A High Sensitive LC-MS/MS Method for the Simultaneous Determination of Potential Genotoxic Impurities Carboxy Phenyl Boronic Acid and Methyl Phenyl Boronic Acid in Lumacaftor

A simple, rapid, and highly sensitive LC-MS/MS method has been developed for the simultaneous and trace level quantification of underivatized boronic acids in lumacaftor active pharmaceutical ingredient. Chromatographic separation of boronic acids and lumacaftor achieved using Agilent Poroshell HPH C18 150 × 4.6 mm 2.7 μ column with 0.1% ammonia in water as mobile phase A and 100% acetonitrile as mobile phase B at a flow rate of 0.25 ml/min. Gradient elution was used with a total method run time of 14 minutes. Boronic acids were successfully ionized and quantified without derivatization using electrospray ionization in negative mode using tandem quadrupole mass spectrometry in multiple reactions monitoring mode. Method validation was performed as per ICH guidelines with good linearity over the concentration range of 0.05 ppm to 5 ppm of Lumacaftor test concentration for both the boronic acids with a correlation coefficient of >0.99. Recoveries were found good at different concentration levels and within the range of 80% 120%. The developed method can be successfully used for the routine quantification of boronic acids at a concentration level of 20 ng/ml (1 ppm with respect to 20 mg/ml lumacaftor).


Introduction
Boronic acids are used as chemical building blocks during the synthesis of organic compounds and are the most common intermediates used in the preparation of biaryl derivatives using Suzuki-Miyaura coupling [1] [2] [3]. Studies have been conducted to evaluate the mutagenic toxicity of commercially available boronic acids resulted in positive Ames test [3]. Boron containing compounds are potential mutagenic impurities and often controlled in drug substances as per International council of Harmonization (ICH) M7 [4] guidelines using highly sensitive analytical techniques like LC-MS/MS for quantification.
Lumacaftor is a pharmaceutical drug used in combination with Ivacaftor sold under the name of ORKAMBI approved by FDA for the treatment of genetic disease cystic fibrosis (CF) [5]. Cystic fibrosis results due to the defects in the cystic fibrosis transmembrane conductance regulator (CFTR) causes dysregulation of epithelial fluid transport in the lungs, gastrointestinal tract, and sweat glands and can cause progressive multi organ failure [6] [7] [8].
The aim of the current research work is to quantify mutagenic boronic acid impurities in Lumacaftor drug substance. During literature search we could find methods published for determination of boronic acids using SIM ionization mode in single quadrupole LCMS [9] [10], however in the present article we have analyzed boronic acids which using highly sensitive and selective triple quadrupole LC-MS/MS in Multiple reaction monitoring (MRM) mode. We could also find article published for derivatized boronic acids using LC-MS/MS [11] however we could analyze underivatized boronic acids in the present work using highly sensitive LC-MS/MS which can reduce the sample preparation time and efforts of derivatization and increase the throughput during analysis of multiple samples of lumacaftor drug substance. Several LCMSMS methods have published for the determination of Lumacaftor in combination with Ivacaftor in biological fluids [12] [13]. Chromatographic methods published for the determination of Lumacaftor in its bulk dosage form [14] [15]. As per the extensive literature survey performed, there is no data published for the quantification of boronic acids in lumacaftor using LC-MS/MS till date.
Both the possible boronic acids were procured based on the results obtained from computational structure analysis for mutagenicity alerts. The concentrations of boronic acid impurities in lumacaftor must be controlled at concentrations lower than 1 ppm considering the maximum allowable dosage. In this paper, we present the LC-MS/MS method development for the simultaneous determination of carboxy phenyl boronic acid and methyl phenyl boronic acid in lumacaftor. The chemical structures of Carboxy phenyl boronic acid, Methyl phenyl boronic acid and Lumacaftor along with molecular formula and mono isotopic mass considering the most abundant isotope of boron are captured in Figure 1. The validation of the method in terms of limit of detection, limit of quantification, repeatability, accuracy, robustness, linearity and specificity is in accordance with ICH guidelines [16].

Reagents and Chemicals
LCMS grade with highest purity of >99.8% solvents and reagents were used. Water, Acetonitrile and Methanol were procured from Honeywell (Charlotte, NC, USA). Ammonium hydroxide solution was purchased from Fluka. Lumacaftor drug substance and boronic acids were procured from PS3 labs LLP, Hyderabad, India.

Mobile Phase Preparation
Mobile phase A was prepared by adding 1 ml of ammonium hydroxide solution in 1000 ml of water and mobile phase B was 100% acetonitrile. Both the mobile phases were sonicated to degas and stored at ambient temperature for further usage. Mobile phases prepared freshly before each set of analysis.

