Shau-Chun Wang

Using orthogonal array to obtain gradient liquid chromatography conditions of enhanced peak intensity to determine geniposide and genipin with electrospray tandem mass spectrometry

The conditions to determine geniposide and genipin using gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS) via electrospray ionization were obtained using fractional factorial experimental design approaches, guided with Taguchi orthogonal arrays to enhance peak intensity. Geniposide, the major iridoid glycoside component of Gardenia herbs, which has been recognized to have choleretic effects, is transformed to genipin in animals. In this paper, the gradient establishment times, ionization source temperatures, and the concentrations of volatile additive ammonium acetate were investigated under the guidance of experimental designs to obtain LC-MS/MS signals of the highest peak intensity. Using geniposide and genipin standards, the methods are validated at the concentration ranges of 0.5-1000ng/mL and 10-5000ng/mL using ammonium adducts. The correlation coefficients of geniposide and genipin standard curves are greater than 0.999. Compared with the sensitivities of previously published LC-MS/MS methods, the methods developed in this work provide 6-fold sensitivity improvement. The lowest concentrations of geniposide and genipin, 0.19 and 2ng/mL, respectively, to generate detectable LC-MS/MS signal peaks are one order of magnitude lower than the repoered values in previous publications. The measurement accuracy and precision of geniposide are within 23% and 15%, respectively. The accuracy and precision of genipin are within 16% and 12.5%, respectively. When the validated calibration curves of geniposide and genipin are used to determine spiked control samples in rat blood dialysates, the geniposide determination errors are within 15% accuracy and within 5.8% precision, respectively, and the genipin determination errors are within 23% accuracy and within 3.6% precision, respectively.

Using femtosecond laser to fabricate highly precise interior three-dimensional microstructures in polymeric flow chip.

This paper reports using femtosecond laser marker to fabricate the three-dimensional interior microstructures in one closed flow channel of plastic substrate. Strip-like slots in the dimensions of 800 μm×400 μm×65 μm were ablated with pulse Ti:sapphire laser at 800 nm (pulse duration of ∼120 fs with 1 kHz repetition rate) on acrylic slide. After ablation, defocused beams were used to finish the surface of microstructures. Having finally polished with sonication, the laser fabricated structures are highly precise with the arithmetic roughness of 1.5 and 4.5 nm. Fabricating such highly precise microstructures cannot be accomplished with nanosecond laser marking or other mechanical drilling methods. In addition, since laser ablation can directly engrave interior microstructures in one closed chip, glue smearing problems to damage molded microstructures possibly to occur during the chip sealing procedures can be avoided too.

Selective dynamic concentration of peptides at poles of cation-selective nanoporous granules

The authors exposed a non-equilibrium dynamic counterion and coion analyte concentration to an AC electric field to selectively concentrate peptides at the poles of a cation-selective granule. The counterion polarization results from the focusing of the electric field show a discontinuous drop in the intra-granule counterion electromigration flux at the pole. The coion concentration polarization is due to the combined external convective and electromigration fluxes toward the pole that neutralize the accumulating counterions. Because the electromigration mobility of the peptide anion analyte depends on the pH, the authors determined a 20 000-fold high concentration factor for a near-neutral pH of 6.0 to 7.7. Because the peptide is protonated at the acidic pole and its absolute charge ranges from -0.3 to -1.9, the concentration factor scales exponentially with the absolute charge, thus allowing extremely selective concentrations of various peptides, which is demonstrated by fluorescein isothiocyanate tagged angiotensin I (pI ∼ 5.8) and Texas red tagged avidin (pI ∼ 10.5). This dynamic concentration effect can substantially enhance the sensitivity of bio-assays.

Tailoring noise frequency spectrum between two consecutive second derivative filtering procedures to improve liquid chromatography-mass spectrometry determinations.

This paper reports a simple chemometric technique to alter the noise spectrum of a liquid chromatography-mass spectrometry (LC-MS) chromatogram between two consecutive second-derivative filter procedures to improve the peak signal-to-noise (S/N) ratio enhancement. This technique is to multiply one second-derivative filtered LC-MS chromatogram with another artificial chromatogram added with thermal noises prior to the other second-derivative filter. Because the second-derivative filter cannot eliminate frequency components within its own filter bandwidth, more efficient peak S/N ratio improvement cannot be accomplished using consecutive second-derivative filter procedures to process LC-MS chromatograms. In contrast, when the second-derivative filtered LC-MS chromatogram is conditioned with the multiplication alteration prior to the other second-derivative filter, much better ratio improvement is achieved. The noise frequency spectrum of the second-derivative filtered chromatogram, which originally contains frequency components within the filter bandwidth, is altered to span a broader range with multiplication operation. When the frequency range of this modified noise spectrum shifts toward the other regimes, the other second-derivative filter, working as a band-pass filter, is able to provide better filtering efficiency to obtain higher peak S/N ratios. Real LC-MS chromatograms, of which 5-fold peak S/N ratio improvement achieved with two consecutive second-derivative filters remains the same S/N ratio improvement using a one-step second-derivative filter, are improved to accomplish much better ratio enhancement, approximately 25-fold or higher when the noise frequency spectrum is modified between two matched filters. The linear standard curve using the filtered LC-MS signals is validated. The filtered LC-MS signals are also more reproducible. The more accurate determinations of very low-concentration samples (S/N ratio about 5-7) are obtained via standard addition procedures using the filtered signals rather than the determinations using the original signals.

