Quality by design compliant strategy for the development of a liquid chromatography-tandem mass spectrometry method for the determination of selected polyphenols in Diospyros kaki.

Diospyros kaki fruits possess great beneficial properties for human health due to their strong antioxidant and antiradical activities related to the high level of bioactive compounds and particularly polyphenols. In this paper a rapid and efficient liquid chromatography-tandem mass spectrometry method for the determination of 38 polyphenolic compounds in Diospyros kaki flesh was developed. The optimization of the chromatographic method was performed applying a Quality by Design approach, which is unexplored in the field of food analysis. The Critical Method Attributes (CMAs) were the critical resolutions of some isobaric compounds and analysis time. The Critical Methods Parameters (CMPs) were related to the characteristics of both the mobile phase and the column: flow rate, temperature, starting organic phase concentration of the mobile phase, formic acid percentage in the eluents, type of organic solvent in the mobile phase and gradient of organic eluents. The effects of the CMPs on the CMAs were evaluated by experimental design, at first carrying out a screening phase by an asymmetric screening matrix and then applying Response Surface Methodology by a Doehlert Design. The quadratic polynomial models postulated to link the CMAs to CMPs were calculated and the Method Operable Design Region was identified with the aid of Monte Carlo simulations as the multidimensional combination of the CMPs that satisfied the requirements for the CMAs with a probability ≥90%. The developed method was applied to real samples obtained by the extraction of Diospyros kaki flesh from two different cultivars (Rojo Brillante and Kaki Tipo), making it possible to obtain extensive information on their polyphenolic profiles.

A comprehensive strategy in the development of a cyclodextrin-modified microemulsion electrokinetic chromatographic method for the assay of diclofenac and its impurities: Mixture-process variable experiments and quality by design.

A comprehensive strategy involving the use of mixture-process variable (MPV) approach and Quality by Design principles has been applied in the development of a capillary electrophoresis method for the simultaneous determination of the anti-inflammatory drug diclofenac and its five related substances. The selected operative mode consisted in microemulsion electrokinetic chromatography with the addition of methyl-ß-cyclodextrin. The critical process parameters included both the mixture components (MCs) of the microemulsion and the process variables (PVs). The MPV approach allowed the simultaneous investigation of the effects of MCs and PVs on the critical resolution between diclofenac and its 2-deschloro-2-bromo analogue and on analysis time. MPV experiments were used both in the screening phase and in the Response Surface Methodology, making it possible to draw MCs and PVs contour plots and to find important interactions between MCs and PVs. Robustness testing was carried out by MPV experiments and validation was performed following International Conference on Harmonisation guidelines. The method was applied to a real sample of diclofenac gastro-resistant tablets.

Enantioseparation and impurity determination of ambrisentan using cyclodextrin-modified micellar electrokinetic chromatography: Visualizing the design space within quality by design framework.

A capillary electrophoresis method for the simultaneous determination of the enantiomeric purity and of impurities of the chiral drug ambrisentan has been developed following the Quality by Design principles. The selected separation system consisted of a micellar pseudostationary phase made by sodium dodecyl sulphate with the addition of γ-cyclodextrin. The effects of critical process parameters (capillary length, temperature, voltage, borate concentration, pH, sodium dodecyl sulphate concentration, γ-cyclodextrin concentration) on enantioresolution of ambrisentan and analysis time were extensively investigated by multivariate strategies involving a screening phase and Response Surface Methodology. The Design Space was defined with a desired probability level π≥90%, and the working conditions, with the limits of the Design Space, corresponded to the following: capillary length, 64.5cm; temperature, 22°C; voltage, 30kV (26-30kV); background electrolyte, 100mM borate buffer pH 9.20 (8.80-9.60), 100mM sodium dodecyl sulphate, 50mM (43-50mM) γ-cyclodextrin. A Plackett-Burman design was applied for robustness testing, and a method control strategy was established. The method was fully validated according to the International Conference on Harmonisation guidelines and was applied to ambrisentan coated tablets.

Quality by design in the chiral separation strategy for the determination of enantiomeric impurities: development of a capillary electrophoresis method based on dual cyclodextrin systems for the analysis of levosulpiride.

