Performance evaluation of a MIP for the MISPE-LC determination of p-[18F]MPPF and a potential metabolite in human plasma.

Within the family of serotonin (5-HT) receptors, the 5-HT1A subtype is particularly interesting as it may be involved in various physiological processes or psychological disorders. The p-[18F]MPPF, a highly selective 5-HT1A antagonist, is used for in vivo studies in human or animal by means of positron emission tomography (PET) [1]. In order to selectively extract p-[18F]MPPF and its main metabolites from plasma, molecularly imprinted polymer (MIP) was prepared against these compounds by using the p-MPPF as template. For the control of the selectivity, non-imprinted polymer (NIP) was also synthesized without template. The MIP sorbent, packed in disposable extraction cartridges (DECs), was then evaluated as molecularly imprinted solid-phase extraction (MISPE) prior to the LC determination. The conditions of extraction were evaluated in order to obtain the highest selective retention of the p-[18F]MPPF and its metabolites on this MIP. The MIP selectivity was exploited in the loading and washing steps by adjusting the pH of plasma samples at a suitable value and by selecting mixtures for the washing step to limit the contribution of non-specific interactions. Other important parameters involved in the conditioning and elution steps were also studied. Finally, a pre-validation was carried out with optimal extraction conditions to demonstrate the performance of this MISPE-LC method as a generic method in the context of evaluation of new MISPE for p-[18F]MPPF and its potential for metabolites extraction from human plasma.

Compromise in uncertainty estimation by modelling and validation approaches for an HPLC-UV method for measurement of biochemical indicators of vitamins A and E.

It is common practice nowadays to associate the measurement uncertainty to the measurand, in order to judge the quality of a result related to the measurement process. However, the improvement of this parameter as well as the adaptation of its estimation modes always remain an analytical challenge, especially in chemical testing. In this paper, we outline a measurement uncertainty estimation mode based on the one-point linear calibration equation to fully establish a "bottom-up" approach for estimating the measurement uncertainty of a multi-point calibration-based HPLC-UV quantitative method. To demonstrate this estimation mode, we have followed as an example of interest the influences resulting from the simultaneous determination of two biochemical indicators, namely the human plasma vitamers retinol and α-tocopherol. Results from this estimation showed consistency when compared to those obtained from the validation-based alternative method, where the relative expanded uncertainties were found, at a 95% confidence level, to be less than 15% for the low concentration ranges of the two molecules. However, the modelling approach shows all the benefits of its use to identify and quantify all the uncertainty contributions arising from the different steps of the analytical process and seems to be quite achievable for comparative HPLC methods.

Fingerprinting and validation of a LC-DAD method for the analysis of biflavanones in Garcinia kola-based antimalarial improved traditional medicines.

African populations use traditional medicines in their initial attempt to treat a range of diseases. Nevertheless, accurate knowledge of the composition of these drugs remains a challenge in terms of ensuring the health of population and in order to advance towards improved traditional medicines (ITMs). In this paper chromatographic methods were developed for qualitative and quantitative analyses of a per os antimalarial ITM containing Garcinia kola. The identified analytical markers were used to establish TLC and HPLC fingerprints. G. kola seeds were analysed by HPLC to confirm the identity of the extract used by the Congolese manufacturer in the ITM. The main compounds (GB1, GB2, GB-1a and Kolaflavanone) were isolated by preparative TLC and identified by UPLC-MS and NMR. For the quantification of the major compound GB1, a simple and rapid experimental design was applied to develop an LC method, and then its validation was demonstrated using the total error strategy with the accuracy profile as a decision tool. The accurate results were observed within 0.14-0.45mg/mL range of GB1 expressed as naringenin. The extracts used in several batches of the analysed oral solutions contained GB1 (expressed as naringenin) within 2.04-2.43%. Both the fingerprints and the validated LC-DAD were found suitable for the quality control of G. kola-based raw material and finished products, respectively.

Towards a full integration of optimization and validation phases: An analytical-quality-by-design approach.

