RAPID DETERMINATION OF SPHINGANINE AND SPHINGOSINE IN URINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING MONOLITHIC COLUMN

Abstract

A rapid high performance liquid chromatography method for the determination of sphinganine (Sa) and sphingosine (So) in urine samples by employing a silica-based monolithic column is described. The samples were first extracted using ethyl acetate and derivatized using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol. C20 sphinganine was used as internal standard. Under the optimized conditions, separation was achieved using a mixture of methanol:water (93:7, v/v), column temperature at 30°C, flow rate of 1 mL min⁻¹, and an injection volume of 10 μL. Good linearity was obtained for Sa and So over the concentration range 20–500 ng mL⁻¹(correlation coefficients ≥0.9978). The detection limits were 0.45 ng mL⁻¹ for Sa and 1.60 n g mL⁻¹ for So in male urine; in female urine the values were 0.85 ng mL⁻¹ and 2.62 ng mL⁻¹ for Sa and So, respectively. Recoveries for spiked urine samples ranged from 98.6 to 108.2% for Sa and 99.9 to 104.0% for So_. A marked reduction in separation time (less than 6 min) was found compared to approximately 14 min by using a conventional C18 particle column. Twenty-two urine samples from the healthy donors and seven samples from the liver cancer patients were analyzed using the method.

Keywords:

• high performance liquid chromatography
• monolithic column
• mycotoxin
• sphinganine
• sphingosine
• urine

ACKNOWLEDGMENTS

Financial support of the work by Universiti Sains Malaysia (USM) Postgraduate Research Grant Scheme (USM-RU-PRGS), 1001/PKIMIA/843052, and research fellowship program is acknowledged. Mr. Tan Wee Jin from General Hospital of Penang and Mr. Ong Chiang Hock are thanked for procuring the urine samples.

Notes

t _R: retention time; k: retention factor; N: number of theoretical plates; R_s: resolution.

^a The value refers to R_s between So and Sa.

^b The value refers to R_s between Sa and C20 Sa.

^a Correlation coefficient.

^b Limit of detection (ng mL⁻¹).

^c Limit of quantification (ng mL⁻¹).

RSD: relative standard deviation.

RSD: relative standard deviation.

M: male; F: female; SD: Standard Deviation.

^a Sample number.