Eco-friendly graphene quantum dots as a novel spectrofluorimetric probe for lamivudine quantification with evaluation of its greenness and blueness profiles.

In this study, graphene quantum dots (GQDs) were employed for quantitatively analyzing lamivudine using a fluorescence quenching technique. This approach allows for sensitive determination of the concentration of lamivudine in different matrices without requiring derivatization. The mechanism behind the fluorescence intensity quenching between GQDs and lamivudine molecules was explored using the Stern Volmer equation, revealing dynamic quenching behavior. Additionally, various factors affecting fluorescence quenching efficiency such as pH, GQDs concentration, and incubation time were carefully tuned. Moreover, our developed method successfully met ICH guidelines for validation parameters including linearity, accuracy, precision, and selectivity demonstrating excellent performance. The results showed good accuracy and precision, with a mean recovery value of 101.91% for method accuracy and a relative standard deviation of 0.682 and 1.489 for intraday and interday precision, respectively. Finally, the greenness and blueness of the developed method were also investigated to assess its environmental friendliness and analytical practicality. Greenness evaluation using the AGREE tool demonstrated that the developed method has a low environmental impact with an AGREE score of 0.75, Besides, the blueness evaluating using the BAGI tool indicated that the developed method is practical, reliable, and well-suited for routine analysis of lamivudine in various samples.

Box-Behnken design aided optimization and validation of developed reverse phase HPLC analytical method for simultaneous quantification of dolutegravir sodium and lamivudine co-loaded in nano-liposomes.

A stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of dolutegravir sodium and lamivudine encapsulated in the nanoliposomal formulation was developed. The chromatographic parameters namely, organic phase ratio, flow rate, and sample injection volume were selected as independent factors and were optimized by multivariate Box-Behnken design. Responses analyzed were retention time, peak area, and resolution. The optimized chromatographic method with Hypersil BDS C8 CN column as stationary phase and methanol and acetonitrile mixture and acidified Milli-Q water (pH 2.8, adjusted with 0.02% v/v orthophosphoric acid) as the mobile phase in an isocratic elution mode was validated according to parameters of International Conference on Harmonization Q1(R2) guidelines. The validated reversed-phase high-performance liquid chromatography method exhibited specificity for both dolutegravir sodium and lamivudine in the presence of degradation products as well as the liposomal matrix. This method was effectively utilized to determine the amount of drug entrapped and drug loading efficiency of dolutegravir sodium and lamivudine in a nano-liposomal formulation.

Islatravir in combination with doravirine for treatment-naive adults with HIV-1 infection receiving initial treatment with islatravir, doravirine, and lamivudine: a phase 2b, randomised, double-blind, dose-ranging trial.

