Practical uses of proficiency testing as valuable tools for validation and performance assessment in environmental analysis

Besides their role as an external quality control tool, PT results or samples could be used as an alternative to fulfil some of the quality assurance requirements such as analytical precision, uncertainty assessment, and internal quality control. This additional use of proficiency testing could help laboratories to reduce the financial impact of their quality assurance process. The purpose of this paper is to highlight some practical uses of PT results or samples in the environmental analytical field, which have been implemented at ISSeP (Institut Scientifique de Service Public), either for method validation or for internal quality control.Presented at the Eurachem PT Workshop September 2005, Portorož, Slovenia     

Chemoselective addition of in situ prepared lithium alkynyl borates to aldehydes: a practical and transition metal free approach toward the synthesis of propargylic alcohols

A convenient synthesis of functionalized propargylic alcohols arising from the 1,2-addition of lithium alkynyl-trimethyl borate onto aldehydes under transition metal free mild conditions is reported. The reaction tolerates a wide range of functional groups, is highly chemoselective and the propargylic alcohols are isolated in good to excellent yields.     

Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend^®) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min⁻¹. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and p^H; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and p^H have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control.     

A need for clearer terminology and guidance in the role of reference materials in method development and validation

 The fact that various definitions and terminology applied to measurements in analytical chemistry are not always consistent and straightforward, by not only answering the question ”what”, but also ”how”, leads to their various interpretations. This results in non-uniform implementation of very basic and essential metrological principles in chemistry. Such a diverse situation is not conducive to the endorsement of harmonised measurements all across the world, to serve as a tool for improving the quality of life in its broadest sense for all its citizens. The discussion in this paper is focused on problems associated with terminology and definitions of ’reference material’ and ’validation’. The role of reference materials in measurement processes for purposes other than calibration and validation principles in analytical chemistry are also discussed in this paper. Where possible, potential solutions are proposed, but more often, questions of essential importance are raised in order to initiate international discussion which will hopefully lead to equally understandable answers. Received: 2 November 2002 Accepted: 3 February 2003 Acknowledgements   The author is grateful to Aleš Fajgelj for his comprehensive comments on the topic described in this paper. Sincere thanks also to Philip Taylor, Ewa Bulska, Emilia Vassileva, Miloslav Suchanek and Margreet Lauwaars for their contribution during fruitful discussions on validation. Presented at the CERMM-3, Central European Reference Materials and Measurements Conference: The function of reference materials in the measurement process, May 30–June 1, 2002, Rogaška Slatina, Slovenia Correspondence to N. Majcen     

A convenient and economic approach to achieve SI-traceable reference values to be used in drinking-water interlaboratory comparisons

Metrologically traceable reference values add an essential benefit to interlaboratory comparisons: unlike consensus values, they can be used to establish national and international comparability. Furthermore, the participating laboratories obtain a reliable and unbiased benchmark to check their results for accuracy. Usually, metrologically traceable reference values are obtained by so-called primary methods which demand excessive efforts at great expense. Within the framework of two national drinking-water interlaboratory comparisons (proficiency testing rounds), a new approach to provide metrologically traceable reference values was applied. It is solely based on existing data which were collected during the comparison itself. Lead (Pb) measurements serve as an example to show how metrologically traceable reference values were derived from the lead amount added during sample preparation and the amount of lead already present in the drinking-water matrix used to prepare these samples. Within this approach, the matrix content is calculated in a way similar to a standard addition experiment. An uncertainty budget for the reference value was set up which describes the link to the corresponding SI units. Isotope dilution mass spectrometry (IDMS) as a primary method was used to validate this approach in the case of cadmium, chromium, copper, lead, and nickel.     

Comparability of analytical results obtained in proficiency testing based on a metrological approach

A “yes–no” type of criterion is proposed for the assessment of comparability of proficiency testing (PT) results when the PT scheme is based on a metrological approach, i.e. on the use of a reference material as the test sample, etc. The criterion tests a null hypothesis concerning the insignificance of a bias of the mean of the results from a traceable value certified in the reference material used for the PT. Reliability of such assessment is determined by the probabilities of not rejecting the null hypothesis when it is true, and rejecting it when it is false (the alternative hypothesis is true). It is shown that a number of chemical, metrological and statistical reasons should be taken into account for careful formulation of the hypotheses, enabling the avoidance of an erroneous assessment of the comparability. The criterion can be helpful for PT providers and laboratory accreditation bodies in analysis of PT results.     

