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International Journal of Pharmaceutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

1 Rapid communication

2 Microbiological assay for analysis of certain macrolides in

3 pharmaceutical dosage forms
4 Q1 A. Mahmoudia,* , R.E.-A. Fourarb , S. Zarkouta
5 a
Laboratory of Research on Bioactive Products and Biomass Valorization (LRPBVB), Ecole Normale Supérieure—Kouba, P.O. Box 92, Kouba,
6 16050 Algiers, Algeria
7 b
Laboratory of Biology of Microbial Systems (LBMS), Ecole Normale Supérieure—Kouba, P.O. Box 92, Kouba, 16050 Algiers, Algeria

A R T I C L E I N F O A B S T R A C T

Article history: Clarithromycin (CLA) and roxithromycin (ROX) are macrolide antibiotics with an expanded spectrum of
Received 22 April 2015 activity that are commercially available as tablets. A microbiological assay, applying the cylinder–plate
Received in revised form 16 June 2015 method and using a strain of Micrococcus luteus ATCC 9341 as test organism, has been used and validated
Accepted 18 June 2015
for the quantification of two macrolide drugs; CLA and ROX in pure and pharmaceutical formulations. The
Available online xxx
validation of the proposed method was carried out for linearity, precision and accuracy. The linear
dynamic ranges were from 0.1 to 0.5 mg/mL for both compounds. Logarithmic calibration curve was
Keywords:
obtained for each macrolide (r > 0.989) with statistically equal slopes varying from 3.275 to 4.038, and a
Clarithromycin
Roxithromycin
percentage relative standard deviation in the range of 0.24–0.92%. Moreover, the method was applied
Microbiological assay successfully for the assay of the studied drugs in pharmaceutical tablet dosage forms. Recovery from
Cylinder–plate method standard addition experiments in commercial products was 94.71–96.91% regarding clarithromycin and
Micrococcus luteus 93.94–98.12% regarding roxithromycin, with a precision (%RSD) 1.32–2.11%. Accordingly, this
microbiological assay can be used for routine quality control analysis of titled drugs in tablet formulation.
ã2015 Elsevier B.V. All rights reserved.

8 1. Introduction Clarithromycin is a semi-synthetic 6-o-methyl derivative of the 26

14-member macrolide antibiotic erythromycin, and the O-methyl 27

9 Macrolide antibiotics have been used clinically for more than group attached to the position six of the lactone makes it more acid 28
10 50 years. They are a very important class of antibacterial stable than erythromycin (Lu et al., 2009). Roxithromycin-9-{O- 29
11 compounds extensively used to treat a wide range of infections, [(2-methoxyethoxy)-methyl]-oxime}-erythromycin is a semi-syn- 30
12 not only in medical but also in veterinary practices. The macrolides thetic, 14-membered ring macrolide antibiotic, in which the 31
13 inhibit RNA-dependent protein synthesis by reversibly binding to erythronolide A lactone ring has been altered to prevent 32
14 the 50S ribosomal subunit of a susceptible microorganism (Omura, inactivation in the gastric environment (Głowka and Kar- 33
15 2002). In addition to erythromycin A, the prototype macrolide, a
zniewicz-Łada, 2007). These new semi-synthetic erythromycin 34
16 several other macrolides, clarithromycin and roxithromycin are derivatives have a better bioavailability and a more favorable 35
17 clinically available (Payne et al., 2007). Besides its antibacterial pharmacokinetic profile than erythromycin. Many dosage forms of 36
18 activity, macrolides exert diverse biological effects including the both drugs, such as tablets, have been developed based on their 37
19 ability to modulate inflammatory and immune responses without wide antibacterial spectrum. 38
20 affecting homeostatic immunity (Mencarelli et al., 2011). More Determination of antibiotics, including macrolides, is mainly 39
21 recently, macrolides have been also considered for other uses in carried out by microbiological assays (Horwitz, 2000). These 40
22 the areas of neurology, gastroenterology, rheumatology, cardiolo- assays excel as a qualitative means by which samples may be 41
23 gy, and cancer therapy, facilitating the development of new screened for residual amounts of antibacterial substances, but they 42
24 synthetic methods as well as the design of new therapeutic agents are often lengthy and lack the specificity. Chemical methods such 43
25 Q2 possessing non-antibacterial activities (Miroshnyk et al., 2008). as high-performance liquid chromatography (HPLC) are 44

