Simultaneous determination of sulforaphane and its major metabolites from biological matrices with liquid chromatography-tandem mass spectroscopy.
A simple, sensitive and specific LC-MS/MS method for the simultaneous determination of sulforaphane (SFN) and its major metabolites, the glutathione (SFN-GSH) and N-acetyl cysteine conjugates (SFN-NAC) from biological matrices was developed and validated. The assay procedure involved solid-phase extratcion of all three analytes from rat intestinal perfusate using C2 extraction cartridges, whereas from rat plasma, metabolites were extracted by solid-phase extraction and SFN was extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation of SFN, SFN-GSH and SFN-NAC was achieved on a C8 reverse phase column with a mobile phase gradient (Mobile Phase A: 10mM ammonium acetate buffer, pH: 4.5 and Mobile Phase B: acetonitrile with 0.1% formic acid) at a flow rate of 0.3 mL/min. The Finnigan LCQ LC-MS/MS was operated under the selective reaction monitoring mode using the electrospray ionization technique in positive mode. The nominal retention times for SFN-GSH, SFN-NAC and SFN were 8.4, 11.0, and 28.2 min,, respectively. The method was linear for SFN and its metabolites with correlation coefficients >0.998 for all analytes. The limit of quantification was 0.01-0.1 microm depending on analyte and matrix, whereas the mean recoveries from spiked plasma and perfusate samples were approximately 90%. The method was further validated according to U.S. Food and Drug Administration guidance in terms of accuracy and precision. Stability of compounds was established in a battery of stability studies, i.e., bench top, auto-sampler and long-term storage stability as well as freeze/thaw cycles. The utility of the assay was confirmed by the analysis of intestinal perfusate and plasma samples from single-pass intestinal perfusion studies with mesenteric vein cannulation in rats.
Agrawal
Shruti
S
Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, 08854, USA.
Winnik
Bozena
B
Buckley
Brian
B
Mi
Lixin
L
Chung
Fung-Lung
FL
Cook
Thomas J
TJ
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007413:Intestinal Mucosa / Q000378:metabolism*; D017879:Isothiocyanates; D013058:Mass Spectrometry / Q000379:methods*; D010539:Permeability; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013454:Sulfoxides; D013861:Thiocyanates / Q000097:blood / Q000378:metabolism*
D017879:Isothiocyanates; D013454:Sulfoxides; D013861:Thiocyanates; C016766:sulforaphane