DL-methionine hydroxy analogue titration: adaptation of the Siggla and Edsberg method for diakyl sulfides (Ind. Eng. Chem. Anal. Ed., 1948, 20, 938-939). Oxidation of the sulfur by bromine in acid medium and titration by potentiometry.

1. DL-methionine-Hydroxy-Analogue Titration

Reagents
- A previously titrated 0.1N potassium bromide/potassium bromate solution.
- Acid medium:
  · Glacial acetic acid 400 ml
  · Distilled water 100 ml
  · Concentrated hydrochloric acid 30 ml.
- Mix well before use.

Material
A pH meter or a specific potentiometer with a scale in millivolts and provided with a system of platinum and glass electrodes.

Procedure
- Accurately weigh a sample of 0.2 - 0.3 g in a beaker (100 ml -or 250 ml)
- Add about 50 ml of acid mixture, introduce a magnetic stirrer bar
- Put the beaker containing the solution on a magnetic stirrer and place it properly so as to have maximum mixing without exposing the electrodes to air bubbles
- Titrate the solution with the potassium bromide/ bromate solution at a flow rate of 1 drop per second using the scale + 700 mV
- Near the end point (which is observed by a slight change in potential), stop the titration, then continue dropwise, noting the changes in potential in mV and the volume of reagent. At the end point, a change in potential of 200 mV or more is observed
- Continue the titration beyond this limit
- The end point will be taken at the volume corresponding to the maximum change in potential. It can also be obtained graphically as the point of inflection of the potentiometric curve as a function of the volume of reagent

Calculation
Let:
- V be the volume in ml de Br- / BrO3- determined according to the procedure.
- N the normality of the Br- / BrO3- solution determined according to the procedure described below.
- E the mass in grams of the sample,

2. Preparation of the titrated bromide/Bromate Solution

Principle
The 0.1N bromide/bromate solution is obtained by dissolving 2.78g of anhydrous potassium bromate of (KBrO3) and 10 g of potassium bromide (KBr) in 1 liter.

Procedure
6 ml of 6N H2SO4 are added to 50 ml of the solution to be assayed in a 250 ml iodine flask. The flask is cooled on an ice bath and 10 ml of a 20% potassium iodine solution are carefully added so that the solution is introduced into the flask without allowing bromine fumes to escape. The flask is stirred for 2 to 3 minutes to allow the bromine to react with the potassium iodide. The iodide liberated is titrated with a standard thiosulphate solution using starch as an indicator.
The exact normality of the bromide/bromate solution is thus obtained according to the conventional formula:

3. Determination of DL-methionine-hydroxy-analogue acid in feeds

Principle
Added methionine hydroxy analogue is extracted from the feed with a water-acetonitrile (90: 10, by volumes) mixture in one or two steps, depending on the incorporation level. Extracted polymers are hydrolyzed into monomer in alkaline conditions and the resulting solution immediately neutralized. Monomer separation is then performed by reverse phase HPLC with UV detection at 214 nm.

Safety precautions
The major risk comes from using orthophosphoric acid (potential chemical burns with solutions). Wear protection glasses as much as possible. Wear clothing that covers exposed skin and work under a fume hood. In case of skin or eye contact, rinse thoroughly with water and seek medical assistance.

Reagents
All reagents must be of analytical grade. Water must be de-ionized or of equivalent quality.
- Acetonitrile (lichrosolv)
  Gradient grade for chromatography.
  · Extraction mixture
    Place 100 ml. acetonitrile (3-1) in a graduated measure. Complete to 1000 ml with de-ionized water.
- 85% Orthophosphoric acid (RP Normapur grade)

- Potassium hydroxyde (RP Normapur)
  · 50% KOH
    Weigh accurately 50 ± 0.1 g potassium hydroxyde in a 100 ml gauged flask. Complete to 100 ml with de-ionized water.

- Methionine Hydroxy Analogue, calcium salt (SIGMA ref. MO 126)
  (DL-2 - hydroxy - 4 - (methylthio) - butanoic acid)
  The methionine hydroxy analogue content is determined by potentiometry via the method Br / BrO3 -
  (SIGGLA and EDSBERG, Anal. Chem. (1948), 20/10, 938 - 939).

Equipment
- Standard laboratory apparatus
- Non - reusable polypropylene 30 ml tubes
- Laboratory Rotating Tube Divider
- Grinder designed for the obtention of particles that completely pass through a sieve of perforation size 0.5 mm (f.i. Ultra-Centrifugal Mill ZM 1000 from RETSCH) or, in default, mortar and pestle
- Balance, readability -0.1 g
- Balance, readability -0.1 mg
- Centrifuge with speed = 6000 rpm (5900 g)
- 100 ml centrifuge pots
- Linear shaking water bath, accuracy ± (0.1)°C
- Millipore filters - cellulose esters - 0.22 µm (ref. GSW 04700)
- H.P.L.C. equipped with automatically controlled gradient pump, auto sampler, column oven, and UV spectrophotometric detector set at 214 nm

Procedure
All preparations are done in duplicate.
- Preparation of the working standard solution
  · Weigh approximately 20 ± -0.01 mg methionine hydroxy analogue, calcium salt (see page 6) of known purity, in a 50 ml gauged flask.
  · Complete to 50 ml with the extraction solution (see page 6).
  · Homogenize.
  · Then, pipette 10 ml of this solution in a 50 ml gauged flask and adjust to 50 ml with the extraction solution (see page 6).

