European Pharmacopoeia Monograph Research Articles

Improving the Current European Pharmacopoeia Enantio-Selective HPLC Method for the Determination of Enantiomeric Purity in Atorvastatin Calcium Salt Drug Substance

Atorvastatin (ATV) is a well-established lipid-lowering agent. ATV has two stereogenic centers, and of the four possible stereoisomers, only the (3R,5R) form is used therapeutically. The European Pharmacopoeia (EP) monograph 2022 for ATV calcium salt describes a normal-phase high-performance liquid chromatography (HPLC) method for the determination of enantiomeric purity in both drug substance and working standard samples, based on a 150 mm × 4.6 mm Chiralpak AD-H column. The main problems with this method are the very long analysis time and the high solvent consumption. Here, an alternative chromatographic protocol was developed using the Chiralpak AD-3 column (250 mm × 4.6 mm) packed with 3 μm silica particles instead of the 5 μm silica particles of the Chiralpak AD-H chiral stationary phase and characterized by the same polysaccharide selector, amylose-tris(3,5-dimethylphenylcarbamate). Using a mobile phase consisting of a mixture of n-hexane-ethanol-formic acid 90:10:0.1 (v/v/v) as the mobile phase and setting the flow rate and column temperature to 1.0 mL min−1 and 35 °C, respectively, a simultaneous stereo-selective separation was achieved within 35 min without observing any overlap between the enantiomeric impurity peak and peaks related to other ATV impurities. Compared to HPLC EP conditions, the analysis time to elute all the potentially related substances was faster and significantly less mobile phase volume was required. The linearity of the method has been demonstrated in the range of 4.4 μg mL−1 to 1000 μg mL−1 (R2 > 0.999). At a concentration of 4.4 μg mL−1, which is 0.075% of the test solution (5.8 mg mL−1, as ATV free acid), the signal-to-noise ratio was found to be 20.

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and RRT for a common hydrolytic degradation product of Fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of Fipronil. This situation causes misidentification, mislabeling and/or false out-of-specifications results for this hydrolytic degradation product of Fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase UPLC method for assay and identification of Fipronil including identification and estimation of its related substances in bulk drug substance batches of Fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by a gradient elution on a Halo C18 column (50 mm × 2.1 mm i.d., 2.0 µm particle size) maintained at 40 °C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a 7 minute total run time (∼40% faster than EP). In this paper, we report the development and validation of a fast, stability indicating reversed phase UPLC method for assay and estimation of related substances of Fipronil in stability samples and bulk batches of Fipronil. The new UPLC method is ∼40% faster than the current EP monograph for Fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of Fipronil.

Solvents in the industrial synthesis of naphazoline nitrate: implementation of the principles of "Green chemistry" and analysis

The aim: implementation of the principles of green chemistry by regenerating the synthesis solvent 1,2,4-trichlorobenzene and reusing it during the synthesis of the Naphazoline nitrate substance. Study of the influence of the regenerated solvent on the quality of the final product by controlling analytical quality parameters. Development of a method for the quantitative determination and validation of synthesis solvents in a substance. Materials and methods: samples of the substance were synthesized according to the optimized proprietary technology of Farmak JSC. The obtained batches of fresh and regenerated 1,2,4-trichlorobenzene were analyzed according to the monograph of the European Pharmacopoeia on Naphazoline nitrate 0147. Results: the possibility of using regenerated 1,2,4-trichlorobenzene for the synthesis of the substance Naphazoline nitrate has been proven. It is shown that the regenerated solvent does not have a negative effect on the profile of impurities and the polymorphic form of the substance. Analytical quality parameters met the requirements of the internal specification and the requirements of the European Pharmacopoeia monograph. The developed and validated method of quantitative determination of synthesis solvents makes it possible to determine them at the required level. Conclusions: the introduction of regenerated 1,2,4-trichlorobenzene into the synthesis scheme made it possible to significantly reduce the amount of waste per 1 kg of product, which in turn significantly reduced the negative impact on the environment. Analytical quality parameters for regenerated 1,2,4-trichlorobenzene meet the requirements of the internal specification. Industrial series obtained on the regenerated solvent meet the requirements of the monograph of the European Pharmacopoeia. The polymorphic form of the substance batches manufactured on regenerated 1,2,4-trichlorobenzene corresponds to the polymorphic form of the substance batches manufactured on the fresh solvent. The obtained results on the influence of the regenerated solvent on the profile of impurities in the finished substance show the similarity of the profile of the series manufactured on both solvents. According to the requirements of ICH Q3C Impurities: Guideline for residual solvents, a method for quantitative determination of the residual content of 1,2,4-trichlorobenzene in the final product by gas chromatography was developed and validated. The absence of synthesis solvents at the limit of detection is shown

