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ISSN: 2161-0444
Medicinal Chemistry

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In vitro Activity and Evaluation of Quality of Some Selected Penicillins on the Ghanaian Market using Developed HPLC Methods

Rita Frema Boadu1, Christian Agyare1*, Martin Adarkwa-Yiadom2, Francis Adu1, Vivian Etsiapa Boamah1 and Yaw Duah Boakye1

1Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

2Forensic Unit, Ghana Standard Authority, Accra, Ghana

*Corresponding Author:
Dr. Christian Agyare
Department of Pharmaceutics
Faculty of Pharmacy and Pharmaceutical Sciences
Kwame Nkrumah University of Science and Technology
Kumasi, Ghana
Tel: +233246369803
E-mail: [email protected]; [email protected]

Received date: December 01, 2014; Accepted date: date: January 09, 2015; Published date: date: January 12, 2015

Citation: Boadu RF, Agyare C, Yiadom MA, Adu F, Boamah VE, et al. (2015) In vitro Activity and Evaluation of Quality of Some Selected Penicillins on the Ghanaian Market using Developed HPLC Methods. Med chem 5:001-014. doi: 10.4172/2161-0444.1000235

Copyright: © 2015 Boadu RF, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The use of antibioticsin health delivery is inevitable since it is one of the most prescribed medications. The quality and efficacy of these medications are crucial in health systems since they can affect the quality of healthcare delivery. The study was designed to determine the quality and activity of some penicillins on the Ghanaian market. A total of 54 samples (29 capsules and 25 suspensions) of different brands and batches were collected from different pharmacies in Accra and Kumasi, Ghana, from October 2011 to May 2012. The activity (zones of inhibition) and minimum inhibitory concentration (MIC) of the samples were determined by the agar-well diffusion and micro-dilution methods respectively against two typed strains of Gram-negative and Gram-positive bacteria. Quality of the samples was determined quantitatively by developed and validated HPLCmethods. The MICs of flucloxacillin and cloxacillin samples were ≥ 1400 μg/mL, whiles that of amoxicillin samples were ≥ 200 μg/mL, with reference to the standard antibiotics which gave MICs of 200 to 800 μg/mL against all the test bacteria with the suspensions exhibiting higher antimicrobial activity. Specificity, linearity, precision and accuracy of the developed HPLC method were determined. HPLC analysis of the samples revealed that 75% of amoxicillin capsule samples and 92.3% of amoxicillin suspension samples contained the right amount of active pharmaceutical ingredient (API) with percentages ranging from 93.2 to 104.3% and 81.0 to 104.1% respectively. For samples of flucloxacillin capsules, 62.5% of the samples showed API content from 96 to 120.5%. All the suspension samples have their API within BP and USP specification of 114.4 to 120.0%. Capsules (58.6%) of all the samples contained the right API whereas 64% of them were recorded for suspensions. Out of the 54 samples evaluated, 61.1% were within the BP and USP specifications. The biological assay revealed higher MIC values for all the penicillin samples evaluated compared with the reference samples. Among the samples evaluated, amoxicillin showed better quality of 82.8% as compared to flucloxacillin (31.3%) and cloxacillin (44.4%) samples. Efforts should therefore be made to improve the quality and storage conditions of these antibiotics and also constant monitoring and surveillance of activity and potency of these antibiotics should be done.

Keywords

Penicillins; HPLC; Minimum inhibitory concentration (MIC); Active pharmaceutical ingredient (API)

Introduction

The World Health Organization (WHO) defines counterfeit products as those which are deliberately and fraudulently mislabeled with respect to identity or source [1,2]. Substandard medicines, on the other hand, are medicines that do not meet official standards and specification for strength, quality, purity, packaging, and labeling and their presence are one of the latest threats facing the pharmaceutical industry and healthcare delivery system globally. As a result of weak or no regulatory systems in many low and middle income countries [3,4], most of the medicines in circulation in these countries do not meet internationally accepted quality and specification and may be detrimental to patients.

The total worldwide trade in counterfeit medicines is estimated to be 5 to 7% of the pharmaceutical market [5]. The problem is more severe in developing countries. More than 30% of all medicines sold in Africa are counterfeit medicines [6]. Counterfeit and substandard medicines are not only available in the developing countries but also in the developed world [7]. In 1999, 22% of the 771 reports of counterfeited medicines Received date: by WHO came from the developed countries, the remaining 78% were from the developing countries [3].

Prevalence of counterfeit and substandard medicines has a major effect on the health delivery system. They can result in treatment failure, toxicity, adverse reaction or severe side effects thereby increasing mortality rate [8]. Counterfeit and substandard medicines may be found in all classes of medicines. The two major classes most counterfeited in the developing countries are anti-parasitic and anti-infective medicines [2]. Exposure of microorganisms to counterfeit and substandard antiinfectives leads to antimicrobial resistance, thereby putting health of patients at risk [9]. Antimicrobial resistance contributes to high cost of healthcare as patients using these counterfeit and substandard medicines do not respond to treatment and have to resort to higher doses and newer medicines. Additionally, patients remain ill for longer period leading to the loss of productivity [1,10]. Infectious diseases are taking lives of people and believed to be the world’s leading cause of death. It is estimated that 50,000 people die a day out of infectious diseases [11].

Medicines need to be of acceptable quality, safety and efficacy, especially antibiotics [12]. The appropriate active pharmaceutical ingredients (API) quantity and its efficacy to effect treatment must be ascertained. This is achieved through analysis and comparison to the manufacturer’s specifications or standard specification in the pharmacopoeias. Consequently, there is the need to sample and evaluate some of the antibiotics on the Ghanaian market to ensure that they meet the required specifications as spelt out in the United States Pharmacopoeia (USP) and British Pharmacopoeia (BP) to avoid all the problems associated with counterfeit and substandard medicines.

Antibiotics are natural or synthetic chemical agents that can inhibit the growth or kill microorganisms [13]. Antibiotics are one class of antimicrobials and they are either referred to as bactericidal or bacteriostatic when they kill or inhibit growth or bacteria respectively [14]. They are heterogeneous and the only common property is that they are all organic in nature. A required feature of any antibiotic is its effect on bacteria at low concentration since that differentiate antibioticsfrom other compounds which have antimicrobial effect at higher concentrations e.g. ethanol. The discovery of antibiotics have significantly reduced mortality resulting from infectious diseases and also facilitated the success rates of many medical procedures such as surgery [15,16]. They are also employed extensively to prevent and treat infectious diseases in humans and animals [17]. These agents are mostly directed against some targets that are peculiar to bacteria, interfering with the growth of sensitive structures or processes that are critical to the survival and growth of the bacteria. Antibiotics inhibit sensitive bacteria by blocking important macromolecules like enzymes and nucleic acid activity which are very important in cell multiplication or division [18]. In effect, they are able to bind to specific site on the macromolecule to form a complex, different from the original entity and are unable to perform its function. The main targets are bacterial cell wall synthesis (peptidoglycan), bacterial protein synthesis (bacterial ribosome), bacterial DNA replication (bacterial enzymes involved in DNA supercoiling) and cytoplasmic membrane function [19]. The aim of this study was to determine the antibacterial activity and develop HPLC methods to analyze API content of various samples of amoxicillin, flucloxacillin and cloxacillin on the Ghanaian market.

Materials

Chemicals and reference drugs

All chemicals used for the HPLC analysis including reference compounds such as amoxicillin trihydtrate (96% HPLC), flucloxacillin (98% HPLC), cloxacillin (98% HPLC), caffeine anhydrous (98% HPLC) and acetaminophen (98% HPLC), solvents etc. were of analytical and chromatographic grade purchased from Sigma-Aldrich, Darmstadt, Germany unless otherwise stated and they were available in the Forensic Laboratory of Ghana Standard Authority, Accra, Ghana. All materials and equipment used in the microbiological evaluation are available in the Microbiology Section, Department of Pharmaceutics, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana.

Test bacteria

Four typed strains of bacteria consisting of two Gram-negative and two Gram-positive bacteria were used for the microbiological evaluation. All organisms were typed cultures stored at the Microbiology Research Laboratory, Department of Pharmaceutics, KNUST, Kumasi, Ghana with the following identities: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 4853, Staphyloccocus aureus ATCC 25923 and Bacillus subtilis NTCC 10073.

Test penicillin samples

Imported and locally manufactured penicillin samples were randomly purchased from different Pharmacies in Accra and Kumasi, Ghana. The reasons for the choice of samples were to compare different brands and different batches within a brand. Sampling of antibiotics was done from October, 2011 to May, 2012.

