Journal of Chromatography B (v.877, #23)

Method validation, comparison and transfer by Serge Rudaz; Philippe Hubert (2179).

Analytical method development should aim at delivering reliable measurements within a given application. This implies that method validation is integrated in the development process, because it enables to establish a method's performance capabilities, and to demonstrate its fitness-for-purpose. Although analytical chemists mostly are familiar with the validation guidelines within the discipline of their responsibility, we believe that they may take advantage of a better acquaintance with recommendations among disciplines. Therefore, we review the guidance given in 4 disciplines (laboratory medicine, pharmacy, environment, and food), with emphasis on the proposed experimental protocols, acceptance criteria and interpretation strategies by statistical significance testing. Last but not least, we give incentive towards a modernized validation design.
Keywords: Analytical performance characteristics; Limit of detection; Limit of quantitation; Linearity; Precision; Trueness; Accuracy; Total error; Experimental protocols; Analytical acceptance criteria; Statistical significance testing;

Calibration curves for ligand-binding assays (LBAs) are inherently non-linear and standard curve fitting algorithms require careful selection. Reference standards for macromolecule LBAs are more complex than are low-molecular-weight reference standards. Specificity of small molecule LBAs, and accuracy of reported study sample data are easier to assess by cross-validation with a chromatographic method than for macromolecule LBAs. Due to the lack of knowledge of the potential interference of unknown products of catabolism, proteolysis or biotransformation of macromolecules (particularly proteins) in LBAs for the parent molecule, the accuracy of reported concentrations and derived pharmacokinetic data for macromolecules, as determined by LBA, should be viewed with caution. In validation of LBAs, the total error and confidence interval approaches to assessment of the acceptability of an assay for routine implementation for the desired purpose should be given due consideration.
Keywords: Non-linear calibration curve; Accuracy; Specificity; Reference standard; Incurred sample re-analysis; Acceptance criteria; Total error; Proteins; Xenobiotics;

Validation of bioanalytical LC–MS/MS assays: Evaluation of matrix effects by Ann Van Eeckhaut; Katrien Lanckmans; Sophie Sarre; Ilse Smolders; Yvette Michotte (2198-2207).
Liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry is currently the method of choice for the quantitative determination of drugs in biological matrices. The advantages of this technique include high specificity, sensitivity and throughput. However, co-eluting matrix components, which are not observed in the chromatogram, can have a detrimental effect on the analysis, since they can cause ion suppression or enhancement of the analyte. The evaluation of matrix effects on the quantitative analysis of drugs in biological fluids is an important and sometimes overlooked aspect of assay validation. In this review, the influence of matrix effects on bioanalytical LC–MS/MS methods is discussed and illustrated with some examples. In addition, possible solutions to reduce or eliminate matrix effects are highlighted. A literature overview of validated LC–MS/MS methods published from January till June 2008 is also included. Although matrix effects are investigated in most papers, there is no consensus on how matrix effects should be evaluated during method validation. In addition, the definition of specificity should be changed for LC–MS/MS based methods.
Keywords: Matrix effects; Liquid chromatography; Tandem mass spectrometry; Bioanalysis;

At any phase of the development of a drug or a vaccine, scientists need to compare different (bio)assays. The objective of this paper is to review different statistical methodologies that could be used to assess the equivalence between methods. Depending on the objective (average or individual equivalence), depending on the design, several approaches are detailed: two one-sided t-tests, concordance correlation coefficient, limits of agreement, tolerance intervals and probabilistic approach.
Keywords: Assay; Equivalence; Acceptance limits; Confidence interval; Tolerance interval; Probability;

Methodologies for the transfer of analytical methods: A review by E. Rozet; W. Dewé; E. Ziemons; A. Bouklouze; B. Boulanger; Ph. Hubert (2214-2223).
The transfer of a method from a laboratory to a production site is an important step in the development cycle of new pharmaceutical products. Method transfers are increasingly implemented due to the economical pressure coming from the rationalization of production sites, analytical subcontracting and fusion of pharmaceutical groups. However, no official guidance regarding study design, data analysis, or decision procedures is present neither in FDA documents nor in ICH documents for method transfers. The experiments performed in such a transfer and the methodology used to accept or reject it should be fitted for purpose. In order to provide to analysts a global view of the problematic of analytical method transfer, this paper reviews the documentation available in the scientific literature about the design of transfer studies and the required sample size. Special focus is also made on the statistical methodologies available for decision making with particular emphasis on risk management. Examples of transfer of pharmaceutical, bio-pharmaceutical and biological methods published in the literature are reviewed in order to illustrate the various possibilities among the strategies for methods transfer.
Keywords: Total error; Accuracy; Analytical method transfer; Cross validation; Tolerance interval;

