GENERAL REFEREE REPORTS Committee on Food Nutrition Sugars and Sugar Products M ARY A N G ODSHALL Sugar Processing Research Institute, Inc., 1100 Robert E.
Lee Blvd, New Orleans, LA 70124, Tel: +1-504-286-4329,
Fax: +1-504-282-5387, E-mail: godshall@srrc.ars.usda.gov Summary Sugars and Sugar Products has 7 subsections: (1) sugars and syrups; (2) molasses and molasses products; (3) confec-
tionery; (4) honey; (5) maple, sap, maple syrup, maple syrup
products; (6) sugar beets; and (7) corn syrups and other
starch-derived sweeteners. This subcommittee is currently un-
der-represented in several areas, and attempts are being made
to correct this situation. Honey Topic Advisor Peter Martin, Q.P. Services, Orchard Cot- tage, Crazies Hill, Reading RG10 BLU, UK, Tel:
44-118-940-2212, Fax: 44-118-940-1235, E-mail: honeysci@aol.com. The stable isotope method for honey
(998.12, “Plant Sugars in Honey, Internal Standard Stable
Carbon Isotope Ratio Method”) is written incorrectly and
Martin requests help rewriting it. (See also Study Director re-
port on Stable Isotope Ratio Methods, below.) Martin also re-
ports that with the adoption of the new Codex Standard, some
AOAC methods are out of date. He plans to compile a list of
the methods that need to be updated. Corn Syrup and Other Starch-Derived Sweeteners A Topic Advisor is needed. Melanie O’Donnell, Corn Re- finers Association, Inc., 1701 Pennsylvania Ave, NW, Suite
950, Washington, DC 20006, Tel: +1-202-331-1634, Fax:
+1-202-331-2054, E-mail: modonnell@corn.org, has been
recommended. She has agreed to review the existing methods
in the Official Methods of Analysis, 17th Ed. The Corn Re-
finers Association, like many professional organizations, has
its own book of methods called the “Analytical and Microbio-
logical Methods of the Member Companies,” so it is possible
that some of the AOAC corn sweetener methods may require
updating. Maple, Sap, Maple Syrup, and Maple Syrup Products A Topic Advisor is needed. Sugar and Sugar Products The International Commission for Uniform Methods of Sugar Analysis (ICUMSA) is the organization within the
sugar industry that is concerned with adopting official meth- ods of analysis. ICUMSA follows the harmonized protocol for
conducting collaborative studies. Methods are reviewed and
updated periodically and older methods repealed. Because of
this active pursuit of methods for the sugar industry within the
ICUMSA organization, it is noted that many of the methods
for sugar products listed in the OMA (subsections 1, 2, and 6)
are out of date. ICUMSA held its 23rd Session June 3–5,
2002, in Pune, India. A number of collaborative studies were
reported, which may be of interest to AOAC. All are found in
the Reports of 2002 ICUMSA 23rd Session and are listed
below. Copies of the reports are available upon request to the
General Referee. Collaborative tests reported at the 23rd Session of ICUMSA: (1) Polarization of sugar products without wet lead clarifi- cation (2) Determination of insoluble matter in white sugar
(3) Determination of acid beverage floc in white cane sugar—10 day floc test (4) Determination of acid beverage floc in white beet sugar—24 h test (5) Comparative study of 2 color methods for highly col- ored specialty sugars and syrups using conventional pH ad-
justment and using TEA buffer (6) Determination of anticaking agents in powdered sugar
(7) Sulfite analysis in brown sugar using an enzyme method (8) Reducing sugars in molasses using Layne and Eynon constant volume procedure (9) Total reducing sugars in molasses after hydrolysis us- ing Layne and Eynon constant volume procedure (10) Modified Ofner method for reducing sugars in white sugar (11) Knight and Allen method for reducing sugars in white sugar (12) Hexokinase method for glucose + fructose in white sugar Polarimetric Methods that Use Lead Acetate A review of methods in Sugars and Sugar Products reveals that several AOAC methods use lead acetate for clarification
in polarization measurement. For some years now, there has
been an effort in the sugar industry to eliminate the use of lead
salts in clarification and to substitute less toxic reagents, such
as aluminum salts, or to use filter aid filtration with
polarimeters that read sugar degrees in the near infrared re-
gion, where solution color does not interfere as much. AOAC
methods in the OMA (16th Ed.) that use lead acetate include: (1) 925.46: Sucrose in sugars and syrups, polarimetric methods G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 139 (2) 925.47: Sucrose in sugars and syrups, polarimetric method before and after inversion with invertase (3) 896.02: Sucrose in sugars and syrups, double dilution method (4) 930.36: Sucrose in sugars and syrups, from reducing sugars before and after inversion (5) 970.57: Sucrose in molasses, polarimetric methods
(6) 948.23: Reducing substances (unfermentable) in mo- lasses, titrimetric method (7) 920.190: Sugars (reducing) in maple products as invert sugar (8) 942.20: Sucrose in sugar beets
It is recommended that a Study Director be appointed to re- view polarimetric methods and efforts should be made to up-
date to methods that do not use lead salts as clarifying agents. Cane and Beet Sugar Products Topic Advisor Gillian Eggleston, SRRC-USDA-ARS, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, Tel:
+1-504-286-4446, Fax: +1-504-286-4367, E-mail: gillian@srrc.ars.usda.gov. In Chapter 44 of OMA, many
AOAC methods for measuring sucrose in sugars and syrups
are based on polarimetric methods, e.g., 925.46. Three out of
the 4 clarifying agents recommended in 925.46 include lead
compounds. Because of the environmentally unsafe nature of
lead compounds, and the high costs to safely dispose of them
after use, there has been a great drive, over the last 10 years in
the international sugar industry, to stop using them. Instead,
clarifying agents based on aluminum compounds are being
used, or in highly colored sugars to use celite as the clarifying
agent and measure the clarified solution at 880 nm (instead of
the standard 589 nm) for improved accuracy. It is recom-
mended that the AOAC polarimetric methods be updated and
brought into line with ICUMSA methods. In general, the AOAC methods for measuring sucrose and invert sugars in cane or beet products are based on
polarimetric and chemical methods. More modern methods of
LC and GC should be included to give the users of AOAC
methods more accurate options. ICUMSA has both LC and
GC methods and the acceptance of Kevin Schaffler’s
ICUMSA method, using ion chromatography, on trace glu-
cose and fructose analysis in sugar products, was a step in the
right direction. Eggleston recently demonstrated that an ion chromatogra- phy method (also known as HPAEC–PAD) that she developed
can simultaneously detect ethanol, mannitol, and oligosaccha-
rides in cane products (1). These compounds are sensitive indi-
cators of different types of enzymatic and microbial deteriora-
tion in cane. This method could be applied to other agricultural
and food commodities, especially sugarbeets. She would be in-
terested in undertaking an interlaboratory study of this method,
if there is enough interest. She co-organized an American Chemical Society sympo- sium in 2002 titled “Oligosaccharides in Food and Agricul-
ture,” and is currently editing a book based on the symposium,
which includes up-to-date methodologies to measure oligo-
saccharides (2). Oligosaccharides are currently a very hot topic, particularly their application as prebiotic nutrients to
stimulate the growth of bifidobacteria in the human intestine.
