Hydroxyproline Assay of Meat, Meat Products, & Sausages According to German Food and Feed Code §64 LFGB 06.00-8

Section Overview

• Introduction
• Experimental
• Reagents, Instruments and Materials
• Analytical Procedure
• Analytical Quality Assurance
• Related Products

Note: Pursuant to the valid copyright regulations this application note contains only a rough description of the content of the official method followed by a detailed description of the specific measurement procedure with the Spectroquant^® Prove Spectrophotometers. A detailed description of the method specific handling steps can be found in the official method of the German Food and Feed Code §64 LFGB 06.00-8.¹

Introduction

Hydroxyproline is a non-essential amino acid predominantly localized within collagen molecules,² thereby serving as a specific biochemical marker for collagen determination in meat and meat products. Collagen, the major structural protein of animal connective tissue, influences both sensory and nutritional attributes of processed meats, with elevated levels being associated with undesirable texture and reduced concentrations of essential amino acids, which are critical indicators of product quality.² Since hydroxyproline constitutes up to 10% of the collagen structure, its quantification provides a reliable estimate of total collagen content.³ Owing to its scarcity in proteins other than collagen, hydroxyproline determination is widely applied as a standardized method for assessing meat quality and for detecting adulteration with low-value raw materials.²

The official procedure described in the German Food and Feed Code §64 LFGB 06.00-8 employs a colorimetric method for the hydroxyproline assay for collagen. This standardized hydroxyproline assay protocol enables accurate quantification of hydroxyproline, and thereby collagen content, in a variety of meat matrices, supporting regulatory compliance, compositional analysis, and product labeling.

Experimental

Hydroxyproline Assay Principle

The Hydroxyproline content is determined after acid hydrolysis of the sample followed by the separation of the fat content and oxidation with Chloramine T. The oxidized form of Hydroxyproline reacts with 4-(Dimethylamino) benzaldehyde to form a red-colored product that is measured photometrically at 430 nm. This method is based on the official method of the German Food and Feed Code §64 LFGB 06.00-8¹ and describes the determination of Hydroxyproline in meat, meat products and sausages.

Measuring Range

Method 2538        Hydroxyproline Meat §64 LFBG 06.00-8           0.000 – 1.000 g/100g

Applicable Sample

Meat, meat products and sausages

Reagents, Instruments and Materials

Reagents

• Chloramine T Trihydrate for analysis (1.02426)
• Hydrochloric acid solution 6.0 mol/L (6.0 N) (1.43007)
• Petroleum benzine boiling range 40-60 °C SupraSolv^® (1.01772)
• Citric acid monohydrate for analysis EMSURE^® (1.00244)
• Sodium hydroxide pellets for analysis EMSURE^® (1.06498)
• Sodium acetate anhydrous for analysis EMSURE^® (1.06268)
• Perchloric acid 60% for analysis EMSURE^® (1.00518)
• 1-Propanol for analysis EMSURE^® (1.00997)
• 2-Propanol for analysis EMSURE^® (1.09634)

Instrument(s) & Devices

For the measurement one of the following Spectroquant^® photometers is necessary

• Spectroquant^® VIS Spectrophotometer Prove 100 plus (1.73026)
• Spectroquant^® UV/VIS Spectrophotometer Prove 300 plus (1.73027)
• Spectroquant^® UV/VIS Spectrophotometer Prove 600 plus (1.73028)

This application note pertains to the above listed photometers and all discontinued instruments from the Spectroquant^® Prove series.

Software for Data transfer

• Optional Spectroquant^® Prove Connect to LIMS software package (Y.11086) to transfer your data into an existing LIMS system.

Instrument Accessories

• Rectangular cells 10 mm (1.14946)

Other Reagents and Accessories

• L-Hydroxyproline for biochemistry (optional, for AQA)
• DURAN^® Laboratory bottles
• Round-bottomed flask or Erlenmeyer flask with NS and return condenser
• Test tube, closable, 10–15 mL
• Heating block, drying cabinet or heating plate
• Water bath (temperature controllable)
• Vacuum
• Funnel
• Folded filter, diameter 15 cm
• Volumetric flasks, 50 mL, 100 mL
• Standard laboratory glassware (e.g., glass beakers) and pipettes
• Analytical balance 

Analytical Procedure

Preparation of Solutions

• Buffer solution pH 6.8: The solution must be prepared according to German Food and Feed Code §64 LFGB 06.00-8.¹
• Oxidizing reagent: The solution must be prepared according to German Food and Feed Code §64 LFGB 06.00-8.¹
• Color reagent solution: The solution must be prepared according to German Food and Feed Code §64 LFGB 06.00-8.¹

Sample Preparation

Homogenize the sample.

Preparation of Measurement Solutions

Acid hydrolysis and fat separation

• Weigh approx. 2 g of the homogenized sample with an accuracy of 1 mg to a DURAN^® laboratory bottle and follow the procedure according to German Food and Feed Code §64 LFGB 06.00-8, chapter 7.1^.1.
• Note the sample weight.
• Use filtrate for the preparation of the measurement solution.