appropriate alternatives. Some review articles published recently 45

have covered literature on the analysis of macrolide antibiotics 46

(Kanfer et al., 1998; Lahane et al., 2014), and several HPLC methods 47
* Corresponding author. Fax: +213 21 2820 67.
E-mail address: mahmoudi_a2003@yahoo.fr (A. Mahmoudi). have been developed. 48

http://dx.doi.org/10.1016/j.ijpharm.2015.06.027
0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Mahmoudi, A., et al., Microbiological assay for analysis of certain macrolides in pharmaceutical dosage forms.
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2 A. Mahmoudi et al. / International Journal of Pharmaceutics xxx (2015) xxx–xxx

49 A large number of reports in the literature have been published Dipotassium hydrogen phosphate was of analytical-reagent 113
50 for the quantitative determination of CLA and ROX in raw material, grade from SIGMA–ALDRICH (Steinheim, Germany). Methanol 114
51 dosage forms and biological fluids. The analytical methods was of HPLC grade from the same source. All other used reagents 115
52 reported include microbiological bioassay, spectrophotometry were of analytical grade. Doubly distilled water was used 116
53 Q3 (Jagtap et al., 2013), and HPLC with fluorescent (Głowka and throughout. 117
54 Kara zniewicz-Łada, 2007; Sastre and Guchelaar, 1998), ultraviolet
55 (UV) (Głowka and Kara zniewicz-Łada, 2007; Li et al., 2007), 2.1. Preparation of standard solutions 118
56 electrochemical (Pappa-Louisi et al., 2001), amperometric
57 Q4 (Taninaka et al., 2000) and mass spectrometric detection Stock standard solutions of CLA and ROX were prepared by 119
58 (Shin et al., 2008; Verlag, 2009). A spectrofluorimetric method dissolving an accurately weighted amount of each compound in 120
59 has been described (Khashaba, 2002) for the analysis of several methanol (1000 mg/mL). Working standard solutions were 121
60 macrolides including CLA and ROX. Also, UV is the most common prepared by diluting the stock solution with 0.025 M dipotassium 122
61 detection system. However, macrolides like clarithromycin and hydrogen phosphate buffer pH 8.0. The solutions were prepared 123
62 roxithromycin lack a suitable chromophore and non-selective freshly every day to be used as working standards at 124
63 low-UV wavelength must be used. Generally, these methods were concentrations of 0.1, 0.3 and 0.5 mg/mL, which were used in 125
64 time-consuming, tedious, and dedicated to sophisticated analytical the assay as reference solutions. 126
65 instruments which is expensive and could not be available in many
66 laboratories. 2.2. Preparation of the tablet samples 127
67 Among drug quantification methods, the microbiological assays
68 are recommended by different pharmacopoeias (United States 2.2.1. Clarithromycin tablets 128
69 Pharmacopeia, 2007; British Pharmacopoeia, 2011) and many The sample preparation was done as follows based on previous 129
70 publications. This experimental assay was the official method papers (Khashaba, 2002; Breier et al., 2002). Ten tablets were 130
71 provided by the Japanese Ministry of Health and Welfare (Official weighed and finely powdered. A weighed portion equivalent to the 131
72 Methods for Residual Substances in Livestock Products, 1994). In weight of one tablet was transferred to a 100 mL volumetric flask, 132
73 majority of studies, paper disk procedure was employed for the sonicated for 20 min with about 10 mL methanol then the solution 133
74 determination of drug antibacterial activity. However, other was completed to volume with the same solvent. The mixture was 134
75 methods including standard dilution assay (Ma et al., 2009), mixed well, allowed any insoluble matter to settle then filtered. A 135
76 standard broth microdilution (Kapi c et al., 2011), and monitoring measured volume of the filtrate was diluted quantitatively with the 136
77 the turbidity (Lange et al., 2006) were used. No official potassium hydrogen phosphate buffer pH 8 solvent to yield a 137
78 microbiological cylinder–plate assays have been reported for the sample solution having a working concentration of 0.1, 0.3 and 138
79 identification and quantification of CLA and ROX in pharmaceutical 0.5 mg/mL. This sample was evaluated in triplicate. This procedure 139
80 formulations. From the different classes of microorganisms was performed two times. 140
81 frequently used to test the presence of residual antibiotics,
82 Micrococcus luteus ATCC 9341, was strong, and its sensitivity of 2.2.2. Roxithromycin tablets 141
83 the drug detection was sufficient. The procedure was followed as mentioned in Section 2.2.1. An 142
84 In the study by Breier et al. (2002), investigation of azithromycin amount equivalent to 150 mg of drug was transferred to 100 mL 143
85 concentration present in pharmaceutical formulations was volumetric flask with 10 mL methanol and shaken for 20 min, 144
86 successfully done using cylinder–plate method and M. luteus ATCC followed by marking up to volume with the same solvent. After 145
87 9341 as test organism. However, there are few reports applying filtration, the dilutions were made, to give the same final 146
88 such as technique described in the literature for macrolides concentrations used above. This sample was evaluated in triplicate. 147
89 determination in tablet dosage forms and, to our knowledge, none This procedure was performed two times. 148
90 of them includes clarithromycin and roxithromycin. All preparations were kept at 4
C, protected from light in amber 149
91 In the present manuscript, quantitative analysis of the new glass vessels and periodically tested. 150
92 semi-synthetic 14-membered macrolide antibiotics; clarithromy-
93 cin and roxithromycin in tablets, as dosage forms of certain local 2.3. Microbiological assay 151
94 markets from different manufacturers, have been studied.
95 Therefore, a simple, linear, precise and accurate cylinder–plate A microbiological assay, adapted from the method described in 152
96 method based on agar diffusion was performed. the US Pharmacopoeia applying the cylinder-plate diffusion 153

