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CHAPTER 4

STUDIES ON SELECTED DRUGS AND THEIR METHOD

DEVELOPMENT AND VALIDATION BY HPLC METHOD IN

PHARMACEUTICAL DOSAGE FORM

4.1. DRUG PROFILE

EFAVIRENZ91-94:

Dosage Form: Capsule, gelatine coated tablet, film coated tablet. (Sustiva - 600mg orally

once daily).

Chemical Name: (4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl)-1H-3,1-

benzoxazin-2-one

Chemical Structure:

Figure 4.1.1: Structure of Efavirenz

Molecular Formula : C14H9ClF3NO2

Molecular Weight : 315.675g/mol

Physical Form : It is a white to off-white crystalline powder.

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Storage: Efavirenz should be stored at room temperature, 15 C to 30 C (59 F to 86 F).

Solubility: Insoluble in water, soluble in lower alcohol (methanol)

Category:

Anti-HIV Agents

Non nucleoside Reverse Transcriptase Inhibitors

Reverse Transcriptase Inhibitors

Clinical Pharmacology:

Mechanism of Action:

Efavirenz inhibits the activity of viral RNA-directed DNA polymerase (i.e.,

reverse transcriptase). Antiviral activity of Efavirenz is dependent on intracellular

conversion to the active triphosphorylated form. The rate of Efavirenz phosphorylation

varies, depending on cell type. It is believed that inhibition of reverse transcriptase

interferes with the generation of DNA copies of viral RNA, which, in turn, are necessary

for synthesis of new virions. Intracellular enzymes subsequently eliminate the HIV

particle that previously had been uncoated, and left unprotected, during entry into the

host cell. Thus, reverse transcriptase inhibitors are virustatic and do not eliminate HIV

from the body. Even though human DNA polymerase is less susceptible to the

pharmacologic effects of triphosphorylated efavirenz, this action may nevertheless

account for some of the drug's toxicity.

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Pharmacokinetics:

Absorption

Peak efavirenz plasma concentrations of 1.6-9.1 μM were attained by 5 hours

following single oral doses of 100 mg to 1600 mg administered to uninfected volunteers.

Dose-related increases in Cmax and AUC were seen for doses up to 1600 mg; the

increases were less than proportional suggesting diminished absorption at higher doses.

Distribution

Efavirenz is highly bound (approximately 99.5-99.75%) to human plasma

proteins, predominantly albumin. In HIV-1 infected patients (n=9) who received

SUSTIVA 200 to 600 mg once daily for at least one month, cerebrospinal fluid

concentrations ranged from 0.26 to 1.19% (mean 0.69%) of the corresponding plasma

concentration. This proportion is approximately 3-fold higher than the non-protein-bound

(free) fraction of efavirenz in plasma.

Metabolism

Studies in humans and in vitro studies using human liver microsomes have

demonstrated that efavirenz is principally metabolized by the cytochrome P450 system to

hydroxylated metabolites with subsequent glucuronidation of these hydroxylated

metabolites. These metabolites are essentially inactive against HIV1. The in vitro studies

suggest that CYP3A and CYP2B6 are the major isozymes responsible for efavirenz

metabolism.

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Efavirenz has been shown to induce CYP enzymes, resulting in the induction of

its own metabolism. Multiple doses of 200-400 mg per day for 10 days resulted in a

lower than predicted extent of accumulation (22-42% lower) and a shorter terminal half-

life of 40-55 hours (single dose half-life 52-76 hours).

Elimination

Efavirenz has a terminal half-life of 52-76 hours after single doses and 40-55

hours after multiple doses. A one-month mass balance/excretion study was conducted

using 400 mg per day with a 14C-labeled dose administered on Day 8. Approximately 14-

34% of the radiolabel was recovered in the urine and 16-61% was recovered in the feces.

Nearly all of the urinary excretion of the radiolabeled drug was in the form of

metabolites. Efavirenz accounted for the majority of the total radioactivity measured in

feces.

Indication:

Efavirenz is used to treat HIV infection. It is never used alone and is always given

in combination with other drugs. The decision on when to start treatment should take into

account CD4 count, HIV viral load, treatment history, resistance profiles and patient

preference.

Adverse Effects:

Psychiatric symptoms, including insomnia, nightmares, confusion, memory loss,

and depression, are common and more serious symptoms such as psychosis may

occur in patients with compromised liver or kidney function.

Rash, nausea, dizziness and headache may occur.

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4.2 DRUG PROFILE:

LAMIVUDINE96-100:

Dosage Form: (Epivir) Tablets – 150mg and 300mg, Oral Solution (10mg per ml).

Chemical Name: 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-

dihydropyrimidin-2-one

Chemical Structure:

N

N

O

O

S

HO

NH2

Figure 4.2.1: Structure of Lamivudine

Molecular Formula : C8H11N3O3S

Molecular Weight : 229.26 g/mol

Physical Form : White crystalline powder.

Storage : Store in tightly closed bottles at 25°C (77°F).

Solubility : Soluble in water and sparingly soluble in methanol.

Category:

Lamivudine has been used for treatment of chronic hepatitis B at a lower dose than

for treatment of HIV. It improves the seroconversion of e-antigen positive hepatitis B and

also improves histology staging of the liver. Long term use of lamivudine unfortunately leads

to emergence of a resistant hepatitis B virus (YMDD) mutant. Despite this, lamivudine is still

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used widely as it is well tolerated. It is a nucleoside analog reverse transcriptase inhibitor

(nRTI).

Clinical Pharmacology:

Mechanism of Action:

Lamivudine enters cells by passive diffusion and is phosphorylated to its active

metabolite, lamivudine triphosphate. Lamivudine triphosphate competes with

deoxycytidine triphosphate for binding to reverse transcriptase, and incorporation into

DNA results in chain termination. It has very low affinity for human alpha and omega

DNA polymerases, moderate affinity for beta DNA polymerase, and higher affinity for

gamma DNA polymerase.

Pharmacokinetics:

Absorption and Bioavailability:

Lamivudine was rapidly absorbed after oral administration in HIV-1-infected

patients. Absolute bioavailability in 12 adult patients was 86% ± 16% (mean ± SD) for

the 150-mg tablet and 87% ± 13% for the oral solution. After oral administration of 2

mg/kg twice a day to 9 adults with HIV-1, the peak serum lamivudine concentration

(Cmax) was 1.5 ± 0.5 mcg/mL (mean ± SD). The area under the plasma concentration

versus time curve (AUC) and Cmax increased in proportion to oral dose over the range

from 0.25 to 10 mg/kg.

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Distribution:

The apparent volume of distribution after IV administration of lamivudine to 20

patients was 1.3 ± 0.4 L/kg, suggesting that lamivudine distributes into extravascular

spaces. Volume of distribution was independent of dose and did not correlate with body

weight.

Binding of lamivudine to human plasma proteins is low ( < 36%). In vitro studies

showed that over the concentration range of 0.1 to 100 mcg/mL, the amount of

lamivudine associated with erythrocytes ranged from 53% to 57% and was independent

of concentration.

Metabolism:

Metabolism of lamivudine is a minor routeofelirhination. Inman, the only known

metabolite of lamivudine is the trans-sulfoxide metabolite. Within 12 hours after a single

oral dose of lamivudine in 6 HIV-1-infected adults, 5.2% ± 1.4% (mean ± SD) of the

dose was excreted as the trans-sulfoxide metabolite in the urine. Serum concentrations of

this metabolite have not been determined.

Elimination:

The majority of lamivudine is eliminated unchanged in urine by active organic

cationic secretion. In 9 healthy subjects given a single 300-mg oral dose of lamivudine,

renal clearance was 199.7 ± 56.9 mL/min (mean ± SD). In 20 HIV-1-infected patients

given a single IV dose, renal clearance was 280.4 ± 75.2 mL/min (mean ± SD),

representing 71% ± 16% (mean + SD) of total clearance of lamivudine.

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Indication:

Lamivudine is a nucleoside analogue indicated in combination with other

antiretroviral agents for the treatment of human immunodeficiency virus (HIV-1)

infection.

Adverse Effects:

Lactic acidosis and severe hepatomegaly with steatosis , Hepatic decompensation

in patients co-infected with HIV-1 and Hepatitis C and Pancreatitis and headache,

nausea, malaise, fatigue, nasal signs and symptoms, diarrhea cough.

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4.3 DRUG PROFILE:

TENOFOVIR DISOPROXIL FUMARATE101-106:

Dosage Form: Tablets (Viread) – 150, 200, 250, 300 mg, Oral Powder: 40mg per 1g.

Chemical Name: ({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-

yl]oxy}methyl)phosphonic acid.

Chemical Structure:

Figure 4.3.1: Structure of Tenofovir disoproxil fumarate

Molecular Formula : C9H14N5O4P

Molecular Weight : 287.213g/mol

Physical Form : White to off-white crystalline powder.

Storage : Store Tenofovir DF at 59°F to 86°F (15°C to 30°C).

Solubility : Soluble in water.

Drug Category:

Anti-HIV Agents

Nucleoside and Nucleotide Reverse Transcriptase Inhibitors

Reverse Transcriptase Inhibitors

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Clinical Pharmacology:

Mechanism of Action:

Tenofovir inhibits the activity of HIV reverse transcriptase by competing with the

natural substrate deoxyadenosine 5’-triphosphate and, after incorporation into DNA, by

DNA chain termination. Specifically, the drugs are analogues of the naturally occurring

deoxynucleotides needed to synthesize the viral DNA and they compete with the natural

deoxynucleotides for incorporation into the growing viral DNA chain. However, unlike

the natural deoxynucleotides substrates, NRTIs and NtRTIs (nucleoside/tide reverse

transcriptase inhibitors) lack a 3'-hydroxyl group on the deoxyribose moiety. As a result,

following incorporation of an NRTI or an NtRTI, the next incoming deoxynucleotide

cannot form the next 5'-3' phosphodiester bond needed to extend the DNA chain. Thus,

when an NRTI or NtRTI is incorporated, viral DNA synthesis is halted, a process known

as chain termination. All NRTIs and NtRTIs are classified as competitive substrate

inhibitors.

Pharmacokinetics:

Absorption:

VIREAD is a water soluble diester prodrug of the active ingredient tenofovir. The

oral bioavailability of tenofovir from VIREAD in fasted subjects is approximately 25%.

Following oral administration of a single dose of VIREAD 300 mg to HIV-1 infected

subjects in the fasted state, maximum serum concentrations (Cmax) are achieved in 1.0 ±

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0.4 hrs. Cmax and AUC values are 0.30 ± 0.09 μg/mL and 2.29 ± 0.69 μg-hr/mL,

respectively.

The pharmacokinetics of tenofovir are dose proportional over a VIREAD dose

range of 75 to 600 mg and are not affected by repeated dosing.

Distribution:

In vitro binding of tenofovir to human plasma or serum proteins is less than 0.7

and 7.2%, respectively, over the tenofovir concentration range 0.01 to 25 μg/mL. The

volume of distribution at steady-state is 1.3 ± 0.6 L/kg and 1.2 ± 0.4 L/kg, following

intravenous administration of tenofovir 1.0 mg/kg and 3.0 mg/kg.

Metabolism and Elimination:

In vitro studies indicate that neither tenofovir disoproxil nor tenofovir are

substrates of CYP enzymes. Following IV administration of tenofovir, approximately 70–

80% of the dose is recovered in the urine as unchanged tenofovir within 72 hours of

dosing. Following single dose, oral administration of VIREAD, the terminal elimination

half-life of tenofovir is approximately 17 hours. After multiple oral doses of VIREAD

300 mg once daily (under fed conditions), 32 ± 10% of the administered dose is

recovered in urine over 24 hours.

Tenofovir is eliminated by a combination of glomerular filtration and active

tubular secretion. There may be competition for elimination with other compounds that

are also renally eliminated.

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Indication:

HIV-1 infection: Tenofovir is indicated in combination with other antiretroviral

agents for the treatment of HIV-1 infection in adults and pediatric patients 2 years of age

and older.This indication is based on analyses of plasma HIV-1 RNA levels and CD4 cell

counts in controlled studies of tenofovir in treatment-naive and treatment-experienced

adults. It is indicated for the treatment of chronic hepatitis B in adults and pediatric

patients 12 years of age and older.

Adverse Effects: Nausea, vomiting, diarrhea, and asthenia.

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4.4. LITERATURE REVIEW

EFAVIRENZ:

Francois G107 et al., (2000) have describes a method for the simultaneous

determination of four licensed HIV protease inhibitors (amprenavir, nelfinavir, saquinavir

and ritonavir) and two novel non-nucleoside reverse transcriptase inhibitors (efavirenz

and delavirdine) in human plasma in a single run. Plasma samples (500 ml) were treated

by liquid–liquid extraction with methyl tert.-butyl ether. The compounds were separated

by reversed-phase liquid chromatography on a C 18 column with spectrophotometric

detection at 260 nm. The method is linear over the specific ranges investigated, accurate

(in accuracy, 11.7%) and showed intra-day and inter-day precision within the ranges of

0.9–7.0 and 1.9–8.8%.

Decosterd L. A108 et al., (2000) have a on-going study on the suitability of a

formal therapeutic drug monitoring (TDM) of antiviral drugs for improving the

management of HIV infection, a high-performance liquid chromatography method has

been developed to quantify simultaneously in plasma five HIV protease inhibitors (PIs)

(i.e., indinavir, amprenavir, saquinavir, ritonavir, nelfinavir) and the novel non-

nucleoside reverse transcriptase inhibitor efavirenz. After viral inactivation by heat (60°C

for 60 min), plasma (600 μl), with clozapine added as internal standard, is diluted 1:1

with phosphate buffer, pH 7 and subjected to a solid-phase extraction on a C18 cartridge.

Matrix components are eliminated with 2×500 μl of a solution of 0.1% H3PO4 neutralised

with NaOH to pH 7. PIs and efavirenz are eluted with 3×500 μl MeOH. The resulting

eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 μl

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50% MeOH. A 40-μl volume is subjected to HPLC analysis onto a Nucleosil 100, 5 μm

C18 AB column, using a gradient elution of MeCN and phosphate buffer adjusted to pH

5.15 and containing 0.02% sodium heptanesulfonate: 15:85 at 0 min→30:70 at 2

min→32:68 at 8 min→42:58 at 18 min→46:54 at 34 min, followed by column cleaning

with MeCN–buffer, pH 5.15 (90:10), onto which 0.3% AcOH is added. Clozapine,

indinavir, amprenavir, saquinavir, ritonavir, efavirenz and nelfinavir are detected by UV

at 201 nm at a retention time of 8.2, 13.0, 16.3, 21.5, 26.5, 28.7 and 31.9 min,

respectively. The total run time for a single analysis is 47 min, including the washing-out

and reequilibration steps. The calibration curves are linear over the range 100–10 000

ng/ml. The absolute recovery of PIs/efavirenz is always higher than 88%. The method is

precise with mean inter-day relative standard deviations within 2.5–9.8% and accurate

(range of inter-day deviations −4.6 to +4.3%). The in vitro stability of plasma spiked with

PIs/efavirenz at 750, 3000 and 9000 ng/ml has been studied at room temperature, −20°C

and +60°C.

