29.

Nonalcoholic Beverages and Concentrates*

Richard D. Thompson, Chapter Editor
Food and Drug Administration

*Methods in this chapter apply to both beverages and concentrates, although not specifically stated in section titles.

29.1.01
AOAC Official Method 950.12
Nonalcoholic Beverages
Preliminary Examination
Procedure

Note and record (a) appearance, whether bright or turbid, or any

sediment; (b) color and depth of color; (c) odor, whether fruity, foreign,
or artificial; (d) taste, whether tart or sweet, fruity, artificial, or foreign,
and whether any synthetic substance can be identified by odor or taste.

29.1.02
AOAC Official Method 950.13
Alcohol in Nonalcoholic Beverages
Final Action

(a) From specific gravity.See 942.06A and B (see 26.1.07).

(b) From refractive index.See 950.04 (see 26.1.10).
(c) By weight.See 945.07 (see 26.1.12).
(d) From gas chromatography (First Action 1973).See 973.23
(see 36.1.01).

29.1.03
AOAC Official Method 950.14
Ash of Nonalcoholic Beverages
Final Action

A. Ash

Proceed as in 900.02A or B (see 44.1.05), using sample containing

10 g solids.
B. Soluble and Insoluble Ash

Proceed as in 900.02D (see 44.1.05), using ash of 950.14A.

C. Alkalinity of Soluble Ash

Proceed as in 900.02E (see 44.1.05), using soluble ash of

950.14B.
D. Alkalinity of Insoluble Ash

Proceed as in 900.02F (see 44.1.05), using insoluble ash of

950.14B.
E. Elemental Analysis of the Ash

See chapter on fruits and fruit products.

29.1.04
AOAC Official Method 950.15
Acidity (Total) in Nonalcoholic Beverages
Final Action

See 940.15A (see 26.2.14).

29.1.05
AOAC Official Method 950.16
Acids (Dibasic) in Nonalcoholic Beverages
Preparation of Sample
First Action

(a) Alcoholic products.See 940.15B (see 26.2.14).

(b) Nonalcoholic products.Use sample containing 30 g solids
and 200 mg acid to be determined, as calculated from acidity.
Evaporate to 30 mL, if necessary, and treat as in 950.17950.20
(see 29.1.0629.1.09).

29.1.06
AOAC Official Method 950.17
Citric Acid
in Nonalcoholic Beverages
First Action
Surplus 1980

See 12.019, 12th ed.

29.1.07
AOAC Official Method 950.18
Malic Acid (Levo
and Inactive) in Nonalcoholic Beverages
First Action
Surplus 1980

See 12.020, 12th ed.

29.1.08
AOAC Official Method 950.19
Malic Acid (Levo)
in Nonalcoholic Beverages
First Action
Surplus 1980

See 11.021, 12th ed.

29.1.09
AOAC Official Method 950.20
Tartaric Acid
in Nonalcoholic Beverages
Final Action 1965
Surplus 1980

See 12.018, 12th ed.

29.1.10
AOAC Official Method 950.21
Acidity (Volatile)
in Nonalcoholic Beverages
Final Action

See 940.19 (see 28.1.31).

29.1.11
AOAC Official Method 950.22
Esters
in Nonalcoholic Beverages
Final Action

Proceed as in 9.125, 12th ed., collecting ca 300 mL distillate.

29.1.12
AOAC Official Method 950.23
Benzaldehyde
in Nonalcoholic Beverages
Final Action

Measure 500 mL beverage, 100 mL flavoring sirup, or 1025

mL flavor into distilling flask. Add 32 mL alcohol, and in case of
sirup or flavor, ca 300 mL H2O, and proceed as in 966.14B (see
36.4.03).
Reference: JAOAC 19, 408(1936).
CAS-100-52-7 (benzaldehyde)

29.1.13
AOAC Official Method 950.24
Monochloroacetic Acid
in Nonalcoholic Beverages
Final Action

See 942.09B(b), 942.09C(b) (see 47.3.23), and 942.10C (see

47.3.24).