technique was used to assay CLA and ROX in tablet dosage forms 154
97 2. Materials and methods (United States Pharmacopeia, 2007). The parallel-line model was 155

chosen, and differences were considered statistically significant if 156

98 Clarithromycin standard is a kind gift from SAIDAL GROUPE P was <0.05. 157
99 (Saidal, Algiers, Algeria). Roxithromycin was obtained from HUP.P.
100 Pharma laboratories (HUP.P.Pharma, Constantine, Algeria) and 2.3.1. Organism and inoculum 158
101 kindly provided by Biochim Company (Tipaza, Algeria), while Briefly, the bioanalytical method was implemented, as 159
102 pharmaceuticals containing these macrolide antibiotics were indicated by Breier et al. (2002), with slight modifications. 160
103 obtained commercially. Claridar1 tablets (Dar Al Dawa, Therefore, the M. luteus strain (ATCC 9341) was grown on selective 161
104 Cheraga—Algiers, Algeria): labeled to contain clarithromycin as agar at culture medium pH 8.0 for 24 h at 35
C. 162
105 500 mg/tablet; Clarimed1 tablets (Saidal, Algiers, Algeria): labelled
106 to contain clarithromycin as 500 mg/tablet; Roxid1 tablets 2.3.2. Cylinder–plate assay 163
107 (Pharmalliance, Ouled.fayet—Algiers, Algeria): labeled to contain For each macrolide, nine stainless steel cylinders of uniform 164
108 roxithromycin as 150 mg/tablet; Roxithromycine HUP1 tablets size (8 mm  6 mm  10 mm) were placed on the solidified surface 165
109 (HUP.P.Pharma, Constantine, Algeria): labeled to contain of the seeded agar. Three alternate cylinders were filled with 166
110 roxithromycin as 150 mg/tablet, respectively. All drugs were used 200 mL of reference solutions (three concentrations) or problem 167
111 as received and their solutions were prepared freshly every day to samples. Each assay was designed to evaluate in the same plate the 168
112 be used as working standards. reference solutions and two different samples. Eight plates were 169

