CHAPTER 10

DARUNAVIR

267

10.1. DRUG PROFILE

Darunavir (brand name Prezista, formerly known as TMC114) is in the protease inhibitor

class used to treat human immunodeficiency virus (HIV) in adults and children 6 years of age

and older. It was approved by the Food and Drug Administration (FDA) on June 23, 2006.[1]

To

overcome the problems with early protease inhibitor (PIs) like severe side effects and drug

toxicities, require a high therapeutic dose, are costly to manufacture, and show a disturbing

susceptibility to drug resistant mutations a second generation protease inhibitor (PIs) Darunavir

is discovered.

Darunavir is used with ritonavir (Norvir) and other medications to treat human

immunodeficiency virus (HIV). It works by slowing the spread of HIV in the body. Darunavir

does not cure HIV infection and may not prevent you from developing HIV-related illnesses.

Darunavir does not prevent you from spreading HIV to other people.

Figure 10.A: Darunavir

Systematic (IUPAC) name : (1R, 5S, 6R)-2, 8-dioxabicyclo [3.3.0] oct-6-yl] N

[(2S, 3R)-4- [(4-aminophenyl) sulfonyl- (2 methylpropyl)

amino]-3-hydroxy-1-phenyl- butan-2 yl] carbamate

Formula : C27H37N3O7S

268

Mol. Mass : 547.665 g/mol

Routes : Oral

Darunavir was designed to form robust interactions with the protease enzyme from many

strains of HIV, including strains from treatment-experienced patients with multiple resistance

mutations to PIs.[2][3]

It blocks HIV protease, an enzyme which is needed for HIV to multiply.

When used with other anti-HIV medicines. It may reduce the amount of HIV in your blood

(called "viral load") and increase your CD4 (T) cell count. HIV infection destroys CD4 (T) cells,

which are important to the immune system. The immune system helps fight infection. Reducing

the amount of HIV and increasing the CD4 (T) cell count may improve your immune system

and, thus, reduce the risk of death or infections that can happen when your immune system is

weak (opportunistic infections).

List of brand names of Darunavir

Table: 10.1

S.NO. BRAND

NAME

FORMULATION COMBINATION AVAILABLE

STRENGTH

Mg

MANUFACTURER

1 PREZISTA Tab Ritonavir 75 JOLLC

2 PREZISTA Tab Ritonavir 150

3 PREZISTA Tab Ritonavir 400

4 PREZISTA Tab Ritonavir 600

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10.2. LITREATURE SURVEY

Several analytical methods have been reported for the determination of Darunavir in pure

drug, pharmaceutical dosage forms and in biological samples using spcetrophotometry liquid

chromatography, electro kinetic chromatography high performance thin layer chromatography

either in single or in combined forms.

Raveendra B.Ganduri et al [4]

has develop a simple, rapid, sensitive, accurate, precise and

reproducible high performance liquid chromatographic method for the determination of

Darunavir in tablet dosage form. Waters, Symmetry shield RP18 (250X4.6mm, 5µm)

column0.1% orthophosphoric acid and acetonitrile (50:50 % v/v) as mobile phase, detection

wavelength of 265 nm, flow rate of 1.0 mLmin-1. The method is linear from 25µg mL-1 to

100µg mL-1, accuracy was found to be 99.54%, mean inter and intraday assay relative standard

deviation (RSD) were less than 1.0%. The method is simple, accurate, specific and precise, can

be used for the determination of Darunavir.

