American Journal of Analytical Chemistry, 2013, 4, 707-714
Published Online December 2013 (http://www.scirp.org/journal/ajac)
Open Access AJAC
Development and Validation of Extraction Method for the
Determination of UC781 in Cervicovaginal Fluid
Naser L. Rezk1,2,3*, Zhiqing Qiao1, Majed Jeriasy3
1Eshelman School of Pharmacy, Division of Pharmacotherapy and Experimental Therapeutics,
University of North Carolina, Chapel Hill, USA
2Andor Labs, Durham, North Carolina, USA
3King Abuallah International Medical Research Center, Riyadh, KSA
Email: *email@example.com, *firstname.lastname@example.org, *email@example.com
Received October 25, 2013; revised November 26, 2013; accepted December 1, 2013
Copyright © 2013 Naser L. Rezk et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Liquid chromatography plays the important and critical role in the field of clinical pharmacology of evaluating drugs in
biological matrices. Studying antiretroviral drugs in the female genital tract has important implications for using drugs
as a vaginal microbicide for prevention of HIV-1 sexual transmission. Accurate measurement of drug levels is ex-
tremely important in optimizing drug concentration in gel formulation. Extracting drugs from small volumes of viscous,
proteinaceous substances like cervicovaginal fluid (CVF) is a practically challenging process. The proposed method
was designed to introduce procedure for sample collection and drug extraction procedure for CVF matrix before the
chromatographic separation. Based on this extraction method, we validated a reverse phase high performance liquid
chromatography with electrospray ionization mass spectrometry assay in order to quantify UC781 in female genital
tract compartment. The LC-MS method was validated based on a novel extraction technique which proved to be effi-
cient in reducing analyte degradation with an average extraction efficiency of 72%. This method is accurate, demon-
strating an average accuracy over three QC (n = 30) concentrations ranging from 99.9% to 106.1%. Average precision
within-day and between-day ranged from 3.1% to 10.2% and 5.1% to 6.4%, respectively. We demonstrated that the
analyte was able to maintain its stability under various conditions using this extraction method. The sample preparation,
extraction, and the powerful liquid chromatography and mass spectrometry can readily be applied for accurate quantifi-
cation of similar drugs in CVF.
Keywords: HIV-Prevention; Cervicovaginal Fluid; Extraction; LC-MS; Nonnucleoside Reverse Transcriptase Inhibitor
Sexual transmission of HIV is the principal mode of
spread of HIV throughout the world . The majority of
HIV-1 infections are acquired sexually, and interventions
to prevent sexual transmission are urgently needed to
curb the growth of the HIV pandemic .
Methods to reduce or prevent sexual transmission of
HIV-1 are urgently needed to sharply reduce the global
HIV-1 epidemic . Understanding the pharmacokinet-
ics of drugs in human body compartments, such as the
female genital tract, is especially important . Among
several classes of HIV inhibitors, many drugs belong to
non-nucleoside reverse transcriptase inhibitors (NNRTIs).
The thiocarboxanilide ((N-[4-chloro-3-(3-methyl-2-bu-
tenyloxy) phenyl]-2-methyl-3-furancarbotthioamide) or
UC-781 (Figure 1(a)) is ranked among the most potent
NNRTI’s [5-8]. Currently, UC781 is being investigated
as a prevention therapy [9,10]. The highly potent non-
nucleoside reverse transcriptase inhibitor UC781 has been
tested as a safe vaginal microbicide gel formulation for
preventing the sexual transmission of HIV-1 virus .
To investigate whether UC781 retained anti-infective
activity following exposure to the female genital tract,
we developed the assay for the analysis UC781 levels
and antiviral activity in cervicovaginal fluid (CVF). Based
on our LC-ESI-MS method  for sensitive and accu-
rate determination of UC781 in blood plasma, we modi-
fied the extraction method to fit with the (CVF) matrix.