Characterization of Degradation Products of Bisoprolol by LC-MS and In-silico Determination of Toxicity and Biological Activity Prediction of its Degradation Product: A Comprehensive Approach during Drug Development

The stability of bisoprolol in tablet and bulk form was developed and validated using chromatographic methods. The bisoprolol was exposed to oxidizing agents like hydrogen peroxide, heat (dry and wet) and photolytic conditions, acid, alkali, and water hydrolysis. However, considerable degradation was observed in acid, alkali, oxidative, and wet heat thermal conditions. The drug’s stability under photolytic and dry heat conditions was established. The drug and its degradation products were separated on ODS C-18 with the specification of (250 mm × 4.6 mm, 5 μm) and the mobile phase containing acetonitrile and phosphate buffer (20 mM, pH 8) (60:40, v/v). According to ICH Q2 (R1) all method validation parameters are verified. Determination of the active pharmaceutical ingredient was not interfered by excipients or degradation products. In the 6–14 μg/mL range, the response was shown to be linear. One degradation product’s LC-MS m/z values and fragmentation patterns matched an impurity mentioned in the drug’s European Pharmacopoeia monograph. The remaining three were unidentified degradation products. LC-MS fragmentation experiments were used to characterize the products. The findings may lead to the suggestion of a more thorough drug degradation mechanism. The Toxtree software (Version 3.1.1) was then used to forecast the toxicity of the degradation products. Additional toxicity determination was carried out by using in-silico method.

Insight into the photolytic degradation products of Rosuvastatin: Full chiral and structural elucidation and conversion kinetics by a combined chromatographic, spectroscopic and theoretical approach

Rosuvastatin (RSV) is a well-established lipid-lowering drug. RSV is susceptible to degradation under various stress conditions and forms two cyclic derivatives by a radical-mediated photolytic mechanism. On a structural basis, these epimeric compounds (reported as FP-B in the European Pharmacopeia monograph Rosuvastatin tablets) retain the configuration of the stereogenic carbons of RSV (3R,5S) and have opposite absolute configurations at the third stereogenic center. Herein, we report the kinetics of formation and the complete structural characterization, including the assignment of the absolute configuration, of each epimer collected after HPLC separation on a chiral stationary phase. The stereochemistry of the epimers was determined by comparison of the experimental circular dichroism data with the corresponding theoretical values. Kinetic studies revealed that RSV degrades completely to FP-B within 3 h at room temperature. Furthermore, through a multi-disciplinary approach involving chromatography (HPLC and UHPLC), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), it was demonstrated that FP-B in turn degrades to the lactones under the mild acidic conditions of the chromatographic mobile phase. The ability of RSV to form multiple degradation products may affect the quantification of RSV-related substances and draw attention to potentially toxic RSV-like species in the environment.

Chemo- and enantio-selective reversed-phase HPLC analysis of rosuvastatin using a cellulose-based chiral stationary phase in gradient elution mode

A direct reversed-phase high-performance liquid chromatographic (HPLC) method was developed for determining the content of the enantiomeric impurity of the chiral statin rosuvastatin calcium salt (RSV) in commercial tablets. The baseline enantioseparation was achieved using the Lux Cellulose-2 column and a binary linear gradient of acetonitrile and trifluoroacetic acid 0.05% in an aqueous solution. The flow rate of the mobile phases and column temperature were set at 1.0 mL min− 1 and 40 °C, respectively. In comparison with the isocratic HPLC method reported in the European Pharmacopoeia (EP) monograph for RSV, the gradient elution method offered improved chemo-and enantio-selectivity and reduced analysis times. The limits of quantitation and detection of the enantiomeric impurity were found to be 0.15 and 0.05 µg mL−1.

