Gas Chromatography-Olfactometry

Chapter 5

Trace Compound Analysis for Off-Flavor Characterization of Micromilled Milk Powder

Downloaded by UNIV OF MICHIGAN ANN ARBOR on September 10, 2015 | http://pubs.acs.org Publication Date: March 1, 2001 | doi: 10.1021/bk-2001-0782.ch005

Martin Preininger and Frank Ullrich Kraft Jacobs Suchard R&D, Unterbiberger Strasse 15, D-81737 Munich, Germany Abstract Aroma compounds causing the off-flavor in micromilled and stored milk powder (MMP) were detected and identified by comparative GC/Olfactometry and GC/MS of aroma distillates from untreated standard milk powder (MP) and MMP. In addition to aldehydes and ketones from lipid peroxidation, 2-aminoacetophenone (ΑΑΡ) was detected only in MMP and was present at high aroma intensity (musty, wet popcorn odor). ΑΑΡ was not found in MP. ΑΑΡ was quantified by GC/MS using internal surrogate standards, and occurred in MMP at high Odor Activity Value (OAV = concentration/odor threshold), indicating its strong contribution to the off-flavor. A method for convenient synthesis of deuterium labeled d -AAP was developed for use in precise GC/MS quantitation (Stable Isotope Dilution Assay). However, the application of d -AAP for quantitation of ΑΑΡ in MP failed because of matrix-effected deuterium-loss. 3

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Introduction After micromilling sweet-dairy smelling MP by high speed collision, a musty off -flavor developed within days of storage. The off-flavor was reminiscent of wet-dog fur odor. The objective of this study was to identify potent aroma compounds causing the off-flavor in MMP by GC/Olfactometry and GC/MS of aroma distillates. In addition, methods should be evaluated for quantitation of a key off-flavor compound in order to analytically measure the off-flavor development. Material and Methods Milk Powder Samples Standard milk powder (MP) MP consisted of 75 % (w/w) skim milk powder and 25 % whey powder. 90 % of the particles had 360 μm particle size. 46

© 2001 American Chemical Societv In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

47 Micromilled milk powder (MMP) MP was micromilled to particle size distribution 90 % < 7.7 μηι and 50 % < 3.6 μιη by high speed collision under controlled atmosphere (88 % N , 12 % 0 ) and stored at 16 °C for 10 months (qualitative analysis) and from one day up to 12 months (quantitative analysis). In the 12 months stored sample, the moisture was 3.44 % (w/w) and the total fat content was 0.9 % at the time of quantitative analysis. MP and MMP were stored with silica gel pouches at 16°C in odorless plastic bags in the dark according to pilot plant conditions.


2

2

Sample Preparation Chemicals All chemicals were purchased from Aldrich (Steinheim, Germany) at the highest purity available, except for deuterated d -2-aminoacetophenone, which was synthesized in this study. 3

Solvent Extraction For qualitative analysis, milk powder (100 g) was power stirred in freshly distilled diethylether (200 mL) in the dark under argon at room temperature for 30 min. Deionized, sonicated water (150 g) was added and stirring continued for an additional 50 min. Solvent Assisted Flavor Evaporation (SAFE®) The slurry from solvent extraction was distilled by SAFE® (1) using high vacuum (ca. 5 χ 10" mbar) for 45 min at 40 °C during sample addition under argon. After sample addition, distillation was continued for an additional 75 min at 50 °C. The distillate was washed with saturated aqueous sodium chloride (40ml), and the etherous phase was dried over sodium sulfate (anhydrous). The dried etherous phase was then concentrated to 2 mL via Vigreux column distillation at 40°C before GC/O and GC/MS analysis. For quantitative analysis, 50 g milk powder, 100ml diethyl ether, 75g water and 20ml sodium chloride solution were used, respectively. Internal standard compounds (3,4-dimethoxyacetophenone, 4-decanone, d -2-aminoacetophenone; see Results) were added to the diethylether during the solvent extraction step. The SAFE® distillate was concentrated to 300 μΐ, before GC/MS analysis. Other parameters in the sample preparation procedure were kept as described above for qualitative analysis. 5

