Analysis of Anka Pigments by Liquid Chromatography with Diode Array Detection and Tandem Mass Spectrometry

S. S. Teng 1'2. / W. Feldheim 1 lInstitute of Human Nutrition and Food Science, University of Kiel, Dtisternbrooker Weg 17-19, 24105 Kiel, Germany 2Current address: Department of Nutrition and Food Science, Fu-Jen University, Taipei, Taiwan

Key Words
Column liquid chromatography Thin layer chromatography Mass spectrometry detection Anka pigments

Summary
Anka is with Monascus sp. fermented rice, which shows a purplish red color. On rice grains Monascus purpureus DSM 1379 synthesized four anka pigments. Through extraction of anka with n-hexane and methanol in succession and through repeated crystallizations from absolute ethanol anka pigments were crystallized into two fractions: orange anka pigments (rubropunctatin and monascorubrin) and yellow anka pigments (monascin and ankaflavin). The treatment of two orange anka pigments with aqueous ammonia solution gave red nitrogen analogues (rubropunctatamine and monascorubramine). The above six pigments were separated into each pigment by two dimensional thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCIMS-MS) and direct probe mass spectrometry (MS) were used to identify these pigments. HPLC-diode array detector (HPLC-DAD) was used to determine UV-VIS spectra of pigments. Pigments were analyzed with HPLC under the following conditions: C18 column, acetonitrile/water (80:20, v/v), 0.5 mL min -1, 233 nm.

Introduction
Anka is with Monascus sp. fermented rice, which shows a purplish red color. In certain regions of Asia such as Taiwan, China, and Japan, anka has been used for hundreds of years as a natural coloring agent for food or as

one of the starter cultures for brewing of red rice wine. Salomon and Karrer [1] initiated the systematic study of anka pigments; they isolated a yellow pigment, elucidated the structure and named it monascin. Until 1973 there were two yellow (monascin and ankaflavin) and two orange pigments (rubropunctatin and monascorubrin) which had been isolated from anka or from mycelia of Monascus spp. by several research groups [1-6]. These four pigments produced by this fungus are named anka pigments. The two orange anka pigments react with aqueous ammonia solution under very mild conditions to give red nitrogen analogues (rubropunctatamine and monascorubramine) [3, 4]. Figure 1 shows the structures of the above six pigments and the chromophoric structures responsible for the color of these pigments. Both yellow monascin and ankaflavin contain the same chromophoric system, and differ from each other only in the length of the saturated side chain (C5 or C7) on ketonic carbonyl group. The same structural relationship was also observed between the two orange anka pigments and the two red nitrogen analogues. Although the four anka pigments were isolated before 1973 [1-6], the yields were very low because the separation methods used were poor. Hiroi et al. [7] used nhexane and benzene to extract pigments from the m y c e lium of Monascus anka H-26. The method he used demanded large volumes of solvents and was very timeconsuming. Therefore, at the beginning of our studies there were neither commercially available pure anka pigments nor satisfactory purification method for these pigments. In the most early studies, no pure pigments were used as reference compounds and no separation technique was applied to differentiate the pigments. The absorbance of a crude ethanol or methanol extract of anka or mycelia of Monascus spp. at 400 nm and 500 nm was determined as a measure of the amount of yellow and orange anka pigments, respectively [8-14]; these quantitative data were unreliable. Lin et al. [15] modified the HPLC system of Sweeny et al. [16] using a C18-column and an UV detector at 400 and 500 nm; Chen and Johns [17] used 392 nm to detect anka pigments. Lin et al. [15] used gradient of acetonitrile and

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5000/33 (Digital Equipment, Unterfoehring, Germany) and ICIS version 8.1 software (Finnigan MAT, Bremen, Germany). Mass spectra were determined directly with a MAT 8230 probe mass spectrometer (Finnigan MAT, Bremen, Germany).

Materials

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Structures of anka pigments and two red nitrogen analogues. Pigments: I ankaflavin, II monascin, III monascorubrin, IV rubropunctatin, V monascorubramine, VI rubropunctatamine.

water to separate six pigments in 40 rain. Chen and Johns [16] separated the six pigments with an isocratic mobile phase of acetonitrile/water (70:30, v/v) in 10 min. However, total run time of 10 rain might result in low resolution of all the pigments. In both the above studies, the choice of the detection wavelengths and the assignment of peaks were not explained. This paper describes methods to purify, separate, and analyze anka pigments.

