American Journal of Analytical Chemistry, 2011, 2, 294-302
doi:10.4236/ajac.2011.22037 Published Online May 2011 (http://www. SciRP.org/journal/ajac)
Occurrence of N-Acyl Homoseri ne L acton es i n Extra cts of
Bacterial Strain of Pseudomonas aeruginosa and in Sputum
Sample Evaluated by G a s Chromatography—Mass
Spectrometry
Susheela Rani1, Ashwini Kumar1, Ashok Kumar Malik1, Philippe Schmitt-Kopplin2
1Department of Chemistry, Punjabi University , Patiala, India
2Institute for Ecological Chemistry, Helmholtz Center Munich, German Research Center for Environmental Health,
Neuherberg, German y
Received October 8, 2010; revised January 10, 2011; accepted May 16, 2011
Abstract
This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry
(GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of
bacterial strain of Pseudomonas aeruginosa and sputum sample of a cystic fibrosis patient. This method in-
volves direct separation and determination of AHLs by using GC-MS as simultaneous separation and cha-
racterization of AHLs were possible without any prior derivatization. Electron ionization resulted in a com-
mon fragmentation pattern with the most common fragment ion at m/z 143 and other minor peaks at 73, 57
and 43. The limit of detection for N-butanoyl, N-hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and
N-tetradecanoy l homoserine lactones was 2.14, 3.59, 2.71, 2.10, 2.45 and 2.34 µg/L, respectively. The pres-
ence of AHLs in the culture of P. aeruginosa strain and sputum of a cystic fibrosis patient was achieved in
selected ion monitoring (SIM) mode by using the prominent fragment at m/z 143.
Keywords: Gas Chromatography—Mass Spect r ometry, N-Acyl Homoser ine Lactone (N-Butanoyl,
N-Hexanoyl, N-Octanoyl, N-Decanoyl, N-Dodecanoyl and N-Tetradecanoyl) Homos er ine
Lactone, Sputum Sample, Bacterial Strain
1. Introducti on
N-acyl homoserine lactones (AHLs) are important inter-
cellular signaling molecules used by many bacteria to
monitor their population density and control a variety of
physiological functions in a cell-density-dependent man-
ner by the process called quorum sensing. Quorum sens-
ing involves synthesis and detection of extra cellular
signals termed as auto inducers. Many Gram-negative
bacteria like Pseudomonas aeruginosa, Vibrio fischeri,
Agrobacterium tumefaciens etc., use acyl homoserine
lactones (AHLs) as cell-cell communication molecules.
When a threshold bacterial density (and corresponding
AHL concentration) is reached, AHLs interact with tran-
scriptional activators to trigger the expression of target
genes. Members of the LuxI family of proteins synthes-
ize these signals. These signal molecules diffuse from
bacterial cells and accumulate in the medium as a func-
tion of cell growth.
Diverse gram-negative bacterial cells communicate
with each other by using N-acyl homoserine lactones
(AHLs) as signal molecules to coordinate gene expres-
sion with cell population density during invasion and
colonization of higher organisms [1-3]. These AHL sig-
nal molecules are commonly referred as quorum sensing
because the system enables a given bacterial species to
sense when a critical population density has been
reached in the host and in response activate expression of
target genes required for succession [4,5]. All AHLs are
characterized by a homoserine moiety and a fatty acyl
group that differ in having various lengths, ranging from
4 to 18 carbons. They also have 3-oxo, 3-hydroxyl, or
fully reduced methylene group at C-3 position or have an
unsaturated bond within the side chain [6,7]. The acyl

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295
group of all natural AHLs of bacterial strain has even
number of carbon atoms.