An optimal extraction method for female genital tract
secretion must include the release of drug candidate from
N. L. REZK ET AL.
Figure 1 (a) Chemical structure of UC781; (b) Chemical
structure of F2591 (IS).
the viscous proteinaceous substances  like CVF. In
this manuscript, the primary objective of this work is to
develop and optimize an extraction method for a sample
clean-up procedure to fit with CVF. The second objective
is to validate the procedure to be a standard assay for
quantification of drug concentration in CVF.
2. Materials and Methods
UC781 (purity 98.7%) was supplied by Regis Technolo-
gies, Inc (Morton Grove, IL, USA). F2951 (purity 99.2%),
used as internal standard, was supplied by Chemtura,
Technology Center (Guelph, Ontario, Canada). Tetrahy-
drofuran (purity 99.9%) was purchased from Aldrich (St.
Louis, MO, USA). HPLC-grade reagents and chemicals
were purchased from Fisher Scientific (Norcross, GA,
USA). Purified compressed nitrogen gas was obtained
from National Welders Supply (Charlotte, NC, USA).
An Eppendorf® Positive Displacement contamination-
free pipette (PDP), adjustable ranges 1 µl - 20 µl and
Positive Displacement Tips obtained from (Eppendorf
North America, Washington DC, USA). This pipette func-
tions according to the positive-displacement principle in
conjunction with the special Positive Displacement Tip
(Figure 2). An Eppendorf 5415D centrifuge (Eppendorf
AG, Hamburg, Germany) was used during sample prepa-
ration. The high-performance liquid chromatography
(HPLC) system consisted of an Agilent Technologies
(Wilmington, DE, USA) HP1100 binary pump, degasser,
and thermostatic auto sampler (programmed at 4˚C). The
HPLC system was connected to a 1100 Series Mass Spec-
trometer. Positive electrospray ionization was the mode
used for analytical compounds. Data analysis was per-
formed using HP ChemStation software (Version A.09.03)
run on a Dell computer (Windows 2000 Professional
2.3. Blank Vaginal Fluid Collection and
The biological matrix, vaginal secretion fluid (CVF) was
collected from five healthy subjects. The collection of
CVF has been approved by the institutional review board
(IRB) at the University of North Carolina at Chapel Hill
(UNC) according the Federal-Wide Assurance (FWA)
#4801, the IRB approval # (45 CFR 46 CFR 46.110), and
all volunteers/patients gave informed consent prior to
Before spiking drug to the pooled CVF, the vaginal
secretion was diluted 1:3 in normal saline. Because the
volume of CVF varies from subject to subject, a similar
approach was used on the patient sample directly after
the collection of CVF secretion and before storage. Based
on the amount of collected CVF, the PDP adjustable
positive displacement pipette should be adjusted on 5 or
10 or 15 or 20, then pipette the possible volume, wipe the
narrow tip. Then transfer the aspirate into a clean tube
containing three times the volume of the aspirate.
2.4. Preparation of Standards
A total of 5.066 mg of UC781 (molecular weight 335.82)
Figure 2. Positive displacement with a special tip.
Open Access AJAC
N. L. REZK ET AL. 709
powder with a purity of 98.7% was accurately weighed
and dissolved in 5 mL of methanol to produce a final
concentration of 1 mg·mL−1. The master stock solution
was prepared by diluting a stock of 1 mg·mL−1 to 500
μg·mL−1 in 50% HPLC-grade methanol/HPLC-grade wa-
ter. This 500 μg·mL−1 master solution was used to pre-
pare six working solutions in methanol/HPLC-grade wa-
ter (1:1) at concentrations of 0.5 - 500 mg·mL−1 of
UC781. CVF calibration samples (50, 100, 500, 1000,
5000, 10,000, 25,000 and 50,000 ng·mL−1 of UC781)
were prepared by using a 1:10 dilution of the respective
working solutions to blank CVF. From an additional 500
ng·mL−1 working stock solution, concentrations of 1.5
μg·mL−1, 15 μg·mL−1 and 150 μg·mL−1 of UC781 were
prepared in methanol/HPLC-grade water (1:1). CVF qual-
ity control samples at concentrations of 150, 1500, and
15,000 ng·mL−1 of UC781 were prepared using a 1:10
dilution of their respective working solutions to blank
2.5. Internal Standard (IS) Preparation
F2951 (Figure 1(b)) powder (5.187 mg; purity 99.2%)
was dissolved in 3 mL methanol, then complete the vol-
ume of methanol (5.0 mL) to achieve a final concentra-
tion of 1.0 mg·mL−1 (stock solution). From this solution,
an aliquot was diluted in HPLC-grade acetonitrile to a
final concentration of 20.0 ng/mL (working solution).
