Food Chemistry 132 (2012) 1040–1045

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Food Chemistry

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Short communication

Natural occurrence of citrinin in widely consumed traditional Chinese food redyeast rice, medicinal plants and their related products

Yan Li a,b, Yu-Chun Zhou a,c, Mei-Hua Yang a,⇑, Zhen Ou-Yang b

a Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, Chinab School of Pharmacy, Jiangsu University, Zhenjiang 212013, Chinac JiangXi University of Traditional Chinese Medicine, Nanchang 330004, China

a r t i c l e i n f o

Article history:Received 22 December 2010Received in revised form 5 October 2011Accepted 8 November 2011Available online 20 November 2011

Keywords:CitrininImmunoaffinity columnHPLC–FLDRed yeast riceMedicinal plantsLC–ESI–MS/MS

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.11.051

⇑ Corresponding author. Tel.: +86 10 57833277; faxE-mail address: yangmeihua15@hotmail.com (M.-

a b s t r a c t

Natural occurrence of citrinin in traditional Chinese food red yeast rice, medicinal plants and their relatedproducts has been investigated for the first time. Samples were extracted by methanol/water, cleaned-upwith an immunoaffinity column (IAC) and quantified by HPLC–FLD. The mean recoveries, spiking withcitrinin at levels ranging from 25 to 200 lg/kg, were 73.4–92.5%, and the coefficients of variations(CVs) were 1.4–7.9%. The limit of detection (LOD) was 0.8 lg/kg. Out of a total of 109 widely consumedsamples analysed, citrinin was detected in 31 samples (28%) ranging from 16.6 to 5253 lg/kg, all of themderived from 59 red yeast rice and related products. None of the remaining 50 medicinal plants sampleswas found to contain citrinin. The positive samples were further confirmed using LC–ESI–MS/MS.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Citrinin is a secondary toxic metabolite produced by several fil-amentous fungal species of Aspergillus, Penicillium and Monascus,originally isolated from Penicillium citrinum (Blanc, Loret, & Goma,1995; Cole, 1986). This mycotoxin commonly contaminates grains,food and feedstuffs, such as wheat, corn, oats, wheat bran and fruitjuices (Dietrich, Schmid, & Märtlbauer, 2001; Kononenko & Burkin,2008; Vrabcheva, Usleber, Dietrich, & Märtlbauer, 2000). Red yeastrice, a traditional Chinese food, is the fermented product fromsteamed rice by Monascus purpureus, and has been served as a nat-ural dietary supplement for thousands of years in some Asiancountries. The present researches have proved that red yeast riceimproves digestion and blood circulation. Red yeast rice and itsrelated products are accepted as functional foods or drugs or asdietary supplements, worldwide. Therefore, the presence of citri-nin in red yeast rice and its related products has already becomea threat to human health (Liu, Wu, Su, Chung, & Yu, 2005). Medic-inal plants and related products are generally used in China to pre-vent and cure human disease, with a long history. Like cereals andother foodstuffs, medicinal plants may be contaminated by thosefungi producing citrinin during the process of growth, harvest,transportation and storage, and citrinin may also be found in the

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: +86 10 62896288.H. Yang).

final products (Molinié, Faucet, Castegnaro, & Pfohl-Leszkowicz,2005).

Citrinin has been known to be nephrotoxic, hepatotoxic andcarcinogenic to humans and animals. It has been reported that, likeochratoxin A (OTA), citrinin is a potential risk factor for human Bal-kan endemic nephropathy (BEN), originally described as a chronictubulointerstitial kidney disease in south-eastern Europe (Bamias& Boletis, 2008; Castegnaro, Chernozemsky, Hietanen, & Bartsch,1990; Krejci, Bretz, & Koechel, 1996). At present, the State Foodand Drug Administration (SFDA) of China has enforced a limit ofcitrinin with level of 50 lg/kg in red yeast rice-based functionalfoods.

Various methods have been developed to determine citrinin in dif-ferent kinds of matrices. These include fluorometric analysis (Tran-tham & Wilson, 1984), thin-layer chromatography (TLC) (Betina,1985), enzyme-linked immunosorbent assay (ELISA) (Abramson, Usle-ber, & Märtlbauer, 1995, 1996), gas chromatography (GC), gas chroma-tography–mass spectrometry (GC–MS) (Shu & Lin, 2002) and liquidchromatography–mass spectrometry (LC–MS) (Rasmussen, Storm,Rasmussen, Smedsgaard, & Nielsen, 2010; Tuomi, Johnsson, Hintikka,& Reijula, 2001). The most common analytical method for citrinin ishigh-performance liquid chromatography, with fluorescence detec-tion (HPLC–FLD).

