This study evaluated plasma concentrations of the antiretroviral drug atazanavir (ATV) in 110 HIV-positive patients receiving either ATV 300 mg with ritonavir (ATV300/r) boosting or unboosted ATV 400 mg. Plasma samples were analyzed by HPLC. ATV300/r achieved higher trough concentrations and longer half-life than unboosted ATV 400 mg. Coadministration of drugs that induce or inhibit CYP3A4, the main metabolic pathway of ATV, impacted ATV exposure. Higher ATV concentrations were associated with elevated bilirubin levels. The study confirmed pharmacokinetic variability with ATV dosing and identified

1. Validation of a rapid and sensitive high-performance liquid
chromatography–tandem mass spectrometry (HPLC–MS/MS)
assay for the simultaneous determination of existing and new
antiretroviral compounds
Laura Elsea a,∗
, Victoria Watsonab
, John Tjia a, Andrew Hughesb
, Marco Siccardia
, Saye Khoo a
,
David Back a
a Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK
b NIHR Biomedical Research Centre, Royal Liverpool Hospital Trust, Liverpool, UK
Journal of Chromatography B, 878 (2010) 1455–1465
Date:07/05/14
Unit of Clinical Pharmacology,
Department of Biomedical and Clinical Sciences,
"Luigi Sacco" University Hospital

4. Abstract
Abstract |
Simple, fast and sensitive HPLC–MS/MS method for determination of
the commonly used protease inhibitors (PI) amprenavir, atazanavir,
darunavir, lopinavir, ritonavir, saquinavir and the non-nucleoside
reverse transcriptase inhibitor (NNRTI) nevirapine, as well as the
more recent antiretrovirals, the CCR5 antagonist maraviroc and the
“second generation” NNRTI etravirine and rilpivirin
The method described is being successfully applied to measure
plasma antiretroviral concentrations from samples obtained from
clinical pharmacokinetic studies.

5. Introduction
Highly active antiretroviral therapy (HAART) has dramatically reduced HIV-1-
associated mortality and morbidity and currently comprises 25 drugs from five
different classes
Various guidelines recommend for treatment naive patients, a combination of
three or more antiretroviral agents; an NNRTI or a ritonavir boosted PI in
combination with a dual NRTI backbone.
An estimated 8% of treatment naive and 33% of experienced patients do not
achieve viral suppression or experience viral rebound within 12 months of
initiating HAART
Forty per cent of therapy-naive people with HIV experience virological failure
during the first two years of antiretroviral therapy (ART)
low plasma concentrations of antiretroviral drugs is the predictive of more rapid
immunological failure and failure to achieve virological success in the first year of
therapy .
Furthermore, a substantial number of people receiving ART discontinue therapy
in the first 45 weeks of treatment, the majority stop therapy because of drug
related toxicity

6. Routine therapeutic drug monitoring (TDM) and pharmacokinetic drug
interaction studies between existing and new antiretrovirals, and with
concomitant medications are essential for the optimization and management of
antiretroviral therapy

8. Drug Therapy Monitoring
Therapeutic drug monitoring (TDM) is the clinical practice of measuring specific
drugs at designated intervals to maintain a constant concentration in a patient's
bloodstream, thereby optimizing individual dosage regimens.
Purpose
TDM is employed to measure blood drug levels so that the most effective dosage can be
determined, with toxicity prevented. TDM is also utilized to identify noncompliant
patients (those patients who, for whatever reason, either cannot or will not comply with
drug dosages as prescribed by the physician).

