NF 31 Official Monographs / Starch 2239

film of stopcock grease to the sealing surfaces of all of • LOSS ON DRYING 〈731〉 the joints except the joint between the separatory fun- Sample: 1 g nel and the boiling flask, and clamp the joints to ensure Analysis: Dry the Sample at 130° for 90 min. tightness. Acceptance criteria: NMT 20.0% Sample: 25.0 g of Potato Starch • PH 〈791〉 Analysis: Add 150 mL of water to the boiling flask. Sample solution: Prepare a slurry by weighing 5.0 g of Close the stopcock of the separatory , and begin Potato Starch, transferring to a suitable nonmetallic the flow of carbon dioxide at a rate of 100 ± 5 mL/min container, and adding 25.0 mL of freshly boiled and through the Apparatus. Start the coolant cooled water. flow. Add 10 mL of Hydrogen peroxide solution to a re- Analysis: Agitate continuously at a moderate rate for 1 ceiving . After 15 min, without interrupting the min. Stop the agitation, and allow to stand for 15 min. flow of carbon dioxide, remove the Determine the pH to the nearest 0.1 unit. from the boiling flask, and transfer the Sample into the Acceptance criteria: 5.0–8.0 boiling flask with the aid of 100 mL of water. Apply stopcock grease to the outer joint of the separatory fun- ADDITIONAL REQUIREMENTS nel, and replace the separatory funnel in the boiling • ◆PACKAGING AND STORAGE: Preserve in well-closed flask. Close the stopcock of the separatory funnel, and containers. No storage requirements specified.◆ add 80 mL of 2 N hydrochloric acid to the separatory funnel. Open the stopcock of the separatory funnel to permit the hydrochloric acid solution to flow into the boiling flask, guarding against the escape of sulfur diox- . ide into the separatory funnel by closing the stopcock Hydroxypropyl Potato Starch before the last few mL of hydrochloric acid drain out. Boil the mixture for 1 h. Remove the receiving test tube, and transfer its contents to a 200-mL wide- necked, conical flask. Rinse the receiving test tube with a small portion of water, add the rinsing to the 200-mL conical flask, and mix. Heat on a water bath for 15 min, and allow to cool. Add 0.1 mL of Bromophenol blue indicator solution, and titrate the contents with 0.1 N sodium hydroxide VS until the color changes from yellow to violet-blue. Per- form a blank determination, and make any necessary correction (see Titrimetry 〈541〉). Calculate the content, in ppm, of sulfur dioxide in the For the Amylose derivative, m is about 300–1000. Sample taken: DEFINITION Result = 1000 × 32.03 × (VN/W) Hydroxypropyl Potato Starch is partially substituted 2-hydroxypropylether obtained from potato starch by a 32.03 = milliequivalent weight of sulfur dioxide chemical modification of etherification with propylene ox- V = volume of titrant consumed (mL) ide. In addition, this starch may be partially hydrolyzed N = normality of the titrant using acids or enzymes to obtain thinned starch. It con- W = weight of the Sample (g) tains NLT 2.0% and NMT 7.0% of hydroxypropyl groups, Acceptance criteria: NMT 50 ppm on the dried basis. • LIMIT OF OXIDIZING SUBSTANCES Sample solution: Transfer 4.0 g to a glass-stoppered, IDENTIFICATION 125-mL conical flask, and add 50.0 mL of water. Insert • A. PROCEDURE the , and swirl for 5 min. Transfer to a glass- Analysis: Examine under a , using NLT 20× stoppered, 50-mL centrifuge tube, and centrifuge to magnification and a mixture of glycerin and water (1:1) clarify. Transfer 30.0 mL of the clear supernatant to a as a mounting agent. glass-stoppered, 125-mL conical flask. Add 1 mL of