Application of novel plasma separation filter cards for quantification of nucleoside/nucleotide reverse transcriptase inhibitor di/triphosphates in dried blood spots using LC-MS.

Background: A rapid and sensitive LC-MS method has been developed and validated for the quantification of nucleoside di/triphosphates using a novel plasma separation card (HemaSep). Materials & methods: Cards were spotted with whole blood and stored at -80°C. Metabolites were extracted using 70:30 MeOH:20% formic acid, followed by weak anion exchange SPE and eluted using a Biobasic-AX column. Quantification was performed using a triple quadrupole mass spectrometer with a calibration range of 1.25-250 pmol/sample. Results: The recovery of metabolites was high (>93%). Precision and accuracy were acceptable and metabolites remained stable on the card after 29 days (stored at ambient temperature). Conclusion: HemaSep dried blood spots are a useful microsampling tool and offer an alternative to liquid plasma as they maintain stability over time.

Undeclared pre-exposure or post-exposure prophylaxis (PrEP/PEP) use among syphilis-positive blood donors, England, 2020 to 2021.

An individualised blood donor selection policy was implemented in the United Kingdom from summer 2021. We have investigated the impact of this policy by comparing the extent of undeclared use of HIV pre-exposure or post-exposure prophylaxis (PrEP/PEP) before and after this change. The rate of PrEP usage in syphilis-positive male blood donors has not changed since individualised donor assessment was implemented but provides continuing evidence of undisclosed PrEP use which may be associated with current or past higher-risk sexual behaviours.

Pharmacokinetics of ß-d-N4-Hydroxycytidine, the Parent Nucleoside of Prodrug Molnupiravir, in Nonplasma Compartments of Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

ß-d-N4-hydroxycytidine (NHC), the parent nucleoside of molnupiravir, a COVID-19 antiviral, was quantified at SARS-CoV-2 transmission sites in 12 patients enrolled in AGILE Candidate-Specific Trial-2. Saliva, nasal, and tear NHC concentrations were 3%, 21%, and 22% that of plasma. Saliva and nasal NHC were significantly correlated with plasma (P < .0001). Clinical Trials Registration. NCT04746183.

Evidence of HIV pre-exposure or post-exposure prophylaxis (PrEP/PEP) among blood donors: a pilot study, England June 2018 to July 2019.

OBJECTIVE: Due to increased use of pre-exposure prohylaxis (PrEP) and its potential to affect HIV screening of blood donors, we undertook antiretroviral residual testing among HIV-negative male donors in England. METHODS: Residual plasma samples were obtainnd from 46 male donors confirmed positive for syphilis and 96 donors who were repeat reactive for HIV antibodies in screening but confirmed as HIV-negative by reference testing. These were tested for concentrations of tenofovir and emtricitabine by high-performance liquid chromatograhpy coupled with mass spectrometry. RESULTS: We found evidence of pre-exposure or post-exposure prophylaxis (PrEP/PEP) use in three male blood donors confirmed positive for syphilis (3 out of 46 screened, 6.5%). Two were estimated to have taken PrEP/PEP within a day of donating, and the third within 2 days. Two were new donors, whereas one had donated previously but acquired syphilis infection after his last donation. CONCLUSIONS: Our findings indicate that a small proportion of blood donors have not been disclosing PrEP/PEP use and therefore donating in non-compliance to donor eligibility criteria.

An Open Label, Adaptive, Phase 1 Trial of High-Dose Oral Nitazoxanide in Healthy Volunteers: An Antiviral Candidate for SARS-CoV-2.