Method validation may be regarded as one of the most well-known areas in analytical chemistry as is reflected in the substantial number of articles submitted and published in peer review journals every year. However, some of the relevant parameters recommended by regulatory bodies are often used interchangeably and incorrectly or are miscalculated, due to few references to evaluate some of the terms as well as wrong application of the mathematical and statistical approaches used in their estimation. These mistakes have led to misinterpretation and ambiguity in the terminology and in some instances to wrong scientific conclusions. In this article, the definitions of various relevant performance indicators such as selectivity, specificity, accuracy, precision, linearity, range, limit of detection, limit of quantitation, ruggedness, and robustness are critically discussed with a view to prevent their erroneous usage and ensure scientific correctness and consistency among publications.
Keywords: Validation; Terminology; Selectivity; Specificity; Accuracy; Precision; Linear function analysis; Range; Limit of detection; Limit of quantitation; Ruggedness; Robustness;

A risk-based analysis of the AAPS conference report on quantitative bioanalytical methods validation and implementation by Bruno Boulanger; Eric Rozet; François Moonen; Serge Rudaz; Philippe Hubert (2235-2243).
The 3rd American Association of Pharmaceutical Scientists (AAPS)/Food and Drug Administration (FDA) Bioanalytical workshop in 2006 concluded with several new recommendations regarding the validation of bioanalytical methods in a report published in 2007. It was aimed to conciliate or adapt validation principles for small and large molecules and an opportunity to revisit some of the major decision rules related to acceptance criteria given the experience accumulated since 1990. The purpose here is to provide a “risk-based” reading of the recommendations of 3rd AAPS/FDA Bioanalytical Workshop. Five decision rules were compared using simulations: the proposed pre-study FDA and Total Error Rules, the rules based on the β-Expectation Tolerance and β-γ-Content Tolerance Interval and, finally, the 4-6-20 rule for in-study acceptance of runs. The simulation results demonstrated that the β-Expectation Tolerance Rule controls appropriately the risk. The β-γ-Content Tolerance Interval was found to be too conservative, depending on the objective, and to lead to a high rate of rejection of procedures that could be considered as acceptable. On the other side, the FDA and the AAPS/FDA workshop Total Error Rule, combined or not, did not achieve their intended objective. With these rules, the risk is high to deliver results in study that would not meet the targeted acceptance criteria. This can be explained because, first, there is confusion between the quality of a procedure and the fitness of purpose of the results it could produce and, second, between the initial performances of a procedure, for example evaluated during pre-study validation and the quality of the future results.
Keywords: Validation; Tolerance interval; Decision rule; Accuracy; Total error; Acceptance criteria; Risk; Fit-for-purpose;

Accuracy, precision and lower limit of quantification (LLOQ) are experimentally achievable key analytical factors by which the quality of analytical methods can be ascribed and objectively evaluated. Endogenous substances (endobiotica) are physiologically present in biological fluids and tissues at varying basal concentration (C 0,Ln). Formally, the definition of accuracy and LLOQ is same for xenobiotica and endobiotica. However, these analytical factors must be determined differently, notably by considering the C 0,Ln value of endobiotica. Often, the impact of the endogeneity on the analytical method is underestimated. This especially applies to the LLOQ, because the LLOQ values for endobiotica are regularly not fixed measures due to the varying C 0,Ln value in biological samples. In order to circumvent these difficulties and for a more reliable and objective evaluation and comparison of analytical methods for endobiotica, this work proposes the use of the relative lower limit of quantification, i.e., rLLOQ. The rLLOQ is defined as the percentage ratio of the LLOQ value, i.e., C LLOQ to C 0,Ln: rLLOQ = (C LLOQ:C 0,Ln) × 100. Thus, the rLLOQ describes that fraction of C 0,Ln that can be still determined with acceptable values for accuracy (e.g., recovery of 100 ± 20%) and precision (e.g., RSD ≤ 20%) or with a total error (i.e., recovery + precision) of ≤30%. Examples from the quantitative analysis of selected endogenous compounds by previously validated GC–MS, GC–MS/MS and LC–MS/MS methods support the appropriateness and expressiveness of the rLLOQ in the quantitative analysis of endobiotica.
Keywords: Accuracy; Comparison; Limit of quantification; Mass spectrometry; Precision; Validation;

