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L-Malic acid
UV-method
for the determination of L-malic in foodstuffs and other
materials
BOEHRINGER MANNHEIM / R-BIOPHARM
Enzymatic BioAnalysis / Food Analysis
For use in in vitro only Store at 2-8°C
Cat. No. 0 139 068
Test-Combination for approx. 25 determinations
Principle (Ref. 1)
L-Malic acid (L-malate) is oxidized to oxaloacetate by nicotinamide-adenine
dinucleotide (NAD) in the presence of L-malate dehydrogenase (L-MDH)
(1).
The equilibrium of this reaction lies on the side of L-malate. Removal of
oxaloacetate from the reaction system causes displacement of the equilibrium
in favour of oxaloacetate. In the reaction catalyzed by the enzyme
glutamate-oxaloacetate transaminase (GOT), oxaloacetate is converted to
L-aspartate in the presence of L-glutamate (2).
The amount of NADH formed is stoichiometric to the amount of L-malate.
The increase in NADH is measured by means of its light absorbance at 334,
340 or 365 nm.
The Test-Combination contains
1. Bottle 1 with approx. 30 ml solution, consisting of:
glycylglycine buffer, pH approx. 10.0; L-glutamic acid, 440 mg
2. Bottle 2 with approx. 210 mg NAD lyophilizate
3. Bottle 3 with approx. 0.4 ml glutamate-oxaloacetate transaminase
suspension, approx. 160 U
4. Bottle 4 with approx. 0.4 ml L-malate dehydrogenase solution, approx.
2400 U
5. L-Malic acid assay control solution for assay control purposes (measurement
of the assay control solution is not necessary for calculating the
results.) Use the assay control solution undiluted. (Expiry date: see pack
label)
Preparation of solutions
1. Use contents of bottles 1, 3 and 4 undiluted.
2. Dissolve contents of bottle 2 with 6 ml redist. water.
Stability of reagents
The contents of bottles 1, 2, 3 and 4 are stable at 2-8°C (see pack label).
Bring solution 1 to 20-25°C before use.
Solution 2 is stable for 3 weeks at 2-8°C and for 2 months at 15 to
25°C.
Procedure
Wavelength1: 340 nm, Hg 365 nm or Hg 334 nm
Glass cuvette2: 1.00 cm light path
Temperature: 20-25°C
Final volume: 2.220 ml
Read against air (without a cuvette in the light path) against water or against
blank3
Sample solution: 0.5-35 μg L-malic acid/assay4 (in 0.100-1.000 ml sample
volume)
* Rinse the enzyme pipette or the pipette tip of the piston pipette with sample solution before
dispensing the sample solution.
** For example, with a plastic spatula or by gentle swirling after closing the cuvette with
Parafilm (trademark of the American Can Company, Greenwich, Ct., USA)
(1) L-Malate + NAD+ L-MDH
oxaloacetate + NADH + H+
(2) Oxaloacetate + L-glutamate
GOT
L-aspartate + 2-oxoglutarate
Pipette into cuvettes Blank Sample
solution 1
solution 2
suspension 3
sample solution*
redist. water
1.000 ml
0.200 ml
0.010 ml
-
1.000 ml
1.000 ml
0.200 ml
0.010 ml
0.100 ml
0.900 ml
Mix**, read absorbances of the solutions (A1) after approx. 3 min (see also
instructions pt. 10). Start reaction by addition of:
solution 4 0.010 ml 0.010 ml
Mix**, after completion of the reaction (approx. 5-10 min) read absorbances of
the solutions immediately one after another (A2) (see pt. 7).
Determine the absorbance differences (A2-A1) for both, blank and sample.
Subtract the absorbance difference of the blank from the absorbance
difference of the sample.
A = (A2-A1)sample - (A2-A1)blank
The measured absorbance differences should, as a rule, be at least 0.100
absorbance units to achieve sufficiently precise results (see “Instructions for
performance of assay” and “Sensitivity and detection limit”, pt. 4).
