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Gas chromatographic determination of isocitric and malic acid in the presence of a large excess of citric acid
165
Analytica Chimica Acta, 239 (1990) 165-170 Elsevier Science Publishers B.V., Amsterdam
Short Communication
Gas chromatographic determination of isocitric and malic acid in the presence of a large excess of citric acid I. Molnar-Per1 Institute of Inorganic and Analytical
*, M. Morvai
and M. Pint&-Szakacs
Chemistry, L. ELitubs University, P.O. Box 123, H-I443 Budapest (Hungary)
M. Petro-Turza Central Food Research Institute, Herman Ott6 lit 15, 1022 Budapest (Hungary) (Received
29th March
1990)
Abstract Derivatization yields of different esters (methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl) of citric, malic and isocittic acids with various acylating agents, such as acetic anhydride, propionic anhydride, trifluoroacetic anhydride and heptafluorobutyric anhydride, were investigated. The formation of acylated malic and isocitric acid esters is rapid and quantitative whereas the acylation of citric acid esters was slow and partial in most instances. It was found that selective analytical conditions can be achieved with the 0-heptafluorobutyryl n-butyl esters. The analytical applicability of the separation and determination of the malic, isocitric and citric acid contents of model solutions and of pressed lemon juice is discussed. Concentrations of 1.2 X lo-‘-9.0 X 10m3 g per 100 g of isocitric acid and 4.2 X 10m3-3.5 X 10W2 g per 100 g of malic acid in the presence of 9.3 x 10-l g per 100 g of citric acid were measured with relative standard deviations of 7.5 and 4.2X, respectively. Keywork
Isocitric
acid; Malic acid; Citric acid; Fruit juice;
There is a considerable interest in methods for detecting adulteration of fruit juices. Numerous papers have been published on this subject [l-16] and mainly statistical methods have been proposed [lo-161 for evaluating the authenticity of fruit juices. In one instance [l-4] the determination of specific acids, i.e., isocitric and malic acids, which are present in measurable amounts only in genuine juices, was recommended. These two acids were measured by enzyme methods of the Boehringer Company. Methods based on gas chromatography (GC) or liquid chromatography do not seem to have been applied to the simultaneous determination of isocitric and malic acids in the presence of a very large excess of citric acid. This work was intended to provide a suitable GC method for the rapid 0003-2670/90/$03.50
0 1990 - Elsevier
Science Publishers
Lemon juice
measurement of citric, isocitric and malic acids in order to establish a general technique for the determination of isocitric acid in the presence of a very large excess of citric acid. Experimental Reagents. Citric acid, malic acid, concentrated sulphuric acid, n-butanol, chloroform, anhydrous sodium sulphate, acetic anhydride (AA) and propionic anhydride (PA) were obtained from Reanal (Budapest, Hungary), heptafluorobutyric anhydride (HFBA) and trifluoroacetic anhydride (TFAA) from Fluka (Buchs, Switzerland) and trisodium DL-isocitrate from Serva (Heidelberg, F.R.G.). Separation of n-butyl ester and/or O-heptajluorobutyryl (HFB) n-butyl ester derivatives. The B.V.
166
temperatures of the injection and detector ports were 320 and 350 o C, respectively. With temperature progr amming from 170 to 320 o C at 8 o C/min 20 min were required to elute the n-butyl ester and the 0-HFB n-butyl ester derivatives. The flow-rate of nitrogen was 120 cm3/min. Preparation of n-butyl ester derivatives. Various amounts of malic (6.6 X 10e4-5.2 x 10e3 g) and isocitric acid (1.8 X 10e4-1.4 X 1O-3 g), and in all instances the same amount of citric acid (1.4 x 10-l g) in model solutions, or 5 cm3 of pressed lemon juice were evaporated to dryness in a rotary evaporator at 50-60°C. The dehydrated residue was esterified with 2 cm3 of n-butanol and 0.2 cm3 of sulphuric acid in the presence of 2 g of anhydrous sodium sulphate using the esterification vessel and stirrer described previously [17,18]. The ground joint of the vessel, wetted with one drop of sulphuric acid, was fitted to the reflux condenser and the apparatus placed in the waterbath. Esterification was continued at 100 o C for 1 h. The solution was cooled to room temperature and transferred into a separating funnel together with 200 cm3 of water and 10 cm3 of chloroform. Extraction was completed with two additional locm3 portions of chloroform. The combined chloroform extracts were evaporated under vacuum in a water-bath, kept at room temperature, to a volume of 2-2.5 cm3. A stock solution of 3 cm3 was then prepared and 5-lo-p1 aliquots were injected into the gas chromatograph. The methyl, ethyl, n-propyl, isopropyl and isobutyl esters were prepared similarly, with the corresponding alcohol in place of n-butanol. Preparation of 0-HFB n-butyl ester derivatives. The n-butyl esters, prepared as detailed above, were mixed with HFBA at volume ratios of ester to anhydride of 1: 2, and kept at 150°C for 10 min in a drying oven. A lo-p1 volume of the acylated solution was injected into the gas chromatograph. The 0-trifluoroacetyl (TFA), 0-acetyl (A) and 0-propionyl (P) esters were prepared with the exceptions that instead of HFBA the corresponding acylating agent was applied and the time of the acylations was varied. Gas chromatography. The gas chromatograph used was a Chromatron Model GCHF 18.3 instrument equipped with a flame ionization detector.