Repurposing approved drugs may rapidly establish effective interventions during a public health crisis. This has yielded immunomodulatory treatments for severe coronavirus disease 2019 (COVID-19), but repurposed antivirals have not been successful to date because of redundancy of the target in vivo or suboptimal exposures at studied doses. Nitazoxanide is a US Food and Drug Administration (FDA) approved antiparasitic medicine, that physiologically-based pharmacokinetic (PBPK) modeling has indicated may provide antiviral concentrations across the dosing interval, when repurposed at higher than approved doses. Within the AGILE trial platform (NCT04746183) an open label, adaptive, phase I trial in healthy adult participants was undertaken with high-dose nitazoxanide. Participants received 1,500 mg nitazoxanide orally twice-daily with food for 7 days. Primary outcomes were safety, tolerability, optimum dose, and schedule. Intensive pharmacokinetic (PK) sampling was undertaken day 1 and 5 with minimum concentration (Cmin ) sampling on days 3 and 7. Fourteen healthy participants were enrolled between February 18 and May 11, 2021. All 14 doses were completed by 10 of 14 participants. Nitazoxanide was safe and with no significant adverse events. Moderate gastrointestinal disturbance (loose stools or diarrhea) occurred in 8 participants (57.1%), with urine and sclera discoloration in 12 (85.7%) and 9 (64.3%) participants, respectively, without clinically significant bilirubin elevation. This was self-limiting and resolved upon drug discontinuation. PBPK predictions were confirmed on day 1 but with underprediction at day 5. Median Cmin was above the in vitro target concentration on the first dose and maintained throughout. Nitazoxanide administered at 1,500 mg b.i.d. with food was safe with acceptable tolerability a phase Ib/IIa study is now being initiated in patients with COVID-19.

The development and validation of a novel LC-MS/MS method for the simultaneous quantification of Molnupiravir and its metabolite ß-d-N4-hydroxycytidine in human plasma and saliva.

In light of the recent global pandemic, Molnupiravir (MPV) or EIDD-2801, developed for the treatment of patients with uncomplicated influenza, is now being trialled for the treatment of infections caused by highly pathogenic coronaviruses, including COVID-19. A sensitive LC-MS/MS method was developed and validated for the simultaneous quantification of MPV and its metabolite ß-d-N4-hydroxycytidine (NHC) in human plasma and saliva. The analytes were extracted from the matrices by protein precipitation using acetonitrile. This was followed by drying and subsequently injecting the reconstituted solutions onto the column. Chromatographic separation was achieved using a polar Atlantis C18 column with gradient elution of 1 mM Ammonium acetate in water (pH4.3) and 1 mM Ammonium acetate in acetonitrile. Analyte detection was conducted in negative ionisation mode using SRM. Analysis was performed using stable isotopically labelled (SIL) internal standards (IS). The m/z transitions were: MPV (328.1→126.0), NHC (258.0→125.9) and MPV-SIL (331.0→129.0), NHC-SIL (260.9→128.9). Validation was over a linear range of 2.5-5000 ng/ml for both plasma and saliva. Across four different concentrations, precision and accuracy (intra- and inter-day) were 15%; and recovery of both analytes from plasma and saliva was between 95% and 100% and 65-86% respectively. Clinical pharmacokinetic studies are underway utilising this method for determination of MPV and its metabolite in patients with COVID-19 infection.

Pharmacokinetic/pharmacodynamic investigation of raltegravir with or without lamivudine in the context of HIV-1 pre-exposure prophylaxis (PrEP).

BACKGROUND: To characterize their potential use in pre-exposure prophylaxis (PrEP) we compared the pharmacokinetics of raltegravir and lamivudine in genital tissue against ex vivo tissue infection with HIV-1. METHODS: Open-label trial of 36 HIV-negative females and males randomized to 7 days raltegravir 400 mg twice daily and 7 days raltegravir 400 mg+lamivudine 150 mg twice daily (after washout), or vice versa. Blood, saliva, rectal fluid, rectal tissue, vaginal fluid and vaginal tissue were sampled at baseline and on and off PrEP during a total of 12 days, for pharmacokinetics and antiviral activity via ex vivo HIV-1BaL challenge. Ex vivo infectivity was compared with baseline. The trial has been registered in https://clinicaltrials.gov/ with the identifier NCT03205566. RESULTS: Steady state for both drugs was reached by day 4. Dosing with raltegravir alone provided modest ex vivo HIV protection with higher drug levels in rectal tissue and vaginal tissue than in plasma on and off PrEP. Off PrEP, plasma and vaginal concentrations declined rapidly, while persisting in the rectum. On PrEP, the highest lamivudine concentrations were in the rectum, followed by vaginal tissue then plasma. Lamivudine washout was rapid in plasma, while persisting in the rectum and vagina. Raltegravir/lamivudine increased ex vivo protection on and off PrEP compared with raltegravir alone, reaching maximum protection at day 2 in rectal tissue and at day 8 in vaginal tissue. CONCLUSIONS: Raltegravir 400 mg+lamivudine 150 mg showed high levels of ex vivo HIV protection, associated with high drug concentrations persisting after discontinuation in vaginal and rectal compartments, supporting further investigation of these agents for PrEP.

