Abstract
Aim: An LC-MS/MS method to quantify drug in dried
cervicovaginal secretions from flocked swab was developed and validated
using the antiretroviral efavirenz as example.
Methods: Cervicovaginal swabs (CVS) were prepared by submerging
flocked swabs in efavirenz-spiked matrix. Time to full saturation,
weight uniformity, recovery and room temperature stability were
evaluated. Chromatographic separation was on a reverse-phase C18 column
by gradient elution using 1mM ammonium acetate in water/acetonitrile at
400 µL/min. Detection and quantification were on a TSQ Quantum Access
triple quadrupole mass spectrometer operated in negative ionisation
mode. The method was used to quantify efavirenz in CVS samples from
HIV-positive women in the VADICT study (NCT03284645).
Results: Swabs were fully saturated within 15 seconds,
absorbing 128 µL of matrix with coefficient of variation (%CV) below
1.3%. The method was linear with a weighting factor (1/X) in the range
of 25-10000 ng/mL with inter- and intra-day precision (% CV) of
7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of
efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix
effect. Efavirenz remained stable in swabs for at least 35 days after
drying and storage at room temperature. Median (range) CVS efavirenz
AUC0-24h was 16370 ng*h/mL (5803-22088),
Cmax was 1618 ng/mL (610-2438) at a Tmaxof 8.0 h (8.0-12), and Cmin was 399 ng/mL (110-981).
Efavirenz CVS:plasma AUC0-24 ratio was 0.41 (0.20-0.59).
Conclusion: Further application of this method will
improve our understanding of the
pharmacology of other therapeutics in the female genital tract,
including in low- and middle-income countries.
Introduction
Heterosexual transmission through the genital mucosa and mother-to-child
transmission (MTCT), mainly during delivery, are major routes of HIV
acquisition in Sub-Saharan Africa. High HIV RNA in genital secretions is
a known risk factor for sexual transmission, independent of plasma HIV
concentration [1]. HIV shedding in the female genital tract has been
shown to increase the risk of MTCT during delivery [2]. Therefore,
adequate penetration of antiretroviral (ARV) drugs into this compartment
to achieve undetectable HIV RNA is critical to minimize the risk of
transmission [3]. For instance, differential accumulation of
tenofovir and emtricitabine in rectal fluid versus female genital
secretions has been observed [4], an observation consistent with
differential expression of efflux transporters [5]. A proper
understanding of ARV pharmacokinetics in the female genital tract is
crucial in developing effective pre-exposure prophylaxis (PrEP) and
prevention of MTCT (PMTCT) interventions.
Cervicovaginal fluid concentration has been shown to be an acceptable
surrogate for tissue concentration [6]. However, developing and
validating a method to quantify drugs in the female genital tract is
challenging and several methods have been reported, including the use of
menstrual cup and cervicovaginal lavage. Limited sample volumes and
difficulty in standardizing dilution practices are major limitations
associated with these methods. The use of cervicovaginal cotton-based
smears and swabs have also been explored, suboptimal analyte extraction
being a major drawback. Polyester based materials offer better analyte
recovery than cotton-based materials. For instance, Bennetto-Hoodet al described an ophthalmic tear strip wicking method in which
standards and quality control samples were prepared by applying 7 µL of
spiked blank cervicovaginal fluid [7]. In many of these methods, the
applied volumes differ from the capacity of the collection devices. In a
method where the applied volumes (25 µL on ophthalmic tear strips, 125
µL on polyester-based swabs) fully saturated the devices, preparation of
standards and quality control samples did not replicate real-life sample
collection. Hence in both scenarios, clinical application of these
methods will require weighing the devices pre- and post-collection to
allow for normalization of fluid collected.
More recently, flocked swabs with perpendicular nylon fibers on the tip
of a solid molded plastic applicator shaft have become available. In
this paper, we report the validation and clinical application of a LC-MS
method for efavirenz quantification in flocked cervicovaginal swabs
(CVS) that accurately mimics real life specimen collection process.
Importantly, we extended the application of this method to settings with
inadequate ultra-low temperature storage facilities by validating room
temperature drying and storage.
