Upreti Vijay V, Khurana Manoj, Cox Donna S, Eddington Natalie D
Pharmacokinetics and Biopharmaceutics Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, 20 Penn Street, Baltimore, MD 21201, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2006 Feb 2;831(1-2):156-62. doi: 10.1016/j.jchromb.2005.11.047. Epub 2005 Dec 27.
SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of -8.92 to 5.61% and -16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.
SB - 424323是一种新型的口服活性抗血栓药物，目前正处于二期临床开发阶段，出血风险有限，其独特的作用机制涉及诱导糖胺聚糖（GAGs）生物合成。本研究的目的是开发一种简单快速的高效液相色谱（HPLC）方法，用于测定SB - 424323二期临床研究血浆样本中内源性GAGs衍生的二糖。样品制备是对稀释后的血浆进行简单热处理，然后用软骨素酶ABC消化内源性GAGs，以产生不饱和二糖，2 - 乙酰氨基 - 2 - 脱氧 - 3 - O -（β - D - 葡萄糖 - 4 - 烯吡喃糖醛酸） - D - 半乳糖（ΔDi - 0S）、2 - 乙酰氨基 - 2 - 脱氧 - 3 - O -（β - D - 葡萄糖 - 4 - 烯吡喃糖醛酸） - 4 - O - 磺基 - D - 半乳糖（ΔDi - 4S）和2 - 乙酰氨基 - 2 - 脱氧 - 3 - O -（β - D - 葡萄糖 - 4 - 烯吡喃糖醛酸） - 6 - O - 磺基 - D - 半乳糖（ΔDi - 6S）。这些二糖通过使用截留分子量为3000的滤器进行离心过滤回收和纯化，并加入外部添加的内标2 - 乙酰氨基 - 2 - 脱氧 - 3 - O -（2 - O - 磺基 - β - D - 葡萄糖 - 4 - 烯吡喃糖醛酸） - D - 半乳糖（ΔDi - UA2S）。在Waters Symmetry C(18)柱（4.6 mm×150 mm，5μm）上进行梯度反相HPLC分离，梯度流动相系统由0.8 mM硫酸四丁铵、2 mM氯化钠和乙腈组成，流速为1.0 mL/min。用设置在230 nm的紫外检测器监测洗脱液。血浆标准曲线在1.0 - 20μg/mL浓度范围内呈线性（r(2)≥0.994），每种二糖的定量下限（LLOQ）为1.0μg/mL。二糖的平均实测质量控制（QC）浓度与标称浓度的偏差在日间为 - 8.92至5.61%，日内为 - 16.3至16.7%。QC样品测量的日间和日内精密度，相对标准偏差（R.S.D.）分别在3.21至18.2%和0.32至20.9%范围内。人对照血浆中内源性GAGs衍生二糖测量的日间和日内精密度，相对标准偏差分别在5.8至15.9%和1.17至7.74%范围内。经处理的样品在自动进样器中长达48小时的稳定性得到确认。该方法简单、可靠，易于适用于临床研究物流条件下大量样品的分析。本方法已用于研究SB - 424323二期临床研究患者血浆中的GAGs水平。
