CHAPTER ONE

Azithromycin
Ahmed H.H. Bakheit*, Badraddin M.H. Al-Hadiya†,
Ahmed A. Abd-Elgalil‡
*Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Kingdom of
Saudi Arabia
†
Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, TAIF, KSU
‡
Research Center, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia

Contents
Background 2
1. Description 2
1.1 Nomenclature 2
1.2 Formulae 3
1.3 Elemental analysis 4
1.4 Appearance 4
2. Methods of Preparation of Azithromycin 4
3. Physical Characteristics 5
3.1 Specific optical rotation 5
3.2 Ionization constant 6
3.3 Solubility characteristics 6
3.4 Partition coefficient 6
3.5 Particle morphology 6
3.6 Crystallographic properties 7
3.7 Hygroscopicity 7
3.8 Thermal methods of analysis 9
3.9 Spectroscopy 12
3.10 Mass spectrometry 17
4. Methods of Analysis 17
4.1 Compendial methods of analysis 17
4.2 Electrochemical methods of analysis 24
4.3 Spectroscopic methods of analysis 25
4.4 Chromatographic methods of analysis 28
4.5 Determination in body fluids and tissues 30
5. Stability 30
6. Clinical Applications 33
6.1 An overview 33
6.2 Antimicrobial spectrum susceptibility 33
6.3 Mechanism of action 33
6.4 Resistance to macrolides 34
6.5 Actions other than antimicrobial effects 34

Profiles of Drug Substances, Excipients, and Related Methodology, Volume 39 # 2014 Elsevier Inc. 1
ISSN 1871-5125 All rights reserved.
http://dx.doi.org/10.1016/B978-0-12-800173-8.00001-5
2 Ahmed H.H. Bakheit et al.

6.6 Clinical uses and dosing 35

6.7 ADME profile 35
6.8 Side effects 36
6.9 Drug interactions 36
References 36

BACKGROUND
A team of researchers at the Croatian pharmaceutical company Pliva,
led by Dr. Slobodan Ðokić, discovered azithromycin in 1980. It was pat-
ented in 1981. Pfizer launched azithromycin under Pliva’s license in other
markets under the brand name Zithromax in 1991. After several years, the
U.S. Food and Drug Administration approved AzaSite, an ophthalmic for-
mulation of azithromycin, for the treatment of eye infections [1].

1. DESCRIPTION
1.1. Nomenclature
1.1.1 Systematic chemical names [2–5]
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-(2,6-dideoxy-3-C-3-O-
dimethyl-a-L-ribo-hexopyranosyloxy)-2-ethyl-3,4,10-trihydroxy-3,5,6,
8,10,12,14-heptamethyl-11-(3,4,6-tride-oxy-3-dimethylamino-b-D-xylo-
hexopyranosyloxy)-1-oxa-6-aza-cyclopentadecan-15-one dehydrate.
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-
3,5,6,8,10,12,14-heptamethyl-15-oxo-11-{[3,4,6-trideoxy-3-
(dimethylamino)-b-D-xylo-]oxy}-1-oxa-6-azacyclopentadec-13-yl
2,6-dideoxy-3-C-methyl-3-O-methyl-a-L-ribo-hexopyranoside.
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-dideoxy-3-C-
methyl-3-O-methyl-a-lribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-tri-
hydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-
(dimethylamino)-b-D-xylo-hexopyranosyl]oxy]-1-oxa-6-
azacyclopentadecan-15-one.
(2R,3S,4R,5R,8R,11R,13S,14R)-11-(((2S,3S,4S,6R)-4-(dimethylamino)-
3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-ethyl-3,4,10-
trihydroxy-13-(((2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimeth-
yltetrahydro-2H-pran-2-yl)oxy)-3,5,6,8,10,14-hexamethyl-1-oxa-6-
azacyclopentadecan-15-one.
N-methyl-11-aza-10-deoxo-10-dihydroerythromycin A; 9-deoxo-9a-
methyl-9a-aza-homoerythromycin A; CP-62993; XZ-450; Azitrocin;
Sumamed; Trozocina; Zithromax; Zitromax.
Azithromycin 3

1.1.2 Nonproprietary names

Generic [6]: azithromycin
Synonyms [2–7]: Atsitromysiini; Azithromycine; Azithromycinum;
Azitromicina; Azitromicinas; Azitromisin; Azitromycin; Azytromycyna;
CP-62993; XZ-450; Azithramycine; Azithromycin Dihydrate;
Azithromycine [French]; Azithromycinum [Latin]; Azitromicina [Spanish].

1.1.3 Proprietary names [3,7,8]

Azacid® (Biosen: TR); Azitromin® (Farmasa: BE); Azitromax® (Pfizer:
NO); Azro® (Eczacibasi: TR); Cronopen® (Elea: AR); Hemomycin®
(Hemofarm: YU); Misultina® (Microsules: AR); Mixoterin® (Roux-Ocefa:
AR); Sumamed® (Pliva: CZ, HR, PL); Triamid® (Beta: AR); Zifin®
(Pratapa: ID); Zithrax® (Dankos: ID); Zithromax® (Pfizer: NL); Zitromax®
(Pfizer: AR, BE, LU, TR); Zitrotek® (Pfizer: TR);
Dihydrate: Arzomicin® (Sintyal: AR); Azadose® (Pfizer: FR); Azatek®
(Biosen: TR); Azithral® (Alembic: IN); Azitrin® (Andromaco: AR);
Azitro® (Deva: TR); Azitrocin® (Pfizer: MX); Azitrocin® (Roerig: IT);
Azitromax® (Pfizer: NO,SE); Azitromicina Richet® (Richet: AR);
Azitrotek® (Deva: TR); Aziwok® (Wockhardt: IN); Azomax® (Kocak:
TR); Azro® (Eczacibasi: TR); Cronopen® (Elea: AR); Goxal® (Pharmacia:
ES); Ribotrex® (Pierre Fabre: IT); Toraseptol® (Lesvi: ES); Triamid® (Beta:
AR); Trozocina® (Sigma-Tau: IT); Ultreon® (Pfizer: DE); Vinzam® (Funk:
ES); Zentavion® (Vita: ES); Zistic® (Bernofarm: ID); Zithromax® (Bayer:
DE); Zithromax® (Mack: DE); Zithromax® (Pfizer: AT, AU, CA, CH, FI,
FR, ID, IE, IN, PT, UK, US); Zitromax® (Pfizer: BE, BR, DK, ES, IT).

1.2. Formulae [2,3]

1.2.1 Empirical formula, molecular weight, CAS number
Empirical formula Molecular CAS number
weight
Anhydrous azithromycin C38H72N2O12 748.984 83905-01-5
Monohydrate azithromycin C38H72N2O12H2O 767.02 121479-24-4
Azithromycin dihydrate C38H72N2O122H2O 785.0 117772-70-0

1.2.2 Structural formula

See Figure 1.1
4 Ahmed H.H. Bakheit et al.

Figure 1.1 Structural formula of azithromycin hydrate.

1.3. Elemental analysis

The theoretical elemental composition of azithromycin is as follows [5]:
Carbon: 60.94 %; Hydrogen: 9.69%; Nitrogen: 3.74%; Oxygen: 25.63%

1.4. Appearance [2]

A white or almost white powder.

