BIOMEDICAL CHROMATOGRAPHY, VOL. 5 , 265-268 (1991)
High Performance Liquid Chromatographic Analysis of Tetroxoprim and Sulphadiazine in Serum and Urine Hanan N. Alkaysi" and Mutaz Sheikh Salem Faculty of Pharmacy, Jordan University of Science and Technology, Irhid, Jordan
Adnan A. Badwan Jordanian Pharmaceutical Manufacturing Company. Naor, Jordan
Quantitative determination of tetroxoprim and sulphadiazine in serum and urine was performed using reversed phase high performanceliquid chromatography.Protein precipitation using 10% perchloric acid was utilized for purification of serum samples while urine samples were diluted prior to analysis. The mobile phase consisted of triethylammonium acetate buffer (85%), acetonitrile (12%) and methanol (3%), with a final pH of 4.2. The eluent was monitored at 280 nm. Benzoic acid was used as an internal standard. Standardization, validation and application of the method is described. Materials. Tetroxoprim and sulphadiazine were provided by JPM (Naor, Jordan). Benzoic acid, perchloric acid (60%), acetic acid and triethylamine were purchased from BDH Combinations of sulpha drugs with derivatives of 2,4(Poole, UK). Acetonitrile and methanol were HPLC grade diamino-5-benzylpyrimidine have been used as syncr(May & Baker, UK). Water was distilled and deionized. The gestically acting antibacterial drugs. Their mechanism dosage form TibiroxB was generously provided by Hoffman la ot action involves blockage of folic acid metabolism. Roche (Basel, Switzerland). One of the clinically effectivc combinations is comThe metabolite N4-acetylsulphadiazine was synthesized by posed of tetroxoprim, 2,4-diamino-S-[3,5-dimethoxy-4acetylation of sulphadiazine using acetic anhydride and con(2-methoxyethoxy)benzyl pyrimidine], and sulphadiacentrated sulphuric acid. The identity of the product was zine. This combination exhibits bactericidal activity confirmed by melting point determination and by spectral against many pathogens (Bywater et al., 1979); howdata (IR and NMR). The purity was checked by thin layer ever it is primarily recommended in the treatment of chromatography (TLC) and HPLC. respiratory a n d urinary tract infections (Pust et al., 1979; Ferber a n d Ahrens, 1979). Chromatographic conditions. The HPLC column used was Methods for t h e analysis of sulphadiazine using packed with Spherisorb 5 p ODS 2 (25 x 0.46 crn2), supplied reversed phase and ion pairing high performance liquid by Phase Separation (Clwyd, UK). The mobile phase conchromatography (HPLC) have been reported (Su et al. sisted of triethylammonium acetate buffer prepared by dis1976; Helboe and Thomsen, 1977; G o e h l et al. 1978). solving 6 mL glacial acetic acid and 3 mL triethylamine in one However, only a few methods are available for the litre of water (0.1 M). The percentage of the buffer was 85% combination of sulphadiazine and tetroxoprim (Vergin and the organic components were acetonitrile 12% and metha n d Fritschi, 1979; Vergin and Bishop, 1980; anol 3%; the pEl was adjusted to 4.2 using triethylamine. A Bishop-Frcudling a n d Vergin, 1981; Springolo and solution of benzoic acid in the mobile phase was used as Coppi, 1989). T h e reported methods require different internal standard. The flow rate was 1.5 mL/min, the detecchromatographic systems or extensive and separate tion wavelength was set at 280 nm and the injection volume sample purification techniques for each component. was 100 pL. This report describes an HPLC analytical method for tetroxoprim and sulphadiazine using the same sample Sample collection. Two tablets of Tibirox@,each containing purification procedure a n d a single mobile phase for 100 mg of tetroxoprim and 250 mg of sulphadiazine, were both components. Application of the method to the administered to six healthy male volunteers. Blood samples analysis of serum a n d urine samples is reported. were withdrawn at time intervals extending over 12 h. The collected serum samples were kept at -20°C until analysis. Urine samples were collected every two hours and stored at -20°C for analysis. The volume was recorded for each EXPERIMENTAL collection.