Peter Langmann109 et al., (2001) have described a new high-performance liquid

chromatographic method for the determination of efavirenz in human plasma.

Quantitative recovery following liquid–liquid extraction with diethyl ether from 200 ml

of human plasma was achieved. Subsequently, the assay was performed with 67 mM

potassium dihydrogen phosphate–acetonitrile as a mobile phase, a XTerraRP 18 column

protected with a Phenomenex C 18 column and UV detection at 246 nm. Linear standard

curves were obtained for concentrations ranging from 25 to 15 000 ng/ ml. The calculated

intra- and inter-day coefficients of variation were below 10%.

193

Marıa Sarasa-Nacenta110 et al., (2001) have developed a simple, high-

performance liquid chromatographic method has been developed and validated for the

quantitative determination of efavirenz in human plasma. The method involved solid-

phase extraction of the drug and the internal standard (L-737,354) from 300 ml of human

plasma. The analysis was via UV detection at 250 nm using a reversed-phase C analytical

column 8 and a isocratic mobile phase consisting of phosphate buffer (pH 5.75)–

acetonitrile that resolved the drug and internal standard from endogenous matrix

components and potential coadministered drugs. Within- and between-day precisions

were less than 8.6% for all quality control samples. The lower limit of quantification was

0.1 mg/ ml. Recovery of efavirenz from human plasma was greater than 83%.

Eda Ross Montgomery111 et al., (2001) have developed a stability-indicating

high performance liquid chromatographic (HPLC) method was developed for the assay of

efavirenz, a non-nucleoside reverse transcriptase inhibitor used in the treatment of AIDS.

The HPLC method, which is used to determine potency in efavirenz capsules and related

substances in efavirenz drug substance and capsules, was validated per ICH guidelines.

This method, which uses a cyano column, is capable of separating efavirenz from its

trans-alkene reduction product.

Woolf E.J112 et al., (2002) have reported for the quantitative determination of

efavirenz in human plasma and the qualitative assessment of the stereochemical integrity

of efavirenz in post-dose human plasma samples are described. After the addition of an

internal standard, plasma samples were extracted with hexane–methylene chloride

194

(65/35, v/v%). The extracts were evaporated to dryness and reconstituted in mobile

phase. Upon exposure to UV light, the analyte was found to form fluorescent products;

the major fluorescent product was isolated and identified as a substituted quinoline. Thus,

the plasma extracts were analyzed via HPLC with post-column photochemical

derivatization and fluorescence detection. Reverse phase chromatography was used for

the quantitative assay, whereas chromatography with a column containing a chiral

stationary phase (dinitrobenzoyl leucine) was used for the stereochemical assessment.

The quantitative assay has been validated in the concentration range of 50–1000 ng/ml

using 0.5 ml samples. Analyte recovery was better than 89% at all points on the standard

curve. Intra-day precision was better than 5% C.V., while accuracy was between 95 and

104% of nominal over the range of the assay.

Kappelhoff B.S113 et al., (2003) have described a simple and rapid high-

performance liquid chromatographic method for the simultaneous quantification of

efavirenz and nevirapine in human plasma suitable for therapeutic drug monitoring is

described. Sample pre-treatment consisted of protein precipitation with acetonitrile and

subsequently dilution with distilled water. The drugs were separated from endogenous

compounds by isocratic reversed-phase high-performance liquid chromatography with

ultraviolet detection at 275 nm. The method was validated over the therapeutically

relevant concentration range of 0.05–15.0 mg/ml and 0.25–15.0 mg/ml for efavirenz and

nevirapine, respectively, using a volume of 100 ml of plasma. The calibration curves

were linear over this concentration range. Carbamazepine was used as internal standard.

The assay proved to be accurate (accuracies varied between 12.7 and 8.5%) and precise

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(intra- and inter-assay precisions were less than 5.9%). The tested batches of control

human plasma and frequently co-administered drugs did not interfere with the described

methodology. Efavirenz and nevirapine were stable under various relevant storage

conditions.

Katharina M. Rentsch114 (2003) have presented quantify a highly specific

method is presented, which is capable of quantifying the different proteinase inhibitors

(amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir) and non-nucleoside

reverse transcriptase inhibitors (efavirenz, nelfinavir). The antiretroviral agents were

separated and detected using LC–MS and atmospheric pressure chemical ionization.

After solid-phase extraction, the antiretrovirals were separated within 21 min using

gradient elution. The calibration range of each drug was linear including the expected

minimum and maximum concentrations measured in plasma after the administration of

the different drugs. All within-day and between-day coefficients of variation were below

10% and the recovery rates were between 34.8 and 124%. The respective quantification

limits were 1 mg/ l (indinavir), 10 mg/ l (amprenavir, efavirenz), 50 mg/ l (saquinavir),

90 mg/ l (nelfinavir), 200 mg/ l (nevirapine, ritonavir) and 250 mg/ l (lopinavir).

Dailly E115 et al., (2004) have described a global method is proposed for

therapeutic drug monitoring of atazanavir, a novel protease inhibitor and of all other

protease inhibitors (PI) and non nucleoside reverse transcriptase inhibitors (NNRTI)

which are currently used to treat HIV patients. All drugs are extracted after a liquid–

liquid extraction and separated on a C18 column with a binary gradient elution except

196

lopinavir which is separated without this gradient. The absorbance is measured at 259 nm

except for lopinavir (205 nm) and nevirapine (320 nm). This method is specific, accurate,

precise (the intra-day and inter-day imprecision and inaccuracy are lower than 15%) and

the limits of quantitation (0.40 mg/L for nevirapine, 0.10 mg/L for indinavir, 0.10 mg/l

for M8, 0.05 mg/L for amprenavir, 0.10 mg/L for nelfinavir, 0.10 mg/L for saquinavir,

0.10 mg/L for ritonavir, 0.10 mg/L for efavirenz, 0.10 mg/L for atazanavir and 0.20 mg/L

for lopinavir).

Dominique Breilh116 et al., (2004) have develop a selective and accurate assay

for the simultaneous quantitation of four protease inhibitors (PIs) (amprenavir (APV),

lopinavir (LPV), ritonavir (RTV) and saquinavir (SQV)) and a non-nucleoside reverse

transcriptase inhibitor (NNRTI) (efavirenz, EFV) in human peripheral blood

mononuclear cells using high-performance liquid chromatography–mass chromatography

(LC/MS) has been developed and validated. After liquid–liquid extraction, the

antiretroviral agents were separated within 15 min. The calibration curves of each drug

showed a good linearity in a range of concentration between 2 and 200 ng/3×106 cells for

amprenavir, lopinavir, efavirenz, 1.60 and 128 ng/3×106 cells for ritonavir and

saquinavir. Mean intra- and inter-assay coefficients of variation over the ranges of the

standard curves were less than 15% and mean extraction recoveries ranged 88.7–112.1%.

The limits of quantification were 2 ng/3×106 cells for amprenavir, lopinavir, efavirenz, 1

ng/3×106 cells for ritonavir and 1.6 ng/3×106 cells for saquinavir.

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Soumya Swaminathan117 et al., (2006) have developed a simple and rapid high

performance liquid chromatographic method for determination of efavirenz in human

plasma was developed. The method involved extraction of sample with ethyl acetate and

analysis using a reversed-phase C18 column (150 mm) with UV detection. The assay was

linear from 0.0625 to 10.0 μg/ml. The method was specific for efavirenz estimation and

the drug was stable in plasma up to one month at −20 °C. The average recovery of

efavirenz from plasma was 101%.

Paolo Ascenzi118 et al., (2006) have developed a HPLC–UV method to quantify

simultaneously seven HIV protease inhibitors (amprenavir, atazanavir, indinavir,

lopinavir, nelfinavir, ritonavir, and saquinavir; PIs), seven nucleoside reverse

transcriptase inhibitors (abacavir, didanosine, emtricitabine, lamivudine, stavudine,

zalcitabine, and zidovudine; NRTIs), and two non-nucleoside reverse transcriptase

inhibitors (efavirenz and nevirapine; NNRTIs) in human plasma. The volume of the

plasma sample was 600μL. This method involved automated solid-phase extraction with

Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone) and evaporation in a

water bath under nitrogen stream. The extracted samples were reconstituted with 100μL

methanol. Twenty microliters of these samples were injected into a HPLC–UV system,

the analytes were eluted on an analytical C18 SymmetryTM column (250mm×4.6mm

I.D.) with a particle size of 5μm. The mobile phase (0.01M KH2PO4 and acetonitrile)

was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single

analysis was 35 min, the anti-HIV drugs were detected by UV at 240 and 260 nm. The

calibration curves were linear up to 10 μg/mL. The absolute recovery ranged between 88

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and 120%. The in vitro stability of anti-HIV drugs (0.005–10 μg/mL) in plasma has been

studied at 24.0 ◦C. On these bases, a two to four analyte method has been tailored to the

individual needs of the HIV-infected patient.

Stefania Notari119 et al., (2008) have quantified a new MALDI-TOF/TOF

technology to abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine in

the plasma of HIVinfected patients, by standard additions analysis. Regression of

standard additions was linear over the whole anti-HIV concentration range explored

(1.00×10−2–1.00 p mol/μL). The absolute recovery ranged between 80% and 110%.

Values of the drug concentration determined by MALDI TOF/ TOF were in the range of

1.00×10−2–1.00 p mol/μL. The limit of quantification value was 1.00×10−2 pmol/μL for

abacavir, amprenavir, didanosine, efavirenz, nevirapine, and stavudine.

Appala Raju N120 et al., (2008) has developed a simultaneous stability indicating

RP-HPLC method for the estimation of Emtricitabine, Tenofovir disoproxil fumerate and

Efavirenz in tablet dosage form. Chromatography was carried on an Inertsil ODS 3V

column using gradient composition of 0.02M sodium dihydrogen orthophosphaste as

mobile phase A and mixture of Methanol and water in ratio of 85:15 as mobile phase B at

a flow rate of 1.5 ml/min with detection at 265 nm. The retention times of the

Emtricitabine, Tenofovir disoproxil fumerate and Efavirenz was about 5.875, 8.800 and

12.020 mins respectively. The detector response is linear from 8- 120μg/ml, 12-

180μg/ml, 20-360μg/ml of test concentration for Emtricitabine, Tenofovir and Efavirenz

respectively. The respective linear regression equation being Y=10175x-76883 for

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Emtricitabine, Y=6280.8x+219800 for Tenofovir disoproxil fumerate and

Y=1883.5x+323060 for Efavirenz. The limit of detection and Limit of quantification was

0.06, 0.07 and 0.08 μg/ml and 0.14, 0.12 and 0.15μg/ml for Emtricitabine, Tenofovir and

Efavirenz respectively. The percentage assay of Emtricitabine, Tenofovir disoproxil

fumerate and Efavirenz was 99.31, 99.77 and 100.20 % respectively and percentage

recovery for average of three different concentrations was 100.87%, 100.04% and

99.52% respectively.

Isadore Kanfer121 et al., (2009) have developed and validation of a quantitative

method for the analysis of EFV in plasma. A simple mobile phase consisting of 0.1 M

formic acid, acetonitrile and methanol (43:52:5) was pumped at a low flow rate of

0.3 ml/min through a reverse phase Phenomenex® Luna C18 (2) (5 μm, 150 mm × 2.0 mm

i.d.) column maintained at 40 °C. Diclofenac sodium was used as an internal standard

(IS) and EFV and IS were monitored at 247 nm and 275 nm, respectively. A simple and

rapid sample preparation involved the addition of mobile phase to 100 μl of plasma to

precipitate plasma proteins followed by direct injection of 10 μl of supernatant onto the

column. The procedures were validated according to international standards with good

reproducibility and linear response (r = 0.9990). The intra- and inter-day accuracies were

between 12.3 and 17.7% at the LLOQ and between −5.8 and 9.1% for the QC samples.

The intra- and inter-day precision of EFV determinations were 5.1 or less and 7.2% RSD

or less, respectively across the entire QC concentration range. Mean recovery based on

high, medium and low quality control standards ranged between 92.7 and 94.1% with

%RSD values better than 3%. Plasma samples were evaluated for short-term (ambient

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temperature for 6 h) and long-term (−10 ± 2 °C for 60 days) storage conditions and were

found to be stable.

Dailly. E122 et al., (2009) have developed a liquid chromatography–tandem mass

spectrometry assay for simultaneous determination of the plasma concentration of 11

antiretroviral agents (nevirapine, indinavir, atazanavir, amprenavir, saquinavir, ritonavir,

lopinavir, efavirenz, tipranavir, darunavir and maraviroc) has been developed. Sample

pre-treatment is limited to protein precipitation with a mixture of methanol and zinc

sulfate. After centrifugation the supernatant is injected in the chromatographic system,

which consists of on-line solid phase extraction followed by separation on a phenyl–

hexyl column.

Sharma R123 et al., (2010) have developed a Three UV spectrophotometric

methods, simultaneous equation method, multicomponent analysis (II) and derivative

spectroscopy method (III). The absorption maxima of the drugs were found to be 247,

259 and 272 nm, respectively for efavirenz, tenofovir disoproxil fumarate and lamivudine

in methanol:water (50:50) solvent system. Efavirenz, tenofovir disoproxil fumarate and

lamivudine obeyed Beer's law in the concentration range of 10-60, 5-30 and 5-30 μg/ml,

respectively. Results of analysis for all the three methods were analyzed an validated for

various parameters according to ICH guidelines.

Michelle Viljoen124 et al., (2010) have determined a novel and robust screening

method for efavirenz (EFV), in human saliva have been developed and validated based

on high performance liquid chromatography tandem mass spectrometry (LC–MS/MS).

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Sample preparation of the saliva involved solid-phase extraction (SPE) on C18 cartridges.

