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IUBMB Life, 61(4): 470–475, April 2009

Research Communication

Simultaneous Determination of Maraviroc and Raltegravir in


Human Plasma by HPLC-UV
Stefania Notari1, Chiara Tommasi1, Emanuele Nicastri1, Rita Bellagamba1, Massimo Tempestilli1,
Leopoldo Paolo Pucillo1, Pasquale Narciso1 and Paolo Ascenzi1,2
1
Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, Roma, Italy
2
Dipartimento di Biologia, Università Roma Tre, Viale Marconi 446, Roma, Italy

INTRODUCTION
Summary Therapeutic drug monitoring (TDM) is a strategy by which
Therapeutic drug monitoring is pivotal to improve the man- the dosing regimen for a patient is guided by measurements of
agement of HIV infection. Here, a new HPLC–UV method to plasma drug levels, enabling physicians to optimize anti-HIV
quantify simultaneously maraviroc and raltegravir levels in drugs efficacy and to avoid drug-related toxicity. Although in
human plasma is reported. Remarkably, this is the first method
for maraviroc determination in human plasma. The volume of daily practice the use of TDM remains controversial, it appears
the plasma sample was 600 lL. This method involved auto- as a useful tool in selected category of patients such as those
mated solid-phase extraction with Oasis HLB Cartridge 1 cc with limited treatment options because of infection with HIV
(30 mg divinylbenzene and N-vinylpyrrolidone) and evaporation strains resistant to currently used antiretroviral drugs (1–9).
in a water bath under nitrogen stream. The extracted samples Resistance has been demonstrated against all currently used
were reconstituted with 200 lL 50/50 of mobile-phase solution
anti-HIV drugs, and hence there is still a high need for new
(0.01 M KH2PO4 and acetonitrile). Twenty microliters of these
samples were injected into a HPLC–UV system, the analytes agents with different resistance profiles or that target different
were eluted on an analytical dC18 Atlantis column (150 mm 3 phases of the virus life cycle (10). Therefore, over the last 2
4.6 mm I.D.) with a particle size of 5 lm. The mobile phase years, the Food and Drug Administration approved new anti-
(0.01 M KH2PO4 and acetonitrile) was delivered at 1.0 mL/min HIV drugs, including maraviroc and raltegravir, for the treat-
with isocratic elution. The total run time for a single analysis
ment of HIV-infected patients who exhibit extensive drug resist-
was 10 min; maraviroc and raltegravir were detected by UV at
197 and 300 nm. The calibration curves were linear up to 2,500 ance and limited treatment options (http://www.fda.gov/).
ng/mL. The absolute recovery ranged between 93 and 100%. Maraviroc, the first slowly reversible and selective antago-
The HPLC–UV method reported here has been validated and is nist of the human chemokine CCR5 receptor, prevents HIV
currently applied to monitor plasma levels of maraviroc and entry into host cells reducing viral load. After oral adminis-
raltegravir in HIV-infected patients. Ó 2009 IUBMB
tration, maraviroc is rapidly absorbed, efficiently cleared, and
IUBMB Life, 61(4): 470–475, 2009
does not significantly influence the activity of drug-metabo-
lizing enzymes. Maraviroc is approved for different dosages,
Keywords anti-HIV drugs; HPLC-UV; maraviroc; raltegravir; thera- according to optimized background therapy: 150 mg twice a
peutic drug monitoring. day (BID) with protease inhibitors, 300 mg BID without pro-
Abbreviations BID, twice a day; HPLC, high-performance liquid tease inhibitors, and 600 mg once a day (QID) with efavirenz
chromatography; LOD, limit of detection; LOQ, limit (11, 12).
of quantification; QID, once a day; SPE, solid-phase Raltegravir, the first HIV integrase strand transfer inhibitor,
extraction; TDM, therapeutic drug monitoring. prevents the insertion of viral DNA into the genome of the host
cell and consequently viral replication. Raltegravir reduces
effectively HIV-RNA in treatment-naı̈ve patients and in patients
infected with multidrug-resistant strains of HIV. Raltegravir is
Received 26 November 2008; accepted 11 December 2008 metabolized by glucuronidation and exhibits poor drug–drug
Address correspondence to: Paolo Ascenzi, Istituto Nazionale per le
Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Via Portuense 292, interaction. At standard dose of 400 mg BID, raltegravir is
Roma, Italy. Tel: 139-06-55170934. Fax: 139-06-5582825. rapidly absorbed, reaching the steady-state level in 2 days (13,
E-mail: ascenzi@uniroma3.it 14).
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.181
MARAVIROC AND RALTEGRAVIR DETERMINATION BY HPLC–UV 471