When using an analytical method, defining an analytical target profile (ATP) focused on quantitative performance represents a key input, and this will drive the method development process. In this context, two case studies were selected in order to demonstrate the potential of a quality-by-design (QbD) strategy when applied to two specific phases of the method lifecycle: the pre-validation study and the validation step. The first case study focused on the improvement of a liquid chromatography (LC) coupled to mass spectrometry (MS) stability-indicating method by the means of the QbD concept. The design of experiments (DoE) conducted during the optimization step (i.e. determination of the qualitative design space (DS)) was performed a posteriori. Additional experiments were performed in order to simultaneously conduct the pre-validation study to assist in defining the DoE to be conducted during the formal validation step. This predicted protocol was compared to the one used during the formal validation. A second case study based on the LC/MS-MS determination of glucosamine and galactosamine in human plasma was considered in order to illustrate an innovative strategy allowing the QbD methodology to be incorporated during the validation phase. An operational space, defined by the qualitative DS, was considered during the validation process rather than a specific set of working conditions as conventionally performed. Results of all the validation parameters conventionally studied were compared to those obtained with this innovative approach for glucosamine and galactosamine. Using this strategy, qualitative and quantitative information were obtained. Consequently, an analyst using this approach would be able to select with great confidence several working conditions within the operational space rather than a given condition for the routine use of the method. This innovative strategy combines both a learning process and a thorough assessment of the risk involved.

Improvement of a stability-indicating method by Quality-by-Design versus Quality-by-Testing: a case of a learning process.

The understanding of the method is a major concern when developing a stability-indicating method and even more so when dealing with impurity assays from complex matrices. In the presented case study, a Quality-by-Design approach was applied in order to optimize a routinely used method. An analytical issue occurring at the last stage of a long-term stability study involving unexpected impurities perturbing the monitoring of characterized impurities needed to be resolved. A compliant Quality-by-Design (QbD) methodology based on a Design of Experiments (DoE) approach was evaluated within the framework of a Liquid Chromatography (LC) method. This approach allows the investigation of Critical Process Parameters (CPPs), which have an impact on Critical Quality Attributes (CQAs) and, consequently, on LC selectivity. Using polynomial regression response modeling as well as Monte Carlo simulations for error propagation, Design Space (DS) was computed in order to determine robust working conditions for the developed stability-indicating method. This QbD compliant development was conducted in two phases allowing the use of the Design Space knowledge acquired during the first phase to define the experimental domain of the second phase, which constitutes a learning process. The selected working condition was then fully validated using accuracy profiles based on statistical tolerance intervals in order to evaluate the reliability of the results generated by this LC/ESI-MS stability-indicating method. A comparison was made between the traditional Quality-by-Testing (QbT) approach and the QbD strategy, highlighting the benefit of this QbD strategy in the case of an unexpected impurities issue. On this basis, the advantages of a systematic use of the QbD methodology were discussed.

Fast method for the simultaneous quantification of toxic polyphenols applied to the selection of genotypes of yam bean (Pachyrhizus sp.) seeds.

The purpose of the research was to develop and validate a rapid quantification method able to screen many samples of yam bean seeds to determine the content of two toxic polyphenols, namely pachyrrhizine and rotenone. The analytical procedure described is based on the use of an internal standard (dihydrorotenone) and is divided in three steps: microwave assisted extraction, purification by solid phase extraction and assay by ultra high performance liquid chromatography (UHPLC). Each step was included in the validation protocol and the accuracy profiles methodology was used to fully validate the method. The method was fully validated between 0.25 mg and 5 mg pachyrrhizin per gram of seeds and between 0.58 mg/g and 4 mg/g for rotenone. More than one hundred samples from different accessions, locations of growth and harvest dates were screened. Pachyrrhizine concentrations ranged from 3.29 mg/g to lower than 0.25 mg/g while rotenone concentrations ranged from 3.53 mg/g to lower than 0.58 mg/g. This screening along with principal component analysis (PCA) and discriminant analysis (DA) analyses allowed the selection of the more interesting genotypes in terms of low concentrations of these two toxic polyphenols.

Determination of 4-aminophenol in a pharmaceutical formulation using surface enhanced Raman scattering: from development to method validation.

A surface enhanced Raman scattering (SERS) method able to quantify 4-aminophenol in a pharmaceutical formulation based on acetaminophen, also called paracetamol, was developed and, for the first time, successfully validated. In this context, silver nanoparticles were synthesized according to the method described by Lee-Meisel and used as SERS substrate. The repeatability of the silver colloid synthesis was tested using different methods to characterize the size and the zeta potential of silver nanoparticles freshly synthesized. To optimize the SERS samples preparation, a design of experiments implicating concentrations of citrate-reduced silver nanoparticles and aggregating agent was performed in order to maximize the Raman signal enhancement. Finally, an approach based on tolerance intervals and accuracy profiles was applied in order to thoroughly validate the method in a range of concentrations comprised from 3 to 15 µg mL(-1) using normalized band intensities. The standard addition method was selected as method calibration. Therefore, measurements were carried out on 4-aminophenol spiked solutions of the pharmaceutical formulation. Despite the well-known stability and reproducibility problems of SERS, the validation was performed using two operators and five batches of nanoparticles, one for each validation day.

Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry.

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC-MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.

Application of an innovative design space optimization strategy to the development of LC methods for the simultaneous screening of antibiotics to combat poor quality medicines.

The poor quality of medicines is a crucial problem of public health. Therefore, it is important to have analytical tools to attend decisions of the legal authorities while combating this offense. In this context, the main objective of this study was to develop generic methods able to trace, screen and determine several antibiotics and common associated molecules by mean of liquid chromatographic techniques. For that purpose, an innovative Design Space optimization strategy was applied, targeting 16 antibiotics and 3 beta-lactamase inhibitors. The robustness of the developed method allowed using its use in an environment where operational factors such as temperature are not easy to control and eased its transfer to Ultra High Performance Liquid Chromatography. To demonstrate its ability to quantify the targeted molecules, the developed and transferred method was fully validated for two active ingredients commonly used in association, sulbactam and ceftriaxone, using the accuracy profile as decision tool. Based on this successful step, the method was then used for the quantitative determination of these two active ingredients in three pharmaceutical brands marketed in the Democratic Republic of Congo. Two out of the three pharmaceutical products did not comply with the specifications.

Experimental in situ exposure of the seagrass Posidonia oceanica (L.) Delile to 15 trace elements.

The Mediterranean seagrass Posidonia oceanica (L.) Delile has been used for trace element (TE) biomonitoring since decades ago. However, present informations for this bioindicator are limited mainly to plant TE levels, while virtually nothing is known about their fluxes through P. oceanica meadows. We therefore contaminated seagrass bed portions in situ at two experimental TE levels with a mix of 15 TEs (Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Mo, Ag, Cd, Pb and Bi) to study their uptake and loss kinetics in P. oceanica. Shoots immediately accumulated pollutants from the beginning of exposures. Once contaminations ended, TE concentrations came back to their original levels within two weeks, or at least showed a clear decrease. P. oceanica leaves exhibited different uptake kinetics depending on elements and leaf age: the younger growing leaves forming new tissues incorporated TEs more rapidly than the older senescent leaves. Leaf epiphytes also exhibited a net uptake of most TEs, partly similar to that of P. oceanica shoots. The principal route of TE uptake was through the water column, as no contamination of superficial sediments was observed. However, rhizomes indirectly accumulated many TEs during the overall experiments through leaf to rhizome translocation processes. This study thus experimentally confirmed that P. oceanica shoots are undoubtedly an excellent short-term bioindicator and that long-term accumulations could be recorded in P. oceanica rhizomes.

Usefulness of information criteria for the selection of calibration curves.

The reliability of analytical results obtained with quantitative analytical methods is highly dependent on the selection of the adequate model used as the calibration curve. To select the adequate response function or model the most used and known parameter is to determine the coefficient R(2). However, it is well-known that it suffers many inconveniences, such as leading to overfitting the data. A proposed solution is to use the adjusted determination coefficient R(adj)(2) that aims at reducing this problem. However, there is another family of criteria that exists to allow the selection of an adequate model: the information criteria AIC, AICc, and BIC. These criteria have rarely been used in analytical chemistry to select the adequate calibration curve. This works aims at assessing the performance of the statistical information criteria as well as R(2) and R(adj)(2) for the selection of an adequate calibration curve. They are applied to several analytical methods covering liquid chromatographic methods, as well as electrophoretic ones involved in the analysis of active substances in biological fluids or aimed at quantifying impurities in drug substances. In addition, Monte Carlo simulations are performed to assess the efficacy of these statistical criteria to select the adequate calibration curve.

Methodology for the validation of analytical methods involved in uniformity of dosage units tests.

Validation of analytical methods is required prior to their routine use. In addition, the current implementation of the Quality by Design (QbD) framework in the pharmaceutical industries aims at improving the quality of the end products starting from its early design stage. However, no regulatory guideline or none of the published methodologies to assess method validation propose decision methodologies that effectively take into account the final purpose of developed analytical methods. In this work a solution is proposed for the specific case of validating analytical methods involved in the assessment of the content uniformity or uniformity of dosage units of a batch of pharmaceutical drug products as proposed in the European or US pharmacopoeias. This methodology uses statistical tolerance intervals as decision tools. Moreover it adequately defines the Analytical Target Profile of analytical methods in order to obtain analytical methods that allow to make correct decisions about Content uniformity or uniformity of dosage units with high probability. The applicability of the proposed methodology is further illustrated using an HPLC-UV assay as well as a near infra-red spectrophotometric method.

