1. INTRODUCTION:

Parabens (p-hydroxybenzoates) are the esters of para-hydroxybenzoic acids. They possess anti-fungal and anti-bacterial properties and are commonly employed as preservatives in pharmaceutical, cosmetics, personal care, food products etc. The antimicrobial property of these compounds increases with increasing alkyl part chain length of ester functional group. To achieve the desired antimicrobial activity, combinations of different parabens are used rather than single one.

The most commonly used parabens are methyl, ethyl, propyl and butyl parabens (Figure:1) Parabens have neutral pH with no odour or taste, they do not produce any discoloration or cause hardening thus making them suitable for their extensive use in different personal care formulations¹. These compounds exhibit hydrophobic character which increases with the increasing chain length of the alkyl part of ester functional group. Parabens also lacks any kind of easily ionizable functionality.

Fig 1: Basic structure of Paraben (R = methyl, ethyl, propyl and butyl)

In the last few years public awareness towards the potential hazard of chemicals used in everyday life has grown manifold. Debate for parabens safety in human beings is initiated in recent years with the documentation of their ability to act in similar nature as of estrogen^2-4. Estrogenic potency of parabens increases with increasing branching and chain length of the alkyl part of the paraben esters. They are also detected in human cancerous breast tissues^5-6. The raised safety issues make analytical identification as well as quantification of parabens of high importance for quality assurance as well as consumer health protection.

Use of parabens as preservatives in cosmetic products is restricted at concentration of 0.4% for single ester or 0.8% for ester mixtures by council directive 76/768/EC of European Community and Bureau of Indian Standards⁷. In food products an acceptable daily intake (ADI) of 0–10mg/kg body weight for the sum of methyl and ethyl esters of p-hydroxybenzoic acid has been suggested by the Joint Food and Agriculture Organization (FAO) and World Health Organization (WHO) Expert Committee on Food Additives⁸.

Literature survey revealed that different methods for identification and quantification of parabens have been reported. The choice of analytical techniques are commonly based on Thin layer chromatography (TLC) using visualization reagent^9-10, high performance liquid chromatography (HPLC)^11-12, gas chromatography- mass spectrometry¹³, liquid chromatography- mass spectrometry (LC-MS)¹⁴ and high performance thin layer chromatography (HPTLC)^15-16. These analytical techniques were applied for the preservative analysis on different matrices like biological¹⁷, water samples¹⁸, food¹⁹, cosmetic products²⁰ and pharmaceutical samples²¹.

Complicated sample pretreatment, limited types of samples, number of preservatives analysed are some of the limitation of the reported RP-HPLC methods. Removal of complex sample matrix like fats, lipids, wax and additives from cosmetics samples is labor intensive and time-consuming procedures²². Kumar et.al.¹² reported a fully validated RP-HPLC method for the analysis of studied preservatives but have its limitation in terms of applicability towards commercial formulation. Other reported RP-HPLC- DAD methods during recent years have sample limitations and are applicable only to specific types of samples²³. Thus a requirement of a single method with ease of application for identification and quantification of parabens in wide range of cosmetics, personal care and pharmaceutical formulations always exist. With this aim through the present work we report a simplified sample pretreatment procedure applicable for extraction of parabens from different types of sample matrix and validated RP-capillary HPLC- DAD method for simultaneous determination and quantification of methyl, ethyl, propyl and butyl parabens in wide range of pharmaceuticals, cosmetics and personal care formulations. The developed method was successfully applied to different commercially available products in Indian market.

2. EXPERIMENTAL:

2.1 Chemicals and reagents:

Certified reference standards of Methyl 4-hydroxybenzoate (MP, 99%), Ethyl 4- hydroxybenzoate (EP, 99%), Propyl 4-hydroxybenzoate (PP, 99%) and Butyl 4-hydroxybenzoate (BP, 99%) were procured from Alfa Aesar (Heysham, England). Other chemicals were obtained from the following sources: Acetonitrile (HPLC grade, 99%) from Merck Specialities (Mumbai, India) and Water (HPLC grade) from Fisher Scientific (Mumbai India), Hydrochloric acid (HCl), sodium chloride (NaCl), anhydrous sodium sulphate and diethyl ether (all analytical grades) from Loba Chemie (Mumbai India).