Nowadays, the determination of AHLs is of great in-
terest, and development of reliable and sensitive analyti-
cal methods for their structural characterization can pro-
vide the information to elucidate their biological signi-
ficance and activities. A wide variety of analytical pro-
cedures for the analysis of N-acyl homoserine lactones
was developed and summarized in the literature [8-14,
20-25]. Screening for AHLs production from bacterial
strains is based on bacteriological monitoring system,
alone [8-10] or in simultaneous combination of these
monitoring systems [11]. Using high performance liquid
chromatography with tandem mass spectrometry in con-
junction with chemical synthesis, Throup et al. [12] iden-
tified two signal molecules and showed them to be
N-hexanoyl-L- homoserine lactone (HHL) and
N-3-oxo-hexanoyl-L- homoserine lactone (OHHL). The
production of OHHL was confirmed by Jacobi et al. [13]
by thin layer chromatography in combination with an
Escherichia coli AHL biosensor. A robust method based
on solid-phase extraction (SPE) followed by ultra high-
pressure liquid chromatography is proposed for the de-
termination of five derivatives of N-acyl homoserine
lactones [14]. Pseudomonas aeruginosa is a highly rele-
vant opportunistic pathogen and is the most common
gram-negative bacterium found in nosocomial, compli-
cated and life threatening infections of immunosup-
pressed patients [15]. Patients with cystic fibriosis are
especially disposed to P. aeruginosa infections and for
these persons; the bacterium is responsible for the high
rates of morbidity and mortality [16,17]. It can also co-
lonize implanted devices, catheters, heart valves or den-
tal implants [18]. Using different assays, a broad range of
AHLs was detected in cell-free supernatants of bacterial
cultures of P. aeruginosa [19 -22]. Shaw et al. [23] re-
ported the occurrence and separation of 3-hydroxy forms
of N-hexanoyl, N-octanoyl, and N-decanoyl-L-homose
rine lactones in the supernatant of A. tumefaciens cul-
tures with thin layer chromatography. AHLs are the dif-
ficult compounds analyzed by the UV detectors as they
have absorbance maxima at low wavelength and,
coupled with the high background of commonly used
solvents [24]. Moreover, these molecules are produced at
very low concentrations, so the conventional techniques
cannot be used. For this reason, Charlton et al. repo r ted a
GC/MS method for the quantification of 3-oxo AHLs
based on their derivatization with pentafluorobenzyloxime
derivatives [25]. The capillary separation techniques were
also developed in conjunction with mass spectrometric
detection for the analysis of AHLs from small sample vo-
lumes in Burk- holderia cepacia [27 -29]. Analyses of
N-acyl-L-homoserine lactone in some gramnegative bac-
teria were also report ed us ing GC / MS method [30,31].
The main aim of the present work is to develop a pro-
cedure for the direct separation and detection of AHLs
by GC/EI-MS. The method was applied for the analysis
of Pseudomonas aeruginosa extracts and sputum sample
of a Cystic fibrosis patient when a prominent fra gment
ion at m/z 143 was selected as a marker in selected ion
monitoring (SIM) mode of mass detection.
2. Experimental
2.1. Chemicals and Reagents
N-Acyl homoserine lactone standards (N-butanoyl, N-
hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and
N-tetradecanoyl homoserine lactones) were obtained
from Sigma-Aldrich (Steinheim, Germany) and kept at
–4˚C. Stock solutions for standards were prepared by
dissolving these samples in acetonitrile at a con c entr a tion
of 1000 mg/L. The stock solutions were kept at – 4˚C
and could be stored over a four week period. Acetonitrile
was purchased from Merck (Mumbai, India). Standard
solutions were prepared by diluting the stock solutions
with acetonitrile. For GC-MS analyses, the stock solu-
tions were mixed and diluted with acetonitrile to the de-
sired concentration. Pure helium gas was delivered to the
GC- MS system as a carrier gas.
2.2. Microorganism’s Growth Conditions and
Sample Extraction
The bacterial strain of P. aeruginosa Microbial Type
Culture was grown in growth media. The composition
used for the growth media was beef extract (1 g/L), yeast
extract (2 g/L), peptone (5 g/L) and sodium chloride (5
g/L). Cultures were grown in an Erlenmeyer flask con-
taining 50 ml growth media at a temp erature of 37˚C for
48 hours. Liquid mediu m were inoculated with the strain
and were sub-cultured every week. Cell suspension was
centrifuged at 4000 rpm for 20 min and 10 ml of cell-free
supernatant harvested during the stationary growth phase
was extracted using the procedure described previously
[30]. In brief, 10 ml of cell-free supernatant solution was
extracted three times with an equal amount of chloro-
form. The combined organic phases were washed with 2
mL of water and taken to dryness in the oven kept at
20˚C. The residue was redissolved in 2 mL of acetoni-
trile. This culture solution was stored at freezing temper-
ature until further use.
