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Spectrophotometric methods for the determination of ampicillin by potassium permanganate and 1-chloro-2,4-dinitrobenzene in pharmaceutical preparations
*Corresponding author at: Center of Excellence for Advanced Material Research, Faculty of Science, King Abdulaziz University, Saudi Arabia draapk@gmail.com (Aftab Aslam Parwaz Khan)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Available online 5 May 2012
Abstract
Two simple and sensitive kinetic methods for the determination of ampicillin (AMP) are described. The first method is based on kinetic investigation of the oxidation reaction of the drug with alkaline potassium permanganate at room temperature for a fixed time of 25 min. The absorbance of the colored manganate ions is measured at 610 nm. The second method is based on the reaction of AMP with 1-chloro-2,4-dinitrobenzene (CDNB) in the presence of 0.1 mol L−1 sodium bicarbonate. Spectrophotometric measurement was achieved by recording the absorbance at 490 nm for a fixed time of 60 min. All variables affecting the development of the color were investigated and the conditions were optimized. Plots of absorbance against concentration in both procedures were rectilinear over the ranges 5–30 and 50–260 μg mL−1, with mean recoveries 99.80 and 99.91, respectively. The proposed methods were successfully applied for the determination of AMP in bulk powder and in capsule dosage form. The determination of AMP by the fixed concentration method is feasible with the calibration equations obtained, but the fixed time method proves to be more applicable.
Keywords
Kinetic determination
Ampicillin
Potassium permanganate
1-Chloro-2,4-dinitrobenzene
1 Introduction
Ampicillin AMP, (6R)-6-(a-phenyl-d-glycylamino) penicillanic acid (Fig 1) is a semisynthetic penicillin (Goodman, 1991). It is prepared from the benzylpenicillin or penicillin-G (Wilson, 1982) which is prepared by a biosynthetic process using various strains of Penicillium notatum and Penicillium chrysogenum (Bentley, 1969). Penicillin-G was the first antibiotic to be used in the chemotherapy. It is a bacterio static drug of choice for the treatment of the infections caused by most of the Gram-positive and Gram-negative bacteria (Delgado and Remers, 1995) and a broad spectrum antibiotic (Rang et al., 1996). AMP is acidic in nature, and it acts by inhibiting the protein synthesis (Satoskar and Bhandarkar, 1990) of the bacterial cell wall. The basic nature of the ampicillin is 6-aminopenicillanic acid, which consists of a thiazolidine ring linked to β-lactam ring. The side chain determines the antibacterial and pharmacological characteristics of this compound (Rang et al., 1998). The several procedures reported for the determination of ampicillin in pure form or in pharmaceutical formulations as well as in biological fluids included by spectrophotometric (Askal et al., 1991; Al-Khamees et al., 1995; Sun et al., 1996; Sastry et al., 1998), polarographic (Belal et al., 1998; El-Sayed et al., 1994), flow injection analysis (Garcia et al., 1994), HPLC methods (Verdon and Couedor, 1999; Ishida et al.,1999) and the complexes of ampicillin with different metal ions have also been studied (Mukherjee and Ghosh, 1995; Alekseev and Samuilova, 2008; Lyle and Yassin, 1993). Kinetic-based methods of pharmaceutical analysis are not widely applied although they do not suffer any interference from additives which probably affects other methods. Furthermore, some specific advantages in the application of the kinetic methods can be expected (Mansilla et al., 1998):
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Selectivity due to the measurement of the evolution of the absorbance with the time of reaction instead of measuring a concrete absorbance.
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Possibility of no interference from the colored/turbid background of the samples.
- Chemical structure of AMP.
In the present work, kinetically based methods are proposed for the determination of AMP by measuring the absorbance at 610 nm after oxidation with alkaline KMnO4 or at 490 nm after addition of CDNB in the prescience of borate buffer. All variables affecting the development of the color were investigated and the conditions were optimized. The proposed methods were successfully applied for the determination of AMP in bulk powder and in capsule dosage form. The determination of AMP by the fixed concentration method is feasible with the calibration equations obtained, but the fixed time method proves to be more applicable.
