Abstract
Objectives
The validation and clinical application of a LC-MS/MS method for the quantification of nevirapine in dried blood spots (DBS) and dried breast milk spots (DBMS) are presented.
Methods
DBS and DBMS were prepared from 50 and 30 µL of nevirapine-spiked whole blood and human breast milk, respectively. Chromatographic separation was achieved on a reverse phase C18 column with 0.1% formic acid in water/acetonitrile using a solvent gradient program at a flow rate of 400 µL/min and detection was by TSQ Quantum Access triple quadrupole mass spectrometer. The clinical application was evaluated in HIV positive nursing mothers and their breastfed infants.
Results
The assay was validated over the concentration range of 50-10000 ng/mL. Accuracy ranged from 93.3 to 113.4% and precision ranged from 1.9 to 12.0%. The mean (percentage coefficient of variation, %CV) recovery of nevirapine from DBS and DBMS was ≥70.7% (≤8.2) and matrix effect was ≤1.04 (≤6.1). Nevirapine was stable in DBS and DBMS for ≥ 15 months at room temperature and -80°C. Mean (SD) AUC0-12, Cmax and Cmin in maternal plasma versus breast milk were 57808 ng.h/mL (24315) versus 55817 ng.h/mL (22368), 6140 ng/mL (2605) versus 5231 ng/mL (2215) and 4334 ng/mL (1880) versus 4342 ng/mL (2245), respectively. The milk-to-plasma concentration ratio over the dosing interval was 0.94 (0.15). Infant plasma concentrations 2 and 8 hours after maternal dose were 580.6 ng/mL (464.7-1607) and 584.1 ng/mL (381.5-1570), respectively.
Conclusions
These methods further extend opportunities for conducting clinical pharmacokinetic studies in nursing mother-infant pairs, especially in resource-limited settings.
Keywords: nevirapine, dried blood spots, dried breast milk spots, mass spectrometry
Introduction
Since its approval by the US Food and Drug Administration in 1996 as the first non-nucleoside reverse transcriptase inhibitor for the treatment of HIV, nevirapine has been used extensively as part of combination antiretroviral therapy (ART). The 2013 World Health Organisation (WHO) consolidated guidelines for the use of antiretroviral (ARV) drugs for treating and preventing mother-to-child transmission of HIV infection highlights the continued significance of nevirapine in HIV treatment and prevention strategies.1
Although several pharmacokinetic studies have been conducted on nevirapine, important gaps remain in our understanding of its disposition in special populations. For instance, studies investigating its pharmacokinetics during pregnancy have reported inconsistent findings.2–4 Fillekes et al. reported high risk of subtherapeutic nevirapine concentrations among children less than 5 months.5 Evaluation of the current WHO dosing recommendations indicated under-dosing in the 3-6 and 6-10 kg weight ranges.6 Therefore, further pharmacokinetic (and pharmacodynamic) studies are needed to address the uncertainties about paediatric nevirapine dosing. In addition, the WHO recommends exclusive breastfeeding for the first 6 months of life, with infant nevirapine post-exposure prophylaxis for 6 weeks and continued lifelong (Option B+) or breastfeeding-limited (Option B) maternal ARV.1 However, its pharmacokinetics in breast milk and in infants doubly exposed through breast milk and post-exposure prophylaxis1 have not been studied. Understandably, pharmacokinetic studies in these special populations are fraught with ethical and practical challenges, including the difficulties associated with traditional pharmacokinetic sampling techniques. Furthermore, new treatment strategies, including new paediatric formulations, nanoformulations, once daily dosing, and dose reduction in treatment-experienced patients, will require an evaluation of impact on pharmacokinetic parameters in relevant populations.
The use of dried matrix spots is increasingly recognised as a suitable alternative that can help overcome many of the practical challenges in conducting such studies. The advantages include its non-invasive nature, low volume requirement, ease of transport, low cost, acceptability by trial subjects, improved biosafety and room temperature stability of most drugs. Here we present the development and validation of a rapid method for the quantification of nevirapine in DBS and DBMS using LC-MS/MS. The validated method was used to investigate plasma and breast milk pharmacokinetics of nevirapine during an entire dosing interval in HIV positive nursing mothers and their breastfed infants.
Materials and Methods
Nevirapine and nevirapine-d5 (internal standard, IS) reference standards were obtained from Toronto Research Chemicals Inc. (North York, ON, Canada). LC-MS grade acetonitrile was obtained from Fisher Scientific (Loughborough, Leicestershire, UK), methanol from VWR International (Lutterworth, Leicestershire, UK), formic acid from Sigma-Aldrich (Gillingham, Dorset, UK) and water was produced from an Elga Option 4 water purifier (Elga Labwater, High Wycombe, Buckinghamshire, UK) and was further purified to 18.2 MΩ with a Purelab Classic UVF (Elga LabWater, High Wycombe, Buckinghamshire, UK). Whatman 903 Protein Saver cards were obtained from Scientific Laboratory Supplies (Hessle, East Yorkshire, UK). Blank whole blood was obtained from drug-free healthy volunteers and blank breast milk samples were obtained (with ethical approval) from Wirral Mothers’ Milk Bank, Clatterbridge Hospital, Wirral, UK.
