Briefing Session on Analysis of Preservatives in Food

Dr. Jasmine LAU Po-kwan Chemist

Food Safety B SectionGovernment Laboratory, HKSAR

23rd May 2008

Contents

Background

Testing Methods

Findings in 2007

Proficiency Tests

BackgroundCap 132BD

Preservatives in Food Regulations

Preservatives: any substance which is capable of inhibiting, retarding or arresting the process of fermentation, acidification or other deterioration of food of masking any of the putrefaction.:

Background testing scope

Benzoic acid, ;

(as sodium, potassium, calcium benzoate);

Sorbic acid, ;

(as sodium, potassium, calcium sorbate);

Salicylic acid, ;

Methyl, Ethyl or Propyl para-hydroxbenzoate, (as sodium salt)

Boric acid,

Background permitted level

Permitted preservatives:

Benzoic acid, ; Sorbic acid, ;Methyl, Ethyl or Propyl para-hydroxbenzoate,

Non-Permitted preservatives:

Salicylic acid, , Boric acid,

FOOD WHICH MAY CONTAIN PRESERVATIVE AND THE NATUREAND PROPORTION OF PRESERVATIVE IN EACH CASE

Specified food Permitted preservatives

Parts per million not exceeding

Beer

Benzoic acid, or Methyl para-hydroxybenzoate or Ethyl para-hydroxybenzoate or Propyl para-hydroxybenzoate or

70707070

Fish ball fish cake, and shredded fish

Sorbic acid, orBenzoic acid, or Methyl para-hydroxybenzoate or Ethyl para-hydroxybenzoate or Propyl para-hydroxybenzoate or

10001000100010001000

Soy or soy sauce soya bean product

()

Sorbic acid, orBenzoic acid, or Methyl para-hydroxybenzoate or Ethyl para-hydroxybenzoate or Propyl para-hydroxybenzoate or

1000550550550550

Food Category

Range of food samples Beverage (soft drinks, juices, coffee) Flour confectionery (bread, biscuits, pies) Pickles or preserved food( prune, plum..) Snacks (candy, chocolates) Fruits (orange, snow pear, lemon, watermelon...) Sauce (oyster sauces, soya sauce,

salad dressing) Meat (beef, lamp chop, pork) Etc

Testing Method: International Standard Methods

AOAC 910.02 Benzoic acid in Food Qualitative Test

AOAC 960.38 Benzoic acid in non-solid food and beverage

Spectrophotometric method

AOAC 974.10 Sorbic acid in dairy products Spectrophotometric method

AOAC 975.10 Sorbic acid in wine Calorimetric method

AOAC 960.38 Benzoic acid in non-solid food and beverage

Spectrophotometricmethod

Testing Method: International Standard Methods

ISO 5518:2007 Benzoic acid in fruits, vegetables and derived products

Spectrophotometricmethod

ISO 6560:1983 Benzoic acid in fruits and vegetable products

Molecular spectrometric method

AOAC 975.31 Salicylic acid in food and beverage

Calorimetric method

AOAC 983.16 Benzoic acid and sorbic acid

GC

Testing Method: methods used by the Government Laboratory

Analysis of Benzoic acid, Salicylic acid, Sorbic acid and Parabens (methyl, ethyl, propyl hydroxybenzoates)

i) Screened and semi-quantified by HPLC method similar to method of J. Assoc. Off. Anal. Chem., 1985, 68, 488-492.

ii) Quantified by HPLC method similar to method of Official Methods of Analysis of the Association of Official Analytical Chemists, 1990, 15th edition, pp 1142 and 1156.

iii) Determination of esters of para-hydroxybenzoic acid by HPLC, method similar to method of J. Assoc. Off. Anal. Chem., 1985,68, 488.

iv) Structural confirmation by GC-MS method similar to AOAC 983.16.

i) Semi-quantified by HPLC method

Weigh approximate 10 g well blended sample in a 50mL centrifuge tube

Add about 20 mL extraction solvent into the tube Shake and sonicate for 10 minutes Filter the extraction through No.542 filter paper Filter the extraction solution through 0.45 m membrane filter

before HPLC analysis

HPLC condition for Benzoic acid, Salicylic acid, Sorbic acid and Parabens (methyl, ethyl, propyl para-hydroxybenzoates)

Column : C-18, 7 m, 250 mm in length and 4.6mm i.d.

