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Clinical Chemistry 46: 73-81, 2000;
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(Clinical Chemistry. 2000;46:73-81.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Simultaneous HPLC Assay for Quantification of Indinavir, Nelfinavir, Ritonavir, and Saquinavir in Human Plasma

Rory P. Remmel1,a, Sagar P. Kawle2, Dennis Weller2 and Courtney V. Fletcher2

Departments of
1 Medicinal Chemistry and
2 Experimental and Clinical Pharmacology, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455.
a Address correspondence to this author at: University of Minnesota College of Pharmacy, 8-174 WDH, 308 Harvard St. SE, Minneapolis, MN 55455. Fax 612-624-0139; e-mail remme001{at}tc.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: HIV protease inhibitors are recommended as part of combination antiretroviral therapy. Dual protease inhibitor therapy is also being used clinically. Consequently, a simultaneous assay for indinavir, nelfinavir, ritonavir, and saquinavir was developed.

Methods: Indinavir, nelfinavir, ritonavir, and saquinavir were extracted from plasma (250 µL) with methyl-t-butyl ether at basic pH after addition of an internal standard (A-86093). The compounds were separated on a Keystone BetaBasic C4 column (250 x 3 mm i.d.) at 40 °C with a mobile phase of acetonitrile-50 mmol/L ammonium formate buffer, pH 4.1 (52:48, by volume) at a flow rate of 0.5 mL/min. Indinavir, nelfinavir, ritonavir, and the internal standard (A-86093) were detected at 218 nm, and saquinavir was detected at 235 nm. The method was validated by analysis of five triplicate analyses of calibrators along with quality-control samples at three different concentrations prepared in human plasma.

Results: The extraction recovery was 87–92%. Within-run accuracy for quality-control samples was 6–8%, with CVs of 2–8%. Limits of quantification were 40–50 µg/L for indinavir, nelfinavir, and ritonavir, and 20 µg/L for saquinavir. Cross-validation with a liquid chromatography-mass spectroscopy method for saquinavir and nelfinavir was conducted with patient samples. Regression analysis revealed a good correlation (r2 >0.94) between methods. Larger variations at concentrations >4000 µg/L were observed with nelfinavir. Interference with drugs commonly used in AIDS patients was not observed. Pharmacokinetic profiles for two patients on dual protease therapy were determined.

Conclusions: A reliable and rugged simultaneous HPLC assay for four HIV protease inhibitors was developed. The assay method is convenient for clinical laboratories involved in therapeutic drug monitoring for HIV protease inhibitors. The assay has enough sensitivity to conduct pharmacokinetic studies in patients taking more than one HIV protease inhibitor along with other antiretroviral medications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhibitors of HIV protease, in combination with nucleoside inhibitors of HIV reverse transcriptase, are the cornerstone of currently recommended therapy for HIV infection (1). A regimen that includes a protease inhibitor and two nucleoside reverse transcriptase inhibitors has been shown to delay HIV disease progression and to improve survival when compared with a regimen of just two nucleosides (2). Unfortunately, not all patients have an equally optimal response to this anti-HIV regimen. Accumulating clinical information suggests relationships between systemic exposure to a protease inhibitor and antiviral effect (3)(4)(5)(6). The cytochrome P450 enzyme system catalyzes the oxidative metabolism of the currently available protease inhibitors, especially CYP3A4, in humans (7)(8)(9)(10). In addition, the HIV protease inhibitors also act as inhibitors of CYP3A4 to varying degrees (ritonavir > indinavir > nelfinavir > saquinavir in terms of inhibitory potency) (8)(9). Thus, the potential for drug-drug interactions is a prominent pharmacologic characteristic of the HIV protease inhibitors. In fact, several combinations of protease inhibitors, such as saquinavir and ritonavir, or indinavir and ritonavir, are under clinical investigation in an attempt to exploit the potent inhibition of metabolism by ritonavir. These pharmacologic characteristics of HIV protease inhibitors suggest there are several scenarios where information on the plasma concentration of a protease inhibitor would be of clinical interest.