This paper describes an attempt to derive a new methodology to determine the significance of effects estimated from a screening design, starting from the algorithm of Dong but overcoming its drawbacks. Especially in situations where effect sparsity does not occur and the number of significant effects approaches 50%, the currently often applied algorithm of Dong leads to many important effects incorrectly considered non-significant, i.e. to false negative results. For these situations, a new methodology is recommended. Based on the algorithm of Dong, several alternative approaches were explored and compared. From all approaches, the one using the 75% lowest absolute factor effects to calculate the initial error estimation s0, i.e. s0  = 1.5 × median|E75%|, resulted in the highest number of correct decisions on effects significance. After its definition, the new methodology was tested on a bioanalysis application data set. This study confirmed the earlier conclusions on literature and semisimulated data. The new methodology is especially interesting to be applied in minimal screening designs, for which other error estimates (e.g. based on interaction or dummy effects) cannot be applied.
Keywords: Screening design; Effects significance; Algorithm of Dong; Effect sparsity;

Statistical methods for assessing long-term analyte stability in biological matrices by David Hoffman; Robert Kringle; Julia Singer; Stuart McDougall (2262-2269).
The objective of a long-term stability experiment is to confirm analyte stability in a given biological matrix, encompassing the duration of time from sample collection to sample analysis for a clinical or preclinical study. While long-term analyte stability has been identified as a key component of bioanalytical method validation, current regulatory guidance provides no specific recommendations regarding the design and analysis of such experiments. This paper reviews and evaluates various experimental designs, data analysis methods, and acceptance criteria for the assessment of long-term analyte stability. Statistical equivalence tests based on linear regression techniques are advocated. Both a nested errors and bivariate mixed model regression approach are suitable for application to long-term stability assessment, and control the risk of falsely concluding stability.
Keywords: Stability; Equivalence; Regression; Bivariate mixed model; Nested errors regression; Bioanalysis;

Many approaches have been proposed for decision criteria to judge whether or not transfer of bioanalytical methods has been successful. Many of these approaches involve mathematical and statistical complexities that limit their use routinely. The FDA accuracy criterion (±15%) without an allowance for imprecision may be used for method transfer and may result in a large numbers of method transfers being judged unacceptable when the method is valid under both conditions. An acceptance criterion should be based on the existing guidance, be convenient and be based on statistical principles that provide consistent and reasonable rejection rates. In the current paper, we propose a “fixed” range acceptance criteria based on the FDA bioanalytical guidance limits on precision and accuracy. While the proposed “fixed” range criteria shares the shortcomings of any other fixed criterion, there are advantages when compared to use of accuracy criterion alone. The proposed criterion is also more user-friendly. Data simulations were performed to assess the probabilities of successful transfer using the proposed criteria. With an experimental design consisting of 3 independent runs with 3 replicates per run, a fixed criterion of ±20% of the reference method mean is proposed.
Keywords: Bioanalytical method transfer; Acceptance criteria; Fixed range; Standard error of mean;

A LC–APPI–MS method was developed and validated for the detection of alprazolam, flunitrazepam and their major metabolites in haemolysed blood. Samples were diluted with water (2:1, v:v) and extracted with a hydrophobic–lipophilic balanced copolymer. The method was fully validated according to ICH guidelines and SFSTP protocols. Deuterated internal standards of both parent drugs were used and good quantitative performance was achieved in terms of trueness and precision (repeatability and intermediate precision) since accuracy profiles were achieved within the acceptance limits (±30% for biological samples). The LC–APPI–MS method was linear over the concentration range of 1–1000 and 3–1000 ng/mL, for alprazolam and flunitrazepam, respectively. Lower limits of quantification as low as 1 ng/mL in haemolysed blood were reached and the method was successfully applied to the quantification of alprazolam, flunitrazepam and their major metabolites in real toxicological samples.
Keywords: Alprazolam; Flunitrazepam; Benzodiazepines; Haemolysed blood; SPE; APPI; LC–APPI–MS; Validation;