Acceptance criteria: It presents granules, either irregu- glacial acetic acid and 0.5–1.0 g of potassium iodide. larly shaped, ovoid or pear-shaped, usually 30–100 µm Insert the stopper, swirl, and allow to stand for 25–30 in size, but occasionally exceeding 100 µm, or rounded min in the dark. Add 1 mL of starch TS. 10–35 µm in size. There are occasional compound Analysis: Titrate with 0.002 N sodium thiosulfate VS to granules having 2–4 components. The ovoid and pear- the disappearance of the starch–iodine color. Perform a shaped granules have an eccentric hilum, and the blank determination, and make any necessary rounded granules have a centric or slightly eccentric hi- correction. Each mL of 0.002 N sodium thiosulfate is lum. All granules show clearly visible concentric stria- equivalent to 34 µg of oxidant, calculated as hydrogen tions. Between crossed nicol prisms, the Hydroxypropyl peroxide. Potato Starch granules show a distinct black cross inter- Acceptance criteria: NMT 1.4 mL of 0.002 N sodium secting at the hilum. thiosulfate is required (20 ppm, calculated as H2O2). • B. PROCEDURE Sample solution: Suspend 1 g of Hydroxypropyl Potato SPECIFIC TESTS Starch in 50 mL of water, boil for 1 min, and cool. • MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR Acceptance criteria: A translucent or clear mucilage is SPECIFIED MICROORGANISMS 〈62〉: The total aerobic formed. microbial count does not exceed 103 cfu/g; the total • C. PROCEDURE combined molds and yeasts count does not exceed 102 Analysis: To 1 mL of the Sample solution obtained in cfu/g; and it meets the requirements of the test for the Identification test B add 0.05 mL of iodine and potas- absence of Escherichia coli. sium iodide TS 2. Acceptance criteria: An orange-red to dark blue color is produced, which disappears upon heating. • D. PROCEDURE Ninhydrin solution: Dissolve 3 g of ninhydrin in 100 mL of a 45.5-g/L solution of sodium metabisulfite. 2240 Starch / Official Monographs NF 31

Diluted sulfuric acid: 98 g/L of H2SO4 and of the methyl groups at 0 ppm of the internal stan- 13 Sample: 100 mg of Hydroxypropyl Potato Starch dard (A1) without C-satellites. Analysis: Transfer the Sample to a 100-mL volumetric Measure the signal coming from the 3 protons of the flask, and add 12.5 mL of Diluted sulfuric acid. Place the methyl group in the hydroxypropyl function. flask in a water bath, and heat until the Sample is dis- Calculate the content of hydroxypropyl groups as a per- solved. Cool, and dilute with water to 100 mL. [CAU- centage (w/w, dried basis): TION—When sulfuric acid is miscible with water, it pro- duces intense heat.] Result = (N × A2/A1) × (Ci × Wi/W) × (Mr2/Mr1) × [100/ Pipet 1 mL of this solution to a glass-stoppered, 25-mL (100 − B)] × 100 graduated test-tube and, with the tube immersed in cold water, add drop-wise 8 mL of sulfuric acid. Mix N = numerical value representing the 3 methyl well, and place the tube in a boiling water bath for groups in the internal standard (sodium exactly 3 min. Immediately transfer the tube to an ice 3-trimethylsilyl-1-propane sulfonate), 3 bath until the solution is chilled. Add 0.6 mL of A2 = area of the methyl groups of hydroxypropyl in Ninhydrin solution, carefully allowing the reagent to run Hydroxypropyl Potato Starch down the walls of the test tube. Immediately shake the A1 = area of the methyl groups in the internal tube well, and place it in a water bath at 25° for 100 standard (sodium 3-trimethylsilyl-1-propane min. Dilute with sulfuric acid to 25 mL [CAUTION—Use sulfonate) sulfuric