Currently, there are four protease inhibitors available for clinical use: indinavir sulfate, nelfinavir mesylate, ritonavir, and saquinavir (Fig. 1 ). Separate analytical procedures have been developed to measure of each of the four protease inhibitors. For indinavir, HPLC-tandem mass spectroscopy (MS/MS)1 and HPLC assays have been published (10)(11)(12). HPLC methods have been described for both nelfinavir and ritonavir (12)(13)(14)(15). Saquinavir has been quantified by HPLC/MS/MS, HPLC, and RIA (16)(17)(18)(19)(20). The objective of this study was to develop an HPLC assay for the simultaneous quantification of indinavir, nelfinavir, ritonavir, and saquinavir in human plasma.



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  		Figure 1. Chemical structures of the HIV protease inhibitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
chemicals and reagents
Indinavir monohydrate (free base), nelfinavir mesylate, ritonavir, and saquinavir mesylate were gifts from Merck Research Laboratories, Agouron Pharmaceutical Inc., Abbott Laboratories, and Roche Products Ltd., respectively. The internal standard, A-86093 {(5S,8S,10S,11S)-9-hydroxy-2-cyclopropyl-5-(1-methylethyl)-1-[(2-1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester} was obtained from Abbott Laboratories. Formic acid (88%, ACS grade) was from Fisher Scientific. Solvents used for assay development were of HPLC grade and were also obtained from Fisher Scientific.

apparatus and chromatographic conditions
The HPLC system consisted of a GBC HPLC system equipped with a GBC model LC1650 Advanced Autosampler, a GBC model LC1150 HPLC pump, and an ERC 3145 solvent degasser (GBC Separations). The compounds were detected with a SpectraFOCUS forward optical scanning ultraviolet (UV) detector (Spectra-Physics). The column used was a BetaBasic C4, 5 µm bead size, 250 x 3 mm from Keystone Scientific. The column was heated to 40 °C in an Eppendorf column heater. A Jouan GR 4 22 centrifuge (Jouan) was used for centrifugation during the extraction process. The mobile phase used for analysis was acetonitrile-50 mmol/L sodium formate buffer (52:48, by volume; pH adjusted to 4.10 with 2 mol/L sodium hydroxide). The flow rate was set to 0.5 mL/min. The detection wavelength was 218 nm for indinavir, nelfinavir, ritonavir, and the internal standard, and 235 nm for saquinavir.

sample preparation and assay
A stock solution containing indinavir sulfate, nelfinavir mesylate, ritonavir, and saquinavir mesylate was prepared in methanol. Different calibration solutions were prepared by dilution from the stock solution such that the final concentration ranges were 48–19 430 µg/L for indinavir (as free base), 21–8550 µg/L for nelfinavir (as free base), 50–20 000 µg/L for ritonavir, and 22–8750 µg/L for saquinavir (as free base). A quality-control stock solution was prepared separately in methanol at a concentration of 0.25 g/L of each drug. The individual quality controls were prepared by adding appropriate volumes of the quality-control stock solution to a 50-mL volumetric flask and diluting with blank human plasma (EDTA-derived; Biological Specialty). Quality-control samples (50 mL each) were prepared at concentrations of 4850, 972, and 194 µg/L for indinavir (as free base); 4275, 855, and 171 µg/L for nelfinavir (as free base); 5000, 1000, and 200 µg/L for ritonavir; and 4375, 875, and 175 µg/L for saquinavir (as free base). The quality-control samples were stored in 1.0-mL aliquots at -80 °C. The internal standard (A-86093) solution was prepared in acetonitrile to give a 5 g/L stock solution. The working internal standard solution concentration was 125 mg/L in acetonitrile.