Methods

Determination of antibacterial activity

The antimicrobialactivity was determined using modified method described by Agyare et al. [20] and Girish and Satish [21]. Twenty (20) milliliters stabilized agar at 45°C was seeded with 100 μL of 105 colony forming units (cfu)/mL of 18 to 24 h broth culture of S. aureus and rolled in the palm for uniform distribution and was aseptically poured into sterilized Petri dish and allowed to set. Four wells were bored with diameter of 10 mm. The wells were filled with 200 μL each of respective concentrations and allowed to stand for 1 h on the bench to allow diffusion of antibiotic. The plate was then incubated at 37°C for 24 h and zones of growth inhibition recorded in millimeter (mm). The method used was performed in triplicate for all test samples using B. subtilis, E. coli and P. aeruginosa. Concentrations used were 0.125 to 1.0 μg/mL for amoxicillinsamples and 1.25 to 10.0 mg/mL for flucloxacillin and cloxacillin samples.

Determination of minimum inhibitory concentration

Minimum inhibitory concentrations (MIC) of the various antibiotic samples were determined using the method described by Agyare et al. [20]. Sterile 96-well microtitre plates were labeled appropriately for S. aureus. Total volume of 200 μL were prepared by dispensing a fixed volume of 100 μL sterile double strength nutrient broth and 20 μL (105 cfu/mL) of 18 h culture was aseptically added to the medium. Amoxicillin samples were evaluated within concentration range of 0.1 to 0.5 mg/mL. The MIC of flucloxacillin and cloxacillin samples was determined within a concentration range of 0.5 to 2.2 mg/mL. Experiments were performed in triplicate under the same conditions for all samples. Reference samples were prepared and the MIC determined under the same conditions as described above.

The plates were incubated at 37°C for 24 h. Microbial growth was determined by addition of 30 μL 3-(4,5-dimethylthiazole -2-yl)-2,5- diphenyltetrazolium bromide (MTT) after incubation and as growth of organism was indicated by purple to blue coloration and yellow coloration indicated no growth of organism. The well with least concentration of test sample without bacterial growth recorded as the MIC. The procedure above was repeated for all test samples using E. coli, B. subtilis and P. aeruginosa respectively.

HPLC analysis of reference and test samples

Reference amoxicillin trihydrate samples were dissolved in 0.1 M hydrochloric acid. Samples were analyzed at concentrations of 5.26, 10.52, 15.78, 21.04 and 26.3 μg/mL with an injection volume of 100 μL. Reference flucloxacillin and cloxacillin samples were dissolved in sterile distilled millipore water. They were analyzed at concentrations of 25.35, 50.7, 101.4, 152.1 μg/mL and 11.72, 23.44, 35.16, 58.6 μg/mL for reference standard and the sample respectively, with an injection volume of 1 mL. All samples were analyzed under isocratic conditions with Shim-Pac CLS ODS (M) C18 column for amoxicillin. Shim- Pac CLC-NH2 C18 column was used in analysis of flucloxacillin and cloxacillin. An internal standard of 1025 μg/mL caffeine anhydrous was used in the development of HPLC method for amoxicillin and analysis of amoxicillin trihydrate samples. Concentrations of 1.4156 μM and 1.3296 μM of acetaminophen (paracetamol) were used for the HPLC method development for flucloxacillin and cloxacillin respectively. The same concentrations were used for the analysis of flucloxacillin and cloxacillin samples.

Preparation of test sample solutions

Concentrations of amoxicillin trihydrate equivalent to 15.78 μg/ mL were prepared. They were dissolved in 0.1M hydrochloric acid and mobile phase consisting of methanol/ 0.01M potassium dihydrogen phosphate (65:35, v/v). Equivalent of 50.7 and 11.72 μg/mL of flucloxacillin and cloxacillin were prepared. Samples were dissolved in sterile distilled water and mobile phase.

Statistical analysis

All graphs were plotted with Excel version 2010 and graph pad prism (Graph Pad Prism 5 Software, San Diego, CA, USA) for all the statistical analysis. Data analysis was by one-way analysis of variance (ANOVA). There is not enough evidence at alpha=0.05 and the model for the method development is not significant since F-value > F-crit and P<0.05 (alpha). ChromQuest and Endnote X6 (Bld 6348) were used to generate HPLC analysis data and references respectively.

Results

Antibacterial activities of samples

The MICs of capsules were within the range of 200 to 800 μg/mL for amoxicillin trihydrate samples and ≥ 800 to 1900 for flucloxacillin and cloxacillin test samples. Reference amoxicillin samples showed lower MICs of 200 μg/mL against E. coli, 500 μg/mL against P. aeruginosa, 300 μg/mL against B. subtilis and 200 μg/mL against S. aureus. MICs of reference flucloxacillin sample were 800 μg/mL against E. coli, 1500 μg/ mL for P. aeruginosa, 1400 μg/mL for B. subtilis and 1400 μg/mL for S. aureus. MICs for reference cloxacillin sample were 800 μg/mL against E. coli, 1500 μg/mL against P. aeruginosa, 1500 μg/mL against B. subtilis and 1500 μg/mL for S. aureus (Table 1).

Sample Organisms/MIC (µg/mL)
  E. coli P. aeruginosa B. subtilis S. aureus
    AMOXICILLIN    
Reference sample 200 500 300 200
01A 200 500 400 400
01B 300 700 500 500
02A 300 700 400 200
02B 400 800 400 300
03A 200 600 300 300
03B 200 700 300 300
03C 200 600 300 300
04A 200 700 300 300
05A 300 600 300 300
06A 400 800 400 400
06B 400 700 500 300
06C 300 700 500 400
07A 200 500 300 200
07B 400 800 400 400
08A 300 700 400 400
09A 300 500 300 200
    FLUCLOXACILLIN    
Reference 800 1500 1400 1400
FLMG01 1300  1900 1500 1500
FLMG02 1200 1700 1400 1500
FLMG02 800 1500    1500 1500
FLLP04 1300 1800 1500 1500
FLLP05 1200 1600 1500 1500
FLLP06 1300 1700 1500 1500
FLAR07 800 1600 1500 1500
FLAR08 800 1600 1500 1500
    CLOXACILLIN    
Reference 800 1500 1500 1500
CLLP01 800 1500 1500 1500
CLLP02 900 1600 1500 1500
CLLP03 800 1500 1500 1500
CLAR04 900 1600 1500 1500
CLAR05 800 1500 1500 1500
CLMG06 800 1400 1500 1400

Table 1: MICs of capsule samples of amoxicillin, flucloxacillin and cloxacillin.

Antibacterial activity of sampled antibiotic suspensions of amoxicillin, flucloxacillin and cloxacillin samples. Evaluation of samples gave MICs within the range of 200 to 700 μg/mL for amoxicillin test samples, 800 to 1600 for flucloxacillin and 500 to 1700 cloxacillin samples (Table 2).

Sample Organisms/MIC (μg/mL)
E. coli P. aeruginosa B. subtilis S. aureus
    AMOXICILLIN    
S01 300 600 300 200
S02A 200 500 300 200
S02B 300 500 300 200
S02C 300 600 300 200
S03A 200 500 300 200
S04A 300 600 300 200
S05A 300 500 300 200
S06A 200 500 300 200
S06B 300 700 400 300
S06C 300 600 300 300
S07A 200 500 300 200
S08A 200 500 300 200
S08B 200 500 300 200
    FLUCLOXACILLIN    
FLSMG01 800 1500 1400 1400
FLSMG02 800 1600 1400 1400
FLSMG03 800 1500 1400 1500
FLSLP04 800 1600 1400 1600
FLSLP05 800 1600 1600 1600
FLSLP06 800 1500 1500 1400
FLSAR07 800 1500 1400 1400
FLSAR08 800 1500 1600 1400
    CLOXACILLIN    
CLSLP01 800 1500 1500 1600
CLSLP02 800 1700 1500 1500
CLSLP03 800 1600 500 1500
CLSMG04 800 1500 1600 1600
CLSMG05 800 1600 1600 1600

Table 2: MICs of suspension of amoxicillin, flucloxacillin and cloxacillin samples.

Antibacterial activity of sampled antibiotic capsules of amoxicillin, flucloxacillin and cloxacillin samples. Evaluation of samples at test concentrations gave mean zones of inhibition within the range of 0.0 to 30.0 mm for amoxicillin test samples, 0.0 to 31.67 mm for flucloxacillin and 0.00 to 29.83 mm for cloxacillin samples (Table 3).