A Novel Validated UHPLC Method for the Estimation of Rosuvastatin and Its Complete Impurity Profile in Tablet Formulations

A key step in the development of medicinal products is the research and validation of selective and sensitive analytical methods for the control of impurities from synthesis and degradation. As most impurities are similar in structure to the drug substance, the achievement of chemo-selective conditions is usually challenging. Herein, a direct and highly selective ultra-high-performance liquid chromatographic method for determining the assay and related substances content in medicinal products containing rosuvastatin calcium salt (RSV) is presented. RSV is used to treat high cholesterol levels and prevent heart attacks and strokes. The most engaging feature of this method was the baseline separation of all organic related substances listed in the European Pharmacopoeia (EP) monograph for the RSV tablets, achieved for the first time in less than 15 min using the Acquity BEH C18 (100 mm × 2.1 mm, 1.7 μm) column under reversed-phase isocratic conditions. The mobile phase adopted for the chemo-selective analysis does not contain buffers but instead contains trifluoroacetic as an acid additive. The chromatographic method was validated according to the guidelines of the International Conference on Harmonization (ICH) and proved to be linear, precise and accurate for determining the content of RSV and related chiral substances in tablet formulations.

Chemical composition of essential oil of common juniper (Juniperus communis L.) branches from Estonia

For the treatment of the urinary system and kidney diseases, common juniper (Juniperus communis L.) fruits are widely used. This raw material is included in the European Pharmacopoeia (Ph.Eur.) and is one of the most popular kinds of official medicinal plant material with diuretic and uroantiseptic activity. However, the main biomass of bushes consists of green branches, which also contain a significant amount of essential oil that can be used in pharmaceutical practice. The branches become waste during bush cutting. So, it is advisable to investigate the chemical composition of essential oil isolated from common juniper branches from different regions of Estonia to prove the possibility of using this essential oil and branches in pharmaceutical practice.
 Aim. Therefore, the aim of the research was to determine the chemical composition of essential oil from common juniper (J. communis L.) branches from Estonia.
 Materials and Methods. The branches of juniper shrubs were collected in the summer months from 27 different habitats in Estonia. The essential oil was isolated from fresh juniper branches by the modified distillation method described in the European Pharmacopoeia monograph of Juniperi pseudo-fructus. GC/MS analysis was carried out using an Agilent 5975 Series MSDMSD, Agilent7890A GC (Agilent Technologies, Inc.) with two detectors (MSMS and FID) on a fused silica capillary column (30 m x 0.25 mm) with a bonded stationary phase: poly(5 %-diphenyl-95 %-dimethyl)siloxane (DB-5). The carrier gas was helium with a split ratio of 1:30, and the flow rate of 1.3 mL/min was applied. The temperature program was from 50°–240˚C at 2˚C/min and the injector temperature was 300˚C. The MS detector was operated in the EIEI mode of 70 eV and at a scan rate of 2 scans/s with a mass acquisition range of 20–400 u.
 Research results. The average amount of juniper essential oil in branches extracted during distillation using the Ph. Eur. method was 0.23±0.04 ml. 103 substances were identified in 27 different samples of juniper branches and quantified by the GC/MS method. The dominant components of Estonian common juniper essential oil are α-pinene (37.5-69.3 %), pinene, sabinene, β-myrcene and β-phellandrene. The juniper essential oils from Estonian raw materials were compared with Serbian, Iran, Portuguese, French and Greek ones. It was established that the common juniper growing in Estonia belongs to the α-pinene chemotype.
 Conclusions. Common juniper growing in Estonia belongs to the α-pinene chemotype. 103 substances were identified, and their assay was established in 27 different samples of juniper branches. The dominant components of Estonian common juniper essential oil are α-pinene (37.5-69.3 %), so it could be used as a source of a-pinene for the pharmaceutical industry.
 As the essential oils of common juniper branches didn’t meet all the requirements of the European Pharmacopoeia for juniper berries oil, so separate regulatory documentation must be developed for the essential oil from the branches

68Ga-DOTATATE Prepared from Cyclotron-Produced 68Ga: An Integrated Solution from Cyclotron Vault to Safety Assessment and Diagnostic Efficacy in Neuroendocrine Cancer Patients.

