Acta Chromatographica 21(2009)1, 57–70

DOI: 10.1556/AChrom.21.2009.1.5

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 Pharma Limited, Srikakulam Dist., Pydibhimavaram-532 409, India
2Department 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 indi-
vidual 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 degrada-
tion 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 be-
low 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 Gram-
positive 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 entero-
coccus, 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

0231–2522 © 2009 Akadémiai Kiadó, Budapest

58 Ch.B.V.N. Raju et al.

drug products [4–9], dosage forms [10–16], and premixes [17, 18]. Some LC
methods with UV detection have been described for separation of side-
chain diastereo isomers [19], C5 epimers of amoxicilloic acids [20, 21],
amoxicillin and amoxicillin dimer [22], and impurities eluted before amox-
icillin [23]. Although an LC method with UV and mass spectrometric detec-
tion for related substances has also been reported [24], no validated stability-
indicating 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), amoxicilloic ac-
ids 1 and 2 (c and d), 6-aminopenicillanic acid (e), L-amoxicillin (f), amoxil-
loic acids 1 and 2 (g and i), (4-hydroxyphenyl)glycylamoxicillin (h), amox-
icillin diketopiperazines 1 and 2 (j and k), (2R)-2-[(2,2-dimethylpropa-
noyl)amino]-2-(4-hydroxyphenyl) acetic acid (l), 3-(4-hydroxyphenyl)pyra-
zine-2-ol (m), amoxicilloic acid dimers 1 and 2 (n and o), amoxiamoxicilloic
acid dimers 1 and 2 (p and r), 6-aminopenicillanic acid amoxicillin amide
(q), and N-pivaloylamoxicillin (s). A gradient LC method [5] based on the
USP method [4] has been proposed for purity control of related substances in
amoxicillin drug substance; it was specified in the European Pharmacopoeia
and further examined in a collaborative study. LC–UV and LC–MS methods
have been reported for separation and identification of only thirteen of the po-
tential impurities of amoxicillin [24]. These methods [5, 24] are neither vali-
dated nor stability indicating.

H NH2
H H H
N S CH3

O N CH3
HO
O
H COOH
(a)

(CH3)3C C HN H H H
H S CH3
N

N CH3
O
HO O H COOH

(b)

Fig. 1. The molecular structures of (a) amoxicillin and (b) N-pivaloylamoxicillin

RP-HPLC Analysis of Related Substances in Amoxicillin 59

This paper reports the development of an LC method for separation of in-

dividual related substances of amoxicillin and a newly identified impurity
(Fig. 1) by an approach similar to that reported earlier [25]. The authors felt it
necessary to develop a stability-indicating method for separation and quantifi-
cation of related substances and known degradation products in amoxicillin
drug substance. Amoxicillin drug substance was exposed to forced degrada-
tion conditions (acid, base, elevated temperature, peroxide, light, and hu-
midity) in accordance with International Conference on Harmonization
(ICH) guidelines [26] and analysed to prove the specificity and stability-
indicating nature of the method. The method was validated in accordance
with ICH method-validation guidelines [27].
The novelty of the proposed method over previously reported meth-
ods [3–23] is its capacity to:

• separate substances (a–s) with resolution suitable for quantification;

• report the retention times of substances (b–s);
• identify without reference material; and
• quantify the individual substances (b–s) using response factors against
the substance (a).

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).

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, Hyderabad, India. Acetonitrile (LC grade) from Merck
(Germany) and potassium dihydrogen orthophosphate (AR grade) from
Rankem (Mumbai, India) were used. High-purity water was prepared by use
of a Millipore Milli Q plus water-purification system.
Standard (Std) and test solutions were prepared from amoxicillin
qualified standard (0.015 mg mL−1) and amoxicillin control sample
(1.5 mg mL−1) spiked with substances b–s (1.0%). Phosphate buffer (0.05 M;
pH 5.0 adjusted with dilute NaOH) was used as diluent.
60 Ch.B.V.N. Raju et al.

