17
Sample processing procedure: the human serum spiked with analytes was protein precipitated by vortex mixing with the crashing solvent at a 1:3 ratio. Then the mixture was centrifuged at 10,000 x g for 3 min and the resulting supernatant was collected and directly injected for LC/MS analysis.
On-line SPE-LC/MS setup
As shown in Figure 2, the on-line SPE-LC/ MS setup consists of a 6-port switching valve and two pumps; one for sample loading and washing, the other for sample elution. To minimise the potential peak broadening from the cartridges, the flow of sample loading/washing and the subsequent elution are in reversed directions.
On-line SPE-LC/MS: Instrument:
Figure 2: Configuration of the on-line SPE-LC/MS system. Results and Discussion:
Shimadzu LCMS-8030 with 2DLC setup
HPLC column: Ascentis Express Biphenyl 10 cm x 2.1 mm (MilliporeSigma Cat# 64065-U)
Mobile phase: (A) Water; (B) MeOH, each with 0.1% acetic acid 70% B% for 10 min 0.3 mL/min
Isocratic: Flow:
Column temperature:
Sample loading/ washing:
35ºC
0.3 mL/min for 2 min, then the valve switches to in-line with HPLC column, before sample loading the cartridge is equilibrated with the loading solvent for 2.5 min.
Sample
loading solvent: 10% methanol Injection Vol: Detection:
2 µL injection MS, ESI(+), MRM mode
The conventional (off-line) sample preparation by SPE typically involves multiple labour-intensive and time- consuming steps, including: conditioning, sample loading, washing, elution, and finally evaporation and reconstitution of the sample in mobile phase. The on-line cartridges were developed to automate the sample preparation process, minimise hands on time and human error, and reduce overall sample processing time. The present work utilised the C8 and RPA on-line cartridges with LC/MS for the detection of thyroids from in human serum with C8 and RPA on-line cartridges, respectively. The human serum samples were simply protein precipitated with methanol containing ammonium formate and then directly injected for on-line SPE and LC/ MS analysis. The sample loading/washing were carried out entirely by the instrument, without any hands-on effort. Additionally, the time-consuming solvent evaporation and reconstitution steps were eliminated. As can
been seen from Figures 3 and 4, both C8 and RPA were capable of capturing a trace amount (100 ng/mL x 2 µL in this case) of thyroids from complicated human serum. All three thyroids are resolved from each other, with a peak width at half height <6s and tailing factor from 1.4-1.8. These indicate sharp and nice peak shapes with the on-line cartridges. The total run time is within 6 min.
Table 1 and 2 shows the ruggedness of the on-line SPE-LC/MS with C8 and RPA cartridge, respectively, from 120 consecutive injections of the dirty human serum samples. As can be seen, the retention time of the thyroid analytes with C8 or RPA is very reproducible, with RSD’s of 0.1%-0.2. The reproducibility (RSD%) of the peak area of the thyroid analytes with C8 and RPA cartridges is 6.2-7.0% and 5.1%-7.7%, respectively, which indicates great reproducibility.
Figure 3: Representative LC/MS chromatogram of thyroids in human serum with C8 on-line cartridge.
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