Analytical purity method development and validation by gas chromatography of l valine methyl ester hydrochloride for production of anti-hypertensive drugs
This document describes the development and validation of a gas chromatography method for analyzing the purity of L-valine methyl ester hydrochloride, which is used to produce anti-hypertensive drugs. The method was validated for precision, recovery, linearity, robustness, and solution stability according to ICH guidelines. Gas chromatography with a flame ionization detector was used to separate and detect L-valine methyl ester hydrochloride peaks from potential impurity peaks such as isoleucine methyl ester hydrochloride. The method demonstrated high recovery and low variability, confirming its suitability for analyzing the purity of L-valine methyl ester hydrochloride in pharmaceutical applications.
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Analytical method development and validation for simultaneous estimationProfessor Beubenz
Brief about analytical method development and validation
Subscribe to the YouTube Channel #Professor_Beubenz
https://www.youtube.com/channel/UC84jGf2iRN5VjwnQqi6qmXg?view_as=subscriber
Bioanalytical RP-HPLC Method Development and Validation for Estimation of Cur...Sagar Savale
The present study was aimed at developing a reversed phase high performance liquid chromatography (RPHPLC) method for determination of curcumin (CRM) in plasma and hydrochlorothiazide was used as an internal
standard. The separation was achieved by using C-18 column (Qualisil BDS C18, 250 mm x 4.6 mm I.D.)
coupled with a guard column of silica, mobile phase was consisting of acetonitrile: water with 0.1% formic acid
(40:60 v/v). The flow rate was 0.3 ml/min and the drug was detected using PDA detector at the wavelength of 423
nm. The experimental conditions, including the diluting solvent, mobile phase composition, column saturation
and flow rate, were optimised to provide high-resolution and reproducible peaks. The developed method was
validated in terms of linearity, recovery, precision, ruggedness, sensitivity (LOD and LOQ) and stability study
(short and long-term stabilities, Freeze/thaw stability and post-preparative).
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Analytical method development and validation for simultaneous estimationProfessor Beubenz
Brief about analytical method development and validation
Subscribe to the YouTube Channel #Professor_Beubenz
https://www.youtube.com/channel/UC84jGf2iRN5VjwnQqi6qmXg?view_as=subscriber
Development and Validation of Novel RP-HPLC method for the estimation of Nalo...Bhavana Gundavarapu
The RP-HPLC method was developed and validated for quantitative determination of Naloxegol in pharmaceutical dosage forms. The method was validated with regard to linearity, accuracy, precision, selectivity, and robustness. The method was applied successfully for the determination of Naloxegol during kinetic studies in aqueous solutions (pH and thermal degradation).
Quality-by-design-based development and validation of a stability-indicating ...Ratnakaram Venkata Nadh
A systematic design-of-experiments was performed by applying quality-by-design concepts to determine
design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography
(UPLC) method in the presence of degradation products. Response surface and central composite
quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The
response variables such as resolution, retention time, and peak tailing were analyzed statistically for the
screening of suitable chromatographic conditions. During this process, various plots such as perturbation,
contour, 3D, and design space were studied. The method was developed through UPLC BEH C18
2.1 � 100 mm, 1.7-μ column, mobile phase comprised of buffer (5 mM K2HPO4 containing 0.1%
triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5 mL min 1 and UV detection at
250 nm. The method was developed with a short run time of 1 min. Forced degradation studies revealed
that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product.
The method was found to be linear in the range of 28–84 μg mL 1, 2.8–22.7 μg mL 1 with a correlation
coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in
the range of 100.1–101.7%. The method was validated according to ICH guidelines.
