A Versatile Detector for the Sensitive and Selective Measurement of Numerous Fat-Soluble Vitamins and Antioxidants in Human Plasma and Plant Extracts

Transcription

1 A Versatile Detector for the Sensitive and Selective Measurement of Numerous Fat-Soluble Vitamins and Antioxidants in Human Plasma and Plant Extracts Ian Acworth, Bruce Bailey, Paul Gamache, and John Waraska Thermo Fisher Scientific, Chelmsford, MA, USA Introduction Fat-soluble vitamins (FSVs) play essential roles in a wide spectrum of biochemical and physiological processes. One FSV, Vitamin E (tocopherol), along with a suite of other fat-soluble antioxidants (FSAs) (e.g., carotenoids, CoQ) are purported to help mitigate the potentially disastrous effects of oxidative stress that has been linked to numerous diseases including cancer, atherosclerosis, and neurodegenerative diseases. These compounds are thought to exert their beneficial effects by acting as chain-breaking antioxidants inhibiting lipid peroxidation of polyunsaturated fatty acids contained within biological membranes, thereby preventing the formation of potentially cytotoxic and highly reactive aldehydes (malondialdehyde and -hydroxynonenal). Although a number of FSVs and FSAs have been measured using HPLC-UV, this approach typically lacks the sensitivity and selectivity required to measure these compounds in biological samples. Electrochemical detection, however, is both sensitive and selective and makes use of the inherent redox activity of these compounds. The Thermo Fisher Dionex CoulArray Coulometric Array Detector the only HPLC electrochemical detector that is fully gradient compatible uses an array of flow-through, highly efficient electrochemical sensors to generate qualitative voltammetric data to help identify analytes and resolve coeluting compounds. The versatility of this detector is illustrated using a variety of examples including: a global gradient method for determination of FSVs and FSAs in plasma; a gradient method for the analysis of carotenoid isomers in carrots and human plasma; an isocratic method for the measurement of reactive nitrogen species damage to biomembranes measuring 5-nitro-g-tocopherol in rat astrocytes; a gradient method for the measurement of tocopherol and tocotrienol isomers in palm oil; and an isocratic method for the determination of reduced and oxidized CoQ9 and CoQ in human plasma. Global FSV and FSA Method The gradient analytical system consisted of two pumps, an autosampler, and a Dionex CoulArray detector. Column: Mobile Phase A: Mobile Phase B: MD5, 5 3 mm, 3 µm C8 Methanol:. M ammonium acetate, ph., (9:) (v/v) Methanol: -propanol:. M ammonium acetate, ph., (78::) (v/v/v) Gradient Conditions: Isocratic % B from to min. Linear increase of phase B from to 8% from to 5 min. Linear increase of phase B from 8 to % from 5 to 5 min. Isocratic % phase B from 5 to 3 min. Linear decrease of phase B from to % from 3 to 35 min. Flow Rate:.8 ml/min Temperature: 37 C Potentials:,, 5, 7, 8, -,, 5 mv vs Pd. Serum or standard (. ml) + diluent (. ml) [ mg/l butylated hydroxyanisole (BHA) in ethanol]. Add µl of µg/ml retinyl acetate (internal standard) 3. Vortex for min. Add ml hexane 5. Vortex min and centrifuge ( g, min) 6. Withdraw.8 ml of supernatant and evaporate to dryness by vacuum centrifugation 7. Dissolve residue in. ml EtOH/BHA diluent by vortex-mixing for 5 min 8. Inject µl

