[Simultaneous determination of tryptophan and its metabolites in plasma by high performance liquid chromatography with on-column derivatization].
A high performance liquid chromatography-ultraviolet/fluorescence detection (HPLC-UV/FLD) with on-column derivatization was established to simultaneously determine tryptophan (Trp), kynurenine (Kyn), 5-hydroxyindole acetic acid (5-Hiaa) and kynurenic acid (Kyna). A Hypersil C-18 column (250 mm x 4.0 mm, 5 microm) was used for the analysis at 30 degrees C. The separation was carried out with the mobile phase consisting of 250 mmol/L zinc acetate (pH 5.5) and acetonitrile (95: 5, v/v) at a flow rate of 0.8 mL/min using 3-nitrotyrosine as internal standard (IS). The excitation (Ex) and emission (Em) wavelengths were set at 278 nm (lambda(ex))/343 nm (lambda(em)) for 5-Hiaa and 244 nm (lambda(ex))/400 nm (lambda(em)) for Kyna, while the wavelengths of ultraviolet detection were set at 360 nm for Kyn and IS, 302 nm for Trp. The recoveries were in the range of 91.62% to 114.17%. The linearities were from 2.50 micromol/L to 320.00 micromol/L for Trp, 0.32 micromol/L to 15.36 micromol/L for Kyn, 3.27 nmol/L to 104.60 nmol/L for 5-Hiaa, and 14.00 nmol/L to 464.80 nmol/L for Kyna. The detection limits were 0.078 micromol/L, 0.056 micromol/L, 0.690 nmol/L and 1.290 nmol/L for Trp, Kyn, 5-Hiaa, and Kyna, respectively. Thirty plasma samples of normal pregnant women and 28 plasma samples of healthy controls were tested, and the results exhibited that the concentrations of Trp, Kyn and Kyna in the plasma of the normal pregnant women were significantly different from those of the control group (all P < 0.01). The method is simple and sensitive with good reproducibility, and it is suitable for clinical measurements.
College of Laboratory Medicine, Chongqing Medical University, Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education, Chongqing 400016, China.
Chengya
C
Feng
Jieying
J
Gao
Qianna
Q
Zhen
Zimian
Z
Fan
Mingsong
M
Zhu
Xiangchun
X
Yang
Min
M
Ding
D000097:Acetonitriles; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005453:Fluorescence; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000097:blood; D007736:Kynurenic Acid / Q000097:blood; D007737:Kynurenine / Q000097:blood; D057230:Limit of Detection; D011247:Pregnancy; D015203:Reproducibility of Results; D014364:Tryptophan / Q000097:blood*; D014443:Tyrosine / Q000031:analogs & derivatives; D019345:Zinc Acetate
D000097:Acetonitriles; D007737:Kynurenine; C002744:3-nitrotyrosine; D014443:Tyrosine; D006897:Hydroxyindoleacetic Acid; D014364:Tryptophan; D019345:Zinc Acetate; D007736:Kynurenic Acid; C032159:acetonitrile