A rapid simple approach to screening pharmaceutical products using ultra-performance LC coupled to time-of-flight mass spectrometry and pattern recognition

The comparison of batches of pharmaceutical product or raw active pharmaceutical ingredients (API) for product release can be time consuming and tedious process. It often requires long analysis times and potentially several liquid chromatography-tandem mass spectrometry (LC-MS-MS) analytical runs to determine the identity of the impurities and their relationship to the active pharmaceutical ingredient. The combination of a high resolution (sub 2 microm porous particle) LC coupled to exact mass MS, principal components analysis (PCA) allowed for the rapid classification of batches of Simvastatin tablets according to their impurity profile. Evaluating the ultra-performance LC-MS exact mass data with PCA allowed for the impurities of Simvastatin to be easily detected and identified. This approach to impurity batch analysis should be applicable to many other forms of batch analysis, fermentation broths, food production, and API manufacturing.     

The rapid detection and identification of the impurities of simvastatin using high resolution sub 2 microm particle LC coupled to hybrid quadrupole time of flight MS operating with alternating high-low collision energy

The profiling and identification of impurities in raw pharmaceuticals or finished drug product is an essential part of the pharmaceutical manufacturing process. Critical to this process is the ability to confirm known, expected impurities and identify new impurities. LC coupled to electrospray MS is a powerful tool that has been employed for the identification of impurities, natural products, drug metabolites, and proteins. In this study, we show how sub 2 microm porous particle LC has been coupled to hybrid quadrupole orthogonal TOF mass spectrometer to profile and identify the impurities of the common cholesterol lowering drug simvastatin. The hybrid quadrupole TOF mass spectrometer was operated by alternating the collision cell energies to allow for the rapid, facile conformation of the identity of impurities. Using this process it was possible to identify all of the common impurities of simvastatin in a single 10 min run. During the analysis a new impurity of simvastatin was detected and identified as the saturated ring form of simvastatin.     

The Determination of Impurities in 233UO2 by Spark Source Mass Spectrometry

Abstract

A method for the determination of impurities in ²³³UO₂ has been developed and applied to the analysis of a product stream of ceramic grade ²³³UO₂ powder. The analytical method has combined isotope dilution and internal standard methods to permit the determination of impurities at concentrations between 0.1 and 500 ppm in a single sample.     

HPLC analysis of aspartame and saccharin in pharmaceutical and dietary formulations

Summary A reversed-phase HPLC method has been developed suitable for a reliable quality control of pharmaceutical and dietary formulations containing the synthetic sweeteners aspartame and saccharin. The proposed method is able to separate acesulfame, aspartame and saccharin, and their impurities such as 5-benzyl-3,6-dioxo-2-piperazineacetic acid (the major degradation product of aspartame) and 4-sulphamoylbenzoic acid,o- andp-toluenesulphonamides (the synthesis impurities of saccharin). A convenient solid-phase extraction (SPE) procedure using C-18 sorbent, was also developed for the determination of potential saccharin impurities.     

Enhancing the detection sensitivity of trace analysis of pharmaceutical genotoxic impurities by chemical derivatization and coordination ion spray-mass spectrometry

Many pharmaceutical genotoxic impurities are neutral molecules. Trace level analysis of these neutral analytes is hampered by their poor ionization efficiency in mass spectrometry (MS). Two analytical approaches including chemical derivatization and coordination ion spray-MS were developed to enhance neutral analyte detection sensitivity. The chemical derivatization approach converts analytes into highly ionizable or permanently charged derivatives, which become readily detectable by MS. The coordination ion spray-MS method, on the other hand, improves ionization by forming neutral-ion adducts with metal ions such as Na⁺, K⁺, or NH₄⁺ which are introduced into the electrospray ionization source. Both approaches have been proven to be able to enhance the detection sensitivity of neutral pharmaceuticals dramatically. This article demonstrates the successful applications of the two approaches in the analysis of four pharmaceutical genotoxic impurities identified in a single drug development program, of which two are non-volatile alkyl chlorides and the other two are epoxides.     