Improving liquid chromatography–tandem mass spectrometry determinations by modifying noise frequency spectrum between two consecutive wavelet-based low-pass filtering procedures

This paper employs one chemometric technique to modify the noise spectrum of liquid chromatography-tandem mass spectrometry (LC-MS/MS) chromatogram between two consecutive wavelet-based low-pass filter procedures to improve the peak signal-to-noise (S/N) ratio enhancement. Although similar techniques of using other sets of low-pass procedures such as matched filters have been published, the procedures developed in this work are able to avoid peak broadening disadvantages inherent in matched filters. In addition, unlike Fourier transform-based low-pass filters, wavelet-based filters efficiently reject noises in the chromatograms directly in the time domain without distorting the original signals. In this work, the low-pass filtering procedures sequentially convolve the original chromatograms against each set of low pass filters to result in approximation coefficients, representing the low-frequency wavelets, of the first five resolution levels. The tedious trials of setting threshold values to properly shrink each wavelet are therefore no longer required. This noise modification technique is to multiply one wavelet-based low-pass filtered LC-MS/MS chromatogram with another artificial chromatogram added with thermal noises prior to the other wavelet-based low-pass filter. Because low-pass filter cannot eliminate frequency components below its cut-off frequency, more efficient peak S/N ratio improvement cannot be accomplished using consecutive low-pass filter procedures to process LC-MS/MS chromatograms. In contrast, when the low-pass filtered LC-MS/MS chromatogram is conditioned with the multiplication alteration prior to the other low-pass filter, much better ratio improvement is achieved. The noise frequency spectrum of low-pass filtered chromatogram, which originally contains frequency components below the filter cut-off frequency, is altered to span a broader range with multiplication operation. When the frequency range of this modified noise spectrum shifts toward the high frequency regimes, the other low-pass filter is able to provide better filtering efficiency to obtain higher peak S/N ratios. Real LC-MS/MS chromatograms, of which typically less than 6-fold peak S/N ratio improvement achieved with two consecutive wavelet-based low-pass filters remains the same S/N ratio improvement using one-step wavelet-based low-pass filter, are improved to accomplish much better ratio enhancement 25-folds to 40-folds typically when the noise frequency spectrum is modified between two low-pass filters. The linear standard curves using the filtered LC-MS/MS signals are validated. The filtered LC-MS/MS signals are also reproducible. The more accurate determinations of very low concentration samples (S/N ratio about 7-9) are obtained using the filtered signals than the determinations using the original signals.

ac electroosmotic pumping induced by noncontact external electrodes

Electroosmotic (EO) pumps based on dc electroosmosis is plagued by bubble generation and other electrochemical reactions at the electrodes at voltages beyond 1 V for electrolytes. These disadvantages limit their throughput and offset their portability advantage over mechanical syringe or pneumatic pumps. ac electroosmotic pumps at high frequency (>100 kHz) circumvent the bubble problem by inducing polarization and slip velocity on embedded electrodes,1 but they require complex electrode designs to produce a net flow. We report a new high-throughput ac EO pump design based on induced-polarization on the entire channel surface instead of just on the electrodes. Like dc EO pumps, our pump electrodes are outside of the load section and form a cm-long pump unit consisting of three circular reservoirs (3 mm in diameter) connected by a 1x1 mm channel. The field-induced polarization can produce an effective Zeta potential exceeding 1 V and an ac slip velocity estimated as 1 mmsec or higher, both one order of magnitude higher than earlier dc and ac pumps, giving rise to a maximum throughput of 1 mulsec. Polarization over the entire channel surface, quadratic scaling with respect to the field and high voltage at high frequency without electrode bubble generation are the reasons why the current pump is superior to earlier dc and ac EO pumps.

Entrainment of DNA and Charged Nano-Particles Using AC Electrospray

We examine our earlier finding that AC electrospray cone can preferentially entrain and concentrate low-mobility ions generated at the cone tip [Chetwani, Maheshwari and Chang, Phys Rev Lett., 101, 204501 (2008)]. Instead of tip ionization, we scrutinize entrainment effects on dispersed ionic species in the spray solution. Fluorescence images of ejected ionic species, including anionic oxide ions, organic dyes, charged nano-particles, and stained DNA from AC electrospray cone generated with a 4–5 kV source. Negatively charged species such as anionic dyes concentrate at the tip of AC spray cone before ejection. Cationic dyes are more homogeneously distributed in the cone during ejection. Larger particles with negative charges, including fluorescence dye-tagged nano-particles (about 50 nm) and dye-stained DNA, have similar spatio-temporal evolutions of anioic dyes within the cone before ejection. These observations extend our previous entrainment and concentration effect to AC spray without ionization. The images will be correlated with tandem Mass Spectrometry and will be used to selectively gate either cationic (protein) or anionic polymers (DNA).Copyright