Analytical method related to authorised feed additive - 1i534

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1 Title DETERMINATION OF MESO-TARTARIC ACID, (D- + L-TARTARIC ACID), OXALIC ACID, MONO- AND DIHYDROXYMALONIC ACID IN mta-solutions Subtitle Liquid chromatography

2 1 SCOPE Determination of mg of meso-tartaric acid, mg of (D- + L-tartaric acid), mg of oxalic acid, mg of monohydroxymalonic acid and mg of dihydroxymalonic acid in 0.6 g of concentrated or 1.5 g of diluted mta-solution (see 8.1). All components are expressed in the H-form on anhydrous base. 2 PRINCIPLE Decomposition of Fe-tartrate complexes by reaction with an excess of hydroxide. Filtration of the formed Fe(OH) 3. Liquid chromatographic separation of the components by using an IOA 1000 Organic Acids column as stationary phase and sulphuric acid, c(h 2 SO 4 ) = 0.01 mol/l, as mobile phase. Detection with a Refractive Index-detector (see 8.6). Calculation versus an external standard. 3 APPARATUS AND EQUIPMENT 3.1 Liquid chromatograph Pump : e.g. Gyrokotek High Precision pump model Injection valve : e.g. Rheodyne 7010 provided with a 20 µl sample loop Column : see Column oven : e.g. Spark Mistral Detector : Refractive Index, e.g. Showa-Denko model RI Output facilities Data acquisition and integration system, e.g. Chromeleon. 3.3 Further requirements Syringe filters, disposable, diameter 30 mm, pore size 0.45 m, PTFE/PP FFL/ML, color code green, Whatman ReZist TM Catalog Number Syringe, 2 ml, disposable Vials, glass, e.g. Alltech No Piston pipette, e.g. Eppendorf Research pipette, ml. 4 REAGENTS 4.1 Mobile phase Sulphuric acid, c(h 2 SO 4 ) = 0.01 mol/l. 4.2 Column Stainless steel tube, length 300 mm, internal diameter 7.8 mm, IOA 1000 Organic Acids, as supplied by Alltech (No. 9646). Page 2 of 8

3 4.3 Calibration substances (see 8.3) Meso-tartaric acid, c(c 4 H 6 O 6..H 2 O) > 98 % (m/m), e.g. Sigma T3259. Determine the meso-tartaric acid concentration ànd the concentration of D-+Ltartaric acid according to this SMA , or applying NMR ( X % (m/m) of meso-tartaric acid.0aq ànd Y % of D-+L-tartaric acid.0aq) D-tartaric acid, c(c 4 H 6 O 6 ) > 99 % (m/m), e.g. Aldrich T L-tartaric acid, c(c 4 H 6 O 6 ) > 99 % (m/m), e.g. Aldrich T Oxalic acid, c(c 2 H 2 O 4.2H 2 O) > 99 % (m/m), e.g. Aldrich Monohydroxymalonic acid (tartronic acid), c(c 3 H 4 O 5 ) 97 % (m/m), e.g. Fluka Store this product in a refrigirator at 4 o C Disodium dihydroxymalonic acid (disodium mesoxalate), c(c 3 H 2 Na 2 O 6.H 2 O) 98 % (m/m), e.g. Aldrich Multi component standard solutions A (2 x; see 8.4). Transfer to a 50 ml flask mg of meso-tartaric acid, C 4 H 6 O 6..H 2 O (4.3.1; = Mcq 1 mg] and mg of D-tartaric acid (4.3.2; = Mcq 2 mg)) or L-tartaric acid (4.3.3), each component weighted to the nearest 0.01 mg. Add 50 ml of sulfuric acid, c(h 2 SO 4 ) = 0.01 mol/l (4.1.1) and dissolve. Determine the mass of the contents to the nearest 0.1 mg (= Mt g). Analyze the solutions on the day of preparation (see 8.10). Prepare 2 separate solutions with slightly different amounts of meso- and L, D- tartaric acid; both calibration samples bracketing the test sample solution (see 6.3) Multi component standard solution B, c(each component in anhydrous H-form) 0.25 mg/ml. Transfer to a 0.5 l flask 250 mg of oxalic acid (4.3.4; = Mcq 3 mg), 250 mg of monohydroxymalonic acid (4.3.5; = Mcq 4 mg) and 250 mg of disodium hydroxymalonic acid (4.3.6; = Mcq 5 mg), each component weighted to the nearest 0.1 mg. Dissolve in and dilute to 0.5 l with sulfuric acid, c(h 2 SO 4 ) = 0.01 mol/l (4.1.1). Determine the mass of the contents to the nearest 1 mg (= Mt g). 4.4 Further requirements Water, doubly distilled or of equivalent purity. For example prepared by the Milli-RO/Milli-Q-System (Millipore) Sodium hydroxide solution, c(naoh) = 5 mol/l. 5 SAMPLE Store the sample in a completely filled brown plastic flask or tube. Close the flask/tube properly to exclude oxygen. If the flask/tube cannot be completely filled cover the liquid with a nitrogen blanket. Avoid sunlight (UV light) and store the vessel in a refrigerator at 4 o C. The sample solutions are stable for at least 2 weeks. See PROCEDURE 6.1 Instrument settings (somewhat dependent on the instrument) Column : IOA 1000 Organic Acids, 300 x 7.8 mm. Temperature column : 10 o C. Mobile phase : sulphuric acid, c(h 2 SO 4 ) = 0.01 mol/l (4.1). Mobile phase flow : 0.3 ml/minute. Injection volume : 20 µl. Detector : RI-detector. Page 3 of 8

