Validated high performance liquid chromatographic (HPLC) method for analysis of zerumbone in plasma

Laboratory of Cancer Research MAKNA-UPM, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Selangor DE, Malaysia. Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 Serdang, Selangor DE, Malaysia. Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University Putra Malaysia, 43400, Serdang, Selangor DE, Malaysia. Department of Biochemistry and Molecular Biology, Faculty of Medicine and Health Sciences, University of Sana’a, Sana’a, Yemen.


INTRODUCTION
Zingiber zerumbet (L.) smith, known as lempoyang, is a member of the family Zingiberaceae, used in the traditional medicine as a cure for swelling, sores, loss of appetite and worm infestation in children (Somchit and Nur-Shakirah, 2003).In some Southeast Asian countries, the rhizomes of the plant are employed in traditional medicine as anti-inflammation, while the young shoots and inflorescence are used as condiments (Murakami et al., 2002).This plant has been shown to have anti-tumor (Sakinah et al., 2007), anti-inflammatory (Murakami et al., 2002) and cyclooxygenase-2 suppressant properties *Corresponding author.E-mail: ahmadbstmm@yahoo.com or adelalzubairi@hotmail.com.Tel: +603-89462124 Fax: +603-89462101.(Tanaka et al., 2001).Zerumbone (ZER) is a bioactive crystalline monocyclic sesquiterpene derived from zingiber zerumbet rhizomes.It exhibits variety of interesting reactions, such as region-and regeoselective conjugates additions, transannular ring contraction, cyclization and several regeospecific reactions which cleave the 11-membered ring (Kitayama et al., 1999(Kitayama et al., , 2001;;Ohe et al., 2000).This bioactive component has its unique structure, with crossconjugated ketone in an 11-membered ring, as well as remarkable biological activities.It has been reported that ZER constitutes about 37% of Z. zerumbet (Matthes et al., 1980;Sakinah et al., 2007).Moreover, this compound showed a potential candidate for the development of anticancer treatment of natural origin.
ZER has been previously identified as distinct suppressor of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation in Raji cells (Murakami et al., 1999).Murakami et al. (2004) reported that ZER inhibits the proliferation of colon and skin cancer cells through apoptosis, while having less effect on normal cells proliferation (Murakami et al., 2002;Murakami et al., 2004).Recently, ZER was reported to suppress the development of cervical intraepithelial neoplasia in mouse (Abdul et al., 2008).ZER has gained a great attention due to its activity towards many diseases in vitro and in vivo.Very recently, Sung et al. (2009) reported ZER as modulator for osteoclastogenesis induced by Receptor Activator for Nuclear Factor κ B Ligand (RANKL) and breast cancer (Sung et al., 2009).In addition ZER was reported to effectively suppress mouse colon and lung carcinogenesis through multiple modulatory mechanisms (Kim et al., 2009).
However, to the best of our knowledge, no chromatographic method has been described in literature for ZER determination in human plasma as well as other biological fluids.Here, we present for the first time an easy, HPLC method for determination of ZER in human plasma which requires minor laboratory efforts.This method is one step plasma protein precipitation followed by injection into HPLC with Photodiode Array (PDA) detector which is available in most analytical laboratories.The method will reduce the experimental work to almost 50%.

Chemicals
ZER was extracted from Zingiber zerumbet rhizomes obtained from the wet market in Kuala Lumpur, Malaysia.The purity of the extracted ZER was > 98%.Structurally similar, α-Humulene (HUM) (Figure 1 (A) and (B)) was used as internal standard (IS) and was purchased from Sigma Chemical Co.(Street Louis, MO, USA).HPLC grade acetonitrile, methanol and potassium dihydrogen orthophosphate monobasic were purchased from Fisher Scientific (USA).Ultra pure water was used throughout the experiment.

Standard preparation and quality control
Stock and working solutions of ZER and HUM (100 µg/ml) was prepared by dissolving in acetonitrile.Standard solutions of ZER in human plasma were prepared by spiking the diluted stock solution, to give the final concentrations of 2, 4, 6, 8, 10 and 15 µg/ml.The internal standard HUM solution 50 µg/ml was also prepared.

