1.INTRODUCTION:

Alkyl esters of p-hydroxybenzoic acid are commonly named as parabens. Parabens are widely used as antimicrobial agents and preservatives in pharmaceutical preparations, cosmetics and food products¹. Methyl Paraben (MP), Ethyl Paraben (EP), Propyl Paraben (PP) and Butyl Paraben (BP) are the most commonly used parabens. Low toxicity, neutral pH, no perceptible odour and possessing anti-bacterial and anti-fungal properties facilitates the use of parabens as preservatives². Parabens does not cause hardening or produce any kind of discoloration and their antimicrobial properties increases with increase in alkyl chain length. Parabens are often used in two or more combinations to achieve desired results. Parabens have hydrophobic character which increases with increasing alkyl chain length and also lacks any kind of easily ionizable functionality³.

In the recent years numerous studies has documented the estrogenic activities of the parabens^4-6. Due to estrogenic activities of certain parabens secretion of testosterone and functioning of male reproductive system are effected⁷. Concern towards safe use of parabens has emerged after their detection in human cancerous breast tissues⁸. In cosmetic products the permissible limits for parabens is 0.4% w/w for individual paraben and for mixture of parabens it is fixed at 0.8 % w/w⁹.

With increase in debates on the effects of parabens numerous methods for their detection and quantification have been developed and reported by various research groups in different matrices such as food¹⁰, water¹¹, cosmetics products^12-13, environmental samples [14], biological samples^15-16 and pharmaceuticals¹⁷. The choice of analytical techniques covers a wide range like Thin Layer chromatography (TLC)¹⁸, high performance thin layer chromatography (HPTLC)¹⁹, high performance liquid chromatography (HPLC)^20-22, liquid chromatography- mass spectrometry (LC-MS)²³, micellar electrokinetic chromatography (MEKC)²⁴, Gas Chromatography (GC)²⁵ and gas chromatography- mass spectrometry (GC-MS)^26-27. Most of the reported methods using GC as separation technique involve the step of derivatization before detection of the target analyte. Methods employed to determine parabens without derivatization are limited. The mass detector used were either single^28-29 or triple-quadrupole (GC-MS/MS)³⁰. Nontraditional extraction procedures like solid phase extraction (SPE), solid phase micro extraction (SPME), development and optimization were attempted as a final objective by different research groups in the methods using GC as separation technique for determination of parabens³¹.

In this background requirement for an easy method suitable for routine analysis always exist. With this aim a method has been tailored for analysis of parabens over a wide range of personal care products, cosmetics, and pharmaceutical formulations. In the proposed method liquid extraction (LE) has been employed and seems to be versatile enough in terms of application on different types of sample matrices. A GC-MS method was developed and validated for the simultaneous analysis of MP, EP, PP and BP and satisfactorily applied to the commercial formulations which can be procured over the counter from the local market.

2. EXPERIMENTAL:

2.1 Instrumentation and operational conditions:

The detection and quantification of analytes were performed on Finnigan Trace GC Ultra interfaced with a Thermo DSQ Quadrupole MS and thermo auto sampler AS 3000 (Thermo Fisher, USA). The chromatographic separation was carried out using a cross-linked BP-5 (5% diphenyl- 95% dimethyl polysiloxane 30 m×0.32 mm i.d×0.5 μ film thickness) capillary column (Trajan Scientific and Medical, Australia). Helium of purity 99.99% was used as carrier gas at constant flow rate of 1.2 mL/min. The Programmable injector port was kept at 250°C in constant temperature split mode with split ratio of 30. The temperatures of Ion source and MS transfer line were 200°C and 300°C respectively. Initial oven temperature was 90°C (hold for 0.7min) increased to 280°C at a rate of 35°C/min, with a final hold time of 2 min (total run time: 8.13 min). The MS was operated in electron impact ionization (EI) positive mode at -70 eV. Full scan data was obtained in mass rage of 50-550 amu and was processed on Xcalibur 1.4 (Thermo Fisher, USA). Injection volume was kept constant at 0.5 μL throughout the experiment.

