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A High-Performance Liquid Chromatographic Assay for Dideoxyinosine in Monkey Plasma and Urine
A High-Performance Liquid Chromatographic Assay for Dideoxyinosine in Monkey Plasma and Urine RASHID J.
RAVASCO'*,
JASHVANT D. UNADKAT'~',AND CHE-CHUNG TSAI~
Received March 4, 1991, from the 'Department of Pharmaceutics, School of Pharma the *School of Public Health, and the *Regional Primate Accepted for p u b y h o n August 15,1991. Research Center, Universiv of Washington, Seattle, WA 98795.
Abstract 0 A simple, reliable, reproducible, rapid, and sensiUve method for high-performance liquid chromatographic assay of dideoxyinosine (DDI) in macaque plasma and urine is descripd. The method is capable of detecting 25 ng of DDI on-column.
Dideoxyinosine (DDI), a new anti-human immunodeficiency virus (HIV) agent, has shown promising efficacy and toxicity profiles in phase I clinical trials for patients with the acquired immunodeficiency syndrome (AIDS) and is currently undergoing phase I1 clinical trials.' The Food and Drug Administration has accelerated the schedule for approval of anti-HIV drugs, meaning that many preclinical studies must run concurrently with human clinical trials. An AIDS-like syndrome can be experimentally induced in macaques infected with the simian immunodeficiency virus.z~3This model makes it possible to test the in vivo efficacy and toxicity of new antiviral agents. To study the pharmacokinetics of DDI in the pigtailed macaque (Macaca nenestrinu), we have developed a high-performance liquid chromatographic (HPLC) assay to determine the concentration of DDI in macaque plasma and urine. Methods for determining DDI in biological fluids for mice and cats" and humans7.8 have been reported, but the procedures are relatively complicated.For example, Knupp et al.7 and Carpen et al.8 used ion-pairing reagents in the mobile phase and an extraction step in the preparation of urine samples. We describe a simple, reliable, and reproducible assay for DDI in plasma and urine samples by using a simpler, ion-pair-reagent-fiee chromatographic system that allows direct injection of urine samples after dilution. In addition, we describe the use of this assay for analysis of samples obtained from monkeys.
samples to yield a final concentration of 2 pg/mL. Then, 50 pL of this mixture was injected onto the HPLC system. The calibrators (DDI at 0.25-2.0 @mL) and the controls were prepared as previously described, except that the calibrators were prepared in deionized water and the controls (0.25 and 2.0 @mL) were prepared in diluted (1:50)blank macaque urine. A straight line was then fitted, by using linear regression, to the peak height ratio (DDYIS) versus the concentration of DDI. The concentration of DDI in the unknown samples was determined from the fitted calibration line by inverse regression. Chromatography-The HPU: system consisted of a C18 analytical column (Ultrasphere, 250 x 4.6 mm; Rainin, San Ramon, CA) preceded by a C18 guard column (30x 2.1 mm; Alltech, San Jose, CAI, and a model 501 pump (Waters, Milford, MA). The analytes were eluted from the column at a rate of 1 d m i n with acetonitri1e:ammonium phosphate (0.06M,pH 4.0)(6:94)and detected at 254 nm by a model 440 UV detector (Waters).
