4.2 Materials and Methods .1 Reagents .1 Reagents
5.3.1 Quantitation of absorbers
In this work, six organic absorbers and two physical absorbers were quantified. Some of the products contained other absorbers but these were not quantified because standards were not readily available.
However, all were present in the HPLC chromatograms. Each organic UV filter was quantified at the wavelength of its maximum absorption and physical blockers, TiO2 and ZnO, were quantified at their preferred wavelengths of emission. The chromatogram in Fig. 5.2 shows a typical separation of the OCT, MBBT and BEMT standards and the corresponding UV spectra used in the identification. This chromatogram was obtained with a mobile phase of methanol-acetonitrile (90:10 % v/v). The calibration curves of the UV filters were linear in the investigated concentration ranges (R2 0.99).
The analytical parameters of representative calibration curves are summarised in Table 5.2. To validate this method the mean recovery of the UV filters from the spiked samples was calculated and results are shown in Table 5.3. To check on instrument signal stability during the period of analysis an intra- and inter-day analysis of authentic standards was done. A high level of precision was realized ≥ 99 % for the intra- and inter-day analysis (Table 5.4). The statistical limit of detection (LOD) is defined as the analyte concentration that gives a signal equal to yb + 3.3 Sb, where yb is the signal of the blank and Sb is its standard deviation. Similarly, the limit of quantitation (LOQ) is given as yb + 10 Sb. However, for the un-weighted least-squares method it is recommended in practice to use the standard deviation of the slope (Sy/x) (Miller and Miller 1984) instead of Sb
Thus
= . ⁄ ⁄
and
= ⁄ ⁄
where b is the slope of the regression line.
The amounts of the UV filters found in this work were all within the allowed maxima set by COLIPA and FDA. However, some samples had much lower amounts than anticipated. For instance in B1,
100
B2, B3 and B4 the amount of BMDBM was below the limit of quantitation and so the respective amounts are indicated as zero (Table 5.5). The amount of BP3 was in the range of 2.69 – 5.37 % (m/m), EHMC ranged from 0.28 – 3.62 % (m/m), OCT ranged from 0.68 – 4.02 % m/m, MBBT ranged from 2.65 – 7.93 % (m/m) and BEMT ranged from 3.79 – 6.07 % (m/m). The sunscreen products; P1 – P3 containing plant extracts had much lower concentration of the UV filters, EHMC and BMDBM. The levels of BEMT and MBBT for those products that had them were sufficiently comparable to the allowed amounts by COLIPA and FDA. The amount of TiO2 in these samples ranged from 0.72 – 12.60 % (m/m) an average slightly higher than those found in skin-lightening preparations (see Chapter Six). The quantitation of ZnO in other sunscreens products served as a control because the percentage composition was indicated on the packs unlike those that contained TiO2. The experimental values were very close to the packet label an indication that the packet labels represented the actual concentration of ZnO (Table 6). The levels of organic absorbers in the samples containing ZnO had previously been reported by Lyambila (2003) and therefore not included in this work, however, levels of ZnO were not done and in this work they ranged from 5.03 – 8.61 % (m/m).
Figure 5.10: A typical HPLC chromatogram of OCT at retention time 2.309 minute, MBBT at 11.431 minute, and BEMT at 13.186 minutes on a reversed-phase C-12 Phenomenox synergi column (150 mm × 4.6 mm). The injection volume was 20 µL at a flow rate of 1 mL min-1 in isocratic elution mode of MeOH-ACN (90:10 v/v). The chromatogram was monitored at 304 nm.
OCT MBBT BEMT
Table 5.7: Summary of analytical parameters for representative calibration curves of the UV filters in the sunscreenproducts.
UV filter Retention time/min
Standards
range/µM Slope R2 LOD/
µM
LOQ/
µM
BP3 a 3.73 - 3.83 3.12 - 4 99 1.37 x 1011 0.987 0.1001 0.33
OCT a 8.88 - 8.90 5.90 - 189 1.29 x 1011 9881 0.14 0.470
BMDBM a 9.77 – 10.37 4.37 - 273 3.31 x 1011 0.9998 0.0132 0.044 EHMC a 11.00 - 11.40 4.86 - 778 2.15 x 1011 0.9939 0.076 0.253 MBBT b 11.45 - 11.52 6.11- 196 2.64 x 1010 0.9962 0.066 0.220 BEMT b 13.19 - 13.56 2.45 - 157 4.17 x 1010 0.9784 0.191 0.637 TiO2
c - 0.48 - 2.5 d 4.49 x 104 0.9997 0.0187 0.0623
ZnO c - 10.0 - 100.0 e 1.15 x 105 0.9999 0.0077 0.0256
a Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase: methanol–water (84:16 % v/v).
b Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase: methanol–acetonitrile (90:10 % v/v).
c Determined on a Perkin Elmer Optima 5300 DV ICP-OES in radial view mode monitored at 337.279 nm for TiO2 and 206 nm for ZnO.
d the units are mol dm-3, and e mg dm-3.
The LOD is calculated as 3Sy/xb-1 (Sy/x is the standard error of the slope and b is the slope of the calibration line) and LOQ is given as 3.33(LOD).
Table 5.8: Validation studies of the eight UV absorbers added to the test formulation.
