Simultaneous determination of tramadol and paracetamol in human plasma using LC-MS/MS and application in bioequivalence study of ­fixed-dose combination

Figures & data

Figure 1. Molecular structures of (a) tramadol, (b) tramadol D6, (c) paracetamol and (d) paracetamol D4.

Table 1. Mass spectrometry (MS/MS) conditions of each analyte and IS.

Analyte	Precursor ion [M + H]^+	Type of ion transition	MRM transition for product ion (m/z)	Q1 pre-bias (V)	Collision energy (V)	Q3 pre-bias (V)
Tramadol	264.14	Quantification	58.15	−12.0	−30.0	−22.0
		Confirmation	42.15	−12.0	−55.0	−14.0
Tramadol D6 Hydrochloride	270.25	Quantification	64.15	−14.0	−15.0	−26.0
Paracetamol	152.10	Quantification	110.20	−30.0	−19.0	−18.0
		Confirmation	65.20	−30.0	−32.0	−24.0
Paracetamol D4	156.15	Quantification	114.10	−11.0	−18.0	−22.0
		Confirmation	69.10	−11.0	−28.0	−27.0

Figure 2. Mass chromatograms of the analysis of (a) blank plasma, (b) zero sample with IS and (c) tramadol and paracetamol at LLOQ level with IS (d) tramadol and paracetamol at ULOQ level with IS.

Figure 3. Mass chromatogram with all analytes and IS.

Figure 4. Product ion spectra of (a) paracetamol, (b) paracetamol D4, (c) tramadol D6 and (d) tramadol.

Table 2. Within-, between- and extended run of precision and accuracy.

		Tramadol QC samples (ng/mL)					Paracetamol QC samples (µg/mL)
Analytes		2.50^a	7.50	125.00	250.00	375.00	0.025^a	0.075	5.00	10.00	15.00
Within-run 1 (n = 6)	Mean	2.68	8.00	138.91	273.69	416.31	0.0263	0.0727	4.97	9.93	14.76
	CV (%)	16.83	3.05	0.90	2.66	1.03	16.35	11.83	2.60	2.93	0.89
	Accuracy (%)	107.14	106.64	111.13	109.47	111.02	105.20	96.93	99.47	99.35	98.39
Within-run 2 (n = 6)	Mean	2.52	7.26	118.84	239.39	355.29	0.0263	0.0710	4.88	9.90	14.76
	CV (%)	12.94	3.64	1.76	1.30	1.20	12.93	8.03	1.22	2.37	1.40
	Accuracy (%)	100.66	96.76	95.07	95.75	94.74	105.20	94.67	97.62	99.00	98.42
Within-run 3 (n = 6)	Mean	2.80	7.22	119.76	239.49	354.95	0.0277	0.0722	4.84	9.74	14.51
	CV (%)	10.79	5.29	2.08	2.01	0.83	16.25	7.76	1.73	2.24	2.17
	Accuracy (%)	111.89	96.25	95.81	95.80	94.65	110.80	96.27	96.73	97.41	96.76
Between-run (n = 18)	Mean	2.66	7.49	125.84	250.85	375.51	0.0268	0.0720	4.90	9.86	14.68
	CV (%)	13.63	6.22	7.72	6.92	7.96	14.55	8.89	2.19	2.54	1.69
	Accuracy (%)	106.56	99.85	100.67	100.34	100.13	107.20	96.00	97.94	98.59	97.85
Extended-run (n = 25)	Mean	–	7.11	122.98	245.51	379.31	–	0.0758	4.95	9.91	15.21
	CV (%)	–	5.85	1.93	1.30	1.56	–	9.76	1.48	1.66	1.51
	Accuracy (%)	–	94.76	98.38	98.20	101.15	–	101.07	98.99	99.10	101.43

^aLLOQ QCs at 2.50 and 0.025 ng/mL for tramadol and paracetamol (the acceptance criteria for CV (%) and accuracy were ≤20% and within ±20% from nominals). CV: coefficient of variation; n: number of replicates.

