USER GUIDE. Can You Complete An Amino Acid Analysis In 15:00 Minutes? ...now you can!

Transcription

1 USER GUIDE TM Can You Compete An Amino Acid Anaysis In 15:00 Minutes?...now you can! Patent Pending Amino acid anaysis kits for: Sampe Prep Derivatization GC or LC Anaysis

2 Patent Pending Tabe of Contents Obstaces in Current... 3 Amino Acid Protocos Physioogica & Hydroysate... Amino Acid Testing 50 + Amino Acids... 5 and Reated Compounds Quantified in 15 Minutes Eight Simpe Steps... 6 in 15 minutes Amino Acid Sampe Prep and Anaysis 8 Minutes of Sampe Prep... 8 Accurate Anaysis...9 in 15 Minutes Accurate Quantitation Appications EZ:faast Kit Components Frequenty Asked Questions.. 19 Evauate an EZ:faast kit in your ab for 30 days. If you are not competey satisfied with the performance, return it for a fu refund. Ordering Information

3 Patent Pending USERGUIDE EZ:faast kits provide reagents and suppies for sampe preparation, derivatization and GC/FID, GC/NPD, GC/MS and LC/MS anaysis of over 50 amino acids and dipeptides in compex matrices and protein hydroysates. Additionay the EZ:faast method offers: A standard 8 minute procedure for sampe preparation and derivatization Eimination of abor-intensive protein and urea remova procedures Exceent method reproducibiity (RSD <1%) and quantitation down to 1nmo/mL Estabished methods for amino acid anaysis in serum, urine, beer, wine, feeds, fermentation broths, foodstuffs and protein hydroysates te: Obstaces in Current Amino Acid Protocos Ion-exchange chromatography using ninhydrin derivatization or reversed phase chromatography have traditionay been used on dedicated instruments for amino acid anaysis. Current ion-exchange methods originated in the ate 190s and are known for engthy minute runtimes aong with extensive sampe preparation to remove interfering components (such as proteins or urea). Ion-exchange methods typicay use expensive, dedicated instruments that are difficut to maintain and operate. Other methods, reying on reversed-phase HPLC, have been deveoped that can be run on any HPLC system saving the expense of purchasing and maintaining a dedicated amino acid anayzer. However, reversed phase HPLC methods can suffer from numerous deficiencies incuding: ong anaysis times, aborious sampe preparation protocos, poor peak resoution, margina quantification, and imited stabiity of derivatized amino acids. Laboratories needing a rapid and simpe method to process high voumes of sampes may find traditiona amino acid anaysis aborious and costy. EZ:faast Amino Acid anaysis kits has been deveoped to overcome these obstaces with a simpe kit containing reagents and suppies to perform sampe preparation, derivatization and improved quantitation using any GC/FID, GC/NPD, or GC/MS system in 15 minutes or by LC/MS in 2 minutes. 3