Samples Organisms
Concentrations   (µg/mL) S. aureus E. coli B. subtilis P. aeruginosa
AMOXICILLIN
 01A 1000 22.33 ± 0.82 16.00 ± 0.63 20.50 ± 0.55 21.67 ± 0.52
500 20.83 ± 0.75 12.67 ± 0.52 18.50 ± 0.55 19.33 ± 0.52
250 25.00 ± 0.0 12.00 ± 0.0 18.17 ± 0.41 17.83 ± 0.75
125 0.0 0.0 0.0 0.0
01B 1000 25.83 ± 0.41 26.66 ± 0.52 24.67 ± 0.82 21.67 ± 0.52
500 25.00 ± 0.63 24.67 ± 0.82 23.00 ± 0.63 19.67 ± 0.82
250 22.67 ± 0.52 22.67 ± 0.52 21.00 ± 0.89 18.33 ± 1.37
125 0.0 0.0 0.0 0.0
02A 1000 25.67 ± 1.03 24.00 ± 0.9 19.00 ± 00 23.50 ± 0.55
500 23.33 ± 1.03 17.50 ± 0.55 14.17 ± 0.75 22.50 ± 0.84
250 22.17 ± 0.41 16.17 ± 0.75 17.00 ± 0.0 21.50 ± 0.55
125 0.0 0.0 0.0 0.0
02B 1000 25.33 ± 0.52 23.33 ± 1.21 25.33 ± 0.51 23.00 ± 0.89
500 24.50 ± 1.38 22.50 ± 0.55 24.83 ± 0.98 20.83 ± 1.17
250 22.50 ± 1.05 18.50 ± 1.05 22.67 ± 0.52 18.50 ± 0.84
125 0.0 0.0 0.0 0.0
03A 1000 24.8. ± 0.41 20.83 ± 0.52 24.50 ± 0.84 0.0
500 21.83 ± 0.41 23.83 ± 0.75 24.00 ± 0.89 0.0
250 20.83 ± 0.41 18.83 ± 0.75 22.50 ± 0.84 0.0
125 0.0 0.0 0.0 0.0
           
03B 1000 25.83 ± 0.98 20.83 ± 0.75 24.83 ± 0.75 20.67 ± 1.03
500 22.67 ± 1.21 18.00 ± 0.63 23.83 ± 0.41 17.83 ± 0.75
250 21.17 ± 0.98 12.67 ± 0.52 20.67 ± 0.82 16.33 ± 0.82
125 0.0 0.0 0.0 0.0
03C 1000 24.67 ± 1.00 18.67 ± 0.52 23.50 ± 0.55 0.0
  500 22.17 ± 1.17 16.83 ± 0.98 21.33 ± 1.37 0.0
  250 20.67 ± 1.21 15.00 ± 0.89 20.50 ± 1.38 0.0
  125 0.0 0.0 0.0 0.0
04A 1000 26.57 ± 1.05 20.71 ± 0.36 23.86 ± 0.75 0.00 ± 0.00
500 22.14 ± 0.84 18.43 ± 0.52 21.57 ± 0.52 0.0
250 21.43 ± 1.21 22.50 ± 0.71 20.29 ± 0.82 0.0
125 0.0 0.0 0.0 0.0
05A 1000 30.00 ± 0.89 23.00 ± 0.0 24.50 ± 0.84 0.0
500 27.67 ± 1.03 26.00 ± 0.0 21.33 ± 1.03 0.0
250 25.67 ± 1.03 24.00 ± 0.0 20.17 ± 0.98 0.0
125 0.0 0.0 0.0 0.0
06A 1000 21.00 ± 0.0 18.87 ± 0.4 22.83 ± 0.14 22.00 ± 0.00
  500 20.00 ± 0.0 22.00 ± 0.0 22.30 ± 0.18 21.67 ± 0.18
  250 18.00 ± 0.0 21.00 ± 0.17 21.00 ± 0.18 20.00 ± 0.0
  125 0.0 0.0 0.0 0.0
06B 1000 25.50 ± 0.55 15.50 ± 0.84 16.00 ± 0.82 0.0
500 24.50 ± 0.84 12.67 ± 0.52 12.00 ± 0.82 0.0
250 23.33 ± 1.03 0.0 0.0 0.0
125 0.0 0.0 0.0 0.0
07A 1000 24.50 ± 0.84 20.00 ± 0.0 24.50 ± 0.55 23.17 ± 0.41
500 21.83 ± 1.17 19.83 ± 1.17 22.83 ± 0.75 22.50 ± 1.05
250 20.50 ± 1.22 19.17 ± 1.17 20.67 ± 1.21 19.00 ± 1.10
125 0.0 0.0 0.0 0.0
07B 1000 24.33 ± 0.82 20.17 ± 0.75 23.50 ± 0.84 22.17 ± 0.75
500 21.67 ± 0.52 19.67 ± 1.03 23.00 ± 1.10 0.0
250 20.17 ± 0.75 19.00 ± 0.89 20.50 ± 0.84 0.0
125 0.0 0.0 0.0 0.0
08B 1000 24.33 ± 0.52 19.33 ± 1.03 21.50 ± 0.84 20.50 ± 0.55
500 22.17 ± 0.75 17.5 ± 0.55 18.67 ± 0.82 16.33 ± 0.82
250 21.33 ± 1.03 16.00 ± 0.89 15.53 ± 0.55 22.00 ± 0.0
125 0.0 0.0 0.0 0.0
09A 1000 25.17 ± 0.41 21.83 ± 0.98 25.00 ± 0.89 20.50 ± 1.38
  500 23.50 ± 0.55 21.17 ± 0.98 24.17 ± 0.75 18.83 ± 0.98
  250 22.17 ± 0.75 18.33 ± 0.52 21.50 ± 1.38 17.33 ± 1.37
  125 0.0 0.0 0.0 0.0
FLUCLOXACILLIN
FLMG01 10000 23.17 ± 0.41 26.30 ± 0.28 22.83 ± 0.59 23.17 ± 0.63
  5000 17.00 ± 0.63 20.00 ± 0.22 21.17 ± 0.34 23.83 ± 0.51
  2500 17.00 ± 0.89 20.33 ± 0.18 20.83 ± 0.45 22.67 ± 0.36
  1250 0.0 0.0 0.0 0.0
FLMG02 10000 25.67 ± 1.21 21.33 ± 1.03 28.67 ± 1.03 19.83 ± 0.98
5000 22.50 ± 1.38 19.50 ± 1.22 27.67 ± 1.21 17.00 ± 0.52
2500 20.67 ± 0.81 18.50 ± 0.55 24.50 ± 0.84 16.00 ± 0.0
1250 17.67 ± 1.37 17.00 ± 0.0 19.00 ± 0.69 14.75 ± 0.50
FLMG03 10000 31.67 ± 0.82 18.67 ± 0.52 29.50 ± 1.22 0.0
5000 29.50 ± 0.55 18.17 ± 0.75 27.83 ± 0.98 0.0
2500 28.33 ± 0.82 16.00 ± 0.82 27.67 ± 0.82 0.0
1250 0.0 0.0 0.0 0.0
FLLP04 10000 30.67 ± 1.10 24.17 ± 0.41 30.50 ± 0.55 0.0
5000 27.33 ± 0.52 19.67 ± 0.51 27.83 ± 0.41 0.0
2500 26.83 ± 0.41 0.0 27.00 ± 0.63 0.0
1250 0.0 0.0 0.0 0.0
FLLP05 10000 30.67 ± 1.17 24.00 ± 0.63 30.50 ± 0.84 0.0
5000 27.33 ± 0.52 19.67 ± 1.03 27.83 ± 0.75 0.0
2500 26.83 ± 0.98 0.0 27.00 ± 0.63 0.0
1250 0.0 0.0 0.0 0.0
FLLP06 10000 26.17 ± 0.41 25.50 ± 0.55 24.00 ± 0.63 22.17 ± 0.98
5000 24.33 ± 0.52 23.00 ± 0.63 23.50 ± 0.55 21.17 ± 0.75
2500 22.67 ± 0.52 21.67 ± 0.51 21.00 ± 0.08 16.83 ± 0.75
1250 0.0 0.0 0.0 0.0
FLAR07 10000 24.00 ± 0.50 23.67 ± 1.18 25.50 ± 0.68 20.67 ± 0.89
  5000 21.50 ± 0.68 18.00 ± 0.53 24.30 ± 0.18 24.00 ± 0.0
  2500 17.83 ± 1.00 16.00 ± 0.63 23.30 ± 0.18 20.00 ± 0.0
  1250 0.0 0.0 0.0 0.0
FLAR08 10000 20.00 ± 0.89 22.67 ± 0.82 25.83 ± 0.41 0.0
  5000 18.50 ± 0.55 19.33 ± 0.52 22.50 ± 1.05 0.0
  2500 0.0 16.33 ± 0.07 0.0 0.0
  1250 0.0 0.0 0.0 0.0
CLOXACILLIN
CLLP01 10000 29.83 ± 0.41 21.83 ± 0.41 29.50 ± 0.84 25.17 ± 0.41
5000 27.83 ± 0.98 19.33 ± 0.52 26.67 ± 0.52 25.33 ± 0.82
2500 26.17 ± 0.40 20.17 ± 0.41 25.67 ± 0.52 23.50 ± 0.55
1250 0.0 0.0 0.0 0.0
CLLP02 10000 29.17 ± 0.41 22.00 ± 1.26 28.50 ± 0.55 29.50 ± 0.55
5000 27.50 ± 0.84 21.50 ± 0.55 28.33 ± 0.52 26.00 ± 0.0
2500 25.17 ± 0.41 20.83 ± 0.75 24.33 ± 0.52 25.67 ± 0.51
1250 0.0 0.0 0.0 0.0
CLLP03 10000 29.00 ± 0.89 26.33 ± 1.03 27.33 ± 0.82 22.67 ± 0.82
5000 28.17 ± 1.33 23.67 ± 0.82 26.00 ± 0.89 16.67 ± 0.52
2500 26.33 ± 1.03 22.50 ± 0.84 24.00 ± 0.89 15.17 ± 0.75
1250 0.0 0.0 0.0 0.0
CLAR03 10000 26.50 ± 1.38 14.33 ± 1.37 26.50 ± 0.84 18.50 ± 1.38
5000 23.50 ± 1.0 0.0 24.83 ± 0.75 14.83 ± 0.41
2500 0.0 0.0 23.67 ± 0.82 12.00 ± 0.63
1250 0.0 0.0 0.0 0.0
CLAR04 10000 26.17 ± 0.98 21.00 ± 0.89 24.17 ± 1.17 17.50 ± 0.55
  5000 23.00 ± 0.89 23.17 ± 0.75 25.67 ± 0.52 11.50 ± 0.55
  2500 23.33 ± 0.52 23.17 ± 1.17 20.30 ± 0.52 0.0
  1250 0.0 0.0 0.0 0.0
CLMG 10000 27.50 ± 0.84 20.83 ± 0.98 23.67 ± 0.82 27.67 ± 1.21
5000 25.17 ± 0.41 25.33 ± 0.52 23.50 ± 0.55 24.83 ± 0.41
2500 23.17 ± 0.41 22.33 ± 0.52 22.50 ± 0.55 24.17 ± 0.47
  1250 0.0 0.0 0.0 0.0

