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Pharmacom, C/41 vikash Nagar Maheshwari Dhuruhera Rewari Harayana, Rewari (2026)

Name: Pharmacom
Address: C/41 vikash Nagar Maheshwari Dhuruhera Rewari Harayana, Rewari, IN
Telephone: 9610506222

27/02/2017

PURPOSE :
Analytical Method Development
ANALYTICAL METHOD SELECTION:
Many new technologies are now available for bio pharmaceutical development. These analytical advances and their appropriate application are discussed in detail in the literature, Because these technologies are constantly improving—resulting in shorter testing times and increased throughput, ease of use, sensitivity, selectivity, and precision—at some point existing methods will be replaced with better ones. Automating a procedure, resulting in long-term savings and fewer operator errors, is one reason to follow this process. A more sensitive method may increase the likelihood of observing impurities at an upstream-process stage where corrective action is less expensive.
Qualified personnel should carefully select a new test methodology and its appropriate instrumentation. Changing biological assays. Accuracy is a prime consideration, because any bias in results must be reflected in the release specifications. When replacing existing technologies with automated or more sensitive ones, alert and action levels and associated specifications must be adjusted if needed. In-process and product specifications should reflect production process consistency and analytical capabilities, unless otherwise dictated by regulatory authorities.
.
Current GMP guidelines state that GMP documentation and the detail of validation activities should increase as the production process progresses. Testing upstream stages may actually be more critical than many final container release tests because it provides evidence of fermentation quality and the efficiency of impurity removal—although the tests are more uncertain and variable. Final container testing attests that active and inactive formulation components remain at predicted levels with little variability.
Science- and risk-based testing should carefully evaluate different product quality attributes that can impact overall product quality. Testing for in-process impurities should emphasize overall measurement sensitivity, selectivity, and precision. In other words, the analytical method should detect batch-to-batch variations; whether the measurements are extremely accurate (100% recovery) is not as important.
Some of the most advanced and innovative analytical technologies may be extremely informative for characterization of product, impurities, or the product matrix, but may not be appropriate for product release testing. When selecting an appropriate quality control (QC) method, the pros and cons should be carefully weighed against each other. Solid evidence that the new method will provide equivalent or better results is necessary when submitting a license change to regulatory authorities. The method's requirements should be similar to instrument requirements and based on the expected capability of the new method, as determined by a careful data review and identification of critical assay characteristics.
ANALYTICAL METHOD DEVELOPMENT:
It is the responsibility of the analytical method development (AMD) Sr Analytical chemist to include the test method's details in the standard operating procedure (SOP), including optimization of assay elements (such as mixing volumes, number of replicates, and statistical data reduction). If practical, all AMD data should ideally be generated in a GMP environment. In other words, we should generate all development data with qualified equipment by qualified personnel, and properly document and summarize the data in an AMD report approved by quality assurance (QA).
Often, methods are not developed from the ground up, but are optimized for a particular product and product matrix. In any case, always follow a thorough optimization process, which includes incorporating the best-fit data reduction function.

A well-planned and controlled experimental design that emphasizes QC release-testing suitability will prevent multiple, unsuccessful trial-and-error efforts. Scientific and regulatory concerns must be balanced with potential economical restrictions.

QA approval is required at many points in method development and validation . Ideally, the process does not continue until it has been approved. Data generated using a final, optimized method may be used to set acceptance criteria for the AMV protocol. All instruments and equipment should be qualified and all relevant software should be validated, ensuring that all AMD data and results (summarized later in the AMV protocol) are valid from a compliance perspective.

Results in and outside the product specifications must be reliable. If the boundaries are fuzzy, it is not possible to clearly differentiate between acceptable and unacceptable (out-of-specification) results, and material may be improperly accepted or rejected.

Accuracy can be estimated by measuring the recovery of various spiked levels of particular analytes. Many critical assays of product purification efficiency and product quality determine product purity and impurities simultaneously (for example, protein composition by capillary zone electrophoresis [CZE] or high performance size exclusion chromatography [HP-SEC]).

