of cephalosporins in human plasma and a new solid-phase extraction procedure for cefazolin and ceftizoxime. (Received 26 January 1990; revision 9 April 1990 ...
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Clinica Chimica Acru, 190 (1990) 121-124 Elsevier
CCA 04776
Letter
to the Editor
Improved HPLC method for the determination of cephalosporins in human plasma and a new solid-phase extraction procedure for cefazolin and ceftizoxime (Received
26 January
1990; revision 9 April 1990; accepted
15 April 1990)
Dear Editor, Cefazolin and ceftizoxime are widely used antibiotics. The mixed polarity of cephalosporins makes usual solvent extraction procedures inefficient. For cefazolin, serum protein precipitation is inefficient, due to its high protein binding (74-868) and virtual insolubility in methanol [l]. Solid-phase extraction of cefazolin was described using Cl8 columns [2], but the extracts were not clean. The HPLC analysis of antibiotics is reported using reversed-phase [2,3], ion-exchange [4] and normal-phase systems [5]. In this communication, improvements on existing solid-phase extraction and HPLC procedures are presented. Spiked plasma samples (l-5 pg/rnl) were prepared by adding aliquots of drug stock solution (1 mg/ml water) to blank plasma. HPLC: A mobile phase of distilled water-2 mmol/l tetramethyl ammonium hydroxide (TMAH) in methanol-acetic acid (60 : 40 : 0.5), (flowrate: 0.8 ml/mm), was passed through a Cl8 (25 X 0.46 cm) column connected to a 20 ~1 injection system. UV detection: 262 nm Cl8 Bond-Elut columns (1 ml capacity) were conditioned with methanol (2 ml), 8.5% phosphoric acid (2 ml). Spiked plasma sample (0.5 ml), 8.5% HPO, (25 ~1) and internal standard solution (250 ~1, 1 mg/ml coumarin-3carboxylic acid in water) were added. Columns were washed with water (0.5 ml), 8.5% HPO, (1 ml), 5% methanol-8.5% HPO, (20: 1) (1 ml). NH, columns, pre-conditioned with hexane (1 ml) were placed beneath the Cl8 cartridges. Methanol-8.5% HPO, (60 : 40) (1 ml) was passed through. Cl8 cartridges were removed and NH, columns directly washed with hexane (1 ml), acetpnitrile (1 ml). The drugs were eluted using water-lo% ammonium sulphate (wyv) (95 : 5) (1 ml), and analysed directly. Percentage drug recovery was determined by comparing extract peak areas with those of drug standards. The HPLC system was suitable for determining cefazolin, ceftizoxime, cefaclor, cefalexin and cefaloridine. The relationship between peak area and four drug concentrations was calculated between l’ and 10 pg/ml, giving maximum CV values of 4.2% (within-day) and 6.6% (between-day). The use of TMAH at low concentra0009-8981/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
122
Cl8
02
4 (a)
Cl8
6
II
CEFTIZOXIME
IO (b)
0
12
CEFAZOLIN
(c)
+ Nll2
2
4
6
8
CUUMWN-3-C/WlUXYLIC
only
Cl8
10
12
ACID
+ NIV
II
0
?1._ l-_-k 2
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6
8
tb)
(c)
10
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RETENTION
0
2
TIME
(tulnuter)
4
6
8
10
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Fig. 1. Ceftizoxime and cefazolin extracted from plasma using Cl8 (non-polar) and ClI/NH, polar/polar) solid-phase extraction columns.
(non-
tion blocked residual silanol groups on the Cl8 column rather than acting specifically as an ion-pairing agent, resulting in excellent peak shapes. Impurities still present following single Cl8 extraction were significantly removed after NH, extraction (Fig. 1). Cefazolin recovery was 94.8 f 5.1%; ceftizoxime 97.34 + 3%; and internal standard 100.8 f 4.8% (n = 10). The extraction exploits the non-polar and polar properties of the drugs. A single polar extraction is inefficient and a single non-polar extraction is not clean. When applied to other cephalosporins under investigation, rather than using direct protein precipitation, the recoveries were: cefaclor (57.8 f 25%), cefalexin (86.1 f 5.8%), cefaloridine (0%). This shows the widely differing degree of polarity within the cephalosporin group and the difficulties involved in developing a single
123
extraction procedure for all cephalosporins. The extraction procedure system described are useful in clinical and toxicological investigations.
and HPLC
Acknowledgement
CMM is a post-doctoral fellow supported by the Japan Society for the Promotion of Science and the Royal Society, who helped to finance this work. Christine M. Moore *, Keizo Sato *, Hideki Hattori 2 and Yoshinao Katsumata ’ ’ Department
of Legal Medicine, Nagoya University School of Medicine, Showa-ku, Nagoya, Japan and ’ Department of Legal Medicine, Aichi Medical University, Aichi, Japan
References 1 2 3 4 5
Kirby WMM, Regamey C. J Infect Dis 1973;128 (Suppl):341. Brendel E, Zschunke M, Meineke I. J Chromatogr 1985;339:359. Hayashi Y. Jpn J Antibiotics 1981;XXXIV-3, 440. Tokuma Y, Shiozaki Y, Noguchi H. J Chromatogr 1984;311:339. Ascalone V, Dal Bo L. J Cbromatogr 1983;273:357.
Correspondence to: C.M. Moore, Department of Legal Medicine, Nagoya University School of Medicine, 65 Tsunnnai-cho, Showa-ku, Nagoya 466, Japan.