Many approaches have been utilised to purify samples contain-ing citrinin. Both liquid–liquid extraction (LLE) and solid-phaseextraction (SPE) are the typically applied clean-up methods. SPE

Fig. 1. HPLC chromatograms: (A) blank red yeast rice (No. 1); (B) citrinin standard (4 ng/ml); (C) fortified red yeast rice (No. 1) at 200 lg/kg; (D) red yeast rice (No. 26,naturally contaminated with citrinin at 178 lg/kg).

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has commonly been used because of its superior precision andrecoveries, with C8, polyamide columns, molecularly imprintedpolymers (MIPs) (Guo et al., 2010; Meister, 2004; Tuomi et al.,2001). There are also LC–MS/MS-based methods that attempt toanalyse crude extracts (Spanjer, Rensen, & Scholten, 2008; Sulyok,Krska, & Schuhmacher, 2007). However, LLE and some SPE meth-ods are complicated in their procedure, and many interfering peaksmay exist in the chromatogram. In contrast, immunoaffinity

column (IAC) clean-up has been widely used for most of the myco-toxins because of its high specificity for antibodies of a single toxin(Alvito, Sizoo, Almeida, & van Egmond, 2010; Czerwiecki,Wilczynska, & Kwiecien, 2006; He, Li, & Zhou, 2009). In this study,a simple and sensitive HPLC–FLD method based on IAC clean-up isdeveloped, to determine citrinin in red yeast rice, medicinal plantsand their related products in order to evaluate natural occurrenceof citrinin in these widely consumed samples in China.

Table 1Mean recoveries from blank samples spiked with citrinin at different levels (n = 3).

Spiking levels (lg/kg) Mean recovery, RSD (%)

American genseng Coix seed Red yeast rice Red yeast rice product

25 89.3 (3.3) 91.0 (4.7) 88.0 (6.0) 89.4 (7.9)50 82.4 (3.4) 92.5 (2.6) 85.7 (2.6) 81.2 (6.6)

100 79.2 (1.4) 81.0 (3.4) 89.0 (3.0) 73.4 (4.5)200 86.5 (4.8) 87.0 (1.4) 84.9 (4.9) 77.8 (2.1)

Table 2Citrinin levels in 109 analysed samples (n = 3).

Samples No. Incidence Conc. range (lg/kg)a Median conc. (lg/kg) Positive (%)

RFAs 1–11 8/11 127–4960 230 73RMMs 12–30 19/19 18.2–5253 1409 100RFMPs 31–59 4/29 16.6–62.5 24.8 14

TCMPsRoot and rhizomes 60–90 0/31 –b – –Fruits 91–97 0/7 – – –Seeds 98–101 0/4 – – –Others 102–105 0/4 – – –TCPPs 106–109 0/4 – – –Total 1–109 31/109 16.6–5253 1060 28

a Concentration.b <LOD; RFAs, red yeast rice food additives; RMMs, red yeast rice medicinal materials; RFMPs, red yeast rice functional food and medicine products; TCMPs, traditional

Chinese medicinal plants; TCPPs, traditional Chinese medicine patent prescriptions.

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2. Materials and methods

2.1. Materials and chemicals

Standard (citrinin) was ordered from Fermentek (Jerusalem, Is-rael). Acetonitrile (HPLC grade) was purchased from Fisher Scien-tific (Waltham, USA). CitriTest™ immunoaffinity columns wereordered from Vicam (Watertown, MA, USA). Microfibre filters werepurchased from Vicam (Watertown, MA, USA). Quantitative filterpapers were supplied by Hangzhou Special Paper Industry Co.,Ltd. (Zhejiang, China). Tween-20 was analytical grade and pur-chased from Shantou Xilong (Guangdong, China). Other reagentswere of analytical grade and water was double-distilled.

2.2. Samples collection

In total, 109 widely consumed samples were purchased fromdifferent markets of China during 2008–2009, comprising 5 catego-ries as follows: red yeast rice food additives (No. 1–11, RFAs,n = 11); red yeast rice medicinal materials (No. 12–30, RMMs,n = 19); red yeast rice functional food and medicinal products(No. 31–59, RFMPs, n = 29); traditional Chinese medicinal plants(roots and rhizomes, No. 60–90; fruits, No. 91–97; seeds, No. 98–101; other species, No. 102–105, TCMPs, n = 46); traditional Chi-nese medicine patent prescriptions (No. 106–109, TCPPs, n = 4).All of the samples were ground to particles of less than 1 mm witha pulveriser and mixed, and stored with refrigeration at �20 �Cbefore analysis.