- Test solution for feed at low concentration -(<0.1%)
  · Weigh approximately 5 g ± 0.1 mg of previously divided and ground feed in a 100 ml centrifuge pot.
  · Bottletop dispenser (Dispensette), add in the pot 50 ml of the extraction solution (see page 6).
  · Stop the pot.
  · Stir for 1 hour in the water bath at 45°C.
  · Centrifuge 10 minutes at 6000 rpm.
  · Take the supernatant.

- Test solution for feed at high concentration (<0.1%)
  · Repeat procedure steps described on page 8.
  · To the centrifuge pot residue, add another 50 ml of the extraction solution (see page 8) by means of the Dispensette.
  · Repeat procedure steps described on page 8.
  · Take the supernatant and mix it with the first one obtained in (see page 8).

- Polymers hydrolysis:
  Proceed with the feed as follows:
  · Place 10 ml of the solution in a non - reusable polypropylene 30 ml tube.
  · Add 1 ml 50% KOH (see page 6); stir for 1 minute.
  · Add 1 ml 85% H3PO4 (see page 6); stir for 30 seconds.
    Should the solution be cloudy, then centrifuge 10 minutes at 10000 rpm.
  · Pass (the supernatant) on 0.22 µm filter.

- Chromatographic analysis:
  Inject working standard solution and the test solution into the HPLC system using the conditions described in appendix 1.

Note: Preparing the mobile phase at the pump level makes it possible to adjust it to the type of feed.

Expressing the results
- Methionine hydroxy analogue acid incorporation rate of the feed
  Peak areas are calculated by the software and the results are expressed as follows.

Variables:
AE: Peak area for methionine hydroxy analogue in the test solution
AR: Peak area for methionine hydroxy analogue in the working standard solution (average of 2
determinations)
MR: Mass of the test taking for the working standard, expressed in mg. (approx. 20 mg)
PR: Purity of the working standard of methionine hydroxy analogue
ME: Mass of the test taking for the sample, expressed in g
VE: Volume of dilution of the sample, expressed in ml (50 or 100 ml depending on the expected
concentration)
VR: Volume of dilution of the working standard, expressed in ml (here, 250 ml)
F: Dilution factor for the hydrolysis = 12/10 = 1.2

- Rhodimet™ AT 88 incorporation rate of the feed
  Rhodimet™ AT 88 % (w/w) = HMB % (w. /w.) / 0.88

Remarks: Indications for improving the separation
It is advised in case of encountering a separation problem to adjust the composition of the mobile phase. In addition, when the peaks are very flat, column temperature must be increased. However, you should not exceed a temperature of 55°C as above this level the product composition may change.
- Impact of temperature
  Should temperature increase, then retention times are decreasing and resolution is improving.
- Impact of pH of the mobile phase
  When pH increases, the retention times are decreasing. However acidity must stay in the range of pH 2 to 4.
- Impact of composition of the mobile phase
  Should the content of the acetonitrile solution (see page 6) be increased, then the retention time decreases.

Fidelity
See appendix 2 and 3.
Accuracy is approximately 4 % for feedstuffs.

Appendix
- Appendix 1: Chromatographic conditions.
  · Apparatus:
    Isocratic chromatograph equipped with automatically controlled gradient pump able to deliver a flow rate of -0.8 ml / min., autosampler with RHEODINE injector, column thermostat, UV spectrophotometric detector set at 214 nm and integrator
  · Column: HIBAR pre- pack RT 250 - 4 Lichrosorb RP 18 (5 µm)
  · Mobile Phase: (% V/V) De-ionized water acidified at pH 2 with H3PO4
    (see page 6) 92
    Acetonitrile (see page 6) 8
    Washing solution (% V./ V.): De-ionized water 50
    Acetonitrile (see page 6) 50
  · Detection: 214 nm
  · Flow rate:0.8 ml / min
  · Injection Loop: 20 µl
  · Frequency of calibration: once per day
  · Data system: IDA MULTICHROM
  · Retention time (approx.): 7.5 min
  · Total duration of the chromatogram: 20 min
  · Total duration of the analysis: 45 min

- Analytical cycle:
  · From 0 to 15 min: Mobile phase; flow rate = 0.8 ml / min
  · From 15 to 16 min: Transition step
  · From 16 to 22 min: Rinse step; flow rate = 0.6 ml / min
  · From 22 to 30 min: Transition step
  · From 30 to 45 min: Mobile phase; flow rate = 0.8 ml / min

- Appendix 2: Fidelity - Repeatability of measuring instrument with the working standard solution

- Appendix 3: Fidelity - Repeatability of measuring instrument and method with test solution (of a Broiler Grower feed)

- Appendix 4: Chromatogram of the working standard solution of DL-methionine-hydroxy-analogue acid

- Appendix 5: Chromatogram of the test solution (of the Broiler Grower feed).

Warnings
This analytical procedure is based upon proprietary experimental results. When employed by a competent analyst, it should yield accurate and reproductible results. No representation is expressed or implied and nothing herein shall be construed as permission or recommendation to practice a patented invention. Permission to reproduce or to publish this method in whole or in part should be sought in writing from Adisseo, Antony, France.