A novel, simple, validated stability indicating HPLC method was developed for determination of Koptrizon and Tinosorb S. Stability indicating power of the method was established by forced degradation study. The chromatographic separation was achieved with Waters X Bridge
Frequent exposure to UV radiation causes pronounced harmful effects on human health. UV radiation-induced effects are manifested as acute responses, namely, sunburn, hyperplasia, and immunosuppressant, and as chronic responses, primarily photo carcinogenesis and photo ageing [
The molecules employed in cosmetic products to protect skin from the sun are classified as physical and chemical sunscreens. Physical sunscreens are represented mainly by zinc and titanium oxides which interrupt the path of UV light by scattering or reflection. Chemical sunscreens are generally aromatic compounds conjugated with an electron-donating group in “ortho” or “para” position and an electron acceptor group. This chemical structure favours electron delocalization and therefore helps excitation of molecules from ground state to an excited state. The energy required for this transition corresponds to the energies of ultraviolet A (UVA) and Ultraviolet B (UVB) radiations [
Chronic exposure to UVB (280–320 nm) wavelengths induces damage to human skin, such as burns and erythema, but increases evidence of demonstrates that UVA radiation (320–400 nm) contributes to photo aging, which results in the accumulation of massive amounts of abnormal elastic material in the dermis of photo-aged skin and modification in collagen structure [
The use of sunscreen products is a widely accepted way of primary prevention against the harmful effects of solar radiation. To limit sun exposure, one is advised to wear loose fitting, tightly woven clothing, to stay in the shade between 11 a.m. and 3 p.m., and to use a sunscreen with a sun protection factor (SPF) of 15 or higher liberally reapplying every 2 h or after working, swimming, playing, or exercising outdoors [
The necessity to provide high SPF and screening efficiency against both UVA and UVB wavelengths has led to the development of sunscreen formulations with multiple added sunscreen chemicals [
The literature survey reveals that several techniques have been reported such as derivative spectrophotometry [
Koptrizon (KPT) or Ethylhexyl triazone chemically known as 4-[[4,6-bis[[4-(2-ethylhexoxy-oxomethyl)phenyl]amino]-1,3,5-triazin-2-yl]amino] benzoic acid 2-ethylhexyl ester (Figure
Chemical structure of (a) Koptrizon and (b) Tinosorb S.
Tinosorb S (TIN) or Bemotrizinol chemically known as 2,2′-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl] bis
There is not any method reported to estimate sunscreen agents KPT and TIN simultaneously in sunscreen formulations. It is essential to have a good analysis method to provide assurance of quality and effectiveness of the products. The purpose of this research work is to develop simple, accurate, and stability indicating method for simultaneous determination of Koptrizon and Tinosorb S in sunscreen formulations by HPLC. Design of experiment (DOE) technique was employed to study the effect of critical factors on the method performance.
The drug product stability guideline Q1A (R2) issued by the International Conference on Harmonization (ICH) [
Sunscreen topical formulation and placebo were provided by Dr. Reddy’s Lab., India. Koptrizon (Potency 99.7%) and Tinosorb S (Potency 99.1%) working standards were provided by BASF, India. HPLC grade acetonitrile and tetrahydrofuran were purchased from Rankem, India. 0.2
HPLC (Allaince Waters, with Empower 2 software), photo stability chamber (Sanyo, Leicestershire, UK), dry air oven (Cintex, Mumbai, India), XS205 dual range balance (Mettler Toledo), and Cintex digital water bath were used for specificity studies. All chromatographic experiments were performed in the isocratic mode. Separation was achieved on Waters X Bridge C18 (50 × 4.6
The stress degraded samples and the solution stability samples were analyzed using a PDA detector covering the range of 200–400 nm.
An accurately weighed 15 mg each of KPT and TIN working standards was taken into 100 mL volumetric flasks. About 70 mL of diluent was added to this and sonicated in an ultrasonic bath to dissolve. We made up the volume with diluent, mixed well.