Table 3: Antibacterial activity (mean zones of inhibition ± SEM) of test samples (capsules).

Antibacterial activity of sampled antibiotic suspensions of amoxicillin, flucloxacillin and cloxacillin samples. Evaluation of samples at test concentrations gave mean zones of inhibition within the range of 0.0 to 28.67 mm for amoxicillin test samples, 0.0 to 37.83 mm for flucloxacillin and 0.0 to 33.83 mm for cloxacillin samples (Table 4).

  Organism
Sample Concentration   (μg/mL) S.   aureus E. coli B. subtilis P. aeruginosa
      AMOXICLLIN    
S01A 1000 21.83 ± 1.22 18.00 ± 0.68 22.50 ± 0.81 0.0
500 19.67 ± 0.91 15.83 ± 0.31 21.33 ± 0.76 0.0
250 18.67 ± 0.91 15.00 ± 0.0 18.83 ± 0.42 0.0
125 0.0 0.0 0.0 0.0
S02A 1000 14.00 ± 0.22 19.83 ± 0.14 16.17 ± 0.14 13.00 ± 0.31
500 15.50 ± 0.19 19.50 ± 0.29 15.00 ± 0.22 11.50 ± 0.19
250 13.50 ± 0.19 18.00 ± 0.0 13.33 ± 0.18 0.0
125 0.0 0.0 0.0 0.0
S02B 1000 19.33 ± 0.18 19.50 ± 0.29 19.83 ± 0.26 0.0
500 11.67 ± 0.18 16.83 ± 0.26 18.33 ± 0.60 0.0
250 15.17 ± 0.14 15.67 ± 0.28 15.38 ± 0.34 0.0
125 0.0 0.0 0.0 0.0
S02C 1000 18.33 ± 0.17 18.00 ± 0.22 18.17 ± 0.14 0.0
500 15.80 ± 0.29 16.33 ± 0.18 15.17 ± 0.14 0.0
250 12.50 ± 0.19 12.00 ± 0.0 12.17 ± 0.14 0.0
125 0.0 0.0 0.0 0.0
S03A 1000 20.00 ± 0.0 17.67 ± 0.28 20.33 ± 0.56 22.67 ± 0.28
500 18.67 ± 0.28 17.00 ± 0.0 18.50 ± 0.57 20.33 ± 0.36
250 19.17 ± 0.45 14.67 ± 0.28 18.00 ± 0.38 19.67 ± 0.18
125 0.0 0.0 0.0 0.0
S04A 1000 17.33 ± 0.18 16.83 ± 0.14 21.17 ± 0.26 18.33 ± 0.18
500 15.83 ± 0.14 14.67 ± 0.17 20.17 ± 0.14 17.33 ± 0.28
250 15.00 ± 0.0 13.00 ± 0.0 19.00 ± 0.0 14.67 ± 0.18
125 0.0 0.0 0.0 0.0
05A 1000 16.67 ± 0.18 24.83 ± 0.26 17.00 ± 0.22 26.33 ± 0.18
500 16.00 ± 0.22 23.00 ± 0.53 15.33 ± 0.28 24.50 ± 0.19
250 14.67 ± 0.28 22.33 ± 0.78 14.00 ± 0.22 21.67 ± 0.18
125 0.0 0.0 0.0 0.0
S06A 1000 20.00 ± 0.00 16.83 ± 0.14 17.83 ± 0.14 28.67 ± 0.18
500 16.33 ± 0.18 16.00 ± 0.22 14.67 ± 0.18 25.00 ± 0.38
250 14.00 ± 0.00 12.67 ± 0.18 12.67 ± 0.18 22.17 ± 0.34
125 0.0 0.0 0.0 0.0
S06B 1000 19.50 ± 0.19 20.67 ± 0.18 18.33 ± 0.18 20.62 ± 0.18
500 17.50 ± 0.29 20.50 ± 0.29 16.83 ± 0.14 17.00 ± 0.00
250 14.83 ± 0.14 21.67 ± 0.36 16.00 ± 0.30 16.83 ± 0.45
125 0.0 0.0 0.0 0.0
S06C 1000 19.83 ± 0.14 19.83 ± 0.40 15.17 ± 0.34 22.50 ± 0.42
500 15.50 ± 0.19 19.00 ± 0.31 14.50 ± 0.19 21.33 ± 0.36
250 16.67 ± 0.18 17.17 ± 0.14 13.83 ± 0.14 16.33 ± 0.18
125 0.0 0.0 0.0 0.0
S07A 1000 24.67 ± 0.86 19.67 ± 0.36 20.00 ± 0.0 13.17 ± 0.40
500 19.50 ± 0.36 19.0 ± 0.22 18.67 ± 0.18 17.17 ± 0.63
250 17.67 ± 0.41 18.33 ± 0.52 18.50 ± 0.29 0.0
125 0.0 0.0 0.0 0.0
S08A 1000 20.33 ± 0.18 19.33 ± 0.18 20.00 ± 0.0 24.67 ± 0.36
500 19.17 ± 0.14 18.16 ± 0.14 18.50 ± 0.48 22.50 ± 0.19
250 18.50 ± 0.29 16.00 ± 0.22 17.17 ± 0.40 20.33 ± 0.18
125 0.0 0.0 0.0 0.0
S08B 1000 22.00 ± 0.00 17.67 ± 0.56 20.17 ± 0.14 25.83 ± 0.34
500 20.33 ± 0.18 16.50 ± 0.19 18.67 ± 0.35 24.17 ± 0.14
250 19.67 ± 0.18 16.00 ± 0.0 17.17 ± 0.14 20.33 ± 0.18
125 0.0 0.0 0.0 0.0
FLUCLOXACILLIN
FLSMG01 10000 27.17 ± 0.41 20.17 ± 0.41 32.83 ± 0.75 29.67 ± 0.52
5000 22.23 ± 0.52 18.00 ± 0.63 31.17 ± 1.32 28.83 ± 0.41
2500 11.17 ± 0.41 0.0 30.17 ± 0.40 27.83 ± 0.41
1250 0.0 0.0 0.0 0.0
FLSMG02 10000 25.50 ± 0.55 18.83 ± 0.75 25.60 ± 1.05 25.00 ± 0.89
  5000 21.17 ± 0.75 17.67 ± 0.82 25.33 ± 1.03 25.5 ± 0.55
  2500 0.0 0.0 25.00 ± 0.63 24.17 ± 0.41
  1250 0.0 0.0 0.0 0.0
FLSMG03 10000 24.67 ± 1.03 19.50 ± 0.84 27.33 ± 0.51 18.50 ± 0.55
5000 20.17 ± 0.41 15.50 ± 0.55 26.00 ± 0.63 15.67 ± 0.52
2500 14.83 ± 0.98 0.0 24.17 ± 0.75 0.0
1250 0.0 0.0 0.0 0.0
FLSLP04 10000 34.67 ± 0.52 21.67 ± 0.82 30.50 ± 0.55 20.33 ± 0.52
5000 29.83 ± 0.41 18.50 ± 0.55 26.00 ± 0.63 16.67 ± 0.52
2500 29.33 ± 0.52 0.0 25.00 ± 0.0 11.00 ± 0.0
1250 0.0 0.0 0.0 0.0
FLSP05 10000 30.17 ± 0.41 25.50 ± 0.84 37.83 ± 0.75 29.17 ± 0.75
5000 28.67 ± 0.52 24.50 ± 0.84 37.17 ± 0.41 25.33 ± 1.03
2500 28.0.00 ± 0.0 20.33 ± 0.51 33.67 ± 0.52 20.67 ± 0.81
1250 0.0 0.0 0.0 0.0
FLSP06 10000 32.33 ± 0.41 20.50 ± 1.23 30.33 ± 0.52 20.33 ± 0.52
5000 29.17 ± 0.75 17.83 ± 0.98 26.50 ± 0.55 0.0
2500 29.17 ± 0.75 0.0 25.00 ± 0.63 0.0
1250 0.0 0.0 0.0 0.0
FLSAR07 10000 27.83 ± 0.75 20.83 ± 0.41 29.50 ± 0.55 0.0
5000 25.50 ± 0.55 16.33 ± 0.52 28.00 ± 0.63 0.0
2500 24.00 ± 0.63 0.0 26.33 ± 0.52 0.0
1250 0.0 0.0 0.0 0.0
FLSAR08 10000 25.67 ± 0.52 28.83 ± 0.75 29.67 ± 1.03 0.0
5000 24.17 ± 0.98 26.67 ± 0.52 27.50 ± 1.05 0.0
2500 21.83 ± 0.41 25.17 ± 0.41 25.50 ± 0.55 0.0
1250 19.83 ± 0.75 22.83 ± 0.41 24.50 ± 1.22 0.0
CLOXACILLIN
CLSLP01 10000 33.83 ± 0.41 14.17 ± 0.75 20.67 ± 0.82 15.67 ± 0.52
5000 32.00 ± 0.00 11.17 ± 0.41 16.17 ± 0.41 14.33 ± 0.52
2500 31.00 ± 0.0 0.0 11.00 ± 0.0 0.0
1250 0.0 0.0 0.0 0.0
CLSLP02 10000 20.33 ± 0.52 19.17 ± 0.41 20.33 ± 0.52 20.17 ± 0.41
5000 0.0 14.83 ± 0.41 14.67 ± 0.52 12.00 ± 0.00
2500 0.0 0.0 0.0 0.0
1250 0.0 0.0 0.0 0.0
CLSLP03 10000 19.83 ± 0.41 14.83 ± 0.41 30.17 ± 0.41 20.33 ± 0.52
5000 16.17 ± 0.41 13.83 ± 0.41 26.00 ± 0.0 13.83 ± 0.41
2500 14.67 ± 0.52 0.0 24.83 ± 0.41 0.0
1250 0.0 0.0 0.0 0.0
CLMGS04 10000 33.33 ± 0.82 20.83 ± 0.98 31.17 ± 0.75 20.00 ± 0.63
5000 30.17 ± 0.41 11.17 ± 0.41 30.17 ± 0.41 14.83 ± 0.75
2500 27.67 ± 0.52 0.0 29.50 ± 0.55 0.0
1250 0.0 0.0 0.0 0.0
CLMG 10000 17.00 ± 0.0 21.17 ± 0.75 17.33 ± 0.52 24.00 ± 0.63
5000 15.83 ± 0.75 23.00 ± 0.0 15.17 ± 0.41 20.50 ± 0.55
2500 15.00 ± 0.89 22.50 ± 0.58 14.17 ± 0.41 22.00 ± 0.0
1250 0.0 0.0 0.0 0.0

