Precision of the reportable result. Simultaneous optimisation of number of preparations and injections for sample and reference standard in quantitative liquid chromatography.

In pharmaceutical analysis, the precision of the reportable result, i.e. the result which is to be compared to the specification limit, is relevant for the evaluation of the suitability of the analytical procedure. But also for other applications, the precision of the result is important and an optimisation often of interest. However, increasing the number of determinations (e.g. injections or preparations) will reduce only the variability (or standard error) of the corresponding precision level. Therefore, the knowledge of the individual variance contributions, obtained from reliable precision studies is important to determine on a scientific basis which format of the (reportable) result, i.e. the number of injections and sample preparations (or even series), should be used. In case of relative analytical procedures such as LC, the calibration model and format, i.e. the number of determinations of the reference standard is one of the factors (besides instrument, operator, reagents, etc.) affecting the between-series variance contribution at intermediate precision/reproducibility level. Consequently, the precision of the reportable result is only valid for the calibration format used to obtain intermediate precision/reproducibility. Instead of repeating the whole precision study to optimize the calibration format, the present paper describes a statistical approach using variability results from the original precision study.

The transfer of analytical procedures.

Analytical method transfers are certainly among the most discussed topics in the GMP regulated sector. However, they are surprisingly little regulated in detail. General information is provided by USP, WHO, and ISPE in particular. Most recently, the EU emphasized the importance of analytical transfer by including it in their draft of the revised GMP Guideline. In this article, an overview and comparison of these guidelines is provided. The key to success for method transfers is the excellent communication between sending and receiving unit. In order to facilitate this communication, procedures, flow charts and checklists for responsibilities, success factors, transfer categories, the transfer plan and report, strategies in case of failed transfers, tables with acceptance limits are provided here, together with a comprehensive glossary. Potential pitfalls are described such that they can be avoided. In order to assure an efficient and sustainable transfer of analytical procedures, a practically relevant and scientifically sound evaluation with corresponding acceptance criteria is crucial. Various strategies and statistical tools such as significance tests, absolute acceptance criteria, and equivalence tests are thoroughly descibed and compared in detail giving examples. Significance tests should be avoided. The success criterion is not statistical significance, but rather analytical relevance. Depending on a risk assessment of the analytical procedure in question, statistical equivalence tests are recommended, because they include both, a practically relevant acceptance limit and a direct control of the statistical risks. However, for lower risk procedures, a simple comparison of the transfer performance parameters to absolute limits is also regarded as sufficient.

Comparison of the recovery spread in analytical development and routine quality control--based on the ICH quality guideline Q2B.

In the present study, a simulation was performed for the ICH Q2B guideline for assessing the accuracy. By means of an experimental data set a permutation has been performed to investigate in which interval experimental mean recovery can be expected to scatter just by random effects. A good agreement has been found between the experimental intervals obtained by means of a permutation and the statistically derived confidence intervals. These findings could be confirmed with additionally generated virtual data sets with a true mean of 100% and a true standard deviation of 0.7%.

Wide concentration range investigation of recovery, precision and error structure in liquid chromatography.

Using a typical HPLC assay, the characteristics of recovery, system precision and repeatability were investigated over a wide concentration range. In the presence of a constant amount of typical tablet excipients, the antidiabetic drug glibenclamide was analyzed in the range from 0.24 to 0.005mg/mL (18 concentration levels, 6 independent sample preparations each). On the basis of a typical concentration for an HPLC glibenclamide assay of 0.2mg/mL, this corresponds to a relative amount of 120-0.025% label claim. In the range from 120 to 0.075%, the recovery was found to be quite constant and systematically heightened mainly due to the evaporation from vials during centrifuging and the displacement of solvent volume by the added matrix. Both system precision and repeatability remain almost constant in the interval from 120 to 10% at a R.S.D.% of 0.31 and 0.70%, respectively, indicating that the sample preparation is the major error source in this range (0.63%). Between 10 and 0.25%, a linear relationship between the logarithmized concentration and the repeatability was noted. However, for lower amounts close to the limit of quantitation, the R.S.D.% of measurements increases much more distinctly. This increase is caused by a strong rise of the system precision. At this concentration range, system precision and repeatability are not significantly different any longer. This leads to the conclusion that with the injection error being constant the peak integration error becomes the dominating error source at low concentrations, e.g. at concentrations below the five-fold of the LOQ. The results obtained here agree well with earlier published data. As the quantitation limit of 0.05% can be regarded as typical for a pharmaceutical impurity control test, generalizations of these findings from this extensive data set should be possible. In this context, peak integration and improvements of the signal-to-noise ratio are the most promising measures to improve an unsatisfactory precision in LC.

