Hyphenation of Hadamard Encoded Multiplexing Liquid

Nov 9, 2015 - The hyphenation of HPLC and circular dichroism (CD) detection is a useful analytical tool that can significantly facilitate the analysis...
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Hyphenation of Hadamard Encoded Multiplexing Liquid Chromatography and Circular Dichroism Detection to Improve the Signal-to-Noise Ratio in Chiral Analysis Alexander F. Siegle and Oliver Trapp* Organisch-Chemisches Institut, Ruprecht-Karls Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany ABSTRACT: The hyphenation of HPLC and circular dichroism (CD) detection is a useful analytical tool that can significantly facilitate the analysis (e.g., the assignment of the configuration) and quantitation of chiral compounds. HPLCCD chromatograms often exhibit a low signal-to-noise ratio compared to chromatograms obtained by conventional UV detection. In this study we demonstrate for the first time the hyphenation of Hadamard encoded multiplexing HPLC with circular dichroism detection where positive and negative signals overlap. Here, a macro control of the HPLC instrument that was developed for conventional HPLC was implemented in HPLC-CD. In the chiral analysis of racemic samples, exemplified for warfarin, the signal-to-noise ratio could be enhanced by an order of magnitude. The presented results highlight the great modularity of the software controlled implementation of multiplexing and its facile transfer to other detection techniques.

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Hadamard transform were used to improve signal-to-noise ratio (S/N) in various field such as time-of-flight mass spectrometry,25−27 ion mobility spectrometry,28,29 and capillary electrophoresis.30−35 Lin and co-workers constructed injection devices for the application of Hadamard transform techniques to gas chromatography/mass spectrometry (GC/MS)36−41 and liquid chromatography−mass spectrometry (LC−MS).36 Trapp developed an algorithm for multiplexing GC that allows achieving very high sample-throughputs of up to 453 samples/h using structured pseudorandom modulation sequences.42−45 Recently it was shown that the application of correlation techniques improves the S/N and enables continuous monitoring of volatile pollutants in a micro-GC system.46 We demonstrated the implementation of Hadamard encoded multiplexing on commercially available HPLC instrumentation by simple software modification.47 Here we report the first application of our macro control approach in Hadamard transform multiplexing HPLC coupled with circular dichroism detection.

ecause of the often significant differences in pharmacokinetic activity between two enantiomers of a chiral drug, a great part of modern drug development focuses on single enantiomer pharmaceuticals. With its ability to selectively detect chiral compounds, circular dichroism (CD) provides a useful analytical tool in the pharmaceutical industry. Since CD detectors for liquid chromatography were introduced1−3 and became commercially available, the importance of CD detection for the analysis of chiral compounds has increased. The hyphenation of HPLC and CD detection has successfully been utilized in the chiral analysis of pharmacologically active compounds.4,5 Other applications of HPLC-CD include the assignment of absolute configurations of enantiomers by elution order6 and the analysis of enantiomeric purities on achiral stationary phases.7,8 Mikami9,10 and Reetz11 developed high-throughput screening techniques for enantioselective reactions utilizing HPLC-CD analysis. Nevertheless, the CD signal strongly depends on the acquisition wavelength and CD detection often suffers from low sensitivity compared to conventional UV-detection.12,13 Therefore, efforts have been made to improve sensitivity in HPLC-CD, for instance by low-pass electronic noise filtering of the CD signal.14 A mathematical approach to improve sensitivity in separation sciences is the application of multiplexing strategies. Modulation of sample injection by pseudorandom binary sequences and subsequent deconvolution by cross correlation with the multiplexed signal has first been employed in correlation separation techniques.15−24 In a very similar approach, multiplexing techniques based on deconvolution by inverse © 2015 American Chemical Society

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EXPERIMENTAL SECTION Materials. Warfarin and HPLC-grade solvents were purchased from Sigma-Aldrich (Taufkirchen, Germany) and used as received. The warfarin sample was dissolved in ethyl acetate (520 μg/mL). Received: October 1, 2015 Accepted: November 9, 2015 Published: November 9, 2015 11932

DOI: 10.1021/acs.analchem.5b03705 Anal. Chem. 2015, 87, 11932−11934

Article

Analytical Chemistry Instrumentation. HPLC-CD multiplexing experiments were carried out on a commercially available Agilent Infinity 1200 system equipped with a high-performance autosampler model HiP-ALS SL+, a photodiode array detector (DAD), and a Jasco CD-2095 circular dichroism detector. The sample was injected according to n-bit (m = 2n − 1 matrix elements) Hadamard modulation sequences, which were applied by use of a macro that was executed as a prerun command in ChemStation. The macro and a description of its functionality are subject of previous studies.47 In a typical experiment, 1 μL of sample was injected though the full injection range of the autosampler. Separation of the warfarin enantiomers was conducted on a Chiralpak IB-3 column (particle size, 3 μm, 4.6 mm × 150 mm, n-hexane/ethyl acetate/TFA 70:30:0.1, v/ v/v, 1 mL/min) purchased from Chiral Technologies (Illkirch, France). Data Acquisition and Deconvolution. Multiplexed HPLC-CD traces were acquired as ASCII files with a frequency of 10 Hz, using a LabJack U6-Pro data card with 22-bit resolution (LabJack Corporation, Lakewood, CO) connected to the Jasco CD-2095 detector. The data acquisition was controlled by software written in Delphi (Embarcadero Technologies, South San Francisco, CA). Construction of Hadamard modulation sequences by use of a virtual shift register48 and inverse Hadamard transformation of the multiplexed traces was performed by a program written in Delphi. Signal-to-noise (S/N) ratios were directly determined, using the implemented S/N feature of the Agilent ChemStation software package.