% Compound = C (H/H) (V/V) 0.1

29.1.14
AOAC Official Method 979.08
Benzoate, Caffeine,
and Saccharin in Soda Beverages
Liquid Chromatographic Method
First Action 1979
Final Action 1984

A. Principle

Saccharin, benzoate, and caffeine are simultaneously determined in soda beverages by isocratic LC, using CH 3COOH
solution as mobile phase with UV detection at 254 nm. Artificial
colors and sorbates may interfere. Beverage blanks containing color
or sorbate should be run to assure non-interference by these compounds. In absence of beverage blanks, analyze samples with 2
different mobile phases with different amounts of isopropanol and/or
CH3COOH or by method of standard additions. Adjustment of
mobile phase (% acid and/or % isopropanol) can resolve interfering
peaks.
B. Apparatus and Reagents

(a) Liquid chromatograph.Equipped with suitable injection

device, solvent delivery system, UV detector, 10 mV strip chart
recorder or equivalent electronic integrator and Bondapak C18
column, 300 3.9 (id) mm (Waters/Millipore) or equivalent. Operating conditions: flow rate 2 mL/min; injection volume 10 L;
detector wavelength 254 nm; temperature ambient.
(b) Mobile phase.20% CH3COOH (volume/volume) buffered
to pH 3.0 with saturated sodium acetate solution. Modify with 02%
isopropanol to obtain baseline resolution and retention times of
standards from mixed standard solution in ca 10 min. Solution is
stable 23 days. Degas prior to use. (Alternatively, lower CH3COOH
concentration may be used, and, for some columns, may be necessary to obtain retention and/or resolution. Lower CH3COOH concentration solutions give longer retention times, e.g. 5% CH3COOH
elutes compounds in 35 min.)
(c) Standard solutions.Prepare individual standard solutions
from compounds of known purity to give following concentrations
in H2O: sodium saccharin, 0.50 mg/mL; caffeine, 0.050 mg/mL; and
sodium benzoate, 0.50 mg/mL. Use these solutions to determine
sensitivity for detector response and retention times of individual
standards.
(d) Mixed standard solution.Prepare solution containing 0.50
mg/mL sodium saccharin, 0.50 mg/mL sodium benzoate, and 0.050
mg/mL caffeine in H2O. Use this solution to optimize LC conditions
for complete resolution of the 3 compounds and to quantitate.
C. Preparation of Sample

(a) Carbonated beverages.Decarbonate by agitation or ultrasonic treatment. If free of particulate matter, inject directly.
(b) Beverages containing particulate matter.Filter through a
suitable membrane filter (0.45 m), discarding first few mL filtrate.
If large amount of particulate matter is present, centrifuge prior to
filtering. Inject filtered solution directly.
D. Determination

Inject known volume (ca 10 L) of mixed standard solution in

duplicate. Peak heights should agree within 2.5%. Inject known
volume (ca 10 L) of prepared sample in duplicate. Measure peak
heights of standard and sample components.

where C = concentration of standard in mg/mL; H and H = average

peak height of sample and standard, respectively; V and V = volume
injected in L of sample and standard, respectively.
Reference: JAOAC 62, 1011(1979).
CAS-58-08-2 (caffeine)
CAS-128-44-9 (saccharin sodium)
CAS-532-32-1 (sodium benzoate)

29.1.15
AOAC Official Method 955.27
Essential Oils
in Nonalcoholic Beverages
First Action

See 942.08B (see 36.6.03).

29.1.16
AOAC Official Method 960.22 was repealed.

29.1.17
AOAC Official Method 962.13
Caffeine
in Nonalcoholic Beverages
First Action 1962

Method II
A. Reagents

(a) Reducing solution.Dissolve 5 g Na2SO3 and 5 g KSCN in

H2O and dilute to 100 mL.
(b) Dilute phosphoric acid solution.Dilute 15 mL H3PO4 to 85
mL with H2O.
(c) Sodium hydroxide solution.Dissolve 25 g NaOH in 75 mL H2O.
(d) Caffeine standard solution.1 mg/mL. Dissolve 100 mg in
CHCl3 and dilute to 100 mL with CHCl3. (Caution: See Appendix B,
safety notes on chloroform).
B. Preparation of Standard Curve