Resuits and Discussion Plasma-Elution times for DDI and IS were 7.2 and 14.0 min, respectively. Chromatograms obtaihed after extraction of blank macaque plasma indicate that no interfering peaks are present at the positions where both DDI and IS are expected to elute (Figure 1). However, at buffer pH 5-7, the peak eluting immediately after DDI merged with the DDI peak. When extracted, the recovery of DDI is 87.3 2 12.6% over a concentration range of 0.5-5 crg/mL. Because DDI is unstable at acidic pH, the on-column stability was confirmed by direct injection of standards containing various concentrations of DDI, with the pH of the ammonium phosphate buffer in the mobile phase fixed at pH 4.0or 6.8.In addition, stability of DDI in the reconstituting fluid was also ascertained over a 2 4 h period a t room tem-
Experimental Section P l a s m e T o 25-400 p L of macaque plasma was added 50 p L of the internal standard (IS)solution (3-hydroxyacetamidophenol, 5 pg/mL; Aldrich, Milwaukee, WI) diesolved in deionized water. Sufficient deionized water was added to bring the total volume up to 500 pL. This mixture was placed on a preconditioned (with one volume each of methanol and deionized water), solid-phaae extraction column (C,,, Bond Elut, 3 mL, Analytichem International, Harbor City, CAI. The sample was washed with two volumes (2.0 mL each) of deionized water, and the analytes were eluted from the column with one volume of methanol. The methanol fraction was evaporated to dryness under a gentle stream of nitrogen at 3 0 4 0 "C. The sample was recomtituted with 100 p L of acetonitri1e:water (6941,and 50 pL of the mixture was injected onto the HPLC. Calibrators (0.02fLO.26pg of DDI per sample) and controls (0.025 and 0.25 pg per sample) were processed as previously described, except that the matrix was blank macaque plasma (200 p L ) . A straight line was fitted, by using linear regreseion, to the peak height ratio of the calibrators (DDHS) versus the amount of DDI per sample. The concentration of DDI in the unknown samplea was determined from the fitted calibration line by inverse regression. Urine-IS (100 pg/mL) was added to the diluted (1:50)urine 690 I Journal of Pharmaceutical Sciences Vol. 81, No. 7, July 1992
I
'4O
I
I
0 . W AUFS
I
0
I
5 10 15 MINUTES
20 0
IS
DDI 7.2
I
5 10 15 MINUTES
f
20
Figure l-chromatograms obtained after extracting (Left) 200 pL of blank macaque plasma and (Right) 25 pL of plasma obtained after an iv bolus dose of DDI (3.2mgkg) to a male macaque. Numbers indicate retention times in minutes. AUFS. absorbance unit full scale. 00~-3549/92/0700-0690$02. MI0 0 7992, American Pharmaceutical Association
Table Untmday CVa of the Asmy
h
2
sample
DDI Concentration'
cv, %b
Plasma
0.025 0.075 0.250 0.25 0.5 2.0
5.1 4.2 5.3 2.1 1.5 0.5
Urine
2
10.000
v
'Concentrations in plasma are expressed in micrograms per sample;
those in urine are expressed in micrograms per milliliter. n = 5.
a n D
DDI
0
0
50
1
100
150
0 200
250
1
300
7
350
400
Figure 3-Profile of concentration of DDI in plasma versus time after administration of an iv bolus dose (3.2 mg/kg) of DDI to a female macaque.
0.001AUFS
Is 15.2
MINUTES
.
TIME (MIN)
8.0
I
o
MINUTES
Flgum2-Chromatogramsobtained after injecting onto the HPLC (Left) 50 pL of diluted (150 in distilled water) blank macaque urine and (Right) 50 pL of diluted urine (150) obtained after administration of an iv bolus dose of DDI (10 mgkg) to a male macaque. Numbers indicate retention time in minutes. AUFS, absorbance unit full scale.
perature. No significant decrease in DDVIS peak height ratio was observed over this period (data not shown). The excellent reproducibility of the assay is demonstrated by the small interday coefficient of variability (CV)in the slope (5.5%) and the small intraday coefficient of variations in the DDVIS peak height ratio (Table I). The mean percent deviation of the predicted concentration of DDI in the control samples from the expected concentration was 5.8 k 4.2% at 0.25 pglsample and -0.35 2 7.8% a t 0.025 &sample. The lower limit of the detection of DDI is 50 p L of plasma a t a concentration of 0.5 CcglmL. Urine-Elution times for DDI and IS were 8.0 and 15.2 min, respectively. Chromatograms obtained after direct injection of diluted blank macaque urine (1:50) indicate the absence of interfering peaks at the positions where both DDI and Is are expected to elute (Figure 2). DDI was stable in the urine samples at mom temperature for at least 24 h. The
excellent reproducibility of the assay is demonstrated by the small interday variability in the slope (CV,5%) and the small intraday variation in the DDVIS peak height ratio (Table I). The mean percent deviation of the predicted concentration of DDI in the control samples from the expected concentration was 0.30 f 4.8% and -0.30 f 3.0% for the low and high concentrations, respectively. The assay described here has been used to analyze plasma and urine samples obtained from pharmacokinetic studies conducted in macaques. A typical plot of the concentration of DDI in plasma versus time obtained afler administration of DDI at 3.2 mg k g [intravenous (iv) bolus1 to a female macaque is illustrated in Figure 3. In addition, the percent dose recovered as DDI in urine over a 24-h period was 64%. In conclusion, the assay described for DDI in monkey urine and plasma is simple, reliable, and reproducible and should be applicable to preclinical studies conduded in macaques.