Small aliquots of working solution must be stored at
2.6. Extraction Procedure
The extraction of UC781 from the CVF matrix was per-
formed with liquid-liquid extraction, using diethylether.
Prior to extraction, CVF protein was precipitated. The
precipitation occurred by adding 10 µL aliquot of CVF
blank, calibrators, and QCs into 2.0 mL tube containing
20 µL the internal standard made in acetonitrile. All ma-
trix aliquots were pipette using positive displacement
pipette with narrow end tip (Figure 2) which easy to
wipe off the remaining outside sticky fluid for better ac-
curacy. The tips are narrow, transparent and long enough
to monitor the volume. The solutions were mixed via
vortex-mixing for 30 seconds. After vortex-mixing, 10 µl
of (0.1 N) NaOH was added to each tube, followed by
1.7 mL of the extraction liquid. All tubes were immedi-
ately capped and gently mixed for 30 min at low speed.
All tubes were placed in a dry ice/acetone bath for ap-
proximately 1 minute; the aqueous portion of the sample
was frozen and the organic layer was immediately trans-
ferred to a centrifuge tube and evaporated until dry under
a nitrogen stream at 35˚C for approximately 8 mints.
Finally, the residue was reconstituted with 100 µL of
methanol/water (50/50). The resulting solutions were
carefully vortexed for 30 s and centrifuged at 14,000 rcf
for 3 mints. The supernatants were transferred to 200 µL
HPLC micro-vials (Agilent Technologies), and 10 µL of
each sample was injected for LC-MS analysis.
2.7. Chromatography Separation Conditions
1) High performance liquid chromatography condi-
Chromatographic separation was performed using gra-
dient elution. Separation was conducted using an Allure
C-18 (100 × 2.1 mm, 3.0 µm particle size, Restek, Belle-
fonte, PA, USA) analytical column with an Allure C-18
(10 × 2.1 mm, 5.0 µm particle size, Restek) guard col-
umn. Two mobile phase components were utilized
throughout the study. Mobile phase A consisted of 10
mM of ammonium formate in water, and mobile phase
B was composed of LC-MS-grade methanol containing
0.01% tetrahydrofuran (THF). A linear gradient was pro-
grammed as 80% mobile phase B to 100% B over the
first 5 minutes, followed by 0.5 minutes at 100% mobile
phase B, then 6 minutes at 80% B, and finally 4 minutes
of re-equilibration. The analysis was performed at 30˚C
with a mobile phase flow rate of 0.3 mL·min−1.
2) Mass spectrometry detection conditions
Mass spectral analysis was performed on an Agilent
Quadrupole 1100 Mass Spectrometer fitted with elec-
trospray ionization (ESI) source and operated in the posi-
tive ionization mode. The vaporizer was operated at
300˚C, the nebulizer gas pressure was set to 40 psi and
the capillary voltage was set to 3000 V. The IS and
UC781 were detected by their positive ion (m/z 326.0
and 336.1, respectively) using the single ion monitoring
2.8. Assessment of Performance Characteristics
An equal weighted regression was performed to assess
linearity. The deviation in the mean calculated concen-
trations over five runs was required to be within 15% of
the nominal concentration for the non-zero calibration
2.8.2. Accuracy and Precision
Accuracy was calculated as the percent deviation from
the nominal concentrations. All intra- and inter-day pre-
cision was required to be within a coefficient of variation
(CV%) of 15% or less. Sample ranges included a low QC
where the concentration was three times of the LOQ
[13,14], a medium QC and a high QC.
