Validated determination method of tramadol and its desmethylates in human plasma using an isocratic LC-MS/MS and its clinical application to patients with cancer pain or non-cancer pain
Separation and selectivity
Figure 2 shows the LC-MS/MS chromatograms of tramadol, ODT, NDT, NODT, and IS in human plasma.
No peaks interfering with tramadol, ODT, NDT, NODT, or IS in six independent drug-free
plasma specimens in cancer and non-cancer patients were observed (Fig. 2a). Tramadol, ODT, NDT, NODT, and IS were eluted at 6.1, 3.4, 7.4, 3.9, and 6.0 min,
respectively, with a total run time of 10 min (Fig. 2b). In addition, no peaks interfering with detection in tramadol non-treated patients
with cancer pain or non-cancer pain were observed (Fig. 2c and d).
Fig. 2. MS/MS chromatograms of human drug-free plasma (a), human drug-free plasma spiked with 200 ng/mL tramadol, 40 ng/mL O-desmethylate, 40 ng/mL?N-desmethylate, and 40 ng/mL?N,O-didesmethylate, (b) a plasma specimen at 8 h after evening dosing in cancer pain (c) and non-cancer pain (d) patients treated with oral tramadol. (1) Tramadol, (2) O-desmethylate, (3) N-desmethylate, (4) N,O-didesmethylate, and (5) tramadol-d6 as internal standard
Calibration curve, sensitivity, recovery, and matrix effect
The calibration curves of tramadol, ODT, NDT, and NODT in human plasma were linear
over the concentration ranges of 12.5–1600, 2.5–320, 2.5–320, and 2.5–320 ng/mL, respectively.
Their correlation coefficients were greater than 0.999. The LLOQ of tramadol, ODT,
NDT, and NODT in human plasma were 12.5, 2.5, 2.5, and 2.5 ng/mL, respectively (n?=?6).
The pretreatment recoveries including deproteinization of tramadol, ODT, NDT, and
NODT were mean?±?standard deviation (SD), 86.0?±?3.4 %, 85.5?±?1.8 %, 106.3?±?2.9 %,
and 93.9?±?0.8 %, respectively. The analytes and IS did not exhibit any matrix effects
in human plasma (mean?±?SD, 88.3?±?4.1 % for tramadol, 89.9?±?5.6 % for ODT, 105.1?±?2.7 %
for NDT, 98.8?±?6.4 % for NODT, and 91.7?±?4.9 % for IS, n?=?5).
Assay accuracy and precision in human plasma
Table 2 shows the intra- and inter-assay accuracies and precisions in human plasma. The intra-assay
and inter-assay accuracies of tramadol, ODT, NDT, and NODT were 102.0–106.2 % and
95.1–103.4 %, 93.4–102.0 % and 94.9–100.8 %, 89.2–105.2 % and 92.7–101.6 %, and 92.5–102.5 %
and 97.5–99.8 %, respectively. The intra-assay and inter-assay precisions of tramadol,
ODT, NDT, and NODT were 1.6–8.2 % and 4.6–6.3 %, 3.6–4.8 % and 2.7–5.1 %, 3.4–7.9 %
and 3.2–6.3 %, and 6.2–8.7 % and 4.2–10.2 %, respectively.
Table 1. Patient characteristics
Table 2. Intra- and inter-assay precisions and accuracies of tramadol and its desmethylates
in human plasma
Stability tests
The stock solutions of tramadol, ODT, NDT, NODT, and IS were stable at 4 °C (% of
initial value, 88.3–99.1 %) for up to 3 months. Tramadol, ODT, NDT, and NODT in plasma
specimens were stable at room temperature (% of initial value, 88.1–113.3 %) for up
to 24 h. Tramadol, ODT, NDT, and NODT in plasma specimens were stable at ?80 °C (%
of initial value, 89.9–111.8 %) for up to 1 month. Tramadol, ODT, NDT, NODT, and IS
in injection solutions were stable at 4 °C (% of initial value, 92.6–104.6 %) for
up to 24 h.
Plasma concentrations of tramadol and its desmethylates
Figure 3 shows the plasma concentrations of tramadol and its desmethylates in cancer pain
and non-cancer pain patients treated with oral tramadol. The plasma concentrations
of tramadol, ODT, NDT, and NODT in patients with cancer pain ranged from 18.2 to 564,
11.8 to 137, 4.9 to 250, and 6.1 to 147 ng/mL, respectively. In patients with non-cancer
pain, the plasma concentrations of tramadol, ODT, NDT, and NODT ranged from 32.8 to
670, 7.0 to 84.8, 5.1 to 317, and 6.7 to 85.2 ng/mL, respectively. The plasma concentration
ranges of tramadol and its desmethylates were measurable within their calibration
curves in cancer and non-cancer patients.
Fig. 3. Plasma concentrations of tramadol, O-desmethylate, N-desmethylate, and N,O-didesmethylate obtained from patients with cancer pain (a) or non-cancer pain (b) just before treatment of oral tramadol on day 4 or later. (1) Tramadol, (2) O-desmethylate, (3) N-desmethylate, and (4) N,O-didesmethylate
Variations in plasma exposure and metabolic ratio
The median and interquartile range (IQR) of dose-adjusted plasma concentrations for
tramadol, ODT, NDT, and NODT were 73.6 and 33.4–88.2, 14.8 and 9.1–34.0, 14.0 and
7.9–26.1, and 12.3 and 7.5–18.8 ng/mL per mg/kg in patients with cancer pain, respectively.
In patients with non-cancer pain, the median dose-adjusted plasma concentrations of
tramadol, ODT, NDT, and NODT were 122 (IQR, 96.2–180), 19.2 (10.3–20.7), 29.1 (10.6–78.5),
and 10.4 (5.2–20.7) ng/mL per mg/kg, respectively. The median metabolic ratios to
ODT, NDT, and NODT were 0.30 (IQR, 0.22–0.36), 0.25 (0.15–0.43), and 0.23 (0.12–0.32)
in patients with cancer pain, respectively. In patients with non-cancer pain, the
median metabolic ratios to ODT, NDT, and NODT were 0.15 (IQR, 0.09–0.22), 0.19 (0.13–0.51),
and 0.09 (0.06–0.19), respectively.