Prepare diluted standard solutions containing 0.10, 0.25, 0.50,

1.00, 1.50, and 2.00 mg caffeine/100 mL CHCl3. Determine A of all
solutions at ca 276 nm against CHCl3 in 1 cm matched cells. Plot A
against concentration or use linear regression analysis of data points.
C. Determination

Remove any gas by agitation or ultrasonic treatment. Pipet 10 mL

sample into 125 mL separator, add 5 mL 1.5% KMnO4 solution, and
mix. After exactly 5 min, add 10 mL reducing solution and mix. Add
1 mL dilute H3PO4 solution, mix, add 1 mL NaOH solution, mix,
and extract with 50 mL CHCl3 1 min. After separation, drain lower
layer through 7 cm paper into 100 mL glass-stoppered volumetric
flask. Add 23 mL CHCl3 to separator and drain through paper to
rinse separator stem. Wash paper with 23 mL CHCl3. Re-extract
solution with 40 mL CHCl3 and wash stem and paper as before.
Dilute to volume with CHCl3. Determine A of this solution at
wavelength of maximum A against CHCl3. Determine concentration
of caffeine from standard curve or by linear regression and calculate
mg/100 mL beverage.
References: JAOAC 41, 617(1958); 45, 252(1962).

Reference: JAOAC 50, 857(1967).

29.1.18
AOAC Official Method 967.11
Caffeine in Nonalcoholic Beverages
Spectrophotometric Method
First Action 1967
Method III

[Method is preferable to 962.13 (see 29.1.17) when only few samples are to be analyzed].
A. Apparatus

Chromatographic tubes.Glass, ca 25 250 mm.

B. Reagents

(Use CHCl 3 and ether washed with 12 volume H 2O throughout.

Caution: See Appendix B, safety notes on ether and chloroform.)
Caffeine standard solutions.(1) Stock solution.See
962.13A(d) (see 29.1.17). (2) Working solutions.Prepare dilutions
of stock solution containing 0.25, 0.50, and 0.75 mg caffeine/50 mL
CHCl3. Check factor, 967.11F, frequently to minimize instrument
variations.
C. Preparation of Sample

Remove any gas by agitation or ultrasonic treatment. Pipet

appropriate amount sample (5 or 10 mL) into 250 mL beaker.
Evaporate to near dryness on steam bath. Dissolve residue in 5
mL NH4OH (1 + 2) and heat 2 min on steam bath. Remove from
heat, add 6 g Celite 545, and mix carefully to homogeneity before
preparing column.
D. Preparation of Columns

(a) Column I.Carefully mix 2 mL 4N H2SO4 with 2 g Celite

545, place in chromatographic tube over glass wool plug, and tamp
to uniform mass with moderate pressure. Top with glass wool plug
to minimize damage to column surface.
(b) Column II.(1) Layer 1.Carefully mix 3 g Celite 545 with
2 mL 2N NaOH, transfer to tube over glass wool plug, and tamp to
uniform mass. (2) Layer 2.Transfer prepared sample and Celite
mixture to column directly over layer 1 and tamp. Dry-wash beaker
with 12 g dry Celite and transfer to column. Tamp to uniform mass
and top column with glass wool pad.
E. Determination

Mount column II over column I. Pass 150 mL ether through

column II and into column I, using initial portion of ether to rinse
sample beaker. Drain well and remove column II. Pass additional 50
mL ether through column I and drain well. Pass 50 mL CHCl3
through column I, using initial portion to wash tip of column II.
Collect eluate in 50 mL volumetric flask and adjust volume to 50
mL. Read A of CHCl3 solution at 276 nm against CHCl3 reference
with 1 cm silica cells. If necessary, adjust concentrations to acceptable levels by diluting with CHCl3.
F. Calculations

Factor F = mg caffeine in 50 mL standard solution/A

mg Caffeine in sample = F A
Express results as mg caffeine/100 mL beverage.

