INTRODUCTION:
Potassium Clavulanate, Potassium (2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo [3.2.0]heptane-2-carboxylate 1,2.
It is a potassium salt of clavulanic acid produced by the growth of certain strains of Streptomyces clavuligerus or obtained by any other means, is a powerfull β- lactamase (responsible for degradation of β-lactum antibiotics) inhibitor. Potassium Clavulanate is commonly used in combination with β-lactum antibiotics like amoxicillin, cefuroxime, cefpodoxime etc.
Various high-performance liquid chromatography methods have been developed and validated for the assay of potassium clavulanate in combination of β-lactum antibiotic in pharmaceutical preparations and biological fluids using special techniques such as precolumn derivatization, derivatization followed by solid phase extraction, post column derivatization, β-cyclodextrin stationary phase, amperometric detection and ion pair technology 3–10. Capillary electrophoresis with UV detection was also used for simultaneous determination of amoxicillin and clavulanic acid in pharmaceutical formulations 11. There were also methods reported for the determination potassium Clavulanate in biological fluids 12-13.
Potassium Clavulanate is official in IP 2007 14, BP 2009 1, USP XXX 15; in which isocratic method is reported for its assay and gradient method for its chromatographic purity. But till now to the best of our knowledge no stability indicating method has been reported for it.
In our research we developed a fast and cost effective HPLC method for the assay of potassium Clavulanate in bulk drug and in dosage forms and validate it as per ICH guidelines 16. Developed method was also able to resolve the degradation peaks from the peak of potassium Clavulanate and amoxicillin (as potassium Clavulanate is most commonly used in combination with it).
MATERIALS AND METHODS:
Potassium clavulanate was kindly provided by Medreich pharmaceuticals. Methanol (HPLC grade) and Water (HPLC grade) were purchased from Merck. (Mumbai, India).
Instrumentation:
JASCO HPLC system (2000 series) comprising of JASCO PU – 2080 plus intelligent pump, JASCO MD- 2010 plus multi wavelength detector and Rheodyne 7725i injector fitted with 20 μl capacity loop was used. Separations and quantitation was done on a Nucleosil C18 (250 mm x 4.6 mm) column.
Chromatographic conditions:
The mobile phase was consisted of methanol and 0.7% sodium dihydrogen phosphate (10: 90 v/v). The mobile phase was filtered through 0.45 µm filter and degassed by ultrasonic vibrations prior to use. The flow rate was 1.0 mL min-1.
Fig. 1: Representative Chromatogram of Potassium Clavulanate (50 µg mL-1) and Amoxicillin using optimized chromatographic conditions showing retention time of 4.21 and 6.76 min respectively.
Fig. 2: Chromatogram of potassium clavulanate after exposing it to acidic stress condition 5 N HCl for 20 min shows retention time of pot clav and its degreded product D1, D2 and D3 respectively at 4.13, 3.64, 3.85 and 5.0 min respectively.
Selection of detection wavelength:
Potassium clavulanate show good absorbance at 230 nm so it was selected as the wavelength of detection.
Preparation of dilution:
25 mg of drug was weighed and transferred to 25 ml volumetric flask containing about 15 mL water, ultrasonicated for 10 min to dissolve the drug and then the volume was made up to 25 mL with water. The solution was filtered using whatmann filter paper No.41. 2.5 mL of this solution was diluted to 25 mL with water and was used as stock solution.
Method development:
Method development was started with the mobile phase combination of 80:20:: water: methanol v/v, here retention time of drug was 2.8 min, also the peak was merging with that of the blank peak. So to increase the retention time content of methanol was decreased to 10% i.e. ratio used was 90: 10 v/v. here no significant increase in retention time was obtained still the drug peak was merging with that of the blank peak.
Fig. 3: Chromatogram of potassium clavulanate after exposing it to Basic stress condition A) 0.1 N NaOH for 10 min. B) 1 N NaOH for 20 min shows retention time of pot clav and its degreded product D4 and D5 respectively at 4.21, 9.55 and 7.51 min respectively.