4 6.2 Calibration samples Meso, D-+L-tartrate Analyze both standard solutions A (4.3.7) according to Decomposition products Prepare the calibration solutions (I - VII) as follows: transfer, using a piston pipette (3.3.4), the respective volume (= Vc g) of the multi standard-solution B (4.3.8) to seven 50 ml flasks, as stated in the next Table: Solution I II III IV V VI VII in ml in ml in ml in ml in ml in ml in ml Multi standard-solution (4.3.8) Add 50 ml of sulfuric acid, c(h 2 SO 4 ) = 0.01 mol/l (4.1.1). Determine the mass of the contents to the nearest 0.1 mg (= Maq g). Proceed according to Test sample Dilute 600 or 1500 mg (= Ms mg, weighted to the nearest 0.1 mg) of test portion with 25 ml of water in a 50 ml flask. Add an excess (see 8.8) of 1 ml of sodium hydroxide solution (4.4.2) and let the iron hydroxide precipitate for at least 1 hour (see 8.9). Determine the total mass of the contents to the nearest 0.1 mg (= M 2 g). Proceed according to Measurement Fill a syringe (3.3.2) with calibration/test sample solution and pass - for the test samples only - the solution through the clean-up filter (3.3.1) into a glass vial (3.3.3). Inject 20 µl of filtered (calibration) sample into the liquid chromatograph. Record the chromatogram with the RI-detector using identical integrator settings for the calibration and test samples, and determine (3.2.1) the peak areas of the components (= A). 7 EXPRESSION OF RESULTS 7.1 Calculation Calibration functions (see 10.4) Calculate according to the calibration functions: where: y = aq + bq.x aq = intercept of the calibration line for component q; bq = slope of the calibration line for component q; y = peak area (Ac) of component q in the calibration sample; x = concentration (= c) of mg of component q/g of calibration sample, calculated from (see formulae (01) (02) in ): Mt = mass, in g, of each multicomponent standard solution prepared in 4.3.7; or Mt = mass, in g of the multicomponent standard solution prepared in ànd Vc = aliquot, in g taken in 6.2.2; Maq = mass, in mg, of each calibration solution (6.2.2). Mcq = mass, in mg, of component q in the H-form on an anhydrous base (see 8.5), in the standard solutions (4.3.7; 4.3.8), using the formulae (03) (07) in Page 4 of 8

5 Formulae for the calculation of x Meso- and D+L-tartaric acid x = Mcq Mt mg/g (01) Oxalic acid, mono- and dihydroxymalonic acid x = Mcq Vc. Mt Maq mg/g (02) Formulae for the calculation of Mcq meso-tartaric acid: D-+L-tartaric acid: Mcq = Mcq X (03) Mcq = Mcq 2 + [Mcq Y. ] (04) X = % (m/m) of meso-tartaric acid.0aq in the calibration substance 4.3.1; Y = % (m/m) of D-+L-tartaric acid.0aq in the calibration substance 4.3.1; Mcq 1 = mg of meso-tartaric acid.1aq in standard solution 4.3.7; Mcq 2 = mg of D- or L-tartaric acid.0aq in standard solution Oxalic acid: Mcq = Mcq (05) Monohydroxymalonic acid: Mcq = Mcq 4 (06) Dihydroxymalonic acid: Mcq = Mcq (07) Test sample Calculate the concentration of each component q, c(q), in the test sample using the formula: Asq - aq. c(q) = Ms. bq M % Page 5 of 8

