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Identification
A: Prepare a 1% solution of Gellan Gum by hydrating 1g in 99 mL of deionized water. Stir the mixture for about 2 hours, using a motorized stirrer and a propeller-type stirring blade. Draw a small amount of the solution obtained into a wide-bore pipet, and transfer it into a 10% calcium chloride solution. [Note: Reserve the remaining portion of this solution for Identification test B.]
A tough, wormlike gel will form instantly.
B: To the remaining solution prepared for Identification test A, add 0.5 g of sodium chloride, heat the solution to 80℃, stirring constantly, and hold at 80℃ for 1 minute. Stop heating and stirring the solution, and allow it to cool to room temperature. A firm gel will form.
ASSAY ( CO2 Yields)
APPARATUS
The required apparatus (see Figure 2) contains a capillary metering valve, A, followed by a flowmeter, B, to control and monitor the flow of nitrogen through the system. Halogenated vinyl plastic tubing and a rubber fitting, C, are used to connect the flow meter to a sidearm of a reaction flask, D. Flask D is a 250-mL round-bottom, boiling flask, resting in a suitable heating mantle, E. Flask D is provided with a 225-mm Hopkins coil reflux condenser, F. The condenser terminates in a U-shaped trap, G, which contains two 25-g bands of 20-mesh zinc, the bands being bounded and separated by three 3-inch plugs of glass wool. The trap terminates in an adapter, H, that by means of a halogenated vinyl plastic tubing and a twistcock connector, I, connects with a 250-mL gas washing bottle, J. The inlet (bubbling) tube extends almost to the bottom of the gas washing bottle, and it terminates in a fritted disk having a coarse porosity. The size of all glass joints is 24/40, except for the 45/50 joint of the gas washing bottle.
SYSTEM SUITABILITY
Using D-glucuronolactone as the standard, proceed as directed for Procedure, but do not perform preboiling steps. The system is suitable if the following criteria are met: (1) a blank determination results in a net titration value C, between 0.02 and 0.06 mEq of 0.1 N hydrochloric acid, calculated as follows:
Ab – Bb
in which Ab is the number of mEq of 0.25 N sodium hydroxide in the 25 mL used, and Bb is the number of mEq of 0.1 N hydrochloric acid used in the blank titration; and (2) the percentage of carbon dioxide, CO2, obtained from the standard is between 24.7% and 25.3%.
PROCEDURE
transfer a specimen of about 1.2g, accurately weighed, into the reaction flask, D, add 50 mL of 0.1 N hydrochloric acid, insert several boiling chips, and connect the flask to the reflux condenser, F, using phosphoric acid as a lubricant. [NOTE—Stopcock grease may be used for the other connections.] Connect the nitrogen line to the sidearm of the flask, and adjust the flow of cooling water to about 2 L per minute.
[NOTE—The following preboiling steps, outlined in this paragraph, are optional and need only be performed when the presence of inorganic carbonates is suspected.] Maintain the flow of nitrogen through the apparatus at 90 to 100 mL per minute. Raise the heating mantle, E, to the flask, heat the specimen to boiling, and boil gently for 2 minutes. Turn the heat off, lower the mantle, E, and allow to cool for about 10 minutes.
Connect the empty gas washing bottle assembly, J, and sweep the system with nitrogen at a rate of 90 to 100 mL per minute for 5 minutes. Reduce the nitrogen flow to 60 to 65 mL per minute, add 10 drops of butyl alcohol, 25.0 mL of 0.25 N sodium hydroxide VS, and 50 mL of distilled water into the bottle, rinsing down the inside of the gas washing bottle, and replace the cap. Detach the rubber fitting, C, from sidearm, and add 46 mL of hydrochloric acid through the sidearm of the boiling flask. Reattach the nitrogen line, raise the heating mantle, and heat the reaction mixture to boiling. After 2 hours of boiling, increase the nitrogen flow to 90 to 100 mL per minute, discontinue the heating, and lower the mantle. Allow to cool for 10 minutes. Disconnect, and disassemble the gas washing bottle. Using a directed stream of distilled water, thoroughly rinse all parts of the bubbling tube and cap, collecting the washings in the gas washing bottle. Use nitrogen to gently force all water out of the bubbling tube. To the bottle immediately add 10 mL of 10% barium chloride solution and a stirring bar. Insert a tight stopper, and stir gently for 1 minute. Allow to stand for at least 5 minutes. Add three drops of phenolphthalein TS, and titrate with 0.1 N hydrochloric acid VS. Perform a blank determination. Calculate the percentage of carbon dioxide, CO2, by the formula:
2200[(A B) C] / (1000W) (1 D)
in which A is the number of mEq of 0.25 N sodium hydroxide in the 25 mL used; B is the number of mEq of 0.1 N hydrochloric acid used for the titration of the sample or the standard; C is the net titration value calculated in the blank determination; W is the weight, in g, of the sample or the standard taken; and D is the percentage expressed as a decimal (1 decimal place), obtained in the test for Loss on drying for the sample or for the standard.
