RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.

12, 317327 (1998)

Quantitative Analysis of Antibiotics by Matrixassisted Laser Desorption/Ionization Time-of-ight Mass 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), 5,10,15,20tetrakis(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 (R2 b 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 conrm 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 1998 Rapid Commun. Mass Spectrom. 12, 317327 (1998)

Antibiotics are administered to animals to promote growth and to reduce the incidence of infectious disease. The majority of all animal protein consumed in Taiwan originates from animals fed with antibiotics at some time in their lives. Antibiotic residues may consequently occur in food products such as milk, eggs or tissues. The potential for abuse from failure to adhere to prescribed treatment protocols and sufficient withdrawal times exaggerates the public concern about antibiotic residues in animal products. The misuse of antibiotics or other antimicrobial drugs, which hasten the evolution of resistant microbes, is held partially responsible for the re-emergence of infectious diseases as major threats to human health.1 The use of reliable and rapid methods for antibiotic residues, to find misuse and abuse of antibiotics, is urgently needed for protecting the public health. The ROC Department of Health has issued standard methods for the analysis of sulfonamide antibiotic residues in food for human consumption.2 Considering the number and variety of antibiotics used in animal breeding, the need for simple, inexpensive and sensitive methods for identifying multiple residues of a wide range of antibiotics, and for providing confirmatory or quantitative information, is apparent. This demand is currently met by using microbiological or immunochemical methods for screening tests and physico-chemical methods such as gas chromatography (GC) and high-pressure liquid
*Correspondence to: Y.-C. Ling, Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043. Contract/grant sponsor: National Science Council at the Republic of China; Contract/grant number: NSC 87-2113-M-007-037. CCC 09514198/98/06031711 $17.50

chromatography (HPLC) for confirmatory tests. The microbiology based methods are advantageous in batch throughput, sample preparation and cost. The chromatographic methods are advantageous in specificity and confirmation, i.e. they could reduce the possibility of false-positive or false-negative results caused by matrix effects generally observed in traditional screening methods.3 Recent applications of mass spectrometric techniques for the analysis of antibiotics usually employ chromatographic techniques such as GC,4,5 HPLC612 to separate the antibiotics before introducing them into the mass spectrometer. These hyphenated techniques offer the most reliable results often at the cost of sample throughput, which make them less suitable for screening tests. This shortcoming might be improved by using an appropriate pretreatment technique and exploring the direct analysis and informationrich advantages of desorption ionization mass spectrometry. Matrix-assisted laser desorption/ionization (MALDI), combined with time-of-flight mass spectrometry (TOFMS)13,14, has become a powerful and increasingly popular tool for the analysis of bio- and synthetic polymers.1517 The applications of MALDI for the analysis of low molecular weight compounds have been limited, however.1820 The advantages of MALDI include relatively inexpensive hardware, high detection sensitivity and high speed of analysis and sample throughput. These features, along with the potential of providing quantitative information, have stimulated us to evaluate MALDI-TOFMS for the direct analysis of antibiotic compounds. Several matrices were studied first to evaluate their potential to desorb and ionize antibiotics. Important parameters concerning the quantifica# 1998 John Wiley & Sons, Ltd.

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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) Sulfaguaniline (SG) Sulfadiazine (SDA)

C8H9NO2 C7H10N4O2S C10H10N4O2S

151.16 214.24 250.28

Sulfamethoxazole (SMX)

C10H11N3O3S

253.31

Sulfamerazine (SMR) Sulfamethizole (SMTZ) Sulfamethazine (SMZ)

C11H12N4O2S C9H10N4O2S2 C12H14N4O2S

264.3 270.33 278.32

Sulfaquinoxaline (SQX)

C14H12N4O2S

300.33

Sulfadimethoxine (SDM)

C14H14N4O4S

310.33

Clopidol (CLP)

C7H7Cl2NO

255.32

Morantel (MRT)

C12H16N2S

220.33

Nitrofurazone (NFZ) Furazolidone (FZD)

C6H6N4O4 C8H7N3O5

198.14 225.16

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

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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

tion were studied, using the most prevalent sulfonamide antibiotics as model compounds. The use of a structurally analogous internal standard, the means to calculate the measured response, the linearity and dynamic range of the calibration curves, the detection limits and precision, were discussed and measured. In addition, an acylation derivatization process was developed for confirmatory analysis to overcome the limitation that MALDI-TOFMS could provide only molecular mass information.
# 1998 John Wiley & Sons, Ltd.