Rapidly recomputable EEG forward models for realistic head shapes.

With the increasing availability of surface extraction techniques for magnetic resonance and x-ray computed tomography images, realistic head models can be readily generated as forward models in the analysis of electroencephalography (EEG) and magnetoencephalography (MEG) data. Inverse analysis of this data, however, requires that the forward model be computationally efficient. We propose two methods for approximating the EEG forward model using realistic head shapes. The 'sensor-fitted sphere' approach fits a multilayer sphere individually to each sensor, and the 'three-dimensional interpolation' scheme interpolates using a grid on which a numerical boundary element method (BEM) solution has been precomputed. We have characterized the performance of each method in terms of magnitude and subspace error metrics, as well as computational and memory requirements. We have also made direct performance comparisons with traditional spherical models. The approximation provided by the interpolative scheme had an accuracy nearly identical to full BEM, even within 3 mm of the inner skull surface. Forward model computation during inverse procedures was approximately 30 times faster than for a traditional three-shell spherical model. Cast in this framework, high-fidelity numerical solutions currently viewed as computationally prohibitive for solving the inverse problem (e.g. linear Galerkin BEM) can be rapidly recomputed in a highly efficient manner. The sensor-fitting method has a similar one-time cost to the BEM method, and while it produces some improvement over a standard three-shell sphere, its performance does not approach that of the interpolation method. In both methods, there is a one-time cost associated with precomputing the forward solution over a set of grid points.

Validation in pharmaceutical analysis. Part I: an integrated approach.

The ICH guidelines achieved a great deal in harmonising the definitions of the required validation characteristics and their basic requirements. However, they provide only a basis for a general discussion of the validation parameters, their calculation and interpretation. It is the responsibility of the analyst to identify parameters which are relevant to the performance of the given analytical procedure as well as to design proper validation protocols including acceptance criteria and to perform an appropriate evaluation. In order to fulfil this responsibility properly, the background of the validation parameters and their consequences must be understood. In this part, the general concept of an integrated validation is discussed. The interdependencies to other ICH guidelines and topics during drug development (e.g. impurities and degradants, stability and specification design) must be taken into account to define the required acceptance criteria. Evaluation of the results in order to prove the suitability of the analytical procedure must be based on the specification limits. Important parameters and aspects are discussed for the individual validation characteristics. In the following parts, these parameters will be discussed in detail. Examples will be given for their interpretation in order to facilitate the selection of parameters which are relevant to the performance and suitability of the given analytical procedure.

Applications of hyphenated LC-MS techniques in pharmaceutical analysis.

In pharmaceutical analysis, ie the analytical development and quality control of drug substances and dosage forms, mass spectrometry (MS) combined with chromatographic separation is perhaps the most powerful technique for the monitoring, characterization and identification of impurities. The addition of further dimensions to chromatographic separations by hyphenated techniques offers unique possibilities of efficiently supporting pharmaceutical development and ensuring the quality and safety of pharmaceuticals. However, the ionization process in MS involves some characteristics which have to be recognized and taken into account for an appropriate application as well as the evaluation of the results. Chromatographic method development and validation can be supported very effectively by MS detection, eg in the investigation of coelution and peak purity. Chiral amino acid analysis is largely facilitated by the mass-specific detection of the derivatized amino acid enantiomers, which ignores all other interfering substance peaks. Examples are presented for the use of LC-MS-MS fragmentation and high-resolution MS in the structural elucidation of degradation products and impurities. LC-MS is systematically applied to monitor impurity profiles during pharmaceutical development and scaling up and supports the safety evaluation of batches used in clinical studies.

