Quality Assurance of Vitamins
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1 Quality Assurance of Vitamins Ronda Greaves Medical Scientist: The Royal Children s Hospital Melbourne Chair: AACB Vitamins working party Ronda.greaves@rch.org.au APCCMS 14th Jan 2010
2 Outline Definition External QA Biological Variation Standardization Common Reference Intervals
3 The Vital Amine Definition: organic compound required as a nutrient, which cannot be synthesized in adequate amounts, and therefore must be obtained in the diet
4 The Vitamin Alphabet A - Retinol B - group of 8 C - Ascorbic acid D - Ergocalciferol - Cholecalciferol E - Tocopherol K - Phylloquinone B1 Thiamine B2 Riboflavin B3 Niacin (B4 Adenine) B5 Pantothenic acid B6 Pyridoxine B7 (H) Biotin (B8 Inositol) B9 Folate (B10 PABA) (B11 Choline) B12 Cobalamin Total: 13 = 4 fat soluble + 9 water soluble ( ) indicates B group compound no longer classified as vitamins
5 Methods NOTE: Presentation of the commercial methods in the following slides do not constitute endorsement by the AACB Vitamins Working Party or the RCPA-QAP
6 Method principles Immunoassay Folate Vitamin B12 Vitamin D HPLC Fat soluble vitamins A, D, E, K, β carotene + other carotenoids (& coenzyme Q10) Water soluble - B1, B2, B6, C Spectrophotometric Carotenoids Microbiological Vitamin B1 Activity Measurements Vitamin B1 & 6
7 Vitamin A & E: Commercial Chromatograms Chromsystems Bio-Rad δ γ α VitA IS VitE Recipe: Varian application note
8 Fat soluble vitamins with MS e.g. Waters application UPLC & PDA +MS Gradient elution MP A = 10%ACN + 90%H 2 0 MP B = 50%ACN + 50%H min per sample Vitamin D routinely performed by MS or TMS Vitamin A & E no RCPA-QAP enrolled laboratories Vitamin K not part of RCPA-QAP program Vitamin E Potential future direction for analysis Standard Butter Waters application note: Analysis of fat soluble vitamins using UPLC-PDA-SQD
9 Vitamin B1 & B6: - Example method Commercial method: Chromsystems Gradient elution Wavelengths: Start: EX 320 nm, EM 415 nm - Vitamin B6 (PLP) 4 min switch: EX 367 nm, EM 435 nm - Vitamin B1(TPP) Internal standard
10 Vitamin C example method Commercial method: Chromsystems Isocratic HPLC system UV detection 245nm Sample preparation involves protein precipitation with a stabilizer. Controls and Calibrators available for purchase separately.
11 External QA Vitamin A, E and the carotenoids Vitamin B1 & B6 Vitamin C Vitamin D
12 EQA programs worldwide Program Country Vitamins CAP USA DEQAS UK 25D & 1,25D DGKL Germany A, B6, B12, D, E & folate NEQAS UK A, E, β carotene & K SKML Netherlands A, E, B1, B6 & β carotene RCPA-QAP Australia A, E, B1, B6, C, β carotene & total carotenoids Vital USA A This does not include vitamins that may fall under endocrine / immunoassay programs
13 RCPA-QAP Vitamin program Countries: Australia New Zealand South Africa Thailand USA Singapore Israel analyte Vitamin B1 Vitamin B2 Vitamin B6 Vitamin C Vitamin K Red Cell Folate Coenzyme Q10 1,25 Vit D 2006 questionnaire: n = 66 Analytes: Pilot Intro 2000 Vitamin A Vitamin E Beta carotene Total carotenoids Pilot Intro 2009 Vitamin B1 Vitamin B6 Pilot Intro 2010 Vitamin C (Endo program) Vitamin D
14
15 2010 Vitamin method classification
16 RCPA QAP: Vitamin A interim report
17 RCPA QAP: Vitamin E end of cycle report
18 RCPA QAP: Vitamin B1 interim report/methods
