ACCELERATED AMINO ACID ANALYSIS USING HPLC WITH POST-COLUMN DERIVATIZATION Maria Ofitserova, Ph.D., Wendy Rasmussen, Michael Pickering, Ph.D. CATALYST FOR SUCCESS BACKGROUND METHODS The traditional, and to date most accurate, method of detecting Each chromatogram was performed using a Linear Citrate-Buffer free amino acids in solution is the use of ion-exchange separation Gradient (Lithium Citrate for Physiologic samples, Sodium Cit- followed by post-column derivatization with Ninhydrin. No other rate for Hydrolysate samples) along with a Column Temperature technique has been shown to match the reproducibility of the Gradient to effect the elution. The derivatization of the amino analysis from simple solutions to the most challenging matrices. acids was performed using Post-Column Derivatization with Trione® Ninhydrin Reagent at 130° C and with a reactor volume Nevertheless, with run times of typically 1-2hrs, this method of 0.5ml. can be very time-consuming in terms of laboratory throughput. While the sample preparation is very and the instrumentation All chromatograms were generated using an HPLC pump, au- relatively simple for the analyst, the long turn around time in tosampler, Pickering Pinnacle PCX todays busy laboratory environment can be viewed as a disadvan- post-column derivatization instrument, and HPLC UV/Vis tage. If a laboratory can guarantee faster turn around, but main- detector*. tain accurate results, this will enable them to remain competitive All reagents, columns, and standards are produced by Pickering in the market. For this reason, we have been working to acceler- Laboratories, Inc. ate Amino Acid Analysis while at the same time maintaining the high reproducibility and sensitivity that the industry expects from Real samples were provided by independent laboratories. Ninhydrin derivatization. Here we present new Accelerated Amino Acids Analysis methods for both Physiological and Protein Hydrolysate samples. Our method utilizes a combination of temperature and eluant gradi- ents followed by post-column derivatization. Lithium Amino Acids (typically Physiologic Fluids) can be ana- lyzed in 88 minutes inject-to-inject and Sodium Amino Acids (typically Protein Hydrolysates) can be analyzed in 39 minutes inject-to-inject. Phenylalanine Phosphoserine AEC Alanine Taurine Cystine Cystathionine Glycine Glutamine Acid Allo-Isoleucine a-Amino-n-butyric Acid Citrulline Glycine Glutamine Alanine Methionine Ornithine Serine Valine aline Threonine Leucine V Isoleucine Lysine Glutamic Acid Homocystine Aspartic Acid Acid Phosphoethanolamine Glucosaminic Acid Hydroxylysines Histidine Tyrosine ysine Phenylalanine a-Amino-n-butyric L 1-Methylhistidine 3-Methylhistidine aurine a-Aminoadipic Acid Acid T Serine Threonine Asparagine Arginine Leucine Glutamic Tryptophan Histidine Ammonia Ornitine Ethanolamine Ammonia gamma-Aminobutyric Acid Isoleucine Citrulline yrosine Carnosine Anserine Arginine T Urea b-Alanine Methionine b-Amino-i-butyric Acid Aspartic Sarcosine Proline Asparagine Phosphoserine Proline Cystine Urea Hydroxyproline 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Amino Acid standard for physiological fluids, containing 2 internal A plasma sample of patient with PKU. Eluants: 1700-1125, Li365, Li375, standards – Glucosaminic Acid and 2-Aminoethyl-L-Cysteine (AEC). RG003, column 0354675T (Pickering Laboratories, Inc), temperature Eluants: 1700-1125, Li365, Li375, RG003, column 0354675T (Pickering gradient from 32 °C to 70 °C Laboratories, Inc), temperature gradient from 32 °C to 70 °C (Pickering Laboratories, Inc), temperature gradient from 46°Cto70 gradientfrom (Pickering Laboratories,Inc),temperature as internalstandard.Eluants:Na315,Na740,RG011,column1154110T containingNorleucine hydrolysate, Amino Acidstandardforprotein 32°Cto70 gradientfrom temperature Li375, RG003,column0354675T(PickeringLaboratories,Inc), A plasmasampleofpatientwithMSUD.Eluants:1700-1125,Li365, 0 0 *This method can be used with virtually ANY manufacturer’s HPLC systemwhenused inconjunctionwithPinnaclePCX methodcan be usedwithvirtually*This ANY manufacturer’sHPLC products. biotechnology and pharmaceuticals in control quality ensuring and beverages, and food in levels nutritional monitoring disease, detecting for worldwide inlaboratories utilized are Our protocols gradients. column temperature analyststocombineeluantgradientallows capabilitiesof modern instrumentswith HPLC auniqueopportunitywhich systemprovides Our PinnaclePCXpost-columnderivatization ouranalyticalcolumnwithout thelossof protecting all thewhile peakresolution. separation; afast,reliable toachieve able are thenewGARD™, we System.With Protection theNewGARD™Column possibilitiesandcreated lookedtootherguard we thisreason, For hadadeleteriouseffect ontheseparation. which band-spreading of creating requirements thespeedandseparation were match guards thetraditionalion-exchange Inotherwords, thechromatogram. When thespeedof to itwasdeterminedthetimenotadequate theaminoacidrunwasincreased, modelat guard thecurrent that R Phosphoserine ES Taurine Urea U LTS LTS Aspartic Acid Threonine 10 Serine Asparagine AND Glutamic Acid Aspartic Acid Glutamine Threonine Serine D Proline Glycine IS Alanine Citrulline a-Amino-n-butyric Acid 20 CU Glutamic Acid Valine 10 Cystine Proline Methionine SSI Allo-Isoleucine Isoleucine Leucine Tyrosine ON Glycine Alanine Phenylalanine Cystine 30 Valine Methinone Isoleucine Leucine Norleucine Tyrosine 40 Phenylalanine 20 Lysine A Histidine Ammonia 50 Ammonia CC Ornithine ELE Tryptophan Lysine Histidine 60 RA Arginine 30 TE 70 Arginine D WITH P Laboratories, Inc), temperature gradient from 32°Cto70 gradient from Laboratories, Inc),temperature Eluants: 1700-1125,Li365,Li375,RG003,column0354675T(Pickering A plasmasampleofpatientwithArgininosuccinicaciduria(ASA). 0 from 46°Cto70 from gradient column 1154110T(PickeringLaboratories,Inc),temperature ing Norleucineasinternalstandard.Eluants:Na315,Na740,RG011, accordingtoAOACMethod994.12,contain A feedsample,hydrolyzed AMI 0 Phosphoserine Taurine Urea Aspartic Acid Threonine 10 Serine NO Asparagine Glutamic Acid Glutamine O Aspartic Acid A Threonine Proline ST-C Serine Glycine Alanine Citrulline C 20 a-Amino-n-butyric Acid Valine Methionine Glutamic Acid Cystathionine I 10 Isoleucine D Leucine Proline Argininosuccinic Acid (ASA) Tyrosine O ANA 800-654-3330 /Fax:650-968-0749 /650-694-6700 [email protected] /[email protected] 94043 CA / Mountain View, 1280 Space ParkWay Phenylalanine Glycine L 30 Alanine U Cystine Valine M Methinone LYSIS N Isoleucine ASA anhydride II Leucine 40 Norleucine DE Tyrosine ASA anhydride I Phenylanine 20 R Ammonia 50 U Lysine IV Histidine SI Ornithine A Ammonia Lysine TIZ Histidine NG 60 A H TI P ON - 30 Arginine 70 Arginine L C C.