Cyclotron production of 68Ga is a promising approach to supply 68Ga radiopharmaceuticals. To validate this capability, an integrated solution for a robust synthesis of 68Ga-DOTATATE prepared from cyclotron-produced 68Ga was achieved. A retrospective comparison analysis was performed on patients who underwent PET/CT imaging after injection of DOTATATE labeled with 68Ga produced by a cyclotron or eluted from a generator to demonstrate the clinical safety and diagnostic efficacy of the radiopharmaceutical as a routine standard-of-care diagnostic tool in the clinic. Methods: An enriched pressed 68Zn target was irradiated by a cyclotron with a proton beam set at 12.7 MeV for 100 min. The fully automated process uses an in-vault dissolution system in which a liquid distribution system transfers the dissolved target to a dedicated hot cell for the purification of 68GaCl3 and radiolabeling of DOTATATE using a cassette-based automated module. Quality control tests were performed on the resulting tracer solution. The internal radiation dose for 68Ga-DOTATATE was based on extrapolation from rat biodistribution experiments. A retrospective comparison analysis was performed on patients who underwent PET/CT imaging after injection of DOTATATE labeled with cyclotron- or generator-produced 68Ga. Results: The synthesis of 68Ga-DOTATATE (20.7 ± 1.3 GBq) with high apparent molar activity (518 ± 32 GBq/μmol at the end of synthesis) was completed in 65 min, and the radiopharmaceutical met the requirements specified in the European Pharmacopoeia monograph on 68Ga-chloride (accelerator-produced) solution for radiolabeling. 68Ga-DOTATATE was stable for at least 5 h after formulation. The dosimetry calculated with OLINDA for cyclotron- and generator-produced 68Ga-DOTATATE was roughly equivalent. The SUVmean or SUVmax of tumoral lesions with cyclotron-produced 68Ga-DOTATATE was equivalent to that with generator-produced 68Ga. Among physiologic uptake levels, a significant difference was found in kidneys, spleen, and stomach wall, with lower values in cyclotron-produced 68Ga-DOTATATE in all cases. Conclusion: Integrated cyclotron production achieves reliable high yields of clinical-grade 68Ga-DOTATATE. The clinical safety and imaging efficacy of cyclotron-produced 68Ga-DOTATATE in humans provide supporting evidence for its use in routine clinical practice.

Study of the Adsorption of Bacillus subtilis and Bacillus cereus Bacteria on Enterosorbent Obtained from Apricot Kernels

This paper presents the results of scientific research on the structural parameters and the adsorption capacity of activated carbon obtained from apricot kernels (AC-A) in a fluidized layer. The obtained results highlight the fact that the described procedure allows obtaining a mesoporous carbon adsorbent with increased adsorption capacities (SBET = 1424 m2/g) and with quality indices corresponding to the requirements of the carbon enterosorbents imposed by the European Pharmacopoeia Monograph. Adsorption kinetics studies of the bacteria Bacillus subtilis and Bacillus cereus have shown that the time to establish the adsorption equilibrium is 75–90 min. The adsorption of the mentioned bacteria on the carbon enterosorbent AC-A was studied depending on the temperature (26 and 36 °C) and pH of the solution (1.97–4.05). Scanning Electron Microscopy (SEM) showed that the immobilization of bacteria takes place on the outer surface of the carbon adsorbent due to the fact that the geometric dimensions of the bacteria are often larger than the macro diameter of the activated carbon pores. FTIR investigations also indicated the presence of bacteria on the surface of the activated carbon.

Effective Isolation of Picrocrocin and Crocins from Saffron: From HPTLC to Working Standard Obtaining.

