NVP was obtained from Boehringer Ingelheim Pharmaceuticals (Ridgefield, CT). Human plasma was obtained from Biologic Specialty Corp. (Colmar, PA). Thin-layer chromatography plates, cartridges for solid-phase extraction (SPE), and all other chemicals were purchased from Fisher Scientific Co., L.L.C. (Pittsburg, PA).
A 500-μg/mL NVP stock solution was prepared by weighing 5 mg of NVP into a 10-mL volumetric flask and bringing to volume with methanol. The solution was vortexed and then sonicated for 5 minutes to ensure dissolution. Two- and 4-μL aliquots of NVP stock solution were added to separate 10-mL volumetric flasks and brought to volume with drug-free human plasma to produce limit of detection (LOD) NVP plasma standards of 100 ng/mL and 200 ng/mL, respectively.
NVP was extracted from 4 mL of plasma using SPE modified from a previously published procedure. 6 Briefly, SPE cartridges (PrepSep C 18, 500 mg/6 mL) were conditioned with 3 mL methanol, followed by 3 mL water, and finally with 2 mL of a 66.7-m M potassium phosphate buffer, pH 7.0, under vacuum (2 inches Hg). Each plasma sample was mixed with 4 mL of the phosphate buffer, vortexed thoroughly, and centrifuged at 500 ×g for 5 minutes before loading onto the cartridges (6 inches Hg vacuum). After loading, cartridges were washed with 2 mL water followed by 2 mL phosphate buffer, and allowed to dry under vacuum (10–15″ Hg) for at least 30 minutes. Once cartridges were dry, samples were eluted with 1 mL methanol and collected into 1.5-mL microfuge tubes. Collected samples were allowed to dry under a stream of air. Four milliliters of drug-free (blank) plasma were also extracted to serve as a negative control.
Selecto Scientific (Norcross, GA) silica gel 60 TLC plates (F-254, 200 μm) were used to separate NVP from plasma components. Plates were cut in half from their original length of 20 cm because NVP spots begin to dissipate at longer lengths and resolution is lost. TLC plates were marked using a soft-leaded pencil with an origin line 2 cm from the base of the plate. All samples were blotted 1 μL at a time using a micropipette, allowing each microliter to dry before adding the next. This ensures the spot on the plate is concentrated. The 2 outermost lanes on each side of the plate were blotted with 5 μL of NVP stock solution. Extracted samples were reconstituted in 20 μL of methanol and were blotted onto the remaining lanes. All spots were blotted 1 cm apart.
After all samples had been blotted and the spots had dried, the plate was developed in ethyl acetate in a covered TLC tank. The plate was removed when the solvent front reached within 1 cm of the top of the plate. The solvent front was marked with a soft-leaded pencil and the plate was allowed to air dry. Once dried, NVP was visualized by the absence of fluorescence when held under a short-wave UV lamp. Dark spots were outlined by pencil. The retention factor (Rf) values were determined for the innermost NVP stock spots and were compared with the spots in the sample lanes. Rf values were calculated by dividing the distance from the origin to the sample spot by the distance from the origin to the solvent front. A horizontal line was drawn through the middle of the NVP standards to help with this comparison. Rf values were generally between 0.52 and 0.54. The observed LOD for this assay using 4 mL of plasma is 100 ng/mL. A spot was observed in all lanes except for the blank plasma and the absence of a spot in the NVP control standard sample lane was interpreted as a negative result.
One hundred thirty-five plasma samples from cord blood with previously measured NVP concentrations, ranging from nondetectable (lower limit of quantitation <25 ng/mL) to 1000 ng/mL were either pooled together or diluted with healthy plasma. Each pooled sample contained 400 μL from 10 samples to yield a 4-mL aliquot. Using the SPE method described here, NVP was extracted and detected by TLC. Sample lanes contained spots corresponding to the Rf values of the NVP standards. No spots on the TLC plates were visible at the NVP Rf using clinical samples containing no previously measurable NVP.
Table 1 lists the TLC results for blank plasma samples and clinical plasma samples with detectable and undetectable NVP concentrations. Although the TLC assay was able to detect NVP at 60 ng/mL, 100 ng/mL was chosen as the LOD due to the robustness of the spot detection at this concentration compared with the faint spot observed at 60 ng/mL.
TLC is a simple and relatively inexpensive assay technique. This method uses no expensive hardware, few consumable items, and has a turnaround time of approximately 2 hours. The estimated initial setup cost was approximately $3000, including $540 for consumables. Sixteen patient samples can be run per TLC plate, which means 800 total samples could be completed using $540 in consumables ($1.50 per sample). A standard HPLC system costs approximately $45,000 and the cost of a single patient sample is in the range of $50. More importantly, this assay can reliably detect NVP concentrations of ≥100 ng/mL in plasma, making it an appropriate qualitative measure of adherence in field settings.
There are several limitations to this methodology. Concomitant zidovudine (ZDV) use will confound the results of this assay. ZDV is extracted by this same method and has an Rf value equal to the NVP reference standards; thus interfering with the NVP TLC results. A history of the patient's ZDV use should be obtained before subjecting the plasma to this assay. A problem may also arise from obtaining the patient blood sample too soon after dosing, which could yield a concentration below the LOD. The median (range) NVP maximum plasma concentration (Cmax) following a single 200-mg dose is approximately 1663 (447–2639) ng/mL and occurs at a median time of 3.0 hours after the dose. 4 This TLC assay should adequately capture most NVP levels if the subject indeed took the dose and the sample was collected at least 1 hour later. Furthermore, the blood volume required for this assay makes detecting NVP by this methodology impractical for infants but is still applicable for maternal and cord blood concentrations.
This TLC assay could have considerable utility in the monitoring of MTCT program effectiveness and possibly as a surveillance tool for NVP adherence. In a study we conducted in Zambia, 37 of 188 (20%) of women who received a directly observed single dose of NVP in labor had a cord blood concentration <100 ng/mL. 7 Based on these results alone, our NVP TLC assay could have a false-negative detection rate of 20%. Another study examining NVP in cord blood following observed dosing found that 8 of 109 cord blood samples (7%) had NVP concentrations <100 ng/mL, suggesting an even lower potential false-negative detection rate. 8 Combining these studies, we estimate that the field sensitivity of this TLC assay would be in the range of 80–93%; however, this needs to be confirmed prospectively. This crude estimate of field sensitivity may vary considerably depending on the interval between maternal dosing and delivery, such that if delivery occurs <1–2 hours after maternal NVP dosing, the false-negative rate may be increased. A caveat in applying this methodology is that timing of maternal NVP dosing relative to delivery must be taken into consideration.
We have observed that as many as 26% of HIV-infected pregnant women in resource-poor settings who receive NVP via self-administration at labor onset might not actually ingest the drug. 5 In addition, many MTCT prevention programs currently do not use NVP exposure to estimate population effectiveness. Using this TLC methodology, MTCT prevention programs could test seropositive specimens from exposed infants to determine the proportion of at-risk infants who received prophylaxis. This method would also potentially allow for the screening of cord blood specimens in HIV-infected pregnant women; an undetectable NVP by TLC (<100 ng/mL) might indicate that the infant should receive an NVP dose as soon as possible after delivery, although this would require further study.
In conclusion, we have developed a TLC assay for field use that can reliably detect the presence of NVP in human plasma at ≥100 ng/mL. NVP is becoming more readily available to mothers and clinicians throughout the developing world. Accessibility of the drug, however, does not ensure it will be taken. The assay described in this paper will allow the clinician to monitor NVP use in a cost-effective and timely manner.
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