Masaaki Takahashi et al [5]

has described a HPLC method to validate the determination

of plasma DRV concentrations, a simple procedure for simultaneous determination of seven HIV

protease inhibitors and efavirenz. The calibration curve was linear (range of 0.13 to 10.36

μg/ml). The average accuracy ranged from 100.7 to 105.6%. Both the interday and intraday

coefficients of variation were less than 6.7%, which was similar to or much lower than

previously reported values by the LC/MS/MS method. It is concluded that HPLC can be used to

determine plasma DRV concentrations and routinely in the clinical setting; thus, this HPLC

method enables further study of DRV pharmacokinetics in conventional hospital laboratories

Lauriane Goldwirt et al [6]

has developed and validated a precise and accurate high-

performance liquid chromatography (HPLC) method with UV detection for Darunavir, a peptidic

protease inhibitor. An internal standard, methylclonazepam, was added to 100 μL of plasma

before a solid-phase extraction on C18 Bond Elute column. The eluted solutions were evaporated

to dryness and reconstituted with 100 μL of mobile phase before being injected in the

chromatographic system. The separation was performed on a C8 column using an acetonitrile

270

and ultrapure water mixture (40:60, v/v) as mobile phase. All compounds were detected at a

wavelength of 266 nm. The method was linear and validated over a concentration range of 0.25–

20 mg/L. The within-day precision, ranged from 3.0 to 7.9%, while the within-day accuracy

ranged from −11.4 to 0.5%. The between day precision and accuracy were respectively less than

13.7 and −11.4%. The mean recovery was 75.7% for Darunavir and 66.7% for

methylclonazepam. This method provides a useful tool for therapeutic drug monitoring in HIV

patients.

Ivy Song et al [7]

has conducted a pharmacokinetic work to determine the Effect of

Lopinavir/Ritonavir and Darunavir/Ritonavir on the HIV Integrase Inhibitor S/GSK1349572 in

Healthy Participants. S/GSK1349572 is an unboosted, once-daily integrase inhibitor with a novel

resistance profile. As standard of care for patients infected with HIV is combination therapy, the

potential interaction between S/GSK1349572 and ritonavir-boosted protease inhibitors was

evaluated. In an open-label, repeat-dose, 2-period, 2-sequence crossover study in healthy

participants, S/GSK1349572 was administered at 30 mg once daily for 5 days, followed by

randomization to lopinavir/ritonavir 400/100 mg twice daily or Darunavir/ritonavir 600/100 mg

twice daily co administered with S/GSK1349572 30 mg once daily for 14 days. There was no

washout between periods. Serial pharmacokinetic (PK) samples and safety assessments were

obtained throughout the study. Thirty of 31 participants completed the study (15 participants per

group). Treatment comparisons of steady-state S/GSK1349572 PK parameters demonstrated that

co administration of lopinavir/ritonavir had no significant effect on steady-state PK of

S/GSK1349572. Coadministration of Darunavir/ritonavir resulted in a nonclinically significant

reduction in steady-state plasma S/GSK1349572 exposures. Plasma S/GSK1349572 AUC (0-τ),

Cmax, and Cτ decreased by 22%, 11%, and 38%, respectively, on average. S/GSK1349572 was

well tolerated with no serious adverse events (AEs) or withdrawals due to drug-related AEs. The

most frequent drug-related AEs were diarrhea, dizziness, and headache. No dosage adjustment

for S/GSK1349572 is required when used with lopinavir/ritonavir or Darunavir/ritonavir.

D'Avolio A et al[8]

has developed and validated a new method using high performance

liquid chromatography coupled with electrospray mass spectrometry (HPLC-MS) for the

quantification of plasma concentration of the new protease inhibitors Darunavir (DRV) and other

11 antiretroviral agents (ritonavir, amprenavir, atazanavir, lopinavir, saquinavir, indinavir,

271

nelfinavir and its metabolite M-8, nevirapine, efavirenz and tipranavir). A simple protein

precipitation extraction procedure was applied on 50 microl of plasma aliquots and

chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a

gradient (acetonitrile and water with formic acid 0.05%) on a C-18 reverse phase analytical

column with 25 min of analytical run. Calibration curves were optimized according to expected

ranges of drug concentrations in patients, and correlation coefficient (r2) was higher than 0.998

for all analytes. Mean intra- and inter-day precision (relative standard deviation %) for all

compounds were 8.4 and 8.3%, respectively, and mean accuracy (% of deviation from nominal

level) was 3.9%. Extraction recovery ranged within 93 and 105% for all drugs analyzed. This

novel HPLC-MS methodology allows a specific, sensitive and reliable determination of DRV

and 11 other antiretrovirals. In our hand, it was used to measure DRV and ritonavir plasma

concentration in HIV-positive patients, and it is now successfully applied for routine therapeutic

drug monitoring and pharmacokinetics studies.