2 Experimental
2.1 Apparatus
A Shimadzu UV–visible 1601 spectrophotometer was used for all spectral measurements, pH-metric measurements were done with Elico-Li 120 pH meter and a water bath shaker NSW 133, India was used to control the temperature.
2.2 Materials
AMP was purchased from Sigma (New Delhi, India). The KMnO4, NaOH and 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from Merck (Mumbai, India). Pharmaceutical preparations containing the studied compounds were purchased from commercial sources in the local market. Double distilled, de-ionized water was used throughout. The chemicals used were of analytical grade. Stock solutions of the compounds were wrapped with carbon paper to protect them from photodecomposition.
2.3 Method A
Transfer aliquots equivalent to 5–30 μg mL−1 AMP (solution A) into a series of 50 mL volumetric flasks. Add to each flask 1 mL of 0.5 mol L−1 NaOH and 2 mL of 5 × 10–3 mol L−1 KMnO4, mix well, dilute to volume with water, and leave to stand for 25 min. Measure the absorbance of the resulting solution at 610 nm at 5-min intervals at ambient temperature (25 °C) against a blank solution prepared simultaneously. To obtain the standard calibration curve, plot the values of absorbance against the drug concentration in μg mL−1 after 25 min.
2.4 Method B
Transfer aliquots equivalent to 50–260 μg mL−1 AMP (solution B) into a series of 10-mL volumetric flasks. Add to each flask 2 mL of 3.5 × 10−3 mol L−1 CDNB with 0.2 mol L−1 borate buffer of shake well, dilute to volume with distilled water, and leave to stand for 60 min. Measure the absorbance of the reaction mixture at 490 nm at 10-min intervals at ambient temperature (25 °C) against a blank solution prepared simultaneously. To obtain the standard calibration curve, plot the values of absorbance against the concentration of AMP after 60 min.
2.5 Procedures for formulations
The entire content of 20 capsules containing AMP were weighed and mixed well. Amount of the powder equivalent to 500 mg of AMP was weighed into a 100 mL volumetric flask containing about 75 mL of distilled water. It was shaken thoroughly for about 15–20 min, filtered through a Whatman filter paper No. 40 to remove the insoluble matter and diluted to the mark with distilled water. A volume of 25 mL of the filtrate was diluted to 100 mL and a suitable aliquot was analyzed using the general procedures followed in the concentration ranges mentioned above.
3 Results and discussion
3.1 Optimization of the reactions conditions
3.1.2 Method A (oxidation with KMnO4)
The reaction between AMP and KMnO4 in alkaline solution yields a green color as a result of the manganate species, which absorbs at 610 nm (Fig. 2). The intensity of the color produced increases gradually reaching its maximum after 25 min, when it remains stable for at least 1 h. As the intensity of color increases with time, it was deemed useful to elaborate a kinetically based method for the determination of AMP in bulk and in pharmaceutical forms. The reaction was investigated under various conditions of reagent concentration and alkalinity. Water was used to dissolve the drug since KMnO4 oxidizes with the production of green manganate ions. At room temperature the reaction increased substantially with time, as revealed by the intensification of the developed color and subsequent increase in the slope of the calibration graph (Table 1) indicating high analytical sensitivity.
Absorption spectrum of AMP (25 μg mL−1) after reaction with KMnO4: (a) oxidation product, (b) manganate ions (Method A).