Validation of a strategic approach to high-performance liquid chromatographic method selection

Summary A recently reported chromatographic method selection strategy has been validated using fifteen drug formulations selected randomly from the Belgium Compendium, 1992. On the basis of the hydrophobic and the acidic — basic properties of the sample components, reversed-phase was recommended as the first choice mode for all formulations. For two multicomponent formulations the range of analyte polarity dictated the need for gradient elution. The commerical software DryLab G/plus^® was used for selection of optimum gradient conditions. The results obtained by both isocratic and gradient chromatography are discussed, as is the usefulness of a tailing suppressor in both modes.     

A practical approach to the preparative purification of peptides using analytical instrumentation with analytical and semipreparative columns

Summary We have examined the effect of particle size of silica-based reversed-phase packings and column packing techniques on the reversed-phase analytical separation of a peptide mixture. A C₁₈ packing of 15–20 μm average particle size produced satisfactory peptide resolution, allowing a relatively inexpensive scale up to the preparative purification of peptides. A shallow gradient (0.1% acetonitrile/min) elution procedure was developed for the preparative purification of closely related decapeptides (differing by one methyl group) on analytical (250×4.1 mm I.D.) and semipreparative (250×10 mm I.D.) columns. Up to 30 mg and 225 mg of the two-peptide mixture was efficiently resolved, with high yields of homogeneous peptides, on analytical and semipreparative columns, respectively, containing the 15–20 μm packing. We have also demonstrated the potential of our purification procedure for resolving more complex multicomponent mixtures by efficiently separating a total of 22 mg of three closely-related peptides on analytical columns containing 7 μm or 15–20 μm particle size reversed-phase packings. The use of the inexpensive 15–20 μm packing, coupled with the ability to pack efficient columns with analytical HPLC instrumentation, offers great cost saving potential.     

Nonparametric assessment of comparability of analytical results obtained in proficiency testing based on a metrological approach

A nonparametric sign test is implemented for assessment of comparability of proficiency testing (PT) results when their distribution differs from the normal or other known distribution. It allows testing the null hypothesis about insignificance of the bias of median of results obtained in PT from the traceable certified value of the reference material used in PT as test items, i.e., the hypothesis stating that comparability of the PT results is successful. Probability of type I error of rejecting the null hypothesis when it is true, and probability of type II error of it not rejecting when it is false (the alternative hypothesis about unsuccessful comparability is true) are considered. The test can be helpful for PT providers and laboratory accreditation bodies in analysis of PT results when the comparability criterion developed for a normal results distribution (Accred. Qual. Assur. 10:466–470) is not applicable.     

Determination of acyclovir in plasma by high performance liquid chromatography with UV detection. Method development and method validation

A HJPLC method for the determination of acyclovir in plasma is described. The method is simple and sensitive enough for bioequiva-lence studies, where a large number of plasma samples with low acyclovir concertration are involved. The procedure is based on the deproteinization of plasma with perchloric acid and separation of acyclovir on a Hypersil ODS Column at pH 5.6 with UV detection. The calibration standards are linear up to at least 4000 ng/mL and the limit of quantification is 10 ng/mL.     

Development and validation of a UPLC method for quantification of antiviral agent,Acyclovir in lipid-based formulations

PurposeThe objective of the current study is to evaluate the Ultra Performance Liquid Chromatography (UPLC) method for quantification of Acyclovir in lipid-based formulations.MethodA simple, rapid, reliable and precise reversed phase UPLC method has been developed and validated according to the regulatory guidelines, which composed of isocratic mobile phase; 0.25% formic acid (FA) in Milli-Q water with a flow rate of 0.5 ml/min, and column BEH C18 (2.1 × 50 mm, 1.7 μm). The detection was carried out at 254 nm.ResultsThe developed UPLC method was found to be rapid (1.2 min run time), selective with well resoluted Acyclovir peak (0.89 min) from different lipid matrices and sensitive (Limit of Detection (LOD) was 0.3 ppm and Lower Limit of Quantification (LLOQ) was 1 ppm). The accuracy and precision were determined and were perfectly matching with the standard FDA limits.ConclusionThe study showed that the proposed UPLC method can be used for the assessment of drug purity, stability, solubility and lipid-formulation release profile with no interference of excipients or related substances of active pharmaceutical ingredient.     