The analytes were separated by high performance liquid chromatography (Phenomenex

Kinetex C18, 150mm×3mm internal diameter, 2.6μm particle size) and detected with

tandem mass spectrometry in electrospray positive ionization mode with multiple

reaction monitoring. Gradient elution with increasing methanol (MeOH) concentration

was used to elute the analytes, at a flow-rate of 0.4 mL/min. The total run time was 8.4

min and the retention times for the internal standard (reserpine) was 5.4 min and for EFV

was 6.5 min. The calibration curves showed linearity (r2, 0.989–0.992) over the

concentration range of 3.125–100μg/L. Mean intra- and inter-assay relative standard

deviation, accuracy, mean extraction recovery, limit of detection (LOD) and limit of

quantification (LOQ) were 0.46–9.43%, 80–120%, 60% (±7.95), 1.84 and 6.11μg/L

respectively. The working range was defined as 6.25–100μg/L.

Lindsay B. Avery125 et al., (2010) have developed a combined UPLC–tandem

mass spectrometric (UPLC–MS/MS) technique have been validated for quantitation of

protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and

seminal plasma. The analytical method possesses capabilities for concentration

measurements of EFV ranging from 0.5 to 10,000 ng/ml with an accuracy (%dev) of

−5.2–8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients

of variation (r2) >0.98. The method employs a racemic fluorinated analog of EFV (F-

EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC

column via gradient elution with detection via negative ion multiple reaction monitoring

(MRM). EFV and F-EFV, respectively, were detected via the following MRM

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transitions: m/z 314.0 > 244.1 and m/z 298.0 > 227.9. The time required for the analysis

of each sample was 8.0 min. The analytical technique is capable of a reliable detection

limit of -15–20 fmol of EFV injected on column.

Poonam Tandon126 et al., (2010) have studied structural and spectral

characteristics of efavirenz by methods of Raman spectroscopy and quantum chemistry.

The optimized geometries, electronic charge distribution, dipole moments and

electrostatic potential surface were calculated by Hartree–Fock and Density functional

B3LYP method with the 6-311++G(d,p) basis sets. Electrostatic potential surfaces have

been mapped over the electron density isosurfaces to obtain information about the size,

shape, charge density distribution and chemical reactivity of the molecules.

Purnima D.H127 et al., (2010) have been established a stability-indicating high

performance liquid chromatographic (HPLC) method for analysis of Efavirenz in the

presence of the degradation products generated in the stress degradation study. The drug

was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal

decomposition. Extensive degradation was found to occur in alkaline medium and under

thermal stress. Minimum degradation was observed under acidic medium, in the

photolytic conditions and oxidative stress. Successful separation of drug from

degradation products formed under stress conditions was achieved on a C-8 column using

acetonitrile: potassium dihydrogen phosphate (pH 2.9, 25mM) - (60:40% v/v) as the

mobile phase. The flow rate was 1 mL min-1 and the detector was set at in a range of

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wavelength between 220nm to 390nm. The method was validated for linearity, range,

precision, accuracy, limit of quantification and limit of detection.

Anand Kumar Y128 et al., (2010) have developed a simple, sensitive, accurate,

precise and rapid ultraviolet (UV) spectrophotometric method for the estimation of

efavirenz in pure form, its formulations and stability samples. For the estimation of

efavirenz, solvent system employed was 1% w/v sodium lauryl sulphate (SLS) and

wavelength of detection was 247 nm. The developed method was used to estimate the

total drug content in two commercially available oral formulations of efavirenz and

recovery studies were also carried out. Sample recovery in both the formulations using

the above method was in good agreement with their respective labeled claims.

Deirdre Fox129 et al., (2011) have determined a simple and rapid isocratic, high

performance liquid chromatography (HPLC) assay employing solid phase extraction

(SPE) for the simultaneous determination of the anti HIV drug, efavirenz, the anti-

tuberculosis drug, rifampicin and the desacetyl metabolite of rifampicin in plasma from

HIV/tuberculosis infected patients has been developed. Using a Zorbax SB-Phenyl

reverse-phase analytical column with UV detection, good separation and detection of the

drugs was attained within a 10 min run time. Intra- and inter-assay precision RSD values

were found to be less than 15% at the concentrations examined (0.1–20 μg/mL). The

LOQ was found to be 0.1 μg/mL for each agent and the assay was found to generate a

linear response up to 20 μg/mL.

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Yazen Alnouti130 et al., (2011) have reported a simple, rapid, and sensitive ultra

performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method

for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir

(IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney,

lung, and spleen). These antiretroviral drugs are currently the cornerstones of common

therapeutic regimens for human immunodeficiency virus (HIV) infection.

Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile

in methanol and 7.5 mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC®BEH

Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used

as an internal standard. Detection was achieved by dual positive and negative ionization

modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray

ionization (ESI) source. The dynamic range was 0.2–1000 ng/mL for RTV, IDV, and

ATV, and 0.5–1000 for EFV. The method was validated and showed high and consistent

intra-day and inter-day accuracy and precision for all analytes. This method is used to

support the preclinical development studies of targeted- and sustained-release

combination ART (nanoART). The current data demonstrate a 1.5–4 fold increase in

serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice

when compared to native drug.

Pradeep kumar131 et al., (2011) have developed a rapid, precise, accurate,

specific and simple RP-HPLC method for the estimation of Efavirenz in its tablet form. A

High performance liquid chromatograph 10AT SHIMADZU- SPD10A, using

Phenomenex - Luna RP-18(2),250X4.6mm, 5 μm column, with mobile phase

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composition of Acetonitrile: Phosphate Buffer [58:42 %(v/v)] was used. The flow rate of

1.0 ml min-1 and effluent was detected at 247 nm. The retention time of Efavirenz was

4.611 minutes. Linearity was observed over concentration range of 500-10000ng ml/ml.

The Limit of detection and limit of quantification was found to be 157.63ng ml/ml and

477.68ng ml/ml respectively. The accuracy of the proposed method was determined by

recovery studies and found to be 98.240 to 101.170 %. The proposed method was

validated for various ICH parameters like linearity, limit of detection, limits of

quantification, accuracy, precision, range and specificity.

Pedro Jose R. N132 et al., (2011) have developed and validated a simple assay

method by high performance liquid chromatography for efavirenz tablets. The physical

chemical characteristics of efavirenz were investigated to developing the method.

Analyses were performed by an ultraviolet detector at a 252 nm wavelength, on a

reverse-phase column (C18, 250 mm x 3.9 mm, 10 μm), using an isocratic mobile phase

containing acetonitrile/water/orthophosphoric acid (70:30:0.1). The validation parameters

used were: selectivity, linearity, precision, accuracy, robustness, detection and

quantification limits, and all resulting data were treated by a statistical method.

Balamuralikrishna K133 et al., (2011) have developed a Reverse phase high

performance liquid chromatography methods for the simultaneous estimation of

Efavirenz, Lamivudine and Zidovudine in tablet dosage form. In reverse phase high

performance liquid chromatography analysis is carried out using Acetonitrile, Methanol

and 0.05M dipotassium hydrogen orthophosphate in the ratio of 40:40:20 v/v/v (pH was

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adjusted to 4.0 with o-phosphoric acid) as the mobile phase and Luna C18 (4.6 x 250

mm) column as stationary phase with detection wavelength of 259 nm. Linearity was

obtained in the concentration range of 100-200, 15-45 and 40-120 μg/ml for Efavirenz,

Lamivudine and Zidovudine, respectively.

Bhavsar D S134 et al., (2012) have developed a simple, precise, accurate, and

rapid reverse phase-high performance liquid chromatography (RP-HPLC) method with

UV-Visible detector and subsequently validated for the simultaneous determination of

tenofovir disoproxil fumarate (TDF), lamivudine (LAMI), and efavirenz (EFV) in their

combined tablet dosage form. The separation was based on the use of a Kromasil C18

analytical column (150 x 4.6 mm, i.d., 5 μm). The mobile phase consisted of a mixture of

70 volumes of methanol and 30 volumes of 10 mM phosphate buffer (pH 5.0). The

separation was carried out at 40°C temperature with a flow rate of 1 ml/min. Quantitation

was achieved with UV detection at 254 nm, with linear calibration curves at

concentration ranges of 1–6 μg/ml for TDF and LAMI and 2–12 μg/ml for EFV. The

recoveries obtained were 99.46-101.36% for LAMI, 99.57-101.42% for TDF, and 99.96-

100.87 for EFV. The method was validated according to International conference of

harmonisation guidelines in terms of accuracy, precision, specificity, robustness, limits of

detection and quantitation, and other aspects of analytical validation.

Venkata Raju Y135 et al., (2012) have developed a reverse phase stability

indicating HPLC method for the analysis of efavirenz bulk and pharmaceutical

formulations. The developed method was also utilized for in-vitro dissolution studies of

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efavirenz formulations. Acetonitrile and acetate buffer pH 3.4 was the mobile phase

(75:25% v/v), with retention time of 4.007 min at a flow rate of 1.5 mL/min detected at

292 nm wavelength. Linear regression analysis calibration plot showed an excellent

linearity between response and concentration in the range of 50-300 μgmL-1. The

regression co-efficient was 0.999 and the linear regression equation was y =

7780x+11159. Limits of detection (LOD) and quantification (LOQ) were 0.238 and 0.793

μgmL/ml respectively. The method was validated for accuracy, precision, specificity,

robustness, detection and quantification limits, in accordance with ICH guidelines. The

specificity of the method was ascertained by forced degradation studies by acid, alkali

hydrolysis, oxidation and thermal degradation methods. The degraded products were well

resolved from the analysis peak with significant differences at their retention time values.

Naga Sandhya B136 et al., (2012) have been devlopede a simple, specific,

accurate and precise reverse phase high performance liquid chromatography (RP-HPLC)

method, which can separate and quantitatively estimate lamivudine and efavirenz in

pharmaceutical dosage form. The chromatographic separation for lamivudine and

efavirenz was achieved with mobile phase containing 0.1 % triethylamine (pH adjusted to

5.11 with 0.1% orthophosphoric acid) and acetonitrile (30:70 % v/v), reverse phase

phenomenexR (Luna 5μ C18(2) 100A (250 × 4.60 mm i.d) column in isocratic mode at

room temperature and UV detection at 245 nm. The compounds were eluted at a flow rate

of 1.0 ml/min. The retention times of lamivudine and efavirenz were found to be 2.271 ±

0.177 min and 7.267 ± 0.513 min respectively. The above method was validated in

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terms of linearity, accuracy, precision, Limit of Detection (LOD), Limit of Quantification

(LOQ) etc. in accordance with ICH guide lines.

Nalini S137 et al., (2013) have developed and validated a simple, rapid, and

stability-indicating RP-HPLC method for a combination of tenofovir disoproxil fumarate

(TDF), emtricitabine (FTC), and efavirenz (EFV) with the help of a suitable statistical

sofware as an application tool for the quality by design. The drugs individually, and in

combination, were subjected to forced degradation (thermal, photolytic, hydrolytic, and

oxidative stress conditions) and accelerated stability studies (40 ± 1°C/75 ± 3% RH for

three months). Successful separation of combined drugs from degradation products was

achieved by gradient elution on a reverse-phase C18 column, using a mobile phase

containing phosphate buffer (pH 3.5): acetonitrile at 1.5 mL min−1 flow rate, detection

wavelength 256 nm, column oven temperature 250C, and injection volume 10 L.

Linearity was established in the range of 20–300 g mL−1, 24.5–367.5 g mL−1 and 60–

900 g mL−1 for FTC, TDF, and EFV, respectively.

Naga Sandhya B138 et al., (2013) have developed a simple, specific, accurate and

precise reversed phase high performance liquid chromatography (RP-HPLC) method for

the simultaneous estimation of lamivudine, didanosine and efavirenz in pharmaceutical

dosage form. The chromatographic separation for lamivudine, didanosine and efavirenz

was achieved with mobile phase containing water: tetrahydrofuran: acetonitrile (45.83:

20.83: 33.34 % v/v/v), Oyster BDS premium C18 (250 mm × 4.6 mm, 5 μm) analytical

column in isocratic mode at room temperature and UV detection at 245 nm. The

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compounds were eluted at a flow rate of 1.15 ml min-1. The retention times of

lamivudine, didanosine and efavirenz were found to be 2.01 ± 0.003, 3.01 ± 0.001 and

8.61 ± 0.002 min respectively. Different analytical performance parameters such as

linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantification

(LOQ), robustness and ruggedness were determined according to ICH guidelines. The

linear dynamic ranges were obtained from 15-90, 12.5-75 and 30-180 μg mL-1 for

lamivudine, didanosine and Efavirenz respectively. The mean recoveries obtained for

lamivudine, didanosine and efavirenz were 99.85, 99.78 and 99.94 % respectively. Limit

of detection and quantification for lamivudine 0.61 and 1.85 μg mL-1, for didanosine

0.43 and 1.31 μg mL-1 and for efavirenz 0.65 and 1.97 μg mL-1 respectively. The

developed method was free from interferences due to excipients present in formulation.

Sreekanth N139 et al., (2013) have develop a stability indicating RP-HPLC

method for quantitative determination of Emtricitabine ,Tenofovir DF and Efavirenz in

Pharmaceutical Dosage Form. Chromatographic separation was achieved through

gradient elution. Detection wavelength was monitored at 262nm. The retention time of

the Emtricitabine ,Tenofovir DF and Efavirenz was about 2.6, 5.4, 7.9 min respectively.

The developed method was validated as per ICH guidelines.

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4.5 LITERATURE REVIEW:

LAMIVUDINE:

Jean-Pierre Sommadossi140 et al., (2000) have developed and validated a high-

performance liquid chromatography (HPLC) method utilizing triple quadrupole mass

spectrometry (MS) detection for the simultaneous measurement of the intracellular

nucleoside 59-triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP),

and stavudine (d4T-TP). These compounds were extracted from patient peripheral blood

mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. Ion-

exchange solid phase extraction (SPE) followed by enzymatic digestion with alkaline

phosphatase was utilized to yield the measurable nucleoside forms of the nucleotides.

Reversed phase C-18 SPE with addition of a nucleoside internal standard, 39-azido-

29,39- dideoxyuridine (AzdU) allowed for the indirect measurement of the original 59-

triphosphate concentration by HPLC/MS/MS. Quantitation was performed from

calibration curves generated from authentic 59-triphosphate standards spiked in PBMCs

from healthy volunteers. Analytical range for the three 59-triphosphates was equivalent to

50–45,000 pg. Mean interassay accuracies for 3TC-TP, d4T-TP, and ZDV-TP (n . 90)

were 99.4%, 100.1%, and 108.0%, respectively. Mean interassay precisions (%C.V.) for

3TC-TP, d4T-TP, and ZDV-TP (n . 90) were 8.8%, 10.4%, and 8.2%, respectively.

Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3TC-TP, d4T-TP,

and ZDV-TP, respectively.

