Supplementary Information

Method validation

Preparation of standard stock and working solution

Primary solutions of warfarin prepared in methanol at the concentration of 1 mg·mL -1. Stock
solutions of warfarin at the concentrations of 200 and 20 μg·mL -1were diluted from the primary
solution with methanol, and further diluted to working solutions(0.05, 0.10, 0.20, 0.50, 1.50, 3.00
and 6.00 μg·mL-1) were diluted from the stock solution with methanol. The stock solution of
Gliclazide (IS) was prepared in methanol at the concentration of 200 μg·mL -1and the working
solution (1.0 μg·mL-1) was diluted from the stock solution with methanol. All the standard
solutions were stored at 4 °C without direct light.

Specificity

Full-scan mass spectrum of warfarin (a) and IS (b) was shown in the Figure S2. Blank plasma
from six volunteers (Figure S3-A), Warfarin and IS standard solution (Figure S3-B,C); blank
plasma spiked with warfarin at (Figure S3-D), and plasma sample from No.14 volunteer at 72
after 2.5mg warfarin (Figure S3-E) was extracted using the above liquid-liquid extraction
procedure. The supernatants of the six different plasma samples were analyzed for 30 min to
investigate whether the impurity in blood influenced the next two or three samples. The overall
run time was 11 min. The retention times of warfarin and IS were around 6.10 and 7.59 min,
respectively. No chromatographic peaks interfering with quantitation were observed under the
above conditions.

Matrix effect and extraction recovery

The matrix effect and extraction recovery of warfarin and IS were evaluated with five replicates
of the QC samples at three concentration levels (10, 50, and 500 ng·mL-1) and five replicates of
the IS samples (1.0 μg·mL-1). The average matrix effect ranged from 93.5 to 98.6% for warfarin
and 96.3% for the IS. Thus, ionization enhancement or suppression is negligible under the
current conditions.

The mean extraction recoveries of warfarin ranged from 60.3 to 62.7% and that of IS was 61.9%,
suggesting that the present liquid-liquid extraction method is appropriate and reliable with good
reproducibility (Table S1).

Stability

The stabilities of warfarin in the biological matrix were evaluated as follows and the results were
expressed as percentage recoveries (Table S3). The stabilities of warfarin in plasma sample s at
different concentrations were examined under different study conditions, i.e. storing the samples
at -20°C for 1, 28 and 63 days. Freeze-thaw stability was determined after freezing (-20 °C) and
thawing (25°C) QC samples for two cycles. The stability of post-extracted samples on the bench
top and within the HPLC-MS auto-sampler was also observed for 12 and 24 h at room
temperature.

Table S1. The matrix effect and extraction recovery(n = 5)

Compoun Concentration Matrix effect(%) Extract recovery(%)

d (ng/mL) Mean±SD RSD Mean±SD RSD

10 98.6±12.2 12.4 62.4±4.1 6.7

Warfarin 50 100.2±5.7 5.7 60.3±2.8 4.6

500 93.5±2.0 2.2 62.7±4.4 7.1

IS IS 96.3±5.3 5.5 61.9±5.4 8.7

Table S2. The intra-day and inter-day precision and accuracy (n = 5).

Intra-day Inter-day
concentration
(ng/mL) Accuracy(%
Mean±SD RSD(%) Accuracy(%) Mean±SD RSD(%)
)

10 10.01±0.2 1.7 100.1 10.03±0.5 4.6 100.3

50 45.98±0.7 1.4 92.0 48.78±3.4 7.1 97.6

500 460.04±4.6 2.2 92.0 489.46±28.1 5.7 97.9

 Table S3. Stabilities under different conditions (n = 5).

10 ng·mL-1 50 ng·mL-1 500 ng·mL-1

Conditions Mean±S RSD(% Mean±S RSD(% RSD(%
Mean±SD
D ) D ) )

10.62±0. 48.46±4. 502.41±31.

25°C/6h 4.0 9.8 6.3
4 7 4
49.75±2. 500.55±28.
-20°C/1d 9.90±0.2 1.7 4.2 5.7
1 6
10.11±0. 48.25±2.
-20°C/7d 4.9 4.3 501.63±8.6 1.7
5 1
10.29±0. 50.86±1. 516.33±33.
-20°C/28d 4.9 1.9 6.4
5 0 2
10.36±0. 49.03±2. 495.63±36.
-20°C/63d 6.5 5.5 7.3
7 7 0
-20°C/1 freeze-thaw 10.25±0. 49.38±1. 494.90±46.
7.0 2.4 9.5
cycle 7 2 9
-20°C/2 freeze-thaw 10.49±0. 50.63±3. 482.83±23.
5.0 7.0 4.9
cycle 5 5 7
25°C/12h (extracted 10.35±0. 49.38±1. 494.90±46.
7.0 2.4 9.5
samples) 7 2 9
25°C/24h (extracted 10.50±0. 50.63±3. 482.83±23.
5.0 7.0 4.9
samples) 5 5 7
 Figure S1. The chemical structure of Warfarin

Figure S2. Full-scan mass spectrum of warfarin (a) and IS (b).

Figure S3. Typical MRM chromatograms blank human plasma sample (A); warfarin and IS standard
solution (B,C); blank human plasma spiked with warfarin and IS (D); sample obtained from No.14
volunteer at 72h after administration of 2.5 mg warfarin.
Figure legends for supplement figures:

Figure S1. The chemical structure of Warfarin.

Figure S2. Full-scan mass spectrum of warfarin (a) and IS (b).

Figure S3. Typical MRM chromatograms blank human plasma sample (A); warfarin and IS standard
solution (B,C); blank human plasma spiked with warfarin and IS (D); sample obtained from No.14
volunteer at 72h after administration of 2.5 mg warfarin.