1. MOLNdrR-PERL
ET AL.
Chromatographic peak-area determinations were made with a Chinoin Model Digint 34 microcomputing integrator. Stainless-steel columns (2 m X 4 mm i.d.) were used. The packing materials were 15% Dexsil GC 300 on 80-lOO-mesh Chromosorb W AW DMCS, purchased from Applied Science Labs. (State College, PA). Results and discussion In order to separate and determine isocitric acid in the presence of a very large excess of citric acid, the main task was to find suitable derivatives for GC analysis. The model esterifications and subsequent acylations of citric, isocitric and malic acid provided the following results. The esters of malic and citric acid could be separated and measured equally well as the methyl, ethyl, iso- and n-propyl and iso- and n-butyl esters as single peaks; any of the six esters of isocitric acid emerged together with the corresponding citric acid ester as a wide “double twin”
250
290
320
10
15
19
Temp.(‘C 1 c Ret.lim(mml
Fig. 1. Gas cluomatograms of citric acid n-butyl ester acylated with HFBA for (A) 5, (B) 10, (C) 30 and (D) 60 min. Peaks: 1-3 = O-HFB derivatives of citric acid n-butyl ester; 4 = citric acid n-butyl ester.
GC DETERMINATION
OF ISOCITRIC
5
10
AND
15
MALIC
ACID
167
19 Re;eme
Fig. 2. Gas chromatograms of citric acid together with various amounts of malic and isocitric acid n-butyl esters after acylation with 0-HFBA for 10 min. Detailed data in Table 2. Peaks: 1, 3 and 4 as in Fig. 1; 5, 2+ and 6 = 0-HFB n-butyl esters of (5) malic acid, (6) isocitric acid alone and (2+ ) citric and isocitric acids together; a and b = unknown derivatization products; c = butyl sulphate.
TABLE Acylation time
250
290
5
10
15
c 320 Templ'C1 19 Ret.Tlme (mm)
Fig. 3. Gas chromatograms of the 0-HFB n-butyl pressed (A) lemon juice with (B-D) various additional of isocitric and malic acid. Detailed data in Table l-6 and a-c as in Fig. 2.
yield of different
esters of citric acid obtained
Acylation Time.
with various
acylating
agents (AA, PA, HFBA)
as a function
of the acylation
yield (W) of citric acid esters a
Methyl
Ethyl
n-Propyl
Isopropyl
n-Butyl
Isobutyl
10 30 60 10 30 60 10 30
13 40 54 41 65 100
6 _ 59 8 10 90
8 13 56 6 15 30 8
44 100 100 5 15 100 19 70
20 22 54 2 8 35 4 16
16 21 52 5 13 56 5 _
60
96
96
85
100
33
33
(mm) AA
PA
HFBA
a
esters of amounts 3. Peaks:
1
Acylation Reagent
210
-, No data available.
_
1. MOLNAR-PERL
168 TABLE
2
Reproducibility of determinations n-butyl ester a Sample
of O-HFB
malic- and isocitric
(PLP) A B C D E Mean SD. R.S.D. (9)
0.73 1.45 2.91 4.36 5.81
a Detailed data (6) = calculations for each.