Time (min)
Regression equation
Correlation coefficient (r)
Method A (oxidation with KMnO4)
5
A = −0.05554 + .04126 C
0.9912
10
A = −0.04602 + .04406 C
0.9945
15
A = −0.02212 + 0.05130 C
0.9975
20
A = −0.00122 + 0.0286 C
0.9932
25
A = −0.00015 + 0.0311 C
0.9982
Method B (reaction with CDNB)
10
A = −0.04041 + 0.03805 C
0.9968
20
A = −0.02110 + 0.04102 C
0.9959
30
A = −0.0076 + 0.0032 C
0.9982
40
A = −0.00078 + 0.0032 C
0.9991
50
A = −03.05 × 10−3 + 0.03826 C
0.9989
60
A = 2.12 × 10−3 + 0.03932 C
0.9994
3.2 The influence of KMnO4
The reaction rate and absorbance increases with increasing KMnO4 concentration. The absorbance was studied in the range 1 × 10−4 to 1 × 10−3 mol L−1 keeping all other parameter constant. It was found that 7.5 × 10−4 mol L−1 KMnO4 is the optimum concentration for the absorbance of AMP as shown in (Fig 3). The effect of the color development was investigated by adding different volumes (0.1–2.0 mL) of 7.5 × 10−4 mol L−1 potassium permanganate to a drug. The maximum absorbance of the green color was attained with 1.8 mL of the reagent, and remained constant even when higher volumes were added (Fig 4). Therefore, 2 mL of the reagent was used throughout the experimental investigations.
Effect of the concentration ranges 1 × 10−4 to 1 × 10−3 mol L−1 of KMnO4 on the intensity of the color produced during the reaction (AMP 25 μg mL−1; 1 mL of 0.5 mol L−1 NaOH).
Effect of the volume of 2 mol L−1 KMnO4 on the intensity of the color produced during the reaction (AMP 25 μg mL−1; 1 mL of 0.5 mol L−1 NaOH).
3.3 The influence of the NaOH
The reaction rate and absorbance increases with increasing KMnO4 concentration on the formation of
was also examined at constant concentration of drug, permanganate ion and varying volume (0.2–2.0 mL) of 0.5 mol L−1 NaOH at 25 °C. The optimum absorbance was obtaine with 0.9 mL of 0.5 mol L−1 NaOH, after which increase in volume of NaOH caused no changed in absorbance. Hence 1 mL of 0.5 mol L−1 NaOH was used throughout the experimental investigations (Fig. 5).
Effect of the volume of 0.5 mol L−1 NaOH on the intensity of the color produced during the reaction (AMP 25 μg mL−1; 2 mL of 7.5 mol L−1 KMnO4).
3.4 Method B (complexation with CDNB)
The reaction between the investigated drugs and CDNB in slightly alkaline borate buffer produces an orange-yellow color with maximum absorbance at 477 nm (Fig. 6). This bathochromic shift from 370 nm CDNB to 490 nm may be attributed to the formation of a charge transfer complex through n–π∗ interaction, where the electron donor is the amine moiety of the drug and the π-acceptor is the CDNB moiety (Amin et al., 2002).The possibility of the reaction of AMP with CDNB was investigated under various conditions. It was found that the reaction proceeds in alkaline medium and at room temperature. The absorbance of the colored adducts remains stable for at least one and half hour. The extent of formation of this species depends on the concentration of reactants, alkalinity, temperature, pH and therefore the effects of these variables were carefully studied.
Absorption spectrum of AMP (250 μg mL−1) after reaction with CDNB at pH 7.8 (Method B).
3.5 The influence of the CDNB
The effect of CDNB concentration on the absorbance of yellow colored Meisenheimer complex was studied in the range of 1 × 10−4 to 4 × 10−3 mol L−1. It was found that 3.5 × 10−3 mol L−1 CDNB is the optimum concentration for the absorbance of AMP as shown in Fig. 7. Therefore, the optimum concentration of 3.5 × 10−3 mol L−1 CDNB was chosen for further work.
Effect of the concentration ranges 1 × 10−4 to 4 × 10−3 mol L−1of CDNB on the absorbance value of the reaction product of AMP (250 μg mL−1) with 0.2 mol L−1 borate buffer.
