Roda A, Gioacchini A M, Cerrè C, Baraldini M
Dipartimento di Scienze Farmaceutiche, Università de Bologna, Italy.
J Chromatogr B Biomed Appl. 1995 Mar 24;665(2):281-94. doi: 10.1016/0378-4347(94)00544-f.
The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.
本研究描述了配备电喷雾接口的高效液相色谱 - 质谱系统用于胆汁酸定量分析的开发。使用C18反相柱（粒径3微米，内径70×4.6毫米），以甲醇 - 15 mM醋酸铵作为等度和梯度模式的流动相，流速为0.3毫升/分钟，实现了游离胆汁酸以及甘氨酸和牛磺酸共轭胆汁酸的良好分离。该系统允许对洗脱液进行柱后分流，以便由两种不同的检测器进行分析：（1）流速为18微升/分钟的电喷雾质谱仪；（2）互补的蒸发光散射质量检测器。当胆汁盐在负离子模式下的电喷雾接口中离子化时，仅产生[M - H]-分子离子；对于所有研究的胆汁酸，检测限为注入15皮克。在第二个系统中，使用了半微量预柱分流装置（Acurate，LC Packings）：通过该装置，高效液相色谱泵的流速降低至1.4微升/分钟，使用与上述相同的流动相，用微径C18柱（粒径3微米，内径150×0.30）分离胆汁酸。对于后一个系统，可以使用柱头富集技术：注入量可以从60纳升增加到200纳升，从而将检测限提高到注入5皮克。描述了高效液相色谱 - 电喷雾 - 质谱法在胆汁和血清胆汁酸分析中的应用；还对第一个系统测定富含13C标记胆汁酸的血清中13C/12C同位素比的能力的初步数据进行了批判性讨论。
