LingYC98_Quantitative Analysis of Antibiotics by Matrix

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Quantitative Analysis of Antibiotics by Matrix-assisted Laser Desorption/Ionization Time-of-flightMass Spectrometry

Yong-Chien Ling, Lihnian Lin and Yi-Ting Chen

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043

SPONSOR REFEREE: Professor Liang Li, Chemistry Department, University of Alberta, Edmonton, Canada.

Comparative studies of the matrix-assisted laser desorption/ionization (MALDI) of 30 antibiotics were

made using a-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 5,10,15,20-

tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin and meso-tetra(N-methyl-4-pyridyl)porphyrin matrices.

Most antibiotics generated intense protonated molecules in HCCA and DHB matrices, and sodium or

potassium adduct ions in porphyrin matrices. Using sulfonamide antibiotics as model compounds, DHB was

found to be the most suitable matrix for quantitative analysis. Detection limits were about 1 picomole.

Linear correlation (R2b 0.97), between the sample quantity over the range 1 to 100 picomole and the signal

response, was obtained when ratios of the sum of peak areas of protonated molecules and sodium adduct

ions, with reference to that of a structurally analogous internal standard (acetaminophen), were measured.

The precision was found to be in the range of 4 to 32 % relative standard deviation, dependent on the type

and concentration of the analyte. A simple acylation derivatization process was developed to confirm the

presence of suspected antibiotic residues. It is demonstrated that MALDI is a viable technique for the

quantitative analysis of low molecular weight antibiotics. # 1998 John Wiley & Sons, Ltd.

Received 19 January 1998; Accepted 21 January 1998Rapid Commun. Mass Spectrom. 12, 317327 (1998)

Antibiotics are administered to animals to promote growth

and to reduce the incidence of infectious disease. Themajority of all animal protein consumed in Taiwanoriginates from animals fed with antibiotics at some timein their lives. Antibiotic residues may consequently occur infood products such as milk, eggs or tissues. The potential forabuse from failure to adhere to prescribed treatmentprotocols and sufficient withdrawal times exaggerates thepublic concern about antibiotic residues in animal products.The misuse of antibiotics or other antimicrobial drugs,which hasten the evolution of resistant microbes, is heldpartially responsible for the re-emergence of infectiousdiseases as major threats to human health.1 The use ofreliable and rapid methods for antibiotic residues, to find

misuse and abuse of antibiotics, is urgently needed forprotecting the public health. The ROC Department ofHealth has issued standard methods for the analysis ofsulfonamide antibiotic residues in food for human con-sumption.2

Considering the number and variety of antibiotics used inanimal breeding, the need for simple, inexpensive andsensitive methods for identifying multiple residues of a widerange of antibiotics, and for providing confirmatory orquantitative information, is apparent. This demand iscurrently met by using microbiological or immunochemicalmethods for screening tests and physico-chemical methodssuch as gas chromatography (GC) and high-pressure liquid

chromatography (HPLC) for confirmatory tests. The

microbiology based methods are advantageous in batchthroughput, sample preparation and cost. The chromato-graphic methods are advantageous in specificity andconfirmation, i.e. they could reduce the possibility offalse-positive or false-negative results caused by matrixeffects generally observed in traditional screening meth-ods.3

Recent applications of mass spectrometric techniques forthe analysis of antibiotics usually employ chromatographictechniques such as GC,4,5 HPLC612 to separate theantibiotics before introducing them into the mass spectro-meter. These hyphenated techniques offer the most reliableresults often at the cost of sample throughput, which make

them less suitable for screening tests. This shortcomingmight be improved by using an appropriate pretreatmenttechnique and exploring the direct analysis and information-rich advantages of desorption ionization mass spectrometry.Matrix-assisted laser desorption/ionization (MALDI), com-bined with time-of-flight mass spectrometry (TOFMS)13,14,has become a powerful and increasingly popular tool for theanalysis of bio- and synthetic polymers.1517 The applica-tions of MALDI for the analysis of low molecular weightcompounds have been limited, however.1820

The advantages of MALDI include relatively inexpensivehardware, high detection sensitivity and high speed ofanalysis and sample throughput. These features, along withthe potential of providing quantitative information, have

stimulated us to evaluate MALDI-TOFMS for the directanalysis of antibiotic compounds. Several matrices werestudied first to evaluate their potential to desorb and ionizeantibiotics. Important parameters concerning the quantifica-

*Correspondence to: Y.-C. Ling, Department of Chemistry, NationalTsing Hua University, Hsinchu, Taiwan 30043.Contract/grant sponsor: National Science Council at the Republic ofChina; Contract/grant number: NSC 87-2113-M-007-037.

