Introduction to the ACQUITY UPLC H-Class Amino Acid Analysis System

Transcription

1 Introduction to the ACQUITY UPLC H-Class Amino Acid Analysis System Cys Waters Corporation 1

2 Amino Acid Analysis Required in many disciplines and applications Components of proteins abolic intermediates Bound vs. Free Nutritional value of foods and feeds Difficult analytical problem Separation o Wide range of properties o Slight differences between pairs Detection o No chromophore o Wide concentration range 2012 Waters Corporation 2

3 Separation of Amino Acids Cation-exchange Original method for amino acid analysis Large historical data base of uncommon amino acids Realistic run times near 30-60min Reversed-phase HPLC of derivatized amino acids Best separation selectivity Best sensitivity Shortest run times 2012 Waters Corporation 3

4 Detection of Separated Amino Acids Post-column Reaction Ninhydrin or OPA with ion-exchange most common ESI-MS with reversed phase HPLC and no derivatization Pulsed Amperometric Detection Pre-column derivatization Many reagents used successfully; some commercialized Add UV or fluorescence detectability Improve chromatography Usually faster and more sensitive Successful use requires care 2012 Waters Corporation 4

5 Design Considerations Base new method on AccQ-Tag Derivatization Established chemistry Well understood control of reaction Simple manual derivatization of batches Base new method on ACQUITY UPLC H-Class System Highest resolution Well-controlled column chemistry Quality-tested eluents Established and available instrumentation Base new method on Empower Software Fully-compliance ready hod templates Custom calculations 2012 Waters Corporation 5

6 Chemistry of AQC Derivatization O O N H N O N O O AQC Reagent O NH 2 OH O NH 1 o Amino Acid; t 1/2 <<1 s OH 2 o Amino Acid; t 1/2 <<1 s N H N O NH OH Derivatized Amino Acids H 2 O t 1/2 ~ 15 s N H N O N HO O N-Hydroxy Succinimide + CO 2 + N NH 2 6-Aminoquinolone () N H N O H N bis-aminoquinoline urea (derivatization peak) N 2012 Waters Corporation 6

7 Ultra Performance LC Technology A class of separation science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small particles Improves Resolution, Sensitivity, and Speed with no compromises Suitable for chromatographic applications in general 2012 Waters Corporation 7

8 UPLC Amino Acid Analysis Application Solution 2012 Waters Corporation 8

9 Amino Acids Analysis Solution ACQUITY and H-Class ACQUITY Hy Asn Gln GABA Hy1 Hy2 AABA Trp Orn Cys H-Class Asn Hy Gln GABA Hy1 Hy2 AABA Orn Cys Trp Waters Corporation 9

10 ACQUITY UPLC H-Class and H-Class Bio Amino Acid Analysis System 2012 Waters Corporation 10

11 Detector Comparison GABA Hy Asn Gln Cys Trp Orn Cell Culture, TUV 0.10 Cell Culture, PDA GABA Hy Cys Trp Asn Gln Hy1 Hy2 EU AABA Orn Hy Asn Gln GABA Hy2 AABA Hy1 Hy2 AABA Hy1 Trp Cys Orn Cell Culture, FLR Waters Corporation 11

12 Comparison of Application Kit UPLC H-Class System CD User s Guide Empower projects AccQ Tag Ultra Chemistry Package AccQ Tag Ultra Eluent A Concentrate AccQ Tag Ultra Eluent B AccQ Tag Ultra Column AccQ Tag Ultra Derivatization Kit Amino Acid Hydrolysate Standard Low flow inlet tubing Column in-line filter kit 150mm column stabilizer 2μL sample loop System CD User s Guide Empower projects AccQ Tag Ultra Chemistry Package AccQ Tag Ultra Eluent A Concentrate AccQ Tag Ultra Eluent B AccQ Tag Ultra Column AccQ Tag Ultra Derivatization Kit Amino Acid Hydrolysate Standard Low flow inlet tubing Column in-line filter kit 2012 Waters Corporation 12

13 ACQUITY UPLC H-Class and H-Class Bio Amino Acid Analysis System Turn-key UPLC Amino Acid Analysis application Optimized for the ACQUITY UPLC H- Class Systems Dedicated QC-tested column and reagents Application-specific Performance Installation Test and Training Same result day-to-day, instrument-to-instrument, lab-tolab, around the world Target Applications tein and peptide identification and quantitation Monitoring cell culture media Analysis of Foods and Feeds 2012 Waters Corporation 13

14 H-Class Amino Acid Analysis System Cell Culture Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn Waters Corporation 14

15 Retention Time Reproducibility 2012 Waters Corporation 15

16 Area Reproducibility 2012 Waters Corporation 16

17 Quantitative Linearity Derivatized Amino Acids Peak Conc (pmol/ul), Ave Peak Areas Name CM Cys PE Cys D-Peak Cys , Area vs. Conc y = x R² = Waters Corporation 17

18 Analysis of tein Hydrolysates Purified teins or Simple Pharmaceutical Formulations Confirmation of tein Identity Determination of tein Amount Requirements Unequivocal peak identification Two order-of-magnitude linear dynamic range Limitations Destruction of Tryptophan, aragine, and tamine Variable, but poor recovery of sulfur-containing amino acids Other variables in hydrolysis procedure Matrix interference Compositional accuracy on the order of 5% 2012 Waters Corporation 18