Acid
units, to 1 /.kg ’
of the partly
acylated
citric acid
acid (6) b
Injected
Integrator equivalent
(PP)
1400 1447 1568 1466 1452 1467 61.9 4.2
0.19 0.37 0.14 1.12 1.49
units, to 1 pg ’
1135 1206 1218 1005 1183 1149 86.7 7.5
of Fig. 2, chromatograms A-E. The amount of citric acid injected was 100 ng in all instances. b (5) and based on peaks 5 and 6 in Fig. 2. ’ Integrator units for samples A-E are the means of at least three parallel tests
ably slower (Fig. 1, Table 1). With methyl and isopropyl esters, after reaction times of 30-60 min quantitative acylations could be achieved, but the corresponding citric and isocitric acid derivatives are not resolvable; the retention time differences between the acylated citric and isocitric acid esters increase in the order O-A derivatives (19 s), O-P derivatives (20 s) and 0-HFB derivatives (45 and 65 s) between peaks from 2+ to 6 and from 6 to 3, respectively (Figs. 2 and 3). The 0-HFB derivatives of citric acid n-butyl ester, after acylation for 10 min time (Table 1, Figs. l-3) do not affect the determination of the
3 study of malic and isocitric
acids measured
as O-HFB
Amount in lemon juice
Amount added
(g per ICQ 9)
(g per 100 g)
Malic Isocitric
Sample A 0.476 0.092
Malic Isocitric
0.416 0.092
Malic Isocitric
0.476 0.092
Sample B 0.451 0.112 Sample C 0.915 0.224 Sample D 1.372 0.336
a Detailed
esters in the presence
Isocitric Integrator equivalent
peak, probably owing to the four possible components of the esters of the rhreo-Ds, three-L,, e~~ythro-D, and e~Uhro-~, forms of isocitric acid. Thus, for satisfactory resolution of citric and isocitric acid only the acylated esters could be considered. Therefore, extensive acylation studies were performed with all six esters applying AA, PA and HFBA as acylating agents. The results obtained revealed that the formation of O-A, -P and -HFB malic and isocitric acid esters is rapid; the reactions were quantitative with all six esters of both acids after 10 min. The acylation of citric acid esters is consider-
Recovery
acid n-butyl
Malic acid (5) b Injected
TABLE
ET AL.
data of Fig. 3.
n-butyl
esters added
to natural
pressed
lemon juice a
Calculated
Found
Recovery
(g per IO0 8)
(g per 100 g)
(%)
0.933 0.204
0.946 0.210
101.4 102.9
1.391 0.318
1.414 0.324
101.7 101.9
1.848 0.428
1.845 0.427
99.8 99.8
GC DETERMINATION
TABLE
OF ISOCITRIC
AND
MALIC
ACID
169
4
Citric, malic and isocitric
acid contents
of citrus juice concentrates
Sample
Concentration
(g per 100 g concentrate)
Grape Orange Orange Orange Orange Orange Orange Orange Orange Lemon Lemon Lemon
a
fruit a a bi b2 b3 b4 Cl c2 d a ’ ’
a Samples unknown.
Mass ratio
Citric acid
Malic acid
Isocitric acid
Citric to malic acid
Citric to isocitric acid
12.15 11.85 3.39 3.44 3.47 3.58 13.57 9.63 4.60 12.04 13.57 18.54
0.41 0.38 0.064 0.019 0.011 0.040 0.13 0.21 0.76 0.37 0.13 0.476
0.11 0.11 0.023 0.019 0.025 0.040 0.11 0.037 0.025 0.094 0.11 0.092
30 32 53 102 315 90 143 46 6 33 143 40
110 108 136 181 139 90 104 260 48 128 104 200
were obtained from The Netherlands (a), Israel (bl-b4), Greece (cl, c2) and Brazil (d). The producers Sample (s), laboratory-pressed lemon juice, detailed data in Table 3 and Fig. 3.