Much interest is being shown in the analysis of oligosaccha-
rides for many applications, and robust and accurate analytical
methods are essential for future progress. AOAC should be
providing these. There are a number of techniques to measure
oligosaccharides. Included here is an adapted review section
written by Eggleston for the introductory chapter of the afore-
mentioned book. Separation and Analysis of Oligosaccharides (2) Until the last 15 years, paper chromatography and thin-layer chromatography (TLC) were the most frequently
used methods to separate oligosaccharides, but these have
largely been replaced with more rapid and powerful separa-
tion and analytical techniques. High-performance size exclu-
sion chromatography (HPSEC) and gel permeation chroma-
tography (GPC) with laser light scattering (LLS) or refractive
index detection allows the separation and direct detection of
oligosaccharides and provides molecular weight distribution
information. Oligosaccharides can be separated by liquid chromatogra- phy (LC) including the use of reversed-phase and calcium col-
umns. High-performance anion exchange chromatography
(HPAEC) with pulsed amperometric detection (PAD) is now
frequently used to separate and directly detect oligosaccharides
at alkaline pH using gradient methods. HPAEC offers high sep-
aration resolution of oligosaccharides and even oligosaccharide
isomers, coupled with very sensitive detection. However, one
stated problem of using PAD to detect oligosaccharides of in-
creasing DP (degree of polymerization) is that the mass sensi-
tivity of PAD decreases with the increase of DP (3). High-performance capillary electrophoresis (HPCE) with laser-induced fluorescence (LIF) detection also provides high
separation resolution of oligosaccharides, but a precolumn
derivitization is required to produce spectroscopically active
compounds. HPCE can also be coupled with PAD to separate
oligosaccharide and alditol mixtures. McPherson and Jane (1999), using HPAEC–PAD on en- zyme digested starches, were able to detect maltooligosaccharides up to DP 85 (4). In comparison, in a re-
cent comparative study of oligosaccharides by Kuhn et al.
(1999; 5) of capillary electrophoresis, matrix-assisted laser
desorption ionization–time of flight mass spectroscopy
(MALDI–TOF MS) and HPAEC–PAD, dextran oligosaccha-
rides up to 45 DP were detected by HPCE and HPAEC–PAD,
whereas MALDI–TOF MS allowed detection from DP 4 to
DP 60. HPAEC–PAD was observed to be the most sensitive
technique, but the separation resolution performance was
better in HPCE and MALDI–TOF MS. Drawbacks of
MALDI–TOF MS are that quantitation is usually limited by
poor reproducibility, and it is a destructive technique; there-
fore, preparative work cannot be undertaken. Fluorophore assisted carbohydrate electrophoresis (FACE) technology is also being currently used to separate
and detect oligosaccharides, particularly from glycoconjugates. Analysis involves 4 steps: release, labeling 140 G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 with a fluorescent tag, separation using precast polyacrylamide gels, and imaging. Although FACE technol-
ogy is simple and reliable, quantitative accuracy is limited and
also limited by available standards. Another technique also being currently used to separate and detect oligosaccharides includes the automated use of
modern planar chromatography. Furthermore, despite the im-
provement of LC techniques, gas chromatography (GC) still
continues to have a place in oligosaccharide analysis, particu-
larly for structural studies, although prederivatization is re-
quired. Nuclear magnetic resonance (NMR) is also still seen
as a powerful technique to elucidate the structure of oligosac-
charides. Selected Study Director Topics Visual Appearance of Sugar and Sugar Products Study Director Mary An Godshall, Sugar Processing Re- search Institute, Inc., 1100 Robert E. Lee Blvd, New Orleans,
LA 70124, E-mail: godshall@srrc.ars.usda.gov. SD recom-
mends that 954.10, Color of Raw Cane Sugars, be repealed
[JAOAC 37, 292(1954)]. The method is not scientifically
valid, according to accepted modern methods of color mea-
surement of raw cane sugar, studied and reported for many
years by the International Commission for Uniform Methods
of Sugar Analysis (ICUMSA). Modern methods of raw sugar
solution color analysis are standardized at pH 7.0, 0.45 µ membrane filtration, and read at 420 nm. This method, which
does not adjust pH, reads at 560 nm, and uses filter aid filtra-
tion, is based on obsolete methods used before the days of
standardized color measurement. Color measurement at
560 nm gives much lower values than at 420 nm, and in a col-
laborative test showed poor precision (6). Other methods for the measurement of the color of raw cane sugar are available. ICUMSA Method GS1/3-7 (The De-
termination of Raw Sugar Solution Color at pH 7.0 and the
Determination of Solution Colors of Partly Refined and
Brown Sugars and Colored Syrups at pH 7.0) underwent a col-
laborative test using IUPAC protocols in 1990 with satisfac-
tory results for raw sugar (6). Subsequently, a collaborative
test on colored specialty sugars was performed, again with sat-
isfactory results (7). A second method was developed and tested in 1998, using a pH 7.0 buffer (3-(N-morpholino) propanesulphonic acid)
(MOPS). The collaborative study was satisfactory and the
method was accepted as ICUMSA Method GS1-8, The Deter-
mination of Raw Sugar Solution Color at pH 7.0 by the MOPS
Method (8). The raw data and statistics for both methods were pub- lished in the referenced proceedings and can be made avail-
able to the AOAC Statistics Committee. It is recommended
that these methods be adopted as ICUMSA–AOAC methods,
after the appropriate documentation is provided. The individ-
uals who conducted the various tests referenced above are
agreeable to having the work presented to AOAC. Chromatographic Methods for Sugar and Sugar Products Study Director Kevin Schaffler, Sugar Milling Research Institute, University of Ntal, King George V Ave, Durban
4001, South Africa, Tel: +27-31-261-6882, Fax: +27-31-261-6886, E-mail: kschaffler@smri.org. SD recom-
mends that 996.04, “Sugars in Cane and Beet Final Molasses,
Ion Chromatographic Method,” (cross referenced to ICUMSA Official Method No. GS7/8/4-24), progress to Final
Action. This method received First Action in 1996. Because
of changes in General Referees during the 1998 period, it is
possible this was overlooked. In a survey conducted by
Schaffler on the use of this method, it was noted that it is in
routine use in several sugar industry laboratories (9). Schaffler also recommends that 2000.17, “Determination of Trace Glucose and Fructose in Raw Cane Sugar, High-Per-
formance Anion-Exchange Chromatography,” (cross refer-
enced to ICUMSA Official Method No. GS1/2/3-4) receive
Final Action. In a survey conducted by Schaffler on the use of
this method, it was noted that the method is in routine use in
several sugar industry laboratories (10). Schaffler further informs that 2000.17 was also tested for glucose and fructose in white sugar, but the Statistics Commit-
tee felt the HORRAT for white sugar were too high (average
2.8). For this reason, the method was accepted only for raw
sugar. The method was published in J. AOAC Int. (Janu-
ary/February 2002), in which the First Action recommenda-
tion includes the analysis of refined beet sugar and refined
cane sugar, along with raw cane sugar (9). Schaffler notes that
other AOAC methods have been accepted with higher
HORRAT, illustrated by an AOAC collaborative study of an
LC method for food products, in which it was decided that the
HORRAT was exceeded due to the use of different instru-
ments and columns (11). Formaldehyde in Maple Syrup by a Spectrofluorimetric
Method Study Director Nathalie Martin, Centre ACER, 3600 Boul. Casavant Ouest, Saint-Hyacinthe, Quebec, Canada J2S 8E3,
Tel: 450-773-1105, Fax: 450-773-8461, E-mail: nathaliemartin@centreacer.qc.ca. SD reports that a manu-