9. Why TDM in ART
The PI and NNRTI undergo cytochrome P450 mediated metabolism via CYP3A4
and to a lesser extent by CYP2B6, CYP2D6 and CYP2C19 which renders them prone to
variable pharmacokinetics and extensive drug–drug interactions when given in
combination or with other concomitant medications .
All PI inhibit CYP3A4, ritonavir being the most potent and is used exclusively at
subtherapeutic doses to “boost” other PI.
In addition, ritonavir, lopinavir and amprenavir have CYP enzyme inducing properties.
The first generation NNRTI nevirapine and efavirenz are substrates and inducers of
CYP3A4 and CYP2B6 .
The second generation NNRTI rilpivirine is metabolized primarily by CYP3A4, and
etravirine by CYP3A4, CYP2C9, CYP2C19 .
Maraviroc is a substrate for both CYP3A4 and the efflux transporter P-
gycloprotein, and has shown clinically significant interactions with both PI and NNRTI,
rendering mandatory maraviroc dosage adjustments with some associations

10. Thus,
•Antiretroviral Shows Pharmacokinetics and pharmacodynamic relationship
•Antiretroviral Shows Pharmacokinetics and toxicity relationship
Characterisation of this relationship is a key to the selection of an optimal dose
for a drug, understanding inter- and intra-subject variability, and to design
strategies to optimize response and tolerability while avoiding unwanted toxicity.
Here, authors developed fast and sensitive HPLC–MS/MS method for the
determination of the commonly used PI [amprenavir (APV), atazanavir (ATV),
darunavir (DRV), lopinavir (LPV), ritonavir (RTV), saquinavir (SQV)] and NNRTI
[nevirapine (NVP)], as well as recently licensed CCR5 antagonist maraviroc
(MVC) and the second generation NNRTI etravirine (ETV) and rilpivirine (RPV).

11. Materials and methods
1. Chemicals
APV : Glaxo Wellcome Research and Development (Middlesex, UK)
ATV (atazanavir sulphate) : Bristol-Myers Squibb (Hounslow, UK)
SQV: by Roche Discovery (Welwyn, UK),
LPV, RTV : Abbott Laboratories (Chicago, IL, USA)
NVP: Boehringer Ingelheim Pharmaceuticals, Inc. (Berkshire, UK).
DRV: (darunavir ethanolate), ETV, RPV (rilpivirine hydrochloride): Tibotec (Mechelen, Belgium)
MVC: Pfizer (Sandwich, Kent, UK).
2. Equipment
The HPLC system consisted of a variable loop Accela autosampler (200 vial capacity set at a
temperature of 15 C) and an Accela LC pump (Thermo Electron Corporation, Hemel Hempstead,◦
UK).
A reverse-phase AscentisTM C18 column (3m: 100mm×2.1mm) at an temperature of 26 C.◦
The HPLC system coupled with a triple-quadrupole TSQ Quantum Ultra mass spectrometer (Thermo
Electron Corporation, with a heated-electrospray ionization (H-ESI) source.
TSQ Tune Software (Thermo Electron Corporation, Hemel Hepstead, UK) was used for the
optimization of tuning parameters.
LC QuanTM software (Version 2.5.6, Thermo Electron Corporation, Hemel Hepstead, UK) was used
for data acquisition and processing.

12. Chromatographic and mass spectrometric conditions
•Chromatographic separation was achieved using a rapid stepwise gradient
[ACN:water (0.05% formic acid) 5:95 and 80:20,v/v] mobile phase at a flow rate
of 400 μL /min over a total run time of 5 min.
•The triple-quadrupole mass spectrometer was operated in positive ionization
mode and detection and quantification was performed using selective reaction
monitoring (SRM).

13. Preparation of calibrators, quality controls and internal Standard
Calibration point range
•10 to 10,000 ng/ml (APV, ATV, NVP, RPV and SQV)
• 5 to 5000 ng/ml (ETV and RTV)
• 5 to 1000 ng/ml (MVC)
•15 to 15,000 ng/ml (DRV and LPV)
APV, ATV, NVP,
RPV, SQV
ETV, RTV, MVC DRV LPV
LLQC 25 ng/ml 12.5 ng/ml 40 ng/ml
LQC 150 ng/ml 100 ng/ml; MVC 50
ng/ml
250 ng/ml),
MQC 1500 ng/ml 800 ng/ml; MVC 400
ng/ml
3500 ng/ml
HQC 8000 ng/ml 4000 ng/ml; MVC 700
ng/ml
12,000 ng/ml
Quality Controls

