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ISSN 10619348, Journal of Analytical Chemistry, 2015, Vol. 70, No. 12, pp. 1512–1520. © Pleiades Publishing, Ltd., 2015.

ARTICLES

An Improved High Performance Liquid Chromatography Method for the Separation of Carotenoids Extracted from Phaffia rhodozyma1 Lijun Lia ,b, c, Yue Yua, Xiping Dua, b, c, Zedong Jianga, b, c, Feng Chena, d, and Hui Nia, b, c, * aCollege

of Food and Biological Engineering, Jimei University Xiamen Fujian Province 361021 China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering Xiamen Fujian Province 361021 China c Research Center of Food Biotechnology of Xiamen City Xiamen 361021 China d Department of Food, nutrition and package Science, Clemson University South Carolina 29634 United States *email: [email protected]

b

Received September 15, 2014; in final form, March 23, 2015

Abstract—Separation of analytes is an important factor affecting the effectiveness of carotenoids analysis from a complex extraction. In this study, a high performance liquid chromatography procedure with improved separation was developed to determine carotenoids extracted from P. rhodozyma. Mixture design experiment was employed to optimize the organic solvent constitution, which showed that the optimal organic phase was composed (v/v) of 61% of methanol, 32% of tetrahydrofuran, and 7% of acetonitrile. Fur thermore, the gradient elution procedure optimization demonstrated that the gradient elution started at the optimal organic phase concentration of 60% lead to the best separation effect. Meanwhile, 18 compounds in total 25 detected peaks were separated. Based on their absorption spectra, astaxanthin, βcarotene, zeaxan thin, βcryptoxanthin and lycopene were identified by matching their retention times with standards. Inter estingly, our improved HPLC method was effective to classify P. rhodozyma strains and monitor the degrada tion of astaxanthin in accordance with their carotenoids composition. Keywords: Phaffia rhodozyma, carotenoids, astaxanthin, improved HPLC method, classification of species DOI: 10.1134/S1061934815120102

ó1 The red fermenting yeast Phaffia rhodozyma has received considerable attention in the last three decades as a nature source of carotenoid astaxanthin (3,3dihydroxyβ,β'carotene4,4'dione) [1–5], which possesses potent antioxidant activity that protects cytoplasm and lipid membrane from the damage of oxidation and helps cure degenerative diseases [6–9]. P. rhodozyma has a complex synthetic pathway of car otenoids [2, 10–12]. It has been reported that at least 14 kinds of colorful carotenoids, including lycopene, βcarotene and astaxanthin, were involved in the bio synthetic pathway of astaxanthin [1, 3, 4]. Further more, carotenoids are easy to degrade in the presence of oxygen and metal ions, or under acidic and basic conditions [2], leading to generate several other caro tenoids with their degradation products [13].

tenoid profiles in different plants [15], such as marine macro algae, tomatoes, cauliflowers, pummelo, and sour cherries [16–21]. Although the HPLC analysis on plants’ carotenoids was studied and reported widely, there is still few available information on improving the separation of carotenoids extracted from P. rhodozyma so far, leading to difficult to analyze the individual carotenoids in the extract of P. rhodozyma. The restrictions of separation technol ogy obstruct the further studies on classifying P. rhodozyma strains according to their carotenoids constituent and monitoring the degradation of astax anthin [15, 22]. Thus, it is very important to determine each carotenoid and improve the separation of caro tenoids in the extract of P. rhodozyma.

Although spectrophotometric method was reported for rapid determination of carotenoids, it can not quantify an individual carotenoid in the extract of P. rhodozyma [14]. Recently, HPLC has been reported to be the most common method for determining caro

In our previous studies, an HPLC method was employed to determine astaxanthin in the acidic extraction from P. rhodozyma [23, 24]. In this study, we aim to improve the separation of astaxanthin and other carotenoids in P. rhodozyma extract to classify P. rhodozyma strains based on their carotenoids pro files and monitor the degradation of astaxanthin based

1 The article is published in the original.

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on the analysis of the concentration of astaxanthin and its degradation products.