script, Spectrofluorimetric Determination of Formaldehyde in
Maple Syrup by L. Lagacé, J. Dumont, G. Brazeau, A. Soucy,
J. Haché, and V. Marquis, has been submitted to J. AOAC Int.
It is proposed that this method may replace 964.21, “Formal-
dehyde in Maple Syrup, Spectrophotometric Method.” As justification, Martin offers the following: To control microbial growth in the tap holes of maple trees,
paraformaldehyde was used with a tolerance for residue of
2 mg/kg in maple syrup set by Health Canada and the U.S.
Food and Drug Administration. Since long term damage to
maple trees is associated with this practice, paraformaldehyde
is no longer recommended for maple syrup production. Regis-
tration of the pesticide was not renewed in 1990 and the toler-
ance of 2 mg/kg residue was revoked in 1999 by the U.S. En-
vironmental Protection Agency. This new regulation status of
no tolerance of paraformaldehyde residue in maple syrup has G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 141 revived interest in formaldehyde determination since a certain
amount of formaldehyde can be naturally present in maple
syrup without using paraformaldehyde in the tap holes. When
the use of paraformaldehyde was introduced, a method for
formaldehyde determination in maple syrup was adopted as
AOAC Method 964.21, Formaldehyde in maple syrup, spec-
trophotometric method. However, this method was later de-
scribed as tedious and found to have a low recovery and a
great variability. As yet, no method has been adopted as an of-
ficial method to replace the original one. The method we are
proposing (Spectrofluorimetric determination of formalde-
hyde in maple syrup) is a quick and simple method for the de-
termination of formaldehyde in maple syrup with suitable per-
formance. In this method, formaldehyde reacts with Fluoral P
to form a complex which is chemically extracted from maple
syrup by isobutanol and determined by spectrofluorimetry.
Performance, as gauged by the limits of detection
(0.16 mg/kg) and quantitation (0.21 mg/kg) as well as recov-
ery (over 79%) and variability (1.9 to 16.1%, depending on
fortification level and class of syrup) were superior to the cur-
rent official AOAC standard method. A limited collaborative
test with 3 laboratories showed good performance. We recom-
mend that the new method undergo a collaborative study pro-
cess (AOAC Official Methods Program) in order to replace
the old method. Sugar Alcohols Study Director Jeff Rohrer, Dionex Corp., 500 Mercury Dr, Sunnyvale, CA 94088, E-mail: Jeff.Rohrer@dionex.com.
SD served as the AOAC representative for the CEN/TC275/WG 2, a European committee reviewing meth-
ods for intense sweeteners and sugar alcohols. He reviewed
the proposed methods and the report and resolutions from that
meeting. He reports that, with his job responsibilities, he does
not foresee having the time to initiate and conduct collabora-
tive studies. In a February 2002 interim report, previous GR
Raffaelle Bernetti recommended that the topic of sugar alco-
hols be transferred to the General Referee, Food Additives,
Committee C. Stable Isotope Ratio Methods Study Director Réal Paquin, Laboratoire de Spectrométrie de Masse de Rapports Isotopiques, MAPAQ-DLEAA, 2700
Einstein, C2.105, Sainte-Foy (Québec) G1P 3W8, Canada,
Tel: 418-266-4440 #232, Fax: 418-266-4440, E-mail:
rpaquin@hertz.phy.ulaval.ca. SD reports that he submitted
corrections to Bernetti that were included in the latest edition
of the Official Methods of Analysis of AOAC INTERNA-
TIONAL, 17th Ed. At that time, he suggested rewriting those
methods concerning stable isotope ratio analysis, and he feels
that this still should be done. Paquin is presently writing a
manuscript for an internal standard method for maple syrup,
and he intends to submit it as an official method. He also re-
ports that he has done much work on new methods of combus-
tion for the analysis of carbon isotope ratio analysis and is now
ready to begin publishing the results of this research. It would be of interest to include these newest methods of sample prep-
aration in official methods. Paquin states that minor errors still exist in the latest edition of OMA: 984.23, “Corn Syrup and Cane Sugar in Maple
Syrup, Carbon Ratio Mass Spectrometric Method” and
998.12 , “C4 Plant Sugars in Honey, Internal Standard Stable Carbon Isotope Ratio Method.” He will send the modifica-
tions at a later date. With this in mind, 998.12, which received
First Action in 1998, will not be recommended for Final Ac-
tion until the necessary modifications are made. Recommendations (1) The topic of sugar alcohols be transferred to the Gen- eral Referee, Food Additives. (2) 996.04 be progressed to Final Action (Sugars in Cane and Beet Final Molasses, Ion Chromatographic Method). In a
recent survey conducted by Schaffler on the use of this
method, it was noted that it is in routine use in several sugar in-
dustry laboratories (9). (3) 2000.17 be progressed to Final Action (Determination of Trace Glucose and Fructose in Raw Cane Sugar, High-Per-
formance Anion-Exchange Chromatography). In a survey re-
cently conducted by Schaffler on the use of this method, it was
noted that the method is in routine use in several sugar indus-
try laboratories (9). (4) Repeal 954.10 (Color of Raw Cane Sugars). The method is no longer scientifically valid, based on modern
methods of color measurement of raw cane sugar. This
method, which does not adjust pH, reads at 560 nm, and uses
filter aid filtration, is based on obsolete methods used before
the days of standardized sugar solution color measurement. (5) Pursue alternative, modern methods of raw sugar solu- tion color measurement in cooperation with ICUMSA. Ap-
point a Study Director for this topic. (6) Appoint a Study Director to review AOAC polarimetric methods that use lead clarification, and make ef-
forts to update to methods that do not use lead salts as clarify-
ing agents. (7) Conduct a collaborative study on the new spectrofluorimetric method for the determination of formalde-
hyde in maple syrup. (8) The SNIF–NMR method for beet or cane sugar in ma- ple syrup (2000.19), which received First Action in 2000, be