14. Sample pre-treatment
Calibrators and QC samples in duplicate (100µl) QX (20µl and 1 µg/ml )
protein precipitation with ACN (500µl) vortexed and left to stand at room
temperature (15 min) re-vortexed and centrifuged supernatant
decanted into correspondingly labelled 5ml glass tubes followed by the addition of 0.05%
formic acid (200µl) re-vortexed and transferred to autosampler vials
injection (10µl) onto the HPLC column.
Validation of calibrators and quality controls
•A minimum of 10 calibration curves on separate days to ascertain the concentration at
each calibrator level for all 10 drugs.
•Calibrator curves were constructed using a 1/concentration weighted quadratic regression
equation of analyte:internal standard peak area ratios versus target concentration, from
which unknown drug concentrations were interpolated
• Minimum of 10 QC samples (at LLQC, LQC, MQC and HQC) were treated as unknown
values and run in duplicate on separate days alongside a validated calibration curve in order
determine the final QC concentration and inter assay precision and accuracy
• Intra-assay variation was ascertained by running six LLQC, LQC, MQC and HQC samples
within a single analytical run.

15. Standard curve range
10 standard curves run to ascertain mean target calibrator and QC concentrations
Drugs Mean calibration curve
range (ng/ml)
Correlation Coefficient
(r2
)
APV 11-10,063
> 0.998ATV 11-10,017
DRV 16-15,062
ETV 5-5000
LPV 16-15,083
MVC 5-1,009
NVP 11-10,056
RPV 11-10,045
RTV 5-5018
SQU 10-10,087
Mean QC Concentration
QC Levels % variation
LLQC <14
LQC, MQC,HQC 2-11

16. Validation of calibrators and quality controls and Precision (Inter and Intra assay)

17. Assay lower and upper limits of quantification and limit of detections
LOD LLQ ULQ
APV 1.3 11 10,063
ATV 0.2 11 10,017
DRV 1.0 16 15,062
ETV 2.7 5 5000
LPV 2.0 16 15,083
MVC 0.7 5 1009
NPV 0.3 11 10,056
RPV 2.7 11 10,045
RTV 1.3 5 5018
SQV 0.3 10 10,087

18. Stability study

19. Detection and chromatography
•Triple-quadrupole mass spectrometer was operating in positive SRM mode set to a
narrow scan width (0.01 m/z) and scan time (0.01 s) for all transitions.
•The capillary temperature and vaporising temperature within the H-ESI source were
maintained at 300 and 350 C throughout an assay◦

21. Recovery and matrix effects

23. Discussion
The developed bioanalytical method successfuly applied to measure antiretroviral
plasma concentration for clinical pharmacokinetic studies.
This HPLC–MS/MS method was fully validated with accuracy (% bias) and precision
(CV%), which did not exceed 13% and 10% for all compounds with Percentage
recovery greater than 90% for all analytes.
Stability data suggest that all analytes remained sufficiently stable under our current
storage conditions (−20 ◦C) for up to 1 month.
Assay describes very short run time of 5 min per sample with a quick and simple
sample pre-treatment procedure and hence , suitable for high-throughput TDM
purposes
This method requires small volume of plasma for analysis (100µl). This is
advantageous when quantifying drug in patients such as children and neonates, or
from alternative matrices

24. Conclusion
A simple and rapid assay for the quantification of all currently approved PI (APV,
ATV, DRV, LPV, RTV and SQV) and NNRTI (NVP), as well as recently licensed
antiretroviral classes (MRC) and the second generation NNRTI (ETV and RPV) in
human plasma has been developed and validated.
This method is proven to be specific, accurate, precise and robust.
The assay is highly sensitivity for all analytes and the step-wise gradient potentially
allows addition of new analytes into the same analytical run.
Due to the high signal-to-noise ratio at the LLQ level, this method could be adopt to
measure low antiretroviral concentrations in sites such as the genital tract and in
cerebrospinal fluid.

