Simple and Sensitive Determination of Citrinin in Monascus by GC-Selected Ion Monitoring Mass Spectrometry

Abstract

IntroductionCitrinin, a fungal metabolite, was first isolated from Penicillium citrinum in 1931 1 and has been detected as a natural contaminant of wheat, rye, barley, oat and Monascus. In fact, the mycotoxic citrinin is a secondary metabolite, which is produced by several fungal species of the genera Penicillium and Aspergillus. This mycotoxin, which could be present either naturally or artificially, at enhanced levels, may cause a public health problem, 2-5 such as the "yellowed rice" problem in Japan. Thus far, several TLC multi-mycotoxin methods have been proposed, but they exhibit relatively low sensitivities. [6][7][8] Reversed-phase HPLC methods with either UV absorbance or fluorescence detection have also been applied. [9][10][11] At this time, GC/MS is currently a reliable method of detection in many fields. However, the majority of mycotoxins are not volatile and must be derivatized in order to make them amenable to GC. 12 Even though several derivatization methods have been developed, this procedure is frequently time-consuming and is sometimes lacking in specificity.To overcome these limitations, a cold on-column injection technique coupled with GC/MS was developed for the direct analysis of metabolites produced by several Fusarium organisms without any chemical derivatization. 13In this study, we report on a new and highly sensitive method for the detection of citrinin in Monascus using GC-selected ion monitoring (SIM) mass spectrometry. This method, which does not require a derivatization procedure, was successfully applied to the analysis of citrinin in commercial Monascus products. Experimental Citrinin standard and reagentsCommercially pure citrinin [IUPAC: (3R,4S)-4,6-dihydro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid] and chloroform (99 + %) were purchased from Sigma (St. Louis, MO, USA). Six commercial samples of Monascus red rice were acquired from various drugstores in Taipei, Taiwan. ApparatusA gas chromatograph (GC 5890 Hewlett-Packard, Avondale, PA, USA) equipped with a mass spectrometer (Hewlett-Packard 5973 mass selective detector) was used. A capillary column (30 m × 0.25 mm i.d.) with a piece of HP-5MS (cross-linked 5% PH ME siloxane) bonded stationary phase film 0.25 µm thickness (Agilent Technologies, USA) was used. The inlet temperature was maintained at 200˚C. The column oven was held at 70˚C for 3 min, then programmed from 70 -280˚C at 15˚C/min and, finally, held for 5 min. Helium carrier gas was used at a constant flow-rate of 1 mL/min without splitting mode. Data were collected using the Hewlett-Packard Chem-Station software. The mass spectrometric conditions were as follows: SIM mode; ionization energy, 12 eV; ion source temperature, 230˚C. The mass selective detector was operated in the SIM mode at a scan rate of 1.25 scans per second. Liquid-liquid extraction procedureMonascus samples were finely ground and 2 g of the resulting powder was extracted with 2 mL of chloroform. After 15 min of sonication and a 5 min centrifugation at 5000 rpm at room tempe...

Review on the qualitative and quantitative analysis of the mycotoxin citrinin

“…2), which showed much weaker cytotoxicity than that of citrinin (Hirota, Menta, Yoneyama, & Kitabatake, 2002). It was found that after boiling in water, concentration of citrinin in Monascus was dramatically decreased; 20 minutes of heating could decrease the concentration of citrinin by 50% (Shu & Lin, 2002). These facts indicated that citrinin was unstable and thermolabile in aqueous solution.…”

“…If mycotoxins are sufficiently volatile at the column temperature, or can be converted into volatile derivates, GC technique can be used for their determination. Recently, a new method for the qualitative and quantitative analysis of citrinin in Monascus by gas-chromatographyselected ion monitoring (SIM) mass spectrometry was developed (Shu & Lin, 2002). GC separation of citrinin in Monascus extract was achieved without the need for chemical derivatization, and could be detected as a single peak when the SIM mode selected five prominent fragmentations (m/z of 220, 205, 177, 105 and 91).…”

An Overview of Conventional and Emerging Analytical Methods for the Determination of Mycotoxins

“…Internal standards nitrobenzene [129] or 13 C-labeled patulin [180] were proposed. Finally, a GC-MS method without previous derivatization was proposed for citrinin [237]. …”

Toxicological Properties of Citrinin

“…These new toxins are CTN H 1 and CTN H 2 (12). The concentration of CTN in the extract of Monascus decreases by 50 % after boiling in water for 20 minutes, which proves that CTN is thermally unstable in aqueous solution (13).…”