Determination of Lithium in Pharmaceutical preparations with 1-(2-Arsenophenylazo)-2-Hydroxy 3, 6-Naphthalendisulfonic Acid

Abstract

This paper reports the characteristics, in acetonic medium, of the lithium complex with 1-(2-arsenophenylazo)2-hydroxy 3,6-naphthalenedisulfonic acid (APHNDS).The analytical optimization is also reported. The coloured product was measured spectrophotometrically at 468 nm. The Beer's law was realized at 0.1–4.0 μg ml^?1. The method was used for the determination of lithium in pharmaceutical preparation. The reproducibility of the described method was efficient. The accuracy of the procedure was evaluated measuring the recovery, between 86.0–97.9 %.     

Synthesis of Repaglinide Congeners

This report describes a synthesis of two potent impurities of repaglinide, benzyl repaglinide 1 and repaglinide isomer 2, from commercially available raw materials: 2-fluoro benzonitrile, (S)-3-methyl-1-[2-(piperidin-1-yl)phenyl]butylamine (5), and 3-ethoxy-[4-(ethoxycarbonyl)phenyl]acetic acid (7). These impurities are the crucial components in determining the quality of the drug substance, repaglinide, during its manufacturing.     

Measurement of Interferents from Dosages in Metabolite Analyses

Abstract

Polar metabolites of and impurities from dosage solutions may elute similarly by HPLC and thereby yield inaccurate quantitative results. Therefore, impurities collected with eluate fractions containing metabolites from exposed animals may interfere in metabolite analyses. Marine organisms (shrimp) were exposed to ¹⁴C-labeled naphthalene and the resulting metabolites compared to dosage solution impurities using a ³H-labeled naphthalene internal standard obtained from exposed rats. HPLC purification is shown to remove impurities from dosage solutions prior to evaluation of metabolism. Procedures which test both for impurities and for effects of other compounds on metabolite studies are recommended, including criteria for evaluating when metabolism experiments should be conducted with purified solutions to avoid interferences due to impurities.

Determining metabolites of dosage substances is important in toxicity studies¹. For such bioanalytical measurements, trace impurities in exposure solutions may interfere with accurate analyses and lead to erroneous evaluations of metabolism'. If dosage materials are not pure, it is incumbent upon the investigator to devise purification methods appropriate to the task at hand. These needs seem to be well understood in mammalian toxicology¹. However, the special pharmacokinetics shown by aquatic organisms may require special tests for purity and extensive dosage purifications for metabolism studies using aquatic species. Reviewing the aquatic toxicology literature shows that such extensive evaluations and purifications are done only occasionally.

In aquatic organisms moderately polar aromatic metabolites often tend to accumulate while their parent aromatic compounds are eliminated rapidly^2–4. Measurements of metabolic analytes in systems displaying these toxicokinetics are therefore vulnerable to interferences by polar impurities froin dosage compounds. These impurities may mimic analytes, especially if the polar impurities and the metabolites of interest exhibit similar physiochemical properties.

As an example consider a case where 0.5% of a radiolabeled intraperitoneal dosage is polar impurities which are eliminated from an experimental animal with a half-life of 1 week, while the parent compound is eliminated with a half-life of 12 hours. Residual radioactive compounds isolated from an exposed organism after a short depuration, e.g. 60 hours, may have polar physiochemical properties and therefore be measured as metabolites of the dosage parent compound. However, these compounds may include impurities introduced by the dosage solution which were selectively retained because of their polarity. Impurities in this example may comprise only 0.5% of the original radioactivity, but are bioconcentrated to be 10% of the residual radioactivity. Thus, an apparently minor impurity in the dosage may prove to be a significant interferent, and thereby cause errors in analyses.

For toxicologic experiments, reagent evaluation or purification may use a variety of procedures which separate impurities from the radiolabeled compounds to be employed in biological experiments. Often the supplier performs purifications and typically guarantees that reagent purity exceeds 98% or 99%. However, we have found that radiolabeled compounds purchased for aromatic hydrocarbon metabolism studies, even those which contain low concentrations of polar impurities, may not be sufficiently pure for aquatic organism studies⁵. Thus, dosage evaluations must precede toxicologic experiments which are sensitive to small amounts of impurities, e.g., metabolite determinations.