EXPERIMENTAL Materials Antibiotic compounds including sulfaguaniline (SG), sulfadiazine (SDA), sulfamethoxazole (SMX), sulfamerazine (SMR), sulfathiazole (SMTZ), sulfamethazine (SMZ), sulfaquinoxaline (SQX, sodium salt), sulfadimethoxine (SDM), morantel (MRT, citrate salt), nitrofurazone (NFZ),
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Table 2. Characteristic MALDI-TOFMS ions obtained with HCCA and DHB matrices
Compound [MH] HCCA [MNa] [MK] [MH] DHB [MNa] [MK]

AAP SG SDA SMX SMR SMTZ SMZ SQX SDM CLP MRT NFZ FZD PYR NA CDX CAP TAP GEN STZ OA TMP STR PA PEN LIN TC OTC CTC ERY TYL

100 49 48 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 58 100

100 100 59 21 60 21 20 28 20 8 53 81 34 38 32 44 100

55 26 32 15 55 12 21 12 28 35 32 22 27

100 100 100 100 80 100 100 100 100 100 100 100 100 65 100 100 100 100 100 86 100 100 100 100 15 100

10 71 33 100 60 32 73 25 31 53 100 31 57 100 14 26 19 12 100 13

2 16 51 13 27 12 22 25 54 12 30 46

Not detected; unit is % relative intensity.

furazolidone (FZD), pyrimethamine (PYR), nalidixic acid (NA), carbadox (CDX), chloramphenicol (CAP, water soluble, 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), penicillin G (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 antibiotic used in this study. Acetaminophen (AAP) used as an internal standard was obtained from ICN Biomedicals Inc. (Aurora, OH, USA). The matrices, a-cyano-4-hydroxycinnamic acid (HCCA) 33 mM in acetonitrile/methanol, and 2,5-dihydroxybenzoic acid (DHB) 100 mM in methanol/water, were obtained from Hewlett Packard Co. (Palo Alto, CA, USA). Porphyrin matrix, 5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23Hporphyrin (THPP) was obtained from Aldrich Chemical Co. (Milwaukee, WI, USA) and meso-tetra(N-methyl-4pyridyl)porphyrin (TMPP) was synthesized in-house. The derivatizing agent 4-acetamidobenzenesulfonyl chloride was obtained from TCI (Tokyo, Japan). Solvents n-hexane, ethanol and acetonitrile were of Optima grade from Fisher
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Co. (Fair Lawn, NJ, USA), and acetone, methanol and dimethyl formamide were of HPLC grade from Tedia Co. (Fairfield, OH, USA). Deionized water was obtained by a Millipore water purification system with a resistivity of b18.3 M. MALDI sample preparation The stock solutions were prepared as 1 mg/mL using mostly methanol (MRT, TAP, GEN, STR, and TYL used water; CLP and PA used CH3CN:H2O=1:1) and stored at 0 C, unless otherwise specified. The stock solutions were further diluted and spiked with an appropriate amount of AAP. These mixture solutions were vortexed to give complete dissolution and mixing. A 3 mL aliquot of matrix solution was mixed thoroughly with an equivalent volume of the above mixture solutions to yield 25 ppm analyte (or 25, 12.5, 2.5, 1.25 and 0.5 ppm standard) solutions, each containing 12.5 ppm AAP. A 1 mL aliquot of each solution was then deposited on a gold-plated sample probe and allowed to crystallize using vacuum (102 torr) drying conditions 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 mass spectra. The mass spectrometer was a linear TOFMS with a 1 meter flight tube operated in positive-ion mode. The ions
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Figure 1. The MALDI mass spectra of SMX, SMZ, SDM and AAP in (a) HCCA and (b) DHB matrix.

were formed by irradiating the sample with a pulsed UV laser beam (nitrogen laser, 337 nm, 5 ns pulse length) with an energy of $5 mJ, were accelerated to 30 keV, and detected using a dual channel microchannel plate detector. HP G2025A software 3.00 running on an IBM-compatible 586 PC was used for automated data acquisition, mass spectra processing and quantitation. All analyses were automatically performed by adding ten successive scans, each with a signal-to-noise ratio greater than 15, from 10 different areas in the 1 mL aliquot solution on the sample probe, to generate an averaged MALDI mass spectra. Timeto-mass conversion was achieved by external calibration using the calibration kit (G2051A low molecular weight standards) from Hewlett-Packard.
# 1998 John Wiley & Sons, Ltd.