3

GC/O Analysis The SAFE® distillate (1 μ ί ) was injected into a HRGC Mega Series 5300 (Carlo Erba, Milan Italy) equipped with an on-column injector (Carlo Erba). The sample was separated by a CP-FFAP fused silica capillary (25 m, 0.32 mm i.d., 0.3 μιτι film thickness; Chrompack Inc.) with the following oven and injector program: The initial temperature of 35 °C was held for 2 min and then ramped to 60 °C at a rate of 40 °C/min. The temperature of 60 °C was held for 2 min and then ramped to 240 °C In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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at a rate of 6 °C/min. The final temperature of 240 °C was held for 15 min. The capillary effluent was split (1:1) to a flame ionization detector and a sniffing port held at 200 °C. GC/MS Analysis The SAFE® distillate (1 μ ι ) was injected into a HRGC Mega Series 5300 (Carlo Erba, Milan Italy) equipped with a KA S 3 cold on-column injector (Gerstel, Munich, Germany). The sample was separated by a HP-FFAP crosslinked fused silica capillary (50 m χ 0.32 mm i.d. χ 0.5 μηι film thickness, Agilent, Taufkirchen, Germany) with the following oven program: The initial temperature of 35 °C was ramped to 230 °C at a rate of 6 °C/min. The final temperature of 230 °C was held for 15 min. The sample was analyzed using a mass selective detector, MSD 5973 (Agilent). The MSD 5973 was operated either in ΕΙ-SCAN mode (35-250 amu) or in SIM mode as specified. Data analysis was performed using the HP-Chemstation of the MSD 5973. For the different lots of MMP analyzed, the corresponding MP was also analyzed before micromilling. Synthesis of deuterated 2-aminoacetophenone (d -AAP) ΑΑΡ (7.2 mg) was dissolved in deuterium oxide (5.0 mL), adjusted to pH 14.0 with sodium deuteroxide (40 % in D 0) and vigorously stirred at room temperature for 16 h. After deuteration the mixture was extracted with diethylether (3x7 mL) using a 10 mL Mixxor® (Aldrich) and dried over sodium sulfate (anhydrous). The concentration of the etherous d -AAP solution was determined by GC/MSD 5973 SIM with ΑΑΡ as internal standard and assuming a response factor of 1.0. 3

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ds-AAP Stability Control Water (HPLC grade, 1.0 mL) was added to a solution of ds-AAP (5.13 μg) in ether (4 mL) and adjusted to pH6.27 with hydrochloric acid (0.1 N). The mixture was vigorously stirred at room temperature for 80 min. The etherous phase was then separated, washed with saturated aqueous sodium chloride (1.5 mL) by the Mixxor®, and dried with sodium sulfate (anhydrous) before GC/MS, similar to the procedure of ΑΑΡ quantitation from milk powder. Results and Discussion Identification of Potent Aroma Compounds in MP and MMP The aroma compounds from MP and MMP were isolated by high vacuum distillation of a solvent extract. The distillate of MMP represented the off-flavor well, possessing an intense musty, stale odor. The MP distillate had a weak, sweet odor like caramelized milk. The distillates were analyzed by comparative GC/O and GC/MS for detection and identification of potent aroma compounds which may cause the flavor difference between the samples. 2-Aminoacetophenone (ΑΑΡ) was detected and identified only in the MMP distillate by an intense musty, wet-popcorn odor and by its retention index in GC/O (Table 1, No.l). Based on the GC/O results, the characteristic mass traces of ΑΑΡ (m/z 135, In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


