Determination of pilocarpic acid in human plasma by capillary gas chromatography with mass-selective detection

A novel, highly sensitive method for the determination of pilocarpic acid ( PA) in human plasma is described. In addition, the method provides for the conversion of the lactone, pilocarpine (P), to PA so that a total drug pres ence can be determined. Using novel high-performance liquid chromatographic conditions capable of separating P, isopilocarpine (I-P), PA and isopiloca rpic acid (I-PA) from each other and from endogenous plasma impurities, it was confirmed that P exclusively and quantitatively converts to PA in hepar inized human plasma during storage. For the determination of PA, the select ive extraction of PA from protein-free plasma was accomplished using two di fferent solid-phase extraction (SPE) cartridges in two consecutive SPE step s. After extraction, PA was lactonized with trifluoroacetic acid back to P and both P and an internal standard were acylated using heptafluorobutyric anhydride (HFBA). The trifluoroacetylated derivatives were monitored using gas chromatography (GC) with mass spectrometric (MS) detection. This proced ure allowed the sensitive and reliable determination of PA with a limit of quantification (LOQ) of 1 ng/ml, which could not be achieved using previous ly described methods. The assay was validated in the concentration range of 1 to 10 nglml with an intra-day precision (expressed as the coefficient of variation, C.V.) ranging from 9.9 to 0.5%. Inter-day precision for the qua lity control standard at 2.5 ng/ml showed a C.V. of 10.2%. Accuracy ranged from 94 to 102%. The assay was used to monitor the maximum systemic exposur e to P, administered by the ocular route, in terms of total plasma PA (P an d PA). (C) 1998 Elsevier Science B.V. All rights reserved.