Saffron is widely cultivated and used as a spice. Recently published data on the chemical composition and pharmacological potential of saffron determine its use in pharmacy and medicine. The proposed high-performance thin-layer chromatography (HPTLC) method allows good separation of 11 analytes. The saffron quality (Iran, Ukraine, Spain, Morocco samples) assessment was based on the European Pharmacopoeia monograph and ISO 3632. The HPTLC method for the safranal, crocin, and picrocrocin quantification was proposed and validated. The crocins content in Ukrainian saffron was from 17.80% to 33.25%. Based on qualitative and quantitative assessment results, the saffron sample from Zaporizhzhia (Ukraine) had the highest compounds content and was chosen to obtain the working standards of picrocrocin and crocins (trans-4GG, trans-2G, trans-3Gg) by preparative chromatography. The compounds were isolated from lyophilized extract of saffron using a Symmetry Prep C18 column (300 × 19 mm × 7 µm), and identified by spectroscopic techniques (HPLC-DAD, UPLC-ESI-MS/MS). The purity of crocins and picrocrocin was more than 97%. A novel method proposed to obtain working standards is simple and reproducible for the routine analysis of saffron quality control.

Critical Evaluation of Specific Efficacy of Preparations Produced According to European Pharmacopeia Monograph 2371.

European Pharmacopoeia monograph 2371 describes the production of homeopathic preparations. A specific efficacy of these preparations in high dilution levels is questionable in view of basic scientific principles. There is empirical evidence for such effects, for example in a Lemna-intoxication bioassay published 2010. To test the replicability and robustness of this bioassay, we conducted two experimental series (five independent blinded and randomised experiments each). The specimen of Lemna gibba L., clone-number 9352, were stressed in arsenic solution for 48 h (158 mg/L AsNa2HO4 (250 mg/L in series 2)), then grew in either As2O3 preparations produced according to Eu. Pharm. Monogr. 2371 or control solution. Comparing the area-related relative growth rate of day 3–9 (rgr 3–9) between treatment and control groups for each series showed differences that were not significant in series 1 (p = 0.10), significant in series 2 (p = 0.04) and significant in the pooled data of both series (p < 0.01). The effect direction (rgr 3–9 increase) was comparable to experiments of 2010, but the effect size was smaller, likely due to a changed light cycle. These results are not compatible with the hypothesis that the application of European Pharmacopoeia monograph 2371 results in pharmaceutical preparations without specific effects. Further studies are needed to investigate a potential mode of action explaining these effects.

Development and Validation of a GMP-Compliant High-Pressure Liquid Chromatography Method for the Determination of the Chemical and Radiochemical Purity of [18F]PSMA-1007, a PET Tracer for the Imaging of Prostate Cancer.

For the PET imaging of prostate cancer, radiotracers targeting the prostate-specific membrane antigen (PSMA) are nowadays used in clinical practice. [18F]PSMA-1007, a radiopharmaceutical labeled with fluorine-18, has excellent properties for the detection of prostate cancer. Essential for the human use of a radiotracer is its production and quality control under GMP-compliance. For this purpose, all analytical methods have to be validated. [18F]PSMA-1007 is easily radiosynthesized in a one-step procedure and isolated using solid phase extraction (SPE) cartridges followed by formulation of a buffered injection solution and for the determination of its chemical and radiochemical purity a robust, fast and reliable quality control method using radio-HPLC is necessary. After development and optimizations overcoming problems in reproducibility, the here described radio-HPLC method fulfills all acceptance criteria—for e.g., specificity, linearity, and accuracy—and is therefore well suited for the routine quality control of [18F]PSMA-1007 before release of the radiopharmaceutical. Recently a European Pharmacopeia monograph for [18F]PSMA-1007 was published suggesting a different radio-HPLC method for the determination of its chemical and radiochemical purity. Since the here described method has certain advantages, not least of all easier technical implementation, it can be an attractive alternative to the monograph method. The here described method was successfully validated on several radio-HPLC systems in our lab and used for the analysis of more than 60 batches of [18F]PSMA-1007. Using this method, the chemical and radiochemical purity of [18F]PSMA-1007 can routinely be evaluated assuring patient safety.

Simultaneous separation of insulin and six therapeutic analogues on a mixed mode column: HPLC-UV method development and application

Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.

