4.104.10 © Springer-Verlag Berlin Heidelberg 2005 II.4.10 β-Lactam antibiotics by Yuko Ito and Hisao Oka Introduction β-Lactum antibiotics constitute an important class of antibacterial agents being used extensively for both humans and food-producing animals to treat or prevent infections.  e drugs occasionally cause human deaths due to anaphylactic shock during medical treatments, especially when they are parenterally administered without their prior intracutaneous tests.  ese cases are usually handled as medical accidents ( malpractice), and subjected to autopsies and analysis of the drugs used.  ese antibiotics are composed of cephems (> Table 10.1) and penicillins (> Table 10.2), which are naturally occurring or semi-synthetic. Furthermore, two subclasses of cephem anti- biotics are cephalosporins and cephamycins ( > Table 10.1).  e primary structural di erence between the cephalosporins and cephamycins is the methoxy group substituted for the α-hy- drogen in the 7-position on the β-lactam ring. In this chapter, methods for simultaneous analysis of 6 kinds of penicillins and of 4 kinds of cephems by HPLC-UV are described. HPLC-UV analysis of penicillin antibiotics [1] Reagents and their preparation • Benzylpenicillin, phenoxymethylpenicillin, ampicillin, cloxacillin, dicloxacillin, nafcillin and β-hydroxyethyltheophylline can be purchased from Sigma (St. Louis, MO, USA). • Acetonitrile is of HPLC grade, and water is puri ed with a Milli-RO/Q water puri cation system (Millipore, Bedford, MA, USA). • A working internal standard (IS) solution is prepared daily with puri ed water to give a concentration of 20 µg/mL of β-hydroxyethyltheophylline. • Calibration standard are prepared daily with drug-free human serum covering the range of 0.5–50 µg/mL. • 1 mM Ammonium acetate bu er solution (pH 6.4): 77.8 mg of ammonium acetate is dis- solved in puri ed water to prepare 1 L solution, and adjusted to pH 6.4 with either ammonia water solution or acetic acid solution. • 0.2 M Tetrabutylammonium hydrogen sulfate solution: 67.9 g of tetrabutylammonium hydrogen sulfate is dissolved in puri ed water to prepare 1 L solution. It is adjusted to pH 7.7 with the below 0.2 M NaOH solution, and then bu ered with 0.2 M borate bu er solution, pH 7.7 (1:1, v/v). • 0.2 M NaOH solution: 8 g of NaOH is dissolved in puri ed water to prepare 1 L solution. • 0.2 M Borate bu er solution: 12.4 g of boric acid is dissolved in puri ed water to prepare 1 L solution and adjusted to pH 7.7 with 1 M NaOH solution. • 1 M NaOH solution: 40 g of NaOH is dissolved in puri ed water to prepare 1 L solution. 396 β-Lactam antibiotics ⊡ Table 10.1 Chemical structures of cephem antibiotics Cephalosporin Structure of side chain R1 R2 cephalothin cephazolin ceftiofur cephalosporin C cephalexin cephapirin cephaloglycine cefuroxime cephaloridine Cephamycin Complete structure cefoxitin cephamycin A 397 HPLC conditions Instrument: a model 620 solvent delivery system (Kontron AG, Zurich, Switzerland), a Uvikon 720LC UV/VIS variable-wavelength detector (Kontron AG), a model 3390A plotting inte grator (Hewlett-Packard, Avondale, PA, USA) and model 200 programmer (Kontron AG); column: Spherisorb ODS (250 × 4.6 mm i. d., particle size 5 µm, Kontron AG); guard column: Pell ODS (50 × 4.6 mm i. d., Whatmann, Cli on, NJ, USA); mobile phase: A = 1 mM ammonium acetate bu er solution (pH 6.4), B = acetonitrile with a linear gradient from 90 % A/10 % B to 75 % A/25 % B in 15 min;  ow rate: 2 mL/min; UV detection: 208 nm. Procedure i. A 50-µL volume of working IS solution and 2.5 mL dichloromethane are added to 200 µL of a test serum or each calibration standard in a 10 mL screw-top centrifuge tube. ii.  e mixture is vortexed for 30 s, followed by addition of 100 µL of 0.2 M tetrabutylammo- nium hydrogen sulfate solution. iii. A er vortex-mixing for 1 min and centrifugation at 2,800 g for 5 min, the upper aqueous layer is discarded. iv. A 2-mL volume of the organic phase is transferred to a new 10-mL conical test tube and evaporated to dryness at room temperature in a Speed Vac Concentrator (Savant Instru- ments Inc., Farmingdale, NY, USA). v.  e residue is reconstituted in 50 µL acetonitrile/puri ed water (10:90, v/v) and a 20-µL aliquot is injected into the chromatograph. ⊡ Table 10.2 Diagnostic ions of penicillin antibiotics obtained under ESI LC/MS/MS conditions in the negative mode benzylpenicillin R=C 7 H 7 phenoxymethylpenicillin R=C 10 H 7 O oxacillin R=C 10 H 8 ON cloxacillin R=C 10 H 7 ONCI nafcillin R=C 13 H 11 O 2 dicloxacillin R=C 10 H 6 ONCI 2 Penicillins [M–H] – [M–H–CO 2 ] – [M–H–141] – benzylpenicillin 333 289 192 phenoxymethylpenicillin 349 305 208 oxacillin 400 356 259 cloxacillin 434 390 293 nafcillin 413 369 272 dicloxacillin 468 424 327 HPLC-UV analysis of penicillin antibiotics 398 β-Lactam antibiotics Assessment and some comments on the method  e recoveries of the six penicillins were 79.4 to 95.7 % for serum specimens.  