Proença Paula, Mustra Carla, Marcos Mariana, Franco João Miguel, Corte-Real Francisco, Vieira Duarte Nuno
National Institute of Legal Medicine, Largo da Sé Nova, 3000-213 Coimbra, Portugal.
J Forensic Leg Med. 2013 Aug;20(6):655-8. doi: 10.1016/j.jflm.2013.03.002. Epub 2013 Apr 24.
The use of synthetic phosphodiesterase type 5 (PDE-5) inhibitors for the treatment of erectile dysfunction: sildenafil citrate (Viagra(®)), tadalafil (Cialis(®)) and vardenafil hydrochloride (Levitra(®)) has increased dramatically over the past 2 years. These substances are prescription drugs and must be used under medical supervision. However, they can easily be obtained over the internet from illegal sites, being a potential for a threat to public health. The development of an electrospray ionisation (ESI) ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) procedure for the simultaneous identification and quantification of three PDE5 inhibitors in blood samples was desired. Samples were prepared using Oasis(®) HLB solid-phase cartridges (3 cc, 60 mg) and chromatographic separation was achieved on an Acquity UPLC(®) HSS T3 (100 × 2.1 mm i.d., 1.8 μm particles) column with a gradient mobile phase of 0.1% formic acid and acetonitrile at a 0.5 mL/min flow rate. Quantification was achieved by multiple reaction monitoring (MRM) of two transitions per compound: m/z 475.1 > 58 e m/z 475.1 > 311.1 for sildenafil; m/z 389.9 > 267.9 e m/z 389.9 > 134.8 for tadalafil and m/z 489 > 71.9 e m/z 489 > 150.9 for vardenafil. Zolpidem-d6 (m/z 314.5 > 235.3) was used as the internal standard. Calibration curves were linear over the concentration range of 5-1000 ng/mL, with a coefficient of determination better than 0.997. The lower limits of detection and quantification for these substances were ≤ 3 ng/mL and ≤ 8 ng/mL, respectively. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. A rapid, selective and sensitive UPLC-MS/MS method using solid-phase extraction was developed for the simultaneous determination and quantification of sildenafil, vardenafil and tadalafil in blood samples.
在过去两年中，用于治疗勃起功能障碍的合成5型磷酸二酯酶（PDE-5）抑制剂——枸橼酸西地那非（万艾可®）、他达拉非（希爱力®）和盐酸伐地那非（艾力达®）的使用量急剧增加。这些物质是处方药，必须在医疗监督下使用。然而，它们很容易从非法网站在互联网上获取，对公众健康构成潜在威胁。因此需要开发一种电喷雾电离（ESI）超高效液相色谱串联质谱（UPLC-MS/MS）方法，用于同时鉴定和定量血样中的三种PDE5抑制剂。使用Oasis® HLB固相萃取柱（3 cc，60 mg）制备样品，并在Acquity UPLC® HSS T3柱（100×2.1 mm内径，1.8μm颗粒）上进行色谱分离，流动相为0.1%甲酸和乙腈的梯度溶液，流速为0.5 mL/min。通过对每种化合物的两个跃迁进行多反应监测（MRM）实现定量：西地那非为m/z 475.1 > 58和m/z 475.1 > 311.1；他达拉非为m/z 389.9 > 267.9和m/z 389.9 > 134.8；伐地那非为m/z 489 > 71.9和m/z 489 > 150.9。唑吡坦-d6（m/z 314.5 > 235.3）用作内标。校准曲线在5-1000 ng/mL的浓度范围内呈线性，决定系数优于0.997。这些物质的检测下限和定量下限分别≤ 3 ng/mL和≤ 8 ng/mL。该方法具有令人满意的灵敏度、精密度、准确度、回收率和选择性。开发了一种使用固相萃取的快速、选择性和灵敏的UPLC-MS/MS方法，用于同时测定和定量血样中的西地那非、伐地那非和他达拉非。
