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.

Analytical method transfer using equivalence tests with reasonable acceptance criteria and appropriate effort: extension of the ISPE concept.

A method development process is commonly finalized by a method transfer from the developing to the routine laboratory. Statistical tests are performed in order to survey if a transfer succeeded or failed. However, using the classic two-sample t-test can lead to misjudgments and unsatisfying transfer results due to its test characteristics. Therefore the International Society of Pharmaceutical Engineering (ISPE) employed a fixed method transfer design using equivalence tests in their Guide for Technology Transfer. Although it was well received by analytical laboratories worldwide this fixed design can easily bring about high beta-errors (rejection of successful transfers) or high workload (many analysts employed during transfer) if sigma(AN) (error due to different analysts) exceeds 0.6%. Hence this work introduces an extended concept which will help to circumvent this disadvantage by providing guidance to select a personalized and more appropriate experimental design. First of all it demonstrates that former t-test related acceptance criteria can be scaled by a factor of 1.15, which allows for a broader tolerance without a loss of decision certainty. Furthermore a decision guidance to choose the proper number of analysts or series at given percentage acceptance limits (%AL) is presented.

Evaluation of monolithic HPLC columns for various pharmaceutical separations: method transfer from conventional phases and batch to batch repeatability.

Methods developed on conventional particle-packed C18 columns for pilocarpine, propranolol, glibenclamide, glimepiride, insulin and their respective degradation products or related compounds were transferred from the conventional Superspher 100RP-18e column to Chromolith Performance RP-18e columns. All transfers were successful applying the same chromatographic conditions, except for insulin where the acetonitrile content of the mobile phase was reduced by 0.5%. The intraday and interday precisions for both retention time and peak area were evaluated over a wide concentration range. Results were found to be equal, or slightly better on Chromolith Performance with RSD%<1.1% in all cases. Monolithic batch to batch repeatability of both retention time and peak area, compared for monolithic columns from different batches gave an RSD% of less than 1.3%. The separation of each drug and its related products was investigated on monolithic columns at flow rates from 1 to 9 ml/min, and superior resolution was always obtained using monolithic over conventional columns at the same flow rate. A total of seven monolithic columns from four different batches were used in this study.

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%.

Evaluation of monolithic C18 HPLC columns for the fast analysis of pilocarpine hydrochloride in the presence of its degradation products.

Monolithic Performance C18 HPLC columns (Chromolith Performance RP-18e, Merck) were applied for the determination of pilocarpine hydrochloride in the presence of its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid. The method was validated using a set of six monolithic columns and compared to a conventional C18 column. The separation of pilocarpine from its degradation products was investigated on monolithic columns at different flow rates from 1 to 9 ml/min. Superior resolution was obtained using monolithic columns over the conventional C18 column at the same flow rate of 1 ml/min with a total run time of 9 min compared to 13.5 min for the conventional C18 column. Comparable resolution to that obtained in the C18 column (but with better peak symmetry) was obtained at a flow rate of 4 ml/min, although the total run time was reduced to 3.5 min. The precision for both retention time and peak area was investigated over a wide concentration range and found to be equal, or slightly better on Chromolith Performance compared to the conventional column. The overall RSDs% ranged from 0.5% to 1.16% for the conventional column, while for monolithic columns ranged from 0.38% to 0.87% at a flow rate of 1 ml/min and from 0.38% to 0.89% at a flow rate of 4 ml/min. Monolithic column to column reproducibility (n = 6) was measured. The RSDs% ranged from 0.34% to 0.68% for retention time and from 0.3% to 0.94% for peak areas. The detection and quantitation limits on monolithic columns at both flow rates (1 and 4 ml/min) were found to be 0.17 microg/ml and 0.5 microg/ml, compared to 0.31 microg/ml and 1 microg/ml on the conventional column. Monolithic silica rods have also shown the advantage of reducing the time to wash and to re-equilibrate the column. The method showed good linearity and recovery and was found to be suitable for the analysis of pilocarpine hydrochloride formulations.

Application of the equivalence test for analytical method transfers: testing precision using the United States Pharmacopoeia concept (1010).