John A. Dunn141 et al., (2000) have developed and validated a high performance

liquid chromatography (HPLC) with tandem mass spectrometry (MS) for the

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simultaneous determination of clinically relevant levels of zidovudine (AZT) and

lamivudine (3TC) in human serum. The method incorporates a fully automated

ultrafiltration sample preparation step that replaces the solid-phase extraction step

typically used for HPLC with UV detection. The calibration range of the dual-analyte

LC-MS: MS method is 2.5–2500 and 2.5–5000 ng/m1 for AZT and 3TC, respectively,

using 0.25 ml of human serum. The lower limit of quantification was 2.5 ng/m1 for each

analyte, with a chromatographic run time of approximately 6 min.

Richard R. Tidwell142 et al., (2000) have developed and validated a HPLC–MS–

MS method to measure lamivudine and zidovudine simultaneously in small volumes of

human seminal plasma. Sample preparation was simple and rapid, requiring 25 ml of

sample, the use of isotopically labeled lamivudine and zidovudine as internal standards

and ultrafiltration through a molecular mass cut-off membrane. Lamivudine and its

internal standard were separated from zidovudine and its internal standard with isocratic

HPLC. Detection was carried out using tandem mass spectrometry.

Joanna J. Zheng143 et al., (2001) have quantify a new method for 29-deoxy-39-

thiacytidine (lamivudine, 3-TC), which incorporated the use of 3-isobutylmethylxanthine

as internal standard (I.S.) was developed and validated in human plasma, using HPLC

with UV absorbance detection. Using solid-phase extraction, 3-TC and I.S. were

selectively extracted from human plasma. Subsequently, chromatographic separation was

performed using a YMC phenyl column with ion-pair chromatography and detection at

270 nm. The method was validated over a concentration range of 10 to 5000 ng/ml using

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0.5 ml of human plasma. The extraction recovery for both 3-TC and I.S. was greater than

95%. The determination of inter- and intra-day precision (RSD) was less than 10% at all

concentration levels, while the inter- and intra-day accuracy (% difference) was less than

6%.

Sibel A. Ozkan144 et al., (2002) have proposed three methods for the

simultaneous determination of lamivudine and zidovudine. The first method depends on

first derivative UV spectrophotometry, with zero-crossing and peak-to-base

measurement. The first derivative amplitudes at 265.6 and 271.6 nm were selected for the

assay of lamivudine and zidovudine, respectively. The second method depends on first

derivative of the ratio-spectra by measurements of the amplitudes at 239.5 and 245.3 nm

for lamivudine and 225.1 and 251.5 nm for zidovudine. Calibration graphs were

established for 1–50μg/ml for lamivudine and 2–100μg/ml for zidovudine. In the third

method (HPLC), a reversed-phase column with a mobile phase of methanol:

water:acetonitrile (70:20:10 (v/v/v)) at 0.9 ml/min flow rate was used to separate both

compounds with a detection of 265.0 nm. Linearity was obtained in the concentration

range of 0.025–50μg/ml for lamivudine and 0.15–50μg/ml for zidovudine. All of the

proposed methods have been extensively validated.

James T. Stewart145 et al., (2002) have developed a new high-performance liquid

chromatography (HPLC) assay for the simultaneous determination of zidovudine

(AZT)/lamivudine (3TC)/nevirapine in human plasma. Plasma samples were treated

using a solid-phase extraction procedure. The compounds were separated using a mobile

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phase of 20 mM sodium phosphate buffer (containing 8 mM 1-octanesulfonic acid

sodium salt)–acetonitrile (86:14, v/v) with pH adjusted to 3.2 with phosphoric acid on an

octylsilane column (150×3.9 mm i.d.) with UV detection at 265 nm. Aprobarbital was

chosen as the internal standard (IS). The method was validated over the range of 57.6–

2880 ng/ml for AZT, 59.0–17 650 ng/ml for 3TC and 53.2–13 300 ng/ml for nevirapine.

The method was shown to be accurate, with intra-day and inter-day accuracy from 0.1 to

11% and precise, with intra-day and inter-day precision from 0.4 to 14%. Extraction

recoveries of the analytes and IS from plasma were higher than 92%.

Daniel R. Doerge146 et al., (2003) have described a transmission of HIV from

mother to infant can be effectively prevented by zidovudine (3-azido-3-deoxythymidine;

AZT) alone or in combination with other anti-retroviral drugs; however, significant

evidence for genotoxicity, including transplacental carcinogenicity in mice, has been

reported for AZT. A method, based upon solid phase extraction (SPE) in the 96-well

format, gradient liquid chromatography (LC), and electrospray mass spectrometry (MS),

was developed and validated to measure serum concentrations in maternal C57BL/6N

and fetal B6C3F1 mice of the nucleoside analogs AZT, lamivudine ((-)2,3-dideoxy-3-

thiacytidine; 3TC), and several metabolites selected based on importance in

detoxification and bioactivation reactions. After intravenous (IV) and oral dosing with

either 400 mg/kg AZT or 200 mg/kg 3TC, pharmacokinetics were determined for AZT,

AZT-5-glucuronide, 3-amino-3-deoxythymidine (AMT), AZT-5-phosphate, 3TC, and

3TC- 5-phosphate in serum of adult female mice. Pharmacokinetics were also determined

in spleen for AZT-5-phosphate and 3TC-5-phosphate following IV dosing. In addition, a

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preliminary assessment was made of placental transfer of AZT and 3TC and the presence

of metabolites in the fetal compartment.

Naser L. Rezk147 et al., (2003) have developed a accurate, sensitive and specific

reversed-phase high-performance liquid chromatography assay for the simultaneous

quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine,

lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside

reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new

Polarity dC C 18 silica column used in this method provides better resolution and peak

shape than all other columns tested. Also, four different ultraviolet wavelengths were

used for accurate and specific quantitation of the analytes. The method was validated

over the range of 10–10 000 ng/ml for all analytes except zalcitabine (10–5000 ng/ml).

This method is accurate (average accuracies of three different concentrations ranged from

97.2 to 105%), and precise (within- and between-day precision measures ranged from 0.5

to 5.1% and 0.5 to 5.6%, respectively).

Michael G. Bartlett148 et al., (2004) have reported a HPLC method for the

quantification of lamivudine (3TC) in rat plasma, amniotic fluid, placental and fetal

tissues has been developed, validated and applied to the study of the placental transport

of this drug in the pregnant rat. Placental and fetal tissues were processed using liquid-

liquid extraction enhanced by salting out the sample using a saturated solution of

ammonium sulfate. Plasma and amniotic fluid samples were processed by protein

precipitation using 2M perchloric acid. Reverse phase chromatography was performed

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using a phenyl column (5 μm, 150mm ×2mm i.d.) under a flow rate of 0.2 ml/min. The

mobile phase consisted of 5% methanol in 20mM dibasic phosphate buffer (pH 6). The

method was validated over the range from 0.1 to 50 μg/ml for plasma and amniotic fluid

and 0.2–50 μg/ml for the placental and fetal tissues.

Valentina Porta149 et al., (2005) have developed a simple, accurate, precise and

sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet

detection was developed to quantificate lamivudine (3-TC) in human plasma samples

from bioequivalence studies. 3-TC and stavudine (internal standard, I.S.) were extracted

from 0.5 ml of human plasma by acetonitrile protein precipitation. The method was

validated over a concentration range of 0.05–3.00 μg/ml and used in a bioequivalence

trial between two lamivudine formulations, to assess its usefulness in this kind of study.

FURP–lamivudine (Fundacao para o Remedio Popular, Brazil, as test formulation) and

Epivir® (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a

single 150 mg oral dose to 24 healthy volunteers of both genders. The dose was

administered after an overnight fast according to a two-way crossover design.

Bioequivalence between the products was determined by calculating 90% confidence

intervals (90% CI) for the ratio of Cmax, AUC0−t and AUC0−inf values for the test and

reference products, using logarithmic transformed data. The 90% confidence intervals for

the ratio of Cmax (0.86–1.06), AUC0−t (0.96–1.04) and AUC0−inf (0.97–1.05) values

for the test and reference products are within the 0.80–1.25 interval proposed by FDA

and EMEA. It was concluded that the two 3-TC formulations are bioequivalent in their

rate and extent of absorption, and thus, may be used interchangeably.

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Gholamreza Bahrami150 et al., (2005) have developed a simple, fast, and

sensitive high performance liquid chromatographic (HPLC) assay for quantitation of

lamivudine in human serum. Lamivudine is polar compound and its extraction from the

human serum in previously published HPLC methods involved either protein

precipitation or solid phase extraction techniques. However, existence of endogenous

peaks which interfere with the drug or appeared as late eluting peaks and lead to long run

time of analysis has been reported. Application of either an ion pairing agent in the

mobile phase or time consuming column purge has been used in the published methods.

Present paper describes liquid – liquid extraction of lamivudine and internal standard

(famotidine) using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent

and salting out approach. The mobile phase was a mixture of phosphate buffer (0.05 M)

containing triethylamine (1 mL/L, v/v; pH 3.5) and methanol (91:9, v/v) at a flow rate of

2.2 mL/min. The analysis was performed on a column (150mm×6mm i.d.) which was

packed with 5μm particles of ODS packing material. Under these conditions no

interference in the assay from any endogenous substance was observed. The limit of

quantification was evaluated to be 5 ng/mL. Accuracy and precision of the method were

also studied and the technique was shown to be selective and linear into the concentration

range of 5–2500 ng/mL.

Sekar R151 et al., (2005) have reported a micellar electrokinetic chromatographic

(MEKC) method for the simultaneous separation and determination of lamivudine

(LMV) and zidovudine (ZDV) in pharmaceutical formulation has been developed.

Factors that affect the separation, such as buffer pH, surfactant concentration (sodium

217

dodecyl sulfate, SDS), organic solvents and applied voltage were optimized. Buffer

consisting of 12.5mM sodium tetraborate decahydrate and 15mM boric acid adjusted at

pH 10.8, containing 90mM SDS and 5% (v/v) acetonitrile (ACN) was found to be

suitable for the separation of the drugs. p-Aminobenzoic acid (PABA) was used as

internal standard (I.S.). Detection of analytes and I.S. was performed at a wavelength of

210 nm. It was observed that both the drugs and I.S. were migrated within 20 min at the

applied voltage of +10 kV. Validation of the method was performed in terms of linearity,

accuracy, precision, limit of detection (LOD) and quantification (LOQ). An excellent

linearity was obtained in the concentration range 10–80 μg/ml for LMV and 10–100

μg/ml for ZDV. The detection limits for LMV and ZDV were found to be 2.5 and

2.0μg/ml, respectively. The optimized method was applied to the simultaneous

determination of LMV and ZDV in pharmaceutical formulation and human plasma

(spiked) samples. Recovery of both the drugs in tablet dosage form and spiked drugs in

plasma were ≥99.72% (relative standard deviation (R.S.D.)≤1.84%) and ≥80.4%

(R.S.D.≤5.4%), respectively. In the electropherogram no interfering peaks were observed

in the region of analytes and I.S. due to inactive ingredients in the tablets and matrices in

plasma.

Philippe Morin152 et al., (2005) have developed a Zalcitabine (ddC), lamivudine

(3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir

(PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are

nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a

specific reversed-phase high-performance liquid chromatography (HPLC) method was

218

developed for their analysis in rat plasma. The method involved a quantitative recovery

of these drugs from rat plasma by solid-phase extraction on Oasis® HLB Waters

cartridges followed by optimised HPLC separation on an AtlantisTM dC18 column with

acetic acid–hydroxylamine buffer (ionic strength 5 mM, pH 7)-acetonitrile elution

gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves

were obtained within a 30–10,000 ng/mL plasma concentration range. Correlation

coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits

of quantification were in 30–90 ng/mL concentration range. Quantitative parameters

(accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory

results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been

tested in HPLC/ESIMS/MS and appears to be an efficient volatile buffer in the medium

5–7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one

hundred times higher to that obtained for the usual acetic acid/ammonia buffer.

Sockalingam Anbazhagan153 et al., (2005) have developed a simultaneous

quantification of stavudine (SV), lamivudine (LV) and nevirapine (NV) in tablets by UV

spectroscopy, reverse phase HPLC (RP-HPLC) and HPTLC methods. In the UV multi-

component spectral method, SV, LV and NV was quantified at 266, 271 and 315 nm,

respectively. In the RP-HPLC method, the drugs were resolved using a mobile phase of

20mM sodium phosphate buffer (containing 8mM1-octanesulphonicacid sodium

salt):acetonitrile (4:1, v/v) with pH adjusted to 3.5 using phosphoric acid on a C18-ODS-

Hypersil (5 _m, 250mm×4.6 mm) column in isocratic mode. The retention time of SV,

LV and NV was 2.85, 4.33 and 8.39 min, respectively. In the HPTLC method, the

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chromatograms were developed using a mobile phase of chloroform:methanol (9:1, v/v)

on precoated plate of silica gel 60 F254 and quantified by densitometric absorbance mode

at 265 nm. The Rf of SV, LV and NV were 0.21–0.27, 0.62–0.72 and 0.82–0.93,

respectively. Recovery values of 99.16–101.89%, percentage relative standard deviation

of <0.7 and correlation coefficient (linear dynamic range) of 0.9843–0.9999 shows that

the developed methods were accurate and precise.

Verweij-van Wissen C.P.W.G.M154 et al., (2005) have developed a reversed

phase high performance liquid chromatography method for the simultaneous quantitative

determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine,

didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-

phase extraction with OasisMAXcartridges from plasma, followed by high performance

liquid chromatography with a Symmetry Shield RP 18 column and ultraviolet detection

set at a wavelength of 260 nm. The assay was validated over the concentration range of

0.015–5 mg/l for all five NRTIs. The average accuracies for the assay were 92–102%,

inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries

were higher than 97%.

Ramesh P155 et al., (2006) were developed and validated a high-performance

liquid chromatographic and an UV spectrophotometric method for the quantitative

determination of three antiretroviral drugs viz. Lamivudine, Stavudine and Nevirapine

that constitute one of the first line regimens in antiretroviral therapy. Chromatography

was carried out by isocratic technique on a reversed-phase C-18 SYMMETRY column

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with mobile phase based and optimized depending on the polarity of the molecules. The

UV spectrophotometric determinations were performed at 270, 265 and 313 nm for

Lamivudine, Stavudine and Nevirapine, respectively. The linearity of the calibration

curves for each analyte in the desired concentration range is good (r2 > 0.999) by both

the HPLC and UV methods. Both the methods were accurate and precise with recoveries

in the range of 97 and 103% for all the three drugs and relative standard deviation

(R.S.D.) <5%.

