Development of a Validated HPLC/Photodiode Array Method for the Determination of Isomenthone in the Aerial Parts of Ziziphora tenuior L.

Background: Ziziphora tenuior L. known as Kakuti in Persian, is used in traditional medicine for fever, dysentery, uterus infection and as an analgesic. It is used also in the treatment of gastrointestinal disorders as carminative, or remedy of diarrhea or nausea. Major components of plant essential oil including pulegone, isomenthone, thymol, menthone, and piperitone are suggested to be responsible for the mentioned medicinal properties. Objectives: In the present study, a normal high performance liquid chromatography (HPLC)/photodiode array validated method for quantification of isomenthone, one of the major constituents of Ziziphora, was established for the first time with a simple, rapid and accurate method. Materials and Methods: HPLC analysis was done on a Waters system, equipped with 515 HPLC pump and waters 2996 photodiode array detector. The column was a Nova-Pak Silica (3.9 × 150 mm), and Empower software was used for the determination of the compounds and processing the data. The method was validated according to USP 32 requirements. Results: A selective method for the resolution of isomenthone from two nearest peaks, thymol, and carvacrol was obtained with gradient system of hexane (A), and hexane: ethyl acetate (9:1) (B), starting with A: B (100:0) for 2 minutes, then 0-20% B in 5 minutes, A:B (80:20) for 5 minutes, then 20-30% B in 3 minutes, 30-100% B for 5 minutes, A:B (0:100) for 4 minutes following with equilibrating for 10 minutes. The flow rate was 1 mL/min at 22˚C and the injection volume for the standards and the samples was 20 μL. The retention time for isomenthone was found to be 7.45 minutes. The regression equation was y = 143235x - 2433 with the correlation co-factor R2 = 0.9992 and the percent recovery of 65.4 ± 3.85%. The sample obtained from 5 g of Z. teniour dried powder in 6 mL extract was standardized to contain 1.14 ± 0.030 μL/mL isomenthone which is equivalent to % 1.37 μL/g of the dried powdered plant. Limit of detection (LOD) and Limit of Quantification (LOQ) were 0.037, and 0.122 μL/mL determined by using the formula based on the signal to noise ratio. Conclusions: Due to this fact that plant extracts may cause irreversible damages to the capillary GC columns, using validated HPLC method for the analysis of these compounds in cruse plant extracts is recommended.