Letter pubs.acs.org/ac
Identification of Isomeric Ephedrines by Cold Ion UV Spectroscopy: Toward Practical Implementation Vladimir Kopysov,† Alexander Makarov,‡ and Oleg V. Boyarkin*,† Laboratoire de Chimie Physique Moléculaire, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland Thermo Fisher Scientific, Hanna-Kunath Str. 11, 28199 Bremen, Germany
† ‡
S Supporting Information *
ABSTRACT: Ephedrine and pseudoephedrine are stimulant drugs whose use is prohibited in athletic competition by the World AntiDoping Agency (WADA) at very different threshold doping violation concentrations. We use a recently developed universal approach that integrates UV photofragmentation spectroscopy of cold ions with Orbitrap mass spectrometry (MS) for highly selective and highly sensitive identification of these diastereomers. Both species can be selectively detected at a solution concentration of a few tens of ng/mL, which is almost 3 orders of magnitude lower than the threshold concentration required by WADA. Relative concentrations of the isomers in solutions have been determined with the standard deviation of 3.1%, when the ions were cooled in an ion trap maintained at T = 6 K. Considering practical implementation of the method, we evaluated its performance for a simplified instrumentation. At an affordable elevated temperature of ∼70 K and with a low-maintenance midbandwidth optical parametric oscillator, a few second measurement should yield nearly the same selectivity and only ten times lower sensitivity than with the current research grade instrument.
F
nervous system stimulants, their use is prohibited in sport by the World Anti-Doping Agency (WADA).7 The nonequivalent potency of the ephedrine isomers is reflected in the decision limits for reporting an Adverse Analytical Finding by WADA. For ephedrine (the pair of (1R,2S) and (1S,2R) enantiomers), this limit is only 11 μg/mL, while for pseudoephedrine (the (1R,2R) and (1S,2S) enantiomers) it extends to 170 μg/mL.8 It is challenging to separately identify the two isomers by mass spectrometry alone, because of the high degree of similarity of their fragmentation mass spectra, which differ only slightly by the intensities of a few peaks (see HCD mass spectra in Figure S1). Several highly selective and sensitive chromatographic methods have been developed specifically for the identifications of ephedrine and pseudoephedrine in biological samples.9−13 The use of specific LC conditions in these targeted methods however drastically limits their universality. In contrast, the 2D UV-MS approach is based on direct measurements of fundamental characteristics of ions (UV photofragmentation yield vs wavelength and m/z), such that the same instrumentation and data treatment protocols can also be used for detecting any (small to midsize) UV-absorbing peptides and nonpeptide species, including ephedrines. Our experimental apparatus and the data treatment procedure are described elsewhere.5,6,14 Briefly, singly proto-
unctionality and activity of many biomolecules in vivo crucially depend on their isomeric forms. Isomers of, for instance, many drugs exhibit different pharmacokinetic and/or pharmacodynamic properties, such as rates of absorption,1 volumes of distribution,2 metabolic pathways,3 and pharmacological activity.4 Identification and quantification of isomers and conformers of the same molecules are therefore in high demand in a variety of applications, including proteomics, pharmacology, doping control, and forensics. Coupling of mass spectrometry (MS) to high performance liquid chromatography (LC) enables separation of many sufficiently different isomeric species but may require the compound-specific conditions of LC separation. Such specificity limits the universality of the method in the separation of isomers simultaneously for a broad range of species. We recently demonstrated the use of a combination of cold ion UV spectroscopy with high-resolution mass spectrometry (2D UV-MS) for library-based identifications of isomeric peptides in their mixtures, as well as for library-free (blind) determination of the number and relative abundances of isomers and conformers of cryogenically cooled peptides.5,6 Here, we apply the 2D UV-MS technique with the same instrumentation for library-based quantitative identifications of two diastereomers of the ephedrine molecule, (+)-ephedrine and (+)-pseudoephedrine, and study some practical aspects of the method. Ephedrine is an alkaloid found in plants of Ephedra type. This drug has been used for treatment of asthma, nasal congestion, and obesity, but because ephedrines are central © XXXX American Chemical Society
Received: October 26, 2016 Accepted: December 19, 2016 Published: December 19, 2016 A
DOI: 10.1021/acs.analchem.6b04182 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
