Konieczna Lucyna, Kaźmierska Katarzyna, Roszkowska Anna, Szlagatys-Sidorkiewicz Agnieszka, Bączek Tomasz
Department of Pharmaceutical Chemistry, Medical University of Gdańsk, Hallera 107, 80-416 Gdańsk, Poland.
The Medicinal Entity, Copernicus Hospital, Nowe Ogrody 1-6, 80-803 Gdańsk, Poland.
J Pharm Biomed Anal. 2016 May 30;124:374-381. doi: 10.1016/j.jpba.2016.03.021. Epub 2016 Mar 15.
Cystic fibrosis (CF) is one of the most common genetic diseases in children and affects mainly respiratory and digestive system functions. Despite the prolonged supplementation of vitamins, malnutrition manifested by poor growth and weight loss in children is a major complication in CF related to pancreatic insufficiency and difficulty in absorbing fat-soluble vitamins. In the present study, we have developed and validated a sensitive and accurate high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method for the simultaneous quantification of three fat-soluble vitamins (A, E and K1) and two vitamin D3 active metabolites: 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 in serum samples obtained from pediatric patients with CF. In optimized conditions, the LC-MS method was highly sensitive and presented excellent linearity with a regression coefficient higher than 0.999. The accuracy was in the range of 87.55-95.58 % for all analytes. The precision of the method, expressed as% RSD, ranged from 1.36 % to 3.74 % as the intra-day variability and from 2.35 % to 7.98 % as the inter-day precision for all the studied compounds. Sample preparation included a protein precipitation step with the use of methanol followed by liquid-liquid extraction with n-hexane. The statistical analysis (t-test and principal component analysis (PCA)) of the obtained results revealed significant changes in the plasma level of the analyzed compounds, with 25-hydroxyvitamin D3, vitamin E and K1 present at extremely low concentrations in patients with cystic fibrosis in comparison to healthy controls. The elaborated method reached the expectations for the fast and reliable assessment of fat-soluble vitamin status in children with cystic fibrosis in order to diagnose the disease and monitor the treatment process.
囊性纤维化（CF）是儿童中最常见的遗传疾病之一，主要影响呼吸和消化系统功能。尽管长期补充维生素，但儿童生长发育不良和体重减轻所表现出的营养不良仍是CF的主要并发症，这与胰腺功能不全以及脂溶性维生素吸收困难有关。在本研究中，我们开发并验证了一种灵敏且准确的高效液相色谱-质谱联用（LC-MS）方法，用于同时定量检测从患有CF的儿科患者血清样本中获取的三种脂溶性维生素（A、E和K1）以及两种维生素D3活性代谢物：25-羟基维生素D3和1,25-二羟基维生素D3。在优化条件下，LC-MS方法具有高度灵敏性，呈现出出色的线性，回归系数高于0.999。所有分析物的准确度在87.55 - 95.58%范围内。该方法的精密度以%RSD表示，所有研究化合物的日内变异性范围为1.36%至3.74%，日间精密度范围为2.35%至7.98%。样品制备包括使用甲醇进行蛋白沉淀步骤，随后用正己烷进行液-液萃取。对所得结果的统计分析（t检验和主成分分析（PCA））显示，所分析化合物的血浆水平有显著变化，与健康对照相比，囊性纤维化患者体内的25-羟基维生素D3、维生素E和K1浓度极低。所阐述的方法达到了快速可靠评估囊性纤维化儿童脂溶性维生素状态的预期，以便诊断疾病并监测治疗过程。
