Identification of phenylbutyrate-generated metabolites in Huntington disease patients using parallel liquid chromatography/electrochemical array/mass spectrometry and off-line tandem mass spectrometry

Abstract

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB.

Figure 1

LC-EC-array/UV/F method showing full 14-channel LC-EC-array/UV/Fluorescence-detected chromatograms of plasma from a patient, taken (A) before treatment and (B) on visit 6 after SPB-treatment. Metabolites of interest (M), phenylacetate (PA) and phenylbutyrate (PB) are labeled in B. This figure was generated directly from the CoulArray software.

Figure 2

Flow chart showing the course of samples from initial offline LC-EC-array screening through sample fractionation, parallel LC-EC-array-MS analysis and high resolution and MS/MS characterization.

Figure 3

LC-EC-array chromatograms of the 40% ACN fraction collected from the Diazam C-18 column. Elution of a metabolite of interest is indicated at the retention time of 32 minutes. PB is not EC active.

Figure 4

(A) QStar Q-o-TOF MS total ion chromatogram of the 40% ACN plasma fraction containing phenylbutyrate (PB) and an unknown SPB metabolite. Other panels show single ion chromatograms of (B) m/z 163, corresponding to the [M-H]⁻ of PB, and (C) m/z 117 and (D) m/z 161) which both displayed maxima at the retention time (32 min) of the unknown metabolite.

Figure 5

Orbitrap nanospray MS spectrum of 40% ACN fraction collected from Diazam C-18 column at 32 min. Ions assigned to the unknown SPB metabolite (m/z 161.0613, 117.0713) are circled. The ([M-H]⁻m/z 163.0769) peak indicates presence of a the leading edge of the peak corresponding to the parent drug PB which reached its maximum in the following LC fraction. Inset shows Orbitrap MS/MS spectrum of the unknown metabolite, [M-H]⁻m/z 161.0609. Proposed structures are indicated for the metabolite selected as precursor and its abundant fragment ions.

Figure 6

Structures and fragments of metabolites found in SPB-treated HD patient plasma and urine. Structures and fragments correspond to those listed in Tables 1 and 2.