individual isomers at the m/z of the main fragment. The observed spectral difference in the UV fragmentation patterns of the cold species creates a basis for a highly selective identification of the two isomers, while the use of a mass spectrometer for detecting the fragments makes this identification highly sensitive. In order to assess the performance of the method in quantitative identifications of the two isomers, we prepared a set of 30 different mixtures in a water−methanol (50:50) solvent with the total concentrations of analytes ranging from 40 ng/mL (2.4·10−7 M) to 16.8 μg/mL (1.0·10−4 M) (Table S1). For each mixture, we measured a 2D UV-MS spectrum within the 37 540−37 650 cm−1 spectral range and performed a numerical decomposition of the respective 2D data array in the basis set of the two measured library components. This range was selected as a compromise between the need to keep several nonoverlapped UV peaks of the two isomers to ensure high selectivity of the decomposition and the desire to shorten the time of the measurements. The quality of the matrix-based decomposition can be assessed by comparing the UV spectrum of a mixture and of its fit by a linear combination of the UV spectra of the isomers. Figure 1b displays an example of a perfect match obtained for the decomposition of an equimolar mixture of the two isomers. Every single peak is clearly resolved and fitted by the corresponding peak of either ephedrine or pseudoephedrine. For the entire set of mixtures, relative concentrations of the two isomers were calculated with the root-mean-square error (RMSE) of 3.1%, implying ±6.2% accuracy for determining their relative concentrations at the 95% confidence level. Assuming that the total concentration of the two isomers (known in our measurements) can be accurately determined by standard MS methods,16 Figure 2a shows the results of our analysis in terms of absolute concentrations. For the analyte concentrations below 100 ng/mL, the RMSEs are 14.8 and 26.8 ng/mL for ephedrine and pseudoephedrine, respectively. Together with the analysis of blank samples17 (samples containing only one of the isomers), these numbers allow for an evaluation of the limits of detections to 29 and 52 ng/mL for ephedrine and pseudoephedrine, respectively. These results demonstrate the capability of the UV-MS approach to measure absolute concentrations of the two analytes in their mixtures on a sub-100 ng/mL concentration scale. The current research-grade instrumentation of the 2D UVMS method employs a high-resolution dye laser, which requires the use of several different dyes to cover the whole UV/VUV spectral range. This is, apparently, not the most convenient procedure for a commercial instrument. Also, a typical laser scan may take more than half an hour, which might be too slow for practice, in particular for coupling the method to LC preseparation. Finally, 6 K temperature of the ion trap is often considered as a challenge for implementation of the method. Respectively, from the point of view of practical feasibility of the method, one would desire to achieve a similar high performance as that demonstrated above but (i) at a mild cooling, (ii) using low-maintenance laser sources, and (iii) with fingerprinting of a sample mixture on a time scale of few seconds. Below, we address these three issues. Figure 3a shows the plot of concentrations that are calculated with the fragment mass spectra, sliced only at three "specific" (to the isomers) wavelengths from the fingerprints, measured at 6 K. These wavelengths were preselected on the basis of the analysis of the library 2D UV-MS fingerprints. Moreover, in
nated ions are produced from a methanol/water solution of one of the ephedrine isomers or of a mixture of both of them using a nanoelectrospray ionization source. The ions are preselected by a quadrupole mass filter, stored, and cooled in collisions with He buffer gas in a linear octupole ion trap,15 maintained at a desired temperature within T = 6−64 K. Once cooled, the ions are fragmented by a UV laser pulse. Both fragment and parent ions are then guided into the Orbitrap mass analyzer (Exactive mass spectrometer, Thermo Scientific). The 2D UV-MS spectra are measured by continuously recording the fragmentation mass spectra on the Orbitrap while scanning the UV laser. The recorded and rectified 2D data arrays of individual isomers are stored as a library and are subsequently used to fit the 2D data arrays of mixtures of the two isomers (see ref 5 and the Supporting Information for details). The two coefficients of the best linear fit represent relative concentrations of the two isomers in a mixture. The 2D UV-MS fingerprints of individual isomers, measured within the 37 500−38 500 cm−1 spectral range (Figure 1a),
Figure 1. (a) Wide-range UV absorption spectra of (+)-ephedrine and (+)-pseudoephedrine obtained by integrating the respective 2D UVMS spectra within ±0.05 Th window around the dominant fragment (m/z = 58.066 Th) and normalized to the maximum signal; (b) UV spectrum of an equimolar mixture of the two isomers and its fit by a linear combination of the two library spectra. The decomposition yields 53:47 relative concentrations of the two isomers.