Whenever relative percentages of various analytes are estimated using a single assay, response factors must be established and integrated (normalized) in the calculations in order to accurately report purity and impurity levels. Using different detectors to measure analyte signals (for example, HP-SEC with ultraviolet detection to measure protein aggregation versus laser-light scattering or refractive-index detection) affects these relative percentages and should be thoroughly evaluated during AMD. A simple way to directly compare response factors from various detectors during AMD is to connect all detectors in-parallel (or inline).10
SYSTEM SUITABILITY:
The test system must be properly controlled to ensure reliable release-testing results. The system suitability criteria should be established during the AMD and optimization phase. This is usually accomplished by running a set of control points. For each test, system suitability will be satisfied (valid test results generated) if all control points are in established limits. A test system must be able to reproduce measurable results of a homogeneous sample (control) to allow examination of differences between batches. Small differences in batches are normal and acceptable, but the sources of variation should be identified. Ultimately, we will have more certainty when we can separate differences in production batches from assay variability.

SAMPLE SUITABILITY:
Technically, sample suitability is part of system suitability so these parameters can be evaluated together. Sample suitability should be established during AMD and should ideally ensure that samples, controls, and standards are prepared identically and run simultaneously. In addition, sample suitability should include a statistical analysis of the number of replicates needed to generate significant release results. Single measurements may be acceptable if the production-process sampling can deliver truly batch representative samples and the precision of assay repeatability is high compared to the product specifications—and therefore high compared to the batch-to-batch variation on which these specifications are based.
ROBUSTNESS:
Robustness, defined as the lack of a significant effect when small changes are deliberately introduced into the test system, should ideally be addressed during the method optimization phase and not as part of AMV. We should know the degree of robustness of a method before starting the formal AMV phase. Critical test system parameters (for example, the acceptable range of diluting the test sample) must be identified and controlled with appropriate operational limits. These limits should be described in the AMD report and documented in the method SOP. The SOP will then contain operational limits which are in the context of the overall system suitability criteria and which are adhered to during the validation phase. In addition, robustness should be tested in the AMD phase during or after method optimization because significant differences in the AMV results (from challenging the critical operational limits) may be difficult to explain in the AMV report.
We must remember that AMV is the formal evidence that this method is suitable to be used under strictly controlled QC testing conditions. The AMV protocol should be set up to deliver this evidence through appropriate acceptance criteria by varying sample batches and concentrations, operators, instruments, days, and other factors that are expected to vary during routine testing—in established sample and system suitability conditions and operational limits.

