Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol82.Issue3-4.Dec1999:

Scientia Horticulturae 82 (1999) 193±201

Development of an automated system for pyruvic
acid analysis in onion breeding
Kil Sun Yoo*, Leonard M. Pike
Vegetable Improvement Centre, Department of Horticultural Sciences,
Texas A&M University, College Station, TX 77843, USA
Accepted 5 May 1999

Abstract
An automated system for pyruvic acid analysis was developed in order to screen a large number
of onion bulbs in our breeding program. The system includes two high performance liquid
chromatography (HPLC) pumps, an autosampler, a column heater, a spectrophotometric detector,
and an integrator. With this new method, human error can be eliminated with great repeatability
(CV ˆ 0.9%), and we can run one sample per min. In a test using five cultivars, this automated
system showed highly significant correlations with the HPLC and the spectrophotometric method
(r2 > 0.99), and estimated about 7% and 17% more pyruvic acid over the HPLC and
spectrophotometric methods, respectively. Interference by sugars (48 mg mlÿ1) and quercetin
compounds (68 mg mlÿ1) was about 0.1 mmoles mlÿ1 as pyruvic acid. # 1999 Elsevier Science
B.V. All rights reserved.
Keywords: Allium cepa L.; Flavor; Pungency; Breeding; Screening

1. Introduction
Onion tissues produce distinctive volatile sulfur compounds when the cells are
damaged mechanically. S-alk(en)yl-L-cysteine-sulfoxides, the flavor precursor
compounds, are hydrolyzed by the action of alliinase to produce thiopropanal
S-oxide (tear-causing factor), pyruvic acid, and ammonia (Whitaker, 1976). The
* Corresponding author. Tel.: +1-409-847-8848; fax: +1-409-862-4522
E-mail address: k-yoo@tamu.edu (K.S. Yoo)
0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 7 5 - 8

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thiopropanal S-oxide degrades into mono-, di-, or tri-sulfides through complex
chemical reaction, such as via thiosulfinates. Onion pungency has been estimated
by measuring one of the enzymatic reaction products by gas chromatography,
high performance liquid chromatography (HPLC), or spectrophotometric
methods. Because pyruvic acid measurement is simple and fast, it has been

widely used to estimate onion pungency since the 1960's.
The original method by Schwimmer and Weston (1961) (SW) includes
blending onions with water, filtering, centrifuging, and diluting before analysis.
This method was slow and tedious when used in a breeding program requiring
analysis of many samples. Several modifications are made to the original method
for fast analysis. Randle and Bussard (1993) developed a fast juice extraction
method by using a press instead of blending bulb tissues with water, and reported
that 120 samples could be extracted per day. Yoo et al. (1995) (Yoo) extracted
juice by blending onion tissues without adding water, then reacted the juice with
reagents without two steps of dilution. We saved time by eliminating dilution
steps and by reduced dish-washing work. Using frozen juice samples, two
technicians could process up to 250 samples a day by this method.
To improve the current analysis system, we developed an automated system to
avoid pipetting error and to reduce labor costs. This new method uses the same
principle as the original Schwimmer and Weston (1961) method, but is automatic
and should reduce human error.

2. Materials and methods
2.1. Apparatus
Two HPLC pumps (Model Acuflow II, Fisher Scientific, Pittsburgh, USA)

were used to deliver dinitro phenyl hydrazine (DNPH) and NaOH solutions
(Fig. 1). Each pump delivered 0.7 ml minÿ1 DNPH solution (25 mg DNPH and
25 ml phosphoric acid per liter) or 1.4 ml minÿ1 NaOH solution (20 g lÿ1). Onion
juice sample was filtered through a 0.45 mm nylon filter (microfilter) and a 5 ml
sample was injected into the DNPH solution by an autosampler (Model 570,

Fig. 1. A flow diagram of the automated system for pyruvic acid analysis.

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Alltech, Deerfield, IL, USA) at an interval of 1 min. The mixture of onion juice
and DNPH was heated in a 3 m long HPLC tubing coiled in a column heater set
to 708C. The NaOH solution was mixed with the DNPH/onion juice at the end of
the heating coil. The final mixture was passed through a column
(4.6 mm  150 mm) packed with glass beads (0.2 mm). Detection was made
by a UV/Vis detector set to 485 nm and peak area was recorded by an integrator
or a Perkin-Elmer data collection system. Pyruvic acid standards between 0 and
12 mmoles mlÿ1 were used to quantify unknown samples. The DNPH solution