CCC 09514198/98/06031711 $17.50 # 1998 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 12, 317327 (1998)


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Table 1. Chemical structure, chemical formula and molecular mass information of the internal standard and antibiotic compounds

Compound Chemical structure Formula Molecular mass

Acetaminophen (AAP) C8H9NO2 151.16

Sulfaguaniline (SG) C7H10N4O2S 214.24

Sulfadiazine (SDA) C10H10N4O2S 250.28

Sulfamethoxazole (SMX) C10H11N3O3S 253.31

Sulfamerazine (SMR) C11H12N4O2S 264.3

Sulfamethizole (SMTZ) C9H10N4O2S2 270.33

Sulfamethazine (SMZ) C12H14N4O2S 278.32

Sulfaquinoxaline (SQX) C14H12N4O2S 300.33

Sulfadimethoxine (SDM) C14H14N4O4S 310.33

Clopidol (CLP) C7H7Cl2NO 255.32

Morantel (MRT) C12H16N2S 220.33

Nitrofurazone (NFZ) C6H6N4O4 198.14

Furazolidone (FZD) C8H7N3O5 225.16

Pyrimethamine (PYR) C12H13ClN4 248.71

Nalidixic Acid (NA) C12H12N2O3 232.23

Carbadox (CDX) C11H10N4O4 262.23

Chloramphenicol (CAP) C11H12Cl2N2O5 323.14

Thiamphenicol (TAP) C12H15Cl2NO5S 356.23

Gentamicin C1 (GEN) C21H43N5O7 477

Sulfathizole (STZ) C9H9N3O2S2 255.32

Oxolinic Acid (OA) C13H11NO5 261.24

Rapid Communications in Mass Spectrometry, Vol. 12, 317327 (1998) # 1998 John Wiley & Sons, Ltd.

318 QUANTITATION OF ANTIBIOTICS BY MALDI-TOFMS


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tion were studied, using the most prevalent sulfonamideantibiotics as model compounds. The use of a structurallyanalogous internal standard, the means to calculate themeasured response, the linearity and dynamic range of thecalibration curves, the detection limits and precision, were

discussed and measured. In addition, an acylation deriva-tization process was developed for confirmatory analysis toovercome the limitation that MALDI-TOFMS couldprovide only molecular mass information.

EXPERIMENTAL

Materials

Antibiotic compounds including sulfaguaniline (SG), sulfa-

diazine (SDA), sulfamethoxazole (SMX), sulfamerazine(SMR), sulfathiazole (SMTZ), sulfamethazine (SMZ),sulfaquinoxaline (SQX, sodium salt), sulfadimethoxine(SDM), morantel (MRT, citrate salt), nitrofurazone (NFZ),

Table 1. Chemical structure, chemical formula and molecular mass information of the internal standard and antibiotic compounds. cont.