Calculation
According to the general equation for calculating the concentration:
V = final volume [ml]
v = sample volume [ml]
MW = molecular weight of the substance to be assayed [g/mol]
d = light path [cm]
ε = extinction coefficient of NADH at:
340 nm = 6.3 [l × mmol-1 × cm-1]
Hg 365 nm = 3.4 [l × mmol-1 × cm-1]
Hg 334 nm = 6.18 [l × mmol-1 × cm-1]
It follows for L-malic acid:
If the sample has been diluted on preparation, the result must be multiplied
by the dilution factor F.
When analyzing solid and semi-solid samples which are weighed out for
sample preparation, the result is to be calculated from the amount weighed:
1. Instructions for performance of assay
The amount of L-malic acid present in the assay has to be between 1 g and
35 g (measurement at 365 nm) or 0.5 g and 20 g (measurement at 340,
334 nm), respectively. In order to get a sufficient absorbance difference, the
sample solution is diluted to yield a L-malic acid concentration between 0.08
and 0.35 g/l or 0.04 and 0.2 g/l, respectively.
Dilution table
If the measured absorbance difference (A) is too low (e.g. 0.100), the
sample solution should be prepared again (weigh out more sample or dilute
less strongly) or the sample volume to be pipetted into the cuvette can be
increased up to 1.000 ml. The volume of water added must then be reduced
to obtain the same final volume in the assays for sample and blank. The new
sample volume v must be taken into account in the calculation.
1 The absorption maximum of NADH is at 340 nm. On spectrophotometers, measurements
are taken at the absorption maximum; if spectralline photometers equipped with a mercury
vapor lamp are used, measurements are taken at a wavelength of 365 nm or 334 nm.
2 If desired, disposable cuvettes may be used instead of glass cuvettes.
3 For example, when using a double-beam photometer
4 See instructions for performance of assay
c =
V × MW
× A [g/l]
ε × d × v × 1000
c =
2.220 × 134.09
× A =
2.977 × A [g L-malic acid/l
ε × 1.00 × 0.100 × 1000 ε sample solution]
ContentL-malic acid =
cL-malic acid [g/l sample solution]
× 100 [g/100 g]
weightsample in g/l sample solution
Estimated amount of
L-malic acid per liter
measurement at
Dilution
with water
Dilution
factor F
340 or 334 nm 365 nm
0.02 g
0.2-2.0 g
2.0-20 g
> 20 g
0.35 g
0.35-3.5 g
3.5-35 g
> 35 g
-
1 + 9
1 + 99
1 + 999
1
10
100
1000
For recommendations for methods and standardized procedures see
references (2)
0101.1. 248 673rb bz
2
2. Technical information
2.1 In carrying out the calculation, a clear indication should be given as to
whether the results are to be given as L-malic acid (molar mass 134.09 g/mol)
or as L-malate (molar mass 132.07 g/mol). (In enzymatic determinations,
the L-malate ion is measured.)
2.2 In evaluating the analytical results, it should be taken into account that
in the acidimetric determination of “total acid calculated as L-malic
acid” protons are measured and in enzymatic determinations the
L-malate ions are measured. It is thus not possibile to compare such
results directly.
3. Specificity (Ref. 1)
The method is specific for L-malic acid. The D-isomer does not react. Also
L-lactic acid, D-Lactic acid, L-aspartic acid and fumaric acid are not converted.
L-Malic acid esters do not react (Ref. 3.3).
In the analysis of commercial L-malic acid, results of approx. 99 % have to
be expected.
4. Sensitivity and detection limit (Ref. 1.2)
The smallest differentiating absorbance for the procedure is 0.005 absorbance
units. This corresponds to a maximum sample volume v = 1.000 ml
and measurement at 340 of a L-malic concentration of 0.25 mg/l sample
solution (if v = 0.100 ml, this corresponds to 2.5mg/l sample solution).
The detection limit of 0.5 mg/l is derived from the absorbance difference of
0.010 (as measured at 340 nm) and a maximum sample volume v = 1.000 ml.
5. Linearity
Linearity of the determination exists from 0.5 g L-malic acid/assay (0.5 mg Lmalic
acid/l sample solution; sample volume v = 1.000 ml) to 35 g L-malic
acid/assay (0.35 g L-malic acid/l sample solution; sample volume v = 0.100 ml).