was kept at 58C in a refrigerator and there was no degradation for seven days.
2.2. Reproducibility test
Twenty identical juice samples were analyzed by both the Yoo and the
automated methods. Coefficients of variation (CV) were calculated from peak
areas or absorbency.
2.3. Background pyruvic acid and interfering compounds in onion juice
Onion bulbs (cv. `TG 1015Y' and `Texas Early White' from our storage and an
unknown onion from a local store) were cut in halves longitudinally. One half was
wrapped with plastic film and cooked completely in a microwave oven (5 min).
The tissue was blended in a home mixer for 2 min and juice was collected
through a paper filter. The other fresh half was blended and juice collected. The
juice sample was filtered again by the microfilter and injected into the automated
system.
Compounds in onion juice that may interfere with pyruvic acid analysis were
also tested. Sugar mixture (sucrose, glucose, and fructose mixture of 16 mg mlÿ1
each) and quercetin (68 mg mlÿ1) were injected and peak areas recorded. The
effect of protein compounds was tested by injecting microfiltered juice samples
with or without adding trichloro acetic acid in juice (3%).
2.4. Comparison with other methods using spiked samples
1 ml of 0±16 mmoles mlÿ1 pyruvic acid was added to 1 ml of identical onion

juice and the total pyruvic acid content was measured by the SW, Yoo, automated,
and HPLC methods. In the SW method, 49-fold diluted juice was used, while the
other three methods used undiluted juice. The HPLC method used an Alltech IOA
1000 organic acid column (300  7.8 mm) and solvent was 0.01 N sulfuric acid
at a rate of 0.5 ml minÿ1. A 20 ml sample was injected after microfiltration.
Column temperature was maintained at 458C and detection was made at 210 nm.
Sodium pyruvate standards were run with the samples and used to calculate the
unknown.

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2.4.1. Five cultivar samples
Longday onions with red, yellow, and white colors (cultivars unknown) bought
from a local store, `TG 1015Y', and `Texas Early White' (both from cold storage)
were used. Five bulbs of onions were used from each cultivar as replications.
Each onion bulb was cut into two pieces longitudinally and half was cooked as in
Section 2.3. The fresh and cooked halves were blended with the same weight of
water and juice extracted. The juice sample was filtered through a microfilter and

analyzed by the Yoo, automated, and HPLC methods. Correlation between the
Yoo method and the other two was calculated.
2.5. Application to screening of a breeding line
Forty onion bulbs of a breeding line were blended without adding water, and
juice samples were collected. These samples were frozen at ÿ208C, defrosted,
microfiltered, and analyzed by both Yoo and the automated methods. Correlation
between pyruvic acid concentrations by these two methods was calculated.

3. Results and discussion
The new automated system, without a true HPLC column, produced very
smooth and symmetrical peaks of a chromatograph, resembling a true
chromatogram (Fig. 2). After much trial and error, flow rates of 0.7 and
1.4 ml minÿ1 for the DNPH and NaOH, respectively, were found to be the
optimum condition for an analysis. A batch of 96 samples could be loaded to the
autosampler and about 500 samples could be analyzed per day. Because this
method needs no tubes or pipetting for mixing chemicals, we could save time and
eliminate human error in the analysis. On the other hand, microfilters must be
used to clean the juice, adding to the cost of analysis.
3.1. Reproducibility
The CV of the automated method was 0.9%, as compared with 1.7% of the Yoo

method. This result demonstrated that this new automated method is superior to a
conventional method in consistency because of automation and elimination of
human error.
3.2. Background pyruvic acid and interfering compounds
Background pyruvic acid content in `TG 1015Y', `Texas Early White', and a
long day onion were 0.2, 0.3, and 0.3 mmoles mlÿ1, while total pyruvic acid

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Fig. 2. A representative chromatogram of the automated pyruvic acid analysis system, with eight
levels of pyruvic acid standard and 10 onion samples. Numbers on the chromatograph are retention
times.

content were 3.3, 4.1, and 3.3 mmoles mlÿ1, respectively. These results indicate
that the automated system can accurately measure low levels of pyruvic acid
without much interference in onion juice. These background contents were
similar to our previous results using undiluted onion juice sample (Yoo et al.,
1995).

A total sugar mixture of 48 mg mlÿ1 and quercetin solution (68 mg mlÿ1)
resulted in peak areas equivalent to about 0.1 mmoles mlÿ1 pyruvic acid. Removal
of protein substances in the onion juice reduced its pyruvic acid content by about
0.1 mmoles mlÿ1, which was regarded as negligible. Therefore, this automated
system may over-estimate pyruvic acid content by about 0.3 mmoles mlÿ1 in
onions with a high sugar or quercetin concentration. This problem was thought to
be avoided by blending onion tissue with one or two volumes of water and
thereby reducing the concentration of interfering compounds. Microfiltration is
always required in sample preparation to avoid clogging the tubing of an
autosampler and a UV/Vis detector.

