| AIM: To develop and validate a rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for simultaneous quantification of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma. Then apply this method in a bioavailability study after an oral administration of a capsule containing 300 mg paracetamol, 25 mg caffeine, 30 mg pseudoephedrine, 2.5 mg chlorpheniramine and 12 mg cloperastine to each of the 22 healthy volunteers.STUDY METHOD: Paracetamol, caffeine, pseudoephedrine, chlorpheniramine, cloperastine and the internal standard (I.S.) diphenhydramine were extracted from human plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Venusil Mp-C18 (4.6×50 mm I.D.,5μm ) column using methanol-10 mM ammonium acetate-formic acid (60:40:0.4 v/v/v). Detection was carried out by multiple reaction monitoring (MRM) on a Q-trapTM LC/MS/MS system with an ESI interface in the positive ion mode. MRM was performed at unit resolution using the mass transition ion-pairs m/z 152.1→m/z 110.1, m/z 195.1→m/z 138.1, m/z 166.2→m/z 148.1, m/z 275.2→m/z 230.1, m/z 330.2→m/z 201.0 and m/z 256.3→m/z 167.1 for analytes and I.S., respectively. The linearity, specificity, precision, accuracy, recovery and stability were estimated in the method of validation for human plasma.The validated method was applied to determine the plasma concentrations of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine from a clinical trial in which 22 healthy male volunteers received an oral dosage (two capsules containing 300 mg paracetamol, 25 mg caffeine, 30 mg pseudoephedrine, 2.5 mg chlorpheniramine and 12 mg cloperastine). Blood samples were collected into heparinized glass tubes before administration and at 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, 24.0, 36.0, 48.0 and 72.0 h post-dose. Plasma was separated immediately by centrifugation at 3500 rpm for 10 min and stored at–20°C prior to analysis.The concentrations of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in health volunteers'plasma samples were measured, respectively. After the plasma concentration–time profile was shown, the pharmacokinetic parameters were derived from these curves. Based on these, the bioavailability of two capsule formulations was estimated by a two one-sided t-test and a 90% confidence intervals of test to reference ratio (after log-transformed) of the AUC0-t and that of Cmax (after log-transformed).RESULTS: A simple, rapid and sensitive LC/MS/MS method has been developed and validated for the determination of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in human plasma. This method was linear in the concentration range of 5.0–2000 ng/ml for paracetamol,10.0–4000 ng/ml for caffeine, 0.25–100 ng/ml for pseudoephedrine , 0.05–20 ng/ml for chlorpheniramine and 0.10–40 ng/ml for chlorpheniramine with the limit of quantitation (LOQ) of 5 ng/ ml, 10 ng/ ml, 0.25 ng/ ml, 0.05 ng/ ml and 0.10 ng/ ml, respectively. In the range of the standard curve, there is no co-eluting endogenous substances significantly influenced the determination of analytes in human plasma. The accuracy was <4.64 % and the intra- and inter-day precision was <11.27%. The sample preparation was simple and cost-effective. The short-term test indicated reliable stability under the experimental conditions of the analytical runs. The results of the freeze/thaw stability test indicated that the analytes were stable in human plasma for three cycles when stored at–20°C and thawed to room temperature. The findings from the long-term test indicated that storage of plasma samples containing the analytes at–20°C was adequate when maintained for 30 days. Thus, no stability-related problems are expected during the routine analyses for our study. Moreover, the devised method fully meets the conformance to relevant standards of Pharmacopoeia of People's Republic of China, and suites for bioequivalence studies of the compound paracetamol caffeine and pseudoephedrine hydrochloride capsules in clinical trials.The method was applied to evaluate the bioequivalence of two capsule formulations of compound paracetamol caffeine and pseudoephedrine hydrochloride in 22 healthy adult male volunteers who received a single dose (containing 300 mg paracetamol, 25 mg caffeine, 30 mg pseudoephedrine, 2.5 mg chlorpheniramine and 12 mg cloperastine) in a two-period randomized crossover design with a one-week washout period between doses. After the concentrations of paracetamol, caffeine, pseudoephedrine, chlorpheniramine and cloperastine in health volunteers'plasma samples were measured, the mean plasma concentration–time curves for two compound paracetamol caffeine and pseudoephedrine hydrochloride capsules were shown, and the pharmacokinetic parameters were derived from these curves. Based on the data of mean AUC0-t, the relative bioavailability of the test formulation was 98.2±11.6 %, 98.6±25.2 %, 113.5±42.4 %, 101.9±34.1 % and 106.9±21.7 %, respectively.There were no significant differences between the two formulations on the basis of assessment by a two one-sided t-test (P>0.05). The 90% confidence intervals of test to reference ratio (after log-transformed) of the AUC0-t (93.68–101.76%) and AUC0-∞(93.71–101.83%) for paracetamol, (86.01–105.44%) and (84.42–106.26%) for caffeine, (97.58–120.59%) and (97.70–120.83%) for pseudoephedrine, (89.08–107.60%) and (89.06–107.64%) for chlorpheniramine, (99.84–110.25%) and (97.97–109.97%) for cloperastine were within the bioequivalence criteria range of 80–125%, and that of Cmax (after log-transformed) (86.66–102.05%) for paracetamol, (83.92–104.29%) for caffeine, (87.40–111.80%) for pseudoephedrine, (87.72–104.14%) for chlorpheniramine, (87.14–102.35%) for cloperastine were within 70–143%. Based on these, the two tablet formulations were found to be bioequivalent. |
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