Validation and clinical application of a method to quantify efavirenz in cervicovaginal secretions from flocked swabs using liquid chromatography tandem mass spectrometry.

Background: A liquid chromatography tandem mass spectrometry method to quantify drugs in dried cervicovaginal secretions from flocked swabs was developed and validated using the antiretroviral efavirenz as an example. Methods: Cervicovaginal swabs (CVS) were prepared by submerging flocked swabs in efavirenz-spiked matrix. Time to full saturation, weight uniformity, recovery and room temperature stability were evaluated. Chromatographic separation was on a reverse-phase C18 column by gradient elution using 1mM ammonium acetate in water/acetonitrile at 400 µL/min. Detection and quantification were on a TSQ Quantum Access triple quadrupole mass spectrometer operated in negative ionisation mode. The method was used to quantify efavirenz in CVS samples from human immunodeficiency virus (HIV)-positive women in the VADICT study (NCT03284645). A total of 98 samples (35 paired intensive CVS and DBS samples, 14 paired sparse CVS and DBS samples) from 19 participants were available for this analysis. Results: Swabs were fully saturated within 15 seconds, absorbing 128 µL of matrix with coefficient of variation (%CV) below 1.3%. The method was linear with a weighting factor (1/X) in the range of 25-10000 ng/mL with inter- and intra-day precision (% CV) of 7.69-14.9%, and accuracy (% bias) of 99.1-105.3%. Mean recovery of efavirenz from CVS was 83.8% (%CV, 11.2) with no significant matrix effect. Efavirenz remained stable in swabs for at least 35 days after drying and storage at room temperature. Median (range) CVS efavirenz AUC 0-24h was 16370 ng*h/mL (5803-22088), C max was 1618 ng/mL (610-2438) at a T max of 8.0 h (8.0-12), and C min was 399 ng/mL (110-981). Efavirenz CVS:plasma AUC 0-24 ratio was 0.41 (0.20-0.59). Conclusions: Further application of this method will improve our understanding of the pharmacology of other therapeutics in the female genital tract, including in low- and middle-income countries.

Infant Exposure to Dolutegravir Through Placental and Breast Milk Transfer: A Population Pharmacokinetic Analysis of DolPHIN-1.

BACKGROUND: Rapid reduction in human immunodeficiency virus (HIV) load is paramount to prevent peripartum transmission in women diagnosed late in pregnancy. We investigated dolutegravir population pharmacokinetics in maternal plasma, umbilical cord, breast milk, and infant plasma samples from DolPHIN-1 participants (NCT02245022) presenting with untreated HIV late in pregnancy (28-36 weeks gestation). METHODS: Pregnant women from Uganda and South Africa were randomized (1:1) to daily dolutegravir (50 mg/d) or efavirenz-based therapy. Dolutegravir pharmacokinetic sampling (0-24 hours) was undertaken 14 days after treatment initiation and within 1-3 weeks after delivery, with matched maternal and cord samples at delivery. Mothers were switched to efavirenz, and maternal and infant plasma and breast milk samples were obtained 24, 48, or 72 hours after the switch. Nonlinear mixed-effects modeling was used to describe dolutegravir in all matrices and to evaluate covariates. RESULTS: A total of 28 women and 22 infants were included. Maternal dolutegravir was described by a 2-compartment model linked to a fetal and breast milk compartment. Cord and breast milk to maternal plasma ratios were 1.279 (1.209-1.281) and 0.033 (0.021-0.050), respectively. Infant dolutegravir was described by breast milk-to-infant and infant elimination rate constants. No covariate effects were observed. The median predicted infant dolutegravir half-life and median time to protein-adjusted 90% inhibitory concentration (0.064 mg/L) for those above this threshold were 37.9 (range, 22.1-63.5) hours and 108.9 (18.6-129.6) hours (4.5 [0.8-5.4] days) (n = 13), respectively. CONCLUSIONS: Breastfeeding contributed relatively little to infant plasma exposure, but a median of 4.5 days of additional prophylaxis to some of the breastfed infants was observed after cessation of maternal dolutegravir (3-15 days postpartum), which waned with time postpartum as transplacental dolutegravir cleared.

