Below are two methods that we advise for Sample Preparation of a Physiological Fluid Sample and have been taken from the Biochrom 20 manual.

Deproteinisation of physiological fluid samples prior to amino acid analysis is usually accomplished using the compound 5-sulphosalicylic acid (SSA) which needs to be of the highest purity to avoid contamination of the resin - minimum of 99% purity.  However, the conditions employed during the deproteinisation vary according to the different concentration of protein in the sample.  Consequently, no single deproteinisation method is suitable for all physiological samples hence, the two methods described below are most suitable for general use with the Biochrom 20.  In the preparation of all samples, for the best results the following precautions must be observed.

1. The sample must be completely free of proteins and large peptides.

2. Where possible maintain the molar concentration of salts in the sample below the level where they interfere with the application of sample to the analytical column.

3. The pH of the sample should be controlled so that chromatography of the acidic amino acids is not affected.

4. When heat labile amino acids are being measured the sample preparation must be performed at +4°C.

5. If possible, all samples should be filtered using a 0.2µm membrane filter before injection into the sample capsule.

6. Centrifugation must always be performed at the highest speed, greater than 10⁴g.

Of the two methods described below, method 1 has the advantage that no pH adjustment of the sample is normally necessary before injection.  Method 1 has the disadvantage that if significant amounts of protein are present in the sample then complete deproteinisation is not achieved.  To prevent protein contamination of the analytical column, when large amounts of protein are present in the sample, use method 2.   After using method 2 the sample pH must be adjusted prior to injection.

Urine samples can be deproteinised using either method except in cases where pathological abnormalities such as proteinuria are suspected.  In these cases and in the analysis of serum, plasma and CSF, method 2 must be used.

Method 1

1. Make up a 10% solution of SSA in water and add 1 ml to a conical centrifuge tube.

2. Cool the sample to 4°C and add 1ml of this to the conical centrifuge tube. Immediately mix the sample and SSA then allow this mixture to stand for 1 hour at 4°C.

3. Centrifuge the mixture for 5 minutes in a high speed centrifuge to spin down the precipitate.

4. Remove supernatant and filter through a 0.2µm filter. Place in a sample tube for storage at -20°C. Before analysis remove the sample from the cold store and carefully raise the temperature from -20°C to +4°C before loading the sample into the vial.

Method 2

1. Prepare a conical centrifuge tube for each sample, placing 50mg of solid SSA in each tube. Cool the tubes to 4°C.

2. Cool the sample to 4°C and add 1ml of this to the conical centrifuge tube. Immediately mix the sample and SSA then allow this mixture to stand for 1 hour at 4°C.

3. Centrifuge the mixture for 15 minutes at 4°C in a high speed centrifuge to spin down the precipitate.

4. Remove the supernatant and filter through a 0.2µm filter. Adjust the pH of this supernatant to pH 2.2 using 0.3M lithium hydroxide. Approximately 10µl to 20µl of hydroxide will be required for each 20µl of sample. If sufficient sample volume is available then use a pH meter or narrow range pH paper to determine the correct pH.

Using both methods a small proportion of the amino acids will be precipitated during deproteinisation, depending on the amount of SSA used, hence the results obtained from a sample prepared using method 2 cannot be compared directly with the results obtained using method 1.

To obtain adequately sized peaks for quantitation with the Biochrom 20 operating in the range 0-1 OD a 40µl sample of serum, plasma and urine is required, an 80µl sample of CSF being required.

References:

Ohara, L. and Ariyoshi, S. Agric Biol Chem 43, 1473 (1979)

Mondino, A. et al J Chromatogr 74, 255 (1972)

De Wolfe, M.S. et al Clin Biochem 1, 75 (1967)

Bech-Andersen et al Z. Tierphysiol., Tierernrg. v. Futtermittelkde 63, 188 (1990)