2.3. Sputum Sample Extraction
Sputum sample was collected from a cystic fibrosis pa-

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296
tient from a local clinic. The sample was centrifuged
1000 rpm for the 10 minutes. The supernatant was col-
lected and stored at –4˚C. Extraction was done with 4 ml
of chloroform using a separating funnel and then the or-
ganic layer was washed with equal amount of water to
remove any matrix impurity and was taken to dryness at
room temperature. The residue redissolved in 2 mL of
methanol. This sample was further extracted with solid
phase extraction using 7020-01 (100 mg/mL) monomer
octadecyl cartridge (J. T. Baker, USA) and SPE process
was conducted on Visiprep SPE vacuum manifold sys-
tem Supelco (Bellefonte, PA, USA). For optimal proce-
dure of RP-C18 SPE, cartridge was pre-conditioned with
2 ml of methanol, 2 mL of acetonitrile on an SPE mani-
fold. Then, sample aliquots of 1 mL were passed through
the cartridge at a flow rate of 1 mL/min. After that, the
cartridges were dried by vacuum for 3 min and finally
eluted with 2 mL of acetonitr ile.
2.4. GC/MS Instrumentation and Conditions
Gas chromatographic mass spectrometric (GC-MS) sys-
tem with model GC-MS-QP2010 Plus (Shimadzu Cor-
poration, Kyoto, Japan) was used for the analysis. The
capillary column used in the GC was Rxi-1ms (30 m ×
0.25mm ID × 0.25μm) supplied by Restek U.S. (Belle-
fonte, PA, U.S.A.). Chromatographic data were collected
and recorded by GC-MS Real Time Analysis software.
Sample injection was done in split mode (split ratio 1 0:1).
Helium was used as the carrier gas at a flow rate of 1
mL/min. The GC injector temperature was set at 270˚C.
The column oven temperature was optimized to hold at
100˚C for 1 minute and then to increase by 10˚C/min up
to 200˚C, then increase by 15˚C/min up to 260˚C and
then by 30˚C /min up to 300˚C. Mass spectrometry con-
ditions were as follows: electron ionization source set at
70 eV, MS source temperature 200˚C and solvent cut
time was 3.5 minutes. The mass spectrometer was run in
full scan mode (m/z 20-500) and in (SIM) mode at 143
m/z. The quantitation of samples was done by using the
SIM mode.
3. Results and Discussion
3.1. Optimization of GC-MS Conditions
Standard solutions of AHL were assayed by GC/MS
without derivatization using full scan acquisition mode.
Using different oven temperature programs, the condi-
tions were optimized. The best results in terms of selec-
tivity and analysis time were obtained using an oven
temperature gradient starting from 100˚C to 300˚C. De-
tection was performed in selected ion monitoring (SIM)
mode by using fragment at m/z 143. A chromatogram of
a mixture of six AHLs separated under the optimized
experimental conditions is shown in Figure 1. As it can
be seen, a very good separation of all AHLs was achieved
in analysis time of 19 minutes (Table 1). All peaks of
AHLs in the standard solution were detected and identi-
fied from the respective mass spectra (Figure 2.). The
mass spectrum of each compound shows a molecular ion
[M]+. that was characteristic of each homoserine lactone
(Table 1). A common fragmentation abundant ion was
observed for all AHLs at m/z 143 and other minor peaks
at 73, 57 and 43. A similar mass spectrum was reported
by Pearson et al. [32] for the N-butyryl homoserine lac-
tone (mol. wt. = 171) purified from P. aeruginosa.
As shown in Scheme 1, the fragment ion at 143 m/z is
the most likely due to McLafferty rearrangement, which
is a typical of carbonyl groups having a hydrogen atom
in the γ-position. This rearrangement gives rise to an
enolic fragment and an olefin. Two major fragmentation
Figure 1. Typical GC/MS chromatogram of a mixture of BHL, HHL, OHL, DHL, dDHL and tDHL in acetonitrile at a con-
centration of 1 mg/L.

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297
Figure 2. Representative GC/EI-MS spectrum recorded for N-Acyl homoserine lactone.
Table 1. Data of the N-acyl homoserine lactones investigated.
Homoserine lactones (HL) Acronym Melting point (˚C) Molecular ion [M]+ Retention time (min)
N-butanoyl HL BHL 89.8 171 8.629
N-hexanoyl HL HHL 102.6 199 11.178
N-octanoyl HL OHL 105.8 227 13.351
N-decanoyl HL DHL 112.5 255 15.108
N-dodecanoyl HL dDHL 115.3 283 16.740
N-tetradecanoyl HL tDHL NA 311 18.602
Scheme 1. Electron impact formation of ion at m/z 143 due to cleavage of an acyl homoserine lactone by a McLafferty rear-
rangement.
processes involving an inductive effect and α-cleavage
produce the other fragment ions at 73, 57 and 43.