BACKGROUND: Islatravir is a nucleoside reverse transcriptase translocation inhibitor in development for the treatment and prevention of HIV-1 infection. We aimed to assess the efficacy and safety of islatravir-based regimens for the treatment of HIV-1. METHODS: We did a phase 2b, randomised, double-blind, comparator-controlled, dose-ranging trial at 24 clinics or hospitals in four countries (Chile, France, the UK, and the USA). Treatment-naive adults (≥18 years) with plasma HIV-1 RNA concentrations of at least 1000 copies per mL, CD4 T-cell counts of at least 200 cells per mL, and a calculated creatinine clearance of at least 50 mL/min (all within 60 days before study treatment) were eligible for inclusion. Participants were randomly assigned (1:1:1:1) with a block size of four via an interactive voice and web response system to receive oral treatment with one of three doses of islatravir (0·25 mg, 0·75 mg, or 2·25 mg) plus doravirine (100 mg) and lamivudine (300 mg) or to doravirine (100 mg) plus lamivudine (300 mg) plus tenofovir disoproxil fumarate (TDF; 300 mg) once daily with placebo (part 1). Treatment groups were stratified according to screening HIV-1 RNA concentration (≤100 000 copies per mL or >100 000 copies per mL). After at least 24 weeks of treatment, participants taking islatravir who achieved an HIV-1 RNA concentration lower than 50 copies per mL switched to a two-drug regimen of islatravir and doravirine (part 2). All participants and study investigators were masked to treatment in part 1; in part 2, the islatravir dose was masked to all participants and investigators, but the other drugs were given open label. The primary efficacy outcomes were the proportions of participants with an HIV-1 RNA concentration lower than 50 copies per mL at weeks 24 and 48 (US Food and Drug Administration snapshot approach). The primary safety outcomes were the number of participants experiencing adverse events and the number of participants discontinuing study drug owing to adverse events. All participants who received at least one dose of any study drug were included in the analyses. This trial is ongoing, but closed to enrolment of new participants; herein, we report study findings through 48 weeks of treatment. This trial is registered with ClinicalTrials.gov, NCT03272347. FINDINGS: Between Nov 27, 2017, and April 25, 2019, 121 participants (mean age 31 years [SD 10·9], 112 [93%] male, 92 [76%] white, 27 [22%] with HIV-1 RNA concentration >100 000 copies per mL) were randomly assigned: 29 to the 0·25 mg, 30 to the 0·75 mg, and 31 to the 2·25 mg islatravir groups, and 31 to the doravirine, lamivudine, and TDF group. At week 24, 26 (90%) of 29 participants in the 0·25 mg islatravir group, 30 (100%) of 30 in the 0·75 mg islatravir group, and 27 (87%) of 31 in the 2·25 mg islatravir group achieved HIV-1 RNA concentrations lower than 50 copies per mL compared with 27 (87%) of 31 in the doravirine plus lamivudine plus TDF group (difference 2·8%, 95% CI -14·9 to 20·4, for the 0·25 mg islatravir group; 12·9%, -1·6 to 27·5, for the 0·75 mg islatravir group; and 0·3%, -17·9 to 18·5, for the 2·25 mg islatravir group). At week 48, these data were 26 (90%) of 29 in the 0·25 mg islatravir group, 27 (90%) of 30 in the 0·75 mg islatravir group, and 24 (77%) of 31 in the 2·25 mg islatravir group compared with 26 (84%) of 31 in the doravirine plus lamivudine plus TDF group (difference 6·1%, 95% CI -12·4 to 24·4, for the 0·25 mg islatravir group; 6·2%, -12·2 to 24·6, for the 0·75 mg islatravir group; and -6·1%, -27·1 to 14·8, for the 2·75 mg islatravir group). 66 (73%) of participants in the islatravir groups combined and 24 (77%) of those in the doravirine plus lamivudine plus TDF group reported at least one adverse event. Two participants in the 2·25 mg islatravir group and one participant in the doravirine plus lamivudine plus TDF group discontinued owing to an adverse event. No deaths were reported up to week 48. INTERPRETATION: Treatment regimens containing islatravir and doravirine showed antiviral efficacy and were well tolerated regardless of dose. Doravirine in combination with islatravir has the potential to be a potent two-drug regimen that warrants further clinical development. FUNDING: Merck, Sharp, & Dohme Corp, a subsidiary of Merck & Co., Inc.

A simple alternative and improved HPLC method for the estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in solid oral dosage form.

A novel method was developed for the simultaneous estimation of the doravirine, lamivudine, and tenofovir disoproxil fumarate in the pharmaceutical dosage form. The chromatogram was run through an Ascentis C18 column (150 × 4.6 mm, 2.7 µm), with the mobile phase consisting of a phosphate buffer and acetonitrile in the ratio of 50:50 (v/v). The mobile phase was pumped through the column at a flow rate of 1 mL/min. The column temperature was maintained at 30°C. The optimized wavelength for doravirine, lamivudine, and tenofovir disoproxil fumarate was 230.0 nm. The retention times for doravirine, lamivudine, and tenofovir disoproxil were 2.222, 2.764, and 3.403 min, respectively; the relative standard deviation (%) values of method precision for doravirine, lamivudine, and tenofovir disoproxil were 0.6, 0.6, and 0.1, respectively. The % recovery was 100.20%, 100.15%, and 100.36% for doravirine, lamivudine, and tenofovir disoproxil fumarate, respectively. The limit of detection and limit of quantification values were obtained from regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate, and were 0.24 and 0.73 ppm, 0.53 and 1.60 ppm, and 0.47 and 1.43 ppm, respectively. The regression equations of doravirine, lamivudine, and tenofovir disoproxil fumarate were y = 17,541x + 117,303, y = 15,555x + 10,791, and y = 15,250x + 31,663, respectively. The method developed was accurate, simple, precise, sensitive, and economical. Hence, it could be adopted for regular quality control for estimation of doravirine, lamivudine, and tenofovir disoproxil fumarate in pharmaceutical industries.

Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach.

Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors' knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.

Factors influencing hair lamivudine concentration among people living with HIV in Guangxi, China.

BACKGROUND: Hair antiretroviral concentration has served as an innovative and objective measure of antiretroviral adherence. However, some factors (for example, pharmacokinetics and hair characteristics) may contribute to the variability of hair antiretroviral concentration that may threaten the validity and reliability of the hair measure as a biomarker of adherence. This study aimed to examine the potential factors that may influence the measure of hair antiretroviral concentration. METHODS: Hair samples from a cohort of 372 people living with HIV (PLHIV) receiving lamivudine (300 mg/day) in Guangxi, China. Lamivudine concentration was analysed using liquid chromatography-tandem mass spectrometry. Multivariable linear regression was used to evaluate the associations of hair lamivudine concentration with age, sex, ethnicity, height, weight, body mass index, duration of HIV diagnosis, duration of current regimen, dosing schedule, concomitant antiretroviral medications, frequency of hair washing, hair care products use, hair cosmetic treatment and self-reported adherence. RESULTS: Multivariable models revealed that frequency of hair washing (ß=-0.221, P=0.001), dosing schedule (ß=0.141, P=0.036) and self-reported adherence (ß=0.160, P=0.002) were associated with hair lamivudine concentration. CONCLUSIONS: We observed that, among those potential factors, hair lamivudine concentration was influenced by frequency of hair washing and dosing schedule. Therefore, frequency of hair washing and dosing schedule should be considered in future research using hair lamivudine concentration as a measure of lamivudine exposure and biomarker of adherence.

Simple thin layer chromatography-ultraviolet spectrophotometric method for quality assessment of binary fixed-dose-combinations of lamivudine/tenofovir disoproxil fumarate and lamivudine/zidovudine in tablet formulations.

Antiretroviral fixed-dose-combination drugs are best assayed with high-performance liquid chromatography, or liquid chromatography-tandem mass spectrometry. However, most scientists in developing nations have no access to these expensive instruments. A more affordable quantitative technique is the use of ultraviolet-visible spectroscopy-where often the absorption spectra of these antiretrovirals are overlapping; thus complex derivative methodologies are required for quantification. A simple, rapid, and accurate thin layer chromatography-ultraviolet spectrophotometric method for the quantification of binary mixtures of lamivudine, zidovudine, and tenofovir-disoproxil-fumarate in tablet formulations was developed. Lamivudine/tenofovir-disoproxil-fumarate and lamivudine/zidovudine were extracted and separated on glass thin-layer chromatography plates. Drugs were identified in ultraviolet light at 254 nm and quantified in acidic medium using ultraviolet spectrophotometry. The retardation factors were 0.43, 0.79, and 0.81 for lamivudine, tenofovir-disoproxil-fumarate, and zidovudine, respectively, with corresponding absorption maxima at 270, 260, and 265 nm. Linearity ranged from 1 to 40 µg/mL for all drugs (R = 0.9998-0.9999), while recovery studies were 95.10-102.11% and amount in formulations ranged from 97.99 ± 0.63 to 101.47 ± 2.39%. The paired t-test (n = 5) indicated no significant difference between the proposed and high-performance liquid chromatography methods, hence comparable and can be used as an alternative method in routine quality determination of antiretroviral medicines.

[Chemical and toxicological determination of lamivudine in biological substances]. / Khimiko-toksikologicheskoe opredelenie lamivudina v biologicheskikh ob''ektakh.