acid cautiously.], and mix by inverting the tube Ci = concentration of the internal standard in the several times. Do not shake. Internal standard solution (mg/g) Acceptance criteria: A violet color develops within 5 Wi = weight of the Internal standard solution in the min due to the presence of hydroxypropyl groups NMR tube (g) (starch ether). W = weight of the washed and dried Hydroxypropyl Potato Starch in the NMR ASSAY tube (mg) • PROCEDURE FOR HYDROXYPROPYL GROUPS Mr1 = molecular weight of the internal standard, Deuterium chloride solution: Dilute 1 mL of deuterium 218.32 g/mol chloride (38% w/w) with 5 mL of deuterium oxide. Mr2 = molar mass of hydroxypropyl group, 59.09 g/ Internal standard solution: Dissolve 50.0 mg of so- mol dium 3-trimethylsilyl-1-propane sulfonate in about 5 g B = moisture content of the washed and dried of deuterium oxide, weighed to the nearest 0.1 mg. Hydroxypropyl Potato Starch used in the Store in a sealed . Sample solution, as a percentage (w/w) Sample solution: Disperse 20 g of Hydroxypropyl Po- Acceptance criteria: The content of hydroxypropyl tato Starch in 200.0 mL of carbon dioxide-free water at groups is 2.0%–7.0% on the dried basis. room temperature. Agitate for 15 min, and filter. Re- peat the operation two more times. If poor dispersibility IMPURITIES or slow filtration is observed, use refrigerated carbon Inorganic Impurities dioxide-free water for the washing operation. Dry the • RESIDUE ON IGNITION 〈281〉: NMT 0.6%, determined on a washed starch for NLT 4 h in vacuum at 30 ± 5°. Deter- 1.0-g test specimen mine the moisture content (B) on 5 g of the washed • LIMIT OF IRON and dried starch following the Loss on Drying test. Standard iron stock solution: Prepare a solution Weigh 12.0 mg of the washed and dried starch in a containing the equivalent of 10 µg/mL of iron, as 5-mm NMR tube. Add 0.75 mL of deuterium oxide and directed under Iron 〈241〉. 0.1 mL of Deuterium chloride solution. Cap the tube, Diluted standard iron solution: Immediately before mix, and place it in a boiling water bath until a clear use, dilute an accurately measured volume of Standard solution is obtained. [NOTE—It may take 3 min–1 h.] iron stock solution quantitatively with water to obtain a When a clear solution is obtained, allow to cool to solution containing the equivalent of 1 µg/mL of iron. room temperature. Dry the exterior of the tube, and Standard solution: Transfer 10 mL of the Diluted weigh to the nearest 0.1 mg. Add 0.05 mL of Internal standard iron solution to a test tube. Add 2 mL of citric standard solution, and weigh to the nearest 0.1 mg. De- acid solution (2 in 10) and 0.1 mL of thioglycolic acid, termine the mass of the Internal standard solution and mix. Add 10 N ammonium hydroxide until the added. Mix thoroughly. solution is distinctly alkaline to litmus, dilute with water Nuclear magnetic resonance spectrometry to 20 mL, and mix. (See Nuclear Magnetic Resonance 〈761〉, Quantitative Ap- Sample solution: Shake 1.0 g of Hydroxypropyl Potato plication.) Starch with 20 mL of 2 N hydrochloric acid, and filter. Apparatus: FT-NMR spectrometer at minimum Transfer 10 mL of the filtrate to a test tube. Add 2 mL 300 MHz of citric acid solution (2 in 10) and 0.1 mL of Acquisition of 1H NMR spectra: The following param- thioglycolic acid, and mix. Add 10 N ammonium eters may be used. hydroxide until the solution is distinctly alkaline to Sweep width: 8 ppm (about −1.0 to +7 ppm) litmus, dilute with water to 20 mL, and mix. Irradiation frequency offset: None Acceptance