In the late 1970s the atypical metabolite of ornithine, ornithine-lactam, has been observed in urine samples of patients suffering from hyperornithinemia. However, not least due to insufficient analytical methods, until now there are no data available about ornithine-lactam in human plasma. Here, we describe a new method, which is, for the first time, suitable to identify and quantify ornithine-lactam in human EDTA-plasma. The method was validated according to the requirements of the FDA guidance for bioanalytical method validation. The analytes were extracted on mixed mode cation exchange SPE columns, separated on a silica analytical HPLC column working in the HILIC mode and detected on a tandem mass spectrometer equipped with an ESI ion source. As internal standard newly synthesized stable isotope labeled D6-ornithine-lactam was used. The calibration function was linear in the range of 0.1–5 μM. Intra- and inter-day precision and accuracy was better than 14% at all concentration levels. In EDTA-plasma samples from 30 volunteers ornithine-lactam concentrations ranging from 0.136 to 0.653 μM were determined. These concentrations correlated significantly (p  < 0.001, R 2  = 0.784) to those of ornithine in EDTA-plasma.
Keywords: Ornithine-lactam; Ornithine; HILIC; LC–MS/MS; Plasma;

A robust, sensitive and selective method to quantify budesonide epimers in human plasma using solid-phase extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and fully validated. The drug was first isolated from the biological matrix by automated solid-phase extraction (SPE) on disposable extraction cartridges (C-2). The methanolic eluate was then collected and evaporated to dryness. The residue was dissolved in mobile phase and an aliquot was injected onto a Phenomenex Luna octadecylsilica (C-18) column (50 mm × 4.6 mm i.d., 3 μm). The mobile phase is composed of water containing 10 mM ammonium acetate adjusted to pH 3.2 with glacial acetic acid and acetonitrile (65:35, v/v). The flow-rate was 1.00 ml/min. Hydrocortisone acetate was used as internal standard (IS). Detection of the analytes was achieved using negative atmospheric pressure chemical ionization (APCI) tandem mass spectrometry in selected reaction monitoring (SRM) mode. The MS/MS ion transitions monitored were m/z 489.3 → 357.3 and 463.3 → 403.2 for budesonide epimers and hydrocortisone, respectively. The method was validated using SFSTP (2003) proposal based on total measurement error and accuracy profiles as a decision tool. The most appropriate regression model for the response function as well as the limit of quantitation was first selected during the prevalidation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for budesonide epimers. The method was validated with respect to stability, recovery, linearity, precision, trueness and accuracy. Risk and uncertainty were also evaluated. The validated method was finally applied successfully to investigate the plasma concentration of budesonide epimers in a pharmacokinetic study.
Keywords: Budesonide; LC–MS/MS; Pharmacokinetics; Solid-phase extraction; Validation strategy;

Validation and long-term evaluation of a modified on-line chiral analytical method for therapeutic drug monitoring of (R,S)-methadone in clinical samples by Nicolas Ansermot; Serge Rudaz; Marlyse Brawand-Amey; Sandrine Fleury-Souverain; Jean-Luc Veuthey; Chin B. Eap (2301-2307).
Matrix effects, which represent an important issue in liquid chromatography coupled to mass spectrometry or tandem mass spectrometry detection, should be closely assessed during method development. In the case of quantitative analysis, the use of stable isotope-labelled internal standard with physico-chemical properties and ionization behaviour similar to the analyte is recommended. In this paper, an example of the choice of a co-eluting deuterated internal standard to compensate for short-term and long-term matrix effect in the case of chiral (R,S)-methadone plasma quantification is reported. The method was fully validated over a concentration range of 5–800 ng/mL for each methadone enantiomer with satisfactory relative bias (−1.0 to 1.0%), repeatability (0.9–4.9%) and intermediate precision (1.4–12.0%). From the results obtained during validation, a control chart process during 52 series of routine analysis was established using both intermediate precision standard deviation and FDA acceptance criteria. The results of routine quality control samples were generally included in the ±15% variability around the target value and mainly in the two standard deviation interval illustrating the long-term stability of the method. The intermediate precision variability estimated in method validation was found to be coherent with the routine use of the method. During this period, 257 trough concentration and 54 peak concentration plasma samples of patients undergoing (R,S)-methadone treatment were successfully analysed for routine therapeutic drug monitoring.
Keywords: Methadone; Enantiomer; Therapeutic drug monitoring; HPLC–MS; Method validation; Long-term evaluation; Matrix effects;