Table 3. Re-injection reproducibility results.

Analyte	Nominal concentration	Initial injection	Re-injection reproducibility		Re-injection reproducibility
			(First re-injection)		(Second re-injection)
		Mean initial calculated concentration^a	Mean calculated concentration	Difference from initial (%)^a	Mean calculated concentration^a	Difference from initial (%)^a
Tramadol (ng/mL)	2.50	2.49	2.50	−0.40	2.90	−16.44
	7.50	7.31	7.73	−5.73	7.46	−2.06
	125.00	124.10	121.27	2.28	122.61	1.20
	250.00	255.08	246.69	3.29	252.79	0.90
	375.00	363.08	361.84	0.34	356.89	1.71
	500.00	496.99	496.43	0.11	501.55	−0.92
Paracetamol (µg/mL)	0.025	0.0253	0.0250	1.19	0.0236	6.72
	0.075	0.0705	0.0706	−0.14	0.0743	−5.39
	5.00	4.91	5.02	−2.21	4.95	−0.71
	10.00	10.27	10.41	−1.38	10.19	0.79
	15.00	14.72	14.99	−1.88	14.54	1.24
	20.00	20.37	20.84	−2.29	19.88	2.43

^aCalculated as [(mean initial calculated concentration − mean calculated re-injection concentration)/mean initial calculated concentration] × 100%.

Table 4. Stability study results of tramadol, paracetamol and IS under different conditions (n = 3).

Analyte	Tramadol	Paracetamol
Short-term stability; 25 ± 4 °C, 24 h
LQC	110.72 ± 1.43	90.44 ± 12.93
HQC	101.29 ± 2.17	99.38 ± 1.88
Post-preparative stability in autosampler; 25 ± 3 °C, 48 h
LQC	90.15 ± 5.27	91.73 ± 11.57
HQC	98.07 ± 2.80	99.27 ± 0.25
Post-preparative stability at room temperature; 25 ± 4 °C, 48 h
LQC	93.11 ± 8.89	94.93 ± 6.02
HQC	99.97 ± 1.48	100.11 ± 3.83
Freeze and thaw stability; −20 ± 10 °C, 7 cycles
LQC	111.26 ± 10.85	102.22 ± 13.40
HQC	107.90 ± 1.71	106.53 ± 1.53
Long term plasma stability; −20 ± 10 °C, 74 days
LQC	99.48 ± 6.15	108.71 ± 15.40
HQC	108.02 ± 3.76	111.25 ± 4.46
Working solution stability (analyte); 25 ± 4 °C, 74 days
LLOQ	102.00 ± 1.65	101.19 ± 2.16
ULOQ	105.32 ± 0.52	107.20 ± 1.83
Working solution stability (analyte); 5 ± 3 °C, 74 days
LLOQ	94.36 ± 7.20	97.84 ± 3.87
ULOQ	103.77 ± 1.52	104. 04 ± 0.93
Stock solution stability (analyte); 25 ± 4 °C, 74 days
LLOQ	101.76 ± 3.59	94.70 ± 2.19
ULOQ	105.51 ± 0.76	107.55 ± 2.45
Stock solution stability (analyte); 5 ± 3 °C, 74 days
LLOQ	98.64 ± 1.04	95.76 ± 3.95
ULOQ	106.41 ± 0.35	105.52 ± 1.02
Working solution stability (IS); 25 ± 4 °C, 74 days	97.75 ± 0.68	95.74 ± 0.37
Working solution stability (IS); 5 ± 3 °C, 74 days	101.42 ± 0.08	95.53 ± 0.78
Stock solution stability (IS); 25 ± 4 °C, 74 days	98.45 ± 1.02	97.47 ± 1.84
Stock solution stability (IS); 5 ± 3 °C, 74 days	98.82 ± 0.68	96.71 ± 1.26

LLOQ: lower limit of quantification; LQC: low-quality control; HQC: high-quality control; ULOQ: upper limit of quantification; n: number of replicates.

