Clinica Chimica Acta 382 (2007) 20 – 24

www.elsevier.com/locate/clinchim

Simultaneous determination and pharmacokinetic study of roxithromycin and

ambroxol hydrochloride in human plasma by LC-MS/MS
Tai-jun Hang a,⁎, Meng Zhang a , Min Song a , Jian-ping Shen b , Yin-di Zhang b
a
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, PR China
b
Institute of Clinical Pharmacology, Nanjing Medical University, Nanjing 210009, PR China
Received 28 October 2006; received in revised form 27 February 2007; accepted 9 March 2007
Available online 24 March 2007

Abstract

Background: Although roxithromycin and ambroxol HCl were often administered concomitantly for the treatment of respiratory infections, the
pharmacokinetic interactions between them have not been reported. We investigated the interactions between these drugs in health male Chinese
volunteers by LC-MS/MS in human plasma.
Methods: The pharmacokinetics were studied in 12 healthy male Chinese volunteers after an overnight fast by a single oral dose, 4-way crossover
design with a period of 7-day washout. Each subjects was randomized to receive a single oral dose of 1 compound roxithromycin (150 mg) and
ambroxol HCl (30 mg) dispersible tablet (test formulation, treatment A), one 150 mg roxithromycin dispersible tablet together with one 30 mg
ambroxol HCl tablet (combined reference formulations, treatment B), one 150 mg roxithromycin dispersible tablet (reference formulation I,
treatment C), or one 30 mg ambroxol HCl tablet (reference formulation II, treatment D) with 250 ml of water. Venous blood was collected at pre-
dose (0 h) and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 48, 72 h after dosing. The plasma concentrations of roxithromycin and ambroxol HCl
were simultaneously determined by using a validated internal standard LC-MS/MS method.
Results: No significant differences were observed for the major pharmacokinetic parameters such as Cmax, Tmax, t1/2 and AUC of both
roxithromycin and ambroxol HCl between different treatments.
Conclusion: The pharmacokinetics of both roxithromycin and ambroxol HCl are not affected by their concomitant oral administration. Therefore,
there are no obvious pharmacokinetic interactions between roxithromycin and ambroxol HCl after oral administration. Roxithromycin and
ambroxol HCl dispersible tablets were bioequivalent with reference to the roxithromycin dispersible tablets and ambroxol HCl tablets in
combination usage.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Roxithromycin; Ambroxol HCl; Pharmacokinetics; LC-MS/MS

1. Introduction The concomitant usage of roxithromycin and ambroxol

HCl is often needed in the treatment of respiratory infections
Roxithromycin [1,2] is a semisynthetic macrolide antibiotic [5,6]. But the pharmacokinetic interactions between them
derived from erythromycin. It is more effective than other have not been reported up until now. Therefore, the
macrolides for a wide range of infections and has been clinically pharmacokinetics and bio-equivalency (BE) of the tested
used for the treatment of respiratory infections. Ambroxol HCl roxithromycin and ambroxol HCl compound formulation and
[3,4], an active metabolite of bromhexine, is used as an two single component reference formulations were investi-
expectorant and bronchodecretolytic in the treatment of gated in this study by a single oral dose, 4-way crossover
bronchial asthma and chronic bronchitis. Ambroxol is also design with a period of 7-day washout in 12 healthy male
used in pulmonary alveolar proteinosis and infant respiratory Chinese volunteers after an overnight fast.
distress syndrome. Although several methods such as HPLC and LC-MS have
been reported for the determination of roxithromycin [7–11] or
⁎ Corresponding author. Fax: +86 25 83271090. ambroxol HCl [12–14] in body fluids, no reference has been
E-mail address: [email protected] (T. Hang). found for the simultaneous determination of them in human
0009-8981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2007.03.015
 T. Hang et al. / Clinica Chimica Acta 382 (2007) 20–24 21