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. The LC sys-
tem 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. Molecular mass was de-
termined by use of a Perkin–Elmer API2000, PESCIEX triple-quadrupole mass
spectrometer with Analyst software. A Humidity-Photo stability chamber
(Thermo Lab, Mumbai, India) was used for light and humidity degradation
studies.

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 sub-
stance (75 mg). The conditions used were:

• temperature (the sample was stored at 105ºC for 3 h);

• acid (1 M aqueous HCl (1 mL) was added and the sample was
stored at room temperature for 30 min);
• base (0.5 M aqueous NaOH (1 mL) was added and the sample was
stored at 25°C for 2 min);
• oxidation (10% v/v H2O2 (1 mL) was added and the sample was
stored at 85°C for 2 min);
• light (the sample was illuminated at 12,000 lux for 144 h at 25°C);
and
• relative humidity (RH; the sample was stored at 92% RH and 25ºC
for 144 h).

The degraded samples were neutralized if necessary, and sample solution

(0.75 mg mL−1) was prepared and injected. 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.

Method Validation

The method was validated for precision, limits of detection (LOD) and
quantification (LOQ), linearity, accuracy, robustness, and solution stability.
RP-HPLC Analysis of Related Substances in Amoxicillin 61

Precision

The precision of the LC instrument (system precision), the method (method

precision), and the ruggedness (intermediate precision) are important as-
pects of method validation. System precision was evaluated by calculating
RSD (%) for the area counts obtained from six injections of standard solu-
tion to prove the consistency of the output signal produced by the LC sys-
tem. Method precision was evaluated by calculating RSD (%) for the results
obtained from six individual determinations using standard and test solu-
tions to prove the consistency of the results produced by the method. The
intermediate precision was evaluated using the same solutions with differ-
ent columns, instruments, and analysts.

LOD and LOQ

LOD and LOQ were obtained by study of linearity for five low concentra-
tions (0.05, 0.075, 0.1, 0.125, and 0.15 μg mL−1) of the individual sub-
stances. The RSD (%) of the area counts was calculated for six replicates at the
predicted LOQ level.

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 speci-
fication (2.0% for substances c and d and 1.0% for substances b and e–s.
The slope, y-intercept, residual sum of squares, and correlation coefficient
were calculated. The response factors were calculated by comparing the
slopes of the calibration plots for substances b–s with that for amoxicillin
(a). The regression equations were of the type y = mx + b (x is the concentration
and y is peak area). 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).

Accuracy

The control sample solution (1.5 mg mL−1) was spiked with substances b–
s at 50, 100, and 150% of specification level (1.0%). Recovery (%) was calcu-
lated using the results obtained.
62 Ch.B.V.N. Raju et al.

Robustness

The chromatographic conditions flow rate, acetonitrile (organic) content of

mobile phase, mobile phase pH, detector wavelength, and column oven
temperature were deliberately altered, one at a time, by ±10%, ±1%,
±0.1 unit, ±5 nm, and ±5º, respectively, and test solution was injected under
each condition. The effect of these changes on USP resolution, USP tailing,
and USP plate count, and on selectivity for the individual substances was
checked.

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. The
differences (%) between the area counts for each substance at hourly inter-
vals were calculated.

