Nanoarchitectonics of Electrically Activable Phosphonium Self-Assembled Monolayers to Efficiently Kill and Tackle Bacterial Infections on Demand.

Prosthetic implants are widely used in dentistry and orthopedics and, as a result, infections can occur which cause their removal. Therefore, it is essential to propose methods of eradicating the bacteria that remain on the prosthesis during treatment. For this purpose, it is necessary to develop surfaces whose antibacterial activity can be controlled. Herein, we designed innovative and smart phosphonium self-assembled monolayer (SAM) interfaces that can be electrically activated on demand for controlling bacterial contaminations on solid surfaces. Upon electroactivation with a low potential (0.2 V for 60 min., conditions determined through a DOE), a successful stamping out of Gram-positive and Gram-negative bacterial strains was obtained with SAM-modified titanium surfaces, effectively killing 95% of Staphylococcus aureus and 90% Klebsiellapneumoniae. More importantly, no toxicity towards eukaryotic cells was observed which further enhances the biocompatible character of these novel surfaces for further implementation.

Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one-pot/two-step biocatalytic whole-cell system.

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 ).

Electromagnetic sunscreen model: design of experiments on particle specifications.

We report a numerical study on sunscreen design and optimization. Thanks to the combined use of electromagnetic modeling and design of experiments, we are able to screen the most relevant parameters of mineral filters and to optimize sunscreens. Several electromagnetic modeling methods are used depending on the type of particles, density of particles, etc. Both the sun protection factor (SPF) and the UVB/UVA ratio are considered. We show that the design of experiments' model should include interactions between materials and other parameters. We conclude that the material of the particles is a key parameter for the SPF and the UVB/UVA ratio. Among the materials considered, none is optimal for both. The SPF is also highly dependent on the size of the particles.

Optimization of capacitive acoustic resonant sensor using numerical simulation and design of experiment.

Optimization of the acoustic resonant sensor requires a clear understanding of how the output responses of the sensor are affected by the variation of different factors. During this work, output responses of a capacitive acoustic transducer, such as membrane displacement, quality factor, and capacitance variation, are considered to evaluate the sensor design. The six device parameters taken into consideration are membrane radius, backplate radius, cavity height, air gap, membrane tension, and membrane thickness. The effects of factors on the output responses of the transducer are investigated using an integrated methodology that combines numerical simulation and design of experiments (DOE). A series of numerical experiments are conducted to obtain output responses for different combinations of device parameters using finite element methods (FEM). Response surface method is used to identify the significant factors and to develop the empirical models for the output responses. Finally, these results are utilized to calculate the optimum device parameters using multi-criteria optimization with desirability function. Thereafter, the validating experiments are designed and deployed using the numerical simulation to crosscheck the responses.

Self-assembled liquid crystalline nanoparticles as an ophthalmic drug delivery system. Part I: influence of process parameters on their preparation studied by experimental design.

To develop self-assembled liquid crystalline nanoparticles as a drug delivery system for keratoconus treatment, a formulation containing riboflavin a water-soluble drug, two surfactants (poloxamer 407 and mono acyl glycerol - monoolein-) and water was optimized and prepared by emulsification and a homogenization process. A fractional factorial design was applied to estimate the main effects and interaction effects of five parameters on two responses, namely particle size and encapsulation efficiency. The five parameters are the temperature of the two phases, the duration of emulsification, the presence of heating during homogenization, the number of passes and pressure. The most influent parameters are the presence of heating during the homogenization and the pressure that led to the production of nanoparticles with an average size of 145 nm and an average encapsulation efficiency of 46%.

Self-assembled liquid crystalline nanoparticles as an ophthalmic drug delivery system. Part II: optimization of formulation variables using experimental design.

In the field of keratoconus treatment, a lipid-based liquid crystal nanoparticles system has been developed to improve the preocular retention and ocular bioavailability of riboflavin, a water-soluble drug. The formulation of this ophthalmic drug delivery system was optimized by a simplex lattice experimental design. The delivery system is composed of three main components that are mono acyl glycerol (monoolein), poloxamer 407 and water and two secondary components that are riboflavin and glycerol (added to adjust the osmotic pressure). The amounts of these three main components were selected as the factors to systematically optimize the dependent variables that are the encapsulation efficiency and the particle size. In this way, 12 formulas describing experimental domain of interest were prepared. Results obtained using small angle X-rays scattering (SAXS) and cryo-transmission electron microscopy (cryo-TEM) evidenced the presence of nano-objects with either sponge or hexagonal inverted structure. In the zone of interest, the percentage of each component was determined to obtain both high encapsulation efficiency and small size of particles. Two optimized formulations were found: F7 and F1. They are very close in the ternary phase diagram as they contain 6.83% of poloxamer 407; 44.18% and 42.03% of monoolein; 46.29% and 48.44% of water for F7 and F11, respectively. These formulations displayed a good compromise between inputs and outputs investigated.

