Blood Brain Barrier (BBB) permeability is frequently compromised in the course of diseases affecting the central nervous system (CNS). Sucrose is a low molecular weight, hydrophilic marker with slow permeability at the naive BBB and therefore one of the widely used indicators of barrier integrity. Our laboratory recently developed a highly sensitive UPLC–MS/MS method for stable isotope labeled [¹³C₁₂]sucrose in biological matrices. Correction of total brain concentration for contribution of intravascular space is required in such experiments in order to accurately measure BBB permeability, and it is often accomplished by vascular perfusion with buffer solutions prior to brain sampling. The purpose of the present study was to develop a UPLC–MS/MS method, which allows simultaneous analysis of two different stable isotope labeled sucrose variants, one of which can be utilized as a vascular marker. The first analyte, [¹³C₁₂]sucrose, serves to quantify brain uptake clearance as a measure of BBB permeability, while the second analyte, [¹³C₆]sucrose, is administered just before termination of the animal experiment and is considered as the vascular marker. [²H₂]sucrose is used as the internal standard for both ¹³C labeled compounds. Because the majority of recent studies on CNS diseases employ mice, another objective was to validate the new technique in this species. The UPLC–MS/MS method was linear (r² ≥ 0.99) in the tested concentration ranges, from 10 to 1000 ng/mL for both analytes in plasma, from 2 to 400 ng/g [¹³C₁₂]sucrose in brain and from 10 to 400 ng/g [¹³C₆]sucrose in brain. It was also validated in terms of acceptable intra and inter run accuracy and precision values (n = 5). The dual analyte technique was applied in a study in mice. One group received intravenous bolus injections of 10 mg/kg [¹³C₁₂]sucrose at time 0, and 10 mg/kg [¹³C₆]sucrose at 14.5 min, and subsequent terminal blood and brain sampling was performed at 15 min. For comparison, another group received an intravenous bolus dose of 10 mg/kg [¹³C₁₂]sucrose and was submitted to transcardiac perfusion with buffer after 15 min. We demonstrate that the two alternative techniques to correct for intravascular content deliver equivalent values for brain concentration and brain uptake clearance.

在影响中枢神经系统（CNS）的疾病过程中，经常会损害血脑屏障（BBB）的通透性。蔗糖是一种低分子量，亲水性标记物，在幼稚的BBB上具有较慢的渗透性，因此是屏障完整性的广泛使用的指标之一。我们的实验室最近开发了一种高灵敏度的UPLC–MS / MS方法，用于标记[ ¹³ C ₁₂蔗糖在生物基质中。为了准确地测量BBB的通透性，在此类实验中需要校正总脑部浓度对血管内空间的影响，这通常是通过在脑部采样之前用缓冲液对血管进行灌注来实现的。本研究的目的是开发一种UPLC-MS / MS方法，该方法可以同时分析两种不同的稳定同位素标记的蔗糖变体，其中一种可用作血管标记。第一种分析物[ ¹³ C ₁₂ ]蔗糖用于量化脑摄取清除率，作为BBB通透性的量度，而第二种分析物[ ¹³ C ₆蔗糖是在动物实验终止前给药的，被认为是血管标记物。[ ² H ₂ ]蔗糖用作两种¹³ C标记化合物的内标。由于最近有关中枢神经系统疾病的大多数研究都使用小鼠，因此另一个目标是验证该物种中的新技术。在UPLC-MS / MS法是线性关系（r ² 在所测试的浓度范围≥0.99），从10至1000毫微克/毫升血浆中两种分析物，从2至400毫微克/克[ ¹³ Ç ₁₂ ]蔗糖的脑和10至400 ng / g [ ¹³ C ₆大脑中的蔗糖。还通过了可接受的内部和内部运行精度和精度值（n = 5）进行了验证。双重分析物技术已应用于小鼠研究中。一组在时间0接受10毫克/千克[ ¹³ C ₁₂ ]蔗糖静脉推注，在14.5分钟接受10毫克/千克[ ¹³ C ₆ ]蔗糖静脉注射，随后在15分钟进行末次血液和脑采样。为了进行比较，另一组接受了10 mg / kg的静脉推注剂量[ ¹³ C ₁₂蔗糖]，并在15分钟后用缓冲液进行心内灌注。我们证明了纠正血管内含量的两种替代技术可提供相等的大脑浓度和大脑摄取清除率值。