Development and validation of a capillary electrophoresis method with ultraviolet detection for the determination of the related substances in a pharmaceutical compound

A capillary electrophoresis (CE) method was successfully developed to quantify the impurity profile of a new substance of pharmacological interest: LAS 35917. CE method was developed in order to separate the chloromethylated, monomethylated and hydroxylated impurities (molecules with very similar chemical structures) having the three coelution in the reversed-phase LC method initially established. Taking into account the structure of the impurities of LAS 35917, separation by conventional liquid chromatography (LC) methods would be longer and tedious than separation by CE, which is an appropriate and versatile technique giving easier and quicker methods. Among the three potential impurities mentioned of LAS 35917, two are due to the synthesis route of this drug, and the third arises from degradation. These drug-related impurities were separated using a capillary of 56 cm of effective length and 50 microm I.D., a 60 mM tetraborate buffer, at pH 9.2, and a positive voltage of 20 kV. The optimised CE method was preliminary validated with regard to specificity, linearity, limits of detection and quantitation, repeatability and solution stability. The method allows the detection and quantitation of impurities above 0.04 and 0.08% level, respectively. All three related substances were separated, detected and quantified from their parent drug in the analysis of real samples of LAS 35917, stressed or not stressed, with this simple and fast CE method.     

Development and validation of a high-performance liquid chromatography-tandem mass spectrometric method for quantification of daunorubicin in rat plasma

A high-performance liquid chromatography-tandem mass spectrometric method (LC-MS/MS) has been developed and validated for the determination of daunorubicin in K₃EDTA rat plasma. The 100 μL plasma samples were extracted by a methanol:acetone protein precipitation step in the presence of additional 50 μL of 70% (w/v) zinc sulfate, and subsequently analyzed by LC/MS/MS using positive turbo-ion spray ionization mode. The LC/MS/MS instrument was operated in the multiple-reaction-monitoring (MRM) mode. Doxorubicinol was better than doxorubicin as the internal standard because its recovery and absolute matrix effect data exactly matched with those for daunorubicin. In addition, HPLC gradient condition was optimized to thoroughly separate daunorubicin from the background interference. The validated concentration range was from 0.250 to 100 ng/mL. The true recoveries of daunorubicin and doxorubicinol were 93.2% and 93.6%, respectively. In addition, the ion-suppression data of daunorubicin and doxorubicinol were 78.2% and 78.4%, respectively. Absence of the relative matrix effect from six unique lots was confirmed. Results obtained from the GLP validation study demonstrated very good accuracy (95-105%) and precision (less than 10% CV).     

A tutorial on the validation of qualitative methods: From the univariate to the multivariate approach

This tutorial provides an overview of the validation of qualitative analytical methods, with particular focus on their main performance parameters, for both univariate and multivariate methods. We discuss specific parameters (sensitivity, specificity, false positive and false negative rates), global parameters (efficiency, Youden's index and likelihood ratio) and those parameters that have a quantitative connotation since they are usually associated to concentration values (decision limit, detection capability and unreliability region).     

Ion chromatographic determination of lithium at trace level concentrations: Application to a tracer experiment in a high-mountain lake

The water residence time of a high-mountain seepage lake in the Austrian Alps was derived from the flushing rate of a tracer substance. A diluted lithium chloride solution was injected into the lake during holomictic conditions in order to favour the homogeneous distribution of the tracer. The exponential decline of the mass of lithium in the lake revealed a water residence time of 1.5 to 3 months for summer and almost no lake water exchange during winter. Lithium concentrations ranged from background values of 0.06 μg l⁻¹ to about 3 μg l⁻¹ immediately after the tracer injection. Lake water samples were analyzed with ion-exchange chromatography using a Dionex device with a CS 12A separation column. The method detection limit determined according to the definition of the US Envirinmental Protection Agency amounted to 0.009 μg l⁻¹.     