CAS-58-08-2 (caffeine)

29.1.19
AOAC Official Method 969.13
Dihydroanethole, Dihydrosafrole,
Isosafrole, Methyl Salicylate, and Safrole
in Nonalcoholic Beverages
Gas Chromatographic Method
First Action 1969
Final Action 1972

(Quantitative for methyl salicylate; semiquantitative for other compounds.)

A. Reagents

All solvents must be chromatographically pure and meet following test: Concentrate 500 mL solvent to 10 mL in evaporative
concentrator and chromatograph on prepared column with instrument set at maximum sensitivity to be used during analysis; 5 L
concentrated solvent must not show any trace of peaks beyond
solvent front. (Distilled-in-glass solvents generally conform to test
standards.) Purify solvents not passing test as in (a) and (b).
(a) Methanol.Anhydrous. Reflux 1 L with 10 g KOH and 25 g
Zn dust 3 h. Distil, discarding first 100 mL. (Caution: See Appendix
B, safety notes on distillation, potassium hydroxide, and methanol.)
(b) Chloroform.Redistil at bp. Add 1% methanol if stored.
(Caution: See Appendix B, safety notes on chloroform.)
(c) Flavor standard solutions.(1) Stock solution.79 mg/mL
(10,000 ppm). Dilute 7.90 g compound to 100 mL with methanol.
(2) Working solution.7.9 mg/mL (1000 ppm). Dilute 1 mL stock
solution to 10 mL with methanol.
(d) Internal standard solutions.Use n-decyl alcohol for methyl
salicylate determination and m-tolyl acetate for determination of
other compounds. Prepare stock and working standard solutions as
in (c).
(e) Defoaming agent.Dow Corning Antifoam A, or equivalent.
B. Apparatus

(a) Steam distillation apparatus.500 mL round-bottom

flask with long neck, standard taper 24/40 joint; adapter, standard
taper 24/40 inner joint at bottom and at side at 75 angle and with
standard taper 24/40 joint at top; gas inlet tube, 30 cm, standard
taper 24/40 inner joint and perforated 8 mm bulb at dispersion
tip; condenser, 200 mm standard taper 24/40 joint; and adapter,
vacuum takeoff type with extended lower tube, straight joint
standard taper 24/40.
(b) Concentrator.Evaporative concentrator, Kuderna-Danish
type, 500 mL, with 10 mL receiving flask.
(c) Gas chromatograph.With flame ionization detector, and
operating parameters as follows: 1.8 m (6) 4 mm id glass column,
coiled or U-shaped, packed with 15% polypropylene glycol adipate
(Reoplex 400) on 6080 mesh Gas-Chrom P (weight/weight); temperatures ()column 130 (isothermal), detector 220, injection port
180; N2 carrier gas 40 psi at ca 90 mL/min. Column may be
programmed over range 90150 at 2/min.
C. Determination of Relative Retention Time (RT)

(a) Methyl salicylate.Pipet 1 mL working standard solution

and 1 mL n-decyl alcohol working internal standard solution into 10
mL volumetric flask, and dilute to volume with methanol.
(b) Other flavor compounds.Pipet 1 mL appropriate working standard solution and 5 mL m-tolyl acetate working internal

standard solution into 10 mL volumetric flask, and dilute to volume

withmethanol.
Inject 35 L into gas chromatograph. Set sensitivity and attenuation controls to provide peak height 80% of chart scale.
Calculate RT = time for standard component peak to emerge/time
for internal standard peak to emerge. (Time is measured from
emergence of solvent front.) Use RT to identify component peak in
sample. Calculate peak area (PA).
D. Determination