An accurately weighed sample equivalent to 15 mg of KPT and TIN was taken into 100 mL volumetric flask. About 70 mL of diluent was added to this and sonicated in an ultrasonic bath for 15 min with intermittent shaking. We made up the volume with diluent, mixed well. We filtered a portion of solution through 0.2
Prime objective of an RP-HPLC method development for determination of KPT and TIN in topical dosage form was that the method should be able to determine assay of drug in single run and should be accurate, reproducible, robust, and stability indicating. All degradation products from stress conditions should be well separated from each other. Method should be simple so that it can be useful in analytical research and quality control laboratory for routine use. Furthermore, primary developed method was challenged by forced degradation as a prevalidation.
Column selection and mobile phase selection were done simultaneously. A method development was started with Waters Symmetry C18 50 × 3.9 mm, 5
A typical chromatogram of (a) placebo (b) standard (c) sample (d) base degrdation sample and (e) peroxide degradation sample.
After satisfactory development of method, it was subjected to method validation as per ICH guideline [
System suitability parameters were measured so as to verify the system, method, and column performance. The % RSD (relative standard deviation) of KOP and TIN was calculated from peak area count of five replicate injections (standard preparation) to be below 0.20%. Low values of % RSD of replicate injections indicate that the system is precise. Results of other system suitability parameters such as theoretical plates tailing factor are presented in Table
System suitability results (precision, intermediate precision, and robustness).
Parameter | Theoretical plates for KPT > 2000 | Tailing factor for KPT ≤ 2.0 | % RSD* of standard Area (KPT) ≤ 2.0 | Theoretical plates for TIN > 3000 | Tailing factor for TIN ≤ 2.0 | % RSD* of standard Area (TIN) ≤ 2.0 |
---|---|---|---|---|---|---|
Precision | 3512 | 1.0 | 0.3 | 4363 | 0.9 | 0.3 |
Intermediate precision | 3912 | 1.0 | 0.2 | 4578 | 1.0 | 0.4 |
At 1.7 mL min−1 flow rate | 3163 | 1.0 | 0.3 | 3917 | 0.9 | 0.4 |
At 1.3 mL min−1 flow rate | 3802 | 1.0 | 0.2 | 4789 | 0.9 | 0.2 |
At 55°C column temp. | 3128 | 1.0 | 0.3 | 3938 | 0.9 | 0.1 |
At 45°C column temp. | 3757 | 1.0 | 0.3 | 4594 | 0.9 | 0.3 |
Mobile phase THF +5% | 3117 | 1.0 | 0.4 | 4070 | 0.9 | 0.5 |
Mobile phase THF −5% | 3701 | 1.0 | 0.1 | 4416 | 0.9 | 0.3 |
*Determined on five values. THF: tetrahydrofuran.
Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities [
Data of forced degradation study for Koptrizon and Tinosorb S.
Stress conditions | Koptrizon | Tinosorb S | ||||||
---|---|---|---|---|---|---|---|---|
PA | PTH | % Deg. | Purity flag | PA | PTH | % Deg. | Purity flag | |
Acidic hydrolysis | 0.088 | 0.326 | 2.5 | No | 0.097 | 0.302 | 1.4 | No |
(5(N) HCl at 70°C, 5 hrs) | ||||||||
Alkaline hydrolysis | 0.145 | 0.381 | 43.7 | No | 0.130 | 0.367 | 39.6 | No |
(5(N) NaOH at 70°C, 5 hrs) | ||||||||
Oxidation | 0.076 | 0.311 | 14.5 | No | 0.094 | 0.299 | 9.2 | No |
(30% H2O2 at 70°C, 2 hrs) | ||||||||
Thermal exposed | 0.071 | 0.306 | ND | No | 0.091 | 0.307 | ND | No |
(At 105°C, 6 hrs) | ||||||||
Photolytic exposed | 0.089 | 0.341 | ND | No | 0.084 | 0.325 | ND | No |
(1.2 million lux hr visible |
PA: purity angle; PTH: purity threshold; Deg.: degradation; ND: no degradation.
