Trace Analysis of Potentially Mutagenic Boronic Acids and Esters in

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Trace Analysis of Potentially Mutagenic Boronic Acids and Esters in Drug Substance by ICP-MS Ila Patel, C. J. Venkatramani, Andreas Stumpf, Larry Wigman, and Peter Yehl Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00325 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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Organic Process Research & Development

Trace Analysis of Potentially Mutagenic Boronic Acids and Esters in Drug Substance by ICP-MS Ila Patel*, C.J. Venkatramani, Andreas Stumpf, Larry Wigman, Peter Yehl Small Molecule Pharmaceutical Sciences, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

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ABSTRACT: Boronic acids and boronate esters are commonly used reagents in Suzuki-Miyaura coupling chemistry which are known to be weakly mutagenic in microbial assays. In this paper, we describe an inductively coupled plasma mass spectrometer method (ICP-MS) for the analysis of boron at low parts per million levels. Boronic acid and ester levels are then calculated stoichiometrically in drug substance based on the determination of boron with good sensitivity (LOQ of 0.8 ppm). The paper outlines some of the important aspects of the method and instrument features like use of organic diluent, spray chamber configuration and its optimization which are critical for analysis of trace inorganic species in drug substance.

KEYWORDS: Boronic acid and esters, ICP-MS, trace analysis, drug substance, mutagenic impurities.

INTRODUCTION The analysis of mutagenic impurities in drug substance is a regulatory expectation and requires methodology capable of trace detection down to low parts-per-million (ppm). Boronic acids and esters are commonly used in C-C bond formation for the convergent synthesis of new drug substances by the palladium-catalyzed Suzuki cross-coupling reaction.1-12 Donovan et. al.13 showed some boronic acids to be weakly mutagenic in microbial assays. Thus; potentially mutagenic boronic acids and esters require highly sensitive testing methods for drug substance analysis. Developing methods with sensitivity in the low ppm range has been challenging.


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In general, quantitative analysis of residual mutagenic impurities is very challenging due to their high reactivity, low volatility and high polarity, requiring an array of analytical techniques. Strategies for the analysis of mutagenic impurities is discussed at length in literature14-22, however, analysis of boronic acids have not been published. Chromatographic analysis of mutagenic cyclopropyl boronic acid (AMES positive) is extremely challenging. GC with flame ionization detection is an option; however, the technique lacks sensitivity. The detection limit is about few tenths of a percent (0.2% = 2000 ppm) that is few orders of magnitude higher than the levels desired for mutagenic impurities. Lack of chromophores and poor ionization of cyclopropyl boronic acid makes it difficult to analyze it by HPLC with UV and or MS detection. While highly specific, quantitative 1H-NMR (qNMR) is not practical for trace analysis. ICP-MS is highly sensitive and specific for trace elemental boron. Hence, accurate stoichiometric calculation of the corresponding boronic acids and esters is an approach that addresses the sensitivity limitations of other techniques discussed above. An ICP-MS method was developed and validated for accuracy, precision, linearity and sensitivity. The method is linear from 0.1 ng/mL to 20 ng/mL with correlation coefficient of 0.9996 and with a limit of quantitation (LOQ) of 0.8 ppm. EXPERIMENTAL Background. Acid digestion involving aqueous solvents is an option to solubilize the sample, however, most pharmaceutical compounds show poor solubility especially at high concentration (10 mg/mL or higher). In general, acid digestion is time consuming and laborious. On the contrary, pharmaceutical compounds are fairly soluble in organic solvents like dimethyl sulfoxide (DMSO) and hence are ideal for ultra-trace analysis of residual boron using ICP-MS.


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Historically, direct analysis of trace metals in organic solvents like DMSO has been challenging due major short-comings of the nebulizers and spray chambers used in ICP-MS. Burning of carbon rich organics in the plasma resulted in deposition of carbon particles or soot on the sampler cone. The deposits block the nebulizer orifice; shift the background signal by producing Swann bands and decrease sensitivity. The deposits also lead to carbon-carbon polyatomic species such as such as

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12 14

C NH and

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C-14N+ which interference with signals for elements

Al. Limiting the total carbon entering the plasma significantly reduces the carbon

deposits. The carbon buildup can be eliminated by addition of oxygen flow into the nebulizer stream through an external gas module allowing a continuous oxygen supply into the plasma. Optimizing the flow of oxygen is critical, as low flow rate (sub-optimal) will result in carbon buildup on the cones and the torch whereas excessive flow will destabilize the plasma and promote the formation of oxides resulting in reduced intensities for elements of interest. Most commercially available organic interfaces are geared towards solvents that have higher vapor pressure than water (e.g. hexane, ethanol, benzene, xylene, chloroform and acetone). This requires sub-ambient operation to lower the solvent mass going into the plasma. However, the lower operation temperature precludes the use of lower vapor pressure solvents like DMSO due to freezing in the nebulizer tip. To circumvent this problem, we collaborated with ESI (Elemental Scientific, Inc, Omaha, NE, USA) and Perkin Elmer (Waltham, MA, USA) to modify the first generation organic solvent spray chamber by incorporating heating capability and continuous temperature monitoring. This modified cyclonic spray chamber contains a Peltier heated controlled unit marketed as the PC3x unit. This is the ideal spray chamber for trace level analysis of boron in drug substance which are soluble in solvents such as DMSO.


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INSTRUMENTATION All experiments were performed using a Perkin Elmer Nexion 300 ICP-MS (Perkin Elmer, Shelton, CT) with an AS-91 autosampler. The instrument was optimized using a Perkin Elmer Nexion tuning solution containing multiple elements (1 µg/ml solution of Be, Ce, Fe, In, Li, Mg, Pb and U) to enhance its sensitivity and selectivity. The instrumental conditions and operation parameters are shown in Table 1. To decrease the matrix effect, the ICP-MS was operated at a higher power (1600 W) and low nebulizer flow rate. The nebulizer gas flow and auxiliary gas flows were adjusted to minimize interferences (CeO++/Ce+ 15,000 cps

79657.7 cps

>35,000 cps

74318.1 cps

>30,000 cps

Precision

Trace Analysis of Potentially Mutagenic Boronic Acids and Esters in

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