A Validated RP-HPLC Method for the Determination of Citrinin in Xuezhikang Capsule and other Monascus-Fermented Products

Citrinin is a toxic product usually produced during the Monascus fermentation. The presence of citrinin in xuezhikang capsule has been a concern due to its ingredient which is derived from monascus-fermented rice. A rapid and sensitive RP-HPLC method with fluorescence detection at λex = 331 nm and λem = 500 nm for analysis of citrinin in Monascus-fermented products was developed to analyze citrinin in Monascus-fermented products. The chromatography was performed with mobile phase containing acidified water and acetonitrile. The calibration curve was linear (r = 0.9999) over a range of 0.01070.537 μg/mL. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.187 ng/mL and 0.6 ng/mL respectively. The analysis of xuezhikang capsules using the developed method suggested that the product does not contain detectable citrinin and the result has been further confirmed using independent LC-MS/MS analysis. The proposed method has also been applied to analyze 11 samples of other Monascus-fermented products. The results suggested that there were no detectable citrinin in 4 of the 11 samples, however citrinin with the levels between 0.10-594 ng/kg has been detected in the other 7 samples. It indicates that the proposed method can also be applied to carry out the quantitative detection of citrinin for other Monascus-fermented products.

In this work, we developed a selective and sensitive PR-HPLC method that can conveniently detect the lowest possible amount of 0.187 ng/mL citrinin, which is more sensitive than the recent report of 0.5 ng/mL detection limit 4 .Further, how much citrinin is lost in the pretreatment process was investigated by recovery studies.

Experimental
Citrinin was purchased from Sigma (Shanghai, China).Xuezhikang capsule (Xuezhikang) was produced by Beijing WBL Peking University Biotechnology Co., Ltd., China.The commercially available Monascus-fermented products were obtained from the local market.Acetonitrile and methanol were of HPLC grade and all other reagents were analytical grade.

Instrumentation
The chromatographic system consisted of Shimadzu LC-20A separations module and a Shimadzu RF-20A Fluorescence Detector.Fluorescence detection was performed with the Shimadzu RF-20A fluorescence detector with λ ex = 331 nm and λ em = 500 nm.A column of Aichrombond-AQ C 18 (250 mm × 4.6 mm i.d., 5 µm) was used and the column temperature was set at 25 o C.An Agilent 1200 HPLC separation system was used to deliver samples into a Bruker micrOTOF-QII mass spectrometer.

Preparation of standard solution
The standard stock solution was prepared by dissolving accurately weighed 5.37 mg of citrinin standard in 100 mL of methanol (final concentration, 53.7 µg/mL).Different calibration standards ranging from 0.01074, 0.0537, 0.1074, 0.2685 and 0.537 µg/mL were prepared by appropriate dilution of standard stock solution with methanol.

Preparation of sample solution
An assay sample (Xuezhikang or other Monascus-fermented products) was milled and passed through 80-mesh sieve.Accurately weighed 1.5 g of sample powder was transferred to a 15 mL PTEE centrifuge tube, and ultrasonic-extracted with 10 mL toluene-ethyl acetateformic acid = 7:3:1 for 20 min, 3 times.The combined extract solution was centrifuged to remove undissolved residue and the supernatant was evaporated under vacuum to afford citrinin-enrichment crude extract.The crude extract was dissolved with 10 mL methanol and filtered through a 0.45 µm membrane filter for detection.

Chromatographic conditions
The mobile phase A was acetonitrile and mobile phase B was acidified water (pH 2.5 adjusted with phosphoric acid).The gradient elution program has been developed and optimized for better, accurate and consistent results.The gradient eluting system was shown in Table 1.The injection volume was 20 µL and the flow rate was 1.0 mL/min.

Results and Discussion
The applied chromatographic conditions permitted a good resolution of citrinin in standard solution (A) and in sample solution (B) (Figure 2).The LC method was validated for the parameters reported below.Retention Time

Linearity
The calibration standards were chromatographed using the mobile phase, and the linearity of peak areas versus corresponding concentrations was studied from 0.01074 -0.537 µg/mL for citrinin.A linear response was observed over the examined concentration range.The results are tabulated in Table 2.

Limit of detection and limit of quantitation
The limit of quantitation (LOQ) was the lowest concentration of the sample assayed when the signal/noise ratio was at least 10:1.The limit of detection (LOD) was defined as a signal/noise ratio of 3:1.The LOQ and LOD were found to be 0.6 ng/mL and 0.187 ng/mL respectively.

Precision and recovery
The precision of the method was evaluated by analyzing the standard solution of 0.1074 µg/mL citrinin with five replicates.The results are shown in Table 3.The recovery tests were conducted to evaluate the extraction method aforementioned.They were performed by adding known amounts of standard stock solutions to the xuezhikang samples and preparing solutions according to the preparation of sample solution.The percentage of recovery was calculated by comparing the determined amount of citrinin standard with the added amount.The calculation of % recovery is tabulated in Table 4.

Detection of citrinin in Xuezhikang and other Monascus-fermented products
Natural occurrence of citrinin in Xuezhikang and other Monascus-fermented products (including red yeast rice and Monascus pigment) was studied.A total of 18 samples were analyzed by HPLC for citrinin.The results revealed that 11 samples were negative for containing citrinin and 7 samples were positive for containing citrinin with the levels between 0.10 and 594 µg/kg (Table 5).

LC-MS/MS analysis
Chromatographic separation was modified based on the HPLC condition.The same column was used and the mobile phase A was acetonitrile and mobile phase B was 0.4% formic acid.The gradient eluting system is shown in Table 6.The injection volume was 20 µL.The flow rate was 1.0 mL/min, with the flow rate split down to 0.25 mL/min into the MS source.

Conclusion
The proposed reverse phase HPLC method has been evaluated over the linearity, precision and recovery and proved to be convenient and effective for the detection of citrinin in Monascusfermented products as well as other routine quality control procedure.Under the reported HPLC conditions, a detection limit of citrinin achieved was as low as 0.187 ng/mL.Quantitative analysis of citrinin in xuezhikang using the proposed protocol suggested that there were no detectable citrinin and the result has been double-confirmed by LC-MS/MS.The method can also be applied for routine quality control analysis of citrinin in Monascus-fermented products.

Table 1 .
Gradient elution system in HPLC

Table 2 .
Results of the data analysis for the linearity of citrinin

Table 3 .
The result of precision assay

Table 4 .
The result of recovery analysis

Table 5 .
Detection results of citrinin in 18 samples

Table 6 .
Gradient elution system in LC-MS/MS detection