Quality by design (QbD) concepts, in accordance with International Conference on Harmonisation Pharmaceutical Development guideline Q8(R2), represent an innovative strategy for the development of analytical methods. In this paper QbD principles have been comprehensively applied in the set-up of a capillary electrophoresis method aimed to quantify enantiomeric impurities. The test compound was the chiral drug substance levosulpiride (S-SUL) and the developed method was intended to be used for routine analysis of the pharmaceutical product. The target of analytical QbD approach is to establish a design space (DS) of critical process parameters (CPPs) where the critical quality attributes (CQAs) of the method have been assured to fulfil the desired requirements with a selected probability. QbD can improve the understanding of the enantioseparation process, including both the electrophoretic behavior of enantiomers and their separation, therefore enabling its control. The CQAs were represented by enantioresolution and analysis time. The scouting phase made it possible to select a separation system made by sulfated-ß-cyclodextrin and a neutral cyclodextrin, operating in reverse polarity mode. The type of neutral cyclodextrin was included among other CPPs, both instrumental and related to background electrolyte composition, which were evaluated in a screening phase by an asymmetric screening matrix. Response surface methodology was carried out by a Doehlert design and allowed the contour plots to be drawn, highlighting significant interactions between some of the CPPs. DS was defined by applying Monte-Carlo simulations, and corresponded to the following intervals: sulfated-ß-cyclodextrin concentration, 9-12 mM; methyl-ß-cyclodextrin concentration, 29-38 mM; Britton-Robinson buffer pH, 3.24-3.50; voltage, 12-14 kV. Robustness of the method was examined by a Plackett-Burman matrix and the obtained results, together with system repeatability data, led to define a method control strategy. The method was validated and was finally applied to determine the enantiomeric purity of S-SUL in pharmaceutical dosage forms.

An integrated quality by design and mixture-process variable approach in the development of a capillary electrophoresis method for the analysis of almotriptan and its impurities.

The development of a capillary electrophoresis (CE) method for the assay of almotriptan (ALM) and its main impurities using an integrated Quality by Design and mixture-process variable (MPV) approach is described. A scouting phase was initially carried out by evaluating different CE operative modes, including the addition of pseudostationary phases and additives to the background electrolyte, in order to approach the analytical target profile. This step made it possible to select normal polarity microemulsion electrokinetic chromatography (MEEKC) as operative mode, which allowed a good selectivity to be achieved in a low analysis time. On the basis of a general Ishikawa diagram for MEEKC methods, a screening asymmetric matrix was applied in order to screen the effects of the process variables (PVs) voltage, temperature, buffer concentration and buffer pH, on critical quality attributes (CQAs), represented by critical separation values and analysis time. A response surface study was then carried out considering all the critical process parameters, including both the PVs and the mixture components (MCs) of the microemulsion (borate buffer, n-heptane as oil, sodium dodecyl sulphate/n-butanol as surfactant/cosurfactant). The values of PVs and MCs were simultaneously changed in a MPV study, making it possible to find significant interaction effects. The design space (DS) was defined as the multidimensional combination of PVs and MCs where the probability for the different considered CQAs to be acceptable was higher than a quality level π=90%. DS was identified by risk of failure maps, which were drawn on the basis of Monte-Carlo simulations, and verification points spanning the design space were tested. Robustness testing of the method, performed by a D-optimal design, and system suitability criteria allowed a control strategy to be designed. The optimized method was validated following ICH Guideline Q2(R1) and was applied to a real sample of ALM coated tablets.

Development and characterization of functionalized niosomes for brain targeting of dynorphin-B.