Compound Chemical structure Formula Molecular mass

Trimethoprim (TMP) C14H18N4O3 290.32

Streptomycin (STR) C21H39N7O12 581.58

Piromidic Acid (PA) C14H16N4O3 288.31

Pennicillin G (PEN) C16H18N2O4S 334

Lincomycin (LIN) C18H34N2O6S 406.56

Tetracycline (TC) C22H24N2O8 444.43

Oxytetracycline (OTC) C22H24N2O9 460.44

Chlortetracycline (CTC) C22H23ClN2O8 478.88

Erythromycin (ERY) C37H67NO13 733.92

Tylosin (TYL) C46H77NO17 916.14

# 1998 John Wiley & Sons, Ltd. Rapid Communications in Mass Spectrometry, Vol. 12, 317327 (1998)

QUANTITATION OF ANTIBIOTICS BY MALDI-TOFMS 319


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furazolidone (FZD), pyrimethamine (PYR), nalidixic acid(NA), carbadox (CDX), chloramphenicol (CAP, watersoluble, balanced with 2-hydroxypropyl-b-cyclodextrin),thiamphenicol (TAP), oxolinic acid (OA), trimethoprim(TMP) and piromidic acid (PA) were obtained from Sigma(St. Louis, MO, USA). Gentamicin C1 (GEN, sulfate),sulfathizole (STZ), streptomycin (STR, sulfate), penicillinG (PEN, sodium salt), lincomycin (LIN, hydrochloride),tetracycline (TC, free base, trihydrate), oxytetracycline(OTC, dihydrate), chlorotetracycline (CTC, hydrochloride),erythromycin (ERY) and tylosin (TYL, 500 mg/ml), were

obtained from ICN Biomedicals Inc. (Aurora, OH, USA).Clopidol (CLP) was obtained from Kanto Chemical Co.(Tokyo, Japan). Table 1 lists the chemical structure,chemical formula and molecular mass of each antibioticused in this study.

Acetaminophen (AAP) used as an internal standard wasobtained from ICN Biomedicals Inc. (Aurora, OH, USA).The matrices, a-cyano-4-hydroxycinnamic acid (HCCA)33 mM in acetonitrile/methanol, and 2,5-dihydroxybenzoicacid (DHB) 100 mM in methanol/water, were obtained fromHewlett Packard Co. (Palo Alto, CA, USA). Porphyrinmatrix, 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphyrin (THPP) was obtained from Aldrich ChemicalCo. (Milwaukee, WI, USA) and meso-tetra(N-methyl-4-

pyridyl)porphyrin (TMPP) was synthesized in-house. Thederivatizing agent 4-acetamidobenzenesulfonyl chloridewas obtained from TCI (Tokyo, Japan). Solvents n-hexane,ethanol and acetonitrile were of Optima grade from Fisher

Co. (Fair Lawn, NJ, USA), and acetone, methanol anddimethyl formamide were of HPLC grade from Tedia Co.(Fairfield, OH, USA). Deionized water was obtained by aMillipore water purification system with a resistivity ofb18.3 M.

MALDI sample preparation

The stock solutions were prepared as 1 mg/mL using mostlymethanol (MRT, TAP, GEN, STR, and TYL used water;CLP and PA used CH3CN:H2O=1:1) and stored at 0C,

unless otherwise specified. The stock solutions were furtherdiluted and spiked with an appropriate amount of AAP.These mixture solutions were vortexed to give completedissolution and mixing. A 3 mL aliquot of matrix solutionwas mixed thoroughly with an equivalent volume of theabove mixture solutions to yield 25 ppm analyte (or 25,12.5, 2.5, 1.25 and 0.5 ppm standard) solutions, eachcontaining 12.5 ppm AAP. A 1 mL aliquot of each solutionwas then deposited on a gold-plated sample probe andallowed to crystallize using vacuum (102 torr) dryingconditions with a HP G024A Sample Prep Accessory.

MALDI-TOFMS analysis

A G2025A MALDI-TOFMS instrument (Hewlett-Packard,Palo Alto, CA, USA) was used to obtain the MALDI massspectra. The mass spectrometer was a linear TOFMS with a1 meter flight tube operated in positive-ion mode. The ions

Table 2. Characteristic MALDI-TOFMS ions obtained with HCCA and DHB matrices

HCCA DHBCompound [MH] [MNa] [MK] [MH] [MNa] [MK]