2.2 Chemicals and reagents:

Certified references of Methyl 4-hydroxybenzoate (MP), Ethyl 4- hydroxybenzoate (EP), Propyl 4-hydroxybenzoate (PP) and Butyl 4-hydroxybenzoate (BP), all of purity 99% were procured from Alfa Aesar (Heysham, England). Methanol (HPLC grade, 99%) was purchased from Merck Specialities (Mumbai, India), Hydrochloric acid (HCl), sodium chloride (NaCl), anhydrous sodium sulphate and diethyl ether (all analytical grades) are obtained from Loba Chemie (Mumbai India).

2.3 Sample collection:

Different types of commercially available products including baby lotion, baby cream, baby oil, hair remover, gels, moisturizers, toothpaste, shampoo and pharmaceutical preparations of generic and branded formulations available in tablet and liquid form were procured from local market to undertake the study. The samples were selected to include different types of matrices without preference for any particular type of matrices or brand. All the samples were stored at room temperature before taken up for analysis.

2.4 Calibrators and Sample Preparation:

A standard stock solution of mixture of MP, EP, PP and BP of concentration 1000 µg/mL was prepared in Methanol. Working standard solutions for calibration containing MP, EP, PP and BP were prepared by diluting the appropriate volume of the stock solution with methanol.

Sample preparation method developed in-house¹⁸ was broadly followed. In brief accurate quantity of samples of different commercial formulations were thoroughly mixed and dissolved in 10 mL saturated solution of NaCl (pH 2) adjusted using 0.5 M HCl, followed by ultrasonication for 20 minutes and filtration through Whatmann No.1 filter paper (GE Healthcare, UK). LE was carried out with filtrates with 10 mL diethyl ether (3 times). The organic phases were passed through anhydrous sodium sulphate before collection and evaporated to dryness at room temperature. The extracts were reconstituted in 2 mL Methanol and filtered through 0.45 µ nylon membrane filter (Millipore) before taken up for GC-MS analysis.

3. RESULTS AND DISCUSSION:

3.1. Method development and optimization:

A LE technique followed by GC-MS method has been developed and optimized for the simultaneous determination of MP, EP, PP and BP in cosmetics, personal care formulations and pharmaceuticals preparations real samples of different types of matrices. The assay includes isolation of all four analytes from real samples by LE and their identification by GC-MS.

The sample preparation method was investigated using NaCl solution of different pH and found that it is most appropriate to adopt pH 2. At this pH potential interferences from the other ingredients of the cosmetics and pharmaceutical formulations are minimal and can be easily evaded. Chromatographic conditions for GC-MS analysis mainly temperatures for programmable injector port (220-280^oC), MS transfer line (280-330^oC), column oven including initial (70-150^oC) and final (250-320^oC) temperatures, ramp rate (15-40^oC), ion source (180-220^oC) and carrier gas flow (0.9-1.3 mL/min) were investigated and optimized. The investigated ranges are given in brackets and optimized conditions are briefed in instrumentation and operational conditions section. The retention times for MP, EP, PP and BP were 4.94, 5.20, 5.54 and 5.90 minutes respectively under optimized conditions.

3.2 Method Validation Studies:

The investigated analytical methodology was suitable for the determination of MP, EP, BP and PP simultaneously. It was further validated for, stability, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), robustness and precision including repeatability. Each sample and standards were analysed five times (n=5) for whole study.

Stability of each analytes was evaluated from the results obtained at different temperature conditions during GC-MS analysis. None of the studied compound shows any type of variations in results. For specificity studies final hold time in ramp rate was increased and data was recorded for additional 10 minutes and no potential interferences from the other ingredients of the cosmetics and pharmaceutical formulations were observed indicating the extraction method is specific enough for extraction of parabens from commercial formulations. Calibration curves were obtained by analyzing standard calibrators in the concentration level of 1 µg/mL to 1000 µg/mL. Linearity of the method was found excellent with regression coefficient values (R²) > 0.993 for all four analytes. The LOD and LOQ for each analyte were determined as the lowest concentration yielding signal-to-noise ratios of S/N x 3 and S/N x 10 respectively.