INTRODUCTION
Instruments. All analyses were performed using a high performance liquid chromatograph consisting of a Beckman (114M) solvent delivery system, a JASCO variable wavelength detector (875-UV) and a Rheodyne injection head (7125) fitted with a 100 WLloop. *
Author to whom correspondence should be addressed
0269-3879191 1060265-04 $OS.OO 01991 by John Wiley & Sons, Ltd
Sample preparation. Serum samples, (i) Sulphadiazine. 100 pL serum and 200 pL internal standard solution (7.5 pg mL) were placed in a centrifuge tube. 100 pL 10% perchloric acid was added. The tube was vortex-mixed for one minute and centrifuged at 15,000 X g for 5 min. 100 pL of the supernatant was injected. (ii) Tetroxoprim. 100 FL serum Received 20 August 1990 Accepted (revised) 24 January 1991
H. N. ALKAYSI, M. S. SALEM AND A.A. BADWAN
266
and 100 yL internal standard solution (100 yg/rnL) were placed in a centrifuge tube. The procedure was completed as for sulphadiazine. Urine samples. (i) Sulphadiazine. 100 yL urine was diluted to 2 mL with the mobile phase after the addition of internal standard solution. (ii) Tetroxoprim. 100 pL urine was diluted to 1mL with the mobile phase after the addition of internal standard solution. The variation in the final volumes and the concentrations of the internal standard was necessary to accomodate the expected difference in concentration between sulphadiazine and tetroxoprim in biological fluids.
Standardization and calculation. Stock solutions, 2 mg/mL tetroxoprim in the mobile phase and 0.5 mg/mL sulphadiazine in methanol were used for spiking serum at concentrations of 10 yg/mL for tetroxoprim and 25 pg/mL for sulpha-
iI
II
'
diazine. Serial dilutions were prepared covering the concentration ranges 1-6 pg/mL and 2.5-25 pglmL for tetroxoprim and sulphadiazine, respectively. The standard solutions were prepared in pooled serum and stored at -20°C for further analysis. A stock solution of the internal standard was prepared by dissolving benzoic acid in the minimum volume of methanol and completed with the mobile phase. All serum analyses for sulphadiazine were performed at a range setting of 0.16 aufs. The range was set at 0.01 aufs for the detection of tetroxoprim. Analysis of both components in urine was calibrated over concentrations ranging from 12.5-100 yglrnL for sulphadiaLine and 2-20 y g h L for tetroxoprim. Calculations of interand intra-day variations for urine analysis were performed at concentrations of 4 and 16 pg/rnL for tetroxoprim and 25 and 100 pg/mL for sulphadiazine. Calibration curves were constructed by plotting peak height ratio (drug/IS) vs. concentration. Relative recovery was determined by comparing peak height ratios of injected serum samples with aqueous solutions containing equivalent amounts of analysed components, internal standard and perchloric acid. Absolute recovery was calculated in a Fimilar manner but using peak heights rather than ratios.
.1.
"
l--rTll 0
4
12
0
1G
(inin.)
( I3
I
r,
.s.
m 0
4
8
12
(min.
16
1
l--rTTl U
4
72
0 ( min.
16
1
Figure 1. Representative chromatogram of sulphadiazine (I), tetroxoprim (11) and benzoic acid (1.S.) in (A) volunteer's serum containing 17.6 pg/mL sulphadiazine (I) and 50 pg/mL I.S. (0.16 aufs); (6)blank serum at 0.16 aufs; (C) volunteer's serum containing 5.2 pg/mL tetroxoprim (11) and 7.5 pg/mL I.S. (0.01 aufs); (D) blank serum at 0.01 aufs.
l -4 l Ul -12 l 16l
U
(iiii ii
.