The purpose of the study is to develop a method for detecting, isolating and quantifying lamivudine in biological substances. Lamivudine was isolated by liquid-liquid and solid phase extraction. The conditions for isolating lamivudine (extractant, pH of the medium, electrolyte, time and frequency of extraction) from aqueous solutions were selected and methods were developed for isolating it from biological substances, including urine, saliva and liver, using liquid-liquid and solid phase extraction methods. Qualitative and quantitative analysis of lamivudine in extracts from urine, saliva and liver was performed by thin layer chromatography, UV spectrophotometry and high-performance liquid chromatography. A validation assessment of the developed techniques indicates their suitability for chemical and toxicological analysis of lamivudine.

Zidovudine and Lamivudine as Potential Agents to Combat HIV-Associated Neurocognitive Disorder.

The central nervous system has been identified as an anatomical reservoir for HIV due the difficulties in delivering therapeutic agents into the brain and this complication results in HIV-associated neurocognitive disorder that persists in infected patients. The brain regions that are potentially exposed to tissue deficits due to HIV have been reported in previous reports; therefore, it is important to determine the drugs that can enter and localize in brain regions that are known to be susceptible to HIV neurodegeneration. Sprague-Dawley rats received intraperitoneal doses of zidovudine and lamivudine (50 mg kg-1). Mass spectrometry methods were used to determine the pharmacokinetics, of zidovudine and lamivudine, in the brain using liquid chromatography tandem mass spectrometry and mass spectrometry imaging (MSI), respectively. Zidovudine and lamivudine displayed complementary pharmacokinetic curves indicating a rapid absorption and blood-brain barrier penetration of both drugs reaching Cmaxat 0.5 h after single dose. MSI of coronal brain sections showed that zidovudine and lamivudine are mostly distributed in corpus callosum, globus pallidus, striatum, and the neocortex region. Mass spectrometry techniques were used to demonstrate that zidovudine and lamivudine drugs are able to reach and localize in brain regions that are targets of HIV neurodegeneration in the brain.

Total drug quantification in prodrugs using an automated elemental analyzer.

Polymeric prodrugs have become an increasingly popular strategy for improving the pharmacokinetic properties of active pharmaceutical ingredients (API). Therefore, identifying a robust method for quantification of the API in these prodrug products is a key part of the drug development process. Current drug quantification methods include hydrolysis followed by reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC)-based molecular weight determination, and mass spectrometry. These methods tend to be time-consuming and often require challenging method development. Here, we present a comparative study highlighting the automated elemental analyzer as a facile approach to drug quantification in this up-and-coming class of therapeutics. A polymeric prodrug using poly(L-lysine succinylated) (PLS) and the drug lamivudine (LAM) was prepared and analyzed using the elemental analyzer in comparison to the traditional approaches of hydrolysis followed by RP-HPLC and SEC using multi-angle light scattering (MALS) detection. The elemental analysis approach showed excellent agreement with the conventional methods but proved much less laborious, highlighting this as a rapid and sensitive analytical method for the quantitative determination of drug loading in polymeric prodrug products.

Risk of false conformity decisions of multicomponent items controlled by correlated measurement results due to the sharing of analytical steps.

The assessment of the conformity of some items, such as medicines, food products or drinking waters, with limits set for several of their components, involves the determination of these components using multi-analyte measurement procedures. Since these determinations involve the sharing of relevant analytical steps, such as the sample preparation and analytical instrument run, the measurement results of the various components become correlated (i.e. 'between components metrologically correlated'). The closeness of the values of the components to the respective tolerance limits, the measurements uncertainty and the correlation of the measurements results affects the risk of false conformity decisions of the analysed item. This correlation can either increase or decrease the risk of false conformity decision and is relevant to decide if the item should be considered conform or not conform with the regulation. This work presents a methodology to estimate the 'between components metrological correlation' of results of the analysis of an item subsequently used to assess the impact of this correlation on the risk of false conformity decisions. The methodology was successfully applied to the assessment of the conformity of pharmaceutical tablets against tolerance limits for lamivudine (3TC) and zidovudine (AZT) determined from the analysis of the same analytical portion in the same HPLC-UV/Vis run. The correlation of measurement results was determined from Monte Carlo simulations of shared analytical operation (linear correlation coefficient of 0.53) being their impact on conformity decisions relevant. For instance, for measurement results of 3TC and AZT equal to the upper limit and lower limit, respectively, the risk of a wrong acceptance of the medicines is 84% while if it is assumed that measurement results are independent this risk would be wrongly considered as 75%. The Excel® spreadsheet used for this assessment is made available as supplementary material.