Ramesh P156 et al., (2006) have described for the simultaneous determination of

lamivudine (3TC) and stavudine (d4T) in combined pharmaceutical tablets. The first

method depends on first derivative UV-spectrophotometry with zero-crossing

measurement technique. The first derivative absorbances at 280 and 300 nm were

selected for the determination of stavudine and lamivudine, respectively. The second

method is based on the separation of both drugs by high performance liquid

chromatography using methanol:water (20:80) as the mobile phase at 0.6 ml/min on a

reverse phase column with detection at 270 nm. Both the methods showed good linearity,

reproducibility and precision. No spectral or chromatographic interferences from the

tablet excipients were found.

Brian L. Robbins157 et al., (2007) have developed a cartridge-LC–MS/MS

method. The quantitation range was 2.5–250 pg/μl for 3TC-TP, 0.1–10.0 pg/μl for ZDV-

TP and 0.05–5.00 pg/μl for CBVTP. This corresponds to 0.1–11.0 pmol 3TC-TP per

million cells, 4–375 fmol ZDV-TP per million cells and 2–200 fmol CBV-TP per million

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cells, extracted from 10 million cells. Patient samples demonstrated measured levels in

the middle regions of our standard curves both at pre-dose and 4 h post-dose times.

Pranav Shrivastav158 et al., (2007) have developed and validated a selective and

high throughput liquid chromatography/tandem mass spectrometry (LC–MS/MS) method

to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and

nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After

solid phase extraction (SPE), the analytes and the IS were chromatographed on a

Symmetry C18 (150mm×3.9mm i.d., 5 μm particle size) column using 5 μL injection

volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial

acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The

precursor and product ions of these drugs were monitored on a triple quadrupole mass

spectrometer, operating in the multiple reaction monitoring mode (MRM) without

polarity switch. The method was validated over the range of 25–3000 ng/mL for 3TC,

20–2000 ng/mL for d4T and 50–5000 ng/mL for NVP. The absolute recoveries for

analytes (≥86%) and IS (98.12%) achieved from spiked plasma samples were consistent

and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs

(LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels

was within ±8% in terms of relative error.

Miriam C. Poirier159 et al., (2008) have described a antiretroviral nucleoside

reverse transcriptase inhibitor (NRTI) toxicity is currently under investigation in

Erythrocebus patas monkeys, and whereas NRTI pharmacokinetics have been studied in

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other monkey species, pharmacokinetics for Zidovudine plus Lamivudine (AZT/3TC)

dosing have not been reported in the patas. Here we present 24 h serum pharmacokinetic

parameters after a single oral exposure to the combination of AZT (40 mg) and 3TC (24

mg), doses equivalent to a human daily dose of Combivir®. The patas (n=3) AZT/3TC

pharmacokinetic profiles were similar to those seen in other primate species. Average

maximum serum concentrations (Cmax) for AZT and 3TC were 2.35 and 2.65 μg/ml,

respectively, and were observed at 0.83 h (Tmax). Cmax was 13.34 μg/ml for the AZT-

glucuronide (AZT-G) and was 0.023 μg/ml for the potentially toxic minor metabolite 3′-

amino-3′-deoxythymidine (AMT), both occurring at about 1 h after dosing. Similar

elimination half-times, 0.70 and 0.68 h−1, were found for AZT and AZT-G, respectively,

while 3TC was eliminated about half as fast (0.33 h−1) resulting in AUC(0–∞) values of

6.97 μg/ml h for 3TC, 2.99 μg/ml h for AZT, 20.5 μg/ml h for AZT-G and 0.002 for

AMT 6.97 μg/ml h.

Adams E160 et al., (2009) have developed a liquid chromatographic method to

analyse a tablet containing three anti-human immunodeficiency virus (HIV) compounds:

lamivudine, zidovudine and a compound with the code name TMC278.HCl. Due to the

presence of UV absorbing chromophores in the three active components, a single LC

method with UV detection was developed. A Hypersil BDS C18 column was used as

stationary phase and the assay was performed with gradient elution using mobile phases

containing acetonitrile, 0.2M potassium dihydrogen phosphate and water. The sample

pretreatment is performed by treating the formulation with dimethyl sulfoxide–water

(1:1) followed by filtration. After method development, the influence of the different

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chromatographic parameters on the separation, the interference of other active

compounds and excipients, the repeatability and the linearity were investigated.

Srikar A161 et al., (2009) have developed a simple and sensitive

spectrophotometric method for the estimation of Lamivudine in both pure and tablet

dosage form. This was based on the condensation reaction of Lamivudine with carbonyl

reagent such as p-dimethylaminocinnamaldehyde (PDCA) in acidic condition to orange

yellow colored chromogen with absorption maxima at 496 nm. Beer’s law is valid in the

concentration range of 2-10 μg/ml. This method was validated for precision, accuracy,

ruggedness and robustness. Statistical analysis proves that the method is reproducible and

selective for the estimation of the said drug.

Sudha T162 et al., (2010) has been developed and validated a simple, precise,

accurate and rapid high performance thin layer chromatographic method for the

simultaneous estimation of Lamivudine and Abacavir sulphate in combined dosage

forms. The stationary phase was precoated silica gel 60F254. The mobile phase used was

a mixture of (Acetone: chloroform: methanol 4: 4: 2 v/v/v). The detection of spot was

carried out at 265nm. The method was validated in terms of linearity, accuracy, precision

and specificity. The calibration curve was found to be linear between 500 to 3000 ng with

regression coefficient of 0.9998.

Anandakumar Karunakaran163 et al., (2010) have describes validated First

Derivative Spectrophotometric method for the simultaneous estimation of Lamivudine

and Tenofovir disoproxil fumerate in pure and in formulation. The solutions of standard

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and sample were prepared in distilled water. Quantitative determination of the drugs was

performed at 287 nm and at 249 nm (N = 1; λ = 1) for Lamivudine and Tenofovir

disoproxil fumerate, respectively. Proposed method was evaluated for the different

validation parameters. The specificity test showed that there was no interference from

excipients commonly found in the commercial pharmaceutical formulations at the

analytical wavelengths of LAM and TDF. Quantification was achieved over the

concentration range of 5 – 30 μg/ ml for Lamivudine and 10 – 60 μg/ ml for Tenofovir

disoproxil fumerate. The mean recovery was 100.27 ± 1.2511 and 100.70 ± 1.0604 % for

LAM and TDF, respectively.

Anandakumar Karunakaran164 et al., (2012) has been developed and validated

a simple, rapid reverse - phase high performance liquid chromatographic method for the

simultaneous estimation of lamivudine and tenofovir disoproxil fumarate in pure and in

tablet dosage form. The estimation was carried out on a Phenomenax Luna C18 (150 mm

x 4.6 mm i.d., particle size 5μm) column with a mixture of acetonitrile: methanol: water

in the ratio of 30:50:20 (v/v) as mobile phase. UV detection was performed at 258 nm.

The method was validated for linearity, accuracy, precision, specificity and sensitivity as

per ICH norms. The developed and validated method was successfully used for the

quantitative analysis of commercially available dosage form. The retention time was 3.27

and 4.15 min. for lamivudine and tenofovir disoproxil fumarate, respectively. The flow

rate was 1.0 mL min-1. The calibration curve was linear over the concentration range of 2

–12 μg mL-1 for both lamivudine and tenofovir disoproxil fumarate. The LOD and LOQ

values were found to be 0.0099 and 0.0299 μg mL-1 for lamivudine and 0.0328 and

225

0.0994 μg mL-1 for tenofovir disoproxil fumarate, respectively. The high percentage of

recovery and low percentage coefficient of variance confirm the suitability of the method

for the simultaneous estimation of lamivudine and tenofovir disoproxil fumarate in pure

and in tablet dosage form.

Panzade P S165 et al., (2013) have developed and validated a simple, precise,

selective and rapid reverse phase high-performance liquid chromatographic method for

the simultaneous estimation of Tenofovir and Lamivudine in bulk and combined dosage

form. The mobile phase used was mixture of phosphate buffer (6.5 mM) adjusted to pH

2.5 with orthophosphoric acid and acetonitrile (50:50 v/v). The Inertsil C18 column (15

cm x 4.6 mm, 5 μm) was used and drugs were detected by UV detector at 260 nm. The

retention time of Lamivudine and Tenofovir were found to be 2.04 and 3.54 min

respectively. The method was linear in the concentration range of 60-140 μg/ml and 180-

420 μg/ml with correlation coefficient (r2) of 0.998 and 0.999 for Lamivudine and

Tenofovir respectively. The method was validated according to ICH guidelines with

respect to accuracy, precision, specificity.

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4.6 LITERATURE REVIEW:

TENOFOVIR DISOPROXIL FUMARATE:

Fernandez C166 et al., (2003) have developed a new high-performance liquid

chromatography assay for the determination of tenofovir, a nucleotide analogue, in

plasma. A solid–liquid extraction procedure was coupled with a reversed-phase HPLC

system. The system requires a mobile phase containing Na2HPO4 buffer,

tetrabutylammonium hydrogen sulfate and acetonitrile for different elution through a C18

column with UV detection. The method proved to be accurate, precise and linear between

10 and 4000 ng/ ml.

Elisabeth Rey167 et al., (2003) have developed a sensitive high-performance

liquid chromatography method with spectrofluorimetric detection for the determination

of tenofovir, a new HIV reverse transcriptase inhibitor, in human plasma. After

precipitation of 200 μl of plasma samples by methanol and evaporation of the

supernatant, fluorescent derivatized compounds were obtained by a 40-min incubation at

80°C with chloroacetaldehyde 0.34% at pH 4.5. The assay was performed isocratically

using 5 mM Na2HPO4 (pH 6), containing tetrabutylammonium (TBA) chloride 5 mM,

and acetonitrile (85:15, v/v) as mobile phase, and a Cluzeau C8 plus satisfaction column

maintained at 35°C. Detection was performed at excitation and emission wavelengths set

at 236 and 420 nm, respectively. In these conditions, tenofovir can be separated from

adefovir, the internal standard, and endogenous substances. The method was found to be

linear and has been validated over a concentration range of 5–1000 mg/ l. The average

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coefficient of the limit of quantification (5 mg/ l) was 5.38% and at this concentration, a

signal-to-noise ratio of 500 was measured.

Rolf W. Sparidans168 et al., (2003) has been developed and validated a sensitive

and selective reversed-phase liquid chromatographic assay for tenofovir in human

plasma. Tenofovir was isolated from a 200 ml plasma sample using protein precipitation

with trichloroacetic acid. The fluorescent 1,N6 -etheno derivative is formed at 98°C in the

buffered extract with chloroacetaldehyde. This derivative was analysed using gradient

ion-pair liquid chromatography and fluorescence detection at 254 nm for excitation and

425 nm for emission. In the evaluated concentration range (20–1000 ng/ml), the intra-day

precision was 4% and the inter-day precision was 5–6%. An accuracy of between 97 and

110% was determined. The lower limit of quantification was 20 ng/ml with an inter-day

precision of 11%, an intra-day precision of 12% and an accuracy of 103%.

Naser L. Rezk169 et al., (2005) has described a accurate, sensitive and simple

reverse-phase (RP) high-performance liquid chromatography (HPLC) assay for the

simultaneous quantitative determination of emtricitabine and tenofovir in human blood

plasma. Using 200 μL of plasma and BOND ELUT-C18 Varian columns, the solid phase

extraction (SPE) method results in a clean baseline and high extraction efficiencies

(100% for emtricitabine and 98.6% for tenofovir). An AtlantisTM dC-18 analytical

column is used along with an 18 min linear gradient elution of phosphate buffer (pH 5.7)

and methanol to provide sharp peaks for emtricitabine at 280 nm, tenofovir at 259 nm,

and the internal standard 2,3-didoxyuridine (DDU) at 262 nm. The method was validated

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over the range of 10–10,000 ng/mL for both analytes, and is accurate (average accuracies

of three different concentrations ranged from 98 to 105% for emtricitabine and 97 to

103% for tenofovir) and precise (within- and between-day precision ranged from 1.7 to

3.7% and 3.7 to 5.2%, respectively).

Courtney V. Fletcher170 et al., (2006) have described to facilitate the evaluation

of drug safety, virologic activity, and pharmacokinetics, an anion exchange isolation of

tenofovir-diphosphate (TFVDP) from human peripheral blood mononuclear cells

(hPBMCs), coupled with dephosphorylation, desaltation, and detection by LC–MS–MS

was validated. hPBMCs were harvested from whole blood, lysed, and a suspension of

intracellular tenofovir moieties was produced. TFV-DP was isolated from TFV-

monophosphate (TFV-MP) and tenofovir (TFV), dephosphorylated with acid

phosphatase to form TFV and then desalted and concentrated, making it possible for

tandem mass spectral detection. An LC–MS–MS methodology was developed and

validated for the determination of TFV concentrations, which directly correspond with

the intra-hPBMC TFV-DP concentration. The assay was linear in the range of 50–10,000

μmol per sample. The lower limit of quantitation (LLOQ) of the method is 10 μmol per

million cells with 5 million hPBMCs used.

Tom Delahunty171 et al., (2006) have been developed and validated an

LC/MS/MS assay for the determination of tenofovir (TNF) for use with the EDTA

anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were

also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was

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used to produce a protein-free extract. Chromatographic separation was achieved with a

Polar-RP Synergi, 2.0mm×150 mm, reversedphase analytical column. The mobile phase

was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was

achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions,

respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable

limit of 10 ng/ml when 250 μl aliquots were analyzed.

Marie-Claude Gagnieu172 et al., (2007) have developed a sensitive high-

performance liquid chromatography method coupled to UV and single mass spectrometry

(MS) detection for the determination of tenofovir in human plasma. A solid phase

extraction procedure (Bond-Elut® C18 Varian cartridges) provided high extraction

efficiency (91% for tenofovir and 68.8% for the internal standard, 3-methylcytidine). An

atlantis®-dC-18 analytical column is used with an isocratic mode elution of a mixture

(pH 2.5) of ammonium acetate/methanol (98.5:1.5, v/v). Detection was performed at 260

nm and by using the ion at m/z 288. The signals from both detectors were validated over

the range of 10–1000 ngmL−1 and were found to be linear, accurate and precise. At the

lowest limit of quantification, 10 ngmL−1 for UV and 5 ngmL−1 for MS, the average

coefficient of variation was 6.9 and 3.9%, respectively. To investigate the potential of the

validated method for clinical studies, more than 170 samples from HIV-infected adult

patients were then analyzed with this assay. A good correlation was observed between the

results obtained with both detectors. However, in several cases discordant results were

observed between UV and MS detections. Therefore, tenofovir can sometimes suffer

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from interferences using either UV or single MS detection. We concluded that the double

detection allows obtaining a more specific quantification of tenofovir.