The precision of the method was evaluated by three different calibrators (25, 100, 250 µg/mL) both inter-day and intra-day. Data reflecting analytical performance for precision is summarized in table 1. The robustness of the method proposed was evaluated by minor changes in several method parameters like pH of NaCl, GC temperature parameters (injector temperature, column oven, ramp rate and MS transfer line) and carrier gas flow rate. No major anomaly was observed with change in single parameter or combinations which proved that the method is sufficiently robust.

Table 1: Data of Inter-day and Intra-day Precision studies:

Analytes	Concentration (μg mL^-1)	Inter-day Precision % RSD (n=5)	Intra-day Precision % RSD (n=5)
MP	25	0.59	0.11
	100	0.20	1.04
	250	0.85	0.16
EP	25	1.07	0.26
	100	0.11	0.93
	250	0.22	0.29
PP	25	1.02	0.72
	100	0.65	0.45
	250	0.21	0.45
BP	25	0.34	0.86
	100	0.26	0.09
	250	0.10	0.14

3.3 Method Application on Real Samples:

The developed method was successfully applied to the qualitative and quantitative analysis of four commonly used preservatives over a range of personal care, cosmetics and pharmaceutical formulations. The results obtained were satisfactory. The confirmation for presence on MP, EP, PP and BP in any of the samples and calibrators were carried out using mass spectral libraries. Chromatogram of a standard calibrator and baby cream sample is depicted in fig: 1 with structure of paraben in inset.

Fig 1: Chromatograms of parabens (A) Standard mixture at low concentration with basic structure of paraben in inset where R = methyl, ethyl, propyl and butyl (B) Representative sample

Comparison of method reported in the present work with recent previously reported without derivatization GC-MS methods in similar types of matrices revealed that the sensitivity of the proposed method is comparable with others. More over in most of the previously reported GC-MS methods use of complicated and labor intensive sample pretreatment is common when compared with present work in which simple extraction procedure is adopted without any special sample pretreatment like derivatization commonly used in GC and GC-MS methods. Also the variety of the sample matrices is on higher side in the present study when compared with others. An overview of the comparison is depicted in table 2.

4. CONCLUSION:

This work demonstrates a validated GC-MS analysis method combined with LE as an accurate and simple technique for the simultaneous determination of MP, PP, EP and BP cosmetics, personal care and pharmaceutical formulations. The method presented here authenticates its applicability in variety of sample matrices (solid, liquids, gels, creams and oils). The sample preparation method was found to be effective for target analytes. The analytical parameters of the proposed method were acceptable. In nutshell the method is suitable for laboratories that perform everyday routine analysis of target compounds at low cost.

5. ACKNOWLEDGEMENT(S):

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

6. REFERENCES:

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Received on 23.07.2017 Modified on 20.08.2017

Research J. Science and Tech. 2017; 9(3): 308-312.

DOI: 10.5958/2349-2988.2017.00055.9

Method	Types of Sample	Analytes	Extraction Method	Limit of Detection LOD)	Reference
GC-MS	baby lotion, baby cream, baby oil, hair remover, gels, moisturizers, toothpaste, shampoo, pharmaceutical preparations	MP,EP,PP,BP	LE	MP – 0.7 μg/mL EP – 0.5 μg/mL PP – 0.5 μg/mL BP – 0.4 μg/mL	Present Study
GC-MS	Hair sprays, perfumes, deodorants, cream, lotion	MP,EP,PP,BP	SPE	10 - 200 μg kg^-1	Shen HY. et al.²⁸
GC-MS/MS	Cosmetics	MP,EP,PP,BP	LE	Not Provided	Wang PG and Zhou W.³⁰