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170 used in each assay. After incubation (24 h at 35
C) the zone 2.5. Methods comparison 229
171 diameter (in mm) of the growth inhibition were measured using a
172 caliper. It was analyzed four samples using the proposed proceeding 230

and by HPLC. The methods used in determination by HPLC were 231

173 2.3.3. Calculation of assay results described by Lu et al. (2009) and Chepkwony et al. (2001). 232
174 The general technique used for data evaluation is the
175 analysis of variance (ANOVA). A theoretical basis for this 3. Results and discussion 233
176 technique is detailed in Ref. Commissariat à l’Energie Atomique
177 (1978). The aim is to determine if a controlled factor had a 3.1. Analytical method 234
178 significant influence on the dispersion of the results access by
179 comparing mean squares through a statistical test. The response The development of analytical methods for the determination 235
180 used was the assay result. The data population must be of drugs has received considerable attention in recent years 236
181 Gaussian, which is a necessary condition for the regression because of their importance in pharmaceutical analysis. Analytical 237
182 analysis to be applicable. methods used for the quantitative determination of substances are 238
183 The assay was statistically calculated by the linear parallel the key determinants in generating reproducible and reliable data 239
184 model and by means of Student’s t-test with a P < 0.05 level which in turn are used in quality control of the medicines 240
185 considered statistically significant as mentioned above (United (Shah et al., 2000). In this context, the choice of a suitable 241
186 States Pharmacopeia, 2007; Hewitt, 1977). analytical methodology is fundamental for quality control of an 242

active substance. Moreover, the availability of equipments and 243

187 2.4. Validation of method reagents should be considered for the development of accessible 244

and useful methodologies. 245

188 The method was validated according to frequently Considering that the potency of an antibiotic may be 246
189 recommended references (United States Pharmacopeia, 2007; demonstrated under suitable conditions by comparing the 247
190 British Pharmacopoeia, 2011; ICH—Guidelines Validation of inhibition of growth of susceptible microorganisms induced by 248
191 Analytical Procedures, 2011). These documents describe the known concentrations of the antibiotic to be tested and the 249
192 validation of linearity, recovery, precision and curacy for a reference standard (United States Pharmacopeia, 2007; British 250
193 quantitative confirmation method. Pharmacopoeia, 2011), a microbiological assay was proposed 251

as a suitable method for determination of clarithromycin and 252

194 2.4.1. Linearity roxithromycin in tablets. 253
195 Standards at concentrations ranging from 0.1 to 0.5 mg/mL of In microbiological agar diffusion method for antibiotics, two 254
196 CLA and ROX were prepared from stock standard solution. In order phenomena occur simultaneously: (a) the diffusion of antibiotic; 255
197 to study linearity, each sample was tested six times in agreement to and (b) microbiological growth. The inhibition zone diameters 256
198 the International Conference on Harmonization (ICH). Calibration depend on these two phenomena. The susceptibility of microor- 257
199 graphs were obtained by measurement of diameters of the ganism-test may significantly affect the inhibition zone diameters 258
200 inhibition zones. The proposed method was evaluated by its (Hodges, 2001). The experimental conditions of the proposed 259
201 correlation coefficient and intercept value, calculated in the method were tested and adjusted to accurately determine the 260
202 corresponding statistical study. performance of the assay. Some parameters were tested earlier to 261

establish the conditions, and a strain of M. luteus ATCC 9341 was 262
203 2.4.2. Precision shown to be an appropriate test microorganism because of its 263
204 Precision was assessed by performing replicate analysis of sensitivity to CLA and ROX and ability to form sharply defined 264
205 tablet samples. Intra-day repeatability of the described method inhibition growth zones, allowing accurate measurements. For 265
206 was determined using different-independent test solutions at the handling microorganisms, all safety procedures (wearing masks, 266
207 same day. The intermediate precision of the assay method was gloves and goggles) were adopted. All assays were performed 267
208 also evaluated using different analyst on three different days in a laminar air flow cabinet, and the infected material was 268
209 (eight replicates per concentration). The precision was expressed decontaminated before being discarded. 269
210 as RSD. The assay of antibiotics must be designed in a way that will 270

permit examination of the validity of the mathematical model on 271

211 2.4.3. Accuracy which the potency equation is based. If a parallel-line model is 272
212 The accuracy was carried out by adding known amounts of chosen, the two-log dose–response line of the preparations to be 273
213 macrolide reference substance to the samples at the beginning of examined and the standard preparation must be linear over the 274
214 the process. Amounts of 3, 3 and 15 mg of macrolide were placed in range of doses used in the calculation (United States Pharmacopeia, 275
215 300, 100, and 300 mL volumetric flask, respectively, where 1.0, 2007). The microbiological assay described in this work was 276
216 1.0 and 5.0 mL of macrolide reference solution (300 mg mL1) were performed in 3  3 designs (three dose levels of reference and 277
217 added (in this order). Dilutions were made in potassium phosphate three dose levels of sample), according to the United States 278
218 buffer, pH 8.0, to give final concentrations of 0.11, 0.33 and Pharmacopeia (2007). 279
219 0.55 mg/mL. The solutions were submitted the cylinder–plate assay
220 described above. The percentage recovery of macrolide reference 3.2. Validation parameters 280
221 added was calculated using the equation proposed by AOAC
222 (Horwitz, 2000). Analytical methods need to be validated and compared with 281