The precision of the assay method was verified by repeatability and by intermediate precision. Precision was investigated using sample preparation procedure for six real lotion samples and analysing by proposed method. The average % assay values (
Method precision, intermediate precision result, LOD, LOQ evaluations, and linearity data for KPT and TIN.
Parameter | Koptrizon | Tinosorb S |
---|---|---|
Precision Day-1/repeatability ( |
101.3 ± 0.39; 0.38; 0.31 | 100.2 ± 0.51; 0.51; 0.41 |
(% Assay ± SD; % RSD; 95% C.L.) | ||
Intermediate precision/reproducibility ( |
100.2 ± 0.53; 0.53; 0.42 | 99.9 ± 0.29; 0.29; 0.24 |
(% Assay ± SD; % RSD; 95% C.L.) | ||
LOD ( |
0.024 | 0.048 |
LOQ ( |
0.08 | 0.16 |
Linearity range ( |
0.08–225.8 | 0.16–225.1 |
Correlation coefficient | 0.99999 | 0.99999 |
Intercept ( |
2103.15 | 2032.08 |
Slope ( |
27767.747 | 13163.737 |
Bias at 100% response | 0.050 | 0.102 |
95% C.L.: 95% confidence interval.
To confirm the accuracy of the proposed method, recovery experiments were carried out by standard addition technique. Three different levels (50%, 100%, and 150%) of standards were added to preanalysed placebo samples in triplicate. The percentage recoveries of KOP and TIN at each level and each replicate were determined. The mean of percentage recoveries (
Accuracy results for Koptrizon and Tinosorb S (
Active |
Amount added |
Amount recovered |
% Recovery ± SD; |
---|---|---|---|
74.9 | 75.4 | 100.7 ± 0.63; 0.62 | |
KPT | 149.8 | 153.0 | 102.1 ± 0.49; 0.48 |
224.7 | 220.4 | 98.1 ± 0.53; 0.54 | |
| |||
75.6 | 75.5 | 99.0 ± 0.00; 0.00 | |
TIN | 151.2 | 154.7 | 102.3 ± 1.56; 1.52 |
226.8 | 222.7 | 98.2 ± 0.33; 0.34 |
The LOD and LOQ were determined at a signal-to-noise ratio of 3 : 1 and 10 : 1, respectively, by injecting a series of dilute solutions with known concentrations. The limit of detection and limit of quantification values of KOP and TIN are reported in Table
Linearity was demonstrated from 50% to 150% of standard concentration using minimum five calibration levels (50%, 75%, 100%, 125%, and 150%) for the KOP and TIN compounds, which gave us a good confidence on analytical method with respect to linear range. The response was found linear for all KOP and TIN from 50% to 150% of standard concentration, and correlation coefficient was greater than 0.999. Bias was also found within ±0.5. The results of correlation coefficients,
As defined by the ICH, the robustness of an analytical procedure describes its capability to remain unaffected by small and deliberate variations in method parameters [
Design Expert Software (Stat-Ease Inc., Statistic made easy, Minneapolis, MN, USA, version 7.0.0) was used for the experimental design throughout this screening process study. The full factorial design requires fewer measurements than the classical one-at-a-time experiment to give the same precision. At the same time, it detects and estimates any interaction between the factors. In order to study the simultaneous variation of the factors on the considered responses, a multivariate approach using design of experiments is recommended in robustness testing. However, if an analytical method is fast and requires the testing of a few factors (three or less), a good choice for robustness testing may be design expert, widely employed because of its high efficiency with respect to lesser number of runs required in full factorial mode. In order to study the three variables at two levels, the design used in robustness testing of KOP and TIN retention times and theoretical plate number for TIN was a 23 full factorial design. ANOVA with linear model was applied to estimate the model coefficients and also check the robustness of the method. Three factors and two levels caused full factorial design 23, in addition to that two centre points resulted total of ten experimental points—which were carried out in random order. Effect of two factors in the resulting RT responses of KPT, TIN and plate count of KPT shown in Pareto chart (Figure
The range and levels of the variables in the 23 full factorial design.