References and Notes 1. Yarchoan, R.; Mitauya, H.;Thomas, R. V.; Pluda, J. M.; Hartman,
N.R.; Perno,.C.-F.; Marcz k, K. S.; Allain, J.-P.; Johns, D. G.;
Broder, S. Sczence 1989,2&, 412415. 2. Benveniste, R. E.; Morton, W.R.; Clark, E. A.; T F i , C.-C.; &he,
H. D.; Ward,J. M.; Kuller, L.;Knott, W. B.; Hill, R. W.; Gale, M. J.; Thouless, M. E. J. Viml.1988,62,2091-2101. 3. Kuller, L.;Morton, W. R.; Benveniste, R. E.; Teai, C.-C.; Clark, E. A.; Gale, M. J.;Hu, S.-L.; Thouless, M. E.; Katze, M. G.J.Med. Primutol. 1990,19,367-380. 4. Blau, P.A.; Hinea, J. W.; Voyksner, R. D. J. Chmmatogr. 1987,
420, 1-12. 6. Kalin, J. R.; Hill, D. L.J. Chromutogr. 1988,431, 18p191. 6. Ruseell, J. W.; Klunk, L.J. Biochem.Pharmacol. 1989,38,13851388. 7. Knu p,. Catherine, A.; Stancato, Frank A.; Pap Eu ene A.; Barbimya, Rashmi H. J. Chmmutogr. 1990,533, %82-280 8. Carpen, M. E.; Balis, F.M. J. Chromatogr. 1990,526,69-76.
Acknowledgments We thank Dr. C. A. Knupp, Bristol-Myers Company, for her advice. This work was su ported by National Institutes of Health grant NO1 A1 62526. Rashii J. Ravasco is supported by the Fogarty International Fellowshi and is on leave from the Clinical Research Center, Kenya Medical kxiearch Institute, P.O. Box 20778, Nairobi, Kenya.
Journal of Pharmaceutical sciences 1 691 Vol. 87, No. 7, July 1992
RAVASCO'*,
JASHVANT D. UNADKAT'~',AND CHE-CHUNG TSAI~
Received March 4, 1991, from the 'Department of Pharmaceutics, School of Pharma the *School of Public Health, and the *Regional Primate Accepted for p u b y h o n August 15,1991. Research Center, Universiv of Washington, Seattle, WA 98795.
Abstract 0 A simple, reliable, reproducible, rapid, and sensiUve method for high-performance liquid chromatographic assay of dideoxyinosine (DDI) in macaque plasma and urine is descripd. The method is capable of detecting 25 ng of DDI on-column.
Dideoxyinosine (DDI), a new anti-human immunodeficiency virus (HIV) agent, has shown promising efficacy and toxicity profiles in phase I clinical trials for patients with the acquired immunodeficiency syndrome (AIDS) and is currently undergoing phase I1 clinical trials.' The Food and Drug Administration has accelerated the schedule for approval of anti-HIV drugs, meaning that many preclinical studies must run concurrently with human clinical trials. An AIDS-like syndrome can be experimentally induced in macaques infected with the simian immunodeficiency virus.z~3This model makes it possible to test the in vivo efficacy and toxicity of new antiviral agents. To study the pharmacokinetics of DDI in the pigtailed macaque (Macaca nenestrinu), we have developed a high-performance liquid chromatographic (HPLC) assay to determine the concentration of DDI in macaque plasma and urine. Methods for determining DDI in biological fluids for mice and cats" and humans7.8 have been reported, but the procedures are relatively complicated.For example, Knupp et al.7 and Carpen et al.8 used ion-pairing reagents in the mobile phase and an extraction step in the preparation of urine samples. We describe a simple, reliable, and reproducible assay for DDI in plasma and urine samples by using a simpler, ion-pair-reagent-fiee chromatographic system that allows direct injection of urine samples after dilution. In addition, we describe the use of this assay for analysis of samples obtained from monkeys.