LC-MS/MS systems and conditions
The LC-MS/MS system and conditions were very similar to those previously described for efavirenz with a few modifications.7 In brief, it consisted of a reverse phase Fortis™ C18 column: 3 µm, 100 x 2.1 mm (Fortis Technologies Ltd, Neston, Cheshire, UK) with a 2 μm C18 Quest column-saver (Thermo Electron Corporation, Hemel Hempstead, Hertfordshire, UK) on a HPLC connected to a TSQ Quantum Access triple quadrupole mass spectrometer (Thermo Electron Corporation, Hemel Hempstead, Hertfordshire, UK) equipped with a heated electrospray ionisation source. Xcalibur™ Software and LCquan™ (version 2.6.1, Thermo Fisher Scientific, Hemel Hempstead, UK) were used for method setup, data acquisition, data processing, and reporting.
A solvent gradient program (flow rate of 400 µL/min) with 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) was used for chromatographic separation. The gradient programme started with 95% mobile phase A, decreasing to 20% over 1.5 minutes. This was maintained for 2 minutes, followed by column equilibration to the initial conditions over 1.5 minutes. The total run time was 5 minutes. Injection volume was 10 µL and the needle was washed twice with 2 mL acetonitrile:water (80:20 v/v) between injections. The MS was operated in positive ionization mode to produce characteristic fragmentation patterns. Product ion characterisation was done by directly infusing 1 µg/mL solutions of nevirapine and nevirapine-d5 separately into the MS using a syringe at a flow rate of 5 µL/min. Nevirapine and nevirapine-d5 precursor ions ([M+H]+) were monitored by selective reaction monitoring and tube lenses were 75.09 V and 77.6 V, respectively.
Stock solutions, calibration standards (STD) and quality controls (QC)
Nevirapine and nevirapine-d5 stock solutions were prepared from their respective reference standards in 100% methanol to obtain a final concentration of 1 mg/mL and refrigerated at -20°C until use. Whole blood working stock of nevirapine (20 µg/mL) was prepared, tumbled for 60 minutes and used to make eight whole blood STDs in the range of 50-10000 ng/mL by serial dilution. Whole blood low quality control (LQC, 150 ng/mL), medium quality control (MQC, 4000 ng/mL) and high quality control (HQC, 8500 ng/mL) samples were similarly prepared from a separate 20 µg/mL stock. Working stocks of nevirapine in human breast milk were similarly prepared and used to make breast milk STDs and QCs. Working solutions of nevirapine at concentrations equivalent to the three QC levels and 250 ng/mL of nevirapine-d5 (internal standard, IS) were prepared in methanol:water (50:50, v/v) from their respective 1 mg/mL stock solutions.
DBS and DBMS STD and QC samples preparation
Nevirapine DBS STDs and QCs were prepared by carefully spotting 50 µL of whole blood STDs and QCs on each circle of Whatman 903 Protein Saver cards. DBMS STDs and QCs were similarly prepared by spotting 30 µL of breast milk STDs and QCs. Spotted cards were left to dry at room temperature overnight and stored with desiccant sachets in ziplock bags.
Sample pre-treatment
A section of each DBS sample was punched into a 7 mL screw cap tube using a 6mm hole punch. For DBMS samples, the entire spot was removed using a 13 mm hole punch and folded into a 7 mL screw cap tube. In each case, the samples were extracted with 1 mL of 0.1% formic acid in acetonitrile:water (80:20, v/v) by tumbling for 30 minutes in the presence of 20 µL of IS. A 350 µL aliquot of the extract was transferred into autosampler vials.
Calibration curves, accuracy and precision
More than ten separate assays, each consisting of a zero blank, eight STDs in the range of 50-10000 ng/mL (n = 2 for each level), and QCs (n = 6 for each level) were run for each of DBS and DBMS. Calibration curves were constructed using a linear regression equation of analyte/IS peak area ratio versus nominal concentrations with a 1/concentration weighting. Accuracy was defined as percentage deviation of measured concentration from the nominal value and precision was defined as the percentage coefficient of variation (%CV). According to guidelines, not less than 75% of all STDs and 67% of all QCs (50% at each level) in any batch were required to have percentage deviation within ±15%, except for the lower limit of quantification (LLOQ) and the LQC which were allowed to be ±20% of the nominal value.8, 9
Recovery, matrix effect, dilution integrity and spot stacking
Recovery was assessed as recommended by Matuszewski et al.10 by comparing peak area from extracted QCs (n = 6) with the peak area from solutions of nevirapine of equivalent concentrations in mobile phase (n = 6). To evaluate matrix effect, six drug-free whole blood and breast milk samples from different donors were spotted on Whatman 903 Protein Saver cards and extracted as previously described. Blank DBS or DBMS extracts (n = 6) were spiked with the appropriate nevirapine solution (150 ng/mL, 4000 ng/mL or 8500 ng/mL) to obtain final concentrations equivalent to extracted nevirapine DBS or DBMS at LQC, MQC or HQC (n = 6). The recovery at each QC level was defined as the absolute peak area response of extracted samples expressed as a percentage of the peak area response of mobile phase samples spiked with nevirapine at equivalent concentration. Matrix effect was defined as the ratio of the peak area response of blank extracts spiked post-extraction to the peak area response of mobile phase samples spiked with nevirapine at equivalent concentration. A %CV of ≤ 15% was set as the level of acceptance for both recovery and matrix effect in line with the FDA and EMA guidelines8, 9, and 85-115% was set as a further acceptance criterion for recovery in both matrices.