Std. | Run | Factor 1 | Factor 2 | Factor 3 | Response 1 | Response 2 | Response 3 |
---|---|---|---|---|---|---|---|
A: flow rate | B: column temp | C: THF comp | RT of KPT | RT of TIN | Plate count of KPT | ||
(mL min−1) | (°C) | (%) | (min) | (min) | (min) | ||
0 | 2 | 1.5 | 50 | 38 | 2.8 | 4.3 | 3476 |
0 | 7 | 1.5 | 50 | 38 | 2.8 | 4.2 | 3346 |
7 | 1 | 1.3 | 55 | 43 | 2.2 | 3.2 | 2806 |
4 | 3 | 1.7 | 55 | 33 | 3.1 | 4.7 | 3200 |
2 | 4 | 1.7 | 45 | 33 | 3.9 | 6.0 | 4108 |
6 | 5 | 1.7 | 45 | 43 | 2.2 | 3.2 | 2767 |
8 | 6 | 1.7 | 55 | 43 | 1.8 | 2.6 | 2204 |
3 | 8 | 1.3 | 55 | 33 | 3.8 | 5.8 | 4114 |
5 | 9 | 1.3 | 45 | 43 | 2.8 | 4.1 | 3336 |
1 | 10 | 1.3 | 45 | 33 | 5.1 | 7.9 | 4804 |
0 | 2 | 1.5 | 50 | 38 | 2.8 | 4.3 | 3476 |
Pareto chart effect of factor (a) C > A > B in retention time of KPT, (b) C > B > A in retention time of TIN (c), and C > A >B in plate count of KPT.
Single factor interaction: (a) flow rate in RT of KPT; (b) column temperature in KPT; (c) THF composition in KPT; (d) flow rate in RT of TIN; (e) column temperature in RT of TIN; (f) THF composition in RT of TIN; (g) flow rate in USP plate count of KPT; (h) column temperature in USP plate count of KPT; (i) THF composition in USP plate count KPT.
The ANOVA statistical test was employed to determine the significant and most contributing factors where they were ranked on the basis of degree of
ANOVA for 23 full factorial design: response: RT of Koptrizon (min).
Source | Sum of squares | df | Mean square |
|
|
|
---|---|---|---|---|---|---|
Model | 0.073284126 | 3 | 0.024428 | 743.3259616 | <0.0001 | Significant |
A—flow | 0.008862903 | 1 | 0.008863 | 269.6911034 | <0.0001 | |
B—column temp. | 0.009766119 | 1 | 0.009766 | 297.1752743 | <0.0001 | |
C—THF comp. | 0.054655104 | 1 | 0.054655 | 1663.111507 | <0.0001 | |
Curvature |
|
1 |
|
2.68560136 | 0.1622 | Not significant |
Residual |
|
5 |
|
|||
Lack of fit | 0.000164316 | 4 |
|
|||
Pure error | 0 | 1 | 0 | |||
Cor total | 0.073536699 | 9 | ||||
Std. dev. | 0.006488107 |
|
0.996565345 | |||
Mean | 0.591672677 | Adjusted |
0.994848018 |
Values of prob >
ANOVA for 23 full factorial design: response: RT of Tinosorb S (min).