Hydra bioassay for the evaluation of chlordecone toxicity at environmental concentrations, alone or in complex mixtures with dechlorinated byproducts: experimental observations and modeling by experimental design.

In chlordecone (CLD)-contaminated soils of the French West Indies, if microbial remediation or a physicochemical remediation process, e.g., in situ chemical reduction, is implemented, concentrations of degradation byproducts, such as hydrochlordecones, are expected to increase in the ecosystems. To study their impact in mixtures with CLD, bioassays were carried out. They consisted in evaluating the regenerative capacity of hydra polyps, from a clone whose phylogenetic analysis confirmed that it belonged to the species Hydra vulgaris Pallas, 1766. Hydra gastric sections were exposed to CLD alone or CLD plus dechlorinated byproducts (CLD-BP) for 96 h to assess regeneration. Based on chromatographic analysis, the CLD-BP mix was composed of the 5-monohydrochlordecone isomer (CAS nomenclature), four dihydrochlordecone isomers, and one trihydrochlordecone isomer representing 50%, 47%, and 3% of the total chromatographic area, respectively. A total of 18 mixtures of CLD and CLD-BP were tested. Six environmental concentrations of CLD (2.10-4 µM to 4.10-2 µM) and a similar range of CLD-BP were used. Results from exposures to CLD alone showed the following: (i) a significant decrease in the regenerative capacity of hydra, except at the lowest concentration (2.10-4 µM); (ii) a concentration-independent deleterious effect. The regeneration scores obtained after the exposure to the addition of CLD-BP were not significantly different from those obtained after exposure to CLD alone. Using an experimental design, a modeling of the regeneration scores of hydra exposed to mixtures is proposed. Interpreted carefully, since they are limited to only one type of bioassay, the present results suggest that the situation in the aquatic environments should not become worse in terms of toxicity, if soil remediation programs resulting in the formation of hydrochlordecones are put in place.

Global sensitivity analysis of bandpass and antireflection coating manufacturing by numerical space filling designs.

We present the effectiveness of global sensitivity analyses of optical coatings manufacturing to assess the robustness of filters by computer experiments. The most critical interactions of layers are determined for a 29 quarter-wave layer bandpass filter and for an antireflection coating with eight non-quarter-wave layers. Two monitoring techniques with the associated production performances are considered, and their influence on the interactions classification is discussed. Global sensitivity analyses by numerical space filling designs give clues to improve filter manufacturing against error effects and to assess the potential robustness of the coatings.

Design of Experiments for Matrix-Assisted Laser Desorption/Ionization of Amphiphilic Poly(Ethylene Oxide)-b-Polystyrene Block Copolymers.

Matrix-assisted laser/desorption ionization (MALDI) has become a very popular ionization technique for mass spectrometry of synthetic polymers because it allows high throughput analysis of low amounts of sample while avoiding the complexity introduced by extensive multiple charging of electrospray ionization. Yet, fundamental mechanisms underlying this ionization process are not fully understood, so development of sample preparation methods remains empirical. Reliable prediction for the optimal matrix/analyte/salt system is indeed still not possible for homopolymers and it becomes even more challenging in the case of amphiphilic block copolymers where conditions dictated by one block are not compatible with MALDI requirements of the second block. In order to perform MALDI of copolymers composed of poly (ethylene oxide) (PEO) and polystyrene (PS) blocks, it was postulated here that experimental conditions suitable for both species would also be successful for PEO-b-PS. Accordingly, designs of experiments based on Quantitative Structure Activity Relationship (QSAR) analysis were first implemented, studying the influence of 19 matrices and 26 salts on the laser fluence requested for successful MALDI. This analysis first permitted to highlight correlations between the investigated 10 descriptors of matrices and salts and the analytical response, and then to construct models that permits reliable predictions of matrix/salt couples to be used for one or the other homopolymer. Selected couples were then used for MALDI of a PEO-b-PS copolymer but no general trend was observed: experimental conditions expected to work often failed whereas ionic adducts of the copolymer were clearly detected with some matrix/salt systems that were shown to badly perform for constituting homopolymers. Overall, this rules out the working assumption stating that the MALDI behavior of chains composed of PEO and PS segments should combine the behavior of the two polymeric species. Yet, although requiring a dedicated design of experiments, MALDI of the amphiphilic PEO-b-PS copolymer was achieved for the first time.