Before the extraction procedure, the 13 x 100 mm borosilicate glass test tubes (Kimax-51®; VWR Scientific) were rinsed with 2 mL of HPLC-grade methanol on a vortex-type mixer to remove interferences. The calibrators were prepared by adding 20 µL of stock solution in methanol to 250 µL of blank plasma. A 250-µL sample of patient plasma was used for analysis. Twenty microliters of the internal standard was added to the plasma, followed by 250 µL of 0.05 mol/L sodium hydroxide. The solution was mixed on a vortex-type mixer, and 2 mL of methyl-tert-butyl ether was added. The samples were mixed on a vortex-type mixer for 30 s and centrifuged at 2500g for 5 min, and the aqueous layer was frozen for 5 min in a dry-ice-isopropanol bath. The methyl-tert-butyl ether layer was decanted into 12 x 75 mm Kimax-51 borosilicate glass test tubes (VWR) that had been rinsed previously with 2 mL of HPLC-grade methanol on a vortex-type mixer. The organic layer was dried under nitrogen at 40 °C on a TurboVap LV evaporator (Zymark). The residue was reconstituted in 200 µL of HPLC mobile phase, and 50 µL of the reconstituted extract was injected onto the HPLC system for analysis.

method validation, comparison, and patient studies
The assay was validated by assaying calibrators and quality controls in triplicate on 5 different days. Quality controls were assayed in quintuplets on the same day to estimate the intraday coefficient of variation (CV) and the accuracy. A comparison of results obtained with this simultaneous HPLC procedure and "reference" methods (done by LC/MS) was accomplished with plasma samples that had been obtained previously from children participating in a trial of the combination of saquinavir and nelfinavir. The concentrations of nelfinavir and saquinavir were determined, and linear regression was used to evaluate the performance of the simultaneous procedure vs that of the reference method used by Roche Laboratories. To investigate the clinical utility of this simultaneous method, protease inhibitor concentrations in plasma were quantified in two adults: one receiving a combination of ritonavir (400 mg twice daily) and saquinavir soft gelatin capsule (400 mg twice daily); the other receiving a combination of nelfinavir (750 mg three times daily) and saquinavir soft gelatin capsule (800 mg three times daily). Blood samples were obtained at the following times: predose and 0.5, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h after observed doses of the combinations. The plasma was harvested and frozen at -80 °C for subsequent analysis. The procedure was approved by the Human Subjects Committee of the University of Minnesota, and each subject gave his or her written consent before participation. Concentrations of the protease inhibitors were determined, and pharmacokinetic parameters were calculated with statistical moment theory methods (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Typical chromatograms of blank plasma, blank plasma with internal standard, plasma supplemented with a medium-range calibrator, and patient plasma containing saquinavir and nelfinavir are shown in Fig. 2 . Two interfering peaks, one eluting before nelfinavir and one eluting under nelfinavir, were removed by rinsing the borosilicate glass tubes with methanol. The identity of these compounds was not investigated further. Table 1 presents the accuracy and precision data for the calibrators. Accuracy for the four drugs ranged from -11% to 14%. The total CV for the four drugs was 2–21%, whereas the within-run CV was 2–24%. The peak height ratios of the analytes were linear throughout the calibration range and encompassed the therapeutic concentrations in plasma. The mean (± SD) weighted linear regression slopes, intercepts, and coefficients of determination (r2 values) were as follows: for indinavir, slope = 0.1854 ± 0.0054, y-intercept = 0.00406 ± 0.00201, r2 = 0.9977 ± 0.0021; for nelfinavir, slope = 0.2978 ± 0.0077, y-intercept = 0.0104 ± 0.0099, r2 = 0.9976 ± 0.002; for saquinavir, slope = 0.7001 ± 0.0264, y-intercept = 0.00371 ± 0.0108, r2 = 0.9975 ± 0.0023; for ritonavir, slope = 0.1452 ± 0.0063, y-intercept = 0.00096 ± 0.00149, r2 = 0.9982 ± 0.0022. The 95% confidence interval of the y-intercept included zero for all four compounds. Table 2 contains the accuracy and precision data for the quality controls. The intrarun variability for the quality controls is shown in Table 3 . Extraction efficiencies (mass recoveries) are presented in Table 4 . The recovery was 86–89% for indinavir, 88–91% for nelfinavir, 87–90% for saquinavir, and 89–93% for ritonavir. The extraction recovery for the internal standard was 86.8% ± 2.1% (n = 3). The detection limit at a signal-to-noise ratio of 5:1 was 22 µg/L nelfinavir, 5.9 µg/L for saquinavir, and 13.5 µg/L for ritonavir. The limit of quantification (defined as a CV <=20% and an accuracy within ±20% of the true value) was 40–50 µg/L for indinavir, nelfinavir, and ritonavir, and 20 µg/L for saquinavir. The nelfinavir calibrator at 21 µg/L could not be determined reproducibly, and the data were omitted from Table 1 .



