ECAMA Specifications for Trisodium Citrate Dihydrate (E 331 iii) INTRODUCTION These specifications have been prepared by the Applications and Technology Working Group of ECAMA (the European Citric Acid Manufacturers Association). The ECAMA specifications have been ratified by all ECAMA members and have attempted to harmonise universal Food Additive and Pharmaceutical compendial requirements. Where the European Pharmacopoeia, Food Chemicals Codex or the United States Pharmacopoeia did not offer a Quantitative Method of Analysis ECAMA have substituted alternative or modified procedures to produce Quantitative analytical data. The ONLY exception has been for the determination of Sulphate which still relies on the Qualitative Barium Chloride precipitation procedure. In the test the only Quantitative result is when Sulphate is shown to be absent. ECAMA members have recommended a variety of Quantitative instrumental methods for the Determination of Sulphate at the specification recommendation of 150ppm (mg/kg) which might not be routinely available to the end user of these products. ECAMA SPECIFICATIONS TRISODIUM CITRATE DIHYDRATE DETERMINAND PROPOSED LIMIT QUANTITATIVE METHOD Appearance Translucent to white crystals. EP 3rd Edition Free from visible foreign FCC 4th Edition * matters and odours* Identification Citrates EP 3rd Edition Colour of Solution 98% min. Spectrophotometric 1 cm Cell Transmission 420 nm 40% w/v solution. Clarity of Solution 4 NTU max. Turbidity 40% w/v solution Assay 99% min. EP 3rd Edition Iron 5 mg/kg max. Spectrophotometric 1 cm Cell 40% w/v solution 360 nm 85% min Transmission. Heavy Metals as Pb 5 mg/kg max. FCC 4th Edition Lead 1 mg/kg max. FCC 4th Edition Arsenic 1 mg/kg FCC 4th Edition * Mercury 1 mg/kg max. Atomic absorbtion/graphite furnace pH 5% w/v aqueous solution 7.5 to 9 Use freshly boiled water cooled to ambient in air tight flasks. * Sulphate 150 mg/kg max. 40 % w/v solution. Turbidity with BaCl2 * Chloride 50 mg/kg max. Silver nitrate titration Oxalate as Oxalic Acid 100 mg/kg max. EP 3rd Edition Dihydrate Spectrophotometric 1 cm Cell Measured at 520 nm Loss on Drying 12 ± 1% 2g, 2 hours at 180°C E 331 iii Sulphuric Acid Test 80% min. Spectrophotometric 1 cm Cell (Carbonisables) Transmission 470 nm 1 g sample 1 hour 90 ± 1°C 10 ml 98% min H2SO4 APPENDIX 1 ECAMA / CEFIC HARMONISED METHODS OF ANALYSIS CITRIC ACID 1) COLOUR OF SOLUTION The presence of Particulate Turbidity will interfere with the results of this Test Procedure. It is recommended that in the presence of Particulate Turbidity (Greater than 1.0 NTU) the test solution should be membrane filtered (0.2 micron porosity) and the particulate contamination examined under a microscope. 2) IRON i) Principle Colourimetric reaction between Iron and a mixed ferro-ferri cyanide reagent measured quantitatively at 670 nm in a 1 cm Cell against a water reference cell using Iron Calibration Standards (0 to 10 ppm) prepared in Iron-Free 40% w/v Citric Acid Solutions. ii) Reagents Solution A Dissolve 13 g of Sodium Ferrocyanide (Na4 [Fe(CN)6] • 10H2O) in a 200ml volumetric flask with distilled/deionised water and make up to volume. Solution B Dissolve 7.5 g of Potassium Ferricyanide (K3 [Fe(CN)6] • H2O) in a 200ml volumetric flask with distilled/deionised water and make up to volume. Combine solutions A & B and mix well. iii) Analytical Procedures Using a 150 ml beaker prepare 100 ± 1 ml of 40% w/v Citric Acid test solution. Add 5 ± 0.2 ml of the Combined Reagent mix well and allow to stand for 10 ± 2 minutes to permit Colour Development. Measure the Absorbance at 670 nm against a