O-HFB isocitric acid n-butyl ester; as can be seen, with HFBA citric acid esters give three (Figs. 1-3, peaks 1, 2 and 3) and isocitric acid esters two derivatives (Figs. 2 and 3, peaks 6 and 2+). The determination of isocitric acid on the basis of peak 6 gives good results, both in model solutions (Fig. 2, Table 2) and in natural matrices (Fig.3, Table 3). Recovery investigations on fresh lemon juice, also in the presence of additional amounts of 0.457-1.372 g per 100 g of malic acid and 0.1120.336 g per 100 g of isocitric acid, showed a recovery of 99.8102.9% (Table 3, Fig. 3). The amount of citric acid was evaluated from stock solutions of esters with a relative standard deviation of 1.5% or less, The usefulness of the method is shown by the determination of the citric, malic and isocitric acid contents of citrus juice concentrates, mainly products from pure fruits (Table 4: grapefruit, orange and lemon samples of various origins). The smallest amounts of isocitric acid (0.019 g per 100 g) and malic acid (0.011 g per 100 g), measured in orange concentrates (Table 4, orange samples b2 and b3) can be regarded as characteristic of the limit of determination. Concerning the composition of adulterated juice concentrates, it should to be noted that their citric
of the samples
were
acid contents agree with those for concentrates obtained from the corresponding pure fruits, but the mass ratios calculated from their citric, isocitric and malic acid contents vary from 200 to 1000, depending on the extent of their adulteration (data not available for publication). This work was partly supported by the Hungarian Ministry of Agriculture and Food through project G 8.
REFERENCES
7 8 9 10 11
B. Lang, Dtsch. Lebensm.-Rundsch., 68 (1972) 176. B. Bergner-Lang, Dtsch. Lebensm.-Rundsch., 70 (1974) 431. B. Bergner-Lang, Dtsch. Lebensm.-Rundsch., 71 (1975) 348. B. Bergner-Lang, Dtsch. Lebensm.-Rundsch., 73 (1977) 211. T. Philip and F.E. Nelson, J. Food Sci., 38 (1973) 18. A. Sasson, Y. Emer and S.P. Monselise, J. Agric. Food Chem., 24 (1976) 562. A. Sasson and S.P. Monselise, J. Am. Sot. Hort. Sci., 102 (1977) 331. P.E. Shaw and C.W. Wilson, J. Sci. Food Agric., 32 (1981) 1242. B.S. Buslig, C.W. Wilson and P.E. Shaw, J. Agric. Food Chem., 30 (1982) 342. C.E. Vandercook, J.L. Navarro, D.C. Smolensky, D.B. Nelson and G.L. Park, J. Food Sci., 48 (1983) 636, 655. P.E. Shaw and C.W. Wilson, J. Sci. Food Agric., 34 (1983) 1285.
170 12 P.E. Shaw, B.S. Buslig and C.W. Wilson, J. Agric. Food Chem., 31 (1983) 182. 13 P.R. Monk and P.G. Iland, Food Technol. Aust., 36 (1984) 18. 14 R. Schwarzenbach, Mitt. Geb. Lebensmittelunters. Hyg., 75 (1984) 51. 15 E.D. Coppola, Food Technol., 38 (1984) 88.
1.MOLNbrR-PERL
ET AL.
16 M.B. Brown and E. Cohen, J. Assoc. Off. Anal. Chem., 66 (1983) 781. 17 I. Molnar-Per1 and M. Pint&-Szakacs, J. Chromatogr., 365 (1986) 171. 18 I. Molnfir-Perl, V. Fabian-Vonsik and M. Pint&-Szakks, Chromatographia, 18 (1984) 673.