Monascus purpureus DSM 1379 was purchased from "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH" (Braunschweig, Germany). The fungus was grown on agar slant containing 2 % yeast extract,5 % glucose,0.1% KH2PO4, 0.5 % MgSO4" 7H20, 0 . 1 % CaCI2, and 1.5 % agar [18]. After incubation at 30 ~ for 5 days, slants were harvested. Spore suspension was prepared with sterilized distilled water. Anka was produced by inoculation of autoclaved long-grain rice (Master Foods, Belgium) with a freshly prepared spore suspension (2 x 10 7 spores 10 g-1 rice). Fermentation was performed in a 500 mL Erlenmeyer flask plugged with cotton wool. After incubation at 30 ~ for 14 days the fermentation was terminated. Fermented red rice was freeze-dried at -60 ~ for 24 h and then stored at -26 ~ untill analysis. For analysis, HPLC-grade solvents such as methanol, acetonitrile, n-hexane, acetone, and ethyl acetate were purchased from Merck (Darmstadt, Germany). Water for the HPLC analysis was double distilled. Solvents used for preparation of pigments such as methanol, ethanol, acetone, diethyl ether, petroleum benzine (60-80 ~ and n-hexane were analytical grade (Merck, Darmstadt, Germany). Pre-coated silica gel TLC plates (Si 60/Kieselguhr F254, 0.25 mm, 20 x 20 cm) were purchased from Merck (Darmstadt, Germany).

Experimental
Instrumentation
The HPLC instrument consisted of a Waters 6000 A pump (Milford, MA, USA), a Waters WISP 710 B autosampler (Milford, MA, USA), a Nucleosil R 7 C18 column (250 x 4.6 mm I.D., Macherey Nagel, D0ren, Germany) and a Waters 481 spectrophotometer (Milford, MS, USA). A Maxima 820 chromatography workstation was used for the data acquisition (Dynamic solutions, Millipore, Ventura, CA, USA). The HPLC with the diode array detector (HPLC-DAD) consisted of a Jasco PU-980 pump with DG-1300 degassed system (Tokyo, Japan), a Jasco AS-851 autosampler (Tokyo, Japan), a Nucleosil R 7 C18 column (250 x 4.6 mm I.D., Macherey Nagel, Dtiren, Germany), a Jasco MD-910 multiwavelength detector (Tokyo, Japan) and DAD software (Jasco, Tokyo, Japan). The HPLC-MS-MS consisted of a Knauer 64 pump (Berlin, Germany), a Rheodyne 7725 injector (injection loop 5 pL), a Nucleosil R 7 C18 column (250 x 4.6 mm I.D., Macherey Nagel, Dtiren, Germany), a Knauer variable wavelength detector and a TSG 7000 tandem mass spectrometer with atmospheric pressure chemical ionization (APCI) interface (Finnigan MAT, Bremen, Germany). Data acquisition and mass spectrometric evaluation were on a Personal DEC station 530

Thin-Layer Chromatography (TLC)

A two dimensional TLC method was developed to separate the anka pigments and the two nitrogen analogues. A methanol extract of anka (3 mg anka mL -1 methanol), anka.pigments solution (1 mg pigment mL -1 methanol) and red nitrogen analogues were applied to the TLC plates, which were developed with n-hexane/ethyl acetate (7:3, v/v). After 60 rain development in the first eluent, the plate was dried at room temperature, then was developed with n-hexane/acetone (2:1, v/v) for further 60 rain. The pigments were separated and their RF-Values were recorded. The spots were scrapped off, extracted with methanol, and identified by comparison of retention times in HPLC and UV-VIS spectra with those of pure anka pigments. The two red nitrogen analogues were not identified independently.

High Performance Liquid Chromatography (HPLC)

A mobile phase of acctonitrile/water (80:20, v/v) and a C1s column (250 4.6 mm I.D.) were used to separate the anka pigments and the nitrogen analogues. The flow rate was 0.5 mL rain -1 with detection at 233 nm. The anka pigments were identified by both off-line direct Original