Gel Strength Determination
APPARATUS
Thermo-tank ( 5 ~ 50℃)
Electronic balance (accurate to 0.001g)
Gel Strength Meter
Water bath ( 10 ~100℃)
PROCEDURE
Weigh 0.6g sample ( accurate to 0.01g ), transfer to 250mL beaker and add 120mL distilled water, weigh the total weight of beaker and solution and note it. Put the beaker in water bath ( 80℃) and stir it by glass bar for three times, 30 seconds for each time, heat for 20 minutes to ensure that the gellan gum dissolved totally, then add 2.7% calcium chloride ( CaCl2 ) 2mL, then take the beaker out, swab up the outer wall of the beaker, add the water to make up a loss. Agitate to make the solution evenly, then pour the solution into the flat bottom container, the height of the liquid is 2cm, cool the solution to form the gel, then store the gel for 20h in Thermo-tank ( 20℃). Make three samples parallelly, determine their gel strength and take the average value.
Transmittance Determination
APPARATUS
Electronic balance (accurate to 0.001g)
Spectrophotometer
Thermo-tank ( 5 ~ 50℃)
Water bath ( 10 ~100℃)
PROCEDURE
Weigh 0.6g sample ( accurate to 0.01g ), transfer to 250mL beaker and add 120mL distilled water, weigh the total weight of beaker and solution and note it. Put the beaker in water bath ( 80℃) and stir it by glass bar for three times, 30 seconds for each time, heat for 20 minutes to ensure that the gellan gum dissolved totally, then add 2.7% calcium chloride ( CaCl2 ) 2mL, then take the beaker out, swab up the outer wall of the beaker, add the water to make up a loss. Agitate to make the solution evenly, pour the solution into 10mm cuvette and put the cuvette in Thermo-tank immediately, stand it for 15 minutes, using Spectrophotometer to determine the transmittance at 497nm, against the blank. Make three samples parallelly, determine their transmittance and take the average value.
pH Value
APPARATUS
High speed agitator
pH meter
Electronic balance (accurate to 0.001g)
PROCEDURE
Weigh 2.5g sample ( accurate to 0.01g ), transfer to 1000mL beaker and add 500mL distilled water, agitate the solution by the high speed agitator to ensure that all the gellan gum dissolved totally, then determine the pH value with the pH meter under the temperature of 20℃ ( accurate to 0.1pH unit ).
Arsenic
APPARATUS
The apparatus (see illustration) consists of an arsine generator (a) fitted with a scrubber unit (c) and an absorber tube (e) with standard-taper or ground glass ball-and-socket joints (b and d) between the units. However, any other suitable apparatus, embodying the principle of the assembly described and illustrated, may be used.
Arsenic Trioxide Stock Solution— Dissolve 132.0 mg of arsenic trioxide, previously dried at 105 for 1 hour and accurately weighed, in 5mL of sodium hydroxide solution (1 in 5) in a 1000-mL volumetric flask. Neutralize the solution with 2 N sulfuric acid, add 10mL more of 2 N sulfuric acid, then add recently boiled and cooled water to volume, and mix.
Standard Arsenic Solution— Transfer 10.0mL of Arsenic Trioxide Stock Solution to a 1000-mL volumetric flask, add 10mL of 2 N sulfuric acid, then add recently boiled and cooled water to volume, and mix. Each mL of Standard Arsenic Solution contains the equivalent of 1 µg of arsenic (As). Keep this solution in an all-glass container, and use within 3 days.
