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Sensors and Materials, Vol. 36, No. 3 (2024) 823–830
MYU Tokyo
S & M 3564

Determination and Imaging of Lactones in Beef
by Girard’s Reagent T Derivatization TechniqueHitomi Shikano,1 Kazunori Matsumoto,2 Fumika Honma,2
Naoaki Obana,3 Kazuaki Yoshinaga,1 Yuka Takada,1Naoto Ishikawa,1 Meguru Hara,4 Daisaku Kaneko,5 and Shu Taira1*
1Faculty of Food and Agricultural Sciences, Fukushima University, Kanayagawa, Fukushima 960-1248, Japan
2National Livestock Breeding Center, 1 Odakurahara, Nishigo, Fukushima 961-8511, Japan

3National Livestock Breeding Center Tottori Station, 14 Idekami, Kotoura, Tottori 689-2511, Japan
4Agricultural Promotion Division, Agriculture, Forestry and Fishery Department,

Fukushima Prefecture Government, 2-16 Sugitsuma-cho, Fukushima City, Fukushima 960-8670, Japan
5The Key Laboratory of Synthetic and Biological Colloids, Ministry of Education,
School of Chemical and Material Engineering, Jiangnan University, Wuxi, Jiangsu 214122, China(Received December 5, 2023; accepted January 9, 2024)

Keywords: imaging, lactone, derivatization, Wagyu beef

Derivatization methods improve ionization efficiency and chemical properties such as
molecular weight, polarity, and boiling point. Nonderivatized standard lactones evaporate overtime due to their volatility. Thus, optimal lactone detection should be performed without delay.
In this study, Girard’s reagent T-labeled-γ-nonalactone (m/z 270.2), γ-decalactone (m/z 284.2),and γ-undecalactone (m/z 298.3) were stably detected by matrix-assisted laser desorption/
ionization mass spectrometry regardless of analysis delay. In a mass spectrometry imaging
experiment, we analyzed the localization of lactones in beef meat (Japanese Black Wagyu) using
the derivatization method and identified a border between lactones and fatty acids such as freeoleic acid.

1.

Introduction

Derivatization methods have been used to improve volatility, thermal stability, and ionization
efficiency in liquid and gas chromatography as well as mass spectrometry (MS). An example ofa well-known derivatization reagent that is commonly used is (hydrazinocarbonylmethyl)
trimethylammonium chloride, also called Girard’s Reagent T (GirT).(1) GirT is the same asconventional carbonyl reagents such as hydrazines and semicarbazides, and forms a Schiff-basetype aqueous condensate (hydrazone inductor) with various aldehydes and ketones in an acetic
acid alcohol solution. After derivatization of the target molecule by GirT, the GirT-labeled (GirT-)
target molecule gains a hydrophilic property and quaternary ammonium group. The quaternaryammonium group imparts a cationic property. Thus, in MS measurements, we can easily detect
the target molecule even if the original chemical structure is difficult to ionize. In addition,*Corresponding author: e-mail: staira@agri.fukushima-u.ac.jp

https://doi.org/10.18494/SAM4813

ISSN 0914-4935 © MYU K.K.
https://myukk.org/

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Sensors and Materials, Vol. 36, No. 3 (2024)

another derivatization advantage of GirT is that volatile molecules such as flavor compounds
become stable due to an increase in their boiling point.(2)Japanese Black (Wagyu) cattle account for more than 90% of the beef cattle raised in Japanand are characterized by a high degree of marbling. Intramuscular fat is the fat deposited
between and within muscle cells and is also called Marbling or Sashi.
Recently, Wagyu beef has become increasingly popular in Japan and throughout the world,
where it is prized for its tenderness, juiciness and Wagyu aroma, three of its most desirable
qualities. Wagyu flavor is characterized by a sweet and fatty aroma that is produced when thebeef is heated and is considered to be unique to Wagyu beef. Lactones are the main components
considered to be responsible for the distinctive peach or coconut-like sweet aroma unique to
Wagyu beef.(3,4)
Although breeding and fattening methods are being actively studied to produce high-quality
Wagyu beef,(5) the location and number of lactones in the meat have not been investigated indetail. Therefore, we aimed to elucidate the localization of lactones using imaging mass
spectrometry. However, normal lactones evaporate at atmospheric pressure as well as undervacuum conditions. Thus, it was necessary to identify a derivatization method that permits
detection while avoiding evaporation under vacuum. Lactones have ketone groups in theirchemical structures that react with GirT, resulting in an increase in their boiling point following
derivatization. Derivatization methods are commonly used for chromatography analysis tochange the physical characteristics of target molecules, namely, altering their volatility
temperature and thermal stability, and to avoid column adsorption in gas chromatography(GC),(6) as well as to lower the detection limit in liquid chromatography.(7) In addition, GirT canalso yield ions that are detectable in positive mode due to the positive ions in its structure.
Mass spectrometry imaging (MSI) is an analytical method that can clarify the localization ofsubstances in tissue sections by performing matrix-assisted laser desorption/ionization(MALDI)-MS in two dimensions,(8,9) and it has been attracting increasing attention inagricultural food-related fields in recent years.(10,11) In addition, the fatty acid composition is anindicator of beef quality and is one of the most important parameters of Wagyu beef. We havealready reported a fatty acid analysis with a near-infrared fiber-optic detection method.(12)In this study, we aimed to derivatize lactones on beef sections using GirT and visualize thederivatized lactones using MALDI-MSI. Since oleic acid (OA) content is particularly important,localization analysis of OA was also conducted using MSI.

