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A Single Laboratory Validation of Test Method for the Quantitation of Rosmarinic Acid inRosemary Leaf, Rosemary Leaf Extract, Lemon Balm Leaf and Lemon Balm Leaf Extract by HPLC

Larry Zeng1, Yolanda Xiong1, Leo Li1, Yanjun Zhang2, Peter Chang2, Gary Swanson2

1Herbalife Nutrition, 1318 Kaiyuan East Road, Changsha, Hunan, 410100, China2Herbalife Nutrition, 950 West 190th Street, Torrance, CA 90502, USA

AbstractAn HPLC method was developed for the determination of rosmarinic acid inrosemary leaf, rosemary leaf extract, lemon balm leaf and lemon balm leaf ex-tract. Rosemary leaf and lemon balm leaf were extracted with 80% ethanol bysonication, and rosemary extract and lemon balm extract were extracted withwater by sonication. The amount of rosmarinic acid was determined by HPLC.The precision, accuracy, linearity, specificity, ruggedness of the proposed methodwere validated based on AOAC Guidance for Single laboratory Validation Pro-cedure. The linearity of the test method ranged from 0.013 mg/mL to 0.250mg/mL, and the recoveries of rosmarinic acid from rosemary leaf, rosemary leafextract, lemon balm leaf and lemon balm leaf extract were between 90% and110%. The method is reliable for the quantitation of rosmarinic acid in rosemaryleaf, rosemary leaf extract, lemon balm leaf and lemon balm leaf extract and issuitable for routine analysis.

IntroductionRosmarinic acid is a well-known natural product with health benefits [1], derivedfrom rosemary (Rosmarinus officinalis), lemon balm (Melissa officinalis), andother Lamiaceae. Accurate quantitation of rosmarinic acid is important for thequality control of related material. However, a limited number of papers havebeen published on the determination of rosmarinic acid in these botanicals byHPLC [2]. No report has been found on determination of rosmarinic acid inboth rosemary leaf, lemon balm leaf and their extracts using HPLC.

Materials and MethodsMaterial: Rosmarinic Acid, Chemical Reference (CR), NIFDC, Catalog #111871, Lot#111871-201706. Standard Preparation: 5.0 mg of rosmarinic acid chemical referencewas accurately weighted into a 100 mL volumetric flask. Added about 50 mL of 80% ethanol,swirled to dissolve. Diluted to volume with 80% ethanol. Sample Preparation: For rawmaterial, a sample with about 5.0 mg of rosmarinic acid was weighted into a 100 mL vol-umetric flask. Added about 70 mL of 80% ethanol, sonicated for 30 min, cooled to roomtemperature. Diluted to volume with 80% ethanol. Filtered a portion of the sample througha 0.45µm syringe filter, discarded the first few drops of the filtrate and then filled into anHPLC vial. For extract, a sample with about 5.0 mg of rosmarinic acid was weighted intoa 100 mL volumetric flask. Added about 70 mL of water, sonicated for 10 min, cooled toroom temperature. Diluted to volume with water. Filtered a portion of the sample through a0.45µm syringe filter, discarded the first few drops of the filtrate and then filled into an HPLCvial.

Equipment

Waters 2690/2695 Alliance Separa-tions Module consisting of pumpand autosampler coupled with WatersPDA detectorColumn: Phenomenex Synergi 4µHydro-RP 80 A (Catalog # 00G-4375-E0)Mobile Phase: 0.1% Phosphoric acidsolution/acetonitrile [3]

Table 1: HPLC operating conditions

Item Parameter

Column temp 30°CSample temp ambientWavelength 330 nmFlow rate 1.0 L/minRun time 40 minInjection Volume 10µL

Method ValidationSpecificity: No significant peak was present in the chromatogram of the blankat the retention time of the rosmarinic acid peak from CR solution.

The rosmarinic acid peak in sample solutionsand standard solution had the same retentiontime. The UV spectra of the rosmarinic acidpeak from sample solutions were identical tothat of the rosmarinic acid peak from standardsolution.

Linearity/Range: Working standard solu-tions containing about 0.013, 0.025, 0.050, 0.125and 0.250 mg/mL of rosmarinic acid were pre-pared by making dilutions from the CR stocksolution at concentration (1.2459 mg/mL of ros-marinic acid). Three (3) replicate injectionswere made for each of the five (5) solutions. Thepeak areas of rosmarinic acid obtained for eachsolution were plotted against their correspond-ing theoretical concentration. Linear regressionanalyses on the five coordinates were performed.The R2 of the linear curve was 0.99992.

Table 2: Linearity/RangeTest Results

LinearityLevels

Standard Conc.(mg/ml) Area

Level 1 (25%) 0.0124591

353904

348831

352588

Level 2 (50%) 0.0249183

716296

721739

725189

Level 3 (100%) 0.0498365

1461559

1465605

1451784

Level 4 (250%) 0.1245914

3652372

3687826

3687482

Level 5 (500%) 0.2491827

7451839

7448898

7480454

Figure 1

Method Validation (Continued)Precision: Six (6) replicate samples of each sample were prepared. The RSDof rosemary leaf test results was 4%, the RSD of lemon balm leaf test resultswas 3%, the RSD of rosemary leaf extract test results was 0% and the RSD oflemon balm leaf extract test results was 1%.