Asymmetric dimethylarginine (ADMA, N G,N G-dimethyl-l-arginine) is an endogenous inhibitor of nitric oxide (NO) synthesis, a potential risk factor for cardiovascular diseases and a powerful biochemical parameter in clinical studies. In our previous work we have reported on a GC-tandem MS method for the accurate and precise quantification of ADMA in biological fluids using de novo synthesized [2H3]-methyl ester ADMA (d3Me-ADMA) as internal standard (IS). This method provides basal ADMA concentrations in biological fluids that agree with those obtained by other groups using other validated methods for ADMA. Unanimously, de novo synthesized stable-isotope labeled analogues are considered not ideal IS, because they must be prepared in a matrix different from the biological sample. Recently, [2,3,3,4,4,5,5-2H7]-ADMA (d7-ADMA) has become commercially available and we took this opportunity to test the reliability of the de novo synthesized d3Me-ADMA as an IS for ADMA in GC-tandem MS. In this article, we report on the re-validation of the previously reported GC-tandem MS method for ADMA in human plasma and urine using d7-ADMA as IS, and on comparative quantitative analyses of ADMA by GC-tandem MS using d7-ADMA and d3Me-ADMA. After thorough standardization of d7-ADMA and methods validation, we obtained by GC-tandem MS very similar ADMA concentrations in plasma and urine using d7-ADMA and d3Me-ADMA. The present study gives a proof of evidence for the aptitude of 2H3-ADMA as IS in GC-tandem MS and suggests that de novo synthesis of stable-isotope labeled alkyl esters of amino acids and amino acid derivates may be a generally applicable method in mass spectrometry-based methods for amino acids. This approach is especially useful for amino acids for which no stable-isotope labeled analogues are commercially available.
Keywords: Amino acids; Comparison; De novo synthesis; O-Methylation; Nitric oxide; Stable-isotopes; Standardization; Validation;

Validation and performance comparison of two carbon isotope ratio methods to control the misuse of androgens in humans by Christophe Saudan; Caroline Emery; François Marclay; Emmanuel Strahm; Patrice Mangin; Martial Saugy (2321-2329).
Carbon isotope ratio of androgens in urine specimens is routinely determined to exclude an abuse of testosterone or testosterone prohormones by athletes. Increasing application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) in the last years for target and systematic investigations on samples has resulted in the demand for rapid sample throughput as well as high selectivity in the extraction process particularly in the case of conspicuous samples. For that purpose, we present herein the complimentary use of an SPE-based assay and an HPLC fractionation method as a two-stage strategy for the isolation of testosterone metabolites and endogenous reference compounds prior to GC/C/IRMS analyses. Assays validation demonstrated acceptable performance in terms of intermediate precision (range: 0.1–0.4‰) and Bland–Altman analyses revealed no significant bias (0.2 ‰). For further validation of this two-stage analyses strategy, all the specimens (n  = 124) collected during a major sport event were processed.
Keywords: Method validation; Isotope ratio mass spectrometry (IRMS); Testosterone misuse; Doping control;