One-by-one imprinting in two eccentric layers of hollow core-shells: Sequential electroanalysis of anti-HIV drugs.

Double layered one-by-one imprinted hollow core-shells@ pencil graphite electrode was fabricated for sequential sensing of anti-HIV drugs. For this, two eccentric layers were developed on the surface of vinylated silica nanospheres to obtain double layered one-by-one imprinted solid core-shells. This yielded hollow core-shells on treatment with hydrofluoric acid. The modified hollow core-shells (single layered dual imprinted) evolved competitive diffusion of probe/analyte molecules. However, the corresponding double layered one-by-one imprinted hollow core-shells (outer layer imprinted with Zidovudine, and inner layer with Lamivudine) were found relatively better owing to their bilateral diffusions into molecular cavities, without any competition. The entire work is based on differential pulse anodic stripping voltammetry at double layered one-by-one imprinted hollow core-shells. This resulted in indirect detection of electro inactive targets with limits of detection as low as 0.91 and 0.12 (aqueous sample), 0.94 and 0.13 (blood serum), and 0.99 and 0.20 ng mL-1 (pharmaceutics) for lamivudine and zidovudine, respectively in anti-HIV drug combination.

Simultaneous quantification of four antiretroviral drugs in breast milk samples from HIV-positive women by an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method.

The primary strategy to avoid mother-to-child transmission of human immunodeficiency virus (HIV) through breastfeeding is administration of highly active antiretroviral therapy (HAART) to HIV-positive pregnant women. Because significant changes in the pharmacokinetics of antiretroviral (ARV) drugs occur during pregnancy, quantifying HAART and the viral load in breast milk in this population is essential. Here, we developed an analytical assay for the simultaneous quantification of four ARV drugs in breast milk using ultra-performance liquid chromatography coupled to tandem mass spectrometry. We validated this method following Mexican and international guidelines. ARV drugs. We extracted the ARV drugs from 200 µL samples of breast milk and detected these drugs in a triple quadrupole mass spectrometer with positive electrospray ionization. The validated concentration ranges (ng/mL) for zidovudine, lamivudine, lopinavir, and ritonavir were 12.5-750, 50-2500, 100-5000 and 5 to 250, respectively. Additionally, the absolute recovery percentages (and matrix effects) were 91.4 (8.39), 88.78 (28.75), 91.38 (11.77) and 89.78 (12.37), respectively. We determined that ARV drugs are stable for 24 h at 8°C and 24°C for 15 days at -80°C. This methodology had the capacity for simultaneous detection; separation; and accurate, precise quantification of ARV drugs in human breast milk samples according to Mexican standard laws and United States Food and Drug Administration guidelines.

Processo de fabricação de comprimidos de lamivudina e zidovudina (150+300mg): avaliação retrospectiva da variabilidade e desenvolvimento de metodologia analítica por espectroscopia no infravermelho próximo com transformada de Fourier (FT-NIR) aplicada a avaliação da homogeneidade da mistura de pós / Lamivudine and zidovudine tablet manufacturing process (150 + 300mg): retrospective evaluation of the variability and development of analytical methodology by Fourier transform (FT-NIR) near infrared spectroscopy applied to the evaluation of homogeneity of the powder mixture

