Recently, several simultaneous determination methods of macrolide antibiotics have been developed by mass spectrometry coupled with HPLC [18-20].. The aim of this study is to develop a m
Trang 1Ma cro lid e s are fre qu e n tly u se d in v e te rin a ry
m e dic in e a s th e ra pe u tic a n d p re ve n tive a ge n ts for
various diseases It is difficult to determ ine m ac rolide s
sim u ltan e o u sly w ith c on v e n tion a l m e th od s d u e to
th e ir sim ilar stru ctu re s A sim u lta n e ou s an a lys is for
e ry th rom y cin , rox ith rom y cin , tiam u lin an d tylo sin
w ith LC/MS h a s be e n de v e lo pe d Se pa ratio n w a s
pe rfo rm e d on C18 re v e rs e d p h as e co lu m n Mo bile
ph a se w as g rad ie n tly flow e d w ith 10 m M am m on iu m
ac e ta te a n d m e th an o l Th e m a ss s pe c tro m e te r w as
ru n in th e po sitiv e m od e a n d s e le ctiv e ion m o n itorin g
m od e Th e m ole cu lar ion s w e re [M+H]+ form a t m /z
837.5 for erythromycin, at m/z 859.5 for rox ith rom y cin ,
at m /z 494.2 for tia m u lin a n d a t m /z 916.7 for tylo sin
Lim its of d e te c tion w e re in th e ran g e fro m 0.001 to
0.01 ㎍/g low e r th an th e ir MRLs.
Ke yw ard s : simultaneous determination; liquid
chromato-graphy/mass spectrometry; macrolides antibiotics
1 Introduction
Macrolide antibiotics have 12-, 14-, 16- or 17-membered
macrocyclic lactone ring, which is bound to several amino
and/or neutral sugars (fig 1) Because of their effective
an tim icrobial activity again st Gr am -posit ive bacteria,
my-coplasma, chlamydia, they are frequently used in industrial
animals to treat and prevent diseases or as growth
promotants [1]
Incorrect use of these antibiotics may leave residues in
edible tissues causing toxic effects on consumers, e.g.,
allergic reactions in hypersensitive individuals, or indirectly,
problems through the induction of resistant strains of
bacteria [2] Therefore, the Sourth Korea has set maximum
residue limits (MRLs) for macrolide antibiotics in edible
tissues of food-producing animals The MRLs of erythromycin
and tylosin are 0.1 g/kg in bovine and porcine In case of
＊Corresponding author: Hyo-In Yun
Tel: +82-42-821-6759 Fax: +82-42-822-5780
E-mail: hiyun@hanbat.chungnam.ac.kr
poultry, those are 0.125 g/kg for erythromycin and 0.1 g/kg for tylosin In order to monitor macrolide residues, simple, confirmatory and simultaneous analytical methods are required
Microbiological assays were widely used for determination
of macrolide antibiotics [3, 4] Unfortunately, these methods could not be used for simultaneous analysis due to lacks of their specificities Gas chromatography-mass spectrometry (GC-MS) supplies good sensitivity and selectivity [5], but direct analysis for macrolides antibiotics is difficult because
of their thermal labile property and low volatility
Liquid chromatographic methods have been reported for the determination of macrolide antibiotics: UV absorption [6-11], fluorimeteric [12-14], ch e m ilu m in e s cen ce [1 5] a n d ele ct r och e m ica l d e t ect ion [1 6, 17 ] m et h od s h a ve be en u s ed for d et e r m in a t ion , b u t t h e s e m e t h od s h a ve s h ow n h igh lim it s of d et ect ion
Recently, several simultaneous determination methods of macrolide antibiotics have been developed by mass spectrometry coupled with HPLC [18-20] The determination methods of macrolides by LC/MS have advantages such as high specificity and selectivity due to each molecular mass The aim of this study is to develop a more simple, rapid and effective method for the simultaneous determination of three macrolide antibiotics (erythromycin, roxithromycin and tylosin) and a pleuromultlin antibiotic (tiamulin) by LC/MS with electrospray interface Although tiamulin does not belong to a group of macrolide antibiotics, we determined this drug due to its similar structure to tylosin
2 Materials and methods 2-1 Ch e m ic als an d re ag e n ts
Erythromycin, roxithromycin and tylosin were supplied
by S in il C h em ica ls (S e ou l, K or ea ) T ia m u lin w a s s u p p lie d b y
D a es u n g M icr obia ls (S eou l, K or ea ) H P L C gr a d e w a t er a n d
m et h a n ol w er e p u r ch a s e d fr om J T & B a k e r (N e w J er s e y,
U S A) R e a ge n t gr a d e a m m on iu m a ce t a t e w a s p u r ch a s ed
fr om S igm a (M is s ou r i, U S A).
