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Proforma for registration of subjects for dissertation

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“ABHIJIT” Opp. J.K. Classes,

Mahadevnagar, Islampur.

Sangli- 415409





Chikkabellandur, Carmelaram Post,

Varthur Hobli, Sarjapur road;

Bangalore- 560035




Master of Pharmacy

( Quality Assurance )



11th June 2010.






    1. Need of the study:

Rosmarinic acid, C18H16O8, is a natural polyphenol antioxidant carboxylic acid found in many Lamiaceae herbs used commonly as culinary herbs such as lemon balm, rosemary, oregano, sage, thyme and peppermint. Rosmarinic acid has antioxidant, anti-inflammatory and antimicrobial activities.

The antioxidant activity of rosmarinic acid is stronger than that of vitamin ‘E’. Rosmarinic acid helps to prevent cell damage caused by free radicals, thereby reducing the risk for cancer and atherosclerosis. Rosmarinic acid is used to treat peptic ulcers, arthritis, cataract, cancer, rheumatoid arthritis and bronchial asthma.

Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. It is also the process of verifying that a method is fit for purpose.

Method validation is an important element of quality control. Validation helps provide assurance that a measurement will be reliable.

It is known that there is variation in content of Rosmarinic Acid (RA) in the different plant extract. So it was thought of interest to determine the Rosmarinic Acid from Rosemary and validate analytically.

So as to determine the quality and quantity of Rosmarinic Acid various performance characteristics i.e., parameters are to be evaluated. Quality parameters, assay, etc., will be validated to assure quality of Rosmarinic Acid.

Different guidelines like ICH, WHO etc. suggest that analytical methods used for the analysis must be stability indicating and validated.

Various chromatographic methods such as HPLC, HPTLC, and GC etc. are used because it fulfills requirement of various guidelines by effectively separating degraded and other substances likely to be present with phytochemical.

So the interest of work is emphasized on the HPLC technique, because in the modern pharmaceutical industry, high performance liquid chromatography (HPLC) is the major and integral analytical technique applied in all stages of drug discovery, development, and production.

HPLC is very useful tool for the analysis of many drugs. HPLC technique is very simple, highly specific and highly sensitive.

Hence it was thought of interest to develop a simple, rapid, precise, accurate and economic HPLC method for study of Rosmarinic acid from Rosemary extract.

The aim of this study is to develop a more reliable, selective, precise and accurate gradient HPLC method, validating it for the determination of Rosmarinic acid (RA).

6.2 Review of Literature:

1) According to this review, rosmarinic acid content was determined in eight commercial tinctures derived from fresh and dried Melissa officinalis herb. The column used was a Luna C18, 5 μm (150 x 4.6 mm I.D., Phenomenex) maintained at ambient room temperature. The HPLC system consisted of a Shimadzu SCL-6B controller, Shimadzu LC-6A pumps, Shimadzu SPD-6A UV single wavelength spectrophotometric detector set to 320 nm and Shimadzu SIL-6B autosampler. Gradient elution of the samples and standard were performed using ammonium formate (0.02 M; pH 6.25 at 27°C; eluent A) and methanol (eluent B). The flow rate was 1 mL/min. The assay was validated for sensitivity, accuracy and reproducibility.

2) This review of literature shows that, the simple method for the determination of rosmarinic acid (RA) by using a gradient high-performance liquid chromatography (HPLC) was developed. An HPLC system consisting of a 600 E model HPLC pump, a 717 plus Autosampler, a 996 photodiode array detector (PAD), and a Millenium 32 data processor was used for the HPLC analysis from Waters Corp.; an Octadecylsilane (ODS, C18) Ultrasphere column from Teknokroma (100× 4.6 mm inner diameter, particle size of 3 μm) was utilized for the HPLC analysis.

The analysis was performed by utilizing a two solvents system [A: methanol/water/formic acid (10:88:2; v: v: v); B: methanol/water/formic acid (90:8:2; v: v: v)] on a reverse-phase column. The flow rate and injection volume were 1 ml min-1 and 10 μl, respectively. Signals were detected at 280 nm. In addition, an internal standard (IS) technique was applied for the analysis of RA to increase precision, and propyl paraben was employed for this purpose. The method was evaluated for a number of validation characteristics i.e., repeatability, linearity, Limit Of Detection, Limit Of Quantitation. The method was applied to the extracts of certain Lamiaceae plants (Salvia candidissima Vahl. subsp. candidissima, S. sclarea L., S. verticillata L. subsp.verticillata and R. officinalis L.), and reasonable results were obtained.

3) This review shows that, the HPLC method for the determination of Rosmarinic acid and caffeic acid in several aromatic herbs, namely, rosemary, sage, thyme, and balm was developed and validated. The separation system consisted of a C18 reversed-phase column, a gradient elution system of methanol/water containing orthophosphoric acid, and a photodiode array detector. The content of rosmarinic acid was found to be 2.0–27.4 mg/g, in the aromatic herbs analysed.

