Research & Reviews: Journal of Pharmaceutical Analysis
Menue-ISSN: 2320-0812
e-ISSN: 2320-0812
1Bharat Institute of Technology, Jawaharlal Nehru Technological University Hyderabad (JNTUH), India
2BCDA College of Pharmacy & Technology, Kolkata, West Bengal University of Technology (WBUT), West Bengal, India
Received date: 21/02/2019; Accepted date: 09/03/2019; Published date: 26/03/2019
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A new method for the determination of paraben by High-Performance Liquid Chromatography (HPLC) and determination of Neolone 950 by High- Performance Thin Layer Chromatography (HPTLC) was developed. Skin creams usually consist of many components, including preservatives. Consumer health and safety is the main reason for including preservatives as antimicrobial additives in skin cream formulations. Preservatives are often used as a multi-component mixture to increase the range of microorganisms against which the product is protected. Ingredients in cosmetic products are labelled in accordance with (European Union) EU legislation. Here we established a quantitative method to estimate the concentration of preservative in Herbal skin cream. Their methods were based on High- Performance Liquid Chromatography (HPLC) analysis and performed under different chromatographic conditions. The proposed method was successfully applied to the assay of methylparaben, propyl paraben and neolone 950 in cosmetic products with minimal sample preparation
Cream, Methyl Paraben, Propyl Paraben, Preservative, Niolone 950, HPLC, HPTLC.
The concept of beauty and cosmetics is as ancient as mankind and civilization. Indian herbs and its significance are popular worldwide. Cream is a semi-solid emulsion, which is a mixture of oil and water in presence of a suitable emulsifying agent [1]. Skin creams usually consist of many components, including preservatives. Consumer health and safety is the main reason for including preservatives as antimicrobial additives in skin cream formulations. Preservatives are often used as a multi-component mixture to increase the range of microorganisms against which the product is protected. However, preservatives are also important causes of allergic contact dermatitis [2]. Skin-care products are used daily by many people, correct and complete ingredient labelling is important, and the use of preservatives in skin products is strictly regulated. Ingredients in cosmetic products are labelled in accordance with EU legislation. Correct labelling means that people with sensitivities can be aware of any preservatives in a product formulation that could trigger an allergic reaction. Several studies of skin cream preservative content regarding correct ingredient labelling as well as maximum allowed concentration have been performed earlier. Many thousands of cosmetic, food and pharmaceutical products would deteriorate or lose their perfume, flavor and/or quality in a short time without any preservative.
Paraben As Preservative
To conserve the quality of food, pharmaceuticals and cosmetics for a relatively long time, different preservatives are added to them. Parabens (4-hydroxybenzoic acid esters), as oestrogenic chemicals, can protect against micro-organisms like bacteria and moulds (Table 1).
Table 1. Target Preservatives in the present study
| Preservative | Maximum allowed concentration% (m/m) |
|---|---|
| Parabens (esters of 4-hydroxy benzoic acid): | 0.4% as 4-hydroxy benzoic acid for a paraben, 0.8% as 4-hydroxy benzoic acid for a mixture of parabens |
| Methyl paraben, | |
| Ethyl paraben | |
| Propyl paraben | |
| Isobutyl paraben | |
| Butyl paraben | |
| Benzyl paraben |
Due to their broad antimicrobial spectra with relatively low toxicity, good stability and non-volatility, parabens are commonly used as preservatives to prevent alteration and degradation of cosmetics, pharmaceuticals and foods from microbial and fungal contamination and to protect the consumers [3].
Neolone 950 As Preservative
Neolone bactericides. These broad-spectrum bactericides are particularly suitable as replacements for formaldehyde donors in leave-on applications such as skin care formulations and sun care formulations, Zinc pyrithione-based antidandruff shampoos.
Neolone 950 preservative is effective at very low use levels, is compatible with existing fungicides like parabens and is more stable in difficult to preserve high pH formulations. And Neolone 950 is supported by extensive toxicology studies and global regulatory dossiers [4].
The active ingredient of Neolone preservative is an isothiazolinone identified by the Chemical Abstract and IUPAC system of nomenclatures as 2-Methyl-4-isothiazolin-3-one and 2-Methyl-3(2H) isothiazolinone. The recommended use level for Neolone 950 is 0.05%-0.1% (48-95 ppm of active ingredient) of Neolone [5,6] (Table 2).
