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Printed by: USP NF Official Date: Official as of 01-Jun-2023 Document Type: USP & NF @2024 USPC
Do Not Distribute DOI Ref: lu5rh DOI: https://doi.org/10.31003/USPNF_M61250_02_01
1

A0 = A0-TS − (A0-EB + A0-TB)

Pectin
A10 = A10-TS − (A10-EB + A10-TB)

Calculate the quantity of unsaturated product produced:

Result = (A10 − A0)/(ε235 × L)

Pectin CAS RN®: 9000-69-5. ε235 = molar extinction coefficient of the reaction
product (4600 M-1 · cm-1)
DEFINITION L = path length of the reaction cuvette, 1 cm
Pectin is a purified carbohydrate polymer consisting mainly
of a linear backbone of partially methoxylated alpha (1-4) Acceptance criteria: The amount of unsaturated product
linked D-galacturonic acid. It is obtained from the dilute acid is NLT 0.5 × 10-5 M.
extract of the rind of citrus fruits or from apple pomace. No
organic solvents other than methanol, ethanol, and ASSAY
isopropanol are used during its production. Pectin yields NLT • DEGREE OF ESTERIFICATION
74.0% of galacturonic acid (C6H10O7), calculated on the Sample: 5.0 g
dried basis. Analysis: Transfer the Sample to a suitable beaker, and stir
[NOTE—Commercial pectin for the production of jellied for 10 min with a mixture of 5 mL of hydrochloric acid and
food products is standardized to the convenient “150 100 mL of 60% alcohol. Transfer to a sintered-glass filter
jelly grade” by addition of dextrose or other sugars, and (30- to 60-mL crucible or Büchner type, coarse), and wash
sometimes contains sodium citrate or other buffer salts. with six 15-mL portions of the hydrochloric acid–60%