Figure 2. Chromatogram of urine samples collected from a human volunteers. (A) Urine sample containing 49.5 pg/mL sulphadiazine (I)and 100 pg/mL IS.; (B) urine sample containing 6.7 pg/mL tetroxoprim (11) and 25 Fg/mL I.S.; ( C ) blank urine
sample.
‘TETROXOPRlM AND SULPHADIAZTNE 1N SERUM AND URINE
267
Table 1. Statistical data for the calibration curves of tetroxoprim and sulphadiazine in serum and urine Drug (pg/mL)a
Tetroxoprirn: Serum (1-6 pglmL) Urine (2-20 hg/mL) Sulphadiazine: Serum (2.5-25 pg/rnL) Urine (12.5-100 pglmL) a
Correlation coefficient
Slope
0.997 f 1.65 X 10 - 3 0.998i8.33X10-4
0.33 f7.66 X -5.99X 10-*+3.54X 5 . 5 2 X 1 0 ~ 2 ~ 1 . 3 4 X 1 0 ~-2.77X10 3 2+6.68X 10
0.997+ 1 . 1 6 ~10 0.997f2.54~
8 . 8 6 ~1 0 - 2 ? 2 . 1 9 ~ 1.331 ~0 - * f 3 . 6 5 x
Intercept
9.46x 10-2+5.1 X - 4 . 6 9 ~10-*+9.1 x
lo-*
Each value is an average of five determinations F S.D.
Table 2. Intra- and inter-day precision of tetroxoprim and sulphadiazine assays in serum lntraday
Tetroxoprim: Concentration (KglrnL) Mean ( n = 15) SD
cv ( % I
2 1.97 7.7 x 3.9 - 1.5
lnterday
2 2.01 9.55X 4.75 0.5
5 4.96 8.06X10 1.73 - 0.8
Bias (%P Sulphadiazine: Concentration ( WglmL) 5 20 5 Mean ( n = 15) 4.94 19.75 4.91 SD 1.02 X 0.396 0.159 cv (Yo) 2.06 2.01 3.24 Bias (YoP - 1.2 - 1.25 - 1.8 measured concentration -target concentration x 100. a Percentage bias = target concentration
RESULTS AND DISCUSSION
The difference in the chemical nature and the solubility of the two active ingredients imposed problems on the use of liquid and solid phase extraction of both components with comparable efficiency. However, since both components are soluble in aqueous acidic media, a
.-
1
20 20.12 1.01 5.01 0.6
relatively strong acid (10% perchloric acid) was used to serve two purposes: solubilization and serum protein precipitation. Small volumes of serum (100 pL) were required to carry out the analytical procedure as compared to previously reported methods in which 1 mL of serum was used for the analysis (Vergin and Fritschi, 1979; Vergin and Bishop, 1980; Bishop-Freudling and Vergin, 1981; Springolo and Coppi, 1989). Sulphadiazine (pg/ml) 1 20
letroxoprir (pq/ml)
4
5 5.07 11.84X10-* 2.33 1.4
/
/*
\
kl’
A
‘ 0
I
I
I
I
I
1
1
0
2
4
6
8
10
12
Time
0
14
(HRS.)
Figure 3. Time is plotted vs. average serum concentrations of tetroxoprim (0) and sulphadiazine ( A )for six volunteers following the administration of 200 mg tetroxoprirn and 500 mg sulphadiazine.