Paired MEG data set source localization using recursively applied and projected (RAP) MUSIC.

An important class of experiments in functional brain mapping involves collecting pairs of data corresponding to separate "Task" and "Control" conditions. The data are then analyzed to determine what activity occurs during the Task experiment but not in the Control. Here we describe a new method for processing paired magnetoencephalographic (MEG) data sets using our recursively applied and projected multiple signal classification (RAP-MUSIC) algorithm. In this method the signal subspace of the Task data is projected against the orthogonal complement of the Control data signal subspace to obtain a subspace which describes spatial activity unique to the Task. A RAP-MUSIC localization search is then performed on this projected data to localize the sources which are active in the Task but not in the Control data. In addition to dipolar sources, effective blocking of more complex sources, e.g., multiple synchronously activated dipoles or synchronously activated distributed source activity, is possible since these topographies are well-described by the Control data signal subspace. Unlike previously published methods, the proposed method is shown to be effective in situations where the time series associated with Control and Task activity possess significant cross correlation. The method also allows for straightforward determination of the estimated time series of the localized target sources. A multiepoch MEG simulation and a phantom experiment are presented to demonstrate the ability of this method to successfully identify sources and their time series in the Task data.

Pharmacokinetics of promethazine hydrochloride after administration of rectal suppositories and oral syrup to healthy subjects.

The pharmacokinetics of promethazine hydrochloride after administration of rectal suppositories at three dosage strengths and oral syrup were studied. The study had an open-label, randomized, crossover design. At intervals of five to nine days, healthy volunteers were given two 12.5-mg promethazine rectal suppositories, one 25-mg suppository, one 50-mg suppository, or 50 mg (10 mL) of promethazine oral syrup. Blood samples were collected before each dose and at intervals from 0.5 to 48 hours afterward. Promethazine concentration was determined by high-performance liquid chromatography, and pharmacokinetic values were calculated with noncompartmental methods. Thirty-six subjects (18 men and 18 women) completed the study. Absorption was highly variable for all the formulations. On average, absorption was more rapid and the maximum plasma concentration (Cmax) higher for the syrup than for the suppositories. Cmax was significantly lower for the 50-mg suppository (mean, 9.04 ng/mL) than for the syrup (19.3 ng/mL). The time to Cmax (tmax) was significantly shorter for the syrup (mean, 4.4 hours) than for the suppositories (6.7-8.6 hours). There were no significant differences in dose-normalized Cmax among the three suppository treatments. Area under the concentration-versus-time curve (AUC) was comparable between the syrup and the 50-mg suppository and between the treatments with two 12.5-mg suppositories and the 25-mg suppository. Elimination profiles were similar among all treatments (mean half-life [t1/2], 16-19 hours). There were no significant differences in pharmacokinetics on the basis of sex or race. The mean relative bioavailability for the three suppository treatments ranged from 70% to 97%. Individual relative bioavailabilities ranged from 4% to 343%. The pharmacokinetics of promethazine administered in oral syrup and rectal suppositories were highly variable, but, in general, the suppositories produced a lower Cmax and later tmax than the syrup. All formulations were comparable in terms of dose-normalized AUC and t1/2, and the three suppository treatments were comparable in terms of dose-normalized Cmax.

Metabolic disposition of 14C-bromfenac in healthy male volunteers.