6. Precision
In a double determination using one sample solution, a difference of 0.005 to
0.010 absorbance units may occur. With a sample volume of v = 0.100 ml
and measurement at 340 nm, this corresponds to a L-malic acid concentration
of approx. 2-5 mg/l. (If the sample is diluted during sample preparation, the
result has to be multiplied by the dilution factor F. If the sample is weighed in
for sample preparation, e.g. using 1 g sample/100 ml = 10 g/l, a difference of
0.02-0.05 g/100 g can be expected.)
The following data have been published in the literature:
x = 2.97 g/l wine CV = 1.8 % n = 12 (Ref. 1.1, 1.2)
Fruit juice: r = 0.014 + 0.030 × x R = 0.032 + 0.070 × x
x = content of L-malic acid in g/l (Ref. 2.6)
Wine: r = 0.03 + 0.034 × xi R = 0.05 + 0.071 × xi
xi = content of L-malic acid in g/l (Ref. 2.10, 2.11)
7. Interference/sources of error
Traces of glutamate dehydrogenase (GIDH) in GOT result in reagent-dependent
creep reactions. An extrapolation of the measuring value with A2 is not
necessary if the absorbances of blank and sample are read immediately one
after another.
8. Recognizing interference during the assay procedure
8.1 If the conversion of L-malic acid has been completed according to the
time given under “Procedure” it can be concluded in general that no
interference has occurred.
8.2 On completion of the reaction, the determination can be restarted by
adding L-malic acid (qualitative or quantitative): if the absorbance is
altered subsequent to the addition of the standard material, this is also
an indication that no interference has occurred.
8.3 Operator error or interference of the determination through the
presence of substances contained in the sample can be recognized by
carrying out a double determination using two different sample
volumes (e.g. 0.100 ml and 0.200 ml): the measured differences in
absorbance should be proportional to the sample volumes used.
When analyzing solid samples, it is recommended that different quantities
(e.g. 1 g and 2 g) be weighed into 100 ml volumetric flasks. The
absorbance differences measured and the weights of sample used
should be proportional for identical sample volumes.
8.4 Possible interference caused by substances contained in the sample
can be recognized by using an internal standard as a control: in
addition to the sample, blank and standard determinations, a further
determination should be carried out with sample and assay control
solution in the same assay. The recovery can then be calculated from
the absorbance differences measured.
8.5 Possible losses during the determination can be recognized by carrying
out recovery tests: the sample should be prepared and analyzed with
and without added standard material. The additive should be recovered
quantitatively within the error range of the method.
9. Reagent hazard
The reagents used in the determination of L-malic acid are not hazardous
materials in the sense of the Hazardous Substances Regulations, the
Chemicals Law or EC Regulation 67/548/EEC and subsequent alteration,
supplementation and adaptation guidelines. However, the general safety
measures that apply to all chemical substances should be adhered to.
After use, the reagents can be disposed of with laboratory waste, but local
regulations must always be observed. Packaging material can be disposed
of in waste destined for recycling.
10. General information on sample preparation
In carrying out the assay:
Use clear, colorless or slightly colored and practically neutral liquid
samples directly, or after dilution according to the dilution table, and of a
volume up to 1.000 ml;
Filter turbid solutions;
Degas samples containing carbon dioxide (e.g. by filtration);
Adjust acid samples which are used undiluted for the assay, to pH 8-10 by
adding sodium or potassium hydroxide solution and incubate for approx. 30 min;
Measure “colored” samples (if necessary adjusted to pH 8-10) against a
sample blank (= buffer or redist. water + sample), adjust the photometer to
0.000 with the blank in the beam, especially, if there is a creep reaction
before the addition of solution 4 (L-MDH);
Treat “strongly colored” samples that are used undiluted or with a higher
sample volume with polyvinylpolypyrrolidone (PVPP; e.g. 1 g/100 ml);
Crush or homogenize solid or semi-solid samples, extract with water or
dissolve in water and filter if necessary.
11. Application examples
Determination of L-malic acid in wine
Free L-malic acid can be determined in white or red wine directly or after a
dilution acc. to the dilution table normally without prior decolorization.