AAP 100 100

SG 49 100 55 100 10 2

SDA 48 100 26 100 71 16

SMX 100 59 32 100 33 51

SMR 100 21 15 80 100 13

SMTZ 100 60 55 100 60 27

SMZ 100 21 100 32 12

SQX 100 20 12 100 73 22

SDM 100 28 21 100 25

CLP 100 100 31 25

MRT 100 100

NFZ 100

FZD 100

PYR 100 100

NA 100 20 12 100 53 54

CDX

CAP

TAP

GEN 65 100

STZ 100 8 28 100 31 12

OA 100 53 35 100 57 30

TMP 100 100

STR 100 100

PA 100 81 32 100

PEN 86 100

LIN 100 34 22 100 14

TC 100 38 100 26

OTC 100 32 100 19

CTC 100 44 100 12

ERY 58 100 27 15 100 46

TYL 100 100 13

Not detected; unit is % relative intensity.




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were formed by irradiating the sample with a pulsed UVlaser beam (nitrogen laser, 337 nm, 5 ns pulse length) withan energy of $5 mJ, were accelerated to 30 keV, anddetected using a dual channel microchannel plate detector.HP G2025A software 3.00 running on an IBM-compatible586 PC was used for automated data acquisition, massspectra processing and quantitation. All analyses wereautomatically performed by adding ten successive scans,each with a signal-to-noise ratio greater than 15, from 10different areas in the 1 mL aliquot solution on the sample

probe, to generate an averaged MALDI mass spectra. Time-to-mass conversion was achieved by external calibrationusing the calibration kit (G2051A low molecular weightstandards) from Hewlett-Packard.

RESULTS AND DISCUSSION

Selection of matrix

The application of MALDI mass spectrometry to a widevariety of analytes is made possible by the advances inuseful matrix materials.1431 Other factors related to the co-matrix, matrix solution conditions, matrix pH and cationsavailability, the nature of sample crystallization, the analytedistribution in the matrix and matrix structure/acidity, on

ion formation in MALDI, have been reported.32,33,34,35,36,37Most matrices have been evaluated based on their efficiencyin desorption and ionization of macromolecules, however.

We therefore carried out a preliminary study to find the

Figure 1. The MALDI mass spectra of SMX, SMZ, SDM and AAP in (a) HCCA and (b) DHB

matrix.




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best matrix for the antibiotics selected. Matrices HCCA andDHB were tested first because they are popular and havebeen shown to be useful matrices for low molecular weightcompounds.1820 The characteristic ions in the MALDI-TOF mass spectra obtained with either matrix aresummarized in Table 2. The ions include protonatedmolecules [MH], and [MNa], [MK] adduct ions.Most spectra are dominated by intense [MH] ions.Figure 1 shows the typical MALDI-TOFMS spectra of

SMX, SMZ and SDM at a concentration of 25 ppm usingHCCA and DHB matrices. CDX, CAP and TAP failed toproduce detectable ions in both matrices. GEN and PENproduced detectable ions only in DHB matrix. NFZ and

FZD produced detectable ions in HCCA matrix only. Theseresults show that the matrix played an important role inMALDI analysis of antibiotics.

The limited resolving power of low-cost linear MALDI-TOFMS systems, such as the one used in this study,combined with interferences from low-mass ions introducedby the matrix, were previously considered to limit theapplication of MALDI in the low-mass region. We thereforeinvestigated the matrix effect upon the mass resolution,

calculated as m/D m (D m is the peak width at half-height).The mass resolution for most compounds is between 100and 300. A significant improvement in mass resolution wasobserved for SDM, STR and TMP in the HCCA matrix, for

Figure 2. The MALDI mass spectra of (a) THPP and (b) TMPP matrix.




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which the mass resolution was found to be 395, 530 and 380,respectively. SDM/matrix and TMP/matrix show similaroptical microgaphs (not shown). The thickness of theanalyte/matrix complex, measured using a profiler, wasabout 0.2mm. The STR/HCCA co-crystals were small(several mm) and distributed evenly throughout the probe

surface. The STR/DHB co-crystals are irregular in shape,and appear as large flat sheets ($25 mm). The better massresolution obtained with HCCA matrix might be partiallyattributed to the smaller crystals and more uniformdistribution of these analyte/matrix crystals on the sampleprobe surface.36