Türkiye piyasasında satılan Şahtere (Fumaria officinalis L.) bitkisi üzerinde incelemeler

Bu çalışmada Ankara, Sivas, Tokat ve Konya piyasasından şahtere adı ile satılan toplam 10 farklı örnek satın alınmıştır. Piyasa’dan satın alınan bütün örneklerin morfolojik özellikleri ve saflıkları incelenmiştir. Tedavi edici değeri olduğu düşünülen bitki kısımlarına ait olmayan organik ve inorganik maddeler belirlenmiştir. Piyasadan satın alınan örneklerin standart olarak kullanılan herbaryum örneği ile karşılaştırılması sonucunda, Fumaria officinalis bitkisine ait olduğu tespit edilmiştir. Örneklerin içeriğindeki organik ve inorganik yabancı maddeler ve oranları belirlenmiştir. Piyasada satılan "şahtere" örneklerinin doğru bitkiden elde edildiği ancak büyük bir kısmının Avrupa ve Türk Farmakopeleri monograflarına uymadığı görülmüştür. Örneklerin toplama ve saklama sırasında, organik ve inorganik maddelerle kontamine olduğu ve bu nedenle de halk sağlığı açısından uygun nitelikler taşımadığı gösterilmiştir.

Design and characterization of spring water hydrogels with natural inorganic excipients

An essential first step to deep into the knowledge of the mechanisms of action that underlie the use of clay-based spring water gels is their design and development according to standardized protocols and procedures. Hydrogels with two spring waters and two clay excipients, sepiolite (PS9) and palygorskite (G30), were studied. The excipients were fully characterized as regards their identity, purity and richness, as well as their textural features, cation exchange capacities and zeta-potential. Conductivity, pH and ionic composition of spring waters were also assessed. Rheological and thermal properties of hydrogels were monitored through time. PS9 complied with identity, richness and purity descriptions of “Magnesium silicate” (or “Magnesium trisilicate”) United States and European Pharmacopoeia monographs', whereas G30 satisfied “Attapulgite” monograph. Cooling rates of 10% w/w spring water hydrogels were adequate for topical application as thermotherapic agents. Gel-like systems obtained with PS9 showed higher viscosities and yield values than G30 hydrogels, the type of spring water not exerting significant influence. The properties remained almost constant during a period of 2 months, ensuring quality standards. Longer periods resulted in unforeseeable changes in properties ascribed to water losses.

An Improved Reversed-Phase High-Performance Liquid Chromatography Method for the Analysis of Related Substances of Prednisolone in Active Ingredient.

Prednisolone, an important active pharmaceutical ingredient, is a synthetic glucocorticoid used for the preparation of various pharmaceutical products with anti-inflammatory and immunosuppressive properties. It is a challenge in high-performance liquid chromatography (HPLC) to separate the prednisolone peak and its structurally related substance (hydrocortisone), which only differs in a double bond at the C-1 position. Successful application of the HPLC method according to the European Pharmacopoeia monograph for related substances of prednisolone is very often limited to the chromatographic system available. This is due to the nonbaseline separation of the prednisolone and hydrocortisone peaks, which is strongly influenced by the instrument parameters and the chosen C18 column. First, an adjusted European Pharmacopoeia method for related substances of prednisolone was developed within the allowable adjustments. Next, an improved stability-indicating reversed-phase HPLC method for related substances of prednisolone was developed and validated for use in quality control laboratories for routine analysis. The optimized separation was performed on a Phenomenex Gemini C18 column (150 mm × 4.6 mm, 3 μm) using a gradient mobile-phase system consisting of acetonitrile/tetrahydrofuran/water (15:10:75 v/v/v), acetonitrile/water (80:20 v/v), and ultraviolet detection at 254 nm. A baseline separation was achieved, and stability indicating capability was demonstrated by a forced degradation study. A full validation procedure was performed in accordance with International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines.

