Acta Chromatographica 21(2009)1, 57–70 DOI: 10.1556/AChrom.21.2009.1.

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RP-HPLC Method for Analysis of Related Substances in Amoxicillin Drug Substance
CH.B.V.N. RAJU1,*, H.K. SHARMA1, CH.S. RAO1, AND G.N. RAO2
1Aurobindo 2Department

Pharma Limited, Srikakulam Dist., Pydibhimavaram-532 409, India of Inorganic and Analytical Chemistry, Andhra University, Visakhapatnam-530 003, India E-mail: chbvn_5@yahoo.co.in

Summary. Linear gradient HPLC on a C8 column has been used for separation of individual related substances of amoxicillin listed in the European Pharmacopoeia and a newly identified degradation impurity. The USP plate count for the amoxicillin peak was more than 3000 and USP tailing for the same peak was less than 2.0. Forced degradation studies were conducted on amoxicillin drug substance using ICH stress study guidelines to demonstrate the specificity and stability-indicating nature of the method. A new impurity observed after thermal and alkaline degradation was identified as N-pivaloylamoxicillin. The LOD and LOQ for individual related substances were below 0.045 and 0.086% (w/w), respectively. The method was fully validated in accordance with ICH analytical method validation guidelines. The results of the study prove the method is specific, precise, linear, robust, and can be used for evaluation of the stability of amoxicillin drug substance. Key Words: amoxicillin, N-pivaloylamoxicillin, forced degradation, method validation

Introduction
Amoxicillin (Fig. 1) is bactericidal compound with activity against Grampositive and Gram-negative organisms. Most strains of meningococcus, pneumococcus, and gonococcus are sensitive to amoxicillin. Amoxicillin has been reported to be more active in vitro than ampicillin against enterococcus, Helicobacter pylori, and salmonella spp. but less active against shigella spp. [1, 2]. Several LC methods have been reported for assay of amoxicillin in drug substance [3–5] and for analysis of related substances in drug substance and
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6-aminopenicillanic acid amoxicillin amide (q).2-dimethylpropanoyl)amino]-2-(4-hydroxyphenyl) acetic acid (l). 3-(4-hydroxyphenyl)pyrazine-2-ol (m). drug products [4–9]. amoxicillin diketopiperazines 1 and 2 (j and k).N. and premixes [17. 18]. (4-hydroxyphenyl)glycylamoxicillin (h). 21]. and N-pivaloylamoxicillin (s). Although an LC method with UV and mass spectrometric detection for related substances has also been reported [24]. amoxicilloic acid dimers 1 and 2 (n and o). and impurities eluted before amoxicillin [23]. 1. Some LC methods with UV detection have been described for separation of sidechain diastereo isomers [19]. A gradient LC method [5] based on the USP method [4] has been proposed for purity control of related substances in amoxicillin drug substance.V. LC–UV and LC–MS methods have been reported for separation and identification of only thirteen of the potential impurities of amoxicillin [24]. it was specified in the European Pharmacopoeia and further examined in a collaborative study.58 Ch. H NH2 H H N H S N O H (a) O (CH3)3C C HN H H H N N O H H S CH3 CH3 COOH CH3 CH3 COOH O HO O HO (b) Fig. Raju et al. amoxicilloic acids 1 and 2 (c and d).B. These methods [5. (2R)-2-[(2. amoxicillin and amoxicillin dimer [22]. The molecular structures of (a) amoxicillin and (b) N-pivaloylamoxicillin . 24] are neither validated nor stability indicating. dosage forms [10–16]. 6-aminopenicillanic acid (e). L-amoxicillin (f). amoxilloic acids 1 and 2 (g and i). no validated stabilityindicating LC method has been reported for separation and quantitative determination of amoxicillin (a) and its related substances including isomers (2R)-2-amino-2-(4-hydroxyphenyl) acetic acid (b). C5 epimers of amoxicilloic acids [20. amoxiamoxicilloic acid dimers 1 and 2 (p and r).