To investigate dilution integrity for samples with nevirapine concentrations above 10000 ng/mL, 50 µL of whole blood or 30 µL breast milk containing 20 µg/mL of nevirapine were spotted on each circle of Whatman 903 card. The samples were dried and extracted as previously described. The extracts were diluted 5x and 10x using blank DBS or DBMS spots similarly extracted in the same batch. The application of the method to samples below the LLOQ of 50 ng/mL was also investigated by extracting multiple 25 ng/mL spots (3x and 5x) as previously described in the same 7 mL screw cap tube. The corresponding factor (e.g. 10.0 for 10x dilution and 0.2 for 5x stacking) was entered on the LCquan assay sequence file.
Stability and re-injection reproducibility
We evaluated the short-term and long-term stability of nevirapine in DBS and DBMS under different storage and processing conditions. Short-term stability was evaluated by storing extracted QC samples at room temperature and in the autosampler (4°C) for 24 hours and over the weekend (72 hours). For long-term stability at room temperature and -80 °C, unextracted DBS and DBMS QC samples were stored at these temperatures for 15 months. The concentrations of the stored samples were determined using freshly made STDs and QCs. An accepted validation assay batch (STDs and QCs) was re-injected after 24 hours in the autosampler to evaluate re-injection reproducibility.
Spotting practice, inter-card and spot volume variability
To evaluate the effect of poor spotting practice, the wraparound covers of DBS cards spotted with 50 µL of spiked whole blood at LQC, MQC and HQC were closed and pressed against the sample collection areas immediately after spotting. Spot volume variability was evaluated by spotting 20 µL and 35 µL of spiked whole blood at LQC, MQC and HQC on Whatman 903 cards. Variability associated with card type was investigated using Perkin Elmer, FTA-C, and Agilent cards. The purpose was to investigate the possibility of analysing samples collected on different card types in the same assay batch, not to test card quality. For each card type, equal volumes of spiked whole blood at LQC, MQC and HQC were spotted on sample collection areas. All samples were dried overnight at room temperature. Each spot was punched into a 7 mL screw cap tube using a 6mm hole punch and extracted as previously described. Extracted samples (n = 6) were quantified using a calibration curve constructed from 50 µL STDs spots and compared with 50 µL QCs standard spots (n = 6) from Whatman 903 cards.
Clinical application
The validated method was applied in a pharmacokinetic study to evaluate plasma and breast milk pharmacokinetic profiles of nevirapine in HIV positive nursing mothers (n = 5) receiving regimens containing 200 mg nevirapine every 12 h. Patients were recruited from two hospitals in Benue State, Nigeria: Bishop Murray Medical Centre, Makurdi and St Monica’s Hospital, Adikpo. Written informed consent was obtained from each patient prior to enrolment. The study protocol and the material transfer agreement (MTA) were approved by the National Health Research and Ethics Committee (NHREC), Abuja, Nigeria (approval number: NHREC/01/01/2007-13/11/2012). Those taking drugs with known or uncertain interaction with nevirapine were excluded. Paired DBS and DBMS were collected at 0.5, 1, 2, 4, 8, and 12 h after an observed dose of 200 mg nevirapine taken about 30 minutes after a standard local meal. Whole blood samples were collected as DBS after sterile skin cleaning and finger prick using a 2mm safety lancet (BD, Oxford, Oxfordshire, UK). The first drop of blood was discarded and subsequent blood drops were collected on the sample collection areas of Whatman 903 cards. Within 2 minutes of DBS collection, about 5 mL of breast milk was manually expressed by the mother and a 30 µL aliquot was immediately spotted on each sample collection area of Whatman 903 cards. Only one infant was less than 6 weeks old and still receiving nevirapine post-exposure prophylaxis.1 DBS samples were collected from infants at 2 h and 8 h after maternal nevirapine dose. All infants were breastfed on demand to reflect real-life situations. DBS and DBMS were dried, stored and shipped at room temperature to the Department of Molecular and Clinical Pharmacology, University of Liverpool, United Kingdom for analysis. Nevirapine in DBS and DBMS was quantified using the method described above. Plasma nevirapine concentrations were estimated using [DBS[NVP]/(1-HCT)]*0.6 = Plasma[NVP], where DBS[NVP] is nevirapine concentration in DBS, HCT is the patient-specific haematocrit and 0.6 is the fraction of nevirapine bound to plasma protein.11
Results
LC-MS/MS conditions
The transitions were 267.103 → 198.108 and 226.09 m/z for nevirapine and 272.131 → 199.060 and 227.053 m/z for nevirapine-d5 with optimal collision energies of 34 and 28, 36 and 29, respectively. Representative chromatograms are presented in Figure S1, with retention time of 2.48 minutes for both nevirapine and the IS.