2. METHODS OF PREPARATION OF AZITHROMYCIN

Azithromycin (5) was prepared from erythromycin A [9,10] by treating
the erythromycin (1) in methanol with hydroxylamine hydrochloride and a
base at reflux temperature for 10 h to form oxime (2). The oxime was isolated,
purified, and subjected to Beckmann’s rearrangement to obtain the interme-
diary (6,9-iminoether) (3) (Scheme 1.1) in aqueous acetone in the presence of
p-toluenesulfonyl chloride and base for 2 h at 5
C (and 2 h more at room
temperature). The iminoether was reduced to the secondary amine (4) with
sodium borohydride in methanol [11,12] or by catalytic hydrogenation in the
presence of platinum dioxide and acetic acid as solvents [13].
Another alternate synthetic method for azithromycin is reported [14] The
iminoether (3) was prepared in single step (Scheme 1.2) from erythromycin
A (1), by treating the erythromycin A (1) solution in acetone with O-mesi-
tylene-sulfonylhydroxylamine, to form the mesitylenesulfonyloxime “in situ”
from erythromycin A, which was treated with an aqueous base (sodium bicar-
bonate) at 0
C, and then the intermediary 6-9-iminoether (3) (Scheme 1.2)
was produced by a Beckmann’s rearrangement. The iminoether (3) was
reduced with reductive methylation using common techniques [15] to obtain
azithromycin (5).
Azithromycin 5

O NMe2 HON NMe2

OH OH
HO O HO O
HO O NH2OH HCl HO O

HO BASE HO

O OCH3 O OCH3
O O

O OH O OH
O O

(1)
pTsCl, BASE

NMe2 NMe2
H
N OH N
HO O O
HO O HO O
HO O
Reductor
HO
Agent HO

O OCH3 O OCH3
O O
O OH O OH
O O

(4) (3)
Formaldehyde
Formic acid Rh/C, H2,
Formaldehyde, acetic acid

NMe2
H3C
N OH
HO O
HO O

HO

O OCH3
O

O OH
O

(5)
Scheme 1.1 Beckmann’s rearrangement to obtain the intermediary (6,9-iminoether) for
preparation of Azithromycin.

Azithromycin is a semisynthetic 15-memberedmacrolide antibiotic, which

is derived from erythromycin A by a sequence of oximation, Beckmann
rearrangement, reduction, and N-methylation [16–19]. Rengaraju et al. [20]
improved process for preparing nonhygroscopic azithromycin dehydrate.

3. PHYSICAL CHARACTERISTICS
3.1. Specific optical rotation [5,21]
[a] 20
45
to 49
(anhydrous substance) (C ¼ 1 in anhydrous ethanol R)
[a]20 37
(C ¼ 1 in CHCl3)
6 Ahmed H.H. Bakheit et al.

NMe2 NMe2
O
N
OH O
HO HO O
HO O O O HO O

HO S ONH2 HO
O
O OCH3 O OCH3
O O
NaHCO3
O OH O OH
O O
(3)
(1)

NMe2
H3C
N OH
HO O
HO O

HO

O OCH3
O

O OH
O
(5)
Scheme 1.2 An alternate synthetic method for Azithromycin.

3.2. Ionization constant [22]

pKa ¼ 7.34

3.3. Solubility characteristics [21]

Azthiromycin is practically insoluble in water and freely soluble in anhy-
drous ethanol and methylene chloride.

3.4. Partition coefficient

The octanol/water partition coefficient (Kow) of azithromycin was 0.65 at
20
C and pH 7 [23]. Adsorption isotherm studies indicated that the ther-
modynamic data revealed that the adsorption of azithromycin on the surface
of zinc was endothermic, spontaneous, and consistent with the adsorption
model of Langmuir [24].

3.5. Particle morphology

Gandhi et al. [25] viewed the commercial sample, dehydration and mon-
ohydration of azithromycin by scanning electron microscopy (Jeol electron
Azithromycin 7

microscope, D-6000, Japan). The samples were sputter coated with gold
before examination, and they found that the internal crystal structure appears
to be the same, as is evident from the similar enthalpy of fusion for all three
samples [26].

3.6. Crystallographic properties

3.6.1 Single crystal structure
Data were collected at room temperature using Bruker X-ray diffractome-
ters equipped with copper radiation and graphite monochromators. Struc-
tures were solved using direct methods. The SHELXTL computer library
provided by Bruker AXS, Inc. facilitated all necessary crystallographic com-
putations and molecular displays (SHELXTL™ Reference Manual, Version
5.1, Bruker AXS, Madison, WI, USA) [14].
The molecular structure of the anhydrous crystalline azithromycin
obtained is shown in Figure 1.2, and the structure was elucidated by single
crystal X-ray diffraction, finding that it coincides with the anhydrous crys-
talline form, with a tetragonal crystal system and the space group P42212.
These and other crystallographic data from the diffraction analysis are com-
pared with data reported for the dihydrated crystalline form. A displays peaks
at 9.3, 13.0 and 18.7 degrees of 2-theta [27].

3.6.2 X-ray powder diffraction pattern [28]

The X-ray powder diffraction pattern of azithromycin has been measured
using a Bruker D5000 diffractometer (Madison, Wis.) equipped with copper
radiation, fixed slits (1.0, 1.0, 0.6 mm), and a Kevex solid state detector. The
pattern obtained is shown in Figure 1.3, and Azithromycin dehydrate dis-
plays peaks at 7.2, 7.9, 9.3, 9.9, 11.2, 12.0, 12.7, 13.0, 14.0, 15.6, 16.0,
16.4, 16.8, 17.5, 18.2, 18.7, 19.1, 19.8, 20.5, 20.9, 21.2, 21.6, 21.8, and
24.0, the data was collected from 3.0 to 40.0 degrees in 2-theta using a step
size of 0.04 degrees and a step time of 1.0 seconds.

3.7. Hygroscopicity
Azithromycin was found to exhibit pseudopolymorphism and can exist as
monohydrate and dihydrate. The anhydrous form of AZI seemed to be
unstable since it converted to dihydrate on storage at room temperature.
On the other hand, monohydrate in the presence of moisture can convert
8 Ahmed H.H. Bakheit et al.

Figure 1.2 An X-ray crystal structure of the crystalline anhydrous azithromycin.

to the more stable dihydrate form. Therefore, the most stable form of AZI is
dehydrate [25].
Allen et al. [29] found that azithromycin was obtained as hygroscopic
monohydrate when it was prepared by crystallization from ethanol and
water, and nonhygroscopic dehydrate azithromycin was prepared by crys-
tallization from tetrahydrofuran and aliphatic (C5–C7) hydrocarbon in the
presence of at least two molar equivalents of water.
Azithromycin 9

100,000

80,000

60,000

40,000

20,000

0
3 10 20 30 40
2-Theta-scale
Figure 1.3 An experimental powder X-ray diffraction pattern of azithromycin
dehydrate [8].

3.8. Thermal methods of analysis

3.8.1 Melting behavior
The melting range of azithromycin crystals is between 113 and 115
C [5].

3.8.2 Differential scanning calorimetry [25]

The differential scanning calorimetry (DSC) thermogram of azithromycin
was recorded on a DSC (Mettler, Toledo DSC 821 Switzerland) using
Mettler Star system. The curve shown in Figure 1.4 was collected from
0 to 250
C with nitrogen purging (80 ml/min), and using a heating rate
of 10
C/min. DSC thermograms of anhydrous, dehydrate, and mono-
hydrate are shown in Figure 1.4, and the thermal characteristics are shown
in Table 1.1.

