Laboratory procedures

3.6.1 Preparation of mercury standards

Mercury standard stock (1000 mgL-1)solution was used to prepare serial standard solutions; 10 ppm, 20 ppm, 40 ppm, 60 ppm, 80 ppm and 100 ppm. Standard solutions of stannous chloride solution was prepared by dissolving 20 g of the stannous chloride (SnCl2.2H20) salt in 40 mL of

1 M hydrochloric acid and then the solution diluted to 200 mL total volume. This solution was used as a reducing agent.

35 3.6.2 Preparation of other standards

Magnesium ascorbyl phosphate (20.0 mg/mL) was prepared by dissolving 2 g of magnesium ascorbyl phosphate in 100 millilitre of water, ascorbyl glucoside (10.0 mg/mL) by dissolving 0.5 g of ascorbyl glucoside in 50 millilitre of water, arbutin (10.0 mg/mL) 1 g in 100 millilitre of water, hydroquinone (5.0 mg/mL) 0.5 g of hydroquinone in 100 millilitre of water, and kojic acid (2.0 mg/mL) dissolving 0.2 gin 100 millilitre of water, All these were prepared as the standard stock solutions. The stock solutions were diluted using de-ionized water to prepare a series of standard solutions; magnesium ascorbyl phosphate (200, 400, 600, 800 and 1000), ascorbyl glucoside (100, 200, 300, 400, and 500), kojic acid (20, 40, 60, 80 and 100), arbutin (100, 200, 300, 400 and 500) while series standard solutions of hydroquinone were (50,100, 150, 200 and 250 (Shou-chieh et al., 2004). Internal standard stock solution of pyridoxine with a concentration of 1.0 mg/mL was prepared by dissolving 1 g of pyridoxine solid in 1000 millilitre of water.

3.6.3 Method validation

The accuracy of CV-AAS was investigated by using the spiking method. Spiking is an addition method where the standard is directly added to the aliquots of the sample to be analyzed. This method is used in situations where sample matrix also contributes to the analytical signal (matrix effect) thus making it impossible to compare the analytical signal between sample and standard in the calibration curve approach. In this study samples were spiked with a known amount of standards. Table 1 shows the concentration of unspiked samples, the known concentration of the standard added to each sample and the concentration of the spiked samples. Results were used to calculate the percentage recovery (Equation 3.1).

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R%= (Cf – Cu /Ca) 100...Eq 3.1

Where:

R- % recovery

Cf- Concentration of the sample after spiking

Cu- Concentration of the sample before spiking

Ca-Concentration of standard used for spiking

Table 3:1 Concentration of spiked, unspiked and standards added

Skin lightener Concentration of unspiked sample Mean ± SE(ppm) Concentration of Standard added to sample(ppm) Concentration of spiked sample. Mean±SE.(ppm) MAP Ag 1.65±0.01 0.95±0.01 300 150 301.000± 0.01 149.112±0.02 HQ 0.51±0.00 75 75.100±0.01 KA 0.96±0.05 30 31.202±0.01 AR 1.33±0.01 150 148.122±0.02 Hg 47.87±0.01 10 58.099±0.00

Recovered amount after digestion of the spiked samples was used to calculate percentage recovery (Borosova et al, 2002). The mean recovery of the matrix was evaluated at 95% confidence level (Miller and Miller, 1988). Limit of detection (LOD) was calculated using equation (3.2) (EURACHEM guide,1998) using the determined absorbance values for 10 replicates of the blank solution, then transformed into concentration values in order to be compared with the data obtained from the calibration curve.

LOD x blank + 3sblank...(3.2)

x blank –mean absorbance obtained with the blank solutions: sblank-standard deviations of the

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High performance liquid chromatography (HPLC) was validated using calibration and precision. Calibration is a general method for determining the concentration of a substance in an unknown

sample by comparing the unknown to a set of standards samples of known concentration. Precision is the degree to which repeated measurement under unchanged conditions show same results. In calibration, five different concentrations of standard solutions were prepared from the stock solutions and 40 μg/mL of an internal standard was added and analyzed, respectively. Linear regression equations and correlation coefficients were obtained from the plots of concentration versus peak area ratio of standard to internal standard solutions. In precision, the standard stock solution and the internal standard stock solution were quantified precisely and diluted with distilled water to three different concentrations in µg/ML within the standard calibration range, and then 40 μg/mL of internal standard was added to each standard solution. The samples were analyzed in triplicates by HPLC. The standard deviation and relative standard deviation were then calculated.

3.6.4 Sample preparation

For analysis of mercury, 1.000 g of each cream and each soap was weighed accurately into a conical flask. A 20 mL acid mixture of concentrated nitric and hydrochloric acid in the ratio 3:1 was added to the sample. The conical flask was covered and mixture heated at 200 oC until there were no more brown fumes produced. The solution was cooled, filtered through Whatman paper (Number 1) into a 50 mL volumetric flask and then made up to the mark using distilled water and used for analysis.

For analysis hydroquine, arbutin, kojic acid, magnesium ascorbyl phosphate and ascorbyl glucoside, 1.000 g of each cream and each soap was weighed precisely, mixed with an

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appropriate amount of the internal standard stock solution and diluted with twenty-fold of 0.05 M KH2PO4 buffer (pH 2.5). A homogeneous suspension was obtained after 30 min of

sonification. The suspension was filtered and the filtrate further diluted with 0.05 M KH2PO4

buffer (pH 2.5) until the final concentrations of the whitening ingredients was within the standard calibration range and the internal standard 40 μg/mL before HPLC analysis. The ratio of the peak area of the sample to the internal standard was compared to determine the concentration of each sample.

The mean levels of skin lighteners analysed by HPLC, were transformed from mg/g into percentage since their results are always recorded in percentage (Shou-chieh et al., 2002). The transformation was done using the following formula Xmg/1000mg×100. This was done for ascobyl glucoside, arbutin, magnesium ascorbyl phosphate, hydroquinone and kojic acid in skin lightening soaps. To determine the final concentration of mercury in the samples, the concentration values from the calibration curve in appendix 6 was multiplied by the dilution factor which was 50.