Feasibility of an inhaled antibiotic/adjuvant dry powder combination using an experimental design approach.

The global increase of multidrug resistant bacteria and the lack of new classes of antibiotic especially those targeting Gram-negative pathogens are leaving the clinicians disarmed to treat numerous bacterial infections. Recently, the design of adjuvants able to enhance antibiotics activities appears to be one of the most promising investigated solutions to circumvent this problem. In this context, we have recently identified a new polyamino-isoprenyl derivative NV716 able to potentiate, at a very low concentration the activity of doxycycline against resistant P. aeruginosa bacterial strains by increasing its intracellular concentration. In this study we will report an experimental protocol to optimize a dry powder for inhalation ensuring the simultaneous delivery of an antibiotic (doxycycline) and an adjuvant (the polyaminoisoprenyl derivative NV716 since aerosol therapy could allow a rapid drug administration and target the respiratory system by avoiding the first pass effect and minimizing undesirable systemic effects. Thus, an experimental design was carried out permitting to identify the influence of several factors on the aerosolization efficiency of our combination and allowing us to find the right composition and manufacture leading to the best optimization of the simultaneous delivery of the two compounds in the form of an inhalable powder. More precisely, the powders of the two active ingredients were prepared by freeze drying and their aerosolization was improved by the addition of carrier particles of lactose inhalation grade. Under these conditions, the best formulation was defined by combining the optimal factors leading to the best aerodynamic properties' values (the lowest MMAD (Mass Median Aerodynamic Diameter) and the highest FPF (Fraction of Fine Particles)) without even using sophisticated engineering techniques. Finally, our results suggest that these molecules could be successfully delivered at the requested concentration in the lungs and then able to decrease drug consumption as well as increase treatment efficacy.

Evaluation of PIG-A-mutated granulocytes and ex-vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans.

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.

Preclinical optimization of an enterotoxigenic Escherichia coli adjuvanted subunit vaccine using response surface design of experiments.

Enterotoxigenic E. coli (ETEC) is a leading cause of moderate-to-severe diarrhoea. ETEC colonizes the intestine through fimbrial tip adhesin colonization factors and produces heat-stable and/or heat-labile (LT) toxins, stimulating fluid and electrolyte release leading to watery diarrhoea. We reported that a vaccine containing recombinant colonization factor antigen (CfaEB) targeting fimbrial tip adhesin of the colonization factor antigen I (CFA/I) and an attenuated LT toxoid (dmLT) elicited mucosal and systemic immune responses against both targets. Additionally, the toll-like receptor 4 ligand second-generation lipid adjuvant (TLR4-SLA) induced a potent mucosal response, dependent on adjuvant formulation. However, a combination of vaccine components at their respective individual optimal doses may not achieve the optimal immune profile. We studied a subunit ETEC vaccine prototype in mice using a response surface design of experiments (DoE), consisting of 64 vaccine dose-combinations of CfaEB, dmLT and SLA in four formulations (aqueous, aluminium oxyhydroxide, squalene-in-water stable nanoemulsion [SE] or liposomes containing the saponin Quillaja saponaria-21 [LSQ]). Nine readouts focusing on antibody functionality and plasma cell response were selected to profile the immune response of parenterally administered ETEC vaccine prototype. The data were integrated in a model to identify the optimal dosage of each vaccine component and best formulation. Compared to maximal doses used in mouse models (10 µg CfaEB, 1 µg dmLT and 5 µg SLA), a reduction in the vaccine components up to 37%, 60% and 88% for CfaEB, dmLT and SLA, respectively, maintained or even maximized immune responses, with SE and LSQ the best formulations. The DoE approach can help determine the best vaccine composition with a limited number of experiments and may accelerate development of multi-antigen/component ETEC vaccines.

Biological wheat straw valorization: Multicriteria optimization of Polyporus brumalis pretreatment in packed bed bioreactor.