3.8.3 Thermogravimetric analysis

Thermogravimetric analysis was carried out using TGA (Mettler, LMI JM
Switzerland) apparatus. The samples (1–5 mg) were placed in aluminum
pans and heated up to 250
C at a rate of 1
C/min under nitrogen purge
(40 ml/min). The TGA thermogram obtained with the AZC sample is
given in Figure 1.5. This thermogram exhibited the transition from the
dihydrate to the anhydrous form of azithromycin. The observed weight loss
10 Ahmed H.H. Bakheit et al.

∧exo

10
mW

40 60 80 100 120 140 160 180 200 220 240 ⬚C

0 2 4 6 8 10 12 14 16 18 20 min

Temperature (⬚C)
Figure 1.4 DSC thermograms of different forms of AZI. Upper curve depicts the com-
mercial sample of AZI; middle curve shows the endotherm of dihydrate (DH) and lower
curve shows the endotherm of monohydrate (MH). The thermograms were generated
using a sealed pan.

Table 1.1 Thermal analysis and KFT of different forms of AZI

Sample name DSC analysis Water content
Temperature Heat fusion
range (
C) (J/g) TGA (%) KFT (%)
Anhydrous 132.29–143.09 62.73  7.13 4.452  0.18 4.57  0.03
azithromycin 149.83–155.48 30.41  2.93
Monohydrate 134.65–155.48 92.99  8.58 4.155  0.41 4.35  0.28
azithromycin
azithromycin 139.88–156.31 92.83  2.41 2.472  0.41 2.39  0.79
dihydrate

of 4.38% corresponded to the stoichiometric weight loss of two water

molecules [30].
Gandhi et al. [25] were found a stoichiometric weight loss of two water
molecules (theoretical weight loss 4.58%) for CS and DH by TGA, while
MH showed a weight loss corresponding to one molecule of water (theo-
retical weight loss 2.29%) as shown in Figure 1.6. The results were in good
Azithromycin 11

101
100
99
98 4.3816%
Mass %

97
AZC
96
95
94
93
92
25 45 65 85 105 125 145 165 185
Temperature (⬚C)
Figure 1.5 TGA thermogram of AZC.

2
mg

40 60 80 100 120 140 160 180 200 220 240 °C

0 2 4 6 8 10 12 14 16 18 20 min

Figure 1.6 TGA thermograms of different forms of AZI. Upper curve indicates stoichio-
metric weight loss of two water molecules in CS, middle curve indicates weight loss
of two water molecules in DH, and lower curve indicates weight loss of one water
molecule in MH.

agreement with those found by Karl Fischer titration KFT as shown in

Table 1.1.

3.8.4 Karl Fischer titration (KFT) [25]

Water content in different forms of AZI was determined using a Karl Fischer
titrimeter (Metrohm, 716 DMS, Switzerland). Samples (20–25 mg) were
12 Ahmed H.H. Bakheit et al.

accurately weighed and quickly transferred to the titration vessel containing

anhydrous methanol.

3.8.5 Boiling point, enthalpy of vapor, flash point, and vapor pressure
The calculated value of the boiling point of azithromycin under a pressure of
760 mmHg was 822.1
C. The enthalpy of vapor calculated value was
135.99 KJ/mol. The value of flash point was found to be 451
C, and the
vapor pressure was calculated to be 251 1031 mmHg at 25
C [31].

3.9. Spectroscopy
3.9.1 UV/Vis spectroscopy
The ultraviolet spectrum of azithromycin dihydrate in methanol and mobile
phase (methanol: acetonitrile: phosphate buffer pH 6.7: tetrahydrofuran,
15:25:60:2.5, v/v) shown in Figure 1.7. The figures were recorded using
a double beam Model GBC 916UV VIS spectrophotometer (GBC Scien-
tific Equipment Pty Ltd., Melbourne, Victoria, Australia). The values of
wavelength maximum in nanometer (lmax) are 201.6 nm on methanol
and 199.2 nm on mobile phase. The spectra of azithromycin in methanol
(Figure 1.7A) and mobile phase (Figure 1.7B) were shown below.

3.9.2 Vibrational spectroscopy [32]

The infrared absorption spectrum of azithromycin is shown in Figure 1.8. It
was obtained in a KBr disc using a FT-IR Nicolet® Imoact 410 instrument,
infrared spectrophotometer. The principal peaks were 3561, 3496, 13344,
1282, 1269, 1251, and 1083.

3.9.3 Nuclear magnetic resonance spectrometry [33]

3.9.3.1 1H NMR spectrum [33]
The 1H nuclear magnetic resonance (NMR) spectrum of AZI was recorded
using an internal deuterium lock at ambient probe temperatures on the fol-
lowing instruments: Bruker DPX-400 (400 MHz), Bruker Avance DRX-
400 (400 MHz), Bruker Avance 500 BB-ATM (500 MHz), and Bruker
Avance 500 Cryo Ultrashield (500 MHz). An internal reference of dH
7.26 was used for the residual CHCl3 in CDCl3. Data are represented as fol-
lows: chemical shift (in ppm to the nearest 0.01 ppm), integration, multiplic-
ity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling
constant (J in Hz to the nearest 1 Hz), and assignments, which were deter-
mined either on the basis of unambiguous chemical shift or coupling pattern,
Azithromycin 13

A
0.8
201.6

0.6
Absorbance

0.4

0.2

0
192 202 212 222 232 242
Wavelength (nm)

0.2 199.2

0.16
Absorbance

0.12

0.08

0.04

0
195 205 215 225 235 245
Wavelength (nm)
Figure 1.7 (A) The ultraviolet spectrum of AZI dihydrate in methanol. (B) The ultraviolet
spectrum of AZI dihydrate in mobile phase.
14 Ahmed H.H. Bakheit et al.

75

70

65
% Transmittance

1251.57

1282.43
60
1268.93

55

50 3496.31
3561.88
45 1344.14
1083.80

3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm-1)
Figure 1.8 Infrared absorption spectrum of azithromycin.

10A 9 8 7 6 5 4 3 2 1
1
Figure 1.9 Full H NMR spectrum of azithromycin in CDCl3.

by patterns observed in 2D experiments (1H–1H COSY and HMQC) or by

analogy to fully interpreted spectra for related compounds.
Azithromycin is shown in Figure 1.9 (full 1H NMR spectrum). The
assignments for the observed bands are provided in Table 1.2.

13
3.9.3.2 C NMR spectrum [33]
13
The C NMR spectrum of azithromycin was recorded by broadband pro-
ton spin decoupling at ambient probe temperature using an internal
Azithromycin 15

Table 1.2 Assignments for the resonance bands observed in the 1H NMR spectrum of
azithromycin in CDCl3
See Figure 1.10 reference 33
Chemical shift Number of Multiplicity and coupling
(ppm) protons constant (J) Assignment
9.44 1 (1H, C2dOH)
5.13 1 Doublet (5 Hz) C100 dH
4.74 1 Doublet (6 Hz) C11dOH
4.68 1 Doublet of doublets (10 Hz) C13dH
4.43 1 Doublet (7 Hz) C10 dH
4.29 1 Doublet of doublets C3dH
(5, 2 Hz)
4.08 1 Doublet of quartet C50 dH
(10, 6 Hz)
3.68 1 Doublet (5 Hz) C11dH
3.65 1 Doublet (7 Hz) C5dH
3.55-3.47 1 Multiple C50 dH
3.35 1 Singlet C12dOH
3.34 3 Singlet C30 dOCH3
3.23 1 Doublet of doublets C20 dH
(10, 7 Hz)
3.03 1 app. triplet (10 Hz) C40 dH
2.89 1 Singlet C6dOH
2.78–2.72 1 Multiple C2dH
2.69 1 Quartet (7 Hz) C10dH
2.52 1 Doublet (10 Hz) C9dH
2.46–2.41 Multiple C30 dH
2.35 1 Doublet (15 Hz) C20 dH
2.31 3 Singlet C1dH3
2.28 6 Singlet C30 dN(CH3)2
2.15 1 Doublet (11 Hz) C40 dOH
2.07–1.95 2 Multiple C4dH and
C8dH
Continued
16 Ahmed H.H. Bakheit et al.

