An ultra-high performance liquid chromatography method for simultaneous determination of tacrolimus impurities in pharmaceutical dosage forms has been developed. Appropriate chromatographic ...separation was achieved on a BEH C
column using gradient elution with a total run time of 14 min. The method was applied to analyses of commercial samples and was validated in terms of linearity, precision, accuracy, sensitivity and specificity. It was found to be linear, precise and accurate in the range of 0.05 to 0.6 % of the impurities level in pharmaceutical dosage forms. Stability indicating power of the method was demonstrated by the results of forced degradation studies. The forced degradation study in solution revealed tacrolimus instability under stress alkaline, thermal, light and photolytic conditions and in the presence of a radical initiator or metal ions. The drug was stable at pH 3–5. Solid-state degradation studies conducted on amorphous tacrolimus demonstrated its sensitivity to light, elevated temperature, humidity and oxidation.
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•Nitrocellulose blister material can be a source of N-nitrosamines in drug products.•Nitrocellulose primer in a lidding foil acts as a nitrosating agent.•N-Nitrosylation of secondary ...amines, present in printing ink, yields N-nitrosamines.•N-Nitrosamines transfer from foil to drug product during the blistering operation.•Nitrocellulose-free blister material should be considered to avoid N-nitrosamines.
The recent focus of pharmaceutical regulatory authorities has been oriented towards the mitigation of carcinogenic N-nitrosamines in drug products and different sources of N-nitrosamines have been revealed. Within this work, the elucidation of a further source of N-nitrosamines in drug products is reported. A case was investigated where traces of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were detected in a finished dosage form, whereas they were not found in the bulk drug product. This led to an in-depth study of blister material as a potential source, wherein nitrocellulose primer in a lidding foil was identified as a risk factor. Nitrocellulose acts as a nitrosating agent for secondary amines, present in printing inks, forming N-nitrosamines in lidding foil. Their formation was confirmed by the addition of printing ink containing dimethylamine and diethylamine, or diethylamine alone, to lidding foil containing nitrocellulose primer. Their transfer to drug product during the blistering operation was demonstrated by solid-phase microextraction sampling of N-nitrosamine vapors on two commonly used types of pharmaceutical blistering machines, operating with plate sealing or roller sealing technology. Higher vapor amounts were detected on plate sealing equipment, where N-nitrosamine contamination was additionally confirmed in film-coated tablets and blister cavities of the finished dosage form.
N-nitrosamines are genotoxic compounds which can be found as impurities in drug substances and drug products used in the pharmaceutical industry. To date, several possible nitrosamine sources in drug ...products have been reported and this study aims to illuminate another one. A case of afatinib drug product was investigated, in which up to 50 ppb N-nitrosodimethylamine (NDMA) traces were detected. Afatinib was found to degrade to the secondary amine dimethylamine (DMA), forming NDMA with traces of nitrite in crospovidone. Two series of film-coated tablets were prepared with crospovidone from two different manufacturers, containing different levels of nitrites. Tablets were subjected to an accelerated stability study (40 °C/75% relative humidity) or stored at room temperature and levels of NDMA, DMA and nitrite in tablets were monitored. NDMA and nitrite were found on ppb levels, whereas DMA was detected on ppm levels. NDMA formation in the drug product was found to be time, temperature and nitrite dependent and it was emphasized that DMA and nitrite should be reduced. The accelerated stability study proved to be a useful tool for predicting nitrosamine formation in the drug product.
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Fast analysis of pravastatin in production media Kocijan, Andrej; Grahek, Rok; Bastarda, Andrej ...
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences,
08/2005, Letnik:
822, Številka:
1
Journal Article
Recenzirano
High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle ...column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20
mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-
epi pravastatin) and runtimes are shorter than 1
min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5
mL
h
−1. The most sensitive was the method using particle column (LOD was about 10
−5
mg
mL
−1), followed by the system using monolith column (LOD was 2
×
10
−4
mg
mL
−1) and the MECK method (LOD was about 0.02
mg
mL
−1).
Despite the fact that it was put on the market more than 60 years ago, hydrochlorothiazide (HCT) is still one of the most important antihypertensive drugs. Due to its chemical structure, which ...contains the secondary aryl-alkyl-amino moiety, it is vulnerable to the formation of N-nitrosamine drug substance-related impurity (NDSRI) N-nitroso-hydrochlorothiazide (NO-HCT). In our study, we reveal that NO-HCT degrades rapidly at pH values 6 to 8. The main degradation products identified are formaldehyde, thiatriazine, and aminobenzenesulfonic acid derivative. Interestingly, degradation of NO-HCT at pH values from 5 to 1 is significantly slower and provides a different impurity profile when compared to the profile generated between pH 6 and 8. Specifically, between pH 1 and 5, HCT is observed as one of the key degradation products of NO-HCT in addition to formaldehyde and aminobenzenesulfonic acid. Moreover, at pH 1, the aminobenzenesulfonic acid derivative is transformed to the corresponding diazonium salt in approximately 3% yield with the nitrosyl cation, which is released during the decomposition of NO-HCT to HCT. This diazonium is highly unstable above pH 5. To verify that degradation of NO-HCT does not produce the corresponding diazonium salt that could be formed via metabolic activation of NO-HCT, this diazonium salt and its hydrolytic and reduction degradation products were synthesized and used as standards for the identification of species formed during the degradation of NO-HCT. This enabled us to confirm that the corresponding aryl diazonium salt, which would be obtained from metabolic activation of NO-HCT, is not observed in the NO-HCT degradation pathway. Our study also demonstrates that this diazonium salt is stable only in the presence of a large excess of strong mineral acid under anhydrous conditions. In the presence of water, it is instantaneously converted to an aminobenzenesulfonic acid derivative. These findings suggest that the NO-HCT should not be considered as a typical compound belonging to the cohort of the concern.