Empirical investigation of preparations produced according to the European Pharmacopoeia monograph 1038

According to the European Pharmacopoeia monograph 1038 (Praeparationes homoeopathicae), homeopathic preparations are produced by successive dilution and succussion steps. Dilution levels beyond Avogadro's limit, however, render specific effects implausible according to standard scientific knowledge. Accordingly, we were interested in a critical empirical investigation of preparations produced according to this monograph.Within a precursor study we developed a bioassay based on a fingerprint metabolomic analysis of Lepidium sativum seeds germinated in vitro in either homeopathic preparations or controls in a blinded and randomized assignment. Results of the precursor study were not consistent with the hypothesis that the effects of a Stannum metallicum 30x preparation are identical to placebo.In the present study we investigated the reproducibility of these effects after scrutinizing and optimizing experimental procedures. Ten independent experiments were performed in a blinded and randomized assignment in two independent laboratories. Additionally, 10 systematic negative water control experiments were performed in both laboratories to critically assess the stability of the experimental set-up.The effects of the Stannum metallicum 30x treatment were reproduced. The systematic negative control experiments did not yield false-positive results, indicating a stable experimental set-up.We thus repeatedly observed biological effects conflicting with the assumption that Stannum metallicum 30x is identical to placebo. We therefore wish to discuss whether these findings are to be considered a scientific anomaly or whether they might stimulate further investigations to clarify whether application of the European Pharmacopoeia monograph 1038 may result in pharmaceutical preparations with specific effects.

An improved automated one-pot synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) based on a purification by cartridges

IntroductionO-(2-[18F]Fluoroethyl)-L-tyrosine ([18F]FET) is an established radiotracer used for oncology investigations by Positron Emission Tomography (PET). Main limitations to its widespread use are the synthesis itself (time; cost; radiochemical yield; complexity) and a troublesome and time-consuming HPLC purification. Aim of this work was to improve the preparation overall efficiency and, most important, to achieve an efficient and reliable purification by means of disposable cartridges. Methods[18F]FET was synthesized by direct nucleophilic radiofluorination of O-(2-tosyloxy-ethyl)-N-trityl-L-tyrosine t-butylester (TET) followed by acid hydrolysis with HCl. Several conditions and materials were tested for the synthesis and purification step. For the latter, a number of different commercial cartridges, varying in amount, particulate size and adsorbent, were examined. Best results were obtained by a combination of STRATA-X, tC18 and QMA cartridges. ResultsStarting from only 5 mg of TET, up to 11 GBq of injectable solutions of [18F]FET were produced within 36 min with 54–65% radiochemical yields and radiochemical purities >99%. No D-form was observed by chiral HPLC. Chemical purity was 1–2 order of magnitude below the limits imposed by the European Pharmacopoeia's monograph on [18F]FET.A radiochemical purity decrease by radiolysis, observed only on relatively large batches of [18F]FET, was efficiently suppressed by preloading in the receiving final vial a small amount of ethanol (<2% v/v). ConclusionsBy combining improvements to a known synthetic route with a novel cartridge-based purification, [18F]FET was obtained in a very efficient and reproducible way. The whole process was easily implemented on a commercial automated module presently used for [18F]FDG production. Advances in knowledge and implications for patient careA few drawbacks regarding the HPLC conditions recommended in the European Pharmacopoeia were highlighted. An alternative method able to cope with them is herein proposedThe simplified preparation herein described is expected to encourage a more widespread clinical use of [18F]FET.

Prospects to the formation and control of potential dimer impurity E of pantoprazole sodium sesquihydrate

Pantoprazole sodium, a substituted benzimidazole derivative, is an irreversible proton pump inhibitor which is primarily used for the treatment of duodenal ulcers, gastric ulcers, and gastroesophageal reflux disease (GERD). The monographs of European Pharmacopoeia (Ph. Eur.) and United States Pharmacopoeia (USP) specify six impurities, viz.; impurities A, B, C, D, E and F, respectively for its active pharmaceutical ingredient (API). The identification and synthesis of all impurities except impurity E are well described in the literature; however, there is no report related to impurity E. The prospects to the formation and controlling of impurity E up to ≤0.03% in the synthesis of pantoprazole sodium sesquihydrate (PAN) were discussed in detail for the first time. The present work described the journey towards the successful development of an optimal preparation procedure of dimer impurity E. The most plausible mechanism involved in the formation of impurity E has been proposed.