2.8.3. Extraction Efficiency (%)
Extraction efficiency was calculated by dividing the area
Open Access AJAC
N. L. REZK ET AL.
response of three pre-spiked QC levels (low, medium,
and high in mobile phase) by the area response of ex-
tracted blank plasma that was post-spiked with the same
three QC concentrations.
The stability of UC781 during sample handling was veri-
fied by subjecting samples to three freeze-thaw cycles
and storage for 2 days in refrigerator 4˚C prior to analy-
ses. An additional test was performed to verify drug sta-
bility in the final extract for 48 hours in autosampler
while tubes waiting for HPLC analysis. The samples
were left at room temperature for 6 hours prior to analy-
ses. Two concentrations medium and high (QC) samples
were utilized in the stability tests.
2.8.5. Applying the Method on the Clinical Samples
The patient samples will be diluted 1:3 before pipetting
10 µL using positive displacement pipette. An SOP was
developed describing the sample collection procedure
and submitted to participating clinical centers. After the
measurement in order to obtain the final concentration
calculated concentration will be multiplied*3.
3.1. Chromatographic Separation and Selectivity
The approximate retention times for UC781 and IS were
2.0 and 4.3 min, respectively. As depicted in Figures
3(a)-(c) (chromatograms of extracted drugs from CVF,
with internal standard at low, medium and high QC’s
respectively), none of the endogenous substances from
the blank CVF extracts interfered with the analyte or
3.2. Linearity and Limit of Quantification
The peak area of the UC781: IS ratio for calibration
standards were proportional to the level of drug in CVF
over the range of tested concentrations. The calibration
curves were fitted by performing weighted least-squares
linear regression. The linear regression data for the cali-
bration curves obtained from this method (n = 5) consis-
tently demonstrated a coefficient of determination ≥0.999.
Using this method, we produced data that were linear
from 50 - 50,000 ng·mL−1. The low limit of quantifica-
tion of UC781 was 50 ng·mL−1. As shown in Table 1(a),
this concentration demonstrated high accuracy and preci-
sion. Linearity was also tested without the internal stan-
dard to determine the direct proportionality of the UC781
peak area to the corresponding concentrations. The cal-
culated regression coefficient (r2) of all calibration curves
Figure 3. (a) LC-MS chromatogram of the low QC (150
ng·mL−1); (b) LC-MS chromatogram of the high QC (1500
ng·mL−1); (c) LC-MS chromatogram of the high QC (15,000
3.3. Accuracy and Precision
Results from the method validation in human CVF (Ta-
ble 1) were acceptable. All observed intra- and inter-day
precision (CV %) data were at or below 15% and in ac-
cordance with the FDA guidelines . Chromatograms
of the three QC concentrations are illustrated in Figure
3(a) (150 ng·mL−1), Figure 3(b) (1500 ng·mL−1) and Fig-
ure 3(c) (15,000 ng·mL−1). UC781 concentrations are pre-
sented as a percent deviation from the nominal concen-
trations for both within-day and between-day analyses.
Using our method, precision for UC781 determinations
was always ≤10.2% for both within- and between-day
analyses. Throughout the range of control sample con-
Open Access AJAC
N. L. REZK ET AL.
Open Access AJAC
Table 1. Summary of accuracy and precision (%) during method validation at low, medium and high QC concentration.
QC Concentration (ng·mL−1) Accuracy (%) Precision (%) within-day (n = 5) Precision (%) between-day (n = 26)
Low 150 101.0 3.06 5.08
Medium 1500 102.3 10.20 6.20
High 15,000 99.6 4.60 6.36
centrations, the intra-day precision was always lower than
10.2%. Overall, the mean inter-day precision was 6.4%,
with mean RSDs ranging from 5.1% - 6.4%.