Chromatographia Vol.47, No. 9/10, May 1998

MS determination and the on-line HPLC-MS-MS system with an APCI interface. The nitrogen analogues were identified only by the on-line analysis. The MS analysis of each sample was performed in duplicate. For the off-line direct MS analysis, solution of anka pigments (4 mg pigments 100 mL -1) was first injected into HPLC. The differentiated anka pigments were collected individually several times at the outlet of the HPLC instrument. The eluent was evaporated to dryness under a stream of nitrogen at room temperature. Before MSdetermination, the collected pigments were injected into HPLC again to verify the collection and to ensure a sufficient amount for MS analysis. Electron impact, positive chemical ionization with isobutane, and chemical ionization (methane) along with elementary analysis were conducted to determine the molecular weights, fragmentation patterns, and molecular formulas of the pigments. A methanol extract of anka (3 mg anka 3 mL-1 methanol) and the nitrogen analogues were separated by HPLC into four peaks and two peaks, respectively. The MS of these pigments were determined on-line by HPLC-MS-MS with an APCI interface. For the mass spectrometer the following parameters were used: vaporizer temperature 300 ~ heated capillary serving simultaneously as repeller electrode (20 V) 180 ~ corona voltage 4 KV; electron multiplier voltage 1.6 KV. Nitrogen served both as sheath (50 psi) and auxiliary gas, and argon served as collision gas at a pressure of 1.9 mTorr. The mass spectrometer was operated in daughter ion scanning, which was chosen to detect positive ions rrdz 354.0 (rubropunctatamine), m/z 355.1 (rubropunctatin), m/z 359.1 (monascin), m/z 382.0 (monascorubramine), m/z 383.1 (monascorubrin), and m/z 387.1 (ankaflavin) for total scan durations of 1.0 s. The offset voltage was -18 V. The ions represented the protonated molecular ion [M+H] + for each of these pigments. In order to find the proper wavelength to detect the anka pigments, UV-VIS spectra of the six pigments were determined in the range from 200 to 650 nm on-line by HPLC-DAD. The anka pigments were quantified by absolute calibration. The crystal mixtures of anka pigments with the same color were used as reference compounds, each of which consisted of two pigments, monascin and ankaflavin, or, rubropunctatin and monascorubrin. The amount of each pigment in these crystal mixtures was calculated after the ratio of peak area of the pigments in the stock solutions, which were prepared by dissolving 1.24 mg of yellow crystal mixture in 50 mL methanol (i.e. monascin 21.3 ng pL -1, ankaflavin 3.5 ng pL-1) and 1.90 mg of orange crystal mixture in 25 mL methanol (i.e. rubropunctatin 25.6 ng pL-1, monascorubrin 50.4 ng pL-1). The calibration graph was constructed with the results of three consecutive injections of five concentrations of pigments; the response had good linearity (correlation coefficient = 0.99). Original

Purification of Pigments
Anka pigments were purified in two different forms: crystals of two orange anka pigments and two yellow anka pigments. For purification of the two orange pigments, anka powder was extracted with n-hexane until the extract was no longer highly coloured, after which the extract was concentrated to dryness in a rotary evaporator (Rotavapor RE, with cooler bath Julabo F40, Buechi) at 45 ~ under reduced pressure. Evaporation of solvent gave an orange solid, which was then rinsed with a small amount of cool absolute ethanol. Absolute ethanol was added to the orange residue and the mixture was warmed to 70 ~ to dissolve this residue. Repeated recrytallizations from ethanol were conducted to yield needle crystals of the both orange anka pigments (rubropunctatin and monascorubrin). After the exhaustive extraction of anka powder with nhexane, the residue was successively extracted with methanol. The extraction was terminated when the extract became almost colorless, after which the pooled extracts were concentrated in a rotary evaporator at 45 ~ under reduced pressure. The resultant dark concentrate was stored at 4 ~ for one week until black material was formed. This black material was then rinsed in succession with small amount of diethyl ether and acetone. It was then dissolved in acetone, filtered through Whatman No. 1 filter paper (Whatman, England) and the solvent was evaporated under reduced pressure. Absolute ethanol was added to the residue. In order to dissolve the residue, the ethanol mixture was warmed to 70 ~ The warm ethanol solution was cooled slowly by standing at room temperature until crystals were formed. The procedures of recrystallization from ethanol were repeated several times to give a yellow crystal mixture of both monascin and ankaflavin.

Preparation of Nitrogen Analogues

Both red pigments, rubropunctatamine and monascorubramine, were prepared according to the papers of Haws et al. [3] and Fielding et al. [4]. Solution of orange pigments, rubropunctatin and monascorubrin, in diethyl ether (10 mg 10 mL -1) was shaken for one min with 7.5 mL 8 % aqueous ammonia solution, the color of the ether layer changing from orange to red. After addition of concentrated hydrochloric acid (480 pL), the ether layer was collected and dried under nitrogen at room temperature. The prepared red pigments were dissolved in 8 mL methanol. The solution was subjected to TLC, HPLC, HPLC-MS-MS, and HPLC-DAD analyses.