Calibration curves, accuracy and precision
The method was linear in the range of 50-10000 ng/mL, with accuracy and precision within the acceptance criteria from FDA and EMA guidelines (Table 1).8, 9 The mean regression coefficient (%CV) of the IS response was 0.997 (7.6) for DBS and 0.998 (4.8) for DBMS.
Table 1. Accuracy (%) and precision (%) for the quantification of nevirapine in DBS and DBMS.
| Standards and QCs (ng/mL) | Inter-day (measured concentration) | Intra-day (measured concentration) | ||||||
|---|---|---|---|---|---|---|---|---|
| mean | SD | precision (%CV) | accuracy (%) | mean | SD | precision (%CV) | accuracy (%) | |
| DBS (nominal concentration) | ||||||||
| 50 | 49.7 | 3.9 | 7.8 | 99.4 | 50.7 | 3.7 | 7.3 | 101.5 |
| 100 | 99.4 | 5.6 | 5.7 | 99.4 | 101.4 | 6.8 | 6.7 | 101.4 |
| 200 | 200.1 | 14.2 | 7.1 | 100.1 | 194.1 | 12.7 | 6.6 | 97.0 |
| 500 | 485.9 | 34.5 | 7.1 | 97.2 | 466.3 | 35.4 | 7.6 | 93.3 |
| 1000 | 1016.3 | 41.3 | 4.1 | 101.6 | 1034.7 | 32.9 | 3.2 | 103.5 |
| 2000 | 2060.0 | 66.7 | 3.2 | 103.0 | 2045.5 | 73.7 | 3.6 | 102.3 |
| 5000 | 4982.0 | 311.9 | 6.3 | 99.6 | 5029.0 | 396.2 | 7.9 | 100.6 |
| 10000 | 9950.5 | 597.3 | 6.0 | 99.5 | 9928.2 | 744.3 | 7.5 | 99.3 |
| 150 (LQC) | 145.1 | 15.8 | 10.9 | 96.7 | 146.7 | 17.6 | 12.0 | 97.8 |
| 4000(MQC) | 3912.9 | 283.1 | 7.2 | 97.8 | 3799.3 | 152.9 | 4.0 | 95.0 |
| 8500 (HQC) | 8382.8 | 395.7 | 4.7 | 98.6 | 8460.3 | 493.5 | 5.8 | 99.5 |
| DBMS (nominal concentration) | ||||||||
| 50 | 51.1 | 3.6 | 6.9 | 102.3 | 51.9 | 2.9 | 5.6 | 103.8 |
| 100 | 96.8 | 5.7 | 5.8 | 96.8 | 98.5 | 5.2 | 5.2 | 98.5 |
| 200 | 202.0 | 9.9 | 4.9 | 101.0 | 206.2 | 13.3 | 6.5 | 103.1 |
| 500 | 514.5 | 26.1 | 5.1 | 102.9 | 508.6 | 30.2 | 5.9 | 101.7 |
| 1000 | 1007.5 | 45.3 | 4.5 | 100.8 | 984.9 | 56.4 | 5.7 | 98.5 |
| 2000 | 1909.6 | 71.5 | 3.7 | 95.5 | 1864.8 | 79.4 | 4.3 | 93.2 |
| 5000 | 5007.5 | 158.7 | 3.2 | 100.1 | 4980.9 | 93.1 | 1.9 | 99.6 |
| 10000 | 10060.9 | 363.6 | 3.6 | 100.6 | 10154.0 | 243.5 | 2.4 | 101.5 |
| 150 (LQC) | 169.7 | 8.0 | 4.7 | 113.1 | 170.1 | 6.0 | 3.5 | 113.4 |
| 4000(MQC) | 4156.2 | 140.5 | 3.4 | 103.9 | 4091.0 | 140.2 | 3.4 | 102.3 |
| 8500 (HQC) | 9145.6 | 364.6 | 4.0 | 107.6 | 9091.9 | 384.9 | 4.2 | 107.0 |
Recovery, matrix effect, dilution integrity and spot stacking
The mean recovery (%CV) of nevirapine from DBS and DBMS were 83.2% (≤8.2) and 70.7% (≤6.1) respectively. The matrix effects in DBS and DBMS ranged between 0.99 and 1.04 with a %CV of ≤6.1. After 5x and 10x dilutions the mean (%CV) back calculated concentrations for 20 µg/mL DBS samples were 94.4 (3.2) and 99.3% (5.4) of nominal values, respectively. The corresponding values for DBMS were 102.6 (2.5) and 99.6% (4.8), respectively. In the spot stacking experiments, the back calculated concentrations for the 25 ng/mL DBS samples were 93.6 (9.7) and 109.9% (5.1) of nominal values for the 3x and 5x stacks, respectively.