Determination of anabolic steroids in bovine urine by liquid chromatography–tandem mass spectrometry by George Kaklamanos; Georgios Theodoridis; Themistoklis Dabalis (2330-2336).
A liquid chromatography–tandem mass spectrometric (LC–MS/MS) multi-method has been developed for the determination of 15 anabolic steroids in bovine urine (diethylstilbestrol, dienestrol, hexestrol, β-estradiol, ethynylestradiol, α/β-boldenone, α-nortestosterone, α/β-zearalenol, α/β-zaeralanol, zearalenone, stanozolol and 16β-OH-stanozolol). The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, a washing step with hexane and final clean-up with SPE with Oasis HLB and Amino cartridges. The analytes were quantified by liquid chromatography coupled to a tandem mass spectrometer (LC-TSQ Quantum AM) operating in both positive and negative atmospheric pressure chemical ionisation (APCI). Data acquisition was performed in multiple reaction monitoring (MRM) mode quantifying two diagnostic product ions from a chosen precursor. The method was validated according to the Commission Decision 2002/657/EC, for the detection and confirmation of residues in products of animal origin. The method specificity, sensitivity, accuracy and precision were evaluated. The decision limits CCα ranged from 0.06 to 0.26 ng/ml and the detection capabilities CCβ ranged from 0.11 to 0.49 ng/ml. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in bovine urine and can be used for residue control programs.
Keywords: Steroid; Anabolic; Urine; Validation; Liquid chromatography–tandem mass spectrometry (LC–MS/MS);

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography–negative chemical ionization tandem mass spectrometry (GC–NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347 → 163 (for the quantification) and m/z 347 → 119 (for the identification) for EtG, and m/z 352 → 163 for EtG-d5 used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r 2) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.
Keywords: Ethyl glucuronide; Hair; GC–NCI-MS/MS; Quality control; Alcohol marker;

A simple high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection has been developed and validated for simultaneous identification and quantification of three antivitamin K drugs (acenocoumarol, warfarin and phenprocoumon) in whole blood. The aim of this development was to propose an analytical technique adapted to the situations of forensic toxicology, i.e. intoxication with massive anticoagulant doses, when the usual coagulation tests could not be used. The blood sample, after spiked with prazepam as an internal standard (IS), was submitted to a liquid–liquid extraction (LLE) prior to HPLC analysis. A chromatographic separation was achieved on a C8 Symmetry column with a mobile phase consisting of an acetonitrile and phosphate buffer (pH 3.8) mixture in a gradient mode. Detection was carried out at a wavelength between 200 and 400 nm. This method has been validated with the concept of total error as decision criterion. Trueness ranged from 99.1% to 105.0% and precision was good with RSD between 1.3% and 6.7%. Consequently, this rapid and simple chromatographic technique is well adapted to focus intoxications with most important coumarinic drugs available on pharmaceutical market and is now routinely used in our laboratory for forensic “general unknown” screening.
Keywords: Anticoagulant drugs; HPLC; Total error;

In order to quantify oxytetracycline (OTC) in nasal secretions of healthy pigs after intramuscular injection of OTC at doses of 10, 20 and 40 mg/kg bodyweight, an original method based on ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) was developed and fully validated. Sample preparation consisted in protein precipitation preceded by the addition of a releasing protein reagent. Metacycline (MTC) was used as internal standard. Separation was carried out at 65 °C in the gradient elution mode on a short analytical column filled with Acquity BEH C18 stationary phase. The mobile phase consisted in a mixture of water and acetonitrile containing 1 mM of oxalic acid and 0.1% (v/v) of formic acid. The triple quadrupole mass spectrometer operated in the positive electrospray ionization mode; OTC and MTC were detected using multiple reaction monitoring. The pre-study and in-study validation of this bioanalytical method was performed by applying a novel strategy based on total measurement error and accuracy profiles. The maximum risk of observing future measurements falling outside the acceptance limits during routine as well as the measurements uncertainty were also estimated.
Keywords: Oxytetracycline; Nasal secretions; Healthy pigs; UPLC–MS/MS; Validation; Uncertainty;

Nicarbazin, a coccidiostat, is used as a feed additive in poultry but not in laying hens. Feed contamination may however occur resulting in residues being present in eggs. As a Maximum Residue Limit (MRL) does not exist for nicarbazin residues in eggs a “Differential Action Level” (DAL) of 100 μg/kg has been established by the Veterinary Medicines Directorate (VMD). We have studied a commercial ELISA kit validated to detect and quantify nicarbazin in eggs with a sensitivity of 3 μg/kg. We used the total error approach to assess the performance of and validate the kit at the DAL level. The accuracy profile has been successfully obtained for the ELISA kit. The method cannot however be validated as a semi-quantitative method and we have consequently determined a cut-off based on 5% false negative rate according to European Decision 2002/657 on blank and spiked samples (70 μg/kg). The cut-off value established was 20 μg/kg using the 95th percentile.
Keywords: Elisa; Eggs; Nicarbazin; Accuracy profile; Validation;

