Biotransformation of cyclizine in greyhounds. 1: identification and analysis of cyclizine and some basic metabolites in canine urine by gas chromatography-mass spectrometry

Abstract

1. The partial in vivo biotransformation of Marezine [(cyclizine.HCl); 1-diphenylmethyl-4-methylpiperazine hydrochloride] in the racing greyhound and the excretion of the unconjugated and conjugated (Phase II) basic metabolites of cyclizine in canine urine are reported. 2. Using copolymeric bonded mixed-mode solid-phase extraction cartridges, the basic isolates from both unhydrolysed and enzyme hydrolysed urine samples were isolated, derivatized as trimethylsilyl ethers and analysed by positive-ion electron ionization gas chromatography-mass spectrometry (EI(+)-GC-MS). Selected samples were analysed by positive-ion methane chemical ionization (CI(+))-GC-MS to aid structure elucidation of the putative metabolites. 3. Cyclizine was the major component excreted in post-administration urine. Five substrate-related basic compounds (M1--> M5) were tentatively identified by EI(+)- and CI(+)-GC-MS. The major Phase I metabolite was identified as norcyclizine [1-diphenylmethylpiperazine] (M1), the other metabolites (M2 --> M5) were tentatively identified as monohydroxylated products based on MS data. 4. Cyclizine and the N(4)-desmethyl metabolite (M1) are excreted unconjugated; the other four hydroxylated metabolites are excreted as Phase II conjugates (glucuronides and/or sulphates). Structures of the putative basic metabolites are presented. At least four other basic metabolites were also detected in post-administration urine, but could not be characterized from GC-MS data. 5. All unhydrolysed post-administration urine samples were analysed by selected ion monitoring EI(+)-GC-MS to quantify cyclizine and norcyclizine (M1) using authentic cyclizine as the analyte and chlorcyclizine as the internal standard. The level of M1 is expressed as 'cyclizine equivalents'. The duration of urinary elimination of cyclizine and M1 was obtained from their excretion profiles. 6. From these studies, cyclizine and norcyclizine (M1) would be the target compounds of choice in the development of screening and confirmatory methods for the detection of cyclizine administration to racing greyhounds. Information on any of the other metabolites may also be of some value for confirmatory analysis.

An LC–MS–MS method for the determination of cyclizine in human serum

“…Assay methods for CYC in both serum and urine have used colorimetric detection methods [4], derivatization with tritiated acetic anhydride [5], gas-liquid chromatography [6,7], GC-MS [8,9] and HPLC with ultraviolet detection [10,11]. Limitations of some of these methods include the lack of the requisite sensitivity and selectivity necessary for accurate assessment of the pharmacokinetics of the drug.…”

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

“…For all three compounds the m/z 167 fragment was dominant and was therefore used for quantification. This fragment is likely to be a diphenylmethyl cation [5] as indicated in Fig. 1.…”

“…Other routes include N-glucuronidation resulting in the formation of quaternary N + -glucuronides, which are polar and non-volatile metabolites that are not easy to detect [4]. In greyhounds several urinary metabolites have been identified including conjugated hydroxy-metabolites [5,6].…”

N-Deethylation and N-oxidation of etamiphylline: identification of etamiphylline-N-oxide in greyhound urine by high performance liquid chromatography–mass spectrometry

“…The prepared samples were extracted off-line using XtracT ® mixed mode SPE columns as described previously [6,7]. The dry residues were derivatised by the addition of MSTFA in toluene (1:1, v/v, 50 l; 80 • C, 0.5 h), the derivatising reagents removed under nitrogen at 60 • C, re-dissolved in toluene (50 l) and analysed by GC/EI+/MS.…”