Matrices THPP (molecular mass 675 Da) and TMPP(molecular mass 678 Da) were studied, to avoid low-massion interferences occurring with HCCA and DHB.38,39,40

The absence of low-mass ion interferences with THPP andTMPP matrices is evident in the MALDI mass spectra (Fig.2). We therefore used these matrices to investigateantibiotics with molecular masses less than 300 Da, which

were known to be subject to interference by the low-massions generated by the HCCA and DHB matrices. Table 3lists the characteristic ions in the MALDI-TOF mass spectraobtained with THPP (and TMPP) matrix as well as withoutany matrix. The ions observed include protonated molecules[MH], sodium adduct ions [MNa], and potassiumadduct ions [MK]. It was noticed that, unlike whenHCCA and DHB were used, most spectra obtained usingTHPP and TMPP matrices were dominated by intense[MNa] or [MK] ions. In the absence of a matrix, i.e.under laser desorption ionization (LDI) conditions, the LDImass spectra were similar to the MALDI mass spectraobtained using THPP and TMPP matrices. However, thesignal fluctuations were more pronounced due to the overall

low intensity. The [MNa] ion became the dominantpeak, presumably due to the universal presence of sodium asa contaminant. SMTZ, CLP, PYR and PA failed to produceany detectable ions in either of these two high-mass

matrices. This is contrary to their behavior in HCCA andDHB matrices, when they all produced characteristic ions.TMP produced detectable ions in THPP matrix only. SDAand OA produced detectable ions only in TMPP matrix. It isnoted that CDX, which failed to produce detectable ions ineither HCCA or DHB matrix, also produced characteristic

ions in both THPP and TMPP matrices. The advantage ofthe absence of low-mass ion interferences with THPP andTMPP matrices is evident in the MALDI mass spectra ofMRT (Fig. 3). Spectral reproducibility from shot-to-shotis generally inferior to that obtained using HCCA andDHB matrices. The signal intensity obtained from thesematrices followed the order LDI` THPP` TMPP` DHB HCCA. Hence, subsequent quantitative analysis wascarried out using HCCA and DHB matrices.

Selection of internal standard

MALDI is an established method for qualitative analysis.

Examples of quantitative applications of MALDI areincreasing.1820,4157 Quantitative analysis using MALDIis still difficult due to the poor point-to-point repeatability,sample-to-sample reproducibility, and shot-to-shot signaldegradation.56,57 We tried to overcome these difficulties byusing an internal standard. An ideal internal standard shouldbe chemically similar to the analyte, chemically stableduring the analysis, completely resolved from the analytes,and close to the analytes in mass and concentration to avoidinstrumental errors.58 The evaluation of quantitation wascarried out using SMX, SMZ and SDM sulfonamideantibiotics as model compounds, which are the mostfrequently encountered antibiotic residues in animal pro-ducts in Taiwan.

An isotopically labeled analog of the analyte is an idealinternal standard. The possible overlap between the analytesand their isotopically labeled internal standards due to theinsufficient mass resolution of the linear TOF instrument

Table 3. Characteristic MALDI-TOFMS ions for internal standard and antibiotic compounds with molecular mass less than 400 Da,obtained with THPP, or TMPP matrix, or without any matrix

THPP TMPP no matrixCompound [MH] [MNa] [MK] [MH] [MNa] [MK] [MH] [MNa] [MK]

AAP 8 67 100 5 78 100

SG 98 100 46 100 100 82

SDA 53 100 100 59

SMX 51 100 45 100 100 88SMR 4 100 79 37 100

SMTZ 7 100 39

SMZ 70 100 81 100 46 100

SQX 7 100 48 16 100 35 100 8

SDM 80 100 60 100 49 100 16

CLP

MRT 100 100 100

NFZ 100 41 100 41 100 32

FZD 64 100 16 100 35 85 100

PYR 100

NA 100 15 3 100 37 100 13

CDX 100 26 32 100 31 52 100 19

STZ 11 100 53 89 100 20 100 70

OA 100 33 100 33

TMP 100 30 22 100 96

PA 11 100 91

Not detected; unit is % relative intensity.