The metabolic disposition of 14C-bromfenac, an orally active, potent, nonsteroidal, nonnarcotic, analgesic agent was investigated in six healthy male subjects after a single oral 50-mg dose. The absorption of radioactivity was rapid, producing a mean maximum plasma concentration (Cmax) of 4.9 +/- 1.8 microg x equiv/mL, which was reached 1.0 +/- 0.5 hours after administration. Unchanged drug was the major component found in plasma, and no major metabolites were detected in the plasma. Total radioactivity recovered over a 4-day period from four of the six subjects averaged 82.5% and 13.2% of the dose in the urine and feces, respectively. Excretion into urine was rapid; most of the radioactivity was excreted during the first 8 hours. Five radioactive chromatographic peaks, a cyclic amide and four polar metabolites, were detected in 0- to 24-hour urine samples. Similarity of metabolite profiles between humans and cynomolgus monkeys permitted use of this animal model to generate samples after a high dose for structure elucidation. Liquid chromatography/mass spectrometry (LC/MS) analysis of monkey urine samples indicated that the four polar metabolites were two pairs of diastereoisomeric glucuronides whose molecular weight differed by two daltons. Enzyme hydrolysis, cochromatography, and LC/MS experiments resulted in the identification of a hydroxylated cyclic amide as one of the aglycones, which formed a pair of diastereoisomeric glucuronides after conjugation. Data also suggested that a dihydroxycyclic amide formed by the reduction of the ketone group that joins the phenyl rings formed the second pair of diastereoisomeric glucuronides. Further, incubation of various reference standards in control (blank) urine and buffer with and without creatinine indicated that the hydroxy cyclic amide released from enzyme hydrolysis can undergo ex vivo transformations to a condensation product between creatinine and an alpha-keto acid derivative of the hydroxy cyclic amide that is formed by oxidation and ring opening. Further experiments with a dihydroxylated cyclic amide after reduction of the keto function indicated that it too can form a creatinine conjugate.

The sensory profile: a discriminant analysis of children with and without disabilities.

OBJECTIVES: The purpose of this study was to determine which factors on the Sensory Profile, a measure of children's responses to commonly occurring sensory experiences, best discriminate among children with autism or pervasive developmental disorder (PDD), children with attention deficit hyperactivity disorder (ADHD), and children without disabilities. METHOD: Data for three groups of children 3 to 15 years of age were used: 38 children with autism or PDD, 61 with ADHD, and 1,075 without disabilities. The researchers conducted a discriminate analysis on the three groups, using group membership as the dependent variable and the nine factors of the Sensory Profile as independent variables. RESULTS: The analysis yielded two discriminant functions: one that differentiated children with disabilities from children without disabilities and another that differentiated the two groups of children with disabilities from each other. Nearly 90% of the cases were correctly classified with these two functions. CONCLUSION: The Sensory Profile is useful for discriminating certain groups of children with disabilities. Children with disabilities are accurately classified into disability categories with the factors described by previous authors. This suggests that patterns of behavior associated with certain developmental disorders are reflected in populations of children without disabilities. It may be the frequency or intensity of certain behaviors that differentiate the groups.

The use of hyphenated LC-MS technique for characterisation of impurity profiles during drug development.

As part of an integrated quality concept for impurities during drug development, the multidimensional evaluation of impurity profiles by LC MS coupling is presented using peptide drugs as an example. This quality concept can be regarded as an adaptation of the ICH-requirements to the special situation during the drug development process. The primary goal is to obtain qualitative molecular weight information for impurity peaks detected at the same UV wavelength as for the impurity test procedure. The approach is focused on the investigation, if the impurities in a clinical batch were also present in the toxicologically qualified batch(es). Depending on the relevance of individual impurities in further batches or as degradation products, the molecular weight can be used as a starting point for further characterization and identification. Often, eluents with volatile buffers required for MS result in different selectivities and/or inferior chromatographic separation and sensitivity compared with nonvolatile buffers (e.g. phosphates). In these cases, peak 'tracking' especially for small peaks can become critical. A procedure is presented for on-line coupling of LC methods with non-volatile eluents to mass spectrometry.