O uso de ferramentas estatísticas no ciclo de vida de um produto farmacêutico permite verificar e controlar o processo tendo como objetivo a sua melhoria contínua. No presente estudo foi avaliada a estabilidade e a capacidade estatística do processo de fabricação dos comprimidos revestidos de lamivudina 3TC e zidovudina AZT (150 + 300 mg) fabricados pela Fundação para o Remédio Popular "Chopin Tavares de Lima" (FURP). Esse medicamento, distribuido gratuitamente pelo programa DST/AIDS do Ministério da Saúde, e fabricado por compressão direta, processo rápido que permite a implementação futura da tecnologia analítica de processo (Process Analytical Technology - PAT). No Capítulo I foi realizada avaliação retrospectiva da variabilidade de atributos criticos da qualidade de 529 lotes dos comprimidos fabricados de acordo com a RDC ANVISA 17/2010 e as monografias oficiais, sendo tais atributos: peso médio, uniformidade de dose unitária e % m/v de fármaco dissolvido, antes e após o revestimento. O objetivo foi identificar eventuais causas especiais de variabilidade dos processos que permitam melhorias contínuas. No Capitulo II foi desenvolvida metodologia analítica empregando a espectroscopia no infravermelho próximo com transformada de Fourier para a avaliação da homogeneidade da mistura dos pós. Nesse estudo foram analisadas amostras de misturas dos fármacos lamivudina 3TC e zidovudina AZT e mistura excipiente, empregando como método de referência a CLAE, para a quantificação desses dois fármacos. No Capitulo I, a avaliação do processo para o peso médio revelou a necessidade de investigação das causa especiais de variabilidade, evidenciada por meio das cartas de controle. Os resultados do ano de 2015 indicaram necessidade de centralização e de consistência do processo, com redução de probabilidade de falha. As cartas de controle para uniformidade de dose unitária, no ano de 2013, revelaram menor variabilidade do processo. Porem, nesse ano, a análise estatística para a dissolução revelou processo descentralizado e sem consistência, com maior evidência para o fármaco 3TC que demonstrou menor desempenho, Cpk<1,0. A avaliação da estabilidade e da capacidade do processo de fabricação de comprimidos de lamivudina + zidovudina (150+300 mg), no período de 2012 a 2015, permitiu o maior entendimento de suas fontes de variação. Foi possível detectar e determinar o grau dessa variação e seu impacto no processo e nos atributos críticos de qualidade do produto com evidentes oportunidades de melhoria do processo, reduzindo os riscos para o paciente. No capítulo II, no desenvolvimento do método, as estatísticas de validação revelaram que os menores valores de BIAS foram observados para a 3TC, 0,000116 e 0,0021, respectivamente para validação cruzada e validação. Os valores de BIAS próximos a zero indicaram reduzida porcentagem de variabilidade do método. O presente estudo demonstrou a viabilidade do uso do modelo desenvolvido para a quantificação da 3TC e AZT por FT-NIR apos ajustes que contribuam para a elevação de R, R2 e RPD para valores aceitáveis. Valores de RPD acima de 5,0 que permitem o uso do modelo para uso em controle de qualidade

The use of statistical tools in the life cycle of a pharmaceutical product allows verifying and controlling the process aiming at its continuous improvement. In the present study, the stability and statistical capacity of the lamivudine coated tablets 3TC and zidovudine AZT (150 + 300 mg) manufactured by the Chopin Tavares de Lima Foundation (FURP) were evaluated. This drug, distributed free of charge by the Ministry of Health's DST/AIDS program, is manufactured by direct compression, a rapid process that allows the future implementation of Process Analytical Technology (PAT). In Chapter I, a retrospective evaluation of the variability of critical quality attributes of 529 batches of tablets manufactured was carried out, such attributes being: mean weight, unit dose uniformity and % m/v of dissolved drug substances, before and after coating. The objective was to identify possible special causes of variability of the processes that allow continuous improvements. In Chapter II an analytical methodology was developed employing the near infrared spectroscopy with Fourier transform for the evaluation of the homogeneity of the powder mixture. In this study, samples of mixtures of the drugs lamivudine 3TC and zidovudine AZT and excipient mixture were analyzed, using as reference method the HPLC, for the quantification of these two drugs. In Chapter I, the evaluation of the process for the mean weight revealed the need to investigate the special cause of variability, as evidenced by the charts. The results of the year 2015 indicated the need for centralization and process consistency, with a reduction in the probability of failure. The control charts for unit dose uniformity, in the year 2013, revealed less process variability. However, in that year, the statistical analysis for dissolution revealed a decentralized process with no consistency, with greater evidence for the 3TC drug that showed lower performance, Cpk<1.0. The evaluation of the stability and capacity of the lamivudine + zidovudine tablet manufacturing process (150 + 300 mg) in the period from 2012 to 2015 allowed a better understanding of its sources of variation. It was possible to detect and determine the degree of this variation and its impact on the process and the critical quality attributes of the product with evident opportunities to improve the process, reducing risks for the patient. In Chapter II, in the development of the method, the validation revealed that the lowest values of BIAS were observed for 3TC, 0.000116 and 0.0021, respectively for cross validation and validation. BIAS values close to zero indicated a reduced percentage of variability of the method. The present study demonstrated the feasibility of using the model developed for the quantification of 3TC and AZT by FT-NIR after adjustments that contribute to the elevation of R, R2 and RPD to acceptable values. RPD values above 5.0 that allow the use of the model for use in quality control