HPLC apparatus and chromatographic conditions
HPLC system consist of Alliance separation module model e2695 with PDA detector, the signals were processed by Empower™ software (Waters, Milford, MA, USA).The mobile phase composed of acetonitrile: methanol: 0.01 M potassium dihydrogen orthophosphate (25: 55: 20).The analytical column used was C18 (Symmetry, 250 X 4.6 mm ID, 5 µm particles size) at an ambient temperature.The elute was monitored by PDA detector at a flow rate of 1 ml /min in wavelength of 254 nm.

Sample preparation
Ten microliters of internal standard solution 50 µg/ml were added to 150 µl plasma sample in microcentrifuge tube.The tube was vortex for 1 min and 200 µl of acetonitrile was added and vortexed for 2 -4 min to precipitate the plasma protein, then the sample was centrifuged at 10000 rpm for 15 min.The supernatant was withdrawn in 300 µl insert vials and 10 µl was injected into HPLC system.

Validation procedure
The validation parameters obtained were specificity, linearity, accuracy, precision, stability and robustness.The method was validated according to FDA guidelines.

Specificity
Specificity was obtained by comparing chromatograms of 6 different batches of blank plasma obtained from 6 different subjects and plasma samples spiked with ZER and HUM.

Linearity and lower limit of quantification (LOQ)
Calibration plots were constructed from blank plasma spiked with HUM and six concentrations of ZER (2 -15 µg/ml).The linearity of each calibration curve was determined by plotting the peak area ratio(y) of ZER to HUM vs the nominal concentration (x) of ZER.The calibration curves were constructed by weighted (1/x) leastsquare linear regression method.The lower limit of quantitation (LOQ) is the lowest concentration of the analyte that can be determined with acceptable precision and accuracy.

Precision and accuracy
The intra-day and inter-day precision and accuracy of the method were determined by percent coefficient of variation (% CV) and percentage relative error (% RE) respectively, according to the reported guidelines (FDA guidance for industry, 2001; Shah et al., 2000).Quality Control (QC) samples containing 3, 7 and 12 µg/ml concentrations were spiked for determination of precision and Where: CC = Calculated concentration, AC = Added concentration

Recovery
The recovery of ZER was determined for QC samples at the three samples pools 3, 7, 12 µg/ml.Three replicates of each QC sample were treated as mentioned in the sample preparation previously and injected into HPLC system.The extraction recovery was calculated by the following equation: Recovery = [(peak area after extraction) / (peak area after direct injection)] X 100

Stability
The stability studies were carried out at the three different concentrations of the QC values (3, 7, 12 µg/ml) stored at room temperature (bench top stability) was evaluated for 24 h and compared with freshly prepared extracted samples.Freeze and thaw stability for three cycles was determined at the same concentrations (QC), by thawing at room temperature for 2 -6 h and then refreezing at -20°C for 12 -24 h.The long term stability of ZER at the same QC values was assessed by carrying out the experiment after 15 days of the storage in -30°C.The concentration of ZER after each storage period was related to the initial concentration as determined for the samples that were freshly prepared.

Robustness
The robustness of a method is its capability to remain unaffected by small, but deliberate variations in method parameters.The effect of percent organic strength as well as the buffer on resolution was studied by varying acetonitrile from -1 to +1%, methanol from -2 to +2% and 0.01 M KH2PO4 from -1 to +1% while the other mobile phase components were remained constant as stated in chromatographic condition.The flow rate was changed by ± 0.2 units while the other mobile phase components were remained constant as stated in chromatographic condition.The column temperature was also studied in such a way that the temperature was studied at ambient, 25 and 30°C.