Analytica Chimica Acta, 239 (1990) 165-170 Elsevier Science Publishers B.V., Amsterdam
Short Communication
Gas chromatographic determination of isocitric and malic acid in the presence of a large excess of citric acid I. Molnar-Per1 Institute of Inorganic and Analytical
*, M. Morvai
and M. Pint&-Szakacs
Chemistry, L. ELitubs University, P.O. Box 123, H-I443 Budapest (Hungary)
M. Petro-Turza Central Food Research Institute, Herman Ott6 lit 15, 1022 Budapest (Hungary) (Received
29th March
1990)
Abstract Derivatization yields of different esters (methyl, ethyl, n-propyl, isopropyl, n-butyl and isobutyl) of citric, malic and isocittic acids with various acylating agents, such as acetic anhydride, propionic anhydride, trifluoroacetic anhydride and heptafluorobutyric anhydride, were investigated. The formation of acylated malic and isocitric acid esters is rapid and quantitative whereas the acylation of citric acid esters was slow and partial in most instances. It was found that selective analytical conditions can be achieved with the 0-heptafluorobutyryl n-butyl esters. The analytical applicability of the separation and determination of the malic, isocitric and citric acid contents of model solutions and of pressed lemon juice is discussed. Concentrations of 1.2 X lo-‘-9.0 X 10m3 g per 100 g of isocitric acid and 4.2 X 10m3-3.5 X 10W2 g per 100 g of malic acid in the presence of 9.3 x 10-l g per 100 g of citric acid were measured with relative standard deviations of 7.5 and 4.2X, respectively. Keywork
Isocitric
acid; Malic acid; Citric acid; Fruit juice;
There is a considerable interest in methods for detecting adulteration of fruit juices. Numerous papers have been published on this subject [l-16] and mainly statistical methods have been proposed [lo-161 for evaluating the authenticity of fruit juices. In one instance [l-4] the determination of specific acids, i.e., isocitric and malic acids, which are present in measurable amounts only in genuine juices, was recommended. These two acids were measured by enzyme methods of the Boehringer Company. Methods based on gas chromatography (GC) or liquid chromatography do not seem to have been applied to the simultaneous determination of isocitric and malic acids in the presence of a very large excess of citric acid. This work was intended to provide a suitable GC method for the rapid 0003-2670/90/$03.50
0 1990 - Elsevier
Science Publishers
Lemon juice
measurement of citric, isocitric and malic acids in order to establish a general technique for the determination of isocitric acid in the presence of a very large excess of citric acid. Experimental Reagents. Citric acid, malic acid, concentrated sulphuric acid, n-butanol, chloroform, anhydrous sodium sulphate, acetic anhydride (AA) and propionic anhydride (PA) were obtained from Reanal (Budapest, Hungary), heptafluorobutyric anhydride (HFBA) and trifluoroacetic anhydride (TFAA) from Fluka (Buchs, Switzerland) and trisodium DL-isocitrate from Serva (Heidelberg, F.R.G.). Separation of n-butyl ester and/or O-heptajluorobutyryl (HFB) n-butyl ester derivatives. The B.V.
166
temperatures of the injection and detector ports were 320 and 350 o C, respectively. With temperature progr amming from 170 to 320 o C at 8 o C/min 20 min were required to elute the n-butyl ester and the 0-HFB n-butyl ester derivatives. The flow-rate of nitrogen was 120 cm3/min. Preparation of n-butyl ester derivatives. Various amounts of malic (6.6 X 10e4-5.2 x 10e3 g) and isocitric acid (1.8 X 10e4-1.4 X 1O-3 g), and in all instances the same amount of citric acid (1.4 x 10-l g) in model solutions, or 5 cm3 of pressed lemon juice were evaporated to dryness in a rotary evaporator at 50-60°C. The dehydrated residue was esterified with 2 cm3 of n-butanol and 0.2 cm3 of sulphuric acid in the presence of 2 g of anhydrous sodium sulphate using the esterification vessel and stirrer described previously [17,18]. The ground joint of the vessel, wetted with one drop of sulphuric acid, was fitted to the reflux condenser and the apparatus placed in the waterbath. Esterification was continued at 100 o C for 1 h. The solution was cooled to room temperature and transferred into a separating funnel together with 200 cm3 of water and 10 cm3 of chloroform. Extraction was completed with two additional locm3 portions of chloroform. The combined chloroform extracts were evaporated under vacuum in a water-bath, kept at room temperature, to a volume of 2-2.5 cm3. A stock solution of 3 cm3 was then prepared and 5-lo-p1 aliquots were injected into the gas chromatograph. The methyl, ethyl, n-propyl, isopropyl and isobutyl esters were prepared similarly, with the corresponding alcohol in place of n-butanol. Preparation of 0-HFB n-butyl ester derivatives. The n-butyl esters, prepared as detailed above, were mixed with HFBA at volume ratios of ester to anhydride of 1: 2, and kept at 150°C for 10 min in a drying oven. A lo-p1 volume of the acylated solution was injected into the gas chromatograph. The 0-trifluoroacetyl (TFA), 0-acetyl (A) and 0-propionyl (P) esters were prepared with the exceptions that instead of HFBA the corresponding acylating agent was applied and the time of the acylations was varied. Gas chromatography. The gas chromatograph used was a Chromatron Model GCHF 18.3 instrument equipped with a flame ionization detector.