2.2 Samples:

In order to examine the parabens concentration, 20 commercially available products like baby cream, baby lotion, toothpastes, shampoos, hair removers, shaving cream, skin care creams, moisturizers, and pharmaceutical preparations in liquid and tablet forms were procured over the counter from the local market. The samples were randomly selected irrespective of the brands. The samples were stored at room temperature before taken up for analysis.

2.3 Working Standard stock solution:

Standard stock solution of mixture of MP, EP, PP and BP of concentration 1000 µg mL^-1 was prepared by dissolving each of the reference standards in mobile phase and further dilution was carried out with this stock solution throughout the experimental procedure.

2.4 Sample preparation:

An in-house developed sample preparation method⁹ was broadly followed. In brief different commercially available cosmetics and personal care formulations samples depending upon matrix were accurately weighed in the range of 200 mg to 700 mg and 50 mL of saturated solution of NaCl (pH adjusted to 2 by addition of 0.5 M HCl) added to each of the samples. The samples were ultra sonicated for 30 minutes followed by filteration through Whatmann No.1 filter paper (GE Healthcare, UK). Each filtrate was subjected to liquid-liquid extraction (3 times) with diethyl ether. The organic phase was filtered through anhydrous sodium sulphate and evaporated to dryness at room temperature. The samples were reconstituted in 2 mL Acetonitrile and filtered through a 0.45 µ nylon membrane filter (Millipore). The filtrate was used for the HPLC analysis.

2.5 Instrumentation and chromatographic conditions:

Agilent 1100 Series LC system (Agilent Technologies, Santa Clara USA) equipped with an auto sampler, degasser, a binary gradient pump, a column oven coupled with Diode Array Detector (DAD) was used for analysis. The column used was Agilent Zorbax SB C-18 narrow bore RR (2.1 mm x 100 mm x3.5 μm) protected with an Agilent C-18 guard column. The column oven was maintained at 40⁰C throughout the analysis.

The injection volume was 1 μL set with a mobile phase needle wash throughout the study. Acetonitrile: water (50:50: v/v) in isocratic mode with the flow rate of 150 μL min^-1 was used for analysis. Necessary precautions for degassing and filtration of mobile phase were maintained during analysis. Total run time was 10 minutes. The elution was monitored at 273 nm, 254 nm and 210 nm simultaneously. Quantification was carried out using wavelength of 254 nm. The data acquisition and processing was carried out on ChemStation B.02.01 software supplied by Agilent Technologies, Santa Clara USA.

2.6 Method Validation Studies:

The proposed method was validated for linearity, limit of detection (LOD), limit of quantification (LOQ), precision including repeatability, specificity and robustness. Stability of standards under different temperature conditions was also evaluated. The validation was carried out by analysing each concentration five times (n=5) throughout the study.

2.6.1 Linearity, Limit of Detection and quantification:

For validation of linearity a series of eight concentrations of standard mixture of MP, EP, PP, and BP were prepared by diluting the stock solution with mobile phase. The concentrations of the four parabens were kept in the range of 1000 µg mL^-1 to 5 µg mL^-1. Mean peak area taken in consideration for calibration curve. Slope and the other statistics of the calibration curves were determined using linear regression equation. The signal to noise (S/N) ratio of S/N x 3 was calculated for determination of limit of detection (LOD) and limit of quantification (LOQ) were determined using S/N x 10.

2.6.2 Precision, Robustness and Specificity:

The precision of the method was evaluated for both inter-day and intraday on the basis of repeatability studies by analyzing three different concentrations ( 25, 100 and 500 µg mL^-1) in the linearity range of four parabens. The results were evaluated as Relative Standard Deviation Percentage (% RSD) for peak area (PA) and retention time (Rt) of each paraben. Robustness of the proposed method was examined from analysis of 100 µg mL^-1 concentration of analytes by making deliberate, variations in critical LC parameters such as the composition of mobile phase (± 2%), flow rate (± 1 µL) and column temperature (± 1^oC) to monitor its effects. The specificity of the analytical method was evaluated from the chromatogram where complete separation of four parabens were achieved and against no potential interferences from other components that may be present. The sample preparation method was also evaluated for extraction of parabens from the real samples with minimum interference of other ingredients.