(Analyze samples on same day and under same conditions used for
determination of RT. Caution: See Appendix B, safety notes on
distillation, and chloroform.)
(a) Methyl salicylate.Place 200 mL sample, degassed if necessary, in 500 mL round-bottom flask; add 10 mL methanol, few SiC
chips, and small amount defoamer. Add 1 mL n-decyl alcohol
working internal standard, (d), and attach to distilling apparatus not
connected to steam generator. Bring sample to incipient boil, connect
steam generator, and collect 90 mL distillate at ca 2 mL/min in 100
mL Nessler tube or graduate containing 10 mL methanol to cover
delivery tube outlet. Rinse condenser and delivery tube into distillate
twice with 5 mL methanol. Quantitatively transfer distillate to 250
mL separator. Add 50 mL saturated NaCl solution and extract with
25, 25, and 10 mL CHCl3 by carefully shaking 5 min for each
extraction. Filter CHCl3 through 12.5 cm Whatman No. 30 paper, or
equivalent, containing 35 g Na2SO4 into 500 mL evaporative
concentrator. Wash separator and paper with 1015 mL CHCl3 into
concentrator. Add few SiC chips and evaporate on steam bath to ca
7 mL. Cool, and dilute to 10.0 mL with CHCl3. Determine methyl
salicylate by GC as in 969.13C.
mg methyl salicylate/mL or ppm = (PA/PA)(PAI/PAI) C F
where PA and PA are peak areas of sample and standard solutions,
respectively; PAI and PAI are peak areas of internal standard in
standard and sample solutions, respectively; C = concentration
methyl salicylate in working standard solution (7.9 mg/mL or 1000
ppm); and F = dilution factor (1/20).
(b) Other flavor compounds.Proceed as in (a) except use 5.0
mL m-tolyl acetate working internal standard solution, (d), and
collect 150 mL distillate at 5 mL/min.
References: JAOAC 52, 481(1969); 54, 900(1971).
CAS-104-45-0 (dihydroanethole)
CAS-94-58-6 (dihydrosafrole)
CAS-120-58-1 (isosafrole)
CAS-119-36-8 (methyl salicylate)
CAS-94-59-7 (safrole)

29.1.20
AOAC Official Method 950.25
Methyl Anthranilate
in Nonalcoholic Beverages
Colorimetric Method
Final Action

(Applicable to samples containing <500 mg/L.)

A. Reagents

(a) Dilute hydrochloric acid.Dilute 83 mL HCl to 1 L with H2O.

(b) Sodium nitrite solution.Dissolve 2 g NaNO2 in 100 mL
H2O.
(c) Hydrazine sulfate solution.Dissolve ca 3 g N2H4H2SO4
(Caution: See Appendix B, possible carcinogen, see safety notes on
carcinogens) in 100 mL H2O.
(d) Sodium--naphthol-2-sulfonate solution.Dissolve 5 g of
the sulfonate in 100 mL H2O.
(e) Sodium carbonate solution.Dissolve 25 g Na2CO3 in 75 mL
H2O.
(f) Methyl anthranilate standard solution.1 mg/mL. Dissolve
0.25 g methyl anthranilate in 60 mL alcohol and dilute to 250 mL
with H2O.
B. Apparatus

(a) Steam generator filled with H2O.Oil can holding 1 gal. (ca
4 L) serves purpose.
(b) Distillation flask.Kjeldahl flask, ca 750 mL, with shortened
neck, ca 25 cm over-all height.
(c) Spray tube.Glass tube with small perforated bulb at end,
passing through rubber stopper and reaching to bottom of distillation
flask.
(d) Connecting bulb.Kjeldahl bulb with bent connecting tube.
(e) Worm condenser.With H2O jacket 2530 cm long, and
outlet tube reaching bottom of 500 mL Erlenmeyer receiving
flask.
C. Determination

Place just enough H2O in receiving flask to seal end of extended

condenser tube. Place 10100 mL sample in distillation flask and add,
if necessary, H2O to make 100 mL. Insert stopper carrying spray tube
and connecting bulb, and connect with condenser and receiving flask.
Immerse distillation flask in H2O bath near bp to level of contents.
When sample reaches temperature of bath, connect to steam generator
with H2O boiling and rapidly pass steam through sample until ca 300
mL distillate collects.
Disconnect apparatus and wash out condenser with little H2O. Add
25 mL dilute HCl and 2 mL NaNO2 solution to distillate, mix well, and
let stand exactly 2 min. Add 6 mL N2H4H2SO4 solution and mix well
1 min, so that liquid comes in contact with all parts of flask that solution
may have touched when it contained free HNO2. Keep liquid in flask in
rapid motion, quickly add 5 mL sodium -naphthol-2-sulfonate solution, and then immediately add 15 mL Na2CO3 solution. Dilute colored
solution to 500 mL with H2O, mix, and compare color of portion with
color of standard, or set of standards, prepared as nearly as possible at
same time. Calculate results as mg methyl anthranilate/L.
References: J. Agric. Research 33, 301(1926). JAOAC 11, 46, 505
(1928).
CAS-134-20-3 (methyl anthranilate)