Fig. 4: Chromatogram of potassium clavulanate after exposing it to oxidative stress condition 30% H2O2 for overnight shows retention time of pot clav and its degreded product D2 and D3 respectively at 4.24, 3.90 and 5.12 min respectively.
To have resolution of potassium clavualnate peak and the blank peak buffer was added to the mobile phase.
After step by step optimization final optimized chromatographic conditions were as listed below:
Mobile phase: A solution of 0.7% sodium dihydrogen phosphate and methanol 90: 10 v/v.
Flow rate: 1ml/mim
Wave length: 230nm
Temperature: Ambient.
Column: Nucleosil C18 (250 mm x 4.6 mm, 5µm)
The chromatogram obtained using above mentioned chromatographic condition was as shown in Fig. 1.
System suitability parameters Potassium Clavulanate and amoxicillin were tabulated in table 1.
Method Validation:
The method validation was done as per the ICH guidelines, and accordingly the parameters evaluated were:
1. Linearity and range.
2. Precision
3. Accuracy
4. Specificity
5. Limit of Detection
6. Limit of Quantification
7. Robustness
Table 1: System suitability parameters.
|
Parameters |
Potassium Clavulanate |
Amoxicillin |
|
Rt |
4.21 |
6.76 |
|
Assymetry |
0.85 |
0.80 |
|
Plate No. |
9158 |
7162 |
|
Resolution |
0 |
10.30 |
Table 2: Summary of degradation result.
|
Parameters |
Peaks obtained |
% Degradation |
|
Neutral |
No |
29.22 |
|
Acidic |
D1; D2 and D3 |
80.71 |
|
Basic |
D4; D5 |
90.3 |
|
oxidative |
D2; D3 |
90.8 |
Linearity and Range:
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. It was studied by analyzing five concentrations of the drug in the range of 1-6 µg mL-1 and process was repeated for five times.
Precision:
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The precision of the method was demonstrated by
1. Repeatability: Repeatability expresses the precision under the same operating conditions over a short interval of time. Repeatability is also termed intra-assay precision, was studied by injecting three concentrations (2, 3, 4 µg mL-1) of the drug, and process was repeated three times each.
2. Intermediate: Intermediate precision expresses within-laboratory variations:
Analysis on different days: was studied by injecting three concentrations (2, 3, 4 µg mL-1) of the drug, and process was repeated three times each, on three consecutive days.
Accuracy:
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found.
1. Recovery experiments. The recovery studies were carried out by standard addition method at three levels of 80, 100 and 120% and the percentage recovery was calculated.
2. Assay. Study was carried out using twenty tablets, each containing 75 mg potassium clavulanate. Tablets were weighed and finely powdered. A quantity of powder equivalent to 10 mg was weighed and transferred to 25 mL volumetric flask containing about 15 mL methanol, ultrasonicated for 10 min and then the volume was made up to 25 mL with methanol. The solution was filtered using Whatman filter paper No.41. From the filtrate appropriate dilutions were made in mobile phase to obtain concentration of 4 µg mL-1. The tablet sample solution was injected and chromatogram was obtained.
Table 3: Validation Parameters.
|
Parameter |
Obtained results |
|
|
Range |
10-50 µg mL-1 |
|
|
Linearity |
Y=10585x – 996.2 |
|
|
R2 |
0.997 |
|
|
Precision (%RSD) |
Intraday |
0.44 – 0.80% |
|
Interday |
0.83 – 1.76% |
|
|
Assay |
97.91% w/w |
|
|
Recovery |
80% |
100.54 |
|
100% |
101.06 |
|
|
120% |
100.89 |
|
|
LOD |
401 ng mL-1 |
|
|
LOQ |
1325 ng mL-1 |
|
Specificity:
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. Here study was done stress degradation studies.
Limit of Detection (LOD):
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value. Based on the standard deviation of the response and the slope of linear calibration data, the detection limit (DL) may be expressed as:
3.3 σ
DL=
S
Where, σ = the standard deviation of the response for the lowest conc. in the range, S = the slope of the calibration curve.
