The effect of irradiation of 20 mg/mL of CPZ on the antimicrobial activity of CPZ.

<p>(A).CPZ @ 20 mg/mL irradiated for 1 minute. B). CPZ @ 20 mg/mL irradiated for 240 minutes. Amounts of “CPZ” of sample applied to disks: 1 (100 µg); 2 (200 µg); 3 (300 µg); 4 (400 µg); 5 (500 µg); 6 (600 µg). Note that the # 4 disk containing 400 µg of “CPZ” 20 mg/mL sample irradiated 240 minutes produces a zone of inhibition whereas the disk that contains the same amount of “CPZ” 20 mg/mL irradiated 1 minute does not produce a zone of inhibition. These results suggest that the antimicrobial activity of the CPZ exposed to a 266 nm laser beam for 240 min. is increased.</p

Absorption spectra of the irradiated products per unit period of time.

<p>Irradiation of 2 mg/mL of CPZ for intervals of A) 30 minutes up to 240 minutes at E = 1 mJ and B) Irradiation of 2 mg/mL of CPZ from 1 to 240 minutes at E = 6.5 mJ. Note: After irradiation, an aliquot of the contents of the cuvette was diluted to 0.2 mg/mL and the shown absorption spectra were measured. Due to the fact that the absorption peaks are quite broad and the experimental errors to assign their wavelength are associated with the peaks spectral widths, the peak absorbance of control, un-irradiated CPZ varied from 254 to 256 nm.</p

Experimental Set-up.

<p>The laser beam incident on the sample is emitted by a Nd:YAG pulsed laser and is crossing an OPO system that allows obtaining a laser beam at 266 nm; the grating G is utilized to separate the second harmonic (532 nm) with respect to the fourth (266 nm) so that on the Cuvette only the 266 nm beam falls. The liquid in the cuvette is agitated by a Magnetic stirrer and the beams that are not useful are blocked by a Beam Stop unit. The laser induced fluorescence emission by the sample is collected with an Optical fiber, and analyzed with a Spectrometer – PC system. The sample exposed/modified to/by laser radiation is characterized by the absorption spectrum measured with a spectrophotometer and by Thin Layer Chromatography (TLC) measurements.</p

HPLC MS/MS.

<p>Multiple reactions monitoring (MRM) was performed for [M+H+]  = 285, 301, 317, 319 and 335, in order to obtain selective chromatograms for PZ (3), hydroxypromazine or PZ sulfoxide (1), hydroxypromazine sulfoxide (2), CPZ (4) and CPZ sulfoxide, respectively (A). Identity of PZ sulfoxide over hydroxypromazine for peak 1 was proven based on the on-peak UV spectrum obtained from the HPLC-DAD chromatogram; the four maxima are characteristic for phenothiazine sulfoxides. UV chromatogram recorded during the MRM experiment at 240 nm is also shown (B).</p

Dynamics of CPZ photolysis at a 2 (A), 5 (B), 10 (C) and 20 (D) mg/mL starting concentration expressed as percentage present in the sample compared with the 100% initial amount.

<p>Kinetic curves were fitted according to the one-phase decay exponential model of GraphPad Prism 5.0.</p

HPLC-DAD chromatograms of un-irradiated 2 mg/mL of CPZ (A) and 2 mg/mL of CPZ irradiated for time periods of 1 (B), 5 (C), 15 (D), 30 (E), 60 (F), 120 (G) and 240 (H) minutes, recorded at the maximum absorbance in the wavelength range of λ = 230–450 nm.

<p>Note: intensity scales are not identical; overall amount of UV-active material is gradually decreasing with the irradiation time.</p

Absorption spectra of irradiated CPZ at concentrations of A) 5 mg/mL, B) 10 mg/mL and C) 20 mg/mL during 240 minutes.

<p>E = 6.5 mJ. Note: After irradiation, an aliquot of the contents of the cuvette was diluted to 0.2 mg/mL and the shown absorption spectra were measured.</p

Structures of CPZ (A) and PZ (B).

<p>Structures of CPZ (A) and PZ (B).</p

HPLC-DAD chromatograms of 10 mg/mL of CPZ irradiated for time periods of 1 (A), 5 (B), 15 (C), 30 (D), 60 (E), 120 (F) and 240 (G) minutes, recorded at the maximum absorbance in the wavelength range of λ = 230–450 nm.

<p>Note: intensity scales are not identical; overall amount of UV-active material is gradually decreasing with the irradiation time.</p

HPLC-DAD chromatograms of 20 mg/mL of CPZ irradiated for time periods of 1 (A), 5 (B), 15 (C), 30 (D), 60 (E), 120 (F) and 240 (G) minutes, and 16 (H) and 24 (I) hours, recorded at the maximum absorbance in the wavelength range of λ = 230–450 nm.

<p>Note: intensity scales are not identical; overall amount of UV-active material is gradually decreasing with the irradiation time.</p