identify without reference material. The method can be used for routine quality control and stability evaluation of amoxicillin drug substance stored in different packing and under different storage conditions and to quantify the substances (b–s) at low concentrations (μg mg−1 levels). pH 5. and quantify the individual substances (b–s) using response factors against the substance (a). High-purity water was prepared by use of a Millipore Milli Q plus water-purification system.5 mg mL−1) spiked with substances b–s (1. light. base.RP-HPLC Analysis of Related Substances in Amoxicillin 59 This paper reports the development of an LC method for separation of individual related substances of amoxicillin and a newly identified impurity (Fig. . report the retention times of substances (b–s). peroxide.015 mg mL−1) and amoxicillin control sample (1. Hyderabad. elevated temperature. Standard (Std) and test solutions were prepared from amoxicillin qualified standard (0. The authors felt it necessary to develop a stability-indicating method for separation and quantification of related substances and known degradation products in amoxicillin drug substance. and humidity) in accordance with International Conference on Harmonization (ICH) guidelines [26] and analysed to prove the specificity and stabilityindicating nature of the method. The novelty of the proposed method over previously reported methods [3–23] is its capacity to: • • • • separate substances (a–s) with resolution suitable for quantification. India) were used.0 adjusted with dilute NaOH) was used as diluent. Amoxicillin drug substance was exposed to forced degradation conditions (acid. India. The method was validated in accordance with ICH method-validation guidelines [27]. Phosphate buffer (0. Acetonitrile (LC grade) from Merck (Germany) and potassium dihydrogen orthophosphate (AR grade) from Rankem (Mumbai. 1) by an approach similar to that reported earlier [25].0%).05 M. Experimental Chemicals and Solutions Samples of amoxicillin drug substance and its related substances were obtained from the chemical research and analytical research departments of Aurobindo Parma.

accuracy. A Humidity-Photo stability chamber (Thermo Lab. the sample was stored at 92% RH and 25ºC for 144 h). robustness. . PESCIEX triple-quadrupole mass spectrometer with Analyst software. Raju et al. Molecular mass was determined by use of a Perkin–Elmer API2000. The conditions used were: • • • • • • temperature (the sample was stored at 105ºC for 3 h).75 mg mL−1) was prepared and injected. Mumbai. Peak purity for substances (a–s) in test solution and for amoxicillin in the presence of degradation products obtained in degradation studies was evaluated by use of the PDA detector.N. acid (1 M aqueous HCl (1 mL) was added and the sample was stored at room temperature for 30 min). Instrumentation The LC system used for method development and specificity studies was a Waters Alliance 2695 separation module with thermostatic compartment and photodiode-array (PDA) detector with Empower software.V. limits of detection (LOD) and quantification (LOQ). oxidation (10% v/v H2O2 (1 mL) was added and the sample was stored at 85°C for 2 min). linearity. Forced Degradation/Specificity Study The specificity of the method was evaluated using amoxicillin test solution and samples from forced degradation of a solution of amoxicillin drug substance (75 mg). and relative humidity (RH.000 lux for 144 h at 25°C). and sample solution (0. The LC system used for validation and to study intermediate precision was a Waters alliance 2695 separation module with thermostatic compartment and a 2487 dual-wavelength detector with Empower software. and solution stability.5 M aqueous NaOH (1 mL) was added and the sample was stored at 25°C for 2 min).60 Ch. The degraded samples were neutralized if necessary. India) was used for light and humidity degradation studies. light (the sample was illuminated at 12. Method Validation The method was validated for precision.B. base (0.

LOD and LOQ LOD and LOQ were obtained by study of linearity for five low concentrations (0. 0. 0. Accuracy The control sample solution (1. instruments. the method (method precision). and the ruggedness (intermediate precision) are important aspects of method validation. y-intercept. and 150% of specification level (1. System precision was evaluated by calculating RSD (%) for the area counts obtained from six injections of standard solution to prove the consistency of the output signal produced by the LC system.15 μg mL−1) of the individual substances. The regression equations were of the type y = mx + b (x is the concentration and y is peak area). . and 0. The intermediate precision was evaluated using the same solutions with different columns.05. 100.0%).5 mg mL−1) was spiked with substances b– s at 50. and analysts. 0. residual sum of squares. Linearity Calibration plots were constructed for substances a–s by plotting peak-area counts against concentration in the range from the LOQ to 150% of specification (2. The response factors were calculated by comparing the slopes of the calibration plots for substances b–s with that for amoxicillin (a). Method precision was evaluated by calculating RSD (%) for the results obtained from six individual determinations using standard and test solutions to prove the consistency of the results produced by the method.1.RP-HPLC Analysis of Related Substances in Amoxicillin 61 Precision The precision of the LC instrument (system precision).0% for substances c and d and 1.0% for substances b and e–s. Recovery (%) was calculated using the results obtained.125. Substances b–s were quantified against area count of amoxicillin in a standard solution multiplied by their response factors to obtain the result (% w/w). The RSD (%) of the area counts was calculated for six replicates at the predicted LOQ level. and correlation coefficient were calculated.075. The slope.

