In this contribution, an overview on the current legal requirements regarding limits for heavy metals in plant material is given, drawing particular attention to the limits for lead, cadmium and mercury in herbal drugs proposed for the European Pharmacopoeia (Ph. Eur.). A new set of data resulting from about 7100 samples of herbal drugs (fresh and dried) tested for lead and cadmium and about 2500 samples tested for mercury between 2002 and 2007 is presented. Based on this evaluation the proposed limits for the Ph. Eur. can in principle be regarded as acceptable provided that exemptions are included for several herbal drugs, e.g. those accumulating cadmium. Such exemptions might be included either in the general monograph Herbal drugs or in individual Ph. Eur. monographs. For these herbal drugs, respective limits are proposed based on recent experiences from daily practice.

Measurement of somatropin charged variants by isoelectric focusing was replaced with capillary zone electrophoresis in the January 2006 European Pharmacopoeia Supplement 5.3, based on results from an interlaboratory collaborative study. Due to incompatibilities and method-robustness issues encountered prior to verification, a number of method parameters required optimisation. As the use of a diode array detector at 195 nm or 200 nm led to a loss of resolution, a variable wavelength detector using a 200 nm filter was employed. Improved injection repeatability was obtained by increasing the injection time and pressure, and changing the sample diluent from water to running buffer. Finally, definition of capillary pre-treatment and rinse procedures resulted in more consistent separations over time. Method verification data are presented demonstrating linearity, specificity, repeatability, intermediate precision, limit of quantitation, sample stability, solution stability, and robustness. Based on these experiments, several modifications to the current method have been recommended and incorporated into the European Pharmacopoeia to help improve method performance across laboratories globally.

The detection of a contamination of heparin with oversulphated chondroitin sulphate (OSCS) was first analysed in an unfractionated heparin batch supplied to the US API-market in April 2006. OSCS is a semi-synthetic derivative of the natural occuring glycosaminoglycan chondroitin sulphate. Moreover some spectroscopic characteristics of the substance overlap with those of heparin, so that the infrared (IR) spectra are visually difficult to distinguish whereas (1)H-NMR (Nuclear Magnetic Resonance) spectroscopy or capillary electrophoresis (CE) provides identification by a simple visual inspection of either the spectrum or the electropherogram respectively. However, applying special tools of Multivariate Data Analysis (MVA) to the IR spectra an identification of the contaminated samples is possible. In detail a rapid Attenuation Total Reflectance-Infrared (ATR-IR) measurement was selected, which does not require any sample preparation. The result (contaminated or not contaminated) is predicted within a few minutes. A method transfer to mobile ATR-IR spectrometers seems to be possible. The analysis is based on the fact that the fingerprint of the OSCS IR spectrum (1st derivative) complies with a theoretically calculated principal component in the MVA.

This paper presents a rapid method based on MALDI-TOF-MS peptide mass finger printing (PMF) for identification of somatropin. The protein was digested with either trypsin or endoproteinase Lys-C prior to MALDI-MS analysis. The identification was then performed by comparing the measured mass spectra with those predicted in-silico. The cysteine residues at positions 182, 190 and 165 in the protein were identified by carboxyamidomethylation of the cysteine residues with iodoacetamide. One of the two disulfide bridges connecting C182 and C189 was observed in the peptide mass mapping of the unmodified tryptic digest. Sequence coverage obtained from individual trypsin and Lys-C was 79 % and 60 %, respectively. Sequence coverage of both maps together was 98 %.

In pharmacopoeial monographs for herbal drugs and herbal preparations, conventional assay methods such as colorimetry or spectophotometric assays are often replaced by modern, more specific and reliable methods, e.g. liquid chromatography (LC). However, existing dosage recommendations in the monographs on efficacy and safety of herbal medicinal products which are an important basis for licensing procedures do not refer to the mandatory new methods but to the existing photometric methods. The laboratory comparison of the determination of silymarin of Milk Thistle extract shows that a conversion factor can be calculated which allows a correlation between the new and the existing method. It is suggested that this factor should be included in the Ph. Eur. monograph on Milk Thistle extract, allowing reference to dosages given in official monographs (e.g. ESCOP, HMPC). The solution to use a conversion factor should also be applied to other herbal drugs and herbal preparations, especially for standardised extracts.

Quality of homoeopathic mother tinctures is assured by the definition of the starting material, the manufacturing process and the analytical characteristics described in the monograph. Traditionally analytical characterisation of the mother tincture comprises appearance, odour, identity, density and dry residue. According to annex I of directive 2001/83/EC an assay is only performed in case of a health hazard due to toxic compounds. The concept of marker substances as usually used in phytotherapy cannot be transferred to mother tinctures without research effort. For example the marker substances echinacoside, apigenin-7-glucoside and rosmarinic acid found in dried underground parts of Echinacea pallida Nutt., dried flower heads of Matricaria recutita L. and dried herb of Pulmonaria officinalis L. cannot be found in homoeopathic mother tinctures prepared from fresh material thereof.