e detection limits were 0.05 µg/mL for benzylpenicillin, 0.10 µg/mL for phenoxymethylpenicillin and cloxacillin, 0.15 µg/mL for ampicillin and dicloxacillin, and 0.20 µg/mL for nafcillin (signal- to-noise ratio = 5). Over the concentration range studied (0.5–50 µg/mL), a good linear response was found for all penicillins assayed (correlation coe cients for calibration curves > 0.996).  e only penicillin antibiotic, which overlaps benzylpenicillin in the chromatogram, is amoxicillin; but the latter does not interfere with the assay, because it is not extracted with this procedure.  e separation of the six drugs was relatively good. Penicillins can be separated using acetonitrile/water alone as a mobile phase. However, when biological samples spiked with penicillins are analyzed, there is a considerable shi in retention times between biological samples and the standards due to matrix e ects. To avoid such a phenomenon, in many HPLC methods, various bu ers (pH around 7), such as phos- phate and acetate solutions, are used as mobile phases. Because many penicillins do not show speci c strong ultraviolet absorption, several HPLC methods utilized pre-column [2–5] and post-column [6–11] derivatization techniques for de- tection with enhanced selectivity and sensitivity. In addition, some of these methods require the use of mercury (II) chloride, a toxic environmental pollutant. For highly sensitive determi- nations, LC/MS with ESI can be recommended; but these methods were developed for the analysis of residual penicillins in foods [12–19]. Benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin give three kinds of product ions by ESI LC/MS/ MS, which is useful for both identi cation and quantitation as shown in > Table 10.2 [12, 15]. Because this technique is highly selective, it seems useful for the determination of penicillins in both pharmaceutical and biomedical specimens. HPLC-UV analysis of cephem antibiotics in plasma with a column-switching system [20] Reagent and their preparation • Cephalexin, cefoxitin, cefuroxime and cefotaxime sodium (IS) can be purchased from Sigma. Cephaloridine can be obtained with request from Eli Lilly & Co., Indianapolis, IN, USA. • Standard solutions of the 4 cephem drugs are prepared by dissolving each compound in puri ed water and diluted to appropriate concentrations with 0.01 M acetate bu er solu- tion (pH 3.5). • IS solution is prepared by dissolving 5 mg cefotaxime in 10 mL of puri ed water and by diluting 100 times with 0.01 M acetate bu er solution (pH 3.5). • 0.01 M Acetate bu er solution (pH 3.5): 778 mg of ammonium acetate is dissolved in puri-  ed water to prepare 1 L solution, and it is adjusted to pH 3.5 with 1 M acetic acid solu- tion. • 0.02 M Acetate bu er solution (pH 4.3): 1.6 g of ammonium acetate is dissolved in puri ed water to prepare 1 L solution, which is adjusted to pH 4.3 with acetic acid. 399 HPLC conditions Instrument: a model M 501 pump (for washing solvent, Waters, Milford, MA, USA), a 10-port multifunction valve (Valco, Houston, TX, USA), a model SP 8800 pump (for mobile phase, Spectra Physics, Santa Clara, CA, USA) a Reodyne 7125 injector with a 10 mL loop (Cotati, CA, USA), a model SP 8450 UV/VIS detector (Spectra Physics) and a model SP 4270 comput- ing integrator (Spectra Physics); precolumn: Corasil RP C18 (40 × 2.0 mm i.d., particle size 37–50 µm, Waters); guard column: Lichrosorb RP-8 (20 × 4.0 mm i.d., particle size 25–40 µm, Merk, Darmstadt, Germany); analytical column: Partisil ODS-3 (250 × 4 mm i.d., Whatman, Cli on, NJ, USA); mobile phase: acetonitrile/0.02 M acetate bu er solution (pH 4.3) (15:85, v/v); washing solvent: 0.01 M acetate bu er (pH 3.5):  ow rate: 1 mL/min each; UV detection: 254 nm. Column switching system; step I (0–4 min): the sample solution is injected onto the pre- column a , step II (5–8 min): the retained compounds are eluted from the pre-column b to the guard column/analytical column with the mobile phase, step III (9–25 min): the eluted drugs are separated with the analytical column. Procedure A 100-µL volume of the spiked plasma and 300 µL of IS in 0.01 M acetate bu er solution (pH 3.5) (5.0 µg/mL cefotaxime) are mixed, and 100 µL of the mixture is injected c into the HPLC system. Assessment of the method  e recoveries of the  ve cephem drugs ranged from 72.3 to 85.6 % for plasma specimens.  