Source | Sum of squares | df | Mean square |
|
|
|
---|---|---|---|---|---|---|
Model | 0.057184881 | 3 | 0.019061627 | 630.2536737 | <0.0001 | Significant |
A—flow | 0.006317517 | 1 | 0.006317517 | 208.8823992 | <0.0001 | |
B—column temp. | 0.007126362 | 1 | 0.007126362 | 235.6260641 | <0.0001 | |
C—THF comp. |
|
1 | 0.043741001 | 1446.252558 | <0.0001 | |
Curvature |
|
1 |
|
0.055223918 | 0.8235 | Not significant |
Residual |
|
5 |
|
|||
Lack of fit |
|
4 |
|
2.071326494 | 0.4746 | Not significant |
Pure error |
|
1 |
|
|||
Cor total | 0.057337773 | 9 | ||||
Std. dev. | 0.005047971 |
|
0.997333484 | |||
Mean | 0.484279065 | Adjusted |
0.996000226 |
Values of prob >
ANOVA for 23 full factorial design: response: Koptrizon plate count.
Source | Sum of squares | df | Mean square |
|
|
|
---|---|---|---|---|---|---|
Model | 5139776.375 | 3 | 1713258.792 | 111.8731376 | <0.0001 | Significant |
A—flow | 966745.125 | 1 | 966745.125 | 63.12695487 | 0.0005 | |
B—column temp. | 905185.125 | 1 | 905185.125 | 59.1071825 | 0.0006 | |
C—THF comp. | 3267846.125 | 1 | 3267846.125 | 213.3852755 | <0.0001 | |
Curvature | 65.025 | 1 | 65.025 | 0.004246031 | 0.9506 | Not significant |
Residual | 76571.5 | 5 | 15314.3 | |||
Lack of fit | 68121.5 | 4 | 17030.375 | 2.015428994 | 0.4800 | Not significant |
Pure error | 8450 | 1 | 8450 | |||
Cor total | 5216412.9 | 9 | ||||
Std. dev. | 113.0166101 |
|
0.985308578 | |||
Mean | 3416.1 | Adjusted |
0.977962866 |
Values of prob >
Three-dimensional plot of the fully factorial for predicted response RT of KPT plotted on
DOE revolves around the concept of the design space, the multidimensional combination, and interaction of input variables and process parameters that have been demonstrated to provide assurance of quality. Working within the design space is not considered as a change. Movement out of the design space is considered to be a change and would normally initiate a regulatory postapproval change process. Design space is proposed by the applicant and is subject to regulatory assessment and approval [
Graphical plot of the fully factorial design space for (a) changing factors A (flow rate) and B (column temperature); fixed factor: C (% Tetrahydrofuran) (b) changing factors A (flow rate) and C (% Tetrahydrofuran); fixed factor: B (column temperature).
Stability of sample solution was established by its storage at ambient temperature for 24 hrs. The assays of KOP and TIN were analysed. It was found that % labelled amounts of KOP at 0, 12, and 24 hrs were 101.3, 100.7, and 101.6, respectively, and % labelled amounts of TIN were 100.2, 99.8, and 99.7, respectively.
Filter compatibility was performed for nylon 0.2
The experimental design describes the key HPLC method components including column temperature, mobile phase flow rate, and % tetrahydrofuran composition in mobile phase. The interrelationships are studied and the preliminary optimized conditions are obtained for each combination. Here a better understanding of the factors influencing chromatographic separation and greater confidence in the ability of the methods to meet their intended purposes is done. Moreover, this approach ensures better design of product. A gradient RP-HPLC method was successfully developed for the estimation of KOP and TIN in topical dosage form. The method validation results have proved that the method is selective, precise, accurate, linear, robust, filter compatible, and stability indicating. The drug is stable in thermal and photolytic conditions and degrades in acidic, basic, and oxidative conditions. The method robustness was demonstrated using experimental design techniques, taking into consideration the selectivity of the RP HPLC method. The short run time (6.0 min) enables rapid determination of drug. Moreover, it may be applied for determination of KOP and TIN in the study of content uniformity, tube homogeneity, and
The authors of the paper declare that there is no direct financial relation with the commercial identities mentioned in the paper that might lead to a conflict of interests.
The authors would like to thank M/s. Dr. Reddy’s Laboratories Ltd. for supporting this work. The authors’ Intellectual Property Management (IPM) department has given this paper internal publication no. PUB00228-13.