Limit of Quantification (LOQ):
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Based on the standard deviation of the response and the slope of linear calibration data, the quantitation limit (QL) may be expressed as:
10 σ
QL=
S
Where, σ = the standard deviation of the response for the lowest conc. in the range, S = the slope of the calibration curve.
Robustness:
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage and was done by observing the effect of following parameter on peak area and retention time:
- Influence of variations in mobile phase composition;
- Flow rate.
Fig. 5: Chromatogram of potassium clavulanate (30 µg mL-1) after keeping it overnight in water showing significant decrease in peak ares but no degradation peaks was found.
Stability study:
Potassium Clavulanate was exposed to stress degradation by conditions as recommended by ICH guideline i.e. hydrolytic, oxidative and dry heat degradation (table 2).
With Acid:
5 ml of stock solution was transferred in the 10 ml volumetric flask. To it 1 ml of 5 N HCl was added and the mixture was diluted to 9 ml with water, sample was kept as such for 20 min and then neutralized by adding 1 ml of 5 N NaOH. Prepared sample was injected under stabilized HPLC condition; the obtained chromatogram was shown in Fig. 2.
With Base:
5 ml of stock solution was transferred in the 10 ml volumetric flask. To it 1 ml of 1 N NaOH was added and the mixture was diluted to 9 ml with water, sample was kept as such for 20 min and then neutralized by adding 1 ml of 1 N HCl. Prepared sample was injected and the obtained chromatogram was shown in Fig. 3B. in this condition Pot Clav totally degraded so milder condition was used (0.1 N NaOH for 10 min) fig. 3A.
With H2O2:
5 ml of stock solution was transferred in the 10 ml volumetric flask. To it 1 ml of 30% H2O2 was added and the mixture was diluted to 10 ml with water, sample was kept as such for overnight. Prepared sample was injected in HPLC the obtained chromatogram was shown in Fig. 4.
Neutral hydrolysis:
Significant decrease in area of peak was obtained but there is no degradation peaks obtained after keeping the 50 µg ml-1 aqueous solution of potassium Clavulanate in water for overnight.
Table 4: Resolution between peaks under different conditions
|
Chromatographic condition |
Resolution between |
Value |
|
Acidic |
D1 and D2 |
1.79 |
|
D2 and Pot clav |
2.07 |
|
|
Pot Clav and D3 |
5.60 |
|
|
Basic |
Pot Clav and D2 |
26.93 |
|
D1 and D2 |
6.22 |
|
|
H2O2 |
D1 and Pot Clav |
2.01 |
|
D1 and D2 |
4.69 |
Table 5: Robustness study with flow rate.
|
Concentration (µg mL-1) |
Flow rate |
Rt |
% Change in Assay |
|
50 |
1.1 |
3.97 |
3.37% |
|
50 |
0.9 |
4.34 |
4.79% |
Dry heating:
No significant decrease in area was observed by exposing the potassium clavulanate blend with microcrystalline cellulose for 4 hr at 100 0C
RESULTS:
Linearity and Range:
The data obtained in the linearity experiments was subjected to linear-regression analysis. A linear relationship between peak areas and concentrations was obtained in the range of 10- 50 µg mL-1 with r2 0.997 for isocratic (Table 3).
Precision:
The developed method was found to be precise as the % RSD value for repeatability studies was less than 1%, where as the %RSD for interday precision was higher than that of intraday repeatability study, but was within the acceptable limit
Accuracy:
The results of recovery studies and assay for accuracy determination are depicted in Table 3. Good recoveries and assay of the drug was obtained at each added concentration, indicating that the method was accurate.
Specificity:
Potassium clavulanate mixture with amoxicillin was analyzed under HPLC (as is most popular combination); the obtained chromatogram shows two separate peaks for each drug with resolution grater than 10.
Potassium clavulanate was subjected to the stress conditions and it was observed that peaks of product of degraded product were well resolved from peak of potassium clavulanate (fig 3 and 5). The method were considered to be specific since there was no interfering peak at the retention time of potassium clavulanate and also the peak was well resolved from the peaks of degradants, also the resolution between each peak were more than 2 (Table 4). The peak purity profile by PDA detector also confirmed the specificity in which peak purity in front and tail have to be more than 950 (limit is not less than 900).
