1. INTRODUCTION:

Parabens (esters of para-hydroxybenzoic acid) also known as alkyl p-hydroxybenzoates are class of chemical compounds with antimicrobial properties. These compounds and their salts are most commonly employed preservatives in cosmetic, personal care, pharmaceutical, food and beverage formulations to prevent the development of micro organisms which causes deterioration of these products thus increasing their shelf life. The antimicrobial and antifungal properties of parabens were found to be good enough to use them as preservatives in various preparations. The antimicrobial activities of the parabens found to increase with increasing chain length. Two or more types of parabens are often simultaneously used to achieve desired effects.

The simplicity and effectiveness of parabens as preservatives, in combination with their low cost and the long history of their use probably explains why they are so commonplace, exposing human population to parabens from a wide variety of sources on a daily basis. The most common parabens are methyl, ethyl, propyl and butyl parabens (Figure:1)

Fig 1: Structure of Paraben, where R is the alkyl group (methyl, ethyl, propyl and butyl).

Increase in the public awareness of the potential hazard for the chemicals used as raw material in the products of everyday life has raised concern and attracted much attention towards their safety issues and parabens is one of them.

The ability of certain parabens to act in similar nature as estrogen^1-³initiated the debate of parabens toxicity. Detection of methyl paraben at the highest concentration in breast tumours has raised safety issues in the uses of parabens in the different products of everyday life. Average levels of 20 nanograms/gram of parabens have been detected in a sample of 20 breast tumors⁴ made some scientists to conclude that the presence of parabens may be associated with the occurrence of breast cancer, but due to non availability of sufficient scientific findings, no reasonable link can be established between the parabens and cancer. 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.

Literature survey revealed that several matrices have been analysed for these compounds: Biological^5-6, pharmaceutical samples⁷, water samples⁸, food⁹ and cosmetic products¹⁰. The choice of techniques for analysis of different matrices are mainly High Performance Liquid Chromatography (HPLC)^11-12, gas chromatography- mass spectrometry (GC-MS)^13-14, liquid chromatography- mass spectrometry (LC-MS)¹⁵, capillary electrophoresis (CE)^16-17 and High Performance Thin layer Chromatography (HPTLC)^18-21. All the reported methods of HPTLC were associated with the identification of these compounds by densitometry. Only one method describes the use of Vibrio fischeri luminescence bacteria with video densitometry²². The methods employing the use of TLC followed by detection with visualisation reagent is rare^23-24. The previously reported methods using the TLC as separation technique for parabens employs the mobile phase in acidic medium and the methods employing mobile phase in basic medium is scarce and only reported method for the separation of four parabens in mobile phase of basic medium uses cyanopropyl bonded stationary phases²². No method describes the separation of four commonly used parabens with normal phase silica gel F₂₅₄ TLC plates using mobile phase of basic medium.

The alkyl part of the ester functional group of parabens plays the principal role in the separation of homologous parabens. Due to the similar chemical properties and identical structures, the separation and identification methods should be very selective for these compounds. The method reported through present work is totally new from the previously reported ones and first of its kind in respect to the mobile phase, conditions and chromogenic visualisation reagent. The separation shows the advantage of the method over the existing. Also a new chromogenic visualisation reagent was developed for the compounds and the mechanism for the colour complex formed has been proposed. The application of the developed method was successfully carried out on different personal care products and pharmaceutical preparations available in Indian market. The proposed method is of great significance and use for various quality control and testing laboratories encountering these compounds.

2. EXPERIMENTAL:

2.1 Chemicals and Materials:

Methyl 4-hydroxybenzoate (MP, 99%), Ethyl 4- hydroxybenzoate (EP, 99%), Propyl 4-hydroxybenzoate (PP, 99%) and Butyl 4-hydroxybenzoate (BP, 99%) were purchased from Alfa Aesar (Heysham, England). Analytical grade Hexane, dioxane, dichloromethane, methanol, ethanol, diethylamine and triethylamine were procured from Merck Specialities (Mumbai, India). Methyl red, sodium nitrite, hydrochloric acid, sodium chloride, anhydrous sodium sulphate, diethyl ether, all analytical grade were purchased from Loba Chemie (Mumbai India). HPLC grade Water was purchased from Fisher Scientific (Mumbai India). Silicagel 60 F₂₅₄ TLC plates were procured from Merck KGaA (Darmstadt, Germany). Twin-trough chamber for TLC development from Camag, (Switzerland).