This paper shows the use of ultra-performance liquid chromatography (UPLC) coupled to orthogonal acceleration time of flight mass spectrometry (TOF MS) for the comprehensive screening of 150 veterinary drugs residues in raw milk. An easy sample preparation based on protein precipitation associated with ultrafiltration was hyphenated to fast chromatography. This method enabled the screening for more than 50 samples per day and searched for 150 drugs and metabolites including avermectines, benzimidazoles, beta-agonists, beta-lactams, corticoides, macrolides, nitroimidazoles, quinolones, sulfonamides, tetracyclines and some others. Identification of contaminants is based on accurate mass measurement. UPLC–TOF also showed very good performances for quantitation and allowed the determination of majority of compounds below MRL. An in-house validation procedure was conducted based on European directive 2002/657/EC with measurement of response function, accuracy, repeatability, limits of detection (LOD), decision limit (CCα) and detection capability (CCβ).
Keywords: Milk; Veterinary drugs; Fast liquid chromatography; UPLC; Time of flight; Mass spectrometry;

A semi-quantitative immunological method (BIACORE method) for detecting sulfamethazine in bovine milk was developed and validated using the total error approach. The acceptance limits were set at ±40% and the risk of procedure of (1−β) proportion measurements falling outside the acceptance limits was chosen at 5%. Different response functions were tested on the basis of the accuracy index (I A). The best model was a weighted (1/X 2) quadratic regression and the simplest one was an unweighted quadratic regression. This approach identified the weak point of the method, which was precision. Finally this BIACORE method was able to detect positive samples containing sulfamethazine in the dosing range between 50 and 150 ng/ml.
Keywords: Validation; Error total; Immunoassay; Sulfamethazine; Milk;

This paper describes an application of multivariate and multilevel quality control charts with the aim of improving the internal quality control (IQC) procedures for the monitoring of dioxins and dioxin-like PCBs analysis in food. Dioxin analysts have to use the toxic equivalent concept (TEQ) to assess the toxicity potential of a mixture of dioxin-like compounds. The TEQ approach requires quantifying individually 29 dioxin-like compounds. Monitoring the congeners separately on univariate QC charts is misleading owing to the increase of false alarm rate. We propose to subdivide the TEQ value into 3 sub-groups and to control simultaneously the 3 variables in a T 2 chart. When a T 2 exceeds the upper control limit, it acts as a warning to trigger additional investigations on individual congeners. We discuss the minimum number of runs required to reliably estimate the QC chart parameters and we suggest using data from multilevel QC charts to properly characterize the standard deviations and the correlation coefficients. Moreover, the univariate QC chart can be sensitised to detect systematic errors by using exponentially weighted moving average (EWMA) technique. The EWMA chart provides an additional guidance on setting appropriate criteria to control the method bias and to support trend analysis. Finally, we present an estimate of measurement uncertainty by computing the accuracy profile in a retrospective way with the QC data generated and we discuss assessment of compliance with regulatory maximum levels.
Keywords: Multivariate quality control; Hotelling's T 2; Exponentially weighted moving average (EWMA); Measurement uncertainty; Polychlorinated dibenzo-p-dioxin (PCDDs); Polychlorinated dibenzofurans (PCDFs); Polychlorinated biphenyls (PCBs);

Determination of complex polysaccharides by HPAE-PAD in foods: Validation using accuracy profile by Max Feinberg; Jinadevi San-Redon; Audrey Assié (2388-2395).
The increasing supplementation of foods with carbohydrates substitutes and the growing regulatory requirements for controlling these products, turn into the necessary development and validation of accurate analytical control techniques. This paper presents the simultaneous validation of two close analytical procedures for the determination of sucralose and fructooligosaccharides (FOS) in fruit juices using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). This study consisted in applying the accuracy profile procedure with a three-level validation experimental design. Decision criteria, namely acceptability limits (±10%) and proportion of result contained in the calculated tolerance intervals (80%), were decided on a consensus basis with end-users, whereas no official references were available. In conclusion, the proposed analytical procedures were validated over the selected validation domains for fruit juices and came out on very capable techniques. Validation strategy was purposely oriented towards the ease of use in routine and the liability of the methods rather than extreme performances. This objective is consistent with this of contract laboratories which need to reach a known level of guarantee for the results which they produce. In that respect, accuracy profile represents a very convenient tool to ascertain such a goal.
Keywords: Sucralose; Fructooligosaccharides; FOS; Accuracy profile; High-performance anion-exchange chromatography with pulsed amperometric detection; HPAE-PAD; Validation;

