HPLC/ICP-MS in combination with "reverse" online isotope dilution in drug metabolism studies.
During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel ((14)C or (3)H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisible, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach. However, the method lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with "reverse" online isotope dilution ("reverse" online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a (81)Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and "mixed" species, based on their isotopic "fingerprint". A good repeatability (relative standard deviation of 4.2%) and limit of detection (0.35 mg of drug compound L(-1) of feces extract), of the same order of magnitude as those observed for "normal" online ID HPLC/ICP-MS and HPLC/radiodetection, were achieved.
Department of Analytical Chemistry, Ghent University, Krijgslaan 281-S12, 9000 Ghent, Belgium.
Björn
B
Meermann
Anne
A
Hulstaert
Aline
A
Laenen
Cis
C
Van Looveren
Maarten
M
Vliegen
Filip
F
Cuyckens
Frank
F
Vanhaecke
D000818:Animals; D000995:Antitubercular Agents / Q000032:analysis / Q000378:metabolism; D001966:Bromine / Q000737:chemistry; D002250:Carbon Radioisotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005243:Feces / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D007554:Isotopes / Q000737:chemistry; D004364:Pharmaceutical Preparations / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D014316:Tritium / Q000737:chemistry
D000995:Antitubercular Agents; D002250:Carbon Radioisotopes; D007554:Isotopes; D004364:Pharmaceutical Preparations; D014316:Tritium; D001966:Bromine