Bromfenac disposition in patients with impaired kidney function.

OBJECTIVES: To compare the pharmacokinetics of bromfenac among normal subjects and renally compromised patients and patients with end-stage renal disease. METHODS: Bromfenac pharmacokinetics were examined after a single 50 mg oral dose in 18 subjects with normal kidney function, 12 subjects with decreased kidney function, and 10 dialysis-dependent subjects. Protein binding was assessed by equilibrium dialysis. RESULTS: Mean peak concentrations and areas under the concentration versus time curve ranged from 3.3 to 3.9 micrograms/ml and 5.1 to 6.9 micrograms.hr/ml, respectively. The mean unbound fraction in the subjects receiving dialysis (0.29%) was nearly twice that in the subjects with normal kidney function (0.17%) and in the subjects with impaired kidney function (0.16%), but no differences were detected in clearance, volume of distribution, or their free fraction-corrected counterparts. Bromfenac half-life nearly doubled in the impaired and dialysis groups but was shorter than the anticipated 8-hour dose interval. Eight subjects had a total of 11 study events; none were serious and all were self-limited. CONCLUSIONS: These findings suggest that no dosage adjustment is necessary in patients with impaired kidney function, but clinical monitoring appropriate for their individual condition is recommended.

Pharmacokinetic-pharmacodynamic relationships of bromfenac in mice and humans.

The relationship between pharmacodynamic effect and plasma concentrations of the analgesic bromfenac was assessed retrospectively. The drug was administered in single doses of 5, 10, 25, 50, or 100 mg to patients with oral surgery pain. Concentration-effect curves were generated by a semiparametric pharmacokinetic-pharmacodynamic procedure. The bromfenac EC50 (the effect site concentration giving 50% of the maximum effect) was estimated to be 0.36 microgram/ml in patients when all five dose groups were combined, and an Emax model was used for pharmacodynamic response. A similar EC50 value, 0.40 microgram/ml, was obtained when bromfenac was tested in a mouse pain model. On the basis of combined-dose data, effect site concentrations were predicted to be above the analgesic EC50 for approximately 7-8 hours after a 50-mg bromfenac dose was taken in the fasting state. Predictions based on a pharmacokinetic-pharmacodynamic modeling procedure were in reasonable agreement with the clinical observations.

Quality control of peptide drugs. Chiral amino acid analysis versus standard for icatibant acetate.

A method of chiral amino acid analysis versus standard is presented. By treating the peptide sample and a chiral standard whose intrinsic chiral composition is known in the same way, i.e. simultaneous hydrolysation and analysis, exact corrections can be made for the sequence-related and many hydrolysation-related racemisations. The accuracy obtained in this way permits use of the results for quality control of the chiral purity of peptide drugs. Using the bradykinin antagonist Icatibant acetate (INNM) the method was validated with respect to its precision, accuracy, specificity, limit of detection, and robustness.

Concomitant etodolac affects neither the unbound clearance nor the pharmacologic effect of warfarin.

Potential interactions between the nonsteroidal anti-inflammatory etodolac and the anticoagulant warfarin were studied in 18 healthy subjects by use of a randomized, three-period crossover design. Each treatment lasted 2 1/2 days and consisted of warfarin, etodolac, or both drugs. Prothrombin time was determined daily during each warfarin period to measure pharmacologic effect. Total serum concentration and unbound fraction of both drugs were determined over the dose interval after the last dose of the study drug(s). Concomitant etodolac did not affect the prothrombin time response or the unbound clearance of warfarin. During concomitant etodolac administration, the median peak concentration of total warfarin was significantly decreased by 19% (p = 0.005), median total clearance was significantly increased by 13% (p = 0.0123), and the unbound fraction tended to increase (median unbound fraction of warfarin, 1.245% with etodolac and 1.045% without etodolac; p = 0.0979; not statistically significant). These observations suggest a small displacement of warfarin from serum protein by etodolac or a metabolite of etodolac. No etodolac pharmacokinetic parameter was significantly affected by concomitant warfarin administration. Thus etodolac does not appear to alter the unbound clearance of warfarin or augment its pharmacologic effect. Nevertheless, it is prudent that clinical monitoring be done for individuals taking these two compounds concomitantly.