plasma. A validated LC-MS/MS method was established for the 2.3. Determination of roxithromycin and ambroxol HCl concentration
simultaneous determination of roxithromycin and ambroxol in plasma
HCl in human plasma for this study.
A validated LC-MS/MS method was established for the simultaneous
determination of roxithromycin and ambroxol HCl in human plasma. Briefly, to
2. Materials and methods an aliquot of 0.2 ml plasma sample in a 1.5 ml polypropylene tube, 50 μl of
internal standard solution (4 μg/ml erythromycin in methanol) and 0.4 ml of
2.1. Chemicals and reagents acetonitrile were added and vortex-mixed for 1 min, followed by centrifugation
at 12,500 ×g force for 10 min. The top limpid layer was then transferred to an
Compound roxithromycin and ambroxol HCl dispersible tablets (150 mg auto-sampler vial for LC-MS/MS analysis.
roxithromycin and 30 mg ambroxol HCl) were from Jiangsu YaBang AiPuSeng The same sample handling process was used for the determination of
Pharmaceutical Co. Ltd. (Changzhou, PR China). Roxithromycin dispersible linearity, recovery, precision and accuracy in a concentration range from 0.01 to
tablets (150 mg roxithromycin) were from Jiangsu HengRui Medicine Co. Ltd.
20 μg/ml for roxithromycin and from 2 to 200 ng/ml for ambroxol HCl, where
(Lianyungang, PR China). Ambroxol HCl tablets (30 mg ambroxol HCl) were the control plasma sample was prepared by mixing aliquot of 0.2 ml blank
from Changzhou SiYao Pharmaceutical Co. Ltd. (Changzhou, PR China). plasma with 50 μl standard solution in methanol containing a proper amount of
Chemical reference substances of roxithromycin, ambroxol HCl and erythro- both roxithromycin and ambroxol HCl.
mycin (an internal standard for LC-MS/MS determination) were from the
The LC-MS/MS system consisted of a Surveyor LC pump, a Surveyor auto-
National Institute for the Control of Pharmaceutical and Biological Products sampler, a TSQ Quantum Ultra AM triple-quadrupole tandem mass spectrometer
(Beijing, PR China). HPLC grade methanol and acetonitrile were purchased with an ion max source, and the Xcalibur 1.1 software for data acquisition and
from Tedia Company Inc. (Fairfield, OH). Ammonium acetate was from
analysis (Thermo Finnigan, San Jose, CA). HPLC separation was performed on a
Nanjing Chemical Reagent (Nanjing, PR China). Water used for all procedures
Phenomenex-C18 analytical column (150 mm× 4.6 mm, 5 μm) maintained at 30 °C
and for the preparation of all solvents and reagents was prepared with double with a mixture of methanol and ammonium acetate (0.2%) (68:32, v/v) as a mobile
distillation. phase which was delivered at 1.0 ml/min. Thirty percent of the eluent was split into
the inlet of the MS spectrometer using an electrospray ionization (ESI) source.
2.2. Subjects and study design The TSQ Quantum parameters were optimized and set as following: positive
electrospray ionization with spray voltage of 5000 V, capillary temperature of
Twelve healthy male Chinese volunteers, ranging in age from 20 to 25 years 350 °C, nitrogen sheath gas pressure of 35 psi, auxiliary gas pressure of 5 psi,
(22.9 ± 1.4 years), in weight from 60 to 74 kg (65.9 ± 4.4 kg), and in height from argon collision gas pressure of 1.5 mTorr and collision energy of 25 eV. Typical
167 to 180 cm (173 ± 4 cm) were recruited. All subjects gave their written product ion scan spectra for the analytes and the internal standard are shown in
consent for their participation in the study after having been informed all aspects Fig. 1. Quantifications were performed by using selected reaction monitoring
of the study, especially the potential risks. The study protocols were approved by (SRM) with ion transitions of m/z 837.4 to 679.4 for roxithromycin, m/z 378.9
the relevant Ethical Review Committee, in accordance with the principles of the to 263.8 for ambroxol HCl, and m/z 734.4 to 576.2 for erythromycin.
Declaration of Helsinki and the recommendations of the State Food and Drug The roxithromycin and ambroxol HCl calibration curves were created by
Administration of China. plotting the peak area ratios of the corresponding analyte to erythromycin versus
The study was carried out with a single oral dose, four-way crossover the corresponding analyte concentrations.
design with a period of 7-day washout. Subjects were randomized and after an Inter- and intra-day precision for the assay were characterized by the
overnight fast each received a single oral dose of one compound roxithromycin performances of 3 levels of quality control samples run at 3 different days each
(150 mg) and ambroxol HCl (30 mg) dispersible tablet (test formulation, in 5 replicates. The concentrations of quality control samples for roxithromycin
treatment A), one 150 mg roxithromycin dispersible tablet together with one at low, medium and high levels were 0.02, 5 and 20 μg/ml, respectively; those
30 mg ambroxol HCl tablet (combined reference formulations, treatment B), for ambroxol HCl were 5, 50 and 200 ng/ml, respectively. The accuracy of the
one roxithromycin (150 mg) dispersible tablet (reference formulation I, method was expressed using relative error (RE%) and the precision was denoted
treatment C), or one ambroxol HCl (30 mg) tablet (reference formulation II, by the coefficient of variance (%CV). The absolute recoveries of roxithromycin
treatment D) with 250 ml of water. Subjects fasted overnight before and 3 h and ambroxol HCl were also determined at 3 quality control levels by comparing
after drug administration. Venous blood samples of 3.5 ml were collected in the peak areas obtained from plasma samples with those from standard solutions
heparinized tubes at pre-dose (0 h) and 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, of the same concentrations. Long-term storage stability and freeze–thaw
48, 72 h after dosing. Plasma samples were separated immediately by stability of roxithromycin and ambroxol HCl plasma samples were investigated
centrifugation at 1500 ×g for 10 min and stored at − 20 °C until analysis at intermediate concentration of 5 μg/ml and 50 ng/ml, respectively.
(Table 1).
2.4. Pharmacokinetic analysis