Hydroxyproline determination

Reagent blank

• Mix 0.100 mL of distilled water with 5 mL of oxidizing reagent in a closable test tube and incubate for 20 min at room temperature.
• Add 2 mL of Color reagent, close the tube, mix and incubate in a water bath at 60 °C for 15 min.
• Cool down the vessel to room temperature with running water within 3 min.
• Incubate for 30 min at room temperature.

Sample

• Mix 0.100 mL of the resulting filtrate after acid hydrolysis and fat extraction of the sample with 5 mL of oxidizing reagent in a closable test tube and incubate for 20 min at room temperature.
• Add 2 mL of Color reagent, close the tube, mix and incubate in a water bath at 60 °C for 15 min.
• Cool down the vessel to room temperature with running water within 3 min.
• Incubate for 30 min at room temperature.

Measurement

Note: It is advisable to measure the reagent blank and the sample using the same cell as the one used for the zero adjustment or else a cell with identical optical characteristics and an identical absorption (matched pair).

• Open the methods list (<Methods>) and select Method No. 2538 "Hydroxyproline Meat §64 LFGB 06.00-8".
• The instrument automatically prompts a "Zero adjustment".
• For the zero adjustment, fill a clean and dry 10 mm rectangular cell with distilled water.
• After prompting, insert the filled rectangular cell into the cell compartment. The zero adjustment is performed automatically.
• Confirm the performance of the zero-adjustment procedure by clicking on <OK>.
• A window with an input field to enter the sample weight pops up.

Enter the weight of the sample in grams (g), accurate to 0.001 grams (g), confirm with <OK>, and click on <START> to switch to the measurement procedure. 
Note: It is possible to enter a sample weight in a range of 0.010 to 5.000 g.

• Fill the prepared reagent blank into a clean and dry 10-mm rectangular cell. Insert the cell into the cell compartment. The measurement is performed automatically. A (✓) symbol appears behind the cue "Insert Reagent Blank".
• Confirm the measurement by clicking on <OK>.
• Finally fill the prepared sample solution into a clean and dry 10-mm rectangular cell. Insert the cell into the cell compartment. The measurement is performed automatically. A (✓) appears behind the cue “Insert Sample”.
• Confirm the measurement by clicking on <OK>
• Read off the result in g/100g and the absorption for the reagent blank (A_RB) and the sample (A_Sample) from the display.
• Tap the <START> button to start the measurement procedure for the next sample. 

Analytical quality assurance

• The method can be checked using L-Hydroxyproline as a standard substance
• Prepare a stock solution with 600 mg/L resp. 60 mg/100 mL L-Hydroxyproline by dissolving 60.0 mg L-Hydroxyproline in approx. 80 mL distilled water. Transfer the solution completely to a 100 mL volumetric flask and fill up to the mark with distilled water.
• Dilute the stock solution to 4.8 mg/100 mL L-Hydroxyproline by pipetting 4 mL of the 60 mg/100 mL LHydroxyproline stock solution into a 50 mL measuring flask and fill up to the mark with distilled water.
• Mix 0.100 mL of the prepared standard solution with 5 mL of the Oxidizing reagent in a closable test tube and incubate for 20 min at room temperature.
• Add 2 mL of Color reagent, close the tube, mix and incubate in a water bath at 60 °C for 15 min.
• Cool down the vessel to room temperature with running water within 3 min.
• Incubate for 30 min at room temperature.
• Measure this solution versus a reagent blank as described in the section "Measurement". Hereby enter a weight of 2.00 g.

Note: Due to the different sample preparation procedure and determination procedure of the standard solution compared to a sample analysis, it is necessary to recalculate the displayed result manually as follows:

Measured Concentration standard [mg/100 mL] = Displayed result [g/100 g] × F1/F2 = Displayed result [g/100 g] × 1000/50

Measured Concentration standard [mg/100 mL] = Displayed result [g/100 g] × 20

Where, F1 = 1000 = recalculation g/100 g to mg/100 mL

                  F2 = 50 = Factor sample preparation for real sample

Adjustment

• In case of significant deviations in the method control procedure, the preprogrammed factor of 10.55 or the current factor used in the calculation of the displayed results can be adjusted by the user.
• The corrected factor must be recalculated as follows: 
  Factor corrected = Current factor x (target value standard / measured and recalculated value standard)
• To edit the preprogrammed factor, select method 2538 from <Methods>.
• Close the window for the “Zero adjustment” by clicking on <X>.
• Close the input field for the sample weight by clicking on <X>.
• Click <Settings> and select the list “FACTORS”.
• Tap on the input field “Factor”, enter the corrected factor and confirm by clicking on <OK>.
• Close the window for the “Zero adjustment” by clicking on <X>.
• For the next measurement restart the method by selecting the method anew from <Methods>.

Note:

To find the used factor, select Method 2538 from <Methods>.

• Close the window for the “Zero adjustment” by clicking on <X>.
• Close the input field for the sample weight by clicking on <X>.
• Click <Settings> and select the list “FACTORS”.