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This monograph refers to the pectin to which no such alcohol mixture, followed by 60% alcohol until the filtrate
additions have been made.] is free from chlorides. Finally wash with 20 mL of alcohol,
dry for 1 h at 105°, cool, and weigh. Transfer exactly
IDENTIFICATION one-tenth of the total net weight of the dried Sample
• PROCEDURE (representing 500 mg of the original unwashed Sample)
Sample stock solution: Transfer a quantity of Pectin,
equivalent to 0.05 g on the dried basis, to a suitable
container, and moisten with 250 µL of 2-propanol. Add
50 mL of water to the container, and mix the solution
ci to a 250-mL conical flask, and moisten with 2 mL of alcohol.
Add 100 mL of carbon dioxide-free water, insert the
stopper, and swirl occasionally until the Pectin is completely
dissolved. Add 5 drops of phenolphthalein TS, and titrate
using a magnetic stirrer. Use 0.5 N sodium hydroxide to with 0.1 N sodium hydroxide VS. Perform a blank
adjust the pH of the solution to 12, stop the stirrer, and determination, and make any necessary correction. Record
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allow the solution to stand undisturbed at room the results as the initial titer, VI (mL). Add 20.0 mL of 0.5 N
temperature for 15 min. Adjust with 0.5 N hydrochloric sodium hydroxide VS, insert the stopper, shake vigorously,
acid to a pH of 7.0, and dilute with water to 100 mL. and allow to stand for 15 min. Add 20.0 mL of 0.5 N
Tris buffer solution: Transfer 6.055 g of hydrochloric acid VS, and shake until the pink color
tris(hydroxylmethyl)aminomethane and 0.147 g of calcium disappears. Add phenolphthalein TS, and titrate with 0.1 N
chloride (CaCl2 · 2H2O) to a 1000-mL volumetric flask sodium hydroxide VS to a faint pink color that persists after
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containing 950 mL of water. Adjust with 1 N hydrochloric vigorous shaking. Perform a blank determination, and make
acid to a pH of 7.0, and dilute with water to volume. any necessary correction. Record this value as the
Enzyme solution: Mix pectate lyase1 with Tris buffer saponification titer, VS (mL). Calculate the degree of
solution to make a solution (1 in 100). esterification:
Sample blank: Mix 0.5 mL of Tris buffer solution, 1.0 mL of
Sample stock solution, and 1.0 mL of water in a quartz Result = [VS/(VI + VS)] × 100
cuvette.
Enzyme blank: Mix 0.5 mL of Tris buffer solution, 1.5 mL of VS = saponification titer (mL)
water, and 0.5 mL of Enzyme solution in a quartz cuvette. VI = initial titer (mL)
Sample solution: Mix 0.5 mL of Tris buffer solution, 1.0 mL
of Sample stock solution, 0.5 mL of water, and 0.5 mL of Acceptance criteria: The value for Degree of Esterification is
Enzyme solution in a quartz cuvette. within the range stated on the label.
Analysis • GALACTURONIC ACID: Each mL of 0.1 N sodium hydroxide
Samples: Sample blank, Enzyme blank, and Sample solution used in the total titration (the initial titer added to the
Perform the test with the Samples using a suitable UV/visible saponification titer) in the Assay for Degree of Esterification
spectrophotometer (see Ultraviolet-Visible Spectroscopy is equivalent to 19.41 mg of C6H10O7. Calculate the
á857ñ) and using water as a blank. Measure the percentage of galacturonic acid in the portion of Pectin
absorbance at 235 nm immediately after mixing the taken:
solutions well, and record the value at time 0 for the
Enzyme blank, A0-EB; for the Sample blank, A0-TB; and for the Result = 19.41 × [(VI + VS)/W] × 100
Sample solution, A0-TS. After incubation at room
temperature for 10 min, determine the absorbance again VI = initial titer (mL)
at 235 nm for the Enzyme blank, A10-EB; for the VS = saponification titer (mL)
Sample blank, A10-TB; and for the Sample solution, A10-TS. W = weight of the original unwashed and dried Pectin
Calculate the corrected absorbance A0 at time 0 and the taken to prepare the solution for titration (mg)
corrected absorbance A10 at 10 min:
Acceptance criteria: NLT 74.0%
1A • METHOXY GROUPS: Each mL of 0.1 N sodium hydroxide
suitable pure enzyme is available from Megazyme International Ireland
Ltd., Bray Business Park, Bray, Co. Wicklow, Ireland
used in the saponification titer in the Assay for Degree of
(www.megazyme.com). Esterification is equivalent to 3.10 mg of –OCH3. Calculate

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Printed on: Fri Jan 05 2024, 08:53:49 PM(EST) Status: Currently Official on 06-Jan-2024 DocId: GUID-377EC6C9-55A9-4B86-B4AF-42C937A81430_2_en-US
Printed by: USP NF Official Date: Official as of 01-Jun-2023 Document Type: USP & NF @2024 USPC
Do Not Distribute DOI Ref: lu5rh DOI: https://doi.org/10.31003/USPNF_M61250_02_01
2

