Evaluation of the Effects of Sample Dilution and Volume in Hydrophilic Interaction Liquid Chromatography

Isokawa, Muneki; Funatsu, Takashi; Tsunoda, Makoto

doi:10.1007/s10337-014-2748-z

Cited by 12 publications

(14 citation statements)

References 14 publications

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“…After incubation for 1 h at 60°C, the solution was mixed with 25 µL of 1 M hydrochloric acid and cooled in ice to quench the reaction. The resulting solution was diluted 5 times with acetonitrile for injection to avoid deterioration of the peak shape [30,31]. Five microliters of the sample were injected into the HPLC system for analysis.…”

Section: 3preparation Of Standard and Biological Samplesmentioning

confidence: 99%

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Isokawa

Shimosawa

Funatsu

et al. 2016

Journal of Chromatography B

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Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.

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Section: 3preparation Of Standard and Biological Samplesmentioning

confidence: 99%

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Isokawa

Shimosawa

Funatsu

et al. 2016

Journal of Chromatography B

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“…Thiols in the supernatant were derivatized with SBD-F, followed by 5× dilution with acetonitrile for injection to avoid deterioration of peak shape [13,20]. For the analysis of Hcy in CBS-KO samples, an additional 4× dilution was conducted and final samples were composed of 80% acetonitrile.…”

Section: Sample Preparationmentioning

confidence: 99%

“…Recently, we developed an analytical method utilizing ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) as a fluorogenic derivatization reagent and hydrophilic interaction liquid chromatography (HILIC) as a separation mode to tackle these problems [12][13][14]. Since HILIC is more suitable for retention and separation of highly polar compounds than RPLC, and the fluorescence of SBD-thiols is enhanced in the acetonitrile-rich mobile phase of HILIC [12,15,16], this newly developed method enables simultaneous quantification of all biological thiols described above in human and mouse plasma samples.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Biological Thiols in the Plasma of a Homocystinuria Model with Cystathionine β-Synthase Deficiency Utilizing Hydrophilic Interaction Liquid Chromatography and Fluorescence Detection

et al. 2016

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Biological thiols -homocysteine (Hcy), cysteine (Cys), γ-glutamylcysteine (γGluCys), glutathione (GSH), and cysteinylglycine (CysGly) -in plasma samples of cystathionine β-synthase (CBS, EC 4.2.1.22)-deficient mice were quantified using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection. Mice deficient in CBS provide a model mouse of homocystinuria, an inherited metabolic disease characterized by the abnormal accumulation of Hcy in urine and blood. For quantification, the thiols were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by separation with an amide column containing a mobile phase of acetonitrile-40 mM ammonium formate buffer (pH 3.0) (75/25, v/v). The total concentrations of Hcy in the plasma samples of CBS-wild type (-WT), -heterozygous (-Hetero), and -knockout (-KO) mice were 24.7, 29.3, and 237.7 μmol/L, respectively; 236.4, 178.7, and 78.5 μmol/L for Cys; 5.80, 4.87, and 0.75 μmol/L for γGluCys; 64.6, 74.0, and 51.2 μmol/L for GSH; and 2.48, 3.98, and 0.90 μmol/L for CysGly. The concentration of Hcy was significantly increased, while Cys, γGluCys, and CysGly concentrations were significantly decreased in CBS-KO mice compared to those in CBS-WT mice. The results indicated that biological thiols other than Hcy could also be utilized for the evaluation and investigation of homocystinuria pathologies.

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“…[36] A crucial parameter that we had to optimize was the nature of the dissolution solvent that usually affects the chromatographic peak shape in HILIC and it appears to be more critical than in reversed-phase LC. [37][38][39] During method development we realized that the peak shapes of cephalosporins and alfuzosin (ISTD) were distorted and split depending on the percentage of the biological sample in the injection sample and on the nature of the dissolution solvent. Thus, different proportions of breast milk and human plasma in the solvent mixture were tested, to define the maximum amount of biological material that could be used for analyses without deteriorating the peak shape of the compounds.…”

Section: Sample Preparationmentioning

confidence: 99%

Quantification of three beta‐lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive‐ion electrospray ionization mass spectrometry

Kiriazopoulos

Zaharaki

Vonaparti

et al. 2016

Drug Testing and Analysis

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The use of cephalosporins during breast feeding raises several issues, including the risk of drug exposure through breast milk for the infant. In this paper, a hydrophilic interaction liquid chromatography/positive ion electrospray mass spectrometric assay (HILIC/ESI-MS) was developed for the quantitation of cefuroxime, cefoxitin, and cefazolin in breast milk and human plasma. The assay was based on the use of small sample size, 25 μL of biological samples, following acetonitrile precipitation of proteins and filtration that enabled injection into the HILIC/ESI-MS system. All analytes and the internal standard, alfuzosin, were separated by using a ZIC®-HILIC analytical column (150.0 × 2.1 mm i.d., particle size 3.5 µm, 200 Å) with isocratic elution. The mobile phase was composed of a 6% 12.5 mM ammonium acetate water solution in acetonitrile and pumped at a flow rate of 0.25 mL min . The assay was linear over a concentration range of 0.2 to 5 µg mL and 0.4 to 20 µg mL for all the analytes in breast milk and in human plasma, respectively. Intermediate precision was found to be less than 4.2% over the tested concentration ranges. A run time of less than 12 min for each sample made it possible to analyze a large number of biological samples per day. The method is the first reported application of HILIC in the analysis of antibiotics in breast milk and human plasma and it can be used to support a wide range of clinical studies. Copyright © 2016 John Wiley & Sons, Ltd.

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Evaluation of the Effects of Sample Dilution and Volume in Hydrophilic Interaction Liquid Chromatography

Cited by 12 publications

References 14 publications

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Quantification of Biological Thiols in the Plasma of a Homocystinuria Model with Cystathionine β-Synthase Deficiency Utilizing Hydrophilic Interaction Liquid Chromatography and Fluorescence Detection

Quantification of three beta‐lactam antibiotics in breast milk and human plasma by hydrophilic interaction liquid chromatography/positive‐ion electrospray ionization mass spectrometry

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