Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 42)

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Drugs and Poisons in Humans - A Handbook of Practical Analysis (Part 42)

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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. The drugs occasionally cause human deaths due to anaphylactic shock during medical treatments, especially when they are parenterally administered without their prior intracutaneous tests. These cases are usually handled as medical accidents (malpractice), and subjected to autopsies and analysis of the drugs used. These 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 antibiotics are cephalosporins and cephamycins (...

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  1. 4.10 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. The drugs occasionally cause human deaths due to anaphylactic shock during medical treatments, especially when they are parenterally administered without their prior intracutaneous tests. These cases are usually handled as medical accidents (malpractice), and subjected to autopsies and analysis of the drugs used. These 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). The primary structural difference 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 purified with a Milli-RO/Q water purification system (Millipore, Bedford, MA, USA). • A working internal standard (IS) solution is prepared daily with purified 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 buffer solution (pH 6.4): 77.8 mg of ammonium acetate is dis- solved in purified 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 purified water to prepare 1 L solution. It is adjusted to pH 7.7 with the below 0.2 M NaOH solution, and then buffered with 0.2 M borate buffer solution, pH 7.7 (1:1, v/v). • 0.2 M NaOH solution: 8 g of NaOH is dissolved in purified water to prepare 1 L solution. • 0.2 M Borate buffer solution: 12.4 g of boric acid is dissolved in purified 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 purified water to prepare 1 L solution. © Springer-Verlag Berlin Heidelberg 2005
  2. 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
  3. HPLC-UV analysis of penicillin antibiotics 397 ⊡ Table 10.2 Diagnostic ions of penicillin antibiotics obtained under ESI LC/MS/MS conditions in the negative mode benzylpenicillin R=C7H7 phenoxymethylpenicillin R=C10H7O oxacillin R=C10H8ON cloxacillin R=C10H7ONCI nafcillin R=C13H11O2 dicloxacillin R=C10H6ONCI2 Penicillins [M–H]– [M–H–CO2]– [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 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 integrator (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, Clifton, NJ, USA); mobile phase: A = 1 mM ammonium acetate buffer solution (pH 6.4), B = acetonitrile with a linear gradient from 90 % A/10 % B to 75 % A/25 % B in 15 min; flow 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. The mixture is vortexed for 30 s, followed by addition of 100 µL of 0.2 M tetrabutylammo- nium hydrogen sulfate solution. iii. After 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. The residue is reconstituted in 50 µL acetonitrile/purified water (10:90, v/v) and a 20-µL aliquot is injected into the chromatograph.
  4. 398 β-Lactam antibiotics Assessment and some comments on the method The recoveries of the six penicillins were 79.4 to 95.7 % for serum specimens. The 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 coefficients for calibration curves > 0.996). The 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. The 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 shift in retention times between biological samples and the standards due to matrix effects. To avoid such a phenomenon, in many HPLC methods, various buffers (pH around 7), such as phos- phate and acetate solutions, are used as mobile phases. Because many penicillins do not show specific 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 identification 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 purified water and diluted to appropriate concentrations with 0.01 M acetate buffer solu- tion (pH 3.5). • IS solution is prepared by dissolving 5 mg cefotaxime in 10 mL of purified water and by diluting 100 times with 0.01 M acetate buffer solution (pH 3.5). • 0.01 M Acetate buffer solution (pH 3.5): 778 mg of ammonium acetate is dissolved in puri- fied 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 buffer solution (pH 4.3): 1.6 g of ammonium acetate is dissolved in purified water to prepare 1 L solution, which is adjusted to pH 4.3 with acetic acid.