the percentage of methoxy groups in the portion of Pectin Analysis: Place the Sample in a 500-mL flask, moisten with
taken: 3–5 mL of alcohol, rapidly pour in 100 mL of water, shake,
and allow to stand until the solution is complete. To this
Result = 3.10 × (VS/W) × 100 solution add 100 mL of alcohol containing 0.3 mL of
hydrochloric acid, mix, and filter rapidly. Measure 25 mL of
VS = saponification titer (mL) the filtrate into a tared dish, evaporate the liquid on a steam
W = weight of the original unwashed and dried Pectin bath, and dry the residue in a vacuum oven at 50° for 2 h.
taken to prepare the solution for titration (mg) Acceptance criteria: The weight of the residue does not
exceed 20 mg, corresponding to NMT 16% of sugars and
Acceptance criteria: The percentage of methoxy groups is organic acids.
within the range stated on the label. • METHANOL (METHYL ALCOHOL), ETHANOL (ALCOHOL), AND
IMPURITIES ISOPROPANOL (2-PROPANOL)
[NOTE—Residual alcohols are volatile. They should be
Change to read: stored in a cool and dry place. When preparing the
Standard stock solution, Standard solution, and
• ▲ARSENIC á211ñ, Procedures, Procedure 2▲ (CN 1-Jun-2023): NMT Internal standard stock solution for residual alcohols,
3 ppm mix thoroughly and keep the solutions at 20° when
• LEAD diluting with water to volume.]
Standard stock solution: 1000 µg/mL of lead. [NOTE—Use Internal standard stock solution: 5000 µg/mL of USP
a commercially available certified solution.] 2-Butanol RS. [NOTE—This solution can be stored at 5°–8°
Standard solution: 2 µg/mL of lead, prepared immediately for 3 months.]
before use by pipetting 0.10 mL of Standard stock solution Standard stock solution: Use USP Methyl Alcohol RS, USP
into a 50-mL volumetric flask containing 30 mL of water, 2-Propanol RS, and alcohol to prepare a solution having

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4 mL of 20% hydrochloric acid, and 4 mL of 0.1 M EDTA. known concentrations of 5000 µg/mL each for methyl
Dilute with water to volume, and mix. alcohol, 2-propanol, and alcohol.
Reference solution: 0.4 µg/mL of lead, prepared by Standard solution: To a 250-mL volumetric flask add
pipetting 5.0 mL of the Standard solution into a 25-mL 2.5 mL of the Standard stock solution and 2.5 mL of the
volumetric flask containing 10 mL of water, 2 mL of 20% Internal standard stock solution. Dilute with water to
hydrochloric acid, and 2 mL of 0.1 M EDTA. Dilute with
water to volume, and mix.
Sample solution: Transfer 2.0 g of Pectin to a clean, 100-mL
ci volume, and mix. This solution contains 50 µg/mL each of
methyl alcohol, 2-propanol, and alcohol. [NOTE—This
solution can be stored at 5°–8° for 3 months.] Transfer 1.0 g
glass beaker, add 25 mL of 70% nitric acid, cover with a of this solution to a 10-mL headspace vial.
watch glass, and heat at low to moderate heat on a hot Sample solution: Transfer 1.0 g of Pectin and 5 g of sucrose
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plate in a fume hood for 2 h. Remove the watch glass, and to a stoppered 100-mL Erlenmeyer flask containing 90 mL
continue to heat until the sample is dry with no visible of water, add 1.0 mL of the Internal standard stock solution,
fumes. Add 0.5 mL of 70% nitric acid, and heat to dryness. and dilute with water to 100 mL. Mix the solution using a
Cool to room temperature, and add 2 mL of 20% magnetic stirrer. Continue stirring until all of the Pectin has
hydrochloric acid and 2 mL of 0.1 M EDTA. Quantitatively been completely dissolved; typically it takes about 1–2 h.
transfer the solution to a 25-mL volumetric flask, dilute with This solution contains 50 µg/mL of USP 2-Butanol RS.
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water to volume, and mix. Transfer 1.0 g of this solution to a 10-mL headspace vial.
Blank solution: Add 30 mL of water, 4 mL of 20% Chromatographic system
hydrochloric acid, and 4 mL of 0.1 M EDTA into a 50-mL (See Chromatography á621ñ, System Suitability.)
volumetric flask. Dilute with water to volume, and mix. Mode: Headspace GC
Analysis: Lead is determined using an inductively coupled Detector: Flame ionization
plasma–atomic emission spectrometer (ICP–AES) (see Column: 0.32-mm × 30-m capillary column; 1.8-µm layer
Plasma Spectrochemistry á730ñ) by measuring the emission of phase G43. [NOTE—An alternative column such as a
at 220.35 nm with the settings optimized as directed by the 0.32-mm × 25-m capillary column bonded with a 5-µm
manufacturer. Instrument performance must be verified to layer of phase S3 can be used as long as the system
conform to the manufacturer’s specifications for resolution suitability requirements are met.]
and sensitivity. Before analyzing samples, the instrument Temperatures
must pass a suitable performance check. Calibrate the Detector: 280°
instrument with the Blank solution and the Standard Column: 70°
solution. Then analyze the Reference solution and the Sample Injection port: 200°
solution. Carrier gas: Nitrogen
Acceptance criteria: The concentration in the Sample Flow rate: 1.5 mL/min
solution is NMT that in the Reference solution, Make up gas: Nitrogen
corresponding to NMT 5 ppm of lead. Split flow rate: 30 mL/min
• SULFUR DIOXIDE, Method V á525ñ Injection volume: 1 mL (the gaseous headspace)
Sample: 100 g Injection type: Split ratio 20:1
Analysis: Suspend the Sample in 500 mL of methanol, then Balanced pressure automatic headspace sampler
transfer this mixture to the flask (C). Prepare a mixture of Equilibration time: 10 min
20 mL of hydrochloric acid and 10 mL of water, and transfer Equilibration temperature: 70°
it to the separatory funnel (B). Add 10 mL of hydrogen Agitation speed: 500 rpm
peroxide solution to the vessel (G). Perform the refluxing Agitation on time: 5 s
for 2 h before removing the vessel (G). Agitation off time: 90 s
Acceptance criteria: NMT 50 ppm Syringe temperature: 80°
• SUGARS AND ORGANIC ACIDS Syringe size: 2.5 mL
Sample: 1 g Fill speed: 100 µL/s
Pull-up delay: 2.0 s