3.4. Extraction Efficiency (%)
The extraction efficiencies for UC781 and IS from CVF
using the described liquid-liquid extraction method were
calculated using the ratio of the concentration of analyte
in CVF to the identical concentration of the analyte pre-
pared in mobile phase without extraction. The absolute
recovery of analyte from CVF using the diethyether (liq-
uid-liquid extraction) procedure was investigated (Table
2). This extraction method reliably eliminated interfering
material from CVF and demonstrated high recovery (88.3%)
for three (QC) concentration levels.
Stability data for UC781 under various conditions are
provided in Table 3. For all conditions tested, UC781
proved to be stable. All results were within the accep-
tance criteria of ±15% deviation from the nominal con-
Liquid chromatography separation hyphenated to mass
spectrometry (LC-MS) has been developed into an im-
portant application in clinical pharmacology—not only
for research purposes but also for routine use. At present,
most important application fields are target analyses in
drug measurements in a variety of biological matrices for
therapeutic drug monitoring (TDM), metabolic disorders
diagnosis, and many other applications. The essential
strengths of LC-MS include a potentially high analytical
specificity, a wide range of applicability to small or large
molecules, and the opportunity to develop powerful as-
says with a high degree of flexibility.
There has been more than 25 million people have al-
ready died because of HIV since the discovery of the
virus in 1981. This makes AIDS is one of the most disas-
trous epidemics in human history [15,16]. In year 2008
2.7 million people got infected by the HIV virus, mainly
due to hetero sexual HIV transmission [17,18]. Despite
the enormous efforts made, the development of a pro-
phylactic AIDS-vaccine will likely not be available in the
recent years to come . This leads researchers to focus
more on the importance of microbicides (i.e. chemical
entities that can prevent or reduce the transmittance viral
infection) which can applied locally (e.g. vaginally and
rectally) as an alternative approach in preventing HIV-
Our research group at the University of North Carolina
was first research team to focus on investigating antiret-
roviral (ARV); protease inhibitors (IP), nucleoside re-
verse transcriptase inhibitors (NRTI) and non-nucleoside
reverse transcriptase inhibitors (NNRTI) in female geni-
tal tract (FGT) [23,24]. It was the first method to quantify
drugs in direct aspirates of cervicovaginal fluids (CVF). In
that study we found large deference in CVF drugs pene-
tration (from ≤10% to ≥100%) of blood plasma concen-
tration . Since then, we went over to the evaluation
of many other ARV medications in FGT [4,26,27]. Most
of these studies supported the usage of several ARV
which could be an excellent pre-exposure/post-exposure
prophylaxis (PrEP/PEP) candidate.
The nonnucleoside reverse transcriptase inhibitor UC781
proved to be a potential microbicide to prevent sexual
transmission of human immunodeficiency virus type
(HIV-1). Several gel formulations of UC781 were evalu-
ated in a range of preclinical safety assessments, includ-
ing systemic absorption analysis following topical appli-
cation in human.
In this bioanalytical work, we are facing major chal-
lenges. First obstacle is the technical difficulties of UC781
in terms of solubility and stability which discussed in
details elsewhere . Yet, the challenge remains in
dealing with CVF secretion. In brief, the vaginal fluid
contains water, pyridine, squalene, urea, acetic acid, lac-
tic acid, cholesterol, lipids, mucin, carbohydrates, amino
acids, proteins, inorganic ions complex alcohols and
glycols, ketones, and aldehydes. It can vary in consis-
tency, texture, taste, color, and odor, depending on sexual
arousal, the phase of the menstrual cycle, the presence of
an infection, certain drugs (legal or illegal), genetic fac-
tors, and diet. Vaginal fluid is slightly acidic and can be-
come more acidic with certain sexually transmitted dis-
eases. The normal pH of vaginal fluid is between 3.8 and
4.5, whereas male semen is typically between 7.2 and 8.0
(a neutral substance has a pH of 7.0).
CVF is vital element of the immune system of the fe-
male genital tract. CVF is made of 1) vulvar secretions
from sebaceous, sweat, Bartholins and Skene glands; 2)
plasma transudate through the vaginal wall; 3) exfoliated
cells; 4) bacterial products; 5) cervical mucus 6) endo-
N. L. REZK ET AL.
Table 2. Summary of the assay extraction efficiency %.