Table 2. Formulation design for o/w cream 500 g.
| Ingredients | Amount (g) | Ingredients | Amount (g) |
|---|---|---|---|
| Alovera powder ext | 0.005 | Rose Water | 15 |
| Herbal distillate | 50 | Neolone 950 | 0.5 |
| Methyl paraben | 1.03 | Potassium hydroxide | 3.5 |
| Propyl paraben | 0.51 | DM water | 350 |
| Propylene glycol | 2.5 | Zinc Oxide (Lab) | 1.5 |
| Nicinamide | 7.5 | Tapioca Starch | 20 |
Materials Use for Analysis
Acetic acid (HPLC grade) (MERCK)
Acetone (MERCK)
Methanol (MERCK)
Demineralized Water (Prepared in the laboratory)
Chloroform (MERCK)
Methyl Paraben (MERCK)
Propyl Paraben (MERCK)
Absolute Ethanol (MERCK)
Ethanol 95% (MERCK)
Acetonitrile (MERCK)
Neolone 950 (MERCK)
Instrument Used for Analysis
Camag HPTLC system with camag TLC scanner 4 with (i) TLC aluminium sheet Silica gel 60 F 254 plate (10/10 cm) and (ii) CamagLinomet 5 applicator (syringe size 100 μl).
Ultra-violate spectroscopy (Shimadzu model 1800).
High performance liquid chromatography with (i) Syringe filter (nylon, PVDF, 0.45 m porosity) (ii) HPLC detector diode array (190-950 nm) and (iii) Reversed phase analytical column (Nucleosil 5C18 or equivalent, stainless steel, 25 cm x 4.6 mm) with 20 L loop Micro syringe.
Software Used
LC Solution (Shimadzu high performance liquid chromatography).
Win cats ‘planar chromatography manager (software system).
UV probe 2.33.
Microsoft excels 2007.
Estimation Methyl Paraben, Propyl Paraben, in The O/W Skin Cream by HPLC
This method specifies a procedure for the determination of methyl 4-hydroxy benzoate (methyl paraben), propyl 4-hydroxybenzoate (Propyl Paraben), in cosmetic products.
The amounts of the preservatives determined by this method are expressed as percentage by mass.
Procedure for Preparing Standard
Stock solution contains the preservatives in ethanol/water 9:1 V/V. Weight 0.05 g of methyl Paraben/propyl Paraben and made the volume up to 100 by water. Transfer 0.5, 1, 2, 5, 10 and 20 ml stock solution (0.5 μg/μl) respectively into 50 ml volumetric flasks. To each flask and fill up to the line with ethanol-water (9:1 V/V) mixture. (Use a solvent mixture to fill up each flask made e.g. as follows: 270 ml absolute ethanol + 30 ml water.) Prepare these solutions fresh for every experiment [7,8].
Procedure for Sample Preparation
Weigh 1.0 g of cream into a 100 ml volumetric flask (20 ug/μl). Add 1.0 ml 2 mol sulphuric acid solution. Add 50.0 ml ethanol-water mixture (9:1 V/V; use a solvent mixture made in the following way: 90 ml absolute ethanol + 10 ml water). Shake the closed tube for at least one minute, to suspend the sample in the solvent mixture. Put the tube in a water bath kept at 60 ± 1◦C. Leave for five minutes to facilitate the extraction of the parabens into the liquid phase. Immediately cool the tube in a stream of cold water and store the extract in the refrigerator for one hour. Filter the extract using a filter paper with 0.45 μm porosity. Transfer approximately 2 ml of the filtrate into a 5 ml volumetric flask. Store the extracts in the refrigerator and perform the HPLC determination within 24 hours [9].
Procedure for HPLC Separation
For HPLC separation, mix and filter the mobile phase (water-acetonitrile (50:50)). Adjust the flow rate of the mobile phase (1.5 ml min-1) and the UV detector (290 nm).
The injected volume should be 20 μl of each solution (standard and sample). In all cases, record the chromatogram and measure the peak areas and peak heights. Calculate the amount of preservative present [10,11].
Estimation of Neolone 950 (2-Methyl-4-Isothiazolin-3-One (Mi)
This method is used for the determination of neolone 950 (2-methyl-4-isothiazolin-3-one (Mi) in cream. Estimation process used HPTLC (High Performance Thin Layer Chromatography). Chloroform: methanol (90:10) taken as Mobile phase and Stationary phase used was TLC Aluminium sheet Silica gel 60 F 254 plate (10/10 cm).
Preparation of Standard Curve
0.1 g of neolone 950 standard was weight accurately in a 100 ml volumetric flask (1 μg/μl) dissolved it by methanol: 0.4% Acetic acid (20:80) and make up to the mark with same solvent mixture (Stock solution is stable for 1 week, when stored at 4◦C). Filter the solution by 0.45 μl, 1 μl, 2 μl, 3 μl, 4 μl above solution was injected (contain 1 μg, 2 μg, 3 μg, 4 μg) on TLC Aluminium sheet Silica gel 60 F 254 plate (10/10 cm) by using Camag Linomet 5 applicator (syringe size 100 μl).