exhibit essentially identical UV fragmentation mass spectra with a single prominent peak at m/z = 58.066 Da. This peak does not appear in the CID/HCD mass spectra and corresponds to the β-bond cleavage (Figure S1). The observed similarity of the UV fragmentation mass spectra does not allow identification of the two isomers by mass spectrometry alone. In contrast, the UV fragmentation spectra of protonated ephedrine and pseudoephedrine, cooled to Tvib = 10 K, exhibit an apparent difference in the onset of UV absorption and in the positions of well-resolved vibronic transitions. Figure 1a shows such spectra, obtained by slicing the respective 2D data arrays of the B
DOI: 10.1021/acs.analchem.6b04182 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 2. Calculated (a) relative and (b) absolute (log scale) concentrations of ephedrine (red squares) and pseudoephedrine (blue circles) as a function of their respective concentrations in solution for 30 different mixtures. The calculations include the entire UV-MS data arrays of the isomers and their mixtures, measured at T = 6 K with the ultimate spectral resolution of 0.15 cm−1. Solid lines show the ideal 1:1 dependence, and the area between two dashed lines in (a) corresponds to the 95% confidence band (±2·RMSE) for calculating the concentrations. Figure 3. Calculated relative concentrations of ephedrine as a function of their relative concentration in solution (a) for the same 30 different mixtures as in Figure 2 measured at T = 6 and (c) for 9 different mixtures with 2.5 μg/mL total solution concentrations (Table S2) measured at T = 64 K. Both calculations include only the fragmentation mass spectra obtained by integrating the fingerprints within ±2.5 cm−1 spectral intervals around three “specific” wavelengths: 37 543, 37 550.7, and 37 575.6 cm−1. A solid line is the ideal 1:1 dependence; the areas between the dashed lines in (a) and (c) correspond to the 95% confidence band (±2·RMSE) for calculating concentrations. (b) UV absorption spectra of ephedrine and pseudoephedrine obtained from the measurements at temperature of 64 K (red and blue traces); the spectra of the isomers measured at the trap temperature of 6 K (Figure 1) are shown in black for a comparison.
order to simulate broadband laser sources, the integrating slicing “window” in this calculation was increased for both the library and the mixture fingerprints from 0.15 cm−1 (ultimate resolution of our dye laser) to 5 cm−1. The 5−7 cm−1 line width is typical for commercial midresolution UV optical parametric oscillators (UV OPO) that employ critical phasematching in nonlinear crystals.18 These all-solid-state OPOs, which have no diffraction gratings, are, eventually, widely tunable low-maintenance devices. Compared with the calculations for the full 2D UV-MS data arrays, measured at 0.15 cm−1 spectral resolution (Figure 2a), the results in Figure 3a exhibit slightly lower RMSE of 2.9%. We attribute this improvement to the averaging of the absorption bands, which makes the calculations insensitive to the high-frequency noise. The full matrix, measured over ∼50 min, contains ∼750 wavelengths. In contrast, a measurement of the mass spectra only at three wavelengths at 20 Hz repetition rate of the experiment with the average of 10 shots per wavelength may take less than 2 s, realistically assuming