existing traditional methods that are approved by various 282

223 2.4.4. Specificity bodies. The validation parameters for the microbiological 283
224 The previous tests proposed with the samples in comparison to method were therefore addressed according to various 284
225 the standard should detect excipients and/or probable impurities documents (United States Pharmacopeia, 2007; British 285
226 and in this case the contents would propositional to the Pharmacopoeia, 2011; ICH—Guidelines Validation of 286
227 microbiological inhibition. The Student’s t-test was performed to Analytical Procedures, 2011). The parameters validated were 287
228 compare the macrolide standard and sample values. linearity, accuracy, precision. According to the recommended 288

Please cite this article in press as: Mahmoudi, A., et al., Microbiological assay for analysis of certain macrolides in pharmaceutical dosage forms.
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289 Pharmacopoeias the limits of detection and quantification are Calibration graphs were constructed by plotting the diameters 309
290 not required for this category of assay. of the inhibition zones (mm) against the logarithm of the drug 310

concentrations (mg/mL) (Fig. 2). 311

291 3.3. Linear working range Table 1 enlists the calibration curve parameters for the both 312

drugs. A good linear relationship was found, as described by the 313

292 With the present study, microbiological assays of CLA and ROX following linear regression equations: y = 3109 Ln x + 8090 and 314
293 were performed by cylinder–plate method as described previously. y = 4053 Ln x + 6910 for the ROX and CLA, respectively. In the 315
294 The results are expressed as diameters of the inhibition zones. proposed method, satisfactory correlation coefficients (greater 316
295 Least square regression analysis was carried out for getting the than 0.989) were obtained in the concentration ranges selected. 317
296 slope, intercept, correlation coefficient and the relative standard
297 deviation values. The results of mean zone diameters for reference 3.4. Accuracy and precision evaluation 318
298 solutions were: 15.22–16.31 mm (RSD = 0.24–0.30) for low dose
299 (0.1 mg/mL), 18.76–20.49 mm (RSD = 0.55–0.71) for medium dose Performing replicate analysis of tablet samples of macrolides 319
300 (0.3 mg/mL) and 20.19–22.91 mm (RSD = 0.89–0.92) for high dose assessed precision and accuracy. Precision was evaluated by 320
301 (0.5 mg/mL) for ROX and CLA, respectively. studying repeatability and intermediate precision. 321
302 According to the experimental values obtained, it appears that
303 ROX possess better activity against M. luteus ATCC 9341 than CLA. 3.4.1. Precision 322
304 These results suggested that introduction of the N-substituent Precision is the measure of the degree of repeatability of an 323
305 moiety into the 9-position (Fig. 1) can enhance antibacterial analytical method under normal operation and is normally 324
306 activity against test strain resistant comparing to CLA. expressed as the percent relative standard deviation (RSD) for a 325
307 Linearity was determined for clarithromycin and for statistically significant number of samples. According to the 326
308 roxithromycin in the concentration range of 0.1–0.5 mg/mL. ICH—Guidelines Validation of Analytical Procedures (2011), 327

y = 3,109 Ln x + 8,090 (A)

Mean diameter zone of inhibition (mm)
20
R² = 0,998

15

10

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0
Log of concentration (µg/mL)

y = 4,053 Ln x + 6,910 (B)

Mean diameter zone of inhibition (mm)

R² = 0,997

20

15

10

0
0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0
Log of concentration (µg/mL)

Fig. 2. Calibration curves for clarithromycin (A) and roxithromycin (B) obtained by
Fig. 1. Chemical structure of clarithromycin (A) and roxithromycin (B). the microbiological cylinder–plate assay.