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prevents us from using such an internal standard in practice.We therefore used a structurally analogous compound,AAP, as an internal standard. The MALDI-TOFMS spectraof SMX, SMZ and SDM, at concentrations of 25 ppm usingHCCA and DHB matrices, are shown in Fig. 1. The[MH] ions and [MNa] ions of all three analytes werepresent in either matrix (Fig. 1(b)). The characteristic ionsfrom AAP, including [AAPH] (m/z 152) and[AAPNa] (m/z 174), appeared in both spectra. The peakarea of the [SMXH] (m/z 254) ion was used for

quantitation. It was susceptible to interference from the[HCCAKNa-H] (m/z 250) ion in HCCA matrix.Subsequent quantitation studies were therefore performedusing DHB matrix.

Quantitative analysis

A series of standard solutions containing SMX, SMZ andSDM, at concentrations of 25, 12.5, 2.5, 1.25 and 0.25 ppm,and an internal standard (AAP) at 12.5 ppm concentration,were subjected to MALDI-TOFMS analysis. The absoluteamounts of analyte in 1 mL of these solutions, based on themolecular mass of 250 Da, were 100, 50, 10, 5, and 1picomole (pmol), respectively. We collected five averagedmass spectra for each solution. To reduce the effect of

instrumental fluctuations on quantitation results, we dis-carded two mass spectra which yielded the maximum andminimum values of the (Aanalyte/Ai.s) peak area ratio. Toevaluate the suppression effect on the signal of the less

Figure 3. The MALDI mass spectra of MRT in (a) THPP and (b) TMPP matrix.




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concentrated components by the more concentrated com-ponents, Aanalyte is measured either as the [MH]

ion peakarea or the peak area sum of [MH] and [MNa] ions.

Ai.s is the peak area of the [AAPNa] (m/z 174) ion from

the AAP internal standard.The comparative results of these two quantitation means

are presented in Table 4. The linear correlation coefficients(R2) ranged from 0.973 to 0.985 when using the peak area

sum of [MH] [MNa] to represent Aanalyte, whichare better than the values 0.962 to 0.977 obtained using thepeak area of [MH] alone. This might be due to the factthat the signal intensities of either [MH] or [MNa]

ions alone are highly matrix-dependent during MALDI.Proton transfer for [MH] and a gas-phase mechanism forformation of [MNa] are the two competing mechanismsfor the ionization step.59 The signal intensity of [MH] or[MNa] ions alone rapidly fluctuates. The sum of thesignal intensities of [MH] [MNa] ions remainedrelatively constant over a sufficiently wide range of analyteconcentrations, and thus better R2 values were obtained

when the peak area sum for ([MH] [MNa]) wasused to represent Aanalyte. These values of linear correlationcoefficient (R2) were greater than 0.97 over two decades ofanalyte concentrations in every case. The values of the

Figure 4. The MALDI mass spectra of SMZ in HCCA matrix (a) before, and (b) after,

acylation derivatization reaction.




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sensitivity factor (obtained from the slopes of the calibrationcurves) were similarly greater when the peak area sum of([MH] [MNa]) was used to represent Aanalyte. Thelimit of detection (quantitation) LOD (LOQ) was definedhere as the analyte concentration giving a peak in theMALDI spectrum with an area equal to the (meanbaselineN standard deviation), where N=3 for theLOD and 10 for the LOQ. The mean baseline (standarddeviation) is the measured average (fluctuations) measuredfrom a baseline located far away from the analyte peak. The

LOD and LOQ values follow the order of SDM`SMZ` SMX, and are in the low picomole range.In order to evaluate the signal suppression effect by the

more concentrated components on the less concentratedcomponents, the precision was obtained using two differentquantitation means similar to those used in Table 4. Theresults are summarized in Table 5. At larger concentrations,i.e. above 2.5 ppm, the results appear similar for the twomeans whereas a significant difference was observed at lowconcentrations. The precision is in the range of 4 to 32 %RSD, dependent on the type and concentration of theanalyte. For a higher concentration range, the averageprecision appears to be better and is about 10 % RSD. Thisresult suggests that the primary source of uncertainty in

MALDI quantitative analysis lies in the sample hom-ogeneity.