3.6 The influence of pH
The influence of pH on the absorbance value of the reaction product was evaluated. Maximum absorbance value was obtained at pH 7.8 after which the absorbance of the reaction product began to decrease gradually until pH 9.5. Therefore, pH of 7.8 was chosen as the optimum pH (Fig. 8). Other buffers having the same pH value such as phosphate buffer and hexamine buffer were tried and compared with 0.2 mol L−1 borate buffer. The borate buffer was found to be superior to the phosphate and hexamine buffers having the same pH value since it gave the highest absorbance value.
Effect of the concentration of CDNB (3.5 × 10−3 mol L−1) on the absorbance of the colored product keeping AMP (250 μg mL−1).
3.7 Calibration graphs
After optimizing the reaction conditions, the fixed time was applied to the determination of AMP in pure form over the concentration ranges 5–30 and 50–260 μg/mL for both methods, respectively.
Analysis of the data gave the following regression equations:
A = 0.1606 + 0.01054 C (r = 0.9906) Method A
A = 0.3068 + 0.001126 C (r = 0.9919) Method B
The calibration graphs were shown in (Figs. 9 and 10).
Spectrophotometric calibration curves for Method A.
Spectrophotometric calibration curves for Method B.
3.8 Kinetic study of the reactions
The rate of the reactions was also found to be dependent on the concentration of AMP. The rates were followed at room temperature with various concentrations of AMP:
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In the range 5–30 μg mL−1, keeping KMnO4 and NaOH constant at high concentration as described in the general procedures, applying method A; and
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In the range 50–260 μg mL−1, keeping the other reactants, and CDNB constant at high concentration as described in the general procedures, applying method B.
From the graphs shown in Figs. 11 and 12, obtained by applying methods A and B, respectively, it is clear that the rate increases as the AMP concentration increases, indicating that the reactions rates obey the equation:
Plots of absorbance vs. time for the oxidation of AMP with alkaline KMnO4 (Method A) concentration of AMP: (1) 1.4 × 10−5, (2) 2.8 × 10−5, (3) 4.2 × 10−5, (4) 5.6 × 10−5, (5) 7.0 × 10−5, (6) 8.4 × 10−5 mol L−1.
Plots of absorbance vs. time for the reaction of AMP with CDNB (Method B) concentration of AMP: (1) 1.5 × 10−4, (2) 3 × 10−4, (3) 4.5 × 10−4, (4) 6 × 10−4, (5) 7.5 × 10−4 mol L−1.
Taking logarithms of rates and concentration, as shown in Table 2, Eq. (1) is transformed into:
Log ΔA/Δt
Log[AMP], (mol L−1)
Method A (oxidation with KMnO4)
−3.942
−4.854
−3.690
−4.553
−3.491
−4.376
−3.360
−4.244
−3.281
−4.155
−3.150
−4.075
Method B (reaction with CDNB)
−4.066
−3.824
−3.750
−3.523
−3.552
−3.251
−3.500
−3.222
−3.452
−3.125
log (rate) = −0.9172 + 0.9923 log C
r = 0.9936 Method A
log (rate) = −0.6790 + 0.8808 log C
r = 0.9914 Method B
Hence K′ = 0.121 S−1 or 0.209 S−1, applying methods A or B, respectively, and the reactions can be approximated to first order (n ≈ 1) with respect to AMP concentration.
3.9 Evaluation of the kinetic methods
The quantitation of drug under the optimized experimental conditions outlined above would result in a pseudo-first order with respect to their concentrations where KMnO4 concentration was at least 50 times of the initial concentration of AMP and NaOH concentration was at least 100 times the initial concentration of AMP applying method A; and CDNB concentration was at least four times of the concentration of AMP applying method B.
