Robustness testing, using experimental design, of a flow-through dissolution method for a product where the actives have markedly differing solubility properties.

The use of experimental design for the robustness testing of a flow-through dissolution method (Ph Eur/USP Apparatus 4) for atovaquone, one of the drug substances in a dual-active anti-malarial tablet formulation, Malarone tablets, is described. This procedure was developed to overcome the suppression of the atovaquone solubility, caused by the presence of the co-drug proguanil hydrochloride and potential imprecision due to the poor solubility of the coating material in the basic dissolution media employed. For this testing a quarter fractional two-level factorial design was applied, assessing six factors in sixteen experiments, with a further six centre points to assess natural experimental variation. Results demonstrate that the method is robust to small changes in all the main factors evaluated at sample times of 30 min or greater. At 15 min, variations in the concentration of sodium hydroxide in the dissolution media, peristaltic pump speed and flow rate were assessed as statistically significant. This observation is a result of the initial steepness of the dissolution release curve and hence these factors are now controlled routinely in the method. Release of this poorly soluble drug is limited at the 45 min time point (Q=75%) according to pharmacopoeial guidelines. The approach may be applied for other dissolution procedures.

The determination of the preservative, chlorocresol, in a pharmaceutical formulation by flow injection analysis.

A flow injection analysis (FIA) procedure is described for the determination of chlorocresol in a parenteral pharmaceutical formulation. The product is directly injected into a carrier stream of water and subsequently reacted with a reagent stream of nitrous acid. The resulting brown nitro-derivative is determined spectrophotometrically at 400 nm. The method has been validated and should be applicable to chlorocresol in other pharmaceutical products and to compounds containing a phenolic ring, assuming absence of matrix interference.

The determination of busulphan in dissolution samples by flow injection analysis.

A robust colourimetric flow injection analysis (FIA) procedure is described for the determination of busulphan in dissolution samples of a 2 mg tablet formulation. The sample solution is injected directly into a reagent stream containing 4-(4-nitrobenzyl)pyridine/potassium hydrogen phthalate. An on-line heating stage allows the formation of a coloured pyrridinium salt species, which following stabilisation is detected spectrophotometrically at 570 nm. The method has been fully validated and is linear over the concentration range 0.004-0.024 mg of busulphan ml(-1). The method can also been applied to uniformity of content and bulk assay testing.

Use of experimental design to optimise a flow injection analysis assay for L-N-monomethylarginine.

A flow injection analysis (FIA) method to determine L-N-monomethylarginine, based on the reaction with ortho-phthalaldehyde in the presence of a suitable thiol-group, was optimised using experimental design. Two different approaches were followed wherein, (i) critical factors were identified in a screening design, and (ii) the simplex algorithm was used for further optimisation. In the first approach, the chemical reaction was optimised off-line and the optimal chemical conditions were transferred to the FIA-system. In the second approach the reaction and the FIA-system parameters were optimised together. The on-line approach is preferred.

The determination of pentavalent antimony in sodium stibogluconate in a pharmaceutical formulation by flow injection analysis.

A flow injection analysis (FIA) procedure is described for the determination of pentavalent antimony (Sb5+) in the drug, sodium stibogluconate, in a parenteral pharmaceutical formulation. The sample solution is injected directly into a carrier stream of iodide ion which is then mixed with an acid stream in situ. Sb5+ is determined by the redox reaction with acidified iodide to liberate iodine, which is monitored spectrophotometrically at 350 nm. The closed conditions prevent interference from atmospheric oxygen and the rapid reaction time assists in minimizing interference from side reactions. The use of tartaric acid as a solvent for sample and standard solutions ensures obedience of Beer's law over the Sb5+ concentration range 0.01-0.2% (w/v). The method is specific for the higher oxidation state in an ionic mixture of Sb5+ and Sb3+, and has been fully validated for use in a pharmaceutical preparation. Assuming absence of matrix interference it is applicable to Sb5+ from other sources and should be applicable to other reducible ionic species.

Development and optimisation of a flow injection assay for fluticasone propionate using an asymmetrical design and the variable-size simplex algorithm.

A flow injection analysis method is described to determine fluticasone propionate, based upon a novel adaptation of the reaction of o-phthalaldehyde with a thiol and a primary amine. The method, which allows both UV and fluorescence detection, has been optimised using experimental design. First a screening is executed to select the significant factors and in a second step these factors are optimised with the variable-size simplex algorithm. In the screening step, a two-level fractional factorial design is compared with an asymmetrical design containing the same number of experiments, but in which one factor is at three levels. It was found that in both designs the same significant variables are detected for the two-level factors, but that for the three-level factor the asymmetrical design confirms an expectation of having a (local) optimum in the examined domain, whilst from the two-level design this is not at all apparent. Complete optimisation was carried out for both UV and fluorescence detection. The two detection methods did not have the same significant variables. For the UV detection, the temperature and the pH adjustment on-line (concentration of sodium hydroxide and amount of boric acid) were the most critical parameters. For the fluorimetric detection the temperature and the fraction of methanol were critical. Moreover the conditions found to be optimal are different for both detection methods.

Validation of a fluorimetric assay for 4-aminophenol in paracetamol formulations.