Table 4: Antibacterial activity (mean zones of inhibition ± SEM) of suspension samples.

Antibacterial activity of reference antibiotic of amoxicillin, flucloxacillin and cloxacillin samples. Evaluation of samples at test concentrations gave mean zones of inhibition within the range of 0.00 to 30.83 mm for amoxicillin test samples, 0.00 to 38.00 mm for flucloxacillin and 0.00 to 30.00 mm for cloxacillin samples (Table 5).

Concentration (μg/mL) S.   aureus E.  coli B. subtilis P. aeruginosa
AMOXICILLIN
5000 30.83 ± 0.34 27.00 ± 0.0 24.83 ± 0.14 24.50 ± 0.89
2500 27.17 ± 0.14 24.67 ± 0.28 24.00 ± 0.31 21.67 ± 0.18
1250 25.67 ± 0.18 21.67 ± 0.47 21.33 ± 0.18 20.00 ± 0.0
625 0.0 0.0 0.0 0.0
FLUCLOXACILLIN
10000 35.17 ± 0.14 26.33 ± 0.28 38.00 ± 0.31 29.67 ± 0.36
5000 31.50 ± 0.89 20.00 ± 0.22 35.17 ± 0.14 24.33 ± 0.18
2500 29.67 ± 0.18 20.33 ± 0.18 32.83 ± 0.14 22.67 ± 0.36
1250 0.0 0.0 0.0 0.0
CLOXACILLIN
10000 30.00 ± 0.22 23.67 ± 0.34 25.50 ± 0.68 26.00 ± 0.22
5000 28.00 ± 0.26 18.00 ± 0.53 24.33 ± 0.18 28.33 ± 0.28
2500 25.67 ± 0.28 19.67 ± 0.36 23.33 ± 0.18 25.67 ± 0.36
1250 0.0 0.0 0.0 0.0

Table 5: Antibacterial activity (mean zones of inhibition ± SEM) of reference antibiotic samples.

HPLC analysis of amoxicillin samples

The active pharmaceutical ingredients (APIs) in the samples were determined using the developed and validated HPLC method. The chromatographic conditions for the analysis of amoxicillin trihydrate were made up of mobile phase consisting of methanol: 0.01M potassium dihydrogen phosphate (65:35, v/v) yielded maximum sensitivity and separation. Flow rates between 0.5 and 1.2 mL/min on a Shim-pack CLS-ODS C18 (M) 250 x 4.6 mm, 5 microns column were studied and a flow rate of 1.0 mL/min gave an optimal signal to noise ratio with a reasonable separation time of 1.42 min for amoxicillin when injected alone.

HPLC chromatogram of amoxicillin (Figure 1) as reference sample alone and reference amoxicillin and caffeine as internal standard (Figure 2). The running time of the reference sample and the internal standard was less than 3 min. The major peak at 1.421 min is for amoxicillin whereas that for caffeine is 2.974 min (Figure 1).

medicinal-chemistry-HPLC-chromatogram-amoxicillin

Figure 1: HPLC chromatogram of amoxicillin trihydrate as reference standard at wavelength (λ) 230 nm. AUC=Area under curve.

medicinal-chemistry-chromatogram-amoxicillin-trihydrate

Figure 2: HPLC chromatogram of amoxicillin trihydrate as reference standard and caffeine anhydrous as internal standard at wavelength (λ) 230 nm. Amox: Amoxicllin.

A five-point calibration curve was generated for amoxicillin in the concentration range of 5.26 to 263.0 μg/mL (Figure 3). The calibration curve provided a linear relationship between the peak area (y-axis) and the concentrations of amoxicillin trihydrate with the regression equation of y=194.41x + 0.004, R2=0.9995 (Figure 3). The residual points of the calibration curve were well distributed within acceptable limits (Figure 4).

medicinal-chemistry-calibration-curve-amoxicillin

Figure 3: HPLC calibration curve of amoxicillin trihydrate (reference standard).

medicinal-chemistry-Residual-calibration-amoxicillin

Figure 4: Residual plot of the HPLC calibration curve of amoxicillin trihydrate (reference standard).