Bhavini N. Patel et al

[9] has developed and validated a quantitative high-performance

thin-layer chromatography (HPTLC) method for determination of Darunavir ethanolate (DRV)

in tablets. DRV from the formulations was separated and identified on silica gel 60 F254 HPTLC

plates with toluene-ethyl acetate-methanol 7.0:2.0:1.0 (ν/ν) as mobile phase. The plates were

developed to a distance of 8 cm. Quantification was performed at λ = 267 nm. Well-resolved

bands were obtained for DRV. The method was validated for specificity, precision, robustness,

and accuracy. The calibration plot for DRV standard was linear in the range 250–1750 ng per

band with r = 0.9994, slope = 0.4253, and intercept = 44.81. The limits of detection and

quantification were 15.28 and 45.84 ng per band, respectively. The method is selective, sensitive,

and specific, with potential application in pharmaceutical analysis.

Jose Molto et al[10]

has described a Open-label, randomized pilot study in HIV-infected

patients on Darunavir/ritonavir 600/100 mg twice daily (viral load <50 copies/ml; Darunavir vIQ

>2). Thirty patients were randomized to Darunavir/ritonavir 900/100 mg once daily (once-daily

group, n=15) or 600/100 mg twice daily (twice-daily group, n=15). Viral load, blood chemistry,

and Darunavir and ritonavir trough plasma concentrations (Ctrough) were determined up to 48

weeks. If the Darunavir vIQ fell to <1.5, the dosage was switched to 600/100 mg twice daily.

The primary end point was the percentage of 48-week treatment failure. Patients had taken a

272

mean 11.6 (SD ±3.9) antiretroviral regimens before Darunavir/ritonavir administration. The

proportion of patients without 48-week treatment failure was 86.7% in both groups. The median

(interquartile range [IQR]) Darunavir Ctrough decreased from 3.09 mg/l (IQR 2.43–3.93) at

baseline to 1.60 mg/l (IQR 1.25–2.04) at week 48 (P=0.001) in the once-daily group. Three

once-daily group patients switched to Darunavir/ritonavir 600/100 mg twice daily. Fewer

patients had triglyceride levels >200 mg/dl at week 48 in the once-daily group (20.0%) than in

the twice-daily group (20.0% versus 57.1%; P=0.046). Treatment simplification to Darunavir/-

ritonavir 900/100 mg once daily guided by the Darunavir vIQ in treatment-experienced HIV-

infected patients receiving Darunavir/ritonavir 600/100 mg twice-daily seems to be safe enough

to be tested in adequately powered clinical trials.

273

10.3. EXPERIMENTAL

10.3.1. Instrumentation

Peak HPLC containing LC 20AT pump and variable wavelength programmable UV-

Visible detector and Rheodyne injector was employed for investigation. The chromatographic

analysis was performed on a Chromosil C18 column (250 mm × 4.6 mm, 5µm). Degassing of the

mobile phase was done using a Loba ultrasonic bath sonicator. A Denwar Analytical balance was

used for weighing the materials.

10.3.2. Chemicals and Solvents

The reference sample of Darunavir (API) was obtained from Jollc. The Formulation

PREZISTA (Darunavir) was purchased from the local market. Methanol, Water used were of

HPLC grade and purchased from Merck Specialties Private Limited, Mumbai, India.