H. N . ALKAYSI, M. S. SALEM AND A.A. BADWAN
268
Furthermore, detection and purification procedures were standardized by using benzoic acid, and readily available and stable compound. Mobile phase The percentage of the organic constituents in the mobile phase was important for the resolution of the analytes from endogenous components. Retention times were 1 3 . 4 f 0 . 2 m i n for tetroxoprim and 5.2k 0.3 min for sulphadiazine. The final pH determined the retention time of the internal standard, which was 11.0 & 0.5 min using the reported chromatographic conditions. Chromatograms for tetroxoprim and sulphadiazine in collected human serum and urine are shown in Fig. 1 (A and C) and 2 (A and B), respectively. Analysis of blank serum (Fig. 1B and D) and urine samples (Fig. 2C) showed the lack of intcrference with endogenous components. Specificity The major metabolite of sulphadiazine is the N4-acetyl derivative. The peak corresponding to the metabolite appeared at R, = 7.7 f0.3 min in the chromatograms obtained for spiked and collected serum samples. Tetroxoprim metabolism represents only 5% of the total adminsitered dose, which presented difficulties in detection using chromatographic analytical procedures (Korn et at. 1979). The chromatograms of both components under sample purification conditions did not change following storage overnight at room temperature and 72 h at 4 "C. The specificity of the analysis in the presence of other drugs was tested. The following drugs were studied without interference with the analytes: (a) antimicrobials-sulphamethoxazole, trimethoprim, metronidazole, erythromycin, ampicillin, cloxacillin, chloramphenicol and tetracycline; (b) analgesicsacetaminophen, acetylsalicylic acid, ibuprofen, ketoprofen and indomethacin; (c) common alkaloidscaffeine and theophy llin. Linearity and reproducibility
for calibration curves conducted using spiked urine samples. The reproducibility of the procedure was verified by repeatedly analysing spiked serum samples at two different concentrations of tetroxoprim iind sulphadiazine. Inter- and intra-day Coefficients of variation ("/o CV) were calculated for both components. The results are listed in 'Table 2. Inter- and intra-assay coefficients of variation were determined for urine control samples and did not exceed 2.86% for tetroxoprim and 3.4% for sulphadiazine. Recovery The mean absolute and relative recoveries were greater than 95% for both drugs and 97% for the internal standard. The lowest detectable concentrations using reported chromatographic conditions were 0.5 yg/mL for tetroxoprim and 1pg/mL for sulphadiazine. Lower concentrations of sulphadiazine were detectable, but standardization was conducted at a higher concentration range to match the expected serum levels following the oral administration of the drug combination. Application Serum and urine samples collected from six healthy male volunteers were analysed following the administration of two tablets of [email protected] serum concentrations of tetroxoprim and sulphadiazine at each time-point were plotted vs. time as shown in Fig. 3 .
CONCLUSION In conclusion, the proposed method of anlaysis offers the advantages of being simple, precise and applicable to pharmacokinetic studies on tetroxoprim and sulphadiazine drug combinations. Acknowledgements
Linearity was established over concentrations in the ranges 1-6 pg/mL for tetroxoprim and 2.5-25 pg/mL for sulphadiazine in serum. Standard curve statistics are listed in Table 1. This table also presents linearity data
The authors would like to thank thc Deanship of Scientific Research at Jordan University of Scicnce and Technology and the Jordanian Pharmaceutical Manufacturing Company. Thanks are also extended to Hoffman la Roche for providing the dosage form.
REFERENCES Bishop-Freudling, G. B. and Vergin, H. (1981). J . Chromatograph. Biomed. Appl. 224, 301. Bywater, M. J., Holt, H. A. and Reeves, D. S. (1979). J . Antimicrob. Chemother. 5 (Suppl. B), 51. Ferber, H. and Ahrens, K. H. (1979).J . Antimicrob. Chemother. 5 (Suppl. B), 231. Goehl, T. J., Marthur, L. K., Strum, J. D., Jaffe, J. M., Pitlick, W. H., Shah, V. P., Poust, R. 1. and Colaizzi, J. L. (1978). J. Pharm. Sci. 67, 404. Helboe, P. and Thornsen, M. (1977).Arch. Pharrn. Chem. Sci. Ed.
5, 453.