29.1.21
AOAC Official Method 950.26
Methyl Anthranilate
in Nonalcoholic Beverages
Gravimetric Method
Final Action

(Applicable to samples containing 500 mg/L.)

A. Reagents and Apparatus

(a) -Naphthol solution.Dissolve 0.2 g -naphthol in 100 mL

30% alcohol.
(b) Sodium bicarbonate solution.Dissolve 8.4 g NaHCO3 in
100 mL H2O.
(See 950.25A [see 29.1.20] for reagents and 950.25B [see 29.1.20]
for apparatus.)
B. Determination

Place sample containing 50125 mg methyl anthranilate in distillation flask and dilute, if necessary, to 100 mL with H2O. Steam distil
as in 950.25C (see 29.1.20), collecting ca 400 mL distillate.
Wash out condenser with little H2O and dilute distillate to 500 mL.
Mix, and to 200 mL aliquot add 5 mL dilute HCl and 5 mL NaNO2
solution. Mix well and let stand 1 min. Mix 25 mL -naphthol
solution and 6 mL NaHCO3 solution, pour diazotized solution into
mixture, and let stand 10 min. Fold 2 Whatman No. 1 or S&S No.
595 papers, 12.5 cm diameter, and determine difference in their
weights by placing one on each pan of balance and counterpoising
with added weights. Place heavier inside lighter paper, fit into funnel,
and moisten. Pour mixture through this filter and wash precipitate 7
or 8 times, using total of ca 100 mL H2O. Fill filter only to ca 1 cm
from top. Place funnel carrying filter and washed precipitate in oven,
and dry ca 10 min at 100. Separate and dry filter papers ca 1 h at
same temperature. Determine difference in weights, dry again,
weigh again, and repeat until difference in weights remains constant.
(Constant difference in weights original difference in weights of
2 papers) 0.4935 = weight anthranilic acid ester, as methyl anthranilate. Calculate as g methyl anthranilate/L.
References: Ind. Eng. Chem. 15, 732(1923). JAOAC 11, 47,
505(1928).
CAS-134-20-3 (methyl anthranilate)

29.1.22
AOAC Official Method 950.27
Solids (Total)
in Nonalcoholic Beverages
Final Action

See 925.45C or D (see 44.1.03).

29.1.23
AOAC Official Method 950.28
Specific Gravity
of Nonalcoholic Beverages
Final Action

See 945.06C (see 26.1.06).

29.1.24
AOAC Official Method 950.29
Sucrose
in Nonalcoholic Beverages
Final Action

A. By Polarization

Determine by polarizing before and after inversion as in 925.47

(see 44.1.08) or 925.48 (see 44.1.09).
B. By Reducing Sugars Before and After Inversion

See 930.36 (see 44.1.13).

29.1.25
AOAC Official Method 950.30
Sugars (Reducing)
in Nonalcoholic Beverages
Final Action

Use value obtained for reducing sugars before inversion, 950.29B

(see 29.1.24).

29.1.26
AOAC Official Method 950.31
Glucose (Commercial)
in Nonalcoholic Beverages
Procedure (Approximate)

See 930.37B (see 44.1.14).

29.1.27
AOAC Official Method 950.32
Quaternary Ammonium Compounds
in Nonalcoholic Beverages
Final Action

See 942.13EF (see 47.3.31).

29.1.28
AOAC Official Method 950.33
Gamma Undecalactone
in Nonalcoholic Beverages
Final Action
Surplus 1970

See 8.030, 10th ed.

29.1.29
AOAC Official Method 960.23
Alginates
in Chocolate Beverages
Final Action

See 959.06A (see 31.5.11).