In this work, the performance of the USP (1010) concept for comparing precision has been investigated. A diagram has been constructed to relate common variance ratios, sample sizes and the corresponding powers. The choice of the upper acceptable limit of variance ratios strongly influences the power. Small upper limits, such as 2.25, are not practical. The proposed upper limit of 4 requires sample sizes of 14 or higher to achieve a power of 80%. If the precision of a method is very good, higher ratios seem to be acceptable, with a significant reduction in measurements. For example, using n = 6 is sufficient to obtain a power of 90%, if the variances are in fact the same and the acceptable variance ratio is 16.

Application of the equivalence test according to a concept for analytical method transfers from the International Society for Pharmaceutical Engineering (ISPE).

The performance of the equivalence test in the context of analytical method transfers was investigated by means of a simulation study. An ISPE design proposal and typical error contributions for pharmaceutical routine control have been used for the testing of accuracy. Acceptable results (probability of a correct decision) have been obtained here. For total variations above 0.4% R.S.D. the basic design was not sufficient. An overview for the number of additional series needed corresponding to higher variations has been developed based on further simulations. An alternative approach may be the choice of wider acceptance criteria, which was also evaluated.

A sensitive TLC method to identify Echinaceae pallidae radix.

In this work a fast, simple and sensitive qualitative TLC method was developed to identify Echinaceae pallidae radix and to distinguish this drug from similar ones. The TLC method is based on the lipophilic compounds of E. pallida. Three mobile phases provided good separation, e.g. toluene/ethylacetate 7 + 3 (v/v). A marker substance was found which shows a blue fluorescence at an excitation wavelength of 366 nm after detection with a spray agent containing 95 volume parts ethanol 96%, 5 parts trifluoroacetic acid 99% and zinc ions in 0.15 molar concentration. After spraying the chromatogram was heated at 110 degrees C for 7 min. This method is superior to HPLC methods to characterise mixtures of Echinacea extracts in terms of selectivity due to this post-chromatographic derivatisation and subsequent fluorescence detection.

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.

Improvements in pentosan polysulfate sodium quality assurance using fingerprint electropherograms.

Complex samples from polymer production, plant extracts or biotechnology mixtures can be characterized by fingerprints. Currently, the standard approach for sample characterization employs near-infrared (NIR) spectroscopy fingerprinting. Up to now, however, fingerprints obtained by chromatography or electrophoresis could only be visually evaluated. This type of inspection is very labor-intensive and difficult to validate. In order to transfer the use of fingerprints from spectroscopy to electrophoresis, spectra-like properties must be obtained through a complete alignment of the electropherograms. This has been achieved by interpolation and wavelet filtering of the baseline signal in the present work. The resulting data have been classified by several algorithms. The methods under survey include self-organizing maps (SOMs), artificial neural networks (ANNs), soft independent modeling of class analogy (SIMCA) and k-nearest neighbors (KNNs). In order to test the performance of this combined approach in practice, it was applied to the quality assurance of pentosan polysulfate (PPS). A recently developed capillary electrophoresis (CE) method using indirect UV detection was employed in these studies [1]. All algorithms were well capable of classifying the examined PPS test batches. Even minor variations in the PPS composition, not perceptible by visual inspection, could be automatically detected. The whole method has been validated by classifying various (n = 400) unknown PPS quality assurance samples, which have been correctly identified without exception.

The lectin from the mushroom Pleurotus ostreatus: a phosphatase-activating protein that is closely associated with an alpha-galactosidase activity. A part of this paper has been presented as a preliminary report at the 17th Interlec. Meeting 1997 in Würzburg, Germany.