T h e in d ivid u a l s t ock s t a n d a r d s olu t ion s w er e p r e p a r e d a s
1 m g/㎖ in methanol and working standard solutions were prepared weekly by dilution of stock standard solutions with
Simultaneous Determination of Various Macrolides by Liquid Chromatography/Mass Spectrometry
Youn-Hwan Hwang, Jong-Hwan Lim, Byung-Kwon Park and Hyo-In Yun*
Division of Veterinary Pharm acology and Toxicology, C ollege of Veterinary Medicine, C hungnam National U niversity
Rece ived J a n 4, 2002 / Accept ed Ap r 29, 2002
Trang 2m e t h a n ol All s t a n d a r d s olu t ion s w er e s t or e d a t 4℃ and
were stable for at least 1 month under this condition
Deionized or distilled water of 18.2 ㏁ cm-1 resistivity was
used throughout the experiment
2-2 Instrumentation and chrom atographic co n ditio n s
Samples were analyzed by a Hewlett-Packard 1100 series
LC/MSD system It consisted of a G1322A degasser, a
G1312A binary pump, a G1315A photo-diode-array detector,
a 59987A electrospray interface and a 5989B mass spectrometer
The separation was performed on Nova-Pak C18 reverse
phase column (4 ㎛, 3.9 mm x 150 mm I.D., Waters, USA)
Analytical system was operated with a gradient elution at
flow rate of 0.5 ㎖/min The mobile phase consisted of 10
mM ammonium acetate (A) and methanol (B) Gradient
runs were programmed as follows: 100% B for 3 min,
decrease from 100% to 90% B for 6 min, decrease from 90%
to 5% B for 6 min, 5% B for 5 min, re-equilibration with
100% B for 5 min, post-run with 100% B for 10 min, until
the next sample injection
The nebulizer gas was flowed at 45 p.s.i., 350℃ and 9.0
l/min and quadrupole was heated to 100℃ Mass spectrometer
was run in the positive mode and scan mode from m/z 100
to 1000 Fragmentation voltage was 100 V Analysis was
carried out at the room temperature
2-3 Calibratio n c u rve s an d th e lim it o f de te ctio n
Calibration curves have been constructed by plotting area
against the standard concentrations of macrolides in the
range of 0.001 ㎍/㎖ ~ 5 ㎍/㎖
Limit of detection (LOD) and limit of quantitation (LOQ) were based on the signal-to-noise ratio based on their areas The signal-to-noise ratio of 3 was accepted for the LOD and that of 10 for the LOQ
3 Results 3-1 Ch ro m ato gra ph ic s e p ara tion
All drugs used for the experiment were separated under the adopted conditions within 18 min (Fig 2) Each separation of erythromycin (15 min), roxithromycin (16 min), tylosin (12 min) and tiamulin (14 min) was achieved successfully, on the same chromatogram
3-2 Ma ss s pe c tra
For each molecule, the produced ions on mass spectra were the molecular related ion [M+H]+, two adduct ions [M+Na]+ and [M+K]+, and several fragmentation ions (Fig
3) The molecular ions, [M+H]+, at m / z 734.5, 837.5, 494.4
and 916.5 for erythromycin, roxithromycin, tiamulin and tylosin were represented dominantly Except tiamulin, other drugs produced two adduct ions, [M+Na]+ and [M+K]+, at
m / z 756.5 and 772.5 for erythromycin, at m / z 859.5 and
875.5 for roxithromycin, at m / z 938.5 and 954.5 for tyloisin
(Fig 3) The appearance of fragmentation ions was due to the dissociation of amino or sugar moieties on the structure
of drugs The m / z 576.5 and 679.5 in mass spectra of
erythromycin and roxithromycin were corresponding with
F ig 1 The structure of erythromycin (A), roxithromycin (B), tylosin (C) and tiamulin (D).
