Zein
[9010-66-6].
• C.
It is insoluble in water, but 100 mg/mL in a mixture of isopropyl alcohol and water (17:3), at a temperature of 37
, yields a clear to cloudy solution.NF30
• C. SDS-Polyacrylamide Gel Electrophoresis
Organic Impurities
NF30
• Labeling:
Label it to indicate the corn source from which it is derived.NF30
(zee' in).
[9010-66-6].DEFINITION
Zein is a prolamine derived from corn [Zea mays Linné (Fam. Gramineae)].
IDENTIFICATION
• A.
Sample solution:
Dissolve 0.1 g in 10 mL of 0.1 N sodium hydroxide.
Analysis:
To the Sample solution add a few drops of cupric sulfate TS. Warm in a water bath.
Acceptance criteria:
A purple color develops.
• B.
Sample solution:
In a test tube add 1 mL of nitric acid to 25 mg of Zein.
Analysis:
Agitate the Sample solution vigorously.
Acceptance criteria:
The solution becomes light yellow. Further addition of about 10 mL of 6 N ammonium hydroxide produces an orange color.
Delete the following:
• C.
It is insoluble in water, but 100 mg/mL in a mixture of isopropyl alcohol and water (17:3), at a temperature of 37, yields a clear to cloudy solution.NF30
Add the following:
• C. SDS-Polyacrylamide Gel Electrophoresis
Solvent:
55% isopropyl alcohol with 2% beta mercaptoethanol
Sample loading buffer:1
0.5 M Tris hydrochloride pH 6.8, 20% glycerin, 4% sodium dodecyl sulfate (SDS), and 0.005% bromophenol blue
Gel running buffer stock solution:2
0.25 M Tris base pH 8.6, 1.92 M glycine, and 1.0% SDS
Gel running buffer:
Gel running buffer stock solution and water (1:9)
Gel staining solution:
A suitable Coomassie blue-based solution3
Molecular weight marker:
Use a suitable molecular weight marker4 containing protein bands at 10–190 kDa. [Note—A molecular weight marker with protein bands at 10–100 KDa can also be used. ]
Molecular weight standard solution:
Dilute the Molecular weight marker (1:1) with the Sample loading buffer. Incubate the mixture in a closed microcentrifuge tube for 10 min at 95. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
Sample stock solution:
10 mg/mL of Zein in Solvent. Mix on a vortex mixer until the sample is fully dissolved. Centrifuge at 10,000–12,000 rpm in a microcentrifuge with a fixed rotor for 10 min to pellet any undissolved material.
Sample solution:
Dilute the Sample stock solution (1:1) with the Sample loading buffer. Incubate the mixture in a closed microcentrifuge tube for 10 min at 95. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
. After incubation, allow the tube to flash-cool on ice. Place the tube in a microcentrifuge, spin at a top speed for a few seconds, and allow to stop on its own to collect any condensation on the sides and top of the tube.
Electrophoretic system
(See Electrophoresis 
726
.)
726.)
SDS-PAGE gel and apparatus setup:
Following the manufacturer's instructions, assemble and fill a precast 16% Tris-Glycine Gel5 in an appropriate electrophoresis module.
Running buffer:
Gel running buffer
Voltage:
100 V
Run time:
2.5 h or until the upper dye front is at the bottom of the gel. [Note—The total run time may need to be altered, depending on the molecular weight standards as well on as laboratory equipment variability, because the dye front may co-migrate with or close to the lowest bands of the set. ]
Analysis
Samples:
Molecular weight standard solution and Sample solution
Gel loading:
Load 25 µL of the Molecular weight standard solution. Load a volume of the Sample solution, equal to approximately 25 µg of calculated Zein. [Note—The actual amount of Zein extracted from the starting material cannot be quantified. Therefore, an estimated amount is derived. ]
Gel staining:
After electrophoresis, carefully remove the gel from the plates. Rinse the gel three times with water. Stain the gel by following the manufacturer’s directions for the stain used.
Destaining:
After staining as directed by the manufacturer, destain as directed by the manufacturer.
Gel scan procedure:
Set up a gel scanner according to the manufacturer’s instructions. Place the gel in the detector, and obtain a single image of all loaded lines of the gel.