An HP 1100 series liquid chromatograph system comprising vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, and diode array detector was used. The flow rate was 1.0 ml min-1. Detection wavelength was 330 nm. The sample injection volume was 10 µl. The linearity range for rosmarinic and caffeic acid was determined to be 2–100 µg/ml and 0.4-100µg/ml respectively.

4) In this review, A simple HPLC method was developed to determine the content of Rosmarinic acid in the lemon balm (Melissa officinalis L.). The influence of the plant development phase at harvest time on the content of rosmarinic acid was studied in Melissa leaf samples of Slovak origin.

The HPLC system consisted of a gradient programmer (GP3), a micropump (LCP3001) and a variable UV-VIS detector (LCD 2082). The analytical column was a reversed phase SGX C18, 4 mm × 250 mm, 5 µm. For sample preparation, an ultrasonic bath, a pH meter and a centrifuge were used. The mobile phase consisted of methanol and water (pH 2, adjusted with phosphoric acid). The flow rate was 0.5 ml/min and injection volume was 20 µl. Spectra were recorded at 320nm.

5) This review shows that, reversed-phase high-performance liquid chromatography (HPLC) method was developed for analyzing phenolic compounds in fennel (Foeniculum vulgare). The method was validated for the major phenolic compounds present in fennel plant material: 3-O-caffeoylquinic acid, chlorogenic acid, 4-O-caffeoylquinic acid, eriocitrin, rutin, Rosmarinic acid, etc. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.05 to 1.0 µg/mL and from 0.15 to 2.5 µg/mL, respectively.

The analyses were performed using a Finnigan Surveyor HPLC system equipped with a UV–visible detector. The monolithic column in use was Chromolith Performance RP-18e (octadecylsilyl silica). UV detection was performed at 330 nm. The flow rate was 3.0 mL/min and the injection volume was 5µL. Mobile phase A consisted of 0.1% formic acid and 5% acetonitrile (v/v), while mobile phase B consisted of 0.1% formic acid and 90% acetonitrile (v/v).

6) In this review, the polyphenols such as rosmarinic acid, caffeic acid, cichoric acid, sinesetine etc., identified and determined from plant Orthosiphon stamineus Benth by using HPLC. A Shimadzu Class-VP chromatograph was used coupled with a diode array UV/Vis detector. An RP-18 LiChrosphere 5 µm, 4X4 mm guard column and an RP-18 LiChrosphere 5 mm, 125x4 mm column was used as stationary phase. The mobile phase was a gradient made by changing the content of solvent A (acetonitrile/phosphoric acid 99.9:0.1, v/v) from 15 to 100% in 32 min, the solvent B being water/phosphoric acid (98:2, v/v). The flow rate was 0.5 ml/min and the column temperature 40°C. The detection was performed at 340 nm. The results obtained were reliable.

7) This review shows that, polyphenolic compounds like rosmarinic acid, ferulic acid, caffeic acid and its derivatives etc. were identified and determined from Hungarian Thymus species by using High-performance liquid chromatography (HPLC) coupled with diode array detector (DAD) and on-line mass spectrometry (ESI-MS) method. The method was evaluated for a number of validation characteristics i.e., repeatability and intermediate precision, LOD, LOQ, calibration range, and recovery.

Chromatographic separations were performed on an Ascentis Express C18 column (50mm×2.1mm, 2.7µm,) at 50°C. For the separations, a gradient of mobile phase A (2% (v/v) acetic acid in water) and mobile phase B (2% (v/v) acetic acid in methanol) was used. The flow rate was 0.2mL min-1. UV detection wavelengths were 280 nm, 320 nm, and 360nm. The results obtained were reliable.

8) According to this review, HPLC-PAD method for the determination of rosmarinic acid in Rosmarinus officinalis L. was established by an ultrasonically aided method. The separation was performed at the following conditions: Kromasil C18 column (4.6mm×250mm, 5μm) at 25℃, with methanol-0.2% phosphoric acid solution (v/v, 45∶55) as the mobile phase, a flow rate of 0.8mL/min, and the detection wavelength at 331nm. The recovery was 97.8% and RSD was 1.2 %( n=6).RSD of the precision test and repeatability were 0.6% and 2.1%.The contents of rosmarinic acid were 0.11549~0.20157%. This method was accurate, simple for the determination of rosmarinic acid in Rosmarinus officinalis L.