We have observed and measured interferences in chromato-graphic analyses for metabolites⁵ due to small amounts of impurities in experiments, and herein illustrate effective procedures using HPLC which may be used to avoid errors in analyses. Small amounts of dosage solutions may be used for purity assessment. Up to 75 mg of exposure substance may be purified using commercially available analytical HPLC columns. Other advantages of these purifications include: (a) excellent separation of similar compounds due to the high resolution of HPLC, (b) similarity between the purification method used to test and prepare the dosage solution and the HPLC methods used to separate and detect metabolites, and (c) lower cost than performing purifications by other techniques.     

Recent advances in analysis of hazardous genotoxic impurities in pharmaceuticals by HPLC,GC, and CE

Nowadays, genotoxic impurities in pharmaceuticals at lower levels are of increasing concerns not only to pharmaceutical industries but also for the regulatory agencies due to their risks for human carcinogenesis and, thus, requiring manufacturers to pay extra attention for their analysis and control. The need to determine these impurities at trace levels, based on the threshold of toxicological and daily dose, taking into consideration the often reactive and labile nature of genotoxic impurities, which poses significant analytical challenges. Therefore, sensitive and sophisticated analytical methodologies are deemed necessary in order to be able to test and control genotoxic impurities in drug substances. This review demonstrates the approaches reported in the literature for the analysis of the hazardous genotoxic impurities and the strategies used to enhance the sensitivity such as using ion spray-mass spectrometry and the separation techniques for the analysis of such impurities.     

Synthesis of potential impurities of dabigatran etexilate

The present work describes the origin, control, and synthesis of two potent impurities of dabigatran etexilate 1, dabigatran dimer 2, and dabigatran n-propyl ester 3 from the commercially available raw materials 2-[(4-cyanophenyl)amino]acetic acid (4) and N-[3-amino-4-(methylamino)benzoyl]-N-2-pyridinyl-β-alanine ethyl ester (5). These impurities are the process-related impurities and may affect the quality of drug substance, during its manufacturing in large scale. These impurities are not only the crucial components in determining the quality and safety of the drug substance 1 but also provide a better understanding of impurity profiling.     

Application of two‐dimensional selective‐TOCSY HMBC for structure elucidation of impurities in mixture without separation

In the pharmaceutical industry, regulatory expectations driven by patient safety considerations make structure elucidation of impurities at levels greater than 0.1% in the active pharmaceutical ingredient (API) of primary interest. Impurities can be generated from isomers in starting materials, or produced from different process steps toward the final API. Proton peaks belonging to different impurities could be potentially identified in the one‐dimensional ¹H NMR spectrum, when evaluated in combination with two‐dimensional (2‐D) COSY and HSQC data. However, in 2‐D HMBC data, correlation responses from different impurities may overlap with those from the major component, causing uncertainty of long‐range proton to carbon correlations and quaternary carbon assignments. This observation prompts us to design the 2‐D selective‐TOCSY HMBC experiment to distinguish responses from different impurities in mixtures to obtain 2‐D NMR data for each impurity, thus eliminating the use of a chromatographic isolation step to obtain material for NMR analysis. This methodology is demonstrated for structure elucidation of impurities ranging from 8.2% in the raw material to 0.4% in the API in this study, and would be particularly useful for industrial samples in which the solubility and availability of material are not an issue. Copyright © 2012 John Wiley & Sons, Ltd.     

Using Electron Induced Dissociation (EID) on an LC Time-Scale to Characterize a Mixture of Analogous Small Organic Molecules

LC ESI FTICR MS of a sample of cediranib identified this pharmaceutical target molecule plus an additional 10 compounds of interest, all of which were less than 10% total ion current (TIC) peak intensity relative to cediranib. LC FTICR tandem mass spectrometry using electron induced dissociation (EID) has been achieved and has proven to be the best way to generate useful product ion information for all of these singly protonated molecules. Cediranib [M + H]⁺ fragmented by EID to give 29 product ions whereas QTOF-CID generated only one very intense product ion, and linear ion trap-CID, which generated 10 product ions, but all with poor S/N. Twenty-six of the EID product ions were unique to this fragmentation technique alone. By considering the complementary LC-EID and LC-CID data together, all 10 unknown compounds were structurally characterized and proven to be analogous to cediranib. Of particular importance, EID produced unique product ion information for one of the low level cediranib analogues that enabled full characterization of the molecule such that the presence of an extra propylpyrrolidine group was discovered and proven to be located on the pyrrolidine ring of cediranib, solving an analytical problem that could not be solved by collision induced dissociation (CID). Thus, it has been demonstrated that EID is in harmony with the chromatography duty-cycle and the dynamic concentration range of synthetic compounds containing trace impurities, providing crucial analytical information that cannot be obtained by more traditional methodologies.     