2.2 Working Standard stock solutions:

Standard stock solutions of 1 mg/ml concentration of MP, EP, PP and BP were prepared by dissolving each of the reference standards in ethanol and further dilution was carried out as per the experimental requirements.

2.3 Samples:

A total of 26 different types personal care products including baby lotion, baby cream, hair remover, moisturizer, toothpaste, shampoo and different pharmaceutical preparations available in tablet and liquid form were undertaken for the study.

2.4 Sample preparation:

10 mg of each sample is dissolved in saturated solution of sodium chloride acidified with 0.5M HCl by adjusting the pH: 2. The solution is filtered and the liquid-liquid extraction (4 times) is carried out with diethyl ether. The organic phase was filtered through anhydrous sodium sulphate and evaporated to dryness at room temperature. The residue was re-dissolved with ethanol and used for the analysis.

2.5 Mobile phase:

The optimised mobile phase, Hexane: 1, 4 dioxane: methanol: triethylamine (7.5:1:1:0.5, v/v) was mixed and centrifuged. 20 ml of the said mobile phase was taken in a twin trough TLC chamber and kept for optimized saturation time of 30 minutes.

2.6 Sample application and development:

The reference standards as well as the extracted samples were spotted using micro syringe on a silica gel F₂₅₄ TLC plate (20x20 cm) at a distance of 1.5 cm from the base and the elution was carried out up to 16 cm. After elution the plate was taken out and kept for drying at room temperature. The developed plate was visualised under UV (254 nm) and the corresponding spots of standards as well as the samples were noted and the Rf values were calculated.

2.7 Visualisation using chromogenic reagent:

A new chromogenic visualisation reagent was developed for parabens. The developed plate was heated at 100^oC for 30 minutes for activation and sprayed with 2N sodium nitrite solution prepared in 0.5M HCl, dried at room temperature followed by spraying with 0.4% methyl red solution prepared in 80% ethanol.

2.8 Determination of Limit of detection (LOD):

The minimum detection limit for parabens using the new chromogenic visualisation reagent as well as UV (254nm) has been determined by spotting different concentrations of each of the reference standards.

3. RESULTS AND DISCUSSION:

3.1 Development of mobile phase:

Various experiments were conducted using different combinations of organic solvents as mobile phases (Table 1) for the successful separation of the compounds.

Table 1: Different mobile phase compositions experimented

Mobile phase	Composition(v:v)
Chloroform: ethylacetate: acetic acid	8:1.5:0.5
Dichloromethane: ethylacetate: formic acid	7:2.5:0.5
Hexane: acetone:diethylamine	7.5:2:0.5
Hexane: Dichloromethane:methanol:triethylamine	7.5:1:1:0.5
Hexane: 1,4-Dioxane:methanol:triethylamine	7.5:1:1:0.5

The first three mobile phase compositions were not satisfactory as in all the cases resolution was poor and elution was too slow. But the fourth and fifth systems found to be better than before and gave good resolution for the compounds. Varying ratios of the fifth system were tried and the mobile phase was optimised in the ratio of 7.5:1:1:0.5 (v/v) with optimized chamber saturation time of 30 min at room temperature (25 ± 2 ^oC) which gave good resolution with better spot shape.

Figure 2 depicts the UV chromatogram obtained under the optimised conditions for paraben standards which clearly shows the compounds are well resoluted and can be specifically identified.

Fig 2: UV Chromatogram of Standard Parabens.

The newly developed TLC method is the first one applied for the separation of four different parabens using mobile phase under basic medium on normal stationary phase.

The developed method was applied to different types of personal care products and pharmaceutical preparations available in tablet and liquid form which gave reproducible results. Figure 3 depicts the

Fig 3: Chromatogram visualised with new chromogenic reagent.

Previously reported methods for separation of four parabens suggest the use of acidic medium in the mobile phase with various rigorous conditions of drying the TLC plates for 2 hrs at 160^oC followed by storing over potassium hydroxide in evacuated desiccator²⁵ or use of modified stationary phases (Table 2). Only method which uses silica gel stationary phase and mobile phase in basic medium was applied for separation of MP, PP²⁰ as methyl and propyl esters can be relatively easily separated²⁵.

3.2 New visualization reagent:

The new visualization reagent developed was found to be selective for the identification of parabens. The reddish pink colour obtained with the respective parabens clearly confirms the applicability of the new reagent. The preparation of the new reagent involves simple procedure. The reaction mechanism (figure 4) involved in formation of reddish pink color is proposed to be formation of azo dye.

