Journal of Chromatography A,815(1998)213–223
Determination of antibiotics in different water compartments via liquid chromatography–electrospray tandem mass spectrometry
a ,a a
b b
*Roman Hirsch ,Thomas A.Ternes ,Klaus Haberer ,Armin Mehlich ,Frank Ballwanz ,
c
Karl-Ludwig Kratz a
ESWE Institute for Water Rearch and Water Technology ,Soehnleinstras 158,D -65201Wiesbaden ,Germany
b
StVUA Detmold ,Westernfeldstras 1,D -32758Detmold ,Germany c
Institute for Nuclear Chemistry ,Strassmannweg 2,D -55099Mainz ,Germany
Received 9December 1997;received in revid form 17April 1998;accepted 17April 1998
居住证明怎么开Abstract
For the determination of 18antibiotics in water samples down to the lower ng/l range,an analytical multi method is prented.The analytes belong to different groups of antibiotics such as penicillins,tetracyclines,sulfonamides and macrolid antibiotics.Samples were enriched using a universal freeze–drying procedure or a solid-pha extraction facultatively.Analysis was performed by liquid chromatography with electrospray–tandem MS detection.Chromatography required different columns and eluents.Mean recovery rates were in excess of 70%,however,with one exception and a quantitation limit of 50ng/l for the tetracyclines and 20ng/l for all other antibiotics were t.©1998Elvier Science B.V .All rights rerved.
Keywords :Water analysis;Mass spectrometry;Antibiotics;Tetracyclines;Penicillins;Macrolid antibiotics;Sulfonamides
1.Introduction
the appearance of bacterial strains that are resistant to antibiotics which are important drugs for the One of the most relevant topics in today’s en-treatment of many rious infections.
一模一样的反义词vironmental analytical chemistry is water quality.In In Germany the annual production of antibiotics is order to characterize water quality,concentrations of in the range of 2000tons per year.One such main polar organic pollutants have to be determined.In group are the penicillins,with production rates in addition to pesticides,industrial chemicals and their Germany of approximately 900tons/year [4].Tetra-metabolites,pharmaceutical substances have ex-cyclines,sulfonamides and macrolid antibiotics are perienced a fast growing interest.Recent studies some additional important groups of antibiotics for have shown that a multitude of drugs are prent in which a proper analytical method in water down to aquatic systems [1–3].The interest in the analysis of the ng/l range has to be designed (e Fig.1).Intake antibiotic residues in the environment aris from the pathways into the aquatic environment result from fact that they are suspected of being responsible for
their applications in both human and veterinary medicine.After consumption the active compounds are often metabolized only partially and are then *ed via urine or feces [5].Thus,they are
0021-9673/98/$19.00©1998Elvier Science B.V .All rights rerved.PII:S0021-9673(98)00335-5
214R.Hirsch et al./J.Chromatogr.A815(1998)213–223
Fig.1.Structures of the investigated antibiotics.
R.Hirsch et al./J.Chromatogr.A815(1998)213–223215
Fig.1.(continued)
presumably prent in raw wage and may even Therefore,no estimation for the environmental in-leave the wage treatment plants(STPs)as they take of the substances can be made.As the have probably not been fully eliminated[2,3,6].compounds are most likely to be found at very low The u of tho substances as feed additives in concentrations in the different compartments of the veterinary medicine underlies only minor legal reg-aquatic environment,like STP influents and ef-imentation.Application amounts are not available.fluents,surface,ground and drinking water[7,8],it is
夸女孩子216R.Hirsch et al./J.Chromatogr.A815(1998)213–223
necessary to develop a nsitive procedure which has at2208C.They were always renewed after two the capability to confirm the prence of antibiotics months.All solvents were utilized in gradient grade down to the lower ng/ higher quality.
Most of the continuously monitored water con-
taminants are determined via gas chromatography– 2.1.Preconcentration
mass spectrometry(GC–MS).A paration of the
antibiotics mentioned herein via GC requires de-For the extraction of the analytes from water two rivatization of the polar moieties.In literature some alternative methods have been developed bad on methods for the determination of e.g.,chloram-solid-pha extraction(SPE)and lyophilization as phenicol and sulfamethazine via GC–MS have been described below.
described[9,10].However,as the analyte groups
show different properties concerning number and 2.1.1.Lyophilization
kind of functional groups(e Fig.1),it ems quite Prior to the lyophilization procedure,water with difficult to develop a universal derivatization pro-high natural organic matter(NOM),like STP ef-cedure suitable for all the substances.High-perform-fluents and surface water samples,werefiltered ance liquid chromatography(HPLC)coupled to a through a0.45-m m glassfibrefilter.Then100ml of tandem MS detector is a powerful technique for water wasfilled into a250-ml round-bottomedflask. paration,identification and quantitation of polar In order to prevent the formation of solid calcium compounds and was therefore chon as a preferable and magnesium carbonate,the cations were com-method.As,macrolid antibiotics,which are made up plexed by the addition of100mg of EDTA(di-of three structure elements,the erythronolide agly-sodium salt,purchad from Riedel-de Haen).After-
cone and two sugar moieties(desosamine and cladin-wards,the samples were frozen in an ethanol bath at o as described in Fig.1),show no measurable UV2308C.During the freezing procedure theflasks absorption in the spectrum of conventional deuterium were rotated to obtain a large ice surface and thus lamps,most publications concerning the determi-significantly reduce the overall drying time.Samples nation of the substances describe electrochemical were then mounted on a freeze-dryer for about15h. detection[11].Generally,most of the published Finally,the lyophilized samples were redissolved in methods concerning the determination of antibiotic1ml of phosphate buffer(c520mmol/l)and the drugs are designed for complex matrices like meat,resulting extracts were stored at2208C until mea-milk,blood etc.,and achieve only relatively high surement.Prior injection,the extracts werefiltered detection limits in the range of veral hundred through0.45-m m PTFE syringefilters(Schleicher ng/kg or ng/l,respectively[12–16].In addition and Schuell)to remove precipitated EDTA.The most of the applied extraction procedures are espe-phosphate buffer was prepared by mixing two solu-cially designed for only one special class of anti-tions of KH PO and K HPO,both20mmol/l,
2424
,tetracyclines.until a pH value of6.0was obtained.