2.6.3 Stability of Standards:

Paraben standards were prepared in duplicate. The stability of each standards were investigated by storing preparations at room temperature and under refrigeration for temperature range of 2-8^oC. The two sets of standards were stored up to 10 days and three randomly selected dilutions from the each set were analyzed by the proposed method against the freshly prepared standards on daily basis and the results were evaluated for the stability.

2.7 Method Application:

The developed method was successfully applied for analysis of four preservatives in broad range of commercially available products and the obtained results were analysed for presence of paraben as per international protocols.

3. RESULTS AND DISCUSSION:

The chromatographic conditions and instrumental parameters for study were optimized on the basis of physical characteristics of parabens. Under the stated HPLC experimental conditions, separation of the MP, EP, PP and BP were achieved with good resolution. Sample preparation method was developed keeping in consideration to keep it as simple, cheap and possibility of its application to wide range of personnel care, cosmetics and pharmaceutical formulation.

The pH was maintained to evade the potential interferences from the other ingredients of the cosmetic and pharmaceutical products. The sample preparation method was tested for extraction from different formulations which does not contain the target analytes. The results were satisfactory and proposed sample preparation method was found to be selective enough for parabens. Linearity of the proposed method was found to be excellent with regression coefficient values (R²) > 0.997 for all analytes. Results for the Retention time (Rt) and other calibration values are presented in table 1.

Table 1: Validation Data of Parabens: Retention time (Rt), linear regression equation and Regression value

Analytes	Retention Time (Rt)	Linear Regression equation y = mx + c	Regression value (R²)
MP	3.4 min	y = 21.438x + 110.68	0.9976
EP	4.2 min	y = 21.756x + 78.732	0.9978
PP	5.5 min	y = 19.686x + 157.49	0.9973
BP	7.7 min	y = 18.444x + 148.64	0.9971

The quantification for each of the parabens was carried out and LOD and LOQ for MP, EP, PP and BP were successfully determined. Results for LOD and LOQ are given in table 2.

Table 2: Validation Data of Parabens: Limit of detection (LOD) and Limit of quantification (LOQ) studies

Analytes	LOD	LOQ
(µg mL^-1)
MP	1	3
EP	2	6
PP	2	6
BP	2.5	8

Parabens show significant absorption at their corresponding wavelengths of maximum absorption (λ_max) which was found to be 254 nm. The representative chromatogram obtained for mixture of four parabens standards at detection wavelength of 254 nm is shown in figure 2.

Fig 2: Typical chromatogram of standard mixture of parabens obtained with described method (MP – Methyl Paraben, EP- Ethyl Paraben, PP- Propyl Paraben and BP- Butyl Paraben)

Table 3 Validation Data of Parabens: Precision Studies

Analytes	Concentration (µg mL^-1)	Inter-day Precision %RSD (n=5)		Intraday Precision %RSD (n=5)
		Rt	PA	Rt	PA
MP	25	1.23	2.44	0.59	1.72
	100	1.58	0.80	2.21	0.97
	500	1.02	1.04	0.14	1.42
EP	25	1.69	2.42	0.45	2.26
	100	1.70	1.94	1.95	2.67
	500	1.62	1.09	0.22	1.49
PP	25	2.33	2.93	0.31	2.05
	100	2.28	2.47	1.55	3.05
	500	2.46	1.24	0.33	1.75
BP	25	3.08	2.34	0.15	2.27
	100	3.27	1.64	1.21	1.88
	500	3.45	1.06	0.40	1.48

Results of inter-day and intraday precision were presented in table 3. The %RSD for four analytes were in the range of 0.1-3 % for both Rt and PA for intraday studies and the values were less then 3.5 % for inter-day studies. The low %RSD values clearly indicate the good precision of the proposed analytical method and its suitability to carry out the quantitative determination of four analytes from personnel care and pharmaceuticals formulations. Robustness studies of proposed method indicated that deliberate changes in critical LC parameters does not have any significant influence on the results neither as a single parameter nor in combination, proving the method to be sufficiently robust. The data on robustness studies is shown in table 4.