Development, validation and clinical application of a method for the simultaneous quantification of lamivudine, emtricitabine and tenofovir in dried blood and dried breast milk spots using LC-MS/MS.

OBJECTIVES: To present the validation and clinical application of a LC-MS/MS method for the quantification of lamivudine (3TC), emtricitabine (FTC) and tenofovir (TFV) in dried blood spots (DBS) and dried breast milk spots (DBMS). METHODS: DBS and DBMS were prepared from 50 and 30µL of drug-spiked whole blood and human breast milk, respectively. Following extraction with acetonitrile and water, chromatographic separation utilised a Synergi polar column with a gradient mobile phase program consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection and quantification was performed using a TSQ Quantum Ultra triple quadrupole mass spectrometer. The analytical method was used to evaluate NRTI drug levels in HIV-positive nursing mothers-infant pairs. RESULTS: The assay was validated over the concentration range of 16.6-5000ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2-1250ng/mL. Intra and inter-day precision (%CV) ranged from 3.5-8.7 and accuracy was within 15% for all analytes in both matrices. The mean recovery in DBS was >61% and in DBMS >43% for all three analytes. Matrix effect was insignificant. Median AUC0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165-6057) and 6050 (5217-6417)ngh/mL for 3TC, 3312 (2259-4312) and 4853 (4124-6691)ngh/mL for FTC and 1559 (930-1915) and 56 (45-80)ngh/mL for TFV. 3TC and FTC were quantifiable (>16.6ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants. CONCLUSIONS: DBS and DBMS sampling for bioanalysis of 3TC, FTC and TFV is straightforward, robust, accurate and precise, and ideal for use in low-resource settings.

Lamivudine Concentration in Hair and Prediction of Virologic Failure and Drug Resistance among HIV Patients Receiving Free ART in China.

BACKGROUND: The assessment of adherence to antiretroviral therapy (ART) is important in order to predict treatment outcomes. Lamivudine (3TC) is one of the most widely used NRTIs in China, but its concentrations in hair and association with virologic failure and drug resistance have not been studied. METHODS: We conducted a cross-sectional survey to investigate 3TC concentrations in hair as a predictor of virologic failure and drug resistance among HIV patients receiving free ART. We also compared the capacity of hair 3TC concentrations with self-reported adherence in predicting virologic responses. Hair 3TC concentrations were detected through the LC-MS/MS system. RESULTS: In patients without HIV drug resistance (HIVDR), with a threshold hair 3TC concentration of 260 ng/g, the sensitivity and specificity in predicting virologic suppression were 76.9% and 89.9%, respectively. Some factors, including CD4+ cell counts, initial treatment regimens with 3TC, and current regimens with second-line drugs, influenced the association between hair 3TC concentrations and virologic suppression. In patients who experienced virologic failure with HIVDR, with a threshold of 180 ng/g, the sensitivity and specificity were 70.0% and 74.4%, respectively. Hair 3TC concentrations had higher sensitivity and specificity in predicting virologic failure and drug resistance than self-reported adherence. CONCLUSIONS: The hair 3TC concentration was a stronger indicator than self-reported adherence in predicting virologic failure and drug resistance in HIV patients receiving free ART.

A simultaneous determination of related substances by high performance liquid chromatography in a drug product using quality by design approach.