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  		Figure 2. Chromatograms from simultaneous HPLC assay of HIV protease inhibitors.

(A), extracted blank EDTA-derived plasma; (B), extracted plasma calibrator (calibrator C) containing 964 µg/L indinavir (IND), 438 µg/L saquinavir (SAQ), 428 µg/L nelfinavir (NEL), and 1000 µg/L ritonavir (RIT). (C), extracted low-concentration quality-control sample containing 194 µg/L indinavir (IND), 175 µg/L saquinavir (SAQ), 171 µg/L nelfinavir (NEL), and 200 µg/L ritonavir (RIT). (D), patient sample containing 309 µg/L saquinavir (SAQ) and 1253 µg/L nelfinavir (NEL). The peak eluting before saquinavir is AG1402 (M8 metabolite of nelfinavir). Arrows indicate the time of the detector wavelength switch. I.S., internal standard.


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  		Table 1. Accuracy and precision data for the calibrators.


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  		Table 2. Accuracy and precision data for the quality controls.


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  		Table 3. Intraday data for the quality controls.


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  		Table 4. Extraction efficiencies.

The relative retention factors (k' values) for several other drugs that are commonly used as either antiviral drugs (DMP 266, delavirdine, and nucleosides) or drugs that are used to prevent or treat opportunistic infections (azole antifungals, macrolides, atovaquone, rifabutin, rifampin, trimethoprim, and sulfamethoxazole) are listed in Table 5 .


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  		Table 5. Capacity factor (k') values of drugs commonly used in HIV patients.