distilled/deionised water reference using 1cm cells. Calculate the Iron concentration by comparing with a Calibration Curve (0 to 10 ppm) or a known Standard (5 ppm) prepared in 40% w/v Iron- Free Citric Acid Solution and analysed at the same time as the Test Samples. iv) Analytical Procedure Detection Limit 0.2 ppm (mg/kg) Analytical Tolerance ± 0.3 at 5 ppm (mg/kg) 3) CALCIUM i) Principle Atomic Absorption Spectroscopy (AAS) utilising a 10% w/v Citric Acid Solution measured against a Calibration Curve prepared with 10% w/v Calcium-Free Citric Acid Solutions. ii) Analytical Procedures Prepare duplicate Test Solutions by adding the following aliquots to 100 ml volumetric flasks:- A) 30 ± 0.2ml of 1M Hydrochloric Acid (Calcium Free) B) 10 ± 0.1g of Citric Acid Sample Finally make up to volume with distilled/deionised water and mix well. Calibrate the AAS Instrument with the following solutions made up in Calcium-free 10% w/v Citric Acid Solution:- Blank, 2.0, 5.0 and 10.0 ppm Calcium (mg/kg) and analyse the duplicate Test Solutions against the above Calibration Curve. iii) Calculation The Calcium content of the Citric Acid Sample is equivalent to:- Test Solution ppm x 10 iv) Analytical Performance Detection Limit 2 ppm (mg/kg) Analytical Tolerance ± 3 at 75 ppm (mg/kg) 4) SULPHATE ECAMA members have demonstrated that the Barium Chloride Precipitation Procedure used in the European Pharmacopoeia and many other compendial specifications can only be used as a Qualitative assessment of the Sulphate content of Citric Acid and the Citrates. Typically at 150 ppm a variance of ± 50 ppm was not uncommon. Only when this type of Test Procedure demonstrates an absence of Sulphate can the result be treated as Quantitative. This would indicate levels of Less than 10 ppm (mg/kg) ECAMA members would recommend the use of the following analytical procedures where there is a need to measure the Quantitative level of Sulphate in Citric Acid or the Citrates. Inductively Coupled Plasma Emission Spectroscopy X-ray Fluorescence Ion Chromatography Elemental Analysis by Combustion 5) OXALATE as OXALIC ACID DIHYDRATE i) Principle Oxalate is determined using the European Pharmacopoeia 3rd Edition Colourimetric Procedure using Phenlyhydrazine hydrochloride. The Calibration Curve (0 to 150 ppm) is prepared from Oxalate-Free Citric Acid and Oxalic Acid Dihydrate with absorption measured at 520 nm in a 1 cm cell against a water reference. ii) Reagents See European Pharmacopoeia 3rd Edition. iii) Analytical Procedure See European Pharmacopoeia 3rd Edition. Following the 30 ± 5 minutes Colour Development period measure the Absorption at 520nm in a 1 cm cell. The Oxalate concentration is calculated from the Calibration Curve or from the Absorption of a known Standard (100ppm) prepared from Oxalate-Free Citric Acid and analysed at the same time as the Test Samples. iv) Analytical Performance Detection Limit 20 ppm (mg/kg) 6) SULPHURIC ACID TEST (CARBONISABLES) ECAMA members have demonstrated that the Colour Standard, Fluid K used in the various Compendial Specification Tests for Carbonisables has a Colour Intensity which varies with ambient temperature. Fluid K colour increases with temperature. Consequently ECAMA recommend a Test Procedure which monitors the Test Sample against Fixed Wavelength Absorbance/Transmission of Fluid K at 25°C. Thus the compliance limits for any Test Solution are at 470 nm in a 1 cm Cell. Absorbance must not exceed 0.52 Transmission must be equal to or exceed 30% Please note the colour of the Sulphuric Acid Test Solutions are NOT Temperature sensitive. i) Principle When