The lectin from the mushroom Pleurotus ostreatus described earlier [F. Conrad, H. Rüdiger, The lectin from Pleurotus ostreatus: purification, characterization and interaction with a phosphatase, Phytochemistry 36 (1994) 277-283] was further characterized. Determination of the isoelectric point by capillary electrophoresis gave a value of 7.6. The dissociation constant of the lectin-alpha-lactose-1-phosphate complex determined by capillary electrophoresis is 3 mM. The activation of an endogenous phosphatase by the lectin as found earlier for the pseudosubstrate p-nitrophenylphosphate was confirmed also for naturally occurring substrates as ADP and ATP. We observed that at all purification steps the lectin is accompanied by an alpha-galactosidase activity. Both activities could neither be resolved by electrophoresis under non-denaturing conditions nor by affinity chromatography. However, carbohydrate binding by the lectin and carbohydrate processing by the enzyme are not due to the same site since: (i) the lectin accepts both alpha- and beta-glycosides whereas the enzyme activity is restricted to the alpha-anomer; (ii) the interaction with erythrocytes leads to a stable agglutinate, i.e. no 'clot-dissolving activity' [C.N. Hankins, J.I. Kindinger, L.M. Shannon, Legume alpha-galactosidases which have hemagglutinin properties, Plant Physiol. 65 (1980) 618-622] is observed; (iii) the alpha-galactosidase activity is inhibited by galactose but not by a beta-galactoside. Therefore, lectin and enzymatic activities are either properties of two tightly associated proteins, or of just one molecule. The kinetic parameters of the lectin-associated alpha-galactosidase activity for p-nitrophenyl-alpha-galactopyranoside are: K(M)=2.5 mM, k(cat)=66 s(-1), and K(I)=20mM for the inhibitor D-galactose.

Studies on the structural variations of pentosan polysulfate sodium (NaPPS) from different sources by capillary electrophoresis.

Commercial pentosan polysulfate sodium salts (NaPPS) are highly sulfated polysaccharides derived from beechwood hemicellulose by sulfate esterification with a Mrel range of 1500-6000. The polysaccharide backbone of NaPPS consists of repeating linear units of 1-4 linked beta-D-xylopyranose with laterally substituted 4-methylglucopyranosyluronic acid units glycosidically linked to the 2 position of the main chain at every 10th xylopyranose unit on average. For many years NaPPS has been used for antithrombotic prophylaxis in Europe and interstitial cystitis in the USA and Australia. More recently NaPPS has found veterinary application for the treatment of osteoarthritis and related conditions in domestic animals and is registered for this use in Australia, New Zealand, Canada, UK, Eire, and several Scandanavian countries. At present the use of NaPPS for human disorders is confined to material manufactured by one company. However, for veterinary applications, NaPPS from three manufactures have been described. Since it is well known that the biological activities of sulfated polysaccharides are dependent on their molecular structures we considered it important to characterise these various NaPPS preparations using an established method of analysis. Unfortunately, traditional analytical techniques such as TLC, OR, UV/Vis spectroscopy, and size exclusion chromatography were incapable of providing structural information which would distinguish these NaPPS preparations from each other. In contrast, a capillary zone electrophoresis (CZE) method facilitated characterisation of the different NaPPS by a highly reproducible fingerprint, using a benzene-1,2,4-tricarboxylic acid buffer (8.75 mmol/L, pH = 4.9) with indirect UV detection (lambda = 217 nm) and a special capillary pre-treatment (1 M NaOH for 1 h at 25 degrees C, then running buffer for 120 min at 25 degrees C applying -20 kV). In the present study more than 20 batches of NaPPS from the three manufacturers have been investigated and compared. Minor batch variations were observed to exist for each manufacturer's product however significant differences were detected between NaPPS synthesised by the different manufacturers. Moreover, some preparations showed fingerprint profiles that indicated a more heterogeneous mixture, probably containing other polysaccharides as well.

Possibilities to improve automation, speed and precision of proteome analysis: a comparison of two-dimensional electrophoresis and alternatives.

Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two-dimensional gel electrophoresis (2-DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF-MSn) and preparative IEF followed by size-exclusion chromatography, hyphenated with MS (PIEF-SEC-MS). These two approaches provide a similar separation pattern as the established 2-DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2-DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

Unexpected surface chemistry in capillaries for electrophoresis.