After drying the TLC plate put into the mobile phase and run time is 10 min. Retention factor, peak height, peak area was measured by scanning the plate using camag TLC scanner 4 at 275 nm [12,13] (Table 3).
Table 3. Standard curve for Methyl Paraben.
| (According to Area) | |||
|---|---|---|---|
| Track | Retention time | Amount (μg) | Area |
| 1 | 4.884 | 0.20 | 1181329 |
| 2 | 4.865 | 0.4 | 2295050 |
| 3 | 4.850 | 1 | 5617441 |
| 4 | 4.839 | 2 | 11072487 |
| 5 | 4.828 | 4 | 22750992 |
Estimation of Neolone 950 in Sample
Weigh accurately 2.00 g sample in a 25 ml volumetric flask and add 20 ml methanol/0.4% acetic acid (20:80). Treat the sample by ultrasound for 10 min. Fill the measuring flask up to the mark and shake well. Filter the sample using a membrane filter (4.5). 10 ml above solution and condensed it 5 times, (2 ml). Apply the above solution in on TLC Aluminium sheet Silica gel 60 F 254 plate (10/10 cm) by using Camag Linomet 5 applicator (syringe size 100 μl). Concentration of sample is 400 ug/μl. After drying the TLC plate put into the mobile phase and run time is 10 min. Qualitative and quantitative estimation is done by observing Retention factor, peak height, pick area measured by scanning the plate using camag TLC scanner 4 at 275 nm.
Estimation of Methyl Paraben in O/W Skin Cream
Methyl paraben in different concentration are scanned by HPLC detector diode array (190-950 nm) in reversed phase analytical column. λmax is of methyl paraben was determined in 190 nm. The standard retention time of methyl paraben is 4.8 shown in Figure 1.
Figure 1: HPLC chromatogram of Standard Methyl Paraben.
Methylparaben is eluting out in this time. In different concentration methyl paraben shown in Table 4 having same identical retention time. To determine the amount of an ingredient, present in the formulation at different concentration standard curve were calibrated shown in Figures 2-5.
Figure 2: HPLC Standard curve of Methyl Paraben (According to Area). Regression via Area y = 54,93,379.8469 x + 97,534.8878.
Figure 3: HPLC chromatogram of sample from batch 1. It shows the HPLC spectrum of sample (o/w skin cream). Where the identifying peak for methyl Paraben is come on retention time of 4.813.
Figure 4: HPLC chromatogram of sample from batch 2. It shows the HPLC spectrum of sample (o/w skin cream). Where the identifying peak for methyl Paraben is come on retention time of 4.768.
Figure 5: HPLC chromatogram of sample from batch 3. It shows the HPLC spectrum of sample (o/w skin cream) where the identifying peak for methyl Paraben is come on retention time of 4.741.
Table 4. Estimation of percentage of Methyl Paraben in sample (According to area). The percentage of methyl paraben present in 3 different batch are 0.225, 0.247 and 0.220, when calculated according to area.
| Track | Batch | volume applied (µl) | Amount applied (μg) | Area | Ret time | Amount of Methyl Paraben (μg) | % of Methyl Paraben |
|---|---|---|---|---|---|---|---|
| 1 | 1 | 20 | 400 | 5053259 | 4.813 | 0.9021 | 0.225 |
| 2 | 2 | 20 | 400 | 5533220 | 4.761 | 0.9894 | 0.247 |
| 3 | 3 | 20 | 400 | 4940002 | 4.741 | 0.8815 | 0.220 |
Table 4. Estimation of percentage of Methyl Paraben in sample (According to area). The percentage of methyl paraben present in 3 different batch are 0.225, 0.247 and 0.220, when calculated according to area.
According to formulation 0.2% of Methyl Paraben is given in this o/w base cream. We analysed the percentage of Methyl Paraben in 3 batch of the cream. And the calculated amount of Methyl Paraben from HPLC analysis is shown in Table 5.
Table 5. Percentage of Methyl Paraben in Different Batch.
| Batch No. | % of Methyl Paraben (according to Area) |
|---|---|
| 1 | 0.225 |
| 2 | 0.247 |
| 3 | 0.220 |
Table 4. Estimation of percentage of Methyl Paraben in sample (According to area). The percentage of methyl paraben present in 3 different batch are 0.225, 0.247 and 0.220, when calculated according to area.