Acceptance criteria:
Zein has two major bands: the 
band is at 21–25 kDa, and the 
band is at 17–18 kDa.NF30
band is at 21–25 kDa, and the band is at 17–18 kDa.NF30
IMPURITIES
Inorganic Impurities
• Residue on Ignition 281:
NMT 2.0%, using an ignition temperature of 800 ± 25
281:
NMT 2.0%, using an ignition temperature of 800 ± 25
• Heavy Metals, Method II 231:
NMT 20 ppm
231:
NMT 20 ppm
Add the following:
Organic Impurities
• Limit of Hexane-Soluble Matter
Sample:
15 g of Zein
Solvent:
Alcohol and water (17:3, w/w)
Analysis:
Dissolve the Sample in 150 mL of Solvent. Stir the mixture, using a magnetic stirrer, and heat the solution to 30. Once the Sample is dissolved, transfer the solution to a 500-mL separatory funnel.
. Once the Sample is dissolved, transfer the solution to a 500-mL separatory funnel.
Add 60 mL of n-hexane. Shake the mixture, and allow the phases to separate. Discharge the bottom layer (alcohol) to a beaker, and transfer the top layer (hexane) to a first 500-mL flask. Weigh the first 500-mL flask, and record the weight. Pour the bottom layer of alcohol back into the separatory funnel. Repeat this step four more times.
After the five 60-mL hexane solutions have been added to the first 500-mL flask, attach it to a rotary evaporator to distill the hexane. Collect the hexane in a second 500-mL flask.
The first 500-mL flask contains a yellow to reddish oil. Record the weight of the flask containing this oil.
Calculate the percentage of hexane-soluble matter in the portion of Zein taken:
Result = [(WT 
WF)/W] × 100
WF)/W] × 100| WT | = | = weight of the flask (g) |
| WF | = | = weight of the first 500-mL flask (g) |
| W | = | = weight of the Sample (g) |
Acceptance criteria
For Zein from normal dent corn:
NMT 12.5% for hexane-soluble matter
For Zein from waxy corn:
NMT 16.0% for hexane-soluble matter
NF30SPECIFIC TESTS
• Loss on Drying 731:
Dry a sample at 105 for 2 h: it loses NMT 8.0% of its weight.
731:
Dry a sample at 105 for 2 h: it loses NMT 8.0% of its weight.
• Microbial Enumeration Tests 61 and Tests for Specified Microorganisms 62:
The total bacterial count does not exceed 103 cfu/g, and the tests for Salmonella species and Escherichia coli are negative.
61 and Tests for Specified Microorganisms 62:
The total bacterial count does not exceed 103 cfu/g, and the tests for Salmonella species and Escherichia coli are negative.
Change to read:
• Protein Content
Analysis:
Proceed as directed in Nitrogen Determination, Method I 461. Calculate the weight percentage of the protein content in Zein by multiplying the percentage of nitrogen found by 6.25.
461. Calculate the weight percentage of the protein content in Zein by multiplying the percentage of nitrogen found by 6.25.
Acceptance criteria:
81.9%–100.0%NF30 on the dried basis
81.9%–100.0%NF30 on the dried basis
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in well-closed containers, and store at room temperature.
Add the following:
• Labeling:
Label it to indicate the corn source from which it is derived.NF30
1
Available from Invitrogen as Tris-Glycine SDS Sample Buffer (2X), catalog number LC2676.
2
Available from Invitrogen as Tris-Glycine SDS Running Buffer (10X), catalog number LC2675.
3
Available from Invitrogen as SimplyBlue Stain, catalog number LC6065.
4
Available from Invitrogen as BenchMark Prestained Protein Ladder, catalog number 1074810.
5
Available from Invitrogen, catalog number EC6495. However, these are readily available from several other manufacturers.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Robert H. Lafaver, M.S.
Scientific Liaison 1-301-816-8335 |
(EXC2010) Monographs - Excipients |
| 61 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
| 62 |
Radhakrishna S Tirumalai, Ph.D.
Principal Scientific Liaison 1-301-816-8339 |
(GCM2010) General Chapters - Microbiology |
USP35–NF30 Page 2019
Pharmacopeial Forum: Volume No. 36(6) Page 1641