2.3. Apparatus

A Shimadzu technologies LC-20AT series system equipped withFLD detector (Shimadzu, Kyoto, Japan) was used. The analytical col-umn was a Phenomenex (Torrance, CA, USA) Gemini 5u C18

reversed-phase column (4.6 mm � 250 mm, 5 lm). The columntemperature was set at 25 �C. The excitation wavelength was setat 331 nm and emission wavelength at 500 nm. The mobile phase

consisted of 0.1% phosphoric acid with water/acetonitrile (55:45,v/v) at a flow-rate of 1.0 ml/min. The injection volume was 50 ll.

2.4. Preparation of standard and reagent solutions

Citrinin stock solution was prepared by dissolving the solid tox-in in methanol (100 lg/ml). The solution was stored at �20 �C in ameasuring flask and left at room temperature before use. PBS buf-fer solution was prepared by dissolving 8.0 g of sodium chloride,1.2 g of sodium hydrogen phosphate, 0.2 g of potassium chlorideand 0.2 g of potassium dihydrogen phosphate in 900 ml of distilledwater. The pH was adjusted to 7.0, and it then was diluted to1000 ml with water.

2.5. Sample extraction and IAC clean-up

Finely homogenised and ground sample (1.0 g) and 5 ml ofmethanol/water (70:30, v/v) were placed in a centrifuge tube.The sample was extracted for 3 min by a high speed vortex oscilla-tor (Changzhou, China), then centrifuged at 6000 rpm for 20 min,3 ml of clear supernatant was diluted to 30 ml by PBS buffer solu-tion. After filtration through a microfibre filter, the filtrate wasreserved for the IAC clean-up procedure below.

The immunoaffinity column was pre-washed with 2 ml of PBS buf-fer solution before the addition of the sample extract. Then 2 ml of thepreceding obtained filtrate (by pipette) was passed through the columnat a flow rate of about 1 drop per 5 s; 10 ml of PBS–Tween 20 (0.05%)solution, recommended by Vicam, were added to wash the remainingnonspecific adsorbing impurities. Finally, the target object citrininbound to the column was eluted by 2 ml of methanol/0.1% phosphoricacid aqueous solution (70:30, v/v) and the eluate was transferred to anautomatic sampling amber vial for the further HPLC analysis.

2.6. LC–ESI–MS/MS confirmation

A Shimadzu LC-20AD series HPLC system (Shimadzu, Kyoto, Ja-pan) coupled to an Applied Biosystems API-4000 mass spectrometer

Fig. 2. LC/MRM-chromatograms: (A) product-ion spectra of citrinin for the determination of the MRM-transitions; (B) extracted ion current (XIC) chromatograms of citrininstand solution (1 ng/ml) with MRM mass transitions (MT) 251.1–233.0 and 251.1–205.1; (C) XIC of positive sample (No. 26) with MT 251.1–233.0 and 251.1–205.1.

Y. Li et al. / Food Chemistry 132 (2012) 1040–1045 1043

(Concord, Ontario, Canada), via an electrospray ionisation (ESI)source, was used for the analysis. Applied Biosystems Analyst soft-ware, version 1.4.2 package was used to control the LC–MS/MS sys-tem and for data acquisition and processing. Citrinin was separated

on a Phenomenex Gemini C18 column (20 mm � 2.00 mm, 3 lm,Torrance, CA, USA) maintained at 20 �C. Mobile phase A (100%H2O) and B (100% acetonitrile) both contained 0.1% formic acidand the flow-rate was set at 0.5 ml/min. The injection volume was

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10 ll. A fast gradient was used for the confirmation analysis: 0.01–0.60 min, 90% A; 0.60–1.20 min, from 90% A to 5% A; 1.20–2.50 min, 5% A; 2.50–2.51 min, from 5% A to 90% A; 2.51–4.00 min,90% A. A two phase switching valve was used to divert the pre-eluentfrom entering the ion source.

The mass spectrometer was operated in the positive ESI mode,with multiple reaction monitoring (MRM) at unit resolution. Nitro-gen was used as the nebuliser, heater and curtain gas, as well as thecollision activation dissociation gas. Two precursor-to-product iontransitions were simultaneously monitored at m/z 251.1 ? 233.0and m/z 251.1 ? 205.1 for citrinin. Optimum parameters were asfollows: nebuliser (GS1), heater (GS2) and curtain gas flow rates60, 60 and 12 units, respectively; ionspray needle voltage5500 V; heater gas temperature 550 �C; collision gas N2; decluster-ing potential 56 V both for m/z 251.1 ? 233.0 and m/z251.1 ? 205.1; collision energies 23 eV for m/z 251.1 ? 233.0;36 eV for m/z 251.1 ? 205.1; cell exit potential 12 V.