18/02/2017

Jay Prakash Tiwari
Mob :+91- 9610506222
E-mail : [email protected]
Development and Validation of RP-HPLC Method for
Simultaneous Estimation of Clonazepam and Propranolol
Hydrochloride in Bulk and Pharmaceutical Dosage Forms
Abstracts: A simple, precise, accurate, reproducible RP-HPLC method was developed and validated for the simultaneous estimation of Clonazepam and Propranolol hydrochloride in bulk and in pharmaceutical dosage forms. Chromatographic separation was carried out on a Thermo-hypersil C8 column (250mm×4.6mm i.d,5μm) utilizing a mobile phase consisting of acetonitrile and 0.01M ammonium phosphate buffer (pH adjusted to 3.0 with ortho phosphoric acid) in the ratio of 60:40 v/v at a flow rate of 1ml/min with UV detection at 238nm. The retention times of Clonazepam and Propranolol hydrochloride were
3.362 min. and 2.524 min. respectively. The developed method was validated in terms of accuracy, precision, linearity, specificity, limit of detection and limit of quantification. The linear range was found to be 0.5-3.0μg/ml and 20-120μg/ml for Clonazepam and Propranolol hydrochloride respectively. The proposed method can be used for estimation of these drugs in combined dosage forms.
INTRODUCTION
Clonazepam (CNZ) is an anticonvulsant used for several types of seizures, including myotonic or atonic seizures,
photosensitive epilepsy and absence seizures. Chemically, Clonazepam is 5-(2-chlorophenyl)-7-nitro-2,3-dihydro-1H-
1,4-benzodiazepin-2-one 1.
Propranolol hydrochloride (PRH) is a non selective beta- blocker mostly used in the treatment of hypertension and also used in the treatment or prevention of many disorders including acute myocardial infarction, arrhythmias, angina pectoris, hyperthyroidism, migraine and anxiety. Chemically, Propranolol hydrochloride is (RS)-1-(1-methylethylamino)-
3-(1-naphthyloxy) propan-2-ol hydrochloride 2.
Literature survey reveals that methods such as RP- HPLC 1-8, HPTLC 9-10, spectrophotometric methods 11-13 were reported for the determination of Propranolol hydrochloride and Clonazepam individually, in combination with other drugs and in human plasma.
single HPLC method 14 was reported for the simultaneous estimation of these two drugs in combined dosage form. The present investigation is to develop a simple, precise, accurate, reproducible and economical RP-HPLC method for simultaneous determination of Propranolol hydrochloride and Clonazepam in combined tablet dosage form. The validation was done in accordance with the ICH guidelines 15.
MATERIALS AND METHOD Instrumentation
Chromatographic separation was performed on HPLC (Make: Waters-alliance 2695 separation module equipped with UV detector controlled by Empower® software. Thermo-hypersil C8 column with 250 mm × 4.6 mm i.d, particle size 5μm was used for separation
Reagents and Chemicals
Analytical pure drugs of PRH and CNZ were obtained as gift samples from M/S. Simson Life Sciences Pvt Ltd., Hyderabad, India. The combined tablet formulation (Clotas Plus) with a labeled claim of CNZ 0.5mg and PRH 20mg respectively, were obtained from local drug store. Dibasic ammonium phosphate and ortho phosphoric acid of analytical grade and Acetonitrile of HPLC grade were procured from Merck
Chromatographic Conditions
The analysis was carried out on a HPLC system using a C8column (250mm×4.6mm i.d, Particle size 5μm) with a mobile phase consisting of acetonitrile and 0.01M ammonium phosphate buffer (pH adjusted to 3.0 with ortho phosphoric acid) in the ratio of 60:40 v/v and the eluents were monitored at 238nm. An injection volume of
20μl was used, keeping the flow rate at 1.0ml/min.
Mobile Phase
Acetonitrile and 0.01M ammonium phosphate buffer (adjusted to pH 3.0 with ortho phosphoric acid) in the ratio of 60:40 v/v was used for separation of these drugs after filtering through 0.45μm membrane filter and sonicated for
10 min.
Preparation of Mixed Standard Stock Solution
Accurately weighed 0.5 mg of CNZ and 20 mg of PRH working standards were transferred into a 25ml clean, dry volumetric flask and was dissolved and diluted with the mobile phase and then sonicated for 10 min, to obtain a final concentration of 20μg/ml of CNZ and 800μg/ml of PRH.
Calibration
The standard stock solution was further diluted to obtain solutions in the concentration range of 0.5-3μg/ml of CNZ and 20-120μg/ml of PRH. A 20μl sample was injected into the HPLC system. The flow rate was maintained at 1ml/min and the eluents were monitored at 238nm. The separation was done on C8 column using a mobile phase consisting of acetonitrile and buffer (60:40%v/v) and the chromatograms were recorded.
Analysis of Formulation
Twenty tablets were accurately weighed and average weight was calculated. A quantity of tablet powder
equivalent to 0.5 mg of CNZ and 20 mg of PRH was weighed and taken into 25 ml volumetric flask. The drugs were extracted into mobile phase and the volume was adjusted to 25 ml and sonicated for 15 min. The solution was filtered through 0.45μ membrane filter and from the filtrate, further dilutions were made using mobile phase to get a final concentration of 2μg/ml of CNZ and 80μg/ml of PRH and this solution was used for estimation. The amount of each active ingredient was calculated from the respective calibration curves and the results are presented in Table 2.
RESULTS AND DISCUSSION
Using the above chromatographic conditions, the method developed was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantification.
Linearity
The linearity of the method was determined at six concentration levels ranging from 0.5 to 3.0μg/ml for CNZ and 20 to 120μg/ml for PRH respectively. The plots of peak
area versus respective concentrations of PRH and CNZ were
found to be linear in the above concentration range with a correlation coefficient (r2) of 0.999 for CNZ and 1.0 for PRH.
Precision
The system precision was established by six replicate injections of the standard solution containing analytes of
interest. Method precision was carried out using six
different sample preparations from same homogenous blend of marketed sample. The %RSD values for the
responses were found to be 0.48 for CNZ and 0.3 for PRH
for system precision and 0.31 for CNZ and 0.29 for PRH for system precision.
Accuracy
The accuracy of the method was determined by recovery studies. The preanalysed samples were spiked with 50%,
100%, 150% of mixed standard solution and were analysed
by the proposed method. The study was carried out in triplicate. The mean percentage recovery values were
found to be 99.65% and 99.58% for CNZ and PRH
respectively as shown in the Table 1.
Specificity
Specificity was performed to exclude the possibilities of interference with excipients in the region of elution of CNZ and PRH. The specificity and selectivity of the method was tested under normal conditions and the results of the tests proved that the components other than the drug did not produce a detectable signal at the retention place of CNZ and PRH.
Limit of Detection (LOD) and Limit of Quantification
(LOQ)
The LOD and LOQ values were found to be 0.04501μg/ml and 0.1404μg/ml for CNZ and 0.7894μg/ml and
2.4637μg/ml for PRH. The LOD and LOQ showed that the
method is sensitive for CNZ and PRH.
Robustness
This was evaluated by small deliberate variation in the chromatographic conditions. The factors selected were
flow rate and the % organic content in the mobile phase.
The results obtained remain unaffected by small variations in these parameters. The %RSD values thus obtained showed that the method is robust.
System Suitability
The system suitability test was performed to check the various parameters such as column efficiency, resolution, peak tailing and retention time. The number of theoretical plates for CNZ and PRH were 5847 and 3836 respectively. All these parameters were evaluated with the background of regulatory requirements, which also suggest good chromatographic condition. The results were shown in Table 2.
CONCLUSION
The proposed method for the determination of CNZ and PRH in combined dosage form is accurate, precise, linear, robust, simple and rapid. Hence the present RP-HPLC method is suitable for ascertaining the quality control of the raw materials and formulations in combined dosage forms