UV absorber Spiked mass/g Recovery/% (m/m)
BMDBM 0.095 ± 0.021 100.5 ± 1.32
BP3 1.007 ± 0.147 99.7 ± 0.59
EHMC 0.0428 ± 0.0125 99.8 ± 1.01
OCT 0.035 ± 0.009 101.5 ± 4.39
MBBT 0.041 ± 0.011 99.5 ± 1.93
BEMT 0.020 ± 0.002 98.3 ± 2.48
TiO2 0.021 ± 0.005 98.8 ± 0.46
ZnO 0.032 ± 0.0041 99.5 ± 0.99
(n = 5) Each determination is mean ± SD.
102
Table 5.9: A summary of intra- and inter-day instrumental response analysis.
Sunscreen
absorber Conc./µM Intra-day (n = 5) Inter-day (n = 5)
Found/µM RSD/% Accuracy/% Found/µM RSD/% Accuracy/%
BMBDMa 30 30.23 ± 0.95 3.14 100.8 29.57 ± 1.01 3.42 98.6
BP3a 60 59.87 ± 0.33 0.55 99.8 60.1 ± 0.56 0.93 100.2
EHMCa 50 50.12 ± 0.05 0.10 100.2 49.68 ± 0.96 1.93 99.4
BEMTb 20 19.99 ± 0.11 0.55 100.0 20.02 ± 0.12 1.00 100.1
MBBTb 40 39.98 ± 0.35 0.88 100.0 40.12 ± 0.85 0.60 100.3
OCTa 70 70.55 ± 0.46 0.65 100.8 69.85 ± 1.04 1.49 99.8
TiO2 2 d 2.01 ± 0.02 0.10 100.5 2.01 ± 0.02 0.10 100.5
ZnO 80 d 80.80 ± 0.40 0.50 101.0 79.39 ± 0.06 0.08 99.2
Recovery/% = (mean of found concentration/theoretical amount) × 100 % and RSD/% = (SD/mean concentration) × 100 %, n is the sample population, SD is standard deviation, and RSD is the relative standard deviation.
a Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase, methanol–water (84:16 v/v).
b Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase, methanol–
acetonitrile (90:10 v/v).
c Determined on Perkin Elmer Optima 5300 DV ICP-OES in a radial view mode monitored at 337.279 nm for TiO2 and 206 nm for ZnO.
d the units are mg dm-3.
Table 5.10: The percentage levels of sunscreen agents in commercial sunscreen products containing plant extracts determined by HPLC.
SAMPLE BMDBMa/%
(m/m)
BP3a/%
(m/m)
EHMCa/%
(m/m)
OCTa/%
(m/m)
MBBTb/%
(m/m)
BEMTb/%
(m/m)
TiO2c
/% (m/m)
XD 2.33 ± 0.21 4.25 ± 0.32 3.11 ± 0.45 - - - -
PB 3.05 ± 0.65 2.69 ± 0.12 3.62 ± 0.92 4.02 ± 0.46 - 3.85 ± 0.63 -
B1 0 - 3.02 ± 0.08 - 2.65 ± 0.19 - 7.70 ± 0.09
B2 0 - 0 - 7.93 ± 1.90 - 8.81 ± 0.03
B3 0 - - - 2.81 ± 0.64 - -
B4 0 - 0 - 3.15 ± 1.23 - 12.60 ± 0.21
P1 0.59 ±0.002 - 0.28 ± 0.0003 0.68 ± 0.002 - 5.62 ± 0.01 0.70 ± 0.01
P2 0.124 ± 0.004 5.37 ± 0.29 0.41 ± 0.0003 3.61 ± 0.19 - - 7.43 ± 0.02
P3 0.26 ± 0.0004 5.01 ± 0.15 0.33 ± 0.007 2.65 ± 0.03 - 3.79 ± 0.04 1.50 ± 0.01
P4 2.49 ± 0.03 4.41 ± 0.15 - 2.75 ± 0.02 - 6.07 ± 0.11 0
P6 2.38 ± 0.81 - - 2.47 ± 0.05 - - 0.72 ± 0.04
(n = 5) Each measurement is average value ± SD.
a Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase: methanol–water (84:16 % v/v).
b Chromatographic conditions: A reversed-phase C-12 Phenomenex Synergi 4µ Max-RP 80 Å column (150 mm × 4.6 mm); mobile phase: methanol–
acetonitrile (90:10 % v/v).
c Determined on a Perkin Elmer 5300 DV ICP-OES in a radial view mode monitored at 337.279 nm for TiO2.
The amounts of sunscreen absorber detected but below the quantitation limit in this method are indicated as zero (0) and those without particular sunscreen absorber are indicated -.
104
Table 5.11: The percentage composition of zinc oxide in some selected sunscreen products in the market.
Sample ID Product
name SPF Zinc oxide/% (m/m) Packet labelled Zinc oxide % (m/m)
SAU4 Banana
boat ultra 30+ 8.33 ± 0.63 8
SAU5
Banana boat sensitive skin
30+ 8.74 ± 0.41 8
SAU6 Banana
boat faces 30+ 8.40 ± 0.39 8
SAU8 Triplegard 7.09 ± 0.17 7
SAU12 Solar block 30+ 6.97 ± 0.08 6
SAU14 Banana
boat kids 30+ 8.61 ± 0.64 8
SAU16
UV triplegard kids lotion
30+ 7.19 ± 0.95 6.06
SAU17
UV triplegard kids
30+ 6.93 ± 0.10 7.07
SAU19
The cancer council of Australia - children
30+ 6.17 ± 0.55 7
SAU21 Triplegard
sun stick
30+ 4.84 ± 0.54 5
SAU24 Triplegard
lip balm 30+ 5.03 ± 0.27 5
(n = 5) Each determination is mean ± SD.
The amounts of organic absorbers in these products and their photostabilities had previously been reported by Lyambila (2003).