicrobial activity increases with increasing C no.) Act as an effective preservatives.
Disadvantages, can causes undesirable effects range from mild skin irritation to estrogenic
activity. Could potentially induce human breast tumors.

Table 1. cosmestic directive on parabens and maximum allowed concentration.

Organization Directive Highest permissible concentration

European European Regulation (EC) No. 0.4% (as acid for single ester)
Union 1223/2009 0.8% (as acid for mixture of esters)
Denmark Danish Ministry of the Environment. 0.4% (as acid for single ester)
Statutory Order, amending the Statutory 0.8% (as acid for mixture of esters)
Order on cosmetic products Propylparaben, butylparaben,
isopropylparaben and salt thereof must
not be used in products for children
under 3 years.
United State Federal Food, Drug and Cosmetic Act 0.4% (as acid for single ester)
0.8% (as acid for mixture of esters)
Japan Japan Pharmaceutical Affairs Law 1% single paraben
1% for a mixture of parabens
Cananda Food and Drug Act No restriction

Methyl paraben Ethyl paraben Propyl paraben Butyl paraben.

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 SQC7008 ADVANCE LABORATORY SKILL I

In this study, an approach method by using a High Performance Liquid Chromatography (HPLC) to
determine the amount of 4 ester from p-hydroxybenzoid acid( methyl-, ethyl-, propyl- and butyl-) in
cosmetics cream in local market. Does the amount of -hydroxybenzoid acid have comply the
permission value from international authorities.

3.0 Material and method.

3.1 Cosmetics
In this experiment, five (5) selected brands from local market were analyzed and labeled as Cocoa
Butter lotion (A), Sunplay Watery Cool sunscreen (B), Nivea Express Hydration (C), Banana Boat
Sport Sunscreen (D), and Vaselin Intensive care (E).

3.2 Reagents

Nanopure dionized water is obtain from laboratory, methylparaben (MP), ethylparaben (EP),
butylparaben (BP) and propylparaben (PP) were obtain from sigma aldrich. A methanol with mass
spect grade from Merck.

3.3 Chromatographic conditions

3.3.1 Apparatus
HPLC was performed using a Shimadzu liquid chromatography equipped with LC -
201 solvent delivery pump, a SPD-M20A ultraviolet detector operating at 254nm and
a CTO-10AS VP column oven. Injection were perform by SIL-20A HT auto sampler
system.

3.3.2 HPLC condition

A Shimadzu, shim-pack GIST C18 column (4.4 x250mm, 5µm) and Shimadzu, shim-
pack GIST guard column (4.0 x10mm x 5µm) were used. Column temperature at
420C and injection volume at 10µl. flow rate was 1.0ml/min. the determination was
done at room temperature.

3.4 Preparation of standard and calibration series.

Stock solutions of 1000ppm for all 4 parabens were prepared. 10mg of each parabens were
added into a 10ml volumetric flask and topped up with methanol. For every calibration
series, such amount of volume will be taken from stock solutions i.e 0.02 ml, 0.1 ml, 0.5 ml,
1 ml, 2 ml and 5 ml respectively. The stock solution were mixed and diluted using methanol
to 0.2ppm, 1ppm, 5ppm, 10ppm, 20ppm, 50ppm and 100ppm respectively for 7 calibration
points. The mixture solutions were kept in refrigerator with temperature 4-10 oC.

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 SQC7008 ADVANCE LABORATORY SKILL I

Table 2. series of calibration mixture parabens

Pipetting volume into 10ml Concentration (µg/ml)

Standard
volumetric flask MP EP PP BP
1 0.02 0.02 0.02 0.02 0.02
2 0.1 0.1 0.1 0.1 0.1
3 0.5 0.5 0.5 0.5 0.5
4 1 1 1 1 1
5 2 2 2 2 2
6 5 5 5 5 5
7 100 100 100 100

3.5 Sample preparation

 0.2g of cosmetic lotion sample was weighed into 50ml polypropylene tube fitted with
screwcap.
 5ml of methanol was added and sonicated for 30minutes.
 Centrifuged for 10minutes at 5000rpm.
 The supernatant was filtered using 0.25µm membrane and transferred to HPLC vial.