The standard deviation between three batches is 0.0079 and average % of methyl paraben in three batches is 0.225, 0.247 and 0.220. From the above experiment percentage of Methyl Paraben in 3 batches is come in between 0.20-0.24.
Quantitative Estimation of Propyl Paraben in O/W Skin Cream (by HPLC)
Propyl paraben in different concentration are scanned by HPLC detector diode array (190-950 nm) in reversed phase analytical column. λmax is of propyl paraben was determined in 190 nm. The standard retention time of propyl paraben is 8.6 shown in the Figure 6 and Table 6. Propyl paraben is eluting out in this time. In different concentration propyl paraben shown same retention time [13,14].
Figure 6: HPLC chromatogram for standard propyl paraben.
Table 6. Standard curve for Propyl paraben.
| (According to Area). | |||
|---|---|---|---|
| Track | Retention time | Amount (μg) | Area |
| 1 | 8.657 | 0.1 | 538446 |
| 2 | 8.649 | 0.2 | 1086943 |
| 3 | 8.643 | 0.4 | 2160759 |
| 4 | 8.625 | 1 | 5394568 |
| 5 | 8.608 | 2 | 10535781 |
| 6 | 8.594 | 4 | 21183637 |
To determine the amount of active ingredient, present in the formulation at different concentration standard curve were calibrated by HPLC shown in the Figures 7-16.
Figure 7: HPLC Standard curve of Propyl Paraben (According to Area). Regression via Area y = 52,83,010.9901x + 36,824.8960.
Figure 8: HPLC chromatogram of sample from batch 1. It shows the HPLC spectrum of sample (o/w skin cream) where the identifying peak for propyl Paraben is come on retention time of 8.159.
Figure 9: HPLC chromatogram of sample from batch 2. It shows the HPLC spectrum of sample (o/w skin cream) where the identifying peak for propyl Paraben is come on retention time of 8.091.
Figure 10: HPLC chromatogram of sample from batch 3. It shows the HPLC spectrum of sample (o/w skin cream) where the identifying peak for propyl Paraben is come on retention time of 8.078. Here the amount of propyl paraben in 3 different batch’s sample are calculated by the regression equation of pure propyl paraben. Retention time of propyl paraben in sample is 8.1.
Figure 11: HPLC Standard curve of Neolone 950 (According to Area).
Figure 12: HPLC Standard curve of Neolone 950 (According to height). Regression via Area Y=958.2+1417*X Regression via Height =Y=958.2+48.68*X.
Figure 13: TLC plate of Neolone 950 (HPTLC). In this figure plate shown the standard and sample point for Neolone 950, With same Rf value. It helps to identify the presence of neolone 950 in sample.
Figure 14: The chromatogram of standard Neolone 950.
Figure 15: The chromatogram of Neolone 950 in sample.
Figure 16: Spectrum scan of neolone 950. It shows the comparison between the standard and sample for spectrum matching of neolone 950 spectrum for sample and standard shows the super imposable image.
According to formulation 0.1% of Propyl paraben is given in this o/w base cream. We analysed the percentage of Propyl paraben in 3 batch of the cream. And the calculated amount of Propyl paraben from HPLC analysis are shown in Tables 7 & 8.
Table 7. Estimation of % of Propyl paraben in sample (According to area).
| Track | Batch | volume applied(µl) | Amount applied (μg) | Area | Ret time | Amount of Propyl paraben (μg) | % of Propyl paraben |
|---|---|---|---|---|---|---|---|
| 1 | 1 | 20 | 400 | 2086488 | 8.159 | 0.3942 | 0.098 |
| 3 | 2 | 20 | 400 | 2435782 | 8.091 | 0.4603 | 0.115 |
| 4 | 3 | 20 | 400 | 2001240 | 8.078 | 0.3781 | 0.094 |
Table 8. Percentage of Propyl paraben in different batch.
| Batch | % of Propyl paraben from Area |
|---|---|
| 1 | 0.098 |
| 2 | 0.115 |
| 3 | 0.094 |
Standard deviation between the 3 batches is 0.011. From the above experiment percentage of Propyl paraben in 3 batches is in between 0.097-0.011, and the standard curve for Neolone shown in Table 9.
Table 9. Standard curve for Neolone 950.
| (According to Area) | (According to Height) | ||||||
|---|---|---|---|---|---|---|---|
| Track | Rf | Amount (μg) | Area | Track | Rf | Amount (μg) | Height |
| 1 | 0.73 | 1 | 2462.25 | 1 | 0.73 | 1 | 97.04 |
| 2 | 0.72 | 2 | 3459.33 | 2 | 0.72 | 2 | 134.98 |
| 3 | 0.72 | 3 | 5614.21 | 3 | 0.72 | 3 | 207.11 |
| 4 | 0.72 | 4 | 6467.49 | 4 | 0.72 | 4 | 235.27 |
Quantitative Estimation of Neolone 950 in O/W Skin Cream (by HPTLC)
The retention factor (Rf) of sample and standard are estimated by spectrum scan, that is Rf of sample is 0.73 and Rf of standard is 0.72. It help to identify the presence of neolone 950.
