1
Quantitative Analysis of Carotenoids in Human Plasma
Researcher: Qiyun Zhou
Supervisors: Dr. Marion Priebe
Dr. Han Roelofsen Nicolai Kraut
Mentor: Prof. Dr. Roel Vonk
Departments: Centre of Medical Biomics University: University of Groningen Date: 25-Aug-2010
February – August 2010
2 CONTENTS
ABSTRACT ……….……… 3
1. INTRODUCTION ………..….… 4
2. MATERIALS AND METHODS ...………...……….……. 7
2.1 Chemicals ……….………. 7
2.2 Stock solutions and protein precipitation solvents ……….…....…. 7
2.3 Columns and guard columns ……….……….……… 7
2.4 Collection of plasma samples ………. 7
2.5 Extraction of carotenoids from plasma samples ……….………. 7
2.6 Carotenoids identification by mass spectrometer ……… 8
2.7 Sample analysis by high performance liquid chromatography (HPLC) UV/VIS system ………...…. 8
2.8 Data processing ……….... 9
3. RESULTS ……….. 10
3.1 Establishment the analytic methods of carotenoids ………... 10
3.2 Analysis of carotenoids by LC-MS ………....…………..… 11
3.3 Issues of the temperature ……….……… 13
3.4 Issues of the mixer in HPLC ……….……… 15
3.5 Calibration curves of the lutein / zeaxanthin concentration in human-plasma ……… 16
3.6 Determination of lutein and zeaxanthin concentration from two subjects . 18 3.7 Improvement of recovery of the carotenoids ……….………. 19
4. DISSCUSSIONS & CONCLUSION ……….. 25
REFERENCES ………. 28
APPENDIX ………..……….. 30
ACKNOWLEDGEMENTS ………... 33
CURRICULUM VITAE ………. 34
3 ABSTRACT
Lutein and zeaxanthin are two major hydroxycarotenoids (xanthophylls) which are responsible for the health of human eyes. A HPLC method using YMC C-30 column has been developed and validated for the determination of these two compounds. After protein precipitation and re-extraction, lutein, zeaxanthin and the internal standard echinenone in human plasma were baseline separated using this HPLC method. The analytic method was reproducible, specific and precise. In addition, the recoveries of carotenoids from human plasma have been investigated in this study. Important factors that influence the recovery rates were investigated and the extraction protocol was optimized. The practical applicability of the methods developed here was demonstrated by a pilot experiment to determine the lutein and zeaxanthin concentration in plasma from two subjects. The level of lutein and zeaxanthin in the non-fasted subject was significant higher than in the fast subject. This work can serve as the basis for the future large-scale clinical experiments of bioavailability of carotenoids in human after consuming xanthophylls-rich foods such as kale, spinach and some kinds of potatoes.
4 1. INTRODUCTION
Carotenoids are plant pigments of various colors such as yellow, orange and red, which are found in almost all plants and are taken up into the human body mainly from consumed fruits and vegetables.1
Two major hydroxycarotenoids (xanthophylls) are lutein and zeaxanthin, which are entirely of dietary origin and are the only carotenoids present in the macula,2 the side in the human eye providing the visual acuity. It is generally thought that the two compounds may play an important role in protection against the age-related macular degeneration (AMD).1,2
A
B
Figure 1. The chemical structures of lutein and zeaxanthin.
A. Lutein. B. Zeaxanthin
In the whole carotenoids family, lutein and zeaxanthin are more hydrophilic than the hydrocarbon carotenes because they have oxygen atoms in their molecular structures.3 (Figure 1) Lutein and zeaxanthin are isomers and lutein can be converted into meso- zeaxanthin, both in plants and in the retina.4,5
The absorption or bioavailability of the carotenoids can be investigated by measuring their plasma concentrations after consumption. Plasma concentrations of carotenoids are determined by the rates of absorption, removal from plasma to tissues, and efflux from tissues. Several processes are necessary in the absorption of carotenoids,6 which include sufficient digestion, 7 formation of lipid micelles in the small intestine and uptake of carotenoids by intestinal mucosal cells. (Figure 2) After the absorption by intestinal cells, carotenoids are mostly incorporated into chylomicrons in the intestinal mucosa cells and secreted into the lymph.8 Several studies have proven that the dietary fat is important or sometimes essential for the carotenoids absorption.9 The dietary fat may serve at least two functions in the absorption: it provides a hydrophobic domain within which carotenoids are solubilized, and it stimulates bile flow from the gall bladder, which is essential in the presence of bile salt micelles. For the hydrolysis of carotenoid esters, dietary fat can stimulate the digestion by stimulating the pancreatic enzyme secretion.10,11
5 Figure 2. The conceptional absorption route of carotenoids. (C-carotenes, X-xanthophylls) Competition between carotenoids in the intestinal absorption happens in transport and metabolism because they follow the same absorptive pathway. Reductions of lycopene or /and lutein were observed in response to a high dose (from 12 mg to 100 mg) of ß- carotene intake (from 6 days to 6 weeks), which suggested that the carotenoids were competing for absorption or /and transport.12,13,14 Conversely, if the carotenoids level is boosted by ß-carotene (20 mg per day) for 24 months, the serum concentrations of lycopene (available only in certain food items such as tomatoes, tomato products and watermelon) and α-carotene increased.15 It is still not clear whether these increments are due to the interactions among different carotenoids during absorption or there is a regulatory mechanism controlling the balance or equilibrium of biological concentrations of ß-carotene and other carotenoids.
In case of the digestive phase of lutein and zeaxanthin, it was proven that zeaxanthin was preferentially solubilized in the polar lipid surface of the biological emulsions while the nonpolar ß-carotene was preferentially solubilized in the biological emulsion triglyceride core.16 This may be supported by their different polarity.