However, the rate will be directly proportional to drug concentration in a pseudo-first rate equation as follows:
4 Rate-constant method
Graphs of log (absorbance) versus time over the concentration ranges 1.4 × 10−5 to 8.4 × 10−5 mol L−1, and 1.5 × 10−4 to 7.5 × 10−4 mol L−1 AMP, were plotted by applying methods A or B, respectively. The pseudo-first order rate constants corresponding to different AMP concentrations were then calculated from the slopes multiplied by −2.303; they are presented in Table 3.
K′ (s−1)
Log[AMP], (mol L−1)
Method A (oxidation with KMnO4)
−9.4773 × 10−4
1.4 × 10−5
−7.9414 × 10−4
2.8 × 10−5
−5.6421 × 10−4
4.2 × 10−5
−4.9483 × 10−4
5.6 × 10−5
−5.3669 × 10−4
7.0 × 10−5
−4.8899 × 10−4
8.4 × 10−5
Method B (reaction with CDNB)
−4.9344 × 10−4
1.5 × 10−4
−4.6647 × 10−4
3.0 × 10−4
−3.7702 × 10−4
4.5 × 10−4
−1.5545 × 10−4
6.0 × 10−4
−1.0255 × 10−4
7.5 × 10−4
Regression of (C) versus K′ gave the equations:
K′ = −4.5 × 10−3 + 76.96 C (r = 0.9831) Method A
K′ = −4.1 × 10−3 + 0.5119 C (r = 0.8739) Method B
The value of r is indicative of poor linearity, probably because of inconsistency of K′.
4.1 Fixed-concentration method
Reaction rates were recorded for different AMP concentrations in the range 5.6 × 10−5 to 8.4 × 10−5 mol L−1, and 4.5 × 10−4 to 7.5 × 10−4 mol L−1, applying methods A or B, respectively. A pre-selected value of the absorbance was fixed and the time was measured in seconds. The reciprocal of time (i.e. 1/t) versus the initial concentration of AMP (Table 4) was plotted. The following equations for calibration graphs were obtained by linear regression:
1/t = −4.5 × 10−3 + 76.97 C (r = 0.9831) for Method A
1/t = −4.1 × 10−3 + 1.862 C (r = 0.9962) for Method B
| 1/t (s−1) | Log[AMP], (mol L−1) |
|---|---|
| Method A (oxidation with KMnO4) | |
| 1.11 × 10−3 | 5.6 × 10−5 |
| 8.40 × 10−4 | 7.0 × 10−5 |
| 6.70 × 10−4 | 8.4 × 10−5 |
| Method B (reaction with CDNB) | |
| 3.34 × 10−4 | 4.5 × 10−4 |
| 3.03 × 10−4 | 6.0 × 10−4 |
| 2.78 × 10−4 | 7.5 × 10−4 |
4.2 Fixed-time method
Reaction rates were determined for different concentrations of AMP. At a preslected fixed time, which was accurately determined, the absorbance was measured. Calibration graphs of absorbance versus initial concentration of AMP were established at fixed time of 5, 10, 15, 20 and 25 min applying method A and a fixed times of 10, 20, 30, 40, 50 and 60 applying method B with the regression equation shown in Table 1.
It is clear that the slope increases with time and the most acceptable values of the correlation coefficient (r) and the intercept were obtained for a fixed times interval for measurements when applying methods A and B, respectively (Table 5).
Parameters
Method A
Method A
Optical characteristics
λmax (nm)
610
490
Linearity range (μg mL−1)
5–30
50–260
Regression equation
Intercept (a)
0.0863
0.0994
Standard deviation of intercept (Sa)
0.6814
0.5814
Slope (b)
0.0138
0.0022
Standard deviation of slope (Sb)
0.002
0.0004
Correlation coefficient (r)
0.9977
0.9919
LOD (μg mL−1)
0.162
0.866
LOQ (μg mL−1)
0.493
0.2625
4.3 Mechanism of the reaction
The stoichiometry of the reaction was studied adopting the limiting logarithmic method (Rose, 1964). The ratio of the reaction between (log Abs versus log[AMP], log[KMnO4] and log[CDNB] were calculated by dividing the slope of KMnO4 and CDNB over the slope of the drug curve. It was found that, the ratio was 1:2 (AMP to KMnO4) while for (AMP to CDNB) the ratio was 1:1. The proposal pathway of the reaction is presented in scheme 1.