2.

Data, Materials, and Methods

One Japanese Black steer (31.6 months of age) obtained from the Japanese meat market wasused in this study. After slaughter, the longissimus thoracis muscle was collected and cooled for
14 days before being used as a sample for aging.(13) The beef samples were embedded into supercryo embedding medium (Section Lab Co., Ltd., Hiroshima, Japan), flash frozen in liquid N2,
and stored at −80 °C until use.

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2.1

825

MALDI-MS and MSI analysis of lactone standards

The following GirT-target solutions were prepared: 10 mM γ-nonalactone (γ-NL),γ-decalactone (γ-DL), and γ-undecalactone (γ-UdL) (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan) in methanol. A 100 μL aliquot of each sample solution was mixed with 100 μL of 100 mM
GirT (Tokyo Chemical Industry Co., Ltd.) in 20% acetic acid in a sealed 1.5 mL tube. The
mixture was allowed to react at room temperature for 10 min. After the reaction, each sample
was prepared at 5 nM, 50 nM, 500 nM, 5 µM, and 50 µM in methanol. For comparison,
individual nonderivatized target solutions were prepared. A 1.0 μL aliquot of GirT-lactones andnonderivatized target solutions were placed on a target plate using a pipette. A 15 mg/mL
solution of the matrix 2,5-dihydroxybenzoic acid (DHB) was suspended in 6 mL of 90%
acetonitrile and sprayed onto each spot using an automated pneumatic sprayer (TM-Sprayer;
HTX Tech., Chapel Hill, NC, USA). A total of 14 passes were sprayed using the following
conditions: flow rate of 130 μL/min, air flow of 10 psi, and nozzle speed of 800 mm/min.
Ionization of the GirT-lactones and nonderivatized targets was confirmed by MALDI-TOF-MS(rapifleX; Bruker Daltonics GmbH, Bremen, Germany). Signals between m/z 150 and 700 werecorrected in positive ion detection mode. The laser power was optimized to minimize the insource decay of targets. The same measurements were also taken 60 min after introduction to
the mass spectrometer to check for changes over time. To examine the detection efficiency of
GirT-lactones and nonderivatized lactones, spectral data from 20 locations were randomly
selected from each 50 μM sample spot, and the signal-to-noise ratio was calculated. We
calculated the limit of detection (LOD) and coefficient of determination (R2) from semilog plotsof the signal intensity versus amount to compare the sensitivity between GirT-lactones. LOD iscalculated as 3.3 × (s/a), where s is the standard deviation of the signal intensity and a is the slopeof the calibration curve.
2.2
2.2.1

MSI of beef sections
Preparation of beef sections for MSI detection of lactones

Beef samples were cut into serial sections (10 μm) using a cryostat (NX70; Thermo Fisher
Scientific, Waltham, MA, USA). The beef sections were transferred by thaw mounting onto
indium tin oxide glass slides as the conductive transparent electrode (Bruker Daltonics GmbH)for MSI. In the same manner, serial sections were prepared on glass slides for hematoxylin andeosin (H&E) staining. H&E staining was performed according to a standard protocol.
For MSI, a 60 mM GirT solution in 20% acetic acid was sprayed onto the beef sections using
an artistic airbrush (Procon Boy FWA Platinum 0.2 mm caliber airbrush; Mr. Hobby, Tokyo,
Japan). To enhance the reaction efficiency of GirT on sections, the GirT sprayed sections wereallowed to react at room temperature for 10 min. After derivatization, DHB was sprayed onto thesections using an automated pneumatic sprayer under the same condition as above. For themeasurement of OA, we used serial sections. A 6.7 mg/mL solution of the matrix 9-aminoacridine(9AA) was suspended in 6 mL of 70% ethanol and sprayed onto each spot using an automated826