Table 3: Precision Test Results

Replicate

RosemaryLeaf

Lemon BalmLeaf

RosemaryLeaf Extract

Lemon BalmLeaf Extract

Result(g/100g) RSD Result

(g/100g) RSD Result(g/100g) RSD Result

(g/100g) RSD

1 0.9981

4%

1.4222

3%

1.2812

0%

3.7864

1%

2 1.0953 1.3545 1.2763 3.7372

3 1.1184 1.3480 1.2775 3.7495

4 1.0890 1.3881 1.2715 3.7651

5 1.0263 1.3739 1.2787 3.7655

6 1.0797 1.4333 1.2774 3.7239

Accuracy: Nine (9) replicates of each sample at the normal test concentrationwere prepared. Known quantities of the CR stock solution were added to thesample solutions as described in Table 4 and 5. These spiked samples (three con-centrations and three replicates of each concentration) were analyzed accordingto the test method. At the same time, three (3) additional samples from thesame lot of each sample were prepared and analyzed. The additional amountsof rosmarinic acid found in the spiked samples versus the expected amount werecalculated as %Recovery. All spiked recoveries were within 90–110%.

Table 4: Accuracy Test Results for Rosemary Leaf and Lemon Balm Leaf

Spike level Sample #

Added Rosemary Leaf Lemon Balm Leaf

(mg) FoundRecovery

FoundRecovery(mg) (mg)

50%

1 1.2459 1.2401 100% 1.2818 103%

2 1.2459 1.3003 104% 1.3683 110%

3 1.2459 1.3017 104% 1.3248 106%

100%

1 2.4918 2.4366 98% 2.3788 95%

2 2.4918 2.5012 100% 2.7182 109%

3 2.4918 2.548 102% 2.4069 97%

150%

1 3.7377 3.6664 98% 3.8409 103%

2 3.7377 3.6924 99% 3.8144 102%

3 3.7377 3.7368 100% 3.7344 100%

Method Validation (Continued)Table 5: Accuracy Test Results for Rosemary Leaf Extract and

Lemon Balm Leaf Extract

Spike level Sample #

Added Rosemary Leaf Extract Lemon Balm Leaf Extract

(mg) FoundRecovery

FoundRecovery(mg) (mg)

50%

1 1.2459 1.1925 96% 1.2542 101%

2 1.2459 1.1760 94% 1.2518 100%

3 1.2459 1.1997 96% 1.2532 101%

100%

1 2.4918 2.3945 96% 2.4346 98%

2 2.4918 2.3720 95% 2.4718 99%

3 2.4918 2.3793 95% 2.4414 98%

150%

1 3.7377 3.5861 96% 3.6727 98%

2 3.7377 3.5364 95% 3.6498 98%

3 3.7377 3.5418 95% 3.6606 98%

Ruggedness: The same lots of all 4 products were tested (in duplicate) by asecond analyst on a different day. The RSD were within 10%.

Table 6: Ruggedness Test Results

Replicate Performed By

RosemaryLeaf

Lemon BalmLeaf

RosemaryExtract

Lemon BalmExtract

Result(g/100g)

Result(g/100g)

Result(g/100g)

Result(g/100g)

1

Analyst 1

0.9981 1.4222 1.2812 3.7864

2 1.0953 1.3545 1.2763 3.7372

3 1.1184 1.3480 1.2775 3.7495

4 1.0890 1.3881 1.2715 3.7651

5 1.0263 1.3739 1.2787 3.7655

6 1.0797 1.4333 1.2774 3.7239

7Analyst 2

1.2597 1.4686 1.2883 3.9193

8 1.2778 1.4994 1.2028 3.8794

RSD% 9% 4% 2% 2%

ConclusionA simple and sensitive HPLC analytical method for determination of rosmarinicacid in rosemary leaf, rosemary leaf extract, lemon balm leaf and lemon balmleaf extract was validated. This method is reliable and suitable. This methoddemonstrated excellent coefficient of determination (R), precision, accuracy,ruggedness, and specificity.

References[1] H Wang. “Determination of rosmarinic acid and caffeic acid in aromatic herbs by HPLC”.

In: Food Chemistry 87.2 (2004), pp. 307–311.

[2] Ying Zhang et al. “Degradation Study of Carnosic Acid, Carnosol, Rosmarinic Acid,and Rosemary Extract (Rosmarinus officinalis L.) Assessed Using HPLC”. In: Journalof Agricultural and Food Chemistry 60.36 (2012), pp. 9305–9314.

[3] Ai-Hua Liu et al. “Simultaneous quantification of six major phenolic acids in the rootsof Salvia miltiorrhiza and four related traditional Chinese medicinal preparations byHPLC–DAD method”. In: Journal of Pharmaceutical and Biomedical Analysis 41.1(2006), pp. 48–56.