A detailed mechanistic scheme of the oxidation and reaction of AMP.
5 Validation of the proposed method
5.1 Accuracy and precision of the proposed methods
Accuracy and precision was checked according to USP validation guidelines (TUSP, 2002) at three concentration levels within the specified range, six replicate measurements were recorded at each concentration levels. The results are summarized in Table 6.
Amount taken (μg mL−1)
Amount found (μg mL−1)
% Recovery ± S.D.
±RSDa (%)
SAEb
Method A
10
9.96
99.86
0.520
0.02
20
19.2
99.34
0.478
0.04
30
30.05
100.06
0.368
0.05
Method B
55
54.95
99.97
0.432
0.02
150
150.02
100.04
0.428
0.03
260
259.05
99.72
0.424
0.06
5.2 Limit of detection (LOD)
LOD was calculated based on standard deviation of response and the slope of calibration curve. The limit of detection was expressed as: where σ is the standard deviation of intercept, S is the slope of calibration curve. The results were summarized in Table 5 indicating good sensitivity of the proposed method. According to USP validation guidelines (TUSP, 2002), the calculated LOD values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated drug concentration by LOD equations were actually detected in these experiments.
5.3 Limit of quantitation (LOQ)
LOQ was calculated based on standard deviation of intercept and slope of calibration curve. In this method, the limit o quantitation is expressed as: The results were summarized in Table 5 indicating good sensitivity of the proposed method. According to USP validation guidelines (TUSP, 2002), the calculated LOQ values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated drug concentration by LOQ equations were actually quantitated in these experiments.
6 Application to pharmaceutical dosage forms
The rate constant and fixed time methods of the proposed kinetic spectrophotometric method for determination of investigated AMP have been tested on commercial pharmaceutical dosage forms. The concentration of investigated AMP was computed from its responding regression equations. The results of proposed methods were statistically compared with those of reported methods (Saleh et al., 2003; Ayad et al., 1999; Taha, 2003), in respect to accuracy and precision. The obtained mean recovery values of the obtained amount were 99.80 and 99.91 respectively, which ensures that there is no interference of other active compounds present in the capsule. The calculated and theoretical value of both the proposed and the reported methods at 95% confidence level. This indicates good precision and accuracy in the analysis of investigated of AMP in pharmaceutical dosage forms.
7 Conclusion
Different methods were established to determine AMP concentration kinetically, the reaction rate method, rate constant and fixed time methods were applied. Applying the fixed time method, it is clear that the slope increased with time and the most acceptable values of correlation coefficients (r) and intercepts were obtained for a fixed time, which was therefore chosen as the most suitable time interval for measurements. The proposed method is sensitive enough to enable determination of lower amounts of drug, these advantage encourage the application of proposed method in routine quality control of investigated AMP in industrial laboratories. Finally our method provide advantages of improving selectivity, avoiding interference of colored and turbidity background of samples as our methods measure the increase in absorbencies with time against blank treated similarly and possibility avoiding interference of other active compound present in commercial product.
References
- Complex formation in systemscobalt(II)–glycine–beta-lactam antibiotics. Russ. J. Inorg. Chem.. 2008;53:327.
- [Google Scholar]
- Kinetic method for the quantitation of ampicillin trihydrate in bulk and in drug formulations. Sci. Pharm.. 1995;63:191.
- [Google Scholar]
- Colorimetric determination of β-blockers in pharmaceutical formulations. J. Pharm. Biomed. Anal.. 2002;30:1347.