Noel A. Gomes173 et al., (2008) have developed and validated a rapid and

sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for

simultaneous quantification of Tenofovir (TEN) and Emtricitabine (EMT) in human

plasma using Chromolith Speed Rod RP18. The mass transition ion-pair has been

followed as m/z 288.10→176.10 for TEN, m/z 248.20→130.20 for EMT and m/z

230.10→112.10 for Lamivudine (LAM). The method involves solid phase extraction

from plasma, simple isocratic chromatographic conditions and mass spectrometric

detection using an API 5000 instrument that enables detection at nanogram levels.

Lamivudine was used as the internal standard. The proposed method has been validated

with a linear range of 10–600 ng/ml for TEN and 25–2500 ng/ml for EMT. The intrarun

and interrun precision values are within 12.0% for TEN and 15.6% for EMT at their

respective LOQ levels. The overall recoveries for TEN and EMT were 84.3% and 68.5%,

respectively. Total elution time was as low as 2 min.

Courtney V. Fletcher174 et al., (2009) have described a LC/MS/MS method for

medication has prompted us to develop and validate a convenient assay to determine

simultaneously TFV and FTC plasma concentrations. In view of their chemical similarity

to the analytes, stable isotope internal standards (IS) were chosen. These consisted of

TFV labeled uniformly with 13C in the adenine moiety (Iso-TFV) and FTC labeled with

13C and 15N in the cytosine moiety (Iso-FTC). Trifluoroacetic acid was added to the

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patient’s EDTA plasma (containing the IS) to produce a de-proteinated extract after high

speed centrifugation. The extracts were directly injected into the mobile phase (3%

acetonitrile/1% acetic acid, aq.) stream flowing at 200_L/min. A Synergi Polar-RP,

2.0mm×150mm, reversed-phase analytical column was used to achieve the

chromatographic separation. Detection of the analytes was achieved by ESI positive

ionization tandem mass spectrometry. The precursor/product transitions (m/z) in the

positive ion mode were 288/176 and 293/181 ions for TFV and Iso-TFV, respectively and

the precursor/product transitions (m/z) were 248/130 and 251/133 ions for FTC and Iso-

FTC, respectively. When the analyte/IS abundance ratios were plotted against the

specified concentrations, the linearity of the concentration curves were in the range 10

ng/mL to 1500 ng/mL for both analytes (250μL plasma extracted), with a minimum

quantifiable limit of 10 ng/mL for both analytes. The inter- and intra-day accuracy and

precision for both TFV and FTCwere within±20% at the LLOQ and±15% at the other QC

levels.

Robert S. Jansen175 et al., (2010) have developed and validated for the

simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP),

TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood

mononuclear cells. Reference compounds and internal standards were obtained by

thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable

isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v)

and the extracted nucleotides were analyzed using weak anion-exchange chromatography

coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727

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to 36.4, 1.33 to 66.4 and 1.29 to 64.6nM for FTC-MP, FTC-DP and FTC-TP and from

1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127nM for TFV, TFV-MP and TFV-DP,

respectively, were validated. Accuracies were within −10.3 and 16.7% deviation at the

lower limit of quantification at which the coefficients of variation were less than 18.2%.

At the other tested levels accuracies were within −14.3 and 9.81% deviation and the

coefficients of variation lower than 14.7%.

Anandakumar K176 et al., (2011) have developed and validated a simple,

efficient, precise and accurate simultaneous equation for the estimation of emtricitabine

and tenofovir disoproxil fumerate in pure and in fixed dose combination. The method is

based on the ultraviolet absorbance maxima of the above two drugs at 281 nm and 210

nm, respectively. Both the drugs obeyed Beer’s law in the concentration range of 4 – 24

μg/ ml. The validity of the proposed method was assessed by applying the standard

addition technique where the percentage recovery of the added standard was found to be

99.15 ± 0.2840 and 99.11 ± 0.2732 for emtricitabine and tenofovir disoproxil fumerate,

respectively.

Vishnu P C177 et al., (2011) have described a three simple, economical, precise,

and accurate methods for the simultaneous determination of Tenofovir disoproxil

fumarate (TE) and Emtricitabine (EM) in combined tablet dosage form. The first method

is ratio derivative spectra, second is first-order derivative spectrophotometry and third is

absorption corrected method. The amplitudes at 271.07 and 302.17 nm in the ratio

derivative method, 224.38 and 306.88 nm in the first order derivative method were

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selected to determine Tenofovir disoproxil fumarate (TE) and Emtricitabine (EM),

respectively, in combined formulation. Beer's law is obeyed in the concentration range of

3-21 μ/ml for TE and 2-14 μ/ml for EM for first two methods and range for third method

was 6-30 μ/ml of TE and 4-20 μ/ml of EM. The percent assay for commercial

formulation was found to be in the range 98.91%–101.72% for both the analytes by the

proposed three methods. Absorption corrected method was successfully applied to carry

out dissolution study of commercial tablet formulation by using USP II dissolution test

apparatus. The methods were validated with respect to linearity, precision, and accuracy.

Recoveries by proposed methods were found in the range of 99.06 %-101.34 % for both

the analytes.

Sharma T178 et al., (2012) have developed a simple, economic, accurate, reverse

phase isocratic HPLC method for quantitation of Tenofovir disoproxil fumarate in tablet

dosage form. The quantification was carried out using Reverse phase column Inertsil

ODS-3 (150×4.6 mm), 5μm. The detection was carried out at the wavelength of 260nm.

The elution was achieved isocratically with a mobile phase comprising a mixture of

Sodium dihydrogen orthophosphate buffer pΗ 2.3 and Methanol (49:51v/v). The flow

rate was 1.0 mL/min. The procedure was validated as per ICH rules for accuracy,

precision, detection limit, linearity, reproducibility, and quantitation limit. The linearity

concentration range was 50-300 μg/mL with the correlation coefficient of 0.9995. The

percentage recovery for Tenofovir disoproxil fumarate was found to be 99.98-100.14.

Limit of detection and limit of quantitation values were found to be 0.28 μg/mL and 0.85

μg/mL.

234

Narendra D179 et al., (2012) have developed and validated a simple, rapid

reverse phase high-performance liquid chromatographic method for the simultaneous

estimation of Tenofovir and Emtricitabine in bulk and pharmaceutical dosage forms.

Chromatography was carried out by using Chromosil C-18,column having 250 x 4.6mm

internal diameter with a mixture of methanol, acetonitrile and TEA in the ratio of

46:50:04 (v/v/v) as mobile phase. Determination of the different analytical parameters

such as linearity, precision, accuracy, and specificity, limit of detection (LOD) and limit

of quantification (LOQ) was done. The calibration curve was found to be linear for each

analyte in the desired concentration range. The % recovery was found to be 99.59 and

99.61 for Tenofovir and Emtricitabine respectively. The proposed method is highly

sensitive, precise and accurate, which was evident from the LOD value of 0.05 and 0.02

ppm for Tenofovir and Emtricitabine respectively.

Soumya B180 et al., (2012) have developed a simple and rapid UV

spectrophotometric method for simultaneous estimation of Tenofovir disoproxil fumarate

(TDF) and Lamivudine (LAM). The absorption maxima of both drugs were found at

260nm and 280nm and obeyed Beer’s law in the range of 5-45μg/ml (y = 0.021x + 0.002;

r2 = 0.999) and 2-16μg/ml (y = 0.061x + 0.004; r2 = 0.998) respectively for TDF and

LAM in acetonitrile : 0.1N HCl (20:80) solvent system. Accuracy and reproducibility of

the proposed method was statistically validated by recovery studies. This method is found

to be precise and accurate.

Declan P. Naughton181 et al., (2013) have developed and validated a sensitive

and reproducible method for the simultaneous quantification of the key antiretroviral

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drugs abacavir and tenofovir in hair using LC–MS/MS. Hair samples (50 mg) were

decontaminated and subjected to methanolic extraction, where 1 ml methanol was added

along with the internal standard abacavir-d4 at a final concentration of 0.15 ng/mg hair.

After 16 h, the drugs were recovered by liquid–liquid extraction using ammonium acetate

buffer and a mixture of methyl tert-butyl ether:ethyl acetate (1:1). The samples were

reconstituted with 200 μl acetonitrile:water (1:1) prior to injection for LC–MS/MS. The

LOD and LOQ values were 0.06 and 0.12 ng/mg (drug/hair) for both drugs. Calibration

curves were linear in the concentration range of 0.12–4.0 ng/mg of drug/hair with

regression coefficient (r2) value of 0.999 for both drugs. The data for accuracy, precision

and recovery were within the FDA limits. The concentrations of the drugs in the hair

samples ranged from 0.12 ng/mg to 4.48 ng/mg and 0.32 ng/mg to 1.67 ng/mg for

tenofovir and abacavir, respectively.

Syed Sajjad hussen182 et al., (2013) have develop a novel stability-indicating

high performance liquid chromatographic (HPLC) method for Tenofovir Disproxil

fumarate (TEN) with photodiode array (PDA) detection and validated as per International

Conference on Harmonisation (ICH) guidelines. The developed method was successfully

applied for assay of Tenofovir Disproxil fumarate to nanoparticle formulation. A

Lichrocart (C18) (250mm × 4.6mm, 5 μm particle size) column and a mobile phase

composed of acetonitrile and 0.025M potassium di hydrogen phosphate buffer (pH 3.0

adjusted by using 10% v/v Orthophosphoric acid) in the ratio 35:65 (v/v) was used, and

the detection wavelength of 260 nm. The method was validated with the parameters like

specificity, linearity, precision, accuracy, limits of detection and quantification as per

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ICH guidelines. Forced degradation studies under different stress conditions like Acid,

Base and Oxidation was successfully achieved, TEN was found to degrade significantly

in alkaline and acidic conditions, PDA peak purity test confirmed the specificity of the

developed method. The method was found to be precise and accurate with a linearity

range of 0.1 μg mL-1 to 50 μg mL-1 (r2 > 0.999).

237

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF

INVITRO DISSOLUTION STUDIES OF EFAVIRENZ, LAMIVUDINE

AND TENOFOVIR DISOPROXIL FUMARATE IN PHARMACEUTICAL

DOSAGE FORM

4.7. EXPERIMENTAL PROTOCOL

4.7.1. Chemicals and Reagents

Working stands of Efavirenz (99.7%), Lamivudine (99.9%) and Tenofovir

Disoproxil Fumarate (98.3%) were donated by Herero Healthcare Ltd (Hyderabad,

India).

The pharmaceuticals, TENOLAM E tablets containing Efavirenz – 600mg,

Lamivudine – 300mg and Tenofovir Disoproxil Fumarate – 300mg purchased from

Hetero HC (GenX).

The reagents, sodium dihydrogen phosphate and ortho phosphoric acid were of

analytical reagent grade supplied by M/S SD Fine chemicals (Mumbai, India). The

solvents, acetonitrile (MeCN) and methanol (MeOH) were of HPLC grade supplied by

M/S SD Fine chemicals (Mumbai, India). HPLC grade water was obtained following

distillation in glass and passage through a Milli-Q system (Millipore, Bangalore, India)

and was used to prepare all the solutions.

238

4.7.2. Selection of Initial Conditions for Method Development

4.7.2.1. Determination of absorption maxima by UV-Visible Spectroscopy:

Efavirenz:

Accurately weighed and transferred about 100 mg of Efavirenz into a 100 ml

volumetric flask, added to it about 70 ml of methanol and sonicated for 2minutes to

dissolve and diluted up to the mark with Milli-Q water and mixed well. Further diluted 1

ml of the above solution to 100 ml with Milli-Q water and mixed well. (Concentration of

Efavirenz is about 10µg/ml).

Figure 4.7.1: UV Spectrum of Efavirenz

Lamivudine:

Accurately weighed and transferred about 100 mg of Lamivudine into a 100 ml

volumetric flask, added to it about 70 ml of methanol and sonicated for 2minutes to

dissolve and diluted up to the mark with Milli-Q water and mixed well. Further diluted 1

ml of the above solution to 100 ml with Milli-Q water and mixed well. (Concentration of

Lamivudine is about 10µg/ml).

239

Figure 4.7.2 : UV Spectrum Lamivudine

Tenofovir Disoproxil Fumarate:

Accurately weighed and transferred about 100 mg of Tenofovir Disoproxil

Fumarate into a 100 ml volumetric flask, added to it about 70 ml of methanol and

sonicated for 2minutes to dissolve and diluted up to the mark with Milli-Q water and

mixed well. Further diluted 1 ml of the above solution to 100 ml with Milli-Q water and

mixed well. (Concentration of Tenofovir Disoproxil Fumarate is about 10µg/ml).

Figure 4.7.3: UV Spectrum Tenofovir disproxil fumarate

240

Figure 4.7.4: UV overlain Spectrum of EFV, LAM and TDF

Isobestic Point:

It is the point at which all the drugs in a particular combination will have same

absorbance at a single wavelength. The Isobestic point of Efavirenz, Lamivudine &

Tenofovir disoproxil fumarate is found to be 260nm.

4.7.3. HPLC instrumentations and conditions

Chromatographic measurements were made on Waters 2695 separation module,

Quaternary gradient type pump, water 2489 PDA/UV Visible Detector, Waters Auto

injectors. The system was controlled through a system controller (Empower 2) and a

personal computer using chromatographic software installed on it. Chromatographic

separations were carried out on Hypersill BDS C-18 analytical column (150 mm X

4.6mm i.d., 5µm) and XTERRA Symmetry C 18 column (150mmX 4.6mm, 5µm).

Before the analysis, the mobile phase was filtered through a 0.2 µm filter (Gelman

science, India) and degassed using Branson sonicator (Branson Ultrasonics Corporation,

241

USA) at the flow rate of 1.0ml per minutes. Sample solutions were also filtered through a

0.2 µm filter and aliquots of 10 µL were injected into the chromatographic system. The

HPLC system was used in air-conditioned laboratory atmosphere (20±2°C). The mobile

phase consisted of mobile phase A as pH 4.0 phosphate buffer and Mobile phase B as

water and acetonitrile (30:70) by gradient elution. A UV wavelength of 260nm was

selected for detection (Fig 4.7.4).

4.8 METHOD DEVELOPMENT OF EFAVIRENZ, LAMIVUDINE &

TENOFOVIR DISOPROXIL FUMARATE:

4.8.1 TRIAL 1:

Chromatographic Conditions:

Column : Hypersil BDS C-18, 100 mm X 4.6 mm X 3.6

Flow rate : 1.5 mL/min

Wave length : 260 nm

Injection volume : 10 L

Column oven temp : 35°C

Sample temp : 25°C

Run time : 18 min

242

Gradient time program:

Time (in min) Mobile Phase-A in % Mobile Phase-B in %0.0 90.0 104.0 90.0 107.0 10.0 9011.0 10.0 9015.0 90.0 1018.0 90.0 10

Procedure:

Mobile phase A Preparation:

3.12 g of Sodium dihydrogen ortho phosphate in 1000ml Milli-Q water sonicated

and dissolved.