Table 4 Validation Data of Parabens: Robustness Studies

Analytes	Parameters
	Column Oven Temperature (± 1^oC)			Flow Rate (± 1 µL)		Mobile Phase (± 2%)
	%RSD (n=5)
	Rt	PA	Rt		PA	Rt	PA
MP	1.13	1.02	1.23		1.38	2.45	1.60
EP	1.06	1.70	1.18		1.72	4.44	1.43
PP	1.28	2.43	1.44		2.35	3.60	1.93
BP	1.83	1.56	1.99		1.87	4.57	1.14

Specificity studies show that sample pretreatment and chromatographic method was specific enough for the analysis of target analytes as no potential interferences were observed from the other components present in commercial formulations. The samples were well separated and no potential interferences from other ingredients or excipients were observed in the chromatograms for the commercial samples at the Rts of studied compounds. Stability studies confirmed that the standard solutions of MP, EP, PP and BP stored under normal conditions and refrigerated conditions were stable for the studied period as no significant deviations were observed in the chromatographic results in terms of both Rt and PA.

Analysis of real samples gave satisfactory results. Peak identification in the samples for the target preservatives was based on the comparison between the Rts of the standards and that of the samples. It was further confirmed by comparing the obtained DAD spectra of both standards and samples. Representative chromatogram of a sample is depicted in fig 3.

Fig 3: Representative chromatogram of sample of moisturizer (Brand 5) obtained with described method

On comparing the present work with methods reported in the literature we found that the sensitivity of the present method is comparable with existing HPLC methods. In the present study, different types of sample matrices were on the higher side than that of methods reported by other research groups. The present method is applicable for both personal care and pharmaceutical formulations whereas the other HPLC methods were meant for analysis of the target analytes in either cosmetics²⁰ or pharmaceuticals formulations^11,21, and food stuffs¹⁹. Table 5 outlines the details for the some of the types of samples analysed and presence of parabens in them. All the positive samples contain parabens in the range of 0.04- 0.35 % with no sample infringing the legal directives about their use. MP (92%) was found to be most commonly used preservative followed by PP (78%) and BP (30%) respectively in cosmetics and personal care formulations. EP was not found in any of the samples. Forty two percent (42%) of samples analysed were found to contain only single type of ester and remaining samples contain two types of parabens except the one sample of baby cream found to contain mixture of three different paraben esters. Two samples were found to contain one paraben in addition to the labelled content on the packaging.

Table 5: Representative data for different types of samples analysed

Sample	Type	Paraben Present	Total Percentage (%) found (as per permissible limits)⁷
Brand 1	Hair Colouring	MP, PP	0.06
Brand 2 (A)	Baby Cream	MP,PP, BP	0.2
Brand 2 (B)	Baby Lotion	MP, BP	0.35
Brand 3	Body Gel	PP	0.11
Brand 4	Deep Cleansing Milk	MP, PP	0.08
Brand 5	Moisturizer	MP, PP	0.3
Brand 6	Hair remover	MP, BP	0.09
Brand 7	Shaving Cream	MP, PP	0.23
Brand 7	Cold Cream	MP PP	0.31
Brand 8	Body lotion	MP	0.13
Brand 9	Hair Mask	MP	0.04
Brand 10	Diabetic Tablets	MP	0.13

CONCLUSION:

In the nutshell, a fast and sensitive method for analysis of parabens was developed. The separation of all the studied analytes was obtained in less than 10 mins. The method shows good result in terms of linearity, LOD, LOQ, inter-day and intraday precision, specificity, robustness and stability of analytes. The sample preparation procedure was short, selective and resulted in minimum interferences from complex sample matrix. RP-capillary HPLC method was found effective for determination of MP, EP, PP and BP in personal care, cosmetics and pharmaceutical formulations. Less prerequisites and low quantity of sample and chemicals required are additional benefits and thus the proposed method seems to be economical. The method has far reaching applicability to determine the studied components in variety of sample matrices. The proposed method is of great significance and use for various quality control and testing laboratories encountering these compounds and can be easily put on board for routine testing.

ACKNOWLEDGEMENT:

The authors wish to express their gratitude to Director, Central Forensic Science Laboratory, Chandigarh, India for constant support, encouragement and providing necessary facilities to carry out the study.

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Received on 18.07.2017 Accepted on 10.09.2017

Asian J. Pharm. Ana. 2017; 7(4): 229-234.

DOI: 10.5958/2231-5675.2017.00037.0