Table 1.2 Assignments for the resonance bands observed in the 1H NMR spectrum of
azithromycin in CDCl3—cont'd
Chemical shift Number of Multiplicity and coupling
(ppm) protons constant (J) Assignment
2.02 1 Doublet (10 Hz) C9dH
1.93–1.85 1 Multiple C13dCH2dCH3
1.79 1 Doublet (15 Hz) C7dH
1.67–1.63 1 Multiple C40 dH
1.58 1 Doublet of doublets C200 dH
(15, 5 Hz)
1.51–1.43 1 Multiple C13dCH2dCH3
1.32 3 Doublet (5 Hz) C500 dCH3
1.31 3 Singlet C6dCH3
1.24 3 Singlet C300 dCH3
1.26–1.21 2 Multiple C7dH and
C40 dH
1.22 3 Doublet (6 Hz) C50 dCH3
1.19 3 Doublet (7 Hz) C2dCH3
1.08 1 Doublet (7 Hz) C10dCH3
1.08 3 Singlet C12dCH3
1.04 3 Doublet (7 Hz) C4dCH3
0.90 3 Doublet (7 Hz) C8dCH3
0.89 3 Triplet (7 Hz) C13dCH2dCH3

deuterium lock. All chemical shift values are reported in ppm to the
nearest 0.01 ppm. An internal reference of dC 77.0 was used for CDCl3.
Assignments were supported by DEPT editing and determined either on
the basis of unambiguous chemical shift, by patterns observed in 2D exper-
iments (HMQC) or by analogy to fully interpreted spectra for related
compounds.
Azithromycin is shown in Figure 1.11 (full 13C NMR spectrum). The
assignments for the observed bands are provided in Table 1.3, which are
consistent with the 13 carbon contents of azithromycin.
Azithromycin 17

HO NMe2
N 9 7 3⬘
O 1⬘
HO 5⬘
HO O
11 5
HO 13 3 OMe
O 1 3⬙
O 1⬙ OH
5⬙
O O

Figure 1.10 AZI proton assignment.

3.10. Mass spectrometry [34]

The mass spectrum of azithromycin was obtained utilizing A Thermo Sci-
entific TSQ Quantum mass spectrometer systems feature HyperQuad quad-
rupoles. The MS analysis was performed with electro-spray ionization (ESI)
interface in both negative and positive ion modes.
TSQ Quantum Ultra AM Instrument Conditions: Ionization mode and
source: Positive and negative ESI; electrospray voltage: (þ) 3.5 kV, ()
2.5 kV; sheath gas: 1; auxiliary gas: 0; ion transfer tube temperature:
270
C; ion transfer tube offset: 35 V; tube lens offset: 77 V; collision energy:
25 eV (famotidine), 23 eV (azithromycin); collision pressure: 1.2 mTorr; Q1/
Q3 resolution: 0.1 Da FWHM; accurate mass mode: internal; micro scans: 2.
Figure 1.12 shows the detected mass fragmentation pattern of azithromycin.
The major peaks in the spectrum occur at m/z 749, 591, 574, 434, 158.
For protonated azithromycin, elimination of H2O and successive loss of
the two sugar moieties were the major fragmentation pathways. The CID
mass spectrum of the [M  H] of azithromycin was very similar to that
of the protonated species and showed successive loss of the two sugar moi-
eties as the major dissociation pathways as shown in Figure 1.13.

4. METHODS OF ANALYSIS
4.1. Compendial methods of analysis
4.1.1 Identification
4.1.1.1 IR spectrum of Azithromycin
The IR spectra of the drug were obtained in the solid state using 90 g/l solu-
tions in methylene chloride [21,34]
 200 175 150 125 100 75 50 25
13
Figure 1.11 Full C NMR spectrum of azithromycin in CDCl.
Azithromycin 19

Table 1.3 Assignments for the resonance bands observed in the 13C NMR spectrum of
azithromycin
Chemical shift (ppm) Assignments Chemical shift (ppm) Assignment
178.9 (C1) 42.3 (2C, C4 and C7)
0
102.9 (C1 ) 40.3 (C30 dN(CH3)2)
94.5 (C100 ) 36.2 (C1)
83.3 (C5) 34.7 (C200 )
78.1 (C400 ) 28.7 (C40 )
77.6 (C3) 27.6 (C6dCH3)
77.4 (C13) 26.7 (C8)
73.6 (C6) 22.0 (C8dCH3)
73.6 (C11) 21.6 (C300 dCH3)
72.9 (C300 ) 21.3 (C50 dCH3)
70.8 (C20 ) 21.3 (C13dCH2)
70.1 (C9) 18.2 (C500 dCH3)
68.7 (C50 ) 16.2 (C12dCH3)
65.9 (C30 ) 14.6 (C2dCH3)
65.5 (C500 ) 11.2 (C13dCH2dCH3)
62.4 (C10) 9.0 (C4dCH3)
49.4 (C300 -OCH3) 7.3 (C10dCH3)
45.3 (C2)

4.1.1.2 HPLC drug chromatogram

The principal peak in the HPLC drug chromatogram obtained with test
solution was similar in retention time and size to the principal peak in the
chromatogram obtained with reference solution [21,34].

4.1.2 Impurity Analysis [47]

The specified impurities (A, B, C, D, E, F, G, H, I, K, J, L, M, N, O, and P)
of azithromycin were determined using liquid.
20 Ahmed H.H. Bakheit et al.

[M-H-desosamine]-
571.1700
100
[M-H]-
90 747.4300
80
Relative abundance

70
[M-H-desosamine-cladinose]-
60

50 396.1000 499.6300
40
668.6800
30
212.9100
20

10

0
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z

[M(d5)-D]-
[M(d5)-D-DNSO2ND2]+ 751.4675
100
243.1004 [M(d5)-D-desosamine]-
90

80 573.2704
Relative abundance

70

60 [M(d5)-D-desosamine-cladinose]-
50
397.1729
40

30

20

10

0
100 150 200 250 300 350 400 450 500 550 600 650 700 750
m/z

Figure 1.12 Negative ion ESI mass spectra of azithromycin (MW ¼ 748): CID product ion
spectra (MS/MS) of [M  H] at m/z 747 and the fully exchanged [M(d5)-D] at m/z 751.
Deuteration was achieved by liquid phase H/D exchange method. MS and MS/MS exper-
iments were performed on a TSQ Quantum Ultra AM mass spectrometer.
Azithromycin 21

OH(D)
O O

O
:O O O H HO
O O

:
H (D)HO O
O O O +
O N (D) HO
(D)HO
O HO HO
N (D) N
(D)
HO HO N
(D) (D) desosamine (neutral)
N m/z = 571 (574) 176 (177)
m/z = 747 (751)

O
OH (D) O
O
O
(D) HO
+
HO
O N
HO
cladinose (neutral) (D)
175 (176)
m/z = 396 (398)

Figure 1.13 Proposed CID fragmentation mechanisms for the major fragment ions from
deprotonated azithromycin at m/z 747 determined from H/D exchange patterns, high-
resolution mass measurements, and MS/MS experiments. Numbers in parentheses refer
to deuterated fragmentations. The proposed site of deprotonation is based on the most
acidic proton of the lactone ring.