Compared to carotenes, it seems that lutein and zeaxanthin need a shorter time to reach their peak plasma concentrations after consumption, and are cleared more rapidly from plasma.17 This probably due to the fact that lutein and zeaxanthin are easier to go to the surface of chylomicrons when carotenes going into the internal core of chylomicrons，
11, 18
which suggested that xanthophylls are earlier to be absorbed and released from chylomicrons than carotenes. Another possibility is that there may be an intestinal
hydrolysis s
Emulsified particles c x
x
x
c c
c x
Bile salts
Micelle s
x x
x
x x
x c c
c c
c c
Unstirred water layer
Intestinal mucosal cell
c x
Chylomicrons x c
x c
x c x c Food
matrix c
x x c
x c
x c VLDLs
6 discrimination in favor of xanthophylls by intestinal carotenoid binding proteins which is not clear yet.19 This information may provide an advice that when study the bioavailability of xanthophylls in the future, the plasma samples should be taken at earlier stage of ingestion.
Since lutein and zeaxanthin are thought to associate with the surface layer of the lipoprotein, it can be assumed that the larger particles can carry more xanthophylls.
Most studies showed the lutein and zeaxanthin are evenly distributed between HDL and LDL, with small amounts associated with VLDL, since the specific surface area of HDL is larger than other lipoproteins. In 2007, the first study of the relationships between the number and the size of LDL and HDL particles and plasma lutein concentrations was performed. It was reported that the size of LDL and HDL particles were positively correlated with circulating lutein.20 The amounts of lutein molecules per lipoprotein are approximate 2 lutein molecules per VLDL particle, 3 lutein molecules per 10 LDL particles, and 7 lutein molecules per 1000 HDL particles. In another study, HDL was the major lipoprotein transporter of lutein (52%) and zeaxanthin (44%). LDL was the major transporter of α-carotene (50%), ß-carotene (55%), and lycopene (57%).21
Most health effects of lutein and zeaxanthin reported in literature are claimed to be due to their powerful antioxidant and eye health effects. A number of epidemiological studies indicated that antioxidants, especially lutein and zeaxanthin, which can be accumulated by the retina and form the yellow macular pigment (MP) are protectors against AMD.
1,2,3,4
According to their antioxidant properties and anti-inflammatory properties,22 carotenoids including lutein and zeaxanthin may be used to prevent tissues from oxidative stress.
Nowadays, these compounds have been suggested to prevent cardiovascular diseases (CVD)23,24 and protect skin from damage caused by ultraviolet light (UV).25
The aim of this project was to optimize the protocol of carotenoids extraction from human plasma samples, with the goal to improve the recovery rate and the reproducibility of the extractions. Moreover, a HPLC method is established, which can separate and qualify the carotenoids within 45 min. A comparison of lutein and zeaxanthin between one fasted and one non-fasted subjects also need to be tested by a confirmed HPLC gradient.
7 2. MATERIALS AND METHODS
2.1 Chemicals
Acetone (for HPLC, ≥99.9%), methanol (MeOH) (for LC/MS, ≥99.9%), tert-butyl methyl ether (MTBE) (for HPLC, ≥99.9%) and acetonitrile (ACN) (for LC/MS, ≥99.9%) were obtained from Sigma-Aldrich/Fluka, Germany. Ultra-pure water (for UPLC/MS) was purchased from Biosolve, USA. Dichloromethane (for HPLC, ≥99.8%) was purchased from VWR-Prolabo, France. Ammonium acetate (for analysis, ≥98%) was from Merck and Butylated hydroxytoluene (≥99.9%) was from SAFC, Germany.
Analytical standards, including echinenone 1 mg (HPLC 98%, synthetic, crystal), lutein 1 mg (HPLC 94%, isolated, crystal) and zeaxanthin 1 mg (HPLC 97%, synthetic, crystal) were obtained from CaroteNature GmbH.
2.2 Stock solutions and protein precipitation solvents
The stock solutions of the standard compounds were prepared as following: 1 mg of echinenone was dissolved in ethanol; 1 mg of lutein was dissolved in dichloromethane; 1 mg of zeaxanthin was dissolved in dichloromethane. All the solutions were filled up to 50 mland stored in 50 mL amber glassvolumetric flasks at -20 °C until the further use.
The most commonly used protein precipitation solvent in the experiments was MeOH:
ACN: acetone (1:1:1, v/v/v) with 0.1% (m/v) BHT, unless stated otherwise.
Dichloromethane and acetone were chosen in the “modification of extraction protocols”
study.
2.3 Columns and guard columns
The following YMC C30 columns and guard columns were used: YMC C30 250 x 4.6 mm i.d. S-3µm, with the guard column YMC C30, 10 x 4.6 mm i.d. S-3µm and its direct connectable holder (YMC Europe); YMC C30 250 x 2.1 mm i.d. S-3µm, with the guard column YMC C30, 10 x 2.0 mm i.d. S-3µm.
2.4 Collection of plasma samples
Venous blood samples were collected in EDTA vacutainers (10 mL, K2E 18.0 mg, Beliver Industrial Estate, UK) from each subject, kept at 0 °C (less than 20 min) and centrifuged at 3000g at 4 °C for 10 min (X-22R Centrifuge, Beckman Coulter, USA).
The plasma samples were divided into 0.5 mL aliquots in each glass tube and frozen at - 20°C until further analysis.
8 2.5 Extraction of carotenoids from plasma samples
Plasma extraction was preformed under subdued light. Each tube of plasma (0.5 mL) was thawed at room temperature, and 100 µ L of echinenone standard solution was added as an internal standard before adding 2.0 mL of the protein precipitation solvent (kept at 0 °C). The solutions were mixed by MS2 minishaker (IKA) immediately for 15 sec and kept still at 0°C for 30 min.
After centrifugation at 3000g at 4 °C for 10 min, 2 mL of the supernatant was taken from each tube without disturbing the pellet. Subsequently, the organic solvents were dried under N2 gas by using “Heating / Stirring Module” and “Evaporation Unit” (Thermo Scientific, Bremen, Germany), with or without heating in different temperatures in a dark air-flow chamber.
Samples were extracted twice with 2.0 mL hexane containing 0.01% (m/v) BHT (kept at 0 °C). In each extraction step, the samples and hexane were mixed by vortexing for 15 seconds and then centrifuged at 200 g at 4 °C for 5 min. The combined hexane extracts were dried under N2 gas and reconstituted in 200 µL of the eluent A and kept in amber glass vials at -20 °C.