Figure 5. The mean plasma of (a) tramadol and (b) paracetamol concentration-time profiles of Ultracet tablet and Tramicet F.C. tablet.

Table 5. Pharmacokinetic parameters of tramadol and paracetamol following oral administration of tramadol and paracetamol (37.5/325 mg × 2) tablet formulations (test and reference) in 23 healthy volunteers under fasting conditions.

Parameter	Tramadol		Paracetamol
	Test	Reference	Test	Reference
Cmax
Mean ± SD	283.17 ± 51.96 ng/mL	271.94 ± 52.93 ng/mL	8.34 ± 2.64 µg/mL	7.98 ± 1.80 µg/mL
Range	217.56–421.10 ng/mL	168.94–424.67 ng/mL	5.60–11.63 µg/mL	5.08–16.83 µg/mL
Tmax
Mean ± SD	2.04 ± 0.60 h	2.09 ± 0.58 h	1.11 ± 0.67 h	1.05 ± 0.59 h
Range	1.00–4.00 h	1.00–3.00 h	0.50–3.00 h	0.50–2.50 h
t1/2
Mean ± SD	6.59 ± 1.09 h	6.43 ± 1.12 h	3.62 ± 0.70 h	3.74 ± 0.64 h
Range	5.10–9.62 h	5.06–9.63 h	2.42–6.04 h	2.85–5.43 h
ke
Mean ± SD	0.11 ± 0.02 h^−1	0.11 ± 0.02 h^−1	0.20 ± 0.04 h^−1	0.19 ± 0.03 h^−1
Range	0.07–0.14 h^−1	0.07–0.14 h^−1	0.12–0.29 h^−1	0.13–0.24 h^−1
MRT
Mean ± SD	10.38 ± 1.73 h	10.23 ± 1.73 h	5.00 ± 0.79 h	4.97 ± 0.75 h
Range	8.21–15.44 h	8.21–15.44 h	3.40–6.17 h	3.40–6.17 h
AUC0–24
Mean ± SD	2678.76 ± 665.44 h.ng/mL	2572.61 ± 665.48 h.ng/mL	35.70 ± 8.41 h.µg/mL	35.16 ± 8.65 h.µg/mL
Range	1871.21–4098.17 h.ng/mL	1628.78–4268.63 h.ng/mL	23.48–63.45 h.µg/mL	25.56–46.16 h.µg/mL
AUC0–∞
Mean ± SD	2974.79 ± 836.79 h.ng/mL	2846.57 ± 823.92 h.ng/mL	36.31 ± 8.73 h.µg/mL	35.74 ± 8.93 h.µg/mL
Range	1958.82–4806.82 h.ng/mL	1762.10–4818.04 h.ng/mL	23.77–64.98 h.µg/mL	25.79–47.04 h.µg/mL

Table 6. Comparison between current method and previously published method for simultaneous quantification of tramadol and paracetamol in human plasma.

Reference	Analyte	Calibration range	Column	Mobile phase	Internal standard	Flow rate	Run time	Application
Present method	Tramadol Paracetamol	2.50–500.00 ng/mL 0.025–20.00 µg/mL	Poroshell 120 EC-C18 Fast LC column	Methanol:water:formic acid (30:70:0.1, v/v/v)	Tramadol D6 Paracetamol D4	440 µL/min	3.5 min	Bioequivalence study
Zhu et al. [39]	Tramadol Paracetamol	5–600 ng/mL 0.03–16 µg/mL	Hanbon LiChrospher CN column	10 mM ammonium acetate buffer with 0.5% formic acid:methanol (40:60, v/v)	Phenacetinum	1000 µL/min	5 min	Pharmacokinetic study

Zhu T, Ding L, Guo XF, et al. Simultaneous determination of tramadol and acetaminophen in human plasma by LC-ESI-MS. Chroma. 2007;66(3–4):171–178. doi: 10.1365/s10337-007-0268-9.

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