A Single Laboratory Validation of Test Method for the Quantitation of Rosmarinic Acid in

Rosemary Leaf, Rosemary Leaf Extract, Lemon Balm Leaf and Lemon Balm Leaf Extract by HPLC
1 1 1 2 2 2
Larry Zeng , Yolanda Xiong , Leo Li , Yanjun Zhang , Peter Chang , Gary Swanson
1
Herbalife Nutrition, 1318 Kaiyuan East Road, Changsha, Hunan, 410100, China
2
Herbalife Nutrition, 950 West 190th Street, Torrance, CA 90502, USA

Abstract Equipment Method Validation (Continued) Method Validation (Continued)

An HPLC method was developed for the determination of rosmarinic acid in Table 1: HPLC operating conditions Precision: Six (6) replicate samples of each sample were prepared. The RSD Table 5: Accuracy Test Results for Rosemary Leaf Extract and
Waters 2690/2695 Alliance Separa-
rosemary leaf, rosemary leaf extract, lemon balm leaf and lemon balm leaf ex- of rosemary leaf test results was 4%, the RSD of lemon balm leaf test results Lemon Balm Leaf Extract
tions Module consisting of pump Item Parameter
tract. Rosemary leaf and lemon balm leaf were extracted with 80% ethanol by was 3%, the RSD of rosemary leaf extract test results was 0% and the RSD of
and autosampler coupled with Waters Added Rosemary Leaf Extract Lemon Balm Leaf Extract
sonication, and rosemary extract and lemon balm extract were extracted with
PDA detector Column temp 30°C lemon balm leaf extract test results was 1%.
water by sonication. The amount of rosmarinic acid was determined by HPLC. Spike level Sample #
Column: Phenomenex Synergi 4µ Sample temp ambient (mg) Found Found
The precision, accuracy, linearity, specificity, ruggedness of the proposed method Table 3: Precision Test Results Recovery Recovery
Hydro-RP 80 A (Catalog # 00G-4375- Wavelength 330 nm (mg) (mg)
were validated based on AOAC Guidance for Single laboratory Validation Pro- Flow rate 1.0 L/min Rosemary Lemon Balm Rosemary Lemon Balm
E0) 1 1.2459 1.1925 96% 1.2542 101%
cedure. The linearity of the test method ranged from 0.013 mg/mL to 0.250 Leaf Leaf Leaf Extract Leaf Extract
Mobile Phase: 0.1% Phosphoric acid Run time 40 min Replicate
2 1.2459 1.1760 94% 1.2518 100%
mg/mL, and the recoveries of rosmarinic acid from rosemary leaf, rosemary leaf 50%
solution/acetonitrile [3] Injection Volume 10µL Result Result Result Result 3 1.2459 1.1997 96% 1.2532 101%
extract, lemon balm leaf and lemon balm leaf extract were between 90% and RSD RSD RSD RSD
(g/100g) (g/100g) (g/100g) (g/100g)
110%. The method is reliable for the quantitation of rosmarinic acid in rosemary 1 2.4918 2.3945 96% 2.4346 98%
leaf, rosemary leaf extract, lemon balm leaf and lemon balm leaf extract and is 1 0.9981 1.4222 1.2812 3.7864
2 2.4918 2.3720 95% 2.4718 99%
100%
suitable for routine analysis. Method Validation 2 1.0953 1.3545 1.2763 3.7372 3 2.4918 2.3793 95% 2.4414 98%
3 1.1184 1.3480 1.2775 3.7495
Specificity: No significant peak was present in the chromatogram of the blank 1 3.7377 3.5861 96% 3.6727 98%
4 1.0890 4% 1.3881 3% 1.2715 0% 3.7651 1%
at the retention time of the rosmarinic acid peak from CR solution. 2 3.7377 3.5364 95% 3.6498 98%
Introduction 5 1.0263 1.3739 1.2787 3.7655
150%
3 3.7377 3.5418 95% 3.6606 98%
Rosmarinic acid is a well-known natural product with health benefits [1], derived The rosmarinic acid peak in sample solutions 6 1.0797 1.4333 1.2774 3.7239
and standard solution had the same retention Table 2: Linearity/Range
from rosemary (Rosmarinus officinalis), lemon balm (Melissa officinalis), and Test Results Ruggedness: The same lots of all 4 products were tested (in duplicate) by a
other Lamiaceae. Accurate quantitation of rosmarinic acid is important for the time. The UV spectra of the rosmarinic acid
peak from sample solutions were identical to Linearity Standard Conc. second analyst on a different day. The RSD were within 10%.
quality control of related material. However, a limited number of papers have Levels (mg/ml)
Area
Accuracy: Nine (9) replicates of each sample at the normal test concentration
been published on the determination of rosmarinic acid in these botanicals by that of the rosmarinic acid peak from standard 353904 were prepared. Known quantities of the CR stock solution were added to the Table 6: Ruggedness Test Results
HPLC [2]. No report has been found on determination of rosmarinic acid in solution. 348831 sample solutions as described in Table 4 and 5. These spiked samples (three con-
Level 1 (25%) 0.0124591
Rosemary Lemon Balm Rosemary Lemon Balm
both rosemary leaf, lemon balm leaf and their extracts using HPLC. 352588 centrations and three replicates of each concentration) were analyzed according Leaf Leaf Extract Extract
Linearity/Range: Working standard solu- Replicate Performed By
716296 to the test method. At the same time, three (3) additional samples from the
tions containing about 0.013, 0.025, 0.050, 0.125 721739 Result Result Result Result
Level 2 (50%) 0.0249183 same lot of each sample were prepared and analyzed. The additional amounts
and 0.250 mg/mL of rosmarinic acid were pre- 725189 (g/100g) (g/100g) (g/100g) (g/100g)
Materials and Methods of rosmarinic acid found in the spiked samples versus the expected amount were
pared by making dilutions from the CR stock 1461559
calculated as %Recovery. All spiked recoveries were within 90–110%. 1 0.9981 1.4222 1.2812 3.7864
Material: Rosmarinic Acid, Chemical Reference (CR), NIFDC, Catalog #111871, Lot solution at concentration (1.2459 mg/mL of ros- Level 3 (100%) 0.0498365
1465605