progressed to Final Action. References (1) Eggleston, G. (2002) G. Food Chem. 78, 99–107 (2) Eggleston, G., & Cote, G.L. (Eds) (2002) in Oligosaccha- rides in Food and Agriculture, ACS, Washington DC (in
press) (3) Larew, L.A., & Johnson, D.C. (1988) Anal. Chem. 60, 1867–1872 (4) McPherson, A.E., & Jane, J. (1999) Carbohydr. Polym. 40, 57–70 142 G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 (5) Kuhn, R., Weide, F., & Schollaert, W. (1999) BIOforum 1–2, 29–32 (6) McCowage, R.J. (1990) General Subject 1, Raw Sugar, ICUMSA Proc. 20th Session, pp 1–27 (7) Burge, M.L. (2002) General Subject 3, Specialty Sugars, Re- ports of 23rd Session of ICUMSA, pp 28–50 (8) Urquhart, R.M. (1998) General Subject 1, Raw Sugar, ICUMSA Proc. 22nd Session, pp 90–107 (9) Schaffler, K. (2002) Subject 8, Chromatographic techniques for sugars, Reports of 23rd Session of ICUMSA, pp 163–179 (10) Schaffler, K. (2002) J. AOAC Int. 85, 95–106 (11) Bugner, E., & Feinburg, M. (1992) J. AOAC Int. 75, 443–464 Nonvitamin Micronutrients H ARVEY I NDYK NZMP, No. 1 Main Rd, Waitoa, New Zealand (shipping
address: PO Box 7, Waitoa, New Zealand), Tel: +64 7889
3989, Fax: +64 7887 1502, E-mail: harvey.indyk@nzmp.com
(work), indyk@clear.net.nz (home) Summary Nonvitamin micronutrients encompass innumerable nutri- tionally active substances present in foods at low levels that
may have beneficial and protective properties. These include
nonvitamin carotenoids, essential fatty acids, amino acids,
phospholipid components (including choline, inositol), and any
compounds with antioxidant or antimicrobial potential. Other
conditionally essential, or pseudo-vitamin dietary components
also are attracting increasing attention such as the important
lipid carrier L -carnitine. These analytes are currently under-rep- resented in the Official Methods of Analysis (OMA) and it is
hoped that activities in these areas will increase. In the case of
amino acids, it is disappointing that there has been no replace-
ment of 960.47 (Amino Acids in Vitamin Preparations, Micro-
biological Assay) with a chromatographic procedure. A new method for isoflavones in soy-based foods (2001.10) was approved during 2001 for First Action by the Food Nutrition
Committee E and Official Methods Board. These phytoestrogenic flavonoids are naturally occurring in a variety of
plants with high levels in soybeans and have been connected with
inhibition of certain cancers, reduction in menopausal symptoms
and improvements to bone density. At least 15 isoflavones are
found in food, usually as glycosides, while aglycones are found
in fermented soy products. The new method is based on hydroly-
sis of glycoside esters and determination as isoflavone glycosides
and the corresponding aglucones. Methods for many of the botanical extracts have been transferred to the new Committee K which specializes in such
validations. One method, for ginsenosides in panax species,
has remained with Committee E. The method is based upon
LC determination of extracted analytes (R g1 , R e , R f , R g2 , R b1 , R c , R b2 , and R d ) using UV detection. The ginsenosides have been collectively well-researched but are also believed to have
some individual nutritional properties. Standardized extracts
need a reliable method of analysis to substantiate label claims, so a validated method is currently under review for First Ac-
tion status. Nucleotides play important roles in major biochemical functions and recent evidence suggests that dietary nucleo-
tides are semi-essential for newborns. The nucleotides and nu-
cleosides are present in human milk at relatively high levels so
bovine milk-based infant formulas are increasingly supple-
mented with the 5 ′ monophosphate nucleotides (CMP, UMP, AMP, GMP, and IMP). A complicating problem for such
products is the potential conversion of nucleotides to nucleo-
sides during production. As far as analytical methodology is
concerned, the 2 main approaches would seem to be either si-
multaneous determination of both nucleotides and nucleo-
sides, or conversion of nucleotides to nucleosides during sam-
ple preparation and target the latter as the sum of nucleotide
and nucleosides. A protocol LC method for determining total
nucleoside content in milk and infant formulas is currently be-
ing drafted. Study Director Bruce Molitor has contributed the
following summary of this approach: The addition of nucleotides to nutritional formulas and the quantitative confirmation of fortified amounts can be a com-
plex determination. Most proteins contain inherent amounts
of nucleotides and/or nucleosides. Alkaline phosphatase pres-
ent in the milk proteins can reduce nucleotides to nucleosides
during the manufacturing process. Adenosine deaminase in
the protein can convert adenosine to inosine. Hydrolyzed pro-
teins can contain large amounts of nucleic acid components as
background amounts. This method can evaluate total nucleo-
tides and total nucleosides, and track conversion of adenosine
to inosine. The first step involves enzymatic hydrolysis to
their corresponding nucleosides of adenosine, cytidine,
guanosine, inosine, and uridine. Samples are then covalently
bonded to a boronic acid gel, which virtually eliminates all
nonsugar, non-nucleotide, and non-nucleoside components
prior to LC quantification. This LC quantification method has
already been extensive used to evaluate American, Asian, and
European breast milk; infant and adult nutritionals; soy for-
mulas, cow’s milk, and hydrolyzed proteins; various process-
ing stages during manufacturing; and multiple international
infant nutritionals. Since no current AOAC method exists for
quantification of nucleotides, a collaborative international
AOAC of this method will be used to verify this as a universal
method that is accurate, precise, rugged, and appropriate for
routine use. The antioxidant potential of foods and dietary supplements is an important nutritional property to gauge the potential ben-
efit against oxidative damage and age-related diseases. Rather
than attempting to sum the antioxidant properties of all com-
pounds, an assessment is better achieved using one of various
indicating reagents. A common reagent for free radical detec-
tion is 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulphonate
(ABTS), a well-known substrate for enzymatic peroxide tests.