Determination of Etodolac in Commercial Formulations by HPLC-UV Methodijtsrd
The aim of this study was to develop and verify a simple, rapid and sensitive high performance liquid chromatography method coupled with UV detector HPLC UV method for the quantitative determination of etodolac in bulk and pharmaceutical dosage forms. Chromatographic separation was performed at ambient conditions on a reverse phase ACE C8 analytical column 250 mm x 4.6 mm ID, 5 umm using the mobile phase containing acetonitrile water 80 20, v v at a flow rate of 1.0 mL min 1. A wavelength of 272 nm was used for etodolok and paracetamol IS . A retention time of 4.21 min and 2.02 min were obtained for etodolac and IS, respectively. The method showed linearity in the range of 0.08 10 µg mL 1 for etodolac R = 0.9999 . The linear regression equations obtained by least square regression method were the ratio of peak area of etodolac and IS =1.559 concentration etodolac µg mL 0.139. The intra day and inter day RE and RSD values of the method were =10.0 and =2.65 , respectively. Limit of detection LOD and limit of quantification LOQ were found to be 0.04 and 0.06 µg mL 1 for etodolac, respectively. A new, simple and sensitive high performance liquid chromatography method was developed and validated for etodolac. The method can be applied for the quantification of etodolac without derivatization in bulk solutions and commercial formulations using the internal standard. Tugrul Cagri Akman | Yucel Kadioglu "Determination of Etodolac in Commercial Formulations by HPLC-UV Method" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29452.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29452/determination-of-etodolac-in-commercial-formulations-by-hplc-uv-method/tugrul-cagri-akman
Inspirational quotes from the thinkers of the world. I have also provided my personal comments to aid acquiring the meanings of these quotes in modern parlance.
Development and Validation of Novel RP-HPLC method for the estimation of Nalo...Bhavana Gundavarapu
The RP-HPLC method was developed and validated for quantitative determination of Naloxegol in pharmaceutical dosage forms. The method was validated with regard to linearity, accuracy, precision, selectivity, and robustness. The method was applied successfully for the determination of Naloxegol during kinetic studies in aqueous solutions (pH and thermal degradation).
Quality-by-design-based development and validation of a stability-indicating ...Ratnakaram Venkata Nadh
A systematic design-of-experiments was performed by applying quality-by-design concepts to determine
design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography
(UPLC) method in the presence of degradation products. Response surface and central composite
quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The
response variables such as resolution, retention time, and peak tailing were analyzed statistically for the
screening of suitable chromatographic conditions. During this process, various plots such as perturbation,
contour, 3D, and design space were studied. The method was developed through UPLC BEH C18
2.1 � 100 mm, 1.7-μ column, mobile phase comprised of buffer (5 mM K2HPO4 containing 0.1%
triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5 mL min 1 and UV detection at
250 nm. The method was developed with a short run time of 1 min. Forced degradation studies revealed
that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product.
The method was found to be linear in the range of 28–84 μg mL 1, 2.8–22.7 μg mL 1 with a correlation
coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in
the range of 100.1–101.7%. The method was validated according to ICH guidelines.
Determination of Etodolac in Commercial Formulations by HPLC-UV Methodijtsrd
The aim of this study was to develop and verify a simple, rapid and sensitive high performance liquid chromatography method coupled with UV detector HPLC UV method for the quantitative determination of etodolac in bulk and pharmaceutical dosage forms. Chromatographic separation was performed at ambient conditions on a reverse phase ACE C8 analytical column 250 mm x 4.6 mm ID, 5 umm using the mobile phase containing acetonitrile water 80 20, v v at a flow rate of 1.0 mL min 1. A wavelength of 272 nm was used for etodolok and paracetamol IS . A retention time of 4.21 min and 2.02 min were obtained for etodolac and IS, respectively. The method showed linearity in the range of 0.08 10 µg mL 1 for etodolac R = 0.9999 . The linear regression equations obtained by least square regression method were the ratio of peak area of etodolac and IS =1.559 concentration etodolac µg mL 0.139. The intra day and inter day RE and RSD values of the method were =10.0 and =2.65 , respectively. Limit of detection LOD and limit of quantification LOQ were found to be 0.04 and 0.06 µg mL 1 for etodolac, respectively. A new, simple and sensitive high performance liquid chromatography method was developed and validated for etodolac. The method can be applied for the quantification of etodolac without derivatization in bulk solutions and commercial formulations using the internal standard. Tugrul Cagri Akman | Yucel Kadioglu "Determination of Etodolac in Commercial Formulations by HPLC-UV Method" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-4 | Issue-1 , December 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29452.pdfPaper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/29452/determination-of-etodolac-in-commercial-formulations-by-hplc-uv-method/tugrul-cagri-akman
Inspirational quotes from the thinkers of the world. I have also provided my personal comments to aid acquiring the meanings of these quotes in modern parlance.