Materials and methods
Materials
Reference standards of efavirenz and 13C-labelled
efavirenz (internal standard, IS) 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), and
formic acid from Sigma-Aldrich (Gillingham, Dorset, UK). Water was
produced from an Elga Option-S water purification unit (Elga Labwater,
High Wycombe, Buckinghamshire, UK) and further purified to 18.2 MΩ with
the Purelab Ultra (Elga LabWater, High Wycombe, Buckinghamshire, UK).
FLOQSwabs® were obtained from
COPAN Diagnostics Inc. (Murrieta, CA, USA). Blank plasma was obtained
from drug-free healthy volunteers.
LC-MS/MS systems and conditions
The LC-MS/MS system and conditions were as previously described for the
quantification of efavirenz in dried blood spot [8] and dried
breastmilk spot [9]. In brief, chromatographic separation was on a
reverse phase Fortis™ C18 column 3 µm, 10cm 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). The mobile phase consisted of 1mM ammonium acetate
in water (mobile phase A) and 1mM ammonium acetate in acetonitrile
(mobile phase B) in gradient elution at a flow rate of 400µL/min over 5
minutes. The total injection volume was 10 µL and 3 mL MeOH-water (1:1,
v/v) was used as wash solvent between injections. Detection was on the
TSQ Quantum Access (Thermo Electron Corporation, Hemel Hempstead,
Hertfordshire, UK) with a heated electrospray ionisation source operated
in the negative ionisation mode and selective reaction monitoring.
Xcalibur™ was used for compound tuning and optimisation while the
LCquan™ (version 2.7.0, Thermo Fisher Scientific, Hemel Hempstead, UK)
was used for sequence acquisition and processing.
Swab saturation and weight uniformity
The weight uniformity of the swabs was evaluated by individually
weighing 20 new swabs. The percentage deviation from the mean weight was
computed in each case. Time to complete saturation was assessed by
inserting previously weighed swabs in plasma for different lengths of
time from 5 to 120 sec and 12 h (n = 10 per duration). The weight
uniformity of completely saturated swabs was assessed.
Preparation of stock solutions, calibration standards (STD)
and quality controls (QC) samples
Efavirenz and 13C-labeled efavirenz stock solutions
were prepared from their respective reference standards in methanol to
obtain a final concentration of 1 mg/mL and stored at -20 °C until use.
Working stock of efavirenz in plasma were prepared by spiking an
appropriate volume of the 1 mg/mL stock solution into blank plasma to
obtain final concentrations of 10, 30 and 34 µg/mL. Plasma calibration
standards in the range of 25-10000 ng/mL were prepared from the 30 µg/mL
working stock by serial dilution. Plasma lowest limit of quantification
(LLOQ, 25 ng/mL), low quality control (LQC, 75 ng/mL) and medium quality
control (MQC, 4500 ng/mL) samples were prepared from the 10 µg/mL
working stock while the high quality control (HQC, 8500 ng/mL) samples
was prepared from the 34 µg/mL working stock. A 5 µg/mL working stock of13C-labeled efavirenz in plasma was prepared by
spiking an appropriate volume of its intermediate stock (100 µg/mL in
methanol-water (50:50, v/v) prepared from the 1 mg/mL stock) in plasma
and used as IS.
Efavirenz CVS STDs and QCs samples were prepared by completely inserting
each swab in the corresponding plasma STDs and QCs until full
saturation. Each swab was transferred into 1.8 mL cryovials and stored
at -80 °C until analysis.
Sample pre-treatment
Each swab was transferred into a 7 mL screw cap tube and extracted with
1 mL of methanol by tumbling for 30 min in the presence of 20 µL of IS.
The tubes were centrifuged at 4000 rpm for 10 min and 500 µL of the
extract was transferred into 5 mL glass tube and evaporated to dryness
under a gentle stream of nitrogen gas. The residue was reconstituted in
500 µL of mobile phase A and B. After centrifugation for 5 min at 4000
rpm, 250 µL was transferred into a new 5 mL glass tube and diluted with
250 µL of mobile phase. This was followed by further centrifugation at
4000 rpm for 5 min and 300 µL was transferred into autosampler vial for
injection.
Calibration curves, accuracy and precision
Five separate validation assay batches were run, each consisting of a
zero blank, ten calibrators in the range of 25-10000 ng/mL (n = 2 for
each level), and QCs (n = 6 for each level). Calibration curves were
constructed using a linear regression equation of analyte/IS peak area
ratio versus nominal concentrations with a 1/concentration weighting.