A niosomal formulation, functionalized with N-palmitoylglucosamine, was developed as potential brain targeted delivery system of dynorphin-B. In fact, this endogenous neuropeptide, selective agonist of k opioid receptors, is endowed with relevant pharmacological activities on the central nervous system, including a marked antinociceptive effect, but is unable to cross the blood brain barrier (BBB), thus requiring intracerebroventricular administration. Statistical design of experiments was utilized for a systematic evaluation of the influence of variations of the relative amounts of the components of the vesicle membrane (Span 60, cholesterol and SolulanC24) on vesicle mean diameter, polydispersity index and drug entrapment efficiency, chosen as the responses to optimize. A Scheffé simplex-centroid design was used to obtain the coefficients of the postulated mathematical model. The study of the response surface plots revealed that variations of the considered factors had different effects on the selected responses. The desirability function enabled for finding the optimal mixture composition, which represented the best compromise to simultaneously optimize all the three responses. The experimental values obtained with the optimized formulation were very similar to the predicted ones, proving the validity of the proposed regression model. The optimized niosomal formulation of dynorphin-B administered intravenously to mice (100mg/kg) showed a pronounced antinociceptive effect, significantly higher (P<0.05) than that given by i.v. administration of the simple solution of the peptide at the same concentration, proving its effectiveness in enabling the peptide brain delivery. These positive results suggest that the proposed approach could be successfully extended to other neuro-active peptides exerting a strong central action, even at low doses, but unable to cross the BBB.

Direct resolution and quantitative analysis of flurbiprofen enantiomers using microcrystalline cellulose triacetate plates: applications to the enantiomeric purity control and optical isomer determination in widely consumed drugs.

Flurbiprofen enantiomers have very different pharmacological properties, since the (S)-(+) form has a much higher anti-inflammatory activity than the (R)-(-) isomer, the latter being responsible for very undesirable side effects, such as gastrointestinal irritation. Based on the different biological properties of flurbiprofen enantiomers, the development of chiral chromatographic methods for the control of the enantiomeric purity is a very important topic. In this study the separation of flurbiprofen enantiomers was achieved using for the first time noncommercial MCTA layers with polyvinyl alcohol as binder, which gives to these plates a mechanical stability equivalent to that of marketed ones. Baseline resolution (α = 1.31; RS = 2.0) was obtained with ethanol-acetic acid solution (pH 3.0 ± 0.1; 60:40, v/v) as eluent and a migration distance of about 14.5 cm. Under these experimental conditions, the thin-layer chromatography determination of the enantiomeric purity of the pharmacologically active (S)-(+)-flurbiprofen in the presence of 1% of the undesired (R)-(-) form was demonstrated. Moreover, the quantitative analysis of flurbiprofen enantiomers was achieved, obtaining quantification limits and detection limits of 50 and 25 ng of each enantiomer applied to the plate, respectively. The method was succesfully applied to the enantiomer determination in widely consumed drugs, obtaining results consistent with the flurbiprofen content declared in the drug facts.

Quality by Design approach in the development of a solvent-modified micellar electrokinetic chromatography method: finding the design space for the determination of amitriptyline and its impurities.

A solvent-modified micellar electrokinetic chromatography method was set up for the simultaneous determination of the tricyclic antidepressant amitriptyline (AMI) and its main impurities. The method was developed following Quality by Design (QbD) principles according to ICH Guideline Q8(R2). QbD approach made it possible to find the design space (DS), where quality was assured. After a scouting phase, aimed at selecting a suitable capillary electrophoresis pseudostationary phase, risk assessment tools were employed to define the critical process parameters (CPPs) to be considered in a screening phase (applied voltage, concentration and pH of the background electrolyte, concentration of the surfactant sodium dodecyl sulphate, of the cosurfactant n-butanol and of the organic modifiers acetonitrile and urea). The effects of the seven selected CPPs on critical quality attributes (CQAs), namely resolution values between critical peak pairs and analysis time, were investigated throughout the knowledge space by means of a symmetric screening matrix. Response surface study was then carried out on four selected CPPs by applying a Doehlert Design. Monte-Carlo simulations were performed in order to estimate the probability of meeting the desired specifications on CQAs, and thus to define the DS by means of a risk of failure map. Additional points at the edges of the DS were tested in order to verify the requirements for CQAs to be fulfilled. A control strategy was implemented by defining system suitability tests. The developed method was validated following ICH Guideline Q2(R1), including robustness assessment by Plackett-Burman design, and was applied to the analysis of real samples of amitriptyline coated tablets.

Mixture-process variable approach to optimize a microemulsion electrokinetic chromatography method for the quality control of a nutraceutical based on coenzyme Q10.

