Securityguard guard column

Chemical analysis of total urinary paracetamol and p-aminophenol was performed at the Wadsworth Center, New York State Department of Health, Albany, NY. Specifically, 300 µL of 1 M ammonium acetate containing 30 U of β-glucuronidase (pH=5.5) was added to 500 µL of urine sample, followed by incubation at 37°C for 12 h. Target analytes were extracted thrice with ethyl acetate and were quantified by ultra-high performance liquid chromatography (Acquity I Class; Waters, Milford, MA) coupled with an electrospray triple quadrupole mass spectrometry (API 5500; AB SCIEX, Framingham, MA) (UPLC-ESI-MS/MS). Separation of target analytes was carried by a Kinetex C18 (1.3 µ, 100A, 50 × 2.1 mm) column (Phenomenex; Torrance, CA) with a SecurityGuard guard column (Phenomenex) with positive ionization, multiple reaction monitoring mode of detection. Quality assurance and quality control parameters included procedural blanks, matrix spikes and duplicate analysis of samples. Labelled internal standards (d₇-p-aminophenol and ¹³C₂-¹⁵N- paracetamol) were spiked into all samples and quantification was by isotope dilution. The method limits of quantitation (LOQ) for p-aminophenol and paracetamol were 0.25 and 0.5 ng/mL, respectively.

Method 2 is an alternative method for fipronil sulfone quantification and for independent evaluation of the accuracy of method 1. Analysis was carried out with a method reported by McMahen et al. (2015) (link). Briefly, rat urine (100μL) was precipitated with 900 μL of cold acetonitrile and centrifuged for 8 min at 12,500 ×g. An aliquot of the supernatant was extracted and mixed 50:50 with 10 mM ammonium acetate buffer before LC/MS analysis. Quantification analysis (LC/triple-quad) was carried out using an Agilent 1100 HPLC interfaced with a Sciex 3000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex) fitted with an ESI operated in the negative ionization mode. Fipronil sulfone specific transitions used for quantification were 451.1/415, 451.1/281.9, 451.1/243.9. The HPLC system consisted of a Phenomenex Luna C18 column (50 × 3 mm, 5 μm; Torrance, CA, USA) with a Security-guard guard column (Phenomenex). The method consisted of the following: 0.4 mL/min flow rate which increased to 0.75 mL/min at time = 2 min; temperature: 30 °C; mobile phases — A: ammonium acetate buffer (0.2 mM) and DI water:methanol (95:5, v/v), and B: ammonium acetate buffer (0.2 mM) and acetonitrile:DI water (95:5, v/v); gradient: 0–2 min 50% A and 50% B; 2.1–4 min, a linear gradient from 50:50 A:B to 10:90 A:B; 4–6 min 10% A and 90% B; 6.1–10 min re-equilibration to 50% A and 50% B.