Percentage deviation of measured concentrations from nominal values was
used to define accuracy and the percentage coefficient of variation
(%CV) defined precision. In any batch, at least 75% of STDs and 67%
of QCs (and at least 3 at each level) were required to have percentage
deviation within ±15%, with an additional ±5% permitted for the lower
limit of quantification (LLOQ) and the LQC [10].
Evaluation of room temperature drying and efavirenz stability
in dried CVS
To assess the feasibility of room temperature drying, CVS loaded with
blank plasma were weighed and kept at room temperature (n = 6) or in the
oven at 45 °C (n = 6) and weighed at regular intervals until a constant
weight was obtained. Efavirenz stability in CVS after drying at room
temperature was assessed using CVS QC samples dried at room temperature
and assayed immediately, 1 week or 1 month after storage at room
temperature in ziplock bags with desiccants. The concentrations were
determined using freshly made CVS STDs and QCs.
Recovery and matrix effect
Recovery was assessed by comparing peak areas from extracted CVS QC
samples (n = 6 per level) with corresponding extracts of drug-free swabs
spiked with the efavirenz solution post-extraction which represented
100% recovery. A %CV within ±15% in replicate responses at each level
was set as acceptance threshold to ensure consistency and
reproducibility. To assess matrix effect, CVS samples were collected
from six different healthy volunteers (n = 9 per volunteer) who have not
taken any drug during the 2 weeks prior to sampling. The CVS samples
were collected and stored in cryovials at -80°C. Each CVS was brought to
room temperature and extracted as described under sample pre-treatment.
Matrix effect was calculated at LQC, MQC and HQC for each lot of matrix
using the ratio of the peak area in the presence of matrix (measured by
analysing blank matrix spiked with efavirenz after extraction), to the
peak area in the absence of matrix (measured by analysing pure solution
of the efavirenz in mobile phase).
Clinical application
To evaluate its clinical utility, the validated method was used to
quantify efavirenz in intensive CVS pharmacokinetic samples collected
from some of the participants in the VADICT study (ClinicalTrials.gov
Identifier: NCT03284645) who received 600 mg efavirenz daily as part of
antiretroviral therapy. Study centres were the Federal Medical Centre
Makurdi, Benue State, Nigeria. The protocol and material transfer
agreement (MTA) were approved by the National Health Research and Ethics
Committee, Abuja, Nigeria (approval number:
NHREC/01/01/2007-05/06/2017). A detailed description of the study
protocol has been published elsewhere [11].
CVS sample collection procedure
In brief, participants were required to refrain from unprotected sexual
intercourse for at least 12 h before sample collection. Participants
laid down for 5 min before each sample collection to allow pooling of
fluid in the back of the vagina. To collect the CVS sample, the head of
each FLOQSwab® was gently inserted approximately 3 inches into the
vagina. While separating the labia with one hand, the other hand was
used to hold the FLOQSwab® between the thumb and forefinger. The head of
the FLOQSwab® was inserted further until it touches the back of the
posterior fornix. The swab was gently rubbed against the mid-vaginal
walls for at least 30 sec, withdrawn and immediately transferred into a
cryovial. A total of 35 intensive CVS samples from 5 women were
collected at 0.5, 1, 2, 4, 8, 12 and 24 h after an observed evening dose
of a fixed-dose combination tablet containing 600 mg efavirenz. Paired
DBS samples were collected at each time point on Whatman 903 protein
saver cards following finger prick with a safety lancet. Samples were
stored at -80 °C at the Federal Medical Centre, Makurdi, until transfer
to the Translational Pharmacokinetics Research Laboratory, Obafemi
Awolowo University, Ile-Ife using Arctic Express® Dry Shipper (Thermo
Scientific, Waltham, MA, USA) where they were stored at -80 °C until
analysis.
To demonstrate the feasibility of performing assay on CVS samples dried
and stored at room temperature, some sparse CVS samples from additional
participants in the VADICT study were removed from the -80 °C freezer
and left to dry at room temperature overnight at the Translational
Pharmacokinetic Research Laboratory, Obafemi Awolowo University,
Nigeria. They were transferred into new vials and individually packed in
ziplock bags with desiccants along with paired DBS samples and posted at
room temperature to the University of Liverpool for analysis.