Sensors and Materials, Vol. 36, No. 3 (2024)

pneumatic sprayer. A total of 8 passes were sprayed using the following conditions: flow rate of
150 μL/min, air flow of 10 psi, and nozzle speed of 1000 mm/min.
Ionization and imaging of the GirT-lactones and OA were confirmed by MALDI-TOF-MS(rapifleX; Bruker Daltonik GmbH). To detect the laser spot areas, the sections were scanned,
and the laser spot areas (200 shots) were detected with a spot-to-spot center distance of 80 μm ineach direction of the beef sections. Signals between m/z 200 and 1000 were corrected. The
section surface was irradiated with YAG laser shots in the positive ion detection mode forlactones and negative ion mode for OA. The laser power was optimized to minimize the insource decay of targets. The obtained MS spectra were reconstructed as MS images with a mass
bin width of m/z 0.1 from the exact mass using Flex Imaging 5.1 software (Bruker Daltonics
GmbH). Optical images of beef sections were obtained using a scanner (Nanozoomer,
Hamamatsu Photonics, Hamamatsu, Japan), followed by MALDI-TOF-MSI of the sections.

3.

Results and Discussion

3.1

Measurement of GirT-lactones by MALDI-TOF-MS and MS/MS analysis

The detected masses of GirT-standards, γ-NL (m/z 270.2), γ-DL (m/z 284.2), and γ-UdL (m/z298.3) increased by 114.1 Da compared with the original masses (MW 156.1, 170.1, and 184.2,
respectively) (Fig. 1). Tandem MS detected a characteristic signal representing a GirT-derived
fragment from GirT-lactones on both the standards and sections (Table 1). Representative MS

Fig. 1.

Schematic of (a) targeted lactones, (b) GirT, and (c) GirT-lactones and product ions. BP: boiling point

Table 1
Precursor ions and product ions for GirT-targets by tandem MS analysis
TargetPrecursor ion (m/z)
GirT-γ-nonalactone (standard)
270.2
GirT-γ-nonalactone (on section)
270.2
GirT-γ-decalactone (standard)
284.2
GirT-γ-decalactone (on section)
284.2
GirT-γ-undecalactone (standard)
298.3
GirT-γ-undecalactone (on section)
298.3

Product ion (m/z)
211.1
211.1
225.1
225.1
239.2
239.2

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spectra of GirT-γ-NL, GirT-γ-DL, and GirT-γ-UdL, are shown in Fig. 2. Unlabeled γ-NL was
marginally detected with high noise spectra (data not shown). Non-GirT-γ-DL and γ-UdL were
not detected. Product ions of GirT-γ-NL (m/z 211.1), GirT-γ-DL(m/z 225.1), and GirT-γ-UdL (m/z239.2) were correlated with the minus chlorine region (Δ59.1), indicating that GirT successfully
reacted with the target molecules on the standards and sample sections.
Next, the detection efficiencies of GirT-lactones and nonderivatized lactones were calculated
from the signal-to-noise (S/N) ratio (Table 2). The results showed that both GirT-γ-NL andnonderivatized γ-NL were detected, and the S/N ratio was 2.5-fold higher for GirT-γ-NL than for
the nonderivatized γ-NL. While GirT-γ-DL and GirT-γ-UdL were detected, nonderivatized γ-DL
and γ-UdL were not detected. This result suggests that the ionization efficiencies of γ-DL andγ-UdL are quite low. Although the S/N ratios for GirT-γ-DL and -γ-UdL were 8.2- and 2.9-foldlower, respectively, than that for GirT-γ-NL, GirT derivatization appears to enable the detectionof lactone family members with long carbon chains.
The detection efficiency over time was also examined. GirT-γ-NL showed no change in S/Nratio after 60 min compared with that at 0 min, whereas the S/N ratio for nonderivatized γ-NL
decreased by about 60%. For GirT-γ-DL and GirT-γ-UdL, no change in S/N ratio was observed
over time (Table 2).
Organic compounds with a boiling point (BP) below 523 K are defined as volatile organic
compounds. Thus, nonderivatized γ-NL, γ-DL, and γ-UdL (BP: 470, 493, and 516 K) were notpresent or their concentration decreased due to volatilization during measurement. In contrast,
the detection of GirT-lactones was stable over time. Using chemical information software

Fig. 2.