Mobile Phase B Preparation:

Methanol: Water (85:15)

Diluent Preparation:

Dissolution media used as diluent.

Standard solution:

60.5 mg of Efavarienz, 30.3 mg of Lamivudine and 30.7 mg of Tenofovir were

weighed accurately to 100 mL volumetric flask. 70 mL of diluent was added and

sonicated for 10 min under cold condition (2º to 8º). Make up to the volume with diluent.

Filter through 0.45µ nylon membrane filter.

243

Figure 4.8.1: Chromatogram of Efavirenz, Lamivudine & Tenofovir disoproxil

fumarate Trial - 1

Observation:

1) Retention time of Lamivudine is = 6.0 min.

2) Retention time of Tenofovir is = 8.3 min.

3) Retention time of Efavirenz is = 12.0 min.

Conclusion:

Lamivudine & Tenofovir failed in peak purity so the next trial was taken by

changing the mobile phase B.

244

4.8.2 TRIAL 2:

Changed Conditions:

Mobile Phase B Preparation: Acetonitrile: Water (70:30)

Figure 4.8.2: Chromatogram of Efavirenz, Lamivudine & Tenofovir disoproxil

fumarate Trial - 2

Observation:

1) Retention time of Lamivudine is = 4.4 min.

2) Retention time of Tenofovir is = 8.9 min.

3) Retention time of Efavirenz is = 12.5 min.

Conclusion:

Lamivudine, Tenofovir, Efavirenz failed in peak purity so the next trial was taken

by changing the column, flow & gradient program.

245

4.8.3 TRIAL 3:

Changed Conditions:

Column : Hypersil BDS C-18 150 mm X 4.6 mm X 5

Flow rate : 1.0 mL min-1

Run time : 20 min

Gradient time program:

Time (in min) Mobile Phase-A in % Mobile Phase-B in %0.0 93.0 74.0 93.0 75.0 20.0 8010.0 20.0 8011.0 93.0 720.0 93.0 7

Figure 4.8.3: Chromatogram of Efavirenz, Lamivudine & Tenofovir disoproxil fumarate Trial - 3

Observation:

1) Retention time of Lamivudine is = 4.1 min.

2) Retention time of Tenofovir is = 7.9 min.

3) Retention time of Efavirenz is = 12.6 min.

Conclusion:

Lamivudine, Tenofovir and Efavirenz peak shape is good, and good RT is seen

but tenofovir fails in peak purity, so by changing gradient programme next trial is done.

246

4.8.4 TRIAL 4 (OPTIMISED METHOD):

Changed Conditions:

Gradient time program:

Time (in min) Mobile Phase-A in % Mobile Phase-B in %0.0 93.0 74.0 93.0 75.0 20.0 8012.0 20.0 8013.0 93.0 720.0 93.0 7

Figure 4.8.4: Chromatogram of Efavirenz, Lamivudine & Tenofovir disoproxil fumarate Trial - 4

Observation:

1) Retention time of Lamivudine is = 4.153 min.

2) Retention time of Tenofovir is = 7.922 min.

3) Retention time of Efavirenz is = 12.755 min.

Conclusion:

The peaks of Efavirenz, Lamivudine & Tenofovir are well separated. All the

peaks passed in peak purity. The peaks are well resolved from each other and from blank.

Hence this trial is finalized in method development.

247

4.9 METHODOLOGY:

4.9.1 Dissolution:

Dissolution is the process by which a solid, liquid or gas forms a solution in

a solvent. For the dissolution of solids, the process of dissolution can be explained as the

breakdown of the crystal lattice into individual ions, atoms or molecules and their

transport into the solvent. Dissolution is the process by which a solid solute enters in to a

solution. In the pharmaceutical industry, it may be defined as “the amount of drug

substance that goes into solution per unit time under standardized conditions of

liquid/solid interface, temperature and solvent composition”.

The dissolution test was carried out using an Electro lab Dissolution Test System.

A Waters HPLC system equipped with a 2695 solvent delivery system, Waters auto

injector, thermostatted column compartment and photo diode array detector was used.

Waters column (Hypersil BDS C-18 150 mm X 4.6 mm X 5 particle size) was used for

the analysis.

Media Preparation:

200g of SLS is accurately weighed & transferred into 10L Demineralized water.

Mixed well & sonicated to Dissolve.

Dissolution Parameters:

Medium : Water+2%SLS

Volume : 1000ml

Apparatus : Paddle

Agitation : 75RPM

248

Time : 60min

Temperature : 37°C

Volume Withdrawn : 10ml

HPLC Chromatographic Conditions:

Column : Hypersil BDS C18 150×4.6mm, 5µm

Flow Rate : 1.0ml/min

Wavelength : 260nm

Injection Volume : 10 µl

Column Oven Temperature : 35°C

Sample Temperature : 25°C

Run Time : 20min

Detector : PDA

Gradient Programme:

Time (in min) Mobile Phase-A in % Mobile Phase-B in %0.0 93.0 74.0 93.0 75.0 20.0 8012.0 20.0 8013.0 93.0 720.0 93.0 7

4.9.2 Preparation of Standard solution:

60.5 mg of Efavarienz, 30.3 mg of Lamivudine and 30.7 mg of Tenofovir were

weighed accurately to 100 mL volumetric flask. 70 mL of diluent was added and

sonicated for 10 min under cold condition (2º to 8º). Make up to the volume with diluent.

Filter through 0.45µ nylon membrane filter.

249

4.9.3 Preparation of Sample Formulation:

Each Tablet (1775mg) is transferred into each of six Dissolution Vessels

containing 1000ml of dissolution media, which is at 37°C. Run the apparatus as per the

dissolution conditions mentioned. Sample is withdrawn at 60min. The sample is filtered.

Figure 4.9.1: Chromatogram of Blank

Figure 4.9.2: Chromatogram of standard Efavirenz, Lamivudine & Tenofovir

disoproxil fumarate

250

Name RT Area Resolution Plate Count TailingLamivudine 4.7 6090946 -- 5914 1.0Tenofovir DF 8.0 3790045 15.0 30811 1.0Efavirenz 13.7 4411505 22.0 28796 1.0

Figure 4.9.3: Chromatogram of Sample of Efavirenz, Lamivudine & Tenofovir disoproxil fumarate

Name RT Area Resolution Plate Count TailingLamivudine 4.7 6030896 -- 5872 1.0Tenofovir DF 8.0 3629635 14.5 26251 1.0Efavirenz 13.7 4351013 21.2 27641 1.0

Observation:

The proposed dissolution test method for Efavirenz, Lamivudine & Tenofovir

Disoproxil Fumarate in tablets provides sink condition and is able to satisfactorily

discriminate drug release between formulations differing in terms of hardness, exposure

to heat and moisture and composition of excipients. The analysis was carried out by a

HPLC method which was validated. Hence the proposed dissolution method may be used

as a single test method for the routine dissolution testing of tablets containing Efavirenz,

Lamivudine & Tenofovir Disoproxil Fumarate.

251

4.10 RESULT AND DISCUSSION

4.10.1 Validation

In Drug investigation method validation, which ensures the method is suitable for

its intended use, is imperative according to regulation such as those of the FDA, EMEA

& ICH. Method validation requires appraisal of a variety of method characteristics, for

instance system suitability, linearity, accuracy, precision, selectivity & specificity in the

matrix of interest.

4.10.1.1 Specificity:

Blank and Placebo are injected to check interference at the Retention Time of

main peaks.

4.10.1.1.1 Blank interference:

2% SLS in water is used as blank (dissolution medium) and injected into HPLC

system in duplicate. The interference of the blank to that of the retention time of the

analyte peak is evaluated.

4.10.1.1.2 Placebo interference:

Accurately weighed and transferred 575.5 mg of placebo in 1000ml volumetric

flask. Few ml of diluent is added and sonicated for 10min. Make upto the mark with the

diluent and mixed well. The sample is filtered through 0.45µ filter. Inject this solution in

to the HPLC system in duplicate. The interference of the placebo to that of the retention

time of the analyte peak is evaluated.

252

Figure 4.10.1: Chromatograms of blank in Specificity studies

Figure 4.10.2: Chromatograms of Placebo in Specificity studies

Data Interpretation:

On the basis of these chromatograms we can say that there is no interference of

blank and placebo with the Retention time of active component.

4.10.1.2 PRECISION:

4.10.1.2.1 System precision

The system precision is checked by using standard to ensure that the analytical

system is precise. Five injections of standard Lamivudine, Tenofovir & Efavirenz were

253

taken and the retention time and area of five determinations was measured and RSD was

calculated

Procedure for System Precision:

Standard solution:

60.5 mg of Efavirenz, 30.3 mg of Lamivudine and 30.7 mg of Tenofovir were

weighed accurately to 100 mL volumetric flask. 70 mL of diluent was added and

sonicated for 10 min under cold condition (2º to 8º). Make up to the volume with diluent.

Filter through 0.45µ nylon membrane filter. Inject the standard solution into HPLC

system. Record the % RSD of the five injections.

4.10.1.2.2 Method Precision:

Sample Preparation:

Each Tablet (1775mg) is transferred into each of six Dissolution Vessels

containing 1000ml of dissolution media, which is at 37°C. Run the apparatus as per the

dissolution conditions mentioned. Sample is withdrawn at 60min. The sample is filtered.

Calculate the release in % for Lamivudine, Tenofovir & Efavirenz at the time specified,

average of six units & % RSD for the six observations.

254

.

Figure 4.10.3A: Chromatogram of System Precision studies EFA, LAM and TDF

Figure 4.10.3B: Chromatogram of System Precision studies EFA, LAM and TDF

255

Figure 4.10.3C: Chromatogram of System Precision studies EFA, LAM and TDF

Figure 4.10.3D: Chromatogram of System Precision studies EFA, LAM and TDF

256

Figure 4.10.3E: Chromatogram of System Precision studies EFA, LAM and TDF

Table 4.1 Results of System Precision

Injections Efavirenz ( Area)Lamivudine (

Area)Tenofovir ( Area)

1 4416022 6105371 3810374

2 4415373 6089848 3803005

3 4417435 6103935 3802905

4 4411915 6090946 3790045

5 4411915 6088055 3794309

Mean 4414450 6095632 3800127

SD 2614 8315 8007.8

% RSD 0.1 0.1 0.2

257

Data interpretation for System Precision

It is observed from the data tabulated above, that the area response is consistent as

evidenced by the values of relative standard deviation. Hence, the %RSD is not more

than 2% so it can be concluded that the system precision parameter meets the requirement

of method validation.

Figure 4.10.4A: Chromatogram of Method Precision studies EFA, LAM and TDF

Figure 4.10.4B: Chromatogram of Method Precision studies EFA, LAM and TDF

258

Figure 4.10.4C: Chromatogram of Method Precision studies EFA, LAM and TDF

Figure 4.10.4D: Chromatogram of Method Precision studies EFA, LAM and TDF

259

Figure 4.10.4E: Chromatogram of Method Precision studies EFA, LAM and TDF

Figure 4.10.4F: Chromatogram of Method Precision studies EFA, LAM and TDF

260

Table 4.2 Results of Method Precision

InjectionEfavirenz Lamivudine Tenofovir

Area% Drug Release

Area% Drug Release

Area% Drug Release

1 4279717 97.4 6018478 99.6 4030670 106.72 4491013 102.2 6230896 103.1 4029635 106.73 4227749 96.2 5953108 98.5 4050181 107.24 4431115 100.8 6157577 101.9 3920251 103.85 4208517 95.7 5946405 98.4 4018080 106.46 4161298 94.7 5912285 97.9 4033598 106.8

Average -- 97.8 -- 99.9 -- 106.27SD -- 3.0031 -- 2.1232 -- 1.2356

% RSD -- 3.1 -- 2.1 -- 1.2

Data interpretation for Method Precision

From the above results, it was concluded that the method is precise as the % RSD

is not more than 5%

4.10.1.3 Accuracy:

The accuracy of an analytical method is established across its range. Accuracy is

performed in four different levels for Lamivudine, Tenofovir & Efavirenz. The known

quantity of Lamivudine/Tenofovir/Efavirenz is spiked at 25%, 50%, 100% and 125%

level into the placebo. The samples is analysed in triplicate for each level. From the

results, % recovery was calculated.

Preparation of Accuracy Concentrations for Lamivudine/Tenofovir/Efavirenz:

Accurately weigh the amount of Lamivudine, Tenofovir, Efavirenz & placebo

equivalent to 25%. 50%,100% & 125% concentrations level. Transfer them to into

261

1000ml volumetric flask & few ml of diluent is added & sonicated to dissolve. Make upto

the mark with diluent and filtered through 0.45µ filter.

