Accelerated 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 Cystathionine Glycine Glutamine Acid Allo- a-Amino-n-butyric Acid Glycine Glutamine Alanine Serine aline Threonine V Isoleucine 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 Proline Asparagine Phosphoserine Proline Cystine Urea

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 Accelerated Amino Acid analysis using HPLC with Post-Column Derivatization Citrulline Acid (ASA) Leucine Alanine Argininosuccinic Acid Glycine aline V Glutamic Glutamine Glycine aline V aurine Acid Alanine T Glutamine anhydride I Acid ysine ASA L Ornithine Isoleucine Acid ysine Threonine L Leucine Serine Histidine Threonine a-Amino-n-butyric Serine Glutamic Acid Arginine anhydride II Ammonia Ornithine Histidine aurine a-Amino-n-butyric Acid T yrosine Ammonia yrosine Phenylalanine Arginine T Allo-Isoleucine ASA T Isoleucine Phenylalanine Methionine Urea Urea Methionine Aspartic Citrulline Asparagine Cystathionine Phosphoserine Cystine Asparagine Proline Proline Aspartic Phosphoserine

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 A plasma sample of patient with MSUD. Eluants: 1700-1125, Li365, A plasma sample of patient with Argininosuccinic aciduria (ASA). Li375, RG003, column 0354675T (Pickering Laboratories, Inc), Eluants: 1700-1125, Li365, Li375, RG003, column 0354675T (Pickering temperature gradient from 32 °C to 70 °C Laboratories, Inc), temperature gradient from 32 °C to 70 °C Leucine Histidine Cystine Norleucine Acid Alanine ysine L Methinone Glutamic Acid Ammonia aline Serine V Leucine aline Isoleucine Acid V Glycine Threonine yrosine Norleucine T Glycine Alanine Aspartic Serine Acid Phenylalanine Aspartic Threonine Isoleucine Histidine Glutamic Arginine Phenylanine ysine L yrosine T Ammonia Arginine Methinone ryptophan T Cystine Proline Proline

0 10 20 30 0 10 20 30 Amino Acid standard for protein hydrolysate, containing Norleucine A feed sample, hydrolyzed according to AOAC Method 994.12, contain- as internal standard. Eluants: Na315, Na740, RG011, column 1154110T ing Norleucine as internal standard. Eluants: Na315, Na740, RG011, (Pickering Laboratories, Inc), temperature gradient from 46 °C to 70 °C column 1154110T (Pickering Laboratories, Inc), temperature gradient from 46 °C to 70 °C

results and discussion When the speed of the amino acid run was increased, it was determined that the current guard model at the time was not adequate to match the speed and separation requirements of the chromatogram. In other words, the traditional ion-exchange guards were creating band-spreading which had a deleterious effect on the separation. For this reason, we looked to other guard possibilities and created the New GARD™ Column Protection System. With the new GARD™, we are able to achieve a fast, reliable separation; all the while protecting our analytical column without the loss of peak resolution. Our Pinnacle PCX post-column derivatization system provides a unique opportunity which allows analysts to combine eluant gradient capabilities of modern HPLC instruments with column temperature gradients. Our protocols are utilized in laboratories worldwide for detecting disease, monitoring nutritional levels in food and beverages, and ensuring quality control in pharmaceuticals and biotechnology products.

*This method can be used with virtually ANY manufacturer’s HPLC system when used in conjunction with Pinnacle PCX

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