Characterization of disposable optical sensors for heavy metal determination.

This paper presents the development, characterization and quality control of analytical methods based on the use of disposable optical sensors for determination of heavy metals. Chromogenic reagents such as 1-(2-pyridylazo)-2-naphthol, (2-pyridylazo)resorcinol, Zincon, Ferrozine, and Chromazurol S were used to develop optical sensors of heavy metal ions found as contaminants in pharmaceutical substances and products, such as Zn(II), Cu(II), Ni(II), Fe(II), and Fe(III). The chromogenic reagents were immobilized in polymeric membranes by spin-coating from cocktails containing all reagents needed. The methods were prevalidated using a comprehensive quality control strategy based on a system of mathematical/statistical testing and diagnosis of each prevalidation step. This system involved characterization of analytical groups; checking of two limiting groups; testing of data homogeneity; recognition of outliers; and determination of analytical functions, limiting values, precision and accuracy. The prevalidation strategy demonstrated the reliability of the proposed method and pointed out some limitations. Combining the optical sensors with multicomponent linear regression allowed simultaneous determination of multiple metals in synthetic mixtures with different compositions. Good agreement between experimental and theoretical amounts of heavy metals in the mixtures was obtained for the majority of sensors and metals. Even better agreement was obtained between the experimental and theoretical total amounts of metals in the mixtures. The proposed analytical methods were successfully applied to the determination of zinc in pharmaceutical preparations of insulin and the determination of metal mixtures in a commercial nasal spray of isotonic seawater. The reliable and sensitive individual optical sensors developed in this study may be useful for designing a multimembrane optical tongue that with appropriate further optimization can be used for screening heavy metals in various matrices.

Development and validation of a sensitive and fast solid-phase spectrophotometric procedure for phenol determination in pharmaceuticals.

A very sensitive, simple, and fast solid-phase spectrophotometric procedure for the determination of phenol using the p-nitrobenzenediazonium reagent (DAR reagent) was developed. This procedure is based on the simultaneous concentration of the orange product on a Dowex 1-X2 anion exchanger within 15 min, and a direct absorbance measurement of the sorbed species at both 530 nm (the absorption maximum of the phenol-DAR in the resin phase) and 700 nm (the range where only the resin absorbs light). Quality control and evaluation of the analytical parameters was carried out using a comprehensive prevalidation strategy. The linearity of the method was confirmed within an analyte working range from 0.01 to 0.10 µmol (0.2 to 2.0 nmol mL(-1)). The precision ranged from ±1.17 to ±9.61%; the accuracy ranged from -17.50 to +17.81%. The evaluated limiting values were L(D) = 0.0013 µmol and L(Q) = 0.0082 µmol. The DAR-SPS method was successfully applied to the determination of phenol in a pharmaceutical sample of salicylic acid (98.0-100.0%) and vaccines (98.0-103.4%).

Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.

Quality assessment of the procedure for determination of heavy metals on reactive carriers.

Quality control and validity check of the solid-phase spectrophotometric procedure (Me-SPS) for determination of heavy metals important in pharmaceutical practice: zinc (Zn-SPS), lead (Pb-SPS), and cobalt (Co-SPS) were performed using a very simple and informative prevalidation concept. Further, metrological characteristics of the Me-SPS procedure together with the diagnosis of particular prevalidation steps were evaluated. Favourable prevalidation characteristics, i.e., ideal analytical function, homogeneity of data material, low limit of quantitation, and acceptable accuracy verified the quality of the Me-SPS procedure and pointed to the limitations of some investigated systems.

Prevalidation in pharmaceutical analysis. Part I. Fundamentals and critical discussion.

A complete prevalidation, as a basic prevalidation strategy for quality control and standardization of analytical procedure was inaugurated. Fast and simple, the prevalidation methodology based on mathematical/statistical evaluation of a reduced number of experiments (N < or = 24) was elaborated and guidelines as well as algorithms were given in detail. This strategy has been produced for the pharmaceutical applications and dedicated to the preliminary evaluation of analytical methods where linear calibration model, which is very often occurred in practice, could be the most appropriate to fit experimental data. The requirements presented in this paper should therefore help the analyst to design and perform the minimum number of prevalidation experiments needed to obtain all the required information to evaluate and demonstrate the reliability of its analytical procedure. In complete prevalidation process, characterization of analytical groups, checking of two limiting groups, testing of data homogeneity, establishment of analytical functions, recognition of outliers, evaluation of limiting values and extraction of prevalidation parameters were included. Moreover, system of diagnosis for particular prevalidation step was suggested. As an illustrative example for demonstration of feasibility of prevalidation methodology, among great number of analytical procedures, Vis-spectrophotometric procedure for determination of tannins with Folin-Ciocalteu's phenol reagent was selected. Favourable metrological characteristics of this analytical procedure, as prevalidation figures of merit, recognized the metrological procedure as a valuable concept in preliminary evaluation of quality of analytical procedures.

Determination of aucubin and catalpol in Plantago species by micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) was used for the separation and determination of two iridoid glycosides, aucubin and catalpol, in several Plantago species growing in Croatia: P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. (subsp. depauperata, subsp. holosteum and subsp. scopulorum), P. lagopus L., P. lanceolata L., and P. maritima L. Hot water extraction (HWE) was applied for the isolation of iridoid substances. Significant differences appeared between the iridoid contents in the examined species. The yield of aucubin and catalpol was up to 0.27% and 1.81% of the dry mass of the leaves, respectively. Besides aucubin and catalpol, two related compounds were determined in the plant samples.