- [Google Scholar]
- Utility of certain pi-acceptors for the spectrophotometric determination of some penicillins. Analyst. 1991;116:387.
- [Google Scholar]
- Spectrophotometric and atomic absorption spectrometric determination of certain cephalosporins. J. Pharm. Biomed. Anal.. 1999;18:975.
- [Google Scholar]
- Polarographic determination of some penicillins through nitrosation. J. Pharm. Biomed. Anal.. 1998;17:275.
- [Google Scholar]
- Text Book Pharmaceutical Chemistry (eighth ed.). London: Oxford University Press; 1969. pp. 793
- Wilson & Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry (tenth ed.). Philadelphia, PA: Lippincott; 1995. pp. 7
- DC polarographic determination of ampicillin in pharmaceutical dosage forms. Anal. Lett.. 1994;27:2515.
- [Google Scholar]
- Determination of ampicillin or amoxicillin in pharmaceutical samples by flow injection analysis. J. Pharm. Biomed. Anal.. 1994;12:1585.
- [Google Scholar]
- The Pharmacological Basis of Therapeutics Vol vol. 2. (eighth ed). New York: Pergamon Press; 1991. pp. 1076
- Simple high performance liquid chromatographic determination of ampicillin in human serum using solid-phase extraction cartridge. J. Chromatogr.. 1999;727:245.
- [Google Scholar]
- Chemical Analysis (second ed.). New York: McGraw-Hill; 1975.
- Polarographic studies of metal ion complexes of Ampicillin and Amoxycillin. Anal. Chim. Acta. 1993;274:225.
- [Google Scholar]
- Kinetic determination of ansamicins in pharmaceutical formulations and human urine. Manual and semiautomatic (stopped-flow) procedures. Anal. Chim. Acta.. 1998;376:365.
- [Google Scholar]
- Metal ion interaction with penicillins part VII: mixed-ligand complex formation of cobalt(II), nickel(II), copper(II), and zinc(II) with ampicillin and nucleic bases. J. Inorg. Biochem.. 1995;59:827.
- [Google Scholar]
- Pharmacology (third ed.). Churchill Livingstone Publications; 1996. pp. 725
- Pharmacology (fourth ed.). Chuchill Livingstone Publications; 1998. pp. 691
- Advanced Physicochemical Experiments. London. UK: Pitman; 1964.
- Spectroscopic analytical study for the charge-transfer complexation of certain cephalosporins with chloranilic acid. Anal. Sciences. 2003;19:281.
- [Google Scholar]
- New spectrophotometric method for the determination of some drugs with iodine and wool fast blue. Talanta. 1998;45:1227.
- [Google Scholar]
- Pharmacology and Pharmacotherapeutics Vol vol. II. (eleventh ed). Bombay: Popular Prakashan; 1990. pp. 549
- Amodification of B.P network and application for simultaneous determination of four drugs. Fenxi Shinyanshi. 1996;15:39.
- [Google Scholar]
- Kinetic spectrophotometric methods for the determination of dothiepin hydrochloride in bulk and in drug formulation. Anal. Bioanal. Chem.. 2003;376:1131.
- [Google Scholar]
- The United States Pharmacopoeia XXV and NF XX., 2002. American Pharmaceutical Association. Washington. DC.
- Multiresidue analytical method for the determination of eight penicillin in muscle tissue by ion pair-reversed phase HPLC after precolumn derivatization. J. Assoc. Off. Anal. Chem.. 1999;82:1083.
- [Google Scholar]
- Techniques of Organic Chemistry. Part 1. Vol vol. 3. New York: Interscience; 1953.
- Text Book of Organic Medicinal and Pharmaceutical Chemistry (eighth ed.). Philadelphia: J.R. Lipponcot Company; 1982. pp. 237
- Kinetic Methods of Analysis. Pergamon Oxford Press; 1966.