Stability and re-injection reproducibility
Table 2 shows the short-term stability of nevirapine in processed samples and its long-term stability in DBS and DBMS. The stability of extracted samples in the autosampler (at 4°C) for 72 h was demonstrated. Nevirapine remained stable in DBS and DBMS when stored for 15 months at room temperature (about 25°C) or at -80°C (Table 2). In repeat analyses of accepted validation batches of DBS and DBMS, all assay validation parameters were within acceptable limits, demonstrating re-injection reproducibility.
Table 2. Short-term stability of nevirapine in processed samples and long-term stability in DBS and DBMS.
| % Stability (%CV) |
|||
|---|---|---|---|
| Storage condition | QC level | DBS | DBMS |
| Bench-top stability of extracted samples (24 h at 25 °C) | LQC | 99.7 (2.1) | 101.7 (3.2) |
| MQC | 103.0 (1.7) | 98.6 (1.8) | |
| HQC | 98.8 (2.5) | 103.9 (4.1) | |
| Autosampler stability of extracted samples (24 h at 4 °C) | LQC | 100.6 (1.1) | 114.0 (5.1) |
| MQC | 108.7 (3.6) | 103.9 (1.6) | |
| HQC | 111.4 (4.3) | 103.6 (3.0) | |
| Extracted samples stability (72 h at 4 °C) | LQC | 102.4 (9.3) | 100.8 (2.5) |
| MQC | 96.8 (5.2) | 97.6 (3.8) | |
| HQC | 99.3 (2.7) | 107.2 (7.1) | |
| Long-term stability of dried spots (15 months at 24°C) | LQC | 106.0 (4.5) | 109.2 (2.0) |
| MQC | 106.3 (3.0) | 102.5 (2.6) | |
| HQC | 114.5 (6.3) | 105.1 (3.6) | |
| Long-term stability of dried spots (15 months at -80°C) | LQC | 111.6 (9.0) | 112.4 (1.6) |
| MQC | 109.6 (4.2) | 100.1 (1.2) | |
| HQC | 113.4 (6.0) | 105.7 (3.4) | |
Spotting practice, inter-card and spot volume variability
Bad spotting practice, simulated by deliberately pressing the wraparound cover against the sample collection areas immediately after spotting, significantly affected nevirapine concentration in DBS, with back calculated percentage (%CV) of nominal values of 82.8% (18.7), 79.3% (15.5) and 84.8% (7.6) at LQC, MQC and HQC respectively. There were no significant differences between the four card types evaluated, with back calculated nevirapine concentrations at the QC levels within 93.1-115.1% of nominal values and %CV of ≤6.5. Nevirapine concentrations from spots volumes of 20 and 35 µL were comparable to those of 50 µL, giving 94.4-102.1% of nominal values and %CV of ≤8.2 across the three QC levels (Table 3).
Table 3. Effects of variation in card type and spot volume.
| Variable | LQC (150 ng/mL) |
MQC (4000 ng/mL) |
HQC (8500 ng/mL) |
|
|---|---|---|---|---|
| % of nominal concentration (%CV) | ||||
| Card Type | Whatman 903 | 100.0 (5.2) | 93.1 (2.0) | 100.1 (0.62) |
| Perkin Elmer1 | 103.3 (4.9) | 103.0 (3.8) | 100.6 (6.3) | |
| FTA DMPK-C1 | 114.6 (5.8) | 107.0 (5.9) | 108.7 (3.5) | |
| Bond Elut (Agilent1) | 115.1 (6.5) | 105.0 (5.4) | 108.5 (3.6) | |
| Spot Volume | 20 µL | 97.6 (7.1) | 94.4 (6.1) | 95.9 (8.0) |
| 35 µL | 102.1 (8.2) | 95.2 (6.0) | 94.5 (6.2) | |
Spots made with 35 µL of blood.
Clinical application
The median (range) age of the five mothers included in the pharmacokinetic study was 33 years (25-38) and weight was 67 kg (64-79). All have been treated with regimens containing 12 hourly 200 mg nevirapine for duration of 52 months (29-75) and the latest CD4 count was 508 cells/mm3 (422-762). Samples were collected at steady state and 136 days (15-183) after delivery. Infant weight was 7.0 kg (4.0-7.1). Only one infant was less than six weeks old and still taking 2 mg/kg nevirapine for post-exposure prophylaxis. The full pharmacokinetic profiles of nevirapine in plasma (DBS predicted) and DBMS are shown in Figures 1 and S2, showing considerable inter-individual variability in plasma and breast milk concentrations. The mean (SD) AUC, Cmax and Cmin were 57808 ng.h/mL (24315), 6140 ng/mL (2605) and 4334 ng/mL (1880), respectively in plasma. Corresponding values in breast milk were 55817 ng.h/mL (22368), 5231 ng/mL (2215) and 4342 ng/mL (2245), respectively. Apparent plasma clearance (CL/F) was 3.89 l/h (1.34). The average (range) milk-to-plasma concentration (M/P) ratio over the dosing interval was 0.94 (0.84-1.2), with moderate intra- (%CV, 13.5) and inter-individual (%CV, 15.7) variability (Figure 1 and S2). The only infant still taking nevirapine post-exposure prophylaxis had plasma concentrations of 1607 and 1570 ng/mL. Plasma concentrations 2 and 8 hours after maternal dose were 529.1 ng/mL (464.7-836.4) and 555.6 ng/mL (381.5-738.2), respectively, in the other four infants.
