Improved detection of cefonicid with 2D HPLC

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  • Published: Oct 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Improved detection of cefonicid with 2D HPLC

Standard method for cefonicid HPLC unsuitable for MS

It is important to characterise impurities in pharmaceuticals, and often LC-MS is the best way to do so. However, standard pharmacopeia HPLC methods often use non-volatile salts, such as phosphate buffers, which clog up mass spectrometers. Abandoning such a method by, for instance, replacing phosphate with ammonium formate risks changing the elution order of impurities or failing to separate them altogether. There may also be difficulties in comparing UV traces from the initial HPLC method with the total ion current (TIC) output from LC-MS.

The antibiotic cefonicid sodium is commonly used in many countries, typically being administered by injection. The Chinese Pharmacopeia HPLC analysis method for this compound uses phosphate, so cannot be used with standard LC-MS. The Hangzhou scientists employed a trap-free heart-cutting two-dimensional HPLC system to identify new impurities in cefonicid. The first column used a phosphate buffer, while the second used ammonium formate, allowing mass spectrometry by an ion trap time-of-flight instrument (IT-TOF). Six 20 μL loops were employed to collect output from the first column prior to injection onto the second column.

Two-dimensional HPLC and MS detect new cefonicid impurities

HPLC was carried out using a Shimadzu two-dimensional Nexera-XR system. The first column was a Grace Alltima C18, eluted with methanol and 0.02 M aqueous ammonium dihydrogen phosphate (adjusted to pH 7.0 with ammonia solution), using a gradient ranging from 16% to 60% methanol. A Shimadzu SPD-M20A photodiode array detector (PDA or DAD) was employed. The second column was a Shimadzu Shim-pack GISS C18, eluted with methanol and 0.01 M aqueous ammonium formate, using a gradient ranging from 5 to 95% methanol. A Shimadzu SPD-20A UV/visible detector was used for detection from the second column. Particular peaks from the first column were taken up in the loops and injected onto the second column. The phosphate from the first column was eluted early on from the second column and diverted to waste prior to elution of the peaks of interest.

Mass spectrometry was carried out with a Shimadzu IT-TOF using electrospray ionisation (ESI). The peaks were identified in both positive and negative ion modes, giving complementary fragment ions. Seven impurities were identified from cefonicid, one of which was already noted in the Chinese Pharmacopeia and six of which were novel. Cefonicid that had been subjected to heat treatment gave an additional unknown impurity peak, making seven new impurities in all.

Close examination of the mass spectral output and comparison with the data from cefonicid allowed all seven new impurities to be identified. The instrument gave accurate masses for both molecular and daughter ions. The new impurities included one oxidation product (ring sulfur converted to sulfoxide), an isomer (double bond position changed), a synthetic intermediate (acetate on the benzylic alcohol) and four hydrolysis/degradation products.

Powerful 2D-HPLC MS system successfully identifies impurities

The combination of two-dimensional HPLC and IT-TOF MS proved a powerful method to identify new impurities. The impurities were initially separated in line with the pharmacopeia method, but the use of a second column allowed mass spectrometry to be carried out, with the unwanted phosphate being diverted to waste. Although more costly than standard methods, such a setup could be usefully applied to identify impurities in many other pharmaceuticals and could also be used for other organic compounds, such as agrochemicals or polymer additives.

Related Links

Rapid Communications in Mass Spectrometry, 2017, 31, 1541-1550. Wang et al. Separation and characterization of unknown impurities in cefonicid sodium by trap-free two-dimensional liquid chromatography combined with ion trap time-of-flight mass spectrometry.

Chromatographia, 2013, 76, 5-11. Lee et al. Application of heart-cutting 2D-LC for the determination of peak purity for a chiral pharmaceutical compound by HPLC.

Analytical Chemistry, 2015, 87, 5310-5317. Pursch et al. Loop-based multiple heart-cutting two-dimensional liquid chromatography for target analysis in complex matrices.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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