Good and reproducible capillary quality is needed to develop robust methods and to facilitate method transfer in CE. Physical surface defects no longer play a major role in variability of fused-silica capillaries. Nevertheless, problems are frequently being reported when buffers in the pH range between 4 and 7 are used. Thus the surface chemistry has been studied by X-ray photoelectron spectroscopy. Silicon-carbon bindings have been found on inner capillary surfaces for electrophoresis. This binding type is not completely removed by pre-conditioning with 1 M NaOH for 30 min. This corresponds to the result, that capillaries provide more stable migration times, especially in the pH range 4-7, when they are pre-conditioned for longer than 1 h. The origin of this Si-C bond is still not quite clear. They could be caused by graphite which is used during the fabrication of the raw cylinders prior to capillary drawing. Further investigations are intended in order to understand if there are any differences in surface carbon content from batch to batch and if this can influence experimental results in CE. A better understanding of the surface chemistry should not only improve robustness in CE, but also help to facilitate and accelerate capillary pre-conditioning and rinsing procedures to remove strongly adsorbed analytes or matrices.

Quantitation and validation of cis-camphoric acid 3-methyl ester and cis-camphoric acid 1-methyl ester using CE.

The 1- and 3-methyl esters of cis-camphoric acid, the active agents of a mild laxative (Flubilar) have been simultaneously assayed using capillary electrophoresis (CE). The compounds are completely separated using a sodium acetate buffer pH 4.0, 40 mmol/l. In order to obtain reproducible results, [+]-naproxen has been used as internal standard (IS). Initially migration times changed over 50% within a series of 20 runs. This problem has been overcome by using an overnight capillary preconditioning (1 mol/l NaOH, 1.5 h) and subsequent equilibrating (running buffer, 12 h). Thereby a precision corresponding to a CV %, of about 1.17 and 1.42 for the cis-camphoric acid methyl esters has been obtained (six series of n = 10 runs each). The method has been validated regarding specificity, accuracy, precision, linearity and robustness. In order to test robustness, all key parameters have been considered. The result of the validation is given in nine tables. In the case under investigation, lamp age and wavelength accuracy are the most critical parameters. Therefore, the lamp age should be limited to about 1000 h. The wavelength accuracy can be indirectly controlled using quality assurance samples. According to the fundamental mechanisms in CE, changes in the voltage and in the temperature influence migration times and peak areas. However, these effects are very well compensated using an IS. Slight variations of the parameters buffer pH and molarity rinsing times, storage conditions of buffers and samples as well as the capillary material had little or no influence on the analytical results.

Micellar electrokinetic capillary chromatography as a powerful tool for pharmacological investigations without sample pretreatment: a precise technique providing cost advantages and limits of detection to the low nanomolar range.

A number of pharmaceuticals (e.g., acetaminophen, salicylic acid, sulfamethoxazole, theophylline, tolbutamide and trimethoprim) have been determined in human plasma by micellar electrokinetic chromatography (MEKC), without sample pretreatment, using underivatized fused-silica capillaries. The total analysis time was only 10 min. A sodium dodecyl sulfate (SDS)-containing borate buffer (60 mM with 200 mM SDS) at pH 10 was used. Between runs, proteins adsorbed to the capillary wall are removed by rinsing with SDS buffer and either acetonitrile (e.g., 50% v/v) or isopropanol (e.g., 10% v/v). Other rinsing procedures are discussed (salts, enzyme-containing solutions, organic solvents, sodium hydroxide, hydrofluoric acid). The separation system is tested in a concentration range between 10 ng/mL and 100 microg/mL; a detection limit of about 20 ng/mL can readily be obtained. The sensitivity was substantially improved using isopropanol as buffer additive. A day-to-day precision for relative peak areas of 1-2% relative standard deviation (RSD, n > 40) was reached in the upper concentration range. Under repeatability conditions, these values could also be obtained for low microg/mL concentrations. Thus, not only drug monitoring but also pharmacokinetic investigations from blood plasma become possible without further sample pretreatment.

Fused-silica capillaries for capillary electrophoresis and gas chromatography: inner surface corrosion, within-batch differences, and influence of drawing parameters studied by atomic force microscopy.

Fused-silica capillaries for capillary electrophoresis (CE) and gas chromatography (GC) were investigated by atomic force microscopy (AFM). Differences from batch to batch and within one batch were often observed. Surface heterogeneity can be caused by bulk material, manufacturing parameters, or by aging effects. One batch of a fused-silica capillary was stored in water for three years at room temperature. The significant increase in surface roughness (measured as rms = root mean square) during this time is demonstrated. The effect of different drawing temperatures was investigated. Other drawing parameters were kept constant using one capillary batch. If the chosen drawing temperature was too low, the roughness values more than doubled. This increase in roughness did not affect the separation efficiency. However, the relative standard deviation (RSD%) of migration times and peak areas increased at the same time. Three capillary batches for gas chromatography of different inner diameters (250 microm, 320 microm, 530 microm) were also investigated. In all cases the higher rms values for surface roughness could be found at the beginning of the drawing process, although all values were close to atomic flatness.

