Luceri F, Fattori S, Luceri C, Zorn M, Mannaioni P, Messeri G
Laboratory of Clinical Biochemistry, Azienda Ospedaliera Careggi, Firenze, Italy.
Clin Chem Lab Med. 2001 Dec;39(12):1234-9. doi: 10.1515/CCLM.2001.198.
The urinary 6beta-OH-cortisol/cortisol ratio is a specific, non-invasive marker for evaluating inductive or inhibitory effects on cytochrome P450 3A activity. We propose a new quantitative gas chromatography-mass spectrometry with isotope dilution (GC-ID-MS) method for the simultaneous determination of urinary free cortisol (UFC) and 6beta-OH-cortisol (6beta-OHC). The method utilizes the following: (a) addition of internal standard (2H2 cortisol) to 1 ml of urine; (b) loading on to an Extrelut column and elution with dichloromethane; (c) derivatization to dimethoxime tri-(trimethyl-silyl)ether (MOX-TMS); (d) separation and identification by GC-ID-MS. The detection limit for cortisol was 22 pg injected (signal-to-noise ratio 10:1) and for 6beta-OH-cortisol 123 pg injected (signal-to-noise ratio 10:1). The intra-assay and the inter-assay imprecision were 4.69% and 7.4% for 6beta-OHC and 2.44% and 3.53% for cortisol, respectively. We used this method to analyze 57 morning urine samples of healthy volunteers and patients under different conditions. We found that chronic alcoholics had a significantly higher ratio of 6beta-OHC/UFC compared to controls (p<0.0001), whereas adults undergoing methadone therapy and patients with acute alcohol intoxication exhibited a significantly lower urinary 6beta-OHC/UFC ratio (p<0.05 and p<0.01, respectively). The proposed method allows a rapid and accurate assessment of the 6beta-OHC/UFC ratio.
尿中6β-羟基皮质醇/皮质醇比值是评估细胞色素P450 3A活性诱导或抑制作用的一种特异性、非侵入性标志物。我们提出了一种新的带同位素稀释的定量气相色谱-质谱联用（GC-ID-MS）方法，用于同时测定尿游离皮质醇（UFC）和6β-羟基皮质醇（6β-OHC）。该方法采用以下步骤：（a）向1 ml尿液中加入内标（2H2皮质醇）；（b）加载到Extrelut柱上并用二氯甲烷洗脱；（c）衍生化为二甲氧基肟三（三甲基硅基）醚（MOX-TMS）；（d）通过GC-ID-MS进行分离和鉴定。皮质醇的检测限为进样22 pg（信噪比10:1），6β-羟基皮质醇的检测限为进样123 pg（信噪比10:1）。6β-OHC的批内和批间不精密度分别为4.69%和7.4%，皮质醇的批内和批间不精密度分别为2.44%和3.53%。我们用该方法分析了57份不同状态下健康志愿者和患者的晨尿样本。我们发现，与对照组相比，慢性酒精中毒者的6β-OHC/UFC比值显著更高（p<0.0001），而接受美沙酮治疗的成年人和急性酒精中毒患者的尿6β-OHC/UFC比值显著更低（分别为p<0.05和p<0.01）。所提出的方法能够快速、准确地评估6β-OHC/UFC比值。