The development and validation of a novel LC-MS/MS method for the quantification of cenicriviroc in human plasma and cerebrospinal fluid.

A high-performance liquid chromatography tandem mass spectrometric method was developed and validated for cenicriviroc (CVC) quantification in human plasma and cerebrospinal fluid (CSF). The method involved precipitation with acetonitrile and injecting supernatants onto the column. Separation was achieved on an XBridge C18 column with a gradient elution of 0.1% formic acid in water and acetonitrile. Analyte detection was conducted in positive ion mode using selected reaction monitoring. The m/z transitions were: CVC (697.3 → 574.3) and CVC-d7 (704.4 → 574.3). Calibration curve ranged from 5 to 1000 ng/mL for plasma and from 0.241 to 15.0 ng/mL for CSF. The intra- and inter-day precision and accuracy were <15% for both plasma and CSF across four different concentrations. CVC recovery from plasma and artificial CSF was >90%. The method was utilized for the measurement of patients' plasma and CSF samples taking a dose of 50, 150 and 300 mg q.d.

Safety and pharmacokinetics of dolutegravir in pregnant mothers with HIV infection and their neonates: A randomised trial (DolPHIN-1 study).

BACKGROUND: The global transition to use of dolutegravir (DTG) in WHO-preferred regimens for HIV treatment is limited by lack of knowledge on use in pregnancy. Here we assessed the relationship between drug concentrations (pharmacokinetics, PK), including in breastmilk, and impact on viral suppression when initiated in the third trimester (T3). METHODS AND FINDINGS: In DolPHIN-1, HIV-infected treatment-naïve pregnant women (28-36 weeks of gestation, age 26 (19-42), weight 67kg (45-119), all Black African) in Uganda and South Africa were randomised 1:1 to dolutegravir (DTG) or efavirenz (EFV)-containing ART until 2 weeks post-partum (2wPP), between 9th March 2017 and 16th January 2018, with follow-up until six months postpartum. The primary endpoint was pharmacokinetics of DTG in women and breastfed infants; secondary endpoints included maternal and infant safety and viral suppression. Intensive pharmacokinetic sampling of DTG was undertaken at day 14 and 2wPP following administration of a medium-fat breakfast, with additional paired sampling between maternal plasma and cord blood, breastmilk and infant plasma. No differences in median baseline maternal age, gestation (31 vs 30 weeks), weight, obstetric history, viral load (4.5 log10 copies/mL both arms) and CD4 count (343 vs 466 cells/mm3) were observed between DTG (n = 29) and EFV (n = 31) arms. Although DTG Ctrough was below the target 324ng/mL (clinical EC90) in 9/28 (32%) mothers in the third trimester, transfer across the placenta (121% of plasma concentrations) and into breastmilk (3% of plasma concentrations), coupled with slower elimination, led to significant infant plasma exposures (3-8% of maternal exposures). Both regimens were well-tolerated with no significant differences in frequency of adverse events (two on DTG-ART, one on EFV-ART, all considered unrelated to drug). No congenital abnormalities were observed. DTG resulted in significantly faster viral suppression (P = 0.02) at the 2wPP visit, with median time to <50 copies/mL of 32 vs 72 days. Limitations related to the requirement to initiate EFV-ART prior to randomisation, and to continue DTG for only two weeks postpartum. CONCLUSION: Despite low plasma DTG exposures in the third trimester, transfer across the placenta and through breastfeeding was observed in this study, with persistence in infants likely due to slower metabolic clearance. HIV RNA suppression <50 copies/mL was twice as fast with DTG compared to EFV, suggesting DTG has potential to reduce risk of vertical transmission in mothers who are initiated on treatment late in pregnancy. TRIAL REGISTRATION: clinicaltrials.gov NCT02245022.

Pharmacokinetics of dolutegravir with and without darunavir/cobicistat in healthy volunteers.