The combination of Abacavir, Lamivudine and Dolutegravir is an anti-retroviral formulation that displays high efficacy and superiority in comparison to other anti-retroviral combinations. Analysis of related substances in this combination drug product was very challenging due to the presence of nearly thirty peaks including the three active pharmaceutical ingredients (APIs), eleven known impurities and other pharmaceutical excipients. Objective of this study was to develop a single, selective, and robust high performance liquid chromatography method for the efficient separation of all peaks. Initially, one-factor-at-a-time (OFAT) approach was adopted to develop the method. But, it could not resolve all the critical peaks in such complex matrix. This led to the advent of two different HPLC methods for the determination of related substances, one for Abacavir and Lamivudine and the other for Dolutegravir. But, since analysis of a single sample using two methods instead of one is time and resource consuming and thus expensive, an attempt was made to develop a single and robust method by adopting quality by design (QbD) principles. Design of Experiments (DoE) was applied as a tool to achieve the optimum conditions through Response surface methodology with three method variables, pH, temperature, and mobile phase composition. As the study progressed, it was discovered that establishment of the design space was not viable due to the completely distant pH requirements of the two responses, i.e. (i) retention time for Lamivudine carboxylic acid and (ii) resolution between Abacavir impurity B and unknown impurity. Eventually, neglecting one of these two responses each time, two distinguished design spaces have been established and verified. Edge of failures at both design spaces indicate high probability of failure. It therefore, becomes very important to identify the most robust zone or normal operating range (NOR) within the design space with low risk of failure and high quality assurance. For NOR establishment, Monte Carlo simulation was performed on the basis of which process capability index (Cpk) was derived. Finally, the selectivity issue problem faced due to the pH dependency and the dissimilar pH needs of the two critical responses was resolved by introducing pH gradient into the program. This new ternary gradient program has provided a single robust method. Thus, two HPLC methods for the analysis of the combination drug product have been replaced with a selective, robust, and cost effective single method.

Photolysis of three antiviral drugs acyclovir, zidovudine and lamivudine in surface freshwater and seawater.

Photodegradation is an important elimination process for many pharmaceuticals in surface waters. In this study, photodegradation of three antiviral drugs, acyclovir, zidovudine, and lamivudine, was investigated in pure water, freshwater, and seawater under the irradiation of simulated sunlight. Results showed that zidovudine was easily transformed via direct photolysis, while acyclovir and lamivudine were mainly transformed via indirect photolysis. We found that in freshwater, nitrate enhanced the photodegradation of the three antiviral drugs, bicarbonate promoted the photodegradation of acyclovir, and dissolved organic matter (DOM) accelerated the photolysis of acyclovir and lamivudine. In seawater, the photolysis of acyclovir was not susceptible to Cl(-), Br(-) and ionic strength; however, the photolysis of zidovudine was inhibited by Cl(-) and Br(-), and the photolysis of lamivudine was enhanced by Cl(-), Br(-) and ionic strength. Second-order reaction rate constants for the three antiviral drugs with (1)O2 (k1O2) and OH (kOH) were also measured. These results are important for fate and ecological risk assessment of the antiviral drugs in natural waters.

Spectra resolution for simultaneous spectrophotometric determination of lamivudine and zidovudine components in pharmaceutical formulation of human immunodeficiency virus drug based on using continuous wavelet transform and derivative transform techniques.

In the present paper, two spectrophotometric methods based on signal processing are proposed for the simultaneous determination of two components of an anti-HIV drug called lamivudine (LMV) and zidovudine (ZDV). The proposed methods are applied to synthetic binary mixtures and commercial pharmaceutical tablets without the need for any chemical separation procedures. The developed methods are based on the application of Continuous Wavelet Transform (CWT) and Derivative Spectrophotometry (DS) combined with the zero cross point technique. The Daubechies (db5) wavelet family (242 nm) and Dmey wavelet family (236 nm) were found to give the best results under optimum conditions for simultaneous analysis of lamivudine and zidovudine, respectively. In addition, the first derivative absorption spectra were selected for the determination of lamivudine and zidovudine at 266 nm and 248 nm, respectively. Assaying various synthetic mixtures of the components validated the presented methods. Mean recovery values were found to be between 100.31% and 100.2% for CWT and 99.42% and 97.37% for DS, respectively for determination of LMV and ZDV. The results obtained from analyzing the real samples by the proposed methods were compared to the HPLC reference method. One-way ANOVA test at 95% confidence level was applied to the results. The statistical data from comparing the proposed methods with the reference method showed no significant differences.