The accurate measurement of maraviroc and raltegravir plas- Sample Preparation


matic levels is crucial for the management of HIV-infected According to the protocol approved by the Ethics Committee
patients. Here, the development and the validation of a new of the Istituto Nazionale per le Malattie Infettive I.R.C.C.S.
HPLC-UV method for the simultaneous determination of mara- ‘Lazzaro Spallanzani’ (Roma, Italy) and with the written
viroc and raltegravir in human plasma are reported. Remark- informed consent of the patients, blood samples were drawn
ably, this is the first method for maraviroc determination in from HIV-infected patients. Patients were instructed not to take
human plasma. their morning pills prior to the consultation. The patient selec-
tion criteria were pharmacological steady-state and efficient
response to the therapy.
MATERIALS AND METHODS Blood samples (6.0 mL) were collected in monovetters Li
heparinate and centrifuged at 3000 rpm for 20 min at 24.0 8C.
Chemicals Then, human plasma was separated from blood cells and stored
Maraviroc and raltegravir were obtained through the NIH at 220.0 8C. Human plasma samples were cleaned-up by off-
AIDS Research Reagent Program, Division of AIDS, NIAID, line solid-phase extraction using Oasis HLB Cartridge 1 cc (30
National Institute of Health (Bethesda, MD). Both anti-HIV drugs mg divinylbenzene and N-vinylpyrrolidone) (Waters). The car-
were of analytical grade and used without further purification. tridges were conditioned with 1.0 mL methanol followed by 1.0
Acetonitrile, methanol, and KH2PO4 (from Carlo Erba Reagenti, mL 0.01 M KH2PO4. Six hundred microliters of human plasma
Rodano, Milano, Italy) were of HPLC grade. Deionized water were loaded in the cartridge. Then, cartridges were washed with
(18.2 mO, total organic carbon \ 100 ppb) was produced on-site. 1.0 mL of water Milli-Q. Analytes were eluted by washing car-
tridges with 1.0 mL methanol. The eluate was evaporated in a
water bath at 60.0 8C under a stream of nitrogen. The extracted
Chromatographic System sample was reconstituted with 200 lL 50/50 of mobile-phase
The chromatographic system consisted of a Waters 600 solution (0.01 M KH2PO4 and acetonitrile) and transferred to an
pump and a Waters autosample 717 PLUS equipped with a injection vial.
spectrophotometric UV–vis dual-wavelength system Waters
2487 set at 197 and 300 nm (Milford, MA). Maraviroc and Calibration Curves
raltegravir separation was performed at 24.0 8C on an analyti- The calibration curves of maraviroc and raltegravir were
cal dC18 Atlantis column (150 mm 3 4.6 mm I.D.) with a established over the 19.53–2500 ng/mL concentration range.
particle size of 5.0 lm (Waters) equipped with a Waters Sen- Under all the experimental conditions, the response/amount
try guard column (20 mm 3 3.9 mm I.D.) filled with the same ratio was linear.
packing material (Waters). The ‘‘Millenium’’ software
(Waters) was used to pilot the HPLC–UV instrument and to
Recovery
process the data (i.e., area integration, calculation, and plotting
of chromatograms) throughout the method validation and The efficiency of solid-phase extraction was determined with
sample analysis. control samples at 19.53, 156.25, and 2500 ng/mL. The absolute
recovery of maraviroc and raltegravir from plasma was obtained
as the peak-area response of the processed samples, expressed
Mobile Phase Solutions as the percentage of the response of the anti-HIV drugs con-
The mobile phase is composed of solution A (0.01 M tained in the 20-lL injection volume and not subjected to solid-
KH2PO4) and B (acetonitrile). Both solutions were degassed by phase extraction.
sparging with helium. The injection volume was 20 lL. The
mobile phase was delivered at 1.0 mL/min. The isocratic condi-
RESULTS
tion was 60/40 in 10 min.
Chromatograms
The HPLC method here reported provides a simple procedure
Stock, Working, and Plasma Solutions to determine simultaneously the concentration of maraviroc and
Stock solutions of maraviroc and raltegravir (1.0 mg/mL) raltegravir in the plasma of HIV-infected patients by UV detec-
were prepared by dissolving 5.0 mg of each drug in 5.0 mL of tion at 197 nm (maraviroc and raltegravir) and 300 nm (ralte-
methanol. Stock solutions were appropriately diluted with meth- gravir). The retention time of maraviroc and raltegravir was 3.3
anol for the preparation of working solutions (final concentra- and 6.1 min, respectively. Figure 1 shows the chromatogram of a
tion ranging between 19.53 and 2500 ng/mL). The anti-HIV standard mixture of maraviroc and raltegravir (156.25 ng/mL)
drug concentration in plasma calibration samples ranged (panel A), of a drug-free human plasma sample from a healthy
between 19.53 and 2500 ng/mL. All working solutions were donor (panel B), of a healthy donor plasma sample spiked with
stored at 14.0 8C and were stable for at least 4 months. 100 lL of maraviroc and raltegravir (156.25 ng/mL) (panel C),
472 NOTARI ET AL.