(A) R1 ¼ OH, R2 ¼ R6 ¼ H, R3 ¼ R4 ¼ R5 ¼ CH3: 6-

demethylazithromycin,
(B) R1 ¼ R6 ¼ H, R2 ¼ R3 ¼ R4 ¼ R5 ¼ CH3: 3-deoxyazithromycin
(azithromycin B),
(C) R1 ¼ OH, R2 ¼ R3 ¼ R5 ¼ CH3, R4 ¼ R6 ¼ H: 300 -O-
demethylazithromycin (azithromycin C),
(D) R1 ¼ OH, R2 ¼ R3 ¼ R4 ¼ CH3, R5 ¼ CH2OH, R6 ¼ H:
14-demethyl-14-(hydroxymethyl)azithromycin (azithromycin F),
(F) R1 ¼ OH, R2 ¼ R4 ¼ R5 ¼ CH3, R3 ¼ CHO, R6 ¼ H: 30 -N-
demethyl-30 -N-formylazithromycin,
(I) R1 ¼ OH, R2 ¼ R4 ¼ R5 ¼ CH3, R3 ¼ R6 ¼ H: 30 -N-
demethylazithromycin,
(O) R1 ¼ OH, R2 ¼ R3 ¼ R4 ¼ R5 ¼ R6 ¼ CH3: 2-desethyl-
2-propylazithromycin,
22 Ahmed H.H. Bakheit et al.

(E) 30 -(N,N-didemethyl)azithromycin (aminoazithromycin),

(G) 30 -N-demethyl-30 -N-[(4-methylphenyl)sulphonyl]azithromycin,

(H) 30 -N-[[4-(acetylamino)phenyl]sulphonyl]-30 -N-demethylazithro-

mycin,

(J) 13-O-decladinosylazithromycin,
Azithromycin 23

(L) azithromycin 30 -N-oxide,

(M) 30 -(N,N-didemethyl)-30 -N-formylazithromycin

(N) 30 -de(dimethylamino)-30 -oxoazithromycin,

(K) C14, 100 -epoxyazithromycin (azithromycin E),

Unknown structure.

4.1.3 Other tests

4.1.3.1 Water [47]
1.8–6.5%, determined on 0.200 g.

4.1.3.2 Sulphated ash [47]

Maximum 0.2%, determined on 1.0 g.
24 Ahmed H.H. Bakheit et al.

4.1.4 Assay method [47]

4.1.4.1 Liquid chromatography
Solution A: Mix 60 volumes of acetonitrile R1 and 40 volumes of a 6.7 g/l
solution of dipotassium hydrogen phosphate R adjusted to pH 8.0 with
phosphoric acid R.
• Test solution: Dissolve 53.0 mg of the substance to be examined in 2 ml
of acetonitrile R1 and dilute to 100.0 ml with solution A.
• Reference solution (a): Dissolve 53.0 mg of azithromycin CRS in 2 ml
of acetonitrile R1 and dilute to 100.0 ml with solution A.
• Reference solution (b): Dissolve 5 mg of the substance to be examined
and 5 mg of azithromycin impurity A CRS in 0.5 ml of acetonitrile R1
and dilute to 10 ml with solution A.
• Column size: l ¼ 0.25 m, Ø ¼ 4.6 mm; stationary phase: octadecylsilyl
vinyl polymer for chromatography R (5 mm); temperature: 40
C.
• Mobile phase: Mix 60 volumes of acetonitrile R1 and 40 volumes of a
6.7 g/l solution of dipotassium hydrogen phosphate R adjusted to pH
11.0 with a 560 g/l solution of potassium hydroxide R.
• Flow rate, 1.0 ml/min; UV spectrophotometry detection at l 210 nm;
injection, 10 ml; run time, 15 min; retention time of Azithromycin,
10 min; resolution: minimum 3.0 between the peaks of impurity A
and azithromycin.

4.2. Electrochemical methods of analysis

4.2.1 Voltammetry
Studies on the electrochemical oxidation and determination of azithromycin
on glassy carbon and modified glassy carbon electrodes have been frequently
published. The voltammetric determination of azithromycin at a carbon
paste electrode [48], the adsorptive stripping voltammetric determination
of azithromycin at a glassy carbon electrode modified by electrochemical
oxidation [49], and the voltammetric assay of azithromycin in pharmaceu-
tical dosage forms [50] have been published. Also, studies on the electro-
chemical oxidation of azithromycin and its interaction with bovine serum
albumin [51], identification of azithromycin by abrasive stripping
voltammetry [52], and the mathematical modeling of the electrode process
of azithromycin using cyclic voltammetry at a hanging mercury drop elec-
trode [53] can be found in the literature. There is also some data concerning
a validated LC method for in vitro analysis of azithromycin using
Azithromycin 25

electrochemical detection with dual glassy carbon electrodes operating in

the oxidative screen mode [37]. The electro-chemical oxidation of
azithromycin using voltammetry and in situ FTIR spectroscopy to obtain
mechanistic information about the overall process on a platinum electrode
in acetonitrile has also been reported [54].
Avramov et al. [55] examined the oxidative properties and assay of
azithromycin at a gold electrode in neutral electrolyte using cyclic linear sweep
voltammetry. The maximum value of the current of the oxidation peak of pure
azithromycin and azithromycin from tablet dosage form (Hemomycin®,
Hemofarm, Vrsac, Serbia and Montenegro) at 0.6 V versus SCE in 0.05 M
NaHCO3 and in a mixture methanol0.05 M NaHCO3 (1:1) at a scan rate
of 50 mV s1 is a linear function of the concentration in the range of
0.235–0.588 mg/cm3. HPLC analysis of the bulk of electrolyte confirmed
the data obtained by analysis of the values of the current peak concerning
the concentration of antibiotic in the investigated concentration range.
For the determination of the azithromycin concentration, only the first
cycle was recorded, which will be presented later for different concentra-
tions. It is obvious from the cyclic voltammograms of pure azithromycin
in 0.05 M NaHCO3 (Figures 1.14)

4.2.2 Coulometry
A variant of the Karl Fischer water determination was described [25]. By
heating the drug substance, the contained water was transferred into a titra-
tion cell by a carrier gas. The automated system consisted of an oven sample
processor and a coulometer.

4.3. Spectroscopic methods of analysis

4.3.1 Spectrophotometry
Azithromycin was determined in its pharmaceutical dosage forms by the for-
mation of an ion pair between this drug and an inorganic complex of (Mo
(V)-thiocyanate) followed by its extraction with dichloroethane. This ion-
association complex shows an orange color and exhibits a maximum absor-
bance at 469 nm [56].
Su-Ying [57] established a UV spectrophotometry method to determine
the contents of azithromycin tablets quickly and exactly. The concentration
of azithromycin was determined according to related drug dissolubility stan-
dard procedures in pharmacopoeia. Ethanol and 0.1 mol/L hydrochloric
26 Ahmed H.H. Bakheit et al.

