A Newly Developed HPLC-UV/Vis Method Using Chemical Derivatization with 2-Naphthalenethiol for Quantitation of Sulforaphane in Rat Plasma

Sulforaphane (SFN), a naturally occurring isothiocyanate, has received significant attention because of its ability to modulate multiple biological functions, including anti-carcinogenic properties. However, currently available analytical methods based on high-performance liquid chromatography (HPLC)-UV/Vis for the quantification of SFN have a number of limitations, e.g., low UV absorbance, sensitivity, or accuracy, due to the lack of a chromophore for spectrometric detection. Therefore, we here employed the analytical derivatization procedure using 2-naphthalenethiol (2-NT) to improve the detectability of SFN, followed by HPLC separation and quantification with UV/Vis detection. The optimal derivatization conditions were carried out with 0.3 M of 2-NT in acetonitrile with phosphate buffer (pH 7.4) by incubation at 37 °C for 60 min. Separation was performed in reverse phase mode using a Kinetex C18 column (150 mm × 4.6 mm, 5 μm) at a flow rate of 1 mL/min, with 0.1% formic acid as a mobile phase A, and acetonitrile/0.1% formic acid solution as a mobile phase B with a gradient elution, with a detection wavelength of 234 nm. The method was validated over a linear range of 10–2000 ng/mL with a correlation of determination (R2) > 0.999 using weighted linear regression analysis. The intra- and inter-assay accuracy (% of nominal value) and precision (% of relative standard deviation) were within ±10 and <15%, respectively. Moreover, the specificity, recovery, matrix effect, process efficiency, and short-term and long-term stabilities of this method were within acceptable limits. Finally, we applied this method for studying in vivo pharmacokinetics (PK) following oral administration of SFN at doses of 10 or 20 mg/kg. The Cmax (μg/mL), Tmax (hour), and AUC0–12h (μg·h/mL) of each oral dose were 0.92, 1.99, and 4.88 and 1.67, 1.00, and 9.85, respectively. Overall, the proposed analytical method proved to be reliable and applicable for quantification of SFN in biological samples.

[1]  S. Nazzal,et al.  Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation. , 2019, Journal of chromatographic science.

[2]  M. Shabanian,et al.  2-Naphthalenthiol derivatization followed by dispersive liquid-liquid microextraction as an efficient and sensitive method for determination of acrylamide in bread and biscuit samples using high-performance liquid chromatography. , 2018, Journal of chromatography. A.

[3]  S. Nabavi,et al.  Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment , 2018, Critical reviews in food science and nutrition.

[4]  P. Liu,et al.  Sulforaphane exerts anti-angiogenesis effects against hepatocellular carcinoma through inhibition of STAT3/HIF-1α/VEGF signalling , 2017, Scientific Reports.

[5]  F. Elbarbry,et al.  A new validated HPLC method for the determination of sulforaphane: application to study pharmacokinetics of sulforaphane in rats. , 2016, Biomedical chromatography : BMC.

[6]  N. Weerapreeyakul,et al.  Simultaneous quantification of sulforaphene and sulforaphane by reverse phase HPLC and their content in Raphanus sativus L. var. caudatus Alef extracts. , 2016, Food chemistry.

[7]  Stephanie M. Tortorella,et al.  Dietary Sulforaphane in Cancer Chemoprevention: The Role of Epigenetic Regulation and HDAC Inhibition. , 2015, Antioxidants & redox signaling.

[8]  Á. Gil-Izquierdo,et al.  A new ultra-rapid UHPLC/MS/MS method for assessing glucoraphanin and sulforaphane bioavailability in human urine. , 2014, Food chemistry.

[9]  R. Brigelius-Flohé,et al.  A derivatization method for the simultaneous detection of glucosinolates and isothiocyanates in biological samples. , 2013, Analytical biochemistry.

[10]  E. Marchioni,et al.  Improvement in determination of isothiocyanates using high-temperature reversed-phase HPLC. , 2012, Journal of separation science.

[11]  K. Row,et al.  Separation and Purification of Sulforaphane from Broccoli by Solid Phase Extraction , 2011, International journal of molecular sciences.

[12]  E. Marchioni,et al.  Simultaneous Determination of Various Isothiocyanates by RP-LC Following Precolumn Derivatization with Mercaptoethanol , 2011, Chromatographia.

[13]  Tao Zhang,et al.  Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells , 2010, Clinical Cancer Research.

[14]  A. Kong,et al.  Molecular Targets of Dietary Phenethyl Isothiocyanate and Sulforaphane for Cancer Chemoprevention , 2010, The AAPS Journal.

[15]  B. Ramírez-Wong,et al.  HPLC method validation for measurement of sulforaphane level in broccoli by-products. , 2009, Biomedical chromatography : BMC.

[16]  C. Ioannides,et al.  Absolute bioavailability and dose-dependent pharmacokinetic behaviour of dietary doses of the chemopreventive isothiocyanate sulforaphane in rat , 2008, British Journal of Nutrition.

[17]  K. Nakagawa,et al.  Evaporative light-scattering analysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphane contents. , 2006, Journal of agricultural and food chemistry.

[18]  T. Shapiro,et al.  Quantitative determination of dithiocarbamates in human plasma, serum, erythrocytes and urine: pharmacokinetics of broccoli sprout isothiocyanates in humans. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[19]  B. P. Klein,et al.  Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea. , 2001, Journal of agricultural and food chemistry.