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

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