Fluorimetric determination of D-lactate in urine of normal and diabetic rats by column-switching high-performance liquid chromatography

Jen Ai Lee, Yih Chiao Tsai, Hsiang Yin Chen, Chih Chun Wang, Shih Ming Chen, Takeshi Fukushima, Kazuhiro Imai

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

A highly sensitive method for the fluorimetric determination of d-lactate in urine of normal and diabetic rats was developed using column-switching high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column connected to a chiral column, an amylose tris(3,5- dimethylphenylcarbamate) coated on silica gel (Chiralpak AD-RH). During the separation step on the ODS column, the peak fraction of the (d+l)-lactate derivative with a fluorescence reagent, 4-nitro-7-piperazino-2,1,3- benzoxadiazole (NBD-PZ), was introduced directly to the chiral column by changing the flow of the eluent via a six-port valve. The d-lactate derivative was separated enantiomerically from the l-lactate derivative, and the enantiomeric ratio was determined from the chromatogram. The accuracy values for the determination of d-lactate in 20 μL of rat urine were 96.93-104.85%, and the intra- and inter-day precision values were within 0.80 and 14.44%, respectively. The detection limit for d-lactate was approximately 10 nM (with a signal-to-noise ratio of 3). The proposed HPLC method was applied to the urine of normal and diabetic rats induced by intraperitoneal administration of streptozotocin, and significant increases in d-lactate excreted into the urine were observed in diabetic rats compared to normal rats. In diabetic rats, d-lactate concentrations showed a rising tendency from the seventh day and then remained stable from the 28th day after induction, suggesting that urinary d-lactate may be used as an indicator to determine the diabetic stage and the level of kidney damage.

Original languageEnglish
Pages (from-to)185-191
Number of pages7
JournalAnalytica Chimica Acta
Volume534
Issue number2
DOIs
Publication statusPublished - Apr 8 2005

Fingerprint

High performance liquid chromatography
urine
Rats
liquid chromatography
Lactic Acid
High Pressure Liquid Chromatography
Urine
Derivatives
signal-to-noise ratio
fluorescence
gel
silica
Silica Gel
damage
Signal-To-Noise Ratio
Streptozocin
Limit of Detection
Signal to noise ratio
Fluorescence
Kidney

Keywords

  • Chiralpak AD-RH
  • Column-switching HPLC
  • D-Lactate
  • Enantiomeric separation
  • Fluorescence derivatization
  • NBD-PZ

ASJC Scopus subject areas

  • Biochemistry
  • Analytical Chemistry
  • Spectroscopy
  • Environmental Chemistry

Cite this

Fluorimetric determination of D-lactate in urine of normal and diabetic rats by column-switching high-performance liquid chromatography. / Lee, Jen Ai; Tsai, Yih Chiao; Chen, Hsiang Yin; Wang, Chih Chun; Chen, Shih Ming; Fukushima, Takeshi; Imai, Kazuhiro.

In: Analytica Chimica Acta, Vol. 534, No. 2, 08.04.2005, p. 185-191.

Research output: Contribution to journalArticle

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abstract = "A highly sensitive method for the fluorimetric determination of d-lactate in urine of normal and diabetic rats was developed using column-switching high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column connected to a chiral column, an amylose tris(3,5- dimethylphenylcarbamate) coated on silica gel (Chiralpak AD-RH). During the separation step on the ODS column, the peak fraction of the (d+l)-lactate derivative with a fluorescence reagent, 4-nitro-7-piperazino-2,1,3- benzoxadiazole (NBD-PZ), was introduced directly to the chiral column by changing the flow of the eluent via a six-port valve. The d-lactate derivative was separated enantiomerically from the l-lactate derivative, and the enantiomeric ratio was determined from the chromatogram. The accuracy values for the determination of d-lactate in 20 μL of rat urine were 96.93-104.85{\%}, and the intra- and inter-day precision values were within 0.80 and 14.44{\%}, respectively. The detection limit for d-lactate was approximately 10 nM (with a signal-to-noise ratio of 3). The proposed HPLC method was applied to the urine of normal and diabetic rats induced by intraperitoneal administration of streptozotocin, and significant increases in d-lactate excreted into the urine were observed in diabetic rats compared to normal rats. In diabetic rats, d-lactate concentrations showed a rising tendency from the seventh day and then remained stable from the 28th day after induction, suggesting that urinary d-lactate may be used as an indicator to determine the diabetic stage and the level of kidney damage.",
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AU - Chen, Shih Ming

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AU - Imai, Kazuhiro

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