Results and Discussion

Optimization of Chromatographic Conditions

When the test solution was prepared and injected using the method [3] re-
ported elsewhere, compound j merged with i, k merged with n, and p and r
merged with q. To achieve better chromatographic separation 0.05 M potas-
sium dihydrogen orthophosphate buffer (pH 5.0) and acetonitrile were se-
lected as mobile phase components A and B, respectively. Trials were
conducted using C8 and C18 stationary phases from different manufac-
turers to achieve selectivity for substances a–s by changing pH, flow rate,
composition of mobile phase components A and B at different times (linear
gradient program), and column oven temperature. Good separation of a–s
was eventually achieved by use of a 150 mm × 4.6 mm, 5-μm particle,
Agilent Zorbax SB-C8, column with the linear gradient program given in
Table I at a flow rate of 2.0 mL min−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. The column oven temperature was maintained at 40°C
for good selectivity and sharp peaks. The USP resolution between sub-
stances k and l of not less than 1.5 was fixed as defining system suitability.
Substances b–r were potential impurities reported in the European
Pharmacopoeia [3]. Substance s is formed by acylation of the amino group
of the side chain with pivaloyl chloride, which is used in the manufactur-
ing process; it is also formed in thermal and alkaline degradation studies.
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, Switzerland) Avance DPX-300M spec-
trometer). Mass spectra, and NMR spectra with chemical shifts, are given
in Figs 2 and 3, respectively. Retention times (tR) and MS m/z values for
individual substances a–s are given in Table II. Chromatographic separation
of a–s is illustrated in Fig. 4.

Table I. Linear mobile phase gradient program

Time (min) Component A Component B

0.01 100.0 0.0
5.00 100.0 0.0
25.00 94.0 6.0
40.00 84.0 16.0
50.00 84.0 16.0
51.00 100.0 0.0
60.00 100.0 0.0

Component A was potassium dihydrogen orthophosphate buffer (pH 5.0) and

component B was acetonitrile

Fig. 2. Mass spectrum of N-pivaloylamoxicillin. A is the molecular ion peak, [M + H]+,

B is the ammonium adduct of the molecular ion peak ([M + H]+ + NH4),
and C is the sodium adduct ([M + H]+ + Na)
64 Ch.B.V.N. Raju et al.

Fig. 3. Proton NMR spectrum of N-pivaloylamoxicillin

Fig. 4. Chromatogram obtained from amoxicillin drug substance spiked with its related
substances
 RP-HPLC Analysis of Related Substances in Amoxicillin 65

Table II. Summary of mass values, chromatographic retention times, and method
validation data

LOD LOQ Re- Pu- Purity Residual

Sub- m/z tR y-
(% (% sponse rity thresh- Slope sum of
stance (M+) (min) intercept
w/w) w/w) factor angle old squares
a 365 4.800 – – 1.00 0.036 0.235 15441 2301 1407
b 167 0.853 0.005 0.008 0.54 0.326 1.748 28703 2630 2328
c 383 1.755 0.018 0.037 1.19 0.773 14.904 13005 18 1820
d 383 2.314 0.018 0.037 1.19 0.149 3.692 13005 18 1820
e 216 3.181 0.045 0.086 4.37 0.472 8.507 3531 51 308
f 365 3.837 0.011 0.021 0.97 0.197 4.176 15984 797 822
g 339 12.542 0.021 0.037 1.05 0.551 10.502 14702 1141 1166
h 514 15.468 0.015 0.032 0.95 0.189 4.384 16252 497 1596
I 339 16.902 0.021 0.037 1.05 0.481 8.825 14702 1141 1166
j 365 18.758 0.013 0.027 1.02 3.033 77.353 15173 846 1634
k 365 19.765 0.013 0.027 1.02 0.234 4.797 15173 846 1634
l 251 20.593 0.020 0.039 0.82 1.280 5.215 18779 1602 2275
m 188 21.012 0.015 0.023 0.89 0.960 3.895 17255 −800 2504
n 748 22.871 0.031 0.054 1.57 0.762 13.452 9848 756 822
o 748 22.681 0.031 0.054 1.57 1.336 20.158 9848 756 822
p 730 30.559 0.016 0.030 1.15 0.136 3.018 13438 86 1400
q 563 30.951 0.028 0.049 1.99 0.232 5.033 7753 −55 821
r 1095 34.865 0.018 0.037 1.56 0.256 4.502 9919 343 692
s 449 39.432 0.020 0.038 1.40 0.190 4.558 11063 241 853

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 ab-
sence of co-eluting peaks. Degradation products were obtained in tempera-
ture, oxidation, and acid and basic degradation studies only. The results
from the forced degradation studies are given in Table III. The peak-purity
angle was found to be less than the purity threshold for amoxicillin in all
the degradation studies, which confirms that no degradation product was
co-eluted with amoxicillin. This study proves the specificity and stability-
indicating nature of the method.
66 Ch.B.V.N. Raju et al.