The reaction product is a green colored radical cation. Another
is the stable free radical 2,2-diphenyl-1-picrylhydrazyl
(DHPH). A collaborative study for total antioxidant capacity
of foods using DHPH is currently at the protocol submission G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 143 stage. The following has been submitted by Topic Advisor
Aruna Prakash: Antioxidants such as phenolic compounds, flavonoids, ca- rotenoids, vitamin C, vitamin E, and phytoestrogens are pres-
ent in fruits, vegetables, and whole grains. Scientific evidence
suggests that antioxidants in food scavenge free radicals and
inhibit oxidative reactions, thereby reducing risk for chronic
diseases. The qualitative analysis of foods to determine its free
radical scavenging capacity is necessary in order to find its ef-
ficacy and nutritional value and also help us understand the
functional properties of food. Antioxidant capacity and anti-
oxidant activity are used interchangeably. The free radical
scavenging activity of antioxidants in foods have been sub-
stantially investigated using various methods and reported in
the literature. The antioxidant activity of food is determined
by reacting the sample with the stable DPPH radical in 50%
aqueous methanol for 4 h at 35 ° C. DPPH radical is purple in color and has an intense absorption at 517 nm in 50% aqueous
methanol solution. On reaction with an antioxidant, the purple
color of the DPPH solution changes to pale yellow. This
change in absorbance is related to the antioxidant activity. A
common reference standard, (S)-(-)-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox), a water solu-
ble analogue of vitamin E is used. Antioxidant activity of
foods is expressed as Trolox equivalents (TE)/100 g sam-
ple. Antioxidant activity for a wide variety of food samples
can be conveniently determined using the rapid, simple DPPH
method. Colostral immunoglobulins, specifically IgG, confer passive immunity to the neonate until the immune system is developed.
There has been an increase in the global availability of
colostrum-based functional foods and supplements, which are
claimed to improve gastrointestinal health and stimulate the im-
mune system. In the absence of a reference analytical method, it
is becoming increasingly important to standardize analysis
techniques for IgG in such materials. While commercial RID
kits are available, they are generally quite variable in response.
An affinity LC method based on specific binding of bovine IgG
with immobilized Protein G has been validated within the labo-
ratory of the prospective Study Directors, Don Otter and Colin
Hughes, and is currently at the protocol development stage.
Biosensor technology may provide an alternative approach to
IgG analysis and a method is currently under development
based on the interaction between an anti-bovine-IgG ligand and
analyte utilizing an optical transducer. The General Referee suggests 3 nonvitamin micronutri- ents, carnitine, inositol, and conjugated linoleic acid (CLA)
need to be strategically targeted for collaborative study since
they are not covered in the Official Methods of Analysis by any
technique. Carnitine is available through limited de novo syn-
thesis, although deficiency is recognized, particularly in in-
fants. Thus, infant formulas are commonly supplemented with
carnitine and reliable analytical techniques are needed. A pub-
lished enzymatic methodology [Food Chem. 66 , 121–127(1999)] will hopefully be subjected to collaborative
study in the near future. Similarly, inositol is considered a con- ditionally essential pseudo-vitamin and although microbio-
logical or gas chromatographic techniques are generally used,
neither approach has been subjected to the highest level of val-
idation. However, a Nestlé sponsored interlaboratory trial is
currently evaluating a GLC-based assay for inositol. Accept-
able performance may result in submission of this technique
to the official methods program. Conjugated linoleic acid has
attracted much attention recently due to the reportedly benefi-
cial biological properties of some of its 56 geometrical and po-
sitional isomers [Roach et al. (2002) Anal Chim. Acta. 465,
207–226]. A gas chromatographic approach will undoubtedly
be sought to provide a validated methodology for CLA sup-
plements and dairy products, in which the principal active iso-
mer is thought to be the 9<i>c,11<i>t-18:2 and 10<i>c,12<i>t-18:2 forms. Where other organizations involved with method valida- tion are active in these analyte areas, it may be timely to con-
sider AOAC policy regarding joint adoption in order to avoid
duplication of scarce scientific resources. Major difficulties
may occur when validation protocols do not entirely meet the
AOAC INTERNATIONAL, ISO 5725, and IUPAC harmonized protocols. Whether such studies need either to be
repeated in full using OMA protocol, or perhaps preferably,
publish the method within OMA at a lower level of validation
requires clarification at OMB level. Selected Study Director Topics Determination of Ginsenosides (ginseng saponins) in Dry
Root Powder from Panax Ginseng, Panax Quinquefolius
and Selected Commercial Products by LC Method Study Directors Ebeneezer Asafu-Adjaye, FDA, Rockville, MD 20857, and Siu Kay Wong, Hong Kong Gov-
ernment Laboratory, Hong Kong. Study completed and wait-
ing First Action approval by Methods Committee and Official
Methods Board. The study may be in violation of the Harmo-
nized Protocol for Collaborative Studies. 2001.10 Determination of Isoflavones in Soy and Foods
Containing Soy by Extraction, Saponification, and LC Study Directors Stephen P. Klump and John L. MacDonald, Ralston Analytical Laboratories, Saint Louis,
MO, E-mail: sklump@ralston.com. Study completed and
method approved First Action. Nucleotides and Nucleosides in Infant Formulas and Milk Study Director Bruce Molitor, Ross Abbott Laboratories, Columbus, OH, E-mail: Bruce.Molitor@RossNutrition.com.
New method; study protocol in process. Nucleotides in Infant Formulas Study Director to be appointed. Antioxidant Activity in Foods New Study Director to be appointed. 144 G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 Determination of IgG Levels in Milk and Colostrum Study Directors Donald Otter and Colin Hughes, New Zea- land Dairy Research Institute, Palmerston North, New Zea-
land, E-mail: don.otter@nzdri.org.nz and colin.Hughes@nzdri.org.nz. New method; study protocol in
progress. Recommendations (1) Determination of Ginsenosides (ginseng saponins) in Dry Root Powder from Panax Ginseng, Panax Quinquefolius
and Selected Commercial Products, LC Method: Move to
First Action and continue study. (2) 2001.10, Determination of Isoflavones in Soy and Foods Containing Soy by Extraction, Saponification, and LC:
First Action approved by the OMB. (3) Nucleotides in Infant Formulas and Milk: Study Direc- tor Bruce Molitor now appointed. Prepare the method proto-
col for AOAC approval at the earliest opportunity. (4) Nucleotides in Infant Formulas: SD (Christine Rose Sallin, Nestle, Lausanne, Switzerland) to be appointed. (5) Antioxidant Activity in Foods: Appoint new Study Di- rector and prepare the method protocol for AOAC approval at
the earliest opportunity. (6) Determination of IgG Levels in Milk and Colostrum: Study Directors Donald Otter and Colin Hughes to be ap-
pointed. Prepare the method protocol for AOAC approval at
the earliest opportunity. Fat-Soluble Vitamins C HRISTOPHER J. B LAKE Nestlé Research Center, Quality and Safety Assurance
Department, Vers Chez-les-Blanc, Lausanne 1000,
Switzerland, Tel: 0041 21 785 8348, E-mail:
christopher-john.blake@rdls.nestle.com Summary Fat Soluble Vitamins: Review of Recent Analytical
Developments The analysis of the individual fat-soluble vitamins A, E, D, and K 1 by LC are covered by the AOAC Official Methods in Chapters 45 (vitamins and other nutrients) and 50 (infant for-
mula, baby foods, and enteral products). These methods are
rather product-specific and often dated. Modern methods gen-
erally focus on LC separations which allow multi-analyte de-
terminations and detection of individual isomers. This ad-
vance in methodology has a greater opportunity to address
biopotency differences between various vitamers. Recent ‘horizontal’ versions of these methods, applicable to all of the 9 sectors of the food matrix, are under study. One
such LC method has been evaluated for vitamins A and E (1)
and given First Action approval (2001.13) for vitamin A, but
vitamin E requires a second collaborative study. A joint NMKL–AOAC method for vitamin D has recently been ap-
proved as Official Method 2002.02. An LC method for β -carotene has been collaboratively studied for a variety of foods and the statistical analysis of the results has been made.