A short powerpoint on the importance of protein and several amino acids including proline, glutamine, cysteine, valine, tryptophan, arginine and glycine. In case you're wondering, we used this PPT while presenting the report in a Salamat Dok way.
The Information Content of DNA and the Language of Life The code used to translated DNA to RNA to Protein could not have evolved but must have been created.
This Course is included in the syllabus of Bachelor in Science Agriculture level study in Tribhuvan University. The course belongs to 1h lecture.This slide include general introduction of amino acid. It describes about structure, function , type and role of amino acid.
This presentation describes about the syllabus of Agriculture Microbiology for B.Sc agriculture student of second semester in Tribhuvan University, Nepal. It is helpful to understand the student about the courses and guide the students to focus in the related topics.
Similar to Analytical purity method development and validation by gas chromatography of l valine methyl ester hydrochloride for production of anti-hypertensive drugs
Development and Validation of Reverse Phase Liquid Chromatography Method for ...IOSR Journals
A simple, precise and reversed phase liquid chromatographic method was developed and it is validated for estimation of losartan in bulk drug. Losartan is use for treatment of hypertension. The separation was achieved on Acquity BEH C18 1.7μ, (2.1 X 100) mm, analytical column with mobile phase consisted of buffer (adjust pH 3.0 of water with dilute formic acid) : Acetonitrile (50:50 v/v) at isocratic flow of 0.3ml/min with UV detection wavelength was at 230 nm. The method was successfully validated in accordance to ICH guidelines for accuracy, precision, specificity, linearity. The linear regression analysis data for calibration plots showed good linear relationship in the concentration range 25-75μg/mL for losartan. The % Recovery/Accuracy was within the range between 98% and 102%. The percentage RSD for precision method was found to be less than 2%. The method was successfully applied for routine analysis of losartan in bulk samples
Development and Validation of RP HPLC Method for Estimation of Vortioxetine i...ijtsrd
A rapid and precise reverse phase high performance liquid chromatographic method has been developed for the validated of Vortioxetine in its pure form as well as in tablet dosage form. Chromatography was carried out on ODS C18 4.6 x 250 mm, 5µm column using Acetonitrile And Methonal 70 30 as the mobile phase at a flow rate of 1.0 mL min, the detection was carried out at 274nm. The retention time of the Vortioxetine was 2.922 ±0.02min. The method produce linear responses in the concentration range of 20 µg ml of Vortioxetine. The method precision for the determination of assay was below 2.0 RSD. The method is useful in the quality control of bulk and pharmaceutical formulations. Rathod K. G | Bargaje G. S | Rathod G. R | Deshpande O. V "Development and Validation of RP-HPLC Method for Estimation of Vortioxetine in Bulk and Pharmaceutical Dosage Form" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-6 , October 2019, URL: https://www.ijtsrd.com/papers/ijtsrd28040.pdf Paper URL: https://www.ijtsrd.com/pharmacy/analytical-chemistry/28040/development-and-validation-of-rp-hplc-method-for-estimation-of-vortioxetine-in-bulk-and-pharmaceutical-dosage-form/rathod-k-g
RP-HPLC Method Developed for the Estimation of Etodolac and Thiocolchicoside ...ijtsrd
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Analytical purity method development and validation by gas chromatography of l valine methyl ester hydrochloride for production of anti-hypertensive drugs
1. Chemical and Process Engineering Research www.iiste.org
ISSN 2224-7467 (Paper) ISSN 2225-0913 (Online)
Vol.11, 2013 - Selected from International Conference on Recent Trends in Applied Sciences with Engineering Applications
1
Analytical Purity Method Development and Validation by gas
Chromatography of L-valine Methyl Ester Hydrochloride for Production of
Anti-hypertensive Drugs
Anita shinde1
, Suman Malik2
, Amit Asati2
sanita757@gmail.com, drsumanmalik@gmail.com, amit.asati29@gmail.com
1-Department of chemistry G.M.L.B.G.P.G.A.college, Bhopal
2-Department of chemistry Sadhu Vaswani college Bairagarh, Bhopal
Abstract
Gas chromatography is the most widely used technique in pharmaceutical industry. Analytical chemistry
research is largely driven by performance of sensitivity, selectivity, robustness, linear range, accuracy, precision.