The pharmacokinetic parameters were calculated via the noncompartmental

Table 1
method, with the aid of the DAS program (ver. 2.1, The Chinese Mathematical
Drug administration schedule
Pharmacology Society). The peak plasma concentration (Cmax) and the time of its
Subject no. Period I Period II Period III Period IV occurrence (Tmax) were obtained directly from the observed concentration–time
data. The terminal elimination rate constant (λz) was estimated by linear
1 A B C D
regression of the terminal portion of the concentration–time curve, and the
2 A B C D
elimination t1/2 was calculated as 0.693/λz. The area under the plasma
3 A B C D
concentration–time curve (AUC0–τ) was calculated by use of the linear
4 B C D A
trapezoidal rule and extrapolated to infinity (AUC0–∞). The apparent oral
5 B C D A
clearance (CL/F) was calculated as Dose/AUC0–∞.
6 B C D A
7 C D A B
8 C D A B 2.5. Statistical analysis
9 C D A B
All results from the study were expressed as mean ± SD. The pharmacoki-
10 D A B C
netic parameters between different treatment groups were compared by using
11 D A B C
general linear model ANOVA. P ≤ 0.05 was considered statistically significant
12 D A B C
for all tests. All analyses were performed with SPSS software (ver. 11.5; SPSS,
A = Treatment A; B = treatment B; C = treatment C; D = treatment D. Chicago, IL). The bioequivalence of Cmax and AUC0–τ for roxithromycin or
22 T. Hang et al. / Clinica Chimica Acta 382 (2007) 20–24

specific with typical chromatograms shown in Fig. 2. The

typical retention time for roxithromycin is about 6.1 min, for
ambroxol HCl about 5.6 min, and for erythromycin about
3.7 min. Calibration curves were established in two segments
for both roxithromycin (from 0.01–1 μg/ml, and 1–20 μg/ml)
and ambroxol HCl (from 2–50 ng/ml, and 50–200 ng/ml), and
good correlations were obtained. The LLOQ for roxithromycin
was 10 ng/ml and that for ambroxol HCl was 2 ng/ml based on