criteria: After 5 min, any pink color in the Time domain: NLT 64 K Sample solution is not more intense than that in the Pulse width: 90 degree Standard solution, corresponding to a limit of 20 µg/g Pulse delay: 10 s of iron. Dummy scans: 0 • LIMIT OF SULFUR DIOXIDE, Method IV 〈525〉: NMT 50 ppm Number of scans: 8 Organic Impurities Use the CH3 signal of the internal standard for shift • PROCEDURE 1: LIMIT OF OXIDIZING SUBSTANCES referencing. Set the shift of the peak of the singlet to Sample: 4.0 g of Hydroxypropyl Potato Starch 0 ppm. Record the FID signal. Analysis: Transfer the Sample to a glass-stoppered, Analysis 125-mL conical flask, and add 50.0 mL of water. Insert Samples: Internal standard solution and Sample solution the stopper, and swirl for 5 min. Transfer to a glass- Call the integration sub-routine after phase corrections stoppered, 50-mL centrifuge tube, and centrifuge to and baseline correction between −0.5 and +6 ppm. clarify. Transfer 30.0 mL of the clear supernatant to a Measure the peak areas of the doublet from the methyl glass-stoppered, 125-mL conical flask. Add 1 mL of groups of the hydroxypropyl function at +1.2 ppm (A2), glacial acetic acid and 0.5–1.0 g of potassium iodide. NF 31 Official Monographs / Starch 2241

Insert the stopper, swirl, and allow to stand for 25–30 flask in a water bath, and heat until the Sample is dis- min in the dark. Add 1 mL of starch TS, and titrate solved. Cool, and dilute with water to 100 mL. [CAU- with 0.002 N sodium thiosulfate VS to the TION—When sulfuric acid is miscible with water, it pro- disappearance of the starch-iodine color. Perform a duces intense heat.] blank determination, and make any necessary Pipet 1 mL of this solution to a glass-stoppered 25-mL correction. Each mL of 0.002 N sodium thiosulfate VS graduated test tube and, with the tube immersed in is equivalent to 34 µg of oxidant, calculated as cold water, add dropwise 8 mL of sulfuric acid. Mix hydrogen peroxide. well, and place the tube in a boiling water bath for Acceptance criteria: NMT 1.4 mL of 0.002 N sodium exactly 3 min. Immediately transfer the tube to an ice thiosulfate VS is required (20 µg/g, calculated as H2O2). bath until the solution is chilled. Add 0.6 mL of • PROCEDURE 2: FOREIGN MATTER Ninhydrin solution, carefully allowing the reagent to run Sample: 50 mg/mL of Hydroxypropyl Potato Starch in down the walls of the test tube. Immediately shake the a mixture of glycerin and water (1:1) tube well, and place it in a water bath at 25° for 100 Analysis: Examine under a microscope, using NLT 20× min. Dilute with sulfuric acid to 25 mL. [CAUTION—Use magnification and a mixture of glycerin and water sulfuric acid cautiously.] Mix by inverting the tube sev- (1:1) as a mounting agent. eral times. Do not shake. Acceptance criteria: NMT traces of matter other than Acceptance criteria: A violet color develops within 5 Hydroxypropyl Potato Starch granules are present. min due to the presence of hydroxypropyl groups (starch ether). SPECIFIC TESTS • MICROBIAL ENUMERATION TESTS 〈61〉 and TESTS FOR ASSAY SPECIFIED MICROORGANISMS 〈62〉: The total aerobic • ASSAY FOR HYDROXYPROPYL GROUPS microbial count does not exceed 103 cfu/g, the total Deuterium chloride solution: Dilute 1 mL of deuterium combined molds and yeasts count does not exceed 102 chloride (38% w/w) with 5 mL of deuterium oxide. cfu/g, and it meets the requirements of the test for the Internal standard solution: Disperse 50.0 mg of so- absence of Escherichia coli. dium 3-trimethylsilyl-1-propane sulfonate in about 5 