Confirmatory analysis

Confirmatory analysis is used to identify the residues withthe highest confidence and is generally mandatory in aregulatory environment. Typical confirmatory analysisemploys complex and expensive procedures which involvemass spectral analysis of the tentatively identified residues.MALDI is a soft ionization technique which uses molecularweight information to identify residues. The lack of MS/MScapability in a low-cost linear TOFMS does not allow us to

explore controlled dissociation and fragmentation forconfirmatory analysis. We therefore propose to selectivelychange the m/z value of the analytes through the followingchemical derivatization reaction and by inspecting the

presence of the targeted m/z to confirm the presence of thetentative residues.

The derivatives were obtained by acylating the sulfona-mides with 4-acetamidobenzenesulfonyl chloride underalkaline conditions. Figure 4 shows the MALDI massspectra of STZ in HCCA before and after derivatization.The base peak has shifted from the original peak ( m/z 256, inFig. 4(a)) to the targeted peak (m/z 453, in Fig. 4(b)), therebydemonstrating the feasibility of this approach.

CONCLUSIONS

Important parameters have been studied for quantitativeanalysis of antibiotics using MALDI-TOFMS. Most anti-

biotics generate a stable protonated molecule in HCCA andDHB matrices and sodium adduct ions in porphyrinmatrices. Carbadox, chloramphenicol and thiamphenicolfailed to generate any characteristic ions in any matrix. Toobtain quantitative results for sulfonamide antibiotics, wesuggest using DHB matrix and incorporating a structurallyanalogous internal standard acetaminophen (AAP) to over-come shot-to-shot and sample-to-sample variability. Theprecision of the method needs to be improved if it is to beapplied to a lower concentration range, however. Using anacylation derivatization process for confirmatory analysis,the limitation that MALDI-TOFMS provides only molecu-lar mass data could be overcome. We conclude that thelimited resolving power of the low-cost linear MALDI-

TOFMS systems is not an obstacle to obtaining usefulquantitative data. However, more work is required to searchfor new matrix materials and appropriate internal standardsspecific for analyzing different classes of low molecular

Table 5. Precision from MALDI analysis of SMZ, SMX and SDM of various concentrations with two different quantitation means

SMZ SMX SDMConcentration (ppm) [MH] [MH] [MNa] [MH] [MH] [MNa] [MH] [MH] [MNa]

25 12 8 18 8 9 9

12.5 10 9 15 11 13 10

2.5 10 5 24 21 5 4

1.25 31 16 13 13 38 25

0.25 28 28 32 32

the analyte signal is too low; unit is % relative standard deviation.

Table 4. Quantitative results for SMZ, SMX and SDM, calculated using two different quantitation means, and the corresponding LOD andLOQ values

[MH] [MH] [MNa] LOD LOQAntibiotic R2 SF R2 SF (pmol) (pmol)

SMZ 0.962 10.7 0.983 17.9 0.6 0.8

SMX 0.970 2.7 0.973 4.6 0.8 1.0

SDM 0.977 8.1 0.985 15.1 0.4 0.5

R2 linear correlation coefficient; SF : sensitivity factor; LOD : limit of detection; LOQ : limit of quantitation. See text for details.




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mass compounds. While the quantitative analysis of realsamples necessitates the incorporation of clean-up andconcentration pretreatment, the capability of MALDI-TOFMS to provide data on low picomole amounts ofantibiotics within a few minutes at low cost indicates thatMALDI-TOFMS continues to present itself as a viabletechnique for the quantitative analysis of antibiotics. Itcould serve as a sensitive and specific screen, and possiblyalso as final-step detection device.

Acknowledgement

We are grateful to Professor Chung-Sun Chung of the Department ofChemistry, National Tsing Hua University for providing the TMPPmatrix. The research was supported by the National Science Council ofthe Republic of China under grant No. NSC 87-2113-M-007-037.

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LingYC98_Quantitative Analysis of Antibiotics by Matrix

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