Table 1
Anti-HIV regimens and maraviroc and raltegravir plasma
concentration of HIV-infected patientsa
Plasma
Anti-HIV concentration
Patient drug Dose (mg) (ng/mL)
1 Maraviroc 150BID 69.462
Raltegravir 400BID 74.301
2 Maraviroc 150BID 21.506
Raltegravir 400BID 95.808
3 Maraviroc 150BID 58.111
Raltegravir 400BID 127.328
4 Maraviroc 150BID 74.052
Raltegravir 400BID 219.941
5 Maraviroc 300BID 128.05
Raltegravir 400BID 192.836
6 Maraviroc 150BID 49.496
Raltegravir 400BID 147.667
7 Maraviroc 300BID 146.010
Raltegravir 400BID 167.050
8 Maraviroc 150BID 28.028
Raltegravir 400BID 151.330
9 Maraviroc 300BID 51.685
Raltegravir 400BID 131.619
10 Maraviroc 300BID 21.409
Raltegravir 400BID 66.481
11 Maraviroc 300BID 204.160
Raltegravir 400BID 104.559
12 Maraviroc 150BID 37.981
Raltegravir 400BID 108.948
13 Maraviroc 150BID 40.327
Raltegravir 400BID 119.540
14 Maraviroc 150BID 38.704
Raltegravir 400BID 97.958
15 Maraviroc 150BID 20.393
Raltegravir 400BID 96.153
BID
twice a day.
a
Data referring to the HIV-infected patients 10 and 13 correspond to those
reported in panels D and E, respectively, of Fig. 1.

Figure 1. Simultaneous detection of maraviroc and raltegravir


by HPLC–UV. Chromatogram of a standard mixture of mara-
viroc and raltegravir (156.25 ng/mL) (panel A). Chromatogram
of a drug-free human plasma sample from a healthy donor
(panel B). Chromatogram of a healthy donor plasma sample
spiked with 100 lL of maraviroc and raltegravir (156.25 ng/
mL) (panel C). Chromatogram of plasma samples from two
HIV-infected patients (panels D and E). Data shown in panels
D and E correspond to those of the HIV-infected patients 10
and 13 reported in Table 1. maraviroc, 1; raltegravir, 2. For
details, see text.
MARAVIROC AND RALTEGRAVIR DETERMINATION BY HPLC–UV 473

Table 2
Maraviroc and raltegravir recovery after extraction from
human plasma
Recovery (% 6 S.D.)a

Anti-HIV drug 19.53 ng/mL 156.25 ng/mL 2500 ng/mL


Maraviroc 94.07 6 7.2 99.25 6 5.3 100 6 1.7
Raltegravir 95.10 6 5.6 93.67 6 6.1 97.45 6 2.7
a
Results are the mean of six experiments.

