Technical Note pubs.acs.org/ac
Flow-Injection MS/MS for Gas-Phase Chiral Recognition and Enantiomeric Quantitation of a Novel Boron-Containing Antibiotic (GSK2251052A) by the Mass Spectrometric Kinetic Method Lianming Wu,* Frederick G. Vogt, and David Q. Liu Analytical Sciences, Product Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, United States S Supporting Information *
ABSTRACT: The present work demonstrates, for the first time, the application of the mass spectrometric kinetic method for quantitative chiral purity determination by automatic flowinjection MS/MS. The particular compound analyzed is GSK2251052A, a novel boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections. Chiral recognition and quantitation of GSK2251052A was achieved based on the competitive dissociation kinetics of the Cu II -bound trimeric complex [Cu II (A)(ref*) 2 −H]+ (A = GSK2251052A or its R-enantiomer, ref* = L-tryptophan) that gives rise to CuII-bound dimeric complexes. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow chiral purity determination of pharmaceutical compounds during enantioselective synthesis. By using flow-injection MS/MS, enantiomeric quantitation of GSK2251052A by the kinetic method proved to be fast (2 min for analysis of each sample) and to have accuracy comparable to chiral LC−MS/MS and LC−UV methods as well as the method using chiral derivatization followed by LC−MS/MS analysis. This flow-injection MS/MS method represents an alternative approach to commonly used chromatographic techniques as a means of chiral purity determination and is particularly useful for rapid screening of chiral drugs during pharmaceutical development.
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However, chiral separation is often achieved using a chiral column that is typically more delicate and expensive and might provide less rugged separation conditions than those used in other forms of chromatography.15 In addition, development and validation of an “impurity-tolerant” chiral chromatographic method can take extensive time and resource even when automated screening systems are employed. Faster methods for chiral analysis are always desirable, especially in drug development settings where large numbers of drug candidates in multiple batches need to be examined. As such, mass spectrometry (MS) has attracted much interest in chiral recognition and quantification due to its unparalleled speed, intrinsic sensitivity, molecular specificity, tolerance of impurities, and capability of probing chiral interactions in a solventfree environment. Because enantiomers have identical mass, MS has often been thought of as a “chirally blind” technique. However, chiral molecules can be distinguished by MS via reactions with chiral reference molecules.16 Recently, several purely MS-based methods for chiral analysis have been developed, including (i) the kinetic method based on the dissociation of cluster
he accelerating trend toward the use of enantiomerically pure compounds as drugs has attracted much interest in the pharmaceutical industry.1−3 Enantiomeric forms of drugs can produce different therapeutic (or adverse) effects and may be metabolized by different pathways.4,5 As a result, the development and marketing of single enantiomer drugs have grown rapidly6 following the release of new U.S. Food and Drug Administration (FDA) guidelines that recommend the use of enantioselective identity and stability tests, as well as assays to determine the contributions of individual enantiomers to pharmacological and toxicological activities.7 Therefore, methods for quantitative analysis of enantiomeric mixtures of drugs and their metabolites are required from early clinicalphase research to the filing of a New Drug Application (NDA) as well as during postapproval manufacturing and control.8,9 Enantiomeric forms of chiral molecules can be recognized and quantified only in an asymmetric environment, whether the analysis is by a traditional chemical or instrumental method.10 Solution-phase spectroscopic techniques (such as circular dichroism (CD)11 and nuclear magnetic resonance (NMR) using chirally pure shift reagents12) can be employed for chiral analysis, but they are not tolerant of impurities. Chiral chromatography, including liquid chromatography (LC)13 and supercritical fluid chromatography (SFC),14 is most commonly utilized for chiral analysis in the pharmaceutical industry. © 2013 American Chemical Society
Received: March 6, 2013 Accepted: April 19, 2013 Published: April 19, 2013 4869
dx.doi.org/10.1021/ac401079x | Anal. Chem. 2013, 85, 4869−4874
Analytical Chemistry
Technical Note
