High-Performance Liquid Chromatographic Micro-Assay for Cefradine in Biological Fluids

440(204) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 Mar. 1981

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC MICRO-ASSAY FOR CEFRADINE IN BIOLOGICAL FLUIDS

YASUYUKIHAYASHI Department of Clinical Pathology, University of Juntendo, School of Medicine, Tokyo, Japan (Receivedfor publication December 11, 1980)

Recently, cephalosporins have been developed progressively that the clinical experiences in

new cephalosporins have been reported one after another; some of them are widely used in

daily medicine. The microbiological studies and other metabolic studies; i.e. absorption , dis- tribution, and excretion, have undertaken simultaneously during the clinical trials of new anti-

biotics. On the other hand, the necessity of the optimum dose regimen for not only antibiotics

but also other drugs has been emphasized to achieve the maximum pharmacological effects with

minimal dosage or to prevent the side-effects and sequelae. In such cases, the monitoring of

the blood level is essential and the drug concentration is necessary to be measured as soon as

possible. The chemical assay has an advantage in this point over the bioassay, and has become available for the routine analysis recently. With cephalosporins, the blood and urine levels of

cephalothin (CET), cefoxitin (CFX), cephalexin (CEX)2,3), cefazolin (CEZ)4), cefuroxime

(CXM)5), cephaloridine (CER)6) and cefradine (CED)7) determined by high-performance liquid chromatography (HPLC) have been reported.

The author describes a new HPLC method using a reversed phase column which found to

be applicable to the routine analysis of CED in serum and urine comparing with bioassay .

Experimental

Samples

Ten healthy male adults received orally 500 mg of CED in a day according to the three different protocols; a single dose of 500 mg, a duplicate doses of 250 mg after meal, and a single dose of 500 mg before meal. Blood samples were obtained after 1, 2, 3, 4, 6 and 8 hours. Urine samples, voided every 2 hours, were also collected up to 8 hours.

The volunteers administered 500 mg of CED two or three times after 2•`4 weeks interval . Thus from 5 to 10 volunteers were employed in each study as shown in Table 1 . They had been physically examined in 62 items totally prior to the study; internal examination, general blood tests and routine clinical tests. Each serum sample was separated within 1•`3 hours after the sampling, and divided into two portions; one was used for the bioassay and another was for the HPLC assay . Each urine sample was also divided into two portions and stored at-80•Ž in caps. Bioassay

The following two microbiological methods, a double layer cup or disc method , were used. Conditions are; Test organism: Micrococcus luteus ATCC 9341. Culture media: Nutrient agar (Difco).

Petri plate: The basic layer was 20 ml and the seed layer was 4 ml of agar to each plate . Samples: Serum sample was diluted to the estimated concentration ranging from 0.5 to 8 .0 μg/ml using 1% phosphate buffer (pH6.0). Urine was also diluted to the same concentration range with 1% phosphate buffer . Standard solution: The standard CED (965ƒÊg/mg of potency) was weighed and diluted to Mar. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 441(205) 1981

Table 1. Subject characteristics and assignment to each study Study-I: A single oral administration of 500 mg of CED per a day after meal Study-II: Twice oral administration of 250 mg of CED per a day after meal Study-III: A single oral administration of 500 mg of CED per a day before meal

a concentration of 1,000ƒÊg (potency)/ml with the phosphate buffer, and then prepared the standard curve concentration, 0.5, 1.0, 2.0, 4.0 and 8.0ƒÊg/ml solution by dilution.

Preparation of unbound sample: A 1.0 ml of serum was centrifuged in a Centriflo membrane cone (Amicon) at 1,200 r.p.m. for 5 minutes and the filtrate was used for the microbioassay after dilution.

HPLC assay

Apparatus: A HPLC system including a Model 6,000A pump and a U6K injector (Waters

Assoc.) equipped with a Model 440 UV 254 nm detector (Waters) was used. The HPLC analysis was made with a reversed phase ƒÊ-Bondapak C-18 column (30cm•~4mm i.d., Waters).

Chromatographic conditions: The mobile phase was prepared by degassing a mixture of

200 ml of methanol with 800 ml of 0.01 M phosphate buffer (pH 6.8). The analysis using above solution was carried out at a flow rate of 2.0 ml/min (1,500 psi), and the sample size was set at 15•`20ƒÊl/inj.

Sample preparation: The filtrate using a Millipore filter (0.45 pm type-HA, Millipore) and

the deproteinized sample by addition of 1.5 ml of methanol to 0.5 ml of plasma were prepared

prior to the analysis. Analysis time: The retention time of CED was found to be 5.6 to 5.7 min.

Preparation of calibration curves for HPLC assay: The standard CED (965ƒÊg/ml of potency)

was weighed, and diluted in the concentration of 20, 40, 60, 80, and 100ƒÊg/ml of CED using

water, serum water, and fresh serum to prepare the separate three calibration curves.