The initial step of heating the developed plate at 100^oC for making the stationary phase of neutral character was optimized for 30 minutes after trying the heating for different time intervals (15, 30, 45 and 60 minutes). Treating the TLC plate with acidified sodium nitrite at room temperature produces o-nitrosoderivative, a transition state which in turn converted to the corresponding diazonium salt²⁶. Development of pink red chromatographic zone on yellowish background is a result of the coupling of diazonium salt with methyl red. The diazonium coupling usually happens at position meta to the acid functional group of methyl red.

The use of methyl red as a spray reagent was not previously reported for parabens. The preparation of reagent and its application is much easy when compared with Sodium Hydroxide- 4 Amino antipyrine- Potassium Hexacyanoferrate (III) reagent²³ and Denige’s reagent²⁴. The information on R_f values and detection limits of the parabens by newly developed spray reagent is given in Table 3.

Table 3: R_f values and Limit of Detection by developed method

Name of the compound	R_f value	LOD (µg/spot) Spray reagent
Methyl paraben	0.43	0.8
Ethyl paraben	0.49	0.8
Propyl paraben	0.55	0.6
Butyl paraben	0.59	0.4

The results clearly suggest the better sensitivity for parabens identification in the range of 0.4-0.8 (µg/spot) with the proposed new reagent and can be adopted for the routine analysis of these preservatives using the simple TLC method.

4. CONCLUSION:

Due to the similar chemical properties and identical structures of p- hydroxybenzoates the proposed TLC method was found to be suitable for the determination of parabens which acts as preservatives in personal care products and pharmaceutical preparations. The proposed method presented in this paper has advantages of ease and simplicity and first one for separation of MP, EP, PP and BP by simplest form of chromatography i.e. TLC using basic medium for mobile phase on normal silica gel F₂₅₄ plates and does not requires any special modifications for stationary phases or temperature control. The newly developed reagent also provides ease for preparation and application and eliminates the toxic effects of mercury during the use of Denige’s reagent and also the method is simple, fast and gives reproducible results with better sensitivity. Thus the proposed TLC method can be applied for routine analysis of MP, EP, PP and BP in laboratories encountering the same compounds.

5. ACKNOWLEDGEMENT:

The authors are highly thankful to the Director, Central Forensic Science Laboratory, Chandigarh, India for his constant encouragement and valuable suggestions.

6. REFERENCES:

1. Routledge E.J, et.al. Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic. Toxicology and Applied Pharmacology. 153(1); 1998: 12– 19.

2. Darbre P.D, et.al. Oestrogenic activity of isobutylparaben in vito and in vivo. Journal of Applied Toxicology. 22(4); 2002: 219-226.

3. Byford J.R, et.al. Oestrogenic activity of parabens in MCF7 human breast cancer cells. The Journal of Steroid Biochemistry and Molecular Biology. 80(1); 2002: 49-60.

4. Darbre P.D., et.al Concentrations of Parabens in Human Breast Tumours. Journal of Applied Toxicology. 24(1); 2004: 5-13.

5. Zotou A., et.al., LC-determination of five paraben preservatives in saliva and toothpaste samples using UV detection and a short monolithic column. Journal of Pharmaceutical and Biomedical Analysis. 53(3); 2010: 785-789.

6. Frederiksen H., et.al., Parabens in Urine and seminal plasma from healthy Danish men determined by Liquid chromatography- tandem mass spectrometry ( LC-MS/MS). Journal of Exposure Science and Environmental Epidemiology. 21(3); 2011: 262-271.

7. Boonleang J., Tanthana C., Simultaneous Stability- indicating HPLC method for the determination of cisapride, methylparaben and propylparaben in oral suspension. Songklanakarin Journal of Science and Technology. 32(4); 2010:379-385.

8. Pedrouzo M., et. al., Ultra-high-performance liquid chromatography-tandem mass spectrometry for determining the presence of eleven personal care products in surface and wastewaters. Journal of Chromatography A. 1216(42); 2009: 6994-7000.

9. Saada B., et.al., Simultaneous determination of preservatives ( benzoic acid, sorbic acid, methyl paraben and propy paraben) in foodstuffs using high-performance liquid chromatography. Journal of Chromatography A. 1073; 2005: 393-397.