25. Atazanavir (ATV) is an azapeptide protease inhibitor.
Protease inhibitors ATV is not associated with abnormal lipid profiles .
ATV has a unique pharmacokinetic profile which makes it suitable for once-daily oral
administration.

26. Method
This is a observational study carried out at St. Vincent’s Hospital which evaluated TDM data from
a cohort of 110 people (Treatment experienced ) with HIV who received ATV over a 5-month
period.
The mean age and number of the participants in the study
•(ATV300/r), n=92, was 46± 9 years
•(ATV400), n=26 46 ± 11 years
Plasma samples Collection : ATV300/r: 0–8 h (n = 5), 8–16 h (n=20) and 16–30 h (n=67) and
ATV400: 8–16 h ( n = 2) and 16–30 h ( n = 24).
ATV Plasma concentration were quantified by HPLC on a phenyl hexyl column (250 x 4.6 mm; 5
mm) with ultraviolet detection at 205 nm.
The ATV calibration standards ranged from 50 to 10 000 µg/l and the lower limit of ATV
quantification was 50 µg/l
.

27. Results and Discussion :
Out of 20 patient 11 with through plasma ATV concentration below the limit of detection
(25 µg/l) participated in Steady-state pharmacokinetic analysis
•8 received 400 mg ATV daily and 3 received ATV300/r daily
• Plasma samples were collected at 0, 3, 6, 9 and 24 h
• 2nd
Dose after 24h with Coca Cola and a single 3 h blood sample was collected to
observe the effect on ATV concentrations.

28. ATV 400 ATV 300/r
Median
C min Conc
30 µg/l (range < 25–390
µg/l )
476 µg/l (range
< 25–2108 µg/l)
Half Life 1.9 to 4.1 h 2.3 to 11.2 h
(half life < 4h
in 13% of
patient)
ATV Trough 25
µg/l
(below
detection limit)
13 (8 interviewed for
adherence, 2 patient
coadministered ,
efavirenz,
colchicines.nandrolone
and esomeprazole )
7 (3
interviewed for
adherence.
Coadministered
esomeprazole,
fluticasone ,
rtv,)
Elevated
Bilirubin
35 % patient
(median 15.0; range 4–
49 µmol l-1
)
49 % patient
All ATV Cmin plasma concentrations >500
mg l-1
were associated with abnormal
serum bilirubin results (median 55.5;
range 24–119 µmol l-1

30. ATV Concentration-time Profile of 8 patient receiving 400 mg ATV daily

31. Pharmackinetic profile of the patient
suffering from hypertension and asthama
•Fluticasone accuhaler 500 µg bid
•Lopinavir/ritonavir for 11 month before
switching on to ATV 300/r
•First pharmacokinetic study
•Diagnosed with Cushing syndrome after 7
month
•Fluticasone replaced with montelukast
•Second pharmacokinetic study after 2 week
wash out period
•The AUC for ATV increased from 21,447 mg
h-1
l 38,384 mg h-1
l when fluticasone was
ceased
•No acidic beverage effect

32. Conclusion
Substantial Pharmacokinetic variability with the ATV dose of 300/r and ATV 400
This study confirmed a relationship between elevated serum bilirubin concentration
and higher ATV concentrations
Study describes possible drug interaction between RTV, fluticasone and ATV.
Coadministration of RTV and fluticasone is not recommended due to the ability of
RTV to inhibit CYP3A4 mediated fluticasone metabolism causing iatrogenic Cushing
Syndrome
Reduced plasma concentrations of ATV are expected when the drug is used in
combination with antacids, buffered medications, H2-receptor antagonists and
proton-pump inhibitors

33. Thank you