9) HPTLC-densitometric and HPLC–UV techniques were used for qualitative and quantitative determination of luteolin-7-O-glucuronide, lithospermic acid, rosmarinic acid and caffeic acid in several herbal drugs from the Lamiaceae family. Unmodified silica gel (HPTLC Si60) and silica gel chemically modified with aminopropyl groups (HPTLC NH2) were used. Among HPTLC methods the best resolution and selectivity was achieved with mobile phases: diisopropyl ether–acetone–formic acid–water (50:30:10:10, v/v/v/v) and acetone–formic acid (85:15, v/v), respectively. Plates were densitometrically evaluated. Contents of analyzed compounds in the studied aqueous extracts prepared from herbal drugs were established using both techniques. The results from the HPTLC-densitometric analysis were compared with those from HPLC–UV on a C18 column with acetonitrile–water–formic acid as a mobile phase. The chromatographic methods were validated for linearity, LOD, LOQ, repeatability, intermediate precision and recovery. An analysis of variance showed that the HPTLC-densitometric and HPLC–UV methods are equivalent and sufficiently precise for the estimation of polyphenolic compounds mentioned above. All of the suggested methods (HPTLC NH2, HPTLC Si60 and HPLC RP18) give results with good agreement.
6.3 Objective of study: -

1) The primary objective of this study is to develop HPLC method and validate it for determination of rosmarinic acid in Rosemary extract.

  • Extraction of rosmarinic acid from Rosemary

  • Isolation of rosmarinic acid

  • Standardization of rosmarinic acid

2) The secondary objective is to validate developed HPLC method

  • Accuracy

  • Precision

  • Repeatability

  • Linearity

  • Limit of detection

  • Limit of quantification

  • Robustness

  • Ruggedness



    1. Source of Data :

Data will be collected from literature surveys, abstracts, journals, various related websites, Industries, research publication and from libraries of Krupanidhi college of pharmacy, Indian institute of sciences, Government college of Pharmacy etc.

7.2 Method of collection of data :

Data will be collected from the following stepwise experimental procedure proposed in the study.

1) Development of analytical method for estimation of Rosmarinic acid by HPLC

  • By performing experiment in the laboratory from the information obtained from the above source.(7.1)

  • Perform trials with different stationary and mobile phase in different phase with suitable column and sample concentration.

  • Determine parameters of HPLC i.e., Retention, degree of separation, column efficiency.

  • Conclude various results from above experiment to conduct the following study.

2) Validation of the above developed HPLC method.

  • Protocol validation

  • Purpose, scope and application

  • Performance parameter

  • Perform raw material qualification studies.

  • Perform system suitability and analytical quality control checks for the routine

  • Perform method validation experiment and report the result.

7.3 Does the study require any investigation of intervention to be conducted

On patient or other human or animal? If so please describe briefly:


7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Not Applicable


1. accessed on 27th Oct 2010

2. accessed on 27th Nov 2010.

3. accessed on 27th Oct.2010

4. Medina AS, Etheridge CJ, Hawkes GE, Hylands PJ, Pendry BA, Hughes MJ et al. Comparison of rosmarinic acid content in commercial tinctures produced from fresh and dried lemon balm. J Pharm Pharmaceut Sci 2007; 10(4):455-463.

5. Nilgun O, Muzzafer T, Ulku DU, Elif MO, Onur K. “Determination of rosmarinic acid by High Performance Liquid Chromatography and its application to certain Salvia species and Rosemary.” Food Anal Method 2008; 1, 2010; 3.

6. Huafu W, Provan GJ, Helliwel K., “Determination of rosemarinic acid and caffeic acid in aromatic herb by HPLC.” Food Chem 2004; 87: 307-311.

7. Toth J, Mrlianova M, Tekelova D, Korenova M. “Rosmarinic acid an important phenolic active compound of Lemon balm (Melissa officinalis L.).” Acta Facult Pharm Univ Comenianae 2003;50:139-146.

8. Krizman M, Bericevic D, Prosek M. “Determination of phenolic compounds in fennel by HPLC and HPLC –MS using monolithic reversed –phase column.” J Pharm Biomed Anal 2007; 43:481-485.

9. Olah NK, Radu L, Mogosan C, Hanganu D, Gocan S. “Phytochemical and Pharmacological studies on Orthosiphon stamineus Benth. (Lamiaceae) Hydroalcoholic extract.” J Pharm Biomed Anal 2003; 33:117-123.

10. Boros B, Jokobova S, Dornyei A, Horvath G, Pluhar Z, Kilar F, et al. “Determination of polyphenolic compound by liquid chromatography-Mass spectrometry in Thymus species.” J Chromatogr. A 2010,DOI: 10.1016/j.chroma.2010.07.042

11. Sheng-quinl ZOU, Xiao-quing SUN. “Determination of rosmarinic acid in Rosmarinus officinalis L. from different places using Ultrasound assisted extraction and RP-HPLC.” China Food Additives 2010-03.

12. Fecka I, Raj D, Baranowska MK. “Quantitative determination of four Water-soluble compounds in herbal drugs from Lamiaceae using different Chromatographic techniques.” Chromatographia 2007; 66:87-93.

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