Plasma level monitoring of the major metabolites of diacetylmorphine (heroin) by the "chasing the dragon" route in severe heroin addicts.

The objective of the present study was to verify if severe physical health problems frequently encountered in heroin addicts and the concomitant use of alcohol and legal or illegal drugs other than heroin influenced the pharmacokinetics of the major metabolites of heroin. We conducted a 90 minutes follow-up of the plasma concentrations of the pharmaceutical heroin, named diacetylmorphine (DAM), in patients recruited in a DAM assisted treatment centre. TADAM (Traitement Assisté par DiAcétylMorphine) aimed to compare the efficacy of heroin-assisted treatment (HAT) compared with methadone maintenance treatment (MMT) for heroin users considered as treatment resistant patients and who have severe physical and mental health problems. Eleven patients were recruited. Blood samples were collected at baseline and 15, 45 and 90 minutes after DAM administration. All patients received DAM by the "chasing the dragon" route. Plasma samples were analyzed by a previously described ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC/MS-MS) method. A principal component analysis (PCA) was performed and 8 metabolite concentrations ratios were calculated to evaluate the influence of various factors (DAM dose, patient pathologies, concomitant use of medications, methadone, street heroin, alcohol and cocaine) on heroin metabolite pharmacokinetics. It seemed to be not affected by the DAM dose, patient pathologies and the concomitant use of medications, methadone, street heroin and alcohol. Cocaine use was the only parameter which showed differences in heroin pharmacokinetics.

Reply to the responses on the comments on "Uncertainty profiles for the validation of analytical methods" by Saffaj and Ihssane.

Saffaj and Ihssane, recently proposed an uncertainty profile for evaluating the validity of analytical methods using the statistical methodology of γ-confidence ß-content tolerance intervals. This profile assesses the validity of the method by comparing the method measurement uncertainty to a predefined acceptance limit stating the maximum uncertainty suitable for the method under study. In this letter we comment on the response (T. Saffaj, B. Ihssane, Talanta 94 (2012) 361-362) these authors have made to our previous letter (E. Rozet, E. Ziemons, R.D. Marini, B. Boulanger, Ph. Hubert, Talanta 88 (2012) 769-771). In particular, we demonstrate that ß-expectation tolerance intervals are prediction intervals, we show that ß-expectation tolerance intervals are highly useful for assessing analytical methods validation and for estimating measurement uncertainty and finally we show what are the differences and implications for these two topics (validation and uncertainty) when using either the methodology of ß-expectation tolerance intervals or the γ-confidence ß-content tolerance tolerance interval one.

Validation of analytical methods involved in dissolution assays: acceptance limits and decision methodologies.

Dissolution tests are key elements to ensure continuing product quality and performance. The ultimate goal of these tests is to assure consistent product quality within a defined set of specification criteria. Validation of an analytical method aimed at assessing the dissolution profile of products or at verifying pharmacopoeias compliance should demonstrate that this analytical method is able to correctly declare two dissolution profiles as similar or drug products as compliant with respect to their specifications. It is essential to ensure that these analytical methods are fit for their purpose. Method validation is aimed at providing this guarantee. However, even in the ICHQ2 guideline there is no information explaining how to decide whether the method under validation is valid for its final purpose or not. Are the entire validation criterion needed to ensure that a Quality Control (QC) analytical method for dissolution test is valid? What acceptance limits should be set on these criteria? How to decide about method's validity? These are the questions that this work aims at answering. Focus is made to comply with the current implementation of the Quality by Design (QbD) principles in the pharmaceutical industry in order to allow to correctly defining the Analytical Target Profile (ATP) of analytical methods involved in dissolution tests. Analytical method validation is then the natural demonstration that the developed methods are fit for their intended purpose and is not any more the inconsiderate checklist validation approach still generally performed to complete the filing required to obtain product marketing authorization.

A rapid validated UHPLC-PDA method for anthocyanins quantification from Euterpe oleracea fruits.