Table 1. Mixture design and optimization of the organic phase composition*

EXPERIMENTAL Chemicals and reagents. Astaxanthin and βcaro tene standards were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Lycopene, βcryptoxan thin and zeaxanthin standards were purchased from TAKARA in Reagent Co. (Dalian, China). They were all stored at –20°C under nitrogen environment. Eth anol and dimethyl sulfoxide (DMSO) of analytical grade were purchased from Sinopharm chemical Reagent Co. Ltd. (Shanghai, China). Methanol, tet rahydrofuran and acetonitrile of HPLC grade were produced by TEDIA Company Inc. (Ohio, USA). Stock solutions of astaxanthin and βcarotene were prepared at the concentration of 100 μg/mL in ethanol. Stock solutions of zeaxanthin (100 μg/mL) and βcryptoxanthin (100 μg/mL) were prepared with methanol. Lycopene was dissolved in chloroform at a concentration of 10 μg/mL. All the stock solutions were added with 10 mg/mL of butylated hydroxytolu ene, filled with nitrogen and kept at –20°C until use. Working solutions for HPLC analysis were prepared by serial dilution to desirable concentrations. Yeast strains and carotenoid extracts. P. rhodozyma 7B12 originated from P. rhodozyma Past1 that was generously provided by Professor Ulf Stahl, Berlin Industrial College, Germany [23]. P. rhodozyma MVPJMU14, ly322, B4M, B1R, and 15r strains were mutant strains induced from P. rhodozyma 7B12 by the treatment of ethyl methane sulfonate followed by strain scanning using the selective pressure of H2O2. Cells of P. rhodozyma were fermented and har vested according to the method as described previ ously [23]. Pigment extraction was conducted by the DMSO method [25] followed by storage at –20°C under nitrogen in the dark. Optimization of organic phase. The optimization of organic phase composition was started from the HPLC procedure as reported by Ni et al. [23] with some modifications by using a Waters HPLC system, equipped with a 2695 pump, a NovaPak C18 column (3.9 × 150 mm, 4 μm), and a 2478 UV detector (Waters corporation, Milford, MA, USA). The operation was conducted from an injection of sample (20 μL), and the flow rate was kept at 1 mL/min, the column tem perature at 35°C, and the column pressure at 0– 3000 psi. The absorbance of each fraction was mea sured at the wavelength of 474 nm. The mobile phase was composed of ultrapure water (A) and organic phase (B), containing methanol, ace tonitrile and tetrahydrofuran. The gradient elution was begun with 80% B followed by maintaining at 80% for 3 min, ascending to 100% in the next 12 min, main taining at 100% for 12 min, descending to 80% in 8 min, and finally maintaining 80% for 5 min. JOURNAL OF ANALYTICAL CHEMISTRY

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Runs

X1

X2

X3

COF

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1.000 0.000 0.000 1.000 0.500 0.500 0.167 0.667 0.333 0.000 0.500 0.000 0.167 0.000 0.000

0.000 0.000 0.000 0.000 0.000 0.000 0.167 0.167 0.333 0.500 0.500 0.500 0.667 1.000 1.000

0.000 1.000 1.000 0.000 0.500 0.500 0.667 0.167 0.333 0.500 0.000 0.500 0.167 0.000 0.000

221.490 105.900 94.930 220.750 232.700 232.440 253.010 283.420 283.840 178.970 294.610 178.710 126.490 73.100 73.080

* X1, X2, X3 are volume ratios of methanol, tetrahydrofuran and acetonitrile in organic phase, respectively.

The proportions of methanol, acetonitrile and tet rahydrofuran were investigated by using a mixture experiment design (Table 1). All the mixtures of the three solvents were used to run the mixture of the car otenoids extracts isolated from these strains. After sig nals were detected, the runs were recorded for the retention time values and the numbers of the separated peaks, which were further used to calculate the resolu tion of the adjacent peaks and the chromatography optimization function (COF) according to the method as described previously [26] with equations (1) and (2). Finally, the relationship between COF and organic phase composition (the proportion of metha nol, acetonitrile and tetrahydrofuran) was modeled to predict the optimal organic phase composition with the highest value of COF using formulas showed as follow: 2 ( t Ri + 1 – t Ri ) , R i = Wi + 1 + Wi

(1)

where t Ri is the retention time of peak i and Wi is the peak width of peak i. In any case, a Ri value larger than 2 is designated at 2. n