2.6 Carotenoids identification by mass spectrometer
Carotenoids were identified by a triple quadrupole mass spectrometer (TQ Vantage, Thermo Scientific). The molecular mass and the fragmentations of lutein, zeaxanthin and echinenone were identified and the data were processed by using the programme:
“Thermo Xcalibur” (Version 2.1.0). The order of appearance of carotenoids peaks was identified by testing the corresponding standard solutions.
The ion source was operated at 5.5 kV of capillary voltage, vaporizer temperature was set at 300 °C, capillary temperature was 300 °C, sheath gas pressure maintained at 0.0 arbitrary units, aux gas pressure was 10 arbitrary, S-lens RF amplitude: SRM table, and declustering voltage was nil.
2.7 Sample analysis by high performance liquid chromatography (HPLC) UV/VIS system
The extracts of samples were analysed by reverse-phase HPLC. The analysis was performed on a Shimadzu HPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with a degasser, a communications bus module, an autosampler and an UV/VIS detector. Two types of YMC C30 columns with their guard columns were used. The analysis of carotenoids was carried out at room temperature, with detection at 447 nm.
The system “Solutions” was used to collect and integrate the data.
The eluents used in the LC-MS were: eluent A: mixture of MeOH, MTBE, H2O and 1M Ammonium acetate in MeOH [81:15:4:1 (v/v/v/v)]; eluent B: mixture of MeOH, MTBE and 1M Ammonium acetate in MeOH [6:90:1 (v/v/v)]. The eluents used in the HPLC
9 UV/VIS were: eluent A: mixture of MeOH, MTBE, H2O [81:15:4 (v/v/v)]; eluent B:
mixture of MeOH, MTBE [1:15 (v/v)]. In two HPLC UV/VIS studies, the former eluents was used and will be mentioned in the RESULTS.
2.8 Data processing
To determinate the concentration of lutein and zeaxanthin in plasma samples, the ratio of peak area between lutein / zeaxanthin and the internal standard-echinenone was used. To investigate the extraction efficiency, this formula was used:
Extraction efficiency = (Avg. peak area E* from extraction / Avg. peak area E from standard solution) / (C’E* / Concentration of standard E) x 100%,
*E: echinenone,
*C’E: expected concentration of echinenone in plasma-extraction samples.
The data were processed by EXCEL.
10 3 RESULTS
3.1 Establishment the analytic methods of carotenoids
To separate and identify lutein, zeaxanthin and the internal standard echinenone from plasma-extraction samples, HPLC analysis was used. In the pilot study, a plasma- extraction sample was tested by a full-screen (eluent B: 0%--100% in 90 min, see Appendix. Figure 1) reverse-phase HPLC UV/VIS measurement. YMC C30 column (250 x 4.6 mm i.d. S-3µm) was used and the flow was 1 mL/min.
The result was compared to a profile from www.ymc-europe.com (YMC Europe GmbH) to identify the sequence of appearance of the different carotenoids (Figure 3).
Figure 3. HPLC profile of a plasma-extraction sample with additional echinenone.
Lutein
Echinenone Zeaxanthin
11 This reverse-phase HPLC profile showed that the order of carotenoids peaks was: lutein (Retention Time: 14~15 min), zeaxanthin (Retention Time: 17~18 min) and echinenone (Retention Time: 27~28 min), and all of them could be eluted out before the gradient reached 30 % of eluent B.
Figure 4. HPLC profile of a 20 µL standard solution with 0.44 µM lutein, 0.44 µM zeaxanthin and 0.45 µM echinenone in eluent A, tested by a gradient shown in Appendix. Figure 2.
After the success of the initial experiment, one gradient elution program was set up (see Appendix. Figure 2, flow: 1.0 mL/min) to optimize the separation of the compounds.
The targeting carotenoids (lutein, zeaxanthin and echinenone) could be eluted out by this method (Figure 4) with better resolution. This gradient was commonly used unless mentioned otherwise.
3.2 Analysis of Carotenoids by LC-MS
In order to confirm the identification of carotenoids in the liquid chromatography, the MS profile of lutein, zeaxanthin and echinenone authentic standards were tested.
After optimising the parameters, the parent mass and the product mass of lutein, zeaxanthin and echinenone were identified by Electrospray ionisation (ESI)-MS.
(Table 1)
Table 1: The parent mass and the product mass of carotenoids by using ESI-MS.
Carotenoids Lutein Zeaxanthin Echinenone
Parent mass 568.3 568.3 551.2
Product mass 476.4, 338.3 476.4, 338.3 459.6, 203.3
Optimized settings: Spray Voltage: 5500 V, Vaporizer Temperature: 300, Sheath Gas Pressure:
0.0, Aux Gas Pressure: 10, Capillary Temperature: 300, S-lens RF Amplitude: SRM Table, Dedustering Voltage: 0.
The reliability of the HPLC analysis was examined. Standard solution of lutein and zeaxanthin were injected 5 times individually. The analyses were performed with a YMC C30 column (250 x 4.6 mm i.d. S-3µm). The carotenoids were separated by a gradient elution at the flow rate of 1 mL /min. The injection of volume of each sample was 10 µL.
Lutein
Zeaxanthin
Echinenone
12
Area of lutein peaks
0 10000 20000 30000 40000 50000 60000
1 2 3 4 5
Manually Area
Area of zeaxanthion peaks
0 10000 20000 30000 40000 50000 60000
1 2 3 4 5
manually Area
The peak areas (PA) of lutein and zeaxanthin were shown in Figure 5. The standard deviation (STDEV) of the peaks in 5 runs of lutein was 1066 and the STDEV of the zeaxanthin peaks in 5 runs was 1756.
A B
Figure 5. LC-MS results (PA) of Lutein and Zeaxanthin standard solutions.
A. The peak areas of lutein in 5 runs.
B. The peak areas of zeaxanthin in 5 runs.
Figure 6. HPLC profile of lutein standard solution by LC-MS.