Plasma samples (n = 51) that had been analyzed previously for nelfinavir and saquinavir by a separate method (HPLC/MS) were analyzed with our simultaneous procedure. These LC/MS methods were used by industrial sponsors for previous phase II and III studies but should not be considered equivalent to a "gold standard" method for these compounds or a true reference procedure because information on these procedures has not been published. The r2 value for the comparison was 0.94 for nelfinavir and 0.95 for saquinavir. The slope of the regression line between the comparison methods and the simultaneous procedure was 0.87 (P <0.0001; SE, 0.03) for nelfinavir and 0.88 (P <0.0001; SE, 0.03) for saquinavir. The y-intercepts for both comparisons were not statistically different from zero. These comparisons are illustrated in Fig. 3 . Bland-Altman plots (Fig. 4 ) were examined to further determine the difference between the two analytical procedures (this method vs the comparison HPLC/MS method) for both saquinavir and nelfinavir. The difference between the two analytical methods was plotted vs the mean concentration (22). For saquinavir, there was weak (r2 = 0.016) but statistically significant relationship. The line of best fit was described by the equation: y = 3.01 + 0.10x; the intercept was not statistically different from 0. Removal of single point at the highest mean concentration from the analysis produced a nonsignificant relationship. Similarly, for the comparison of the nelfinavir methods, there was a weak but significant relationship (r2 = 0.18). The line of best fit was y = -92.2 + 0.114x, and the intercept was not different from 0. Figs. 5 and 6 show the concentration-time profiles of saquinavir, ritonavir, and nelfinavir measured in the two adults receiving combination protease inhibitor therapy for >=4 weeks. The pharmacokinetic parameter values for saquinavir when given (400 mg twice daily) with ritonavir were as follows: maximum concentration (cmax), 2350 µg/L; 12-h postdose concentration (c12 h), 485 µg/L; area under the curve (AUC) from time 0 to 12 h, 17.5 mg · h/L; and elimination half-life (t1/2), 3.6 h. The saquinavir parameter values when given (800 mg three times daily) with nelfinavir were as follows: cmax, 1754 µg/L; c12 h, 127 µg/L; AUC, 7.3 mg · h/L; and t1/2, 2.1 h. The pharmacokinetic parameters for ritonavir were as follows: cmax, 6203 µg/L; c12 h, 2600 µg/L; AUC, 51.2 mg · h/L; and t1/2, 5.2 h. The pharmacokinetic characteristics for nelfinavir were as follows: cmax, 4381 µg/L; c12 h, 1096 µg/L; AUC, 27 mg · h/L; and t1/2, 3.3 h. In patients taking nelfinavir at high doses, some peak concentrations were greater than the highest calibrator (8550 µg/L). Patient samples above the highest calibrator were diluted 1:4 in blank plasma and re-analyzed. Subsequent experiments have indicated that the nelfinavir calibration curve is linear to 20 000 µg/L (validation data not shown).



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  		Figure 3. Correlation of the simultaneous HPLC method with a comparison HPLC/MS method for nelfinavir (top) and saquinavir (bottom; data provided by Roche Laboratories).

The solid line is the line of identity. The dashed line indicates the slope of the linear regression analysis.



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  		Figure 4. Bland-Altman plot comparison of the difference between the simultaneous HPLC method and the comparison HPLC/MS methods for nelfinavir (top) and saquinavir (bottom).



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  		Figure 5. Concentration-time profile for saquinavir (•) and nelfinavir ({blacksquare}) in a patient on combination therapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A simultaneous assay for the currently approved HIV protease inhibitors is both convenient and important. Therapeutic regimens with nucleosides in combination with dual protease inhibitors currently are being evaluated, especially in patients who are failing typical triple therapy regimens (two nucleosides plus a protease inhibitor). Theoretically, dual protease therapy may prevent the emergence of resistant viral strains, and practically, the potent inhibition of CYP3A4 by ritonavir dramatically increases the plasma concentrations and half-lives of the other protease inhibitors, especially nelfinavir and saquinavir. For example, the AUC of saquinavir is increased up to 60-fold when it is coadministered with ritonavir (6). This dramatic increase is a result of substantial inhibition of saquinavir first-pass metabolism catalyzed by intestinal CYP3A4 and a reduction of hepatic clearance (also catalyzed by CYP3A enzymes in the liver) (8)(23).

The initial approach to the problem was to develop a rapid HPLC assay with a total run time <20 min while maintaining suitable sensitivity and selectivity. Initial attempts at developing a separation were done on a 250 x 4.6 mm Spherisorb C8 column with a mobile phase consisting of acetonitrile-0.05 mol/L phosphate buffer, pH 3.1 (35:65, by volume) previously used in this laboratory for an assay of indinavir (Remmel et al., unpublished results). Under these conditions, the retention time for indinavir was ~7 min, but the most nonpolar of the protease inhibitors, ritonavir, eluted at later than 60 min. Consequently, less hydrophobic stationary phases (C1 and C4) were examined to reduce the retention times of ritonavir, saquinavir, and nelfinavir relative to that of indinavir, the least lipophilic protease inhibitor. Indinavir retention is highly pH dependent. Increasing the mobile phase pH from 3.1 to 4.1 substantially increased retention of indinavir on either a C1 or C4 column, and thus the total separation time of <20 min was achieved for all four protease inhibitors with mobile phases containing 500 mL of acetonitrile per liter of mobile phase.