Regression analysis cannot minimize the distance for all points simultaneously but does it for most of the points. The residual plot of points shows maximum points closer to line for amoxicillin (Figure 4).

The developed HPLC methods were validated using the International Conference on Harmonization guidelines and the parameters therein. It was performed using a well-designed experiment and statistically relevant methods in accordance with International Conference on Harmonization (ICH) guidelines on validation of analytical procedures [22,23].

The linearity of the detector response for amoxicillin was confirmed from 5.26 to263.0 μg/mL. The calibration curve (Figure 3) and the residuals (Figure 4) were inspected to asses linearity (Table 6).

Parameter Amoxicillin trihydrate
Concentration range 5.26 to 263.0 µg/mL
Number 5
Average values 0.001315
Correlation coefficient 0.9995
Relative standard deviation (%) 0.7483
Calibration equation y=194.41x + 0.004
Limit of detection (LOD) 1.6703 × 10-5
Limit of quantification (LOQ) 5.0617 × 10-5
System suitability 0.002
Method precision 0.58%

Table 6: Statistical validation of the calibration data for quantitative determination of amoxicillin.

The internal standard yielded accurate results as increase or decrease in peak area of analyte also affected area of internal standard. Peak ratios were directly proportional to concentrations (Table 7).

IS (AUC) RS (AUC) IS:RS (AUC ratio)
165429 478918 0.3454
164384 472481 0.3478
166733 479600 0.3477
165828 474066 0.3498
166732 474678 0.3513
172047 493711 0.3484
Mean=0.3484
SDEV=0.00201
%RSD=0.58%

Table 7: Analysis of homogenous reference amoxicillin solution for system suitability and precision analysis.

HPLC analysis of flucloxacillin and cloxacillin samples

HPLC method was developed and validated for the evaluation of flucloxacillinand cloxacillin samples. Analysis was carried out in an ambient temperature (25°C) with Shim pack CLC-NH2 C18 column 150 × 4.6 mm, 5 microns column and a Finnigan Spectra System HPLC. A mobile phase consisting of acetonitrile: 0.01M potassium dihydrogen phosphate, KH2PO4, with a ratio of 60:40 (v/v) yielded maximum sensitivity and separation with sample detection at UV wavelength of 225 nm.

HPLC analysis of reference flucloxacillin

HPLC chromatograms of flucloxacillin as reference sample (Figure 5) and with acetaminophen (paracetamol) as an internal standard (Figure 6) were developed. The running time for the reference sample and the internal standard were within four (4) min. The peak at 3.146 min is for flucloxicillin whereas that for acetaminophen is 1.953 min.

medicinal-chemistry-chromatogram-flucloxacillin-wavelength

Figure 5: HPLC chromatogram of flucloxacillin as reference at wavelength (λ ) of 225 nm.

medicinal-chemistry-chromatogram-flucloxacillin-acetaminophen

Figure 6: HPLC chromatogram of flucloxacillin as reference sample and acetaminophen as internal standard at wavelength (λ) 225 nm.

A four-point calibration curvewas generated for flucloxacillin in the concentrations range of 25.35 to 152.10 μg/mL (Figure 7). The calibration curve provided a linear relationship between the area under curve (y) and the concentrations of flucloxacillin with the regression equation of y=156.94x + 0.0699 (R2=0.995) (Figure 7). The residual points of the calibration curve were well distributed within acceptable limits (Figure 8).

medicinal-chemistry-calibration-curve-flucloxacillin

Figure 7: HPLC calibration curve of flucloxacillin (reference standard).

medicinal-chemistry-Residual-calibration-flucloxacillin

Figure 8: Residual plot of the HPLC calibration curve of flucloxacillin.

The methods were validated using the International Conference on Harmonization guideline and the parameters therein. It was performed using a well-designed experiment and statistically relevant methods in accordance with International Conference on Harmonization (ICH) guidelines on validation of analytical procedures [22,23]. The linearity of the detector response for flucloxacillin was confirmed within 25.35 to 152.10 μg/mL (Figure 7).

Calibration curves were analyzed using a linear regression model and linear co-efficients (Table 8). The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the signal–to– noise ratio ICH-Q2B, 1996] and were found to be 1.2837 × 10-4 and 3.89 × 10-4 μg/mL [23].

Parameter Flucloxacillin
Concentration range 25.35 – 152.10 μg/mL
Number 4
Average values 0.0066
Correlation coefficient (R2) 0.995
Relative standard deviation (%) 0.9262
Calibration equation y=156.94x + 0.0699
Limit of Detection 1.2837 × 10-4 μg/mL
Limit of Quantification  3.89 × 10-4 μg/mL
System suitability 0.00253
Method precision 0.25%

Table 8: Statistical validation of the calibration data for quantitative determination of flucloxacillin.

Areas under curve ratios were directly proportional to concentrations as increase or decrease in peak area of analyte also affected area of internal standard (Table 9).

IS (AUC) RS (AUC) IS:RS (AUC ratio)
780955 799289 1.0235
812336 830814 1.0227
801131 823499 1.0279
822182 843224 1.0256
797503 814643 1.0215
    Mean = 1.02424
SDEV = 0.00253
% RSD = 0.25%

Table 9: System suitability and precision parameters for reference flucloxacillin.

Accuracy for flucloxacillin was determined by the mean and SDV of the percentage recovery studies (Table 10).

Number (n) % Recovery
1 92.36
2 99.02
3 107.87
4 94.71
Mean 98.49
SDEV 6.834486

Table 10: Standard and internal standard recovery studies of reference flucloxacillin.

HPLC analysis of cloxacillin

HPLC chromatograms of cloxacillin as reference sample (Figure 9) and acetaminophen (paracetamol BP) as internal standard (Figure 10). The cloxacillin peak is at 2.874 min and that of acetaminophen is 1.933 min.

medicinal-chemistry-HPLC-chromatogram-cloxacillin

Figure 9: HPLC chromatogram of cloxacillin as reference at λ 225 nm.

medicinal-chemistry-HPLC-chromatogram-wavelength

Figure 10: HPLC chromatogram of cloxacillin as reference and acetaminophen as internal standard at wavelength 225 nm.

A four-point calibration curve was generated for cloxacillin in the concentration range of 11.72 to58.6 μg/mL. The calibration curve provided a linear relationship between the peak area (y) and the concentrations of amoxicillin injected (x) with the regression equation of y=787.78x + 0.0839 (R2=0.9986) (Figure 11). The residual points of the calibration curve were well distributed within acceptable limits (Figure 12).

medicinal-chemistry-calibration-cloxacillin-reference

Figure 11: HPLC calibration curve of cloxacillin (reference standard).

medicinal-chemistry-Residual-calibration-cloxacillin

Figure 12: Residual plot of the HPLC calibration curve of cloxacillin (reference standard).

The methods were validated using the International Conference on Harmonization guidelines and the parameters therein. It was performed using a well-designed experiment and statistically relevant methods in accordance with International Conference on Harmonization (ICH) guidelines on validation of analytical procedures (Q2A and Q2B). The linearity of the detector response for cloxacillin was from 11.72 to 58.6 μg/mL. The calibration curve (Figure 11) and the residuals (Figure 12) were inspected to asses linearity.

Calibration curves were analyzed using a linear regression model and linear coefficients (Table 11). The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the signal– to– noise ratio and were found to be 9.5246× 10-6 μg/mL and 2.8861 × 10-5 μg/mL respectively.

Parameter Cloxacillin 
Concentration range  μg/mL
Number 4
Average values 0.0025784
Correlation coefficient 0.9986
Relative standard deviation (%) 1.1340
Calibration equation y=787.78x + 0.0839
Limit of detection (LOD)  9.5246 × 10-6 μg/mL
Limit of quantification (LOQ) 2.8861 × 10-5 μg/mL
System suitability 0.00275
Method precision 0.0336%

Table 11: Statistical validation of the calibration data for quantitative determination of reference cloxacillin.

Peak ratios were directly proportional to concentrations as increase or decrease in peak area of analyte also affected area of internal standard (Table 12).

IS (AUC) RS (AUC) IS:RS (AUC ratio)
232461 195259 0.8391
237534 200609 0.8391
238890 185172 0.8445
230526 187178 0.7751
232653 190099 0.8171
Mean=0.81754, SDEV = 0.0275, % RSD = 0.0336

Table 12: Internal standard, system suitability and precision parameters for reference cloxacillin.