Korn, A,, Ferber, H., Hitzenberger and Vergin, H. (1979). J . Antirnicrob. Chemother. 5 (Suppl. 8). 139. Pust, R., Ferber, H., Weinder. W. and Rothaug, C. F. (1979). J . Anfirnicrob. Chemother. 5 (Suppl. B), 171. Springolo, V., and Coppi, G. (1989). J. Pharm. Biorned. Anal. 7 , 57. Su, S. C., Hartkopf, A. V. and Karger, 6.L. (1976).J . Chrornatogr. 119, 523. Vergin, H. and Bishop, G. 6.(1980). Arzneim. Forsch. 30, 317. Vergin, H. and Fritschi, E. (1979). J. Anfirnicrob. Chemother. 5 (Suppl. B), 103.
High Performance Liquid Chromatographic Analysis of Tetroxoprim and Sulphadiazine in Serum and Urine Hanan N. Alkaysi" and Mutaz Sheikh Salem Faculty of Pharmacy, Jordan University of Science and Technology, Irhid, Jordan
Adnan A. Badwan Jordanian Pharmaceutical Manufacturing Company. Naor, Jordan
Quantitative determination of tetroxoprim and sulphadiazine in serum and urine was performed using reversed phase high performanceliquid chromatography.Protein precipitation using 10% perchloric acid was utilized for purification of serum samples while urine samples were diluted prior to analysis. The mobile phase consisted of triethylammonium acetate buffer (85%), acetonitrile (12%) and methanol (3%), with a final pH of 4.2. The eluent was monitored at 280 nm. Benzoic acid was used as an internal standard. Standardization, validation and application of the method is described. Materials. Tetroxoprim and sulphadiazine were provided by JPM (Naor, Jordan). Benzoic acid, perchloric acid (60%), acetic acid and triethylamine were purchased from BDH Combinations of sulpha drugs with derivatives of 2,4(Poole, UK). Acetonitrile and methanol were HPLC grade diamino-5-benzylpyrimidine have been used as syncr(May & Baker, UK). Water was distilled and deionized. The gestically acting antibacterial drugs. Their mechanism dosage form TibiroxB was generously provided by Hoffman la ot action involves blockage of folic acid metabolism. Roche (Basel, Switzerland). One of the clinically effectivc combinations is comThe metabolite N4-acetylsulphadiazine was synthesized by posed of tetroxoprim, 2,4-diamino-S-[3,5-dimethoxy-4acetylation of sulphadiazine using acetic anhydride and con(2-methoxyethoxy)benzyl pyrimidine], and sulphadiacentrated sulphuric acid. The identity of the product was zine. This combination exhibits bactericidal activity confirmed by melting point determination and by spectral against many pathogens (Bywater et al., 1979); howdata (IR and NMR). The purity was checked by thin layer ever it is primarily recommended in the treatment of chromatography (TLC) and HPLC. respiratory a n d urinary tract infections (Pust et al., 1979; Ferber a n d Ahrens, 1979). Chromatographic conditions. The HPLC column used was Methods for t h e analysis of sulphadiazine using packed with Spherisorb 5 p ODS 2 (25 x 0.46 crn2), supplied reversed phase and ion pairing high performance liquid by Phase Separation (Clwyd, UK). The mobile phase conchromatography (HPLC) have been reported (Su et al. sisted of triethylammonium acetate buffer prepared by dis1976; Helboe and Thomsen, 1977; G o e h l et al. 1978). solving 6 mL glacial acetic acid and 3 mL triethylamine in one However, only a few methods are available for the litre of water (0.1 M). The percentage of the buffer was 85% combination of sulphadiazine and tetroxoprim (Vergin and the organic components were acetonitrile 12% and metha n d Fritschi, 1979; Vergin and Bishop, 1980; anol 3%; the pEl was adjusted to 4.2 using triethylamine. A Bishop-Frcudling a n d Vergin, 1981; Springolo and solution of benzoic acid in the mobile phase was used as Coppi, 1989). T h e reported methods require different internal standard. The flow rate was 1.5 mL/min, the detecchromatographic systems or extensive and separate tion wavelength was set at 280 nm and the injection volume sample purification techniques for each component. was 100 pL. This report describes an HPLC analytical method for tetroxoprim and sulphadiazine using the same sample Sample collection. Two tablets of Tibirox@,each containing purification procedure a n d a single mobile phase for 100 mg of tetroxoprim and 250 mg of sulphadiazine, were both components. Application of the method to the administered to six healthy male volunteers. Blood samples analysis of serum a n d urine samples is reported. were withdrawn at time intervals extending over 12 h. The collected serum samples were kept at -20°C until analysis. Urine samples were collected every two hours and stored at -20°C for analysis. The volume was recorded for each EXPERIMENTAL collection.