Results and Discussion

At the beginning of our studies, there were neither commercially available anka pigments nor satisfactory purification methods of these pigments. Anka prepared in our laboratory was extracted with different solvents. The color of these solvent extracts were remarkably dif531

Chromatographia Vol.47, No.9/10, May 1998

Table I, RF-values of pigments eluated with n-hexane/ethyl acetate (7:3,v/v) and n-hexane/acetone (2:1,v/v). First dimension Pigment Ankaflavin (yellow, C7) a Monascin (yellow, C5) a Rubropunctatin (orange, C5) a Mortascorubrilb(orange, C7) ~ Red pigment Red pigment 2 b n-hexane/ethyl acetate (7:3, v/v) 0.45 0.40 0.38 0.38 0.01 0.01 Second dimension n-hexane/ethyl acetone (7:3, v/v) 0.49 0.44 0.50 0.45 0.19 0.17

a identified anka pigment (color, saturated side chain on ketonic carbonyl group). bpigment not identified.

ferent. When two dimensional T L C was used to separate the pigments, it was found that the pigment patterns in these solvent extracts were different. With some solvents, such as methanol and acetone two yellow and two orange pigments were extracted but with n-hexane or petroleum benzine more of the two orange pigments was extracted than the yellow pigments. Based on the above observation, a series of purification methods for a fractional separation of pigments from anka was designed. A n k a was extracted first with n-hexane to obtain the orange pigments principally. The anka residue was then extracted with methanol to obtain the remaining yellow pigments. The following procedures to rinse or to crystallize the pigments were based on those published by Salomon and K a r r e r [1]. Thus, crystal mixtures of two orange pigments and two yellow pigments were obtained. A H P L C m e t h o d was developed to differentiate six pigments. These pigments were identified by comparison of molecular weights, molecular formulas, and fragmentation patterns analyzed by direct MS and H P L C - A P C I MS-MS with those reported in earlier studies [1-6]. With help of H P L C - D A D , UV-VIS spectra of pigments were determined on-line. From these spectra, it was possible to find a suitable wavelength to simultaneously detect all pigments at one wavelength. Along with the purified crystals, it was possible not only to qualitatively analyze but also quantitatively analyze anka pigments. The extraction of anka by various solvents and the solubility of the pigments in various solvents were analyzed. An excellent agreement between the results obtained by T L C or H P L C was observed and supported the purification procedures carried out. H P L C - A P C I - M S - M S was also used to investigate whether Monascus purpureus produced any of the nitrogen analogues, rubropunctatamine and monascorubramine, or if these two red compounds were only products of orange anka pigments with dilute aqueous ammonia solution. Only very small amount of the nitrogen analogues was extracted from purplish red anka. When

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Chromatographia Vol.47, No. 9/10, May 1998

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the methanol extract of anka reacted with dilute aqueous ammonia solution, the orange pigments disappeared and the two red nitrogen analogues should be formed. The above results along with the other results of our studies provide a new understanding of the pigments produced by M. purpureus on rice and support the proposition of Carels and Shepherd [11] who suggested that M. purpureus biosynthesize only four anka pigments, i.e., two yellow and two orange anka pigments; whereas, the red nitrogen analogues should be the products of the orange anka pigments with NH-groups in rice or in the mycelium of fungus.

Analysis of Pigments by HPLC

The six pigments were separated by a C18 column with an isocratic mobile phase of acetonitrile/water (80:20, v/v) at 0.5 mL min-1 in 18 min. The compounds were detected at 233 nm. The four anka pigments were quantitatively analyzed by absolute calibration methods but because no pure reference nitrogen analogues were available they were only qualitatively analyzed. Figure 2 gives the UV-VIS spectra of the anka pigments and the nitrogen analogues determined on-line with a DAD. A saturated side chain of C5 or C7 on the ketonic carbonyl group did not influence the color or the UVVIS spectra of the pigments and both yellow anka pigments (monascin and ankaflavin) had the same spectrum. This was also observed for the two orange pigments (rubropunctatin and monascorubrin) and for the red nitrogen analogues (rubropunctatamine and monascorubramine). In this study, mixtures of each of the two anka pigments with the same color were used as reference compounds for quantative analysis. The optimal detection wavelengths for the yellow, orange, and red pigments were 391,472, and 307 nm, respectively. However, simultaneous detection of all these pigments at wavelength of 233 nm was a suitable alternative. Figure 3 compares the chromatograms of six pigments detected with DAD at the wavelength of 233 and 533

