PKKK/300/UP/054 Identification and Quantitation of Methyl, Ethyl, Propyl and Butyl 4-Hydroxybenzoate in Cosmetic Products by HPLC

All reagents must be of analytical quality or HPLC grade.

Water used must be Ultrapure water, 18 mega ohm

Methanol (HPLC) Grade

Tetrahydrofuran

Acetonitrile

Ethanol

Sulfuric acid 2M

Mobile phase - mixture of Tetrahydrofuran : Water : Methanol : Acetonitrile 1:12:2:5 (v:v:v:v)

Diluent - Ethanol : Water (9:1) (v:v)

Reference material:

Methyl 4-hydroxybenzoate primary/secondary reference standard

Ethyl 4-hydroxybenzoate primary/secondary reference standard

Propyl 4-hydroxybenzoate primary/secondary reference standard

Butyl 4-hydroxybenzoate primary/secondary reference standard

Isopropyl 4-hydroxybenzoate primary/secondary reference standard

High Pressure Liquid Chromatography

Erlenmeyer flask

HPLC column: Cosmosil C18 250 mm x 4.6 mm (5 µm) or equivalent

UV Detector, with detection capability at 280 nm

Column Oven temperature : 25oC

Flow rate : 1.5 mL/min

UV detector wavelength : 280 nm

Injection volume : 20 µL

Instrument run time : 30 min

Internal standard solution: Isopropyl 4-hydroxybenzoate (500 µg/mL)

Accurately weigh 50 mg Isopropyl 4-hydroxybenzoate into 100 mL volumetric flask. Dissolve it and make up to volume with diluent.

Standard solution for identification: Methyl 4-hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, Butyl 4-hydroxybenzoate and Isopropyl 4-hydroxybenzoate (100 µg/mL)

Accurately weigh 2 mg of Methyl 4-hydroxybenzoate into 20 mL volumetric flask. Dissolve in 10 mL of diluent by shaking it, and if necessary sonicate for 1-2 minutes. Make up to volume with diluent. Repeat the same process for Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate, Butyl 4-hydroxybenzoate and Isopropyl 4-hydroxybenzoate in separate volumetric flasks.

Accurately weigh 12.5 mg of Methyl 4-hydroxybenzoate, Ethyl 4-hydroxybenzoate, Propyl 4-hydroxybenzoate and Butyl 4-hydroxybenzoate into 25 mL volumetric flask. Dissolve in 10 mL of diluent by shaking it, and if necessary sonicate for 1-2 minutes. Make up to volume with diluent.

Pipette 4 mL of standard stock solution into a 10 mL volumetric flask. Add 2 mL of internal standard solution into the volumetric flask. Add 0.2 mL of sulfuric acid 2M and shake to homogenize it. Add diluent to volume. Filter through a 0.45 µm filter into auto sampler vial.

Internal Quality Control (IQC)

LOD solution prepared in 6.3.4 will be injected for every 5 samples in a single run. The sequence for IQC is as follows:

Calculate the %RSD for retention time of LOD solutions injected

% RSD retention time for LOD solution injection should not be more than 2.0%

6.3.5.3 System Suitability Test (SST) criteria :

Asymmetric factor/Tailing factor in the range of 0.9 to 1.5

Resolution NLT 0.9

Requirements for standard and sample solutions

Resolution NLT 0.9

Asymmetric factor/Tailing factor in the range of 0.9 to 1.5

A steady baseline

Note: Mobile phase composition can be adjusted so that the requirements above can be met.

Results Interpretation

The identification test for preservatives are positive if the retention time is similar to that obtained from the standard solution and the peak area of sample is more than the peak area of LOD solution.

If preservatives peak is detected in the sample, the actual percentage (%w/w) of the preservatives in the sample will be determined according to the procedure below.

Test Method (Quantitation)

The reagents and HPLC condition is the same as in the procedure for identification of methyl, ethyl, propyl and butyl 4-Hydroxybenzoate.

Standard solution for calibration curve:

Pipette separately 0.2 mL, 0.4 mL, 1 mL, 2 mL and 4 mL of mix standard stock solution into a 10 mL volumetric flask. Add 2 mL of internal standard solution into each volumetric flask. Add 0.2 mL of Sulfuric acid 2M and shake to homogenize it. Add diluent to volume. Filter through a 0.45 µm filter into auto sampler vial.

These solutions must be freshly prepared.

Sample solution preparation

Note: The amount of sample used can be adjusted depending on the availability and condition of sample received. Sample solution prepared for identification test can be used.

System suitability test

Acceptance criteria :

The asymmetric factor/tailing factor in the range of 0.9 to 1.5, resolution NLT 0.9 and the value of RSD should not be more than 2%.

Calibration curve of each preservative were performed (concentration ratio vs. peak area ratio). Check the coefficient correlation of the graph. The R2 should be >0.995. Preservatives concentration in the sample is determined from the calibration curve.

Sequence of injection is shown in the table below:

Requirements for standard and sample solutions

Resolution NLT 0.9

Tailing factor in the range of 0.9 to 1.5

A steady baseline

Note: Mobile phase composition can be adjusted so that the requirements above can be met.

Results Interpretation

Five different peak area ratio from chromatogram of standard solutions representing five different concentration levels of standard solutions. The peak area ratio of the sample solution obtained from the standard calibration curve is directly proportional to the concentration of preservatives in the sample.

Note: The concentration of sample solution obtained from calibration curve is the concentration ratio of sample to concentration of internal standard.

The peak area ratio of sample solutions must fall within the standard calibration range. Therefore, a dilution process shall be carried out if the peak area ratio of sample solutions is more than the peak area ratio of the highest standard solution concentration.

Determine the amount of preservatives in the portion of sample taken by using the following formula. The amount of preservatives present in the sample will be reported as percentage of weight per sample weight (%w/w)

Notes :

Specification for Preservatives content: