Report on

Two Days Hands on Training (Workshop)

On

“Analytical Method Development on High Performance Liquid

Chromatography (HPLC)”

09-10 October 2017

Organized by

RUSA Centre for Herbo Medicinal Studies

Swami Ramanand Teerth Marathwada University,
Nanded (Maharashtra)

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About Hands on Training (Workshop):
High performance liquid chromatography (HPLC) is the fasted growing analytical
technique for analysis of drugs. Its simplicity, high specificity and wide range of sensitivity
make it ideal for analysis of many drugs in both dosage form and biological fluids. The rapid
growth of HPLC has facilitated by development of reliable, moderately precise
instrumentation and efficient columns.
Analytic method development, validation, and transfer are key elements of any
pharmaceutical development program. Methoddevelopment is a continuous process that
progresses in parallel with the evolution of the drug product. HPLC is a potent analytical tool,
allowing for the separation, identification and quantification of drug substances. It also
presents many challenges, from the correct use of instrumentation, to analysing results, to the
fulfillment of the regulatory requirements of method validation.

Key feature of Training:Over two days, this hands on training will offer participants the
opportunity to learn the important aspects of HPLC theory and method development,
including troubleshooting and participants will examine quantitation and method validation.

Eligibility:All graduate, Post graduates students, Research Scholars, Faculty and Industry
Persons who are interested and willing to participate .

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Inauguration:

Two days’ workshop was inaugurated by Hon’ble Vice Chancellor Prof. Pandit Vidyasagar
sir. Dr. R. M. Mulani, Registrar, Dr. G. B. Katalakute, Finance and Accounts Officer,
Convener Dr. S. J. Wadher, Dr. C. N. Khobragade and Dr. R. S. Khairnar were present for
the inauguration.

Inauguration atHon’ble Vice Chancellor Prof. Pandit Vidyasagar sir

Twenty eight (28) students registered for workshop hands on training. Dr. Santosh Chhajad,
Associate Professor, Mumbai Education Trust College of Pharmacy, Nashik and Mr. Ram
Sakhare, Assistant Professor, School of Pharmacy, SRTM University delivered talk on Basic
of HPLC system and application of HPLC in Pharma field.

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Lecture by Dr. Santosh Chhajad Lecture by Mr. Ram Sakhre

In the training workshop on HPLC following work has been

assigned to each students
RP-HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS
ESTIMATION OF AMOXICILLIN TRIHYDRATE AND METRONIDAZOLE IN
BULK AND IN-HOUSE TABLET
Materials and method
Pharmaceutically pure sample of amoxicillin trihydrate and metronidazole was obtained from
Aarati drugs ltd. Tarapur as gift samples. All other chemicals (water, acetonitrile, methanol)
used in the analysis were HPLC grade.
Instrumentation
Chromatographic separation was performed on HPLC system (model Shimadzu SCL – 10),
C18 column (250mm × 4.6mm; 5 µm), UV Detector, equipped with a solvent delivery pump,
sample injector and column thermostats. Lab solution (version 1.25) software was applied for
data collecting and processing. Mobile phase: water: acetonitrile: methanol in the ratio of
70:20:10 (v/v/v).
Selection of analytical wavelength
Accurately weighed 10 mg AMT and MET were transferred into 100 ml volumetric flasks
separately and dissolved in mobile phase. This solution was sonicated for 20 minute and
volume was made up to mark with mobile phase. Pipetted out 2 ml of stock solution and

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diluted to 10 ml to get a concentration of 20 µg/ml of AMT and MET. Both the solutions
were scanned in the spectrum mode over the range of 200-400 nm. The overlain spectra
showed iso-absorptive point at 237.3 nm and this wavelength was selected for further
detection.
Preparation of standard solution
Accurately weighed 100 mg of AMT and 80 mg of MET working standard was transferred
into 100 ml volumetric flask and dissolved in mobile phase. This solution was sonicated for
20 minute, then volume was made up to mark with mobile phase to get concentration of 1000
µg/ml for AMT and 800 µg/ml for MET. From the standard stock solution, mixed standard
solution was prepared to contain 20 µg/ml of AMT and 16 µg/ml of MET.
Optimized chromatographic conditions
Mobile phase : Water: ACN: Methanol (70:20:10 v/v/v)
Column : C18 column
Detector wavelength : 237.3 nm
Injection volume : 20 µl
Flow rate : 1.4 ml /min
Run time : 15 minutes
System Suitability test
System suitability testing is used to verify that the resolution and reproducibility of the
system are adequate for the analysis to be performed, mixed standard solution of AMT and
MET was injected into HPLC system and their system suitability parameters were recorded.
Validation of the method
Method was validated according to ICH guidelines for linearity, accuracy, precision, LOD,
LOQ, robustness and specificity.
Linearity
Linearity was studied by preparing standard solution at different concentration levels.
Standard stock solution of AMT and MET was further diluted to get concentration in the
range of 20-100 µg/ml for AMT and 16-80 µg/ml for MET. Each dilution of both the drugs
was injected into the HPLC system and peak areas were determined. Standard calibration
curves were constructed by plotting peak areas versus concentrations of drug and the
regression equations were calculated Accuracy:
The accuracy of the proposed method was assessed by recovery studies at three different
levels i.e. 80%, 100%, 120%. The recovery studies were carried out by adding a known
amount of standard drug to pre-analyzed sample. The resulting solutions were then
reanalyzed by proposed method. Whole analysis procedure was repeated to find out the
recovery of the added drug sample. This recovery analysis was repeated at three replicate of
three concentrations levels.
Precision:

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Precision of an analytical method is usually expressed as the standard deviation or relative
standard deviation (coefficient of variation). The precision was determined at different
parameter like repeatability, intermediate precision (intra-day, inter-day). Repeatability was
determined by analyzing AMT (100 µg/ml) and MET (80 µg/ml) three times. Intraday
precision was determined by analyzing same concentration of solution for three times within
the day and Interday precision was determined daily for three days. Then % RSD was
calculated and it was within limit (less than 2 %). Robustness:
The robustness of the method was determined as a measure of the analytical method
capability to be unaffected by small variations in method parameters and the different
variants such as variation in flow rate by ± 0.2 ml/minute; variation in wavelength by ± 2 nm
were selected. At these changed conditions, the standard and sample solutions were injected.
The system suitability was evaluated in each varied condition.
Limit of detection (LOD) and Limit of quantification (LOQ):
LOD is the lowest concentration of analyte in sample that can be detected but not necessarily
quantified. LOQ is the lowest concentration of analyte in sample that can be quantitatively
determined with precision and accuracy. LOD and LOQ was calculated from linear curve
using following formulae
LOD = 3.3 σ/slope, LOQ = 10 σ/slope
(Where σ = the standard deviation of the response and S = Slope of calibration curve).
Specificity:
Specificity was checked for the interference of impurities in the analysis of blank solution
and injecting sample solution under optimized chromatographic conditions to demonstrate
separation of both amoxicillin trihydrate and metronidazole from impurities.
Analysis of in-house tablet
Due to unavailability of tablet containing amoxicillin trihydrate and metronidazole in local
Indian market, in-house tablets were prepared via direct compression technique using
commonly used excipients containing 250 mg of amoxicillin trihydrate and 200 mg of
metronidazole.
Twenty tablets were taken and their average weight was determined, crushed to fine powder;
powder equal to 50 mg of AMT and 40 mg MET was taken in 100 ml volumetric flask. Then
80 ml of mobile phase was added and the flask was sonicated for about 20 minute to
solubilize the drug present in tablet, after that solution was filtered through Whatman filter
paper No. 41. The filtrate was collected and the volume was made up to the mark with mobile
phase. Further dilution was done in 10 ml volumetric flask to get final concentration of 20
µg/ml of AMT and 16 µg/ml of MET. The standard and sample solution was injected into
HPLC system and peak areas were measured. The content of AMT and MET was calculated
by using following formula.
Amount of drug (mg) = At/As x Ds/Dt x Ws/Wt x A ……... (i)
% Estimation = At/As x Ws/Wt x Avg. wt (A)/Label claim x 100 ……… (ii)
Where,
At = Area count for sample solution

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As = Area count for standard solution
Ds = Dilution factor for standard
Dt = Dilution factor for sample
Ws = Weight of standard (mg)
Wt = Weight of sample (mg)
A = Average weight of mixture

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE

ESTIMATION OF SIMVASTATIN IN BULK AND TABLET DOSAGE FORM

MATERIALS & METHODS

Pure simvastatin was procured as a gift sample from IPCA laboratory, Mumbai India. The
marketed pharmaceutical tablet dosage form of simvastatin SIMVAS 10 by Micro Labs,
India was purchased from local market, and used within its shelf life period. Acetonitrile,
methanol and water was used in the method development of HPLC-grade. 0.1 N HCl, 0.1 N
(NaOH), 30% H2O2 prepared in lab. Buffer materials and all other chemicals were of AR
grade was purchased from Merck Specialties Private Limited.
The chromatographic system model SHIMADZU SCL-10 VP equipped with UV-Visible
detector controlled by LC solution (version 1.25) software and column enable C18
(250×4.5mm) was used as a stationary phase.