4 USERGUIDE Patent Pending Economica, Hasse-Free Amino Acid Anaysis Free (Physioogica) Amino Acids Free amino acids are routiney anayzed in physioogica fuids, feeds, fermentation broths and other sources for basic research, nutritiona abeing and more recenty for patient diagnosis. Metaboic deficiencies are diagnosed based on the eves of particuar amino acids in body fuids. Whie some protocos may provide adequate chromatographic methods and derivatization procedures, sampe throughput remains sow due to engthy sampe ceanup and preparation requirements. Using the EZ: faast Free (Physioogica) Amino Acid GC and LC/MS kits, sampe preparation time is ony 7 minutes regardess of sampe matrix. Bood, pasma, urine, cerebra spina fuid, wine or grain sampes contain interfering proteins, urea and other impurities that can ead to poor chromatographic resuts. In 7 minutes, EZ:faast can remove interfering components eaving a highy pure and derivatized amino acid sampe ready for injection. Anaysis time is dramaticay reduced and quantitation significanty improved over traditiona methods; this makes for a rapid and sensitive method for any ab (Figure 1). For abs with a arge number of sampes, EZ:faast truy provides rapid throughput. Without the need for engthy sampe preparation, abor cost is dramaticay decreased, making EZ:faast a much more economica method. Protein and Peptide Hydroysates Numerous abs perform quaity contro assays of both natura and synthetic proteins and peptides by quantifying the hydroyzed amino acid composition of their fina protein product. Protein core abs wi find the EZ:faast kits exceent for confirming protein identity and purity quicky. Using an EZ:faast GC kit, resuts are obtainabe in about 15 minutes and in 2 minutes when using the EZ:faast LC/MS kit. For abs anayzing arge number of sampes the short anaysis time dramaticay increases productivity and reduces the cost of sampe anaysis. Proteins are ong inear strings of amino acids connected to each other by peptide bonds. Hydroysis of the peptide bonds reeases the individua amino acids. The EZ:faast system aows rapid purification and derivatization of the reeased amino acids. Composition and quantity of hydroyzed amino acids can then be determined quicky and accuratey (Figure 2). The overa system provides both a rapid and simpe sampe preparation and anaysis procedure, and is amenabe to imited automation. More sampes can now be anayzed over a given amount of time for ess money as abor cost is dramaticay decreased by this simpe and rapid procedure. Figure 1 Free Amino Acid Standards Anayzed by EZ:Faast 60 0 pa ALA SAR GLY ABA VAL ß-ALA IS LEU aile ILE SER THR PRO ASN MET TPR ASP HYP GLU PHE AAA APA ORN GPR LYS HIS TYR TRP PHP CTH App ID 133 C-C 20 GLN HLY 0 Figure 2 Protein Amino Acids Standards Anayzed by EZ:faast 2 3 LEU PRO HYP PHE GLY ALA VAL NORV ILE SER 5 6 min C-C THR GLU ASP MET LYS HIS HYL TYR App ID 137 AAA min te:

5 Patent Pending USERGUIDE Tabe 1 Anayze Over 50 Amino Acids Abbreviation At. Abbreviation: Chemica Name SIM ALA A Aanine 130, 70 SAR SA Sarcosine 130, 58 GLY G Gycine 116, 7 ABA α-aminobutyric acid 1, 102 VAL V Vaine 158, 72 ß-ALA ß-Aanine 217, 129, 116 ß-AIB BAIBA ß-Aminoisobutyric acid 172, 13, 8 ß-ABA ß-Amino-n-butyric acid 216, 172, 1 NORV Norvaine 158, 72 LEU L Leucine 172, 86 ILE I Isoeucine 172, 130 aile Ao-Isoeucine 172, 130 HSER Homoserine 101, 128, 13 NLE Noreucine 172, 86 THR T Threonine 160, 101 SER S Serine 16, 203 PRO P Proine 156, 23 GABA γ-amino-n-butyric acid 130, 1, 17 ASN N Asparagine 155, 69 TPR Thiaproine 17, 17 ASP D Aspartic acid 216, 130 MET M Methionine 203, 277 HYP OHPro -Hydroxyproine 172, 86 GLU E Gutamic acid 230, 170 PHE F Phenyaanine 206, 190 AAA α-aminoadipic acid 2, 98 CYS C Cysteine 28, 162, 206 PABA -Aminobenzoic acid 265, 206, 163 HCYS Homocysteine 12, 203 APA α-aminopimeic acid 198, 258, 286 HA Histamine 180, 168, 9 THE Theanine MET-SO Methionine Sufoxide GLN Q Gutamine 8, 187 DABA 2,-Diamino-n-butyric acid 203, 12, 25 GLY-GLY Gycine-gycine (dipeptide) 117, 1, 201 MET-SO 2 Methionine Sufone ORN O Ornithine 156, 70 GPR Gycine-proine (dipeptide) 70, 300 LYS K Lysine 170, 128 THR-ASP Threonine-aspartic acid (dipeptide) 218, 360, 130 HIS H Histidine 282, 168 Se-CYS Seenocystine HLY OHLys Hydroxyysine (2 isomers) 129, 169 TYR Y Tyrosine 206, 107 DAP Diaminopimeic acid 256, 168, 196 PHP Proine-hydroxyproine (dipeptide) 156, 11 TRP W Tryptophan 130 NTYR 3-Nitrotyrosine LYS-ALA Lysine-aanine (dipeptide) 170, 22, 153 DA Dopamine 179, 136, 123 CTH Cystathionine 203, 272 DOPA 3,-Dihydroxyphenyaanine 222, 123 C-C (Cys) 2 Cystine 28, 216 HC-HC (Hcys) 2 Homocystine 230, 188, 128 ARG-SUC Arginino-succinic acid 1, 326 Et(OH)NH 2 Ethanoamine 116, 117 ETH Ethionine 203, 291, 13 PA Pipecoic acid 50 Amino Acids & Reated Compounds Quantified in 15 Minutes The EZ:faast method is currenty designed to anayze over 50 amino acids and reated compounds (Tabe 1). A peaks can be accuratey and reproduciby quantified. Additiona amino acids can be anayzed with itte to no modification of the standard methodoogy. Pease contact Phenomenex for additiona amino acids anayzed using the EZ:faast method. Additiona Amino Acids anayzed by the EZ:faast LC/MS kit Abbreviation At. Abbreviation: Chemica Name SIM ARG Arginine 303 CIT Citruine 287 SDMA Dimethy arginine (Symmetrica) ADMA Dimethy arginine (Asymmetrica) 1MHIS 1-Methy-histidine 298 3MHIS 3-Methy-histidine 298 te:

6 USERGUIDE Patent Pending Coors added for iustration purposes ony : 0 1 : 0 0 1P 1S 00:00:00 Step 2. 1 min Pipette sampe through SPE sorbent tip. Advantage: No engthy protein or urea remova. Step 1. 0 min Pipette sampe and combine interna standard (yeow). Advantage: Interna standard for improved quantitation. 8 Simpe Steps 00:00:00 ndh. Spannung [mv] GLY ALA ABA BAIB VAL IS THR ILE LEU SER PRO ASN ASP MET HYP PHE GLU CYS AAA GLN APA HCY ORN GPR LYS HIS TYR HYL TRP PHP CTH C-C App ID 1168 HC-HC 800:15: min Step min Anayze the chromatogram Advantage: Fu resoution of over 50 amino acids and reated compounds in a 7 minute GC run. 700:07:00 * 15 minute sampe prep and anaysis time appies to GC kits ony. Sampe prep and anaysis time by LC/MS is 2 minutes. 6 Step 7. 7 min Sampe preparation compete inject onto GC/FID, GC/NPD, GC/MS or LC/MS system. Advantage: Very stabe derivatized sampe reduces osses due to degradation. te:

7 Patent Pending USERGUIDE 3 00:02:00 Step 3. 2 min Draw wash soution (bue) through SPE sorbent tip. Advantage: Outstanding sampe purity for easy and accurate quantitation. in 15 Minutes! The EZ:faast procedure s greatest benefit is the rapid sampe preparation and derivatization method. For compex matrices ike pasma, urine, fermentation broths or feeds this procedure can provide resuts equa to traditiona protein and urea remova methods in ess time. Even for reativey cean sampes ike protein hydroysates, this method shows improvement in quantitation due to ceaner sampes and more stabe amino acid derivatives. Shorter sampe preparation times cut abor time and cost, making this a highy cost effective assay for amino acid anaysis (ess than $ a sampe). Beow is a simpified diagram iustrating each of the major steps within the EZ:faast protoco. S 00:03:00 Step. 3 min Expe amino acids (bue) with SPE sorbent from tip with Euting Medium (cear). Advantage: Amost no oss of amino acids. 55P 00:0: :06:00 Step 5. min Add organic derivatizing reagent (orange). Advantage: Derivatization is compete in 2 min and can be done in aqueous phase. Step 6. 6 min Extract amino acid derivatives from aqueous ayer (ight green) into organic ayer (orange). Advantage: Additiona purification step. te:

8 USERGUIDE Patent Pending 8 minutes of Sampe Prep No Protein or Urea Remova Required The anaysis of free amino acids in physioogica fuids, grains, fermentation broths and other compex matrices differs from the anaysis of protein hydroysates due to the high concentration of interfering compounds. A reativey arge amount of proteins, peptides, urea and other matrix components have to be removed prior to free amino acid anaysis by GC or HPLC, otherwise coumns deteriorate rapidy, quantitation is poor, and anaysis resuts are not reproducibe. Current procedures for de-proteinization and urea remova are abor intensive and recoveries are ow for some amino acids. Aso, reagents used for de-proteinization can interfere in the amino acid profie. The EZ:faast method, however, does not require traditiona de-proteinization or urea remova methods to be foowed; proteins are excuded from the sampe as it is passes through an SPE sorbent tip. The SPE sorbent binds amino acids whie proteins and urea are washed away eaving ony the free amino acids. A comparison of the EZ:faast method to other common de-proteinization methods shows as good or better resuts using the EZ:faast method. EZ:faast transates into major cost and time savings. Hydroyzed protein sampes are often in a cean sampe matrix. Hydroysis under strong acidic conditions at eevated temperatures causes proteins and peptides to breakdown into their component amino acids. Other components ike hydroyzed carbohydrates and ipids, are excuded from the sampe as it passes through the SPE sorbent tip. With purer sampes and better derivative stabiity quantitation and method reproducibiity can be improved. Tabe 2: Data representing a series of comparative tests on the same pasma sampe with and without protein remova; anaysis of a sampes was performed using the EZ:faast kit. The data represents the cacuated amino acid concentration in µmo/l after the sampe has undergone three common de-proteinization procedures (SSA = sufosaicyic acid; TCA = trichoroacetic acid; ORG = acetonitrie:ethano 2:1) compared to the EZ:faast procedure. The comparative data (mean vaues for 12 measurements) is presented in the tabe beow. Resuts for amino acid content show no significant difference between sampes with or without protein remova. Tabe 2 Without SSA TCA ORG De-proteinization (Recommended for OPA (Recommended for PITC (Standard EZ:faast method) derivatized sampes) derivatized sampes) Procedure Time: 7 minutes 3 hours 3 hours 3 hours GLY 290 ( ) 288 ( ) 259 ( ) 261 ( ) ALA 21 (15-27) 22 (17-27) 380 (357-02) 393 (365-21) ABA 23 (22-2) 23 (20-26) 22 (21-22) 22 (21-23) LEU 165 ( ) 16 ( ) 162 ( ) 163 ( ) ILE 7 (72-75) 70 (69-72) 71 (69-72) 73 (72-73) MET 30 (29-30) 32 (31-33) 31 (30-31) 30 (29-30 PRO 209 ( ) 207 (20-210) 212 ( ) 206 (197-21) ASP 18 (17-19) 16 (15-17) 16 (1-17) 19 (18-20) GLU 0 (38-1) 35 (25-) 35 (29-0) 35 (30-39) THR 176 ( ) 166 ( ) 153 (15-161) 177 ( ) SER 12 (136-17) 133 ( ) 129 ( ) 136 ( ) HYP 1 (13-1) 12 (11-12) 13 (12-13) ASN 3 (32-35) 3 (33-35) 31 (27-3) 31 (28-33) GLN 50 (95-58) 588 ( ) 553 (51-592) 569 (55-58) C-C 1 (39-2) 1 (39-2) 5 (1-9) 30 (21-39) PHE 58 (56-60) 58 (56-59) 63 (59-67) 60 (57-63) TYR 62 (59-6) 62 (58-66) 63 (59-66) 60 (57-63) TRP 8 (6-9) (1-7) 5 (2-8) 50 (3-56) ORN 60 (58-61) 68 (62-73) 6 (60-67) 6 (3-9) LYS 179 ( ) 200 ( ) 18 ( ) 118 ( ) HIS 8 (78-89) 89 (82-96) 90 (85-95) 82 (73-91) 8 te:

9 Patent Pending USERGUIDE Derivatization Simpified After the very short sampe ceanup procedure, the amino acids are quicky derivatized at room temperature with the addition of two reagents. The proprietary reagents modify both the carboxy and amino groups of the amino acids forming derivatives stabe at room temperature for severa hours and stabe at ºC for severa days. With good derivative stabiity, amino acid quantitation is more accurate and reiabe; amost no amino acid sampe is ost in sampe preparation or through sampe degradation. Reagents and suppies for sampe preparation aow for the anaysis of 38 sampes per kit. Reagents, when stored according to abe directions, are guaranteed for up to 12 months. Rapid Chromatographic Anaysis Traditiona ion exchange and reversed phase methods for the anaysis of amino acids utiize ong minute runtimes. The EZ:faast method takes advantage of the fast separation capabiity and high resoution of GC/FID, GC/NPD, GC/MS to anayze over 50 amino acids in about 8 minutes. The LC/MS chromatographic runtime is a quick 12 minutes. High coumn efficiencies and resoution provides compete baseine separation for easy quantitation. Interfering compounds such as drug metaboites, proteins and urea that can be present in the sampe matrix have been removed during sampe preparation. With traditiona methods, a arge number of potentia interfering compounds, ike drugs and drug metaboites may co-migrate with amino acids thus affecting quantitation. The high resoution GC method aso aows for additiona rare amino acids not isted in Tabe 1 to be anayzed without compromising throughput or resoution of other amino acids. In traditiona HPLC based methods, rare amino acids may be anayzed but often resuts in a substantia increase of the anaysis times this is not the case with EZ:faast. The GC protoco aows for adjustments in carrier gas fow, oven temperature and the temperature gradient to ensure for the anaysis of additiona amino acids with a minor increase to the very short anaysis time. High separation power and speed of the GC protoco aso ensures method fexibiity for the rapid anaysis of additiona amino acids with ony sight (<5 min) increases in runtime. Figure 3 Simpified diagram iustrating EZ:faast derivatization reactions N H 2 R OH O C O R OR' + 2 cat. + 2HC + CO OR' OR' NH O O 2 te:

10 USERGUIDE Patent Pending EZ:faast kits provide reiabe and accurate quantitation of over 50 fuy baseine resoved amino acids. The ower imit of detection for EZ:faast is 1 nmo/ml and the precision in quantitation is <10% for most amino acids (a few amino acids show precisions in quantitation of +/- 15%). Sampe oss and method precision is iustrated in the comparison of 5 injections of free amino acids extracted from pasma by a traditiona ion exchange (IEX) method run on a dedicated amino acid anayzer and by the EZ:faast method (Tabe 3). EZ:faast provides improved quantitation due to severa factors. Both sensitivity and reproducibiity are improved with EZ:faast. Fu baseine resoution accounts for some of the improvement, and the sampe preparation procedure insures neary a interfering compounds are removed without sacrificing amino acid recovery. Once derivatized, amino acids are stabe for severa hours at room temperature and for severa days if refrigerated, preventing sampe oss by degradation. Tabe 3. Comparative data for the anaysis of pasma free amino acids by ion exchange chromatography (on a dedicated amino acid anayzer with ninhydrin detection), and by GC/FID (EZ:faast). Concentrations are given in nmo/ml (statistica evauation by Horn). P L stands for mean, L L is ower imit; L U is upper imit; IEX is ion exchange chromatography. Tabe 3 GLY ALA ABA VAL LEU ILE MET PRO IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast P L L L L U THR SER ASP GLU GLN C-C PHE TYR IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast P L L L L U TRP ORN LYS HIS Accuracy and Precision IEX EZ:faast IEX EZ:faast IEX EZ:faast IEX EZ:faast P L L L L U Detection Limit Precision in Quantitation, %RSD Reproducibiity of Retention Time, % RSD <1% 1nmo/mL by GC/FID 1nmo/mL by GC/MS +/-15% (<10% for most amino acids) te:

11 Patent Pending USERGUIDE Sampe: Derivatized amino acids in human serum (0.1mL), Norvaine is the interna standard added at a concentration of 200 µmo/l 1. Aanine 2. Gycine 10. Proine 11. Asparagine 19. Lysine 20. Histidine Free Amino Acids in Human Serum by GC/FID 3. α-aminobutyric acid. Vaine 5. Norvaine (IS) 6. Leucine 7. Isoeucine 8. Threonine 9. Serine 12. Aspartic Acid 13. Methionine 1. -Hydroxyproine 15. Gutamic Acid 16. Phenyaanine 17. Gutamine 18. Ornithine 21. Tyrosine 22. Tryptophan 23. Cystine Kit: Order No.: EZ:faast GC/FID Free (Physioogica) Amino Acid Kit KG App ID 1169 Injection: Spit 250 C, 2.5µL 200 Carrier Gas: Pressure Rise: Oven Program: Heium 1.5mL/minute ( C 6 kpa/min 30 C/min from 110 to 320 C, hod at 320 for 1 minute Detector: 320 C [mv] min te:

12 USERGUIDE Patent Pending Free Amino Acids in Human Urine by GC/FID Sampe: Derivatized amino acids in human urine (0.1mL). Norvaine is the interna standard added at a concentration of 200 µmo/l 1. Aanine 2. Gycine 9. Serine 10. Proine 17. Lysine 18. Histidine 3. Vaine. ß-Aminoisobutyric acid 5. Norvaine (IS) 6. Leucine 7. Isoeucine 8. Threonine 11. Asparagine 12. Aspartic Acid 13. Gutamic Acid 1. Phenyaanine 15. α-aminoadipic acid 16. Gutamine 19. Tyrosine 20. Proine hydroxyproine (dipeptide) 21. Tryptophan 22. Cystine App ID 1170 Kit: EZ:faast GC/FID Free (Physioogica) Amino Acid Kit Order No.: Injection: KG Spit 250 C, 2.5µL Carrier Gas: Heium 1.5mL/minute ( C Pressure Rise: 6 kpa/min Oven Program: 30 C/min from 110 to 320 C, hod at 320 for 1 minute Detector: 320 C [mv] min 12 te:

13 Patent Pending USERGUIDE Sampe: Derivatized amino acids in potato tissue (0.1mL). Norvaine is the interna standard added at a concentration of 200 µmo/l 1. Aanine 2. Gycine 12. Aspartic Acid 13. Methionine Free Amino Acids in Potato Tissue by GC/FID 3. α-aminobutyric Acid. Vaine 5. Norvaine (IS) 6. Leucine 7. Isoeucine 8. Threonine 9. Serine 10. Proine 1. Gutamic Acid 15. Phenyaanine 16. Gutamine 17. Ornithine 18. Lysine 19. Histidine 20. Tyrosine 21. Tryptophan 11. Asparagine Kit: Order No.: Injection: EZ:faast GC/FID Free (Physioogica) Amino Acid Kit KG Spit 250 C, 2.5µL App ID 13 Carrier Gas: Pressure Rise: Heium 1.5mL/minute ( C Constant pressure Oven Program: 30 C/min from 110 to 320 C, hod at 320 for 1 minute Detector: 320 C pa min te:

14 USERGUIDE Patent Pending Free Amino Acids in Corn Mea by GC/FID Sampe: Derivatized amino acids in corn mea (0.1mL). Norvaine is the interna standard added at 200 µmo/l 1. Aanine 2. Gycine 3. Vaine. Norvaine (IS) 5. Leucine 6. Isoeucine 7. Threonine 11. Aspartic Acid 12. Gutamic Acid 13. Phenyaanine 1. Lysine 15. Histidine 16. Tyrosine 17. Tryptophan 8. Serine 9. Proine 10. Asparagine App ID 135 Kit: Order No.: EZ:faast GC/FID Free (Physioogica) Amino Acid Kit KGO-7165 Injection: Spit 250 C 2.5µL Carrier Gas: Heium 8psi (60 kpa) Pressure Rise: Constant pressure Oven Program: 32 C/min from 110 to 320 C, hod at 320 C for 1 minute Detector: 320 C min 1 te:

15 Patent Pending USERGUIDE Sampe: Derivatized amino acids in wine (0.1mL). Norvaine is the interna standard added at a concentration of 200 µmo/l 1. Aanine 2. Gycine 11. Aspartic Acid 12. Methionine Free Amino Acids in Wine by GC/MS 3. Vaine. α-aminoisobutyric acid 5. Norvaine (IS) 6. Leucine 7. Isoeucine 8. Threonine 9. Proine 13. -Hydroxyproine 1. Gutamic Acid 15. Phenyaanine 16. Ornithine 17. Lysine 18. Tyrosine 19. Tryptophan 10. Asparagine Kit: Order No.: EZ:faast GC/MS Free (Physioogica) Amino Acid Kit KGO-7166 App ID 1610 Injection: Carrier Gas: Pressure Rise: Spit 250 C, 2.5µL Heium 1.1mL/ minute@110 C 6 kpa/min 9 Oven Program: 30 C/min from 110 to 320 C, hod at 320 C for 1 min. 1 Detector: 300 C min te:

16 USERGUIDE Patent Pending Free Amino Acids in Beer by GC/FID Sampe: Derivatized amino acids in beer. Norvaine is the interna standard added at 200 µmo/l 1. Aanine 2. Gycine 3. Vaine. Norvaine (IS) 5. Leucine 6. Isoeucine 7. Serine 8. Proine 9. Asparagine 10. Aspartic Acid 11. Gutamic Acid 12. Phenyaanine 13. Ornithine 1. Histidine 15. Tyrosine 16. Tryptophan App ID 1623 Kit: Order No.: EZ:faast GC/FID Free (Physioogica) Amino Acid Kit KGO-7165 Injection: Spit 250 C 2.5µL 8 Carrier Gas: Pressure Rise: Heium 8psi (60 kpa) Constant pressure Oven Program: 32 C/min from 110 to 320 C, hod at 320 C for 1 minute 1 Detector: 320 C min 16 te:

17 Patent Pending USERGUIDE Sampe: Derivatized amino acids from a corn mea hydroysate sampe. Norvaine is the interna standard added at 200 µmo/l 1. Aanine 2. Gycine 3. Vaine. Norvaine (IS) 5. Leucine 6. Isoeucine 7. Threonine 8. Serine 10. Aspartic Acid 11. Methionine 12. Hydroxyproine 13. Gutamic Acid 1. Phenyaanine 15. Lysine 16. Histidine 17. Tyrosine Amino Acids in Corn Mea Hydroysate by GC/FID 9. Proine Kit: Order No.: Injection: EZ:faast GC/FID Protein Hydroysate Kit KG Spit 250 C, 2.5µL App ID 136 Carrier Gas: Pressure Rise: Heium 1.5mL/ minute ( C 6kPa/min 5 Oven Program: 30 C/min from 110 to 320 C, hod at 320 for 1 minute Detector: 320 C min te:

18 USERGUIDE Patent Pending Microdispenser for Organic Reagents and 5 Interna Standard Washing Soution (Reference Sampe Cean-up) Euting Medium I (Sampe Cean-up) Euting Medium II (Sampe Cean-up) Organic Soution I (Derivatization) Organic Soution II (Derivatization) Acid Soution (Derivatization) Amino Acids Standard Soution 00 Sampe Preparation Vias 0.6mL Syringe for SPE amino acid eution Via Rack for Sampe Preparation Zebron ZB-AAA GC Coumns or AAA HPLC Coumn 1.5mL Syringe for SPE oading & washing EZ:faast workstation SPE Sorbent Tips SGE GC FocusLiners (Not Shown) te:

19 Patent Pending USERGUIDE Frequenty Asked Questions Q: For how ong are the derivatized amino acids stabe? A: Amino acids derivatized by the EZ:faast method are stabe for up to a day at room temperature and for severa days if refrigerated. Q: How many sampes can be anayzed with the EZ:faast kits? A: 38 sampes can be anayzed with the reagents and suppies of one kit. Q: How ong wi the EZ:faast GC and LC coumn ast? A: We have found that the GC and LC coumns wi ast for the duration of the kit. For best resuts coumns shoud be repaced with each new kit. Q: What is the shef ife of the reagents? A: EZ:faast reagents have a guaranteed shef ife of 12 months if propery stored as indicated on the botte in a refrigerator or freezer. Q: What are the derivatization reagents and reaction? A: The derivatization reagents and reaction are proprietary. The reaction derivatizes both the amine and carboxy groups of the amino acids forming a highy stabe derivative. Amino Acid Anaysis Kits Each kit incudes: ZB-AAA GC coumn, or AAA LC coumn, sampe prep and derivatization reagents, sampe prep vias, AA standards, SPE pipette tips, via rack, and microdispenser. Order No. Description Unit Price KG GC/FID Free (Physioogica) Amino Acid Anaysis Kit ea KG GC/MS Free (Physioogica) Amino Acid Anaysis Kit ea KG GC/FID Protein Hydroysate Kit ea KG GC/MS Protein Hydroysate Kit ea KH LC/MS Free (Physioogica) Amino Acids Kit with 250 x 2.0mm coumn ea KH LC/MS Free (Physioogica) Amino Acids Kit with 250 x 3.0mm coumn ea KH LC/MS Protein Hydroysates Kit with 250 x 2.0mm coumn ea KH0-730 LC/MS Protein Hydroysates Kit with 250 x 3.0mm coumn ea AG0-718 Free (Physioogica) Amino Acid Standards (SD1, 2, 3) 2mL/via x 2 ea AG Protein Hydroysate Standard (SD) 2mL/via x 2 ea EZ:faast is a trademark of Phenomenex, Inc Phenomenex, Inc. A rights reserved. te:

20 11 Madrid Avenue Torrance, CA U.S.A. PRSRT STD U.S. POSTAGE PAID PERMIT #185 TORRANCE, CA Can You Compete An Amino Acid Anaysis In 15:00 Minutes?...now you can! 3300_U