Precaution Steps
 Solvent used must a MS Grade, no need to filter. Otherwise, all solvent used must be
filtered to avoid blockage inside column during separation.
 Ultra nanopure water is used, therefore is not needed to filter.
 Sample must be weight in crucible bowl to get a better accuracy in weighing.
 Before transfer all samples in vial, all samples must be filtered by using a syringe
with a filter (various size).
 Before running the experiment, the HPLC have to be purged first to remove any
contaminants.

4.0 Result and discussion.

From separation processes, retention time for methylparaben (5.24 minutes), ethylparaben (7.14
minutes), propylparaben (10.71 minutes) and butylparaben (17.67 minutes) were obtained. In
this experiment, a isocratic elution 60% methanol and 40% water were used as mobile phase.
The separation ratio Rs give a value more than 1.5, it’s the ratio to use for mobile phase.

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4.1 Optimization studies

An isocratic approach is used for this method. The suitable mobile give a good separation
and resolution Rs >1.5.

Stationary phase:
Shim-pack GIST (G) (4.0 x 10mm, 5µm) C18 column, temperature 42⁰C [1]
Mobile phase:
Isocratic Elution; 60% Methanol: 40%Water, Flow rate at 1ml/min

`
Figure 1. All peaks are resolved with Rs>1.5, mobile phase condition is suitable to be
used

Table 3. Result obtain from calibration curve for blank to mixture from 0 ppm to 100 ppm (area vs
concentration).

Concentration (ppm) Methylparaben Ethylparaben Propylparaben Butylparaben

Blank 0 0 0 0
0.2 26761 25268.5 25044 17415
1 168903 181757 153124.5 115792
5 851626 838846.5 773406.5 584308.5
10 1583426 1765045 1434550 1085879
20 3180111 3122109 2883999 2184544
50 9790850 9653111 8929383 6769846
100 16254765 16123574 15003065 11412559

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Methyl Paraben Ethyl Paraben

20000000
20000000
y = 167408x + 97862

Area Response
y = 165488x + 127965
15000000 R² = 0.9903 15000000
R² = 0.991
Area

10000000 10000000

5000000 5000000

0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Concentration Concentration (ppm)

(a) (b)

Propyl Paraben Butyl Paraben

20000000 15000000
Area Response

Area Response
15000000 y = 154344x + 66245 y = 117378x + 44934
R² = 0.9914 10000000 R² = 0.9916
10000000
5000000
5000000
0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Concentration (ppm) Concentration (ppm)

(c) (d)

Figure 2. Calibration curve for four types of parabens, methylparabens(a), ethylparaben

(b),butylparaben (c) and propylparaben (d).

Table 4. Calibration Curve parameters obtain by linear regression

Parabens
Parameters
MP EP PP BP
Slope 167408 165448 154344 117378
Intercept 97862 127965 66245 44934
Regression Coefficient (R2) 0.990 0.991 0.991 0.991
Standard Deviation
132.7323 225.1761 107.5294 20.15606
LOD, mg/L 0.002616 0.00449 0.002299 0.000567
LOQ, mg/L 0.007929 0.013607 0.006967 0.001717

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 SQC7008 ADVANCE LABORATORY SKILL I

Limitation of qualitative and quantitative for every method can be elaborated from the table
given. Three methods approach to determine the limit for both, qualitative and quantitative is by
blank, y-intercept and from residuals itself.

Table 5. Limit of detection for qualitative and quantitative.