The aim of this work was to establish an effective
羊的成语大全and simple multi-method in order to determine the 2.1.2.Solid-pha extraction
amounts of veral antibiotic substances via LC–As an alternative to the lyophilization method,a MS–MS down to the lower ng/l range.SPE method for the antibiotics(except tetracyclines)
was developed bad on methods that are currently
儿童绘画基础in u for a variety of medium polar substances.One 2.Experimental liter of the sample was glassfibrefiltered(e above)
and the pH was adjusted to3.0with H SO(c53
24 Reference compounds were purchad from Sigma mol/l).The solid pha material(100mg of Lichro-except for roxithromycin and clarithromycin which lute EN and250mg of Lichrolute C,both pur-
18
were supplied courtesy of the manufacturers(Rous-chad from Merck,Darmstadt)was manuallyfilled
l Uclaf,France and Abbott,Germany).Standard into glass cartridges and was concutively con-solutions were stored in phosphate buffer(e below)ditioned with332ml n-hexane,332ml methanol
R.Hirsch et al./J.Chromatogr.A815(1998)213–223217微不足道的意思
Table1
and632ml water(pH3.0).The sample was sucked
Mobile pha compositions for the three paration methods through the cartridge with aflow rate of approxi-
a a mately20ml/min.The cartridges were then dried
Time(min)Solvent A(%)Solvent B(%) for1h with nitrogen and eluted four times with1ml Method TET
methanol.The extracts were reduced to approximate-09010
1.79010
ly20m l in a gentle nitrogen stream and thenfilled
2.77525
up to1ml with phosphate buffer and stored at
153862
2208C until measured.
202377
210100
2.2.HPLC conditions250100
269010
359010
The HPLC system consisted of a Merck–Hitachi
L-6200pump connected to an AS-2000a autosampler a a
Method PEN Solvent C(%)Solvent D(%) and a D-6000interface.The LC conditions required09010
three types of columns and gradients.Tetracyclines89010
107030
were parated using a12533mm Macherey and
267030
Nagel Lichrospher100RP-8(end-capped)column(5
280100
m m)with a mobile pha consisting of20mmol/l
320100
oxalic acid in water–acetonitrile(method TET).Due349010
to the low pH of the gradient buffer,EDTA might419010
qq信箱precipitate after injection of the freeze-dried extract
a a
Method ANT Solvent C(%)Solvent E(%) and clog the capillary of the MS interface.To
07426 circumvent such problems,the HPLC solvent should
26238
be nt to waste during thefirst3min of the run as76238
the EDTA was hardly retained on the chromato-104852
150100
graphic columns and therefore elutes before the
保护眼睛的手抄报210100 analytes.Alternatively,the lyophilizate for the tetra-
257426
cycline determination should be reconstituted in the
337426
oxalic acid buffer to prevent a precipitation during
a Solvent A:1800ml10mmol/l oxalic acid1200ml acetonitrile; measurement.Penicillins were chromatographed
solvent B:800ml10mmol/l oxalic acid11200ml acetonitrile; utilizing a12533mm Merck LiChrospher100RP-solvent C:acetic acid was added to900ml water containing20
18(end-capped)column(5m m)with a mobile pha mmol/l NH until a pH of5.7was obtained.To this100ml of
3
consisting of10mmol/l ammonium acetate in
acetonitrile was added;solvent D:400ml of solvent C1600ml
acetonitrile;solvent E:200ml of solvent C1800ml acetonitrile. water–acetonitrile(method PEN).For the remaining
antibiotics a12533mm Merck LiChrospher RP-18
column(5m m)with a mobile pha containing10flow-rate of1l/min synthetic air with a pressure of
mmol/l ammonia acetate in water–acetonitrile250kPa and an approximateflow-rate of0.7l/min
(method ANT)was utilized.The injection volume was utilized as nebulizer gas.The interface had a
was50m l.Solvents and gradients are shown in temperature of608C.The HPLCflux was split1:10
Table1.in the interface resulting in an approximate spray
flux of40m l/min.Orifice voltages varied generally 2.3.MS–MS parameters from60to70V,depending on the best mass signal
of the ionization products.
The system utilized was a Perkin-Elmer Sciex API MS–MS parameters were optimized in continuous
III plus triple stage quadrupole mass spectrometerflow mode as follows:after determination of the best
with electrospray ionization.Except for chloram-conditions for the isolation of the precursor ion
phenicol,the analysis was performed in positive ion(mostly proton or ammonium adduct of the respec-
mode.Nitrogen was ud as curtain gas with a tive analyte)the ion spray voltage,quadrupole and