19 RCPA QAP: Vitamin B6 end of cycle report/methods
20 Biological Variation
21 Biological Variation Desirable specification CVw CVg I(%) B(%) TE(%) S- α-carotene S- α-tocopherol S- Ascorbic Acid S- β-carotene S- β-criptoxantin S- Lycopenene P- Retinol S- Retinol P- Vitamin B B- (B)Eryt (B)Eryt h - h - Vitamin B2 (Riboflavin) Vitamin B2 (Riboflavin) Vitamin B2 status (gluthation reductase activation) (B)Eryt h - (B)Eryt Vitamin B h - Vitamin B B- (B)Eryt Vitamin B6 Vitamin B6 status h - (AST activation) (B)Eryt (B)Eryt h - h - Analyte Vitamin E (α- Tocopherol) Vitamin K (Phylloquino ne)
22 Is the assay fit for purpose?: Uncertainty of Measurement Formula Vit A Vit E Analytical Imprecision (CVa) CVa = SD/mean * % Mean =1.26 µmol/l 5.1% Mean =16.9 µmol/l n = 23 n = 23 MU MU = 2 x CVa 12.2% 10.2% Biological Variation (CVw) From literature* 13.8% 13.6% Total variation (CVt) CVt = [(Cva)2 + (CVw)2] 15.1% Desirable = <16.5%* 14.5% Desirable = <17.1%* Fit for purpose Optimum: CVa 0.25 x CVw Desirable: CVa 0.5 x CVw Minimum: CVa 0.75 x CVw = 6.1/ =5.1/13.8 Outcome Desirable Desirable [i] Fraser CG. Biological Variation: From principles to practice. AACC Press 2001 [ii] White GH, Farrance I: Uncertainty of Measurement in Quantitative Medical testing A Laboratory Implementation Guide. Clin Biochem Rev 2004;25 S1-4 [iii] Westgard JO. (Accessed 27/08/09)
23 Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis Clinica Chimica Acta, 2009; 408: 8-13 Dietmar Stöckl, Patrick M. Sluss and Linda M. Thienpont
24 Standardization
25 Why should we pursue standardization?.. Fundamental concept for clinical biochemists! Method basis similar Reference intervals are similar With standardization, results from one lab can be directly compared to another lab - improved patient care.
26 DEQAS: Vitamin D
27 Now 968d NIST human serum SRM
28 New SRM for vitamin D NIST Released 2009 SRM 972 Vitamin D in human serum Allows for a common primary calibrator
29 Vitamin A & E: Commercial reagent sources Standards Bio-Rad Chromsystems Sigma NIST Other Internal QC Bio-Rad Chromsystems Recipe In house Other Commercial Kits Bio-Rad Chromsystems Recipe Other Columns Alltech Phenomenex Varian Waters Other
30 RCPA QAP vitamin A
31 Vitamin A compared to SRM968c Level Raw Corr NIST Median values µmol/l COMPARISON OF BIORAD CALIBRATOR STANDARDISED DATA WITH TARGET VALUES VITAMIN A umol/l TARGET VALUES SRM 968c STANDARDISED VALUES USING BIORAD CALIBRATOR MEDIAN LAB VALUES AACB ASM September, 2008
32 Study conclusions The use of a common calibrator influenced and improved the spread of results seen between participants of the RCPA-QAP vitamin program. However, this standardisation experiment does not account for all the dispersion seen and other factors including vitamin recovery and chromatographic separation need to be considered. A pilot of a common calibrator by all participants enrolled in the RCPA-QAP Vitamin program will be considered by the AACB vitamins working party.
33 Summary RCPA QAP program provides a wide range of vitamins to participants AACB vitamins working party aiming to improve laboratory performance Consensus method Standardization Peer support
34 Members of the AACB vitamins working party 2009 Chris Salonikas Lisa Jolly Kirsten Hoad Ronda Greaves Trevor Walmsley Lambro Johnson Gerald Woollard Scott Briscoe (not pictured)
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