to-noise (S/N) ratio of the CD trace, the UV trace shows a S/ N ratio that is by a factor of about 45 better. For the (R) and (S) enantiomer, a S/N ratio of 3.8 and 2.8 were determined from the single injection chromatogram, respectively. The warfarin sample was subsequently multiplexed utilizing a macro that was executed in the ChemStation software of the HPLC instrument. Depending on the selected modulation sequence and injection interval between two elements of the sequence, the macro creates a list of instructions that are transmitted to the autosampler. Here modulation of the sample injection was performed according to 6-bit (m = 63 matrix elements) to 9-bit (m = 511) pseudorandom binary sequences while the injection interval was varied between Δt = 18 s and Δt = 30 s. The results are presented in Table 1. Table 1. S/N Enhancement Depending on Sequence Length and Injection Interval S/N enhancement sequence length m

Δt (s)

(R)

(S)

acquisition time (min)

63 127 255 511

30 30 30 18

3.9 5.4 7.9 11.2

3.6 5.2 7.7 10.4

44 76 140 165

For shorter sequence lengths, the best gain in S/N was achieved using the larger injection interval whereas for 9-bit modulation the shorter injection interval offered the best performance. This can be correlated to the increasing probability of the instability of the CD signal for longer acquisition times. The acquisition time for a 9-bit modulation sequence with an injection interval of Δt = 18 s is comparable to that of an 8-bit sequence with Δt = 30 s while offering significantly better improvement of the S/N. The results of this 9-bit multiplexing experiment are shown in Figure 2. Because of the overlap of positive and negative peaks, the multiplexed trace depicted in Figure 2a is severely convoluted with peaks partially canceling out each other. Surprisingly, inverse Hadamard transformation of the multiplexed chromatogram yields a chromatogram containing the signals of both enantiomers with accurate peak shapes (Figure 2b). The S/N in the deconvoluted chromatogram is significantly improved in comparison to the single injection chromatogram shown in Figure 1. For (R) and (S) warfarin an S/N-enhancement of 11.2 and 10.4 could be observed, respectively. This matches well with the theoretical prediction of an 11.3-fold gain.48 The ratio of the peak areas of the enantiomers remains at 50:50 after inverse Hadamard transformation, which shows that the information on the conventional single injection chromatogram is conserved in the deconvolution process. Thus, the implementation of Hadamard encoded multiplexing in HPLC-CD provides a clear improvement in sensitivity without sacrificing accuracy in the quantitation of chiral compounds.

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RESULTS AND DISCUSSION In the here presented study, we used the chiral anticoagulant warfarin as a model substrate. Separation was achieved on a Chiralpak IB-3 column with a mobile phase containing hexane, ethyl acetate (70:30, v/v), and 0.1% (v/v) trifluoroacetic acid as modifier. To avoid eluent absorption due to the presence of ethyl acetate in the eluent, a CD detection wavelength of λ = 270 nm was chosen. The resulting chromatogram is depicted in Figure 1. At the selected wavelength, the CD signal is rather weak in comparison to the UV signal and thus significant noise can be observed in the CD trace. In comparison to the signal-

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CONCLUSION Our experiments show the broad and general applicability and modularity of the macro control approach that allows the use of multiplexing with circular dichroism detection on commercially available HPLC instrumentation. For the chiral separation of a racemic warfarin sample, the S/N could be improved by an order of magnitude. Multiplexing presents an intrinsic

Figure 1. Chromatogram of a racemic warfarin sample obtained by circular dichroism detection at λ = 270 nm. The (R) enantiomer is eluted first. Significant noise can be observed under the given experimental conditions. 11933

DOI: 10.1021/acs.analchem.5b03705 Anal. Chem. 2015, 87, 11932−11934

Article

Analytical Chemistry

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Figure 2. (a) Multiplexed HPLC-CD chromatogram obtained by 9-bit (m = 511 matrix elements) modulation with an injection interval of Δt = 18 s at λ = 270 nm. (b) Deconvoluted chromatogram.

advantage in speed compared to signal averaging of single chromatographic runs, and future advances in autosampler injection speed will further decrease the amount of time necessary for acquiring the multiplexed data.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +49-6221-544904. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the European Research Council under Grant Agreement Nos. StG 258740 and PoC 310196 and the Agilent Foundation, Santa Clara, California, for financial support.

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REFERENCES

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DOI: 10.1021/acs.analchem.5b03705 Anal. Chem. 2015, 87, 11932−11934