A study report should be available in the near future. The Committee of European Normalization (CEN) has re- leased LC methods for vitamins A, D, and E and β -carotene similar to the AOAC procedures. However, their validation
was not always up to internationally accepted protocols and
covered only selected foods. CEN (2–5), ISO (6), and
ISO/IDF (7) issued LC methods specifically for vitamins A
and D in skimmed milk powder. A product-specific method
for vitamin A in supplemented liquid milks is being proposed
for adoption as a First Action method (F-26) which avoids the
use of saponification . International standard methods are currently lacking for vi- tamin K 3 (used mostly in pet foods) and for some pro-vitamin carotenes like β -crytoxanthin. Methods for nonvitamin carot- enoids with nutritional significance, such as lycopene, are also
in need of further validation. In addition a method for the sepa-
ration of cis- and trans-phylloquinone (K 1 ) using a C30 re- versed-phase column has been reported (8) which has poten-
tial regulatory use. The natural biologically active form of
vitamin K 1 (phylloquinone) is the trans-isomer. The inactive cis-isomer is found in synthesized vitamin K 1 . There is a need for reliable data on vitamin K content because of recent
changes in U.S. legislation on nutritional labeling. Rodrigo et
al. (9) described the analysis of the various tocopherols using
normal-phase LC in infant formulas to assess total vitamin E
although such separations are now possible using C30 re-
versed-phase columns (10) A number of new developments to speed up sample prepa- ration have been reported. An on-line supercritical fluid ex-
traction (SFE) procedure/immobilized lipase hydrolysis was
proposed by Turner et al. (11) for the determination of vita-
mins A and E esters in dairy and meat products. This tech-
nique should also be applicable to other fat-soluble vitamins.
The method was collaboratively studied for vitamins E and A
and $-carotene (12) for a wide variety of food matrixes. It was reported that sample throughput was at least 12 per day, about
double that of the conventional LC methods. This promising
technique should be further examined. Another approach involves direct solvent extraction avoid- ing saponification (13, 14) which is applicable to vitamin E in
margarine and reduced fat products and for total vitamin E and
$-carotene in reduced-fat mayonnaise. A similar approach was
also reported for extraction of all-trans-retinyl palmitate, $-car- otene, and vitamin E in fortified foods (15). However it was re-
ported by Paixao and Stamford (16) that this technique gave
poor recoveries of vitamins A, D, and E from milk-based prod-
ucts and these authors obtained better recoveries using a classi-
cal overnight saponification followed by solvent extraction. Chase (17) described the use of accelerated solvent extrac- tion (ASE) to reduce extraction time. ASE was combined with
matrix solid-phase dispersion (MSPD) for the analysis of vita-
min K 1 in medical foods. Coupling the 2 techniques automates G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 145 the MSPD. The vitamin K 1 in the ASE was analyzed by re- versed-phase liquid chromatography. Since the extraction methods are similar for the vitamins A, D, and E, it would be advantageous to determine several vita-
mins in one chromatographic run. Eitenmiller and Landen (18) reviewed multi-analyte methods for FSV. A
novel approach was described by Gomis et al. (19) in which A,
D 2 , D 3 , E, K 1 , retinyl acetate, retinyl palmitate, tocopherol ac- etate, and provitamins D 2 and D 3 milk were separated simulta- neously by reversed-phase fused-silica microcolumn chroma-
tography with UV detection. Recoveries of each vitamin spike
were in the range 89–107%, however this method needs fur-
ther validation. Qian and Sheng (20) described the simulta-
neous determination of vitamins A, D, and E and pro-vitamin
D 2 in animal feeds by a one-step extraction and LC analysis. Some attempts have been made to automate sample prepara- tion and LC separation using robotics. Gamiz Gracia et al. (21)
described an automated analysis of vitamins A and E based on
AOAC procedures. Although not in routine or regulatory use for the fat-soluble vitamins (FSV), liquid chromatography–mass spectrometry
(LC–MS) and tandem LC–MS/MS are beginning to be
recognized as important tools for the future. The analytes are
not easily ionized so the harsher treatment of APCI (atmo-
spheric pressure chemical ionization) is currently preferred to
ESI (electrospray ionization). Several papers describe the
analysis of individual vitamins—tocopherols and caroten-
oids (22) and vitamin E (23). Conditions for vitamin K 1 in foods are also reported in supplier application notes
(Shimadzu Application 036). If a multi-analyte method can be
developed, significant productivity gains will be possible. A
difficulty is to find suitable internal standards for each vita-
min. A possible approach to analyze FSV could be to combine
the SFE extraction with SPE cleanup and LC–MS or tandem
LC–MS/MS analysis. Scheiber et al. (24) described the simultaneous determina- tion of carotenes and tocopherols in vitamin supplemented
juices and nectars by LC–UV. The method allowed the com-
plete separation of α -, $-, (-, and *-tocopherol, α -tocopherol acetate, all-trans α -carotene, all-trans $-carotene, and the 9- and 13-cis isomers of $-carotene in about 50 min. Majchrzak et al. (25) reported the determination of the carotenoid profile
and retinol content of baby foods. Capillary electrophoresis (CE) is as yet poorly exploited for analysis of fat soluble vitamins. Sanchez and Salvado (26) described the use of micellar and microemulsion electrokinetic chromatography for the analy-
sis of both water and fat-soluble vitamins. Very few applications of NIR/FTIR have been described for analysis of vitamins in food products. However, an inter-
esting application is for controlling FSV in vitamin premixes.