19 Analysis of tein Hydrolysate Bovine um Albumin Cys Waters Corporation 19

20 Quantitative Linearity BSA Hydrolysate, μg/mL Cys Waters Corporation 20

21 Quantitative Linearity BSA Hydrolysate, μg/mL Cys Waters Corporation 21

22 Analysis of Alkylated Cysteines CM Cys PE Cys Cys Waters Corporation 22

23 Analysis of Cell Culture Media Growing cell culture media Measure free amino acids rather than hydrolysates Nutritional status Waste products Larger array of amino acids Measure composition of media batches Measure changes over time 2012 Waters Corporation 23

24 H-Class Amino Acid Analysis System Cell Culture Amino Acids Additional Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn Waters Corporation 24

25 Cell Culture Sample 6 day Gln 3 day 1 day Waters Corporation 25

26 Amino Acid Analysis Foods and Feeds Nutritional Content Total amount of amino acids Optimize blended feeds Identification/Adulteration Unusual amino acids Marker amino acids prietary constituents cess monitoring Changes during processing Changes during storage 2012 Waters Corporation 26

27 Amino Acid Analysis Classes of Analysis Components of proteins - Bound amino acids abolic intermediates - Free amino acids Building blocks for protein Source of nitrogen for the synthesis of compounds such as nucleotide bases or vitamins Biomarkers in metabolic and genetic studies Surplus amino acids are used as metabolic fuel 2012 Waters Corporation 27

28 Amino Acids as Biomarkers Free Amino Acids Nutrients abolic intermediates Waste products Qualitative and Quantitative file Genotype of food Origin Practical Application cess Monitoring Identity and adulteration 2012 Waters Corporation 28

29 H-Class Amino Acid Analysis System Free/Cell Culture Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn Waters Corporation 29

30 Free Amino Acids Orange Juice Gln AABA Orn Cys Trp 0.10 Asn GABA Hy Asn Gln GABA Hy1 Hy2 AABA 0.10 Cys Orn Trp Free Amino Acid Standard Orange Juice Waters Corporation 30

31 Free Amino Acids Different Juices Gln AABA Orn Cys Trp GABA Asn GABA Trp Asn Asn Gln AABA Orn GABA Trp Grape Juice 0.10 Apple Juice Orange Juice Waters Corporation 31

32 Free Amino Acids Different Brands of Grape Juice Asn Gln Orn GABA Trp Asn Gln Orn GABA Trp Grape Juice # Grape Juice # Waters Corporation 32

33 Free Amino Acids Popular Energy Drink GABA 0.10 Hy Asn Gln GABA Hy1 Hy2 AABA 0.10 Cys Orn Trp Free Amino Acid Standard Energy Drink Waters Corporation 33

34 Free Amino Acids Additives in Infant Formula Free Amino Acid Standard Hy Gln Orn Asn Trp Gln Orn Trp Asn Hy Asn Gln 0.10 GABA Hy1 Hy2 AABA Orn Cys Trp Fat free milk Reconstituted Infant formula Waters Corporation 34

35 Amino Acid Analysis Foods and Feeds Nutritional Content Total amount of amino acids portions of essential amino acids Optimize blended feeds 2012 Waters Corporation 35

36 Food and Feed Hydrolysate hod Different Separation Conditions Cya SO2 AABA Cys 0.10 IIe Different Gradient Table Higher Temperature 2012 Waters Corporation 36

37 Amino Acid Analysis Hydrolyzed Feeds Food and Feed Amino Acid Standard Cys Cys Cys 0.10 Cya SO2 AABA Cys Swine feed Poultry feed Soy Meal Hydrolysate Waters Corporation 37

38 Hydrolysis of Feeds Acid Hydrolysis 6N/Constant Boiling HCl, 105 for 24 hours Sample suspended in fold weight excess of acid Sealed and heated Best method for complex samples Destroys some amino acids Oxidation before hydrolysis for cysteine and methionine Alkaline hydrolysis for tryptophan Cumbersome Long hydrolysis time Bulky apparatus requirements, especially for multiple samples 2012 Waters Corporation 38

39 Microwave Hydrolysis Faster cycle time Controlled hydrolysis conditions Automated sample handling 2012 Waters Corporation 39

40 Workflow Hydrolysis and Analysis 2012 Waters Corporation 40

41 Microwave Acid Hydrolysis Performic Acid Oxidation Food and Feed Amino Acid Standard Cya SO2 AABA Cys IIe Trp Oxidized Soy Flour Oxidized Turkey Starter Oxidized Nutritional Supplement Waters Corporation 41

42 Complete Amino Acid Analysis Turkey Starter 0.15 Food and Feed Amino Acid Standard Cya SO2 AABA Cys IIe Trp Alkaline Hydrolysis Acid Hydrolysis Oxididation with Acid Hydrolysis Waters Corporation 42

43 Application Classes Same Separation Conditions Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn Cell Culture, TUV Waters Corporation 43 Cys 0.10 Hydrolysate, TUV 0.05 CM Cys PE Cys Cys 0.10 Alkylated Cysteines, TUV Feed Hydrolysate, TUV Different hod 0.05 Cya SO2 AABA Cys 0.10 IIe

44 ACQUITY UPLC H-Class and H-Class Bio Amino Acid Analysis System Turn-key UPLC Amino Acid Analysis application Optimized for the ACQUITY UPLC H- Class Systems Dedicated QC-tested column and reagents Application-specific Performance Installation Test Same result day-to-day, instrument-to-instrument, lab-tolab, around the world Target Applications tein and peptide ID and quantitation Monitoring cell culture media Analysis of Foods and Feeds 2012 Waters Corporation 44