0.16

0.08
i-(mAcm–2)

–0.08

–0.16

–1200 –800 –400 0 400 800 1200

E (mV)
Figure 1.14 Cyclic voltammogram of an Au electrode in 0.05 M NaHCO3 (- - -) and after
the addition of 0.588 mg/cm3 of pure azithromycin dihydrate, 30th sweep (full line),
sweep rate: 50 mV/s.

acid were used as dilute solvents and 75 ! 100 sulfuric acid was used as col-
ored solvent. The samples were analyzed at the wavelength of 482 nm.
Huakan et al. [58] developed a spectrophotometric method for the deter-
mination of azithromycin based on the charge transfer reaction between
azithromycin as donor and alizarin as acceptor in ethanol solution. The com-
position ratio and stability constant of charge transfer complex were 1∶1 and
4.8  103, respectively. The apparent molar absorptivity of complex at
546 nm is 5.79  103 L mol1 cm1.
Suhagia et al. [59] developed a simple and sensitive spectrophotometric
method for the determination of azithromycin in its pharmaceutical dosage
forms. In the method, azithromycin is oxidized with potassium permanga-
nate to liberate formaldehyde, which is determined in situ using acetyl ace-
tone, in the presence of ammonium acetate. An yellow colored chromogen
was obtained, having an absorption maxima at 412 nm. The method is
found to be linear in the concentration range of 10–75 mg/ml, with regres-
sion coefficient of 0.9978.
Shu-xia et al. [60] established a method for the determination of
azithromycin tablets dissolution based on charge–transfer reaction with aliz-
arin red. Methods of the dissolution test were conducted, using phosphate
Azithromycin 27

buffer solution as medium, with a stirring speed of 100 rpm/min. The solu-
tion was withdrawn after 45 min. The absorbance of dissolution solutions
was measured at 538 nm. Dissolution limit is 75% of the labeled amount.
Result Good linear correlation was achieved at the range of 50–250 mg/ml
azithromycin (r ¼ 0.9996).
Paula et al. [61] proposed a new method for simple and fast spectropho-
tometric determination of azithromycin in pharmaceutical formulations.
The method is based on the charge transfer reaction between the azithromycin
and quinalizarin in methanol medium. In order to achieve maximum sensi-
tivity, the effect of some chemical variables such as the type of solvent, reagent
concentration, and reaction time was evaluated. The reaction was character-
ized in terms of stability of the product formed and its stoichiometry, and the
apparent molar absorptivity and association constant were derived. Best con-
ditions for the analytical determination of azithromycin were observed in
methanol medium with a quinalizarin concentration of 50 mg L1. At these
conditions, the radical anion (absorbing species) was formed in the medium
immediately after mixing of the reagents and showed maximum absorption
at 564 nm. The method presented a limit of detection of 0.35 mg L1 and
a limit of quantification of 1.2 mg L1.
Sayed et al. [62] developed two simple, accurate, precise, and rapid spec-
trophotometric and conductometric methods for the estimation of erythro-
mycin thiocyanate(I), clarithromycin (II), and azithromycin dihydrate(III) in
both pure and pharmaceutical dosage forms. The spectrophotometric proce-
dure depends on the reaction of rose Bengal and copper with the cited drugs to
form stable ternary complexes which were extractable with methylene chlo-
ride, and the absorbances were measured at 558, 557, and 560 nm for (I), (II),
and (III), respectively. The conductometric method depends on the forma-
tion of an ion-pair complex between the studied drug and rose Bengal.
Ashour et al. [63] developed and validated new, simple, and rapid spec-
trophotometric for the assay of two macrolide drugs, azithromycin (AZT)
and erythromycin (ERY), in pure and pharmaceutical formulations. The
method was based on the reaction of AZT and ERY with sodium 1,2-
naphthoquinone-4-sulphonate in alkaline medium at 25
C to form an
orange-colored product of maximum absorption peak at 452 nm.

4.3.2 Spectrofluorimetry
El-Rabbat et al. [64] described a simple spectrofluorometric method for the
analysis of four macrolide antibiotics. The method is based on the conden-
sation of 10% (w/v) malonic acid and acetic acid anhydride under
28 Ahmed H.H. Bakheit et al.

the catalytic effect of tertiary amine groups of the studied macrolides. The
relative fluorescence intensity of the condensation product was measured at
397/452 nm (excitation/emission) for azithromycin dihydrate and at 392/
445 nm (for clarithromycin, erythromycin ethylsuccinate, and
roxithromycin.
Almeida et al. [65] proposed a fluorescence method for azithromycin
determination in pharmaceutical formulations. The method is based on
the synchronous fluorescence (Dl ¼ 30 nm, 482 nm) produced when
azithromycin is derivatized in strong acidic medium (9.0 mol L1 HCl).
Khashaba [66] analyzed the macrolides (erythromycin, erythromycin
esters, azithromycin dihydrate, clarithromycin, and roxithromycin) by a
simple spectrofluorimetric method based on the oxidation by cerium (VI)
in the presence of sulfuric acid and monitoring the fluorescence of cerium
(III) formed at lex 255 nm and lem 348 nm.

4.3.3 Colorimetry
Hunfeld et al. [67] used a newly developed colorimetric microdilution
method to analyze the activity of 12 antimicrobial agents against nine Borrelia
burgdorferi isolates, including all three genospecies pathogenic for humans. In
addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and
Borrelia bissettii tick isolates were investigated. The applied test system is
based upon color changes that occur in the presence of phenol red and result
from the accumulation of nonvolatile acid produced by actively metaboliz-
ing spirochetes. After 72 h of incubation, minimal inhibitory concentrations
(MICs) were determined from the decrease of absorbance by software-
assisted calculation of growth curves. MIC values were lowest for azlocillin
(MIC, 0.125 mg/ml), ceftriaxone (MIC range, 0.015–0.06 mg/ml), and
azithromycin (MIC range, 0.015–0.06 mg/ml). Whereas tobramycin
(MIC range, 8–64 mg/ml) exhibited little activity, spectinomycin (MIC
range, 0.25–2 mg/ml) showed in vitro antimicrobial activity against
B. burgdorferi.
Haleem et al. [68] developed a simple, accurate, and rapid spectropho-
tometric method for the estimation of azithromycin by the acidic hydrolysis
of the drug with sulfuric acid and monitoring the absorbance at 482 nm.

4.4. Chromatographic methods of analysis

4.4.1 Electrophoresis
Kumar et al. [69] described the use of AZM as a chiral selector for the enan-
tiomeric separations of five chiral drugs and one amino acid (tryptophan) in
Azithromycin 29

capillary electrophoresis (CE). The enantioseparation is carried out using

polar organic mixtures of acetonitrile (ACN), methanol (MeOH), acetic
acid, and triethylamine as run buffer. The influences of the chiral selector
concentration, ACN/MeOH ratio, applied voltage, and capillary tempera-
ture on enantioseparation are investigated. The results shown that AZM is a
viable chiral selector in CE for the enantioseparation of the type of chiral
drugs investigated.
Lebedeva et al. [70] described successful use of macrolide antibiotic
azithromycin for enantioseparation of tetrahydrozoline. The procedure
was proposed for the analysis of tetrahydrozoline in pharmaceuticals. Line-
arity was achieved in the concentration range 5  102 to 1 mg/ml. The
azithromycin stability in the background electrolyte and the antibiotic
adsorption on the fused-silica capillary were studied. Best enantioseparation
with resolution factor 1.6 was achieved in less than 10 min.