13 Figure 7. HPLC profile of zeaxanthin standard solution by LC-MS.
As expected, the retention time (RT) of lutein was shorter than the RT of zeaxanthin. For lutein, a minor peak after the main peak was detectable (Figure 6, 7). It is proposed that this minor peak could be zeaxanthin, which can be converted from dietary lutein.4 Other observations from the MS analysis of zeaxanthin standard sample showed that a minor lutein peak before the main peak was also detectable, could be supported by the RT.
Figure 8. The shift of retention time (RT) of lutein and zeaxanthin by LC-MS.
The curve of the shifting of retention time (Figure 8) accounted 5 runs of lutein samples and 5 runs of zeaxanthin samples. Both the main peaks and the minor peaks were taken into account. It shows the RTs of both lutein and zeaxanthin were decreasing continuously. This phenomenon might due to the shifting of temperature.
Retention time of L / Z
15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00
1 2 3 4 5 6 7 8 9 10
Retetion time
lutein peak Zeaxanthin peak Poly. (Zeaxanthin peak) Poly. (lutein peak)
14 In this project, HPLC UV/VIS method was most commonly used to analyze the samples.
LC-MS can be another supplementary method to measure the carotenoids and it is expected to cost less time. In this way, an LC-MS method needs to be established further on.
3.3 Issues of the Temperature
In order to investigate how much the shifting of temperature can influence the RT in the HPLC analysis, one study was done by HZPC. Three temperatures were set up in the oven: 20 °C, 25 °C and 30 °C. Standard solution of lutein and zeaxanthin was injected and analyzed 5 times with each temperature setting. The average RT and PA were showed in Table 2, Figure 9, 10. It is clear that when the temperature rose in the column, the RT of peaks of carotenoids was shorter. There was no significant difference of PAs between the groups set with temperature 20 °C and 25 °C, but there was significant difference of PAs between the groups set with temperature 25 °C and 30 °C. (Table 2) Table 2: The RT and PA of lutein and zeaxanthin peaks. (Analyzed by LC-MS in HZPC.)
RT 1 PA 1 RT 2 PA 2
Temperature Avg. STDEV Avg. STDEV Avg. STDEV Avg. STDEV
20 °C 5,90 0,005 50,34 1,895 6,67 0,006 250,54 12,753
25 °C 5,54 0,004 52,01 0,156 6,04 0,004 242,87 0,396
30 °C 5,02 0,040 47,82 0,202 5,52 0,049 264,49 1,575
Between the groups: 20 °C and 25 °C, p value of PA 1 = 0.130, p value of PA 2 = 0.251;
Between the groups: 25 °C and 30 °C, p value of PA 1 < 0.001, p value of PA 2 < 0.001;
Figure 9. The average RT analyzed at 20 °C, 25 °C and 30 °C by LC-MS in HZPC.
Average of Retention Time
0 1 2 3 4 5 6 7 8
20 °C 25 °C 30 °C
Tem.
RT Lutein
Zeaxanthin
Average of Peak areas
100 150 200 250 300
PA Lutein
Zeaxanthin
15
Figure 10. The average PA analyzed at 20 °C, 25 °C and 30 °C by LC-MS in HZPC.
3.4 Issues of the mixer in HPLC
In order to investigate whether the mixer in the HPLC could cause an influence of the HPLC results, one study designed of using one eluent (8 0% of eluent A +20 % eluent B, pre-mixed before the analysis) or two eluent with a constantly 20 % B gradient was carried out (Table 3 , 4). A standard solution with zeaxanthin and echinenone was injected.
Table 3. The HPLC results of a standard solution by using a pre-mixed eluent (80% of eluent A +20% eluent B).
RT (Z) PA (Z) RT (E) PA (E)
1 7.200 138845 15.566 116337
2 7.174 133974 15.516 114323
3 7.173 139001 15.522 120103
4 7.172 140838 15.540 117318
5 7.192 143943 15.587 122413
6 7.215 147021 15.663 124709
Average 7.188 140604 15.566 119201
STDEV 0.017694 4516.684 0.054702 3924.581
Table 4. The HPLC results of a standard solution by using multiple pumps mixing eluent A and eluent B.
RT (Z) PA (Z) RT (E) PA (E)
1 7.375 131783 16.079 112987
2 7.387 134573 16.085 115694
3 7.361 135965 16.040 115132
4 7.369 134664 16.060 114284
5 7.390 137232 16.139 114715
16
6 7.411 134838 16.160 113389
7 7.398 132929 16.128 111356
8 7.393 127614 16.106 109539
9 7.386 129177 16.107 110872
Average 7.386 133197 16.100 113108
STDEV 0.015282 3163.916 0.038494 2110.982
The STDEV of the second method was not higher than the first one, which means the mixer showed good repeatability.
17 3.5 Calibration curves of the lutein / zeaxanthin concentration in human- plasma
To measure the lutein and zeaxanthin concentration in human-plasma extractions, the calibration curves of these two compounds in plasma samples (from one subject) were prepared by using the HPLC UV/VIS analysis. The ratio of peak areas between the targeting carotenoids and the internal standard was used. The calibration curves were shown in Figure 11 and Figure 12.
In the extraction of the plasma samples, 20 µL standard solution of lutein [0 µM, 7.5µM, 15 µM, 30 µM in ethanol, prepared from stock solution, with 0.1% BHT (m/v)] or 10 µ L standard solution of zeaxanthin [0 µM, 5µM, 10 µM, 20 µM in CH2Cl2, prepared from stock solution, with 0.1% BHT (m/v)] were added individually after thawing the plasma samples (0.5 mL). 100 µL internal standard (1.815 µM echinenone in ethanol into zeaxanthin-added samples and 18.15 µM echinenone in ethanol into lutein-added samples) was added in each plasma samples before the extraction.
Each lutein spiked-sample was measured twice immediately by HPLC UV/VIS without keeping at -20 °C (Table 5). HPLC analysis of zeaxanthin spiked-samples were conducted 9 days after the extraction and each sample was measured once (Table 6). All the HPLC analysis was performed with an YMC C30 column (250 x 4.6 mm i.d. S-3µm) by the commonly used gradients (Appendix. Figure 2) and 20 µL of each sample was injected.