10. Gao W., Legido-Quigley C., Fast and sensitive high performance liquid chromatography analysis of cosmetic creams for hydroqninone, phenol and six preservatives. Journal of Chromatography A. 1218(28); 2011: 4307-4311

11. Kumar S., et.al Development and validation of a single RP-HPLC assay method for analysis of bulk raw material batches of four parabens that are widely used as preservatives in pharmaceutical and cosmetic products. Journal of Chromatographic Science. 53; 2011: 405-411.

12. Shabir G.A., Determination of combined p-hydroxybenzoic acid preservatives in liquid pharmaceutical formulation by HPLC Journal of Pharmaceutical and Biomedical Analysis. 34(1); 2004: 207-213.

13. Shanmugama G., et.al., GC-MS method for the determination of paraben preservatives in the human breast cancerous tissue. Microchemical Journal. 96(2); 2010: 391-396.

14. Saraji M., Mirmahdieh S., Single- drop microextraction followed by in-syringe derivatization and GC-MS detection for the determination of parabens in water and cosmetic products. Journal of Separation Science. 32(7); 2009: 988-995.

15. Nieto A., et.al., Determination of personal care products in sewage sludge by pressurized liquid extraction and ultra high performance liquid chromatography- tandem mass spectrometry. Journal of Chromatography A 1216; 2009: 5619-5625.

16. Blanco E., et. al., Simultaneous determination of p-hydroxybenzoic acid and parabens by capillary electrophoresis with improved sensitivity in nonaqueous media. Electrophoresis. 29(15); 2008: 3229-3238.

17. Blanco C.C., et.al., Simultaneous determination, by capillary zone electrophoresis, of multiple components of different industrial products. Chromatographia. 53(7-8); 2001: 414-418.

18. Thomassin M., et.al., Comparision of quantitative high performance thin layer chromatography and the high performance liquid chromatography of parabens. Journal of Pharmaceutical and Biomedical Analysis. 15(6); 1997: 831-838.

19. Tomankova H., Pinkasova M., Determination of parabens and their degradation product p-hydroxy benzoic acid in pharmaceutical dosage forms by HPTLC densitometry. Analytical Letters. 23(7); 1990: 1319-1332.

20. Cakar M., et.al., Simultaneous HPTLC determination of Imidazole Antimycotics and Parabens in creams. Journal of Planer Chromatography. 17; 2004: 177-180.

21. Mirzaie A., et.al., TLC Quantification of Methylparaben on a n Inorganic Ion- Exchanger in Presence of Other Food Additives. Journal of Planer Chromatography. 20(2); 2007: 303-306.

22. Seigel A., et.al., Quantification of parabens by diode-array thin layer chromatography coupled with a Vibrio fischeri bioluminescence assay Journal of Planer Chromatography 26(2); 2013: 119-124.

23. Gossele J.A.W., Modified thin-layer chromatographic separation of preservatives. Journal of Chromatography A. 63; 1971: 433-437

24. Manual of Methods of Analysis of Foods-Food Additives, Directorate General of Health Services, Ministry of Health and Family Welfare, Government of India. 2005.

25. Stahl E, Thin Layer Chromatography- A laboratory hand book. Springer, India. 2009.

26. Philpot J.S.L., Small P.A., The action of nitrous acid on p-cresol and tyrosine, Biochemical Journal. 32; 1938: 534-541.

Received on 04.05.2015 Modified on 15.05.2015

Asian J. Research Chem 8(6): June 2015; Page 394-398

DOI: 10.5958/0974-4150.2015.00065.6

Mobile Phase composition	Stationary Phase	Visualisation	Number of Parabens analysed
n-pentane: glacial acetic acid (88:12, v/v)¹⁹	Silica gel	Densitometry	MP,EP,PP,BP
n-hexane: ethyl methyl ketone: acetic acid 8:2:0.3 (v/v)²¹	Inorganic ion-exchanger stannic silicate	Densitometry	MP,EP,PP
Water: acetonitrile: dioxane: Ethanol: Ammonia (8:2:1:1:0.05, v/v)²²	Cyanopropyl bonded	Video Densitometry	MP,EP,PP,BP
Petroleum ether(40/60): carbon tetrachloride : chloroform :formic acid : glacial acetic acid (50:40:20:8:2, v/v²³	SILCEL-Mix-25 (mixture of silica and cellulose)	Sodium Hydroxide- 4 Amino antipyrine- Potassium Hexacyanoferrate(III) reagent	MP, EP, PP
Toluene: methanol: acetic acid (90: 16: 8)²⁴	Silica gel G	UV and Denige’s reagent	Not specified