29 A B
Here, we’ve developed a method for purity analysis of 2-butynoic acid, along with trace level impurity identification, to facilitate successful synthetic processes in the pharmaceutical industry. 2-butynoic acid, containing a weak chromophore, typically yields poor sensitivity in HPLC-UV analytical measurements, making it a good candidate to validate the robustness of IC-MS. In this method, anionic separation coupled with ESI MS employing negative ionisation was used to analyse impurities present in 2-butynoic acid samples. Limits of quantification and linearity of detection by both IC-sCD and MS were evaluated. The developed method was then used to study a range of organic acids potentially present as impurities at pharmaceutically relevant levels.
Materials and methods
Standards, materials and instruments
Figure 2. Additives influence the m/z response. Extracted ion chromatograms of m/z 127 (A) and m/z 171 (B) analysed using MeCN: H2
O (black), MeCN: H2 mM ammonia (pink) as make-up flow [2].
of the acids is gained through ion-exchange of the negatively charged acidic group with the ammonium resin on the column with a basic eluent [9].
IC is inherently better suited for analysing polar molecules: the separation is highly dependent on the analyte’s pKa and size-to-charge ratio. However, IC analysis with conductivity detection does not offer absolute identification of the components corresponding to each peak, in turn, not allowing the accurate quantitation of all trace impurities present if they partially or fully coelute. Consequently, as peak purity tracking is not directly possible with just IC-sCD, it hinders peak confirmation for identifying unknowns. Despite its promise, IC methods, therefore, have been underutilised in organic acid analysis.
One way to improve the overall selectivity is to couple IC with MS. The mass information obtained for each peak allows another level of resolution of the corresponding components, increasing the specificity and selectivity of the analytical approach. However, the strong basicity of eluents used
in anionic analysis by IC are incompatible with electrospray ionisation (ESI) MS due to their possible corrosive effects on the stainless steel components of the MS ion source, adding another challenge to overcome. In recent times, eluent suppressors have made MS coupling easier [9,10] as they suppress the eluent by removing the counter ions before introducing them into the MS ion source as water, thereby enabling ionisation of the analyte to happen.
Hyphenating IC with MS along with eluent suppressors can address the lack of obtainable information on peak purity. This IC-MS approach to analyse polar organic acids, when appropriately developed and optimised, can serve as a superior alternative to HPLC and GC methods in terms of sensitivity and robustness. In addition to sensitive measurements, quality control and quality assurance laboratories across different industries need reliable and reproducible methods, with quantitative capabilities in the required dynamic range, to routinely detect organic acid impurities to minimise risks upfront.
O + 12.5 mM ammonium acetate (blue) and MeCN: H2 O + 25
Standards of 2-butynoic acid and other organic acids (namely, acetic acid, propinoic acid, formic acid, butanoic acid, crotonic acid, pentanoic acid, propiolic acid, pentynoic acid) were obtained from Sigma-Aldrich Company Ltd (Gillingham, United Kingdom). Samples were diluted with 18.2MΩ-cm ultrapure water obtained from a MilliQ unit (Watford, Hertfordshire). HPLC grade 0.25 M ammonia and 0.25 M ammonium acetate were obtained from Fisher Scientific (Loughborough, UK). Samples of 2-butynoic acid were obtained from FAR Chemical (Florida, USA), Boropharm (Michigan, USA) and AstraZeneca (Macclesfield, UK).
IC-MS was performed on a Thermo Scientific Dionex Integrion High Pressure Ion Chromatograph (Hemel Hempstead, UK) coupled to an ISQ EC mass spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK). The IC-MS workflow was set up as shown in Figure 1.
IC conditions
The IC systems comprised of a guard column (Dionex IonPac™ AG11-HC-4 µm 2 ×50 mm), an IC column (Dionex IonPac™ AS11-HC-4 µm 2 ×250 mm) and suppressor (initially, Dionex AERS 500e 2 mm and then changed to Dionex ADRS 600 2 mm) with conductivity detector. The Dionex AERS 500e suppressor applies a direct fixed current to suppress the hydroxide to water, and the potassium counter-ions are vented to waste as they are pulled through the exchange membrane. A suppressor current
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