The purpose of this work was to optimize the pretreatment process of wheat straw by Polyporus brumalis_BRFM985 in order to improve carbohydrate accessibility for more efficient bioconversion. Indeed, there is growing demands to develop sustainable routes for lignocellulosic feedstocks valorization into value-added products in energy, chemicals, materials, and animal feed fields. To be achieved, implementation of cheap and ecofriendly biomass pretreatment processes is necessary. In this frame, white rot basidiomycetes, well known for their ability to degrade lignin efficiently and selectively, are of great interest. The pretreatment of wheat straw by Polyporus brumalis_BRFM985 was performed in packed bed bioreactor and optimized using response surface methodology. The four pretreatment parameters optimized were metals addition (Cu, Mn, and Fe), time of culture, initial water content, and temperature. Multicriteria optimization highlighted that wheat straw pretreatment by Polyporus brumalis_BRFM985 in the presence of metals with high initial water content of 3.6 g H2 O/g at 27°C for 15-16 days led to an improvement of carbohydrate accessibility with minimal matter loss.

Chemometric approach to evaluate the parameters affecting electrospray: application of a statistical design of experiments for the study of arginine ionization.

The effects of different experimental parameters on arginine electrospray ionization have been investigated with response surface modelling design. This chemometric technique allows a study of the effects of selected experimental variables and their interactions on the response of an experiment by performing a limited number of analyses. Six variables were studied: methanol content in the liquid phase, formic acid concentration, electrospray voltage, orifice voltage, mobile phase flow rate, and sheath gas flow rate. Signal abundance and signal-to-noise ratio of the protonated molecule and the protonated dimer were measured from the electrospray mass spectra and these four responses were tested by the design. The factor that exhibits the greatest influence on MH+ abundance is shown to be the liquid flow rate whereas the formation of protonated dimer is mainly controlled by the percentage of methanol in the mobile phase. A strong synergic effect of methanol content and formic acid concentration in the liquid has also been demonstrated in the study of noise level. Moreover, the capabilities of the multicriteria optimization method have been demonstrated through a successful prediction of a set of optimal experimental conditions.

Impact of organic pollutants on metal and As uptake by helophyte species and consequences for constructed wetlands design and management.

Various industrial processes and anthropogenic activities in urban areas induce a release of metals, metalloids and organic pollutants. Phytoremediation of co-contaminated waters in constructed wetlands is a promising solution for reducing the impact on natural environments. In order to improve the design and management of constructed wetlands, more knowledge is needed concerning the effect of organic pollutants on plant metal and metalloid uptake. In this study, the effects of a mixture of organic pollutants commonly found in industrial effluents (hydrocarbons, polycyclic aromatic hydrocarbons, anionic detergent) on the uptake of ten metals and metalloids (MM), i.e. Al, As, Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn, by five helophytes having a wide European distribution were studied. Main effects of plant species and pollutant conditions on metal uptake and interactions between factors were determined by a statistical treatment of a microcosm experiment. Overall, the order of element uptake in plants was Fe > Al > Mn > Cr, Ni, Zn, > Cu > As, Cd, Pb, which was consistent with relative concentrations in the rhizosphere environment of microcosms. Larger amounts of metals were retained in belowground biomass of plants than in aboveground parts. Statistical analysis showed that organic pollutants enhanced the accumulation of Mn in whole plants and the retention of Fe in belowground parts, while they reduced the accumulation of Cd, Ni, and Zn in whole plants and the retention of Cu in belowground parts. For the other MM (Al, As, Cr, Pb), effects were variable, depending on the plant species. Among the five plants tested, Carex cuprina generally removed the highest quantities of MM, which was the result of both a high metal accumulation capacity and high biomass production. Nevertheless, no significant proportion of the MM total loading could be removed in plants' aboveground parts.

In vitro study of GDNF release from biodegradable PLGA microspheres.

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction-evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using (125)I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.

Assessment of potential positive effects of nZVI surface modification and concentration levels on TCE dechlorination in the presence of competing strong oxidants, using an experimental design.

Nanoscale zero-valent iron (nZVI) particles are efficient for the remediation of aquifers polluted by trichloroethylene (TCE). But for on-site applications, their reactivity can be affected by the presence of common inorganic co-pollutants, which are equally reduced by nZVI particles. The aim of this study was to assess the potential positive effects of nZVI surface modification and concentration level on TCE removal in the concomitant presence of two strong oxidants, i.e., Cr(VI) and NO3(-). A design of experiments, testing four factors (i.e. nZVI concentration, nZVI surface modification, Cr(VI) concentration and NO3(-) concentration), was used to select the best trials for the identification of the main effects of the factors and of the factors interactions. The effects of these factors were studied by measuring the following responses: TCE removal rates at different times, degradation kinetic rates, and the transformation products formed. As expected, TCE degradation was delayed or inhibited in most of the experiments, due to the presence of inorganics. The negative effects of co-pollutants can be palliated by combining surface modification with a slight increase in nZVI concentration. Encouragingly, complete TCE removal was achieved for some given experimental conditions. Noteworthily, nZVI surface modification was found to promote the efficient degradation of TCE. When degradation occurred, TCE was mainly transformed into innocuous non-chlorinated transformation products, while hazardous chlorinated transformation products accounted for a small percentage of the mass-balance.

