Stability-indicating RP-HPLC method for assay of silver lactate

Article abstract of DOI:10.1155/2011/234640

A simple, economic and time-efficient stability-indicating, reversephase high-performance liquid chromatographic (RP-HPLC) method has been developed for analysis of silver lactate in the presence of degradation products generated by decomposition. When si

Full text of DOI:10.1155/2011/234640

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2011, 8(2), 483-490
http://www.e-journals.net
Stability-Indicating RP-HPLC
Method for Assay of Silver Lactate
V. SRINIVASAN§*, H. SIVARAMAKRISHNAN and B. KARTHIKEYAN§
Department of Analytical Development
Piramal Healthcare Ltd, Ennore, Chennai-600 057, India
§Department of Chemistry
Annamalai University, Annamalainagar-608002, India
vsvnreddy@yahoo.com
Received 27 March 2010; Revised 31 May 2010; Accepted 17 July 2010
Abstract: A simple, economic and time-efficient stability-indicating, reverse-
phase high-performance liquid chromatographic (RP-HPLC) method has been
developed for analysis of silver lactate in the presence of degradation products
generated by decomposition. When silver lactate was subjected to acid
hydrolysis, base hydrolysis, oxidative, photolytic, humidity and thermal stress,
degradation was observed during base hydrolysis, oxidation, humidity and
thermal stress. The drug was found to be stable to other stress conditions.
Successful chromatographic condition of the drug from the degradation
products formed under stress conditions was achieved on a Phenomenex Gemini
column with potassium dihydrogen phosphate buffer, pH adjusted to 2.2 with
orthophosphoric acid, as mobile phase. The method was validated for linearity,
precision, specificity and robustness and can be used for quality-control during
manufacture and assessment of the stability of samples of silver lactate. To the
best of our knowledge, a validated stability-indicating LC assay method for
silver lactate based on lactic acid is reported for the first time.
Keywords: RP-HPLC, Silver lactate, Lactic acid, Degradation products
Introduction
Silver lactate chemically 2-hydroxypropanoic acid silver salt1 has the molecular formula
C3H5AgO3 and molecular weight 196.94. Silver lactate is a drug which is used as
antimicrobial agent in the external application2. Its application in the treatment of thermal
burn has been reported. Silver lactate is an ideal base for bactericidal agents in the treatment
of full thickness thermal wound3-4. With the experience accumulated from more than a
century of silver applications in biology and medicine, physical development has become a
powerful bio-analytical tool for marker amplification in blotting procedures, in situ hybridization

484
V.SRINIVASAN et al.
and histochemistry. To accommodate the modern demand for accurate physical development,
various strategies have been devised; one among them is changing to silver lactate5. Silver
lactate also finds application as stainer in the study of fungal by electron microscope6.
The different analytical techniques reported so far for analysis of lactic acid in
biological samples and in pharmaceutical formulations includes electrophoresis7, NMR8,
NIR9. Several HPLC methods for assay and LC-MS-MS method for analysis of lactic acid
have previously been published10-13
.
This paper describes a simple isocratic reverse-phase HPLC method that separates
silver, lactic acid and degradation products. Assay of silver lactate is calculated based on the
lactic acid peak. To the best of our knowledge no such report on the validation of assay of
silver lactate has been taken, hence we have taken this study. The structures of silver lactate
and chromatographic separation of the compounds are illustrated in Figure 1.
OH
+
Ag
-
COO
.
Mol. Wt.: 196.94
Figure 1. Chemical structure of silver lactate
The process for the synthesis of silver lactate has recently been developed in our
laboratory; an RP-HPLC method was developed for analysis of silver lactate. The
specificity, linearity, precision and robustness of the method were determined in accordance
with ICH guidelines14. This paper reports, for the first time, a rapid, efficient, simple and
validated stability-indicating RP-HPLC method for assay of silver lactate.
Experimental
Sample of silver lactate was synthesized in our laboratory and characterized by use of IR
and NMR. All the reagents used were of analytical reagent grade unless stated otherwise.
Orthophosphoric acid spectroscopic grade was purchased from Merck. Lactic acid from
Lancaster synthesis grade having 98+% is used as reference standard.
Instrumentation and chromatographic conditions
HPLC was performed with quaternary gradient pumps, an auto sampler and an auto injector
(model 1200;Agilent, USA) connected to a photodiode-array detector, all were controlled by
Empower software (Waters). Chromatographic separation was achieved on a Phenomenex
Gemini C18, 250x4.6 mm, 5 µm particle, with potassium dihydrogen phosphate, pH
adjusted to 2.2 with orthophosphoric acid, as mobile phase. Before use, the mobile phase
was filtered through a 0.2 µm PTFE filter and degassed by ultrasonication for 15 min. The
injection volume was 10 µL, the mobile phase flow rate was 1.0 mL min-1, the column
temperature was 30 °C and detection was at 210 nm.
Preparation of working solutions
The standard and test concentration for the assay were 7 mg mL-1 and 15 mg mL-1
respectively. Standard and test dilutions were prepared in water.
Forced degradation studies (stress testing)
Silver lactate was deliberately subjected to stress to establish the stability-indicating nature
and specificity of the method. The drug was exposed to UV light (254 nm), heat (80 °C),