Figure 1.
Nevirapine pharmacokinetic profile (mean, SD) in plasma (DBS predicted) and breast milk of HIV positive nursing mothers (n = 5) taking regimens containing 200 mg of nevirapine every 12 h. See individual patient profiles in Figure S2.
Discussion
A simple, rapid, accurate and precise method for the quantification of nevirapine in DBS and DBMS has been developed, validated and its clinical application demonstrated. Although different methods have been described for the quantification of nevirapine in DBS or dried plasma spots 11–14, this is the first report of nevirapine quantification in DBMS. We recently described and used related methods for efavirenz in DBS15, 16 and DBMS.7 In addition, full pharmacokinetic profiles of nevirapine in plasma and breast milk of nursing mothers on continuous HAART has been described using these micro volume sampling techniques for the first time.
The low sample volume requirements (20-50 µL) will facilitate pharmacokinetic studies in special populations like neonates, children, pregnant women and nursing mothers. Variable spot volumes associated with the finger prick method did not introduce significant variability in nevirapine concentration, making venepuncture unnecessary. The long-term stability of nevirapine in both matrices at room temperature will facilitate clinical studies in settings with inadequate cold storage facilities required for traditional sampling strategies. The possibility of analysing samples collected on different card types was also demonstrated using four card types. The bias often associated with the DBS method14, 17 was previously shown to be adequately addressed using the formula [DBS[NVP]/(1-HCT)]*0.6 = Plasma[NVP].11, 18 However, bad spotting practice that results in transfer of sample from the collection area to the wraparound cover was shown to cause significant underestimation of nevirapine concentration. It should be noted that this could easily be minimised or eradicated with pre-trial sampling education. The reliability of the DBMS method depends on accurate pipetting from expressed breast milk and spotting on the marked sample collection areas on the cards because breast milk is colourless.
Koal et al. described the first method for the quantification of ARV in DBS (nevirapine and 8 others) using 5 µL of anticoagulated blood collected by venepuncture.12 A DBS method later described by ter Heine et al. with a run time of 5 minutes19 was recently used in a study that confirmed the reliability of nevirapine (and efavirenz) plasma concentrations derived from DBS concentrations.11 Furthermore, a dried plasma spot method was described for the quantification of 9 ARV (including nevirapine)13 and later used in a study that evaluated the influence of genetic factors on nevirapine pharmacokinetics.14 However, the complex extraction involving drying and reconstitution steps and the 19-minute run time in the latter methods constitute major disadvantages. In addition, drug stability was evaluated for 30 minutes, 7 days and 3 months, respectively, compared with the 15-month stability in the present study. Only one other fully validated method has been described for the quantification of nevirapine in human breast milk (as a liquid), with single time-point M/P ratio of 0.82.20, 21 However, the extraction procedure is complicated, involving a combination of liquid–liquid extraction and solid-phase extraction. Two other studies reported single time-point M/P ratios of 0.67 (range, 0.24-1.01)22 and 0.75 (IQR, 0.64-0.89).23 As observed in the present study, the M/P ratio varies during the dosing interval and a time-averaged value is known to be more reliable.24, 25 Using this approach in the present study we obtained time-averaged M/P ratio of 0.94 (range, 0.84-1.2).
The 2013 WHO consolidated guidelines for the use of ARV for treating and preventing HIV infection recommend nevirapine, used in combination with two NRTIs (e.g. zidovudine and lamivudine or tenofovir and emtricitabine), as an alternative to efavirenz-based first-line regimens.1 Immediate initiation of ART in all infants up to 5 years of age regardless of clinical staging or CD4 count was also recommended. The advantages of this approach were highlighted by the long-term remission achieved in the ‘Mississippi baby’ treated with a nevirapine-containing regimen, started within a few hours after birth.26 However, paediatric ART coverage (34% vs 64% in adults in 2012) remains poor and most eligible children are not being treated.27 Despite the superiority of lopinavir/ritonavir-based regimens,28, 29 a nevirapine-based regimen is proposed as an alternative in resource-limited settings because of the challenges presented by lopinavir/ritonavir cold storage requirement, taste-associated suboptimal adherence, and high cost.1, 30 These data highlight the continued importance of nevirapine in HIV treatment. Although several studies have reported the relationship between nevirapine efficacy and adequate plasma concentrations,31–34 data in special populations are still inadequate.
In conclusion, micro volume sampling strategies like the DBS and DBMS methods described here present techniques for overcoming the inherent challenges with conducting clinical studies in special populations, especially in settings with limited resources.
Supplementary Material
Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
Acknowledgements
We would like to thank the participating patients, staff and management of Bishop Murray Medical Centre, Makurdi, Nigeria for their support in the proof of concept pharmacokinetic study. We also thank staff at Liverpool Bioanalytical Facility for their input during the method development and validation.