In recent years, multivariate optimization has played an increasing role in analytical method development. ICH guidelines recommend using statistical design of experiments to identify the design space, in which multivariate combinations of composition variables and process variables have been demonstrated to provide quality results. Considering a microemulsion electrokinetic chromatography method (MEEKC), the performance of the electrophoretic run depends on the proportions of mixture components (MCs) of the microemulsion and on the values of process variables (PVs). In the present work, for the first time in the literature, a mixture-process variable (MPV) approach was applied to optimize a MEEKC method for the analysis of coenzyme Q10 (Q10), ascorbic acid (AA), and folic acid (FA) contained in nutraceuticals. The MCs (buffer, surfactant-cosurfactant, oil) and the PVs (voltage, buffer concentration, buffer pH) were simultaneously changed according to a MPV experimental design. A 62-run MPV design was generated using the I-optimality criterion, assuming a 46-term MPV model allowing for special-cubic blending of the MCs, quadratic effects of the PVs, and some MC-PV interactions. The obtained data were used to develop MPV models that express the performance of an electrophoretic run (measured as peak efficiencies of Q10, AA, and FA) in terms of the MCs and PVs. Contour and perturbation plots were drawn for each of the responses. Finally, the MPV models and criteria for the peak efficiencies were used to develop the design space and an optimal subregion (i.e., the settings of the mixture MCs and PVs that satisfy the respective criteria), as well as a unique optimal combination of MCs and PVs.

Quality by design approach for developing chitosan-Ca-alginate microspheres for colon delivery of celecoxib-hydroxypropyl-ß-cyclodextrin-PVP complex.

The aim of the present work was to develop a new multiparticulate system, designed for colon-specific delivery of celecoxib for both systemic (in chronotherapic treatment of arthritis) and local (in prophylaxis of colon carcinogenesis) therapy. The system simultaneously benefits from ternary complexation with hydroxypropyl-ß-cyclodextrin and PVP (polyvinylpyrrolidone), to increase drug solubility, and vectorization in chitosan-Ca-alginate microspheres, to exploit the colon-specific carrier properties of these polymers. Statistical experimental design was employed to investigate the combined effect of four formulation variables, i.e., % of alginate, CaCl2, and chitosan and time of cross-linking on microsphere entrapment efficiency (EE%) and drug amount released after 4h in colonic medium, considered as the responses to be optimized. Design of experiment was used in the context of Quality by Design, which requires a multivariate approach for understanding the multifactorial relationships among formulation parameters. Doehlert design allowed for defining a design space, which revealed that variations of the considered factors had in most cases an opposite influence on the responses. Desirability function was used to attain simultaneous optimization of both responses. The desired goals were achieved for both systemic and local use of celecoxib. Experimental values obtained from the optimized formulations were in both cases very close to the predicted values, thus confirming the validity of the generated mathematical model. These results demonstrated the effectiveness of the proposed jointed use of drug cyclodextrin complexation and chitosan-Ca-alginate microsphere vectorization, as well as the usefulness of the multivariate approach for the preparation of colon-targeted celecoxib microspheres with optimized properties.

Mixture experiment methods in the development and optimization of microemulsion formulations.

Microemulsion formulations represent an interesting delivery vehicle for lipophilic drugs, allowing for improving their solubility and dissolution properties. This work developed effective microemulsion formulations using glyburide (a very poorly-water-soluble hypoglycaemic agent) as a model drug. First, the area of stable microemulsion (ME) formations was identified using a new approach based on mixture experiment methods. A 13-run mixture design was carried out in an experimental region defined by constraints on three components: aqueous, oil and surfactant/cosurfactant. The transmittance percentage (at 550 nm) of ME formulations (indicative of their transparency and thus of their stability) was chosen as the response variable. The results obtained using the mixture experiment approach corresponded well with those obtained using the traditional approach based on pseudo-ternary phase diagrams. However, the mixture experiment approach required far less experimental effort than the traditional approach. A subsequent 13-run mixture experiment, in the region of stable MEs, was then performed to identify the optimal formulation (i.e., having the best glyburide dissolution properties). Percent drug dissolved and dissolution efficiency were selected as the responses to be maximized. The ME formulation optimized via the mixture experiment approach consisted of 78% surfactant/cosurfacant (a mixture of Tween 20 and Transcutol, 1:1, v/v), 5% oil (Labrafac Hydro) and 17% aqueous phase (water). The stable region of MEs was identified using mixture experiment methods for the first time.