Mobile phase : Tetrahydrofuran/CH3CN/acetic acid/water(13+25+1+70, v/v)

Flow rate : 1.1 mL/min.

PDA wavelength : 254 nm

Injection volume : 20 L

HPLC Chromatograms of sorbic, benzoic and salicylic acid

HPLC Chromatograms of methyl, ethyl, propyl p-hydroxybenzoates

ii) Quantification by HPLC method

Comminute all chopped sample in a blender. For liquid sample, mix the sample by shaking or by stirring with a glass rodWeigh about 12.5 g well mixed sample in a 500 mL steam distillation tube. Add about 50 g magnesium sulphate heptahydrate and about 25 mL water. Acidify with 10 mL 85% phosphoric acid and carry out steam distillation.

For sorbic acid, collect ~ 200 mL distillate in a 250 mLvolumetric flask containing 5 mL 1 M sodium hydroxide solution

For benzoic acid, collect ~ 450 mL distillate in a 500 mLvolumetric flask containing 10 mL 1 M sodium hydroxide solution

For both sorbic acid and benzoic acid, collect ~ 450 mLdistillate in a 500 mL volumetric flask containing 10 mL 1 M sodium hydroxide solution

Make up to volume with distillated water

Filter the extraction through No.542 filter paper Filter the extraction solution through 0.45 mm membrane filter before HPLC analysis Record the retention time and peak area of sorbic or benzoic acid

HPLC condition for Benzoic acid and Sorbic acid

Column : C-18, 5 or 7 m, 250 mm in length and 4.6mm i.d.

Mobile phase : Tetrahydrofuran/CH3CN/acetic acid/water(13+25+1+70, v/v)

Flow rate : 1.2 mL/min.

PDA wavelength : Benzoic acid: 272 nm

: Sorbic acid: 254 nm

Injection volume : Benzoic acid: 20 L

: Sorbic acid: 10 L

HPLC Chromatograms of sorbic acid

HPLC Chromatograms of benzoic acid

iii) Quantitative Test for ester of para-Hydroxybenzoic acid

Weigh 25 g sample in a 200 mL centrifuge bottle

Add 30 mL methanol, homogenise for 1 minute

Centrifuge at 1500 rpm for 3 minutes

100 ml v-flaskhomogenise for 1 minutecentrifuge and decant

extracts +30 mL methanol/water(3+2)

Filter through No. 542 filter paper

Transfer to vial

Inject 10 uL of the resultant into HPLC for analysis

iv) Structural confirmation by GC-MS method

Centrifuge the tubes at 1500 rpm for 10 minutes Transfer the ethyl layer into a 250 mL separating funnel (repeat

the extraction process twice)

Internal standard solution (2000 mg/L): Weight 500 mg of phenylacetic acid and caproic acid, respectively, dissolve in 250.0 mL 4% aqueous potassium hydroxide solution

Weigh 5 g homogenized sample+ 3.0 mL internal standard solution

+1.5 mL 3M sulphuric acid & 5 g sandCentrifuge tube

Extract the ethyl layers twice with 15 mL 0.5M sodium hydroxide solution and 10 mL saturated chloride solution each time. Collect the aq. layers in a 250 mL separating funnel

Add 2 drops of methyl orange, acidify the aqueous layer with 10 mL 6 M hydrochloric acid

Extract the aq. layer with successive portion of CH2Cl2 Drain the dichloromethane extracts through filter paper containing

15 g anhydrous sodium sulphate into a 250 mL round-bottom flask Evaporate the CH2Cl2 solution at 40 C just to dryness

iv) Structural confirmation by GC-MS methodDerivatisation with MSTFA

Pipette 10.0 mL chloroform to re-dissolve the residue in the RB flask Transfer 1.00 mL of the chloroform solution accurately to test tube with