Preparation of Sample and Standard Solutions
Weighed accurately 10 mg each of carboxy phenyl boronic acid and Methyl phenyl boronic acid and transferred in 10 ml volumetric flasks and made up to mark with 100% acetonitrile to obtain final concentration of 1000 µg/ml. A 10 µg/ml mixture of boronic acids were further prepared by addition of appropriate stock volumes and diluted with 80% Acetonitrile solution. Performed further dilution to 1 µg/ml mixture. Calibration standards were prepared from 1 µg/ml to achieve the final concentrations of 100, 80, 60, 40, 10, 5 and 1 ng/ml.
Recovery experiment required spiking solutions which were prepared by weighing accurately 100 mg of lumacaftor drug substance in 5 ml volumetric flask and then add appropriate volume of boronic acids impurity mix stock to obtain a concentration of 20 ng/ml (1.0 ppm) and 5 ng/ml (0.25 ppm) with respect to test concentration of 20 mg/ml of lumacaftor. To ensure repeatability experiment performed in triplicates.

Method Validation
Successful validation of the developed method in terms of Specificity, reproducibility, linearity, LOD, LOQ, robustness and solution stability executed, and the validation parameters conducted using ICH guidelines [16].

Chromatographic Method Development
This study was conducted to develop highly sensitive and selective analytical method that could separate and quantify both the mutagenic boronic acid impurities in Lumacaftor drug substance.

Optimization of MSMS Parameters
Mass spectrometric conditions optimization aimed at developing simple, selective, highly sensitive and robust method for the determination of determination of underivatized boronic acids in lumacaftor drug substance. 1.0 µg/ml impurity mix solution was used to carry out MSMS method development. During initial stages of development negative mode ionization was found to be more sensitive due to the nature of the impurities, limiting the method development to negative ionization only. Critical compound dependent parameters like capillary, Nozzle and fragmentor voltages (V) were optimized for boronic acids to obtain the desired response for parent molecular ions considering the most abundant isotope of Boron which are presented in Table 1.

Method Validation
As per ICH recommended guidelines, the developed method was successfully validated and established all the critical parameters required to demonstrate the method efficiency.

Specificity
Lumacaftor with boronic acid impurities mix solution was prepared at required specification level in the diluent and then subjected to LCMSMS analysis. The results obtained shown that there was no interference of Lumacaftor API with carboxy phenyl and methyl phenyl boronic acid impurities. The chromatogram acquired was presented in Figure 2.

Linearity
Method linearity was established from 1 ng/ml to 100 ng/ml (0.05 -5 ppm) for both the boronic acid impurities. The regression coefficient, slope, and intercept values were derived using least squares linear regression analysis of average peak areas versus concentration of impurities. Good correlation between peak areas and concentrations of impurities observed as can be seen in Table 2 and linearity figures captured in Figure 6 & Figure 7.

LOD and LOQ
The LOQ and LOD values for both the boronic acid impurities were determined based on S/N ratios of 10.0 and 3.0 respectively, by injecting known standard concentrations and the results are captured in Table 2. S/N ratio values are derived using peak to peak algorithm for both the boronic acid impurities. Recovery and reproducibility were also evaluated at LOQ level using triplicate injections. Reproducibility data at LOQ concentration for Carboxy phenyl and Methyl phenyl boronic acids also captured in Table 3.

Accuracy and Recovery
Deviation from linearity standard concentrations was referred to as accuracy and was evaluated by injecting impurity mixture from LOQ which is 25% of specification limit and on 5 times the specification limit. The acceptance criteria for accuracy is between 80% -120% for all the linearity standards except LOQ which could be 70% -130% for such a low concentration range. Accuracy values observed at all levels for both the boronic acid impurities were within 15% which are well within the required acceptance criteria. Recovery was evaluated by standard addition method in triplicate at two concentrations at 0.25 ppm and 1.0 ppm levels in Lumacaftor API. The acceptance criteria for recovery is 80% -120%. The percentage recoveries for both the boronic acid impurities presented in Table 4.

Robustness
Method robustness was evaluated changing different conditions of the method including the flow rate and composition of the mobile phase and column oven temperatures. The optimized flow rate of the mobile phase was 0.25 mL/min and the same was altered from 0.225 to 0.275 ml/min. The effect of column oven temperature on resolution was studied at 35˚C and 45˚C (altered by 5.0˚C).
There was no impact on chromatographic performance of both the boronic acid impurities due to the mentioned changes proving the method robustness based American Journal of Analytical Chemistry on the obtained results.

Repeatability and Solution Stability
Repeatability of the developed method was evaluated by injecting six replicate injections at 20 ng/ml (1.0 ppm) mixture of boronic acid impurities and observed the %RSD after including multiple bracketing standards. The acceptance criteria for cumulative %RSD is less than 15%. The RSD values achieved for both the boronic acid impurities are less than 6.1% which are well within the acceptance criteria which are presented in Table 5. Repeatability overlaid chromatogram for both the boronic acid impurities captured in Figure 8. The solution stability study of lumacaftor and boronic acid mixture was evaluated by placing spiked and un spiked sample solutions at 25˚C for 24 h and measured against freshly prepared standard solutions and there were no significant changes observed for any of the impurities. Therefore, we confirmed the stability of impurities in sample solution for at least 24 hours.

Conclusion
In summary, the work presented here is novel in terms of simultaneous deter-

Conflicts of Interest
To the best of our knowledge, this is the first method published for simultaneous determination of underivatized Carboxy phenyl and Methyl phenylboronic acids in Lumacaftor drug substance and hold no conflicts to declare.