Fig. 1. Product ion scan mass spectra of roxithromycin (A), ambroxol HCl (B)
and erythromycin (C).

ambroxol HCl between the compound dispersible tablets and two single
component reference formulations in combination or in single usage were
determined by multiple comparisons ANOVA based on least-significant
difference assumption after their natural log-transformation.

3. Results and discussion

3.1. LC-MS/MS method validation Fig. 2. Representative chromatograms for the simultaneous determination of
roxithromycin and ambroxol HCl, A: human blank plasma; B: plasma spiked
Roxithromycin and ambroxol HCl plasma samples stored at with 5 μg/ml roxithromycin, 50 ng/ml ambroxol HCl, and 1 μg/ml
− 20 °C were stable for at least 40 days and no significant erythromycin; C: plasma sample from a subject 4 h after the administration of
one compound roxithromycin and ambroxol HCl dispersible tablet, in which the
changes were found after three freeze–thaw cycles. The concentrations of roxithromycin and ambroxol HCl were found to be 5.3 μg/ml
simultaneous determination of roxithromycin, ambroxol HCl and 56.4 ng/ml, respectively. The retention time for roxithromycin is about
with erythromycin as internal standard was sensitive and 6.1 min, for ambroxol HCl about 5.6 min, and for erythromycin about 3.7 min.
 T. Hang et al. / Clinica Chimica Acta 382 (2007) 20–24 23

Table 2
Recoveries of roxithromycin and ambroxol hydrochloride from human plasma
(n = 5)
Analyte Spiked concentration Recovery (%) RSD (%)
Roxithromycin (μg/ml) 0.02 98.2 5.1
5 105.3 3.3
20 104.7 0.9
Ambroxol hydrochloride 5 106.2 1.3
(ng/ml) 50 110.1 2.9
200 110.1 2.9