Teflon-lined screw cap Add 0.20 mL silylating agent MSTFA to the test tube. Screw the cap and let

it stand for 15 minutes in an oven at 60 C Inject the residue solution to the GC for analysis

GC condition for Benzoic acid and Sorbic acid

Column : SPB-1, 30 m, 0.53 mm i.d., 1.5 mm film thickess or equivalent

Injector : Temperature: 200 CMode: splitlessPurge flow: 1.0 mL/min

Oven temperature : Initial: 80 C for 7 minutes: Rate: 8 C/min: Final: 210 C for 1 minute

Carrier gas flow rate : 7 mL/min, helium gasDetection (FID) : Temperature: 280 C

: Air flow: 300 mL/min: Hydrogen flow: 30mL/min: Makeup flow: 30 mL/min

Injection volume : 1 L

GC-MS condition for Benzoic acid and Sorbic acidColumn : HP-5ms, 30 m, 0.25 mm i.d., 0.25 m film thickess

or equivalentInjector : Temperature: 200 C

Mode: splitlessPurge flow: 1.0 mL/min

Oven temperature : Initial: 80 C for 7 minutes: Rate: 8 C/min: Final: 210 C for 1 minute

Carrier gas flow rate : 0.7 mL/min, helium gasDetection (MS) : Transfer line heater temperature: 280 C

: Acquistion mode: Scan: Solvent delay: 3 minutes: Scan mass: 50 to 550 amu: Threshold: 150

Injection volume : 1 L

GC chromatograms of sorbic and benzoic acid

Caproic Acid

Sorbic acid

Benzoic acid

Phenylacetic acid

GC-MS chromatograms of sorbic and benzoic acid

Reporting Limits

Sorbic acid: 50 (mg/kg)

Benzoic acid: 50 (mg/kg)

Salicylic acid: 50 (mg/kg)

Methyl p-hydroxybenoate: 30 (mg/kg)

Ethyl p-hydroxybenoate: 30 (mg/kg)

Propyl p-hydroxybenzoate: 30 (mg/kg)

Testing Method: methods used by the Government Laboratory

Analysis of Boric acid

i) Semi-quantitative Determination of Boric acid in Food, method similar to AOAC method (17th edition 2000) 47.3.09

ii) Quantitative determination of Boric acid in Food by UV-VIS & microwave digestion followed by ICP-OES analysis, similar to method of J. Assoc. Off. Anal. Chem., 1978,61, 894-897 & J. Assoc. Off. Anal. Chem., 1982,65, 234-237

i) Semi-quantitative determination of Boric acid in Food

Comminute the chopped solid sample

Weigh 25g well-blended sample in a beaker

Add 50 mL distilled water

Boil the sample

Cool to room temperature

Add 7 mL of 6M HCl

Immerse the turmeric

paper into the resulted

solution and air dry the

paper

Results of tested samples

iia) Determination of Boric acid in Food by UV-VIS Techniques

Weigh 10 g homogenised sample in platinum dish

Add 10 mL 0.1M NaOH

Heat it on water bath until the sample is charred

Ignite it over a burner

Furnace at 550 oC for 2 hours

Cool to room temp with further addition of 10 mL 0.5M HCl and triturate the residue

Heat the solution on water bath for 30 minutes

Cool, filter and collect to 50 mL v-flask

X 2

Pipette 1 mL aliquot to 10 ml v-flask.

Add 3 mL azomethine H solution and then make-up.

Transfer the solution to 1 cm cell Measure the absorbance at 420 nm ( slit width = 1 nm )

iib) Determination of Boric acid in Food by microwave digestion followed by ICP-OES analysis

Weigh 0.5 gram of homogenised sample

into PTFE digestion vessel

Add 4 mL nitric acid

Cap the PTFE digestion vessel tightly

Connect to Microwave unit.