Determination of free and esterified L-malic acid in wine (Ref. 3.3)
To determine the total L-malic acid content (the sum of free and esterified
L-malic acid), white wine or red wine, respectively, should be treated as
follows:
Heat 20 ml of wine and 6 ml sodium hydroxide (2 M) for 30 min under a
reflux condenser while stirring (do not use ammonia for alkaline hydrolysis
since an excessively high concentration of ammonium ions inhibits the
reaction!), allow to cool to 20-25°C, and neutralize with sulfuric acid (1 M)
(indicator paper).
Transfer quantitatively into a 50 ml volumetric flask and fill up to the mark
with water. Use the sample for the assay according to the standard procedure
(this results in total L-malic acid content = the sum of free and
esterified L-malic acid).
Determination of L-malic acid in fruit juice, concentrates and in
beverages
Use clear, liquid, almost neutral samples directly or after dilution with water
(concentration of L-malic acid approx. 0.04-0.35 g/l) for the assay.
Filter turbid juices and dilute to obtain an L-malic acid concentration of
approx. 0.04 to 0.35 g/l. The diluted solution can be used for the assay even
if it is slightly colored.
only intensely colored juices require previous decolorization when they are
used undiluted for the assay. In such cases, proceed as follows:
Mix 10 ml of juice and approx. 0.1 g of polyamide powder or polyvinylpolpyrrolidone
(PVPP), stir for 1 min, and filter. Use the clear, slightly colored
solution for the assay.
When using colored juices or red wine as sample material which is used
undiluted for the assay, occasionally it may occur that the absorbance A1 is not
constant because of the change in pH value (sample is acidic; assay mixture
shows pH 10). In this case it is recommended to adjust the sample to pH 10
before assaying and to incubate for approx. 30 min.
Determination of L-malic acid in beer
To remove the carbonic acid, stir
using a glass rod or filter; dilute the largely CO2-free sample according to
the dilution table. Alternatively, alkalize the beer sample by the addition of
solid sodium or potassium hydroxide.
Determination of L-malic acid in solid foodstuffs
Homogenize solid or semi-solid samples (e.g. fruit and vegetable products)
with a mortar, meat grinder, or homogenizer. Weigh out a well mixed sample
and extract with water - heated to 60°C, if necessary. Transfer quantitatively
into a volumetric flask and fill up to the mark with water. Filter and use the
clear solution for the assay. Dilute solution depending on the L-malic acid
content, if necessary (see dilution table)
12. Further applications
The method may also be used in research when analyzing biological
samples.
For details of sampling, treatment and stability of the sample see Gutmann, I.
& Wahlefeld, A. W. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.
U., ed.) 2nd ed., vol. 3, pp. 1586-1587, Verlag Chemie, Weinheim/Academic
Press, Inc., New York and London, as well as Ref. 1.2, pp. 43-44
The method may also be used in the examination of cosmetics and
pharmaceuticals , e.g. infusion solutions.
References
1.1 Möllering, H. (1974) in Methoden der enzymatischen Analyse (Bergmeyer, H. U., Hrsg.)
3. Aufl., Bd. 2, S. 1636-1639; Verlag Chemie, Weinheim and (1974) in Methods of
Enzymatic Analysis (Bergmeyer, H. U., ed.) 2nd ed., vol. 3, pp. 1589-1593, Verlag
Chemie, Weinheim/Academic Press, Inc., New York and London
1.2 Möllering, H. (1985) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.) 3rd. ed.,
vol. VII, pp. 39-47, Verlag Chemie, Weinheim, Deerfield Beach/Florida, basel
2.1 International Federation of Fruit Juice Producers (IFU, Methods of Analysis, no. 21-
1985) contained in "Code of Practice for evaluation of Fruit and Vegetable Juices"
(1996) edited by Association of the Industry of Juices and Nectars from Fruits and Vegetables
of the European Economic Community (A.I.J.N.)