10.3.3. The mobile phase

A mixture of Acetontrile: Methanol in the ratio of 90:10 v/v was prepared and used as

mobile phase.

10.3.4. Standard solution of the drug

For analysis 100 ppm standard solution was prepared, required concentrations were

obtained from 100 ppm solution by appropriate dilution.

10.3.5. Sample (Capsule) solution

The formulation tablets of Darunavir (PREZISTA - 75 mg) were crushed to give finely

powdered material. From the Powder prepared a 30 ppm solution with mobile phase and then

filtered through Ultipor N66 Nylon 6, 6 membrane sample filter paper.

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10.4. METHOD DEVELOPMENT

For developing the method (as described in chapter 1 and 2), a systematic study of the

effect of various factors was undertaken by varying one parameter at a time and keeping all other

conditions constant. Method development consists of selecting the appropriate wave length and

choice of stationary and mobile phases. The following studies were conducted for this purpose.

10.4.1. Detection wavelength

The spectrum of 10ppm solution of the Darunavir in methanol was recorded separately

on UV spectrophotometer. The peak of maximum absorbance wavelength was observed. The

spectra of Darunavir were showed maximum absorbance at 271nm.

10.4.2. Choice of stationary phase

Preliminary development trials have performed with octadecyl columns with different

types, configurations and from different manufacturers. Finally the expected separation and peak

shapes were obtained on Chromosil C18 (250 mm x 4.6 mm, 5μm) column.

10.4.3. Selection of the mobile phase

In order to get sharp peak, low tailing factor and base line separation of the separation of

the components, a number of experiments were carried out by varying the composition of

various solvents and flow rate. To have an ideal separation of the drug under isocratic conditions,

mixtures of solvents like methanol, water and Acetonitrile with or without different buffers

indifferent combinations were tested as mobile phases on a Chromosil C18 column. A mixture of

Acetonitrile : Methanol in the ratio of 90:10 v/v was proved to be the most suitable of all the

combinations since the chromatographic peak obtained was better defined and resolved and

almost free from tailing.

10.4.4. Flow rate

Flow rates of the mobile phase were changed from 0.5 – 1.5 mL/min for optimum

separation. A minimum flow rate as well as minimum run time gives the maximum saving on the

275

usage of solvents. It was found from the experiments that 1.0 mL/min flow rate was ideal for the

successful elution of the analyte.

10.4.5. Optimized chromatographic conditions

Chromatographic conditions as optimized above were shown in Table: 10.2 these

optimized conditions were followed for the determination of Darunavir in bulk samples and in its

Formulations. The chromatogram of standard (3ppm) shown in Figure 10.B

Mobile phase Acetonitrile : Methanol 90:10 v/v

Pump mode Isocratic

Mobile phase PH 6.2

Diluent Mobile phase

Column Chromosil C18 column (250 mm x 4.6

mm, 5μ)

Column Temp Ambient

Wavelength

271 nm

Injection Volume 20 μl

Flow rate 1.0 mL/min

Run time 5 min

Retention Time 2.59 min

Table 10.2: Optimized chromatographic conditions for estimation Darunavir

276

Figure10.B: Chromatogram of standard solution

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10.5. VALIDATION OF THE PROPOSED METHOD

The proposed method was validated (as described in the chapter 1 and 2) as per ICH

guidelines . The parameters studied for validation were specificity, linearity, precision, accuracy,

robustness, system suitability, limit of detection, limit of quantification, and solution stability.

10.5.1. Specificity

The specificity of method was performed by comparing the chromatograms of blank,

standard and sample (Prepared from Formulation). It was found that there is no interference due

to excipients in the tablet formulation and also found good correlation between the retention

times of standard and sample. The specificity results are shown in Table 10.3

NAME OF THE SOLUTION Retention Time in Min

Blank No peaks

Darunavir (Standerd) 2.59

Darunavir (Sample) 2.56

Table 10.3: Specificity study

10.5.2 Linearity

Linearity was performed by preparing mixed standard solutions of Darunavir at different

concentration levels including working concentration mentioned in experimental condition i.e.