An HPLC/MS/MS method was developed for identification of impurities in gentamicin. The HPLC was performed on a Synergy Hydro-RP column using 50
mM trifluoroacetic acid (TFA), pH 2 adjusted with ...ammonium solution and methanol as mobile phase. All impurities in gentamicin were separated from main gentamicin components. Atmospheric pressure chemical ionization (APCI) was used and product mass spectra of protonated molecules were acquired. Seventeen impurities were detected in gentamicin. Reference compounds: gentamicins: C
2b, B, B
1, G-418, sisomicin, garamine and gentamines: C
1, C
1a, C
2, C
2a were used for spectra interpretation and impurities identification. All MS/MS spectra were interpreted and fragmentation transitions for gentamicins and in general for aminoglycoside antibiotics (AG) were proposed. All impurities were identified. More than one isomere were proposed for three impurities.
•Solid state compatibility study.•Isolation and characterization of new tacrolimus degradation impurity.•Structural elucidation using NMR and MS.•Degradation pathway of tacrolimus in the presence of ...divalent cations proposed.
Tacrolimus is macrolide drug that is widely used as a potent immunosuppressant. In the present work compatibility testing was conducted on physical mixtures of tacrolimus with excipients and on compatibility mixtures prepared by the simulation of manufacturing process used for the final drug product preparation. Increase in one major degradation product was detected in the presence of magnesium stearate based upon UHPLC analysis. The degradation product was isolated by preparative HPLC and its structure was elucidated by NMR and MS studies. Mechanism of the formation of this degradation product is proposed based on complementary degradation studies in a solution and structural elucidation data. The structure was proven to be alpha-hydroxy acid which is formed from the parent tacrolimus molecule through a benzilic acid type rearrangement reaction in the presence of divalent metallic cations. Degradation is facilitated at higher pH values.
Methods were developed for the preparation and isolation of four oxidative degradation products of atorvastatin. ATV-FX1 was prepared in the alkaline acetonitrile solution of atorvastatin with the ...addition of hydrogen peroxide. The exposition of aqueous acetonitrile solution of atorvastatin to sunlight for several hours followed by the alkalization of the solution with potassium hydroxide to pH 8–9 gave ATV-FXA. By the acidification of the solution with phosphoric acid to pH 3 ATV-FXA1 and FXA2 were prepared. The isolation of oxidative degradation products was carried out on a reversed-phase chromatographic column Luna prep C18(2) 10
μm applying several separation steps. The liquid chromatography coupled with a mass spectrometer (LC-MS), high resolution MS (HR-MS), 1D and 2D NMR spectroscopy methods were applied for the structure elucidation. All degradants are due to the oxidation of the pyrrole ring. The most probable reaction mechanism is intermediate endoperoxide formation with subsequent rearrangement and nucleophilic attack by the 5-hydroxy group of the heptanoic fragment. ATV-FX1 is 4-1b-(4-Fluoro-phenyl)-6-hydroxy-6-isopropyl-1a-phenyl-6a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropaainden-3-yl-3-(R)-hydroxy-butyric acid and has a molecular mass increased by two oxygen atoms with regard to atorvastatin. ATV-FXA is the regioisomeric compound, 4-6-(4-Fluoro-phenyl)-6-hydroxy-1b-isopropyl-6a-phenyl-1a-phenylcarbamoyl-hexahydro-1,2-dioxa-5a-aza-cyclopropaainden-3-yl-3-(R)-hydroxy-butyric acid. Its descendants ATV-FXA1 and FXA2 appeared without the atorvastatin heptanoic fragment and are 3-(4-Fluoro-benzoyl)-2-isobutyryl-3-phenyl-oxirane-2-carboxylic acid phenylamide and 4-(4-Fluoro-phenyl)-2,4-dihydroxy-2-isopropyl-5-phenyl-3,6-dioxa-bicyclo3.1.0hexane-1-carboxylic acid phenylamide, respectively. Quantitative NMR spectroscopy was employed for the assay determination of isolated oxidative degradation products. The results obtained were used for the determination of the UV response factors relative to atorvastatin.
The purification of pravastatin, simvastatin and lovastatin in the sodium salt or lactone form and of mevastatin in the lactone form by reversed-phase displacement chromatography is presented. The ...mobile phases consisted of water or mixtures of water–methanol and water–acetonitrile. Six different displacers were successfully used. Up to 0.14 g of raw sample per gram of stationary phase was loaded on a column packed with silica-based octadecyl phase. Crude substances from 85 to 88% chromatographic purity were purified and at least 99.5% purity was achieved.
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