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Printed by: USP NF Official Date: Official as of 01-Jun-2023 Document Type: USP & NF @2024 USPC
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3

GC run time: 10.5 min CS = concentration of the respective residual alcohol

[NOTE—These GC conditions should be optimized (methanol, ethanol, or 2-propanol) in the
according to the instruments used.] Standard solution (µg/mL)
System suitability W = weight of Pectin taken to prepare the Sample
Sample: Standard solution solution (g)
[NOTE—See Table 1 for the relative retention times.] F = conversion factor (10-6 g/µg)
V = volume of the Sample solution, 100 mL
Table 1
Relative Acceptance criteria: NMT 1% for total methanol, ethanol,
Retention and isopropanol
Component Time
SPECIFIC TESTS
Methanol 0.5 • MICROBIAL ENUMERATION TESTS á61ñ and TESTS FOR
Ethanol 0.6 SPECIFIED MICROORGANISMS á62ñ: The total aerobic
microbial count is NMT 103 cfu/g, and the total combined
2-Propanol 0.7 molds and yeasts count is NMT 102 cfu/g.
2-Butanol 1.0 • LOSS ON DRYING á731ñ
Analysis: Dry a sample at 105° for 3 h.
Acceptance criteria: NMT 10.0%
Suitability requirements
Resolution: NLT 1.5, between each pair of analytes ADDITIONAL REQUIREMENTS
Relative standard deviation: NMT 10%, determined • PACKAGING AND STORAGE: Preserve in tight containers.
from each analyte Store in a cool and dry place.

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Analysis • LABELING: Label it to indicate whether it is of apple or of
Samples: Standard solution and Sample solution citrus origin. Label it to indicate the range of the degree of
Calculate the percentage of methanol, ethanol, and esterification and the range of the percentage of methoxy
2-propanol in the portion of Pectin taken: groups. The labeling also indicates the presence of sulfur
dioxide if the residual sulfur dioxide concentration is greater
Result = (RU/RS) × (CS/W) × F × V × 100 than 10 ppm.
RU = internal standard ratio (peak response of the
respective alcohol/peak response of the internal
ci • USP REFERENCE STANDARDS á11ñ
USP 2-Butanol RS
USP Methyl Alcohol RS
standard) from the Sample solution USP 2-Propanol RS
RS = internal standard ratio (peak response of the
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respective alcohol/peak response of the internal
standard) from the Standard solution
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