The treatment of infants and small children with nebuliser-based therapy is commonplace. However, the breathing pattern proposed for general chapter 2.9.44 covering preparations for nebulisation, in development for the European Pharmacopoeia, is that of an adult at rest. This position paper is a concise summary of recommendations for 3 breathing patterns that would be applicable to a neonate, a 12-month-old infant, and a child approximately 4 years old. The information presented herein is intended to inform the development of the proposed general chapter with the intention that information be provided to assist those involved with the evaluation of preparations for paediatric use in the choice of more appropriate breathing patterns for the assessment of active substance delivery rate and total active substance delivered.

The European Pharmaceutical Aerosol Group (EPAG) has undertaken investigations with the aim of developing robust methods for the droplet size analysis of nebuliser-produced aerosols in support of the proposed European Pharmacopeia general chapter 2.9.44 covering preparations for nebulisation. A multi-centre study was designed to investigate the effects of cooling the Next Generation pharmaceutical Impactor (NGI) before sample collection, as a means of reducing bias and variability caused by heat transfer-related evaporation. Droplets containing salbutamol were sized from 3 different nebulisers chosen to offer fundamentally different modes of aerosol generation: AeroNeb Go, a vibrating mesh nebuliser; PARI LC Plus, a breath-enhanced jet nebuliser; and MistyMax, a constant-output jet nebuliser. Each laboratory undertook determinations at ambient temperature, using an NGI pre-cooled in a refrigerator (5 degrees C for at least 90 min). The corresponding measurements were made using an ambient NGI as a benchmark. Salbutamol solution 5 mg/2 ml (Teva, Runcorn, UK) was used throughout the study. Analysis of individual and pooled results from 5 of the participants showed a similar trend insofar as the cooled NGI yielded a coarser nebulised aerosol than that obtained by the ambient NGI. Mass Median Aerodynamic Diameter (MMAD) was on average reduced by 9.5-21.9 % and the Fine Droplet Fraction < 5 microm (FDF) increased on average by 5.5-17.4 % for all the nebuliser designs when comparing ambient to cooled NGI. Despite the more laborious procedure of cooling the NGI, variability in data was generally similar to that obtained with the ambient NGI. We conclude that it is beneficial to cool the NGI when sizing nebulised aerosol. Furthermore, occasional findings during this study revealed a build-up of solute deposits within the interior of the NGI, and a more rigorous impactor cleaning/drying procedure is therefore recommended.

The European Pharmaceutical Aerosol Group (EPAG) has been undertaking a series of investigations in support of developing robust methods for the droplet size analysis of nebuliser-produced aerosols for the proposed European Pharmacopoeia general chapter 2.9.44 covering preparations for nebulisation. The present two-centre study, which was designed to confirm whether or not coating the collection cups of the Next Generation pharmaceutical Impactor (NGI, the recommended impactor for the proposed monograph) is required, forms part of these investigations. The study evaluated NGI-measured aerodynamic droplet-size distributions of aerosols of a generic salbutamol solution for nebulisation generated by two different jet nebulisers. The results demonstrated that there was no indication of biased stage deposition when silicone oil was used to coat the impaction surfaces, as compared to uncoated surfaces. The conclusion was that there is no need to coat the NGI cups when nebulised products are investigated.

Ethyl methanesulfonate (EMS) is a potential human mutagenic and carcinogenic compound which has been found by Roche laboratories in nelfinavir mesylate, the active pharmaceutical ingredient of Viracept. In order to verify the quality of the medicinal product, a gas chromatographic method using mass spectrometry detection was developed for the trace analysis of EMS in Viracept 250 mg tablets from Roche laboratories. Combined with suitable sample preparation including a liquid/liquid extraction this method allows the EMS quantification with a reporting limit of 5 ppm. The extract is injected on a gas chromatographic system with a CP624-CB capillary column. Selected Ion Monitoring mode was used for the EMS quantification. Some validation elements of the method are reported. The validation study was performed over a range from 5 ppm to 100 ppm.

Glycosylation of proteins may, or may not, affect biological activity, either directly or indirectly. In addition, variability in glycosylation arises within the manufacturing procedure. Therefore, glycosylation would be an important parameter when assessing product quality. Regulatory authorities require biotechnological and biological products to be characterised, including determination of their biological activity, physicochemical and immunochemical properties, their purity and their impurity profiles. Here we outline how we can decide if and which types of glycan analysis and methodologies to use for glycoprotein therapeutics to answer the scientific questions as well as meeting regulatory requirements that already exist or are going to be developed/implemented.

Proton NMR was evaluated as an alternative to amino acid analysis as an identity test for peptides. Proton NMR can readily distinguish and identify all peptides currently described in the European Pharmacopoeia (Ph. Eur.). A comparison with amino acid analysis as an identity test is presented.

Evaporative light-scattering detection (ELSD) is an increasingly important technology for the control of substances for pharmaceutical use. A LC-ELSD method has recently been introduced in the Ph. Eur. monograph for sesame oil, refined to control the composition of triglycerides. A collaborative study was run to assess the reproducibility of the new method and to confirm that it was suitable for pharmacopoeial use.

This article describes some recent improvements to simplify the test for heavy metals, avoiding loss of analytes and increasing sensitivity.