METHOD I
Standard Preparation— Pipet 3.0mL of Standard Arsenic Solution into a generator flask, and dilute with water to 35mL.
Test Preparation— Unless otherwise directed in the individual monograph, transfer to the generator flask the quantity, in g, of the test substance calculated by the formula:
3.0/L
in which L is the arsenic limit in ppm, dissolve in water, and dilute with water to 35 mL.
Procedure— Treat the Standard Preparation and the Test Preparation similarly as follows. Add 20mL of 7 N sulfuric acid, 2mL of potassium iodide TS, 0.5mL of stronger acid stannous chloride TS, and 1mL of isopropyl alcohol, and mix. Allow to stand at room temperature for 30 minutes. Pack the scrubber tube (c) with two pledgets of cotton that have been soaked in saturated lead acetate solution, freed from excess solution by expression, and dried in vacuum at room temperature, leaving a 2-mm space between the two pledgets. Lubricate the joints (b and d) with a suitable stopcock grease designed for use with organic solvents, and connect the scrubber unit to the absorber tube (e). Transfer 3.0mL of silver diethyldithiocarbamate TS to the absorber tube. Add 3.0 g of granular zinc (No. 20 mesh) to the mixture in the flask, immediately connect the assembled scrubber unit, and allow the evolution of hydrogen and the color development to proceed at room temperature for 45 minutes, swirling the flask gently at 10-minute intervals. Disconnect the absorber tube from the generator and scrubber units, and transfer the absorbing solution to a 1-cm absorption cell. Any red color produced by the Test Preparation does not exceed that produced by the Standard Preparation. If necessary or desirable, determine the absorbance at the wavelength of maximum absorbance between 535 and 540 nm, with a suitable spectrophotometer or colorimeter, using silver diethyldithiocarbamate TS as the blank.
Interfering Chemicals— Metals or salts of metals, such as chromium, cobalt, copper, mercury, molybdenum, nickel, palladium, and silver, may interfere with the evolution of arsine. Antimony, which forms stibine, produces a positive interference in the color development with silver diethyldithiocarbamate TS; when the presence of antimony is suspected, the red colors produced in the two silver diethyldithiocarbamate solutions may be compared at the wavelength of maximum absorbance between 535 and 540 nm, with a suitable colorimeter, since at this wavelength the interference due to stibine is negligible.
Particle Size
APPARATUS
Standard sieves: 80 mesh
Electronic balance (accurate to 0.001g)
PROCEDURE
Weigh 50g sample ( accurate to 0.01g ), transfer to standard sieve with aperture of 0.175mm, shake the sieve until no sample drop from the sieve. Weigh the remained matters in sieve.
CALCULATION
M – M1
Particle Size = --------------------- X 100
M
Where: M is the sample weight
M1 is the weight of remained matters in sieve
Loss on Drying
Mix and accurately weigh the substance to be tested, and, unless otherwise directed in the individual monograph, conduct the determination on 1 to 2 g. If the test specimen is in the form of large crystals, reduce the particle size to about 2 mm by quickly crushing. Tare a glass-stoppered, shallow weighing bottle that has been dried for 30 minutes under the same conditions to be employed in the determination. Put the test specimen in the bottle, replace the cover, and accurately weigh the bottle and the contents. By gentle, sidewise shaking, distribute the test specimen as evenly as practicable to a depth of about 5 mm generally, and not more than 10 mm in the case of bulky materials. Place the loaded bottle in the drying chamber, removing the stopper and leaving it also in the chamber. Dry the test specimen at the temperature of 105℃ and for 2.5 hours: it loses not more than 14.0%. [NOTE—The temperature to be regarded as being within the range of ±2℃ of the stated figure.] Upon opening the chamber, close the bottle promptly, and allow it to come to room temperature in a desiccator before weighing.