Validation is founded on but not specifically prescribed by regulatory requirements and is best viewed as an
important and integral part of GMP (Good Manufacturing Practice). Gas chromatography method has been
developed for L-valine methyl ester hydrochloride. It is used for production of anti-hypertensive drug. The Gas
Chromatography system was used for method development and method validation with an auto injector and
detection was performed by means of flame ionization detector (FID) with capillary column DB-624, 30m length,
0.53mm diameter and 1.0µm thickness. Nitrogen an inert gas was used as carrier gas. The method was validated
for precision (system precision and method repeatability), recovery, linearity range, robustness and sample
solution stability. The high recovery and low relative standard deviation confirms the suitability of the method
for purity of L-valine methyl ester hydrochlride. It has been found from data of validation criteria that the
proposed method has adequate reproducibility and specificity therefore suitable in pharmaceutical industry.
Key words: Validation, Gas chromatography, FID, GMP, Pharmaceutical industry.
INTRODUCTION
L-valine methyl ester Hydrochloride ((S)-2-AMINO-3-METHYL-BUTYRIC ACID METHYL
ESTERHYDROCHLORIDE) is intermediate product for production of Valsartan (Anti-hypertensive drug).[1-2]
L-Valine methyl ester structure was shown in Figure-I. Validation is a rapidly growing and evolving subject and
is a requirement that has always made sense from both regulatory and quality perspective. It determined the
quality purity of the final products.[3-4]
Analytical methods rely on scrupulous attention to cleanliness, sample
preparation accuracy and precision. A standard method for analysis of concentration involves the creation of
calibration curve. In the concentration of elements of compound in a sample is too high for the detection range of
a technique, it can simply be diluted in a pure solvent. If the amount in sample is below an instruments range of
measurement, the method of addition can be used. In this method a known quantity of the elements or compound
under study is added, and the concentration observed in the amount actually in the sample.[5-7]
Analytical
chemistry research is largely driven by performance of sensitivity, selectivity, robustness, linear range, accuracy,
precision.[8-9]
Validation is founded on but not specifically prescribed by regulatory requirements and is best
viewed as an important and integral part of GMP (Good manufacturing Practice). The high recovery and low
relative standard deviation confirms the suitability of the method for purity of compound.[10-11]
Literature survey
indicates that there is no GC-FID method available for the determination of L-valine methyl ester hydrochloride
and thus we aimed to develop it. Gas chromatography (GC) is a common type of chromatography used
in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition.[12-
13]
Principle of gas chromatography is similar to column chromatography typical uses of GC include testing the
purity of a particular substance, or separating the different components of a mixture (the relative amounts of such
components can also be determined).[14-15]
In gas chromatography, the mobile phase (moving phase) is a
carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a
microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a
column.[16]
Experimental details, methods and materials
Gas chromatograph with split auto injector and flame ionised detector (FID) is used. Chemicals and Reagents
Isoleucine methyl ester HCl Methanol (AR grade) and L-Valine methyl ester HCl are used. The GC system used
for method development and method validation was with a auto sampler. The detection was performed by means
of flame ionization detector (FID). DB-624, 30m length, 0.53mm diameter and 1.0µm thickness capillary
column has been procured from Agilent technologies and used for the method development and method
validation study. The column oven programme as follows: initial column oven temperature, 150°C hold for
15 min. The run time of analysis was 15 min. The injector and detector temperature was kept at 180°C and
2. Chemical and Process Engineering Research www.iiste.org
ISSN 2224-7467 (Paper) ISSN 2225-0913 (Online)
Vol.11, 2013 - Selected from International Conference on Recent Trends in Applied Sciences with Engineering Applications
2
200°C, respectively. Nitrogen was used as a carrier gas with a constant flow rate of 5.0ml/min. The split ratio
was set at 1:50 deactivated open-glass tube liner packed with fused silica wool was employed. Sample was
injected injection volume 0.5µL. Solutions of Isoluecine and L-valine methyl ester HCl were prepared in
methanol. Inject system blank, QL and Sample solutions. Determine the area of all peaks in each solution.
