Ph. Hubert

Advances in validation, risk and uncertainty assessment of bioanalytical methods

Bioanalytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in order to trust the critical decisions that will be made with them. Even if several guidelines exist to help perform bioanalytical method validations, there is still the need to clarify the meaning and interpretation of bioanalytical method validation criteria and methodology. Yet, different interpretations can be made of the validation guidelines as well as for the definitions of the validation criteria. This will lead to diverse experimental designs implemented to try fulfilling these criteria. Finally, different decision methodologies can also be interpreted from these guidelines. Therefore, the risk that a validated bioanalytical method may be unfit for its future purpose will depend on analysts personal interpretation of these guidelines. The objective of this review is thus to discuss and highlight several essential aspects of methods validation, not only restricted to chromatographic ones but also to ligand binding assays owing to their increasing role in biopharmaceutical industries. The points that will be reviewed are the common validation criteria, which are selectivity, standard curve, trueness, precision, accuracy, limits of quantification and range, dilutional integrity and analyte stability. Definitions, methodology, experimental design and decision criteria are reviewed. Two other points closely connected to method validation are also examined: incurred sample reproducibility testing and measurement uncertainty as they are highly linked to bioanalytical results reliability. Their additional implementation is foreseen to strongly reduce the risk of having validated a bioanalytical method unfit for its purpose.

Enantiomeric purity determination of propranolol by cyclodextrin-modified capillary electrophoresis

Abstract A capillary electrophoretic method for the enantiomeric purity determination of either enantiomer of propranolol was developed using cyclodextrins as chiral additives and uncoated fused-silica capillaries thermostated at 15°C. The effect of the type and concentration of cyclodextrin added to a triethanolamine-phosphate buffer (pH 3.0) on chiral resolution and migration times was studied. The propranolol enantiomers could be separated with all cyclodextrins tested (β-cyclodextrin and seven of its derivatives), except dimethyl-β-cyclodextrin. A particularly high resolution value of 4.4 was obtained for propranolol enantiomers with a buffer containing 10 mM carboxymethyl-β-cyclodextrin. This buffer was selected for testing the enantiomeric purity of propranolol, making it possible to reach detection limits of less than 0.1% for the minor enantiomer. The R enantiomer of propranolol (second migrating) could be quantified at the 0.5% level with good precision (intra-day R.S.D. = 1.7%) in samples of the S enatiomer (first migrating), while the limit of quantification of the latter, when present as an impurity in the R enantiomer, was 0.1%. The method also gave good results in terms of linearity and accuracy.

Innovative high-performance liquid chromatography method development for the screening of 19 antimalarial drugs based on a generic approach, using design of experiments, independent component analysis and design space

An innovative methodology based on design of experiments (DoE), independent component analysis (ICA) and design space (DS) was developed in previous works and was tested out with a mixture of 19 antimalarial drugs. This global LC method development methodology (i.e. DoE-ICA-DS) was used to optimize the separation of 19 antimalarial drugs to obtain a screening method. DoE-ICA-DS methodology is fully compliant with the current trend of quality by design. DoE was used to define the set of experiments to model the retention times at the beginning, the apex and the end of each peak. Furthermore, ICA was used to numerically separate coeluting peaks and estimate their unbiased retention times. Gradient time, temperature and pH were selected as the factors of a full factorial design. These retention times were modelled by stepwise multiple linear regressions. A recently introduced critical quality attribute, namely the separation criterion (S), was also used to assess the quality of separations rather than using the resolution. Furthermore, the resulting mathematical models were also studied from a chromatographic point of view to understand and investigate the chromatographic behaviour of each compound. Good adequacies were found between the mathematical models and the expected chromatographic behaviours predicted by chromatographic theory. Finally, focusing at quality risk management, the DS was computed as the multidimensional subspace where the probability for the separation criterion to lie in acceptance limits was higher than a defined quality level. The DS was computed propagating the prediction error from the modelled responses to the quality criterion using Monte Carlo simulations. DoE-ICA-DS allowed encountering optimal operating conditions to obtain a robust screening method for the 19 considered antimalarial drugs in the framework of the fight against counterfeit medicines. Moreover and only on the basis of the same data set, a dedicated method for the determination of three antimalarial compounds in a pharmaceutical formulation was optimized to demonstrate both the efficiency and flexibility of the methodology proposed in the present study.

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.

An improved HPLC-UV method for the simultaneous quantification of triterpenic glycosides and aglycones in leaves of Centella asiatica (L.) Urb (APIACEAE)☆

The simultaneous quantification of madecassoside, asiaticoside, madecassic acid and asiatic acid in Centella asiatica by HPLC-UV is proposed. Asiaticoside was used as reference for the quantification of heterosides and asiatic acid for aglycones. The evaluation of the extraction efficiency of the four molecules led to use Soxhlet extraction for 8 h. The method was validated and was found to be accurate in the concentration range of 1.0-3.0 mg/ml for asiaticoside and 0.5-2.0 mg/ml for asiatic acid with CV <3% for all investigated compounds. LOD and LOQ were, respectively, 0.0113 and 1.0 mg/ml for asiaticoside and 0.0023 and 0.5 mg/ml for asiatic acid. This method was shown to be convenient for routine analysis of samples of C. asiatica.

Critical analysis of several analytical method validation strategies in the framework of the fit for purpose concept.

