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29. Nonalcoholic Beverages and Concentrates*

Richard D. Thompson, Chapter Editor

Food and Drug Administration

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 950.17–950.20(see 29.1.06–29.1.09).

29.1.06

AOAC Official Method 950.17Citric Acid in Nonalcoholic Beverages

First Action

Surplus 1980

See 12.019, 12th ed.

29.1.07

AOAC Official Method 950.18Malic Acid (Levo and Inactive) in Nonalcoholic Beverages

First Action

Surplus 1980

See 12.020, 12th ed.

29.1.08

AOAC Official Method 950.19Malic Acid (Levo) in Nonalcoholic Beverages

First Action

Surplus 1980

See 11.021, 12th ed.

29.1.09

AOAC Official Method 950.20Tartaric 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 10–25 mL flavor into distilling flask. Add 32 mL alcohol, and in case of sirup or flavor, ca 300 mL H 2 O, 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).

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 deter- mined in soda beverages by isocratic LC, using CH 3 COOH 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 com- pounds. In absence of beverage blanks, analyze samples with 2 different mobile phases with different amounts of isopropanol and/or CH 3 COOH 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 C 18

column, 300 3.9 (id) mm (Waters/Millipore) or equivalent. Oper- ating conditions: flow rate 2 mL/min; injection volume 10 L; detector wavelength 254 nm; temperature ambient.

(b) Mobile phase.—20% CH 3 COOH (volume/volume) buffered

to pH 3.0 with saturated sodium acetate solution. Modify with 0–2%

isopropanol to obtain baseline resolution and retention times of standards from mixed standard solution in ca 10 min. Solution is stable 2–3 days. Degas prior to use. (Alternatively, lower CH 3 COOH concentration may be used, and, for some columns, may be neces- sary to obtain retention and/or resolution. Lower CH 3 COOH con- centration solutions give longer retention times, e.g. 5% CH 3 COOH elutes compounds in 35 min.)

(c) Standard solutions.—Prepare individual standard solutions

from compounds of known purity to give following concentrations in H 2 O: 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 H 2 O. 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 ultra-

sonic 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.

% Compound = C

(H/H )

(V /V)

0.1

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:

CAS-58-08-2 (caffeine) CAS-128-44-9 (saccharin sodium) CAS-532-32-1 (sodium benzoate)

JAOAC 62, 1011(1979).

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 Na 2 SO 3 and 5 g KSCN in

H 2 O and dilute to 100 mL.

(b) Dilute phosphoric acid solution.—Dilute 15 mL H 3 PO 4 to 85

mL with H 2 O.

(c)

Sodium hydroxide solution.—Dissolve 25 g NaOH in 75 mL H 2 O.

(d)

Caffeine standard solution.—1 mg/mL. Dissolve 100 mg in

CHCl 3 and dilute to 100 mL with CHCl 3 . (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 CHCl 3 . Determine A of all solutions at ca 276 nm against CHCl 3 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% KMnO 4 solution, and mix. After exactly 5 min, add 10 mL reducing solution and mix. Add 1 mL dilute H 3 PO 4 solution, mix, add 1 mL NaOH solution, mix,

and extract with 50 mL CHCl 3 1 min. After separation, drain lower

layer through 7 cm paper into 100 mL glass-stoppered volumetric flask. Add 2–3 mL CHCl 3 to separator and drain through paper to rinse separator stem. Wash paper with 2–3 mL CHCl 3 . Re-extract solution with 40 mL CHCl 3 and wash stem and paper as before. Dilute to volume with CHCl 3 . Determine A of this solution at wavelength of maximum A against CHCl 3 . 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).

29.1.18

Reference:

JAOAC 50, 857(1967).

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 sam- ples are to be analyzed].

A. Apparatus

Chromatographic tubes.—Glass, ca 25 250 mm.

CAS-58-08-2 (caffeine)

B. Reagents

(Use CHCl 3 and ether washed with 1 2 volume H 2 O throughout. Caution: See Appendix B, safety notes on ether and chloro- form.)

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 CHCl 3 . 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 NH 4 OH (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 H 2 SO 4 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 1–2 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 CHCl 3

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 CHCl 3 solution at 276 nm against CHCl 3 reference with 1 cm silica cells. If necessary, adjust concentrations to accept- able levels by diluting with CHCl 3 .