Increasing the mobile phase pH necessitated a change in the buffer because phosphate (pKa1 = 2.1, pKa2 = 7.2) is a poor buffer at pH 3.5–6. A formate buffer was selected because of its good buffering capacity at pH 4.1 and its volatility, which could be useful for LC/MS applications if enhanced sensitivity was required. Acetonitrile was selected as the organic modifier of choice because of its low UV absorbance cutoff and improved peak widths compared with methanol. A 3-mm inner diameter column was used rather than the commonly used 4.6-mm (i.d.) columns to increase sensitivity and reduce mobile phase usage while retaining ruggedness.

Two extraction methods were evaluated. The previously mentioned indinavir assay was developed based on extraction with methyl-tert-butyl ether (11). Extraction recoveries with this solvent were 85–92% (Table 4Up ) at basic pH. The extraction efficiencies were increased after the addition of 250 µL of 0.05 mol/L NaOH compared with extraction at neutral pH. This procedure will also reduce the recovery of other acidic drugs and potential acidic interfering substances such as sulfamethoxazole, a commonly used agent in AIDS patients to prevent Pneumocystis carinii pneumonia. Solid-phase extraction on C18 Empore extraction disk cartridges (3M) and elution with HPLC mobile phase was also examined; however, the lower limit of quantification was higher than that obtained with liquid-liquid extraction, and interfering endogenous substances in serum eluting near the internal standard, A86093, were observed. Consequently, the liquid-liquid extraction with methyl-tert-butyl ether at basic pH was selected for subsequent validation of the method.

Some small interfering peaks that elute very close to nelfinavir were observed in extracts of blank plasma. These peaks increased when the source of the test tubes used for extraction was changed from Kimax-51 to Kimble borosilicate glass test tubes. This suggested that the impurities were present on the glassware, and a series of experiments demonstrated that rinsing the test tubes with 2 mL of methanol before extraction eliminated the interferences and increased the detectability of nelfinavir at low concentrations. This observation was confirmed when mobile phase was injected after exposure to the test tubes. Therefore, we routinely rinse all test tubes with methanol before the extraction and reconstitution steps. Hemolysis of patient samples does not appear to affect the extraction of the compounds and does not introduce any additional chromatographic interference.

Initially, the assay was developed with a commercially available compound, 6,7-dimethyl-2,3-di(pyridyl)quinoxaline, that had been used previously as an internal standard for a published assay for nelfinavir (14). This compound elutes before nelfinavir under our conditions; thus, there was a concern that metabolites from the protease inhibitors could possibly interfere with the compounds of interest. Selectivity testing (Table 5Up ) demonstrated that the nelfinavir M8 metabolite elutes close to this potential internal standard, as does delavirdine, a non-nucleoside HIV reverse transcriptase inhibitor. To overcome this problem, we selected A86093, a ritonavir analog available from Abbott, as an internal standard because this compound elutes after all of the compounds of interest.

The assay is both rugged and reliable. We have analyzed >1000 patient samples in the assay, primarily for nelfinavir, ritonavir, and saquinavir. The analytical column is protected by an in-line 0.5 µm filter and a guard column that is changed every 100–200 injections. The Keystone C4 column has been more rugged and has less column-to-column variability than C1 columns from the same manufacturer. The assay is selective, as shown in Table 5Up , which reports the retention times of potentially interfering drugs used in AIDS patients. The limit of quantification of the assay is comparable to other HPLC-UV methods in the literature and can be used for either therapeutic drug monitoring or pharmacokinetic analyses (see Figs. 4Up and 5Up ). Saquinavir sensitivity is enhanced three- to fourfold when monitored at 235 nm compared with 218 nm. This requires the use of a time-programmable variable wavelength UV detector or perhaps a diode array detector. The limit of quantification of this assay for indinavir is ~50 µg/L compared with 20 µg/L with our previously developed indinavir assay, but it is still sensitive enough for monitoring indinavir trough concentrations (typically 100–200 µg/L).