Efavirenz in CVS was quantified using the newly developed and validated
method while efavirenz in DBS was quantified using a previously
described method and plasma concentrations were estimated using the
equation: [DBS[EFV]/(1-HCT)]*0.995, where
DBS[EFV] is efavirenz concentration in DBS, HCT is
the patient-specific haematocrit and 0.995 is the fraction of efavirenz
bound to plasma protein [8]. Both the CVS and DBS methods were
initially set up at the Bioanalytical Facility, University of Liverpool,
UK and were later successfully transferred to the Obafemi Awolowo
University Bioanalytical Laboratory, Ile-Ife, Nigeria where the TSQ
Quantum Access LC-MS/MS system is now located and the analysis of the
intensive pharmacokinetic samples was implemented. The area under the
concentration-time curve over a 24-h dosing interval
(AUC0-24h) was determined using the trapezoidal rule,
the apparent clearance (Cl/F) was calculated by dividing dose by
AUC0–24 while the maximum (Cmax),
minimum concentration (Cmin), and time to reach maximum
concentration (Tmax) were determined by visual
inspection.
Results
CVS saturation and weight uniformity
The average (SD) weight of an empty swab was 0.7378 g (0.0069) with a
relative standard deviation of 0.93%. The swabs were fully saturated
within 15 sec as no further weight gain was observed after this time
(15-120 sec and 12 h), the average weight being 0.8704 g (0.0111) with a
relative standard deviation of 1.28%. This is equivalent to 128 µL of
plasma (density, 1.1200 g/mL) per swab. Hence, insertion for a duration
of not less than 15 ssec was considered adequate and used for the
preparation of standards and QC samples during method validation and
incorporated into the SOP for collection of CVS samples in the VADICT
study for pharmacokinetic assays.
LC-MS/MS conditions
Representative chromatograms are presented in Figure 1 showing efavirenz
at the LLOQ, LQC and a patient CVS. The total runtime was 7 min and the
retention time was 2.8 min for both efavirenz and the IS,
efavirenz-13C6. The MS transitions were 314.042 →
242.083 and 244.087 m/z for efavirenz and 320.099 → 247.970 and 249.990
m/z for the IS with optimal collision energies of 21 and 20, 20 and 18,
respectively.
Linearity, accuracy and precision
The method was linear with a weighting factor \((1x)\) in the range of
25-10000 ng/mL with inter- and intra-day precision (% CV) was between
7.69 and 14.9%, and accuracy (% bias) ranged from 99.1 to 105.3%.
(Table 1). These values are within the acceptance criteria established a
priori as per FDA guidance [10]. The mean regression coefficient
(r2) was > 0.99.
Room temperature drying
and efavirenz stability in dried CVS
Drying for two hours resulted in a 12.9% weight loss at room
temperature and 12.3% at 45 °C, with additional 0.03 and 0.04%
respectively after 3 h. Constant weight was achieved after 8 h at room
temperature and 5 h at 45 °C. Hence, drying at room temperature for 8 h
or more was considered optimal. Efavirenz remained stable in CVS after
drying at room temperature and storage at same for 24 h (LQC, 96.8%;
MQC, 110%; HQC, 108%) and 35 days (LQC, 98.5%; MQC, 96.9%; HQC,
104%).
Efavirenz recovery from CVS and matrix effect
The pre-treatment method described above resulted in an average (SD)
recovery of efavirenz from CVS of 83.8% (9.4) with %CV of 11.2; 74.1%
at LQC, 84.4% at MQC and 92.9% at HQC. The overall matrix effect was
92.7% (5.8), 98.6% at LQC, 92.4% at MQC and 87.1% at HQC, with an
overall % CV of 6.2.
Clinical application
A total of 108 samples (40 paired intensive CVS and DBS samples, 14
paired sparse CVS and DBS samples) from 20 participants were available
for this analysis. The five postpartum women who contributed the
intensive pharmacokinetic samples had a mean (SD) age of 29.3 years
(7.3), weight was 70.2 kg (11.8), and they were at 46.2 weeks (6.2)
postpartum. Duration on fixed dose combination of efavirenz with
lamivudine and tenofovir was 4.5 years (1.9) and samples were collected
at seven time points during a dosing interval. The fifteen pregnant
women who participated in the sparse pharmacokinetic sampling were 31.5
years (5.9) old, weight was 74.4 kg (16.0), duration on fixed dose
combination of efavirenz with lamivudine and tenofovir was 3.7 years
(3.3), and samples were collected 14.2 hours (0.75) after the last dose
and at 34.1 weeks (4.2) of gestation.