Table 3
Intra-day (n = 5) and inter-day (n = 3 ⁎ 5) precision and accuracy
Analyte Concentration Precision (RSD%) Accuracy Fig. 4. Mean plasma concentration–time profiles of ambroxol HCl following a
(RE%) single oral dose of one compound roxithromycin (150 mg) and ambroxol HCl
(30 mg) dispersible tablet (test formulation, treatment A), one 150 mg
Intra-day Inter-day (n = 5) roxithromycin dispersible tablet together with one 30 mg ambroxol HCl tablet
(n = 5) (n = 3⁎5) (combined reference formulations, treatment B), or one 30 mg ambroxol HCl
Roxithromycin 0.02 13.8 14.8 − 3.1 tablet alone (reference formulation II, treatment D) to 12 volunteers. Each point
(μg/ml) 5 7.2 9.5 − 6.6 represents the mean ± SD.
20 5.1 8.7 − 8.0
Ambroxol 5 10.3 14.2 2.0
hydrochloride (ng/ml) 50 4.6 12.0 2.3 HCl are shown in Figs. 3 and 4. The main pharmacokinetic
200 4.0 9.0 1.4 parameters obtained with different treatments are shown in
Tables 4 and 5). Multiple comparisons ANOVA show (Table 6)
that no significant differences in roxithromycin pharmacokinetics
signal-to-noise ratio of 10.0. The recoveries of roxithromycin
and ambroxol HCl ranged from 98% to 110% (Table 2). The Table 4
intra-day and inter-day precision at 3 quality control concentra- Pharmacokinetics for roxithromycin found in treatments A, B and C
(mean ± SD, n = 12)
tions for roxithromycin ranged from 5.1–14.8%, while the
accuracy range from − 3.1% to − 8.0%. The corresponding Parameter Treatment A Treatment B Treatment C
results for ambroxol HCl were 4.0–14.2% for precision, and Cmax (μg/ml) 6.99 ± 1.55 6.80 ± 1.50 7.51 ± 2.11
1.4–2.3% for accuracy (Table 3). Tmax (h) 2.0 ± 0.9 2.7 ± 1.9 2.3 ± 1.0
MRT (h) 14.3 ± 2.5 15.4 ± 3.3 13.7 ± 1.9
t1/2 (h) 13.3 ± 2.6 13.1 ± 4.4 12.5 ± 2.1
3.2. Pharmacokinetics AUC0–τ (h μg/ml) 84.6 ± 26.2 90.7 ± 21.3 86.7 ± 27.0
AUC0–∞ (h μg/ml) 86.6 ± 27.6 93.1 ± 22.7 88.4 ± 28.2
All 12 subjects completed the study. The mean plasma CL/F(l/h) 1.88 ± 0.51 1.71 ± 0.46 1.90 ± 0.73
concentration-time profiles of roxithromycin and ambroxol
Table 5
Pharmacokinetics for ambroxol hydrochloride found in treatments A, B and D
(mean ± SD, n = 12)
Parameter Treatment A Treatment B Treatment D
Cmax (ng/ml) 53.9 ± 22.4 60.7 ± 23.9 60.3 ± 18.3
Tmax (h) 2.0 ± 0.7 1.9 ± 0.9 2.1 ± 0.8
MRT (h) 8.0 ± 0.5 7.8 ± 1.2 7.7 ± 0.7
t1/2 (h) 8.2 ± 2.0 7.9 ± 1.8 7.7 ± 2.3
AUC0–τ (h ng/ml) 448 ± 139 452 ± 138 495 ± 113
AUC0–∞ (h ng/ml) 517 ± 152 521 ± 165 569 ± 158
CL/F (l/h) 62 ± 15 64 ± 22 56 ± 14

Table 6
ANOVA of roxithromycin pharmacokinetics in treatments A, B and C
Parameter df Mean F Significance Conclusion
Fig. 3. Mean plasma concentration–time profiles of roxithromycin following a square probability
single oral dose of one compound roxithromycin (150 mg) and ambroxol HCl
Cmax 2 1.63 0.540 0.592 P N 0.05 no significant
(30 mg) dispersible tablet (test formulation, treatment A), one 150 mg
Tmax 2 1.34 1.098 0.354 differences
roxithromycin dispersible tablet together with one 30 mg ambroxol HCl tablet
t1/2 2 5.96 2.123 0.147
(combined reference formulations, treatment B), or one 150 mg roxithromycin
MRT 2 21.4 3.074 0.070
dispersible tablet alone (reference formulation I, treatment C) to 12 volunteers.
AUC0–τ 2 115 0.351 0.708
Each point represents the mean ± SD.
24 T. Hang et al. / Clinica Chimica Acta 382 (2007) 20–24

Table 7 and ambroxol HCl after oral administration. Roxithromycin and

ANOVA of ambroxol hydrochloride pharmacokinetics in treatments A, B and D ambroxol HCl dispersible tablets were bioequivalent with
Parameter df Mean F Significance Conclusion reference to the roxithromycin dispersible tablets and ambroxol
square probability HCl tablets in combination usage.
Cmax 2 173 1.295 0.297 P N 0.05 no significant
Tmax 2 0.132 0.307 0.739 differences Acknowledgement
t1/2 2 0.409 0.206 0.816
MRT 2 0.984 0.235 0.792
AUC0–τ 2 7383 1.203 0.322 The LC-MS/MS analyses of this study were conducted in the
Key Laboratory of Drug Quality Control and Pharmacovigi-
lance (China Pharmaceutical University), Ministry of Education
Table 8 of the People's Republic of China.
Multiple comparisons ANOVA of log-transformed Cmax and AUC0–τ for
roxithromycin between treatments A, B and C
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