LOD using SD of blank 0.002616 0.00449 0.002299 0.000567

LOQ using SD of blank 0.007929 0.013607 0.006967 0.001717

LOD using SD y-intercept 0.300491 0.687227 0.333227 0.29929

LOQ using SD y-intercept 0.910578 2.082506 1.009779 0.906941

LOD using SD of y-
residuals 0.056759 0.25717 0.044259 0.067592
LOQ using SD of y-
residuals 0.171996 0.779304 0.134118 0.204825

4.2 Analysis of samples

Five products were selected randomly at local market, to determine the amount of parabens
present from brands taken. Table xx is shown a type of parabens present in the cosmetic
creams that is Cocoa Butter, Sunplay Watery Cool, Nivea Express Hydration, Banana Boat
sport Sunscreen and Vaseline Intensive Care. The result shown that most of cosmetic creams
were complies regulation stated.
Table 6. Result after analysis of parabens
Product Product Ingredients Result after analysis Concentration
(µg/ml)
A Methylparaben, Methylparabens, Ethylparabens 4.365
Ethylparabens
B Methylparaben Methylparabens 7.896
C Metylparabens, Methylparabens,Propylparabens 4.731
Propylparabens
D Methylparabens Methylparabens 3.657
E Metylparabens, Methylparabens, Propylparabens 2.170
Propylparabens

5.0 Conclusion.
By using a High Performance Liquid Chromatography, all the parabens amount were collected.
Its show that Cocoa Butter cream show methyl and propyparaben present. It’s give the
concentration 1.680 mg/ml and 2.685 mg/ml respectively. Sunplay Watery Cool only show a
methyl-paraben with concentration 7.896 mg/ml. Nivea Express Hydration indicate the present
of methyl and propylparaben with concentration 3.848 mg/ml and 0.883 mg/ml. Banana Boat
Sport Suncreen only present of methylparaben with the concentration 3.657 mg/ml. Meanwhile

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 SQC7008 ADVANCE LABORATORY SKILL I

for Vaseline Intensive Care, methyl and propylparaben were present and give a concentration
1.345 mg/ml and 0.825 mg/ml.

Methyl Paraben
Products Propyl Paraben (µg/ml)
(µg/ml)
Cocoa Butter 1.680 2.685
Sunplay Watery Cool 7.896
Nivea Express Hydration 3.848 0.883
Banana Boat Sport
Suncreen 3.657
Vaseline Intensive Care 1.345 0.825
Table 7. result of analysis samples

Amount of parabensin cosmetic creams

9.000
7.896
8.000
7.000
6.000
5.000
3.848 3.657
4.000
2.685
3.000
1.680 1.345
2.000
0.883 0.825
1.000
0.000
-1.000 Cocoa Butter Sunplay Watery Nivea Express Banana Boat Vaseline
Cool Hydration Sport Suncreen Intensive Care

Methyl Paraben Propyl Paraben

Figure 3.Comparison data of parabens present in cosmetic creams.

6.0 References

[1] I. R. L. D. F. C. C. V. R. I. T. a. V. O. MANUELA M. MINCEA1, "Determination of

methylparaben from cosmetic products by ultra performance liquid
chromatography," Journal of the Serbian Chemical Society, vol. 74, no. 6, pp. 669-
676, 2009.

[2] M. S. a. E. B. Belma Imamovic, "HPLC Determination of Some frequently used

Parabens in Sunscreens," International Journal of Pharmacy Teaching & Practices,
vol. 3, no. 1, pp. 219-224, 2012.

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[3] C. L.-Q. Wenhui Gao, "Fast and sensitive high performance liquid chromatography
analysis of cosmetic cream for hydroquinone, phenol and six preservatives," Journal
of Chromatography A, vol. 1218, no. 28, pp. 4307-4311, 2011.

[4] S. L. L. Z. N. W. A. W. I. a. A. A. N. M. Marsin Sanagi, "Comparison of Signal-to-noise,

Blank Determination, and Linear Regression Methods for the Estimation of
Detection and Quantification Limits for Volatile Organic Compounds by Gas
Chromatography," Ibnu Sina Institute for Fundamental Science Studies, Department
of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Skudai,
Johor, Malaysia .

[5] N. l. CalleC.BendichoI.Lavilla, "An overview of sample preparation for the

determination of parabens in cosmetics," TrAC Trends in Analytical Chemistry, vol.
57, pp. 34-46, 2014.