4.4.2 Thin-layer chromatography

Kwiecie n et al. [71] established a thin-layer chromatographic (TLC) method
with densitometric detection for quantification of azithromycin in pharma-
ceutical preparations. Silica gel plates with fluorescence indicator F254 were
used with chloroform–ethanol–ammonia 6:14:0.2 (v/v) as mobile phase.
Chromatograms were visualized by spraying with 1:4 (v/v) sulfuric
acid–ethanol and heating at 120
C for 5 min. Scanning and densitometric
analysis was performed at 483 nm. The RF of azithromycin under these con-
ditions was 0.53. The method was characterized by high sensitivity
(LOD ¼ 40 ng/zone and LOQ ¼ 80 ng/zone), wide linear range (from
0.08 to 1.2 mg/zone, r ¼ 0.9965)
Khedr et al. [72] described a validated stability-indicating TLC method of
the analysis of azithromycin (AZT) in bulk and capsule forms. Both AZT
potential impurity and degradation products can be selectively and accurately
estimated in both raw material and product onto one precoated silica-gel TLC
plate 60F254. The development system used is n-hexane–ethyl acetate
diethylamine (75:25:10, v/v/v). The separated bands are detected as brown
to brownish red spots after spraying with modified Dragendorff’s solution.
The RF values of AZT, azaerythromycin A, and the three degradation prod-
ucts are 0.54, 0.35, 0.40, 0.20, and 0.12, respectively.

4.4.3 High-performance liquid chromatography

Liquid chromatography with UV detection has been already employed for
the analysis of azithromycin in azithromycin tablets [39], raw material, and
30 Ahmed H.H. Bakheit et al.

azithromycin tablets [36,73]. A high-performance liquid chromatography

(HPLC) method with UV detector was developed for the determination
of azithromycin and other related compounds, impurities, degradation
products in raw material as well as in new pharmaceutical formulations:
dry suspension and capsules. Validation of the method was performed
according to the requirements of USP for assay determination, which
included accuracy, precision, specificity, linearity, and range.
An HPLC method was developed that confirmed the photodegradation
of azithromycin in environmental waters, under simulated solar
radiation [74].
A stability-indicating HPLC method has been described for
azithromycin in raw materials, capsules, and suspension [75].
Other HPLC methods for the analysis of AZI in raw material, dosage
forms, and biological samples that have been reported in the literature are
listed in Table 1.4.

4.5. Determination in body fluids and tissues

Azithromycin concentrations versus time profiles in extracellular space of
muscle and subcutaneous adipose tissue, also in plasma and white blood cells,
were determined at days 1 and 3 of treatment as well as 2 and 7 days after end
of treatment. Of all compartments, azithromycin concentrations were
highest in white blood cells, attesting for intracellular accumulation. How-
ever, azithromycin concentrations in both soft tissues were markedly lower
than in plasma both during and after treatment. Azithromycin concentra-
tions were measured by subinhibitory at all time points in both soft tissues
and at the large majority of observed time points in plasma [76].

5. STABILITY
El-Gindy et al. [75] developed a validated stability-indicating HPLC
method for the analysis of azithromycin (AZ) and its related compounds
in raw materials and capsules. The stability of AZ was studied under accel-
erated acidic, alkaline, and oxidative conditions. The major peak detected
from the degradation of AZ in alkaline and acidic conditions was
decladinosylazithromycine, while azithromycin N-oxide was detected from
the oxidative degradation. Long-term stability studies for capsule and oral
suspension were also carried out.
Table 1.4 HPLC methods for the analysis of azithromycin
Column Sample matrix Mobile phase composition Detection References
Gamma-alumina Raw material Phosphate buffer–acetonitrile, Amperometric guard: [35]
adjusted to pH 11.0 with potassium þ0.70 V screen:
hydroxide þ0.85 V
LiChroCART® C18, 5 mm Raw material Phosphate buffer–acetonitrile– UV 215 nm [36]
methanol, adjusted to pH 8.0 with
phosphoric acid
Nova-Pack C18, 4 mm Raw material Ammonium acetate–acetonitrile– Amperometric guard: [37]
methanol tetrahydrofuran, mobile þ0.7 V screen: þ0.8 V
phase pH 7.2–7.4
XTerra RP C18, 5 mm Raw material Phosphate buffer–water– UV 215 nm [38]
acetonitrile, adjusted to pH 6.5
with potassium hydroxide
Phenomenex Synergi® C18, 4 mm Raw material, dosage Gradient elution, phosphate UV 210 nm [39]
forms buffer–acetonitrile–methanol,
adjusted to pH 7.0 with potassium
hydroxide
YMC-Park ODS-AP C18, 5 mm Rat’s plasma Phosphate buffer–acetonitrile, Amperometric detect: [40]
adjusted to pH 7.2 with potassium þ0.95 V
hydroxide
Continued
Table 1.4 HPLC methods for the analysis of azithromycin—cont'd
Column Sample matrix Mobile phase composition Detection References
Nova-Pack C18, 4 mm Human tears and Phosphate buffer–sodium Amperometric guard: [41]
plasma perchlorate–acetonitrile–methanol, þ0.7 V screen:
adjusted to pH 7.0 with phosphoric þ0.85 V
acid
Radial-Pak Resolve Silica Rat’s blood plasma, Ammonium acetate–acetonitrile– Coulometric guard: [42]
cartridge, 5 mm serum, and human methanol, adjusted to pH 7.0 with þ0.90 V
urine acetic acid
C18 (250 mm  4.6 mm, 5 mm) Azithromycin syrup Acetonitrile 0.067 mol L1 UV 210 nm [43]
K2HPO3 (pH adjusted to 6.5)
(40∶60)
ODS-C18 column Eye drops Acetonitrile and 0.1 mol L1 UV 215 nm [44]
(150 mm  4.6 mm, 5 mm) KH2PO4 as the mobile phase
(30∶70)
CAPCELL PAK C(18) Azithromycin Acetonitrile phosphate buffer UV 210 nm [45]
MGIIcolumn (250 mm  4.6 mm, capsules (to dissolve dipotassium hydrogen
5 mm) phosphate 8.7 g diluting to 1000 ml
with water, and adjust pH to 8.2
with phosphoric acid) (60:40)
Dikma Technologies Diamonsil Ammonium dihydrogen phosphate UV 210 nm [46]
C18 column (150 mm  4.6 mm, (0.045 M, pH 3.0 adjusted by
5 mm) phosphoric acid):acetonitrile 47:15
(v/v)
Azithromycin 33

Moreno et al. [77] were developed a stability study of azithromycin in

ophthalmic preparations by submission to different types of light, tempera-
ture, and pH, using the biodiffusion assay (cylinder 3  3) for the quantifi-
cations. Bacillus subtilis, ATCC 9372, was used as test organism. The used
concentration range was of 50–200 mg/ml.

6. CLINICAL APPLICATIONS
6.1. An overview
Azithromycin is the member of macrolide antibiotics. It is semisynthetic
derivatives of erythromycin. Azithromycin differs from erythromycin by
the addition of a methyl-substituted nitrogen atom into the lactone ring.
This structural modification improved acid stability and tissue penetration
and broaden the spectrum of activity. Macrolides generally cover a wide
range of Gram-positive and Gram-negative bacterial species including intra-
cellular pathogens such as Chlamydia and Legionella. They express their
antibiotic activity by binding to the 50S ribosome subunit and inhibit pro-
tein synthesis [78,79].
Azithromycin pharmacology and therapeutics aspects were given below
in more details.