In the lutein added-samples, the expected concentrations of additional lutein in plasma were 0 µ M, 0.3µM, 0.6µM, 1.2 µM. The expected concentration of echinenone in plasma was 3.63 µM. In the zeaxanthin-added samples, the expected concentrations of additional zeaxanthin in plasma were 0 µM, 0.1µ M, 0.2µM, 0.4 µ M. The expected concentration of echinenone in plasma was 0.363 µM.
Table 5. The HPLC UV/VIS results of plasma-extraction samples with additional lutein.
Sample* RT of L
(min) PA of L RT of Z (min)
PA of Z RT of E (min)
PA of E L/E (PA)
Avg.
L/E
Z/E (PA) 0 L 14,910 65922 17,542 9823 33,984 53654 1,229
1,169 0,183 15,003 96974 18,019 13339 34,179 87352 1,110 0,153 0.3L 14,876 135757 17,841 13456 33,938 79451 1,709
1,698 0,169 14,983 130463 18,013 14383 34,117 77307 1,688 0,186 0.6L 14,824 179594 17,797 17916 33,914 75613 2,375
2,407 0,237 14,968 204926 17,970 20603 34,167 83997 2,440 0,245 1.2L 14,912 223373 17,901 17468 34,014 77028 2,900
2,930 0,227 14,989 295877 17,995 24766 34,157 99939 2,961 0,248
*4 tubes of plasma sample were used in this experiment in total.
[Abbreviations: 0 L: sample with 0 µM additional lutein, 0.3L: sample with 0.3 µM additional lutein, 0.6L:
sample with 0.6 µM additional lutein, 1.2L: sample with 1.2 µM additional lutein, RT of L: Retention Time of lutein, PA of L: Peak Area of lutein, RT of Z: Retention Time of zeaxanthin, PA of Z: Peak Area of zeaxanthin, RT of E: Retention Time of echinenone, PA of E: Peak Area of echinenone, L/E (PA): ratio of PA between lutein and echinenone, Avg. L/E: average of L/E (PA), Z/E (PA): ratio of PA between zeaxanthin and echinenone.]
18 Calibration curve of additional Lutein
y = 1,4761x + 1,2763 R2 = 0,9523
0 0,5 1 1,5 2 2,5 3 3,5
0 0,3 0,6 0,9 1,2
C of addtional L (µM)
L/E (Peak area)
Calibration Curve of additional zeaxanthin
y = 13,871x + 1,176 R2 = 0,9928
0 1 2 3 4 5 6 7 8
0 0,1 0,2 0,3 0,4
C of addtional Z (µM)
Z /E (Peak Area)
Table 6. The HPLC UV/VIS results of plasma-extraction samples with additional zeaxanthin.
Sample
*
RT of L
(min) PA of L RT of Z (min)
PA of Z RT of E (min)
PA of E L/E (PA)
Z/E (PA)
Avg.
Z/E 0Z A 15,043 84870 18,138 9991 34,659 8726 9,726 1,145
1,135 0Z B 14,983 43434 18,076 4685 35,651 4163 10,433 1,125
0.1Z A 14,905 68219 17,843 19711 34,101 8027 8,499 2,456
2,788 0.1Z B 15,038 75563 18,137 21958 34,651 7038 10,736 3,120
0.2Z A 14,495 99639 17,298 38381 33,589 10793 9,232 3,556
3,695 0.2Z B 14,986 104217 18,025 38105 34,524 9938 10,487 3,834
0.4Z A 14,475 107110 17,293 71970 33,556 10322 10,377 6,972
6,796 0.4Z B 15,035 77230 18,074 50977 34,598 7701 10,029 6,620
*8 tubes of plasma sample were used in this experiment totally. In each concentration of additional zeaxanthin, 2 samples were prepared.
[Abbreviations: 0 Z: sample with 0 µM additional zeaxanthin, 0.1Z: sample with 0.3 µM additional zeaxanthin, 0.2Z: sample with 0.6 µM additional zeaxanthin, 0.4Z: sample with 1.2 µM additional zeaxanthin, Avg. Z/E: average of Z/E (PA).]
Figure 11. Calibration curve of additional lutein (0 µM~1.2 µM) in plasma extraction samples.
Figure 12. Calibration curve of additional zeaxanthin (0 µM~0.4 µM) in plasma extraction samples.
19 The interceptions of these two figures showed the backgrounds of lutein and zeaxanthin in plasma as we expected. Both of these figures had good linearity in the expected range, which suggested that in further studies, the concentration of lutein can be investigated up to 1.2 µM and the concentration of zeaxanthin can be tested up to 0.4 µM in plasma.
3.6 Determination of lutein and zeaxanthin concentration from two subjects The aim of this study was to investigate the different levels of lutein and zeaxanthin in one male subject (P1) and one female subject (P2). The male subject was fasted and the female subject was not fasted before the experiment.
The plasma samples were taken in Aug 2009 and were kept at -20 °C. 3 samples (0.5 mL) from each subject were used. In the sample preparation protocols, 10 µL standard solution of zeaxanthin [5µM, 10 µM, 20 µM in CH2Cl2, prepared from stock solution, with 0.1% BHT (m/v)] was added individually into every 3 samples and 100 µ L internal standard of echinenone (1.815 µM in ethanol) was added in each sample before the extraction steps. In this way, the additional concentration of zeaxanthin in each plasma sample can be calculated:
Cadd Z1 = 5 µM x 0.01 mL / 0.5 mL = 0.1 µM Cadd Z2 = 10 µM x 0.01 mL / 0.5 mL = 0.2 µM Cadd Z3 = 20 µM x 0.01 mL / 0.5 mL = 0.4 µM
The extractions were kept at -20°C. HPLC UV/VIS analysis of these zeaxanthin-added samples was carried out 4 days later, which was not tested by the commonly used gradient but eluent B: 0%-40% in 55 min (Appendix. Figure 3), column: YMC C30 column (250 x 4.6 mm i.d. S-3µm), flow: 1.0 mL/min. 10 µL of each sample was injected and was measured once.
