Clinical Laboratory needed for Gas Chromatography of Human Urine Samples.
The protocol using Agilent equipment is below. We do not require the exact same equipment - we do require the data to be standardized at the output stage. We are open to suggestions on protocol improvements and modifications.
1. Derivatization of urine samples
a. deplete excess urea with urease - Incubate with urease for 30 min
b. extract with (add) ethanol and/or methanol - mix, centrifuge and collect supernatant and dry under nitrogen or freeze dry
c. derivatize with MOX and MSTFA - add methoxamine hydrochloride and incubate (up to 2 hours), derivatize using MSRFA with 1% TMCS (for 30 to 60 minutes)
Use helium as the carrier gas at 1.5 ml min- 1 and set the injector split ratio to 1:20. Maintain the injector, transfer line and ion source temperature at 220 °C, 200 °C and 250 °C, respectively, throughout each analysis. Program oven temperature at 70 °C for 0.2 min, increase at 10 °C min- 1 to 270 °C and hold at 270 °C for 10 min. Operate the MS EI mode (70 eV). Perform data acquisition in the full scan mode from m/z 40 to 600 with a solvent cutoff of 360 s and acquisition rate of 20 Hz. Set manual mass defect to 0 and detector voltage to between 1,700 and 1,800 V, depending on the detector's age and condition. Perform sample injections using an autosampler. Perform three and five cycles of syringe washes before and after sample injection, respectively, using toluene as the wash solvent.
A GC/TOFMS instrument equipped with an Agilent 7890 GC, Pegasus IV TOFMS (LecoCorp) and CTC CombiPAL autosampler
A DB-1 (Agilent J&W Scientific; cat. no. 122-1032G) capillary column with activated fused silica column (30 m × 250 µm (i.d.) × 0.25 µm) or an equivalent, such as the Rtx-1 (Restek Corp.) capillary column
ChromaTOF software (version 4.21, LecoCorp.) for data pre-processing such as baseline correction, smoothing, noise reduction, deconvolution, library matching and peak area calculation
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