Limit of Detection (LOD) and Limit of Quantitation (LOQ):
The LOD and LOQ were found to be 401 ng mL-1 and 1325 ng mL-1 respectively, for potassium clavulanate (Table 3).
Robustness:
The results obtained by making small, deliberate change in some parameters are as follows:
· Influence of mobile phase composition: 2 % change in mobile phase composition leads to no significant change the retention time.
· Flow rate: changing the flow rate by 0.1 mL/min affects the method with retention time by ±0.23 min and area by ± 4.79% (Table 5).
DISCUSSION:
A stability indicating HPLC method, under hydrolytic conditions, has been developed for the determination of Potassium Clavulanate with fulfilling all the parameters as specified in the USP XXX (like resolution, RRTs, Plate no., tailing factor etc.). The method is also able to determine degraded products under above mentioned conditions. The major advantage of the developed method over the official one was no need to monitor the pH and temperature.
ACKNOWLEDGEMENT:
The authors wish to express their gratitude to Medreich pharmaceuticals, for providing gift sample of Potassium Clavulanate. The authors are also thankful to Principal, AISSMS College of Pharmacy for providing necessary facilities to carry out the research work.
REFERENCES:
1. British Pharmacopoeia. 2009; CD ROM version.
2. Sweetman SC, Martindale, 2005 The Complete Drug Reference, The Pharmaceutical Press, London.
3. Abounassif MA et al. Liquid chromatographic determination of amoxicillin and clavulanic acid in pharmaceutical preparations. J. Pharm. Biomed. Anal. 1991; 9: 731–735.
4. Tsou TL et al. Simultaneous determination of amoxycillin and clavulanic acid in pharmaceutical products by HPLC with β-cyclodextrin stationary phase. J. Pharm. Biomed. Anal. 1997; 15: 1197–1205.
5. Ellis H et al. A fast and simple method for the determination of clavulanic acid in human plasma using derivatisation reaction kinetics. J. Pharm. Biomed. Anal. 2000; 22: 933–937.
6. Foulstone M, Reading C. Assay of amoxicillin and clavulanic acid, the components of Augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob. Agents Chemother. 1982; 22: 753–762.
7. Martin C et al. Comparison of concentrations of two doses of clavulanic acid (200 and 400 milligrams) administered with amoxicillin (2,000 milligrams) in tissues of patients undergoing colorectal surgery. Antimicrob. Agents Chemother. 1995; 39: 94–98.
8. Chulavatnatol S, Charles BG. Determination of dose-dependent absorption of amoxycillin from urinary excretion data in healthy subjects. J. Chromatogr. 1993; 615: 91–96.
9. Cass QB et al. Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography. J. Chromatogr. A 2003; 987, 235–241.
10. Aghazadeh A, Kazemifard G. Simultaneous determination of amoxycillin and clavulanic acid in pharmaceutical dosage forms by LC with amperometric detection. J. Pharm. Biomed. Anal. 2001; 25: 325–329.
11. Pajchel G, Pawlowsky K, Tyski S (2002) J. Pharm. Biomed. Anal. 29: 75–81.
12. Hoizey G et al. Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC with UV detection. J. Pharm. Biomed. Anal. 2002; 30: 661–666.
13. Foroutan SM et al. Simultaneous determination of amoxicillin and clavulanic acid in human plasma by isocratic reversed-phase HPLC using UV detection J. Pharm. Biomed. Anal. 2007; 45: 531–534.
14. Indian Pharmacopoeia (2007).
15. USP XXX. CD ROM Version.
16. ICH Validation of Analytical Procedures: Text and Methodology Q2 (R1) (2005) International Conference on Harmonization IFPMA, Geneva.
Received on 08.08.2009 Modified on 25.09.2009
Accepted on 20.10.2009 © RJPT All right reserved
Research J. Pharm. and Tech. 3(1): Jan. - Mar. 2010; Page 141-145