Purpose: This study describes the development of a novel and sensitive UPLC-MS/MS method for the simultaneous determination of diethyl phthalate (DEP) and its major metabolite, monoethyl phthalate (MEP), in rat plasma, urine, and 11 different tissues.
Methods: The analytes were separated using 0.1% (v/v) aqueous formic acid and acetonitrile containing 0.1% (v/v) formic acid as a mobile phase by gradient elution at a flow rate of 0.25 mL/min, equipped with a KINETEX core-shell C18 column (50 mm × 2.1 mm i.d., 1.7 μm particle size). Quantitation of this analysis was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring. The mass transitions were m/z 223.20→149.00 for DEP, 192.90→121.10 for MEP, and 226.80→153.00 for DEP-d4 as internal standard. Liquid-liquid extraction and protein precipitation with ethyl acetate-acetonitrile (1:9, v/v) were used in the sample extraction.
Results: Chromatograms showed high resolution, sensitivity, and selectivity without interference by plasma, urine, and 11 different tissue constituents. The assay achieved lower limit of quantification of 0.04 ng/mL of DEP, 0.1 ng/mL of MEP for plasma, urine, and all tissues. Calibration curves of DEP and MEP in rat plasma, urine, and all tissues ranged from 0.04 to 4000 ng/mL, and 0.1 to 4000 ng/mL, respectively. Calibration curves of each analyte displayed excellent linearity, with correlation coefficients greater than 0.99. For all analytes, both intra- and inter-day precisions (CV%) were less than 5.99%. The accuracy was 95.60-103.30% for DEP, and 95.81-102.72% for MEP. The disposition of DEP was characterized by short half-life (0.72-1.34 h) and a high clearance (11.44-12.28 L/h/kg). It was rapidly metabolized to MEP, with their levels consistently exceeding the DEP levels. The concentration-time curves of the DEP and MEP in rats are shown in Figure 1. Figure 2 illustrates the cumulative urinary excretion-time profiles of DEP and MEP, after oral and IV administration of DEP (0.1, 0.5, 2, and 10 mg/kg) to rats. The distribution of DEP and MEP to tissues of liver, kidney, gastro-intestinal tract, spleen, testis, lung, brain, muscle, thymus, heart, and adipose was determined after 24 h of drug administration. For DEP, the tissue to plasma partition coefficient was the highest for kidney (16.72) followed by liver (10.04), spleen (1.35), and adipose (1.18). In contrast, for MEP, it was the highest for liver (2.18) but was less than unity for all other tissues. The tissue to plasma partition coefficients (Kp) of DEP and MEP in IV administration are summarized in Table 1. In addition, there was no significant difference in pharmacokinetic parameters between male and female rats for DEP intravenous doses of 2 mg/kg.
Conclusion: The developed analytical method satisfied the criteria of international guidance and could be successfully applied to the toxicokinetic study of DEP after oral and IV administration of DEP to rats. In addition, findings of this study may be useful to evaluate the exposure and toxic potential of DEP and its metabolite in risk assessment.