COF =

⎞ + 10n, ∑ ⎛⎝ R ⎠ i

Ri

(2)

id

where Ri is the resolution (R) of peak i calculated by equation (1) [26] and Rid is the resolution value of 2 that meant baseline separation of two adjacent com pounds; n is the number of separated peaks. No. 12

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Optimization of the gradient elution procedure. For optimizing the gradient elution procedure, the opti mal organic phase (B), which was composed (v/v) of 61% methanol, 32% tetrahydrofuran and 7% acetoni trile, was used to run the sample starting from different concentrations (i.e., 60, 65, 70, 75 and 80%), followed by linearly ascending to 100% in 40 min, descending to the start proportion in 5 min and maintaining for 3 min. After recoding the retention time and the peak numbers, COFs of each runs were calculated to com pare their separations among the runs, as described above. Qualification of carotenoids. Qualification of caro tenoids was conducted on an Agilent 1200 HPLC series equipped with a diode array detector (DAD) (Agilent Technologies, California, USA). The chemi cals were qualified by matching the retention time and absorption spectra to those of standards. Plotting calibration curves and measuring limits of detection (LOD) and quantification (LOQ). For plot ting the calibration curve, the stock solutions were diluted to a desirable concentration followed by filtra tion through a 0.22 μm membrane prior to HPLC analysis. The peak area at 474 nm was recorded for regressive analysis against the concentration. LOD was estimated as the sample concentration corresponded to 3 times of the noise level, while LOQ was considered to be the sample concentration giving a signal has 10 times to the noise. Investigation of the accuracy and the repeatability. Astaxanthin standard solutions at the concentrations of 1, 3, 5, 10 and 30 μg/mL and βcarotene standard solutions at the concentrations of 1, 10, 20 and 30 μg/mL were analyzed by the procedure, describe above. The recovery was then computed by equation (3) to estimate the accuracy. c Recovery = d × 100%, ct

(3)

where cd is the detected concentration, and ct is the theoretical concentration. Classification of P. rhodozyma strains based on car otenoids profiles. Carotenoids from different P. rhodozyma strains, named P. rhodozyma JMU MVP14, P. rhodozyma ly322, P. rhodozyma B4M, P. rhodozyma B1R, P. rhodozyma 15r and P. rhodozyma 7B12 were analyzed by a spectrophoto metric method to measure the total carotenoids con tent and the HPLC procedure to estimate contents of individual carotenoids, respectively. Investigation of the degradation of astaxanthin and βcarotene. Ten milliliters of astaxanthin solution (50 μg/mL) and βcarotene solution (50 μg/mL) were transported into plates, followed by exposure under sunlight and oxygen at a temperature 30 ± 1°C, and each sample was taken in every 30 min to analyze the carotenoids profile until 7 days exposure.

Statistical analysis. The result of mixture design was statistically analyzed by using the Statistics Analy sis System (SAS) software (SAS Institute Inc., Cary, NC). The means and standard deviations were calcu lated by using Excel 2007. A difference was considered significant at p < 0.05. RESULTS AND DISCUSSION Optimization of organic phase and elution proce dure. The organic phases and the elution procedure are known to be the most important factors for improving separation efficiency in the HPLC analysis [27]. Although any of the three parameters could be used to evaluate the separation, COF was adopted as the criteria to optimize the organic phase composition due to that it was able to combine both the number of separated peaks and total resolution. Moreover, COF was considered better depending on either peak num ber or total resolution: 1—in the case of an equal number of separated peaks, higher total resolution, meant a higher COF value; 2—if the total resolution was equal, while more peaks were observed, meant a higher COF value [25]. In the study, mixture design, provide a maximal value mathematically, was applied to investigate the effects of methanol, acetonitrile and tetrahydrofuran on separation of carotenoids from P. rhodozyma. The chromatographic optimization functions were calcu lated according to the separation results of the fifteen groups of organic phase compositions (Table 1). Then, the relationship between COF and the proportion of methanol (X1), tetrahydrofuran (X2) and acetonitrile (X3) were regressively analyzed. The results demon strated that X *1 X2, X *1 X3, and X *2 X3 showed significant effects on COF (Table 1). Furthermore, equation (4), a formula related COF to X1, X2 and X3, was simulated and applied to predict the maximal COF. Based on equation (4), the contour plot of X1, X2, and X3 was constructed, suggesting that the maximal COF value exists inside the triangle area (Fig. 1). Moreover, the organic phase composed (v/v) of 61% methanol, 32% tetrahydrofuran and 7% acetonitrile was predicted to produce the maximal COF (288.18) with 27 peaks and a total resolution of 18.18. The confirmation of pre dicted organic phase showed 27 peaks separated from the extract of P. rhodozyma (Fig. 2a), and a COF of 286.33, which was very close to the predicted COF value (288.18), verifying reliability of the optimiza tion. Y = 22.29X 1 + 60.81X 2 + 108.35X 3 (4) + 533.36X *1 X 2 + 310.44X *1 X 3 + 364.67X *2 X 3 with significant levels of 0.22% for X *1 X2, 1.65% for X *1 X3 and 0.72% for X *2 X3 (p < 0.05). Although more compounds were able to separate with the improved organic phase, most peaks were