Response surface methodology in the optimization of chitosan-Ca pectinate bead formulations.

This study describes the application of a multi-varied experimental design methodology to the optimization of a bead formulation based on a mixed network of Ca pectinate and chitosan. The effect of varying the relative percent of the three components used for the bead production, i.e. pectin, chitosan and CaCl(2), has been systematically investigated with the aim of identifying their best levels to optimize drug encapsulation efficiency (considered as the experimental response to be maximized), as well as to highlight possible interactions among the components. The study was applied to two different drugs, i.e. prednisone and theophylline, selected, respectively, as model insoluble and relatively soluble drugs, in order to evaluate the influence of this parameter as well. Different bead batches were prepared according to Doehlert and D-optimal design and randomly evaluated for drug encapsulation efficiency. Analysis of response surface plots allowed identification of the best combination of the three bead components in order to maximize drug encapsulation efficiency. The most effective compositions were chitosan 3% (w/v), pectin 9% (w/v), CaCl(2) 4% (w/v) for the theophylline-loaded beads and chitosan 0.75% (w/v), pectin 6% (w/v), CaCl(2) 7.9% (w/v) for the prednisone-loaded ones. The good correspondence between calculated and experimental values indicated in both cases the validity of the generated statistical models for the prediction of microsphere encapsulation efficiency. The different results obtained for the two drugs indicated the importance of the greater or lesser drug lipophilicity in determining the optimal bead composition with the highest encapsulation efficiency.

Simultaneous effect of cyclodextrin complexation, pH, and hydrophilic polymers on naproxen solubilization.

The effect of pH variation on complexation and solubilization of naproxen (pK(a) 4.2) with natural betaCyclodextrin (betaCyD) and various neutral, cationic and anionic betaCyD-derivatives has been investigated. The combined effect of pH variation and hydrophilic polymer addition on CyD solubilizing and complexing efficiency has also been determined. Phase-solubility analysis in buffered aqueous solutions (pH from 1.1 to 6.5) was used to study the interaction of the drug with each CyD, in the presence or not of the water-soluble polymer. A clear influence of the substituent type was observed, the methylderivative being the most efficient agent; on the contrary, unexpectedly, no influence of the CyD charge in the interaction with the ionizable drug was detected. As expected, total drug solubility increased with increasing pH; however, the solubility increment with respect to drug alone obtained by CyD complexation progressively decreased, with a parallel reduction of the complex stability, attributed to the reduced affinity of charged drug for the hydrophobic CyD cavity. The addition of the polymer in part counterbalanced the destabilizing effect obtained with increasing pH, by improving the CyD complexation power towards naproxen. In particular, the presence of PVP allowed an increase of the complex stability constant with hydroxypropyl betaCyD up to 60% with respect to the corresponding drug-CyD binary system. Therefore, the combined strategy of pH control and polymer addition to the CyD complexing medium can be successfully exploited to improve naproxen solubilization and reduce the amount of CyD needed. The construction of theoretical drug solubility curves as a function of pH for any given CyD and polymer concentration enables selection of the best experimental conditions for obtaining the desired drug solubility value.

Interaction of naproxen with ionic cyclodextrins in aqueous solution and in the solid state.