30ppm. Twenty micro liters of each concentration was injected in duplicate into the HPLC

system. The response was read at 271 nm and the corresponding chromatograms were recorded.

From these chromatograms, the mean peak areas were calculated and linearity plots of

concentration over the mean peak areas were constructed individually. The regressions of the

plots were computed by least square regression method. Linearity results were presented in Table

10.4.

278

Level Concentration of Darunavir

in ppm

Mean peak area

Level -1 10 66073

Level -2 20 112573.0

Level -3 30 169196.8

Level -4 40 214545.7

Level -5 50 281291.2

Range: 10-30ppm Slope

Intercept

Correlation coefficient

5324.09

9013.21

0.997

Table 10.4: Linearity results

Figure 10.C: On X axis concentration of sample, On Y axis peak area response

10.5.3. Precision Precision is the degree of repeatability of an analytical method under normal Operational

conditions. Precision of the method was performed as intraday precision, Inter day precision.

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10.5.3.1. Intraday precision

To study the intraday precision, six replicate standard solutions (50ppm) of Darunavir

were injected. The percent relative standard deviation (% RSD) was calculated and it was found

to be 1.068, which are well within the acceptable criteria of not more than 2.0. Results of system

precision studies are shown in Table 10.5

SAMPLE

CONC(PPM) INJECTION

no.

PEAKS

AREA

R.S.D(Acceptance

criteria ≤ 2.0%)

Darunavir

50

1 280344.9

1.068

2 281291.2

3 287555

4 286832.8

5 286435.8

6 284917.2

Table 10.5: System Precision (Intra Day)

10.5.3.2. Inter Day precision

To study the interday precision, six replicate standard solution (50ppm) of Darunavir was

injected on third day of sample preparation. The percent relative standard deviation (% RSD)

was calculated and it was found to be 0.560, which are well within the acceptable criteria of not

more than 2.0. Results of system precision studies are shown in Table 10.6

SAMPLE

CONC(PPM) INJECTION

No.

PEAKS AREA R.S.D(Acceptance

criteria ≤ 2.0%)

Darunavir

50

1 288032.7

0.560

2 287629.1

3 290361.9

4 290419.7

5 291211.5

6 287647.5

Table 10.6: System Precision (Inter Day)

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10.5.4. Accuracy

The accuracy of the method was determined by standard addition method. A known

amount of standard drug was added to the fixed amount of pre-analyzed tablet solution. Percent

recovery was calculated by comparing the area before and after the addition of the standard drug.

The standard addition method was performed at 50%, 100% and 150% level of 20ppm. The

solutions were analyzed in triplicate at each level as per the proposed method. The percent

recovery and % RSD was calculated and results are presented in Table 10.7 Satisfactory

recoveries ranging from 99.0 to 102.0 were obtained by the proposed method. This indicates that

the proposed method was accurate.

Level Amount of Darunavir

spiked (ppm)

Amount of Darunavir

recovered(ppm)

% Recovery % RSD

50 %

30 29.95 99.83

0.440 30 29.9 99.66

30 29.7 99.00

100%

40 39.8 99.5

0.318 40 40.02 100.05

40 39.8 99.5

150%

50 49.5 99.0

0.696 50 49.5 99.0

50 50.1 100.2

Mean % of

recovery

99.52

Mean

RSD =

0.484

Table 10.7: Percentage RSD and % Recovery

10.5.5. Robustness

The robustness study was performed by slight modification in flow rate of Mobile phase,

pH of the buffer and composition of the mobile phase. Darunavir at 40 ppm concentration was

analyzed under these changed experimental conditions. It was observed that there were no

marked changes in chromatograms, which demonstrated that the developed method was robust in

nature. The results of robustness study are shown in Table 10.8

281

Condition Mean area % assay % difference

Unaltered 214545.7 100.0 0.0

Flow rate at 0.8 mL/min

Flow rate at 1.2mL/min

215328.6

215124.8

100.36

100.26

0.36

0.26

Mobile phase:

MEOH: Water

75% 25%

85% 15%

213845.5

214853.0

99.67

100.14

0.33

0.14

pH of mobile phase at 7.3 2140537.5 99.77 0.23

pH of mobile phase at 7.7 213452.9 99.49 0.51

Table 10.8: Robustness

10.5.6. System suitability

System suitability was studied under each validation parameters by injecting six

replicates of the standard solution 2 ppm). The results obtained were within acceptable limits

(Tailing factor ≤2 and Theoretical plate’s ≥2000) and are represented in Table 10.9. Thus, the

system meets suitable criteria.

Parameter Tailing factor Theoretical plates

Specificity study 1.23 5069.89

Linearity study 0.95 5368.86

Precision study 1.21 4709.73

Table 10.9: System Suitability

10.5.7. Limit of detection and Limit of quantification

Limit of detection (LOD) is defined as the lowest concentration of analyte that gives a

detectable response. Limit of quantification (LOQ) is defined as the lowest Concentration that

can be quantified reliably with a specified level of accuracy and Precision. For this sample was

dissolved by using Mobile Phase and injected until peak was disappeared. After 0.6ppm dilution,

Peak was not clearly observed. So it confirms that 0.6ppm is limit of Detection and 0.198ppm

282

dilution is Limit of Quantification. For this study six replicates of the analyte at lowest

concentration were Measured and quantified. The LOD and LOQ of Darunavir are given in

Table 10.10

parameter Measured volume

Limit of Quantification 0.198ppm

Limit of Detection 0.6ppm

Table 10.10: LOD And LOQ

Formulation:

For assay Darunavir (PREZISTA - 75 mg) 20 tablets were weigh and calculate the

average weight. Accurately weigh and transfer the sample equivalent to 10mg of Darunavir in to

a 10ml volumetric flask. Add diluent and sonicate to dissolve it completely and make volume up

to the mark with diluents. Mix well and filter through 0.45um filter. Further pipette 1ml of the

above stock solution into a 10ml volumetric flask and dilute up to mark with diluents and finally

10 ppm were prepared. Mix well and filter through 0.45um filter. An aliquot of this solution was

injected into HPLC system. Peak area of Darunavir was measured for the determination.

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10.6. RESULTS AND DISCUSSION

Having optimized the efficiency of a chromatographic separation the quality of the

chromatography was monitored by applying the system suitability tests. It was assessed by

replicate analysis of two injections of the drug at a concentration of 30ppm. The acceptance

criterion was ± 2% for the percent coefficient of variation (%CV) for the peak area and retention

times for Darunavir. The number of theoretical plates should not be less than 2500 and the tailing

factor should not be more than 2.0. The peak purity of Darunavir was assessed by comparing the

retention time (Rt) of standard and the sample. Good correlation was obtained between the Rt of

standard and sample. A chromatogram obtained from reference substance solution is presented.

System suitability parameters were shown in Table: 10.9

Linearity was studied by preparing standard solutions at different concentration

levels. The linearity y ranges for Darunavir found to be 10-50ppm. The regression equation for

LP and AT were found to be y = 9013x+5324 with coefficient of correlation, (r) 0.997. The

results of Linearity studies are shown in Table: 10.4 and the linearity curve were shown in

Figure: 10.C. Precision was evaluated by carrying out six independent sample preparation of a

single lot of formulation. The sample solution was prepared in the same manner as described in

sample preparation. Percentage relative standard deviation (%RSD) was ( for Intradey-1.068, For

Interday-0.560) found to be less than 2% for within a day and day to day variations, which

proves that method is precise.