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X1 1.00

250.15

0

0

250.15

174.41

212.28 136.54

174.41

136.54

1.00 X2

1.00 X3

Fig. 1. The contour plot of X1, X2, X3 against COF (Y1) based on the mixture design results.

overlapped and insufficiently separated (Fig. 2a). To further improve the separation efficiency, the linear elution program was investigated by prolonging the linear elution time and starting the elution at series of organic phase concentration from 60 to 80%. As showed in Table 2, starting the gradient elution at the optimal organic phase concentration of 60% produced the best separation, which showed 33 peaks and a COF value of 349.06. Most peaks distributed evenly within the gradient ascending step (from 16 to 37 min) (Fig. 2b). Furthermore, 15 of the detected peaks were estimated to have resolution values above 2, revealing that these 15 kinds of corresponding compounds were separated completely (Fig. 2b). Obviously, the com bined improvement in both the organic phase and the elution program (Fig. 2b) showed better separation

than those of solo improvements in either organic phase (Fig. 2a) or elution program (Figs. 2c and 2d), indicating the validity of the improvement. Most of carotenoids have strong absorption within the wavelength range of 400–500 nm in relation to their conjugated double bonds, which was used to pri marily identify carotenoids [27, 28]. In addition, the structure characteristics or molecular weights of some carotenoids, such as phoenicoxanthin and astaxan thin, echinone and hydroxyechinone, were too similar to be distinguished by the spectrum. Furthermore, some carotenoids with various stereomers were also difficult to distinguished by the spectrum, for instance, astaxanthin had two chiral centers and consisted of three different optical R/S isomers [29]. To further qualify and quantify the carotenoids, a DAD was

Table 2. Effect of initial proportion of organic phase on gradient elution of carotenoids extracted from P. rhodozyma* Proportion of organic solvent in mobile phase at different time, min

Run 1 2 3 4 5

0

40

45

48

60.0 65.0 70.0 75.0 80.0

100.0 100.0 100.0 100.0 100.0

60.0 65.0 70.0 75.0 80.0

60.0 65.0 70.0 75.0 80.0

Number of peaks

Total resolution

COF

33 29 28 27 26

38.12 33.78 34.93 33.92 30.33

349.06 306.89 297.46 286.96 275.17

* The organic phase was composed of 61% methanol, 32% tetrahydrofuran and 7% acetonitrile. The aqueous phase was pure water. JOURNAL OF ANALYTICAL CHEMISTRY

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AU (474 nm)

(a) 0.30 0.25 0.20 0.15 0.10 0.05 0

100 80 60 40 20 0

8

4

16 24 20 Retention time, min

12

28

32

36

0 40

AU (474 nm)

(b) 0.18 0.15 0.12 0.09 0.06 0.03 0

100 80 60 40 20 0

4

8

12

16

20 24 28 32 Retention time, min

36

40

44

0 48

AU (474 nm)

(c) 100

0.18 0.55 0.12 0.09 0.06 0.03 0

80 60 40 20 0

4

8

12

16


36

40

44

0 48

AU (474 nm)

