Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards

Maya Kamao, Yoshitomo Suhara, Naoko Tsugawa, Toshio Okano

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51 Citations (Scopus)

Abstract

A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K1, phylloquinone, PK and K2, menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R2 = 0.988), PK (R2 = 0.979) and MK-7 (R2 = 0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.

Original languageEnglish
Pages (from-to)41-48
Number of pages8
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume816
Issue number1-2
DOIs
Publication statusPublished - 2005 Feb 25
Externally publishedYes

Fingerprint

Vitamin K
High performance liquid chromatography
Vitamin K 1
Fluorescence
Chromatography
Plasmas
Assays
Derivatives
Vitamin K 2
Hexanes
Phase separation
Ethanol
Lipids
Recovery

Keywords

  • Fluorescence detection
  • Internal standards
  • Vitamin K

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards",
abstract = "A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K1, phylloquinone, PK and K2, menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92{\%} and the inter- and intra-assay R.S.D. values were 5.7-9.2{\%} for MK-4, 4.9-9.6{\%} for PK and 6.3-19.3{\%} for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R2 = 0.988), PK (R2 = 0.979) and MK-7 (R2 = 0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.",
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author = "Maya Kamao and Yoshitomo Suhara and Naoko Tsugawa and Toshio Okano",
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T1 - Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards

AU - Kamao, Maya

AU - Suhara, Yoshitomo

AU - Tsugawa, Naoko

AU - Okano, Toshio

PY - 2005/2/25

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N2 - A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K1, phylloquinone, PK and K2, menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R2 = 0.988), PK (R2 = 0.979) and MK-7 (R2 = 0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.

AB - A HPLC fluorescence determination method for Vitamin K derivatives (Vitamin K1, phylloquinone, PK and K2, menaquinones, MK-4 and MK-7) using post-column reduction and internal standards was developed. Selectivity and reproducibility were increased by optimized chromatography conditions and satisfactory precision and accuracy were attained by using synthetic internal standards. After addition of internal standards to plasma samples, lipids were extracted with ethanol and hexane. Chromatography was performed by isocratic reverse phase separation on a C18 column. Vitamin K derivatives were detected at 430 nm with excitation at 320 nm for MK-4 and 240 nm for PK and MK-7. The detection limits for MK-4, PK and MK-7 were 4, 2 and 4 pg, respectively. The recoveries of MK-4, PK and MK-7 were greater than 92% and the inter- and intra-assay R.S.D. values were 5.7-9.2% for MK-4, 4.9-9.6% for PK and 6.3-19.3% for MK-7. The data showed good correlation between proposed method and LC-APCI/MS method for MK-4 (R2 = 0.988), PK (R2 = 0.979) and MK-7 (R2 = 0.986). The method allows the determination of Vitamin K for evaluating their clinical and nutritional status.

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