g • PH 〈791〉 of deuterium oxide, weighed to the nearest 0.1 mg. Sample solution: Suspend 5.0 g of Hydroxypropyl Store in a sealed bottle. Potato Starch in 25.0 mL of carbon dioxide-free water, Sample solution: Determine the moisture content (B) and shake for 60 s. Allow to stand for 15 min. on 5 g of Pregelatinized Hydroxypropyl Potato Starch Acceptance criteria: 4.5–8.0 following the Loss on Drying test. Weigh 12.0 mg of the • LOSS ON DRYING 〈731〉: Dry about 1 g at 130° for 90 min: Pregelatinized Hydroxypropyl Potato Starch in a 5-mm it loses NMT 20.0% of its weight. NMR tube. Add 0.75 mL of deuterium oxide and 0.1 mL of Deuterium chloride solution. Cap the tube, ADDITIONAL REQUIREMENTS mix, and place it in a boiling water bath until a clear • PACKAGING AND STORAGE: Preserve in well-closed solution is obtained. [NOTE—This may take from 3 min containers. Store at room temperature. to 1 h.] When a clear solution is obtained, allow it to cool to room temperature. Dry the exterior of the tube, and weigh to the nearest 0.1 mg. Add 0.05 mL of the Internal standard solution. Weigh to the nearest 0.1 mg. . Determine the mass of the Internal standard solution Pregelatinized Hydroxypropyl Potato added. Mix thoroughly. Starch Instrumental conditions (See Nuclear Magnetic Resonance 〈761〉, Quantitative Ap- DEFINITION plications.) Pregelatinized Hydroxypropyl Potato Starch is prepared from Mode: Nuclear magnetic resonance spectrometry Hydroxypropyl Potato Starch by mechanical processing in Apparatus: FT-NMR spectrometer at minimum the presence of water, with or without heat, to rupture all 300 MHz or some of the starch granules, and is subsequently dried. Acquisition of 1H NMR spectra: The following param- It contains NLT 2.0% and NMT 7.0% of hydroxypropyl eters may be used: groups on the dried basis. Sweep width: 8 ppm (about −1.0 to +7 ppm) Irradiation frequency offset: None IDENTIFICATION Time domain: NLT 64 K • A. TEST FOR PREGELATINIZED STATE Pulse width: 90° Sample: 1 g Pulse delay: 10 s Analysis: Disperse the Sample in 50 mL of water at a Dummy scans: 0 temperature NMT 25°. Shake vigorously until lumps Number of scans: 8 completely disperse/solubilize or until lumps disappear. Use the CH3 signal of the internal standard for shift refer- Allow to stand for 20 min. encing. Set the shift of the peak of the singlet to Acceptance criteria: A translucent or clear mucilage 0 ppm. Record the FID signal. without precipitate is formed. Analysis • B. TEST FOR STARCH Samples: Internal standard solution and Sample solution Analysis: Disperse 0.5 g in 2 mL of water without heat- Call the integration subroutine after phase corrections ing, and add 0.05 mL of iodine and potassium iodide and baseline correction between −0.5 and +6 ppm. TS2. Measure the peak areas of the doublet from the methyl Acceptance criteria: A reddish-violet or blue color is groups of the hydroxypropyl function at +1.2 ppm (A2), produced. and of the methyl groups at 0 ppm of the internal stan- 13 • C. NINHYDRIN TEST dard (A1) without C-satellites. Ninhydrin solution: Dissolve 3 g of ninhydrin in Measure the signal originating from the 3 protons of the 100 mL of a 45.5-g/L solution of sodium metabisulfite. methyl group in the hydroxypropyl function. Diluted sulfuric acid: 98 g/L of H2SO4 Calculate the content of hydroxypropyl groups as a per- Sample: 100 mg centage (w/w, dried basis): Analysis: Transfer the Sample to a 100-mL , and add 12.5 mL of Diluted sulfuric acid. Place the Result = (N × A2/A1) × (CI × WI/W) × (Mr2/Mr1) × [100/ (100 − B)] × 100