4-aminophenol (4-AP) is the primary degradation product of paracetamol (PARA). According to the European Pharmacopoeia, 50 ppm 4-AP/PARA is the specification limit of 4-aminophenol in paracetamol drug substance. For drug products, often higher specification limits, such as 1000 ppm 4-AP/PARA are applied. This paper describes a fluorimetric method to quantify the low amount of this degradant (50 ppm) in a pharmaceutical preparation, i.e. in paracetamol tablets. The fluorimetric method was validated and the linearity, precision, trueness, range, limit of detection and limit of quantification were determined. They were found acceptable to assay the low amounts of 4-aminophenol in paracetamol tablets.

Comparison of Plackett-Burman and supersaturated designs in robustness testing.

An optimized FIA assay of L-N-monomethylarginine (LNMMA) was validated. The linearity, precision, accuracy and range of the analytical method were evaluated and robustness testing was performed. Several experimental designs for robustness testing containing different numbers of experiments (N) were compared. Both Plackett-Burman (N=8 or 12) and supersaturated designs (N=6) were examined. The latter design results were analyzed with the Fixing Effects and Adding Rows (FEAR) method, based on the initial addition of zero effect rows to the model matrix, which then are iteratively replaced by fixed effects. It was evaluated whether by reducing the number of experiments from 12 to 8 or 6, similar effects are estimated and considered (non-)significant. The FIA method was found linear in a range of 70-130% of the LNMMA concentration in the samples, and precise and accurate in a range of 80-120%. The estimated factor effects and the critical effects were found comparable for all examined designs, though there also are some indications that some from the supersaturated designs tend to be overestimated. The method was considered robust, since no significant effects occurred for the response describing the quantitative aspect of the method. For other responses, such as peak height and residence time, significant effects occur. However, only the most important effects are found with all designs. The effects reported from a supersaturated design based on the FEAR method still can be subject of further research.

The spectrophotometric determination of hydroperoxide and peroxide in a lipid pharmaceutical product by flow injection analysis.

A sensitive, rapid and automatable flow injection analysis procedure is described for the determination of total hydroperoxides and peroxides in lipid products. All unsaturated lipids are susceptible to degradation by oxidation, and the quantification of these major oxidation products is an essential measure of lipid product stability. In this methodology a lipid emulsion is dispersed and injected into an acidic solution of propan-2-ol, which is then merged with iodide ion in situ in a two-stream manifold. The lipid hydroperoxide oxidises acidified iodide to iodine, which is detected spectrophotometrically at 350 nm. The closed conditions prevent interference from atmospheric oxygen and the short reaction time minimises interference from side reactions. Conditions were optimised, using experimental design, for a lipid product under development at GlaxoWellcome. A two-level half-fractional factorial design was applied to screen for the critical factors, followed by a multi-level central composite design to optimise these variables. The resulting method was fully validated and is linear down to 0.1 nmol ml-1. This approach should be applicable to other lipid formulations and offers significant advantages in terms of speed, automation and precision compared with existing manual procedures.

A sensitive and rapid assay for 4-aminophenol in paracetamol drug and tablet formulation, by flow injection analysis with spectrophotometric detection.

4-Aminophenol (4AP) is the primary degradation product of paracetamol which is limited at a low level (50 ppm or 0.005% w/w) in the drug substance by the European, United States, British and German Pharmacopoeias, employing a manual colourimetric limit test. The 4AP limit is widened to 1000 ppm or 0.1% w/w for the tablet product monographs, which quote the use of a less sensitive automated HPLC method. The lower drug substance specification limit is applied to our products, (50 ppm, equivalent to 25 mug 4AP in a tablet containing 500-mg paracetamol) and the pharmacopoeial HPLC assay was not suitable at this low level due to matrix interference. For routine analysis a rapid, automated assay was required. This paper presents a highly sensitive, precise and automated method employing the technique of Flow Injection (FI) analysis to quantitatively assay low levels of this degradant. A solution of the drug substance, or an extract of the tablets, containing 4AP and paracetamol is injected into a solvent carrier stream and merged on-line with alkaline sodium nitroprusside reagent, to form a specific blue derivative which is detected spectrophotometrically at 710 nm. Standard HPLC equipment is used throughout. The procedure is fully quantitative and has been optimised for sensitivity and robustness using a multivariate experimental design (multi-level 'Central Composite' response surface) model. The method has been fully validated and is linear down to 0.01 mug ml(-1). The approach should be applicable to a range of paracetamol products.

A rapid and precise assay for peroxide as 'active oxygen' in products, by flow injection analysis in a high pressure system with spectrophotometric detection.

A simple, rapid and automated assay for 'active oxygen' originating from hydrogen peroxide, or other organic peroxides, in products is presented employing flow injection (FI) analysis. The product is dispersed and peroxide dissolved in a solvent of 5% (v/v) acetic acid, which constitutes the carrier stream. Ammonium molybdate can be added to this carrier stream to increase sensitivity as required. The sample solution is injected into the acid carrier stream, which is then merged with iodide ion in situ in a two-stream manifold. The 'active oxygen' in the product oxidises acidified iodide to iodine, which is detected spectrophotometrically at 350nm. The closed conditions prevent interference from atmospheric oxygen and the short reaction time minimises the potential for interference from side reactions. Standard HPLC equipment is used throughout, employing a back-pressure to improve precision (high pressure flow injection). Conditions have been investigated using screening multivariate experimental design (two-level quarter fractional factorial design incorporating centre points) to identify and optimise the critical variables. The method has been fully validated (with sample solution R.S.D.s typically < 0.5%, LOQs of 0.04 or 0.006mugml(-1) as 'active oxygen' for acid or acid/molybdate carriers respectively) and is quicker and simpler than the currently employed manual titration approach. It should be applicable to a range of 'active oxygen' products.