Category Archives: Amino Acid Analysis

Chromatograpy Quiz No. 15

Chromatography Quiz #14 Results

We would like to congratulate our grand prize winners of our last newsletter’s Carbamate/Glyphosate Crossword Puzzle Chromatography Quiz: Jim Balk from DHHS Public Health Environmental Laboratory, Amanda Comando and Dan Raphaely from SCWA, June Black from the Pennsylvania Department of Environmental Protection, Narjes Ghafoori from LA County Environmental Toxicology Laboratory, Helene Lachance from Shur-Gain Nutreco, Holger Franz from Thermo Fisher Scientific, Matthew Hartz from Underwriters Laboratories, Hossein Hajipour from Texas Department of State Health Services, and Daniel Durham from Georgia Department of Natural Resources!!!

rum cake picThey have each won and will shortly be receiving: a Tortuga To Go Suitcase Gift Pack from tortugarumcakes.com!  This “vacation in a box” includes three rum cakes and an 8oz package of coffee, shipped directly from the Cayman Islands!

We would like to thank all of you for your submissions!

The correct answers for the Carbamate/Glyphosate crossword puzzle:

Across:

1. Pickering column protection system: GARD
6. Heated or Ambient: REACTOR
10. ChlorAC buffer for sample ________: PRESERVATION
11. Over-pressure ________ valve: RELIEF
12. Internal Standard: BDMC
13. Faulty check valves yield a noisy ________: BASELINE
14. Solution to late, broad Glyphosate and AMPA peaks: RESTORE

Down:

1. Glyphosate oxidized by hypochlorite: GLYCINE
2. Pickering Catalog number 3700-2000: THIOFLUOR
3. Reagent one: HYDROLYSIS
4. Fourth peak: METHOMYL
5. Glyphosate herbicide: ROUNDUP
7. _______ detection: FLUORESCENCE
8. o-phthaladehyde: OPA
9. Gradient, water and _______ : METHANOL

Thank you!
Pickering Labs

Chromatography Quiz #15:

Identify the error made when running the Amino Acids chromatogram below and win a prize!  Simply email your answer as well as your full contact information to Rebecca at rlsmith@pickeringlabs.com by May 1, 2014 in order to win.  You will receive email confirmation that your submission has been received.  The answer to the quiz and winner congratulations will be published in the next issue (to be anonymous, please notify Rebecca in submission).

Amino Acid Analysis – Baseline Noise

Pinnacle PCX post-column instrument is being used, in a traditional HPLC setup as recommended by Pickering Labs.  The chromatogram is expanded to show the baseline noise.  The quiz question is: what is causing this problem?

Post-column conditions for amino acid analysis:
Reagent 1: Trione
Reactor 1: 130 °C, 0.5 mL
Reagent flow rate: 0.3 mL/min

Detection: UV-Vis Detector, 570 nm for primary amino acids, 440 nm for secondary amino acids

Problem Chromatogram:

problem chromatogram
Please use our website to view any reference Amino Acid chromatogram – the problem for this quiz is not application-specific, so any of our AAA methods could be applicable.  If you have any questions, please don’t hesitate to email Rebecca at rlsmith@pickeringlabs.com.

Amino Acid Analysis of Monoclonal Antibodies

Method Abstract 373, Amino Acid Analysis of Monoclonal Antibodies

The peptide and protein based pharmaceuticals are a rapidly expanding class of therapeutical agents that are used to treat a wide variety of health conditions, including cancer, metabolic and auto-immune diseases, HIV and more. Biologic drugs, such as monoclonal antibodies, are derived from living organisms and are usually very expensive. As many biologics are coming off of patents, the market is ready for cost-saving biogenerics. But all proteins, including monoclonal antibodies, have complex structures that determine their function. Differences in structure would alter biological activity leading to changes in safety and efficacy of the drug.

ICH Q6B is a guidance document that provides a set of internationally accepted specifications for biotechnological and biological products to support new marketing applications. It establishes the set of criteria to which a drug substance, drug product or material should conform to be considered acceptable for intended use.

aaa chromatograms from MA373Determining Amino Acid composition following hydrolysis is listed in ICH Q6B as a way to characterize the protein and to confirm its identity by comparing with Amino Acid composition deduced from the gene sequence of the desired product. Amino Acid Analysis data is also used to accurately determine the protein content.