ions,17−20 (ii) the formation of diastereomeric host−guest adducts21 or Cu(II)−chiragen complexes22 in single-stage MS, (iii) host−guest ion/molecule reactions,23 (iv) collisioninduced dissociation (CID) of diastereomeric adducts formed from an analyte and a chiral reference in a MS/MS experiment,24 and (v) kinetic resolution in solution phase followed by MS analysis.25 Practical applications of chiral analysis, especially quantitative analysis, require several characteristics that are not often simultaneously available. These requirements are (i) a large chiral selectivity to facilitate quantitative analysis, (ii) chiral recognition which is independent of the relative concentration of an analyte and chiral reference reagent, (iii) high tolerance to impurities, (iv) no requirement for isotopically labeled reagents, and (v) implementation using a commercial instrument. All these features are available, however, by employing a massspectrometric method in which the kinetics of dissociation of transition metal-bound complex ions is measured in a MS/MS experiment. The procedure represents a particular application of the kinetic method26 that has been successfully utilized to recognize and quantify different classes of pharmaceutical compounds, including antiviral nucleoside analog agents,27 antibiotics,28 Parkinson, and potassium channel opener drugs,29,30 as well as β-blockers and stimulants.31 The objective of this study is to demonstrate the extension of the kinetic method to the rapid determination of enantiomeric compositions using flow-injection MS/MS without chromatographic separation. The particular case of interest is the practical application of the gas-phase mass spectrometric method for rapid chiral purity analysis of GSK2251052A [(S)-3-(aminomethyl)-7-(3-hydroxypropoxy)-3H-benzo[c][1,2]oxaborol-1-ol, Scheme 1., structure a] that is a novel
(Figure S1 of the Supporting Information). The CuCl2 salt, L-tryptophan (L-Trp), trifluoroacetic acid (TFA), perchloric acid (HClO4), triethylamine (TEA), and 2,3,4,6-tetra-O-acetylβ-D-glucopyranosyl isothiocyanate (GITC) were purchased from Sigma-Aldrich (St. Louis, MO). The HPLC-grade solvents, including water and acetonitrile, were purchased from Honeywell Burdick and Jackson (Morristown, NJ). Formation of CuII-bound Trimeric Complexes of GSK2251052A and Its R-enantiomer by the Automatic Flow Injection. The 50/50 water/acetonitrile solutions containing a mixture of GSK2251052A and its R-enantiomer, chiral reference ligand L-Trp, at a concentration of 100 μM each, and 25 μM CuCl2 salt were prepared in 2 mL HPLC vials. Such sample solutions of 100 μL were introduced into the electrospray ionization (ESI) source using the flow injection (Figure 1a) at a flow rate of 100 μL/mL. The sequential runs
Figure 1. Schematic diagram of (a) the automatic flow injection used in this study and (b) the conventional infusion injection.
were performed by flow injection on an autosampler without connecting a column (that is typically used for chromatographic separation). This is in contrast to the conventional infusion (Figure 1b) injection that is incapable of a sequential run. The gas-phase Cu II-bound trimeric complexes of GSK2251052A and its R-enantiomer were generated during ESI. Dissociation of Trimeric Transition Metal Ion Complexes of GSK2251052A and Its R-enantiomer. The flowinjection MS and MS/MS experiments were performed on an Agilent 1100 LC system (Agilent Technologies, Wilmington, DE, USA) coupled to a Waters Q-TOF Premier quadrupole orthogonal acceleration time-of-flight mass spectrometer with LockSpray controlled by MassLynx 4.1 (Waters Corporation, Manchester, U.K.). The ESI source was operated in the positive ion mode under the following conditions: spray voltage, −3.5 kV; sample cone voltage, 30 V; source and desolvation gas temperatures, 120 and 300 °C, respectively; desolvation gas flow rate, 600 L/hour; argon (Ar) collision gas flow rate in the T-Wave Mark II collision cell, 0.45 mL/min; collision energy to cause CID of the Cu(II)-bound trimeric complexes of GSK2251052A and its R-enantiomer, 10 eV. The accurate masses were measured using the internal reference ion of m/z 556.2771 (protonated leucine-enkephalin) introduced via the Lockspray. Theoretical Calculations. The DFT calculations were performed using Gaussian 03 (revision D.02).37 Standard orientations for the Cu(II)-complex ions were generated by building the structures in Gaussview 4.1; these coordinates were then utilized for geometry optimization of the structures. Geometry optimizations were performed using Becke’s threeparameter hybrid functional B3LYP38 with the 6-31+G(d) basis
Scheme 1. Structures of GSK2251052A and Its REnantiomer
boron-containing systemic antibiotic for the treatment of multidrug-resistant Gram-negative bacterial infections.32 Since GSK2251052A is a very polar water-soluble molecule, it is difficult to be separated by normal-phase chiral separation. While reversed-phase chiral separation can be the method of choice, it is challenging to separate GSK2251052A from other process impurities. This novel automatic flow-injection MS/MS method will provide an alternative approach to the current chiral chromatographic method that is challenging and labor intensive.
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EXPERIMENTAL SECTION Chemicals. Samples of GSK2251052A and its R-enantiomer [(R)-3-(aminomethyl)-7-(3-hydroxypropoxy)-3H-benzo[c][1,2]oxaborol-1-ol] (Scheme 1., structure b) were enantioselectively synthesized and purified at GlaxoSmithKline (King of Prussia, PA). The absolute R- and S-chiral conformations were characterized by comparing the experimental electronic circular dichroism (ECD) spectra with the predicted ones calculated by time-dependent density functional theory (TD-DFT)33−36 4870
dx.doi.org/10.1021/ac401079x | Anal. Chem. 2013, 85, 4869−4874
Analytical Chemistry
Technical Note
The larger the difference between Rchiral and unity, the higher the degree of chiral recognition. Energy differences between the diastereomeric product ions represent the fundamental basis for chiral distinction. Two competitive dissociation channels, even when they display only very small differences (