A definitive RNAA method for determination of selenium in biological samples: uncertainty evaluation and assessment of degree of accuracy

This article describes work on the development of a highly accurate RNAA method for determination of selenium in biological samples. The analytical post-irradiation procedure is based on a combination of cation-exchange and extraction chromatography with final selective and quantitative fixation of selenium on a column packed with 3,3′-diaminobenzidine (DAB) supported on Amberlite XAD4, followed by gamma-ray spectrometric measurement. The suitability and accuracy of the method was demonstrated by analysing CRMs with certified selenium content. The uncertainty budget for Se determination in standard reference material Peach Leaves NBS 1547 was estimated; the combined standard uncertainty was calculated as 1.7%. The described method fulfils all the criteria for definitive methods. It was subsequently used for determination of selenium in biological materials intended as new CRMs and proficiency test samples.     

Unauthorised antibiotic treatments in beekeeping: Development and validation of a method to quantify and confirm tylosin residues in honey using liquid chromatography–tandem mass spectrometric detection

A liquid chromatographic–tandem mass spectrometric (HPLC-MS/MS) method is proposed for the identification and quantification of tylosin in honey. Sample treatment involves an extraction in a Tris buffer at pH 10.5, followed by a solid-phase clean up step on an Oasis HLB column. Roxithromycin was used as the internal standard. Chromatographic separation of tylosin and roxithromycin was performed on an XTerra MS C₁₈ column (100 mm × 2.1 mm i.d., 5 μm) using a gradient of aqueous 0.01 M ammonium acetate pH 3.5 and acetonitrile as the mobile phase, at a flow rate of 0.25 ml min⁻¹. The method was validated according to the guidelines laid down by the Commission Decision 2002/657/EC. Tylosin residues were confirmed by MS/MS experiments considering the appropriate identification points. All validation parameters such as Cc (lower than 3 ng g⁻¹), Ccβ (lower than 5 ng g⁻¹), recovery and precision were assessed on the basis of the “critical ion” (less intense ion permitting unambiguous identification of the analyte).     

Preparation of lithium stannide mixtures in organic solvents. A alternate source of lithium in organolithium chemistry

Lithium stannides were prepared from lithium naphthalenide and tin (II) chloride or tin (0) powder in THF solvent at room temperature under dry argon atmosphere. They were characterized with elemental analysis, XRD, and solid ^6,7Li NMR. Stabilities and reactivities of lithium stannides prepared from different conditions were tested and showed they were stable for a limited time at low temperatures. Best reactivity was obtained when they were prepared from tin (II) chloride and an excess of lithium naphthalenide. The lithium stannide mixture can reductively cleave carbon-halogen bonds and yield pinacol coupling with aldehydes. Organolithium compounds prepared from lithium stannides and organic halides add to ketones or aldehydes under Barbier conditions.     

Characterization of a novel crown ether-bearing near-infrared heptamethine cyanine dye. A study of fluorescence quenching by lithium

The spectral characteristics of a crown ether-bearing heptamethine cyanine dye (JCM-15C5) and its quenching by lithium ion are reported. The absorbance maximum of the dye is at 776 nm in acetonitrile. This value matches the output of a commercially available laser diode (780 nm), thus making use of such a source practical for excitation. The emission wavelength of the dye in acetonitrile is at 799 nm. It was found that Li⁺ ion selectively quenches the fluorescence intensity of JCM-15C5 by the static quenching mechanism. The Stern-Volmer quenching constant (K_sv) was calculated from the Stern-Volmer plot and found to be 1.17×10⁷ M⁻¹ at room temperature. The detection limit for Li⁺ ion was found to be 7.43×10⁻² ppb. The stability constant (K_s) of the metal-dye complex was determined by fluorometric titration and found to be 5.40×10⁷ M⁻¹.