#111871-201706. Standard Preparation: 5.0 mg of rosmarinic acid chemical reference marinic acid). Three (3) replicate injections 1451784
Table 4: Accuracy Test Results for Rosemary Leaf and Lemon Balm Leaf 2 1.0953 1.3545 1.2763 3.7372
3652372
was accurately weighted into a 100 mL volumetric flask. Added about 50 mL of 80% ethanol, were made for each of the five (5) solutions. The
3687826
swirled to dissolve. Diluted to volume with 80% ethanol. Sample Preparation: For raw peak areas of rosmarinic acid obtained for each Level 4 (250%) 0.1245914 Added Rosemary Leaf Lemon Balm Leaf 3 1.1184 1.3480 1.2775 3.7495
3687482 Analyst 1
material, a sample with about 5.0 mg of rosmarinic acid was weighted into a 100 mL vol- solution were plotted against their correspond- 7451839 Spike level Sample #
umetric flask. Added about 70 mL of 80% ethanol, sonicated for 30 min, cooled to room ing theoretical concentration. Linear regression (mg) Found Found 4 1.0890 1.3881 1.2715 3.7651
7448898
Level 5 (500%) 0.2491827 Recovery Recovery
temperature. Diluted to volume with 80% ethanol. Filtered a portion of the sample through analyses on the five coordinates were performed. 7480454 (mg) (mg)
5 1.0263 1.3739 1.2787 3.7655
a 0.45µm syringe filter, discarded the first few drops of the filtrate and then filled into an The R2 of the linear curve was 0.99992. 1 1.2459 1.2401 100% 1.2818 103%
HPLC vial. For extract, a sample with about 5.0 mg of rosmarinic acid was weighted into 6 1.0797 1.4333 1.2774 3.7239
2 1.2459 1.3003 104% 1.3683 110%
a 100 mL volumetric flask. Added about 70 mL of water, sonicated for 10 min, cooled to 50%
room temperature. Diluted to volume with water. Filtered a portion of the sample through a 3 1.2459 1.3017 104% 1.3248 106% 7 1.2597 1.4686 1.2883 3.9193
0.45µm syringe filter, discarded the first few drops of the filtrate and then filled into an HPLC Analyst 2
1 2.4918 2.4366 98% 2.3788 95% 8 1.2778 1.4994 1.2028 3.8794
vial.
2 2.4918 2.5012 100% 2.7182 109%
100% RSD% 9% 4% 2% 2%
3 2.4918 2.548 102% 2.4069 97%
References 1 3.7377 3.6664 98% 3.8409 103%
[1] H Wang. “Determination of rosmarinic acid and caffeic acid in aromatic herbs by HPLC”. 2 3.7377 3.6924 99% 3.8144 102%
In: Food Chemistry 87.2 (2004), pp. 307–311. 150% Conclusion
[2] Ying Zhang et al. “Degradation Study of Carnosic Acid, Carnosol, Rosmarinic Acid, 3 3.7377 3.7368 100% 3.7344 100%
and Rosemary Extract (Rosmarinus officinalis L.) Assessed Using HPLC”. In: Journal
A simple and sensitive HPLC analytical method for determination of rosmarinic
of Agricultural and Food Chemistry 60.36 (2012), pp. 9305–9314. acid in rosemary leaf, rosemary leaf extract, lemon balm leaf and lemon balm
[3] Ai-Hua Liu et al. “Simultaneous quantification of six major phenolic acids in the roots leaf extract was validated. This method is reliable and suitable. This method
of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by demonstrated excellent coefficient of determination (R), precision, accuracy,
HPLC–DAD method”. In: Journal of Pharmaceutical and Biomedical Analysis 41.1
Figure 1
ruggedness, and specificity.
(2006), pp. 48–56.