The novel simultaneous method we developed for the quantification of indinavir, nelfinavir, ritonavir, and saquinavir was accurate and precise. The accuracy for the quality controls was -6% to 9%. The interrun CV of the quality controls for the four drugs processed on a single day was 1.7–10%. The extraction efficiencies calculated for the four drugs in the high- and medium-concentration calibrators were excellent. The limit of quantification was 48.6 µg/L for indinavir, 42.8 µg/L for nelfinavir, 50 µg/L for ritonavir, and 21.9 µg/L for saquinavir. The plasma volume requirement was 250 µL. These characteristics indicate that this assay is suitable for use in clinical pharmacologic studies of the four protease inhibitors in adults as well as in children.

The simultaneous procedure performed quite favorably when compared with other LC/MS methods used for measurement of nelfinavir and saquinavir with high correlations (r >0.94). The Bland-Altman plot and the difference between the line of regression and the line of identity may indicate a bias or a nonlinearity between the two methods, although the correlations in the Bland-Altman plots were weak (r2 <0.2). Because information on the LC/MS method used as the comparison method has not been published and was not available to us, the data indicate that one of the methods may be nonlinear. The calibration curves for the simultaneous method for saquinavir and ritonavir indicate a high degree of linearity throughout the measurement range. Examination of the Bland-Altman plot for the nelfinavir regression analysis revealed a larger disparity between the two methods at concentrations >4000 µg/L. Population pharmacokinetic studies of nelfinavir at a dose of 750 mg every 8 h demonstrated a cmax of 3150 µg/L and a trough concentration of 1500 µg/L. Nelfinavir trough concentrations may be more clinically relevant with regard to prevention of resistance. At typical trough concentrations, the two methods have a strong correlation with low variability (see Figs. 3Up and 4Up ). The small differences between the two methods at the typical trough concentrations of nelfinavir are not likely to produce substantial differences in dosage regimen calculations for individual patients. The concentration-time information obtained in the two patients receiving the nelfinavir-saquinavir and ritonavir-saquinavir combinations provides additional support for the clinical utility of this simultaneous assay. Pharmacokinetic parameters calculated for the protease inhibitors in these two patients were comparable with published data (16)(23)(24).

Contemporary treatment of the HIV-infected person is a complex, long-term undertaking that entails unavoidable polypharmacy. Scenarios where knowledge of the concentration of a protease inhibitor may be clinically useful include (a) lack of initial response, (b) loss of response or a new toxicity in a previously stable patient, (c) management of drug-drug interactions, and (d) documentation of medication compliance. In these settings, the simultaneous HPLC procedure we have developed would appear to offer several potential advantages over individual analytical procedures to facilitate the collection and application of concentration information to the pharmacotherapy of HIV disease.



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  		Figure 6. Concentration-time profile for saquinavir (•) and ritonavir ({blacksquare}) in a patient on combination therapy.


   Acknowledgments
 
This work was supported in part by Grants RO1-AI33835–05, UO1-AI41089, and UO1-AI38858 from the National Institute of Allergy and Infectious Diseases. The protease inhibitors were gifts of Merck Research Laboratories (West Point, PA), Agouron Pharmaceutical Inc. (La Jolla, CA), Abbott Laboratories (Abbott Park, IL), and Roche Products Ltd. (Nutley, NJ). We would like to acknowledge the technical expertise of Shao-Mei Han and the helpful advice of Lane Bushman.


   Footnotes
 
Presented in part at the 5th Conference on Retroviruses and Opportunistic Infections, February 1, 1998, Chicago, IL.

1 Nonstandard abbreviations: MS, mass spectroscopy; UV, ultraviolet; LC, liquid chromatography; cmax, maximum concentration; c12 h, 12-h postdose concentration; AUC, area under the curve; and t1/2, elimination half-life.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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