1. MOLNdrR-PERL
ET AL.
Chromatographic peak-area determinations were made with a Chinoin Model Digint 34 microcomputing integrator. Stainless-steel columns (2 m X 4 mm i.d.) were used. The packing materials were 15% Dexsil GC 300 on 80-lOO-mesh Chromosorb W AW DMCS, purchased from Applied Science Labs. (State College, PA). Results and discussion In order to separate and determine isocitric acid in the presence of a very large excess of citric acid, the main task was to find suitable derivatives for GC analysis. The model esterifications and subsequent acylations of citric, isocitric and malic acid provided the following results. The esters of malic and citric acid could be separated and measured equally well as the methyl, ethyl, iso- and n-propyl and iso- and n-butyl esters as single peaks; any of the six esters of isocitric acid emerged together with the corresponding citric acid ester as a wide “double twin”
250
290
320
10
15
19
Temp.(‘C 1 c Ret.lim(mml
Fig. 1. Gas cluomatograms of citric acid n-butyl ester acylated with HFBA for (A) 5, (B) 10, (C) 30 and (D) 60 min. Peaks: 1-3 = O-HFB derivatives of citric acid n-butyl ester; 4 = citric acid n-butyl ester.
GC DETERMINATION
OF ISOCITRIC
5
10
AND
15
MALIC
ACID
167
19 Re;eme
Fig. 2. Gas chromatograms of citric acid together with various amounts of malic and isocitric acid n-butyl esters after acylation with 0-HFBA for 10 min. Detailed data in Table 2. Peaks: 1, 3 and 4 as in Fig. 1; 5, 2+ and 6 = 0-HFB n-butyl esters of (5) malic acid, (6) isocitric acid alone and (2+ ) citric and isocitric acids together; a and b = unknown derivatization products; c = butyl sulphate.
TABLE Acylation time
250
290
5
10
15
c 320 Templ'C1 19 Ret.Tlme (mm)
Fig. 3. Gas chromatograms of the 0-HFB n-butyl pressed (A) lemon juice with (B-D) various additional of isocitric and malic acid. Detailed data in Table l-6 and a-c as in Fig. 2.
yield of different
esters of citric acid obtained
Acylation Time.
with various
acylating
agents (AA, PA, HFBA)
as a function
of the acylation
yield (W) of citric acid esters a
Methyl
Ethyl
n-Propyl
Isopropyl
n-Butyl
Isobutyl
10 30 60 10 30 60 10 30
13 40 54 41 65 100
6 _ 59 8 10 90
8 13 56 6 15 30 8
44 100 100 5 15 100 19 70
20 22 54 2 8 35 4 16
16 21 52 5 13 56 5 _
60
96
96
85
100
33
33
(mm) AA
PA
HFBA
a
esters of amounts 3. Peaks:
1
Acylation Reagent
210
-, No data available.
_
1. MOLNAR-PERL
168 TABLE
2
Reproducibility of determinations n-butyl ester a Sample
of O-HFB
malic- and isocitric
(PLP) A B C D E Mean SD. R.S.D. (9)
0.73 1.45 2.91 4.36 5.81
a Detailed data (6) = calculations for each.
Acid
units, to 1 /.kg ’
of the partly
acylated
citric acid
acid (6) b
Injected
Integrator equivalent
(PP)
1400 1447 1568 1466 1452 1467 61.9 4.2
0.19 0.37 0.14 1.12 1.49
units, to 1 pg ’
1135 1206 1218 1005 1183 1149 86.7 7.5
of Fig. 2, chromatograms A-E. The amount of citric acid injected was 100 ng in all instances. b (5) and based on peaks 5 and 6 in Fig. 2. ’ Integrator units for samples A-E are the means of at least three parallel tests
ably slower (Fig. 1, Table 1). With methyl and isopropyl esters, after reaction times of 30-60 min quantitative acylations could be achieved, but the corresponding citric and isocitric acid derivatives are not resolvable; the retention time differences between the acylated citric and isocitric acid esters increase in the order O-A derivatives (19 s), O-P derivatives (20 s) and 0-HFB derivatives (45 and 65 s) between peaks from 2+ to 6 and from 6 to 3, respectively (Figs. 2 and 3). The 0-HFB derivatives of citric acid n-butyl ester, after acylation for 10 min time (Table 1, Figs. l-3) do not affect the determination of the
3 study of malic and isocitric
acids measured
as O-HFB
Amount in lemon juice
Amount added
(g per ICQ 9)
(g per 100 g)
Malic Isocitric
Sample A 0.476 0.092
Malic Isocitric
0.416 0.092
Malic Isocitric
0.476 0.092
Sample B 0.451 0.112 Sample C 0.915 0.224 Sample D 1.372 0.336
a Detailed
esters in the presence
Isocitric Integrator equivalent
peak, probably owing to the four possible components of the esters of the rhreo-Ds, three-L,, e~~ythro-D, and e~Uhro-~, forms of isocitric acid. Thus, for satisfactory resolution of citric and isocitric acid only the acylated esters could be considered. Therefore, extensive acylation studies were performed with all six esters applying AA, PA and HFBA as acylating agents. The results obtained revealed that the formation of O-A, -P and -HFB malic and isocitric acid esters is rapid; the reactions were quantitative with all six esters of both acids after 10 min. The acylation of citric acid esters is consider-
Recovery
acid n-butyl
Malic acid (5) b Injected
TABLE
ET AL.