(d) 0.12 0.10 0.08 0.06 0.04 0.02 0

100 80 60 40 20 0

4

8

12

16


36

40

44

0 48

Fig. 2. Mixture design and the organic phase optimization of HPLC analysis. Elution profile of carotenoids by original elution procedure (a) or optimized elution procedure (b) with optimal organic phase, and elution profile of carotenoids by original elu tion procedure with methanol (c) or optimized elution procedure with acetonitrile (d). Where solid line is the eluted peak, and dotted line is the proportion change of organic phase.

employed to analyze the absorptive spectra of the sep arated peaks. As the result, 25 peaks were detected and 18 kinds of them showed strong absorption within 400–500 nm (Figs. 3, 4; Table 3), indicating the pre liminary identification of carotenoids. By comparing

their retention time with standards, five kinds of caro tenoids (astaxanthin, zeaxanthin, βcryptoxanthin, lycopene and βcarotene) were qualified at the reten tion times of 19.2, 23.2, 33.9, 37.8 and 38.3 min, respectively (Table 3). Further structure analysis


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AN IMPROVED HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD 200 150 100 50

800 700 600 500 400 5

10

40 45 30 35 25 15 20

mAU 100 80 60 40 20 0 0

approaches are necessary to identify the structures of the others. An HPLC method has been established by Ni et al. [23] to determine astaxanthin produced by P. rhodozyma. VilasoaMartínez et al. [30] detected astaxanthin, βcarotene and two astaxanthin deriva tives in shells with a mobile phase consisting of meth anol, acetonitrile and hexanedichloromethane. Sachindra et al. proposed an HPLC analysis, which was able to simultaneously determine astaxanthin, zeaxanthin, βcarotene, and astaxanthin monoester and astaxanthin diester in crabs [31]. Loto et al. devel oped an HPLC method that could determine 11 kinds of carotenoids in P. rhodozyma [32]. Our results reveal that the improved HPLC system are able to separate more carotenoids than that reported in previous stud ies described above, and possess better resolution than previous methods with respect to determination of the carotenoids from P. rhodozyma. Quantification of carotenoids. The regressive equa tions were calculated by standards and showed in Table 4, y = 81.014x + 10.232 (R2 = 0.9991) for astax anthin, y = 47.218x + 0.4276 (R2 = 0.9995) for βcar otene, y = 2.0214x + 0.2589 (R2 = 0.9994) for zeaxan thin, and y = 12.895x + 1.375 (R2 = 0.9992) for βcryptoxanthin. Our results demonstrated that the LODs (μg/mL) were 0.05 for astaxanthin, 0.01 for βcarotene, and 0.37 for βcryptoxanthin, respec tively, according to the S/N of 3; and the LOQs (μg/mL) were 0.17 for astaxanthin, 0.03 for βcaro tene, and 1.2 for βcryptoxanthin, respectively, according to the S/N of 10 (Table 4). Astaxanthin was detected to possess the recovery ranges from 95.4 to 101.7% and the relative standard deviations (RSDs) ranges from 1.1 to 3.2%; βcaro tene showed recovery ranges from 101.0 to 104.8% and RSDs ranges from 1.2 to 3.0% (Table 5). All these results are consistent with the previous studies [23, 30, 31], indicating that the present HPLC method is accu rate and precise for the determination of the caro tenoids. Analysis of carotenoids composition extracted from P. rhodozyma strains. Carotenoids, including βcaro tene, astaxanthin, zeaxanthin, βcrptoxanthin, echi JOURNAL OF ANALYTICAL CHEMISTRY

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(a)

5

10

mAU 40 32 24 16 8 0

Fig. 3. Full wavelengths scan 3D figure of substances fer mented from P. rhodozyma JMUMVP14.