3.2. Method Validation
Calibration curves of bacterial culture and sputum sam-
ple spiked with AHLs standards were performed in the
range 1 - 500 µg/L on GC-MS with six concentration
levels. The calibration curves were described by the li-
near regression equation:
y = mx + c
where y is peak area, x is the concentration, m is the
slope and c is the intercept. The correlation coefficient
was in the range of 0.987 - 0.999. The limit of detection
(LOD) was set at the concentration when the signal/noise
ratio was equal to 3:1 and found in the rang e of 2.1 - 3.6
µg/L (Table 2). The quality control (QC) samples were
prepared with the concentration of 100 µg/L for AHLs.
The accuracy and precision were calculated for the QC
samples, both within and between days. The experiments
were done six times during six different days. The RSDs
for quality control samples were less than 6.24%. The
extraction recoveries of the AHLs were calculated by
comparing the peak areas of extracted QC samples from

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298
Table 2. GC-MS charact eristics of N-acyl homoserine lactones in standard solutions.
N-AHL BHL HHL OHL DHL dDHL tDHL
R2 0.9876 0.9979 0.9992 0.9889 0.9925 0.9957
Regression
Equation Y=7.7479x + 30.007 Y = 11.91x + 133.44 Y = 16.068x
– 100.71
Y=20.988x
– 281.12
Y = 22.627x
– 489.4
Y = 33.898x
– 1443.6
Slope (m) 7.7479 11.91 16.068 20.988 22.627 33.898
calibration range (µg/L) 10 - 1000 10 - 1000 10 - 1000 10 - 1000 10 - 1000 10 - 1000
LOD* = 3*S/N (10 µg/L) 2.14 3.59 2.71 2.10 2.45 2.34
Intra-day R.S.D*. (%) 2.66 1.56 1.82 1.91 2.43 1.58
Inter-day R.S.D*. (%) 2.98 1.57 2.13 2.27 2.57 1.94
Retention Factor 1.86 2.72 3.45 4.03 4.58 5.20
Selectivity factor 1.45 1.26 1.16 1.13 1.13
Resolution 26.27 17.52 15.48 16.07 12.01
*Each value is a mean of three measurements.
the bacterial culture and sputum sample to the peak areas
of analyte standard solutions.The recovery of all AHLs
in the sputum sample was found in the range of 94% -
99% and in the Pseudomonas aeruginosa was 93% -
98%. The statistical data (Table 3) reveals that the pro-
posed method is acceptable for the quantification of
AHLs in the real samples.
3.3. Real Sample Analysis
Using the optimized experimental conditions, the analy-
sis of P. aeruginosa extract was performed. Three su-
pernatant samples were extracted and injected into the
GC-MS system. Chromatogram for the extracts of P.
aeruginosa in full scan mode and corresponding SIM
mode for m/z 143 has been shown in Fig ure 3(a) and (b),
respectively. The chromatograms recorded in full scan
mode shows the interference in the identification of
AHLs by intense signals of matrix. Analysis of bacterial
extract in SIM mode provides signals for five AHLs effi-
ciently. From the retention time and the characteristic
fragment ion at m/z 143, the occurrence of BHL, OHL,
DHL, dDHL and tDHL was confirmed and the respective
concentrations of all five AHLs were obtained from real
samples and RSD values were less than 6.5% (Table 4).
These values of concentrations obtained were below the
other reported methods. Therefore, the developed me-
thod is highly sens itive and is suitable for the determina-
tion and quantification of AHLs highly expressed by the
bacteria.
AHLs were extracted from the sputum sampl e with the
same procedure as mentioned above. The chromatograms
in full scan mode (Figure 4(a)) and SIM mode (Figure
4(b)) were obtained for the sputum extract and the same
common ion peak was obtained at m/z 143. In the spu-
tum extract, only two AHLs i.e. OHL and DHL were
obtained and the amount recovered were 3.47 and 3.44
µg/L, respectively (Table 4).
Significant differences in the AHL recovery profile
were observed in this study and already published data
for the same bacterial culture (Table 5). Most studies to
detect P. aeruginosa in vivo have focused on detection of
3-oxo-C12 AHL and C14 AHL using reporters that are not
completely specific for these AHLs. Middleton et al. [21]
reported that the predominant AHL present in one spu-
tum sample was C6 AHL. Erickson et al. [26] found
Table 3. Results for determination of N-acyl homoserine lactones from spiked Sputum samples and extracts of bacterial
strain of Pseudomonas aeruginosa.