Table III. Summary of results from forced degradation studies conducted using
0.75 mg mL−1 sample solution and analyzed using the proposed chromatographic
conditions

Not de-
Acid Basic Thermal Peroxide Photolytic Humidity
Sub- graded
degrada- degrada- degrada- degrada- degrada- degrada-
stance (%)
tion (%) tion (%) tion (%) tion (%) tion (%) tion (%)

a 99.33 89.57 89.86 77.99 88.47 99.26 99.25

b 0.21 0.24 0.27 0.20 0.25 0.23 0.24

c – 0.78 0.08 0.15 0.07 – –

d – 8.60 8.78 0.95 2.97 0.09 0.09

e – – – 0.83 0.05 – –

f – – – 0.23 7.33 – –

g – – – 1.02 – – –

h 0.23 0.18 0.22 0.55 0.20 0.21 0.23

i – – – 0.78 – – –

j – – – – – – –

k – 0.04 0.02 2.80 – 0.03 0.02

l – – – – – – –

m – – – – – – –

n – – – – – – –

o – – – – – – –

p 0.23 0.16 0.24 13.14 0.15 0.18 0.16

q – – – 0.30 – – –

r – – – – – – –

s – – 0.18 0.73 – – –
RP-HPLC Analysis of Related Substances in Amoxicillin 67

Method Validation

Precision

RSD for system precision, method precision, and intermediate precision

was less than 2.0, 1.5, and 2.0%, respectively, which confirm the high preci-
sion of the method.

LOD and LOQ

LOD and LOQ for each substance are given in Table II. RSD for six replicate
injections at the LOQ level was <10%.

Linearity

Correlation coefficients (r) for calibration plots for the individual substances
were >0.998. The slope, y-intercept, residual sum of squares, and response
factors are given in Table II.

Accuracy

Recovery of the individual substances at 50, 100, and 150% of specification

concentrations were between 90 and 110%, which proves the accuracy of
the method.

Robustness

When the chromatographic conditions were deliberately altered, system-

suitability results remained within acceptance limits and selectivity for in-
dividual substance was not affected. The results of the study, given in
Table IV, prove the robust nature of the method.
 68 Ch.B.V.N. Raju et al.

Table IV. Effect of deliberately altered chromatographic conditions on USP plate count
and USP tailing for substance a, and on USP resolution between substances k and l

Column
oven
Organic Detector Mobile USP USP USP
Altera- Flow tem-
strength wavelength phase plate tail- resolu-
tion (mL min−1) pera-
(%) (nm) pH count ing tion
ture
(ºC)

Not al-
2.0 ** 230 40 5.0 3471 0.8 1.8
tered

Flow 1.80 # # # # 3951 0.8 2.2

rate 2.20 # # # # 3740 0.8 1.5

Organic # +1 # # # 3635 0.8 1.5

strength # -1 # # # 3889 0.8 1.9

Detector # # 225 # # 3473 0.8 1.8

wave-
length # # 235 # # 3471 0.8 1.8

Column # # # 35 # 3675 0.8 1.5

oven
tem- # # # 45 # 3302 0.8 2.0
perature

Mobile # # # # 4.9 3700 0.8 1.9

phase
pH # # # # 5.1 3741 0.8 1.8

#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.

Conclusions
This method is rapid, precise, accurate, and selective. All the results from
method validation were satisfactory. This validated stability-indicating
method can be used for quality control and studies for assessment of the stabil-
ity of amoxicillin drug substance.
RP-HPLC Analysis of Related Substances in Amoxicillin 69

Acknowledgements
The authors wish to thank the management of APL group for supporting
this work. The authors wish to acknowledge the process research group for
providing the samples for this work. They also thank colleagues in APL Re-
search and Development Analytical Research Division for their co-
operation in carrying out this work.

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