Ash
Accurately weigh a quantity of the Test Sample, representing 2 to 4 g of the air-dried material, in a tared crucible, and incinerate, gently at first, and gradually increase the temperature to 675 ± 25℃, until free from carbon, and determine the weight of the ash. If a carbon-free ash cannot be obtained in this way, extract the charred mass with hot water, collect the insoluble residue on an ashless filter paper, incinerate the residue and filter paper until the ash is white or nearly so, then add the filtrate, evaporate it to dryness, and heat the whole to a temperature of 675 ± 25℃. If a carbon-free ash cannot be obtained in this way, cool the crucible, add 15 mL of alcohol, break up the ash with a glass rod, burn off the alcohol, and again heat the whole to a temperature of 675 ± 25℃. Cool in a desiccator, weigh the ash, and calculate the percentage of total ash from the weight of the drug taken.
Microbiological test
Total plate count: Using aseptic technique, disperse 1 g of sample into 99 ml of phosphate buffer and use a Stomacher, shaker or stirrer to fully dissolve. Limit dissolving time to about 10 min and then pipette 1 ml of the solution into separate, duplicate, appropriately marked petri dishes. Pour over the aliquot of sample in each petri dish 12-15 ml of Plate Count Agar previously tempered to 44-46℃. Mix well by alternate rotation and back and forth motion of the plates, allow the agar to solidify. Invert the plates and incubate for 48±2 h at 35±1℃.
After incubation count the growing colonies visible on each plate and record the number of colonies. Take the average of both plates, and multiply by the sample dilution factor, 100. Where no colonies are visible, express the result as less than 100 cfu/g.
E. coli determination: Using aseptic technique, disperse 1 g of sample in 99 ml of Lactose broth using either a Stomacher, shaker or stirrer to fully dissolve the sample. Limit the dissolving time to about 15 min and then lightly seal the container and incubate the broth for 18-24 h at 35±1℃. Using a sterile pipette, inoculate 1 ml of the incubate into a tube containing 10 ml GN broth. Incubate for 18-24 h and then streak any GN broths showing positive growth or gas production onto duplicate plates of Levine EMB agar. Incubate the plates for 24±2 h at 35±1℃ and then examine for colonies typical of E. coli i.e. showing strong purple growth with dark centre and a green metallic sheen sometimes spreading onto the agar. Record any typical E. coli colonies as presumptive positive, otherwise negative.
Streak any well isolated suspect colonies onto a plate of PCA and incubate for 18-24 h at 35±1℃. Perform a Gram stain on any growth to confirm it is Gram negative. If so, disperse any colony growth into a small volume of 0.85% saline and perform chemical tests to confirm the identity of the bacterial growth. This can most conveniently be done by using API 20E or Micro ID strips or equivalent systems.
After completion of the tests, identify the organism from the Identification manual of the system used and record the final result.
Media
GN Broth (Gram Negative Broth) |
| Peptone |
20.0 g |
| Dextrose |
1.0 g |
| Mannitol |
2.0 g |
| Sodium citrate |
5.0 g |
| Sodium deoxycholate |
0.5 g |
| Potassium phosphate (dibasic) |
4.0 g |
| Potassium phosphate (monobasic) |
1.5 g |
| Sodium chloride |
5.0 g |
Make up to 1 litre with distilled or de-ionised water, pH 7.0±0.2 at 25℃.
Salmonella determination: Using aseptic technique, disperse 5 g of sample into 200 ml of sterile lactose broth using either a Stomacher, shaker or stirrer to maximise dissolution over a 15 min period. Loosely seal the container and incubate at 35±1℃ for 24±2h.
Continue as per method on page 221 of FNP 5/Rev. 2 (1991). Identification can be more conveniently done using API or Micro ID systems or equivalent.
Yeasts and moulds: Using aseptic technique, disperse 1 g of sample into 99 ml of phosphate buffer and use a Stomacher, shaker or stirrer to fully dissolve. Limit dissolving time to about 10 min and then pipette 1 ml of the solution into separate, duplicate, appropriately marked petri dishes. Pour over the aliquot of sample in each petri dish 15-20 ml of Potato dextrose Agar (either acidified or containing antibiotic) previously tempered to 44-46℃. Mix well by alternate rotation and back and forth motion of the plates, and allow the agar to solidify. Invert the plates and incubate for 5 days at 20-25℃.
After incubation, count the growing colonies visible on each plate using a colony counter and record the number of colonies. Separate the yeasts from the moulds according to their morphology and count them separately. Take the average of both plates and multiply by the sample dilution factor, 100. Where no colonies are visible, express the result as less than 100cfu/g.
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The Specification of Gellan Gum