The possible role of the cyclodextrin charge in the interaction with an acidic drug such as naproxen (pKa 4.8) has been evaluated. Sulfobutylether-beta-cyclodextrin (SBE-betaCyd) and trimethylammonium-beta-cyclodextrin (TMA-betaCyd) were selected as, respectively, anionically and cationically charged carriers and their performance was compared with that of the parent beta-cyclodextrin (betaCyd) and of its methyl-derivative (Me betaCyd) previously found as the best partner for the drug. Interactions in solution were investigated by phase-solubility, fluorescence and circular dichroism analyses. Equimolar drug-carrier products prepared by different techniques (blending, cogrinding, sealed-heating, colyophilization) were characterized by differential scanning calorimetry and X-ray powder diffractometry and tested for drug dissolution properties. Anionic charges of SBE-betaCyd did not negatively influence interactions in unbuffered aqueous solutions (pH approximately 5) with the acidic drug. In fact, it was a very effective carrier, exhibiting solubilizing and complexing properties considerably better than the parent betaCyd and comparable to those of Me betaCyd. On the contrary, the positive charges of TMA-betaCyd did not favour interactions with the counter-ionic drug (despite the presence of about 60% ionised drug) and it was less efficacious also than native betaCyd. Therefore, the role of the Cyd charge on the complexing and solubilizing properties towards naproxen was not important whereas other factors, such as steric hindrance effects and favourable hydrophobic interactions were significant in determining the drug affinity for the Cyd inclusion. Solid state studies evidenced similar amorphizing properties of both charged Cyds towards naproxen. On the other hand, dissolution tests, in agreement with solution studies, showed that all products with SBE-betaCyd exhibited significantly better dissolution properties than the corresponding ones with TMA-betaCyd. A clear influence of the preparation method of drug-Cyd solid systems on the performance of the end product was also observed. Colyophilization was the most effective technique, followed by the cogrinding one. Colyophilized product with SBE-betaCyd allowed a 10-times increase in drug dissolution efficiency (D.E.) (with respect to the five-times increase obtained with the corresponding coground product) and a reduction of t(50%) from about 60 min (for the coground product) to less than 2 min.

Determination of stability constant values of flurbiprofen-cyclodextrin complexes using different techniques.

Three new experimental approaches for calculating the stability constant (K(st)) of complexes of flurbiprofen with natural beta-cyclodextrin (betaCyd) and the hydroxyethyl- (HEbetaCyd) and the methyl- (Me betaCyd) derivatives were tested and compared to the classic phase-solubility procedure: (a) the membrane permeation technique through a lipophilic synthetic membrane permeable to the drug but not to the Cyd molecules, by analysing the permeation profiles with a non-linear least-squares method; (b) the affinity capillary electrophoresis (ACE) technique, where K(st) were calculated from the relationship between Cyd concentration in solution and drug electrophoretic mobility, using three different linear plotting methods; (c) the molecular modeling technique, based on the relationship between the docking energies and the experimental K(st) values. The study allowed evaluation of the advantages and limits of each examined method, providing a useful guide for the choice of the most suitable one depending on the kind of host-guest system to be investigated. The K(st) values obtained with the various techniques were rather different, probably due to the very different experimental conditions required by each one. However, all the methods indicated the methyl-derivative as the most powerful complexing agent for the drug, showing the general trend: K(st)(Me betaCyd)>>K(st)(HEbetaCyd)>K(st)(betaCyd). Only in the case of the ACE method was an inversion of the trend found between HEbetaCyd and betaCyd; this was probably due to the lower molecular weight of the natural Cyd, which, in this case, became more important in determining the complex electrophoretic mobility than the different affinity of the drug for these two Cyds.

Optimization of glibenclamide tablet composition through the combined use of differential scanning calorimetry and D-optimal mixture experimental design.

A systematic analysis of the influence of different proportions of excipients on the stability of a solid dosage form was carried out. In particular, a d-optimal mixture experimental design was applied for the evaluation of glibenclamide compatibility in tablet formulations, consisting of four classic excipients (natrosol as binding agent, stearic acid as lubricant, sorbitol as diluent and cross-linked polyvinylpyrrolidone as disintegrant). The goal was to find the mixture component proportions which correspond to the optimal drug melting parameters, i.e. its maximum stability, using differential scanning calorimetry (DSC) to quickly obtain information about possible interactions among the formulation components. The absolute value of the difference between the melting peak temperature of pure drug endotherm and that in each analysed mixture and the absolute value of the difference between the enthalpy of the pure glibenclamide melting peak and that of its melting peak in the different analyzed mixtures, were chosen as indexes of the drug-excipient interaction degree.