QC Pre-spike Post-spike % recovery Mean SD %CV
(ng·mL−1) Peak area Peak area
150 41,827 47,947 87 90 3.6 4.0
52,324 55,601 94
52,749 59,237 89
1500 589,493 648,324 91 92 1.7 1.9
516,108 546,940 94
597,420 692,801 92
15,000 5,023,683 6,589,592 76 83 6.2 7.4
5,885,670 6,834,217 86
6,156,078 7,026,467 88
Table 3. Stability of UC781 in spiked rabbit vaginal fluid and final extract
Conc. (ng·mL−1) Three freeze-thaw cycles 48 h at 4˚C 8 h at 25˚C One week matrix stability
1500 10.6 2.5 −2.6 3.3
15000 7.7 1.3 −2.8 5.8
All values are represented as the mean of the deviation from the nominal concentration. All samples were performed in triplicate.
metrial and oviductal fluids and 7) secretions from vagi-
nal immune cells. The latter three are influenced by sex
steroid hormones e.g. during the menstrual cycle and
pregnancy [28,29]. It covers the lower female genital
tract pH and hydrates the mucosa, creating a physical
barrier for microbial invasion. The important part of ac-
curate quantification of drug in FGT is the extraction
Due to sample collection limitations with a small sam-
ple size, CVF collection is a challenging process. It was
important to use dilution just right after aspiration in or-
der to prevent fluid from forming a clot. Average sample
size of normal CVF secretion ranged between 0 to 0.7
mL. In this study, we developed and optimized liquid-
liquid extraction method and introduced a simple proce-
dure allowing to accurately sampling from the collected
fluid before it turns to clot. Using the positive displace-
ment pipette with special tips (Figure 2) was important
to dilute the sample before storage, similarly when trans-
ferring diluted sample for extraction. In the day of vali-
dation samples were brought to room temperature and
treated for the extraction as described in the method.
CVF sample dilution immediately after collection when
the temperature near to 37˚C (body temperature) is nec-
essary, because it keeps the secretion in the fluid state.
Using this procedure of sample collection and extraction,
the validation data proved to be accurate and precise for
any minute sample collected.
However, normal hexane was the optimal organic sol-
vent for blood plasma , we found 100% of diethylether
releases higher amount of UC781 compared with the
other three solvents (chloroform/ether 50/50, hexane/
ether 50/50 and 100% hexane) and SPE. This could be
explained as the higher amount of lipids in blood plasma
than CVF which required highly non polar solvent.
We have successfully developed and validated an LC-
MS bioanalytical method for UC781 in the CVF matrix
using the sub-2 µ column with a powerful mobile phase.
The method proved to be accurate meeting all validation
criteria. For the rare matrix, it exhibits good linearity,
precision and accuracy over a wide range of drug con-
centrations (50 - 50,000 ng·mL−1). This novel sample
preparation associated with liquid-liquid extraction pro-
cedure has been proved to be an excellent sample han-
dling option for such a sensitive compound in minute
amount of vaginal secretion. The method sample prep,
extraction, and the powerful liquid chromatography and
mass spectrometry can readily be used for accurate quan-
tification of any similar drugs in CVF.
The authors thank the CVF donors for their participation,
my analytical team at the University of North Carolina at
Chapel Hill for assisting in the validation of the method,
Dr. David Friend from Conrad, for providing the stan-
dard and internal standard for the method, Mrs. Soheir
Hagras at the Drug Research Center, National Center of
Research & Radiation Technology (NCRRT), Egypt, and
Dr. Chris Sheehan at Badirbio, Riyadh, KSA for review
of this manuscript.
 S. Taylor, M. Boffito and P. L. Vernazza, “Antiretroviral
Open Access AJAC
N. L. REZK ET AL. 713
Therapy to Reduce the Sexual Transmission of HIV,”
Journal of HIV Therapy, Vol. 8, No. 3, 2003, pp. 55-66.