The Amino Acids Analysis with post-column derivatization is a very sensitive, reproducible and rugged method and it has been a preferred approach for laboratories running biological samples, protein, peptides and foods analysis. Pickering Laboratories Inc. offers many Amino Acids Analysis products including post-column derivatization instruments, columns, eluants, reagents and standards. All products are designed to work together to deliver optimum results for any chosen sample.

METHOD
Analytical conditions
Column: High-efficiency Sodium cation-exchange column, 4.6 x 110 mm, P/N 1154110T
Flow Rate: 0.6 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditionsgradient table for AAA monoclonal AB
Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids
Injection Volume: 10-50 uL

You can download this abstract, as well as our product catalog and other notes from our website: www.pickeringlabs.com

 

AOAC International Meeting

fountainThe AOAC International held their annual meeting in Chicago, Illinois from August 25-27, 2013.

Pickering Laboratories were on hand to share our latest methods and instruments.  We also gave an Exhibitor Presentation titled “An Automated Highly Sensitive Method for Aflatoxin B/G or Ochratoxin A Clean-up and Analysis – A Novel Approach in Mycotoxin Analysis”

The purpose of the talk was to showcase our newest method and instrument:

Building on the FREESTYLE Automated Sample Preparation Instrument, the new ThermELUTE module and columns enable rapid, sensitive analysis of Aflatoxin or Ochratoxin A. The ThermELUTE module uses a thermal denaturation technique to release jellybean4toxin from a new column format requiring only 20% of usual solvent volumes, and FREESTYLE quantitatively transfers the eluate into the HPLC. In combination with online injection, analytical sensitivity can be increased without manual manipulation. The increase in sensitivity provides a reduction of sample load, while maintaining excellent chromatographic results. The increase of sensitivity allow analysis of mycotoxin levels well below regulatory limits.

The ThermELUTE takes the separate steps of cleanup and analysis, and combines them into one automated system. Using a FREESTYLE robotic system equipped with the

FREESTYLE Automated Sample Preparation System
FREESTYLE Automated Sample Preparation System

ThermELUTE module, we can now clean up a sample for mycotoxins, and then directly inject the cleaned sample onto the HPLC column for analysis!

Automating Mycotoxin Analyses from Cleanup to Detection:

  • Results 3 times faster
  • 80% less solvent
  • Direct connect to HPLC

The brochure for ThermELUTE can be found HERE. To view the slides from our presentation, click HERE.

 

Calculation of Whey Protein Fraction in Milk-Based Infant Formula

A method for the calculation of whey protein fraction in milk-based formula products was developed and validated by a group of researchers from Abbott Nutrition, Covance Laboratories and AOAC International. The method is based on calculating the ratio of (Asx + Ala)/(Pro+Phe) determined from the amino acid profile of the hydrolyzed sample.

The method was approved as AOAC Official First Action Method 2012.07 and is appropriate for accessing compliance with whey content requirements of GB 10765-2010 (National Food Safety Standard Infant Formula)*. It is applicable to finished products containing both intact and partially hydrolyzed whey proteins.

According to this method, the Amino Acid Profile of hydrolyzed samples is determined using cation-exchange chromatography with post-column derivatization and UV/Vis detection. The researchers used Pickering Laboratories post-column derivatization system, our patented Trione Ninhydrin reagent and Pickering Sodium analytical column and buffers.

The method performance was evaluated using non-fat dry milk and products with whey levels from 32% to 63%, including NIST Infant Formula Standard Reference Material. The repeatability ranged between 0.3 and 2.5 %, while intermediate precision were between 2.6 and 3.4%. Average recoveries ranged from 97 to 100%.

The unmatched accuracy and precision of amino acid analysis by HPLC with post-column derivatization allowed the researches to develop a robust mathematical model for calculating whey protein fraction that can be successfully applied to a wide range of products. This proved once again that post-column derivatization analysis of amino acids is still a method of choice for laboratories that require highly accurate results.

Pickering Laboratories offers a total solution for amino acids analysis, including post-column derivatization systems, reagents, columns, buffers and standards.

The reference to the published study is below. The First Action Official Method 2012.07 can also be found online at http://www.eoma.aoac.org/.

Calculation of Whey Protein Fraction in Milk-Based Infant Formula: First Action 2012.07

Authors: Wesley Jacobs, Paul Johns, Phillip Haselberger, Joseph J. Thompson, Darryl Sullivan, Steve Baugh.