0

15 20 25 30 35 Retention time, min

40

45

(b)

5

10

15 20 25 30 35 Retention time, min (c)

40

45

5

10

15 20 25 30 35 Retention time, min

40

45

mAU 20 16 12 8 4 0 0

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Fig. 4. The degradation chromatogram of astaxanthin by using the improved HPLC method. Elution profile of astaxanthin exposed to sunlight and oxygen at initial time (a), after 30 min (b), and after 7 days (c).

none, hydroechinone, lyconene, torulene, HDCO (3hydroxy3',4'β, βcarotene4one), DCD (3'di hydroxyβ, β'carotene4,4'dione) and so on, might exist in the extract of P. rhodozyma [1, 3, 4]. To evalu ate the practical value for classifying the strains in terms of their carotenoids, six P. rhodozyma mutant strains were analyzed by their carotenoids with the improved HPLC method. The results showed in Table 6 demonstrated that nine major carotenoids were determined with great differences in their profile and yield among these mutant strains. The mutant strains ly322, B1R and B4M were incapable of syn thesizing astaxanthin, instead of astaxanthin, their major carotenoids were βcryptoxanthin, βcarotene and an unknown compound corresponding to peak 7 with a retention time of 37.5 min (Table 6). Other pro files of carotenoids, contained in mutant strains of 7B12 and 15r, were very simple, in which only two kinds of carotenoids were detected, and the majority of the both were astaxanthin. Moreover, the strain of JMUMVP14 was observed to synthesize nine caro tenoids, including the highyield production of astax anthin, βcryptoxanthin and βcarotene. These results agreed with previous studies that P. rhodozyma No. 12

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Table 3. The retention time and maximum absorption wavelength of substances Peak Retention no. time, min

Maximum absorption, nm

Standard matching

Identifica tion Astaxanthin Unknown Unknown Zeaxanthin Unknown Unknown Unknown Unknown Unknown Unknown Unknown βCryptox anthin Unknown Unknown Unknown Unknown Lycopene βCarotene

1 2 3 4 5 6 7 8 9 10 11 12

19.2 21.3 21.9 23.2 24.7 26.4 30.8 32.0 32.8 33.0 33.5 33.9

480 476 372, 468 482 368, 472 480 496, 522 476 462 446, 474 451, 480 474

+ – – + – – – – – – – +

13 14 15 16 17 18

34.6 35.5 36.1 37.5 37.8 38.3

478 492 448, 480 490, 521 432, 460, 481 439, 461, 484

– – – – + +

+ Matching with a standard; –Lack of a standard.

was able to synthesize astaxanthin via a complex syn thesis pathway [2, 10–12], and suggested that the improved HPLC method is effective in the classifica tion of P. rhodozyma strains. Investigation of the degradation of astaxanthin. Astaxanthin is one of the most important carotenoids produced by P. rhodozyma due to its high antioxidant potential and healthpromoting properties. Astaxan thin is a highly unsaturated molecule that decomposes easily when exposed to heat, light and oxygen [33⎯36]. Many strategies has been attempted to improve the solubility and stability of astaxanthin, such as inclu

sion complexation with hydroxypropylβcyclodex trin, calcium ions [37, 38], microencapsulation into chitosan matrix [39], and embedding into halfcenti meterchitosan beads [40] or liposomes [41]. How ever, the difficulty in analyzing the degradation pro cess hinders indepth studies of astaxanthin degrada tion and in developing more effective approach to protect it from cleavage. By using the improved HPLC method, the amount of astaxanthin was detected to decrease rapidly while exposed to sunlight and oxygen (Fig. 4a). After 30 min exposure, 54.6% of tested astaxanthin was degraded (Fig. 4b). After 7 days expo sure, the tested astaxanthin completely disappeared and an unknown compound was detected at 28.9 min by improved HPLC analysis (Fig. 4c). These results were in a good agreement with a previous study, which suggested that sunlight plays an important role in the degradation process of astaxanthin [42]. The results also indicate that our new improved method is a desir able candidate for exploring the degradation process of astaxanthin and other carotenoids due to its potential capability of separating carotenoids. *** An efficient reversed phase HPLC method was developed to improve the separation of carotenoids, which could separate 18 kinds of carotenoids from the extract of P. rhodozyma, including astaxanthin, βcar otene, βcryptoxanthin, zeaxanthin and lycopene. The determination of astaxanthin and βcarotene indicate the method was accurate and precise to simultaneously determine the carotenoids. Moreover, this method was effective to classify P. rhodozyma strains and monitor the degradation of astaxanthin. Our results also provide a feasible analytical procedure for classifying different P. rhodozyma strains and inves tigating the depredating process of astaxanthin. The improved HPLC method could facilitate indepth studies on astaxanthin and other carotenoids. ACKNOWLEDGMENTS This research could have not been accomplished without the financial support provided by the National