 T. Lalani and C. Hicks, “Does Antiretroviral Therapy
Prevent HIV Transmission to Sexual Partners?” Current
HIV/AIDS Reports, Vol. 10, No. 2, 2007, pp. 80-82.
 J. M. Baeten and J. Overbaugh, “Measuirng the Infec-
tiousness of Peresons with HIV-1: Opportunities for Pre-
venting Sexual HIV-1 Transmission,” Current HIV Re-
search, Vol. 1, No. 1, 2003, pp. 69-86.
 J. A. Talameh, N. L. Rezk and A. Kashuaba, “Quantify-
ing the HIV-1 Integrase Inhibitor Raltegravir in Female
Genital Tract Secretions Using High-Performance Liquid
Chromatography with Ultraviolet Detection,” Journal of
Chromatography B, Vol. 878, No. 1, 2010, pp. 92-106.
 J. Balzarini, M. J. Perez-Perez, S. Velazquez and A. San-
Felix, “Suppression of the Breakthrough of Human Im-
munodeficiency Virus Type 1 (HIV-1) in Cell Culture by
Thiocarboxanilide Derivatives When Used Individually
or in Combination with Other HIV-1-Specific Inhibitors
(i.e., TSAO Derivatives),” Proceeding of the National
Academy of Sciences of the United States of America, Vol.
92, 1995, pp. 5470-5474.
 J. Balzarini, W. G. Brouwer, D. C. Dao, E. M. Osika and
E. De Clercq, “Identification of Novel Thiocarboxanilide
Derivatives That Suppress a Variety of Drug-Resistant
Mutant Human Immunodeficiency Virus Type 1 Strains
at a Potency Similar to That for Wild-Type Virus,” An-
timicrobial Agents and Chemotherapy, Vol. 40, No. 6,
1996, pp. 1454-1466.
 R. W. J. Buckheit, T. L. Kinjerski, V. Fliakas-Boltz, J.
Russell, T. L. Stup, L. A. Pallansch, et al., “Structure-
Activity and Cross-Resistance Evaluations of a Series of
Human Immunodeficiency Virus Type-1-Specific Com-
pounds Related to Oxathiin Carboxanilide,” Antimicro-
bial Agents and Chemotherapy, Vol. 39, No. 12, 1995, pp.
 J. B. MacMahon, R. W. J. Buckheit, R. J. Gulakowski, M.
J. Currens, D. T. Vistica, R. H. Schoemaker, et al., “Bio-
logical and Biochemical Anti-Human Immunodeficiency
Virus Activity of UC 38, a New Non-Nucleoside Reverse
Transcriptase Inhibitor,” Journal of Pharmacology and
Expermental Therapeutics, Vol. 276, No. 1, 1996, pp.
 J. Vingerhoets, H. Azijn, E. Fransen, I. De Baere, L.
Smeulders, D. Jochmans, et al., “TMC125 Displays a
High Genetic Barrier to the Development of Resistance:
Evidence from in Vitro Selection Experiments,” Journal
of Virology, Vol. 79, No. 20, 2005, pp. 12773-12782.
 R. E. Haaland, T. Evans-Strickfaden, A. Holder, C. P.
Pau, J. M. Mcnicholl, S. Chaikummaao, et al., “UC781
Microbicide Gel Retains Anti-HIV Activity in Cervico-
vaginal Lavag Fluids Collected Following Twice-Daialy
Vaginal Application,” Antimicrobial Agents and Chemo-
therapy, Vol. 56, No. 7, 2012, pp. 3592-3596.
 N. Rezk, “Development and Validation of LC-ESI-MS
Method for Sensitive, Accurate and Rabid Determination
of UC-781 in New Zealand White Rabbit Plasma,” Ta-
lanta, Vol. 85, No. 4, 2011, pp. 2074-2079.
 D. H. Owen and D. F. Katz, “A Vaginal Fluide Simu-
lant,” Contraception, Vol. 59, No. 2, 1999, pp. 91-95.
 V. P. Shah, K. K. Midha, J. W. Findlay, et al., “Bio-
analytical Method Validation—Revisit with a Decade of
Progress,” Pharmaceutical Research, Vol. 17, No. 12,
2000, pp. 1551-1557.