Digest the target sample with pre-set operation condition of the microwave unit

After complete digestion,

make up to 50 mL with D.D. water

Sample solutions are then processed by ICP-OES

Reporting Limits

Boric acid: 50 (mg/kg)

Findings in 2007

Parameter tested Total samples testedNo. of

Positive samples

Sorbic acid 3553 219

Benzoic acid 3572 229

Salicylic acid 2592 0

Methyl para-hydroxbenzoate 2483 1

Ethyl para-hydroxbenzoate 2483 0

Propyl para-hydroxbenzoate 2483 0

Boric acid 1086 0

Findings in 2007

Parameter tested Types of samples with positive findingsNo. of positive

samplesConcentration range

found

Sorbic acid Beverage 16 130 ppm to 240 ppm

Cooked food 4 59 ppm to 340 ppm

Dairy products 5 57 ppm to 490 ppm

Flour confectionery 40 60 ppm to 1,400 ppm

Marine products 5 120 ppm to 650 ppm

Meat and meat products, fresh, frozen

40 62 ppm to 570 ppm

Preserved fruits and vegetables

52 70 ppm to 2,300 ppm

Sauces 53 180 ppm to 680 ppm

Other items 4 180 ppm to 390 ppm

Findings in 2007

Parameter tested Types of samples with positive findings

No. of positive samples

Concentration range found

Benzoic acid Beverage and fruit juice 87 63 ppm to 470 ppm

Cooked food 6 64 ppm to 420 ppm

Flour confectionery 5 51 ppm to 450 ppm

Meat and meat products, fresh, frozen

10 59 ppm to 2,200 ppm

Preserved fruits and vegetables

96 48 ppm to 2,100 ppm

Sauces 22 120 ppm to 500 ppm

Other items 3 110 ppm to 640 ppm

Methyl para-hydroxbenzoate Sauces 1 100 ppm

British Pharmacopoeia

Food Chemcials Codex

Joint FAO/WHO Expert Committee on Food Additives(JECFA)

Standards Validation

http://www.fao.org/ag/agn/jecfa-additives/search.html?lang=en

Standard Reference Material

Assay of reference materials

Characterization of reference materials

Infra-red Reference spectra

Proficiency Test

i) FAPAS

ii) LGC Standards Proficiency Testing Group

Proficiency Test Start Date Matrix Analytes T2056 24/10/2008 Jam -benzoic acid

-Brix-Citric Acid-pH-Sorbic Acid

T0381 23/10/2008 Soft Drink -Brix-pH-Citric Acid-Sorbic Acid-Cyclamate-Saccharin

http://www.fapas.com

http://www.lgcpt.com/

http://www.fapas.com/http://www.lgcpt.com/default.aspx

Thank You

The End

Briefing Session on Analysis of Preservatives in FoodContentsBackgroundBackground testing scopeBackground permitted levelFOOD WHICH MAY CONTAIN PRESERVATIVE AND THE NATUREAND PROPORTION OF PRESERVATIVE IN EACH CASEFood CategoryTesting Method: International Standard MethodsTesting Method: International Standard MethodsTesting Method: methods used by the Government Laboratoryi) Semi-quantified by HPLC methodHPLC condition for Benzoic acid, Salicylic acid, Sorbic acid and Parabens (methyl, ethyl, propyl para-hydroxybenzoates)HPLC Chromatograms of sorbic, benzoic and salicylic acidHPLC Chromatograms of methyl, ethyl, propyl p-hydroxybenzoatesii) Quantification by HPLC method 16 17HPLC condition for Benzoic acid and Sorbic acidHPLC Chromatograms of sorbic acidHPLC Chromatograms of benzoic acid iii) Quantitative Test for ester of para-Hydroxybenzoic acid iv) Structural confirmation by GC-MS method 24iv) Structural confirmation by GC-MS methodGC condition for Benzoic acid and Sorbic acidGC-MS condition for Benzoic acid and Sorbic acidGC chromatograms of sorbic and benzoic acidGC-MS chromatograms of sorbic and benzoic acidReporting LimitsTesting Method: methods used by the Government Laboratoryi) Semi-quantitative determination of Boric acid in Food 33Results of tested samples iia) Determination of Boric acid in Food by UV-VIS Techniques 36 37 iib) Determination of Boric acid in Food by microwave digestion followed by ICP-OES analysis 39 40 41Reporting LimitsFindings in 2007Findings in 2007Findings in 2007Standards Validation 47 48 49Proficiency Test 51