Analysis of Pigments by TLC

Table I shows the RF-values of the anka pigments and the nitrogen analogues by two dimensional TLC with n-hexane/ethyl acetate (7:3, v/v) and n-hexane/acetone (2:1, v/v) as eluents in the two dimensions. The first solvent system resulted in good separation of yellow ankaflavin and monascin. The remaining four pigments, rubropunctatin, monascorubrin, and the red nitrogen analogues, overlapped after the first development. To overcome this problem, a second elution at the right angles to the first was conducted to separate these pigments individually. Original

Chromatographia Vol.47, No. 9/10, May 1998

Table II. Retention times of anka pigments and red nitrogen analogues (n = 30). Pigments 1) Red nitrogen analogues rubropunctatamine monascorubramine 2) Anka pigments monascin (yellow) rubropunctatin (orange) ankaflavin (yellow) monascorubrin (orange)
a Retention time of pigments

Amount (ng) a n.d. b n.d. b 17.0-594.4 5.1-178.8 2.9-100.0 10.1-352.7

Retention time (min) 7.35 _+0.05 8.96 0.04 10.72 _+0.08 11.77 -+0.11 14.45 0.10 16.58 _+0.12

was determined in this weight range.

bn. d.= not determined.

Table III. Identification of anka pigments and nitrogen analogues. Retention time in HPLC 7.4 min 9.0 min 10.7 min 11.8 min 14.5 min 16.6 min Molecular weight 353.1 a 381.1 a 358.1 a, 358.1779 b 354.1 a, 354.1466 b 386.1 a, 386.2090 b 382.1 a 382.1779 b Molecular formula C21Hz3NO4a C23H27NO4a
C2lH2605

Identified pigments rubropunctatamine (red) monascorubramine (red) monascin (yellow) rubropunctatin (orange) ankaflavin (yellow) monascorubrin (orange)

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determined by HPLC-APCI-MS-MS. b determined by MS.

392, 400, and 500 nm, respectively, which were used in the early studies [15, 16]. As expected, the chromatograms detected at 392 nm or 400 nm are similar. The remarkable difference between chromatograms at 233 nm and 400 nm was the low sensitivity of two red nitrogen analogues. The sensitivity of the U V detector at 233 n m was much higher than at 400 nm. At a wavelength of 500 nm, the two yellow anka pigments were not detectable. Reproducibility tests showed a variation of less than 1.0 % in the retention times (Table II). The standard solution of anka pigments, the methanol extract of anka, and a mixture of these two solutions were analyzed separately five times in one day to determine the precision of the H P L C method. The resulting C.V. for the yellow pigments was less than 1.5 %; and less than 4 % for the orange pigments. The recovery of yellow pigments was 100.7 and 100.2; and 102.6 and 103.9 for the orange compounds.

lar formulas. From the MS (electron impact,70 eV) (Figure 4), it can be seen that pigments with the same color exhibited closely similar fragmentation patterns, including the same base peak (m/z 162 and m/z 298, respectively) and most of the product ion peaks. The greatest difference between the mass spectra of monascin and ankaflavin was the molecular ion [M peaks, which showed a difference of 28 m.u.; the same relationship was also observed between the orange compounds, monascorubrin and rubropunctatin. The chromatogram of the anka pigments obtained by daughter scan is given in Figure 5, and that of the two nitrogen analogues in Figure 6. From these spectra, the order of elution in H P L C was identified as rubropunctatamine, monascorubramine, monascin, rubropunctatin, ankaflavin, and monascorubrin.

Acknowledgement
This work has been supported in part by the GottliebDaimler- and Carl-Benz-Foundation. The authors would like to express their thanks to Prof. Schreier, Institute of Food Chemistry, University of Wtirzburg, Dr. E. Hoberg, Bundesanstalt for Pflanzenziachtung Quedlinburg, for the kind lending of H P L C - A P C I - M S MS and HPLC-DAD, respectively. We are indebted to

Identification of Pigments
A n k a pigments and nitrogen analogues were identified by MS, including comparison of the determined molecular weights, molecular formulas, and fragmentation patterns with those reported in the early studies [3-6]. Table III shows the various molecular weights and molecu-

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Chromatographia Vol.47, No. 9/10, May 1998

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Chromatographia Vol. 47, No. 9/10, May 1998

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Dr. M. H e r d e r i c h , Institute of Food Chemistry, University of WSrzburg, for p e r f o r m i n g the m e a s u r e m e n t s of H P L C - A P C I - M S - M S , to Miss J. Lin and Prof. B. H. Chen, D e p a r t m e n t of Nutrition and F o o d Science, FuJen University for the correction of this text.

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