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

Introduction to the ACQUITY UPLC H-Class Amino Acid Analysis System 0.22 0.20 0.18 0.16 0.14 0.12 Cys 0.10 0.08 0.06 0.04 0.02 1.5 0 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 2012 Waters Corporation 1

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

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

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

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

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

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

UPLC Amino Acid Analysis Application Solution 2012 Waters Corporation 8

Amino Acids Analysis Solution ACQUITY and H-Class ACQUITY Hy Asn Gln GABA Hy1 Hy2 AABA Trp Orn Cys H-Class 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 Asn Hy Gln GABA Hy1 Hy2 AABA Orn Cys Trp 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 2012 Waters Corporation 9

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

Detector Comparison GABA Hy Asn Gln Cys Trp Orn Cell Culture, TUV 0.10 Cell Culture, PDA 0.05 2.50 5.00 7.50 GABA Hy Cys Trp Asn Gln Hy1 Hy2 EU AABA Orn Hy Asn Gln GABA Hy2 AABA Hy1 Hy2 AABA Hy1 Trp Cys Orn 0.10 0.05 Cell Culture, FLR 160 80 2.50 5.00 7.50 2012 Waters Corporation 11

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

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

H-Class Amino Acid Analysis System Cell Culture Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn 0.10 0.05 2.50 5.00 7.50 2012 Waters Corporation 14

Retention Time Reproducibility 2012 Waters Corporation 15

Area Reproducibility 2012 Waters Corporation 16

Quantitative Linearity Derivatized Amino Acids Peak Conc (pmol/ul), Ave Peak Areas Name 0.05 0.075 1.0 2.5 25 50 6033932 6020972 6101437 6056268 5069175 3648294 4064 4810 9094 14396 86830 155693 167 304 4064 9618 104287 204119 334 330 4052 9459 102749 199653 230 320 3994 9516 102770 201123 591 682 4260 9608 100540 196412 211 272 4053 9704 101645 190396 238 303 3988 9330 100969 190784 238 329 4115 9617 104228 201799 CM Cys 197 310 4110 9726 105289 211792 269 336 4029 9447 102180 195455 PE Cys 240 339 4281 10014 109782 224055 219 308 3807 8916 96538 187201 D-Peak 94086 84767 94312 89565 62635 46575 Cys 172 262 3566 8315 91527 179095 334 499 6810 15852 173792 329877 217 320 4177 9874 106770 209182 176 268 4049 9354 104998 204492 274 374 4127 9765 105725 204246 219 331 3893 8911 99315 198500 373 435 4323 9899 106378 205866 225 300 4084 9521 103758 201006 237 348 4312 9970 106180 208031 250000 200000 150000 100000 50000 0, Area vs. Conc y = 4098.3x + 54.943 R² = 0.9999 0 5 10 15 20 25 30 35 40 45 50 2012 Waters Corporation 17

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

Analysis of tein Hydrolysate Bovine um Albumin 0.28 0.26 0.24 0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 Cys 0.02 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 2012 Waters Corporation 19

Quantitative Linearity BSA Hydrolysate, 20-1000μg/mL 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 Cys 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 2012 Waters Corporation 20

Quantitative Linearity BSA Hydrolysate, 20-1000μg/mL 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 Cys 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 2012 Waters Corporation 21

Analysis of Alkylated Cysteines CM Cys PE Cys Cys 0.10 0.05 2.50 5.00 7.50 2012 Waters Corporation 22

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

H-Class Amino Acid Analysis System Cell Culture Amino Acids Additional Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn 0.10 0.05 2.50 5.00 7.50 2012 Waters Corporation 24

Cell Culture Sample 6 day Gln 3 day 1 day 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 2012 Waters Corporation 25

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

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

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

H-Class Amino Acid Analysis System Free/Cell Culture Amino Acids Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn 0.10 0.05 2.50 5.00 7.50 2012 Waters Corporation 29

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 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 30

Free Amino Acids Different Juices Gln AABA Orn Cys Trp 0.10 0.10 GABA Asn GABA Trp Asn Asn Gln AABA Orn GABA Trp Grape Juice 0.10 Apple Juice Orange Juice 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 31

Free Amino Acids Different Brands of Grape Juice Asn Gln Orn GABA Trp Asn Gln Orn GABA Trp Grape Juice #1 0.10 Grape Juice #2 0.10 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 32

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 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 33

Free Amino Acids Additives in Infant Formula Free Amino Acid Standard Hy Gln Orn 0.10 0.10 Asn Trp Gln Orn Trp Asn Hy Asn Gln 0.10 GABA Hy1 Hy2 AABA Orn Cys Trp Fat free milk Reconstituted Infant formula 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 34

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

Food and Feed Hydrolysate hod Different Separation Conditions Cya SO2 AABA Cys 0.10 IIe 0.05 2.50 5.00 7.50 Different Gradient Table Higher Temperature 2012 Waters Corporation 36

Amino Acid Analysis Hydrolyzed Feeds Food and Feed Amino Acid Standard Cys 0.10 0.10 0.10 Cys Cys 0.10 Cya SO2 AABA Cys Swine feed Poultry feed Soy Meal Hydrolysate 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 2012 Waters Corporation 37

Hydrolysis of Feeds Acid Hydrolysis 6N/Constant Boiling HCl, 105 for 24 hours Sample suspended in 10-100-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

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

Workflow Hydrolysis and Analysis 2012 Waters Corporation 40

Microwave Acid Hydrolysis Performic Acid Oxidation Food and Feed Amino Acid Standard 0.15 0.10 0.05 Cya SO2 AABA Cys IIe Trp 0.20 0.15 Oxidized Soy Flour 0.10 0.05 0.15 Oxidized Turkey Starter 0.10 0.05 0.06 Oxidized Nutritional Supplement 0.04 0.02 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 2012 Waters Corporation 41

Complete Amino Acid Analysis Turkey Starter 0.15 Food and Feed Amino Acid Standard 0.10 0.05 Cya SO2 AABA Cys IIe Trp 0.20 0.15 Alkaline Hydrolysis 0.10 0.05 0.08 0.06 Acid Hydrolysis 0.04 0.02 0.15 0.10 Oxididation with Acid Hydrolysis 0.05 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 2012 Waters Corporation 42

Application Classes Same Separation Conditions Hy Asn Gln GABA Hy2 AABA Hy1 Trp Cys Orn Cell Culture, TUV 0.10 0.05 2.50 5.00 7.50 2.50 5.00 7.50 2012 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 0.05 2.50 5.00 7.50 2.50 5.00 7.50

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