The ratio of peak areas between carotenoids and internal standard was used. The RT and PA were showed in Table 7 and the calibration curves were shown in Figure 13.
Table 7. The HPLC UV/VIS results of plasma-extraction samples with additional zeaxanthin.
Sample Lutein
[RT(min)/PA]
Zeaxanthin [RT(min)/PA]
Echinenone [RT(min)/PA]
L/E (PA)
Z/E (PA) P2 + 0.1M Z 14,745/24076 17,701/18226 33,908/7738 3,111 2,355 P2 + 0.2M Z 14,832/27490 17,813/24665 34,117/9011 3,051 2,737 P2 + 0.4M Z 14,718/25952 17,641/30567 33,876/8578 3,025 3,563 P1 + 0.1M Z 14,690/15552 17,662/5450 33,881/6667 2,333 0,817 P1 + 0.2M Z 14,805/18303 17,799/8961 34,059/8481 2,158 1,057 P1 + 0.4M Z 14,726/16672 17,686/10473 33,921/6286 2,652 1,666
20
Figure 13. Calibration curves of additional zeaxanthin in human-plasma extraction samples from two subjects.
The curves did not start from 0 because the backgrounds were not measured. After calculating the average ratio of PA between lutein and echinenone (L/E):
L/E (PA)P2 = (3.111+3.051+3.025) /3 = 3.06, L/E (PA)P1 = (2.333+2.158+2.652) /3 = 2.38,
The lutein level in P2 was higher than in P1. (p=0.005) This result suggested that the not-fasted subject had more lutein and zeaxanthin in the plasma than the fasted subject.
However, we cannot conclude that the difference between the fasted group and the not- fasted group due to the limited amount of subjects.
3.7 Improvement of recovery of the carotenoids 3.7.1 Recovery of the carotenoids
After establishing the HPLC analysis of the carotenoids, we further examined the efficiency of the extraction protocol. In this study, the recovery was tested twice by using the same extraction protocol (see Page 21, Table 9, without heating in the evaporation steps).
In the first experiment, the standard solution of echinenone [100 µ L of 1.815 µM E +200 µL eluent A, C’(E) = 0.605 µM] was prepared just before analysis. The gradient was shown in Appendix. Figure 4. 20 µL sample was injected. The column was YMC C30 column (250 x 4.6 mm i.d. S-3µm) and the flow was 1.0 mL/min. The average PA was calculated from 3 HPLC runs.
Avg. peak area E from standard solution = 12144.
8 samples were analyzed (the same as Table 6) 2 days after the extraction with the same method which was used in the analysis of standard solution of echinenone. 20 µL of each
Calibration curve
y = 2,86x + 0,5127 R2 = 0,9968 y = 4,0417x + 1,9423
R2 = 0,9996
0 0,5 1 1,5 2 2,5 3 3,5 4
0 0,1 0,2 0,3 0,4
C of additional Z (µM)
PA (Z) / PA (E)
MP N Linear (N) Linear (MP)
21 sample was injected and was measured once. The peak area of echinenone (PA of E) from these 8 samples was used to calculate the “Avg. peak area E from extraction” (see
“materials and methods”).
Avg. peak area E from extraction = 6743.
The expected concentration of echinenone in the 8 plasma samples can be calculated by the formula: C’E = 1.815 µM x 0.1 mL x (2/2.6)/0.2 mL = 0.698 µM. (2/2.6 was the extraction factor.)
Recovery 1 = (6743 / 12144) / (0.698 µM / 0.605 µM) x 100% = 48.1%
In another study, the data “PA of E” from Table 5 was used to calculated the Avg. peak area E from extraction, which was 79293. The expected concentration of echinenone in those 4 samples was 6.98 µM, according to the extraction protocols.
The 3 standard solutions of lutein and echinenone were prepared to measure the Avg. PA of echinenone (Table 8) just before analysis. The HPLC method and the column were same as used in Table 5. 40 µL of each sample was injected. Each sample was measured twice.
Table 8. Standard solutions and HPLC results of echinenone.
Sample preparation Concentration of L
Concentration of E
PA of E
200 µL 18.15 µM E + 66.7 µL 7.5 µM L
+ 233.3 µL eluent A 1 µM
7.26 µM
233453 364892 200 µL 18.15 µM E + 66.7 µL 15 µM L
+ 233.3 µL eluent A 2 µM 246314
275494 200 µL 18.15 µM E + 66.7 µL 30 µM L
+ 233.3 µL eluent A 4 µM 332490
375396 Avg. peak area E from standard solution = 304673.
Recovery 2 = (79293 / 304673) / (6.98 µM / 7.26 µM) x 100% x (20/40)* = 54.1%
(20/40)* was accounted here because of the different volume of samples which were injected into HPLC.
In this study, only the data of echinenone were used in the calculations. The outcome was only from 48.1 % to 54.1 % but reasonably reproducible. The low recovery rate may due to several reasons, which will be investigated individually in further experiments.
22 3.7.2 Carotenoids loss in the extraction process
Since the recovery was only less than 55% in the previous studies, the loss of carotenoids in each extraction step was investigated. In this study, internal standard was added before the different steps to check which step is essential for the carotenoids loss.
5 tubes of plasma (0.5 mL each) from one subject were used in this study. 100 µ L of echinenone (1.815 µM) was added into the tubes before the steps (Table 9) below individually: 1 (1), 1 (4), 1 (6), 2 (2), 2 (9). The samples were named as 1 (1), 1 (4), 1 (6), 2 (2), 2 (9).
The expected concentration of Echinenone in each sample was:
1 (1): 0.698 µM, 1 (4): 0.698 µM, 1 (6): 0.907 µM, 2 (2): 0.907 µM, 2 (9): 0.907 µM.
Before the HPLC UV/VIS analysis, the extraction samples were kept at -20 °C for 15 days. YMC C30 column (250 x 2.1 mm i.d. S-3 µm) was used at the flow: rate of 0.25 mL/min and the gradient was shown in Appendix. Figure 5. 10 µ L of each sample was injected. In the sample: 1 (1), 1 (4), 1 (6), 2 (2), one HPLC result (PA of echinenone) was used. In the sample 2 (9), 2 runs were used to calculate the average PA of echinenone.
