کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1212787 1494041 2016 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Fast and sensitive quantification of human liver cytosolic lithocholic acid sulfation using ultra-high performance liquid chromatography–tandem mass spectrometry
ترجمه فارسی عنوان
اندازه گیری سریع و حساس از سولفات اسید لیتوفولیک سیتوزووی کبد انسان با استفاده از کروماتوگرافی مایع کروماتوگرافی فوق العاده بالا
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی


• UPLC–MS/MS analysis of human liver cytosolic LCA sulfation.
• The matrix (human liver cytosol) did not interfere with the assay.
• The assay was optimized for Mg, DTT, 2-mercaptoethanol, and PAPS concentrations.
• The assay was linear with respect to amount of enzyme and incubation time.
• Faster and more sensitive than existing LC–MS or radiometric methods.

Detoxification of lithocholic acid (LCA) to lithocholic acid sulfate (LCA-S) is catalyzed by sulfotransferases, mainly SULT2A1. We developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify human liver cytosolic-dependent LCA sulfation. Chromatographic separation was achieved on an UPLC C18 column (2.1 × 50 mm, 1.7 μm) and a gradient elution of 0.1% formic acid in water and acetonitrile. Negative electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify LCA-S (455.3 → 97.0) and cholic acid (407.2 → 343.3; internal standard). The retention time was 3.51 min for LCA-S and 3.08 min for cholic acid. The lower limit of quantification of LCA-S was 0.5 nM (or 0.23 ng/ml in 400 μl total volume) and the assay was linear from 0.2 to 200 pmol. Intra-day and inter-day accuracy and precision were <14%. The quality control samples were stable at room temperature for 4 h, 4 °C for 24 h, −20 °C for 14 days, and after three freeze–thaw cycles. The matrix (20–100 μg cytosolic protein) did not affect LCA-S quantification. This is the first UPLC–MS/MS method applied to optimization of the human liver cytosolic LCA sulfation assay. The optimal levels of MgCl2 and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Addition of reducing agents (2-mercaptoethanol and DL-dithiothreitol) did not affect LCA-S formation. Human liver cytosolic LCA sulfation was linear with 20–100 μg of cytosolic protein and 5–30 min incubation time. This UPLC–MS/MS approach offers a specific, sensitive, fast, and direct approach for quantifying human liver cytosolic LCA sulfation.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volume 1011, 1 February 2016, Pages 171–178
نویسندگان
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