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.

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 com- pounds.)

A. Reagents

All solvents must be chromatographically pure and meet follow-

ing test: Concentrate 500 mL solvent to 10 mL in evaporative

concentrator and chromatograph on prepared column with instru- ment 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 75angle 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 60–80 mesh Gas-Chrom P (weight/weight); tem- peratures ()—column 130 (isothermal), detector 220, injection port 180; N 2 carrier gas 40 psi at ca 90 mL/min. Column may be programmed over range 90–150at 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 work-

ing standard solution and 5 mL m-tolyl acetate working internal

standard solution into 10 mLvolumetric flask, and dilute to volume withmethanol. Inject 3–5 L into gas chromatograph. Set sensitivity and attenu- ation 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 neces-

sary, 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 CHCl 3 by carefully shaking 5 min for each

extraction. Filter CHCl 3 through 12.5 cm Whatman No. 30 paper, or

equivalent, containing 3–5 g Na 2 SO 4 into 500 mL evaporative concentrator. Wash separator and paper with 10–15 mL CHCl 3 into concentrator. Add few SiC chips and evaporate on steam bath to ca 7 mL. Cool, and dilute to 10.0 mL with CHCl 3 . Determine methyl salicylate by GC as in 969.13C.

mg methyl salicylate/mL or ppm = (PA/PA )(PA I /PA I ) C F

where PA and PA are peak areas of sample and standard solutions,

respectively; PA I and PA I 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 H 2 O.

(b)

Sodium nitrite solution.—Dissolve 2 g NaNO 2 in 100 mL

H

2 O.

(c)

Hydrazine sulfate solution.—Dissolve ca 3 g N 2 H 4 H 2 SO 4

(Caution: See Appendix B, possible carcinogen, see safety notes on

carcinogens) in 100 mL

(d) Sodium- -naphthol-2-sulfonate solution.—Dissolve 5 g of

the sulfonate in 100 mL H 2 O.

H 2 O.

 

(e)

Sodium carbonate solution.—Dissolve 25 g Na 2 CO 3 in 75 mL

H

2 O.

(f)

Methyl anthranilate standard solution.—1 mg/mL. Dissolve

0.25 g methyl anthranilate in 60 mL alcohol and dilute to 250 mL with H 2 O.

B. Apparatus

(a) Steam generator filled with H 2 O.—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 H 2 O jacket 25–30 cm long, and

outlet tube reaching bottom of 500 mL Erlenmeyer receiving flask.

C. Determination

Place just enough H 2 O in receiving flask to seal end of extended condenser tube. Place 10–100 mL sample in distillation flask and add,

if necessary, H 2 O to make 100 mL. Insert stopper carrying spray tube

and connecting bulb, and connect with condenser and receiving flask.

Immerse distillation flask in H 2 O bath near bp to level of contents.

When sample reaches temperature of bath, connect to steam generator

with H 2 O boiling and rapidly pass steam through sample until ca 300

mL distillate collects.

Disconnect apparatus and wash out condenser with little H 2 O. Add 25 mL dilute HCl and 2 mL NaNO 2 solution to distillate, mix well, and let stand exactly 2 min. Add 6 mL N 2 H 4 H 2 SO 4 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 HNO 2 . Keep liquid in flask in rapid motion, quickly add 5 mL sodium -naphthol-2-sulfonate solu- tion, and then immediately add 15 mL Na 2 CO 3 solution. Dilute colored solution to 500 mL with H 2 O, 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 NaHCO 3 in

100 mL H 2 O.

(See 950.25A [see 29.1.20] for reagents and 950.25B [see 29.1.20] for apparatus.)

B. Determination

Place sample containing 50–125 mg methyl anthranilate in distil- lation flask and dilute, if necessary, to 100 mL with H 2 O. Steam distil as in 950.25C (see 29.1.20), collecting ca 400 mL distillate. Wash out condenser with little H 2 O and dilute distillate to 500 mL. Mix, and to 200 mL aliquot add 5 mL dilute HCl and 5 mL NaNO 2 solution. Mix well and let stand 1 min. Mix 25 mL -naphthol solution and 6 mL NaHCO 3 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 H 2 O. 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 anthra- nilate. 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 942.13E–F (see 47.3.31).

29.1.28

AOAC Official Method 950.33Gamma 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).