RESULTS AND DISCUSSION Selection of matrix The application of MALDI mass spectrometry to a wide variety of analytes is made possible by the advances in useful matrix materials.1431 Other factors related to the comatrix, matrix solution conditions, matrix pH and cations availability, the nature of sample crystallization, the analyte distribution in the matrix and matrix structure/acidity, on ion formation in MALDI, have been reported.32,33,34,35,36,37 Most matrices have been evaluated based on their efficiency in desorption and ionization of macromolecules, however. We therefore carried out a preliminary study to find the
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Figure 2. The MALDI mass spectra of (a) THPP and (b) TMPP matrix.

best matrix for the antibiotics selected. Matrices HCCA and DHB were tested first because they are popular and have been shown to be useful matrices for low molecular weight compounds.1820 The characteristic ions in the MALDITOF mass spectra obtained with either matrix are summarized in Table 2. The ions include protonated molecules [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 using HCCA and DHB matrices. CDX, CAP and TAP failed to produce detectable ions in both matrices. GEN and PEN produced detectable ions only in DHB matrix. NFZ and
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FZD produced detectable ions in HCCA matrix only. These results show that the matrix played an important role in MALDI analysis of antibiotics. The limited resolving power of low-cost linear MALDITOFMS systems, such as the one used in this study, combined with interferences from low-mass ions introduced by the matrix, were previously considered to limit the application of MALDI in the low-mass region. We therefore investigated 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 100 and 300. A significant improvement in mass resolution was observed for SDM, STR and TMP in the HCCA matrix, for
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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
Compound [MH] THPP [MNa] [MK] [MH] TMPP [MNa] [MK] [MH] no matrix [MNa] [MK]

AAP SG SDA SMX SMR SMTZ SMZ SQX SDM CLP MRT NFZ FZD PYR NA CDX STZ OA TMP PA

8 4 7 100 100 100 100 11 100

67 98 51 100 70 100 80 41 64 15 26 100 30

100 100 100 79 100 48 100 100 3 53

5 100 16 32

78 46 53 45 37 81 16 60 100 100 100 100 89 100

100 100 100 100 100 100 100 100 41 35 31 100

7 35 49 100 100 37 52 20 33 22 11

100 100 100 100 46 100 100 100 85 100 100 100 100 100 100

82 59 88 39 100 8 16 32 100 13 19 70 33 96 91

Not detected; unit is % relative intensity.

which the mass resolution was found to be 395, 530 and 380, respectively. SDM/matrix and TMP/matrix show similar optical microgaphs (not shown). The thickness of the analyte/matrix complex, measured using a profiler, was about 0.2 mm. 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 mass resolution obtained with HCCA matrix might be partially attributed to the smaller crystals and more uniform distribution of these analyte/matrix crystals on the sample probe surface.36 Matrices THPP (molecular mass 675 Da) and TMPP (molecular mass 678 Da) were studied, to avoid low-mass ion interferences occurring with HCCA and DHB.38,39,40 The absence of low-mass ion interferences with THPP and TMPP matrices is evident in the MALDI mass spectra (Fig. 2). We therefore used these matrices to investigate antibiotics with molecular masses less than 300 Da, which were known to be subject to interference by the low-mass ions generated by the HCCA and DHB matrices. Table 3 lists the characteristic ions in the MALDI-TOF mass spectra obtained with THPP (and TMPP) matrix as well as without any matrix. The ions observed include protonated molecules [MH], sodium adduct ions [MNa], and potassium adduct ions [MK]. It was noticed that, unlike when HCCA and DHB were used, most spectra obtained using THPP 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 LDI mass spectra were similar to the MALDI mass spectra obtained using THPP and TMPP matrices. However, the signal fluctuations were more pronounced due to the overall low intensity. The [MNa] ion became the dominant peak, presumably due to the universal presence of sodium as a contaminant. SMTZ, CLP, PYR and PA failed to produce any detectable ions in either of these two high-mass
# 1998 John Wiley & Sons, Ltd.