Trang 3a [M-desosamine+H]+ The m / z 158.1 of erythromycin and
roxithromycin was corresponding with a [desosamine + H]+,
and the m / z 115.1 of roxithromycin was corresponding with
a [cladinose-OCH3 + H]+ The fragment ions of tiamulin,
m / z 192.1 was a moiety of [2-(diethylamino)-ethyl, thio]
acetic acid dissociating from m olecu la r ion T h e fr a gm e n t
ion s of t ylos in , m / z 74 2.5 a n d 77 2.5 w er e cor r e s p on d in g
w it h [M -m ycin os e+H ]+ a n d [M -m yca r os e +H ]+, r e s p e ct ively.
Th es e r es u lt s w e r e s u m m a r ized in T a b le 1
3 -3 L i n e a r i t y a n d t h e li m i t o f d e t e c t i o n
All exp e r im en t e d d r u gs in t h e r a n ge of 0.0 01 ㎍/g ~ 5㎍/g
showed good linearity, with correlation coefficient of
0.99(Ta ble 2) Th e lim it of d et ect ion a n d lim it of q u a n t it a t ion
r a n ge d fr om 0 0 01 t o 0 0 1 ㎍/g and from 0.005 to 0.05 ㎍/g
(Ta ble 3 ), r e s p e ct ively T h e s e figu r es w er e m u ch low e r t h a n
t h e M R L s s et u p b y t h e S ou t h K or e a
4 D is cu s si o n
LC /M S w a s h igh ly s e n s it ive a n d s ele ct ive for t h e
s im u lt a n eou s d et er m in a t ion of m a cr olid es com p a r in g w it h
ot h er p u b lis h ed m et h od s S e ver a l m et h od s w er e r e p or t ed for
s im u lt a n eou s d e t er m in a t ion of m a cr olid e s S im u lt a n eou s
d e t e r m in a t ion m et h od s by H P L C w it h U V d et ect or [6-11 ]
h a ve be en d eve lop e d , b u t t h es e m e t h od s a r e d ifficu lt t o
d e t e ct m a cr olid e s s u ch a s e r yt h r om ycin a n d r oxit h or m ycin
d u e t o t h e ir w ea k U V a b s or b a n ce Th e flu or im et r ic d et ect ion
w it h p r e-colu m n d e r iva t iza t ion p r oce d u r es r e qu ir e s lon g
s ep a r a t ion t im es a n d is le s s s en s it ive t h a n L C /M S [1 2-14 ].
In a ddit ion , flu or im et r ic det ect ion is lim it ed for s im u lt a n e ou s
d et er m in t ion b eca u s e of t h e d iffe r e n t d er iva t iza t ion m et h od
of e a ch d r u g K ee s et a l [16 ] a n d D r ea s s i et a l [1 7] h a ve
r e p or t ed for t h e d e t e r m in a t ion m et h od s of er yt h r om ycin a n d
r oxit h r om ycin u s in g H P LC w it h e lect r och em ica l d e t ect or ,
w h ich is m or e s en s it ive t h a n U V d e t e ct or B u t , t h es e
m et h od s a r e d ifficu lt t o s et u p a n a lyt ic con d it ion b eca u s e
t h e d et er m in a t ion m e t h od s b y ele ct r ochemical detection are very sensitive to environmental condition
The determination method by gas chromatography-mass spectrometry (GC-MS) has been reported [5] This method needs the derivatization procedures for each macrolide, thus taking a long time for the determination of macrolides LC/
MS which omits the derivatization procedures was successfully applied to determine several macrolides Simultaneous de-termination methods by HPLC with UV detector [6-11] have been developed, but these methods are difficult to detect macrolides such as erythromycin and roxithormycin due to their weak UV absorbance The fluorimetric detection with pre-column derivatization procedures requires long separation times and is less sensitive than LC/MS [12-14] In addition, fluorimetric detection is limited for simultaneous determintion
because of the different derivatization method of each drug
Kees et al [16] and Dreassi et al [17] have reported for the
determination methods of erythromycin and roxithromycin
F i g 2 T ot a l ion ch r om a t ogr a p h y (TI C , A) of m a cr olid e a n d t ia m u lin E xt r a ct ion s ch r om a t ogr a p h y (E I C ) of t ia m u lin (B ),
t ylos in (C ), er yt h r om ycin (D ) a n d r oxit h r om ycin (E ).