An improved HPLC-UV method for the simultaneous quantification of triterpenic glycosides and aglycones in leaves of Centella asiatica (L.) Urb (APIACEAE) by M.H. Rafamantanana; E. Rozet; G.E. Raoelison; K. Cheuk; S.U. Ratsimamanga; Ph. Hubert; J. Quetin-Leclercq (2396-2402).
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.
Keywords: Centella asiatica; Madecassoside; Asiaticoside; Madecassic acid; Asiatic acid; Method validation; Quantification;

Total error profiling of a proinsulin time-resolved fluorescence immunoassay by P.E. De Pauw; R.B. Mackin; P. Goubert; C. Van Schravendijk; F.K. Gorus (2403-2406).
We applied total error profiling to evaluate the conversion of a known proinsulin (PI) enzyme-linked immunosorbent assay (ELISA) into a time-resolved fluorescence immunoassay (TRFIA). The formula and acceptance criteria proposed by the Ligand Binding Assay Bioanalytical Focus Group (LBABFG) of the American Association of Pharmaceutical Scientists (AAPS) were applied. We found that the expected dynamic range enlargement with TRFIA compared to ELISA ([0.5–240] versus resp. [0.7–98] pmol/L) is limited by an interference of C-peptide when present in the sample at high concentrations (>7000 pmol/L).
Keywords: Validation; Immunoassays; Total error profiling; Proinsulin; Time-resolved fluorescence; C-peptide; Diabetes;

Use of Total Error concept in the validation of viral activity in cell cultures by N. Gibelin; D. Dupont; S. Imbert; E. Rozet (2407-2411).
Due to the high variability inherent of experimental recipients, validating biological methods is often a complex exercise, and following ICH Q2R1 recommendations is not always feasible and/or meaningful. Linking systematic error and random error to obtain a unique criterion, as defined in ISO guideline, could be of interest to capture the total variability in biological assays. In this paper, the use of Total Error concept in the validation of biological assays was for the first time investigated and compared to a conventional interpretation of the ICH guideline. Both decision methodologies concluded that the assay was valid from 2.13 to 5.83 log10(CCID50/ml). However, only the Total Error approach using accuracy profile as decision tool allowed to guarantee that accurate and reliable results will be obtained during the future routine application of the assay. In addition, the risk to obtain out of acceptance limits results was estimated using this approach and was found out to be at the most 3.1% irrespective of the concentration level, thus demonstrating the reliability of the biological assay.
Keywords: Biological assay; Analytical validation; Accuracy; Total Error; Tolerance interval;

Analytical validation based on total error measurement and cut-off interpretation of a neonatal screening TSH-immunoassay by François Boemer; Vincent Bours; Roland Schoos; Philippe Hubert; Eric Rozet (2412-2417).
To prevent the severe developmental and physical morbidities associated with congenital hypothyroidism, we developed a home-made Enzyme-Linked Immunosorbent Assay (ELISA) method to quantify Thyroid Stimulating Hormone (TSH) levels on newborn dried blood spots. In order to agree with actual clinical laboratory quality referential (ISO 15189), we desired to update our analytical validation protocol. For this purpose, an approach using accuracy profiles based on tolerance intervals for the total error measurement was for first time applied to an immunological assay. According to acceptance limits fixed at ±30%, the method was found accurate over a concentration range from 17.48 to 250 mIU/L. Based on 99.5 percentile of a 16,459 newborn population, cut-off was fixed at 20.1 mIU/L and validated against normal and pathologic neonatal populations. Additionally, uncertainty regions around this value were obtained applying four different approaches. Finally, we demonstrated here our in-house immunological technique fulfils criterions of a neonatal screening policy.
Keywords: Neonatal screening; Congenital hypothyroidism; Validation; Total error; Accuracy profile;