What is the role of current mass spectrometry in pharmaceutical analysis?

The role of mass spectrometry (MS) has become more important in most application domains in recent years. Pharmaceutical analysis is specific due to its stringent regulation procedures, the need for good laboratory/manufacturing practices, and a large number of routine quality control analyses to be carried out. The role of MS is, therefore, very different throughout the whole drug development cycle. While it dominates within the drug discovery and development phase, in routine quality control, the role of MS is minor and indispensable only for selected applications. Moreover, its role is very different in the case of analysis of small molecule pharmaceuticals and biopharmaceuticals. Our review explains the role of current MS in the analysis of both small-molecule chemical drugs and biopharmaceuticals. Important features of MS-based technologies being implemented, method requirements, and related challenges are discussed. The differences in analytical procedures for small molecule pharmaceuticals and biopharmaceuticals are pointed out. While a single method or a small set of methods is usually sufficient for quality control in the case of small molecule pharmaceuticals and MS is often not indispensable, a large panel of methods including extensive use of MS must be used for quality control of biopharmaceuticals. Finally, expected development and future trends are outlined.

Analytical Quality by Design-Compliant Development of a Cyclodextrin-Modified Micellar ElectroKinetic Chromatography Method for the Determination of Trimecaine and Its Impurities.

In 2022, the International Council for Harmonisation released draft guidelines Q2(R2) and Q14, intending to specify the development and validation activities that should be carried out during the lifespan of an analytical technique addressed to assess the quality of medicinal products. In the present study, these recommendations were implemented in Capillary Electrophoresis method development for the quality control of a drug product containing trimecaine, by applying Analytical Quality by Design. According to the Analytical Target Profile, the procedure should be able to simultaneously quantify trimecaine and its four impurities, with specified analytical performances. The selected operative mode was Micellar ElectroKinetic Chromatography employing sodium dodecyl sulfate micelles supplemented with dimethyl-ß-cyclodextrin, in a phosphate-borate buffer. The Knowledge Space was investigated through a screening matrix encompassing the composition of the background electrolyte and the instrumental settings. The Critical Method Attributes were identified as analysis time, efficiency, and critical resolution values. Response Surface Methodology and Monte Carlo Simulations allowed the definition of the Method Operable Design Region: 21-26 mM phosphate-borate buffer pH 9.50-9.77; 65.0 mM sodium dodecyl sulfate; 0.25-1.29% v/v n-butanol; 21-26 mM dimethyl-ß-cyclodextrin; temperature, 22 °C; voltage, 23-29 kV. The method was validated and applied to ampoules drug products.

Nitrites as precursors of N-nitrosation in pharmaceutical samples - A trace level analysis.

The ultra-performance liquid chromatography (UPLC) method, which involves pre-column derivatization of nitrite with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotriazole (NAT), offers the advantages of easy sample preparation, simple derivatization, stable derivatives, rapid analysis, high sensitivity and specificity and lack of interferences for determining nitrite in pharmaceutical samples. Determination of NAT was performed on a an Acquity UPLC HSS T3 column using a gradient elution of 0.1% formic acid with acetonitrile at flow rate of 0.4 mL/min and temperature at 45 °C. The single-quadrupole mass detector was operated in the positive ion mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 170 as [M+H]+. The UPLC-MS method was validated as per International Council on Harmonization (ICH) guidelines in terms of linearity, limit of detection, limit of quantification, selectivity, accuracy, precision, intermediate precision and stability. The UPLC-MS method was demonstrated to be applicable for the determination of nitrite in various pharmaceutical samples. The proposed UPLC-MS method was used to study the effect of nitrite content in pharmaceutical products on the formation of N-nitrosamines. The high importance of nitrites in relation to the N-nitrosation reaction was discussed. As deduced from theory and justified by the presented results, reducing the nitrite concentration could definitely solve the N-nitrosamine contamination. Nitrites, unlike secondary and tertiary amines, are universal precursors to any N-nitrosamine, so this solution is easily transferable to any relevant pharmaceutical product.

N-Nitrosation in the absence of nitrosating agents in pharmaceuticals?

The possibility of N-Nitrosation in the absence of nitrosating agents was studied on model solutions and film coated tablets containing metformin. N-nitrosodimethylamine (NDMA) and N-nitrosation precursors (dimethylamine and nitrites) were determined using previously published fully validated analytical methods. Alternative routes to N-nitrosation were found. Dimethylamine can undergo an oxidation to nitrite in the presence of strong oxidants (e.g., H2O2), as was observed during wastewater treatment in several published works. The resulting nitrite can consecutively act as a nitrosating agent. We proved that the described reaction indeed leads to N-nitrosation (NDMA formation in case of dimethylamine precursor) in model solutions made of dimethylamine and H2O2. An experiment was designed in order to prove those reactions take place in dosage forms. Film coated tablets present a highly heterogenous system with several solid phases and low water activity, which is in stark contrast to the liquid wastewater, where this reaction was originally studied. Despite that, the described reaction took place even in the tablets, but only to a small degree. The amount formed via this alternative route corresponds to less than 10 % of the total formed NDMA. The pH optimum of this alternative route lies in the alkaline range which was confirmed by the determined NDMA concentration in model solutions. The solid phase system (i.e., tablets) was found to behave differently. The addition of Na2CO3 into the tablets during manufacture resulted in tablets without NDMA (cNDMA < LOQ) even in batches spiked with both dimethylamine and H2O2. Thus, adjusting the pH of the solid dosage forms remains a sufficient measure of controlling N-nitrosamines in the product, even in product with limit amounts of oxidating agent (H2O2) and N-nitrosation precursor (dimethylamine).