Trang 4(C )
(D )
ig 3 T h e m a s s s p ect ra of erythromycin (A), roxithromycin (B), tylosin (C) and tiamulin (D)
Trang 5using HPLC with electrochemical detector, which is more
sensitive than UV detector But, these methods ar e difficu lt
to set up a nalytic con dition beca use th e determination
methods by electrochemical detection are very sensitive to
environmental condition
As described in above as to the simultaneous determination
of macrolides, there are several problems such as weak UV
absorption, long separation time and difficult derivatization
procedure Our method has solved previous problems by
application of liquid chromatorygraphy/mass spectrometry
(LC/MS) LC/MS minimizes chromatographic separation and
method development time in confirming the molecular
identities of the target substance
The partially overlapped peak in erythromycin observed
in Fig 3 needs some discussion in this study Macrolides
were generally composed of more than one structural
component In determination of macrolides, major components
were generally used as indicators to evaluate the residue
levels [7] However, minor components could be also
remained in edible tissues This peak in erythromycin
indicates a major component combined with a minor
component Its mass spectrum pattern was different from
that of major component Even changing the mobile phase, the minor component was not separated and was moved together with the major component The chromatographic property of partially overlapped peak in erythromycin is similar to that of the major component As this overlapped peak resulted from the addition of the minor component, LC/MS based on their molecular weight could identify the minor component
5 Conclusion
LC/MS with electrospray is a simple, rapid and effective technique for the simultaneous determination of macrolides The fragmentation patterns provide the confirmatory information of macrolides The relevance of these studies for the determination of macrolide in biomatrices remains further investigated
Reference
1 P re s co tt, J F an d B ag go t, J D. Antimicorbial Therapy in Veterinary Medicine (2nd ed.), Iowa State
D ru gs Mole cu lar m as s Mo le c u la r io n
[M + Na ]+
Add u ct ion s [M + N a]+ ; [M + K]+ Fra gm e n ta tion io n s Ery th ro m y cin
Ro xith ro m yc in
Tia m u lin
Ty los in
733.5 836.5 493.4 915.5
734.5 837.5 494.4 916.5
756.5; 772.5 859.5; 875.5
－ 938.5; 945.5
158.1; 558.3; 576.5 115.1; 158.1; 679.5 192.1
742.5; 772.5; 794.5
Ta ble 2 The linearity of the drugs
D ru gs
Equ a tion a
S lo pe (106) In te rce p t(104) Ery th rom y cin
Rox ith rom y cin
Tiam u lin
Ty lo sin
7.02 4.90 43.3 3.9
-1.93 -73.49 -65.12 -32.2
1.45 8.43 4.2 4.2
0.99 0.99 0.99 0.99
a is used for the back-calculation of the drug concentration in the sample, y=mx + b (m, the slope; b, the intercept; x, the amount; y, the area)
b is the residual standard deviation of calibration curve in the regression analysis and is represented as 105
Ta ble 3 LOD, LOQ and reproducibility of four drugs
Ery th ro m y cin
Ro xith ro m yc in
Tia m u lin
Ty los in
0.005 0.01 0.001 0.001
0.02 0.05 0.005 0.01
0.99 0.99 0.99 0.99
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