M. R. Sevenants R. A. Sanders The Procter & Gamble Company Winton Hill Technical Center 6071 Center Hill Rd. Cincinnati, Ohio 45224
Anatomy afan Off- Flavor Investigation:
A superior experimental cake mix product was beset with an elusive, intermittent off-flavor problem which was delaying its introduction to the marketplace. The problem was first recognized during panel testing hy about 10% of the panelists. Another cake was baked from the ingredients associated with the complaint cakes, and our flavor experts evaluated it. Most found it t o he perfectly acceptable. However, they changed their minds when they observed sensitive people cough, contort, and generally react violently after tasting the cake. Everyone was convinced that we had a major problem-one complicated by the fact that our technical flavor experts, the authors included, were not sensitive to the offensive component(.$. The first step in the investigative sequence was to establish a coordinator. This person was a technical services representative who had access to the process, raw materials, and product development personnel. He could get appropriate samples quickly and 0003-2700/84/0351-293A$O 1S O / O 0 1984 American Chemical Society
relay our suggestions to process development personnel.
Analysis of the Complaint Sample We began hy finding an extraction procedure which removed the off flavor efficiently enough to obtain a reaction from the sensitive people. The cake mix was blended with methanol to remove the flavors from the fat, and the methanol fraction was reextracted with chloroform. The chloroform fraction caused a reaction among sensitive panelists similar to that caused by the off-flavored cakes. At this point we knew the following about the off-flavor compound Its odor and flavor were medicinal; it was anosmic, that is, it did not elicit a response from some people due to the lack of certain receptor sites in the olfactory system. The compound was reasonably volatile and was soluble in organic solvents. When extracted into base, the off flavor vanished. When the base fraction was acidified, the off flavor was regenerated, suggesting that the compound or compounds
were acids or phenols. The sample preparation scheme used a series of solvents to selectively remove the nonvolatile solids and concentrate the volatile off-flavor fraction. The initial methanol extraction dissolved the flavor and left behind the hulk of carbohydrate, protein, and lipid. Mixing equal portions of NaCIsaturated HzO and CHC13 with the MeOH fraction in a separatory funnel resulted in a methanol/H20-free CHCIs phase (100%recovery of CHCM. The addition of NaCI-satu-
ANALYTICAL CHEMISTRY, VOL. 56. NO. 2, FEBRUARY 1984
293 A
Hgure 1. Gas chromatogram of complaint cake mix extract
rated HzO provided the driving force to favor the partitioning of polar organics into the CHC4 phase. In some cases, polar lipids (emulsifiers etc.) were extracted into the CHC1, phase. These could be removed by concentratiug the sample to -1 mL followed by precipitation at low temperatures, for example -10 "C. The fat-free concentrate could have been analyzed b..
chromatographicmethods a t this point. However, our problem dictated that we continue with the classical separation method to obtain neutral, acid, and hasic fractions. Our focus was on the acidic fraction. The concentrate was analyzed by gas chromatography (GC) using a 170-m X 0.75-mm capillary column, wall-coated with P--h-.*.-- Onn" m-
(minx) 14:15
2051
2606 31:x) 3635 41:49 47:M 5239 5733 6248
Figure 2. GClMS chromatogram of extract of a mixture of minor ingredients 294A * ANALYTICAL CHEMISTRY, VOL. 56, NO. 2. FEBRUARY 1984
ure 1shows the flame ionization chromatogram of the extract from a suspect cake mix. People known to he sensitive to the off flavor were cajoled to the gas chromatograph where they profiled the aromas evolvingfrom a sniff port following separation on the 100 000-plate column. In the present case, a 5050 splitter allowed half the sample to go to the sniff port while the other half went to the flame ionization detector or the mass spectrometer (MS). The arrow in Figure 1denotes the region where great excitement among the sniffers suggested the elution of the off odor. Thorough examnation of the corresponding region in the GC/MS chromatogram convinced us that the aroma was not caused by anything even faintly resembling a peak in this chromatogram. Shortly afterward, our technical services people made a discovery which helped greatly in our efforts to clean up the chromatogram in the region where the off odor was detected. They found that minor ingredients of the complaint mixes, i.e., everything but shortening, flour, and sugar, when mixed together had the same objectionable odor and flavor, but much stronger than that detected in the cake mix itself. Even nonsensitive people could detect the aroma in the mixture of minor ingredients-salt, leavening agents, flavors, nutrients, and colors. Cakes made with these offflavor minor ingredients inevitably duplicated the cake off flavor in the complaint samples. Figure 2 shows the GC/MS chromatogram resulting from injection of an extract of a mixture of minor ingredients. The off odor eluted as indicated by the arrow. As we had come to expect, the arrow points at a valley rather than a peak. At the low pph levels involved in this problem, the offending component, if detectable a t all, was