matrices. This is contrary to their behavior in HCCA and DHB matrices, when they all produced characteristic ions. TMP produced detectable ions in THPP matrix only. SDA and OA produced detectable ions only in TMPP matrix. It is noted that CDX, which failed to produce detectable ions in either HCCA or DHB matrix, also produced characteristic ions in both THPP and TMPP matrices. The advantage of the absence of low-mass ion interferences with THPP and TMPP matrices is evident in the MALDI mass spectra of MRT (Fig. 3). Spectral reproducibility from shot-to-shot is generally inferior to that obtained using HCCA and DHB matrices. The signal intensity obtained from these matrices followed the order LDI ` THPP ` TMPP ` DHB HCCA. Hence, subsequent quantitative analysis was carried out using HCCA and DHB matrices. Selection of internal standard MALDI is an established method for qualitative analysis. Examples of quantitative applications of MALDI are increasing.1820,4157 Quantitative analysis using MALDI is still difficult due to the poor point-to-point repeatability, sample-to-sample reproducibility, and shot-to-shot signal degradation.56,57 We tried to overcome these difficulties by using an internal standard. An ideal internal standard should be chemically similar to the analyte, chemically stable during the analysis, completely resolved from the analytes, and close to the analytes in mass and concentration to avoid instrumental errors.58 The evaluation of quantitation was carried out using SMX, SMZ and SDM sulfonamide antibiotics as model compounds, which are the most frequently encountered antibiotic residues in animal products in Taiwan. An isotopically labeled analog of the analyte is an ideal internal standard. The possible overlap between the analytes and their isotopically labeled internal standards due to the insufficient mass resolution of the linear TOF instrument
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Figure 3. The MALDI mass spectra of MRT in (a) THPP and (b) TMPP matrix.

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 spectra of SMX, SMZ and SDM, at concentrations of 25 ppm using HCCA and DHB matrices, are shown in Fig. 1. The [MH] ions and [MNa] ions of all three analytes were present in either matrix (Fig. 1(b)). The characteristic ions from AAP, including [AAPH] (m/z 152) and [AAPNa] (m/z 174), appeared in both spectra. The peak area 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 performed using DHB matrix.
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Quantitative analysis A series of standard solutions containing SMX, SMZ and SDM, 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 absolute amounts of analyte in 1 mL of these solutions, based on the molecular mass of 250 Da, were 100, 50, 10, 5, and 1 picomole (pmol), respectively. We collected five averaged mass spectra for each solution. To reduce the effect of instrumental fluctuations on quantitation results, we discarded two mass spectra which yielded the maximum and minimum values of the (Aanalyte/Ai.s) peak area ratio. To evaluate the suppression effect on the signal of the less
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Figure 4. The MALDI mass spectra of SMZ in HCCA matrix (a) before, and (b) after, acylation derivatization reaction.

concentrated components by the more concentrated components, Aanalyte is measured either as the [MH] ion peak area 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 (R 2) ranged from 0.973 to 0.985 when using the peak area sum of [MH] [MNa] to represent Aanalyte, which are better than the values 0.962 to 0.977 obtained using the peak area of [MH] alone. This might be due to the fact that the signal intensities of either [MH] or [MNa]
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ions alone are highly matrix-dependent during MALDI. Proton transfer for [MH] and a gas-phase mechanism for formation of [MNa] are the two competing mechanisms for the ionization step.59 The signal intensity of [MH] or [MNa] ions alone rapidly fluctuates. The sum of the signal intensities of [MH] [MNa] ions remained relatively constant over a sufficiently wide range of analyte concentrations, and thus better R 2 values were obtained when the peak area sum for ([MH] [MNa]) was used to represent Aanalyte. These values of linear correlation coefficient (R 2) were greater than 0.97 over two decades of analyte concentrations in every case. The values of the
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Table 4. Quantitative results for SMZ, SMX and SDM, calculated using two different quantitation means, and the corresponding LOD and LOQ values
Antibiotic R2 [MH] SF R2 [MH] [MNa] SF LOD (pmol) LOQ (pmol)

SMZ SMX SDM

0.962 0.970 0.977

10.7 2.7 8.1

0.983 0.973 0.985

17.9 4.6 15.1

0.6 0.8 0.4

0.8 1.0 0.5

R 2 linear correlation coefcient; SF : sensitivity factor; LOD : limit of detection; LOQ : limit of quantitation. See text for details.