Source: Journal of AOAC International, Volume 96, No. 3, 2013, pp. 502-507

 

(*) this is a Chinese Standard

Amino Acid Analysis of Cell Culture Media

We have a new Method Abstract! MA371, Amino Acid Analysis of Cell Culture Media

Cell cultures are widely used to produce biopharmaceuticals and other biologically active compounds. The composition of the cell culture media affects the yield and structure of the desired products and must be carefully optimized. Cell culture media is typically composed of mixtures of amino acids, vitamins, carbohydrates, inorganic salts as well as different peptides, proteins and other compounds. As the cells grow, they consume nutrients and release target biopharmaceuticals as well as waste products.

Amino Acids serve as the building blocks of proteins, as well as intermediates in many metabolic pathways. Amino Acids are typically added to cell culture media to provide nutritional requirements for the cells. Monitoring and adjusting Amino Acid composition is an essential part of optimizing the manufacturing process to ensure high quality and optimum yield of the final product.

Amino Acid Analysis using cation-exchange chromatography with post-column Ninhydrin derivatization allows for easy determination of Amino Acid concentrations in many complex matrices, including cell culture media. The post-column method is very sensitive, reproducible and rugged. It has been and continues to be a method of choice for laboratories running biological samples, protein, peptides and foods analysis. Most chemical compounds present in the media do not interfere with analysis, so the majority of samples only need diluting with citric buffer and filtering before analysis. If serum is added to the media, then the proteins need to be precipitated using either Seraprep™ solution or ultrafiltration.

Pickering Laboratories, Inc. offers the complete solution for Amino Acid Analysis, including post-column derivatization instruments, columns, eluants, reagents and standards. The Pinnacle PCX derivatization system has a programmable column oven to allow for shorter run times and easy method optimization.

YChromatograms of Cell Culture Mediaou can download this application note, and many others from our website: www.pickeringlabs.com

METHOD
Analytical conditions
Column: High-efficiency Lithium cation-exchange column, 4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 1

Post-Column Conditions
Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Detection: UV/VIS 570 nm for primary amino acids, 440 nm for secondary amino acids
Injection Volume: 10-50 uL

gradient table for AAA

2013 Pickering Laboratories North American Catalog

Our latest Catalog for North America is now available!

Click on the link to download the latest product Catalog from Pickering Laboratories, Inc.

Pickering Catalog: http://pickeringlabs-retentiontimes.com/wp-content/uploads/2013/08/2013_PickeringLabs_Catalog_NorthAmerica_LowRes.pdf

Inside our catalog you will find a complete product listing for all instruments and consumables we currently have available. From Sample Cleanup to Post-Column Derivatization, to artificial Perspiration and Saliva, we hope you find something you will find useful!

Pickering Laboratories in 2013

By Wendy Rasmussen

In recent years, we have released several new products and applications, and with still more on the horizon, it occurred to me that now would be a great time to summarize the Pickering of today – our mindset and our wide variety of products & applications.

Acai Berries
Acai Berries

No longer are we simply the “Post-Column Company”. We are the “Automated-Sample Antioxidants” company.  Think of us as the new “super fruit.” The Acai berry, or perhaps the new Chia Seeds (incidentally, we do have a post-column application for the identification & quantitation of  Antioxidants in a variety of matrices).

We are still very active, and we as a company plan to be here for many years to come. We are still the company founded on chemistry and a desire to to teach, to spread our technical expertise, to support our customers.

It’s been a few years now since Pickering began distributing and supporting our LCTech Product line.  The products have shown an ever increasing interest here in the US and in Canada (our official Sales Territory for this product line). We are very proud to offer these products and we hope we can develop it further in the future.

Historically, we have provided the back-end of an analysis (post-column derivatization). Nowadays, we can provide the front-end of analysis as well (the sample-cleanup).

In thinking about our product offerings, I realized that a simple list does not effectively show the scope of the products we have to offer in 2013 – primarily because we have a lot of overlap between products and product lines.  We’re not a vertical company in that regard.  I suppose one could say our product offerings are more circular in that many do not fall into a single distinct category.  I am a very visual person, and for me, a Venn diagram and our overall “product scale” really helped to understand and clarify our products:

Venn Diagram of Pickering Laboratories
Venn Diagram of Pickering Laboratories
General "Product Scale" for Pickering Offerings
General “Product Scale” for Pickering Offerings
For those of who like lists, you can find one Here, on Pickering’s website, and on LCTech’s Website

For any Questions, please feel free to contact us:

Pickering Laboratories, Inc.
Mountain View, California
Email: sales@pickeringlabs.com
Phone: (direct) 650-694-6700 or (toll-free) 800-654-3330

 

Image of Acai Berries:  http://acaiberryeducation.com/