Disregard peaks observed in blank and report the results by area normalization. Resolution between L-valine
methyl ester HCl and Isoluecine methyl ester HCl should be more than 5.0.
Observations results and Discussion
Specificity: The ICH documents define specificity as the ability to assess unequivocally the analyte in the
presence of components that may be expected to be present, such as impurities, degradation products, and matrix
components.
The specificity of the method was carried out by injecting the blank, System suitability and sample solution (un-
spiked and spiked), determined the resolution factors between analyte peak (of L-Valine Methyl ester) and the
nearest peak. Sample of L-Valine methyl ester HCl at about 200mg/mL spiked with about 0.2mg/mL of
Isoleucine methyl ester HCl. Un-spiked will be injected once.
Linearity:- The linearity of an analytical procedure is its ability to elicit test results that are directly, or by a well-
defined mathematical transformation, proportional to the concentration of analyte in sample within a given range.
It should be established initially by visual examination of a plot of signals as a function of analyte concentration
of content. If there appears to be a linear relationship, test results should be established by appropriate statistical
methods (e.g., by calculation of a regression line by the method of least squares). In some cases, to obtain
linearity between the response of an analyte and its concentration, the test data may have to be subjected to a
mathematical transformation. Data from the regression line itself may be helpful for providing mathematical
estimates of the degree of linearity. The correlation coefficient, y-intercept, slope of the regression line, and
residual sum of squares should be submitted. Linearity curve was shown in Figure-VI and Regression analysis of
the calibration curve was given in Table-I.
Detection limit:- The detection limit is a characteristic of limit tests. It is the lowest amount of analyte in a
sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Thus, limit
tests merely substantiate that the amount of analyte is above or below a certain level. The detection limit is
usually expressed as the concentration of analyte (e.g., percentage, parts per billion) in the sample. Detection
limit of L-Valine methyl ester HCl and Isoleucine methyl ester HCl are about 40µg/ml.
At DL level peaks are detected.
Quantification limit:- The quantification limit is a characteristic of quantitative assays for low levels of
compounds in sample matrices, such as impurities in bulk drug substances and degradation products in finished
pharmaceuticals. It is the lowest amount of analyte in a sample that can be determined with acceptable precision
and accuracy under the stated experimental conditions. Quantitation limit of L-Valine methyl ester HCl and
Isoleucine methyl ester HCl are about 100µg/ml. These QL solutions are injected in six replicate.
At QL level S/N ratio of peaks are more than 10.0
Precision:- The precision of an analytical method is the degree of agreement among individual test results when
the method is applied repeatedly to multiple samplings of a homogeneous sample. The precision of an analytical
method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) of a
series of measurements. Precision may be a measure of either the degree of reproducibility or repeatability of the
analytical method under normal operating conditions. The ICH documents recommend that repeatability should
be assessed using a minimum of nine determinations covering the specified range for the procedure (i.e., three
concentrations and three replicates of each concentration, or a minimum of six determinations at 100% of the test
concentration).