INTRODUCTION
Instruments. All analyses were performed using a high performance liquid chromatograph consisting of a Beckman (114M) solvent delivery system, a JASCO variable wavelength detector (875-UV) and a Rheodyne injection head (7125) fitted with a 100 WLloop. *
Author to whom correspondence should be addressed
0269-3879191 1060265-04 $OS.OO 01991 by John Wiley & Sons, Ltd
Sample preparation. Serum samples, (i) Sulphadiazine. 100 pL serum and 200 pL internal standard solution (7.5 pg mL) were placed in a centrifuge tube. 100 pL 10% perchloric acid was added. The tube was vortex-mixed for one minute and centrifuged at 15,000 X g for 5 min. 100 pL of the supernatant was injected. (ii) Tetroxoprim. 100 FL serum Received 20 August 1990 Accepted (revised) 24 January 1991
H. N. ALKAYSI, M. S. SALEM AND A.A. BADWAN
266
and 100 yL internal standard solution (100 yg/rnL) were placed in a centrifuge tube. The procedure was completed as for sulphadiazine. Urine samples. (i) Sulphadiazine. 100 yL urine was diluted to 2 mL with the mobile phase after the addition of internal standard solution. (ii) Tetroxoprim. 100 pL urine was diluted to 1mL with the mobile phase after the addition of internal standard solution. The variation in the final volumes and the concentrations of the internal standard was necessary to accomodate the expected difference in concentration between sulphadiazine and tetroxoprim in biological fluids.
Standardization and calculation. Stock solutions, 2 mg/mL tetroxoprim in the mobile phase and 0.5 mg/mL sulphadiazine in methanol were used for spiking serum at concentrations of 10 yg/mL for tetroxoprim and 25 pg/mL for sulpha-
iI
II
'
diazine. Serial dilutions were prepared covering the concentration ranges 1-6 pg/mL and 2.5-25 pglmL for tetroxoprim and sulphadiazine, respectively. The standard solutions were prepared in pooled serum and stored at -20°C for further analysis. A stock solution of the internal standard was prepared by dissolving benzoic acid in the minimum volume of methanol and completed with the mobile phase. All serum analyses for sulphadiazine were performed at a range setting of 0.16 aufs. The range was set at 0.01 aufs for the detection of tetroxoprim. Analysis of both components in urine was calibrated over concentrations ranging from 12.5-100 yglrnL for sulphadiaLine and 2-20 y g h L for tetroxoprim. Calculations of interand intra-day variations for urine analysis were performed at concentrations of 4 and 16 pg/rnL for tetroxoprim and 25 and 100 pg/mL for sulphadiazine. Calibration curves were constructed by plotting peak height ratio (drug/IS) vs. concentration. Relative recovery was determined by comparing peak height ratios of injected serum samples with aqueous solutions containing equivalent amounts of analysed components, internal standard and perchloric acid. Absolute recovery was calculated in a Fimilar manner but using peak heights rather than ratios.
.1.
"
l--rTll 0
4
12
0
1G
(inin.)
( I3
I
r,
.s.
m 0
4
8
12
(min.
16
1
l--rTTl U
4
72
0 ( min.
16
1
Figure 1. Representative chromatogram of sulphadiazine (I), tetroxoprim (11) and benzoic acid (1.S.) in (A) volunteer's serum containing 17.6 pg/mL sulphadiazine (I) and 50 pg/mL I.S. (0.16 aufs); (6)blank serum at 0.16 aufs; (C) volunteer's serum containing 5.2 pg/mL tetroxoprim (11) and 7.5 pg/mL I.S. (0.01 aufs); (D) blank serum at 0.01 aufs.
l -4 l Ul -12 l 16l
U
(iiii ii
.