Disposal of animal by-products by wet air oxidation: performance optimization and kinetics.

This paper describes the application of subcritical wet oxidation to the disposal of sheep animal by-products originating from slaughterhouse. Animal by-products (ABPs) from categories 1 and 3 (gall, head, tail, spinal cord, offal, ileum and blood) were oxidized at high pressure and moderate temperature (P=12.5-20 MPa, T=200-320 degrees C). The oxidation experiments were performed on individual samples or on a reconstituted mixture representing the ABPs of a slaughtered sheep. The oxidation kinetics of a representative sample was studied and the apparent activation energy was found to be 42.9 kJmol(-1). The chemical by-products were also identified and quantified in the final oxidized solution: acetic acid and ammonia were identified in all samples as the major by-products representing around 31% and 69%, respectively, of residual TOC and initial nitrogen after the oxidation of a representative sample of ABPs containing initially 5 gL(-1) of total organic carbon (TOC). The contribution of the experimental factors temperature, reaction time and concentration of the feed solution to remove the organic matter was assessed and optimized using an experimental design based on the response surface methodology. Fitting of the experimental data showed that the 2nd order polynomial model represented the data best. A multicriteria optimization, using the desirability function, allowed the determination of the best region of the experimental domain to optimise the TOC removal and the energy consumption.

Optimisation of the chemical generation of singlet oxygen (1O2, 1Deltag) from the hydrogen peroxide-lanthanum(III) catalytic system using an improved NIR spectrometer.

A near-IR chemiluminescence spectrometer designed to study chemical sources of singlet oxygen ((1)O(2), (1)Delta(g)), was built by coupling a reactor compartment to a nitrogen-cooled Ge diode through a bundle of optical fibres. This device was used to optimise the generation of (1)O(2) from the hydrogen peroxide-lanthanum(iii) catalytic system. The reaction kinetics were studied with a 2(3)3(3)//12 screening experimental design comprising twelve experiments. The influence of six factors was examined: the nature of the lanthanum salt (hydroxide, oxide or nitrate) and its concentration (0.05 or 0.1 mol L(-1)), the pH value (5, 7 or 9), the concentration of H(2)O(2) (0.5, 1 or 2 mol L(-1)), the temperature (20 or 30 degrees C) and the concentration of EDTA (0 or 5 mmol L(-1)). Two responses were measured: the rate of H(2)O(2) disproportionation and the intensity of the luminescence of (1)O(2) at 1270 nm. The essential factor is the nature of the lanthanum salt since La(NO(3))(3) induces the disproportionation of H(2)O(2) about 60 x faster than La(2)O(3) or La(OH)(3). Other influencing factors are the pH value, the concentration of H(2)O(2), the temperature and the concentration of the lanthanum salt whereas the concentration of EDTA has no effect on the reaction. The catalytic activity of La(NO(3))(3) was then investigated in further detail by studying the influence of two factors (pH and [H(2)O(2)]) thanks to a Doehlert design.

Optimization of the assay of naphthodianthrones in dry St John's wort extract by reversed-phase liquid chromatography.

An optimization procedure for the reversed-phase liquid chromatography (RPLC) assay of naphthodianthrones in St John's wort extract from the European Pharmacopoeia project is described. The results obtained from two screening designs showed that light exposure, recommended in the monograph as sample pretreatment, does not permit one to obtain a reproducible quantification of the main ingredients. Improvement of the method robustness implies the need to overcome the problem of light exposure, to subsequently quantify protonaphthodianthrones and to perform the separation on octadecyl (ODS)-bonded phase at optimized flow rate. The method robustness was checked by using a bifurcation sequential approach investigating the influence of 13 factors. The eluent recommended in the monograph is a ternary mixture of methanol, phosphate buffer and ethyl acetate. For the sake of simplicity, the phosphate buffer was substituted by an acetate buffer. The best composition of the ternary mixture was determined by a combined design including three mixture variables and the temperature as an independent variable. Chromatographic parameters were modelled in terms of analysis time, resolution and asymmetry. Desirability functions permit one to cope with these parameters and to determine the best compromise. The naphthodianthrones were separated on a conventional endcapped octadecyl silica gel column eluted by a ternary mobile phase at 40 degrees C in 10 min.