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Fig. 3. Pyruvic acid concentrations in onion juice as measured by Schwimmer and Weston (1961),
and Yoo et al. (1995), the automated, and HPLC methods after adding known amounts of pyruvic
acid.

3.3. Comparison with other methods using spiked samples

In the test of adding a known amount of pyruvic acid into onion juice, all
methods resulted in very linear increases (Fig. 3). The Yoo, automated, and
HPLC methods estimated very similar concentrations of pyruvic acid in juice.
The SW method requiring two-step dilution to obtain 49-fold dilution, however,
estimated about 0.7±1.0 mmoles mlÿ1 less than the other three methods. The
sticky nature of onion juice was thought to cause sample loss during pipetting, as
compared to a non-sticky standard solution, resulting in reduced concentrations
against the standard. Measuring pyruvic acid within a range of 8 mmoles mlÿ1
seemed to be more accurate than at the higher levels. This can be easily
accomplished by using 2- or 3-fold diluted onion juice. In this result, the
automated method proved to be a reliable way to accurately estimate pyruvic acid
content in onion juice.
3.4. Five cultivar samples
Onion samples with wide genetic backgrounds could be accurately measured
by the automated method. The automated and HPLC methods estimated about
17% and 7% over the Yoo method, respectively (Fig. 4(A) and (B)). Because the
HPLC method is believed to measure pyruvic acid specifically, as compared to
the non-specific Yoo method reacting with any carbonyl compounds, the actual
over-estimation was thought to be about 10%. Background pyruvic acid in the
cooked sample again were less than 0.8 mmoles mlÿ1, as similarly mentioned in

Section 3.2. In all `TG 1015Y' and `Texas Early White' onions, the background

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199

Fig. 4. Comparison of pyruvic acid analysis by the automated and HPLC methods with the Yoo et
al. (1995) method using juice extracted from paired fresh and cooked halves of five onion cultivars
(25 bulbs).

levels were less than 0.5 mmoles mlÿ1 and was considered negligible in a
breeding program.
3.5. Application to screening of a breeding line
In the actual screening of a breeding line, the automated system showed highly
significant correlation with the Yoo method (Fig. 5). The automated method,
using undiluted juice, estimated about 15% higher pyruvic acid than the Yoo
method, probably due to combined interference of sugars, quercetin, or other

Fig. 5. Comparison of pyruvic acid analysis by the automated and Yoo et al. (1995) methods in a
screening of an onion breeding line (N ˆ 40).

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compounds. Since the interference proportionally increased with pyruvic acid
concentration, some by-products of the alliinase reaction was thought to be the
compound. This deviation could not be eliminated even if diluted juice were used,
as in Sections 3.3 and 3.4.
3.6. Comparison with a commercial autoanalyzer
Because actual analysis has not been performed by an autoanalyzer (Scientific
Instruments, Westco, CT), a brief subjective comparison between the two systems
was made. The new automated system looks simpler in structure and is more
flexible to change flow rate, as compared with the autoanalyzer which has a
complex tubing system and fixed flow rate determined by diameter of a tubing.
The peak shape in our method resembled an ordinary HPLC peak with a smooth
baseline, and an ordinary integrator or a data collection system can be used to
record peak areas. The autoanalyzer, however, produced step- or wave-like
chromatograms, and peak height data can be measured manually or with a special
computer program. Over all, our automated system was thought to be equal or
more versatile and efficient than a commercial autoanalyzer.
3.7. Costs
Two HPLC pumps for $4000; an autosampler for $7000; a column heater for
$1500; a UV/Vis detector for $8000; an integrator for $1500; and tubing and
other materials for $500. Total cost is about $22 500.

4. Conclusion
A new automated system using two HPLC pumps, an autosampler, and a
detector was demonstrated to be a consistent and accurate way to measure
pyruvic acid content in onion juice. The advantages of this system were the
elimination of pipetting error and the speed of analysis. Using this system, we
could run about 500 samples per day, compared to 250 samples by our current
method employing two people. This system has proven to be effective in a mass
screening of onion bulbs in a breeding program and could be used in a
commercial analysis laboratory where fast, efficient, highly uniform results are
required.

References
Randle, W.M., Bussard, M.L., 1993. Streamlining onion pungency analysis. HortScience 28, 60.

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Schwimmer, S., Weston, W.K., 1961. Enzymatic development of pyruvic acid in onion as a measure
of pungency. J. Agric. Food Chem. 9, 301±303.
Whitaker, J., 1976. Development of flavor, odor, and pungency in onion and garlic. Adv. Food Res.
22, 73±133.
Yoo, K.S., Pike, L.M., Hamilton, B.K., 1995. A simplified pyruvic acid analysis suitable for onion
breeding programs. HortScience 30, 1306.