3.2 Method validation
This method performed stable baseline in analytical channel (m/z
586.3→513.2) and IS channel (595.4→514.2). The selectivity was
acceptable since there were no interference peak in these channels. The
total run time was 2.5 min, which was shorter than most methods to
analyze TGC in bio-samples [10, 11]. Respectively,
the retention time of TGC in serum samples and in CSF samples was
1.464±0.005 min and 1.466±0.005 min with five continuously reduplicate
injections. The chromatograms obtained from blank serum and CSF, blank
bio-samples spiked with TGC and IS, and the samples from patients were
described in Figure 1. The serum and CSF samples spiked with TGC at
lower limit of quantification (LLOQ, 5 ng/mL) also depicted unique and
prominent peak at retention time.
Calibration curves were Y=0.019X+0.182 for serum samples (5-2000 ng/mL)
and Y=0.0322X+0.53 for CSF (5-2000ng/mL) (a weighting of 1/x was used).
Linear coefficients (r2) were 0.9999 and 0.9997
respectively, which could absolutely achieve accurate samples analysis.
Precision and accuracy data of TGC analysis in serum and CSF samples
were tabulated in Table 2. At three QC concentrations of TGC in serum,
the accuracy bias was ranged from -4.69% to 3.00%, with the intra-day
and inter-day RSD ranged from 2.85% to 7.86%. For the CSF samples at
concentration of 10.0 ng/mL, 400 ng/mL and 800 ng/mL, the accuracy
expressed in terms of percentage bias was within -1.85% to 6.40%, and
the precision varied from 3.06% to 13.72%. The data of matrix effect
was shown in Table 3. The IS normalized matrix factors (MFs) for TGC
ranged from 79.04% to 109.64% at all levels, and the RSD ranged from
4.93% to 9.43%. All of the data met the requirement of bio-samples
quantification.
The results utilized to evaluate the stability of the method were listed
in Table 4. There was no significant change of analyte in varied storage
conditions within the prescribed time. The accuracy bias of TGC in
bio-samples at low, medium and high concentration was ranged from
-9.30% to 3.92%, and the RSD was ranged from 2.18% to 8.33%.
Simultaneously, the variations of CSF and serum samples spiked with
standard solution in auto-sampler for 90 min were acceptable. And the
stability of TGC also not affected by three cycles of freezing and
thawing significantly.