Shipkova M, Schütz E, Armstrong V W, Niedmann P D, Oellerich M, Wieland E
Abteilung Klinische Chemie, Zentrum Innere Medizin, Georg-August-Universität Göttingen, Germany.
Clin Chem. 2000 Mar;46(3):365-72.
The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated.
The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents.
The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C.
The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.
已发现霉酚酸（MPA）的酰基葡糖醛酸（AcMPAG）在体外具有药理活性和潜在的促炎活性。为确定其在体内的药理和毒理学相关性，对反相高效液相色谱法进行了改进，以同时测定MPA、酚性MPA - 葡糖醛酸（7 - O - MPAG）和AcMPAG。此外，还研究了AcMPAG在MPA的Emit测定法中的交叉反应性。
该方法采用简单的样品制备，使用Zorbax Eclipse - XDB - C8柱进行分离，并进行梯度洗脱。AcMPAG以7 - O - MPAG当量进行定量。
该测定法对于MPA在高达50 mg/L时呈线性，对于7 - O - MPAG在高达250 mg/L时呈线性，对于AcMPAG在高达10 mg/L时呈线性（r>0.999）。MPA、7 - O - MPAG和AcMPAG的检测限分别为0.01、0.03和0.04 mg/L。MPA的回收率为99 - 103%，7 - O - MPAG的回收率为95 - 1�3%，AcMPAG的回收率为104 - 107%。日内精密度对于MPA（0.2 - 25 mg/L）<5.0%，对于7 - O - MPAG（10 - 250 mg/L）<4.4%对于AcMPAG（0.1 - 5 mg/L）≤14%。日间精密度对于MPA、7 - O - MPAG和AcMPAG分别<6.2%、<4.5%和≤14%。当从微粒体中分离出来时，纯化的AcMPAG（1 - 10 mg/L）在用于测定MPA的Emit测定法中显示出浓度依赖性交叉反应性，范围为135%至185%。这与接受霉酚酸酯治疗的肾移植受者的270份样本中HPLC和Emit之间的偏差一致，该偏差更大（中位数为151.2%），高于通过HPLC测定的相应AcMPAG浓度。发现当样品在室温下储存长达24小时或在4℃或 - 20℃下储存长达30天时，AcMPAG会发生水解。酸化样品（pH 2.5）在 - 20℃下长达30天稳定。
本文所述的AcMPAG的HPLC和Emit方法可能有助于研究其与霉酚酸酯治疗相关的免疫抑制和副作用的相关性。
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