Background: Dolutegravir combined with darunavir/cobicistat is a promising NRTI-sparing and/or salvage strategy for the treatment of HIV-1 infection. Methods: This Phase I, open-label, 57 day, crossover, pharmacokinetic (PK) study, enrolled healthy volunteers aged 18-65 years, who were randomized to one of two groups. Group 1 received dolutegravir (50 mg) once daily for 14 days followed by a 7 day washout, then a 14 day dolutegravir/darunavir/cobicistat (DTG/DRV/COBI) once-daily co-administration period followed by a 7 day washout and finally a 14 day period of darunavir/cobicistat (800/150 mg) once daily. Group 2 followed the same sequence starting with darunavir/cobicistat and concluding with dolutegravir. Each group underwent intensive PK sampling over 24 h on day 14 of each drug period and DTG/DRV/COBI concentrations were measured using validated LC-MS/MS methods. Results: Twenty participants completed all PK phases. Thirteen were female and median age and BMI were 33.5 years and 27 kg/m2. Dolutegravir geometric mean ratios (GMR, DTG/DRV/COBI versus dolutegravir alone) and 90% CI for Cmax, AUC0-24 and C24 were 1.01 (0.92-1.11), 0.95 (0.87-1.04) and 0.9 (0.8-1.0), respectively. Darunavir GMR (DRV/COBI/DTG versus darunavir/cobicistat alone) and 90% CI for Cmax, AUC0-24 and C24 were 0.90 (0.83-0.98), 0.93 (0.86-1.00) and 0.93 (0.78-1.11), respectively. No grade 3 or 4 adverse events or laboratory abnormalities were observed. Conclusions: Concentrations of dolutegravir and darunavir, when boosted with cobicistat, decreased by <10% during co-administration, suggesting this combination can be prescribed safely in the treatment of HIV-1, with no adjustment to current guideline-recommended dosages.

Drug Interactions between Dolutegravir and Artemether-Lumefantrine or Artesunate-Amodiaquine.

Across sub-Saharan Africa, patients with HIV on antiretrovirals often get malaria and need cotreatment with artemisinin-containing therapies. We undertook two pharmacokinetic studies in healthy volunteers, using standard adult doses of artemether-lumefantrine or artesunate-amodiaquine given with 50 mg once daily dolutegravir (DTG) to investigate the drug-drug interaction between artemether-lumefantrine or artesunate-amodiaquine and dolutegravir. The dolutegravir/artemether-lumefantrine interaction was evaluated in a two-way crossover study and measured artemether, dihydroartemisinin, lumefantrine, and desbutyl-lumefantrine over 264 h. The dolutegravir/artesunate-amodiaquine interaction was investigated using a parallel study design due to long half-life of the amodiaquine metabolite, desethylamodiaquine and measured artesunate, amodiaquine, and desethylamodiaquine over 624 h. Noncompartmental analysis was performed, and geometric mean ratios and 90% confidence intervals were generated for evaluation of both interactions. Dolutegravir did not significantly change the maximum concentration in plasma, the time to maximum concentration, and the area under the concentration-time curve (AUC) for artemether, dihydroartemisinin, lumefantrine, and desbutyl-lumefantrine, nor did it significantly alter the AUC for artesunate, dihydroartemisinin, amodiaquine, and desethylamodiaquine. Coadministration of dolutegravir with artemether-lumefantrine resulted in a 37% decrease in DTG trough concentrations. Coadministration of dolutegravir with artesunate-amodiaquine resulted in 42 and 24% approximate decreases in the DTG trough concentrations and the AUC, respectively. The significant decreases in DTG trough concentrations with artemether-lumefantrine and artesunate-amodiaquine and dolutegravir exposure with artesunate-amodiaquine are unlikely to be of clinical significance since the DTG trough concentrations were above dolutegravir target concentrations of 300 ng/ml. Study drugs were well tolerated with no serious adverse events. Standard doses of artemether-lumefantrine and artesunate-amodiaquine should be used in patients receiving dolutegravir. (This study has been registered at ClinicalTrials.gov under identifier NCT02242799.).

Validation and clinical application of a novel LC-MS method for quantification of dolutegravir in breast milk.

Aim: A novel, sensitive and reproducible method for quantification of dolutegravir (DTG) in dried breast milk spots (DBMS) was developed and validated for use in clinical studies. Its application enabled measurement of DTG pharmacokinetics in breastfeeding mothers and their infants. Results/methodology: Sample extraction was by liquid-liquid extraction using tert-butyl methy-ether, with DTG-d5 as an internal standard. DTG was eluted on a reverse phase C18 Waters XBridge (3.5 µm: 2.1 × 50 mm) column using a gradient mobile phase consisting of 0.1% formic acid in deionised water or methanol. The assay was validated over a calibration range of 10-4000 ng/ml. Conclusion: Stability, inter and intra-assay variability were acceptable according to FDA and EMA bioanalytical method guidelines. The assay is robust, accurate, precise and can be reliably applied for analysis of clinical samples in trials from low resource settings.