2 FIGURE. Analysis of: A) extracted standard; B) National Institute of Standards and Techology (NIST) medium level serum. A 7 B 7 IS IS Peaks:. Retinol. Lutein 3. γ-tocopherol. α-tocopherol 5. Vitamin K 6. Retinyl Palmitate (All Trans) 7. Lycopene 8. α-carotene 9. β-carotene. Coenzyme Q IS. (Internal Standard) Retinyl Acetate Carotenoid Isomers The gradient analytical system consisted of two pumps, an autosampler, and a Dionex CoulArray detector. Column: C3,.6 5, 5 µm Mobile Phase: M ammonium acetate, ph.: methyl-tert-butyl ether: methanol, :35:63 (v/v/v) Flow Rate: ml/min Temperature: Ambient Potentials: + to +5 mv (vs Pd) in 6 mv increments Sample was mixed with ethanol/bha extracted with hexane, dried down, and reconstituted in-phase Only some channels (Ch) are shown for clarity. Ch = mv; Ch = mv; Ch = 7 mv; Ch 7 reoxidation at mv following reduction on Ch 6 at - mv. 833 Table. Observed vs Expected Values Obtained from NIST a Sera Low Concentration Medium Name Obs. Exp. Diff. % Obs. Exp. Diff. % Retinol Lutein γ-tocopherol α-tocopherol ,5, 3.96 Retinly Palmitate Lycopene α-carotene β-carotene Coenzyme Q 59.8 NA NA 95.7 NA NA Vitamin K <.98 NA NA <.98 NA NA NA-value not available in control. a Control sera obtained from NIST Standard Reference Material 968b. A Versatile Detector for the Sensitive and Selective Measurement of Numerous Fat-Soluble Vitamins and Antioxidants in Human Plasma and Plant Extracts

3 FIGURE. Analysis of human plasma and raw and cooked carrot samples Raw Carrot trans- -Carotene Lutein trans-ß-carotene Human Plasma Cooked Carrot 9-cis- -Carotene Lutein -Carotene ß-Carotene trans-ß-carotene 9-cis-ß-Carotene 5-Nitro-γ-Tocopherol (An Indicator of Membrane Damage) 83 The isocratic analytical system consisted of one pump, an autosampler, and a PDA preceding a -channel Dionex CoulArray detector. Column: TosoHaas, ODS-8TM C8,.6 5 mm, 5 µm Mobile Phase: 3 mm Lithium acetate: acetonitrile: methanol: and acetic acid (5:83::.) (v/v/v/v) Flow Rate:. ml/min Temperature: Ambient Inj. Volume: 6 µl Potentials: +, +3, +, +55, +6, +65, +65, +675, +7, +75, +85, +9 mv vs Pd Rat brain astrocytes were prepared according to Hensley, K.; Williamson, K. S.; Floyd, R. A. Measurement of 3-Nitrotyrosine and 5-Nitro-γ-Tocopherol by High- Performance Liquid Chromatography with Electrochemical Detection. Free Radical Biol. Med., 8 (), 5 58 and Williamson, K. S. et al. The Nitration Product 5-Nitro-γ-tocopherol is Increased in the Alzheimer Brain. Nitric Oxide, 6 (), 7. FIGURE 3. Analysis of standards; 5 pm each on column (reproduced with permission of Dr. Kenneth Hensley, Oklahoma Medical Res. Fdn.). δ-tocopherrol α-tocopheryl quinone γ-tocopherol Tocol α-tocopherol Response (na) 5-NO-γ-Tocopherol 3 9 mv 85 mv 75 mv 675 mv 65 mv 65 mv 6 mv 55 mv mv 3 mv mv 835 FIGURE. Primary rat astrocytes 36 h following stimulation with endotoxin. (The 5-nitro-g-tocopherol peak is equivalent to.5 pmol on column.) Only one channel is shown for clarity. (Reproduced with permission of Dr. Kenneth Hensley, Oklahoma Medical Res. Fdn.) Tocol δ- Tocopherrol γ-tocopherol α-tocopherol 5-NO -γ-tocopherol 3 6 mv 836 3