30/07/2016

GENERAL SEPARATION PROCESS (HPLC)
M. S.Tswet defined the fractional adsorption process, with the explanation that
molecules of different analytes have different affinity (interactions) with the
adsorbent surface, and analytes with weaker interactions are less retained [3].
In modern high-performance liquid chromatography the separation of
the analytes is still based on the differences in the analyte affinity for the
8 INTRODUCTION
Figure 1-3. Components of performance as defined by C. Horvath. (Reprinted from
reference 12, with permission.)
Figure 1-4. Separation of fatty acids on pellicular graphitized carbon black from the
mixture of ethanol and 10−4M aqueous NaOH. Refractive index detection. (Reprinted
from reference 13, with permission.)
stationary phase surface, and the original definition of the separation process
given at its inception almost 100 years ago still holds true.
Liquid chromatography has come a long way with regard to the practical
development of HPLC instrumentation and the theoretical understanding of
different mechanisms involved in the analyte retention as well as the devel-
opment of adsorbents with different geometries and surface chemistry.

30/07/2016

HPLC HISTORY

HISTORY OF DISCOVERY AND EARLY DEVELOPMENT
(1903–1933)
Chromatography as a physicochemical method for separation of complex
mixtures was discovered at the very beginning of the twentieth century by
Russian–Italian botanist M. S. Tswet. [2]. In his paper “On the new form of
adsorption phenomena and its application in biochemical analysis” presented
on March 21, 1903 at the regular meeting of the biology section of the Warsaw
Society of Natural Sciences, Tswet gave a very detailed description of the
newly discovered phenomena of adsorption-based separation of complex mix-
tures, which he later called “chromatography” as a transliteration from Greek
“color writing” [3]. Serendipitously, the meaning of the Russian word “tswet”
actually means color. Although in all his publications Tswet mentioned that
the origin of the name for his new method was based on the colorful picture
of his first separation of plant pigments (Figure 1-2), he involuntarily incor-
porated his own name in the name of the method he invented.
The chromatographic method was not appreciated among the scientists at
the time of the discovery, as well as after almost 10 years when L. S. Palmer
[4] in the United States and C. Dhere in Europe independently published the
description of a similar separation processes. More information on history of
early discovery and development of chromatography could be found in refer-
ence 5.
Twenty-five years later in 1931, Lederer read the book of L. S. Palmer and
later found an original publications of M. S. Tswett, and in 1931 he (together
with Kuhn and Winterstein) published a paper [6] on purification of
xantophylls on CaCO3 adsorption column following the procedure described
by M. S. Tswet.
In 1941 A. J. P. Martin and R. L. M. Synge at Cambridge University, in UK
discovered partition chromatography [7] for which they were awarded the
Noble Prize in 1952. In the same year, Martin and Synge published a seminal
paper [8] which, together with the paper of A.T. James and A. J. P. Martin [9],
laid a solid foundation for the fast growth of chromatographic techniques that
soon followed.
6 INTRODUCTION
Figure 1-1. Classification of chromatographic modes.
Chromatography was discovered by Tswet in the form of liquid–solid chro-
matography (LSC), but its development continued for over 50 years primar-
ily in the form of gas chromatography and partially as thin-layer and
liquid–liquid chromatography. Rebirth of liquid chromatography in its modern
form and its enormously fast growth had driven this to be the dominant ana-
lytical technique in the twenty-first century which can be attributed in the most
part to the pioneering work of Prof. C. Horvath at Yale University. In the mid-
1960s Prof. Horvath, who previously worked on the development of a porous
layer open-tubular columns for gas chromatography, had decided to use for
liquid chromatography small glass beads with porous layer on their surface to
facilitate the mass transfer between the liquid phase and the surface. Columns
packed with those beads developed a significant resistance to the liquid flow,
and Prof. Horvath was forced to build an instrument that allowed develop-
ment of a continuous flow of the liquid through the column [11]. This was the
origin of high-performance liquid chromatography (HPLC), and the actual
name for this separation method was introduced by Prof. Horvath in 1970 at
the Twenty-first Pittsburgh Conference in Cleveland, where he gave this tit

08/06/2016

We all remember the feeling we had in school as we
learned math, wondering how it would actually come into
practical use. Scientists have to learn more math than
many professionals, and this section reminds us why.
Here, we will briefly review the equations and theory behind many of the concepts that drive chromatography.
Understanding these concepts will help you to get the best results, and to troubleshoot if you encounter
problems.
We start with fundamentals of performance:
• Efficiency
• Retention
• Selectivity
• Resolution
• Pressure
These are all key to understanding how to optimize results and successfully develop methods.
We also explore a few more complex concepts:
• van Deemter curves
• The gradient equation
These two topics are also important for method development.

03/12/2015

How Does High Performance Liquid Chromatography Work?

The components of a basic high-performance liquid chromatography [HPLC] system are shown in the simple diagram in Figure E.

A reservoir holds the solvent [called the mobile phase, because it moves]. A high-pressure pump [solvent delivery system or solvent manager] is used to generate and meter a specified flow rate of mobile phase, typically milliliters per minute. An injector [sample manager or autosampler] is able to introduce [inject] the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column. The column contains the chromatographic packing material needed to effect the separation. This packing material is called the stationary phase because it is held in place by the column hardware. A detector is needed to see the separated compound bands as they elute from the HPLC column [most compounds have no color, so we cannot see them with our eyes]. The mobile phase exits the detector and can be sent to waste, or collected, as desired. When the mobile phase contains a separated compound band, HPLC provides the ability to collect this fraction of the eluate containing that purified compound for further study. This is called preparative chromatography [discussed in the section on HPLC Scale].

Note that high-pressure tubing and fittings are used to interconnect the pump, injector, column, and detector components to form the conduit for the mobile phase, sample, and separated compound bands.

hplc system

Figure E: High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that records the electrical signal needed to generate the chromatogram on its display and to identify and quantitate the concentration of the sample constituents (see Figure F). Since sample compound characteristics can be very different, several types of detectors have been developed. For example, if a compound can absorb ultraviolet light, a UV-absorbance detector is used. If the compound fluoresces, a fluorescence detector is used. If the compound does not have either of these characteristics, a more universal type of detector is used, such as an evaporative-light-scattering detector [ELSD]. The most powerful approach is the use multiple detectors in series. For example, a UV and/or ELSD detector may be used in combination with a mass spectrometer [MS] to analyze the results of the chromatographic separation. This provides, from a single injection, more comprehensive information about an analyte. The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.

