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.
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.
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 %.
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.
Proton NMR was evaluated as an alternative to amino acid analysis (AAA) as an identity test for peptides. Proton NMR can readily distinguish and identify all peptides currently described in the European Pharmacopoeia. A comparison with AAA as an identity test is presented.
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.
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.
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.
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.
The European Pharmacopoeia (Ph.Eur.) monograph for Cefamandole Nafate (CFN) and the revised monograph prescribe the identification of the antibiotic in solid state by infrared (IR) absorption spectrophotometry using potassium bromide (KBr) disc technique. But, this technique may cause unwanted solid-solid transformations in the crystalline structure of the beta-lactam antibiotic CFN. The latter is a drug with proven polymorphism/pseudopolymorphism. In this context we have examined the suitability of the two techniques (KBr disc and Nujol mull) for IR spectral analyses to identify the antibiotic CFN in solid state. The results of our examinations show that KBr disc technique alters the crystalline state of CFN during the preparation of its KBr disc samples by the tribomechanical treatments (grinding and compression pressure). On the contrary, the Nujol mull technique does not cause such transformations and it is estimated as a better, more suitable technique to be employed for identification of CFN. For a greater precision, in solving the possible difficulties due to the KBr disc technique a second record of IR spectra is necessary to be provided by the Nujol mull technique with use of an internal standard (the stretching vibration of vC = 0 of the condensed beta-lactam cycle).
With the ICH harmonisation of the chapters 'Microbiological examination of non-sterile products: microbial enumeration tests' 'Test for specified micro-organisms' and 'Microbiological quality of pharmaceutical preparations' between Ph. Eur., USP and JP, altered specifications and test methods for pharmaceutical preparations are applicable in Europe since 2007 From this results the necessity to check and, where appropriate, adapt microbiological limit values in the individual monographs of the Ph. Eur.- provided that they contain such values. The present examination, which gives an evaluation of 3387 examinations in the period from 1997 to 2006 of the microbiological quality of 40 different pharmaceutical raw materials, is to provide a database for this purpose. It was shown that the existing limit values were not exceeded for 93% of the examined raw materials. 5.5% of the examinations were within the specification after using the valid tolerance factor of 5 of the old methods. Only 1.5% of all examinations led to OOS results. In comparison an evaluation of these data against the new, harmonised limits is leading to an increase in OOS results to a total of 6%. Most raw materials were of the required microbiological quality but a few, predominantly of plant origin, exceeded the limits. Regular incoming goods inspections are indispensable here.
This article describes some recent improvements to simplify the test for heavy metals, avoiding loss of analytes and increasing sensitivity.
The discriminatory power of the new and the old requirements are compared on a mathematical basis and by the simulation of data. The probability of passing the requirements of the test for uniformity of dosage units versus the tests for uniformity of content and uniformity of mass are compared by the simulation of different batches. These batches are characterised by a true content and true standard deviation under the prerequisite of normal distribution. Furthermore, a mathematical evaluation of the newly introduced acceptance value AV is presented.
Aerosol inhalers are widely used to deliver drug directly to the respiratory system in patients with respiratory disease. Currently available nebulisers only deliver a proportion of the loaded or charged dose to the patient. The proportion of the charged dose received by the patient can be considered as the inhalable dose and is that part of the dose generated during inhalation and breathed in by the patient. As such, it is important to fully characterise the performance of new nebuliser brands to assess the proportion of the charged dose actually delivered to the patient. Despite the large variety of nebulisers, driver units and formulations currently on the market, often little is known about the performance of new nebulisers in terms of inhalable dose under a variety of breathing patterns and with different drug formulations. The results presented here highlight the variation in performance in terms of inhalable dose and droplet distribution of a breath-enhanced jet nebuliser when tested with a number of formulations and under different breathing patterns. Based on the results presented here we propose that a standard protocol for evaluating the performance of established and developmental nebulisers should include breathing patterns appropriate to the intended use and a variety of test formulations to capture those currently available for nebulisation.
