56 August / September 2019
Figure 2. Thermo Scientific HyperSep Verify cation exchange SPE protocol.
prior to analysis. However, this approach is non-selective and can result in the extraction of other matrix materials, such as urea, hormones and other substances that urine might contain. Furthermore, LLE is not always suitable for the extraction of all analytes and does not involve a concentration step, which can impact measurement sensitivity. In SPE you start with 3 ml sample and end up with 50 µl of sample prior to injection after drying down.
In the workflow reported here, a sample preparation step utilising either a C18 or cation exchange solid phase extraction (SPE) cartridge was used, depending on the substance class to be analysed. To provide the cleanest results possible, silica-based materials were employed, as polymers can release substances that can interfere with the measurement and lead to higher limits of detection (LODs) and limits of quantitation (LOQs) [3].
Figure 1 and Figure 2 highlight the SPE protocols using the C18 and mixed-mode cation exchanger, respectively. Silica-based C18 and mixed-mode cation exchangers were used as these resulted in the cleanest extracts for the GC-MS analysis. The figures show generic protocols, which were employed for the majority of the analytes studied. However, modifications to the method were required in some cases. Additionally, for the horse urine samples, it was necessary to freeze the samples in liquid nitrogen prior to the SPE step. Freezing opens up the cells and gets the urine back in a liquid form. Adding other chemicals to get the urine in a liquid form can reduce the activity of the beta-Glucuronidase.
SPE yields cleaner extracts and can offer a concentration factor of up to 60-fold, resulting in enhanced LODs. As explained above, the starting point is 3000 µl urine and the final sample volume is 50 µl prior to injection. LLE does not provide concentration enhancement, and the
Table 1. GC and GC-MS parameters.
Thermo Scientific TRACE 1310 GC System Parameters Analytical column: Injection volume: Liner: Inlet:
Inlet module and mode: SSL in Split mode Split flow:
18 mL/min
Carrier gas, carrier mode: He, programmed flow 3.66 mL/min (0.3 min), 50 mL/min2 0.843 mL/min (1 min)
Oven Temperature Program Temperature 1: Hold time: Rate:
Temperature 2: Hold time: Rate:
Temperature 3: Hold time: Rate:
Temperature 4: Hold time:
140°C 0 min
40°C/min 180°C 0 min
3°C/min 230°C 0 min
40°C/min 300°C 2 min
Thermo Scientific TSQ 8000 Evo Triple Quadrupole Mass Spectrometer Parameters Acquisition mode: Transfer line:
Ionisation type: Ion source:
Electron energy:
Timed-SRM 300°C EI
320°C 30 eV
to
100% dimethylpolysiloxane (17m, 0.2 mm id, 0.1 µm) 2 µL
Single Taper Liner with Quartz Wool (p/n 453A1925) 280 °C
extracts are not clean, which can lead to higher detection limits when analysed by GC-MS.
After SPE, the samples were evaporated and reconstituted and/or derivatised. Silylation was used for those substances that contained, COOH-, NH- or NH2
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