MS spectra of (a) GirT-γ-NL, (b) GirT-γ-DL, and (c) GirT-γ-UdL on section.

Table 2Comparison of detectable time between GirT-lactones and unlabeled lactones.
S/N ratios for derivatized standard lactones
0 min.
60 minTargetNonderivatization
GirT
Nonderivatization
γ-Nonalactone
123 ± 61305 ± 121
51 ± 32γ-Decalactone
N.D.
37 ± 18
N.D.
γ-Undecalactone
N.D.
103 ± 41N.D.

GirT
322 ± 15575 ± 3188 ± 39828

Sensors and Materials, Vol. 36, No. 3 (2024)(ChemDraw Prime 19, Hulinks, Tokyo, Japan), we estimated the theoretical boiling points of
GirT-γ-NL, GirT-γ-DL, and GirT-γ-UdL to be 786, 809, and 832 K, respectively, indicating that
the GirT-lactones did not volatilize during measurement.
The LOD for the GirT-lactones was 355-768 fmol (Table 3). Additionally, the coefficient ofdetermination (R 2) obtained from semilog plots of the signal intensity versus quantity indicatedhigh precision. These results indicate that the sensitivity is higher for GirT-lactones than for thenonderivatized lactones. Therefore, we investigated the localization of lactones on beef sections
using GirT-derivatized MSI.
3.2

MSI of beef sections

GirT-γ-NL, GirT-γ-DL, GirT-γ-UdL, and OA were detected on beef sections and imaged at
m/z 270.2, 284.2, 298.3 and 281.2, respectively. H&E staining and MSI images of GirT-lactones
and OA in beef sections are shown in Fig. 2. In this research, we imaged a small section toinvestigate whether there is a correlation between the production of lactones and adipose tissue.
The muscle and intramuscular fat regions were observed from the H&E images [Fig. 3(a)]. All
lactones were imaged in fat regions [Figs. 3(b)–3(d)]. On the other hand, free OA was mainly
localized in muscle regions [Fig. 3(e)]. OA is a precursor molecule of lactones and is considered
to be converted to lactones through aging and cooking;(14) however, the evidence supporting thisremains unclear.(15) If the OA present in fat tissues is used in the synthesis of lactones, theobserved OA intensity should be lower in fat tissues versus muscle regions. In fact, lactones
imaged at adipose region although OA marginally imaged at adipose region. The MSI datasuggests that unsaturated fatty acids, such as OA stored in intramuscular fat as neutral fat, areconverted to the lactones that are detected in the adipose regions on the sections. Moreover, mostfatty acids in muscle are incorporated in triacylglycerols and not as free fatty acids, althoughsome fatty acids are also present as phospholipids and free fatty acids. In addition, the plasmaTable 3
GirT-lactones for correlation coefficients and detection limits.
LactoneLOD (fmol)
GirT-γ-nonalactone
442
GirT-γ-decalactone
355
GirT-γ-undecalactone
768

Linearity (R2)
0.998
0.999
0.997

Fig. 3. (Color online) GirT-based imaging mass spectrometry of lactones and oleic acid. (a) H&E-stained image ofbeef section. Distributions of (b) γ-NL, (c) γ-DL, (d) γ-UdL, and (e) OA.

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borne long-chain free fatty acids readily enter skeletal muscle cells, after which they are oxidized
or esterified and a fraction remains free (non-esterified). Hence, free fatty acids in muscle are
preferentially used for life-sustaining energy production.(16)4.

Conclusions

To the best of our knowledge, this is the first study on the direct and simultaneous
visualization of several major aroma molecules in beef. This approach demonstrates the utility ofderivatization in MS measurements. Although quantification by MALDI-MSI can bechallenging because the signal intensity depends on many factors (e.g., ionization efficiency,
extraction efficiency from tissues, and sample preparation), the GirT reagent prevents thevolatilization of molecules such as lactones. MSI is a powerful tool for the direct visualization of
biomolecules in biological tissues. Our imaging results for the localization of lactones in fat
tissues suggest that OA is a precursor molecule of lactones, whereas free OA is mainly localized
in muscle. We anticipate that the clarification of the localization of lactones and OA, both ofwhich are important in Wagyu beef, in beef tissues will contribute to improvements in breeding
and fattening methods.

Acknowledgments
This work was supported by “Reconstruction and Revitalization of the Agriculture, Forestry
and Fisheries in Fukushima Prefecture” project, Grant-in-Aid for Scientific Research B(21H02133 to S.T.) and the LOTTE Foundation.

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