Stability-Indicating RP-HPLC Method for Assay of Silver Lactate
485
acid (5.0 N HCl, 60 °C), alkali (1.0 N NaOH, 25 °C), oxidation (30.0% H2O2 for 12 H)
and Humidity (95% RH, 25 °C) to evaluate the ability of the method to separate silver
lactate from its degradation products. For heat and light stress the study period was 72 h;
for acidic 30 min, alkaline 20 min and oxidative stress it was 12 h. Peak purity testing
was performed for the lactic acid peak obtained from stress samples by use of the PDA
detector. Assay studies of stressed samples were performed by comparison with lactic
acid reference standard.
Precision
The precision of the method was assessed by performing six independent assays of silver
lactate test sample (15 mg mL-1) in comparison with lactic acid reference standard (7 mg mL-1)
and calculating the RSD (%) of the assay. The intermediate precision was assessed for the
method by performing assay by a different analyst using a different instrument with different
lot number HPLC column in the same laboratory.
Linearity
Solutions for testing the linearity of the assay method were prepared for silver lactate
sample and lactic acid reference standard at five concentrations from 80 to 120% of the
nominal assay concentration of the sample (12, 13.5, 15, 16.5 and 18 mg mL-1) and lactic
acid standard (5.6, 6.3, 7.0,7.7 and 8.4 mg mL-1) respectively. Peak area and concentration
data were treated by least squares linear regression analysis. These tests were conducted and
correlation coefficient was calculated between concentration verses area. Linearity
calculations were performed with Microsoft Excel.
Robustness
To assess the robustness of the method, experimental conditions were deliberately altered
and assay was recorded. The effect of flow rate was studied by varying the flow rate by
0.1 mL min-1. The effect of column temperature was studied by varying the temperature by
5.0 °C. The effect of pH was studied by varying the pH by 0.2 units. The effect of
wavelength was studied by varying the wavelength by 5.0 nm. In all tests the other
conditions were held constant.
Solution stability
The solution stability of silver lactate in the assay method was carried out by leaving test
solutions of sample and reference standard in water in tightly capped volumetric flasks at
room temperature for 24 h. Area of the lactic acid in sample solution and in the standard
solution were determined every 4 h. Cumulative RSD was calculated.
Results and Discussion
Optimization of chromatographic conditions
The main objective of the chromatographic method was to determine the assay of silver lactate.
Chromatographic separation was achieved on a phenomenex Gemini C18, 250x4.6 mm, 5 µm
particle, with potassium dihydrogen phosphate, pH adjusted to 2.2 with orthophosphoric acid, as
mobile phase. The injection volume was 10 µL, the mobile phase flow rate was 1.0 mL min-1, the
column temperature was 30 °C and detection was at 210 nm. Peak shape of lactic acid was
symmetrical. Under the optimized conditions typical retention times of lactic acid was
approximately 3.0 minutes (Figure 2). The method was specific for silver lactate.

486
V.SRINIVASAN et al.
Time min
Figure 2. Chromatogram showing resolution of silver ion and lactic acid
Analysis of stressed samples
Degradation was not observed when silver lactate exposed to acid hydrolysis. Silver
lactate was degraded when exposed to alkali, peroxide, thermal, humidity and UV
(Figure 3a-d). Results from peak purity testing confirmed the lactic acid peak obtained
by analysis of all the stress samples was homogenous and pure. Assay of silver lactate
was unaffected by the presence of its degradation products, confirming the stability-
indicating nature of the method. The results from forced degradation studies are
summarized in Table 1.
(a) Peroxide degradation
Minutes
(b) Peroxide degradation peak purity
Minutes

Stability-Indicating RP-HPLC Method for Assay of Silver Lactate
487
(c) Thermal degradation
Minutes
(d) Thermal degradation peak purity
Minutes
Figure 3. Chromatograms (a) and (c) for samples obtained from peroxide and humidity
stress testing, (b) and (d) peak purity of peroxide and humidity stress testing
Table 1. Summary of results from forced degradation
Assay, %w/w on as is
basis
Sample without
treatment
(as such)
Peak purity
Purity angle Purity Threshold
99.0%
0.770
2.280
Stress Conditions
Peak Purity
Samples
Condition
Purity Purity
angle Threshold
2 mL 5 N HCl/ 60 °C
/30 min
Acid Degradation
97.1
89.2
81.8
2
0.223
0.168
0.237
0.306
1.871
0.294
1 mL 1N NaOH/
20 min
Alkali Degradation
Peroxide Degradation
10
17
2 mL 30%w/w
H2O2/12 H
60 °C/72 H
Thermal Degradation
Humidity Degradation
UV light Solid (Shorter
wave length)
89.6
93.4
9
6
0.776
0.801
2.328
3.140
25 °C /95% RH/ 72 H
72 H
72 H
90.5
98.4
9
1
0.760
0.744
2.496
2.061
UV light Solution
(Shorter wave length)
White light Solid
72 H
72 H
92.7
98.2
6
1
0.668
1.024
1.884
3.028
White light Solution