Sample Nominal (µg/L) Amount of BHL found
(µg/L)
Amount of
HHL found
(µg/L)
Amount of
OHL found
(µg/L)
Amount of
DHL found
(µg/L)
Amount of
dDHL found
(µg/L)
Amount of
tDHL found
(µg/L)
Sputum sample 100 95.59 97.60 96.57 98.75 94.23 95.38
Pseudomonas
aeruginosa culture 100 93.78 95.32 97.89 97.34 96.45 96.74
RSD% 6.23 5.67 5.98 4.67 5.91 5.54

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299
(a)
(b)
Figure 3. G C-MS chromatogram of an extract of cell-free supernatant of Pseudomonas aeruginosa in the TIC mode (a) and in
SIM mode at m/z 143 (b).
Table 4. Results for determination of N-acyl homoserine lactones in extracts of bacterial strain of Pseudomonas aeruginosa
and Sputum sam ples.
Sample Amount of BHL
found (µg/L) Amount of HHL found (µg/L) Amount of OHL found (µg/L) Amount of DHL found (µg/L) Amount of dDHL found (µg/L) Amount of tDHL found (µg/L)
Sputum sample _ _ 3.47 3.44 _ _
Pseudomonas aeru-
ginosa culture 2.78 _ 4.89 2.34 2.75 3.51
RSD % 6.45 5.86 5.62 5.88 5.39
the significant amounts of 3-oxo-C8 AHL and 3-oxo-C12
AHL in one of the sputum sample. All these studies
suggest that it is important to identify specific AHLs
present in clinical specimens and in a bacterial species
that often produce more than one AHL, thus, the use of
reporters to detect the AHLs produced in the bacterial
culture is very complicated and problematic. Thus, de-
finitive identification of AHLs would require mass spec-
trometry analysis. M. Frommberger et al. [28] detected
N-acyl homoserine lactones using mass spectrometry
along with capillary zone electrophoresis by studying the
alkaline hydrolytic product of the same. In our study, six

S. RANI ET AL.
300
(a)
(b)
Figure 4. GC-M S chromatogram of the sputum sample of a cystic fibrosis patient in the TIC mode (a) and in SIM mode (b) at
m/z 143.
Table 5. Comparison of AHLs recovered in developed method with literature data.
Sample In our optimized method In Published data Reference
Sputum sample of
Cystic fibriotic patient OHL, DHL HHL, OHL, DHL, ODHL, [20]a
Pseudomonas
aeruginosa
BHL, OHL, DHL, d DH L,
tDHL
BHL, HHL, OHHL, OHL, ODHL, OdDHL [19]a
OdDHL, ODHL, OHL [20]b
HHL, OdHL [21]c
OtDHL, ODHL, OOHL [25]d
BHL, DHL, d-DHL [31]e
Method of analysis employed: abiosensor strain Escherichia coli; bLC-tral-luxCDABE-based reporter; cLC-MS TLC; dGC-MS with derivatization; eDirect
analysis on GC-MS.

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different AHLs were separated and identified in the
spiked samples by GC-MS without any chemical mod-
ification. Five of these AHLs were identified in real
sample of P. aeruginosa culture and two AHLs were
found in the sputum sample of cystic fibrosis patient.
Cataldi et al. [31] found the three-homoserine lactones
i.e. N-butanoyl, N-decanoyl and N-dodecanoyl homose-
rine lactones in the P. aeruginosa culture in the range
between 0.92-1.07 mg/L. In our study, AHLs are deter-
mined at lower concentration levels than the other re-
ported me- thod [31]. Therefore, the developed GC-MS
method is relatively rapid, reliable and almost 1,000
times more sensitive than the previous methods and
could be used for assessing the patterns of AHLs produc-
tion duri n g gr owth of bacteria at different growth states.
4. Conclusions
A primary aspect of this work is to establish an assay for
analyzing the AHLs from bacterial cultures and sputum
samples. A major goal is to keep a minimal extraction
procedure to enable rapid, reproducible and GC com-
patible sample prepar ation. SPE is used fo r the ex traction
of AHLs in the sputum sample. This is a reliable and
convenient procedure for indicating the presence of AHL
in real sample without us ing any chemical derivatizatio n.
It provides more comprehensive alternative to current
bacteriological techniques for the detection of AHLs in
biological extracts. The GC-MS technique can be used
for characterizing AHLs on a routine basis, a necessary
requirement for assessing potential health risks asso-
ciated with microbial spoilage and to improve the under-
standi ng of A HL s behavior.
5. Acknowledgements
The authors are pleased to acknowledge the financial
support from UGC, New Delhi vide Project No. F-33-
293(2007).
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