| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
The University of Alabama at Birmingham,
1 Division of Clinical Pharmacology and
2 Department of Obstetrics and Gynecology, Birmingham, AL
aaddress correspondence to this author at: University of Alabama at Birmingham, School of Medicine, Division of Clinical Pharmacology, 1530 3rd Ave. South, VH 116, Birmingham, AL 35294-0019; fax 205-934-6201, e-mail EAcosta{at}uab.edu
In sub-Saharan Africa, India, Southeast Asia, and other resource-poor regions of the world, the antiretroviral nonnucleoside reverse transcriptase inhibitor nevirapine (NVP) is widely prescribed for HIV treatment and prevention because of its relative safety, long half-life, and low cost. Furthermore, pregnant women are often prescribed NVP therapy to decrease the rate of mother-to-child HIV transmission or as part of a combination therapeutic regimen in more developed countries. As one example, measuring NVP concentrations in pregnant women and their infants, in particular, has increased as clinics in developing countries attempt to better assess NVP "coverage" (e.g., universal treatment regardless of HIV status vs targeted treatment) (1). Because most developing countries currently do not have state-of-the-art sample collection, processing, and/or storage capabilities, more data are needed on how various procedures in these countries may affect NVP stability.
In clinical chemistry, drug stability during sample processing and storage is an important consideration for the interpretation of drug concentrations. If drugs do not remain stable in a given matrix under the conditions used for storage and/or processing, inaccurate drug concentrations will be obtained. Blood samples taken in resource-poor regions are often sent to laboratories in the developed world for drug measurements. However, these samples may not be processed and handled in the same manner as they would be in the US. In resource-poor regions, blood is often collected in red-top (no additive) tubes to separate serum from cells. Samples may not be placed on ice immediately but instead left on the laboratory bench for a period of up to 24 h before packaging and shipping for antiretroviral analysis. Furthermore, because of clinical settings in tropical climates, samples may be exposed to high temperatures for an undisclosed amount of time. Currently, NVP stability data under these conditions are not available. The objective of the present study was to assess the short-term stability of NVP in human whole blood and serum as a function of storage time, concentration, and temperature.
Four drug-free samples of EDTA-whole blood, heparin-whole blood, and anticoagulant-free serum (25 mL) were obtained, with each sample coming from a different patient. NVP was then added to all three sample types at low (250 µg/L) and high (2500 µg/L) concentrations. The NVP-enriched samples were subsequently divided and transferred to three environments: the laboratory bench at room temperature (20–25 °C), the refrigerator (4 °C), and an incubator (37 °C) for a 24-h period. At each time point, (0, 1, 2, 4, 8, or 24 h), 1 mL of NVP-enriched blood product (EDTA and heparin) was removed from the samples stored at each temperature and centrifuged (15 000g for 5 min) to separate the plasma. Three 100-µL aliquots of the separated plasma from whole blood or anticoagulant-free serum were taken, and 50-µL of internal standard was added to each aliquot. Internal standards were 20 mg/L and 2 mg/L for the high and low NVP concentrations, respectively. We added 1 mL of extraction solution (methylene chloride–methyl-tert-butyl ether, 25:75 by volume) to each tube, and the tubes were vortex-mixed for 10 min and then centrifuged (5 min at 8000g) to separate the aqueous and organic layers. The samples were then placed in an ultra-low-temperature freezer (-80 °C). When the aqueous layer was frozen, the organic layer was decanted into clean microcentrifuge tubes and left overnight on the laboratory bench to evaporate. Dried residues were reconstituted in 100 µL of mobile phase, transferred to injector vials, and injected into the HPLC system.
HPLC analysis was performed with a Waters Alliance chromatography system, including a Model 2695 Separations Module and a Model 2487 Dual Wavelength ultraviolet detector. The samples were subjected to reversed-phase liquid chromatography on a Microsorb MV C8 analytical column [250 x 4.6 (i.d.) mm; 5-µm particle size]. The column temperature was maintained at 25 °C with a column heater to decrease variability between runs. A SupelguardTM Discovery® C8 [20 x 4 (i.d.) mm; 5-µm particle size] in-line guard column was used to extend the life of the analytical column. The ultraviolet detector was set to monitor 284 nm. The isocratic mobile phase was 3 g/L triethylamine in water–methanol (60:40 by volume) at a flow rate of 1.0 mL/min. This assay is a modification of a previously published method (2). This assay was validated over the range 50–10 000 µg/L with inter- and intraday variability (CV) of 9.8% and 7.8%, respectively. The limit of quantification for this assay is 25 µg/L.