Development of a chromatographic bioreactor based on immobilized beta-glucuronidase on monolithic support for the determination of dextromethorphan and dextrorphan in human urine.

We here reported the development and application of an immobilized enzyme reactor (IMER) based on beta-glucuronidase to the on-line determination of urinary molar ratios of dextromethorphan (DOMe)/dextrorphan (DOH) for the assessment of the metabolic activity of CYP2D6, a genetically variable isoform of cytochrome P-450 (CYP). beta-Glucuronidase was immobilized on an HPLC monolithic aminopropyl silica support. Catalytic activity and stability of the chromatographic reactor were evaluated using 8-hydroxyquinoline glucuronide (8-HQG) as substrate. The IMER was coupled through a switching valve to a reversed-phase column (C8) for the simultaneous determination of dextromethorphan and its main metabolite dextrorphan. On purpose a selective reversed-phase ion pair HPLC method coupled with fluorescence detection was developed. Urine samples were first centrifuged to remove insoluble materials and then aliquots of the supernatants were injected into the coupled-column analyser. Linearity, precision and accuracy of the method were established. The method reliability was verified by comparing our data with previous data of a phenotyping study carried out by the Poison Control Centre of Pavia-Clinical Toxicology Division.

Micellar electrokinetic chromatography for the simultaneous determination of ketorolac tromethamine and its impurities. Multivariate optimization and validation.

A simple, fast and selective micellar electrokinetic chromatographic (MEKC) method for the simultaneous assay of ketorolac tromethamine and its known related impurities (1-hydroxy analog of ketorolac, 1-keto analog of ketorolac and decarboxylated ketorolac), in both drug substance and coated tablets, is described. The compounds were detected at 323 nm, and flufenamic acid (FL) and tolmetin (TL) were chosen as internal standards to quantify ketorolac tromethamine and impurities, respectively. The multivariate optimization of the experimental conditions was carried out by means of the response surface study, considering as responses the resolution values and analysis time. The optimized background electrolyte (BGE) consisted of a mixture of 13 mM boric acid and phosphoric acid, adjusted to pH 9.1 with 1 M sodium hydroxide, containing 73 mM sodium dodecyl sulfate (SDS). Optimal temperature and voltage were 30 degrees C and 27 kV. Applying these conditions, all compounds were resolved in about 6 min. The related substances could be quantified up to the 0.1% (w/w) level. Validation was performed, either for drug substances and drug product, evaluating selectivity, robustness, linearity and range, precision, accuracy, detection and quantitation limits and system suitability.

How experimental design can improve the validation process. Studies in pharmaceutical analysis.

A critical discussion about the possibility of improving the method validation process by means of experimental design is presented. The reported multivariate strategies concern the evaluation of the performance parameters robustness and intermediate precision, and the optimisation of bias and repeatability. In particular, accuracy and precision improvement constitutes a special subset of experimental design in which the bias and the relative standard deviation of the assay are optimised. D-optimal design was used in order to plan experiments for this aim. The analytical methods considered were capillary electrophoresis, HPLC, adsorptive stripping voltammetry and differential pulse polarography. All methods were applied to real pharmaceutical analysis problems.

Lipases for biocatalysis: development of a chromatographic bioreactor.

The development of a new chromatographic reactor based on immobilized Candida rugosa lipase (CRL) is described. The chromatographic system has been used to evaluate the rate differences by which the product enantiomers of esterolytic reactions catalyzed by immobilized CRL are obtained. The method has been applied to a series of racemic 2-aryloxyalkanoic acids and isosteric analogous methyl esters and to some non-steroidal antiinflammatory drugs 2-arylpropanoic acids methyl esters in order to study the structure effects on reaction rate and enantioselectivity. Lipase from C. rugosa has been non-covalently and covalently immobilized on HPLC chromatographic silica supports to develop an immobilized enzyme reactor (IMER). The reactor was connected through a switching valve to an analytical reversed-phase column, which was used for the on-line determination of the hydrolysis rate by peak area integration. The enantiomeric excess of the hydrolytic reaction products was determined off-line on a CSP utilizing immobilized penicillin G acylase (PGA-CSP).