Table 9. Extraction steps and numbering.
Number Step
1 (1) Add 2.0 mL protein precipitation solvent 1 (2) Vortex the mixture at 4°C for 15 sec 1 (3) Leave still at 0 °C for 30 min
1 (4) Centrifuge at 3000 g at 4 °C for 10 min
1 (5) Take 2.0 mL of the supernatant without disturbing the pellet
1 (6) Evaporate the organic phase under nitrogen stream at room temperature 2 (1) Add 2 mL of hexane into each tube
2 (2) Vortex the mixture at 4 °C for 15 sec 2 (3) Centrifuge at 200 g at 4 °C for 5 min 2 (4) Take out most of the upper organic layer 2 (5) Add 2 mL of hexane into each tube again 2 (6) Vortex the mixture at 4 °C for 15 sec 2 (7) Centrifuge at 200 g at 4 °C for 5 min
2 (8) Take out most of the upper organic layer and combine the upper organic layers 2 (9) Evaporate the organic extraction under N2 at room temperature
2 (10) Redissolve the pellet in 200 µL eluent A
23 Figure 14. The remaining of carotenoids in extraction samples
The peak area of echinenone was used to calculate the remaining of carotenoids in extraction samples. These data needed to be divided by the expected concentration of echinenone. Figure 14 showed the analysis of the remaining echinenone. The percentage of its loss was not calculated because the standard solution of echinenone was not measured by using the same gradient in the same day. However, after comparing the ratios between these samples, we can conclude that the essential steps for the carotenoids loss were step 1 (4) & 1 (5). This result indicated that the echinenone lost mostly in the pellet.
3.7.3 Modifications of the extraction protocol from plasma
The temperature in the two evaporating steps [1 (6) and 2 (9), see Table 9] and the protein participated solvent were the two factors to improve the extraction protocol in this study.
Based on the results above (3.7.2), the carotenoids are lost most in the pellet. The aim of the this study was to look for another protein precipitation solvent, which can both precipitate the impurity, especially proteins in human plasma samples and keep more carotenoids in the liquid layer than using the general protein precipitation solvent: MeOH:
ACN: acetone (1:1:1, v/v/v).
In the second study design, the protein participation solvent was changed into dichloromethane (CH2Cl2) or acetone in replacement of MeOH: ACN: acetone (1:1:1, v/v/v). After adding 100 µL of echinenone (1.815 µM in ethanol), three extractions from 3 tubes of plasma (0.5 mL each, from one subject) were carried out by using these solvents and followed by the same extraction protocol (Table 9). All the extraction samples were kept at -20°C until the HPLC analysis.
All the HPLC runs were performed with a YMC C30 column (250 x 2.1 mm i.d. S-3µm) by the two gradients (Appendix. Figure 6, 7) and 10 µL of each sample was injected.
The flow rate was 0.25 mL/min. Using the first gradient, the carotenoids peaks could not be separated well (see Appendix. Figure 8) and most peaks were lost in the sample prepared by using CH2Cl2 as protein precipitation solvent.
PA of Echinenone/expect C of E
0 10000 20000 30000 40000 50000 60000
1(1) 1(4) 1(6) 2(2) 2(9)
24 A
B
C
Figure 15. HPLC result of 3 plasma-extraction samples by using the gradient showed in Appendix. Figure 7 (with echinenone as an internal standard).
A) HPLC profile of a plasma-extraction sample by using CH2Cl2 as protein precipitation solvent.
B) HPLC profile of a plasma-extraction sample by using acetoneas protein precipitation solvent.
C) HPLC profile of a plasma-extraction sample by using MeOH: ACN: acetone (1:1:1, v/v/v)as protein precipitation solvent.
According to the HPLC profiles showed in Figure 15, the plasma-extraction samples were analyzed with the column YMC C30 (250 x 2.1 mm i.d. S-3µm) by the gradient shown in Appendix. Figure 7. Based on these HPLC results, the samples prepared by acetone (named as “by acetone”) and MeOH: ACN: acetone (1:1:1, v/v/v)(named as “by 1:1:1”) as protein precipitation solvent were taken into analysis. The sample “by acetone” was measured 3 times and the sample “by 1:1:1” was measured 5 times. Since the detection of zeaxanthin in these samples was insignificant, the peak areas of lutein and echinenone were taken into the calculation (Table 10).
25 Table 10. HPLC results of samples “by Acetone” and “by 1:1:1”.
Sample Average PA of lutein STDEV Average PA of echinenone STDEV
by acetone 108041 6409 33870 1161
by 1:1:1 137318 7069 24270 1197
The average PA of lutein in the sample “by acetone” was lower than in the sample “by 1:1:1”, but the average PA of echinenone in the sample “by acetone” was higher than in the sample “by 1:1:1”. This result indicated that acetone is a better solvent to extract more echinenone than MeOH: ACN: acetone (1:1:1, v/v/v), but the latter solvent is more suitable for lutein extraction.
In the second study design, 4 tubes of human plasma from one subject were needed. All the extractions should follow the protocol shown in Table 9 but the temperature in the two evaporating steps [1 (6) and 2 (9)] was changed to “no-heating”, “heating at 25°C”,
“heating at 30°C” and “heating at 35°C”. 100 µ L of echinenone (1.815 µM in ethanol) was added into the tubes before the step 1(1). The time cost of these evaporation steps has been recorded (by checking the evaporation unit in every 10 min, see Table 11).
After the extractions, these samples were kept at -20°C for 13 days before the HPLC analysis.
Table 11. The time cost of the evaporation steps.
Sample Time in step 1 (6) Time in step 2 (9)
“no-heating” 140 min 60 min
“heating at 25 °C” 60 min 50 min
“heating at 30 °C” 40 min 40 min
“heating at 35 °C” 40 min 40 min
In the HPLC UV/VIS measurement, parameters were the same as the former study.