6 where: aq, bq = see 7.1.1; Asq = peak area of component q in the test sample solution; Ms = mass, in mg, of the test portion; M 2 = mass, in g, of the contents of the 50 ml flask (see 6.3). Report the results to the nearest 0.01 %. 7.2 Statistical data Repeatability Product Component c n k p r Sample A D+L tartaric acid Meso tartaric acid Sample B D+L tartaric acid Meso tartaric acid Sample C D+L tartaric acid Meso tartaric acid c n k p r : overall mean value, in % (m/m); : number of replicates per series; : number of laboratories; : number of series; : repeatability limit, in % (m/m) Reproducibility Product Component c n k p R Sample A D+L tartaric acid Meso tartaric acid Sample B D+L tartaric acid Meso tartaric acid Sample C D+L tartaric acid Meso tartaric acid c : overall mean value, in % (m/m); n : number of replicates per series; k : number of laboratories; p : number of series; R : intermediate reproducibility limit, in % (m/m) Limit of quantitation (LOQ) The limit of quantitation is not relevant for meso-, D+L-tartrate. For the 3 decomposition products the LOQ is calculated based on standard error of estimation, s e, of each of the calibration functions. Page 6 of 8

7 8 NOTES 8.1 mta-solutions Next to meso-tartaric acid and D- and L-tartaric acid the aqueous mta-solutions contain NaCl and Fe and various concentrations of decomposition products: oxalic acid, mono-and dihydroxymalonic acid. Diluted mta-solution: 0.58 % Fe, 1.6 % meso-tartaric acid, 0.7 % D- + L-tartaric acid. Concentrated mta-solution: 2.2 % Fe, 5.8 % meso-tartaric acid, 2.9 % D- + L-tartaric acid. The molar ratio meso : Fe = 1 : 1. Total tartaric acid: 67 % meso-tartaric acid + 33 % (D- + L-) tartaric acid. 8.2 Calibration substances Commercially available meso-tartaric acid can be contaminated with D+L-tartaric acid. Check the quality of the calibration substance using this HPLC-analysis, NMR- or ICanalysis. Performing additionally an acid-base titration and a KF-titration for the water concentration, can be required too. 8.3 Calibration A 2-point bracketing calibration is performed for quantifying the meso- and the D- +L-tartrate concentration. o The calibration equation is only valid within the small bracketing range. o An alternative is to use the average of the calibration factor (F q = x/y) for each component in further calculations. o A 7-points calibration procedure is used to quantify decomposition products. 8.4 D-, L-tartaric acid The retention time and sensitivity of D-tartaric acid and L-tartaric acid are the same. 8.5 Molecular masses C 4 H 6 O 6.H 2 O [meso-tartaric acid.h 2 O] : 168.1; C 4 H 6 O 6 [(meso)-tartaric acid] : 150.1; C 4 H 4 O 6 [(meso)-tartrate] : 148.1; C 2 H 2 O 4 [oxalic acid] : 90.0; C 2 H 2 O 4.2H 2 O [oxalic acid.2h 2 O] : 126.1; C 3 H 4 O 5 [monohydroxymalonic acid] : 120.1; C 3 H 4 O 6 [dihydroxymalonic acid] : 136.1; C 3 H 2 Na 2 O 6.H 2 O [disodium dihydroxy malonic acid] : Detector Instead of the RI-detector an UV-detector ( = 210 nm) can be used. 8.7 Accuracy Determine if Mcq < 100 mg its mass to the nearest 0.01 mg or incorporate a dilution step based on weights. 8.8 ph of the test sample solution A typical ph-value for mta-solutions is ph 4. Page 7 of 8

8 8.9 Fe-free filtrate The filtrate should be iron-free: c(fe) < 1 g in 25 ml filtrate % removal efficiency. A Fe-removal efficiency of > 99 % is found in daily practice Stability multi-component standard solutions A Slightly alkaline standard solutions A have an increased stability. 9 REFERENCES None Page 8 of 8