49 120 and 92) were extracted from the total ion chromatograms after GC/MS-SCAN analysis of the MP and MMP distillates (Figure 1 and 2). Peaks of ΑΑΡ mass traces occurred only in the mass chromatogram of the MMP distillate. The mass traces of ΑΑΡ were overlayed by a peak of m/z 243 which originated from an unknown compound. This inhibited direct identification of ΑΑΡ by MS library search (Figure 3). After background substraction the identity of ΑΑΡ was subsequently confirmed by its mass spectrum (Figure 4). ΑΑΡ was not found in MP by GC/O and by GC/MS-SCAN (Figure 1) and GC/MS-SIM (data not shown). Metallic, green, fatty smelling aldehydes and ketones, which derive from lipidperoxidation of milk-fat, were detected by GC/O either only in MMP, or at much higher aroma intensity in MMP, compared to MP (Table 1, No.6-22). Alkanals were found by GC/MS at much higher level in MMP than in MP, using acetic and octanoic acid as naturally occurring internal reference compounds (Figure 5 and 6). The aroma intensity of sweet caramelized smelling Furaneol® was found by GC/O to be much lower in MMP compared to MP (Table 1, No.23). In summary GC/O data were interpreted in the following way. In MMP, the sweet, caramelized smelling compounds (Table 1, No.23-27) may be lower in concentration and may be masked by increased concentrations of musty, green, metallic smelling off-flavor compounds, compared to MP. The off-flavor compounds may have derived from lipid and amino acid degradation (2, 3, 4) during micromilling and storage of milk powder. Quantitation of 2-Aminoacetophenone (ΑΑΡ) via Surrogate Standards In order to measure the off-flavor intensity, ΑΑΡ was quantified by GC/MS-SIM from M M P using the internal surrogate standards, 3,4-dimethoxyacetophenone (490 μg/kg) and 4-decanone (510 μg/kg). MS response factors for each standard were determined by GC/MS-SIM. Recovery factors were determined by applying the quantitative GC/MS-SIM analysis to MP which had been spiked with ΑΑΡ (396 μg/kg), and which did not contain detectable amounts of ΑΑΡ naturally. The recovery factors were calculated as quotients of the actually added amount of ΑΑΡ and the amount of ΑΑΡ measured via the internal surrogate standards, employing the MS response factors. The recovery factors (Table 2) for ΑΑΡ determined via 3,4-dimethoxyacetophenone and via 4-decanone varied greatly by the factor of 151, showing the importance of determining the recovery factors in order to obtain correct analyte concentration data of ΑΑΡ. ΑΑΡ was found in 12 months stored MMP at a level of ca. 13 μg/kg (Table 3). In different lots of MMP the ΑΑΡ concentrations were 9.3 μg/kg after 10 months storage and 6 μg/kg after only one day of storage. No ΑΑΡ was detectable in MP from which each of the different MMP lots were produced. ΑΑΡ is a highly potent off-flavor compound in MMP due to its musty smell and high Odor Activity Value.

In Gas Chromatography-Olfactometry; Leland, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

50 Abundance (counts)

Figure 1


1000

500

m/z 120

m/z 135

m/z 243 30.01

100 29.70 29.80 29.90 30.00 30.10 30.20 30.30 30.40 Time (min)

Abundance (counts) Figure 2

1000

500

29.70 29.80 29.90 30.00 30.10 30.20 30.30 30.40 Time (min)

Figure 1 and 2: Extracted ion chromatogram (GC/MS-SCAN) of distillate from standard milk powder (MP, Figure 1) and micro-milled and stored milk powder (MMP, Figure 2). 2-Aminoacetophenone (ΑΑΡ) detected by its mass traces (m/z 135, 120 and 92) only in MMP Mass trace m/z 243 stemsfromunknown overlaying compound.


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Rel. Abundance 45 00%

Figure 3 Scan 2753 (29.987 min)

50

100 Rel. Abundance 00%

120

140

160

180

200

220

240 (m/z)

120 Figure 4

2-aminoacetophenone (ΑΑΡ)

135 m/z 243 from unknown compound

50 92

243

65 162 38 51 ••1Ί •' 40 60

77

106

145

,Λ.,Ιι 80

100

120

ι 172 187

, IJ •• •• \l

140

160

2 0 8

223

.III • ι . Λ •• • ,J. L

180

200

I

[, , J , . | l l , l , l l , J l

220

240 (m/z)

Figure 3 and 4: ΕΙ-MS of 2-aminoacetophenone (ΑΑΡ) detected in distillate from micromilled a stored milk powder without (Figure 3) and with (Figure 4) background subs from GC/O information.


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Abundance (counts)

Figure 5


2000000

1500000 C2-acid 1000000

15.67 acetoin

500000

100000

hexanal

heptanal nonanal 12.05 7.43 9.66 I 14.37

Κ]

Χ ^ί» ij ι, Ψ* Λ, ••• ••», L •»•»» |4V

4.00

Abundance (counts) 737

30.00 Time (min)

20.00

10.00

hexanal

Figure 6

nonanal 14.36 3000000

C8-acid 2000000

26.98 octanal 12.01

1000000

200000 4.00

10.00

C2-aci

Gas Chromatography-Olfactometry

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