Please cite this article in press as: Mahmoudi, A., et al., Microbiological assay for analysis of certain macrolides in pharmaceutical dosage forms.
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Table 1 Table 3
Standard curve of clarithromycin and roxithromycin. Experimental values obtained in the recovery test for clarithromycin and Q7
roxithromycin in samples, by the microbiological cylinder–plate assay.
Drug Equation of line Correlation coefficient
Sample Amount of reference (mg) %Recoverya
CLA y = 3109 Ln x + 8090 0.998
ROX y = 4053 Ln x + 6910 0.997 Added Recovered
y: diameter of the inhibition zone in mm; x: logarithm of the drug concentration Claridar1 0.01 0.009776 97.76
in mg/mL. 0.03 0.030414 101.38
0.05 0.049005 98.01

328 Clarimed1 0.01 0.009785 97.85

precision should be performed at different levels such as 0.03 0.028521 95.07
329 repeatability and intermediate precision. Repeatability is the 0.05 0.051525 103.05
330 results of the method operating over a short time interval under
331 the same conditions (intra-assay precision). For a pharmaceutical Roxithromycine HUP1 0.01 0.009991 99.91
332 0.03 0.029292 97.64
formulation, acceptable variance ratio test limits were fixed at
333 0.05 0.048260 96.52
p < 0.05 (5%).
334 The intra-day precision of the method was determined by Roxid1 0.01 0.009725 97.25
335 preparing the tablet samples with three concentrations and eight 0.03 0.028701 95.67
336 replicate each. The RSD of the assay results, expressed as a 0.05 0.047205 94.41
337 percentage of the label claim, was used to evaluate the method a
Each value is the mean of eight analysis.
338 precision. The inter-day precision was also determined by assaying
339 the tablets in eight replicate per day for consecutive 3 days.
340 remain, in general as the standard for resolving doubt with respect 363
The intra- and inter-day variability or precision data are
341 to possible loss of activity (United States Pharmacopeia, 2007). 364
summarized in Table 2. The coefficient of variation values were
342 The results of analysis of the commercial tablets and the 365
1.32–1.96% for intra-day assay and 1.47–2.11% for inter-day assay,
343 recovery study suggested that there is no interference from any 366
respectively.
excipients, which are present in pharmaceutical samples. Further- 367
344 more, the Student’s t-values of CLA and ROX calculated for assay, 368
3.4.2. Accuracy
345 The accuracy of an analytical procedure expresses the 1.57 and 1.85, respectively, are below tabulated values at a = 0.05 369
346 (ttheoretical 2.06). These results showed that the microbiological 370
agreement between the experimental and the conventional true
347 assay was specific and the excipients or the probable impurities did 371
value or an accepted reference value. Accuracy represents the sum
348 not interfere in the capacity of the method to assess the analyte. 372
of systematic (trueness) and random errors and therefore,
349 In summary, the analysis of the obtained results with the 373
expresses the inherent total error of the result.
350 described method for the quantification of CLA and ROX in 374
Accuracy of the assay method was evaluated by recovery
351 pharmaceutical dosage forms demonstrate good linearity, 375
studies at three different quantities levels (low, medium and high)
352 precision and accuracy over the concentration range selected. 376
by standard addition method. The mixtures prepared as described
353 All results are within the ranges acceptable for bioanalytical 377
above in Section 2.4.3, and were analyzed. The mean percentage
354 purposes and coefficient of variation was found to be less than 5% 378
recoveries obtained were in the range of 94.41–103.05% for four
355 (Table 2). Furthermore, the analytical reagents are inexpensive, 379
different tablet samples of clarithromycin and roxithromycin
356 have good shelf life, and are available in any analytical laboratory. 380
(Table 3).
Therefore, this method is practical and valuable for its routine 381
357 application in the analysis of these drugs. 382
3.5. Specificity

358 3.6. Comparison of microbiological assay and HPLC 383

Microbiological methods are advantageous because the
359 parameters that are measured with these techniques and the
360 The quantification of antibiotic components by chemical 384
properties for the drug used are the same. Thus, impurities and the
361 methods such as HPLC, although precise, cannot provide a true 385
related substances do not interfere, maintaining the precision of
362 indication of microbiological activity. Attempts to correlate 386
the analytical method (Hodges, 2001). Therefore, those assays
antibiotic microbiological assay results with those from chemical 387

Table 2
Intra- and inter-day precision for clarithromycin and roxithromycin in samples by the microbiological cylinder–plate assay.