Figure 4.10.5A: Chromatogram of Accuracy studies EFA, LAM and TDF (25%)

Figure 4.10.5B: Chromatogram of Accuracy studies EFA, LAM and TDF (25%)

262

Figure 4.10.5C: Chromatogram of Accuracy studies EFA, LAM and TDF (25%)

Figure 4.10.6A: Chromatogram of Accuracy studies EFA, LAM and TDF (50%)

263

Figure 4.10.6B: Chromatogram of Accuracy studies EFA, LAM and TDF (50%)

Figure 4.10.6C: Chromatogram of Accuracy studies EFA, LAM and TDF (50%)

264

Figure 4.10.7A: Chromatogram of Accuracy studies EFA, LAM and TDF (100%)

Figure 4.10.7B: Chromatogram of Accuracy studies EFA, LAM and TDF (100%)

265

Figure 4.10.7C: Chromatogram of Accuracy studies EFA, LAM and TDF (100%)

Figure 4.10.8A: Chromatogram of Accuracy studies EFA, LAM and TDF (125%)

266

Figure 4.10.8B: Chromatogram of Accuracy studies EFA, LAM and TDF (125%)

Figure 4.10.8C: Chromatogram of Accuracy studies EFA, LAM and TDF (125%)

267

Table 4.3 Results of Accuracy studies of Efavirenz

ConcentrationAmount

added (mg)Area

Amount Recovered

(mg)

% Recovery

Average Recovery

% RSD

25 150.1 1103316 149.0 99.399.63 0.825 150.2 1121487 149.1 99.2

25 149.6 1112487 150.2 100.450 299.0 2242652 302.9 101.3

100.93 0.250 299.1 2248563 303.0 100.950 299.6 2253672 304.3 100.6\100 598.4 4401678 594.4 99.3

99.30 0.2100 598.6 4400968 593.8 99.1100 598.2 4412326 595.9 99.6125 747.7 5437929 734.4 98.2

98.16 0.1125 747.8 5438257 734.5 98.1125 747.9 5438495 734.4 98.2

Table 4.4 Results of Accuracy studies of Lamivudine

ConcentrationAmount

added (mg)Area

Amount Recovere

d (mg)

% Recovery

Average Recovery

% RSD

25 75.2 1566451 75.8 100.8100.86 0.125 75.3 1568531 75.8 100.9

25 75.1 1567252 75.8 100.9

50 150.2 3102386 150.1 99.999.90 0.050 150.3 3103130 150.1 99.9

50 150.3 3103130 150.1 99.9

100 300.1 6168183 298.4 99.499.66 0.3100 300.2 6195193 299.7 99.8

100 300.2 6195193 299.7 99.8

125 374.7 7686609 371.8 99.299.16 0.1125 374.7 7686609 371.8 99.2

125 375.3 7685464 371.8 99.1

268

Table 4.5 Results of Accuracy studies of Tenofovir

ConcentrationAmount

added (mg)Area

Amount Recovered

(mg)

% Recovery

Average Recovery

% RSD

25 74.2 931860 72.9 98.298.2 0.125 74.1 930920 72.7 98.4

25 74.5 932898 73.0 98.0

50 148.2 1876141 146.8 99.198.5 0.650 148.2 1859946 145.5 98.2

50 148.2 1859946 145.5 98.2

100 295.6 3815679 298.6 101.0101.1 0.3100 295.6 3815679 298.6 101.0

100 295.1 3824324 299.3 101.4

125 369.3 4720384 369.4 100.0100.3 0.4125 369.5 4781263 370.2 100.3

125 369.3 4747334 371.5 100.6

Data interpretation:

From the above results, it can be concluded that the recovery is well within the

limit. Hence, the method is accurate.

4.10.1.4 Linearity:

Individually, samples equivalent to 25%, 50%, 60%, 80%, 90%, 100%,110%,

120%, 140%, 150% concentrations are taken from stock solution individually. A graph of

concentration taken versus chromatographic area was plotted for Lamivudine, Tenofovir

& Efavirenz peaks. The regression line obtained should be linear. From the data obtained,

co-relation coefficient was calculated. Ideally co-relation coefficient should be around 1.

269

Stock Preparation:

Accurately weighed & transferred 120.6mg, 60.5mg & 60.5mg of Efavirenz,

Lamivudine & Tenofovir respectively into 100ml volumetric flask. Few ml of diluent is

added & sonicated to dissolve. Make upto the mark with diluent & mixed well. This

stock solution is diluted to different dilutions.

270

Fig 4.10.9 Linearity plots of Efavirenz, Lamivudine, Tenofovir Disoproxil Fumarate

Table 4.6 Results of Linearity Studies

S No Efavirenz Lamivudine Tenofovir

Concentration (µg/ml)

Average area

Response

Concentration (µg/ml)

Average area

Response

Concentration (µg/ml)

Average area

Response1 150 1192027 75 1551348 75 6558702 300 2336345 150 3103275 150 15844623 360 2725449 180 3652098 180 19340604 480 3589580 240 4882408 240 28329675 540 4048512 270 5539840 270 33348216 600 4453528 300 6153149 300 37490337 660 4887987 330 6754174 330 41388098 720 5326678 360 7419842 360 45287789 840 6051923 420 8543907 420 524210410 900 6593820 450 9370579 450 5761175

Correlation coefficient

0.9997 0.9997 0.9993

Slope 7106.2262 20483.5110 13845.5558Intercept 172764.2668 -28749.3350 -431703.7575

271

Data Interpretation:

Correlation coefficient obtained is 0.9997, 0.9997 & 0.9993 for Efavirenz,

Lamivudine & Tenofovir Disoproxil Fumarate respectively. Hence the method is linear.

4.10.1.5 Robustness:

The robustness of an analytical method is a measure of its capacity to remain

unaffected by small but deliberate variations in method parameters and provides an

indication of its reliability during normal usage. Robustness was done by changing the

column temperature (± 5°C), flow rate (± 10%), Changing the wavelength (± 5 nm).

Figure 4.10.10: Chromatograms with increase & decrease in temperature

272

Figure 4.10.11: Chromatograms with increase & decrease in flow rate

Figure 4.10.12: Chromatograms with increase & decrease in wavelength

Table 4.7 Results of Robustness Studies

ParametersEfavirenz Lamivudine Tenofovir

Rt Area Rt Area Rt AreaOriginal

12.099 2519249 4.000 1036495 7.860 1991771

Column Temperature

(35˚c±5 ˚)

12.362 2762168 3.761 2909209 7.843 1886929

12.921 2738189 4.236 3122530 7.856 1869780

Mobile Phase Flow Rate

(1.0±0.1ml/min)

11.906 737431 3.663 832814 7.555 504160

13.522 638247 4.459 718282 8.194 431852

Detection Wavelength(260±5nm)

12.190 1199623 3.974 7043339 7.851 3529511

12.191 1185437 3.974 5248607 7.850 3679869

273

Data interpretation:

From the above data, it is concluded that the method is robust.

4.5.1.6 Ruggedness

Ruggedness is a measure of reproducibility of test results under the variation in

conditions normally expected from laboratory to laboratory and from analyst to analyst.

This is to prove the lack of influence of operational and environmental variables of the

test results by using the method. The average of the six preparations and % RSD for the

six observations was calculated and recorded. The method precision was carried out as

described above using different analyst, different column and different instrument. The %

RSD for the six determinations shall be NMT 5.0.

Figure 4.10.13A: Chromatogram in Waters HPLC for Ruggedness studies

274

Figure 4.10.13B: Chromatogram in Waters HPLC for Ruggedness studies

Figure 4.10.13C: Chromatogram in Waters HPLC for Ruggedness studies

275

Figure 4.10.13D: Chromatogram in Waters HPLC for Ruggedness studies

Figure 4.10.13E: Chromatogram in Waters HPLC for Ruggedness studies

276

Figure 4.10.13F: Chromatogram in Waters HPLC for Ruggedness studies

Fig 4.10.14: Chromatograms in Agilent for Ruggedness studies

277

Data Interpretation:

From the data tabulated above it was concluded that the method is rugged.

4.10.1.7 Solution Stability:

A solution of standard and sample was prepared and stored at room temperature

for 36 hrs. The stability of solutions was checked for 36 hours. The different time

intervals are 0, 12, 22, 30, 36hr. This is to check whether the solution is stable throughout

the analysis. The difference in responses shall not be more than 2%

Table 4.8 Results of solution stability of Efavirenz

Efavirenz:

Solution Stability of Standard preparation

Solution Stability of Test preparation

Time in Hours

Area Response

% Difference

w.r.t. initial response

Time in Hours

Area Response

% Difference

w.r.t. initial response

Initial 4415698 NA Initial 4279179 NA

12 4418337 0.1 12 4272644 0.2

22 4430768 0.3 22 4284194 -0.1

30 4425754 0.2 30 4270452 0.2

36 4439119 0.5 36 4274359 0.1

278

Table 4.9 Results of solution stability of Lamivudine

Lamivudine:

Solution Stability of Standard preparation

Solution Stability of Test preparation

Time in Hours

Area Response

% Difference

w.r.t. initial response

Time in Hours

Area Response

% Difference

w.r.t. initial response

Initial 6137369 NA Initial 6018478 NA

12 6114247 0.4 12 6033927 -0.3

22 6139202 0.0 22 6045326 -0.4

30 6135862 0.0 30 6040380 -0.4

36 6145584 -0.1 36 6060048 -0.7

Table 4.10 Results of solution stability of Tenofovir

Tenofovir:

Solution Stability of Standard preparation

Solution Stability of Test preparation

Time in Hours

Area Response

% Difference

w.r.t. initial response

Time in Hours

Area Response

% Difference

w.r.t. initial response

Initial 3806690 NA Initial 4030670 NA

12 3773369 0.9 12 3993660 0.9

22 3749520 1.5 22 3958976 1.8

30 3724931 2.1 30 3919310 2.8

36 3696665 2.9 36 3906729 3.1

279

Data Interpretation:

The standard & Samples containing the ELT tablets in dissolution medium (water

+ 2%SLS) are found to be stable upto 22hours only.

4.10.1.8 Filter Interference:

To establish the suitability of filter and to validate the interference of the filters

with the sample or standard, the study was conducted using three different filters namely

0.45 μm PVDF filters, 0.45 μm PTFE and 0.45 nylon filters. The unfiltered standard

solutions and the centrifuged sample solutions were compared with the filtered standard

and samples. The interference of various types of filters with the standard & sample

solution is checked. The percentage difference for unfiltered and filtered Solutions should

not be more than 2.

Table 4.11 Results of Filter interference of Efavirenz

Efavirenz:

Injection no

Standard Solution Test Solution

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

1 4429164 4398763 4434750 4431731 4348750 4339922 4359533 42627212 4426606 4426920 4449668 4422192 4338271 4332436 4345702 4264919Average 4427885 4412842 4442209 4426962 4343511 4336179 4352618 4263820

% Difference

NA 0.3 -0.3 0.0 NA 0.2 -0.2 1.8

280

Table 4.12 Results of Filter interference of Lamivudine

Lamivudine:

Injection no

Standard Solution Test Solution

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

1 6107080 6083228 6120354 6120147 6087637 6080840 6081416 60366892 6125165 6122156 6154702 6103492 6075593 6082346 6089412 6016863Average 6116123 6102692 6137528 6111820 6081675 6083096 6085414 6026776% Difference

NA 0.2 -0.3 0.1 NA 0.0 -0.1 0.9

Table 4.13 Results of Filter interference of Tenofovir

Tenofovir:

Injection no

Standard Solution Test Solution

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

Centrifuged0.45µ Nylon

0.45µ PVDF

0.45µ PTFE

1 3792477 3775080 3794752 3801638 4059693 4057705 4035944 40098262 3798679 3796501 3813323 3788726 4049535 4057000 4036732 3992272Average 3795578 3785791 3804038 3795182 4054614 4057353 4036338 4001049% Difference

NA 0.3 -0.2 0.0 NA -0.1 0.5 1.3

Data Interpretation:

From the above results it is concluded that there is no interference of filters with

standard & sample solutions as the difference in responses is within the limit (NMT 2%)

4.10.1.9 System Suitability:

Five replicate injection of standard solution are injected and the chromatograms

were recorded. The system is suitable for analysis if

The relative standard deviation of area counts in the five replicate injections for

each peak should not be more than 2.0 %

281

The USP plate count of peak should not be less than 2000 theoretical plates for

HPLC

The tailing factor for each peak should not be more than 2.0

System suitability tests are used to verify the reproducibility of the

chromatographic System. To ascertain its effectiveness, system suitability tests were

carried out on freshly prepared stock solutions.

Table 4.14 Results of System Suitability

S. No

System Suitability Parameter

Observations Proposed Acceptance

CriteriaEfavirenz Lamivudine Tenofovir

1% RSD for six replicate

injections of analyte peak in standard solution

0.1 0.1 0.2 NMT 2

2Tailing factor for analyte peak

in standard solution1.0 1.0 1.0 NMT 2

3USP plate count for analyte peak in standard solution

30168 6391 26398 NLT 2000

4 Resolution 22.0 - 15.0 -

Data interpretation:

It was observed from the data tabulated above that the method complies with

system suitability parameters. Hence, it was concluded that the system suitability

parameter met the requirement of method validation.

282

Table 4.15 Optical Characteristic of the Proposed Method

Parameters Efavirenz Lamivudine Tenofovir DFλmax (nm) 260Linearity range (μg/ml)

150 – 900 75 – 450 75 – 450

Regression Equation Y=7106.2262x+17264.2668

Y= 20483.5110x-28749.3350

Y = 13845.5558x-431703.7575

Slope 7106.2262 20483.5110 13845.5558Intercept 17264.2668 -28749.3350 -431703.7575Correlation Coefficient (r2)

0.9997 0.9997 0.9993

%RSD 0.1 0.1 0.2Retention Time(min)

13.7 4.7 8.0

% Recovery 98.10 – 101.30 99.10 – 100.90 98.20 – 101.40Tailing Factor 1.0 1.0 1.0Plate Count 30168 6391 26398Resolution 22.0 - 15.0

4.11 SUMMARY

In this study an gradient RP-HPLC method for the simultaneous determination of

Efavirenz, Lamivudine and Tenofovir DF in formulation was developed and optimized.

The results of the study demonstrate the benefit of applying this approach in selecting

optimum conditions for the determination of drugs in formulation. The validation study

supported the selection of the assay conditions by confirming that the assay was specific,

accurate, linear, precise and robust. Therefore the proposed HPLC method can be used as

a routine quality control analysis in a pharmaceutical environment.

283

Validation Parameters

Acceptance Criteria HPLC Results

SpecificityThe peaks of diluent and

placebo should not interfere with the main peak

The peaks of diluent and placebo are not interfering with the main peaks of

Efavirenz, Lamivudine, Tenofovir DF.

System Precision

The %RSD of peaksobtained from the 10replicate injections

should be NMT 2.0%

Efavirenz Lamivudine Tenofovir

0.1 0.1 0.2

Method Precision

The % RSD for the sixdeterminations shall be

NMT 5.0.

Efavirenz Lamivudine Tenofovir

3.1 2.1 1.2

Accuracy

The % recovery at eachspike level shall be NLT98.0% and NMT 102.0%

of the added amount.

Efavirenz Lamivudine Tenofovir

99.7 99.9 99.6

LinearityThe Correlation

coefficient shall be NLT0.999

Efavirenz Lamivudine Tenofovir

0.9997 0.9997 0.9993

RobustnessAll the system suitabilityparameters should passfor all the conditions.

The system suitability parameters passed for all the Conditions.

RuggednessAll the system suitabilityparameters should passfor all the conditions.

The system suitability parameters passed for all the Conditions.

Solution Stability

The difference in responses shall not be more than 2.0%

Standard and sample solutions are stable at 25°C for 22hours

Filter Interference

The difference in responses shall not be more than 2.0%

Filter Interference was found to less below 2.0%

System Suitability

For 6 replicate injections Efavirenz Lamivudine TenofovirThe %RSD NMT 2.0% 0.1 0.1 0.2

Tailing factor NMT 2.0% 1.0 1.0 1.0Plate Count NLT 2000 30168 6391 26398

Resolution 22.0 - 15.0