Figure 2. Chromatogram of urine samples collected from a human volunteers. (A) Urine sample containing 49.5 pg/mL sulphadiazine (I)and 100 pg/mL IS.; (B) urine sample containing 6.7 pg/mL tetroxoprim (11) and 25 Fg/mL I.S.; ( C ) blank urine
sample.
‘TETROXOPRlM AND SULPHADIAZTNE 1N SERUM AND URINE
267
Table 1. Statistical data for the calibration curves of tetroxoprim and sulphadiazine in serum and urine Drug (pg/mL)a
Tetroxoprirn: Serum (1-6 pglmL) Urine (2-20 hg/mL) Sulphadiazine: Serum (2.5-25 pg/rnL) Urine (12.5-100 pglmL) a
Correlation coefficient
Slope
0.997 f 1.65 X 10 - 3 0.998i8.33X10-4
0.33 f7.66 X -5.99X 10-*+3.54X 5 . 5 2 X 1 0 ~ 2 ~ 1 . 3 4 X 1 0 ~-2.77X10 3 2+6.68X 10
0.997+ 1 . 1 6 ~10 0.997f2.54~
8 . 8 6 ~1 0 - 2 ? 2 . 1 9 ~ 1.331 ~0 - * f 3 . 6 5 x
Intercept
9.46x 10-2+5.1 X - 4 . 6 9 ~10-*+9.1 x
lo-*
Each value is an average of five determinations F S.D.
Table 2. Intra- and inter-day precision of tetroxoprim and sulphadiazine assays in serum lntraday
Tetroxoprim: Concentration (KglrnL) Mean ( n = 15) SD
cv ( % I
2 1.97 7.7 x 3.9 - 1.5
lnterday
2 2.01 9.55X 4.75 0.5
5 4.96 8.06X10 1.73 - 0.8
Bias (%P Sulphadiazine: Concentration ( WglmL) 5 20 5 Mean ( n = 15) 4.94 19.75 4.91 SD 1.02 X 0.396 0.159 cv (Yo) 2.06 2.01 3.24 Bias (YoP - 1.2 - 1.25 - 1.8 measured concentration -target concentration x 100. a Percentage bias = target concentration
RESULTS AND DISCUSSION
The difference in the chemical nature and the solubility of the two active ingredients imposed problems on the use of liquid and solid phase extraction of both components with comparable efficiency. However, since both components are soluble in aqueous acidic media, a
.-
1
20 20.12 1.01 5.01 0.6
relatively strong acid (10% perchloric acid) was used to serve two purposes: solubilization and serum protein precipitation. Small volumes of serum (100 pL) were required to carry out the analytical procedure as compared to previously reported methods in which 1 mL of serum was used for the analysis (Vergin and Fritschi, 1979; Vergin and Bishop, 1980; Bishop-Freudling and Vergin, 1981; Springolo and Coppi, 1989). Sulphadiazine (pg/ml) 1 20
letroxoprir (pq/ml)
4
5 5.07 11.84X10-* 2.33 1.4
/
/*
\
kl’
A
‘ 0
I
I
I
I
I
1
1
0
2
4
6
8
10
12
Time
0
14
(HRS.)
Figure 3. Time is plotted vs. average serum concentrations of tetroxoprim (0) and sulphadiazine ( A )for six volunteers following the administration of 200 mg tetroxoprirn and 500 mg sulphadiazine.