samples to yield a final concentration of 2 pg/mL. Then, 50 pL of this mixture was injected onto the HPLC system. The calibrators (DDI at 0.25-2.0 @mL) and the controls were prepared as previously described, except that the calibrators were prepared in deionized water and the controls (0.25 and 2.0 @mL) were prepared in diluted (1:50)blank macaque urine. A straight line was then fitted, by using linear regression, to the peak height ratio (DDYIS) versus the concentration of DDI. The concentration of DDI in the unknown samples was determined from the fitted calibration line by inverse regression. Chromatography-The HPU: system consisted of a C18 analytical column (Ultrasphere, 250 x 4.6 mm; Rainin, San Ramon, CA) preceded by a C18 guard column (30x 2.1 mm; Alltech, San Jose, CAI, and a model 501 pump (Waters, Milford, MA). The analytes were eluted from the column at a rate of 1 d m i n with acetonitri1e:ammonium phosphate (0.06M,pH 4.0)(6:94)and detected at 254 nm by a model 440 UV detector (Waters).
Resuits and Discussion Plasma-Elution times for DDI and IS were 7.2 and 14.0 min, respectively. Chromatograms obtaihed after extraction of blank macaque plasma indicate that no interfering peaks are present at the positions where both DDI and IS are expected to elute (Figure 1). However, at buffer pH 5-7, the peak eluting immediately after DDI merged with the DDI peak. When extracted, the recovery of DDI is 87.3 2 12.6% over a concentration range of 0.5-5 crg/mL. Because DDI is unstable at acidic pH, the on-column stability was confirmed by direct injection of standards containing various concentrations of DDI, with the pH of the ammonium phosphate buffer in the mobile phase fixed at pH 4.0or 6.8.In addition, stability of DDI in the reconstituting fluid was also ascertained over a 2 4 h period a t room tem-
Experimental Section P l a s m e T o 25-400 p L of macaque plasma was added 50 p L of the internal standard (IS)solution (3-hydroxyacetamidophenol, 5 pg/mL; Aldrich, Milwaukee, WI) diesolved in deionized water. Sufficient deionized water was added to bring the total volume up to 500 pL. This mixture was placed on a preconditioned (with one volume each of methanol and deionized water), solid-phaae extraction column (C,,, Bond Elut, 3 mL, Analytichem International, Harbor City, CAI. The sample was washed with two volumes (2.0 mL each) of deionized water, and the analytes were eluted from the column with one volume of methanol. The methanol fraction was evaporated to dryness under a gentle stream of nitrogen at 3 0 4 0 "C. The sample was recomtituted with 100 p L of acetonitri1e:water (6941,and 50 pL of the mixture was injected onto the HPLC. Calibrators (0.02fLO.26pg of DDI per sample) and controls (0.025 and 0.25 pg per sample) were processed as previously described, except that the matrix was blank macaque plasma (200 p L ) . A straight line was fitted, by using linear regreseion, to the peak height ratio of the calibrators (DDHS) versus the amount of DDI per sample. The concentration of DDI in the unknown samplea was determined from the fitted calibration line by inverse regression. Urine-IS (100 pg/mL) was added to the diluted (1:50)urine 690 I Journal of Pharmaceutical Sciences Vol. 81, No. 7, July 1992
I
'4O
I
I
0 . W AUFS
I
0
I
5 10 15 MINUTES
20 0
IS
DDI 7.2
I
5 10 15 MINUTES
f
20
Figure l-chromatograms obtained after extracting (Left) 200 pL of blank macaque plasma and (Right) 25 pL of plasma obtained after an iv bolus dose of DDI (3.2mgkg) to a male macaque. Numbers indicate retention times in minutes. AUFS. absorbance unit full scale. 00~-3549/92/0700-0690$02. MI0 0 7992, American Pharmaceutical Association
Table Untmday CVa of the Asmy
h
2
sample
DDI Concentration'
cv, %b
Plasma
0.025 0.075 0.250 0.25 0.5 2.0
5.1 4.2 5.3 2.1 1.5 0.5
Urine
2
10.000
v
'Concentrations in plasma are expressed in micrograms per sample;
those in urine are expressed in micrograms per milliliter. n = 5.
a n D
DDI
0
0
50
1
100
150
0 200
250
1
300
7
350
400
Figure 3-Profile of concentration of DDI in plasma versus time after administration of an iv bolus dose (3.2 mg/kg) of DDI to a female macaque.