Known amounts of standard Darunavir added to pre-analyzed samples and were

subjected to the proposed HPLC method at 50%, 100% and 150% to evaluate the degree of

accuracy. . Results of recovery studies are shown range 99.00-101.45%. The mean recovery data

obtained for each level as well as for all levels combined (Table 10.7) were within 2.0% of the

label claim for the active substance with an R.S.D. < 2.0%, which satisfied the acceptance

criteria set for the study.

To evaluate the robustness of the developed RP-HPLC method, small deliberate

Variations in the optimized method parameters were done. The effect of change in flow rate, pH

and mobile phase ratio on the regent ion time and tailing factor were studied. The values for

284

proposed method are well within acceptance limits of 98-102%, with a RSD of less than 2.0%.

Above experiments indicated that the method is rugged and provides consistent and reliable

results. Limit of detection (LOD) and Limit of quantification (LOQ) were 0.65ppm and 24 ng/ml

respectively. Tablets were evaluated for the amount of Darunavir present in the formulation.

Each sample was analyzed in triplicate and the amount of was Darunavir 21.2 %.

A rapid, specific isocratic HPLC method has been developed for the determination of

Darunavir using a UV detector. The method was validated for accuracy, precision, linearity,

specificity & stability, limit of detection & limit of quantization and robustness & ruggedness.

The method uses a simple mobile phase composition, easy to prepare with little or no variation.

The rapid run time of 10 min and the relatively low flow rate allows the analysis of large number

of samples with less mobile phase that proves to be cost-effective. Efficient UV detection at 271

nm was found to be suitable without any interference from injectable solution excipients or

solvents. Statistical analysis of the results has been carried out revealing that the proposed

method is simple, sensitive and reproducible and hence can be used in routine for simultaneous

determination of Darunavir in bulk as well as in pharmaceutical preparations.

285

10.7. BIBILOGRAPHY

1. Rodger D MacArthura; “Darunavir: promising initial results”, doi:10.1016/S0140-

6736(07)60499-1

2. Ghosh AK, Dawson ZL, Mitsuya H "Darunavir, “A conceptually new HIV-1

protease inhibitor for the treatment of drug-resistant HIV". Bioorg. Med. Chem;

2007; 15(24):7576–80.

3. “Darunavir-ritonavir more effective than Lopinavir-ritonavir in HIV infected,

treatment-experienced patients”, The Lancet; 2007, 370, article URL

4. “New RP-HPLC method for the determination of Darunavir in Tablet dosage

form”. Asian J. Pharm. Res; 2011; 1(1): 10-14.

5. “The Validation of Plasma Darunavir Concentrations Determined by the HPLC

Method for Protease Inhibitors”, Biological & Pharmaceutical Bulletin; 2007;30(

10): 1947-1949.

6. “Quantification of Darunavir (TMC114) in human plasma by high-performance

liquid chromatography with ultra-violet detection”; Journal of Chromatography

B” 2007; 857(2, 1): 327-331.

7. “The Effect of Lopinavir/Ritonavir and Darunavir/Ritonavir on the HIV Integrase

Inhibitor S/GSK1349572 in Healthy Participants”; J Clin Pharmacol; 2011; 51(2):

237-242.

8. “HPLC-MS method for the simultaneous quantification of the new HIV protease

inhibitor Darunavir, and 11 other antiretroviral agents in plasma of HIV-infected

patients”; J Chromatogr B Analyt Technol Biomed Life Sci; 2007; 859(2): 234-40.

9. “A simple and sensitive HPTLC method for quantitative analysis of Darunavir

ethanolate tablets”; JPC - Journal of Planar Chromatography - Modern TLC;

2011; 24: 232-235.

10. “Treatment simplification to once daily Darunavir/ritonavir guided by the

Darunavir inhibitory quotient in heavily pretreated HIV-infected patients”,

Antiviral Therapy; 2010; 15:219-25.