The Measurement of Dextran in Raw Sugars Using 1H NMR1

Abstract

In the cane sugar industry the purchase price of raw cane sugar, the product of sugar cane processing, is determined by polarimetric measurement of sucrose content in raw sugar solutions, expressed as Pol. Raw sugar generally contains more than 96% sucrose, but also contains other saccharides and non-sugars which can contribute to Pol. Dextrans, one class of polysaccharides often found in raw sugar, effect an increase in Pol and interfere with subsequent refining. The U.S. sugar refining industry can impose a penalty on the raw sugar purchase price for high dextran content. While there are several wet chemical methods for the determination of dextran in raw sugar, the results of these analyses are rarely in agreement. The existing wet chemical methods for the determination of dextran in raw cane sugar are reviewed and the results of these wet chemical analyses are compared with the results obtained from the physical measurement of dextran in raw sugar by ¹H NMR spectroscopy.     

Identification,control strategies,and analytical approaches for the determination of potential genotoxic impurities in pharmaceuticals: A comprehensive review

Potential genotoxic impurities in pharmaceuticals at trace levels are of increasing concern to both pharmaceutical industries and regulatory agencies due to their possibility for human carcinogenesis. Molecular functional groups that render starting materials and synthetic intermediates as reactive building blocks for small molecules may also be responsible for their genotoxicity. Determination of these genotoxic impurities at trace levels requires highly sensitive and selective analytical methodologies, which poses tremendous challenges on analytical communities in pharmaceutical research and development. Experimental guidance for the analytical determination of some important classes of genotoxic impurities is still unavailable in the literature. Therefore, the present review explores the structural alerts of commonly encountered potential genotoxic impurities, draft guidance of various regulatory authorities in order to control the level of impurities in drug substances and to assess their toxicity. This review also describes the analytical considerations for the determination of potential genotoxic impurities at trace levels and finally few case studies are also discussed for the determination of some important classes of potential genotoxic impurities. It is the authors’ intention to provide a complete strategy that helps analytical scientists for the analysis of such potential genotoxic impurities in pharmaceuticals.     

Spectrophotometric Determination of Sparfloxacin in Pharmaceutical Preparations by Ternary Complex Formation with Pd(II) and Eosin

Abstract

A simple, sensitive, and direct spectrophotometric method has been developed for the assay of sparfloxacin in bulk and pharmaceutical preparations. The proposed method is based on the formation of ternary complex between an investigated drug, palladium(II) ion and eosin in the presence of methylcellulose as surfactant and acetate buffer of pH 4.2. Spectrophotometrically, under the optimum conditions, the ternary complex showed absorption maximum at 550 nm, with apparent molar absorptivity of 2.69×10⁴ l mol^?1 cm^?1, Sandell's sensitivity of 0.01458 µg ml^?1 and linearity in the concentration range 1.6–16 µg ml^?1. The composition of the ternary complex was studied by Job's method of continuous variation and the result indicated that the molar ratio of SPFX: Pd: eosin is 1∶1∶1. The optimum reaction conditions and other analytical parameters are evaluated. The proposed method was successfully applied for the determination of SPFX in its pharmaceutical product with mean percentage recoveries of 99.71%. The observed data has been subjected to statistical analysis, which revealed high accuracy and precision.     

Determination of Benzylpenicillin In Pharmaceuticals By Capillary Zone Electrophoresis

Abstract

A rapid and direct method is described for the determination of benzylpenicillin (penicillin G) in pharmaceutical preparations. the method involves very little sample preparation and total analysis time for duplicate results is less than 30 minutes per sample.