Shi et al. (27) described the quantitative determination of vita-
min E by NIR. Flow-injection analysis is particularly suitable where a large number of analyses are required for quality control or
monitoring purposes. An on-line microwave-assisted saponification of vitamin A to retinol and direct in-line spec- trophotometric/FIA determination was reported by Perez and
Haswell (28). References (1) DeVries, J.W., & Silvera, K.R. (2002) J. AOAC Int. 85, 424–434 (2) CEN (2000a) Foodstuffs. Determination of vitamin A by high performance liquid chromatography. II. Measurement of
beta-carotene, EN 12823-2 (3) CEN (2000b) Foodstuffs. Determination of vitamin A by high performance liquid chromatography. I. Measurement of
all-trans-retinol and 13-cis-retinol, EN 12823-1 (4) CEN (2000c) Foodstuffs. Determination of vitamin E by high performance liquid chromatography. Measurement of alpha-,
beta-, gamma-, and delta-tocopherols, EN 12822 (5) CEN (2000d) Determination of vitamin D by high perfor- mance liquid chromatography. Measurement of
cholecalciferol (D3) and ergocalciferol (D2), EN 12821 (6) ISO (2000) Dried skimmed milk. Determination of vitamin A content. Part 2. Method using high-performance liquid chro-
matography. ISO 12080-2:2000(E) (7) ISO/IDF (2002) Dried skimmed milk. Determination of vita- min D content using high-performance chromatography. ISO
14892:2002(E)/IDF 177:2002(E) (8) Woollard, D.C., Indyk, H.E., Fong, B.Y., & Cook, K.K. (2002) J. AOAC Int. 85, 682–691 (9) Rodrigo, N., Alegria, A., Barbera, R., & Farre, R. (2002) J. Chromatogr. A 947, 97–102 (10) Abidi, S. (2000) J. Chromatogr. A 881, 197–216 (11) Turner, C., King, J.W., & Mathiasson, L. (2001) J. Agric. Food Chem. 49, 553–558 (12) Mathiasson, L., Turner, C., Berg, H., Dahlberg, L., Theobald, A., Anklam, E., Ginn, R., Sharman, M., Ulberth, F., &
Gabernig, R. (2002) Food Addit. Contam. 19, 632–646 (13) Ye, L., Landen, W.O., & Eitenmiller, R.R. (1998) J. Liq. Chromatogr. A 21, 1227–1238 (14) Ye, L., Landen, W.O., & Eitenmiller, R.R. (2001) J. Food Sci. 66, 78–82 (15) Ye, L., Landen, W.O., & Eitenmiller, R.R. (2000) J. Agric. Food Chem. 48, 4003–4008 (16) Paixao, J.A., & Stamford, T.L.M. (2002) J. Liquid. Chromatogr. Rel. Technol. 25, 217–239 (17) Chase, G.W. (2000) J. AOAC Int. 83, 407–410 (18) Eitenmiller, R.R., & Landen, W.O. (1999) Multi-analyte methods for analysis of fat-soluble vitamins in Vitamin Anal-
ysis for the Health and Food Sciences, CRC Press, Boca
Raton, FL (19) Gomis, D.B., Fernández, M.P., & Guitiérrez-Alvarez, M.D. (2000) J. Chromatogr. A 891, 109–114 (20) Qian, H., & Sheng, M. (1998) J. Chromatogr. A 825, 127–133 (21) Gamiz Gracia, L., Velasco-Arjona, A., & Luque de Castro, M.D. (1999) Analyst 124, 801–804 (22) Rentel, C., Strohschein, S., Albert, K., & Bayer, E. (1998) Anal. Chem. 70, 4394–4400 (23) Stoeggl, W.M., Huck, C.W., Scherz, H., Popp, M., & Bonn, G.K. (2001) Chromatographia 54, 179–185 (24) Scheiber, A., Marx, M., & Carle, R. (2002) Food Chem. 76, 357–362 146 G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 (25) Majchrzak, D., Frank, U., & Elmadfa, I. (2000) Eur. Fd. Res. Technol. 210, 407–413 (26) Sanchez, J.M., & Salvado, V. (2002) J. Chromatogr. A 950, 241–247 (27) Shi, Y.H., Xu, G.M., Shen, X.Y., & Xu, Z.D. (2000) Fenxi-Huaxue 28, 587–589 (28) Perez, E.L., & Haswell, S.J. (1995) Anal. Proceedings 32, 85–89 Selected Study Director Topics 2001.13 Vitamins A and E in Foods by LC Study Director Jonathan W. de Vries. Vitamin A approved as First Action 2001.13. Vitamin E was not approved [J.
AOAC Int. 85, 424–434(2002)]. Collaborative study for vitamin E will be re-run using an international collaborative study protocol. Study Directors
Jonathan W. de Vries/Karlene Silvera. Carotene in Foods Study Director Lynn Hagemann. Continue with prepara- tion of study report. Submit to Food Nutrition Committee at
earliest opportunity. Determination of Cholecalciferol in Selected Foodstuff by
LC In the absence of a GR for FSVs until the current appoint- ment, Harvey Indyk (GR nonvitamin micronutrients) has
acted as GR for this study. The Study Director is Anders
Staffas (NMKL). The principle of the method is saponification of food product, extraction of vitamin D 3 and vitamin D 2 (internal standard) into n</i>-heptane. The fraction containing vitamin D 2 /D 3 is separated by preparative nor- mal-phase LC, and then determined quantitatively by re-
versed-phase LC with UV detection at 265 nm. This proce-
dure provides a more general horizontal technique, applicable
to a wider range of food products than current AOAC meth-
ods. This method was validated in a study by the Nordic Com-
mittee for Food Analysis using the Harmonized Protocol. It
therefore becomes a joint AOAC–NMKL method . The re- vised study report is now under review for recommendation to
First Action approval. Determination of Vitamin K 1 in Foods Using C30 Re- versed-Phase LC, Separation of cis- and trans-Phylloquinone Study Director Vacant. New topic. Determination of Vitamins K 3 in Human and Pet Foods Using Reversed-Phase LC Study Director Vacant. New topic. Recommendations (1) Vitamins A and E in Foods by LC: Collaborative study for vitamin E to be re-run using an international collaborative
study protocol. (2) Carotene in Foods: Continue with preparation of study report. Submit report to Food Nutrition Committee at earliest
opportunity. (3) Determination of Cholecalciferol in Selected Food- stuff by LC: The revised study (NMKL procedure) for
cholecalciferol should be recommended for acceptance as a
joint NMKL–AOAC method as First Action. (4) Determination of Vitamin K 1 in Foods Using C30 Re- versed-Phase LC: Separation of cis- and trans-phylloquinone.