and column oven temperature were deliberately altered.5 was fixed as defining system suitability.V.1 unit. acetonitrile (organic) content of mobile phase.62 Ch. one at a time.6 mm. detector wavelength. Good separation of a–s was eventually achieved by use of a 150 mm × 4. column with the linear gradient program given in Table I at a flow rate of 2. The differences (%) between the area counts for each substance at hourly intervals were calculated. flow rate. Raju et al. mobile phase pH. Substances b–r were potential impurities reported in the European Pharmacopoeia [3]. and ±5º. The USP resolution between substances k and l of not less than 1. composition of mobile phase components A and B at different times (linear gradient program).N. The effect of these changes on USP resolution. The column oven temperature was maintained at 40°C for good selectivity and sharp peaks. and on selectivity for the individual substances was checked. 5-μm particle. and USP plate count.B. Results and Discussion Optimization of Chromatographic Conditions When the test solution was prepared and injected using the method [3] reported elsewhere. k merged with n. .0 mL min−1. Trials were conducted using C8 and C18 stationary phases from different manufacturers to achieve selectivity for substances a–s by changing pH. Robustness The chromatographic conditions flow rate. Substance s is formed by acylation of the amino group of the side chain with pivaloyl chloride. ±1%. At 254 nm [3] UV absorbance of d is low whereas that of b is high. Good response to substances a–s was achieved at 230 nm. Solution Stability The stability of the sample solution was evaluated by analyzing the test solution at hourly intervals for 24 h during storage at ~25 and ~6°C. it is also formed in thermal and alkaline degradation studies. ±0. which is used in the manufacturing process. ±5 nm. USP tailing. respectively. and p and r merged with q.0) and acetonitrile were selected as mobile phase components A and B. by ±10%. Agilent Zorbax SB-C8. and column oven temperature. To achieve better chromatographic separation 0. compound j merged with i. respectively.05 M potassium dihydrogen orthophosphate buffer (pH 5. and test solution was injected under each condition.

0 84.00 25. Chromatographic separation of a–s is illustrated in Fig.0 Component B 0.00 40.00 50. Mass spectrum of N-pivaloylamoxicillin.01 5.0 94.00 60.0) and component B was acetonitrile Fig.00 51. A is the molecular ion peak. Table I.0 6. Retention times (tR) and MS m/z values for individual substances a–s are given in Table II. 4.0 16. are given in Figs 2 and 3. Linear mobile phase gradient program Time (min) 0.0 100. Mass spectra. and C is the sodium adduct ([M + H]+ + Na) .0 100.0 84.RP-HPLC Analysis of Related Substances in Amoxicillin 63 It was isolated by preparative LC and characterized by LC–MS and NMR spectrometry (Bruker (Faellanden.00 Component A 100. B is the ammonium adduct of the molecular ion peak ([M + H]+ + NH4). respectively.0 0.0 100.0 0.0 0.0 16. Switzerland) Avance DPX-300M spectrometer).0 Component A was potassium dihydrogen orthophosphate buffer (pH 5. and NMR spectra with chemical shifts. 2. [M + H]+.

Proton NMR spectrum of N-pivaloylamoxicillin Fig.64 Ch. Raju et al. 3. 4.N.B.V. Chromatogram obtained from amoxicillin drug substance spiked with its related substances . Fig.

This study proves the specificity and stabilityindicating nature of the method.045 0.551 0.031 0.82 0.472 0.037 0.190 Purity threshold 0.018 0.692 8.018 0.97 1.015 0.021 0.37 0.158 3.960 0.256 0. Degradation products were obtained in temperature.54 1.149 0. and method validation data Substance a b c d e f g h I j k l m n o p q r s m/z (M+) 365 167 383 383 216 365 339 514 339 365 365 251 188 748 748 730 563 1095 449 tR (min) 4.56 1.232 0.468 16.027 0.353 4.502 4.384 8.15 1.336 0.033 0.027 0.452 20. Summary of mass values.197 0.189 0.086 0.030 0.021 0.054 0.507 4.825 77.895 13.542 15.57 1.765 20. The results from the forced degradation studies are given in Table III.013 0.800 0. oxidation.904 3.19 4.681 30.05 1.RP-HPLC Analysis of Related Substances in Amoxicillin Table II.020 LOQ (% w/w) – 0. chromatographic retention times.558 Slope 15441 28703 13005 13005 3531 15984 14702 16252 14702 15173 15173 18779 17255 9848 9848 13438 7753 9919 11063 yintercept 2301 2630 18 18 51 797 1141 497 1141 846 846 1602 −800 756 756 86 −55 343 241 65 Residual sum of squares 1407 2328 1820 1820 308 822 1166 1596 1166 1634 1634 2275 2504 822 822 1400 821 692 853 Forced Degradation/Specificity Study The smaller purity angles than purity thresholds for substances a–s in the test solution (Table II) are indicative of the spectral homogeneity and absence of co-eluting peaks.235 1.037 0.00 0. The peak-purity angle was found to be less than the purity threshold for amoxicillin in all the degradation studies.502 4.012 22.05 0. which confirms that no degradation product was co-eluted with amoxicillin.837 12.95 1.005 0.037 0.02 1.755 2.326 0.008 0.02 0.559 30.234 1.89 1.018 5.038 Response factor 1.015 0.797 5.865 39.023 0.016 0.773 0.176 10.19 1.036 0.031 0.57 1.762 1.871 22.037 0.951 34.853 1.054 0. .013 0.020 0.136 0.037 0.032 0.314 3.021 0.018 0.99 1.902 18.049 0.40 Purity angle 0.481 3.215 3.033 4.593 21.028 0.748 14.011 0.039 0.280 0. and acid and basic degradation studies only.758 19.432 LOD (% w/w) – 0.181 3.