Accuracy for cloxacillin was determined by the mean and SDEV of the percentage recovery studies (Table 13).

Number % Recovery
1 91.17
2 91.51
3 96.46
4 113.41
Mean 98.1375
SDV 10.46475
SDEV= Standard deviation  

Table 13: Standard and internal standard recovery studies of reference cloxacillin (n=4).

HPLC analysis show that 75% amoxicillin capsules and 92.3% of suspension were within USP specification of 93.2 to 104.3% and 81.0 to 104.1% respectively. Sample of flucloxacillin capsuleshad 62.5% of the samples within specification of 96 to 120.5%. All suspension samples were below the required USP specification. None of cloxacillin capsule samples were within the USP specification. All the suspensionsamples, however, were within USP specification of 114.4 to 120.0%. The USP specifications for amoxicillin trihydrate and flucloxacillin are 92.5 to 110% and 80 to 120% of stated amount for capsules and suspensions, respectively. Cloxacillin samples had 90 to 120% of API for both capsules and suspensions.

Discussion

The samples of the three different penicillins evaluated varied slightly from the standard reference samples in the microbiological evaluation. Suspensions had lower MICs as compared to the capsule samples. All samples in general showed higher MIC compared to the reference standards. The developed and validated HPLC methods were suitable for the intended purpose. HPLC analysis of the samples showed some of the samples contained the right amount of active pharmaceutical ingredients as stated in the USP [24] and BP [25] but they had higher MICs against the test bacteria.

Antibacterial activities of penicillin samples

Most of the penicillinsamples were active against all the organisms but the mean zones of inhibition varied with different bacteria and sample as well as different concentrations. The pattern of zones of inhibition were not consistent as, in some cases, lower concentrations of the same sample had bigger or same sizes of zones of inhibition as compared to higher concentrations. This could be attributed to the fact that the antibiotic had to diffuse through the solid medium and the more concentrated they are, the higher the viscosity, hence, less diffusion rate. Consequently, the micro-dilution method was selected and used in the determination of the MIC as the test organisms are in direct contact with the antibiotic [26].

Helegbe et al. [27] reported that some selected antibiotics were active against some bacteria and recommended further studies on a larger scale. The current study, however, revealed higher MIC for the samples and this may be due to insufficient amount in the penicillin samples analyzed. A typical example is the report by Rahman et al. [28] which showed that zones of inhibition of amoxicillin samples against selected bacteria at 100 μg/mL were 19.5 mm for E. coli, 15.3 mm for B. subtilis and 17.0 mm for S. aureus. The current study on the other hand had no zones of inhibition at concentration below 250 μg/mL. The amoxicillin samples had MIC of 125, 180 and 220 μg/mL against E. coli, S. aureus and B. subtilis respectively and the current study, amoxicillin had MICs of 200, 200 and 300 μg/mL against E. coli, S. aureus and B. subtilis respectively.

There are differences between the literature values and that obtained from this study, but samples showed some level of sensitivity towards the test bacteria. Generally, there were differences in the sensitivity of Gram-negative and Gram-positive bacteria which could be due to the composition of the cell wall of two types of bacteria [29-31].

Some samples exhibited variations in the MIC. The antibacterial activity and MIC of samples varied from bacteria to bacteria which were similar to that of the reference sample. It was observed that, there were also variations among various brands and even batches within the same brand but variations were not significant (p>0.05).

Other reason that could account for differences in literature values and that of present study is the inoculum size of test organisms. Gbedema et al. [32] reported MIC of 0.46, 640, 0.29 and 0.26 mg/ mL against E. coli, P. aeruginosa S. aureus and B. subtilis 105 cfu/mL using the agar diffusion method. The inoculum size used in the present study was 106 cfu/mL and it is higher than the inoculum size used by Gbedema et al. [32]. This might have resulted in the higher MICs recorded for the samples compared to the values reported by earlier workers [28,32]. Besides that, the micro-dilution method used in the determination of the MIC is reported to be a better approach than the agar diffusion technique [20,21].

Beta-lactams are inhibited by the beta lactamases produced by bacteria and the size of inoculum will have direct influence on the performance of the antibacterial agent. The inoculum size will determine the amount of beta-lactamase present to deactivate the beta lactam ring [33].

Comparison results from the biological and chemical method revealed that some of the samples passed the chemical assay but had higher MIC values. For this reason higher doses of these samples of amoxicillin are required for the treatment of infections due to these bacteria. Amoxicillin has enantiomers with its mirror image having the same chemical structure. A compound and its enantiomer show different activity with only one of its enantiomers usually biologically active [34].

Antibacterial activities of samples were similar but not the same as those of the reference standard. In general, flucloxacillin and cloxacillin samples were much active against S. aureus and B. subtilis compared to E. coli and P. aeruginosa. This could be due to the simple reason that isoxazolyl antibiotics are not very active against Gram-negative bacteria [27]. Samples in suspension forms showed higher activity as compared to the capsules against Gram-negative and Gram-positive bacteria. The possible reason could be due to the nature of formulation and the type of experimental design (In vitro) used. Capsules are to be swallowed and an acidic environment is required to enhance dissolution and release of API.

The isoxazolyl antibiotics such as flucloxacillin are not sensitive to penicillinase enzymes secreted by many penicillin-resistant bacteria, but able to bind to penicillin-binding proteins (PBPs) and inhibit peptidoglycan cross-linkage. This is made possible due to the presence of the isoxazolyl group on the side-chain of the penicillin nucleus which facilitates the  resistance, since they are relatively intolerant of side-chain steric hindrance but it is not inactivated by β-lactamases. They are acid stable and have proven to be effective against S. aureus [35,36].

There are some antibiotics that have been found to be substandard and counterfeited [37,38]. Substandard and counterfeit antibiotics are also noted to be one of the main causes of bacterial resistance to antibiotics [39]. Reports on substandard and/or counterfeit antibiotics on various markets have triggered investigations into their quality and activity. Different approaches, both biological and chemical analysis are used in the evaluations. The unavailability of specific materials such as the type of column and solvent systems to be used in chemical analysis in some laboratories in some developing countries and comparison of the results with specifications in standard reference books such as United State Pharmacopoeia (USP) and the British pharmacopoeia (BP) have made it necessary for the modification and validation of the existing methods with materials readily available to suit the type of analysis being performed especially in resource restrain areas or settings.

HPLC analysis of penicillin samples

The internal standard (IS), caffeine, was selected based on the fact that caffeine did not interact with the sample and absorbs at the same wavelength as the sample but it did not have the same retention time as the sample.

HPLC method with a good linearity depicts the direct proportionality between concentration of analytes and the area under curve of the peaks. With correlation coefficient (r) of 0.9997 and R2 of 0.9995 from the regression analysis of the calibration curve shows the direct proportional relationship between concentrations and peak area ratios. This represents an excellent linearity between them and how precise the HPLC method is. The method was shown to be linear. Observation of the calibration curve also confirms the linearity of the method developed (Figure 3).

The ability for the analyte of interest as far as this study is concerned, to elute in the presence of other compounds was ensured. A specific method is able to distinguish analyte even in the presence of other similar compounds. The ability of the amoxicillin to elute at the same retention time when spiked with the internal standard (Figure 2) attests to the fact that the method was specific for the samples. The method can be used in the assessment of caffeine the analyte of interest. The internal standard was able to achieve the purpose for which it was intended (Table 12). Changes that could not be or difficult to control such as variations from run to run temperature and pressure during the run time were monitored by the internal standard. Relationship between the area under curve for the internal standard and area under curve for the reference standard yielded consistent area ratios (Table 13). The internal standard method is therefore considered the ideal as it yields accurate and precise results [40].