2.2 Norme Française Homologuée NF V 76-104 (1980) Jus de Fruits et Jus des
Légumes: Détermination de la Teneur en Acides Carboxyliques (L-malique, tartrique,
citrique, et isocitrique)
2.3 Schweizerisches Lebensmittelbuch, Kapitel 61B (Enzymatische Bestimmungen)/3.4.
(1981); Kapitel 28A (Frucht- und Gemüsesäfte u.a.)/7.5 (1988), Kapitel 30A (Wein aus
Trauben)/6.4 (1993)
2.4 Gombocz, E., Hellwig, E., Vojir, F. & Petuely, F. (1981) Deutsche Lebensmittel-Rundschau
77, 7
2.5 Brautechnische Analysenmethoden, Band III, S. 556-559 (1982), Methodensammlung
der Mitteleuropäischen Brautechnischen Analysenkommission (MEBAK), herausgegeben
von F. Drawert im Selbstverlag der MEBAK, Freising
2.6 Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG; Untersuchung von
Lebensmitteln: Bestimmung von L-Äpfelsäure (L-Malat) in Fruchtsäften, 31.00-15
(November 1984); Enzymatische Bestimmung des Gehaltes an L-Äpfelsäure (L-Malat)
in Frucht- und Gemüsesäften, 31.00-15 (Januar 1997); Enzymatische Bestimmung des
Gehaltes an L-Äpfelsäure (L-Malat) in Gemüsesäften, L 26.26-13 (Januar 1997)
2.7 Ministero dell' Agricoltura e delle Foreste (1986) Approvazione dei "Metodi ufficiali di
analisi per i mosti, i vini, gli agri di vino (aceti) e i sottoprodotti della vinificazione".
Gazzetta Ufficiale della Repubblica Italiana, n. 161 del 14 luglio 1986
2.8 Nederlandse Norm NEN 2849 (1e druk, september 1987) Vruchtesappen: Bepaling van
het L-appelzuurgehalte; Enzymatische methode (Fruit juices - Determination of the Lmalic
acid content - Enzymatic method)
Concentration: see bottle label
L-Malic acid assay control solution is a stabilized aqueous solution of Lmalic
acid. It serves as assay control solution for the enzymatic determination
of L-malic acid in foodstuffs and other materials.
Application:
1. Addition of L-malic assay control solution to the assay mixture:
Instead of sample solution the assay control solution is used for the assay.
2. Restart of the reaction, quantitatively:
After completion of the reaction with sample solution and measuring of A2,
add 0.050 ml assay control solution to the assay mixture. Read absorbance
A3 after the end of the reaction (approx. 10 min). Calculate the concentration
from the difference (A3-A2) according to the general equation for
calculating the concentration. The altered total volume must be taken into
account. Because of the dilution of the assay mixture by addition of the
assay control solution, the result differs insignificantly from the data stated
on the bottle label.
Also available:
Test-Combination D-Malic acid, Cat. No. 1215 558
2.9 RSK-Values, The Complete Manual, Guide Values and Ranges of Specific Numbers for
Fruit Juices and Nectars, Including the Revised Methods of Analysis (1987), 1st ed.,
Verlag Flüssiges Obst/Liqiud Fruit, D-56370 Eschborn, pp. 114-117
2.10 Recueil des méthodes internationales d'analyse des vins et des moûts, Complément
n° 1 à l'édition officielle de juin 1990, OFFICE INTERNATIonAL DE LA VIGNE ET DU
VIN, pp. 195-197
2.11 Amtsblatt der Europäischen Gemeinschaften L 272 (3. Oktober 1990), Rechtsvorschriften:
Verordnung (EWG) Nr. 2676/90 der Kommission vom 17. September 1990 zur
Festlegung gemeinsamer Analysenmethoden für den Weinsektor (S. 103-105); Official
Journal of the European Communities L 272 (3 October 1990), Commission Regulation
(EEC) No 2676/90 of 17 September 1990 determining Community for the analysis of
wines (pp. 103-105)
2.12 AOAC Official Methods of Analysis (1990) 15th Ed., 5th Suppl. (1994), p. 274