Funding
The method development and validation was carried out as part of our routine work at Liverpool Bioanalytical Facility, Department of Molecular and Clinical Pharmacology, University of Liverpool, UK. Adeniyi Olagunju received funding from Tertiary Education Trust Fund, Nigeria, for the pharmacokinetic study carried out as part of his PhD. Catriona Waitt was funded by an Academy of Medical Sciences Starter Grant for Clinical Lecturers subsequently by a Wellcome Clinical Postdoctoral Training Fellowship WT104422MA.
Footnotes
Transparency declarations
David Back, Saye Khoo, Andrew Owen and Laura Else have received research grants and/or travel bursaries from Merck, Bristol Myers and Squibb, GlaxoSmithKline, Pfizer, Abbott, ViiV Healthcare, Boehringer Ingelheim and Janssen Pharmaceuticals. The remaining authors have none to declare.
References
- 1.World Health Organisation. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Available at: http://www.who.int/iris/bitstream/10665/85321/1/9789241505727_eng.pdf.
- 2.von Hentig N, Carlebach A, Gute P, et al. A comparison of the steady-state pharmacokinetics of nevirapine in men, nonpregnant women and women in late pregnancy. Br J Clin Pharmacol. 2006;62:552–9. doi: 10.1111/j.1365-2125.2006.02664.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Mirkuzie AH, Hinderaker SG, Sisay MM, et al. Current status of medication adherence and infant follow up in the prevention of mother to child HIV transmission programme in Addis Ababa: a cohort study. J Int AIDS Soc. 2011;14:50. doi: 10.1186/1758-2652-14-50. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Lamorde M, Byakika-Kibwika P, Okaba-Kayom V, et al. Suboptimal nevirapine steady-state pharmacokinetics during intrapartum compared with postpartum in HIV-1-seropositive Ugandan women. J Acquir Immune Defic Syndr. 2010;55:345–50. doi: 10.1097/QAI.0b013e3181e9871b. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Fillekes Q, Mulenga V, Kabamba D, et al. Pharmacokinetics of nevirapine in HIV-infected infants weighing 3 kg to less than 6 kg taking paediatric fixed dose combination tablets. AIDS. 2012;26:1795–800. doi: 10.1097/QAD.0b013e32835705fd. [DOI] [PubMed] [Google Scholar]
- 6.Foissac F, Bouazza N, Frange P, et al. Evaluation of nevirapine dosing recommendations in HIV-infected children. Br J Clin Pharmacol. 2013;76:137–44. doi: 10.1111/bcp.12069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Olagunju A, Bolaji OO, Amara A, et al. Development, validation and clinical application of a novel method for the quantification of efavirenz in dried breast milk spots using LC-MS/MS. J Antimicrob Chemother. 2015;70:555–61. doi: 10.1093/jac/dku420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.European Medicines Agency. Guideline on Validation of Bioanalytical Methods. London: Committee for Medicinal Products for Human Use; http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf. [Google Scholar]
- 9.Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validation. Maryland: Food and Drug Administration, US Department of Health and Human Services; http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070107.pdf. [Google Scholar]
- 10.Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem. 2003;75:3019–30. doi: 10.1021/ac020361s. [DOI] [PubMed] [Google Scholar]
- 11.Kromdijk W, Mulder JW, Rosing H, et al. Use of dried blood spots for the determination of plasma concentrations of nevirapine and efavirenz. J Antimicrob Chemother. 2012;67:1211–6. doi: 10.1093/jac/dks011. [DOI] [PubMed] [Google Scholar]
- 12.Koal T, Burhenne H, Romling R, et al. Quantification of antiretroviral drugs in dried blood spot samples by means of liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19:2995–3001. doi: 10.1002/rcm.2158. [DOI] [PubMed] [Google Scholar]
- 13.D'Avolio A, Simiele M, Siccardi M, et al. HPLC-MS method for the quantification of nine anti-HIV drugs from dry plasma spot on glass filter and their long term stability in different conditions. J Pharm Biomed Anal. 2010;52:774–80. doi: 10.1016/j.jpba.2010.02.026. [DOI] [PubMed] [Google Scholar]
- 14.Calcagno A, D'Avolio A, Simiele M, et al. Influence of CYP2B6 and ABCB1 SNPs on nevirapine plasma concentrations in Burundese HIV-positive patients using dried sample spot devices. Br J Clin Pharmacol. 2012;74:134–40. doi: 10.1111/j.1365-2125.2012.04163.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Amara AB, Else LJ, Tjia J, et al. A validated method for quantification of efavirenz in dried blood spots using high-performance liquid chromatography-mass spectrometry. Ther Drug Monit. 2015;37:220–8. doi: 10.1097/FTD.0000000000000127. [DOI] [PubMed] [Google Scholar]
- 16.Olagunju A, Bolaji O, Amara A, et al. Pharmacogenetics of pregnancy-induced changes in efavirenz pharmacokinetics. Clin Pharmacol Ther. 2015;97:298–306. doi: 10.1002/cpt.43. [DOI] [PubMed] [Google Scholar]