Table 4. Standard curves, LOD and LOQ of astaxanthin, βcarotene, βcryptoxanthin and zeaxanthin Compound Astaxanthin βCarotene βCryptoxanthin Zeaxanthin

Standard curve Y = 81.014X + 10.232 Y = 47.218X + 0.4276 Y = 12.895X + 1.375 Y = 2.0214X + 0.2589

R1

Linear range, μg/mL

LOD, μg/mL

LOQ, μg/mL

0.9991 0.9995 0.9992 0.9994

2–50 2–50 2–10 5–70

0.05 0.01 0.37 0.03

0.17 0.03 1.23 0.11

The organic phase was composed of 61% methanol, 32% tetrahydrofuran, and 7% acetonitrile. The aqueous phase was pure water. For the gradient elution, organic phase raise from 60 to 100% in 40 min, decrease from 100 to 60% within 5 min, and maintain at 60% for 3 min. JOURNAL OF ANALYTICAL CHEMISTRY

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Table 5. Results of recovery and RSDs of detection of astaxanthin and βcarotene of P. rhodozyma Compound

Mean, μg/mL

Astaxanthin

Detected, μg/mL

3 5 10 30 1 10 20 30

βCarotene

2.86 4.91 9.85 30.53 1.05 10.35 20.66 30.31

n

Recovery, %

RSD, %

6 6 9 9 9 6 3 9

95.4 98.3 98.5 101.7 104.8 103.5 103.3 101.0

1.8 1.4 3.2 1.1 1.3 1.5 1.4 3.0

Table 6. The determination of carotenoids extracted from several P. rhodozyma strains using the improved HPLC method

Strain

15r JMUMVP14 ly322 7B12 BlR B4M

Peak 6 Peak 9 Peak 2 Peak 1 Peak 8 Peak 5 Peak 4 Peak 3 Peak 7 astaxan zeaxan βcrypo βcaro lycopene 30.8 min, 32.0 min, 32.8 min, toxanthin 37.5 min, thin thin tene 37.8 min, % % % % 19.2 min, 23.2 min, 33.9 min, 38.3 min, % % % % % 85.65 31.84 – 97.73 – –

– 2.58 – – – –

– 8.73 – – – –

– 7.19 – – – –

Nature Science Foundation of China (20702019), Fujian Provincial National Nature Science Founda tion (2010N5009) and foundation for innovative research team of Jimei University (2010A006). REFERENCES 1. Andrews, A.G., Phaff, H.J., and Starr, M.P., Phy tochemistry, 1976, vol. 15, p. 1003. 2. Johnson, E.A. and An, G.H., Crit. Rev. Biotechnol., 1991, vol. 11, p. 297. 3. An, G.H., Cho, M.H., and Johnson, E.A., J. Biosci. Bioeng., 1999, vol. 88, p. 189. 4. Visser, H., Ooyen, A.J., and Verdoes, J.C., FEMS Yeast Res., 2003, vol. 4, p. 221. 5. Guerin, M., Huntley, M.E., and Olaizola, M., Trends Biotechnol., 2003, vol. 21, p. 210. 6. Kennedy, T.A. and Liebler, D., J. Biol. Chem., 1992, vol. 267, p. 4658. 7. Chew, B., Park, J., Wong, M., and Wong, T., Anticancer Res., 1998, vol. 19, p. 1849. 8. Mayne, S.T., FASEB J., 1996, vol. 10, p. 690. 9. Lysenko, V.S., Chistyakov, V.A., Zimakov, D.V., Soier, V.G., Sazykina, M.A., Sazykina, M.I., Sazykin, I.S., and Krasnov, V.P., J. Anal. Chem., 2011, vol. 66, p. 1281. 10. Misawa, N. and Shimada, H., J. Biotechnol., 1998, vol. 59, p. 169. JOURNAL OF ANALYTICAL CHEMISTRY

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– 14.09 – – – –

14.35 4.62 67.73 – 66.24 60.64

– 5.50 13.35 – 8.82 21.80

– 10.95 7.61 – 7.32 –

– 14.49 11.31 2.27 17.61 17.56

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