 US Department of Health and Human Services, Food and
Drug Administration Center for Drug Evaluation and Re-
search (CDER), Center of Veterinary Medicine (CVM),
“FDA Guidance for Industry,” Pharmaceutical Research,
 UNAIDA, “AIDS Epidemic Update,” Geneva, 2005.
 UNAIDA, “AIDS Epidemic Update,” Geneva, 2009.
 UNAIDS, “Report in the Global AIDS Epidemic,” Ge-
 “Will There Be an HIV Vaccine in the Next Decade?”
Nature Medici ne, Vol. 13, 2007, pp. 518-519.
 L. C. Rohan and A. B. Sassi, “Vaginal Drug Delivery
System for HIV Prevention,” American Association of
Pharmaceutical Scientists, Vol. 11, No. 1, 2009, pp. 78-
 B. Gulter and J. Justman, “Vaginal Microbicides and
Prevention of HIV Transmission,” Lancet Infection Dis-
ease, Vol. 8, No. 11, 2008, pp. 685-697.
 A. B. Moscicki, “Vaginal Microbicides: Where Are We
and Where Are We Going?” Journal of Infection Chemo-
therapy, Vol. 14, No. 5, 2008, pp. 337-341.
 S. M. Iqbal, T. B. Ball, P. Levinson, L. Maranan, W. Jaoko,
C. Wachihi, et al., “Elevated Elafin/Trappin-2 the Female
Genital Tract Is Associated with Protection against HIV
Acquisition,” AIDS, Vol. 23, No. 13, 2009, pp. 1669-
 R. M. Grant, D. Hamer, T. Hope, R. Johnston, J. Lange,
M. M. Lederman, et al., “Whither or Wither Microbi-
cides?” Science, Vol. 321, No. 5888, 2008, pp. 532-534.
 S. Min, A. Corbett, N. Rezk, S. Cu-Uvinc, S. Fiscus, et
al., “Protease Inhibitor and Nonnucleoside Reverse Tran-
scriptase Inhibitor Concentration in Genital Tract of HIV-
1 Infected Women,” Journal of Acquired Immune Defi-
ciency Syndrome, Vol. 37, No. 5, 2004, pp. 1577-1580.
 D. Julie, R. Yeh, K. Paterson, A. Corbett, B. Jung, N.
Rezk, et al., “Antiretroviral Drug Exposure in the Female
Genital Tract: Implications for Oral Pre- and Post Expo-
sure Prophylaxis,” AIDS, Vol. 21, No. 14, 2007, pp.
 R. Yeh, N. Rezk, A. Kashuba, et al., “Genital Tract, Cord
Blood, and Amniotic Fluid Exposures of Seven Antiret-
Open Access AJAC
N. L. REZK ET AL.
Open Access AJAC
roviral Drugs during and after Pregnancy in Human Im-
munodeficiency Virus Type 1-Infected Women,” Antim-
icrobial Agents and Chemotherapy, Vol. 53, No. 6, 2009,
pp. 2367-2374. http://dx.doi.org/10.1128/AAC.01523-08
 A. Kawara, A. Delong, N. Rezk, et al., “Antiretroviral
Drug Concentrations and HIV RNA in the Genital Tract
of HIV-Infected Women Receiving Long-Term Highly
Active Antiretroviral Therapy,” Clinical Infectious Dis-
eases, Vol. 46, No. 5, 2008, pp. 719-725.
 G. R. Huggins and G. Preti, “Vaginal Odors and Secre-
tions,” Clinical Obstetrics and Gynecology, Vol. 24, No.
2, 1981, pp. 355-377.
 L. L. Klein, K. R. Jonschr, M. J. Heerwagen, R. S. Gibbs
and J. L. McManaman, “Reprod, Shotgun Proteomic
Analysis of Vaginal Fluid from Women in Late Preg-
nancy,” Science, Vol. 15, No. 3, 2008, pp. 263-273.