- 17.Denniff P, Spooner N. The effect of hematocrit on assay bias when using DBS samples for the quantitative bioanalysis of drugs. Bioanalysis. 2010;2:1385–95. doi: 10.4155/bio.10.103. [DOI] [PubMed] [Google Scholar]
- 18.Li W, Tse FL. Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules. Biomed Chromatogr. 2010;24:49–65. doi: 10.1002/bmc.1367. [DOI] [PubMed] [Google Scholar]
- 19.ter Heine R, Rosing H, van Gorp EC, et al. Quantification of protease inhibitors and non-nucleoside reverse transcriptase inhibitors in dried blood spots by liquid chromatography-triple quadrupole mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2008;867:205–12. doi: 10.1016/j.jchromb.2008.04.003. [DOI] [PubMed] [Google Scholar]
- 20.Rezk NL, Abdel-Megeed MF, Kashuba AD. Development of a highly efficient extraction technique and specific multiplex assay for measuring antiretroviral drug concentrations in breast milk. Ther Drug Monit. 2007;29:429–36. doi: 10.1097/FTD.0b013e318074db39. [DOI] [PubMed] [Google Scholar]
- 21.Rezk NL, White N, Bridges AS, et al. Studies on antiretroviral drug concentrations in breast milk: Validation of a liquid chromatography-tandem mass spectrometric method for the determination of 7 anti-human immunodeficiency virus medications. Ther Drug Monit. 2008;30:611–9. doi: 10.1097/FTD.0b013e318186e08e. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Shapiro RL, Holland DT, Capparelli E, et al. Antiretroviral concentrations in breast-feeding infants of women in Botswana receiving antiretroviral treatment. J Infect Dis. 2005;192:720–7. doi: 10.1086/432483. [DOI] [PubMed] [Google Scholar]
- 23.Mirochnick M, Thomas T, Capparelli E, et al. Antiretroviral Concentrations in Breast-Feeding Infants of Mothers Receiving Highly Active Antiretroviral Therapy. Antimicrob Agents Chemother. 2009;53:1170–6. doi: 10.1128/AAC.01117-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Wilson JT, Brown RD, Cherek DR, et al. Drug excretion in human breast milk: principles, pharmacokinetics and projected consequences. Clin Pharmacokinet. 1980;5:1–66. doi: 10.2165/00003088-198005010-00001. [DOI] [PubMed] [Google Scholar]
- 25.Ito S. Drug therapy for breast-feeding women. N Engl J Med. 2000;343:118–26. doi: 10.1056/NEJM200007133430208. [DOI] [PubMed] [Google Scholar]
- 26.Shiau S, Kuhn L. Antiretroviral treatment in HIV-infected infants and young children: novel issues raised by the Mississippi baby. Expert Rev Anti Infect Ther. 2014;12:307–18. doi: 10.1586/14787210.2014.888311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Joint United Nations Programme on HIV/AIDS, Global Report: UNAIDS report on the global AIDS epidemic 2013. UNAIDS; Geneva: 2013. http://www.unaids.org/sites/default/files/media_asset/UNAIDS_Global_Report_2013_en_1.pdf. [Google Scholar]
- 28.Palumbo P, Lindsey JC, Hughes MD, et al. Antiretroviral treatment for children with peripartum nevirapine exposure. N Engl J Med. 2010;363:1510–20. doi: 10.1056/NEJMoa1000931. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Violari A, Lindsey JC, Hughes MD, et al. Nevirapine versus ritonavir-boosted lopinavir for HIV-infected children. N Engl J Med. 2012;366:2380–9. doi: 10.1056/NEJMoa1113249. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Alvarez-Uria G, Pakam R, Naik PK, et al. Induction with Lopinavir-Based Treatment Followed by Switch to Nevirapine-Based Regimen versus Non-Nucleoside Reverse Transcriptase Inhibitors-Based Treatment for First Line Antiretroviral Therapy in HIV Infected Children Three Years and Older. PLoS One. 2014;9:e108063. doi: 10.1371/journal.pone.0108063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Wang J, Kou H, Fu Q, et al. Nevirapine plasma concentrations are associated with virologic response and hepatotoxicity in Chinese patients with HIV infection. PLoS One. 2011;6:e26739. doi: 10.1371/journal.pone.0026739. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Duong M, Buisson M, Peytavin G, et al. Low trough plasma concentrations of nevirapine associated with virologic rebounds in HIV-infected patients who switched from protease inhibitors. Ann Pharmacother. 2005;39:603–9. doi: 10.1345/aph.1E563. [DOI] [PubMed] [Google Scholar]
- 33.Gunda DW, Kasang C, Kidenya BR, et al. Plasma Concentrations of Efavirenz and Nevirapine among HIV-Infected Patients with Immunological Failure Attending a Tertiary Hospital in North-Western Tanzania. PLoS One. 2013;8 doi: 10.1371/journal.pone.0075118. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Veldkamp AI, Weverling GJ, Lange JMA, et al. High exposure to nevirapine in plasma is associated with an improved virological response in HIV-1-infected individuals. AIDS. 2001;15:1089–95. doi: 10.1097/00002030-200106150-00003. [DOI] [PubMed] [Google Scholar]
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