The Waters Millennium32® software stored all data and performed the chromatographic integrations. Peak area ratios (PARs) were calculated as detector response of analyte vs detector response of the internal standard from baseline. The mean, SD, and CV of the PAR for each time point were calculated. The difference for each time point (tx) from the initial concentration (t0) in that temperature environment was also calculated as (mean tx - mean t0)/t0, expressed as a percentage. Nonparametric analysis was performed for comparison of matrix lots. Differences between storage temperatures within each matrix were assessed by the Kruskal–Wallis test. Finally, NVP was considered to be stable in a given environment and blood product if there was a <10% change from t0 over the 24-h period.
NVP was stable in whole blood and serum at 4 °C, ambient temperature (20–25 °C), and 37 °C during the entire observation period at both concentrations studied. The measured NVP concentrations for the low and high samples over the 24-h period are presented in Table 1
as percentage of initial concentration. The PARs of NVP in EDTA-whole blood, heparin-whole blood, and serum were not significantly different over 24 h within or between temperature environments when analyzed by the Kruskal–Wallis test. The change from initial concentration (
t0) was <10% at all time points, for each blood product, in each of the three temperature environments, with the exception of EDTA-whole blood with the low NVP concentration at the 8- and 24-h time points at 4 °C and the 24-h time point at 25 °C. These values were slightly beyond a 10% change from the initial concentration (mean, 11.5%). However, calculated peak-height ratios showed that NVP was stable at these time points (<10% difference from baseline; data not shown). The imprecision for each time point was also calculated and is expressed as the CV calculated as the SD divided by the mean PAR. Throughout the course of this experiment, the CV remained <15%, demonstrating the good reproducibility of the assay.
|
Data on the stability of antiretrovirals in plasma and whole blood at various temperature-controlled settings are limited. Most studies to date have evaluated short-term antiretroviral plasma stability at room temperature or long-term stability during storage at very low temperatures. The available stability data for NVP show that it is stable in plasma when stored at -20 °C for up to 6 months (3). Furthermore, NVP is stable in serum when stored at 4 °C for 15 days (4), at room temperature for 7 days (5), or when heated to 56 °C for HIV deactivation for just over 1 h (5). Although the available stability data for NVP are encouraging, most studies have been performed on plasma samples. Testing antiretroviral stability in whole blood is unusual because plasma samples are commonly used for drug quantification. However, whole blood was used as a medium in this experiment because blood samples obtained in resource-poor regions are commonly collected in red-top tubes to separate serum from cells. Only limited data exist on the stability of medications in whole blood. For example, four of the protease inhibitors (indinavir, nelfinavir, saquinavir, and ritonavir) have been shown to be stable in plasma and whole blood for up to 5 days at 20 °C (6). The present study is the first to examine the stability of NVP in whole blood and serum under typical and atypical conditions, and the results suggest that standard sample collection, processing, and storage procedures are suitable in developing countries with limited resources.
Two different NVP concentrations were used in this experiment to reflect actual patient sample concentrations. The high NVP concentration (2500 µg/L) approximates trough concentrations that may be obtained during steady-state conditions (7) and is similar to single-dose cmax concentrations (8). The low NVP concentration (250 µg/L) was used to cover the lower end of expected concentrations for single-dose use.
NVP is commonly used in combination with other antiretrovirals for the treatment of HIV infection. In resource-poor regions, however, it is widely used as monotherapy, particularly in the treatment of HIV-infected pregnant women. Although other drug regimens, such as zidovudine alone or in combination with lamivudine, may be more efficacious (9)(10)(11)(12), NVP therapy is more cost-effective (13)(14)(15)(16) and has dramatically decreased the rate of mother-to-child HIV transmission (10). The results of our study are relevant to various clinical or research situations in which NVP concentrations are quantified, but they could potentially be particularly useful in developing countries.
Acknowledgments
This study was supported in part by Grants UO1-AI-41089 and AI-32775 from the National Institutes of Allergy and Infectious Diseases.
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