Chromatographic conditions
The chromatographic conditions were performed using following:
Mobile phase: Acetonitrile: phosphate buffer (pH4.5) (25:75 v/v)
Run time: 20 min
Wavelength of scanning: 238 nm
Diluent: Mixture of 400 ml phosphate buffer (pH 4.5) and 600 ml acetonitrile in the ratio of
40:60% v/v was taken.

Preparation of standard solution

A standard solution was prepared by transferring 50 mg of simvastatin in 100 ml of
volumetric flask. To it diluent was added and sonicated for 20 min to dissolve completely,
and then volume was made up to the mark with diluent to get concentration 1000 µg/ml. It
was filtered through Whatman filter paper No. 41. It was further diluted to get concentration
of 10 µg/ml.

Selection of mobile phase and chromatographic conditions

Trials were carried out using different solvent combinations including methanol, water,
acetonitrile in various proportions. Acetonitrile : phosphate buffer 25:75 v/v (pH: 4.5 adjusted
with O-phosphoric acid) was selected as optimum mobile phase with in a run time of 20 min
and filtered by a millipore vacuum filter system equipped with 0.45 µm high vacuum filter,
which gave resolution and sharp peak for the drug. Other chromatographic conditions like run
length, sample injection volume, flow rate, detection wavelength, was optimized to give Rt
and symmetrical peak shape for the drug.

Selection of detection wavelength

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The standard solution was scanned over the range of 200-400 nmand the spectrum was
obtained.

Analysis of marketed formulation

Twenty tablets (SIMVAS 10 labelled to contain 10mg of simvastatin)were weighed
accurately, average weight was determined and finely powdered. A quantity of powder
equivalent to 10 mg of simvastatin was weighed and transferred to a 200 ml volumetric flask
to it 140 ml of diluent was added and sonicatedfor 30 min with intermediate shaking. It was
diluted up to the mark with diluent. It was further diluted to get concentration of 10 µg/ml of
simvastatin. The analytical procedure was repeated six times for the powder sample.

VALIDATION PARAMETERS
The HPLC method was validated on accuracy, precision, LOD, LOQ, linearity, range and
robustness as per ICH guidelines 17.

Accuracy
To check the accuracy of proposed method was determined by performing recovery studies
by standard addition method in which pre-analyzed sample of tablet powder was taken and
known amount of pure was added at three different levels (80%, 100% and 120%) and the %
recovery of simvastatin ranging from 98.70 to 102.6 % was calculated.

Precisions
This parameter was evaluated by carrying out six independent test samples. RSD (%) of six
assay values obtained which was calculated. The system precision and method precision was
carried out by analysing the sample in different days. The RSD (%) values for method
precision and system precision where less than 2% indicating high degree of precision of
developed method.

Linearity and range

Five different concentrations ranging from 50, 75,100, 125 and 150 µg/ml of simvastatin was
prepared in 100 ml volumetric flask. The peak areas of those solutions were measured at 238
nm.

Specificity
This method was carried out by injecting the working standard, blank and stressed samples
into the chromatograph to check the co-elution; at the retention time of simvastatin peak.

Limit of Detection
The limit of detection (LOD) for simvastatin was determined from standard deviation of the
response and slope of the curve by using equation 1.
LOD= σ/S X 3.3 - - - - - - - - - (1)

Limit of Quantitation
Limit of quantitation (LOQ) for simvastatin was determined from standard deviation of the
response and the slope of curve by using equation 2.
LOQ= σ/S X 5- - - - - - - - - (2)

Robustness
Robustness was carried out by changed in chromatographic conditions such as changed in
wavelength of detection (±2nm), flow rate (±0.1ml/min), pH (±0.5) & acetonitrile content in
mobile phase (±2%).

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System suitability parameters
The system suitability test parameters was carried out to evaluate resolution and
reproducibility of the system for the performed analysis, using five replicate injections of
reference solution containing 100 μg/ml of simvastatin. The parameters like resolution,
retention time, theoretical plates and tailing factor were measured.

Participants of Workshop

Hon’ble Vice Chancellor Prof. Pandit Vidyasagar with Participants of workshop

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 Valedictory program and certificate distribution at the Hand of Hon’bleVice Prof.
Pandit Vidyasagar

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