swamped by coeluents at higher concentrations despite the acidbase cleanup. We began a scan-hyscan evaluation of the mass spectra in the region of interest, looking for anything suspicious. Given the medicinal aroma and previous off-flavor experience with chlorophenols, we looked for halogenated phenols. Chlorine and bromine give unique isotopic distributions that are easy to spot-hut there were none. However, examination of the accurate mass results revealed an ion at nominal mass 234, which was very suspicious due to its negative mass defect. This ion was less than 2% of the base peak in the spectrum a t which it peaked; nevertheless, careful subtraction of coeluting peaks before and after revealed a spectrum similar to the one shown in Figure 3. The best formula fit to the measured mass of 233.951 was C7H701,and the presence (continued on p . 298 A )
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Structure, Synthesis, and Biological Activities
I
&a Figure 3. Mass spectrum of suspected offflavor Frank M. Unger, Editor Sandoz-Forschungsinstituf- in Concentrates on lipopolysaccharides, a significant biological component of the outei membrane of Gram-negative bacterial cells. Explores the relationship between the structure and the biological activity of LPS. Emphasizes chemical synthesis of particular structures of various domains of the LPS molecule. Examines the potential of synthetic production and of modified natural materials in preventative medicine and therapy. CONTENTS Structural Principles of Lipopolysaccharides and Biological Properlies of Synthetic Partial Structures * Lipopolysaccharide Antigens of Pseudwnones aeruginosa * Synthesis of Shigella llexnen 0-Antigenic Repealing Unils Pathways tor the Polymerizalion of
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Components * Chemical Sinthesis of Lipid A for Elucidation of SlrUCtUre-Activity Relationships * Convergent Approach to Synthesis 01 Lipid A and Its Analogs Synthetic Studies on Lipid A Component 01 Bacterial Lipopolysaccharide Synthetic Studies on Structural Elements of Hydrophobic Region Present in Bacterial Endotoxins
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ACS Symposium Series NO.231 330 Pages (1983) Clothbound LC 8515828 ISEN 08412-0080-X US &Canada 144.95 Export $53.95
of iodine was confirmed by the (M - I) fragment at M - 127. Iodocresol was proposed as a structure consistent with this mass spectrum, consistent with the measured molecular formula, and consistent with the observed aroma. Confirmation and Source of the Off Flavor
We needed to confirm that iodocresol was the culprit. This was accomplished by crudely synthesizing it. Molecular I p and p-cresol were heated together in carbon disulfide, producing a product which behaved in aroma, GC retention, and effect on cake flavor precisely as the off odor extracted from complaint mixes. So we were quite confident of the structure of the component responsible for the off flavor. Where did it come from? In addition to the analytical problems caused by iodonesol's anosmic nature and by its low odor threshold, wbicb caused it to affect the product even at levels undetectable by usual techniques, this problem continued to be annoyingly elusive for another reason. A selected-ion-monitoring GCIMS procedure for measuring iodocresol a t part-per-trillion levels in individual cake mix minor ingredients
Table 1. Determination of lodocresol in Extracts of Cake Mix Components Sunpl.
Cake mix Mixture of minor ingredients Minor ingredients with iodized salt Minor ingredients withoUt
iodized salt
298A
ANALYTICAL CHEMISTRY, VOL. 56. NO. 2, FEBRUARY 1984
Ioms,HoI (PFW
0.2 5.0 2.4
0.093
and in packaging materials was developed. Analyses of the individual ingredients and packaging materials revealed no iodocresol. And yet, combined mixtures of minor ingredients contained iodocresol at low ppb levels. We began to realize that a reaction was occurring after the mixing of ingredients. From previous GCIMS work, we knew that significant levels of cresol (a natural, approved flavor ingredient) were present in our lemon flavor and that iodine (at least in ionic form) was present in iodized salt. So we made our own mixture of minor ingredients, with and without iodized salt, and analyzed the extracts for iodocresol. The results are shown in Table I. Mixes made with iodized salt contained, on the average, about 25 times more iodocresol than those made with uniodized salt. And cakes baked with uniodized salt tasted fine. A major source, if not the only source. of the off flavor was identified. We recommended that uniodized salt be used in the product since elimination of cresol was judged to he nearly impossible without drastically affecting product flavor. Our technical services organization ensured that this policy was implemented. As a result, the problem was solved and bas not resurfaced. In many ways, the problem could not have been tougher. The off flavor was due to an anosmic compound which was not readily detectable organoleptically by our aroma experts; it affected the product in a noticeable way a t levels so low that analytical measurements were very difficult; and it was not present in any of the raw materials, but rather formed in situ due to a reaction of two otherwise innocuous components. Resolution of this difficult problem gives great confidence that practically any off-flavor problem can be solved.