HPLC Waters Alliance

Figure F: A Typical HPLC [Waters Alliance] System

HPLC Operation
A simple way to understand how we achieve the separation of the compounds contained in a sample is to view the diagram in Figure G.

Mobile phase enters the column from the left, passes through the particle bed, and exits at the right. Flow direction is represented by green arrows. First, consider the top image; it represents the column at time zero [the moment of injection], when the sample enters the column and begins to form a band. The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band. [In reality, this sample could be anything that can be dissolved in a solvent; typically the compounds would be colorless and the column wall opaque, so we would need a detector to see the separated compounds as they elute.]

After a few minutes [lower image], during which mobile phase flows continuously and steadily past the packing material particles, we can see that the individual dyes have moved in separate bands at different speeds. This is because there is a competition between the mobile phase and the stationary phase for attracting each of the dyes or analytes. Notice that the yellow dye band moves the fastest and is about to exit the column. The yellow dye likes [is attracted to] the mobile phase more than the other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase. The blue dye band likes the packing material more than the mobile phase. Its stronger attraction to the particles causes it to move significantly slower. In other words, it is the most retained compound in this sample mixture. The red dye band has an intermediate attraction for the mobile phase and therefore moves at an intermediate speed through the column. Since each dye band moves at different speed, we are able to separate it chromatographically.

chromatographic column bands

Figure G: Understanding How a Chromatographic Column Works – Bands

What Is a Detector?
As the separated dye bands leave the column, they pass immediately into the detector. The detector contains a flow cell that sees [detects] each separated compound band against a background of mobile phase [see Figure H]. [In reality, solutions of many compounds at typical HPLC analytical concentrations are colorless.] An appropriate detector has the ability to sense the presence of a compound and send its corresponding electrical signal to a computer data station. A choice is made among many different types of detectors, depending upon the characteristics and concentrations of the compounds that need to be separated and analyzed, as discussed earlier.

What Is a Chromatogram?
A chromatogram is a representation of the separation that has chemically [chromatographically] occurred in the HPLC system. A series of peaks rising from a baseline is drawn on a time axis. Each peak represents the detector response for a different compound. The chromatogram is plotted by the computer data station [see Figure H].

chromatogram separation representation

Figure H: How Peaks Are Created

In Figure H, the yellow band has completely passed through the detector flow cell; the electrical signal generated has been sent to the computer data station. The resulting chromatogram has begun to appear on screen. Note that the chromatogram begins when the sample was first injected and starts as a straight line set near the bottom of the screen. This is called the baseline; it represents pure mobile phase passing through the flow cell over time. As the yellow analyte band passes through the flow cell, a stronger signal is sent to the computer. The line curves, first upward, and then downward, in proportion to the concentration of the yellow dye in the sample band. This creates a peak in the chromatogram. After the yellow band passes completely out of the detector cell, the signal level returns to the baseline; the flow cell now has, once again, only pure mobile phase in it. Since the yellow band moves fastest, eluting first from the column, it is the first peak drawn.

A little while later, the red band reaches the flow cell. The signal rises up from the baseline as the red band first enters the cell, and the peak representing the red band begins to be drawn. In this diagram, the red band has not fully passed through the flow cell. The diagram shows what the red band and red peak would look like if we stopped the process at this moment. Since most of the red band has passed through the cell, most of the peak has been drawn, as shown by the solid line. If we could restart, the red band would completely pass through the flow cell and the red peak would be completed [dotted line]. The blue band, the most strongly retained, travels at the slowest rate and elutes after the red band. The dotted line shows you how the completed chromatogram would appear if we had let the run continue to its conclusion. It is interesting to note that the width of the blue peak will be the broadest because the width of the blue analyte band, while narrowest on the column, becomes the widest as it elutes from the column. This is because it moves more slowly through the chromatographic packing material bed and requires more time [and mobile phase volume] to be eluted completely. Since mobile phase is continuously flowing at a fixed rate, this means that the blue band widens and is more dilute. Since the detector responds in proportion to the concentration of the band, the blue peak is lower in height, but larger in width.

Pharmacom

27/02/2017

18/02/2017

30/07/2016

30/07/2016

08/06/2016

03/12/2015

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