Analytical method validation is a mandatory step at the end of the development in all analytical laboratories. It is a highly regulated step of the life cycle of a quantitative analytical method. However, even if some documents have been published there is a lack of clear guidance for the methodology to follow to adequately decide when a method can be considered as valid. This situation has led to the availability of several methodological approaches and it is therefore the responsibility of the analyst to choose the best one. The classical decision processes encountered during method validation evaluation are compared, namely the descriptive, difference and equivalence approaches. Furthermore a validation approach using accuracy profile computed by means of beta-expectation tolerance interval and total measurement error is also available. In the present paper all of these different validation approaches were applied to the validation of two analytical methods. The evaluation of the producer and consumer risks by Monte Carlo simulations were also made in order to compare the appropriateness of these various approaches. The classical methodologies give rise to inadequate and contradictory conclusions which do not allow them to answer adequately the objective of method validation, i.e. to give enough guarantees that each of the future results that will be generated by the method during routine use will be close enough to the true value. It is found that the validation methodology which gives the most guarantees with regards to the reliability or adequacy of the decision to consider a method as valid is the one based on the use of the accuracy profile.

Reliable low-cost capillary electrophoresis device for drug quality control and counterfeit medicines

The proportion of counterfeit medicines is dramatically increasing these last few years. According to numerous official sources, in some pharmaceutical wholesalers in African countries, the proportion has reached 80%. Unfortunately, this situation is far to be improved due to lack of suitable analytical equipment allowing rapid actions of the Regulatory Agencies based on scientific consideration, at affordable cost and all over the drug supply chain. For that purpose, a network group considered that mater by building a low-cost original capillary electrophoresis (CE) equipment equipped with a new deep UV detector based on LED technology. The generic conditions for analysis were investigated: capillary zone electrophoresis (CZE) performed at acidic pH for basic drug molecules (i.e., quinine, highly used as the last antimalarial rampart), basic pH for compounds such as furosemide (a common diuretic drug) and at neutral pH for a well known antibiotic combination, trimethoprim/sulfamethoxazol. To evaluate the ability of the CE equipment for quantification, a full validation and a method comparison study were carried out for the CZE method dedicated to quinine determination. The validation involved the use of accuracy profile and total error concept to monitor the adequacy of the results obtained by the new prototype. The method comparison was based on the Bland and Altman approach by comparing results obtained by the low-cost CE and a conventional set-up. Subsequent validation studies were realized with neutral and acidic drug molecules, each focusing on a single concentration level calibration curve in order to maintain as low as possible the expenses due to reagents and thus the cost of analysis, as important advantages of CE for drug quality control.

Determination of fenofibric acid in human plasma using automated solid-phase extraction coupled to liquid chromatography

The pharmacokinetic studies of fenofibrate require a rapid, selective and robust method to allow the determination of fenofibric acid, its active metabolite, in different biological matrixes (such as plasma, serum or urine). A new fully automated method for the determination of fenofibric acid in plasma has been developed, which involves the solid-phase extraction (SPE) of the analyte from plasma on disposable extraction cartridges (DECs) and reversed-phase HPLC with UV detection. The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with octadecyl silica was first conditioned with methanol and pH 7.4 phosphate buffer. A 0.8-ml volume of diluted plasma sample containing the internal standard (sulindac) was then applied on the DEC. The washing step was performed with the same buffer (pH 7.4). Finally, the analytes were successively eluted with methanol (1.0 ml) and 0.04 M phosphoric acid (1.0 ml). After a mixing step, 100 microl of the resultant extract was directly introduced into the HPLC system. The liquid chromatographic (LC) separation of the analytes was achieved on a Nucleosil RP-8 stationary phase (5 microm). The mobile phase consisted of a mixture of methanol and 0.04 M phosphoric acid (60:40, v/v). The analyte was monitored photometrically at 288 nm. The method developed was validated. In these conditions, the absolute recovery of fenofibric acid was close to 100% and a linear calibration curve was obtained in the concentration range from 0.25 to 20 microg/ml. The mean RSD values for repeatability and intermediate precision were 1.7 and 3.9% for fenofibric acid. The method developed was successfully used to investigate the bioequivalence between a micronized fenofibrate capsule formulation and a fenofibrate Lidose formulation.

Direct liquid chromatographic enantioseparation of sotalol and other β-blockers using an α1-acid glycoprotein-based chiral stationary phase

The behaviour of an α1-acid glycoprotein-based chiral stationary phase (Chiral AGP) towards changes in pH and organic modifier in the mobile phase was investigated in order to deduce suitable conditions for the liquid chromatographic enantioseparation of a series of β-adrenoreceptor blocking drugs. The effects of the pH of the mobile phase on retention, selectivity and resolution were studied. Methanol was the only non-ionic modifier tested in the mobile phase while different aliphatic carboxylic acids (C4 to C8) and alkanesulfonic acids (C6 to C8) were used as ionic modifiers. The influence of the nature and concentration of these modifiers on retention and enantioselectivity was investigated. Under these conditions, enantiomeric separations could be obtained for more than 70% of the β-blocking agents examined. The use of heptanoic acid as an ionic additive in the mobile phase has permitted the resolution of sotalol enantiomers. An enantioselective assay for sotalol was then developed and validated.

Data processing of vibrational chemical imaging for pharmaceutical applications.

Vibrational spectroscopy (MIR, NIR and Raman) based hyperspectral imaging is one of the most powerful tools to analyze pharmaceutical preparation. Indeed, it combines the advantages of vibrational spectroscopy to imaging techniques and allows therefore the visualization of distribution of compounds or crystallization processes. However, these techniques provide a huge amount of data that must be processed to extract the relevant information. This review presents fundamental concepts of hyperspectral imaging, the basic theory of the most used chemometric tools used to pre-process, process and post-process the generated data. The last part of the present paper focuses on pharmaceutical applications of hyperspectral imaging and highlights the data processing approaches to enable the reader making the best choice among the different tools available.