Rashed M S, Al-Ahaidib L Y, Aboul-Enein H Y, Al-Amoudi M, Jacob M
Metabolic Screening Laboratory, King Faisal Specialist Hospital and Research Centre, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia.
Clin Chem. 2001 Dec;47(12):2124-30.
L-pipecolic acid (L-PA), an important biochemical marker for the diagnosis of peroxisomal disorders, is usually determined as the racemate. We developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of L-PA in plasma.
We used a narrow bore chiral macrocyclic glycopeptide teicoplanin column for the enantioseparation of D-pipecolic acid (D-PA) and L-PA and interfaced the column directly to the electrospray source of a tandem mass spectrometer. We used phenylalanine-d5 as internal standard added to 50 microL of plasma followed by deproteinization, evaporation, and injection. The analysis was performed in the selected-reaction monitoring mode using two transitions: m/z 130-->m/z 84 for PA, and m/z 171-->m/z 125 for phenylalanine-d5. L-PA eluted at 7 min, and D-PA eluted at 11.7 min, whereas phenylalanine-d5 eluted at 6 min. The turnaround time for the assay was 20 min.
Linear calibration curves were obtained in the range of 0.5-80 micromol/L. At a plasma concentration of 1.0 micromol/L, the signal-to-noise ratio was 50:1. The intra- and interassay variations were 3.1-7.9% and 5.7-13%, respectively, at concentrations of 1-50 micromol/L. Mean recoveries of L-PA added to plasma were 95% (5 micromol/L) and 102% (50 micromol/L). The method clearly distinguished between healthy individuals and peroxisomal disease patients.
The novel LC-MS/MS method is simple, rapid, and stereoselective, and uses only 50 microL of plasma, no derivatizing reagents, and a commercially available internal standard. Sample preparation is not complex and is faster than for all other methods.
L-哌可酸（L-PA）是诊断过氧化物酶体疾病的重要生化标志物，通常以消旋体形式测定。我们开发了一种用于分析血浆中L-PA的手性液相色谱-串联质谱（LC-MS/MS）方法。
我们使用窄内径手性大环糖肽替考拉宁柱对D-哌可酸（D-PA）和L-PA进行对映体分离，并将该柱直接连接到串联质谱仪的电喷雾源。我们将苯丙氨酸-d5作为内标添加到50微升血浆中，随后进行去蛋白、蒸发和进样。分析在选择反应监测模式下进行，使用两个跃迁：PA的m/z 130→m/z 84，以及苯丙氨酸-d5的m/z 171→m/z 125。L-PA在7分钟时洗脱，D-PA在11.7分钟时洗脱，而苯丙氨酸-d5在6分钟时洗脱。该检测方法的周转时间为20分钟。
在0.5 - 80微摩尔/升范围内获得线性校准曲线。在血浆浓度为1.0微摩尔/升时，信噪比为50:1。在1 - 50微摩尔/升的浓度下，批内和批间变异分别为3.1 - 7.9%和5.7 - 13%。添加到血浆中的L-PA的平均回收率分别为95%（5微摩尔/升）和102%（50微摩尔/升）。该方法能够清晰地区分健康个体和过氧化物酶体疾病患者。
这种新型的LC-MS/MS方法简单、快速且具有立体选择性，仅使用50微升血浆，无需衍生试剂，且使用市售内标。样品制备不复杂，比所有其他方法都更快。