3. Results and discussion

3.1. Method validation

The calibration curve was obtained by analysing calibrationstandards, in the range 0.1–1000 ng/ml, on the eight calibrationstandards (0.1, 0.5, 1, 5, 10, 100, 400, 1000 ng/ml). The standardsolutions were prepared by the serial dilution of the stock solu-tions. The correlation coefficient is above 0.9999 and fulfils therequirement for a linear method. The method was considered tobe linear in the studied range. Regression analysis was performedfor peak area (Y) and the quantity of standard solution injected(X), using the following calibration curve equation, Y = 4650.7X + 8121.1.

The recovery experiments were determined by spiking blanksamples with citrinin at four different levels (25, 50,100 and200 lg/kg) to ensure the accuracy of the method. The data forrecovery were calculated by comparing the absolute responses ofcitrinin from the spiking samples to the absolute response ofcitrinin standard solutions. Four sorts of samples, red yeast rice,red yeast rice related product, American ginseng and coix seed,were selected for the determination of recoveries, since these sam-ples represented different matrices, and also had no interferingpeaks in their HPLC chromatograms (Fig. 1). The mean recoveries,spiking with citrinin, ranged from 73.4% to 92.5% and the RSDswere 1.4–7.9% within the spiking levels of 25–200 lg/kg (Table 1).

Limits of detection and quantification were defined as the low-est concentration giving a response of three and ten times the aver-age of the baseline noise obtained from diluted spiked blanksamples. By this approach, the LOD of citrinin was calculated as0.8 lg/kg, based on a signal-to-noise (S/N) ratio of 3:1, and theLOQ was 2 lg/kg, based on a S/N ratio of 10:1.

3.2. Analysis of samples

The contents of citrinin in 109 analysed samples are summa-rised in Table 2. A few of the analysed samples with high contentcitrinin (>500 lg/kg) were discovered during the experiment; thusthe citrinin contents, in these samples extracts added to the col-umn, were higher than the column capacity (20 ng). Aimed at thesesamples, optimised clean-up procedures were performed by dilut-ing the solution added to the column to proportional concentra-tions by PBS buffer solution, to avoid overload of the IAC.

According to the method described above, citrinin was detectedin 31 samples (28%), ranging from 16.6 to 5253 lg/kg. All of thesepositive samples belonged to 59 red yeast rice and related prod-ucts, and none of them belonged to the rest of the 50 TCMPs and

TCPPs. Twenty seven of 30 (90%) red yeast rice samples, includingRFAs and RMMs, had citrinin concentration ranging from 18.2 to5253 lg/kg, the highest levels of all positive samples. Only 4 of29 (14%) red yeast rice related products (RFMPs) had citrinin,and the maximum content was 62.5 lg/kg. The high incidence ofcitrinin in red yeast rice indicated that the red yeast rice commod-ities marketed in China were widely contaminated. Red yeast riceis the fermented product of steamed rice by Monascus species.Some strains of Monascus could produce citrinin and might con-taminate red yeast rice and related products. Efforts to decreasecitrinin content in red yeast rice should be made (according tothese results). Therefore, to avoid citrinin contaminating red yeastrice and related products, or to keep citrinin concentration low,screening of some strains of Monascus with non-producing/low-producing citrinin is very important.

3.3. Confirmation result

Two parameters from LC–ESI–MS/MS analysis were used toconfirm the positive samples: retention times and fragment ionsof the analytes of interest. The characteristic product ions m/z233 ([M+H–H2O]+), m/z 205 ([M+H–H2O–CO]+) of the precursorion, and m/z 251 ([M+H]+) for citrinin, can be observed againstthe noisy background. Therefore, the transitions of m/z251.1 ? 233.0 and 251.1 ? 205.1 were used to confirm the pres-ence of citrinin. The intensity ratios of the two extracted ion cur-rent chromatograms (XICs) may also used as an additionalcriterion to confirm a positive result. The ratio observed in thesample has to agree with that of the citrinin standard within cer-tain limits (EC, 2002). The retention time of citrinin of the positivesample was 2.18 min and was consistent with that of citrinin stan-dard. The results obtained from LC–ESI–MS/MS in Fig. 2 indicatethat the positive sample contained citrinin.

4. Conclusion

A simple and accurate HPLC–FLD method, based on IAC clean-up, has been developed to determine citrinin in different matrices,including red yeast rice, medicinal plants and their related prod-ucts. It was found that most of the red yeast rice materials of dif-ferent origins marketed in China were contaminated with citrininat different levels. In the context of food and drug quality andsafety, further studies are necessary to decrease citrinin contentin red yeast rice. This method can be used for monitoring citrininlevels in widely consumed traditional Chinese food red yeast rice,medicinal plants and their related products to prevent hazard tohuman health.

Acknowledgements

This work was supported by the Important New Drug ResearchProject of the Ministry of Science and Technology of China(2009ZX09502-025) and the Scientific Research Project of Tradi-tional Chinese Medicine Vocation in 2008 (200807042).

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