Sample Claimed conc. (mg/tablet) Experimental amounta (mg) %Levelb %RSD

1 2 3 4 5 6
Intra-assay
Claridar1 500 465.83 487.10 472.34 475.20 476.30 473.83 95.02 1.46
Clarimed1 500 484.50 481.60 485.60 479.98 497.34 478.28 96.91 1.41
RoxithromycineHUP1 150 146.52 147.12 148.38 145.86 150.33 144.87 98.12 1.32
Roxid1 150 143.61 144.90 141.13 136.90 140.40 141.30 94.25 1.96

Inter-assay
Claridar1 500 463.75 484.15 468.95 471.85 478.10 474.50 94.71 1.68
Clarimed1 500 479.20 477.90 482.05 466.95 473.65 489.75 95.65 1.54
RoxithromycineHUP1 150 146.16 142.98 141.25 145.27 140.94 141.54 95.35 1.47
Roxid1 150 141.40 138.84 139.95 138.13 139.08 148.05 93.94 2.11
a
Each value is the mean of eight analysis.
b
Results are compared with that of standard calibration curve.

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concerning selectivity and the efficiency of quantitation. In 434

4
industry, quantification of CLA and ROX by HPLC method is precise 435
3.5 but rather expensive and inconvenient. For example, HPLC 436

apparatus is quite expensive and a great deal of organic solvents 437

3
HPLC, ×10¯¹µg/mL

will also be used during analysis, and the analysis time is also quite 438
2.5 439
long (about 10 min to 1 h per sample), a lot of money and time
2 would be cost. On the other hand, using the microbiological assay, 440

all the samples could be tested in many plates at one time with 441
1.5
cheap cost and the analytical results could be obtained within 24 h. 442
1
0.5 4. Conclusion 443

0
In conclusion, a microbiological assay, applying the 444
0 0.5 1 1.5 2 2.5 3 3.5 4
cylinder–plate method, was reported in this study, for the 445
Microbiological.Assay, ×10¯¹µg/mL
quantitative determination of clarithromycin and roxithromycin 446
Fig. 3. Correlation between HPLC and microbiological methods corresponding to in pharmaceutical tablet dosage forms of certain commercial lots 447
clarithromycin tested in tablet as a representative example of the studied from different manufacturers. 448
macrolides antibiotics: y = 0.992x  0.020; r = 0.98; n = 12. 449
The results of the validation have shown acceptable precision,
accuracy and adequate linearity at concentration ranging from 450

0.1 to 0.5 mg/mL. Hence, the described method can be easily 451
388 methods have proved disappointing. Therefore, microbiological 452
adopted for the routine quality control analysis of CLA and ROX in
389 assays continue to play an essential role in manufacturing and 453
pharmaceutical formulations.
390 quality control of antibiotic medicines, and yet still demand
391 considerable skill and expertise to assure success (Baird and
392 Acknowledgements 454
Hodges, 2000). To our knowledge, no comparative study of HPLC
393 and microbiological methods to measure CLA and ROX in tablets
394 The authors would like to thank SAIDAL GROUPE of Algiers, 455
has been reported previously (Kanfer et al., 1998; Lahane et al.,
395 and HUP.P.Pharma laboratories of Constantine for kindly 456
2014). Thus, the performance of the proposed method was judged
396 supplying macrolide standards. Department of Natural 457
by comparing with other reported methods, which could indicate
397 Sciences and Microbiology Laboratory of Ecole Normale 458
the acceptable sensitivity of the microbiological method, with no
398 Supérieure—Kouba, Algiers, Algeria are gratefully acknowledged 459
significant difference. For method comparison purposes,
399 for support and providing necessary facilities to doing this 460
pharmaceutical samples of those drugs were then analyzed by
400 research work. 461
the HPLC method and the microbiological cylinder–plate assay
401 method in which the strain of M. luteus ATCC 9341 was used.
402 The results obtained with the cylinder-plate assay were 462
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Please cite this article in press as: Mahmoudi, A., et al., Microbiological assay for analysis of certain macrolides in pharmaceutical dosage forms.
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