Based on the forty CVS efavirenz concentration-time data contributed by
5 postpartum women, median (range) CVS efavirenz
AUC0-24h was 16370 ng*h/mL (5803-22088),
Cmax was 1618 ng/mL (610-2438) at a Tmaxof 8.0 h (8.0-12), and
Cmin was 399 ng/mL
(110-981). The corresponding plasma (DBS-derived)
AUC0-24h was 34772 ng*h/mL (25841-43987),
Cmax was 4752 ng/mL (3602-5363) at a
Tmax of 2.0 h (2.0-4.0), and Cmin was
1728 ng/mL (1127-2514). Efavirenz CVS:plasma AUC0-24ratio was 0.41 (0.20-0.59). The combined and individual patient CVS and
plasma concentration-time profiles are presented in Figure 2. Efavirenz
Cmin in CVS was above the protein binding adjusted
IC95 of 126 ng/mL for wild-type HIV-1, but below the 470
ng/mL trough plasma effective concentration threshold established in the
ENCORE 1 study in 74% of patients.
Efavirenz was successfully quantified in dried CVS samples (n = 14), the
median (range) concentration was 1144 ng/mL (173-6379) and the
corresponding plasma concentration (DBS derived) was 1619 ng/mL
(891-7049). The CVS:plasma concentration ratio was 0.58 (0.13-1.4) for
these paired samples collected at 14.2 hours (0.75) after the last dose.
A good correlation was observed between CVS and plasma efavirenz
concentrations (Pearson r = 0.62; R = 0.38) in this limited number of
paired sparse samples.
Discussion
To facilitate intensive pharmacokinetic studies to better understand the
pharmacology of therapeutics in the female genital tract, we developed
and validated a simple, accurate and precise method for drug
quantification in cervicovaginal fluid collected with flocked swab
(using efavirenz as example). The utility of the new method was
demonstrated in a small pharmacokinetic study of efavirenz in CVS
samples from pregnant and postpartum HIV-infected women taking
efavirenz-based antiretroviral therapy. Additionally, the feasibility of
room temperature drying and storage was demonstrated, further extending
its application to settings with limited ultra-low temperature storage
facilities.
In this cohort of postpartum women, mean efavirenz CVS:plasma
AUC0-24 ratio was 0.41 (range: 0.20-0.59), individual
patient profiles indicated substantial variability within the dosing
interval and between individuals. Importantly, efavirenz
Cmin in CVS was above the protein binding adjusted
IC95 for wild-type HIV-1 of 126 ng/mL in all patients.
The preparation of standards and QCs in the CVS method described in this
paper closely mimics the procedure for specimen collection in patients.
Importantly, this departs from previously reported methods in which
specific volumes of spiked matrix were applied to swabs using pipette.
The latter method requires obtaining the weight of each swab pre- and
post-collection, otherwise the resulting calibration curves will
underestimate drug concentration in patient samples. This new method
effectively eliminates this requirement, significantly simplifies and is
expected to improve pharmacokinetic studies in female genital tract.
To illustrate, the female genital tract constitutes a major route of
heterosexual and mother-to-child transmission of HIV during delivery.
Suppressive antiretroviral therapy reduces transmission risk. The
development of precise and accurate bioanalytical method to accurately
determine drug concentration in the female genital tract is important to
understand antiretroviral distribution pattern in this compartment. For
example, in HIV prevention trials among women, characterisation of drug
distribution in this compartment for different delivery technologies
will facilitate proper assessment of antiretroviral exposure-response
relationships. This will enable the selection of the best in class
technologies and therapeutics for optimal clinical benefits. An
important consideration in this regard is the reliability of female
genital fluid drug concentration as a surrogate for tissue
concentration. Moderate to high correlations of cervicovaginal fluid
with mucosal tissue concentration has been reported for four
antiretrovirals (r2 = 0.37 for maraviroc, 0.45 for
tenofovir, 0.50 for emtricitabine and 0.74 for raltegravir; P
< 0.001). Hence, intensive pharmacokinetic data from the
genital fluid could supplement sparse genital tissue data where
quantitative assessment of drug concentration is needed. Hence, the
method reported here will facilitate studies that aim to establish
pharmacokinetic targets for PrEP efficacy in women. With increasing
interests in long-acting antiretroviral formulations, such targets will
ensure an evidence-based approach in selecting an optimal dose and
dosing frequency. Interestingly, the utility of efavirenz for PrEP
repurposing was recently demonstrated using population
pharmacokinetics-pharmacodynamic modelling [12, 13].