Pharmacokinetic investigations with direct injection of plasma samples: possible savings using capillary electrophoresis (CE).

Capillary electrophoresis (CE) is often regarded as a separation technique of choice because of its high selectivity and its cost advantages compared to LC.RSD% of 0.5% have become standard for quality control assays. Using CE, sample pretreatment can often be significantly reduced, leading to notable savings of labor and reagent costs. Moreover, errors from sample pretreatment steps are avoided. A number of pharmaceuticals (e.g. acetaminophen, salicylic acid, sulfamethoxazole, theophylline, tolbutamide, and trimethoprim) have been determined in human plasma on underivatized fused silica capillaries by MEKC without sample pretreatment, the total analysis time being only 10 min. An sodium dodecyl sulfate-containing borate buffer (60 mM with 200 mM SDS) at pH 10 has been used. Between runs, proteins adsorbed to the capillary wall are removed by a rinsing regimen consisting of SDS buffer and either acetonitrile (e.g. 50% v/v) or isopropanol (e.g. 10% v/v). Other rinsing approaches are discussed (salts, enzyme containing solutions, organic solvents, sodium hydroxide, hydrofluoric acid). The separation system is tested in a concentration range between 10 ng/mL and 100 micrograms/mL, the detection limit being about 5 ng/mL. The sensitivity has been substantially improved compared to preceding work using field-amplified injection mechanisms and efficient computer algorithms that take advantage of multiwavelength detection. Correlations between the limit of quantitation (LOQ), the limit of detection (LOD) and the signal/noise ratio are discussed. A day-to-day precision for relative peak areas of 1 to 2% relsdv (n > 40) has been reached in the upper concentration range. Thus, not only drug monitoring but also pharmacokinetic investigations from blood plasma have become possible without further sample pretreatment.

Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications.

This review is in support of the development of selective, reproducible and validated capillary electrophoretis (CE) methods. Focusing on pharmaceutical and biological applications, the successful use of CE is demonstrated by more than 800 references, mainly from 1994 until 1998. Approximately 80 recent reviews have been catalogued. These articles sum up the existing strategies for method development in CE, especially in the search for generally accepted concepts, but also looking for new, promising reagents and ideas. General strategies for method development were derived not only with regard to selectivity and efficiency, but also with regard to precision, short analysis time, limit of detection, sample pretreatment requirements and validation. Standard buffer recipes, surfactants used in micellar electrokinetic capillary chromatography (MEKC), chiral selectors, useful buffer additives, polymeric separation media, electroosmotic flow (EOF) modifiers, dynamic and permanent coatings, actions to deal with complex matrices and aspects of validation are collected in 20 tables. Detailed schemes for the development of MEKC methods and chiral separations, for optimizing separation efficiency, means of troubleshooting, and other important information for key decisions during method development are given in 19 diagrams. Method development for peptide and protein separations, possibilities to influence the EOF and how to stabilize it, as well as indirect detection are considered in special sections.

Quality control of pentosane polysulfate by capillary zone electrophoresis using indirect detection.

Pentosane polysulfate sodium salt (PPS) is a mixture of multiply charged anionic polysaccharides, used for urological treatment. Several constituents of the polysaccharide can be characterized by a highly reproducible fingerprint. In comparison with earlier approaches the separation efficiency has been further improved using an anionic benzene-1,2,4-tricarboxylic acid buffer (8.7 mmol l-1, pH = 4.9) with indirect UV detection (lambda = 217 nm) and a special capillary pretreatment (1 M NaOH for 10 h at 25 degrees C applying -20 kV). The method has been optimized with regard to buffer concentration and pH. The robustness was tested on several capillaries. PPS was separated from all major synthetic impurities such a sulfate, chloride and acetate. Twelve PPS batches from two manufactures were measured and compared.