Quantitative estimation of Neolone 950 in Sample
λmax is of Neolone 950 was determined from UV Visible spectrophotometer by scanning through the entire range (190-1100). The λmax was found to be 275 nm against the reagent blank. To determine the amount of an ingredient, present in the formulation at different concentration standard curve were calibrated. In this method area and height of the HPTLC spectrum curve was measured in different concentration [15].
The percentage of neolone in sample is measure by area and height of the HPTLC peak, which is estimated by spectrum scanning, was shown in Table 10.
Table 10. Estimation of % of Neolone 950 in sample (According to area). Average % of neolone in 1, 2 and 3 is respectively 0.11, 0.12 and 0.13, and their standard deviation are 0.014,0 and 0.014 according to area.
| Batch | volume applied (µl) | Amount applied (μg) | Area | Rf | Amount of Neolone 950 (μg) | % Neolone 950 | Std dev | Avg |
|---|---|---|---|---|---|---|---|---|
| 1 | 3 | 1200 | 3379.05 | 0.72 | 1.708 | 0.12 | ||
| 5 | 2000 | 4560.19 | 0.72 | 2.542 | 0.10 | 0.0141 | 0.11 | |
| 2 | 3 | 1200 | 3077.25 | 0.73 | 1.495 | 0.11 | ||
| 5 | 2000 | 4069.47 | 0.73 | 2.192 | 0.11 | 0 | 0.11 | |
| 3 | 3 | 1200 | 2990.79 | 0.74 | 1.344 | 0.14 | ||
| 5 | 2000 | 4200.23 | 0.72 | 2.287 | 0.12 | 0.014 | 0.13 |
According to formulation 0.1% of Neolone 950 is given in this o/w base cream. We analysed the % of Neolone 950 in 3 batch of the cream.
And the calculated amount of Neolone 950 from HPTLC analysis is shown in Tables 11 & 12.
Table 11. Estimation of % of Neolone 950 in sample (According to Height). Average % of neolone in 1, 2 and 3 is respectively 0.14, 0.13 and 0.13 and their standard deviation are 0.0141, 0.0070and 0. 012 according to height.
| Track | Batch | volume applied (µl) | Amount applied (μg) | Height | Rf | Amount of Neolone 950 (μg) | % of Neolone 950 | Std dev | Avg |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 29 | 3 | 1200 | 135.82 | 0.73 | 1.827 | 0.15 | ||
| 2 | 29 | 5 | 2000 | 177.94 | 0.73 | 2.692 | 0.13 | 0.0141 | 0.14 |
| 3 | 30 | 3 | 1200 | 130.98 | 0.74 | 1.727 | 0.14 | ||
| 4 | 30 | 5 | 2000 | 178.97 | 0.74 | 2.711 | 0.13 | 0.0070 | 0.13 |
| 5 | 31 | 3 | 1200 | 139.00 | 0.73 | 1.892 | 0.15 | ||
| 6 | 31 | 5 | 2000 | 172.99 | 0.72 | 2.594 | 0.12 | 0.0212 | 0.13 |
Table 12. Percentage of Neolone 950 in Each Batch. The average percentage of Neolone 950 in 3 batch are 0.12, 0.12 and 0.13. standard deviation within the batch are 0.012247, 0 and 0.01.standerd deviation between 3 batch is 0.005774 [16].
| Batch | Percentage of Neolone 950 according to Area | Percentage of Neolone 950 according to Height | Std dev | Average | Std dev |
|---|---|---|---|---|---|
| 1 | 0.11 | 0.14 | 0.012247 | 0.12 | 0.005774 |
| 2 | 0.11 | 0.13 | 0.01 | 0.12 | |
| 3 | 0.13 | 0.13 | 0 | 0.13 |
In conclusion, the proposed method allows a rapid and sound quantification of methylparaben, propayl paraben and neolone 950 in cosmetic samples, being based on a simple and rapid sample preparation procedure and a very fast and reliable chromatographic separation. The method can be used to monitor the occurrence at trace level of this preservative in cosmetic.
Conflict of Interest
The author does not have a commercial or other association that might pose a conflict of interest with any organization.
The Authors are thankful to the management authoritarians of Emami limited Kolkata for providing necessary facilities like chemical and instruments to carry out this study.