data of Fig. 3.
n-butyl
esters added
to natural
pressed
lemon juice a
Calculated
Found
Recovery
(g per IO0 8)
(g per 100 g)
(%)
0.933 0.204
0.946 0.210
101.4 102.9
1.391 0.318
1.414 0.324
101.7 101.9
1.848 0.428
1.845 0.427
99.8 99.8
GC DETERMINATION
TABLE
OF ISOCITRIC
AND
MALIC
ACID
169
4
Citric, malic and isocitric
acid contents
of citrus juice concentrates
Sample
Concentration
(g per 100 g concentrate)
Grape Orange Orange Orange Orange Orange Orange Orange Orange Lemon Lemon Lemon
a
fruit a a bi b2 b3 b4 Cl c2 d a ’ ’
a Samples unknown.
Mass ratio
Citric acid
Malic acid
Isocitric acid
Citric to malic acid
Citric to isocitric acid
12.15 11.85 3.39 3.44 3.47 3.58 13.57 9.63 4.60 12.04 13.57 18.54
0.41 0.38 0.064 0.019 0.011 0.040 0.13 0.21 0.76 0.37 0.13 0.476
0.11 0.11 0.023 0.019 0.025 0.040 0.11 0.037 0.025 0.094 0.11 0.092
30 32 53 102 315 90 143 46 6 33 143 40
110 108 136 181 139 90 104 260 48 128 104 200
were obtained from The Netherlands (a), Israel (bl-b4), Greece (cl, c2) and Brazil (d). The producers Sample (s), laboratory-pressed lemon juice, detailed data in Table 3 and Fig. 3.
O-HFB isocitric acid n-butyl ester; as can be seen, with HFBA citric acid esters give three (Figs. 1-3, peaks 1, 2 and 3) and isocitric acid esters two derivatives (Figs. 2 and 3, peaks 6 and 2+). The determination of isocitric acid on the basis of peak 6 gives good results, both in model solutions (Fig. 2, Table 2) and in natural matrices (Fig.3, Table 3). Recovery investigations on fresh lemon juice, also in the presence of additional amounts of 0.457-1.372 g per 100 g of malic acid and 0.1120.336 g per 100 g of isocitric acid, showed a recovery of 99.8102.9% (Table 3, Fig. 3). The amount of citric acid was evaluated from stock solutions of esters with a relative standard deviation of 1.5% or less, The usefulness of the method is shown by the determination of the citric, malic and isocitric acid contents of citrus juice concentrates, mainly products from pure fruits (Table 4: grapefruit, orange and lemon samples of various origins). The smallest amounts of isocitric acid (0.019 g per 100 g) and malic acid (0.011 g per 100 g), measured in orange concentrates (Table 4, orange samples b2 and b3) can be regarded as characteristic of the limit of determination. Concerning the composition of adulterated juice concentrates, it should to be noted that their citric
of the samples
were
acid contents agree with those for concentrates obtained from the corresponding pure fruits, but the mass ratios calculated from their citric, isocitric and malic acid contents vary from 200 to 1000, depending on the extent of their adulteration (data not available for publication). This work was partly supported by the Hungarian Ministry of Agriculture and Food through project G 8.
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