Cerebrospinal fluid drug concentrations and viral suppression in HIV-1-infected patients receiving ritonavir-boosted atazanavir plus lamivudine dual antiretroviral therapy (Spanish HIV/AIDS Research Network, PreEC/RIS 39).

This study aimed to assess cerebrospinal fluid (CSF) drug concentrations and viral suppression in HIV-1-infected patients on ritonavir-boosted atazanavir (ATV/r) plus lamivudine (3TC) dual therapy. HIV-1-infected adults with suppressed plasma HIV-1 RNA who switched to ATV/r plus 3TC were studied. Total ATV and 3TC concentrations at the end of the dosing interval (C24h), using a validated LC-MS/MS method, and HIV-1 RNA were measured in paired CSF and plasma samples 12 weeks after switching. Ten individuals were included. Median (range) age was 42.5 (33-70) years, time on ART was 39.5 (11-197) months, and time with plasma HIV-1 RNA < 40 copies/mL was 15.5 (6-46) months. At baseline, CSF HIV-1 RNA was < 40 copies/mL in all patients. Twelve weeks after switching to ATV/r plus 3TC, HIV-1 RNA remained at < 40 copies/mL in both plasma and CSF in 9/10 patients. One patient with suboptimal adherence to ART had HIV-1 RNA rebound in both plasma and CSF. The median CSF-to-plasma concentration ratios of ATV and 3TC were 0.013 and 0.417, respectively. Median ATV C24h in CSF was 10.4 (3.7-33.4) ng/mL (in vitro ATV IC50 range, 1-11 ng/mL). Median 3TC C24h in CSF was 43.4 (16.2-99.3) ng/mL (in vitro 3TC IC50 range, 0.68-20.6 ng/mL). Most patients maintained HIV-1 RNA in CSF < 40 copies/mL despite CSF ATV C24h close to or within the IC50 range in the majority. ATV PK data in CSF should be considered and rigorous patient selection is advisable to assure effective CSF viral suppression with this two-drug simplification regimen.

Comparison of Dried Blood Spots Versus Conventional Plasma Collection for the Characterization of Efavirenz Pharmacokinetics in a Large-Scale Global Clinical Trial-The ENCORE1 Study.

BACKGROUND: The aim of this study was to determine the utility of dried blood spots (DBS) compared with conventional plasma collection methods for characterization of efavirenz pharmacokinetics, in the setting of a large-scale, global clinical trial (ENCORE1). METHODS: Six hundred thirty patients were recruited from 38 sites and had single matched whole blood DBS and plasma samples (mid-dose interval) taken at weeks 4 and 12 of treatment. In addition, a subgroup of patients underwent intensive DBS and plasma sampling (0-24 hours) to provide full-profile data for pharmacokinetic parameters. Efavirenz concentrations were determined by validated high-performance liquid chromatography-mass spectrometry methods. A DBS-predicted plasma concentration was derived and linear regression and Bland-Altman plots were used to compare DBS-predicted plasma concentrations with that of measured plasma concentrations. RESULTS: Efavirenz DBS and plasma concentrations were significantly correlated (R = 0.904, P < 0.001; n = 1094), and DBS concentrations were, on average, 53% ± 9.5% lower than plasma. In the main study, the DBS-predicted plasma values significantly underestimated the true measured concentration of efavirenz in plasma; the mean difference (95% confidence interval) between efavirenz DBS-predicted concentrations and measured plasma concentrations was -0.451 mg/L (-0.504 to -0.398) at week 4 (n = 561). However, in the intensive study, the mean difference was only 0.086 mg/L (-0.006 to 0.178) at 12 hours after dose (n = 46) and was not statistically significant. CONCLUSIONS: Our data show a high correlation between measurements of efavirenz concentrations in plasma and in DBS. However, DBS concentrations significantly underestimated the true measured plasma concentrations in the sparse samples taken in this large multinational ENCORE1 trial.

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.