4 Tocopherols and Tocotrienols The gradient HPLC-ECD system consisted of two pumps, a refrigerated autosampler, a thermal chamber, and an 8-channel Dionex CoulArray detector. Column: Hypersil BDS, 5 3. mm, 3 µm, C8 Phase A: Acetonitrile: water, 9: (v/v) containing mm sodium perchlorate and 5 mm perchloric acid Phase B: Acetonitrile: -propanol, 65:35 (v/v) containing mm sodium perchlorate and mm perchloric acid Gradient: Isocratic % B from to min. Linear increase of phase B from to % B for min. Isocratic % B for 9 min before returning to initial conditions for 5 min. Total run time was min. Flow Rate:.6 ml/min Temperature: 3 C Inj. Volume: µl Potentials: -7,, 75, 5, 5, 3, 375, and 5 mv vs Pd Palm oil samples were obtained from Drs. Sumit Shah and Paul Sylvester of University of Louisiana, College of Pharmacy. Samples were weighed and diluted with ethanol prior to injection. FIGURE 5. Analysis of tocotrienols (T) and tocopherols (toc) in palm oil extract. Note expanded time scale for palm oil extract chromatogram Retinol Acetate δ-t3 γ-t3 α-t3 δ-t3 δ-toc γ-toc γ-t3 α-t3 α-toc Standards Palm Oil Extract α-toc β -Carotene CoQ 5 mv 375 mv 3 mv 5 mv 5 mv 75 mv mv 5 mv 375 mv 3 mv 5 mv 5 mv 75 mv mv 837 Oxidized and Reduced CoQ9 and CoQ (Ubiquinone and Ubiquinol Speciation) The isocratic HPLC-ECD system consisted of two pumps, a refrigerated autosampler, a thermal chamber, and a 3-channel Dionex CoulArray detector. Column: Capcell Pak C8 MG (.6 5 mm; 3 µ) Mobile Phase: Methanol: -propanol:. M ammonium acetate, ph., (78::) (v/v/v) Flow Rate: ml/min Temperature: 3 C Inj. Volume: 5 µl (tray at C) Applied Potentials: Model 5A: +7 mv (vs Pd); Model 5A: E = -7 mv; E = +5 mv A Versatile Detector for the Sensitive and Selective Measurement of Numerous Fat-Soluble Vitamins and Antioxidants in Human Plasma and Plant Extracts

5 Blood must be collected in a heparinized Vacutainer tube. Following centrifugation for min at C, the plasma was immediately removed and stored at -8 C until analyzed. A µl volume of plasma was vortexed with 5 µl internal standard and µl (cold) -propanol. After centrifugation, the supernatant was transferred to an autosampler vial and analyzed promptly. Discussion Coulometric array detection is both sensitive (pg) and selective. Analytes are identified both by retention time and by voltammetric behavior across the electrode array. This versatile detector can be used for either global or targeted analysis of FSVs and FSAs in both mammalian and plant tissues. FIGURE 6. Analysis of extracted human plasma. For clarity, only the final oxidative analytical channel is shown. 3 CoQ6 IS Response (na) CoQ red Co Q ox CoQ9 red [5 mv] Table. Analysis of Several NIST Human Plasma Samples Comparing This Data to That Obtained Using Other Analytical Methods NIST Sample ID TOTAL CoQ NIST-Assigned Value (µg/l) ESA CoQ Red ESA CoQ Ox ESA CoQ total [ΣRed + Ox] (% red) Other CoQ Red Other CoQ Ox Other CoQ total [ΣRed +Ox] (% red) 6 ± 5 68 ± 3 68 ± ± 35 6 ± 9 77 ± ± 99 9 ± 9 7 ± 7 35 ± (3) 59 (63) 59 (9) 75 (79) 75 (85) 6 (8) 93 (58) 57 (9) 76 (9) 6 (7) nd (-) 5 (8) 68 (8) 735 (6) 579 (78) 59 () 85 (7) 9 (7) 89 (88) 35 (57) Total CoQ amounts were in good agreement to NIST values. Although NIST values do not report levels of reduced and oxidized forms separately, data from our method show considerable oxidation of the NIST samples. When sample preparation and storage were controlled appropriately, levels for normal patient samples (n = 3) were found to be: CoQ x = 8 ± 3 (µg/l); CoQ Red = 775 ± 9; CoQ Total = 87 ± 33; 9% in reduced form. 5

6 Thermo Fisher Scientific, Inc. Standard Reference Material is a registered trademark of The National Institute of Standards and Technology. Vacutainer is a registered trademark of Becton Dickenson and Co. Capcell Pak is a registered tracemark of Shiseido Co., Ltd. Hypersil is a registered trademark of Agilent Technologies. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Dionex Products: 8 Titan Way, PO Box 363, Sunnyvale, CA , (8) North America: U.S./Canada (87) South America: Brazil (55) Europe: Austria (3) , Benelux (3) (3) Denmark (5) , France (33) 39 3, Germany (9) Ireland (353) 6 6, Italy (39) , Sweden (6) , Switzerland () , United Kingdom () Asia Pacific: Australia (6) 9 533, China (85) 8 38, India (9) , Japan (8) , Korea (8) , Singapore (65) 689 9, Taiwan (886) LPN 93- /