Citric Acid is heated at 90°C with concentrated Sulphuric Acid a yellowish-brown colour develops especially in the presence of non-Citric based organic impurities or Nitrates. Compliance is demonstrated by comparison with a Standard Colour Solution Fluid K (Aqueous Solution of Cobalt and Iron Chlorides). ii) Reagents Sulphuric Acid 98% minimum Reagent Grade Sulphuric Acid (low Nitrate less than 1 ppm max) Fluid K Prepare as per European Pharmacopoeia 3rd Edition. iii) Analytical Procedure Carefully add 10 ± 0.1 ml of 98% minimum Sulphuric Acid to 1.00± 0.01 g of Citric Acid in a test tube (18 mm o.d, 150 mm length). These must be pre-cleaned with 98% minimum Sulphuric Acid at 90°C before use or previously reserved for Carbonisable Tests. Place the test tube in a water bath maintained at 90 ± 1°C for 1 hour. Occasionally remove and CAREFULLY shake the tube to ensure that the Citric Acid is dissolved and gaseous decomposition products are allowed to escape to atmosphere. After 1 hour cool to Ambient Temperature, shake to ensure all dissolved gases are removed and measure the Absorbance/Transmission at 470 nm in a 1 cm Cell. iv) Analytical Performance Product meets the ECAMA Specification if the Colour generated in the Sulphuric Acid meets the following criteria at 470 nm in a 1 cm Cell:- Absorbance does not exceed 0.52 Transmission is equal to or exceeds 30% APPENDIX 2 ECAMA / CEFIC HARMONISED METHODS OF ANALYSIS TRIPOTASSIUM CITRATE MONOHYDRATE 1. COLOUR OF SOLUTION See Appendix 1 CITRIC ACID Section 1. 2. IRON There is a colourimetric reaction between Tripotassium Citrate and Iron which gives rise to an Absorption at 360 nm. Typically for a 40%w/v solution 5 ppm of Iron in the Citrate will give at least 80% Transmission at 360 nm in a 1 cm Cell. Transmissions less than 80% indicate an Iron Concentration in excess of 5 ppm. Please note that turbidity in the Test Solution will effect the result (See Appendix 1 CITRIC ACID, Section 1). 3. OXALATE See Appendix 1 CITRIC ACID Section 5. 4. SULPHURIC ACID TEST (CARBONISABLES) For the Analytical Procedure see Appendix 1 CITRIC ACID Section 6 taking 1.00 ± 0.01 g sample of Tripotassium Citrate Dihydrate where by compliance is demonstrated if Transmission at 470 nm in a 1 cm Cell is equal to or exceeds 80%. APPENDIX 3 ECAMA / CEFIC HARMONISED METHODS OF ANALYSIS TRISODIUM CITRATE DIHYDRATE 1) COLOUR OF SOLUTION See Appendix 1 CITRIC ACID Section 1. 2) IRON There is a colourimetric reaction between Trisodium Citrate and Iron, which gives rise to an Absorption at 360 nm. Typically for a 40% w/v Solution 5ppm of Iron in the Citrate will give at least 85% Transmission at 360 nm in a 1 cm Cell. Transmissions less than 85% indicate an Iron concentration in excess of 5 ppm. Please note that turbidity in the Test Solution will affect the results. (See Appendix 1 CITRIC ACID Section 1). 3) OXALATE See Appendix 1 CITRIC ACID Section 5. 4) LOSS ON DRYING i) Principle Heating approximately 2g ± 1mg of Trisodium Citrate Dihydrate at 180 ± 5°C for 2 hours. Please note ECAMA members have demonstrated that 180° for 2 hours is sufficient to dehydrate 2g of Trisoium Citrate Dihydrate to constant weight as opposed to 4 hours recommended in 96/77/EC of December 1996 for E331 (iii). ii) Analytical Procedure As per E331 (iii) from 96/77/EC with heating restricted to 2 hours only. 5) SULPHURIC ACID TEST (CARBONISABLES) For the Analytical Procedure see Appendix 1 CITRIC ACID Section 6 taking a 1.00 ± 0.01 g sample of Trisodium Citrate Dihydrate whereby compliance is demonstrated if Transmission at 470 nm in a 1 cm Cell is equal to or exceeds 80%.