The method takes advantage of the speed and separating power of capillary zone electrophoresis (CZE). Detection of penicillin is by absorption at 228 nm. an internal standard is employed to reduce sample injection error. the method was applied successfully to both tablets and injectable preparations.

larger on longer aging. It was the authors' hypothesis that this peak was due to formation of penicilloate. When this degradation product was synthesized according to an established procedure¹⁴, CZE retention time of the synthesized product was the same as that final peak in the aged penicillin samples. UV spectra on both solutions was further evidence we had properly identified the degradation product. Thus the relative amount of active and degraded compound in a sample can be estimated readily. Also, this indicates fresh solutions should be used for analytical work.

In conclusion, CZE of penicillin G was shown to not only be possible, but useful in a practical, rapid assay for the level of the antibiotic in pharmaceutical samples. Extension of this work to other types of penicillin and to the variety of samples in which the antibiotic is found in the next logical step.     

Assay determination by mass spectrometry for oligonucleotide therapeutics

Phosphorothioate oligonucleotide drugs typically contain product‐related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co‐eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry‐based purity assessment of the main chromatography peak can be used to quantify co‐eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co‐eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co‐sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry‐based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R² = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications.     

Separation,Characterization, and Quantification of Atorvastatin and Related Impurities by Liquid Chromatography-Electrospray Ionization Mass Spectrometry

A method coupling high-performance liquid chromatography with diode-array detector and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MSⁿ) has been developed for the separation and characterization of atorvastatin and its related impurities. The results obtained using positive ion mode showed some diagnostic fragments that are useful for the identification of atorvastatin related impurities in real samples. Quantitative analysis of drug impurities was performed in the multiple reaction monitoring mode. Quantification limits for impurities were in the ranges 21.5–70.8 ng mL^?1. The method was successfully applied to the drug purity evaluation and quantitative determination of atorvastatin related impurities in bulk drugs and pharmaceutical formulations.     

A Rapid,Simple, and Sensitive Spectrophotometric Determination of Traces of Vanadium (V) in Foodstuffs,Alloy Steels,and Pharmaceutical,Water, Soil,and Urine Samples

Abstract

A rapid, simple, sensitive, and selective spectrophotometric method is investigated for the determination of traces of vanadium (V) in foodstuffs, alloy steels, and pharmaceutical, water, soil, and urine samples in aqueous DMF medium. The metal ion forms a green colored complex with 2-hydroxy-3-methoxy benzaldehyde thiosemicarbazone (HMBATSC) in an acidic buffer of pH 6.0. The green colored solution, having an absorbance maximum at 380 nm, is stable for more than 72 hours. Beer's law is obeyed in the range of 0.051–2.037 µg ml^?1. The molar absorptivity and Sandell's sensitivity of the method are found as 2.75 × 10⁴ l mol^?1 cm^?1 and 0.0018 µg cm^?2, respectively. The green colored complex has 1:2 [V(V)-HMBATSC] stoichiometry. The stability constant of the complex is determined as 3.267 × 10¹¹ by Job's method. The optimum reaction conditions and other analytical parameters are studied. A sensitive and selective second-order derivative spectrophotometry has also been proposed for the determination of V(V). The interference of various cations and anions are studied. The present method is successfully applied to the determination of vanadium (V) in foodstuffs, alloy steels, and pharmaceutical, water, soil, and urine samples.     

Ion mobility spectrometry of talarozole, a new azole drug, in cleaning quality control

Ion mobility spectrometry (IMS) gains increased pharmaceutical interest as an analytical technique for the verification of equipment cleaning. Using a fractional factorial design, we developed an IMS method for talarozole, which represents a new generation of retinoic acid metabolism blocking agents (RAMBA) that can be used for the treatment of different dermatological diseases, such as psoriasis and acne. Using a Smiths Detection Ionscan-LS and the optimal IMS settings obtained, talarozole showed a drift time of 16.648 ms, corresponding to a reduced ion mobility K₀ of 1.072 cm²V^?1s^?1. Total analysis times below 1 min were achieved. Talarozole was well separated in the plasmagram from other azole compounds and the limit of detection was found to be 43 ng/ml. Swab samples collected from steel and glass plates were successfully analyzed, thereby showing that IMS is indeed a suitable technique for the quantitative analysis of talarozole in cleaning quality control.