  5. HPLC-UV analysis of cephem antibiotics in plasma with a column-switching system 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, Clifton, NJ, USA); mobile phase: acetonitrile/0.02 M acetate buffer solution (pH 4.3) (15:85, v/v); washing solvent: 0.01 M acetate buffer (pH 3.5): flow 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-columnb 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 buffer 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 The recoveries of the five cephem drugs ranged from 72.3 to 85.6 % for plasma specimens. The 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. The 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. The mean coefficients 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). The detection limit was equally about 0.5 µg/mL (signal-to-noise ratio = 3). The calibration curves with peak-area ratios were linear in the range of 1–100 µg/mL, respectively. The correlation coefficients were better than 0.999. The 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 caffeine. The total analysis time per sample was less than 25 min.
  6. 400 β-Lactam antibiotics ⊡ Figure 10.1 a b 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. 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. The anaphylactic shock caused by parenteral administration is not so rare. The 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.
  7. Poisoning symptoms, and toxic and fatal concentrations 401 Notes a) Polar interfering plasma components are passed through the pre-column. Then, the wash- ing solvent is passed through the pre-column to remove interfering impurities. The guard column and the analytical column should be equilibrated with the mobile phase before analysis. b) The pre-column is re-equilibrated with the washing solvent for the next injection. c) The 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. Biomed Chromatogr 5:78–82 2) Haginaka J, Wakai J (1985) High-performance liquid-chromatographic assay of ampicillin, amoxicillin and cicla- cillin in serum and urine using a pre-column reaction with 1,2,4-triazole and mercury(II) chloride. Analyst 110:1277–1281 3) Rogers ME, Adlard MW, Saunders G et al. (1984) Derivatization techniques for high-performance liquid-chro- matographic analysis of beta-lactams. J Chromatogr 297:385–391 4) Rogers ME, Adlard MW, Saunders G et al. (1983) High-performance liquid-chromatographic determination of penicillins following derivatization to mercury-stabilized penicillenic acids. J Liq Chromatogr 6:2019–2031 5) Miyazaki K, Ohtani K, Sunada K et al. (1983) Determination of ampicillin, amoxycillin, cephalexin and cephradine in plasma by high-performance liquid-chromatography using fluorimetric detection. J Liq Chromatogr 276: 478–482 6) Haginaka J, Wakai J (1988) Liquid-chromatographic determination of penicillins by post-column alkaline degradation using a hollow-fibre membrane reactor. Anal Biochem 168:132–140 7) Selavka CM, Krull IS, Bratin K (1986) Analysis for penicillins and cefoperazone by HPLC-photolysis-electrochemical detection (HPLC-hv-EC). J Pham Biomed Anal 4:83–93 8) Haginaka J, Wakai J (1985) Liquid-chromatographic determination of penicillins by post-column alkaline de- gradation. Anal Biochem 57:1568–1571 9) Kok WT, Halvax JJ, Voogt WH et al. (1985) Determination of thioethers of pharmaceutical importance by liquid chromatography with online generated bromine. Anal Chem 57:2580–2583 10) Rogers ME, Adlard MW, Saunders G et al. (1983) High-performance liquid-chromatographic determination of beta-lactam antibiotics, using fluorescence detection following post-column derivatization. J Chromatogr 257:91–100 11) Lee TL, Darconte L, Brooks MA (1979) High-pressure liquid-chromatographic determination of amoxicillin in urine. J Pharm Sci 68:454–458 12) Ito Y, Goto T, Oka H et al. (2004) Application of ion-exchange cartridge clean-up in food analysis. VI. Determina- tion of six penicillins in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectro- metry. J Chromatogr A. 1042:107–111 13) Fagerquist CK, Lightfield AR (2003) Confirmatory analysis of beta-lactam antibiotics in kidney tissue by liquid chromatography/electrospray ionization selective reaction monitoring ion trap tandem mass spectrometry. Rapid Commun Mass Spectrom 17:660–671 14) Holstege DM, Puschner B, Whitehead G et al. (2002) Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS-MS. J Agric Food Chem 50:406–411 15) Ito Y, Ikai Y, Oka H et al. (2001) Application of ion-exchange cartridge clean-up in food analysis. IV. 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
  8. 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

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