Results

HPLC Assay

Figs. 1-1 and -2 show the HPLC chromatograms of serum and urine. The calibration

curves obtained by peak height method are linear as shown in Figs. 2-1 and -2. The injection

volume for the deproteinized serum samples was set to 20ƒÊl, and for the other samples was

set at 15ƒÊl considering the dilution. The concentration range of 0•`100ƒÊg/ml with respect to

peak height, absorbance, was linear both untreated and deproteinized samples. In case of the

quantitation, the separate calibration curve should be used due to the slope differences. The

precision of HPLC method is shown in Table 2. 442(206) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 Mar. 1981

Table 2. Precision of CED determination by HPLC

Table 3. Correlation between HPLC and bioassay in CED determination y=ax+b,bioassay: cup method, HPLC-1: deproteinization by methanol, HPLC-2: direct injection

The comparison of HPLC and bioassay Table 4. Comparison of CED concentration in the determination of CED in serum and determined by HPLC and bioassay bioassay: cup method, HPLC-1: depro- urine after a single administration of 500mg teinization by methanol, HPLC-2: direct before meal is shown in Tables 3 and 4. injection The good correlation (r), 0.838 to 0.998, was obtained between HPLC and bioassay in each case as shown in Table 3. The HPLC assay gave higher values than the bioassay in the case of deproteinized serum samples as shown in Table 4. Both assays showed a good accordance with the other samples.

Time Course of Blood Level after a Single Administration of CED

Figs. 3-1 and -2 show the time course of CED in serum with 5 volunteers after a 500mg administration before meal. In this case, HPLC assay was carried out with untreated serum samples and the ultrafiltrate for the total and free CED determination respectively. CED in serum showed almost the same time course pattern and half-life time (T1/2) both determined by HPLC and bioassay.

Comparison of Urinary Recovery

Fig. 4 shows the urinary recovery of CED obtained by HPLC and bioassay in the three studies. High urinary excretion was observed in each case, and the CED concentration both determined by HPLC and bioassay gave a good accordance. The urinary accumulative excretion curves of CED were found to be different between the dose schedules as shown in Fig. 4.

Discussion

The comparison of HPLC assay and bioassay for CED in biological fluids is described. The Mar. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 443(207) 1981

Fig. 1-1. Chromatogram of CED in serum (a) standard solution of CED and CEX, (b) control serum, (c) addition of standard CEX and CED to control serum; 3.5ƒÊg/ml, 20 ƒÊl/inj.

Fig. 1-2. Chromatogram of CED in urine

(d) standard solution of CED and CEX, (e) control urine (f) addition of standard CEX and CED to control urine; 3.5ƒÊg/ml, 20ƒÊl/inj. 444(208) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 Mar. 1981

Fig. 2-1, -2. Calibration curves for HPLC assays of CED 2-1: AUFS 0.02, 20ƒÊl/inj., 2-2: AUFS 0.05, 15ƒÊl/inj. (1) in water, (2) in serum water (pretreatment), (3) in serum (pretreatment) (4) in water (pretreatment), (5) in serum water; (6) in water, (7) in serum

Fig. 3-1, -2. Time course of total and free CED in blood after an oral administration of 500 mg of CED in 5 healthy volunteers before meal 3-1: HPLC, 3-2: bioassay, Pharmacokinetic parameters were calculated with total CED in serum: Ka (rate constant of absorption), Kel (rate constant of elimination) and T1/2 (half-life time)=0.693/Kel Mar. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 1981 445(209)

UV photometry (at 261 nm)7) and HPLC Fig. 4. Urinary recovery of CED after an oral method8) for CED determination had been administration of 500 mg of CED in various reported. However, these methods had been manners limited only to the experimental application, and not to the routine analysis for the (I) 500mg of CED after meal (n-10) clinical purposes. The photometry7) was complicated, especially in the purification step. The HPLC method reported was a methodology for the separation of CEX and CED in urine using another HPLC conditions. The separation of our HPLC assay for CED in urine and serum was found to be satisfactory, and no interferences from endogenous constituents were eluting at the (II) 250mg of CED twice after breakfast and lunch(n-8) retention time of CED. Of course CED showed perfect separation from CEX in this method. The deproteinization using methanol was found to be inadequate on account of the decrease in peak height. Especially in urine analysis, CED concentration calculated by this calibration curve gave higher values.

Moreover, CED in aqueous solution was (III) 500mg in fasting (n-5) reported to be decomposed by the addition of methanol according to the heat of dilu- tion7). It is more convenient method to be able to inject serum samples directly into HPLC. However, it became necessary to check the change of retention time of CED peak in this method. The reversed phase column was found to be decreasing its resolution after the continuous injections of serum samples without pretreatment. The author did not apply this method for the analysis of patient's samples, so that it was unnecessary to check the interferences by the other thera- peutic drugs in this study. The time course of blood level and the accumulative urinary excretion determined by HPLC showed a good accordance with those reported by bioassay9) . The HPLC method was found to be applicable to the routine analysis from the followings; the time course of blood level and the amounts of urinary excretion of CED obtained were in good accordance with those obtained by bioassay.

Conclusion

The routine analysis for CED in biological fluids has been established using HPLC . The analytical conditions were optimized for the analysis of CED in serum and urine after a 500mg of oral administration to 10 healthy volunteers, and the results were compared with bioassay . The data obtained by HPLC showed a good correlation with those by bioassay . The conditions of this HPLC assay for CED was found to be satisfactory . This method is more convenient than bioassay and also will be applicable to the routine clinical analysis from the following backgrounds; the sample size is small , the procedure is simple, and the result comes out rapidly. 446(210) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXIV-3 Mar. 1981

Acknowledgement

The author is grateful to Dr. TOSHIMASA KOJIMA of Product development laboratories, Sankyo Co., Ltd. for his help in bioassay.

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