2.13 Deutsche Norm DIN EN 1138 (Dez. 1994) Frucht- und Gemüsesäfte; Enzymatische
Bestimmung des Gehaltes an L-Äpfelsäure (L-Malat); Spektrophotometrische Bestimmung
von NADH (Fruit and vegetable juices; Enzymatic determination of L-malic acid
(L-malate) content; NADH spectrophotometric method)
2.14 European Standard EN 1138 (Dec. 1994) Fruit andvegetable juices; Enzymatic determination
of L-malic acid (L-malate) content by the NADH spectrometric method
2.15 Standard der Russischen Föderation / Standard of the Russian Federation / GOSSTANDART
ROSSII GOST R 51239-98 (1998) Fruit and vegetable juices. Determination of Lmalic
acid content
3.1 Mayer, K. & Pause, G. (1969) Äpfelsäure-, Milchsäure- und Zitronensäure-Gehalte in
Schweizer Weinen, Vitis 8, 38-49
3.2 Möhler, K. & Looser, S. (1969) Enzymatische Bestimmung von Säuren in Wein,
Zeitschrift für Lebensmittel-Untersuchung und -Forschung 140, 94-100
3.3 Olschimke, D., Niesner, W. & Junge, Ch. (1969) Bestimmung der Äpfelsäure in Weinen
und Traubensäften, Deutsche Lebensmittel-Rundschau 65, 383-384
3.4 Gorin, N. (1976) Differences in L-Malate Determined Enzymatically or Titrimetrically in
Golden Delicious Apples, Zeitschrift für Lebensmittel-Untersuchung und -Forschung
162, 259-261
3.5 Wallrauch, S. (1978) Äpfelsäurebestimmung in Fruchtsäften und Weinen, Die industrielle
Obst- und Gemüseverwertung 63, 488-492
3.6 Beurteilung des Saftgehaltes niedersafthaltiger Getränke (1979) Der Mineralbrunnen
29, 126-132
3.7 Klempp, J., Regula E. & Wassermann, L. (1982) Veränderung der Malat- und
Citratgehalte bei der Herstellung von Sauerteigbroten, Zeitschrift für Lebensmittel-
Untersuchung und -Forschung 175, 403-405 and (1984) 178, 187-191
3.8 Piendl, A. & Wagner, I. (1984) Physiologische Eigenschaften der organischen Säuren
des Biers; 4. L-Malat, Brauindustrie 69, 36-38 und 40
3.9 Klopper, W.J., Angelino, S.A.G.F., Tuning, B. & Vermeire, H.A. (1986) Organic acids and
glycerol in beer, J. Inst. Brew. 92, 225-228
3.10 Elkins, E. R. & Heuser, J. R. (1994) Detection of Adulteration in Apple Juice by L-Malic/
Total Malic Acid Ratio: Collaborative Study, J. Assoc. Off. Anal. Chem.Intern. 77, 411-415
3.11 Saalfeld, U. & Freund, W. (1999) Charakterisierung pulverisierter Sauerteige und Möglichkeiten
ihrer qualitativen Bestimmung im Brot - Teil 1: Säuregehalt und Abbauvermögen
für L-Malat und Citrat, Deutsche Lebensmittel-Rundschau 95, 209-219
3. Internal standard:
The assay control solution can be used as an internal standard in order to
check the determination for correct performance (gross errors) and to see
whether the sample solution is free from interfering substances:
:
The recovery of the standard is calculated according to the following
formula:
Pipette into
cuvettes
Blank Sample Standard Sample +
Standard
solution 1
solution 2
suspension 3
sample solution
assay control sln.
redist. water
1.000 ml
0.200 ml
0.010 ml
--
1.000 ml
1.000 ml
0.200 ml
0.010 ml
0.100 ml
-
0.900 ml
1.000 ml
0.200 ml
0.010 ml
-
0.100 ml
0.900 ml
1.000 ml
0.200 ml
0.010 ml
0.050 ml
0.050 ml
0.900 ml
Mix, and read absorbances of the solutions (A1) after approx. 5 min.
Continue as described in the pipetting scheme under "Procedure". Follow
the instructions given under "Instructions for performance of assay" and
the footnotes.
recovery =
2 × Asample + standard - Asample × 100 [%]
Astandard
L-Malic acid assay control solution
R-BIOPHARM GmbH
Dolivostraße 10
64293 Darmstadt/Germany
L-苹果酸检测试剂盒