78 8.25 0.26 0.02 0.15 0. Raju et al.86 0.04 – – – – 0.16 – – – Basic degradation (%) 89. Table III.N.78 – 2.20 – – – – – – – 0.80 – – – – 13.24 – – 0.95 0.97 0.18 – – – Humidity degradation (%) 99.99 0.18 Thermal degradation (%) 77.16 – – – .33 0.21 – – – – – 0.B.25 0.18 – – 0.75 mg mL−1 sample solution and analyzed using the proposed chromatographic conditions Not degraded (%) 99.24 0.02 – – – – 0.22 – – 0.05 7.33 – 0.55 0.07 2.23 – – – Substance a b c d e f g h i j k l m n o p q r s Acid degradation (%) 89.23 – 0.09 – – – 0.83 0.20 0.78 – – – 0.57 0.23 – – 0.66 Ch.23 – – – – – – – 0.60 – – – 0.08 8.30 – 0.21 – – 0.09 – – – 0. Summary of results from forced degradation studies conducted using 0.15 – – – Photolytic degradation (%) 99.14 0.27 0.03 – – – – 0.47 0.73 Peroxide degradation (%) 88.24 – 0.V.23 1.02 – – – – 0.

Accuracy Recovery of the individual substances at 50. RSD for six replicate injections at the LOQ level was <10%. Robustness When the chromatographic conditions were deliberately altered.RP-HPLC Analysis of Related Substances in Amoxicillin 67 Method Validation Precision RSD for system precision. method precision. and intermediate precision was less than 2. and 2. The slope. respectively. y-intercept. which proves the accuracy of the method. 100. LOD and LOQ LOD and LOQ for each substance are given in Table II. The results of the study. prove the robust nature of the method. and 150% of specification concentrations were between 90 and 110%.998. residual sum of squares. Linearity Correlation coefficients (r) for calibration plots for the individual substances were >0. and response factors are given in Table II. given in Table IV. systemsuitability results remained within acceptance limits and selectivity for individual substance was not affected.5.0%. 1. .0. which confirm the high precision of the method.

Raju et al.8 0.80 2.0 1. precise.V.20 # # # # # # # # ** # # +1 -1 # # # # # # 230 # # # # 225 235 # # # # 5.8 0. and on USP resolution between substances k and l Column oven temperature (ºC) 40 # # # # # # 35 45 # # Alteration Flow (mL min−1) Organic strength (%) Detector wavelength (nm) Mobile phase pH USP plate count USP tailing USP resolution Not altered Flow rate Organic strength Detector wavelength Column oven temperature Mobile phase pH 2.5 1.8 2. All the results from method validation were satisfactory.8 1.5 2.8 1.8 0.9 1.8 0. and selective.5 1.8 0. .0 1.1 3471 3951 3740 3635 3889 3473 3471 3675 3302 3700 3741 0.N.0 # # # # # # # # 4.8 0.8 #Chromatographic conditions not altered **Acetonitrile strength at different times are given in Table I Solution Stability The test solution is stable for at least for 4 h at 25°C and 24 h at 6°C. This validated stability-indicating method can be used for quality control and studies for assessment of the stability of amoxicillin drug substance. Table IV.8 0.8 0. Conclusions This method is rapid. accurate. Effect of deliberately altered chromatographic conditions on USP plate count and USP tailing for substance a.68 Ch.9 1.8 1.8 0.B.9 5.8 0.2 1.

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