Figure 2. Calibration curves for the determination of maraviroc Limits of Detection and Quantification
(circles), and raltegravir (squares). Calibration curves obtained The limit of detection in plasma of maraviroc and raltegravir
dissolving maraviroc and raltegravir in methanol and plasma are was defined as the concentration that yields a signal-to-noise
superimposable. The calibration curve parameters are as follows: ratio of 3:1. For the concentration to be accepted as the lowest
maraviroc: y 5 229.0x 2 8354.0 and r2 5 0.990; raltegravir: y limit of quantification (LOQ), the percent deviation from the
5 142.3x 1 1089.0 and r2 5 0.992. Where not shown, standard nominal concentration (measure of accuracy) and the relative
deviation is smaller than the symbol. For details, see text. standard deviation (measure of precision) have to be less than
20% (http://www.fda.gov/cher/guidelines.htm). The LOQ value
for maraviroc and raltegravir was 19.53 ng/mL.
and plasma samples from two HIV-infected patients (panels D
and E). Table 1 shows the antiretroviral regimens and maraviroc
and raltegravir plasma concentration of HIV-infected patients. Recovery
Values of raltegravir plasma levels here determined agree with The absolute recovery was calculated by comparing the peak
those reported in the literature (http://aidsinfo.nih.gov). areas obtained from standard working solutions with the peak
areas from standard extracts. Recovery experiments were carried
out at 19.53, 156.25, and 2500 ng/mL anti-HIV maraviroc and
Specificity and Selectivity raltegravir concentration in plasma samples. Unspiked samples
Blank samples showed no interfering endogenous substances were used as a control. Results are shown in Table 2.
eluting at the retention time of maraviroc and raltegravir. The
selectivity was determined by injecting onto the HPLC column Precision and Accuracy
all currently prescribed anti-HIV drugs and/or employed in the
Intra- and inter-day precision and accuracy were studied at
treatment/prophylaxis of opportunistic infections. Note that all
six different concentrations. The precision was calculated as the
patients were under multidrug therapeutic regimens. The follow-
relative standard deviation within a single run (intra-day) and
ing anti-HIV drugs were coadministered with raltegravir and
between different assays (inter-day):
maraviroc to the enrolled patients: tenofovir/emtricitabine 300/
200 mg QID (patients 1, 2, 4, 13, and 14 in Table 1), darunavir Precision ð%Þ ¼ ðS.D.=meanÞ 3 100;
600 mg BID (patients 1, 2, 8, 12, 14, and 15 in Table 1), ritona-
vir 100 mg BID (patients 1, 2, 3, 4, 6, 8, 12, 13, 14, and 15 in where S.D. is the standard deviation. The accuracy was calcu-
Table 1), lamivudine 300 mg QID (patients 3 and 6 in Table 1), lated as the percentage of the deviation between the nominal
saquinavir 1,000 mg BID (patients 3, 4, 5, 6, 10, and 13 in Ta- and the found concentration:
ble 1), enfuvirtide 90 mg BID (patients 7 and 11 in Table 1),
zidovudine 300 mg QID (patient 9 in Table 1), and TMC-125 Accuracy ð%Þ ¼ ðð½found  ½nominalÞ=½nominalÞ 3 100:
200 mg BID (patient 10 in Table 1).
Results are shown in Table 3. Both precision and accuracy
were \20%, according to the literature (http://www.fda.gov/
Linearity cher/guidelines.htm).
The calibration curves for the determination of maraviroc
and raltegravir concentration are shown in Fig. 2. The response/
amount ratio was linear between 19.53 and 2500 ng/mL. The DISCUSSION
linearity of standard curves was very good (r2 [ 0.990). Cali- Here, we report the first HPLC–UV method for the simulta-
bration curves obtained dissolving maraviroc and raltegravir in neous determination of maraviroc and raltegravir in human
methanol and plasma are superimposable. plasma from HIV-infected patients, note that maraviroc and ral-
474 NOTARI ET AL.

Table 3
Intra- and inter-day determination of maraviroc and raltegravir
Intra-daya Inter-daya

Nominal Found Nominal Found


concentration concentration Accuracy concentration concentration Precision Accuracy
Anti-HIV drug (ng/mL) (ng/mL) Precision (%) (ng/mL) (ng/mL) (%) (%)
Maraviroc 19.53 22.60 6 1.57 6.64 15.7 19.53 18.32 6 0.61 3.28 26.2
156.25 156.28 6 0.05 0.032 0.031 156.25 155.52 6 0.21 0.13 20.47
2500 2419.06 6 3.23 0.13 23.23 2500 2489 6 0.19 0.007 20.44
Raltegravir 19.53 19.58 6 0.12 0.61 0.25 19.53 20.32 6 0.74 3.6 4.04
156.25 148.55 6 9.88 6.65 24.92 156.25 162.12 6 1.93 1.19 3.75
2500 2714.32 6 8.57 0.31 8.57 2500 2469 6 0.60 0.02 21.24
a
Results are the mean of six experiments.

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