488
V.SRINIVASAN et al.
Precision
In the study of the precision of the assay method RSD of the peak area for assay of silver
lactate was 0.20% or better. RSD of peak area in the intermediate precision study was 0.26%
or better confirming the high precision of the method. Results are summarized in Table 2.
Table 2. Summary of precision results
S.No.
1
Area of lactic acid peak
3179030
3177782
3177938
3175906
3173636
3173746
3176340
2285
2
3
4
5
6
Mean
SD
% RSD
0.07
Linearity
In the assay method a linear relationship was obtained between response and concentration
over the calibration range tested, ie. 12 - 18 mg mL-1 for silver lactate sample and 5.6-8.5 mg mL-1
for lactic acid standard. The correlation coefficient was >0.999 for both sample and standard.
Results are summarized in Table 3.
Table 3. Linearity of lactic acid peak in silver lactate sample
Concentration of Silver
lactate sample, mg/mL
Mean area of silver lactate
peak
2372220
Sample Name
Linearity, 80%
Linearity, 90%
Linearity, 100%
Linearity, 110%
Linearity, 120%
Slope
12.02
13.52
15.02
16.53
18.03
2679345
3026444
3324259
3633338
210719
-158716
0.9997
Intercept
Correlation coefficient
Robustness
When the chromatographic conditions flow rate, column temperature, mobile phase pH and
wavelength were deliberately varied, cumulative RSD was 0.28% for the sample. The
asymmetry of the lactic acid peak was always found less than 2.0. This illustrates that the
method is robust. Results are summarized in Table 4.
Table 4. Summary of results from robustness studies
Parameter
Set - 1
Set - 2
Set - 3
Set - 4
Set - 5
Condition
Method precision
Flow plus
Assay, %w/w. % sfab*
100.9
101.6
101.7
101.3
101.4
Flow minus
Temp.plus
Temp.minus
Contd…

Stability-Indicating RP-HPLC Method for Assay of Silver Lactate
489
Set - 6
Set - 7
Set - 8
Set - 9
pH plus
pH minus
Wavelength plus
Wavelength minus
Mean
101.7
101.8
101.3
101.3
101.4
0.28
SD
% RSD
*solvent free as is basis
0.28
Where,
Set-1 = As per method
Set-2 = Variation in flow by +10%
Set-3 = Variation in flow by - 10%
Set-4 = Variation in column oven temperature by + 5 °C
Set-5 = Variation in column oven temperature by – 5 °C
Set-6 = Variation in pH of the mobile phase as +0.2.
Set-7 = Variation in pH of the mobile phase as –0.2.
Set-8 = Variation in detection wave length by + 5 nm
Set-9 = Variation in detection wave length by - 5 nm
Solution stability
RSD for assay of lactic acid reference and silver lactate sample during stability experiments
was 0.25% and 0.33% respectively. The results from solution stability conformed that
sample solution of lactic acid and silver lactate in water for assay analysis was stable for at
least 24 h. Results are summarized in Table 5
Table 5. Sample Solution stability
Time, H
Initial
4 H
Area of Lactic acid
3044537
3042888
Cumulative, %RSD
-
0.04
0.04
0.20
0.19
0.25
0.33
8 H
3041973
3055221
12 H
16 H
20 H
24 H
3050461
3060962
3068735
System suitability
Tailing factor less than 2.0, similarity factor between 0.98 and 1.02 and RSD for lactic acid
peak from the replicate injections less than 2.0% for all the system suitability study. Results
are summarized in Table 6
Table 6. System suitability
Experiment
Repeatability, Linearity
(Silver lactate)
Tailing factor
1.6
Similarity factor %RSD
1.00
1.00
0.99
0.07
0.03
0.06
Robustness
1.5
1.5
(Flow,pH & Temp minus)
Robustness (Temp plus) and Solution
stability
Intermediate Precision
Degradation, Linearity (Lactic acid)
1.3
1.5
1.00
1.01
0.29
0.09

490
V.SRINIVASAN et al.
Conclusion
The HPLC method developed for quantitative assay of silver lactate is precise, rapid and
specific. Full validation of the method furnished satisfactory results for all the method
validation data tested. The method was stability-indicating and can be conveniently used by
quality control departments for analysis of assay of silver lactate. Moreover, the lower
solvent consumption along with the short analytical run time of 10.0 min leads to cost
effective chromatographic method
Acknowledgment
The authors would like to thank the management of Research and Development, Piramal
Healthcare Ltd. for supporting this work. Cooperation from colleagues Analytical Research
and Department is appreciated.
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