New topic. (5) Determination of Vitamins K 3 in Human and Pet Foods Using Reversed-Phase LC: New topic. Water-Soluble Vitamins E RIK J.M. K ONINGS Inspectorate for Health Protection and Veterinary Public
Health, PO Box 2280, 5202 CG’s-Hertogenbosch, The
Netherlands, Tel: +31 40 2911500, Fax: +31 40 2911600,
E-mail: Erik.Konings@kvw.nl Summary In the 2001 General Referee Report, it was stated that prog- ress in establishing new vitamin methods for foods is rather
slow. This situation has not been changed. In general, micro-
biological assays (MBA) are the primary reference methods in
the Official Methods of Analysis, and should be changed for
chromatographic separations preferably or other modern tech-
niques. Apart from AOAC, the Committee of European
Normalization (CEN) is also in progress with the standardiza-
tion of methods of analysis for vitamins. However, there is
some question whether the validation procedures are compa-
rable. Because validation procedures are expensive and time
consuming, it should be encouraged to align validation activi-
ties around the world in order to obtain good analytical meth-
ods in an effective way. Dietary Supplements Vitamins in food supplements are a large and growing mar- ket that cannot be ignored. In nearly all European countries,
vitamins in food supplements fall under food law instead of,
e.g., pharmaceutical regulations. The importance of economi-
cal and safety aspects (e.g., vitamins A, D, and B 6 ) in food supplements is higher than for food. The degree of standard-
ization in methods of analysis for vitamins in food supple-
ments is less than for food (e.g., companies and commercial
laboratories use a large number of different methods leading
to a wide range of results). Considering these observations,
members of CEN Working Group 9 have decided to start ac- G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 147 tivities to standardize methods of analysis. Collaboration with
industry will be sought. In the Official Methods of Analysis, few methods are avail- able for the analysis of vitamins in food supplements (932.16,
936.14, 940.33, 944.12, 948.26, 970.65, 975.43, 989.09 ). Fur- thermore, the available methods are outdated: 932.16 (Vita-
min D 3 in Poultry Feed Supplements: Chick Bioassay); 975.43 (Identification of RRR- or all-rac-alpha-Tocopherol in Drugs and Food or Feed Supplements, Polarimetric Method). Vitamin B 6 Currently, the FDA is running a collaborative study on the determination of vitamin B 6 in infant formula based on the method of Bergaentzle et al. [Food Chem. (1999)]. However,
this method falls under the scope of the Infant Formula and
Medical Diets Committee. Within CEN, the group is working on 3 methods for vita- min B 6 (2 LC and one microbiological method). The microbi- ological method might be withdrawn in the future because it is
rarely used within European laboratories. In one LC method
(ENV 14164), after dephosphorylation, pyridoxamine is
transformed into pyridoxal, which is then reduced to
pyridoxine. Pyridoxine is then quantified fluorimetrically.
This method is based on the method published by Bergaentzle
et al. [Food Chem. (1999)]. β -Glycosylated forms are ex- cluded, while in the other method (doc. CEN/TC 275/WG 9 N
121rev) vitamin B 6 is the sum of the individual dephosphorylated vitamers pyridoxine, pyridoxal, and pyridoxamine calculated as pyridoxine including the β -glycosylated forms. This analysis is based on the work of Bognár et al. [Z. Lebensm. Unters. Forsch. (1985 and 1995)].
This procedure may be considered as an official method. Folic Acid So far, only microbiological analysis, which measures total folate, is accepted as reference method for determining the
level of this vitamin (992.05, Folic acid in infant formula;
CEN: EN 14131 Determination of folate by microbiological
assay). Apart from the urgent need to measure dietary folates
with a higher specificity, one would like to be able to measure
the ‘added’ folic acid in order to enforce the allowed additions
and the accompanying maximum intake level of 1 mg folic
acid per day. Within CEN, efforts are employed to set up a
collaborative study on folic acid (used for enrichment) and
5-methyltetrahydrofolic acid (the most abundant natural fo-
late form) based on an LC method [J. AOAC Int. 82,
119–127(1999)] and the Harmonized Protocol. Biotin Indyk and Woollard are interested in looking at a biotin method for foods using BIA techniques. However, there are
some technical issues. Collaboration with other scientists will
be sought. CEN is working on a draft for the determination of
biotin in foods. D-biotin and D-biocytin are extracted from
food after an enzymatic treatment and quantified by LC with
postcolumn derivatization. This procedure is based on the work of Lahély et al. [Food Chem. (1999)] and Arella et al.
[Ann. Fals. Exp. Chim. (2000)]. Vitamin B 12 Indyk et al. [J. AOAC Int. (2002)] have published a paper on the determination of vitamin B 12 in milk products and se- lected foods by optical biosensor protein-binding assay. The
proposed method was compared with reference microbiologi-
cal and radioisotope protein-binding methods for a range of
food samples. Other Vitamins In Europe, methods for the determination of vitamins B 1 (prEN 14122) and B 2 (prEN 14152) are about to be adopted as final drafts. However, these methods do not meet the AOAC
or ISO 5725 validation criteria. This is also the case for a
method to determine vitamin C (prEN 14130). Selected Study Director Topics Vitamin C in Foods, LC Method Study Director Allen Brause, Analytical Services of Co- lombia Inc., 9151 Rumsey Rd, Suite 190, Columbia, MD
21045, E-mail: atplabs@netscape.net. A collaborative study
was completed in 1995, which was extended to other food
products. This collaborative study will be repeated at a later
date after consultation with the International Fruit Juice Un-
ion. The completed method is not satisfactory for AOAC First
Action because of analyte instability but will be reported in J.
AOAC INTERNATIONAL. Additional foods will also be with-
drawn from study (as originally intended) because LC–UV
techniques are not suitable for low level detection. Determination of Niacin in Foods by Capillary Electropho-
resis and Liquid Chromatography: Acid and Alkaline Ex-
traction Study Director Norbert Strobel, Australian Government Analytical Laboratories, 51-65 Clarke St, S. Melbourne, Vic-
toria 3205, Australia. The original Study Director, Craige
Trenerry, has moved to another laboratory but it was expected
that this study would continue. However, there was no re-
sponse from the Study Director for 2 years. So this study will
be canceled. In France a collaborative study for the determina-
tion of niacin in foods will be finalized by the end of 2002.
With this method niacin vitamers are extracted from food with
an enzymatic treatment and quantified by LC with a
fluorimetric detection after a postcolumn derivatization with
UV irradiation. However, this collaborative study does not
follow the harmonized protocol. Determination of Calcium Pantothenate in Multivitamin
Premixes by LC Study Director Gerald A. Woollard, Auckland Hospital, Department of Clinical Biochemistry, Park Rd, Auckland,
New Zealand, E-mail: geraldw@adhb.govt.nz. The study
would be extended to include certain supplemented products. 148 G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 Further progress is dependent upon charges imposed by
AOAC for collaborative studies. Sponsorship of this study
may be required. Recommendations (1) Vitamin C in Foods, LC Method: Repeat collaborative study in fruit juices. Prepare the method protocol for AOAC
approval at the earliest opportunity. (2) Determination of Niacin in Foods by Capillary Elec- trophoresis and Liquid Chromatography: Acid and Alkaline
Extraction: Discontinue study. (3) Determination of Calcium Pantothenate in Vitamin Premixes and Tablets Using LC: Prepare the revised method
protocol for AOAC approval at the earliest opportunity. (4) Determination of Folic Acid and 5-Methyltetrahydrofolic Acid in Foods by LC: Appoint Erik
J.M. Konings as SD. Prepare the method protocol for AOAC
approval at the earliest opportunity. G ENERAL R EFEREE R EPORTS : J OURNAL OF AOAC I NTERNATIONAL V OL . 86, N O . 1, 2003 149