H. N . ALKAYSI, M. S. SALEM AND A.A. BADWAN
268
Furthermore, detection and purification procedures were standardized by using benzoic acid, and readily available and stable compound. Mobile phase The percentage of the organic constituents in the mobile phase was important for the resolution of the analytes from endogenous components. Retention times were 1 3 . 4 f 0 . 2 m i n for tetroxoprim and 5.2k 0.3 min for sulphadiazine. The final pH determined the retention time of the internal standard, which was 11.0 & 0.5 min using the reported chromatographic conditions. Chromatograms for tetroxoprim and sulphadiazine in collected human serum and urine are shown in Fig. 1 (A and C) and 2 (A and B), respectively. Analysis of blank serum (Fig. 1B and D) and urine samples (Fig. 2C) showed the lack of intcrference with endogenous components. Specificity The major metabolite of sulphadiazine is the N4-acetyl derivative. The peak corresponding to the metabolite appeared at R, = 7.7 f0.3 min in the chromatograms obtained for spiked and collected serum samples. Tetroxoprim metabolism represents only 5% of the total adminsitered dose, which presented difficulties in detection using chromatographic analytical procedures (Korn et at. 1979). The chromatograms of both components under sample purification conditions did not change following storage overnight at room temperature and 72 h at 4 "C. The specificity of the analysis in the presence of other drugs was tested. The following drugs were studied without interference with the analytes: (a) antimicrobials-sulphamethoxazole, trimethoprim, metronidazole, erythromycin, ampicillin, cloxacillin, chloramphenicol and tetracycline; (b) analgesicsacetaminophen, acetylsalicylic acid, ibuprofen, ketoprofen and indomethacin; (c) common alkaloidscaffeine and theophy llin. Linearity and reproducibility
for calibration curves conducted using spiked urine samples. The reproducibility of the procedure was verified by repeatedly analysing spiked serum samples at two different concentrations of tetroxoprim iind sulphadiazine. Inter- and intra-day Coefficients of variation ("/o CV) were calculated for both components. The results are listed in 'Table 2. Inter- and intra-assay coefficients of variation were determined for urine control samples and did not exceed 2.86% for tetroxoprim and 3.4% for sulphadiazine. Recovery The mean absolute and relative recoveries were greater than 95% for both drugs and 97% for the internal standard. The lowest detectable concentrations using reported chromatographic conditions were 0.5 yg/mL for tetroxoprim and 1pg/mL for sulphadiazine. Lower concentrations of sulphadiazine were detectable, but standardization was conducted at a higher concentration range to match the expected serum levels following the oral administration of the drug combination. Application Serum and urine samples collected from six healthy male volunteers were analysed following the administration of two tablets of [email protected] serum concentrations of tetroxoprim and sulphadiazine at each time-point were plotted vs. time as shown in Fig. 3 .
CONCLUSION In conclusion, the proposed method of anlaysis offers the advantages of being simple, precise and applicable to pharmacokinetic studies on tetroxoprim and sulphadiazine drug combinations. Acknowledgements
Linearity was established over concentrations in the ranges 1-6 pg/mL for tetroxoprim and 2.5-25 pg/mL for sulphadiazine in serum. Standard curve statistics are listed in Table 1. This table also presents linearity data
The authors would like to thank thc Deanship of Scientific Research at Jordan University of Scicnce and Technology and the Jordanian Pharmaceutical Manufacturing Company. Thanks are also extended to Hoffman la Roche for providing the dosage form.
REFERENCES Bishop-Freudling, G. B. and Vergin, H. (1981). J . Chromatograph. Biomed. Appl. 224, 301. Bywater, M. J., Holt, H. A. and Reeves, D. S. (1979). J . Antimicrob. Chemother. 5 (Suppl. B), 51. Ferber, H. and Ahrens, K. H. (1979).J . Antimicrob. Chemother. 5 (Suppl. B), 231. Goehl, T. J., Marthur, L. K., Strum, J. D., Jaffe, J. M., Pitlick, W. H., Shah, V. P., Poust, R. 1. and Colaizzi, J. L. (1978). J. Pharm. Sci. 67, 404. Helboe, P. and Thornsen, M. (1977).Arch. Pharrn. Chem. Sci. Ed.
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