Table 5. Precision from MALDI analysis of SMZ, SMX and SDM of various concentrations with two different quantitation means
Concentration (ppm) [MH] SMZ [MH] [MNa] [MH] SMX [MH] [MNa] [MH] SDM [MH] [MNa]

25 12.5 2.5 1.25 0.25

12 10 10 31 28

8 9 5 16 28

18 15 24 13 32

8 11 21 13 32

9 13 5 38

9 10 4 25

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

sensitivity factor (obtained from the slopes of the calibration curves) were similarly greater when the peak area sum of ([MH] [MNa]) was used to represent Aanalyte. The limit of detection (quantitation) LOD (LOQ) was defined here as the analyte concentration giving a peak in the MALDI spectrum with an area equal to the (mean baseline N standard deviation), where N=3 for the LOD and 10 for the LOQ. The mean baseline (standard deviation) is the measured average (fluctuations) measured from 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 concentrated components, the precision was obtained using two different quantitation means similar to those used in Table 4. The results are summarized in Table 5. At larger concentrations, i.e. above 2.5 ppm, the results appear similar for the two means whereas a significant difference was observed at low concentrations. The precision is in the range of 4 to 32 % RSD, dependent on the type and concentration of the analyte. For a higher concentration range, the average precision appears to be better and is about 10 % RSD. This result suggests that the primary source of uncertainty in MALDI quantitative analysis lies in the sample homogeneity. Conrmatory analysis Confirmatory analysis is used to identify the residues with the highest confidence and is generally mandatory in a regulatory environment. Typical confirmatory analysis employs complex and expensive procedures which involve mass spectral analysis of the tentatively identified residues. MALDI is a soft ionization technique which uses molecular weight information to identify residues. The lack of MS/MS capability in a low-cost linear TOFMS does not allow us to explore controlled dissociation and fragmentation for confirmatory analysis. We therefore propose to selectively change the m/z value of the analytes through the following chemical derivatization reaction and by inspecting the
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presence of the targeted m/z to confirm the presence of the tentative residues.

The derivatives were obtained by acylating the sulfonamides with 4-acetamidobenzenesulfonyl chloride under alkaline conditions. Figure 4 shows the MALDI mass spectra of STZ in HCCA before and after derivatization. The base peak has shifted from the original peak (m/z 256, in Fig. 4(a)) to the targeted peak (m/z 453, in Fig. 4(b)), thereby demonstrating the feasibility of this approach. CONCLUSIONS Important parameters have been studied for quantitative analysis of antibiotics using MALDI-TOFMS. Most antibiotics generate a stable protonated molecule in HCCA and DHB matrices and sodium adduct ions in porphyrin matrices. Carbadox, chloramphenicol and thiamphenicol failed to generate any characteristic ions in any matrix. To obtain quantitative results for sulfonamide antibiotics, we suggest using DHB matrix and incorporating a structurally analogous internal standard acetaminophen (AAP) to overcome shot-to-shot and sample-to-sample variability. The precision of the method needs to be improved if it is to be applied to a lower concentration range, however. Using an acylation derivatization process for confirmatory analysis, the limitation that MALDI-TOFMS provides only molecular mass data could be overcome. We conclude that the limited resolving power of the low-cost linear MALDITOFMS systems is not an obstacle to obtaining useful quantitative data. However, more work is required to search for new matrix materials and appropriate internal standards specific for analyzing different classes of low molecular
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mass compounds. While the quantitative analysis of real samples necessitates the incorporation of clean-up and concentration pretreatment, the capability of MALDITOFMS to provide data on low picomole amounts of antibiotics within a few minutes at low cost indicates that MALDI-TOFMS continues to present itself as a viable technique for the quantitative analysis of antibiotics. It could serve as a sensitive and specific screen, and possibly also as final-step detection device. Acknowledgement
We are grateful to Professor Chung-Sun Chung of the Department of Chemistry, National Tsing Hua University for providing the TMPP matrix. The research was supported by the National Science Council of the Republic of China under grant No. NSC 87-2113-M-007-037.

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