Every 2 HPLC runs of each sample were used to calculate the average PA of lutein and echinenone (Table 12) except the sample 1(1).
Table 12. The average peak area of lutein and echinenone in samples.
Sample Average PA of lutein Average PA of echinenone
“no-heating” 87106 11170
“heating at 25 °C” 96375 22385
“heating at 30 °C” 143380 30999
“heating at 35 °C” 110248 23033
As shown in Table 12, 30 °C is the best evaporating temperature for the extraction of lutein.
26 4. DISCUSSIONS & CONCLUSION
The aims of this project were to optimize the extraction protocol of carotenoids from human plasma and to establish an analytic method of two main xanthophylls (carotenoids with hydroxyl groups), lutein and zeaxanthin, by using reverse-phase liquid chromatography.
Reverse-phase HPLC is an easy and reliable analytical method to separate and quantify the carotenoids. To determine the lutein and zeaxanthin concentrations in plasma, the first step is to build up the calibration curves. The set of concentrations of the standards should be near the expected concentration of the targeting compounds and must lie within the working range of the instrument. In this study, the linear ranges of the additional lutein and zeaxanthin are up to 1.2 µM and 0.4 µM, respectively. The PAs of the compounds are not directly used. Instead, the PA ratio between the carotenoids and internal standard were plotted against the concentration to make the calibration curve.
Therefore, the concentration of the added internal standard should be considered. The PA of the internal standard should be comparable with the PA of the carotenoids and should not be changed in the whole study. After establishing the calibration curve, the concentrations of the carotenoids in the human plasma extraction samples can be calculated. For real concentration, the results need to be corrected according to the recovery rate.
In the HPLC analysis, several factors influence the results and need to be concerned.
This HPLC analytical method is sensitive to the temperature. The shifting of column temperature would result in the alteration of both RT and PA. Due to the lack of temperature controlling device in our lab, the intra-day temperature variation could reach up to 2 degrees, which is likely the main cause for the RT shifting in this study. In this case, a column oven will be necessary to the control of the temperature and important in further studies.
Pressure is another parameter that should be considered. Stable pressure is required for the good reproducibility of the HPLC analysis. However, in this study, occasional pressure decreasing was observed, which may be caused by the leakage of connecting jacket between the pre-column and column.
For the future clinical studies of human plasma samples, time cost of the analytical method is crucial. Much more samples need to be analyzed within one or two days, since xanthophylls are unstable compounds. Therefore, to shorten the analytical time is one of the most important targets for the optimization of the protocol. The run time was already shortened to 45 min and established by using a YMC C30 column and still can be optimized by raising the gradient more quickly after the zeaxanthin peak appears. While further optimizations are expected, the current protocol could fulfill the demand of future clinical studies.
Besides the traditional HPLC analysis, we also established a LC-MS (QQQ) protocol for carotenoids analysis. LC-MS analytical method possesses advantages in higher
27 sensitivity and providing more specific information. In this study, the parameters of LC- MS have also been optimized to measure lutein, zeaxanthin and echinenone. The carotenoids were well separated and the mass information was obtained. Due to the limited time-scale of the project, this method was not used for analyzing real plasma samples. However, it could be served as an alternative analytical method in future studies.
The recovery rate of the carotenoids from the human plasma has been tested in this project was only 48.1 % and 54.1 %. The reasons for these low recovery rates may include the oxidation or degradation of carotenoids during the extractions and the loss in the pellet. The higher recovery rate suggested that the potential problems in the extraction were circumvented. Meanwhile, the repeatability is also critical for judging the protocols, which means the recovery rate of one carotenoid should be statistical constant.
The precision of the method should be examined by adding the targeting compounds (lutein and zeaxanthin) or the internal standard. In this project, the internal standard was added to calculate the recovery rate and only the result from one concentration was accounted in each test. In future, for intra-day accuracy, different replicated quality- control samples at each concentration should be assayed on the same day.xxvi To the best of our knowledge, the highest recovery of lutein from the literatures was 98.3%.xxvii In that study, ethanol was used to precipitate the proteins and hexane was used for the extraction. However, the details of the recovery test were not mentioned in that article.
The protein precipitation solvent MeOH: ACN: acetone (1:1:1, v/v/v) was chosen initially, since it is suggested that MeOH/ACN/acetone could display superior protein removal property and gave the most favorable intravariability results.28 Two other solvents were used in this project to compare the influence of the precipitation agents, which include commonly used lipophilic solvent acetone and CH2Cl2. The extraction sample prepared by using CH2Cl2 as the protein participation solvent did not give any significant peaks of all carotenoids. This might due to the fact that the sample was kept for 2 weeks before the analysis. While the recovery rate of lutein from the acetone precipitated sample was lower than the MeOH/ACN/acetone treated sample, the recovery rate of echinenone showed the opposite result. Since lutein is more polar than echinenone, the recovery rates of the two compounds could be variable by using different solvents. This result indicated that it is better to choose MeOH/ACN/acetone (1:1:1) as the protein precipitation solvent, since the research mainly focus on the xanthophylls. In future, ethanol can also serve such a role according to the high recovery rate result from the literature. In addition, this result suggested that the recovery rate of the internal standard can not represent the recovery rates of the other carotenoids.
The commonly used extraction protocol from human plasma samples would take 3-4 hours (see Table 9). In this long preparation, the loss of carotenoids could be significant.
In the modification of the extraction protocol, the temperature of the evaporating steps was proven to be an important factor for the recovery rate. The tubes could not be kept at room temperature under N2 in a no-heating condition, since the boiling points of the organic solvents are very low. By heating the tubes, the time cost of evaporating steps could be significantly shortened from 200 min to 80 min. Although the high temperature
28 may associate with carotenoids degradation, the recovery rates of the shorter protocol were still higher.
In conclusion, a HPLC UV/VIS analytical method was built up to measure the concentrations of carotenoids extracted from human plasma samples. Two factors of the extraction protocol were optimized. In the human pilot study, the difference of lutein and zeaxanthin level can be investigated. In future, this extraction protocol and this analytical method can be used.
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