Laser-induced acoustic desorption (LIAD) was successfully coupled to a conventional atmospheric pressure chemical ionization (APCI) source in a commercial linear quadrupole ion trap mass spectrometer (LQIT). Model compounds representing a wide variety of different types, including basic nitrogen and oxygen compounds, aromatic and aliphatic compounds, as well as unsaturated and saturated hydrocarbons, were tested separately and as a mixture. These model compounds were successfully evaporated into the gas phase by using LIAD and then ionized by using APCI with different reagents. From the four APCI reagent systems tested, neat carbon disulfide provided the best results. The mixture of methanol and water produced primarily protonated molecules, as expected. However, only the most basic compounds yielded ions under these conditions. In sharp contrast, using APCI with either neat benzene or neat carbon disulfide as the reagent resulted in the ionization of all the analytes studied to predominantly yield stable molecular ions. Benzene yielded a larger fraction of protonated molecules than carbon disulfide, which is a disadvantage. A similar but minor amount of fragmentation was observed for these two reagents. When the experiment was performed without a liquid reagent (nitrogen gas was the reagent), more fragmentation was observed. Analysis of a known mixture as well as a petroleum cut was also carried out. In summary, the new experiment presented here allows the evaporation of thermally labile compounds, both polar and nonpolar, without dissociation or aggregation, and their ionization to predominantly form stable molecular ions.

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Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC.

Second order nonlinear optical imaging of chiral crystals (SONICC) and two-photon excited fluorescence measurements [both autofluorescence and two-photon excited UV-fluorescence (TPE-UVF)] were assessed for the selective detection of APIs relative to common pharmaceutical excipients. Active pharmaceutical ingredients (APIs) compose only a small percentage of most tabulated formulations, yet the API distribution within the tablet can affect drug release and tablet stability. Complementary measurements using either UV-SONICC (266 nm detection) or TPE-UVF were shown to generate signals >50-fold more intense for a model API (griseofulvin) than those produced by common pharmaceutical excipients. The combined product of the measurements produced signals >10 4 -fold greater than the excipients studied. UV-SONICC or TPE-UVF produced greater selectivity than analogous measurements with visible-light detection, attributed to the presence of aromatic moieties within the API exhibiting strong one and two photon absorption at ~266 nm. Complementary SONICC and fluorescence measurements allowed for the sensitive detection of the three-dimensional distribution of tadalafil within a Cialis ® tablet to a depth of >140 µm.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction for combined single-platform analysis, examining the viability of NLO microscopy as an alternative to the conventional X-ray raster scan for the purposes of sample centering. Second-harmonic generation microscopy and two-photon excited ultraviolet fluorescence microscopy were evaluated for crystal detection, and assessed by X-ray raster scanning.