6.2. Antimicrobial spectrum susceptibility

Azithromycin is less active against Gram-positive bacteria than erythromy-
cin but is considerably more effective against some Gram-negative organ-
isms such as Haemophilus influenzae, Moraxella catarrhalis as well as having
activity against some of the Enterobacteriaceae such as Escherichia coli and
Salmonella and Shigella species; Also activity against Legionella pneumophila,
B. burgdorferi, Mycoplasma pneumoniae. Also it is owes enhanced activity
against Mycobacterium avium-intracellulare, as well as against some protozoa
(e.g., Cryptosporidium, and Plasmodium species, an excellent action against
Toxoplasma gondii, killing the cysts). Azithromycin more active than eryth-
romycin against Chlamydia trachomatis and Ureaplasma urealyticum, [79,80].

6.3. Mechanism of action

It is a bacteriostatic agent that inhibits protein synthesis by binding reversibly
to 50S ribosomal subunits of sensitive microorganisms. Cells are consider-
ably more permeable to the unionized form of the drug, which probably
explains the increased antimicrobial activity at alkaline pH [79].
34 Ahmed H.H. Bakheit et al.

6.4. Resistance to macrolides

Generally, resistance to macrolides results from the following mechanisms:
• Drug efflux by an active pump mechanism
• Ribosomal protection by inducible or constitutive production of meth-
ylase enzymes, mediated by expression of (ermA), (ermB), and (ermC),
which modify the ribosomal target and decrease drug binding
• Hydrolysis by esterases produced by Enterobacteriaceae
• Chromosomal mutations that alter a 50S ribosomal subunit protein
(found in B. subtilis, Campylobacter species, mycobacteria, and Gram-
positive cocci) [79].

6.5. Actions other than antimicrobial effects

• Studies were indicated the activity of azithromycin as promising medi-
cation for the treatment of gastroparesis and gastrointestinal dysmotility
[81,82].
• One study showed that azithromycin may be effective against late-onset
asthma [83].
• Azithromycin has been shown to be effective against malaria when used
in combination with artesunate or quinine. Azithromycin–artesunate,
even when given only once daily for 3 days, and azithromycin–quinine,
given 3 times daily, are safe and efficacious combination treatments for
uncomplicated falciparum malaria, and they deserve additional study in
special patient populations [84].
• Ischaemic heart disease: Macrolide antibacterials, including azithromycin,
clarithromycin, and roxithromycin, have been investigated in the preven-
tion of ischaemic heart disease, based on a suggested link between athero-
sclerosis and infection with Chlamydophila pneumoniae (Chlamydia
pneumoniae). Although preliminary results from some pilot studies were
promising, longer-term studies in large numbers of patients were disap-
pointing and none of the three macrolides decreased ischemic events or
provided clinical benefit; indeed, in one study, an unexpected increase in
cardiovascular mortality was seen in those taking clarithromycin [79,86,87].
• Gingival hyperplasia: Azithromycin improves cyclosporin-associated gin-
gival hyperplasia, especially when administered early in the process [88].
• Cystic fibrosis: Long-term azithromycin is widely used in cystic fibrosis,
with evidence demonstrating a reduction in lung function decline and
exacerbation rate. This immunomodulatory therapy probably disrupts
Pseudomonas aeruginosa biofilm growth [89].
Azithromycin 35

6.6. Clinical uses and dosing

Clinically, azithromycin used for many as follows: respiratory-tract infec-
tions, otitis media, skin and soft-tissue infections, uncomplicated genital
chlamydial infections and nongonococcal urethritis, mild or moderate
typhoid due to multiple-antibacterial-resistant organisms, prophylaxis of
group A streptococcal infection and as prevention therapy of bacterial endo-
carditis in patients undergoing dental procedures who are at high risk for
endocarditis, pertussis, mycobacterial infections [79,88,90].
• Treatment or prophylaxis of Mycobacterium avium-intracellulare infection
in AIDS patients requires higher doses: 600 mg daily in combination
with one or more other agents for treatment, or 1200 mg once weekly
for primary prevention.
• Azithromycin is useful in the treatment of sexually transmitted diseases,
especially during pregnancy when tetracyclines are contraindicated. The
treatment of uncomplicated nongonococcal urethritis presumed to be
caused by C. trachomatis consists of a single 1-g dose of azithromycin.
This dose also is effective for chancroid [79].
• Azithromycin (1 g per week for 3 weeks) is an alternative regimen for the
treatment of granuloma inguinale or lymphogranuloma venereum [79].
• Uncomplicated genital chlamydial infections and nongonococcal
urethritis, 1 g as a single dose [90].
• Typhoid, 500 mg once daily for 7 days [90].

6.7. ADME profile

Azithromycin administered orally is absorbed rapidly and distributes widely
throughout the body, except to the brain and cerebrospinal fluid. Peak
plasma concentrations occur 2–3 h after an oral dose. A 500-mg loading dose
will produce a peak plasma drug concentration of 0.4 g/ml. When this
loading dose is followed by 250 mg once daily for 4 days, the steady-state
peak drug concentration is 0.24 g/ml. Azithromycin also can be adminis-
tered intravenously, producing plasma concentrations of 3–4 g/ml after a
1-h infusion of 500 mg. Absorption from capsules, but not tablets or suspen-
sion, is reduced by food. Azithromycin’s unique pharmacokinetic properties
include extensive tissue distribution and high drug concentrations within
cells (including phagocytes), resulting in much greater concentrations of
drugs in tissue or secretions compared to simultaneous serum concentra-
tions. Tissue fibroblasts act as the natural reservoir for the drug in vivo. Data
from animal studies indicate that azithromycin crosses the placenta. Protein
36 Ahmed H.H. Bakheit et al.

binding is 50% at very low plasma concentrations and less at higher concen-
trations. Azithromycin undergoes some hepatic metabolism (demethylation)
to inactive metabolites, but biliary excretion is the major route of elimina-
tion. Only 12% of drug is excreted unchanged in the urine. The elimination
half-life (t1/2), 40–68 h, is prolonged because of extensive tissue sequestra-
tion and binding [79,85].

6.8. Side effects

Anorexia, dyspepsia, flatulence, dizziness, headache, drowsiness, convul-
sions, arthralgia, and disturbances in taste and smell; rarely constipation, hep-
atitis, hepatic failure, syncope, insomnia, agitation, anxiety, asthenia,
paraesthesia, hyperactivity, thrombocytopenia, haemolytic anaemia, inter-
stitial nephritis, acute renal failure, photosensitivity, tooth and tongue
discoloration [90].

6.9. Drug interactions

• Azithromycin, generally appear to be free of drug interactions. Caution
is advised, nevertheless, when using azithromycin in conjunction with
drugs known to interact with erythromycin [80].
• Giving azithromycin with antacids containing aluminum or magnesium
salts can reduce the rate, but not the extent, of its absorption;
azithromycin should be given at least 1 h before or 2 h after the
antacid [85].
• Azithromycin serum concentrations are markedly increased when it is
given with nelfinavir [85].
• Azithromycin capsules should not be administered with food because it
will result in reduced absorption.
• Azithromycin possibly enhances anticoagulant effect of coumarins [90].
• Azithromycin possibly increases plasma concentration of theophylline [90].

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