0.001AUFS
Is 15.2
MINUTES
.
TIME (MIN)
8.0
I
o
MINUTES
Flgum2-Chromatogramsobtained after injecting onto the HPLC (Left) 50 pL of diluted (150 in distilled water) blank macaque urine and (Right) 50 pL of diluted urine (150) obtained after administration of an iv bolus dose of DDI (10 mgkg) to a male macaque. Numbers indicate retention time in minutes. AUFS, absorbance unit full scale.
perature. No significant decrease in DDVIS peak height ratio was observed over this period (data not shown). The excellent reproducibility of the assay is demonstrated by the small interday coefficient of variability (CV)in the slope (5.5%) and the small intraday coefficient of variations in the DDVIS peak height ratio (Table I). The mean percent deviation of the predicted concentration of DDI in the control samples from the expected concentration was 5.8 k 4.2% at 0.25 pglsample and -0.35 2 7.8% a t 0.025 &sample. The lower limit of the detection of DDI is 50 p L of plasma a t a concentration of 0.5 CcglmL. Urine-Elution times for DDI and IS were 8.0 and 15.2 min, respectively. Chromatograms obtained after direct injection of diluted blank macaque urine (1:50) indicate the absence of interfering peaks at the positions where both DDI and Is are expected to elute (Figure 2). DDI was stable in the urine samples at mom temperature for at least 24 h. The
excellent reproducibility of the assay is demonstrated by the small interday variability in the slope (CV,5%) and the small intraday variation in the DDVIS peak height ratio (Table I). The mean percent deviation of the predicted concentration of DDI in the control samples from the expected concentration was 0.30 f 4.8% and -0.30 f 3.0% for the low and high concentrations, respectively. The assay described here has been used to analyze plasma and urine samples obtained from pharmacokinetic studies conducted in macaques. A typical plot of the concentration of DDI in plasma versus time obtained afler administration of DDI at 3.2 mg k g [intravenous (iv) bolus1 to a female macaque is illustrated in Figure 3. In addition, the percent dose recovered as DDI in urine over a 24-h period was 64%. In conclusion, the assay described for DDI in monkey urine and plasma is simple, reliable, and reproducible and should be applicable to preclinical studies conduded in macaques.
References and Notes 1. Yarchoan, R.; Mitauya, H.;Thomas, R. V.; Pluda, J. M.; Hartman,
N.R.; Perno,.C.-F.; Marcz k, K. S.; Allain, J.-P.; Johns, D. G.;
Broder, S. Sczence 1989,2&, 412415. 2. Benveniste, R. E.; Morton, W.R.; Clark, E. A.; T F i , C.-C.; &he,
H. D.; Ward,J. M.; Kuller, L.;Knott, W. B.; Hill, R. W.; Gale, M. J.; Thouless, M. E. J. Viml.1988,62,2091-2101. 3. Kuller, L.;Morton, W. R.; Benveniste, R. E.; Teai, C.-C.; Clark, E. A.; Gale, M. J.;Hu, S.-L.; Thouless, M. E.; Katze, M. G.J.Med. Primutol. 1990,19,367-380. 4. Blau, P.A.; Hinea, J. W.; Voyksner, R. D. J. Chmmatogr. 1987,
420, 1-12. 6. Kalin, J. R.; Hill, D. L.J. Chromutogr. 1988,431, 18p191. 6. Ruseell, J. W.; Klunk, L.J. Biochem.Pharmacol. 1989,38,13851388. 7. Knu p,. Catherine, A.; Stancato, Frank A.; Pap Eu ene A.; Barbimya, Rashmi H. J. Chmmutogr. 1990,533, %82-280 8. Carpen, M. E.; Balis, F.M. J. Chromatogr. 1990,526,69-76.
Acknowledgments We thank Dr. C. A. Knupp, Bristol-Myers Company, for her advice. This work was su ported by National Institutes of Health grant NO1 A1 62526. Rashii J. Ravasco is supported by the Fogarty International Fellowshi and is on leave from the Clinical Research Center, Kenya Medical kxiearch Institute, P.O. Box 20778, Nairobi, Kenya.
Journal of Pharmaceutical sciences 1 691 Vol. 87, No. 7, July 1992