The aim of this work is to develop the first validated UHPLC-PDA method for major anthocyanins quantification in Euterpe oleracea fruits after fast extraction procedures and samples preparation. The separation was performed on HSS C18 column (1.8 µm) using a gradient elution with acetonitrile and 5% formic acid in a total run time of only 17 min. Total error and accuracy profiles were used as criteria for the validation process. Calibration in the matrix was found to be more accurate than calibration without matrix. Trueness (<6.76% relative bias), repeatability (<4.6% RSD), intermediate precision (<5.3% RSD), selectivity, response function and linearity for major anthocyanins, cyanidin-3-glucoside and cyanidin-3-rutinoside, were evaluated. The concentration range validated was 1-48 µg/mL for both compounds. In addition two cyanidin-di-O-glycosides were detected for the fist time in this fruit. We also showed that a first extraction of the fruits with ethyl acetate removes the lipophilic compounds and allows an easier extraction by methanol and quantification of anthocyanins in this extract.

Application of an innovative design space optimization strategy to the development of liquid chromatographic methods to combat potentially counterfeit nonsteroidal anti-inflammatory drugs.

In the context of the battle against counterfeit medicines, an innovative methodology has been used to develop rapid and specific high performance liquid chromatographic methods to detect and determine 18 non-steroidal anti-inflammatory drugs, 5 pharmaceutical conservatives, paracetamol, chlorzoxazone, caffeine and salicylic acid. These molecules are commonly encountered alone or in combination on the market. Regrettably, a significant proportion of these consumed medicines are counterfeit or substandard, with a strong negative impact in countries of Central Africa. In this context, an innovative design space optimization strategy was successfully applied to the development of LC screening methods allowing the detection of substandard or counterfeit medicines. Using the results of a unique experimental design, the design spaces of 5 potentially relevant HPLC methods have been developed, and transferred to an ultra high performance liquid chromatographic system to evaluate the robustness of the predicted DS while providing rapid methods of analysis. Moreover, one of the methods has been fully validated using the accuracy profile as decision tool, and was then used for the quantitative determination of three active ingredients and one impurity in a common and widely used pharmaceutical formulation. The method was applied to 5 pharmaceuticals sold in the Democratic Republic of Congo. None of these pharmaceuticals was found compliant to the European Medicines Agency specifications.

Flexibility and applicability of ß-expectation tolerance interval approach to assess the fitness of purpose of pharmaceutical analytical methods.

An innovative versatile strategy using Total Error has been proposed to decide about the method's validity that controls the risk of accepting an unsuitable assay together with the ability to predict the reliability of future results. This strategy is based on the simultaneous combination of systematic (bias) and random (imprecision) error of analytical methods. Using validation standards, both types of error are combined through the use of a prediction interval or ß-expectation tolerance interval. Finally, an accuracy profile is built by connecting, on one hand all the upper tolerance limits, and on the other hand all the lower tolerance limits. This profile combined with pre-specified acceptance limits allows the evaluation of the validity of any quantitative analytical method and thus their fitness for their intended purpose. In this work, the approach of accuracy profile was evaluated on several types of analytical methods encountered in the pharmaceutical industrial field and also covering different pharmaceutical matrices. The four studied examples depicted the flexibility and applicability of this approach for different matrices ranging from tablets to syrups, different techniques such as liquid chromatography, or UV spectrophotometry, and for different categories of assays commonly encountered in the pharmaceutical industry i.e. content assays, dissolution assays, and quantitative impurity assays. The accuracy profile approach assesses the fitness of purpose of these methods for their future routine application. It also allows the selection of the most suitable calibration curve, the adequate evaluation of a potential matrix effect and propose efficient solution and the correct definition of the limits of quantification of the studied analytical procedures.

Critical review of near-infrared spectroscopic methods validations in pharmaceutical applications.

Based on the large number of publications reported over the past five years, near-infrared spectroscopy (NIRS) is more and more considered an attractive and promising analytical tool regarding Process Analytical Technology and Green Chemistry. From the reviewed literature, few of these publications present a thoroughly validated NIRS method even if some guidelines have been published by different groups and regulatory authorities. However, as any analytical method, the validation of NIRS method is a mandatory step at the end of the development in order to give enough guarantees that each of the future results during routine use will be close enough to the true value. Besides the introduction of PAT concepts in the revised document of the European Pharmacopoeia (2.2.40) dealing with near-infrared spectroscopy recently published in Pharmeuropa, it agrees very well with this mandatory step. Indeed, the latter suggests to use similar analytical performance characteristics than those required for any analytical procedure based on acceptance criteria consistent with the intended use of the method. In this context, this review gives a comprehensive and critical overview of the methodologies applied to assess the validity of quantitative NIRS methods used in pharmaceutical applications.