The determination of two analogues of 4-(azidomethyl)-1,1'-biphenyl as potential genotoxic impurities in the active pharmaceutical ingredient of several sartans containing a tetrazole group.

4'-(azidomethyl)-[1,1'-biphenyl]-2-carbonitrile (GTI-azide-1) and 5-(4'-(azidomethyl)-[1,1'-biphenyl]-2-yl)-1H-tetrazole (GTI-azide-2) are potentially genotoxic impurities that can be present at trace levels in the active pharmaceutical ingredients and drug products of sartans containing a tetrazole group. A method of high-performance liquid chromatography coupled with mass spectrometry, that allows the determination of those genotoxic impurities at sub-ppm level relative to the active pharmaceutical ingredient, was developed. The method utilises a very efficient liquid chromatograph Waters Acquity I-Class coupled with a highly sensitive tandem mass spectrometer Xevo TQ-XS. The separation was achieved on a column Acquity UPLC BEH Shield RP18 1.7 µm employing a linear elution gradient. The mass spectrometer was used with a heated electrospray ionization. The method was found to be sufficient in terms of sensitivity, linearity, precision, accuracy, selectivity and robustness and is easily applicable in the pharmaceutical quality control environment. The method allows for accurate quantification of both impurities GTI-azide-1 and GTI-azide-2 at levels below 1/10th of the specification limit, which is crucial in the context of pharmaceutical analysis. The limit of quantification was determined to be 0.033 ppm and 0.025 ppm for GTI-azide-1 and GTI-azide-2, respectively.

Insight into the formation of N-nitrosodimethylamine in metformin products.

An effective analytical method for the quantification of N-nitrosodimethylamine (NDMA) using a liquid chromatography coupled with tandem mass spectrometry was developed and applied to a process optimization study of the production of metformin film coated tablets in order to identify the key factors behind the NDMA formation in metformin products. The method uses a linear gradient elution with mobile phases 0.1 % formic acid in water for chromatography and methanol for chromatography and a column Acquity UPLC HSS T3 1.8 µm. The use of the tandem mass spectrometry in a positive ion mode with an atmospheric pressure chemical ionization allows for the use of an isotopically labelled internal standard and an external calibration standard. The method was validated according to the guidelines of International Council for Harmonization in terms of limit of detection and quantification, linearity, precision, accuracy and method selectivity. To further justify the effectiveness of the method, a comparison between two laboratories was performed using a linear regression testing. Both methods give comparable results. 469 samples of both metformin active pharmaceutical ingredient and film coated tablets were analysed and the key factors behind NDMA formation were identified. Hypotheses explaining the mechanism were formulated and confronted with measurements and scientific literature. Protective measures to prevent NDMA contamination in metformin products were drawn.

HILIC-MS determination of dimethylamine in the active pharmaceutical ingredients and in the dosage forms of metformin.

A sensitive and specific hydrophilic interaction chromatography (HILIC) method for the separation and determination of dimethylamine (DMA) in active pharmaceutical ingredients (APIs) and in dosage forms of metformin (MET) has been developed and validated. A feasible analytical method based on HILIC coupled with mass spectrometry detection (HILIC-MS) was established using a simple sample preparation. The separation of MET was achieved on a Cortecs HILIC column using a mixture of 10 mmol/L ammonium formate adjusted to pH 4.8 and acetonitrile (25:75, v/v) at 0.8 mL/min flow rate. The a single-quadrupole mass detector was operated in positive ion mode. Quadrupole mass analyser was employed in selected ion monitoring mode using a target ion at m/z = 46 as [M+H]+. The HILIC-MS method was validated as per International Council on Harmonization (ICH) guidelines in terms of linearity, limit of detection, limit of quantification, selectivity, accuracy, precision and intermediate precision. The main benefit of the HILIC-MS method is a simple sample pretreatment and a quick and sensitive HILIC-MS analysis. The method was demonstrated to be applicable for the determination of DMA in routine quality control evaluation of commercial samples of metformin of both API and dosage forms. The HILIC-MS method was developed as a simpler and faster alternative to compendial method for determination of DMA (as specific impurity F) in MET described in European Pharmacopoeia.

Detection and structure elucidation of the new degradation impurities in the pharmaceutical formulations of ruxolitinib hydrobromide.

New degradation impurities at m/z 327.15 and m/z 311.16 using gradient UHPLC method with UV detection and highly selective QDa mass detection were observed during the ruxolitinib hydrobromide (RUX.HBr) : excipient binary mixture degradation study. High mass resolution LC-MS and nuclear magnetic resonance (NMR) techniques were employed to identify and fully characterize the degradation compounds. The degradation impurities were unambiguously identified as (R)-4-amino-6-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)pyrimidine-5-carboxylic acid and (R)-3-(4-(6-amino-5-formylpyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile and mechanism of their formation was proposed. It has been confirmed that the degradation products are formed in mixtures of RUX.HBr with some excipients in the presence of oxygen. Based on the forced degradation study, the chemically stable of pharmaceutical formulations were prepared to eliminate the formation of these impurities.