RESULTS AND DISCUSSION
The chromatographic conditions and sample preparation for the proposed analytical procedure were optimized to be suitable for preclinical pharmacokinetic studies.The main challenge here is to use the smallest plasma volume as much as you can in order to avoid the risk of contamination and poor recovery as well.Unlike solid phase extraction for purification of plasma samples for HPLC analysis that needs more plasma volumes (Cao et al., 2008), protein precipitation by acetonitrile is simple, not tedious and less risky for the analyst.As shown in the chromatograms of human plasma (Figure 2), the retention times were 4.5 and 8.89 min for HUM and ZER respectively, with a total run time of less than 10 min.The analytical process of ZER and HUM were resolved with good symmetry.No interfering peaks were observed in individual blank plasma at the retention time of ZER and HUM, thus confirming the specificity of the method.System suitability parameters for the method were as follows: the theoretical plates of ZER and HUM were 8530, 4608, respectively.Tailing factors was less than 1.6 for both ZER and HUM and resolution between ZER and HUM was 11.6.The peak area ratio of ZER to HUM was used for the quantification of ZER in human plasma samples.Figure 3, the mean of five calibration curves made over a period of 14 days, each calibration curve originating from a new set of extraction.The mean calibration curve was linear in the concentration range of 2 -15 µg/ml and equation of the six points was y = 0.1948X -0.0862 with correlation coefficient (r) of 0.9978.The accuracy and precision were evaluated with QC samples at concentrations of 3, 7 and 12 µg/ml.The intra-day precision (expressed as percentage error RE) was deter-mined by the analysis of three replicates of QC samples at three different concentrations.The inter-day accuracy and precision were determined on three different days and the results were shown in Table 1.The intra-day and inter-day of the QC values were satisfactory with C.V% less than 10% and accuracy with RE within ±3%.
The lower LOQ was calculated by determining the concentration of four spiked calibration standards and was found to be 2 µg/ml for ZER in human plasma with CV of less 20% and the accuracy of 88 -112%.The limit of detection (LOD) was determined to be 1.5 µg/ml based on signal to noise ratio (s/n) ratio of 3:1.The extraction recovery was determined by standard addition at three different concentrations (3, 7 and 12 µg/ml) for ZER and one concentration (10 µg/ml) for HUM and was found to be 95 ± 3, 97 ± 1 and 96 ± 2 for ZER and 92 ± 3 for HUM, Table 2.The recovery of ZER using the described    method was consistent and efficient.Analysis of the stock solution was performed at 200 µg/ml and after storage for two weeks at 4°C and at room temperature for 24 h, more than 99% of ZER remained unchanged, based on the peak areas in comparison with freshly prepared solution of ZER (200 µg/ml).Thus, ZER in standard solution was stable at least 14 days when store at 4°C and for 24 h at room temperature.Bench top stability of ZER was investigated at the concentration of 3, 7 and 12 µg/ml and the results revealed that the ZER in plasma was stable for at least 24 h with average percentage of 96, 95 and 88% respectively.Repeated freezing and thawing (three cycles) of plasma samples spiked with ZER at three levels (3, 7, 12 µg/ml) showed a mean percentage concentration of 97, 94 and 90%, respectively.Long term stability of ZER in plasma at -30 was also performed after 15 days of storage at three levels (3, 7, 12 µg/ml), which showed mean percentage concentration of (non-tested, vial was broken), 93, 87% respectively.The results of the stability study was presented in Table 3 and indicated that ZER was stable in the studied conditions.
The robustness results are shown in Table 4.It can be seen that the chromatographic conditions in accordance with established value (Shah et al., 2000).A change in mobile phase composition (acetonitrile 25 ± 1% v/v), (methanol 55 ± 2% v/v), (0.01 M KH 2 PO 4 20 ± 1% v/v), flow rate (1 ± 0.2 units) and column temperature (ambient, 25 and 30°C) had no impact on chromatographic performance.The tailing factor for both ZER and HUM was less than 2 and the two analytes (ZER and HUM) were well separated under the conditions carried out.The resolution between ZER and HUM was ranged 9.0 -14.6.Considering the result of modifications in the system suitability parameters and the specificity of the method, it could be concluded that the method conditions are robust.

Conclusion
The newly developed HPLC method for determination of ZER in micro-volumes human plasma (| 1.5 ml) was found to be simple, accurate, specific and robust.The method consisted of one step plasma protein precipitation using acetonitrile, followed by chromatographic separation in photodiode detector.No interference peaks were observed at the retention time of both ZER and HUM.This method could therefore be recommended for in vivo analysis of ZER for preclinical pharmacokinetic study and may also be applied for the estimation of ZER in other biological fluids such as serum and tissues after partial validation.

Table 1 .
Intra-and Inter-day precision and accuracy of ZER in human plasma.

Table 2 .
ZER recovery and accuracy of the assay.

Table 3 .
Stability of ZER in three different concentrations at three different conditions (bench top, freezing and thawing cycles and long term storage).
d Not tested.

Table 4 .
The robustness data of the assay method.