Subsite affinities of Aspergillus niger glucoamylase II determined with p-nitrophenylmaltooligosaccharides.

Kinetic parameters were obtained for glucoamylase catalysed hydrolysis of substrates of an alpha-(1,4)-maltooligosaccharide series and of a p-nitro-phenyl-alpha-maltooligosaccharide series. p-Nitrophenyl substrates of chain length 11 and 17 were synthesized in 97% and 95% purity, respectively, to test the significance of binding at remote subsites. The affinities of the subsites > 4 are demonstrated to be insignificant. The subsite binding contributions for D-glucopyranosyl and for p-nitrophenyl residues were calculated.

The catalytic power of pyruvate decarboxylase. A stochastic model for the molecular evolution of enzymes.

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate anion by a factor of around 10(12), compared with the non-enzymic decarboxylation by thiamine, under standard state conditions of 1 mM pyruvate and thiamine diphosphate (TDP), pH 6.2. Free-energy diagrams constructed on the basis of earlier measurements for the enzymic and non-enzymic reactions give some information on catalysis by PDC. PDC stabilizes the reactant state preceding TDP addition to pyruvate by 76 kJ mol-1 and the transition state for the addition by 83 kJ mol-1. PDC stabilizes the reactant state preceding decarboxylation (presumably alpha-lactyl-TDP) by 27 kJ mol-1 and the transition state for decarboxylation by 68 kJ mol-1. In addition, the free-energy diagrams reveal a leveling of reactant-state free energies in the enzymic reaction compared with the non-enzymic reaction, in that the former are nearly equal to each other. The enzyme-bound transition-state energies are similarly leveled. The energetic leveling of reactant states has been noted by Albery, Knowles and their coworkers in many enzymic reactions and termed 'matched internal thermodynamics.' They showed that the result would arise naturally (and inevitably) in the 'evolution to perfection' of enzymes, when the evolutionary process was treated by a deterministic model. The critical assumption of this model was the validity of a Marcus-type or Brønsted-type linear free-energy relationship between rate and equilibrium constants for reactions occurring wholly within enzyme complexes. Here a completely stochastic simulation of molecular evolution, with no deterministic assumptions, is shown to reproduce both 'matched internal thermodynamics' and the 'matched internal kinetics' or leveling of transition-state energies noted here. The Albery-Knowles result is thus more general than might have been supposed.

Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.

Accumulation and washout kinetics of valproic acid and its active metabolites.

There is growing evidence that the metabolites of valproic acid (VPA) may be pharmacologically active and could contribute to both the therapeutic and toxic effects of the drug. The accumulation and washout kinetics of VPA and its oxidative metabolites were, therefore, examined in five healthy volunteers. Valproic acid (250-mg capsules) was administered bid for 15 days. Blood samples were obtained periodically during the 15 days of drug administration and for seven days following termination of treatment. Urine was also collected over the final dosing interval. Steady-state serum concentrations of VPA were achieved within three to four days of treatment. The accumulation of all metabolites in serum lagged behind that of the parent compound, with the mono-desaturated metabolites accumulating more slowly than the hydroxylated species. Furthermore, the apparent washout half-life of each metabolite was longer than the elimination half-life of VPA. In general, the unsaturated metabolites were eliminated more slowly than the hydroxylated metabolites. The serum and urinary metabolite profiles of VPA observed in the healthy volunteers were comparable with those reported for epileptic patients. The differences in the disposition kinetics of VPA and of its potentially active metabolites may explain the previously observed dissociation between the pharmacokinetics and pharmacodynamics of the drug in epileptic patients.