Determination of Tramadol in Urine using LC-MS/MS

Dr Patrik Appelblad, Senior Principal Scientist, Advanced Analytical R&D, Merck Life Science

Introduction: Analysis of Tramadol and N-desmethyl-tramadol in Human Urine

Tramadol is a weak µ-opioid receptor agonist and inhibitor of serotonin/noradrenalin re-uptake used for pain treatment and other indications such as treatment for restless leg syndrome and fibromyalgia. Tramadol is structurally similar to the natural opiate codeine. Tramadol is reported to have a lower risk of developing dependence than codeine. In addition, tramadol is extensively metabolized, where a total of 24 metabolites have been identified. This application presents an LC-MS/MS method for the analysis of tramadol in urine using reversed-phase LC-MS/MS aided by isotope internal standards.

Figure 1 Chemical structures of Tramadol and N-desmethyl-tramadol

The experimental conditions for Tramadol and N-desmethyl-tramadol analysis in urine using reversed-phase LC-MS/MS is given in Table 1, LC gradient conditions in Table 2, and performance data in Table 3.

Table 1. LC-MS/MS experimental conditions for Tramadol and N-desmethyl-tramadol analysis

Column	Purospher® STAR RP-18 endcapped (2 µm) Hibar® HR 50-2.1 mm ID	Injection volume	5 µL
Detection	LC-MS/MS ESI; MRM transitions: m/z 264/58 (Tramadol) and m/z 250/44 (N-desmethyl-tramadol)	Flow rate	See table
Mobile Phase	A: 0.1% formic acid in Milli-Q® water B: 0.1% formic acid in acetonitrile	Temperature	Column: 40 °C
Gradient:	See table	Pressure	170 bar (2448 psi) at start of gradient
Diluent	Mobile Phase A:B (50:50)
Sample	Urine samples treated according to in-house sample preparation protocol (not disclosed)

Table 2. LC gradient conditions

Time (min)	A (%)	B (%)	Flow rate (mL/min)
0.00	95	5	0.40
0.20	95	5	0.40
2.00	50	50	0.40
2.50	10	90	0.40
2.80	10	90	0.40
3.00	95	5	0.50
4.50	95	5	0.50

Chromatograms for Tramadol and N-desmethyl-tramadol, and corresponding internal standards, are shown in Figure 2.

Figure 2 Chromatograms for Tramadol (left) and N-desmethyl-tramadol (right), and corresponding internal standards.

Table 3. LC and MS/MS performance data for Tramadol and N-desmethyl-tramadol

	Compound	Retention Time (min)	Precursor ion (m/z)	Product ions (m/z)
	Void volume	0.2	-
1	Tramadol	2.07	264.0	58.0
2	N-desmethyl-tramadol	2.09	254.0	44.0.2

Conclusions

Human patient samples were analyzed along with standards and control samples. The linear range was found to be 25-1500 ng/mL.