With respect to the suitability of a method, the USP [24] states that the percent relative standard deviation (%RSD) from a six replicates runs of homogenous samples must not be more than 2. The current method developed yielded RSD of 0.58% which is less than 2% and this is an indication of the suitability and precision of the method. The limits of detection and quantification values (Table 6) were indicative of how sensitive the method is. The attributes of the validation parameters considered shows that the method could be used to analyze amoxicillin samples within a considerable time using the readily available materials. The retention time of caffeine (internal standard) was 2.97 min whereas that of amoxicillin was 1.42 min at wavelength of 230 nm (Figure 2). The maximum absorption of the two compounds was detected at the same wavelength. Penicillins have no specific chromophore [41] and eluent must be maintained at wavelength less than 230 nm to obtain a meaningful detection limits. In this study, however amoxicillin was detected at wavelength of 230 nm. The reason for the possible difference in retention time could be due to the different types of columns used and flow rates used. This was the method described by Ashnager and Naseri [42] to analyze amoxicillin samples at wavelength of 230 nm using Spherimage-80, ODS, 2-5 mm C18 column. A similar study of amoxicillin gave a retention time of 10 min for amoxicillin using the same buffer system and temperature whereas retention time of 1.42 min was recorded for amoxicillin in this current study. Abreu and Ortiz [43] also had a retention time of 5.2 min for amoxicillin using the C18 column at wavelength of 229 nm with mobile phase of phosphate buffer and acetonitrile. The limits of detection and quantification values as (Table 6) were indicative of how sensitive the method was. The specificity of the method was confirmed when the internal standard and reference standard were spiked with different concentrations of the same samples and they gave distinctive peaks of the two compounds at their respective retention times (Figure 2).

Analysis of the samples revealed that the content of all 16 different samples of the capsules were in the range of 81.53 to 104.34% (Tables 14 and 15). Twelve samples had their content within the USP [24] specification of 92.5 to 110.0%. The sample with API of 93.2% was analyzed just 2 years before its expiry and few months after manufacturing and this means that the probability of the product failing later analysis before its expiry may be high.

Sample / Amount / % API
92.5 to 110 (USP, 2011) 92.5 to 110 (USP, 2011) 90-120 (USP, 2011)
Amoxicillin capsules 250 mg Flucloxacillin capsules 250 mg Cloxacillin capsules 250 mg
Sample code Amount (mg) % API Sample code Amount (mg) % API Sample code Amount (mg) % API
01A 260.85 104.34 FLMG01 276.10 110.44 CLLP01 156.00 62.40
01B 227.80 91.12 FLMG02 161.63 64.65 CLLP02 177.75 71.10
02A 255.95 102.38 FLMG03 111.85 44.74 CLLP03 145.18 58.07
02B 244.83 97.93 FLLP04 269.08 107.63 CLAR04 139.60 55.84
03A 203.83 81.53 FLLP05 250.98 100.39 CLAR05 201.95 80.78
03B 240.15 96.06 FLLP06 239.90 95.96 CLAR06    
03C 244.53 97.81 FLAR07 301.13 120.45 CLMG    
04A 230.07 92.03 FLAR08 147.65 59.06      
05A 237.45 94.98            
06A 217.20 86.88            
06B 253.48 101.39            
06C 238.58 95.43            
08A 232.97 93.19            
Amoxicillin capsules 500mg            
07A 480.00 96.00            
07B 481.85 96.37            
09A 493.15 98.63            

Table 14: HPLC analysis of amoxicillin, flucloxacillin and cloxacillin capsule samples.

Sample / Amount / % API
80 to 120 (USP, 2011)                                80 to 120 (USP, 2011)                                 90 to 120 (USP, 2011)
Amoxicillin (125 mg/5 mL) Flucloxacillin (125 mg/5 mL) Cloxacillin (125 mg/5 mL)
Sample code Amount % API Sample code Amount % API Sample code Amount % API
S01 117.56 94.05 FLMG01 52.66 42.13 CLSLP01 140.28 112.22
S02A 101.29 81.03 FLMG02 47.51 38.01 CLSP02 149.96 119.97
S02B 114.15 91.32 FLMG03 48.03 38.42 CLSLP03 143.05 114.44
S02C 101.66 81.33 FLLP04 48.91 39.13 CLSMG04 143.86 115.09
S03A 120.56 96.45 FLLP05 58.21 46.57 CLSMG05    
S04A 117.30 93.84 FLLP06 62.59 50.07      
S05A 98.38 78.70 FLAR07 45.05 36.04      
S06A 125.53 100.42 FLAR08 45.35 36.28      
S06B 126.75 101.40            
S06C 127.23 101.79            
S07A 130.14 104.11            
S08A 121.20 96.96            
S08B 110.53 88.42            

Table 15: HPLC analysis of amoxicillin, flucloxacillin and cloxacillin suspension samples.

The amount of API in suspension samples was 92.3% and these values are below the acceptable limit [24]. Percentages of active ingredient range of the suspension samples were from 81.03 to 104.1%. Two batches were found to contain 81.0 and 81.33% active ingredient respectively and these samples have their API fall below the USP [24] specification. The fact that they were analyzed few months after their manufacture may indicate the samples may breakdown before expiry or did not contain the right amount of API. Almost 8% of the samples had their APIs below the USP [24] range.

After observing flow rates between 0.5 and 1 mL/min, the later was found to give an optimal signal-to-noise ratio with a reasonable separation and retention. In the quest of finding internal standard, various reference standards were used including amoxicillin cloxacillin and flucloxacillin. Injection of flucloxacillin and cloxacillin gave peaks with almost the same retention time and hence could not be used as the internal standard. Acetaminophen gave a retention time different from that of cloxacillin and flucloxacillin. Hence, it was used as internal standard for the analysis of cloxacillin and flucloxacillin samples. Environmental changes that could not be or difficult to control such as variations from run to run, temperature, pressure and power fluctuations during the run time were also monitored by the use of the internal standard in the analysis of the samples (Tables 9 and 12).

The limit of detection and limit of quantitative of the analysis indicate the sensitivity of the method. The direct proportional relationship between concentrations and peak area ratios with correlation coefficient R2 of 0.995 for flucloxacillin and 0.9986 for cloxacillin from the regression analysis of the calibration curves and these indicate the level of linearity. For five runs of the same homogenous reference solution (Tables 9 and 12) the suitability and precision of the method were in the acceptable limit as stated in USP [24] with SDEV of 0.0025 and %RSD of 0.25 for flucloxacillin and standard deviation of 0.028 and %RSD of 0.034 for cloxacillin. All these values were less than 2% in the USP [24].

The range of recovery for flucloxacillin and cloxacillin were 92.4 to 107.9% and 91.2 to 113.4% respectively with an average percentage recovery of 98.5% for flucloxacillin and 98.1% for cloxacillin. These represent a high level of accuracy of the methods.

In the evaluation of flucloxacillin samples (capsules) using the acceptance limit of 92.5 to 110 % as stated in USP [24], 5 out of 8 samples evaluated were within the specification of USP [24] with percentage of 95.96 to 120.45 representing 62.5% of samples. The remaining samples had API of 44.7 to 64.7% which did not meet the specification in USP [24].

All the samples of flucloxacillin suspension analyzed were in the range of 36.0 to 50.1%. These content are outside the USP [24] and BP [25] range of acceptance limit of 80 to 120%. These low amounts of APIs may be due to insufficient active ingredients or poor storage conditions of the samples leading to the degradationof the API.

Antibiotics of this quality are threat to patients, the nation, and the world at large. Patients receiving such antibiotics would obviously not respond to minimum doses and would have to resort to higher doses. The activity of these antibiotic samples that failed the various evaluations may lead to antibiotic resistance in previously susceptible organisms.

Ensuring the quality, efficacy and safety of antibiotics would go a long way to prevent the problems associated with substandard and counterfeit antibiotics. The regulatory authorities that are mandated to regulate medicines must intensify their effort to monitor the quality and conditions of storage conditions of these antibiotics in especially developing countries.

Conclusion

All the penicillin samples (amoxicillin, flucloxacillin and cloxacillin) evaluated showed activity against test bacteria (E. coli, P. aeruginosa, S. aureus and B. subtilis). The level of activity and concentrations of penicillin samples gave different zones of inhibitions against these bacteria. Amoxicillin was observed to have broad spectrum activity showing activity against all bacteria used in the evaluation. Flucloxacillin and cloxacillin samples were observed to have higher activity against Gram-positive bacteria as compared to Gram-negative bacteria. P. aeruginosa was found to be most resistant bacteria to the penicillin samples. Suspension samples exhibited higher activity compared to capsule formulations. The MICs of 200 to 800 μg/mL were recorded for amoxicillin samples whereas flucloxacillin and cloxacillin samples had MIC of 500 to 1900 μg/mL. All samples of flucloxacillin suspensions and cloxacillin capsules had their API below the USP specification. Almost 83% of amoxicillin samples contained the right amount of API compared to 32.1 % of flucloxacillin and 44.4% of cloxacillin samples having the right amount of API.

Acknowledgements

We are grateful to Mr. Samuel Bekoe and Mr. James Oppong Kyekyeku of Department of Pharmaceutical Chemistry, and Dr. Edmund Ekuadzi, Department of Pharmacognosy, and Mr. Newman Osafo, Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana for their technical assistance.

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