e probable reason for their relatively low recoveries is interaction between drug molecules and proteins; minor parts of the drug molecules might have been lost during the pre-column wash- ing. However, the use of cefotaxime as IS can compensate such losses upon calculation.  e precision and the accuracy for the assays of cephalexin, cefuroxime, cefoxitin and cephalori- dine using the IS were evaluated over the concentration range of 1–100 µg/mL in plasma.  e mean coe cients of variation for intra- and inter-assay were both less than 4.9 %, and the rela- tive recoveries ranged from 96 to 105 %. > Figure 10.1 shows HPLC chromatograms of hu- man blank plasma and blank plasma spiked with the 4 cephem drugs (20 µg/mL each).  e detection limit was equally about 0.5 µg/mL (signal-to-noise ratio = 3).  e calibration curves with peak-area ratios were linear in the range of 1–100 µg/mL, respectively.  e correlation coe cients were better than 0.999.  e following drugs did not interfered with the assays of the above 5 cephem drugs: cefotiam, cefadroxil, cefazolin, cefoperazone, cephalothin, cefaman- dole, aspirin, diclofenac, alcofenac, lonazolac, piroxicam, ibuprofen, indomethacin, ketopro- fen, naproxen, phenylbutazone, mefenamic acid and ca eine.  e total analysis time per sample was less than 25 min. HPLC-UV analysis of cephem antibiotics in plasma with a column-switching system 400 β-Lactam antibiotics Poisoning symptoms, and toxic and fatal concentrations Since β-lactam antibiotics are considered least toxic among all antibacterial agents, their fatal doses are not clear. However, the presence of high blood levels of these antibiotics can cause seizures, nephritis, leukopenia and bleeding disorders [21]. It is well-known that β-lactam antibiotics occasionally cause allergy reactions.  e anaphylactic shock caused by parenteral administration is not so rare.  e sensitivity test is, therefore, essential before parenteral administration of β-lactam antibiotics. When such a test is neglected or overlooked, resulting fatality due to anaphylactic shock, such a case is regarded as malpractice. HPLC chromatograms of human blank plasma (a) and blank plasma spiked with cefoxitin, cefuroxime, cephalexin and cephaloridine (each 20 µg/mL) (b). Peaks: 1 = cephalexin; 2 = cefotaxime (IS); 3 = cefuroxime; 4 = cefoxitin; 5 = cephaloridine. Reproduced from reference [21] with permission of Friedr. Vieweg & Sohn Verlagsgesellshaft mbH. ⊡ Figure 10.1 a b 401 Notes a) Polar interfering plasma components are passed through the pre-column.  en, the wash- ing solvent is passed through the pre-column to remove interfering impurities.  e guard column and the analytical column should be equilibrated with the mobile phase before analysis. b)  e pre-column is re-equilibrated with the washing solvent for the next injection. c)  e prepared samples should be kept at 4 °C before injection. References 1) Mendez-Alvarez E, Soto-Otero R, Sierra-Paredes G et al. (1991) Reversed-phase liquid-chromatographic me- thod for the simultaneous determination of several common penicillins in human serum. 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Confirmatory assay of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr A 911:217–223 16) Bruno F, Curini R, di-Corcia A et al. (2001) Solid-phase extraction followed by liquid chromatography-mass spectrometry for trace determination of beta-lactam antibiotics in bovine milk. J Agric Food Chem 49:3463– 3470 Poisoning symptoms, and toxic and fatal concentrations 402 β-Lactam antibiotics 17) Riediker S, Stadler RH (2001) Simultaneous determination of five beta-lactam antibiotics in bovine milk using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Anal Chem 73:1614– 1621 18) Daeseleire E, de-Ruyck H, van-Rentergham R (2000) Confirmatory assay for the simultaneous detection of penicillins and cephalosporins in milk using liquid chromatography-tandem mass spectrometry. Rapid Commun Mass Spectrom 14:1404–1409 20) Blanchflower WJ, Hewitt SA, Kennedy DG (1994) Confirmatory assay for the simultaneous detection of five penicillins in muscle, kidney and milk using liquid chromatography-electrospray mass spectrometry. Analyst 119:2595–2601 21) Lee YJ, Lee HS (1990) Simultaneous detection of cefoxitin, cefuroxime, cephalexin and cephaloridine in plasma using HPLC and a column-switching technique. Chromatographia 30:80–84 22) Parry MF (1987) The penicillins. Med Clin North Am 71:1093–1112 . Haginaka J, Wakai J (1985) High-performance liquid-chromatographic assay of ampicillin, amoxicillin and cicla- cillin in serum and urine using a pre-column. resulting fatality due to anaphylactic shock, such a case is regarded as malpractice. HPLC chromatograms of human blank plasma (a) and blank plasma spiked