CVS efavirenz concentrations in this study are more than the 18.4 ng/mL
(6.95, 48.73) in 13 women at 8-12 h previously reported by Kwara et al
[14]. The CVS:plasma concentration ratio of 0.01 (0.00-0.03)
obtained from the sparse pharmacokinetic data in the same study is
significantly less than the CVS:plasma efavirenz AUC0-24ratio of 0.41 (0.20-0.59) observed in the present study. An earlier
study reported a ratio of 0.25 (0.06, 1.05) at 3-4 h and 0.08 (0.01,
0.53) at 8-12 h after dose[15]. Both studies used directly aspirated
genital fluid samples. Alternatively, flocked swabs were used for
cervicovaginal fluid collection in this study and efavirenz was
quantified using a method specifically validated for CVS. Hence, the use
of different sampling techniques makes direct comparison across
different studies difficult and impractical. Additionally, it is unclear
if patients in previous studies were pregnant or non-pregnant women.
Patients who contributed intensive pharmacokinetic samples in this study
were postpartum women while those who contributed sparse samples were
pregnant (34.1 weeks of gestation). The influence of changes in CVF
volume associated with the menstrual cycle [16] and pregnancy on
drug concentration in the CVF require further investigation as the
present study was not designed to address these.
Adequate penetration of tenofovir and lamivudine (taken in combination
with efavirenz by patients in the present study) has been reported
[14, 17], with CVF:plasma concentration ratio of 3.2 (1.2-8.5) for
lamivudine and 5.2 (1.2-22.6) for tenofovir in one study at 8-12 h after
dosing [14]. In that study, samples were collected at two time
points and CVF:plasma concentration ratios at 3-4 h were less than at
8-12 h after dosing for all 13 antiretrovirals evaluated. An extensive
review of the pharmacokinetics of antiretrovirals in the female genital
tract was previously published [18], indicating the use of direct
aspirate in most studies and a trend for class-specific differences:
nucleoside reverse transcriptase inhibitors > nonnucleoside
reverse transcriptase inhibitors > protease inhibitors.
CVF:plasma concentration ratios of 0.06 for dolutegravir [19], and
above 1.0 for rilpivirine [20] have been reported. Population and
physiologically based pharmacokinetic models of antiretrovirals in
female genital tract have also been described [17, 21, 22].
Complimentary to these are recently described deterministic [23] and
quantitative structure activity relationship [24] models of vaginal
drug distribution describing sources of variability and potential
application across different delivery systems and species. In addition
to generating data to validate such models, the application of the new
method reported here to other drug candidates could facilitate more
clinical studies and accelerate progress towards the establishment of
reliable exposure-response relationships for interventions aimed at
preventing heterosexual and mother-to-child transmission of diseases
across the vaginal mucosa.
The use of drug-free plasma in the preparation of calibration standards
and QC samples instead of drug-free cervicovaginal fluid due to sample
inaccessibility is one of the limitations of the present study. However,
no significant matrix effect was observed. Some authors have reported
diluting cervicovaginal fluid up to twenty times with water to obtain
sufficient volume for assay development and validation [25]. Another
limitation is non-availability of cervicovaginal fluid aspirate or
lavage from the same patients who contributed CVS for cross-validation
with the CVS method. Though efavirenz is known to be stable in DBS at
room temperature for at least 18 months, assessment of its stability in
dried CVS over a longer period than the 35 days evaluated in this study
is still desirable to further build confidence. This method is now being
used for the assay of sparse and intensive pharmacokinetic samples in
the VADICT study [11]. The associated standard operating procedure
for the collection of CVS for pharmacokinetic and viral load assessments
is available upon request.