The system precision for the method was assessed by three preparation of different concentration for L-valine
methyl ester HCl standard about QL, 100% and 150% of nominal concentration and for Isoleucine methyl ester
HCl standard about QL, 100% and 150% of nominal concentration. System repeatability RSD data is given in
Tbale-II. Method repeatability was performed by injecting six different preparation of L-valine methyl ester HCl.
If sample was not containing specified impurity (Isoleucine valine methyl ester HCl), again prepare six different
solutions with spike 0.1 % Isoleucine methyl ester HCl (100% of specification level).
Recovery:- The ICH documents recommend that accuracy be assessed using a minimum of nine determinations
over a minimum of three concentration levels, covering the specified range (i.e., three concentrations and three
replicates of each concentration). Recovery data was given in Table-III.
Solution stability:- The sample solution prepared by spiking known concentration (0.1%) of L-valine methyl
ester HCl with respect to sample concentration 200mg/mL in methanol was stored at 25 ± 2°C temperature
conditions, and was injected into chromatographic system at different time intervals with fresh preparation. At
each interval, the sample solution was found to be stable over a period of 36 hours.
3. Chemical and Process Engineering Research www.iiste.org
ISSN 2224-7467 (Paper) ISSN 2225-0913 (Online)
Vol.11, 2013 - Selected from International Conference on Recent Trends in Applied Sciences with Engineering Applications
3
Robustness:- To assess robustness of the method, the experimental conditions were deliberately altered and
system suitability parameter was evaluated. Helium was used as a carrier gas with a constant flow rate
5.0 mL/min. To study the effect of flow rate on the resolution, the same was altered by 0.5 units that are from 4.5
to 5.5mL/min. The effect of column temperature was studied at 140°C and 160°C instead of 150°C. The effect of
changing the split ratio by ±10% (1:45 and 1:55 instead of 1:50) was also studied. All the other chromatographic
conditions were held constant as described above. In all the deliberate varied chromatographic conditions (flow
rate, column temperature, and split ration), the all system suitability criteria were within the limits.
CONCLUSIONS:
The proposed GC methods provide simple, accurate and reproducible for purity and impurity profile of L-valine
methyl ester HCl. The method was validated by testing its precision, linearity and recovery, limit of detection,
limit of quantitation, robustness and specificity as per ICH guideline.
Figure-I (L-Valine Methyl Ester HCl Structure)
Figure-II (Chromatogram of sample with impurity for specificity)
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Figure-III (System suitability Chromatogram)
Figure-IV {Blank (Methanol) Chromatogram}
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Figure-V (QL level Chromatogram)
Linearity of L-valine methyl ester HCl Linearity of Isoleucine methyl ester HCl
Figure-VI (Linearity curve for L-valine methyl ester HCl and L-Isoluecine HCl)
L-Valine methyl ester HCL Isoleucine methyl ester HCL
Parameters Results Parameters Results
Intercept value at 0.1% 3.9% Intercept value at 0.1% 0.7%
Slop 79692 Slope 85866
Intercept 653 Intercept 131
Correlation factor r2
0.999 Correlation factor r2
0.998
Table-I Regression analysis of the calibration curve
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Precision-System Repeatability
L-Valine methyl ester HCL Isoleucine methyl ester HCL
Concentration level % RSD Concentration level % RSD
QL 0.9% QL 2.6%
100% 2.4% 100% 2.3%
150% 1.2% 150% 1.1%
Table-II Data of system repeatability precision
Accuracy/Recovery
L-isoluecine methyl ester HCl QL 100% 150%
Spike amount mg/ml 0.1022 0.2051 0.3026
Average Recover amount mg/ml 0.1054 0.203 0.2945
% recovery 103.1 99 97.3
Table-III Recovery data
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